1 Trp Channels and Human Diseases

VANILLOID RECEPTOR TRPV1 IN DRUG DISCOVERY

Targeting Pain and Other Pathological Disorders

Edited by

ARTHUR GOMTSYAN CONNIE R. FALTYNEK Abbott Laboratories

Published by John Wiley & Sons, Inc., Hoboken, New Jersey

Published simultaneously in Canada

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Library of Congress Cataloging-in-Publication Data

ISBN: 978-0-470-17557-6

Printed in the United States of America

10 9 8 7 6 5 4 3 2 1 Arthur wishes to dedicate this book to his wife Natalia, daughters Anahit, Lusine, Ani, and parents, especially father, with love and gratitude.

Connie dedicates this book with love to her husband Robert, who is her lifelong dearest friend and soul mate.

CONTENTS

PREFACE AND ACKNOWLEDGMENTS ix FOREWORD xiii CONTRIBUTORS xv

PART I INTRODUCTION TO THE TRP CHANNELS 1

1 TRP Channels and Human Diseases 3 Bernd Nilius and Rudi Vennekens 2 Role of TRP Channels in Pain: An Overview 68 Arpad Szallasi 3 Biochemical Pharmacology of TRPV1: Molecular Integrator of Pain Signals 101 Carol S. Surowy, Philip R. Kym, and Regina M. Reilly 4 TRPV1 Genetics 134 Ruslan Dorfman, Hubert Tsui, Michael W. Salter, and H.-Michael Dosch

PART II ROLE FOR TRPV1 IN PAIN STATES 151

5 TRPV1 and Inﬂ ammatory Pain 153 Anindya Bhattacharya, Sonya G. Lehto, and Narender R. Gavva 6 Role of TRPV1 Receptors in Osteoarthritic Pain 175 Shailen K. Joshi and Prisca Honore 7 TRPV1 and Bone Cancer Pain 191 Juan Miguel Jimenez-Andrade and Patrick Mantyh 8 TRPV1 in Visceral Pain and Other Visceral Disorders 206 António Avelino and Francisco Cruz

vii viii CONTENTS

9 TRPV1 Receptors and Migraine 239 Philip R. Holland and Peter J. Goadsby 10 TRPV1 in Neuropathic Pain and Neurological and Neuropsychiatric Disorders 260 Enza Palazzo, Katarzyna Starowicz, Sabatino Maione, and Vincenzo Di Marzo

PART III TRPV1 ANTAGONISTS AND AGONISTS AS NOVEL ANALGESIC DRUGS 293

11 Aryl-Urea Class and Related TRPV1 Antagonists 295 Arthur Gomtsyan 12 2-Pyridinylpiperazine Carboxamide Class and Related TRPV1 Antagonists 311 Natalie A. Hawryluk and Nicholas I. Carruthers 13 TRPV1 Agonist Approaches for Pain Management 325 Keith R. Bley

PART IV ROLE FOR TRPV1 IN OTHER PHYSIOLOGICAL PROCESSES BESIDES PAIN TRANSMISSION 349

14 The TRPV1 Channel in Normal Thermoregulation: What Have We Learned from Experiments Using Different Tools? 351 Andras Garami, Maria C. Almeida, Tatiane B. Nucci, Tamara Hew-Butler, Renato N. Soriano, Eszter Pakai, Kazuhiro Nakamura, Shaun F. Morrison, and Andrej A. Romanovsky 15 The Role of TRPV1 in Respiratory Diseases 403 Serena Materazzi, Alain Tchoimou, Romina Nassini, Marcello Trevisani, and Pierangelo Geppetti 16 The Role of TRPV1 in Diabetes 423 Hubert Tsui, Ruslan Dorfman, Michael W. Salter, and H.-Michael Dosch

AFTERWORD 449 INDEX 451 PREFACE AND ACKNOWLEDGMENTS

The intended purpose of this book is to summarize available data related to the therapeutic utility for transient receptor potential vanilloid 1 (TRPV1) ligands. Many of the world ’ s leading experts on TRPV1 contributed to this book, not only by reviewing the most relevant information on the subject but also by providing personal opinions on the critical issues. The history of the TRPV1 receptor is, for the most part, the history of chili pepper, a popular hot spice that has been cultivated in South America for over 6000 years and in the rest of the world since the sixteenth century. However, food fl avoring has not been the only benefi t chili peppers have offered. Chili preparations have been used as treatments for ulcers, backaches, and coughs, among other maladies. The ingredient that is responsible for the initial burning effect of chili pepper, as well as for its medicinal properties, is a compound called capsaicin, which constitutes approximately 0.14% w/w of chili peppers. Capsaicin was isolated in 1876, and its structure was determined shortly thereafter. The homovanillyl fragment in the structure of capsaicin was the basis for naming the putative cell surface receptor vanilloid receptor 1 (VR1). An amazing breakthrough occurred in 1997, when VR1 was molecularly cloned by David Julius ’ team. VR1 later was renamed the TRPV1 to underscore its membership in a larger family of transient receptor potential (TRP) ion channels. The Foreword by Professor Julius presents an illuminating perspective on the entire fi eld. The two introductory chapters in this volume (Nilius and Vennekens; Szallasi) are designed to familiarize the reader with the multifunctional TRP family of ion channels and, at the same time, to lay the foundation for the remaining chapters, which focus on one member of that family, TRPV1. Members of the TRP superfamily have been shown to be polymodal molecular ix x PREFACE AND ACKNOWLEDGMENTS sensors, with the important roles of detecting a variety of thermal, mechanical, and chemical stimuli. These ion channels have been shown to contribute to sensory processes such as pain transmission, vision, taste, and hearing. The complex nature of TRPV1 modulation is described in the chapter by Surowy et al., which provides basic information about the structure of the receptor, numerous regulators (activators and sensitizers), and their modes and sites of action. The chapter on TRPV1 genetics (Dorfman et al.) reviews the impact of TRPV1 genetic variability on the pharmacological properties of TRPV1 in different species and summarizes available data on identifi cation of the binding sites for biological substrates. Several key studies with TRPV1 knockout mice and with TRPV1 antagonists, support the conclusion that TRPV1 plays an important role in pain pathways. Indeed, most of the advances with TRPV1 as a new molecular target for novel medications have been in the development of new analgesics. The next chapters discuss in detail the connection between TRPV1 and various types of pain. Preclinical studies provide a strong case for the benefi cial effects of TRPV1 blockade for the treatment of infl ammatory, osteoarthritic, and bone cancer pain. The effi cacy of TRPV1 antagonists in animal models of infl ammatory pain is discussed in the chapter by Bhattacharya et al. Several preclinical studies have shown the benefi t of TRPV1 antagonists in treating the pain associated with osteoarthritis (Joshi and Honore). It has also been shown that bone cancer pain, which is often poorly controlled by existing analgesics, can be successfully attenuated in mice by treatment with TRPV1 antagonists (Jimenez- Andrade and Mantyh). TRPV1 has been actively investigated as a potential target for conditions such as visceral pain and the treatment of lower urinary tract symptoms, including urinary frequency, urgency, and incontinence (Avelino and Cruz). Evidence for the role of TRPV1 in migraine (Holland and Goadsby) and in neuropathic pain (Palazzo et al.) is limited and inconclusive. To conclude the theme of TRPV1 antagonists and pain, two medicinal chemistry chapters summarize structural and structure – activity relationship information on the two major classes of TRPV1 antagonists: aryl- ureas (Gomtsyan) and pyridinylpiperazine carboxamides (Hawryluk and Carruthers). While the blockade of receptor activation by TRPV1 antagonists attenuates pain transmission, TRPV1 agonists have also been shown to be effective in pain management, especially for peripheral neuropathies (Bley). Antinociception induced by TRPV1 agonists is the result of receptor desensitization/defunctionalization after prolonged administration of the agonist. In fact, capsaicin cream has been used for many years to treat peripheral neuropathies, and new topical and injectable formulations of capsaicin with better safety margins are undergoing late - stage clinical trials. This volume also presents experts’ opinions on the links between TRPV1 and disease states other than pain. Some of these links are well established. For example, high expression of TRPV1 on sensory nerves in airways led PREFACE AND ACKNOWLEDGMENTS xi scientists to explore the potential for TRPV1 antagonists in treating chronic cough, asthma, and other respiratory diseases (Materazzi et al.). A number of such compounds have shown potent antitussive activity in preclinical models of cough, and at least one compound (MK - 2295) has entered clinical testing. The role of TRPV1 in some other diseases is less well validated. Expression of functional TRPV1 receptors in the brain suggests the possibility of novel strategies for the treatment of neurological and perhaps neuropsychiatric disorders (Palazzo et al.), while recent observations with non - obese diabetic mice suggest a potentially important role for TRPV1 in the development of diabetes (Tsui et al.). One of the properties of TRPV1 and other members of the TRP family is the ability to detect thermal stimuli within a specifi c intensity range. The chapter by Garami et al. discusses the role of TRPV1 in thermoregulation, presenting both a mechanistic point of view and the implications for the development of safe TRPV1 antagonist - based therapies. Recent setbacks in the clinical development of TRPV1 antagonists due to the induction of transient hyperthermia and a defi cit in perception of potentially injurious heat make this chapter especially relevant. Our hope is that this book successfully highlights the role of TRPV1 as one of the best- known integrators of multiple painful stimuli and serves as a good reference regarding current and potential future applications of TRPV1 ligands in treating pain as well as other pathophysiological conditions.

The editors are very grateful to the authors for their excellent contributions and their understanding of efforts to keep the delicate balance between appreciation of the busy schedules of the authors and the deadlines set by the publisher. We also express our indebtedness to Jonathan Rose at John Wiley & Sons for inviting us to serve as editors for this book and for patiently providing guidance throughout the process.

A rthur Gomtsyan C onnie R. Faltynek

FOREWORD

The use of plant extracts in folk medicine can be traced back thousands of years to geographically and ethnically diverse cultures. Such accumulated wisdom has served as the starting point for the development of important experimental and therapeutic drugs. This process is perhaps best exemplifi ed by pain medications, so many of which — particularly those of the opiate and NSAID class — were inspired by the active ingredients in opium poppies and in willow bark, respectively. While some natural plant products (such as opiates) suppress pain, others produce it. This is certainly the case for capsaicin, the pungent agent in “ hot ” chili peppers that elicits that familiar and intense sensation of burning pain. Just as morphine and aspirin have served as chemical keys for unlocking cellular mechanisms of pain suppression, capsaicin and other pungent natural products have helped to defi ne the neurons, molecules, and signaling pathways that initiate or enhance pain under normal (acute) and pathological (chronic) circumstances. The culinary and medicinal wonders of capsaicin and related vanilloid compounds can be traced to Central and South America, where Capsicum plants have their indigenous roots. Thousands of years later, Christopher Columbus and other explorers introduced chili peppers to European, Asian, and African continents, where they have been similarly and widely exploited for sensorial and medicinal applications ranging from food preservation to hypothermic cooling. In more recent times, seminal work from Jansco and colleagues in Hungary showed that capsaicin functions as a highly selective excitatory agent for a subset of primary afferent sensory neurons, making sensitivity to capsaicin a defi ning functional hallmark of the nociceptor. Consequently, the goal of delineating a mechanism of capsaicin action, and in particular, identifying a “ vanilloid receptor, ” became a holy grail of pain xiii xiv FOREWORD research, much in the same way that molecular identifi cation of the T- cell receptor was seen as a watershed in the genetic and functional characterization of the lymphocyte. Leading up to this goal were the fundamental contributions of Rang, Bevan, Wood, and colleagues at the Sandoz Institute, as well as those of Blumberg and coworkers at the U.S. National Cancer Institute, whose early electrophysiological and pharmacological studies helped to better defi ne and to validate a bona fi de “ vanilloid site.” Still, this putative receptor defi ed genetic identifi cation for quite some time, and thus its existence and molecular nature remained enigmatic. The cloning and molecular characterization of TRPV1 (n é VR1) by Michael Caterina and by other members of my group at once resolved these issues while providing a defi nitive molecular explanation for the highly selective nature of capsaicin action. In doing so, this accomplish- ment gave molecular validation to the so- called specifi city theory of nociception fi rst espoused by Sherrington over a century ago. From a more general perspective, cloning of the vanilloid receptor put a spotlight on TRP channels as important new players in vertebrate sensory systems. In retrospect, this should not have come as a great surprise given the well - established role of TRP channels in fl y phototransduction, but for whatever reason, their relevance to sensory signaling in higher systems had not been fully appreciated. The study of TRPV1 has established several important new paradigms that are of general relevance to sensory signaling but also hold special signifi cance for nociception and pain. Of course, one of these relates to the discovery that TRPV1 functions as a thermosensor, which has provided a molecular and cellular framework for understanding how ion channels sense heat or cold, and how primary afferent neurons detect changes in ambient temperature. Additionally, these studies set the stage for understanding how TRP channels function as polymodal signal detectors that can integrate information from both physical and chemical stimuli to modulate neuronal excitability in the face of changing environmental or physiological conditions. In the context of the primary afferent nociceptor, this has great signifi cance for understanding how tissue injury and infl ammation produce pain hypersensitivity. The molecular analysis of TRPV1 has opened up exciting new vistas in the study of somatosensation, nociception, and pain. Of course, the full therapeutic impact of this work will come with the development of analgesic agents that target TRP channels on primary afferent nociceptors. Should this come to pass — as I expect it will — then we can add capsaicin to the list of natural products that have inspired novel classes of pain medicines.

D avid Julius San Francisco CONTRIBUTORS

Maria C. Almeida, Systemic Inﬂ ammation Laboratory, Trauma Research, St. Joseph ’ s Hospital and Medical Center, Phoenix, AZ

Ant ó nio Avelino, Institute of Histology and Embryology, Faculty of Medi- cine of Porto, Alameda Hernani Monteiro, Porto, Portugal; IBMC, University of Porto, R. Campo Alegre, Porto, Portugal; Email: aavelino@ med.up.pt

Anindya Bhattacharya, Department of Pain and Related Disorders, Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 3210 Merryﬁ eld Row, San Diego, CA 92121

Keith R. Bley, NeurogesX, Inc., 2201 Bridgepointe Parkway, San Mateo, CA 94404; Email: [email protected]

Nicholas I. Carruthers, Johnson & Johnson Pharmaceutical Research & De- velopment, L.L.C. San Diego, CA; Email: [email protected]

Francisco Cruz, Institute of Histology and Embryology, Faculty of Medicine of Porto, Alameda Hernani Monteiro, Porto, Portugal; IBMC, University of Porto, R. Campo Alegre, Porto, Portugal; Department of Urology, Hospital Sã o Joã o and Faculty of Medicine of Porto, Porto, Portugal; Email: [email protected]

Vincenzo Di Marzo, Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Naples, Italy; Email: [email protected] xv xvi CONTRIBUTORS

Ruslan Dorfman, University of Toronto, The Hospital For Sick Children, Research Institute, Genetics & Genome Biology Program, Toronto, Ontario, Canada

H. - Michael Dosch, University of Toronto, The Hospital For Sick Children, Research Institute, Neurosciences & Mental Health Programs, Toronto, Ontario, Canada; Email: [email protected]

Connie R. Faltynek, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL; Email: [email protected]

Andras Garami, Systemic Inﬂ ammation Laboratory, Trauma Research, St. Joseph ’ s Hospital and Medical Center, Phoenix, AZ

Narender R. Gavva, Department of Neuroscience Amgen Inc., Thousand Oaks, CA; Email: [email protected]

Pierangelo Geppetti, Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy; Center of Excellence for the Study of Inﬂ ammation, University of Ferrara, Ferrara, Italy; Email: pierangelo. geppetti@uniﬁ .it

Peter J. Goadsby, Headache Group Department of Neurology, University of California, San Francisco, CA; Email: [email protected]

Arthur Gomtsyan, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL; Email: [email protected]

Natalie A. Hawryluk, Johnson & Johnson Pharmaceutical Research & Development, L.L.C. San Diego, CA

Tamara Hew - Butler, Systemic Inﬂ ammation Laboratory, Trauma Research, St. Joseph ’ s Hospital and Medical Center, Phoenix, AZ

Philip R. Holland, Headache Group Department of Neurology University of California, San Francisco, CA

Prisca Honore, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL; Email: [email protected]

Juan Miguel Jimenez - Andrade, Department of Pharmacology, University of Arizona, Tucson, AZ

Shailen K. Joshi, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL; Email: shailen. [email protected] CONTRIBUTORS xvii

David Julius, Department of Physiology and Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA Email: [email protected] Philip R. Kym, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL Sonya G. Lehto, Department of Neuroscience Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA Sabatino Maione, Endocannabinoid Research Group, Department of Experimental Medicine, Section of Pharmacology “ L. Donatelli,” Faculty of Medicine and Surgery, Second University of Naples, Naples, Italy; Email: [email protected] Patrick Mantyh, Department of Pharmacology, University of Arizona, Tucson, AZ; Research Service, VA Medical Center, Minneapolis, MN Email: [email protected] Serena Materazzi, Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy Shaun F. Morrison, Division of Neuroscience, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR Kazuhiro Nakamura, Division of Neuroscience, Oregon National Primate Research Center, Oregon Health & Science University, Beaverton, OR Romina Nassini, Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy. Bernd Nilius, KU Leuven, Department of Molecular Cell Biology, Laboratory Ion Channel Research, Campus Gasthuisberg, Leuven, Belgium; Email: [email protected] Tatiane B. Nucci, Systemic Infl ammation Laboratory, Trauma Research, St. Joseph ’ s Hospital and Medical Center, Phoenix, AZ Eszter Pakai, Systemic Infl ammation Laboratory, Trauma Research, St. Joseph ’ s Hospital and Medical Center, Phoenix, AZ Enza Palazzo, Endocannabinoid Research Group, Department of Experimental Medicine, Section of Pharmacology “ L. Donatelli,” Faculty of Medicine and Surgery, Second University of Naples, Naples, Italy Regina M. Reilly, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL Andrej A. Romanovsky, Systemic Infl ammation Laboratory, Trauma Research, St. Joseph’ s Hospital and Medical Center, Phoenix, AZ; Email: [email protected] xviii CONTRIBUTORS

Michael W. Salter, University of Toronto, The Hospital For Sick Children, Research Institute, Neurosciences & Mental Health Programs, Toronto, Ontario, Canada Renato N. Soriano, Systemic Inﬂ ammation Laboratory, Trauma Research, St. Joseph ’ s Hospital and Medical Center, Phoenix, AZ Katarzyna Starowicz, Department of Pain Pharmacology, Institute of Pharmacology PAS, 12 Smetna str 31343 Cracow Poland; Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Naples, Italy Carol S. Surowy, Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL; Email: [email protected] Arpad Szallasi, Departments of Pathology and Laboratory Medicine, Monmouth Medical Center, Long Branch, NJ; Drexel University College of Medicine, Philadelphia, PA; Email: [email protected] Alain Tchoimou, Department of Preclinical and Clinical Pharmacology, University of Florence, Florence, Italy Marcello Trevisani, Center of Excellence for the Study of Inﬂ ammation, University of Ferrara, Ferrara, Italy; present address: PharmEste srl, Ferrara, Italy Hubert Tsui, University of Toronto, The Hospital For Sick Children, Research Institute, Neurosciences & Mental Health Programs, Toronto, Ontario, Canada Rudi Vennekens, KU Leuven, Department of Molecular Cell Biology, Laboratory Ion Channel Research, Campus Gasthuisberg, Leuven, Belgium PART I

INTRODUCTION TO THE TRP CHANNELS

1 TRP CHANNELS AND HUMAN DISEASES

Bernd Nilius and Rudi Vennekens

1.1 INTRODUCTION

Transient receptor potential (TRP) cation channels have been described in detail as polymodal cell sensors in many reviews (Nilius and Voets, 2005 ; Pedersen et al., 2005 ; Voets et al., 2005 ; Nilius and Mahieu, 2006 ; Ramsey et al., 2006 ; Nilius, 2007 ; Nilius et al., 2007 ; Venkatachalam and Montell, 2007 ). The mammalian TRP superfamily consists of 28 mammalian TRP cation channels, which can be subdivided into six main subfamilies: the TRPC (“ Canonical ” ), TRPV ( “ Vanilloid ” ), TRPM ( “ Melastatin ” ), TRPP ( “ Polycystin ” ), TRPML ( “ MucoLipin ” ), and the TRPA ( “ Ankyrin ” ) groups. In the current review, we attempt to create an overview of the currently available data on the functional role of TRP channels and their possible signifi cance for human disease. We will extend and complement the very comprehensive reviews on this topic published just recently (Jordt and Ehrlich, 2007 ; Kiselyov et al., 2007 ; Nilius, 2007 ; Nilius et al., 2007 ). For that reason, and due to space limitations, it is inevitable that some topics will not be covered at this occasion (Fig. 1.1 ). Several TRP channelopathies are known, that is, diseases with identifi ed defects in the gene encoding the channels. Channel dysfunctions caused by regulatory proteins can also contribute to the genesis of several diseases and may result in changes in channel abundance, channel gating or modulation, and inadequate responses to various stimuli. Abnormal endogenous production of various agents during the development of a disease (e.g., in infl ammation conditions) can affect channel function and may determine the progression

3 4 TRP CHANNELS AND HUMAN DISEASES

TRPM3 TRPM6 “M, melastatin” “C, canonical” TRPM1 TRPM7 TRPM2 TRPC7 TRPM8 TRPC3 TRPM4 TRPC6 TRPM5 TRPC5 “P, polycystin” TRPP3 TRPC4 (PKD2L1) TRPC1 TRPP2 (PKD2) TRPV6 TRPP5 (PKD2L2) TRPV5 TRPML1 TRPV2 TRPML2 “V, vanilloid” TRPML3 TRPV1 TRPV4 TRPV3 “ML, mucolipin” Figure 1.1 A phylogenetic tree of the mammalian members of the TRP family. of the disease. This review will list all known mammalian TRP channels and will indicate links to possible diseases based on data from human patients, genetically manipulated organisms, and pharmacological studies. The increasing evidence that TRP channels are involved in human diseases has created a huge interest for these channels as novel drug targets. Comprehensive reviews have been published on the modulation of TRP channels by novel modulators and the general role of TRPs as pharmaceutical targets (see Okuhara et al., 2007 ; Szallasi et al., 2007 ; Landry and Gies, 2008 ). This topic will only sporadically be discussed.

1.2 THE “ CANONICAL” TRPC S

The mammalian TRPC (canonical) channels can be subdivided into four subclasses: TRPC1, TRPC2, TRPC3/6/7, and TRPC4/5 (Vazquez et al., 2004 ). All TRPC channels are nonselective Ca 2+ - permeable cation channels. TRPC1, TRPC4, and TRPC5 can form heteromers, and current properties are signiﬁ - cantly different between TRPC5 and TRPC1/TRPC5 - expressing cells. Similarly, TRPC3, TRPC6, and TRPC7 also can form heteromers (Strubing et al., 2001, 2003 ; Goel et al., 2002 ; Hofmann et al., 2002 ). TRPC activation occurs mainly via different isoforms of phospholipases (PLCs) (Venkatachalam et al., 2002 ). TRPC1, TRPC4, and TRPC5 are activated by receptor- induced PLC, but, in contrast to TRPC3, TRPC6, and TRPC7, are completely unresponsive to diacylglycerol (DAG) and show a quite complicated activation pattern including changes in membrane potential (voltage dependence), binding of multivalent cations to the pore region, and interaction with phosphatidylinositol phosphates (Hofmann et al., 1999 ; Venkatachalam et al., 2003 ; THE “CANONICAL” TRPCS 5

Chromosomal location of Homo sapiens TRP channel genes

85.20 (ML3) p p MCOLN3 q p p MCOLN2 p q p q p q q (ML2) q p P2 135.58 q 85.11 q A1 C3 C7 137.26 142.09 C1 PKD2L2 V6 M8 (P5) V5 Chr 1 Chr 2 Chr 3 Chr 4 Chr 5 Chr 6 Chr 7142.13 Chr 8 M5 P1 p p p p p q q q q p q M1 p M3 q C4 q M6 M7 Mg PKDL (P3) C6 V4

Chr 9 Chr 10 Chr 11 Chr 12 Chr 13 Chr 14 Chr 15 Chr 16 V3 36.67 V1 34.19 MCOLN1 V2 p (ML1) p p p q p q p p q q p q q q q C5 M4 PKDREJ M2 (P4) Chr 17 Chr 18 Chr 19 Chr 20 Chr 21 Chr 22 Chr X Chr Y Figure 1.2 Schematic overview of the chromosomal location of human TRP genes (reproduced from Abramowitz and Birnbaumer [2006 ] with the permission of Springer- Verlag and the authors) . (See color insert.)

Otsuguro et al., 2008 ). TRPC channels have been linked to cardiovascular disease (Yao and Garland, 2005 ; Dietrich et al., 2007 ; Firth et al., 2007 ; Kwan et al., 2007 ; Ohba et al., 2007 ), pulmonary disease (Meng et al., 2008 ), inﬂ ammation (Brechard et al., 2008 ), cancer (Fraser and Pardo, 2008 ), skin disease (Pani and Singh, 2008 ), and proliferative diseases via dysregulation of the cell cycle (Quadid - Ahidouch and Ahidouch, 2007 ) (Fig. 1.2 ).

1.2.1 TRPC1 1.2.1.1 Cardiovascular Cardiac hypertrophy is coupled to an increased Ca2+ entry and a dysfunction of Ca 2+ signaling (Bers and Guo, 2005 ) (for a review including the role of TRPs, see Watanabe et al. [ 2008 ]). Two lines of evidence suggest that TRPC1 may be involved in the development of cardiac hypertrophy. First, TRPC1 expression is signiﬁ cantly increased in the hearts of abdominal aortic- banded (AAB) rats, which develop pressure- induced hypertrophy, compared to sham - operated rats. It is also increased in cultured myocardial cells pretreated with hypertrophic factors such as endothelin - 1 (ET - 1), brain natriuretic peptide (BNP), and atrial natriuretic factor (ANF). Silencing of the TRPC1 gene via small interfering RNA (siRNA) prevents 6 TRP CHANNELS AND HUMAN DISEASES

Hypertrophic agonists: ATII ET-1 TRPC1 PE TRPC3 2+ Ca L-type Ca2+ channel TRPC6

2+ Δϕ Gαq DAG Ca 2+ PLC Ca2+ Ca IP3

Calcineurin ER Ca2+ store P NFAT

Positive feedback system NFAT

MEF2 NFAT GATA TRPC Cardiac growth and remodeling genes

Figure 1.3 Hypertrophic agents such as angiotensin II, endothelin- 1, and phenyleph- rin promote hypertrophy of the cardiomyocyte by causing excess Ca infl ux, which stimulates NFAT- driven gene expression of specifi c genes. Ca infl ux either occurs directly through TRP channels or the membrane potential is set by these channels, infl uencing Ca infl ux through L- type Ca channels. Expression of several TRPC channels is stimulated by NFAT, generating a positive feedback loop. See text for more details and references . ER: endo/sarcoplasmic reticulum; GATA, MEF2, and NFAT: nuclear transcription factors; IP3: inositol- 1,4,5 - trisphosphate. (See color insert.)

ET - 1 - , angiotensin II (ATII) -, and phenylephrine (PE) - induced cardiac hypertrophy. Second, ATII also induces hypertrophy of vascular smooth muscle cells (VSMCs). TRPC1 is overexpressed in these cells, and TRPC1 siRNA prevents the development of ATII- induced hypertrophy (Takahashi et al., 2007 ). The induction of TRPC1 expression is probably mediated through the nuclear factor of activated T cell (NFAT). The general mechanism for hypertrophy development might be that proliferative stimuli activate TRPC1, inducing depolarization and activation of L - type Ca2+ channels, which subse- 2+ quently increase cardiac [Ca ]i , activate calcineurin, and trigger NFAT translocation to the nucleus, which in turn results in an increased transcription of TRPC1 channels (Bush et al., 2006 ) (Fig. 1.3 ). Occlusive vascular disease is often a lethal complication in myocardial infarction, stroke, atherosclerosis, and clinical procedures such as angioplasty and grafting in bypass surgery. It can be caused by a switch in smooth muscle cell phenotype to an invasive and proliferative mode, leading to neointimal THE “CANONICAL” TRPCS 7 hyperplasia. TRPC1 might be involved in this switching of cell fate, which is associated with enhanced calcium entry and cell cycle activity. A speciﬁ c E3 - targeted (pore blocking) antibody to TRPC1 reduced neointimal growth in human veins, indicating that this might be a new avenue in the treatment of occlusive vascular diseases (Kumar et al., 2006 ; van Breemen et al., 2006 ).

1.2.1.2 Skeletal Muscle A role for TRPC1 in Duchenne muscular dystrophy (DMD) has been suggested extensively (Gailly, 2002 ; Hopf et al., 2007 ). In dystrophic myocytes, an increased Ca 2+ infl ux is apparent, which can be suppressed by antisense TRPC1, TRPC4, and TRPC6 oligonucleotides. Furthermore, a direct interaction between α 1 - syntrophin and TRPC1 was recently shown. Apparently, normal regulation of calcium infl ux in skeletal muscle depends on the association between TRPC1 channels and α 1 - syntrophin that may anchor the store - operated channels (SOCs) to the dystrophin - associated protein complex (DAPC). The loss of this molecular association could contribute to the calcium alterations observed in DMD cells (Vandebrouck et al., 2007 ). A further indication for the role of TRPC1 in a myopathy comes from homer 1− / − mice. Mice lacking homer 1 show decreased muscle fi ber cross - sectional area and decreased skeletal muscle force generation. Homer 1− / − myotubes displayed altered cation infl ux, which can be blocked by TRPC1 downregulation. Diminished Homer 1 expression in mouse models of DMD suggests that loss of Homer 1 scaffolding of TRP channels may contribute to the increased stretch- activated channel activity observed in mdx (dystrophin - defi cient) myofi bers (Stiber et al., 2008 ). Also, reactive oxygen species (ROS), which are increased in DMD, activate an src kinase, which in turn can activate TRPC1 (Gervasio et al., 2008 ).

1.2.1.3 Skin Darier ’ s disease (DD) is caused by mutations of SERCA2. Keratinocytes from DD patients show increased TRPC1 expression levels and increased Ca inﬂ ux. The latter might be involved in reinforcement of NFkB- mediated upregulation of antiapoptotic genes, for example, BclxL. TRPC1 therefore could increase cell proliferation, a hallmark of lesional keratinocytes (Beck et al., 2008 ; Pani and Singh, 2008 ).

1.2.1.4 Immunodefense TRPC1 is involved in antibody recognition in B lymphocytes. Suppression of TRPC1 results in reduced B - cell antigen receptor 2+ (BCR) - mediated oscillations in [Ca ]i and consequently depressed activation of the NFAT (Mori et al., 2002 ).

1.2.1.5 Neurological Disorders TRPC channels seem to be crucially involved in the signaling via metabotropic glutamate receptors (Kim et al., 2003 ) and in the actions of brain- derived neurotrophic factor (BDNF) (Li et al., 1999 ) that include netrin - 1 and BDNF - mediated growth cone guidance (Greka et al., 2003 ; Li et al., 2005c ; Shim et al., 2005 ). Recent studies indicate 8 TRP CHANNELS AND HUMAN DISEASES a potential link between TRPC1 and neurotoxicity induced by the exogenous agent 1 - methyl - 4 - phenylpyridium (MPP). The latter causes selective nigral dopaminergic lesions and induces Parkinson’ s disease- like syndromes. (Bollimuntha et al., 2005 ). Apparently, TRPC1 may execute a neuroprotective role in dopaminergic neurons. TRPC1 (and probably also TRPC3) are activated by the peptide hormone orexin A via the GPCR OX 1 (Larsson et al., 2005 ). This activation mode may link TRP channels to important physiological functions, because orexin regulates sleep/wakefulness states, alertness, and food intake (appetite). In addition, the orexin knockout mouse is a very useful model of human narcolepsy, a disorder that is characterized primarily by rapid eye movement (REM) sleep dysregulation.

1.2.2 TRPC2 The TRPC2 gene trpc2 is a pseudogene in humans and will not be discussed further in this review.

1.2.3 TRPC3 1.2.3.1 Cardiovascular TRPC3 might be critically involved in heart hypertrophy . RNAi - mediated knockdown of TRPC3 decreases expression of hypertrophy- associated genes such as the A- and B- type natriuretic peptides (ANP and BNP) in response to numerous hypertrophic stimuli. Overexpres- sion of TRPC3 increases BNP expression (Brenner and Dolmetsch, 2007 ). Increased Ca 2+ causes cell death in response to ischemia – reperfusion (I/R) . After I/R, apoptosis was signiﬁ cantly increased in TRPC3 overexpressing cardiomyocytes compared to control. TRPC3 overexpression increased not only apoptosis but also calpain- mediated proteolysis resulting from I/R injury, as well as sensitivity to Ca 2+ overload in cardiomyocytes (Shan et al., 2008 ). It is noteworthy to mention that Epo activates TRPC3. TRPC3 is expressed in human erythroid progenitor cells and may regulate Ca 2+ inﬂ ux during erythroid differentiation (Tong et al., 2008 ). Importantly, Epo secretion is critically regulated by the hypoxia - induced factor HIF - 1 , which in turn upregulates two other TRPCs, TRPC1 and TRPC6 (Wang et al., 2006 ).

1.2.3.2 Immunodefense Interestingly, mutations in TRPC3 induce a reduction of T - cell receptor activation - induced Ca 2+ entry in T lymphocytes, which may parallel the defective immune responses from patients with severe combined immunodeﬁ ciency (SCID) (Philipp et al., 2003 ).

1.2.3.3 Neurological Disorders TRPC3 seems to be critically involved in brain development. BDNF shapes synapses in hippocampal neurons. It also activates a nonselective cationic current (IBDNF ) in CA1 pyramidal neurons. Activation requires phospholipase C, IP3 receptors, and the respective stores THE “CANONICAL” TRPCS 9

2+ and depends on extracellular Ca . CA1 neurons express TRPC3, and I BDNF is absent after silencing with TRPC3 siRNA. A sustained phase of IBDNF depends on phosphatidylinositol 3- kinase, which is required for plasma membrane insertion of TRPC3. TRPC3 channels are necessary for BDNF to increase dendritic spine density(Amaral and Pozzo - Miller, 2007 ). Furthermore, the Δ 202 mouse line, which has defective TRPC3 expression, develops severe neurological problems, including paralysis and atrophy starting at week 4. Mice die around week 12. The neurologic syndrome of the Δ202 mice appears to be a monogenic recessive neuromotor disease caused by the interruption of the trpc3 gene, leading to a failure in the postnatal development of the central nervous system (CNS) (Rodriguez - Santiago et al., 2007 ). There may be a connection between TRPC3 and Parkinson ’ s disease : Parkinsonian movement disorders are often associated with abnormalities in the GABA neuron fi ring pattern in the substantia nigra pars reticulata. These neurons express TRPC3 channels, which are tonically active and mediate an inward Na+ current, leading to a substantial depolarization in these neurons. Inhibition of TRPC3 channels induces hyperpolarization, decreases fi ring frequency, and increases fi ring irregularity, all of which may contribute to movement disorders (Zhou et al., 2008a ). Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in protein kinase PKCg. This kinase normally inhibits TRPC3. The mutant PKCγ cannot phosphorylate TRPC3, resulting in sustained Ca2+ entry. This alteration in Ca 2+ homeostasis in Purkinje cells may contribute to neurodegeneration in SCA14 (Adachi et al., 2008 ).

1.2.4 TRPC4 1.2.4.1 Cardiovascular TRPC4 has been associated with regulation of vascular tone (Freichel et al., 2001 ) and endothelial permeability (Tiruppathi et al., 2002 ). Recently, TRPC4 has also been implicated in angiogenesis. The angiogenesis inhibitor thrombospondin - 1 (TSP1) controls the switch in renal cell carcinoma (RCC) toward an angiogenic phenotype. This factor is highly secreted in normal kidney cells, whereas RCC cells secrete little TSP1. Ca2+ inﬂ ux through TRPC4 may be critical for TSP1 secretion since expression of TRPC4 is very low in RCC cells, and TRPC4 silencing in normal kidney cells causes TSP1 retention and impaired secretion (Veliceasa et al., 2007 ).

1.2.4.2 Neurological Disorders Alzheimer ’ s disease has multiple genetic variants. A genome - wide screen of two extended pedigrees identifi ed a gene related to TRPC4, TRPC4- associated protein (TRPC4AP), on chromosome 20q11.22, as relevant for the disease. Multiple signifi cant single - nucleotide polymorphisms (SNPs) in this gene were found with an initial genome scan and were confi rmed by haplotype analysis. TRPC4AP might be involved 10 TRP CHANNELS AND HUMAN DISEASES with the disease in these late- onset Alzheimer’ s families, but the mechanism is unclear (Poduslo et al., 2009 ). TRPC4 is also present in F2 - synaptic terminals of the thalamic network. These terminals provide a GABAergic input into the dorsal lateral geniculate nucleus. GABA release from such F2 terminals depends on Ca 2+ infl ux initiated by metabotropic receptors. In trpc4 − /− mice, the 5- hydroxytryptamine (5- HT) - induced release of GABA from the thalamic interneurons is dramatically reduced (Munsch et al., 2003 ). This GABAergic component is critical for the control of the sleep/wake cycle, processing of visual information, and may also be important for the control of retinogeniculate transmission.

1.2.4.3 Skin Aberrant keratinocyte differentiation causes hyperproliferative skin diseases, including basal cell carcinoma (BCC). Ca2+ inﬂ ux plays a crucial role in this differentiation process. Knocking down of both TRPC4 and TRPC1 expression prevents Ca2+ - dependent differentiation of keratinocytes, and the expression of both channels is signiﬁ cantly decreased in BCC, suggesting a critical role for both channels in the process controlling normal keratinocyte differentiation (Beck et al., 2008 ).

1.2.5 TRPC5 1.2.5.1 Gastrointestinal Quite recently, TRPC5 and TRPC6 have been mentioned in infantile hypertrophic pyloric stenosis (IHPS) (OMIM #179010). This disease has an incidence of one to eight per 1000 live births and is inherited as a complex sex - modiﬁ ed multifactorial trait with a striking male pre- ponderance. The disease is characterized by projectile vomiting due to gastric outlet obstruction caused by hypertrophy of the smooth muscle of the pylorus. In a genome- wide SNP- based high- density linkage scan, two genomic loci were identiﬁ ed, which harbor TRPC6 (11q21- 22) and TRPC5 (Xq23) (Everett et al., 2008 ).

1.2.5.2 Joints The identifi cation of endogenous agents interacting with TRP channels is a major priority in the fi eld. Recently, it has been shown that activation of TRPC5 homomultimers and TRPC5 – TRPC1 heteromultimers can be mediated by extracellular reduced thioredoxin (Xu et al., 2008 ). Thioredoxin is an endogenous redox protein, which has several intracellular functions in cancer, ischeamic reperfusion injury, infl ammation, and aging. Also, thioredoxin is highly secreted in rheumatoid arthritis . TRPC5 and TRPC1 are expressed in secretory fi broblast- like synoviocytes (FLSs) (FLS cells). FLS cells secrete matrix metalloproteinases (MMPs), which promote the progression of arthritis. Thioredoxin decreases MMP secretion in these cells. (Q: is this OK?) Block of TRPC5- containing channels enhances secretion of MMPs and prevents suppression of MMP secretion by thioredoxin. Thus, TRPC5/TRPC1 – TRPC5 activation could represent a novel strategy in the treatment of rheumatoid arthritis (Xu et al., 2008 ). THE “CANONICAL” TRPCS 11

1.2.6 TRPC6 1.2.6.1 Cardiovascular Several lines of evidence point to a role for TRPC6 in multiple cardiovascular conditions. For instance, TRPC6 might play a role in idiopathic pulmonary arterial hypertension (IPAH) , which can lead to right heart failure. The crucial event for IPAH is excessive pulmonary artery smooth muscle cell proliferation, which is connected to overexpression of TRPC3 and TRPC6 (Yu et al., 2004 ). TRPC6 is also involved in angiotensin- induced heart hypertrophy. ATII induces NFAT activation, and cardiac hypertrophic responses have been correlated with TRPC6 activity (Onohara et al., 2006 ). TRPC6 may contribute to the development of hypoxic pulmonary hypertension (Wang et al., 2006 ). Finally, transgenic overexpression of TRPC3 and TRPC6 induces heart hypertrophy (Kuwahara et al., 2006 ; Nakayama et al., 2006 ). Apparently, NFAT consensus sites in the promoter of the TRPC6 gene confer responsiveness to cardiac stress. TRPC6 has been found to mediate membrane depolarization in smooth muscle cells and subsequent vasoconstriction induced by elevated intravascu- lar pressure, the important myogenic constriction response (Bayliss effect) in small arteries and arterioles (Welsh et al., 2002 ). However, somewhat paradoxically, trpc 6− / − mice display elevated blood pressure, which may be explained by a compensatory increase in the expression of the constitutively active TRPC3 channel in smooth muscle cells obtained from trpc6− / − mice (Dietrich et al., 2005 ). TRPC6 is activated by DAG and its derivatives (Dietrich and Gudermann, 2007 ). Cellular DAG levels are controlled by DAG kinase (DGK), which catalyzes DAG phosphorylation and acts as a regulator of GPCR signaling. DGKε acts speciﬁ cally on DAG produced by inositol cycling. Interestingly, a transgenic mouse with cardiac- speciﬁ c overexpression of DGK ε (DGK - TG ) was resistant to cardiac hypertrophy and progression to heart failure under chronic pressure overload. Increases in heart weight after PE infusion and thoracic aortic vasoconstriction (TAC) were abolished in DGK- TG mice. Cardiac dysfunction after TAC was prevented in DGK- TG mice, and survival rate after TAC was higher in DGK - TG mice. Also, upregulation of TRPC6 expression after TAC was attenuated in DGK - TG mice (Niizeki et al., 2008 ). These data further underscore a role for TRPC6 activity in the development of cardiac hypertrophy.

1.2.6.2 Kidney TRPC6 is one of the few TRP channels to which a real channelopathy can be assigned. Six mutations of the trpc6 gene are linked to the human proteinuric kidney disease focal and segmental glomerulosclerosis (FSGS) of the late- onset type (Kriz, 2005 ; Reiser et al., 2005 ; Winn et al., 2005 ). TRPC6 is expressed in the podocytes in the kidney glomerular ﬁ lter (Reiser et al., 2005 ; Winn et al., 2005 ). Podocyte foot processes and the glomerular slit diaphragm form the glomerular ﬁ lter and are an essential part of the permeability barrier in the kidney, which is defective in FSGS (Kriz, 2005 ). 12 TRP CHANNELS AND HUMAN DISEASES

This results in proteinuria and progressive kidney failure, leading to end - stage renal failure (Kriz, 2005 ). However, it is currently unclear how mutant forms of TRPC6 contribute to disease development. Three of the TRPC6 mutations in FSGS patients are gain- of - function mutations of TRPC6. Thus, enhanced Ca2+ entry through TRPC6 may constitute the pathogenic trigger, initiating cell death or disregulation that compromises the integrity of the permeability barrier (Reiser et al., 2005 ). On the other hand, a lack of nephrin, a central component of the slit diaphragm, induces increased TRPC6 expression in podocytes and leads to an altered localization of TRPC6 (Reiser et al., 2005 ). Vice versa, TRPC6 may be important for guidance of proteins such as nephrin and podocin, which are required to maintain the ﬁ ltration barrier (Winn et al., 2006 ). It is tempting to speculate that blocking TRPC6 channels could be of therapeutic beneﬁ t in idiopathic FSGS. Interestingly, in this regard, preliminary experiments reveal that the commonly used immunosuppres- sive agent FK - 506 can inhibit TRPC6 activity in vivo (Winn, 2008 ).

1.2.6.3 Pulmonary During regional alveolar hypoxia, a local vasoconstriction shifts the blood ﬂ ow from hypoxic to normoxic areas, a mechanism known as “ hypoxic pulmonary vasoconstriction ” (HPV) (Euler – Liljestrand mechanism). This mechanism excludes blood ﬂ ow from poorly ventilated areas. For acute HPV, TRPC6 is apparently indispensable since it is absent in trpc6 − / − mice. Hypoxia causes DAG production, which in turn activates TRPC6. This mechanism may be extremely important in pulmonary gas exchange disturbances (Weissmann et al., 2006 ).

1.2.6.4 Neurological Disorders An interesting connection between TRPC6 and depression has been suggested. TRPC6 is activated by hyperforin, a bicyclic polyprenylated acylphloroglucinol derivative, which is the main active principle of St. John’ s wort extract. This extract is used as an antidepressant. Hyperforin inhibits the neuronal serotonin and norepinephrine reuptake similarly to synthetic antidepressants such as Prozac. However, hyperforin also increases synaptic serotonin and norepinephrine concentrations by an + indirect mechanism, that is, increase in [Na ]i , which inhibits the neuronal Na – amino acid pump. Hyperforin thus has a dual effect on neurotransmitter regulation. TRPC6 channels could be a novel target for a new class of antidepressants (Leuner et al., 2007 ). Some data also suggest a role for TRPC6 in Alzheimer ’ s disease. Mutations in the presenilin (PS) genes are linked to the development of early - onset Alzheimer ’ s disease. Transient expression of PS mutants (N141I and M239V) with TRPC6 in HEK - 293 cells results in a strong inhibition of agonist - induced Ca2+ entry (Lessard et al., 2005 ). Interestingly, TRPC6 is localized to excitatory synapses and promotes their formation via a CaMKIV - Kinase - CREB - dependent pathway. Overexpression of TRPC6 increases the number of spines in hippocampal neurons and TRPC6 gene silencing decreases their number. Transgenic mice overexpressing THE TRPV SUBFAMILY 13

TRPC6 showed enhancement in spine formation and better spatial learning and memory in Morris water maze. These results reveal the role of TRPC6 in synaptic plasticity and the behavioral consequences (Zhou et al., 2008b ).

1.2.7 TRPC7 To our knowledge, no data are available in the literature linking TRPC7 to human disaeses.

1.3 THE TRPV SUBFAMILY

The TRPV family comprises six mammalian genes, TRPV1– TRPV6 . Members of the TRPV family contain six ankyrin repeats in their cytosolic N - termini, which have now been crystallized (Jin et al., 2006 ; Lishko et al., 2007 ; Phelps and Gaudet, 2007 ). TRPV1 – TRPV4 are polymodal thermo - and chemosensitive channels that are nonselective for cations and are modestly permeable to Ca 2+. In contrast, TRPV5 and TRPV6 are the only highly Ca 2+ - selective 2+ channels in the TRP family, and both channels are tightly regulated by [Ca ]i (Vennekens et al., 2000 ; Yue et al., 2001 ) (for more detailed reviews, see Liedtke [2005 ], Nijenhuis et al. [2005a ], O’ Neil and Heller [2005 ], and Vennekens et al. [ 2008 ]).

1.3.1 TRPV1 The involvement of TRPV1 in pain and other disorders is the topic of this book. For more details on this intriguing subject, we refer to the proceeding chapters in this volume. Apart from those topics, however, it might be interesting to coin a few other leads to a role for TRPV1 in human disease. For instance, one might consider a role for TRPV1 in obesity. TRPV1 is expressed in preadipocytes and in visceral adipose tissue from mice and humans. Oral administration of capsaicin for 120 days prevented obesity in male wild - type (WT) mice but not in trpv1− /− mice under high fat diet (Zhang et al., 2007a ). Also, an important role of TRPV1 has been suggested in bladder diseases (Birder, 2005 ). In the urinary bladder, TRPV1 is expressed on sensory nerve terminals and in the epithelial cells (urothelium) lining the bladder lumen (Birder et al., 2001 ). Overactive bladder symptoms due to various etiologies have been successfully treated with capsaicin or resiniferatoxin (RTX) (Kim and Chancellor, 2000 ; Cannon and Chancellor, 2002 ). Mice lacking TRPV1 display a higher frequency of low - amplitude (spontaneous low - volume spot- ting) non - voiding bladder contractions in comparison to WT animals (Birder et al., 2002 ). This gain of function was accompanied by reduction in both spinal cord signaling and refl ex voiding during bladder fi lling. TRPV1 might also be 14 TRP CHANNELS AND HUMAN DISEASES required for bladder stretch detection, which involves stretch- evoked release of ATP and nitric oxide (NO). Release of both mediators is reduced in bladders from trpv1− / − mice. Moreover, the trpv1− /− mouse does not develop bladder overactivity during acute bladder infl ammation, indicating that TRPV1 is involved in bladder hyperrefl exia in infl ammation (Daly et al., 2007 ). A role for TRPV1 in bladder overactivity is also supported by clinical observations. In patients suffering from neurogenic detrusor overactivity (NDO), TRPV1 immunoreactivity in the urothelium and the number of nerve fi bers expressing TRPV1 are increased (Brady et al., 2004 ; Apostolidis et al., 2005 ). For those patients who benefi ted from intravesicle RTX therapy, TRPV1 urothelial immunoreactivity decreased following treatment. In addition, in biopsies from the same patients, suburothelial TRPV1 - expressing nerve fi bers were reduced in number following therapy with RTX. Apparently, successful therapy using RTX leads to reduced TRPV1 expression in both urothelial and neuronal cells (Apostolidis et al., 2005 ). TRPV1 is activated in patients with gastroesophageal refl ux disease (GERD), which is characterized by heartburn and chest pain. TRPV1 is expressed in sensory nerves within the mucosa of the esophagus, and its expression is upregulated in esophagitis patients (Matthews et al., 2004 ). TRPV1 is activated by acid pH and its activation is sensitized by ethanol, which all trigger the burning pain characteristic of GERD (Bhat and Bielefeldt, 2006 ). Also, TRPV1 is a potential target for novel therapies for functional bowel disorders (FBDs) (Holzer, 2004 ; Schicho et al., 2004 ). TRPV1 seems to be important for several skin diseases. Importantly, TRPV1 is involved in the control of human hair growth. TRPV1 activation by capsaicin in native human hair follicle cells causes inhibition of hair shaft prolongation, suppression of hair follicle cell proliferation, induction of apoptosis, and premature hair follicle cell regression (Bodo et al., 2005 ). In parallel, intrafollicular transforming growth factor (TGFβ 2) and other known endogenous hair growth inhibitors (e.g., interleukin 1 β ) were upregulated, and hair growth promoters (e.g., insulin - like growth factor 1, hepatocyte growth factor , stem cell factor) were downregulated (Bodo et al., 2005 ; Biro et al., 2006 ). TRPV1 may also be a target for modulating epithelial cell growth disorders. 2+ 2+ Cell layers in the skin are characterized by a Ca gradient and [Ca ]i is sen- sitively regulated in keratinocytes, which express TRPV1. Finally, TRPV1 might be linked to memory and synaptic plasticity and consequently to neuropsychiatric and cognitive disorders, for example, epilepsy and schizophrenia (Newson et al., 2005 ; Li et al., 2008 ). The TRPV1 channel is a potential target to facilitate long- term potentiation (LTP) and to suppress long- term synaptic depression (LTD) (Li et al., 2008 ). TRPV1 activation is necessary and suffi cient to trigger LTD. Excitatory synapses onto hippocampal interneurons were depressed either by capsaicin or an endogenously released eicosanoid, 12 - (S) - HPETE. TRPV1 receptor antagonists prevented interneuron LTD. In brain slices from TRPV1− / − mice, LTD was absent (Gibson et al., 2008 ). THE TRPV SUBFAMILY 15

1.3.2 TRPV2 TRPV2 is involved in pain transmission. The channel is activated by very noxious temperatures and is expressed, among other locations, in dorsal root ganglia (DRGs), in medium - sized neurons. Intraplantar injection of complete Freund ’ s adjuvant in rat induces overexpression of TRPV2, which is connected to peripheral sensitization during infl ammation and thermal hyperalgesia (Shimosato et al., 2005 ). Expression of either TRPV1 and/or TRPV2 infl uences heat sensitization to painful thermal stimuli in deep and superfi cial C - and A δ - nociceptors in skin (Rau et al., 2007 ).

1.3.2.1 Muscle A mouse model with speciﬁ c overexpression of TRPV2 in the heart displays cardiomyopathy, left ventricular dilation, decreased systolic performance, disorganized myocyte arrangement, and interstitial ﬁ brosis. Thus TRPV2 may play a role in cardiac hypertrophy (Iwata et al., 2003 ). Furthermore, the expression of TRPV2 is increased in the sarcolemma of skeletal/cardiac muscle in dystrophic patients and in an animal model (the BIO14.6 strain of the Syrian hamster) that lacks functional δ - sarcoglycan and displays reduced abundance of dystrophin (Iwata et al., 2003 ).

1.3.2.2 Bladder Both the full - length TRPV2 (hTRPV2) and a short splice variant (s - TRPV2) are detected in normal human bladder, in normal human urothelial cells, and in urothelial carcinomas (UCs). Enhanced hTRPV2 expression was found in high - grade UC specimens and in UC cell lines. On the other hand, a progressive decline of s - TRPV2 was associated with progressive UC (Caprodossi et al., 2007 ).

1.3.3 TRPV3 TRPV3 is highly expressed in the skin, both in keratinocytes and in cells surrounding hair follicles (Chung et al., 2004 ; Moqrich et al., 2005 ; Asakawa et al., 2006 ). TRPV3 functions as a voltage- dependent thermoTRP channel. Trpv3− / − mice exhibit deﬁ cits in innoxious and noxious heat perception. Hair abnormalities have been reported in the TRPV3 knockout mouse (Moqrich et al., 2005 ). TRPV3 gain- of - function mutations cause an autosomal dominant hairless phenotype with dermatitis (Xiao et al., 2008 ). Interestingly, TRPV3 is also a target for antidepressants. Incensole acetate, a plant compound from Boswellia, has been used as an antidepressant and is now described as a strong activator of TRPV3 (Moussaieff et al., 2008 ).

1.3.4 TRPV4 TRPV4 is a polymodal channel activated by a variety of stimuli, including heat, cell swelling, shear stress, and ligands such as the non- PKC - activating phorbol 4α PDD. Several studies in the literature imply a role for TRPV4 in neuropathic pain. For instance, in several neuropathy models (vincristine 16 TRP CHANNELS AND HUMAN DISEASES chemotherapy, alcoholism, diabetes, and human immunodefi ciency virus/ acquired immunodefi ciency syndrome), mechanical hyperalgesia was reduced by spinal intrathecal administration of antisense oligonucleotides to TRPV4 (Alessandri -Haber et al., 2004, 2006). Mechanical hyperalgesia induced by paclitaxel, vincristine, or diabetes was strongly reduced in the TRPV4 knockout mice (Alessandri - Haber et al., 2008 ). Taxol - induced painful peripheral neuropathy could be treated by gene silencing of TRPV4 (Alessandri - Haber et al., 2004 ), and the induction of osmotic and mechanical hyperalgesia was absent in trpv4− / − mice (Alessandri - Haber et al., 2006 ). TRPV4 might play a crucial role in chronic dorsal root compression (CCD) - induced mechanical allodynia (Zhang et al., 2007b ). TRPV4 is expressed in DRG. After CCD, TRPV4 expression is signifi cantly increased, and administration of TRPV4 antisense oligonucleotides partly reversed the CCD- induced mechanical allodynia. TRPV4 is also involved in the development of infl ammatory mechanical and thermal hyperalgesia induced by intraplantar injection of carrageenan, a powerful infl ammogen, or a cocktail of infl ammatory mediators (Todaka et al., 2004 ). During infl ammation, the protease- activated receptor 2 (PAR 2 ) is activated. PAR 2 agonists sensitize activation of TRPV4 by 4 α - phorbol 12,13 - didecanoate (4 α PDD) and hypotonic cell swelling. Development of − / − mechanical hyperalgesia by PAR2 activation is absent in trpv4 mouse (Sipe et al., 2008 ). Ca2+ entry via TRPV4 causes release of substance P (SP) and calcitonin gene related peptide (CGRP), which may underlie, at least partially, the path omechanism of infl ammatory mechanical hyperalgesia (Grant et al., 2007 ). TRPV4 has also been linked to visceral pain. Mechanosensory responses of colonic serosal and mesenteric afferents were enhanced by a TRPV4 agonist and were dramatically reduced by targeted deletion of TRPV4. Behavioral responses to noxious colonic distention were also substantially reduced in mice lacking TRPV4 (Brierley et al., 2008 ). TRPV4 expression is increased in sinus mucosa from patients with a specifi c form of chronic rhinosinusitis (CRS), which may indicate a potential role for TRPV4 in mucus homeostasis and in CRS pathogenesis (Bhargave et al., 2008 ).

1.3.4.1 Cardiovascular The analysis of the functional effects of TRPV4 (and other TRP channels) has been hampered by the poor speciﬁ city of agonists or antagonists. Recently, GSK1016790A was reported to be a novel speciﬁ c TRPV4 activator. Injection of this compound in mice induced a dose - dependent reduction in blood pressure, followed by profound circulatory collapse. On the other hand, the compound had no effect in the trpv4 − / − mouse. GSK1016790A had no effect on rate or contractility in the heart of normal mice. Instead, it produced a potent endothelium - dependent relaxation of rodent - isolated vascular ring segments in vitro that was abolished by NO synthase (NOS) inhibition (L- NAME), ruthenium red, and eNOS gene deletion. Nevertheless, the in vivo circulatory collapse caused by GSK1016790A THE TRPV SUBFAMILY 17 was not altered by NOS inhibition (L - NAME) or eNOS gene deletion but rather was associated with (concentration and time dependent) profound vascular leakage and tissue hemorrhage in the lung, intestine, and kidney (Willette et al., 2008 ). Thus, GSK1016790A may help to determine the role of TRPV4 in disorders associated with edema and with microvascular congestion. Several other studies have suggested a role for TRPV4 in vascular disease. For instance, TRPV4 is indirectly activated by 5- HT in pulmonary arterial smooth muscle cells (Ducret et al., 2008 ). TRPV4 surface expression is downregulated by WNK kinases (Fu et al., 2006 ). Several WNK kinases, in particular WNK4, have been put forward as candidate genes for essential hypertension, familial hyperkalemia and hypertension (FHH or Gordon’ s syndrome, or pseudohypoaldosteronism type II, PHA- II). The proposed causal link is the downregulation of the expression of the Na+ /Cl − cotransporter (NCC) in the kidney by WNK kinases. However, this effect does not occur in some disease - causing WNK mutants (Wilson et al., 2003 ; Yang et al., 2003 ). It is intriguing to hypothesize that the osmoregulatory channel TRPV4 might also be involved in the pathogenesis of hypertension (Cope et al., 2005 ).

1.3.4.2 Pulmonary TRPV4 is widely expressed in the airway system and seems to be involved in bronchial hyperresponsiveness , an asthma symptom (Liedtke and Simon, 2004 ). Asthma is accompanied by a denudation of the epithelial lining of the bronchi and bronchioli. Bronchial smooth muscle cells, as well as nerve endings, can become exposed to the hypotonic bronchial fl uid, which could contribute to bronchial hyperresponsiveness through activation of TRPV4. Activation of TRPV4 might also be involved in cilia movement in bronchial epithelial cells, as shown for oviductal cells (Andrade et al., 2005 ). TRPV4 is important for regulation of the lung barrier. Elevation of lung 2+ microvascular pressure increases endothelial [Ca ] i via activation of TRPV4. This increase elevates the fi ltration coeffi cient via activation of myosin light - chain kinases and simultaneously stimulates NO synthesis. In TRPV4 - defi cient 2+ mice, pressure - induced increases in endothelial [Ca ]i , NO synthesis, and lung wet/dry weight ratio are greatly reduced. Endothelial NO formation limits the permeability increase by a cGMP - dependent attenuation of the pressure - 2+ induced [Ca ]i , via inactivation of TRPV4 in pulmonary microvascular endothelial cells in a negative feedback loop (Yin et al., 2008 ). TRPV4 activation can disrupt the alveolar septal barrier. TRPV4 activators increase lung endothelial permeability and induce breaks in the epithelial layer of the alveolar septal wall. This disruption leads to acute lung injury, patchy alveolar fl ooding, and hypoxemia (Alvarez et al., 2006 ). Stretch- activated cation channels initiate the acute pulmonary vascular permeability increase in response to high peak infl ation pressure (PIP) ventilation. This stretch- activated channel may be identical to TRPV4. Permeability was assessed by measuring the fi ltration coeffi cient (Kf) in isolated perfused lungs of WT mice. Pretreatment with the inhibitors of TRPV4 (ruthenium red), arachidonic acid production (meth- 18 TRP CHANNELS AND HUMAN DISEASES anandamide), or P450 epoxygenases (miconazole) prevented the increases in Kf. In trpv4 − / − knockout mice, the high PIP ventilation protocol did not increase Kf. Lung distention, which causes Ca2+ entry in isolated mouse lungs, did not occur in trpv4− / − and in ruthenium red - treated lungs. Alveolar and perivascular edema were also signifi cantly reduced in trpv4 − / − lungs. Rapid calcium entry through TRPV4 channels is a major determinant of the acute vascular permeability increase in lungs following high PIP ventilation (Hamanaka et al., 2007 ). Interestingly, aquaporin- 5 (AQP5), a water- permeable channel expressed in the lung, in the cornea, and in various secretory glands, is downregulated in hypotonic media. In mouse lung epithelial cells, the downregulation of AQP5 can be blocked by pharmacological inhibitors of TRPV4, suggesting that TRPV4 is involved in the water and osmolyte homeostasis in various epithelial cells of the lung (Sidhaye et al., 2006 ).

1.3.4.3 Skin TRPV4 is highly expressed in the skin and inﬂ uences the epidermal permeability barrier. Skin forms a water- impermeable barrier and prevents transcutaneous water loss. A defect in barrier function often accompanies human diseases. Damage of the stratum corneum (e.g., by “ tape strip- ping” ) can impair the barrier function.. At temperatures from 36 to 40 ° C, barrier recovery is accelerated in a hairless mouse model and in human skin. 4α PDD accelerates barrier recovery, whereas ruthenium red, a nonselective blocker of TRPV4, delays barrier recovery. Capsaicin, an activator of TRPV1, delayed barrier recovery, whereas capsazepin, an antagonist of TRPV1, blocked this delay. TRPV4 might be activated by osmotic pressure to fulﬁ ll the role of a sensor to restore skin barrier function (Denda et al., 2007 ).

1.3.4.4 Bone TRPV4 is expressed in chondrocytes and in osteoblasts. It also has been implicated in mechanosensing in bone. Mechanical stress determines the levels of bone mass. Hind limb unloading induces osteopenia in WT but not in TRPV4 - deﬁ cient mice. Unloading induces an increase in the number of osteoclasts in the primary trabecular bone, which is suppressed by TRPV4 knockout. Unloading - induced reduction in the longitudinal length of primary trabecular bone is also suppressed by TRPV4 deﬁ ciency (Mizoguchi et al., 2008 ).

1.3.4.5 Bladder TRPV4 as a putative mechanosensor may also be involved in the regulation of the normal bladder voiding, since trpv4 − / − mice manifest an incontinent phenotype (Gevaert et al., 2007 ). Cystometric experiments revealed that trpv4− /− mice exhibit a lower frequency of voiding contractions as well as a higher frequency of non - voiding contractions. Decreased stretch - evoked ATP release occurs in isolated whole bladders from trpv4− / − mice. Thus, TRPV4 appears to play a role in voiding behavior and in urothelium- mediated transduction of intravesical mechanical pressure. (Gevaert et al., 2007 ). THE TRPV SUBFAMILY 19

Additionally, infusion of the potent TRPV4 agonist GSK1016790A into the bladders of trpv4+/+ mice induces bladder overactivity (Thorneloe et al., 2008 ). These data suggest that TRPV4 antagonists may be beneﬁ cial against bladder overactivity and may increase the storage volume of the bladder (Thorneloe et al., 2008 ). Interestingly, several families have been identiﬁ ed with nocturnal enuresis/ incontinence. Four linkages to chromosomal regions were described, two of which refer to the location of TRP channel genes, namely, trpc4 (13q13) and trpv4 (12q) (Loeys et al., 2002 ).

1.3.4.6 Hearing TRPV4 is expressed in the cochlea within hair cells, the stria vascularis, and the spiral ganglion (Liedtke et al., 2000 ). It has been proposed that TRPV4 is involved in sensorineural hearing impairment . Indeed, trpv4− / − mice, older than 24 weeks, demonstrate a higher hearing threshold as evaluated by the auditory brainstem response. TRPV4 might thus be associated with a delayed - onset hearing loss and with an increased cochlea vulnerability to acoustic injury (Tabuchi et al., 2005 ). One of the gene loci for autosomal dominant nonsyndromic hearing loss (ADNSHL) has been mapped to a small region in chromosome 12q21- 24 where the trpv4 gene is located (Greene et al., 2001 ). The TRPV4 gene locus has also been discussed as the human ortholog of the region affected in the bronx waltzer (bw) mutant mice, which are characterized by waltzing behavior, deafness, and the loss of cochlear inner hair cells during early development.

1.3.5 TRPV5 and TRPV 6 The Ca 2+ - selective channels, TRPV5 and TRPV6, seem to play an essential role in the maintenance of a constant extracellular Ca2+ concentration. The important role of TRPV5 and TRPV6 in the processes of transcellular Ca 2+ reabsorption in the kidney and intestine is well established. TRPV5 is mainly expressed in the apical membrane of kidney distal convoluted tubule (DCT) and of connecting tubule (CNT) cells in the bone and intestine, whereas TRPV6 is widely expressed in the brush border of the apical membrane in the intestine but also in the kidney. An excellent review on the molecular and functional aspects of these channels has been published recently (Hoenderop and Bindels, 2008 ). trpv5− / − mice exhibit a diminished Ca2+ reabsorption in the kidney, which causes severe hypercalciuria. However, compensatory hyperabsorption of dietary Ca2+ was measured in trpv5− /− mice (Hoenderop et al., 2003 ). The urine from trpv5− / − mice is much more acidic than from WT animals, which might be a defense mechanism against kidney stone formation. TRPV5 dysfunction could also be involved in diseases such as osteoporosis (Hoenderop et al., 2003 ). A possible TRPV5 channelopathy is autosomal dominant idiopathic hypercalciuria (IH) (Muller et al., 2002 ). Although the coding sequence of the TRPV5 gene is not altered in this disease, three SNPs were detected in the 20 TRP CHANNELS AND HUMAN DISEASES

5′ - fl anking region. Inactivation of the trpv6 gene causes decreased intestinal Ca2+ reabsorption (Bianco et al., 2004 ). Some Ca2+ - related disorders are associated with alterations in TRPV5 and/ or TRPV6 (van Abel et al., 2005 ): (1) Vitamin D defi ciency rickets type I (VDDR - I) : This autosomal recessive disease is characterized by low 1,25(OH)2D3 levels, hypocalcemia, rickets, osteomalacia, growth retardation, and failure to thrive. Both Ca2+ reabsorption channels, TRPV5 and TRPV6, are downregulated, which is consistent with a pathogenic role in this disease. (2) Postmenopausal osteoporosis : Estrogen has stimulatory effects on TRPV5 and TRPV6 expression. This type of osteoporosis is coupled to estrogen defi - ciency and decreased Ca2+ reabsorption via both channels. (3) IH : This autosomal disease is caused by either excessive intestinal Ca2+ reabsorption or defective renal Ca 2+ reabsorption. Both forms of IH increase the risk of kidney stone formation. It is currently unknown whether mutations in either TRPV5 or TRPV6 are involved in the pathogenesis of this disorder. (4) Parathyroid hormone (PTH) - related disorders: Reduced serum PTH levels downregulate TRPV5 expression, which contributes to hypocalcemia. (5) Tacrolism : Immunosuppressant drugs, like tacrolimus, induce increased bone turnover with hypercalciuria. Renal Ca2+ wasting may be due to decreased TRPV5 expression (Nijenhuis et al., 2004 ). (6) Thiazide diuretics : These diuretics increase Ca 2+ reabsorption and cause hypocalciuria. A similar phenotype occurs in mutations of the NCC leading to Gitelman’ s syndrome. An upregulation of TRPV5 has been excluded in this disease (Nijenhuis et al., 2005b ). However, an increased paracellular reabsorption of Ca2+ has been measured (Nijenhuis et al., 2003 ) and a clear downregulation of TRPM6 has been observed. The trpv6 gene was completely sequenced in 170 renal calcium stone patients. The ancestral trpv6 haplotype consisting of three nonsynonymous polymorphisms (C157R, M378V, and M681T) occurred in signifi cantly more calcium stone- forming patients than in the general population, which does not have the ancestral genotype. Expression of the mutated protein (157R + 378V + 681T) in Xenopus oocytes showed enhanced calcium permeability. Thus, the gain of function of TRPV6 might play a role in calcium stone formation (see also Hughes et al., 2008 ; Suzuki et al., 2008b ).

1.3.5.1 Cancer TRPV5 and TRPV6 are both expressed in the prostate. Expression of TRPV6 is signiﬁ cantly increased in prostate adenocarcinoma in comparison to benign prostate hyperplasia. Moreover, expression levels of TRPV6 correlate with tumor grade (Peng et al., 2001 ; Wissenbach et al., 2001 ). TRPV6 expression is decreased by dihydrotestosterone and is conversely increased by an androgen receptor antagonist (Peng et al., 2001 ). Prostate carcinoma is a hormone - sensitive malignancy, and nonsteroidal antiandro- gens, such as ﬂ utamide, are frequently used in prostate cancer treatment. Furthermore, estrogens, which are also used in the therapy of prostate cancer, positively regulate TRPV5 and TRPV6 expression. Hence, TRPV6 might be THE TRPV SUBFAMILY 21 a molecular mediator involved in the anticancer effects of these compounds. The channel is consistently overexpressed not only in prostate cancer but also in breast , thyroid , colon , and ovarian carcinomas (Zhuang et al., 2002 ; Bolanz et al., 2008 ). Ca 2+ entry through TRPV6 may increase the rate of Ca2+ - dependent cell proliferation and thus may be directly linked to tumor growth (Schwarz et al., 2006 ). It has to be noted that hormonal effects are more widespread than just modulating TRPV6 expression, which might exlain complex interaction patterns with tumor growth.

1.3.5.2 Age TRPV5 knockout mice develop age - related osteoporosis and hyperparathyroidism much earlier than WT mice (van Abel et al., 2006 ). Aging is associated with changes in Ca 2+ homeostasis, which contribute to age- dependent osteoporosis and hyperparathyroidism. It has been recently shown that the impaired Ca 2+ reabsorption in the elderly is caused by a downregulation of TRPV5 and TRPV6.

1.3.5.3 Placenta Function Maternal - fetal Ca 2+ transport is crucial for fetal Ca 2+ homeostasis and bone mineralization. The Ca 2+ concentrations in fetal blood and amniotic ﬂ uid are signiﬁ cantly lower in trpv6 − / − fetuses than in WT fetuses (Suzuki et al., 2008a ). The transport activity of radioactive Ca2+ ( 45 Ca) from mother to fetuses was 40% lower in trpv6 − / − mice than in WT mice. It was recently proposed that cyclophylin B (CypB) is a novel TRPV6 accessory protein, which apparently has a stimulatory effect on TRPV6 channel activity. In the human placenta, TRPV6 and CypB are mainly located intracellularly in the syncytiotrophoblast layer, but a small amount of TRPV6 and CypB is also expressed in microvilli apical membranes, the feto- maternal barrier (Stumpf et al., 2008 ).

1.3.5.4 Skin Silencing of TRPV6 impairs cell differentiation, as shown by decreased expression of differentiation markers such as involucrin, transglu- taminase- 1, and cytokeratin- 10. TRPV6 silencing also affects keratinocyte morphology, the development of intercellular contacts, and the ability of cells to stratify. 1,25 - Dihydroxyvitamin D3, which is a well - known cofactor of kera- 2+ tinocyte differentiation, upregulates TRPV6, thereby increasing [Ca ]i and promoting cellular differentiation (Lehen ’ kyi et al., 2007 ).

1.3.5.5 Hearing TRPV5 and TRPV6 are expressed in the inner ear (Yamauchi et al., 2005 ). The channels are likely required for maintaining the endolymph Ca2+ concentration in the μ M range. pH- dependent inhibition of TRPV5 and TRPV6 might be responsible for the vestibular dysfunction in Pendred Syndrome due to endolymph acidiﬁ cation, which occurs by knocking −− out of the Cl HCO3 exchanger SLC26A4 (pendrin) (Nakaya et al., 2007 ; Wangemann et al., 2007 ). Pendred syndrome is an inherited disorder that accounts for as much as 10% of hereditary deafness. 22 TRP CHANNELS AND HUMAN DISEASES

1.4 THE TRPM SUBFAMILY

Members of the TRPM family, on the basis of sequence homology, fall into three subgroups: TRPM1/3, TRPM4/5, and TRPM6/7. TRPM2 and TRPM8 represent structurally distinct channels. TRPM channels exhibit highly variable permeability to Ca2+ and Mg 2+, ranging from Ca 2+ - impermeable (TRPM4 and TRPM5) to highly Ca 2+ and Mg2+ permeable (TRPM6 and TRPM7). In contrast to TRPCs and TRPVs, TRPMs do not contain ankyrin repeats within their N - terminal domain. The following reviews summarize the key features of the members of the TRPM subfamily (Aarts and Tymianski, 2005 ; Chubanov et al., 2005 ; Kraft and Harteneck, 2005 ; Kuhn et al., 2005 ; McNulty and Fonfria, 2005 ; Reid, 2005 ; Scharenberg, 2005 ).

1.4.1 TRPM 1 1.4.1.1 Cancer TRPM1 is possibly a tumor suppressor. The melanocyte- specifi c gene trpm1 is exclusively expressed in melanoma cells and is downregulated during the development of metastasis in cutaneous malignant melanoma. Malignant melanoma is a tumor developing from moles or normal- looking skin, as well as in eyes and the meninges, and constitutes the most aggressive skin tumor (Duncan et al., 2001 ). The inverse correlation between TRPM1 transcript expression and metastatic potential represents one of the most reliable differential diagnostic markers to discriminate between non - metastatic and metastatic melanomas. TRPM1 has at least fi ve splice variants. A potential, but disputed, mechanism of the regulation of TRPM1 may involve a short cytosolic variant (TRPM1- S) binding to, or interacting with, the full- length variant (TRPM1- L) to suppress its translocation to the plasma membrane. Upon a specifi c yet unidentifi ed stimulus, TRPM1- S might dissociate from TRPM1 - L and might enable plasma membrane insertion of the latter where subunit proteins can associate and form functional Ca 2+ infl ux channels (Xu et al., 2001 ). This model suggests that retention of TRPM1 - L in an intracellular compartment is critical in regulating Ca 2+ infl ux.

1.4.2 TRPM 2 1.4.2.1 Cardiovascular Blocking TRPM2 may represent a novel therapeutic approach to protect against oxidant - induced endothelial barrier disruption

(Dietrich and Gudermann, 2008 ; Hecquet et al., 2008 ). H2 O2 stimulates ADP - ribose formation, which activates TRPM2. Expression of TRPM2 increases endothelial permeability and endothelial dysfunction by oxidative stress. siRNA depleting of TRPM2 or antibody blocking of TRPM2 attenuates the increased endothelial permeability induced by H 2 O2 (Hecquet et al., 2008 ).

1.4.2.2 Cell Death TRPM2 activation is implicated in β - cell death after application of radicals, such as H2 O2 (Ishii et al., 2006 ). TRPM2 is activated THE TRPM SUBFAMILY 23 by ROS, and its activation induces necrotic cell death. Insertion of TRPM2 into A172 human glioblastoma cells enhances cell death induced by H2 O2 . Proliferation, migration, and invasion activities were not affected by the expression of TRPM2 (Ishii et al., 2007 ). Death of hematopoietic cells through activation of caspases and poly (ADP - ribose) polymerase (PARP) cleavage might also be mediated by TRPM2 (Zhang et al., 2005 ). 1.4.2.3 Immunology and Infl ammation TRPM2 is highly expressed in granulocytes and cells of the monocytic lineage, including macrophages (Perraud et al., 2004 ). When such cells are exposed to oxidative stress and become activated, they produce ROS in a process known as the respiratory burst. An involvement of ROS in the fi ght against invading pathogens has been postulated (Babior, 2000 ). Ca2+ signaling via TRPM2 could play an important role during this process: activation of TRPM2 by ROS induces Ca 2+ entry, which in turn potentiates activation of this channel, leading to a positive feedback mechanism. Similar to the situation in lymphocytes, the oxidant- induced activation of the transcription factors NF - kB and AP - 1 is thought to be of importance in the production of cytokines in macrophages (Iles and Forman, 2002 ). ROS induce chemokines responsible for the recruitment of infl ammatory cells to sites of injury or infection. Recently, it has been shown that TRPM2 controls ROS - induced chemokine production in monocytes. ROS evoke Ca 2+ infl ux through TRPM2, which subsequently activates the Ca2+ - dependent tyrosine kinase Pyk2 and amplifi es Erk signaling via Ras GTPase. This causes nuclear translocation of NFκ B, which is essential for the production of the chemokine interleukin - 8. This mechanism is impaired in TRPM2 - defi cient mice (Yamamoto et al., 2008 ). 1.4.2.4 Neurological Disorders The pathophysiology of bipolar disorder has been connected to a variety of TRP channels. TRPM2 is expressed in several human tissues, and abundantly so in the brain, lymphocytes, and hematopoietic cells (Nagamine et al., 1998 ; Sano et al., 2001 ; Heiner et al., 2003 ). A truncated variant of TRPM2 is expressed in the striatum (i.e., caudate nucleus and putamen) (Nagamine et al., 1998 ; Uemura et al., 2005 ). Both TRPM2 and TRPM7 have been directly implicated in neuronal cell death pathways and have been proposed as potential factors in neurodegenerative diseases including Alzheimer’ s disease, amyotrophic lateral sclerosis (ALS), Parkinson’ s disease, stroke, and diseases associated with oxidant- mediated neuronal damage (Li et al., 2004, 2005a; McNulty and Fonfria, 2005 ). For instance, inhibition of TRPM2 by the selective poly(ADP - ribose) polymerase inhibitor SB- 750139 (GlaxoSmithKline) attenuates hydrogen peroxide and amyloid β - peptide - induced cell death in rat striatum neurons (Fonfria et al., 2005 ). This may suggest a direct involvement of TRPM2 in Alzheimer ’ s disease. The role of TRPM2 in neuronal cell death seems to be evident. TRPM2 expression is elevated in a rat stroke model. It is localized in microglial cells, which play a key role in pathology produced following ischemic injury in the CNS (Fonfria et al., 2006 ). 24 TRP CHANNELS AND HUMAN DISEASES

TRPM2 might be involved in bipolar disorder I (BD- I) and bipolar disorder II (BD - II). A putative susceptibility locus of BD- I is within the chromosomal regions 12q23– q24.1, which encodes the Ca 2+ - ATPase, SERCA , and 21q22.3, to which the TRPM2 gene has been mapped (Straub et al., 1994 ; Aita et al., 1999 ; Liu et al., 2001 ). In addition to these fi ndings, it has been shown that SNPs in the promoter region of TRPM2 are signifi cantly associated with BD- II, suggesting that TRPM2 polymorphisms contribute to the risk for BD - II (Xu et al., 2006 ). Recently, a very interesting pathology has been described in more detail, that is, Guamanian amyotrophic lateral sclerosis (ALS- G) and Parkinsonism dementia (PD- G or Parkinsonism dementia complex [PDC]) . These are related neurodegenerative disorders that are found at a relatively high incidence on the Pacifi c Islands Guam and Rota (Plato et al., 2002 ). The hyper- endemic ALS - G/PDC foci in the Western Pacifi c (including Guam) have been extensively studied over the years. Results of these studies suggest that a complex interaction between genetic predisposition and environmental factors is involved. However, the etiology of these disorders remains elusive (Plato et al., 2003 ). Recent work has proposed TRPM2 and TRPM7 as candidate susceptibility genes (Hermosura et al., 2005, 2008). The trpm2 gene is located on chromosome 21 (21q22.3), which has been associated with familial ALS (Rosen et al., 1993 ). Recently, a heterozygous variant of trpm2 in a subset of ALS- G and PDC cases has been identifi ed. This variant, TRPM2P1018L, produces a missense mutation in the channel protein, changing proline 1018 (Pro1018) to leucine (Leu1018). Functional studies using a heterologous expression system revealed that, unlike WT TRPM2, P1018L channels rapidly inactivate and are unable to maintain sustained ion infl ux (Hermosura et al., 2008 ).

1.4.3 TRPM 3 TRPM3 is expressed in the kidney, brain, pancreas, testis, and spinal cord. The gene is transcribed in at least 12 splice variants (Grimm et al., 2003 ; Lee et al., 2003 ; Oberwinkler et al., 2005 ), which have been shown to be functionally distinct (Oberwinkler et al., 2005 ). Importantly, TRPM3 has now been indentiﬁ ed as a steroid- modulated channel; for example, pregnenolone sulfate activates the channel. In addition, TRPM3 is highly expressed in the pancreas and may play an important role in insulin release (Nilius and Voets, 2008 ; Wagner et al., 2008 ).

1.4.4 TRPM 4 and TRPM 5 TRPM4 and TRPM5 represent the only known TRP channels that are directly 2+ gated by elevated [Ca ]i . Both TRPM4 and TRPM5 are essentially impermeable to Ca2+ but form ion channels permeable to monovalent cations with single - channel conductances of approximately 20 – 25 pS. They are depolariz- THE TRPM SUBFAMILY 25 ing channels that reduce the inward driving force for Ca 2+ . TRPM4 proteins are detected in the following: heart, pancreas, intestine, lung, thymus, uterus, vomeronasal organ, brain, fat tissue, adrenal gland, kidney, spleen, cultured aortic endothelial cells, and bone marrow- derived mast cells (Vennekens and Nilius, 2007 ). TRPM5, which is expressed in taste receptor cells, is a downstream depolarizing signal transducer of G protein - coupled receptors in taste buds. TRPM5 appears to be important, though not indispensible, for the transduction of sweet, amino acid, and bitter stimuli (Perez et al., 2002 ; Zhang et al., 2003 ; Damak et al., 2006 ).

2+ 1.4.4.1 TRPM4 in Immune Cells TRPM4 exerts control over [Ca ]i in mast cells by maintaining a relatively depolarized membrane potential, which limits the driving force for Ca2+ entry (Vennekens and Nilius, 2007 ; Vennekens et al., 2007 ). trpm4 − /− bone marrow - derived mast cells activate a larger Ca2+ entry after Fc εI stimulation than trpm4+/+ mast cells and release more histamine, leukotrienes, and tumor necrosis factor. trpm4 − / − mice also exhibit a more severe IgE - mediated acute passive cutaneous anaphylactic response. These results may suggest a new mechanism of allergic hypersensitivity and may provide a new therapeutic target (Vennekens and Nilius, 2007 ; Vennekens et al., 2007 ). A similar mechanism may regulate Ca2+ oscillations after T- lymphocyte activation, which is required for NFAT- dependent interleukin- 2 (IL- 2) production (Launay et al., 2004 ). However, it is unclear whether TRPM4 is actually expressed in T cells (Vennekens and Nilius, 2007 ; Vennekens et al., 2007 ). Nevertheless, it seems likely that defects in the function of TRPM4 will result in the inappropriate release of cytokines, triggering immunological hyperresponsiveness , proinﬂ ammatoty conditions , or allergy .

1.4.4.2 Cardiovascular TRPM4 - like currents have been observed in many tissues, including cardiomyocytes (Guinamard et al., 2004a,b ). TRPM4 is overexpressed in cardiomyocytes from spontaneously hypertensive rats, which also show cardiac hypertrophy, kidney sclerosis, and increased bone calciﬁ cation and are characterized by cardiac arrhythmias caused by delayed after- depolarization. TRPM4 - like channels may be involved in the myogenic constriction response in small arteries (Bayliss effect) (Earley et al., 2004 ). Alteration of the arterial myogenic response associated with stroke causes cerebral autosomal dominant arteriopathy with subcortical infarcts and leu- koencephalopathy (CADASIL) (Folgering et al., 2008 ). TRPM4 may also affect blood vessel tone indirectly by virtue of its expression in the endothelium. Agonist stimulation of endothelial cells activates a nonselective cation channel with characteristics similar to those of TRPM4 (Suh et al., 2002 ). This channel is regulated by NO and ATP. NO donors such as S - nitroso - N - acetylpenicillamine (SNAP) and 3 - morpholinosydnonimine (SIN - 1) inhibit TRPM4. Inhibitors of NO synthases potentiate the TRPM4 - like current, whereas superoxide dismutase (SOD), which inhibits the breakdown of NO, causes inhibition. This mechanism indicates a role for TRPM4 26 TRP CHANNELS AND HUMAN DISEASES in sensing the metabolic state of the cell and NO in endothelial cells (Suh et al., 2002 ).

1.4.4.3 Neurological Disorders An intriguing function for TRPM4 can be proposed based on experiments in which the GABA receptor antagonist bicuculline was employed to initiate spontaneous epileptic activity in neocortical slices. TRPM4- like channels are activated during paroxysmal depolarization shift (PDS) discharges and appear to play a role in maintaining subsequent sustained after - depolarization waveforms. The latter effect depends on an 2+ increase in [Ca ]i and can be blocked by maneuvers that inhibit TRPM4 (Schiller, 2004 ). Neuronal damage evoked by reduced blood supply to the brain ( “ vascular stroke ” ), which induces severe hypoxia and hypoglycemia, is very often accompanied by a phenomenon in which susceptible neurons slowly lose their membrane potential and then suddenly enter a transient state of complete depolarization, known as spreading depression (SD) - like hypoxic 2+ depolarization. SD is associated with an increase in [Ca ]i . Intriguingly, TRPM4 (and TRPM5) could be a candidate for triggering this dramatic event, although there is at present no direct experimental evidence in support of this conjecture (Somjen, 2001 ; Anderson and Andrew, 2002 ). It is also worthwhile mentioning that activation of a current with features reminiscent of TRPM4 is involved in the generation of slow ( <1 Hz) sleep oscillations (Crunelli et al., 2005 ). Another example that hints at a contribution of TRPM4 in the pathogenesis of brain swelling during stroke is the discovery in hypoxic gliotic tissue of a 2+ nonselective Ca - activated channel (NCCa - ATP) that also opens under conditions of [ATP] i depletion (Chen and Simard, 2001 ; Simard and Chen, 2004 ). This channel, which shares many properties with TRPM4 (i.e., single- channel 2+ conductance, concentration range of activation by [Ca ]i , submicromolar block by ATP, and voltage - dependent open probability), is found in astrocytes in the adult brain and is regulated by sulfonylurea receptor 1 (SUR1), similar to the KATP channel (Chen et al., 2003 ). Activation of NCCa - ATP causes complete membrane depolarization and cell swelling in astrocytes from injured brains (Chen and Simard, 2001 ; Simard and Chen, 2004 ). Many diseases in the brain, including traumatic conditions, edema, ischemia, and stroke are exacerbated by cerebral edemas. Importantly, these events induce an upregulation of

NCCa - ATP (Simard et al., 2006 ). Cerebral edema after occlusion of the middle cerebral artery (MCA) stroke is responsible for a high mortality of ∼ 80% of the patients (Ayata and Ropper, 2002 ). Edemas in MCA stroke are paralled by an increased expression of the SUR1 driven by an upregulation of the transcription factor Sp 1. Glibenclamide attenuates the development of cerebral edema by MCA stroke. It would be interesting to determine whether TRPM4 is indeed the channel regulated by SUR1 and involved in the pathogenesis of cerebral edemas. If so, TRPM4 might provide a new and promising therapeutic approach to stroke. The trpm5 gene maps to the chromosomal area 11p15.5 (Prawitt et al., 2000 ). A genetic disorder involving a loss of imprinting in this region is the THE TRPM SUBFAMILY 27

Beckwith – Wiedemann syndrome (BWS) , a disease that is mainly characterized by exomphalus, macroglossia, gigantism, and taste abnormalities. Moreover, BWS patients also demonstrate a strong predisposition for neopla- sias, especially for Wilms tumor and the aggressive rhabdomyosarcomas . Therefore, it seems likely that this area contains at least one gene with oncogenic potential and/or a tumor suppressor gene, and impairment in their function, or regulation, will potentially result in disease development. A possible connection between gain - of - function TRPM5 and BWS is anticipated. So far, no mutations in the TRPM5 gene that relate to BWS are known. Interestingly, possibly affected genes are insulin - like growth factor 2 (IGF2), cyclin- dependent kinase inhibitor 1C (CDKN1C), cyclin- dependent kinase inhibitor p21 (p21CIP1), KCNQ1OT1 (potassium channel KCNQ1), and ZNF215 (putative transcription factor), which are located in the same chromosomal region. Such a complex situation might be expected also for other TRPs, which are involved in syndromic diseases. Interestingly, BWS patients have persistent hypoglycemia and hyperinsulinism, features that are similar to diseases caused by mutations in the SUR1 subunits of the KATP channel, which is crucial in glucose - regulated insulin release (Fournet et al., 2001 ; Munns and Batch, 2001 ). Because TRPM5 is highly expressed in pancreatic β cells (Prawitt et al., 2003 ), it is tempting to hypothesize that the TRPM5 channel plays a unique regulatory role in islet β - cell function. It can be assumed that the upregulation of insulin secretion has an oncogenic potential indicating a possible connection between functional TRPM5 and BWS that has not yet been discovered. It remains to be shown whether TRPM5 might play a role in dysfunction of insulin secretion.

1.4.5 TRPM 6 TRPM6 and TRPM7 (see below) combine channel and enzymatic activities (“ chanzymes ” ) and are involved in Mg 2+ homeostasis. TRPM6 is highly expressed in the kidney and in the intestine, speciﬁ cally in association with the apical membrane of the DCT and the brush border membrane, respectively (Voets et al., 2004 ). TRPM6 constitutes a cation channel with high permeability to Mg2+ , and to a lesser extent Ca 2+. TRPM6 functions as a homo- tetramer, but also as a heterotetramer in association with TRPM7 (Li et al., 2006 ). An atypical serine/threonine protein kinase ( α - kinase family) is common to the C- terminal domains of TRPM6 and TRPM7 and is subject to autophos- phorylation (Runnels et al., 2001 ; Schmitz et al., 2003 ).

1.4.5.1 TRPM6 in the Kidney and in the Intestine The trpm6 gene has been mapped to chromosome 9q21.13, an area identifi ed as the “ hypomagnesemia with secondary hypocalcemia (HSH) - critical region. ” Defects in this chromosomal locus have been associated with the disease HSH (Schlingmann et al., 2002 ; Walder et al., 2002 ). HSH is an autosomal recessive disorder that is characterized by very low serum levels of Mg2+ and Ca 2+. The primary defect 28 TRP CHANNELS AND HUMAN DISEASES is an impaired intestinal Mg 2+ absorption. The defect in intestinal transport distinguishes HSH from all other known forms of hereditary hypomagnesemia. Detailed analysis of the HSH - critical region revealed a variety of mutations in the trpm6 gene in all tested HSH patients. Most mutations resulted in a truncated protein through the introduction of stop codons, although single point mutations, frame shift mutations, exon deletions, and mutations affecting alternative splicing have also been described (Schlingmann et al., 2002, 2005; Walder et al., 2002 ). Recently, a new missense mutation was described, which is located in the pore region of TRPM6, P1017R. Because neither surface expression of TRPM6 nor hetereomerization with TRPM7 is affected, the disease- causing mechanism seems to be a defect in the channel pore (Chubanov et al., 2007 ). Intestinal Mg2+ uptake in the brush border epithelia occurs in a curvilinear manner and is regulated by two independent pathways: (1) passive paracellular absorption, which rises linearly with increasing luminal Mg2+ concentrations, and (2) transcellular transport, driven by secondary active transport, reaching saturation at high luminal Mg 2+ levels (Konrad et al., 2004 ). TRPM6 represents an essential molecular component of the active transcellular Mg2+ uptake, being the Mg 2+ infl ux channel at the apical membrane (Voets et al., 2004 ). Saturation at high luminal Mg 2+ concentrations is necessary to prevent the cell from becoming overloaded with Mg 2+ . This feedback mechanism is provided by TRPM6 itself, which is highly regulated by the intracellular concentration of Mg2+ (Voets et al., 2004 ). On the basolateral aspect of the epithelial cell, a yet unidentifi ed Na + /Mg 2+ exchanger accounts for onward transport of Mg 2+ into the interstitial space and blood. The situation in the distal convoluted tubule (DCT) cells of the nephron is different. Although most reabsorption of Mg2+ in the nephron occurs via a paracellular pathway in the thick ascending limb of the loop of Henle, it is the DCT that determines the fi nal reabsorption of fi ltered Mg 2+ from the lumen to the blood and thus the fi nal urinary Mg2+ excretion. In the DCT, paracellular transport of Mg 2+ does not occur, and TRPM6, located on the apical membrane of the epithelial cells, is the obligate pathway for the active reabsorption of Mg 2+ . If this pathway is defective, as in HSH, no further Mg2+ can be reabsorbed, resulting in the urinary Mg2+ leak that has often been observed in HSH patients. Apart from HSH, TRPM6 has also been shown to play a critical role in the etiology of isolated autosomal recessive renal hypomagnesemia . A homozygosity - based mapping strategy in affected patients led to the identifi cation of a chromosomal area containing the EGF gene . Subsequent work showed that a single mutation in this gene (P1070L) is responsible for the development of this disease (and actually established for the fi rst time EGF as a magnesiotropic hormone). The mutation causes mislocation of pro- EGF at the basolateral membrane of the DCT cell, resulting in reduced EGF production at the basolateral membrane, reduced activation of EGFR, and, through an unknown mechanism, reduced TRPM6 activity. This ultimately results in Mg2+ wasting through the urine (Groenestege et al., 2007 ; Muallem and Moe, 2007 ). THE TRPM SUBFAMILY 29

1.4.6 TRPM 7 The closest relative of TRPM6 is TRPM7 (Nadler et al., 2001 ; Runnels et al., 2001 ). In contrast to the rather restricted expression pattern of TRPM6, TRPM7 is ubiquitously distributed. TRPM7 provides an ion channel pathway for Mg2+ and Ca2+ entry into cells and is also responsible for trace metal ion uptake (Monteilh -Zoller et al., 2003 ). Mg 2+ inﬂ ux through TRPM7 is indispensable for cellular viability. Targeted deletion of TRPM7 in DT - 40 chicken B lymphocytes causes rapid cell growth arrest followed by cell death within 2 – 3 days (Nadler et al., 2001 ). However, viability can be maintained and the arrest of cell proliferation reversed by supplementation of the culture medium with excess Mg2+ (> 10 mM) or by reintroduction of WT, or functional mutants, of TRPM7 (Nadler et al., 2001 ; Schmitz et al., 2003 ). An intriguing potential mechanism is a downregulation of the G1 - S transition in the cell cycle (Wolf and Cittadini, 1999 ).

1.4.6.1 Cardiovascular A novel role for TRPM6 and TRPM7 in vascular dysfunction has recently been proposed. It is well documented that increased 2+ [Mg ]i in VSMCs causes vasodilation and reduces agonist- induced vasocon- 2+ striction. By contrast, low [Mg ]i contributes signifi cantly to increased vascular tone, enhanced responses to vasoconstrictors, defective vasodilation and vascular remodeling, and elevated blood pressure (Touyz, 2003 ). Increased 2+ [Mg ]i is also involved in cell cycle activation and growth of VSMCs (Touyz 2+ and Yao, 2003 ). The infl uence of [Mg ]i on VSMCs appears to be tightly connected to Mg2+ infl ux via TRPM7 (He et al., 2005 ). Both ATII and aldosterone increase TRPM7 expression (He et al., 2005 ; Sontia et al., 2008 ). Long - term 2+ ATII application results in an increase in [Mg ]i and in the growth of VSMC, 2+ which are attenuated when TRPM7 is knocked down. Furthermore, [Mg ]i is signifi cantly reduced in VSMCs isolated from spontaneous hypertensive rats (SHR), in parallel with a signifi cantly reduced TRPM7 expression, in comparison to normotensive Wistar Kyoto (WKY) rat controls (Touyz et al., 2005 ). These results suggest an important role for TRPM7 in blood pressure regulation and in VSMC growth.

1.4.6.2 Neurological Disorders TRPM7 has been put forward as a potential factor in neurodegenerative diseases including Alzheimer ’ s disease, ALS, Parkinson ’ s disease, and stroke (McNulty and Fonfria, 2005 ). Earlier, we mentioned ALS- G and PD- G (see under TRPM2) (Plato et al., 2002, 2003). A TRPM7 missense mutation, T1482I, which is located between the channel and the kinase domain, has been described in a subgroup of both ALS- G and PD - G patients (Hermosura et al., 2005 ). The T1482I mutant has no detectable alteration in α - kinase activity but has an increased sensitivity to inhibition by intracellular Mg2+ within a physiologically relevant range. It is known that the incidence of both ALS- G and PD- G is increased in environments, such as in the west Pacifi c, that are defi cient in Ca2+ and Mg 2+ . Therefore, increased 30 TRP CHANNELS AND HUMAN DISEASES sensitivity of TRPM7 to inhibition by Mg 2+ could even worsen the Mg 2+ homeostasis in a Mg2+ - defi cient environment, leading to a reduced intracellular Mg2+ concentration (Schmitz et al., 2003 ; Hermosura et al., 2005 ). Although both ALS- G and PD- G have a multifactorial etiology, the functional changes of the missense mutations may hint at a contribution of TRPM7 malfunction, that is, a channelopathy, to the pathogenesis of both neurodegenerative diseases (Hermosura et al., 2005 ). TRPM7 plays a universal role in Mg2+ homeostasis associated with basic cellular metabolism and activities such as cell viability and proliferation. Cases of anoxic neuronal death have been described that include the involvement of TRPM7 in cellular damage due to an imbalance of the normal physiological processes (Aarts et al., 2003 ; Wei et al., 2007 ). In situations of brain ischemia, oxygen– glucose deprivation (OGD) and excitotoxicity mediate neuronal death (Aarts and Tymianski, 2005 ). The key processes involve high Ca2+ infl ux as a consequence of the excitotoxicity. Subsequent production of ROS/reactive nitrogen species consecutively activates another Ca 2+ conductance, named 2+ IOGD, which is mediated by TRPM7, and results in cellular Ca overload and cell death. In models of ischemic stroke (oxygen and glucose deprivation, NaCN chemical anoxia), the activation of NMDA receptors (NMDA- R) provides a route for toxic Ca2+ infl ux, but TRPM7 probably provides an additional pathway (Macdonald et al., 2006 ). TRPM7 is activated by products of nNOS (free radicals, ROS) and by transient depletion of extracellular Ca2+ and Mg2+ 2+ (e.g., by a decrease in [Mg ]i ). In addition, depolarization by TRPM7 activation will relieve voltage -dependent block of NMDA- R by Mg 2+, inducing a positive feedback on NMDA - R - mediated Ca 2+ entry (for a detailed review, see Macdonald et al. [ 2006 ]).

1.4.6.3 Immunodefense Mg 2+ is also essential for the normal functioning of the immune response, suggesting a potential role of TRPM7 (and TRPM6). Mg2+ defi ciency affects the immune system, for example, by causing a decrease in serum IgA and IgG levels (Zimowska et al., 2002 ; Perraud et al., 2004 ) Under conditions of infl ammation, Mg 2+ defi ciency leads to an exacerbated immune stress response and to a decrease of specifi c immune responses (Petrault et al., 2002 ).

1.4.7 TRPM 8 TRPM8 is widely expressed with high levels in sensory neurons, but also in the bladder and in the prostate (Andersson et al., 2004 ; Bandell et al., 2004 ; Stein et al., 2004 ; Zhang and Barritt, 2004 ; Tsukimi et al., 2005 ). TRPM8 is activated by cooling (< 22 – 26 ° C) and by pharmacological agents evoking a “ cool ” sensation, such as ( - ) - menthol and the “ supercooling ” agent, icilin (McKemy et al., 2002 ; Peier et al., 2002 ; Xu et al., 2002 ). Structurally diverse agents that act as antagonists, or blockers, of TRPV1, namely, BCTC, CPTC , THE TRPM SUBFAMILY 31 capsazepine, and ruthenium red, also suppress the activation of TRPM8 (Weil et al., 2005 ; Liu et al., 2006 ).

1.4.7.1 Pain TRPM8 has been implicated in cool sensation and cold pain by recent knockout mouse studies. TRPM8 appears to exert a novel analgesic gating control over noxious inputs in chronic pain states (Fleetwood- Walker et al., 2007 ). In vivo activation of TRPM8 with cutaneous or intrathecal application of menthol or icilin produces a profound analgesic effect in chronic pain models. Activation of TRPM8 causes the release of glutamate from a subpopulation of DRG neurons, which affects group II/III metabolic glutamate receptors in dorsal horn neurons or in presynapses of DRG neurons in an inhibitory fashion, and results in an analgesic effect (Proudfoot et al., 2006 ). L5 DRG sections from rats with chronic constrictive nerve injury (CCI) show increased expression of TRPM8. Ca2+ fl ux and ion currents induced by cold and menthol are increased in these studies. These changes occur in capsaicin- sensitive neurons, a subpopulation of nociceptive- like neurons. The gain of TRPM8- mediated cold sensitivity on nociceptive afferent neurons provides a mechanism of cold allodynia (Xing et al., 2007 ). In mice expressing a genetically encoded axonal tracer in TRPM8 neurons, it has been shown that TRPM8 neurons have the neurochemical hallmarks of both C- and A δ - fi bers (afferent markers IB4, NF200, peripherin), and presumptive nociceptors and non- nociceptors. TRPM8 axons diffusely innervate the skin and oral cavity, terminating in peripheral zones that contain nerve endings mediating distinct perceptions of innocuous cool, noxious cold, and fi rst - and second - cold pain. Cold temperatures generate a variety of distinct sensations that range from pleasantly cool to painfully aching, prickling, and burning. Pulpal fi bers perceive cold allodynia via TRPM8 sensitization. CCI is a neuropathic pain model, which is characterized by cold allodynia in the hind limbs. The cold allodynic response was signifi cantly reduced by capsazepine, which is a blocker for both TRPM8 and TRPV1. Cold fi bers, caused by the diverse neuronal context of TRPM8 expression, use a single molecular sensor to convey a wide range of cold sensations (Takashima et al., 2007 ).

1.4.7.2 Respiratory A functional, but truncated, TRPM8 variant is expressed in human bronchial epithelial cells. Activation of the TRPM8 variant in lung cells is coupled with enhanced expression of the inﬂ ammatory cytokines IL- 6 and IL- 8 . This may partially explain the asthmatic respiratory hypersensitivity to cold air (Sabnis et al., 2008 ). Breathing cold air induces strong respiratory autonomic responses, including cough, airway constriction, and mucosal secretion. Such a situation can be dangerous in asthmatics and can directly trigger an asthma attack. This cold - induced respiratory response is caused through an autonomic nerve reﬂ ex via vagal afferent nerves expressing TRPM8. Thus, TRPM8 contributes to cold - induced exacerbation of asthma and other pulmonary disorders (Xing et al., 2008 ). 32 TRP CHANNELS AND HUMAN DISEASES

1.4.7.3 Bladder An intriguing example of the possible role of TRPM8 in bladder function has recently been postulated (Tsukimi et al., 2005 ; Jiang et al., 2008 ), although experimental observations suggesting such a role date back to the early 1990s (Lindstrom and Mazieres, 1991 ). Filling of the bladder with ice - cold water induces a rapid detrusor refl ex and voiding in patients with supraspinal lesions, but these effects do not occur in patients with peripheral lesions or in normal subjects. Experimental studies have revealed that the bladder cooling refl ex originates from TRPM8 receptors in the bladder wall (Mukerji et al., 2006 ). In cats (Lindstrom and Mazieres, 1991 ), guinea pigs (Tsukimi et al., 2005 ), and rats (Nomoto et al., 2008 ), inclusion of menthol within an intravesicular infusion of cold saline greatly facilitates the detrusor refl ex, presumably by sensitization of TRPM8. Indeed, Lindstrom and Mazieres (1991) commented with great prescience that “ [the effect of menthol] could be described as a shift of the temperature- response curve for the cooling refl ex towards higher temperatures. ” The bladder specimens from patients with painful bladder syndrome (PBS) and idiopathic detrusor overactivity (IDO), but not asymptotic microscopic hematuria, show marked increases in the number of TRPM8 immuoreactive sensory nerve fi bers of small caliber, in the absence of any change in urothelial TRPM8 immunoreactivity. Thus, TRPM8 may provide a novel target for painful and overactive bladder diseases (Mukerji et al., 2006 ).

