The Cardiovascular and Antiarrhythmic Actions of A
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THE CARDIOVASCULAR AND ANTIARRHYTHMIC ACTIONS OF A SERIES OF KAPPA OPIOID AGONISTS AND RELATED COMPOUNDS by MICHAEL KENNETH PUGSLEY B.Sc., The University of British Columbia, Vancouver, B.C., Canada, 1989 M.Sc., The University of British Columbia, Vancouver, B.C., Canada, 1992 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PHARMACOLOGY & THERAPEUTICS FACULTY OF MEDICINE We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1995 © Michael Kenneth Pugsley In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis br scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed withoót my written permission. (Signature) PARTuEwr OF PI4ARMAOOLO a THERAPEUTICS Facully of MedTcIna Th Unverity of Bntish Colun*1 217ê Health Scf.nc.. Uai1 Department of GII*d VTfZ3 The University of British Columbia Vancouver, Canada Date SEP rt I99 DE-6 (2188) Abstract The cardiovascular and antiarrhythmic actions of the kappa (K) opioid receptor agonists are not well characterized. This may be the result of the limited role opioids play in the regulation of cardiovascular function and the fact that pharmaceutical companies concentrate on their analgesic properties. The studies described in this thesis attempt to characterize the cardiovascular and antiarrhythmic properties of a novel series of arylacetamide Ic receptor agonists and related compounds. The compounds examined included U-62,066E (spiradoline), U-50,488H, (-)PD129,290 and its inactive enantiomer, (+)PD129,289, (±)PDI 17,302 and its inactive enantiomer, (+)PD123,497. Studies were conducted to determine whether the K receptor is involved in the antiarrhythmic actions of these compounds and if it is not, attempt to determine a mechanism by which these compounds may confer antiarrhythmic protection against both electrically-induced and ischaemic arrhythmias. Studies were therefore conducted in rats in the absence and presence of the opioid antagonists, naloxone and Mr2266 or, when possible, with inactive enantiomers of ic receptor agonists. Six novel compounds sharing the arylacetamide structure were examined for their actions on haemodynamic and EGG actions in intact pentobarbitone-anaesthetised rats. The previously unpublished parts of the thesis focuses on U-62,066E (spiradoline) and also provides results obtained in isolated cardiac myocytes for (±)PDI 17,302 and its inactive enantiomer, (+)PD123,497. Previously published data contained in the appendices covers the other three compounds. All compounds produced similar actions. All the compounds examined produced a dose-dependent reduction in heart rate, blood pressure, and prolonged the P-R, QRS duration, and Q-aT intervals with an accompanying increase in RSh (a measure of sodium channel block in the rat). The effects of the arylacetamides, exemplified by spiradoline, were assessed using a modified Langendorff isolated heart preparation. Spiradoline and related compounds reduced the sinus beating rate and contractility of hearts in a concentration-dependent III manner. ECG effects in isolated hearts included prolongation of the P-R interval and QRS width. The effects of the compounds on the ability of electrical stimulation to stimulate the heart were examined in pentobarbitone-anaesthetised rats. Spiradoline and related compounds dose-dependently increased the current and duration of stimulus required to stimulate the heart and also increased ventricular fibrillation threshold. All compounds prolonged effective refractory period and reduced maximum following frequency. In an attempt to determine effectiveness against ischaemic arrhythmias in rats after coronary occlusion a dose of 2.5pmol/kg/min spiradoline was given in the absence and presence of 2.5pmol/kg/min naloxone. Spiradoline reduced the incidence of VT from 100% in controls to 33 and 44% in the absence and presence of naloxone. The incidence of VF was reduced from 100% to 22% and 0% in the absence and presence of naloxone. All chemically related arylacetamides were similarly antiarrhythmic. In order to delineate a mechanism by which these compounds may be antiarrhythmic their effects on sodium and potassium currents were examined in isolated rat cardiac myocytes. Spiradoline blocked these currents in a concentration-dependent and reversible manner. The EC50 for half-maximal sodium current block was 66pM. Spiradoline also produced a hyperpolarizing shift in the voltage-dependence of inactivation of the channel but did not alter activation kinetics. The block