Zurich Open Repository and Archive University of Zurich Main Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2015 Reversal of chronic pain by activation of single GABAA receptor subtypes Ralvenius, William T Abstract: SUMMARY The ability to perceive pain is a basic property of humans and of all other higher order animals. The primary role of pain sensation and nociception (that is, the neuronal activity encoding pain) is to protect against potentially harmful threats arriving from the environment or the body interior. Pain can however also become dysfunctional and persist for extended periods of time without an appar- ent benefit, instead becoming a major burden that requires medical attention. Chronic pain isamajor socio-economic challenge, which – despite scientific advances in the understanding of its causes – remains poorly responsive to the drugs available on the market today. Recent insights into the mechanisms of chronic pain states suggest that a common factor for many kinds of persistent pain states is a loss of inhibition in spinal cord circuits that normally control nociceptive input to the brain. The so- called benzodiazepines – first marketed by Hoffmann-La Roche in the 1960s – are drugs that facilitate synaptic inhibition throughout the CNS and have as such the potential to reverse pathological disinhibition. Ben- zodiazepines facilitate inhibition by increasing the activity of ฀-aminobutyric acid (GABA) at its receptor, a heteropentameric anion permeable ion channel. Although rodent studies have shown that pathologically increased pain sensitivity can be normalized by intrathecal (spinal) injection of benzodiazepines, these drugs do not exert clinically relevant analgesia in human patients, at least not after systemic application. In this thesis, I have tested the hypothesis that benzodiazepines reverse pathological pain after systemic application if their action is restricted to well-defined subtypes of GABAA receptors. Using GABAA receptor point-mutated mice, I was able to demonstrate that selective targeting of GABAA receptors that contain the ฀2 subunit (฀2-GABAA) receptors evoke pronounced pain relief in the absence of con- founding and undesired sedation. I could also confirm previous findings that had proposed that activation of the same GABAA receptors induces anxiolysis and muscle relaxation. Importantly, selective target- ing of ฀2- GABAA receptors avoided several unwanted effects of classical non-selective benzodiazepines including sedation, impairment of motor coordination, and the progressive loss of therapeutic efficacy over time. Using mice in which the action of classical benzodiazepine agonists was restricted to only a single GABAA receptor subtype, I could also propose a new hypothesis explaining why classical benzodi- azepines lack clinically relevant analgesic properties. For two clinically used benzodiazepines (diazepam and midazolam), I could demonstrate that strong ฀1-GABAA receptor-mediated sedation occurs already at doses. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-164464 Dissertation Published Version Originally published at: Ralvenius, William T. Reversal of chronic pain by activation of single GABAA receptor subtypes. 2015, University of Zurich, Faculty of Science. 2 REVERSAL OF CHRONIC PAIN BY ACTIVATION OF SINGLE GABAA RECEPTOR SUBTYPES Dissertation zur Erlangung der naturwissenschaftlichen Doktorwürde (Dr. sc. nat.) vorgelegt der Mathematisch-naturwissenschaftlichen Fakultät der Universität Zürich von William T. Ralvenius aus Schweden Promotionskomitee Prof. Dr. Hanns Ulrich Zeilhofer (Leitung der Dissertation) Prof. Dr. Stephan Neuhauss Prof. Dr. Carsten Wagner Zürich, 2015 TABLE OF CONTENTS SUMMARY……………………………………………………………………….. 1 ZUSAMMENFASSUNG…………………………………………………………. 3 ABBREVIATIONS AND CHEMICAL NAMES………………………………. 5 GENERAL INTRODUCTION………………………………………….………. 7 PAIN………………………………………………………………………… 7 CURRENTLY AVAILABLE ANALGESICS…………………………… 24 TESTS FOR MEASURING PAIN IN LABORATORY ANIMALS…… 26 RESTORING THE SPINAL PAIN GATE: GABAA RECEPTORS AS TARGETS FOR NOVEL ANALGESICS……………………………….. 33 AIMS……………………………….…………………………………………..…. 57 EXPERIMENTAL SECTION………………………………………………..…. 59 ANALGESIA AND UNWANTED BENZODIAZEPINE EFFECTS IN POINT-MUTATED MICE EXPRESSING ONLY ONE BENZODIAZEPINE-SENSITIVE GABAA RECEPTOR SUBTYPE…. 61 N-DESMETHYL CLOBAZAM IS AN α2 PREFERING BENZODIAZEPINE THAT PRODUCES ANTIHYPERALGESIA IN MICE IN THE ABSENCE OF OBVIOUS SEDATION………………… 89 GENERAL DISCUSSION…………………………………………………….…. 107 REFERENCES……………………………….……………..……………………. 111 CURRICULUM VITAE…………………………………………………………. 121 PUBLICATIONS……………………………….………...………………………. 