1.4.7.4 Cancer It has been convincingly shown that signifi cant differences exist in the level of expression of TRPM8 between malignant and nonmalig- nant prostate . TRPM8 is a much more specifi c marker for prostate cancer than other markers (prostate- specifi c antigen [PSA], kallikrenin 2 [hK2], or prostate stem cell antigen [PSCA]) (Fuessel et al., 2003 ). In the androgen - responsive prostate epithelial cell line (LNCaP), TRPM8 localizes to the endoplasmic reticulum and the plasma membrane, and TRPM8 has been reported to form a Ca2+ - permeable channel at both locations (Zhang and Barritt, 2004 ). However, in another study performed on LNCaP cells, TRPM8 was found to be largely restricted to the endoplasmic reticulum and to function as an SOC (Thebault et al., 2005 ). In any case, TRPM8 regulates Ca2+ homeostasis in prostate epithelial cells and is required for cell survival, thus defi ning a potential drug target in the management of prostate cancer. (Zhang and Barritt, 2004 ; Thebault et al., 2005 ). Sustained activation of TRPM8 by menthol, however, induces cell death in the LNCaP cell line (Zhang and Barritt, 2004 ). Moreover, expression of TRPM8 appears to be lost as prostate tumors progress to androgen independence and to more severe diseases (Henshall et al., 2003 ). TRPM8 is also expressed in human melanoma cells. The viability of melanoma cells is apparently decreased, in a dose- dependent fashion, by the TRPM8 activator menthol. TRPM8 may be involved in mechanisms underlying tumor progression and may represent a novel target of drug development for malignant melanoma. (Yamamura et al., 2008 ). TRPA 33

Finally, the trpm8 gene may be a promising candidate for gene therapy for cancer. One extracellular region in TRPM8, GLMKYIGEV, has been identi- ﬁ ed as an activating domain for cytotoxic T lymphocytes (Kiessling et al., 2003 ). Thus, TRPM8 may provide an endogenous defense mechanism against cancer growth (for a detailed review, see Zhang and Barritt [2006 ]). It has been shown that TRPM8 is expressed in neuroendocrine tumors (NET). Ca2+ inﬂ ux via TRPM8 regulates the secretion of neurotensins from such tumor cells. This regulation of neurotransmitter secretion in NETs by TRPM8 may have a potential clinical implication in the diagnosis or therapy of cancer (Mergler et al., 2007 ).

1.5 TRPA

The TRPA family currently comprises one mammalian member, TRPA1, which is expressed among others in DRG and trigeminal neurons, in hair cells (Story et al., 2003 ; Corey et al., 2004 ; Nagata et al., 2005 ), and in vagal sensory nerves innervating the airways (Brooks, 2008 ; Nassenstein et al., 2008 ). TRPA1 exhibits 14 NH2 terminal ankyrin repeats (Story et al., 2003 ), an unusual structural feature that is relevant to a proposed mechanosensory role of the channel (Nagata et al., 2005 ). Initial reports (Story et al., 2003 ; Bandell et al., 2004 ) claimed TRPA1 to be activated by noxious cold (< 17 ° C), although such a mode of stimulation has subsequently been disputed (Jordt et al., 2004 ; Nagata et al., 2005 ; Bautista et al., 2006 ). Initially identifi ed chemical activators of TRPA1 include isothiocyanates (the pungent compounds in mustard oil [MO], wasabi, and horseradish) (Bandell et al., 2004 ; Jordt et al., 2004 ), methyl salicylate (in winter green oil) (Bandell et al., 2004 ), cinnamaldehyde (CA) (in cinnamon) (Bandell et al., 2004 ), allicin and diallyl disulfi de (in garlic) (Macpherson et al., 2005 ; Bautista et al., 2006 ), acrolein (an irritant in exhaust fumes and tear gas) (Bautista et al., 2006 ), and Δ 9 tetrahydrocannabinol (Δ 9THC, the psychoactive compound in marijuana (Jordt et al., 2004 ) (for a more detailed review, see McMahon and Wood [ 2006 ]). TRPA1 is now considered as a universal chemosensor. Electrophilic compounds activate TRPA1 through covalent modifi cation of cysteine residues in the N- terminus of TRPA1 (Hinman et al., 2007 ; Macpherson et al., 2007 ; Andersson et al., 2008 ). In addition, formaldehyde, which is common ly used to induce experimental chemical pain, activates TRPA1 channels. The endogenous aldehyde 4- hydroxynonenal (4- HNE; an α , β - unsaturated aldehyde that accumulates in membranes during infl ammatory or oxidative stress), 15dPGJ2 , and other prostaglandins activate TRPA1 (Trevisani et al., 2007 ; Tai et al., 2008 ). 4- HNE is produced by lipid peroxidation in cells following infl ammation or tissue injury. At higher concentrations, 4 - HNE induces apoptosis and necrosis, and is involved in Alzheimer ’ s disease , cataracts , atherosclerosis , and cancer . Importantly, it has recently been established that menthol is also a TRPA1 activator, confounding conclusions 34 TRP CHANNELS AND HUMAN DISEASES from previous studies that used menthol as a TRPM8 - specifi c stimulus (Karashima et al., 2007 ).

1.5.1 TRPA 1 1.5.1.1 Pain TRPA1 has been implicated in several forms of pain sensation, including infl ammatory pain (thermal and mechanical hyperalgesia), neuropathic pain, mechanical pain (Bautista et al., 2006 ; Frederick et al., 2007 ), pungent pain caused by chemical agents, for example, MO and tear gas (Bautista et al., 2006 ; McMahon and Wood, 2006 ; Albin et al., 2008 ; Brô ne et al., 2008 ) and even hangover pain (Bang et al., 2007 ). Recent results obtained with two independent trpa1− / − mouse models (Bautista et al., 2006 ; Kwan et al., 2006 ) underscore an important role for TRPA1 in the pain response to endogenous infl ammatory mediators and to diverse exogenous irritants, including MO, acreolin in tear gas (and a metabolite of cyclophosphamide and fosfamide, which are widely used chemothera- peutic agents), and allicin from garlic and onions. In WT mice, topical application of MO constitutes an acute noxious stimulus and evokes neurogenic infl ammation causing thermal and mechanical hyperalgesia. By contrast, in trpa1− / − mice, the acute effects of MO (e.g., paw licking and fl inching) are abolished (Bautista et al., 2006 ) or reduced (Kwan et al., 2006 ), and hypersensitivity is either absent (Bautista et al., 2006 ) or blunted (Kwan et al., 2006 ). Of considerable signifi cance, the algesic peptide bradykinin activates TRPA1 in a G protein- dependent manner (Bandell et al., 2004 ), and in trpa1− / − mice, the development of hyperalgesia in response to injections of bradykinin is greatly reduced (Bautista et al., 2006 ; Kwan et al., 2006 ). TRPA1 has also been linked to cold hyperalgesia, a symptom of infl ammatory and neuropathic pain. Infl ammation and nerve injury both increase the expression of TRPA1 (and TRPV1) in DRG and in trigeminal neurons without affecting the abundance of TRPM8 (Katsura et al., 2006 ; Diogenes et al., 2007 ). Such overexpression appears to be driven by NGF engaging the p38 mitogen - activated protein kinase (MAPK) pathway (see Mizushima et al., 2006 ), and also involves ERK5 (Katsura et al., 2007 ). TRPA1 appears to contribute to injury - induced cold hyperalgesia since TRPA1 knockdown by siRNA strategies suppresses cold hyperalgesia in a rat nerve injury model (Obata et al., 2005 ). Therefore, TRPA1 may represent a new target for treatment of cold hyperalgesia caused by infl ammation and by nerve damage. Some (allegedly) specifi c compounds have been shown to reduce pain behaviors in mice. For instance, a specifi c small - molecule TRPA1 inhibitor (AP18) can reduce CA - induced nociception in vivo . AP18 can also reverse complete Freund’s adjuvant (CFA) - induced mechanical hyperalgesia in mice. Although TRPA1 - defi cient mice develop normal CFA- induced hyperalgesia, AP18 is ineffective in the knockout mice, consistent with an on- target mechanism (Petrus et al., 2007 ). Probably, compensatory mechanisms may account for the normal hyperalgesia in TRPA1 - defi cient mice; for example, TRPV4 upregulation would be an intruiging candidate (Petrus et al., 2007 ). Also, the TRPA 35 cannabinoid agonists R - (+) - (2,3 - dihydro - 5 - methyl - 3 - [(4 - morpholinyl)methyl] pyrol[1,2,3 - de] - 1,4 - b enzoxazin - 6 - yl) - (1 - naphthalenyl) methanone mesylate [WIN 55,212 - 2 (WIN)] and (R , S ) - 3 - (2 - iodo - 5 - nitrobenzoyl) - 1 - (1 - methyl - 2 - piperidinylmethyl) - 1H - indole (AM1241) exert peripheral antihyperalgesia in infl ammatory pain models. The mechanisms of action are not well understood, but both WIN and AM1241 activate TRPA1. It has been proposed that TRPA1 activation in sensory neurons and the subsequent Ca 2+ infl ux desensitizes TRPV1 thereby exerting their antihyperalgesic effects. The knockdown of TRPA1 activity in neurons completely eliminates the desensitizing effects of WIN and AM1241 on TRPV1. Furthermore, the WIN- or AM1241- induced inhibition of capsaicin- evoked nocifensive behavior is reversed in trpa1− / − mice (Akopian et al., 2008 ).

1.5.1.2 Cardiovascular Raw garlic, with its active component, the TRPA1 activator allicin, protects against the development of right ventricular hypertrophy in monocrotaline - induced pulmonary hypertensive rats (Sun and Ku, 2006 ). This protective effect is due to an action on vascular endothelium, preventing endothelial cell dysfunction. Boiled or aged garlic, which lack the active component allicin, was without effect. Due to the remarkable activation of TRPA1 by garlic, it is intriguing to speculate that this channel might be involved in these beneﬁ cial effects. TRPA1 is expressed in the heart (Stokes et al., 2006 ). However, it has been shown recently that the cardiovascular active compound derived from garlic might be the gaseous messenger H2 S, which induces vasorelaxation by K ATP - induced hyperpolarization in smooth muscles, prevents leukocyte adhesion, reduces cardiac arrhythmias, and acts as a cardioprotective agent (see a concise review in Lefer [ 2007 ]).

1.5.1.3 Irritation Airways continously sense multiple stimuli. Nasal trigeminal nerve endings are particularly sensitive to oxidants formed in polluted air and during oxidative stress, as well as to chlorine, which is frequently released in industrial and domestic accidents. Oxidant activation of airway neurons induces respiratory depression, nasal obstruction, sneezing, cough, and pain. While normally protective, chemosensory airway refl exes can provoke severe complications in patients affected by infl ammatory airway conditions like rhinitis and asthma. Hypochlorite, the oxidizing mediator of chlorine, and hydrogen peroxide, a ROS, activate Ca2+ infl ux and membrane currents in an oxidant- sensitive subpopulation of chemosensory neurons in WT mice, but not in trpa1 − /− mice (Bessac et al., 2008 ). TRPA1 is found preferentially on nociceptive sensory neurons, including capsaicin- sensitive TRPV1 - expressing vagal bronchopulmonary C - fi bers. Oxidative stress, a pathological feature of many respiratory diseases, causes the endogenous formation of a number of reactive electrophilic alkenals via lipid peroxidation. 4- oxononenal (4- ONE), a very electrophilic compound, induced substantial action potential discharge and tachykinin release from bronchopulmonary C - fi ber terminals. 4- ONE also caused a large TRPV1- dependent response. 4 - ONE is a relevant endogenous activator of vagal C - fi bers via an interaction 36 TRP CHANNELS AND HUMAN DISEASES with both TRPA1 and TRPV1 to induce bronchopulmonary dysfunction (Taylor- Clark et al., 2008 ). Irritant- induced cough might be caused by TRPA1 activation and by repeated or severe irritant- induced airway epithelial injury (e.g., reactive airway dysfunction syndrome [RADS]) is probably induced by persistent TRPV1 and TRPA1 activation (Brooks, 2008 ).

1.5.1.4 Bladder Immunohistochemical studies revealed the expression of TRPA1 in sensory nerve fi bers innervating the bladder, that is, in unmyelinated nerve fi bers within the urothelium, the suburothelial space, and the muscle layer as well as around blood vessels throughout the bladder (Streng et al., 2008 ). A majority, but not all TRPA1 nerves also expressed immunoreactivity for CGRP or TRPV1. Low expression was reported in the bladder mucosa (Du et al., 2007 ). Interestingly, TRPA1 expression in human bladder mucosa might be correlated with bladder outlet obstruction (BOO) or benign prostatic hyperplasia (BPH) (Du et al., 2007 ). Recent reports also showed TRPA1 in the urothelium. Apparently TRPA1 was observed in basal urothelial cells, whereas TRPV1 was located to the outer layers (Gratzke et al., 2009 ; Streng et al., 2008 ). Several indications point to a functional role of TRPA1 in the bladder. Activation of TRPA1 with allyl isothiocyanate (MO) or CA causes a contraction of the rat urinary bladder in vitro, which shows desensitization after repeated application. This occurs most likely through sensory fi ber stimulation. Ca2+ infl ux may induce release of tachykinins and may produce cyclooxygenase metabolites, probably prostaglandin E2 (Andrade et al., 2006 ). Intravesical perfusion of trans- CA and allyl isothiocyanate (MO) signifi cantly decreased the intercontraction interval and pressure threshold in rat urinary bladder (Du et al., 2007 ). After precontraction with PE, MO, CA, and NaHS caused concentration- dependent relaxations of urethral strip preparations (Gratzke et al., 2009 ). A new TRPA1 agonist, H2 S, increased micturi tion frequency and reduced the voiding volume in female rats (Streng et al., 2008 ). TRPA1 might also be involved in infl ammation in the bladder. The action of acrolein to induce infl ammation and bladder overactivity seems to be mediated via coop- eration of α 1 - adrenoreceptor and TRPA1 (Geppetti et al., 2008 ). Finally, a short remark should be added to these results. It has been shown recently that some overlap exists between TRPA1 and TRPV1 agonists. In a high concentration range, “ TRPA1 - specifi c ” compounds such as allicin also activate TRPV1 (Salazar et al., 2008 ). Thus, whether to assign a specifi c effect to TRPA1 or TRPV1 activity might be critically dependent on obtaining data from the respective knockout mice.

1.6 THE TRPML SUBFAMILY

The TRPML family contains three mammalian members (TRPML1– TRPML3) that are relatively small proteins consisting of less than 600 amino acid resi- THE TRPML SUBFAMILY 37 dues. TRPML1 is widely expressed and appears to reside in late endosomes/ lysosomes (Qian and Noben - Trauth, 2005 ). It contains a nuclear localization signal and a putative late endosomal/lysosomal targeting signal (Sun et al., 2000 ). The loop between TM1 and TM2 contains a lipase domain of unknown function, although it may be speculated that this region is enzymatically active or represents a binding site for lipids that could potentially exert a regulatory inﬂ uence on TRPML1 (Bach, 2005 ). To date, TRPML2 and TRPML3 have not been adequately functionally characterized (for a more detailed review, see Qian and Noben - Trauth [ 2005 ]).

1.6.1 TRPML 1 TRPML1 (or mucolipin 1 [MLN1]) is a transmembrane protein encoded by the gene mcoln1. TRPML1 is a high- conductance nonselective cation channel that is permeable to Ca 2+ and is regulated by pH changes (Raychowdhury et al., 2004 ). TRPML1 probably assembles into complexes that demonstrate variable single- channel conductance. Channel activity is reduced at low pH probably due to an assembly defect (Cantiello et al., 2005 ). The protein contains a serine– lipase domain in the amino part of the protein between the ﬁ rst two transmembrane domains that might function either enzymatically as a lipase, or as a binding domain, or as a transporter of lipids that might act as channel regulators. TRPML1 would thus appear to be a Ca2+ - permeable channel regulated by pH (Qian and Noben - Trauth, 2005 ).

1.6.1.1 Neurological Disorders Mutations in mcoln1 cause mucolipidosis type IV (MLIV), an autosomal recessive, neurodegenerative, lysosomal storage disorder. MLIV is clinically characterized by severe psychomotor retardation, ophthalmologic abnormalities including corneal opacity, retinal degeneration, strabismus, agenesis of the corpus callosum, blood iron defi - ciency, and achlorohydria (Bach, 2005 ). The disease normally presents itself within the fi rst year of life and progresses slowly, with patients remaining in an apparent steady state for two to three decades (Bach, 2001 ). The pathological mechanisms underlying MLIV are incompletely understood. Abnormal storage of amphiphilic lipids (phospholipids, gangliosides, neutral lipids, and mucopolysaccharides) and membranous materials in multi- concentric lamella together with granulated, water- soluble substances in late endosomes and lysosomes has been visualized by electron microscopy (Bach, 2001 ). However, there is a considerable variability in the composition of the stored materials between different organs and tissues. Such abnormal storage has been hypothesized to be due to altered membrane fusion and fi ssion events in the late endocytic pathway (Chen et al., 1998 ). Mutants of TRPML1 found in human patients (i.e., V446l and Δ F408) retain channel function, but unlike WT, TRPML1 are unaffected by pH changes, suggesting that these mutations somehow affect control of clustering and complexing of TRPML1 (Raychowdhury et al., 2004 ). There is no defec- 38 TRP CHANNELS AND HUMAN DISEASES tive hydrolytic activity, but rather a defective endocytic process in MLIV. An elevation of approximately 1 pH unit was determined in the storage vacuoles of affected cells, which is typical of late endosomes and prelysosomal vacuoles (Luzio et al., 2003 ; Maxfi eld and McGraw, 2004 ). Defective TRPML1 channels induce changes in the endocytotic transport of membrane components, such as block of the endocytotic route to the fi nal lysosome. In cells from MLIV patients, a disturbed Ca2+ signaling has been described. In addition, large acidic organelles appeared consisting of late endosomes and lysosomes. It would appear that the Ca2+ - dependent fusion between late endosome/lysosome hybrid vesicles is defective. TRPML1 could play a key role in Ca2+ release from the endosome/lysosome hybrid, which triggers the fusion and traffi cking of these organelles. Perhaps TRPML1 is important in decreasing the intravesicular concentration of Ca2+ (LaPlante et al., 2004 ). In addition to this mechanism, TRPML1 may constitute a H + leak that limits lysosomal acidifi cation. It has been recently suggested that with TRPML1 mutants, the lysosomes are chronically overacidifi ed because of the dysfunctional TRPML1 (Kiselyov et al., 2005 ).

1.6.2 TRPML 2 and TRPML 3 TRPML1 and TRPML2 are lysosomal, homomeric channels, whereas TRPML3 is a heteromer localized in the endoplasmic reticulum. When coexpressed with TRPML1 or TRPML2, TRPML3 is translocated to lysosomes. Distribution of TRPML3 is impaired when lysosomal trafﬁ cking of TRPML1 and TRPML2 is disrupted (Venkatachalam et al., 2006 ). Thus, localization of TRPML3 is regulated by TRPML1 and TRPML2 and not vice versa. Possibly, trafﬁ c of TRPML3 to lysosomes is required for the normal function of the mucolipidosis inducing TRPML1. It is not currently clear whether mutants of TRPML1 causing MLIV disrupt TRPML translocation, resulting in a decreased number of TRPML1 channels in the patient ’ s lysosomes. Recently, the Scharenberg group showed that TRPML regulates the special lysosomal compartment in B lymphocytes. Lysosomes function normally in the absence of either TRPML1 or TRPML2. However, if the C- terminus of TRPML1 is fused to TRPML2 or vice versa, the function of these channels is antagonized, and large ventricular bodies appear together with enlarged lysosomes. TRPMLs are required for the normal regulation of specialized lysosomal compartments (multivesicular bodies [MVBs]) in B lymphocytes (Song et al., 2006 ).

1.6.2.1 Hearing Extensive work in recent years has elucidated that the long - known varitint - waddler phenotype in mice is caused by mutations in the TRPML3 gene (for a review, see Cuajungco and Samie [2008 ]). The varitint- waddler mouse is characterized by hearing loss, vestibular dysfunction (circling behavior, waddling), and coat color dilution. Two genetic variants THE TRPP SUBFAMILY 39 exist: Va and VaJ . Va/Va homozygosity is lethal, Va/+ and Va J /Va J represent an intermediate form, and VaJ /+ represents the less severe form (normal vestibular function, partial hearing, and very little pigmentation deﬁ ciency). In the cochlea, Va and VaJ mice show progressive cytoplasmic abberation and highly distinctive disorganization of stereociliary bundle formation of the inner and the outer hair cells (Cuajungco and Samie, 2008 ) A decisive breakthrough was established when it was shown that the Va genotype represents a single amino acid substitution (A419P) in the TRPML3 protein, while the VaJ genotype carries an additional mutation in the trpml3 gene (I362T/A419P). TRPML3 is widely expressed in mouse tissues, and speciﬁ c immunohistochemical staining was shown in hair cell bodies and stereocilia (DiPalma et al., 2002 ). Heterologous overexpression studies have shown that TRPML3 represents a cation channel, which is highly permeable to Ca but to a lesser extent also to Na, K, and Mg (Grimm et al., 2007 ; Kim et al., 2007, 2008; Xu et al., 2007 ; Nagata et al., 2008 ). The exact electrophysiological properties of the TRPML3 channel remain controversial in the literature. Apparently, TRPML3 is regulated both by Ca2+ ; that is, high Ca2+ levels inactivate the channel, and by extracystolic protons via three histidine residues (Kim et al., 2008 ). The Va and VaJ mutations lead to a gain of function of the TRPML3 channel (Grimm et al., 2007 ; Kim et al., 2007, 2008; Xu et al., 2007 ; Nagata et al., 2008 ). A419P, such as found in the Va mutant, lies within the predicted cytoplasmic face of the α - helical transmembrane segment 5, and its presence probably induces a “ kink, hinge, or swivel, ” locking the channel in a permanently open state. Additionally, this mutation also abolishes the regulation of the channel by extracellular protons (Kim et al., 2008 ). This is important because mutations in three His residues mimick the Va channel phenotype, indicating that the A419P mutation affects the gating mechanism of the channel. The additional I362T mutation apparently reduces membrane expression of the mutated protein (Grimm et al., 2007 ; Kim et al., 2007 ). Death of melanocytes in the inner ear (and other tissue), leading to the devastating Va phenotype, probably occurs through Ca2+ overloading of the cell and/or loss of the cation gradient in hair cells (Grimm et al., 2007 ). Va J partly ameliorates this phenotype through limiting the number of channels at the plasma membrane. However, to our knowledge, similar mutations in hTRPML3, attributing to deafness and other inner ear - related disorders in humans, are unknown at the present time.

1.7 THE TRPP SUBFAMILY

The TRPP family is very heterogeneous and can be divided, based on structural criteria, into PKD1 (TRPP1) - and PKD2 (TRPP2) - like proteins. PKD1 - like members comprise TRPP1 (previously termed PKD1), PKDREJ, PKD1L1, PKD1L2, and PKD1L3. TRPP1 consists of 11 transmembrane domains, a very long and complex ∼3000 amino acid extracellular domain 40 TRP CHANNELS AND HUMAN DISEASES and an intracellular C - terminal domain that interacts with the C - terminus of TRPP2 through a coiled - coil domain (Qian and Noben - Trauth, 2005 ). The N- terminal domain of TRPP1 contains numerous structural motifs including several adhesive domains that are likely to participate in cell – cell and cell – matrix interactions. TRPP1- like proteins possess a large extracellular loop, containing conserved “ polycystin motifs ” of unknown function, between the sixth and seventh presumed transmembrane segments. The latter region is homologous to the loop interposed between the putative ﬁ rst and second transmembrane region in TRPP2- like family members (Qian and Noben- Trauth, 2005 ). The PKD2- like members structurally resemble other TRP channels in that they are predicted to have intracellular N - and C - termini, six transmembrane- spanning domains, and a pore region. The members of this subgroup comprise PKD2 (TRPP2), PKD2L1 (TRPP3), and PKD2L2 (TRPP5). All PKD2- like members possess a coiled- coil structure in their C- terminus and form polymodal multiprotein/ion channel complexes (Delmas, 2005 ). TRPP2 and TRPP3 additionally feature a Ca2+ - binding EF hand motif in the C- terminus (Koulen et al., 2002 ). In heterologous expression systems, TRPP2 and TRPP3 form constitutively active cation selective channels of relatively large conductance (Somlo and Markowitz, 2000 ; Delmas, 2004 ; Delmas et al., 2004 ). Both channels are permeable to Ca2+ , with TRPP3 displaying modest selectivity toward divalent cations (Delmas, 2005 ) (for more detailed reviews, see Delmas [2005 ], Qian and Noben- Trauth [2005 ], and K ö ttgen et al. [ 2008 ]).

1.7.1 TRPP 1 ( PKD 1) and TRPP 2 1.7.1.1 Kidney Mutations in TRPP1 and TRPP2 lead to autosomal dominant polycystic kidney disease (ADPKD), which is the most prevalent inherited form of (PKD). PKD consists of a group of conditions with progressive development of large cysts in the kidney as their major common characteristic (for a more detailed review, see Kö ttgen et al. [2008 ]). In this condition, epithelial- lined cysts are formed, fi lled with fl uid, and occupying much of the mass of the abnormally enlarged kidneys, thereby compressing and destroying normal renal tissue and impairing kidney function. Approximately 50% of patients with the primary form of PKD will progress to kidney failure, or end - stage renal disease (ESRD) (Grantham, 1993 ). ADPKD is the result of a two - hit mechanism whereby somatic inactivation of the normal allele in individual polarized epithelial cells results in loss of heterozygosity and initiates cyst formation (Somlo and Markowitz, 2000 ). Early cyst formation, which can occur in any segment of the nephron, is associated with an increase in the number of cells in the circumference of already dilated renal tubules (Boletta and Germino, 2003 ). Also, lengthening of tubules, with a characteristic mitotic orientation of cells along the tubule axis, is lacking in epithelial cells from PKD models (Fischer et al., 2006 ). Cysts are initially contiguous with the THE TRPP SUBFAMILY 41 nephron from which they pouch, but well - developed large cysts exist as independent sacs formed by epithelial cell proliferation. They are fi lled with fl uid, perhaps via active secretion mediated by the cyst epithelium (Sutters and Germino, 2003 ). Although ADPKD is characterized by kidney cysts and renal failure, it should be regarded as a systemic disorder with cysts also occurring in other organs (pancreas, brain, and liver) and a range of cardiovascular abnormalities (Kö ttgen et al., 2008 ). Indeed, TRPP1 and TRPP2 are also expressed in smooth muscle cells and seem to be important for the integrity of the vessel wall. ADPKD patients with TRPP1 and/or TRPP2 mutations also suffer from aneurysms, vessel rupture, and internal bleeding (Dietrich et al., 2006 ). TRPP2 mutations are also related to structural defects in the heart, for example, defective septum formation (Wu et al., 2000 ). Intriguingly, cultured endothelial cells from pkd1− /− or Tg737( orpk / orpk) mutant mice are unable to respond to extracellular shear stress and do not mediate shear dependent nitric oxide synthesis, which might implicate TRPP1 in blood pressure regulation (Nauli et al., 2008 ). Disease - causing mutations have been identifi ed in both the TRPP1 and the TRPP2 genes. Eighty to eighty - fi ve percent of cases are caused by mutations in the TRPP1 gene; 56 disease - causing mutations are known. Forty - fi ve disease - causing mutations in TRPP2 are also known, but they lead to less severe diseases. As mentioned above, it is unclear what the actual physiological role of TRPP1 and TRPP2 actually is. TRPP1 has been reported to participate in various signaling pathways, including PI3 kinase, JAK/STAT, Wnt, and NFAT pathways. Subcellular localization of TRPP2 is a matter of debate. The protein has been reported in the plasma membrane, in the ER membrane, and in the primary cilium, where it could serve distinct functions and where its localization could be dynamically regulated (Kö ttgen et al., 2008 ). It is therefore hard to speculate exactly how both proteins actually contribute to the development of the disease. Nevertheless, it has been shown recently that both basal and EGF - stimulated kidney cell proliferation are upregulated in cells that lack TRPP2, indicating that TRPP2 might act as a negative regulator of cell growth. TRPP2 is also a regulator of tubulogenesis: TRPP2 - lacking kidney cells form branching and multicellular tubules, suggesting a role in cyst formation (Grimm et al., 2005 ). TRPP2 overexpression in mice leads to anomalies in tubular function caused by defective tubule morphogenesis (Burtey et al., 2007 ). Another possible explanation includes TRPP1- and TRPP2 - dependent transfer of the helix- loop - helix (HLH) protein Id2 (a crucial regulator of cell proliferation and differentiation) into the nucleus. Enhanced nuclear localization of Id2 in renal epithelial cells from AKPKD patients could be a mechanism for the hyperproliferative phenotype and may cause cyst formation (Benezra, 2005 ; Li et al., 2005b ). The various mechanisms by which TPKD1 (TRPP1)/TRPP2 are involved 2+ in controlling [Ca ]i and the transcription of genes controlling cell growth are summarized in Fig. 1.4 . 42 TRP CHANNELS AND HUMAN DISEASES

Cilium 3 COOH NH2 1 NH TRPP2 PKD1 2 + COOH Ca2+ Na PKD domain TRPP2 2 2+ + Ca Na GPS-cleaved NH TRPC1 REJ domain 2 TRPP2TRPV4 PKD1 6 Plasma membrane NH Id2 NH2 NH2 2 Ankyrin PKD1 repeats COOH G P C-terminus COOH COOH PLC AC Ca2+ COOH 5 InsP3 DAG cAMP NH2 2+ ER 4 Ca PKC PKA PI3K NFAT AP-1Ras/B-Raf TRPP2 Id2-E-protein IP R MEK/ERK 2+ 3 2+ Ca dimer Ca AP-1 E protein 2+ Ca Ca2+

Ca2+ Ca2+ Nucleus Growth-suppressive genes

Figure 1.4 Compartment - speciﬁ c functions of TRPP2. Various models for the localization- dependent functions of TRPP2 are depicted. (1,2,4) In the ER, TRPP2 may function as a Ca 2+ - regulated Ca 2+ release channel. TRPP2 channel activity in the ER might be regulated by interaction with polycystin- 1 (PC- 1) in the plasma membrane or by interaction with the inositol 1,4,5 - trisphosphate receptor (IP3 - R) in the ER. (B) In the basolateral plasma membrane TRPP2 is thought to function in a complex with polycystin- 1. This complex resides either at the basal cell– matrix interface or at cell – cell junctions in the lateral membrane. (3) In the primary cilium TRPP2 may function as a sensor for mechanical or chemical cues. Flow- mediated bending of the cilium was shown to trigger cytosolic Ca 2+ signals, in a process that was proposed to require the TRPP2– polycystin - 1 complex. Other members of the TRP family (TRPV4 and TRPC1) may also be involved in ciliary mechanosensation. (5,6) PKD1 can undergo a proteolytic cleavage. The carboxy- terminal tail is released and is translocated to the nucleus where the transcription factor AP1 is activated. Phosphorylated TRPP2 sequesters Id2 in the cytoplasm and prevents it from entering the nucleus. Id2 binds to a helix - loop - helix protein E - protein, which normally activated growth suppression genes. This decreased transcription of growth suppressors may induce cyst forming. (ERK , extracellular signal - regulated kinase; Id2, inhibitor of DNA binding 2; MEK , mitogen - activated protein kinase/ERK kinase; REJ , receptor for egg jelly). Reprinted by permission from Macmillan Publishers Ltd: Giamarchi, A., Padilla, F., Coste, B., Raoux, M., Crest, M., Honoré , E. and Delmas, P. EMBO Reports 7, 787 – 793 (2006). (See color insert.) REFERENCES 43

Due to the complexity of the pathogenesis of PKD, a strategy for treating this disease is not obvious. However, a recent study has shown that a TRPP2 activating compound might be interesting in this regard. Triptolide is the active diterpene in the traditional Chinese medicine Lei Gong Teng. This natural product can be isolated from the medical vine Tripterygium wilfordii Hook F (the Thunder God Vine). It is used in Chinese medicine for the treatment of fever, chills, edema, and carbuncle. Triptolide induces Ca2+ release via a TRPP2- dependent mechanism. In a murine model of ADPKD (a trpp1− / − kidney), triptolide arrests cellular proliferation and attenuates overall cyst formation. This rescue is due to reactivation of Ca2+ signaling in the tubular epithelial cells. Small- molecule activation of TRPP2 causing Ca2+ release may become a valid therapeutic strategy for ADPKD (Leuenroth et al., 2007 ).

1.8 CONCLUSIONS

We have shown that TRP channels are connected to fundamental cell functions in a variety of tissues. A plethora of links and possible links to diseases has been listed. It is obvious that in most of the cases, the mechanism by which TRP dysfunction induces and modifi es the progression of diseases is poorly understood, and the connection is occasionally tentative. Nevertheless, we are convinced that TRP channels are and will remain in the focus of fundamental scientists, physicians, and medical pharmacists in search of innovative therapeutic strategies for dreadful diseases. Other diseases that can be directly linked to TRP channel dysfunction will likely be identifi ed when more molecular biological and functional information becomes available. The lack of selective pharmacological agents, as well as specifi c antibodies for detection of TRP channels in the native cells, however, still hampers progress in this fi eld. Nonetheless, considering the large amount of information already available from different approaches, TRP channels are without any doubt important tools to identify new pathomechanisms and will remain very promising targets for new pharmacological developments.

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Arpad Szallasi

2.1 INTRODUCTION

Despite the investment of signifi cant resources by the pharmaceutical industry to identify novel analgesic drugs, chronic pain, which is most commonly defi ned as pain lasting longer than 3 months (i.e., outlasting the usual healing process), still represents a diffi cult treatment challenge in a large sector of the population, consisting of an estimated 50 million Americans (http:// www.painfoundation.org). Patients suffering from disabling painful conditions often need complex and aggressive treatment that combines medical and surgical approaches (Campbell et al., 2006 ; Gidal and Billington, 2006 ; Katz and Berkin, 2008 ). The mainstay of medical pain therapy remains drugs that have been around for decades, like nonsteroidal anti - infl ammatory drugs (NSAIDs), or drugs that have been around even for centuries, such as opiates (Katz and Berkin, 2008 ). Many patients, however, fi nd that over - the - counter NSAID medications are ineffective for pain relief. Opiates are very powerful painkillers, but their clinical use is limited by adverse effects (Gallagher and Rosenthal, 2008 ). Also, many clinicians are concerned about the abuse of opiates (http://www.opiates.com/prescription-painkillers- addiction.html ). Of the newer agents, the COX- 2 inhibitor rofecoxib (sold by Merck under the brand name Vioxx) was withdrawn from the market over concerns of its cardiovascular side effects (Brophy, 2007 ), casting a large cloud over the

68 THE CENTRAL ROLE OF TRP CHANNELS 69 future of this class of drugs (Scanzello et al., 2008 ). Adverse effects also appear to plague the use of other recent additions to the market. For example, pregabalin (marketed as Lyrica), which is an α 2 δ calcium channel blocker, is poorly tolerated by some patients due to its central nervous system (CNS) adverse effects, especially somnolence and dizziness (Owen, 2007 ).The clinical use of ziconotide (brand named Prialt), which is a conopeptide N - type calcium channel blocker, is restricted to intractable (opiate - refractory) pain due to a combination of side effects and the need for intrathecal delivery (Wallace, 2006 ). Consequently, chronic pain is often undertreated and remains a signifi - cant unmet medical need (http://www.aarp.org/health/brain/diseases/chronic_ pain.html ) (Dray, 2008 ; Katz and Berkin, 2008 ). In late 2000, the U.S. Congress declared the 10 - year period that began January 1, 2001 as the Decade of Pain Control and Research (http://www. ampainsoc.org/decadeofpain). Furthermore, the Joint Commission of Accreditation of Healthcare Organizations (JCAHO) has mandated pain as the “ fi fth vital sign” (the other four being blood pressure, respiration, pulse, and temperature). Over the past few years, signifi cant scientifi c progress has been made in our understanding of the mechanisms that underlie infl ammatory and neuropathic pain. Preclinical research has identifi ed new factors and mechanisms that are involved in the development and maintenance of chronic pain, many of which represent potential therapeutic targets (Stucky et al., 2001 ; Cortright et al., 2007 ; Cheng and Ji, 2008 ; Oertel and L ö tsch, 2008 ; Zhuo, 2008 ). A key discovery was the molecular cloning of the vanilloid (capsaicin) receptor transient receptor potential vanilloid subfamily member 1 (TRPV1 ), a polymodal nociceptor on primary sensory neurons (Caterina et al., 1997 ). Targeting TRPV1 represents a new strategy in pain relief (Malmberg and Bley, 2005 ; Roberts and Connor, 2006 ; Szallasi et al., 2007 ; Knotkova et al., 2008 ). In contrast to traditional analgesic agents that either suppress infl ammation (e.g., NSAIDs) or inhibit pain transmission (e.g., opiates), TRPV1 antagonists aim to prevent pain by blocking a receptor where pain is generated (Fig. 2.1 ). As discussed in subsequent chapters, small - molecule TRPV1 antagonists are being evaluated in proof - of - concept pain clinical trials. Other transient receptor potential (TRP) channels on sensory neurons represent emerging therapeutic targets (Cortright et al., 2007 ; Dray, 2008 ; Eid and Cortright, 2009 ; Cortright and Szallasi, 2009b ; Patapoutian et al., 2009 ). The clinical value of TRPV1 antagonists might be the litmus test for the feasibility of this novel approach.

2.2 THE CENTRAL ROLE OF TRP CHANNELS IN NOCICEPTION AND INFLAMMATORY PAIN

Nociceptors were ﬁ rst described by Charles Scott Sherrington more than a century ago. A nociceptor (from the Latin nocere or “ to hurt ” ) is deﬁ ned as a “ pain cell” that is capable of sensing noxious stimuli and transmitting the 70 ROLE OF TRP CHANNELS IN PAIN: AN OVERVIEW

TRPV1 Blood vessel

TRPV4 Neurogenic inflammation

TRPV1 Protons, NGF, and “inflammatory soup” Dorsal horn of the spinal cord Mast cell Histamine

SP CGRP TRPV1 Second-order TRPV1 TRPV1 neuron TRPV3 TRPV3 TRPV4 Transducer TRPV4 Neuropeptide release potential DRG TRPA1 TRPA1 TRPA1 Action potential [TRPM8] Action potential TRPM8 PAR-2 [ ]

Interleukin-1

TRPV1, TRPV3, TRPV4

Keratinocyte

Skin Skin irritants Noxious heat or cold Figure 2.1 Simplifi ed, schematic representation of the complex participation of neuronal and non- neuronal TRP channels in nociception, neurogenic infl ammation, and infl ammatory pain. Temperature (heat - and cold) - sensitive TRP channels, so - called “ thermoTRPs, ” are expressed both in nociceptive neurons and in cells that are in contact with these neurons (e.g., keratinocytes and immune cells in the skin). When nociceptive neurons are activated by noxious environmental stimuli, an action potential is generated (afferent function), which is transmitted to the central nervous system where it is perceived as painful, and, at the same time, proinfl ammatory neuropeptides (e.g., SP and CGRP) are released in the periphery, initiating the biochemical cascade collectively known as neurogenic infl ammation. Epithelial cells (e.g., keratinocytes) are believed to generate interleukins when their TRP channels are stimulated by skin irritants; these interleukins, in turn, sensitize sensory nerve endings. Agents in “ infl ammatory soup ” sensitize or activate nociceptive neurons via both direct and indirect effects on TRP channels (e.g., receptor protein phosphorylation). A third route of nociceptor sensitization is by protease - activated receptor - 2 (PAR - 2 ). Vascular endothelial cells also express TRPV1 and TRPV4; these channels may enhance or block the neurogenic infl ammatory response. TRP channels, in particular TRPV1, expressed on central terminals of primary sensory neurons, are believed to play a role in the process of central sensitization, also known as the “ wind - up ” phenomenon. Of note, following injury, DRG neurons undergo a “ phenotypic change” (also known as “ injury - induced messenger plasticity” ) when the expression of some TRP channels is increased, whereas others are downregulated. SP, substance P; CGRP, calcitonin gene - related peptide; NGF, nerve growth factor; PAR- 2, protease- activated receptor 2; DRG, dorsal root ganglion. THE CENTRAL ROLE OF TRP CHANNELS 71 pain signal (Belmonte and Cervero, 1996 ). In mammals, primary sensory (nociceptive) neurons form an anatomic connection between potentially harmful external and internal agents and the CNS (Fig. 2.1 ) (Moller, 2002 ). Many non- neuronal cells, for example, urothelial cells and keratinocytes, also express nociceptor TRP channels (Fig. 2.1 ), in particular TRPV1 (Denda et al., 2001 ; Birder et al., 2002 ; Southall et al., 2003 ; Wilder- Smith et al., 2007 ), TRPV3 (Facer et al., 2007 ), and TRPV4 (Chung et al., 2003 ; Gevaert et al., 2007 ), and it has been suggested that these cells may also function as pain sensors (Southall et al., 2003 ; Birder 2005, 2006 ). Generally speaking, primary sensory neurons are bipolar cells with somata in the dorsal root ganglion (DRG) and in the trigeminal ganglion (TG). The central axons of these neurons enter the CNS where they form synapses with second- order neurons in the dorsal horn of the spinal cord (DRG neurons) or in the spinal nucleus of the trigeminal tract (TG neurons) (Fig. 2.1 ). Many neurons innervating the viscera are located in the nodose ganglia. Their peripheral fi bers travel with the vagus nerve, whereas their central axons project to the area postrema (Holzer, 1991 ). Most primary sensory neurons possess unmyelinated axons (C - fi bers) and are capsaicin sensitive (Holzer, 1991 ). A small subset of neurons with thin myelinated axons (A δ - fi bers) also expresses TRPV1 receptors. Interestingly, it has been shown that Aδ - fi bers that do not normally express TRPV1 do so under infl ammatory conditions or following injury (Ma, 2002 ; Rashid et al., 2003 ). This abnormal, TRPV1- positive A δ - fi ber population has been suggested to contribute to neuropathic pain in patients with diabetic polyneuropathy (Rashid et al., 2003 ). Indeed, desensitization by TRPV1 agonists (e.g., capsaicin and its ultrapotent natural analogue, resiniferatoxin) relieves chronic pain in these patients (Knotkova et al., 2008 ) despite the degeneration of C - fi bers (Lauria et al., 2006 ; Facer et al., 2007 ). A unifying feature of TRP channels relevant to pain is their sensitivity to temperature, hence the term “ thermoTRPs ” (Dhaka et al., 2006 ; Bandell et al., 2007 ; Talavera et al., 2008 ). Of the currently known 28 TRP channels, seven sense hot and warm temperatures (TRPV1 to TRPV4, TRPM2, TRPM4, and TRPM5), whereas two (TRPA1 and TRPM8) are activated by cold (Levine and Alessandri- Haber, 2007 ). Combined, these channels cover a wide temperature range, with extremes falling between 10 ° C (TRPA1) and 53 ° C (TRPV2). Another shared feature of these channels is their sensitivity to a variety of natural products (Table 2.1 ) (Appendino et al., 2008 ). In fact, the TRPV1 channel was originally termed the capsaicin receptor (capsaicin is responsible for the piquancy of hot chili peppers) or the vanilloid receptor VR1, based on the vanillyl fragment present in capsaicin and resiniferatoxin (a diterpene ester isolated from the latex of the perennial Euphorbia resinifera) (Szallasi and Blumberg, 1999 ). In addition to capsaicin, TRPV1 is also a receptor for pungent compounds in jellyfi sh (Cuypers et al., 2006 ) and for some spider toxins (Siemens et al., 2006 ). TRPA1 is activated by both cinnamaldehyde (from cinnamon) (Bandell et al., 2004 ; Namer et al., 2005 ) and 72 ROLE OF TRP CHANNELS IN PAIN: AN OVERVIEW allicin (an active ingredient in garlic) (Macpherson et al., 2005 ). TRPM8 is also referred to as the menthol receptor (Peier et al., 2002a ), and TRPV3 represents a target for camphor (Moqrich et al., 2005 ). TRPV4 is thought to mediate the actions of bisandrographolide, the bioactive ingredient in the Chinese medicinal plant Andrographis paniculata (Smith et al., 2006 ). While some natural products from plants show selectivity for particular TRP channels (such as resiniferatoxin for TRPV1), others are not as “ picky. ” For example, citral, a bioactive component in lemongrass, which is used both as a taste enhancer and as an insect repellant, functions as a partial agonist for all TRPs in sensory neurons (TRPV1, TRPV3, TRPA1, and TRPM8), with a lasting blockage of TRPV1, TRPV3, and TRPM8 and a transient inhibition of TRPV4 and TRPA1 (Stotz et al., 2008 ). Menthol is an even more interesting compound in that it activates TRPM8 (hence its well - known cooling effect), but, paradoxically, it also stimulates TRPV3, causing a warm sensation, and blocks TRPA1 (Macpherson et al., 2006 ). Furthermore, there is signifi cant “ cross talk” between the TRP channels that modifi es the bioactivity of natural TRP channel agonists. An interesting example of this phenomenon is camphor, which acts as a direct agonist for TRPV3 and then strongly desensitizes both TRPA1 and TRPV1 (Xu et al., 2005 ). Cannabinoids constitute another example since they desensitize capsaicin responses, not via cannabinoid CB1 or CB2 receptors, but rather via TRPA1 activation (Akopian et al., 2008 ). Apparently, the antinociceptive and antihyperalgesic actions of cannabinoids are mediated by distinct biological targets, consistent with the observation that these cannabinoid effects occur at different concentrations (Johanek et al., 2001 ). TRP channels play a central role in thermal nociception and also in detecting noxious chemicals (Fig. 2.1 ) (Liedtke and Heller, 2007 ; Nilius et al., 2007 ). This is interesting biology, but, per se, it would not make these channels potential targets for analgesic drugs. Importantly, TRPV1 is also activated and/or sensitized by agents in “ infl ammatory soup, ” ranging from tissue acidosis (protons) through cytokines (e.g., nerve growth factor [NGF], bradykinin, 12- and 15- hydroxyperoxyeicosatetraenoic acid [HPETE], and other arachidonic acid metabolites [Table 2.1 ]) (Caterina and Julius, 2001 ; Pingle et al., 2007 ; Szallasi et al., 2007 ). These agents act in concert to lower the heat activation threshold of TRPV1 (Di Marzo et al., 2002 ; Szallasi et al., 2007 ). These fi ndings have identifi ed TRPV1 as a promising target to relieve infl ammatory pain (Fig. 2.1 ). Indeed, both genetic deletion (Caterina et al., 2000 ; Davis et al., 2000 ) and pharmacological blockade of TRPV1 ameliorate heat hyperalgesia in rodent models of infl ammatory pain (Malmberg and Bley, 2005 ; Szallasi et al., 2007 ; Gunthorpe and Szallasi, 2008 ). Of relevance is the fi nding that TRPV1 expression is increased in refl ux esophagitis (where “ heartburn ” is due to exposure to acidic gastric contents) and in infl ammatory bowel disease (IBD) (Yiangou et al., 2001 ; Matthews et al., 2004 ; Bhat and Bielefeldt, 2006 ). TRPV1 is also elevated in irritable bowel syndrome (also known as colon irritable), a fairly common condition of unknown etiology THE CENTRAL ROLE OF TRP CHANNELS 73

TABLE 2.1 Thermo TRP Channels: Selected Activators and Relevance to Pain Selected Activators Relevance to Pain TRPV1 Heat ( > 43 ° C) Noxious heat detection Protons Thermal hyperalgesia during Capsaicin, resiniferatoxin, piperine infl ammation Anandamide, NADA , 12 - HPETE Jellyfi sh and spider venoms TRPV3 Warm temperature ( > 33 ° C) Candidate sensor for noxious Camphor, thymol, eugenol stimuli in keratinocytes Incensole acetate Proposed role in neuropathic pain (upregulated after nerve injury) TRPV4 Warm temperature ( > 25 ° C) Key role in mechanical Change in osmolality hyperalgesia under infl am- Candidate mechanosensor matory conditions Important role in colic pain Major player in chemotherapy - induced neuropathy TRPA1 Cold ( < 17 ° C) Major chemosensor in airways Hypertonicity Candidate mechanosensor: Mustard oil, allicin mechanical hyperalgesia in Reactive oxidants (cigarette colitis and overactive bladder smoke, exhaust fumes, tear Mediator of cold hyperalgesia gases, etc.) (pathological cold pain) Target for paradoxical pain by anesthetic drugs TRPM8 Cold ( < 23 ° C) Role in cold allodynia Menthol, icilin Possible contribution to genitourinary hyperalgesia and pain Possible role in colic pain

NADA: N - arachidonoyldopamine. characterized by frequent bowel movements and tenesmus (painful straining at stool) (Chan et al., 2003 ). Because there is no effective medical therapy, irritable bowel syndrome is frustrating for both patients and their physicians. Therefore, it is an exciting possibility that per os TRPV1 antagonists may provide symptomatic relief. Indeed, there is anecdotal evidence that eating hot, spicy food exacerbates symptoms in patients with irritable bowel syndrome. Moreover, TRPV1 is emerging as an indirect, downstream target for various endogenous agents, such as endothelin- 1, that evoke pain (Plant et al., 2007 ). A shared (and controversial) feature of thermoTRP channels, in particular TRPV1 (Prescott and Julius, 2003 ) and TRPM8 (Liu and Qin, 2005 ), is their regulation by phosphatidylinositol 4,5- biphosphate (PIP2) (Brauchi et al., 74 ROLE OF TRP CHANNELS IN PAIN: AN OVERVIEW

2007 ; Qin, 2007 ). TRPV1 possesses PIP2 recognition sites (Lishko et al., 2007 ; Kim et al., 2008 ). It was postulated that TRPV1 is under the inhibitory control of PIP2 (Prescott and Julius, 2003 ), implying a pivotal role for phospholipase C, the enzyme that cleaves PIP2, in TRPV1 activation. However, PIP2 may be either inhibitory or activating, depending on the context (Lukacs et al., 2007 ). Of note, recently it was suggested that ethanol potentiates TRPV1 - mediated responses via the PIP2 – TRPV1 interaction (Vetter et al., 2008 ).

2.3 THE EMERGING ROLE OF TRP CHANNELS IN VISCERAL PAIN

As reviewed elsewhere, the majority of sensory fi bers that project into the viscera possess TRPV1 (Holzer, 2004 ). TRPV1 - positive nerves appear to mediate visceral pain in response to noxious rectal distension (Spencer et al., 2008 ). This is somewhat surprising since TRPV1 is not supposed to have mechanosensitive properties. Although surprising, it is not unprecedented, since TRPV1 ( – / – ) mice exhibit decreased mechanical hyperreactivity of the bladder during cystitis (Wang et al., 2008 ). Moreover, silencing by RNA interference of TRPV1 has been reported to ameliorate visceral pain in rats (Christoph et al., 2006 ), implying a role for TRPV1 in visceral pain during colitis. Indeed, the fi rst generation TRPV1 antagonist capsazepine diminishes discomfort to colorectal distension in mice (Sugiura et al., 2007 ), similar to the decrease seen in TRPV1 ( – / – ) animals (Jones et al., 2005 ). Increased TRPV1 immunoreactivity was observed in colonic sensory afferents in patients with IBD (both Crohn ’ s disease and ulcerative colitis [Yiangou et al., 2001 ]) and in rectal sensory fi bers with rectal hypersensitivity and fecal urgency (Chan et al., 2003 ). Currently, it is unclear whether these changes in TRPV1 expression are pathogenic or adaptive. In a rat model of irritable bowel syndrome, TRPV1 antagonists prevent the development of visceral hypersensitivity initiated by acetic acid treatment during the neonatal period (Winston et al., 2007 ). These fi ndings imply a pathogenic role for the dysfunction of TRPV1 - positive colonic fi bers in irritable bowel syndrome. In accord, a correlation has been described between the number of TRPV1 - immunoreactive fi bers in the rectosigmoid colon and the abdominal pain score in patients with irritable bowel syndrome (Akbar et al., 2008 ). Taken together, these observations suggest that TRPV1 is a relevant therapeutic target for the treatment of visceral pain. In a rat model of IBD, topical capsaicin treatment reduces bowel ulceration in response to trinitrobenzene sulfonic acid (TNBS) (Goso et al., 1993 ). In this model, the small- molecule TRPV1 antagonist JYL1421 suppresses microscopic colitis and signifi cantly reduces (but does not completely abolish) visceromotor response to colorectal distension (Miranda et al., 2007 ). TRPV1 also appears to be involved in the post- infl ammatory hyperalgesia that occurs THE EMERGING ROLE OF TRP CHANNELS IN VISCERAL PAIN 75 after resolution of dextran sodium sulfate (DSS)- induced experimental colitis (Eijkelkamp et al., 2007 ). Nonetheless, it may be a premature conclusion that TRPV1 is exclusively responsible for the benefi cial effect of capsaicin in preclinical models of colitis. In a murine model of visceral pain, TRPV1 ( – / – ) mice show a 60% reduction in pain response magnitude compared to wild - type controls (Jones et al., 2005 ). So, what is responsible for the remaining 40% of the pain behavior? The amiloride- sensitive acid- sensing ion channels (ASICs) may be a major contributor (Sugiura et al., 2007 ). Indeed, ASIC (– / – ) mice display a reduction in pain behavior, which is similar in magnitude to that observed in the TRPV1 knockouts (Jones et al., 2005 ). Furthermore, new evidence shows that TRPA1 (presumably present on TRPV1- positive fi bers, Fig. 2.1 ) is markedly upregulated during TNBS - evoked colitis (Yang et al., 2008 ). Consistent with a pathogenic role of the increased TRPA1, intrathecal administration of TRPA1 antisense oligodeoxynucleotide reverses hyperalgesia to colonic distension (Yang et al., 2008 ). It has been known for decades that capsaicin desensitization prevents neurogenic infl ammation by cigarette smoke (Lundberg and Saria, 1983 ; Lundberg et al., 1983 ), although it is now known that these responses are initiated by TRPA1 rather than TRPV1 (André et al., 2008 ; Simon and Liedtke, 2008 ). One cannot help but wonder if a similar phenomenon may also play at least a partial role in the benefi cial effect of capsaicin desensitization in the TNBS colitis model (Goso et al., 1993 ). In man, capsaicin exerts a protective effect against gastric mucosal damage by ethanol (Mó zsik et al., 2007 ), suggesting that functional TRPV1 is protective in the gastrointestinal tract during infl ammation or chemical damage (Eysselein et al., 1991 ). If this hypothesis holds true, a potential side effect for TRPV1 antagonists given per os could be exacerbation of gastric ulcer formation. Confusingly, genetic deletion of TRPV1 has a protective action against gastric ulcers (P. Reeh, pers. comm.). Clearly, more research is needed in this area. In summary, the exact contribution of TRPV1 to visceral pain is still being debated (Hicks, 2006 ). As discussed above, ASIC and other acid- sensitive ion channels may also be involved in visceral pain (Holzer, 2003 ). Recently, TRPA1 (Mitrovic and Holzer, 2008 ; Yang et al., 2008 ) and TRPV4 (Brierley et al., 2008 ) have emerged as molecular mechanotransducers on visceral afferents, suggesting these TRP channels may also play an important role in visceral pain. TRPV4 appears to be preferentially expressed in high levels in colonic sensory neurons (Brierley et al., 2008 ). Behavioral responses to painful colonic distension are signifi cantly reduced in TRPV4 ( – / – ) mice (Brierley et al., 2008 ), as is the mechanical hyperalgesia that occurs in response to protease - activated receptor 2, (PAR - 2) (Grant et al., 2007 ). Of note, PAR - 2 also sensitizes TRPV1 (Amadesi et al., 2004 ). Thus, PAR - 2 appears to function as a regulator of TRP channels (Surprenant, 2007 ). Since gut bacteria produce high amounts of PAR- 2, TRPV4 is an attractive pharmacological target to relieve visceral pain. Unfortunately, as discussed below, pharmacological blockade of TRPV4 may have severe adverse effects. 76 ROLE OF TRP CHANNELS IN PAIN: AN OVERVIEW

2.4 CONTRIBUTION OF TRP CHANNELS TO NEUROPATHIC PAIN, CANCER PAIN, AND MIGRAINE

There is good experimental evidence that sensory neurons expressing TRP channels, in particular TRPV1, are important mediators of pathological pain (Fig. 2.1 ). For instance, rats desensitized to resiniferatoxin are devoid of the thermal hyperalgesia and guarding behavior that develops following mechanical damage of the sciatic nerve (Bennett model) (A. Szallasi, M. Tal, and G. Bennett, unpublished data ). Strikingly, resiniferatoxin also abolishes pain behavior when given to rats in a “ therapeutic fashion,” that is, to animals already in discomfort following the operation. As detailed in the chapter by Jimenes- Andrade and Mantyh in this volume, cancer pain is a promising indication for TRPV1 blockade. Here it suffi ces to mention that TRPV1 expression is enhanced in DRG neurons ipsilateral to bone cancer (osteosarcoma) in the mouse (Niiyama et al., 2007 ). In mice and dogs, treatment with resiniferatoxin to desensitize TRPV1 - containing neurons ameliorates bone cancer pain (Brown et al., 2005 ; Menendez et al., 2006 ). In mice, this effect was mimicked by both genetic disruption of the TRPV1 gene and pharmacological TRPV1 blockade by the selective antagonist JNJ- 17203212 (Ghilardi et al., 2005 ). In man, capsaicin - containing topical patches (e.g., NGX - 4010 by NeurogesX) and injectable capsaicin preparations (e.g., Adlea by Anesiva) were reported to provide relief from pain associated with diabetic neuropathy, AIDS - related neuropathy, and postherpetic neuralgia (Knotkova et al., 2008 ). Other indications for topical capsaicin treatment include migraine, cluster headache, osteoarthritis, lateral epicondylitis (e.g., “ tennis elbow ” ), Morton ’ s neuroma, and postsurgical pain (e.g., bunionectomy and hernia repair) (Knotkova et al., 2008 ). The therapeutic value of capsaicin and other TRPV1 agonists is discussed in the chapter by Bley in this volume. The rationale for using potent, selective small - molecule TRPV1 antagonists to relieve infl ammatory pain is the recognition that TRPV1 is directly activated by agents in the “ infl ammatory soup, ” including the so - called “ endovanilloids” (Fig. 2.1 and Table 2.1 ) (Di Marzo et al., 2002 ; Szallasi et al., 2007 ; see also the chapter by Bhattacharya et al. in this volume). In other words, TRPV1 antagonists prevent the binding of endovanilloids to TRPV1. No such straightforward explanation exists for the mechanism of capsaicin desensitization. It is well established that capsaicin “ silences ” TRPV1 - expressing neurons via ill - defi ned molecular processes (Szallasi and Blumberg, 1999 ). Indeed, neurons desensitized to capsaicin are also unresponsive to mustard oil (Jancsó et al., 1985 ; Patacchini et al., 1990 ), although this chemical agent acts on TRPA1 rather than TRPV1 (Jordt et al., 2004 ). Since TRPV1 agonists like capsaicin and resiniferatoxin block neuropathic pain whereas TRPV1 antagonists apparently do not, it is a reasonable assumption that other receptors present on capsaicin -sensitive neurons besides TRPV1 are directly involved in neuropathic pain. In accord, evidence was presented at the 2008 World CONTRIBUTION OF TRP CHANNELS 77

Pharmaceutical Congress by investigators at Glenmark that the TRPV3 antagonist GRC15133 is capable of inhibiting neuropathic pain (Gullapalli et al., 2008 ). A second mechanism of capsaicin desensitization was described as “ vanilloid - induced messenger plasticity ” (Szallasi and Blumberg, 1999 ). This reversible and longlasting process was suggested to involve downregulation of TRPV1 and of neuropeptides that are proalgesic (e.g., substance P [SP] and calcitonin gene- related peptide [CGRP]) as well as upregulation of peptides (e.g., galanin), enzymes (e.g., nitric oxide synthase [NOS]), and receptors (e.g., cholecystokinin [CCK - 1] receptors) that are analgesic (Szallasi and Blumberg, 1999 ). TRPV1 is now well established as a major mediator of thermal hyperalgesia. The link between TRPV1 and mechanical hyperalgesia is much weaker. In the skin, the expression of TRPV1 appears to be restricted to mechanically insensitive nerve fi bers (Lawson et al., 2008 ). In accord, perineural resiniferatoxin administration blocks thermal, but not mechanical, hyperalgesia during infl ammation (Neubert et al., 2008 ). Resiniferatoxin was previously reported to cause some decrease in mechanical hyperalgesia, presumably mediated by a spinal site, but this effect was very transient compared to the lasting blockade of thermal hyperalgesia (Xu et al., 1997 ). This is a problem for TRPV1 antagonists because many pain clinicians consider mechanical allodynia and hyperalgesia more signifi cant than thermal hyperalgesia. Recently, a specifi c small - molecule TRPA1 antagonist was reported to reverse complete Freund ’ s adjuvant (CFA) - induced mechanical hyperalgesia in wild - type, but not in TRPA1 - defi cient, mice (Petrus et al., 2007 ). As discussed in the chapter by Holland and Goadsby in this volume, the relationship between migraine and TRPV1 remains controversial. There is anecdotal evidence that capsaicin applied to the nasal mucosa is benefi cial in patients with cluster headache (Sicuteri et al., 1989 ). Moreover, ethanol is known to worsen migraine symptoms (Szallasi et al., 2006 ), and it has been suggested that ethanol sensitizes TRPV1 via PIP2 (Vetter et al., 2008 ). CGRP released from sensory neurons has been postulated to play an important role in migraine (Geppetti et al., 2005 ). Indeed, CGRP antagonists prevent migraine attacks, although these compounds are much less effective when given during the attacks (Goadsby, 2005, 2008 ). These fi ndings imply a therapeutic value for TRPV1 antagonists in migraine patients (Szallasi et al., 2006 ). The clinical trials, however, have proved very disappointing. In fact, GSK terminated its migraine clinical trials with TRPV1 antagonists due to lack of clinical effi cacy (Gunthorpe and Szallasi, 2008 ). With the benefi t of hindsight, the negative clinical trial is not unexpected. It is unclear what endovanilloid could be generated during migraine. Moreover, in the trigeminal system, the colocalization of TRPV1 and CGRP is limited. In contrast, CGRP is highly coexpressed with TRPV4. Based on this observation, some neurologists advocate the local injection of TRPV4 antagonists directly into TG for migraine refractory to conventional medical therapy (Liedtke, 2008 ). 78 ROLE OF TRP CHANNELS IN PAIN: AN OVERVIEW

2.5 DIFFERENTIAL TRP CHANNEL EXPRESSION DEFINES FUNCTIONAL SENSORY NEURON SUBTYPES: IMPLICATIONS FOR DRUG DEVELOPMENT

Primary sensory neurons are heterogenous in several aspects, including their anatomy, neurochemistry, and function. For example, these neurons differ in the myelin sheet that protects their axons (myelinated A β - fi bers, thin myelinated A δ - fi bers, and unmyelinated C- fi bers); they use different mediators (e.g., peptidergic and non - peptidergic); and they convey different somatosensory information to the CNS (e.g., touch, pain, itch, and temperature). One way to subclassify primary sensory neurons is by the TRP channels that they express. A major population of neurons with C- fi bers, as well as a minor subset of A δ neurons, coexpresses TRPV1 with the related channels TRPV3 and TRPV4 and also with TRPA1 (Kobayashi et al., 2005 ). TRPV1, TRPV3, and TRPV4 are heat - activated channels so their presence on the same neurons is not unexpected. It is more diffi cult to explain why these heat receptors are coexpressed with the cold receptor TRPA1. Adding to the complexity, TRPA1 seems to be present on both peptidergic and non- peptidergic neurons (Hjerling - Leffl er et al., 2007 ). A second major subset of primary sensory neurons, encompassing both A - and C - fi ber neurons, is characterized by their TRPM8 expression (Kobayashi et al., 2005 ). The minimal overlap between TRPV1 and TRPM8 expression suggests that TRPV1 - positive neurons and TRPM8 - expressing neurons are fundamentally different, although TRPA1 appears to be present on both TRPV1- and TRPM8- expressing populations (Kobayashi et al., 2005 ). In keeping with this concept, TRPV1- like immunoreactivity is elevated, whereas TRPM8 is, by contrast, reduced in injured human brachial plexus nerves (Facer et al., 2007 ). Based on these fi ndings, it has been postulated that TRPV1 may be a more relevant therapeutic target than other thermoTRPs for pain related to posttraumatic neuropathy (Facer et al., 2007 ). Intriguingly and in contrast to expression in DRG, TRPM8 is coexpressed with TRPV1 in vagal sensory neurons innervating the mouse lung (Nassenstein et al., 2008 ). It has been suggested that TRPV1 forms heteromultimers with other TRP channels (Liapi and Wood, 2005 ; Szallasi et al., 2007 ). If this hypothesis holds true, antagonists that do not distinguish between thermoTRPs may have a therapeutic value by targeting TRP heteromultimers. The shared TRP domain in these channels may represent a target for such inhibitors (Garcia- Sanz et al., 2007 ). Of note, N - (4 - tertiarybutylphenyl) - 4 - (3 - chloropyridin - 2 - yl)tetrahydropyrazine - 1(2H) - carboxamide (BCTC), originally described as a TRPV1 antagonist, also functions as a potent inhibitor of TRPM8 (Weil et al., 2005 ). Of note, thermoTRP channels are also colocalized with other receptors involved in pain transmission. In an innovative study, capsaicin has been used to deliver sodium channel blockers into neurons expressing TRPV1. QX - 314 is a quaternary derivative of lidocaine that is ineffective when administered alone because it is not capable of crossing the membrane. However, when TAKING A SHORT TR(i)P BEYOND PAIN 79 coadministered with capsaicin, QX- 314 enters the sensory neuron through the open TRPV1 pore and gains access to its binding site on the sodium channel (Binshtok et al., 2007 ). This elegant approach affords selective targeting of TRPV1 - expressing sensory neurons (Binshtok et al., 2007 ). The peripheral terminals of TRPV1- expressing primary sensory neurons are sites of release for a variety of proinfl ammatory neuropeptides (e.g., SP and CGRP) that initiate the biochemical cascade collectively known as neurogenic infl ammation (Fig. 2.1 ) (Geppetti and Holzer, 1996 ). Neurogenic infl ammation is thought to play a central role in the pathogenesis of various disease states that range from migraine (chapter by Holland and Goadsby in this volume) through asthma (chapter by Materazzi et al. in this volume) to IBD and cystitis (chapter by Avelino and Cruz in this volume). Obviously, diseases with a prominent neurogenic infl ammatory component are potential therapeutic indications for TRP channel blockers.

2.6 TAKING A SHORT T R ( i ) P BEYOND PAIN

Neuropeptides released from sensory neurons have been linked to various conditions encompassing pruritus, cough, emesis, neuroimmune regulation (e.g., type- 1 diabetes), glucose control (metabolic syndrome and type- 2 diabetes), obesity, and sepsis. The participation of TRP channels in these disorders has been exhaustively reviewed elsewhere (Birder, 2007 ; Jordt and Ehrlich, 2007 ; Kim and Baraniuk, 2007 ; Nilius, 2007 ; Nilius et al., 2007 ; Venkatachalam and Montell, 2007 ; Cortright and Szallasi, 2009a ; see also the chapter by Nilius and Vennekens and chapter by Tsui et al. in this volume). Clearly, these topics go beyond the scope of this chapter, namely, pain, but a brief recapitulation of these observations might be useful to the degree the fi ndings imply novel innovative uses for drugs targeting TRP channels. For example, the TRPV1 agonist resiniferatoxin is a powerful antiemetic agent in ferrets (Andrews and Bhandari, 1993 ), implying a potential for this class of compounds to inhibit intractable nausea and vomiting secondary to radiation and/or chemotherapy (Sharkey et al., 2007 ). Inhaled capsaicin is a standard agent to evoke cough response in human studies, and potent small- molecule TRPV1 antagonists are being tested in the clinics as promising antitussive drugs (McLeod et al., 2008 ; see also the chapter by Mazeratti et al. in this volume). TRP channels other than TRPV1, in particular TRPA1, are also potential targets for antitussive drugs (Kim and Baraniuk, 2007 ; Brooks, 2008 ). Pruritus is another promising indication for drugs acting on TRP channels (Paus et al., 2006 ; B í r ó et al., 2007 ). On an empirical basis, both capsaicin and menthol have been in clinical use to relieve itch for decades, identifying TRPV1 and TRPM8 as relevant pharmacological targets for novel antipruritic agents (reviewed in B í r ó et al. [ 2007 ]). The “ supercooling ” agent icilin, which is several hundredfold more potent than menthol, reduces the degree of excoriations by scratching at least 50% in rats on a Mg2+ - defi cient diet (Bí r ó et al., 2007 ). It can 80 ROLE OF TRP CHANNELS IN PAIN: AN OVERVIEW be also argued that TRPV3 and TRPV4 in keratinocytes participate in the pathomechanism of pruritus. In fact, TRPV3 is a known target for skin sensitizers, and activation of TRPV3 in murine keratinocytes by eugenol was reported to release the proinfl ammatory substance interleukin- 1 (IL- 1) (Xu et al., 2006 ). The emerging role of TRPV1 in neuroimmune regulation in general (Cortright and Szallasi, 2009a ) and in type - 1 diabetes in particular (Suri and Szallasi, 2008 ) was recently reviewed elsewhere and is also the subject of the chapter by Tsui et al. in this volume. Of note, TRPV1 has also been linked to obesity, metabolic syndrome, and type- 2 diabetes (Gram, 2003 ; Suri and Szallasi, 2008 ). TRPV1 (– / – ) mice on high fat diet are protected from visceral obesity, the type of “ pear - shaped ” obesity that has been linked to metabolic syndrome in man (Motter and Ahern, 2008 ). Type- 2 diabetes has been suggested to have a signifi cant low - grade infl ammatory component mediated by capsaicin- sensitive nerves (Gram, 2003 ). Indeed, the small- molecule TRPV1 antagonist BCTC has been reported to improve glucose tolerance in a mouse model of type - 2 diabetes (Gram and Hansen, 2007 ).

2.7 DISEASE - RELATED CHANGES IN TRP CHANNEL EXPRESSION: A NEW SPIN COMPLICATING DRUG DEVELOPMENT

TRP channels not only show bidirectional changes during disease states (up- or downregulation) but can be also expressed in cells that do not normally express such channels (Szallasi et al., 2007 ). Representative examples are discussed below. These observations have important practical implications for drug development. For example, animal experiments suggest that TRPM8 may be a relevant target to ameliorate cold hyperalgesia that develops following nerve injury (Katsura et al., 2006 ; Ji et al., 2007 ; Xing et al., 2007 ). In support of this hypothesis, mRNA encoding TRPM8 is increased in the rat DRG following chronic constriction injury (Frederick et al., 2007 ). However, in man, TRPM8 appears to be downregulated after nerve injury (Facer et al., 2007 ) and in painful dental pulp (Alvarado et al., 2007 ). Indeed, no evidence for the involvement of TRPM8 in cold allodynia has been found in neuropathic pain patients (Namer et al., 2008 ). This is a worrisome example of the species - related differences in TRP channel biology that hinder extrapolation of animal experiments to patients. In contrast, TRPA1 appears to be upregulated in human DRG after nerve injury (Anand et al., 2008 ). In rats, antisense knockdown of TRPA1 alleviates cold hyperalgesia after spinal nerve ligation (Katsura et al., 2006 ). However, the relevance of these observations is unclear, since cold allodynia appears to be independent of TRPA1 in neuropathic pain patients (Namer et al., 2008 ). In rodents, the expression of TRPV4 is increased at both the mRNA and protein levels following mechanical nerve injury, induced by CCD (chronic compression of dorsal root ganglia [DRG]) (Zhang et al., 2008 ). TRPV4 has TRPS ON NOCICEPTIVE NEURONS AS TARGETS 81 also been linked to chemotherapy (e.g., taxol- or vincristine)- induced neuropathy (Alessandri- Haber et al., 2004, 2008). When given intrathecally, TRPV4 oligodeoxynucleotide antisense reverses mechanical allodynia induced by CCD (Zhang et al., 2008 ) and ameliorates mechanical hyperalgesia in animal models of neuropathy of various etiologies, such as diabetes, alcoholism, and chemotherapy (Alessandri - Haber et al., 2008 ). TRPV4 appears to be also involved in infl ammatory pain, as implied by the reduced response to “ infl ammatory soup ” in TRPV4 ( – / – ) mice (Chen et al., 2007 ). This fi nding is consistent with the role of TRPV4 as an osmosensor and with the hypotonic nature of the infl ammatory soup. TRPV1 shows bidirectional expression changes in various disease states. During infl ammation and in bone cancer, TRPV1 levels increase substantially (Niiyama et al., 2007 ). Conversely, TRPV1 expression is downregulated in neuropathic pain secondary to injury (Lauria et al., 2006 ). It has been hypothesized that the downregulation of TRPV1 expression in diabetic skin is related to the diminished NGF levels (Facer et al., 2007 ). As reviewed elsewhere (Knotkova et al., 2008 ), a traditional indication for capsaicin- containing topical preparations is diabetic neuropathy. However, the clinical experience with capsaicin is confl icting, with some studies reporting signifi cant pain relief, whereas others have been unable to replicate these results. In the skin of patients with diabetic neuropathy, TRPV1- expressing epidermal nerve fi bers are markedly reduced, accompanied by decreased TRPV3 expression in keratinocytes (Facer et al., 2007 ). Although strictly speaking not a disease, it should be mentioned that chronic morphine administration upregulates TRPV1 expression in the spinal cord in a MAP kinase - dependent manner (Chen et al., 2008 ). This is intriguing because morphine tolerance is often associated with the development of thermal hyperalgesia. In fact, intrathecal pretreatment with the TRPV1 antagonist SB366791 (N - [3 - methoxyphenyl] - 4 - chlorocinnamide) has been shown to attenuate morphine tolerance and to prevent thermal hyperalgesia (Chen et al., 2008 ). It is hoped that TRPV1 antagonists will reduce the need for opioids and, as an added benefi t, will also prevent tolerance to opioids. Interestingly, acute morphine administration has the opposite effect since it negatively modulates TRPV1 via inhibition of adenylate cyclase (Vetter et al., 2006 ).

2.8 TRPS ON NOCICEPTIVE NEURONS AS TARGETS FOR NOVEL ANALGESIC DRUGS: ATTRACTIVE BUT NOT SO INNOCENT

2.8.1 The Capsaicin Receptor TRPV 1 As discussed above, the role of TRPV1 in infl ammatory pain was confi rmed by genetic deletion (Caterina et al., 2000 ; Davis et al., 2000 ) and pharmacological blockade experiments (Gunthorpe and Szallasi, 2008 ). The initial enthusi- 82 ROLE OF TRP CHANNELS IN PAIN: AN OVERVIEW asm for TRPV1 antagonists was generated by two basic tenets. First, the expression of TRPV1 was believed to be fairly selective for primary sensory neurons (Holzer, 1991 ). And second, TRPV1 was thought to be “ silent ” under physiological conditions (Holzer, 1991 ). Sadly, neither postulate turned out to be true. Now it is clear that the tissue expression of TRPV1 is extremely wide, ranging from CNS neurons through epithelial cells (e.g., keratinocytes and urothelial cells), vascular endothelium, and immune cells (mast cells and lymphocytes) to hepatocytes and fi broblasts (Nilius, 2007 ; Cortright and Szallasi, 2009a ; Gunthorpe and Szallasi, 2008 ). Compared to DRG neurons, the expression of TRPV1 is fairly low in these other cell types. Nevertheless, TRPV1 appears to be functional in a variety of tissues. Hypotheses are abundant regarding the role of TRPV1 in these other cell types, but experimental evidence is scarce. Notable suggestions include a role for brain TRPV1 in memory formation and in mood disorders (Gibson et al., 2008 ) and a contribution of TRPV1 on keratinocytes to hair growth and dermatologic disorders (Bodó et al., 2005 ). It was speculated that TRPV1 - expressing brain neurons may play a role in various neurological and psychiatric disorders including schizophrenia (Chahl, 2007 ), Parkinson ’ s disease (Szallasi et al., 2007 ), Huntington chorea, and Alzheimer’ s disease (Yamamoto et al., 2007 ). Of note, other than some spotty incontinence (Birder et al., 2002 ), the phenotype of TRPV1 knockout mice is fairly unremarkable. Knockout animals, however, often compensate for the missing protein. Therefore, conditional TRPV1 knock- downs would be better models to evaluate the potential role of non - DRG TRPV1 receptors. TRPV1 involvement in body temperature regulation seems to have an endogenous tone, as implied by the hyperthermic action of TRPV1 antagonists (Gavva, 2008 ; see also the chapter by Garami et al. in this volume). It has been known for over a century that capsaicin evokes the opposite effect, that is, hypothermia (Holzer, 1991 ; Szallasi and Blumberg, 1999 ). Currently, this concept is still controversial. Several classes of structurally unrelated TRPV1 antagonists evoke hyperthermia (Gavva, 2008 ; Gunthorpe and Szallasi, 2008 ). In fact, this adverse effect can be so severe that Amgen decided to discontinue the clinical trials with its lead compound after body temperature had reached 40 ° C in one patient (Gavva, 2008 ; Gunthorpe and Szallasi, 2008 ). Other potent TRPV1 antagonists (e.g., GRC6211 by Glenmark/Lilly) have no effect on body temperature (S. Narayanan, pers. comm.) or, conversely, cause hypothermia following a very mild and transient initial hyperthermic response (e.g., A - 425619). Clearly, more research is needed to resolve these confl icting fi ndings.

2.8.2 TRPV3, a Close Relative of TRPV 1 TRPV3 is a warm- sensitive ( >33 ° C) channel that, in contrast to TRPV1, is insensitive to acid or capsaicin (Peier et al., 2002b ; Smith et al., 2002 ; Xu et al., 2002 ; Chung et al., 2004 ). The preclinical proof of concept for the role of TRPV3 in thermal nociception and hyperalgesia was furnished by knockout TRPS ON NOCICEPTIVE NEURONS AS TARGETS 83 experiments (Moqrich et al., 2005 ). Indeed, GRC15133, which is a selective TRPV3 antagonist developed at Glenmark, was shown to relieve both infl ammatory and neuropathic pain in animal models (Gullapalli et al., 2008 ). Similar to TRPV1, TRPV3 is expressed in keratinocytes (Peier et al., 2002b ; Chung et al., 2004 ) where it has been suggested to mediate the release of IL- 1, a proinfl ammatory agent that, in turn, may sensitize nociceptive neurons (Xu et al., 2006 ). In the human skin, TRPV3 shows interesting disease- related changes in expression. For example, TRPV3 is downregulated in the skin of patients with diabetes (Facer et al., 2007 ), whereas TRPV3- like immunoreactivity is increased in skin biopsies obtained from the breasts of women with mastalgia secondary to macromastia or other conditions that cause breast tenderness (Gopinath et al., 2005 ). While the existence of warm - activated TRPV3 in keratinocytes is well established (Peier et al., 2002b ; Chung et al., 2004 ), the presence of TRPV3 in nerve fi bers innervating the skin is controversial. In human skin samples, no TRPV3- like immunoreactivity was detected in the epidermal nerve endings (Gopinath et al., 2005 ). However, strong TRPV3- like immunoreactivity was found in the brachial nerve plexus following nerve injury (Facer et al., 2007 ). In rodents, TRPV3 is not only highly colocalized with TRPV1 but may also compensate for TRPV1. Indeed, increased TRPV3 expression was observed in mice when TRPV1 was genetically inactivated by “ knockdown ” via RNA interference (transgenic short hairpin RNA, shRNAtg, animals), although TRPV3 expression was not increased in TRPV1 “ knockout ” mice (Christoph et al., 2008 ). TRPV3 knockout mice have a fairly unremarkable phenotype with only mild alterations in hair texture (G. Story, pers. comm.). This is in sharp contrast to animals with constitutively active, gain - of - function TRPV3 mutations that suffer from severe alopecia (Asakawa et al., 2006 ) and a skin condition that mimics human atopic dermatitis (Imura et al., 2007 ; Xiao et al., 2008 ). Most recently, incensole acetate, an incense ingredient, was shown to exert potent anxiolytic - like and antidepressant - like behavioral activity in wild - type, but not in TRPV3 knockout, mice (Moussaieff et al., 2008 ). These fi ndings were interpreted to imply a role for brain TRPV3 in emotional life (at least in the mouse). It remains to be seen if this observation has relevance for humans. However, some caution is no doubt appropriate, especially since brain TRPV1 has been implicated in rimonabant- induced mood disorders (Gibson et al., 2008 ). An attractive clinical indication for TRPV3 antagonists that block brain TRPV3 is neuroprotection via hypothermic effects (Guatteo et al., 2005 ; Lipski et al., 2006 ).

2.8.3 TRPV4, a Mechanosensor TRP with Multiple Functions TRPV4 was originally deﬁ ned as an osmosensor, hence the alternative name VR- OAC (osmotically activated channel) (Table 2.1 ). Indeed, TRPV4 is essential for the normal response to changes in osmotic pressure (Liedtke and Friedman, 2003 ) and in mechanical pressure (Suzuki et al., 2003 ). 84 ROLE OF TRP CHANNELS IN PAIN: AN OVERVIEW

Subsequently, TRPV4 has been shown to be warm - sensitive (25 – 34 ° C) and is therefore a thermoTRP channel (Gu " ler et al., 2002 ). Experiments with TRPV4 (– / – ) mice suggest that TRPV4 plays a role in normal warm sensation (Lee et al., 2005 ) and also participates in thermal hyperalgesia following infl ammation (Todaka et al., 2004 ). Warm temperatures have been shown to activate TRPV4 in keratinocytes (Chung et al., 2003, 2004). Consistent with the role of TRPV4 as a mechanosensor (Suzuki et al., 2003 ), TRPV4 knockout mice show reduced mechanical hyperalgesia (Chen et al., 2007 ). Arterial response to shear is mediated by TRPV4 expressed on vascular endothelial cells (Hartmannsgruber et al., 2007 ). In the kidney, TRPV4 serves a double role, both as a fl ow sensor (mechanosensation) and as an osmosensor (Wu et al., 2007 ). TRPV4 has been proposed to play a pivotal role in visceral hypersensitivity (Cenac et al., 2008 ). Of relevance, TRPV4 is sensitized by PAR - 2 to cause mechanical hyperalgesia in mice (Grant et al., 2007 ; Sipe et al., 2008 ). This is signifi cant since gut bacteria produce large quantities of PAR- 2. Consequently, it has been postulated that TRPV4 is an important mediator of colic pain in patients with infl ammatory bowel conditions (Brierley et al., 2008 ). Many pain experts believe that mechanical hyperalgesia is a more important player than thermal hyperalgesia in chronic human pain. Unfortunately, TRPV4 knockout mice have a severe phenotype (incontinent [Gevaert et al., 2007 ] and deaf [Tabuchi et al., 2005 ]) that casts a big dark cloud over the clinical utility of TRPV4 antagonists. Even worse, TRPV4 (– / – ) animals have impaired osmoregulation due to abnormal antidiuretic hormone (ADH) secretion, and pharmacological TRPV4 blockade has been suggested to cause a sicca syndrome- like condition (Liedtke, 2008 ). Given the essential role of TRPV4 in osmotransduction and in mechanosensation (Liedtke, 2007 ), the deleterious adverse effects of TRPV4 blockade are hardly unexpected. Consequently, drug discovery activity directed toward TRPV4 has been marginalized. New fi ndings, however, have rekindled interest in drugs targeting TRPV4 that do not get absorbed into the systemic circulation. It has been suggested that inhaled TRPV4 agonists may be benefi cial in cystic fi brosis patients and that TRPV4 - containing eye drops may protect the cornea of patients with sicca syndrome (Sjogren ’ s) (Liedtke, 2008 ). In theory, enemas containing TRPV4 antagonists may relieve colic pain, and TRPV4 antagonists injected directly into the TG via the foramen ovale are expected to ameliorate migraine pain (Liedtke, 2008 ). Parenthetically, TRPV4 is essential for the structural integrity of the broncho - alveolar unit (Alvarez et al., 2006 ; Reiter et al., 2006 ). TRPV4 is negatively regulated by cGMP. Since activation of TRPV4 was shown to cause endothelial failure and circulatory collapse (Willette et al., 2008 ), TRPV4 blockade is predicted to exert a protective action in pulmonary circulation. Last, TRPV4 defi ciency suppresses bone loss in animal experiments (Mizoguchi et al., 2008 ). This fi nding implies that TRPV4 inhibitors may be of clinical value to prevent osteoporosis in postmenopausal women. TRPS ON NOCICEPTIVE NEURONS AS TARGETS 85

2.8.4 TRPA 1, a Sensor of Reactive Oxidants and a Potential Target to Block Pathological Cold Pain Of thermoTRPs, TRPA1 is unique in that it is activated by reversible covalent modifi cation of the sulfhydryl (SH) groups of cysteine residues, rather than by conventional ligand– receptor interaction (Macpherson et al., 2007a ). In fact, TRPA1 has emerged as a major chemosensor for reactive oxidants (e.g., unsaturated dialdehydes) in airways (Andersson et al., 2008 ; Bessac et al., 2008 ) where, among other noxious stimuli, it is activated by cigarette smoke (Andr é et al., 2008 ), exhaust fumes, and tear gases (McMahon and Wood, 2006 ). Similar to TRPV4, TRPA1 is a candidate mechanosensor with postulated roles in mechanonociception (Andrade et al., 2008 ), colitis (Penuelas et al., 2007 ; Yang et al., 2008 ), and overactive bladder (Du et al., 2007, 2008). Indeed, TRPA1 knockout mice have impaired bradykinin- induced mechanical hyperalgesia (but no hearing defi cits). TRPA1 is also a cold thermosensor that is active when temperature drops below 17 ° C (Story et al., 2003 ; Bandell et al., 2004 ). Antisense knockdown of TRPA1 results in reduced cold hyperalgesia but has no infl uence on normal cold sensing (Katsura et al., 2006 ). TRPA1 is upregulated during infl ammation, an effect most likely mediated by NGF (Diogenes et al., 2007 ), and after nerve injury (Frederick et al., 2007 ; Ji et al., 2008 ). It has been postulated that TRPA1 antagonists (Petrus et al., 2007 ) may reduce infl ammatory pain caused by prostaglandins and by other fatty acid metabolites (Trevisani et al., 2007 ; Taylor - Clark et al., 2008 ). Interestingly, TRPA1 is a target for irritation by the commonly used antifun- gal agent clomitrazol (Meseguer et al., 2008 ) and may be also responsible for the paradoxical postoperative pain caused by anesthetics (Matta et al., 2008 ). TRPA1 antagonists, however, may prove a double- edged sword. They may be benefi cial by relieving pain and neurogenic infl ammation, but, at the same time, they may be potentially dangerous by blocking a major sensor for noxious environmental chemicals (Macpherson et al., 2007b ; Tai et al., 2008 ). In fact, mice whose TRPA1 channel has been deleted by genetic manipulation show defi ciencies in respiratory behavior to oxidants (Bessac et al., 2008 ).

2.8.5 TRPM 8, a Cool Receptor Although TRPM8 is best known as the menthol receptor (Bautista et al., 2007 ; Patel et al., 2007 ), the “ M ” stands not for the menthol but for melastatin, a protein identiﬁ ed by comparing benign nevi to malignant melanoma (Nilius et al., 2007 ). In fact, TRPM8 activation suppresses the viability of human melanoma (Yamamura et al., 2008 ). Parenthetically, TRPM8 also plays a role in the differentiation of prostatic epithelium (Bidaux et al., 2007 ), and it has been suggested that TRPM8 ligands may be of clinical value in controlling the growth of prostatic carcinoma (Prevarskaya et al., 2007 ). TRPM8 was the ﬁ rst cold receptor to be cloned (McKemy et al., 2002 ; Dhaka et al., 2007 ). TRPM8 is upregulated following mechanical nerve injury 86 ROLE OF TRP CHANNELS IN PAIN: AN OVERVIEW

(Frederick et al., 2007 ), and it has been postulated to play a role in cold allodynia (Xing et al., 2007 ). TRPM8 agonists have been suggested to induce analgesia to mechanical and thermal allodynia (Proudfoot et al., 2006 ) and to relieve pruritus (Biró et al., 2007 ); in fact, menthol is a traditional treatment for itch. Mouse genetics has shown that TRPM8 is required for cold hypersensitivity after nerve injury and inﬂ ammation (Colburn et al., 2007 ). New functions of TRPM8 seem to include cold sensation in airways in response to inhaled air (Sabnis et al., 2008 ; Xing et al., 2008 ) and control of bladder activity (Du et al., 2008 ; Lashinger et al., 2008 ).

2.9 CONCLUDING REMARKS

Heat - and cold- sensitive TRP channels, so- called thermoTRPs, are in the focus of attention as potential targets for novel analgesic drugs (Fleetwood- Walker et al., 2007 ; Levine and Alessandri - Haber, 2007 ; Cortright and Szallasi, 2009b ; Patapoutian et al., 2009 ;). These channels are expressed on nociceptive neurons where they play a pivotal role in sensing and integrating noxious stimuli (Fig. 2.1 ). Some of these channels, as exemplifi ed by TRPV1, are not only polymodal (i.e., they react to an array of seemingly unrelated stimuli) (Table 2.1 ), but they also have a dynamic threshold of activation. For example, agents in “ infl ammatory soup ” act in concert to sensitize TRPV1 in order to reduce its activation threshold to heat (Fig. 2.1 ) (Szallasi et al., 2007 ). TRPV1 is also a downstream target for bradykinin, NGF, and other endogenous proalgesic substances. Therefore, TRPV1 functions as a “ molecular gateway to the pain pathway ” (Caterina and Julius, 2001 ). Targeting TRP channels on nociceptors is an attractive new and logical strategy in drug development. TRP channel antagonists aim to prevent pain by blocking a receptor where pain is generated (Fig. 2.1 ). TRPV1, arguably the most important signal integrator in nociceptive neurons, has many “ fi rsts ” in this fi eld. TRPV1 was the fi rst thermoTRP to be discovered on sensory neurons (Caterina et al., 1997 ). Genetic deletion (Caterina et al., 2000 ; Davis et al., 2000 ) and pharmacological blockade of TRPV1 (Gunthorpe and Szallasi, 2008 ) furnished the fi rst proof of concept that TRP inhibitors may relieve hyperalgesia and pain. Most important, potent and selective small- molecule TRPV1 antagonists were the fi rst to move into clinical trials as potential analgesic drugs (Szallasi et al., 2007 ). TRPV1 also turned out to be a receptor with many unsuspected assets. There is emerging evidence that TRPV1 may play an important role in various disease states, ranging from type- 1 diabetes (Suri and Szallasi, 2008 ) through neurological and psychiatric disorders (Chahl, 2007 ) to obesity and cancer (Prevarskaya et al., 2007 ). These observations have opened up new avenues for drug development but also serve as warning signals for unforeseen adverse effects. TRPA1 and TRPV3 are now emerging as intriguing new targets for drug development. TRPA1 is believed to function as a sensor of tissue damage by REFERENCES 87 noxious chemicals, including reactive oxidants in inhaled air (Bessac et al., 2008 ; Simon and Liedtke, 2008 ). TRPA1 is a target for both algesic and analgesic prostaglandin metabolites (Trevisani et al., 2007 ), and it has been linked to both mechanical and cold hyperalgesia. TRPV3 is upregulated following neuronal injury (Frederick et al., 2007 ). Indeed, small- molecule TRPV3 antagonists relieve neuropathic pain in preclinical models (Gullapalli et al., 2008 ). In summary, TRP channel antagonists are predicted to inhibit various pain modalities from post - infl ammatory heat or cold hyperalgesia to spontaneous (ongoing) pain. Since these channels are preferentially (though not exclusively) expressed on nociceptors, TRP channel inhibitors are hoped to block pain without the mechanistic limitations that plague the use of existing analgesic compounds. Preclinical experiments and clinical trials are ongoing, and it remains to be seen if TRP channel antagonists will live up to these expectations.

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Carol S. Surowy , Philip R. Kym , and Regina M. Reilly

3.1 INTRODUCTION

The transient receptor potential vanilloid 1 (TRPV1) receptor is a nonselective cation channel, which is highly permeable to calcium. TRPV1 is expressed on peripheral and central terminals of small- and medium- sized primary sensory neurons, as well as in discrete areas of the brain, and plays a key role in the detection and modulation of nociceptive stimuli (Caterina and Julius, 2001 ). Over the past few years, the ﬁ eld has yielded a plethora of information on the biochemical pharmacology of this channel. This review will elaborate upon this knowledge and will summarize the current understanding of how TRPV1 functions as a molecular integrator of pain signals.

3.2 TRPV 1 STRUCTURE

TRPV1 is the ﬁ rst discovered mammalian member of the TRP superfamily. Members of this superfamily are predicted to contain six transmembrane helices (TM1– TM6) with a pore domain between helices 5 and 6 and both the N- and C- termini on the cytosolic side of the cell membrane (Fig. 3.1 ). TRPV1 channels are opened or closed by conformational changes induced by ligand binding or by other modiﬁ cations of the protein.

101 102 BIOCHEMICAL PHARMACOLOGY OF TRPV1

Figure 3.1 Representation of TRPV1 highlighting the six transmembrane domain ion channel topology and key residues involved in ligand- mediated activation (blue), proton- mediated activation (V538/Val538, T633/Thr633, E648/Glu648) or potentiation (E600/Glu600) (green), and heat activation (red oval). A indicates the ankyrin repeat domains in the N- terminus. Phosphatidylinositol (4,5)- bisphosphate (PIP) is reported to tonically inhibit TRPV1 via the indicated region (dashed square) in the C - terminus. The blue circle represents the TRP domain and the overlapping PIP activation domain. CaM indicates calmodulin- binding sites in both the N- and C- terminal domains of the channel. The receptor is activated by capsaicin, endogenous vanilloid lipids such as N - arachidonyl dopamine (NADA), heat > 42 ° C, or protons (pH < 5.5). (See color insert.)

It is generally accepted that the topology of TRPV1 is similar to that reported for other ion channels for which high- resolution X- ray crystal structures (Kcsa [Doyle et al., 1998 ], Kv1.2 [Long et al., 2005 ], MthK [Jiang et al., 2002 ]) have been obtained. The fi rst low - resolution (19 Å ) three - dimensional structure of TRPV1, determined by single - particle electron microscopy, has revealed a protein that is arranged in two distinct domains: a relatively compact domain that is consistent with a six- helix transmembrane protein structure and a large, open, basketlike domain that resides in the intracellular compartment (Moiseenkova- Bell et al., 2008 ). Another feature of TRPV1 architecture confi rmed by this structure is the symmetrical assembly of four TRPV1 subunits to form the channel pore. TRPV1 contains three ankyrin repeat domains (ARDs) in its long N - terminal region. Calmodulin (CaM) has been shown to interact with TRPV1 by binding to the fi rst ARD. The crystal structures of ARD domains for TRPV1 (Lishko et al., 2007 ), TPRV2 (Jin et al., 2006 ), and TPRV6 (Phelps et al., 2008 ) have been solved. The 1.7- Å crystal structure of the TRPV6 ARD (Phelps et al., 2008 ) reveals structural features that are ACTIVATION OF TRPV1—A POLYMODAL RECEPTOR 103 unique to TRPV proteins. Conserved TRPV residues induce a pronounced twist in the ARD and defi ne a specifi c orientation of fi nger loop 3 that may create access to a putative regulatory phosphorylation site. These features make TRPV ARDs distinct from other ARDs. The functional role of the unique TRPV ARD in determining binding sites for specifi c interacting partner proteins has not yet been reported. The large intracellular C- terminal region of TRPV1 acts as a modulatory domain for the receptor and includes the transient receptor potential (TRP) domain, phosphatidylinositol 4,5- bisphosphate (PIP2) binding sites, a CaM- binding site, and a number of key sites of phosphorylation. The highly conserved TRP domain, comprising the region spanning Glu 696 to Arg 722 (rat TRPV1 sequence numbering, Swiss - Prot O35433), has a high probability of adopting an α- helical, coiled- coil, secondary structure. The domain acts as an association domain of the protein by contributing to the tetramerization of TRPV1 to form functional channels (Garcia - Sanz et al., 2004 ). It also plays a role in channel gating, through intersubunit interactions near the channel gate that contribute to the coupling of stimulus sensing to channel opening (Garcia - Sanz et al., 2007 ). In addition, the TRP domain functions in the requirement of PIP2 for TRPV1 activation (Brauchi et al., 2007 ). These fi ndings support a signifi cant role for the TRP domain in several aspects of TRPV1 function. The distal C - terminal domain (amino acids 777 – 820) is also important in modulating TRPV1 activity by several mechanisms. This domain not only contains the proposed site involved in tonic inhibition by PIP2 but also contains Ser 800, which is a critical site for protein kinase C (PKC) - dependent phosphorylation, and overlaps the 35 amino acid segment necessary for C- terminal CaM binding.

3.3 ACTIVATION OF TRPV 1 — A POLYMODAL RECEPTOR

TRPV1 as a polymodal receptor can be activated by numerous exogenous and endogenous agonists, by noxious heat ( > 42 ° C), by extracellular protons (pH < 6.0), and by voltage. The physiological relevance of TRPV1 activation by several endogenous agonists, including the endocannabinoid anandamide, N - arachidonyl dopamine (NADA), N - oleoyldopamine (OLDA), metabolites from the lipoxygenase pathway, such as 12- hydroperoxyeicosatetraenoic acid, and lipids, such as diacylglycerol (DAG), is still being investigated (Caterina et al., 1997 ; Tominaga et al., 1998 ; Hwang et al., 2000 ; Smart et al., 2000 ; Huang et al., 2002 ). The ability of TRPV1 to act as a molecular integrator of multiple stimuli, as well as evidence that differing regions of the receptor are involved in activation by the various stimuli, underscores the complexity and dynamic nature of TRPV1 activation.

3.3.1 Best Characterized Activators of TRPV 1 3.3.1.1 Activation by Capsaicin TRPV1 is widely known for its ability to be activated by capsaicin, the pungent ingredient in hot chili peppers 104 BIOCHEMICAL PHARMACOLOGY OF TRPV1

(Caterina et al., 1997 ; Tominaga et al., 1998 ; Szallasi et al., 2007 ). Capsaicin

(EC50 ∼ 100 nM − 1 μ M, dependent on assay conditions) potently activates TRPV1. Activation of TRPV1 by capsaicin induces robust inward currents (when held at −60 mV) with a short latency that, upon washout, are quickly recovered as the channel deactivates. Current– voltage relations show that capsaicin responses exhibit outward rectiﬁ cation with a reversal potential close to 0 mV. Based on Hill coefﬁ cients of ∼2, it appears that full activation of TRPV1 involves the binding of more than one agonist molecule (Caterina et al., 1997 ). TRPV1 is a nonselective cation channel that demonstrates very high permeability to calcium ions, (PCa /PNa ) ∼ 10. This permeability for calcium ions has implications with respect to function of TRPV1, as well as its desensitization. Activation of TRPV1 by capsaicin appears to require prior phosphorylation by Ca 2+ /CaM - dependent kinase (CaMKII) (Jung et al., 2004 ). TRPV1 activation is also subject to dual regulation by the membrane phospholipid PIP2. In the absence of an agonist or at low concentrations of capsaicin (and other moderate stimuli such as heat), PIP2 exerts a tonic and partial inhibition of TRPV1 (Chuang et al., 2001 ; Lukacs et al., 2007 ), and hydrolysis of PIP2 by phospholipase C (PLC) can activate TRPV1 (Chuang et al., 2001 ). At higher concentrations of agonists, PIP2 contributes to activation of TRPV1 and is required to maintain channel activity (Stein et al., 2006 ; Lukacs et al., 2007 ). The balance between the inhibitory and stimulatory effects of PIP2 appears to depend on the strength of receptor stimulation.

3.3.1.2 Activation by Toxins Resiniferatoxin (RTX), a toxin derived from the plant Euphorbia resinifera, is the most potent of the known TRPV1 agonists, with approximately 10- to 20- fold greater potency than capsaicin. Like capsaicin, RTX induces robust currents, but kinetic studies demonstrate slower activation by RTX and lack of rapid deactivation upon washout (Caterina et al., 1997 ; Raisinghani et al., 2005 ). RTX- induced currents show less outward rectifi cation than currents induced by capsaicin. Thus, RTX induces sustained currents, and the open channel probability at all membrane potentials is signifi cantly more than that obtained with capsaicin. A number of animal toxins known to cause pain have recently been identifi ed as potent TRPV1 agonists. These include three Cys knot peptides from a tarantula toxin (Siemens et al., 2006 ) and several jellyfi sh venoms (Cuypers et al., 2006 ).

3.3.1.3 Lipid Agonists Just as capsaicin and other exogenous agonists described above are highly lipophilic, there are numerous endogenous lipid- like molecules that can directly activate TRPV1. Endogenous fatty acid- like molecules, including the endocannabinoid anandamide, and arachidonic acid metabolites, such as NADA and OLDA, are the most well- characterized putative endogenous agonists of TRPV1 (Zygmunt et al., 1999 ; Huang et al., 2002 ; Chu et al., 2003 ). Compared with capsaicin, anandamide has lower potency (in the range of 0.3 – 5.0 μ M) as an ACTIVATION OF TRPV1—A POLYMODAL RECEPTOR 105 agonist at TRPV1 (Ross, 2003 ). Moreover, anandamide acts as a partial agonist at TRPV1 in many systems, particularly when receptor reserves are limiting, as in native systems. When receptor reserves are not as limited, such as in recombinant expression systems, anandamide can demonstrate full effi - cacy (Ross, 2003 ). The low intrinsic agonist effi cacy of anandamide can have important physiological implications, including the ability to attenuate the effects of a full agonist (Ross, 2003 ). Coapplication of anandamide with capsaicin to trigeminal neurons signifi cantly reduces the currents induced by capsaicin (Roberts et al., 2002 ). However, anandamide has complex pharmacology, including its roles as an endogenous cannabinoid agonist and as a substrate for fatty acid amide hydrolase, in addition to its effects on TRPV1. Depending on the local environment, anandamide binding to TRPV1 may have the potential to enhance or to counteract pain. In this context, anandamide has been shown to mediate calcitonin gene - related peptide (CGRP) release from capsaicin - sensitive sensory neurons in part through its action on TRPV1 (Ahluwalia et al., 2003 ). The increased expression of TRPV1 in dorsal root ganglion (DRG) neurons of undamaged Aδ - and C - fi bers in a neuropathic pain model may provide an environment where the effect of anandamide at TRPV1 receptors is increased, thus contributing to enhanced pain. Another fatty acid - like molecule, NADA, is more potent than anandamide, with a potency similar to capsaicin in some systems. Like capsaicin, NADA appears to be a full agonist (Huang et al., 2002 ). NADA potently activates native TRPV1 on DRG neurons and induces the release of substance P and CGRP from spinal cord slices. Signifi cantly, intradermal administration of NADA causes TRPV1- mediated hyperalgesia. Intraplantar NADA enhances spontaneous and heat- evoked activity in spinal nociceptive neurons via its action on TRPV1 (Huang and Walker, 2006 ), consistent with a role for this endogenous agonist in pain. The related molecule OLDA also produces thermal hyperalgesia (Chu et al., 2003 ). In contrast, another fatty acid amide, oleoylethanolamide, appears to be an antagonist at TRPV1 and produces antinociceptive effects on visceral and infl ammatory pain (Suardiaz et al., 2007 ). Moreover, a minor modifi cation to OLDA, to produce 3- methyl OLDA, maintains agonist activity at TRPV1, whereas the related 4 - methyl OLDA is an antagonist (Almasi et al., 2008 ). Thus, minor differences in endogenous fatty acid amides may have quite diverse or opposite effects on TRPV1 activity in vivo . The related fatty acid - like molecules, N - acyl taurines, have also been reported to activate TRPV1 with EC50 values similar to that of anandamide (Saghatelian et al., 2006 ), although a connection to pain has not yet been reported. Various lipoxygenase products of arachidonic acid are activators of TRPV1, with 12 - (S) - and 15 - (S) - hydroxyperoxyeicosatetraenoic acid and leukotriene B4 as the most potent (Hwang et al., 2000 ). Interestingly, 12- (S) - hydroxyperoxyeicosatetraenoic acid can be induced by bradykinin (Shin et al., 2002 ), thus implicating it in infl ammatory pain. 106 BIOCHEMICAL PHARMACOLOGY OF TRPV1

DAG directly activates TRPV1, with an EC50 of ∼ 40 μ M (Woo et al., 2008 ). DAG, which is involved in several signaling pathways via activation/translocation mechanisms, is produced from the action of PLC. A membrane - permeable analogue of DAG, 1 - oleoyl - 2 - acetyl - sn- glycerol, is a partial agonist of TRPV1, inducing Ca2+ inﬂ ux in rat DRG neurons. Activation of rat TRPV1 by DAG requires the Tyr 511 residue also required by capsaicin. It has been proposed that DAG may represent a direct physiological activator of TRPV1, although its primary effect is often considered to be indirect via activation of PKC. The omega- 3 polyunsaturated fatty acids (n- 3 PUFAs) can also directly activate TRPV1 (in a PKC - dependent manner). In some cases, n - 3 PUFAs, such as linolenic acid (LNA) and eicosapentanoic acid (EPA), competitively antagonize TRPV1 activation by vanilloids (Matta et al., 2007 ). Interestingly, n- 3 PUFAs have been shown to produce analgesic effects in humans, possibly by antagonizing the effects of endovanilloids on TRPV1. The antagonistic effects of LNA and EPA occur in the concentration range of 1– 10 μ M, suggesting potential utility in pain management. In fact, EPA shows profound effects on capsaicin - induced pain behaviors in mice (Matta et al., 2007 ). Although the potency of these endogenous lipid - like molecules is generally not as high as capsaicin or RTX, it may be signiﬁ cantly increased under pathological conditions. For example, the potency of NADA in inducing TRPV1 current and in depolarizing DRG neurons via TRPV1 activation is dramatically increased when the receptors are sensitized by PKC (Premkumar et al., 2004 ).

