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The cardiovascular and antiarrhythmic actions of a series of kappa opioid agonists and related compounds Pugsley, Michael Kenneth 1995

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THE CARDIOVASCULAR AND ANTIARRHYTHMIC ACTIONS OF A SERIES OFKAPPA OPIOID AGONISTS AND RELATED COMPOUNDSbyMICHAEL KENNETH PUGSLEYB.Sc., The University of British Columbia, Vancouver, B.C., Canada, 1989M.Sc., The University of British Columbia, Vancouver, B.C., Canada, 1992A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESDEPARTMENT OF PHARMACOLOGY & THERAPEUTICSFACULTY OF MEDICINEWe accept this thesis as conformingTHE UNIVERSITY OF BRITISH COLUMBIASeptember 1995to the required standard© Michael Kenneth PugsleyIn presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis br scholarly purposes may be granted by the headof mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed withoót my writtenpermission.(Signature)PARTuEwr OF PI4ARMAOOLOa THERAPEUTICSFacully of MedTcInaTh Unverity of Bntish Colun*1217ê Health Scf.nc.. Uai1Department ofGII*d VTfZ3The University of British ColumbiaVancouver, CanadaDate SEP rt I99DE-6 (2188)AbstractThe cardiovascular and antiarrhythmic actions of the kappa (K) opioid receptoragonists are not well characterized. This may be the result of the limited role opioids playin the regulation of cardiovascular function and the fact that pharmaceutical companiesconcentrate on their analgesic properties. The studies described in this thesis attempt tocharacterize the cardiovascular and antiarrhythmic properties of a novel series ofarylacetamide Ic receptor agonists and related compounds. The compounds examinedincluded 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 wereconducted to determine whether the K receptor is involved in the antiarrhythmic actions ofthese compounds and if it is not, attempt to determine a mechanism by which thesecompounds may confer antiarrhythmic protection against both electrically-induced andischaemic arrhythmias. Studies were therefore conducted in rats in the absence andpresence of the opioid antagonists, naloxone and Mr2266 or, when possible, with inactiveenantiomers of ic receptor agonists.Six novel compounds sharing the arylacetamide structure were examined for theiractions on haemodynamic and EGG actions in intact pentobarbitone-anaesthetised rats.The previously unpublished parts of the thesis focuses on U-62,066E (spiradoline) andalso provides results obtained in isolated cardiac myocytes for (±)PDI 17,302 and itsinactive enantiomer, (+)PD123,497. Previously published data contained in theappendices 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 anaccompanying increase in RSh (a measure of sodium channel block in the rat).The effects of the arylacetamides, exemplified by spiradoline, were assessed usinga modified Langendorff isolated heart preparation. Spiradoline and related compoundsreduced the sinus beating rate and contractility of hearts in a concentration-dependentIIImanner. ECG effects in isolated hearts included prolongation of the P-R interval and QRSwidth.The effects of the compounds on the ability of electrical stimulation to stimulate theheart were examined in pentobarbitone-anaesthetised rats. Spiradoline and relatedcompounds dose-dependently increased the current and duration of stimulus required tostimulate the heart and also increased ventricular fibrillation threshold. All compoundsprolonged effective refractory period and reduced maximum following frequency.In an attempt to determine effectiveness against ischaemic arrhythmias in rats aftercoronary occlusion a dose of 2.5pmol/kg/min spiradoline was given in the absence andpresence of 2.5pmol/kg/min naloxone. Spiradoline reduced the incidence of VT from100% in controls to 33 and 44% in the absence and presence of naloxone. The incidenceof 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 beantiarrhythmic their effects on sodium and potassium currents were examined in isolatedrat cardiac myocytes. Spiradoline blocked these currents in a concentration-dependentand reversible manner. The EC50 for half-maximal sodium current block was 66pM.Spiradoline also produced a hyperpolarizing shift in the voltage-dependence of inactivationof the channel but did not alter activation kinetics. The block produced by spiradoline onsodium channels was both tonic and use-dependent. Additional studies with (±)PDI 17,302and its inactive enantiomer, (+)PD123,497 showed that the block produced by thesearylacetamides is pH-dependent and that block is consistent with activity at an extracellularsite on the sodium channel.These results indicate that spiradoline and the arylacetamides examined produceantiarrhythmic actions independent of the K receptor in the rat. Our results demonstratethe sodium channel, and to a lesser extent, potassium channels blocking actions of thesecompounds.ivTABLE OF CONTENTSCHAPTER PageAbstract iiTable of Contents ivList of Figures viiList of Tables ixList of Abbreviations xList of Appendices xiiDedication xivAcknowledgements xvI Introduction I1.1 Antiarrhythmic Drugs I1.1.1 A Brief History of Antiarrhythmic Drugs I1.1.2 Utility of Antiarrhythmics in lschaemia Supraventricular Tachyarrhythmias Ventricular Arrhythmias 61.1.3 Classification of Antiarrhythmic Drugs The Vaughan-Williams Classification Subclassification of Class I Antiarrhythmic Agents The Sicilian Gambit 131.2 Mechanisms of Arrhythmogenesis 141.2.1 The lschaemic Myocardium 141.2.2 Abnormal Impulse Conduction 171.2.3 Early Afterdepolarizations (EAD) 191.2.4 Delayed-Afterdepolarizations (DAD) 201.2.5 Re-entry 211.3 Cardiac Electrophysiology 231.3.1 The Cardiac Action Potential 231.3.2 The Sodium Channel Gating Kinetics Activation Kinetics Inactivation Kinetics 251.3.3 The Potassium Channels Channel Diversity The Transient Outward Potassium Current The Sustained Outward Delayed-Rectifier Current 301.4 Models of Sodium Channel Blockade 311.4.1 The Strichartz-Courtney Model 311.4.2 The Modulated Receptor Hypothesis 321.4.2.1 Simplified Versions of the Model 331. Kappa-Repriming Model 331. Guarded Receptor Hypothesis 341.5 Opiold Receptor heterogeneity 351.5.1 Historical perspective 361.5.2 Classification of Opiold Receptors 371.5.3 Antiarrhythmic Actions 39V1.6 The Kappa (K) Receptor 421.6.1 Chemical Diversity of kappa agonists 441.6.2 Pharmacological actions of arylacetamides 461.6.2.1 Analgesia 461.6.2.2 Systemic Activity 491. Diuresis 491. Cardiovascular Actions 501.7 Objectives and Outline of Experiments Performed 561.7.1 In this Thesis and Those in the Appendix 562 Methods 582.1 Cardiac preparations 582.1.1 Intact rat studies 582.1.2 Surgical preparation 582.1.3 Experimental Design 592.2 Isolated rat hearts 622.2.1 Perfusion apparatus 622.3 Electrically-induced arrhythmias 642.3.1 Surgical preparation 652.3.2 Thresholds for capture (i1) 652.3.3 Threshold Pulse Width (tt) 662.3.4 Ventricular Fibrillation Threshold (VFt) 662.3.5 Maximum Following Frequency (MFF) 662.3.6 Effective Refractory Period (ERP) 672.4 lschaemia-induced arrhythmias 672.4.1 Surgical preparation in acute studies 672.4.2 Experimental Design 682.4.3 Pre- and post-occlusion ECG changes 692.4.4 Analysis of arrhythmias 702.5 Isolated ventricular myocytes 712.5.1 Patch-clamp apparatus 712.5.2 Cell isolation 712.5.3 Recording solutions 722.5.4 Microelectrode Preparation 732.5.5 Patching Ventricular Myocytes 742.5.6 Patch-Clamp Experiments 752.5.6.1 Sodium Currents 752.5.6.2 Potassium Currents 782.5.7 Drugs 782.6 Statistical Analysis 793 Unpublished Results for other Arylacetamides 803.1 Studies with U-62,066E (Spiradoline) 803.1.1 Isolated Heart studies- Contractility and ECG effects 803.1.2 Haemodynamic and ECG actions of Spiradoline 853.1.3 Electrical Stimulation studies 903.1.4 Coronary Artery Occlusion studies 993.1.5 Electrophysiological actions on Sodium currents 1013.1.5.1 Concentration-response curves 1013.1.5.2 Current-Voltage effects 1023.1 .5.2.1 Activation kinetics 1023. Inactivation kinetics 105vi3.1.5.3 Tonic and Use-dependent components of 108spiradoline block of sodium currents3.1.6 Electrophysiological actions on Potassium currents 1133.1.6.1 Transient Outward and Sustained Delayed- 113Rectifier Potassium Currents3.2 Studies with (±)PDII7,302 and (+)PD123,497 1133.2.1 Electrophysiological actions on Sodium currents 1163.2.1 .1 Concentration-response curves 1163.2.1.2 Intracellular vs. extracellular locus of action 1163.3 Summary of Results obtained in previous studies - manuscripts 124in Appendices4 Discussion 1294.1 Cardiovascular actions of arylacetamides 1304.1 .1 Blood Pressure 1304.1.2 Heart Rate 1314.1.3 ECG 1314.2 The effects of arylacetamides- opioid receptor dependent 133mechanism or ion channel blockade in the heart4.3 An electrophysiological basis for the channel blockade seen 136with arylacetamides in the heart4.4 Are the ion channel blocking actions of the arylacetamides 146responsible for the antiarrhythmic actions of these compounds?4.4.1 Electrical Stimulation Studies 1464.4.2 lschaemic Arrhythmia Studies 1504.5 Projections regarding the value arylacetamides have in the 157study and/or treatment of arrhythmias and ion channelblocking drugs5 Summary 1586 References 1607 Appendices 194Appendix 1 196Appendix 2 202Appendix 3 207Appendix 4 214Appendix 5 221Appendix 6 224Appendix 7 232Appendix 8 239vi’LIST OF FIGURESFigure PageI Chemical structure of U-62,066E (spiradoline) and other 82arylacetamides.2 Concentration-dependent effects of spiradoline in the absence and 84presence of 1 pM naloxone on (A) heart rate and (B) the P-R and QRSintervals of the ECG in Langendorff-perfused isolated rat hearts.3 Effect of spiradoline on (A) peak systolic pressure in the absence and 87presence 1 pM naloxone and (B) rate of contration and relaxationin Langendorff-perfused isolated rat hearts.4 Dose-related effects of spiradoline on blood pressure (A) and heart rate 89(B) in pentobarbitone-anaesthetised rats in the absence and presence of2.5 pmol/kg/min naloxone.5 Effects of spiradoline on P-R interval (A) and QRS duration (B) in 92pentobarbitone-anaesthetized rats in the absence and presence of2.5 pmol/kg/min naloxone.6 Effects of spiradoline on RSh (A) and the Q-aT interval (B) of 94pentobarbitone-anaesthetized rats in the absence and presence of2.5 pmol/kg/min naloxone7 In vivo effects of spiradoline, in the absence and presence of 962.5 pmol/kg/min naloxone, on threshold current for capture (it)(A) and ventricular fibrillation threshold (VFt) (B).8 In vivo effects of spiradoline, in the absence and presence of 982.5 pmol/kg/min naloxone, on effective refractory period (ERP)(A) and maximum following frequency (MFF) (B).9 Effect of spiradoline block on (A) sodium currents, and (B) 104concentration-response curve for blockade of sodium current inrat isolated cardiac myocytes10 Effect of spiradoline on the current-voltage relationship (A) and 107inactivation (B) of sodium currents.II The frequency-dependent block of sodium currents by spiradoline. 10912 The concentration-dependent effects of spiradoline on (A) the 112frequency-dependence of block and (B) time to peak sodiumcurrent amplitude reduction.13 The effects of spiradoline on transient outward (ito) and sustained 118VIIIoutward delayed rectifier (Iksus) potassium currents.14 Effect of (±)PDII7,302 on (A) sodium currents, and (B)the 118concentration-response curve for block of sodium current inrat isolated cardiac myocytes15 The intracellular effect of (±)PDI 17,302 block of sodium currents in rat 121cardiac myocytes.16 Effect of (+)PD123,497 (A) blockade of sodium current, (B) applied 123inside the cell and (C) at pH 6.4 and 7.4 in rat cardiac myocytes.ixLIST OF TABLESTable PageI The cardiovascular and ECG effects of 2.5 pmol/kg/min spiradoline 100(U-62,066E), in the absence and presence of 2.5 pmol/kg/minnaloxone- antiarrhythmic studyII Antiarrhythmic effect of spiradoline (2.5 pmol/kg/min) in the 100absence and presence of 2.5 pmol/kg/min naloxone againstcoronary artery occlusion-induced arrhythmias in pentobarbitoneanaesthetised rats.Ill The actions of 2.5 pmol/kg/min spiradoline, in the absence and 101presence of naloxone (2.5 pmol/kg/min) on ECG changes, mortalityand serum potassium induced by coronary artery occlusion inpentobarbitone-anaesthetised rats.IV Summary of Results which can be found in Appendices 128ixxLIST OF ABBREVIATIONScL-level level of significanceANOVA analysis of varianceANS autonomic nervous systemAP action potentialAPD action potential durationAS arrhythmia scoreAVJ atrio-ventricular junctionAVP arginine vasopressinB.P. blood pressureCAST Cardiac ArrhythmiaSuppression Trialdegree CelciusCNS central nervous systemDa daltonsECG electrocardiogramED50 dose of drug producinghalf-maximal responseEKC ethylketocyclazocineEOP endogenous opioid peptideERP effective refractory period9 gramGRH Guarded ReceptorHypothesishr hourHz hertzi.P. intraperitoneallyI.V. intravenousxikg kilogramless thanpM micromolarmg milligramMl myocardial infarctionmm minutemL millilitremmHg millimetres of mercurymM millimolarMRH Modulated ReceptorHypothesismsec millisecondMW molecular weightOZ occluded zonepH hydrogen ion concentrationPVC premature ventricularcontraction% percentages.e.mean standard error of the meansec secondSAR Structure-Activity RelationshipTTX tetrodotoxinVF ventricular fibrillationVT ventricular tachycardiaxl’LIST OF APPENDICESThe following papers comprise the latter part of the thesis and are found in the Appendix.Appendix IPugsley, M.K., Penz, W.P. Walker, M.J.A. and Wong, T-M. Cardiovascular actions ofthe kappa receptor agonist, U-50,488H, in the absence and presence of opioidreceptor blockade. Br. J. Pharmacol. 105: 521-526, 1992.Appendix 2Pugsley, M.K., Penz, W.P. Walker, M.J.A. and Wong, T-M. Antiarrhythmic effects ofU-50,488H in rats subject to coronary artery occlusion. Eur. J. Pharmacol. 212: 15-19, 1992.Appendix 3Pugsley, M.K., Saint, D.A., Penz, W.P. and Walker, M.J.A. Electrophysiological andantiarrhythmic actions of the kappa agonist PD129290, and its R,R (+) enantiomer,PD 129289. Br. J. Pharmacol. 110: 1579-1585, 1993.Appendix 4Pugsley, M.K., Saint, D.A., Walker, M.J.A. An electrophysiological basis for theantiarrhythmic actions of the ic-opioid receptor agonist U-50,488H. Eur. J.Pharmacol. 261: 303-309, 1994.Appendix 5Pugsley, M.K., Hayes, E.S., Saint, D.A. and Walker, M.J.A. Do related kappaagonists produce similar effects on cardiac ion channels? Proc. West. Pharmacol.Soc. 38: 25-27, 1995.xl”Appendix 6Penz, W.P., Pugsley, M.K., Hsieh, M.Z. and Walker, M.J.A. A new measure (RSh) fordetecting possible sodium channel blockade in vivo in rats. J. Pharmacol. Toxicol.Meth. 27(1): 51-58, 1992.Appendix 7Hayes, E., Pugsley, M.K., Penz, W.P., Adaikan, G. and Walker, M.J.A. Relationshipbetween Q-aT and RR intervals in rats, guinea pigs, rabbits and primates J.Pharmacol. Toxicol. Meth. 32(4): 201-207, 1994.Appendix 8Pugsley, M.K., Penz, W.P. and Walker, M.J.A. Cardiovascular actions of U-50,488Hand related kappa agonists. Cardiovasc. Drug Rev. 11(2): 151-164, 1993.xivDEDICATIONThis thesis is dedicated to my beloved parents, my brother and his family, my sister,grandparents and to my SharonxvACKNOWLEDGMENTSI wish to thank all the members of my thesis committee (Drs. MacLeod, Pang, Sutter andRabkin) for all their guidance during my thesis work and for their valuable discussionsabout my thesis - and pharmacology in general. As well I want to thank all the members ofthe department: George and Christian for teaching me all they know about lab preparationfor undergraduates, Elaine, Janelle, Wynne and Margaret for their friendship and helpduring the “fun” Scholarship application season and everyone else!. I also want to thankDr. SSM Karim, Dr. R Tabrizchi and Dr. D.M.J. Quastel for guidance and the Friday nightsocials, Cheers! I am indebted forever to Dr. D.A. Saint, and Dr. S.H. Chung, my mentorswhile in Oz, and everyone at the Australian National University for their friendship,assistance and willingness to teach me while in Australia. Again I want to express mysincere gratitude to my parents, Ken and Mary, my brother, David and his family, sisterChristina and the rest of my family for having faith in me and my career in heart research.To Dr. L. Guppy, Eric, Wei and all others in Lab 413, both past and present; as well asMilah and Andalee and Dr. A. “AB” Bain from RSD, Ltd., I say “thanks guys” for everything!I wish to express my sincere thanks to the Science Council of B.C., the B.C. MedicalServices Research Foundation and to everyone at the Heart and Stroke Foundation ofB.C. & Yukon for providing me with financial support throughout my studies. I am alsoindebted to my Sharon for your continued support, encouragement and understanding,especially during the writing of this thesis. I also want to thank my supervisor, Dr. M.J.A.Walker, for his guidance and wisdom in teaching about both pharmacology, and life’sphilosophy. You are a true teacher and as H. Krebs (1967) stated “Scientists are not somuch born as made by those who teach them research”. I have truly been fortunate tohave been your student.II Introduction1.1 Antiarrhythmic Drugs1.1.1 Brief History of Antiarrhythmic DrugsThe models of ion channel blockade currently used to quantify and qualify theactions of antiarrhythmic drugs in cardiac tissue under ischaemic or normal conditions, arebased upon many key observations. Distinctions are based primarily upon differences inelectrophysiology in tissues subjected to the two conditions. In addition many studies wereconducted in neuronal, rather than cardiac, tissue. However, most findings in neuronshave been applied to, and accurately describe, drug actions in the heart.Hodgkin and Huxley (1952) first examined the electrical properties of the sodiumcurrent using the squid giant axon. The results of this work provided the first implicit modelfor ion channel function whereby the sodium channel may exist in three states: resting(closed), active (open) and closed (inactive). These channel states are a function ofvoltage and time and are dependent upon membrane potential. Hodgkin and Huxleyinvestigated the kinetics of activation and inactivation of this channel and proposed thatthese properties were due to “rn” and “h” gates, respectively. Weidmann in 1955 foundthat in the presence of antiarrhythmic drugs (sodium channel blockers) the voltage-functionof the maximum rate of depolarization (Vmax) was shifted to more negative potentials.Weidmann suggested that this was due to drug interaction with the inactivation (h) gate(according to Hodgkin and Huxley formalism) of the sodium channel. These drugs did notalter activation kinetics.The next observation regarding drug interaction with the sodium channel was madeby Johnson and MacKinnon (1957). The action of quinidine on Vmax was minimal withlong diastolic intervals. When the frequency of channel activation increased sodium2channel blockade was enhanced. In 1970 Jensen and Katzung demonstrated that theantiarrhythmic drug diphenyihydantoin (DPH) acted synergistically with elevated externalpotassium to decrease Vmax in rabbit atrial preparations. Singh and Vaughan Williams thefollowing year showed, in rabbit atrial and ventricular muscle, a similar action for lidocaineand concluded that this drug may have preferential actions in ischaemic conditions.The findings of Johnston and MacKinnon in 1957 became important to ourunderstanding of antiarrhythmic drug action in 1973 when Strichartz showed that at anincreased stimulation frequency the block produced by quaternary local anaestheticsincreased. He also showed that these drugs interact with open sodium channels and thatboth the drug-free and drug-associated channels obey Hodgkin and Huxley kinetics. Thus,a model of drug interaction with the sodium channel was beginning to emerge which hadwide applicability to ion channels. Additional studies by Hondeghem et al. (1974) andHope et al. (1974) showed that many antiarrhythmic agents (such as quinidine, lidocaineand procainamide) selectively depress electrical activity in hypoxic tissue, and ischaemicmyocardium, respectively, as had been suggested by Jensen and Katzung earlier.Chen et al. (1975) suggested that the selective actions of antiarrhythmic drugs inischaemic or hypoxic tissue were due to their voltage-dependent properties. They foundthat in depolarized tissue recovery of channel function from drug block is slow compared tothat of normal or highly polarized cells. At the same time Courtney (1975) extended theStrichartz model (1973) to include the interaction of tertiary compounds and sodiumchannels, and explored the drug-associated channel interaction and its voltagedependence. It was at this time that Courtney coined the term “use-dependence” todescribe the increase in block associated with increased frequency of stimulation.Hille (1977) and Hondeghem and Katzung (1977) concurrently, but independently,formulated the Modulated Receptor Hypothesis (MRH) in an attempt to describe the effectsof local anaesthetics in nerve and cardiac muscle, respectively. The Strichartz-Courtneymodel could easily account for use-dependent development and recovery from block, It3could not, however, explain the voltage-dependence of recovery described by Chen et al.(1975); the MRH however, could account for this action.Starmer et al. (1984, 1985) have proposed the Guarded Receptor Hypothesis(GRH) in an attempt to simplify the complex mathematics involved in the global calculationof drug on-rate and off-rate constants (at least 16 in all) for the many states of drug-associated and drug-free channel association. The above outline briefly describes thedevelopment of antiarrhythmic drug models that have gained widespread acceptance indescribing how antiarrhythmic drugs may effectively suppress supraventricular andventricular-derived arrhythmias.1.1.2 Utility of Antiarrhythmics in lschaemiaWith the advent of models describing local anaesthetic and antiarrhythmic druginteraction with the sodium channel, an explosion in the development of antiarrhythmicdrugs occurred in the early 1970’s. The study of sodium channel antiarrhythmics continuedat a high level until 1989 when it was found that in post myocardial infarction (Ml) patients,the class I agents, flecainide and encainide, did not prevent, but rather increased mortality(CAST investigators, 1989).Initial development of antiarrhythmic drugs was fueled by the need for agents whichwould selectively abolish arrhythmias, possess greater cardiac efficacy and exhibit fewerside-effects. The majority of drugs were local anaesthetics or, according to the VaughanWilliams classification scheme (see below), class I agents, i.e. those which reduce theinflux of sodium ions during phase 0 of the action potential (AP).Sudden cardiac death is responsible for more than 35,000 deaths in Canada eachyear (Reeder et al., 1993). The majority of such deaths are due to ventricular fibrillationthat may or may not preceded by ventricular tachycardia (VT) or premature ventricularcontractions (PVC). The Cardiac Arrhythmia Suppression Trial (CAST or CAST-I) was a4large, randomized, multicentre, placebo-controlled study undertaken to examine whetherthe incidence of cardiac death in patients with asymptomatic or mild ventriculararrhythmias, post-MI, could be reduced with class I antiarrhythmic drugs (CASTinvestigators, 1989). Clinical trials with flecainide, encainide and later moricizine in CAST-Il(CAST-Il investigators, 1992) were stopped due to an abnormally high incidence of deathin drug treated groups. Additional evidence in human and animal arrhythmia studiesshows that class I antiarrhythmic drugs may potentially have proarrhythmic actions (Velebitet al., 1982; El Sherif, 1991; Starmer et al., 1991). Thus the CAST findings merelyhighlighted clinically what was already known by cardiac pharmacologists andelectrophysiologists for many years (Hondeghem, 1987; Tamargo et al., 1992).Local anesthetics or class I sodium channel blocking antiarrhythmic drugs possessseveral properties that characterize the actions of this class of drugs. A review of the largenumber of class I agents developed reveals that most are weak bases i.e., have a PKabetween 7.0-10.0. Thus their protonated:unprotonated ratio varies with pH (Narahashi etal., 1970). In the charged form the compound is hydrophilic and according to HilIe (1977)has limited access to the sodium channel through the pore (or hydrophilic pathway).However, in the uncharged form the compound can readily move into the lipid bilayer of thecell, accessing the channel independent of the channel state (Hille, 1977; Hondeghem andKatzung, 1977).Under ischaemic conditions the acidosis and hyperkalemia (see below for completediscussion) favor the interaction of the charged form of drug with the sodium ion channel.These changes provide the drug with “ischaemic-dependence” and hence a greater degreeof sodium channel block is possible compared to that found in the normal myocardium.Drug asymmetry is also an interesting feature of antiarrhythmic drug actions inischaemia. The stereochemical properties of drugs result in optically active stereoisomers,which may or may not exhibit similar properties in cardiac tissue. For example the (R)enantiomer of mexiletine, a class lb antiarrhythmic agent, is a more potent inhibitor of5sodium currents (Grant, 1990) than its (S)-enantiomer, but is limited in action due to its fastrate of clearance from the body (lgwemezie et al., 1991). The (R)-enantiomer has alsobeen shown to be more effective at reducing ischaemia-induced VF in rats subject tocoronary artery occlusion (lgwemezie et al., 1992).New antiarrhythmic drugs must be developed which show greater selectivity forconditions of ischaemia, possess fewer side effects (i.e., are not proarrhythmic) and whichhave a greater therapeutic efficacy. The arylacetamide compounds, exemplified by U62,066E (spiradoline), have properties that favour these desired actions and provide anovel chemical structure which may be explored and antiarrhythmics developed basedupon it. Supraventricular Tachyarrhythmias (SVT)Atrial arrhythmias are defined by the requirement that either the atria or atrioventricular junction (AVJ) is involved in initiation, and maintenance of the arrhythmia.Supraventricular arrhythmias involving the AVJ can be subdivided into those requiringconduction in the AV node (AVN) and those mediated by accessory pathways. Thesetypes have been extensively reviewed by Waldo and Wit (1993) and Ganz & Friedman(1995) therefore will not be elaborated upon in detail here. Briefly, it should be understoodthat AVN re-entrant tachycardia is the most common cause of SVT. The majority of SVT’sare due to anomalous bands of tissue that conduct abnormal impulses between atria andventricle. Usually conduction in this accessory pathway occurs alongside conduction in thenormal AV system (Ganz and Friedman, 1995). This causes the Wolff-Parkinson-White(WPW) syndrome characterized electrocardiographically as a delta wave accompanied byan ECG with a short P-R interval (Wolff et al., 1930).Pharmacological intervention is used in order to both relieve symptoms and toprevent stroke which can accompany atrial fibrillation. Treatment includes the use of6intravenous adenosine, an A1 agonist which rapidly suppresses more than 90% of all SVTarrhythmias (Camm and Garrett, 1991); the calcium antagonists verapamil and diltiazem;the cardiac glycoside digitalis; the B-antagonists, such as propranolol and nadolol andclass I antiarrhythmics such as intravenous (i.v.) procainamide, lidocaine and oral quinidine(the drug of choice for treatment of non-life threatening SVT) (Sokolow and Edgar, 1950;Fenster et al., 1983; Toulboul et al., 1991; Pritchett, 1992). It is suggested that lidocainemay be beneficial since it blocks conduction in accessory pathways but correspondinglymay further depress the poor haemodynamic status associated with a rapid ventricular rateoccurring during atrial fibrillation (Akhtar et al., 1993). Quinidine is preferred as it can begiven both to treat arrhythmias and with continued administration after arrhythmicsuppression may reduce the incidence of recurrence (Pritchett, 1992).Thus many pharmacological treatments exist for the effective reduction of SVT.Radiofrequency catheter ablation is the method of choice by cardiac electrophysiologistsfor the prevention of atrial arrhythmias (Ganz and Friedman, 1995). Ventricular ArrhythmiasVentricular arrhythmias resulting from myocardial infarction (Ml) are responsible forapproximately one-half of the deaths from coronary heart disease in Canada (Reeder etal., 1993). Arrhythmias originating in the ventricle have complicated etiologies and may bethe result of many factors. The responsible arrhythmogenic stimuli have been examined indetail and have been the focus of many reviews and therefore will not be elaborated uponin this thesis (see reviews by Botting et al., 1985 and Curtis et al., 1993). Briefly, thesestimuli include coronary artery spasm causing angina and its variant forms, plateletthrombosis and embolization, ruptured atheromatous plaques with or without emboli as wellas many local ionic (an imbalance of sodium, potassium and calcium) or metabolicsubstances including phospholipids and eicosanoids. The autonomic nervous system,7especially the sympathetic, may also play an important role in arrhythmogenesis (Boiling etal., 1983, 1985). Regardless of which stimuli or factors are involved, the relativeimportance