123 ACKNOWLEDGEMENTS……………………………………………………… 125 SUMMARY The ability to perceive pain is a basic property of humans and of all other higher order animals. The primary role of pain sensation and nociception (that is, the neuronal activity encoding pain) is to protect against potentially harmful threats arriving from the environment or the body interior. Pain can however also become dysfunctional and persist for extended periods of time without an apparent benefit, instead becoming a major burden that requires medical attention. Chronic pain is a major socio-economic challenge, which – despite scientific advances in the understanding of its causes – remains poorly responsive to the drugs available on the market today. Recent insights into the mechanisms of chronic pain states suggest that a common factor for many kinds of persistent pain states is a loss of inhibition in spinal cord circuits that normally control nociceptive input to the brain. The so- called benzodiazepines – first marketed by Hoffmann-La Roche in the 1960s – are drugs that facilitate synaptic inhibition throughout the CNS and have as such the potential to reverse pathological disinhibition. Benzodiazepines facilitate inhibition by increasing the activity of γ-aminobutyric acid (GABA) at its receptor, a heteropentameric anion permeable ion channel. Although rodent studies have shown that pathologically increased pain sensitivity can be normalized by intrathecal (spinal) injection of benzodiazepines, these drugs do not exert clinically relevant analgesia in human patients, at least not after systemic application. In this thesis, I have tested the hypothesis that benzodiazepines reverse pathological pain after systemic application if their action is restricted to well-defined subtypes of GABAA receptors. Using GABAA receptor point-mutated mice, I was able to demonstrate that selective targeting of GABAA receptors that contain the α2 subunit (α2-GABAA) receptors evoke pronounced pain relief in the absence of confounding and undesired sedation. I could also confirm previous findings that had proposed that activation of the same GABAA receptors induces anxiolysis and muscle relaxation. Importantly, selective targeting of α2- GABAA receptors avoided several unwanted effects of classical non-selective benzodiazepines including sedation, impairment of motor coordination, and the progressive loss of therapeutic efficacy over time. Using mice in which the action of classical benzodiazepine agonists was restricted to only a single GABAA receptor subtype, I could also propose a new hypothesis explaining why classical benzodiazepines lack clinically relevant analgesic properties. For two clinically used benzodiazepines (diazepam and midazolam), I could demonstrate that strong α1-GABAA receptor-mediated sedation occurs already at doses - 1 - more than 20 times lower that those required for significant analgesia. Dose limiting sedation is therefore the likely reason for the lack of clinically relevant analgesia in human pain patients. The second part of the thesis project has been designed to foster the translation of the results of the first project to clinical application. Within recent years, drug companies have developed benzodiazepine site agonists with improved subtype specificity (that is, with reduced activity at α1-GABAA receptors). So far, none of these compounds is generally available for tests in humans, prompting for alternative strategies to obtain proof-of-concept evidence. A previous report has suggested that N-desmethyl clobazam (NDMC), a major metabolite of the clinically used benzodiazepine clobazam, may have a pharmacological profile more favorable than that of most classical benzodiazepines. We found that NDMC indeed possesses an improved α2 versus α1-GABAA receptor selectivity in vitro, and pronounced antihyperalgesic efficacy at doses that do not induce apparent sedation or impairment of motor coordination in mice. In the light of this favorable pharmacological profile and given the fact that new unexpected side effects are highly unlikely to occur in a metabolite of a clinically used benzodiazepine, NDMC should be well-suited for proof-of- concept trials in human volunteers or pain patients. The results of this thesis thus provide further support for a potential use of α2-selective GABAA receptor modulators as novel analgesics. - 2 - ZUSAMMENFASSUNG Schmerzwahrnehmung ist eine grundlegende Fähigkeit des Menschen und aller höheren Tiere. Die primäre Funktion des Schmerzes und der Nociception (d.h. der neuronalen Kodierung von Schmerz) ist der Schutz vor potentieller Gewebeschädigung durch exogene oder endogene Einflüsse. Schmerzen können sich jedoch auch zu einer Fehlfunktion des Körpers entwickeln und über längere Zeit ohne offensichtlichen Nutzen bestehen bleiben. Schmerzen werden dann zu einer schweren Belastung