3.3.1.4 Activation by Heat TRPV1 is also a heat- gated channel, being activated by temperatures >42 ° C at a resting membrane potential of − 60 mV (Caterina et al., 1997 ). TRPV1 currents induced by heat are quite large (about 25% the magnitude of currents evoked by capsaicin), are outwardly rectifying, and can desensitize during stimulus application. Single - channel recordings using excised membrane patches from cells expressing TRPV1 show signifi - cant currents induced by heat, strongly suggesting that heat gates TRPV1 directly (Tominaga et al., 1998 ). Cation permeability evoked by heat is similar to that evoked by capsaicin, although the P Ca /PNa ratio of 3.8 is smaller than with capsaicin - induced activation (Tominaga et al., 1998 ). Despite its well - established role as an activator of TRPV1, the molecular mechanisms involved in heat gating are presently unclear, and the amino acid residues involved in heat activation are not well defi ned. However, TRPV1 activation by both heat and capsaicin is blocked by capsazepine and ruthenium red (Tominaga et al., 1998 ), as well as by several novel antagonists recently described in the literature (El Kouhen et al., 2005 ; Gavva et al., 2005 ; Doherty et al., 2007 ; Surowy et al., 2008 ). As might be anticipated for a thermosensor, TRPV1 exhibits steep temperature dependence, with a Q10 value of 20 (Liu et al., 2003 ). TRPV1 knockout mice show decreased thermal hyperalgesia under infl ammatory conditions (Caterina et al., 2000 ; Davis et al., 2000 ). The extent to which TRPV1, versus other heat - sensitive channels, contributes to detection of noxious thermal stimuli remains to be determined, but the studies with TRPV1 ACTIVATION OF TRPV1—A POLYMODAL RECEPTOR 107 knockout mice suggest that TRPV1 plays a major role. Importantly, the conductance and rectifi cation properties of recombinant TRPV1 resemble those characterized for native heat- activated currents in sensory neurons (Cesare and McNaughton, 1996 ).

3.3.1.5 Activation of TRPV1 by Protons One of the most well- characterized endogenous agonist responses of TRPV1 is that induced by protons. The fi rst indication that protons directly gate the TRPV1 receptor came from the studies of Bevan and Yeats (1991) , who identifi ed a proton- activated, sustained, and slowly inactivating inward current in DRG neurons clamped at negative holding potential. This current appeared to be restricted to those cells that responded to capsaicin, about 45% of the DRG population. This observation, together with the similar ion selectivity of the proton - activated conductance and the capsaicin- activated currents, raised the possibility that these distinct agents activated the same ion channels. This was confi rmed by the studies of Tominaga et al. (1998) , who demonstrated that protons directly gate recombinant TRPV1. Local acidosis (pH ≤ 6) that occurs during infl ammation, ischemia, and infection contributes to the pain and hyperalgesia under these conditions (Steen et al., 1992 ; Steen and Reeh, 1993 ). There is compelling evidence that the slow, sustained response of primary afferents to acidic pH observed with infl ammation or injury is, in fact, mediated by TRPV1 (Caterina et al., 2000 ). TRPV1 activation by protons shows a concentration- dependent increase in inward current, with an EC50 value of ∼ pH 5.4 (Bevan and Yeats, 1991 ; Tominaga et al., 1998 ; Neelands et al., 2005 ). TRPV1 currents induced by protons are relatively rapidly induced and show rapid deactivation after washout (Tominaga et al., 1998 ; Neelands et al., 2005 ). Activation rates of TRPV1 by protons are similar to those induced by capsaicin, although the deactivation rates after proton activation are signifi cantly faster (Neelands et al., 2005 ). However, the maximal current induced by protons is not as large as that induced by capsaicin and, as a result, protons have been proposed to act as partial agonists of TRPV1 (Tominaga et al., 1998 ). Despite different binding sites, both proton- and capsaicin- induced TRPV1 currents are blocked by the capsaicin- site competitive antagonist capsazepine (Tominaga et al., 1998 ), as well as by several other chemically distinct TRPV1 antagonists (El Kouhen et al., 2005 ; Gavva et al., 2005 ; Doherty et al., 2007 ; Surowy et al., 2008 ). However, TRPV1 antagonists that do not block proton activation of TRPV1 have also been reported (Lehto et al., 2008 ). These fi ndings suggest some commonality in gating mechanism by these different agonists and reveal aspects of the polymodal nature of this receptor.

3.3.2 Amino Acids Involved in TRPV 1 Activation Although mutagenesis data have identifi ed a number of key residues (Tyr 511, Ser 512, Thr 550, and Tyr 666) (rat sequence numbering) important for TRPV1 agonist binding (Fig. 3.1 ), diverging hypotheses have emerged on the precise 108 BIOCHEMICAL PHARMACOLOGY OF TRPV1 orientation of agonist binding. Tyr 511 has been shown to be essential for capsaicin and anandamide activation of TRPV1, but is not critical for activation by acidic pH or heat (Jordt and Julius, 2002 ). In the capsaicin - binding model proposed by Julius and Jordt, the vanillyl functionality of capsaicin is proposed to interact with Tyr 511 located in the cytosolic region linking TM2 and TM3. An alternative model has been proposed by Gavva and coworkers, in which capsaicin binds in a manner such that the vanillyl functionality has a favorable electrostatic interaction with Thr 550 (Gavva et al., 2004 ). Alanine scanning mutagenesis of the inner pore region of the TRPV1 channel has shown that replacement of Tyr 666 results in loss of activation by both capsaicin and heat (Susankova et al., 2007 ). Ser 512 has been shown to be important for TRPV1 activation by both capsaicin and protons. Intracellular agonist recognition sites in both the N - (Arg 114) and C - (Glu 761) terminal regions (rat sequence numbering) have also been identifi ed (Jung et al., 2002 ). Point mutation of these amino acids results in the loss of sensitivity to capsaicin but does not affect channel activation by heat. Thus, while multiple groups have provided data on key amino acids involved in TRPV1 activation by capsaicin, consensus does not exist on whether the agonist binds in transmembrane or in cytoplasmic binding sites. Moreover, differing binding orientations of agonists (typically, capsaicin) in putative transmembrane sites have been proposed (Conway, 2008 ). Based on their structural similarity, it is generally believed that endogenous lipid- like agonists such as anandamide and NADA bind the same, or an overlapping, region of TRPV1 as capsaicin and RTX. In some cases, this has been confi rmed by binding studies (Ross et al., 2001 ; Shin et al., 2002 ; Toth et al., 2003 ). The binding sites for other molecules required for capsaicin, RTX, and lipid - based agonist activation have also been defi ned to some extent. The contribution of PIP2 to activation of TRPV1 by capsaicin requires the TRP domain, including two positively charged amino acids (Arg 701 and Lys 710) within the C - terminus of the rat receptor (Brauchi et al., 2007 ). Amino acids 777– 810 (rat sequence numbering) have been proposed as a PIP2 tonic inhibitory site (Prescott and Julius, 2003 ). The requirement for CaMKII- mediated phosphorylation prior to TRPV1 agonist - induced activation depends on the phosphorylation of Ser 502 and Thr 704 (Jung et al., 2004 ). Site - directed mutagenesis has shown that Glu 648, located on the putative extracellular domain adjacent to the pore - forming loop between TM5 and TM6, plays an important role in TRPV1 activation by protons (Tominaga and Tominaga, 2005 ). Single - residue mutations in both the pore helix (Thr633Ala) and the TM3 – TM4 linker domain (Val538Leu) have been shown to abrogate activation of TRPV1 by protons but have no effect on activation by capsaicin or heat (Ryu et al., 2007 ) (Fig. 3.1 ). For heat activation, the conformational changes that lead to channel opening at temperatures >42 ° C are not known. Moreover, there have been no reported mutations of TRPV1 that selectively block activation by heat, relative to activation by other stimuli. However, substituting the entire ACTIVATION OF TRPV1—A POLYMODAL RECEPTOR 109

C- terminal domain of TRPV1 with the C- terminal domain of the cold receptor TRPM8 confers cold sensitivity to the chimeric protein, suggesting that the intracellular C- terminal domain plays a key role in the activation of the TRPV1 channel by heat (Brauchi et al., 2006 ). A proposed α - helical segment of the proximal part of the C - terminus of rat TRPV1 located just downstream of the TRP domain and extending from Glu 727 to Trp 752 has been deﬁ ned as the minimal portion of TRPV1 that confers heat sensitivity (Brauchi et al., 2007 ) (Fig. 3.1 ).

3.3.3 Other Agents Reported to Activate TRPV 1 3.3.3.1 Activation by Other Naturally Occurring Molecules Several other naturally occurring molecules are also TRPV1 agonists. These include piperine, the pungent ingredient in black pepper, which activates TRPV1 with threefold greater effi cacy and faster desensitization than capsaicin. Piperine is structurally similar to capsaicin but lacks the vanilloid moiety and instead contains a methylene dioxy moiety (McNamara et al., 2005 ). Camphor, isolated from the camphor laurel tree, activates TRPV1 less effectively and also desensitizes TRPV1 more rapidly and completely than capsaicin. Camphor activation of TRPV1 appears to be mediated via a different region of the receptor than capsaicin since activation is not blocked by capsazepine (Xu et al., 2005 ). Camphor - induced desensitization of TRPV1 likely contributes to its analgesic action. However, camphor is not selective for TRPV1 since it also activates TRPV3 (Moqrich et al., 2005 ) and blocks TRPA1 (Xu et al., 2005 ). Another naturally occurring compound methylsalicylate (oil of wintergreen) activates TRPV1 (Ohta et al., 2009 ) and, like camphor, markedly desensitizes the receptor with continuous application. The activity of methylsalicylate is maintained when capsaicin - or allicin - sensitive mutants of TRPV1 are used, suggesting that methylsalicylate- induced TRPV1 activation is mediated via a distinct region of the receptor. Other naturally occurring nonselective TRPV1 agonists recently discovered include citral (Stotz et al., 2008 ) and diallyl sulfi des from garlic such as allicin, which are signifi cantly less potent than capsaicin (Koizumi et al., 2009 ). A single Cys in the N- terminal domain of TRPV1 appears necessary for activation by allicin and related compounds (Salazar et al., 2008 ). This mode of activation, through interaction with a specifi c Cys residue, parallels activation of the TRPA1 receptor via several noxious chemicals (Macpherson et al., 2007 ). As such, it provides new insights into the diversity of mechanisms that may be involved in the detection of noxious stimuli by TRPV1.

3.3.3.2 Activation by Phorbol Esters Phorbol esters such as phorbol 12 - myristate 13 - acetate (PMA) can directly activate TRPV1, albeit relatively weakly. Direct activation of TRPV1 by PMA, in addition to the indirect activation via PKC- mediated phosphorylation of the receptor, was conﬁ rmed by activation with 4α - PMA, an analogue that does not activate PKC (Premkumar 110 BIOCHEMICAL PHARMACOLOGY OF TRPV1 and Ahern, 2000 ; Bhave et al., 2003 ). In addition to direct activation of TRPV1 by phorbol esters, PKC- mediated TRPV1 phosphorylation modulates activation by other agents.

3.3.3.3 Activation by Synthetic Pharmacological Agents Several synthetic small- molecule pharmacological agents have recently been shown to exert at least some of their effects through activation of TRPV1. Some of these agents have been used as anesthetic or as analgesic drugs for some time. For example, the local anesthetic lidocaine directly activates TRPV1, likely accounting for the burning pain sensation that occurs upon application (Lefﬂ er et al., 2008 ). Activation of TRPV1 by lidocaine requires amino acid residues in the vanilloid binding domain and Arg 701 (rat sequence) in the proximal C- terminal TRP domain, as well as the presence of PIP2. Like capsaicin, lidocaine- induced TRPV1 activation causes CGRP release and may thus promote neurogenic inﬂ ammation. The analgesic tramadol, which reduces pain through multiple mechanisms, including activation of mu opioid receptors, inhibition of neurotransmitter reuptake, and inhibition of various ligand- and voltage - gated ion channels, also activates TRPV1. Activation of the TRPV1 by tramadol may account for the initial local side effects of burning pain and erythema induced by this drug (Marincsak et al., 2008 ).

3.3.3.4 Activation by Basic pH Recently, TRPV1 has been shown to be directly activated by intracellular basic pH (pH 7.8 – 9.5) (Dhaka et al., 2009 ). This activation of TRPV1 occurs via a distinct mechanism, different from activation by vanilloids, lipids, heat, or protons, and depends on an intracellular histidine in the N- terminal domain of the receptor. TRPV1 appears to be the ﬁ rst ion channel reported with the ability to be activated by both acidic and basic pH. Of potential relevance, neuronal activity causes changes in pH, both alkaline and acidic, which can affect ion channel or receptor function and neural transmission if of sufﬁ cient magnitude (Chesler, 2003 ).

3.3.3.5 Activation by Cations Various cations besides protons have also been reported to activate TRPV1. The divalent cations Mg 2+ and Ca 2+ (Ahern et al., 2005 ) and the polyvalent cations gadolinium (Gd3+ ) (Tousova et al., 2005 ) and various polyamines (Ahern et al., 2006 ) gate TRPV1. Like protons, polyamines and other cations appear to require the extracellular Glu 648 for TRPV1 activation.

3.3.3.6 Voltage-Dependent Activation Several recent studies have indicated that TRPV1 activity is regulated to some extent in a voltage- sensitive manner, contradicting the long- standing dogma that, despite its structural similarity to voltage- gated potassium channels, TRPV1 gating is voltage - independent. It was initially demonstrated that depolarization alone can activate TRPV1 at room temperature but with a high threshold (Voets et al., 2004 ), suggesting ACTIVATION OF TRPV1—A POLYMODAL RECEPTOR 111 that these channels are weakly voltage - dependent. However, binding of various ligands or physical stimuli, such as heat, shift this voltage dependence toward physiologically relevant membrane potentials (Nilius et al., 2005 ). A hypothesis was proposed, based on thermodynamic principles, that small changes in the free energy of activation of the TRPV1 channel result in a large shift in the voltage- dependent activation curve, and thus gating, of the channel by different agents. More recent characterization has offered a somewhat different view and has defi ned TRPV1 as partially activated by voltage (Matta and Ahern, 2007 ). This is based on fi ndings that maximum open channel probability is not attained with voltage activation alone and that higher concentrations of agonists such as capsaicin or RTX, as well as heat, protons, and PKC- mediated phosphorylation of TRPV1, enhance both the effi cacy and sensitivity of voltage - dependent activation. Consequently, an allosteric model of channel gating has been proposed in which voltage acts distinctly, but in concert with other mechanisms, to regulate channel activation (Matta and Ahern, 2007 ). Despite these recent developments, the precise location of the voltage sensor within TRPV1 has not yet been clearly defi ned. Although charged amino acids within the TM4 (S4) domain may play a role, as they do in other voltage- gated channels, these are reduced in number in TRPV1. In combination, these data provide new support for voltage sensitivity of TRPV1. This may have direct implications for pain, since neurons involved in sensing or transmitting pain become depolarized by multiple mechanisms, including via TRPV1 activation itself. Recently, a competitive TRPV1 antagonist (SB - 705498) has been shown to inhibit recombinant human TRPV1 activity with a degree of voltage dependence, being more potent at − 70 mV than at +70 mV (Gunthorpe et al., 2007 ). To date, this property has not been reported for other TRPV1 channel antagonists and awaits further elucidation of mechanism of action and any connection to the voltage - sensitive gating described above.

3.3.4 Dynamic Ion Selectivity In addition to Na + and Ca 2+ permeability, TRPV1 has been reported to be permeable to larger cations, although the underlying mechanism has not been defi ned. Recently, the dogma of static ion selectivity during TRPV1 activation has been challenged by the intriguing fi nding of dynamic ionic selectivity during sustained agonist stimulation (Chung et al., 2008 ). These effects can span several seconds to several minutes. Activation of either native or recombinant TRPV1 by capsaicin or by protons leads to time- and agonist concentration - dependent changes in permeability to large cations and changes in Ca2+ permeability. The effective size of the pore is apparently increased following initial channel gating. These changes appear to be a result of alterations in the TRPV1 selectivity fi lter in the inner pore of the channel and defi ne a new role for this region in not only quantitative but also qualitative aspects of channel gating. Moreover, the various TRPV1 agonists change ion selectivity 112 BIOCHEMICAL PHARMACOLOGY OF TRPV1 differentially. Both capsaicin and NADA produce biphasic responses, albeit with different kinetics, whereas heat evokes a transient current without a robust second phase (Chung et al., 2008 ). Activation- dependent changes in 2+ TRPV1 Ca selectivity causes an up to fi vefold increase in PCa /PNa , depending on concentrations of both capsaicin and Ca2+ . Phosphorylation of TRPV1 by PKC at Ser 800 enhances ionic selectivity. The mode of activation of TRPV1 also affects the calcium current. The fraction of current carried by Ca2+ is smaller for proton- and heat- induced activation (Tominaga et al., 1998 ; Samways et al., 2008 ) than it is for capsaicin - induced activation. The differences in proton - induced ion selectivity during activation are the result of protonation of three acidic amino acids, including Glu 648, that line the mouth of the pore (Samways et al., 2008 ). Interestingly, conformational changes in the outer pore region appear to be critical for determining the balance between open and closed states, providing evidence for a general role for this domain in TRP channel activation (Myers et al., 2008 ). The potential consequences of increased pore size and permeability to Ca2+ and large cations are severalfold. Ca 2+ infl ux has numerous downstream effects, including Ca 2+ - dependent desensitization of TRPV1 or other channels, activation of signaling molecules, and release of infl ammatory mediators and neurotransmitters (Chung et al., 2008 ). The enhanced Ca2+ permeability induced by PKC- mediated phosphorylation of TRPV1 suggests that this may be one mechanism by which infl ammatory mediators that sensitize TRPV1 contribute to pain hypersensitivity. Moreover, since prolonged exposure to capsaicin can selectively destroy TRPV1- expressing neurons, the enhanced permeability to Ca 2+ or other large cations may initiate apoptotic pathways, including during the use of capsaicin as an analgesic where neuronal death is a mechanism of action of the analgesia that occurs with topical treatment. As such, dynamic ion selectivity adds a new and important dimension to the impact of TRPV1 activation and its role as an integrator of pain signals.

3.3.5 Desensitization of TRPV 1 Desensitization of TRPV1 after activation can occur by one of two mechanisms: acute desensitization and tachyphylaxis. Tachyphylaxis is loss of sensitivity to repeated stimulation and appears to occur due to failure to recover from acute desensitization (Liu and Simon, 1996 ). Physiological consequences of TRPV1 desensitization include adaptation of neurons, which decreases pain perception. The process of acute desensitization is initiated by the entry of Ca 2+ into cells upon TRPV1 receptor activation (Koplas et al., 1997 ). A prominent mechanism responsible for this desensitization is the Ca2+ /CaM - dependent phosphatase calcineurin (Docherty et al., 1996 ; Mohapatra and Nau, 2005 ). However, no single mechanism appears to be solely responsible for desensitization. Both dephosphorylation, by calcineurin, of two CaMKII phosphorylated residues, Ser 502 and Thr 704 (rat sequence numbering) (Jung et al., ACTIVATION OF TRPV1—A POLYMODAL RECEPTOR 113

2004 ), as well as depletion of PIP2 (implicating Ca 2+ - dependent PLC) contribute to TRPV1 desensitization (Liu et al., 2005 ; Yao and Qin, 2009 ). Tyr 671 within the internal pore of the channel plays a role in the structural rearrangements that occur during desensitization (Mohapatra et al., 2003 ), and the whole process is accompanied by a change in voltage dependence (Piper et al., 1999 ; Gunthorpe et al., 2000 ). Arg 701 (rat sequence numbering), which is part of a stringent CaMKII - binding site within the TRP box and is critical for PIP2 activation of TRPV1, is also strongly implicated in the slowed gating kinetics of the desensitized channel, suggesting that acute desensitization may alter coupling between the capsaicin - binding site and the PIP2 - sensitive gating mechanisms of TRPV1 (Novakova - Tousova et al., 2007 ). Addition of CaMKII reverses the Ca 2+ - dependent TRPV1 desensitization induced by capsaicin (Jung et al., 2004 ), as anticipated from the role of CaMKII - mediated phosphorylation in capsaicin - induced TRPV1 activation. Thus, desensitization of TRPV1 may occur when the receptor loses its ability to bind ligands after dephosphorylation. Acute Ca2+ - mediated desensitization can also be decreased by protein kinase A (PKA) - or PKC - mediated phosphorylation at discrete sites on TRPV1 (Bhave et al., 2002 ; Mohapatra and Nau, 2003 ; Mohapatra et al., 2003 ; Mandadi et al., 2004, 2006 ). Thus, sensitization of the channel can counteract acute desensitization and can enhance TRPV1 activation in response to tissue injury or infl ammation. The predominant sites involved in PKA- mediated decreased desensitization appear to be the amino acid Ser 116 and, to a lesser extent, Thr 370 (rat TRPV1 sequence numbering) (Mohapatra and Nau, 2003 ). PKC ε - mediated decrease in desensitization occurs via phosphorylation of Ser 800 (rat TRPV1 sequence numbering) (Mandadi et al., 2006 ). In addition to Ca 2+ - dependent desensitization, there is evidence of Ca2+ - independent desensitization by some agonists such as camphor (Xu et al., 2005 ) and, to some extent, piperine (McNamara et al., 2005 ). Finally, TRPV1 agonists can cross desensitize (Tominaga et al., 1998 ; Vlachova et al., 2001 ). For example, capsaicin - induced Ca2+ - dependent desensitization is accompanied by a decreased response to both heat and voltage. Tachyphylaxis of TRPV1 requires the activity of the calcium- binding protein CaM, whereas either ATP or PIP2 can prevent this process (Lishko et al., 2007 ). CaM interacts with the C - terminal domain in a Ca2+ - independent manner but with the N- terminal domain in a Ca 2+ - dependent manner (Numazaki et al., 2003 ; Rosenbaum et al., 2004 ). Mutations in the ARD prevent tachyphylaxis, and it appears that ATP and CaM may compete for binding to an ARD- binding site on TRPV1 to prevent or to induce tachyphylaxis, respectively. PIP2 also causes a partial reduction in tachyphylaxis in the absence of ATP, although neither the mechanism nor binding site for this effect has been fully defi ned (Lishko et al., 2007 ). Although adaptation is an important feature of many sensory receptors, allowing them to continuously respond to varying intensity of stimuli, adaptation has not yet been clearly demonstrated in nociceptive systems. During 114 BIOCHEMICAL PHARMACOLOGY OF TRPV1 desensitization, Ca2+ infl ux through TRPV1 causes depletion of PIP2. The depletion of PIP2 has a time course that is synchronous with TRPV1 desensitization and reaches a level where there is a signifi cant shift in agonist sensitivity while still maintaining a maximal response. Thus, the level of interaction of TRPV1 with PIP2 through the Ca 2+ - mediated desensitization process may confer adaptation onto the TRPV1 receptor (Yao and Qin, 2009 ).

3.4 TRPV 1 AS A MOLECULAR INTEGRATOR

TRPV1 activation by specifi c stimuli/agonists when applied alone often produces only submaximal activation. A maximal response is achieved through the synergistic interaction of two or more stimuli, which work in tandem to open the channel at lower activation thresholds (Tominaga et al., 1998 ). This polymodal activation of TRPV1 plays a major role in its function as a molecular integrator of pain signals. At 37 ° C, an abrupt but relatively small decrease in extracellular pH (pH 6.4) evokes signifi cant TRPV1 currents, demonstrating that protons can activate the channel at normal body temperature. With a more signifi cant but still moderate decrease in pH (pH ≤ 5.9), protons can activate TRPV1 at room temperature (Tominaga et al., 1998 ). Protons thus substantially decrease the temperature threshold for TRPV1 activation and, because different regions of TRPV1 are involved in responses to the various activators, are likely do so in a synergistic manner. A molecular determinant of proton - induced potentiation of TRPV1 activation is the extracellular amino acid Glu 600, localized in the region linking TM5 and the pore- forming region of the channel (Jordt et al., 2000 ). Based on electrophysiological data using mildly acidic solutions at pH 6.6, it has been proposed that protons may act by increasing the probability of channel opening caused by other ligands rather than by altering unitary conductance (Baumann and Martenson, 2000 ). Protons potentiate capsaicin action by increasing potency without altering effi cacy (Caterina et al., 1997 ; Tominaga et al., 1998 ). When the pH is lowered from pH 7.4 to pH 5.5, capsaicin shows a > 10 - fold decrease in EC50 (Neelands et al., 2005 ). In kinetic studies, acidic pH causes both an increase in activation rate and a decrease in deactivation rate of capsaicin - activated currents. The acid - induced increase in activation rate is dependent on the concentration of protons, with a modest increase at pH 6.8 and a greater increase at pH 5.0. In the reverse protocol, capsaicin increases the activation rate for protons almost 10- fold, suggesting that capsaicin binding can increase the affi nity of protons for TRPV1 (Neelands et al., 2005 ). The decrease in the deactivation rate of capsaicin - evoked currents caused by acidic pH, as assessed using paired - pulse and single - pulse protocols, is quite signifi cant. It is possible that the presence of protons results in an allosteric change that prevents the release of capsaicin from its binding site (Neelands et al., 2005 ). Synergistic interaction also occurs when TRPV1 is activated by an agonist at elevated temperatures (Vlachova et al., 2001 ; Neelands et al., 2008 ). TRPV1 AS A MOLECULAR INTEGRATOR 115

Investigation of such interactions using freshly isolated DRG neurons has demonstrated profound effects on membrane excitability (Neelands et al., 2008 ). Increasing the temperature causes larger membrane depolarization and an altered pattern and onset of the action potential train evoked by capsaicin. Moreover, anandamide, which does not normally cause action potentials but rather causes a slow depolarization, at elevated temperature causes rapid and signiﬁ cant depolarization accompanied by a short burst of action potentials. The changes in ﬁ ring properties of DRG neurons produced by heat effects in the presence of another agonist may be important in the temporal coding of pain (Neelands et al., 2008 ).

3.4.1 Sensitization of TRPV 1 Sensitization of TRPV1 is central to its role as an effective transducer of pain signals and contributes substantially to the development of persistent pain. At the cellular level, sensitization reduces the thresholds for activation and heightens responsiveness to multiple stimuli, leading to increased neuronal fi ring. In infl ammatory pain, hypersensitivity arises from release of proalgesic agents, including neurogenic peptides, cytokines, chemokines, prostaglandins, and growth factors, which are released at the injured site from sensory nerve terminals or from infi ltrating immune cells. Together these mediators reduce the nociceptive threshold by binding to their cognate receptors, ultimately activating multiple protein kinase cascades that phosphorylate TRPV1 and elicit dynamic changes in the activity and localization of TRPV1. The sensitized form of the channel exhibits enhanced activity and prevents desensitization to subsequent, persistent stimulation by agonists. At the molecular level, TRPV1 is sensitized by phosphorylation of Ser 502 by PKA or PKC in response to infl ammatory mediators, such as prostaglandins (Moriyama et al., 2005 ), bradykinin (Tominaga et al., 2004 ; Moriyama et al., 2005 ), and other molecules. PKC - dependent phosphorylation of Ser 502 and Ser 800 potentiates capsaicin or proton- evoked TRPV1 activation and reduces the temperature threshold for activation (Vellani et al., 2001 ; Numazaki et al., 2002 ; Bhave et al., 2003 ).

3.4.2 TRPV1 and Infl ammatory Mediators Bradykinin, a neurogenic peptide processed from a precursor at the site of injury or infl ammation, is a potent sensitizer of TRPV1. Stein et al. (2006) propose that TRPV1 sensitization by bradykinin is due to the generation of DAG, which, in turn, has the potential to activate both PKC (Cesare et al., 1999 ; Ferreira et al., 2005 ) and TRPV1 directly (Woo et al., 2008 ). Activation of the PKC and PLC pathways by bradykinin can result in generation of lipoxygenase products (e.g., anandamide and NADA), which directly activate or sensitize TRPV1 on sensory neurons (Premkumar and Ahern, 2000 ; Shin et al., 2002 ; Ferreira et al., 2004 ; Tang et al., 2004 ). Thus, upon injury or 116 BIOCHEMICAL PHARMACOLOGY OF TRPV1 infl ammation, mediators like bradykinin and arachidonic acid metabolites reduce the threshold for activation of TRPV1 by noxious stimuli. Substance P is a tachykinin released from postsynaptic dorsal horn and sensory neurons, which contributes to neurogenic infl ammation by binding to the neurokinin - 1 (NK - 1) receptor on endothelial and immune cells to modulate pain transmission and sensitization. The recent demonstration that NK- 1 and TRPV1 are colocalized on DRG neurons suggests the potential for cross talk (Zhang et al., 2007 ). Binding of substance P to NK- 1 enhances TRPV1 currents (Sculptoreanu et al., 2008 ) and induces translocation of PKC ε to the plasma membrane of sensory neurons where it is responsible for phosphorylation of TRPV1. Conversely, inhibition of PKCε prevents substance P - mediated activation and translocation of TRPV1 (Zhang et al., 2007 ). Involvement of the proinfl ammatory cytokine tumor necrosis factor (TNF)α in pain is well established, but the role of TNFα is complex and is incompletely understood. Recent studies suggest that the hyperalgesic effect of TNFα occurs through mechanisms involving TRPV1. Compelling evidence is provided by studies that demonstrate a thermal hyperalgesic response to intraplantar injection of TNFα in wild - type, but not in TRPV1 null, mice (Jin and Gereau, 2006 ; Russell et al., 2009 ). Greater than 90% of rat TRPV1 - positive trigeminal neurons also express TNFα receptors (Khan et al., 2008 ). TNF α enhances the activation of sensory neurons by capsaicin through both tran- scriptional and posttranscriptional mechanisms. The sensitization and upregulation of TRPV1 expression by TNFα appears to contribute to the proalgesic effect of the cytokine. Prostaglandin E2 (PGE2) is an infl ammatory mediator, critical to pain sensation. PGE2 sensitizes TRPV1 responses, appreciably reducing the temperature threshold in a PKC - dependent manner, as demonstrated in both TRPV1 recombinant cells and cultured mouse DRG neurons (Moriyama et al., 2005 ). This sensitization appears to be a result of EP1 receptor activation. A PKA pathway for sensitization of TRPV1 via EP4 receptors also appears to exist in mouse DRG neurons (Moriyama et al., 2005 ). Intraplantar injection of PGE2 induces edema in rodent paws through activation of TRPV1; the response is abated by administration of a selective TRPV1 antagonist. The use of selective pharmacological agents has indicated that in this case, PGE2 sensitizes TRPV1 through a PKC - dependent mechanism involving the EP3 receptor, the NK - 1 receptor, and the mitogen - activated kinases (MAPKs) (Claudino et al., 2006 ).

3.4.3 Kinases and Sensitization TRPV1 lies at the nexus of a network that directs the function, subcellular localization, and sensitization state of the channel (Cesare and McNaughton, 1996 ; Shin et al., 2002 ; Sugiura et al., 2002 ; Bonnington and McNaughton, 2003 ; Moriyama et al., 2003 ; Amadesi et al., 2004 ; Carlton et al., 2004 ; Dai et al., 2004 ; Ferreira et al., 2004 ; Zhang et al., 2005a,b ). The dynamic phosphorylation status of TRPV1 imparts the ability of this channel to respond TRPV1 AS A MOLECULAR INTEGRATOR 117 instantaneously to environmental changes or to maintain conditions conducive to peripheral sensitization and persistent pain. The human TRPV1 sequence (Swiss- Prot Q8NER1) encodes at least 11 putative phosphorylation sites (Table 3.1 ), similarly reﬂ ected across orthologs, inviting engagement and possible convergence of multiple kinase cascades. Activation of PKA by cAMP is critical to the induction and persistence of hyperalgesia and maintenance of TRPV1 in its sensitized state, as demonstrated by the peripheral sensitization and hyperalgesia elicited by intradermal injection of cAMP analogues, adenylate cyclase activators, or PKA inhibitors and by the use of genetic mutants of PKA (Kress et al., 1996 ; Malmberg et al., 1997 ; Aley and Levine, 1999 ; Hu et al., 2001 ). The effects of speciﬁ c PKA activators and inhibitors have demonstrated that the PKA pathway

TABLE 3.1 Consensus Phosphorylation Sites of TRPV 1 Consensus Sites for Phosphorylation of TRPV1 Modiﬁ cation Site in Modiﬁ cation Site Kinase References Human TRPV1 in Rat TRPV1 Ser 6 Ser 6 PKA Bhave et al. (2002) Ser 117 Ser 116 PKA Bhave et al. (2002) Thr 145 Thr 144 PKA Bhave et al. (2002, 2003) PKC Tyr 200 Tyr 199 Src Lishko et al. (2007) Thr 371 Thr 370 PKA Bhave et al. (2002) Thr 407 Thr 406 Cdk5 Pareek et al. (2007 ) Ser 502 Ser 502 PKA Zhang et al. (2007) , PKC ε Novakova - Tousova CaMKII et al. (2007) , Jung et al. (2004) , Bhave et al. (2003, 2002) , Numazaki et al. (2002) , and Mandadi et al. (2006) Thr 705 Thr 704 PKC Bhave et al. (2003) and CaMKII Jung et al. (2004) Ser 775 Ser 774 PKA Bhave et al. (2002, 2003) PKC Ser 801 Ser 800 PKC ε Zhang et al. (2007) , Bhave et al. (2003) , Ahern (2003) , and Numazaki et al. (2002) Ser 821 Ser 820 PKA Bhave et al. (2002)

Residues assigned on the basis of rat TRPV1 sequence recorded as O35433 and extrapolated to human TRPV1 sequence represented by Q8NER1 in the Swiss - Prot database. 118 BIOCHEMICAL PHARMACOLOGY OF TRPV1 modulates hyperexcitability of injured primary sensory neurons (Hu et al., 2001 ) and functions in TNFα - induced fi ring of rat L4 and L5 DRG neurons (Zhang et al., 2002 ). While the literature is replete with reports on phosphorylation of TRPV1 by multiple kinases (summarized in Table 3.1 ), until recently, relatively little has been known about the molecular organization of the associated scaffolding proteins involved in ensuring appropriate targeting (Jeske et al., 2008 ; Schnizler et al., 2008 ). PKA phosphorylation of TRPV1 is facilitated by A - kinase anchoring proteins (AKAPs) that bind the regulatory subunit of PKA and mediate interactions between PKA and its substrates (Wong and Scott, 2004 ; Dell ’ Acqua et al., 2006 ; Beene and Scott, 2007 ). Experiments that demonstrate effects of an AKAP inhibitory peptide (Rathee et al., 2002 ; Sugiuar et al., 2004 ) and colocalization of scaffolding proteins with TRPV1 in mouse DRG and rat trigeminal neurons identify AKAP150 as the operative scaffolding protein in rodents (Jeske et al., 2008 ; Schnizler et al., 2008 ). Recently, prevention of AKAP150/79 binding to a C- terminal region of TRPV1 has been shown to abrogate sensitization by either bradykinin or PGE2, suggesting that this protein is a common element in infl ammatory mediator action on TRPV1 (Zhang et al., 2008 ). Activation of PKC specifi cally sensitizes thermal responses by amplifying heat - activated currents and by reducing thresholds for detecting heat stimuli as noxious (Cesare and McNaughton, 1996 ; Vellani et al., 2001 ). PKCε is translocated to the plasma membrane in cultured rat DRG following stimulation with bradykinin (Cesare et al., 1999 ) and is responsible for phosphorylation of TRPV1 at Ser 502 and Ser 801 (human sequence numbering) (Numazaki et al., 2002 ). The rapid response, within 5 s of exposure to bradykinin, is consistent with the time course of sensitization and suggests that PKC ε is responsible for bradykinin - induced sensitization of TRPV1 to heat. A subsequent study confi rms that TRPV1 and PKCε are coexpressed in rat DRG neurons and further demonstrates that infl ammation induces upregulation of PKC ε expression, suggesting a critical role of PKCε in TRPV1 function (Zhou et al., 2003 ). This is consistent with the phenotype of PKCε null mice, which exhibit reduced hyperalgesic responses (Khasar et al., 1999 ). Development of a novel viral delivery system for a dominant negative form of PKCε confi rms the direct functional relationship between PKC ε and TRPV1 sensitization (Srinivasan et al., 2008 ). The observation that these recombinant constructs are analgesic in capsaicin - infl amed rat hind paws further emphasizes the signifi cance of PKCε in the activation of TRPV1. In rat DRG neurons, PKCε appears to be essential for maintenance of basal phosphorylation and agonist - induced responses of TRPV1. Nerve growth factor (NGF) rapidly sensitizes nociceptive sensory neurons that express the NGF receptor TrkA to noxious thermal stimuli and activates second messenger signaling cascades leading to sensitization of TRPV1 (Lewin et al., 1993 ; Shu and Mendell, 2001 ). Although some aspects of the specifi c mechanisms underlying these processes remain unresolved, a consensus is TRPV1 AS A MOLECULAR INTEGRATOR 119 emerging that explains the observed sensitization of TRPV1 by NGF in the context of the phosphatidylinositol- 3 - kinase (PI3K), PKC, and CaMKII pathways. Wortmannin, a selective PI3K inhibitor, completely prevents the sensitizing effects of NGF on TRPV1, as monitored by ratiometric Ca 2+ imaging of neonatal mouse DRG neurons. Similarly, inhibition of PKC and CamKII also blocks TRPV1 sensitization by NGF. These elegant studies by Bonnington and McNaughton (2003) conclude that PI3K is essential in the early stages of NGF- facilitated sensitization of TRPV1, while PKC and CaMKII are involved later. Recent studies confi rm that PI3K, but not PLC, is integrally involved in NGF sensitization of TRPV1 (Zhu and Oxford, 2007 ). Along with PI3K, Ras and PLC have been implicated in downstream effects following NGF binding to TrkA. Ras does not seem to affect TRPV1 directly but rather binds to the catalytic domain of PI3K, enhancing its activity. In contrast to the fi ndings with recombinant HEK293 cells (Chuang et al., 2001 ), PLC appears not to be involved in NGF- mediated sensitization of TRPV1 on mouse DRG neurons, perhaps owing to differences in the expression levels and distribution in the host cells or to posttranslational modifi cation or the oligomerization status of the channel (Bonnington and McNaughton, 2003 ). MAPKs are activated by infl ammatory mediators in a cascade of sequential phosphorylations in sensory and dorsal horn neurons as well as in spinal glial cells. MAPKs contribute signifi cantly to the development of hyperalgesia and sensitization through transcription, translation, and posttranslational modifi - cation. Inhibition of any of the three principal MAPK pathways (extracellular signal- regulated protein kinase [ERK], p38 MAPK, and c- Jun N- terminal kinase [JNK]) can abate infl ammatory or neuropathic pain without affecting the protective ability to detect noxious stimuli in the absence of injury. Conversely, intrathecal administration of inhibitors of ERK, p38, and JNK reverses the associated mechanical allodynia in the rat spinal nerve ligation (SNL) model of neuropathic pain (Obata et al., 2004 ). SNL induces activation of all three kinases in distinct DRG neurons, but their activation is differentially regulated. Levels of phospho - p38 are increased in SNL rats in the ipsilateral lumbar region of the spinal cord and in small- to medium- diameter capsaicin- responsive DRG neurons. Phospho- p38 levels peak at 1 day post- ligation in DRG neurons and at 3 days in the spinal cord (Jin et al., 2003 ; Schafers et al., 2003a,b,c ). During this period, phospho - p38 propagates pain signaling and sensitization by phosphorylating transcription factors, some of which regulate production of infl ammatory mediators, including TNF α. By monitoring the time course for p38 activation, Ji et al. (2002) established a causal link and chronology between the effectors, p38 and TNF α, and the induction of neuropathic pain; TNF α is engaged early in the process. Whereas the basal level of phospho - p38 is low in lumbar DRG neurons of naive rats (Ji et al., 2002 ; Kim et al., 2002 ; Schafers et al., 2003a,b,c ), the TRPV1 agonist, capsaicin, causes a rapid (2 min), transient (10 min) elevation, especially in small- to medium- diameter neurons (Mizushima et al., 2005 ). 120 BIOCHEMICAL PHARMACOLOGY OF TRPV1

Likewise, heating of the rat hind paw by immersion in hot (54 ° C) water induces phosphorylation of p38; the majority of the neurons showing activated p38 coexpress TRPV1. Intrathecal injection of a small- molecule p38 inhibitor reduces phospho- p38 levels in DRG neurons and thermal hypersensitivity, but does not affect mechanical allodynia. These observations suggest that activation of TRPV1 by capsaicin or heat induces activation of p38. Inhibition of p38 prevents thermal hypersensitivity and upregulation of TRPV1 following infl ammation and mechanical allodynia associated with nerve injury (Milligan et al., 2003 ). The ERK pathway transduces sensory input into cellular responses through sequential phosphorylation events. NGF activates ERK in DRG neurons and contributes to pain hypersensitivity and TRPV1 sensitization by both tran- scriptional and posttranslational mechanisms. The L5 SNL model induces phosphorylation of ERK predominantly in large - diameter DRG neurons and satellite glial cells (Obata et al., 2004 ). Stimulation of C - fi ber nociceptors by capsaicin induces phosphorylation of ERK and its downstream substrate, the kinase AKT, within 2 min in dissociated cultures of rat DRG neurons. Elevated levels of both phosphorylated ERK and AKT peak at 10 min and are sustained for > 90 min (Zhuang et al., 2004 ). Use of selective pharmacological reagents, including wortmannin, confi rms that these phosphorylation events require Ca2+ infl ux and are positioned downstream of PI3K. Inhibition of the ERK pathway reduces hyperalgesia induced by capsaicin (Dai et al., 2002 ) and NGF (Zhuang et al., 2004 ). Immunofl uorescent monitoring of rat hind paw sections has demonstrated that intraplantar injection of capsaicin elicits increased phosphorylation of AKT in small C - fi ber neurons (Zhuang et al., 2004 ). JNK1 is involved in nociceptive signaling in both neuropathic and infl ammatory pain and appears to play a role in TRPV1- induced signaling. However, the role for JNK activation in TRPV1 - mediated nociception is not yet clearly understood. Levels of phosphorylated JNK1 and its associated transcription factor, c- Jun, increase rapidly in damaged rat small- diameter DRG neurons and in astrocytes in the dorsal horn, and remain transiently elevated after the nerve ligation. In addition, inhibitors that block activation prevent mechanical allodynia (Ma and Quirion, 2002 ; Obata et al., 2004 ; Daulhac et al., 2006 ; Zhuang et al., 2006 ). Similarly, inhibition of JNK also reduces mechanical hypersensitivity and thermal hyperalgesia associated with peripheral nerve and tissue injury induced upon intraplantar injection of irritants, such as capsaicin, bee venom, complete Freund’ s adjuvant, endothelin, or NGF, into rat paws (Guo et al., 1999 ; Jin et al., 2003 ; Doya et al., 2005 ; Motta et al., 2006 ; Zhuang et al., 2006 ; Cao et al., 2007 ; Plant et al., 2007 ).

3.5 TRAFFICKING

In addition to expression on the plasma membrane, TRPV1 is also expressed in intracellular compartments including the endoplasmic reticulum and the cytoplasmic vesicles (Guo et al., 1999 ; Morenilla- Palao et al., 2004 ). Cytoplasmic SUMMARY 121 reserves of TRPV1 can be quickly translocated to the cell surface following cellular activation by several mechanisms, generally involving specifi c phosphorylation (Morenilla - Palao et al., 2004 ). The transport of TRPV1 from intracellular vesicles to the plasma membrane is facilitated through synapto- somal- associated protein 25 receptor (SNARE)- dependent exocytosis (Nakata et al., 1998 ; Ahmari et al., 2000 ), a process that is sensitized by PKA (Chheda et al., 2001 ) and PKC (Zhu et al., 2002 ). PKC and MAPK signaling thereby increases the surface density and sensitization state of TRPV1 expressed on nociceptive terminals (Ji et al., 2002 ; Morenilla- Palao et al., 2004 ). This mobilization of TRPV1 serves as an effective means of developing and propagating infl ammatory hyperalgesia. In the course of an infl ammatory response, when cAMP levels are elevated, PKA activation causes rapid mobilization of inactive TRPV1 monomers from intracellular stores and insertion of multimers into the plasma membrane (Vetter et al., 2008 ). Src - mediated tyrosine phosphorylation also induces a dramatic translocation of TRPV1 to the surface of HEK293 recombinant cells (Zhang et al., 2005b ). Fibronectin can activate src kinase in select cellular models (Meerschaert et al., 1999 ; Ren et al., 2005 ; Tvorogov et al., 2005 ), and a role for fi bronectin as a dynamic modulator of TRPV1 translocation to the plasma membrane of trigeminal neurons has recently been defi ned (Jeske et al., 2009 ). Fibronectin- induced enhancement in TRPV1 sensitivity to activation results from increases in both levels of TRPV1 expression in the plasma membrane and tyrosine phosphorylation of the channel.

3.5.1 Antagonism of TRPV 1 Extensive site - directed mutagenesis of the human ether - a - go - go (hERG) potassium channel (Sanguinetti and Mitcheson, 2005 ) has been used to infer the binding site of antagonists in structurally related channels. Many groups have mutated residues in the pore region and examined the changes in ion currents in cells containing the overexpressed channels. The general conclusion from these studies is that most channel antagonists bind in the pore region, interacting with residues from all four monomers of the tetrameric channel. A pharmacophore model for TRPV1 antagonist binding has recently been proposed in which antagonists bind in an orientation that achieves hydrogen bond interactions with Tyr 667, as well as interactions with a lipophilic binding site in the pore region of the channel (Kym et al., 2009 ). In this model, multiple TRPV1 antagonists can be superimposed such that they optimally ﬁ t in the TRPV1 pore.

3.6 SUMMARY

TRPV1 was fi rst cloned from rat DRG neurons over a decade ago (Caterina et al., 1997 ). Since that time, a signifi cant amount of research in the fi eld has defi ned a key role for this receptor in the detection, transmission, and 122 BIOCHEMICAL PHARMACOLOGY OF TRPV1 integration of diverse pain signals. Some of the recent highlights include the fi rst three- dimensional structure of TRPV1, identifi cation of a domain involved in activation by heat, positive modulation of channel activation by PIP2, the existence of dynamic ion selectivity with prolonged or repeated exposure to agonists with potential consequences on pain signaling, insights into mechanisms and regulation of tachyphylaxis, demonstration of profound effects on membrane excitability caused by the synergistic action of two agonists, sensitizing effects of key infl ammatory mediators TNF α and NGF on TRPV1, and increase in plasma membrane traffi cking of TRPV1 as a result of infl ammatory mediator action. These discoveries underscore the complexity of TRPV1 activation and regulation and the dynamic nature of TRPV1 as a molecular integrator of pain signals.

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Ruslan Dorfman , Hubert Tsui , Michael W. Salter , and H. - Michael Dosch

4.1 OVERVIEW

In this chapter, we will summarize how TRPV1 genetic variability impacts the pharmacological properties of TRPV1 in different species. We will also review site- directed mutagenesis experiments that identify binding sites for biological and pharmacological substrates and modify conductivity of the channel. These studies have been essential for unraveling the complex structure– function paradigm of TRPV1. With this background information, we will then discuss the genetic heterogeneity of the human trpv1 gene, which likely will be a major element in our current progression toward translating the insights gained to better understanding and, hopefully, to new therapeutic strategies, some already effective in animals.

4.2 trpv1 GENE STRUCTURE IN DIFFERENT SPECIES

The human trpv1 gene1 is found on the small arm of chromosome 17 at p13.2, coordinates 3,415,493- 3,447,085 (ENSEMBL assembly release 48, National Center for Biotechnology Information [NCBI] version 36). Human trpv1 is polymorphic, with 76 validated single - nucleotide polymorphisms (SNPs), 9 of which map to coding regions of the gene.2

1 http://www.ensembl.org/Homo_sapiens/geneview?gene=ENSG00000196689 . 2 http://www.ncbi.nlm.nih.gov.myaccess.library.utoronto.ca/SNP/snp_ref.cgi?locusId=7442&choo seRs=het .

134 ALTERNATIVE SPLICING OF trpv1 RESULTS 135

The mouse gene3 maps to chromosome 11 (band B4) at location 73,047,794 – 73,074,744. The mouse gene is also highly polymorphic,4 with a large number of SNPs (111 in total, 6 in coding sequences) that differ across 17 mouse strains genotyped by different consortia. This genetic variability was used to map a large number of quantitative trait loci (QTL) by using collections of recombinant inbred mouse lines derived from different parental strains.5 The rat trpv1 gene on chromosome 10 maps to location 60,109,656 – 60,135,405 (Chr10q24). Several rat QTLs (23) were mapped to this region, conferring potential roles of trpv1 polymorphisms in the regulation of blood pressure, risk of arthritis, bone mineral density, stress responses, resistance to toxoplasma infection and thermal sensitivity.6 However, there is also a high degree of conservation at the genomic level, where the exonic sequences are the most conserved and where intron 1 of the trpv1 gene includes a large conserved region. In the intronic sequences of the human trpv1 gene, there are several highly conserved binding sites for AP1, SRF, ARP1, NRSF, CDP, FOXO1, RP58, PPARG, and PPARA transcription factors, which are predicted by the multiple alignments of noncoding genomic DNA sequences from different species.7 These transcription factors likely play signiﬁ cant roles in the regulation of TRPV1 expression.

4.3 ALTERNATIVE SPLICING OF trpv1 RESULTS IN DIFFERENT FUNCTIONAL VARIANTS

There are at least six different splice variants of the human trpv1 gene; four variants utilize alternative promoters, with the most distant promoter lying in the 3 ′UTR of the CARKL gene. However, these four alternative variants with different promoters give rise to an identical protein sequence of 839 amino acids in the canonical TRPV1 protein. There are other alternative splice variants of the trpv1 gene that differ in the length of mature protein, varying from 510 to 849 amino acid residues. The trpv1 genes of mouse and rat use at least three different promoters. Such diversity in promoters most probably plays a role in the regulation of TRPV1 expression and function in different tissues. In addition, at least three dominant- negative splicing variants of different sizes have been described. One variant, TRPV1beta, cloned from murine dorsal root ganglia (DRGs), codes for 829 amino acids and has an alternative intronic recognition signal within exon 7 of the trpv1 gene (Wang et al., 2004 ). TRPV1beta was not functional by itself, but its coexpression inhibited the function of TRPV1 (Wang et al., 2004 ). The second dominant- negative splice

3 http://www.ensembl.org/Mus_musculus/geneview?gene=ENSMUSG00000005952 . 4 http://phenome.jax.org/pub-cgi/phenome/mpdcgi?rtn=snps/retrieve&gregion=gene&genesym= Trpv1 . 5 http://www.webqtl.org/ . 6 http://www.ensembl.org/Rattus_norvegicus/geneview?gene=ENSRNOG00000019486 . 7 http://genome.ucsc.edu/ . 136 TRPV1 GENETICS

TABLE 4.1 Splice Number Transcript Protein Function Variant of Exons length (bps) length (aa) TRPV1 - 201 16 4,044 839 Canonical promoter TRPV1 - 202 14 4,233 779 TRPV1b TRPV1 - 203 10 2,060 510 Possibly dominant - negative TRPV1 - 204 14 4,483 837 Alternative promoter TRPV1 - 205 16 4,446 850 Includes alternative 3rd coding exon that is skipped in all other splice variants TRPV1 - 206 16 4,087 839 Alternative promoter TRPV1 - 207 15 3,964 839 Shortest promoter variant TRPV1(VAR) was identiﬁ ed in rat kidney (Tian et al., 2006 ). This variant was predicted to encode a form of TRPV1, which was truncated at amino acid residue 253, but utilized the same promoter as canonical TRPV1. Upon heterologous expression, TRPV1(VAR) had a dominant- negative effect, partially blocking TRPV1- dependent resiniferatoxin (RTX) responsiveness in the COS- 7 epithelial cell line, but an opposite effect was observed when TRPV1(VAR) was expressed in HEK293 cells (Tian et al., 2006 ). A third human TRPV1 splice variant, TRPV1b of 779 amino acids, was also cloned from DRG. TRPV1b lacks exon 7, which encodes the third ankyrin domain (Vos et al., 2006 ). Human TRPV1b also inhibited normal TRPV1 channel function in response to capsaicin, acidic pH, heat, and endogenous vanilloids (Vos et al., 2006 ). The EMBL protein database contains two additional, alternatively spliced TRPV1 protein sequences of 667 and 510 residues, respectively, both of which lack the full third ankyrin domain and the pore- forming sequences, and presumably may also represent dominant- negative variants (Table 4.1 ).

4.4 FUNCTIONAL TRPV 1 DIFFERENCES IN NONCONSERVED AMINO ACID POSITIONS

Although highly conserved, overall, substantial differences do exist in TRPV1 protein sequences, which affect the function of TRPV1. The evolutionary changes in different species led to remarkable variability of TRPV1 responses to distinct agonists and antagonists. A number of in vitro studies, based on TRPV1 channels derived from various species through reciprocal site- directed mutagenesis, led to identiﬁ cation of key residues essential for binding of capsaicin and other agonists (Fig. 4.1 ). Jordt and Julius (2002) focused attention on the avian TRPV1, which was known to be insensitive to capsaicin but had a normal response to low pH and heat. The cloning of TRPV1 from chicken DRG and comparison with the rat sequence identiﬁ ed a number of changes in the TM2 and TM4 domains. Site - directed mutagenesis of the rat TRPV1 channel led to establishment of the FUNCTIONAL TRPV1 DIFFERENCES 137

(a) Cladogram

human chimp cow dog guinea_pig mouse rat chicken rabbit

(b) CLUSTAL 2.0.3 multiple sequence alignment of TRPV1 proteins mouse ------MEKWASLDSDES----EPPAQENSCPDPPDRDPNSKPPPAKPHIFAT-RSRTR 48 rat ------MEQRASLDSEES----ESPPQENSCLDPPDRDPNCKPPPVKPHIFTT-RSRTR 48 guinea_pig ------MKKRASVDSKES----EDPPQEDYSLDPLDVDANSKTPPAKPHTFSVSKSRNR 49 cow ------MKKWGSSESRES----QDLPQEDSCPDPLDGDPNYRPAPTKPHSFPTAKSRSR 49 dog ------MKNWGSSDSGGS----EDPPQEDSCLDPLDGDPNSRPVPAKPHIFPTAKSRSR 49 human ------MKKWSSTDLGAA----ADPLQKDTCPDPLDGDPNSRPPPAKP-QLSTAKSRTR 48 chimp ------MKKWSSTDLGAA----ADPLQKDTCPDPLDGDPNSRPPPAKP-QLSTAKSRTR 48 rabbit ------MKRWVSLDSGES----EDPLPEDTCPDLLDGDSNAKPPPAKPHIFSTAKSRSR 49 chicken MSSILEKMKKFGSSDIEESEVTDEHTDGEDSALETADNLQGTFSNKVQPSKSNIFARRGR 60

mouse LFGKGDSEEASPMDCPYEEGGLASCPIITVSSVVTLQRSVDGPTCLRQTSQDSVSTGV-E 107 rat LFGKGDSEEASPLDCPYEEGGLASCPIITVSSVLTIQRPGDGPASVRPSSQDSVSAG--E 106 guinea_pig LFGKSDLEESSPIDCSFREGEAASCPTITVSSVVTSPRPADGPTSTRQLTQDSIPTSA-E 108 cow LFGKGDSEDTSLMDCSYEEGQLASCPAITISPVVIIQRSGDGPTCVRQLSQDSAAT---- 105 dog LFGKCDSEEAS-MDCSYEEGQLASCPAITVSPVVMIPKHEDSPTCARQPSQDSVTAG-SE 107 human LFGKGDSEEAFPVDCPHEEGELDSCPTITVSPVITIQRPGDGPTGARLLSQDSVAAS-TE 107 chimp LFGKGDLEEAFPVDCPHEEGELDSCPTITVSPVITIQRPGDGPTGARLLSQDSVAAS-TE 107 rabbit LFGKGDSEETSPMDCSYEEGELAPCPAITVSSVIIVQRSGDGPTCARQLSQDSVAAAGAE 109 chicken FVMGDCDKDMAPMDSFYQMDHLMAP------SVIKFHANMERGKLHKLLSTDSITGCS-E 113

mouse TPPRLYDRRSIFDAVAQSNCQELESLLSFLQKSKKRLTDSEFKDPETGKTCLLKAMLNLH 167 rat KPPRLYDRRSIFDAVAQSNCQELESLLPFLQRSKKRLTDSEFKDPETGKTCLLKAMLNLH 166 guinea_pig KPLKLYDRRSIFDAVAQNNCQDLDSLLPFLQKSKKRLTDTEFKDPETGKTCLLKAMLNLH 168 cow ENLKLYDRRKIFEAVAQNNCEELESLLLFLQKSKKHLMDSEFKDPETGKTCLLKAMLNLH 165 dog KSLKLYDRRKIFEAVAQNNCEELQSLLLFLQKSKKHLMDSEFKDPETGKTCLLKAMLNLH 167 human KTLRLYDRRSIFEAVAQNNCQDLESLLLFLQKSKKHLTDNEFKDPETGKTCLLKAMLNLH 167 chimp KTLRLYDRRSIFEAVAQNNCQDLESLLLFLQKSKKHLTDNEFKDPETGKTCLLKAMLNLH 167 rabbit KPLKLYDRRRIFEAVAQNNCQELESLLCFLQRSKKRLTDSEFKDPETGKTCLLKAMLNLH 169 chicken KAFKFYDRRRIFDAVARGSTKDLDDLLLYLNRTLKHLTDDEFKEPETGKTCLLKAMLNLH 173

mouse NGQNDTIALLLDIARKTDSLKQFVNASYTDSYYKGQTALHIAIERRNMALVTLLVENGAD 227 rat NGQNDTIALLLDVARKTDSLKQFVNASYTDSYYKGETALHIAIERRNMTLVTLLVENGAD 226 guinea_pig NGQNDTISLLLDIARQTNSLKEFVNASYTDSYYRGQTALHIAIERRNMVLVTLLVENGAD 228 cow NGQNDTIPLLLEIARQTDSLKELVNASYTDSYYKGQTALHIAIERRNMALVTLLVENGAN 225 dog DGQNDTIPLLLEIARQTDSLKELVNASYTDSYYKGQTALHIAIERRNMALVTLLVENGAD 227 human DGQNTTIPLLLEIARQTDSLKELVNASYTDSYYKGQTALHIAIERRNMALVTLLVENGAD 227 chimp DGQNNTIPLLLEIARQTDSLKEFVNASYTDSYYKGQTALHIAIERRNMALVTLLVENGAD 227 rabbit SGQNDTIPLLLEIARQTDSLKEFVNASYTDSYYKGQTALHIAIERRNMALVTLLVENGAD 229 chicken DGKNDTIPLLLDIAKKTGTLKEFVNAEYTDNYYKGQTALHIAIERRNMYLVKLLVQNGAD 233

Figure 4.1 Multiple sequence alignment for TRPV1 protein by CLUSTAL 2.0. (a) Cladogram of homology for TRPV1 orthologs. (b) Conserved ankyrin domains are boxed; the transmembrane domains are underlined in bold. The bold Lys at positions 155 and 160 are essential for ATP binding and are marked with diamonds, as well as Gln at 202 and Tyr at 199, which also is a phosphorylation site. The pentagon marks Cys, which can be reduced either by DTT or by GST and affects the TRPV1 activity. The stars at the TM6 indicate the amino acids where mutation generates dominant - negative mutant proteins; other pore-forming amino acids are highlighted in bold. The pore loop is underlined in a dashed line. Polymorphic amino acids in relevant species are bold - faced and boxed. 138 TRPV1 GENETICS mouse VQAAANGDFFKKTKGRPGFYFGELPLSLAACTNQLAIVKFLLQNSWQPADISARDSVGNT 287 rat VQAAANGDFFKKTKGRPGFYFGELPLSLAACTNQLAIVKFLLQNSWQPADISARDSVGNT 286 guinea_pig VQAAANGDFFKKTKGRPGFYFGELPLSLAACTNQLAIVKFLLQNSWQPADISARDSVGNT 288 cow VQAAANGDFFKKTKGRPGFYFGELPLSLAACTNQLGIVKFLLQNSWQPADISARDSVGNT 285 dog VQAAANGDFFKKTKGRPGFYFGELPLSLAACTNQLGIVKFLLQNSWQPADISARDSVGNT 287 human VQAAAHGDFFKKTKGRPGFYFGELPLSLAACTNQLGIVKFLLQNSWQTADISARDSVGNT 287 chimp VQAAAHGDFFKKTKGRPGFYFGELPLSLAACTNQLGIVKFLLQNSWQTADISARDSVGNT 287 rabbit VQAAANGDFFKKTKGRPGFYFGELPLSLAACTNQLAIVKFLLQNSWQPADISARDSVGNT 289 chicken VHARACGEFFRKIKGKPGFYFGELPLSLAACTNQLCIVKFLLENPYQAADIAAEDSMGNM 293 mouse VLHALVEVADNTADNTKFVTNMYNEILILGAKLHPTLKLEELTNKKGLTPLALAASSGKI 347 rat VLHALVEVADNTVDNTKFVTSMYNEILILGAKLHPTLKLEEITNRKGLTPLALAASSGKI 346 guinea_pig VLHALVEVADNTADNTKFVTSMYNEILILGAKLYPTLKLEELTNKKGFTPLALAASSGKI 348 cow VLHALVEVADNTADNTKFVTSMYNEILILGAKIHPTLKLEELTNKKGLTPLALAARSGKI 345 dog VLHALVEVADNTADNTKFVTSMYNEILILGAKLHPTLKLEGLTNKKGLTPLALAARSGKI 347 human VLHALVEVADNTADNTKFVTSMYNEILMLGAKLHPTLKLEELTNKKGMTPLALAAGTGKI 347 chimp VLHALVEVADNTADNTKFVTSMYNEILILGAKLHPTLKLEELTNKKGMTPLALAAGTGKI 347 rabbit VLHALVEVADNTPDNTKFVTSMYNEILILGAKLHPTLKLEELINKKGLTPLALAAGSGKI 349 chicken VLHTLVEIADNTKDNTKFVTKMYNNILILGAKINPILKLEELTNKKGLTPLTLAAKTGKI 353 mouse GV------LAYILQREIHEPECRHLSRKFTEWAYGPVHSSLYDLSCIDTCEKNSVL 397 rat GV------LAYILQREIHEPECRHLSRKFTEWAYGPVHSSLYDLSCIDTCEKNSVL 396 guinea_pig GV------LAYILQREIPEPECRHLSRKFTEWAYGPVHSSLYDLSCIDTCEKNSVL 398 cow GV------LAYILQREIQEPECRHLSRKFTEWAYGPVHSSLYDLSCIDTCEKNSVL 395 dog GVGGGVLPELGVLAYILQREIQEPECRHLSRKFTEWAYGPVHSSLYDLSCIDTCEKNSVL 407 human GV------LAYILQREIQEPECRHLSRKFTEWAYGPVHSSLYDLSCIDTCEKNSVL 397 chimp GV------LAYILQREIQEPECRHLSRKFTEWAYGPVHSSLYDLSCIDTCEKNSVL 397 rabbit GV------LAYILQREILEPECRHLSRKFTEWAYGPVHSSLYDLSCIDTCERNSVL 399 chicken GI------FAYILRREIKDPECRHLSRKFTEWAYGPVHSSLYDLSCIDTCEKNSVL 403 mouse EVIAYSSSETPNRHDMLLVEPLNRLLQDKWDRFVKRIFYFNFFVYCLYMIIFTTAAYYRP 457 rat EVIAYSSSETPNRHDMLLVEPLNRLLQDKWDRFVKRIFYFNFFVYCLYMIIFTAAAYYRP 456 guinea_pig EVIAYSSSETPNRHDMLLVEPLNRLLQDKWDRFVKRIFYFNFFIYCLYMIIFTMAAYYRP 458 cow EVIAYSSSETPNRHDMLLVEPLNRLLQDKWDRFVKRIFYFNFFVYCLYMIIFTTVAYYRP 455 dog EVIAYSSSETPNRHDMLLVEPLNRLLQDKWDRFVKRIFYFNFFIYCLYMIIFTTAAYYRP 467 human EVIAYSSSETPNRHDMLLVEPLNRLLQDKWDRFVKRIFYFNFLVYCLYMIIFTMAAYYRP 457 chimp EVIAYSSSETP------408 rabbit EVIAYSSSETPNRHDMLLVEPLNRLLQDKWDRVVKRIFYFNFFVYCLYMIIFTTAAYYRP 459 chicken EIIAYSS-ETPNRHEMLLVEPLNRLLQDKWDRFVKHLFYFNFFVYAIHISILTTAAYYRP 462 mouse VE--GLPPYKLNNTVGDYFRVTGEILSVSGGVYFFFRGIQYFLQRRPSLKSLFVDSYSEI 515 rat VE--GLPPYKLKNTVGDYFRVTGEILSVSGGVYFFFRGIQYFLQRRPSLKSLFVDSYSEI 514 guinea_pig VD--GLPPYKMKNTVGDYFRVTGEILSVIGGFHFFFRGIQYFLQRRPSVKTLFVDSYSEI 516 cow AG--GRPPFKPKHTVGDYFRITGEIISVAGGIYFFSRGIQYFLQRRPSLKTLFVDSYSEM 513 dog VD--GLPPYKLKHTVGDYFRVTGEILSVLGGVYFFFRGIQYFLQRRPSLKTLFVDSYSEM 525 human VD--GLPPFKMEKT-GDYFRVTGEILSVLGGVYFFFRGIQYFLQRRPSMKTLFVDSYSEM 514 chimp ------PPFKMEKT-GDYFRVTGEILSVLGGVYFFFRGIQYFLQRRPSMKTLFVDSYSEM 461 rabbit VD--GLPPYKLRNLPGDYFRVTGEILSVAGGVYFFFRGIQYFLQRRPSMKALFVDSYSEM 517 chicken VQKGDKPPFAFGHSTGEYFRVTGEILSVLGGLYFFFRGIQYFVQRRPSLKTLIVDSYSEV 522 mouse LFFVQSLFMLVSVVLYFSHRKEYVASMVFSLAMGWTNMLYYTRGFQQMGIYAVMIEKMIL 575 rat LFFVQSLFMLVSVVLYFSQRKEYVASMVFSLAMGWTNMLYYTRGFQQMGIYAVMIEKMIL 574 guinea_pig LFFVQSLFLLASVVLYFSHRKEYVACMVFSLALGWTNMLYYTRGFQQMGIYAVMIEKMIL 576 cow LFFMQSLFMLATVVLYFCHRKEYVASMVFSLAMGWTNMLYYTRGFQQMGIYAVMIEKMIL 573 dog LFFVQSLFMLGTVVLYFCHHKEYVASMVFSLAMGWTNMLYYTRGFQQMGIYAVMIEKMIL 585 human LFFLQSLFMLATVVLYFSHLKEYVASMVFSLALGWTNMLYYTRGFQQMGIYAVMIEKMIL 574 chimp L------MIL 465 rabbit LFFVQALFMLATVVLYFSHCKEYVATMVFSLALGWINMLYYTRGFQQMGIYAVMIEKMIL 577 chicken LFFVHSLLLLSSVVLYFCGQELYVASMVFSLALGWANMLYYTRGFQQMGIYSVMIAKMIL 582

Figure 4.1 Continued FUNCTIONAL TRPV1 DIFFERENCES 139

mouse RDLCRFMFVYLVFLFGFSTAVVTLIEDGKNNSLPVESPPHKCRGSACRPG-NSYNSLYST 634 rat RDLCRFMFVYLVFLFGFSTAVVTLIEDGKNNSLPMESTPHKCRGSACKPG-NSYNSLYST 633 guinea_pig RDLCRFMFVYLVFLFGFSTAVVTLIEDGKNESLSAE--PHRWRGPGCRSAKNSYNSLYST 634 cow RDLCRFMFVYLVFLFGFSTAVVTLIEDEKNDSVSVELSQHRWRGHGCRSADS-YNSLYST 632 dog RDLCRFMFVYLVFLFGFSTAVVTLIEDGKNNSVPTESTLHRWRGPGCRPPDSSYNSLYST 645 human RDLCRFMFVYIVFLFGFSTAVVTLIEDGKNDSLPSESTSHRWRGP-CRPPDSSYNSLYST 633 chimp RDLCRFMFVYVVFLFGFSTAVVTLIEDGKNDSLPSESTSH------YNSLYST 512 rabbit RDLCRFMFVYLVFLFGFSTAVVTLIEDGKNSSTSAESTSHRWRGFGCRSSDSSYNSLYST 637 chicken RDLCRFMFVYLVFLLGFSTAVVTLIED--DNEGQDTNSSEYARCSHTKRGRTSYNSLYYT 640

mouse CLELFKFTIGMGDLEFTENYDFKAVFIILLLAYVILTYILLLNMLIALMGETVNKIAQES 694 rat CLELFKFTIGMGDLEFTENYDFKAVFIILLLAYVILTYILLLNMLIALMGETVNKIAQES 693 guinea_pig CLELFKFTIGMGDLEFTENYDFKAVFIILLLAYVILTYILLLNMLIALMGETVNKIAQES 694 cow CLELFKFTIGMGDLEFTENYDFKAVFVILLLAYVILTYILLLNMLIALMGETVNKIAQES 692 dog CLELFKFTIGMGDLEFTENYDFKAVFIILLLAYVILTYILLLNMLIALMGETVNKIAQES 705 human CLELFKFTIGMGDLEFTENYDFKAVFIILLLAYVILTYILLLNMLIALMGETVNKIAQES 693 chimp CLELFKFTIGMGDLEFTENYDFKAVFIILLLAYVILTYILLLNMLIALMGETVNKIAQES 572 rabbit CLELFKFTIGMGDLEFTENYDFKAVFIILLLAYVILTYILLLNMLIALMGETVNKIAQES 697 chicken CLELFKFTIGMGDLEFTENYRFKSVFVILLVLYVILTYILLLNMLIALMGETVSKIAQES 700

mouse KNIWKLQRAITILDTEKSFLKCMRKAFRSGKLLQVGFTPDGKDDFRWCFRVDEVNWTTWN 754 rat KNIWKLQRAITILDTEKSFLKCMRKAFRSGKLLQVGFTPDGKDDYRWCFRVDEVNWTTWN 753 guinea_pig KNIWKLQRAITILDTEKSFLKCMRKAFRSGKLLQVGYTPDGKDDYRWCFRVDEVNWTTWN 754 cow KNIWKLQRAITILDTEKSFLKCMRKAFRSGKLLQVGYTPDGKDDYRWCFRVDEVNWTTWN 752 dog KNIWKLQRAITILDTEKSFLKCMRKAFRSGKLLQVGYTPDGKDDYRWCFRVDEVNWTTWN 765 human KNIWKLQRAITILDTEKSFLKCMRKAFRSGKLLQVGYTPDGKDDYRWCFRVDEVNWTTWN 753 chimp KNIWKLQRAITILDTEKSFLKCMRKAFRSGKLLQVGYTPDGKDDYRW-FRVDEVNWTTWN 631 rabbit KSIWKLQRAITILDTEKGFLKCMRKAFRSGKLLQVGYTPDGKDDCRWCFRVDEVNWTTWN 757 chicken KSIWKLQRAITILDIENSYLNCLRRSFRSGKRVLVGITPDGQDDYRWCFRVDEVNWSTWN 760

mouse TNVGIINEDPGNCEGVKRTLSFSLRSGRVSGRNWKNFALVPLLRDASTRDRHSTQPEEVQ 814 rat TNVGIINEDPGNCEGVKRTLSFSLRSGRVSGRNWKNFALVPLLRDASTRDRHATQQEEVQ 813 guinea_pig TNVGIINEDPGNCEGVKRTLSFSLRSGRVSGRNWKNFALVPLLRDASTRDRHSAQPEEVH 814 cow TNVGIINEDPGNCEGIKRTLSFSLRSSRVAGRNWKNFALVPLLRDASTRERHPAQPEEVH 812 dog TNVGIINEDPGNCEGIKRTLSFSLRSGRVSGRNWKNFSLVPLLRDASTRERHPAQPEEVH 825 human TNVGIINEDPGNCEGVKRTLSFSLRSSRVSGRHWKNFALVPLLREASARDRQSAQPEEVY 813 chimp TNVGIINEDPGNCEGVKRTLSFSLRSSRVSGRHWKNFALVPLLREASARDRQSAQPEEVY 691 rabbit TNVGIINEDPGNCEGVKRTLSFSLRSGRVSGRNWKNFALVPLLRDASTRDRHPXPPEDVH 817 chicken TNLGIINEDPGCSGDLKRNPSYCIKPGRVSGKNWK--TLVPLLRDGSRREETPKLPEEIK 818

mouse LKHYTG-SLKPEDAEVFKDSMAPGEK 839 rat LKHYTG-SLKPEDAEVFKDSMVPGEK 838 guinea_pig LKHFSG-SLKPEDAEVFKDSAVPGEK 839 cow LRHFTG-SLKPEDAEIINDSVALGEK 837 dog LRHFAG-SLKPEDAEIFKDPVGLGEK 850 human LRQFSG-SLKPEDAEVFKSPAASGEK 838 chimp LRQFSG-SLKPEDAEVFKSPAASGEK 716 rabbit LRPFVG-SLKPGDAELFKDSVAAAEK 842 chicken LKPILEPYYEPEDCETLKESLPKSV- 843 Figure 4.1 Continued essential roles of Y511 and adjacent S512, since changes in either position abolished the response to capsaicin, while the response to pH and heat remained intact (Jordt and Julius, 2002 ). Replacement of R491 with 491E also reduced the response of rat TRPV1 to vanilloids (Jordt and Julius, 2002 ). Later reports indicated that the S512Y mutation in human TRPV1 also resulted in severely compromised activation by capsaicin (Sutton et al., 2005 ; Johnson et al., 2006 ). Similar comparative genetic approaches were later used to analyze which amino acid replacements in the rodent TRPV1 make the channel responsive 140 TRPV1 GENETICS to inhibition by capsazepine, that effectively blocks the human and guinea pig TRPV1 variants (Correll et al., 2004 ; Phillips et al., 2004 ; Ohta et al., 2005 ). Several key amino acid differences were identifi ed between human and rat in the TM3 - TM4 domains. The evolutionary changes of M in position 514 to I in rat (M514I), L at 518 to V (L518V) and L at 547 to M (L547M) were determined to underlie the insensitivity of the rat channel to capsazepine. Exchange of these amino acids in the rat TRPV1 to human sequences (514M, 518L, and 547L) led to a dramatic increase in the capsazepine sensitivity of the mutated rat TRPV1 (Phillips et al., 2004 ). The sensitivity of human TRPV1 to the phorbol 12- phenylacetate 13- acetate 20- homovanillate (PPAHV) agonist also differed from that in rat. Although a single change in position 547 was again found to be critical, the sensitivity differed between the PPAHV and capsaicin agonists. When the rat sequence at this position was changed to L, as in the human/guinea pig protein, the sensitivity of the channel to PPAHV was reduced 20 - fold, while the sensitivity to capsaicin remained unchanged. Reciprocal change in the human or guinea pig channel in position 547 to the rat variant M led to specifi c PPAHV responsiveness in both human and guinea pig channels, again without affecting capsaicin sensitivity (Phillips et al., 2004 ). Another evolutionary change in the rabbit TRPV1 sequence allowed for identifi cation of an essential role of the neighboring T550 in response to capsaicin. Rabbit and chicken TRPV1 channels are nonresponsive to capsaicin but respond to pH and heat; the single substitution of I to T in position 550 made the rabbit TRPV1 - I550T as responsive as other mammalian TRPV1 channels to capsaicin (Jordt and Julius, 2002 ; Gavva et al., 2004 ). Local anesthetics act through Na+ channel blockade. However, at high concentrations, they increase intracellular levels of Ca2+ , thus causing neurotoxicity (Lambert et al., 1994 ). Such neurotoxicity was suspected to involve activation of TRP channels. Recently, the genetic differences between rabbit and rat TRPV1 in response to lidocaine have helped elucidate the key role of capsaicin- binding domains for the action of lidocaine, which indeed binds and robustly activates TRPV1 at concentrations above 3 mM (Leffl er et al., 2008 ). Lidocaine activates rat but not rabbit TRPV1; transplanting the key rat domain for capsaicin binding (S505- T550) to the rabbit channel resulted in strong activation of the chimera (Leffl er et al., 2008 ). However, lidocaine was able to activate rat TRPV1- Y511A mutant channels, which are insensitive to capsaicin. The activation of TRPV1- positive neurons by high concentrations of lidocaine resulted in release of calcitonin gene- related peptide (CGRP), a physiological response pathway (Leffl er et al., 2008 ). Species - specifi c TRPV1 response variants were also discovered using the highly potent agonist RTX. In dogs, a single administration of RTX resulted in prolonged blockade of experimental infl ammatory hyperalgesia (Karai et al., 2004 ). The phosphorylation of TRPV1 by protein kinases PKC and PKA was known to desensitize the channel (Cesare and McNaughton, 1996 ; Cesare et al., 1999 ; Bhave et al., 2002 ). When dog TRPV1 was cloned, it was found to lack a conserved PKA phosphorylation site (S117), which is present in other STRUCTURE–FUNCTION ANALYSIS 141 orthologs (Phelps et al., 2005 ). The reversion of the dog K117 to S resulted in increased sensitivity to capsaicin and prolonged activation of dog TRPV1 - K117S, compared to wild - type dog TRPV1 (Phelps et al., 2005 ).

4.5 STRUCTURE – FUNCTION ANALYSIS

The N - terminal domain of TRPV1 contains six ankyrin repeat domains (ARDs), which are structurally similar to the six ankyrin repeats of TRPV2 (Lishko et al., 2007 ). However, the TRPV1 ARD is more positively charged and, more importantly, is capable of binding ATP, whereas the TRPV2 ARD is not. The ATP- binding site of the TRPV1 ARD is different from the typical Walker B structure. Three essential amino acids for ATP binding were identifi ed. Replacement of K155 or K160 by A completely abolished ATP binding; replacement of Y199 or Q202 with A signifi cantly reduced ATP binding (Lishko et al., 2007 ). A functional consequence of ATP binding to TPRV1 ARD is loss of the attenuation of TRPV1 activity that usually occurs following repeated activation, a process known as tachyphylaxis. Repeated stimulation of wildtype TRPV1 by capsaicin in the presence of ATP prevents tachyphylaxis, with full activation maintained. Unexpectedly, mutation of any of the ATP - binding amino acids completely abolished tachyphylaxis and increased sensitivity to capsaicin, prompting authors to look for another binding partner that normally promotes tachyphylaxis, but interaction with which could be abolished as well by mutations in the ARD (Lishko et al., 2007 ). Two ARD binding partners were found to have opposite effects on channel activation: the phosphatidylinositol 4,5-bisphosphate (PIP2 ) binding to TRPV1 ARD mimicked the action of ATP, while binding of calmodulin had an opposite effect— its binding promoted tachyphylaxis, thus providing a model for TRPV1 desensitization by increased levels of Ca2+ following channel activation. Deletion analyses of either N - or C - termini of TRPV1 indicated that these domains carry additional agonist recognition sites. Mutation of D114 or E761 abolishes capsaicin and RTX sensitivity (Jung et al., 2002 ; Vlachova et al., 2003 ). Recent genetic screening for gain- of - function mutations in rat TRPV1 revealed essential roles of pore helix- regulating TRP channel gating (Myers et al., 2008 ). Several constitutively active point mutations were identifi ed at distinct domains, including replacement of lysine with glutamic acid (K155E and K160E) in the ankyrin repeats, as well as M581T mutation in the S5 domain and F640L in the pore helix domain (Myers et al., 2008 ). The same positions in the ankyrin domain were previously identifi ed to be regulated by calmodulin and were essential for suppressing TRPV1 activity after repeated activation (Lishko et al., 2007 ). F640L as well as F640I mutations produced the strongest basal channel activity and altered basal versus proton- evoked currents. Indeed, the basal activity was so strong with these mutations that additional stimulation by low 142 TRPV1 GENETICS pH did not alter the current. The enhanced basal activity of the F640L mutant resulted in toxicity when expressed in mammalian HEK293 cells. The same mutant also had increased sensitivity to capsaicin and temperature, but it was still sensitive to capsazepine -mediated inhibition, suggesting that this mutation shifted the equilibrium to the open state rather then refl ecting an inability of the channel to close (Myers et al., 2008 ). The same genetic screen identifi ed several mutations with potentiated TRPV1 activity, although not constitutively active: H166R, I352N or T, Q560R, N652D, E684G or V, and L792P (Myers et al., 2008 ). Comparison of the TM6 sequence in TRPV1 to Na+ channels indicated that there is a highly conserved motif (NML positions 676, 677, 678), which is essential for pore conductivity. Mutations at these three positions (NML676FAP) result in loss of TRPV1 activation by capsaicin and RTX, and the activation by low pH is also severely affected (Kuzhikandathil et al., 2001 ). Moreover, the TRPV1- NML676FAP variant had a dominant- negative effect, indicating that these amino acids function in pore formation of the multimeric complex. Site - directed mutagenesis studies were performed, using an alanine scan of the entire portion of the TM6 inner helix domain of TRPV1 between Y666 and G683 (Susankova et al., 2007 ). Mutation of 16 of the 17 residues affected the functionality of the TRPV1 channel with respect to at least one stimulus modality; only mutation of T670 did not affect channel activity. (Susankova et al., 2007 ). As mentioned above, the posttranslational modifi cation of TRPV1 phosphorylation by PKC and PKA plays a key role in the regulation of channel activity (Cesare and McNaughton, 1996 ; Cesare et al., 1999 ; Bhave et al., 2002 ). Several phosphorylation sites have been identifi ed. S116 (S117 in dog TRPV1) is the phosphorylation site for PKA (Bhave et al., 2002 ). T704 and S800 have been identifi ed as substrates for PKC phosphorylation; mutation at either position abolished phosphorylation and activation induced by phorbol myristate acetate (PMA). Mutation of S502 to A reduced the PMA - enhanced response to capsaicin (Bhave et al., 2003 ). Conversely, mutation of T704 to A did not affect enhancement of the response to capsaicin by PMA but dramatically reduced direct activation of TRPV1 by PMA (Bhave et al., 2003 ). Intriguingly, a later report demonstrated that mutation of T704 to A, but not to I, in conjunction with the S502A mutation, generated a fully functional TRPV1 channel (Novakova - Tousova et al., 2007 ). R701 may play a key role in the phosphorylation of T507, since it is part of the conserved consensus sequence for CaMKII kinase. In addition, a role of

R701 has been proposed for PIP2 - TRPV1 ligation. Mutation of R701 strongly affected heat, capsaicin, and pH - evoked currents, but not voltage sensitivity of TRPV1 (Novakova -Tousova et al., 2007 ). These authors noted that R701A also affected dissociation kinetics for capsaicin, pointing to a potential role of this amino acid in capsaicin binding. TRPV1 sensitization occurs following nerve growth factor (NGF) binding to its receptor (Zhang et al., 2005 ). TRPV1 phosphorylation by Src kinase was TRPV1 FUNCTION AND CAUSE DIABETES 143 demonstrated to be a downstream effect of TrkA activation by NGF through activation of PI3 kinase. Phosphorylation of TRPV1 at Y200 (but not of Y195/ Y199/Y383) increased the rate of plasma membrane insertion of TRPV1. TRPV1 activity is modifi ed by reducing agents such as dithiothreitol (DTT) and glutathione, which increase TRPV1 sensitivity to heat. Extracellular C621 has been identifi ed as the residue responsible for the extracellular modulation of TRPV1 by these reducing agents (Susankova et al., 2006 ). Glycosylation is another form of posttranslational protein modifi cation. Rat TRPV1 was found to be glycosylated at N604, and mutation of this site affected the channel sensitivity to capsaicin. The mutant N604T channel was more sensitive to capsaicin, but it exhibited a decreased maximal capsaicin - induced current. The absence of glycosylation also diminished the response to capsazepine, as well as the dependence of the effect of capsaicin on extracellular pH (Wirkner et al., 2005 ). In summary, the structure – function relationships of TRPV1 from different species and experimental mutagenesis models are extremely complex and remain incompletely understood; a unifi ed theory does not currently exist. The study of the effi cacy of novel pharmacological agonists or antagonists targeting TRPV1 would benefi t from the use of transgenic mice that exclusively carry fully human TRPV1 protein sequences, but the choice of promoter would still represent a challenge for the generation of a fully humanized TRPV1 system. And, as discussed below, the great polymorphic diversity of human TRPV1 alleles still represents a major challenge toward this goal.

4.6 MISSENSE MUTATIONS IN NON - OBESE DIABETIC ( NOD ) MICE AFFECT TRPV 1 FUNCTION AND CAUSE DIABETES

The study of the role of TPRV1 in diabetes (Razavi et al., 2006 ; Tsui et al., 2007 ) (see Chapter 16 in this book) revealed that in diabetes - prone NOD mice, TRPV1NOD nociceptive behavioral responses (biting or licking) evoked by intradermal capsaicin were markedly depressed in NOD as compared to nonobese resistant (NOR) mice. Moreover, the maximum Ca 2+ response to capsaicin of DRG neurons of NOD mice was dramatically smaller than that of NOR DRG neurons. In addition, there was a shift in the capsaicin concentration– response relationship, with about 10- fold higher capsaicin concentrations required for NOD DRG neurons, compared with NOR DRG neurons. The differences in capsaicin- evoked responses in DRG neurons from NOD mice related to decreased TRPV1 expression and/or decreased function of TRPV1 that could be attributed to the two missense mutations found in the NOD allele . The sequence changes corresponding to those in TRPV1NOD have been engineered into human TRPV1 (P322A, D734E) (Xu et al., 2007 ). Under deﬁ ned expression conditions, hTRPV1P322A,D734E showed a markedly abnormal capsaicin concentration – response relationship compared to wild - type 144 TRPV1 GENETICS human TRPV1. The variant channel was hyporesponsive to capsaicin at low doses (< 10 nM) but was hyperresponsive at high doses (> 10 nM), with a markedly elevated Hill slope. The genetic changes in the primary sequence of NOD mouse TRPV1 produces changes in TRPV1 function that were directly responsible for the development of diabetes in NOD mice, ultimately, a deﬁ ciency of TRPV1 - dependent neuropeptides in the endocrine pancreas (Razavi et al., 2006 ; Tsui et al., 2007 ).

4.7 FUNCTIONAL POLYMORPHISMS IN THE HUMAN trpv1 GENE

Existing evidence emphasizes the key role of TRPV1 in pain, in infl ammation, and in a growing number of other disease- related physiological pathways. However, there is currently little evidence whether the high degree of polymorphism in the human trpv1 gene per se plays a role in any disease. Attempts to associate temperature sensitivity and acute pain have been largely inconclusive due to small - sized study cohorts: a common challenge for genetic studies of complex traits. One report, based on an ethnically diverse collection of 500 normal subjects, pointed to a functional role for a frequent polymorphism, I585V. This polymorphism was associated with longer cold withdrawal times in females of European/American descent, who were homozygous for the V585 allele (Kim et al., 2004 ). This study also concluded that gender and personality traits, as well as a polymorphism in the orpd1 gene, are primary determinants of sensitivity to heat - induced pain. The same research group followed up this study with a larger collection of 735 individuals, focusing on polymorphisms in the trpv1 , trpa1 , trm8 , orpd1 , comt , and faah genes (Kim et al., 2006 ). Again, gender and several genetic variants were found to infl uence pain sensitivity to thermal insults. However, the previously identifi ed polymorphisms were not found to be associated with either cold- or heat - induced pain responses in the second cohort (Kim et al., 2006 ). The authors used different statistical approaches in the two studies, and it is unclear whether and to what extent the study cohorts were unique or overlapping. Temperature sensitivity emerges as a complex trait, modifi ed by several genes besides trpv1 . An association study of such a highly diverse phenotype must be adequately powered to derive signifi cant effects and to attribute them to gene polymorphisms; academic funding policies for such activities appear reluctant to acknowledge these realities. Another strategy to identify the functional impact(s) of allelic variation is to model the effects of missense mutations in vitro. There are seven nonsynonymous polymorphisms in the human trpv1 gene reported in the dbSNP database;8 one of these (K2N) was not polymorphic in the four major ethnic human cohorts. One of the recent systematic studies of functional impact for

8 http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?chooseRs=coding&go=Go&locusId=7442 . RARE VARIANTS OF THE HUMAN trpv1 GENE 145 missense variants has indicated that the I315M and P91S variants exhibit a greater maximal response to capsaicin and anandamide, an endogenous cannabinoid and TRPV1 agonist (Xu et al., 2007 ). However, this increase in response may reﬂ ect a higher expression level of these TRPV1 variants (Xu et al., 2007 ). Interestingly, the I585V variant responds to both capsaicin and anandamide similarly to the wildtype channel, suggesting that this variant may be a functionally neutral polymorphism. Resequencing of the human trpv1 gene revealed that there is very high allelic heterogeneity in the gene,9 indicating that each missense mutation is occurring on multiple haplotypes. There is substantial linkage disequilibrium (LD) ( r2 = 0.66 – 0) between several missense mutations: the highest LD is observed between R719K (rs877610) and S680L (rs17706245) (r 2 = 0.66), while M315I (rs222747) has high LD with T469I (rs224534) (r 2 = 0.55). Thus, complex alleles that combine at least two mutations on the same allele exist in population, while other mutations are virtually independent of each other. Since the TRPV1 channel is composed of several subunits, the channel’ s activity could vary dramatically due to assembly of different protein subunits coded by different alleles. Thus, in vitro studies of functional consequences of missense variants on TPV1 activity are further complicated by the need to address allelic heterogeneity at the expression level.

4.8 SELECTION PRESSURE ON HUMAN trpv1 GENE?

Missense mutations in the human trpv1 gene are extremely rare in Africans and are most frequent in Han Chinese and Japanese populations. These polymorphisms have intermediate allele frequencies in Caucasians (minor allele frequency 0.05– 0.342 ) (Xu et al., 2007 ). A bioinformatic modeling study of the evolutionary rates in genes involved in the endocannabinoid system found evidence for signiﬁ cant positive selection pressures in the trpv1 gene only in African populations (McPartland et al., 2007 ). However, the presence of a large number of homozygous and heterozygous allele combinations in non - African populations must now be considered an accepted fact. This reality impacts negatively on our ability to use standard gene scans for TRPV1 disease - associated genome regions in non - African populations.

4.9 RARE VARIANTS OF THE HUMAN trpv1 GENE

An interesting single case report of a person with total insensitivity to capsaicin has been reported (Park et al., 2007 ). In this patient, the expression levels of TRPV1 protein and mRNA in buccal mucosa were greatly reduced. No

9 http://gvs.gs.washington.edu/ . 146 TRPV1 GENETICS changes in the coding regions of the trpv1 gene were found, but several point mutations were identifi ed in the second intron, which is also a 5 ′ UTR for alternative splice variant 4. Although studies of splicing effi ciency were impossible, such very low mRNA levels point to a tissue- selective induction of accelerated RNA decay, which is often induced by either a nonsense mutation or aberrant gene splicing. This patient illustrates the complexities that future TRPV1 functional studies in humans must overcome: this individual showed normal temperature sensitivity but hypersensitivity to garlic extract (allicin) upon exposure of buccal membranes, despite the fact that expression levels of the TRPA1 channel (allicin receptor) were normal (Park et al., 2007 ). A cytogenetic study of children with developmental delay, dysmorphism, and growth defects identifi ed a patient with de novo balanced translocation of chromosomes 17p13.3 and 20q13.33 (Walter et al., 2004 ). Fluorescence in situ hybridization (FISH) mapping of this translocation identifi ed a number of candidate genes near the break points: aspa , trpv3 , trpv1, and ctns at 17p13.3 and three genes of unknown function at 20q13.33. Detailed mapping revealed that the translocation occurred at the end of trpv1 and the entire trpv3 gene, as well as other transcription sites on chromosome 20 (Walter et al., 2004 ). This report may point to a homology between the trpv3 and trpv1 genes and unidentifi ed regions in 20q13.33, which can lead to translocations or deletions in chromosome 17, a wholly unexplored aspect of TRPV1 genetics. Other genes may infl uence TRPV1 function through modulating the level of activity and expression of TRPV1 in multiple ways. The cellular localization and TRPV1 traffi cking is probably a key target function. One recent report demonstrates that patients with Bardet – Biedl syndrome have altered thermal and mechanical sensitivity. The BBS1 and BBS4 genes mutated in Bardet – Biedl syndrome encode basal body proteins involved in TRPV1 traffi cking in humans (OSM - 9 in Caenorhabditis elegans ) (Tan et al., 2007 ). Phosphoinositide interacting regulator of TRP (Pirt ) was recently identifi ed as a core regulator of TRPV1, acting through PIP 2 in the peripheral nervous system (Kim et al., 2008 ). As was mentioned above, PIP2 regulates TRPV1 activity through binding to its C - terminal domain and through reducing activation thresholds (Lishko et al., 2007 ). Together with PIP2 , PIRT binds and activates TRPV1 (Kim et al., 2008 ). Surprisingly, knockout of Pirt in mice generated a phenotype similar to TRPV1 – / – mice (Kim et al., 2008 ). Collectively, the current status of TRPV1 genetics emphasizes complex interactions between polymorphisms in the trpv1 gene and other polymorphisms in the rather large group of genes, gene polymorphisms, gene products, transcription factors, and ligands that impact TRPV1. TRPV1 is, in that sense, at a busy crossroad of heterogeneous physiological response- and - control pathways, with afferent and efferent signaling, which would make it a natural player in complex, polygenic diseases, in particular conditions that combine genetic and acquired causes. As discussed in the chapter by Dosch et al. of this book, type 1 and possibly type 2 diabetes (Razavi et al., 2006 ; Tsui et al., 2007 ) may be but early examples of the role for TRPV1 in such complex, REFERENCES 147 polygenic diseases. However, it is already clear that the genetic diversity of human TRPV1 is almost certain to defeat classical gene scan strategies to identify disease risk associations, and it will remain challenging to comprehend the plausible impact of this diversity on diseases.

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PART II

ROLE FOR TRPV 1 IN PAIN STATES

5 TRPV 1 AND INFLAMMATORY PAIN

Anindya Bhattacharya , Sonya G. Lehto , and Narender R. Gavva

5.1 INTRODUCTION

The management of pain associated with infl ammation, injury, osteoarthritis, and cancer represents a signifi cant unmet medical need with a large impact on health and employment ability. Recently, much attention has been focused on the polymodal nocisensor called the “ vanilloid receptor ” (VR1) or transient receptor potential receptor vanilloid 1 (TRPV1), which is a nonselective cation channel highly expressed on sensory neurons, as a promising molecular target for the treatment of acute and chronic pain (reviewed in Holzer [ 2004, 2008], Immke and Gavva [2006 ], Roberts and Connor [2006 ], Szallasi et al. [ 2007 ], and Gunthorpe and Szallasi [ 2008 ]). Several types of data have triggered such interest: (1) intradermal or topical exposure to capsaicin, an agonist of TRPV1, causes an intense burning pain, erythema, thermal and mechanical hyperalgesia (increased pain response to a painful stimulus), and allodynia (pain response to a nonpainful stimulus) (Szallasi and Blumberg, 1999 ); (2) other painful stimuli, such as heat (> 42 ° C), low pH (pH < 5.9 at room temperature), and endogenous ligands such as endocannabinoids and lipoxygenase products, which are increased during infl ammation, also activate TRPV1; (3) expression of TRPV1 is upregulated in painful infl ammatory conditions in humans; (4) blocking TRPV1 gene expression leads to reduced infl ammation - induced pain (thermal hyperalgesia) in mice; and (5) TRPV1 antagonists block both thermal and/or mechanical hyperalgesia in acute and subacute infl ammatory pain models (Pomonis et al., 2003 ; Walker et al., 2003 ; Gavva et al., 2005c ;

153 154 TRPV1 AND INFLAMMATORY PAIN

Ghilardi et al., 2005 ; Honore et al., 2005 ; Cui et al., 2006 ; Rami et al., 2006 ; Gomtsyan et al., 2008 ). In addition, TRPV1 integrates signals from receptors for a variety of proinfl ammatory agents, such as bradykinin, prostaglandins, proteases, histamine, serotonin, and prokineticin, resulting in sensitization of nociceptors, which play a critical role in infl ammatory pain. This chapter discusses the role of TRPV1 in infl ammatory pain.

5.2 EXPRESSION OF TRPV 1 DURING INFLAMMATION

TRPV1 is expressed in key areas of the pain transduction pathway: skin nerve endings, dorsal root ganglia (DRGs), nodose ganglia (NGs), trigeminal ganglia (TGs) in the peripheral nervous system, as well as lamina II of the dorsal horn and, to a lesser extent, in the hippocampus, cortex, olfactory bulb, and cerebellum in the central nervous system (CNS) (Acs et al., 1996 ; Szallasi and Blumberg, 1999 ; Toth et al., 2005 ). In the TG and DRG, TRPV1 expression is restricted to a subset of small - to medium - sized neurons that are putative nociceptors (unmyelinated C - fi ber and lightly myelinated A δ - fi bers). A majority of the TRPV1 - expressing neurons also express the infl ammatory neuropeptides calcitonin gene - related peptide (CGRP) and substance P (SP) as well as colocalize with the nerve growth factor (NGF) receptor TrkA. Expression of TRPV1 in nociceptive nerve terminals supports an important role for this channel in the cellular mechanisms underlying neurogenic infl ammation. TRPV1 is additionally expressed in non - neuronal cells such as endothelium, immune cells (lymphocytes, dendritic cells, and mast cells), keratinocytes, smooth muscle cells, and urothelium, of which some are known to be involved in infl ammation. In the laboratory, painful peripheral insults such as complete Freund’ s adjuvant (CFA) and carrageenan upregulate TRPV1 expression in DRGs and sometimes in the neuronal projections that innervate tissues affected by infl ammation (Ji et al., 2002 ; Matthews et al., 2004 ). For example, at the level of DRG, ∼ 40% of articular afferents from mouse joints express TRPV1, and the majority of them are peptidergic, as revealed by simultaneous immunostaining for the neuropeptide CGRP (Cho and Valtschanoff, 2008 ). Similar expression or upregulation of TRPV1 in various locations of the pain pathway and tissues has been reported in humans. For example, increased TRPV1 expression within the pulps of hypomineralized teeth may be indicative of an underlying pulpal infl ammation and may help to explain the heat sensitivity experienced by some patients with this condition (Rodd et al., 2007 ). Increased TRPV1 expression has been shown in sensory fi bers of patients with infl amed esophagus (gastroesophageal refl ex disease) and infl amed bowel (both ulcerative colitis and Crohn ’ s disease), as well as chronic breast tenderness and pain (Yiangou et al., 2001 ). There is also an increase in TRPV1 expression in sensory fi bers of patients with rectal hypersensitivity and fecal urgency, and this increase is directly correlated with thermal and mechanical sensitivity (Chan et al., 2003 ). Similarly, the increased innervation of TRPV1 - expressing TRPV1 MEDIATES INFLAMMATORY PAIN 155 nerve fi bers in the vulva may contribute to the painful burning sensation as well as to thermal and mechanical sensitivity experienced by patients with vulvodynia (Tympanidis et al., 2004 ). Finally, TRPV1 expression is signifi - cantly increased in sensory nerve terminals that innervate the bladder in patients with detrusor overactive bladder and interstitial cystitis (Brady et al., 2004 ; Apostolidis et al., 2005 ), as well as in injured brachial plexus fi bers after trauma (Facer et al., 2007 ). Together, the aforementioned studies summarize that TRPV1 is expressed in critical locations of infl ammatory pain pathways.

5.3 TRPV 1 MEDIATES INFLAMMATORY PAIN

One day after injection of CFA, both wildtype and TRPV1 knockout mice exhibited decreased von Frey hair thresholds, suggesting that TRPV1 is not required for development of CFA- induced tactile allodynia (Caterina et al., 2000 ; Davis et al., 2000 ). In contrast, CFA reduced the radiant heat - evoked paw withdrawal latency (thermal hyperalgesia) in wild - type mice by 49%, but no change was observed in mice lacking TRPV1, suggesting that the thermal hyperalgesia induced by CFA is TRPV1 mediated (Caterina et al., 2000 ). Four hours after injection of carrageenan into the hind paw, both wildtype and heterozygous mice exhibited a highly signifi cant decrease in paw withdrawal latencies to a thermal stimulus, compared to baseline pre - infl ammation responses (Scheff é test, p < 0.001 and p < 0.001, respectively). In TRPV1 knockout animals, withdrawal latencies of infl amed paws were indistinguish- able from those measured before carrageenan injection (Scheff é test, p = 0.955) (Davis et al., 2000 ), suggesting that development of thermal hyperalgesia after carrageenan also requires TRPV1. In agreement with the previous study that demonstrated CFA- induced tactile allodynia was not altered in TRPV1 knockout mice (Caterina et al., 2000 ), it was also reported that carrageenan - evoked infl ammatory mechanical hyperalgesia was not altered in TRPV1 knockout mice (Bolcskei et al., 2005 ). Furthermore, analysis of the degree of carrageenan- induced hind paw infl ammation showed no signifi cant differences in carrageenan -induced edema between TRPV1 knockout and wildtype or heterozygous animals (Davis et al., 2000 ). Several studies have reported that TRPV1 mediates the pain that develops after joint infl ammation. Intraplantar injection of CFA into the hind paw and injection at the root of the tail induces swelling and other arthritis features. Histological examination and scoring of the tibiotarsal joints revealed marked arthritic changes in wild - type mice. However, in TRPV1 knockout animals, edema, histological score, and mechanical allodynia were signifi cantly lower (Szabo et al., 2005 ), suggesting that TRPV1 mediates some critical features of infl ammation and infl ammatory pain in adjuvant- induced arthritis. Intra- articular injection of CFA increased levels of knee swelling, hyperpermeability, and thermal hyperalgesia in wildtype mice (a model of joint infl ammation), but all three symptoms developed to a signifi cantly lower extent in TRPV1 156 TRPV1 AND INFLAMMATORY PAIN knockout mice (Keeble et al., 2005 ). In a similar study, knee joints of TRPV1 knockout or wildtype mice were injected with CFA, which resulted in infl ammation and hyperalgesia as measured for 35 days. However, TRPV1 knockout mice exhibited less joint swelling and a lower weight bearing difference between hind limbs, suggesting that TRPV1 receptors are important for the development of joint infl ammation and the associated mechanical hypersensitivity observed in this model (Barton et al., 2006 ). In summary, knockout experiments revealed that TRPV1 plays a signifi cant role in infl ammatory pain.

5.4 ACTIVATION MECHANISMS OF TRPV 1 DURING INFLAMMATION

Anandamide, oleoyldopamine, N - arachidonyl dopamine, 12 - hydroperoxyeicosatetraenoic acid, and low pH have been identifi ed as putative endogenous agonists of TRPV1 (reviewed in Van Der Stelt and Di Marzo [ 2004 ], Immke and Gavva [2006 ], and Szallasi et al. [2007 ]) based on their ability (1) to activate TRPV1 channels expressed in mammalian cells, (2) to compete with [3 H] - resiniferatoxin (RTX) binding to cell membranes prepared from TRPV1 - expressing cell lines, and/or (3) to cause release of CGRP and SP in a TRPV1- dependent manner. Phosphorylation of certain residues in the intracellular loops and cytoplasmic domains sensitizes TRPV1 such that it can be activated at body temperature (Premkumar and Ahern, 2000 ). Activation of TRPV1 during infl ammation appears to be a dynamic process that results from a combination of putative endogenous ligands, phosphorylation, low pH, and body temperature (Tominaga et al., 1998 ). A recent review highlights infl ammatory mediators that modulate TRPV1 as potential targets to treat infl ammatory pain (Ma and Quirion, 2007 ). Here, we highlight some mechanisms that result in TRPV1 activation during infl ammation and injury. An early study demonstrated that bradykinin- induced thermal hyperalgesia was absent in TRPV1 knockout mice, suggesting that TRPV1 mediates bradykinin - induced infl ammatory pain (Chuang et al., 2001 ). Pain sensation triggered by prokineticin was more recently shown to be attenuated in TRPV1 knockout mice (Negri et al., 2006 ). Thrombin and trypsin, which are increased following tissue injury, activate protease- activated receptors (especially PAR2 ). The latter can cause hyperalgesia via activation of TRPV1 channels (Amadesi et al., 2004 ; Dai et al., 2004 ). Prostaglandins, such as prostaglandin E2 (PGE2 ) and prostacyclin I 2 (PGI2 ) produced during tissue injury, are responsible for a signifi cant component of CFA- and carrageenan - induced infl ammatory pain in animal models. These prostaglandins enhance capsaicin - induced currents in DRG neurons (Pitchford and Levine, 1991 ) and reduce the temperature threshold for TRPV1 activation (Moriyama et al., 2005 ). Cross talk of the receptors for prostaglandin E (EP) and/or prostacyclin (IP) with TRPV1 has been demonstrated in experiments using TRPV1 knockout mice (Moriyama et al., 2005 ). NGF, whose levels have been reported to increase during CFA- induced infl ammation, regulates and sensitizes the EFFICACY OF TRPV1 ANTAGONISTS IN THE CFA MODEL 157

TRPV1 receptor (Ji et al., 2002 ). In summary, TRPV1 integrates many proinfl ammatory stimuli, often via activation of protein kinases and recruitment of modulatory scaffolding proteins (Zhang et al., 2007 ). Since TRPV1 integrates many of the infl ammatory pain cascades, TRPV1 antagonists have been considered as potential therapeutics for infl ammatory pain.