of each is still uncertain.While cardiac rhythm disturbances may be the result of a variety ofpathophysiological conditions, it is ischaemia of the myocardium which dominates.Myocardial ischaemia or lack of blood flow to ventricular tissue disrupts a balance thatexists between myocardial demand for, and coronary delivery of ions oxygen, metabolicsubstrates and energy (Hearse and Dennis, 1982). Even more damaging is the lack ofremoval of toxic cellular and metabolic waste products such as protons, carbon dioxide andlactate. The condition of ischaemia produces profound alterations in normal cardiacelectrophysiology and cellular metabolism and as a consequence, the establishment of anectopic focus or perhaps re-entrant patterns of excitation or after-depolarization can arisewhich precipitate ventricular arrhythmias or fibrillation.Many models designed specifically to describe the production of arrhythmias havebeen developed. Models of electrically and chemically-induced arrhythmias induced in avariety of species have been extensively reviewed by Szekeres (1979). As well, Winslow(1984) carefully detailed electrical stimulation, chemical and pathological models ofarrhythmia induction while Walker et al. (1991) and Cheung et al. (1993) summarized theuse of the rat as a model of arrhythmogenesis.These models have been essential in the development of drugs for the treatment ofventricular arrhythmias. Effective treatment relies upon the conditions associated withischaemia and this allows antiarrhythmic drugs to be selective. The required actions ofsuch drugs have been extensively reviewed by Vaughan Williams (1989); they involverendering infarcted or ischaemic tissue electrically silent, producing greater refractorinessin normal ventricular tissue and reducing or silencing ectopic electrical activity in the borderzone between infarcted/ischaemic and normal ventricular tissue. Vaughan Williamsdetermined that blockade of sodium channels met all these criteria. However, more8variability would exist as to the effects of the particular agent or group of agents due totheir effects on APD or refractoriness. Drugs such as lidocaine, which do not prolong APD,may not enhance refractoriness in normal tissue, but instead possess marked use-dependent properties which then directs its “ischaemia-selectivity” (Hondeghem et a!.,1974; Clarkson et aL, 1988; Hondeghem and Snyders, 1990).Despite the large number of experiments that have been conducted remarkably fewsodium channel blocking antiarrhythmic drugs are actually used clinically to suppressarrhythmias. The low number of drugs accentuates the findings from the CardiacArrhythmia Suppression Trial (CAST or CAST-I) and CAST-Il trial (CAST investigators1989; CAST-Il investigators, 1992). Many previous studies conducted with a variety ofsodium channel blocking antiarrhythmic agents suggest that these drugs can effectivelysuppress arrhythmias but this does not necessarily translate into an improved survival rateafter a myocardial infarction (Myerburg et al., 1994). Quinidine, for example, was shown toincrease the incidence of mortality as compared with mexiletine in patients with potentiallylethal arrhythmias (Morganroth and Coin, 1991).Lidocaine prevents the occurrence of VF but does not significantly improve survivalin post-MI patients (Hine et al., 1989). In 1983 Campbell, after examining many clinicaltrials and experimental evidence in animals, suggested that lidocaine prophylaxis therapywas effective against ventricular arrhythmias, but pointed out that in humans the risk-benefit ratio of such treatment was not known.The class II (f’-blocking) antiarrhythmic agents, typified by propranolol and nadolol,are the only drugs that have been consistently shown to produce an increased time toonset of VT (Rosenfeldt et al., 1978), reduction in arrhythmia incidence (Paletta et al.,1989) and to improve survival post Ml (Yusuf et al., 1985). In a recent survey of U.S.cardiologists, B-blockers were the most frequently chosen class of antiarrhythmic drugsused to suppress arrhythmias in newly diagnosed patients (Morganroth et al., 1990).9The outcomes of both CAST-I and CAST-Il represented a marked set-back for thedevelopment of class I antiarrhythmic drugs. Focus has shifted to the development ofselective class Ill antiarrhythmic agents which prolong refractoriness (see Hondeghem,1992, 1994; Janse, 1992). Studies with amiodarone and sotalol have shown that thesedrugs produce an effective reduction in PVC incidence and mortality (Cairns et al., 1991).A recent clinical study which involves amiodarone, is CAMIAT or the Canadian AmiodaroneMyocardial Infarction Arrhythmia Trial. The results of this study are currently awaited.However, the recent Electrophysiologic Study versus Electrocardiographic Monitoring(ESVEM) trial compared sotalol to six class I antiarrhythmic drugs and showed that the riskof arrhythmia occurrence was lower with sotalol than the other agents (Mason, 1993).Despite the marked effectiveness of amiodarone and sotalol in clinical trials, the numerousother class Ill agents under development are “reverse use-dependent” or are essentiallyless effective at elevated heart rates associated with high frequency arrhythmias such asVT or VF (Hondeghem 1994; Janse, 1992). As well, they may induce bradycardicdependent arrhythmias such as torsades de pointes (Sasyniuk et al., 1989; Binah andRosen, 1992).Thus neither class I or Ill antiarrhythmic agents currently in use is ideal. The drugsdiscussed above may be potent, but are not selective, and possess a mixture ofpharmacological actions despite their classification as either sodium or potassium channelblockers. Thus it is worthwhile describing the systems of classification in current use.1.1.3 Classification of Antiarrhythmic Drugs.Drugs with desired antiarrhythmic qualities in ischaemic tissue should possess littleaction in normal cardiac tissue including action potentials. To be an effective agent adrug’s action must be confined to those action potentials which are involved in the genesisof abnormal cardiac arrhythmias (Singh and Courtney, 1990). As this area of10pharmacology expands it becomes necessary to provide a rational framework in which tointerpret the mechanism of action of currently used agents, and perhaps more importantly,newer agents. The schemes by which antiarrhythmic agents have been classified havebeen controversial (Singh and Hauswirth, 1974) and new propositions (such as the SicilianGambit) are no exception to this criticism. It was with the development of anelectrophysiological understanding of the cardiac action potential (which will be reviewedbelow), and its components, that the antiarrhythmic drugs could be reasonably separatedinto discrete groups based on their electrophysiological actions. These groups, accordingto Vaughan Williams (1984a, 1984b), were not a categorization but rather a means bywhich to “describe four putative ways in which abnormal cardiac rhythms can be correctedor prevented”. Classification schemes will be discussed briefly below. The Vaughan Williams ClassificationTwo classification schemes for antiarrhythmic drugs were developed in the early1970’s. The first scheme was developed by Singh and Vaughan Williams in 1970a. Theycategorized a series of drugs, based upon electrophysiological actions, into four distinctgroups. Class I agents were local anaesthetics which reduced the maximum rise rate ofdepolarization (‘1max) by reducing sodium currents in heart cells. Class II agents reducedsympathetic nervous system effects on the heart while class Ill agents, such as sotalol andamiodarone, prolonged action potential duration (APD) and increased refractoriness incardiac tissue. Subsequently, studies with verapamil provided a fourth class ofantiarrhythmic drugs which blocked calcium currents in cardiac tissue (Singh and VaughanWilliams, 1970a, 1972). Hoffman and Bigger (1971) based their scheme on studies withseveral novel antiarrhythmic agents for that time. They proposed two groups of drugs withdistinct properties. Those in group I included agents such as quinidine and procainamidewhich reduced Vmax by inhibiting sodium currents in cells and which also prolonged APD.11Those in group II did not reduce Vmax, or inhibit sodium influx into cells. However group Ilcompounds shortened APD.The current classification of antiarrhythmic drugs is based primarily upon the Singhand Vaughan Williams categorization, but also is a hybridization of the two schemes. In1974, Singh and Hauswirth modified the Vaughan Williams scheme by subdividing class Iagents into Ia, those drugs which reduce Vmax, increase APD and which are effective innormal cardiac tissue (e.g., quinidine), and Ib, those agents such as lidocaine which reduceVmax, decrease APD and also seem to be most effective in ischaemic or partiallydepolarized tissue. With the development of potent sodium channel blockers such asflecainide, Harrison (1981) added a third subclassification, class Ic. This group of drugsblocked sodium channels both in normal and ischaemic myocardium.The possible addition of a fourth subclass to the class I agents which wouldcharacterize the electrophysiological actions of the antiarrhythmic agent transcainide hasbeen proposed (Bennett et al., 1987). It blocks sodium channels with little time or voltage-dependence. As well, a fifth class of antiarrhythmic agents which are bradycardic in naturehas been identified, and it includes drugs such as alinidine which act to depresspacemaker cell depolarization and increase the threshold potential for spontaneousdepolarization in these cells (Kobinger and Lille, 1987).The inhibition of sodium currents by class I agents increases with frequency ofstimulation and results in elevation of electrical thresholds of excitability and depressedcardiac conduction. These properties are consistent for most drugs of this class butproperties vary with individual agents. Subclassification of the large number of agentsfound with these properties was, as noted above, begun. Campbell (1983b) also definedsubgroups on the basis of kinetics of drug onset and offset. Subclassification of class I antiarrhythmic agents12The original Singh and Vaughan Williams classification of antiarrhythmic drugs wasmodified by Harrison in 1981. He attempted to make the classification more useful to bothclinicians as well as basic researchers (Harrison, 1985a, 1985b).Studies with individual agents had shown that rate-dependence was common to allcompounds with class I activity (Courtney, 1980; Campbell, 1989). Campbell, using adifferent set of criteria to Harrison, examined numerous class I agents and suggested thatgroups of drugs sharing common properties corresponded to the sub-classificationdescribed by Harrison (Campbell, 1983a, 1983b). Campbell showed that class I agents fellinto three distinct groups based on their rapidity of response to increases in frequency andrate of onset and recovery from rate-dependent block. Drugs with class Ia properties wereintermediate in kinetics, class lb were rapid and class Ic slow (Campbell, 1983b). Inaddition, the same groups showed differential effects on APD. Class Ia agents prolongedAPD, lb shortened it, and the response to class Ic was variable. Campbell also showedthat these same groups altered the effective refractory period (ERP) relative to APD. Forclass Ia agents there was a moderate increase in ERP relative to APD, while class lbmarkedly prolonged ERP relative to APD. The class Ic agents had little effect on ERP-APD(for an in-depth view of these studies and the ERP-APD relationship see Campbell, 1989and Vaughan Williams, 1984a, 1991).The implications of subgroup classification included the ability to suggest putativemechanisms for ischaemic and ventricular tissue-dependence of antiarrhythmic drugs(Campbell, 1989). lschaemia selectivity is displayed predominantly by the class lb agents.Lidocaine was shown by Hondeghem et al. (1974) to exhibit selectivity. Results from thekinetic studies by Campbell (1983a, 1983b) showed that drug binding occurs during theaction potential and unbinding occurs during diastole, in agreement with the suggestion byHondeghem and Katzung (1977). Lidocaine depresses ischaemic, but not normalmyocardium, and is believed to bind to the closed (inactive or I) state of the sodiumchannel. During depolarization, and in ischaemia-depolarized tissue (see below), this state13of the channel is favoured (Hondeghem and Katzung, 1977) resulting in drug selectivity.This same argument can be used to define the differential selectivity which exists betweenatrial and ventricular tissue selectivity. The brevity of the atrial action potential dictates thatthe sodium channel spends liftle time in the inactive state. This, in turn, reduces the timefor drug-inactive channel association and hence less block results during the AP for thesuppression of ectopic pacemakers (Campbell, 1989).Briefly, the other groups within the Vaughan Williams classification scheme includeclass II (B-blockers). These act to inhibit sympathetic nervous activity on the heart but donot alter normal cardiac function such as conduction velocity at resting membranepotentials. Class Ill agents control cardiac arrhythmias by slowing repolarization. Thiscorresponds to an increase in ERP and reduction in arrhythmias (Singh and Courtney,1990). Many class Ill antiarrhythmics exist and are very potent blockers of cardiacpotassium currents (Hondeghem and Snyders, 1990; Colatsky et al., 1990). The last groupof antiarrhythmic agents in this classification scheme are the class IV calcium channelblockers. There are a number of antiarrhythmic agents of diverse chemical nature in thisgroup. However, the common antiarrhythmic mechanism is blockade of the cardiac L-typecalcium channel (see review by Walker and Chia, 1989). The Sicilian GambitThe most recent framework for classifying antiarrhythmic drugs was postulated inthe Sicilian Gambit (Task Force of the Working Group on Arrhythmias of the EuropeanSociety for Cardiology, 1991; reviewed by Rosen et al., 1992, 1995). The premise for thisclassification scheme was that antiarrhythmic drug effectiveness could be assessed bydetermining the ability of individual drugs to alter arrhythmogenic mechanisms. This couldbe accomplished by identifying “vulnerability parameters”. These parameters includeconduction, phase 4 depolarization, and excitability. This scheme requires that14antiarrhythmic drugs undergo a thorough study of their pharmacological profile for actionson ion channels (and receptors) before an attempt can be made to resolve the mechanismof antiarrhythmic action. The action of a particular drug could then be matched to themechanism underlying the abnormal cardiac rhythm and be prescribed to treat the patient.According to Katritsis and Camm, (1993, 1994) it is the next logical step following theVaughan Williams classification scheme. Colatsky (1992) has been quick to criticize thisnew scheme. He suggests that the Gambit does not offer any new paradigms by which toexamine antiarrhythmic drugs, or aid in their development, and criticizes the targeting ofspecific cellular sites for rational drug choice based on mechanisms which are not wellunderstood. Both Harrison (1992) and Vaughan Williams (1995) also criticized the SicilianGambit. They suggest that with this scheme we pretend to know more than we actually doand state that simplicity is best as exemplified by the original Vaughan Williams (1970)classification.1.2 Mechanisms of Arrhythmogenesis1.2.1 The lschaemic MyocardiumCommon causes of arrhythmias in humans are myocardial ischaemia, myocardialinfarction, or reperfusion of a previously ischaemic myocardium, These conditions can bereadily reproduced in both intact and isolated rat hearts.The rat heart, similar to that of primates and pigs, does not have extensive coronarycollaterals (Johns and Olson, 1954; Maxwell et al., 1984), i.e. rat coronary arteries are endarteries (Winkle et al., 1984). Thus, when a coronary artery is occluded an area isrendered uniformly ischaemic (Schaper, 1971; Schaper et al., 1986). However, ischaemiais not absolute and a residual blood flow of approximately 5% is seen following completeligation of the artery (Maxwell et al., 1984). If occlusion of an artery persists for more than15ten minutes, irreversible damage occurs and infarction results (Saint et al., 1992). Thetime-dependency of the onset of arrhythmias after occlusion is quite characteristic for manyspecies. Arrhythmias first occur in conscious chronically prepared rats 5-15 minutes afterocclusion (Walker et al., 1991; Johnston et al., 1983). The most common arrhythmias seeninclude premature ventricular contractions (PVC), ventricular tachycardia (VT) andventricular fibrillation (VF). A second arrhythmic period occurs after 1-2 hours of occlusionand consists of PVC, VT and VF. Various factors influence the severity and incidence ofarrhythmic outcomes after occlusion. These include both the size of the ischaemic zoneand the serum potassium concentration (which has an inverse log-linear relationship toarrhythmia score) (Curtis et al., 1986a; Curtis et al., 1987; Podrid, 1990; Saint et al., 1992).Well-defined electrocardiographic (ECG) changes also occur in the rat model ofcoronary artery occlusion. The ECG shows an increase in the R-wave height withinminutes after occlusion followed by an elevation in the S-T segment (Johnston et al., 1983;Normann et al., 1961). The exact mechanism by which S-T segment elevation occurs isnot known (Walker et al., 1991). ECG responses to ischaemia are well-defined in pigs anddogs (for a review in these species the reader is referred to Hirsche et al., 1982; Benzing etal., 1972; Hill and Gettes, 1980; Gettes et al., 1989).lschaemia produces changes in the extracellular milieu of the cell. These changeswere first reported for potassium by Harris et al. (1954) who showed that coronaryocclusion was associated with an increase in extracellular potassium. In addition, Benzinget al. (1972) and Case et al. (1979) showed that the change in potassium is accompaniedby changes in pH,°2 and CO2 levels within the ischaemic zone. These changes in ionconcentrations have become better defined with improved experimental techniquesincluding ion sensitive electrodes, nuclear magnetic resonance (NMR) and voltagesensitive dyes (Gettes et al., 1989).The intracellular events which occur as a result of ischaemia include a reduction inpH from 7.2 to 6.0 (Garlick et al., 1979), a slight elevation in sodium from 5 to 20 mM, due16to partial suppression of the sodium/potassium ATPase pump (Wilde and Kleber, 1986),and an increase in calcium (Steenbergen et aL, 1987). Yan and Kleber (1992) haverecently shown that the pH change is not homogeneous within the ischaemic myocardiumdue to local variations in accumulation and diffusion of CO2 This may have implications inthe development of arrhythmogenic circuits (see below). Accompanying the intracellularchanges are extracellular changes. Within the ischaemic myocardium there is a triphasicincrease in potassium which, unless reversed, results in an irreversible loss in membraneintegrity (Hill and Gettes, 1980). A reduction in extracellular pH occurs which parallels thechanges in extracellular potassium levels. In a similar manner intracellular events arevaried and contribute to the heterogeneity within ischaemic tissue (see review by Orchardand Cingolani, 1994; Janse and Opthof, 1995). In addition to the local microinhomogeneities of ions which occur within the myocardial extracellular space, Hill andGettes (1980) have shown in pigs that a disparity exists between the centre and the borderzone of the developing ischaemic tissue and still yet between the myocardialsubepicardium and subendocardium. These transmural differences have been associatedwith the wave-like spread of ischaemia from the endocardium to the epicardium (Reimerand Jennings, 1979).The suggested mechanism producing these changes is based upon biochemicalstudies of anaerobic metabolism which occurs within cardiac tissue after coronaryocclusion (Gettes et al., 1989). Anaerobic metabolism results in glycolysis and a reductionin high energy phosphates which in turn produces lactic acid and reduces pH. The rise inpotassium is attributed to the passive movement of potassium with lactate to maintainelectrical neutrality across the membrane (Benzing et al., 1972). The lack of blood flow tothe ischaemic tissue does not permit a washout of substances within this area therebycontributing to accumulation.Electrophysiologically, the changes in potassium concentration result indepolarization of the cell membrane according to the Nernst potential. Kodama et al.17(1984) have shown that the changes in pH and CO2 augment such depolarization. Thereduction in resting membrane potential slows the maximum rise rate of depolarization andprolongs recovery of cell excitability by altering sodium channel inactivation (Gettes andReuter, 1974). Thus, refractoriness is prolonged beyond the effective refractory period(ERP) of the APD (which is shortened itself due to the increase in extracellular potassium)(Yan et al., 1993). The large inhomogeneity which exists at all levels in the ischaemictissue results in heterogeneous conduction and refractoriness. The development of “injury”currents which flow between non-ischaemic and ischaemic cardiac tissue may precede orgenerate arrhythmias (Han and Moe, 1964; Janse et al., 1980).1.2.2 Abnormal Impulse ConductionRecent advances in cardiac electrophysiological methods have improved ourunderstanding of the mechanisms of cardiac rhythm disturbances. Initially cardiacarrhythmias were classified as being due to an interference with either cardiac impulsegeneration, conduction or both (Hoffman and Cranefield, 1964; Hoffman, 1981). In 1981Hoffman and Rosen altered the classification scheme by expanding abnormal impulsegeneration to include normal/abnormal automaticity and triggered arrhythmias. As well,conduction arrhythmias were expanded to include delayed or blocked impulse propagationand uni-directional conduction block modeled by re-entry. This section will be confined to abrief discussion on the arrhythmic mechanism(s) which are likely due to myocardialischaemia.In order to understand how abnormal impulse conduction relates to abnormalautomaticity the basis for generation of the action potential and its various phases must bebriefly reviewed. Normal automaticity occurs during Phase 4 of the cardiac action potentialas a result of a slow diastolic depolarization in (or of) cardiac nodal cells. This inwardcurrent is due to the slow opening of f, a non-specific cation channel which is activated by18membrane hyperpolarization (DiFrancesco, 1981; Ho et at., 1994). Pacemaker activity incardiac tissue is hierarchical in that the sinus node dominates and suppresses subsidiarypacemaker sites, reducing the likelihood of the production of ectopic impulses. This istermed overdrive suppression (Vassalle, 1970).Phase 0 of the action potential is due to the rapid inward movement of sodium ions(‘Na) and this causes cell membrane depolarization. Sodium currents account for the rapidupstroke and amplitude characteristics of atrial, ventricular and Purkinje cells. Phase Irepolarization is thought to be due to inactivation of‘Na’ and the concomitant activation of atransient outward potassium current (Ito) (Coraboeuf and Carmeleit, 1982). The plateau (orPhase 2) of the action potential is due predominantly to calcium current influx via L-typecalcium channels at depolarized membrane potentials (Bean, 1985). These “slow inwardcurrents” (i51) are required for excitation-contraction coupling and result in contraction ofcardiac muscle (see review by Hess, 1988). However, Coraboeuf et al. (1979) alsoshowed that, in cardiac tissue, not all sodium channels inactivate during depolarization.Thus a sodium “window current” also contributes to the maintenance of the action potentialplateau duration. Phase 3 repolarization can be electrophysiologically complex due to thelarge number of voltage- and ligand-activated potassium channels found in cardiac tissue(Carmeleit, 1993). However, this stage of ventricular repolarization is due predominantly tothe opening of delayed rectifier (‘K) potassium channels (McAllister and Noble, 1966).Repolarization of the membrane activates an inward rectifier (IKI) potassium current whichensures that the membrane returns to its resting potential (Carmeleit, 1993). Thus, with theunderstanding of the components of the action potential and hence normal automaticity,one can discuss abnormal automaticity.There are many factors that may either suppress normal sinus node pacemakerfunction or enhance overdrive suppressed pacemaker automaticity and produce an ectopicbeat. However, in the ventricle the main factor that results in latent pacemaker activity isdamage as a result of stretch, scar formation or ischaemia. lschaemia results in19depolarization of ventricular tissue, inactivation of sodium channels, and reduction inrepolarizing potassium currents due to inactivation of the sodium/potassium ATPaseexchanger. This enzyme, under normal conditions, hyperpolarizes the membrane andsuppresses ectopic pacemaker sites (Binah and Rosen, 1992).Abnormal impulse generation can arise from oscillations in the membrane potentialand has been characterized as triggered rhythms (Cranefield, 1977; Binah and Rosen,1992). These triggered rhythms occur in two forms: early or late-afterdepolarizations (EADor DAD).1.2.3 Early Afterdepolanzations (EAD)Early afterdepolarizations interrupt either Phase 2 or 3 repolarization of the actionpotential. If these afterdepolarizations attain sufficient thresholds they may producetriggered responses and induce single or multiple extrasystoles and even VT (Cranefield,1977). The EAD is an oscillatory potential which is sensitive to frequency, often occurs atslow stimulation rates (Davidenko et al., 1989); the amplitude of the EAD increases at lowrates. High stimulation rates, however, abolish EAD’s (Roden and Hoffman, 1986). EADactivity has been shown in vitro using many types of isolated cardiac muscle and variouscell types including mid-myocardial cells (M-cell) (Antzelevitch and Sicouri, 1994).Induction of EAD activity can be induced by a variety of drugs including class I and Illantiarrhythmic agents (Binah and Rosen, 1992; see review by Antzelevitch and Sicouri,1994), the calcium channel opener, Bay K 8644 (January and Riddle, 1989), andcatecholamines (Priori and Corr, 1990). Experimentally, ischaemic conditions also result inEAD-induced triggered activity (El Sherif, 1991).The ionic basis for EAD development is unclear (January et aL, 1991). However,studies suggest the involvement of the slow inward calcium current (i5i) of the cardiac Ltype calcium channel during the plateau of the action potential. Theoretical modeling20studies conducted by Zeng and Rudy (1995) suggested calcium involvement in sustainingEAD activity as shown experimentally by Marban et al. (1986), January and Riddle (1989),and Priori and Corr (1990). Essentially, i5 re-activation acts as a depolarizing chargecarrier during the depolarizing phase of the EAD. Prolongation of the plateau phase of theaction potential allows for an increased time for calcium channel recovery which enhancesthe inward current thereby depolarizing the membrane and sustaining the EAD (Zeng andRudy, 1995). Other proposed mechanisms include a reduction of outward potassiumcurrents resulting in slow repolarization and an increase in sodium window currentassociated with a prolonged plateau (Coulombe et al., 1984). Ultimately, arrhythmiaswhich result include the long Q-T syndrome and torsades de pointes (Antzelevitch andSicouri, 1994); however, the genesis and maintenance of these arrhythmias by an EADmechanism remains unclear.1.2.4 Delayed Afterdepolarizations (DAD)Transient depolarizations which occur during Phase 4 of the cardiac action potentialare dependent upon the rate of the preceding action potential (Binah and Rosen, 1992).Unlike EAD’s the amplitudes of DAD’s increase with decreasing cycle lengths (Cranefield,1977). DAD have been observed under a variety of experimental conditions all of whichhave a similar end result, i.e. intracellular calcium overload (Tsien and Carpenter, 1978).High intracellular calcium concentrations saturate the sarcoplasmic reticulum sequestrationmechanism resulting in calcium oscillations due to calcium-induced calcium release (Binahand Rosen, 1992). The ionic currents which contribute to this mechanism are not known.The DAD is a self-sustaining rhythm and remains either sub-threshold or reaches thresholdand initiates a premature response (Ferrier et al., 1973). lschaemia, digitalis andcatecholamines can directly produce DAD by enhancing calcium entry into cells (Januaryand Fozzard, 1988; Antzelevitch and Sicouri, 1994). Thus calcium channel blockers, such21as verapamil, abolish DAD’s and studies show that sodium channel blockers includingquinidine, lidocaine, amiodarone and the potassium channel activator, pinacidil, may alleffectively suppress DAD and DAD-induced triggered activity (Rosen et al., 1974; Rosenand Danilo, 1980; Spinelli et al., 1991; Antzelevitch and Sicouri, 1994). Arrhythmias whichresult from DAD triggered rhythms in vitro include single and multiple PVC’s, andtachyarrhythmias (Cranefield, 1977). No direct evidence is available as to the existence ofDAD-induced triggered rhythms in vivo (Antzelevitch and Sicouri, 1994).1.2.5 