NEUROKININ 1 RECEPTORS AND THEIR ROLE IN OPIOID-INDUCED HYPERALGESIA, ANTINOCICEPTIVE TOLERANCE AND REWARD

Item Type text; Electronic Dissertation

Authors Largent- Milnes, Tally Marie

Publisher The University of Arizona.

Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.

Download date 02/10/2021 21:25:44

Link to Item http://hdl.handle.net/10150/193763

NEUROKININ 1 RECEPTORS AND THEIR ROLE IN OPIOID-INDUCED HYPERALGESIA, ANTINOCICEPTIVE TOLERANCE AND REWARD

by

Tally Marie Largent-Milnes

______Copyright © Tally M. Largent-Milnes 2010

A Dissertation Submitted to the Faculty of the

DEPARTMENT OF PHARMACOLOGY

In Partial Fulfillment of the Requirements For the Degree of

DOCTOR OF PHILOSOPHY WITH A MAJOR IN MEDICAL PHARMACOLOGY

In the Graduate College

THE UNIVERSITY OF ARIZONA

2010 2

THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Tally Marie Largent-Milnes entitled, “Neurokinin1 Receptors and Their Role in Opioid-Induced Hyperalgesia, Antinociceptive Tolerance, and Reward” and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy

______Date: 04/15/2010 Todd W. Vanderah, Ph.D.

______Date: 04/15/2010 Frank Porreca, Ph.D.

______Date: 04/15/2010 Victor J. Hruby, Ph.D.

______Date: 04/15/2010 Edward D. French, Ph. D.

______Date: 04/15/2010 Tamara King-Deeny, Ph.D.

Final approval and acceptance of this dissertation is contingent upon the candidate‟s submission of the final copies of the dissertation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement.

______Date: 04/15/2010 Dissertation Director: Todd W. Vanderah 3

STATEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the copyright holder.

SIGNED: Tally Marie Largent-Milnes

4

ACKNOWLEDGEMENTS

In preparing this manuscript, I have had the opportunity to reflect upon the last 5 years of work and assistance along the way. I have to thank my mentor and friend, Dr. Todd

W. Vanderah for all of his support; without his guidance and patience in all arenas, the completion of this dissertation would not have come to fruition. I must acknowledge the input of all of my committee members, past and present. My committee members, present and past: Dr. Victor J. Hruby, Dr. Frank Porreca, Dr. Tamara King-Deeny, and Dr.

Edward D. French, and Dr. Henry I. Yamamura, all of whom spent immeasurable time with me expanding my scientific knowledge.

My work could not have been completed without the ingenious work of Takashi

Yamamoto, who designed many compounds evaluated within. I must thank Milena De

Felice, Janice Oyarzo, and Nadia S. Corral-Frias for their friendship and support over the last 5 years, pulling me through the tough times and finding a way to encourage me. I also thank Peg Davis for her assistance in preparing this and many other manuscripts, bettering each of my publications.

Thank you to David S. Herman, Timothy M. Marshall, Beatriz Fioravanti-Lindstrom,

Alysia N. Ondua, Katherine E. Hanlon and all members of the Tucson Pain Group for allowing me to be part of your research and for being part of mine. I must acknowledge my funding sources that have made all of this work possible: grants from the USDHS,

National Institute on Drug Abuse, DA-13449 and DA-06284 and Pain Therapeutics,

Incorporated (San Francisco, CA). 5

DEDICATION

I dedicate this manuscript to Philippe, my parents (Dale and Terri Largent), my siblings and all of my family members who are present and those who have gone on to glory…

“You have to look for the light of God that is hitting your life, and you will find sparkles you didn’t know were there.”

~ Anonymous

You have all made my life a priority and a much brighter than I could have imagined.

Without your dedication and support to me, I would not be here now. I love you so much and am so grateful that God has allowed you to be part of my life. Thank you. 6

TABLE OF CONTENTS

LIST OF FIGURES ...... 10

LIST OF TABLES ...... 12

ABSTRACT ...... 13

LIST OF ABBREVIATIONS ...... 15

CHAPTER 1: ...... 18 INTRODUCTION AND BACKGROUND ...... 18

1.1 Pain ...... 18 1.2 Opioids: Receptors and Their Ligands ...... 19 1.3 Opioids: Antinocieptivce Tolerance and Opioid-Induced Hyperalgesia .... 22 1.4 Opioids: Physical versus Psychological Dependence ...... 23 1.5 Tachykinins and Their Receptors ...... 26 1.6 Therapeutic Approaches ...... 29 1.7 Significance and Hypothesis ...... 30

CHAPTER 2: ...... 32 MATERIALS AND METHODS ...... 32

2.1 Animals ...... 32 2.2 In vitro Assays ...... 32 Cell Lines ...... 32 Radioligand Labelled Binding Assay ...... 32 [35S]GTPγS Activity ...... 35 Guinea Pig Isolated Ileum (GPI) Assay ...... 36 Mouse Isloated Vas Deferens (MVD) Assay...... 36 Metabolic Stability in Rat Plasma...... 37 In situ Brain Perfusion ...... 37 2.3 Surgical procedures ...... 39 Intracerebroventricular Cannulation (i.c.v.)...... 39 Intrathecal Cannulation (i.t.) ...... 40 Jugular Cannulation (j.v.c.) ...... 40 Spinal Nerve Ligation ...... 41 2.4 Injection Procedures ...... 41 Intracerebroventricular ...... 41 7

TABLE OF CONTENTS - CONTINUED Intrathecal ...... 42 Intraperitoneal (i.p.) ...... 42 Intravenous (i.v.) ...... 42 Oral Gavage ...... 43 Subcuteanous (s.c.) ...... 44 2.5 Compounds for in vivo ...... 44 Anesthesia ...... 44 Control Compounds ...... 44 Experimental Compounds ...... 45 Fixatives ...... 46 2.6 Behavioral Testing Protocols ...... 46 Antinociception ...... 46 Carrageenan ...... 47 Formalin ...... 47 Rotarod/ Motor Skills ...... 47 Substance P-Induced Flinching, Biting, and Scratching ...... 48 Tactile Hypersensitivity ...... 48 Thermal Hypersensitivity...... 49 Aninociceptive Tolerance/ Paradoxical Hypersensitivity ...... 49 Conditioned Place Preference/ Conditioned Place Aversion (CPP/CPA) ...... 50 2.7 Tissue Collection ...... 52 2.8 Direct Coupling Protocols ...... 53 2.9 Immunolabeling ...... 53 2.10 Electrophysiology ...... 54 2.11 Mathematical Calculations ...... 56 Dissociation Constant ...... 56 Inhibition Constant...... 56 [35S]GTPγS Activity ...... 56 Ratio of Radioactivity in Brain (RBr) ...... 57 Antinociception (Thermal, 55 °C water bath)...... 57 Antinociception (Thermal, Radiant Heat) ...... 57 SNL-operated Tactile Hypersensivitiy (von Frey) ...... 57 SNL-operated Thermal Hypersensivitiy (Radiant heat) ...... 57 Sham-operated Tactile Hypersensivitiy ...... 58 Sham-operated Thermal Hypersensivitiy ...... 58 Antinociceptive Tolerance ...... 58 2.12 Data and Statistical Analyses ...... 58 Radioligand Labeled Binding Assay ...... 58 [35S]GTPγS Activity ...... 58 Analysis of GPI and MVD Assay ...... 58 Direct Coupling Analysis ...... 59 Behavioral Analysis ...... 59 Electrophysiological Analysis ...... 59 8

TABLE OF CONTENTS - CONTINUED

CHAPTER 3: ...... 61 BEHAVIORAL AND MOLEUCLAR MODUALTION OF NEUROBIOLOGICAL REMODELING WITH ULTRA-LOW-DOSE (ULD) OPIOID ANTAGONISM ...... …….61

3.1 Introduction ...... ….61 3.2 SNL Induces MOR Coupling to Gαs in Ipsilateral Spinal Dorsal Horn…..61 3.3 Chronic Oxycodone (i.t.) enhanced SNL Induced MOR Coupling to Gαs in Spinal Cord Dorsal Horn is attenuated by co-administration of ULD NTX ………………………………………………………….……………..……………62 3.4 ULD NTX Enhances the Effect of Intrathecal Oxycodone in SNL- animals …………………………………………………………………………………...…68 3.5 ULD NTX Enhances the Effects of Oral Oxycodone in SNL-animals ...... 76

CHAPTER 4: ...... 86 MODULATION OF PAIN WITH MULTIFUNCTIONAL LIGANDS ...... 86

4.1 Introduction ...... 86 4.2 Selection of Opioid Agonist/ NK1 Antagonist Peptides for in vivo Screening ………………………………………………………………………………...... 86 4.3 Opioid Agonist/ NK1 Antagonist Peptides in Acute pain…………………...89 4.4 Receptor Activity of Multifunctional Compounds In Vivo………………..100 4.5 Opioid Agonist/ NK1 Antagonist Peptidomimetics for Neuropathic Pain 111 4.6 Activity of Systemically Administered Opioid Agonist/ NK1 Receptor Antagonist ...... 127 4.7 A New Lead Compound: TY027 ...... 136 4.8 Activity of TY027 in vivo: Acute Pain ...... 142 4.9 Activity of TY027 in vivo: Neuropathic Pain ...... 152

CHAPTER 5: ...... 161 OPIOID AGONIST/ NK1 ANTAGONISTS LACK UNWANTED SIDE EFFECTS TYPICALLY RELATED TO OPIOIDS ...... 161

5.1 Introduction ...... 161 5.2 TY027: Antiallodynic, Antihyperalgesic, and Antinociceptive Tolerance 161 5.3 Development of Reward/ Abuse Potential Behavioral Assay: Conditioned Place Preference or Aversion………………………………………………………….171 5.4 TY027 does not induce paradoxical hypersensitivity ...... 179 5.5 TY027 and Reward Circuitry ...... 187

9

TABLE OF CONTENTS - CONTINUED

CHAPTER 6: ...... 195 DISCUSSION ...... 195 6.1 MOR- G-protein coupling ...... 197 6.2 ULD NTX attenuated opioid- induced, MOR-Gαs coupling and hypersensitivity ………………………………………………………………………………...... 199 6.3 Rational Development of Opioid Agonists/ NK1 Antagonists …………….202 6.4 Multifunctional peptides and Pain …………………………………………204 6.5 TY027: Antinociceptive Tolerance, Paradoxical hypersensitivity, and Abuse Liability ...... 211 6.6 Conclusions ...... 215

CHAPTER 7: ...... 219 APPENDICE ...... 219 A. List of Publications...... 220 B. Permissions: Not Applicable ...... 224 C. Human/ Animal Subjects Approval ...... 225

REFERENCES: ...... 227

10

LIST OF FIGURES

Figure 1: MOR- Gprotein coupling in SNL- and chronic opioid treated rats..64

Figure 2: Antihyperalgesic Effect of Spinal Oxycodone + ULD NTX in SNL animals

...... 71

Figure 3: Antiallodynic Effect of Spinal Oxycodone in SNL- animals ...... 74

Figure 4: Antihyperalgesic Effect of Oral Oxycodone + ULD NTX ...... 80

Figure 5: Antiallodynic effect of Oral Oxycodone + ULD NTX ...... 83

Figure 6: Antinociceptive effects of JSOH11...... 91

Figure 7: In vivo effects of TY004 ...... 94

Figure 8: Spinal TY005 Produces Antinociception in a Dose-dependent Manner without motor impairment ...... 98

Figure 9: TY005 acts at opioid receptors in vivo ...... 103

Figure 10: TY036 has activity in vivo ...... 106

Figure 11 Intrathecal TY005 reverses SNL-induced hypersensitivities ...... 115

Figure 12: Activities of opioid and NK1 peptide fragments: TY036, TY040, and

TY005 ...... 117

Figure 13: Timeline of chronic administration of spinal TY005 & morphine 123

Figure 14: Chronic, intrathecal TY005 does not produce antinociceptive tolerance in nerve-injured nor paradoxical hypersensitivity in sham-operated animals. . 125

Figure 15 TY005 may cross the BBB and be systemically active ...... 130

Figure 16: Systemic TY005 reverses SNL- hypersensitivities ...... 132

Figure 17: TY027 crosses the BBB ...... 139 11

LIST OF FIGURES - CONTINUED

Figure 18: TY027 is antinociceptive in models of acute pain ..……………….145

Figure 19: TY027 is an opioid agonist in vivo………………………………….149

Figure 20: Intrathecal TY027 acutely attenuated SNL-induced sensitivities. 155

Figure 21: Effects of systemic TY027 on neuropathic pain ...... 159

Figure 22: TY027 (i.t.) maintains efficacy in a neuropathic pain model ...... 164

Figure 23: TY027(i.t.) does not Result in Antinociceptive Tolerance ...... 169

Figure 24: Supraspinal (i.c.v.) TY027 does not result in CPP or CPA ...... 175

Figure 25: Systemic TY027 does not result in CPP or CPA… ...... 177

Figure 26: Spinal TY027 does not induce Paradoxical Hypersensitivity…...... 181

Figure 27: Evaluation of Paradoxical Allodynia after Supraspinal and Systemic

TY027…………………………………………………………………………….185

Figure 28: Schematic of Mesolimbic Reward Circuitry:……………………...189

Figure 29: VTA Immunohistochemistry: TH, GAD65, MOR and NK1……...190

Figure 30: Preliminary Electrophysiological Effects of TY027 in the VTA…193 12

LIST OF TABLES

Table 1: In vitro characterization of Multifunctional Peptides………………..88

Table 2: TY005 (i.t.) blocks SP- induced Nocifensive Behaviors in vivo……110

Table 3: Plasma Half- life of Multifunctional Peptides ...... 135

Table 4: In vitro: Direct comparison of TY005 and TY027 ...... 137

Table 5: SP-induced Flinching and Biting is Blocked by Spinal TY027 ...... 151

Table 6: Daily Paw Withdrawal Threshold Baseline Values for Determining

Paradoxical Hypersensitivity ...... 166

Table 7: Antihyperalgesic Tolerance: Daily Paw Withdrawal Latencies Before

Dosing ...... 167

13

ABSTRACT

Pain is the most common and debilitating sign of a medical problem, with nearly 15 million patients suffering from chronic pain, including neuropathic pain. Widely used therapies for treating neuropathic pain include tri-cyclic antidepressants, opioids, anticonvulsants, non-steroidal anti-inflammatory agents and combinations thereof.

Despite the abundance of treatments, the management of chronic pain remains difficult due to an inability for many patients to achieve appropriate pain relief at doses which are tolerable over long periods of time.

Opiates (natural products), or opioids (synthetic derivatives), are considered the gold standard of analgesic care, though with little efficacy for neuropathic pain. Opioids are associated with unwanted side effects, including paradoxical pain and abuse liability that may result from several nervous system adaptations within the pain modulating neural network. These dose related side effects become more prevalent as clinicians try to overcome analgesic tolerance.

Molecular mechanisms underlying these unwanted side effects have been studied extensively, and the literature purports a variety of contributing factors and neurobiological adaptations. The studies herein describe additional molecular adaptations and novel pharmacological approaches to counteract these changes. First, the contributions of neurobiological remodeling within a single receptor system (the opioid system) were investigated in the spinal dorsal horn after peripheral nerve ligation and chronic exposure to an opioid agonist in combination with an ultra-low-dose of opioid antagonist. The effects of the ultra-low-dose opioid antagonist naltrexone on the efficacy 14

of oxycodone for neuropathic pain were investigated after both central and systemic administration.

Secondly, molecular remodeling occurs across different receptor systems in the pain network, including altered regulation of pronociceptive molecules (e.g. substance P; SP).

Previous studies have reported that opioid-induced hyperalgesia, tolerance and reward can be prevented by a blockade or ablation of SP activity at the neurokinin 1 receptor

(NK1). We have characterized single compounds, rationally designed to act as opioid agonists and an NK1 antagonist using in vitro assays and the efficacy in vivo using rodent models of pain, antinociceptive tolerance and reward. Collectively, these studies validate the concept of targeting multiple neurobiological adaptations as a therapeutic option for neuropathic pain and reducing opioid- mediated side effects. 15

LIST OF ABBREVIATIONS

Ala - Alanine ANOVA - Analysis of variance Arg - arginine BBB - blood brain barrier βFNA - beta funaltrexamine BL - baseline BSA - bovine serum albumin CCK - cholecystokinin CGRP - calcitonin gene related peptide CHO - Chinese hamster ovary C.I. - Confidence nterval CNS - central nervous system CPA - conditioned place aversion CPP - conditioned place preference DA - dopamine DAMGO - [D-Ala2, NMePhe4, Gly5-ol]-enkephalin DMEM - Dulbecco's modified Eagle's medium DMSO- dimethyl sulfoxide DOR- delta opioid receptor DPDPE - c[D-Pen2, D-Pen5]-enkephalin DRG - dorsal root ganglion DSM V- Diagnostic and Statistical Manual of Mental Disorders, 5th revision EC50 - effective concentration, 50 percent Emax - maximal effective concentration EtOH - ethanol GABA - γ-amino butyric acid GAD65 - glutamate decarboxylase or glutamic acid decarboxylase, 65 kDa isoform GDP - guanosine diphosphate Gln -glutamine Gly - glycine GTP -guanosine triphosphate GPI - guinea pig isolated ileum HCBD - hydroxyl-cyclo-β- dextrin HEPES - (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HPLC - high performance liquid chromatography or high pressure liquid chromatography IASP - International Association for the Study of Pain IC50 - inhibitory concentration, 50 percent i.c.v. - intracerebrovrentricular i.p. - intraperitoneal i.pl. - intraplantar i.t. - intrathecal 16

LIST OF ABBREVIATIONS - CONTINUED i.v. – intravenous KOR- kappa opioid receptor Leu - leucine LMMP - longitudinal muscle with myenteric plexus Lys - lysine Met- methionine MOR - mu opioid receptor MS - morphine sulfate MVD - mouse vas deferens NIDA- National Institute of Drug Abuse NK1 - neurokinin 1 receptor NK2 - neurokinin 2 receptor NK3 - neurokinin 3 receptor NKA - neurokinin A NKB - neurokinin B NLX- naloxone NOP- nociceptin receptor NSAID- non-steroidal anti-inflammatory drugs NTI- naltrindole NTX- naltrexone OIH – opioid induced hypersensitivities ORL-1 – opioid receptor like receptor 1 PBS - phosphate buffered solution PEG - polyethylene glycol Phe- phenylalanine PMSF- phenylmethanesulfonylfluoride or phenylmethylsulfonyl fluoride PNS- peripheral nervous system PPTI- preprotachykinin 1 gene PPTII – preprotachykinin 2 gene Pro- proline PWL - paw withdrawal latency PWT - paw withdrawal threshold RBr- ratio in brain RT- room temperature s.c.- subcutaneous SD- standard deviation SEM- standard error of the mean SNL- spinal nerve ligation SP- substance P TFL- tail flick latency TH- tyrosine hydroxylase Trp- tryptophan Tyr- tyrosine 17

LIST OF ABBREVIATIONS - CONTINUED ULD- ultra-low-dose VTA- ventral tegmental area 18

CHAPTER 1: INTRODUCTION AND BACKGROUND

1.1 Pain

Pain, as defined by the International Association for the Study of Pain (IASP), afflicts more than 76.5 million Americans each year and costs the public an estimated 100 billion dollars in healthcare expenses and lost productivity (American Pain Foundation, 2008).

As a sensation comprised of physical and psychological components (e.g. emotional), pain is often the first indication of a medical problem. Acute pain, due to injury, resolves as the injury heals and serves a protective role. Chronic pains, however, are episodes of pain that last longer than the time of „normal‟ healing (Bonica 1953), though this definition has been simplified to “pains that persist for a given length of time” (Mersky et al., 1994). More recently, this definition has been clarified to state that chronic pain

“lasts for at least 3 months in a 6 month periods” or greater than 6 months ( IASP 2008).

Nearly 15 million patients suffer from chronic pain of various etiologies, including neuropathic pain. Neuropathic pain is described as a result of primary nervous system injury or dysfunction, either centrally or peripherally. Patients suffering from diseases such as cancer, arthritis, and diabetes often develop painful neuropathies due to disease progression or medical treatment. Other causes of neuropathic pain include surgery, compression (e.g. pinched nerve), and trauma (Jensen et al., 2009). Associated symptoms of neuropathic pain include not only pain (e.g. burning sensations) but also abnormal sensory perceptions, dysesthesias or total lack of input in some cases. Though many neuropathic pains share symptoms, underlying pathology may differ, making treatment 19

difficult (Jensen and Finnerup, 2009). Common therapies employed for treating neuropathic pains include tri-cyclic antidepressants, gabapentinoids, opioids, anticonvulsants, local anesthetics, acetaminophen, non-steroidal anti-inflammatory drugs

(NSAIDs), as well as combinations of opioids and NSAIDs (Dworkin et al., 2008;

Finnerup et al., 2005). Each treatment must be tailored to the patient; though, two-thirds of these patients still report inadequate relief (Jensen and Finnerup, 2009).

Though many options are available for treating chronic pains, specifically neuropathic pains, no single drug may be used across the patient spectra. Mechanisms which underlie the development of neuropathic pains are largely unknown. Study of these mechanisms have resulted in a signaling system which is increasingly complex, leading basic scientists and clinicians alike to propose a „multifaceted‟ or „multi target‟ approach to pain therapeutics (Jensen and Finnerup, 2009; Woodcock et al., 2007). Opioids are effective in alleviating many pains, including those of neuropathic origin, are limited by the development of adverse effects and efficacy at tolerable doses (Rowbotham et al.,

2003). Targeting the opioid receptors, as well as neuro-cellular adaptations resulting from chronic opioid use may present a rationale approach to enhanced pain relief.

1.2 Opioids: Receptors and Ligands

Pharmacological studies demonstrated the existence of opioid receptors over 25 years ago. Less than twenty years ago the molecular cloning of three isolated genes representing the mu- (MOR), delta- (DOR) and kappa- (KOR) opioid receptors was reported (Zaki et al., 1996). These receptors are seven-transmembrane, G-protein coupled 20

receptors found in both the central and peripheral nervous systems (CNS and PNS, respectively) and are typically coupled to inhibitory Gα- proteins. Subsequent signaling leads to decreased cyclic AMP and hyperpolarization due to reduced calcium influx and increased potassium efflux, blocking pain transmission (Connor and Christie, 1999; Ikeda et al., 2000; Laugwitz et al., 1993; Saegusa et al., 2000). A fourth member of the opioid receptor family was cloned in 1995, the opioid receptor-like receptor 1 (ORL-1), and has similar mechanisms of actions, though opposing effects depending on location (spinal versus supraspinal). Further discussion of ORL-1, now known as nociceptin peptide receptor, will be limited (review, Largent-Milnes and Vanderah, 2010).

Opioid agonists are compounds, both natural and synthetic, which bind to and activate the opioid receptors. Examples of endogenous peptide opioids are the peptides: beta- endorphins, enkephalins (Met- and Leu-), and dynorphins (Reisine and Pasternak, 1996).

Endogenous opioid peptides have a tyrosine (Tyr) at the N-terminal (position 1) which targets the opioid receptor, and removal of this Tyr decreases the peptide‟s opioid receptor selectivity. This phenomenon is exemplified by the ligand for NOP, nociceptin.

Nociceptin and the NOP are structurally similar to the kappa –dynorphin system.

However, nociceptin has a phenylalanine (Phe) at position 1 instead of Tyr. Substitution of Phe- with Tyr restores affinity of nociceptin to the kappa opioid receptor (review

Largent-Milnes and Vanderah, 2010).

Non-peptide ligands for the opioid receptors have also been described. Naturally occurring opioids are known as opiates and include morphine, heroin, and codeine.

Synthetic opioids are derivatives of the naturally occurring opiates and are mostly limited 21

to small molecule formulations (e.g. hydrocodone and oxycodone). Morphine, purified from the opium poppy in 1806, was found to be the ligand for the opioid receptors and is the most characterized (Reisine and Pasternak, 1996). Activation of both supraspinal and spinal opioid receptors produce the analgesic effects by inhibiting pain neurotransmitters such as substance P (SP), glutamate and calcitonin gene related peptide (CGRP) (Ossipov et al., 2004, Reisine and Pasternak, 1996). Mu-opioid receptor selective agonists include peptide (see above) as well as morphine, fentanyl, and heroin. Delta receptor selective agonists include deltorphin (I and II) and [D-Pen2,D-Pen5]Enkephalin (DPDPE), though currently there are no clinically available delta ligands. Kappa opioid receptors are activated by dynorphins and synthetic small molecules. It should be noted that clinical trials are under way with a peripherally selective, peptidic kappa agonist (CR845) designed to alleviate pain without the development of dysphoria, a known side effect of kappa opioid receptor activation in the supraspinal CNS (www.caratherapeutics.com).

Higher doses of opioids result in significant side effects including drowsiness, somnolence, constipation, nausea/ vomiting, and respiratory depression (Pasternak, 1993;

Reisine and Pasternak, 1996). In patients suffering from chronic pain, an increased dosing regimen is often needed to overcome the development of antinociceptive tolerance, further increasing the severity of side effects and, in some cases, the frequency of presentation. 22

1.3 Opioids: Antinociceptive Tolerance and Opioid–Induced Hyperalgesia

Though opioids are the most prescribed drugs to relieve pain, multiple processes can act to diminish opioid effectiveness. Antinociceptive tolerance occurs in many patients receiving opioid therapy and is recognized as a need for increased dosing to maintain the same level of antinociceptive efficacy. The repeated use of opioid analgesics for chronic pain states can be rendered ineffective by the development of analgesic tolerance (Foley,

1995; Way et al., 1969), and in some patients, opioid induced paradoxical hypersensitivity. Though mechanisms contributing to opioid tolerance are poorly understood recent evidence from experimental models show opioid analgesic tolerance may be derived from adaptations occurring at the level of the opioid receptor (Bohn et al.,

2000; Childers, 1991; Wang et al., 2008) and at neural networks involved in pain transmission (Vanderah et al., 2001a).

The mechanisms that underlie these unwanted side effects have been the focus of much research. Sustained exposure to opioids has been reported to induce neurocellular adaptations including opioid receptor internalization and long-term changes such as receptor down-regulation (Koch and Höllt, 2008), adenylyl cyclase superactivation

(Tumati et al., 2009), altered receptor trafficking and activation of descending pain facilitatory pathways from the brainstem (Vanderah et al., 2001a). In addition to these in vitro and in vivo studies, a number of clinical and preclinical studies report that sustained opioid use can unexpectedly produce abnormally heightened pain sensations, characterized by increased sensitivity to normally non-noxious (allodynia) and noxious 23

stimuli (hyperalgesia) that may be behavioral presentations of analgesic tolerance (DuPen et al., 2007).

Opioids can elicit heightened pain sensations in experimental models after acute

(Celerier et al., 2000; Yaksh and Harty, 1988) and chronic (King et al., 2005; Mao et al.,

1995a; Rivat et al., 2009; Trujillo and Akil 1991; Vanderah et al., 2000; 2001a and b; Xie et al., 2005) administration. Proposed mechanisms underlying opioid-induced hyperalgesia (OIH) include “mini-withdrawals” (Gutstein, 1996), activation of NMDA receptors (Mao et al., 1995), activation of descending pain facilitatory pathways (Gardell et al., 2002; Vanderah et al., 2001b), and upregulation in pronociceptive neurotransmitters (e.g. substance P) (King et al., 2005). Approaches inhibiting OIH could also inhibit the behavioral development of antinociceptive tolerance (Powell et al., 2003;

Rivat et al., 2009; Vanderah et al., 2000, 2001a, b; Vera-Portocarrero et al., 2007)

1.4 Opioids: Physical versus Psychological Dependence

Antinociceptive tolerance and OIH occur in many patients but do not, in themselves, preclude patients from continuing therapy. Similarly, physical dependence, characterized by the presence of withdrawal symptoms upon cessation of opioid therapy, occurs with sustained used in nearly all patients. Psychological dependence, which is defined as drug-seeking behavior despite the presences of negative consequences (DSM V), is relatively rare in chronic pain patients. Fears of potential opioid „addiction‟ have made compliance an issue for some patients taking opioid therapy, while physicians have become increasingly reluctant to prescribe long-term opioids for chronic pain patients. 24

Together, these concerns over rewarding behaviors and compulsive drug seeking behaviors have impeded the overall relief of all chronic pain patients and require attention when designing compounds for the treatment of chronic pain.

The mechanism by which opioid exposure induces physical dependence and reward is largely unknown. It has been suggested that opioid reward is due to inhibition of the tonically active GABAergic (gamma-amino-butyric acid: GABA) neurons in the ventral tegmental area (VTA) leading to an increase in dopamine (DA) in the nucleus accumbens. However, this increase in DA is evident under both rewarding and aversive circumstances, including persistent mu and kappa opioid use (Joseph et al., 2003). For example, MOR activation results in rewarding effects while kappa activation is adverse.

Delta opioid agonists do not produce preference or aversion. Furthermore, increasing literature points to interdependency of the opioid receptor system upon different neuromodulatory systems as the underlying mechanism of opioid reward (for review, see

Bryant et al., 2005).

The neuromodulatory systems indicated to alter opioid reward include the adrenergic, cannabinoid, cholecystokinin (CCK), corticotrophin-releasing factor, and the SP-NK1 system (Bryant et al., 2005; Gadd et al., 2003; Gold et al., 1981; Heinrichs et al., 1995;

Le Foll and Goldberg, 2005; Mas-Nieto et al., 2001; Murtra et al, 2000; Ossipov et al.,

2005; Ripley et al., 2002). Specifically, the SP-NK1 system has been implicated in the development of opioid induced physical dependence and reward. NK1 receptors have been found in the VTA and other supraspinal areas including the periaquaductal gray, nucleus accumbens, and rostroventral medial medulla (Franklin 1989; McLean et al., 25

1991; Flores et al., 2006; German and Fields 2007; Budai et al., 2007) suggesting that

NK1 and SP play a role in modulating the opioid system. However, direct interaction of the MOR and NK1 systems using immunohistochemistry and electrophysiology in the

VTA in the development of opioid antinociceptive tolerance and rewarding effects have not been shown.

In cultured, cortical neurons, morphine treatment was found to upregulate the functional expression of the NK1 receptor (Wan et al., 2006). NK1 and MORs have been shown to co-localize in the dorsal horn of the spinal cord (Aicher et al., 2000) and may form heterodimers with each other (Pfeiffer et al., 2003). Spinally, chronic opioids increase SP expression and evoked release as well as increase NK1 internalization (King et al., 2005; Liang et al, 2007; Vanderah unpublished results). In the VTA, controversial evidence is present in the literature regarding NK1 as behavioral studies found NK1 agonists alter DA neuron firing, though imaging studies show low density of NK1 in the

VTA, despite the high levels of SP-like immune reactivity (Elliott et al., 1992; Minabe et al., 1996).

Preclinical data report spinal pretreatment with an NK1 receptor antagonist decreases the naloxone precipitated withdrawal symptoms and SP-like immunoreactivity in morphine tolerant animals (Maldonado et al., 1993; Trang et al., 2002; Powell et al.,

2003; Bryant et al., 2005) while co-administration of an NK1 antagonist can block the development of morphine-induced rewarding behavior using a conditioned place preference paradigm (Powell et al, 2003). Mice lacking the gene for the NK1 receptor show decreased preference to pairing with morphine and heroin, but not cocaine 26

indicating a role for NK1 and SP in opioid mediated reward areas of the CNS (Murtra et

al., 2000; Ripley et al., 2002). NK1 knock-out mice do not self administer opioids, yet

cocaine self administration behavior is intact (Ripley et al., 2002). Such studies support a

role for substance P/NK1 mediated development or maintenance of opioid antinociceptive

tolerance and reward at the level of the VTA.

1.5 Tachykinins and Their Receptors

As chronic opioid therapy leads to changes including altered G-protein coupling and

increases in pronociceptive agents (e.g. dynorphin, CGRP, SP, etcetera) , targeting the

single receptor system for long term care may not be the most efficacious approach. One

opioid-induced adaptation, the increase in SP, is implicated in antinociceptive tolerance,

opioid-induced hyperalgesia, and reward and offers an interesting interaction for study.

Tachykinins are excitatory neuropeptides derived from two distinct preprotachykinin

genes, PPTI and PPTII. Substance P (SP) and neurokinin A (NKA) are derived from

PPTI, while neurokinin B (NKB) derives from PPTII. SP was first discovered in 1931 as

a compound with effects in the gastrointestinal tract of pigs (von Euler and Gaddum,

1931). In 1971, sequencing studies determined that SP is an undecapeptide with the

structure Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met (Chang et al., 1971).

Substance P is found throughout the central and peripheral nervous systems and has

effects on many physiological systems through activation of the neurokinin receptors.

Substance P preferentially binds to and activates the NK1 receptor. Activation of this

pathway results in an increase in intracellular calcium and promotion of depolarization in 27

neuronal tissue. In neonatal neuronal cultures, it has also been shown that neurokinin receptor activation can stimulate activity of adenylyl cyclase, though cyclic AMP formation is not increase. The implication of NK1 receptor mediated activity of this pathway is not known (Seybold, 2009). In addition to the NK1 receptor, the tachykinin family of ligands may bind to NK2 or NK3 receptors. These receptors selectively bind

NKA and NKB, respectively. Like the NK1 receptor, NK2 and NK3 are coupled to the

Gαq/11 pathway and phospholipase C. Upon binding of these ligands to the neurokinin receptors, endocytosis and recycling of the receptors to the plasma membrane occur

(review, Seybold, 2009). All three receptors are localized in the central nervous system, specifically in the dorsal horn of the spinal cord, implicating a role in nociceptive modulation. Other systems affected by SP-NK1 interaction include cardiovascular (e.g. vasodilatation), respiratory (bronchoconstriction), skin (itch), and gastrointestinal

(vomiting and nausea) (Harrison and Geppetti, 2001).

The role of SP has been predominately studied as a pain promoting neuropeptide through NK1 receptor activation. When injected spinally in rodents, SP dose-dependently induces nocifensive behaviors such as biting, scratching, and flinching (Hylden and

Wilcox, 1981; Seybold et al., 1982). Increases in spinal SP levels have been shown in inflammation and after chronic opioid exposure (King et al., 2005, Malcangio et al.,

2000), while redistribution of SP within the spinal dorsal horn has been reported in some models of neuropathic pain (Swamydas et al., 2004). After nerve injury, content and release of SP is decreased in some peripheral nerve injury models, but not in others

(Bisby and Keen, 1986). In models where some nerve fibers are spared, for example, 28

superficial dorsal horn content of SP is decreased but dorsal root ganglion (DRG) immunoreactivity is increased (Ma and Eisenach, 2003; Wallin and Schött, 2002).

Swamydas and colleagues (2004) have shown that SP is redistributed after partial nerve transection, leading to increases in the deeper lamina of the spinal cord. Additionally, the affinity of SP to the NK1 receptor has been reported to be altered after nerve injury

(Aanonsen et al., 1992). It has also been shown that the affinity of SP for NK1 is reduced by half once the NK1 receptor has been recycled to the membrane after initial internalization (Seybold, 2009). Furthermore, NK1 receptor expression has been shown to be increased at protein and mRNA levels after peripheral nerve injury (Goff et al., 1998;

Malmberg et al., 1997; Taylor and McCarson, 2004).

Though opioids are considered to be the gold standard of palliative care, side effects preclude long-term use in many patients and the associated risks of increased dosing have become cause for physician concern. Investigation into three of these side effects: antinociceptive tolerance, opioid-induced hyperalgesia, and opioid-induced reward, has yielded much information as to possible commonalities. The underlying mechanisms of these adverse events may be due to alterations within a single receptor system (the opioid system) as well as between multiple receptor systems, though each must be evaluated individually. Moreover, it has been suggested that combination therapy targeting multiple neurobiological adaptations may result in a decreased opioid side effect profile while maintaining analgesic effect, therefore presenting a promising option for the treatment of chronic pain (Woodcock et al., 2007; Bryant et al., 2005; Jensen and

Finnerup, 2009). Specifically, the studies herein investigate the underlying mechanisms 29

of these adverse events within a single receptor system, as well as interactions between multiple receptor systems: mu-opioid receptor neuroplasticity and the opioid receptor-

NK1 systems, respectively and therapeutic targeting of these adaptations in a rodent model of neuropathic pain.

1.6 Therapeutic Approaches

Specifically, adaptations within the opioid receptor system may play a role in the development of antinociceptive tolerance and OIH. Preclinical studies have shown that chronic morphine paradoxically causes excitatory rather than inhibitory effects on the cell, stimulating pain propagation, purporting an additional contributing factor to antinociceptive tolerance and OIH (Crain and Shen, 2000, 2001; Fan et al., 1991;

Harrison et al., 1998; Szucs et al., 2004; Wang and Gintzler et al, 1997). This enhanced pain and the underlying excitatory signaling have been proposed to be mediated by opioid receptors preferentially coupling to Gs proteins (Chakrabarti and Gintzler, 2003;

Shen and Crain, 1990), or alternatively, via the G dimer of the Gi/o proteins native to opioid receptors (Chakrabarti and Gintzler, 2001; Wang and Gintzler, 1997). Moreover, data have shown that chronic opioid administration leads to a shift in G protein coupling by MOR from Gi/o to Gs (Wang et al., 2005), with the G of this MOR-associated Gs protein also contributing to the excitatory signaling (Wang and Burns, 2006). However, the occurrences of these adaptations within the opioid system have not been investigated in an experimental model of neuropathic pain nor have the effectiveness of therapies that target these changes. 30

In addition to altered coupling of the mu-opioid receptor, the connectivity of the opioid system and that of SP-NK1 is of interest. Sustained opioids have been shown to enhance the content and evoked release of pain neurotransmitters suggesting more opioid may be needed to inhibit this opioid-induced upregulation, translating into antinociceptive tolerance. One important change is the upregulation of endogenous pronociceptive molecules including dynorphin, prostaglandins, CCK, CGRP, and SP. Of these, SP has been studied for its role in pain, antinociceptive tolerance and opioid-induced rewarding behaviors. Studies from Powell and colleagues (2003) have shown that co-administration of morphine and a small molecule NK1 antagonist can attenuate the development of opioid antinociceptive tolerance, further indicating that the NK1 receptor and SP may contribute to the underlying mechanisms of opioid antinociceptive tolerance. Moreover, the SP-NK1 system has been implicated in modulation of opioid reward (Murtra et al.,

2000; Ripley et al., 2002). Therefore, one priority would be to develop compounds that incorporate agonist activity at opioid receptors and antagonist activity at NK1 receptors.