5.5 EFFICACY OF TRPV 1 ANTAGONISTS IN THE CFA MODEL OF INFLAMMATORY PAIN

Intraplantar injection of CFA in the rat hind paw causes development of thermal hyperalgesia and mechanical allodynia that lasts for 1– 7 days and is often used as a surrogate model of chronic infl ammatory pain (Larson et al., 1986 ). TRPV1 drug discovery programs across the industry have been using this model extensively, based on the observation that CFA- induced thermal hyperalgesia was attenuated in TRPV1 knockout mice. Numerous TRPV1 antagonists have been reported to reverse the established hyperalgesia induced by CFA (Pomonis et al., 2003 ; Walker et al., 2003 ; Gavva et al., 2005c ; Honore et al., 2005 ; Rami et al., 2006 ; Gomtsyan et al., 2008 ), as discussed in several recent reviews (Immke and Gavva, 2006 ; Roberts and Connor, 2006 ; Szallasi et al., 2007 ; Gunthorpe and Szallasi, 2008 ; Holzer, 2008 ). Capsazepine is one of the earliest TRPV1 antagonists identifi ed from structure– activity relationship studies of capsaicin and is the most studied (Urban and Dray, 1991 ; Bevan et al., 1992 ). Capsazepine is a unique species - specifi c TRPV1 antagonist in that it produces antihyperalgesia in guinea pigs (CFA- induced thermal hyperalgesia and tactile allodynia) but not in rats (Walker et al., 2003 ). TRPV1 antagonists that have been evaluated in the CFA model are discussed here. Effi cacy data are summarized in Table 5.1 . One of the most potent early TRPV1 antagonists, (N - (4 - tertiarybutylphenyl) - 4 - (3 - cholorphyridin - 2 - yl) tetrahydropyrazine - 1(2H) - carbox - amide (BCTC), was reported to be antihyperalgesic against CFA - induced thermal hyperalgesia; doses of 3 and 10 mg/kg (p.o.) produced comparable effi cacy to indomethacin (30 mg/kg, p.o.) (Pomonis et al., 2003 ). Furthermore, BCTC was also effective against CFA- induced mechanical hyperalgesia at 10 and 30 mg/ kg, with effi cacy comparable to the 30 mg/kg (p.o.) dose of indomethacin. It appeared that a threefold higher dose of BCTC was required to be effective against mechanical hyperalgesia versus thermal hyperalgesia in this model. The TRPV1 antagonist AMG9810 completely reversed thermal hyperalgesia in rats, with a quick onset of action (30 min) and with a duration of action for about 1 h, using doses of 30 and 100 mg/kg, 5 days post- administration of CFA (Gavva et al., 2005c ). The 10 mg/kg dose of AMG9810 did not demonstrate statistical signifi cance, although a trend toward effi cacy was seen at 30 min post - dose, a time the compound was active in a model of capsaicin - induced eye wipe. Similar to BCTC, AMG9810 (100 mg/kg, i.p.) was also partially effective in reversing mechanical hyperalgesia in this model. TABLE 5.1 Summary of In Vivo Pharmacology Results Reported for TRPV 1 Antagonists

Compound In Vitro IC 50 (nM) In Vivo Capsaicin Proton Heat Model

ABT compd - 6 17.0 ± 2.0 N/A N/A CFA - TH CFA - MH ABT - 102 (Abbott) 7.0 6.0 (pH 5.5) CFA - TH CI - TH A - 425619 (Abbott) 9.0 8.6 (pH 5.5) 56.0* CFA - TH

CI - TH A - 784168 (Abbott) 25.0 ± 2.0 N/A N/A CFA - TH CFA - MH CI - TH CI - MA A - 795614 (Abbott) 14.0 ± 1.0 8.6 ± 0.8 N/A CFA - TH AMG0347 (Amgen) 0.7 ± 0.1 0.8 ± 0.3 (pH 5.0) 0.2 ± 0.1 CI - TH CI - MH AMG517 (Amgen) 0.76 ± 0.4 0.62 ± 0.3 (pH 5.0) 1.3 ± 0.1 CFA - TH CI - TH AMG628 3.7 ± 0.8 2.0 ± 0.3 CFA - TH AMG8163 (Amgen) 0.5 ± 0.2 0.5 ± 0.3 (pH 5.0) 0.2 ± 0.1 CFA - TH AMG8562 (Amgen) 1.75 ± 0.5 Potentiator > 4000 CFA - TH AMG9810 (Amgen) 86.0 294.0 (pH 5.0) 21.0 CFA - TH CFA - MH Benzimidazole, compd 0.9 ± 0.6 0.9 ± 0.7 1.1 ± 0.2 CFA - TH 7 (Amgen) BCTC (Neurogen) 0.5 0.65 (pH 5.0) 0.6 CFA - TH CFA - MH

Capsazepine (Sandoz - NVS) 324.0 ± 41.0 355.0 ± 25.0 1000 (100% CFA - MH block) CI - TH GRC 6211 9.9 11.9 (pH 5.3) CFA - MH

IBTU 99.0 ± 23.0 N/A N/A CFA - TH CFA - MH JNJ compound A 3.0 3.0 N/A CI - TH JNJ compound B 10.0 3.0 N/A CI - TH NRGN compound 8 1.5 0.5 N/A CI - TH Quinazolinone compound 105.0 ± 3.0 N/A CFA - MH 26 (Novartis) SB - 782443 100.0 100.0 (pH 5.3) < 1000 CFA - MH SB - 705498 (GSK) 17.0 – 35.0 100.0 6.0 CFA - MA

* Amgen internal data. Additional information can be found in Savidge et al. (2002) , Valenzano et al. (2003) , Magal et al. (2005 ), Norman et al. ( 2005 ), Ognyanov et al. (2006) , Rami et al. (2006) , Gunthorpe et al. (2007) , and Khairatkar - Joshi and Szallasi (2009 ). In Vivo Reference

Measure Max % Maximum Dose E D50 Reversal Administered HB N/A N/A 6 μ mol/kg (p.o.) Brown et al. (2008) N/A N/A N/A 76 μ mol/kg (p.o.) HB 90 30 μ mol/kg (p.o.) 10 μ mol/kg (p.o.) Surowy et al. (2008) H B 21 μ mol/kg (p.o.) HB 62.0 100 μ mol/rat (i.p.) 51 μ mol/kg (i.p.) Honore et al. (2005) 96.0 300 μ mol/rat (p.o.) 40 μ mol/kg (p.o.) and El Kouhen et al. HB 77.8 100 μ mol/rat (i.p.) 50 μ mol/kg (i.p.) (2005) HB N/A N/A 7 μ mol/kg (p.o.) Cui et al. (2006) PP - RS N/A N/A 56 μ mol/kg (p.o.) HB N/A N/A 25 μ mol/kg (p.o.) PP - RS N/A N/A 92 μ mol/kg (p.o.) HB N/A N/A 12 μ mol/kg (p.o.) Cui et al. (2006) HB 40.0 – 46.0 30 mg/kg (p.o.) N/A Gavva et al. (2005a) evF No effect 30 mg/kg (p.o.) N/A HB 40 10 mg/kg (p.o.) 0.3 mg/kg (p.o.) MED Gavva et al. (2008) HB 42 10 mg/kg (p.o.) HB 45 10 mg/kg (p.o.) 1.0 mg/kg (MED) Wang et al. (2007) HB 45.0 – 50.0 30 mg/kg (p.o.) N/A Magal et al. (2005) HB 27.0 ± 9.0 100 mg/kg (p.o.) NA Lehto et al. (2008) HB 100.0 100 mg/kg (i.p.) N/A Gavva et al. (2005a) PP - RS 50.0 100 mg/kg (i.p.) N/A HB 46.0 30 mg/kg (p.o.) N/A Norman et al. (2005) and Ognyanov et al. (2006) HB 60.0 10 mg/kg (p.o.) N/A Pomonis et al. (2003) PP - RS 57.0 30 mg/kg (p.o.) N/A and Valenzano et al. (2003) PP - RS 44.0 30 mg/kg (s.c.) N/A Walker et al. (2003) and HB 54.0 30 mg/kg (s.c.) N/A Savidge et al. (2002) N/A 65.0 10 mg/kg (p.o.) N/A Khairatkar - Joshi et al. ( 2009 ) N/A 40.0 – 55.0 30 μ g/paw (i.pl.) N/A Tang et al. (2007) N/A 50.0 – 70.0 30 μ g/paw (i.pl.) N/A HB 50.0 30 mg/kg (p.o.) N/A Bhattacharya (2008) HB 100.0 10 mg/kg (p.o.) N/A Bhattacharya (2008) HB 125.0 3 mg/kg N/A Blum et al. (2008) PP - RS 60.0 30 mg/kg (p.o.) 4.7 mg/kg Culshaw et al. (2006)

N/A 68.0 ± 9.0 3 mg/kg (p.o.) 0.53 mg/kg Westaway et al. ( 2008 ) N/A 85.0 10 mg/kg (p.o.) 2.1 mg/kg Gunthorpe et al. (2007) , Chizh et al. (2007) , and Rami et al. (2006)

MH, mechanical hyperalgesia; TH, thermal hyperalgesia; PP - RS, paw pressure as measured by the Randall – Selitto method; CI, carrageenan - induced; CFA, complete Freund ’ s adjuvant; N/A, not applicable; ED50, effective dose 50; HB, hot box; evF, electronic von Frey; p.o., oral dosing; i.p., intraperitoneal; s.c., subcutaneous; i.pl., intra-plantar; MED, minimally effective dose. 160 TRPV1 AND INFLAMMATORY PAIN

Other TRPV1 antagonists from Amgen (AMG517, AMG628, and AMG8163) were also effective against CFA- induced thermal hyperalgesia, when nociceptive behaviors were measured 1 day after CFA administration (Gavva et al., 2007a ; Wang et al., 2007 ). These compounds were dosed orally, and effi cacy for reversal of thermal hyperalgesia was measured 2 h later, based on pharmacokinetic profi les of the compounds. Minimum effective doses for AMG517, AMG628, and AMG8163 were 1.0, 1.0, and 0.3 mg/kg, respectively. It is important to note that although AMG517, AMG628, and AMG8163 were signifi cantly more potent than AMG9810, maximal effi cacy was only 40 – 50% (compared to 100% for AMG9810). The partial effi cacy was not due to lack of target engagement by the TRPV1 antagonists, since comparable doses completely inhibited capsaicin - induced fl inching (Gavva et al., 2007a ; Wang et al., 2007 ). Several TRPV1 antagonists from Abbott Laboratories have been reported to effectively reduce CFA - induced thermal hyperalgesia in rats (El Kouhen et al., 2005 ; Honore et al., 2005 ; Cui et al., 2006 ; Brown et al., 2008 ; Surowy et al., 2008 ). A - 425619, the fi rst TRPV1 antagonist reported by Abbott, caused 60– 70% reversal of thermal hyperalgesia at a dose of 100 μmol/kg (i.p. or p.o.), with nearly complete reversal at 300 μmol/kg (p.o.). The in vivo effi cacy was reported to correlate well with A- 425619 attenuation of neuronal activity after thermal stimulation in the CFA - injured paw as recorded by wide dynamic range neurons in vivo (McGaraughty et al., 2006 ). More recently, ABT - 102, a potent TRPV1 antagonist that appeared to have progressed into the clinic, was also reported to be effective in the CFA model with an ED50 value of 10 μ mol/kg. TRPV1 antagonists from GlaxoSmithKline (GSK) have also been reported to be effective in models of infl ammatory pain. SB - 705498, which completed phase I and phase II clinical trials, was effective in the guinea pig model of CFA- induced mechanical hyperalgesia (Rami et al., 2006 ). SB - 705498 was reported to exhibit moderate affi nity for rat TRPV1 (Gunthorpe et al., 2007 ), although a recent report indicated low micromolar affi nity in a radioligand binding assay under true equilibrium conditions (Bhattacharya et al., 2007 ). A more recent TRPV1 antagonist, SB- 782443, was more effective against

CFA- induced mechanical hyperalgesia (ED 50 value, 0.53 mg/kg; Table 5.1 ) than SB - 705498 (Westaway et al., 2008 ). In addition to the TRPV1 antagonists described above, several others have been reported to be effective in the CFA model of inﬂ ammatory pain. For example, quinazolinone compound 26 described by Novartis demonstrated 60% reversal of mechanical hyperalgesia at the maximum dose of 30 mg/kg

(p.o.) with an estimated ED50 value of 4.7 mg/kg (Culshaw et al., 2006 ). A compound from the thiourea class, IBTU , was also reported to be effective in the CFA model (Tang et al., 2007 ). Unlike the other compounds described in this section, N - (4 - chlorobenzyl) - N ′ - (4 - hydroxy - 3 - iodo - 5 - methoxybenzyl) thiourea (IBTU) was delivered locally in the paw near the site of CFA tissue injury, and partial reversal of both thermal hyperalgesia and tactile allodynia was observed. Surprisingly, IBTU was effective for 7 days after local delivery. EFFICACY OF TRPV1 ANTAGONISTS IN THE CARRAGEENAN MODEL 161

Increased efﬁ cacy of TRPV1 antagonists in an osteoarthritis pain model after repeated dosing, which is a clinically relevant dosing paradigm, has recently been reported for ABT- 102 (Honore et al., 2009 ; for details, see the chapter by Joshi and Honore in this book).

5.6 EFFICACY OF TRPV 1 ANTAGONISTS IN THE CARRAGEENAN MODEL OF INFLAMMATORY PAIN

Carrageenan administration into the rat hind paw produces infl ammatory pain, as measured by thermal and mechanical hyperalgesia, which lasts for about 7 – 8 h (Hedo et al., 1999 ). This model is often used to measure the effi cacy of drugs as an acute model of infl ammatory pain (Vinegar et al., 1976 ). When capsazepine was administered locally near the site of carrageenan- induced edema and secondary hyperalgesia, there was a dose- dependent reversal of thermal hyperalgesia, probably due to a high local concentration of the compound (Kwak et al., 1998 ). Contrary to this fi nding, 30 mg/kg capsazepine administered systemically had no effect in rats, although it produced a 54% reversal of thermal hyperalgesia in guinea pigs (Walker et al., 2003 ). Several other TRPV1 antagonists have been reported to be effective in carrageenan- induced thermal hyperalgesia in rats. A- 425619 dose dependently inhibited the infl ammatory hyperalgesia caused by intraplantar injection of carrageenan, with an ED50 value of 50 μmol/kg (i.p.) (Honore et al., 2005 ). At the highest dose of 100 μmol/kg, 78% inhibition of thermal hyperalgesia was observed, which was comparable to that obtained in the CFA - induced thermal hyperalgesia model in rats, as discussed above. AMG0347 and AMG517 have also been tested in this model of acute infl ammatory pain. While both AMG0347 and AMG517 were effective in blocking thermal hyperalgesia, with a minimum effective dose of 3 mg/kg, AMG0347 had no effect, with up to 30 mg/kg on carrageenan - induced mechanical hyperalgesia, as measured by electronic von Frey fi laments (Gavva et al., 2007a ). Very recently, Neurogen scientists disclosed a new class of aminoquinazolines (Blum et al., 2008 ). “ Compound 18” from this class fully reversed thermal hyperalgesia after an oral dose of 3.0 mg/kg, with 0.3 mg/kg as the minimal effective dose. Furthermore, compounds from the diaminothiazolopyrimidine series disclosed by Johnson & Johnson were also reported to be effective in this model (Lebsack et al., 2009 ). While carrageenan- induced thermal hyperalgesia in rats is used widely to assess effi cacy of compounds against infl ammatory pain, due to the short assay time (maximal hyperalgesia and edema occur 2 – 3 h post - carrageenan injection), this model is sometimes used in a prophylactic mode where compounds are dosed prior to the infl ammatory insult. Even if the compound is administered shortly after the carrageenan injection, any resulting effi cacy should be considered partial reversal and partial prevention. In that regard, one can 162 TRPV1 AND INFLAMMATORY PAIN argue that the predictability of effi cacy and therapeutic utility is better modeled by the CFA- induced infl ammation model, since compounds are given 24– 48 h post- CFA injection, when maximal hypersensitivity has fully developed and resulting effi cacy is due only to reversal. When comparing effects in different models, it is important to consider the dosing time of TRPV1 antagonists compared to infl ammatory insult (CFA or carrageenan), as well as T1/2 and Tmax , to achieve maximum plasma and brain exposure of the antagonist.

5.7 EFFICACY OF TRPV 1 ANTAGONISTS IN THE FORMALIN MODEL OF INFLAMMATORY PAIN

Injection of formalin into the hind paw of rodents causes licking, lifting, and fl inching of the paw, which has been used as a pain model to evaluate potential analgesics (Dubuisson and Dennis, 1977 ). Limited information is available regarding evaluation of TRPV1 antagonists in the formalin model. So far, effi cacy of only two TRPV1 antagonists has been reported in this model. In one study, coadministration of IBTU (30 μ g/20 μL, i.pl.), with formalin signifi - cantly inhibited both the early and late phases of the formalin response (Tang et al., 2007 ). In this study, both the number of hind paw fl inches and the time spent in paw fl inching were signifi cantly reduced. In a second study, iodoresiniferatoxin, another potent TRPV1 antagonist, dose dependently and signifi - cantly decreased the number of fl inching responses in the formalin - evoked

fi rst and second phase with ID 50 values (drug dose producing 50% inhibition of response) of 1.0 and 3.8 μg, respectively (Kanai et al., 2006 ). However, reports of formalin injection in TRPV1 knockout mice are confl icting. For example, in one study, formalin evoked similar pain behaviors in both wild - type and TRPV1 knockout animals (Bolcskei et al., 2005 ), suggesting that TRPV1 does not mediate formalin- induced pain. In a second study, it was shown that formalin - induced pain behaviors were enhanced in the TRPV1 knockout mice (Staniland and McMahon, 2009 ), which suggests that TRPV1 may have a suppressive effect on the formalin - induced pain. Recently, it was demonstrated that formalin activates TRPA1 channels expressed in vitro and does not induce characteristic pain behaviors in TRPA1 knockout mice in vivo (McNamara et al., 2007 ), suggesting that the majority of pain behaviors induced by formalin are in fact TRPA1- mediated. It is possible that the TRPV1 antagonists described above may have been effective in the formalin model through a general dampening of excitability of neuronal fi bers involved in formalin pain or through some effect on TRPA1.

5.8 TRPV 1 ANTAGONISTS CAN BE DIVIDED INTO DISTINCT GROUPS

TRPV1 antagonists exhibit differential pharmacology dependent on the mode of activation (McIntyre et al., 2001 ; Gavva et al., 2005b, 2007b; Lehto et al., Profile A

(a) 125 Capsaicin (b) 125 pH 5 (c) 125 Heat

100 100 100 uptake uptake uptake 75 75 2+

2+ 75 2+ Ca Ca Ca 50 45

50 45 45 50 25 25 25 % max % max % max 0 0 0 –11 –10 –9 –8 –7 –6 –11 –10 –9 –8 –7 –6 –11 –10 –9 –8 –7 –6 AMG8163 (log M) AMG8163 (log M) AMG8163 (log M) Profile B (d) (e) (f) 125 125 125

100 100 100 uptake uptake 75 uptake 75 2+ 2+ 75 2+ Ca Ca 50 Ca 50 45 45 45 50 25 25 25 % max % max 0 0 % max 0 –10 –9 –8 –7 –6 –5 –10 –9 –8 –7 –6 –5 –10 –9 –8 –7 –6 –5 AMG8563 (log M) AMG8563 (log M) AMG8563 (log M) Profile C (g) 125 (h) 200 (i) 150 175 125 100 150 uptake

uptake 100 uptake 125 2+ 2+

75 2+

Ca 75 Ca

Ca 100 45 45 50 45 75 50 50 25 25 % max % max % max 25 0 0 0 –11 –10 –9 –8 –7 –6 –5 –11 –10 –9 –8 –7 –6 –5 –11 –10 –9 –8 –7 –6 –5 AMG8562 (log M) AMG8562 (log M) AMG8562 (log M) Profile D 300 (j) 125 (k) 300 (l) 250 100 250 200 uptake uptake

uptake 200

75 2+ 2+ 2+ 150 Ca Ca 150 Ca 45 45

50 45 100 100 25 50 50 % max % max % max 0 0 0 –10 –9 –8 –7 –6 –5 –10 –9 –8 –7 –6 –5 –10 –9 –8 –7 –6 –5 AMG7905 (log M) AMG7905 (log M) AMG7905 (log M) Figure 5.1 Characterization of in vitro profi les of TRPV1 modulators. Effect of AMG8163, a profi le A compound, on TRPV1 activation by capsaicin, pH 5 and heat ( A - C ) (Lehto et al., 2008 ). AMG8563, a profi le B compound, on TRPV1 activation by capsaicin, pH 5 and heat (D - F). AMG8562, a profi le C compound, on TRPV1 activation by capsaicin, pH 5 and heat ( G - I). AMG9705, a profi le D compound, on TRPV1 activation by capsaicin, pH 5 and heat (J - L). Each point in the graph is an average ± S D of an experiment conducted in triplicate. 164 TRPV1 AND INFLAMMATORY PAIN

2008 ). Amgen scientists have defi ned four unique profi les of rat TRPV1 modulators, based on the evaluation of numerous compounds for their ability to modulate three distinct modes of TRPV1 activation, using agonist- induced 45 Ca2+ uptake assays in Chinese hamster ovary (CHO) cells stably expressing rat TRPV1 (Fig. 5.1 ). Compounds that potently inhibit activation of rat TRPV1 by capsaicin, pH 5, and heat represent “ profi le A” or group A (e.g., AMG517, AMG8163, and many others; Fig. 1a – c [Gavva et al., 2007b ). Compounds that inhibit activation by capsaicin and heat but only partially block activation by pH 5 include capsazepine and AMG0610, and represent profi le B or “ group B” antagonists (Gavva et al., 2005b ) (Fig. 1d – f ). Compounds that block activation by capsaicin, do not affect activation by heat, and potentiate activation by pH 5 represent profi le C (e.g., AMG8562; Fig. 1g – i ). Compounds that inhibit activation by capsaicin and potentiate activation by both heat and pH 5 represent profi le D (e.g., AMG7905; Fig. 1j – l ). Whether similar profi les described above with rat TRPV1 will also be observed with human TRPV1 remains to be determined. Profi le C and profi le D molecules (AMG8562 and AMG7905, respectively) by themselves did not induce 45 Ca 2+ uptake into CHO cells expressing rat TRPV1 at physiological pH (pH 7.2), indicating that they are not partial agonists. Effects of TRPV1 modulators representing profi les A through D have been evaluated in rats implanted with radiotelemetry probes to monitor their body temperature. As expected (Gavva et al., 2007b ), profi le A (AMG8163) and profi le B (AMG8563) modulators of TRPV1 caused hyperthermia in rats. Interestingly, AMG8562 (profi le C) did not cause hyperthermia, whereas AMG7905 (profi le D) caused marked hypothermia.

5.9 CRITICAL FEATURES OF ANTAGONISTS REQUIRED FOR EFFICACY IN PAIN MODELS

TRPV1 antagonists that block all modes of activation and those that have good CNS penetration appear to have superior antihyperalgesic activity in pain models. The classic TRPV1 antagonist capsazepine shows species differences in its ability to block the multiple modes of TRPV1 activation. For example, capsazepine, which inhibited activation of rat TRPV1 by capsaicin and heat activation but not by protons (profi le B), was ineffective in reversing infl ammation- based pain behaviors in rats (McIntyre et al., 2001 ; Walker et al., 2003 ). Conversely, capsazepine inhibited all modes of activation of guinea pig TRPV1 (profi le A) and reversed infl ammatory and nerve injury- related hyperalgesia. These results suggested that in vivo effi cacy requires inhibition of activation by protons (Walker et al., 2003 ) and that antagonism of all modes of TRPV1 activation is necessary to achieve signifi - cant antihyperalgesic effects. Several chemically distinct compounds (ABT - 102, AMG9810, AMG8163, AMG517, AMG628, JNJ compounds A and B, Neurogen compound 18, Novartis compound 26, and BCTC), which inhibited PERSPECTIVES 165

TABLE 5.2 Classifi cation of TRPV 1 Antagonists Based on the Ability to Modulate Distinct Modes of Activation Capsaicin PH 5 Heat Effi cacy Profi le A Antagonism Antagonism Antagonism Yes (several) Profi le B Antagonism Partial antagonism Antagonism Yes (A - 425619) Profi le C Antagonism Potentiation No effect Yes (AMG8562) Profi le D Antagonism Potentiation Potentiation Unknown

E f fi cacy refers to the antihyperalgesic effect of at least one compound in a rodent model of infl ammation. all modes of rat TRPV1 activation (profi le A), also inhibited and/or reversed infl ammation - induced hyperalgesia in rats (Pomonis et al., 2003 ; Gavva et al., 2005c ; Honore et al., 2005 ; Lebsack et al., 2009 ). However, it was recently reported that AMG8562, a profi le C modulator (that blocks activation by capsaicin, potentiates activation by pH 5, and does not affect activation by heat), was partially effective as an antihyperalgesic in rodent models of infl ammation, suggesting that antihyperalgesia can be achieved with compounds that do not block all modes of TRPV1 activation (Lehto et al., 2008 ). Evaluation of additional molecules representing profi les B through D should shed more light on the role of antagonism of each distinct mode of TRPV1 activation in the generation of effi cacy (Table 5.2 ). The ability of TRPV1 antagonists to penetrate the CNS appears to be another critical feature. CNS- penetrant TRPV1 antagonists have demonstrated better effi cacy than those that are peripherally restricted (Cui et al., 2006 ). For example, it was reported that in rodent models of pain, including CFA - induced mechanical allodynia, A - 784168, which is a TRPV1 antagonist with good CNS penetration, was much more potent than A - 795614, which is a peripherally restricted TRPV1 antagonist (Cui et al., 2006 ). However, the potency of the compounds was similar in these models after intrathecal administration, suggesting that CNS penetration gives superior effi cacy. The risk – benefi t profi le of TRPV1 blockade in the CNS versus only peripheral TRPV1 blockade is still unknown, and current drug discovery programs across the industry seem to be investigating both.

5.10 PERSPECTIVES

5.10.1 Maximal Effi cacy of TRPV 1 Antagonists and End - Point Measurements in Infl ammatory Pain Models The magnitude of effi cacy observed in infl ammatory pain models, as measured by the reversal of thermal hyperalgesia for different TRPV1 antagonists, has varied widely between different research groups. For example, publications from Abbott have indicated that their TRPV1 antagonists exhibit 60– 95% 166 TRPV1 AND INFLAMMATORY PAIN effects, whereas publications from Amgen have indicated that their molecules exhibit a maximum 30– 50% effect (except for AMG9810, which has shown 100% effect). The Novartis group has reported that its TRPV1 antagonists exhibit a maximum 50– 60% effect, whereas publications from the GSK group have indicated maximal effi cacy in the range of 80– 90%. It is not currently known whether this variability arises from the methodology used in the in vivo experiments, from differing selectivity profi les of the TRPV1 antagonists, or from differences in the level of CNS penetration by the compounds. It appears that compounds which exhibit polypharmacology (such as BCTC and AMG9810, which block other channels in addition to TRPV1) show a higher magnitude of effi cacy. Compounds with signifi cantly improved potency and selectivity, such as AMG517, AMG628, and AMG8163, were less effi cacious (maximal reversal of thermal hyperalgesia was 40– 50%) compared with compounds like AMG9810 (maximal reversal of thermal hyperalgesia was 100%). The partial effi cacy of antagonists such as AMG517, AMG8163, and AMG628 was not due to lack of target coverage because they completely inhibited capsaicin - induced fl inching at comparable doses. Although the experimental paradigms used are often somewhat different, it seems reasonable to suggest that the reported differences in effi cacy can be attributed to the selectivity profi les of compounds and that highly selective TRPV1 antagonists only reverse ∼ 50% of hyperalgesia induced by infl ammation. Although TRPV1 knockout mice did not exhibit attenuation of carrageenan - or CFA - induced mechanical hyperalgesia, several antagonists (A - 425619 [Honore et al., 2005 ], BCTC [Pomonis et al., 2003 ], quinazolinone compound 6 [Culshaw et al., 2006 ], AMG9810 [Gavva et al., 2005c ], and SB- 705498 [Rami et al., 2006 ]) showed moderate effi cacy in this end point. These results suggest a potential role for TRPV1 in mediating nociceptive responses to mechanical stimuli under infl ammatory conditions. It is interesting that all the studies that measured mechanical hyperalgesia by the method of Randall– Selitto reported effi cacy. This is perhaps refl ective of the nature of the Randall– Selitto method, which is reported to represent C- fi ber output, and correlates well with the abundant expression of TRPV1 in small- diameter neurons (C - fi bers).

5.10.2 Potential Limitations of TRPV 1 Blockade One of the signifi cant on- target undesirable effects reported for TRPV1 antagonists is hyperthermia (Bannon et al., 2004 ; Swanson et al., 2005 ; Gavva et al., 2007a,b, 2008 ; Steiner et al., 2007 ; Gavva, 2008 ; Mills et al., 2008 ; Tamayo et al., 2008 ). TRPV1 knockout studies have unequivocally demonstrated that TRPV1 antagonist- elicited hyperthermia is TRPV1 mediated (Steiner et al., 2007 ). The fact that numerous structurally distinct TRPV1 antagonists cause hyperthermia indicates that TRPV1 is tonically active and that body temperature maintenance is an evolutionarily conserved function of TRPV1 in a non- pathophysiological state (Gavva et al., 2007b ; Gavva, 2008 ). TRPV1 REFERENCES 167 antagonist - elicited hyperthermia became a hurdle for one molecule, AMG517, which caused marked and persistent hyperthermia in dental pain patients (Gavva et al., 2008 ). However, several other TRPV1 antagonists have been reported to be moving forward in the clinic. It is not currently known whether hyperthermia is an unmanageable hurdle for all TRPV1 antagonists. Efforts to eliminate hyperthermia led to the profi le C modulators (block activation by capsaicin, potentiate activation by pH 5, and do not affect activation by heat), which lack hyperthermia liability in rodent studies (Lehto et al., 2008 ) However, compounds exhibiting profi le C at human TRPV1 channels have not yet been reported (Lehto et al., 2008 ). Because profi le C modulators potentiate activation of TRPV1 by low pH (Lehto et al., 2008 ), potential tox- icities of chronic dosing of these molecules are unknown. Since TRPV1 knockout mice exhibited decreased heat sensitivity (Caterina et al., 2000 ; Davis et al., 2000 ), it will be crucial to determine whether TRPV1 antagonists affect thermosensation in humans. Affecting the ability to sense noxious heat might increase the potential of thermal injury to humans. In fact, it has been reported that SB- 705498 increased the heat pain threshold in humans (Chizh et al., 2007 ); however, no such information has been reported for AMG517 or for other molecules in the clinic. TRPV1 has been shown to be protective in some infl ammatory conditions, such as endotoxin- induced airway infl ammation in the mouse (Helyes et al., 2007 ) and colonic infl ammation (colitis) induced by dinitrobenzene sulfonic acid (Massa et al., 2006 ). Capsaicin - sensitive afferent neurons participate in the regulation of many aspects of gastrointestinal functions, including increased mucosal blood fl ow through peripheral circuitry and initiation of bicarbonate and mucus secretion (Gunthorpe and Szallasi, 2008 ). It will be important to determine whether TRPV1 blockade results in undesirable side effects in humans due to the normal physiological function of TRPV1. The risk – benefi t profi le of TRPV1 blockade will be better understood when the data from clinical trials of TRPV1 antagonists (such as ABT- 102, AZD1386, GRC 6211, JTS- 653, MK 2295/NGD 8243, PF- 4065463, and SB- 705498) are released.

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Shailen K. Joshi and Prisca Honore

6.1 INTRODUCTION

Osteoarthritis (OA) is the most prevalent form of arthritis affl icting about 14.4 million people in the United States alone (Wieland et al., 2005 ). OA is a musculoskeletal disease responsible for mobility disability in a large proportion of the population over 65 (Hunter and Felson, 2006 ) and is the major cause of joint replacement surgery. The societal burden due to OA is expected to increase with the longer life expectancy in an aging population. Though OA can occur in any joint, it is most commonly present in joints of the knee, hip, hand, and foot. This is in contrast to rheumatoid arthritis, a systemic multijoint infl ammatory disease driven by autoimmune processes (Wieland et al., 2005 ). Clinical treatment of OA is particularly problematic due to its largely unknown etiology. OA has a slow insidious onset with changes in the joint tissue starting long before middle age and often preceding the appearance of symptoms and diagnosis by decades. By the time of diagnosis, the structural joint damage is already quite advanced (Wieland et al., 2005 ). Although OA was traditionally thought to be a disease affecting only the cartilage, it is now considered to be a disease of the whole joint. The current concept considers OA to be a multifactorial process that affl icts the entire joint including the synovium, cartilage, subchondral bone, meniscus, joint capsule, tendons/ligaments, and muscles (Creamer et al., 1996 ; Felson, 2005 ; Wieland et al., 2005 ; Hunter and Felson, 2006 ; Kidd, 2006 ; Dray and Read, 2007 ). The principal morphological

175 176 ROLE OF TRPV1 RECEPTORS IN OSTEOARTHRITIC PAIN characteristic of OA is the degenerative breakdown of cartilage with episodic synovitis. The clinical symptom of OA is chronic joint pain accompanied by functional impairment, including joint stiffness and loss of function. Most OA- related knee pain has a mechanical basis and is associated with certain activities. Currently, there is no disease modifying therapy for OA, and the clinical strategy is focused on symptom management.

6.2 PAIN IN OA

Joint pain is the predominant disabling symptom of OA and is typically described as related to joint use, with an exacerbation of symptoms due to activity and relief provided by rest. Other clinical features of OA are a transient stiffness in the morning or after a period of rest, a reduced range of motion, and swelling (Hunter and Felson, 2006 ). While symptoms are mostly experienced near the affected joint, referred pain or tenderness at distant sites can also occur, implicating central nervous system (CNS) changes in addition to the peripheral pathology (Creamer et al., 1996 ; Kidd, 2006 ). An aim of symptomatic treatment in OA is to normalize the pathological pain processing through modulation of neural excitability. Anti - infl ammatory agents such as acetaminophen or cyclooxygenase (COX) enzyme inhibitors are commonly used to alleviate osteoarthritic pain (Wieland et al., 2005 ; Dray and Read, 2007 ). However, osteoarthritic pain is a signifi cant unmet medical need since current drug therapies provide incomplete pain relief. Moreover, side- effect liabilities are associated with the use of COX inhibitors, including gastrointestinal complications and cardiovascular concerns, the latter associated particularly with selective COX - 2 inhibitors (Petit - Zeman, 2004 ). Other pharmacological treatment options are less effective and/or are also associated with liabilities (Wieland et al., 2005 ; Hunter and Felson, 2006 ). For example, opioid analgesics such as tramadol are associated with adverse liabilities especially in the elderly; the effi cacy of glucosamine and chondroitin products remains controversial; and topical agents (nonsteroidal antiinfl ammatory drugs [NSAIDs] and capsaicin) demonstrate only partial pain relief (Wieland et al., 2005 ). Therefore, the need for a new drug therapy to treat osteoarthritic pain is urgent.

6.2.1 Anatomical/Structural Changes Contributing to Osteoarthritic Pain The relationship between pain and the underlying pathology in OA is not straightforward. There are no pain fi bers in the cartilage, and cartilage loss may occur without any accompanying symptom (Wieland et al., 2005 ; Dray and Read, 2007 ). Cartilage breakdown, driven by a multitude of biological molecules, causes the synovium to become infl amed. Chemical mediators (e.g., cytokines, chemokines, and proteolytic enzymes), which are produced by chondrocytes and are associated with infl ammation, cause further cartilage breakdown (Kidd et al., 2004 ; Kidd, 2006 ). Changes also occur in the underly- PAIN IN OA 177 ing bone that thickens with the formation of bony outgrowth (osteophytes), very often seen in weight bearing zones. Anatomical changes contributing to pain arise from bone (raised intraosseous pressure, growing osteophytes, and chondral fractures) and soft tissue (capsule, ligament, and menisci). It has been reported that osteophyte formation in the human knee joint is directly related to and predictive of pain (Lanyon et al., 1998 ). The vascularization that is seen in the areas of bone remodeling may contribute to pain via the sensory fi bers that invade blood vessels (Ghosh and Cheras, 2001 ).

6.2.2 Role of Infl ammation in OA The contribution of synovial and joint infl ammation to osteoarthritic pain has been demonstrated (Kidd et al., 2004 ; Kidd, 2006 ). However, OA is not a disease driven primarily by infl ammatory processes, although a degree of episodic synovitis is commonly seen in the early stages of the disease. The sensory nerve fi bers that provide innervation to the synovium are peptidergic afferents, which contain substance P and calcitonin gene- related peptide (CGRP) (Wieland et al., 2005 ). These neuropeptides have long been known to have a fundamental role in the process of neurogenic infl ammation that contributes to localized synovitis. Immune modulators such as interleukin - 1β (IL - 1 β ) tumor necrosis factor -α (TNF - α ), bradykinin (BK), and prostaglandin E - 2 (PGE - 2 ) are associated with synovial infl ammation and directly or indirectly produce peripheral nociceptor sensitization (Chen et al., 1999 ).

6.2.3 Peripheral Nervous System and CNS Mechanisms Contributing to Osteoarthritic Pain The structural damage infl icted to the joint in OA leads to a pathologically altered nervous system such as occurs in other chronic pain syndromes, with involvement of both peripheral nerves and components of CNS pain pathways (Dray and Read, 2007 ). Except for cartilage, all other joint structures, including the subchondral bone and synovium, are rich in unmyelinated C- fi ber nociceptors. These afferents respond to mechanical stimuli, for example, stretching of the joint capsules, as well as to various pronociceptive mediators that stimulate nociceptive afferents directly and produce sensitization in a pathological state (Felson, 2005 ). The signifi cant role of peripheral pathology in osteoarthritic pain is supported by clinical observations. For example, injection of a local anesthetic into the knee of OA patients completely abolishes pain in 60% of the patients (Creamer et al., 1996 ). Joint replacement in patients suffering from hip OA alleviates the neurological abnormal sensitivity to pain and noxious stimuli (Ordeberg, 2004 ; Felson, 2005 ). There is also considerable evidence for the role of the CNS in osteoarthritic pain. Clinical observations that osteoarthritic pain can be referred to sites distant from affected joints and the presence of increased tenderness in apparently normal tissues are suggestive of a state of altered CNS processing of 178 ROLE OF TRPV1 RECEPTORS IN OSTEOARTHRITIC PAIN painful information, resulting in an amplifi cation of pain sensation (Creamer et al., 1996 ; Kidd, 2006 ). The mechanisms contributing to the development of central sensitization include enhanced peripheral nociceptive barrage from the sensitized arthritic joint afferents into the spinal cord and the enhanced release of neurotransmitters into the spinal cord dorsal horn. Nociceptive specifi c (NS) and wide dynamic range (WDR) spinal cord neurons that have receptive fi elds in the joint and overlying skin demonstrate excitation (Schaible and Grubb, 1993 ; Schaible, 2004 ; Felson, 2005 ). Other CNS mechanisms implicated in clinical osteoarthritic pain include dysfunction of descending inhibitory control (Kosek and Ordeberg, 2000 ) and altered cortical processing of nociceptive information (Buffi ngton et al., 2005 ).

6.3 ROLE OF TRANSIENT RECEPTOR POTENTIAL VANILLOID 1 ( TRPV 1) IN JOINT PAIN

6.3.1 Expression of TRPV 1 Receptor The TRPV1 receptor, which is a nonselective calcium- preferring cation channel, is a molecular integrator of various noxious stimuli and is an initiator of neurogenic infl ammation (Caterina et al., 1997 ; Numazaki and Tominaga, 2004 ; Immke and Gavva, 2006 ; Szallasi et al., 2007 ). The TRPV1 receptor is highly expressed in peripheral afferent C - fi ber neurons involved in the sensation and transmission of nociceptive information (Caterina et al., 1997 ). TRPV1 receptors are expressed in small- and medium- sized dorsal root ganglion (DRG) neurons, including both peripheral and central projections (Guo et al., 1999 ; Carlton and Coggeshall, 2001 ; Valtschanoff et al., 2001 ). This expression profi le suggests that TRPV1 plays a key role in nociception. Indeed, there is extensive preclinical evidence from studies using TRPV1 agonists/ antagonists as well as TRPV1 knockout mice that supports the role of TRPV1 in various pain states (Honore et al., 2005 ; Barton et al., 2006 ; Cui et al., 2006 ; Szallasi et al., 2007 ). Although originally reported to be primarily expressed on peripheral nociceptive afferents, TRPV1 is also expressed at spinal and supraspinal sites implicated in pain processing in both rats and humans (Mezey et al., 2000 ). In the spinal cord, TRPV1 receptors are found both pre - and postsynaptically. Spinal administration of the TRPV1 receptor antagonist capsazepine has been shown to inhibit evoked activity of WDR neurons (Carlton and Coggeshall, 2001 ; Kelly and Chapman, 2002 ). TRPV1 is expressed in various regions of the brain known for their roles in pain transmission and modulation (Mezey et al., 2000 ; Roberts et al., 2004 ). Microinjection of capsaicin into the periaqueductal gray produces antinociceptive effects, supporting a role for TRPV1 in the brain in pain transmission (McGaraughty et al., 2003 ). A role for central TRPV1 receptors in pain processing has also been shown in studies using small - molecule antagonists (Cui et al., 2006 ). ROLE OF TRPV1 IN JOINT PAIN 179

Changes in TRPV1 receptor expression and sensitization have been reported in pathological pain states (Di Marzo et al., 2002 ; Numazaki and Tominaga, 2004 ; Immke and Gavva, 2006 ; Pingle et al., 2007 ; Szallasi et al., 2007 ). In rat OA models, TRPV1 receptor expression has been shown to increase in the joint capsule (Cho and Valtschanoff, 2004 ). In a separate study, TRPV1 expression has been shown to be increased in knee afferents from osteoarthritic rats relative to naive rats (Fernihough et al., 2005 ). A similar increase in TRPV1 expression has been reported in human arthritic synovial nerve fi bers. In addition to expression in primary afferent neurons innervating the joint structures, TRPV1 has also been reported to be expressed in synovial fi broblasts from patients with symptomatic OA, as well as in peripheral blood mononuclear cells from both healthy individuals and OA patients (Engler et al., 2007 ). In the latter report, evidence has been provided to suggest that stimulation of these TRPV1 receptors leads to increased expression of proinfl ammatory cytokines, which contributes to peripheral hypersensitivity.

6.3.2 Role of Peripheral TRPV 1 Receptors in Osteoarthritic Pain As mentioned above, TRPV1 expression is increased in the peripheral axons of DRG neurons in the joint capsule of osteoarthitic rats (Cho and Valtschanoff, 2004 ). Activation and sensitization of these TRPV1 receptors can occur by a variety of mechanisms in OA. The osteoclastic bone resorption, infl ammation, and tissue hypoxia associated with OA lead to decreases in local pH, which can activate TRPV1 (Baron et al., 1985 ; Blair et al., 1989 ). The structural pathology in osteoarthritic joints can cause excitation of the peripheral nervous system, resulting in neuronal hyperexcitability, sensitization and spontaneous fi ring, events in which TRPV1 has been shown to play an important role (Immke and Gavva, 2006 ; Dray and Read, 2007 ; Szallasi et al., 2007 ). The threshold for activation of TRPV1 is dynamic and can be signifi cantly lowered by a variety of infl ammatory mediators in pathological pain states, resulting in peripheral sensitization (Numazaki and Tominaga, 2004 ; Szallasi et al., 2007 ). Mechanisms of TRPV1 sensitization and activation are discussed in greater detail in the chapter by Surowy et al. of this volume.

6.3.3 Evidence for the Role of TRPV 1 in Joint and Bone Pain The role of TRPV1 in acute and chronic joint inﬂ ammation has been demonstrated using TRPV1 knockout mice, which have decreased knee swelling and hyperpermeability compared with wild - type mice following intra - articular injection of complete Freund’ s adjuvant (CFA) (Keeble et al., 2005 ). Additionally, knee swelling lasts for a shorter time in TRPV1 knockout mice. These mice also exhibit a signiﬁ cant decrease in thermal hyperalgesia compared to wildtype controls (Barton et al., 2006 ). In another preclinical arthritis model, in which carrageenan is injected into the rat knee joint, the potent TRPV1 agonist resiniferatoxin (RTX) produces an analgesic and anti- 180 ROLE OF TRPV1 RECEPTORS IN OSTEOARTHRITIC PAIN

infl ammatory effect (Kissin et al., 2005 ). The role of TRPV1 in pain originating from the bone has also been documented in a bone cancer pain model (Ghilardi et al., 2005 ; Menendez et al., 2006 ). Osteoclasts, which are activated following cancer colonization in the bone, produce an acidic pH that contributes signifi - cantly to bone cancer - associated pain via TRPV1 nociceptors innervating the marrow and mineralized bone. This topic is covered in greater detail in the chapter by Mantyh et al. of this volume. Such fi ndings from the joint and bone tissue, in conjunction with the expression pattern and the well- known pronociceptive mechanisms associated with TRPV1, suggest an important role of TRPV1 receptors in bone - related pain, including osteoarthritic pain.

6.3.4 Effi cacy of TRPV 1 Antagonists in Preclinical Models of Osteoarthritic Pain The use of valid predictive preclinical animal models is critical for identifi cation of novel pharmacotherapies for treating chronic pain syndromes in humans. The development of preclinical osteoarthritic pain models in rodents has been particularly challenging due to an incomplete understanding of the etiology of the human disease (Dieppe and Lohmander, 2005 ; Felson, 2005 ; Wieland et al., 2005 ; Ameye and Young, 2006 ; Hunter and Felson, 2006 ). Historically, the development and characterization of animal OA models has focused on structural damage rather than pain. This is problematic for studies on pain induced by OA because the intensity of osteoarthritic pain in clinical patients does not correlate with the extent of knee damage (Ameye and Young, 2006 ; Dray and Read, 2007 ). While it is clear that no single animal model can precisely replicate human OA, animal models of osteoarthritic pain have been developed. They can be broadly classifi ed into surgically (partial medial menisectomy) or chemically (using monosodium iodoacetate) induced joint degeneration and pain (Fernihough et al., 2004 ). In a comparative study between these two models, pain responses in the iodoacetate - treated animals were found to be more robust and amenable to pharmacological modulation (Fernihough et al., 2004 ). Monosodium iodoacetate injection into the rat knee joint rapidly generates the biochemical/structural changes and pain associated with clinical OA. Iodoacetate leads to inhibition of chondrocyte metabolism in the knee joint. Following death of chondrocytes and cartilage fragmentation, a subchondral bone lesion develops, with active resorption and remodeling of trabecular bone by day 21 (Guzman et al., 2003 ; Combe et al., 2004 ; Fernihough et al., 2004 ; Ameye and Young, 2006 ). A transient infl ammation is initially observed followed by evidence of nerve damage in the later stages. Pain end points such as the difference in weight bearing or the grip force defi cit between the normal and injured OA paws can be reproducibly studied in this model. The model also seems to demonstrate predictive validity since the observed pain end points are reversed by morphine, acetaminophen, NSAIDs, and COX - 2 inhibitors (Combe et al., 2004 ; Pomonis et al., 2005 ). The iodoacetate osteoarthritic ROLE OF TRPV1 IN JOINT PAIN 181

50 40 ** 30 ** 20

Weight bearing difference (g) 0 V 30 100 300 A-425619 (μmol/kg, i.p.) Figure 6.1 Antinociceptive effects of the TRPV1 antagonist A- 425619 in osteoarthritic pain induced by iodoacetate injection into the rat knee joint. Pain was evaluated 30 min following intraperitoneal (i.p.) A - 425619 administration. Circles represent weight bearing difference between injured and noninjured paw in animals that received A- 425619. The square is the weight bearing difference in vehicle- treated animals. A - 425619 signifi cantly and in a dose - related manner decreased iodoacetate - induced increase in weight bearing difference with 24.7 ± 5.5% effect at 100 μ mol/kg and 46.8 ± 5.5% effect at 300 μ mol/kg. Data represent mean ± standard error of the mean (SEM). * * p < 0.01 compared to vehicle- treated animals ( n = 12 per group). Adapted from Honore et al. (2005) . pain model therefore appears to be a model in which joint pain can be elicited and reproducibly quantifi ed. The ability of TRPV1 antagonism to produce antinociception in chronic pain models was initially studied using capsazepine (Walker et al., 2003 ). However, the use of capsazepine to investigate the role of TRPV1 in pain transmission is problematic since it is only moderately potent at TRPV1 and is not pharmacologically selective (Wahl et al., 2001 ). More recently, several novel potent and selective TRPV1 antagonists have been described and have shown effi cacy in preclinical pain models (Szallasi et al., 2007 ). For example, A - 425619, which is a novel potent and selective antagonist at both human and rat TRPV1 receptors (Honore et al., 2005 ), has been shown to be effi cacious in rat models of chronic infl ammatory pain and postoperative pain. In addition, A- 425619, in a dose- dependent manner, normalized the weight bearing difference observed in osteoarthritic rats (Fig. 6.1 ). This was the fi rst demonstration of the antinociceptive effects of a selective TRPV1 antagonist in an osteoarthritic pain model. The role of TRPV1 in osteoarthritic pain has been confi rmed using other structurally different TRPV1 antagonists (Cui et al., 2006 ; Honore et al., 2009 ). These compounds have been shown to normalize both the difference in weight bearing and the decrease in grip force observed in the rat model of osteoarthritic pain induced by iodoacetate. 182 ROLE OF TRPV1 RECEPTORS IN OSTEOARTHRITIC PAIN

(a) (b) 60 70

50 60 50 40 ** 40 30 ** 30 20 20 ** 10 ** 10

Weight bearing difference (g) 0 Weight bearing difference (g) 0 V 3 10 30 V 30 100 300

A-784168 (μmol/kg, p.o.) A-795614 (μmol/kg, p.o.)

50 30 40

20 30

** 20 ** ** 10 ** 10

Weight bearing difference (g) 0 Weight bearing difference (g) 0 V 10 30 100 V 10 30 100 A-784168 (nmol, i.t.) A-795614 (nmol, i.t.) Figure 6.2 Antinociceptive effects of A - 784168 (TRPV1 antagonist with good CNS penetration) and A- 795614 (TRPV1 antagonist with poor CNS penetration) following oral and intrathecal (i.t.) administration in the iodoacetate- induced rat osteoarthritis pain model. Circles represent weight bearing difference between ipsilateral and contralateral paws in animals that received either A - 784168 or A - 795614. The square is the weight bearing difference in vehicle - treated animals. (a) A - 784168 p.o. dose

dependently reduced the weight bearing difference with an effective dose 50 (ED 50 ) of 8 μ mol/kg p.o. with 85.0 ± 4.7% efﬁ cacy at 30 μ mol/kg. (b) A - 795614, following p.o. dosing, also signiﬁ cantly reduced the weight bearing difference, but had a lower potency

(ED 50 of 280 μmol/kg) and limited efﬁ cacy (53.0 ± 10.2% effect at 300 μ mol/kg). (c,d) In contrast to oral administration, intrathecal A - 784168 and A - 795614 showed similar potency with ED50 values of 22 and 26 nmol, respectively. Data represent mean ± SEM . * * p < 0.01 compared to vehicle - treated animals (n = 6 per group). Adapted from Cui et al. (2006) . ROLE OF TRPV1 IN JOINT PAIN 183

6.3.5 Site of Action of TRPV 1 Antagonists in Attenuating Osteoarthritic Pain Since osteoarthritic pain is mediated by both peripheral and central mechanisms, TRPV1 blockade at either the periphery and/or the CNS could be involved in the antinociceptive effects of TRPV1 antagonists in osteoarthritic pain states. To understand the relative roles of peripheral versus central TRPV1 receptors in osteoarthritic pain, the effects of two distinct TRPV1 antagonists with similar in vitro potencies but with different degrees of CNS penetration were compared (Cui et al., 2006 ). Both A- 784168 (good CNS penetration) and A - 795614 (poor CNS penetration) prevented capsaicin - induced acute pain with the same potency, demonstrating that they both block peripheral TRPV1 receptors in vivo. In contrast, when tested in osteoarthritic animals, the compound with good CNS penetration, A- 784168, produced potent and full effects, while A - 795614, the compound with poor CNS penetration, produced only weak partial effects (Fig. 6.2a,b ). However, following intrathecal administration, both compounds produced full effects in the osteoarthritic pain model with similar potency (Fig. 6.2c,d ). These results demonstrate that both peripheral and central TRPV1 receptors play a role in osteoarthritic pain. Although blocking only peripheral TRPV1 receptors does produce some degree of pain relief, blocking both peripheral and central TRPV1 receptors provides greater pain relief at a lower plasma exposure.

6.3.6 Effects of Repeated Dosing of TRPV 1 Antagonists in Osteoarthritic Pain OA and associated pain are chronic conditions. The effects of sustained blockade of TRPV1 receptors by repeated dosing of TRPV1 antagonists have been investigated (Honore et al., 2009 ). The TRPV1 antagonist ABT - 102 fully reverses osteoarthritic pain following acute dosing (Fig. 6.3 ). However, following repeated dosing, ABT- 102 and the structurally distinct TRPV1 antagonist A - 993610 demonstrate increased effi cacy (Fig. 6.4a,b ). The enhanced analgesic activity following repeated dosing is not associated with accumulation of the compound in either the plasma or the brain. The increased analgesic activity of TRPV1 antagonists following repeated dosing is in contrast with the effects observed following repeated dosing with other analgesic compounds. For example, repeated dosing of COX- 2 inhibitor does not alter its potency in the rat osteoarthritic pain model (Fig. 6.5 ), whereas repeated dosing with morphine produces tolerance (Mao et al., 1995 ; Chandran et al., 2009 ). However, in a different report, it was shown that repeated dosing with sub - effi cacious doses of celecoxib resulted in signifi cant effi cacy in reversing OA- induced resting pain (Pomonis et al., 2005 ). These data suggest that different pharmacological effects can be observed in the same pain model depending on the behavioral end point assessed and on the dosing regimen. Although the mechanism for the increased analgesic activity of chronically 184 ROLE OF TRPV1 RECEPTORS IN OSTEOARTHRITIC PAIN

(a) (b)

50 1300 1200 ** 40 1100 1000 ** 30 900 800 ** 20 ** 700 600 10 500 *

** CFmax/kg body weight 400 300 Weight bearing difference (g) 0 V 10 30 100 V 3 10 30 100 ABT-102 (μmol/kg, p.o.) ABT-102 (μmol/kg, p.o.) Figure 6.3 Antinociceptive effects of ABT- 102 following acute dosing in the osteoarthritic pain model. ABT - 102 was dosed p.o. 1 h before behavioral testing. (a)

ABT- 102 dose dependently and fully reversed the difference in weight bearing (ED 50 = 30 μmol/kg, p.o.) induced by iodoacetate injection. (b) ABT- 102 also dose dependently and fully reversed the decrease in grip force (ED 50 of 10 μ mol/kg, p.o.), as measured by recording the compression force (CF) exerted on the hind limbs. Data represent mean ± SEM. * p < 0.05, * * p < 0.01 compared to vehicle - treated animals ( n = 6 – 18 per group). Adapted from Honore et al. (2009) . Used with permission from the International Association for the Study of Pain (IASP). administered TRPV1 antagonists is not known, several hypotheses can be proposed and/or can be ruled out. Chronic dosing with selective TRPV1 antagonists does not alter the expression of TRPV1 receptors in the rat spinal cord or in the DRG. In injured animals, chronic dosing of TRPV1 antagonists does not alter normal pain sensitivity since responses to acute thermal and mechanical stimulation on the side contralateral to the injury remain unal- tered. These results show that the increased effi cacy following repeated dosing with TRPV1 antagonists is not due to a general dampening of the pain pathways. It is possible that levels of neuropeptides (BK , CGRP, substance P) that are present in pain states and sensitize TRPV1 receptors may decrease following repeated dosing of TRPV1 antagonists. Indeed, the calcium infl ux that occurs following activation of TRPV1 receptors results in the release of these neuropeptides, and TRPV1 antagonists have been shown to decrease CGRP release in vitro (Rigoni et al., 2003 ). Therefore, inhibition of TRPV1 receptor activation may reduce the extracellular concentrations of these pronociceptive peptides over time and thereby may increase the effi cacy of chronically administered TRPV1 antagonists. An alternative hypothesis is based on the fact that hyperthermia is observed at analgesic doses of TRPV1 antagonists. An increase in core body temperature, even by one degree, could sensitize the TRPV1 receptor and increase the potency of endogenous TRPV1 agonists. This would result in decreased ability of TRPV1 antagonists to effectively block pain transmission during the period of hyperthermia and would increase the effi cacy of TRPV1 antagonists ROLE OF TRPV1 IN JOINT PAIN 185

(a) ## 1400 ** 1200 Naive ## 1000 **

800 **

600

CFmax/kg body weight 400

Chronic Acute Chronic Acute Chronic vehicle ABT-102 ABT-102 ABT-102 ABT-102 (3 μmol/kg) (3 μmol/kg) (10 μmol/kg) (10 μmol/kg)

(b) 1400

1200 Naive ## 1000 **

800

600

400 CFmax/kg body weight body CFmax/kg 200 Chronic Acute Chronic vehicle A-993610 A-993610 Figure 6.4 Enhanced antinociceptive effects of TRPV1 antagonists following repeated dosing in osteoarthritic pain. (a) The effects of ABT - 102 in osteoarthritic pain were signifi cantly increased following repeated dosing for 12 days. Rats received either vehicle (V), ABT - 102 (3 μ mol/kg, p.o.), or ABT - 102 (10 μ mol/kg, p.o.) twice daily for 11 days and were tested on day 12, 1 h after oral dosing with either vehicle or ABT - 102 (3 or 10 μ mol/kg). The analgesic effects of ABT - 102 were signifi cantly increased following repeated dosing, such that on day 12, a 3 μmol/kg dose produced 62 ± 7% effect compared to only a 5 ± 2% effect following a single dose. A similar increase in analgesic activity with multiple dosing was also found for the 10 μ mol/kg dose (98 ± 7% effect compared with a 47 ± 4% effect following a single dose). (b) Similar to ABT- 102, A - 993610, a structurally distinct TRPV1 antagonist, shows enhanced effi cacy in osteoarthritis pain following repeated dosing. The analgesic effects of A - 993610 were signifi cantly increased following repeated dosing such that a 12 μ mol/kg dose produced a 79 ± 4% effect compared to a 22 ± 2% effect following a single dose. Data represent mean ± SEM. * * p < 0.01 compared to vehicle - treated animals; ## p < 0.01 as compared to acute treatment with the same dose of compound (n = 8 per group). Adapted from Honore et al. (2009) . Used with permission from IASP. 186 ROLE OF TRPV1 RECEPTORS IN OSTEOARTHRITIC PAIN

Celecoxib 120 Chronic 100 Acute ** ++ 80

60 ++

40 ++ ** % Effects for GF (g) for % Effects 20

31030 100 Doses (μmol/kg) Figure 6.5 Antinociceptive effects of celecoxib following repeated dosing in osteoarthritic pain model. Celecoxib was dosed p.o. twice daily for 12 days, beginning 7 days after monoiodoacetate (MIA) injection. Repeated dosing with celecoxib yielded similar efﬁ cacy as a single dose administered on day 20. Data represented as percent effects on grip force (GF) (g) (mean ± SEM; n = 12 per dose group). Vehicle CFmax for this study was 130.83 ± 17.72 g. * * p < 0.01 versus vehicle- treated rats for acute celecoxib dose groups; ++ p < 0.01 versus vehicle- treated rats for chronic celecoxib dose groups. Adapted from Chandran et al. (2009) . when the hyperthermia is attenuated. However, the hyperthermia induced by TRPV1 antagonists is normalized after 2 days, whereas the analgesic effect continues to increase for at least 12 days. The large disconnect in the kinetics between normalization of hyperthermia and increase in analgesic activity does not support this hypothesis. The observation that repeated administration of ABT - 102 increases potency in pain models and end points in which a central site of action of TRPV1 antagonists has been demonstrated leads to yet another hypothesis; that is, the effectiveness of TRPV1 antagonists on central sensitization may increase with persistent TRPV1 receptor blockade.

6.4 CONCLUSIONS

OA is a complex multifactorial disease of unknown etiology. Due to signifi cant clinical prevalence, lack of therapy for disease modifi cation, and poor symptom (e.g., pain) management, OA represents an area of considerable, unmet medical need. Several preclinical animal models, including the iodoacetate knee joint OA model, have been developed for studying pain associated with OA. The iodoacetate model has been recently used to assess the effi cacy of antagonists REFERENCES 187 targeting the TRPV1 receptor. Potent and selective TRPV1 antagonists are effective in blocking osteoarthritic pain. Current data suggest that CNS penetration is needed for TRPV1 antagonists to be potent and fully effi cacious in this model, implicating a role for TRPV1 receptors in the CNS in osteoarthritic pain. Finally, TRPV1 antagonists become more potent in preclinical pain models following repeated dosing. These preclinical fi ndings represent an exciting prospect for the management of osteoarthritic pain in humans.

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mediated by vanilloid receptor - 1 (TRPV1) are blocked by the high affi nity antagonist, iodo - resiniferatoxin . Br J Pharmacol 138 : 977 – 985 . Roberts JC , Davis JB , and Benham CD ( 2004 ) [3H]Resiniferatoxin autoradiography in the CNS of wildtype and TRPV1 null mice defi nes TRPV1 (VR- 1) protein distribution . Brain Res 995 : 176 – 183 . Schaible HG ( 2004 ) Spinal mechanisms contributing to joint pain . Novartis Found Symp 260 : 4 – 22 ; discussion 22 – 7, 100 – 4, 277 – 9 . Schaible HG and Grubb BD ( 1993 ) Afferent and spinal mechanisms of joint pain . Pain 55 : 5 – 54 . Szallasi A , Cortright DN , Blum CA , and Eid SR ( 2007 ) The vanilloid receptor TRPV1: 10 years from channel cloning to antagonist proof - of - concept . Nat Rev Drug Discov 6 : 357 – 372 . Valtschanoff JG , Rustioni A , Guo A , and Hwang SJ ( 2001 ) Vanilloid receptor VR1 is both presynaptic and postsynaptic in the superfi cial laminae of the rat dorsal horn. J Comp Neurol 436 : 225 – 235 . Wahl P , Foged C , Tullin S , and Thomsen C ( 2001 ) Iodo - resiniferatoxin, a new potent vanilloid receptor antagonist. Mol Pharmacol 59 : 9 – 15 . Walker KM , Urban L , Medhurst SJ , Patel S , Panesar M , Fox AJ , and McIntyre P (2003 ) The VR1 antagonist capsazepine reverses mechanical hyperalgesia in models of infl ammatory and neuropathic pain . J Pharmacol Exp Ther 304 : 56 – 62 . Wieland HA , Michaelis M , Kirschbaum BJ , and Rudolphi KA ( 2005 ) Osteoarthritis — an untreatable disease? Nat Rev Drug Discov 4 : 331 – 344 . 7 TRPV 1 AND BONE CANCER PAIN

Juan Miguel Jimenez - Andrade and Patrick Mantyh

7.1 INTRODUCTION

More than 12 million people worldwide are currently diagnosed with cancer every year, and by 2020, it is estimated that 20 million new cases will be diagnosed each year (Stewart and Kleihues, 2003 ; Brennan et al., 2007 ). In 2007, cancer caused 7.9 million deaths worldwide (WHO, 2009 ). In the United States, cancer is a major health problem, being the second leading cause of death; 25% of U.S. deaths are currently cancer related (Jemal et al., 2007 ). Cancer - associated pain can be present at any time during the course of the disease, but the frequency and intensity of pain tends to increase with advanc- ing stages of cancer. In patients with advanced cancer, 62 – 86% experience pain, which is described as moderate to very severe (van den Beuken - van Everdingen et al., 2007 ). Of the pain experienced by cancer patients, pain induced by metastatic invasion of bone is one of the most difﬁ cult to treat since this pain is refractory to standard therapies, and it increases in intensity with the advancement of the disease (Mercadante and Fulfaro, 2007 ). Malignant bone tumors occur in patients with primary bone cancer, but more than 95% of bone cancers occur as a result of the metastasis of non - bone primary tumors, including prostate, breast, and lung, to the bone. Thus, bone is the most common site of chronic cancer pain (Coleman, 2006 ).

191 192 TRPV1 AND BONE CANCER PAIN

Currently, the factors that drive bone cancer pain are poorly understood. However, several recently introduced animal models of bone cancer pain have begun to provide insight into the cellular and molecular mechanisms that drive bone cancer pain (for review, see Mantyh [2006 ]). These models have contributed to the development of novel therapies and the initiation of clinical trials to treat the pain and skeletal remodeling that accompany metastatic bone cancer (Lipton et al., 2007, 2008 ; ClinicalTrials.gov, 2008 ).

7.2 THE CHALLENGE OF BONE CANCER PAIN

Although bone is not a vital organ, most common tumors have a strong pre- dilection for bone metastasis. Tumor metastases to the skeleton are major contributors to morbidity and mortality in metastatic cancer (Mercadante, 1997 ; Singh and Figg, 2005 ). Tumor growth in bone results in pain, hypercal- cemia, anemia, increased susceptibility to infection, skeletal fractures, compression of the spinal cord, spinal instability, and decreased mobility, all of which compromise the patient ’ s survival and quality of life (Coleman, 1997, 2006). Pain originating from skeletal metastases usually increases in intensity with the advancement of the disease and is commonly divided into three categories: ongoing pain, spontaneous breakthrough pain, and movement- evoked breakthrough pain (Portenoy and Hagen, 1990 ; Mercadante and Arcuri, 1998 ). Ongoing pain, which is the most frequent initial symptom of bone cancer, begins as a dull, constant, throbbing pain that increases in intensity with time (Mercadante, 1997 ; Portenoy and Lesage, 1999 ). As bone cancer progresses, intermittent episodes of extreme pain (breakthrough pain) can occur spontaneously, or more commonly, after weight bearing or movement of the affected limb (Mercadante, 1997 ; Portenoy and Lesage, 1999 ). Of these types of pain, breakthrough pain is the most diffi cult to control, since the dose of opioids required to fully control this pain is generally high and is accompanied by signifi cant unwanted side effects, such as sedation, somnolence, depression, cognitive impairment, respiratory depression, and constipation (Mercadante, 1997 ; Portenoy, 1999 ; Portenoy and Lesage, 1999 ; Mercadante and Fulfaro, 2007 ). Currently, the treatment of pain from bone metastases involves the use of multiple complementary approaches, including radiotherapy, surgery, chemotherapy, bisphosphonates, calcitonin, and analgesics (Mercadante, 1997 ; Mercadante and Fulfaro, 2007 ). However, bone cancer pain can be diffi cult to fully control (Mercadante, 1997 ) as the metastases are generally not limited to a single site and the analgesics that are most commonly used to treat bone cancer pain, non - steroidal anti - infl ammatory drugs (NSAIDs) (Mercadante, 1997 ) and opioids (Mercadante, 1997 ; Portenoy and Lesage, 1999 ; Cherny, 2000 ; Hanks et al., 2001 ), are limited by signifi cant adverse side effects (Foley, 1995 ; Weber and Huber, 1999 ; Mercadante and Fulfaro, 2007 ). SKELETAL REMODELING AND ACIDOSIS IN BONE CANCER PAIN 193

7.3 SKELETAL REMODELING AND ACIDOSIS IN BONE CANCER PAIN

Experiments in a murine model of bone cancer pain have demonstrated that osteoclasts play an essential role in cancer - induced bone loss and that osteoclasts can contribute to the etiology of bone cancer pain (Luger et al., 2001 ; Sabino et al., 2002 ). Osteoclasts are terminally differentiated, multinucleated, monocyte lineage cells that resorb bone by maintaining an extracellular microenvironment of acidic pH (4.0– 5.0) at the interface between osteoclasts and mineralized bone (Delaisse and Vaes, 1992 ; Boyle et al., 2003 ). This osteoclast - mediated bone remodeling results in robust production of extracellular protons (Teitelbaum, 2007 ), which are known to be potent activators of nociceptors (Julius and Basbaum, 2001 ), raising the possibility that the acidic microenvironment produced by osteoclasts contributes signiﬁ cantly to bone cancer - associated pain through activation of acid - sensitive nociceptors that innervate the marrow, the mineralized bone, and the periosteum (Ghilardi et al., 2005 ). Biphosphonates, a class of antiresorptive compounds that attenuate osteoclast function and induce osteoclast apoptosis, have been reported to reduce pain in patients with osteolytic skeletal metastases (Fulfaro et al., 1998 ; Major et al., 2000 ; Berenson et al., 2001 ). A study of the effects of the bisphosphonate alendronate in the sarcoma model of bone cancer showed a reduction in the number of osteoclasts and osteoclast activity (Sevcik et al., 2004 ), consistent with a reduction in tumor- induced bone resorption and reduction in the number of osteoclasts at the basal bone- resorbing surface (Horton et al., 2002 ). In this model, alendronate attenuated ongoing and movement- evoked bone cancer pain, decreased neurochemical reorganization of the peripheral and central nervous system, and also promoted tumor necrosis (Sevcik et al., 2004 ). These results suggest that biphosphonates can simultaneously modulate pain, bone destruction, tumor growth, and tumor necrosis in bone cancer. Osteoprotegerin (OPG) is a secreted soluble receptor that is a member of the tumor necrosis factor receptor (TNFR) family (Simonet et al., 1997 ). This decoy receptor and presumably the humanized monoclonal antibody AMG - 162 (also known as denosumab) prevents the activation and proliferation of osteoclasts by binding to and sequestering the OPG ligand (OPGL), which is also known as receptor for activator of NF κ B ligand (RANKL) (Anderson et al., 1997 ; Simonet et al., 1997 ; Yasuda et al., 1998 ; Rodan and Martin, 2000 ). OPG has been shown to decrease pain behaviors in the mouse sarcoma model of bone cancer pain (Luger et al., 2001 ). A substantial part of the analgesic actions of OPG seems to result from inhibition of osteoclast - induced acidosis and bone destruction. This reduction of osteoclast function in turn inhibits some of the neurochemical changes in the spinal cord (Luger et al., 2001 ), which are thought to be involved in the generation and maintenance of bone cancer pain. 194 TRPV1 AND BONE CANCER PAIN

The fi nding that sensory neurons can be directly excited by protons, originating from a variety of cell types including osteoclasts, has generated intense interest in the fi eld of pain research (Sutherland et al., 2000 ; Woolf et al., 2004 ). Subsets of sensory neurons express different acid- sensing ion channels (ASICs) (Olson et al., 1998 ; Julius and Basbaum, 2001 ). The two major ASICs expressed by nociceptors are the transient receptor potential vanilloid receptor 1 (TRPV1) (Caterina et al., 1997 ; Tominaga et al., 1998 ) and the acid - sensing ion channel- 3 (ASIC- 3) (Bassilana et al., 1997 ; Olson et al., 1998 ; Sutherland et al., 2000 ). Both of these channels are sensitized and excited by a decrease in pH. Tumor stroma (Griffi ths, 1991 ) and areas of ischemic necrosis (Deigner and Kinscherf, 1999 ), such as those observed in the 2472 sarcoma or in the ACE - 1 prostate bone cancer model, typically exhibit lower extracellular pH than surrounding normal tissues. As infl ammatory cells invade tumor stroma, they release protons that generate local acidosis (Julius and Basbaum, 2001 ). The extensive apoptosis of tumor cells, as well as the endothelial, immune, and infl ammatory cells that are present in the tumor environment, may also contribute to the acidic environment.

7.4 EXPRESSION OF TRPV 1 IN SENSORY NERVE FIBERS THAT INNERVATE THE FEMUR

TRPV1 is expressed in small- to medium- diameter dorsal root ganglion (DRG) neurons and in the peripheral terminals of small- diameter nerve fi bers (Caterina et al., 1997 ; Guo et al., 1999 ). A signifi cant population of the calcitonin gene- related protein (CGRP) immunoreactive nerve fi bers (primarily unmyelinated) that innervate the normal and tumor - bearing mouse bone express TRPV1 (Fig. 7.1a ). At the leading edge of the tumor, many sensory fi bers exhibit TRPV1 immunoreactivity (Fig. 7.1b ). As tumor growth progresses within the bone, the TRPV1- expressing sensory fi bers come in contact with the tumor and with osteoclasts lining the bone. Eventually, the distal ends of the nerve fi bers in contact with the tumor assume a fragmented appearance, which suggests injury and destruction of the very distal processes of these TRPV1 - expressing sensory fi bers (Ghilardi et al., 2005 ). Previous studies have shown that activating transcription factor (ATF)- 3, a member of the ATF/cAMP response element binding (CREB) family of transcription factors, is upregulated in the cell body of sensory and motor neurons following peripheral nerve injury and can serve as a histochemical marker of injury to sensory neurons (Tsujino et al., 2000 ). In the mouse model of bone cancer pain, ATF- 3 was shown to be expressed in the nucleus of sensory neurons in the ipsilateral DRGs (L1– L3) that innervate the tumor- bearing murine femur; the percentage of sensory neurons expressing ATF- 3 was directly correlated with tumor growth within the bone (Ghilardi et al., 2005 ). In contrast, there was no signifi cant increase in the expression of ATF - 3 in the contralateral DRGs or in motor neurons in the spinal cord of sarcoma- bearing (a) TRPV1

(b)

(c)

25 μm

Figure 7.1 A subpopulation of sensory nerve fi bers that innervate the bone expresses the TRPV1 channel. Confocal photomicrographs showing TRPV1 - positive nerve fi bers in the normal and tumor - bearing bone. In the marrow space of the normal bone, TRPV1 - expressing nerve fi bers are closely associated with blood vessels, similar to observations in other peripheral vascular beds (a). After tumor invasion of the marrow space, TRPV1 - positive sensory fi bers remain associated with blood vessels at the leading edge of the tumor, but these fi bers are not as ramifi ed as those found in the normal bone (b). As tumor growth continues, TRPV1- positive nerve fi bers can still be found deep within the tumor, but, in general, the fi bers begin to have a fragmented appearance, suggesting tumor- induced destruction of the distal processes of the sensory nerve fi bers (c). NM, normal marrow; T, tumor (Ghilardi et al., 2005 ; with permission). 196 TRPV1 AND BONE CANCER PAIN or sham/vehicle animals following surgery, indicating that the increased ATF- 3 expression was confi ned to sensory neurons innervating the tumor- bearing bone. Approximately 30 – 35% of the ATF - 3 - expressing neurons that presumably innervate the tumor- bearing bone also express TRPV1 (Fig. 7.2f ). These results suggest that a signifi cant percentage of sensory neurons that innervate the tumor - bearing bone express TRPV1 and that TRPV1 expression is maintained in the cell body and in the distal nerve fi bers, even as the distal processes of these sensory fi bers are activated and are injured by the invading tumor cells.

7.5 TRPV 1 ANTAGONIST OR DISRUPTION OF THE TRPV 1 GENE ATTENUATES BONE CANCER PAIN - RELATED BEHAVIORS

The mechanisms by which inhibition of osteoclast activity (either by bisphosphonates or OPGL/RANKL binding molecules) attenuates bone cancer pain may involve, at least in part, the reduction of osteoclast- induced acidosis. Tissue acidosis may activate nociceptors that innervate the bone through multiple mechanisms (Julius and Basbaum, 2001 ; Mantyh, 2006 ), but TRPV1 has been proposed to play a major role in acid- induced activation of nociceptors. Acid - evoked excitatory responses are greatly reduced in sensory neurons from TRPV1 - defi cient mice (Caterina et al., 2000 ; Davis et al., 2000 ), substan- tiating a role for this ion channel in the detection of extracellular protons in vivo . Recent pharmacological studies showed that selective TRPV1 antagonists signifi cantly decreased ongoing (JNJ- 17203212, ABT- 102, and SB366791) and ambulatory - evoked (JNJ - 17203212, ABT - 102 but not SB366791) pain - related behaviors at days 14 and 15 post- tumor injection in the mouse model of bone cancer pain, without any observable behavioral side effects, such as ataxia or hypoactivity (Ghilardi et al., 2005 ; Honore et al., 2009 ; Niiyama et al., 2009 ). Chronic administration of JNJ- 17203212 decreased ongoing and movement- evoked pain- related behaviors (Ghilardi et al., 2005 ), demonstrating that the analgesic effi cacy was retained at early, middle, and late stages of tumor growth (Fig. 7.3 ). In agreement with this fi nding, a recent study showed that the analgesic effi cacy of the TRPV1 antagonist ABT - 102 not only was retained following repeated administration, but also was signifi cantly increased compared to acute administration (Fig. 7.4 ). The ability of TRPV1 antagonists to maintain or to increase their analgesic effi cacy with disease progression was supported by the fact that sensory nerve fi bers innervating the tumor - bearing mouse femur maintained TRPV1 expression (Fig. 7.1 ), even as tumor growth and tumor - induced bone destruction progressed. These pharmacological results with TRPV1 antagonists suggest a signifi cant role for TRPV1 in bone cancer pain. To extend these fi ndings, studies were performed to determine whether mice lacking TRPV1 also showed a reduction in pain- related behaviors in this bone cancer model (Fig. 7.5 ). Whereas TRPV1 ANTAGONIST OR DISRUPTION OF THE TRPV1 GENE 197

(a) Contralateral (b) Ipsilateral

TRPV1 TRPV1 (c) (d)

ATF3 (e) (f)

TRPV1/ATF3 TRPV1/ATF3

Figure 7.2 Sensory nerve ﬁ bers that innervate the tumor- bearing mouse femur maintain expression of TRPV1 with disease progression. A population of small - to medium - sized neurons in the contralateral (a) and ipsilateral (b) L2 DRG expresses the TRPV1 channel (red). Note that 14 days after tumor injection, the percentage and size of sensory neurons expressing TRPV1 in ipsilateral L2 DRG (b) that innervate the tumor - bearing femur are the same as contralateral DRG (33.3 ± 1.7% contralateral vs. 32.1 ± 4.3% ipsilateral; n = 4). Fourteen days after tumor injection, when tumor cells have invaded the marrow space and mineralized bone, there is an upregulation of ATF- 3 (blue) in sensory neurons of the ipsilateral DRG (d) but not in the contralateral DRG (c). Double- label immunohistochemistry, merging the images obtained in (a) and (c) (e) or in (b) and (d) (f), suggests that a population of TRPV1- expressing sensory neurons innervates the tumor- bearing bone and exhibits an injured phenotype, as demonstrated by ATF - 3 coexpression. Scale bar: (in f) (a – f) 50 μ m (Ghilardi et al., 2005 ; with permission) . (See color insert.) 198 TRPV1 AND BONE CANCER PAIN

(a) (b) Ongoing Movement evoked

Spontaneous guarding Palpation/guarding 12 12 # 9 # 9

6 6 * * 3 3 a 2-min period (s) during a 2-min period (s) Palpation-induced guarding Palpation-induced Time spent guarding during spent guarding Time 0 0 Naive Sham/ Sarc/ Sarc/ Naive Sham/ Sarc/ Sarc/ veh veh TRPV1 veh veh TRPV1 Antagonist Antagonist

16 # 16 * 12 12 * 8 8

4 4

Number of spontaneous 0 0 Number of palpation-induced flinches during flinches a 2-min period Naive Sham/ Sarc/ Sarc/ during flinches a 2-min period Naive Sham/ Sarc/ Sarc/ veh veh TRPV1 veh veh TRPV1 Antagonist Antagonist

(e) (f) Spontaneous flinching Palpation/flinching # 18 # 24 21 15 # 18 # # # 12 15 * # * 9 * 12 # * * * 9 * 6 * 6 3 3 Number of spontaneous

0 Number of palpation-induced

flinches during flinches a 2-min period 0 flinches during flinches a 2-min period Day 9 Day 11 Day 15 Day 18 Day 9 Day 11 Day 15 Day 18

Sham/vehicle Sarcoma/vehicle Sarcoma/TRPV1 antagonist TRPV1 ANTAGONIST OR DISRUPTION OF THE TRPV1 GENE 199

Figure 7.3 Administration of the TRPV1 antagonist JNJ - 17203212 reduces bone cancer - induced pain - related behaviors and retains its analgesic effi cacy with disease progression. Tumor - induced ongoing pain - related behaviors were evaluated by measuring spontaneous guarding (a) and spontaneous fl inching (c ) over a 2 - min observation period. Movement - evoked pain was assessed by measuring the time spent guarding (b) and fl inching (d) over a 2- min observation period, after normally non- noxious palpation of the distal femur. Note that, in mice with bone cancer, there is a signifi cant increase in the duration and magnitude of guarding (a,b) and fl inching (c,d). Chronic treatment with the TRPV1 antagonist JNJ - 17203212 (30 mg/kg, subcutaneous ; twice daily), administered from 6 to 18 days after tumor injection, signifi cantly reduced parameters of both ongoing and movement - evoked pain - related behaviors compared to sarcoma (sarc)/vehicle (veh) animals (e,f). Note also that, at all time points examined (days 9– 18), JNJ- 17203212 (30 mg/kg, s.c.; twice daily) maintained signifi cant analgesic effi cacy with disease progression, during which the severity of pain- related behaviors increased (e,f). n > 8 for all experimental categories with the exception of (e) and (f), in which n = 4 for the sarcoma/TRPV1 antagonist group. Error bars represent standard error of the mean (SEM). # p < 0.05, sham/vehicle versus sarcoma/vehicle; * p < 0.05, sarcoma/TRPV1 antagonist versus sarcoma/vehicle (a – d, one - way ANOVA; e,f, one - way analysis of variance at each time point) (Ghilardi et al., 2005 ; with permission) .

(a) (b) (c) 100 100 100

75 75 75

** ** 50 50 ** 50

25 * 25 25 pain end points ** 0 0 0 % efficacy on bone cancer % efficacy ABT-102 ABT-102 ABT-102 ABT-102 ABT-102 ABT-102 10 acute 10 chronic 10 acute 10 chronic 10 acute 10 chronic Ongoing pain Spontaneous ambulation Palpation-evoked pain Figure 7.4 The antinociceptive effects of the TRPV1 antagonist ABT - 102 are signifi - cantly increased following repeated administration. Tumor- induced ongoing pain- related behaviors were evaluated by measuring spontaneous guarding (a) over a 2- min observation period. Normal limb use during spontaneous ambulation in an open fi eld was scored on a scale of 5– 0, where 5 is normal use and 0 is a complete lack of limb use (b). Movement- evoked pain- related behaviors were assessed by measuring the time spent guarding (c) over a 2- min observation period, after normally non- noxious palpation of the distal femur. At day 15, postinjection of the cancer cells, the effi cacy of ABT - 102 in the three behavioral end points following repeated administration (started on day 6) is signifi cantly greater than following acute administration. Data represent the mean ± SEM. * p < 0.05, * * p < 0.01 compared to respective vehicle- treated mice (Honore et al., 2009 ). Used with permission. 200 TRPV1 AND BONE CANCER PAIN

(a) Ongoing (b) Movement evoked

Spontaneous flinching Palpation/flinching 18 18 # 16 # 16 14 14 12 12 * # # 10 * # # 10 8 8 6 6 4 4 2 2 Number of spontaneous 0 0 Number of palpation-induced flinches during flinches a 2-min period flinches during flinches a 2-min period

Naive Sham Wild/sarc Wild/TRPV1 ko/sarc ko/TRPV1 antagonist antagonist Figure 7.5 Attenuation of bone cancer- induced pain- related behaviors and lack of additional analgesic effect of a TRPV1 antagonist (Antag) in TRPV1 knock out (ko) mice. Mice lacking a functional TRPV1 show a signiﬁ cant decrease in numbers of ongoing (a) and movement- evoked (b) pain- related behaviors compared to wildtype C3H/HeJ animals (behavioral testing 10– 14 days after tumor injection). Note that the reduction in pain- related behaviors in the TRPV1 null mice is approximately the same as that seen in the tumor - bearing wild - type C3H/HeJ animals treated with JNJ - 17203212 (Fig. 7.3 ) and that TRPV1 null mice treated acutely with 30 mg/kg, s.c. JNJ- 17203212 showed no additional reduction in pain - related behaviors, indicating that this compound is exerting its action by antagonizing the TRPV1 channel. Data for naive and sham experimental categories were obtained using TRPV1+/+ - bred mice. Data represent the mean ± SEM. # p < 0.05 versus sham/vehicle; * p < 0.05 versus sarcoma (sarc)/TRPV1+/+ (one - way ANOVA) (Ghilardi et al., 2005 ; with permission) .

the TRPV1+/+ and TRPV1+/ – mice (Ghilardi et al., 2005 ) exhibited the same pain - related behaviors, TRPV1 – / – mice showed a signifi cant reduction in both ongoing and movement - evoked nocifensive behaviors, similar to that observed in C3H/HeJ mice treated with a TRPV1 antagonist (Fig. 7.5 ). Moreover, administration of the TRPV1 antagonist JNJ - 17203212 to TRPV1 – / – mice caused no further reduction in bone cancer pain - related behaviors, demonstrating that the major target for the analgesic action of this TRPV1 antagonist in the bone cancer pain model was indeed the TRPV1 channel (Fig. 7.5 ). To determine whether TRPV1 antagonists were possibly having a direct effect on the tumor, TRPV1 expression was evaluated in sarcoma cancer cells. Using the same immunohistochemical protocols that showed TRPV1 immunoreactivity in the cell body and nerve terminals in sensory neurons, TRPV1 expression was not detected in the sarcoma cells (Ghilardi et al., 2005 ). Some analgesics can infl uence disease progression by indirect mechanisms. However, treatment with the TRPV1 antagonist JNJ - 17203212 did not signifi cantly affect tumor growth, as determined both by hematoxylin and eosin staining and by POTENTIAL ACTIVATORS OR MODULATORS OF TRPV1 IN BONE CANCER 201 radiographic analysis of total tumor burden within the intramedullary space of the sarcoma - bearing femur (Ghilardi et al., 2005 ).

7.6 TRPV 1 ANTAGONIST POTENTIATES THE ANALGESIC EFFICACY OF MORPHINE IN THE BONE CANCER PAIN MODEL

Humans suffering from bone cancer pain generally require high doses of morphine, which result in unwanted side effects, such as sedation, somnolence, depression, cognitive impairment, respiratory depression, and constipation (Baines and Kirkham, 1989 ; Portenoy and Lesage, 1999 ). In the mouse sarcoma model, the dose of morphine required to block bone cancer pain - related behaviors was 10 times higher than the dose required to block infl ammatory pain behaviors of comparable magnitude induced by hind paw injection of complete Freund ’ s adjuvant (Luger et al., 2002 ). The higher dose of morphine required in the bone cancer pain model was not due to morphine -induced tolerance, since responses to morphine were measured after a single dose in both models. These results suggest that bone cancer pain is more resistant than infl ammatory pain to morphine. The reduced analgesic effi cacy of morphine in bone cancer pain may be due, at least in part, to decreased expression of the μ - opioid receptor in the DRG from mice in this model (Yamamoto et al., 2008 ). Subanalgesic doses of either of two distinct TRPV1 antagonists (SB366791 or M68008) potentiated the analgesic effect of morphine in the bone cancer pain model (Niiyama et al., 2009 ), suggesting that this drug combination may represent a novel pharmacological option to treat severe bone cancer pain.

7.7 POTENTIAL ACTIVATORS OR MODULATORS OF TRPV 1 IN BONE CANCER

Protons from other cell types besides osteoclasts may also contribute to stimulation of TRPV1, resulting in bone cancer pain. Tumor cells themselves have a lower intracellular pH than normal cells. Furthermore, as a solid tumor outgrows its vascular supply, ischemic tumor tissue undergoes necrosis, which is associated with acidosis. In addition to agents that can directly activate TRPV1, other factors produced by tissue injury or by inﬂ ammation can modulate TRPV1 function indirectly, through activation of phospholipase C signaling pathways (Woolf and Salter, 2000 ; Julius and Basbaum, 2001 ), resulting in TRPV1 sensitization. Such agents include bradykinin, ATP, and nerve growth factor (NGF), all of which have been shown to be synthesized by and released from a variety of tumor cells, including the 2472 sarcoma cells used in these studies (Sevcik et al., 2005 ). Since bone has a rich sensory innervation by ﬁ bers that express TRPV1, production of these proalgesic agents may sensitize TRPV1 channels, thereby generating a state of hyperalgesia and/or allodynia in bone cancer pain. 202 TRPV1 AND BONE CANCER PAIN

7.8 SUMMARY

Administration of a TRPV1 antagonist or disruption of the TRPV1 gene results in signifi cant attenuation of both ongoing and movement- evoked pain- related behaviors in a murine model of bone cancer pain. The studies summarized in this review have shown that administration of a TRPV1 antagonist retains or increases its effi cacy at early, middle, and late stages of tumor growth. Additionally, factors released by the tumor, infl ammatory cells and immune cells, likely sensitize TRPV1, making it more responsive to the acidic environment provided by the tumor and osteoclasts. Taken together, these results suggest that the TRPV1 channel plays a signifi cant role in the integration of nociceptive signaling in bone cancer pain and that TRPV1 may be a novel target for pharmacological treatment of chronic bone cancer pain.

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Ant ó nio Avelino and Francisco Cruz

8.1 TRPV 1 EXPRESSION IN VISCERAL NEURONAL STRUCTURES

8.1.1 Urinary Tract After the initial studies of Maggi et al. (1989) showing that capsaicin caused a burning sensation and increased refl ex activity upon application in the human bladder, the visualization of the vanilloid receptor binding sites in the urinary tract was performed in the rat, using radioactive resiniferatoxin (RTX), in the urinary bladder (Szallasi et al., 1993 ; Á cs et al., 1994 ) and urethra (Parlani et al., 1993 ). After the cloning of transient receptor potential vanilloid type- 1 (TRPV1) (Caterina et al., 1997 ), immunohistochemical studies in rodents showed the presence of TRPV1 - immunoreactive (IR) nerve fi bers throughout the mucosa and the muscular layers of the entire urinary tract. (Tominaga et al., 1998 ; Birder et al., 2001 ; Avelino et al., 2002 ). TRPV1 - IR fi bers formed two distinct varicose plexuses in the bladder, renal pelvis, ureter, and proximal urethra. In the mucosa, most fi bers coursed closely to the basal cells of the transitional epithelium, sometimes penetrating it almost to the bladder lumen. In the muscular layer, TRPV1- IR fi bers impinged on the surface of the smooth muscle cells. Under the electron microscope (Fig. 8.1 ), TRPV1 fi bers were visible among urothelial cells and encroached in shallow grooves on the smooth muscle cell surface, separated by a narrow, empty cleft

206 TRPV1 EXPRESSION IN VISCERAL NEURONAL STRUCTURES 207

(a) (b)

Figure 8.1 Ultrastructural visualization of TRPV1 immunoreactivity in the bladder. (a) In the mucosa, most of the immunoreactive profi les are varicosities occurring in the lamina propria near the basement membrane (arrows). (b) Some immunoreactive profi les can also be seen between the epithelial cells, above the basement membrane (arrows). (c,d) Images of the muscular layer, showing immunoreactive varicosities apposing to the surface of smooth muscle cells. Small clear and large dense- core vesicles (arrows) can be seen in the immunoreactive profi les. Neither the epithelial cells (a,b) or the smooth muscle cells (c,d) show TRPV1 immunoreactivity. Scale bars = 0.5 μ m. Adapted from Avelino et al. (2002) .

(Avelino et al., 2002 ). Immunoreaction occurred in the cell membrane, synaptic vesicles, neurofi laments, and mitochondria of nerve fi bers but was absent from smooth muscle cells (Avelino et al., 2002 ). Curiously, no immunoreactivity was observed in the rat kidney parenchyma. In the human urinary bladder, TRPV1- IR was also detected in nerve fi bers coursing in the suburothelial connective tissue and in the muscular layer (Yiangou et al., 2001b ; Brady et al., 2004a ; Lazzeri et al., 2004 ; Apostolidis et al., 2005b ). As in rodents, TRPV1- expressing fi bers were found among human urothelial cells (Lazzeri et al., 2004 ). However, the density of labeled fi bers was much lower in humans than that observed in rodents. 208 TRPV1 IN VISCERAL PAIN AND OTHER VISCERAL DISORDERS

(a) (b)

Figure 8.2 Expression of TRPV1 immunoreactivity in the prostate. Thin varicose ﬁ bers can be seen in the lamina propria and penetrating the urothelium in longitudinal sections of the ventral prostatic urethra (a) or in the periurethral acini (b). Reproduced from Dinis et al. ( 2005 ).

In the human prostate (Fig. 8.2 ), TRPV1- IR nerve fi bers were more abundant in the verumontanum, ejaculatory ducts, and periurethral zone (Dinis et al., 2005 ). TRPV1 - IR nerve fi bers under the urethral epithelium formed a dense varicose network, with some fi bers penetrating the epithelial layer up to the urethral lumen (Dinis et al., 2005 ). TRPV1 immunoreactivity was not observed in peripheral zone of the gland (Dinis et al., 2005 ). In colocalization studies performed in rats (Fig. 8.3 ), the majority of the TRPV1 fi bers in the urinary tract coexpressed the neuropeptides substance P (SP) and calcitonin - gene related peptide (CGRP) (Avelino et al., 2002 ; Streng et al., 2008 ). Such colocalization studies with TRPV1- IR fi bers have not been reported for the human urinary tract. There is currently no defi nitive statement regarding TRPV1 expression in non - peptidergic fi bers. In contrast with other studies (Wang et al., 1998 ; Hwang et al., 2005 ), Avelino et al. (2002) did not detect TRPV1 immunoreactivity in IB4- binding primary afferents in the rat bladder. Since peptidergic and non- peptidergic IB4- binding primary afferents are generally considered to be distinct subsets of sensory neurons with diverse functional properties (Snider and McMahon, 1998 ; Stucky and Lewin, 1999 ; Liu et al., 2004 ), this issue still needs clarifi cation. In the rat bladder, TRPV1 also co - localizes with protease - activated receptors (PARs) (Dattilio and Vizzard, 2005 ) and with the TRPA1 channel (Streng et al., 2008 ), which is relevant to bladder refl ex control during infl ammation. PAR activation triggers rat urinary bladder contractions through the release of prostaglandins from the mucosa (Nakahara et al., 2004 ) and through sensitization of TRPV1 (Amadesi et al., 2004 ; Dai et al., 2004 ). TRPA1 is activated by agents induced by infl ammation (Anderson et al., 1997 ). TRPV1 and TRPV1 EXPRESSION IN VISCERAL NEURONAL STRUCTURES 209

(a) (c) (e)

TRPA1 TRPA1 TRPA1 (b) (d) (f)

TRPV1 CGRP SP Figure 8.3 Immunohistochemical localization of TRPA1 in the bladder wall. TRPA1 - IR nerve ﬁ bers co - localize with TRPV1 (a and b), CGRP (c and d), and substance P (e and f). Reproduced from Streng et al. (2008) . (See color insert.)

P2X3 receptors colocalize in dorsal root ganglia (DRG) cells of rodents (Guo et al., 1999 ). Their colocalization is also likely to occur in the urinary tract, since intravesical RTX, which abolishes TRPV1- IR, also abolishes P2X 3 immunoreactivity (Brady et al., 2004a,b ).

8.1.2 Gastrointestinal Tract ( GI ) TRPV1 has been detected by immunohistochemistry in the GI of mice, rats, guinea pigs, and humans (Ward et al., 2003 ; Faussone - Pellegrini et al., 2005 ). Immunoreactivity was defi nitively observed on nerves and interganglionic fi ber tracts throughout the stomach, small intestine, and colon of rodents (Ward et al., 2003 ). In these species, positive nerves were observed within both the mucosa and muscle layers (Ward et al., 2003 ). In the stomach of humans, TRPV1 was observed in nerve fi bers present in the mucosal and submucosal layers (Faussone- Pellegrini et al., 2005 ). TRPV1 was also detected in nerve fi bers coursing in the muscle, submucosal, and mucosal layers of the large intestine and rectum of humans (Yiangou et al., 2001a ; Chan et al., 2003 ; Akbar et al., 2008 ). TRPV1 expression was reported in nerves coursing in the pancreas both in exocrine and in endocrine tissue. Tract tracing studies in the rat showed that the TRPV1 positive fi bers were primary afferent fi bers (Fasanella et al., 2008 ). 210 TRPV1 IN VISCERAL PAIN AND OTHER VISCERAL DISORDERS

Co- localization studies revealed a high level of coexpression of TRPV1 with CGRP and GDNF family receptor alpha - 3 (GFR -α 3) (Gram et al., 2007 ; Fasanella et al., 2008 ). Concerning ontogeny, neurons immunoreactive for TRPV1 were observed primarily in the mouse DRG at embryonic day 13. TRPV1- IR nerve ﬁ bers were observed in most viscera and gradually increased postnatally at different rates. In the urinary tract and in the rectum, TRPV1 expression appears by the end of the embryonic period (E14 and E15, respectively). Many TRPV1 positive nerve ﬁ bers in these organs were also CGRP positive (Funakoshi et al., 2006 ).

8.2 TRPV 1 EXPRESSION IN NON - NEURONAL STRUCTURES

8.2.1 Urinary Tract The ﬁ rst description of TRPV1 expression in non - neuronal cells was in |urothelial cells of rodents (Birder et al., 2001 ). TRPV1 immunoreactivity was found in basal, intermediate, and large superﬁ cial umbrella cells (Fig. 8.4 ).

Figure 8.4 Confocal image of bladder urothelium in bladder whole mounts stained for TRPV1 and cytokeratin 17, a marker for basal urothelial cells. Diffuse cytoplasmic pattern of TRPV1 staining can be seen in the apical and underlying urothelial layers (nuclei are unstained). Arrows indicate apical cells within the ﬁ eld from a single plane of focus. Adapted from Birder et al. (2001) . (Copyright National Academy of Sciences U.S.A., 2009). (See color insert.) TRPV1 EXPRESSION IN NON-NEURONAL STRUCTURES 211

Like in nerve fi bers, the receptor was shown to be functional, since capsaicin increased cytosolic calcium and nitric oxide release in urothelial cells from wildtype (WT) mice but not from TRPV1 knockout (KO) animals (Birder et al., 2001 ). However, in contrast with neuronal cells, TRPV1 desensitization could not be induced in rodent urothelial cells by capsaicin or RTX application (Birder et al., 2001 ). No explanation for such difference has been previously proposed, but it can be speculated that differences in the intracellular pathways involved in desensitization in the two cell types may account for the difference. TRPV1 is also present in human urothelial cells. Reverse transcriptase- polymerase chain reaction (RT - PCR) studies showed TRPV1 mRNA expression in human urothelial cells, either freshly isolated or grown in culture (Kim et al., 2001 ; Charrua et al., 2006 ; Cruz et al., 2007 ). In contrast to dorsal root ganglion cells (Winter et al., 1988 ; Bevan and Winter, 1995 ), TRPV1 mRNA expression in urothelial cells seemed to be independent of the presence of nerve growth factor (NGF) during cell culture. Interestingly, TRPV1 mRNA expression more than tripled when the human urothelial cells were grown in the presence of infl ammatory mediators such as bradykinin, histamine, prostaglandins, and serotonin (Charrua et al., 2006 ; Cruz et al., 2007 ). The TRPV1 receptor in human urothelial cells appeared to be functional (Fig. 8.5 ), since the cobalt uptake (a surrogate indicator of calcium entry) induced by capsaicin, heat (above 43 ° C), and low pH was inhibited by capsazepine (Cruz et al., 2007 ). Furthermore, capsaicin evoked ATP release from these cells that could be inhibited by capsazepine (Charrua et al., unpublished data). Interestingly, TRPV1 immunoreactivity in human urothelial cells has not been universally observed. The TRPV1 staining in human urothelial cells reported by Lazzeri et al. (2004) and Apostolidis et al. (2005a,b) could not be reproduced by other groups using different antibodies that did produce excellent neuronal staining (Yiangou et al., 2001b ; Brady et al., 2004a ; Dinis et al., 2005 ). The reason for such differences is unknown. Differences in the sensitivity of the antibodies or in the type of fi xation methods may explain these confl icting results. TRPV1 immunoreactivity has been reported in interstitial cells of the human bladder (Ost et al., 2002 ) and prostate (Van der Aa et al., 2003 ). These cells, fi rst identifi ed by Ramon y Cajal (1909) in the gut, form a suburothelial network that may contribute to a fast spread of neuronal- induced smooth muscle contractions (Sui et al., 2004 ). The functional role of TRPV1 in interstitial cells, like the cells themselves, is yet currently unclear. Recently, functional TRPV1 receptors were reported in human epithelial prostate cells. In these cells, capsaicin and RTX were shown to induce a concentration - dependent calcium infl ux that was reversed by capsazepine (Sanchez et al., 2005 ). However, since this publication, no further studies have been reported using human epithelial prostate cells. Several studies have described TRPV1 immunoreactivity in smooth muscle cells and in the endothelium of capillaries and arteries from the human lower urinary tract (Ost et al., 2002 ; Van der Aa et al., 2003 ; Lazzeri et al., 2004 ). However, these data are diffi cult to reconcile with other studies that did not 212 TRPV1 IN VISCERAL PAIN AND OTHER VISCERAL DISORDERS

200 pA (a) 10 s 32 *** 30 28 *** 26 24 22 20 18 Signal intensity 16 14 12 Control CAP CAP CAP CAP 100 nM 100 nM 1 uM 1 uM + CPZ + CPZ

(b) 10°C 32 30

28 200 pA 26 5 s 24 *** 22 *** 20 18

Signal intensity 16 14 12 37°C39°C 41°C 45°C45°C + CPZ

Signal intensity 16 2 14 protein fmol ATP/mg 1 12 0 pH 7 pH 5.4 pH5.4 Control CAP CAP + CPZ + CPZ Figure 8.5 Cobalt uptake induced in human urothelial cells by capsaicin (a), heat (b), or low pH (c). In (d), ATP release induced by capsaicin. Effects are blocked by capsazepine (CPZ). (Charrua et al., unpublished). detect TRPV1 immunoreactivity in these tissues (Yiangou et al., 2001b ; Brady et al., 2004a ; Dinis et al., 2005 ). Even if further studies show speciﬁ c TRPV1 immunolabeling of smooth muscle and endothelial cells, the functional meaning of TRPV1 in these cells remains unclear. TRPV1 AND NORMAL VISCERAL FUNCTION 213

Mast cells present in the human bladder have also been reported to be TRPV1 - IR in one study (Lazzeri et al., 2004 ). In mast cells obtained from bone marrow, calcium uptake was shown to occur after capsaicin and RTX stimulation (Biro et al., 1998 ). Although it is tempting to relate these observations to the inﬂ ammatory response, capsaicin or RTX did not induce degranu- lation of mast cells (Biro et al., 1998 ). TRPV1 mRNA was also found in the genital tract (Stein et al., 2004 ). Human positive structures included the testis, the seminiferous tubules, the corpus cavernosum, the glans penis and its overlying skin, and the scrotal skin. However, the mRNA was extracted from whole tissue homogenates, precluding precise identiﬁ cation of the structures expressing the receptor (Stein et al., 2004 ).

8.2.2 GI TRPV1 expression was detected in non - neuronal cells of the GI using immunohistochemistry and molecular biology methods. Positive structures included parietal cells of the human stomach (Faussone- Pellegrini et al., 2005 ), epithelial cells in the rat stomach (Kato et al., 2003 ), and unidentiﬁ ed cells in the intestinal villi of different rodents (Ward et al., 2003 ). The functional meaning of these ﬁ ndings is still unclear.