produced by spiradoline on sodium channels was both tonic and use-dependent. Additional studies with (±)PDI 17,302 and its inactive enantiomer, (+)PD123,497 showed that the block produced by these arylacetamides is pH-dependent and that block is consistent with activity at an extracellular site on the sodium channel. These results indicate that spiradoline and the arylacetamides examined produce antiarrhythmic actions independent of the K receptor in the rat. Our results demonstrate the sodium channel, and to a lesser extent, potassium channels blocking actions of these compounds. iv TABLE OF CONTENTS CHAPTER Page Abstract ii Table of Contents iv List of Figures vii List of Tables ix List of Abbreviations x List of Appendices xii Dedication xiv Acknowledgements xv I Introduction I 1.1 Antiarrhythmic Drugs I 1.1.1 A Brief History of Antiarrhythmic Drugs I 1.1.2 Utility of Antiarrhythmics in lschaemia 3 1.1.2.1 Supraventricular Tachyarrhythmias 5 1.1.2.2 Ventricular Arrhythmias 6 1.1.3 Classification of Antiarrhythmic Drugs 9 1.1.3.1 The Vaughan-Williams Classification 10 1.1.3.2 Subclassification Agents of Class I Antiarrhythmic 11 1.1.3.3 The Sicilian Gambit 13 1.2 Mechanisms of Arrhythmogenesis 14 1.2.1 The lschaemic Myocardium 14 1.2.2 Abnormal Impulse Conduction 17 1.2.3 Early Afterdepolarizations (EAD) 19 1.2.4 Delayed-Afterdepolarizations (DAD) 20 1.2.5 Re-entry 21 1.3 Cardiac Electrophysiology 23 1.3.1 The Cardiac Action Potential 23 1.3.2 The Sodium Channel 23 1.3.2.1 Gating Kinetics 23 1.3.2.2 Activation Kinetics 25 1.3.2.3 Inactivation Kinetics 25 1.3.3 The Potassium Channels 27 1.3.3.1 Channel Diversity 27 1.3.3.2 The Transient Outward Potassium Current 29 1.3.3.3 The Sustained Outward Delayed-Rectifier Current 30 1.4 Models of Sodium Channel Blockade 31 1.4.1 The Strichartz-Courtney Model 31 1.4.2 The Modulated Receptor Hypothesis 32 1.4.2.1 Simplified Versions of the Model 33 1.4.2.1.1 Kappa-Repriming Model 33 1.4.2.1.2 Guarded Receptor Hypothesis 34 1.5 Opiold Receptor heterogeneity 35 1.5.1 Historical perspective 36 1.5.2 Classification of Opiold Receptors 37 1.5.3 Antiarrhythmic Actions 39 V 1.6 The Kappa (K) Receptor 42 1.6.1 Chemical Diversity of kappa agonists 44 1.6.2 Pharmacological actions of arylacetamides 46 1.6.2.1 Analgesia 46 1.6.2.2 Systemic Activity 49 1.6.2.2.1 Diuresis 49 1.6.2.2.2 Cardiovascular Actions 50 1.7 Objectives and Outline of Experiments Performed 56 1.7.1 In this Thesis and Those in the Appendix 56 2 Methods 58 2.1 Cardiac preparations 58 2.1.1 Intact rat studies 58 2.1.2 Surgical preparation 58 2.1.3 Experimental Design 59 2.2 Isolated rat hearts 62 2.2.1 Perfusion apparatus 62 2.3 Electrically-induced arrhythmias 64 2.3.1 Surgical preparation 65 2.3.2 Thresholds for capture (i1) 65 2.3.3 Threshold Pulse Width (tt) 66 2.3.4 Ventricular Fibrillation Threshold (VFt) 66 2.3.5 Maximum Following Frequency (MFF) 66 2.3.6 Effective Refractory Period (ERP) 67 2.4 lschaemia-induced arrhythmias 67 2.4.1 Surgical preparation in acute studies 67 2.4.2 Experimental Design 68 2.4.3 Pre- and post-occlusion ECG changes 69 2.4.4 Analysis of arrhythmias 70 2.5 Isolated ventricular myocytes 71 2.5.1 Patch-clamp apparatus 71 2.5.2 Cell isolation 71 2.5.3 Recording solutions 72 2.5.4 Microelectrode Preparation 73 2.5.5 Patching Ventricular Myocytes 74 2.5.6 Patch-Clamp Experiments 75 2.5.6.1 Sodium Currents 75 2.5.6.2 Potassium Currents 78 2.5.7 Drugs 78 2.6 Statistical Analysis 79 3 Unpublished Results for other Arylacetamides 80 3.1 Studies with U-62,066E (Spiradoline) 80 3.1.1 Isolated Heart studies- Contractility and ECG effects 80 3.1.2 Haemodynamic and ECG actions of Spiradoline 85 3.1.3 Electrical Stimulation studies 90 3.1.4 Coronary Artery Occlusion studies 99 3.1.5 Electrophysiological actions on Sodium currents 101 3.1.5.1 Concentration-response curves 101 3.1.5.2 Current-Voltage effects 102 3.1 .5.2.1 Activation kinetics 102 3.1.5.2.2 Inactivation kinetics 105 vi 3.1.5.3 Tonic and Use-dependent components of 108 spiradoline block of sodium currents 3.1.6 Electrophysiological actions on Potassium currents 113 3.1.6.1 Transient Outward and Sustained Delayed- 113 Rectifier Potassium Currents 3.2 Studies with (±)PDII7,302 and (+)PD123,497 113 3.2.1 Electrophysiological actions on Sodium currents 116 3.2.1 .1 Concentration-response curves 116 3.2.1.2 Intracellular vs. extracellular locus of action 116 3.3 Summary of Results obtained in previous studies - manuscripts 124 in Appendices 4 Discussion 129 4.1 Cardiovascular actions of arylacetamides 130 4.1 .1 Blood Pressure 130 4.1.2 Heart Rate