1.7 Significance and Hypothesis

Current therapies for the treatment of chronic pain have been restricted to opioids, tricyclic antidepressants, dual acting re-uptake inhibitors, and anticonvulsants or combinations with NSAIDs (Dworkin et al., 2008). However, the analgesic efficacy of such compounds is limited by dose related side effects which become more prevalent as clinicians try to overcome analgesic tolerance. Such increases in doses of analgesics result in physician concerns of increasing severity of side effects, decreased quality of life 31

and potential patient abuse liability. This is matched by patient concern for quality of life and in a subset of patients, addiction potential. While the molecular mechanism(s) for the development of opioid antinociceptive tolerance is unknown, recent data point to several nervous system adaptations which correlate with the emergence of behavioral manifestations. These alterations include alterations within the pain modulating, neural network.

Overall Hypothesis: We have hypothesized that strategies which will oppose opioid- induced adaptive changes in the nervous system (e.g. atypical MOR-G-protein coupling and SP mediated excitation) can produce enhanced pain relief with diminished side-effects (i.e., antinociceptive tolerance and reward) compared to opioids alone.

Presently, evidence supports atypical MOR- G-protein coupling in a model of neuropathic pain which is enhanced in the presences of chronic opioids. Selective targeting of this abnormal coupling with ultra-low-dose naltrexone attenuates the pronociceptive coupling and reduces the degree antinociceptive tolerance observed in neuropathic animals. Furthermore, a single multifunctional molecule designed as an opioid agonist and NK1 antagonist dose dependently reverses nerve injury induced hypersensitivity and does not result in the development of antinociceptive tolerance, paradoxical hypersensitivity, or reward in rodent models. Such dual acting compounds will address the urgent need for chronic pain therapy without resulting in escalating doses that often lead to unwanted side effects and concerns of addiction, resulting in an overall better quality of life for chronic pain patients. 32

CHAPTER 2:

MATERIALS AND METHODS

2.1 Animals

Male, Sprague-Dawley rats (175-300 g; Harlan, Indianapolis, IN), male ICR mice (20-

30g; Harlan, Indianapolis, IN), and male Hartley Guinea Pigs (Harlan, Indianapolis, IN) were maintained on a 12-hr light/dark cycle (lights on 7am / lights off 7pm) with food and water available ad libitum. When possible, animals were housed socially. All procedures were performed in accordance with the policies and recommendations of the

International Association for the Study of Pain, the National Institutes of Health guidelines for the handling and use of laboratory animals, and by the Animal Care and

Use Committees of the University of Arizona.

2.2 In vitro assays

Cell Lines (Yamamoto et al., 2007): For opioid receptors, the cDNA for the human δ opioid receptor (DOR) was a gift from Dr. Brigitte Kieffer (IGBMC, Illkirch, France).

The cDNA for the rat µ opioid receptor (MOR) was a gift from Dr. Huda Akil (University of Michigan, MI). Stable expression of the rat MOR (pCDNA3) and the human DOR

(pCDNA3) in the neuroblastoma cell line, HN9.10 were achieved with the respective cDNAs by calcium phosphate precipitation followed by clonal selection in neomycin.

Expression of the respective receptors was initially verified and the level of expression 33

periodically monitored by radioligand saturation analysis (see below). All cells were maintained at 37˚C, with a 95:5 O2: CO2 humidified atmosphere in a Forma Scientific incubator in Dulbecco's modified Eagle's medium (DMEM) with 10% bovine serum albumin (BSA) and 100 U mL penicillin/100 µg/ mL streptomycin. For NK1 receptor:

The hNK1/CHO and rNK1/CHO cell lines were obtained from Dr. James Krause

(University of Washington Medical School, St. Louis, MI). All cells were maintained at a

37 C, 95% air and 5% CO2, humidified atmosphere, in a Forma Scientific incubator in

Ham‟S F12 with 2.5 mM HEPES, 10% fetal bovine serum and 100 U mL penicillin/100

μg mL streptomycin/ 500 μg mL Geneticin.

Radioligand Labeled Binding Assays: For opioid receptors: Crude membranes were prepared as previously described from the transfected cells that express either the rat

MOR (rMOR) or the human DOR (hDOR) (Lorenzen et al, 1993). The protein concentration of the membrane preparations was determined by the Lowry method and the membranes were stored at -80˚C until use. Membranes were resuspended in assay buffer (50 mM Tris, pH = 7.4, containing 5 mg/ mL bovine serum albumin, 50 μg/mL bacitracin, 10 μM captopril, 100 μM phenylmethylsulfonylfluoride (PMSF)) to a final concentration of 200 μg protein/ 100 μL. The dissociation constant (Kd) of tritiated [D-

Ala2, NMePhe4, Gly5-ol]-enkephalin ([3H]DAMGO) at the rMOR and that of tritiated c[D-Pen2, D-Pen5]-enkephalin ([3H]DPDPE) at the hDOR were as previously described

(Yamamoto et al., 2007). For competition analysis, ten concentrations of a test compound were each incubated, in duplicates, with 50 μg of membranes from MOR or DOR

3 expressing cells and the Kd concentration of [ H]DAMGO (1.0 nM, 50 Ci/mmol), or of 34

[3H]DPDPE (1.0 nM, 44 Ci/ mmol), respectively. Naloxone at 10 μM was used to define the non-specific binding of the radioligands in all assays. All assays were carried out in duplicate and were repeated. The samples were incubated in a shaking water bath at 25˚C for 3 hours, followed by rapid filtration through Whatman Grade GF/B filter paper

(Gaithersburg, MD) pre-soaked in 1% polyethyleneimine, washed 4 times each with 2 mL of cold saline, and the radioactivity determined by liquid scintillation counting

(Beckman LS5000 TD). For the NK1 receptor: Competition binding assays for the human and rat NK1 receptors were carried out on crude membranes prepared from transfected

CHO cells expressing the corresponding NK1 receptor. Ten concentrations of a test compound were each incubated, in duplicates, with 50-100 μg of membrane homogenate and 0.3 - 0.4 nM [3H]SP (135 Ci/mmol, Perkin Elmer) in 1 mL final volume of assay buffer (50 mM Tris, pH 7.4, containing 5 mM MgCl2, 50 μg/mL bacitracin, 30 μM bestatin, 10 μM captopril, 100 μM phenylmethylsulfonylfluoride (PMSF) at 25 C for 20 min. Substance P at 10 μM was used to define the non-specific binding. Membrane concentrations used in the assay were within the tissue linearity range. The [3H] substance P concentration was selected based on the saturation binding experiments which showed high affinity binding with Kd = 0.40 0.17 nM or Kd = 0.16 + 0.03 nM for the hNK1 and rNK1 receptors, respectively. The incubation times correspond to the binding equilibrium as determined from the kinetics experiments. The reaction was terminated by rapid filtration and washed as described above. The filter-bound radioactivity was measured by liquid scintillation counting (Beckman LS 6000SC). Log

IC50 value for each test compound was determined from non-linear regression analysis of 35

data collected from two independent experiments performed in duplicates (40 independent experimental values) using GraphPad Prism 4 software (GraphPad, San

Diego, CA). The inhibition constant, Ki, was calculated from the antilogarithmic IC50 value by the Cheng and Prusoff equation.

[35S]GTPγS Binding Assay: The method was carried out as previously described

(Yamamoto et al., 2007; Lorenzen et al., 1993). Membrane from transfected rMOR or hDOR cells (10 μg) in a final volume of 1 mL reaction mix (50 mM Hepes, pH 7.4, 1 mM EDTA, 5 mM MgCl2, 30 μM GDP, 1 mM dithiothreitol, 100 mM NaCl, 0.1 mM

PMSF, 0.1% BSA, 0.1 nM [35S]GTPγS) was incubated with various concentrations of the test drug, in duplicates, for 90 min at 30 °C in a shaking water bath. Reactions were terminated by rapid filtration through Whatman GF/B filters (pre-soaked in water), followed by 4 washes with 4 mL of ice-cold wash buffer (50 mM Tris, 5 mM MgCl2, 100 mM NaCl, pH 7.4). The radioactivity was determined by liquid scintillation counting as above. The basal level of [35S]GTPγS binding was defined as the amount bound in the absence of any test drug. Non-specific binding was determined in the presence of 10 μM unlabeled GTPγS. Total binding was defined as the amount of radioactivity bound in the presence of test drug (see Section 3.1).

For both radioligand and [35S]GTPγS binding, we adopted two independent experiments as our standard procedure for the initial evaluation of all compounds using cell lines due to the large number of compounds routinely screened on multiple receptor types to determine both affinity and biological activity. These initial analyses were 36

designed to identify trends of structure-activity relationship as rapidly as possible and were not intended for determining geometric mean values of Ki or EC50.

Guinea Pig Isolated Ileum (GPI) Assay: The in vitro tissue bioassays were performed as described previously (Porreca and Burk, 1983). Male Hartley guinea pigs under anesthesia were sacrificed by decapitation and a non-terminal portion of the ileum removed and the longitudinal muscle with myenteric plexus (LMMP) was carefully separated from the circular muscle as described previously (Porreca and Burks, 1983).

These tissues were tied to gold chains with suture silk and mounted between platinum wire electrodes in 20 mL organ baths at a tension of 1 g and bathed in oxygenated (95:5%

O2:CO2) Kreb's bicarbonate buffer at 37°C and stimulated electrically (0.1 Hz, 0.4 msec duration) at supramaximal voltage. Following an equilibration period, compounds were added cumulatively to the bath in volumes of 14-60 μL until maximum inhibition was reached. A baseline for PL-017 was constructed to determine tissue integrity and allow calculation of antagonist activity before opioid analogue testing began. If no agonist activity was observed at 1 μM, a repeat PL-017 dose-response curve was constructed to test for antagonist qualities.

All substance P antagonist compound testing was performed in the presence of 1 μM naloxone to block opioid effects on the tissue. Two minutes after naloxone was added to the bath, the test compound was added. Four minutes after naloxone was added, the test dose of substance P was added to the bath, the peak height was noted and the tissues were washed. Agonist activity of the analog was also observed during this period. Testing stopped at 1 mM concentrations of the test compound and the Ke calculated. 37

Mouse Isolated Vas Deferens (MVD) Assay: The in vitro tissue bioassay was performed

as described previously (Yamamoto et al., 2007). Male ICR mice under CO2 anesthesia were sacrificed by cervical dislocation and the vasa deferentia removed. Tissues were tied to gold chains with suture silk and mounted between platinum wire electrodes in 20 mL organ baths at a tension of 0.5 g and bathed in oxygenated (95:5% O2:CO2) magnesium free Kreb's buffer at 37°C and stimulated electrically (0.1 Hz, single pulses, 2.0 msec duration) at supramaximal voltage as previously described (Porreca et al., 1990).

Following an equilibration period, compounds were added to the bath cumulatively in volumes of 14-60 μL until maximum inhibition was reached. Response to an IC50 dose of

DPDPE (10 nM) was measured to determine tissue integrity before a compound testing.

Metabolic Stability In Rat Plasma (Yamamoto et al., 2009): Stock solution of compounds

(50 mg/mL in DMSO) were diluted 1000-fold into rat plasma (Lot 24927, Pel-Freez

Biologicals, Rogers, AK) to give an incubation concentration of 50 μg/mL. All samples were incubated at 37 °C and 200 μL of aliquots were withdrawn at 1, 2, 4, 6, and 24 h.

Then 300 μL of acetonitrile was added and the proteins were removed by centrifugation.

The supernatant was analyzed for the amount of remaining parent compound by HPLC

(Hewlett Packard 1090 m with Vydac 218TP104 C-18 column; 4.6 × 250 mm, 10 μm,

300 Å). The samples were tested in three independent experiments (n = 3).

In Situ Brain Perfusion: The ability of a drug to cross the blood brain barrier (BBB) allows for both peripheral and central actions of a drug. Additionally, penetration of the

BBB by a drug allows for better preclinical evaluation and possible translation into a clinical therapy. Synthesized peptidic compounds were evaluated for their propensity to 38

cross the blood brain barrier at a single time point by in situ perfusion.

In situ brain perfusion was performed as previously described (Huber et al., 2006;

Campos et al., 2008). Briefly, animals were anesthetized, injected with heparin (10,000

U/kg, i.p.) and with body temperature maintained at 37°C using heating pad. The common carotid arteries were cannulated with silicone tubing connected to a perfusion circuit. The perfusate was erythrocyte-free modified mammalian Ringer‟s solution consisting of 117 mM NaCl, 4.7 mM KCl, 0.8 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM

CaCl2, 10 mM D-glucose, 3.9% (w/v) dextran (MW 60,000), and 1.0 g/L bovine serum albumin (type IV), pH = 7.4, warmed to 37°C and oxygenated with 95: 5% O2: CO2.

Evan‟s blue dye (55 mg/L) was added to the perfusate to serve as a visual marker of BBB integrity. Perfusion pressure and flow rate were maintained at 95-105 mmHg and 3.1 mL/ min respectively. Both jugular veins were severed to allow for perfusate drainage. Using a slow-drive syringe pump (Harvard Apparatus, Holliston, MA), 14C-sucrose (10 μCi/ 20 ml perfusate; purity confirmed by HPLC analysis) was added to the inflowing perfusion solution at a rate of 0.5 mL/ min per cerebral hemisphere. Following a 20 min perfusion, the rat was decapitated and the brain was removed. The meninges and choroid plexus were excised and the cerebral hemispheres were sectioned. TS2 tissue solubilizer (1 mL;

Research Products International Corp., Mount Prospect, IL) was added to the tissue samples, which were solubilized for 2 days at room temperature. To eliminate chemiluminescence, 100 μl of 30% glacial acetic acid was added with 2.0 mL Optiphase

SuperMix liquid scintillation cocktail (PerkinElmer, Boston, MA). Radioactivity was measured using a model 1450 liquid scintillation and Luminescence Counter 39

(PerkinElmer). Results were reported as the ratio of radioactivity in the brain to that in the perfusate (RBr).

Following in vitro characterization of synthesized compounds, in vivo testing candidates were chosen based upon set criteria: mu- and delta- opioid receptor affinity and agonist activity, NK1 receptor affinity and SP antagonism, and the lack of affinity/activity at the kappa opioid and NK2/NK3 receptors. Techniques used to assess the in vivo efficacy of selected compounds are given in subsequent sections.

2.3 Surgical procedures

Intracerebroventricular Cannulation (i.c.v.): Direct injection of solution into the lateral ventricles allowed study of any supraspinal effect; therefore, indwelling cannulation in rats was performed. Under ketamine/xylazine anesthesia (100 mg/kg, i.p.) rats were fixed in a stereotaxic head holder (nose bar set at -3.5 mm) and the skull exposed approximately 2 cm. Two 1 mm holes were then drilled by hand (DH-0 Pin Vise, Plastics

One Inc., Roanoke, VA) on either side of midline, at least 3 mm back from bregma, and stainless steel screws (#MPX-080-3F-1M, Small Parts Inc., Miami Lakes, FL) attached, keeping the screws 2mm from the midline to avoid large blood vessels in the dura. The guide cannula (C131, PlasticsOne) was inserted using the following coordinates from bregma, according to the rat brain atlas (Paxinos and Watson, 1998): Anteroposterior: -

1.5 mm; mediolateral: -1 mm; Dorsoventral: -3 to -3.5 mm from the skull. The cannula was inserted with a slow, smooth motion and superglued carefully to the skull without clogging the inner diameter. Dental cement was applied around the screws and cannula 40

to further secure the placement to the skull and allowed to dry for 5 - 10 min. A dummy cannula was inserted to prevent clogging of the inner diameter prior to experimental test days. Rats were housed individually and allowed 5-7 days recovery before any manipulation was performed.

Intrathecal Cannula Implantation (i.t.): Rats were anesthetized using ketamine/xylazine

100 mg/ kg i.p. (vol/vol: 80/20, Sigma-Aldrich; St. Louis, Missouri USA) and placed in a stereotaxic head holder. The cisterna magna was exposed and incised and an 8 cm catheter (PE-10, Stoelting Cat # 51151; Wood Dale, Illinois) was implanted as described by Yaksh and Rudy (1976) terminating in the lumbar region of the spinal cord. Catheters were sutured (3-0 silk suture) into place in the deep muscle and externalized at the back of the neck. Remaining muscle and skin were closed in two layers. Animals were housed individually and allowed to recover for 5-7 days prior to manipulations. Animals with signs of neurological deficits following surgery or with greater than 10% loss in body weight were not used.

Jugular cannulation: For repeated i.v. injections, a jugular cannula was inserted into the right jugular vein, slightly modified from the method described by Yang et al., 2005.

Under ketamine/xylazine anesthesia (100 mg/kg, i.p.), the right jugular vein was exposed and tied off using 4-0 silk suture. With a 27 gauge needle, the vein was pierced to create an opening for an 8 cm total length of PE-50 tubing inserted 2 cm into the vein. Prior to insertion, the cannula was filled with a 10% heparinized saline solution, and clamped with a hemostat to prevent air bubbles from entering the blood stream. Placement was verified by the presence of blood in the cannula when the connected syringe was drawn 41

back. Once secured, the cannula was then closed until the testing day by melting the tip with a cautery tool. The cannula was then fed subcutaneously and externalized at the back of the neck to prevent the animal from displacing it. Amikacin C (4.4 mg/kg, i.p.) or gentamicin (8 mg/kg, s.c.) was given as a prophylactic and rats allowed 7 days recovery prior to behavioral testing.

Spinal Nerve Ligation (SNL): Peripheral nerve ligation produces signs of neuropathic pain including tactile allodynia and thermal hypersensitivity. All nerve operations occurred 5 days after i.t. catheter implantation. Rats were anesthetized with 2.5% isofluorane in O2 anesthesia delivered at 2 L/min. The skin over the caudal lumbar region was incised and the muscles retracted. The L5 and L6 spinal nerves were exposed, carefully isolated, and tightly ligated with 4-0 silk distal to the dorsal root ganglion, as previously reported (Kim and Chung, 1992). Control animals underwent sham surgeries in which the nerve areas were exposed but not ligated. All animals were allowed 5-7 days to recover prior to any behavioral testing. Any animals exhibiting signs of motor deficiency, infection, or greater than 10% loss in total body weight were euthanized.

2.4 Injection Procedures

Intracerebroventricular (i.c.v.) Drug Administration: Direct injections into the ventricle of mice were performed as described by Lowery et al. (2007). Briefly, 5 μL of experimental compounds were injected at a point 2 mm posterior and 2 mm lateral from bregma at a depth of 3 mm from the skull in anesthetized mice. For rats with indwelling i.c.v. cannula, tubing connected the injection cannula (C313I) to a 10 μl Hamilton syringe that 42

was filled with 5 μL of experimental or control compound followed by 0.5 μl air and a 4

μl saline flush. The saline flush monitored the movement of the fluid through the tubing.

After waiting 30 seconds, the injector was slowly removed and the dummy cannula inserted to prevent any back-flow of drug.

Intrathecal (i.t.) Compound Administration: Direct spinal injection of compounds was performed by lumbar puncture to unanaesthetized mice according to the methods described by Hylden and Wilcox (1980). To summarize, a 30.5 gauge needle was inserted spinally, with proper placement confirmed when the tail flicked upon needle probing. Vehicle or experimental compounds were injected in a volume of 5 μL using a

10 μL Hamilton syringe (Hamilton, Reno, NV, USA) for initial screening of opioid agonist- NK1 antagonist compounds. Rats with indwelling i.t. cannulae to the lumbar level were injected while moving freely in their home cages. Briefly, tubing connected the injection cannula (C313I) to a 25 μL Hamilton syringe filled first with 9 μL saline followed by 0.5 μL air and 5 μL of experimental compound. The saline flush monitored the movement of the fluid through the tubing. Tubing was disconnected from the indwelling cannula after 15 seconds to prevent any back-flow of drug.

Intraperitoneal (i.p.) Compound Administration: Compounds were given intraperitoneally and evaluated after administration. Briefly, animals were lightly restrained by hand and inverted allowing for exposing of the ventral lower quadrants of the body. A 25 gauge needle was inserted at a 45° angle and positioned so the tip pointed toward midline. Volume of injection varied by compound/experiment; these values are given in Section 2.5 Compounds for in vivo. 43

Intravenous (i.v.) Compound Administration: Intravenous injections were performed in unanesthetized animals by placing the animal in an appropriate restrainer (mouse or rat;

VWR International, Tempe, AZ) and holding their tail in warm water for 5 sec to dilate the tail vein. A 30 gauge disposable needle on a disposable 1 cc syringe was inserted into the tail vein. Injections of the compound or vehicle were performed over a 10 sec period and were noted as positive by the presence of blood in the tip of the syringe before injection and the absence of an out-pocketing of the tail-skin at the site of injection. For mice, compounds were made in a 10 mL/kg injection volume; for rats the injection volume was 1 mL/kg. In rats with indwelling jugular cannulae, injections were performed while rats moved freely about their home cages; compounds were made in a 1 mL/kg injection volume. In anesthetized rat preparations for electrophysiological experiments a similar approach was used for tail vein injections. However, a 23.5 gauge needle connected by PE-50 tubing (Stoelting) was inserted into the tail vein as described above. This PE-50 tubing was then connected to another 23.5 gauge needle and disposable syringe (1 mL) to avoid touching the tail when injecting, which causes signal noise during electrophysiological recordings. In this preparation, compounds were made in such a way that total volume injected per dose was 0.1 mL. All compounds were dosed in mg/kg as determined by total body weight.

Oral Compound Administration: To evaluate the oral bioactivity of some compounds in vivo, the oral gavage technique was used. A 3 inch, 18 gauge gavage needle was inserted into the stomach of a manually restrained animal. Proper placement was achieved when the entire needle was „swallowed‟ unobstructed. Incorrect placement into the trachea was 44

monitored, and animals with labored breathing, oral bleeding, or punctured lungs were not used (less than 3% of animals exhibited these signs).

Subcutaneous Compound Administration: Subcutaneous (s.c.) injections were performed by manually holding the animal and inserting a 25 gauge disposable needle on a disposable 1 mL syringe dorsally, assuring that the needle remained between the muscle and the skin of the animal. Injections of compounds or vehicles were performed over a 5 sec period and were noted as positive by the development of an out-pocketing of the skin at the site of injection. All compounds were dosed in mg/kg as determined by total body weight.

2.5 Compounds and Reagents

Anesthesia: Induction of anesthesia was performed using both injected and inhaled general anesthetics. The majority of procedures were performed under ketamine/xylazine

(vol/vol: 80/20, respectively) purchased from Sigma-Aldrich (cat #K-113). The ketamine/xylazine mixture was given in a dose of 100 mg/kg (1 mL/kg injection volume, i.p.). Spinal nerve ligation and sham-operations were performed under inhaled anesthesia with 2.0% halothane (Associated Medical Supply, item no longer carried; Scottsdale, AZ,

USA) or 2.5% isofluorane (Fisher Scientific, cat # NC9171659; Tustin, CA, USA) in O2 delivered at 2 L/min. Electrophysiology was conducted under chloral hydrate (Sigma-

Aldrich, cat # C8383; 350 mg/ kg, i.p.).

Control Compounds: Oxycodone hydrochloride was provided by Noramco (Wilmington,

DE) and dissolved in saline for intrathecal administration (10 μg in 5 μL injection volume) and in an ethanol: dH2O (1:2) vehicle for oral delivery (10 mg/kg, 1 mL/kg 45

volume). Morphine sulfate (MS), a mu-opioid receptor agonist, was obtained from the

National Institutes of Drug Abuse (NIDA) and dissolved in 0.9% physiologic saline

(Caligor, cat # BAX2F7124; Tucson, AZ, USA). The kappa opioid agonist U-50, 488 was purchased from Tocris (cat #0496). The non-selective opioid receptor antagonists naltrexone (NTX; Tocris cat # 0677) and naloxone (NLX; Tocris, cat # 0599) as well as the selective mu- opioid receptor antagonist β-funaltrexamine HCl (β-FNA, 42.5 nM;

Tocris, cat# 0926; Perlikowska et al., 2009) and the delta opioid receptor selective antagonist naltrindole (NTI, 66.0 nM; Research Biochemicals, Inc. cat # N-115; Dawson-

Basoa and Gintzler, 1997) were dissolved in saline when used; specific doses of MS, U-

50, 488, NTX, and NLX varied by experiment. Substance P (American Peptide, cat # 70-

1-10) was prepared as a 1 mM solution in 0.9% saline and injected intrathecally in a 10

μL volume (Seybold et al., 1982).

Experimental compounds: Oxytrex was made as directed by Pain Therapeutics,

Incorporated. Intrathecal administration of Oxytrex was 10 μg oxycodone + .033, 0.1, or

0.33 ng naltrexone (5 μL). Oral dosing of Oxytrex was oxycodone (3 mg/ kg or 10 mg/kg) + naltrexone (0.3, 1.0 or 3.0 μg/ kg) as described above. TY005 (H-Tyr-D-Ala-

Gly-Phe-Met-Pro-Leu-Trp-O-3, 5 Bn(CF3)2) and TY027 (H-Tyr-D-Ala-Gly-Phe-Met-

Pro-Leu-Trp-NH-3, 5 Bn(CF3)2) were provided by Dr. Victor J Hruby at the University of Arizona and synthesized as described by Yamamoto et al. (2007, 2008). Central dosing of TY005 and TY027 ranged between 1.0- 100.0 μg in 5 μL and 3 to 30 mg/kg for experiments with systemic dosing. Unless otherwise stated, TY005 and TY027 were dissolved in 10% dimethyl sulfoxide (DMSO) for intrathecal administration and 20% 46

DMSO for intravenous administration. For i.c.v dosing occurred, compounds were dissolved in either 10% DMSO or 18% hydroxyl-cyclo-β-dextrin (HCBD).

Fixatives: For use in perfusion protocols, the following solutions were made: stock, phosphate buffered solution (0.1 M PBS), 12.5% picric acid/ 4% formaldehyde in PBS

(vol/vol), 4% paraformaldehyde in PBS (weight/vol). The recipe for stock PBS recipes is given below:

Stock PBS- One liter of stock PBS was made by adding (weight/ vol): 9.0g NaCl, 0.122g

KH2PO4, and 0.815g Na2HPO4 to 1 L dH20. PBS was balanced to a pH = 7.4 with 1N

HCl and 1 M NaOH. Most often, this recipe was doubled or tripled to meet experiment requirements.

2.6 Behavioral Testing Protocols

Antinociception: The efficacy of experimental compounds against normally painful stimuli was assessed in the absence of injury as an initial screening process by measuring tail flick latency (mice) or paw withdrawal latency (rats). Tail flick latency: Baseline tail withdrawal latencies (s) to a 55 °C water bath were obtained before and after compound administration. Briefly, the distal third of the tail was immersed in water and a stopwatch started. Once the animal flicks its tail to escape the stimulus, timing is stopped and the time recorded as the Tail Flick Latency (TFL). Drug naïve, TFL values were typically between 1.5 and 3.0 s and animals with a baseline TFL greater than 5 s were excluded. A cut off latency of 10.0 s was established to prevent tissue injury. Paw Flick latency: Rats were allowed to acclimate to the testing room in Plexiglas holders on a glass table (Ugo 47

Basile, Comerio, Italy) for 30 min prior to testing. Baseline paw withdrawal latencies

(PWL) to an infrared radiant heat source (Hargreaves et al., 1988) were measured and typically ranged between 20.0 and 25.0 s. The heat intensity ranged from 28 - 32, which was not statistically different. A cut off of 33.0 s was established to prevent tissue damage. Observed values were obtained at time points appropriate for the chosen route of administration.

Carrageenan: A 2% carrageenan solution was used to induce inflammation in the hind paw (200 μL, i.pl.). Baseline paw withdrawal latencies to a radiant heat source were obtained as described above pre- and post-carrageenan. Testing was performed every 15 minutes for a total of 75 minutes. The infrared, noxious stimuli was aimed at the inflamed paw (left hind), until the animal moved its foot and the timer stopped. Results were quantified into averages, and standard error determined

Formalin: 2% formalin was injected into left hind paw (200 μL, i.pl.) 10 min after experimental compound administration (i.t.). Every 5 minutes after the formalin injection, for a total of 60 minutes, the number of times the animal flinched its paw was recorded. Flinching also included licking of the paw. Observations were quantified for each group. Phase 1 consisted of 0 - 10 min after the formalin injection while phase 2 refers to 35 - 55 min after the injection.

Rotarod: Rats were trained to walk on an automated, rotating rod (8 revolutions/ min;

Rotamex 4/8, Columbus Instrument, Columbus OH) for maximal cut off time of 180.0 s

(Vanderah et al., 2008). Training consisted of placement on a non- moving rod with the instrument off for 180.0 s, placement on the non-moving rod with the machine on for 48

180.0 s, and two training sessions of 180.0 s each with the rod rotating. After 10 min, baseline values were recorded for each animal. If the animal reached the maximal time, a cut off value of 180.0 s was assigned as the observed value. Compounds were administered as described above and assessment occurred every 15 min for the first 60 min.

Substance P-Induced Flinching, Biting, and Scratching: Rats were implanted with an intrathecal cannula as previously stated and allowed to recover for 5-7 days. Testing was consistent with the methods by Seybold and colleagues (1982). SP administration (1mM,

10 μL) was performed at t = 0 min; total doses were similar to those given by Seybold and colleagues (1982) at 13.47 μg per animal. Prior to testing, saline (0.9%) or naloxone

(2 mg/ kg, s.c.) was injected at t = - 15 min to block endogenous opioid and TY005 opioid activity (Sakurada et al., 1999) and TY005 (3, 10, or 30 μg in 5 μL) or 10%

DMSO (5 μL) was injected at t = - 10 min. The total number of flinches, bites, or scratches per minute for the first five minutes was counted.

Tactile Hypersensitivity: Rats were acclimated in suspended, wire mesh cages for 30 minutes prior to baseline von Frey testing (pre- and post-nerve ligation/ sham-operation).

Protocol for the acute exposure experiments consisted of compound or vehicle administration (t = 0) and measurement of responses to calibrated von Frey filaments (0.4

- 15.0 g) probed perpendicularly on the plantar surface of the left hind paw (ipsilateral to the SNL) for 7 seconds, at 15 min intervals for the first 60 min utilizing the up-down method used previously (Chaplan et al., 1994). For the multiple exposure experiment, non-noxious tactile testing occurred 15 min after compound administration. Lifting the 49

paw, licking the paw, or vocalizing counted as positive responses to the calibrated filament. Paw withdrawal thresholds were calculated in grams using the Dixon non- parametric test and expressed as the mean paw withdrawal threshold ± SEM in Prism4 by

GraphPad. Contralateral paw was not tested, as injured animals placed more body weight on the uninjured paw than on the injured side.

Thermal Hypersensitivity: Rats were allowed to acclimate in Plexiglas holders for baseline testing (pre- and post-nerve ligation/exposure) for 30 minutes (Ugo Basile,

Comerio Italy). A mobile radiant heat source was used to direct heat to the plantar surface of the left hind paw as reported (Hargreaves et al., 1988). Paw withdrawal latencies (PWLs) were measured in seconds with an automatic shutoff of the heat source at 33.0 s. Baselines and pre-nerve injury PWLs were established between 20.0 - 25.0 s for antinociception experiments. Post-injury baselines were obtained after the 7- day recovery period. On test days, animals were dosed, then tested using von Frey filaments every 15 min for 60 min. After multiple i.t. exposures, rats were evaluated for responses to noxious thermal heat 20 - 22 min following dosing and 5 - 7 min after non-noxious, tactile testing. Paw withdrawal latencies were recorded for each animal and expressed as the mean withdrawal latency ± SEM in Prism4 by GraphPad. The contralateral paw was not evaluated for hypersensitivity as injured animals placed more body weight on the uninjured paw when compared to the injured side. However, in chronic exposure experiments, the paw contralateral to injury was tested to assess the development of paradoxical allodynia. 50

Antinociceptive Tolerance and Paradoxical Hypersensitivity: Repeated administration of morphine results in signs of abnormal pain including mechanical allodynia and thermal hyperalgesia which may be a manifestation of antinociceptive tolerance (Vanderah et al.,

2000; 2001b). TY005 and TY027 were tested for their ability to produce antihypersensitivity after repeated administration (i.t.). The A90 dose was administered i.t. to nerve-injured and sham-operated animals and the animals were evaluated for thermal and tactile hypersensitivity 15, 30, 45, 60, 90 and 120 min after administration as in the acute studies. Treatment continued twice a day (8:00 am and 5:00 pm) for eleven days

(TY005) or seven days (TY027) with behavioral testing for thermal hyperalgesia 15 min after the 8:00 am administration. It is important to note that behavioral testing was performed while the compound was “on-board” to prevent behavioral results that may be misinterpreted as signs of physical withdrawal. On Day 7, a second time course was completed and a dose response curve (DRC) constructed (Vanderah et al., 2000; 2001b;

2007). The development of antinociceptive tolerance was considered to have occurred if the DRC was shifted to the right. Additionally, the inclusion of sham-operated animals allowed evaluation of the development of paradoxical hypersensitivity due to repeated exposure.

Conditioned Place Preference/ Conditioned Place Aversion (CPP/CPA): The ability of a drug to be positively reinforcing (potentially rewarding) can be measured using conditioned place preferences (Bilsky et al., 1990). The amount of time a rat spends in the putative side of conditioning is recorded before and after drug conditioning occurs.

The animal is exposed to both drug and vehicle, each treatment paired with the separate 51

visual and tactile cues of a two chamber box, over the course of two weeks. Rat CPP boxes from San Diego Instruments (San Diego, CA) have end chambers customized as follows: horizontal black and white striped walls with a smooth floor or grey walls with a rough floor. The center transition chamber was always set to have bright light and a grated floor. Sixteen y-axis sensors and four x-axis sensors allow for record fine motor, ambulatory, and exploratory movements. Each automated recording session was programmed to start all boxes in unison for a total of 20 min, recording at 5 min intervals.

Animals were allowed to habituate to the testing room for 60 min prior to baseline recordings. Two baseline recordings were obtained on days 1 and 4 and the average used as the overall baseline and preconditioning time per chamber. Any animal showing a greater than 80% total time preference prior to conditioning was excluded.

At random, animals were separated into treatment groups and conditioned to associate one of the isolated end chambers with treatment and the opposite end chamber with vehicle. The time course of conditioning was as follows: days 2, 3, 9, and 10 were non- conditioning days, days 5, 7, 11, and 13 were vehicle conditioning days, and days 6, 8,

12, and 14 were drug conditioning days. Balanced treatments proposed for this study included: saline-saline, saline-MS (10 μg), 10% DMSO-10% DMSO, 10% DMSO-

TY027 (20 μg), and 10% DMSO-MS (10 μg). All compounds were given i.c.v. as previously described. Secondly, a systemic dosing (i.v.) protocol was used and included the same treatment groups as i.c.v. administration, though the total amount of DMSO increased (from 10% to 20%) to accommodate the larger amount of compound.

Experimental recordings, free of vehicle or compound, occurred 24 hours after the final 52

drug exposure. In secondary experiments, the treatment protocol was altered to allow end chamber confinement only on drug conditioning day and open box availability on the alternate day.

2.7 Tissue Collection

To assess molecular changes in spinal the nervous system, fresh or perfused lumbar spinal cord and whole brain tissue was collected at the end of select experiments for analysis. For direct coupling experiments, fresh tissue was collected, while immunolabeling experiments required perfusion of the tissue with a fixative. Generalized technique for each approach is given below. Fresh Tissue: Fresh tissue was collected following decapitation. Animals were placed in a CO2 container in a fume hood and exposed to CO2 gas until all respiration and heart rate ceased prior to decapitation. The lumbar spinal cord enlargement was harvested and dissected into the ipsilateral/dorsal, ipsilateral/ventral, contralateral/dorsal, and contralateral/ventral sections. Tissue was flash-frozen in liquid nitrogen within 1 minute of removal and stored at – 80 °C until used. Perfused Tissue Collection: Rats were anesthetized with CO2 for intracardial perfusion with 250- 300 mL 0.1 M PBS followed by 250 - 300 mL of a 4% formaldehyde/12.5% picric acid solution in PBS (vol/vol). Tissue of interest was removed within 30 min and post-fixed in the perfusion fixative for a minimum of 4 hrs.

Harvested tissue was then cryoprotected in 30% sucrose at 4o C for a minimum of 48 hrs until sectioning or the final experiment was performed.

53

2.8 Direct Coupling Protocols

To investigate G-protein coupling to MOR, synaptic membranes were prepared from fresh, spinal dorsal horn tissue ipsilateral and contralateral to the sham-operated or spinal nerve ligation in rats treated with vehicle or experimental compound (n = 5 to 7 per experimental condition). Synaptic membranes (200 μg) were incubated with 1 μM morphine for 5 min at 37°C in Krebs Ringer solution. Membranes were solubilized in immunoprecipitation buffer (25 mM HEPES, pH = 7.5; 200 mM NaCl, 2 mM MgCl2, 1 mM EDTA, 0.2% 2- ercaptoethanol, 50 μg/ mL leupeptin, 25 μg/ mL pepstatin A, 0.01

U/mL soybean trypsin inhibitor, 0.04 mM phenylmethylsulfonyl fluoride) containing

0.5% digitonin, 0.2% sodium cholate, and 0.5% NP-40. G protein-coupled MORs were immunopurified by using immobilized anti-Gα antibodies. MOR protein associated with specific Gα proteins was assessed by Western blot, using an antibody directed against the amino-terminal region of MOR (Santa Cruz Biotechnology, Santa Cruz, CA) according to the methodology obtained from Wang et al. (2005)

2.9 Immunolabeling

Repeated exposure to morphine leads to alterations in MOR, NK1, and SP-like immunoreactivity in the superficial layers of the dorsal horn of the spinal cord and in the

DRG. Localization of MOR and NK1 receptors in the VTA has not been clearly evaluated in the literature and is of interest if NK1 plays a role in modulating opioid antinociceptive tolerance and reward.