Re-entryThe major cause of ventricular arrhythmias is due to re-entry. Re-entry has beensubdivided into either circus-movement excitation or reflection (El Sherif, 1995).The model for re-entrant circus-movement is based on a scheme developed bySchmitt and Erlanger (1929). A bifurcating Purkinje fibre bundle attached to the ventriclegives rise to different anatomical conduction pathways. Re-entry occurs when antegradeconduction of the impulse is extinguished at a site of uni-directional block. This type ofblock may arise from ischaemic damage of previously normal conduction pathways. Ifnormal conduction continues in the other branches of the pathway an impulse canretrogradely enter the area of unidirectional block where its conduction is slowed but notextinguished. The impulse can then emerge from this depressed area and, providing thatthe cells are not refractory, re-excite the tissue proximal to the area of block and generatepremature ventricular complexes which can remain as such, or deteriorate into VT or VF(Moe, 1975; Janse and Kleber, 1981; Binah and Rosen, 1992). This results in two forms ofre-entry. Ordered re-entry occurs when re-entrant excitation occurs along a pre-existingpathway and usually results in VT (Cranefield et al., 1973). Random re-entry of impulsepropagation results when electrophysiological differences exist between areas of cardiacmuscle. The development of ischaemia is dynamic therefore the pathway is not constant22for the impulse which circulates. It may fractionate, produce multiple re-entrant circuits,and result in VF (Hoffman and Rosen, 1981). Allessie et al. (1977) showed that random reentry occurs in the absence of an anatomical pathway and that the propagating impulseproduces a central area of inexcitability around which the impulse circulates. This wastermed the “leading circle hypothesis”.Many studies show that cardiac tissue types can, under conditions which mimicischaemia, generate and maintain re-entrant circuits (Sasyniuk and Mendez, 1971; Wit etal., 1972a, 1972b; El Sherif, 1991). Antiarrhythmic drugs can abolish re-entrantarrhythmias by either converting uni- to bi-directional block within the depressed region orprolonging refractoriness (El Sherif, 1991). Which of these is most important is a matter ofsome debate (Janse, 1992). Since re-entry is only possible if the length (time) of the reentrant path of the circus wave exceeds the normal cellular refractory period (about 300milliseconds) antiarrhythmic drugs prevent re-entry by either prolonging refractoriness (aswith Class Ill agents) or slowing conduction (as with Class I agents) (Varro and Surawicz,1991). Wavelength is the term describing the distance the re-entry impulse travels(mathematically this term is described by conduction velocity multiplied by ERP) and hasbeen suggested as a possible index of differential drug effectiveness (Rensma et al., 1988;Janse, 1992). Spinelli and Hoffman (1989) refute this measure as an index of predictiveusefulness because both conduction velocity and ERP are not constant with rate ofstimulation or with time in ischaemic tissue. The mechanisms suggested forarrhythmogenesis are complex and all, under ischaemic conditions, may play a significantrole (Hoffman, 1981; Binah and Rosen, 1992) . It is most likely that re-entry dominatesduring VT and VF while the mechanisms for PVC’s are less clear.The following sections discuss basic cardiac electrophysiology of cardiac ionchannels. As well, several models of ion channel block by antiarrhythmic drugs areoutlined.231.3 Cardiac Electrophysiology1.3.1 The Cardiac Action PotentialAs discussed above, the action potential (AP) is composed of the upstroke, plateauand repolarization phases. The shape of the AP is governed by ionic current flux via gatedchannels in the membrane for sodium, calcium and potassium. As well, membrane pumpsand exchangers such as for Na/K ATPase and Na/Ca are involved. The properties of theaction potential change moderately amongst tissue type. However the fundamentals ofaction potential generation remain essentially unchanged.1.3.2 The Sodium ChannelHodgkin and Huxley (1952) studied sodium conductance in the squid giant axon.They proposed that the voltage-dependent opening and closing of membrane “gates”resulted in a change in membrane permeability to sodium. The permeability changegenerated the action potential and was responsible for the transmembrane movement ofsodium ions. They proposed “m” as an activation gate particle and “h” as an inactivationgate particle which display distinct kinetic properties highly dependent upon changes inmembrane potential. Hodgkin and Huxley also postulated that for the conformationaltransitions of these gates to be voltage-dependent there must be a voltage-sensor, orcharge movement, during such transitions. They predicted the existence of the “gatingcurrent”. Gating Kinetics24Depolarization of the cell membrane opens sodium channels. However this eventonly occurs after some delay (Armstrong and Bezanilla, 1973). During this short timeperiod (<Imsec) charge movement occurs. This delay was described as a series ofvoltage-dependent, closed-state conformational transitions the macromolecular proteinwhich comprised the sodium channel had to pass through before the channel opened(Hille, 1989). The cause of this delay remained elusive until 1973 when Armstrong andBezanilla first recorded the “gating current”. It was a current of small amplitude (0.l3pA)and fast kinetics (80 psec to reach a maximum) (Armstrong and Bezanilla, 1973; DeFelice,1993). Thus, the majority of this current flows prior to the opening of the m gate foractivation (Hille, 1976).Molecular studies have revealed characteristics of the sodium channel itself as wellas the putative “voltage-sensor” for the gating current. The sodium channel is comprisedof approximately 2000 amino acids, containing 4 homologous internal repeats, each ofwhich has 6 putative transmembrane segments (Catterall, 1986). The c subunit contains aparticular region, S4, which is composed of a number of positively charged amino acidsand has been postulated to be the voltage sensor. Displacement by the change inmembrane potential of these amino acids may be responsible for the gating current (Nodaet al. 1984, Catterall, 1995). The outward gating charge for sodium is due to themovement of 6 charges across the membrane (Noda et al., 1984). This findingcorroborated the proposed charge displacement equivalent to 6 electrons flowing from theextra- to intracellular side of the membrane by Hodgkin and Huxley (1952). However,despite the implication of the S4 region in gating, the mechanism by which activation isinitiated is not known.Until recently local anaesthetics and other drugs were believed to immobilize afraction of the gating charge when the channel was blocked (Armstrong and Bezanhlla,1973; Bekkers et al., 1984). However, Hanck et al. (1994) suggest that local anaestheticdrug-bound cardiac channels gate with altered kinetics such that all channels continue to25gate but with a reduced voltage dependency. In light of the Modulated ReceptorHypothesis (see below) this may alter our perspectives regarding drug interaction with thesodium channel. Thus drug occupancy may reduce the voltage-dependence of gating byinhibition of voltage-sensitive charge movement rather than by drugs producing a shift inchannel states to the favored drug-bound inactive state of the channel (Hanck et al., 1994). Activation KineticsActivation of the sodium channel, not unlike gating currents, occurs rapidly and isvery steeply dependent upon depolarization (Colatsky, 1980; Hille, 1984). Thus, the rate ofactivation increases with membrane depolarization (Hodgkin and Huxley, 1952). Activationgenerally occurs at thresholds between -60 to -70 mV via the voltage-dependent openingof the “rn” gate. The change in voltage opens the channel and allows for a rapid increasein sodium permeability (Hille, 1989; Mitsuiye and Noma, 1992). Activation kinetics can bealtered by plant alkaloids such as veratridine, scorpion or sea anemone toxins, or byinsecticides such as pyrethroids. These kinetics are not altered by most antiarrhythmic orlocal anaesthetic drugs such as quinidine or lidocaine (Honerjager, 1983; Narahashi,1992). These agents shift activation to more negative membrane potentials such that atresting potentials the steady-state depolarization is due to a sustained sodium current(Honerjager, 1983). It was originally proposed (for simplicity) that activation wasindependent of inactivation. However, it was not until Armstrong et al. (1973) perfused thesquid giant axon with the enzyme pronase, and showed that inactivation was selectivelydestroyed and activation was unaltered, that the two processes could be dissociated.Later studies confirmed that activation and inactivation could be separated but that theywere not entirely independent (Armstrong et al., 1973; Stimers et al., 1985).I .3.2.3 Inactivation Kinetics26Ionic conductance of the sodium channel is transient in nature. Prolongeddepolarization results in sodium channel inactivation and prevents the influx of sodium intothe cell. Thus refractoriness is maintained (Hodgkin and Huxley, 1952). As with activation,the rate of inactivation increases with an increase in the rate of depolarization. Hodgkinand Huxley (1952) postulated that decay of sodium currents to resting values wasmonoexponential. However, Chiu (1977) found that the rate of inactivation was muchbetter approximated with a bi-exponential function and described two voltage-sensitivecomponents for inactivation: fast and slow. Studies by Khodorov et al. (1976) describedthe slow component in great detail. Aldrich et al. (1983) used inactivation studies of singlechannel sodium currents to show that decay was biphasic, and largely coupled toactivation. These studies indicate that some fraction of the sodium channels must be openbefore inactivation proceeds.The inactivation gate, “h”, can be selectively destroyed by the internal application ofprotease (Armstrong et al., 1973) and chemicals such as the piperazinyl-indole derivativeDPI 201-106 (Wang et al., 1990). Veratridine and batrachotoxin, alkaloid toxins, alsoinhibit inactivation and produce a steady-state depolarization due to enhanced sodiumpermeability (Brown, 1988; Catterall, 1980, 1986; Honerjager, 1983). At the cellular levelthis results in a prolongation of the AP and positive inotropism.Molecular studies have shown that an intracellular linker between domain Ill and IVof the sodium channel is responsible for fast inactivation kinetics (Stuhmer et al., 1989;Patton et al., 1992; see reviews by Catterall, 1995 and Goldin, 1993). In addition thesemolecular studies provide evidence for the proposed “ball and chain” model of inactivationwhereby this cytoplasmic linker may influence the activation and inactivation couplingprocess (Moorman et al., 1990). This model suggests that a positively charged cytoplasmicprotein particle (the “h” gate using Hodgkin and Huxley formalism) electrostatically interactswith a negatively charged inactivation subunit of the sodium channel (Armstrong andBezanhla, 1977; Khodorov et al., 1976; Carmeleit, 1987).27Local anaesthetics and antiarrhythmic drugs interact with the inactivation gate(Weidmann, 1955; Courtney, 1975; Hille, 1977; Hondeghem and Katzung, 1977). Theinactivation produced by a change in membrane potential and drug block of the channelare interacting processes. These occur as a result of drug binding to a site on or near the“h” gate in a voltage, time and channel state-dependent manner according to theModulated Receptor Hypothesis (HilIe, 1977; Hondeghem and Katzung, 1977).Inactivation is complete with the return of the membrane potential to its pre-depolarizing(resting) level by activation of repolarizing potassium currents.1.3.3 The Potassium Channels1.3.3.1 Channel DiversityOver the last several years interest in the development of drugs which prolongrefractoriness, i.e. possess class Ill antiarrhythmic action, has increased markedly. Severalreasons for this resurgence in interest include the negative results of the CAST trial whereproarrhythmic tendencies were associated with some class I agents, the effectiveness ofsotalol and amiodarone in the clinical setting and the results of long-term studies withamiodarone which suggest that it may, in a manner similar to the p-blockers, decreasepost-infarction arrhythmic death (Vaughan Williams, 1982). Repolarization and theconfiguration of phase 3 of the action potential in cardiac tissue occur as a result of thecomplex interaction of up to 7 different potassium channels (Colatsky and Follmer, 1989;Carmeleit, 1993). These potassium channels are heterogeneous and differ in gating andpermeation properties as well as in susceptibility to modulation by neurotransmitters andintracellular ions such as sodium and calcium (Hume et al., 1990). In essence potassiumchannels regulate cell function by establishing the resting membrane potential andcontrolling cell repolarization processes. Individual potassium currents overlap in their28contribution to the total membrane current during the action potential. The relativeimportance of each may vary under different conditions. During ischaemia changes in cellelectrophysiology may alter the degree to which different channels contribute to the actionpotential (Colatsky et al., 1994).Important species and regional differences exist in the contribution potassiumchannels make to repolarization of the cardiac action potential. Studies have shown thatelectrophysiological distinctions can be made between epi- and endocardial tissue in manyspecies including the dog and rat (Wei et al., 1993). In canine ventricles, epicardial, midmyocardial (M-cells) and endocardial cells display distinct electrical properties and hencedifferent action potential morphologies (Vaughan Williams, 1985; Sicouri and Antzelevitch,1991; Antzelevitch et al., 1995). In the rat ventricle at least three cell types have beendistinguished based on APD (Watanabe et al., 1983). Wang et al. (1991) havecharacterized epi- and endo-cardial differences in APD in atrial tissue and suggest that theionic mechanism for these differences is due to different amplitudes of the transientoutward potassium current (ito). Similar differences are also seen in ventricular tissue(Sicouri and Antzelevitch, 1991).Molecular biologists have recently cloned and characterized voltage-gatedpotassium currents. All potassium currents have a similar primary amino acid sequencewith highly conserved structural regions (Miller, 1991). One region, H5, has beenimplicated in pore formation as well as being a distinctive intracellular binding site for drugaction (Pongs, 1992).The heterogeneity of potassium channels provides a large potential for thedevelopment of diverse compounds with potassium channel blocking properties (Colatskyand Follmer, 1989). The therapeutic potential and benefits of these agents are their abilityto increase the time course for repolarization and inhibit SVT and re-entrant ventriculararrhythmias without an associated slowing of intracardiac conduction in an alreadycompromised heart (Katritsis and Camm, 1993). Sotalol, and amiodarone are typical class29Ill agents which block multiple potassium currents (Singh and Vaughan Williams, 1970a;Colatsky and Follmer, 1989). However, many new agents, at various stages of clinicaldevelopment, are highly potent and channel selective. These include sematilide, E-4031,and dofetilide (Colatsky and Follmer, 1989; Katritsis and Camm, 1993; Hondeghem, 1994).Class Ill agents are effective at maintaining a prolonged APD at low rates ofstimulation. However, at high heart rates the effectiveness of these agents is diminished.This “reverse use-dependence” suggests that these drugs have a high affinity for theclosed state of the channel (Hondeghem and Snyders, 1990; Colatsky et al., 1994;Hondeghem, 1994). Only amiodarone lacks this effect (Singh, 1983). The resultingbradycardia associated with these agents has been shown to precipitate arrhythmias suchas the long Q-T syndrome (Zipes, 1991) and torsades de pointes (Roden, 1994; Katritsisand Camm, 1993).Of the many potassium channels that exist in cardiac muscle we examined only twoin our rat myocytes, i0 and the sustained outward delayed rectifier (Ksus) current. Thesewill be briefly discussed as they contribute predominantly to repolarization of the heart inthis species. The Transient Outward Potassium Currentl is a relatively common current found in a wide variety of species and cell types,except the guinea pig (Campbell et al., 1995). The channel possesses rapid activation andinactivation kinetics (Coraboeuf and Carmeleit, 1982) and is important during early phase Irepolarization. It is coupled to both sodium and calcium and is critical for the classic “spikeand dome” appearance of the ventricular action potential (Katritsis and Camm, 1993).Channel density and distribution differences amongst cell types results in a variable actionpotential morphology in various regions of the heart (Antzelevitch et al., 1991). Escande etal. (1987) showed that ito is composed of a large voltage-activated, calcium-independent30component, itol, and a small calcium-activated component, ito2. The channel activates atmembrane potentials more positive than -7OmV and inactivates around -lOmV (Coraboeufand Carmeleit, 1982). 1to is highly potassium selective, shows little rectification andreaches its peak in 3 msec (Campbell et al., 1993). Inactivation, depending on the species,shows either mono- or bi-exponential rates of decay (Tseng and Hoffman., 1989; Jahnel etal., 1994). In the rat ito is the main repolarizing current and is sensitive to blockade by 4-aminopyridine (4-AP) (Josephson et al., 1984; Castle and Slawsky, 1992). Thebradycardic agent, tedisamil (KC8857) is a selective‘to blocker that has been used toextensively characterize both the electrophysiology (Dukes and Morad, 1989; Dukes et al.,1990) and involvement of this channel in ischaemic (Beatch et al., 1991; Adaikan et al.,1992) and programmed stimulation-induced arrhythmias (Wallace et al., 1995). The Sustained Outward Delayed-Rectifier Potassium Current‘Ksus is a time-dependent outward current which contributes to the initial phase Illrepolarization of the action potential. Wang et al. (1993) have extensively characterizedthis current in atrial cells. However, Escande et al. (1985) described a long lasting outwardcurrent (i10) in human atrial cells and Benz and Kohlhardt (1994) recently described acardiac outward rectifier (iKoutw..rect) current in rat myocytes. Whether these are identicalcurrent has not been clearly defined however current characteristics are similar. Thiscurrent is residual in nature and occurs long after ito has inactivated. Interestingly it can befound in cells which lack classical‘K current properties (Wang et al., 1993). Jahnel et al.(1994) suggest it is a third subtype of ito and call it ito steady-state (ito..ss); however, Wanget al. (1993) successfully isolated‘Ksus from the ito subtypes and characterized itsproperties. The channel is slow to inactivate requiring up to 10 sec to complete theprocess, rapidly activates and is sensitive to tetraethylammonium (TEA) and 4-aminopyridine (4-AP) blockade (Wang et al., 1993).311.4 Models of Sodium Channel BlockadeThe actions of cardiac antiarrhythmic drugs and their interaction with the cardiacsodium channel are based on models developed to describe the action of local anaestheticdrugs in nervous tissue (Grant, 1991). Studies conducted with the marine toxin,tetrodotoxin (TTX), showed that sodium channels were blocked at nanomolarconcentrations of this toxin resulting in inhibition of nerve conduction (Narahashi, 1974)while micromolar concentrations are required to inhibit cardiac conduction (Abraham et al.,1989). However, antiarrhythmic drugs show an inverse potency in cardiac and neuronaltissue. Lidocaine inhibits the cardiac sodium channel at concentrations that are 1000 timesgreater than those which inhibit neuronal sodium channels (Bean et al., 1983). Thedifferences between cardiac and neuronal sodium channels are reflected in single channelstudies of gating currents (Kirsch and Brown, 1983). However, despite these differences alarge number of similar properties are found regarding kinetics, drug interactions, andchannel conducting properties and thus neuronal models have been applied to cardiacchannels with success (Grant, 1991).1.4.1 The Strichartz-Courtney ModelThis model was developed by Strichartz in 1973 after conducting studies withseveral quaternary charged analogs of lidocaine (QX-222 and QX-314) on the frog node ofRanvier. Stimulation in the presence of either drug allowed delineation of two types ofblock, one that was tonic and the other which was voltage-sensitive. The Strichartz modelelaborated upon the Hodgkin and Huxley (1952) model of sodium channel statedependence. Strichartz assumed that the drug-bound channel complex either opened orclosed transitionally following similar states proposed by Hodgkin and Huxley for drug-freechannels. Courtney (1975) redefined the model proposed by Strichartz by studying the32voltage-dependent component of block in the presence of the lidocaine derivative GEA968. In his studies Courtney coined the term “frequency or use-dependence” to describean increase in the rate of block development seen with increasing rates of stimulation inthe presence of the drug. The relevant modifications Courtney made to the model includedthe fact that drugs could dissociate from the channel when the cells were at rest, i.e., whenthe resting membrane potential was at rest. He also deduced that drug-bound channelcomplexes favored the inactive state of the channel which is reflected in a hyperpolarizingshift of the voltage-dependence of inactivation. This action was described by Weidmann(1955) for cocaine; however, Courtney’s observations required that only the drug-boundfraction of the sodium channels produced a shift in sodium inactivation. Those channelswhich were not blocked were not altered.This model was elaborated upon by Hondeghem and Katzung (1977) and Hille(1977) who independently proposed similar models for the interaction of local anaestheticswith cardiac and neuronal sodium channels, respectively. Courtney’s model was based onobservations by Chen et al. (1975) who showed that sodium channel recovery was voltage-dependent however could not account for this observation.The Strichartz-Courtney model could not account for the properties of voltage-dependent sodium channel recovery. This model only described the use-dependentblocking and unblocking of sodium channels with local anaesthetics and antiarrhythmicdrugs.1.4.2 The Modulated Receptor Hypothesis1-lilIe (1977) proposed a model for local anaesthetic action on nerve based upon thepreviously described models. He suggested that there was a single specific binding site forlocal anaesthetics and that drug occupancy (block) alters the inactivation kinetics of thechannel as previously shown by Weidmann (1955), Strichartz (1973) and Courtney (1975).33The proposed location of drug action was intracellular (Hille, 1977). HilIe also postulatedthat multiple pathways existed for drug access to these binding sites, thus, unlike previousmodels it could account for all drug access routes to this binding site. Hondeghem andKatzung (1977) used studies in cardiac muscle to justify the proposal of a similar model forantiarrhythmic drug interaction with cardiac sodium channels. In this cardiac model aseries of equations, or global fitting parameters, were developed which defined bindingparameters for each state of the channel (rest, open, inactive) and accurately describedchannel block by quinidine and lidocaine (Hondeghem and Katzung, 1977; Davis et al.,1986). The general model suggests that as sodium channels change states in a voltage-dependent manner local anaesthetic or antiarrhythmic drugs can associate or dissociatefrom each state (Hondeghem and Bennett, 1989). Thus, each state has a characteristicset of association (k) and dissociation (I) rate constants and binding is modulated byvoltage and time (Hondeghem and Katzung, 1977; Hondeghem, 1994). Since the affinityfor the binding site is modulated by the state of the channel the proposed model was calledthe Modulated Receptor Hypothesis (MRH) (Hille, 1977; Hondeghem and Katzung, 1977,1984; Hondeghem, 1987, 1989; Grant, 1991).Over the years the model has been widely tested with many drugs in both cardiacand neuronal preparations (Hondeghem and Katzung, 1977; Clarkson et al., 1984, 1988;Hondeghem and Matsubara, 1988; Snyders and Hondeghem, 1990). The only limit to theuse of this model was that a number of rate constants were required to be determinedsimultaneously. Several attempts have been made to simplify this model.1.4.2 Simplified Versions of the Model1.4.2.1 Kappa (K) Repriming Model34Courtney (1983) proposed the kappa repriming model in an attempt to simplydescribe the quantity of block which develops during the action potential (K) and thequantity of block which is relieved, due to unblocking, during diastole (k). In order tocalculate both K and . the model requires a fixed action potential duration and restingmembrane potential during diastole. In this way an accurate determination of rate of blockdevelopment, steady-state level of block and rate of diastolic recovery of block can bemade. The use of the model is limited due to the fact that the APD and resting membranepotential are not fixed under normal conditions and that both K and 2. depend on time whichmay be highly variable for various drugs and thus may invalidate results. Guarded Receptor HypothesisThe Guarded Receptor Hypothesis (GRH) was proposed by Starmer et al. (1984,1985) for the interaction of either local anaesthetic drugs with nerve or antiarrhythmicagents with cardiac sodium channels. This model simplifies binding and adopts theHodgkin and Huxley model of the sodium channel as it allows for a simple, single-statemodel (i.e., Closed Open Blocked) on which to base drug action (Starmer et al.,1984). Drugs are theorized to interact with a constant affinity site on the sodium channel.Thus each drug interacts at different rate constants for the open and inactive states of thechannel (Starmer, 1987). The drug-bound channels have the same voltage-dependenceas drug-free channels but the model requires that only changes between resting, open andinactive states of the channel occur when there is a change in membrane potential, thusthe MRH series of global fitting equations simplify into three sets of consecutive first-orderequations (Starmer et al., 1984; Grant, 1991). Unlike MRH, drug binding in GRH onlyoccurs in the open channel state. This new model changes the emphasis of druginteraction from the inactivation (h) gate in MRH to the activation gate (m): the activation(m) gate becomes immobilized by the drug-binding site complex and prevents sodium35influx. The frequency and voltage-dependence of the gates, in turn, dicates the voltageand frequency-dependence of the drug with the sodium channel (Grant, 1991).However despite its simplicity neither the GRH, nor the more complex MRH, canexplain block induced by all antiarrhythmic drugs (e.g., transcainide - see Bennett et al.,1987). Antiarrhythmic drug blockade of cardiac sodium channels may be highly dependentupon the structure of the molecule, a factor not incorporated into the models. In addition,experimental evidence has not yet shown that all antiarrhythmic drugs bind to the samesite as proposed in the GRH and MRH models.A novel class of compounds, which are not structurally-related to prototypicalantiarrhythmic or local anaesthetic agents discussed above show putative sodium andpotassium channel blocking properties. These are the arylacetamide ic opioid receptoragonists, typified by U-62,066E (spiradoline). The properties of these compounds are nowdiscussed.1.5 Opiold Receptor HeterogeneityThe identification of both agonist and antagonist drugs acting upon the variousoploid receptors located in the central and peripheral nervous systems has resulted fromthe large monetary incentive and market potential for analgesics. Thus, thepharmaceutical industry strives to find less toxic analgesics and in doing so has createdmany novel classes of agonist and antagonist drugs with potent analgesic properties butwhich retain many undesired side effects. When this increase in development of drugs iscombined with rapidly improving techniques used to distinguish opioid binding sites intissue, an understanding of the mechanism(s) of opioid analgesia may result. To date, themajority of studies conducted with opioids have been in neuronal tissue in an attempt toelucidate the analgesic mechanism of these agents. The pharmacological profile of actionof most opioid agents on the heart and cardiovascular system remains uncharacterized.361.5.1 Historical PerspectiveOpium is an extract derived from the poppy Papaver somniferum which for manycenturies had been used to produce analgesia and alleviate pain. However, for most ofthis time the exact mechanism of action by which extracts from this plant alleviated painwas not known. The major alkaloid derived from the plant is morphine. Morphine is apotent analgesic but its use as such is limited by its side effects which include respiratorydepression, constipation and physical dependence. The term ‘opioid’ was coined byAcheson (Martin, 1967) to designate drugs whose action resemble morphine but whichmay be chemically distinct from its phenanthrene structure. This definition has now beenbroadened to include antagonists, as well as agonists, which may have a wide spectrum ofaction on the opioid system (see review by Martin, 1967).In 1954 Beckett and Casy hypothesized that synthetic analgesic opioid drugs suchas morphine and related morphinans ‘fit’ at a receptor surface stereospecifically. It wasthrough this interaction at the receptor surface that analgesia resulted. However, it wasonly after studies were conducted in the 1970’s using biochemical binding assays thatopioid receptor delineation commenced (Fowler and Fraser, 1994). The use ofradiolabelled naloxone, which could antagonize morphine or other opioid narcotics such aslevorphanol, and the development of stereospecific binding assays for opioid agonistsprovided for the identification and anatomical localization of the opioid receptors in thebrain of mammals (Goldstein et al., 1971; Pert and Snyder, 1973).In rats it could be shown that electrical stimulation of various brain regions producedanalgesia in animals and that naloxone could reverse the effect (Akil et al., 1974).Subsequent to this, the development of bioassays and binding studies provided thenecessary methods by which to screen extracts from both the brain and pituitary gland foropioid binding affinity. Hughes et al. (1975) showed that endogenous opioid peptides(EOP) could be isolated from brain extracts and that these peptides possessed morphine-37like properties. These EOP include enkephalins, dynorphins and endorphins and as of1983 the list had grown to include over 18 chemically distinct peptides derived frommammalian tissue (see reviews by North, 1986; Pasternak, 1993 and Fowler and Fraser,1994). The effects of these opioid peptides are mediated via specific binding sites orreceptors. Many chemically synthesized non-peptide analogues mimic the actions of thesepeptides.1.5.2 Classification of Opioid ReceptorsThe existence of opioid receptors was postulated in the pioneering work of Beckettand Casy (1954). This, in 1965, allowed Portoghese to postulate the existence of separateopioid receptors by correlating analgesic activity to the chemical structure of many opioidcompounds.The first in vivo evidence of multiple opioid receptors was obtained by Martin et al.