8.3 TRPV 1 AND NORMAL VISCERAL FUNCTION

Deletion of the TRPV1 gene did not induce any other changes in the neurochemical phenotype of nociceptive peripheral sensory neurons (Baiou et al., 2007 ). Expression of genes for NGF, trk A, eNOS, COX- 2, P2X3, BK1, and BK2 was similar in WT and TRPV1 KO mice (Wang et al., 2008 ). Functional differences exist between bladders of WT and TRPV1 KO mice. Stretch - and hypo - osmolality - evoked ATP release is diminished from TRPV1 KO urothelial cells (Birder et al., 2002 ). Birder et al. (2002) concluded that elimination of TRPV1 moderately increased both the frequency of non- void- ing bladder contractions detected during fi lling cystometry and bladder capacity (Birder et al., 2002 ). On the other hand, Charrua and co - workers reported that the frequency and amplitude of expulsive refl ex bladder contractions of WT and TRPV1 KO mice were the same (Charrua et al., 2007 ). In another, more recent report, TRPV1 KO mice had more spontaneous micturitions that WT littermates, in agreement with the higher frequency of non- voiding contractions, but their bladder capacity was identical to WT (Wang et al., 2008 ). Blocking TRPV1 receptor activation with specifi c antagonists has been reported to alter bladder function in some, but not all, studies. Capsazepine, the fi rst TRPV1 antagonist synthesized, had no effect on normal bladder refl ex activity even at very high concentrations (Dinis et al., 2004b ). GRC 6211, an orally active TRPV1 antagonist, also did not change refl ex activity of rat and 214 TRPV1 IN VISCERAL PAIN AND OTHER VISCERAL DISORDERS mice bladders when administered in low concentrations. However, at high concentrations, bladder contractions were transiently suppressed in naï ve rats and WT mice. This effect was, at least in part, mediated by the blockade of TRPV1, since the same high doses of GRC 6211 did not paralyze the bladder of TRPV1 KO mice (Charrua et al., 2009 ). The involvement of TRPV1 in the normal digestive tract has also been investigated. In noninfl ammatory conditions, TRPV1 KO mice were signifi - cantly less sensitive to distension than WT mice (Rong et al., 2004 ; Jones et al., 2005 ). Jejunal distention evoked less afferent response in TRPV1 KO mice than in WT mice (Rong et al., 2004 ). Although phasic colon distension did produce graded behavioral responses in both WT and TRPV1 KO mice, the sensitivity to distension in TRPV1 KO mice was only half that observed in WT littermates (Jones et al., 2005 ). The afferent signaling (Rong et al., 2004 ) and behavioral defi cits observed in KO mice (Jones et al., 2005 ) were mimicked by TRPV1 blockade with capsazepine (Rong et al., 2004 ; Jones et al., 2005, 2007). On the other hand, the visceromotor response to colorectal distension was similar in rats treated with a vehicle solution or with the TRPV1 antagonist JYL1421 (Miranda et al., 2007 ). Thus, although the majority of the available data tends to suggest that TRPV1 in the colon has mechanosensitive properties, this requires further confi rmation. The ability of a TRPV1 antagonist to paralyze the bladder refl ex activity suggests that TRPV1 provides mechanosensitivity. The same conclusion can be inferred from the reduction in afferent fi ber sensitivity to circumferential stretch of the colon observed in TRPV1 KO mice (Jones et al., 2005 ). The mechanism responsible for these effects is still unclear, but heterodimerization of TRPV1 is an appealing hypothesis. For example, co- assembly between subunits belonging to different members of the TRP family (Hellwig et al., 2005 ; Liapi and Wood, 2005 ; Rutter et al., 2005 ), such as TRPV4, which has been shown to exhibit mechanosensitive properties (Suzuki et al., 2003 ; Liedtke, 2005 ), is a possible explanation to this fi nding. However, it cannot be discounted that TRPV1 itself is mechanosensitive. TRPV1 expressed in sensory nerves coursing the renal pelvis may be involved in sodium and fl uid homeostasis (Zhu et al., 2005 ; Feng et al., 2008 ). In rats, capsaicin perfusion in one renal pelvis increased urine fl ow and urine sodium excretion in both kidneys. These changes were abolished by capsazepine or prior ipsilateral kidney denervation (Zhu et al., 2005 ). Capsazepine prevented intrapelvic pressure - dependent afferent renal nerve activation and contralateral diuresis/natriuresis at low (20 mm Hg) but not high (50 mm Hg) intrapelvic pressure. These results suggest that TRPV1 in the renal pelvis may function as a low - pressure baroreceptor and regulate neuropeptide release from primary renal afferent C- fi bers in response to mechanostimulation (Feng et al., 2008 ). TRPV1 immunoreactivity has been shown in transitional cell bladder tumors. Intense labeling was observed in low- grade, low- stage tumors whereas a very faint or absent labeling was found in high - grade, high - stage ones TRPV1 IN VISCERAL DYSFUNCTION 215

(Lazzeri et al., 2005 ). In addition, TRPV1 was reported to be expressed in the human cancer androgen - resistant cell line PC - 3 (Sanchez et al., 2005 ). Capsaicin induced apoptosis in this cell line by a mechanism involving oxidative stress, mitochondrial changes, and activation of caspase 3 (Sanchez et al., 2006 ). However, apoptosis was not prevented by capsazepine, indicating that this effect was not mediated by TRPV1 (Sanchez et al., 2006 ).

8.4 TRPV 1 IN VISCERAL DYSFUNCTION

Several studies have shown that TRPV1 expressed in bladder structures is associated with the generation of noxious bladder sensory input and bladder hyperactivity during cystitis. Following induction of bladder infl ammation with acetic acid or Escherichia coli lipopolysaccharides (LPS), bladder distension markedly increased the expression of the pain- evoked c - fos gene in sacral spinal cord neurons of WT but not TRPV1 KO mice (Charrua et al., 2007 ). In addition, the frequency of bladder refl ex contractions strongly increased in WT but not in TRPV1 KO infl amed mice (Fig. 8.6 ) (Charrua et al., 2007 ). These observations were recently confi rmed by other investigators using the acrolein (cyclophosphamide metabolite) model of bladder infl ammation. Likewise, it was found that cystitis induced bladder mechanical hyperactivity in WT but not in TRPV1 KO mice (Wang et al., 2008 ). TRPV1 antagonists administered to rats with bladder infl ammation confi rmed and expanded the observations obtained in TRPV1 KO mice. Capsazepine decreased the frequency of refl ex contractions in cyclophosphamide - infl amed rat urinary bladders (Dinis et al., 2004b ). GRC- 6211, administered preemptively, decreased the frequency of refl ex bladder contractions during acetic acid infusion in a dose - dependent manner and prevented spinal expression of c - fos (Charrua et al., 2009 ). In addition, in rats with established LPS- induced cystitis, GRC- 6211 completely reversed bladder hyperactivity (Fig. 8.7 ) (Charrua et al., 2009 ).

50 1.40

40 *** 1.20 ***

30 1.00

20 0.80

10 0.60 Fos-IR cells/section Fos-IR o N

0 Bladder contractions/min 0.40 +/+ +/+ LPS –/– –/– LPS +/+ +/+ LPS –/– –/– LPS Figure 8.6 c - fos expression (left) or bladder reﬂ ex activity (right) in TRPV1 KO and WT mice with or without bladder inﬂ ammation. Reproduced from Charrua et al. (2007) . 216 TRPV1 IN VISCERAL PAIN AND OTHER VISCERAL DISORDERS

100 100 Vehicle GRC 6211 sulfate 0.001 mg/kg + acetic acid 0.5% + acetic acid 0.5%

45 45

0 0 0′ 2′30″ 5′ 7′30″ 10 0′ 2′30″ 5′ 7′30″ 10 100 100 GRC 6211 sulfate 0.01 mg/kg GRC 6211 sulfate 0.1 mg/kg + acetic acid 0.5% + acetic acid 0.5%

45 45

0 0 0′ 2′30″ 5′ 7′30″ 10 0′ 2′30″ 5′ 7′30″ 10

2.00

1. 8 0 GRC 6211 sulfate + acetic acid 0.5% infusion 1. 6 0

1. 4 0

1. 2 0 * 1. 0 0 *

Bladder contractions/min 0.80

0.60 Vehicle 0.001 mg/kg 0.01 mg/kg 0.1 mg/kg Figure 8.7 Effect of the TRPV1 antagonist GRC - 6211 in the refl ex activity of acetic acid - infl amed rat bladders. The mean number of refl ex bladder contractions/min of each group is shown in the bar graph. Adapted from Charrua et al. (2009) .

It should be stressed that the involvement of capsaicin- sensitive bladder structures with pain and micturition control had been suggested long before TRPV1 cloning and TRPV1 antagonist synthesis. Intravesical instillation of capsaicin in humans (Maggi et al., 1989 ) produced a warm to burning sensation referred to the suprapubic area and to the urethra. Capsaicin also decreased the bladder volume that triggered voiding in those individuals. In rats, capsaicin- sensitive bladder structures contributed to c - fos activation in the sacral spinal cord (Cruz et al., 1996 ; Vizzard, 2000a ; Dinis et al., 2004a ) and to bladder overactivity accompanying cystitis (Dinis et al., 2004a ; Sculptoreanu et al., 2005a ). TRPV1 IN VISCERAL DYSFUNCTION 217

All these data are in agreement with the role for TRPV1 in infl ammation and may be related to the high levels of neurotrophic factors produced by the chronically infl amed bladder (Vizzard, 2000b ; Bjorling et al., 2001 ; Guerios et al., 2006 ) or bowel (Di Mola et al., 2000 ). NGF enhances TRPV1 translation (Ji et al., 2002 ) and releases TRPV1 activity from the inhibitory control of phosphatidylinositol - 4,5 - bisphosphate (Chuang et al., 2001 ). In addition, activation of protein kinase A (PKA) (De Petrocellis et al., 2001 ), protein kinase C (PKC) (Cesare et al., 1999 ; Premkumar and Ahern, 2000 ), and Ca2+/ calmodulin -dependent kinase II (CaMkII) (Jung et al., 2004 ) by infl ammatory mediators may increase TRPV1 activity by phosphorylation. It should also be mentioned that the increase of PARs 2- 4 in TRPV1- expressing fi bers and urothelial cells of infl amed bladders (Dattilio and Vizzard, 2005 ) may contribute to sensitization of TRPV1 through PKC- mediated phosphorylation (Amadesi et al., 2004 ; Dai et al., 2004 ). Also, the sustained desensitization of primary afferent neurons after capsaicin application in cats suffering from feline interstitial cystitis (IC) has been shown to be due to enhanced activity/ expression of PKC (Sculptoreanu et al., 2005b ). TRPV1 β /TRPV1b, which is the most recently identifi ed splice variant of TRPV1 (Wang et al., 2004 ; Vos et al., 2006 ), has been shown to have a dominant- negative effect on the responsiveness of the TRPV1 channel. Interestingly, cyclophosphamide - evoked cystitis in rats is associated with altered TRPV1/ TRPV1b expression in the L5- L6 DRG, which innervate the urinary bladder. While TRPV1 expression was unchanged, TRPV1b expression was signifi - cantly reduced in L5- L6 DRGs during cystitis. These data suggest that infl ammation may also increase responsiveness of the TRPV1 channel by reducing the expression of a nonactive splice variant (Charrua et al., 2008 ). Capsaicin - sensitive bladder afferents have also been shown to trigger a spinal micturition refl ex under other abnormal conditions, including spinal cord transection (Fowler et al., 1992 ; De Groat, 1997 ), chronic bladder outlet obstruction (Chai et al., 1998 ), and idiopathic bladder overactivity (Silva et al., 2002 ). In many of these conditions, the enhancement of the micturition refl ex was accompanied by an increase in the number of bladder nerve fi bers expressing TRPV1 (Brady et al., 2004b ; Liu et al., 2007 ). The role of TRPV1 in the enhancement of the spinal micturition refl ex after chronic spinalization is now fully supported by the effect of specifi c TRPV1 antagonists. The blockade of TRPV1 by the competitive antagonist GRC - 6211 decreased in a dose - dependent manner the frequency of detrusor contractions seen during cystometry in rats with bladder overactivity caused by chronic spinalization (Silva et al., unpublished data). TRPV1 may also be involved in pain generated during acute renal obstruction. In rats, acute ureteral occlusion induced strong c - fos activation at spinal segments T10 - L4, with the peak at L1 - L2. The activated neurons were concentrated in laminae I, lateral IV - V, medial VII and X. Systemic administration of capsaicin, in doses that are known to destroy capsaicin - sensitive sensory fi bers, completely prevented c - fos expression during ureteral occlusion (Avelino et al., 1997 ). 218 TRPV1 IN VISCERAL PAIN AND OTHER VISCERAL DISORDERS

As to the role of TRPV1 in the intestine, it seems reasonable to state that TRPV1 contributes to nociceptive behavior and possibly peripheral sensitization caused by infl ammation. Colonic infl ammation enhances stretch- evoked afferent fi ber responses in WT mice, but not in TRPV1 KO mice (Jones et al., 2005 ). The TRPV1 receptor may also be crucial, along with acid- sensing ion channel (ASIC) receptors, for the long- term sensitization that persists after resolution of the colonic infl ammation. Zymosan - induced infl ammation of the colon produced visceromotor responses to colorectal distention not only acutely but also for a long time period ( > 7 weeks) after the disappearance of infl ammation in WT mice (Jones et al., 2005 ). Such behavioral hypersensitivity was shown to be at least partially dependent on TRPV1, since the effect was reduced in TRPV1 KO mice (Jones et al., 2005 ). The same fi ndings were observed after TRPV1 blockade with specifi c antagonists. Preemptive administration of the TRPV1 antagonist JYL1421 decreased the visceromotor response to colorectal distention in rats with chemically (trinitrobenzenesul- fonic acid) induced colitis. When administered after development of the colitis, JYL1421 also improved microscopic colitis and signifi cantly decreased the visceromotor response to colorectal distention (Miranda et al., 2007 ). All the above data must, however, be weighed against other observations that point in the opposite direction. For example, TRPV1 stimulation by low concentrations of TRPV1 ligands, such as capsaicin, acids, and alcohol, increased the resistance of the gastric mucosa to chemical injury (Holzer et al., 1990 ; Yamamoto et al., 2001 ). Likewise, selective elimination of capsaicin- sensitive nerve fi bers aggravated chemically induced damage of the gastric mucosa (Szolcs á nyi and M ó zsik, 1984 ). More recently, intrarectal infusion of dinitrobenzene sulfonic acid induced a more intense colonic infl ammation in TRPV1 KO mice than in WT littermates, as shown by macroscopic infl ammatory scoring and myeloperoxidase assays (Massa et al., 2006 ). These fi ndings suggest that TRPV1 receptors may regulate a protective response in the GI during infl ammation. If they are confi rmed in further studies, they may indicate that TRPV1 blockade may increase the deleterious effect brought about by infl ammation to tissues (digestive tract only?), despite the capacity of inducing effective analgesia. TRPV1 has been shown to play a role in infl ammation - evoked pain in experimental models of pancreatitis (Wick et al., 2006 ; Xu et al., 2007 ). L- arginine - induced pancreatitis increased both expression of the pain- evoked gene c - fos in the spinal dorsal horn and nocifensive behavior in rats. Both of these responses could be prevented by treatment with capsazepine (Wick et al., 2006 ). Furthermore, capsaicin- induced currents were upregulated, as well as TRPV1 mRNA and protein, in primary afferent perycaria innervating trinitrobenzene - infl amed pancreas (Xu et al., 2007 ). In accordance, the TRPV1 antagonist SB - 366791 reduced both visceral pain behavior and referred somatic hyperalgesia in rats with pancreatitis but not in control animals (Xu et al., 2007 ). Besides pain perception, sensory TRPV1 also may be involved in the control of pancreatic infl ammation (Noble et al., 2006 ; Romac et al., 2008 ). In rats with cerulein - induced pancreatitis, surgical ablation of the celiac ganglion ENDOGENOUS AGONISTS OF TRPV1 IN CYSTITIS AND COLITIS 219 or treatment with RTX, at doses that destroy capsaicin - sensitive nerve fi bers, inhibited SP release and reduced infl ammation (Noble et al., 2006 ). A very exciting fi nding is the observation that TRPV1 - expressing pancreatic afferents may have a role in regulating insulin secretion from pancreatic islet beta cells (Razavi et al., 2006 ; Gram et al., 2007 ). TRPV1 KO mice showed enhanced insulin sensitivity when compared with WT littermates (Razavi et al., 2006 ). In fact, destruction of TRPV1 - expressing nerve fi bers by systemic capsaicin was shown to increase insulin release and prevent hyperglycemia in rats, suggesting that TRPV1 - expressing fi bers have an inhibitory effect on insulin release (Gram et al., 2007 ).

8.5 ENDOGENOUS AGONISTS OF TRPV1 IN CYSTITIS AND COLITIS

Endogenous TRPV1 agonists include protons (Tominaga et al., 1998 ), N - arachidonoyl - ethanolamine (anandamide) (Zygmunt et al., 1999 ), N - arachidonoyl - dopamine (Huang et al., 2002 ), N - oleoyl - dopamine, (Chu et al., 2003 ), and lipoxygenase products, including eicosanoid acids and leukotrienes (Hwang et al., 2000 ). Bradykinin does not bind to TRPV1 but may activate this receptor indirectly through a bradykinin B2 receptor- mediated mechanism (Reeh and Petho, 2000 ). Other agents like NGF, prostaglandins, oestro- gens, glutamate, and ATP may contribute to TRPV1 activation by inducing posttranslational changes in the receptor (Nagy et al., 2004 ). So far, in the bladder, only anandamide has been thoroughly studied as a TRPV1 endogenous agonist (Dinis et al., 2004b ). Cyclophosphamide- induced cystitis increased the concentration of anandamide in the rat bladder (Fig. 8.8 ). Moreover, exogenous application of anandamide or blockade of endogenous anandamide degradation in naive bladders increased both pain- evoked gene expression in the spinal cord and the frequency of bladder refl ex contractions. Both effects were TRPV1- mediated since they could be prevented by capsazepine (Dinis et al., 2004b ). Interestingly, repeated anandamide applications did not produce TRPV1 desensitization (Dinis et al., 2004b ), which might have provided additional evidence for a fundamental role of TRPV1 activation by anandamide in infl ammatory conditions. The levels of anandamide also have been shown to be elevated in some infl ammatory intestinal conditions. Intraluminal administration of Clostridium diffi cile toxin A increased the concentrations of anandamide in the rat ileum, and intraluminal administration of anandamide caused ileum infl ammation similar to that caused by C. diffi cile toxin A. Interestingly, the effects of anandamide in the rat ileum were signifi cantly inhibited by pretreatment with capsazepine (McVey et al., 2003 ). The concentration of anandamide was shown to be signifi cantly elevated in the duodenal mucosa of active celiac patients and returned to normal after remission of the disease with a gluten- free diet (D ’ Argenio et al., 2007 ). Endocannabinoid levels also increased in 220 TRPV1 IN VISCERAL PAIN AND OTHER VISCERAL DISORDERS

60 * tissue) 50 * dry *

40 (pmol/g Anandamide content

Control 42472 192 Hours Figure 8.8 Average anandamide content of control and inﬂ amed bladders at different time points after intraperitoneal cyclophosphamide injection. Reproduced from Dinis et al. (2004b) .

the jejunum of rats treated with methotrexate, which is a model that reproduces the infl ammatory features of celiac patients. Anandamide returned to basal levels at remission of the infl ammation (D’ Argenio et al., 2007 ). Thus, it is possible that, like in the bladder, anandamide may contribute to intestinal infl ammation through a TRPV1 - dependent mechanism. Recent studies in vitro have further implicated anandamide in TRPV1 activation under infl ammatory conditions. In fact, the infl ammatory mediators bradykinin and prostaglandin E2 increased the excitatory potency of anandamide in nociceptive, capsaicin- sensitive, primary afferent neurons (Singh Tahim et al., 2005 ).

8.6 INCREASED TRPV 1 EXPRESSION IN HUMAN DISEASES

There is now robust evidence that TRPV1 expression is increased in human viscera during several pathological conditions that occur in the bladder and in the digestive tract. In addition, vulvodynia is also associated with increased TRPV1 expression. In the bladder, TRPV1 expression is increased in IC, also known as painful bladder syndrome (PBS) and in patients with involuntary contractions of the bladder (a condition called detrusor overactivity), whether due to a neurogenic (neurogenic detrusor overactivity [NDO]) or an idiopathic (idiopathic detrusor overactivity [IDO]) origin. INCREASED TRPV1 EXPRESSION IN HUMAN DISEASES 221

(a) (b)

Figure 8.9 TRPV1 immunoreactive ﬁ bers in suburothelium of control (a, arrows) and PBS (b) bladders. Reproduced from Mukerji et al. (2006) . (See color insert.)

IC is a chronic debilitating heterogeneous syndrome, characterized by suprapubic pain related to bladder fi lling and accompanied by additional symptoms, such as increased daytime and nighttime urinary frequency without proven urinary infection or another obvious pathological condition. Currently, no etiology or pathophysiology is known for IC. A marked increase in suburothelial nerve fi bers expressing TRPV1 was observed under the urothelium of IC patients compared with control individuals. Interestingly, the visual analog pain score correlated signifi cantly with the relative density of nerve fi bers expressing TRPV1, but not with the density of nerve fi bers not expressing this receptor (Fig. 8.9 ) (Mukerji et al., 2006 ). Patients with NDO and IDO have the urgency to pass urine at each involuntary bladder contraction and, whenever intravesical pressure exceeds that of the urethral sphincter, they suffer from urinary incontinence. The enhancement of the micturition refl ex in NDO patients was shown to be associated with increased expression of neuronal TRPV1 (Brady et al., 2004b ; Apostolidis et al., 2005b ). There is also overexpression of TRPV1 in the bladder mucosa and submucosa of IDO patients (Liu and Kuo, 2007 ). In women with sensory urgency, TRPV1 mRNA expressed in trigonal mucosa was not only increased, but also inversely correlated with the bladder volume at which patients refer their fi rst sensation of bladder fi lling (Fig. 8.10 ), indicating that TRPV1 plays a role in the generation of a premature bladder sensation (Liu et al., 2007 ). The fi rst investigation of the expression of TRPV1 in diseases affecting the digestive tract was made in infl ammatory bowel disease. Specimens were obtained from patients with Crohn’ s disease and from three individuals with ulcerative pancolitis. Abdominal pain was a predominant symptom. Immunoblotting showed an increase in TRPV1 expression. TRPV1 immunoreactivity was greatly increased in colonic nerve fi bers of patients with active infl ammatory bowel disease, but not in control individuals (Fig. 8.11 ) (Yiangou et al., 2001a ). 222 TRPV1 IN VISCERAL PAIN AND OTHER VISCERAL DISORDERS

50 r2 = 0.45, **p < 0.01

30 / μ g RNA) 5

20 TRPV1 mRNA (copies × 10 10

0 100 200 300 400 First sensation of filling (mL) Figure 8.10 Linear regression analysis of data from sensory urgency (SU) patients. The TRPV1 mRNA expression level in trigonal mucosa of SU patients correlated inversely with the bladder volume at ﬁ rst sensation of ﬁ lling. Adapted from Liu et al. (2007) .

5 VR1-immunoreactive fibers (% of area) fibers VR1-immunoreactive

0 Normal Inflamed Figure 8.11 Scattergram showing percentage of VR1 - immunoreactive ﬁ bers in submucosa of normal and inﬂ amed intestine. Reproduced from Yiangou et al. (2001a) . CLINICAL EXPERIENCE WITH TRPV1 AGONISTS IN LOWER DYSFUNCTION 223

Since this pioneering study, other bowel diseases characterized by pain and changes in intestinal activity have also been investigated. An increase in TRPV1 - IR fi bers was observed in the bowel of patients with irritable bowel syndrome (Akbar et al., 2008 ) and in the rectum of patients with fecal urgency (Chan et al., 2003 ). Interestingly, there was a good correlation between the expression of TRPV1 and the pain intensity in patients with irritable bowel syndrome (Akbar et al., 2008 ). In patients with fecal urgency, their lower heat threshold compared to control individuals was positively correlated with the percentage of TRPV1 - IR areas in the rectal mucosa. TRPV1 - IR sensory nerve fi bers are expressed in the human esophageal mucosa both in health and in disease. Increased TRPV1 expression in the infl amed esophagus was suggested to mediate the heartburn in refl ux esophagitis (Matthews et al., 2004 ) The recent demonstration of an excess of TRPV1 in Hirschsprung’ s disease is rather peculiar. Hirschsprung ’ s disease is characterized by the absence of enteric neurons and the presence of adrenergic and presumed cholinergic hypertrophic nerve trunks in both the submucosal and myenteric plexuses in the distal gut. These hypertrophic nerve bundles showed intense TRPV1 immunoreactivity, whereas normoganglionic regions of Hirschsprung ’ s disease patients were similar to control individuals (Facer et al., 2001 ). Vulvodynia is characterized by painful burning sensation, allodynia, and hyperalgesia in the region of the vulval vestibulus. TRPV1- expressing fi bers were found to be signifi cantly increased in the vulval epidermis and superfi cial dermis (Tympanidis et al., 2004 ). One common thread among these pathologies is a peripheral excess of neurothrophins. High levels of NGF were found in the bladder of patients with infl ammatory conditions like IC (Okragly et al., 1999 ), IDO, and NDO (Giannantoni et al., 2006 ; Kim et al., 2006 ), and in the intestine of patients with infl ammatory bowel diseases (Di Mola et al., 2000 ). NGF, once taken up by sensory fi bers, increases TRPV1 translation and transport to the peripheral sensory processes (Ji et al., 2002 ).

8.7 CLINICAL EXPERIENCE WITH TRPV 1 AGONISTS IN LOWER URINARY TRACT DYSFUNCTION

The rationale for TRPV1 desensitization induced by intravesical vanilloid application in patients with detrusor overactivity (DO) lies in the experimental demonstration that capsaicin suppresses involuntary detrusor contractions dependent upon a sacral micturition refl ex (De Groat, 1997 ). This C - fi ber sacral micturition refl ex is usually inactive, but it has been shown to be enhanced after spinal cord transection in cats and other mammals (De Groat, 1997 ). A similar C - fi ber driven refl ex was suggested in patients with chronic spinal cord lesions above sacral segments (Fowler et al., 1992 ; Cruz et al., 1997 ), 224 TRPV1 IN VISCERAL PAIN AND OTHER VISCERAL DISORDERS with chronic bladder outlet obstruction (Chai et al., 1998 ), and with IDO (Silva et al., 2002 ). Therapeutic intravesical application of capsaicin in NDO patients was reported in six noncontrolled (Fowler et al., 1992 ; Fowler et al., 1994 ; Geirsson et al., 1995 ; Das et al., 1996 ; Cruz et al., 1997a ; De Ridder et al., 1997 ) and one controlled clinical trial (De S è ze et al., 1998 ). Capsaicin was dissolved in 30% alcohol, and 100 mL of 1– 2 mM solutions were instilled into the bladder for 30 min. The best clinical results were found among patients with incomplete spinal cord lesions, with clinical improvement in up to 70– 90% of the patients (Fowler et al., 1994 ; Cruz et al., 1997a ; De Ridder et al., 1997 ). Only one randomized controlled study compared capsaicin against the vehicle solution. All patients that received capsaicin had signifi cant regression of incontinence and urge sensation, whereas only one ethanol- treated patient had improved symptoms (De S è ze et al., 1998 ). The pungency of alcoholic capsaicin solutions has prevented the widespread use of this treatment. In particular, the possibility that intravesical capsaicin may trigger severe episodes of autonomic dysrefl exia in patients with high spinal cord lesions has progressively restrained its use. Nevertheless, the utility of capsaicin may have returned, based on a recent observation by De S é ze et al. who conducted a double blind placebo controlled study with a glucidic solution of capsaicin. The glucidic- capsaicin treated group showed improvement above the comparator arm and reported an excellent global tolerance of this capsaicin preparation (De S è ze et al., 2006 ). RTX has the advantage over capsaicin of being practically non - pungent at therapeutic concentrations (Cruz et al., 1997a ). Intravesical RTX application in NDO patients was evaluated in fi ve small, open - label studies (Cruz et al., 1997a ; Lazzeri et al., 1997 ; Lazzeri et al., 1998 ; Silva et al., 2000 ; Kuo, 2003a ). Different RTX concentrations, 10 nM, 50 nM, 100 nM, and 10 μM were tested. RTX brought a rapid improvement or disappearance of urinary incontinence in up to 80% of the treated patients and a 30% decrease in their daily urinary frequency. When compared against a placebo solution, RTX increased the volumes to fi rst involuntary detrusor contraction and maximal bladder capacity, as well as caused a signifi cant improvement in urinary frequency and incontinence (Fig. 8.12 ) (Silva et al., 2005 ). Curiously, patients who responded better to RTX exhibited a higher density of TRPV1 - expressing fi bers (Brady et al., 2004a ; Liu and Kuo, 2007 ). Following RTX application, TRPV1 expression in nerve fi bers (Brady et al., 2004a ) and in urothelial cells (Apostolidis et al., 2005a ) decreases substantially. The fi rst study with intravesical RTX in IDO patients was designed as a proof- of - concept study in order to evaluate the effect of TRPV1 desensitization on bladder function and lower urinary tract symptoms of these patients. One single administration of a 50 - nM RTX solution was associated with an improvement in the bladder volume at which the fi rst involuntary contraction appeared. This was accompanied by a decrease in the number of episodes of urinary incontinence (Silva et al., 2002 ). CLINICAL EXPERIENCE WITH TRPV1 AGONISTS IN LOWER DYSFUNCTION 225

mL 500 p = 0.02 450 400 350 p = 0.03 300 250 200 150 100 50 0 FDC MCC Placebo RTX Figure 8.12 Mean volume to ﬁ rst involuntary detrusor contraction (FDC) and maximal cystometric capacity (MCC) at the end of the study in the RTX and placebo group. Reproduced from Silva et al. (2005) .

Subsequent small, open- label studies confi rmed these observations using either a single high - dose (50 – 100 nM) or multiple low - dose (10 nM) protocol (Kuo, 2003b ; Kuo, 2005 ; Silva et al., 2007 ). The effect of RTX was evaluated more recently in a randomized clinical trial. It involved 54 IDO patients receiving four weekly instillations of a low dose RTX solution (10 nM) or the vehicle solution (10% ethanol in saline) (Kuo et al., 2006 ). Three months after completing the four intravesical treatments, the RTX - treated group had 42.3% and 19.2% of patients feeling much better or improved, respectively, whereas the placebo group had only 14.2% and 7.1% of the patients with these responses. Treatment remained effective at 6 months in 50% in the RTX group but only in 11% in the placebo group (Kuo et al., 2006 , Fig. 8.13 ). The involvement of TRPV1 in IDO led to further investigation on the role of this receptor in the genesis of the urgency sensation to void. Urgency is a distinctive symptom in patients with IDO and is defi ned as an intense desire to void that is diffi cult to defer. Often, urgency leads to an increased number of micturitions per day and to episodes of urinary incontinence. Patients with severe urgency were submitted fi rst to a placebo instillation and 1 month later to a 50- nM RTX treatment. A signifi cant decrease in the number of episodes of urgency was detected after RTX treatment when compared with placebo (Silva et al., 2007 , Fig. 8.14 ). The improvement of urgency after TRPV1 desensitization provides, therefore, indirect evidence that this symptom is generated by sensory input involving TRPV1 signaling. 226 TRPV1 IN VISCERAL PAIN AND OTHER VISCERAL DISORDERS

1. 0 0.9 0.8 0.7 0.6 0.5 0.4 Placebo 0.3 0.2 Cumulated success rate Cumulated 0.1 RTX 0.0 0 12 345 6789101112 Months after RTX treatment Kaplan–Meier survival analysis of cumulative success rates showed that RTX group had significantly better success rate than placebo group. Figure 8.13 Results of a comparative study of RTX versus placebo. Reproduced from Kuo et al. (2006) .

p = 0.02 100

p 80 = 0.002

0 Run-in Vehicle RTX 1 month RTX 3 months Figure 8.14 Number of episodes of urgency at the run - in period, after the instillation of the vehicle solution, and at 1 and 3 months after 50 nM RTX instillation. Reproduced from Silva et al. (2007) .

In a placebo - controlled study of 18 patients with IC/PBS, Lazzeri et al. (2000) reported an improvement in pain and urinary frequency after administration of intravesical 10- nM RTX. Chen and coworkers conducted a dose- escalating study and concluded that the most commonly reported adverse event with RTX was pain during instillation. However, at 10 or 5 nM, RTX was innocuous and could improve bladder pain (Chen et al., 2005 ). Additionally, three noncontrolled studies also reported bladder pain improvement after intravesical RTX (Lazzeri et al., 2004 ; Apostolidis et al., 2006 ; Peng and Kuo, 2007 ). Surprisingly, a randomized, double - blind study involving 163 patients CONCLUDING REMARKS 227 with IC/PBS, in which several doses of intravesical RTX (10 nM, 50 nM, and 100 nM) were compared with placebo, failed to show any advantage for the neurotoxin during a 12 - week follow - up period (Payne et al., 2005 ). Although speculative at this moment, it is appealing to consider that TRPV1 may be involved in pain that characterizes chronic prostatitis. This condition, now better designated as chronic pelvic pain syndrome (CPPS), is an ill- defi ned highly prevalent condition characterized by pelvic pain in the absence of a clear identifi able cause. Very often, this pain is described by patients as a burning pain in the urethra (Litwin et al., 1999 ) and is therefore similar to the pain reported by humans after intravesical application of capsaicin (Maggi et al., 1989 ; Cruz et al., 1997b ). In addition, CPPS patients have increased heat sensitivity in the perineal area (Yang et al., 2003 ). Interestingly, TRPV1 - expressing nerve fi bers are particularly abundant in the human prostate, around the urethra and ejaculatory ducts (Dinis et al., 2005 ). Several factors may contribute to TRPV1 activation in CPPS patients, including the low pH of the prostate tissue (White, 1975 ) and the overexpression of NGF in the semen (Miller et al., 2002 ). Curiously, ingestion of alcohol, which is a known TRPV1 agonist (Trevisani et al., 2002 ), enhances pain sensation in CPPS patients (Litwin et al., 1999 ). At the moment and probably in the near future, a lack of stable preparations of RTX for easy bladder instillation will limit further investigation of this compound. Different sources of RTX, as well as different methods of preparation and storage of the solutions, might have caused substantial variations in the amount of active compound effectively administered to the patients. Another reason for the large discrepancies observed among RTX studies, with some claiming good results and others not demonstrating any superiority of RTX over placebo, could be the propensity of RTX to adhere to plastic.

8.8 CONCLUDING REMARKS

From this review, it seems clear that TRPV1 is a widely expressed receptor in the viscera, both in neuronal and non- neuronal structures. TRPV1 seems to play a modest role in the activity of normal viscera, despite some reports that have indicated that this receptor contributes to the mechanosensitivity of the intestine. TRPV1 KO mice have a benign phenotype. On the contrary, the role of TRPV1 in the pathophysiology of several diseases is indisputable. Visceral infl ammation and bladder neurogenic dysfunction are associated with a marked increase in TRPV1 expression. In addition, TRPV1 gene suppression or receptor blockade by specifi c antagonists prevents pain and visceral overactivity accompanying infl ammation. Taking these data together, it is not surprising that TRPV1 has been actively investigated as a potential target for the treatment of lower urinary tract symptoms, including urinary frequency, urgency, and incontinence. Up to 228 TRPV1 IN VISCERAL PAIN AND OTHER VISCERAL DISORDERS now, the therapeutic approach has been based on the desensitization of the receptor by intravesical agonists like capsaicin or RTX. Unfortunately, the pungency of the former and the instability of the latter in solution have prevented their widespread use. However, even if these inconveniences are overcome, desensitizing agents might face competition from small molecule TRPV1 antagonists. Some of these antagonists already have shown clear utility in animal models of visceral infl ammation, by reducing pain and visceral overactivity.

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Philip R. Holland and Peter J. Goadsby

9.1 INTRODUCTION

Primary headaches are a group of distinct individually characterized attack forms including migraine, tension- type headache (TTH), cluster headache (CH), and other trigeminal autonomic cephalalgias (TACs) (Headache Classifi cation Committee of the International Headache Society, 2004 ). It is now widely believed that primary headaches, although varying in presentation, share some common anatomical basis responsible for the pain component of the attack. Ray and Wolff (1940) initially identifi ed that a variety of stimuli could illicit a nociceptive response from intracranial structures including the dura mater and dural blood vessels. The trigeminal nerve gives rise to the majority of afferent fi bers innervating the head, face, and dural vasculature (Messlinger and Burstein, 2000 ; Go et al., 2001 ) and, for this reason, it is of great importance to primary headaches. Headache disorders are among the most prevalent neurological disorders. Migraine, which is one of the most studied subtypes, is a common (Lipton et al., 2001 ), disabling (Menken et al., 2000 ), and economically costly (Stewart et al., 2003 ) condition. Headache disorders account for over $20 billion in lost labor productivity in the United States annually and € 27 billion in the European Community, ranking it as the most disabling neurological condition. The epidemiology of migraine is similar worldwide with an overall prevalence of about 12% of the adult population and a female- to - male ratio of

239 240 TRPV1 RECEPTORS AND MIGRAINE approximately 3:1. In addition to the gender differences in migraine, age, race, geography, and socioeconomic status play an important role in the prevalence of the disease state (Stewart et al., 1995 ). Gender differences in the occurrence of migraine attacks are not seen in prepubescent children. However, with the onset of females ’ cyclic hormonal changes, migraine becomes more common in women (Epstein et al., 1975 ; Lipton et al., 2001 ). This rise in the prevalence of migraine in women, which peaks with a 1 - year prevalence of about 30% in 30– 39 year olds (Lipton et al., 2001 ), declines after the onset of menopause (Neri et al., 1993 ; Fettes, 1999 ), indicating the inﬂ uence of hormonal changes on migraine occurrence. Given the high global prevalence, enormous societal costs, and substantial suffering related to migraine, the development of new anti - migraine therapeutic targets is essential.

9.2 CURRENT MIGRAINE TREATMENT

In general, migraine therapy can be split into two modalities: prophylactic or acute, depending on the frequency and severity of attacks. In severe cases, both approaches may be employed. Prophylactic treatment is given when the incidence of attacks is greater than four per month and when the severity of attack is suffi cient to impair normal function. Prophylactic treatment may also be given when the patient is unable to cope and when acute therapy fails or causes serious side effects. Possible prophylactic therapies include anticonvulsants, antidepressants, β - blockers, calcium channel antagonists, serotonin antagonists, and nonsteroidal anti - infl ammatory drugs (Goadsby and Raskin, 2008 ). The prophylactic drugs are taken even when the headache is not present in an attempt to reduce the frequency of attacks, and if one single drug is ineffective, combination therapies can be utilized. As with the prophylactic treatment, the frequency and severity of a patient ’ s migraine determines the acute medication. Acute intervention has been rec- ommended at an early stage as this may prevent the attack from worsening, but recent data contradict this view (Goadsby et al., 2008 ). Two forms of acute treatment are available: the nonspecifi c analgesics and the specifi c acute anti - migraine treatments, triptans, serotonin (5 - HT)1B/1D receptor agonists. Nonspecifi c therapies are utilized for the treatment of migraine pain as well as that of other disorders, whereas specifi c acute anti - migraine treatments have no general analgesic properties and only treat the migraine attack.

9.3 MIGRAINE AND TRIPTANS

Since the 1980s and the discovery of triptans, acute treatment of migraine has been revolutionized. Triptans were developed on the basis of clinical observations that 5- hydroxyindoleacetic acid (the main metabolite of serotonin) was GENETICS OF MIGRAINE 241 increased in the urine of patients during migraine attack (Curran et al., 1965 ). Further research also identiﬁ ed that platelet 5- HT levels decreased at the onset of migraine (Curran et al., 1965 ) and that intravenous 5- HT could abort headache (Kimball et al., 1960 ; Anthony, 1968 ; Anthony et al., 1968 ). Triptans target speciﬁ c subclasses of the 5 - HT1 receptors that show a differential distribution in the central and peripheral pathways of migraine. 5 - HT1B , 5 - HT 1D , and 5 - HT 1F receptor mRNA are expressed in the human trigeminal ganglia and in afferents; however, only 5- HT 1D immunoreactivity is found on trigeminal sensory nerve endings and only 5 - HT1B immunoreactivity is found on the cranial blood vessels (Beer et al., 1993 ; Longmore et al., 1997 ). The localization of the 5 - HT1F receptor to the trigeminal nucleus caudalis (TNC) and the differential distribution of the 5- HT 1B/1D receptors make them prime candidates for involvement in the pathophysiology of migraine.

9.4 CALCITONIN GENE - RELATED PEPTIDE ( CGRP ) AND MIGRAINE

Activation of the trigeminovascular system results in an increase in cranial CGRP levels. CGRP- like immunoreactivity is abundant in the trigeminal nuclei and in the nonmyelinated trigeminal afferents (Welch, 2003 ), demonstrating an innervation by CGRP- containing nerves (Edvinsson, 2004 ). Intravenous infusion of CGRP is known to cause a delayed migraine - like headache in patients (Lassen et al., 2002 ), and a correlation has been shown between increased plasma CGRP levels and migraine headache (Juhasz et al., 2003 ). In an animal model of trigeminovascular activation induced by trigeminal nerve stimulation, the observed neurogenic dural vasodilation was inhibited by a CGRP receptor antagonist, suggesting that blockade of the CGRP receptor may be a possible therapeutic target in the treatment of migraine (Williamson and Hargreaves, 2001 ). Recent advances in the study of CGRP receptor antagonism have led to the discovery of a potent nonpeptide CGRP receptor antagonist, BIBN4096BS (olcegepant) (Doods et al., 2000 ), which shows high afﬁ nity for the human α - CGRP receptor. Initial clinical trial results have shown that olcegepant is effective in the treatment of migraine without signiﬁ cant side effects (Olesen et al., 2004 ). Similarly, the orally available CGRP receptor antagonist telcagepant (MK0974; Williams et al., 2006 ) is also effective in the acute treatment of migraine (Ho et al., 2008 ) so that the general principle now seems established and a role for CGRP in acute migraine is certainly established.

9.5 GENETICS OF MIGRAINE

Migraine is a complex, polygenic, multifactorial disorder with an array of potential genetic factors that interact with each other and environmental inﬂ u- ences to produce the clinical heterogeneity observed. Several possible loci 242 TRPV1 RECEPTORS AND MIGRAINE have been identiﬁ ed for migraine with and without aura including 19p13, 1q21- 23, and Xq. One rare form of migraine, familial hemiplegic migraine (FHM), has been mapped to three different loci:

1. FHM 1 locus affecting the CACNA1A calcium channel gene has been mapped to chromosome 19p13 (Ophoff et al., 1998 ), 2. FHM 2 affecting the ATP1A2 gene on chromosome 1q23 (De Fusco et al., 2003 ), and 3. FHM 3 affecting the SCN1A gene on chromosome 2q24 (Dichgans et al., 2005 ; Schwedt and Dodick, 2005 ).

The FHM1 locus accounts for about half of all families demonstrating FHM (Estevez and Gardner, 2004 ) and causes mutations in the α 1A pore - forming unit of P/Q - type voltage - dependent calcium channels, resulting in a neuronal channelopathy. CACNA1A mutations have been shown to alter the density and gating of P/Q- type currents, thus resulting in a gain- of - function mutation that causes altered calcium currents. The ﬁ rst in vivo studies on the effects of CACNA1A mutations have demonstrated enhanced neurotransmission at the neuromuscular junction, with reduced thresholds for triggering and increased velocity of propagation of cortical spreading depression (CSD) (van den Maagdenberg et al., 2004 ). Knockout mice that do not carry the CACNA1A gene are born with severe ataxia and die within a few days (Jun et al., 1999 ); however, mice carrying CACNA1A mutations display distinct phenotypes indicating a role of the P/Q- type channels in the control of neurotransmitter release and neuronal development.

9.6 PATHOPHYSIOLOGY

The rich innervation of the vasculature and meninges of the brain provides a dense plexus of mainly unmyelinated fi bers that arise from the trigeminal ganglion and, to a lesser extent, from the upper cervical dorsal roots. The pharmacology of the trigeminovascular system is somewhat complex, although it is being better understood by studying the anatomy and physiology of the intracranial pain - producing structures. The peripheral branch consisting of the cranial circulation and the dura mater receives sympathetic, parasympathetic, and sensory nerve fi bers, all containing their own characteristic neurotransmitters (Fig. 9.1 ). Sympathetic nerve fi bers arising from the superior cervical ganglion supply the cranial vasculature with neuropeptide Y (NPY), noradrenaline (NA), and adenosine triphosphate (ATP). Parasympathetic nerve fi bers arising from the sphenopalatine (pterygopalatine) and from the otic ganglia as well as the carotid mini - ganglia supply the cranial vasculature with vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), acetyl- cholinesterase (AChE), peptide histidine methionine 27 (PHM, human version), pituitary cyclase- activating peptide (PACAP), nitric oxide synthase TRPV1 RECEPTOR ANTAGONISTS 243

Figure 9.1 The three separate systems of perivascular nerve ﬁ bers innervating the cranial circulation .

(NOS), and other VIP- related peptides (Olesen and Edvinsson, 2000 ). Sensory nerve ﬁ bers arising from the trigeminal ganglion supply the cranial vasculature with substance P (SP), CGRP, neurokinin A (NKA), and PACAP. Bipolar trigeminovascular afferents innervating the cranial structures project centrally and synapse on second- order neurons in the TNC, which is the key relay center for transmission of nociceptive information to higher brain structures (Moskowitz, 1984 ).

9.7 TRPV 1 RECEPTOR ANTAGONISTS AS A NOVEL TREATMENT STRATEGY IN MIGRAINE

Vanilloid TRPV1, which was previously known as the vanilloid receptor VR1, is best known for its activation by capsaicin, which is the pungent ingredient in hot chili peppers. The receptor is also activated by protons and by thermal nociceptive heat greater than 43 ° C, acting as a nonselective cationic channel with high permeability to calcium. An ever- increasing number of ligands that can activate or sensitize the channel include mild pH drop (acidifi cation), nerve growth factor, anandamide, bradykinin, arachidonic acid metabolites, lipoxygenase products, prostaglandins, leukotriene B4, adenosine and ATP, polyamines, prokineticins, prolactin, and ethanol and venoms from certain spiders and jellyfi sh (Tominaga et al., 1998 ; Zygmunt et al., 1999 ; Huang et al., 2002 ; Nagy et al., 2004 ; Tominaga and Caterina, 2004 ; Siemens et al., 2006 ; Cromer and McIntyre, 2008 ; Nicoletti et al., 2008 ). The wide range of possible ligands for TRPV1 makes it ideally placed to interact in a variety of physiological systems including pain and possibly 244 TRPV1 RECEPTORS AND MIGRAINE primary headaches (Goadsby, 2005, 2007). TRPV1 receptors are located in the peripheral and central nervous system in a variety of locations with established nociceptive transmitting properties. TRPV1 receptors are located on small- to medium- sized neurons thought to be unmyelinated C- fi bers or thinly myelinated A - fi bers in the trigeminal and dorsal root ganglia (Joo et al., 1969 ; Guo et al., 1999 ; Ichikawa and Sugimoto, 2001 ; Hou et al., 2002 ), with 16% of human trigeminal ganglion cells demonstrating TRPV1- like immunoreactivity (Hou et al., 2002 ). These small- to medium- sized fi bers transmit painful stimuli from the head including the meninges to the TNC, where they synapse on second - order ascending neurons and are transmitted to higher structures for processing. For some time, it was thought that TRPV1 receptors were exclusively located in the periphery; however, it is now known that intrinsic brain neurons express TRPV1 and that the receptor is widely expressed throughout the brain. A variety of sources have confi rmed high levels of TRPV1 receptors in the hypothalamus, the cerebellum, the periaqueductal gray (PAG), the dorsal root and the trigeminal ganglion, and the dura mater; moderate levels in the cortex, the striatum, the amygdala, and other midbrain structures including the locus coeruleus (LC) and the dorsal raphe; and lower levels in the thalamus, pons, and the hippocampus (Acs et al., 1996 ; Mezey et al., 2000 ; Sanchez et al., 2001 ; Szabo et al., 2002 ; Roberts et al., 2004 ; Liapi and Wood, 2005 ; Toth et al., 2005 ).

9.7.1 Cortex Evidence is accumulating that cortical structures play a more pivotal role in nociceptive processing. The cortex can produce antinociceptive effects via relays in the PAG (Zhang et al., 1997 ; Millan, 1999 ) and in other cerebral structures, and the somatosensory cerebral cortex produces descending modulation of trigeminal somatosensory neurons (Chiang et al., 1990 ). CSD is a wave of depolarization followed by hyperpolarization that spreads across the cortex at a rate of 2– 6 mm/min and is widely accepted to represent the aura in migraine with aura. The neuroelectrical changes are associated with an initial hyperemic phase followed by a oligemic phase (Olesen et al., 1990 ; Lauritzen, 1994 ; Olesen, 1998 ). CSD is widely considered to be a trigger of migraine; indeed headache has been shown to follow aura about 80% of the time and usually commences while cerebral blood fl ow remains diminished (Olesen et al., 1990 ). The localization of TRPV1 receptors to the cortex raises the possibility that they may play a role in the propagation of CSD. It is possible that pH changes seen in synaptic transmission (Krishtal et al., 1987 ) may reach signifi cant levels during CSD, resulting in activation of TRPV1 receptors and thereby aiding in the release of proinfl ammatory mediators and further activation of other receptors. To date, no experimental studies have been carried out to test this theory, and the importance of CSD in migraine remains controversial. Aura is known to exist without pain, and certain compounds have demonstrated to alleviate aura symptoms but have no effect on the TRPV1 RECEPTOR ANTAGONISTS 245 headache phase (for review, see Goadsby [2001 ]). Thus, CSD is likely to be a parallel process to migraine in a percentage of sufferers. Despite this, CSD remains a possible target for the development of new migraine treatments including TRPV1 ligands.

9.7.2 PAG In humans, PAG electrical stimulation can be used for the treatment of intractable somatic pain, and in some cases this has been shown to trigger head pain in previously headache - free individuals. The headaches are similar to migraine and include many of the characteristics including unilateral location, throbbing quality, and associated nausea and vomiting (Raskin et al., 1987 ; Veloso et al., 1998 ). Further evidence for a role of the PAG in migraine was obtained from the development of a migraine- like headache attributed to lesions in the region of the PAG (Haas et al., 1993 ; Goadsby, 2002 ). High- resolution magnetic resonance imaging (MRI) of the PAG has identifi ed a possible impairment of iron homeostasis, which can be indicative of a neuronal dysfunction in migraine both with and without aura (Welch et al., 2001 ). Compelling evidence is also provided by positron emission tomography (PET) studies in humans. Increased perfusion is seen in the rostral brain stem and in the cingulate cortex during spontaneous and triggered migraine attacks. The increased perfusion evident in the rostral brain stem but not in the cortex continues even after pharmacological intervention with headache relief, suggesting that brainstem activation is more than a simple reactive response to the pain (Weiller et al., 1995 ; Bahra et al., 2001 ; Afridi et al., 2005a,b ). The brainstem activation seen during migraine attacks is also thought to be specifi c as it is not seen in other conditions (May et al., 1998, 1999 ). The ventrolateral column of the PAG (vlPAG) is of particular relevance to migraine as it receives input from trigeminovascular afferents (Oliveras et al., 1974 ; Keay and Bandler, 1998 ; Hoskin et al., 2001 ), and stimulation of the vlPAG affects the nociceptive trigeminal - mediated jaw - opening refl ex (Oliveras et al., 1974 ; Dostrovsky et al., 1982 ) in the cat. Recent research has identifi ed that both electrical and chemical activation of the vlPAG can inhibit trigeminovascular specifi c nociception in cats and in rats (Knight and Goadsby, 2001 ; Knight et al., 2002, 2003 ), supporting the fi ndings that vlPAG stimulation can inhibit middle meningeal artery afferents (Strassman et al., 1986 ). Interes- tingly, it has also been demonstrated that microinjection of the 5 - HT1B/1D receptor antagonist naratriptan into the vlPAG selectively inhibits A - and C - fi ber responses to dural electrical stimulation, raising the possibility that triptans may exert part of their antimigraine effi cacy within the PAG (Bartsch et al., 2004 ). Experimental studies investigating the role of TRPV1 receptors in the PAG are contradictory. Capsaicin injection into the PAG has been shown to increase the latency of nociceptive responses, indicative of analgesia (Palazzo et al., 2002 ). In contrast, capsaicin injection into the dorsolateral PAG has been shown to produce responses suggesting hyperalgesia (McGaraughty et al., 246 TRPV1 RECEPTORS AND MIGRAINE

2003 ). In agreement with these ﬁ ndings, elevation of endocannabinoid levels in the vlPAG has been shown to produce both analgesia and hyperalgesia resulting from the activation of CB1 and TRPV1 receptors (Maione et al., 2006 ). A tonic role for endovanilloids in maintaining descending antinociceptive drive from the PAG has also been postulated (Starowicz et al., 2007 ).

9.7.3 LC The LC, the main noradrenergic nucleus in the brain (Amaral and Sinnamon, 1977 ), has gained attention recently as a possible candidate for involvement in the pathophysiology of migraine. It has widespread projections with many areas involved in nociceptive processing as well as pan- sensory processing including the hypothalamus and dorsal raphe nucleus. The LC has also been shown to modulate brain blood ﬂ ow and function, raising the possibility of a link with manifestations of migraine, including aura (Goadsby et al., 1982 ; Goadsby and Duckworth, 1989 ). It is activated by painful stimuli (Ter Horst et al., 2001 ) and is involved in antinociception. Interestingly, TRPV1 receptor activation via systemic capsaicin elicits robust activation of LC neurons (Hajos et al., 1987 ), even after destruction of peripheral capsaicin- sensitive sensory neurons, indicating a central effect. In agreement, capsaicin was shown to stimulate the release of glutamate and adrenaline/NA in vitro (Marinelli et al., 2002 ).

9.7.4 Region of the Posterior Hypothalamic Gray Matter It is now widely accepted that the most posterior part of the hypothalamic gray matter plays a major role in the group of primary headaches, resulting in pain and autonomic involvement termed TACs (Goadsby and Lipton, 1997 ). PET and functional magnetic resonance imaging (fMRI) studies have identifi ed hypothalamic activation during spontaneous as well as triggered cluster attacks, in paroxysmal hemicrania and short lasting unilateral neuralgiform with conjuc- tival injection and tearing (SUNCT) (May et al., 1998, 2000; Sprenger et al., 2004a,b ; Matharu et al., 2006 ), and permanent subtle structural abnormalities have been identifi ed using MRI (May et al., 1999 ). In response to the imaging evidence, the use of deep brain stimulation in the posterior hypothalamus for the treatment of chronic CH has proved a successful intervention strategy providing strong evidence for the involvement of this brain region in CH (Leone et al., 2001, 2003; Franzini et al., 2003 ). As mentioned, the region is known to play an important role in the pathophysiology of CH and of other TACs, as well as of chronic migraine. The same may be true for migraine; the presence of premonitory symptoms up to 48 h preceding the onset of an attack indicates an underlying hypothalamic dysfunction (Giffi n et al., 2003 ; Kelman, 2004 ), as does recent research indicating the presence of similar symptoms following glyceryl trinitrate (GTN)- triggered attacks (Afridi et al., 2004 ). As with CH, migraine attacks demonstrate a striking circadian rythmicity (Solomon, 1992 ; Fox and Davis, 1998 ) and link to hormonal fl uctuation (MacGregor, 2000 ) further implicating the hypothalamus. Experimental evidence for a role of the hypothalamus TRPV1 RECEPTOR ANTAGONISTS 247 in migraine has been provided from a variety of studies. Stimulation of the superior sagittal sinus (SSS) in the cat has demonstrated hypothalamic activation with upregulation of Fos protein - like immunoreactivity in hypothalamic nuclei consistent with a role for hypothalamic structures in the modulation of nociception (Benjamin et al., 2004 ). TRPV1 receptors are located throughout the hypothalamus. Consistent with this, capsaicin induces glutamate release from hypothalamic slices in vitro and enhances postsynaptic currents (Sasamura et al., 1998 ). Direct application of capsaicin in vivo into the preoptic area elicits a hypothermic response and increases the activity of warm sensitive neurons while decreasing the response of cold sensitive neurons, suggesting a possible function in thermal nociceptive processing (Jancso - Gabor et al., 1970 ).

9.7.5 Thalamus Only a very limited number of studies have examined this topic and all have been performed in cats. SSS stimulation results in increased blood fl ow and metabolic activity (as measured by 2 - deoxy - D - [114 C] - glucose uptake) in the thalamus (Goadsby et al., 1991 ). Trigeminovascular nociceptive information is relayed in several thalamic nuclei including the ventral posterior lateral (VPM) and its ventral periphery, POm, the zona incerta (ZI), the intralaminar complex, and the ventrolateral nucleus (VL) (Zagami and Lambert, 1990 ). Topical application of capsaicin and bradykinin to the SSS and the middle meningeal artery also resulted in increased fi ring of thalamic neurons (Davis and Dostrovsky, 1988 ; Zagami and Lambert, 1990, 1991). The posterior part of the ventral medial nucleus (VMpo) appears to be a specifi c relay nucleus for nociceptive and thermoreceptive information in primates and in humans. Nociceptive responsive neurons in the medial thalamus are activated by arterial capsaicin infusion, and topical application or injection results in thalamic activation as measured by fMRI and blood oxygen level dependent (BOLD) (Zambreanu et al., 2005 ; Moylan Governo et al., 2006 ). As detailed above, the TRPV1 receptor is clearly implicated in nociceptive processing, and its central distribution and function is beginning to unravel. Thus, TRPV1 receptors are placed throughout the trigeminovascular system, making them ideal candidates for possible therapeutic interventions. TRPV1 receptors colocalize with the vasoactive peptides NKA, SP, and CGRP. Indeed, activation of TRPV1 receptors with intravenous capsaicin has been shown to promote the release of the proinfl ammatory neuropeptides SP and NKA from trigeminal neurons, resulting in dural plasma protein extravasation (PPE) in rats, which further activates TRPV1 receptors in the dura mater (Markowitz et al., 1987 ). It is thought that this action of capsaicin is C - fi ber dependent as the destruction of C- fi bers in neonate rats prevents this response in adults. PPE has been suggested to underpin the pain of migraine (Williamson and Hargreaves, 2001 ), and certain antimigraine compounds including ergot alkaloids and triptans can block this extravasation, lending initial support to this theory (Markowitz et al., 1988 ; Moskowitz and Buzzi, 1991 ). However, it is clear now that effi cacy in blocking PPE is not entirely predictive of therapeutic 248 TRPV1 RECEPTORS AND MIGRAINE potential, given the failure of specifi c PPE blockers, SP, and neurokinin - 1 antagonists in clinical trials (May et al., 1996 ; Roon et al., 2000 ; Diener, 2003 ).

9.8 TRPV 1 AND CGRP

As mentioned, TRPV1 receptors are also colocalized with CGRP; in agreement with a possible interaction, intravenous capsaicin can create reproducible dural vessel dilations in rats. This dilation is inhibited by capsazepine and the CGRP receptor blocker, CGRP8 - 37 (Akerman et al., 2003 ). The ability of CGRP8 - 37 to block this capsaicin - induced dilation suggests that it is CGRP release from the prejunctional C - fi bers, rather than SP or NKA, that results in vasodilation of the middle meningeal artery. In a similar model, capsazepine was unable to block neurogenic durovasodilation, indicating a minor role for the TRPV1 receptor in the electrically induced release of CGRP. This model has proved predictive of a number of anti - migraine compounds including triptans, dihydroergotamine, and CGRP receptor antagonists (Williamson et al., 1997a,b,c ; Williamson et al., 2001 ; Petersen et al., 2004 ). The failure of TRPV1 antagonism suggests a likely minor role for TRPV1 in trigeminovascular modulation, especially at the prejunctional dural fi bers. Perhaps more signifi cantly, the neurokinin- 1 receptor antagonists also failed in this model before proceeding to unsuccessful clinical trials (Connor et al., 1998 ; May and Goadsby, 2001 ; Diener, 2003 ). Despite the relatively modest effect of TRPV1 receptor antagonism in this model of trigeminovascular activation, it is clear that TRPV1 receptors colocalize with and have some effect on CGRP release in vivo. Given the current excitement and clinical effi cacy surrounding CGRP receptor antagonists, the interaction of these two mechanisms lends support to the possible role of TRPV1 modulation as a novel therapeutic potential for migraine, perhaps as a prophylactic agent.

9.9 TRPV 1 POSSIBLE INTERACTION WITH OTHER SYSTEMS

9.9.1 Alcohol Alcohol has long been reported as a possible trigger for migraine, and there is evidence of a negative association between migraine and chronic alcohol consumption (Aamodt et al., 2006 ). Experimentally, it has been shown that EtOH stimulates TRPV1 receptors, resulting in the release of SP and CGRP from central and peripheral terminals of primary sensory neurons by lowering the temperature threshold of the receptors (Trevisani et al., 2002 ). This lower threshold for TRPV1 receptor activation results in plasma extravasation and arterial vasodilation, which has been proposed as a possible mechanistic explanation for alcohol- induced headache. Given the moderate response of direct TRPV1 receptor activation in dural vasodilation as discussed previously and the lack of clinical efﬁ cacy of the PPE model in migraine, any direct link remains somewhat tenuous. CLINICAL DATA 249

9.9.2 Cannabinoid Receptors Arachidonylethanolamide (AEA or anandamide) is believed to be the endogenous ligand to the cannabinoid CB1 and CB 2 receptors (Matsuda et al., 1990 ; Hoehe et al., 1991 ; Devane et al., 1992 ; Munro et al., 1993 ). The known behavioral effects of anandamide are similar to those of Δ9 - tetrahydrocannabinol (the psychoactive constituent of cannabis) and include antinociception, catalepsy, hypothermia, and depression of motor activity (Dewey, 1986 ; Crawley et al., 1993 ; Smith et al., 1994 ; Adams et al., 1998 ). Using the model of intravital microscopy, it has been shown that anandamide is able to attenuate neurogenic dural vasodilation, including CGRP- and nitric oxide (NO)- induced dural vessel dilation

(Akerman et al., 2004a ). This effect was not blocked by the CB 1 receptor antagonist but was blocked by the TRPV1 receptor antagonist capsazepine (Akerman et al., 2004b ). Anandamide is structurally related to capsaicin and olvanil (N - vanillyl - 9 - oleamide); both are TRPV1 agonists, which are known to have effects in animal models of trigeminovascular activation (Akerman et al., 2003 ). In agreement, intravenous anandamide has been shown to reduce C- ﬁ ber responses of TNC neurons in response to stimulation of the dural vasculature (Akerman et al., 2007 ). Pretreatment with capsazepine potentiates this inhibitory response, resulting in a reduction in A- and C- ﬁ ber responses, indicating a complex interaction between TRPV1 and CB1 receptors in the modulation of pain.

9.9.3 Other Receptor Systems TRPV1 receptors are located on primary sensory neurons that express a wide array of receptors thought to be involved in nociceptive transmission. This includes CB1 receptors (discussed previously) and a variety of other receptors that may be important in the pathophysiology of migraine. Experimental evidence for direct relevant interactions is currently lacking; however, possible interactions include those with acid- sensing ion channels (Ugawa et al., 2005 ), purinergic receptors (P2 X) (Guo et al., 1999 ; Simonetti et al., 2006 ), seroto- nergic receptors (Ohta et al., 2006 ), and nerve growth factor receptors (Zhang et al., 2007 ). Given the wide variety of receptors that exist on peripheral sensory nerves, it is diffi cult to dissect out the degree to which specifi c TRPV1 modulation could prevent sensory throughput; perhaps any signifi cant effects will depend on the central versus peripheral action of specifi c drugs.

9.10 CLINICAL DATA

CH is a strictly unilateral headache that occurs with cranial autonomic features including rhinorrhea, lacrimation, conjunctival congestion, and occasional ocular and temporal burning. It has been shown that intranasal capsaicin can also induce similar symptoms without the induction of a delayed headache. Desensitization in response to capsaicin has been used to treat a variety of conditions, and there are some small studies that suggest it may be efﬁ cacious 250 TRPV1 RECEPTORS AND MIGRAINE

Cortex

Thalamus

Hypothalamus Dural vessels Amygdala

PAG DR

LC TG

SP CGRP

P2X TRPV1 NK1 TrkA 5-HT1DASIC

TCC CG

Figure 9.2 TRPV1 localization in pathways and modulatory centers associated with migraine. Inputs from dural vasculature structures project along the trigeminal nerve via the trigeminal ganglion (TG) to second - order neurons in the trigeminal cervical complex (TCC). Convergent inputs from upper cervical roots also terminate on the same second - order neurons. Second - order neurons then project to higher brain structures including the hypothalamus, the thalamus, and the cortex. Descending modulatory systems arising from the cortex, the periaqueductal gray (PAG), the dorsal raphe (DR), and the locus coeruleus (LC) all infl uence TCC transmission. TRPV1 receptors are located in all the above structures including the trigeminal and cervical root ganglia, suggesting a possible role in the modulation of trigeminovascular nociceptive processing. The insert represents some known and hypothesized receptor colocalizations, which may play an important role in TRPV1 receptor- mediated actions. ASIC, acid sensing ion channel; NK1, neurokinin 1; TrkA, tyrosine kinase A. (See color insert.) REFERENCES 251 in the treatment of CH and of chronic migraine. Sicuteri et al. (1989) demonstrated that local intranasal application of capsaicin evoked a burning sensation on the ipsilateral side immediately following application. A gradual decrease in these adverse effects was observed over time, and attack frequency and intensity were reported to decrease in parallel to the desensitization. Civamide, a cis isomer of capsaicin that potently depletes SP and CGRP but produces less severe side effects, has also been reported to be benefi cial in the treatment of CH and of episodic migraine (Diamond et al., 2000 ; Saper et al., 2002 ). As mentioned above, intranasal TRPV1 receptor activation has been suggested as a possible treatment for migraine. The results obtained are limited from a therapeutic viewpoint, as the benefi t is often transient, and its application produces signifi cant unwanted local effects. There is very little data on the effi cacy of specifi c TRPV1 receptor modulators in the clinical setting. Both GlaxoSmithKline and Winston Laboratories have initiated clinical trials with SB - 705498 and WL - 1001, respectively, with both successfully completing phase I trials. The recent decision of GlaxoSmithKline to terminate its phase II clinical studies with their potent TRPV1 receptor antagonist due to lack of clinical effi cacy (Knotkova et al., 2008 ) casts a shadow over the potential therapeutic benefi ts, certainly in migraine.

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Enza Palazzo , Katarzyna Starowicz , Sabatino Maione , and Vincenzo Di Marzo

10.1 NEUROPATHIC PAIN

Neuropathic pain results from a primary lesion or dysfunction of the peripheral or central nervous system (CNS) and is one of the most diffi cult conditions to treat in clinical neurological practice. Neuropathic pain is associated with various symptoms, including spontaneous and evoked pain; the former may be continuous or paroxysmal, while evoked pain consists of allodynia and hyperalgesia. Allodynia is triggered by normally innocuous stimuli, and hyperalgesia corresponds to an exaggerated response to a stimulus that would not normally cause pain. Most neuropathic pain conditions respond poorly to nonsteroidal anti - infl ammatory drugs (NSAIDs) and opioid analgesics. The latter drugs are unsatisfactory due to their low effi cacy, the potential for the development of tolerance and/or addiction, and the multiplicity of their side effects (Foley, 2003 ). The poor effi cacy of opioids in neuropathic pain treatment may be due to a decrease in the expression of spinal opioid receptors after peripheral nerve injury (Besse et al., 1992 ). Thus, clinically, antidepressants and anticonvulsants remain the only mainstays of neuropathic pain therapy, but unfortunately, these drugs only prove effective in about 50% of the patients.

260 TRPV1 RECEPTORS AND NEUROPATHIC PAIN 261

The use of rodent models of neuropathic pain has made it possible to characterize the molecular and cellular alterations leading to the neuronal sensitization process that results in neuronal plasticity and anatomical reorganization at peripheral, spinal, and brain levels, all of which appear crucial in the pathogenesis of neuropathic pain (Woolf and Mannion, 1999 ; Scholz and Woolf, 2002 ; Campbell and Meyer, 2006 ). The most commonly used models of neuropathic pain are based on partial injury of the sciatic nerve, caused by chronic constriction injury (CCI) using loose ligatures around the sciatic nerve (Bennett and Xie, 1988 ), tight ligature around one- third to one- half of the sciatic nerve trunk (Seltzer et al., 1990 ), or tight ligature of L5 and L6 spinal nerves (Kim and Chung, 1992 ). Besides these widely used peripheral nerve injury- induced neuropathic pain models, additional models have been used, such as diabetic neuropathy induced by streptozotocin, postherpetic neuralgia induced by varicella - zoster virus infection, peripheral neuropathy induced by vincristine or by antiretroviral nucleoside analogue AIDS therapy drugs (2′ ,3 ′ - dideoxycytidine [ddC], 2 ′ ,3 ′ - dideoxyinosine [ddI], and 2 ′ ,3 ′ - didehydro - 3 ′ - deoxythymidine [d4T]) (Courteix et al., 1994 ; Aley et al., 1996 ; Rowbotham et al., 1996 ; Joseph et al., 2004 ). All these models of neuropathic pain are associated with thermal hyperalgesia, as well as cold and mechanical allodynia. While the dominant theme in the research on neuropathic pain has been to understand the roles of neurons in the peripheral nervous system, there is a rapidly growing body of evidence indicating a key role of supraspinal- spinal circuitries, such as the periaqueductal gray (PAG)- rostral ventromedial medulla (RVM)- dorsal horn circuitry, which is the best- characterized modulatory system through which pain is endogenously inhibited (reviewed by Palazzo et al., 2008 ). Furthermore, astrocytes and microglial cells recently have been shown to play important roles in chronic pain states (see Tsuda et al., 2005 ; Scholz and Woolf, 2007 for reviews).

10.2 TRPV 1 RECEPTORS AND NEUROPATHIC PAIN

The transient receptor potential vanilloid type- 1 (TRPV1) channel, formerly known as the vanilloid receptor (VR1), is considered a molecular integrator of physical (heat, > 43 ° C) and chemical (capsaicin, the hot ingredient of chili peppers; resiniferatoxin, RTX, isolated from a cactus - like plant; low pH; and endogenous lipids known as “ endovanilloids ” ) pain stimuli. Endovanilloids (Starowicz et al., 2007b ) include cannabimimetic lipids such as anandamide (De Petrocellis and Di Marzo, 2005 ; Pertwee, 2005 ; Singh Tahim et al., 2005 ), N - arachidonoyl - dopamine (Huang et al., 2002 ), and similar unsaturated N- acylethanolamines (Ahern, 2003 ; Movahed et al., 2005 ), as well as several products of lipoxygenases such as 12- (S) - hydroperoxyeicosatetraenoic acid (12 - [S]HPETE), 15 - (S) - hydroperoxyeicosatetraenoic acid (15 - [S]HPETE), and leukotriene B4 (LTB4) (Hwang et al., 2000 ). The use of TRPV1 receptor gene “ knockout ” (KO) mice has defi nitely demonstrated the role of this 262 TRPV1 IN NEUROPATHIC PAIN receptor in pain- related behaviors, since these mice showed impairment in thermal nociception and loss of infl ammatory thermal hyperalgesia. However, in these same animals, mechanical allodynia associated with infl ammation and nerve injury remained unchanged (Caterina et al., 2000 ; Davis et al., 2000 ).

10.2.1 Role of Peripheral, Spinal, and Supraspinal TRPV 1 Receptors in Neuropathic Pain Most research on the role of TRPV1 in pain facilitation and pain transmission has been carried out at the peripheral and spinal level. Indeed, TRPV1 receptors are widely expressed on small- and medium- sized primary afferent neurons and have a signifi cant role in the transmission of nociceptive signals from the periphery to the spinal cord (Caterina et al., 1997 ; Tominaga et al., 1998 ; Guo et al., 1999 ; Ringkamp et al., 2001 ; Ma, 2002 ; Seabrook et al., 2002 ). Capsaicin activates TRPV1 receptors located on polymodal C- fi bers of primary sensory neurons, leading to a cascade of events such as neural excitation, proinfl ammatory mediator release, receptor sensitization, and neural toxicity (Caterina et al., 1997 ). At the dorsal root ganglion (DRG) neuron level, TRPV1 receptors are upregulated, and their activity enhanced, following injury and infl ammation (Ji et al., 2002 ). Indeed, many intracellular regulatory cascades that mediate pain sensitization converge on TRPV1 receptor phosphorylation and subsequent sensitization. In fact, different proinfl ammatory mediators often act together to enhance TRPV1 receptor activity, as in the case of bradykinins and prostaglandins, which modulate TRPV1 receptor activity via protein kinase A (PKA) - and protein kinase C (PKC)- mediated phosphorylation (Premkumar and Ahern, 2000 ; Chuang et al., 2001 ; Ferreira et al., 2004 ). Accordingly, the intraplantar or intra- DRG administration of TRPV1 receptor antagonists attenuates the hyperalgesic behavior and decreases responses of spinal - wide dynamic range neurons to peripheral stimulation (Kwak et al., 1998 ; Kelly and Chapman, 2002 ; Honore et al., 2005 ; Jhaveri et al., 2005 ; McGaraughty et al., 2006 ). At the spinal level, TRPV1 receptors are mainly found presynaptically in lamina I and postsynaptically in lamina II (Guo et al., 1999 ; Valtschanoff et al., 2001 ). When delivered spinally, TRPV1 receptor antagonists or TRPV1 anti- sera proved to be analgesic in thermal hyperalgesia, in complete Freund’ s adjuvant (CFA) - induced infl ammatory pain, and in thermal hyperalgesia and mechanical allodynia in diabetic mice. Moreover, spinal TRPV1 antagonists reduced formalin - induced behavior and inhibited the evoked activity of spinal - wide dynamic range neurons in both noninfl amed and carrageenan- infl amed rats (Kamei et al., 2001 ; Kelly and Chapman, 2002 ; Honore et al., 2005 ; Kanai et al., 2005 ). TRPV1 upregulation appears to occur also at central sites leading to an enhancement of glutamatergic signaling in the spinal cord (Lappin et al., 2006 ). Similar to peripheral sites, sensitization associated with an upregulation of spinal TRPV1 receptors is thought to contribute to the development of mechanical allodynia following chronic constriction injury of the sciatic nerve (Kanai et al., 2005 ). TRPV1 RECEPTORS AND NEUROPATHIC PAIN 263

The role of supraspinal TRPV1 receptors in modulating pain is only beginning to gain scientiﬁ c interest (Palazzo et al., 2008 ). Increasing evidence demonstrates the expression of TRPV1 receptors throughout brain areas involved in pain transmission and modulation (Mezey et al., 2000 ; Szabo et al., 2002 ; Roberts et al., 2004 ; Cristino et al., 2006 ). Interestingly, the stimulation of TRPV1 receptors in the PAG, which is a well - established component of the pain modulatory circuitry via RVM projections (Millan, 2002 ; Renn and Dorsey, 2005 ), produces antinociceptive effects (Palazzo et al., 2002 ; McGaraughty et al., 2003 ; Starowicz et al., 2007a ). The antinociception is due either to activation of the PAG excitatory output neurons to downstream RVM neurons that mediate analgesia, or to the desensitization of other neurons involved in inducing hyperalgesia (Palazzo et al., 2002 ; McGaraughty et al., 2003 ; Starowicz et al., 2007 ). In particular, capsaicin injection into the dorsolateral PAG elicits analgesia by increasing the release of glutamate, which in turn activates the descending antinociceptive pathway via postsynaptic metabotropic glutamate (mGlu) receptor subtypes 1 and 5 and N - methyl - D - aspartate (NMDA) receptors (Palazzo et al., 2002 ). McGaraughty et al. (2003) reported that capsaicin- induced hyperalgesia is followed by analgesia, the latter possibly due to TRPV1 receptor desensitization. The different concentrations of capsaicin used in the two studies may be responsible for the different effects observed, since extensive TRPV1 receptor stimulation leads to desensitization. More recently, Maione et al. (2006) demonstrated that an increase in endocannabinoids in the ventrolateral PAG (VL- PAG) caused by URB597, which is an inhibitor of fatty acid amide hydrolase (FAAH), the enzyme which catalyzes anandamide hydrolysis, can produce analgesia or hyperalgesia depending on whether TRPV1 receptors or cannabinoid type 1 receptors (CB1 ) are activated on excitatory (very likely glutamatergic) antinociceptive VL- PAG output neurons. In particular, low doses of URB597 produced rapid hyperalgesia due to an increase in 2- arachidonoylglycerol (2-

AG) and subsequent CB1 receptor stimulation, which leads to the inhibition of the antinociceptive PAG - RVM descending pathway. Higher doses of URB597, instead, caused rapid analgesia due to TRPV1 receptor activation, which was blocked by the TRPV1 receptor antagonist, capsazepine. Therefore, endocannabinoids/endovanilloids within the PAG affect the pain descending pathway by acting on either CB 1 or TRPV1 receptor in a way that leads to inhibitory or facilitatory output to the RVM, respectively. Consistent with this ﬁ nding, Maione et al. (2006) found that some neurons within the VL- PAG co - express TRPV1 and CB1 receptors. Extensive neuronal colocalization of TRPV1 and CB 1 receptor- like immunoreactivity has also been shown in several mouse brain areas (Cristino et al., 2006 ), suggesting that anandamide and related endogenous cannabimimetic lipids might be implicated in dual

CB1 and TRPV1 receptor control, often evoking opposite effects (De Petrocellis and Di Marzo, 2005 ; Pertwee, 2005 ). Starowicz et al. (2007a ) reported that the intra- VL - PAG microinjection of capsaicin increased the latency of the nociceptive reaction similar to what had been observed 264 TRPV1 IN NEUROPATHIC PAIN previously in the dorsolateral PAG subregion (Palazzo et al., 2002 ). Conversely, the selective TRPV1 receptor antagonist, 5′ - iodo - resiniferatoxin (I - RTX), facilitated nociceptive responses and, at a per se inactive dose, abolished capsaicin- mediated antinociception, hence suggesting that the effect of capsaicin was mediated by TRPV1 receptors. The antinociceptive effect of intra- VL - PAG capsaicin was accompanied by an increase in glutamate release in RVM microdialysates, blocked by a per se inactive dose of I - RTX. The TRPV1 receptor antagonist instead lowered the release of glutamate, thus suggesting that VL- PAG TRPV1 receptors tonically stimulate glutamatergic output to the RVM and concomitantly inhibit nociception. Thus, the function of TRPV1 in the VL - PAG might be also opposite to that described in sensory afferents and the spinal cord. In this brainstem area, TRPV1- mediated stimulation of glutamatergic signaling might cause antinociceptive actions if exerted in excitatory output neurons that reduce nociception by activating RVM OFF neurons.

10.2.2 TRPV 1 - Based Strategies for the Treatment of Neuropathic Pain Since the discovery that topical application of capsaicin paradoxically alleviates neuropathic pain- related symptoms, the focus of much research has been to elucidate its mechanism of action. The desensitization that occurs following TRPV1 receptor stimulation is at the basis of the analgesic action of vanilloids (Holzer, 1991 ; Szallasi and Blumberg, 1999 ). Hence, stimulation of TRPV1 receptors represents a viable strategy for relieving pathological pain. Indeed, topical application of capsaicin proved effective in some neuropathic pain conditions, including postherpetic neuralgia (Watson et al., 1988 ) and surgical neuropathic pain (Ellison et al., 1997 ), but it was ineffective in chronic distal painful polyneuropathy (Low et al., 1995 ). However, in humans, topical applications of creams, lotions or patches containing capsaicin initially induce irritation, discomfort, and pain due to the activation of sensory neurons expressing TRPV1 receptors. Thus, the use of capsaicin is limited by its strong pungent and irritating nature, and the synthesis of novel vanilloids with an improved desensitization/pungency ratio is an ongoing objective. The mechanism at the basis of the antihyperalgesic action of topically applied capsaicin in neuropathic pain induced in mice by partial sciatic nerve injury was investigated by Rashid et al. (2003) . A novel, more hydrophilic ointment containing capsaicin with a higher rate of absorption was not irritating and did not produce thermal hyperalgesia when applied to the naï ve mouse’ s footpad. However, this treatment was able to reverse both thermal hyperalgesia and mechanical allodynia observed after partial sciatic nerve injury. Expression of TRPV1 receptors on neonatal capsaicin - insensitive ﬁ bers developed after nerve injury and accounted for the antihyperalgesic action of topical capsaicin (Rashid et al., 2003 ). Finally, very encouraging results have been obtained recently with a high- concentration capsaicin dermal patch (NGX- 4010) for the treatment of painful HIV- associated distal sensory polyneuropathy in a double- blind multicenter study with 307 patients (Simpson et al., 2008 ). TRPV1 RECEPTORS AND NEUROPATHIC PAIN 265

Our understanding of the role of TRPV1 receptors in chronic and neuropathic pain has been enhanced by studies with TRPV1 receptor “ KO ” mice. TRPV1− / − mice showed normal responses to noxious mechanical stimuli but did not exhibit vanilloid -evoked pain behavior. Their detection of painful heat was impaired, and they showed limited thermal hypersensitivity in the setting of infl ammation. Therefore, TRPV1 channels appear to be essential for selective modalities of pain sensation and for infl ammation - induced thermal hyperalgesia (Caterina et al., 2000 ; Davis et al., 2000 ). This evidence, together with the fi nding that vanilloid application on certain sensory neurons caused a pathological condition very similar to neuropathic pain, including the development of thermal hyperalgesia and mechanical allodynia (Simone et al., 1987 ; Gilchrist et al., 1996 ), led to the conclusion that TRPV1 receptor blockers could prove to be effective analgesics. However, early studies using the pro- totype TRPV1 antagonist, capsazepine, failed to show an analgesic effect in rat models of acute and chronic pain (Perkins and Campbell, 1992 ), leading to the idea that TRPV1 receptor antagonists were unlikely to be useful analgesics. Subsequent studies showed that capsazepine inhibited noxious heat, low pH, and capsaicin - induced responses by cloned human (McIntyre et al., 2001 ) and guinea pig (Savidge et al., 2002 ) TRPV1 receptors expressed in Chinese hamster ovary cells. However, capsazepine failed to inhibit the responses to low pH by rat TRPV1 receptors. These fi ndings indicated possible species differences in the pharmacology of TRPV1 receptors. Later, Walker et al. (2003) examined the activity of capsazepine in models of infl ammatory and neuropathic pain in rats, mice, and guinea pigs. In these studies, capsazepine reversed capsaicin - , CFA - , and carrageenan - induced thermal hyperalgesia and mechanical allodynia in the guinea pig. Surprisingly, capsazepine was also effective in reversing partial sciatic nerve ligation- induced mechanical hyperalgesia in guinea pigs. Conversely, capsazepine exhibited little analgesic effect in mice or rats with neuropathic or infl ammatory pain. This species specifi city of capsazepine led to the development of new TRPV1 antagonists (Table 10.1 ). Thus, Valenzano et al. in 2003 described N - (4 - tertiarybutylphenyl) - 4 - (3 - chloropyridin - 2 - yl)tetrahydropyrazine - 1(2H) - carbox- amide (BCTC) as a potent, selective, and orally bioavailable antagonist of rat TRPV1, and Pomonis et al. (2003) tested BCTC effi cacy in models of chronic pain in the rat. BCTC proved effective at reducing both mechanical and thermal hyperalgesia induced by intraplantar injection of capsaicin or CFA. BCTC also reduced established mechanical hyperalgesia and tactile allodynia 2 weeks after partial sciatic nerve injury, and did so with a safe side effect profi le. Another novel potent and selective antagonist of both human and rat TRPV1 receptors, the 1 - isoquinolin - 5 - yl - 3 - (4 - trifl uoromethyl - benzyl) urea, A- 425619, proved effective in several models of infl ammatory and postoperative pain. A- 425619 showed effi cacy after either oral and intrathecal administration or local injection into the infl amed paw. Furthermore, A - 425619 also showed partial effi cacy in models of neuropathic pain without altering motor performance (Honore et al., 2005 ). Likewise, (E) - 3 - (4 - t - butylphenyl) - 266

TABLE 10.1 Main Effects of TRPV 1 Agonists and Antagonists in Neuropathic Pain Drug Pain - Related Effect Administration Neuropathic Pain Limits or References Route Model Lack Thereof Capsaicin Excellent pain relief in Topical Postherpetic Initial Watson et al., 1988 (0.025%) 56% of the patients neuralgia in irritation, tested humans discomfort, and pain Capsaicin No effi cacy Topical Chronic distal Initial Low et al., 1995 (0.075%) painful polyneu- irritation, ropathy in discomfort, humans and pain Capsaicin Decrease in postsurgi- Topical Postsurgical Initial Ellison et al., 1997 (0.075%) cal neuropathic pain neuropathic pain irritation, in humans discomfort, and pain Ointment Reversion of thermal Intraplantar Artial ligation of Nonirritating Rashid et al., 2003 containing and mechanical sciatic nerve effect capsaicin hyperalgesia (0.1%) with higher rate of absorption Drug Pain - Related Effect Administration Neuropathic Pain Limits or References Route Model Lack Thereof RTX Prevention of tactile Injection in the Photochemical Tender et al., 2008 allodynia dorsal root ganglia injury to rat of the L3, L4, L5, sciatic nerve and L6 nerve roots (Gazelius model) Capsazepine Reversion in mechani- Subcutaneous Partial ligation of Species Walker et al., 2003 1 – 30 mg/kg cal hyperalgesia in sciatic nerve specifi city guinea pigs with little analgesic effect in mice or rats BCTC Reversion of both Oral Partial ligation of Safe side Pomonis et al., 10 – 30 mg/kg mechanical and sciatic nerve effects 2003 thermal hyperalgesia profi le A - 425619 Partial effi cacy Oral Spinal nerve (L5/ No CNS - Honor è et al., 2005 (5 mg/kg) L6) ligation related effects AA - 5 - HT Reversion of thermal Intraperitoneal Chronic constric- Safe profi le Maione et al., 2007 (5 mg/kg) hyperalgesia and tion of the sciatic mechanical allodynia nerve 267 268 TRPV1 IN NEUROPATHIC PAIN

N - (2,3 - dihydrobenzo[b][1,4]dioxin - 6 - yl)acrylamide, AMG 9810, a competitive antagonist of TRPV1 receptors, prevented capsaicin- induced eye wiping in a dose - dependent manner and reversed thermal and mechanical hyperalgesia associated with CFA - induced infl ammatory pain without any signifi cant effects on motor function (Gavva et al., 2005 ). Recently, in a report by Cui et al. (2006) , two potent TRPV1 receptor antagonists, one with high and the other with low CNS penetration (1 - [3 - (trifluoromethyl)pyridin - 2 - yl] - N - [4 - (trifluoromethylsulfo - nyl)phenyl] - 1,2, 3,6 - tetrahydropyridine - 4 - carboxamide, A - 784168, and N - 1H - indazol - 4 - yl - N ′ - [(1R)- 5 - piperidin - 1 - yl - 2,3 - dihydro - 1H - inden - 1 - yl]urea, A- 795614, respectively), were studied using different administration routes (oral, intrathecal, or intracerebroventricular). The two compounds had similar pharmacokinetic and in vitro pharmacological profi les. Results from these studies suggested that peripheral, spinal, and supraspinal TRPV1 receptors are involved in the analgesic actions of these antagonists in infl ammatory pain conditions. In fact, the centrally penetrant TRPV1 antagonist proved more effi cacious compared with the peripherally restricted agent. Interestingly, TRPV1 receptor is upregulated during chronic pain conditions by components of the “ infl ammatory soup ” such as bradykinins, proteases, and acidifi cation (Steen et al., 1996 ; Chuang et al., 2001 ; Ji et al., 2002 ; Holzer, 2004 ; Szolcs á nyi et al., 2004; Keeble et al., 2005 ; Amadesi et al., 2006 ). Indeed, the role of TRPV1 receptor in the transmission of nociceptive signals from uninjured animals, which appears to occur only with high- intensity stimuli (Davis et al., 2000 ; Kelly and Chapman, 2002 ; McGaraughty et al., 2006 ), seems to be increased in importance in models of pathological pain. This is consistent with an increased density in TRPV1 expression in the superfi cial layers of the spinal cord, and with a strong upregulation of TRPV1 receptors on myelinated (compared to unmyelinated) primary afferent neurons during these conditions (Hudson et al., 2001 ; Amaya et al., 2003 ; Rashid et al., 2003 ; Hong and Wiley, 2005 ; Ma et al., 2005 ). Moreover, pronociceptive/ proinfl ammatory mediators enhance the activity of TRPV1 receptors. Bradykinins, by increasing phospholipase C (PLC) activity, release TRPV1 from the inhibitory control of phosphatidylinositol biphosphate (PIP2 ) (Chuang et al., 2001 ). Sensitization of TRPV1 receptors also involves phosphorylation by PKA and PKC (Morenilla - Palao et al., 2004 ; Suh and Oh, 2005 ), whereas desensitization involves dephosphorylation by phosphatases such as calcineurin (Mohapatra and Nau, 2005). Among the PKC isoenzymes, PKCε seems to be of particular importance since its phosphorylation of Ser800 in TRPV1 strongly contributes to infl ammatory hyperalgesia (Mandadi et al., 2006 ). Furthermore, phosphorylation at Ser116 by PKA inhibits capsaicin- induced desensitization (Bhave et al., 2002 ). Phosphorylation - induced sensitization/desensitization of TRPV1 receptors is the reason why capsaicin is effective in a variety of chronic painful conditions such as surgery - induced neuropathic pain (Low et al., 1995 ; Ellison et al., 1997 ) and HIV - induced distal sensory polyneuropathy (Simpson et al., 2008 ). TRPV1 RECEPTORS AND NEUROPATHIC PAIN 269

The mechanisms through which TRPV1 receptor blockade proves effective in neuropathic pain are still not completely understood. Nerve growth factor (NGF), which plays a major role in the development and maintenance of neuropathic pain following peripheral nerve injury (Campbell and Meyer, 2006 ), has been shown to upregulate TRPV1 receptors (Winston et al., 2001 ). Thus, together with the concerted actions of other proinfl ammatory mediators, NGF is involved in the phenotypic changes leading to neuropathic pain development. Indeed, after peripheral nerve injury, TRPV1 is upregulated in undamaged neurons and downregulated in the damaged ones in several models of neuropathic pain (Hudson et al., 2001 ). Interestingly, the increased expression of TRPV1 occurred not only on C- fi bers but also on the myelinated A - fi bers, which explains the effectiveness of TRPV1 receptor agonist/antagonists in mechanical allodynia. Following tight ligation of the L5 spinal nerve, another model of neuropathic pain, the expression of TRPV1 receptors in the injured L5 dorsal root neurons decreased, whereas it increased in the uninjured L4 dorsal root neurons (Hudson et al., 2002 ). The upregulation of TRPV1 receptors at both the peripheral and CNS levels in neuropathic pain conditions provides morphological evidence that the sensitivity of the vanilloid system is increased in this painful condition (conversely to the opioid system), thus rendering the TRPV1 channel a suitable candidate for the future development of novel neuropathic pain - relieving agents. A possible complication of the use of TRPV1 antagonists in the clinic might be linked to their effect on body temperature. TRPV1 receptors tonically regulate body temperature, such that agonists (capsaicin or RTX) produce hypothermia (Hori, 1984 ; Szallasi and Blumberg, 1999 ), whereas a range of chemically distinct antagonists cause hyperthermia in several species (rats, dogs, and monkeys) (Swanson et al., 2005 ; Gavva et al., 2007 ). Recently, it has been reported that AMG 517, a TRPV1 antagonist that is effective in reversing chronic pain in preclinical studies (Gavva et al., 2007, 2008 ; Tamayo et al., 2008 ), induced hyperthemia up to 1.5 ° C in humans (Gavva et al., 2008 ). Clearly, TRPV1 antagonist- induced hyperthermia represents a potential hurdle for using TRPV1 antagonists as therapeutics for chronic pain conditions, and research is actively ongoing to discover a way to avoid, minimize, or bypass its occurrence.

10.2.3 New TRPV 1 - Based Strategies for the Treatment of Neuropathic Pain Several studies are emerging that support the conclusion that TRPV1 channels participate in the analgesic effects of compounds that interact with the endocannabinoid system. Indeed, AM404, which is an inhibitor of endocannabinoid cellular uptake, is effective against thermal hyperalgesia and mechanical allodynia in models of neuropathic pain, and the effect can be blocked by capsazepine (Rodella et al., 2005 ; Costa et al., 2006 ). Even the effectiveness of cannabidiol, a major component of Cannabis sativa, at alleviating 270 TRPV1 IN NEUROPATHIC PAIN neuropathic pain was attributed by Costa et al. (2007) at least in part to TRPV1 receptor stimulation/desensitization. Since Petrosino et al. (2007) demonstrated that, in neuropathic pain, levels of endocannabinoids are elevated at key sites involved in pain processing, and activation of cannabinoid

CB1 and CB 2 receptors can counteract pain, the inhibition of the metabolism of endocannabinoids by inhibiting FAAH should result in analgesic and antihyperalgesic effects. Indeed, FAAH inhibitors proved to be effective in neuropathic pain models (see Jhaveri et al., 2007 for review). On the other hand, FAAH inhibition, by elevating the levels of anandamide and other N - acylethanolamines that activate TRPV1 receptors, could reduce (via TRPV1 activation) or enhance (via TRPV1 desensitization) the analgesic effects of FAAH inhibitors in models of infl ammatory and neuropathic pain. It is worth noting that in a study carried out by Maione et al. (2007) , N - arachidonoyl - serotonin (AA- 5 - HT), a dual FAAH inhibitor and TRPV1 receptor blocker, proved to be analgesic after repeated administration to rats in the sciatic nerve ligation model of neuropathic pain. When compared with much more potent FAAH inhibitors with no antagonist action at TRPV1 (URB597 and OL135), AA - 5 - HT showed similar or even greater effectiveness, hence underlining the role of TRPV1 receptor blockade in alleviating neuropathic pain symptoms. Therefore, by targeting simultaneously FAAH enzyme and TRPV1 receptors, two different proteins controlling nociception in distinct ways, this hybrid molecule may represent an alternative approach that can be used to treat neuropathic pain. Such strategy might even solve the problem of the hyperthermia caused by some “ pure ” TRPV1 antagonists, since AA - 5 - HT does not cause this side effect (possibly because “ indirect ” activation of CB1 receptors might instead lower body temperature). Another alternative to TRPV1 antagonism is therapeutic nociceptive cell deletion, which exploits the enriched TRPV1 expression in nerve terminals of dorsal root or trigeminal ganglia during neuropathic pain. In fact, persistent activation of TRPV1 receptors induces a strong and prolonged increase in intracellular Ca 2+ concentration. The excess of intracellular Ca 2+ leads to excitotoxicity, which might then selectively compromise and delete TRPV1- expressing cells (Olah et al., 2001 ). Thus, overstimulation of TRPV1 receptor might prove useful in deleting TRPV1- positive neurons, thereby eliminating sensitivity to nociceptive stimuli in hyperalgesic conditions such as infl ammatory or neuropathic pain, without affecting normal sensory transmission involving fi bers that do not express TRPV1- receptors. This possibility was explored by Karai et al. (2004) , who showed that RTX application to dorsal root or trigeminal ganglia selectively ablated vanilloid - sensitive nociceptive neurons, while leaving other adjacent neurons unaffected. The treatment blocked experimental infl ammatory hyperalgesia and neurogenic infl ammation in rats, as well as naturally occurring cancer and debilitating arthritic pain in dogs. Importantly, sensations of touch, proprioreception, and high threshold mechanonociception remained unaffected. Likewise, in rats, perineural RTX application to the sciatic nerve inhibited infl ammatory hyperalgesia in a TRPV1 IN NEUROLOGICAL AND NEUROPSYCHIATRIC DISORDERS 271 dose- and time- dependent manner, while leaving proprioreceptive and nociceptive sensations and motor control unaffected (Neubert et al., 2008 ). In rats already exhibiting neuropathic pain, RTX injection in the dorsal root ganglia of the L3, L4, L5, and L6 nerve roots improved the average withdrawal threshold, thus showing that TRPV1 - positive neurons mediate the most sensitive part of mechanical allodynia. When RTX was administrated into the ipsilateral dorsal root ganglia before the nerve injury, this treatment prevented the development of tactile allodynia in 12 out of 14 rats. Immunohistochemical staining revealed that the TRPV1- positive neurons were eliminated in the rats that did not develop tactile allodynia, whereas they were still present in the allodynic rats. RTX injection in sensory ganglia may therefore represent an effective and broadly applicable strategy for pain management of neuropathies (Tender et al., 2008 ).

10.3 TRPV 1 IN NEUROLOGICAL AND NEUROPSYCHIATRIC DISORDERS

Distant from their best- known function as molecular integrators of painful stimuli, TRPV1 receptors have also been identiﬁ ed in the brain, where their physiological role is still poorly understood and stands as a subject for future studies (for review, see Steenland et al., 2006 ; Starowicz et al., 2008 ). Recent reports feature many potential roles for TRPV1 in various brain regions, such as the aforementioned activation of the antinociceptive descending pathway in the PAG as a novel strategy for producing analgesia (Palazzo et al., 2008 ), or its involvement in physiological and pathological behavioral circuits during learning and epileptic activity (Gibson et al., 2008 ). However, the biological role of these channels in the brain remains elusive, although their CNS distribution clearly indicates that they may be involved in many more functions than just pain perception. It seems, therefore, timely to review here also our current knowledge of the functions of brain TRPV1 receptors, and to consider new directions of investigation of their roles in the CNS, particularly in the ﬁ eld of neurodegenerative disorders.

10.3.1 Calcium - Dependent Neurological Disorders and Alzheimer ’ s Disease ( AD ) In a much broader perspective than just pain perception, TRP channels might play a role in AD, a degenerative and terminal disease for which there is no known cure to date. At a macroscopic level, AD is characterized by loss of neurons and synapses in the cerebral cortex and certain subcortical regions. This results in gross atrophy of the affected regions, including degeneration of the temporal and parietal lobes, as well as parts of the frontal cortex and cingulate gyrus (Wenk, 2003 ). These regions have all been reported to be TRPV1- immunoreactive. However, only a recent review by Yamamoto et al. (2007) 272 TRPV1 IN NEUROPATHIC PAIN correlates TRPV1 receptor distribution with its potential function in this very common form of dementia. TRP channels are involved in Ca 2+ homeostasis disruption. Therefore, emerging evidence of the pathophysiological role of TRPs has suggested that they are promising candidates as molecular entities mediating Ca 2+ homeostasis disruption in AD. This disease has been identifi ed as a protein misfolding disease, due to the accumulation of abnormally folded amyloid- beta and tau proteins in the brains of AD patients (Hashimoto et al., 2003 ). AD plaques are made of small peptides known as beta- amyloid peptides, which are fragments of a larger protein called amyloid precursor protein (APP), critical to neuron growth, survival, and post- injury repair (Priller et al., 2006 ). In AD, an unknown process causes APP to be divided into smaller fragments by enzymatic proteolysis (Hooper, 2005 ). One of these fragments is beta- amyloid, which forms clumps that deposit outside neurons in dense for- mations, known as senile plaques (Ohnishi and Takano, 2004 ). Since neuronal 2+ toxicity is not simply attributable to the increase in [Ca ]i via NMDA receptors, it is proposed that another channel that mediates Ca2+ infl ux is critical for neuronal cell death (Chen et al., 1999 ). The TRPV1 receptor, which is another cation channel that plays an essential role in intracellular Ca2+ homeostasis, may be involved in AD pathogenesis. In addition, reactive oxygen species (ROS) involved in various pathological conditions lead to TRPV1 activation (Ruan et al., 2005, 2006 ), which in turn can further enhance ROS production (Gazzieri et al., 2007 ). Furthermore, the neuroinfl ammatory process triggered by Aβ is essential for the neurodegenerative/infl ammatory mechanisms driven by activated microglia and astrocytes, and for the induction of proinfl ammatory molecules and related signaling pathways, thereby leading to further synaptic and neuronal damage as well as to infl ammatory cell activation. Epidemiologic evidence, as well as clinical trial data, suggests that NSAIDs may decrease the incidence of AD, further supporting the role of infl ammation in AD pathogenesis (Townsend and Praticò , 2005; Sastre et al., 2006 ). Although the precise molecular and cellular relationship between AD and infl ammation remains unclear, it is possible that cytokines may mediate activation of signaling pathways, causing further infl ammation and aggravating neuronal injury (Weisman et al., 2006 ). On the other hand, it is widely accepted that TRPV1 can be sensitized not only by capsaicin but also by signaling pathways downstream of a variety of receptors and mediators involved in infl ammation (see above). Recent evidence suggests a wide distribution of TRPV1 in microglia, astrocytes, pericytes, and neurons in the brain (Doly et al., 2004 ; Tó th et al., 2005; Kim et al., 2006 ). Therefore, taken together, these data suggest that TRPV1 channel activation might contribute to AD- related neurotoxicity and neuroinfl ammatory processes. Accordingly, a recent study by Kim et al. (2005) demonstrated that exposure of mesencephalic dopaminergic neurons to the TRPV1 agonist capsaicin resulted in cell death. This effect, observed both in vitro and in vivo, was inhibited by the TRPV1 antagonists, capsazepine and iodoresiniferatoxin, thus suggesting that this channel is directly involved in neurotoxicity. TRPV1 - induced neurotoxicity was accompanied by increases TRPV1 IN NEUROLOGICAL AND NEUROPSYCHIATRIC DISORDERS 273

2+ in [Ca ]i and mitochondrial damage, and these effects were inhibited by capsazepine and an intracellular Ca 2+ chelator, 1,2 - bis - (o - aminophenoxy) ethane - N,N,N′,N′ - tetraacetic acid tetra - (acetoxymethyl) ester ( BAPTA - AM). Treatment of cells with capsaicin or the endocannabinoid/endovanilloid anandamide induced degeneration of DA neurons, through increase in mitochondrial cytochrome c release and enhanced immunoreactivity to cleaved caspase - 3. All these effects were inhibited by capsazepine but not by AM251, a CB1 receptor antagonist. Furthermore, intranigral injection of capsaicin into the rat brain produced cell death through TRPV1. Taken together, these results indicate that activation of TRPV1 channels contributes to dopaminergic neuron damage via Ca2+ signaling and mitochondrial disruption. Similar results were obtained using microglia treated under similar conditions (Kim et al., 2006 ). Furthermore, both activation of brain TRPV1 and its antagonism with capsazepine exerted protective effects in an in vivo animal model of neurotoxicity, that is, ouabain- induced excitotoxicity (Veldhuis et al., 2003 ). The neuroprotective effect of capsaicin was attributed to possible pharmacological desensitization of TRPV1 receptors, as discussed above for neuropathic pain. Accordingly, activation (and desensitization?) of TRPV1 was also found to exhibit neuroprotective effects in the Mongolian gerbil model of ischemia (Pegorini et al., 2005, 2006 ). Finally, dietary supplementation with omega- 3 polyunsaturated fatty acids (n - 3 PUFAs), which are essential fatty acids, is benefi cial in various psychiatric disorders, including not only AD (Barberger - Gateau et al., 2002 ), but also attention defi cit hyperactivity disorder (ADHD) (Richardson and Puri, 2002 ), schizophrenia (Assies et al., 2001 ), and anxiety (Mamalakis et al., 1998 ), some of which will be discussed later in this chapter. The best- known example of the n- 3 PUFAs is docosahexaenoic acid (DHA; 22:6 n - 3). The abundance of DHA in the brain suggests essential roles for this fatty acid in neuronal function, as does the observation that DHA deprivation results in neurological defects (for review, see Marszalek and Lodish, 2005 ). Interestingly, one potential neuronal target for n - 3 PUFAs is the TRPV1 receptor (Matta et al., 2007 ). PUFAs have been previously shown to activate TRP homologues of Caenorhabditis elegans and Drosophila (Chyb et al., 1999 ; Kahn - Kirby et al., 2004 ). Even though these studies indicated a direct effect of PUFAs at homologues of TRPV channels, it was not known whether n - 3 PUFAs interact with mammalian TRPV1. TRPV1 is expressed in nerve terminals in the brain that are highly enriched with DHA. Consequently, Matta et al. (2007) have raised potentially important new biological implications for TRPV1 by providing evidence for a direct interaction of PUFAs with TRPV1. DHA, incorporated into the membrane phospholipid and released by phospholipase A2 , might reach local concentrations suffi cient to activate TRPV1. The potential for DHA signaling via central TRPV1 channels provides an exciting new approach for exploring the physiological role of TRPV1 in the brain (Matta et al., 2007 ). In summary, TRPV1 may be involved in AD and Ca2+ - dependent neurological disorders, although many pathological aspects of TRPV1 and other TRP channels in neurotoxicity and neuroinfl ammation are still unclear. 274 TRPV1 IN NEUROPATHIC PAIN

10.3.2 Degenerative Movement Disorders: Huntington ’ s and Parkinson ’ s Diseases

Huntington ’ s disease (HD) is an inherited genetic neurological disorder, characterized by progressive cell death affecting principally basal ganglia structures: the caudate nucleus and the putamen (see Reddy et al., 1999 ; Crossman, 2000 for reviews). This disorder is characterized by motor abnormalities, lack of coordination, and psychiatric symptoms (see Berardelli et al., 1999 for review). The mutation of a portion of a protein with unknown functions, called huntingtin (Cattaneo et al., 2001 ), and the subsequent expansion of a polyglu- tamine tract, is the best accepted explanation for HD origin. The presence of this mutated protein increases the rate of neuronal death in select areas of the brain, thereby affecting certain neurological functions; it leads to a gain of function of the protein, which results in toxicity, particularly for γ - aminobutyric acid (GABA)ergic neurons of striatopallidal projections (see Sieradzan and Mann, 2001 for review). The degeneration of these neurons is responsible for the motor abnormalities observed in this disease. There is no available treatment to fully arrest the progression of HD but, interestingly, typical HD signs induced in animal models can be alleviated with some cannabinoid receptor- based therapies (see Fern á ndez - Ruiz et al., 2002 for review). However, endocannabinoid signaling becomes hypofunctional in the basal ganglia of HD animal models, as revealed by the reduction of the tissue concentration of endocannabinoid ligands and, in particular, of the expression level of cannabinoid CB1 receptors (Lastres - Becker et al., 2001 ; Bisogno et al., 2008 ). This possibly explains why cannabimimetic compounds, such as anandamide and AM404 (an inhibitor of endocannabinoid cellular uptake), reduce HD signs mostly by non - CB1 - mediated mechanisms (Lastres - Becker et al., 2003 ). This latter observation suggests a potential role of TRPV1 receptors in the motor symptoms of HD, since these compounds also activate TRPV1 channels (Zygmunt et al., 1999, 2000 ; De Petrocellis et al., 2000 ; Smart et al., 2000 ). Indeed, as mentioned above, TRPV1 is widely expressed in the CNS (Cristino et al., 2006, 2008), where it seems to exert several effects (see Steenland et al., 2006 ; Starowicz et al., 2008 for review). In particular, since TRPV1 is present in the basal ganglia, possibly on nigrostriatal dopaminergic neurons (Mezey et al., 2000 ), and its stimulation causes hypokinetic effects (Di Marzo et al., 2001 ), it might represent an alternative target for the reduction of HD- associated hyperkinesia. In fact, as demonstrated by Lastres- Becker et al. ( 2003 ), AM404 reduces hyperkinesia, and causes recovery from neurochemical defi cits in a rat model of HD generated by bilateral intrastriatal injections of 3- nitropropionic acid (3- NP), a model that reproduces the characteristic mitochondrial complex II defi ciency of HD patients (Brouillet et al., 1999 ; Ouary et al., 2000 ). Subsequently, the same authors (Lastres- Becker et al., 2003 ) reported the mechanism(s) by which AM404 produces its antihyperkinetic effect in 3NP- lesioned rats. The involvement of TRPV1, but not CB 1 , receptors was suggested, based on the observation that the reduction of the TRPV1 IN NEUROLOGICAL AND NEUROPSYCHIATRIC DISORDERS 275 enhanced ambulation in 3 - NP - lesioned rats evoked by AM404 was reversed exclusively by capsazepine and not by the CB1 receptor antagonist SR141716A. Additionally, the fi nding that two synthetic inhibitors of endocannabinoid reuptake and hydrolysis, VDM11 and AM374, respectively, with no activity at TRPV1 receptors, did not reduce hyperkinesia in 3NP- lesioned rats (Lastres- Becker et al., 2003 ), indirectly confi rmed a role of TRPV1 in the observed antihyperkinetic activity of AM404. Finally, when agonists for either receptor were tested, that is, capsaicin and CP55,940 for TRPV1 and CB1 , respectively, they both exhibited antihyperkinetic activity, but only the former was able to attenuate the reductions in dopamine (DA) and GABA transmission in the basal ganglia provoked by the 3- NP - induced lesion (Lastres- Becker et al., 2003 ). The above data, taken together, strongly support the hypothesis that the antihyperkinetic action of AM404 in HD is mainly due to its capability to directly activate the TRPV1 receptor and not to its capability to act as inhibitor of endocannabinoid cellular uptake. They also favor the hypothesis that

3 - NP - induced degeneration of striatal projection neurons strongly affects CB1 receptors, which are located in these neurons, without completely reducing TRPV1 channels, which are also expressed in nigrostriatal dopaminergic neurons. Therefore, compounds with dual activity at both receptors will activate preferentially TRPV1 receptors under these conditions (Lastres- Becker et al., 2003 ). Accordingly, arvanil, a synthetic compound with much higher activity as a TRPV1 agonist than as an endocannabinoid uptake inhibitor or

CB1 agonist, was later described to potently inhibit hyperkinesia in 3- NP - lesioned rats, although this compound also caused hypolocomotion in healthy rats (de Lago et al., 2005). Along with HD, Parkinson’ s disease (PD), and, in particular, L- 3,4 - dihydroxyphenilalanine (L - DOPA) - induced dyskinesia (LID) in PD patients, might become a potential future target for centrally acting TRPV1 agonists. Although L- DOPA alleviates parkinsonian symptoms, its long- term administration is accompanied by ﬂ uctuations in its duration of action and disabling motor complications (Obeso et al., 2004 ). Some experiments carried out in various animal models of PD evidenced the role of the endocannabinoid system as a target for the treatment of L- DOPA - associated motor disturbances (Sieradzan et al., 2001 ; Ferrer et al., 2003 ; van der Stelt et al., 2005 ).