Tissue from naïve animals was collected as described above by transcardial perfusion.

Frozen spinal cord sections (L3-L6 segments, 40 µm thick) and VTA (50 μm) were 54

sectioned with a cryostat, collected serially in PBS, and processed as free floating preparations. Tissue sections were rinsed in PBS then incubated in blocking solution (3% normal donkey serum + 0.3% Triton X-100 in PBS) at room temperature (RT) for 1 hr followed by a 24 hr incubation with the respective primary antibody at optimized concentrations. Primary antibodies used were: MOR (1:4000, Neuromics), rabbit anti-

NK1 (1:4000, gift from Dr. Patrick W. Mantyh), mouse anti-tyrosine hydroxylase (TH,

1:100, Chemicon), and mouse anti-GAD65 (1:5000, Dr. Robert Sloviter). After incubation in the primary antibody, sections were washed in PBS three times for 10 min each and incubated in the appropriate secondary antibodies (donkey anti mouse Cy2

1:200; donkey anti rabbit Cy3 1:600, Jackson labs) for 3 hrs. Sections were mounted onto gelatin-coated slides, dehydrated in alcohol gradients (70, 90, and 100%), cleared in xylenes and cover-slipped with DPX (Fluka, Buchs, Switzerland). All imaging capture and analysis was performed with a BX-61 microscope equipped with the Fluoview 1000 imaging software 5.0 (Olympus America Inc, Melville, NY). This laser scanning confocal microscopy system is equipped with He, Ne and Ar lasers, networked with a

Dell Precision workstation and equipped with Fluoview and Imaris 3D rendering software. Image threshold and channel pseudocolors were adjusted with Adobe

Photoshop CS.

2.10 Electrophysiology

All drugs of abuse are known to alter the mesolimbic dopamine system in some way.

Opioids are thought to inhibit GABAergic cells in the VTA ultimately disinhibiting A10 55

dopamine cells and leading to an increase in the firing rate (spikes/s). To further evaluate the potential rewarding effects of the synthesized peptides, single unit electrophysiological recordings of the dopamine cells in the VTA, part of the reward circuitry, were performed. Preparation of the animals and methods for extracellular recordings from single dopamine neurons in the midbrain VTA were conducted as previously described (French, 1992). Presumptive dopamine neurons were identified by the following criteria: (1) biphasic (or triphasic) positive-negative waveform action potentials: (2) action potential duration > 0.2 ms; (3) firing rates < 10 spikes/s; and (4) regular to bursting patterns of firing (Bunney et al., 1973).

Once a neuron meeting these characteristics was found, the basal activity was monitored for approximately 5 min prior to injections. Vehicle control values were obtained prior to compound dosing and included both saline and 20 % DMSO injections

(100 μL each). Only one cell per animal was evaluated once the test compounds were administered. Morphine and TY027 were given i.v. with interinjection intervals of 2–4 min using one of the following protocols: (1) a cumulative dosing paradigm in which

0.5, 0.5, 1, and 2 mg/kg (total cumulative dose of 5.0 mg/kg) of morphine or 1.0, 1.0, 2.0,

2.0, 4.0 mg/kg TY027 i.v. resulting in a total cumulative dose of 10.0 mg/kg, ensuring equipotent amount of opioid agonist on-board for a total injection volume of 500 μL or

(2) a single i.v. injection of MS (3.0 mg/kg) or TY027 (6.0 mg/kg), given to match the doses used in the CPP/CPA experiments for a total final injection volume of 100 μL for experimental compounds. Apomorphine-induced (20 - 40 μg/kg) inhibition of firing was used to confirm pharmacologically that the recording was from a dopamine neuron at the 56

end of the experiment. Electrode placement was verified upon conclusion of the experiment following injection of 0-7 μA current (on for 12 s, off for 12 s) using a 2% pontamine sky blue dye in 0.5 M Na acetate for 60 min. The animal was then sacrificed and brain tissue collected as described above. Coronal sections of 50 μm through the

VTA were mounted on slides and examined by light microscope for the location of the dye spot at a later date.

2.11 Mathematical Calculations

Dissociation constant

L is the amount of free ligand, R is the amount of free receptor, and LR is the amount of ligand-receptor complex

Inhibition Constant (Cheng-Prusoff, 1973):

Kd and [L] are the dissociation constant and concentration of the radioligand used, respectively. This relationship is only valid when the Kd used for the radioligand represents its value under the experimental conditions of the assay. Thus, we measure the

Kd values for all of the radioligands under the conditions used in the inhibition studies by saturation binding analysis. 57

[35S]GTPγS Activity

Ratio of radioactivity in the Brain (RBr)

[Cbrain] is total amount of radioisotope in the brain (dpm/g tissue) and [Cperfusate] is the amount of radioisotope in the perfusate (dpm/mL).

Non-injured % Activity (Thermal, 55 °C water bath)

Non-injured % Activity (Thermal, infrared)

SNL- operated % Activity (Tactile, von Frey)

58

SNL-operated % Activity (Thermal, infrared)

Sham- operated % Activity (Tactile, von Frey)

Sham- operated % Activity (Thermal, infrared)

Antinociceptive tolerance: The percent activities (Tactile and Thermal) were calculated by the equations above. Dose-response curves were generated from selected time points on day 1 and day 7 of treatment as previously described (Tallarida and Murray, 1987).

2.12 Data and Statistical Analyses

Radioligand Binding: Log IC50 value for each test compound was determined from non- linear regression analysis of data collected from two independent experiments performed in duplicates (40 independent experimental values) using GraphPad Prizm 4 software

(GraphPad, San Diego, CA). The inhibition constant Ki was calculated from the antilogarithmic IC50 value by the Cheng and Prusoff equation (Section 2.11). 59

GTPγS activity: Data were analyzed by non-linear least-squares regression analysis by

GraphPad Prism4 using data collected from 2 independent experiments in duplicates. The

EC50 for a test drug is converted from the logarithmic EC50 value derived from the best fit curve, and the maximum stimulatory effect is expressed as Emax standard error.

Analysis of the GPI and MVD assays: For opioid data analysis, percentage inhibition was calculated using the average tissue contraction height for 1 min preceding the addition of the agonist divided by the stimulated contraction height 3 min after exposure to the dose of agonist. IC50 values represent the mean of not less than 4 tissues. IC50 and

Emax estimates were determined by computerized nonlinear least-squares analysis (the pharmacological statistics package FlashCalc: Dr. Michael Ossipov, University of

Arizona, Tucson, AZ). For substance P data analysis, the height of the maximum peak produced during the unstimulated control substance P dose-response curve was used as a

100 % response and other values calculated as a percentage. Ke values represent the mean of not less than 4 tissues. Ke estimates were determined by computerized nonlinear least- squares analysis (FlashCalc).

Direct Coupling Analysis: Statistical significance for direct-coupling data was determined by analysis of variance (ANOVA) followed by Newman-Keuls comparisons.

Differences were considered to be significant at a value of p ≤ 0.05

Behavioral analysis: All treatment groups were comprised of a minimum n-value of 6.

Each experimental result gives the number of animals used. All data were analyzed by non-parametric two-way analysis of variance (ANOVA; post-hoc: Neuman-Kuels) in

FlashCalc (Dr. Michael H. Ossipov, University of Arizona, Tucson). When appropriate, 60

Student‟s T-test or one way ANOVA was used. Differences were considered to be significant if p ≤ 0.05. All data were plotted in GraphPad Prism4.

Electrophysiology: Action potentials were recorded with single glass capillary electrodes that had been pulled and broken back to a tip diameter of approximately 1 pm and filled with a solution of 2% pontamine sky blue dye in 0.5 M Na acetate. The in vitro impedance of the electrodes ranged from 8 to 15 MΩ when measured at 130 Hz. Unitary action potentials were amplified, filtered from background noise (l-10 kHz), displayed on an oscilloscope and passed through a voltage-gating window discriminator for integration and display on a multichannel recorder in Acute Record and as a ratemeter record. In addition to audio monitoring, the signal was fed into a computer configured for the generation of interspikeinterval histograms with continuous tabulation of the average rate of firing. Data were analyzed using Spike2 and MatLab software for the Acute Record files and in RISI for the rate-meter record.

Two-way ANOVA with a repeated measures design was used to compare the cumulative dose-response data of controls versus experimental compounds. Where statistical comparisons of one treatment group versus the controls were required (e.g. basal firing rates), a two-tailed T-test was used. A one-way ANOVA was also used for comparing the basal firing rates among the groups. Statistical significance was set a p ≤

0.05. 61

CHAPTER 3:

BEHAVIORAL AND MOLEUCLAR MODUALTION OF NEUROBIOLOGICAL

REMODELING WITH ULTRA-LOW-DOSE (ULD) OPIOID ANTAGONISM

3.1 Introduction

Both peripheral nerve injury and chronic opioid treatment can result in hyperalgesia associated with enhanced excitatory neurotransmission at the level of the spinal cord.

Chronic opioid administration has been shown to induce a shift in MOR–G protein coupling from Gi/o to Gs that can be prevented by co-treatment with an ultra-low-dose opioid antagonist (ULD). In this study, we investigated the behavioral outcomes after chronic dosing with oxycodone+ ULD naltrexone in SNL–operated animals and hypothesized that SNL injury could induce MOR linkage to Gs in the damaged

(ipsilateral) spinal dorsal horn without changing Gi/o coupling levels and without changing the overall expression of MOR or Gα proteins.

3.2 SNL Induces MOR Coupling to Gαs in Ipsilateral Spinal Dorsal Horn

Both injury and treatment effects on G protein coupling by MOR were evaluated by co- immunoprecipitation of Gα proteins with MOR from spinal dorsal horn tissue of sham operated animals or nerve-injured animals using the direct-coupling protocol.

Notably, the L5/L6 SNL injury alone induced the recruitment of Gαs proteins to MOR, as indicated by the presence MORs in the Gαs immunoprecipitate from ipsilateral dorsal horn in SNL, but not in sham control animals, receiving vehicle (Figure 1A). This SNL- 62

induced, novel coupling was found only in the spinal dorsal horn ipsilateral to the lesion and may present a potential mechanism underlying neuropathic pain not previously known.

3.3 Chronic Oxycodone (i.t.) Enhanced SNL-Induced MOR Coupling to Gαs in

Spinal Cord Dorsal Horn is Attenuated by Co-Administration of ULD NTX

As previous reports have found that chronic opioid agonist therapy also induces MOR-

Gαs coupling (Crain and Shen, 2001) the effect of chronic oxycodone on MOR-Gα protein coupling alone (10μg in 5μL, i.t.) or when co-administered with ultra-low-dose

NTX (0.33 ng, i.t.) was assessed in SNL-operated animals after 7 days. Tissue from animals treated with saline or NTX alone did not show a statistically significant difference between the contralateral side compared to sham-operated animals.

Oxycodone treatment resulted in an increase in MOR-Gαs coupling on the side ipsilateral to injury. Furthermore, oxycodone also induced a statistically significant increase in the atypical coupling on the contralateral side to injury compared to saline- treated animals (Figure 1A). Co-administration of NTX with oxycodone significantly attenuated the level of MOR-Gαs coupling in SNL animals in both the ipsilateral and contralateral dorsal horn. Interestingly, the oxycodone plus ultra-low dose NTX treatment resulted in MOR- Gαs coupling on the ipsilateral side to injury at levels similar to those of vehicle-treated SNL animals (Figure 1A). The decrease of both ipsilateral and contralateral atypical coupling and the similar levels of ipsilateral coupling indicate that co-administration of ultra-low-dose NTX blocks the oxycodone enhancement of MOR- 63

Gαs coupling in SNL animals but may not alleviate the injury-induced contribution to Gαs coupling.

In all cases, in vitro morphine augmented coupling by MOR with similar magnitude without the patterns of G protein coupling seen for each treatment and spinal cord side.

Fig 1B shows the densitometric quantification of MOR coupling stimulated by in vitro morphine. Basal (Krebs Ringer) coupling levels from Fig 1A are omitted for simplicity.

The top panels of Figs 1A and 1B show sham-operated, vehicle-treated animals; all other panels represent L5/L6 SNL-operated animals. Changes in G protein coupling were not due to changes in protein expression levels of MOR or Gα proteins (Data not shown).

64

1A

65

1B

66

Figure 1: MOR- G protein coupling in SNL- and chronic opioid treated rats (A)

“MOR-G protein coupling in spinal cord from rats with lumbar spinal nerve ligation or sham-operation”: Western blot of MORs (mu opioid receptor) co-immunoprecipitated with Gα proteins using immobilized specific antibodies against Gαs, Gαi, Gαo, Gαq in membrane preparations from dorsal horn ipsilateral or contralateral to nerve injury in rats treated with oxycodone (OXY), naltrexone (NTX), or OXY+NTX intrathecally. All animals underwent intrathecal catheterization, followed by a three-day recovery period.

On the fourth day, animals received a left side, L5/L6 spinal nerve ligation (ipsilateral) except for those indicated by “Sham” surgery as the control. After a 7-day recovery period, animals received twice daily injections (i.t.) of vehicle, oxycodone (OXY), naltrexone (NTX), or OXY+NTX, for seven days. On the eighth day of injections, animals received one i.t. bolus of the respective compound and were sacrificed 30 minutes post administration. Lumbar spinal cord was harvested and quartered into the ipsilateral/dorsal, ipsilateral/ventral, contralateral/dorsal, and contralateral/ventral sections. Tissue was fast-frozen in liquid nitrogen within 1 minute of removal.

Membranes prepared from these tissues were incubated with either vehicle or 1 μM morphine, solubilized and immunoprecipitated with immobilized anti-Gα antibodies.

The MOR content in the anti-Gα immunoprecipitates were then analyzed by Western blotting. Experiments were was performed using lumbar spinal cord tissue either ipsilateral to the side of injury or contralateral as a comparison to the injured side after 8 days of compound or vehicle treatment and 15 days after the nerve injury was induced.

(B) “MOR immunoreactivity from rats with lumbar spinal nerve injury or sham- 67

operation”: Densitometric quantification of Western blots showing in vitro morphine- stimulated coupling in Figure 1, A. ‡p < 0.01 when compared to vehicle-treated, sham- operated animals # p < 0.01 compared to the contralateral side; *p < 0.05, **p < 0.01 when compared to respective SNL-operated, vehicle-treated group; + p < 0.05 compared to respective oxycodone-treated group.

68

3.4 ULD NTX Enhances the Effect of Intrathecal Oxycodone in SNL- animals

Rats with SNL were treated with saline, oxycodone (10 μg), NTX (0.33 ng) or oxycodone plus NTX (0.033, 0.1, or 0.33 ng) twice daily for 7 days. Tactile and thermal hypersensitivities were evaluated 20 min after the morning injections, and some animals were tested an additional 4 days after cessation of treatment.

Prior to injury, all animals had an average paw withdrawal latency of 21.3 0.4 s in response to a noxious, thermal stimulus, and SNL reduced this value to 11.9 0.3 s, indicating thermal hypersensitivity. The peak effect of oxycodone (10.0 µg) over an 11- day administration period occurred on day 2: a mean withdrawal latency of 19.0 ± 1.2 s

(Figure 2A). Animals given the combination of oxycodone (10.0 µg) and NTX (0.33 ng) exhibited prolonged paw withdrawal latencies of 22.6 ± 1.2 s, 22.6 ± 1.6 s, and 23.2 ± 1.8 s on days 3, 4, and 5, respectively. While the peak antihyperalgesic effect occurred on these three days, the reversal of thermal hypersensitivity continued through day 11 of testing (Figure 2A). Vehicle control animals showed no significant antihyperalgesia on any testing day, with a mean withdrawal of 13.8 0.9 s. Animals treated with NTX (0.33 ng) did not withdraw the injured paw at latencies significantly different from postSNL baseline values or those of vehicle treated animals. Additional testing on days 9-11 was not conducted for vehicle or on days 8-11 for NTX animals, as withdrawal latencies of these groups were not significantly different from postSNL paw withdrawal baseline latencies. 69

To quantify the antihypersensitivity observed, percent responses were calculated for each group (Figure 2B). The vehicle control group and the NTX-only group did not demonstrate significant responses throughout the 11 days of testing. The greatest reversal of thermal hypersensitivity for animals receiving oxycodone alone occurred on day 2 (60.8 ± 7.0%) with the rapid development of analgesic tolerance thereafter. The co-administration of ultra-low-dose NTX had no significant effect on oxycodone antihyperalgesia on day 2. However, animals co-treated with oxycodone plus NTX showed a peak, reversal of thermal hypersensitivity on day 4 (76.4 ± 7.9 %), with and continued antihyperalgesia through 7 days of treatment and 4 additional days of testing

(Figure 2B). Preliminary data collected for optimization of NTX dosage given in conjunction with oxycodone was used to analyze a possible dose-dependent effect. A dose-response curve for intrathecally administered NTX combined with 10 μg oxycodone

(i.t.) was plotted from percent activity on day 4 for antihyperalgesia (Figure 2C). The A50 was 0.020 ng NTX. The 95% confidence interval was determined to be 0.001 ng to 0.160 ng NTX with an R-squared value of 0.99, indicating ultra-low dose naltrexone acts in a dose-dependent manner to attenuate SNL-induced thermal hyperalgesia.

To confirm the ultra-low-dose-dependency of NTX enhancement of oxycodone (i.t.) a greater than 1000-fold higher dose of NTX (1.0 μg) was co-administered with 10 μg oxycodone following the same testing paradigm as above (in 5 μL). This higher dose of

NTX acted to antagonize the opioid Gαi, significantly reversing opioid antinociceptive behavior in the SNL animals on all testing days compared to oxycodone alone (p < 0.01). 70

Shown in Figure 2D is the complete blockade of the peak effect of oxycodone (i.t.) on day 2.

Tactile allodynia was assessed using calibrated von Frey filaments. Pre-injury baseline thresholds averaged 14.5 0.2 g. After the nerve ligation, tactile thresholds fell to 3.0

0.3 g. Paw withdrawal thresholds measured post-SNL and over the 7-day testing period did not vary significantly. The peak response of oxycodone-treated animals was a threshold of 4.2 ± 0.5 g on day 1 of testing (Figure 3A). Animals receiving oxycodone +

NTX showed a statistically significant reversal of SNL- induced tactile allodynia compared to oxycodone treatment alone on days 1, 2, and 4 (7.4 ± 1.4 g, 6.5 ± 1.0 g, and

6.3 ± 1.1 g, respectively; Figure 3A). The percent reversal of allodynia seen throughout the testing period was below 20% for all SNL control groups (oxycodone, NTX, and vehicle; Figure 3B). On day 1, animals given oxycodone + NTX showed a 41.2 ± 9.3% return to the pre-injury threshold; on days 2 and 4, the combination therapy produced

30.8 ± 6.9% and 33.6 ± 7.6% anti-allodynia, respectively (Figure 3B). Compared to the oxycodone alone group, these percent activities of the combination therapy were significantly different (p < 0.05). Although the addition of ultra-low dose NTX to oxycodone alleviated thermal hyperalgesia in a dose-dependent manner, SNL-induced tactile allodynia was not affected by the combination. Furthermore, the > 1000 fold dose of NTX (1 μg) had no effect on SNL-induced tactile allodynia.

2A 2B 30 Vehicle (saline) 100 Vehicle (saline) OXY (10ug/5uL) * OXY (10ug/5uL) NTX (0.33ng/5uL) * * * NTX (0.33ng/5uL) 25 80 * OXY + NTX SEM) * * OXY + NTX 20 60

SEM) 15 40

(s 10 20

5 Paw Withdrawal Paw Latency %Antihyperalgesia ( 0 0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 11 12 10 11 12

preSNL Time (days) Time (days) postSNL 2C 2D

100

30 80 SEM) 25

60 20

40 SEM) 15 *

(s 10 20

% Antihyperalgesia ( Antihyperalgesia % 5

0 Latency Withdrawal Paw 0.033 0.1 0.33 0 Dose of NTX (ng/ 5 L, i.t.)

NTX + NTX preSNL postSNL oxycodone

71

72

Figure 2 Antihyperalgesic Effect of Spinal Oxycodone + ULD NTX in SNL- animals

(A) Twice daily i.t. injections of oxycodone (OXY, 10 µg/5 L) + naltrexone (NTX, 0.33 ng/5 L), n = 16, produced a partial and transient antihyperalgesic effect in the paw withdrawal latency compared to a much more subtle effect of OXY (10 µg/5 L, n = 14) alone in L5/L6 SNL rats. Animals treated with vehicle (n = 6) or NTX alone (n = 12) did not vary significantly from post-injury baseline values. Days 1-11 correspond to the days of drug treatment and are equivalent to Days 7-18 after the SNL. (B) Calculated

Antihyperalgesic activity of twice daily i.t. injections of oxycodone (OXY, 10 µg/5 L) + naltrexone (NTX, 0.33 ng/5 L) when compared to a much more subtle effect of OXY

(10 µg/5 L) alone in L5/L6 SNL rats. This effect was statistically significant on days 3-5 and 8-11 (* p < 0.05, ANOVA) compared to animals treated with oxycodone alone. Both oxycodone alone and combination therapy groups were significantly different from vehicle treated animals, p < 0.05. Animals treated with vehicle or NTX did not show any significant variance in calculated percent activity over the testing period, or between each other. (C) The calculated percent activity on day 4 of intrathecally injected oxycodone

(OXY) + ultra-low-dose naltrexone (NTX) showed a dose-dependent reversal of thermal hypersensitivity in nerve-injured animals. The A50 value for the dose of NTX to be used with 10 µg/5 L oxycodone was calculated to be 0.024 ng/5 L (95% confidence interval of 0.007-0.086 ng/5 L; p = 0.05, one-way ANOVA, FlashCalc) (D) A 1000-fold higher 73

dose of NTX (1.0 μg/ 5 μL) significantly blocks oxycodone mediated antihyperalgesia (p

< 0.01). Data is shown after day 2 of co-administration.

3A 3B

15 Vehicle (saline) 100 Vehicle (saline) OXY (10 g/5 L) OXY (10 g/ 5 L) NTX (0.33 ng/5 L) NTX (0.33 ng/ 5 L) 12 80

OXY + NTX OXY + NTX SEM)

9 60 * SEM) * 6 40 * (g * 20

3

% Antiallodynia ( Antiallodynia % Paw Withdrawal Threshold Withdrawal Paw 0 0 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 10 11 10 11

preSNL postSNL Time (days) Time (days)

74

75

Figure 3 Antiallodynic Effect of Spinal Oxycodone in SNL- animals (A) Twice daily i.th. injections of oxycodone (OXY, 10 µg/5 L) + naltrexone (NTX, 0.33 ng/5 L) produced a partial reversal of mechanical allodynia using von Frey filaments compared to a much more subtle effect of OXY (10 µg/5 L) alone in L5/L6 SNL rats. The former was determined to be significantly different than animals treated with oxycodone alone.

Animals treated with vehicle or NTX did not vary from pos-injury baseline thresholds over the testing period. (B) Calculated antiallodynic activity of twice daily i.t. injections of oxycodone (OXY, 10 µg/5 L) + naltrexone (NTX, 0.33 ng/5 L) compared to a much more subtle effect of OXY (10 µg/5 L) alone in L5/L6 SNL rats. This effect was statistically significant on days 1, 2, 4, and 5 (* p < 0.05, ANOVA) when compared to animals that had received oxycodone treatment alone. Treatment with oxycodone or the combination of oxycodone + ultra-low-dose naltrexone showed significant attenuation of

SNL-induced allodynia when compared to vehicle treat animals, p < 0.05. Vehicle and

NTX alone treated animals did not show significant antiallodynia across the testing period. 76

3.5 ULD NTX Enhances the Effects of Oral Oxycodone in SNL-animals

As oxycodone is frequently prescribed by clinicians to be taken orally, the effects of ultra-low-dose NTX on oxycodone were also assessed after oral gavage in the SNL- model of neuropathic pain. Similar to the intrathecal treatment protocol, all compounds were given twice daily with behavioral testing occurring 20 - 30 min after the morning gavage. To determine the most effective oxycodone/ NTX ratio, studies were conducted using oxycodone (3 mg/ kg or 10 mg/ kg) and NTX (0.3, 1.0 or 3.0 μg/ kg).

To determine the dose of oxycodone most illustrative of clinical efficacy, two doses were selected for prescreening. Both 3 mg/ kg and 10 mg/ kg oxycodone (p.o.) resulted in significant antihyperalgesia across the 7 day time course (Figure 4A) against a thermal stimulus. Paradoxically, oxycodone (3 mg/ kg) resulted in great attenuation of thermal hypersensitivity in the SNL animals for a longer duration. To assess the effects of ultra- low-dose NTX, oxycodone (10 mg/ kg) was chosen as the data more closely paralleled clinical practices (Figure 4A).

A dose response curve for NTX combined with 10 mg/kg oxycodone was calculated from percent activity of the oral combinations for antihyperalgesia on day 4. The A50 was determined to be 0.050 µg/ kg NTX (95% C.I. = 0.009 to 2.88 µg/ kg NTX). The R- squared value was 0.998 indicating that oral, ultra-low-dose NTX with 10 mg/ kg oxycodone reverses SNL-induced thermal hyperalgesia in a dose-dependent manner

(Figure 4B).

Thermal sensitivity following oral oxycodone with or without NTX was assessed by latency to withdraw the paw from infrared heat (Figure 4). The mean pre-injury paw 77

withdrawal latency was 21.7 0.6 s, while the mean post-surgery baseline was 11.5 0.7 s in SNL animals. Oxycodone alone (10 mg/ kg) produced a mean withdrawal latency of

20.7 ± 2.3 s on day 1, representing analgesia; however, by day 4 this latency had shortened to 15.5 ± 2.9 s, suggesting the development antinociceptive tolerance (Figure

4C). Oxycodone treatment on days 5 through 11 resulted in a mean withdrawal latency not significantly different from the post-surgery baseline. In contrast, animals receiving the combination gavage of oxycodone (10 mg/ kg) + NTX (3 µg/ kg) showed prolonged paw withdrawal latency, peaking on days 2 and 3 (27.4 ± 2.3 s and 28.9 ± 1.9 s, respectively). Only on the last day of the 7-day testing period did the latency of this group approach the post-surgery baseline with a paw withdrawal latency of 12.6 2.5 s.

Paw withdrawal latencies of animals treated with only NTX (3.0 µg/ kg), as well as those of vehicle-treated animals, did not significantly vary from the post-injury baseline across the 7-day period.

To quantify the antihypersensitivity, percent responses were calculated for each group

(Figure 4D). Oxycodone alone reversed the SNL-induced thermal hypersensitivity by

72.3 ± 12.4%, 52.6 ± 14.1%, and 64.5 ± 13.2% on days 1, 2, and 3, respectively.

Animals co-treated with oxycodone + NTX showed a 100% reversal of injury-induced hyperalgesia on day 2 of treatment and significant reversal of the ligation-induced hyperalgesia continued through day 6, with mean percentages ranging from 98.9 ± 1.4% to 71.7 ± 14.7% (Figure 4D). Neither NTX-treated animals nor vehicle-treated animals showed any significant reversal of thermal hypersensitivity throughout the 7-day time course (< 20% attenuation). 78

The effect of oral oxycodone on SNL-induced tactile hypersensitivity was evaluated in parallel with the thermal hypersensitivity studies. The EtOH:dH2O vehicle did not significantly alter paw withdrawal thresholds at any point in the experiments (Figure 5A).

Oxycodone (3 mg/ kg) did not result in attenuation of tactile allodynia as measured by von Frey. However, oxycodone (10 mg/ kg) did significantly reverse SNL-induced tactile hypersensitivity after the first and second day of dosing (Figure 5A).

A dose-response curve for the antiallodynic activity of ultra-low-dose NTX on oral oxycodone was determined from the percent activity on day 1. The A50 value was 1.0

µg/kg (95% C.I. = 0.67 to 1.49 µg/ kg NTX), and the R-squared value was 0.994 (Figure

5B). It should be noted that the time of peak effect for alleviating tactile and thermal hypersensitivities was not the same and may be reflected in the slope of the dose- response curve.

All animals had mean, pre-injury paw withdrawal threshold of 13.2 0.7 g that dropped to 1.8 0.2 g after nerve ligation. Oxycodone (10 mg/ kg)-treated control animals showed a trend toward pre-injury thresholds on days 1 - 3 (10.7 ± 1.5 g, 8.7 ± 2.0 g, and 6.8 ± 1.8 g, respectively). By day 4 through the end of experiment, the paw withdrawal thresholds in animals treated with of oxycodone alone were not significantly different from post-injury levels (Figure 5C). Animals receiving oxycodone + NTX responded to von Frey probing at thresholds near pre-injury values on days 1, 2, and 3 with the peak effect of 15.0 ± 0.0 g (day 3, Figure 5C). These values were significantly different from post-SNL baseline paw withdrawal thresholds and the same day, paw withdrawal thresholds of animals treated with oxycodone alone, indicating dual therapy 79

evoked an antiallodynic response. By day 6, the withdrawal thresholds of this group had nearly returned to post-injury levels (2.7 ± 0.8 g). No significant effect was observed in either vehicle or NTX-alone groups over the 7-day time course in the nerve-injured animals.

Percent activity was also used to quantify antiallodynia (Figure 5D). Oxycodone alone produced a 68.1 ± 10.9% antiallodynic effect on day 1 that declined to 52.8 ± 14.4% and

40.1 ± 12.8% on days 2 and 3. Animals exhibited less than 20% antiallodynia by day 4.

Oxycodone + NTX completely reversed injury-induced allodynia on day 3 (100.0 ±

0.0%), with percent activities of 89.5 ± 11.5% and 86.1 ± 15.5% on days 1 and 2, respectively. The antiallodynia of the combination treatment declined to 66.8 ± 10.3% on day 4 and was near 20% for the final three days of the experiment. NTX and vehicle- treated animals did not exhibit any significant antiallodynic activity over the testing period

4A 4B 100

120 Vehicle (EtOH: dH2O) Oxy (3 mg/kg)

SEM) 80 100 Oxy (10 mg/kg)

80 60

60

SEM) 40 ( 40

20 20

%Antihyperalgesia % Antihyperalgesia ( %Antihyperalgesia 0 0 1 2 3 4 5 6 7 1 3 .03 0.3 Time (days) Dose of NTX ( g/kg, p.o.)

4C 4D

Vehicle 35 Vehicle (EtOH:dH O) 120 2 OXY (10 mg/kg) OXY (10 mg/kg) 30 100 * * NTX (3 g/kg) NTX (3 g/kg) SEM) * OXY (10mg/kg)

25 OXY (10mg/kg) ( * 80 + NTX (3 g/kg) + NTX (3 g/kg) 20

60 sem) 15

(s 40 10 20

5

Antihyperalgesia Paw Withdrawawl Latency Withdrawawl Paw

% 0 0 1 2 3 4 5 6 7 1 2 3 4 5 6 7

preSNL postSNL Time (days) Time (days)

. 80

81

Figure 4 Antihyperalgesic Effect of Oral Oxycodone + ULD NTX (A) Dose comparison of oral oxycodone: 3 mg/kg versus 10 mg/kg. The higher dose of oxycodone resulted in a significant loss of antihyperalgesic activity compared to the 3 mg/kg dose by day 2 of treatment which remained until the end of the experiment (p < 0.05, ANOVA).

(B) The calculated percent activity on day 4 of orally administered oxycodone (OXY) + ultra-low-dose naltrexone (NTX) showed a dose dependent reversal of thermal hypersensitivity in nerve-injured animals. The A50 value for the dose of NTX to be used with 10mg/kg oxycodone was calculated to be 0.05 μg/kg (95% confidence interval

0.009-0.288 μg/kg, p < 0.02, one-way ANOVA, FlashCalc). (C) Antihyperalgesic effects of oral oxycodone with or without NTX at various doses. The oxycodone (OXY) group alone (n = 12) received 30 mg/kg on day 1, and 10 mg/kg on days 2-7; all OXY + NTX,

OxytrexTM, groups (n = 6) received oxycodone at 10 mg/kg. Statistically significant differences between oxycodone treated animals and OxytrexTM-treated animals were observed on days 2-4 (* p < 0.05, ANOVA, FlashCalc) in the thermal hypersensitivity behavioral test. Vehicle (n = 6) and NTX (n = 6) controls showed no variance from post-

SNL baseline paw withdrawal latencies. (D) “Antihyperalgesic effects of oral oxycodone with or without NTX at various doses. The OXY group alone received 30 mg/kg on day

1, and 10 mg/kg on days 2 - 7; all OXY + NTX groups received oxycodone at 10 mg/kg.

Animals receiving OxytrexTM showed complete reversal of thermal hypersensitivity

(shown as 100% activity) on days 2 and 3. Oxycodone treated animals displayed a maximal reversal of thermal hypersensitivity on days 1-3, with a calculated percent activity of 80%. The calculated percent of antihypersensitivity for noxious stimulation 82

was determined to be statistically different between the two groups (OxytrexTM and oxycodone) on days 2-4 and 6 (* p < 0.05, ANOVA, FlashCalc). Vehicle and NTX control animals did not respond throughout the testing period.

5A 5B

100 120 Vehicle (EtOH: dH2O) Oxy (3 mg/kg)

100 Oxy (10 mg/kg) 80 SEM) 80 * 60 60 *

SEM) 40 ( 40 20

20

%Antihyperalgesia % Antiallodynia ( %Antiallodynia 0 0

1 2 3 4 5 6 7 1 3 0.3 Time (days) Dose of NTX ( g/kg, p.o.) 5C 5D

16 Vehicle (EtOH:dH O) 2 120 Vehicle (EtOH:dH O) OXY (10 mg/kg) 2 OXY (10 mg/kg) NTX(3 g/kg) 100 * * 12 OXY (10 mg/kg) NTX (3 g/kg) + NTX (3 g/kg) SEM) ( 80 * OXY (10 mg/kg) + NTX (3 g/kg) 8 SEM) 60

(g 40 4

20

Antiallodynia %

Paw Withdrawawl Threshold Withdrawawl Paw 0 0

1 2 3 4 5 6 7 1 2 3 4 5 6 7

preSNL Time (days) Time (days) postSNL 83

84

Figure 5 Antiallodynic effect of Oral Oxycodone + ULD NTX (A) Dose comparison of oral oxycodone: 3mg/kg versus 10 mg/kg. The lower dose of oxycodone did not affect

SNL induced allodynia at any point over the treatment time-course. Oxycodone (10 mg/kg) did significantly attenuate SNL-tactile hypersensitivity on days 1 and 2 of treatment but lost efficacy over the full treatment paradigm (p < 0.05). (B) The calculated percent activity on day 1 of orally administered oxycodone (OXY) + ultra-low-dose naltrexone (NTX) showed a dose dependent reversal of mechanical hypersensitivity in nerve-injured animals. The A50 value for the dose of naltrexone to be used with 10mg/kg oxycodone was calculated to be 0.999 g/kg (95% confidence interval 0.669-1.490

g/kg; p<0.02, one-way ANOVA, FlashCalc). (C) Antiallodynic effects of oral oxycodone with or without NTX at various doses. The oxycodone (OXY) group alone received 30 mg/kg on day 1, and 10 mg/kg on days 2-7; all OXY + NTX groups received oxycodone at 10 mg/kg. Statistically significant differences between oxycodone treated animals and OxytrexTM-treated animals were observed on days 3 and 4 (p<0.05, Student‟s t-test, FlashCalc) in the mechanical hypersensitivity behavioral test. The peak effect of

OxytrexTM was observed on day 3. Vehicle and NTX controls showed no variance from post-SNL baseline paw withdrawal thresholds. (D) Calculated Antiallodynic effects of oral oxycodone with or without NTX at various doses. The OXY group alone received

30 mg/kg on day 1, and 10 mg/kg on days 2-7; all OXY + NTX groups received oxycodone at 10 mg/kg. Animals receiving OxytrexTM showed complete reversal of mechanical hypersensitivity (shown as 100% antiallodynia) on day 3. Oxycodone treated 85

animals displayed a maximal reversal of thermal hypersensitivity on day1, with a calculated percent activity of 60%. The calculated percent of antiallodynia for non- noxious stimulation was determined to be statistically different between the two groups

(OxytrexTM and oxycodone) on days 1, 3, and 4 (*p < 0.05, Student‟s t-test, FlashCalc).

Vehicle and NTX- treated animals showed no difference in paw withdrawal thresholds across the seven day testing period. 86

CHAPTER 4:

MODULATION OF PAIN WITH MULTIFUNCTIONAL LIGANDS

4.1 Introduction

In addition to altered G-protein coupling, sustained opiate administration activates mechanisms that promote enhanced pain, in part through activation of the NK1 receptor.

The SP-NK1 system has been implicated in opioid induced antinociceptive tolerance, reward, physical dependence, and nausea/vomiting. We hypothesized that opiate mu/ delta agonists with NK1 receptor antagonist actions would produce effective pain relief without demonstration of opiate-induced hyperalgesia and associated antinociceptive tolerance. Studies herein describe the development of such compounds and the resulting efficacy in acute and chronic pain models.

4.2 Selection of Opioid Agonist/ NK1 Antagonist Peptides for in vivo Screening

First, designed compounds were characterized in vitro, where upon lead peptides were chosen for further in vivo screening in rodent models of pain. Peptides were designed to have an N-terminal structure derived from known opioid peptides, specifically, deltorphins (Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2; Tyr-D-Ala-Phe-Glu-Val-Val-Gly-

NH2) or biphalin, (Tyr-D-Ala-Gly-Phe-NH)2, (Erspamer et al., 1989; Kreil et al., 1989;

Silbert et al., 1991). These sequences were then linked to a C-terminal sequence related to known NK1 receptor antagonists (e.g. spantide 1 or L-732, 138). Radioligand binding 87

as described in chapter 2 was performed with additional binding studies to determine the binding affinity of each peptide to the mu and delta-opioid receptors, as well as the rat

NK1 receptors (Table 1). No binding to the kappa opioid receptors or the NK2 and NK3 receptors was observed in the compounds designed.

Once affinity for the opioid receptors and the NK1 receptor was determined, activities were assessed by measuring the in vitro effects in the GPI and MVD biological assays.

Data are shown in Table 1 for compounds selected for in vivo evaluation. Criteria set for potential candidates included nanomolar (nM) binding affinity to each receptor as well as in vitro activity in the nM range.