(1976) using congeners of morphine. These workers identified three distinct syndromesproduced by these agents in the chronic spinal dog. Thus, it was postulated that thesesyndromes were due to agonist interaction with three related receptors. The morphinesyndrome was mediated by the p (mu) receptor, the ketocyclazocine syndrome by the ic(kappa) receptor and the SKF 10,047 syndrome by the a (sigma) receptor (for a completeside effect profile for each syndrome see Martin et al., 1976). It should be noted thatstudies suggest that the a receptor is not in fact opioid in nature. Therefore this receptorcan no longer be classified as such (Holtzman, 1980; Zukin and Zukin, 1981).The development of in vitro pharmacological bioassays greatly enhanced theelucidation of heterogeneity between and within classes of opioid receptors. The mousevas deferens and guinea pig ileum preparations were used by Hutchinson et al. (1975) toshow that with ketocyclazocine and other related compounds a consistently lower potencyratio for agonism was found in the mouse vas deferens were compared to the guinea pig38ileum. It was later shown by Lord et al. (1977) that there was a higher kappa:mu ratio inguinea pig ileum when compared to the mouse vas deferens. Thus, these tissues appearto have a heterogeneous population of opioid receptors. In addition to having mu (p) andreceptor populations the mouse vas deferens was subsequently shown, using endorphinsand enkephalins as agonists, to possess another profile of response which wasindependent of the other opioid receptors. Thus another opioid receptor, the ö (delta)receptor was postulated to exist (Lord et al., 1977; Hutchinson et al., 1980).Recently, new opioid binding sites have been postulated including the B-endorphinselective (epsilon) receptor (Nock et al., 1990; see review by Pasternak, 1993). Howeverits characteristics have not been clearly defined due to lack of procedures and selectiveagents with which to label the site. As with other receptor systems in pharmacology manyopioid receptor subtypes have been postulated. The subtypes postulated for the preceptor are the p1 and P2 receptors which mediate supraspinal and spinal analgesia(Pasternak and Wood, 1986), respectively. The K receptors are a much more diversegroup and many subtypes exist. The analgesic properties of the 1 receptor arecharacterized with i agonists such as U-50,488H and U-69,593. Use of these compoundshas given rise to two binding sites, 1(a and icib.The pharmacological significance of these binding sites is unknown (Clark et al.,1989). Another K subtype was postulated after it was shown that U-50,488H-insensitivebinding sites could be found in the rat brain (Zukin et al., 1988). The pharmacology ofthese 2 binding sites remains unknown. The most recent U-50,488H-insensitive bindingsite, 1(3, has been identified using a novel opiate derivative naloxone-benzoylhydrazone(NalBzoH). This binding site is thought to mediate supraspinal analgesia (Clark et al.,1989; Zukin et al., 1988 and Gistrak et al., 1989).The delta receptor, defined by Kosterlitz as the enkephalin-preferring receptor (Lordet al., 1977), may have two subtypes which are distinguished by the agonists ([D-Pen2,DPen5J-enk phalin) DPDPE and deltorphin as 6.i and 82, respectively (Mattia et al., 1991).39The pharmacological role of these sites may be related to spinal and supraspinalanalgesia, respectively (see Pasternak, 1993). The heterogeneity of opioid receptors isalso reflected in the diversity of location and disparity between species (Martin, 1984;Mansour et al., 1988). However, despite this, distribution is consistent with their role inphysiological function.Thus, the present classification of opiold receptors is primarily based upon thepharmacological profiles of numerous opioid drugs as they relate to differences in bindingpotency ratios in bioassays, in effects of naloxone and other antagonists and also howthese drugs affect various physiological actions, be it in the central or peripheral nervoussystem.1.5.3 Antiarrhythmic ActionsThe first reported effects of opioid compounds on arrhythmias due to myocardialischaemia were those by Fagbemi et al. (1982). Prior to this it had been observed thatopioids were of benefit in endotoxic and haemorrhagic shock states (Holaday and Faden,1978). Naloxone, when given at doses similar to those used in shock states, reduced theincidence of VT and VF in rats subject to coronary artery occlusion (Fagbemi et al., 1982).It was postulated that EOP such as B-endorphin might have detrimentalelectrophysiological effects on the myocardium by 1) directly interacting with opioid bindingsites, 2) indirectly by modulating the autonomic nervous system (ANS) or 3) that opioidsmay act directly on the myocardium and hence alter cardiac action potentials (Fagbemi etal., 1982). Since that time many studies have been conducted using many differentmodels of arrhythmogenesis in many different species.The direct involvement of the opioid receptor in arrhythmogenesis was examinedusing the stereoisomers of two different opioid antagonists (Parratt and Sitsapesan, 1986).(-)Mr1452 and (-)WIN 44,441-3, both ic agonists, dose-dependently decreased arrhythmic40incidence of ischaemic arrhythmias suggesting that blockade of K receptors in themyocardium was antiarrhythmic. Mackenzie et al. (1986) and Sitsapesan and Parratt(1986) showed that naloxone, at doses which inhibit p and K receptors and the Kantagonist, Mr2266, reduced ischaemic arrhythmias in rats. The quatemary naloxonederivative, MrZ2593, was also effective against ischaemic arrhythmias. Since it did notcross the blood brain barrier (BBB) it was suggested that the antiarrhythmic actions ofopioids were mediated by peripheral opioid receptors. Studies with the p, K and 5 agonists,diamorphine, U-50,488H and leu-enkephalin, respectively, suggest that the antagonism ofp and K receptors, but not 5, is important in inhibiting arrhythmias (Sitsapesan and Parratt,1986).The majority of published studies suggest the involvement of EOP in ischaemicarrhythmias (Wong et aL, 1990; Lin et al., 1991; Lee et al., 1992b). It is thought that EOPare released from cardiac muscle by myocardial ischaemia and that they mediatearrhythmias via their respective opioid receptors (Maslov et al., 1993). Many studies haveconfirmed the antiarrhythmic actions of naloxone and other opioids against the arrhythmiasproduced by many methods including chloroform-hypoxia in rats (Wong and Lee, 1985),and ischaemia and reperfusion in isolated rat hearts (Zhan et at., 1985; Lee and Wong,1987) and in dogs (Huang et at., 1986). In contrast, Rabkin and Roob (1986) found thatnaloxone potentiated digitalis-induced arrhythmias in guinea pigs and suggested thatnaloxone inhibition of EOP may provide unopposed parasympathetic activity in the heart(Rabkin and Roob, 1986). Further studies suggest that inhibition of the degradativeenzymes for EOP potentiate digitalis arrhythmias (Rabkin and Redston, 1989). Recently,Lee et al. (1992b) have shown that naloxone inhibits ischaemia-induced arrhythmias,hypotension and bradycardia in coronary artery occluded rats. Natoxone also attenuatedarrhythmias produced by the K opioid receptor agonist U-50,488H suggesting that the Kreceptor may be involved in the genesis of arrhythmias during ischaemia. These resultscontrast those published by Pugsley et al. (1992a) and Sitsapesan and Parratt (1989)41which showed that U-50,488H reduced arrhythmic incidence in ischaemia. However, theresults of Lee et al. (1991) agree, in part, with the theory that K opioids may have a dualaction in ischaemic arrhythmias. Kaschube and Brasch (1991) and Pugsley et al. (1992b)suggest that the arrhythmogenic actions of K opioids occur at low doses due to activationof the i receptor and that antiarrhythmic actions occur at higher doses due to a direct (nonopioid) interaction with the cardiac membrane.Fagbemi et al. (1983) showed, using the partial opioid agonist, meptazinol, thatopioids directly influence the cardiac action potential. Sagy et al. (1987) and Same et al.(1988) showed that naloxone exerts a direct local effect (positive inotropism) on isolated rathearts. Boachie-Ansah et al. (1989) showed that buprenorphine, an opioid having pagonist and K-antagonist properties depressed maximum diastolic depolarization (Vmax)and increased action potential duration (APD) in sheep Purkinje and rat papillary musclesdue to blockade of sodium and delayed-outward potassium channels. The dual actiondiscussed above has been confirmed by Pugsley et al. (1994) using the stereoisomers (-)PD129,290, an active K agonist, and its inactive enantiomer, (+)PD129,289 and U50,488H in the absence and presence of naloxone (Pugsley et al., 1992a, 1992b). Theantiarrhythmic action of these oploid agonists is due to direct interaction with the sodiumand potassium channels of the cardiac membrane. Possible ion channel effects inprolongation in the P-R, QRS width and Q-aT intervals have been observed with manyrelated arylacetamide K opiold agonists in anaesthetized rats and confirmed in voltage-clamped cardiac myocytes (Pugsley et al., 1992a, 1992b, 1993a, 1994, 1995). In thesepatch-clamp studies the K receptor agonists, U-50,488H, (-)PD129,290, (±)PDII7,302 andthe inactive K agonists (+)PD129,289 and (+)PD123,497 produced a concentrationdependent block of inward sodium and both transient outward and sustained delayedoutward potassium currents in the absence as well as presence of naloxone. Therefore itis suggested that these opioids are antiarrhythmic via blockade of ion channels inmyocardial tissue (Pugsley et al., 1995).421.6 The Kappa (K) ReceptorThe existence of the K receptor, as outlined above, was originally postulated on thebasis of pharmacological studies by Martin in 1976 who characterized sedation, pupilconstriction, and depressed flexor response to ethylketocyclazocine (EKC) in the chronicspinal dog as being representative of K receptor activation. It now appears that responsesdue to K receptor activation are very complex and involve a large number of receptorsubtypes (Wollemann et al., 1993). In this section of the thesis the biochemistry of the Kreceptor system, the arylacetamide agonists which have helped in the description of thisreceptor system, and the pharmacological actions associated with this opioid receptor typewill be discussed.The pharmacological actions of K receptors have been characterized by acombination of binding studies and bioassays but the coupling of these receptors tosecond messenger systems is less well understood. Most studies appear to confirm that p,K and receptor activation inhibits adenylate cyclase (Haynes, 1988; Childers, 1993). Thisinhibition is associated with a G protein and requires guanosine triphosphate (GTP) (Hsiaet al., 1984). In guinea pig cerebellar membranes both dynorphin-A and U-50,488Hproduce potent and specific inhibition of adenylate cyclase (Konkoy and Childers, 1989)suggesting that, at least for K receptors, this may be an important mechanism in cellularfunction. It is unclear whether this action is associated with p receptors (Polastron et al.,1990).The coupling of K opioid receptors to other systems may be different from that seenin cerebellar membranes. Attali et al. (1989) assessed the effects of opiates on the influxof 45Ca2 in rat spinal cord and dorsal root ganglion (DRG) cell co-cultures by an elevationin extracellular potassium or with Bay K 8644, a calcium channel opener. Influx of calciumwas dependent upon activation of the L-type calcium channel. U-50,488H and dynorphinA decreased calcium influx whereas agonists for both p and opioid receptors had no43effect on calcium influx. Further study revealed that this inhibition of L-type calciumchannels by K agonists was mediated by G proteins.Other studies indicate that there may be additional mechanisms by which Kreceptors are coupled to response systems. For example, U-50,488H produced aconcentration-dependent increase in phosphatidyl-inositol (P1) turnover in rat hippocampalslices, an effect blocked by naloxone or Mr2266 (Periyasamy and Hoss, 1990).Molecular biology techniques have resulted in the isolation of complementary DNA(cDNA) sequences for mouse, rat, and guinea pig K receptors (Nishi et al., 1993; Miniami etal., 1993; Xie et al., 1994, see review by Reisine and Bell, 1993). These K receptors arestructurally similar to the cloned p (Chen et al., 1993) and (Evans et al., 1992) receptorsin that they are all members of the highly homologous superfamily of G protein coupled,seven transmembrane domain spanning receptors (Reisine and Bell, 1993). Thepharmacological profile of the cloned K receptor now includes expression in humanembryonic kidney cells (Lai et al., 1995) and the human placenta (Mansson et al., 1994).All cells are associated with pertussis toxin sensitive G proteins which mediate inhibition ofadenylate cyclase and hence cAMP formation (Avidor-Reiss et al., 1995).The cloned K receptor is now used as a probe to screen human and murinegenomic libraries for a genetic link to its expression. Yasuda et al, (1994) successfullyisolated a clone which contained part of the gene encoding the human K opioid receptorlocated on chromosome 8. Recent reports using cloned cDNA from murine K receptorssuggest that the gene for this receptor is on chromosome 1 in this species (Kozak et al.,1994; Giros et al., 1995).Thus the pharmacological and biochemical profile of K agonists provided adescription of how the effects of K receptor activation may be mediated and theinvolvement of second messengers in the translation of the message leading to theeventual response of the cell. The cloning of K (as well as p and 8 receptors) has44advanced our understanding of the relationship of the K receptor family to other wellcharacterized pharmacological receptor families (such as the B-receptors).1.6.1 Chemical Diversity of K AgonistsAs discussed elsewhere, activation of the K receptor produces a distinct profile ofpharmacological action in addition to analgesia (Martin et al., 1976). At analgesic doses Kagonists such as (±)PDII7,302 and U-50,488H produce diuresis and sedation (VonVoightlander et al., 1988; Leighton et al., 1987). They do not however produce theuntoward effects of emesis, respiratory depression or constipation associated with pagonists (Martin, 1984). The pharmacological elucidation of K opioid receptor activationhas resulted in part from synthesis of compounds in the arylacetamide series. Thisincludes drugs such as U-50,488H, U-62,066E (spiradoline), (±)PDII7,302, (-)PD129,290and their inactive K opioid enantiomers (+)PD123,497 and (+)PD129,289, respectively.These agents will be the primary focus of this section (and this thesis) despite the multitudeof other structurally related compounds. Prior to the development of the arylacetamide Kagonists only a limited understanding of K receptors and their function was known.Researchers were limited by the lack of selectivity of the benzomorphan prototype ligandssuch as ethylketocyclazocine (EKC) or bremazocine (Kosterlitz et al., 1981; Zukin andZukin, 1981).Szmuzkovicz and Von Voightlander (1982) first described the synthesis andanalgesic effectiveness of benzamide and benzacetamide structural moieties incorporatedinto the trans-cyclohexane-1 ,2-diamine class of antidepressant agents. The benzamideseries showed an increased analgesic potency, but retained p receptor actions. Thebenzacetamide series, typified by the pyrrolidinyl derivatives, were potent analgesicswhose actions were naloxone-reversible but apparently independent of the p receptor(Szmuzkovicz and Von Voightlander, 1982a; Von Voightiander et al., 1981, 1982a). These45compounds are effective analgesics when given orally. U-50,488H was the first nonpeptide benzacetamide compound selective for the K receptor.The development of U-50,488H opened the way for the chemical synthesis ofnumerous arylacetamides. The cyclohexylbenzacetamide group has become interestingbecause these compounds are analgesics but are structurally dissimilar to morphine (Clarket al., 1988; Hunter et al., 1990). The enantiomer of the cyclohexyl derivative of U50,488H was the first to be developed and characterized as having high affinity (3nM) forthe ic receptor (Lahti et al., 1982; Costello et al., 1991). From this compound a potent Kanalgesic analogue, U-62,066E, was developed. Currently it is in Phase I clinical trials(Von Voightiander and Lewis, 1988; P.F. Von Voightlander, Personal Communication).Further entensive structure-activity relationship (SAR) studies resulted in synthesisof compounds with increased p to K selectivity ratio and with optimal affinity for the icreceptor (Clark et al., 1988). SAR studies next involved examination of the N-[(2-aminocyclohexyl)aryloxy]acetamide and N-[(2-aminocyclohexyl)aryl]acetamide series (Clarket al., 1988; Boyle et al., 1990). Sequential examination of electron-donating and electron-withdrawing aromatic substitutions based on U-50,488H was conducted and it was foundthat the thiophene derivatives increased both ic receptor affinity and selectivity (p/k ratio)(Clark et al., 1988; Horwell et al., 1990). Thus (±)PD1I7,302 showed nM affinity for the icreceptor and only pM affinity for the p receptor (the ratio of EC50 values piic was 110). Atthis same time, (±)PDII7,302 was resolved into its enantiomers, (-) PD123,475 whichretained ic agonist stereoselectivity and (-i-)PD123,497 which was essentially inactive at icreceptors (p/ic = 0.59) (Clark et al., 1988).Derivatives of (±)PDI 17,302, containing a benzo[b]thiophene aromatic ring systemwere synthesized and examined (Halfpenny et al., 1989). Substitution of the cyclohexylring of (±)PDI 17,302 resulted in the development of (-)PD129,290. The analgesic potencyfor this compound was 25 times that of morphine and 17 times that of U-62,066E(Halfpenny et al., 1989). The high selectivity ratio for the ic receptor (p/ic = 1520) makes it46one of the most potent K receptor agonists developed (Halfpenny et al., 1989). Theenantiomer, (+)PD129,289, is neither K selective (p/K = 0.38) nor is it an analgesic(Halfpenny et al., 1989).Thus the novel structure of U-50,488H represented a lead compound which couldbe extensively altered and resulted in the discovery of many potent K opioid analgesicslacking the potential for abuse in humans.1.6.2 Pharmacological actions of arylacetamides1.6.2.1 AnalgesiaThe arylacetamides vary in analgesic potency when compared with morphine. U50,488H produces a species-dependent, equipotent analgesia when compared tomorphine using a variety of thermal (hot plate, tail immersion), pressure (tail pinch) andirritation (HCI or acetylcholine abdominal constriction) assays (Von Voigtlander et al. 1981,1982a, 1982b). However, the analgesic potency of U-50,488H varies markedly with theintensity of the applied nociceptive stimulus (Von Voigtlander et al. 1981, 1988). U62,066E (spiradoline), (±)PDII7,302 and (-)PD129,290 are more potent analgesicscompared with U-50,488H and morphine using these same tests (Von Voigtlander andLewis, 1981; Leighton et al., 1987; Hunter et al., 1990). Selectivity of these agents for Kreceptors is also greater than for p-receptors. U-50,488H is 53 times more selective for Kthan for p while U-62,066E is 84 times more selective (Lahti et al., 1982; Kunihara et al.,1989). The selectivity of (±)PDII7,302 (pIK=110) resides in its active K receptorenantiomer (-)PD123,475 (p/K=100, Kik=9.6 and the K1=1000nM) (Clarke et al., 1988)while its inactive enantiomer (+)PD123,497 has low affinity (Kik=1500 and Ki=880nM) andselectivity (p/K=0.59) (Clark et al., 1988; Meecham et al., 1989). Similar enantiomericpotency and selectivity for the K receptor is found with (-)PD129,290 (Hunter et al., 1990).47The analgesia produced by U-50,488H and its arylacetamide analogues, unlike thatof morphine, is not associated with physical dependence nor does it promote self-administration in animals and hence may lack abuse potential in humans (VonVoightlander et al., 1982b; Lahti et al., 1983; Leighton et al., 1987; Kunihara et al., 1989;Hunter et al., 1990). Interestingly, tolerance develops to arylacetamide analgesia whichcannot be attributed to enhanced metabolism (Von Voigtlander et al. 1982a; Hunter et al.,1990). Cross-tolerance develops between these compounds and many benzomorphan Kagonists such as bremazocine, or other arylacetamides, but not to morphine (VonVoightlander et al., 1982a; Von Voightiander et al., 1988; Colombo et al., 1991). Unlikemorphine and its cogeners, U-50,488H and the arylacetamides do not depress respirationsignificantly when given at analgesic doses (Martin et al., 1976; Dosaka-Akita et al., 1983;Beecham et al., 1989). At high (supra analgesic) doses, many arylacetamides produceactions in anaesthetized rats which include respiratory depression (Pugsley et al., 1992,1 993a, 1994, 1995) but which are not blocked by naloxone.Opioid receptor drugs can act at the level of the central (CNS) or peripheral (PNS)nervous systems. Since both high and low CNS centres possess opioid receptors,stimulation can produce a variety of responses including effects on neuroendocrine andcardiovascular systems (Martin, 1984). As well, there are opioid receptors (p and K) in theperipheral ANS. Studies by Paton (1957) demonstrated the existence of these peripheralactions. He showed that morphine inhibits autonomic neurotransmitters in guinea pigileum. The suggested mechanism of such inhibition in the case of morphine and the Kagonists (including U-50,488H, (±)PDII7,302, and (-)PD129,290) is blockade of calciumchannels pre-synaptically and hence a reduction in the release of neurotransmitters, suchas acetylcholine (ACh) in guinea pig myenteric neurons (Cherubini and North, 1985; Xianget al., 1990; Werling et al., 1988; Mulder et al., 1991; Lambert et al., 1991; Kunihara et al.,1993).48Studies with opioid agonists and antagonists in neuronal tissue suggest that there isa non-opioid receptor mediated action of these compounds on ion channel function.Frazier et al. (1973) first reported that both morphine and naloxone could block squid axonaction potentials by inhibiting sodium and potassium currents. Carratu and Motolo-Chieppa(1982) also showed that naloxone blocks sodium currents when applied intracellularly tofrog sciatic nerves. The sodium channel blocking actions of U-50,488H and U-69,593 onneuronal tissue have been demonstrated by Alzheimer and ten Bruggencate (1990). Usingmicroelectrode techniques they showed that these K agonists had local anaesthetic actionsin neuronal tissue which could not be reversed by opioid receptor antagonists. Zhu et al.(1992) have developed a series of benzamide U-50,488H analogues which possessanticonvulsant properties and which inhibit sodium currents in mouse neuroblastoma cells.Recent binding studies by Fraser and Fowler (1995) suggest that the K-receptor bindingsites are independent from those sites modulating the actions of local anaestheticcompounds.Studies have shown that opioid agonists at low concentrations augment voltage-dependent potassium currents and this suggests a means by which these compoundsmediate antinociceptive properties (Grundt and Williams, 1993; Moore et al., 1994). Athigh concentrations these compounds block the current (Moore et al., 1994). Molecularbiological studies show that Xenopus oocytes express K opioid specific binding sites (Henryet al., 1995). When cells were co-injected with a cRNA coding for a G-protein linked,inwardly rectifying potassium channel (GIRKI), activation of the K binding site by lowconcentrations of U-69,593 resulted in a large potassium current. This increase in currentwas blocked by norbinaltorphimine. Unlike the sodium or calcium channel block producedby these opioid compounds, potassium channel block may be linked to K receptoractivation by means of an endogenous intermediate or undetermined G-protein (Henry etal., 1995).491.6.2.2 Systemic ActivityThe study of opioids and their involvement in the cardiovascular system is fraughtwith difficulties. The primary focus of studies with these compounds involves the CNS andANS. However, in the periphery the locus of opioid neurons and receptors has beendefined more clearly for some organ systems (such as in the gastrointestinal tract)compared to others (such as the heart and smooth muscle of the vasculature). The rolethat opioids, especially K agonists, play in regulation of these systems is difficult todetermine because the highly influential physiological effects are influenced bypharmacological variables such as dose, site and route of administration, receptorspecificity and species. DiuresisOpiates have a long history of modulating fluid and electrolyte balance, asdemonstrated initially with morphine in dogs (Debodo 1944). The diuretic activity of potentK agonists such as bremazocine, U-50,488H, U-62,066E, and others, has been the focusof many studies (Huidobro-Toro and Parada, 1985; Leander et al., 1986; Oiso et al., 1988;Yamada et al., 1989, 1990; Bianchi, 1991). All K agonists studied to date show diureticproperties in many different species, including man (Von Voightlander et al., 1982; 1982a,1982b; Peters et al., 1987; Rimoy et al., 1991). The diuresis which results can be blockedby opioid antagonists including naloxone and Mr2266 (Huidobro-Toro and Parada, 1985;Yamada et al., 1989).Leander (1986) and Yamada et al. (1989) showed that U-50,488H and U-62,066Edo not produce diuresis in rats which genetically lack arginine vasopressin (AVP). Thisinformation led to the suggestion that K agonists either suppress AVP levels at the level ofthe neural lobe secretory process, or inhibit the effect of AVP on the kidney (Leander,501986). In man K agonists induce water diuresis without any changes in renal blood flow orsuppression of AVP levels (Rimoy et al., 1991).In addition to effects on AVP levels, many studies suggest that the K agonistsinfluence the serum levels of other circulating hormones. Iyengar et al. (1985, 1986)showed that U-50,488H and EKC elevated plasma corticosterone but decreased plasmathyroid stimulating hormone (TSH) levels in rats. Thus, although many studies show thediuretic actions of K agonists are likely to be due to a ic opioid receptor effect, cleardelineation of the mechanism of action has not been accomplished. This suggests acomplex involvement of the opioid system in the regulation of the hypothalamic-pituitaryadrenocortical axis. Cardiovascular ActionsThe ic agonists and all opioids in general exhibit a variety of complexpharmacological actions on the cardiovascular system (Holaday, 1983). The CNS effectsof ic agonists such as analgesia are mediated by opioid receptors (Von Voightiander et al.,1988; Lahti et al., 1982; Leighton et al., 1987; Kunihara et al., 1989) but the actions ofthese compounds in peripheral tissues including reduction in cardiac contractility andcentral venous pressure may not be dependent upon opioid receptors and may instead bea direct effect on cardiac muscle and vasculature.The existence of opioid receptors and their importance for cardiac tissue isuncertain and any involvement in regulation of cardiovascular function, speculative.Hughes et