As already mentioned, CB1 receptors are expressed in brain areas such as basal ganglia, cerebellum, and motor cortex (Mackie, 2005 ), and their targeting (both activation and inhibition, depending on the phase of the disorder and experimental conditions) in animals has been shown to ameliorate the locomotor impairments typical of both PD and LID (Fern á ndez - Ruiz et al., 2002 ; Bisogno and Di Marzo, 2007 ). Accordingly, the cannabinoid agonist WIN 55,212 - 2 attenuates L - DOPA - induced abnormal involuntary movements in the 6 - hydroxy - dopamine (6 - OHDA) lesioned rat (Morgese et al., 2007 ), which is a widely used experimental model of PD. However, in the same study, the authors also demonstrated that URB597, a potent FAAH inhibitor, inhibited LID in this model only when coadministered with capsazepine. The authors 276 TRPV1 IN NEUROPATHIC PAIN suggested that pharmacological elevation of anandamide levels caused by the blockade of its catabolism produced both antidyskinetic effects similar to those of WIN 55,212- 2 and pro- dyskinetic movements via TRPV1- mediated mechanisms. This ﬁ nding would suggest that TRPV1 activation contributes to LID and that TRPV1 antagonists, rather than agonists, may be useful for its treatment of disorders associated with PD. On the other hand, another study by Lee et al. (2006) suggested that in another model of PD, the reserpine- treated rat, TRPV1 activation might counteract LID. In this model, L- DOPA treatment of reserpine - treated rats elicits high levels of motor activity in both the horizontal and vertical planes. Horizontal activity was attenuated by capsaicin, but not by URB597, nor by a selective endocannabinoid uptake inhibitor, OMDM- 2. Vertical activity was attenuated by both capsaicin and URB597 but not by OMDM- 2. Importantly, both capsaicin and URB597 reduced locomotion in healthy rats by activating TRPV1 receptors (directly and indirectly, respectively) but did not cause greater reduction in locomotion in reserpine - treated rats. These data suggest that activation of the TRPV1 system suppresses spontaneous locomotion in normal animals and modulates several L - DOPA - induced behaviors in reserpine - treated rats (Lee et al., 2006 ). Clearly, further studies are necessary, perhaps in nonhuman primate models of PD, to conclusively assess whether TRPV1 agonism or antagonism would be effective to treat LID.

10.3.3 Neuropsychiatric Disorders: Anxiety and Schizophrenia Disturbed cellular plasticity in the hippocampus might be a common aspect of several neuropsychiatric diseases. Recently, the roles of TRPV1 in anxiety, conditioned fear, and in parallel, hippocampal long - term potentiation (LTP) have been investigated. As previously reported in the literature, both TRPV1 and CB1 are colocalized within several brain structures, including the hippocampal formation, in which they can be found in close vicinity at the cellular level (Cristino et al., 2006, 2008 ). Several studies have shown that stimulation of CB 1 and TRPV1 often produce opposite effects in various experimental settings, including changes in intracellular Ca2+ concentrations (Szallasi and Di Marzo, 2000 ) and glutamate release in the substantia nigra (SNc) (Marinelli et al., 2003 ). Therefore, it can be suggested that the two receptors might control in different ways some hippocampal functions and have different roles in synaptic strength and, hence, cognition and anxiety (Di Marzo et al., 2001 ;

Cristino et al., 2006 ). Thus, if endocannabinoids and CB 1 receptors are involved in the control of anxiety, as identifi ed not only in animal studies (van der Stelt and Di Marzo, 2003 ; Lafenetre et al., 2007 ) but also in humans (Henness et al., 2006 ), one would expect that activation of brain TRPV1 receptors instead causes anxiogenic effects. This is indeed what was suggested by a preliminary study by Kasckow et al. (2004) , in which olvanil, a TRPV1 agonist, produced an anxiogenic response, whereas capsazepine exerted anxiolytic- like TRPV1 IN NEUROLOGICAL AND NEUROPSYCHIATRIC DISORDERS 277 responses (Kasckow et al., 2004 ). More recently, Marsch et al. (2007) reported a crucial role of TRPV1 in anxiety- related behaviors, conditioned fear, and LTP of excitatory postsynaptic currents (EPSPs) in the hippocampus. Using genetically engineered TRPV1 KO mice in the light – dark and the elevated plus maze tests, the authors demonstrated that these animals adapted faster to the aversive light compartment, spent more time in the light – dark test, and explored the open arms of the elevated plus maze more frequently than wild type (WT) animals, thus demonstrating an overall less anxious- like behavior (Marsch et al., 2007 ). Importantly, the anxiolytic - related phenotype of TRPV1 ablation was not related to alterations in locomotion. TRPV1 KO mice also exhibited less “ freezing ” to a tone after auditory fear conditioning, and less stress sensitization. Finally, TRPV1 KO mice were impaired in the acquisition and/or expression of contextual fear memory, both short- and long- term (1 day and 1 month, respectively). These effects were accompanied by reduced LTP in the CA1 region of the hippocampus. In summary, Marsch et al. (2007) provided evidence that in WT animals, TRPV1 strengthens innate fear and anxiety, nonassociative memory components of auditory- cued fear memories, acquisition and/or expression of contextual fear memories after strong conditioning procedures, and LTP in the dorsal hippocampus. Although the authors did not perform any pharmacological experiments with TRPV1 antagonists, these results suggested that TRPV1 channels might represent a new and attractive therapeutic target for the pharmacological treatment of human psychiatric disorders. Moreover, FAAH inhibitors, which indirectly activate cannabinoid CB1 receptors by prolonging the lifespan of endocannabinoids, exhibit anxiolytic actions (Kathuria et al., 2003 ). Theoretically, a synthetic molecule with the “ dual ” capability to inhibit FAAH and antagonize TRPV1 receptors may be an even more effective anti- anxiolytic. One such “ hybrid ” FAAH/TRPV1 blocker is the aforementioned AA- 5 - HT, reported to be effective against infl ammatory and neuropathic pain (Maione et al., 2007 ; Ortar et al., 2007 ). A very recent report demonstrated that AA- 5 - HT inhibited anxiety- related behaviors in mice in the elevated plus maze at concentrations lower than those expected from its potency at FAAH alone. This effect was antagonized by doses of a CB 1 antagonist and a TRPV1 agonist that were inactive per se, thus suggesting a possible dual mechanism of action for AA- 5 - HT (Micale et al., 2009 ). Accordingly, in at least one of the two mouse strains investigated, AA - 5 - HT was also more effi cacious than URB597 and the selective TRPV1 antagonist SB366791. Immunohistochemical data indicated that

TRPV1 receptors were often co - expressed with CB1 receptors in those brain areas controlling emotions (prefrontal cortex, nucleus accumbens [Acb], amygdala, and hippocampus), thus providing anatomical support for the suggested improved anxiolytic - like actions of simultaneous antagonism of TRPV1 receptors and indirect activation of the CB1 receptor (Micale et al., 2009 ). Also, selective pharmacological blockade of TRPV1 in the ventral hippocampus and dorsolateral PAG was recently found to attenuate anxiety - related 278 TRPV1 IN NEUROPATHIC PAIN behavior (Santos et al., 2008 ; Terzian et al., 2009 ). Rats infused with the TRPV1 antagonist capsazepine showed less open - arm avoidance than controls in the elevated plus maze test, indicating again an anxiolytic - like effect, and suggesting an important role for hippocampal and PAG TRPV1 channels in regulating anxiety behaviors. Finally, selective administration of capsaicin in the prefrontal cortex, as well as a high dose of URB597, were found to enhance anxiety- like behaviors in rats in a way antagonized by capsazepine, thus confi rming the role of TRPV1 in anxiety, at least in animals (Rubino et al., 2008 ). Data are emerging suggesting that TRPV1 receptors might play a role and represent a novel promising pharmacological target, also in schizophrenia, which is a neuropsychiatric disorder characterized by abnormalities in the perception or expression of reality. In fact, the endocannabinoid/endovanilloid anandamide plays a signifi cant neuromodulatory role in CNS pathologies associated with forebrain DA dysfunction, such as schizophrenia itself and ADHD (Giuffrida et al., 1999 ; Di Marzo et al., 2000 ; Gubellini et al., 2002 ; Centonze et al., 2004 ). Therefore, mutant mice lacking the dopamine transporter (DAT) are characterized by hyperdopaminergia and hyperactive behavior, and display disturbed sensorimotor gating and cognitive functions (Giros et al., 1996 ). These mutant mice were used to investigate the role of anandamide and its two main targets in the brain, the CB1 and TRPV1 receptors, in the neurobiological alterations due to hyperdopaminergia (Tzavara et al., 2006 ). These authors reported markedly reduced anandamide levels in the striatum of these transgenic mice, accompanied by elevated expression of binding sites for the high - affi nity TRPV1 ligand [ 3H] - RTX in this brain area. Moreover, they found that the endocannabinoid uptake inhibitors, AM404 and VDM11, and the FAAH/ TRPV1 inhibitor, AA - 5 - HT, all attenuated the spontaneous hyperlocomotion typical of these mice. Coadministration of capsazepine (at a dose that did not affect locomotion in either KO or WT mice), but not the CB1 receptor antagonist SR141716A, blocked the effects of AM404, VDM11, and AA - 5 - HT on locomotion in these mutant mice, similarly to what had been reported previously by Lastres- Becker et al. (2003) in 3- NP - lesioned rats. Since VDM11 and AA- 5 - HT do not have a direct stimulatory action on TRPV1 receptors at concentrations that inhibit anandamide reuptake/hydrolysis (Bisogno et al., 1998 ; De Petrocellis et al., 2000 ), the authors suggested that elevation of anandamide levels, due to inhibition of endocannabinoid inactivation, caused TRPV1 activation and subsequent correction of the hyperlocomotor activity in these mice lacking DAT (Tzavara et al., 2006 ). These data suggest that pharmacological elevation of the levels of endovanilloids might constitute an alternative therapeutic strategy for disorders associated with hyperdopaminergia, where TRPV1 receptors might play a key role. However, except for one study in which TRPV1 was suggested to be the molecular target for the anti - psychotic - like effect of cannabidiol in the prepulse inhibition test in mice (Long et al., 2006 ), no specifi c studies have been carried out with selective TRPV1 agonists and antagonists to further investigate this possibility. CONCLUDING REMARKS 279

10.4 CONCLUDING REMARKS

We have reviewed here the results of some of the studies indicating that TRPV1 may represent a target, not only for new pharmacological therapies for neuropathic pain, but also for novel strategies for the treatment of neurological and, perhaps, neuropsychiatric disorders (Fig. 10.1 ). On the one hand, the ﬁ nding of functional TRPV1 receptors in the brain raises the possibility of adverse clinical events associated with the use of blood – brain barrier - permeable TRPV1 agonists and antagonists, while on the other hand, this may also open the way to new therapeutic applications for these compounds. Bearing in mind that antidepressants and anxiolytics are currently used to treat chronic pain states, the increasing realization that

Prefrontal cortex via glutamate Hippocampus release via glutamate Basal ganglia release via glutamate and dopamine release

Excitotoxicity Anxiety (ischemia, AD) Schizophrenia Anxiety Movement control VL-PAG/RVM Neuromotor disorders via glutamate release Antinociception TRPV1

Thermal and inflammatory pain Thermal and inflammatory pain

Spinal cord via glutamate release Peripheral afferents (C- and δ-fibers) via CGRP and SP release

Figure 10.1 Summary of the role and possible targeting of TRPV1 receptors in neuropathic pain, as well as in neurological and neuropsychiatric disorders in various nervous tissues. As described in the main text, activation of TRPV1 might be beneﬁ cial in neurodegenerative motor (neuromotor) disorders (Huntington ’ s chorea and Parkinson ’ s disease) and, following administration in the PAG, pain. On the other hand, TRPV1 antagonism (or desensitization by agonists) might be used for the treatment of neuropathic pain, excitotoxicity, L - DOPA - induced dyskinesia, anxiety, and schizophrenia. AD, Alzheimer ’ s disease; CGRP, calcitonin gene - related peptide; SP, substance P; RVM, rostral ventromedial medulla; VL- PAG, ventrolateral periaqueductal grey. 280 TRPV1 IN NEUROPATHIC PAIN

TRPV1 channels play a facilitatory role in affective disorders might suggest not only that TRPV1 blockers could be used in the future against these disorders, but also that they might possess an intrinsic “ added value” for the treatment of neuropathic pain. Clearly, further preclinical, and, more importantly, clinical studies are required, on both selective and “ dirty ” drugs targeting TRPV1 receptors, in order to corroborate or discard these exciting new possibilities. Only time will tell if these channels will bring us a new generation of drugs for the therapy of neurological disorders, including and beyond neuropathic pain.

ACKNOWLEDGMENTS

VDM is grateful to Allergan Inc. for partly supporting his work. KS is grateful to Foundation for Polish Science and Iceland, Liechtenstein and Norway through the EEA Financial Mechanism for supporting her work.

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TRPV 1 ANTAGONISTS AND AGONISTS AS NOVEL ANALGESIC DRUGS

11 ARYL - UREA CLASS AND RELATED TRPV 1 ANTAGONISTS

Arthur Gomtsyan

11.1 INTRODUCTION

Two major structural classes of TRPV1 antagonists are the aryl- ureas and pyridine - containing structures commonly known as BCTC - type compounds (Fig. 11.1 ) (for recent reviews, see Westaway [ 2006 ], Gharat and Szallasi [ 2007, 2008 ], and Broad et al. [ 2008 ]). Inspection of these two chemical classes reveals a distinct functional pattern, in which the lipophilic side chain at the right- hand side and the hydrogen bond- forming aromatic group at the left- hand side of the molecule are connected through a linker, which itself serves in many cases as the hydrogen bond donor/acceptor site. However, despite the functional similarity, the structural elements in these two classes of TRPV1 antagonists are not easily interchangeable. Therefore, two classes of compounds will be discussed in two separate chapters. This chapter will discuss the aryl- urea class of TRPV1 antagonists. BCTC- like structures are described in the chapter by Hawryluk and Carruthers. Other structures that do not have easily recogniz- able similarity with either class will be briefl y mentioned in this chapter. In an effort to separate the irritable pungent properties of agonists such as capsaicin (1 ) (Fig. 11.2 ) from their useful analgesic activity, researchers at Sandoz (now Novartis) discovered the fi rst competitive TRPV1 antagonist, capsazepine (2) (Walpole and Wrigglesworth, 1993 ; Walpole et al., 1994 ). Structural modifi - cations of capsaicin that led to capsazepine included the introduction of a terminal aromatic ring in the lipophilic portion of the molecule, replacement

295 296 ARYL-UREA CLASS AND RELATED TRPV1 ANTAGONISTS

O Aryl O Aryl Arylalkyl Cyclic N HN N linker H H N Aryl

O O

HN N Cl N N H H HO N

Aryl-ureas Pyridinylpiperazine carboxamides (BCTC-like) Figure 11.1 Two major structural classes of TRPV1 antagonists. of the amide linker with a thiourea, and conformational restriction around one side of the linker. For a long period of time, capsazepine was the most widely used pharmacological tool compound in studies involving TRPV1 (Kwak et al., 1998 ). However, low potency, poor metabolic stability, lack of selectivity (Szallasi and Blumberg, 1999 ), species - related differences in effi - cacy studies in pain models (Walker et al., 2003 ), and, most importantly, emergence of higher- quality TRPV1 antagonists diminished the role of capsazepine as a useful research tool. For example, N,N′ - dibenzyl thiourea - based compounds JYL1421 (Wang et al., 2002 ) and IBTU (Toth et al., 2004 ; Tang et al., 2007 ) (Fig. 11.2 ) are 60 - and 5 - fold more potent TRPV1 antagonists than capsazepine in blocking capsaicin- induced calcium uptakes. Similar TRPV1 antagonists of the dibenzyl - urea type were developed by Merck (Fletcher et al., 2005 ). Replacement of at least one of the benzyl groups led to potent aryl - ureas 3 – 5 . Rigidifi cation of the benzyl group resulted in chemotype 6, while replacement of the urea with bioisosteric α , β - unsaturated amide and further conformational restriction led to chemotypes 7 and 8 , respectively. In the next several sections, we will discuss these chemotypes and will briefl y describe the properties of their most prominent representatives.

11.2 1,3 - DISUBSTITUTED UREAS

11.2.1 N - Aryl - N ′ - Aminoethyl Ureas ( SB - 705498) Encouraged by analgesic effects of early TRPV1 antagonists and the cloning of TRPV1 (Caterina et al., 1997 ), drug discovery labs launched massive high- 1,3-DISUBSTITUTED UREAS 297

O MeO N H HO Capsaicin (1, agonist)

S N HO N H

HO Capsazepine (2, antagonist)

S S F H CO N N 3 N N O O H H S H H N HO Cl H I JYL1421 IBTU

O O

Br HN N HN N H H H H N N HO CF N CF3 3 O N N

3 4 5

O O NN

HN N HN HN H R R Ar R Ar Ar

6 78 Figure 11.2 Genesis of the aryl - urea class and related TRPV1 antagonists.

throughput screening (HTS) campaigns, which resulted in the identiﬁ cation and development of a number of 1,3 - disubstituted ureas as potent TRPV1 antagonists. Researchers from GlaxoSmithKline (GSK) identiﬁ ed the highly potent aminoethyl urea TRPV1 antagonist SB - 452533 (Fig. 11.3 ), which, however, was metabolically unstable as determined by in vitro intrinsic clearance studies with rat and human liver microsomes (Rami et al., 2006 ). 298 ARYL-UREA CLASS AND RELATED TRPV1 ANTAGONISTS

Br H H N N N O

SB-452533

Br Br H H H H N N N N N N CF3 O O N

9 SB-705498 (3) Figure 11.3 Discovery of SB - 705498.

The high clearance was attributed to N - deethylation. To improve metabolic stability, two cyclic analogues were devised, which differed in the mode of cyclization of the parent molecule. The attachment of the alkyl chain to the aromatic ring resulted in 2,3- dihydroindoline compound 9 , while cyclization of the N- ethyl group to an adjacent aliphatic carbon atom resulted in the 3- aminopyrrolidine class of compounds, from which SB- 705498 ( 3) was identi- ﬁ ed. Both modiﬁ cations improved metabolic stability. In particular, SB - 705498 ( 3) exhibited much lower intrinsic clearance. Pharmacologically, SB- 705498 showed potent and reversible blockade of the multiple modes of TRPV1 activation, namely, by capsaicin, heat, and acid. In addition, the compound demonstrated improved oral biovailability (39– 86%) in rat, guinea pig, and dog. Following encouraging results in animal pain models, SB - 705498 entered clinical trials in 2004. However, despite promising results from studies utilizing a human experimental pain model (Chizh et al., 2007 ), trials in migraine were terminated with no data reported to date.

11.2.2 N - Aryl - N ′ - Arylalkyl Ureas ( A - 425619) Investigators from Abbott Laboratories (Gomtsyan et al., 2005 ) and Johnson & Johnson (Jetter et al., 2004 ) independently developed a class of urea TRPV1 antagonists containing bicyclic aromatic and benzyl groups at positions 1 and 3 of the urea fragment (Fig. 11.4 ). Modifi cations of both the carboxamide agonist 10 and the hydroxynaphthalene urea antagonist 11 led to the identifi cation of the potent and selective TRPV1 antagonist 4 (named as A- 425619 in the Abbott publication). Replacement of the pyridine group in 10 with an isoquinoline was the key modifi cation in the Johnson & Johnson approach, since it switched the profi le from agonist to antagonist. Replacement of the hydroxynaphthalene group in 11 with an isoquinoline improved the metabolic profi le of resulting TRPV1 antagonists in the Abbott approach. Based on comparative charge distribution 1,3-DISUBSTITUTED UREAS 299

O O

HN N HN N SMe H H H N HO CF3 O N N Johnson & Johnson HTS hit 10 (agonist) A-425619 (4) Abbott HTS hit 11

O O O O HN HN N n H2N S O HN N HN H R H R HO O N S R 12 13 14 Figure 11.4 1,3 - Disubstituted ureas . studies, several other 6,6 - fused heterocycles were synthesized to support structure– activity relationship (SAR) studies, but the isoquinoline remained the most optimal 6,6 - fused heterocycle. A - 425619 blocked activation of TRPV1 by capsaicin, anandamide, and N - arachidonoyl - dopamine with potent IC50 values of 3 – 5 nM (El Kouhen et al., 2005 ). A - 425619 demonstrated efﬁ cacy in a number of animal models of inﬂ ammatory and postoperative pain (Honore et al., 2005 ). The nature of the bicyclic aromatic substitution was the key structural difference in the other 1,3 - disubstituted ureas reported in the patent literature. Bayer published the hydroxy - tetrahydronaphthalene derivatives (12 ) (Yura et al., 2003 ; Tajimi et al., 2004 ); AstraZeneca described naphthalenyl ester derivatives of sulfamic acid ( 13 ) (Besidski and Nistrom, 2004 ); and Abbott reported thienopyridine derivatives (14 ) (Turner et al., 2006 ) (Fig. 11.4 ).

11.2.3 1,3 - Disubstituted Ureas with Rigid Arylalkyl Substituents (Indans, Chromans, Tetrahydroquinolines, and Tetralines) (ABT - 102) Although several TRPV1 antagonists from the N- aryl - N ′arylalkyl urea series (arylalkyl is benzyl, phenethyl, phenylpropyl, etc.) displayed excellent in vitro potency and selectivity, no compound suitable for the clinical development was reported from these series. In the case of A - 425619 (4 ), the short half - life and low volume of distribution likely contributed to less than optimal analgesic potency in preclinical studies. An important observation was that methylation at the benzylic carbon atom of 4 (Fig. 11.5 ) increased the half - life of the resulting 15 in rat and dog (Gomtsyan et al., 2007 ). The indazole group in 15 was determined to provide better pharmacokinetic properties than the isoquinoline group (Drizin et al., 2006 ). However, a 10- fold decrease in potency was associated with this pharmacokinetic improvement. Thus, the goal of SAR studies was to increase the TRPV1 antagonist 300 ARYL-UREA CLASS AND RELATED TRPV1 ANTAGONISTS

O O O

HN N HN N HN N H H H CF 3 CF3 N N N N N H A-425619 (4) 15 H ABT-102 (16)

R2 R2 R O O O N 2 O R HN N HN N HN N H H H

R1 R1 Heteroacycle Heterocycle Heterocycle

17 18 19 Figure 11.5 1,3- Disubstituted ureas with conformationally constrained benzyl substituents. potency of α - benzyl - substituted compounds such as 15 and to retain the improved pharmacokinetic proﬁ le associated with the indazole. Rigidiﬁ cation of the benzylic fragment by connecting the methyl group to the benzene ring, thus forming the indan moiety, accomplished that goal (Gomtsyan et al., 2008 ). ABT - 102 ( 16 ) blocked the activation of TRPV1 by capsaicin, pH 5.5, and

N - arachidonoyl - dopamine with IC 50 values of 0.7 – 4 nM (Surowy et al., 2008 ). Full blockade of heat activation (50 ° C) of the receptor was observed with 100 - nM ABT -102. ABT - 102 was effi cacious in animal models of acute and chronic infl ammatory pain, osteoarthritis pain, and bone cancer pain with ED 50 values of 8 – 20 μmol/kg p.o. (Honore et al., 2009 ). Other rigidifi ed arylalkyl substituents attached to the urea linker have also been described. These include chroman derivatives (17 ) from Abbott Laboratories (Gomtsyan et al., 2006a ; Brown et al., 2007 ) and Glenmark Pharmaceuticals (Gharat et al., 2007, 2008 ), tetrahydroquinolines (18 ) from Abbott Laboratories (Bayburt et al., 2008 ), and tetralines (19 ) introduced by Janssen Pharmaceutica (Codd et al., 2003 ). Potent TRPV1 antagonists have been described from each of these series.

11.2.4 Aryl Cinnamides Aryl cinnamides of the general structure 21 can be derived from the corresponding ureas (20 ) by replacing the urea functionality with the bioisosteric α , β- unsaturated amide group (Fig. 11.6 ). Therefore, aryl cinnamides can be considered part of a broader class of aryl - urea TRPV1 antagonists. The ﬁ rst potent and selective aryl cinnamide TRPV1 antagonist SB - 366791 ( 22) was reported by GSK researchers (Gunthorpe et al., 2004 ). The compound was discovered via HTS of a large chemical library. In electrophysiological experiments, SB - 366791 was demonstrated to be a potent blocker of 1,3-DISUBSTITUTED UREAS 301

O O

HN N HN H

R1 R1 Heterocycle Heterocycle

20 21

X O O HN O O N

Cl O N N N H H O CF3

SB-366791 (22) AMG-9810 (23) 24 (X = O) 25 (X = CH2) Figure 11.6 Aryl cinnamides.

TRPV1 when activated by capsaicin, acid, or heat (50 ° ). Shortly after this report, Amgen revealed the results of its own SAR studies in the aryl cinnamide series (Doherty et al., 2005 ). AMG- 9810 ( 23) was described to be a potent competitive antagonist that blocked the capsaicin- , heat- , and pH- induced uptake of 45 Ca 2+ in TRPV1 - expressing cells. AMG - 9810 effectively prevented the eye- wiping response induced by capsaicin and reversed thermal and mechanical hyperalgesia in a complete Freund ’ s adjuvant (CFA) model of infl ammatory pain in rats (Gavva et al., 2005 ). However, AMG- 9810 was characterized by high fi rst- pass metabolism and poor oral absorption in rats. Further SAR investigation led to new potent aryl cinnamides 24 and 25 with good oral bioavailability. Specifi cally, the pharmacokinetic profi le of the piperidine analogue 25 in rats was characterized by low plasma clearance (0.8 L/h/ kg), high volume of distribution (2800 mL/kg), and half - life of 2.9 h. Furthermore, compounds 24 and 25 were signifi cantly more potent than AMG- 9810 in vitro . SAR investigation of AMG- 9810 enhanced the understanding of structural features that infl uence pharmacological and pharmacokinetic properties of this class of compounds. These studies demonstrated that the benzodioxane group can be replaced with heterocycles and that 7- quinoline is the heterocycle of choice. Moreover, unsubstituted amides were shown to be preferred over N - methyl amides and thioamides, and trans - cinnamides were preferred over cis - cinnamides .

11.2.5 Conformationally Restricted Analogues of Aryl Cinnamides ( AMG - 517) Conformational analysis of aryl trans - cinnamides performed by Amgen researchers (Zhu et al., 2005 ) revealed that the s- cis conformation of 23 was preferred over the s - trans conformation by 2.6 kcal/mol (Fig. 11.7 ). Moreover, 302 ARYL-UREA CLASS AND RELATED TRPV1 ANTAGONISTS

B O O C C O O O O O N O N N H H H N CF A OH 3 s-trans-(23) s-cis-(23) 32

A B

O O Z O W Y

O N O V X

(26) (27)

NN NN F O N O N N N CF AcHN AcHN 3 S S

AMG-628(29) AMG-517 (28)

NN N CF3 HN HN O HO CF3 N CF3 (30) (31) Figure 11.7 Conformationally restricted aryl cinnamides. compounds that were better able to adopt the s- cis conformation had higher potency at TRPV1. To take advantage of this correlation between conformation and potency, rigorous SAR studies were conducted to determine whether fi xation of the s -cis conformation in cinnamides would yield potent TRPV1 antagonists (Norman et al., 2007 ; Wang et al., 2007a ). Both formation of lactam 26 by connecting the nitrogen atom to the α - position of cinnamides (process A) and formation of various aromatic rings ( 27) by connecting the carbonyl group to the β - position of cinnamides (process B) accomplished the goal of conformational restriction at the double bond. However, B - type cyclization leading to compounds with the general formula 27 showed more promise in generating bioisosteric replacements for MISCELLANEOUS TRPV1 ANTAGONISTS 303 cinnamides. It should be noted that 27 - like compounds are members of one of the very few classes of potent TRPV1 antagonists that do not incorporate a carbonyl or thiocarbonyl group in the linker fragment of their structures. Systematic optimization of TRPV1 activity, selectivity, and metabolic profi le resulted in the identifi cation of AMG - 517 ( 28) (Doherty et al., 2007 ). AMG- 517 blocked multiple modes of TRPV1 activation in humans, monkeys, rats, and mice with IC50 values < 2 nM (Gavva et al., 2007 ). Pain - relieving properties of AMG - 517 were demonstrated in infl ammatory pain models, in hyperalgesia, as well as in the on - target biochemical challenge model (capsaicin - induced fl inch). AMG - 517 entered human clinical trials for the treatment of infl ammatory pain. However, development of this compound was terminated due to fever of 40.1 ° C in one patient receiving the lowest dose of drug (Gavva et al., 2008 ). The exceptionally long half - life of about 300 h in humans and low aqueous solubility presented additional challenges for the development of AMG - 517. The backup compound AMG - 628 ( 29) displayed a pharmacological profi le comparable to AMG- 517, better solubility presumably due to the ionizable piperazine derivative attached to the pyrimidine core, and shorter half - life in rats, monkeys, and dogs (Wang et al., 2007b ). Compounds 30 and 31 (Fig. 11.7 ) reported by Merck (Blurton et al., 2004 ) and Abbott Laboratories (Gomtsyan et al., 2006b ) are additional examples of TRPV1 antagonists with conformationally rigid linkers. A third type of conformational restriction (process C) in aryl cinnamides (Fig. 11.7 ) is reminiscent of a modifi cation in the aryl - benzyl urea series, in which the benzyl fragment was rigidifi ed to yield indan, chroman, tetraline, and tetrahydroquinoline moieties (Fig. 11.5 ). Compound 32 (Fig. 11.7 ) is the result of such a structural modifi cation in the aryl cinnamide series (Uchida et al., 2007 ). A clinical candidate from Mochida/Wyeth, M68008, is believed to be a member of this chemotype.

11.3 MISCELLANEOUS TRPV 1 ANTAGONISTS

TRPV1 antagonists have been disclosed which do not have obvious resem- blance to and are not derived from aryl- ureas or pyridinylpiperazine carboxamides (discussed in the following chapter). Examples of this group of miscellaneous TRPV1 antagonists are shown in Fig. 11.8 . Most of these structures are speciﬁ c representatives of chemotypes described in the patent literature. For example, Pﬁ zer, which collaborated with Evotec (formerly Renovis), has reported cyclopropane carboxamides such as 33 (Hanazawa et al., 2007 ), and Gruenenthal has disclosed a series of N - benzyl - 2 - phenylpropanamides such as 34 (Frank et al., 2008 ). AstraZeneca has completed phase 1 clinical trials of AZD1386 (structure not disclosed) and has published patent applications featuring benzothiazole carboxamides such as 35 (Brown et al., 2006 ), benzimidazole derivatives like 36 (Brown et al., 2008 ), and spiro- imidazolidines such as 37 (Horoszok et al., 2007 ). 304 ARYL-UREA CLASS AND RELATED TRPV1 ANTAGONISTS

F C O 3 H F N N NH O N 2 O H S N O N OH F 33 34

O N CN N N N H H F N S F O 35 36

H N O O N F3CO NH N N H O N N Cl 37 38 O CF Cl 3

O Cl O

39 Figure 11.8 Miscellaneous TRPV1 antagonists.

The spiro - piperidine 38 represents a lead compound in the TRPV1 antagonist program at Schering - Plough (Xiao et al., 2008 ). This compound blocks activation of TRPV1 in a calcium infl ux assay with an IC 50 = 13 nM and exhibits oral bioavailability in rats. Extensive SAR studies performed at Novartis to improve the physico- chemical and pharmacokinetic properties of their HTS hit compound led to the quinazolinone 39 (Culshaw et al., 2006 ). Although 39 was not the most potent member of the series (IC 50 = 105 nM in rat TRPV1 activated by low pH), its acceptable selectivity, metabolic and pharmacokinetic profi les prompted evaluation in preclinical pain models. In a CFA rat model of infl ammatory pain, compound 39 reversed mechanical hyperalgesia with an ED 50 value of 4.7 mg/kg p.o. It is noteworthy that this compound was also effective REFERENCES 305 in a rat model of neuropathic pain reversing mechanical hyperalgesia in the

Bennett model of sciatic nerve ligation with an ED 50 value of 2.6 mg/kg. In contrast, very few other TRPV1 antagonists have been reported to be potent and efﬁ cacious in models of neuropathic pain.

11.4 CONCLUSION

Aryl - urea derivatives represent one of the two major classes of TRPV1 antagonists; the other consists of pyridinylpiperazine carboxamides as discussed in the following chapter. The aryl- urea class is represented by several chemotypes including the prototypical 1,3 - disubstituted ureas such as N - aryl - N′ - benzyl ureas. Conformational restriction of the benzyl group yields very potent indan, chroman, and tetralin - containing TRPV1 antagonists. The aryl - urea class has also given rise to the aryl cinnamide chemotype, in which the urea functionality is replaced with an α , β - unsaturated amide. The aryl cinnamides have been modiﬁ ed to conformationally restricted analogues, which structurally differ substantially from the original aryl- ureas. However, since these new structures can easily be traced back to the prototypical ureas, they are considered to be members of the aryl- urea class of TRPV1 antagonists. With extensive effort across the pharmaceutical industry to identify and to develop potent TRPV1 antagonists with efﬁ cacy and safety in the clinic, it is likely that new compounds will be discovered, which do not have structural and logical connections to the two largest classes of TRPV1 antagonists. Several compounds from this miscellaneous category as presented in this chapter are high - quality TRPV1 antagonists comparable to more established chemotypes.

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Xiao D , Palani A , Aslanian R , McKittrick BA , McPhail AT , Correll CC , Phelps PT , Anthes JC , and Rindgen D ( 2008 ) Spiro - piperidine azetidinones as potent TRPV1 antagonists . Bioorg Med Chem Lett 19 : 783 – 787 . Yura T , Mogi M , Urbahns K , Fujishima H , Masuda T , Moriwaki T , Yoshida N , Kokubo T , Shiroo M , Tajimi M , Tsukimi Y , and Yamamoto N ( 2003 ) Hydroxy tetrahydro - naphthalenylurea derivatives . WO Patent 2003095420 . Zhu J , Viswanadhan V , Ognyanov V , Bo Y , Chen N , Chakrabarti P , Doherty E , Fotsch C , Gavva N , Han N , Klionsky L , Liu Q , Tamir R , Wang X , Sun Y , Treanor JJS , and Norman MH ( 2005 ) Conformational analyses of N - aryl cinnamides as TRPV1 antagonists . Abstracts of 229th National Meeting of the American Chemical Society, San Diego, CA, March 13 – 17, 2005. 12 2 - PYRIDINYLPIPERAZINE CARBOXAMIDE CLASS AND RELATED TRPV 1 ANTAGONISTS

Natalie A. Hawryluk and Nicholas I. Carruthers

12.1 INTRODUCTION

2 - Pyridinylpiperazine carboxamides ( I) (Fig. 12.1 ) represent one of the fi rst classes of high - affi nity TRPV1 antagonists that did not structurally originate from naturally occurring vanilloids. Major chemotypes within this class of TRPV1 antagonists differ from each other by the structure of the central core. Thus, replacement of the piperazine ring in I with tetrahydropyridine or cyclo- hexene results in generic structures II and III , while replacement of the piperazine ring with an aryl group leads to biarylamides IV. The latter chemotype has been further modifi ed by cyclization of the carbonyl group to the central ring, resulting in quinazolines V and partially saturated bicylic heteroaryls VI . Detailed patent coverage of this class of TRPV1 antagonists can be found in the review by Gharat and Szallasi (2008) .

12.1.1 Medicinal Chemistry and Pharmacology of 2 - Pyridinylpiperazine Carboxamides The ﬁ rst examples of this template were independently discovered and described by Neurogen (Bakthavatchalam, 2002 ; Zheng et al., 2006 ), Purdue Pharma (Kyle and Sun, 2003 ; Pomonis et al., 2003 ; Sun et al., 2003 ; Valenzano et al., 2003 ), and Johnson & Johnson (Carruthers et al., 2005 ; Swanson et al.,

311 312 2-PYRIDINYLPIPERAZINE CARBOXAMIDE CLASS

O R Cl N N H N

N I Piperazines

O O O R R R Y N Y X N Y N H X H H

N N N II III IV Tetrahydropyridines and cyclohexenes Regioisomeric tetrahydropyridines Biarylamides and cyclohexenes

R R R HN HN HN

Y N Y N Y O N N N N N N VI V X = N, Partially saturated bicyclic heteroaryls Quinazolines CH Y = Cl, CF3, Me, etc. Figure 12.1 2 - Pyridinylpiperazine carboxamides and related chemotypes .

2005 ). In each case, the template was identiﬁ ed via high - throughput screening (HTS) of corporate compound collections. 1 Neurogen reported compound 1 (Fig. 12.2 ) (IC50 = 17 nM) (Bakthavatchalam, 2002 ) as a starting lead molecule for structure– activity relationship (SAR) studies and for optimization of oral bioavailability (Zheng et al., 2006 ). Purdue Pharma (Valenzano et al., 2003 ) described the optimization of HTS hit 2

(IC50 = 58 nM) into the compound BCTC ( N - (4 - t - butylphenyl) - 4 - (3 - chloropyridin - 2 - yl) - tetrahydropyrazine - 1(2 H ) - carboxamide) ( 3 ), which showed potent antagonism of the rat TRPV1 activation by both acid

(IC50 = 5 nM) and capsaicin (IC50 = 35 nM). Researchers from Johnson & Johnson elaborated their HTS hit 4 (IC 50 = 74 nM) into 5 (IC50 = 57 nM), which was further modiﬁ ed to the bis - pyridine compound JNJ - 17203212

1 Unless otherwise stated, IC50 refers to blockade of capsaicin - induced calcium ﬂ ux in human TRPV1 channels. INTRODUCTION 313

N N N

CF3 N O CF3 N O Cl N O

HN HN HN

1 CF3 2 3

N N N

CF3 N O CF3 N O CF3 N O HN HN HN N

4 NO2 5 CF3 6 CF3 Figure 12.2 Structures from Neurogen, Purdue, and Johnson & Johnson.

HN N N N NCO NH Cl N O Cl N HN ° Cl NH Cl DMSO, 100 C CH2Cl2, 93% 3

HN N N N F3C NHCO2Ph NH CF N O N 3 Cl N HN N CF3 nBuOH, reflux CF3 NH DMSO, 79% 79% 6 CF3 Scheme 12.1 Representative syntheses of 2 - pyridinylpiperazine carboxamides.

( 6 ) (IC50 = 65 nM) Compound 6 exhibited good oral bioavailability and in vivo activity in animal models of pain (Swanson et al., 2005 ).

12.1.1.1 Chemistry and SAR The target compounds were readily prepared via condensation of piperazine with a halopyridine, followed by reaction of the pyridinylpiperazine with an arylisocyanate. In the case of bis - pyridines, the pyridinylpiperazine was condensed with an aminopyridine phenyl carbamate. The syntheses of 3 (Kyle et al., 2003 ; Sun et al., 2003 ) and 6 (Carruthers et al. 2005 ; Swanson et al., 2005 ) are shown in Scheme 12.1 . This modular approach allowed straightforward generation of compound libraries by using 314 2-PYRIDINYLPIPERAZINE CARBOXAMIDE CLASS

N N N

N N N N O N O N O

HN HN HN

7 CF3 8 CF3 9 Cl

F3C N N N

N F3C N N

CF3 N O N O N O

HN HN HN N

10 Cl 11 Cl 12 Cl Figure 12.3 Pyridine fragment SAR. commercially available starting materials. Although such an approach helped to expeditiously generate many compounds for SAR development, the chemical diversity was limited. Given the intense interest in this template, comprehensive SAR was developed for each of its three structural fragments: pyridine, piperazine, and terminal aryl groups. The 2- position of the pyridine ring is the more favorable site for the piperazine attachment (Fig. 12.3 ). Thus, compounds 7 and 8 display

IC50 values of 1200 and 5000 nM, respectively, while compound 9 is a weak agonist (EC 50 = 7200 nM). The 3 - position is the most preferred for the second substitution on the pyridine ring. Among 3- , 4- and 5- substituted compounds 10 – 12 , only the 3 - substituted pyridine compound 10 is a TRPV1 antagonist

(IC50 = 500 nM), while the two other regioisomers, 11 and 12 , are agonists. Ideal substituents are small and nonpolar groups such as halides (Cl, Br, and

I) or haloalkyls (CHF 2 and CF3 ), with CF3 as the most favorable substituent. Larger alkyl (ethyl and longer) or more polar groups ( – CHO and NO 2 ) signifi cantly reduce the potency. Modifi cations to the central piperazine core confi rm that the 1,3 - diamine is essential for the activity, and only small substituents are tolerated (Fig. 12.4 ). The potencies of variously substituted piperazine compounds 13 – 16 are 64, 2450, > 10,000, and > 10,000 nM, respectively. Either ring expansion of the central core (17 ), replacement of one of the nitrogen atoms in the piperazine ring with a carbon atom (18 , 20 , 21 ), or ring contraction to pyrrolidine (19 ) resulted in loss of potency. Finally, the SAR for substitution on the aniline fragment (Fig. 12.5 ) revealed that a 4 substituent is the most desirable, since compound 5 (IC 50 = 57 nM) is INTRODUCTION 315

N N N

CF3 N O CF3 N O CF3 N O

HN HN HN

13 CF3 14 CF3 15 CF3

N N N

N N N CF N O CF CF3 3 3 N O NH HN HN O NH

CF 17 18 16 3 CF3

CF3

N N N H N N N O CF3 CH3 N O CF3 O HN HN HN

CF3 19 20 CF3 21 CF3 Figure 12.4 Piperazine fragment SAR.

N N N N

N N N N CF3 N O CF N O CF3 N O 3 Cl N O CF3 HN HN HN CF3 HN

5 CF3 22 23 24

Figure 12.5 Aniline fragment SAR.

much more potent than the 3 - and 2 - substituted analogues 22 and 23 with IC 50 values of 422 and >10,000 nM, respectively. Particularly preferred substituents are small electron - withdrawing or alkyl groups such as in compounds 1 – 5 (Fig. 12.2 ). Replacement of the aniline with an appropriately substituted aminopyridine is tolerated, and corresponding compounds such as 6 exhibit improved oral bioavailablity. It should be mentioned that the aromatic group can also be replaced by the trans - 4- t - butylcyclohexane fragment, producing the potent TRPV1 antagonist 24 with IC50 = 3.9 nM. 316 2-PYRIDINYLPIPERAZINE CARBOXAMIDE CLASS

12.1.1.2 In Vitro and In Vivo Evaluation Two members of this class of compounds, BCTC (3 ) (Pomonis et al., 2003 ; Valenzano et al., 2003 ) and JNJ- 17203212 ( 6 ) (Scheme 12.1 ) (Carruthers, 2005 ; Ghilardi et al., 2005 ; Swanson et al., 2005 ; Bhattacharya et al., 2007 ), were evaluated in detail. BCTC is the most prominent early member of the pyridinylpiperazine class of TRPV1 antagonists, showing high potency in blocking activation of TRPV1 by acid and capsaicin. The overall selectivity profi le for BCTC is favorable, although it also blocks activation of another TRP channel, TRPM8, with high nanomolar potency (Behrendt et al., 2004 ). The pharmacokinetic profi le of BCTC in rats is characterized by low oral bioavailability (5– 15%), a short half- life (0.85 h), high clearance (5 L/h/kg), and high volume of distribution (5.95 L/kg). The compound has a blood - to - brain ratio of 1.37:1.0 1 h after a 10 mg/kg i.p . dose. BCTC has been extensively profi led in animal models of infl ammatory and neuropathic pain. It has been shown to be effective in reducing thermal and mechanical hyperalgesia associated with infl ammation (intraplantar injection of complete Freund’ s adjuvant), as well as reducing mechanical hyperalgesia and tactile allodynia associated with nerve injury (partial ligation of the sciatic nerve) (Pomonis et al., 2003 ). However, limited metabolic stability and potent blockade of the human ether - a - go - go related gene (hERG) channel (Tafesse et al., 2004 ) has precluded its further development. Inhibition of the hERG channel suggests that the compound may produce undesired cardiovascular effects, such as prolongation of the cardiac QT interval leading to arrhythmia and fi brillation. Extensive SAR studies aimed at improving the metabolic and cardiovascular safety profi le led to compound 25 (Fig. 12.6 ), which has a methyl group on the piperazine ring, and benzothiazolyl and pyridazinyl groups replacing t- butylphenyl and pyridyl groups, respectively. Compound 25 , while less potent than BCTC at TRPV1, exhibits decreased affi nity for hERG and a longer half - life (Tafesse et al., 2004 ). JNJ - 17203212 ( 6) is a weaker TRPV1 antagonist than BCTC but has higher oral bioavailability in rats (40– 70%), a longer plasma half- life of 3.2 h, low clearance of 0.67 L/h/kg, volume of distribution of 3.8 L/kg, and a blood - to - brain ratio of 1.1:1.0 1 h after 10 mg/kg p.o . dose (Swanson et al., 2005 ). This compound also has little or no effect on hERG as measured in a binding assay

F CF3

O S O HN N CH3 N N N Cl N N H H N N F

N N F F Cl N HO 26 (AMG-2674) 25 OH Figure 12.6 Second - generation structures from Purdue and Neurogen. INTRODUCTION 317

(IC50 > 1 0 μ M ). JNJ - 17203212 (6 ) was effective in rat pain models of both tactile allodynia and thermal hyperalgesia. In a mouse model of bone cancer pain, it signifi cantly attenuated nocifensive behaviors following both acute and chronic administration (Ghilardi et al., 2005 ). Compound 6 was also evaluated for antitussive activity in guinea pig (Bhattacharya et al., 2007 ), after fi rst establishing that it had high affi nity for recombinant guinea pig TRPV1 receptors expressed in Chinese hamster ovary (CHO) cells (pK i = 7.14). Intraperitoneal administration of 6 (20 mg/kg) attenuated capsaicin - induced cough with effi cacy similar to codeine and also demonstrated antitussive effi cacy in a citric acid - sensitized model of experimental cough. Another member of the piperazine carboxamide class of TRPV1 antagonists is the Amgen compound AMG - 2674 (26 ) (Fig. 12.6 ) (Ognyanov et al., 2006 ). The structure of 26 was derived from BCTC by replacement of the phenyl group with the benzimidazole fragment. One of the key modifi cations was the introduction of a polar diol group on the pyridine fragment. The compound blocked the activation of rat TRPV1 by both capsaicin and low pH

(IC 50 = 0.9 nM). When administered orally, AMG - 2674 reduced thermal hyperalgesia in the CFA model of thermal hyperalgesia and blocked capsaicin - induced ﬂ inching.

12.1.2 Tetrahydropyridine Chemotype Efforts to discover a bioisosteric replacement for the piperazine urea portion of compound 1 resulted in a series of carboxamides in which the piperazine ring was replaced with the tetrahydropyridyl moiety (Brown et al., 2008 ). Compound 27 (Fig. 12.7 ), which is a representative of the tetrahydropyridyl carboxamide series of TRPV1 antagonists, potently inhibits TRPV1 receptor- 2+ mediated Ca infl ux induced by various stimuli such as capsaicin (IC 50 = 24 nM), N - arachidonoyldopamine (IC 50 = 20 nM), and low pH (IC 50 = 14 nM). In addition, compound 27 was effective in multiple animal pain models. However, compound 27 demonstrated nonlinear pharmacokinetic properties precluding further advancement. Other nitrogen- containing replacements for the piperazine ring, such as the reversed tetrahydropyridine 28 (Sun and Wen, 2005 ) and fl uoropiperidine 29 (Bayliss et al., 2005 ; Sun et al., 2005 ) (Fig. 12.7 ), were reported in patent literature with no specifi c biological data provided.

O O S O O R O CF3 R Cl N N Cl N N Cl N H H H N F N N N 27 28 29 Figure 12.7 Representative tetrahydropyridine analogues. 318 2-PYRIDINYLPIPERAZINE CARBOXAMIDE CLASS

12.1.3 Biarylamide Chemotype The piperazine fragment has also been replaced with a phenyl ring, generating the biarylamide series of TRPV1 antagonists (Fig. 12.8 ). Compounds 30 – 32 in this series are potent (IC50 values of 6– 26 nM) antagonists of both rat and human TRPV1 (Zheng et al., 2006 ). However, these compounds still exhibit the undesirable properties of the corresponding piperazine carboxamides, such as poor aqueous solubility and bioavailability. The corresponding inverse amides (33 ) (Fig. 12.9 ) are also potent TRPV1 antagonists (Fletcher et al., 2006 ). Efforts to improve the pharmacokinetic properties of the inverse amides resulted in the conformationally restricted benzimidazole 34 and indazolone 35 . In vitro activity of compounds in these series was comparable to that of the inverse amides, and the pharmacokinetic proﬁ le of the indazolones was more favorable. For example, systemic levels of indazolone 35 (R 1 =CF 3 , R 2 =4 - CF3 ) after oral dosing in rats were 70.0- and 2.5- fold higher than for the similarly substituted inverse amide 33 and benzimidazole 34 , respectively (Fletcher et al., 2006 ).

CF O 3 O O Cl N CF3 N CF3 N H H H

N N N 30 31 32 Figure 12.8 Representative biarylamide analogues.

R1 H N R1 O

N 33

H H N N R R R2 R1 2 1 N N O N N 34 35 Figure 12.9 Evolution of benzimidazole and benzimidazolone analogues. INTRODUCTION 319

12.1.4 Quinazoline Chemotype Introduction of a phenolic hydroxyl group at the 3 - position of the central ring,

as in compound 36 (IC 50 = 43 nM) (Fig. 12.10 ), validated the approach of cyclization of the carboxamide group to the central phenyl ring (Zheng et al., 2006 ). Hydrogen bonding between the hydroxyl and the amide carbonyl groups enforced planarity and rigidity. Subsequent heterocyclization of the arylamide to the central phenyl ring resulted in the aminoquinazoline deriva-

tive 37 (IC 50 = 1.1 nM) with a dramatic increase in potency and improved oral bioavailability in rats relative to the ureas and carboxamides (%F = 99% for 37 vs. 27% for 1). However, it should be mentioned that the oral bioavailability of 99% in rats was achieved by using a vitamin E D - alpha - tocopheryl polyethylene glycol succinate (TPGS) dosing vehicle. The plasma levels of 37 were signifi cantly diminished upon dosing as a standard methylcellulose suspension. The quinazoline 37 was potent and highly effi cacious in animal pain models, fully reversing carrageenan- induced thermal hyperalgesia in rats following oral dosing (minimum effi cacious dose of 0.1 mg/kg). Although the structure of the clinical candidate NGD- 8243/MK - 2295 from Neurogen/Merck has not been disclosed, it is believed to be a representative of the aminoquinazoline class of TRPV1 antagonists. Further improvement of oral exposure of compound 37 through optimization of its drug- like properties led to the 2 - methoxymethyl analogue 38 (Blum et al., 2008 ). The latter retains good in vitro and vivo potency of compound 37 and, more signifi cantly, is well absorbed following oral dosing. The SAR of the quinazoline series mimics that of the 2- pyridinylpiperazine carboxamide series. The preferred left- hand aryl substituent is a 3- substituted

(3 - CF 3 , 3 - CH 3, 3- Cl) pyridin- 2 - yl moiety, although a variety of ﬁ ve - and six - membered nitrogen - , oxygen - , and sulfur - containing heterocycles also result in TRPV1 antagonists with submicromolar potencies. A similar SAR trend between the two series has been observed in the aniline portion of

the molecule. Substitution at the 4- position with lipophilic groups (CF 3 , t - butyl, SO 2 - alkyl) is preferable. The 2 - position of the quinazoline moiety offers an additional point of diversity. A variety of substitutions such as 2 - alkyl, 2 - alkoxyalkyl, and 2 - hydroxylalkyl are well tolerated (Blum et al., 2008 ).

CF CF3 CF3 CF3 3

HN HN HN HN

CF N O CF O CF N N 3 3 3 CF N H 3 O O N N N N N N 136 37 38 Figure 12.10 Progression of piperazine core structures to amino quinazolines. 320 2-PYRIDINYLPIPERAZINE CARBOXAMIDE CLASS

CF3 CF3 CF3

HN HN HN

CF3 N CF3 CF3 N N N N N N N N N 39 40 41

CF3 CF3 CF3

HN HN HN N CF3 N CF3 N CF3 N N N N N N N N

42 43 44 Figure 12.11 Additional heteroaromatic bicyclic core replacements.

Similar conformationally restricted 6,6 - fused heterocycle - containing analogues have been disclosed in numerous patent applications (Bakthavatchalam et al., 2004a,b ; Brown et al., 2004 ; Bakthavatchalam et al., 2005a,b,c,d ; Bakthavatchalam et al., 2006a,b ; Caldwell et al., 2006 ). Compounds 39 – 44 (Fig. 12.11 ) with a variety of heterocyclic core replacements for the quinazoline core have been reported to be TRPV1 antagonists with IC50 values < 1000 nM. Additionally, Merck (Brown et al., 2004 ) has described a series of 6,5- fused heterocycle - containing compounds 45 – 53 (Fig. 12.12 ) as TRPV1 antagonists, although no speciﬁ c biological data are available.

12.1.5 Partially Saturated Bicyclic Heteroaryls Patent applications published almost simultaneously by Renovis (Kelly et al., 2005 ) and Amgen (Norman et al., 2005 ) have reported a novel class of bicy- cloheteroarylamine TRPV1 antagonists such as 54 (Fig. 12.13 ). Abbott Laboratories (Lee et al., 2006 ) subsequently disclosed an identical series of compounds without speciﬁ c biological data. The SAR of the three fragments of this molecule resembles that of the aryl carboxamides and quinazoline series. Compounds exhibiting IC 50 values < 100 nM contain the 3 - substituted pyridin - 2 - yl (3 - CF 3 , 3 - CH 3 , or 3 - Cl) moiety, para - substituted aniline ( t - butyl, SO2 CF3 , OCF3 , C(CH3 )2 CN, and 1,2,3,4- tetrahydro - 1,4,4 - trimethylquinoline), and 2 - thiomethyl, 2 - methoxymethyl, or 2 - methoxy substitution on the pyrimidine core. Janssen Pharmaceutica N.V. has also reported a series of tetrahydropyrimi- doazepine antagonists of TRPV1 (Allison et al., 2007 ). More recently, the same group disclosed a series of thiazolopyrimidine TRPV1 antagonists (Branstetter et al., 2008 ). CONCLUSION 321

CF CF 3 CF3 3 HN HN HN N N N CF N CF CF N 3 3 3 N N N N N N N N 45 46 47

CF CF 3 3 CF3 HN HN HN N N N CF3 N CF N CF3 N N 3 N N N N N N N N N 48 49 50

CF3 CF3 CF3 HN HN HN N CF N CF N CF N 3 3 N 3 N N N N N N

51 52 53 Figure 12.12 Representative 6,5 - fused heterocyclic core replacements.

CF3

Cl N N N N 54 Figure 12.13 Partially saturated bicyclic heterocyclic cores.

12.2 CONCLUSION

The 2- pyridinylpiperazine carboxamides are one of the fi rst classes of high- affi nity TRPV1 antagonists identifi ed from HTS campaigns by several research groups. This class of TRPV1 antagonists has provided important tool compounds for the exploration of the therapeutic utility of TRPV1 antagonists and represents a medicinal chemistry starting point toward the discovery of compounds with more suitable pharmaceutical properties. Substitution patterns and various structural requirements found in SAR development of the prototypical pyridinylpiperazines have been successfully applied to the design of new chemotypes such as quinazolines, tetrahydropyridines, and others. 322 2-PYRIDINYLPIPERAZINE CARBOXAMIDE CLASS

REFERENCES

Allison BD , Branstetter BJ , Breitenbucher JG , Hack MD , Hawryluk NA , Lebsack AD , McClure KJ , and Merit JE ( 2007 ) Preparation of 6,7,8,9 - tetrahydro - 5H - pyrimidoazepines as TRPV1 receptor modulators. WO Patent 2007109355 - A2 . Bakthavatchalam R ( 2002 ) Preparation of diarylpiperazines as capsaicin receptor ligands. WO Patent 008221 - A2 . Bakthavatchalam R , Blum CA , Brielmann H , Caldwell TM , Cortright DN , Hodgetts KJ , Peterson JM , and Zheng X ( 2006a ) Preparation of quinazolinamines and analogs as capsaicin receptor agonists. U.S. Patent 194805 - A1 . Bakthavatchalam R , Caldwell TM , Chenard BL , Hodgetts KJ , and Capitosti SM (2006b ) Preparation of substituted biaryl quinolin- 4 - ylamine analogues as capsaicin receptor modulators. WO Patent 042289 - A2 . Bakthavatchalam R , Chenard BL , Peterson JM , and Steenstra CK ( 2005a ) Preparation of arylalkylamino- substituted quinazolines as type VR1 capsaicin receptor modulators. WO Patent 087227 - A1 . Bakthavatchalam R , Blum CA , Brielmann H , Caldwell TM , Cortright DN , Hodgetts KJ , Peterson JM , and Zheng X ( 2005b ) Preparation of benzoimidazole, quinazoline and related N - based heterocyclic analogs as capsaicin receptor agonists. WO Patent 042498 - A2 . Bakthavatchalam R , Blum CA , and Chenard BL ( 2005c ) Substituted bicyclic quinazolin- 4 - ylamine derivatives as capsaicin receptor modulators. WO Patent 023807 - A2 . Bakthavatchalam R , Caldwell TM , Chenard BL , De Lombaert S , and Hodgetts KJ ( 2005d ) Preparation of substituted quinoline and naphthyridine amines as capsaicin receptor modulators. WO Patent 007652 - A2 . Bakthavatchalam R , Blum CA , Brielmann H , Caldwell TM , De Lombaert S , Hodgetts KJ , and Zheng X ( 2004a ) Preparation of carboxylic acid, phosphate, or phospho- nate substituted (quinazolin -4 - yl)amines as capsaicin receptor modulators. WO Patent 055004 - A1 . Bakthavatchalam R , Blum CA , Brielmann H , Caldwell TM , De Lombaert S , Hodgetts KJ , and Zheng X ( 2004b ) Preparation of (quinazolin - 4 - yl)amines as capsaicin receptor modulators. WO Patent 055003 - A1 . Bayliss T , Brown RE , Burkamp F , Jones AB , and Neduvelil JG ( 2005) E - Fluoro - 4 - (pyridin - 2 - yl) - piperidine - 1 - carboxamide derivatives and related compounds which modulate the function of the vanilloid - 1 receptor (VR1) for the treatment of pain. WO Patent 2005051390 . Behrendt HJ , Germann T , Gillen C , Hatt H , and Jostock R ( 2004 ) Characterization of the mouse cold - menthol receptor TRPM8 and vanilloid receptor type - 1 VR1 using a fl uorometric imaging plate reader (FLIPR) assay. Br J Pharmacol 141 : 737 – 745 . Bhattacharya A , Scott BP , Nasser N , Ao H , Maher MP , Dubin AE , Swanson DM , Shankley NP , Wickenden AD , and Chaplan SR ( 2007 ) Pharmacology and antitussive effi cacy of 4 - (3 - trifl uoromethyl - pyridin - 2 - yl) - piperazine - 1 - carboxylic acid (5 - trifl uoromethyl - pyridin - 2 - yl) - amide (JNJ 17203212), a transient receptor potential vanilloid 1 antagonist in guinea pigs . J Pharm Exper Ther 323 : 665 – 674 . REFERENCES 323

Blum CA , Zheng X , Brielman H , Hodgetts KJ , Bakthavatchalam R , Chandrasekhar J , Krause JE , Cortright D , Matson D , Crandall M , Ngo CK , Fung L, Day M , Kershaw M , De Lombaert S , and Chenard BL ( 2008 ) Aminoquinazolines as TRPV1 antagonists: modulation of drug- like properties through the exploration of 2- position substitution. Bioorg Med Chem Lett 18 : 4573 – 4577 . Branstetter BJ , Breitenbucher JG , Lebsack AD , and Xiao W ( 2008 ) Preparation of thiazolopyrimidine derivatives as modulators of TRPV1. U.S. Patent 004253 - A1 . Brown BS , Keddy R , Zheng GZ , Schmidt RG , Koenig JR , McDonald HA , Bianchi BR , Honore P , Jarvis MF , Surowy CS , Polakowski JS , Marsh KC , Faltynek CR , and Lee CH ( 2008 ) Tetrahydropyridine - 4 - carboxamides as novel, potent transient receptor potential vanilloid 1 (TRPV1) antagonists. Bioorg Med Chem 16 : 8516 – 8525 . Brown RE , Burkamp F , Doughty VA , Fletcher SR , Hollingworth GJ , Jones BA , and Sparey TJ ( 2004 ) Preparation of amino heterocycles as vanilloid receptor (VR1) modulators, in particular antagonists, for treating pain and/or inﬂ ammation. WO Patent 074290 - A1 . Caldwell TM , Chenard BL , and Hodgetts KJ ( 2006 ) Preparation of substituted pyridazinyl - and pyrimidinyl - quinolin - 4 -ylamine analogs for use in capsaicin VR1 receptor binding assays. WO Patent 081388 - A2 . Carruthers N ( 2005 ) Targeting the brain: successes and pitfalls . Annual One - Day Meeting on Medicinal Chemistry of SRC & KVCV, Namur, Belgium, November 25 . Carruthers NI , Shah CR , and Swanson DM ( 2005 ) Preparation of pyridyl piperazinyl ureas with VR1 antagonistic activity. WO Patent 014580 - A1 . Fletcher CR , McIver E , Lewis S , Burkamp F , Leech C , Mason G , Boyce S , Morrison D , Richards G , Sutton K , and Jones AB ( 2006 ) The search for novel TRPV1 - antagonists: from carboxamides to benzimidazoles and indazolones. Bioorg Med Chem Lett 16 : 2872 – 2876 . Gharat LA and Szallasi A ( 2008 ) Advances in the design and therapeutic use of capsaicin receptor TRPV1 agonists and antagonists. Expert Opin Ther Pat 18 : 159 – 209 . Ghilardi JR , Roehrich H , Lindsay TH , Sevcik MA , Schwei MJ , Kubota K , Halvorson KG , Poblete J , Chaplan SR , Dubin AE , Carruthers NI , Swanson D , Kuskowski M , Flores CM , Julius D , and Mantyh PW ( 2005 ) Selective blockade of the capsaicin receptor TRPV1 attenuates bone cancer pain. J Neurosci 25 : 3126 – 3131 . Kelly MG , Janagani S , Wu G , Kincaid J , Lonergan D , Fang Y , and Wei ZL ( 2005 ) Preparation of bicycloheteroarylamines like 2,6- naphthyridinamines and pyrido[3, 4- d]pyrimidinamine as ion channel ligands and uses thereof. WO Patent 066171 - A1 . Kyle DJ and Sun Q ( 2003 ) Preparation of pyridin - 2 - ylpiperazine derivatives for treating pain. WO Patent 2003066595 . Lee CH , Brown BS , Keddy RG , Perner RJ , and Koenig JR ( 2006 ) Preparation of pyridopyrimidines as antagonists to the vanilloid receptor subtype 1 (VR1). U.S. Patent 062981 - A2 . Norman MH , Ognyanov VI , and Pettus LH ( 2005 ) Preparation of substituted quinazolines and pyridopyrimidines as vanilloid receptor ligands. U.S. Patent 165032 - A1 . Ognyanov VI , Balan C , Bannon AW , Bo Y , Dominguez C , Fotsch C , Gore VK , Klionsky L , Ma VV , Qian YX , Tamir R , Wang X , Xi N , Xu S , Zhu D , Gavva NR , 324 2-PYRIDINYLPIPERAZINE CARBOXAMIDE CLASS

Treanor JJ , and Norman MH ( 2006 ) Design of potent, orally available antagonists of the transient receptor potential vanilloid 1. Structure - activity relationships of 2 - piperazin - 1 - yl - 1H - benzimidazoles . J Med Chem 49 : 3719 – 3742 . Pomonis JD , Harrison JE , Mark L , Bristol DR , Valenzano KJ , and Walker K ( 2003 ) N - (4 - Tertiarybutylphenyl) - 4 - (3 - chloropyridin - 2 - yl)tetrahydropyrazine - 1(2H) - carboxamide (BCTC), a novel, orally effective vanilloid receptor 1 antagonist with analgesic properties: II. in vivo characterization in rat models of infl ammatory and neuropathic pain. J Pharm Exp Ther 306 : 387 – 393 . Sun G and Wen X ( 2005 ) Heteroaryl - tetrahydropyridyl compounds useful for treating or preventing pain. WO Patent 2005009988 . Sun Q , Tafesse L , Islam K , Zhou X , Victory SF, Zhang C , Hachicha M , Schmid LA , Patel A , Rotshteyn Y , Valenzano KJ , and Kyle DJ ( 2003 ) 4 - (2 - pyridyl)piperazine - 1 - carboxamides: potent vanilloid receptor 1 antagonists. Bioorg Med Chem Lett 13 : 3611 – 3616 . Sun Q , Wen X , and Zhou X ( 2005 ) Piperidine compounds and pharmaceutical compositions containing them. WO Patent 2005009987 . Swanson DM , Dubin AE , Shah C , Nasser N , Chang L , Dax SL , Jetter M , Breitenbucher JG , Liu C , Mazur C , Lord B , Gonzales L , Hoey K , Rizzolio M , Bogenstaetter M , Codd EE , Lee DH , Zhang SP , Chaplan SR , and Carruthers NI ( 2005 ) Identifi cation and biological evaluation of 4- (3 - trifl uoromethylpyridin - 2 - yl)piperazine - 1 - carboxylic acid (5 - trifl uoromethyl - pyridin - 2 - yl)amide, a high affi nity TRPV1 (VR1) vanilloid receptor antagonist. J Med Chem 48 : 1857 – 1872 . Tafesse L , Sun Q , Schmid L , Valenzano KJ , Rotshteyn Y , Su X , and Kyle DJ ( 2004 ) Synthesis and evaluation of pyridazinylpiperazines as vanilloid receptor 1 antagonists . Bioorg Med Chem Lett 14 : 5513 – 5519 . Valenzano KJ , Grant ER , Wu G , Hachicha M , Schmid L , Tafesse L , Sun Q , Rotshteyn Y , Francis J , Limberis J , Malik S , Whittemore ER , and Hodges D ( 2003 ) N - (4 - Tertiarybutylphenyl) - 4 - (3 - chloropyridin - 2 - yl)tetrahydropyrazine - 1(2H) - carboxamide (BCTC), a novel, orally effective vanilloid receptor 1 antagonist with analgesic properties: I. in vitro characterization and pharmacokinetic properties. J Pharm Exp Ther 306 : 377 – 386 . Zheng X , Hodgetts KJ , Brielmann H , Hutchison A, Burkamp F , Jones AB , Blurton P , Clarkson R , Chandrasekhar J , Bakthavatchalam R , De Lombaert S , Crandall M , Cortright D , and Blum CA ( 2006 ) From arylureas to biarylamides to aminoquinazolines: discovery of a novel, potent TRPV1 antagonist. Bioorg Med Chem Lett 16 : 5217 – 5221 . 13 TRPV 1 AGONIST APPROACHES FOR PAIN MANAGEMENT

Keith R. Bley

13.1 INTRODUCTION

Capsaicin and other naturally occurring pungent molecules have been used for centuries as topical analgesics to treat a variety of painful conditions. However, only within the last 12 years has it been appreciated that the selective action of capsaicin and similar compounds on nociceptive sensory nerve fi bers is mediated by agonism of a ligand- gated ion channel called the transient receptor potential vanilloid 1 (TRPV1). The selective expression of TRPV1 on nociceptors— those nerve fi bers specialized for the detection of stimuli associated with tissue injury— in the skin, bladder, joints, and other tissues has resulted in this receptor becoming an important target for analgesic drug development. Two different, but possibly complementary, strategies are being pursued: optimization of TRPV1 agonist - based therapies, which can locally defunctionalize nociceptive nerve fi bers for weeks to months, and identifi cation of TRPV1 receptor antagonists, which would reduce nociceptive fi ber activation by infl ammatory stimuli. This chapter will focus on recent advances in the understanding of drugs and treatments that attempt to use naturally occurring or synthetic TRPV1 agonists to alter the function of nociceptive sensory nerves and consequently cause pain relief. The terms nociceptor and C - fi ber will often be used inter- changeably, even though there is not always a complete overlap of the two sensory nerve fi ber categories. Nociceptors actually consist of not only C - fi bers

325 326 TRPV1 AGONIST APPROACHES FOR PAIN MANAGEMENT

but also some A - ﬁ bers (Johanek et al., 2006 ), which parallels the selective expression of TRPV1 receptors in nerves originating from small- and some medium- sized cell bodies. Evidence for hypotheses regarding the basis of nociceptor hyperactivity in pain syndromes will be reviewed, and the prospects for efﬁ cacy of locally administered therapies against various indications will be evaluated.

13.2 TRPV 1 AGONIST - INDUCED NOCICEPTOR DEFUNCTIONALIZATION

When activated by a combination of heat, acidosis, or endogenous agonists, TRPV1 initiates signal transmission to the spinal cord by depolarizing nociceptive sensory nerve endings and by generating action potentials, which may be experienced by the brain as either warming, burning, stinging, or itching sensations (Fig. 13.1 ). However, if TRPV1 is activated continuously by ongoing exposure to chemically stable exogenous agonists such as capsaicin, a local biochemical signal can also be generated in these nerve ﬁ bers, which can produce long - lasting effects on nociceptive ﬁ ber functionality (Szallasi and

Activators of TRPV1

Brain: burning pain sensation H+

Heat Acidosis Endogenous/ exogenous agonists Sensory neuron Ca2+

Na+

Cell TRPV1 membrane Spinal cord

Action potentials initiated

Local defunctionalization

Figure 13.1 Activation of TRPV1 leads to sensations of heat, burning, stinging, or itching and can also result in localized nociceptor defunctionalization. NOCICEPTOR HYPERACTIVITY 327

Blumberg, 1999 ). The TRPV1 channel is highly calcium permeable, allowing calcium to fl ow down its steep electrochemical gradient into the cell. Furthermore, as TRPV1 is also expressed on intracellular organelles, external capsaicin application can cause release of calcium from the endoplasmic reticulum and can even induce additional intracellular calcium release from internal stores via calcium- dependent calcium release (Han et al., 2007 ). If TRPV1 is activated in this continuous fashion, high levels of intracellular calcium and associated enzymatic and osmotic changes can induce processes that impair local nociceptor function for extended periods (Szallasi and Blumberg, 1999 ). A persistent lack of responsiveness to stimuli that would normally cause nociceptor activation has often been termed “ desensitization. ” The use of this term in TRPV1 agonist literature arises from psychophysical studies of human subjects who display reduced reactions to painful stimuli applied to areas pretreated with TRPV1 agonists and is not narrowly confi ned to a direct desensitization of TRPV1 receptors or their intracellular signaling mechanisms (Szallasi and Blumberg, 1999 ). Hence, the emerging preferred term for the persistent effects of capsaicin is “ defunctionalization ” (Holzer, 2008 ), which allows avoidance of conceptual confusion with the intrinsic desensitization of the TRPV1 receptor. It is important to remember that although evidence from immunohistochemical studies suggests that capsaicin can produce highly localized loss of nociceptive nerve fi bers (e.g., Polydefkis et al., 2004 ), impaired nociceptor functionality is not contingent upon nerve fi ber degeneration but may also be due to reductions of electrical excitability, fast axonal transport, or intrinsic desensitization of TRPV1 receptors.

13.3 NOCICEPTOR HYPERACTIVITY

Defunctionalization of nociceptive nerve fi bers may constitute an important therapeutic intervention if they are spontaneously hyperactive or hypersensitive to stimuli which would normally be innocuous. Following acute injury, the basis for the hyperactivity and/or hypersensitivity of nociceptive nerve endings in affected tissues is well established (Johanek et al., 2006 ), and the protective behaviors that result from nociceptor hyperactivity are considered fundamental to tissue repair and avoidance of additional damage. As discussed below, TRPV1 agonist- based therapies are being developed for acute traumatic pain syndromes because it is widely recognized that many types of acute pain (e.g., following surgery) can interfere with healing processes and require the use of pain medicines with unwanted adverse effects. In chronically painful conditions, particularly neuropathic pain syndromes, clinical and nonclinical research show collectively that the most peripheral aspects of damaged sensory nerves often display aberrant “ pathophysiological” electrical hyperactivity (Michaelis, 2002 ). However, direct correlations between aberrant activity of nociceptors and patient pain reports have proven diffi cult to demonstrate due to the technical complexity of measuring electrical 328 TRPV1 AGONIST APPROACHES FOR PAIN MANAGEMENT activity in small - diameter nerve fi bers. A technique known as microneurography, which measures action potentials extracellularly, can be used, but this diagnostic procedure is somewhat invasive and may cause discomfort (Hagbarth, 2002 ). Moreover, microneurography has the greatest utility for the long nerves of the legs and arms, and thus, those chronic pain syndromes with primary presentations in the trunk or face (e.g., lower back pain or postherpetic neuralgia [PHN]) are diffi cult to analyze. Consequently, there are limited published studies that correlate nociceptive nerve fi ber hyperactivity with patient reports of chronic pain, and they all involve neuropathic pain of the extremities. For instance, a systematic study of hyperactive nociceptors in patients with erythromelalgia (burning pain of the feet due to mutations in voltage- dependent sodium channels) showed altered conduction velocities and spontaneous activity or sensitization in some mechano - insensitive C - fi bers (Orstavik et al., 2003 ). In patients with painful small- fi ber polyneuropathy, both polymodal and mechanically insensitive C- fi bers were hyperexcitable, as indicated by reduced receptor thresholds, spontaneous discharge, and multi- plied responses to stimulation (Ochoa et al., 2005 ). Interestingly, it was suggested that the clinical and electrophysiological profi les of these patients resembled the effects of experimental capsaicin application to the skin. In contrast to the limited clinical data, spontaneous activity of distal nociceptive fi bers following nerve injury has been recorded extensively in nonclinical models and has been correlated directly with pain behaviors. For instance, transection of the sciatic nerve in rodents is a long - standing model in which spontaneous or “ ectopic ” electrical activity of the resulting neuroma (the injured tip of a nerve fi ber) develops, and nerve fi bers terminating in the neuroma become extremely sensitive to stimuli (Wall and Devor, 1983 ). However, although highly instructive regarding basic mechanisms of neural excitability, neuromas in large nerves represent only a very small fraction of clinically presenting peripheral neuropathies. Polyneuropathies in which some innervation of the skin or other target organ remains intact, such as due to diabetes, are much more common and are clinically important (Bennett, 1998 ). Accordingly, several recent nonclinical studies have focused on the excitability of intact nociceptors following mechanical injuries to surrounding nerve fi bers. For instance, 1 day following ligation and transection of the L5 spinal nerve in rats, about one- half of the uninjured C- fi ber nociceptors in the L4 spinal nerve develop spontaneous activity (Wu et al., 2001 ) and a signifi cant proportion of uninjured C - fi bers display altered action potential conduction properties (Shim et al., 2007 ). Similarly, 7 days following rhizotomy of L5 ventral roots (which leads predominantly to degeneration in myelinated fi bers) in rats, a marked decrease in paw withdrawal thresholds occurs concomitantly with increased low - frequency C - fi ber spontaneous activity (Wu et al., 2002 ). Furthermore, after partial denervation of the dorsum of the foot is induced by tight ligations of spinal nerve L6 in primates (Ali et al., 1999 ), there is a signifi cantly higher incidence of spontaneous activity in uninjured single C - fi bers in the superfi cial peroneal nerve, recorded using an in vitro skin/nerve BASES FOR NOCICEPTOR HYPERACTIVITY 329 preparation. Taken together, these data provide substantial evidence that a nerve injury leads to persistent alterations in the properties of adjacent uninjured C - fi bers.

13.4 BASES FOR NOCICEPTOR HYPERACTIVITY

The projections of nociceptors into target organs can be visualized and quantifi ed by immunostaining of antigens selectively expressed in neurons. Protein gene product 9.5 (PGP 9.5) is the most commonly studied marker, although substance P, calcitonin gene- related peptide (CGRP), or others have also been used. Antibodies to PGP 9.5 stain all nerve fi bers, but because nerve fi bers in the epidermis of the skin, in the uroepithelium of the bladder, and in the mucosa are almost exclusively nociceptors (see below), changes in the density of these fi bers may be quantifi ed (Kennedy, 2004 ). In order to understand the mechanisms underlying aberrant activity of nociceptive nerve fi bers in chronic pain syndromes, one key factor may be changes in the density of innervation of the target organ; that is, in most pain syndromes that would be considered neuropathic, sensory neuron axons are lost due to either cell body or nerve fi ber damage following viral, metabolic, traumatic, or chemical insults. In contrast, in chronically painful conditions that do not involve direct injury to axons, there may be increased nociceptor innervation of target organs. Immunohistochemical analyses have indicated that the density of epidermal nerve fi bers in the skin is decreased in a wide range of neuropathic pain syndromes, including PHN (Rowbotham et al., 1996 ; Oaklander, 2001 ), diabetic neuropathy (Kennedy et al., 1996 ) and even metabolic syndrome (Pittenger et al., 2005 ), painful HIV - associated neuropathy (HIV - AN) (Polydefkis et al., 2002 ), Fabry disease (Scott et al., 1999 ), and small- fi ber neuropathy (Pittenger et al., 2004 ; Lauria et al., 2006 ). Moreover, data suggest a positive correlation between the extent of epidermal nerve fi ber loss and the severity of pain in diabetic neuropathy (Kennedy et al., 1996 ) and HIV - AN (Polydefkis et al., 2002 ). This observation also extends to nonclinical models: in one study with rats following a chronic constriction injury, there was a very signifi cant reduction of PGP 9.5- and CGRP- immunoreactive fi bers in the epidermis of the footpad on the same day as maximum pain behavior (Lindenlaub and Sommer, 2002 ). Conversely, there are chronically painful syndromes associated with increased nociceptor density; examples include interstitial cystitis (IC) (Christmas et al., 1990 ), nostalgia paresthetica (Springall et al., 1991 ), vulvodynia (Bohm- Starke et al., 1998 ; Tympanidis et al., 2003 ), rectal hypersensitivity (Chan et al., 2003 ), and gastroesophageal refl ux disease (Matthews et al., 2004 ). These conditions do not refl ect nerve injury per se, but can fall under the rubric of neuropathic pain due to the inclusion of dysfunctional sensory nerves under the defi nition of neuropathic pain (Merskey and Bogduk, 1994 ). 330 TRPV1 AGONIST APPROACHES FOR PAIN MANAGEMENT

Intact axon

Damaged axon

Neurotrophic factors Skin Spinal cord Cytokines

Dermis Epidermis

Figure 13.2 Overexposure to neurotrophins and proinﬂ ammatory cytokines may underlie the hyperactivity or the hypersensitivity of intact nociceptors.

There is evidence that intact nociceptor nerve terminals may develop abnormal electrophysiological properties due to exposure to abnormal concentrations of neurotrophins such as nerve growth factor (NGF) and proinfl ammatory cytokines (Fig. 13.2 ). In chronic pain syndromes associated with deinnervation, pain intensity may correlate with reduced nociceptor immunostaining because when there are only a small number of intact nociceptive endings, it is more likely those endings are pathologically active (Campbell, 2001 ). Although NGF has an important role in controlling the survival and development of small- diameter neurons— both sensory and sympathetic— it has recently become clear that NGF also serves as an important signal for neuroimmune and infl ammatory processes in mature organisms (Petruska and Mendell, 2004 ). In the skin, NGF and other neurotrophins are constantly produced by keratinocytes and possibly Langerhans cells (Pincelli, 2000 ). NGF is also produced by human bladder smooth muscle cells (Tanner et al., 2000 ), and increased levels of NGF have been reported in bladder tissues from both humans and rodents when there is outlet obstruction (Gosling et al., 1986 ). Human chondrocytes synthesize NGF and production is upregulated in osteoarthritic chondrocytes (Iannone and Lapadula, 2003 ; Manni et al., 2003 ). In response to enhanced NGF supply (e.g., in infl amed bladders or in osteoarthritis [OA]), residual nociceptors may respond by becoming hyperactive and possibly sprouting. Support for this TRPV1 AGONIST-INDUCED DISRUPTION OF NOCICEPTOR HYPERACTIVITY 331 hypothesis can be found in the well- characterized immediate and delayed excitatory effects of NGF. As a direct excitatory stimulant, NGF causes immediate excitation of nociceptors (Bonnington and McNaughton, 2003 ), resulting in prolonged hyperalgesia and allodynia (Dmitrieva and McMahon, 1996 ; Bowles et al., 2006 ) and bladder overactivity (Lamb et al., 2004 ). In addition to this direct and rapid effect, retrograde transport of NGF to sensory neuron cell bodies may lead to the upregulation of proexcitatory proteins, such as TRPV1 and voltage - activated sodium channels (Ji et al., 2002 ), and to the downregulation of anti - excitatory proteins, such as voltage - activated potassium channels (Zhang and Li Wan Po, 1994 ). Consistently, neutralization of NGF has been reported to reduce pain behaviors in some rodent models of neuropathic pain (Ramer and Bisby, 1999 ) and bladder overactivity (Jaggar et al., 1999 ). Proinfl ammatory cytokines can also directly activate and modify gene expression in sensory neurons. There are several sources of these molecules in close proximity to peripheral nerves. Schwann cells, which have often been thought of as having only a passive support role for peripheral nerves, are able to secrete proinfl ammatory cytokines (Rutkowski et al., 1999 ; Watkins and Maier, 2002 ). Wallerian degeneration is a posttraumatic process of the peripheral nervous system whereby damaged axons and their surrounding myelin sheaths are phagocytosed by infi ltrating macrophages or leukocytes. During the process of infi ltration of infl amed or damaged peripheral nerves, these immune system cells are known to secrete proinfl ammatory cytokines (Shamash et al., 2002 ). Additionally, in the skin, keratinocytes can also release proinfl ammatory cytokines (Southall et al., 2003 ).

13.5 TRPV 1 AGONIST - INDUCED DISRUPTION OF NOCICEPTOR HYPERACTIVITY

The long- term defunctionalization of nociceptors by TRPV1 agonist exposure may not only reduce the ability of nociceptive nerve endings to initiate electrical signals but may also inhibit axonal transport. Even before capsaicin- induced disruption of axonal transport was observed by video- enhanced microscopy (Kawakami et al., 1993 ), it was known that nociceptor nerve terminals exposed to capsaicin lose the capacity to take up and retrogradely to transport neurotrophic factors such as NGF to the cell body (Miller et al., 1982 ; Taylor et al., 1984 ). Without a constant supply of NGF, many nociceptors lose their ability to maintain a hyperexcitable phenotype (Petruska and Mendell, 2004 ). Hence, TRPV1 agonist treatments may actually alter the phenotype of nociceptors by depriving them of proexcitatory infl uence from their target organs. The onset of effi cacy and duration of pain relief or reduced bladder activity after discontinuation of TRPV1 agonist treatment may be related to the rate at which nociceptive nerve endings lose and then regain functionality (Bjerring 332 TRPV1 AGONIST APPROACHES FOR PAIN MANAGEMENT et al., 1990 ; Nolano et al., 1999 ). Although nociceptors can remain functionally inactive for weeks following the topical application of capsaicin, they eventually return to pretreatment levels of sensitivity. Changes in nociceptor immunostaining also occur with the same time course as the effect. As is the case with impaired functionality, immunostaining reductions are reversible, probably through the natural processes of nerve fi ber elongation and protein recycling.

13.6 POTENTIAL INDICATIONS FOR LOCALLY ADMINISTERED TRPV 1 AGONISTS

Early nonclinical results obtained with TRPV1 agonists such as olvanil (Fig. 13.3 ), which have reduced pungency or are allegedly nonpungent, were encouraging (Brand et al., 1987 ). However, given the ubiquitous distribution of TRPV1 - expressing sensory nerve fi bers throughout the body, systemic TRPV1 agonist- based therapies are unlikely to be viable since widespread and persistent nociceptor defunctionalization may produce an unacceptable side - effect profi le. In contrast, directed TRPV1 agonist therapies, in which nociceptor defunctionalization is restricted to discrete target organs or regions, remain a tenable and potentially attractive means to control localized pain or neurogenic infl ammation. In addition to highly localized defunctionalization of nociceptive terminals in the skin or in other peripheral sites, another approach to a TRPV1 agonist - based therapy is possible: use of TRPV1 agonists in a neurolytic procedure that targets nociceptive neuronal cell bodies with high selectivity. Nonselective neurolytic procedures involving injection of substances such as ethanol and

O O O O N N H H O O H H trans-Capsaicin cis-Capsaicin (civamide)

O H HO HO O H O O N O O H O O HO O

Olvanil (NE-19550) Resiniferatoxin Figure 13.3 Structures of selected TRPV1 agonists. PAINFUL PERIPHERAL NEUROPATHIES 333 phenol were fi rst described in 1931 and are still utilized to control intractable chronic pain syndromes (Candido and Stevens, 2003 ). Nonclinical studies have confi rmed the effi cacy and limited side effects of capsaicin or resiniferatoxin (RTX, Fig. 13.3 ) when these substances are injected into the vicinity of sensory ganglia (Karai et al., 2004 ; Tender et al., 2005 ). To debate the justifi cations for the development of new, highly invasive neurolytic procedures is beyond the scope of this chapter, but there can be no doubt that such a therapy would face intense scrutiny from contemporary regulatory authorities and, even if approved, would almost certainly be relegated to use as a last resort.

13.7 PAINFUL PERIPHERAL NEUROPATHIES

The skin is innervated with approximately one million sensory nerve fi bers, with their cell bodies in sensory ganglia (i.e., dorsal root, trigeminal, or nodose ganglia). The main nerve branches enter the subdermal fatty tissue and then divide into smaller bundles that fan out laterally to form a branching network that ascends— often accompanying blood vessels— to form a mesh of interlac- ing nerves in the superfi cial dermis, with some fi bers extending into the epidermis (Kennedy, 2004 ). The epidermis contains almost exclusively unmyelinated C - fi ber nociceptive nerve endings, which specialize in the detection of noxious stimuli (Simone et al., 1998 ; Dux et al., 1999 ). As there are obviously many nociceptive nerve fi bers also in the dermis, the term cutaneous nociceptor is used to refer collectively to those nociceptors that may terminate in the epidermis or may course through the dermis. As discussed above, pathological activity of cutaneous nociceptors is implicated in chronic pain syndromes. Capsaicin and closely related vanilloid compounds have been used as topical analgesic agents for centuries. The fi rst formal report of the pain - reducing properties of capsaicin in the West appeared in 1850 as a recom- mendation to use an alcoholic hot pepper extract on burning or itching extremities (Turnbull, 1850 ). Creams, lotions, and patches containing capsaicin, generally in the range of 0.025 – 0.1% by weight, are now sold, usually without the requirement of a prescription, for the treatment of neuropathic and musculoskeletal pain. Clinical studies of these low - concentration medications, usually involving three to fi ve topical applications per day for periods of 2– 6 weeks, have often suggested benefi cial effects for the treatment of many disorders, including PHN, diabetic neuropathy, OA, and even psoriasis (Mason et al., 2004 ; Hempenstall et al., 2005 ). Since these low - concentration, capsaicin - based products often result in contamination of the patient ’ s environment (clothing, bedding, contact lenses, etc.) and each application is associated with a burning sensation, poor patient compliance with these products is often cited as a likely contributor to limited effi cacy (Paice et al., 2000 ). Initial support for the utility of single or episodic high- concentration topical capsaicin exposure was derived from compassionate treatment of 10 patients (a) NGX-4010 Control

–10

–20

–30

–40 ************

Percentage change from baseline change from Percentage 1 2345678910 11 12 Weeks NGX-4010, n 206 203 202 202 202 199 198 197 196 190 187 187 185 Control, n 196 189 189 189 190 186 186 185 184 180 177 177 172 *p < 0.05.

(b) NGX-4010 pooled dose groups Control pooled dose groups

0 *** ** *** *** *** **** ***

–10

–20

–30

Percentage change from baseline change from Percentage –40 10 2345678910 11 12 Weeks after treatment visit *p < 0.05; **p ≤ 0.01; ***p ≥ 0.001. (c) 100

40 VAS score VAS

04 h8 hDay 1Day 1 Day 2 Day 2 Day 3 Day 3 Day 4 Morning Evening Morning Evening Morning Evening Morning Figure 13.4 Time course of average pain relief following single treatments with (a) NGX- 4010 in PHN, (b) NGX- 4010 in painful HIV- AN, and (c) ALGRX- 4975 in posth- erniotomy pain. VAS, visual analog scale. TRAUMATIC AND MUSCULOSKELETAL PAIN 335 with intractable pain syndromes (Robbins et al., 1998 ). A high- concentration capsaicin - containing patch designated as NGX - 4010 has completed several Phase 3 studies in pain associated with peripheral neuropathies (NeurogesX, 2008a ). Phase 1 data suggest that a single 60 - min patch application is adequate to induce substantial nociceptor defunctionalization, as measured by reductions in thermal sensitivity and epidermal PGP 9.5 immunostaining (Malmberg et al., 2004 ). Published Phase 3 data suggest signiﬁ cant potential for efﬁ cacy against painful HIV - AN (Simpson et al., 2008a,b ) and PHN (Backonja et al., 2009 ). Figure 13.4 shows the average pain reduction over the course of 12 weeks, which follows a single 30 - to 90 - min administration of the NGX - 4010 patch to patients with PHN or painful HIV- AN. A marketing application for NGX - 4010 (now known as Qutenza™ ) has been approved in the European Union and is under review in the U.S. (NeurogesX, 2009 ). Systemic exposure to capsaicin from this product appears to be very low (Babbar et al., 2009 ).

13.8 TRAUMATIC AND MUSCULOSKELETAL PAIN

Until recently, TRPV1 agonist - induced defunctionalization was not considered as a therapeutic alternative for surgical or traumatic injuries. But now, a liquid formulation containing a high concentration of capsaicin, designated as ALGRX- 4975, is undergoing clinical evaluation for use in a variety of surgical procedures, including bunionectomy, herniotomy, and total knee replacement (Remadevi and Szallasi, 2008 ). During a surgical procedure, this liquid formulation is delivered directly onto the cut surfaces of skin, muscle, and bone, with the expectation that defunctionalization will occur before local and general anesthetics have worn off. In addition, ALGRX - 4975 is being investigated for the management of pain associated with interdigital neuromas (Morton ’ s neuroma) and end - stage OA of the knee (Anesiva, 2008 ). With respect to OA, it has been proposed that the erosion of articular cartilage uncovers subchondral bone, allowing nociceptive nerve fi bers within the bone to be activated by the mechanical forces of normal weight bearing (Niv et al., 2003 ). At this time, there is no published information regarding TRPV1 expression in joints or changes in expression in patients with disease. However, in the rat iodoacetate model of OA, there is increased expression of TRPV1 in both sensory cell bodies and nerve fi bers (Fernihough et al., 2005 ). Given the potential contribution of NGF to OA (Iannone and Lapadula, 2003 ), a role of increased TRPV1 expression and/or C- fi ber sprouting would not be surprising. Interestingly, increases in expression of TRPV1 in synovial fi broblasts from patients with OA and rheumatoid arthritis have been reported (Engler et al., 2007 ). Since this study analyzed both protein and RNA levels, it is not subject to concerns regarding a lack of TRPV1 antibody specifi city; however, these concerns do exist for many reports based on data obtained with commercially available TRPV1 antibodies. In contrast to the injectable formulation of capsaicin that provides for direct delivery to joints, topical low - concentration capsaicin has been evaluated as a 336 TRPV1 AGONIST APPROACHES FOR PAIN MANAGEMENT treatment for OA in four double- blind vehicle- controlled clinical trials. From these data, albeit limited by the potential for inadequate blinding due to the lack of vehicle pungency, it can be inferred that topical capsaicin appears effective, either as a monotherapy or as an adjunctive therapy (Rains and Bryson, 1995 ). Civamide is the generic name for cis- capsaicin (which is also known as zucap- saicin; see Fig. 13.3 ). trans - Capsaicin is the naturally occurring form of capsaicin, whereas cis - capsaicin must be synthesized (Cordell and Araujo, 1993 ). Following systemic administration, civamide has been reported to be active in rodent models of nociceptive and neuropathic pain (Hua et al., 1997 ). Topical 0.075% civamide cream has recently been shown to be more effective than an active control in a large double- blind, placebo- controlled Phase 3 trial for OA (Winston Laboratories, Inc., 2005 ). A marketing application for civamide cream was submitted in the European Union in 2008 (Winston Laboratories, Inc., 2008 ). Assuming that the highly lipophilic capsaicin isomers are not working via transdermal delivery and selective accumulation in joints, the basis for the effi cacy of topical TRPV1 agonists in OA may seem mysterious, unlike clinical conditions with known involvement of nociceptors in the skin. One study provides an intriguing clue by suggesting that cutaneous nociceptors actually are affected in OA (and rheumatoid arthritis). This study found that patients reported regions of both tactile allodynia and hypoesthesia in the skin superfi cial to diseased joints, much like those reported with many peripheral neuropathies (Hendiani et al., 2003 ). This sensitivity of the skin at some dis- tance from the joint resembles the cutaneous allodynia often reported during migraine attacks (see below).

13.9 MIGRAINE AND HEADACHE

Civamide has been investigated clinically for a number of indications, including prophylaxis of migraine and episodic cluster headache. Phase 2 data regarding a 0.025% (w/v) intranasal liquid spray dosed daily for migraine (Diamond et al., 2000 ) and cluster headache (Saper et al., 2002 ) were encouraging. As expected, civamide displays pungency similar to capsaicin, and thus nasal irritation and lacrimation are listed as frequent adverse events. Although called “ an orally active capsaicin analogue” in an early publication (Hua et al., 1997 ), at this time, no peer- reviewed data have appeared that support any signiﬁ cant pharmacological or pharmacokinetic differences between cis- and trans - capsaicin. Indeed, the structure – activity relationship of capsaicin analogues suggests that the double bond at this position on the aliphatic chain should have little impact on interactions with TRPV1 (Janusz et al., 1993 ). Thus, it is not surprising that repeated intranasal capsaicin administrations are also reported to successfully reduce the frequency of migraine attacks (Fusco et al., 2003 ). BLADDER PAIN AND OVERACTIVITY 337

13.10 VULVODYNIA AND MUCOSAL DISORDERS

Vulvodynia is a pain syndrome characterized by painful burning sensations, allodynia, hyperalgesia, and itching, usually localized in the region of the vulvar vestibules (Masheb et al., 2000 ). Vulvar tissue arises from the same urogenital progenitors as the bladder; hence, it might not be surprising to fi nd parallels involving hyperproliferation of nociceptive fi bers. In vulvodynia patients, the hypersensitivity of vulvar C- fi bers is well documented (Sonni et al., 1995 ; Bohm- Starke et al., 2001 ), and immunohistological evaluation of small- diameter nociceptive nerve fi bers shows increased densities relative to normal subjects (Bohm- Starke et al., 1998 ). Moreover, TRPV1 expression appears to be signifi cantly increased in these proliferated nociceptors (Tympanidis et al., 2004 ). Low- concentration topical capsaicin has been evaluated in two open- label studies as a treatment for vulvar vestibulitis, the most common form of vulvodynia. In the fi rst study, improvement of symptoms was recorded in 59% of patients after 30 days of twice - daily treatment, but no complete remission was observed (Murina et al., 2004 ). Symptoms recurred in all patients after the use of capsaicin cream was discontinued. A return to a twice - weekly topical application of the cream resulted in the improvement of symptoms. Severe burning was reported as the only side effect by all the patients. In the other study, following 12 weeks of daily applications, signifi cant improvements in pain, irritation, and ability to engage in sexual intercourse were reported (Steinberg et al., 2005 ). Similar patterns of enhanced TRPV1 expression occur in rectal hypersensitivity syndrome, which includes fecal urgency and incontinence as symptoms (Tympanidis et al., 2003 ). Increases in TRPV1 expression appear to correlate with decreases in heat and distension sensory thresholds. Similar to vulvodynia, topical capsaicin has been used with success to treat intractable anal pruritis (Lysy et al., 2003 ).

13.11 BLADDER PAIN AND OVERACTIVITY

The bladder is richly innervated with nociceptive sensory nerve fi bers, which can detect bladder distension or the presence of irritant chemicals; activation of these fi bers triggers refl ex bladder contraction and emptying (Cruz, 2004 ). However, under normal conditions, C - fi ber - initiated refl exes are not the primary control mechanism for the bladder; instead, a long pathway passing through the pontine micturition center and initiated by activation of capsaicin- resistant A δ - fi bers controls most normal bladder contractions. Although there is a short neuronal pathway contained entirely within the sacral spinal cord and initiated by activation of capsaicin - sensitive bladder C - fi bers, this pathway is usually inhibited in adult mammals unless there are pathologies such as infl ammation or spinal transection (de Groat, 1997 ). Thus, under pathophysiological conditions, bladder contractions triggered by capsaicin- sensitive 338 TRPV1 AGONIST APPROACHES FOR PAIN MANAGEMENT

C - fi bers and mediated by the sacral refl ex are involuntary and can be triggered by small volumes of urine, characteristics that generate an urge to urinate, urinary incontinence, and a high urinary frequency. Moreover, these C - fi ber - initiated contractions lack coordination with urethral sphincter muscle relaxation and can lead to increased intravesicular pressure and potential harm to the upper urinary tract (Cruz, 2004 ). Interestingly, TRPV1 is expressed not only in nociceptive fi bers that form close contacts with bladder epithelial (uroepithelial) cells but also in uroepithelial cells themselves (Birder et al., 2001 ), suggesting that uroepithelial cells may work in concert with underlying afferent nerves to detect the presence of irritating stimuli. In conditions characterized as IC, overactive or neurogenic bladder, and detrusor instability (except due to spinal cord injury), an increased density of afferent/nociceptive nerve fi bers has been reported, based upon increased levels of immunostaining for PGP 9.5 and other markers (Christmas et al., 1990 ; Brady et al., 2004 ). As TRPV1 immunostaining is clearly colocalized with nociceptive fi bers in the bladder (Yiangou et al., 2001 ), it is possible that proliferating nociceptors may also express enhanced levels of TRPV1. Interest in TRPV1 agonists for the treatment of overactive bladder and chronic bladder pain began with the need to treat patients with neurogenic detrusor overactivity, frequently due to spinal cord injury. In order to provide compassionate treatment for patients without alternatives, capsaicin was instilled intravesicularly for the fi rst time in 1989 (Maggi et al., 1989 ). These and subsequent unblinded investigations have used single administrations of a high- concentration (usually 1 – 2 mM) solution for about 30 min. Effi cacy from single capsaicin instillations has been reported to last from 1 to 3 months, with some patients showing clinical benefi t for up to 1 year (Chancellor and de Groat, 1999 ). In a meta - analysis of unblinded treatments for 131 patients, mean bladder capacity increased by ∼50% at various times after treatment and mean symptomatic improvement was ∼70%. The primary acute adverse effects reported were suprapubic pain, increased incontinence or macroscopic hematuria, all of these usually resolved within 1 week of therapy. No long - term adverse events or safety issues have been reported (Chancellor and de Groat, 1999 ). RTX, which is a naturally occurring tricyclic diterpene from Euphorbia cactus, is an ultrapotent TRPV1 agonist (Szallasi and Blumberg, 1999 ) often referred to as a nonpungent TRPV1 agonist, even though clinical experience still shows some pungency (Chancellor and de Groat, 1999 ). RTX was licensed from the U.S. National Institutes of Health in 1995, with the goal of developing a well- tolerated treatment for neurogenic/overactive bladder or IC. Clinical trials suggested that some patients experienced signifi cant relief of incontinence or pain- associated IC with few side effects (Lazzeri et al., 1997 ). Additional studies examined the utility in patients with refractory and stable detrusor hyperrefl exia. Initial results showed improvements in bladder capacity, in the number of incontinent episodes, and in patients’ subjective ratings (Chancellor, 2000 ). Consistent with previous reports, RTX appeared to be much better tolerated than capsaicin with respect to acute discomfort. CONCLUSIONS 339

Subsequently, a Phase 2 trial for IC was completed, but RTX treatment failed to meet the primary end points (ICOS Corporation, 2003 ). However, academic research studies for patients with refractory bladder disorders continue, and RTX still shows promise for that subset of patients (Kim et al., 2003 ). A recent meta- analysis of clinical studies of capsaicin and RTX in patients with spinal cord injury suggests that the effi cacy of the two compounds is comparable (MacDonald et al., 2008 ). Quite similar to effects observed in the skin, single instillations of high- concentration capsaicin also reduced immunohistochemical markers for suburothelial nerve fi bers (Dasgupta et al., 2000 ). Interestingly, there appeared to be reduction of nerve fi ber immunostaining only in patients who responded to the treatment. Similarly, in patients with idiopathic detrusor overactivity, instillation of RTX increased the current perception threshold values of C - fi bers in all patients who showed symptomatic improvements (Yokoyama et al., 2004 ). Finally, in an analysis of biopsy samples from 20 subjects with painful bladder syndromes, nerve fi ber counting and urothelial immunostaining for TRPV1 showed a marked increase in suburothelial nerve fi bers expressing TRPV1 (Mukerji et al., 2006 ). The ratio of TRPV1 to neurofi laments was also signifi cantly increased, suggesting overexpression of TRPV1. Overall, the subjects ’ pain scores correlated signifi cantly with the relative density of TRPV1 - expressing nerve fi bers.

13.12 CONCLUSIONS

Evidence for the hyperactivity and hypersensitivity of nociceptive nerve fi bers in acute and chronic pain syndromes is robust, and thus TRPV1 receptor agonist - mediated defunctionalization is a promising approach to pain management. Instead of focusing on the restoration of damaged or lost sensory nerve fi bers, analgesics of this class will focus on the suppression of the enhanced excitability of residual or intact nociceptors. Chronic pain syndromes seem to be associated with either nociceptor proliferation or reduction in nociceptor densities. In both cases, but perhaps for varying reasons such as excessive exposure to neurotrophins and cytokines, upregulation of TRPV1 and other proexcitatory proteins occurs. When TPRV1 agonists are administered in such a way that they gain immediate access to nociceptive fi bers that have been sensitized by acute trauma, the prospects for effi cacy via the induction of long- term nociceptor defunctionalization are high. For chronic pain conditions involving increased TRPV1 expression and nociceptive fi ber proliferation and sprouting (e.g., vulvodynia and IC), signifi cant effi cacy is also likely, since these fi ber tips appear to display enhanced vulnerability to defunctionalization. Finally, recent clinical data suggest that a substantial portion of patients with painful peripheral neuropathies is likely to respond to topical or localized TRPV1 agonist therapies such as NGX - 4010 or ALGRX - 4975. 340 TRPV1 AGONIST APPROACHES FOR PAIN MANAGEMENT

All commercially available TRPV1 agonist - based products or treatments are based on low - concentration capsaicin, whereas high - concentration capsaicin formulations are in advanced clinical evaluation. The former are appropriate for patient self - administration, whereas the latter will require physician administration. Directed high- concentration TRPV1 agonist therapies, in which episodic nociceptor defunctionalization is restricted to discrete regions, may emerge as an attractive means to control localized pain. However, given the ubiquitous distribution of TRPV1- expressing sensory nerve ﬁ bers throughout the body, systemic TRPV1 agonist- based therapies are unlikely to be viable since the side - effect proﬁ le of widespread defunctionalization is unlikely to be acceptable.