Table 1: Selected Opioid Agonist/ NK1 Antagonist for in vivo Screening: In vitro Affinity and Activity Affinity GTP binding MVD GPI/LMMP hDO hDOR rMOR opioid(delta) opioid(mu) SP ID rMOR rNK1 Peptide R agonist agonist agonist (Ki; (Ki; EC E EC E antagonist (Ki; 50 max 50 max (IC ; antagonist (IC ; antagonist (IC ; nM) nM) (nM) (%) (nM) (%) 50 50 50 (Ke; nM) nM) nM) nM) nM) 2.6 ± 1.4 ± 27.0 ± Biphalin (TyrDAlaGlyPheNH) - 8.8 ± 0.3 2 0.4 0.2 1.5 15 fold 285.4 ± 0% at 1 JSOH9 TyrDAlaDPhePheDTrpLeuMetNH 0.7 606 2940 shift at 10 none 280 ± 88 2 46.2 μM μM 482.5 ± 608.7 ± JSOH11 TyrDAlaDTrpPheDTrpLeuLeuNH 16.5 164 7320 173 79 149 55 2 37.4 118.4 35.0 16.0 26.4 50.4 143 ± 399.0 ± 518.7 ± 3.64 ± TY001 TyrDAlaGlyPheProLeuTrpOBn(CF ) 180 1.9 ± ± ± none 3 2 ± 0.8 31.5 23.4 40.0 1.08 34.5 0.7 5.0 42.5 44.5 5.0 ± 23.3 ± 9.6 ± 39.8 ± 101.2 ± 340.7 ± 19.43 ± TY004 TyrDAlaGlyPheLeuProLeuTrpOBn(CF ) 0.8 ± ± none 3 2 1.5 5.0 6.8 20.4 25.3 71.2 4.96 1.8 1.5 47.6 45.6 2.8 ± 36.3 ± 2.9 ± 31.6 ± 22.3 ± 358.8 ± TY005 TyrDAlaGlyPheMetProLeuTrpOBn(CF ) 0.3 ± ± none 24.7 ± 8.8 3 2 1.1 11.3 1.1 3.4 1.2 126.7 4.5 1.9 61.1 ± TY025 TyrDAlaGlyPheMetProLeuTrpNHBn 0.4 1.8 695.7 2.6 51.7 20.7 46.8 4.8 ± 0.3 none 9.9 ± 2.8 9.6 none at 18.6 ± TY032 DmtDAlaGlyPheMetProLeuTrpNHBn(CF ) 0.1 2.0 2.3 1.2 24 62 74 1.8 ± 0.6 100 7.5 ± 0.5 3 2 4.5 nM TY036 MetProLeuTrpOBn(CF3)2 1280 4550 1.0 [C-terminal fragment for TY001, TY004,

TY005] TY040 TyrDAlaGlyPheMetNH2 0.7 0.5 1096 0.9 7 6.3 14 [N-terminal fragemnt of TY005, TY025,

TY027] TY043 TyrDAlaGlyPheMetProLeuTrpOH 1.1 14.2 0.00 0.5 62 4.6 36 [possible metabolite of TY005]

88

89

4.3 Opioid Agonist/ NK1 Antagonist Peptides in Acute Pain

Efficacy in acute studies was established in a rodent model of nociception as measured by paw withdrawal latency to a normally painful stimulus. The observed data was then converted into percent antinociception for analysis. For some compounds, efficacy in inflammatory models was evaluated in comparison to a paw flick assay.

JSOH11: The first compound evaluated JSOH11 had moderate action at the mu and delta opioid receptors in vitro as an agonist, as well as moderate action as an antagonist of SP.

JSOH11 was derived from the structures of deltorphin and spantide 1. Preliminary data from the formalin and carrageenan tests confirmed that the multifunctional design did produce some antinociception after intrathecal administration (Figure 6A and B).

Formalin (2% i.pl.) was injected 10 min after vehicle or JSOH11 (i.t.). Animals receiving

JSOH11 (10 μg) showed a decrease in total flinching in the second phase of formalin when compared to control animals (1:3 EtOH: dH2O). This indicated some in vivo efficacy. Carrageenan (i.pl.) was injected to quantify the amount of antihyperalgesia induced by JSOH11. The anti-hyperalgesic effect of JSOH11 (10 μg) was determined to be approximately 30% anti-hyperalgesia at 15 min after i.t. injection compared to post- carrageenan values; the effect was completely dissipated 45 min after dosing (Figure 6B).

Together, these data confirmed that multifunctional peptide linking opioid receptor agonists and an NK1 receptor antagonist has potential for in vivo activity. When the dose of JSOH11 was increased to 30µg (in 5 μL, i.t.) in both of these assays, approximately half of the animals under observation died within 10 min of the intrathecal injection (4 of

11). The remaining 7 were heavily sedated, had impaired respiration and showed an 90

increase in the amount of fur shed. The conclusions from studies utilizing JSOH11 influenced synthesis of the second set of opioid agonists/ NK1 antagonists as evidenced by modified structures and activity ratios.

91

6A

150

SEM) Vehicle (1:3 EtOH: dH2O) 125 JSOH11 (10 g) 100

75

50

25

0

# Flinches per phase ( phase per Flinches # 1 2

Formalin Phase

6B

100

SEM) 80

60 Vehicle (1:3 EtOH: dH2O) JSOH11 (10 g) 40

20

%Anti-Hyperalgesia ( %Anti-Hyperalgesia 0 15 30 45 60 75 Time (min)

92

Figure 6 Antinociceptive effects of JSOH11 (A) JSOH11 (10 μg) blocked the second phase of 2% formalin induced flinching compared to vehicle treated animals (p < 0.05).

(B) JSOH11 attenuated carrageenan (i.pl.) induced thermal hypersensitivity by 30%.

Compared to vehicle treatment, this was not significantly. Higher doses of JSOH11 induced toxicity.

93

TY- Compounds: The TY series of compounds were rationally designed derivatives of biphalin and L-732,138 and followed a similar design scaffold as previously mentioned

(N-terminal with opioid activity + linker + C-terminal with NK1 receptor affinity). Over

56 compounds have been synthesized to date, however only a select few have been evaluated in vivo. Within this series of compounds, the first of these compounds to be screened in vivo was TY004. TY004 was selected as an in vivo candidate because of the ratio of affinities and activities at the mu- and delta- receptors specifically. Both doses of

TY004 evaluated, 3 and 10 μg, resulted in attenuated responses to normally noxious stimulus (Figure 7A). However, the in vivo effects observed with TY004 dissipated 45 min after dosing, with a trend toward hypersensitivity emerging by 60 min with paw withdrawal latencies of 14.18 ± 2.19s and 4.16 ± 2.76 s (3 μg and 10μg, respectively).

When TY004 was directly compared at equipotent doses to TY005 this hypersensitivity at the later time-point was negligible, while efficacy was maintained (Figure 7B). It should be noted that the duration of action of TY005 was longer than TY004. These results led to the selection of TY005 as the lead compound for full in vivo characterization.

94

7A 35 vehicle TY004 (3 g) 30 TY004 (10 g)

25

20 SEM)

15 (s 10

5 Paw Withdrawal Latency Withdrawal Paw 0 BL 15 30 45 60 Time (min) 7B

35 vehicle TY004 (10 g) 30 TY005 (10 g) 25

20 SEM) 15

(s 10 5

Paw Withdrawal Latency Withdrawal Paw 0 BL 15 30 45 60 Time (min)

95

Figure 7 In vivo effects of TY004 (A) TY004 (i.t.) results in significant antinociception at two doses 15 min following administration (p < 0.05, ANOVA). (B) TY004 versus

TY005: By 60 min after spinal administration, TY004 showed a trend toward the development of acute hypersensitivity. The derivative, TY005, did not result in hypersensitivity and at an equipotent dose maintained efficacy with an increased duration of action. 96

TY005: Structural modification of TY004 at position 5 changed a leucine (Leu) to a methionine (Met), creating TY005. This modification altered binding affinities at opioid and NK1 receptors modestly, but resulted in a 10 fold higher activity at the delta opioid receptor compared to precursors (Table 1). Additionally, these changes in vivo allowed for further study of the effects of the opioid agonist/ NK1 antagonist construct.

TY005 was evaluated after central administration for the ability to produce antinociception dose dependently. As described above, animals were implanted with an indwelling i.t. cannula and baseline paw withdrawal latencies to a noxious thermal stimulus were calibrated to fall between 20.0 - 25.0 s; the mean value for all animals was found to be 21.5 ± 0.5 s (n = 48). TY005 (1, 3, 10 or 30 μg in 5μL) or vehicle (10%

DMSO in MilliPore water, 5μL) was injected and paw withdrawal latencies recorded every 15 min for 60 min (Figure 8A). Maximal effect of TY005 occurred 15 min after the i.t. injection. The highest dose (30 μg) resulted in a maximal paw withdrawal latency of 32.0 ± 1.0 s. TY005 resulted in paw withdrawal latencies of 32.3 ± 0.7 s and 27.4 ±

1.6 s at doses of 10 and 3 μg respectively at the same time point. Each of these doses resulted in significantly higher paw withdrawal latencies when compared to baseline values or vehicle treatment alone (p < 0.01), but did not differ from each other. TY005 (1

μg) did not significantly alter paw withdrawal latency compared to baseline measurements in non-injured rats (Figure 8A).

Percent antinociception was calculated for each dose of TY005 and a DRC generated

(Figure 8B). TY005 (3, 10, 30 μg) produced significant antinociception in non-injured animals compared to vehicle treatment 15 min after i.t. injection (51.0 ± 13.0 %, 94.9 ± 97

5.0 % and 90.0 ± 9.9 %, respectively). Although the paw withdrawal latency after dosing with 1 μg TY005 was not statistically different from baseline or vehicle treated values, the calculated percent activity was statistically higher than the vehicle treated group (p =

0.02) fifteen min after injection (i.t.). The A50 dose to produce antinociception in response to a thermal stimulus was determined to be 2.3 μg (95% C.I.: 1.4- 3.7 μg; Figure

8B).

Motor skills and antinociceptive effects: To confirm the results in the acute pain studies,

TY005 (i.t.) was evaluated for potential motor impairment/sedation using the rotarod motor skills assay. Animals were trained to walk on a rotating rod for up to 180.0 s prior to compound or vehicle exposure with a mean baseline latency of 168.7 ± 5.8 s (n=17).

Animals were separated into groups of 6 and treated with 10% DMSO vehicle or TY005.

Vehicle-treated animals remained on the rotarod between 158.3 -180.0 s throughout the time course. The highest dose of TY005 used in the antinociception assay (30 μg) resulted in rotarod latencies between 160.2-180.0 s, which were not significantly different from baseline values. A 3-fold higher dose (100 μg) did not result in a significant decrease in latency (142.4 - 180.0 s, p = 0.07; Figure 8C) as has been reported for controls such as KOR agonists, some MOR agonist, and channel blockers (Vanderah et al., 2008; Valenzalo et al., 2005; Boyce et al., 1999).

8A 8B 100 35 ** **

30 ** 80 SEM)

25 ( 60

20 SEM) 15 (s 40 10% DMSO 10 1 g/5 l

Paw Withdrawal Latency PawWithdrawal 3 g/5 l

Antinociception 20

5 10 g/5 l % 30 g/5 l 0 0 BL 15 30 45 60 1 ug 3 ug 10 ug 30 ug Time (min; TY005, i.t.) 8C Dose of TY005 (i.t., in 5 L) 180

120

SEM) (s 60

10% DMSO Rotarod Latency Rotarod 30 g/5 l 100 g/5 l 0 BL 15 30 45 60 Time (min; TY005, i.t.)

98

99

Figure 8 Spinal TY005 Produces Antinociception in a Dose-dependent Manner without motor impairment (A) Intrathecal TY005 was injected at t = 0 min to non- injured animals. Mean paw withdrawal latency responses to noxious thermal stimulus were recorded and the time of peak effect occurred 15 min after the injection for all doses tested. Significance was observed with 10 μg and 30 µg when compared to vehicle and baseline latencies (* p < 0.05, ANOVA). (B) TY005 produced antinociception in a dose- dependent manner at 15 min after i.t. injection. Data are represented as the %

Antinociception ± SEM. (C) Motor skills were assessed using the rotarod assay after i.t. administration of TY005. The total time spent walking on a rotating rod in 180.0 s time period was recorded; data are shown as Latency ± SEM). At doses 3-fold higher than required for maximal antinociception, there was no significant motor impairment observed (p > 0.05, ANOVA). 100

4.4 Receptor Activity of Multifunctional Compounds In Vivo

TY005 acts as an opioid agonist in vivo: Initially, the designed opioid fragment of

TY005, termed TY040, was synthesized and evaluated in vivo for efficacy against acute pain. In vitro, TY040 showed mu- and delta opioid receptor binding affinity and activity but none at NK1 receptors (Table 1). Baseline paw withdrawal latencies were measured to be 20.4 ± 0.5 s. TY040 (10 μg in 5 μL, i.t.) resulted in a paw withdrawal latency of 31.5

± 1.0 s 15 min after dosing, which was similar to an equipotent dose of TY005 (31.5 ±

1.6 s; Figure 9A). Additionally, the duration of action with TY040 was similar to that of

TY005. Both peptides produced responses that were statistically higher when compared to vehicle treated animals at 15 min (p < 0.01) and 30 min (p < 0.001) time-points.

The possibility that the antinociceptive effects of TY005 were mediated via actions at the opioid receptor was further evaluated in non-injured animals. The mean baseline paw withdrawal latency was 22.3 ± 0.5 s (n = 20). Either saline or the non-selective opioid antagonist, naloxone (NLX; 2 mg/ kg, s.c.) was administered 10 min prior to i.t. TY005

(30 μg) or 10% DMSO vehicle injections (5 μL). Animals were evaluated for changes in paw withdrawal latency every 15 min for the first hour after the i.t. injection, corresponding with the time of peak TY005 effect and 25 min after NLX. Neither the paw withdrawal latencies of animals receiving saline-10% DMSO nor naloxone-10%

DMSO significantly differed from baseline measurements (19.1 ± 2.5 s and 20.0 ± 1.20 s, respectively). Animals treated with saline-TY005 (30 μg) had a statistically significant and higher mean paw withdrawal latency of 31.6 ± 1.4 s than those treated with 10%

DMSO (p = 0.004). Naloxone treatment 10 min before TY005 (30 μg) resulted in a paw 101

withdrawal latency of 23.5 ± 3.1 s, significantly lower than TY005 treatment alone (p =

0.04). All data was converted to maximal percent effect at the 15 min time point (Figure

9B).

To further delineate the actions of TY005 at the opioid receptors, selective antagonists for the μ- and δ-opioid receptors were used. As TY005 did not have any κ-opioid receptor affinity in vitro, activity at this receptor was not evaluated. To determine the activity of TY005 at the μ- opioid receptor, rats were pre-treated with β-FNA (42.5 nM in

5 μL volume, i.t.) twenty-four hours prior to TY005 (30 μg in 5μL) dosing. Doses and times were chosen based on a previous publication by Perlikowska and colleagues

(2009). Pretreatment with β-FNA (24 hrs) had no significant antinociceptive effect on vehicle treated animals (5.7 ± 2.9 %; n= 10), though the antinociceptive effect of TY005 was significantly decreased to 36.1 ± 11.2 % confirming mu-opioid activity in vivo (n =

14; Figure 9C). Additionally, delta-opioid receptor activity was determined in the presence of the selective antagonist, naltrindole (NTI; 66 nM, 5 μL i.t.; Dawson-Basoa and Gintzler, 1997) given 30 min before TY005 (30 μg in 5μL) or 10% DMSO (5 μL).

In vehicle treated animals, NTI did not produce significant antinociception (5.3 ± 3.0%; n

= 10). In the presence of NTI, intrathecal TY005 resulted in 41.0 ± 11.8% antinociception that was significantly lower than saline pretreated, TY005 animals (n =

14; Figure 9C). A final set of animals (n = 10) were pre-treated with both β-FNA and

NTI. The co-administration of the selective antagonists significantly and fully blocked

TY005-mediated antinociception (14.1 ± 9.3%, p = 0.002) while not producing any significant effect in animals treated with 10% DMSO (9.5 ± 6.4%). 102

Under these conditions, the designed opioid fragment (TY040) produced maximal effect in vivo, and TY005-induced antinociception was blocked by non-selective and selective opioid receptor antagonists confirming mu- and delta- opioid agonist activity in vivo.

9A 35 Vehicle (10% DMSO) TY005 (10 g) 30 TY040 (10 g) 25

20 SEM)

15 (s 10

5 Paw Withdrawal Latency Withdrawal Paw 0 BL 15 30 45 60 Time (min) 9B 9C 100 100 * *

SEM) 80

SEM) 80

60 ** 60 ** * ** * 40 40 ** 20

20 % % Antinociception ( % Antinociception ( Antinociception % 0 0

10% DMSO + - + - Saline + - - - + - - - TY005 (30 g) - + - + -FNA - + - + - + - + NTI - - + + - - + + Saline NLX

10% DMSO TY005 (30 g) 103

104

Figure 9 TY005 acts at opioid receptors in vivo (A) The spinal administration of

TY040, the opioid fragment of TY005 produces significant antinociception in non- injured animals compared to vehicle treatment ( p < 0.05) at a similar level to TY005. (B)

The non-selective opioid receptor antagonist, naloxone (NLX; 2 mg/kg), given s.c. 10 min prior to an intrathecal injection of TY005, blocks the peak effect TY005-mediated antinociception (*p < 0.05). TY005 (i.t) or vehicle (10% DMSO) was given at t = 0 min.

Peak antinociceptive effect of TY005 occurred at t = + 15 min, as previously shown. (C)

Pre-treatment with selective μ- and δ- opioid receptor antagonists (i.t.), beta- funaltrexamine (β-FNA; t= -24 h; 42.5 nM, 5 μL) or naltrindole (NTI; t = -30 min; 66 nM, 5 μL), significantly attenuated TY005- mediated antinociception compared to saline pretreated animals. When the two selective antagonists were combined the TY005 mediated effect was fully blocked, similar to levels observed with the non-selective opioid antagonist NLX. * p < 0.05 compared to saline-10% DMSO treated controls; ** p

< 0.01 versus saline pre- treated TY005 animals. 105

TY005 acts as an NK1 antagonist in vivo: Evaluation of NK1 receptor antagonism was first investigated by using the C-terminal fragment of TY005, named TY036. In vitro studies confirmed that TY036 didn‟t bind to or activate the opioid receptors and blocked

SP mediated activity at the NK1 receptor (Table 1). TY036 (10 μg) produced a significant increase in paw withdrawal latency compared to vehicle treatment (Figure 10). 106

35 Vehicle (10% DMSO) TY005 (10 g) 30 TY036 (10 g) 25

20 SEM)

15 (s 10

5 Paw Withdrawal Latency Withdrawal Paw 0 BL 15 30 45 60 Time (min)

107

Figure 10 TY036 has activity in vivo. Spinal administration of TY036, the NK1 antagonist fragment of TY005, produced significant antinociception in non-injured animals compared to vehicle treated animals (p < 0.05, ANOVA). 108

To assess NK1 receptor antagonism in vivo, we evaluated the effects of TY005 against

SP–induced scratching and biting. Non-injured animals do not normally exhibit nocifensive behaviors in the absence of SP. However, exogenous spinal administration of SP produces robust flinching and biting behaviors (Hylden and Wilcox, 1982; Seybold et al., 1982). In this study, animals were pretreated at t = - 15 min with either saline (1 mL/ kg) or NLX (2 mg/ kg, s.c.) and 10% DMSO (5 μL) or TY005 (3, 10, or 30 μg in

5μL, i.t.; t = -10 min). All animals received 1 mM SP (10 μL, i.t.) at t= 0 min, and flinching, biting and/ or scratching of the hind paws was prominent. Animals (n = 27) pre-treated with both saline and 10% DMSO flinched a mean total of 25.0 ± 4.2 times after SP (i.t.). When TY005 (30 μg) was given instead of 10% DMSO, that value fell significantly to 11.2 ± 3.6 flinches (Table 2). Since TY005 contains an opioid agonist pharmacophore, naloxone (2 mg/ kg, s.c.) was given before both TY005 and SP to isolate

NK1 receptor mediated activity. Naloxone given to vehicle-treated animals resulted in a mean of 20.1 ± 2.2 flinches, which was not significantly different than saline-vehicle treated animals (Table 2). Animals exposed to TY005 (3 μg) ten min prior to SP, in the presence of naloxone, produced 5.8 ± 1.8 flinches over the five min observation period significantly lower than the SP alone and SP + naloxone control groups (Table 2).

Increasing the TY005 dose to 10 μg resulted in a decrease in the total number of flinches when compared to rats given only SP (3.0 ± 1.0). TY005 (30 μg) reduced the flinching response after SP compared to vehicle with a mean total number of flinches equal to 2.1

± 0.9 (Table 2) over a 5 min period. Collectively, these data support activity of the C- 109

terminal fragment (TY036) at the NK1 receptor and dose-dependent antagonist activity of

TY005 at the NK1 receptor in vivo.

110

Table 2: TY005 acts as an NK1 antagonist in vivo: Effects on SP-induced Flinching p-value to p-value Treatment Mean # flinches in 5 min (±SEM) N saline- to NLX- DMSO DMSO Sa-10% DMSO-SP 25.0 (± 4.3) 27 - - a S -TY00530-SP 11.2 (± 3.6) 18 0.02* - NLXb- 10% 20.1 (± 2.2) 30 0.30 - DMSO-SP b NLX -TY0053-SP 5.8 (± 1.8) 12 0.006** 0.001 b NLX -TY00510-SP 3.0 (± 1.0) 12 0.0002** 0.001 b NLX -TY00530-SP 2.1 (± 0.9) 18 0.00005** 0.0001 aS: 0.9% saline (1 mL/ kg, s.c.) or bNLX: naloxone (2 mg/kg, s.c.) given 15-17 min before SP. 10% DMSO or TY005 (dose indicated by subscripts; μg in 5 μL; i.t.) 10 min prior to SP (10 μL of 1 mM; i.t.). P-values were determined using one-way ANOVA (FlashCalc) with Newman-Kuels post-hoc analysis, and significance was achieved if p ≤ 0.05.

111

4.5 Opioid Agonist/ NK1 Antagonist Peptidomimetics for Neuropathic Pain

Intrathecal TY005 reverses SNL-induced hypersensitivities: TY005 (1 - 30 μg in 5 μL) was given as an acute bolus i.t. to rats that had undergone L5/L6 spinal nerve ligation 7 days earlier. Following administration, behavioral measurements of tactile and thermal hypersensitivities were obtained every 15 min for the first hour. Responses were compared to pre-injury and post-injury paw withdrawal thresholds, and those of vehicle- treated and i.t. morphine-treated animals. Morphine was used for comparison of the peptide to an opioid used clinically for the treatment of neuropathic pain.

Prior to and after injury, all animals were evaluated for mechanical response to non- noxious probing of the left hind paw with calibrated von Frey filaments. The mean paw withdrawal threshold before SNL was 15.0 ± 0.0 g. Seven days after nerve ligation, the mean withdrawal threshold was 3.0 ± 0.3 g, indicating the development of tactile allodynia (n = 58). Animals were separated at random into i.t. treatment groups: 10%

DMSO vehicle, MS (10 μg), and TY005 (1 μg, 3 μg, 10 μg, and 30 μg). TY005 (30 μg) significantly attenuated SNL-induced tactile allodynia 15 and 30 min after a single i.t. injection, with the mean paw withdrawal thresholds being increased to 14.7 ± 0.3 g and

13.4 ± 0.8 g, respectively (n = 6, p = 2.9*10-13; Figure 11 A). The 10 μg dose of TY005 resulted in a similar reversal of allodynia with mean paw withdrawal thresholds of 10.1 ±

1.5 g and 6.6 ± 1.5 g, both 15 and 30 min following administration (n = 9, p = 2.9*10-7).

TY005 (3 μg) had a significant effect on SNL-induced allodynia only 15 min after peptide administration (n = 6, p = 2.1*10-5); similarly, the lowest experimental dose of 112

the experimental peptide evaluated (1 μg, n = 6) resulted in significant antiallodynia

(Figure 11A). Vehicle treatment did not result in a statistically significant reversal of nerve-injury induced allodynia at any time point after injection (n = 26, p = 0.33; Figure

11A). Intrathecal morphine administration (10 μg) did not result in significant attenuation of SNL-induced allodynia at any of the time points tested (n = 6; p = 0.83; Figure 11A).

From the paw withdrawal thresholds obtained 15 min after TY005 (i.t.), a dose response curve was generated (Figure 11B). The A50 value was calculated to be 6.1 μg with a 95% confidence interval of 4.2 - 9.0 μg (R2 value = 0.99).

In non-injured rats, noxious infrared stimulation results in a mean paw withdrawal latency of 21.6 ± 0.3 s. Seven days after peripheral nerve ligation, the same stimulus produced a mean paw withdrawal latency of 12.0 ± 0.3 s, indicating the development of thermal hyperalgesia (n = 71). Animals were separated into treatment groups at random.

TY005 (30 μg) significantly attenuated the enhanced sensitivity to a thermal stimulus compared to post-injury, baseline values at both 15 min (22.3 ± 2.2 s) and 30 min (19.8 ±

3.6 s) after i.t. administration (n = 6, p = 0.58*10-3; Figure 11C). The 10 μg dose of

TY005 resulted in a significantly higher paw withdrawal latency of 17.8 ± 1.2 s only 15 min after the bolus injection (n = 11, p = 1.1*10-4; Figure 11C). The same was observed for TY005 (3 μg and 1 μg) with mean paw withdrawal latencies of 17.5 ± 1.9 s (n = 6) and 17.5 ± 2.5 s (n = 10), respectively (p=0.04; Figure 11C). Morphine-treated animals withdrew the hindpaw in response to the thermal stimulus with a mean latency of 20.8 ±

2.2 s 15 min post-i.t. injection (n = 10, p = 0.4*10-2; Figure 11C). Vehicle treatment did 113

not result in a significant change in paw withdrawal latency when compared to post injury baseline values.

A dose response curve was generated from data collected 15 min after the i.t. injection of TY005. Percent antihyperalgesic activity for each dose was calculated to be: 83.4 ±

8.6 % (30 μg), 53.1 ± 9.5% (10 μg), 51.0 ± 12.8 % (3 μg), and 34.1 ± 12.3 % (1 μg)

(Figure 11D). The A50 value was determined to be 4.0 μg in 5 μL (95% C.I. 1.6 - 9.7 μg;

R2 value = 0.88). The dose of MS evaluated resulted in 69.9 ± 10.5 % antihyperalgesic activity.

The differential effects observed between animals acutely receiving only MS (mu- opioid receptor agonist) versus those administered TY005 (mu-/ delta opioid agonist/

NK1 receptor antagonist) in attenuating SNL-induced hypersensitivities required clarification. Therefore, the individual peptide fragments TY040 and TY036, opioid agonist and NK1 antagonist, respectively, were evaluated in SNL-operated animals in both the radiant heat and von Frey assays. TY036 (10 μg) and TY040 (10 μg) produced significant antihyperalgesia 15 min after dosing, (79.8 ± 8.6 % and 100.0 ± 0.0 %, respectively) when compared to vehicle treated rats. Interestingly, TY040-mediated effects were also significantly higher than TY005 (Figure 12A). Responses to von Frey filaments were significantly attenuated by TY005 (10 μg) as previously observed (63.1 ±

11.2%). The same dose of TY036, C-terminal, NK1 receptor antagonist, produced a similar effect, 63.0 ± 17.6 % attenuation of SNL- induced tactile allodynia (Figure 12B). 114

TY040 (10μg, i.t.) resulted in less than 20 % antiallodynic activity, similar to the observations with morphine.

11A 11B ** 100 15 * **

12 * 80 SEM)

9 ** 60 *** *

6 40 (g ±SEM) (g

3 20 % Antiallodynia ( Antiallodynia %

Paw Withdrawal Threshold Withdrawal Paw 0 g) g g g g 0 BL 1 SNL 3 1ug 3ug 3 10ug 30ug vehicle MS (10 11C 11D Dose of TY005 (i.t. in 5 L) 25 * 100

20 * * * SEM) 80 * Latency 60 15

SEM) 40

s 10 ( 20

5 % Antihyperalgesia ( Antihyperalgesia %

PawWithdrawal 0

0 1ug 3ug 10ug 30ug g g g g BL g) SNL 1 3 10 30 vehicle Dose of TY005 MS (10 (i.t. in 5 L)

115

116

Figure 11 Intrathecal TY005 reverses SNL-induced hypersensitivities Intrathecal

TY005 attenuates nerve-injury induced tactile and thermal hypersensitivities in a dose- dependent manner. Vehicle, TY005 (1 μg, 3 μg, 10 μg, 30 μg), or morphine (10 μg) was injected spinally in SNL animals in a 5 μL volume. Non-noxious probing of the hind paw with von Frey filaments or noxious heat stimulation was performed at 15 min time points.

The peak effect of TY005 and morphine of attenuating response to (A) non-noxious and

(C) noxious stimulation was observed 15 minutes after i.t. injection. Percent activity of

TY005 was calculated for each dose and behavioral test and a graded dose response curve generated (B) Antiallodynia and (D) Antihyperalgesia. Acute, i.t. administration of

TY005 significantly attenuated SNL- induced hypersensitivities dose-dependently when compared to vehicle-treated (* p < 0.05) and morphine-treated animals (** p < 0.05). 117

12A 100 Vehicle (10% DMSO) TY005 (10 g) 80 TY036 (10 g) TY040 (10 g) 60

SEM) 40 (

20 %Antihyperalgesia

0

15 30 45 60 Time (min)

12B

100 vehicle (10% DMSO) 80 TY005 (10 g) TY036 (10 g) TY040 (10 g) 60

SEM) 40 (

% Antiallodynia % 20

0

15 30 45 60 Time (min)

118

Figure 12 Activities of opioid and NK1 peptide fragments: TY036, TY040, and

TY005 A direct comparison of the activities of the individual opioid and NK1 peptide fragment with the linked peptide, TY005 revealed (A) All three compounds produced significant antihyperalgesia in SNL-operated animals compared to vehicle (p < 0.05).

The efficacy was ranked: TY040 > TY005 > TY036. (B) TY005 and TY036 significantly reversed SNL-tactile allodynia at equal levels (p < 0.05). TY040 did not significantly affect SNL-induced allodynia fully attenuated thermal hypersensitivity compared to all other treatments (p > 0.05). All statistics were done in FlashCalc,

ANOVA. 119

TY005 (i.t.) retains antihypersensitivity effects after multiple exposures: In both sham- operated and SNL-operated animals, TY005 was compared to vehicle treated controls and morphine treated rats for effects after multiple exposures. Prior to surgery and seven days after sham or nerve ligation, baseline tactile and thermal paw withdrawal thresholds and latencies were recorded. Compound administration occurred twice daily for 11 days afterwards and behavioral measurements obtained 15 min after the morning injection. It is important to note that on days 12-14 and 16-18, no drug was given. On day 15, each treatment was given as a bolus to animals previously exposed to multiple intrathecal injections (Figure 13).

In sham-operated animals, the mean baseline paw withdrawal threshold before surgery was 14.9 ± 0.2 g. The mean post-sham paw withdrawal threshold value was significantly lower than the pre-sham value at 13.4 ± 0.5 g, (n = 41, p = 0.007). Animals given twice daily i.t. injections of 10% DMSO (5 μL) did not withdraw the hind paw at thresholds significantly different from the post-sham threshold across the 18 d testing period (n =

20, p = 0.18; Figure 14A). Rats treated with MS (10 μg, i.t.) had significantly lower paw withdrawal thresholds by the 9th day of twice daily treatment, and these thresholds ranged between 8.4 ± 1.2 g and 4.1 ± 0.8 g for the remainder of the experiment (n = 7, p

< 0.001; Figure 14A) indicating the development of allodynia to morphine alone. TY005

(10 μg), did not produce a significant decrease in paw withdrawal threshold on days 1-11, nor on day 15 after the i.t. injection (n = 14, p = 0.10; Figure 14A). On days 12-14 and

16-18, when no TY005 was administered, animals withdrew the paw at thresholds 120

between 12.1 ± 1.6 g – 10.8 ± 1.6 g, which was significantly lower than pre-sham and post-sham thresholds only on day 18 (p = 0.0004).

SNL-animals were prepared and exposed to the same treatment paradigm as sham- operated animals to evaluate potential tolerance to the antihyperalgesic and antiallodynic efficacy of TY005. Tactile paw withdrawal threshold before and after nerve ligation were

14.6 ± 0.3 g and 4.5 ± 0.6 g, respectively (n= 42, p < 0.001). Repeated vehicle treatment did not result in a significant increase or decrease in paw withdrawal thresholds throughout the duration of the experiment (n = 14, p = 0.38; Figure 14B). Nerve injured rats given repeated injections of morphine (10 μg in 5 μL) did not have paw withdrawal thresholds significantly different from the mean post-SNL threshold or those of vehicle treated animals across the full time-course of the experiment (n = 12, p = 0.17; Figure

14B). TY005 (10 μg), administered twice daily for 11 days, resulted in a significant increase in paw withdrawal threshold compared to vehicle treated and post-SNL baseline values. Thresholds ranged between 11.1 ± 1.2 g and 10.2 ± 1.4 g throughout the experiment with the time of peak effect occurring on day 3 (12.2 ± 1.1 g). (n = 16, p <

0.001; Figure 14B). When challenged on day 15, the animals receiving TY005 withdrew the hind paw at a mean threshold of 8.0 ± 1.2 g, which was significantly higher than post-

SNL and vehicle treatment alone (p = 0.001) but lower than the pre-injury baseline (p =

1.3*10-5).

After tactile paw withdrawal thresholds were recorded, animals were given 5-7 min to acclimate for thermal testing. Before the sham nerve ligation, the mean thermal paw withdrawal latency was 21.5 ± 0.4 s. After the sham operation, the mean paw withdrawal 121

latency was 20.7 ± 0.6 s, which was not significantly different (n = 41, p = 0.26; Figure

14C). Vehicle (10% DMSO) treatment did not produce a significant increase or decrease in paw withdrawal latency over the course of the experiment (n = 20, p = 0.29). Rats receiving morphine had mean paw withdrawal latencies between 31.3 ± 1.1 s and 23.5 ±

1.8 s on days 1-9, demonstrating antinociception (n = 7, p = 0.001). After 11 days of treatment with morphine, the paw withdrawal latency was 19.2 ± 1.0 s, a value not significantly different from post-sham values (p > 0.05), indicating loss of antinociceptive efficacy but not the development of hyperalgesia in this paradigm. On day 15 a bolus injection of morphine (10 μg, i.t.) did not result in a significantly different paw withdrawal latency compared to the post-sham baseline, indicating antinociceptive tolerance (19.4 ± 1.6 s, p = 0.45; Figure 14C). In the group of animals receiving TY005, antinociception was evidenced throughout the 11 day treatment time-course with paw withdrawal latencies ranging from 28.9 ± 1.6 s - 24.7 ± 1.8 s (n = 14, p < 0.001; Figure

14C; a bolus of TY005 (i.t.) on day 15, resulted in a mean paw withdrawal latency of

24.4 ± 1.3 s, further indicating maintenance of antinociception after multiple exposures in sham-operated animals (p = 0.006, Figure 14C).

SNL-animals were allowed to acclimate for 5-7 min between tactile and thermal testing. As previously observed, nerve injury resulted in a decrease in paw withdrawal latency from 21.6 ± 0.4 s prior to nerve injury to 14.3 ± 0.6 s, evidencing the development of thermal hyperalgesia (n = 39, p < 0.001). Rats treated with vehicle did not show a significant change in paw withdrawal latency from the post-SNL value (n =

14, p = 0.38). Twice daily morphine (10 μg, i.t.) treatment resulted in a significant 122

increase in paw withdrawal latency compared to post-injury latencies on days 1 through 7 with values ranging from 20.0 ± 1.6 s to 25.6 ± 2.6 s (n = 9, p = 0.001; Figure 14D). The peak effect of morphine was observed on day 3 of treatment (25.6 ± 2.6 s) which was significantly higher than both post- injury values and pre-injury paw withdrawal latencies

(p = 0.004), though not as pronounced as in non-injured rats. On day 9, morphine did not produce a significant increase in paw withdrawal latency compared to vehicle (p = 0.06), and this was further observed on day 11 when the mean paw withdrawal latency in the morphine group was 16.7 ± 2.0 s (p = 0.14), demonstrating a decrease in antihyperalgesic efficacy. The morphine challenge on day 15, after three treatment-free days, resulted in a paw withdrawal latency of 20.0 ± 2.6 s (p = 0.002). Animals treated with TY005 withdrew the hind paw at significantly longer latencies compared to the post-injury value and vehicle treated rats on all days of treatment (n = 16, p = 2.4*10-14; Figure 14D); the peak antihyperalgesic effect of TY005 was observed on day 5 of treatment and corresponded to the paw withdrawal latency of 24.5 ± 1.7 s (Figure 14D). After 11 days,

TY005 treatment did not result in the development of antinociceptive tolerance in SNL- operated rats. When treatment was discontinued for three days, paw withdrawal latencies returned to post-injury levels (days 12-14). TY005 (10 μg, i.t.) was administered as a challenge on day 15, and the resulting mean paw withdrawal latency was 21.0 ± 1.7 s.

This was significantly higher than vehicle treated animals, but not when compared to morphine treatment (p = 0.004 and p = 0.75, respectively).

Figure 13

T=- 7d, BL T=- 8d, L /L T= 12- 14 d TY005 T=16 -18, TY005 5 6 VF/IR values SNL or sham withdrawn, vF/IR Withdrawn obtained values obtained

T=0d, Post -BL T=15d, one bolus injection (i.t.) T=1- 11d, twice daily i.t. injections Behavioral testing T= - 12d, i.t. cannula TY005 20 min. post implantation T=1, 3, 5, 7, 9, 11d: vF/ IR testing injection 15-20 min post-injection

123

124

Figure 13 Timeline of chronic administration of spinal TY005 & morphine All animals were treated with 5 μl of either vehicle (10% DMSO), TY005 or morphine (MS) twice daily for 11 days, given 3 days washout (shadowed regions on graph), and challenged again on day 15. Behavioral measurements were obtained at 15-20 min post injection.