al. (1977) were the first to show that endogenous enkephalins occur in rat andrabbit atria. Much uncertainty exists as to the localization of EOP in the heart despite itbeing generally agreed that opioid receptors are differentially distributed between atria andventricles (Holaday, 1983; Lang et al., 1983; Krumins et al., 1985; Weihe et al., 1985; Taiet al., 1991). Studies in the heart are hampered by the lack of appreciable ligand binding51to cardiac membrane fractions. However the most abundant binding for K agonists, suchas U-69,593 and diprenorphine, occurs in the right atrium (Krumins et al., 1985; Tai et al.,1991). It is suggested that rather than globally examining cardiac tissue attention shouldbe given to His-Purkinje and nodal conduction tissue (Holaday, 1983). To date thesebinding studies have not been performed.The physiological significance of peripheral K opioid receptor distribution invasculature is not fully understood. Peripheral binding of EOP, or the arylacetamides,provides an outline of the direct involvement of the opioid system in regulation ofhaemodynamic function and cardiac activity in addition to its actions on the cardiovascularcontrol centres found in the brain (Lang et al., 1983).U-50,488H and other arylacetamides have been examined over a large range ofdoses producing effects on heart rate (HR) and blood pressure (BP). These actions aredose- and species-dependent. U-50,488H, for example, has a different cardiovascularprofile when injected i.v. in rats compared to i.c.v. into the CNS (Feuerstein et al., 1985;Pugsley et al, 1992a; Pugsley et al., 1993b). In anaesthetized dogs U-50,488H produced adose-related decrease in BP, HR, peak systolic pressure and cardiac contractility over thedose-range 0.08-24 pmol/kg, i.v. Prior administration of 8pmol/kg naloxone abolished suchresponses (Hall et al., 1988). U-62,066E produced a similar cardiovascular depression indogs which could be prevented by naloxone (Hall et al., 1988).Studies conducted in our laboratory in anaesthetized rats showed that U-62,066E,(-)PDI 29,290, (+)PDI 29,289, (±)PDI 17,302 and U-50,488H all dose-dependentlydecreased BP and HR. In addition, at the highest doses these compounds prolonged thePR, and Q-aT intervals of the ECG (Pugsley et al., 1992a, 1992b; 1993a, 1994, 1995).Neither Mr2266 nor naloxone reduced these cardiovascular actions of the arylacetamides.Studies in rats, using U-50,488H and U-62,066E, report slight depressant actions on HRand BP at low doses similar to those used by Hall et al. (1988) in dogs and cats.52In intact animals the cardiovascular actions produced by K agonists may bemediated by K receptor-dependent effects in the CNS. This may be especially true at thesupra-analgesic doses used in my studies. Both U-50,488H and U-62,066E, while notnoted for actions at p receptors do suppress respiration, possibly as a result of CNSdepression (Clarke et a!., 1988; Hall et al., 1988; Pugsley et al., 1992a, 1993b).To examine the non-opioid pharmacological actions of ic agonists studies wereperformed in the presence of opioid antagonists, such as naloxone or Mr2266, or by usingenantiomers of K arylacetamides which lack opioid receptor agonist properties. Thecardiovascular responses studied in the presence of naloxone or Mr2266 were notinfluenced by these opioid antagonists, even at doses which block, in addition to preceptors, K and 6 receptors (Brasch, 1986; Kaschube and Brasch, 1991; Pugsley et al.,1992a, 1993a, 1995). It was concluded that since these responses were not blocked byopioid receptor antagonists they were not mediated by opioid receptors. The inactiveenantiomer, (+)PD129,289, produced similar reductions in HR and BP to its enantiomer,the K agonist (-)PD129,290 (Pugsley et aI., 1993a).A number of studies have shown that opioid drugs can modulate the incidence andseverity of arrhythmias (vide supra) induced by coronary artery occlusion (see review byPugsley et al., 1993b). The ECG changes produced by arylacetamides provides a usefulindirect method for determining the effects of antiarrhythmic drugs on cardiac ion channels.The ECG changes produced by arylacetamides at high doses are interpreted as indicatingion channel blockade, particularly the sodium channel. Both (-)PD129,290 and its inactiveenantiomer (+)PD129,289 produce P-R interval prolongation and QRS widening in rats(Pugsley et al., 1993a) together with an increase in RSh, an index of sodium channelblockade in the rat (Penz et al., 1992). These drugs also produced widening of the Q-aTinterval, an index of prolongation of repolarization, indicative of possible potassium channelblockade. Other studies suggest that sodium channel blockade occurs with opioid agonistsand antagonists (Same et al., 1989, 1991).53In vitro studies conducted in rat hearts complement the in vivo studies describedabove. We examined the effects of U-50,488H and other ic agonists in the Langendorffisolated rat heart and showed these compounds prolonged the P-R interval and QRSduration of the ECG and reduced peak left-ventricular pressure in a concentration-dependent manner. Naloxone (1 pM) did not block these actions (Pugsley et al., 1992a,1993a, 1995). Such a spectrum of action suggests sodium channel blockade similar toclass I antiarrhythmics (Abraham et al., 1989). Few other studies have been reportedwhich examine the effects of ic opioid agonists on isolated heart function and ion channelinteraction. The only other report of these effects are by Xia et al. (1994) which show that1 pM U-50,488H reduced HR and contractility.The majority of studies involving the non-opiold actions of ic receptor agonists havebeen conducted in isolated cardiac tissue. These non-opioid actions occur at micromolar(pM) concentrations in the isolated heart, whereas ic agonism usually occurs at nanomolar(nM) concentrations.Studies using a variety of cardiac isolated muscle preparations have shown thatopioid agonists such as morphine and U-50,488H, and antagonists including naloxone andMr1452, exert similar non-opioid properties on cardiac muscle (see Pugsley et al., 1993b).The chemical diversity and actions of these compounds suggest non-opioid receptormediated effects (Rashid and Waterfall, 1979; Frame and Argentieri, 1985; Same, 1989).In isolated guinea pig and rat atrial and ventricular muscle opiates suppress excitability incardiac muscle by both increasing the threshold for stimulation and reducing actionpotential amplitude and the maximum rate of depolarization in a manner similar to localanaesthetics or sodium channel blockade (Alarcon et al., 1993). The results of somestudies suggest an interaction of ic opiolds with either the potassium or calcium channelfound in cardiac muscle. Potassium channel blockade was suggested on the basis ofincreased action potential duration, especially at the terminal phase of repolarization seenwith many opioids (Brasch, 1986; Helgesen and Refsum, 1987; Hicks et al., 1992). The54use of calcium fluorescent techniques for measurement of cardiac myocyte contractilitysuggests that the negative inotropic actions of K opioid agonists are due to inhibition of Ltype calcium currents (Kasper et al., 1992; Lakatta et al., 1992).The ion channel or non-opioid actions of the arylacetamides have been examinedon currents evoked from isolated cardiac myocytes subjected to patch-clamp. duBell andLakatta (1991) and Utz and Trautwein (1994) examined the effect of U-50,488H on theslow inward calcium current (l). Intracellular application of U-50,488H had no effect onthe calcium current (Utz and Trautwein, 1994). U-50,488H inhibited the developed currentin a manner indicative of a receptor-independent mechanism at a site accessible from theexterior of the cell. U-50,488H also inhibited a current which had properties similar to thedelayed rectifier potassium current. The non-opioid cardiac actions of U-50,488H andother arylacetamides have been extensively characterized. In isolated rat cardiacmyocytes U-50,488H, (-)PD129,290, (+)PD123,497, (±)PDII7,302 and U-62,066E have allbeen shown to inhibit both the sodium (‘Na) and transient outward (Ito) and sustainedplateau (lKsus) potassium currents (Pugsley et aI., 1993a, 1994, 1995). Channel block wasnot reversed by opioid receptor antagonists. The mode of channel block will be elaboratedupon later in this thesis, however, blockade is similar to the sodium and potassium channelblockade seen in neuronal tissue (Alzheimer and Ten Bruggencate, 1990).In contrast to the studies conducted on cardiac tissue, few studies have beenperformed to determine the effects of K agonists on isolated vascular tissue. However,non-opioid receptor dependent actions have been observed in several types of vasculartissue. U-50,488H was shown by Altura et aI. (1984) to dose-dependently contract thebasilar and middle cerebral arteries of dogs. The U-50,488H-induced contractions werenot inhibited by naloxone. Illes et al. (1987) showed that U-50,488H andethylketocyclazocine, at concentrations greater than 3pM, depressed isolated rat tail veinscontraction in a manner not inhibited by 10pM naloxone. Verapamil (6pM) has been shownto reduce the inhibitory effect of high concentrations of U-50,488H in abdominal aortic55strips (el Sharkawy et al., 1991) suggesting possible direct interactions of i agonists with Ltype calcium channels on vascular smooth muscle. U-62,066E also produced aconcentration-dependent relaxation of pig circumflex coronary arteries attributable toinhibition of voltage-dependent calcium entry into smooth muscle cells (Harasawa et al.,1991). This relaxation was not inhibited by naloxone, even at concentrations of 300IJM.Thus, the sodium, potassium and calcium channel blocking actions of U-50,488Hand other arylacetamides have been unequivocally demonstrated in both neuronal (seeabove) and cardiac tissue. The direct application of this series of compounds in a clinicalsetting may be limited. However, they provide additional information regardingantiarrhythmic agents.561.7 Objectives and Outline of Experiments Performed1.7.1 In this Thesis and in the AppendixMany studies conducted in rats demonstrate that opioid drugs are antiarrhythmic(Sitsapesan and Parratt, 1989; Pugsley et al., 1992b, 1993, 1995). However, the degreeof protection varies with the opioid drug considered. In addition, both antagonists as wellas agonists, reduce arrhythmias (Same etal., 1991; Pugsley et al., 1992b). The effects ofK receptor agonists and related compounds are not well characterized in the heart andcardiovascular system. The experiments conducted in this thesis with the K agonist,spiradoline (U-62,066E), (±)PDI 17,302 and its inactive enantiomer, (+)PD123,497, as wellas those described in the Appendix for U-50,488H, (-)PD129,290 and its inactiveenantiomer, (+)PD129,289, were designed to answer the following questions:1. Is the K receptor involved in arrhythmias produced by coronary artery occlusion inrats?2. If related arylacetamide K receptor agonists, such as spiradoline (U-62,066E), andrelated compounds have antiarrhythmic actions against ischaemic arrhythmias, are theseindependent of K receptor agonism?3. If this independence exists, what then is the putative mechanism by which thearylacetamide K agonists and related compounds exert their antiarrhythmic effects againstischaemic arrhythmias?Examination of the cardiovascular profile of spiradoline against both electrical andischaemic arrhythmias was performed in the absence and presence of naloxone. In57previous studies (those in the Appendix) with U-50,488H, the selective K antagonist,Mr2266, was used. The use of the inactive enantiomer of K receptor agonists has alsoaided in the delineation of K receptor involvement in arrhythmias. Lastly we characterizedthe electrophysiological properties of the arylacetamides on sodium and potassiumcurrents evoked in ventricular myocytes.)582 Methods2.1 Cardiac preparationsDose-response studies were performed in vivo and in vitro to ascertain theprofile of action of the K receptor agonist and related arylacetamides on the cardiac andcardiovascular system of the rat.2.1.1 Intact rat studiesSince there are few studies examining the cardiovascular actions of K receptoragonists and related compounds (Pugsley et al., 1993b) I examined thehaemodynamic, heart rate and ECG effects of these drugs in acutely preparedanaesthetized rats. The rat allows reliable, accurate measurements of cardiovascularfunction (Schroeder et al., 1981) and is routinely used for drug investigation. As asmall animal, it is relatively inexpensive, readily available, and provides reproducibleresults. An extensive biochemical, physiological and anatomical data base exists forthis species. Furthermore, surgical procedures (such as cannulation of blood vessels)are easily performed in the rat for the study of drug actions on organs and/or systems(Curtis et al., 1987). However, like many models it has some drawbacks and as withmany models it is unclear as to exactly how closely these models resemble disease inman. However, this does not detract from the usefulness of the rat in thepharmacological characterization of drugs. It is no less valid an animal model than anyothers which have been used to assess drug actions in the heart and cardiovascularsystem (Curtis et al., 1987; Walker et al., 1991; Cheung et al., 1993).2.1.2 Surgical preparation59Male Sprague-Dawley rats (250-350 g) were used in accordance with theguidelines established by the University of British Columbia Animal Care Committee.Rats were anaesthetized with sodium pentobarbital (60 mg/kg, i.p.). All animals hadtheir right jugular vein and left carotid artery cannulated for administration of drugs andblood pressure monitoring, respectively. The electrocardiogram (ECG) was recordedusing a unique lead configuration. A needle electrode was placed 0.5 cm from themidline of the trachea at the level of the right clavicle while a second needle electrodewas placed 0.5 cm from the midline at the level of the 9th and 10th ribs (Penz et al.,1992). The trachea was cannulated for artificial ventilation at a stroke volume of 10mL/kg and rate of 60 strokes/mm to ensure adequate blood-gas levels (MacLean andHiley, 1988). Animals were placed in a supine position and body temperature wasmonitored by rectal thermometer and maintained between 37-38°C with a heating lamp.Blood pressure and ECG were recorded on a Grass polygraph (model 7D) at abandwidth of 0.1-40 Hz and a chart speed of 100 mm/sec.2.1.3 Experimental DesignCumulative in vivo dose-response curves for K receptor agonists and theirenantiomers (0.5-32pmol/kg/min, i.v.) were obtained in artificially-ventilated,pentobarbitone anaesthetized rats. All K receptor arylacetamides were initiallysolubilized in distilled water as a stock solution and serial dilutions were made in salinevehicle. Drugs were given in vivo in one of two ways; either as a bolus with dosedoubling every 5 mm, or as infusions with the infusion rate doubled every 5 mm. Foreach case, variables were measured just prior to doubling the dose or rate of infusion.For the infusion studies it was assumed that a pseudo-equilibrium had been achievedby 5 mm. Animals (n=5) were randomly assigned to receive either drug or vehicle60control at the end of a 15 mm control period. All doses were infused and bloodpressure, heart rate and ECG were recorded 5 mm later, immediately prior to additionof the next dose.Accurate analysis of drug-induced changes in the rat ECG presents certaindifficulties. Driscoll (1981) exhaustively outlined the many anomalies with the rat ECG.Briefly, the P, QRS and T waves do not share a common baseline and therefore areference point for determination of the isoelectric line is required. Driscoll (1981)suggests that the point at which the P-R interval terminates and the QRS complexbegins is subject to the least variation. We adopted the ECG measures of Budden etal. (1981) which are similar to those of Driscoll (1981) in an attempt to maintainiriterlaboratory consistency.Another important factor which influences the rat ECG is electrode position. Inall our studies of acutely prepared anaesthetized rats, the positioning of electrodes wasas explained previously. There was always some anatomical differences in the positionof the rat heart but these were minimized by the method of electrode placement used.Before starting our studies with arylacetamides we developed a novel ECGmeasure (RSh) for the detection of possible sodium channel blockade in artificiallyventilated, anaesthetized rats. This measure was of value in our studies since it hasbeen shown that ic receptor agonists and related compounds block many different ionchannels in both the CNS and systemically (Pugsley et at., 1993b). Conventionalmeasures of sodium channel blockade (QRS complex widening and/or P-R intervalprolongation) are limited in their sensitivity in detecting low-dose drug effects. The newmeasure, termed “RSh” or RS-height, quantifies the height from the peak of the R waveto the bottom of the S wave (Figure 1, Appendix 6). It is more sensitive to sodiumchannel blockade than conventional measures (Penz et al., 1992). In order to illustratethis, we compared the ECG effects of various Class I sodium channel blockers withother antiarrhythmics. Representative drugs from the three subclasses of Class I, i.e.61quinidine, lidocaine and flecainide, were tested. In each case, changes in RShoccurred before changes in QRS or P-R (see Figure 3a,b,c, Appendix 6). Otherantiarrhythmics (Class II, Class Ill and Class IV) only influenced RSh if they had sodiumchannel blocking properties and then only at high doses, e.g. propranolol (Class II) andtedisamil (Class Ill) (Table 3, Appendix 6). Other physiological manoeuvres, such aschanging vagal activity, administration of catecholamines, or direct pacing of the rightatrium, did not change RSh. Thus, RSh is a useful measure with which to detectpossible sodium channel blocking actions of cardiovascular drugs in rats.The effects of heart rate on the Q-T interval have been examined in severalspecies (Hayes et al., 1994). It is difficult to measure the repolarizing T-wave in a ratECG since the dominant transient outward current causes a rapid repolarization of theventricle (Detweiler, 1981; Josephson et al., 1984). To aid analysis we chose toexamine the Q-aT interval. This is a more useful measure than that previously used inthe investigation of the physiological and pathological factors that underlie Q-T durationand its prolongation (Taran and Szilagyi, 1947) as well as evaluation of drugs such asclass III antiarrhythmic drugs, which lengthen refractory periods and action potentials.In most species the interpretation of Q-T data is complicated with drugs whichchange heart rate since Q-T depends on rate. It is necessary to correct for this effect.The correction problem has been examined in detail in humans and other experimentalanimals but no consensus has been reached as to which of the many correction factorsis the most useful and appropriate (Browne et al., 1983). We have therefore made asystematic analysis of the effect of variations in heart rate on the Q-T interval in anumber of species (rat, guinea pig, rabbit, and primates) (Hayes et al., 1994). In viewof the difficulty in determining when the T-wave returns to the isoelectric line, theaforementioned measure, Q-aT, was adopted (Chemoff, 1972). Q-aT is the time fromthe negative deflection in the Q-wave of the QRS complex to the peak of the T-wave.In rat, there was no correlation between heart rate and the Q-aT (Figure Ic, Appendix627). As a result, in our studies no correction was made for heart rate effects with respectto Q-aT interval.2.2 Isolated rat heartsThe isolated perfused heart has many advantages in the study of the actions ofdrugs on both the mechanical and electrical properties of the heart. The isolated heartwas first described by Langendorff in 1895 to be a simple preparation with which tostudy the actions of drugs. This preparation resolved many of the problemsencountered in using preparations involving blood perfusion. In the Langendorff heart,a Krebs-Henseleit solution is used to replace the blood. The isolated heart is also freeof both CNS and circulating systemic humoral factors which may alter drug activity(Neely, 1967; Doring and Dehnert, 1988).Wiggers first critically appraised the Langendorff isolated heart method in 1909.More recently, Broadley (1979) carefully discussed the advantages and limitations ofthe method. For example, he showed that the perfusate solution had a low oxygen-carrying capacity and that a lack of patency of the aortic valves (and the ease withwhich they are damaged) allowed perfusion fluid to enter and distend the left ventricle.A perfusion apparatus was developed which reduced or eliminated some of theseproblems (Curtis et al., 1986b).2.2.1 Perfusion apparatusA modified perfusion apparatus for the study of the actions of drugs on themechanical and electrophysiological behaviour of hearts from small animals such asrats and guinea pigs was developed in this laboratory (Curtis et al., 1986b). Ninechambers (each of a 250 mL capacity) were machined into a plexiglass block and63placed into a bath containing circulating warm water (30-37°C maintained by anexternal heater). Krebs-Henseleit perfusate from within individually controlledchambers flows via separate silastic tubes to a common manifold and then to the aorticcannula. Since dead-space for each chamber is less than 0.1 mL this allows for a rapidswitching of perfusate while an external (5% CO2 in O) gas mixture maintains aorticroot pressure between 70-125 mmHg, as required.Male Sprague-Dawley rats (300-400 g) were killed by a blow to the head,exsanguinated and the heart rapidly removed from the chest cavity. Hearts wereperfused with 5 mL of ice-cold Krebs-Henseleit solution to remove remaining blood(Curtis et al., 1986b, Curtis, 1991). Within two minutes of sacrifice, hearts wereperfused via an aortic cannula with an oxygenated Krebs-Henseleit solution at 35°Cand pH 7.4. The composition (mM) of the Krebs-Henseleit solution was: NaCl, 118;KCI, 4.74; CaCl2.2H0, 2.5; KH2PO4, 0.93; NaHCO3, 25; D-Glucose, 10; MgSO4.7H20, 1.2.The left atrium was removed and a small compliant balloon (volume ofapproximately 0.5 mL) made of plastic wrapping film (Saran Wrap) was inserted intothe left ventricle and adjusted to give an initial left ventricular end-diastolic pressure of5-10 mmHg. The aortic root of the heart was perfused at a constant pressure of 100mmHg. Ventricular pressure was measured by a pressure transducer and a GrassPolygraph while the maximum rate of intraventricular pressure development(+dP/dtmax) was obtained by differentiating left ventricular pressure using a GrassPolygraph differentiator (model 7P20C).The ECG was recorded from the epicardial surface of the heart with atraumatic,silver-ball electrodes (Curtis et al., 1986b) placed on the right atrium and left-ventricle,i.e., approximating a Lead II configuration. The variability in the rat ECG makesdetermination of drug effects difficult, since the P-, QRS and T-waves do not share acommon baseline (Detweiler, 1981; Driscoll, 1981).64All K receptor agonists and related compounds were dissolved in distilled waterand serial dilutions prepared in Krebs-Henseleit solution. The hearts were perfusedwith Krebs-Henseleit solution for 15 mm prior to perfusion with the drug for a period of 2mm at each concentration. The exposure time was chosen as that during which asteady-state response to drug occurred.2.3 Electrically-induced arrhythmiasTo determine the effectiveness of antiarrhythmic drugs cardiac arrhythmias areusually produced in experimental animal models. Arrhythmias are routinely induced byelectrical stimulation at a variety of sites including the atria, ventricles and the atrioventricular node. These have been produced in a number of species including rats andhumans (Winslow, 1984; Weissberg et al., 1987). The small size of the rat heart doesnot allow for a highly selective placement of electrodes and as a result usually involvesplacement in the right or left ventricle. Access to the ventricles involves transthoracicplacement of stimulating electrodes. A large variety of stimulation protocols can beused in rat ventricular tissue and can be chosen for the induction of arrhythmias or forindirectly probing the functional status of either sodium or potassium channels. Forexample, sodium channel availability may be gauged by examining excitability (i-t)curves (Antoni, 1971; Moore and Spear, 1975). While ERP also indirectly reflectssodium channel status it is highly dependent upon the potassium channels whichcontrol repolarization (Hoffman and Cranefield, 1960). The influence of drugs on therefractory period is also used for evaluation of antiarrhythmic effectiveness.The types of arrhythmias induced by electrical stimulation include singleextrasystoles, VT and VF (Winslow, 1984). It has been suggested that arrhythmiaswhich result from electrical stimulation represent circus-type re-entrant movement(Antoni, 1971). This method allows for the study of the influence of drugs on65ventricular vulnerability and provides a means to examine drugs with potentialantiarrhythmic properties.2.3.1 Surgical PreparationUsing intact rats, prepared as described in section 2.1.2 of the Methods,electrical stimulation of the left-ventricle was performed using two Teflon-coated silverwire stimulating electrodes which were inserted through the chest wall and implantedinto the left-ventricle as described by Walker & Beatch (1988). This placementtechnique produced an inter-electrode distances of between 1-3 mm. Antoni (1971)showed that the optimal inter-electrode distance for induction of consistent thresholdsis between 2-4 mm. Square-wave stimulation was used to determine threshold current(itpA) and pulse-width (tt-ms) for induction of extrasystoles, ventricular fibrillationthreshold (VFrPA), maximum following frequency (MFF-Hz) and effective refractoryperiod (ERP-ms) according to Howard and Walker (1990). Although MFF is anapproximate inverse measure of ERP we chose to measure both values since it hasbeen shown that there are certain differential sensitivities of each to various drugs(Walker and Beatch, 1988). Control measures were taken 10 mm apart until three setsof consistent measures could be obtained sequentially. Usually this required 30 mm toaccomplish. Measures were repeated in triplicate in the order it, tt, VFt, ERP and MFFin controls and for each dose of drug (Pugsley et al., 1992a).2.3.2 Threshold for Capture (it)The threshold for capture (it) is the minimum current which is required tocapture, or pace the heart, and approximates the rheobase of the i versus t curve forexcitability (Vaughan-Williams and Szekeres, 1961). It is a measure of sodium channel66availability (Pugsley et al., 1992a). This threshold is measured for capture of theventricle at a frequency of 7.5 Hz and occurs when the heart follows a I msec square-wave pulse generated by a stimulator (Grass SD9). Capture was detected from theblood pressure and ECG recordings characterized by an increase in signal size, aregular sinus rhythm at a fast rate (slight tachycardia) and a reduction in blood pressure(Howard and Walker, 1988). Usually a range of values (80-100 pA) can be obtainedfor the rat in controls and up to 1000 pA after drug treatment.2.3.3 Threshold Pulse Width (tt)The threshold pulse width for induction of extrasystoles was determined at 7.5Hz and at twice i and tt according to Walker and Beatch (1988). This measureapproximates the chronaxie of the i versus t curve for ventricular excitability and is alsoa measure of sodium channel availability (Cheung et al., 1993).2.3.4 Ventricular Fibrillation Threshold (VFt)Ventricular Fibrillation threshold (VFt) is the minimum current required toproduce ventricular fibrillation (Winslow, 1984). A single train of square-wave pulses attwice it and tt, (Walker and Beatch, 1988) was delivered at 50 Hz while the currentapplied to the myocardium was smoothly increased until fibrillation resulted. Thismethod ensures that the pulse is given during the vulnerable period of late systole,eliciting sustained VF. Once the applied voltage is stopped the rat (as well as thehearts of many other small animals) heart usually spontaneously reverts to normalsinus rhythm (Cheung et al., 1993; Winslow, 1984).2.3.5 Maximum Following Frequency (MFF)67MFF is an indirect measure of ventricular refractoriness (Antoni, 1971; Pugsleyet al., 1992a). It is more of a measure of ventricular functional refractory periods of theaction potential, and thus can exhibit a different sensitivity to drugs from ERP. MFF isdefined as the frequency at which the ventricle fails to follow on a one-to-one basis withthe stimulus (Walker and Beatch, 1988). It is determined by gradually increasing thepacing frequency (at twice it and tt) from 7.5 Hz until the heart fails to follow.Characteristic changes in blood pressure result. The resulting missed beat producesan abrupt decrease, followed by a large increase in blood pressure because of anincreased ventricular filling time. This event is thus an index of MFF measurement.2.3.6 Effective Refractory Period (ERP)The ERP is determined by pacing the heart at 7.5 Hz at twice the it and ttmeasures determined previously. An extra-pulse is added at an increased delay duringthe pacing train such that once the pulse meets or exceeds the absolute refractoryperiod of the action potential an extra-beat is produced in the heart. This extra-beat isdetected by an increase in amplitude of the ECG signal and transient compensatoryreduction in blood pressure (Antoni, 1971). Thus ERP is the shortest interval betweenthe added stimulus and the pacing train at which an extrasystole appears.2.4 lschaemia-induced arrhythmias2.4.1 Surgical preparation in acute studiesThe surgical procedures used were similar to those employed by Au et al.