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PART IV

ROLE FOR TRPV 1 IN OTHER PHYSIOLOGICAL PROCESSES BESIDES PAIN TRANSMISSION

14 THE TRPV 1 CHANNEL IN NORMAL THERMOREGULATION: WHAT HAVE WE LEARNED FROM EXPERIMENTS USING DIFFERENT TOOLS?

Andras Garami , Maria C. Almeida , Tatiane B. Nucci , Tamara Hew - Butler , Renato N. Soriano , Eszter Pakai , Kazuhiro Nakamura , Shaun F. Morrison , and Andrej A. Romanovsky

14.1 INTRODUCTION

The superfamily of mammalian transient receptor potential (TRP 1 ) channels consists of approximately 30 proteins divided into six subfamilies: ankyrin (TRPA), canonical, melastatin (TRPM), mucolipin, polycystin (TRPP), and

1 BAT, brown adipose tissue; CAP, capsaicin; CCK, cholecystokinin; CPZ, capsazepine; DH, dorsal horn; DMH, dorsomedial hypothalamus; DRG, dorsal - root ganglion (ganglia); GABA,

γ - aminobutyric acid; HLI, heat loss index [HLI = (Tsk – Ta )/(Tb – Ta )]; i.c.v. , intracerebroventricular(ly); i.p. , intraperitoneal(ly), i.t. , intrathecal(ly); i.v. , intravenous(ly); LC, locus coeruleus; LPB, lateral parabrachial nucleus; LPBel, external lateral part of the LPB; LPS, lipopolysaccharide; MnPO, median preoptic nucleus; MPO, medial preoptic area; NADA, N- arachidonoyldopamine; OEA, oleoylethanolamide; OVLT, the organum vasculosum of the lamina terminalis; PG, prostaglandin

(e.g., in PGE2 ); p.o. , per os; POA, preoptic area; PVN, paraventricular nucleus; rRPa, rostral raphe pallidus nucleus; RVLM, rostral ventrolateral medulla; RTX, resiniferatoxin; s.c. , subcutaneous(ly); shRNA, short hairpin RNA; T a , ambient temperature; Tb , body temperature; Tsk , skin temperature; TRP, transient receptor potential (channel); TRPA, TRPM, and TRPV, ankyrin, melastatin, and vanilloid TRP, respectively.

351 352 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION vanilloid (TRPV). Of these, the heat - activated (TRPV1 - V4, TRPM2, M4, M5) and the cold- activated (TRPA1, TRPM8) are highly sensitive to temperature and are often called the thermo- TRP channels (Patapoutian et al., 2003 ; Dhaka et al., 2006 ; Caterina, 2007 ). Thermo - TRP channels possess two unique features. First, activation of all thermo - TRP channels increases the inward nonselective cationic current and, consequently, the membrane potential. This mechanism agrees with a possible role for these channels in peripheral thermosensitivity (Okazawa et al., 2002 ) and hypothalamic thermosensitivity, the latter according to some authors (Hori et al., 1999 ) but not others (Zhao and Boulant, 2005 ). Second, although each individual class of thermo - TRP channels is activated within a relatively narrow temperature range, cumulatively, thermo- TRP channels cover a broad temperature range, from noxious cold to noxious heat. These features suggest that thermo- TRP channels are likely to be those long - sought elements of the thermoregulatory system that are responsible for reception of the thermal signals, especially peripheral ones, that trigger thermal pain, thermal sensation, and body temperature (Tb ) regulation. For some thermo- TRP channels, viz, TRPV3 (Moqrich et al., 2005 ), TRPV4 (Lee et al., 2005 ), and TRPM8 (Bautista et al., 2007 ; Colburn et al., 2007 ; Dhaka et al., 2007 ), physiological roles in thermoreception have been established. But even for these channels, it is unknown if, under what conditions, and to what extent they contribute to Tb regulation. This chapter focuses on one thermo- TRP channel, TRPV1, also known as the capsaicin (CAP) receptor, or vanilloid - 1 receptor. We will ﬁ rst describe how Tb is controlled and where TRPV1 channels are located in the body with respect to the thermoregulatory system. We will then analyze the data that shed light on the involvement of TRPV1 channels in T b regulation. Such data have been obtained with different tools: pharmacological (TRPV1 agonists and antagonists) and genetic (mostly Trpv1 knockout [Trpv1 − / − ] mice). Our analysis will indicate that different tools have pointed to different thermoregulatory involvements of TRPV1 channels and have revealed different mechanisms of these involvements. In the concluding section, we will integrate the information reviewed. By putting the pieces of the puzzle together, we will propose a role (or roles) that TRPV1 channels play in normal T b regulation.

14.2 HOW IS DEEP T b REGULATED?

14.2.1 A Federation of Independent Thermoeffector Loops From the thermal physiology point of view, the body can be divided into two parts: the core (the central nervous system and the thoracic and abdominal viscera) and the shell (the rest of the body, including the skin). The core has a relatively high temperature: ∼ 37 ° C, in both rats (the species that was used extensively in the research reviewed in this chapter) and humans. Compared HOW IS DEEP Tb REGULATED? 353 with the shell, the core is relatively homogeneous, and its temperature is relatively stable (changes within ∼ 1 ° C). The shell, on the other hand, is thermally heterogeneous, and even for a given location, shell temperature, especially skin temperature (Tsk ), fl uctuates widely (within ∼ 10 ° C) under physiological conditions (Romanovsky et al., 2002 ). Not only does the thermoregulatory system maintain core Tb at a relatively stable level, but it also raises or lowers it when needed, for example, during exercise or sleep, respectively. Such regulation is achieved by triggering multiple effector responses, both behavioral and physiological. Thermoregulatory behaviors include cold - and heat - seeking and avoidance, as well as many others: from simple postural changes to complex behavioral programs in humans. The principal physiological cold - defense responses are autonomic ones (viz, skin vasoconstriction and nonshivering thermogenesis in brown adipose tissue [BAT]) and shivering. Although humans have signifi cant BAT deposits (Nedergaard et al., 2007 ), shivering is a more important mechanism for heat production in humans. In rodents, nonshivering thermogenesis is a more important mechanism for heat production than shivering (Cannon and Nedergaard, 2004 ). Physiological (autonomic) heat - defense responses include cutaneous vasodilation and species - specifi c responses aimed at evaporating water from the skin, for example, sweating in humans and thermoregulatory salivation in rats. Earlier models of thermoregulation assumed that thermoreceptors sense deep Tb s and Tsk s, code these temperatures (into neuronal activity codes), and that these codes of core and shell T bs are then integrated by a control network (that consists of several neurons with different roles) into some mean T b . The central control network was also thought to compare this integrated, mean Tb with an external or internal reference signal (a set point or its analogue) and, based on such a comparison, generate individual commands to thermoeffectors in a coordinated fashion. Different portions of these earlier models have been challenged by many authors for years. Arguably, such challenges have now reached a critical mass, resulting in a paradigm shift in our views on how

Tb is regulated (Romanovsky, 2007a, 2007b ). According to the new concept, the thermoregulatory system functions as a federation of relatively independent thermoeffector loops (Satinoff, 1978 ), without a single controller and without a single set point or its equivalent (Werner, 1979 ). Each thermoeffector loop includes a unique efferent pathway driving the corresponding thermoeffector response (Nagashima et al., 2000 ; Morrison et al., 2008 ). Each thermoeffector loop is also sensitive to a unique combination of shell and core T b s (Jessen, 1981 ; Roberts, 1988 ; Sakurada et al., 1993 ), and each, therefore, participates in the defense of a different level of a differently distributed Tb (Romanovsky, 2004, 2007a). This concept requires neither a computation of an integrated Tb nor its comparison with an obvious or hidden set point in a unifi ed system. By acting on thermoreceptive elements within thermosensitive neurons, a local Tb (whether deep T b or Tsk ) can change the activity of these neurons and cause sequential changes in neuronal activity throughout the entire pathway 354 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION controlling a given thermoeffector. This new concept emphasizes the signifi - cance of the thermoreceptive elements of thermosensory neurons and gives these elements a principal role in determining whether a thermoeffector response will be triggered. Of special importance for this chapter is the idea that such thermoreceptive elements are likely to be thermo- TRP channels, at least for those sensory neurons that respond to Tsk s and other shell Tb s. Most of the time, the recruitment of thermoeffectors into a thermoregulatory response looks like a highly coordinated event. Those effectors that affect heat balance in opposite directions are typically not activated simultaneously. Energetically expensive and water- consuming responses are typically triggered after those that do not consume a lot of energy or water. Such coordination between thermoeffectors used to be explained with the help of a coordinator, a specialized module within the unifi ed control network. However, this coordinator has never been found experimentally. It is more likely that coordination between thermoeffectors is achieved through their common (or largely overlapping) controlled variables, Tb s (Romanovsky, 2007a ).

14.2.2 Afferent Neural Pathways In humans, precise neural structures involved in thermoregulatory pathways are largely unidentifi ed. In the rat, neural pathways for BAT thermogenesis and thermoregulatory skin vasoconstriction (described in this chapter; also see Fig. 14.1 ), as well as for shivering and, to a lesser extent, thermoregulatory salivation and thermoregulatory skin vasodilation, have been identifi ed and characterized to various degrees over the last two decades. Although each effector response is controlled by separate neural pathways, these pathways, especially for BAT thermogenesis and skin vasoconstriction, follow similar patterns and will be covered jointly. Within all thermoeffector pathways, the primary afferent neurons are sensory neurons in the dorsal- root ganglia (DRG), as well as the nodose and trigeminal ganglia. Of the three broad physiological types of DRG neurons, viz, mechanoreceptors, nociceptors, and thermoreceptors (Perl, 1992 ), the two latter types respond to thermal stimuli, either noxious (nociceptors) or innocuous (thermoreceptors). Most thermoreceptors are located immediately beneath the epidermis and respond to shell temperatures in the skin and in the oral and urogenital mucosa; most of these superfi cial shell receptors are cold sensitive (reviewed by Nomoto et al. [ 2004 ]). Via T sk s, ambient temperatures (T as) act on thermoreceptors (at least some of which are TRP channels) in the cutaneous nerve endings of the lightly myelinated Aδ (cold - sensitive) or unmyelinated C (warm- sensitive) fi bers of DRG neurons that send their axons to secondary afferent neurons in lamina I of the spinal or medullary (trigeminal) dorsal horn (DH) (Fig. 14.1 ). Andrew and Craig (2001 ) have distinguished thermoreceptive - specifi c lamina- I neurons (i.e., those that are involved in triggering thermoeffector responses to innocuous cooling or warming of the skin) from nociceptive - specifi c and polymodal nociceptive HOW IS DEEP Tb REGULATED? 355

MnPO + + MPO − + − +

Neuronal ergicity Warm-sensitive (principal mediator) neurons Glutamate Tonic GABA ? − Acetylcholine + Noradrenaline DMH

LPB Tonic ? ++ rRPa − + +

? Tonic +

Premotor Skin neurons Innocuous warming DH + + + + Innocuous Preganglionic cooling neurons DRG + + Sympathetic ganglia

Skin BAT vasculature

Figure 14.1 A schematic of the neural pathways underlying the regulation of the sympathetic outfl ows to BAT and cutaneous blood vessels by innocuous warming and cooling of the skin. The neuronal bodies are shown as circles and star - like shapes; dendrites and axons are shown as lines; triangles with plus and minus signs represent excitatory and inhibitory synapses, respectively. The main mediator in each neuron is coded by color. The left portion of the fi gure shows afferent pathways; in this portion, the solid circles show neuronal bodies in the pathway activated by warming, and the empty circles show neurons in the pathway activated by cooling. The right portion of the fi gure shows efferent pathways; in this portion, neuronal bodies shown as the solid shapes belong to the BAT thermogenesis pathway, and neuronal bodies shown as the empty shapes belong to the skin- vasomotion pathway. Please see text for detailed descriptions and abbreviations. (See color insert.) 356 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION cells (that are involved in triggering pain responses to noxious heat and cold). In addition to neurons that respond to innocuous warming and cooling of the shell, there are peripheral deep- tissue thermoreceptors, which respond to core

Tb. The endings of these DRG and nodose- ganglion neurons are located on splanchnic and vagal afferents in the esophagus, stomach, large intra- abdom- inal veins, and other organs (Riedel, 1976 ; Cranston et al., 1978 ; Gupta et al., 1979 ; El Ouazzani and Mei, 1982 ). As illustrated in Fig. 14.1 , lamina - I neurons in the rat spinal cord transmit innocuous thermal signals from the skin (and possibly from other organs within the shell and the core) to neurons in the lateral parabrachial nucleus (LPB) that project to the median preoptic nucleus (MnPO) of the preoptic area (POA) of the anterior hypothalamus (Nakamura and Morrison, 2008b ). Experimental support for this part of the pathway is provided by the data showing that axon terminals from DH neurons make close appositions with postsynaptic structures of POA- projecting neurons in the external lateral part of the LPB (LPBel), and that those LPBel neurons that project directly to the MnPO are activated during skin cooling (Nakamura and Morrison, 2008b ). Furthermore, inactivation of LPBel neurons or antagonizing glutamate receptors in the LPBel completely blocks BAT thermogenesis and shivering triggered by skin cooling. On the other hand, glutamatergic stimulation of LPBel neurons with N - methyl - D - aspartate increases BAT thermogenesis (Nakamura and Morrison, 2008b ). Thus, activation of LPBel neurons, likely by glutamatergic inputs from lamina- I DH neurons, is essential for the initiation of cold - defense effector responses by cutaneous innocuous cold stimuli. A parallel pathway, involving neurons in the dorsal part of the LPB, is proposed for heat - defense responses to innocuous cutaneous warming stimuli (Nakamura and Morrison, 2007b ). Neurons in the LPBel activated by innocuous skin cooling send glutamatergic projections to activate γ - aminobutyric acid (GABA) - ergic MnPO neurons, whereas neurons in the dorsal LPB activated by innocuous skin warming likely send glutamatergic projections to activate glutamatergic MnPO neurons (Fig. 14.1 ). Both populations of MnPO neurons project to warm- sensitive neurons in the medial preoptic area (MPO) of the hypothalamus. This part of the pathway accounts for the experimental ﬁ ndings that BAT thermogenesis triggered by LPBel stimulation is blocked by antagonizing glutamate receptors in the MnPO, and that glutamatergic stimulation of MnPO neurons evokes BAT thermogenic responses (Nakamura and Morrison, 2008a,b ).

14.2.3 Efferent Neural Pathways The fact that inhibition of neurons in the MPO (but not those in the MnPO or lateral POA) increases deep Tb and BAT thermogenesis (Osaka, 2004 ) suggests that the warm- sensitive projection neurons in the POA are located mainly in the MPO and are tonically active to suppress cold- defense responses HOW IS DEEP Tb REGULATED? 357 in either BAT or skin vasculature under normal conditions. These warm- sensitive MPO cells can be regarded as the fi rst efferent neurons within the loops controlling BAT thermogenesis and skin vasoconstriction. The next part of the pathways shown in Fig. 14.1 fi nds experimental support in the fi ndings that local cooling of the POA stimulates thermogenesis (Hammel et al., 1960 ; Imai - Matsumura et al., 1984 ), and that coronal brainstem transections just caudal to the POA increase both BAT thermogenesis (Chen et al., 1998 ) and the sympathetic outfl ow to skin blood vessels (Rathner et al., 2008 ). These fi ndings agree with the idea that projection neurons in the MPO send tonic inhibitory outputs to caudal brain regions such as the dorsomedial hypothalamus (DMH) and the rostral raphe pallidus nucleus (rRPa), both of which provide sympathoexcitatory drive supporting thermoeffector activation (Nakamura and Morrison, 2007a ; Morrison et al., 2008 ; Rathner et al., 2008 ). Because BAT thermogenesis induced by skin cooling (Nakamura and Morrison, 2007a ) or by activation of MnPO neurons (Nakamura and

Morrison, 2008a ) is reversed by GABA A receptor blockade in the MPO, we postulate that MnPO neurons activated by cutaneous innocuous cold signals provide a local GABA input to the inhibitory projection neurons in the MPO, thus reducing their tonic inhibitory infl uence on either DMH or rRPa neurons (that drive BAT thermogenesis or cutaneous vasoconstriction, respectively). The rostral ventrolateral medulla (RVLM) also contains sympathetic premotor neurons infl uencing the sympathetic outfl ow to skin blood vessels; these neurons have been proposed to be involved in cold - induced skin vasoconstriction (Ootsuka and McAllen, 2005 ). However, according to an earlier study, RVLM neurons do not appear to be involved in the thermoregulatory control of the cutaneous vasomotor tone, as they are unlikely to receive a signifi cant inhibitory input from MPO neurons (Tanaka et al., 2002 ). It should be noted that in the efferent branch, separation of the loops controlling BAT thermogenesis and skin vasoconstriction is obvious (see Fig. 14.1 for details). It should also be noted that thermoeffector pathways have been reported to include additional neurons not shown in Fig. 14.1 . These neurons are located in the paraventricular hypothalamic nucleus (PVN) and in several structures in the pons (e.g., the locus coeruleus [LC]) and in the midbrain (viz, the ventral tegmental area, periaqueductal gray matter, retrorubral fi eld, and mesencephalic reticular formation) (for references, see Romanovsky et al., 2005 ; Romanovsky, 2007b ). Whereas the efferent neural pathways to all autonomic thermoeffectors and to skeletal muscles include warm- sensitive MPO neurons (Nagashima et al., 2000 ; Romanovsky, 2007a ), these neurons are not likely to be involved in most thermoregulatory behaviors. In fact, the only mammalian thermoregulatory behavior in which an involvement of the MPO has been fi rmly established is a postural extension in response to heat exposure; such postural extension does not occur in animals with MPO lesions (Roberts and Martin, 1977 ). Other thermoregulatory behaviors, such as moving to a “ reward ” zone or pressing a lever to trigger warming or cooling of the system, remain intact in 358 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION

POA - lesioned animals (Lipton, 1968 ; Carlisle, 1969 ; Satinoff and Rutstein, 1970 ; Schulze et al., 1981 ). In our study (Almeida et al., 2006b ), large lesions in the POA (including the entire MPO) did not change cold- seeking and warmth - seeking responses of rats to a variety of thermal, chemical (TRPV1 and TRPM8 agonists), or infl ammatory stimuli. Whereas MPO neurons are probably not involved in most thermoregulatory behaviors, MnPO neurons may be involved in at least one behavioral thermoregulatory phenomenon: the intensifi cation of an operant thermoregulatory behavior (moving to a reward zone during heat exposure in order to trigger a breeze of cold air) caused by hypertonic saline (Konishi et al., 2007 ). As for other structures, neurons of the DMH and fi bers passing through the PVN are required for the cold- seeking behavior in rats during lipopolysaccharide (LPS) - induced shock (Almeida et al., 2006b ). Overall, not much is known about the neural pathways underlying thermoregulatory behaviors (Nagashima et al., 2000 ; Romanovsky, 2007b ).

14.3 DISTRIBUTION OF TRPV 1 CHANNELS

14.3.1 Afferent Nerves TRPV1 channels are abundant in peripheral terminals of thin myelinated (A δ ) and unmyelinated (C) fi bers of primary afferent neurons in the DRG and the trigeminal ganglion, and of peptidergic sensory neurons in the nodose ganglion (Szallasi et al., 1995 ; Caterina et al., 1997 ; Tominaga et al., 1998 ; Mezey et al., 2000 ). These ganglia innervate both the skin (of the trunk, limbs, and head) and visceral organs, thus resulting in the extremely wide distribution of neuronal TRPV1 channels associated with afferent fi bers. Indeed, the TRPV1 channel is present on the majority of the spinal afferents innervating the stomach, large intestine, and urinary bladder, and on ∼ 30% of the spinal afferents in the skin or skeletal muscles (Schicho et al., 2004 ; Hwang et al., 2005 ; Christianson et al., 2006 ). The TRPV1 channel is also expressed in ∼ 20% of the nodose afferents innervating the upper gastrointestinal tract (Schicho et al., 2004 ; Zhang et al., 2004 ; Bielefeldt et al., 2006 ). Thermoregulatory responses are triggered by thermal exposures extensive enough to affect heat exchange between the body and the environment, and the wide distribution of TRPV1 channels would be compatible with a physiologically signifi cant thermoreceptor function. However, TRPV1 channels are located mostly on polymodal nociceptors, suggesting an involvement of these channels in nociception rather than in T b regulation. Furthermore, both in rodents (Story et al., 2003 ; Kobayashi et al., 2005 ; Hjerling- Leffl er et al., 2007 ) and humans (Anand et al., 2008 ), TRPV1 (high - threshold heat - sensitive channel) is largely co - expressed with TRPA1 (high - threshold cold - sensitive channel), which makes TRPV1- expressing neurons good candidates for detecting noxious thermal stimuli of both “ modalities ” (i.e., heat and cold) rather than innocu- DISTRIBUTION OF TRPV1 CHANNELS 359 ous thermal stimuli that would trigger thermoeffector responses of a particular modality (e.g., heat- defense responses). Yet, experiments with heat- sensitive nociceptors suggest that a predominant mechanism used to detect noxious heat under normal conditions may not involve TRPV1 (Woodbury et al., 2004 ). In addition to being expressed on peripheral terminals, TRPV1 channels are also expressed on the central terminals of the DRG, trigeminal, and nodose neurons, as evident from the intense immunoreactivity in the terminals of unmyelinated fi bers projecting to the laminae I and II of the lumbar DH, to the trigeminal nucleus caudalis, and to the nucleus of the solitary tract and area postrema (Tominaga et al., 1998 ; Guo et al., 1999 ). Autoradiography studies with [3 H]resiniferatoxin (RTX), an ultrapotent agonist of TRPV1 channels, also showed strong specifi c binding in these brain structures (Szallasi et al., 1995 ).

14.3.2 Brain Structures within Thermoregulatory Pathways The presence of CAP- responsive structures in the hypothalamus was proposed by Jancso- Gabor and colleagues nearly 40 years ago (Jancso- Gabor et al., 1970a ; Szolcsanyi et al., 1971 ), as they considered the POA to be the site of the hypothermic action of CAP, the fi rst and most famous TRPV1 agonist. Later, transcripts of the Trpv1 gene were shown to be widely distributed in the rat brain, with high levels present in the hypothalamus (Sasamura et al., 1998 ; Mezey et al., 2000 ); cells with weak expression of TRPV1- like immunoreactivity were detected in rat anterior hypothalamic nuclei (Mezey et al., 2000 ); and RTX was shown to specifi cally bind to membranes prepared from rat or human POA tissue (Acs et al., 1996 ). It is tempting to speculate that CAP - sensitive terminals in the hypothalamus are glutamatergic, because CAP causes glutamate release in hypothalamic slices in vitro (Sasamura et al., 1998 ). Interestingly, glutamatergic neurons in the POA (specifi cally, in the MnPO) are involved in the thermoregulatory pathways stimulated by skin warming (Fig. 14.1 ). TRPV1 channels may also be present in the structures that contain premotor neurons for autonomic thermoeffectors. Such structures include the LC, raphe area, DMH, and PVN (Mezey et al., 2000 ). CAP microinjections in the dorsal raphe have been reported to cause cutaneous vasodilation and hypothermia (Hajos et al., 1987 ). Low doses of systemic CAP have been shown to markedly excite LC neurons, perhaps by acting on these cells directly, because the LC does not seem to receive innervation by primary sensory afferents (Hajos et al., 1988 ). Furthermore, [ 3H]RTX binds to membranes from both rat and human LC (Acs et al., 1996 ). It should be noted, however, that the existence of TRPV1 neurons in extrahypothalamic brain structures has been debated, even though RTX binds with low to moderate affi nity to membranes obtained from various brain structures (Acs et al., 1996 ), and the TRPV1 mRNA has been detected widely throughout the brain (Sasamura et al., 1998 ). 360 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION

The evidence against the presence of TRPV1 in extrahypothalamic brain structures is based on the fact that [ 3H]RTX autoradiography in rat brain sections (not in membrane preparations) failed to detect any speciﬁ c labeling (Szallasi et al., 1995 ), with the exception of the nucleus of the solitary tract, a central termination site for nodose- ganglion neurons. Northern- blot hybridization with total RNA isolated from the whole rat brain also failed to detect TRPV1 mRNA (Caterina et al., 1997 ).

14.3.3 Non - Neural TRPV 1 Channels in Thermoeffector Organs The TRPV1 channel is widely expressed in peripheral non- neural tissues, including, but not limited to, the vascular endothelium and a wide range of epithelia (Szallasi et al., 2006 ). However, the level of TRPV1 channel expression in afferent neurons is at least 30 times higher than that in any other cell population (Sanchez et al., 2001 ), which to some extent calls into question the physiological relevance of TRPV1 channels that are not associated with afferent neurons. Nevertheless, direct action of TRPV1 agonists on vascular smooth muscle cells can dilate blood vessels in the skin and constrict vessels in skeletal muscles (Kark et al., 2008 ), suggesting a potential involvement of TRPV1 channels in thermoregulatory cutaneous vasoconstriction at the thermoeffector- tissue level. As for BAT, no data have been found that would show a direct involvement of the TRPV1 channel in thermogenesis in brown adipocytes. Recent data from Trpv1− /− mice suggest that this channel may be involved in the development of obesity by inhibiting thermogenesis (Motter and Ahern, 2008 ), perhaps nonshivering thermogenesis in BAT. However, the thermoregulatory effects of the genetic deletion of the Trpv1 gene are rather unclear (see below). Furthermore, the exact opposite role for TRPV1 channels in obesity (i.e., the prevention of adipogenesis) has been proposed as well (Zhang et al., 2007 ). TRPV1 channels are also expressed on sensory neural ﬁ bers within intercostal nerves innervating interscapular BAT. These ﬁ bers respond to high BAT temperatures (40– 44 ° C) and mediate the inhibition of BAT thermogenesis (Osaka et al., 1998 ).

14.3.4 Conclusions Drawn from the Distribution Data 1. Considering the extensive expression of TRPV1 channels in primary sensory neurons, one can speculate that their major involvement in physiological processes is likely to be on the afferent side. 2. However, the strong association of TRPV1 with polymodal nociceptive ﬁ bers and the relative abundance of these channels in the viscera (as compared with the skin) suggest an involvement in pain mechanisms (especially visceral pain) rather than in the detection of thermal signals

(especially cutaneous thermal signals) used for deep T b regulation. 3. The presence of functional TRPV1 channels on glutamatergic POA neurons can possibly be a target for vanilloid agonists to cause warmth - TRPV1 AGONISTS 361

defense responses, but whether this mechanism is recruited in T b regulation under normal conditions is unclear. Within the POA, TRPV1- positive neurons may be located in the MPO (in which case the thermoregulatory response to TRPV1 antagonists can be expected to involve autonomic, but not behavioral, thermoeffectors) or in the MnPO (in which case some behavioral effectors may also be involved). 4. Direct action of TRPV1 agonists on thermoeffector tissues is also possible, but based on the relatively low levels of expression in non- neural tissues, such an action may be of limited physiological signiﬁ cance.

Below, we will analyze the evidence produced with pharmacological and genetic tools and see if this analysis can conﬁ rm or disprove any of these preliminary conclusions regarding the role of TRPV1 in thermoregulation.

14.4 TRPV 1 AGONISTS

14.4.1 Pharmacological Agonists and Endogenous Ligands of TRPV 1 The TRPV1 channel is activated by heat, protons, and molecular ligands that are usually referred to as vanilloids. By far the most famous vanilloid is CAP (8 - methyl - N - vanillyl - 6 - nonenamide), the principal irritating constituent of hot peppers (Nelson, 1919 ). Another exogenous TRPV1 agonist, RTX, was later extracted from plants of the genus Euphorbia and was found to be 102 – 10 5 times more potent than CAP (Szallasi and Blumberg, 1989 ). Endogenous TRPV1 agonists also exist, but their physiological relevance is still unclear. Endogenous agonists include arachidonoylethanolamide (anandamide), oleoylethanolamide (OEA), N - arachidonoyldopamine (NADA), N- oleoyldopamine, and others (Movahed et al., 2005 ; Pingle et al., 2007 ). The

EC50 values for most endovanilloids are high (in the micromolar range), but some endovanilloids, for example, NADA, have a potency in the nanomolar range, which is similar to that of CAP (Di Marzo et al., 2002 ; Huang et al., 2002 ; Chu et al., 2003 ). Although it is unlikely that most endovanilloids will reach effective systemic concentrations (because of the high EC50 values), they still may achieve physiologically relevant local concentrations in selected tissues. For example, OEA, a lipid mediator of satiety, is present in the gut wall at concentrations close to its EC 50 value against TRPV1 (Fu et al., 2007 ). Of interest is the fact that endovanilloids cause hypothermia and inhibit BAT thermogenesis. For example, anandamide causes hypothermia that is not mediated by cannabinoid receptors (Di Marzo et al., 2002 ). Peripheral administration of OEA has been reported to decrease Tb in mice (Watanabe et al., 1999 ) and to inhibit metabolism in rats (Proulx et al., 2005 ). NADA has also been shown to cause hypothermia (Bisogno et al., 2000 ). Overall, however, the data obtained with endogenous vanilloids are scarce and provide little insight into the potential role, or roles, of the TRPV1 channel in thermoregu- 362 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION lation. The vast majority of the thermoregulatory studies have been performed with pharmacological TRPV1 agonists, CAP and RTX. Some of these studies are reviewed below.

14.4.2 Use of Agonists to Activate TRPV 1 14.4.2.1 Effects on Body Temperature The immediate effects of the ﬁ rst administration of a TRPV1 agonist on Tb regulation (discussed in this section) differ from the delayed effects and the effects following chronic or repeated administration of the same drug (Section 4.3 ). When administered acutely in a variety of species (including, mice, rats, guinea pigs, rabbits, dogs, and cats) either systemically or into the POA, CAP and RTX readily cause hypothermia (reviewed by Hori [ 1984 ]; Szallasi and Blumberg [ 1999 ]; Szolcsanyi [ 2004 ]; Caterina [2007 ]; also see Figs. 14.2 and 14.3 ). However, because some effects of CAP are not TRPV1- mediated (Dogan et al., 2004 ; Mahmoud et al., 2007 ), it was important to determine in direct experiments whether the hypothermic responses to CAP and RTX require the presence of the TRPV1 channel. Such experiments have been conducted, and they demonstrate that the hypothermic responses to CAP and RTX do not occur in mice in which the Trpv1 gene is knocked out (Caterina et al., 2000 ; Steiner et al., 2007 ) or silenced (Christoph et al., 2008 ).

14.4.2.2 Thermoeffectors Involved The hypothermic response to systemic administration of CAP or RTX can involve both autonomic and behavioral thermoeffectors (Hori, 1984 ; Szolcsanyi, 2004 ). Autonomic responses to CAP include skin vasodilation (Fig. 14.2 a), thermoregulatory salivation, and inhibition of the metabolic heat production (Szolcsanyi and Jancso- Gabor, 1973 ). Behavioral responses to CAP may include cold- seeking behavior in a thermo-

Figure 14.2 Effects of peripheral administration of CAP on deep Tb and autonomic and behavioral thermoeffectors. (a) Injection of CAP (0.4 mg/kg i . p.) evokes a fall in colonic temperature and causes tail skin vasodilation (increases tail T sk) in rats. (b) Injection of CAP (50 mg/kg s . c.) decreases colonic temperature and causes cold-

seeking behavior (lowers preferred Ta in a thermogradient apparatus) in newborn rabbits. Note the nonlinear scale for the ordinate axis showing preferred T a . (c) Injection of CAP (1 mg/kg s . c .) causes hypothermia and skin - cooling operant behavior (increases the frequency of bar pressing; pressing the bar turns off a heater and activates a cooling fan). (d) Injection of CAP (0.4 mg/kg s . c.) leads to a fall in colonic temperature and inhibits skin - heating operant behavior (decreases the frequency of bar pressing; pressing the bar turns off a cooling fan and turns on a heater). The data used in panel (a) are reproduced (with modifi cations) from the paper by Dib (1983) , for which the American Psychological Association is the copyright holder; no permission to reproduce the fi gure is required. The data used in panels (b– d) are reproduced (with modifi cations) from Szekely (1986) with permission by Elsevier (b) and from Hori (1984) with permission by Elsevier (c,d). TRPV1 AGONISTS 363

(a) 39 Capsaicin 0.4 mg/kg I.P.

38 Colonic

temperature (°C) 37 32 31 30 29 Tail skin 28 temperature (°C) 27 Capsaicin (b) 40 50 mg/kg S.C. 38

36 Colonic

34 temperature (°C) 45 40 35 30

25 temperature (°C) Preferred ambient

Capsaicin (c) 1 mg/kg S.C. 38

Colonic 36 temperature (°C) 35 0.6

0.03 Bar pressing

frequency (Hz) 0 Capsaicin (d) 0.4 mg/kg S.C. 35

Colonic 33 32

temperature (°C) 31

0.1 0.06

0.03 Bar pressing

frequency (Hz) 0 03060 Time (min) 364 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION

38 Colonic temperature (ºC) 37 29

27 Tail skin Capsaicin temperature (ºC) μ 25 23 g I.C.V.

15 Time spent bar pressing (%) pressing bar 0 –10 0 10 20 Time (min)

Figure 14.3 Effects of central CAP administration on deep Tb and autonomic and behavioral thermoeffectors. CAP (23 μ g i . c . v.) induces hypothermia (decreases colonic temperature), tail skin vasodilation (increases tail Tsk ), and operant cooling behavior (increases the percentage of time that rats spend pressing a bar that turns on a cooling fan). Reproduced from Dib (1982) with permission by Elsevier. gradient apparatus (Fig. 14.2 b), which is likely an innate behavior, and learned operant behaviors such as an increase in the frequency of pressing a lever (if such pressing induces ambient cooling) (Fig. 14.2 c) or a decrease in the frequency of pressing a lever (if it induces ambient heating) (Fig. 14.2 d). Similarly, RTX- induced hypothermia can occur via skin vasodilation (Woods et al., 1994 ), a reduction in metabolic rate (Woods et al., 1994 ), and cold- seeking behavior (Almeida et al., 2006a ). Some authors (Kobayashi et al., 1998 ; Osaka et al., 2000 ) have reported that a hyperthermic phase may follow CAP- induced hypothermia, and that both CAP and RTX, even though they induce skin vasodilation to increase heat loss, can also activate metabolism via sympathoadrenal mechanisms (Watanabe et al., 1988b ) to increase heat production (Kobayashi et al., 1998 ). However, this atypical increase in metabolic rate is insensitive to ruthenium red (a nonselective TRPV1 antagonist), which suggests that it is not mediated by TRPV1 (Okane et al., 2001 ). TRPV1 AGONISTS 365

14.4.2.3 The Site of Action: Peripheral or Central? There is no agreement on whether the hypothermic response to systemically administered TRPV1 agonists is primarily mediated by their action on peripheral or central TRPV1 channels. Evidence in support of the peripheral mediation hypothesis includes the fact that intravenous (i . v .) CAP causes skin vasodilation and hypothermia with short latencies ( < 10 min), and that it does so at doses as low as 15 μ g/kg (i.e., ∼ 5 μ g per rat) (Donnerer and Lembeck, 1983 ). It is often pointed out in the literature that these i . v . doses are comparable to or even lower than the doses used to cause hypothermia by a central administration of CAP. For example, the intrathecal ( i . t.) (Dib, 1987 ) or the intracerebroventricular (i . c . v.) administration of this drug (into the lateral ventricle [Dib, 1982 ] or the third ventricle [Steiner et al., 2007 ]) at a dose ranging from 23 to 40 μ g causes skin vasodilation, a thermoregulatory operant behavior (lever pressing to decrease T a), and hypothermia; an example of such a response is shown in Fig. 14.3 . Hence, it may be tempting to speculate that TRPV1 agonists can cause hypothermia by acting peripherally, even when they are administered centrally. However, no peripheral targets seem to exist that would account for the hypothermic effect of CAP or RTX. An action in a single thermoeffector tissue (e.g., skin vasculature) may contribute to the mechanisms of CAP- or RTX - induced hypothermia, but it does not explain the involvement of multiple effectors (see Section 4.2.2 ). A peripheral action that would explain the multiple thermoeffector involvement would be an action within the afferent thermoregulatory pathways (e.g., on primary sensory neurons). However, a substantial presence of TRPV1 channels on afferents involved in responses to innocuous heat appears unlikely (Section 3.1 ). As for the central mediation hypothesis, peripherally administered CAP has been shown to readily reach the brain (Saria et al., 1982 ), thus making this hypothesis feasible. The strongest piece of evidence supporting the central mediation hypothesis is that CAP causes hypothermia when administered into the POA not only at doses of a few tens of micrograms, but also at substantially lower doses: 500 ng (Jancso - Gabor et al., 1970b ) or even 200 ng (for references, see Hori [ 1984 ]), that is, 10 – 25 times lower than the minimal effective i . v. dose. Other pieces of evidence that are often cited to support the central mediation hypothesis are less convincing because alternative interpretations are possible. For example, it has been shown that subcutaneous (s . c .) CAP excites warm - sensitive neurons in the POA (Nakayama et al., 1978 ; Hori and Shinohara, 1979 ; Hori, 1984 ), but such an excitation could have occurred secondarily to an action on any part of the afferent thermoeffector pathways, especially since the studies cited used high (> 150 μ g) doses of CAP. Electrolytic lesioning of the POA has also been shown to reduce the hypothermic response to s . c . CAP in the study by Szolcsanyi and Jancso - Gabor (1975) , but this ﬁ nding still does not allow us to distinguish a direct action on POA neurons from a secondary one. Certain data are often interpreted as suggesting that mechanisms of the hypothermic response to systemically administered TRPV1 agonists include 366 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION their action on neurons in midbrain structures (the reticular formation and basal ganglia) or in ponto- medullar structures (the dorsal raphe nucleus). For instance, the mesencephalic reticular formation has been shown to contain neurons which respond to both local (brain) temperature and T sk (Nakayama and Hardy, 1969 ), and neurons in the same area respond to s . c. CAP (reviewed by Hori [ 1984 ]). Other examples include data showing that the administration of CAP into the basal ganglia (viz, the substantia nigra or caudate putamen [Hajos et al., 1988 ]) or the dorsal raphe nucleus (Hajos et al., 1987 ) induces skin vasodilation and hypothermia, and that s . c . administration of CAP increases the electrical activity of neurons in the dorsal raphe (Rabe et al., 1980 ). The latter observation, however, does not imply that CAP acts on these neurons directly. Some authors suggest that the paradoxical effect of peripheral CAP, that is, hyperthermia due to sympathoadrenal activation and an increase in metabolic heat production (see Section 4.2.2 ), is at least partially TRPV1- dependent (Watanabe et al., 1988a ) and may be mediated by CAP action on neurons in the RVLM. Lesions of the RVLM, the area that contains sympathetic premotor neurons, attenuate CAP- induced hyperthermia in anesthetized rats, whereas microinjection of CAP into this structure causes hyperthermia (Osaka et al., 2000 ).

14.4.3 Use of Agonists to Desensitize TRPV 1 14.4.3.1 Effects on Basal Body Temperature Whereas the ﬁ rst, acute administration of TRPV1 agonists results in an activation (opening) of TRPV1 channels, and, in the case of a neuronal TRPV1, in an excitation of the corresponding neurons, the delayed effects and the effects of chronic or repeated administration of TRPV1 agonists are more complex. The latter group of effects is related to the phenomenon of desensitization. The term “ desensitization ” was introduced by Jancso and Jancso (1949) and is generally used to describe a state of CAP- or RTX- induced neuronal insensitivity to TRPV1 agonists and other stimuli that normally activate TRPV1- expressing neurons, for example, noxious heat stimuli (Szallasi and Blumberg, 1999 ). Arguably, the term can be applied not only to TRPV1 - expressing neurons, but also to the TRPV1 channels responsible for the phenomenon, to the neural structures that contain the desensitized neurons, and to the entire animal possessing desensitized neural structures. Further complicating the usage of this term, it unites several different conditions varying from TRPV1 agonist - induced conformational changes in the channel to the death of TRPV1- expressing neurons (Szallasi and Blumberg, 1999 ) complicates the meaning of the term. Regardless of the mechanisms underlying desensitization, the administration of TRPV1 agonists to desensitize TRPV1 channels can be used as a tool to assess the functional importance of these channels (or of the neurons that express these channels). TRPV1 AGONISTS 367

To cause TRPV1 desensitization, CAP or RTX is usually administered systemically (intraperitoneally [ i . p.] or s . c.) at high doses (often several increasing doses are used) to either newborn or adult animals, usually rats. In newborn rats, a cumulative CAP dose of 50 mg/kg is usually used. In adult rats, the cumulative dose is usually 150– 310 mg/kg for CAP and 200 μg/kg for RTX. Depending on the animal’ s age at the time of administration of the initial desensitizing dose of the agonist, different patterns of desensitization occur. In particular, some neurons, both peripheral and within the central nervous system, remain CAP- sensitive when tested in adult animals that were treated with CAP as neonates. It is unknown whether such neurons escape or survive the neonatal CAP treatment, or whether they “ evolve ” at a later stage of ontogenesis (Szolcsanyi, 1990 ; Holzer, 1991 ). Furthermore, as we will discuss below, some (but not all) thermoregulatory responses of an adult TRPV1 - desensitized animal to a TRPV1 agonist or to a thermal stimulus differ drastically, based on whether the initial desensitizing dose of a TRPV1 agonist was applied in the neonatal period or in adulthood.

The delayed effects of treatment with large doses of CAP on core Tb in rats were investigated in several laboratories (Jancso - Gabor et al., 1970a ; Szolcsanyi and Jancso - Gabor, 1973 ; Szekely and Szolcsanyi, 1979 ; Obal et al., 1980 ; Hori and Tsuzuki, 1981 ; Obal et al., 1982 ; Szikszay et al., 1982 ; Benedek et al., 1983 ; Dib, 1983 ; Donnerer and Lembeck, 1983 ; Hajos et al., 1983 ; Gourine et al., 2001 ; Yamashita et al., 2008 ). In four studies (Jancso- Gabor et al., 1970a ; Szolcsanyi and Jancso- Gabor, 1973 ; Szekely and Szolcsanyi, 1979 ; Szikszay et al., 1982 ), the authors performed experiments within the ﬁ rst few days after CAP was administered in adult rats; in all these studies, they found an increased deep Tb (up to 1.9 ° C, but usually by just a few tenths of a degree) and skin vasoconstriction registered as a decreased T sk . In different experiments within the same studies, as well in other studies (Obal et al., 1980 ; Hori and Tsuzuki, 1981 ; Obal et al., 1982 ; Benedek et al., 1983 ; Dib, 1983 ; Donnerer and Lembeck, 1983 ; Hajos et al., 1983 ; Gourine et al., 2001 ; Yamashita et al., 2008 ), measurements were also performed 10 days or later after CAP administration to adult or newborn rats, but at these times, no consistent changes were found. How should these ﬁ ndings be interpreted? One possibility is that some neurons expressing TRPV1 channels are tonically activated, and that such an activation leads to inhibition of skin vasoconstriction (and possibly other thermoeffector responses) and, hence, to a suppression of deep Tb . When these tonically activated neurons become silent as the result of desensitization, skin vasoconstriction and hyperthermia occur. Over the long term, however, compensation can develop (Yamashita et al., 2008 ), and TRPV1 - mediated functions can recover (Jancso et al., 1977 ; Hajos et al., 1983 ), possibly explaining the fact that desensitization produces no consistent long - term changes.

14.4.3.2 Effects on Responses to Heat Exposure In contrast to the small, short- lived, and difﬁ cult to ﬁ nd effects of CAP desensitization on basal Tb , the 368 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION

(a) (b) Capsaicin-pretreated rats 39 Controls C) C) ° 31

37 Colonic temperature ( ° temperature Colonic Heat exposure ( temperature Tail skin 25

0246 38 39 40 Time (h) Colonic temperature (°C)

Figure 14.4 Effects of systemic CAP desensitization on deep Tb and autonomic thermoeffectors in heat- exposed rats. (a) Colonic temperature of CAP (310 mg/kg s . c .) -

pretreated rats and untreated controls during an exposure to Ta of 32 ° C. Note that

CAP- pretreated rats are unable to defend their deep T b against heat. (b) The relationship between tail T sk and colonic temperature in CAP (310 mg/kg s . c .) - pretreated rats and untreated controls during radiant heating of the body. Note that CAP - pretreated rats do not develop skin vasodilation (do not increase T sk) even at high colonic temperatures. Reproduced (with modiﬁ cations) from Obal et al. (1980) with permission by Springer - Verlag. effects on the responses to thermal challenges are profound, seem reproducible, and have been found in several species, including rats (Cabanac et al., 1976 ), guinea pigs (Jancso- Gabor et al., 1970a ), and mice (Szelenyi et al., 2004 ). When exposed to heat, animals desensitized with high systemic doses of CAP, either during the neonatal period or in adulthood, develop severe hyperthermia, while their nondesensitized counterparts successfully defend their deep Tb (Jancso - Gabor et al., 1970a,b ; Szolcsanyi and Jancso- Gabor, 1975 ; Cabanac et al., 1976 ; Obal et al., 1980 ; Hori and Tsuzuki, 1981 ; Szikszay et al., 1982 ; Benedek et al., 1983 ; Obal et al., 1983 ; Szolcsanyi, 1983 ; Szelenyi et al., 2004 ).

The higher values of deep Tb of CAP- pretreated rats during heat exposure are shown in Fig. 14.4 a. When Tsk of heat - exposed rats is plotted against their deep Tb , it becomes obvious that for the same value of deep T b , CAP - pretreated rats have a substantially lower tail Tsk value than their nondesensitized counterparts (Fig. 14.4 b). The latter ﬁ nding suggests that the threshold deep T b for tail skin vasodilation is increased in TRPV1- desensitized rats. Other heat- defense responses, both autonomic, for example, salivation (Cabanac et al., 1976 ), and behavioral, for example, operant behaviors (Hori and Tsuzuki, 1981 ) and the heat- escape locomotor response (Szolcsanyi and Jancso- Gabor, 1975 ; Obal et al., 1979 ; Obal et al., 1987 ), can also be diminished in animals treated with systemic CAP, but the impaired ability to recruit skin vasodilation seems to be the primary cause of the inability to defend deep T b against heat. TRPV1 AGONISTS 369

Indeed, whereas amputation of the tail severely compromises heat defense in normal rats, it has no effect on heat defense in rats with desensitized TRPV1 channels (Obal et al., 1980 ). Most often, the effects of CAP desensitization have been explained by an action on warm - sensitive, peripheral (primary sensory) neurons (Dib, 1983 ; Donnerer and Lembeck, 1983 ; Obal et al., 1987 ; Szallasi and Blumberg, 1990 ; Benham et al., 2003 ; Tominaga and Caterina, 2004 ; Yamashita et al., 2008 ). However, just as no peripheral targets can account entirely for the hypothermic effect of CAP or RTX, it is diffi cult, if not impossible, to explain the effects of TRPV1 desensitization (such as multiple thermoeffector involvement) by peripheral mechanisms. It should be noted, however, that involvement of MnPO neurons in the hypothermic response to TRPV1 agonists would readily explain the recruitment of a broad spectrum of both autonomic and behavioral thermoeffectors. Desensitization of MnPO neurons that receive warm signals from the skin may also explain several other phenomena. Inhibition of these neurons has been found recently in one of our laboratories to facilitate skin vasoconstriction (K. Nakamura and S.F. Morrison, unpublished observations), thus suggesting that these neurons provide a certain level of tonic excitatory input to warm- sensitive MPO neurons, that is, fi rst efferent neurons for all known pathways for autonomic thermoeffectors and shivering (Nagashima et al., 2000 ; Romanovsky et al., 2005 ; Romanovsky, 2007b ; Morrison et al., 2008 ). Hence, sudden and uncompensated inactivation of MnPO neurons may explain an increase in deep Tb during the fi rst few days after the administration of desensitizing doses of CAP (Jancso- Gabor et al., 1970a ; Szolcsanyi and Jancso- Gabor, 1973 ; Szekely and Szolcsanyi, 1979 ; Szikszay et al., 1982 ). Interestingly, lesioning the anterior hypothalamus, including the MnPO, causes pronounced hyperthermia in rats (Romanovsky et al., 2003 ). Inactivation of MnPO neurons may also account for the hyperthermia that is reported by some (Watanabe et al., 1988b ; Kobayashi et al., 1998 ; Osaka et al., 2000 ) to follow the hypothermic response to acute administration of high doses of CAP or RTX. Involvement of MnPO neurons in the mechanisms of both desensitization and of acute hypothermic response to TRPV1 agonists may also account for the otherwise diffi cult - to - explain fi ndings by Jancso - Gabor et al. (1970b) (also see Table 14.1 ). These authors found that rats desensitized by intrahypotha- lamic injections of CAP show a reduced hypothermic response to an acute s . c . administration of CAP. Furthermore, these rats also lose their ability to defend deep T b against ambient heat. Because the MPO neurons tonically activated by MnPO neurons are potentially the same neurons that are inhibited by prostaglandin (PG) E 2 to initiate fever (Nakamura et al., 2005 ; Tsuchiya et al., 2008 ), the desensitization of MnPO neurons could also explain exaggerated fever responses to systemic LPS that have been reported to occur in rats treated with high doses of CAP (Szekely and Szolcsanyi, 1979 ) or RTX (Dogan et al., 2004 ). e,h c,e ↓ — ↔ - Gabor Jancso Induced by a Hypothermia Brain Heating Brain Heating Obal et al. (1983) ; e Cabanac et al. (1976) , d

a,d – f e,f,i c ↓ ↓ ↓ Heat - Defense Body Heating Body Heating Responses to Whole Whole Responses to - Gabor Jancso et al. (1970b) ; c , no change; — , not studied. References: References: not studied. , — no change; , ↔ Hori and Tsuzuki (1981) . i

h c c ↓ ↓ ↔ Desensitization Effects mg/kg) CAP Hajos et al. (1983) ; , attenuation; attenuation; , ↓ b Dib (1983) and h Intracerebral (0.002 – 0.2 Hypothermia Induced by

a – c c b,g,h ↓ ↓ ↓ by Systemic (0.05 – 16 mg/kg) CAP Hypothermia Induced and Route and Route Donnerer and Lembeck (1983) and Hajos et al. (1983) ; g CAP CAP Dose (mg/kg) ., i . p ., s . c intrahypothalamically ., i . p ., s . c Desensitization Model Obal et al. (1987) ; et al. (1970a) , Szolcsanyi and Jancso - Gabor (1973) , and Szikszay et al. (1982) ; ; (1982) and Szikszay et al. , (1973) Gabor - Szolcsanyi and Jancso , (1970a) et al. TABLE 14.1 Thermoregulatory Responses to Systemically or Centrally Applied CAP or Heating in Different Models of or Heating in Different CAP Applied Responses to Systemically or Centrally Thermoregulatory 14.1 TABLE CAP Desensitization effect of desensitization on the response interest is marked as follows: The f Neonate 50 – 450 Adult 0.04 – 0.3 Obal et al. (1979, 1980; 1982) , Benedek et al. (1983) , Szolcsanyi (1983) , Szekely and Romanovsky (1997) , and Szelenyi e t , al. (1997) (2004) ; Szekely and Romanovsky , (1983) Szolcsanyi , (1983) Benedek et al. , 1982) 1980; (1979, Obal et al. Age Group Adult 20 – 310

370 TRPV1 AGONISTS 371

Whereas TRPV1 desensitization severely impairs autonomic heat defense mechanisms, studies in rats treated with different doses of CAP (50– 460 mg/kg s . c .) as neonates or with relatively low doses of CAP (130 – 160 mg/kg s . c. or i . p.) as adults, found normal thermoeffector responses to cold (Jancso- Gabor et al., 1970a ; Szolcsanyi and Jancso - Gabor, 1973 ; Hori and Tsuzuki, 1981 ; Yamashita et al., 2008 ). In contrast, two other studies, in which adult rats were desensitized with high doses of CAP (250– 300 mg/kg s . c .), demonstrated attenuated responses to cold (Benedek et al., 1983 ; Cormareche- Leydier, 1984 ). The author of one of these studies, Cormareche - Leydier, points to the similarity between the thermoregulatory consequences of TRPV1 desensitization and of POA lesions. Indeed, it has been well documented that animals with POA (specifi cally MPO) lesions cannot defend their Tb auto- nomically against either cold or heat (Carlisle, 1969 ; Lipton et al., 1974 ; Satinoff et al., 1976 ; Van Zoeren and Stricker, 1976 ; Schulze et al., 1981 ; Almeida et al., 2006b ). The above proposed action of TRPV1 agonists on MnPO neurons cannot explain the fi nding that rats treated with high systemic doses of TRPV1 agonists as adults lose the ability to recruit both autonomic heat- defense and autonomic cold- defense thermoeffectors. The impairment of cold- defense responses, however, can be readily explained if one assumes that MPO neurons are also desensitized. These neurons participate, as fi rst efferent neurons, in both skin - vasomotion and BAT thermogenesis pathways. Notable differences between animals treated with CAP in the neonatal period and animals treated as adults are the responses to intrabrain CAP. Rats that received high doses of CAP as newborns respond to intrabrain CAP with normal hypothermic (Table 14.1 ) and skin - vasodilation (Hajos et al., 1983 ) responses, whereas such responses are ablated in rats treated with CAP as adults (Hajos et al., 1983 ; also see Table 14.1 ). Intriguingly, the same neurons that are desensitized by CAP treatment in adulthood (but are unaffected by CAP treatment during the neonatal period) may be speculated to be involved in thermoeffector responses to hypothalamic heating. Indeed, when these neurons are preserved (rats desensitized as neonates), the responses to localized POA heating are normal; when these neurons are gone (rats desensitized as adults), the responses to POA heating are abolished (Table 14.1 ). It is also interesting that the POA heating protocols used in these studies resulted in a local (POA) temperature of ∼ 42 ° C (Jancso - Gabor et al., 1970b ; Dib, 1983 ), which is practically the same as the threshold temperature for activation of the TRPV1 channel in transfected cells in vitro (Caterina et al., 1997 ; Tominaga et al., 1998 ). The observation that CAP - sensitive neurons may be involved in thermoregulatory responses to hypothalamic heating is important, because the data obtained in electrophysiological studies give contradictory answers to the question of whether TRPV1 can be responsible for hypothalamic thermosensitivity: some studies support such a possibility (Hori et al., 1999 ), whereas other studies reject it (Zhao and Boulant, 2005 ). 372 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION

14.4.4 Studies in Humans By consuming spicy hot foods, humans have experimented with the per os (p . o .) administration of CAP for centuries, and it is common knowledge that hot red pepper causes gustatory- evoked sweating (Lee, 1954 ). The observation that people living closer to the equator prefer their food hotter than those living in cooler climates (Szallasi and Blumberg, 1999 ) gave rise to the theory that eating spicy food helps combat the environmental heat. However, hot red pepper not only causes sweating, but also exaggerates the thermogenic response to a meal, a phenomenon known as spice- induced thermogenesis (Henry and Emery, 1986 ). Both hot red pepper and capsiate, a nonpungent TRPV1 agonist, have also been reported to increase oxygen consumption and

Tb (Ohnuki et al., 2001 ; Hachiya et al., 2007 ). Because consumption of a TRPV1 agonist produces two opposite effects on human thermoregulation, that is, activation of heat- loss mechanisms (sweating) and activation of heat- production mechanisms (thermogenesis), these human data are difﬁ cult to interpret. Another difﬁ culty is that the acute activation of TRPV1 channels has different thermoregulatory implications than the TRPV1 desensitization, which occurs with repeated consumption of hot pepper. Therefore, even if the effects of hot pepper consumption are determined by the thermoregulatory effects of CAP, it is unclear whether the driving mechanism is an acute increase in heat loss or a chronic desensitization of neural pathways for heat- defense mechanisms.

14.4.5 Conclusions Drawn from Experiments with Agonists 1. Because different autonomic and behavioral thermoeffectors can be involved in the hypothermic response to systemic administration of CAP or RTX, no isolated action in a single thermoeffector tissue could account for TRPV1 agonist- induced hypothermia. To explain the recruitment of multiple effectors, one has to hypothesize either an action on multiple targets within different thermoeffector loops or an action of neural elements that service multiple effectors. Because neural thermoeffector pathways are strongly diverged in the efferent branches of thermoeffector loops downstream of MPO neurons, it is more likely that TRPV1 agonists act on primary sensory neurons or on POA neurons, either in the MnPO or in the MPO, but not on premotor, preganglionic, or sympathetic - ganglia neurons. 2. If TRPV1 agonists were to cause hypothermia by acting primarily on the endings of primary sensory neurons, this would mean a peripheral mechanism of action. However, evidence in favor of a peripheral action is relatively weak. If, nevertheless, CAP- or RTX- induced hypothermia is mediated in part by TRPV1 channels on sensory neurons, this would mean either that a physiologically signiﬁ cant number of true thermoreceptors express TRPV1 channels, or that polymodal nociceptors TRPV1 ANTAGONISTS 373

(i.e., neurons that are not believed to participate in thermoregulatory responses triggered by innocuous thermal signals) can make a signiﬁ cant contribution to triggering thermoregulatory responses. Neither assumption can be ruled out completely. 3. If, on the other hand, TRPV1 agonists cause hypothermia by acting on MnPO or MPO neurons, this would be a central mechanism of action. The central action hypothesis has stronger support, including the fact that, upon intra - POA administration, CAP causes hypothermia at doses up to 25 times lower than the lowest dose reported to cause hypothermia in rats upon a peripheral (i . v .) administration. 4. Involvement of MnPO neurons in the hypothermic response to TRPV1 agonists would explain the recruitment of a broad spectrum of both autonomic and behavioral thermoeffector mechanisms. Furthermore, desensitization of these neurons with TRPV1 agonists would explain the selective loss of sensitivity to environmental heat found in animals treated with CAP or RTX as neonates or as adults. 5. Two facts, however, cannot be explained by an involvement of MnPO neurons. First, hypothermic responses both to intra- POA CAP and to POA heating are attenuated in rats treated with systemic CAP as adults. Second, rats treated with high systemic doses of TRPV1 agonists as adults lose not only the ability to respond with autonomic heat- defense responses to external heating, but also the ability to respond with autonomic cold - defense responses to external cooling. Both facts can be readily explained by an involvement of MPO neurons. These neurons are warm- sensitive, and they participate, as ﬁ rst efferent neurons, in both skin- vasomotion and BAT thermogenesis pathways (Fig. 14.1 ).

14.5 TRPV 1 ANTAGONISTS

14.5.1 TRPV 1 Antagonists: New Pain Therapeutics Based on experiments in rodent models of infl ammation and cancer (Pomonis et al., 2003 ; Asai et al., 2005 ; Gavva et al., 2005b ; Honore et al., 2005 ), the TRPV1 channel has been established as a promising target for analgesic therapy (Szallasi et al., 2007 ). The possibility of making next - generation pain therapeutics has stimulated keen interest, and many pharmaceutical companies have entered the race to synthesize and test new, highly potent, and selective TRPV1 antagonists. Many aspects of these new TRPV1 antagonists of different chemotypes are discussed in detail in several chapters of this book. This chapter is dedicated exclusively to the effects of TRPV1 antagonists on thermoregulation. Table 14.2 lists those antagonists for which effects on Tb have been published. In the following sections, we will analyze what experiments with these TRPV1 antagonists have revealed about the role, or roles, of the TRPV1 channel in deep Tb regulation. b a b b b b b b,c d b b ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ Deep T Effect on kg), Route Route kg), 30 p . o 3 p . o 3 i . v 3 – 10 p . o 3 i . v 0.1 – 10 p . o 0.3 i . v 3 i . v 30 p . o uorophenyl) in Rats b uoromethyl)phenyl)carbamate uoromethyl)phenyl)carbamate nyl)oxy) - 2(1H) quinoxalinone (trifl uoromethyl) - 2 pyridinyl)amino)methyl) fl methanesulfonamide idinyl)oxy) - 1,3 benzothiazol 2 amine (trifl ecarboxamide 4 - (3 - (trifl 4 - (3 (trifl uoromethyl) 2 pyridinyl) N (5 1 piperazin- Johnson Johnson A - 425619 AMG1629 Abbott Amgen AMG2820 1 - (5 isoquinolinyl) 3 (4 (trifl uoromethyl)benzyl)urea Amgen AMG3731 3 - amino 5 ((2 methoxyethyl)amino) 6 (4 (trifl uoromethyl)phenyl) 4 pyrimidi- Amgen 8 - ((6 (4 (trifl uoromethyl)phenyl) 4 pyrimidinyl)oxy) 3 isoquinolinol AMG7988 N - (4 (((3 (6 ((2 amino 1,3 benzothiazol 4 yl)oxy) pyrimidinyl) 6 Amgen AMG8163 Amgen 4 - ((6 (2 ((2 (1 piperidinyl)ethyl)amino) 6 (trifl uoromethyl) 3 pyridinyl) pyrim- AMG9810 Amgen BCTC tert - butyl (2 - (6 ((2 (acetylamino) 1,3 benzothiazol 4 yl)oxy) pyrimidinyl) 5 (2E) - 3 (4 tert butylphenyl) N (2,3 dihydro 1,4 benzodioxin 6 yl) 2 propenamide Neurogen N - (4 tert butylphenyl) 4 (3 chloro 2 pyridinyl) 1 piperazinecarboxamide 30 i . p Comp. Comp. 41 Johnson & TABLE TABLE 14.2 Effects of TRPV 1 Name Antagonists on Deep T Company Name according to the International Union of Pure and Applied Chemistry Dose (mg/ 374 b b h d b b e c,f g b d d Gavva ↓ ↑ ↓ ↑ ↑ ↑ ↓ ↑ ↑ ↔ ↔ c Deep T Effect on kg), Route Route kg), 30 p . o 0.01 – 0.5 i . v 3 i . v 0.3 – 30 p . o 1 – 30 p . o 3 i . v 30 p . o 0.1 – 3 p . o 0.1 i . v 3 – 100 p . o 1 – 10 i . p Gavva et al. (2007b) ; b Suh et al. (2003) . * Also referred to as SC0030 in the h Swanson et al. (2005) ; uorophenyl) a Gavva et al. (2008) ; g uoromethyl) , ↔ , none. References: uoromethyl) , a decrease; a decrease; , ↓ Tamayo Tamayo et al. (2008) ; f , an increase; an increase; , ↑ Steiner et al. (2007) ; e phenyl) - 1,3 thiazole 4 carboxamide (trifl (trifl uoromethyl) - 3 pyridinyl) 2 propenamide (trifl uoromethyl)phenyl) - 2 propenamide phenyl) - 4 pyrimidinyl) 1,3 benzothiazol 6 amine (trifl uoromethyl)phenyl) - 2 propenamide benzimidazol - 2 yl) 1 piperazinyl) 3 pyridinyl) 1,2 ethanediol acetamide methanesulfonamide N - (4 ((((4 tert butylbenzyl)carbamothioyl)amino)methyl) 2 fl is marked as follows: is marked as follows: b Pharma Lehto et al. (2008) ; d Schwarz * Comp. Comp. H Amgen AMG0347 Amgen 2 - ((2,6 dichlorophenyl)amino) N (4 (trifl (2E) - N (7 hydroxy 5,6,7,8 tetrahydro 1 naphthalenyl) 3 (2 (1 piperidinyl) 6 AMG8563 Amgen AMG7905 Amgen (2E) - N ((2S) 2 hydroxy 2,3 dihydro 1H inden 4 yl) 3 (2 (1 piperidinyl) AMG8562 Amgen - 4 (trifl N - (6 (2 ((cyclohexylmethyl)amino) JYL1421 (2E) - N ((2R) 2 hydroxy 2,3 dihydro 1H inden 4 yl) 3 (2 (1 piperidinyl) Comp. Comp. G Amgen 1 - (5 chloro 6 ((3R) 3 methyl 4 (6 (trifl uoromethyl) (3,4,5 trifl uorophenyl) 1H AMG 517 Amgen N - (4 ((6 (trifl uoromethyl)phenyl) 4 pyrimidinyl)oxy) 1,3 benzothiazol 2 yl) Name Company Name according to the International Union of Pure and Applied Chemistry Dose (mg/ The effect on deep T et al. (2007a) ; study by Suh et al. (2003) . . (2003) study by Suh et al. 375 376 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION

14.5.2 Effects on T b It has now been established that many TRPV1 antagonists cause hyperthermia, an unwanted side effect (Swanson et al., 2005 ; Gavva et al., 2007a,b ; Steiner et al., 2007 ; Honore et al., 2009 ; also see Table 14.2 ). This effect was seen for compounds of different chemotypes, including pyrimidines (AMG

(a) AMG0347 (b) AMG 517 0.5 mg/kg I.P. 10 mg P.O. Vehicle Vehicle

38 38

37 37

Mouse (ºC) Tympanic temperature Human Abdominal temperature (ºC) temperature Abdominal 36 060120 012345 Time (min) Time (d)

39 40

38 39

37 38 Body (ºC) temperature Dog Body (ºC) temperature Monkey

02468 02468 Time (h) Time (h) Figure 14.5 TRPV1 antagonists cause hyperthermia in different species. (a) Effect of AMG0347 (0.5 mg/kg i . p .) or its vehicle on abdominal temperature in mice at a neutral Ta of 31 ° C. (b) Effect of multiple daily (starting on Day 0) administration of AMG 517 (10 mg p . o.) or placebo on tympanic temperature in humans at room temperature. Note that the arrow in this panel shows just the fi rst day of drug administration. (c) Effects of JYL1421 (30 mg/kg p . o .) or its vehicle on body temperature (location not specifi ed) in dogs at room temperature. (d) Effects of JYL1421 (30 mg/kg p . o .) or its vehicle on body temperature (location not specifi ed) in cyno- molgus monkeys at room temperature. The data are reproduced (with modifi cations) from Steiner et al. (2007) with permission by the Society for Neuroscience (a), from Gavva et al. (2008) with permission by the International Association for the Study of Pain (b), and from Gavva et al. (2007b) with permission by the Society for Neuroscience (c,d). TRPV1 ANTAGONISTS 377

517), piperazines (AMG2674), ureas (A- 425619 and JYL1421), and cinnamides (SB366791 and AMG9810), administered to different animal species, including mice, dogs, monkeys (Fig. 14.5 ), and rats (Fig. 14.6 ). The same side effect (an increase in T b up to ∼40 ° C) was also presented in clinical trials of one of these antagonists, AMG 517 (Gavva et al., 2008 ; also see Fig. 14.5 ).

(a) Ambient temperature (b) Ambient temperature (c) Ambient temperature 28ºC 24ºC 17ºC 39

37 Colonic temperature (ºC) temperature Colonic

AMG0347 AMG0347 0.6 50 m g/kg I.V. 50 m g/kg I.V. Vehicle Vehicle

0.4

Heat loss index 0.2

AMG0347 30 50 m g/kg I.V. Vehicle

15 Oxygen consumption (mL/kg/min) 060120180 0 60 120 180 0 60 120 180 Time (min) Time (min) Time (min) Figure 14.6 Recruitment of autonomic thermoeffectors in the hyperthermic response of rats to AMG0347 at different T a s. AMG0347 (50 μ g/kg i . v .) or its vehicle was injected in rats at a T a of 28 ° C (the upper end of the thermoneutral zone; a),

24 ° C (the lower end of the thermoneutral zone; b), or 17 ° C (a subneutral T a ; c). The effects on colonic temperature (top), HLI (middle), and oxygen consumption (bottom) are shown. Reproduced from Steiner et al. (2007) with permission by the Society for Neuroscience. 378 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION

It was important to determine whether the hyperthermic effect of TRPV1 antagonists is an on - target effect (i.e., whether it is mediated by the TRPV1 channel), and the answer was found (Steiner et al., 2007 ). Trpv1 − / − mice did not respond to AMG0347 with hyperthermia, although their Tb did increase in response to a TRPV1 - independent stimulus (needle prick). Interestingly, the two antagonists mentioned above, AMG0347 and AMG 517, have been shown to cause hyperthermia in rats at very low doses. The minimum i . v . dose that causes maximum effect on Tb (ED max ) is 50 μ g/kg for AMG0347 (Steiner et al., 2007 ) and 100 μ g/kg for AMG 517 (Gavva et al., 2008 ). For AMG0347, a threshold dose (i.e., the minimum dose that signifi - cantly increases Tb ) is 10 μ g/kg (Steiner et al., 2007 ). The hyperthermic effect observed in experiments involving novel TRPV1 antagonists agrees with the transient hyperthermia that was found in several early studies to occur in rats during the fi rst few days after the administration of desensitizing doses of CAP. Both fi ndings suggest that TRPV1 channels are tonically activated in vivo, thus constantly suppressing deep Tb ; when TRPV1 channels are blocked, this suppression is removed, and Tb increases. Below, we will try to understand the mechanisms of this tonic, TRPV1 - mediated, physiological suppression of deep T b by analyzing the mechanisms of the hyperthermic response to TRPV1 antagonists.

14.5.3 Thermoeffectors Involved The effector pattern of the hyperthermic response of rats to AMG0347 has been determined in our study (Steiner et al., 2007 ). Because the activity of autonomic effectors depends on the thermal environment (Romanovsky et al.,

2002 ), we studied the response to the ED max dose of AMG0347 (50 μ g/kg i . v .) at several Ta s: 28 ° C (at the upper end of the thermoneutral zone in that particular setup), 24 ° C (the lower end of the thermoneutral zone), and 17 ° C (a subneutral temperature). The results of this experiment are shown in Fig. 14.6 . To describe these results, the heat loss index (HLI), which is used to assess skin vasomotion, must be deﬁ ned. The HLI is calculated according to the formula: HLI = (T sk − T a )/(T b − T a ) (Romanovsky et al., 2002 ). Before receiving AMG0347 or its vehicle, the rats exhibited pronounced tail skin vasodilation (a high HLI) at 28 ° C, modest vasodilation (an intermediate HLI) at 24 ° C, and maximum vasoconstriction (the lowest HLI) at 17 ° C. Thermogenesis (oxygen consumption) was lower within the thermoneutral zone (28 ° C and

24 ° C) than at the subneutral T a of 17 ° C. At all T a s, AMG0347 (but not its vehicle) caused a signifi cant T b rise. Although T a affected neither the magnitude nor the time course of the hyperthermic response to AMG0347, it modifi ed the thermoeffector profi le of the response. At 28 ° C, AMG0347 elicited skin vasoconstriction (a signifi cant fall in the HLI) and tended to elevate thermogenesis (Fig. 14.6 a). At 24 ° C, AMG0347 evoked skin vasoconstriction and increased thermogenesis signifi cantly (Fig. 14.6 b). At 17 ° C, AMG0347 strongly activated thermogenesis but did not cause any further tail skin vaso- TRPV1 ANTAGONISTS 379 constriction (Fig. 14.6 c). In conclusion, the hyperthermic response to AMG0347 can be brought about by a combination of two autonomic thermoeffector responses, nonshivering thermogenesis in BAT and tail skin vasoconstriction.

Furthermore, similar to the hyperthermic responses to PGE 2 (Crawshaw and Stitt, 1975 ; Szelenyi et al., 1992 ), cholecystokinin (CCK) octapeptide (Szelenyi et al., 1992 ), or LPS (Szekely and Szelenyi, 1979 ), the contributions of skin vasoconstriction and thermogenesis to the overall rise in deep T b caused by AMG0347 differ at different Ta s. We also investigated the effect of AMG0347 on a behavioral response: the selection of a preferred T a in a thermogradient apparatus (Steiner et al., 2007 ). Rats treated with a high dose (500 μ g/kg i . v .) of AMG0347 developed hyperthermia, but even in response to such a high dose, they did not change their preferred Ta . Summarizing the results of our study with AMG0347 (Steiner et al., 2007 ) and of the recent study by Gavva et al. (2008) with AMG 517, we conclude that the hyperthermic effect of TRPV1 antagonists is brought about by skin vasoconstriction (AMG0347 and AMG 517 in rats) and by the activation of thermogenesis, either nonshivering (AMG0347 and AMG 517 in rats) or shivering (AMG 517 in humans), but that it does not involve thermoregulatory behavior (AMG0347 in rats). This thermoeffector pattern suggests that TRPV1 channels are tonically activated, and that this activation contributes a tonic inhibitory inﬂ uence on the neural circuits that drive thermogenesis and skin vasoconstriction. Because more than one autonomic thermoeffector can be involved in the hyperthermic response to a TRPV1 antagonist, no isolated action in a single thermoeffector tissue could entirely account for this response. Because only autonomic (and not behavioral) effectors are involved, TRPV1 - mediated signals that are blocked by TRPV1 antagonists are likely to impinge on the neural pathways for autonomic thermoregulation downstream from the point at which they become separated from the pathways for thermoregulatory behaviors. This may happen in the MPO, because even large POA lesions (that destroy the MPO completely or nearly completely) do not affect thermoregulatory locomotion in a thermogradient apparatus (Almeida et al., 2006b ), whereas a more upstream (closer to the afferent input) structure, the MnPO, is involved in at least some behavioral thermoregulatory responses (Konishi et al., 2007 ).

14.5.4 What Signals Are Blocked to Cause Hyperthermia? In transfected cells maintained at a physiological pH (7.4), the threshold temperature for activation of the TRPV1 channel is ∼ 43 ° C (Caterina et al., 1997 ;

Tominaga et al., 1998 ). Because Tb s rarely reach such a high value, the TRPV1 channel seems to be an unlikely thermoreceptor candidate, at least not under normal conditions. However, the 43 ° C activation threshold was obtained in vitro . In vivo, concomitant activation (or sensitization) by chemical ligands can affect, via multiple mechanisms, the temperature range in which the channel is activated (Premkumar and Ahern, 2000 ; Bhave et al., 2003 ; Carlton 380 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION et al., 2004 ; Van Der Stelt and Di Marzo, 2004 ; Ahern et al., 2005 ). Such sensitization could be speculated to bring the activation threshold closer to the physiological values of deep T b (Romanovsky, 2007b ). Indeed, Ni et al. (2006) have shown that increasing Tb within the normal physiological range (perhaps as low as 34.5 ° C) can exert a direct stimulatory effect on pulmonary sensory neurons, and this effect is likely mediated by TRPV1. This ﬁ nding makes it difﬁ cult to rule out the possibility that TRPV1 may be involved in normal thermoregulation by serving as a thermoreceptor.

If the TRPV1 channel is indeed activated in vivo by relatively low T b s (Ni et al., 2006 ), it is tempting to hypothesize that the thermoregulatory response to TRPV1 antagonists is related to a suppression of the activation of this channel by physiological Tb s, or in other words, to a suppression of the thermosensory function of the TRPV1 channel. Because higher temperatures would produce a stronger thermal activation of the TRPV1 channel, blocking its thermal activation should cause a stronger response at a higher deep T b (if the responsible TRPV1 channels are located in the body core and are activated by deep Tb ) or at a higher Tsk (if the responsible TRPV1 channels are located in the body surface and are activated by Tsk ). Hence, if AMG0347 - induced hyperthermia (Fig. 14.6 ) is due to the blockade of thermal activation of the TRPV1 channel, there would be a positive correlation between the magnitude of the hyperthermic response and the initial (at the time of drug administration) values of deep Tb and/or Tsk . However, our study (Steiner et al., 2007 ) has rejected this hypothesis. By subjecting the data presented in Fig. 14.6 to correlation analyses, no positive correlation was found. Instead we found a tendency for a negative correlation between the maximal change in deep Tb (between 10 and 180 min after AMG0347 administration) and the initial value of deep T b (Fig. 14.7 a); we also found a weak negative correlation between the maximal change in deep

Tb and the initial Tsk (Fig. 14.7 b). The lack of a positive correlation between the magnitude of AMG0347 -

induced hyperthermia and the rats’ deep T b or Tsk suggests that the normally present, tonic suppression of Tb arises from a tonic activation of TRPV1 channels by nonthermal factors. Such nonthermal factors may include protons, inorganic cations, or endovanilloids (see Section 4.1 ). It is known that many nonthermal signals originating in the abdominal viscera can affect thermoregulation. For instance, either the intraportal infusion of glucose (Sakaguchi and Yamazaki, 1988 ) or the distension of the stomach (Petervari et al., 2005 ) can activate thermogenesis via the appropriate reﬂ exes, whereas colorectal distension can trigger neuroreﬂ exive skin vasoconstriction (Laird et al., 2006 ).

14.5.5 The Site of Action: Peripheral or Central? In the study with AMG0347, which readily crosses the blood – brain barrier (Steiner et al., 2007 ), we also attempted to determine the location of TRPV1 TRPV1 ANTAGONISTS 381

(a) (b) 1.5 1.5

1.0 1.0

0.5 0.5 Maximum change in Maximum change in 0.0 0.0 colonic temperature (ºC) colonic temperature colonic temperature (ºC) colonic temperature 37 38 39 20 25 30 35 Initial colonic temperature (ºC) Initial tail skin temperature (ºC) Figure 14.7 Results of a linear correlation analysis between the magnitude of AMG0347- induced hyperthermia and the values of either initial (at the time of drug administration) colonic temperature (a) or initial tail T sk (b). The data used for the analysis are shown in Fig. 14.6 . Reproduced from Steiner et al. (2007) with permission by the Society for Neuroscience. channels responsible for the hyperthermic effect of AMG0347. We studied whether AMG0347 can cause hyperthermia by acting inside the brain or spinal cord. If one of these sites were a primary site of action, the i . c . v. or i . t . administration of AMG0347 would cause hyperthermia at doses substantially lower (perhaps 1 – 2 orders of magnitude) than the minimally effective i . v . dose of 10 μg/kg. When AMG0347 was administered i . c . v. (into the lateral ventricle),

i . t ., or i . v . at a dose as high as 6 μ g/kg, no signiﬁ cant change occurred in Tb (although a tendency for an increase in T b was observed following either the i . c . v . or i . v . administration). Because i . v . AMG0347 showed a tendency to increase T b at the 6 μg/kg dose (and had a signiﬁ cant effect at 10 μ g/kg), there was no reason to further increase the dose of AMG0347 in this experiment. Clearly, AMG0347 is not more effective in causing hyperthermia when administered into the brain or spinal cord than when administered systemically. It has been recently hypothesized (Gavva et al., 2007b ) that TRPV1 antagonists increase Tb by acting on the brain circumventricular organs, including the organum vasculosum of the lamina terminalis (OVLT) that forms the anterior wall of the third cerebral ventricle. This hypothesis agrees with the fact that the anterior preoptic hypothalamus, which contains the OVLT, is more important for the control of autonomic thermoeffectors than for the control of thermotaxis (Almeida et al., 2006b ), and with the fact that electrolytic lesions of the OVLT and neighboring structures readily cause hyperthermia (Romanovsky et al., 2003 ). However, AMG0347 failed to cause marked hyperthermia when the drug was infused via the route ( i . c . v.) that provides good access to the OVLT and other circumventricular structures (Steiner et al., 2007 ). We propose that TRPV1 antagonists cause hyperthermia by acting in the periphery. 382 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION

As discussed above (Section 3.1 ), the TRPV1 channel is markedly expressed in primary sensory DRG and nodose neurons that innervate the abdominal viscera. Therefore, we tested the hypothesis that TRPV1 antagonists cause hyperthermia by acting at intra- abdominal locations (Steiner et al., 2007 ). For this, we induced the localized desensitization of TRPV1 channels in the abdominal viscera by injecting RTX i . p . at a low dose of 20 μ g/kg. Dogan et al. (2004) have found that TRPV1 desensitization caused by this dose of RTX is restricted to the abdominal cavity. In this desensitization model, CCK does not induce satiety, which is mediated, at least in part, by the CCK1 receptor on sensory vagal fi bers innervating the gastrointestinal tract (South and Ritter, 1988 ; Reidelberger et al., 2004 ). Moreover, there are no signs of systemic desensitization, such as an abolition of the chemosensitivity of the cornea (Szallasi and Blumberg, 1989 ; Craft et al., 1993 ) or an exaggeration of LPS fever (see Section 4.3.2 ). Further confi rmation that TRPV1 desensitization in this model is indeed restricted to the abdominal cavity was obtained by Steiner et al. (2007) through the use of a battery of tests: the eye- wiping test (which determines the sensitivity of TRPV1 channels in the eye); the centrally induced tail vasodilation test (which determines the sensitivity of TRPV1 channels in the brain); the Bezold – Jarisch refl ex test (which determines the sensitivity of TRPV1 channels in the heart and lungs); the hot- plate test (which assesses the sensitivity of TRPV1 channels in the skin, primarily of the paws); and the writhing test (determines the sensitivity of TRPV1 channels in the peritoneal cavity). In rats with localized desensitization of intra- abdominal TRPV1 channels caused by the low i . p . dose of RTX (20 μ g/kg), the writhing test confi rmed a drastic reduction in the responsivity of TRPV1 channels in the abdominal compartment, but no other compartment was affected. Confi rming that the sensitivity of all tests used was suffi cient, the same tests revealed a drastically reduced responsivity of TRPV1 channels in all compartments in rats pretreated with a high (200 μ g/kg i . p .) dose of RTX causing systemic desensitization. When TRPV1 antagonists were tested in rats pretreated with the low (20 μg/kg) dose of RTX, it was found that localized desensitization of TRPV1 channels in the abdominal cavity abolished the hyperthermic responses to i . v . AMG0347 (Steiner et al., 2007 ) and AMG 517 (A. Garami, N.R. Gavva, and

A.A. Romanovsky, unpublished observations), each administered at its EDmax dose. Based on the experiments with localized intra- abdominal desensitization of TRPV1 channels by RTX, we propose that the site of the hyperthermic action of TRPV1 antagonists lies within the abdominal cavity, which is densely innervated by TRPV1- expressing spinal and vagal ﬁ bers (see Section 3.1 ). The hypothesis that AMG0347 causes hyperthermia by blocking TRPV1 channels on afferent ﬁ bers innervating the abdominal viscera also agrees with data showing that the hyperthermic response to this drug involves activation of autonomic thermoeffectors but does not involve thermoregulatory locomotion. As reviewed by Romanovsky (2007b) , the latter TRPV1 ANTAGONISTS 383

behavioral response is triggered almost exclusively by changes in Tsk (detected by cutaneous nerves), whereas core Tb s (detected by visceral and deep somatic nerves) seem relatively more important for triggering autonomic thermoeffectors.

14.5.6 Proposed Neural Pathways

As explained above (Section 5.3 ), TRPV1 - mediated, Tb - suppressing signals may reach the pathways for autonomic thermoeffectors in the MPO. This hypothesis is schematically represented in Fig. 14.8 . The tonic activation of TRPV1- expressing visceral afferents could suppress sympathetic outﬂ ows to BAT and skin blood vessels by contributing to the depolarization of warm -

Abdominal viscera Nonthermal stimuli MPO + + TRPV1 − + − +

DMH Skin − Innocuous warming rRPa Innocuous − cooling

− + Skin vasculature BAT

Figure 14.8 Potential mechanism for suppression of BAT thermogenesis and skin vasoconstriction by nonthermal activation of visceral TRPV1 channels. The afferent pathway that starts with TRPV1- expressing sensory endings is shown in green; the unknown portion of this pathway is shown with a dashed line. For comparison, the afferent pathways that start with cutaneous warm - and cold - sensitive endings are also shown (in red and blue, respectively). The portions of these pathways that cannot be compared to the nonthermal visceral pathways (because the corresponding neurons in the visceral pathway are unknown) are not shown. The efferent pathways are shown in gray. As in Fig. 14.1 , neuronal bodies shown as solid shapes belong to the BAT thermogenesis pathway, and neuronal bodies shown as empty shapes belong to the skin. (See color insert.) 384 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION

sensitive inhibitory projection neurons in the MPO. This depolarization could be mediated through a glutamatergic input. Although the tonic activation of TRPV1 afferents has the same effects on BAT thermogenesis and cutaneous vasoconstriction as the activation of cutaneous warm- sensitive afferents, the pathways activated by nonthermal visceral TRPV1- mediated signals and by innocuous thermal signals from the skin likely differ. Indeed, thermal cutaneous signals, but not nonthermal visceral signals, affect the behavioral selection of Ta (Section 5.3 ). Within the proposed framework for the central pathway mediating the thermoregulatory effects of tonic visceral TRPV1 activation, the hyperthermic responses to TRPV1 antagonists would be mediated by a shift in the balance of synaptic inputs to warm- sensitive neurons in the MPO. That is, a reduction in the glutamatergic excitation driven by the TRPV1 afferent pathway coupled with the ongoing GABA- ergic inhibition driven by skin afferents sensitive to innocuous cold would reduce the activity of the warm- sensitive inhibitory output neurons in the MPO, thereby increasing the activity of the sympathoexcitatory efferent pathways that drive BAT thermogenesis and skin vasoconstriction.

14.5.7 Conclusions Drawn from Experiments with Antagonists 1. Tonic activation of TRPV1 channels in the abdominal viscera by yet unidentiﬁ ed factors inhibits BAT thermogenesis and skin vasoconstric-

tion, thus having a suppressive effect on T b . TRPV1 antagonists cause hyperthermia by removing this tonic activation, and consequently triggering skin vasoconstriction and activating BAT thermogenesis.

2. The TRPV1 -mediated, visceral, Tb - suppressing signals that are blocked by TRPV1 antagonists are nonthermal. TRPV1 channels do not participate in thermoreception; at least, they appear not to be involved in the

detection of those shell and core T b s that regulate the activity of thermoreceptors under physiological conditions.

3. It is unknown at what location the TRPV1 - mediated, Tb - suppressing signals reach the pathways for autonomic thermoeffectors. The only hint we have to answer this question is that these signals modulate the activity of at least two autonomic thermoeffectors (skin vasomotion and BAT

thermogenesis) but do not affect the behavioral selection of Ta . Based on this hint, we speculate, with a large degree of uncertainty, that the

TRPV1 - mediated, Tb - suppressing signals join the thermoeffector pathways in the MPO. 4. The blockade of nonthermal signals at an intra- abdominal location was revealed using AMG0347 and AMG 517 at doses that were two to three orders of magnitude lower than doses of TRPV1 antagonists shown to cause hyperthermia in any other study. It cannot be ruled out that higher doses of TRPV1 antagonists can cause hyperthermia via different mechanisms. GENETIC APPROACH 385

14.6 GENETIC APPROACH

14.6.1 Experiments in Genetically Modiﬁ ed Animals

The Trpv1 gene can be “ knocked out” in mice by deleting the exon that encodes part of the fi fth and all of the sixth transmembrane domains of the TRPV1 channel, together with the intervening pore- loop region (Caterina et al., 2000 ). The thermoregulatory responses to TRPV1 agonists and antagonists, viz, CAP- induced hypothermia (Caterina et al., 2000 ), RTX- induced hypothermia (Steiner et al., 2007 ), and AMG0347 - induced hyperthermia (Steiner et al., 2007 ), do not occur in Trpv1 − /− mice, thus confi rming that both agonist- induced hypothermia and antagonist- induced hyperthermia are indeed mediated by TRPV1. Sensory neurons from Trpv1 − /− mice are defi cient in their responses to several noxious stimuli (Caterina et al., 2000 ). Trpv1− / − mice show no vanilloid - evoked pain behavior, have little thermal hypersensitivity under conditions of infl ammation, and are impaired in the detection of painful heat (Caterina et al., 2000 ). Involvement of TRPV1 in the response to noxious heat has been confi rmed in studies performed in transgenic animals. Mice expressing a short hairpin RNA (shRNA) that permanently knocks down the Trpv1 gene have reduced sensitivity to noxious heat (Christoph et al., 2008 ). In another model, transgenic mice that overexpress artemin (a neuronal survival factor) in the skin have a large increase in the number of DRG neurons and exhibit an overexpression of both TRPV1 and TRPA1, as well as an increased sensitivity of sensory neurons to both noxious heat and noxious cold, but not to mechanical stimuli (Elitt et al., 2006 ). The potential involvement of TRPV1 in thermoregulation was investigated in two studies in Trpv1− /− mice (Szelenyi et al., 2004 ; Iida et al., 2005 ). In both studies, the authors found that neither the ability to regulate T b under thermoneutral conditions nor the response to heat or cold was altered in Trpv1− / − animals. However, Trpv1 − /− mice did exhibit a somewhat exaggerated magnitude of day – night fl uctuations in deep T b without changes in the mean − / − Tb value (Szelenyi et al., 2004 ). In agreement with these studies in Trpv1 mice, shRNA transgenic mice did not exhibit altered Tb (Christoph et al., − /− 2008 ). A recent study reported that Trpv1 mice can defend their Tb against severe environmental cooling better than their Trpv1+/+ controls (Motter and Ahern, 2008 ), although an earlier study found no alterations in the response of Trpv1− / − mice to cold exposure (Iida et al., 2005 ). Trpv1− / − mice have been reported to respond to LPS with an attenuated fever (Iida et al., 2005 ). In contrast, Dogan et al. (2004) reported that neither TRPV1 desensitization with RTX nor pharmacological antagonism with capsazepine (CPZ) altered LPS - induced fever in rats. Even if the TRPV1 channel were involved in LPS fever, such an involvement could be in the mechanisms of immune signaling rather than those regulating T b per se. This proposition seems likely in view of the reports of LPS - induced TRPV1 overexpression in 386 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION rats (Orliac et al., 2007 ) and of the exaggeration of several responses (including the hypothermic one) to shock- inducing doses of LPS in Trpv1− / − mice (Clark et al., 2007 ). In the latter study, however, the knockout and control lines were generated from separated colonies, that is, started out with different background genes and then underwent separate genetic drift within each line; false- positive results are possible in a study using such animals (Lariviere et al., 2001 ). Although thermoregulatory experiments are notoriously diffi cult to conduct − / − in mice (Rudaya et al., 2005 ), further studies of T b regulation in Trpv1 mice may be warranted, even though the studies performed so far have not revealed a clear thermoregulatory phenotype. It should be noted, however, that negative results obtained in knockout animals are typically inconclusive. Various types of compensatory changes may restore the function of interest, even if the knocked out gene is normally responsible for this function; the potential pitfalls of working with knockout mice are analyzed elsewhere (Mogil et al., 2000 ; Lariviere et al., 2001 ; Woodbury et al., 2004 ). It may be interesting to study the regulation of Tb in other genetic models as well, because compensation patterns differ when alternative methods of silencing the Trpv1 gene are utilized (Christoph et al., 2008 ). Several other strategies to silence the Trpv1 gene, for example, antisense oligonucleotides (Christoph et al., 2007 ), small interfering RNA (Christoph et al., 2006 ), and shRNA (Christoph et al., 2008 ), may provide new insights into the role of TRPV1 in thermoregulation.

14.6.2 Studies in Humans At least six nonsynonymous polymorphisms have been identiﬁ ed in the human TRPV1 gene (Xu et al., 2007 ). Kim et al. (2004) have found that American women of European origin, homozygous for the Val 585 allele of the TRPV1 gene, are more tolerant of painful cold stimuli, thus suggesting that factors contributing to individual variation in cold - pain sensitivity interact with TRPV1 single nucleotide polymorphism. However, in a later study, the same group found signiﬁ cant associations of both cold- pain sensitivity and heat- pain sensitivity with variations in the TRPA1 (but not TRPV1 ) gene in a population with a similar ethnic background (Kim et al., 2006 ). Taking into consideration the strong data (including in humans; see Anand et al. [2008 ]) showing that the TRPV1 and TRPA1 channels are coexpressed in polymodal nociceptors, it is likely that TRPV1 is involved in thermal pain in humans.

14.6.3 Conclusions Drawn from Genetic Studies Genetic studies in laboratory animals and, to a lesser extent, in humans, conﬁ rm that TRPV1 channels are involved in thermal pain. However, genetic studies have not yet appreciably enhanced our understanding of TRPV1 involvement in deep Tb regulation. IMPLICATIONS FOR DRUG DEVELOPMENT 387

14.7 IMPLICATIONS FOR DRUG DEVELOPMENT

From the point of view of development of TRPV1 antagonists as novel drugs for pain management, the hyperthermic effect of these compounds poses an undesirable on- target side effect. Several strategies of achieving TRPV1 antagonist- induced analgesia without the hyperthermic side effect have been suggested. Initially, it was hoped that the hyperthermic effect was chemotype- specifi c. This, however, is not the case, as compounds of different chemotypes have been shown to cause hyperthermia (Swanson et al., 2005 ; Gavva et al., 2007a,b ; Steiner et al., 2007 ; Gavva et al., 2008 ). It was then proposed that the initial hyperthermic effect may be manage- able, since the hyperthermia attenuated after repeated administration of the antagonist, whereas the analgesic effect was maintained (Gavva et al., 2007a ). Indeed, these distinct differences between the time course of hyperthermia and analgesia were reported for several TRPV1 antagonists (AMG 517, AMG8163, and ABT- 102) in rats, dogs, and monkeys (Gavva et al., 2007a ; Honore et al., 2009 ). However, when AMG 517, which has an exceptionally long half- life (13– 23 days) in humans, was studied in clinical trials, the hyperthermic effect showed limited attenuation with repeated dosing (Gavva et al., 2008 ). It was conceivable that a TRPV1 antagonist could be combined with another drug, for example, an inhibitor of PG synthesis, to yield analgesia without hyperthermia (Gavva et al., 2007a ). Although acetaminophen blocked the hyperthermic response to AMG8163 in rats, it did so at a very high, hypothermia- inducing dose of 300 mg/kg (Gavva et al., 2007a ), which is equivalent to a 24 - g dose for an 80 - kg human. Steiner et al. (2007) have raised the possibility that the analgesic and hyperthermic effects of TRPV1 antagonists could be dissociated by taking advantage of the fact that the TRPV1 channels responsible for these effects appeared to be located in different body compartments. However, to date, no specifi c experimental recommendations have been proposed. Currently, the most promising strategy seems to be based on the observation that a TRPV1 antagonist can exhibit different pharmacological characteristics depending on the mode of activation of the TRPV1 channel (McIntyre et al., 2001 ; Gavva et al., 2005a ; Lehto et al., 2008 ). In a recent study, Lehto et al. (2008) used four structurally related TRPV1 antagonists with different profi les, all of which inhibited TRPV1 activation by CAP. Hyperthermia was caused in rats by those antagonists that either blocked (AMG8163) or partially attenuated (AMG8563) TRPV1 activation by protons in vitro. In contrast, hypothermia was caused by those antagonists that potentiated TRPV1 activation by protons (AMG8562 and AMG7905). AMG8562, a competitive antagonist at the CAP - binding pocket and a positive allosteric modulator for proton activation, had signifi cant effi cacy in several rodent models of pain, thus providing an example of TRPV1 antagonist - induced analgesia without the hyperthermic side effect in rats. The authors concluded that an antagonist will 388 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION cause hyperthermia if it inhibits TRPV1 activation by CAP and also inhibits activation (fully or partially) by protons. On the other hand, an antagonist that inhibits TRPV1 activation by CAP but potentiates TRPV1 activation by protons will not cause hyperthermia and may cause hypothermia instead (Lehto et al., 2008 ). In connection with the hypothesis by Lehto et al. (2008) , the profi le of CPZ, which was the fi rst TRPV1 antagonist to be identifi ed, deserves attention. Although CPZ has been used extensively in thermoregulation studies in rats and mice (Dogan et al., 2004 ; Jakab et al., 2005 ; Shimizu et al., 2005 ), it has not been reported to cause hyperthermia. Interestingly, CPZ does not block activation of the TRPV1 channel by protons (IC 50 > 4000 nM) in the rat or mouse (McIntyre et al., 2001 ; Savidge et al., 2002 ; Correll et al., 2004 ; Gavva et al., 2005a,b ), although it does block proton activation (IC50 = 360 nM) in the guinea pig (Savidge et al., 2002 ). The study by Lehto et al. (2008) suggests that compounds that potentiate or do not block TRPV1 activation by acidic pH may provide a new strategy for developing hyperthermia - free TRPV1 antagonists. However, it is currently unclear to what extent the data from this study can be generalized. Even if we consider, as a fi rst approximation, that different modes of activation of the TRPV1 channel are independent variables, it will be diffi cult to defi nitively ascribe the in vivo hyperthermic effect of a TRPV1 antagonist to its in vitro effects on various modes of activation. Hence, even though Lehto et al. (2008) have proposed an intriguing possibility, further research is needed.

14.8 THE ROLES OF TRPV 1 CHANNELS IN THERMOREGULATION: OVERALL CONCLUSIONS

Experiments using different tools have revealed two major groups of TRPV1 - expressing cells that are relevant to thermoregulation: primary sensory neurons and POA neurons. Based on the distribution data showing extensive expression of TRPV1 channels in primary sensory neurons, it is likely that the major TRPV1 involvement in physiological processes is on the afferent side. The strong association of TRPV1 with polymodal nociceptive fi bers suggests an involvement in the mechanisms of pain rather than in the detection of thermal signals used for deep T b regulation. Genetic studies in both laboratory animals and humans confi rm that TRPV1 channels, perhaps those on polymodal nociceptive primary sensory neurons, are involved, at least to some extent, in thermal pain. Studies with CAP and RTX produced little evidence to support the possibility that TRPV1 agonists cause hypothermia by acting primarily on the peripheral terminals of primary sensory neurons. Nevertheless, if CAP- or RTX- induced hypothermia is mediated in part by TRPV1 channels on sensory neurons, this would suggest that polymodal nociceptors can make a signifi cant contribution to triggering thermoregulatory responses. THE ROLES OF TRPV1 CHANNELS IN THERMOREGULATION 389

Studies with TRPV1 antagonists are consistent with this possibility. These studies have shown that tonic activation of TRPV1 channels in the abdominal viscera, by as yet unidentiﬁ ed factors, inhibits BAT thermogenesis and skin vasoconstriction, and thus has a suppressive effect on deep T b . TRPV1 antagonists cause hyperthermia by removing this tonic activation and, consequently, triggering skin vasoconstriction and activating BAT thermogenesis. The

TRPV1 - mediated, visceral, T b - suppressing signals that are blocked by TRPV1 antagonists are nonthermal. TRPV1 channels do not appear to be involved in the detection of shell and core Tb s that regulate the activity of thermoreceptors under physiological conditions. Distribution studies have documented the presence of functional TRPV1 channels on glutamatergic neurons in the POA. Several groups of POA neurons, including those in the MnPO and MPO, are involved in the neural pathways for autonomic thermoeffectors. Our analysis of studies with TRPV1 agonists (administered to activate or to desensitize TRPV1- expressing neurons) suggests that the agonists cause a hypothermic response possibly by acting on MnPO neurons. Such an action would explain the recruitment of a broad spectrum of both autonomic and behavioral thermoeffector mechanisms. Furthermore, desensitization of these neurons with TRPV1 agonists would account for the selective loss of sensitivity to environmental heat found in animals treated with CAP or RTX as neonates or as adults. However, studies with TRPV1 agonists have also produced two facts that cannot be explained solely by involvement of MnPO neurons. First, hypothermic responses both to intra- POA CAP and to POA heating are attenuated in rats treated with systemic CAP as adults. Second, rats treated with high systemic doses of TRPV1 agonists as adults lose not only the ability to respond with autonomic heat- defense responses to external heating, but also the ability to respond with autonomic cold - defense responses to external cooling. Both facts can be readily explained by an involvement of MPO neurons, the warm - sensitive neurons that participate, as fi rst efferent neurons, in both skin- vasomotion and BAT thermogenesis pathways. Studies with antagonists have produced no evidence to suggest that TRPV1 channels in the POA participate in normal thermoregulation. This may mean either that TRPV1 channels on POA neurons are not tonically activated, or that higher doses of TRPV1 antagonists are required to reach POA neurons and block their tonic activation. On the drug development side, examples of TRPV1 - antagonist - induced analgesia without hyperthermia have been obtained, at least in rodent models. There is a strong hope, therefore, that the hyperthermic side effect can be avoided and will not stop the development of TRPV1 antagonists as pain therapeutics. It is diffi cult to predict which specifi c strategy will ultimately lead to the creation of a drug that does not have thermoregulatory side effects in humans, but it seems likely that the successful strategy (or strategies) will be based, at least in part, on the knowledge about the role of TRPV1 channels 390 THE TRPV1 CHANNEL IN NORMAL THERMOREGULATION

in T b regulation. Further studies in this area are needed, and such studies are currently in progress in several laboratories, including those of the authors of several chapters included in this book.

NOTE ADDED IN PROOF

While this manuscript was in press, the authors have proposed a more plausible explanation for the two facts listed in conclusion 5 (p. 373) and later on page 389. This new explanation (Romanovsky et al., 2009 ) does not require an action of TRPV1 agonists on MPO neurons. It involves an action of TRPV1 agonists on MnPO neurons only and consequent degeneration of MPO neurons.

ACKNOWLEDGMENTS

The authors ’ research summarized in this chapter has been supported by study agreements with Amgen, Inc. (AAR) and grants from the National Institutes of Health (R01NS41233 to AAR and R01NS40987 to SFM), Arizona Biomedical Research Commission (category II, N° 8016 to AAR), and St. Joseph ’ s Foundation (AAR). We thank Dr. Sonya G. Lehto (Amgen, Inc.) for reading the Genetic Approach section, Dr. Mark H. Norman (Amgen, Inc.) for providing the chemical names of TRPV1 antagonists, Dr. Connie R. Faltynek (Abbott Laboratories) and Julie M. Turko for editing the manuscript, and Daniela L. Oliveira for technical assistance.

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Serena Materazzi , Alain Tchoimou , Romina Nassini , Marcello Trevisani , and Pierangelo Geppetti

15.1 PRIMARY SENSORY NEURONS AND NEUROGENIC INFLAMMATION IN THE AIRWAYS

The upper and lower airways, from the nose to the bronchioles, have a dense sensory innervation that is mainly provided by trigeminal and vagal neurons. In rodents, roughly half of this sensory nerve supply consists of primary sensory neurons uniquely sensitive to capsaicin, the hot principle contained in the plants of the genus Capsicum. This subset of neurons is characterized by small and dark cell bodies with C or A δ unmyelinated or lightly myelinated slow - conducting ﬁ bers and by the content of neuropeptides, namely, the calcitonin gene - related peptide (CGRP) and the tachykinin substance P (SP) and neurokinin A (NKA). Naturally occurring tachykinins exert their biological functions via activation, with different afﬁ nities, of three receptor subtypes, the NK1, NK2, and the NK3 receptors (Regoli et al., 1994 ). The CGRP (calcitonin- like [CL]) receptor has the unique feature to become functional only if associates with the receptor activity modifying protein 1 (RAMP1) (Brain and Grant, 2004 ). Sensitivity of these neurons to capsaicin and capsaicin- like molecules (vanilloids) is conferred by the expression on the neuronal plasma membrane of the transient receptor potential vanilloid 1 (TRPV1) (Caterina et al., 1997 ), which is a cation channel belonging to the TRPV family and to the larger superfamily of TRP ion channels (Caterina et al., 1997 ;

403 404 THE ROLE OF TRPV1 IN RESPIRATORY DISEASES

Clapham, 2003 ; Nilius, 2007 ). The nonselective cation channel TRPV1 is stimulated not only by exogenous xenobiotics such as capsaicin or resiniferatoxin and by noxious temperatures ( >42 ° C) but also by a series of endogenous agents, including low extracellular pH, anandamide, N - arachidonoyl dopamine, certain eicosanoids, and other agents (Bevan and Geppetti, 1994 ; Tominaga et al., 1998 ; Hwang et al., 2000 ; Huang et al., 2002 ). Capsaicin- sensitive primary sensory neurons, including those innervating the airways, express on their plasma membranes, in addition to TRPV1, a variety of ion channels and receptors with excitatory and inhibitory activities that regulate neuronal functioning. A specifi c feature of capsaicin- sensitive sensory neurons is their dual function. The ability to sense and to transmit noxious stimuli and nociceptive information is intrinsically associated with the release of neuropeptides from their peripheral terminals. Neuropeptide secretion results in a series of proinfl ammatory responses that are collectively referred to as “ neurogenic infl ammation” (Geppetti and Holzer, 1996 ). Sir Thomas Lewis in his pioneering studies (Lewis, 1937 ) precisely defi ned the dual “ nocifensor ” role of these neurons, as characterized by the capacity of one portion of the widely branching sensory fi ber to respond to the injury and to generate action potentials, which are then carried antidromically to other branches of the fi ber, resulting in the release of a chemical substance that increases the sensitivity of other sensory axons responsible for pain. There is now a bulk of information suggesting that this phenomenon, fi rst described at the somatic (skin) level, occurs in a variety of visceral organs, including the airways. In addition, we know that sensory neuropeptide release may occur not only from collateral fi bers invaded antidromically by action potentials through a tetrodotoxin - sensitive axon refl ex but also, as in the case of capsaicin, by the stimulated terminal itself via a tetrodotoxin - insensitive mechanism (Szolcsanyi, 1987b ). The present chapter focuses on the role of TRPV1 in the activation of the dual function of sensory neurons in the airways of experimental animals and man. Infl ammatory responses, commonly referred to as “ neurogenic infl ammation, ” mainly occur at the vascular level but are also well represented in many other tissues and organs. These responses show remarkable variability according to the organ/tissue examined and the mammal species under consideration. Vascular neurogenic infl ammatory responses are more stereotyped in rodent tissues (including the airways) and consist of arterial vasodilation (mediated by CGRP and the CL/RAMP1 receptor), plasma protein extravasation, and leukocyte adhesion to the vascular endothelium of postcapillary venules (mediated by SP/NKA and the NK1 receptor). Cardiac positive chro- notropic effects (CGRP, CL/RAMP1 receptor), contraction of the smooth muscle of the iris sphincter (SP/NKA, NK2 receptor), ureter, bladder neck and urethra (SP/NKA, NK2/NK1 receptors), relaxation of bladder dome (CGRP, CL/RAMP1 receptor), and exocrine gland secretion (SP/NKA, NK1 receptor) are some of the neurogenic infl ammatory responses in nonrespira- tory tissue. PRIMARY SENSORY NEURONS AND ACTIVATION OF REFLEX RESPONSES 405

Extravascular neurogenic infl ammation in the guinea pig airways encom- passes excitatory nonadrenergic, noncholinergic (eNANC) contraction of the tracheal and bronchial smooth muscle, mediator release from the airway epithelium, and secretion of mucus from airway glands. Gland secretion is observed in many mammalian species and is always mediated by NK1 receptors (Geppetti et al., 1993 ; Rogers, 2002 ). Release of mediators from the epithelium is also caused by NK1 receptors. In contrast, neurogenic contraction of the airway smooth muscle is much more variable across mammalian species and results from either direct or indirect actions of neuropeptides on the smooth muscle. There is no consistent evidence that capsaicin or other TRPV1 - selective agonists produce neurogenic bronchoconstriction in human airways, or in the airways of large - sized mammals. In the guinea pig, mainly NK2, but also NK1, expressed on the airway smooth muscle directly mediates tachykinin- induced bronchoconstriction. However, the airway smooth muscles of rats and mice do not appear to express tachykinin receptors; in these species, SP and NKA cause an indirect bronchodilatation mediated by epithelial nitric oxide/prostanoid release. Tachykinins, mainly through tachykinin NK2 and NK1 receptors, mediate a remarkable contraction of the human airway smooth muscle both in vitro and in vivo (Amadesi et al., 2001 ; Joos and Pauwels, 2001 ). The action of NK3 receptors is minor and seems to be confi ned to excitation of bronchial postganglionic cholinergic nerve terminals (Myers et al., 2005 ). Studies with capsaicin and TRPV1 have enhanced the current understanding of neurogenic infl ammation in the airways. However, it should be noted that at least fi ve additional TRP channels are expressed on sensory neurons, including those of trigeminal and vagal ganglia. These include the TRPV2, TRPV3, and TRPV4 channels (gated by warm, non- noxious and noxious temperatures and small reductions in tonicity) and the transient receptor potential melastatin 8 (TRPM8) channel (activated by menthol and moderate low temperature) (Caterina et al., 1999 ; Liedtke et al., 2000 ; McKemy et al., 2002 ; Peier et al., 2002 ; Alessandri - Haber et al., 2003 ; Bautista et al., 2007 ). TRPA1 is a more recently identifi ed channel, which is almost entirely coexpressed with TRPV1 on sensory neurons (Story et al., 2003 ; Nagata et al., 2005 ) and is activated by isothiocyanates, thiosulfi nate, and cinnamaldehyde (the pungent ingredients in mustard, garlic, and cinnamon, respectively) (Bandell et al., 2004 ; Jordt et al., 2004 ; Macpherson et al., 2005 ; Hinman et al., 2006 ). Recent evidence underlines a primary role for TRPA1 in airway pathophysiology (Andre et al., 2008 ). However, all these TRP channels and their specifi c stimuli are potentially implicated in the activation of neurogenic infl ammatory responses in the airways.