14A 14B

15 10% DMSO 15 10% DMSO TY005 (10 g) * TY005 (10 g) * 12 MS (10 g) 12 * * * * MS (10 g)

9 9 *

SEM) SEM)

6 * 6

(g (g * * * * *

3 * * * 3

Paw Withdrawal Threshold Withdrawal Paw Threshold Withdrawal Paw 0 0 1 3 5 7 9 1 3 5 7 9 BL 11 12 13 14 15 16 17 18 BL 11 12 13 14 15 16 17 18 SNL Sham Time Time (Days) i.t. bolus (Day) i.t. bolus 14C 14D

35 35 * * 10% DMSO 10% DMSO * * 30 * * TY005 (10 g) 30 * TY005 (10ug) * MS (10 g) * MS (10 g) 25 * 25 * * * * *

20 20

SEM) SEM)

15 15

(s (s 10 10

5 5

Paw Withdrawal Latency Withdrawal Paw Paw Withdrawal Latency Withdrawal Paw 0 0 1 3 5 7 9 1 3 5 7 9 BL 11 12 13 14 15 16 17 18 BL 11 12 13 14 15 16 17 18 SNL Sham Time Time (Days) i.t. bolus (Days) i.t. bolus

Sham-operated SNL-operated

125

126

Figure 14 Chronic, intrathecal TY005 does not produce antinociceptive tolerance in nerve-injured nor paradoxical hypersensitivity in sham-operated animals. (A) Paw withdrawal thresholds of sham-operated animals (B) Paw withdrawal thresholds of SNL- operated animals. (C) Paw withdrawal Latencies of Sham-operated animals. (D) Paw withdrawal latencies of SNL-operated animals. Significance was set at *p < 0.05 compared to baseline values. 127

4.6 Activity of Systemically Administered Opioid Agonist/ NK1 Receptor Antagonist

TY005 has the potential to cross the Blood Brain Barrier: Using in situ perfusion, the potential for TY005 to cross the blood brain barrier (BBB) was assessed prior to the commencement of in vivo studies. After a 15 min perfusion, the amount of [125I] TY005 in brain tissue was measured and the rate determined (Figure 15). Compared to the negative control, sucrose, which does not cross the BBB, and biphalin, the positive peptidic control, it was concluded that TY005 had a great potential to cross the BBB after systemic administration.

Systemic TY005 acutely reverses SNL-induced hypersensitivities: Seven days after nerve injury, TY005 was given intravenously into the tail vein as an acute bolus and behavioral measurements of tactile and thermal hypersensitivities recorded. For both tactile and thermal measurements, three doses were evaluated (3, 10, and 30 mg/ kg) and compared to baseline values, as well as vehicle-treated control animals.

Mean paw withdrawal threshold in response to non-noxious tactile probing with von

Frey filaments before and after nerve ligation for all animals were 14.6 ± 0.4 g and 2.5 ±

0.2 g, respectively (n = 23). Nerve injured animals given TY005 (30 mg/ kg) withdrew the hind paw at the maximal threshold of 15.0 ± 0.0 g 15 min following the tail vein injection and 8.6 ± 1.6 g at the 30 min time-point (n = 6, p < 0.0001; Figure 16A). Paw withdrawal thresholds had returned to post-injury values 45 min after administration.

Similarly, 10 mg/kg treatment with TY005 resulted in paw withdrawal thresholds significantly higher than post-injury values 15 and 30 min after injection; 11.2 ± 2.2 g and 7.7 ± 2.2 g, respectively (n = 5, p < 0.001). The lowest dose evaluated, 3 mg/ kg, 128

significantly increased the paw withdrawal threshold of injured animals only at 15 minutes after i.v. administration to a value of 4.4 ± 0.8 g (n = 6, p = 0.001; Figure 16A).

Vehicle treatment (20% DMSO, 1 mL/ kg) did not alter SNL-induced tactile allodynia throughout the duration of the experiment (n = 6, p > 0.05).

A dose response curve for i.v. TY005 was generated using data from the 15 min time- point. Percent antiallodynic activity of each dose was calculated to be: 100.0 ± 0 % (30 mg/kg), 70.9 ± 16.6 % (10 mg/ kg), and 16.1 ± 6.4 % (3 mg/ kg, Figure 16B). The A50 value determined was 7 mg/ kg (95% C.I.: 5.1 - 9.5 mg/kg; R2 value = 0.98).

In a separate set of animals, the acute effect of TY005 (i.v.) on thermal hyperalgesia was evaluated. As in previous experiments, baseline paw withdrawal latencies were recorded prior to and after peripheral nerve ligation. The mean pre-injury baseline was

22.5 ± 0.5 s; the post injury mean withdrawal latency was 12.8 ± 0.6 s, indicating the development of hypersensitivity to a noxious thermal stimulus (n = 23, Figure 16C).

After i.v. administration of TY005 (30 mg/ kg), paw withdrawal latencies at the 15 min

(20.2 ± 2.9 s) and 30 min (17.0 ± 2.0 s) time-points were significantly higher than the post-injury baseline (n = 5, p = 0.004; Figure 16C). Treatment of SNL rats with 10 mg/ kg of TY005 resulted in paw withdrawal latencies of 18.5 ± 0.8 s and 14.7 ± 1.0 s both 15 and 30 min after the i.v. injection. Only the 15 min measurement was significantly different from post-injury baseline values (n = 6, p = 0.003; Figure 16C). The peak time of effect of TY005 (3 mg/ kg) was observed 15 min post administration, with a mean paw withdrawal latency of 18.1 ± 1.2 s, which was statistically higher than that of the untreated or vehicle treated injury (n = 6, p = 0.04). Vehicle control animal paw 129

withdrawal latencies were not altered from post-injury baseline values over the course of the experiment.

The percent activity of TY005 to attenuate SNL-induced thermal hyperalgesia 15 min after intravenous compound administration was determined. TY005 (30 mg/ kg) had a

72.1 ± 18.3% antihyperalgesic effect, while the 10 mg/ kg dose resulted in a 54.3 ± 8.0% reversal of thermal hyperalgesia. The lowest dose tested (3 mg/ kg) produced 40.1 ±

12.2% antihyperalgesia compared to vehicle treated controls. A dose response curve was constructed (Figure 16D). The A50 valued was calculated to be 6.5 mg/kg with a 95% confidence interval of 2.2 - 19.7 mg/ kg and an R2 = 0.99 (Figure 16D).

Figure 15

100

Potentially 10 available for activity

1 Vascular

component 0.1

130

131

Figure 15 TY005 may cross the BBB and be systemically active. TY005 was

125 125 radiolabeled with I and used for the experiment to detect the trace of I in brain. Kin was calculated via single time point analysis (15 min in situ perfusion). Assay performed by Drs. Richard Egleton and Christopher Campos. Sucrose and insulin served as negative controls. CTAP, DPDPE, and biphalin served as positive controls and peptide controls.

16A 16B 15 * 100 * 12 80

9 60 SEM)

SEM) 40

6 * (

(gram % Antiallodynia % 3 20

Paw Withdrawal Threshold PawWithdrawal 0 0 3 BL 10 30 SNL vehicle Dose of TY005 3.0mg/kg 16C 10.0mg/kg30.0mg/kg 16D (i.v., mg/kg) 30

100 25 * 20 * * 80 60

SEM) 15

SEM) 40 ( (s 10 20

5 Antihyperalgesia %

Paw Withdrawal Latency PawWithdrawal 0 0 3 10 30 BL SNL Dose of TY005 vehicle (i.v., mg/kg) 3.0mg/kg 10.0mg/kg30.0mg/kg

132

133

Figure 16 Systemic TY005 reverses SNL- hypersensitivities Vehicle or TY005 (3, 10, or 30 mg/kg) was injected systemically in SNL animals. Non-noxious probing of the hind paw with von Frey filaments or noxious heat stimulation was performed every 15 minutes. TY005 attenuated pain-like responses 15 minutes after i.v. injection (A) tactile and (C) thermal. Percent activity of TY005 was calculated for each dose and a graded dose response curve generated for both (B) Antiallodynia and (D) Antihyperalgesia.

Acute, systemic administration of TY005 significantly attenuated SNL-induced hypersensitivities dose-dependently when compared to vehicle-treated (* p < 0.05). 134

Metabolic stability (t1/2) of TY005 in vitro: The plasma half life of TY005 was determined as described above. The linkage between the peptidic region of the compound and the NK1 receptor antagonist (Table 1) made the compound susceptible to enzymatic degradation. The half-life of TY005 was determined to be 0.83 min (Table 3).

This was longer than L-732, 138, the small molecule antagonist (0.2 min) but indicated the in vivo activity observed may be that of a metabolite. The proposed metabolite is given in Table 1 (TY043).

Metabolite-mediated Antinociception: TY043 was synthesized by the University of

Arizona, Department of Chemistry as the potential metabolite of TY005. TY043 (10 μg, i.t.) produced 50.7 ± 16.0 % antinociception in non-injured animals, indicating the observations made with TY005 may have been due to TY043 activity and that of an unknown metabolite acting to block the NK1 receptors. As a result, TY005 was modified resulting in the peptidic compound, TY027 (Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-

Bn(CF3)2). 135

Table 3: Plasma Half-life of Peptidomimetics

Compound ID t1/2

L-732, 138 (NK1 reference) 0.20 min TY005 0.83 min TY027 4.83 hr 136

4.7 A New Lead Compound: TY027

In vitro characterization of TY027: Modifying the ester-linkage of TY005 into the amide-linkage of TY027 altered the binding affinities and in vitro affinities (Table 4).

The ratio of activity at the mu- and delta opioid receptors was similar between the compounds despite the 10 fold increase in affinity of TY027 to the delta- receptor. The binding affinity of TY027 to the rNK1 receptor was decreased with the modification by approximately 10 fold; interestingly, the affinity for the hNK1 receptor was increased by the same amount. Overall, TY027 had a similar in vitro profile to TY005.

Table 4: In vitro characterization of TY027 versus TY005 Affinity GTPγS binding MVD GPI/LMMP Opioid Opioid hDOR rMOR hNK rNK hDOR rMOR Substance P 1 1 (δ) (μ) (K ; (K ; (K ; (K ; ID Structure i i i i EC E EC E Agonist Agonist Agonist Antagonist nM) nM) nM) nM) 50 max 50 max

(IC ; (IC ; (IC ; (nM) (%) (nM) (%) 50 50 50 (K ; nM) nM) nM) nM) e

TyrDAlaGlyPheMetPro TY005 2.8 36.3 0.082 0.29 2.9 47.6 31.6 45.6 22.3 358.8 None 24.7 LeuTrpOBn(CF3)2

TyrDAlaGlyPheMetPro TY027 0.66 15.7 0.0064 7.27 8.6 58.0 7.0 55.0 14.5 487.9 None 10 LeuTrpNHBn(CF3)2

137

138

Metabolic stability (t1/2) and BBB penetration of TY027: Prior to any in vivo evaluation with TY027, the metabolic stability and potential to cross the blood brain barrier were assessed. Incubation of TY027 in rat plasma (37°C) resulted in an increased half life of the peptide to 4.83 hr versus the < 1 min for TY005 (Table 3). Given the stability of the new multifunctional peptide and the potential of TY005 to cross the BBB, the ratio of

TY027 in the brain was determined. The ratio of radioactivity in the brain (RBr) was statistically higher for TY027 (4.1 ± 0.8) when compared to sucrose (1.3 ± 0.2; Figure

17A). The amount of TY027 potentially available for activity in the brain after 15 min perfusion was 8.0 ± 0.6 μL/min/g similar to that of TY005 (Figure 17B). 139

17A

5 *

4

3 SEM)

2 (RBr

1 Ratio of TY027 in Brain in TY027 of Ratio

0 sucrose TY027

17B

100

Potentially 10 available for activity

in 1

K Vascular component 0.1

140

141

Figure 17 TY027 crosses the BBB (A) Ratio of drug found in Brain tissue versus perfusion solution after 15 min in situ perfusion to determine BBB penetration (* p <

0.01, Student‟s t-test). (B) TY005 and TY027 were radiolabeled with I125 and used for the

125 experiment to detect the trace of I in brain. Kin was calculated via single time point analysis (15 min in situ perfusion). Sucrose and insulin served as negative controls. CTAP,

DPDPE, and biphalin served as positive controls and peptide controls. Assays performed by Drs. Richard Egleton and Christopher Campos. 142

4.8 Activity of TY027 in vivo: Acute Pain

TY027 (i.t.) produces antinociception in non-injured rats without impairing movement:

Rats were implanted with i.t. cannula as described. Paw withdrawal latencies to noxious, radiant heat were obtained from non-injured rats as in previous studies. Animals withdrew the hind paw with a mean latency of 21.8 ± 0.3 s before dosing (n = 52; Figure

18A). Animals were then administered vehicle (10% DMSO, 5 μL) or TY027 (1, 3, 10, or 30 μg in 5 μL. Vehicle treatment did not produce a significant change in paw withdrawal latency at any time point evaluated. All doses of TY027 resulted in a significant increase in paw withdrawal latency 15 min after the injection (i.t.) compared to vehicle and baseline values (Figure 18A).; specifically, the observed latencies were as follows: 28.7 ± 1.7 s (1 μg), 29.1 ± 1.5 s (3 μg), 29.0 ± 1.3 s (10 μg), and 33.0 ± 0.0 s (30

μg). The peak effect of TY027 was between 15 and 30 min after dosing with 3, 10, and

30 μg, as no significant difference was observed between these groups (p = 0.3 and p =

0.08, respectively). Animals receiving TY027 (1 μg) did not withdrawal the paw at significantly higher latencies than vehicle treated groups or baseline observations after 30 min.

The paw withdrawal latencies obtained at 30 min (after intrathecal injection) were converted to percent activities and a dose-response curve generated (Figure 18B).

Overall, the degree of antinociception in the acute pain model was dose-dependent (R2- value = 0.77). The A50 value was determined to be 1.8 μg (95% C.I. = 0.5 - 5.0 μg in 5

μL). 143

The ability of spinal TY027 to induce motor impairment was assessed as reported for

TY005. Vehicle treated animals (10% DMSO) significantly decreased the rotarod latency 15 min after administration (152.1 ± 12.3 s, p = 0.04), however no other time point evaluated reached a statistically significant difference from baseline values. The highest dose of TY027 (i.t.) tested for antinociception (30 μg) resulted in a similar decrease 15 min post injection, 168.9 ± 10.4 s (p = 0.34), though the latency returned to

180.0 ± 0.0 s by 30 min. As noted with the vehicle and TY027 (30 μg) treated groups, spinally administered TY027 (100 μg) decreased 15 min after the injection, but not significantly compared to baseline or vehicle- control values (158.7 ± 19.7 s, p = 0.37).

TY027 (100 μg) did not significantly alter the walking latency on a rotating rod compared to vehicle treatment at the previously observed time of peak effect, 30 min

(174.6 ± 4.7 s, p = 0.33; Figure 18C). Furthermore, no delayed effects were observed when the test was carried out to 180 min (3 hr) after the bolus injection. TY027 did not result in motor impairment when compared to literary reports of known sedative compounds (Vanderah et al., 2008; Valenzano et al., 2005; Boyce et al., 1999).

TY027 produces antinociception in mice: Additional antinociceptive screening of TY027 was performed in male ICR mice to address the concern of species variability. Five doses of TY027 (0.32, 1.0, 3.2, 10, 32 nM, i.c.v.) were evaluated using the tail flick assay until the observed effect had returned to baseline levels. TY027 (0.32 nM) produced significant antinociception compared to baseline tail flick latencies (21.5 ± 4.6 %) 10 min after administration. When the dose of TY027 (i.c.v.) was increased, the maximal effect was observed 20 min post-injection with tail flick latencies calculated to be 68.1 ± 13.6 144

and 83.9 ± 6.2 % above baseline for 1 nM and 3.2 nM, respectively. Maximal antinociception (100.0 ± 0.0 %) was observed with the 10 nM and 30 nM doses of TY027 as the tail flick latencies reached cut-off values.

The dose-dependency of TY027-mediated antinociception observed in mice also resulted in an increase in the peptide‟s duration of action. As the dose was increased by a third, the duration of action of TY027 was increased by 60 min (Figure 18D). In animals treated with a single bolus of the highest doses (10 and 30 nM), tail flick latencies did not return to baseline levels until 3 hours after the i.c.v. injection.

18A 18B

35 100

30 80

25 SEM)

20 60

± SEM) ± 15 Vehicle (10%DMSO) 40 (s TY027 (1 g) R2: 0.771 10 TY027 (3 g) A50: 1.838 Lower C.I.: 0.65

TY027 (10 g) 20 Upper C.I.: 5.2 Paw Withdrawal Latency Withdrawal Paw 5 TY027 (30 g) ( Antinociception %

0 0 1 3 BL 15 30 45 60 10 30 Time (min) Dose of TY027 ( g , i.t.) 18C 18D 12 180

9

120 6

SEM) SEM)

(s 3 (s

60 Latency Flick Tail

Vehicle (10% DMSO) Rotarod Latency Rotarod 0 TY027 (30 g) 0.32 nM 3.2 nM 30 nM TY027 (100 g) 1 nM 10 nM

0 15 30 60 90 BL 10 20 30 45 60 90 BL 120 150 180 120 150 180

145 Time (min) Time (min)

146

Figure 18 TY027 is antinociceptive in models of acute pain (A) Mean paw withdrawal latencies after spinal TY027 was administered to non-injured animals. Mean paw withdrawal latency responses to noxious thermal stimulus were recorded and converted to % antinociception. The peak effect observed occurred between 15 and 30 min after the injection (i.t.) for all doses evaluated. Significant antinociception was observed at all doses when compared to vehicle (* p < 0.05). (B) Dose response curve of TY027 at 30 minutes after spinal administration. (C) To ensure any effect observed was not due to the animal‟s inability to move, motor skills were assessed over a 3 min time course; at doses

3-fold higher than required for maximal antinociception, there was no significant motor impairment observed between treatment groups (p > 0.05). (D) To confirm that TY027 was activity in multiple species, TY027 was also evaluated in a mouse model of antinociception. TY027 produced significant antinociception in a dose and time dependent manner in this model (p < 0.05). Mouse studies were conducted by Ms. Carla

Cosgrove and Ms. Denise Giuvelis in the laboratory of Dr. Edward J. Bilsky. 147

TY027 is an opioid receptor agonist in vivo: To further confirm that the ester to amide linkage modification of TY005 to TY027 did not alter activity at the opioid receptors in vivo, experiments were performed giving a pretreatment with a non-selective opioid antagonist, in both rats (paw flick latency) and mice (55°C tail flick).

Rats were baselined as described above, and the mean paw withdrawal latency was determined to be 22.0 ± 0.5 s. Saline or NLX (2 mg/kg, i.p.) was given 20 min before

TY027 (30 μg, i.t.; Figure 19A). Neither pretreatment with saline nor NLX had an effect of paw withdrawal latencies when compared to baseline (p < 0.05). TY027 (30 μg) significantly increased the mean paw withdrawal latency 20 min post-injection (30.8 ±

1.4 s; p < 0.001). Naloxone pretreatment significantly reduced the paw withdrawal latency of animals treated with spinal TY027 (18.4 ± 2.9 s; p < 0.001).

Mouse tail flick latencies were obtained as in previous experiments (Figure 19B). The mean tail flick latency prior to compound exposure was determined to be 2.0 ± 0.1 s. In the absence of NLX, TY027 (3.2 nM, i.c.v.) resulted in a significant increase in tail flick latency (8.7 ± 0.6) compared to vehicle and baseline values (p < 0.01). When NLX (10 mg/kg, i.p.) was given 20 min prior to TY027 (3.2 nM, i.c.v.), mice withdrew the tail within 3 s (2.4 ± 0.2 s) 20 min after administration.

TY027 is an NK1 receptor antagonist in vivo: Animals were pretreated at t = - 40 min with either saline (1 mL/kg) or NLX (2 mg/kg, s.c.) and 10% DMSO (5 μL) or TY027

(30 μg in 5μL, i.t.; t = -20 min). All animals receiving 1 mM SP (10 μL, i.t.) at t = 0 min, prominent flinching, biting and/or scratching of the hind paws was observed Animals treated with saline instead of SP did not flinch/ bite /or scratch (data not shown). Animals 148

(n=6) pre-treated with both saline and 10% DMSO flinched a mean total of 37.6 ± 7.9 times after SP (i.t.). When TY027 (30 μg) was given instead of 10% DMSO, that value fell significantly to 10.6 ± 3.6 flinches (Table 5). As in the previous experiment, NLX (2 mg/kg, s.c.) was given before both TY027 and SP to isolate NK1 receptor mediated activity from that mediated by opioid agonism. Naloxone given to vehicle treated animals resulted in a mean of 26.3 ± 5.5 flinches, which was not significantly different than saline-vehicle treated animals (Table 5). Animals exposed to TY027 (30 μg) min, in the presence of naloxone, flinched significantly fewer times over 5 min for a total 8.0 ±

3.0 flinches compared to SP alone and SP + naloxone control groups (Table 5). 149

19A 35

30

25

20 * SEM)

15 (s

10

Paw Withdrawal Latency Withdrawal Paw 5

0 Saline - + - + -

NLX - - + - +

TY027 BL 10% DMSO (30 g, i.t.)

19B 12

9

SEM) 6

(s Tail Flick Latency Flick Tail 3 *

0 BL 10%DMSO - NLX +NLX

TY027 (3.2 nM, i.c.v.)

150

Figure 19 TY027 is an opioid agonist in vivo (A) Rats were pretreated with the non- selective opioid receptor antagonist, naloxone (NLX; 2 mg/kg), given s.c. 10 min prior to an intrathecal injection of TY027, blocks the peak effect TY005-mediated antinociception (* p < 0.05). TY027 (30 μg, i.t) or vehicle (10% DMSO) was given at t =

0 min. Paw withdrawal latencies to infrared heat were obtained at t= + 15 min. (B) Mice were treated with NLX (10 mg/kg i.p.) 20 min prior to i.c.v. injection of TY027 (3.2 nM,

5 μL). Tail flick latency was recorded 20 min following the supraspinal injection of the bifunctional peptide, * p < 0.05.

Table 5: TY027 acts as an NK1 antagonist in vivo: Effects on SP-induced Flinching

p-value to p-value Treatment Mean # flinches in 5 min (± SEM) N saline- to NLX- DMSO DMSO

Sa-10% DMSO-SP 37.6 (± 7.9) 5 - - a S -TY02730-SP 10.6 (± 3.4) 6 0.01 - NLXb- 10% DMSO- 26.3 (± 5.5) 6 0.14 - SP b NLX -TY02730-SP 8.0 (± 3.0) 6 0.004 0.05 aS: 0.9% saline (1 mL/ kg, s.c.) or bNLX: naloxone (2 mg/ kg, s.c.) given 40 min before SP. 10% DMSO or TY027 (dos given in subscript = μg in 5 μL; i.t.) 20 min prior to SP (10 μL of 1 mM; i.t.). P-values were determined using one-way ANOVA (FlashCalc) with Newman-Kuels post-hoc analysis Significance was set at p ≤ 0.05.

151

152

4.9 Activity of TY027 in vivo: Neuropathic Pain

Spinal TY027 attenuates SNL- induced hypersensitivity: TY027 (1 - 30 μg in 5 μL) was evaluated for activity against peripheral nerve injury-induced tactile and thermal hypersensitivities in rats. Seven days after SNL, behavioral responses to tactile and thermal stimuli were obtained and compared to pre-injury baseline values. TY027 was administered (i.t.) and behavioral measurements recorded every fifteen min for the first hour, every 30 min to the second hour, and on the hour until the observed effects had returned to baseline levels or 4 hours was reached. Duration of action was cut-off at 4 hours, as a result of in vitro plasma half life determination, to avoid potential metabolite effects. Observed behavioral thresholds of TY027-treated animals were compared to those of vehicle-treated and morphine-treated animals. As previously stated, morphine was used for comparison of the peptide to an opioid used clinically for the treatment of neuropathic pains.

Spinal nerve ligation induced increased sensitivity to normally innocuous touch

(allodynia) as evidenced by a decreased paw withdrawal threshold to calibrated von Frey filaments from 14.6 ± 0.2 g to 3.1 ± 0.2 g. Fifteen minutes after administration (i.t.),

TY027 (1 μg) significantly increased the paw withdrawal threshold of SNL animals (7.3

± 1.4 g) compared to baseline measurements or vehicle treated rats (Figure 20A).

However, paw withdrawal thresholds in these animals had returned to postSNL levels by the next time point (30 min) and remained between 3.1 ±0.6 g and 4.3 ±0.7 g for the remainder of the experiment. TY027 (3, 10, and 30 μg) had a 30 min time of peak effect when injected spinally with thresholds of 7.6 ± 0.9 g, 12.5 ± 0.9, and 13.8 ± 0.7 g, 153

respectively (Figure 20A). The duration of antiallodynic actions for a single injection

(i.t.) of TY027 (3, 10, 30 μg) was 75 min. It should be noted there was a reemergence of significant attenuation of tactile hypersensitivity 120 min after spinal administration of the highest dose evaluated (30 μg), though the implications are not known.

A dose response curve was generated from data collected 30 min after the acute intrathecal injection of TY027 in SNL-operated rats and transformed into percent activity

2 (Figure 20B). The R -value was calculated to be 0.97 with an A50 of 4.46 μg (95% C.I. =

3.4 - 5.8 μg in 5 μL). These data confirm a dose related attenuation of SNL-induced tactile allodynia by acutely administered spinal TY027.

In a separate set of animals, paw withdrawal latencies to noxious heat were evaluated.

As previously reported, spinal nerve ligation induced thermal hyperalgesia as observed by a decrease in paw withdrawal latency from a mean value of 22.8 ± 0.3 s to 14.1 ± 0.4 s

(Figure 20C). Vehicle-treated animals did not withdrawal their hind-paws at any significantly higher or lower latency over the 240 min time-course (p= 0.20). Similarly, rats injected with TY027 (1 μg, i.t.) did not have significantly different paw withdrawal latencies when compared to post-injury values or those of vehicle treated rats (Figure

21C). However, increasing the dose of TY027 (3 μg, i.t.) resulted in increases in paw withdrawal latency to 20.0 ± 1.7 s and 17.9 ± 1.3 s at 15 min and 30 min, respectively.

TY027 (10 μg) further increased the observed increase in latency at 15 min and 30 min to

23.7 ± 2.1 s and 20.8 ± 1.9 s, respectively (Figure 20C). As recorded in the mouse studies, increasing the dose of TY027 administered to SNL-operated rats also led to an increase in the duration of action. At the two highest doses of the multifunctional peptide 154

evaluated, a significant increase in latency was observed 45 min after compound administration (p < 0.005): 20.4 ± 1.8 s (10 μg) and 21.6 ± 1.8s (30 μg). Though the recorded paw withdrawal latencies were not statistically significantly higher than baseline or vehicle treated values when measured 60-, 75- and 90- min after administration, responses were still above post-SNL values and testing was evaluated out to 240 min.

TY027 (30 μg) produced significant effects compared to post-injury baselines and vehicle treatment at 120- , 180- , 240- mins (20.6 ± 1.6 s, 21.8 ± 1.7 s, and 20.0 ± 1.7 s, respectively), further indicating an increase in the duration of action as a function of the dose (p = 0.0002; Figure 20C).

The time of peak effect for TY027 in attenuation of SNL-induced thermal hyperalgesia was 30 min after spinal administration. A dose response curve was generated from calculated percent activities (Figure 20D). The A50 value was determined to be 7.8 μg in

5 μL (95% C.I. = 3.0 – 19.8 μg); the R2-value was equal to 0.70. Together, these indicate the observed antihyperalgesic effects of TY027 are not strongly dose-related at the time of peak effect.

20A 20B 100 15 Vehicle (10% DMSO) TY027 (1 g) 80 TY027 (3 g) 12

TY027 (10 g) SEM) TY027 (30 g) 60

9 SEM) 40

6 (g

3 ( Antiallodynia % 20 Paw Withdrawal Threshold Withdrawal Paw

0 0 1 3 10 30 BL 15 30 45 60 75 90 SNL 120 180 240 Dose of TY027 Time (min) 20C 20D ( g in 5 l, i.t.)

Vehicle (10% DMSO) 100 30 TY027 (1 g) TY027 (3 g) TY027 (10 g) 80

25 SEM) TY027 (30 g)

20 60

SEM) 15 40 (s 10

20 % Antihyperalgesia ( Antihyperalgesia %

Paw withdrawal Latency withdrawal Paw 5 0 1 3 10 30 0 BL 15 30 45 60 75 90 SNL 120 180 240 Dose of TY027 Time (min) ( g/5 l, i.t.) 155

156

Figure 20 Intrathecal TY027 acutely attenuated SNL-induced sensitivities Vehicle or

TY027 (1.0, 3.0, 10.0, 30.0μg), was injected spinally in SNL animals. Non-noxious probing of the hind paw with von Frey filaments or noxious heat stimulation was performed at 15 min time points & 30 min time points for up to 4 hours. TY027 attenuated the injury induced response thresholds to (A) non-noxious and (C) noxious stimulation, peaking 30 minutes after i.t. injection. Percent activity of TY027 was calculated for each dose and behavioral test and a graded dose response curve generated

(B) tactile allodynia and (D) thermal hyperalgesia. An acute, i.t. bolus of TY027 significantly attenuated SNL- induced hypersensitivities dose-dependently when compared to vehicle-treated (p < 0.05). 157

Systemic TY027 reverses SNL-induced hypersensitivities: The effects of systemically administered TY027 were investigated in a different set of animals. Rats were baselined in both tactile and thermal behavioral assays the day before undergoing L5/L6 spinal nerve ligation. After the recovery period, post-SNL thresholds and latencies were measured and recorded as previously reported. A single dose of TY027 (10 mg/kg) was evaluated after bolus administered into the tail vein by intravenous injection and values compared to vehicle. Notably, two different vehicles were used to dissolve the increased amount of TY027 (PEG 400 and 20% DMSO in dH2O) in this study. No effect on SNL- induced allodynia was observed with either vehicle, nor were the values with TY027 different between vehicles used; subsequently, the data were combined. Future experiments use TY027 dissolved in 20% DMSO only; therefore vehicle is reported as such here for consistency.

As before, peripheral nerve injury resulted in a significant decrease in mean paw withdrawal threshold (baseline: 14.8 ± 0.2 g; SNL: 3.3 ± 0.3 g). Intravenous injection of

TY027 (10 mg/kg) resulted in a significant increase in paw withdrawal threshold, peaking at 90 min after administration, to 11.4 ± 1.2 g (p < 0.00001; Figure 21A). The antiallodynic activity of systemic TY027 was statistically and significantly higher than that of vehicle treated animals starting 30 min after i.v. injection (53.0 ± 10.0%) and remained significant through 90 min (70.6 ± 9.9%). Antiallodynic activity was also observed 210 min post compound administration (49.9 ± 10.2%). 158

The effects of TY027 on thermal hyperalgesia were also measured in SNL-operated animals. Prior to nerve injury, the mean paw withdrawal latency to a noxious infrared stimulus was 21.7 ± 0.4 s; this value was significantly decreased to 11.8 ± 0.8 s after animals were allowed to recover for 7 days. Vehicle treatment did not result in paw withdrawal latencies significantly different from those measured postSNL at any time- point tested. TY027 (10mg/kg, i.v.) resulted in significantly higher paw withdrawal latencies up to 150 min after administration with a time of peak effect occurring 30 min after injection (25.6 ± 3.7 s, p < 0.000001; Figure 21B). The antihyperalgesic activity of

TY027 was determined and ranged from 62.1 ± 15.7% (15 min) to 48.4 ± 11.7% (150 min) peaking at 30 min (86.8 ± 8.8%). Notably, the activity of TY027 remained above that of vehicle 420 min post-injection (35.8 ± 10.9%) though not statistically significant

(p = 0.06). 159

21A 15 * 12

9 SEM)

6 (g

3 Paw Withdrawal Threshold Withdrawal Paw 0 BL SNL

20%DMSO

TY027 (10 mg/kg) 21B

30 * 25

20

SEM) 15

(s 10

5 Paw Withdrawal Latency Withdrawal Paw 0 BL SNL

20% DMSO

TY027 (10 mg/kg)

160

Figure 21 Effects of systemic TY027 on neuropathic pain. Vehicle or TY027 (10 mg/kg) was injected into the tail vein of in SNL animals. Non-noxious probing of the hind paw with von Frey filaments or noxious heat stimulation was performed every 15 minutes. TY027 significantly reversed SNL-induced tactile and thermal hypersensitivities for the first hour after i.v. injection (A) Tactile, 90 min after administration (B) Thermal,

30 min after administration (*p < 0.01 compared to post-SNL and 20% DMSO-treated values, ANOVA).

161

CHAPTER 5:

OPIOID AGONIST/ NK1 ANTAGONISTS LACK UNWANTED SIDE EFFECTS

TYPICALLY RELATED TO OPIOIDS

5.1 Introduction

The development of TY027 has provided a compound for completion of proof-of- concept studies evaluating the multi-target approach for treating chronic pains. Given the antinociceptive and antihyperalgesic properties of TY027 after both central and systemic administration, studies investigating the development of side effects, specifically antinociceptive tolerance, paradoxical hypersensitivity, and abuse liability, were of interest.

5.2 TY027: Antiallodynic, Antihyperalgesic, and Antinociceptive Tolerance

The development of antinociceptive tolerance clinically leads to increases in dosing regiments and therefore in dose-related unwanted side effects (e.g. constipation, nausea/vomiting). The rationale design of TY027 was in part to decrease the development of antinociceptive tolerance. Using a cumulative dosing paradigm, this phenomenon was assessed after central administration of TY027.

Animals were implanted with intrathecal cannula, as described previously, and tactile and thermal baseline measurements obtained. The sham- or SNL-operation was performed and, after 7 days, rats were again assessed for responses to tactile and thermal stimuli. On day 1 of treatment, animals were injected with increasing doses of TY027 at 162

60 min increments (1, 3, 10, 30 μg) with testing occurring 30 min after each dose. Thirty min was chosen for behavioral evaluation as previous experiments determined this to be the time of peak effect of spinal TY027. Assessments of tactile and thermal hypersensitivities were performed prior to daily compound administration. Twice daily injections of vehicle (10% DMSO) or TY027 (20 μg in 5 μL) occurred on days 2 - 6 at

8:00 am and 5:00 pm with paw withdrawal thresholds and latencies measured 30 min after the morning injection. On day 7, the cumulative dosing protocol was repeated and a dose response curve generated from data collected 30 min after each injection. Data collected on days 1 and 7 were analyzed and compared to each other as a determinant of antinociceptive tolerance.

Twice daily TY027 does not result in antiallodynic tolerance: The paw withdrawal thresholds of SNL-operated rats followed the patterns as reported above (baseline: 14.4 ±

0.6 g; SNL: 4.0 ± 0.3 g). Animals treated with vehicle as controls did not withdrawal the paw ipsilateral to injury at any force significantly different than SNL values at any time throughout the experiment (Figure 22A). Ipsilateral paw withdrawal thresholds obtained prior to the 8:00 am injection did not significantly differ from SNL values in any animal throughout the duration of the experiment (Table 6). It should be noted that animals injected twice daily with TY027 (20 μg) withdrew the SNL-operated paw at thresholds statistically higher than SNL values on day 2 of the experiment (12.6 ± 1.7 g, p < 0.001;

Figure 22A), an effect that was maintained through day 6 (14.2 ± 0.9 g). The correlating antiallodynic activity ranged from 84.2 ±11.2 % and 93.2 ± 7.5 % (data not shown).

Increasing doses of TY027 resulted in a dose-dependent attenuation of SNL-induced 163

tactile allodynia on day 1 of treatment (Figure 22B) as previously observed (Figure 20B).

When the dose response curve was reconstructed on day 7, the observed antiallodynic activity of TY027 was: 45.1 ± 14.2 % (1μg); 100.0 ± 0% (3 μg); 93.5 ± 7.5 % (10 μg) and

100.0 ± 0.0 (30 and 100 μg). When directly compared to the activities observed on day 1, the day 7 DRC had shifted to the left (Figure 22B).

TY027: Evaluation of antihyperalgesic tolerance after multiple exposures: Paw withdrawal latencies to a thermal stimulus were obtained before (baseline: 22.8 ± 0.8 s) and after SNL (13.4 ± 1.4 s). Vehicle treated control animals did not have significantly altered responses to radiant heat through the experiment when compared to SNL values

(Figure 22C). Behavioral measurements obtained before daily injections (vehicle or

TY027) did not vary significantly from post-SNL values (Table 7). Increasing doses of

TY027 produced antihyperalgesia using the cumulative dosing protocol on day 1 (Figure

22D). The DRC regenerated on day 7 had a significantly lower slope compared to that calculated on day 1, indicating that the antihyperalgesic effects of TY027 were not dose related after multiple exposures.

22A 22B 100 15 Vehicle (10% DMSO) TY027 80 12

60

9

SEM) SEM)

( 40

(g 6 %Antiallodynia

3 20 Paw Withdrawal Threshold Withdrawal Paw TY027 (D1) TY027 (D7) 0 0 1 3 2 3 4 5 6 1 3 1 3 BL 10 30 BL 10 30 10 30 SNL Day 1 Days 2-6 Day 7 Dose ( g in 5 L, i.t.) 22C 22D

35 100

Vehicle (10% DMSO) 30 TY027 80 25

60

20

SEM) SEM)

15 hyperalgesia (

(s 40

10 %Anti

Paw Withdrawal Latency Withdrawal Paw 20 5 TY027 (D1) TY027 (D7) 0 0 1 3 2 3 4 5 6 1 3 1 3 BL 10 30 BL 10 30 10 30 SNL Day 1 Day 7 164 Days 2-6 Dose ( g in 5 L, i.t.)

165

Figure 22 TY027 (i.t.) maintains efficacy in a neuropathic pain model. Spinal TY027 was administered to SNL-operated rats using a cumulative dosing paradigm on days 1 and 7, and the A90 dose (20 μg in 5 μL) given twice daily on days 2-6. (A) Tactile and

(C) Thermal paw withdrawal responses were obtained 15 min after each administration.