(1979) and Paletta et al. (1989). In brief, rats were initially anaesthetized withpentobarbitone (60 mg/kg, i.p.) and supplemental doses (6mg/kg) were given i.p. when68necessary to ensure an adequate level of anaesthesia. The trachea was cannulatedand all animals artificially ventilated. The left carotid artery was cannulated formeasurement of mean arterial blood pressure and withdrawal of blood samples fordetermination of serum potassium concentrations (lonetics Potassium Analyzer). Theright jugular vein was also cannulated for administration of drugs.The thoracic cavity was opened and a polyethylene occluder placed looselyaround the left anterior descending coronary artery. The chest cavity was closed andbody temperature maintained between 35-37°C using a heating lamp.To obtain the best ECG signal for detection of changes, needle electrodes wereplaced subcutaneously along the suspected anatomical axis (right atrium to apex) ofthe heart determined by palpation, according to the method of Penz et al. (1992). Theanimal was allowed to recover for 30 mm prior to drug administration.2.4.2 Experimental DesignRandom and blind experiments were performed (n=9 per group) in whichanimals received either vehicle, or drug as an i.v. infusion. A control record was taken15 mm before occlusion and 1 mm prior to drug administration. Drug or vehicle wasinfused at a volume of 1.0 mL/hr and traces were taken at I mm intervals over a periodof 5 mm, post-infusion. A blood sample (approximately 0.25 mL) was taken for serumpotassium analysis. Thereafter, the occiuder was pulled so as to produce coronaryartery occlusion. ECG, blood pressure, heart rate, arrhythmias and mortality weremonitored for 30 mm after occlusion.Arrhythmias were classified as ventricular premature beats (VP B), ventriculartachycardia (VT) or ventricular fibrillation (VF) and the number of each was recorded.The overall arrhythmic history was expressed as an arrhythmia score (A.S.) as69described by Curtis and Walker (1988). At the end of a 30 mm period of occlusion and,if the animal survived, a second blood sample was taken.After death, hearts were removed and perfused by the Langendorff technique(Langendorff, 1895) with Krebs-Henseleit solution to wash out all remaining blood. Thiswas followed by perfusion with saline containing I mg/mL indocyanine (Fast green dye,BDH) for 60 sec which revealed the underperfused and occluded zone (zone-at-risk).The occluded zone, which was clearly defined visually as having a distinct border fromthe non-occluded ventricular tissue was then cut away, blotted, and weighed on ananalytical balance. The occluded zone was expressed as a percentage of totalventricular weight.2.4.3 Pre- and post-occlusion ECG changesECG traces were taken 1, 2, 5, 10, 15 and 30 mm post-occlusion. Prior to bothdrug administration and occlusion, the ECG showed a positive ST-segment withrespect to the isoelectric baseline. This allowed for signs of drug-mediated changes inthe RSh measure, indicative of sodium channel blockade, to be measured in the heartprior to occlusion and examination of changes in the S-T segment, post-occlusion.After ligation of the coronary artery, changes occured in the ECG associated withischaemia. Initially, there was a rapid increase in the size of the ECG signal,particularly a large increase in the R-wave amplitude. The maximal R-wave wasmeasured as the maximal deflection of the peak of the R-wave from the isoelectric line.The position of the T-wave of the ECG in rats is not clearly defined and becauseof this determination of the S-T segment is difficult. However, ST%, i.e. the elevation ofthe S-wave as a percent of R-wave amplitude, is a consistent measure in the rat (Curtisand Walker, 1986). The S-T segment initially falls to baseline after the onset ofocclusion (Johnston et al., 1981; Kane et al., 1981) then elevation immediately follows,70reaches a maximum, and is maintained for the duration of the experiment (Curtis et al.,1986c). Since all measures of S-T segment elevation were not constant with time(Johnston et al., 1983) the maximum S-T segment elevation and the time which thisoccurred were determined in an attempt to reflect this fact.2.4.4 Analysis of arrhythmiasThe analysis and quantification of the ischaemic arrhythmias produced byocclusion of the coronary artery is complex while the statistical analysis of sucharrhythmias is dependent on how the arrhythmia data is categorized and treated. As aresult a scoring system was developed with which to summarize the arrhythmic historyin a single value- the Arrhythmia Score (Curtis and Walker, 1988).Arrhythmia appearance is biphasically time-dependent in many speciesincluding the rat (Johnston et al., 1981). In the following studies, the severity andincidence of arrhythmias were quantified during the initial occlusion phase (0-30 mmpost-occlusion).Arrhythmias were categorized according to guidelines established by theLambeth conventions (Walker et al., 1988). Ventricular premature beats (VPB) weredefined as single QRS complexes which occurred before any identifiable P wave.Doublets (bigeminal) or triplets (trigeminal), variations in the single complex, were notclassed as distinct arrhythmias but rather were summed for each group (Curtis andWalker, 1988). Ventricular tachycardia (VT) was defined as 4 or more consecutiveVPB’s and not subclassified according to rate. VT incidence was classified bycharacteristic changes in ECG morphology, elevation in heart rate and fall in mean BP.Ventricular fibrillation (VF) was defined as a chaotic ECG pattern in which nodistinguishable QRS complexes could be discerned accompanied by a precipitous fall71in blood pressure to less than 10 mmHg. Animals were not defibrillated and if VF didnot spontaneously revert, the animal died.2.5 Isolated Ventricular Myocytes2.5.1 Patch-Clamp ApparatusThe patch-clamp was developed in the early 1980’s by Neher and Sakmann torecord membrane ion channel currents. At that time these currents were undetectabledue to the high background noise associated with standard voltage-clamp techniques(Neher and Sakmann, 1976; Hamill et al., 1981). By using a glass micropipette alocalized voltage-clamp could be produced and the electrical activity (channels) in anisolated cell or small area of cell membrane (the patch) could be measured (Cahalanand Neher, 1992). The patch-clamp drastically improved recording techniques. Furtherrefinements included the formation of gigaohm seals with cells rather than megaohmseals recorded previously. These new seal resistence’s improved current amplituderesolution by reducing background noise, reduced leak currents, and stabilized thecurrent recording (Hamill et al., 1981; Hondeghem et al., 1981).Several configurations exist in patch-clamp recording. These include the “cellattached patch”, “whole-cell recording clamp”, “inside-out patch” and “outside-outpatch” described in detail by Hamill et al. (1981). For studies conducted in this thesis,only the whole-cell recording mode was used to elicit sodium and potassium currents.2.5.2 Cell isolationFreshly isolated adult ventricular myocytes from Wistar rats were used toexamine the electrophysiological properties of arylacetamide drug action on ionic72currents. Ventricular myocytes were isolated according to the method of Farmer et al.(1983). Briefly, male Wistar rats (300-400 g) were killed by cervical dislocation followedby exsanguination. The chest was opened, the heart removed and immersed in ice-cold, oxygenated, calcium-free Tyrode solution composed of (mM): NaCl 134; KCI 4;NaH2PO4 1.2; MgCl2 1.2; glucose 11; TES (N-tris-(hydroxymethyl)-methyl-2-aminoethanesulphonic acid) 10, and the solution adjusted to pH 7.4 with 1.0 M NaOH.The heart was then attached, via an aortic cannula, for perfusion with the samecalcium-free Tyrode solution warmed to 37°C to facilitate removal of blood fromventricular chambers and coronary vasculature. After a 5 mm wash, the heart wassubjected to enzymatic dissociation in 25 pM calcium Tyrode solution containingprotease (0.1 mg/mL, Sigma Type XIV), collagenase (1 mg/mL, Worthington CLS Il),and fetal calf serum (1 pg/mL).After 20-25 mm of perfusion the ventricles were removed in one-third sections.Each section was carefully cut into small pieces in fresh 25 pM calcium-Tyrode solutionand triturated to dissociate myocytes. Cell suspensions were then gently centrifugedand washed in a 200 pM calcium-Tyrode solution. Cells were then resuspended in a ImM calcium-containing Tyrode solution and 1-2 hr later plated onto glass coverslips.All cells were prepared and stored at room temperature (25-27°C). The cells used inour studies were rod-shaped, clearly striated and quiescent in the 1 mM Ca-Tyrodesolution.2.5.3 Recording SolutionsAll experiments were performed at room temperature (25-27°C). Cells wereexternally perfused with a Tyrode solution of the following composition (mM): NaCI 70;KCI 5.4; MgCI2 1.0; glucose 10; TES 10; CaCI2 2.0; CoCl2 5.0; CsCI2 5.0; choline Cl60, the solution adjusted to pH 7.4 with 1.0 M NaOH. The pipette solution used for73recording both sodium and potassium currents contained (in mM): KF 140; TES 10;MgCI2 1.0; K-EGTA (ethyleneglycol-bis-(B-amino ethyl ether)N,N,N’,N’-tetraacetic acid);CaCI2 2.0; ATP-disodium 5.0; ATP-Mg 5.0 and pH adjusted to 7.4 with I .OM KQH.When recording sodium currents, the intracellular potassium was replaced withcaesium to inhibit any evoked potassium currents and while recording potassiumcurrents, 20 pM tetrodotoxin was added to the bath solution to inhibit sodium currentswhich may have been evoked.2.5.4 Microelectrode PreparationPatch-clamp electrodes were made from borosilicate glass (A-M Systems,Washington, U.S.A.) with an internal diameter of 1.2 mm and an external diameter of1.6 mm. Electrodes were prepared using a two-stage vertical puller (NarishigeScientific Instruments, Tokyo, Japan). The microelectrode glass used required a fixedpulling length in order to minimize tip diameter (between 2-4 pm) variability. Pipetteswere fire polished using a home-made heating forge. This consisted of a U-shapedplatinum filament connected to a variable DC voltage supply. The pipette was mountedon a rotating holder at a speed of 30 r.p.m.. The heating filament was turned on andthe pipette tip, under a light microscope (IOOX magnification), was moved in closeproximity to the filament. After 1-2 secs the pipette tip was withdrawn, allowed to cool,and placed in a covered petri dish for subsequent use.Pipette tips were initially back-filled by applying suction to the pipette while inthe filtered recording solution and was followed by normal filling with a needle toapproximately one-third the length of the pipette. This ensured removal of all airbubbles and proper immersion of the reference wire (from the head-stage amplifier).The pipette was inserted into a holder on the head-stage amplifier (gain=0.1) which, inturn, was connected to the patch-clamp amplifier (Axopatch 200A, Axon Instruments).74The headstage and pipette could then be moved, in any direction, by amicromanipulator. Only microelectrodes with tip resistances of between 5-10 M2 wereused.2.5.5 Patching Ventricular MyocytesOnce secure in its holder a small positive pressure was applied to the pipette inorder to prevent debris becoming attached to the tip when immersed in the bathingsolution. Pipette tip resistance was determined by the application of a 5mV test pulseto the electrode. The pipette offset on the Axopatch amplifier was adjusted to baselinein order to compensate for the developed junction potential (Barry and Lynch, 1991).Although the junction potential can be compensated for on the amplifier, compensationis never absolute. In our studies the liquid junction potential was usually between 5-10mV at 25°C.Positioning of the electrode above the cell was performed during visualobservation. The pipette tip was slowly lowered onto the cell and once the pipette tiptouched the cell, as indicated visually on the oscilloscope by a reduction in the 5mV testpulse (related to formation of a megaohm seal resistance) a brief suction pulse(negative pressure) was applied to the pipette to produce a gigaohm resistance sealand to rupture the cell membrane. The latter was accomplished using a mouth suctiontube connected to the pipette holder and confirmed by the disappearance of the testpulse and formation of membrane capacitance currents.Current flow across the entire cell membrane were recorded 10 mm afterachieving a whole-cell patch-clamp configuration (Hamill et aL, 1981). Currentrecording was performed using an Axopatch 200A amplifier which allowed for 90%compensation and reduction of both capacitance transients and leak currents fromcomputer-generated voltage commands. Output signals were filtered at 5 kHz,75digitized with a 12-bit AID converter and recorded on a Total Peripherals 3861250p(Marlboro, MA) computer. Final capacitance and leak compensation was performed atthe time of data analysis by subtraction of a 20 mV hyperpolarization pre-pulse currentwhich always preceded the test voltage step.2.5.6 Patch-Clamp ExperimentsOnly cells which were quiescent, rod-shaped and which presented clearstriations were used for studies. Complete sets of current data (control + drug +recovery) were obtained for each cell studied. Experiments were performed in a smalltissue bath mounted on an inverted Nikon microscope. The low volume (0.5-1.0 mL)plexiglass recording bath allowed for rapid exchange (1-2 sec) between control andexperimental solution fed from gravity-flow reservoirs. A suction flow ensured thatsolutions superfused cells at 1-2 mLlmin maintaining a constant fluid level. Evokedsodium and potassium currents were obtained only from myocytes which displayed aminimal reduction in current amplitude (less than 5%) during the control period prior todrug application. Adequate voltage control was achieved by both reducing thetransmembrane sodium concentration and recording currents at reduced temperatures(25-27°C) according to Brown et al. (1981) and Bennett et al. (1988). With this method,the sodium current could be recorded for between 30-60 mm after achieving whole-cellconfiguration. Sodium CurrentsSodium current dose-response curves were evoked by application of 10 msecdepolarizing pulses from a fixed conditioning pre-pulse of -150 mV (which ensuredremoval of sodium channel inactivation) to a test pulse of 0 mV at an interval of one76pulse every 6 sec. Measurements were most accurate when clear capacitance artifactcancellation and compensation was achieved resulting in a distinct separation betweensodium channel activation and the capacitance transient decay.The voltage-dependence of activation (mx) of the peak sodium current wasexamined using a voltage-step to a variable test potential (between -70 mV and +50mV) from a fixed conditioning pre-pulse potential of -150 mV. Test potentials wereevoked at I sec intervals (or greater) to ensure adequate recovery of channel functionbetween pulses. A plot of current amplitude against test pulse potential yields acurrent-voltage relationship for sodium current. Conductance (GNa) can be calculatedusing the Hodgkin and Huxley model where lNa=GNa(V-Erev). The line approximatingthis relationship is the Boltzmann equation lNa{Gmax/[l+eXP((VV’)/k)]}x(VErev), Inthis equation Gmax is the maximal channel conductance for sodium, V’ is the voltagefor half-maximal sodium channel activation, k is a slope factor, and Erev is the reversalpotential for the sodium channel. This equation allows for an examination of drugeffects on channel kinetics of activation.The steady-state voltage-dependence of inactivation (h) was studied bymeasuring the effect of various conditioning pre-pulses between -140 mV and -30 mVon the sodium current elicited at a fixed test potential to -20 mV according to Hodgkinand Huxley (1952). The best-line fit for the isolated myocyte data was determinedusing the Boltzmann equation lNaGmax/[l+eXp((VV’)/k)] for inactivation kinetic valuesas determined above.To elucidate use or frequency-dependence of drug action, studies wereconducted by examining drug effects on evoked sodium currents at variousfrequencies. The arylacetamides were initially examined by bath application of a singledrug concentration followed by the delivery of a train of depolarizing pulses at variousfrequencies or by providing a train of depolarizing pulses at a frequency of 10 Hz tocells. Sodium currents were evoked as above except that the trains of depolarizing77pulses were provided by an external Grass stimulator. The stimulator frequencies anddurations were regulated by custom computer software developed in the laboratory ofDr. D.A. Saint.Several studies were conducted to determine the putative site of action of (±)PDI 17,302 and its inactive enantiomer, (+)PD123,497. Studies on sodium currentsinvolved addition of arylacetamide to the bathing solution or to the pipette solution usedto patch the myocyte. In a comprehensive review Horn and Korn (1992) discuss thepotential for exchange of material between the cell interior and pipette solution. Sincethe speed of the resulting “washout” depends upon both pipette resistance and cell sizeit is nonetheless possible to prevent the rundown of recorded currents bysupplementation of the pipette solution with constituents which maintain ionic currentfunction. No significant rundown of current amplitude was seen during patch-clamprecording. Verification of drug perfusion into the cell interior was achieved byintracellular application of lidocaine in several preliminary experiments which quicklyblocked the evoked sodium current (data not shown).Sodium currents were evoked beginning approximately 4 mm after achievingwhole-cell mode of the patch-clamp configuration. Current amplitude was monitored foran additional 4 mm with currents being evoked every 6 sec. Arylacetamides were thenadded to the bath solution perfusing the cells and currents continuously evoked. Peakcurrent amplitudes were plotted as a function of time.An additional study was conducted in an attempt to determine the effects of pHon drug blockade of sodium currents. (+)PD123,497 (13 pM) was added to bathsolutions at pH=6.4 and pH=7.4. Control currents were obtained following which thepH=7.4 bath solution containing (+)PD123,497 was added. Approximately 2 mm later asustained current block resulted at which time the acidic pH solution containing(+)PD123,497 was exchanged for the pH=7.4 solution. Sodium currents were78continuously recorded for 1.5 mm at which time the pH=7.4 bath solution was reappliedto the cell. Peak current amplitude for the responses were plotted as a function of time. Potassium CurrentsStudies were conducted to determine the effects of spiradoline on potassiumcurrents in isolated myocytes. Potassium currents were evoked by depolarization to+50 mV from a pre-pulse potential of -150 mV for a duration of 300 msec. Theconcentration-dependent effects of bath applied arylacetamides on the transientoutward (ito) and sustained outward plateau (KSUS) potassium currents were examined.The potassium current amplitude for i0 was measured at the peak, approximately 5msec after evoking the outward current. A measure of drug effectiveness on thiscurrent was obtained by subtraction of the current magnitude 300msec after evokingthe outward current from the peak amplitude. At 300 msec the ito current hasinactivated and the sustained outward current, mKSUS’ remains. Compounds wereexamined for blocking effects on these currents. Construction of a line tangential to thedecay curve of tmo approximated drug effect on the rate of decay of this current. Weassumed a monoexponential decay of evoked outward potassium current, although itomay be composed of two components (Coraboeuf and Carmeleit, 1982; Campbell etal., 1995).2.5.7 DrugsSpiradoline (U-62,066E) (a gift from Dr. P.F. Von Voightlander, The Upjohn Co.,Kalamazoo, Ml), naloxone (Sigma Chemical Co., St. Louis, MO) and tetrodotoxin (TTX)were initially dissolved in distilled water prior to dissolution in the external bath solutionfor single cardiac myocytes.792.6 Statistical AnalysisAll in vivo studies were performed according to a randomized blockexperimental design. This block design compensates for heterogeneity in theexperiment and controls for variability which may arise from experimental error.Experimental error reflects a combination of both random error and biological variability.The random block design essentially reduces the variability in the system and hencedecreases experimental error (Montgomery, 1984). The only randomization that occursis confined to treatments within blocks. The blocks then represent a restriction onrandomization and thus yield a simple statistical model (Li, 1964).Experiments with the above design lend themselves to Analysis of Variance(ANOVA) particularly as this test demands that treatments are from as uniform anenvironment as possible (Li, 1964). ANOVA allows one to compare many treatments,thus making it a most useful statistical test (Zar, 1984; Gad and Weil, 1988). Statisticalsignificance was determined at an cL-level of 0.05 using the General Linear ModelANOVA (GLM ANOVA) from the NCSS Statistical Package (Hintze, 1981). Values areshown as the mean±s.e.mean for n experiments.A post hoc or multiple comparison test was performed to determine whichtreatment means differed after ANOVA. A large number of tests exist for this purpose;we chose Duncan’s multiple comparison test (Duncan, 1955). This test comparesgroups of continuous and randomly distributed data of equal sample size and is apowerful test for detecting differences between means (Montgomery, 1984). This testwas used throughout the in viva experiments performed except in arrhythmia studies.Mainland’s contingency tables were used rather than analysis of covariance (ANCOVA)to determine significance between arrhythmic groups. Exclusion criteria for thesestudies were according to Pugsley et al. (1992b). Arrhythmic PVC incidence was log10transformed to a normal distribution (Winkle, 1979; Walker et al., 1988).803 Unpublished Results for other Arylacetamides3.1 Studies with U-62,066E (Spiradoline)The chemical structure of spiradoline, (U-62,066E), and the other arylacetamidesexamined in previously published studies can be found in Figure Isolated Heart studies - Contractility and ECG effectsSpiradoline produced concentration-dependent increases in heart rate, P-R intervaland QRS duration of the isolated rat heart ECG. Figure 2A shows the concentration-related reduction in heart rate produced by 100 iM spiradoline (n=5 hearts). In thepresence of 1 pM naloxone, changes in heart rate with spiradoline paralleled spiradolinealone. Observed ECG changes included a P-R interval prolongation and increased QRSwidth (Figure 2B). The P-R interval changed from 69±4 to 189±15 msec in hearts exposedto 100 pM spiradoline. Likewise, QRS increased from 29±2 msec to 76±14 msec in heartsexposed to the same concentration of drug. For sake of clarity, naloxone pre-treatmentdose-response curves are not shown. In the presence of 1 pM naloxone, spiradoline (100pM) increased the P-R interval from a control of 55±1 to 170±11 msec. There were nodifferences between spiradoline effects in the absence and presence of naloxone.Cardiac contractility was reduced in a concentration-dependent manner byspiradoline. Peak systolic pressure (Figure 3A) and maximum rate of peak systolicpressure development (+dP/dtmax) and relaxation (dP/dtmax) were equally reduced byspiradoline (Figure 3B). The peak systolic pressure was reduced by 54% with 100 pMspiradoline (from a control of 93±5 to 43±11 mmHg). A transient elevation (10%) in enddiastolic pressure occurred at this concentration (data not shown).81Figure 1 Chemical structure of U-50,488H, trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzene-acetamide methane sulphonate, (+)PDI 23,497 (+)-Nmethyl-N-[2-(1 -pyrrolidinyl)cyclohexyl]benzo[b]thiophene-4-acetamide monohydrochloride(the inactive ic enantiomer of (±)PDII7,302), (±)U-62,066E (spiradoline) 5cL,7ct,8-(±)-3,4-dichloro-methyl-N-[7(1 -pyrroliclinyl)-1 -oxaspiro(4,5)dec-8-yl]benzeneacetamide methanesulfonate), (±)PDI 17,302 ((±)-N-methyl-N-[2-(1 -pyrrolidinyl)cyclohexyl]benzo[b]thiophene-4-acetamide), (-)PDI 29,290 (-)[5R-(5ct,7c*,8B)]-N-methyI-N-[7-1 -pyrrolidinyl)-1 -oxaspiro[4.5]dec-8-yl]-4-benzofuran acetamide, (±)PDI 17,302 (±)-N-methyl-N-[2-(1 -pyrrolidinyl)cyclohexyl]benzo[b]thiophene-4-acetamide monohydrochloride and (+)PD129,289 (+)5S-(5cç7c,8()]-N-methyl-N-[7-1-pyrrolidinyl)-1 -oxaspiro[4.5]dec-8-yl]-4-benzofuran acetamide(the inactive enantiomer of (-)PD129,290).82Arylacetamide structuresU-50,488H (+) PD123,497MeQ.HCIU-62,066E (Spiradoline)(±) PDII7,302(-) PD129,290(+)PD129,2890CICIMe0IpMe Me.HCIFigure 183Figure 2 Concentration-dependent effects of spiradoline on heart rate (A) and theECG (B) in Langendorff-perfused rat hearts Panel A shows the effects of spiradoilne onheart rate in the absence () and presence (0) of 1pM naloxone. Panel B shows theeffects of spiradoline alone on P-R interval (z) and QRS duration (A). The effects ofspiradoline tested in the presence of I pM naloxone (data not shown) did not differsignificantly from those obtained in the absence of naloxone. Values are shown asmean±s.e.mean for 5 hearts/group. Pre-drug values are shown to the left of thespiradoline concentration axis. Statistically significant difference is shown as * for p<O.05,as compared with pre-drug values.84A300TA250E200cD150100c= 5001 10 100B250 250*__C.)200 200 a,a) 0O 2150 150- 0100 * 100..2C/)cc cc50 C-I-0 I 01 10 100*******drug concentration (pM)85The decrease in peak systolic pressure in the presence of 1 pM naloxone paralleled theeffects of spiradoline alone. The peak systolic pressure in control hearts was reduced from105±8 to 46±6 mmHg at a concentration of 100 pM spiradoline and in the presence of IpM naloxone. The rate of intraventricular pressure development (+dPldtmax) andrelaxation (dP/dtmax) were also reduced by spiradoline (Figure 3B). Data in the presenceof naloxone is not shown for sake of clarity. In the presence of 1 pM naloxone, +dP/dtmaxwas reduced from a control of 2250±110 to 1130±95 mmHg/sec by 100 pM spiradoline.The reduction in the rate of relaxation in the presence of naloxone also paralleled the curvedescribing spiradoline alone. The rate of relaxation was reduced from a control of1850±225 to 490±45 mmHg/sec with 100 pM spiradoline. Thus, these experiments inisolated hearts suggest that the drug produces a direct depression of cardiac contractilitywhich may be related to ion channel blockade.3.1.2 Haemodynamic and ECG actions of SpiradolineBlood pressure and heart rate were stable over the duration of drug infusion in allvehicle (n=5) and 2.5 pmol/kg/min naloxone treated (n=3, data not shown) animals.Spiradoline, in the absence and presence of naloxone, produced a marked dose-dependent reduction in both blood pressure and heart rate (Fig. 4A and B, respectively).At a dose of 32 pmol/kg/min spiradoline reduced blood pressure from 126±7 to 51±4mmHg and heart rate from 352±14 to 124±7 beats/mm. In the presence of naloxone, bloodpressure was equally reduced (from 118±6 to 57±7 mmHg); however, heart rate wasreduced less than in the absence of naloxone (from 360±19 to 201±17 beats/mm).ECG measures were also influenced in a dose-related manner by spiradoline. Thehighest dose of spiradoline (32 pmol/kg/mmn) produced a 36% increase in the P-R interval(from 61±1 to 95±2 msec). In animals treated with naloxone, the highest dose of86Figure 3 Concentration-dependent effects of spiradoline on peak systolic pressure(A) and rates of change of pressure (B) in Langendorff-perfused rat hearts Panel A showsthe effects of spiradoline on peak systolic pressure in the absence (A) and presence (0) of1 pM naloxone. Panel B shows the effects of spiradoline alone on the maxium rate of riseof ventricular pressure (A) and maximum rate of fall of ventricular pressure (A). The effectsof spiradoline tested in the presence of 1pM naloxone (data not shown) did not differsignificantly from those obtained in the absence of naloxone. Values are shown asmean±s.e.mean for 5 hearts/group. Pre-drug values are shown to the left of thespiradoline concentration axis. Statistically significant difference is shown as * for p<O.05,as compared with pre-drug values.87C)-cEa)000000a)****AT0JBT*..11501005002500200015001000500010 1000a)0C)=ExE•1-’0•C+**250020001500100050000ci)C/)C)ExE•C0***1 10 100drug concentration (pM)88Figure 4 Dose-related effects of spiradoline on blood pressure (A) and heart rate (B)in pentobarbitone-anaesthetized rats in the absence and presence of 2.5 pmol/kg/minnaloxone. Values are mean±s.e.mean for n=5. *lndicates a significant difference frompre-treatment at p<O.05. The pre-treatment means for blood pressure were 126±7, 118±6,135±7 mmHg while those for heart rate were 352±14, 360±19 and 375±11 beats/mm forspiradoline (0), spiradoline+naloxone (.) and saline vehicle (D), respectively. Salinecontrol had no significant effect upon either measure.D)zEa)Cl)Cl)a)h..00C2Cl)ci)ci•1-’ci).C1 TT T*AB*****1I . I . I200100050040030020010001089100.1* * *T.