ANALYSIS OF PHARMACODYNAMICS OF -TYPE CENTRALLY ACTING MUSCLE RELAXANTS IN NON-CLINICAL STUDIES AND RESEARCH INTO PRECLINICAL MODELLING OF MIGRAINE

Dissertation for the degree of Doctor of Philosophy (PhD)

Sándor Farkas, MD

Doctoral School of Pharmaceutical Sciences Neuropharmacology Programme

Doctoral School Leader: Prof. Dr. Erika Pintér Programme Leader: Prof. Dr. Erika Pintér Supervisor: Prof. Dr. Zsuzsanna Helyes

Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pécs

Pécs, 2016 Table of contents

List of Abbreviations ...... 5 1 Preface ...... 7 2 Chapter 1: Pharmacodynamics of tolperisone-type centrally acting muscle relaxants ...... 7 2.1 Introduction ...... 7 2.1.1 General objectives ...... 7 2.1.2 Neuropharmacological studies on efficacy and side effects in mice ...... 10 2.1.2.1 Studies of the therapeutic index ...... 10 2.1.2.2 Study on oral effectiveness and duration of action ...... 11 2.1.3 Instrumental neurophysiological studies in unconscious cats and rats ...... 11 2.1.4 Studies on the molecular mechanism of action of tolperisone-type drugs ...... 13 2.1.5 Drugs used in comparative pharmacological studies ...... 15 2.1.6 Summary of specific objectives ...... 16 2.2 Methods ...... 17 2.2.1 Neuropharmacological studies on efficacy and side effects in mice ...... 17 2.2.1.1 GYKI 20039-induced tremor test ...... 17 2.2.1.2 Morphine-induced Straub tail test ...... 18 2.2.1.3 Rotarod test ...... 19 2.2.1.4 Locomotor activity...... 19 2.2.1.5 Weight-lifting test ...... 19 2.2.1.6 Thiopental sleeping time ...... 20 2.2.2 Instrumental neurophysiological studies in unconscious cats ...... 20 2.2.2.1 Animals ...... 20 2.2.2.2 Standard surgery in cats ...... 20 2.2.2.3 A special technique for quantitative evaluation of triggered electrophysiological events ...... 21 2.2.2.4 Flexor reflex in intact cats ...... 22 2.2.2.5 Flexor reflex in spinal cats ...... 23 2.2.2.6 Patellar reflex in cats ...... 23 2.2.2.7 Study on the reticulospinal control of the patellar reflex in cats ...... 23 2.2.2.8 Spinal root potentials evoked by tibial nerve stimulation in cats ...... 24 2.2.2.9 Intercollicular decerebrate rigidity in cats ...... 25 2.2.3 Instrumental neurophysiological studies in unconscious rats ...... 25 2.2.3.1 Intercollicular decerebrate rigidity in rats ...... 25 2.2.3.2 Studies in anaesthetised spinal rats in vivo...... 26 2.2.4 Studies in the isolated hemisected rat spinal cord in vitro ...... 29 2.2.4.1 Spinal root potential studies (in vitro) ...... 29 2.2.4.2 Spinal cord grease gap method (in vitro) ...... 30 2.2.4.3 Data acquisition and analysis in the in vitro hemisected spinal cord studies ...... 30 2.2.5 Patch-clamp analysis of effects on voltage-gated channels ...... 31 2.2.5.1 Preparation of sensory neurones ...... 31