Dose response curves were generated on Days 1 and 7 for both (B) Antiallodynia and (D)

Antihyperalgesia. In SNL-operated rats, TY027 retained activity after multiple exposures.

166

Table 6: Antiallodynic Tolerance: Daily Paw Withdrawal Thresholds Before Dosing Vehicle TY027 Test Day Mean (g) SEM Mean (g) SEM BL 15.0 0.0 14.1 1.0 SNL 4.3 0.3 3.9 0.5 D1 4.3 0.3 3.9 0.5 D2 7.6 1.5 3.5 0.5 D3 4.3 1.2 3.4 0.4 D4 3.7 0.9 3.6 0.4 D5 3.7 0.4 3.4 0.3 D6 3.5 0.3 3.2 0.2 D7 3.2 0.6 3.5 0.3

167

Table 7: Antihyperalgesic Tolerance: Daily Paw Withdrawal Latencies Before Dosing Vehicle TY027 Test Day Mean (s) SEM Mean (s) SEM BL 25.0 1.2 21.7 0.8 SNL 13.4 1.9 13.4 2.0 D1 13.4 1.9 13.4 2.0 D2 14.2 2.1 14.5 1.7 D3 13.1 1.7 9.9 0.9 D4 11.4 0.9 10.3 1.1 D5 8.6 1.8 10.8 0.9 D6 11.1 1.3 12.4 1.4 D7 9.8 0.8 14.8 2.8 168

Multiple injections of TY027 (i.t.) do not produce antinociceptive tolerance: Sham- operated animals were evaluated for the development of antinociceptive tolerance using the same treatment paradigm as SNL-operated rats. The sham-surgery did not significantly alter the mean paw withdrawal latency on the side ipsilateral to surgery

(baseline: 21.9 ± 0.7s; Sham: 21.3 ± 1.3 s). Vehicle treatment lead to a significant decrease in mean paw withdrawal latency over the course of the experiment as determined by measurement both before the morning injection of 10% DMSO and 30 min after (p < 0.01; Figure 23A). On day 1, animals injected with TY027 (1, 3, 10, 30 μg) removed the ipsilateral paw with increasing latencies (1 μg: 25.3 ± 3.0 s; 3 μg: 28.4 ± 1.9 s; 10 μg: 31.4 ± 1.6 s; 30 μg: 31.2 ± 1.4 s). These values were not significantly different from each other (Figure 23B), but were statistically higher than those obtained from vehicle treated controls (p < 0.01; Figure 23A). Twice daily treatment with TY027 (20

μg) did not change the activity of TY027 (20 μg) 30 min after each morning injection

(Figure 23A). When the dose-response of TY027 was re-evaluated on day 7, the percent antinociception was between 55.6 ± 23.0 (1 μg) and 80.0 ± 20.0 (30 μg) and was not significantly different from day 1.

Together, the percent activities of cumulative TY027 (i.t.) in both SNL- and sham- operated rats indicate that tolerance to the antihypersensitivity effects may not occur after multiple exposures. However, these experiments should be repeated given the standard error of the mean is relatively large (Figures 22 and 23). 169

23A

35

30

25

20 SEM)

15 (s 10 Vehicle

Paw Withdrawal Latency Withdrawal Paw 5 TY027

0 1 3 2 3 4 5 6 1 3 BL 10 30 BL 10 30 Sham Day 1 Days 2-6 Day 7

23B

100

80

SEM) ( 60

40 nociception

20 TY027 (D1)

%Anti TY027 (D7)

0 1 3 10 30

Dose ( g in 5 L, i.t.)

170

Figure 23 TY027 (i.t.) does not result in Antinociceptive Tolerance. Spinal TY027 was administered to Sham-operated rats using a cumulative dosing paradigm on days 1 and 7, and the A90 dose (20 μg in 5 μL) given twice daily on days 2-6. (A) Compared to vehicle treated animals, TY027 retained antinociceptive activity over the 7 day time course. (B) Dose response curves of the calculated antinociceptive activity of TY027 were generated on days 1 and 7 and compared; more data are required. 171

5.3 Development of Reward/ Abuse Potential: Conditioned Place Preference or

Conditioned Place Aversion

A conditioned place preference (CPP) /conditioned place aversion (CPA) protocol was developed as a behavioral measurement of the abuse liability of the lead multifunctional peptide (TY027). Though this assay is well documented in the literature, CPP/CPA was not currently employed at this institution. Four, three chamber automated CPP/CPA boxes were obtained and customized as previously stated (Chapter 2). To verify the tactile/ visual cues were able to instate preference/ aversion related behaviors, control compounds were evaluated including: morphine sulfate, the kappa agonist (-) U50, 488, and saline.

Prior to pairing control compounds with visual and tactile cues, baseline measurements were obtained for both putative and non-associated chambers. Rats (n = 36) spent 582.0

± 58.5 s in the chamber that would be associated with drug during the conditioning phase of the experiment and 548.0 ± 113.0 s in the non paired side. Morphine sulfate (3 mg/ kg) served as the positive control for instating CPP (Bardo et al., 1995) while the kappa opioid receptor agonist (-) U50, 488 (3 mg/kg) served as the positive control for CPA

(Bechara and van der Kooy, 1987). Physiologic saline (0.9%, 1 mL/kg) was used a neutral control. Animals were administered one of the three control drugs every other day for a total of 4 exposures. Behavioral testing occurred 24 hr after the last drug pairing. Rats conditioned with MS spent 833.8 ± 103.0 s in the drug-paired chamber out of a total of 1200.0. Pairing of saline with visual and tactile cues did not affect the time spent in the associated chamber (544.1 ± 137.6 s) as predicted. Multiple exposures to (-) 172

U50, 488 resulted in a decreased total time spent in the drug paired chamber when evaluated 24 hours after the final treatment (396.3 ± 85.0 s). Overall, the visual and tactile cues chosen resulted in CPP and CPA with our control compounds, confirming our reproduction of published protocols (Bardo et al., 1995; Bechara and van der Kooy,

1987).

Supraspinal TY027 does not result in CPP/CPA: To conserve the amount of compound used, the first studies evaluating the potential rewarding properties of TY027 were conducted using intracerebroventricular (i.c.v.) administration. Administration of compound (i.c.v.) was chosen to bathe most components of the mesolimbic reward circuitry. Animals were allowed to acclimate to the testing room for 30 min prior to measurements of BL times, conditioning, experimental times. Two baseline measurements were obtained on consecutive days for each animal; the average served as the overall baseline for all experimental testing (554.1 ± 27.9 s). Animals were exposed to at total of 4 drug-cue pairings (20 min = 1200.0 s) and evaluated 24 hrs after the final conditioning day. Animals treated with saline did not spend a significantly different amount of time in either end chamber when compared to baseline values after 4 injections

(647.1 ± 116.4 s). Morphine sulfate (10 μg) administered directly into the ventricle of rats resulted in a significant increase in the amount of time spent in the pre-conditioned chamber when evaluated (888.3 ± 58.1 s; Figure 24A). Rats pre-conditioned with 10%

DMSO showed a significant decrease in the amount of time they spent on the testing day when compared to BL values (367.1 ± 108.0 s, p = 0.03) but not when compared to saline treated controls (p = 0.09). TY027 (20 μg) did not result in the development of CPP or 173

CPA after four supraspinal injections (654.6 ± 82.1 s; Figure 24A). Since 10% DSMO induced conditioned place aversion with this treatment paradigm, another group of animals was evaluated after conditioning with morphine dissolved in 10% DMSO. The

10% DMSO vehicle attenuated the development of morphine induced CPP as seen by a decrease in total time spent in the pre-conditioned chamber (662.7 ± 143.5 s, Figure

24A). Though place preference was not induced after chronic exposure to TY027, the data indicated that the vehicle (10% DMSO) may have been confounding the results.

To address the problems arising from vehicle- induced CPA, a secondary vehicle for the multifunctional peptide was chosen. Hydroxy-cyclo-β-dextrin (HCBD) was used to solubilize TY027 and brought to a final volume with MPH2O (vol/vol: 18/82). As in the previous experiment, baseline measurements were obtained on consecutive days and the average used per animal (mean: 488.9 ± 33.0 s). Preliminary studies showed that the use of HCBD shifted the development of significant CPP with control compounds to 5 exposures from 4 exposures (data not shown); the protocol for all drug-cue pairings was modified to reflect this finding. Animals conditioned with 18% HCBD did not vary significantly from baseline times 596.8 ± 93.3 s. This time did not vary from those of control rats that were confined to an end chamber but without it being drug-paired (non- injected controls). Together, these data indicated that HCBD (18%) was an acceptable vehicle for continued analysis of the abuse potential of supraspinal TY027 compared to morphine (i.c.v). Pre-conditioning with MS (10 μg, i.c.v) dissolved in HCBD led to a significant increase in total time spent in the drug-paired chamber (827.5 ± 77.5 s; p <

0.01, Figure 24B). Conversely, TY027 (20 μg) did not statistically alter total time spent 174

the drug-paired chamber (712.9 ± 124.9 s) compared to baseline or vehicle-treated controls (p = 0.10 and p = 0.46, respectively; Figure 24B).

Systemic administration of TY027 does not result in CPP/CPA: Rats were prepared with indwelling jugular vein cannula for chronic i.v. administration of compounds. As in previous CPP/CPA experiments, the baseline amount of time rats spent in each of the chambers was recorded prior to conditioning with the drug-end chamber pairings. The mean amount of time spent in the putative chamber of conditioning was 466.7 ± 55.2 s.

Rats were conditioned to associate each compound with the tactile-visual cues for a total of 5 exposures. Non-injected control animals, confined to a chamber but not administered any compound, did not spend an altered amount of time in the conditioning chamber (p = 0.22). Neither saline-conditioned animals nor 20% DMSO exposed rats did not spend a significantly different amount of time in either end chamber when compared to baseline values (p = 0.42 and p = 0.32, respectively, Figure 25). Morphine (3 mg/ kg) preconditioning resulted in a significant increase in total time spent in the drug associate chamber (731.9 ± 33.7 s) after 5 exposures (p = 0.01, Figure 25). Conversely, systemically administered TY027 (6 mg/ kg, i.v.) did not result in an increase or decrease in total time spent in the preconditioned chamber (568.6 ± 95.4 s, p = 0.10). Together, these data indicate that TY027 does not induce conditioned place preference or aversion in non-injured animals after multiple systemic exposures. 175

24A

1000

800

600 SEM)

(s 400

200

0

Total Time Spent in Pre-Conditioned Chamber Pre-Conditioned in Spent Time Total Saline l i.c.v.) l i.c.v.) l i.c.v.) Baseline 24B g/5 10% DMSO g/5 g/5

MS (10 TY027 (20 DMSO-MS (10 1000

800

600 SEM)

(s 400

200

0

Total Time Spent in Pre-Conditioned Chamber Pre-Conditioned in Spent Time Total l i.c.v.) l i.c.v.) Baseline 18% HCBD g/5 g/5 non-injected

MS (10 TY027 (20 176

Figure 24 Supraspinal (i.c.v) TY027 does not result in CPP or CPA. Male SD rats were conditioned using tactile and visual cues to associate one end-chamber of a 3 chamber CPP/CPA apparatus with a given treatment (San Diego Instruments, SDI).

Baseline (BL) values were determined from an average between 2 exposures to an open field of the SDI system prior to compound administration for each animal. On compound administration days, animals were confined to one end chamber (the same throughout the experiment) for 20 min. On the alternate day, animals were allowed access to all chamber of the CPP/CPA box. 24 hours after the 5th exposure, experimental values were obtained. Morphine Sulfate (MS) (A) Saline, MS, vehicle (10% DMSO), TY027 were injected. Compared to baseline values, saline treatment did not result in CPP or CPA while MS, the positive control resulted in a significant development of place preference.

10% DMSO, the vehicle for TY027 produced some, but not significant aversion. TY027 did not induce animals to spend a significant amount of time in the preconditioned chamber compared to baseline. To confirm the lack of CPP observed with TY027 was not due to a masking affect by the vehicle, a final set of animals was treated with MS dissolved in 10% DMSO. These rats did not show a place preference, indicating the vehicle choice was inappropriate. (B) 18% HCBD was chosen as an alternative vehicle for TY027 and MS in follow-up experiments. MS dissolved in 18% HCBD did produce significant CPP after 5 supraspinal injections (p < 0.05). Neither rats injected with TY027 nor those exposed 18% HCBD did not show any preference or aversion. 177

Figure 25

1000 Chamber

ed * 800

ondition 600

C

SEM) Pre-

s 400 (

200

Spent in Spent ime T 0

Total Saline Baseline 20% DMSO

MS (3mg/kg, i.v.) TY027 (6mg/kg, i.v.)

178

Figure 25 Systemic TY027 does not result in CPP or CPA. Male SD rats were conditioned using tactile and visual cues to associate one end-chamber of a 3 chamber

CPP/CPA apparatus with a given treatment (San Diego Instruments). Baseline (BL) values were determined from an average between 2 exposures to an open field of the SDI system prior to compound administration for each animal. On compound administration days, animals were confined to one end chamber (the same throughout the experiment) for 20 min. On the alternate day, animals were allowed access to all chamber of the

CPP/CPA box. 24 hours after the 5th exposure, experimental values were obtained.

Morphine Sulfate (MS). MS (3 mg/ kg induced a significant level of CPP in non-injured rats compared to baseline and vehicle control values (p < 0.05). Conversely, TY027 (6 mg/kg) did not induced CPP or CPA in non-injured rats. Neither saline vehicle nor 20%

DMSO produced significant differences compared to baseline measurements.

179

5.4 TY027 does not induce paradoxical hypersensitivity

Opioids are known to cause abnormal hypersensitivity with prolonged exposure. This is different than antinociceptive tolerance, in that an increased sensitivity to noxious and normally non-noxious stimuli develops in the absence of injury. To conserve the number of animals used in these studies, the hind paw contralateral to injury of rats evaluated in the tolerance studies was tested. Additionally, rats used in abuse liability studies were also evaluated for the development of multifunctional peptide induced hypersensitivity.

Results of these latter animals were directly compared to morphine-treated animals.

Spinal TY027 does not induce paradoxical allodynia: Rats were evaluated for the development of paradoxical allodynia in hind paw contralateral to SNL- or sham- surgery

30 min after the morning injection of either 10% DMSO or TY027.

The paw ipsilateral to the sham surgery was evaluated for development of abnormal tactile hypersensitivity with calibrated von Frey filaments. Before surgery, rats withdrew the ipsilateral hindpaw at a threshold of 15.0 ± 0.0 g and this remained the same 7 days after the sham- surgery (Figure 26A). Neither vehicle treatment nor TY027 resulted in a significant decrease in paw withdrawal threshold for the duration of the experiment after cumulative dosing or twice daily injections of the A90 dose (Figure 26A).

To fully characterize any paradoxical effects of chronically administered TY027 (i.t.), the contralateral paw withdrawal thresholds of SNL- and sham-operated animals were assessed. The mean contralateral paw withdrawal threshold in SNL- rats prior to surgery was 14.3 ± 0.7 g and was not statistically different after the injury (15.0 ± 0.0 g).

Contralateral thresholds of sham- operated control rats were 15.0 ± 0.0 g and 14.6 ± 0.4 g 180

before and after surgery, respectively. The daily mean withdrawal threshold of the contralateral paw did not vary over the 7 day course of treatment from baseline, post-

SNL, or post-sham values (Figure 26B). Vehicle treatment did not significantly alter paw withdrawal Thresholds 30 min after each injection (i.t.). Neither cumulative dosing on days 1 and 7 (1, 3, 10, and 30 μg), nor twice daily injections of TY027 (20 μg) resulted in a decrease in the paw withdrawal threshold to calibrated von Frey filaments in SNL- or sham-operated animals (Figure 26B). 181

26A 15

12

9 SEM)

6 (g

3 Vehicle

TY027 Paw Withdrawal Threshold Withdrawal Paw 0 1 3 2 3 4 5 6 1 3 BL 10 30 BL 10 30 Sham

Day 1 Days 2-6 Day 7

26B

15

12

9 SEM)

6 (g

3 SNL-Vehicle Sham-Vehicle

SNL-TY027 Sham-TY027 Paw Withdrawal Threshold Withdrawal Paw 0 1 3 2 3 4 5 6 1 3 BL 10 30 BL 10 30

SNL/Sham Day 1 Days 2-6 Day 7 182

Figure 26 Spinal TY027 does not induce Paradoxical Hypersensitivity (A) Sham- operated rats were injected with spinal TY027 following the protocol of Figure 22 and evaluated for paw withdrawal responses ipsilateral to surgery 20 min after compound administration. At no point of the experiment did TY027 induce significant hypersensitivity, though day 4 of treatment a dip in withdrawal threshold was observed.

(B) The contralateral paws of both sham and SNL-operated animals treated in the antinociceptive tolerance study were also evaluated for the development of paradoxical allodynia. Neither sham-operated nor SNL-operated rats withdrew the contralateral paw at thresholds significantly difference than pre-surgery values (p > 0.05) 183

Supraspinal TY027 does not induce allodynia: Following either testing or daily conditioning, rats exposed to i.c.v. HCBD, MS, or TY027 were assessed for the development of paradoxical allodynia. Prior to drug- chamber conditioning, baseline paw withdrawal thresholds of the hind paws were obtained and pooled to represent the total hind paw allodynia of each animal. The mean of these values was then calculated

(15.0 ± 0.0 g). Animals exposed 5 times to 18% HCBD (i.c.v.) withdrew the hindpaw in response to calibrated von Frey filaments at significantly lower thresholds after 2 injections (Figure 27A). This allodynia was present throughout the entirety of the study with statistically lower paw withdrawal thresholds ranging from 10.4 ± 1.1 g to 11.5 ±

1.2 g (p = 0.003). Morphine (10 μg, i.c.v.) also induced paradoxical allodynia in rats that was statistically significant after 2 injections (11.2 ± 0.8 g) but was maximal after 5 exposures (7.8 ± 1.5 g, Figure 27A). At no point during the CPP/CPA experiment did supraspinal administration of TY027 (20 μg) result in a significantly decreased paw withdrawal threshold (p = 0.11; Figure 27A).

Systemic TY027 and paradoxical hypersensitivity: Rats receiving a total of 5 i.v. injections of saline (1 mL/kg), 20% DMSO, morphine (3 mg/ kg) or TY027 (6 mg/ kg) were tested for tactile hypersensitivity within 90 min of CPP/CPA measurements or conditioning. The mean paw withdrawal threshold prior to conditioning was 14.0 ± 0.3 g. Animals conditioned with saline did not develop significant tactile hypersensitivity over the duration of the experiment, nor did rats exposed to 20% DMSO (p = 0.12 and p

= 0.48, respectively; Figure 27B). Intravenous MS (3 mg/ kg) produced a significant decrease in paw withdrawal thresholds after 2 administrations (11.1 ± 0.9 g). Behavioral 184

responses to tactile stimuli remained significantly lower than baseline values in these animals throughout the experiment with maximal allodynia (4.5 ± 0.83 g) present 24 hr after the final injection (p = 2.6 *10-18; Figure 27B). Rats injected with TY027 (6 mg/ kg, i.v.) withdrew the hindpaw at significantly lower thresholds than baseline values after

4 exposures to the compound (8.4 ± 1.5 g, p = 0.003). However, 24 hours later, the paw withdrawal threshold had returned to levels statistically similar to baseline (13.0 ± 0.9 g, p > 0.05). This observed effect occurred following the 5th injection of TY027 (8.6 ± 1.5 g) and the following day (10.3 ± 1.6 g). When the area under the curve is compared intravenous TY027 produces significantly less allodynia than morphine with the same treatment paradigm (p = 0.02). It should be noted that the onset of TY027-induced allodynia was delayed compared to that of morphine and no allodynia was observed in the absence of drug (Figure 27B).

Though tactile hypersensitivity did develop after multiple systemic exposures to

TY027, the overall side effect profile of TY027 was reduced when compared to the clinically used opiate, morphine. Specifically, TY027 did not produce antinociceptive/ tolerance in SNL- or sham-operated animals or result in significant CPP/CPA after central or systemic exposure.

185

27A 15

12

9 SEM)

6 (g Vehicle (18% HCBD) 3 TY027 (20 g)

MS (10 g) Paw Withdrawal Threshold Withdrawal Paw

0 1 2 3 4 5 6 7 8 9 BL Day

27B 15

12

9 SEM)

6 (g Saline 20% DMSO 3 TY027 (6 mg/kg) Paw Withdrawal Threshold Withdrawal Paw MS (3 mg/kg)

0 1 2 3 4 5 6 7 8 9 BL Day 186

Figure 27 Evaluation of Paradoxical Allodynia after Supraspinal and Systemic

TY027. Rats injected with TY027 (i.c.v. and i.v.) were used for evaluation of reward potential were also assess for the development of tactile allodynia. Probing with von

Frey filaments occurred between 75 min and 90 min following drug exposure. (A)

Supraspinal administration of both 18% HCBD and MS resulted in significant allodynia compared to baseline values beginning after the second exposure. TY027 exposed animals did not show significant decreases in paw withdrawal thresholds, though on some days a trend in this direction was noted (p = 0.11, ANOVA) (B) Neither systemic

(i.v.) administration of saline nor 20% DMSO resulted in significant decreases in paw withdrawal latency when compare to baseline (p = 0.11 and 0.48, respectively). MS (3 mg/ kg) induced significant tactile allodynia after the second exposure and decreased continually until the final assessment (p < 0.00001). TY027 treated animal showed a delayed development of hypersensitivity with the onset occurring after the 4th exposure.

Unlike MS, TY027-induced hypersensitivity resolved in the absence of compound.

Arrow heads indicate day of drug exposure. 187

5.5 TY027 and Reward Circuitry

To further confirm the behavioral results of the abuse liability of TY027 immunohistochemical (IHC) and electrophysiological studies were performed. The VTA is part of the mesolimbic reward pathway. Morphine and other opioids are known to increase the firing rate of dopaminergic neurons (DA), presumably by disinhibitory mechanisms (Figure 28). The direct effects of NK1 antagonists on opioids in this system are not known and the presence/absence of NK1 receptors in the VTA requires clarification.

Immunohistochemical analysis of VTA: Naïve, male Sprague Dawley rats were perfused as described above. Whole brain and spinal cord tissue were harvested for single staining of the enzymes: tyrosine hydroxylase (TH, dopaminergic marker) and glutamic acid decarboxylase, 65 kDa isoform (GAD65, GABAergic marker), and the mu-opioid

(MOR) and NK1 receptor.

In control tissue, the MOR receptors were visualized using the optimized protocol in

Lamina 1 and 2 of the dorsal horn of the spinal cord. Likewise, NK1 receptors were localized in the deeper lamina (3 - 6), though the antibody dilution was changed from

1:5000 to 1:3000). Furthermore, TH and GAD65 were seen in the substantia nigra pars compacta and hippocampus, respectively (data not shown).

Individual brain sections were stained with each of the selective antibodies to determine localization in the VTA. Representative confocal images of VTA brain section

(Anteroposterior: -5.2 to -6.04 mm) are shown in Figure 29. Sections were positive for

TH immunoreactivity confirming the presence of DA containing cells in the VTA; cells 188

containing the GAD65 enzyme were also visualized (Panel B). Visualization of the NK1 receptor was strong on cell bodies, particularly in the caudal VTA (Figure 29C), though more studies are required for confirmation of this observation. A final set of sections were stained for MOR which was visualized on a small portion of fibers and cell bodies

(Figure 29D). It should be noted that supraspinal labeling of MOR-containing cells was not as strong as that observed in control tissue. There was an increase in the ratio of background to signal intensity. This latter staining in solidarity is inconclusive as to the presence of MOR in the VTA. The data provide evidence that, under our conditions in male Sprague-Dawley rats the enzymes, TH and GAD65, as well as the NK1 receptor are present in the VTA. In addition MOR is most likely expressed, suggesting that dopamine, GABA, SP- NK1, and MOR-endogenous opioids are present in the reward circuitry of the VTA.

Figure 28 Nucleus Nucleus TY027 Accumbens, Accumbens, • inhibits GABA at μOR to disinhibit Amygdala, Amygdala, DA+ Prefrontal •AND inhibits SP-NK mediated Prefrontal Morphine 1 Cortex activity in DA+ toward basal activity Cortex • inhibits GABA to disinhibit DA+ • ↑ DA release in NAc

Dopaminergic Dopaminergic +++ +

TY027 GABA+ GABA+ + +

Glu+ Glu+ ? ?

Ventral Tegmental Area (VTA) Ventral Tegmental Area (VTA)

- GABAA Cl NMDA/ AMPA μOR NK1 GABA Glutamate Opioid agonist Substance P DA

189

190

Figure 29

191

Figure 29 VTA Immunohistochemistry: TH, GAD65, MOR and NK1. Confocal representative images showing (A) tyrosine hydroxylase (TH, 1:300), (B) glutamic acid decarboxylase (GAD65, 1:5000), (C) NK1 receptor (1:3000) and (D) μOR (MOR,

1:4000) staining in the VTA (50 μm). A minimum of 9, non consecutive sections were labeled with each antibody. Secondary antibodies used were donkey, anti-mouse Cy2

(1:200, Jackson) and donkey, anti-rabbit Cy3 (1:600, Jackson). Images were obtained with an Olympus Confocal system at 60x magnification with a 2 μm Z- stack. Color was modified using Adobe Photoshop for distinction between stains. As expected, dopaminergic and GABAergic cells were present in the VTA as marked by TH and

GAD65, respectively. NK1 receptors were observed in the VTA under this protocol.

MOR staining was difficult to discern from the background; however it was observed. 192

Electrophysiology: Study of the electrophysiological responses of DA neurons in the

VTA following systemic TY027 is underway. Preliminary results using a cumulative dosing protocol and treating the cells as a single population are shown in Figure 30. It should be noted that the VTA is a heterogeneous population of cells and responses amongst different populations may also be different. More data are required before conclusions may be drawn. 193

30A 5

4

3

SEM) (

Firing rate Firing 2

1

All animals 0 1 2 4 6 10 BL apo Saline

20% DMSO TY027 - cumulative dose (mg/kg, i.v.) 30B

120

90

60

Firing rate Firing 10

30

SEM) (

0

%Change in A in %Change -30 All animals

-60 1 2 4 6 10 BL apo Saline

20% DMSO TY027 - cumulative dose (mg/kg, i.v.)

194

Figure 30 Preliminary Electrophysiological effects of TY027 in the VTA

Electrophysiological data from a total of nine VTA dopamine neurons were obtained. (A)

Preliminary analysis has revealed that within the single, heterogeneous population, cumulative dosing with TY027 (i.v.) trends toward an increase in firing rate when compared to baseline rates, though significant differences are not observed (p = 0.41). (B)

When the data were transformed into percent change from baseline rates, significance was still not achieved.

195

CHAPTER 6:

DISCUSSION

The need for new therapeutic options for treating chronic pain, specifically neuropathic pain, is great. Chronic pain of various etiologies costs billions of dollars worldwide and affects approximately 10 - 20 % of the population at some point throughout the average lifetime (Noble et al., 2010). In the United States, as many as 75 % of these patients fail to achieve adequate pain relief with currently available therapies (American Pain

Foundation, 2008).

Acute pain, arising from tissue injury or noxious input, is reasonably well-controlled with opioids. Neuroplastic adaptations within nociceptive circuitry lead to an alteration of transmission and ultimately change processing of the pain signal (Seybold, 2009;

Vanderah, 2007). Neuropathic pain arising from injuries to peripheral nerves is often intractable clinically and much effort has been devoted to understanding the underlying mechanisms for this pain state. Treatments for neuropathic pain have been outlined and include tri-cyclic antidepressants, selective serotonin reuptake inhibitors, α2δ1 subunit calcium channel blockers, local anesthetics and opioids (Dworkin et al., 2008, Jensen et al., 2007). Despite the advances which have been made in understanding the mechanisms underlying neuropathic pain and the therapeutic agents available, opioids remain one of few options for many patients.

Opioids are the gold standard for treating both acute and breakthrough pain; however, the efficacy of chronic opioid therapy is limited by the presence of adverse effects. 196

Recent reports have found that opioids achieve greater pain relief than other therapeutic options (e.g. tricyclic antidepressants) for chronic pains but at least 80% experience one adverse effect (e.g. constipation, somnolence, pruritis, etc.) (Dworkin et al., 2008; Jensen et al., 2009), while less than half remained on the same therapy up to 24 months because of these side effects (Jensen and Finnerup, 2007). Adverse effects resulting incessation of opioid therapy were found to be more prevalent in patients receiving daily dosing rather than as needed (Hojsted and Sjogren, 2007). In addition to common side effects

(constipation, nausea/ vomiting, somnolence), the development of antinociceptive tolerance, opioid-induced hyperalgesia, physical dependence and the potential for psychological dependence were noted as contributing factors to the patient‟s decision to continue therapy (Rowbotham et al., 2003; DuPen et al., 2007; Chu et al., 2008; Noble et al., 2010).

Concomitantly, the use of chronic opioids and the risk of addiction has been the focus of much sociopolitical debate. In the U.S., it has been estimated that 48 million people will use prescription drugs for nonmedical reasons with an estimated 4.7 million individuals opioids as their drug of abuse (Volkow, 2004). Clinical investigations have revealed the prevalence of addiction in chronic pain patients receiving opioids to be less than 1 % (Noble et al., 2010); those with psychological dependence reported having prior struggles with substance abuse. Despite the low level of incidence of addiction in chronic pain patients, the concern of abuse for physicians and patients alike is great and is reflected in prescribing practices and cessation of treatment (Ballantyne and LaForge,

2007). 197

The complexities underlying the development of opioid analgesia, antinociceptive tolerance, OIH and opioid side effects have long been studied preclinically and clinically.

Neurobiological plasticity within nociceptive pathways has been implicated as a mechanism underlying impaired efficacy of long-term opioid therapy and strategies that oppose these adaptations may result in enhanced pain relief with diminished side-effects

(i.e., antinociceptive tolerance and reward) compared to opioids alone. Compounds targeting changes within the opioid system (e.g. atypical G protein coupling of the MOR receptor) and opioid-induced increased in pronociceptive modulators (SP- NK1 signaling) have been assessed for efficacy in acute and neuropathic pain models. Furthermore, the consequences of long term exposure to these compounds, specifically antinociceptive tolerance, paradoxical sensitivity, and abuse liability were evaluated.

6.1 MOR- G protein coupling

Presently, the data support L5/L6 SNL-induced MOR coupling to Gαs proteins in the spinal dorsal horn, indicating emergence of excitatory signaling by these receptors instead of the typical inhibition via Gαi/o coupling. This excitatory signaling by MOR may contribute to the hyperalgesia associated with neuropathic injury and the lack of efficacy of chronic morphine administration in some patients. Animal models of neuropathic pain have shown that nerve injury induces a host of pronociceptive, neuroplastic changes including: an upregulation of dynorphin in the spinal cord, enhanced levels of CCK, increased activation of phosphorylated p38 mitogen activiated protein kinase, and increased content and evoked release of substance P and CGRP 198

(Abbadie et al., 1996, 2003, 2005; Allen et al., 1999; Burgess et al., 2002; Gellert and

Holtzman, 1979; Nichols et al., 1997; Svensson et al., 2006; Xu et al., 1993). Up- regulation of cytokine receptor mRNA in the sciatic nerve and dorsal root ganglion after partial spinal nerve ligation has been noted, and SNL has also been reported to decrease

MOR density in injured peripheral axons in the ipsilateral Lamina I of dorsal horn, leading to changes in pre- and post-synaptic evoked responses of MOR-containing fiber

(Abbadie et al., 2003; Allen et al., 1999; Sapunar et al., 2005). These synaptic changes include a decrease in presynaptic MOR-mediated inhibition, reduced inhibition of the miniature excitatory post-synaptic potentials in substantia gelatinosa neurons, a shortened after-hyperpolarization duration in C-fibers in the L5 DRG, and increased repetitive firing of A fibers during sustained depolarization (Kohno et al., 2005; Sapunar et al., 2005).

Here, the data demonstrate a nerve injury-induced neuroplastic alteration in MOR–G protein coupling profile in ipsilateral lumbar spinal cord tissue of SNL animals.

Previously, it has been shown that chronic opioid administration causes a shift in

MOR–G protein coupling from Gαi/o to Gαs (Wang et al., 2005), the present work now shows that SNL injury induces this novel MOR–Gαs coupling ipsilateral to the injury without changing the extent of coupling to Gαi/o proteins. Chronic oxycodone administration to nerve-injured rats contributed to this Gαs coupling, as chronic oxycodone treatment significantly enhanced the level of Gαs coupling compared to that seen in the ipsilateral dorsal horn of vehicle-treated SNL animals. Additionally, MOR–

Gαs coupling was detected in both ipsilateral and contralateral dorsal horn of oxycodone- treated SNL rats, whereas the SNL injury itself induced Gαs coupling only in the 199 ipsilateral dorsal horn. This alternative coupling by MOR and its augmentation by chronic oxycodone treatment may be an indication of the development of opioid-induced hyperalgesia and a decreased efficacy of chronic opioid in neuropathic pain states, observed in animals and in some humans (Ali, 1986; Chu et al., 2006; Devulder, 1997;

Dworin et al., 2007; Finnerup et al., 2005; Fan et al., 1991; Mao, 2002; Ossipov et al.,

2005a,; Simonnet and Rivat, 2003; Vanderah et al., 2001b). Prolonged opioid treatment has also been shown to alter the function of Gβγ subunits of G proteins coupling to

MORs resulting in multiplicative excitatory effects such as: phosphorylation of Gβ and enhanced Gβγ stimulation of adenylyl cyclase, phosphorylation of G protein receptor kinase2/3 and G via protein kinase C, and increased activity of PKA, of which the latter can also be activated by Gαs stimulation of adenylyl cyclase (Chakrabarti et al., 1998a, b,

2001; Chakrabarti and Gintzler, 2003; Chakrabarti et al., 2005a,b; Gintzler and

Chakrabarti, 2006; Rivera and Gintzler, 1998). The G that associates with adenylyl cyclases during opioid tolerance has now been shown to originate from the Gαs protein coupling to MOR (Wang and Burns, 2006). In conjunction with increased excitatory signaling via MOR-associated Gαs in an injury state, the Gβ released by these Gαs heterotrimeric proteins may further enhance the analgesic tolerance and hyperalgesia observed clinically.

6.2 ULD NTX attenuated opioid- induced, MOR-Gαs coupling and hypersensitivity

Chronic co-treatment with ultra-low-dose naloxone diminishes the MOR–Gαs protein coupling induced by chronic opioid administration in intact uninjured rats (Wang et al., 200

2005); here, the MOR–Gαs coupling following SNL injury was significantly attenuated by ultra-low-dose NTX co-treatment. Although ultra-low-dose NTX might only have reduced the chronic opioid-induced MOR–Gαs coupling and may not have specifically reduced the injury-induced MOR–Gαs coupling, the combination treatment resulted in a significant enhancement in antihyperalgesic and antiallodynic behavioral responses when compared to oxycodone treatment alone. Furthermore, rapid analgesic tolerance occurred to the effects of oxycodone alone, while tolerance to the antihyperalgesic and antiallodynic effects of oxycodone + NTX was delayed. Both intrathecal and oral co- treatment with ultra-low-dose NTX enhanced the antihypersensitivity effects and diminished the tolerance seen with oxycodone alone. These doses of NTX alone were without effect, illustrating an interaction with the effects of the opioid agonist and underscoring NTX‟s mechanism via high-affinity binding to the scaffolding protein filamin A (Wang et al., 2008). A dose >1000-fold higher, delivered intrathecally, blocked the opioid effect completely for nerve-injury induced hypersensitivities by directly antagonizing opioid receptors.

The dose-response curve generated from noxious, thermal stimulation had a gradual slope, indicating slight dose-dependency for ultra-low-dose NTX in the reversal of thermal hyperalgesia. Oral delivery of the combination therapy also produced a dose- dependent antiallodynic effect. These data are encouraging since opioid therapy has little efficacy in ameliorating the allodynia of neuropathic pain (Arner and Meyerson, 1988;

Tasker et al., 1983). Previous studies have not noted dose-dependent effects within a fairly wide but effective ultra-low-dose range of NTX for enhanced analgesia in intact 201 animals; literature does support a dose-response with NTX only with increasing doses corresponding to classic receptor antagonism (Burns, 2005; Olmstead and Burns, 2005).

Again, the relatively flat dose response curves can be explained by saturation of the high affinity, filamin A binding sites well before antagonism at opioid receptors can occur

(Wang et al., 2008). Interestingly, the doses of NTX used in this study are 10- to 100- fold higher than the NTX doses shown to enhance analgesia and prevent tolerance in intact animals, yet 100-fold to 1000-fold lower than doses used to block opioid analgesia via MORs (Crain and Shen, 1995; Gellert and Holtzman, 1979; Lysle et al., 1993; Powell et al., 2002; Wang et al., 2005; Young et al., 1991). Though this has been the first study to assess oral delivery of an ultra-low-dose opioid antagonist in animals, a direct comparison with intrathecal NTX was possible with the Powell study (2002). The multitude of neuroplastic changes underlying neuropathic pain, including the extent of

MOR–Gαs coupling now thought to be controlled by filamin A (Wang et al., 2008), may contribute to the need for somewhat higher doses of NTX in alleviating hypersensitivities in nerve-injured animals compared to NTX doses reported to enhance opioid analgesia in intact rodents.

The present study demonstrates that SNL injury, like chronic opioid treatment, induces

Gαs coupling by MOR. This aberrant signaling may contribute to the excitatory neurotransmission in spinal dorsal horn after neuropathic injury as well as to the decreased efficacy of endogenous and exogenous MOR agonists in alleviating neuropathic pain. The MOR–Gαs coupling may also contribute to the development of opioid analgesic tolerance, opioid-induced hyperalgesia, and differences in mu opioid 202 agonist efficacy (Chakrabarti and Gintzler, 2003; Connor and Christie, 1999; Goode and

Raffa, 1997; Szucs et al., 2004; Wang et al., 2005; Wang and Gintzler, 1997). Although the ultra-low-dose NTX attenuation of injury-induced MOR–Gαs coupling was mild compared to the suppression of chronic opioid-induced MOR–Gαs coupling in intact animals, oxycodone + ultra-low-dose NTX produced profound and significant antihypersensitivities in the L5/L6 SNL model. These data suggest a common mechanism of action for certain ultra-low-dose opioid antagonist/opioid combinations in preventing analgesic tolerance and in alleviating hypersensitivities in SNL rats.