*****1 10 100dose (pmol/kg/min)90spiradoline caused a 35% increase in P-R interval prolongation (from 62±2 to 96±3 msec)(Figure 5A). Spiradoline did not affect the QRS width until the highest doses wereadministered (Figure 5B). RSh, a novel measure of sodium channel blockade in the rat,was increased in a dose-dependent manner by spiradoline (Figure 6A). The RSh interval,at 32 pmol/kg/min, was increased by 49%, from 0.46±0.03 to 0.91±0.10 mV, while in thepresence of naloxone the prolongation of this measure was 60% at this equivalent dose(from 0.48±0.09 to 1.20±0.21 mV). From Figure 6B it can be seen that spiradoline dose-dependently increased the Q-aT interval. The Q-aT measure was increased 34 and 39%by spiradoline in the absence and presence of naloxone, respectively. The vehicle-controldid not affect any of the ECG measures over the duration of the experiment.3.1.3 Electrical Stimulation studiesThe patterns of drug action in isolated hearts and in the intact rats indicated thatspiradoline may alter both sodium and potassium channel function. In order to determinewhether or not these properties of spiradoline may confer antiarrhythmic activity weexamined the effectiveness of spiradoline against electrically-induced arrhythmias in therat. Figure 7A and B shows that spiradoline dose-dependently increased thresholds forcapture or induction of extrasystoles (it) and ventricular fibrillation (VFt). The it values forspiradoline, in the absence and presence of naloxone, were increased 64 and 66% (orfrom 80±11 and 95±18 to 220±12 and 247±60 pA), respectively. The dose-dependentchanges in VFt produced by spiradoline were much more marked than those on It. VFtincreased from 147±30 to 323±55 pA (54%) in the absence of naloxone and from 124±15to 525±35 in the presence of naloxone (76%). Changes in tt, the time to threshold,transiently increased with spiradoline administration but this was not significant (p>0.05)(data not shown).91Figure 5 Effects of spiradoline on P-R interval (A) and QRS duration (B) inpentobarbitone-anaesthetized rats in the absence and presence of 2.5 pmol/kg/minnaloxone. Values are mean±s.e.mean for n=5. *lndicates a significant difference frompre-treatment at p<O.05. The pre-treatment means for the P-R interval were 61±1, 62±2,57±3.5 msec while those for the QRS duration were 31±1, 30±0.4 and 28±0.8 msec forspiradoline (0), spiradoline+naloxone (.) and saline vehicle (D), respectively. Salinealone had no significant effects upon either measure.P-Rinterval(msec)0 Cl) CD 3 0 30000000)CDF301000000QRSduration(msec)DIDwIIIIIIDR0 0 0***0 -L 0 0*CD p393Figure 6 Effects spiradoline produced on RSh (a novel index of sodium channelblockade in the rat described in full in Appendix 6) (A) and the Q-aT interval (described infull in Appendix 7) (B) in pentobarbitone-anaesthetized rats. Values are mean±s.e.mean,n=5. *lndicates a significant difference from pre-treatment at p<O.05. The pre-treatmentvalues for RSh were 0.48±0.10, 0.46±0.03, 0.54±0.03 mV while those for the Q-aT intervalwere 35±0.6, 37±1 and 37±1 msec for spiradoline (0), spiradoline+naloxone (•) and salinevehicle (D), respectively. Saline alone produced no significant effects upon eithermeasure.94A1.50*1.20>E- 0.90>ci). 0.60-C 0U)0.300.00I I I I I I I1 10 100B10080ci)Co60Cu*>ci)40Io 200I I I I I I Ii? I I I I I I1 10 100*dose (pmol/kg/min)95Figure 7 Effects of the saline vehicle (D), and spiradoline in the absence (0) andpresence of 2.5 pmol/kg/min naloxone (•) on the threshold current for capture (it) (A) andventricular fibrillation threshold, (VFt) (B) in pentobarbitone-anaesthetized rats. Salinealone had no significant effect upon either measure. Values (mean±s.e.mean for n=5animals/group) were measured 3 mm after completion of infusion. *lndicates a significantdifference from pre-treatment at p<O.05.96A500400—, 300 *T2001000I I I 111111111 10 100B600*T500400 * I** .300T>200 0 TT TT100 -0 It, iii1 10 100dose (pmol/kg/min)97Figure 8 Effects of the saline vehicle (D), and spiradoline in the absence (0) andpresence of 2.5 pmol/kg/min naloxone (•) on the effective refractory period (ERP) (A) andmaximum following frequency (MFF) (B) in pentobarbitone-anaesthetized rats. For thesaline vehicle (D) group, no change resulted over the duration of the experiment. Valuesare mean±s.e.mean for n=5 animals/group. *lndicates a significant difference from pretreatment at p<O.05.0 Co CD-3 0 3 Dmaximumfollowingfrequency(Hz)effectiverefractoryperiod(msec)L01001000000IIIF•ED1wIIIIi€•i*0 0 0**- 0 0 0*(099The effective refractory period, ERP, a direct measure of refractoriness was dose-dependently prolonged by spiradoline with and without naloxone pre-treatment (Figure 8A).Maximum following frequency (MFF) was significantly decreased by spiradoline (with, orwithout, naloxone) at doses greater than 8.0 pmol/kg/min (Figure 8B). Spiradoline(32pmollkg/min) reduced MFF from 15.3±0.5 Hz to 7.0±0.4 Hz in control animals and from14.1±0.6 to 6.0±0.5 Hz in the presence of naloxone. The vehicle control values forthresholds (it and VFt) and refractoriness (MFF and ERP) were stable over the treatmentperiod.3.1.4 Coronary Artery Occlusion studiesIn the coronary occlusion-arrhythmia study a dose of spiradoline (2.5 pmol/kg/min)was chosen which produced changes in haemodynamic and ECG measures in theabsence and presence of naloxone as was seen in vivo (Table 1). None of the changesinduced by spiradoline were prevented by naloxone pre-treatment.Spiradoline (2.5 jimol/kg/min) statistically significantly reduced arrhythmias inducedby coronary occlusion (Table 2). Both ventricular tachycardia and fibrillation incidencewere reduced as exemplified by an arrhythmia score of 2.4±0.4 in the spiradoline treatedgroup compared to 6.3±0.4 in saline controls (n=9 rats/group).Naloxone had no antiarrhythmic effect when administered alone. However, VF wasabolished when naloxone was given with 2.5 pmol/kg/min spiradoline.The reduction in arrhythmia incidence could not be ascribed to either occlusionzone size (zone-at-risk) or serum potassium levels. Table Ill shows that there were nosignificant differences between group occluded zone sizes, hence the arrhythmic insultswere assumed to be the same in all groups. Similarly, serum potassium levels were notinfluenced by drug treatment. The post-occlusion serum potassium levels were only100Table I The cardiovascular and ECG effects of 2.5 pmol/kg/min spiradoline, in theabsence and presence of 2.5 pmol/kg/min naloxone in pentobarbitone-anaesthetised ratssubject to acute coronary artery occlusion.Dose BP HR P-R QRS RSh Q-aT(pmol/kg/min)saline 128±4 383±17 55±1 26±1 0.52±0.04 34±1naloxone(2.5) 116±6 361±12 58±1 29±1 0.55±0.05 35±2spiradoline (2.5) 91±6* 298±13* 61±2* 30±2* 0.62±0.05* 38±1spiradoline(2.5) 85±4* 289±11* 62±1* 30±1* 0.68±0.03* 40±2*+ naloxone (2.5)The effects of spiradoline alone, or in the presence of naloxone (2.5 pmol/kg/min),are expressed as mean±s.e.mean (n=9) for the variable indicated. BP = meanarterial blood pressure in mmHg; HR = heart rate in beats/mm, and the P-R, QRSand Q-aT are EGG intervals in msec while RSh is in mV. *lndicates P<0.05 forcomparison with saline.Table IIpresence ofarrhythmias inAntiarrhythmic effect of spiradoline (2.5 pmol/kg/min) in the absence and2.5 pmol/kg/min naloxone against coronary artery occlusion-inducedpentobarbitone-anaesthetized rats.Group IncidenceThe antiarrhythmic actions of spiradoline, either in the absence or presence ofnaloxone (2.5 pmol/kg/min) are expressed in terms of group incidence of one or moreepisodes of the major arrhythmias of ventricular tachycardia (VT) or ventricularfibrillation (VF). Ventricular premature beats (VPB) were log10 transformed fornormalization. Arrhythmia score (A.S.), which summarizes and grades arrhythmicincidence and severity, was expressed as mean±s.e.mean (n=9). *p<005 whencompared with saline.slightly elevated (no significant difference between treatment groups) however, this did notDrug log VPB VT VF VT and br A. S.(pmol/kg/min) VFSaline 1.9±0.2 9/9 9/9 9/9 6.3±0.4Naloxone(2.5) 1.8±0.1 8/9 8/9 8/9 5.1±0.5Spiradoline (2.5) 1.7±0.1 3/9* 2/9* 3/9* 2.4±0.4*Spiradoline (2.5) 1.7±0.3 4/9 0/9* 4/9 2.2±0.4*+_Naloxone_(2.5)play a role in reducing arrhythmia incidence.101The time to S-T segment elevation and R-wave maximum were prolonged afterspiradoline treatment, with or without naloxone pre-treatment. Although spiradolinedelayed the time for the S-T segment to reach maximum elevation and R-wave maximum,it did not reduce the maxima. Naloxone itself slowed the time to development of an Rwave maximum, indicative of an antiarrhythmic effect. Mortality was abolished in thespiradoline groups regardless of naloxone pre-administration.Table Ill The actions of 2.5 pmol/kg/min spiradoline, in the absence and presence ofnaloxone (2.5 pmol/kg/min), on ECG, mortality and serum potassium changes induced bycoronary artery occlusion in pentobarbitone-anaesthetized rats.Time to Serum K+Dose ST-seg. R-wave S-T R- Pre-drug Post-drug OZ Mortality(pmol/kg/min) max. max. max. max (mM) (mM) size(mm) (sec) (%) (my) (%)Saline 17±3 22±2 81±5 1.3±.3 3.8±0.3 4.4±0.2 38±2 5/9Nal (2.5) 18±2 30±5* 74±7 1.6±.5 3.6±0.5 4.0±0.2 37±1 3/9Spir(2.5) 25±2* 48±4* 89±4 1.2±.2 3.7±0.2 4.2±0.3 39±1 0/9Spir+ Nal 24±4* 42±6* 70±9 1.7±.4 3.3±0.4 3.8±0.4 35±2 0/9The effects of spiradoline (spir) alone, or in the presence of naloxone (nal)(2.5imol/kg/min), are expressed as mean±s.e.mean for n=9 (except for post-drugserum potassium levels which correspond to the group size remaining at the end ofthe experiment as indicated in the mortality column) for the variable indicated.*lndicates significant difference from saline at p<0.05. S-T max. is the maximumheight of the S-T segment expressed as a % of the height of the R-wave.3.1.5 Electrophysiological actions on Sodium Currents3.1.5.1 Concentration-response curvesSpiradoline produced a concentration-dependent reduction in the magnitude of thesodium current (Figure 9A, B) evoked in isolated rat myocytes at concentrations whichproduced marked effects on isolated hearts (as shown in Figures 2A, B and 3A, B).Figure 9A shows data obtained from an experiment in which sodium currents were evoked102by voltage steps to 0 mV from a pre-pulse potential of -150 mV. These voltage steps weregiven at 6sec intervals and the currents generated were plotted as a function of time (on atime scale of 10 msec). Spiradoline (15-100 pM) was added to the bath solution andproduced a concentration-dependent readily-reversible block of sodium current. Theseconcentration-response experiments were repeated in 4 additional cells and similar dose-response relationships were obtained. Figure 9B, describes the concentration-responsedata (n=5 cells) for the block of sodium current by spiradoline. An estimate of the half-maximal sodium current block (EC50) from this data (using the best fits of the equationwas 66 pM. In the presence of 1 pM naloxone there was no change inthe concentration-response curve (data not shown). Current-Voltage effects3. Activation kineticsSpiradoline block of sodium currents was examined by determining whether or not itproduced a change in the voltage-dependence of activation or inactivation (h) (n=6 cellsper study). The effect of 150 pM spiradoline was examined on the voltage-dependence ofactivation of the sodium current. A voltage-step to various test potentials between -70 mVand +50 mV was given from a fixed pre-pulse potential of -150 mV. The peak currentamplitude of‘Na is shown in Figure IOA plotted against the pre-pulse potential. To test forthe effects of the drug on activation kinetics we defined the peak sodium conductance(GNa) versus the reversal potential (Erev) over the potential range of -70 mV to +60 mV.Conductance (GNa) was calculated using the Hodgkin-Huxley modeland approximated by a Boltzmann equation fit producing the relationshiplNa{Gmaxhl+exP[(V)/k]}><(’’rev) where Gmax is the maximal channel conductance forsodium, V’ is the voltage at which GNa is half-maximal, k is the slope factor, and Erev is the103Figure 9. Effect of spiradoline block on sodium currents in rat cardiac myocytes.Panel A shows sodium currents evoked by a voltage-step from a pre-pulse potential of-150 mV to a potential of 0 mV. The voltage step was delivered at 6 sec intervals andspiradoline was added to the bath solution at the concentrations indicated. The re-controlcurrent is indistinguishable from control. Spiradoline was added to the bath solution for 2mm before evoking currents at the concentrations examined. Similar results were obtainedin 4 additional cells.Panel B shows the concentration-response curve for the degree of blockade of thetransient sodium current by spiradoline. Sodium currents were evoked as above. Thedegree of block,1(control)-’(block)”(control)’ is shown as a function of log10 concentration ofspiradoline. Data from five individual cells is plotted with the line of best fit for the equationy=1/(1 +(Ka/[A])) shown.104500 pMA4-.CI..I0B00150 pM50 jiM15 pME— controlfrecontrolI I100-10-20-30-40-50-601 . -4 -2 0 2 4 6 8 10time (ms)..10 100 1000concentration (pM)105reversal potential for sodium. For the rat ventricular myocyte shown in Figure IOA, thepeak Gmax was 1.49 nS, with a V’ of -42 mV, a slope factor (k) of 3.0 mV per e-foldchange in‘Na and a reversal potential (Erev) of 47 mV. After exposure to 150 pMspiradoline the values were 0.22 nS, -42 mV, 3.0 mV per e-fold change in‘Na and Erev of47 mV. Spiradoline only reduced‘Na by decreasing Gmax and did not produce a change inthe voltage-dependence of threshold activation of the current. Inactivation kineticsThe effects of spiradoline on inactivation kinetics were studied by giving a voltage-step to -20 mV from pre-pulse potentials which varied between -140 mV and -30 mV.Spiradoline produced channel blockade by changing the steady-state voltage-dependentinactivation kinetics of the current as shown in Figure lOB. A Boltzmann equation(lNaImax/[exP’”)’k]) where ‘Na is the maximal sodium current, V1 is the voltage atwhich‘Na is half-maximal and k is the slope factor (indicating the steepness of the slope orvoltage dependence of inactivation) was used to derive a curve for the data obtained undercontrol conditions and during the application of 150 pM spiradoline. In the presence of 150pM spiradoline a substantial block of the maximum available current was accompanied bya hyperpolarizing shift in the voltage-dependence of‘Na by 21±2.9 mV and 24±2.2 mV inthe presence of 50 and 150 pM drug concentrations (n=6 cells each, data for 50 pMconcentrations not shown). This shift is only revealed when the curve in the presence ofspiradoline is scaled to the control/re-control maximum. As well, 150 pM spiradolineincreased the slope factor, k, from 7 mV in control to 8.5 mV per e-fold change in‘Na afterdrug exposure. However, from the curves, it can be seen that the maximal currentamplitude is greatly reduced with 150 pM spiradoline, even at very negative values of thepre-pulse potential, which indicates that the shift in the voltage-106Figure 10 Effect of spiradoline on the current-voltage relationship and inactivationkinetics (h) of sodium currents. In panel A the current-voltage relationship for activationof sodium currents is shown. Currents were evoked by a voltage pulse to potentials of -70to +50 mV from a potential of -150 mV. Peak current amplitude is plotted against potentialfor control data (.) and in the presence of 150 pM spiradoline (+). Conductance (GNa)was calculated from the Boltzmann equation lNa{GmaxI{lXP[(VV’/k]}><(VErev) whereGmax is maximal channel conductance for sodium, V’ is the voltage at which ‘Na is halfmaximal, k the slope factor, and Erev is reversal potential for sodium. In control and recontrol, Gmax was 1.49 nS, with a V’ of -42 mV, a slope factor (k) of 3.OmV per e-foldchange in‘Na and a reversal potential (Erev) of 47 mV. In the presence of I 5OpMspiradoline (,) Gmax was 0.22 nS, V’ was -42 mV, k was 3.0 mV per e-fold change in ‘Naand Erev was 47 mV. The only difference seen in the cell was a reduction in maximumchannel conductance, Gmax.In panel B sodium currents were evoked by a voltage step to -20 mV from a prepulse potential which varied between -140 and -30 mV. The peak current amplitude isshown plotted against the pre-pulse potential for control and re-control data and in thepresence of 150 pM spiradoline. The data is shown by the best-fit line(s) using theBoltzmann equation y=G/1+exp[(V-V’)/k)] where V is the pre-pulse potential, V’ is thehalf-maximal voltage for inactivation and k is a slope factor. The best fits for the line wereobtained for values of GmaxO.92 nS, V’=-58.6 mV and k=7.0 mV per e-fold change in ‘Nafor the control solid line curve (•). A curve was obtained in the presence of 150 pMspiradoline (,). The dotted line curve (0) shows the data in the presence of spiradolinescaled to the same maximum as control/re-control data. This curve resulted in values ofGmaxO.l7nS, V’=-78.2 mV and k=8.5 mV per e-fold shift in ‘Na107A200-20-60a)-80h..C.) -100-120-80 -60 -40 -20 0 20 40 60potential (mV)B10080C60C0200--140 -120 -100 -80 -60 -40 -20potential (mV)108dependence of inactivation may not be the principal means by which current block isproduced. Tonic and use-dependent components of spiradoline block of sodium currents.To determine whether or not spiradoline possesses use-dependent blockingproperties, sodium currents were evoked by lOmsec voltage-steps at various frequencies.Figure II is a record of one sec of evoked sodium current. Spiradoline (50 pM) produceda slowly-developing steady state use-dependent block. Peak current was reduced from10.2, 10.0 and 10.1 nA (measured as the initial peak control current in panels a, b, c, ofFigure 11, respectively) to 6.92, 6.0 and 3.6 nA (measured as the last evoked current in thetrace for panels a, b, c, respectively) at (a) 6, (b) 13 and (c) 30 Hz, respectively. Theaverage peak amplitude in (d), control current at 25 Hz, was 10.4±0.2 nA.Figure 12A depicts the frequency-dependence of block by 150 pM spiradoline.Sodium currents were evoked by a voltage step to 0 mV from a pre-pulse potential of -150mV. The voltage steps were delivered as frequency trains of 0.7, 3.3, 6.6 and 16 Hz (n=5cells) with 3 mm of recovery time between trains. The bathing solution contained 50 pMspiradoline and cells were exposed to the drug for 2 mm before providing pulse trains. Thepeak current (normalized to the first current evoked) is shown plotted against the time atwhich it was evoked, for each of the frequencies. In control solution no reduction in peakcurrent was seen at frequencies up to 40 Hz (data not shown, see Figure lId for 25 Hzrepresentation). The time to development and maximal amount of use-dependent blockwere increased as the rate of stimulation increased.Since it has been shown that the rate of block development by antiarrhythmic drugsis strongly concentration-dependent, it was determined whether or not this was true forspiradoline. A plot of peak sodium current amplitude and the number of pulses applied(Figure 12 B) was used to explore the concentration-dependency of rate of109a______Cb dflwfl flfl.flFigure 11 Depicts the frequency-dependent block of the sodium current by spiradolineon a one sec time scale. Sodium currents were evoked by a voltage step to 0 mV for 10msec from a pre-pulse potential of -150 mV at various frequencies. The traces arerepresentative of a one sec train of such stimuli at (a) 6 Hz, (b) 13 Hz and (C) 30 Hz all in• the presence of 50 pM spiradoline. Cells were exposed to the drug for 2 mm beforedelivering frequency pulse trains. For comparison, the one sec train of stimuli shown intrace (d) is the evoked sodium current at 25 Hz in control. Results were obtained in 5additional cells.110development of use-dependent block and whether a tonic component of drug block exists.A train of depolarizing pulses of 10 msec duration to 0 mV from a pre-pulse potential of-150 mV were given at a stimulation frequency of 10 Hz. In control solutions no noticeablereduction of current with successive pulses was seen (data not shown, see Figure lId for25 Hz representation). Spiradoline concentrations were bath applied and trains evoked 2mm after application. Spiradolmne block was not fully complete at either of the lowconcentrations (15 and 50 pM) examined since a steady-state block of current did notresult even after 12 pulses (1.0 sec) were evoked, when delivered at 10 Hz. However, fullydeveloped use-dependent block occurred with 150 pM spiradoline after only 7 pulsesdelivered at this same rate. Although not shown, 500 pM spiradoline blocked all current(Figure 9A) after only 5 pulses.Although not studied directly several observable effects of current behaviour in thepresence of spiradoline suggest that the drug may, in addition to the use-dependentcomponent discussed above, produce a reduction in current by tonic block. The firstsuggestion of such a block is seen in Figure lOB. Sodium channel block by sspiradolinearises at low rates of stimulation (one sec between evoked current pulses) and at verynegative potentials where the channels tend to exist in the resting state (according toHodgkin and Huxley formalism). The average reduction in peak current was 69±16% in ratcardiac myocytes exposed to 150 pM spiradoline (n=6) over the potential range of -140 to-100 mV (Figure lOB). As well, tonic block can be seen in Figure 12B where spiradolineproduced a concentration-dependent reduction of peak current when the amplitude of thesecond pulse generated is compared to the first (or control pulse number 1) current. Thus,in these experiments, if the amplitude of the second evoked sodium current is compared tothe first (control) evoked current, spiradoline cann be seen to reduce the peak current by2.2, 8.4 and 48% at 15, 50 and 150 pM, respectively, before the onset of use-dependentblock.IllFigure 12 Panel A depicts the frequency-dependence of block by spiradoline. Sodiumcurrents were evoked by a voltage step to 0 mV from a pre-pulse potential of -150 mV at0.7 Hz, 3.3 Hz, 6.6 Hz and 16 Hz. The bathing solution contained 50 pM spiradoline andcells were exposed to the drug for 2 mm before delivering pulses. The peak current(normalized to the first record) is shown plotted against the time at which it was evoked, foreach of the frequencies tested. In control solution no reduction in peak current was seenat frequencies up to 40 Hz (data not shown, see Figure lId for 25 Hz representation).Panel B shows the relationship between peak sodium current amplitude and thenumber of pulses applied. A train of depolarizing pulses of 10 msec duration were used.Sodium currents were evoked by depolarizing to 0 mV from a pre-pulse potential of -150mV at a stimulation frequency of 10 Hz. In control solution no noticeable reduction ofcurrent with successive pulses was seen (data not shown, see Figure lId for 25 Hzrepresentation). Spiradoline concentrations (15-50 pM) were added and trains given 2 mmafter application.112A1.00.84-.a)I-.h.0.400.20.0B1.00.84.40 0.6L.. I I I I I I10 12I I0 1 2 3 4 5time (s)15 pM150 pM5OpM0 2 4 6 8pulse number1133.1.6 Electrophysiological actions on Potassium currents3.1.6.1 Transient Outward and Sustained Delayed-Rectifier Potassium CurrentsIn addition to the above effects of spiradoline on sodium currents we examined theeffects on potassium currents in isolated myocytes (Figure 13). The concentration-dependent effect of bath applied spiradoline (50 and 150 pM) was examined on thetransient outward (‘to) and sustained outward plateau (K5US) potassium currents evoked bydepolarization to +50 my from a pre-pulse potential of -150 my for a duration of 300 msec.The potassium current amplitude was measured at the peak (approximately 5 msec afterevoking the outward current). This was obtained by subtraction of the current magnituderemaining at 300 msec from the peak amplitude. After 300 msec complete inactivation ofito occurs at which time the sustained outward current trace remains. Spiradoline produceda concentration-dependent block of Ksus which was reduced from a control current of 4.9to 1.5 and 0.5 nA after 50 and 150 pM spiradoline, respectively. The peak component ofito was reduced by spiradoline. In addition, spiradoline increased the rate of decay of ito.Construction of a line tangential to the decay curve of i0 allowed for an approximation ofthe drug effect on the rate of decay of this current. We assumed a monoexponentialdecay of evoked outward potassium current. In Figure 13 the control rate of decay of ito isapproximately 0.115 nA/msec while at a concentration of 50 pM the rate of decayincreases three-fold to 0.35 nAlmsec and then only marginally increases to 0.38 nAlmsecwith 150 pM spiradoline. The acceleration of inactivation induced by spiradoline was suchthat ito inactivation was almost complete within 50 msec as compared with 100 msec incontrol solution (Figure 13). Similar results were obtained in 4 additional cells.3.2 Studies with (±)PDI 17,302 and (+)PD123,497 (not previously reported)114Figure 13. Effects of spiradoline (50 and 150 pM) on depolarization-induced transientoutward (ito) and sustained outward plateau (KSUS) potassium currents. Currents wereelicited by depolarization to +50 mV from a pre-pulse potential of -150 mV for 300 msec.The concentrations of spiradoline were applied in the presence of 20 pM TTX and currentsevoked 2 mm later. The sustained outward current remained after complete inactivation ofito. The potassium current amplitude was measured at the peak and after 300 msec, atwhich time the sustained outward current trace was essentially flat. Note, however, thatthis sustained component remains essentially unchanged at the end of the pulse even withprolonged depolarization. The re-control current, after 2 mm wash, had a similar timecourse to control (data not shown for clarity). Similar results were obtained in 4 additionalcells.1151210C64-C1 4h.C,20-2300controlV50 pM150 pM0 50 100 150 200 250time (ms)1163.2.1 Electrophysiological actions on Sodium currents3.2.1.1 Concentration-response curvesBoth the racemic arylacetamide (±)PDII7,302 (Figure 14A) and its inactive Kenantiomer (+)PD123,497 (Figure 15A), produced concentration-dependent reductions inthe magnitude of sodium currents in isolated rat myocytes. Figure 14A shows dataobtained from an experiment in which sodium currents were evoked by a voltage step to 0mV from a pre-pulse potential of -150 mV. These voltage steps were given at 6 secintervals. For the evoked sodium currents of 10 msec duration a concentration-responsecurve was constructed. Either (±)PDI 17,302 or (+)PD123,497 was applied for 2 mm priorto generation of the current over the concentration range of 1-30 pM and 1.3-40 pM,respectively. At the highest concentrations applied both drugs abolished evoked sodiumcurrents. Re-control currents were obtained after 3 mm wash. In Figure 14B theseconcentration-response experiments were repeated in 2 additional cells and a similar dose-response relation was obtained for each cell. In these experiments an additional dose of100 pM (±)PDI 17,302 was examined. An estimate of the half-maximal sodium currentblock (EC50)expressed as % control, was determined to be 8.OpM using the best fit of theequation 1-y=1-1/[1+(KAIA)]. For the inactive enantiomer a similar curve resulted in anEC50 of 3.0 pM (concentration-response curve not shown). In the presence of I pMnaloxone there was no change in the concentration-response curve for (±)PDI 17,302(Figure 14B). Intracellular vs. extracellular locus of action.These studies determined the putative site on the sodium channel at whicharylacetamide compounds may exert channel blocking actions. In order to examine the117Figure 14 Effect of (±)PDI 17,302 on sodium currents in rat cardiac myocytes. Panel Ashows sodium currents evoked by a voltage-step from a pre-pulse potential of -150 mV to apotential of 0 mV. The voltage step was delivered at 6 sec intervals and (±) PDI 17,302was added to the bath solution at the concentrations indicated. The re-control (recont.)current is indistinguishable from the control. (±)PDII7,302 was added to the bath solutionfor 2 mm before evoking currents at any of the concentrations examined. Similar resultswere obtained in 2 additional cells.Panel B describes the concentration-response curve for the degree of blockade(expressed as a percent control) of the transient sodium current by (±)PDI 17,302. Sodiumcurrents were evoked as above. The degree of block is shown as a function of the log10concentration of (±)PD117,302. Data from 3 individual cells is plotted with the line of bestfit for the equation 1y1{1/[1+(Ka/[A])]} shown as a % of control. The estimated Ka fromthis equation is 8 pM for (±)PDI 17,302. The effects of I pM naloxone (open circles) werealso examined in the presence of spiradoline.118Acent. 1 3 10 30 recent.-10- IS-20--30-___lOrnsC)-40 --50-B. 100--__.________.