15.2 PRIMARY SENSORY NEURONS AND ACTIVATION OF REFLEX RESPONSES IN THE AIRWAYS

Intravenous bolus injection of capsaicin in experimental animals produces a triple response consisting of hypotension, bradycardia, and apnea, known as 406 THE ROLE OF TRPV1 IN RESPIRATORY DISEASES the pulmonary chemorefl ex or Bezold– Jarisch refl ex (Coleridge and Coleridge, 1984 ). A fi rst study on capsaicin inhalation in man showed that the compound produced cough, most likely mediated by stimulation of TRPV1, but no signifi cant change in the forced expiratory volume in 1 s (FEV1) (Collier and Fuller, 1984 ). Studies in heart and lung transplant patients indicated that aqueous solutions that contained low concentrations of chloride anions acted in the upper airways/larynx to produce cough (Higenbottam, 2002 ). The capsaicin receptor responsible for the cough response was initially localized to nerve terminals situated in the larynx (Collier and Fuller, 1984 ), although capsaicin aerosols that provoke cough diffuse to both central and peripheral airways (Higenbottam, 2002 ). In another study, which used a more sensitive measurement of airway resistance, the effect of capsaicin was ascribed to activation of cholinergic refl ex responses due to inhibition by ipratropium bromine (Fuller et al., 1985 ). These in vivo data are only partially consistent with in vitro fi ndings that capsaicin contracts isolated human bronchi via an atropine- insensitive mechanism, which undergoes rapid tachyphylaxis upon repeated exposure to the agonist (Lundberg and Saria, 1983 ). Neither in vivo nor in vitro data on capsaicin - evoked bronchoconstriction in man have been consistently replicated. Studies in experimental animals indicate that the cough refl ex is initiated by stimulation of rapidly adapting receptors (RARs) that conduct with action potentials in the A δ range. Mechanical perturbation of their receptive fi elds activate RARs, but these receptors are unaffected by a variety of chemical agents or messengers, including bradykinin and capsaicin. In contrast, C - fi bers, which are much less sensitive to mechanical stimulation, are activated by capsaicin and bradykinin (Undem et al., 2002 ). In cats, stimulation of pulmonary C - fi bers by intravenous capsaicin or phenylbiguanide evokes apnea and inhibits, rather than causes or enhances, cough induced by mechanical stimuli (Tatar et al., 1988 ). On the other hand, the threshold for initiating the cough refl ex is markedly reduced by capsaicin and bradykinin (Mazzone et al., 2005 ). Whereas hypertonic saline- induced cough was found to be independent from TRPV1 (Trevisani et al., 2004a ), citric acid has been reported to mediate a tussive response that is inhibited by the TRPV1 antagonists, capsazepine, or iodoresiniferatoxin (Lalloo et al., 1995 ; Trevisani et al., 2004b ). However, electrophysiological studies showed that additional mechanisms, including activation of acid- sensing ion channels (ASICs), which sense low extracellular pH, could trigger tussive responses (Kollarik and Undem, 2002 ). Overall, animal and human studies clearly indicate that TRPV1 is a major molecular entity involved in the initiation of the cough refl ex. The ability of TRPV1 to evoke cough seems to be dependent on the anatomical localization of the sensory nerve terminal expressing the channel, with a clear protussive role for TRPV1 in the larynx/upper airways and a paradoxical inhibitory effect of intrapulmonary TRPV1. However, it should be noted that TRPV2, TRPV3, TRPV4, and TRPA1, coexpressed at varying degrees with TRPV1 on capsaicin- sensitive neurons (Story, 2006 ; Nilius, 2007 ), may be also involved in the NEUROGENIC INFLAMMATION AND TRPV1 IN MODELS 407 cough refl ex. In contrast, TRPM8, which at the neuronal level is not expressed with TRPV1, may serve different functions, including cough inhibition (Morice et al., 1994 ). The nasal mucosa in both rodents and humans possesses a dense sensory innervation (Seki et al., 2006 ). Thus, it is not surprising that application of capsaicin into the nose causes refl ex sneezing discharges that are presumably due to activation of TRPV1 in the trigeminal fi bers of the guinea pig (Lundblad et al., 1984 ) and in the human (Geppetti et al., 1988 ) nasal mucosa.

15.3 NEUROGENIC INFLAMMATION AND TRPV1 IN MODELS OF AIRWAY DISEASES

Sensory nerves and neurogenic infl ammation have been shown to contribute to acute infl ammatory and defensive responses in a variety of models of airway disease. The fi rst evidence that cigarette smoke inhalation, the major causative agent of chronic obstructive pulmonary disease (COPD), induced neurogenic plasma protein extravasation in rodent airways, which was abolished by capsaicin desensitization, was provided by the pioneering studies of Lundberg and Saria (Lundberg and Saria, 1983 ). Further evidence that cigarette smoke causes neurogenic leukocyte recruitment in the infl amed airways was provided by the use of early- generation tachykinin NK1 receptor antagonists (Baluk et al., 1996 ). However, very recently, it has been demonstrated that this effect is entirely mediated by the excitatory action of the α , β - unsaturated aldehydes, crotonaldehyde, and acrolein (abundantly contained in cigarette smoke) on the TRPA1 channel coexpressed with TRPV1 on sensory nerve endings (Andre et al., 2008 ). Tachykinin release from sensory nerve endings has been implicated in various acute responses to antigen challenge in guinea pigs and in rabbits (Ricciardolo et al., 2000 ; Keir and Page, 2008 ). Thus, in guinea pigs treated with phosphoramidon (an inhibitor of neutral endopeptidase that metabolizes tachykinins), a large component of the bronchoconstriction and plasma extravasation of the fi rst phase of the allergic response to ovalbumin is dependent on tachykinins and on sensory nerves (Bertrand et al., 1993 ). The underlying mechanism and the possible role of TRP channels in this response are at present unknown, but preliminary observations seem to exclude a role for TRPV1 in the hyperreactivity that follows the allergic response in mice (S.G. Vincent and J.T. Fisher, pers. comm.). Additional examples of the contribution of sensory nerve endings in acute infl ammation in the airways include the response to ozone (Kaneko et al., 1994 ), cold air (Yoshihara et al., 1995 ) and low pH media (Ricciardolo et al., 1999 ). In cultured rat vagal sensory neurons, lowering the pH of the extracellular solution to 7.0 evoked a small- amplitude, transient, and rapidly inactivating current, which has been ascribed to ASIC activation due to sensitivity to amiloride. However, if the pH is reduced to below 6.5, a sustained current is generated, which is almost completely abolished by capsazepine, indicating a 408 THE ROLE OF TRPV1 IN RESPIRATORY DISEASES critical involvement of TRPV1 in acid- evoked currents in these neurons (Gu and Lee, 2006 ). Thus, a long - lasting effect on TRPV1 contributes to the linger- ing irritant effect of acid on the airways. There is evidence that acid instillation into the airways causes neurogenic infl ammation (Lou and Lundberg, 1992 ) and cough (Trevisani et al., 2004b ), which are sensitive to TRPV1 antagonism, and that the presence of acid in the esophagus may cause neurogenic infl ammation in the respiratory system via an hitherto unknown neuroanatomical pathway (Daoui et al., 2002 ). These observations further point to TRPV1 as an important channel in acid- induced diseases of the airways. In particular, it is possible that acid - driven and TRPV1 - dependent mechanisms contribute to the local and refl ex responses in asthmatic patients since low pH has been detected in the airway tissue during attacks of asthma (Ricciardolo et al., 2004 ). An association between gastroesophageal refl ux disease (GERD) and asthma has been clearly shown (Harding, 2005 ), and the acidic component of the refl ux is considered a major causative agent of the infl ammatory response associated with GERD - induced asthma. If triggering factors are continuously produced over time, neurogenic infl ammation may cause exaggerated responses that in the long term may result in tissue injury and disease. However, as mentioned before, according to the proposal that neuropeptide - containing sensory nerves constitute the “ nocifensor system, ” neurogenic infl ammation is regarded as a defensive mechanism. Recent evidence proposes a novel mechanism that further supports the dual sensory and defensive functions of TRPV1 - expressing neurons. In fact, pharmacological and genetic interventions in endotoxin- induced airway infl ammation showed that somatostatin released from sensory nerve terminals, in response to activation of TRPV1 receptors in the lung, reduced bronchial hyperreactivity (Elekes et al., 2007 ).

15.4 LOCALIZATION AND PLASTICITY OF TRPV 1 IN THE AIRWAYS

Immunohistochemical experiments have provided evidence that TRPV1 is widely distributed in the lungs, where it typically colocalizes with SP and CGRP - containing neurons within vagal C - fi ber sensory nerves (Watanabe et al., 2006 ). In the guinea pig, TRPV1- positive nerve fi bers are localized within the epithelium of the trachea around smooth muscles and blood vessels, within the lower airways in the vicinity of bronchi and bronchioles and around alveolar tissue. Of interest for further discussion is the fi nding that no TRPV1 was found in airway epithelial cells in guinea pigs (Watanabe et al., 2005 ). Previous studies using real time reverse-transcriptase-polymerase chain reaction (RT- PCR) revealed that TRPV1, together with ASIC1a and ASIC3 subunits of proton - gated ion channels, are expressed in immortalized human bronchial epithelial cells, normal human bronchial/tracheal epithelial cells, and normal human small airway epithelial cells from the distal airways (Agopyan et al., 2003 ). TRPV1 activation induces apoptosis in these cells LOCALIZATION AND PLASTICITY OF TRPV1 IN THE AIRWAYS 409

Tissue injury and inflammation

Neurogenic inflammation NKA SP CGRP BK ATP HIS 5HT PGE2 H+

X IL-1b 2 P NGF R BK TRPV1 TrkA ASIC

Sensitization of H1 R TRPs airway sensory terminals ET R 5HT

P IL1 C EP N Figure 15.1 A schematic representation of the inﬂ ammatory mediators and receptors that may affect the functional phenotype of the sensory nerve terminal and the activity of TRPV1 in the airways. TRPs, transient receptor potential channels; TrkA, tyrosine kinase receptor A; EP, prostaglandin E2 receptor; BKR, bradykinin receptor; P2X, ionotropic purinergic receptors; CGRP, calcitonin gene - related peptide; SP, substance P; PGE2, prostaglandin E2; NKA, neurokinin A; HIS, histamine; BK, bradykinin; IL - 1 β , interleukin - 1 β ; 5HT, serotonin .

(Agopyan et al., 2004 ). Mouse larynx epithelial cells (Hamamoto et al., 2008 ) and human nasal epithelial and endothelial cells (Seki et al., 2006 ) have also been found to express TRPV1. However, because no conclusive evidence has been produced regarding the functionality of extraneuronal TRPV1 (including TRPV1 in airway epithelial cells) (Stander et al., 2004 ; Basu and Srivastava, 2005 ; Sanchez et al., 2005 ), this issue will not be further discussed. In addition to its neuronal localization and biological functions, a role for TRPV1 in airway disease is markedly supported by the observations that the expression of mRNA/protein and the function of TRPV1 are upregulated under experimental infl ammatory circumstances and in the course of infl ammatory diseases (Fig. 15.1 ). Nerve growth factor (NGF) is released from mast cells during asthma exacerbations (Bonini et al., 1996 ), and there is now a large body of evidence that NGF upregulates TRPV1 and by this mechanism may contribute to a variety of diseases, including asthma. Survival of newborn rat dorsal root ganglia neurons and physiological expression of the TRPV1 phenotype in adult rat dorsal root ganglia neurons in culture is dependent on NGF (Bevan and Winter, 1995 ). NGF induces both acute and long - lasting hyperalgesic effects. Via p38 mitogen- activated protein (MAP) kinase, NGF increases capsaicin sensitivity of dorsal root ganglion (DRG) nociceptive neurons (Chuang et al., 2001 ) by increasing TRPV1 protein transportation to the peripheral endings of sensory neurons, a phenomenon associated with an increase in heat hypersensitivity (Ji et al., 2002 ). Inhibition of phosphatidylinositol - 3 - kinase (PI3K), which is physically and functionally coupled to TRPV1 and facilitates traffi cking of TRPV1 to the plasma mem- 410 THE ROLE OF TRPV1 IN RESPIRATORY DISEASES brane, abrogates NGF - mediated TRPV1 upregulation (Stein et al., 2006 ). TRPV1 functioning is heavily regulated both by protein kinase A (PKA) and protein kinase C (PKC) and by phospholipase A and C metabolites. For example, anandamide, through a PKC - ε - dependent pathway, reduces the threshold temperature for TRPV1 activation (Premkumar and Ahern, 2000 ). Bradykinin sensitizes TRPV1 by different intracellular mechanisms. These include PKC - ε (Premkumar and Ahern, 2000 ; Sugiura et al., 2002 ), displace- ment of phosphatidylinositol 4,5-bisphosphate (PIP2) from TRPV1 (Chuang et al., 2001 ), and production of 12- and 5- lipoxygenase metabolites (Shin et al., 2002 ; Carr et al., 2003 ). Potentiation of TRPV1 activation by bradykinin has been observed in vagal afferent C - fi bers (Lee et al., 2005 ). Protease - activated receptor 2 (PAR2), which is expressed by a large variety of lung cells including TRPV1- positive sensory neurons, is stimulated through cleavage of its extracellular domains by proteases such as trypsin and mast cell tryptase (Ossovskaya and Bunnett, 2004 ). PAR2 stimulation promotes neurogenic infl ammation and hyperalgesia (Steinhoff et al., 2000 ; Vergnolle et al., 2001 ). PAR2 activation contributes to infl ammatory lung responses, including exaggeration of allergic reaction (Schmidlin et al., 2002 ), bronchoconstriction, and plasma protein extravasation (Su et al., 2005 ), which are effects mediated in part by a neurogenic mechanism. PAR2 stimulation upregulates TRPV1 - mediated responses (Amadesi et al., 2004 ; Dai et al., 2004 ) by a PKC - dependent mechanism (Amadesi et al., 2006 ). PKC is also involved in the augmentation of the cough response to TRPV1 agonists by PAR2 activation in guinea pigs (Gatti et al., 2006 ). In addition, PAR2 upregulates both the pulmonary chemorefl ex sensitivity in vivo and the excitability of isolated pulmonary chemosensitive neurons in vitro. This effect of PAR2 activation by capsaicin or acid is mediated through a PKC- dependent transduction pathway (Gu and Lee, 2006 ). Thus, PAR2, mainly through a PKC - mediated pathway, regulates TRPV1 and controls the neural components of the infl ammatory response in the airways. Sensitization of TRPV1 by PKC and by cAMP - dependent protein kinase (PKA) pathways is promiscuously used by different stimuli, including capsaicin, anandamide, heat, and protons (Premkumar and Ahern, 2000 ; De Petrocellis et al., 2001 ; Vellani et al., 2001 ; Bhave et al., 2002 ). However, TRPV1 sensitization does not appear to be a phenomenon uniquely generated by endogenous agents, as there are examples of exogenous stimuli that cause marked TRPV1 potentiation and that may have some clinical relevance. The demonstration that ethanol excites primary sensory neurons by a selective effect on the thermosensor channel TRPV1 (Trevisani et al., 2004a ) explains the common observation that exposure of mucosal surfaces or wounds to alcoholic tinctures causes burning pain. The threshold temperature for TRPV1 activation is 42 ° C (Caterina et al., 1997 ). However, ethanol lowers this threshold temperature by 8 ° C (Trevisani et al., 2002 ). Thus, in the presence of ethanol, the physiological body temperature of 37 ° C is able to stimulate TRPV1 per se, and ethanol markedly potentiates the action of anandamide and protons (Trevisani et al., 2002 ). Ethanol - induced asthma is a recognized, LOCALIZATION AND PLASTICITY OF TRPV1 IN THE AIRWAYS 411 but still poorly understood, clinical condition (Vally and Thompson, 2002 ). In addition to accumulation of acetaldehyde, ethanol itself, which contracts isolated guinea pig bronchi and causes bronchoconstriction and bronchial microvascular leakage through a capsaicin - sensitive, TRPV1 - dependent mechanism (Trevisani et al., 2004a ), may contribute to the disease. The observation that ethanol potentiated TRPV1 - mediated cough in guinea pigs (R. Gatti, M. Trevisani, and P. Geppetti, pers. comm.) is consistent with the recent report that inhalation of ethanol in subjects with sensory hyperreactivity (SHR), but not in healthy controls, exaggerated the cough response to capsaicin (Millqvist et al., 2008 ). This fi nding may explain the airway symptoms induced by chemicals and scents in SHR patients and also supports the hypothesis that sensitization of TRPV1 by alcoholic beverages contributes to symptoms of ethanol - induced asthma. A variety of pollutants have been shown to activate sensory nerves. Subacute exposure to SO2 increases the number of coughs evoked by the TRPV1 agonist, capsaicin (McLeod et al., 2007 ). The malodorous gas hydrogen sulfi de

(H2S) has been recently described as an endogenous mediator with a variety of biological effects (Li et al., 2005 ). However, H 2 S represents a serious chemical hazard in manufacturing industries as it may produce serious toxic effects, especially in the airways, where the gas causes acute respiratory responses, which include cough, respiratory tract irritation, dyspnea, chest pain (tightness), pulmonary edema, and airway hyperreactivity (Enarson et al., 1987 ;

Hessel et al., 1997 ). In guinea pig airways, H 2S evokes a series of infl ammatory responses mediated by sensory nerve activation and tachykinin release (Trevisani et al., 2005 ). The mechanism of these effects is unknown, although there is evidence that H 2 S effects are inhibited by capsazepine (Trevisani et al., 2005 ), thus suggesting the involvement of TRPV1. Other endogenous mediators, such as the eicosanoid 20 - hydroxy - eicosatetraenoic acid (20- HETE), which is a product of cytochrome P- 450 (CYP- 450) omega- hydroxylase, have emerged as potential TRPV1 agonists with a bronchoconstrictor action (Rousseau et al., 2005 ). Increased expression of TRPV1 has been found in a variety of infl amed tissues, and this increased expression is associated with the severity of the symptoms (Yiangou et al., 2001 ; Chan et al., 2003 ). A similar overexpression has been reported in the respiratory tract. The airway epithelium from patients with chronic cough shows a marked increase in TRPV1- positive nerves, whereas protein gene product 9.5 (PGP 9.5) positive nerve fi bers are not increased (Groneberg et al., 2004 ). Correlation between capsaicin tussive response and the number of TRPV1 - positive nerves has been established in studies with these patients (Groneberg et al., 2004 ). Confi rmation of increased expression of TRPV1 in patients with chronic cough has also been reported by the identifi cation of channel overexpression in the airway smooth muscle of these patients (Mitchell et al., 2005 ). The number of TRPV1 - immunoreactive axons in the guinea pig trachea is increased under allergic infl ammatory conditions (Watanabe et al., 2008 ). 412 THE ROLE OF TRPV1 IN RESPIRATORY DISEASES

15.5 SNEEZING, COUGH, AND TRPV 1 CHANNEL IN AIRWAY DISEASES

As discussed above, activation of TRPV1 results in nociception and pain, in defensive refl ex responses, and in a series of species - related proinfl ammatory local responses mediated by the peripheral release of neuropeptides. Sensory activation in the human respiratory tract is not associated with overt pain, but rather it causes sensations of chest tightness and discomfort and triggers a variety of protective refl ex responses, including cough and sneezing. Neurogenic infl ammation in human airways does not seem to encompass the entire panel of responses described in rodents. Failure of tachykinin receptor antagonists to inhibit different types of challenges in asthmatic patients (Fahy et al., 1995 ; Boot et al., 2007 ) supports this contention. Although tachykinin- mediated neurogenic infl ammation does not appear to play a major role in humans, stimulation of the sensory function of TRPV1 - expressing neurons in the upper and lower airways, with the subsequent activation of refl ex responses, is well described in man, and a large body of evidence supports a role for TRPV1 in human airway diseases (Geppetti et al., 2006 ). A major fi nding that underlines the importance of TRPV1 in airway infl ammation is represented by the reduced threshold for capsaicin - evoked cough in a large number of infl ammatory airway diseases, including asthma, cough- variant asthma, interstitial lung disease (ILD), rhinitis, and COPD (Fujimura et al., 1994 ; Wong and Morice, 1999 ; Doherty et al., 2000 ; Millqvist, 2000 ; Pecova et al., 2005 ). TRPV1 - evoked cough has some peculiar features, including the fact that cough evoked by aqueous solutions low in chloride anions, but not cough evoked by capsaicin, is inhibited by bronchodilators (Higenbottam, 2002 ). In addition, normal and nonasthmatic individuals, studied from the onset of an upper respiratory tract infection (URTI), showed a typical pattern of changes in lung function which ameliorated with regular bronchodilator anticholinergic therapy, but this therapy did not affect the persistent cough that accompanies URTI (Lowry et al., 1994 ). During an URTI, the sensitivity to cough by inhaled capsaicin was potentiated, whereas the cough refl ex from an inhaled ultrasonically generated low - chloride aqueous solution was not altered (Lowry et al., 1994 ). These fi ndings suggested that “ infl ammation ” enhances the refl ex response mediated by the capsaicin receptor (TRPV1) but not by the “ water ” receptors. A further example of the distinction between the “ water ” and capsaicin receptor is the observation that patients who developed a dry persistent cough following treatment with angiotensin- converting enzyme inhibitors showed an increased sensitivity to cough by capsaicin, but not by distilled water (Morice et al., 1987 ). In patients with cough associated with asthma, GERD, or rhinitis, treatment of the underlying disease decreased the response to capsaicin (O’ Connell et al., 1994 ). One possible interpretation is that the amelioration of airway infl ammation restored normal sensitivity to the capsaicin receptors (O ’ Connell et al., 1995 ). It can be concluded that enhanced airway response to TRPV1 stimulation accompanies symptomatic chronic SNEEZING, COUGH, AND TRPV1 CHANNEL IN AIRWAY DISEASES 413 cough associated with common diseases or drug treatment. Upregulation of TRPV1 sensitivity may occur not only by the action of endogenous proinfl ammatory mediators but also by exogenous chemicals. For example, patients who are sensitive to scents and chemicals with development of respiratory symptoms have a signifi cant increase in NGF in the nasal lavage fl uid, a phenomenon associated with an increased tussive response to capsaicin (Millqvist et al., 2005 ). In another study, intranasal capsaicin enhanced the cough response provoked by inhalation of a tussigen in humans (Plevkova et al., 2004 ). Taken together, these studies suggest the intriguing hypothesis that TRPV1 antagonists may be selectively effective in reducing “ pathological ” cough associated with chronic infl ammatory diseases without affecting the “ normal ” cough response. Topical application of capsaicin has been successfully used and is continuously being explored for the treatment of localized pain and infl ammatory conditions (Pappagallo and Haldey, 2003 ). The underlying mechanism of the treatment is that, after an early excitatory effect, capsaicin produces a long - lasting desensitization of the TRPV1 channel and of the entire sensory nerve terminal, which is essentially unable to sense any irritant or painful stimulus (Szolcsanyi, 1987a ; Szallasi and Blumberg, 1999 ). Although this therapeutic strategy cannot be easily applied to the lower airways such as the bronchial tree, it has been successfully adopted in the nasal mucosa. A fi rst pivotal study (Geppetti et al., 1988 ) showed that repeated topical capsaicin applications to the human nasal mucosa resulted in an enduring desensitization to all the acute responses evoked by capsaicin, namely, burning pain, sneezing, and rhinorrhea, followed by a full recovery. Shortly thereafter, a series of studies reported the benefi cial effects of different regimens of topical capsaicin application to the nasal mucosa in patients with perennial rhinitis, variably defi ned as either vasomotor rhinitis (Marabini et al., 1991 ; Filiaci et al., 1994 ), chronic rhinitis (Lacroix et al., 1991 ), or noninfectious rhinitis (Blom et al., 1997 ), with improvements observed even throughout a 6- month follow- up. Even short- term treatments of capsaicin on a single day have been found effective (Van Rijswijk et al., 2003 ). An augmented pain response to capsaicin suggests that sensory nerve hyperresponsiveness may characterize allergic airway disease. (Svensson et al., 1998 ). Capsaicin challenge increases total, epithelial, and neutrophil cell counts in the nasal lavage fl uid of patients with allergic rhinitis but not in healthy subjects (Roche et al., 1995 ). During the symptomatic period, the nasal mucosa of allergic patients is more susceptible to neurogenic stimulation by capsaicin, showing enhanced secretory and infl ammatory (cellular) responses (Kowalski et al., 1999 ). These fi ndings suggest that TRPV1- expressing neurons also contribute to the symptoms of allergic rhinitis. Although a fi rst study showed a positive effect of capsaicin treatment in birch pollen allergic rhinitis patients (Stjarne et al., 1998 ), a successive study in perennial allergic rhinitis failed to demonstrate any benefi t by desensitization with capsaicin, at doses suffi cient to control symptoms in patients with severe nonallergic rhinitis (Gerth Van Wijk et al., 2000 ). A recent Cochrane 414 THE ROLE OF TRPV1 IN RESPIRATORY DISEASES metaphysics analysis collected insuffi cient evidence to assess the use of capsaicin in clinical practice in allergic rhinitis (Cheng et al., 2006 ).

15.6 CONCLUSIONS

Chronic cough is a major medical need. At present, its treatment is unsatisfactory, mainly because of the poor understanding of the underlying mechanisms of cough associated with a variety of diseases. Similarly, perennial rhinitis, a condition that affects a signifi cant proportion of the general population, remains inadequately treated because current therapies are not selective and result in side effects. The hypothesis that TRPV1 plays a major role in cough and rhinitis, and the possibility to intervene by blocking TRPV1 and TRPV1 - expressing neurons, either by defunctionalization of the channel and the sensory nerve terminals or by classical receptor antagonists, offers new opportunities to treat these conditions. In addition, the hypothesis that TRPV1 is selectively upregulated and that its function is enhanced under infl ammatory conditions suggests that patients affected by different airway diseases characterized by TRPV1 hypereactivity might benefi t by specifi cally targeting TRPV1 with high - affi nity antagonists, which could reduce the symptoms, including irritation, chest tightness, breathlessness, and discomfort.

ACKNOWLEDGMENTS

This chapter was supported by a grant from Ministero dell ’Istruzione, dell ’Universit à e della Ricerca, Rome, Associazione per la Ricerca e la Cura dell ’Asma, Padua, and Fondazione Cassa di Risparmio di Firenze, Florence, Italy.

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Hubert Tsui , Ruslan Dorfman , Michael W. Salter , and H. - Michael Dosch

16.1 OVERVIEW

For this chapter, the viewpoints and focus derive from our unexpected discovery that TRPV1 plays a central role in the pathoetiology of type 1 diabetes (T1D) of the non - obese diabetic (NOD) mouse (Razavi et al., 2006 ). In Chapter 4, we discuss the genetics of the human trpv1 gene, with a view toward genetic heterogeneity. Here, we review the immune, endocrine, and neurological roles of TRPV1 and relate these to the consequences of TRPV1 mutation in the diabetes - prone NOD mouse. We describe the impact of this mutation on multiple physiological systems as they initiate and drive prediabetes progression toward overt disease, and we discuss the unexpected potential new therapeutic strategies developed from our observations.

16.2 A NEW MODEL OF A CLASSIC DISEASE GROUP

T1D (Razavi et al., 2006 ), type 2 diabetes (T2D) (Kahn et al., 2006 ), and type 3 diabetes (T3D) (Alzheimer’ s disease) (Craft, 2006 ; de la Monte et al., 2006 ; Pilcher, 2006 ) share insulin resistance as a common, chronic β - cell stress with a broad range of progressively more pathogenic consequences. In the T1D - prone NOD mouse, systemic removal of TRPV1- positive neurons by neonatal capsaicin treatment normalizes insulin resistance, thereby preventing islet

423 424 THE ROLE OF TRPV1 IN DIABETES inﬂ ammation and diabetes (reviewed in Tsui et al. [2007 ]). The same strategy prevents T2D- like disease in Zucker rats (Gram et al., 2007 ). Normalized insulin sensitivity has been reported in obesity- prone B6 mice with the TRPV1null genotype (Razavi et al., 2006 ). The role of sensory afferent neurons in diabetes is a recent discovery. We discuss our changed understanding of pancreatic endocrine function and autoimmunity, including their close interface with sensory afferent neurons, beginning with an overview of T1D.

16.3 T 1 D

T1D is a chronic autoimmune disease characterized by T cell- mediated destruction of pancreatic β cells. The resulting insulin defi ciency, at the usually precipitous onset of overt disease with acute, life- threatening insulin- requiring hyperglycemia, signals the culmination of many years of prediabetes in humans. Elevated insulin resistance develops early in prediabetes and, as described below, is a driver of prediabetes progression. While systemic control of insulin resistance involves many body compartments, our observations stress the central role of islets and their neuronal wiring: there is no substitute for endogenous β cells. As the incidence rates of all forms of diabetes follow a dramatic, nongenetic global rise, and as diabetes economic costs continue to escalate in parallel (presently 130+ billion dollars per year in North America), the need for better, preventative, or curative therapeutic strategies is of high priority. Lifelong insulin treatment prevents death but cannot replace physiological minute - to- minute glycemic control; chronic swings of hypo- and hyperglycemia generate long- term vascular complications including stroke, neuropathy, kidney and heart failure, as well as vision and limb loss (Nathan et al., 2005 ). Innovative diabetes therapies must aim to reestablish the physiological control of insulin release and its effective action. T1D is a polygenic disorder. Its primary genetic risk factor maps to human leukocyte antigen (HLA) (Ounissi- Benkalha and Polychronakos, 2008 ), thereby infl uencing T - and B - cell autoreactivities to a panoply of self - antigens, most of which are widely expressed in the body (Dosch et al., 1999 ; Winer et al., 2003 ). Stressed pancreatic islets of Langerhans attract immune cell infi ltration, fi rst around the peri- islet Schwann cell border (Winer et al., 2003 ; Tsui et al., 2008 ) and with progressive peri- islet Schwann cell kill result in invasion of the islet interior and in β - cell destruction (Foulis et al., 1986 ). Interestingly, kill of both Schwann cells and β cells is strictly islet restricted, although Schwann cell - specifi c CD8+ killer T cells can destroy Schwann cells from any tissue source. Despite a solid understanding of the autoimmune mediation of T1D, it has remained uncertain what initiates prediabetic autoimmunity, what drives its progression, and what explains the tissue selectivity of the process. Target autoantigens are overall not β - cell exclusive, and loss of self - tolerance is broad but not unlimited. It now appears that TRPV1 - positive A MODEL OF T1D PATHOGENESIS 425 afferent sensory neurons lie at the heart of the answers to several of these questions (Tsui et al., 2007 ).

16.4 A MODEL OF T 1 D PATHOGENESIS

Much of our knowledge of the events leading to T1D derives from studies using NOD mice and their many derivative congenic mouse lines (Anderson and Bluestone, 2005 ; Shoda et al., 2005 ). Unfortunately, no germline- competent embryonic stem cell line has been developed. The NOD mouse develops spontaneous autoimmune diabetes, sharing many features with the human disease, including similar polygenetic susceptibility (particularly major histocompatibility complex), islet pathology, and autoantigens (Giarratana et al., 2007 ). A widely held view of prediabetes initiation proposes early activation of autoreactive T cells in draining pancreatic lymph nodes (Hoglund et al., 1999 ; Mathis et al., 2001 ), possibly triggered by developmentally regulated islet remodeling in an autoimmune- permissive host (Trudeau et al., 2000 ; Turley et al., 2003 ). Immune activation in pancreatic lymph nodes appears to be necessary for spontaneous diabetes development in NOD mice (Gagnerault et al., 2002 ). However, the events that initiate and then promote islet infl ammation with priming and expansion of pathogenic T- cell pools remain ill defi ned, and the very slow pace of the process is unexplained. Hyperinsulinemia, associated β - cell stress, and compensatory rises in insulin resistance (Rosmalen et al., 2000 ; Homo- Delarche, 2001 ) are now recognized as core elements of the chronic progressive factors ultimately driving prediabetes in the NOD mouse and mechanistically linked to subnormal pancreatic tissue levels of TRPV1 - dependent neuropeptides (e.g., substance P [SP]) (Razavi et al., 2006 ). The conventional view of organ- specifi c autoimmune disease explains tissue selectivity by autoimmune targeting of tissue - specifi c self - antigens, thereby limiting autoimmune damage to the site of antigen presence (Nakayama et al., 2005 ). However, this view has long been unsatisfactory, particularly in T1D, where the majority of autoantigens are not β - cell exclusive but are expressed widely, including prominently in neuronal tissue (Lieberman et al., 2003 ; Carrillo et al., 2005 ). As such, antigen specifi city alone is not suffi cient to explain the β - cell selectivity in T1D. Other contributions to islet infl ammation needed to be identifi ed. The recent concept of nervous system involvement in the pathogenesis of T1D is supported by multiple lines of evidence:

1. It was discovered that autoimmunity in T1D was not, in fact, β - cell specifi c. Indeed, the earliest victim tissue is peri- islet glia (Winer et al., 2003 ; Tsui et al., 2008 ). 2. A large proportion of early islet - infi ltrating B lymphocytes in NOD mice are specifi c for broadly distributed nervous system elements (Carrillo et al., 2005 ; Puertas et al., 2007 ). 426 THE ROLE OF TRPV1 IN DIABETES

3. The list of T1D target autoantigens is lengthy and includes (pro)insulin, glutamic acid decarboxylase (GAD) 65 and 67, tyrosine phosphatase - like protein IA - 2, islet - specifi c glucose - 6 - phosphatase - related protein (IGRP), islet cell antigen 69 kD (ICA69), glial fi brillar acidic protein (GFAP), and heat shock protein (HsP) 60, to name the most prominent autoantigens (Lieberman et al., 2003 ). Most of these are found in neurons or their supporting cells. Insulin is arguably the most β cell - selective protein. However, the brain and thymus also produce insulin (Devaskar et al., 1994 ), which has broad modulatory effects on central nervous system (CNS) neurotransmission, thereby infl uencing associative learning, memory formation, and higher cognition (Zhao and Alkon, 2001 ). While insulin has been proposed to be the primary core diabetes autoantigen (Nakayama et al., 2005 ), this can be fundamentally questioned by several observations (Jaeckel et al., 2004 ), including a lack of autoimmune targeting of nonislet cells engineered to express insulin ectopi- cally. For example, transgenic insulin- secreting pituitary cells are not destroyed and can rescue diabetic NOD mice (Lipes et al., 1997 ). Genetically engineered, insulin - producing hepatocytes also do not succumb to the diabetic autoimmune response (Tabiin et al., 2004 ). The tissue specifi city of T1D, as explained by unique autoantigen expression, has long required scientifi c revision. 4. A linkage between the nervous system and T1D emerged unexpectedly when we discovered that T1D and multiple sclerosis (MS) patients exhibited overlapping, similar T- cell autoreactivities (Winer et al., 2001 ). T1D patient T cells surprisingly recognized and proliferated when exposed to typical MS antigens such as myelin basic protein and proteo- lipid protein. Moreover, lymphocytes from MS patients often target typical T1D antigens, prominently including proinsulin.

These fi ndings were mirrored in NOD mice, which, along with spontaneous autoimmune targeting of myelin components, developed an MS - like encephalitis precipitated by adjuvant - mediated breaches of the blood – brain barrier (Winer et al., 2001 ). NOD mice also developed peripheral neuropathy after genetic removal of just one costimulatory molecule (Salomon et al., 2001 ) or after deprivation of interleukin- 2 (Setoguchi et al., 2005 ). These observations revealed an inherently neuronal autoimmune bias in the NOD mouse, which primarily manifests as islet pathology, but can be redirected, by relatively simple manipulations, to classical nervous system pathology. This is likely not unique to the NOD mouse. In humans, elevated risks to develop both MS and T1D have been reported, affecting the same family and, in particular, the same patient, with the risk magnitude dependent on the study population (Marrosu et al., 2002 ; Dorman et al., 2003 ; Hussein and Reddy, 2006 ; Laroni et al., 2006 ; Nielsen et al., 2006 ). Collectively, the evidence for neuronal elements in diabetic autoimmunity is quite compelling, but by the fi rst half of this decade, it had failed to generate ISLET-INNERVATING NEURONS 427 serious new mechanistic insight. In fact, work in this area remained rather insular and unconnected. During studies of the peri - islet glia, we were sur- prised by the prominence of TRPV1 - positive sensory neuron terminals, whose 45 ° C activation threshold would not have functional relevance in that tissue (see discussion in Winer et al. [2003 ]). However, these terminals also express insulin receptors, whose ligation had been shown to potentiate TRPV1 currents and dramatically lower activation thresholds from 45 ° C to room temperature (Sathianathan et al., 2003 ; Van Buren et al., 2005 ). In the insulin- rich islet milieu, TRPV1 function would thus have unique, tissue - specifi c characteristics, with tonic activation and neuropeptide release at body temperature (37 ° C). As discussed below, we believe that it is this functional profi le of pancreatic TRPV1- positive terminals that ultimately controls the pancreas specifi city of diabetic islet infl ammation (Tsui et al., 2007 ).

16.5 ISLET - INNERVATING NEURONS

The islets of Langerhans are composed of α , β , δ , and pancreatic polypeptide (PP) cells secreting glucagon, insulin, somatostatin, and PP, respectively. Accompanying these endocrine cell types is a dense network of nerve terminals that are focused at the neuroinsular complex, where they penetrate to the islet interior (Persson- Sjogren et al., 2001 ). Peptidergic, cholinergic, adrenergic, and GABAergic neuronal fi bers are present at this site (Ahren, 2000 ; Akiba et al., 2004 ), where some of these terminals directly synapse with endocrine cells and modulate hormone release (Helman et al., 1982 ). For example, the reciprocal effects of adrenaline on insulin and glucagon release have been well described (Bloom, 1976 ; Dunning and Taborsky, 1991 ). In addition, sympathetic islet innervation contributes to the glucagon response following insulin- induced hypoglycemia (Ahren and Taborsky, 1988 ; Dunning et al., 1988 ; Taborsky et al., 1998 ; Benthem et al., 2001 ). Notably, sympathetic control of the glucagon response is lost early in T1D patients (Gerich, 1988 ; Cryer et al., 1989 ). This may be related to a fi nding in the BioBreeding rat model of diabetes, where an islet - restricted, probably autoimmune, sympathetic neuropathy has been observed soon after the onset of hyperglycemia (Mei et al., 2002 ). In the NOD mouse, disrupted islet innervation has been reported during prediabetic progression of insulitis (Persson - Sjogren et al., 2005 ), but it remains unclear whether pancreatic neurons are directly targeted in T1D (Saravia and Homo- Delarche, 2003 ). In addition to being possible autoimmune targets, dysfunctional pancreatic neurons may contribute to local islet infl ammation, extending diabetes etiology from immunology to include neuroendocrine elements. Indeed, our recent observations demonstrate abnormal sensory neuron function as a major contributor to autoimmune diabetes pathoetiology (Razavi et al., 2006 ). The parasympathetic innervation of islets is postganglionic, originating from the vagus and extending from intrapancreatic ganglia (Brunicardi et al., 428 THE ROLE OF TRPV1 IN DIABETES

1995 ). Activation of these neurons releases acetylcholine, generally raising endocrine output (Van der Zee et al., 1992 ). Noncholinergic parasympathetic activity toward islets is mediated through the neuropeptides vasoactive intestinal peptide (VIP), gastrin - releasing peptide (GRP), and pituitary adenylate cyclase - activating polypeptide (PACAP). Similar to acetylcholine, these neuropeptides exert positive secretory actions on insulin and glucagon. Sympathetic islet innervation is postganglionic, deriving from the celiac or paravertebral sympathetic ganglia (Brunicardi et al., 1995 ). The preganglionic neurons originate in the hypothalamus, exiting the spinal cord between C8 and L3, and reach the celiac or paravertebral ganglia via the lesser and greater splanchnic nerves. Sympathetic ﬁ bers are found in mixed autonomic nerves connecting to islets, but preganglionic sympathetic nerves can also directly innervate islets. Activation of sympathetic neurons inhibits both stimulated and basal insulin secretion, with the former being the result of noradrenaline binding to α 2 - adrenoceptors on β cells (Nilsson et al., 1988 ), whereas the latter is likely peptidergic. Sympathetic activity also increases glucagon and PP release but suppresses somatostatin release (Brunicardi et al., 1994 ). Islet function can also be affected by sympathetic, nonadrenergic mechanisms through galanin (Ahren and Lindskog, 1992 ) and neuropeptide Y (Morgan et al., 1998 ), both of which decrease insulin secretion.

16.6 SENSORY NEURONS

Primary sensory neurons are the major conduits by which peripheral information is relayed to the CNS. The cell bodies of sensory afferents lie within dorsal root ganglia (or trigeminal ganglia for the face) with axon projections into the spinal cord as well as distally to a specifi c tissue. Activation can be triggered by dorsal root refl exes, axonal refl exes, or local depolarization, resulting in release of peptidergic neurotransmitters (Richardson and Vasko, 2002 ). The orthodromic signal toward the spinal cord transmits information to the CNS. A defi ning characteristic of many sensory neurons is the antidromic release of the neurotransmitters SP and calcitonin gene- related peptide (CGRP) at the axon terminals within the innervated tissue. This peripheral efferent release of neurotransmitters confers local activity, confi ned to the terminal distribution of the neuron. C - and Aδ - fi bers are two subpopulatons of unmyelinated sensory neurons responsible for the detection of physical and chemical stimuli. These small- diameter fi bers express TRPV1, thereby conferring the specialized ability to sense various noxious insults such as heat and protons (Tominaga et al., 1998 ). Although the biochemical properties of TRPV1 are increasingly well characterized, the endogenous ligand(s) that control its activity in vivo are more uncertain. Some candidates include lipid mediators from the arachidonic acid family such as anandamide and leukotriene B4 (Hwang et al., 2000 ). ISLET ENDOCRINE FUNCTION OF TRPV1-POSITIVE SENSORY NEURONS 429

Similar to most tissues, the exocrine and endocrine pancreas are innervated by TRPV1- positive sensory neurons. Although we are focused here on immune and endocrine elements, it is noteworthy that the exocrine pancreas is also infl uenced by sensory neurons. For example, primary sensory afferents present in the vagus trunk carry information on luminal content from the intestinal mucosa to the brain stem. Activation of these vagal afferent fi bers results in inhibition of food intake, gastric emptying, and stimulation of pancreatic secretion (Li, 2007 ). This pathway has sensory nerves infl uencing pancreatic function via an external circuit, which is reliant on adrenergic efferents (Karlsson et al., 1994 ). It is also becoming clear that sensory neurons in the portal vein act as important glucose sensors (Fujita et al., 2007 ).

16.7 PANCREATIC SENSORY NEURONS

An extensive network of sensory neurons expressing CGRP and SP can be found in the parenchyma of the pancreas, around blood vessels and innervating islets (Wick et al., 2006 ). These sensory fi bers exit the pancreas with sympathetic fi bers within splanchnic nerves en route to the spinal cord (Brunicardi et al., 1995 ). The cell bodies of these spinal sensory afferents are concentrated in dorsal root ganglia of thoracic segments T9– T12 and are equally distributed between right and left sides. Some sensory afferents also derive from the nodose ganglia, but predominantly on the left side (Fasanella et al., 2008 ). Often, the presence of either of the two principal sensory neuron neurotransmitters, CGRP or SP, is used to identify sensory nerve endings. The presence of CGRP around pancreatic islets was fi rst reported in 1983 (Rosenfeld et al., 1983 ). Although there is a greater density of CGRP - than SP - containing sensory neurons around islets (Karlsson et al., 1992 ), both neurotransmitters are capable of modulating islet function.

16.8 ISLET ENDOCRINE FUNCTION OF TRPV 1 - POSITIVE SENSORY NEURONS

A widely used strategy in studies of the contribution of TRPV1 - positive sensory afferent neurons employs capsaicin (caps) to selectively and irre- versibly (in the periphery) destroy the TRPV1- positive neurons ( caps mice, caps rats) (Jancso et al., 1977 ; Karlsson et al., 1992 ). In the murine pancreas, neonatal capsaicin treatment permanently eliminates the majority of CGRP- positive terminals and a major subset of SP- containing afferents innervating the islets. In caps mice, glucose - induced insulin secretion is enhanced, suggesting that sensory neurons exert inhibitory infl uences on β cells (Karlsson et al., 1994 ). In caps rats, oral glucose challenge shows improved glucose handling, although insulin secretion is unaffected, suggesting sensory afferents can regulate 430 THE ROLE OF TRPV1 IN DIABETES glucose via a mechanism independent of insulin release (Guillot et al., 1996 ). Indeed, it has been postulated that TRPV1 - positive sensory afferents modulate insulin sensitivity (Koopmans et al., 1998 ), but the precise molecular pathways remain undefi ned. Most studies have focused on the effects of CGRP, perhaps refl ecting the extremely short tissue half- life of SP. Here, we review some of the fi ndings, which are at times contradictory, on the effects of both CGRP and SP on islet function.

16.9 EFFECTS OF CGRP ON ISLET FUNCTION

Expression of CGRP in pancreatic nerve fi bers occurs in all mammalian species (Sternini et al., 1992 ), whereas CGRP expression in islet endocrine cells varies among species. In rats, CGRP expression in endocrine cells is primarily confi ned to δ cells, whereas islet amyloid polypeptide (IAPP) is expressed in rat β cells (Ahren and Lindskog, 1992 ). IAPP, which shares 20% amino acid homology with CGRP (Barakat et al., 1993 ), has similar effects to CGRP, including inhibition of insulin release as well as inhibition of glucose uptake by striated muscle (Bretherton - Watt et al., 1992 ). Intravenous CGRP inhibits basal and glucose- stimulated insulin secretion in both pigs (Dunning et al., 1987 ) and mice (Pettersson et al., 1986 ), but has been reported to increase basal plasma glucose and insulin in rats (Pettersson and Ahren, 1988 ; Morishita et al., 1992 ). In contrast, in a separate rat study, IV CGRP had no effect on basal glucose and insulin levels, but hyperglycemia and hyperinsulinemia were observed upon glucose challenge (Tedstone et al., 1990 ). Disparate effects were also noted in isolated rat pancreas, where CGRP failed to infl uence glucose- stimulated insulin secretion (Kogire et al., 1991 ), although it did in isolated rat islets (Ishizuka et al., 1988 ; Pettersson and Ahren, 1990 ). CGRP was also reported to suppress insulin release from rat islets stimulated with CGRP and cholecystokinin - 8 (CCK - 8) (Fujimura et al., 1988 ). The regulation of islet function by sensory neurons is incompletely understood, even when considering CGRP in isolation. Low - dose CGRP (0.1 – 1.0 nM) inhibits insulin release from a β - cell line in vitro (Barakat et al., 1994 ), consistent with fi ndings that CGRP negatively regulates β - cell activity following in vivo glucose challenge in caps animals (Karlsson et al., 1994 ). Yet, at higher concentrations, in vitro CGRP potentiates insulin release (Barakat et al., 1994 ). These dose - dependent CGRP effects have also been observed in the perfused dog pancreas (Hermansen and Ahren, 1990 ), although CGRP appeared to have no effect on human islet function in vitro (Beglinger et al., 1988 ). Differing doses, routes, and study methods presently prevent a clear consensus on the islet action of CGRP. Hence, ascribing CGRP as an inhibitory factor on β- cell function will likely be context and species specifi c. Other EFFECTS OF SP ON ISLET FUNCTION 431 effects of CGRP related to glucose control include the ability of CGRP to modulate the insulin sensitivity of skeletal muscles (Leighton et al., 1989 ; Kreutter et al., 1993 ), to antagonize insulin- mediated glycogen synthesis (Leighton and Foot, 1995 ), and to stimulate lipid utilization in the muscles, liver, and blood (Danaher et al., 2008 ). CGRP has also been documented to decrease pancreatic and islet blood fl ow (Svensson et al., 1994 ). As such, the systemic effects of CGRP, many impacting glucose metabolism, complicate interpretations of CGRP effects at the islet level.

16.10 EFFECTS OF S P ON ISLET FUNCTION

SP is coreleased with CGRP in equimolar concentrations as part of the local, efferent sensory neuron response. The endocrine functions of SP have been evaluated to a far lesser extent than CGRP; yet, like with CGRP, controversy exists. SP is a member of the tachykinin family, which also includes neurokinin A, neurokinin B, and hemokinin - 1 (Zhang et al., 2006 ). SP is an 11 - amino acid neuropeptide that binds preferentially to the neurokinin (NK) - 1 receptor mediating different, even opposing, effects over a very wide concentration range (Kraneveld and Nijkamp, 2001 ). Although SP is generally thought of as a nervous system neurotransmitter, tachykinins and their receptors are expressed in a variety of non- neuronal cells, including endothelial and infl ammatory cells, leading to complex pleiotropic interactions (Pennefather et al., 2004 ). Early experiments with isolated islets found that SP inhibited release of insulin while potentiating glucagon release (Moltz et al., 1977 ). Similar to studies with CGRP, considerable species variation has been noted with SP. Studies using dog islets showed that SP increased secretion of somatostatin, insulin, and glucagon (Hermansen, 1980 ). However, in rats, SP diminished both insulin and glucagon release, with no effects on somatostatin (Chiba et al., 1985 ). In calves, SP increased plasma levels of PP but did not alter insulin levels (Edwards and Bloom, 1994 ). SP also was reported to have inhibitory actions on the exocrine pancreas, decreasing cholecystokinin (CCK) - induced amylase release and secretin- induced juice fl ow (Kirkwood et al., 1999 ). Although SP is not produced by adult rat β cells, it is transiently expressed during embryogenesis by both insulin - and non - insulin - containing pancreatic endocrine cells (McGregor et al., 1995 ). Tachykinin- defi cient mice have been generated, and no overt metabolic phenotypes have been reported (Cao et al., 1998 ; Zimmer et al., 1998 ). However, a rigorous assessment of metabolism and islet function has not yet been performed in these mice, and our understanding of the role of SP in pancreatic endocrine function remains incomplete, but it should be noted that the naked mole rat is severely SP defi cient and is characterized by abnormal glucose metabolism (Park et al., 2003 ; Kramer and Buffenstein, 2004 ). 432 THE ROLE OF TRPV1 IN DIABETES

16.11 TRPV 1 ON β CELLS

TRPV1 has been reported to be expressed at both the transcript and protein levels in pancreatic islets of Sprague Dawley (SD) rats (Akiba et al., 2004 ; Gram et al., 2005 ). The presence of TRPV1 on β cells could suggest that activation of TRPV1 may directly inﬂ uence β cells, independently of sensory afferents. Interestingly, TRPV1 expression was not detected on β cells from Zucker diabetic fatty (ZDF) rats. However, it remains to be determined whether the differences in TRPV1 expression between ZDF and SD rats are simply strain differences or whether these differences have implications for diabetes. TRPV1 expression was not observed in NOD mouse β cells (Razavi et al., 2006 ). No data are available regarding TRPV1 expression in human β cells.

16.12 A ROLE FOR TRPV 1 IN GLUCOSE METABOLISM

Sensory afferent neurons mediate potent control of energy utilization and β- cell physiology, which, collectively, impact islet stress. The following discussion highlights this novel and important aspect of sensory neuron physiology. ZDF rats are perhaps the oldest model of T2D. Capsaicin treatment of adult ZDF rats has been reported to improve glucose tolerance, although this study was marred by a high rate of mortality (up to 70%) associated with the high dose of capsaicin utilized (Gram et al., 2005 ). Similar fi ndings have been reported with the neurotoxin resiniferatoxin (RTX) (Gram et al., 2005 ), an ultrahigh - affi nity TRPV1 agonist (Szolcsanyi et al., 1991 ). In pre - obese ZDF rats, plasma CGRP levels are elevated, suggesting that a disturbance in sensory afferents may be contributing to obesity and its associated metabolic sequelae. Interestingly, elevated levels of CGRP have also been reported in human obesity. Measurement of serum levels of SP is not practical due to its half - life of only a few seconds (Zelissen et al., 1991 ). Insulin secretion and insulin sensitivity are two parameters by which sensory neurons appear to modify glucose metabolism. The former involves pancreas - specifi c events, while the latter may involve several tissues working in unison. The majority of CGRP - containing nerve terminals in the pancreas of ZDF rats are TRPV1 positive (Gram et al., 2007 ). Similarly, SP is present in pancreatic TRPV1 - positive terminals of the NOD mouse. Anecdotal observations in humans suggest that eating spicy food can reduce weight gain. Indeed, capsaicin consumption has been shown to reduce appetite (Westerterp- Plantenga et al., 2005 ), to alter lipid metabolism, and to reduce obesity in humans (Yoshioka et al., 1998, 1999 ; Wahlqvist and Wattanapenpaiboon, 2001 ; Belza et al., 2007 ), in rats (Kawada et al., 1986 ; Cui and Himms- Hagen, 1992 ; Melnyk and Himms- Hagen, 1995 ), and in mice (Ohnuki et al., 2001 ; Masuda et al., 2003 ). However, absorption rates, as well SENSORY NEURONS AND T1D 433 as the molecular pathways and mechanisms whereby capsaicin mediates these systemic effects, are not well understood. They will likely involve a combination of capsaicin - responsive tissues in addition to the activation of sensory neurons (Szallasi and Blumberg, 1999 ). One explanation suggests that enhanced catecholamine release may be responsible for the effects of capsaicin on energy and lipid metabolism (Watanabe et al., 1987 ), but further validation is required (Corry and Tuck, 1999 ). TRPV1 has a defi nitive role in energy metabolism, as demonstrated by TRPV1 - defi cient mice, which gain less weight and adiposity when placed on a high- fat diet (Motter and Ahern, 2008 ). This is associated with increased thermogenesis and higher resting metabolic rate, but it is unclear at what level TRPV1 is mediating these changes; this research will likely require employment of tissue - specifi c knockouts. Even if one considers only adipocytes, the situation is already complex, since they express TRPV1 at low levels but also respond to CGRP (Li Zhang et al., 2007 ; Motter and Ahern, 2008 ). Nevertheless, the relevant phenotype of TRPV1 - defi cient mice closely resembles that of rodents with capsaicin- induced neonatal ablation of sensory afferents, supporting the role of TRPV1 in the control of metabolic function.

16.13 SENSORY NEURONS AND T 1 D

Although the precise function requires further investigation, it is clear that pancreatic sensory nerves can modulate islet physiology via local, efferent neurotransmitter release of SP and CGRP (Ahren, 2000 ). Based on studies targeting pancreatic glia (Tsui et al., 2008 ), autoantibodies against nervous system components in mice and in humans (Winer et al., 2003 ; Carrillo et al., 2005 ), reduced diabetes incidence following transgenic expression of CGRP in NOD β cells (Khachatryan et al., 1997 ), and an increasing appreciation of the importance of neuronal modulation of immune function (Wang et al., 2003 ), we decided to analyze the role of TRPV1 in T1D, initially by generating NODcaps mice. The NOD mouse has TRPV1- positive axons and terminals in endocrine and exocrine pancreas, but has no expression of TRPV1 in islets (Razavi et al., 2006 ). Neonatal removal of TRPV1 sensory afferents by capsaicin dramatically reduced subsequent prediabetic islet infl ammation (insulitis), as well as ultimate T1D. Mechanistically, protection against diabetes was not associated with widespread immunosuppression, since NODcaps mice developed normal salivary gland pathology (sialitis) and, remarkably, islet- reactive T- cell pools. In fact, spleen cells of NOD caps were perfectly capable of rapidly transferring insulitis and diabetes to lymphocyte - free NOD.scid mice, which carry the hypofunctional TRPV1NOD mutation (Razavi et al., 2006 ). NOD.scid mice undergo all the steps of diabetogenesis as their immunocompetent cousins, except that the fi nal islet destruction does not occur in the absence of cognate immunity. Thus, TRPV1- positive terminals 434 THE ROLE OF TRPV1 IN DIABETES are critical to allow for islet infl ammation, attracting or permitting immune invasion in a pancreas - selective fashion. The mere presence of autoreactive T- cell pools with pathogenic potential is insuffi cient to cause autoimmune disease. However, the locale and mechanisms of loss of islet self - tolerance in NODcaps mice remain uncertain.

16.14 TRPV 1 MUTATIONS IN THE NOD MOUSE

During investigations of diabetes pathogenesis in the NOD mouse, we identifi ed an important role of TRPV1 - positive primary afferent neurons in the initiation and progression of islet infl ammation and T1D. Subsequently, we determined that TRPV1 maps to a major NOD diabetes risk sublocus, idd4.1, on mouse chr11. We cloned and sequenced TRPV1 cDNA from NOD dorsal root ganglion (DRG). For a comparison strain, we used, among others, the diabetes- resistant control mouse strain non - obese - resistant (NOR), which carries nearly 90% of the NOD genome including the major histocompatibility complex (MHC) and most other T1D risk loci, but is diabetes - and insulitis resistant. We found that the sequence of TRPV1 in NOD mice was identical to that in wild - type B6 and DBA mouse strains, except that the NOD TRPV1 (TRPV1 NOD ) sequence had two in- frame base exchanges, leading to predicted P322A and D734E amino acid replacements. Both of these replacements fall into regions of TRPV1 that are otherwise conserved across diverse species. In contrast to NOD mice, the TRPV1 sequence in NOR mice is identical to that of B6 and DBA strains. We focused on the possibility that sequence differences in TRPV1NOD might cause abnormalities of overall TRPV1 function, taking advantage of the dermal innervation by TRPV1- expressing primary nociceptive sensory afferents. This innervation mediates characteristic behavioral responses to dermal administration of capsaicin. Peripherally administered capsaicin also evokes local plasma extravasation and tissue swelling, which are characteristics of neurogenic infl ammation caused by release of neuropeptides from the terminals of TRPV1 - positive primary afferents. We compared the effects of skin- applied capsaicin in NOD versus NOR mice. There were no differences in basal thermal or mechanical responses between these strains. However, we found that the nociceptive behavioral responses (biting or licking) evoked by intradermal capsaicin were markedly depressed in NOD as compared to NOR mice. Perhaps most importantly, the paw edema produced by capsaicin was signifi cantly reduced in NOD versus NOR mice. These fi ndings were independent of the autoimmune background in NOD mice since similar results were observed in lymphocyte- defi cient NOD.scid mice. Thus, in whole- animal studies, the TRPV1 NOD sequence abnormality revealed decreased TRPV1 - mediated responses to capsaicin. Because the whole- animal approach can only assess TRPV1 function indirectly, we turned to more direct approaches. We recorded capsaicin- evoked LASTING REVERSAL OF ACUTE T1D BY SP 435

Ca2+ responses in DRG neurons from NOD and NOR mice, and found that the maximum Ca 2+ response to capsaicin of DRG neurons from NOD mice was signifi cantly smaller than that of NOR DRG neurons. In addition, there was a shift in the capsaicin concentration– response relationship, with about 10- fold higher capsaicin concentrations required for the small maximum responses in NOD DRG neurons compared with NOR DRG neurons. In contrast, Ca 2+ responses evoked by KCl were not different between NOD and NOR DRG neurons, indicating that NOD mice do not exhibit a general Ca2+ response abnormality. The most direct readout of TRPV1 function is capsaicin - evoked current. Whole - cell current evoked by capsaicin was signifi cantly smaller in NOD than in NOR DRG neurons. The decreased maximum capsaicin - evoked responses in DRG neurons from NOD mice might have been due to decreased expression of TRPV1 and /or decreased function of TRPV1 molecules. We found that the basal TRPV1 protein level in DRG neurons from NOD mice was lower than that in NOR. Thus, the depression of maximum capsaicin- evoked Ca 2+ and current response in DRG neurons from NOD mice may at least in part refl ect decreased steady- state expression of TRPV1 NOD . However, the shift in the capsaicin concentration – response relationship suggests that the function of the TRPV1NOD protein may also be reduced as compared to TRPV1 wild type . Taken together, these converging lines of evidence show functional abnormalities in nociceptive behavior, TRPV1 channel function, and expression. Based on these data, we defi ned TRPV1 NOD as a hypomorphic mutant. Primary sequence changes corresponding to those in TRPV1NOD have been engineered into human TRPV1 (P322A, D734E) (Xu et al., 2007 ). Under defi ned expression conditions, hTRPV1P322A, D734E showed a markedly abnormal capsaicin concentration – response relationship compared to wild - type human TRPV1. The variant channel was hyporesponsive to capsaicin at low doses (< 10 nM) but was hyperresponsive at high doses (> 10 nM), with a markedly elevated Hill slope. Thus, similar changes in the primary sequence of human and NOD mouse TRPV1 produced changes in TRPV1 function. Determining the molecular mechanisms responsible for the altered TRPV1 function and whether TRPV1 function is altered by these changes in protein sequence across the human TRPV1 genetic polymorphisms (see the chapter by Dorfman et al. in this volume) are important topics under current investigation.

16.15 LASTING REVERSAL OF ACUTE T 1 D BY S P

As discussed above, TRPV1NOD is a hypomorphic mutant, with signifi cant phenotypes in behavioral studies. In light of the reduced TRPV1 function in NOD mice and increased accumulation of SP in DRG and nerve terminals associated with the diminished neuropeptide release, we reasoned that local neuropeptide defi ciency may lie at the heart of deranged islet physiology, 436 THE ROLE OF TRPV1 IN DIABETES survival, and infl ammation. Tissue- selective, submicromolar bolus delivery of SP to the pancreas via the intra- arterial route had dramatic overnight effects, clearing insulitis, normalizing hyperinsulinism and insulin resistance, and rees- tablishing normal glucose control in new- onset diabetic NOD mice with suffi cient β - cell mass at the time of acute T1D declaration. Remarkably, the longevity of these effects following a single SP treatment was considerable, lasting from weeks to several months without insulin therapy and with essentially normal glucose control— the latter remaining the elusive challenge of standard T1D therapy. Upon relapse, a subsequent treatment was again effective, provided there was suffi cient residual β - cell mass, as estimated by subdiabetic fasting blood glucose levels. The acute sequelae of SP delivery were multifold. Activated T cells express NK - 1, which is the major SP receptor. Ligation of this receptor induces apoptosis as well as inhibits clonal T- cell expansion in pancreatic lymph nodes (Tsui et al., 2007 ). On the metabolic front, SP administration quickly improves insulin sensitivity, allowing normalization of blood glucose despite reduced β - cell mass, in fact reducing glucose - dependent β - cell stress. Hence, the TRPV1 axis, via effects of neuropeptides, can infl uence T1D in both immunological and neuroendocrine contexts, with the latter having considerable ramifi cations also in T2D. In fact, these observations support shared pathoetiology in T1D and T2D, with progressive β - cell stress and insulin resistance being the driving forces in both diseases. Together, these fi ndings have led to a new model of T1D initiation. This model centers around a prominent role for TRPV1- positive sensory neuron function in diabetes pathoetiology, where the pathogenic element is subnormal, but not absent, neuropeptide release. This pathogenic process initiates and progresses independently of, but cannot complete, tissue destruction without autoimmunity. Although the hypofunctional TRPV1 NOD mutant gene is systemically expressed, its contribution to a pancreas - specifi c disease stems from its unique situation at the islet level, where high local insulin levels normally potentiate TRPV1 currents (Van Buren et al., 2005 ) and lower its thermal activation threshold (Sathianathan et al., 2003 ). Thus, in the presence of a normal TRPV1 gene, such as in NOD idd4 con- genics, where TRPV1 lies within the wildtype genomic interval (Grattan et al., 2002 ), there is tonic neuropeptide release, providing for a local regulatory circuit between TRPV1 terminals and β cells, roughly along lines originally envisioned nearly two decades ago (Hermansen and Ahren, 1990 ). In this circuit, TRPV1 - dependent neuropeptides, such as SP, provide a central survival function for β- cells, along the lines suggested previously (Bretherton - Watt et al., 1992 ; Barakat et al., 1994 ). The above - mentioned neuropeptide dose – response relationships, with deleterious effects at low concentrations, but positive effects at high concentrations, now explain the coun- terintuitive effect of disease prevention by removal of TRPV1- positive afferents, that is, a deleterious effect of constitutively low neuropeptide release in the T1D - prone NOD mouse. Neonatal removal of TRPV1 - positive LASTING REVERSAL OF ACUTE T1D BY SP 437 afferents does not allow establishment of this circuit, possibly replaced through alternative, TRPV1 - independent mechanisms. Low insulin - dependent neuropeptide release is countered by, and explains, the early hyperinsulinism of the NOD mouse— a peculiar disturbance long recognized also in prediabetic humans (Amendt et al., 1976 ; Buschard, 1991 ). Hyperinsulinemia, in turn, triggers a compensatory loss of insulin sensitivity or elevated insulin resistance, preventing hypoglycemia, but raising β - cell stress and driving prediabetes progression to overt disease. The dramatic depletion of islet infi ltration following intra - arterial injection of SP into the pancreas of newly diabetic NOD mice (or diabetic NOD.scid mice carrying adoptive grafts of diabetogenic splenocytes) emphasizes the important and competent physiological role of normal TRPV1 function in guarding against pancreatic infl ammation. Release of SP through normal TRPV1 function acutely removes the β - cell stress, which would have alerted the immune system to clear a perceived tissue lesion in progressively larger numbers of stressed islets. Indeed, all stages of infl ammation can be observed in parallel in a mid- late stage prediabetic pancreas (Winer et al., 2003 ; Tsui et al., 2008 ). Neuropeptides released following TRPV1 activation thus act at multiple, closely linked levels of insulin homeostasis/glucose control, and the failure of these mechanisms in the face of a hyposecretory TRPV1 mutant generates diabetes pathoetiology. NODcaps mice retain the loss of self - tolerance characteristic of NOD mice, generating pathogenic, islet - reactive T - cell pools that can transfer insulitis to NOD.scid recipients (Tsui et al., 2007 ). This clearly separates autoreactivity from autoimmune disease; only the latter requires TRPV1, which emerges as a central controller of tissue infi ltration. Precedence for this role of sensory afferents comes from observations in patients with the rare “ pain - free ” CIPA syndrome. Individuals with congenital insensitivity to pain with anhidrosis (CIPA) syndrome suffer from and often succumb to tissue - invasive infections, despite adequate immune systems (Indo et al., 1996 ), emphasizing the contribution of sensory neurons in directing local infl ammation. Our model of organ- specifi c autoimmunity sheds light on the old question of how widely expressed self- proteins become important local target autoantigens. Systemic transgenic overexpression of a diabetes- associated islet target antigen does not result in multiorgan leukocytic infi ltration or infl ammation (Geng et al., 1998 ; Song et al., 2003 ). Rather, infl ammation remains confi ned to the pancreas, suggesting that the islet milieu may be particularly conducive to an autoimmune response (von Herrath and Holz, 1997 ). In fact, islet - specifi c infl ammation emerged as a consequence of nonimmune conditions, due to an inherently abnormal sensory neuron– β - cell circuit. In this circuit, the β cell calls for release of more trophic neuropeptides through release of more insulin. The secretory defi cit of mutant TRPV1NOD cannot satisfy the demand, creating a vicious cycle of hyperinsulinism and insulin resistance. The resultant tissue stress represents a progressively proinfl ammatory environment, which, in the presence of a permissive immune system, 438 THE ROLE OF TRPV1 IN DIABETES predisposes to progressive autoimmune attack. This lesion attracts T lymphocytes, whether or not they are autoreactive (Faveeuw et al., 1995 ), but preferentially selecting cells that recognize antigenic epitopes available in the locale (Amrani et al., 2000 ). We thus view immune cell homing to the islet as a natural TRPV1 NOD - dependent response to islet stress, a response that likely also includes macrophages and dendritic cells, which accumulate early around islets (Reddy et al., 1993 ; Rosmalen et al., 2000 ). As expected, the perturbed neuronal β - cell axis is active in lymphocyte- free NOD.scid mice, thereby explaining why processed β- cell autoantigens are present on islet- residing macrophages even in the absence of an adaptive immune system (Burlinson et al., 1995 ; Shimizu et al., 1995 ) and why disease can so readily be transferred to NOD.scid mice by wild - type NOD hematopoietic cells (Winer et al., 2001 ). Possible differential or overlapping neuropeptide effects need further clarifi cation, but available evidence indicates that both CGRP and SP affect antigen presentation by macrophages and dendritic cells (Nong et al., 1989 ; Asahina et al., 1995 ; Seiffert and Granstein, 2006 ). Moreover, locally elevated levels of CGRP (Khachatryan et al., 1997 ) and SP (Razavi et al., 2006 ) have a disease - preventive function. One might wonder whether the impaired dendritic cell maturation peculiar to NOD mice (Strid et al., 2001 ) is also related to hyposecretory TRPV1, but appropriate experiments have not yet been performed to address this possibility. The reduced availability of system - wide SP and CGRP may, in addition, alter the capacity for leukocyte recruitment to other sites of infl ammation in NOD mice (Bouma et al., 2005 ). The central position of TRPV1 and neuropeptides in T1D is not without precedent in other disease states, although the mechanisms differ. For example, TRPV1 and SP are important players in the pathogenesis of pancreatitis, in which premature activation of digestive enzymes leads to auto- digestion of exocrine pancreas tissue resulting in infl ammatory cascades critically dependent on SP release from TRPV1 - positive sensory neurons. Binding of SP to NK- 1 on endothelial cells triggers plasma extravasation and neutrophil migration into the pancreas. Blockade of either TRPV1 or NK- 1 attenuates experimental pancreatitis (Liddle and Nathan, 2004 ). Elevated levels of SP would be islet protective, perhaps explaining the common escape of the endocrine pancreas from the severe infl ammatory stress in the surrounding exocrine pancreas (Campbell- Thompson et al., 2009 ). The role of TRPV1 and neuropeptides in infl ammatory and autoimmune diseases is not limited to the pancreas. Recent fi ndings implicate SP in the pathogenesis of alopecia areata, a dermal autoimmune disorder characterized by leukocytic infi ltration around hair follicles culminating in hair loss (Siebenhaar et al., 2007 ). Release of SP triggers apoptosis and neurogenic infl ammation in the hair follicle (Peters et al., 2007 ). In a broader immunological context, there is a large volume of data linking SP with infl ammatory disease (O ’ Connor et al., 2004 ). REFERENCES 439

16.16 SUMMARY

This chapter has focused on the role of TRPV1 in the pathogenesis of T1D. In NOD mice, a hyposecretory TRPV1 mutation leads to decreased neuropeptide release and drives β - cell stress through hyperinsulinemia. Compensatory insulin resistance sustains a vicious infl ammatory cycle, culminating in β- cell demise. This novel mechanism explains autoimmune diabetes through a constellation of neuronal, immune, and endocrine factors that together generate an organ- specifi c disease. The impact of these fi ndings on the role of TRPV1, particularly on insulin resistance, will guide new therapeutic strategies for the management of both T1D and, likely to some extent, T2D.

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The knowledge gained during a decade of active research in the area of TRPV1 receptor physiology and pharmacology was used to develop a number of TRPV1 antagonists that recently entered human clinical trials. Several pharmaceutical companies, including GlaxoSmithKline, Amgen, Merck, Abbott, Glenmark/Eli Lilly, AstraZeneca, Pfi zer, Mochida/Wyeth, and Japan Tobacco, have advanced TRPV1 antagonists to early clinical development. SB - 705489 from GlaxoSmithKline was the fi rst selective TRPV1 antagonist to enter the clinic (Chizh et al., 2007 ; Gunthorpe and Chizh, 2009 ). In a single- dose placebo- controlled phase 1 study, 400 mg SB- 705489 elevated heat pain thresholds in normal skin and reduced capsaicin - evoked fl are. SB - 705498 also signifi cantly attenuated UVB - evoked hyperalgesia compared to placebo. No hyperthermia/hypothermia adverse events were reported. Phase 2 dental pain trials were completed, but to date, the results have not been presented. AMG - 517 from Amgen successfully completed single - dose (up to 25 mg) and multiple - dose (up to 10 mg) phase 1 studies and entered dental pain clinical trials. However, these trials were terminated because AMG- 517, which has a half- life of about 300 h in humans, was reported to cause a pronounced 3 ° C increase in body temperature in one patient after a repeated 2 mg dose (Gavva et al., 2008 ). Press releases from Merck and Neurogen stated good tolerability and lack of serious adverse events for their TRPV1 antagonist MK -2295 in phase 1 clinical trials. However, anecdotal reports of decreased heat sensation induced

449 450 AFTERWORD by MK - 2295 prompted Merck to conduct a series of single - and multiple - dose placebo- controlled heat perception studies including quantitative sensory testing (QST) (Crutchlow et al., 2009 ). These experiments concluded that MK- 2295 caused a potentially dangerous decrease in heat perception in “ real - world ” scenarios, such as bathing, eating, and drinking, in which a decreased ability to detect heat could result in serious burns. Another TRPV1 antagonist, GRC- 6211, developed by Glenmark and licensed to Eli Lilly, was shown to be safe in single- dose (up to 200 mg) and multiple- dose (up to 100 mg) phase 1 studies. However, this compound was withdrawn from phase 2 osteoarthritis trials for undisclosed reasons. Very recently, AstraZeneca announced the termination of Phase II trials to evaluate the efﬁ cacy, safety, and tolerability of AZD 1386 in patients with osteoarthritis of the knee. Although initial reports from clinical studies with TRPV1 antagonists indicate pharmacological target engagement, to date, no results have been reported from proof - of - concept studies in chronic pain. Clinical data for several other compounds, which recently entered clinical trials, will help to further assess the safety and efﬁ cacy of TRPV1 antagonists and to determine their potential as pain therapeutics. Arthur Gomtsyan and Connie R. Faltynek Abbott Park, IL

NOTE ADDED IN PROOF

The TRPV1 research community mourns the loss of Boris Chizh, who passed away in 2009 after a long illness. He will always be remembered as a strong advocate for the potential of TRPV1 antagonists as therapeutics.

REFERENCES

Chizh BA , O ’ Donnell MB , Napolitano A , Wang J , Brooke AC , Aylott MC , Bullman JN , Gray EJ , Lai RY , Williams PM , and Appleby JM ( 2007 ) The effects of the TRPV1 antagonist SB- 705498 on TRPV1 receptor- mediated activity and infl ammatory hyperalgesia in humans. Pain 132 : 132 – 141 . Crutchlow M , Dong y , Schutz V , Von Hoydonck P , Laethern T , Maes A , Larson P , Eid S , Kane S , Hans G , Murphy G , Chodakewitz J , Greenspan J , and Blanchard R ( 2009 ) Pharmacologic inhibition of TRPV1 impairs sensation of potentially injurious heat in healthy subjects. American Society for Clinical and Pharmacology and Therapeutics Conference, National Harbor, MD, March 18 – 21, 2009 . Gavva NR , Treanor JJ , Garami A , Fang L , Surapaneni S , Akrami A , Alvarez F , Bak A , Darling M , Gore A , Jang GR , Kesslak JP , Ni L , Norman MH , Palluconi G , Rose MJ , Salfi M , Tan E , Romanovsky AA , Banfi eld C , and Davar G ( 2008) Pharmacological blockade of the vanilloid receptor TRPV1 elicits marked hyperthermia in humans. Pain 136 : 202 – 210 . Gunthorpe MJ and Chizh BA ( 2009 ). Clinical development of TRPV1 antagonists: targeting a pivotal point in the pain pathway . Drug Discov Today 14 : 56 – 67 . INDEX

A-425619, 161, 265, 298–299, 377 anandamide, 103–105, 114, 219–220, 270, ABT-102, 183, 196, 300 278, 404 airway disease, 167, 409, 412 animal models, of neuropathic pain, 31, ALGRX-4975, 335, 339 119, 261, 269–270 allodynia cold, 31, 80, 86 anxiety, 277–278 allodynia, mechanical, 77, 160, 317. See apoptosis, 215 also TRPV1 antagonists, allodynia, aryl cinnamides, 300, 305 mechanical aryl-ureas, 295, 305 complete Freund’s adjuvant (CFA)- AZD1386, 303 induced, 155, 157, 165 sciatic nerve injury-induced, 264, 316 BCTC, 30, 164, 265, 312, 316 TRPV1 agonists, 269, 271 BCTC-like compounds, 295, 311, 320 TRPV1 receptor, 120, 155, 262, 271 bladder TRPV4 receptor, 16, 81 disorders, 13, 206, 214–217, 223–224, allodynia, tactile. See allodynia, 338 mechanical function, 224 Alzheimer’s disease, 82, 271, 279 bone cancer pain, 180, 200, 300, 317 AM404, 275 mouse sarcoma model of, 193, 201 AMG517, 161, 164, 167, 301, 303, 376, movement-evoked, 193, 202 378–379, 384 osteoclasts and, 193, 196 AMG628, 164, 303 RTX effect, 76 AMG9810, 268, 301, 377 treatment of, 192, 201 aminoquinazolines, 161 bradykinin, 34, 156, 219, 410

451 452 INDEX brain, 83, 177, 244, 263, 271, 273, 276, 279. capsazepine, 31, 74, 106–107, 109, 140, See also central nervous system 142, 164, 213–215, 249, 265, 273, brown adipose tissue (BAT) 275–276, 278, 295. See also under thermogenesis, 354, 356–357, 360– TRPV1 antagonists 361, 371, 373, 379, 384, 389 carrageenan-induced mechanical hyperalgesia, 155, 161, 166 calcitonin gene-related peptide (CGRP), CB1 and CB2 receptors, 263, 270, 276 77, 79, 110, 154, 177, 184, 208, 210, cell functions, 43 241, 243, 247–248, 408, 429–433, 438. central nervous system, 182, 187, 268– See also under TRPV1, 269. See also brain colocalization with channelopathies, 3 calcium homeostasis, 272 civamide, 251, 336 CaMKII, 104, 108, 110, 113 colitis, 167 camphor, 109 colon, 214 capsaicin. See also under TRPV1 complete Freund’s adjuvant (CFA), 155, activation; TRPV1 desensitization 157, 160–161, 165, 262, 265 aerosols, 406 cough, 406, 411–413 ALGRX-4975, 335, 339 binding model, 108 diabetes, 80, 86, 143, 423, 433, 436, bladder disorders, 13, 206, 216–217, 438–439 223–224, 338 diabetic neuropathy, 71, 81 co-administration with sodium channel diacylglycerol (DAG), 106 blocker QX-314, 78–79 digestive tract, 214 co-application with anandamide, 105 dorsal root ganglia (DRG), 76, 80, 154, cough, 406, 411–413 179, 270, 358–359, 382 diabetic neuropathy, 81 hypothermia, 82, 269, 362, 365, 369, endovanilloids, 76 372, 385, 388 epilepsy, 14 -induced cough model, 317 -induced eye wipe model, 157, 268 formalin, 162 -induced fl inch model, 160, 166, 303, 317 gastroesophageal refl ux disease -induced thermal hyperalgesia, 265 (GERD), 14 induction of apoptosis, 215 glycosylation, 143 injection into periaqueductal grey GRC-6211, 213, 217 (PAG), 245, 263–264 insensitivity to, 145 heteromerization, 214 intranasal administration, 249, 336, human disease, 3–4. See also specifi c 407, 413 types of disease intravenous injection, 405–406 Huntington’s disease (HD), 82, 274 irritable bowel disease, 74 hyperalgesia, mechanical. See also under itch, 79 TRPV1 antagonists liquid formulation, 335 carrageenan-induced, 155, 161, 166 neuroprotective effect, 273 complete Freund’s adjuvant (CFA)- NGX-4010, 76, 264, 335, 339 induced, 34, 77, 157, 160, 166, 268, piquancy, 71, 336 301, 304, 316 postherpetic neuralgia, 264 sciatic nerve ligation-induced, 265, topical application, 264, 332–333, 335, 305 337, 413 TRPA1 receptor, 34, 77, 85 INDEX 453

TRPV1 receptor, 77, 153, 155, 166 morphine, 81, 201 TRPV4 receptor, 16, 75, 81, 84 mouse sarcoma model, of bone cancer hyperalgesia, thermal. See also under pain, 193, 201 TRPV1 antagonists mutagenesis, 108, 136, 142 bradykinin-induced, 156 mutations, 134, 139, 141–142 capsaicin-induced, 265 carrageenan-induced, 155, 161, 265 N-arachidonoyl dopamine (NADA), complete Freund’s adjuvant (CFA)- 103–106, 114, 404 induced, 155, 157, 160–161, 262, 265 nerve growth factor (NGF), 247, 331 TRPA1 receptor, 34 neurogenic infl ammation, 75 TRPV1 receptor, 77, 106, 156, 179, 265 neurological disorders, 86 TRPV4 receptor, 16, 84 neuronal cells, 14, 77, 82, 101, 155, 360, 388 hyperthermia. See under TRPV1 neuropathic pain. See also under TRPV1; antagonists TRPV1 antagonists hypothermia. See under capsaicin; RTX; animal models of, 31, 119, 261, 269–270 TRPV1 antagonists capsaicin, 260, 273 capsazepine, 265 IBTU, 160, 162, 296 CB1 and CB2 receptors, 270 incontinence, 265 diabetic neuropathy, 71, infl ammatory pain. See also TRPV1, nerve growth factor (NGF), 331 infl ammatory pain; TRPV1 resiniferatoxin (RTX), 271 antagonists, infl ammatory pain; treatment of, 260 hyperalgesia, thermal; hyperalgesia, TRPA1 receptor, 34 mechanical TRPM8 receptor, 31, 80 morphine resistance, 201 TRPV1 agonists, 76, 336 TRPA1 receptor, 34, 85 TRPV3 receptor, 77, 83, 87 TRPV4 receptor, 81, TRPV4 receptor, 15 proinfl ammatory agents, 154 NGX-4010, 76, 264, 335, 339 insulin resistance, 439 nociceptors, 323, 325–333, 335–340, 354, iodoresiniferatoxin, 162, 264 359, 386, 388 irritable bowel disease, 74, 221, 223 nodose neurons, 359 islet infl ammation, 434 N-oleoyldopamine (OLDA), 103–105 non-neuronal cells, 210, 213, 360 JNJ17203212, 196, 200, 312, 316–317 JYL1421, 218, 296, 377 obesity, 13, 80, 86 omega-3 polyunsaturated fatty acids knockout and knockdown mice, TRPV1, (PUFAs), 106, 273 34–35, 75, 77, 82–83, 85, 106–107, opioids, 82 146, 155–157, 162, 166–167, 178–179, osteoarthritic pain, 161, 179, 181, 183, 211, 242, 261, 352, 386 187, 300 osteoclasts, 18, 179–180, 193–194, 196, 202 lidocaine, 110, 140 lungs, 408 P2X3 receptor, 209 pancreatitis, 438 mechanical hyperalgesia. See Parkinson’s disease (PD), 82 hyperalgesia, mechanical periaqueductal grey (PAG), 245, 263–264 migraine, 248 peripheral nervous system, 182, 268–269 MK-2295, 319 phosphatidylinositol 4,5-biphosphate mood disorders, 82–83 (PIP2), 103–104, 113, 146 454 INDEX phosphorylation. See TRPV1 thermal nociception, 72 phosphorylation thermoeffectors, 353–354, 357, 359–362, piperine, 109, 113 365, 367, 371–373, 378–379, 381–384, polymorphisms, 144 389 proinfl ammatory agents, 154 thermoregulation, 8, 352, 361, 380, prostatitis, 227 385–386 protein kinase A (PKA), 115, 118, 140, thermoTRP channels, 15, 71, 73, 78, 142, 268 84, 86 protein kinase C (PKC), 103, 109–113, tramadol. 110 115, 140, 142, 217, 262, 268 trigeminal ganglia (TG), 270, 358–359 pruritis, 79 TRP (transient receptor potential) psychiatric disorders, 86, 277. See also channels schizophrenia Alzheimer’s disease (AD), 271 calcium homeostasis, 272 QX-314, 78–79 cell functions, 43 channelopathies, 3 resiniferatoxin (RTX), 71, 142, 228, 367, heat- and cold-sensitive, 86 404. See also under TRPV1 human disease, 3–4 activation; TRPV1 desensitization pruritis, 79 bladder disorders, 206, 224–227, sensitivity to temperature. See also 338–339 thermoTRP channels bone cancer pain, 76 superfamily, 3 hypothermia, 269, 362, 364–365, 369, thermal hyperalgesia, 76 372, 385, 388 thermal nociception, 72 injection in the abdominal viscera, visceral pain,75 382 TRPA (transient receptor potential neuropathic pain, 271 ankyrin) channels, 3, 33 pungency, 224, 338 TRPA1 receptor skin vasodilation, 364 activation, 33–36, 71–72, 85, 162 ruthenium red, 364 agonists, 36 airway pathophysiology, 405 SB366791, 196, 201, 277, 300, 377 antagonists, 77, 85 SB705498, 251, 296, 298 antisense oligonucleotide, 75, 85 schizophrenia, 14, 82, 278 co-expression with TRPV1, 208, 358, sciatic nerve, 264, 265, 305, 316 386, 405–407 shivering and nonshivering, 353–354, 356, cold hyperalgesia, 34, 80 360, 369, 379 expression, 33–36, 146 skin vasoconstriction, 353–354, 357, 367, infl ammatory pain, 34 369, 378–380, 384, 389 knockout and knockdown mice, 34–35, skin vasodilation, 354, 362, 364–366, 77, 85, 162 368, 371 mechanical hyperalgesia, 34, 77, 85 spinal cord, 81, 268 polymorphisms, 144 Src kinase, 142 TRPC (transient receptor potential substance P, 77, 116, 154, 177, 184, 208, canonical) channels, 3–5, 7, 22 243, 247, 408, 425, 431 TRPC1 receptor, 4–8, 10 TRPC2 receptor, 4, 8 tachyphylaxis, 113, 141 TRPC3 receptor, 4, 8–9, 11 thermal hyperalgesia. See hyperalgesia, TRPC4 receptor, 4, 7, 9–10 thermal TRPC5 receptor, 4, 10 INDEX 455

TRPC6 receptor, 4, 7–8, 10–13 genetics, 134, 146, 147 TRPC7 receptor, 4, 14 GI, 209, 218, 231 TRPM (transient receptor potential glycosylation, 143 melastatin) channels, 3, 22, 351 heteromerization, 214 TRPM1 receptor, 4, 22 Huntington’s disease (HD), 82, 274 TRPM2 receptor, 4, 22–24, 29, 71, 352 incontinence, 265 TRPM3 receptor, 4, 24 infl ammation, 217 TRPM4 receptor, 4, 22, 24–26 infl ammatory pain, 72, 81, 105, 154–157 TRPM5 receptor, 4, 22, 24–27, 71 insulin resistance, 439 TRPM6 receptor, 4, 20, 22, 27–30 irritable bowel syndrome, 221, 223 TRPM7 receptor, 4, 22–24, 27–30 islet infl ammation, 434 TRPM8 receptor, 4, 22, 30–34, 71–73, mechanical hyperalgesia, 77, 153, 155, 78–80, 86, 109, 316, 352, 358, 405, 407 166 TRPML (transient receptor potential memory formation, 82 mucolipin) channels, 3, 36, 38, 351 migraine, 248 TRPML1 receptor, 36–38 modulators, 164 TRPML2 receptor, 37–38 mood disorders, 82–83 TRPML3 receptor, 36–39 mutagenesis, 108, 136, 142 TRPP (transient receptor polycystin) mutations, 134, 139, 141–142 channels, 3, 39, 352 neurological disorders, 86 TRPP1 receptor, 39–43 neuropathic pain, 81, 105, 265, 269–270 TRPP2 receptor, 39–43 obesity, 13, 80, 86 TRPP3 receptor, 40, osteoarthritis pain, 179, 181 TRPP5 receptor, 40 pain, 13, 34, 122, 144, 178, 180, 262 TRPV (transient receptor potential pancreatitis, 438 vanilloid) channels, 3, 13, 22, 273, Parkinson’s disease (PD), 82 352 permeability to cations, 104, 111–112 TRPV1 pore conductivity, 142 airway disease, 167, 409, 412 prostatitis, 227 Alzheimer’s disease, 82, 271, 279 pruritis, 79 antisense study, 386 psychiatric disorders, 86, 277 anxiety, 277–278 release of infl ammatory mediators, bladder diseases, 13, 206, 214–215 112 blockade, 153, 167, 182, 185, 214, 218, release of neurotransmitters, 112 227, 269–270, 438 schizophrenia, 14, 82, 278 bone cancer pain, 76, 180, 196, 200, 202 sequence, 117, 136, 434 Ca2+ homeostasis, 272 shRNA, 385–386 cloning, 136, 206, 216 structure, 101–103 colitis, 167 tachyphylaxis, 113, 141 cough, 414 thermal hyperalgesia, 77, 225 depolarization, 114 thermoregulation, 8, 352, 361, 380, detrusor overactivity, 223, 225 385–386 diabetes, 80, 86, 143, 423, 433, 436, tolerance to opioids, 82 438–439 translocation, 121 epilepsy, 14 visceral pain, 74 ethanol, 410–411 TRPV1 activation fecal urgency, 223 12-hydroperoxyeicosatetranoic acid gastroesophageal refl ux disease (12-HPETE), 103 (GERD), 14 abdominal viscera, 384, 389 456 INDEX

TRPV1 activation (Continued) 2-pyridinyl-piperazine carboxamides. acid. See TRPV1 activation, protons See BCTC-like compounds AM404, 275 A-425619, 161, 265, 298–299, 377 anandamide, 103–104, 114, 219–220, ABT-102, 183, 196, 300 270, 278, 404 allodynia, mechanical, 77, 157, 160, bradykinin, 34, 219, 410 165, 269, 317 brain, 276 AMG517, 161, 164, 167, 301, 303, 376, Ca2+, 110 378–379, 384 camphor, 109 AMG628, 164, 303 capsaicin, 14, 104, 106, 108, 113, 116, AMG9810, 268, 301, 377 120, 142, 167, 246, 262, 387–388, 404 aminoquinazolines, 161 diacylglycerol (DAG), 106 aryl cinnamides, 300, 305 endovanilloids, 76 aryl-ureas, 295, 305 formalin, 162 AZD1386, 303 heat, 106, 108–109, 120, 167, 243, 361, BCTC, 30, 164, 265, 312, 316 379, 404, 410 BCTC-like compounds, 295, 311, 320 induction of apoptosis, 408 binding site, 121 intranasal, 251 bladder hyperactivity, 215 lidocaine, 110, 140 bone cancer pain, 196, 200–202, 300, lung, 408 317 Mg2+, 110 capsazepine, 74, 164, 213, 249, 273, 278, modes of, 164–165, 298, 303 295 N-arachidonoyl dopamine (NADA), central nervous system, 165, 183 103–106, 114, 404 chronic administration. See TRPV1 N-oleoyldopamine (OLDA), 103–105 antagonists, repeated administration omega-3 polyunsaturated fatty acids differential pharmacology, 107, 162, (PUFAs), 106, 273 164–165 phorbol esters, 109–110 GRC-6211, 213, 217 phosphatidylinositol 4,5-biphosphate hyperalgesia, mechanical, 153, 157, (PIP2), 103–104, 113, 146 160–161, 166, 265, 268, 301, 304–305, piperine, 109 316 protons, 14, 107–108, 110–111, 114–115, hyperalgesia, thermal, 157, 160–161, 140, 142, 167, 361, 387–388, 404 165–166, 262, 265, 317 reactive oxygen species (ROS), 272 hyperthermia, 82, 164, 166–167, 184, resiniferatoxin (RTX), 142, 404 186, 269–270, 378–382, 384–385, 387– substance P, 116 389. See also TRPV1 antagonists, tonic, 378–381, 384, 389, 427 thermoregulation tramadol, 110 hypothermia., 82, 164, 387. See also voltage, 111 TRPV1 antagonists, TRPV1 agonist-based therapies, 325, 327, thermoregulation 332, 340 IBTU, 162, 296 ALGRX-4975, 335, 339 inﬂ ammatory pain, 76, 105, 153, 157, NGX-4010, 76, 264, 335, 339 160–161, 165, 181, 262, 268, 300–301, TRPV1 agonists, 76, 104, 109, 219, 278, 303–304 325, 332, 336, 338, 330, 360–362, 365– iodoresiniferatoxin, 162, 264 366, 372–373, 388–389, 411. See also JNJ17203212, 196, 200, 312, 316–317 TRPV1 activation JYL1421, 218, 296, 377 TRPV1 antagonists MK-2295, 319 1,3-disubstituted ureas, 298–299, 305 morphine tolerance, 81 INDEX 457

neuropathic pain, 265, 305, 316 C-fi bers. See TRPV1 expression, osteoarthritic pain, 161, 181, 183, 187, nerve fi bers 300 colon, 214 repeated administration, 186, 196, 270, digestive tract, 214 317, 387 dorsal root ganglia (DRG), 76, 154, ruthenium red, 364 179, 270, 358–359, 382 SB366791, 196, 201, 277, 300, 377 infl amed tissues, 411 SB705498, 251, 296, 298 lungs, 408 thermoregulation, 373, 379, 388–389 nerve fi bers, 154, 224, 358 TRPV1, coexpression with. See TRPV1, neuronal cells, 14, 77, 82, 101, 155, colocalization with 360, 388 TRPV1, colocalization with nociceptors, 323 calcitonin gene-related peptide nodose neurons, 359 (CGRP), 208, 210, 247–248, 408 non-neuronal cells, 210, 213, 360 CB1 receptor, 263, 276 peripheral nervous system, 182, nerve growth factor (NGF), 247 268–269 P2X3 receptor, 209 regulation of, 135 substance P, 208, 247, 408 spinal cord, 81, 268 TRPA1 receptor, 78, 208, 386, 405–407 trigeminal ganglia (TG), 270, 358–359 TRPM8 receptor, 78 urothelial cells, 14, 210–211, 224, 338 TRPV2 receptor, 406 viscera, 220, 382 TRPV3 receptor, 406 trpv1 gene, 134–135, 144–146, 386 TRPV4 receptor, 406 TRPV1 knockout mice, 75, 82–83, 106– TRPV1 desensitization 107, 146, 155–157, 166–167, 178–179, abdominal viscera, 382 211, 242, 261, 352, 386 bladder function, 224 TRPV1 phosphorylation camphor, 109 CaMKII, 104, 108, 110, 113 capsaicin, 71, 76, 228, 273, 367, 369, protein kinase A (PKA)-dependent, 407, 413 115, 118, 140, 142, 268 intrinsic, 327 protein kinase C (PKC)-dependent, mechanism, 76–77, 112–114 103, 109–113, 115, 140, 142, 217, 262, neurogenic infl ammation, 75 268 in patients with detrusor overactivity, sites of, 103, 117, 142 223 Src kinase, 142 phosphorylation-induced, 268 TRPV2 receptor, 15, 71, 141, 405–406 piperine, 113 TRPV3 receptor , 15, 71–72, 77–78, RTX, 71, 228, 367, 382, 385 80–83, 86–87, 109, 352, 405–406 thermoregulation, 371–372 TRPV4 receptor, 15–19, 34, 70–72, 75, TRPV1 expression 77–78, 80–81, 83–85, 214, 352, A-fi bers. See TRPV1 expression, 405–406 nerve fi bers brain, 83, 177, 244, 263, 271, 273, 279 urothelial cells, 14, 210–211, 224, 338 cancer cells, 200 central nervous system, 182, 187, 268– viscera, 220, 382, 384, 389 269. See also TRPV1 expression, visceral pain, 74–75, 218, 360 brain

Chromosomal location of Homo sapiens TRP channel genes

85.20 (ML3) p p MCOLN3 q p p MCOLN2 p q p q p q q (ML2) q p P2 135.58 q 85.11 q A1 C3 C7 137.26 142.09 C1 PKD2L2 V6 M8 (P5) V5 Chr 1 Chr 2 Chr 3 Chr 4 Chr 5 Chr 6 Chr 7142.13 Chr 8 M5 P1 p p p p p q q q p q p q M3 q C4 M1 q M6 M7 Mg PKDL (P3) C6 V4

Chr 9 Chr 10 Chr 11 Chr 12 Chr 13 Chr 14 Chr 15 Chr 16 V3 36.67 V1 34.19 MCOLN1 V2 p (ML1) p p p q p q p p q q p q q q q C5 M4 PKDREJ M2 (P4) Chr 17 Chr 18 Chr 19 Chr 20 Chr 21 Chr 22 Chr X Chr Y Figure 1.2 Schematic overview of the chromosomal location of human TRP genes (reproduced from Abramowitz and Birnbaumer [2006] with the permission of Springer- Verlag and the authors).

Hypertrophic agonists: ATII ET-1 TRPC1 PE TRPC3 2+ Ca L-type Ca2+ channel TRPC6

2+ Δϕ Gαq DAG Ca 2+ PLC Ca2+ Ca IP3

Calcineurin ER Ca2+ store P NFAT

Positive feedback system NFAT

MEF2 NFAT GATA TRPC Cardiac growth and remodeling genes

Figure 1.3 Hypertrophic agents such as angiotensin II, endothelin-1, and phenyleph- rin promote hypertrophy of the cardiomyocyte by causing excess Ca infl ux, which stimulates NFAT-driven gene expression of specifi c genes. Ca infl ux either occurs directly through TRP channels or the membrane potential is set by these channels, infl uencing Ca infl ux through L-type Ca channels. Expression of several TRPC channels is stimulated by NFAT, generating a positive feedback loop. See text for more details and references. Cilium

COOH 3 NH 1 TRPP2 2 PKD1 NH2 + COOH Ca2+ Na PKD domain TRPP2 2 2+ + Ca Na GPS-cleaved NH TRPC1 REJ domain 2 TRPP2TRPV4 PKD1 6 Plasma membrane NH Id2 NH2 NH2 2 Ankyrin PKD1 repeats COOH G P C-terminus COOH COOH PLC AC Ca2+ COOH 5 InsP3 DAG cAMP NH2 2+ ER 4 Ca PKC PKA PI3K NFAT AP-1Ras/B-Raf TRPP2 Id2-E-protein IP R MEK/ERK 2+ 3 2+ Ca dimer Ca AP-1 E protein 2+ Ca Ca2+

Ca2+ Ca2+ Nucleus Growth-suppressive genes

Figure 1.4 Compartment-speciﬁ c functions of TRPP2. (See text for full caption.)

(e) (f)

Figure 7.2 Sensory nerve ﬁ bers that innervate the tumor-bearing mouse femur maintain expression of TRPV1 with disease progression. A population of small- to medium- sized neurons in the contralateral (a) and ipsilateral (b) L2 DRG expresses the TRPV1 channel (red). Note that 14 days after tumor injection, the percentage and size of sensory neurons expressing TRPV1 in ipsilateral L2 DRG (b) that innervate the tumor-bearing femur are the same as contralateral DRG (33.3 ± 1.7% contralateral vs. 32.1 ± 4.3% ipsilateral; n = 4). Fourteen days after tumor injection, when tumor cells have invaded the marrow space and mineralized bone, there is an upregulation of ATF-3 (blue) in sensory neurons of the ipsilateral DRG (d) but not in the contralateral DRG (c). Double-label immunohistochemistry, merging the images obtained in (a) and (c) (e) or in (b) and (d) (f), suggests that a population of TRPV1-expressing sensory neurons innervates the tumor-bearing bone and exhibits an injured phenotype, as demonstrated by ATF-3 coexpression. Scale bar: (in f) (a–f) 50 μm (Ghilardi et al., 2005; with permission). (a) (c) (e)

TRPA1 TRPA1 TRPA1 (b) (d) (f)

TRPV1 CGRP SP Figure 8.3 Immunohistochemical localization of TRPA1 in the bladder wall. TRPA1-IR nerve ﬁ bers co-localize with TRPV1 (a and b), CGRP (c and d), and substance P (e and f). Reproduced from Streng et al. (2008). Figure 8.4 Confocal image of bladder urothelium in bladder whole mounts stained for TRPV1 and cytokeratin 17, a marker for basal urothelial cells. Diffuse cytoplasmic pattern of TRPV1 staining can be seen in the apical and underlying urothelial layers (nuclei are unstained). Arrows indicate apical cells within the ﬁ eld from a single plane of focus. Adapted from Birder et al. (2001). (Copyright National Academy of Sciences U.S.A., 2009).

(a) (b)

Figure 8.9 TRPV1 immunoreactive ﬁ bers in suburothelium of control (a, arrows) and PBS (b) bladders. Reproduced from Mukerji et al. (2006). Cortex

Thalamus

Hypothalamus Dural vessels Amygdala

PAG DR

LC TG

SP CGRP

P2X TRPV1 NK1 TrkA 5-HT1DASIC

TCC CG

Neuronal ergicity Warm-sensitive (principal mediator) neurons Glutamate Tonic GABA ? − Acetylcholine + Noradrenaline DMH

LPB Tonic ? ++ rRPa − + +

? Tonic +

Premotor Skin neurons Innocuous warming DH + + + + Innocuous Preganglionic cooling neurons DRG + + Sympathetic ganglia

Skin BAT vasculature Figure 14.1 A schematic of the neural pathways underlying the regulation of the sympathetic outfl ows to BAT and cutaneous blood vessels by innocuous warming and cooling of the skin. The neuronal bodies are shown as circles and star-like shapes; dendrites and axons are shown as lines; triangles with plus and minus signs represent excitatory and inhibitory synapses, respectively. The main mediator in each neuron is coded by color. The left portion of the fi gure shows afferent pathways; in this portion, the solid circles show neuronal bodies in the pathway activated by warming, and the empty circles show neurons in the pathway activated by cooling. The right portion of the fi gure shows efferent pathways; in this portion, neuronal bodies shown as the solid shapes belong to the BAT thermogenesis pathway, and neuronal bodies shown as the empty shapes belong to the skin-vasomotion pathway. Please see text for detailed descriptions and abbreviations. Abdominal viscera Nonthermal stimuli MPO + + TRPV1 − + − +

DMH Skin − Innocuous warming rRPa Innocuous − cooling

− + Skin vasculature BAT

Figure 14.8 Potential mechanism for suppression of BAT thermogenesis and skin vasoconstriction by nonthermal activation of visceral TRPV1 channels. The afferent pathway that starts with TRPV1-expressing sensory endings is shown in green; the unknown portion of this pathway is shown with a dashed line. For comparison, the afferent pathways that start with cutaneous warm- and cold-sensitive endings are also shown (in red and blue, respectively). The portions of these pathways that cannot be compared to the nonthermal visceral pathways (because the corresponding neurons in the visceral pathway are unknown) are not shown. The efferent pathways are shown in gray. As in Fig. 14.1, neuronal bodies shown as solid shapes belong to the BAT thermogenesis pathway, and neuronal bodies shown as empty shapes belong to the skin.