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Sándor Farkas, MD ANALYSIS OF PHARMACODYNAMICS OF TOLPERISONE-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 Page 2 of 122 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 ...........................................................................................................................
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