6.3 Rational Development of Opioid Agonists/ NK1 Antagonists

Sustained administration of opioids can lead to the development of antinociceptive tolerance (Ossipov et al., 2005), which may result in higher doses being administered to achieve adequate pain relief. Repeated exposure of opioids has also been shown to produce OIH both pre-clinically and clinically (Dworkin et al., 2008; Vanderah et al.,

2001b), which may be the behavioral representation of antinociceptive tolerance as a result of altered G-protein coupling and increases in excitatory neurotransmitter content and release (Gu et al., 2005; Vanderah, 2007).

Substance P content and release has been increased in the spinal cord after sustained opioid treatment (King et al., 2005) and inflammation (Malcangio et al., 2000). After peripheral nerve injury, SP levels have been reported as both increased and decreased, as well as redistributed, in the spinal cord and DRG (Ma and Eisenach, 2003; Wallin and

Schött, 2002; Bisby and Keen, 1986, Swamydas et al., 2004). Exogenous administration 203 of SP is pronociceptive in rats and mice (Hylden and Wilcox, 1982; Seybold et al., 1982).

Similarly, changes in NK1 receptor level have been reported in the spinal cord (Malmberg et al., 1997; Goff et al., 1998). Interactions between SP – NK1 receptors may be altered after chronic opioids and nerve injury. An increase in receptor internalization has been reported after sustained opioid treatment (King et al., 2005) while chronic constriction of the sciatic nerve results in an increase in the affinity of SP to NK1 (Aanonsen et al., 1992)

Previous studies using NK1 receptor antagonists alone for the treatment of pain have delivered mixed results. Depending on the stimulus and the intensity thereof, both antinociception and very little effect on acute nociception with the NK1 antagonists have been reported (Garces et al., 1993; Hill, 2000; Rupniak et al., 1993). Despite these differing results and the seeming lack of effect in producing acute antinociception, spinal

NK1 antagonists have been reported to alleviate both injury-induced thermal and tactile hypersensitivities in rodent models (Cahill and Coderre, 2002; Cumberbatch et al., 1998).

Clinically however, the NK1 antagonists failed in pain trials, possibly due to differences between species (Hill, 2000). The lack of NK1 antagonism alone in these models suggests that other pain neurotransmitters including glutamate and CGRP are enough to transmit the message of nociception (Hill, 2000; Woodcock et al., 2007).

The present study demonstrates the effects of rationally designed compounds that take advantage of two distinct mechanisms, opioid receptor activation and NK1 receptor blockade, may attenuate acute and neuropathic pain following acute and chronic exposure. Moreover, two of these multifunctional compounds, TY005 and TY027, have 204 been studied extensively for their lack of the development antinociceptive tolerance, paradoxical hypersensitivity, and/ or abuse liability.

6.4 Multifunctional peptides and Pain

Initial Screening: Over 20 compounds were initially screened in a rodent model of acute nociception. JSOH11 was designed as bivalent combination of deltorphins and spantide, a commercially available NK1 antagonist; the compound had greater DOR selectivity over MOR (1:9) in binding assays and a 1: 2 ratio of functionality at the same receptors.

Notably, JSOH11 had no reported activity at the NK1 receptor, limiting analysis of this peptide to the opioid activity. Literature has reported that greater DOR selectivity may not result in as much antinociceptive activity as a MOR agonist (Beaudry et al., 2009) though in inflammatory and neuropathic models, delta agonists do show efficacy (Bilsky et al., 1995; Gallatine and Meert, 2005; Holdridge and Cahill, 2007). The use of a combined MOR- DOR ligand in vivo, given the underlying complexities in understanding

DOR functionality, has been proposed (Ananthan, 2005; Beaudry et al., 2009). As a general concept for a multifunctional approach to pain therapeutics, JSOH11 was a success.

In the formalin assay of chemical-induced pain, JSOH11 was effective in blocking the second phase of flinching, though this may be due to activity at the MOR and not the

DOR (Barr et al., 2003). In the paw flick assay, spinally administered JSOH11 did not produce significant antinociception. These findings of lower antinociceptive effect with delta selective compounds have been previously reported in the literature, though the 205 same literature supports the use of a mu- / delta- mixed ligand (Gallantine and Meert,

2005; Ananthan, 2005; Dietis et al., 2009). Additionally, toxicities were observed at moderate doses of JSOH11 (30 μg). Motor impairment was observed in some animals.

Interestingly, a biphasic locomotor effect has been observed after acute exposure to both peptidic and small molecule agonists of DOR (Fraser et al., 2000), though most reports support DOR-mediated increases in locomotor activity (Jutkiewicz et al., 2003). JSOH11 also induced convulsions, consistent with previous reports of some delta agonists

(Jutkiewicz et al., 2003). The presence of the dose-limiting and severe side effects in the absence of significant antinociception precluded a full characterization of JSOH11 in vivo.

TY005: After initial testing with JSOH11, the design of the multifunctional peptides was assessed and the opioid and NK1 pharmacophore analogues were altered. The resulting family of compounds, TY-series, was chosen following in-depth analysis of in vitro data including affinity and activity at the opioid receptors and affinity in the absence of activity at the rat and human NK1 receptors. Analysis of functionality in vitro revealed that TY004 and TY005 were candidates for in vivo investigations. Between the two compounds, the DOR/ MOR selectivity ratios were 1:3 and 1:16, respectively.

However, the NK1 antagonist activity of TY004 and TY005 were nearly identical. In vivo comparison of the two compounds showed that both compounds administered spinally produced antinociception. The duration of action of TY005 was longer than that of TY004; therefore, TY005 was chosen for continuing characterization. 206

The data show that, in non-injured rats, targeting the mu- and delta- opioid receptors while blocking the NK1 receptor with TY005 results in a dose-dependent antinociception, following acute, spinal administration. This TY005-mediated antinociception occurred without motor impairment, dissimilar to the previous compound JSOH11. However, a relatively short duration of action (< 30 min) was observed following both spinal and systemic TY005 administration. A search of available literature revealed that the in vivo duration of action, peak effect, and potency of TY005 (i.t.) fall within the range of remifentanil (<10 min, 0.1-10 μg in 10 μL) and alfentanil (10 - 30 min, 10 - 100 μg in 10

μL) after intrathecal administration (Buerkle and Yaksh, 1996). Short term application of opioid agonists, such as remifentanil, can lead to the development of hyperalgesia after acute withdrawal (Angst et al., 2003) and may induce long term potentiation of C-fibers

(Drdla et al., 2009). Though these phenomena were not statistically significant a trend was observed and future investigations may be warranted.

The multifunctional properties of TY005 were shown using in vitro assays, but confirmation of these activities in vivo was required. Using selective antagonists, TY005 had activities at both the MOR and DOR. Spinal TY005 blocked SP-induced nocifensive behaviors confirming NK1 receptor antagonism in vivo. These finding were consistent with the in vitro characterization of TY005 at the MOR, DOR and NK1 receptors.

Additional evaluation of the designed pharmacophore fragments (TY036 and TY040) also confirmed that the individual pieces had effects similar to those of TY005 indicating the short linkage of the pharmacophores minimally impacted overall antinociceptive activity. 207

Given the conflict amongst literature and our own results showing increases in both SP content and release, as well as, NK1 internalization after chronic opioid therapy (King et al., 2005a, b) or peripheral nerve injury (Aanonsen et al., 1992; Malmberg et al., 1997;

Swamydas et al., 2004) the efficacy of this bifunctional peptide in a model of peripheral neuropathic pain was evaluated after spinal and systemic administration. SNL rapidly induces both thermal hyperalgesia and tactile allodynia as previously reported (Kim and

Chung, 1992). Notably, TY005 fully attenuated tactile allodynia, while morphine alone

(i.t.) had less than a 20% antiallodynic effect in a spinal nerve ligated animal, as previously reported (Bian et al., 1995; Lee et al., 1995; Ossipov et al., 1995), though other groups have demonstrated greater attenuation of nerve-injury induced allodynia after morphine (i.t.) (Hwang et al., 2000; Zhang et al., 2005; Zhao et al., 2004).

Interestingly, delta agonism has been reported to be effective in mechanically-mediated pain in various models (Mika et al., 2001; Pereira Do Carmo et al., 2009; Scherrer et al.,

2009). The synergism of TY005 at DOR and MOR may partially explain the full reversal of SNL- induced allodynia. A recent report from Takeda et al., implicates a role of SP at

NK1 in allodynia resulting from temporomandibular joint pain and inflammation (2005), while a role for SP-NK1 is also implicated in other models (Torsney and Mac Dermott,

2006; Zhang et al., 2008).

Chronic administration of MOR agonists is often related to the development of antinociceptive tolerance in preclinical models and more importantly in patients (Ossipov et al., 2005). Previous studies showed that morphine induced antinociceptive tolerance and hyperalgesia could be attenuated or reversed by co-treatment with an NK1 antagonist 208

or prevented when cells expressing the NK1 receptor in the spinal dorsal horn were ablated using a conjugated neurotoxin (Powell et al., 2003; Vera-Portocarrero et al.,

2007). Furthermore, PPTI gene knockout animals, which lack both SP and NKA, do not develop antinociceptive tolerance (Guan et al., 2005). The present studies with TY005 suggest that an opioid agonist in combination with an NK1 antagonist does not result in antinociceptive tolerance compared to morphine following spinal administration in sham or nerve injured animals. Overall, spinally administered TY005 has activity comparable to that of morphine in response to normally painful stimuli in a non injured or injured state but increased efficacy against allodynia induced by nerve injury.

Systemic morphine has been shown to attenuate L5/L6 tactile allodynia, yet spinal morphine has less than significant efficacy in such models (Bian et al., 1995; Joshi et al.,

2006; LaBuda and Little, 2005). TY005 given systemically attenuated SNL-induced hypersensitivities to the same levels observed after intrathecal TY005 administration.

These results suggested that TY005 may cross the blood brain barrier. In a single time point analysis, the data confirm that TY005 has potential to cross the BBB. However, as noticed in the previous assessment of spinally mediated antinociception, systemic TY005 had a time of peak effect around 15 min which was diminished by 30 min. This short duration of action and further chemical analysis revealed the metabolic stability of

TY005 was poor. Determination of plasma half-life confirmed that the parent compound was metabolically unstable, leading the investigation to possible metabolite effects.

Spinal assessment of the proposed metabolite, TY043, provided evidence that the observed effects of TY005 may have been a result of metabolic instability. 209

TY027 and Pain Models: To overcome the challenges incurred with TY005, the multifunctional peptide was restructured, changing the ester linkage to an amide linkage.

This alteration did not significantly lessen the binding or activities of compound in vitro, and in fact, increased the compound affinity for the hNK1 receptor. Additionally, the plasma half life and BBB penetrability were increased indicating a decreased susceptibility to degradation (Yamamoto et al., 2009).

Homology between the rNK1 and hNK1 receptors is approximately 95%, but major differences that affect binding occur in the transmembrane domains (Fong et al., 1992).

Selectivity of small molecule antagonists for each receptor has been a challenge which prohibited clinical success of these compounds for pain (Hill, 2000). The bivalent peptides were able to bind to both the rNK1 and hNK1 receptors with nM selectivity. The cross species affinity and antagonist activity of the peptides at the NK1 receptor may be due to spacing of the peptides, allowing them to interact with particular amino acid residues despite differences in helical packing of the receptor (Fong et al., 1992). Unlike the small molecule antagonists, TY005 and TY027 retain antagonist properties at NK1 receptors in vitro and in vivo, shown presently.

Though the modification was made in the C-terminal of the peptide, the antinociceptive effects of TY027 were reassessed, including the activities at both the opioid and NK1 receptor systems. TY027 dose-dependently produced antinociception without motor impairment in non-injured animals, both rats and mice, similar to TY005.

Antinociception was a result of opioid activity, as the non-selective opioid antagonist, 210

NLX, fully blocked TY027-mediated effects. Individual contributions of MOR and DOR activity were not evaluated with TY027, as the overall affinities at these receptors were not significantly different than those of TY005. Like TY005, TY027 (i.t.) was able to block exogenously applied SP- flinching and biting compared to vehicle treated animals.

However, when compared to each other, blockade SP-induced flinching was greater with

TY005 than TY027. This outcome was somewhat expected, given the differences in affinity at the rNK1 receptor in vitro.

TY027 maintained efficacy when compared to TY005 after spinal administration, with a dose-dependently increased duration of action against normally noxious stimuli in a model of neuropathic pain. Interestingly, the duration of action of TY027 remained less than an hour for alleviating SNL-induced allodynia, but reemerged at 120 min after intrathecal administration with 30 μg. This “second phase” of TY027- antiallodynia was most likely due to formation of a metabolite, though additional studies are required to confirm. However, a similar trend was observed following systemic administration.

Specifically, the peak antiallodynic effect of TY027 (10 mg/kg) occurred 90 min after i.v. injection with a second peak, statistically lower than the first, but higher than post SNL baseline measurements observed at 210 min. Neither the second peak nor an increased duration of action was noticed in hypersensitivity assays after systemic administration.

Differential activities of compounds against tactile allodynia and thermal hyperalgesia have previously been reported (Ossipov et al., 1995; Scherrer et al., 2009). However, the time dependent actions of TY027 following systemic administration may be explained by 211 kinetics, as well as binding to lipid membranes and bioavailability. Further studies are needed to investigate these differences.

Overall, the rationally designed, multifunctional, bivalent peptides targeting the MOR,

DOR and NK1 receptors were effective in rodent models of acute and neuropathic pain.

Translation of these compounds into clinically useful therapies would be assisted by the lack of side effects such as antinociceptive tolerance, OIH, and decreased abuse liability

(DuPen et al., 2007; Chu et al., 2008; Ballantyne and La Forge, 2007). Modifications of

TY005 into TY027 bridged the gap in metabolic stability while maintaining positive effects of pain relief.

6.5 TY027: Antinociceptive Tolerance, Paradoxical Hypersensitivity, and Abuse

Liability

The modifications made to TY005 resulted in a new lead compound, TY027.

Confirmation of retained antinociceptive efficacy and activity in a rodent model of neuropathic pain following acute central and systemic exposures led to the question of

TY027‟s ability to maintain effects after multiple exposures. Antinociceptive tolerance is defined as the need to increase dose to maintain the same therapeutic effect. As reported previously, morphine treatment results in antinociceptive tolerance in preclinical and clinical settings, further increasing the severity of other side effects (Koch and Hollt,

2008). 212

The present studies have shown that TY027 does not lose antiallodynic activity after 7 days exposure in SNL-animals and becomes more potent as shown by a leftward shift in the DRC. However, this may be due to accumulation of peptide in lipid membranes which has been shown to alter activity (Yamamoto et al., 2008) or blockade of NK1 receptor interactions with other systems affecting tactile allodynia (e.g. paracine; Zhang et al., 2008). In the same animals, TY027 retained antihyperalgesic activity over the 7- day treatment paradigm, though overall shifts were difficult to determine. These tolerance studies in SNL-animals should be repeated for further clarification. In sham- operated animals, the chronic exposure to spinal TY027 resulted in some, but not significant, decrease in efficacy by the 7th day. These data confirm previous reports that co-administration of an opioid agonist with an NK1 receptor antagonist prevents the development of antinociceptive tolerance and reverses established morphine hypersensitivity (Powell et al., 2003).

The search for analgesics with a decreased abuse liability has been prodded by the ever increasing abuse of prescription drugs. According to the National Institute of Drug

Abuse, 5.2 million Americans misused prescribed opioid medications in 2006 (NIDA,

2008). The mechanism by which opioid exposure induces physical dependence and reward is largely unknown, though it has been suggested that opioid reward is due to inhibition of the tonically active GABAergic neurons in the VTA leading to an increase in dopamine (DA) levels in multiple brain regions (e.g. nucleus accumbens, prefrontal cortex). However, this increase in DA is evident under both rewarding and aversive circumstances, including persistent mu and kappa opioid agonist exposure (Joseph et al., 213

2003). MOR activation results in rewarding effects while KOR activation is adversive. It is not known at this time if delta opioid agonists produce preference or aversion, though administration of both DOR selective agonists and antagonists reportedly modify morphine-induced CPP (Lenard et al., 2007; Liang et al., 2006). Furthermore, increasing literature points to interdependency of the opioid receptor system upon different neuromodulatory systems as the underlying mechanism of opioid reward (for review, see

Bryant et al., 2005). Additionally, SP administration has been shown to induce CPP

(Hasenohrl et al., 1989; Holzhauer- Oitzl et al., 1988). Pharmacological antagonism at the NK1 receptor has decreased morphine reinforcement in self-administration studies

(Placenza et al., 2006) and NK1 receptor knockout mice do not develop place preference or self-administer morphine (Murtra et al., 2000; Ripley et al., 2002). These genetically modified animals display normal rewarding behaviors in response to cocaine or food, indicating an opioid selective modulatory role. Interestingly, a recent report found that

NK1 knockout mice also displayed a decreased consumption of alcohol (Heilig et al.,

2009; Ciccocioppo et al., 2009) Furthermore, the decrease in opioid reinforcing behavior occurs with intact analgesia (De Felipe et al., 1998) Neither supraspinal nor systemic

TY027 produced significant CPP or CPA compared to negative or positive control compounds, indicating the multifunctional peptide acts neutrally in non-injured animals, supporting previous literature and confirming the rational design of bivalent ligands acting as MOR/ DOR agonists and NK1 receptor antagonists.

In attempts to delineate the actions of TY027 in the mesolimbic reward circuitry provided the lack of CPP or CPA, immunohistochemical and electrophysiological studies 214

were performed. The literature has reported the localization of the NK1 receptor in the rostroventral medial medulla nucleus tractus solitarius and dorsal motor nucleus of the vagus, periaquaductal gray, nucleus accumbens, and the VTA (Le Brun et al., 2008;

Budai et al., 2006; Franklin 1989; Flores et al., 2006; Germann and Fields 2007). Some reports state that NK1 receptors are differentially co-localized with TH and GABA within the VTA (Lessard and Pickel, 2005), while other evidence support NK1 receptors localizing on DA+ neurons (Tamiya et al., 1990). Neither of these are mutually exclusive. The present study confirmed the presence of the NK1 receptor in the VTA, though more investigation is required to determine on which cell-type (s) the NK1 receptor is localized.

Previous reports provide evidence that morphine-mediated increases in DA firing rate results from GABAergic inhibition in the VTA (e.g. disinhibition; review by

Vanderschuren and Kalivas, 2000); while NK1 receptor blockade decreases basal firing rate of A10 dopamine neurons (Minabe et al., 1996). The opioid-induced response seemingly occurs in a variety of brain regions (Morgan and Clayton, 2005; Li et al.,

2007; Caille and Parsons, 2004; Akaishi et al., 2000). Single unit, extracellular recordings of A10 dopamine neurons in the VTA following systemic administration of TY027 produced inconclusive results. Addition of more data should clarify the actions of TY027 in the mesolimbic reward circuitry (Figure 28).

215

6.6 Conclusions

Single, Multi-Target Compounds Versus Co-Administration Paradigms: The use of a single compound, rationally designed to target multiple systems, such as TY005 or

TY027, introduces an alternate approach to co-administration of the individual agents alone as in the Powell et al. study (2003). The standard approach in clinical pain management looks to adjunctive therapies of available agents to optimize pain relief

(Dworkin et al., 2008). However, the available agents must be balanced for each patient, with regards to efficacy and safety within the current treatment schedule. The use of designed, multivalent, chimeric molecules, has advantages over a cocktail of individual drugs for easy administration, a simple ADME property, no drug-drug interaction and a potential higher local concentration is also expected than the co-administration of two drugs, since the expressions of the NK1 and opioid receptors show a significant degree of anatomical overlap in the central nervous system. Furthermore, the multifunctional compounds have the potential to counteract molecular adaptations induced by opioid agonists leading to enhanced potency and efficacy.

Significance: Current therapies for the treatment of chronic pain have been restricted to opioids, tricyclic antidepressants, dual acting re-uptake inhibitors, and anticonvulsants or combinations with NSAIDs (Dworkin et al., 2007). However, the analgesic efficacy of such compounds is limited by dose related side effects which become more prevalent as clinicians try to overcome analgesic tolerance. Such increases in doses of analgesics result in physician concerns of increasing severity of side effects, decreased quality of life 216 and potential patient abuse liability. This is matched by patient concern for quality of life and in a subset of patients, addiction potential.

While the molecular mechanism(s) for the development of opioid antinociceptive tolerance, OIH and reward are largely unknown, recent data point to several nervous system adaptations which correlate with the emergence of behavioral manifestations.

These alterations include alterations within the pain modulating neural network, including altered coupling of the MOR with G proteins and the upregulation of endogenous pronociceptive molecules including dynorphin, prostaglandins, CCK, CGRP, and SP.

The present study provides the initial evidence that a selectively, designed compounds targeting opioid induced changes introduce alternatives to available therapeutics.

Specifically, these studies may be one of the first to demonstrate the use of an opioid for pain yet lack addiction behaviors, helping to diminish the growing epidemic of prescription drug abuse. Co-administration of ultra-low dose NTX with opioid agonists or administration of chimeric peptides targeting multiple mechanisms of action results in the enhancement of antinociceptive efficacy with a decreased side effect profile. These data validate the concept of targeting multiple mechanisms within the pain pathways showing that a multifunctional and rationally designed compound is efficacious in rodent models of acute and neuropathic pain. Our data also indicate a multimodal approach to pain therapy may decrease or ablate the unwanted side effects elicited by long term opioid exposure and maintain efficacy under long term conditions. 217

Future Projects: Continued investigations with rationally designed, multi-target compounds may reveal interesting pathobiology common to both pain and opioid induced adaptations. Several questions have remained unanswered from analysis of the present work and still others gleaned for the available literature. With regard to the altered G protein coupling of the MOR, it would be worth studying the correlation between SP upregulation induced by opioids and the MOR-Gs state in a time dependent manner.

Furthermore, it would be of interest to know if SP release or content was altered in the

VTA following chronic opioid exposure. Together, these studies may provide insight as to a mechanistic convergence/ divergence of SP- NK1 in opioid related adverse effects. It may also be of interest to investigate the changes in SP content and release with the bivalent peptides (e.g. TY027) to determine if molecular changes induced by typical opioids are indeed prevented with the rationally designed compounds. Study of TY027

(or derivatives thereof) may also reveal a significant reduction in the degree of nausea/ vomiting, physical dependence and anxiety compared to opioid therapeutics. A recent publication has indicated that NK1 antagonists may be suitable as adjunctive therapy for alcoholism as NK1 knockout animals show decreased ethanol consumption and there is some effect in human trials (Heilig et al., 2009); study of opioid agonists/NK1 antagonists may be of interest in this population. Finally, complete electrophysiological analysis of the effects of TY027 and/ or ultra-low-dose opioid antagonist in the mesolimibic reward pathways (e.g. VTA) would be beneficial to clarification of mechanism underlying the decrease in side effects. 218

In conclusion, rationally designed “dirty” drugs may present a new approach in the management of pain with a decrease in the level/ prevalence of adverse effects. In a broader sense, multifunctional compounds could be specifically designed to target pathophysiological changes associated with many disease states. These compounds may represent the next generation of drugs across clinically diverse fields. 219

CHAPTER 7

APPENDICES 220

APPENDIX A

LIST OF PUBLICATIONS

Invited Manuscripts: 1. Recently patented and promising ORL-1 ligands: Where have we been and where are we going? T.M. Largent-Milnes and T.W. Vanderah. Expert Opin Ther Pat, 2010 20(3):291-305.

Peer Reviewed Manuscripts: 1. Spinal or systemic TY005, a peptidic opioid agonist/neurokinin 1 antagonist, attenuates pain with reduced tolerance. T. M. Largent-Milnes, T. Yamamoto, P. Nair, V.J. Hruby, H.I. Yamamura, J. Lai, F. Porreca, T. W. Vanderah. Brit J Pharmacol (accepted, April 2010) 2. Sustained morphine -mediated pain sensitization and antinociceptive tolerance are blocked by intrathecal Raf-1-selective siRNA treatment. S. Tumati, W. R. Roeske, H. Yamamura, T. Largent-Milnes, R. Wang, T. W. Vanderah, E. Varga. Brit J Pharmacol (in press, March 2010) 3. A CB2 Receptor Agonist Attenuates Bone Cancer-induced Pain and Bone Loss. A. Lozano, C.Wright, A. Vardanyan, T. King, T.M. Largent-Milnes, M. Nelson*, J- M. Jimenez-Andrade, P.W. Mantyh, T.W. Vanderah. Life Sci, 2010 epub ahead of print doi:10.1016/j.lfs.2010.02.014 4. Novel Peptide Ligands with Opioid Agonist Activity and CCK Antagonist Activity for the Treatment of Neuropathic Pain. K. E. Hanlon, D. S. Herman, T. M. Largent-Milnes, P. Davis, S-W Ma, W. Guo, R. S. Agnes, I. Kumarasinghe, V. J. Hruby, J. Lai, F. Porreca, T. W. Vanderah. Eur J Pharmacol (submitted 2010) 5. Discovery of a Potent and Efficacious Analgesic Peptide Derivative for Opioid Agonist/Neurokinin 1 Antagonist With 2', 6'-Dimethyl-L-Tyrosine (Dmt): In Vitro, In Vivo and NMR-based Structural Studies. T. Yamamoto, P. Nair, T. M. Largent-Milnes, N. E. Jacobsen, P. Davis, S-W. Ma, H. I. Yamamura, T. W. Vanderah, F. Porreca, J. Lai, V. J. Hruby. J Med Chem (submitted 2010) 6. Novel bifunctional peptides as opioid agonists and NK-1 antagonists. Nair P, Yamamoto T, Kulkarni V, Moye S, Navratilova E, Davis P, Largent T, Ma SW, Yamamura HI, Vanderah T, Lai J, Porreca F, Hruby VJ. Adv Exp Med Biol, 2009 611:537-8. 7. Intrathecal Raf-1-selective siRNA attenuates sustained morphine-mediated thermal hyperalgesia. S. Tumati, T. L. Milnes, H. I. Yamamura, T. W. Vanderah, W. R. Roeske, E.V. Varga. Eur J Pharmacol, 2008 601(1-3):207-8 221

8. Oxycodone + Ultra-low-dose Naltrexone Attenuates Neuropathic Pain and Associated Mu Opioid Receptor – Gs Coupling. T. M. Largent- Milnes, W. Guo, H-Y. Wang, L. H. Burns, T. W. Vanderah, J Pain, 2008 9(8):700-13 9. Novel D-Amino Acid Tetrapeptides Produce Potent Antinociception by Selectively Acting at Peripheral κ-Opioid Receptors. T. W. Vanderah, T. M. Largent-Milnes, J. Lai, F. Porreca, R.A. Houghten, F. Menzaghi, K. Wisniewski, J. Stalewski, J. Sueiras-Diaz, R. Galyean, C. Schteingart, J-L. Junien, J. Trojnar, P. J-M. Rivière, Eur J Pharmacol, 2008 583(1):62-72 10. A Structure–Activity Relationship Study and Combinatorial Synthetic Approach of C-Terminal Modified Bifunctional Peptides That Are δ/μ Opioid Receptor Agonists and Neurokinin 1 Receptor Antagonists. T. Yamamoto, P. Nair, J. Vagner, T. M. Largent-Milnes, P. Davis, S-W Ma, E. Navratilova, S. Moye, S. Tumati, J. Lai, H. I. Yamamura, T. W. Vanderah, F. Porreca, and V. J. Hruby. J Med Chem, 2008 51(5):1369-76 11. Exploring New Approaches to the Treatment of Prolonged Pain: Neuroplasticity, Disease, and Drug Design, V. J. Hruby, T. Yamamoto, P. Nair, D. Laddu, S-W Ma, T.M. Largent-Milnes, P. Davis, T. W. Vanderah, J. Lai, R. Egleton, F. Porreca Proceeding Book of the 10th Chinese Peptide Symposium, 2008 12. Small and Local Structural Modifications Induced Large Change in Bioactivity and Membrane-bound Conformation of Dual Acting Opioid Agonist/NK1 antagonist; T. Yamamoto, P. Nair, T. M. Largent-Milnes, N. E. Jacobsen, P. Davis, S-W Ma, E. Navratilova, J. Lai, H. I. Yamamura, T. W. Vanderah, F. Porreca, V. J. Hruby, Peptide Science, 2007 pp 53-54

Abstracts: 1. Dual acting opioid agonist/NK1 antagonist does not produce antinociceptive tolerance or reward in an animal model of neuropathic pain. T. M. Largent- Milnes, T. Yamamoto, C.R. Campos, N.S. Corral-Frias, J. M. Jimenez-Andrade, P. Davis, S-W. Ma, P. W. Mantyh, E. D. French, T.P. Davis, J. Lai, V. J. Hruby, F. Porreca, T. W. Vanderah. Abstract for Society for Neuroscience, 2009: Chicago, Illinois, USA 2. Dual acting opioid agonist/NK1 antagonist does not produce antinociceptive tolerance or reward in an animal model of neuropathic pain T. M. Largent- Milnes, T. Yamamoto, C.R. Campos, N.S. Corral-Frias, J. M. Jimenez-Andrade, P. Davis, S-W. Ma, P. W. Mantyh, E. D. French, T.P. Davis, J. Lai, V. J. Hruby, F. Porreca, T. W. Vanderah. Media materials, “ Hot Topics” Abstract for Society for Neuroscience, 2009: Chicago, Illinois, USA 3. Activation of the CB2 receptor attenuates breast cancer induced bone pain and promotes bone health in a murine model of breast cancer metastases. K. E. Hanlon, A. L. Ondoua, T. M. Largent-Milnes, A. Chandramouli, D. D. Sukhtankar, A. Okun, M. Nelson, T. King, P. W. Mantyh, T. W. Vanderah. Abstract for Society for Neuroscience, 2009: Chicago, Illinois, USA 222

4. Novel strategy for next generation analgesics without showing tolerance T. Yamamoto, T. M. Largent-Milnes, P. Nair, P. Davis, S-W Ma, J. Lai, H. I. Yamamura, Todd W. Vanderah, F. Porreca, V. J. Hruby. Japanese Peptide Symposium, 2009 5. Biological Evaluations of Cyclic Bifunctional Peptide Derivatives Possessing Opioid Agonist and NK1 Antagonist Activities, T. Yamamoto, P. Nair, T. Largent-Milnes, N. E. Jacobsen, P. Davis, S-W. Ma, E. Navratilova, J. Lai, H. I. Yamamura, T. W. Vanderah, F. Porreca, V. J. Hruby. Proc. of the 45th Japanese Peptide Symposium, Peptide Science 2008, M. Nomizu, ed., The Japanese Peptide Society, 137-138 2009 6. Bifunctional molecules for the treatment of pain with diminished side-effects: opioid agonist/NK-1 antagonist strategies, F. Porreca, T. Largent-Milnes, T. Vanderah, H. I. Yamamura, T. Yamamoto, V. J. Hruby. 6th Annual James Black Conference, British Society of Pharmacology 2008: St. Andrews, Scotland, UK 7. Dual acting opioid agonist/NK1 antagonist peptide reverses neuropathic pain in an animal model without opioid side effects T. M. Largent-Milnes, T. Yamamoto, P. Nair, E. Navratilova, P. Davis, V. J. Hruby, H. I. Yamamura, J. Lai, F. Porreca, T. W. Vanderah, Abstract for the International Association for the Study of Pain, 2008: Glasgow, Scotland UK 8. CB2 Agonist Inhibit Breast Cancer Cell Proliferation in Vitro in a Non-IL-6 Dependent Manner, E. Velez-Bonet, T. M. Largent-Milnes, A. Chandramouli, M. Nelson, T. W. Vanderah, Abstract for Annual Biomedical Research Conference for Minority Students, 2008: Tampa, Florida, USA 9. Dual Acting Opioid Agonist/NK1 Antagonist reverses Neuropathic Pain and Does not Produce Tolerance, T. M. Largent-Milnes, T. Yamamoto, P. Davis, S-W. Ma, V. J. Hruby, H. I. Yamamura, J. Lai, F. Porreca, T.W. Vanderah, Abstract for Frontiers in Biomedical research, 2007: Tucson, AZ, USA 10. Dual Acting Opioid Agonist/NK1 Antagonist reverses Neuropathic Pain and Does not Produce Tolerance, T. M. Largent-Milnes, T. Yamamoto, P. Davis, S-W. Ma, V. J. Hruby, H. I. Yamamura, J. Lai, F. Porreca, T. W. Vanderah. Abstract for Society for Neuroscience, 2007: San Diego, California, USA 11. Small and local structural modification induced large change in bioactivity and membrane-bound conformation of dual acting opioid agonist/NK1 antagonist, T Yamamoto, P Nair, T. M. Largent-Milnes, N. E. Jacobsen, P. Davis, S-W. Ma, E. Navratilova, J. Lai, H. I. Yamamura, T.W. Vanderah, F. Porreca, V. J. Hruby. Abstract for Japanese Peptide Symposium, 2007: Toyama City, Toyama, Japan 12. Neuropathic Pain is Associated With MOR – Gs Coupling and is Attenuated by Oxycodone + Ultra-Low-Dose Naltrexone, T. M. Largent-Milnes, H-Y. Wang*, E. Friedman, F. Porreca, L.H. Burns, T. W. Vanderah. Abstract for Society for Neuroscience, 2005: Washington D.C.; American Pain Society, 2006: San Antonio, Texas, USA

223

Website (design and maintenance): 1. The active site of Thymidine Kinase in the Herpes Simplex Virus, 2005 http://teach.biosci.arizona.edu/462aH2003/largentmilnest/ 2. Spring Pain Meeting 2008 in Grand Cayman, Cayman Isles , British West Indies http://www.springpainconference.com 3. Spring Pain Meeting 2010 in Grand Cayman, Cayman Isles , British West Indies http://www.springpainconference.com

224

APPENDIX B

PERMISSIONS: NOT APPLICABLE 225

APPENDIX C

HUMAN/ANIMAL SUBJECTS

APPROVAL 226

227

REFERENCES

Aanonsen, L. M., K. C. Kajander, et al. (1992). "Autoradiographic analysis of 125I-substance P binding in rat spinal cord following chronic constriction injury of the sciatic nerve." Brain Res 596(1-2): 259-268.

Abbadie, C. (2005). "Chemokines, chemokine receptors and pain." Trends Immunol 26(10): 529-534.

Abbadie, C., J. L. Brown, et al. (1996). "Spinal cord substance P receptor immunoreactivity increases in both inflammatory and nerve injury models of persistent pain." Neuroscience 70(1): 201-209.

Abbadie, C., J. A. Lindia, et al. (2003). "Impaired neuropathic pain responses in mice lacking the chemokine receptor CCR2." Proc Natl Acad Sci U S A 100(13): 7947-7952.

Aicher, S. A., A. Punnoose, et al. (2000). "mu-Opioid receptors often colocalize with the substance P receptor (NK1) in the trigeminal dorsal horn." J Neurosci 20(11): 4345-4354.

Akaishi, T., H. Saito, et al. (2000). "Morphine augments excitatory synaptic transmission in the dentate gyrus through GABAergic disinhibition." Neurosci Res 38(4): 357-363.

Ali, N. M. (1986). "Hyperalgesic response in a patient receiving high concentrations of spinal morphine." Anesthesiology 65(4): 449.

Allen, B. J., J. Li, et al. (1999). "Primary afferent fibers that contribute to increased substance P receptor internalization in the spinal cord after injury." J Neurophysiol 81(3): 1379-1390.

Angst, M. S., W. Koppert, et al. (2003). "Short-term infusion of the mu-opioid agonist remifentanil in humans causes hyperalgesia during withdrawal." Pain 106(1-2): 49-57.

Arner, S. and B. A. Meyerson (1988). "Lack of analgesic effect of opioids on neuropathic and idiopathic forms of pain." Pain 33(1): 11-23.

American Pain Foundation (2010). 2002, from http://www.painfoundation.org/.

228

American Psychiatric Association. (2010). "DSM5." Retrieved Feb10, 2010, from http://www.dsm5.org.

Ballantyne, J. C. and K. S. LaForge (2007). "Opioid dependence and addiction during opioid treatment of chronic pain." Pain 129(3): 235-255.

Bardo, M. T., J. K. Rowlett, et al. (1995). "Conditioned place preference using opiate and stimulant drugs: a meta-analysis." Neurosci Biobehav Rev 19(1): 39- 51.

Barr, G. A., E. Limon, et al. (2003). "Analgesia induced by local plantar injections of opiates in the formalin test in infant rats." Dev Psychobiol 42(2): 111-122.

Beaudry, H., A. Proteau-Gagne, et al. (2009). "Differential noxious and motor tolerance of chronic delta opioid receptor agonists in rodents." Neuroscience 161(2): 381-391. Bechara, A. and D. van der Kooy (1987). "Kappa receptors mediate the peripheral aversive effects of opiates." Pharmacol Biochem Behav 28(2): 227-233.

Bian, D., M. L. Nichols, et al. (1995). "Characterization of the antiallodynic efficacy of morphine in a model of neuropathic pain in rats." Neuroreport 6(15): 1981-1984.

Bilsky, E. J., S. N. Calderon, et al. (1995). "SNC 80, a selective, nonpeptidic and systemically active opioid delta agonist." J Pharmacol Exp Ther 273(1): 359-366.

Bilsky, E. J., S. H. Marglin, et al. (1990). "Using antagonists to assess neurochemical coding of a drug's ability to establish a conditioned place preference." Pharmacol Biochem Behav 37(3): 425-431.

Bisby, M. A. and P. Keen (1986). "Regeneration of primary afferent neurons containing substance P-like immunoreactivity." Brain Res 365(1): 85-95.

Bohn, L. M., R. R. Gainetdinov, et al. (2000). "Mu-opioid receptor desensitization by beta-arrestin-2 determines morphine tolerance but not dependence." Nature 408(6813): 720-723.

Bonica, J. J. (1953). "The management of pain of cancer." J Mich State Med Soc 52(3): 284-290.