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2.2.5.2 Whole-cell patch-clamp recording and analysis ...... 31 2.2.6 Materials ...... 32 2.3 Results ...... 33 2.3.1 Neuropharmacological studies in mice ...... 33 2.3.1.1 GYKI 20039-induced tremor test ...... 33 2.3.1.2 Straub tail, rotarod, locomotor and weight-lifting tests and therapeutic indices ..... 35 2.3.1.3 Thiopental sleeping time ...... 36 2.3.2 Instrumental neurophysiological studies in unconscious cats ...... 37 2.3.2.1 Flexor reflex in intact cats ...... 37 2.3.2.2 Flexor reflex in spinal cats...... 41 2.3.2.3 Patellar reflex in cats ...... 43 2.3.2.4 Study on the reticulospinal control of the patellar reflex in cats ...... 44 2.3.2.5 Spinal root potentials evoked by tibial nerve stimulation in cats ...... 45 2.3.2.6 Intercollicular decerebrate rigidity in cats ...... 48 2.3.3 Instrumental neurophysiological studies in unconscious rats ...... 50 2.3.3.1 Investigation of the ventral root reflex potentials ...... 50 2.3.3.2 Neuronal excitability test ...... 51 2.3.3.3 Study of feed-forward inhibition ...... 53 2.3.3.4 Study of recurrent inhibition ...... 53 2.3.3.5 Study of afferent nerve conduction ...... 54 2.3.3.6 Intercollicular decerebrate rigidity in rats ...... 54 2.3.4 Studies in the isolated hemisected rat spinal cord in vitro ...... 55 2.3.4.1 Spinal root potential studies ...... 55 2.3.4.2 Spinal cord grease gap study ...... 61 2.3.5 Patch-clamp analysis of effects on voltage-gated channels ...... 62 2.3.5.1 Effects on voltage-gated sodium channels ...... 62 2.3.5.2 Effects on voltage-gated calcium channels ...... 68 2.3.5.3 Effects on voltage-gated potassium channels ...... 71 2.4 Discussion ...... 72 2.5 Summary of conclusions ...... 85 2.6 Epilogue ...... 87 2.7 References ...... 88 3 Chapter 2: Preclinical modelling of migraine ...... 94 3.1 Introduction ...... 94 3.2 Materials and methods ...... 96 3.2.1 Drug treatments and control vehicles ...... 96 3.2.2 Light aversion test ...... 96 3.2.3 Cranial blood flow experiments ...... 97 3.2.4 Immunohistochemistry study of c-Fos and nNOS in TNC and TRG ...... 97 3.2.5 Thermal hyperalgesia of the paw measured with the hot plate test ...... 98 3.2.6 Mechanical allodynia of the paw measured with von Frey filaments ...... 99

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3.2.7 Orofacial pain sensitivity tested with von Frey filaments ...... 99 3.2.8 Data analysis and statistics ...... 99 3.3 Results ...... 100 3.3.1 Light aversion assay ...... 100 3.3.2 Cranial blood perfusion ...... 101 3.3.3 Immunohistochemistry for c-Fos and nNOS ...... 102 3.3.4 Thermal hyperalgesia of the paw ...... 103 3.3.5 Mechanical allodynia of the paw ...... 104 3.3.6 Orofacial pain sensitivity ...... 104 3.4 Discussion ...... 105 3.5 References ...... 112 4 Publications of the applicant ...... 116 4.1 Publications substantiating the topics of the dissertation ...... 116 4.2 Other scientific publications of the applicant ...... 117 5 Acknowledgements ...... 120

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List of Abbreviations

ACSF artificial cerebrospinal fluid AFP afferent fibre potential AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ATP adenosine triphosphate

C1 cervical segment 1 cf. confer (compare) cGMP cyclic guanosine-monophosphate CGRP calcitonin gene related peptide CMR centrally acting CNS central nervous system CP creatine phosphate CPK creatine phosphokinase C-T interval conditioning – test interval DBPCT double-blind placebo controlled clinical trial(s) DMEM Dulbecco’s Modified Eagle’s Medium DR dorsal root DRG dorsal root ganglion (ganglia) DRP dorsal root potential DRR dorsal root reflex DR-VRP dorsal root stimulation induced ventral root potential DSR disynaptic reflex EGTA ethylene glycol tetraacetic acid EMG electromyographic (or electromyogram) EPSP excitatory postsynaptic potential ES extracellular solution GABA gamma-aminobutyric acid GTP guanosine triphosphate HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid hRAMP1 human receptor activity modifying protein 1 i.d. intraduodenal IC independent citations

IC50 fifty percent inhibitory concentration IF impact factor i.p. intraperitoneal i.v. intravenous

ID50 fifty percent inhibitory dose IPSP inhibitory postsynaptic potential IS intracellular solution LO locomotor activity (test)

LX lumbar segment X (e.g. L5 = lumbar segment 5) MN motoneurone – direct excitation MS monosynaptic excitation MSR monosynaptic reflex NMDA N-methyl-D-aspartate

Page 5 of 122 nNOS neuronal nitric oxide synthase NO nitrogen monoxide NTG nitroglycerin p.o. per os PAF primary afferent excitation PBS phosphate-buffered saline PSR polysynaptic reflex RR rotarod (test)

S1 sacral segment 1 s.c. subcutaneous S.D. standard deviation S.E.M. standard error of mean sGC soluble guanylate cyclase SK channel Small conductance calcium-activated ST Straub tail (test) TI therapeutic index TNC trigeminal nucleus caudalis TR tremor (test) TRG trigeminal ganglion TRP transient receptor potential TTX TTX-R tetrodotoxin resistant TTX-S tetrodotox