Boyce, S., A. Wyatt, et al. (1999). "Selective NMDA NR2B antagonists induce antinociception without motor dysfunction: correlation with restricted localisation of NR2B subunit in dorsal horn." Neuropharmacology 38(5): 611-623. 229

Bryant C.D., Z. P. A., Carroll F.I., and Evans C.J. (2005). "Opioids and addiction: Emerging pharmaceutical strategies for reducing reward and opponent processes." Clinical Neuroscience Research 5: 103-115.

Budai, D., S. G. Khasabov, et al. (2007). "NK-1 receptors modulate the excitability of ON cells in the rostral ventromedial medulla." J Neurophysiol 97(2): 1388-1395.

Buerkle, H. and T. L. Yaksh (1996). "Comparison of the spinal actions of the mu- opioid remifentanil with alfentanil and morphine in the rat." Anesthesiology 84(1): 94-102.

Bunney, B. S., J. R. Walters, et al. (1973). "Dopaminergic neurons: effect of antipsychotic drugs and amphetamine on single cell activity." J Pharmacol Exp Ther 185(3): 560-571.

Burgess, S. E., L. R. Gardell, et al. (2002). "Time-dependent descending facilitation from the rostral ventromedial medulla maintains, but does not initiate, neuropathic pain." J Neurosci 22(12): 5129-5136.

Burns, L.H.(2005). Ultra-low-dose opioid antagonists enhance opioid analgesia while reducing tolerance, dependence, and addictive properties. Research Signpost. C. A. Kerala: 115-136.

Cahill, C. M. and T. J. Coderre (2002). "Attenuation of hyperalgesia in a rat model of neuropathic pain after intrathecal pre- or post-treatment with a neurokinin-1 antagonist." Pain 95(3): 277-285.

Caille, S. and L. H. Parsons (2004). "Intravenous heroin self-administration decreases GABA efflux in the ventral pallidum: an in vivo microdialysis study in rats." Eur J Neurosci 20(2): 593-596.

Campos, C. R., S. M. Ocheltree, et al. (2008). "Nociceptive inhibition prevents inflammatory pain induced changes in the blood-brain barrier." Brain Res 1221: 6-13.

Cara Therapeutics.(2010). from www.caratherapeutics.com.

Celerier, E., C. Rivat, et al. (2000). "Long-lasting hyperalgesia induced by fentanyl in rats: preventive effect of ketamine." Anesthesiology 92(2): 465-472.

230

Chakrabarti, S. and A. R. Gintzler (2003). "Phosphorylation of Gbeta is augmented by chronic morphine and enhances Gbetagamma stimulation of adenylyl cyclase activity." Brain Res Mol Brain Res 119(2): 144-151.

Chakrabarti, S., M. Oppermann, et al. (2001). "Chronic morphine induces the concomitant phosphorylation and altered association of multiple signaling proteins: a novel mechanism for modulating cell signaling." Proc Natl Acad Sci U S A 98(7): 4209-4214.

Chakrabarti, S., A. Regec, et al. (2005). "Biochemical demonstration of mu- opioid receptor association with Gsalpha: enhancement following morphine exposure." Brain Res Mol Brain Res 135(1-2): 217-224.

Chakrabarti, S., A. Regec, et al. (2005). "Chronic morphine acts via a protein kinase Cgamma-G(beta)-adenylyl cyclase complex to augment phosphorylation of G(beta) and G(betagamma) stimulatory adenylyl cyclase signaling." Brain Res Mol Brain Res 138(1): 94-103.

Chakrabarti, S., M. Rivera, et al. (1998). "Chronic morphine augments G(beta)(gamma)/Gs(alpha) stimulation of adenylyl cyclase: relevance to opioid tolerance." Mol Pharmacol 54(4): 655-662.

Chakrabarti, S., L. Wang, et al. (1998). "Chronic morphine augments adenylyl cyclase phosphorylation: relevance to altered signaling during tolerance/dependence." Mol Pharmacol 54(6): 949-953.

Chang, M. M., S. E. Leeman, et al. (1971). "Amino-acid sequence of substance P." Nat New Biol 232(29): 86-87.

Chaplan, S. R., F. W. Bach, et al. (1994). "Quantitative assessment of tactile allodynia in the rat paw." J Neurosci Methods 53(1): 55-63.

Cheng, Y. and W. H. Prusoff (1973). "Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction." Biochem Pharmacol 22(23): 3099- 3108.

Childers, S. R. (1991). "Opioid receptor-coupled second messenger systems." Life Sci 48(21): 1991-2003.

Chu, L. F., M. S. Angst, et al. (2008). "Opioid-induced hyperalgesia in humans: molecular mechanisms and clinical considerations." Clin J Pain 24(6): 479-496.

231

Chu, L. F., D. J. Clark, et al. (2006). "Opioid tolerance and hyperalgesia in chronic pain patients after one month of oral morphine therapy: a preliminary prospective study." J Pain 7(1): 43-48.

Ciccocioppo, R., D. R. Gehlert, et al. (2009). "Stress-related neuropeptides and alcoholism: CRH, NPY, and beyond." Alcohol 43(7): 491-498.

Connor, M. and M. D. Christie (1999). "Opioid receptor signalling mechanisms." Clin Exp Pharmacol Physiol 26(7): 493-499.

Crain, S. M. and K. Shen (2000). "Enhanced analgesic potency and reduced tolerance of morphine in 129/SvEv mice: evidence for a deficiency in GM1 ganglioside-regulated excitatory opioid receptor functions." Brain Res 856(1-2): 227-235.

Crain, S. M. and K. F. Shen (1995). "Ultra-low concentrations of naloxone selectively antagonize excitatory effects of morphine on sensory neurons, thereby increasing its antinociceptive potency and attenuating tolerance/dependence during chronic cotreatment." Proc Natl Acad Sci U S A 92(23): 10540-10544.

Crain, S. M. and K. F. Shen (2000). "Antagonists of excitatory opioid receptor functions enhance morphine's analgesic potency and attenuate opioid tolerance/dependence liability." Pain 84(2-3): 121-131.

Crain, S. M. and K. F. Shen (2001). "Acute thermal hyperalgesia elicited by low- dose morphine in normal mice is blocked by ultra-low-dose naltrexone, unmasking potent opioid analgesia." Brain Res 888(1): 75-82.

Cumberbatch, M. J., E. Carlson, et al. (1998). "Reversal of behavioural and electrophysiological correlates of experimental peripheral neuropathy by the NK1 receptor antagonist GR205171 in rats." Neuropharmacology 37(12): 1535-1543.

Dawson-Basoa, M. and A. R. Gintzler (1997). "Involvement of spinal cord delta opiate receptors in the antinociception of gestation and its hormonal simulation." Brain Res 757(1): 37-42.

De Felipe, C., J. F. Herrero, et al. (1998). "Altered nociception, analgesia and aggression in mice lacking the receptor for substance P." Nature 392(6674): 394- 397.

Devulder, J. (1997). "Hyperalgesia induced by high-dose intrathecal sufentanil in neuropathic pain." J Neurosurg Anesthesiol 9(2): 146-148.

232

Dietis, N., R. Guerrini, et al. (2009). "Simultaneous targeting of multiple opioid receptors: a strategy to improve side-effect profile." Br J Anaesth 103(1): 38-49.

Drdla, R., M. Gassner, et al. (2009). "Induction of synaptic long-term potentiation after opioid withdrawal." Science 325(5937): 207-210.

DuPen, A., D. Shen, et al. (2007). "Mechanisms of opioid-induced tolerance and hyperalgesia." Pain Manag Nurs 8(3): 113-121.

Dworkin, R. H., A. B. O'Connor, et al. (2007). "Pharmacologic management of neuropathic pain: evidence-based recommendations." Pain 132(3): 237-251.

Dworkin, R. H., D. C. Turk, et al. (2008). "Interpreting the clinical importance of treatment outcomes in chronic pain clinical trials: IMMPACT recommendations." J Pain 9(2): 105-121.

Elliott, P. J., G. S. Mason, et al. (1992). "Modulation of the rat mesolimbic dopamine pathway by neurokinins." Behav Brain Res 51(1): 77-82.

Erspamer, V., P. Melchiorri, et al. (1989). "Deltorphins: a family of naturally occurring peptides with high affinity and selectivity for delta opioid binding sites." Proc Natl Acad Sci U S A 86(13): 5188-5192.

Fan, S. F., K. F. Shen, et al. (1991). "Opioids at low concentration decrease openings of K+ channels in sensory ganglion neurons." Brain Res 558(1): 166- 170.

Finnerup, N. B., M. Otto, et al. (2005). "Algorithm for neuropathic pain treatment: an evidence based proposal." Pain 118(3): 289-305.

Flores, J. A., B. Galan-Rodriguez, et al. (2006). "Role for dopamine neurons of the rostral linear nucleus and periaqueductal gray in the rewarding and sensitizing properties of heroin." Neuropsychopharmacology 31(7): 1475-1488.

Foley, K. M. (1995). "Misconceptions and controversies regarding the use of opioids in cancer pain." Anticancer Drugs 6 Suppl 3: 4-13.

Fong, T. M., H. Yu, et al. (1992). "Molecular basis for the species selectivity of the neurokinin-1 receptor antagonists CP-96,345 and RP67580." J Biol Chem 267(36): 25668-25671.

Franklin, K. B. (1989). "Analgesia and the neural substrate of reward." Neurosci Biobehav Rev 13(2-3): 149-154.

233

Fraser, G. L., H. Parenteau, et al. (2000). "The effects of delta agonists on locomotor activity in habituated and non-habituated rats." Life Sci 67(8): 913- 922.

French, E. D. (1992). "Competitive NMDA receptor antagonists attenuate phencyclidine-induced excitations of A10 dopamine neurons." Eur J Pharmacol 217(1): 1-7.

Gadd, C. A., P. Murtra, et al. (2003). "Neurokinin-1 receptor-expressing neurons in the amygdala modulate morphine reward and anxiety behaviors in the mouse." J Neurosci 23(23): 8271-8280.

Gallantine, E. L. and T. F. Meert (2005). "A comparison of the antinociceptive and adverse effects of the mu-opioid agonist morphine and the delta-opioid agonist SNC80." Basic Clin Pharmacol Toxicol 97(1): 39-51.

Garces, Y. I., S. F. Rabito, et al. (1993). "Lack of potent antinociceptive activity by substance P antagonist CP-96,345 in the rat spinal cord." Life Sci 52(4): 353- 360.

Gardell, L. R., R. Wang, et al. (2002). "Sustained morphine exposure induces a spinal dynorphin-dependent enhancement of excitatory transmitter release from primary afferent fibers." J Neurosci 22(15): 6747-6755.

Gellert, V. F. and S. G. Holtzman (1979). "Discriminative stimulus effects of naltrexone in the morphine-dependent rat." J Pharmacol Exp Ther 211(3): 596- 605.

German, P. W. and H. L. Fields (2007). "How prior reward experience biases exploratory movements: a probabilistic model." J Neurophysiol 97(3): 2083-2093.

Gintzler, A. R. and S. Chakrabarti (2006). "Post-opioid receptor adaptations to chronic morphine; altered functionality and associations of signaling molecules." Life Sci 79(8): 717-722.

Goff, J. R., A. R. Burkey, et al. (1998). "Reorganization of the spinal dorsal horn in models of chronic pain: correlation with behaviour." Neuroscience 82(2): 559- 574.

Gold, M. S., A. L. Pottash, et al. (1981). "Clinical utility of clonidine in opiate withdrawal." NIDA Res Monogr 34: 95-100.

234

Goode, T. L. and R. B. Raffa (1997). "An examination of the relationship between mu-opioid antinociceptive efficacy and G-protein coupling using pertussis and cholera toxins." Life Sci 60(7): PL107-113.

Gu, G., I. Kondo, et al. (2005). "Resting and evoked spinal substance P release during chronic intrathecal morphine infusion: parallels with tolerance and dependence." J Pharmacol Exp Ther 314(3): 1362-1369.

Guan, J. S., Z. Z. Xu, et al. (2005). "Interaction with vesicle luminal protachykinin regulates surface expression of delta-opioid receptors and opioid analgesia." Cell 122(4): 619-631.

Gutstein, H. B. (1996). "The effects of pain on opioid tolerance: how do we resolve the controversy?" Pharmacol Rev 48(3): 403-407; discussion 409-411.

Hargreaves, K., R. Dubner, et al. (1988). "A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia." Pain 32(1): 77-88.

Harrison, L. M., A. J. Kastin, et al. (1998). "Opiate tolerance and dependence: receptors, G-proteins, and antiopiates." Peptides 19(9): 1603-1630.

Harrison, S. and P. Geppetti (2001). "Substance p." Int J Biochem Cell Biol 33(6): 555-576.

Hasenohrl, R. U., M. S. Oitzl, et al. (1989). "Conditioned place preference in the corral: a procedure for measuring reinforcing properties of drugs." J Neurosci Methods 30(2): 141-146.

Heilig, M., A. Thorsell, et al. (2009). "Translating the neuroscience of alcoholism into clinical treatments: From blocking the buzz to curing the blues." Neurosci Biobehav Rev.

Heinrichs, C., C. Tsigos, et al. (1995). "Familial adrenocorticotropin unresponsiveness associated with alacrima and achalasia: biochemical and molecular studies in two siblings with clinical heterogeneity." Eur J Pediatr 154(3): 191-196.

Hill, R. (2000). "NK1 (substance P) receptor antagonists--why are they not analgesic in humans?" Trends Pharmacol Sci 21(7): 244-246.

Hojsted, J. and P. Sjogren (2007). "An update on the role of opioids in the management of chronic pain of nonmalignant origin." Curr Opin Anaesthesiol 20(5): 451-455.

235

Holdridge, S. V. and C. M. Cahill (2007). "Spinal administration of a delta opioid receptor agonist attenuates hyperalgesia and allodynia in a rat model of neuropathic pain." Eur J Pain 11(6): 685-693.

Holzhauer-Oitzl, M. S., R. Hasenohrl, et al. (1988). "Reinforcing properties of substance P in the region of the nucleus basalis magnocellularis in rats." Neuropharmacology 27(7): 749-756.

Huber, J. D., C. R. Campos, et al. (2006). "Alterations in blood-brain barrier ICAM-1 expression and brain microglial activation after lambda-carrageenan- induced inflammatory pain." Am J Physiol Heart Circ Physiol 290(2): H732-740.

Hwang, J. H., K. S. Hwang, et al. (2000). "An analysis of drug interaction between morphine and neostigmine in rats with nerve-ligation injury." Anesth Analg 90(2): 421-426.

Hylden, J. L. and G. L. Wilcox (1980). "Intrathecal morphine in mice: a new technique." Eur J Pharmacol 67(2-3): 313-316.

Hylden, J. L. and G. L. Wilcox (1981). "Intrathecal substance P elicits a caudally- directed biting and scratching behavior in mice." Brain Res 217(1): 212-215.

Hylden, J. L. and G. L. Wilcox (1982). "Intrathecal opioids block a spinal action of substance P in mice: functional importance of both mu- and delta-receptors." Eur J Pharmacol 86(1): 95-98.

Ikeda, K., T. Kobayashi, et al. (2000). "Involvement of G-protein-activated inwardly rectifying K (GIRK) channels in opioid-induced analgesia." Neurosci Res 38(1): 113-116.

International Association for the Study of Pain (2010). from http://www.iasp- pain.org.

Jensen, M. P., M. J. Chodroff, et al. (2007). "The impact of neuropathic pain on health-related quality of life: review and implications." Neurology 68(15): 1178- 1182.

Jensen, T. S. and N. B. Finnerup (2007). "Management of neuropathic pain." Curr Opin Support Palliat Care 1(2): 126-131.

Jensen, T. S. and N. B. Finnerup (2009). "Neuropathic pain treatment: a further step forward." Lancet 374(9697): 1218-1219.

236

Jensen, T. S., C. S. Madsen, et al. (2009). "Pharmacology and treatment of neuropathic pains." Curr Opin Neurol 22(5): 467-474.

Joseph, M. H., K. Datla, et al. (2003). "The interpretation of the measurement of nucleus accumbens dopamine by in vivo dialysis: the kick, the craving or the cognition?" Neurosci Biobehav Rev 27(6): 527-541.

Joshi, S. K., G. Hernandez, et al. (2006). "Comparison of antinociceptive actions of standard analgesics in attenuating capsaicin and nerve-injury-induced mechanical hypersensitivity." Neuroscience 143(2): 587-596.

Jutkiewicz, E. M., K. C. Rice, et al. (2003). "Effects of the delta-opioid receptor agonist SNC80 on learning relative to its antidepressant-like effects in rats." Behav Pharmacol 14(7): 509-516.

Kim, S. H. and J. M. Chung (1992). "An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat." Pain 50(3): 355-363.

King, T., L. R. Gardell, et al. (2005a). "Role of NK-1 neurotransmission in opioid-induced hyperalgesia." Pain 116(3): 276-288.

King, T., M. H. Ossipov, et al. (2005b). "Is paradoxical pain induced by sustained opioid exposure an underlying mechanism of opioid antinociceptive tolerance?" Neurosignals 14(4): 194-205.

Koch, T. and V. Hollt (2008). "Role of receptor internalization in opioid tolerance and dependence." Pharmacol Ther 117(2): 199-206.

Kohno, T., R. R. Ji, et al. (2005). "Peripheral axonal injury results in reduced mu opioid receptor pre- and post-synaptic action in the spinal cord." Pain 117(1-2): 77-87.

Kreil, G., D. Barra, et al. (1989). "Deltorphin, a novel amphibian skin peptide with high selectivity and affinity for delta opioid receptors." Eur J Pharmacol 162(1): 123-128.

LaBuda, C. J. and P. J. Little (2005). "Pharmacological evaluation of the selective spinal nerve ligation model of neuropathic pain in the rat." J Neurosci Methods 144(2): 175-181.

Largent-Milnes, T. M. and T. W. Vanderah (2010). "Recently patented and promising ORL-1 ligands: where have we been and where are we going?" Expert Opin Ther Pat 20(3): 291-305. 237

Laugwitz, K. L., S. Offermanns, et al. (1993). "mu and delta opioid receptors differentially couple to G protein subtypes in membranes of human neuroblastoma SH-SY5Y cells." Neuron 10(2): 233-242.

Le Brun, I., A. Dufour, et al. (2008). "Differential expression of Nk1 and NK3 neurokinin receptors in neurons of the nucleus tractus solitarius and the dorsal vagal motor nucleus of the rat and mouse." Neuroscience 152(1): 56-64.

Le Foll, B. and S. R. Goldberg (2005). "Cannabinoid CB1 receptor antagonists as promising new medications for drug dependence." J Pharmacol Exp Ther 312(3): 875-883.

Lee, Y. W., S. R. Chaplan, et al. (1995). "Systemic and supraspinal, but not spinal, opiates suppress allodynia in a rat neuropathic pain model." Neurosci Lett 199(2): 111-114.

Lenard, N. R., D. J. Daniels, et al. (2007). "Absence of conditioned place preference or reinstatement with bivalent ligands containing mu-opioid receptor agonist and delta-opioid receptor antagonist pharmacophores." Eur J Pharmacol 566(1-3): 75-82.

Lessard, A. and V. M. Pickel (2005). "Subcellular distribution and plasticity of neurokinin-1 receptors in the rat substantia nigra and ventral tegmental area." Neuroscience 135(4): 1309-1323.

Li, Y., L. He, et al. (2007). "Changes of micro-opioid receptors and GABA in visual cortex of chronic morphine treated rats." Neurosci Lett 428(1): 11-16.

Liang, J., Y. Li, et al. (2006). "The possible involvement of endogenous ligands for mu-, delta- and kappa-opioid receptors in modulating morphine-induced CPP expression in rats." Peptides 27(12): 3307-3314.

Liang, Y. J., D. F. Wu, et al. (2007). "Interaction of the mu-opioid receptor with synaptophysin influences receptor trafficking and signaling." Mol Pharmacol 71(1): 123-131.

Lorenzen, A., M. Fuss, et al. (1993). "Measurement of guanine nucleotide-binding protein activation by A1 adenosine receptor agonists in bovine brain membranes: stimulation of guanosine-5'-O-(3-[35S]thio)triphosphate binding." Mol Pharmacol 44(1): 115-123.

Lowery, J. J., L. Yeomans, et al. (2007). "Glycosylation improves the central effects of DAMGO." Chem Biol Drug Des 69(1): 41-47. 238

Lysle, D. T., M. E. Coussons, et al. (1993). "Morphine-induced alterations of immune status: dose dependency, compartment specificity and antagonism by naltrexone." J Pharmacol Exp Ther 265(3): 1071-1078.

Ma, W. and J. C. Eisenach (2003). "Intraplantar injection of a cyclooxygenase inhibitor ketorolac reduces immunoreactivities of substance P, calcitonin gene- related peptide, and dynorphin in the dorsal horn of rats with nerve injury or inflammation." Neuroscience 121(3): 681-690.

Malcangio, M., M. S. Ramer, et al. (2000). "Abnormal substance P release from the spinal cord following injury to primary sensory neurons." Eur J Neurosci 12(1): 397-399.

Maldonado, R., D. Girdlestone, et al. (1993). "RP 67580, a selective antagonist of neurokinin-1 receptors, modifies some of the naloxone-precipitated morphine withdrawal signs in rats." Neurosci Lett 156(1-2): 135-140.

Malmberg, A. B., C. Chen, et al. (1997). "Preserved acute pain and reduced neuropathic pain in mice lacking PKCgamma." Science 278(5336): 279-283.

Mao, J. (2002). "Opioid-induced abnormal pain sensitivity: implications in clinical opioid therapy." Pain 100(3): 213-217.

Mao, J., D. D. Price, et al. (1995a). "Experimental mononeuropathy reduces the antinociceptive effects of morphine: implications for common intracellular mechanisms involved in morphine tolerance and neuropathic pain." Pain 61(3): 353-364.

Mao, J., D. D. Price, et al. (1995b). "Mechanisms of hyperalgesia and morphine tolerance: a current view of their possible interactions." Pain 62(3): 259-274.

Mas-Nieto, M., B. Pommier, et al. (2001). "Reduction of opioid dependence by the CB(1) antagonist SR141716A in mice: evaluation of the interest in pharmacotherapy of opioid addiction." Br J Pharmacol 132(8): 1809-1816.

Merskey, H. (1994). "Logic, truth and language in concepts of pain." Qual Life Res 3 Suppl 1: S69-76.

Mika, J., R. Przewlocki, et al. (2001). "The role of delta-opioid receptor subtypes in neuropathic pain." Eur J Pharmacol 415(1): 31-37.

239

Morgan, M. M. and C. C. Clayton (2005). "Defensive behaviors evoked from the ventrolateral periaqueductal gray of the rat: comparison of opioid and GABA disinhibition." Behav Brain Res 164(1): 61-66.

Murtra, P., A. M. Sheasby, et al. (2000). "Rewarding effects of opiates are absent in mice lacking the receptor for substance P." Nature 405(6783): 180-183.

Nichols, M. L., Y. Lopez, et al. (1997). "Enhancement of the antiallodynic and antinociceptive efficacy of spinal morphine by antisera to dynorphin A (1-13) or MK-801 in a nerve-ligation model of peripheral neuropathy." Pain 69(3): 317- 322.

NIDA. (2010). "Topics in Brief, National Institue on Drug Abuse." Prescription Drug Abuse Retrieved March 2008, 2008, from http://www.drugabuse.gov/tib/prescription.html.

Noble, M., J. R. Treadwell, et al. (2010). "Long-term opioid management for chronic noncancer pain." Cochrane Database Syst Rev(1): CD006605.

Olmstead, M. C. and L. H. Burns (2005). "Ultra-low-dose naltrexone suppresses rewarding effects of opiates and aversive effects of opiate withdrawal in rats." Psychopharmacology (Berl) 181(3): 576-581.

Ossipov, M. H., J. Lai, et al. (2004). "Antinociceptive and nociceptive actions of opioids." J Neurobiol 61(1): 126-148.

Ossipov, M. H., J. Lai, et al. (2005a). "Underlying mechanisms of pronociceptive consequences of prolonged morphine exposure." Biopolymers 80(2-3): 319-324.

Ossipov, M. H. and F. Porreca (2005b). "Challenges in the development of novel treatment strategies for neuropathic pain." NeuroRx 2(4): 650-661.

Pasternak, G. W. (1993). "Pharmacological mechanisms of opioid analgesics." Clin Neuropharmacol 16(1): 1-18.

Paxinos G, W. C., Ed. (1997). The Rat Brain in Stereotaxic Coordinates. Sydney, Academic Press.

Pereira Do Carmo, G., G. W. Stevenson, et al. (2009). "Effects of pain- and analgesia-related manipulations on intracranial self-stimulation in rats: further studies on pain-depressed behavior." Pain 144(1-2): 170-177.

240

Perlikowska, R., K. Gach, et al. (2009). "Biological activity of endomorphin and [Dmt1]endomorphin analogs with six-membered proline surrogates in position 2." Bioorg Med Chem 17(11): 3789-3794.

Pfeiffer, M., S. Kirscht, et al. (2003). "Heterodimerization of substance P and mu- opioid receptors regulates receptor trafficking and resensitization." J Biol Chem 278(51): 51630-51637.

Placenza, F. M., P. J. Fletcher, et al. (2006). "Effects of central neurokinin-1 receptor antagonism on cocaine- and opiate-induced locomotor activity and self- administration behaviour in rats." Pharmacol Biochem Behav 84(1): 94-101.

Porreca, F. and T. F. Burks (1983). "Affinity of normorphine for its pharmacologic receptor in the naive and morphine-tolerant guinea-pig isolated ileum." J Pharmacol Exp Ther 225(3): 688-693.

Porreca, F., D. LoPresti, et al. (1990). "Opioid agonist affinity in the guinea-pig ileum and mouse vas deferens." Eur J Pharmacol 179(1-2): 129-139.

Powell, K. J., N. S. Abul-Husn, et al. (2002). "Paradoxical effects of the opioid antagonist naltrexone on morphine analgesia, tolerance, and reward in rats." J Pharmacol Exp Ther 300(2): 588-596.

Powell, K. J., R. Quirion, et al. (2003). "Inhibition of neurokinin-1-substance P receptor and prostanoid activity prevents and reverses the development of morphine tolerance in vivo and the morphine-induced increase in CGRP expression in cultured dorsal root ganglion neurons." Eur J Neurosci 18(6): 1572- 1583.

Reisine T., G. Pasternak. (1996). "Opioid analgesics and antagonists". In: JG Hardman, LE Limbird, PB Molinoff, RW Ruddon Goodman and Gilman‟s The Pharmacological Basis of Therapeutics. New York McGraw- Hill. 521-577.

Ripley, T. L., C. A. Gadd, et al. (2002). "Lack of self-administration and behavioural sensitisation to morphine, but not cocaine, in mice lacking NK1 receptors." Neuropharmacology 43(8): 1258-1268.

Rivat, C., L. P. Vera-Portocarrero, et al. (2009). "Spinal NK-1 receptor- expressing neurons and descending pathways support fentanyl-induced pain hypersensitivity in a rat model of postoperative pain." Eur J Neurosci 29(4): 727- 737.

241

Rivera, M. and A. R. Gintzler (1998). "Differential effect of chronic morphine on mRNA encoding adenylyl cyclase isoforms: relevance to physiological sequela of tolerance/dependence." Brain Res Mol Brain Res 54(1): 165-169.

Rowbotham, M. C., L. Twilling, et al. (2003). "Oral opioid therapy for chronic peripheral and central neuropathic pain." N Engl J Med 348(13): 1223-1232.

Rupniak, N. M., S. Boyce, et al. (1993). "Antinociceptive activity of NK1 receptor antagonists: non-specific effects of racemic RP67580." Br J Pharmacol 110(4): 1607-1613.

Saegusa, H., T. Kurihara, et al. (2000). "Altered pain responses in mice lacking alpha 1E subunit of the voltage-dependent Ca2+ channel." Proc Natl Acad Sci U S A 97(11): 6132-6137.

Sakurada, T., M. Yuhki, et al. (1999). "Opioid activity of sendide, a tachykinin NK1 receptor antagonist." Eur J Pharmacol 369(3): 261-266.

Sapunar, D., M. Ljubkovic, et al. (2005). "Distinct membrane effects of spinal nerve ligation on injured and adjacent dorsal root ganglion neurons in rats." Anesthesiology 103(2): 360-376.

Scherrer, G., N. Imamachi, et al. (2009). "Dissociation of the opioid receptor mechanisms that control mechanical and heat pain." Cell 137(6): 1148-1159.

Seybold, V. S. (2009). "The role of peptides in central sensitization." Handb Exp Pharmacol(194): 451-491.

Seybold, V. S., J. L. Hylden, et al. (1982). "Intrathecal substance P and somatostatin in rats: behaviors indicative of sensation." Peptides 3(1): 49-54.

Shen, K. F. and S. M. Crain (1990). "Cholera toxin-A subunit blocks opioid excitatory effects on sensory neuron action potentials indicating mediation by Gs- linked opioid receptors." Brain Res 525(2): 225-231.

Silbert, B. S., A. W. Lipkowski, et al. (1991). "Analgesic activity of a novel bivalent opioid peptide compared to morphine via different routes of administration." Agents Actions 33(3-4): 382-387.

Simonnet, G. and C. Rivat (2003). "Opioid-induced hyperalgesia: abnormal or normal pain?" Neuroreport 14(1): 1-7.

Svensson, C. I., M. Schafers, et al. (2006). "Covariance among age, spinal p38 MAP kinase activation and allodynia." J Pain 7(5): 337-345. 242

Swamydas, M., A. M. Skoff, et al. (2004). "Partial sciatic nerve transection causes redistribution of pain-related peptides and lowers withdrawal threshold." Exp Neurol 188(2): 444-451.

Szucs, M., K. Boda, et al. (2004). "Dual effects of DAMGO [D-Ala2,N-Me- Phe4,Gly5-ol]-enkephalin and CTAP (D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr- NH2) on adenylyl cyclase activity: implications for mu-opioid receptor Gs coupling." J Pharmacol Exp Ther 310(1): 256-262.

Tallarida R.J. and R.B. Murray. (1987). Manual of pharmacologic calculations with computer programs. 2 ed., Springer-Verlag, New York. 297.

Tamiya, R., M. Hanada, et al. (1990). "Substance P afferents have synaptic contacts with dopaminergic neurons in the ventral tegmental area of the rat." Neurosci Lett 110(1-2): 11-15.

Taylor, B. K. and K. E. McCarson (2004). "Neurokinin-1 receptor gene expression in the mouse dorsal horn increases with neuropathic pain." J Pain 5(2): 71-76.

Torsney, C. and A. B. MacDermott (2006). "Disinhibition opens the gate to pathological pain signaling in superficial neurokinin 1 receptor-expressing neurons in rat spinal cord." J Neurosci 26(6): 1833-1843.

Trang, T., M. Sutak, et al. (2002). "The role of spinal neuropeptides and prostaglandins in opioid physical dependence." Br J Pharmacol 136(1): 37-48.

Trujillo, K. A. and H. Akil (1991). "Opiate tolerance and dependence: recent findings and synthesis." New Biol 3(10): 915-923.

Tumati, S., H. I. Yamamura, et al. (2009). "Sustained morphine treatment augments capsaicin-evoked calcitonin gene-related peptide release from primary sensory neurons in a protein kinase A- and Raf-1-dependent manner." J Pharmacol Exp Ther 330(3): 810-817.

Valenzano, K. J., L. Tafesse, et al. (2005). "Pharmacological and pharmacokinetic characterization of the cannabinoid receptor 2 agonist, GW405833, utilizing rodent models of acute and chronic pain, anxiety, ataxia and catalepsy." Neuropharmacology 48(5): 658-672.

Vanderah, T. W., L. R. Gardell, et al. (2000). "Dynorphin promotes abnormal pain and spinal opioid antinociceptive tolerance." J Neurosci 20(18): 7074-7079.

243

Vanderah, T. W., T. Largent-Milnes, et al. (2008). "Novel D-amino acid tetrapeptides produce potent antinociception by selectively acting at peripheral kappa-opioid receptors." Eur J Pharmacol 583(1): 62-72.

Vanderah, T. W., M. H. Ossipov, et al. (2001). "Mechanisms of opioid-induced pain and antinociceptive tolerance: descending facilitation and spinal dynorphin." Pain 92(1-2): 5-9.

Vanderah, T. W., R. B. Raffa, et al. (1998). "Orphanin-FQ/nociceptin: lack of anti nociceptive, hyperalgesic or allodynic effects in acute thermal or mechanical tests following intracerebroventricular or intrathecal administration to mice or rats." Eur J Pain 2(3): 267-278. Vanderah, T. W., N. M. Suenaga, et al. (2001). "Tonic descending facilitation from the rostral ventromedial medulla mediates opioid-induced abnormal pain and antinociceptive tolerance." J Neurosci 21(1): 279-286.

Vanderschuren, L. J., A. N. Schoffelmeer, et al. (2000). "Dissociable effects of the kappa-opioid receptor agonists bremazocine, U69593, and U50488H on locomotor activity and long-term behavioral sensitization induced by amphetamine and cocaine." Psychopharmacology (Berl) 150(1): 35-44.

Vera-Portocarrero, L. P., E. T. Zhang, et al. (2007). "Spinal NK-1 receptor expressing neurons mediate opioid-induced hyperalgesia and antinociceptive tolerance via activation of descending pathways." Pain 129(1-2): 35-45.

Volkow, N. D. (2004). "A note from NIDA's director: blending clinical practice and research." Sci Pract Perspect 2(2): 2.

Wallin, J. and E. Schott (2002). "Substance P release in the spinal dorsal horn following peripheral nerve injury." Neuropeptides 36(4): 252-256.

Wan, Q., S. D. Douglas, et al. (2006). "Morphine upregulates functional expression of neurokinin-1 receptor in neurons." J Neurosci Res 84(7): 1588- 1596.

Wang, H. Y. and L. H. Burns (2006). "Gbetagamma that interacts with adenylyl cyclase in opioid tolerance originates from a Gs protein." J Neurobiol 66(12): 1302-1310.

Wang, H. Y., M. Frankfurt, et al. (2008). "High-affinity naloxone binding to filamin a prevents mu opioid receptor-Gs coupling underlying opioid tolerance and dependence." PLoS One 3(2): e1554.

244

Wang, H. Y., E. Friedman, et al. (2005). "Ultra-low-dose naloxone suppresses opioid tolerance, dependence and associated changes in mu opioid receptor-G protein coupling and Gbetagamma signaling." Neuroscience 135(1): 247-261.

Wang, L. and A. R. Gintzler (1997). "Altered mu-opiate receptor-G protein signal transduction following chronic morphine exposure." J Neurochem 68(1): 248- 254.

Way, E. L., H. H. Loh, et al. (1969). "Simultaneous quantitative assessment of morphine tolerance and physical dependence." J Pharmacol Exp Ther 167(1): 1-8.

Woodcock, J., J. Witter, et al. (2007). "Stimulating the development of mechanism-based, individualized pain therapies." Nat Rev Drug Discov 6(9): 703-710.

Xie, J. Y., D. S. Herman, et al. (2005). "Cholecystokinin in the rostral ventromedial medulla mediates opioid-induced hyperalgesia and antinociceptive tolerance." J Neurosci 25(2): 409-416.

Xu, X. J., M. J. Puke, et al. (1993). "Up-regulation of cholecystokinin in primary sensory neurons is associated with morphine insensitivity in experimental neuropathic pain in the rat." Neurosci Lett 152(1-2): 129-132.

Yaksh, T. L. and G. J. Harty (1988). "Pharmacology of the allodynia in rats evoked by high dose intrathecal morphine." J Pharmacol Exp Ther 244(2): 501- 507.

Yaksh, T. L. and T. A. Rudy (1976). "Chronic catheterization of the spinal subarachnoid space." Physiol Behav 17(6): 1031-1036.

Yamamoto, T., P. Nair, et al. (2007). "Design, synthesis, and biological evaluation of novel bifunctional C-terminal-modified peptides for delta/mu opioid receptor agonists and neurokinin-1 receptor antagonists." J Med Chem 50(12): 2779-2786.

Yamamoto, T., P. Nair, et al. (2008). "The importance of micelle-bound states for the bioactivities of bifunctional peptide derivatives for delta/mu opioid receptor agonists and neurokinin 1 receptor antagonists." J Med Chem 51(20): 6334-6347.

Yamamoto, T., P. Nair, et al. (2009). "The biological activity and metabolic stability of peptidic bifunctional compounds that are opioid receptor agonists and neurokinin-1 receptor antagonists with a cystine moiety." Bioorg Med Chem 17(20): 7337-7343.

245

Yamamoto, T., P. Nair, et al. (2008). "A structure-activity relationship study and combinatorial synthetic approach of C-terminal modified bifunctional peptides that are delta/mu opioid receptor agonists and neurokinin 1 receptor antagonists." J Med Chem 51(5): 1369-1376.

Yang, J., J. M. Maarek, et al. (2005). "In vivo quantitative assessment of catheter patency in rats." Lab Anim 39(3): 259-268.

Young, A. M., S. R. Mattox, et al. (1991). "Increased sensitivity to rate-altering and discriminative stimulus effects of morphine following continuous exposure to naltrexone." Psychopharmacology (Berl) 103(1): 67-73.

Zaki, P. A., E. J. Bilsky, et al. (1996). "Opioid receptor types and subtypes: the delta receptor as a model." Annu Rev Pharmacol Toxicol 36: 379-401.

Zhang, H., C. L. Cang, et al. (2007). "Neurokinin-1 receptor enhances TRPV1 activity in primary sensory neurons via PKCepsilon: a novel pathway for heat hyperalgesia." J Neurosci 27(44): 12067-12077.

Zhang, S. H., Q. X. Sun, et al. (2008). "Paracrine-like excitation of low-threshold mechanoceptive C-fibers innervating rat hairy skin is mediated by substance P via NK-1 receptors." Brain Res Bull 75(1): 138-145.

Zhang, Y., D. R. Conklin, et al. (2005). "Intrathecal morphine reduces allodynia after peripheral nerve injury in rats via activation of a spinal A1 adenosine receptor." Anesthesiology 102(2): 416-420.

Zhao, C., J. M. Tall, et al. (2004). "Antiallodynic effects of systemic and intrathecal morphine in the spared nerve injury model of neuropathic pain in rats." Anesthesiology 100(4): 905-911.