__. .8060-40I20C.)I I•0 -i-ii...i I liii I I I 111110 100concentraUon (pM)119intracellular actions of these arylacetamides either (±)PDI 17,302 or (+)PD123,497 wereadded in the patching pipette at concentrations of 3 pM or 150 i.iM, respectively.Figure 15 shows the effects of 30 pM (±)PDII7,302 applied intracellularly. (±)PDI 17,302 showed no significant reduction in sodium currents after 5 mm of exposure ofdrug to the cell interior (as in current a of Figure 15). When a 3 pM concentration of (±)PDI 17,302 was bath applied the peak sodium current was reduced by 23% (from a controlof 45 nA control peak to 34 nA after drug exposure) as is seen in current trace b in Figure15. Two mm washing readily-reversed the block (see current trace c in Figure 15).Figure 16A shows the bath-applied concentration-dependent block of the sodiumcurrent. In Figure 16B the effects of 150 pM (+)PD123,497 were examined mntracellularly.Peak sodium currents were evoked every 3 sec beginning 4 mm after patch rupture andexposure of the cells interior to the pipette containing (+)PD123,497. After 4 mm ofexposure only a slight reduction (about 4 nA) in peak current amplitude was recorded.When the same concentration of (+)PD123,497 was added to the perfusate (marked by thehorizontal bar) a rapid and readily reversible inhibition of sodium current was observed.In another experiment we examined the effects of pH on sodium current block(Figure 16C). Current amplitudes in the pH=7.4 bath solution was approximately 48 nA(n=3 cells). In an acidic solution (pH 6.4) the amplitude was reduced to 42 nA (data notshown). When 13 pM (i-)PD123,497 was added to the bath solution at pH 7.4 sodiumcurrent was reduced by 83% (from a control current of 49 nA to 8 nA after drug application)within 1.5 mm. When the same concentration of (+)PD123,497 was added to the bathsolution at pH 6.4 some reversal in current block was seen. After 1.5 mm at the low pHapproximately 22% of the current (or 10 nA) recovered from block. When 13 pM(+)PD123,497 in the pH=7.4 bath solution was immediately re-applied to the cells, theevoked sodium current was blocked to a similar degree as at pH 7.4, previously.120Figure 15 The intracellular effectiveness of (±)PDII7,302 block of the sodium currentwas examined in rat cardiac myocytes. Sodium currents were evoked by a voltage-step to0 mV from a pre-pulse potential of -150 mV, repeated at 6 sec intervals. (±)PDII7,302was added to the pipette solution at a concentration of 50 pM. Recording was begun 5 mmafter patch rupture and whole-cell patch-clamp configuration was attained. The 10 msecsample current taken at (a) indicated that relatively little current block was produced by 5.5mm with the intracellular application of (±)PD1 17,302 (maintained for the duration of theexperiment as indicated by the dotted horizontal line). The current trace taken at time (b)indicated the reduction in peak current amplitude after the bath application of 3 pM (±)PDI 17,302. The time of application is seen as the solid horizontal bar. Current trace (c)is the re-control sodium current after 2.5 mm of wash.121-10.18____________-27 C.a0!_time (minutes)a b cI 10 ms122Figure 16 Effects of (+)PD123,497 on sodium currents. Panel A shows concentration-dependent block of the sodium current in rat cardiac myocytes by (÷)PD123,497 at theconcentrations (in pM) indicated. Sodium currents were evoked at 6 sec intervals.(+)PD123,497 was added to the bath solution for 2 mm before evoking currents at theconcentrations examined. Re-control current shows partial recovery after 2 mm washPanel B shows the effects of (+)PD123,497 (130pM) on sodium currents whenapplied to the inside of the cell via the pipette solution. Sodium currents were elicited 4 mmafter patch rupture. After 4 mm 130 pM (+)PD123,497 was applied for 30 sec via the bathperfusate (as indicated by horizontal bar).In panel C the effects of pH on block of sodium currrent by (+)PD123,497 areshown. A concentration of 13 pM (+)PD123497 was added to the cell bath solution at apH=7.4 and pH=6.4 and sodium currents evoked before and after drug in pH 7.4, a pH 6.4,followed by a return to pH 7.4. Time of drug application is shown by the solid horizontalbars.123control 1.3 4.0 13.0 40 recontrol0-.10-b.o-20 10mg-30-B3020.10 \ —00•—10 II4 5 6 7 8 S 10Time after patch rupture (mm)C60 I— 48 pH 7.4 pH L4 pH 7.4.38o 24-12 It.0I I0 1 2 3 4 5time (mm)1243.3 Summary of Results obtained in previous studies - manuscripts in AppendicesStudies for this thesis began with a characterization of the pharmacologicalproperties for the arylacetamide, U-50,488H in rat isolated perfused hearts, and inanaesthetized rats at concentrations or doses greater than those required to produce K-receptor mediated effects. Studies were conducted in the absence and presence of theopioid receptor antagonists naloxone (at either 1 pM or 8 pmollkg) or Mr2266 (8 pmol/kg).Neither antagonist significantly reduced the cardiovascular actions of U-50,488H in vivo orin vitro.U-50,488H dose-dependently reduced blood pressure and heart rate in vivo anddecreased beating rate and left-ventricular peak systolic pressure in vitro. Over aconcentration range in vitro of 1-30 pM and a dose-range in vivo of 0.5-32 pmol/kg, P-R,QRS and 0-aT intervals of the ECG were prolonged. Over the dose-range of 0.5-32pmol/kg, U-50,488H had a biphasic action on thresholds for induction of VF. Thresholdswere reduced at low doses (0.5-4 imol/kg) but increased at higher doses (8-32 pmol/kg).Naloxorie blocked the low dose effects. Both ERP, MFF and threshold pulse widthincreased with dose (Pugsley et al., 1992a).The antiarrhythmic actions of a low and high dose of U-50,488H were examined inpentobarbitone-anaesthetized rats subjected to occlusion of the left coronary artery. Ahigh dose (16 pmol/kg) reduced the incidence of VT from 100% in controls to 40% and VFfrom 67% in controls to 7%. The cardiovascular actions of U-50,488H were notantagonized by naloxone (8 pmol/kg). Naloxone alone may have (p>0.05) reducedarrhythmia incidence but to a much lesser extent than U-50,488H. The low dose of U50,488H (0.2 pmol/kg), in the absence or presence of naloxone, did not affect arrhythmias(Pugsley et al., 1992b).Another series of experiments examined the actions of U-50,488H on voltageactivated sodium and potassium currents in isolated rat cardiac myocytes. U-50,488H125produced a concentration-dependent block of sodium current at an ED50 of 15 pM. Athigher concentrations, (50 pM) blockade of the plateau potassium current (KSUS) and anincrease in the rate of decay of the transient outward potassium current (ito) was observed.At the EC50 for sodium channel blockade, U-50,488H produced a 15 mV hyperpolarizingshift in the inactivation curve (h) for the sodium current. No changes in voltagedependence for activation were seen. U-50,488H had no effect on the voltagedependence of inactivation or activation of the potassium currents studied. The ic receptorantagonist Mr2266 (1 pM) did not influence sodium or potassium currents and did notchange the current blocking properties of U-50,488H (Pugsley et al., 1994).In an attempt to resolve the mechanisms by which ic agonists may be antiarrhythmicwe examined the effects of (-)PD129,290, a potent ic agonist and its inactive enantiomer,(+)PD129,289. It was hoped that exploration of the pharmacological and antiarrhythmicprofile of the two enantiomers may elucidate putative mechanism(s) by which structurallysimilar, but pharmacologically different, arylacetamide drugs exert antiarrhythmic actions.(-)PD129,290 and its corresponding (+) enantiomer, PD129,289, were studied in ratisolated hearts and in intact rats for their cardiovascular and antiarrhythmic actions. Usingthe whole-cell mode of the patch-clamp, the effects of (-) PD129,290 were examined onthe sodium channel of cardiac myocytes.In isolated rat hearts both arylacetamides, at 2-16 pM, dose-dependently reducedpeak left ventricle systolic pressure, and heart rate and prolonged the P-R interval andQRS width of the EGG.In intact rats 1-32 pmol/kg of both enantiomers reduced heart rate and bloodpressure. The RSh, P-R, QRS and Q-aT intervals of the ECG were prolonged. Inelectrical stimulation studies thresholds for capture and VF were dose-dependentlyincreased by both enantiomers. However, (-)PD129,290, the K agonist, reduced thresholdsat low doses (0.5-4 pmol/kg). The reduction in thresholds was blocked by naloxone (8pmol/kg). Both enantiomers increased ERP. Naloxone did not affect any of the actions of126(-)PD129,290. When examined in rats subject to coronary artery occlusion, bothenantiomers (at 2 and 8 pmol/kg) effectively reduced arrhythmia incidence. When (-)PD129,290 was examined in cardiac myocytes, a concentration of 20 pM produced a half-maximal block of the sodium current in the absence and presence of 1 pM naloxone. Noeffect on the voltage-dependence of activation or inactivation on this current was seen withthis compound (Pugsley et al., 1993a).Our studies show that K receptor agonists and related compounds possessantiarrhythmic properties in the rat associated with blockade of cardiac sodium andpotassium currents. Other studies suggest that drugs with mixed channel blockingproperties may have potential therapeutic benefits (Hondeghem and Katzung, 1980) andthe arylacetamides may possess these properties. A comparison was made between the Kagonist drugs, (-)PD129,290 and (±)PDII7,302 and the inactive enantiomer of (-)PD129,290, (+)PD129,289 by using D25 values (potency measures). D25 values wereestimated from dose-response curves for these compounds over the dose range 0.1-32pmol/kg/min. D25 values for drug effects on blood pressure, heart rate and ECG measuresare outlined in Table I of Appendix 5. The K agonist compounds reduced blood pressureat a D25 of 0.5 pmol/kg/min while for the inactive enantiomer, (+)PD 129,289, it was 8pmol/kg/min. In Table 2, Appendix 5, the drugs examined did show some differentialeffects on sodium compared to potassium channels using the D25 value as a measure ofcurrent selectivity. At lower doses, the K agonists affected sodium channels more thanpotassium channels. Thus the patterns of drug response to ECG, blood pressure, heartrate and electrical stimulation indicate that these drugs are antiarrhythmic by virtue ofcardiac ion channel blockade. The measure of drug sensitivity, D25, may be useful as amethod by which to quantify and validate the examination of ion channel sensitivity to drugblockade.127Table IV The studies conducted in the appendicised papers have been published invarious pharmacology Journals. The Summary Results Table indicates which of thearylacetamide compounds were studied in which papers. For the in vivo studies whichwere conducted the following abbreviations were used: C.V. = cardiovascular studiescomprising heart rate, blood pressure and ECG (P-R, QRS, Q-aT and RSh) changes; E.S.= electrical stimulation studies where i, tt, VFt, ERP and MFF were examined and CD. =coronary occlusion studies. For the in vitro studies, l.H. indicates the studies which wereconducted in rat isolated Langendorff hearts for effects on heart rate, contractility and ECGmeasures. P.C. indicates those studies which were conducted in isolated ventricularmyocytes for effects on sodium (Naj current amplitudes, kinetics and use-dependence orpotassium (Kj for effects on i0 and KSUS blocking properties. Studies were conducted inthe absence and presence of the K opioid receptor antagonists naloxone or Mr 2266.TableIV-SummaryofResultswhichcanbefoundintheAppendices- L\)AppendixDrugInvivoFigureTableAntagonistInvitroFigureAntagonistC.V.E.S.C.O.l.H.P.C.NaKU-50,488H3a,b1naloxonela,bnaloxoneMr22662a,bj4a,bnaloxonenaloxone5a,b2U-50,488HqInaloxoneI2,3naloxone3U-50,488Hq1,2,3,Mr22664,5‘.17,8Mr22664PD129,289q21la,bq3I2PD129,290q21naloxonela,bnaloxone•413naloxone..44,5,6naloxone-42naloxone5PDII7,302ITextq2PDI29,289qIq2PDI29,2901TextJ21294 DiscussionThe major focus of this thesis was to examine the antiarrhythmic actions of a seriesof arylacetamide K agonists and related inactive enantiomers. The specific hypothesisbeing tested was that the antiarrhythmic actions of arylacetamide K agonists is not relatedto actions on opioid (particularly ic) receptors. The results clearly indicated that theantiarrhythmic actions of these compounds was independent of actions on opioid receptorsbut dependent upon channel blocking actions, particularly the block of sodium andpotassium channels. In addition to testing this hypothesis these studies also offeredinsights into a number of related topics. The following discussion will summarize theresults of these studies and discuss related topics. This will be accomplished byconsidering the following questions:1) What cardiovascular actions were common to arylacetamide ic agonists andrelated compounds?2) Were these cardiovascular actions, particularly those at high doses orconcentrations, opioid receptor dependent, or were they dependent on ion channelblockade?3) What is the nature of the channel blocking actions of the arylacetamidecompounds?4) Do these channel blocking actions account for the antiarrhythmic actions ofthese compounds?5) Can any projections be made from these studies regarding the value ofarylacetamides in the treatment of arrhythmias and study of ion channel blocking drugs?The discussion will be concerned with the results obtained with spiradoline, (±)PDI 17,302 and its inactive enantiomer, (+)PD123,497; all reported for the first time in this130thesis and how they relate to the previously published manuscripts concerning U-50,488H(Pugsley et al., 1992a, 1992b, 1994), (-)PD129,290, and its inactive (+) enantiomer,(+)PD129,289 (Pugsley et al., 1993, 1995).4.1 Cardiovascular actions of arylacetamides.All of the compounds tested lowered mean arterial blood pressure, heart rate,widened the P-R, QRS, Q-aT intervals and increased RSh measures of the ECG.In the foregoing, spiradoline (U-62,066E) was studied for effects on haemodynamicand ECG responses in anaesthetized rats. The Appendix section contains reports ofstudies with U-50,488H (Appendix 1-3), (-)PD129,290 and its inactive enantiomer,(-i-)PD129,289 (Appendix 4) and (±)PDI 17,302 (Appendix 5). The dose-response studiesconducted for the arylacetamides provided us with important pharmacological data. Itenabled us to select appropriate pharmacological doses for later antiarrhythmic studies andprovided toxicological information.4.1.1 Blood PressureThe arylacetamides reduced mean arterial blood pressure in rats. Studiesconducted in dogs by Hall et al. (1988) showed that U-50,488H and spiradoline dose-dependently reduced blood pressure in this species.The depressor actions of arylacetamides are consistent with those seen with classIa antiarrhythmic agents such as quinidine. Quinidine moderately reduces blood pressureas a result of a combined peripheral vasodilation and direct negative inotropic action on theheart (Block and Winkle, 1983; Legrand and Collignon, 1985). Class lb agents, includinglidocaine and mexiletine, are better tolerated and they produce no significant changes inblood pressure at therapeutic doses (Legand and Collignon, 1985). Class Ic agents, such131as flecainide, appear to be also well-tolerated haemodynamically; however, they exertmoderate negative inotropic effects and may potentially worsen ventricular function incompromised myocardial tissue such as occurs with congestive heart failure. Severalstudies suggest that K agonists depress contraction in rat tail veins (Illes et al., 1987) andporcine coronary circumflex arteries (Harasawa et al., 1991). It is suggested that thisaction on smooth muscle cells, since it is not inhibited by naloxone, may be due to directinhibition of voltage-gated channels. The resulting peripheral vasodilation which mayoccur, in addition to the direct myocardial depression may be the mechanism by which bothheart rate and blood pressure are reduced. Further studies are required to explore theseactions of the arylacetamides.4.1.2 Heart RateHeart rate was also reduced in a dose-dependent manner by all of thearylacetamides examined. The bradycardia produced by spiradoline (Figure 4B) wassimilar to that seen with IJ-50,488H (Figure 3a, Appendix 1). Isolated heart studies suggestthat the bradycardia produced by these compounds involves a direct action on themyocardium which may or may not be related to the depression or contractility (see below).Over a similar dose-range (0.5-32 pmol/kg/min) the arylacetamides produced adose-dependent decrease in cardiac conduction represented by an increase in the P-Rinterval and QRS width of the ECG. As well, the Q-aT interval, a measure ofrefractoriness, was also prolonged in vivo. These measures provide a simple, indirectmeans by which to examine drug action on cardiac ion channels (Cheung et al., 1993).4.1.3 ECG132In the course of studying the antiarrhythmic actions of many different drugs in therat a measure of sodium channel blockade (RSh) was developed for this species (Penz etal., 1992). The conventional measures of P-R interval and QRS width prolongation arelimited in sensitivity and can be supplemented by RSh. It is the most sensitive measure forsodium channel blockade in vivo in the rat (Pugsley et al., 1995). Spiradoline (Figure 6A)as wefl as (±)PDII7,302, (+)PD129,289 and (-)PD129,290 produce changes in RSh atdoses approximately 7.5-20 times less than those widening the QRS width and 3 times lessthan those increasing the P-R interval (See Table 1, Appendix 5).In addition to producing signs of sodium channel blockade the arylacetamidesprolong the Q-aT index which is related to an increase in the refractory period of ventricularcells. Spiradoline dose-dependently prolonged the Q-aT interval (Figure 6B). As well, U50,488H (Figure 3B, Appendix 1), and both (-)PD129,290 and its inactive enantiomer,(+)PD129,289 prolonged the Q-aT interval (Figure 2D, Appendix 3).Consistent with the results observed in vivo, spiradoline and the otherarylacetamides examined in isolated hearts reduced contractility, and heart rate andprolonged the P-R and QRS intervals of the ECG. The isolated hearts provide a means bywhich to directly assess the effects of drugs on myocardial contractility and electricalactivity, free of blood borne constituents and autonomic nervous control (Broadley, 1979).Studies with arylacetamides suggest sodium channel blockade similar to class Iantiarrhythmic agents and TTX (Howard et aI., 1991; Abraham et al., 1989). Ourexperiments showed that spiradoline (Figure 2A, B, 3A, B), U-50,488H (Figure Ia, Ib,Appendix 1), (i-)PD129,289 and (-)PD129,290 (Figure Ia and Ib, Appendix 4) all producedthe described contractility and ECG effects expected of class I antiarrhythmic agents. TheP-R interval prolongation which occurs is unlikely to be due to calcium channel blockade inthese hearts (Boiling et al., 1985).In conclusion, all arylacetamide compounds produced a similar profile ofcardiovascular actions.1334.2 The effects of arylacetamides - opioid receptor dependent mechanisms or ionchannel blockade in the heart?The cardiovascular profile of these compounds was determined in the absence andpresence of opioid antagonists, and with inactive K opioid receptor enantiomers, whenavailable, It is difficult to examine the profile of opioid activity in the cardiovascular systemat relevant opioid doses because of the complex interaction between opioid receptors andregulation of hormones such as those of the endocrine system (Holaday, 1983). At thesupra-analgesic doses used in these studies these actions may not be important (Pugsleyet al., 1992a). The doses of the opioid antagonists used in these studies, to inhibit drugeffects on the opioid system, were 50-100 times the pA2 for antagonism at the K receptor(Martin, 1984). This ensured examination of non-opioid properties of arylacetamides.The involvement of opioid receptor subtypes, Kla, 1(lb and the U-50,488H-insensitive 1(2 and 1(3 sites, and cardiovascular function is not known. Selective agonistsand antagonists for these receptors have yet to be developed. Therefore the involvementof these subtypes in my results will not be discussed.Hall et al. (1988) examined the cardiovascular actions of spiradoline and U-50,488Hin dogs. These compounds produced a dose-related decrease in blood pressure whichwas abolished by naloxone (8 pmol/kg) pre-treatment and thus could be considered to bedue to kappa agonism. In our studies the doses for all arylacetamides were supraanalgesic and furthermore naloxone did not abolish the effects of the active enantiomers(Pugsley et al., 1992a, 1992b, 1993a, 1994). While the arylacetamide compounds usedhave low affinity for p receptors, and thus are selective, they were administered atapproximately 1000 times the minimal dose for K agonism. As a result spontaneousrespiration ceased in animals at only the highest doses of the active K receptor agonistarylacetamides. Respiratory depression resulted from a non-specific opioid receptormediated depression in the CNS (Martin, 1984).134The actions of spiradoline (Figure 5 A,B and 6B), U-50,488H (Figure 3b, Appendix1) as well as (+)PD129,289 and (-)PD129,290 (Figure 2b, 2c, 2d, Appendix 4) on the P-R,QRS and Q-aT measures were not significantly affected by naloxone pre-treatment ateither the 2 or Bpmol/kg/min dose. It is generally accepted that increases in the QRS widthof the ECG reflect depression of phase 0 sodium currents and reduced ventricularconduction velocity (see review by Nattel, 1991). Thus, the sodium channel blockadeproduced by class I agents results in a widened QRS complex, although the degree towhich this occurs depends upon heart rate and the subclass of class I being administered(Vaughan-Williams, 1984a; Harrison, 1985). Since all arylacetamides examined widenedthe QRS complex sodium channel blockade appeared to be a common action of thesecompounds. P-R interval prolongation can be produced by a variety of different drugsincluding calcium channel antagonists and class I antiarrhythmics. Prolongation can resultfrom depressed conduction through atrial tissue due to sodium channel blockade ordepression of slow inward A-V node calcium currents (Walker and Chia, 1989). Ourstudies suggest the former, rather than the latter, occurs. This reflects, in part, the fact thatin small animal hearts calcium channel blockade does not play the primary role in P-Rinterval prolongation and rather that sodium channel blockade dominates (Bofting et al.,1985).Spiradoline (Figure 6B) dose-dependently prolongs refractoriness, especially athigh doses, indicating possible potassium channel blockade. Since the major repolarizingcurrent in the rat is the transient outward potassium current, this is the channel most likelyto be affected by these drugs (Josephson et al., 1984; Beatch et al., 1991). In the rat wehave shown that, for a similar degree of widening (25%) in the Q-aT interval, the dosesrequired of (+)PD129,289, (±)PDII7,302 or (-)PD129,290 are 6-25 times greater thanthose which produce sodium channel blockade (as assessed by a 25% increase in RSh)(see Table 1, Appendix 5).135Thus the patterns of haemodynamic and ECG changes which occur in vivo indicatethat spiradoline and the other arylacetamides examined produce their actions by virtue ofcardiac sodium and potassium ion channel blockade (Pugsley et al., 1992a, 1993a, 1995).In order to determine whether or not these mixed actions occur by a direct action on theheart we conducted studies in isolated Langendorff perfused hearts.Spiradoline and the other arylacetamides examined produced a naloxone-resistantdepression in cardiac contractility, heart rate, and affected the P-R and QRS intervals ofthe ECG. As with the results in vivo, these measures indicate sodium channel blockadewhereby the reduction in contractility may be due to inhibition of the fast sodium currentresponsible for depolarization.In addition to transient inward sodium channels it has been postulated, andconfirmed, that there are additional cardiac sodium channels in heart tissue (Coraboeuf etal., 1979; Carmeleit, 1993; Fozzard et al., 1985; Saint et al., 1992; Ju et al., 1992, 1995).At concentrations of TTX which do not inhibit the inward sodium current, these sodiumchannels are blocked. The first is the pacemaker or f current which has a small amplitudeand activates during hyperpolarization at voltages around -40 and -110 mV correspondingto the diastolic range of cardiac cell depolarizations (DiFrancesco, 1987). It is postulatedthat this “window current” may be electrogenically produced by membrane pumps orexchangers (Fozzard et al., 1985) and is actually a mixed current, carried by sodium andpotassium (DiFrancesco, 1981; Ho et al., 1991). Another type of sodium current is thepersistent sodium current which is found in rat ventricle and is also activated over thediastolic range of cell depolarization (Ju et al., 1992; Saint et al., 1992). This current isthought to contribute to the pacemaker current (Ju et al., 1995). This persistent sodiumcurrent is blocked by low doses of quinidine and lidocaine and may be involved inarrhythmogenesis (Ju et al., 1992). Therefore, in addition to the sodium current which isblocked by arylacetamide compounds, these other additional sodium currents may also beblocked. This possibility has not been explored.136Honerjager et al. (1986) examined the negative inotropic actions of several class Iantiarrhythmic drugs, including TTX, in an attempt to relate this drug action to sodiumchannel blockade. The study compared drug concentrations which produced half-maximalreductions in Vmax to those producing the same decrease in contractility. The resultssuggest that some drugs (quinidine, mexiletine) were more potent sodium channel blockersthan negative inotropic agents. Therefore properties inherent to the drug, in addition tosodium channel blockade, are involved in reducing contractility. The arylacetamidesreduced contractility at the doses which prolonged P-R interval indicating that negativeinotropism occurs over the same dose range that these compounds block sodiumchannels.It is difficult to measure the action of arylacetamides on repolarizing potassiumcurrents in rat hearts due to the inherent variability in the Q-aT interval itself and itsrelationship to the isoelectric line of the ECG (Driscoll, 1981). Thus this measure cannot beused to compare drug effects in vitro to in vivo responses. The isolated heart resultsstrongly support the suggestion that the actions of spiradoline, U-50,488H and otherarylacetamides can be ascribed to effects unrelated to ic agonism. It would be reasonableto suggest that sodium, and to a limited degree at high doses (concentrations), potassiumchannel blockade occurs with these compounds. This would account for thehaemodynamic, contractile and EGG responses observed in vivo and in vitro.4.3 An electrophysiological basis for the channel blockade seen witharylacetamides in the heartElectrophysiological studies conducted in isolated cardiac ventricular myocytesshowed unequivocally that spiradoline, (-)PD129,290 and U-50,488H blocks sodium andpotassium currents and that such an action is not blocked by naloxone or Mr2266.Blockade occurs at concentrations which produce ECG effects in rat isolated hearts and137which are consistent with doses administered to intact rats to produce similar ECG effects.These doses produced patterns of changes in electrical thresholds and refractorinessentirely in keeping with the above.The arylacetamides produced a concentration-dependent block of sodium currentsin single cells. For spiradoline (Figure 9A,B), (±)PDII7,302 (Figure 14 A,B), (+)PD123,497(Figure 15A), U-50,488H (Figure 1 and 2, Appendix 3), and (-)PD129,290 (Figure 4,Appendix 4) the EC50 blocking concentrations were 66, 8, 3, 15 and 2OpM, respectively.The effects of spiradoline, U-50,488H, and (-)PD129,290 were examined on the current-voltage relationship for sodium currents revealing that these compounds only blocked thechannel and did not alter the voltage-dependence of activation (see Figure IOA; Figure 4,Appendix 3 and Figure 4, Appendix 4, respectively). As well the compounds produced,although to varying degrees, a shift in the voltage-dependence of inactivation (h) for thesodium channel. For spiradoline (Figure lOB), U-50,488H (Figure 3, Appendix 3) and (-)PD129,290 (Figure 6, Appendix 4) the relative hyperpolarizing shifts were -20, -15 and -7mV, respectively, at the approximate EC50 concentrations for these compounds. Severalof the arylacetamide compounds showed a tonic block component in addition to afrequency or use-dependent block which occurred at frequencies expected to beencountered during VT or VF in the rat. The voltage and frequency-dependent blockade ofsodium currents by these arylacetamide compounds is best explained by the ModulatedReceptor Hypothesis (Hondeghem and Katzung, 1977, 1984).When sodium channels are maintained at membrane potentials more negative to-lOOmV all of the channels, according to the Hodgkin and Huxley (1952) are in the R orrested state. When depolarization occurs, the channels open from the rested (closed)state, however, when the membrane potential becomes more positive than -5OmV thesodium channels become inactivated, as in the following schematic:138I - ID (j Membrane PotentialsA... ADR () Membrane PotentialsAs discussed earlier, Hille (1977) and Hondeghem and Katzung (1977) formalizedthe Modulated Receptor Hypothesis shown above where R A I indicate the drug freechannel states, resting, active and inactive, while RD AD ID indicate drug-associatedchannel states. The model describes the fraction of channels in each state and canaccount qualitatively and quantitatively for channel behaviour in the absence and presenceof drug (Hondeghem, 1989, 1990). Therefore drug affinity for the binding to the sodiumchannel is modulated by channel state.Studies have shown that useful antiarrhythmic drugs with class I properties have alow affinity for resting channels and high affinity for open or inactive channels (Chen et al.,1975; Hondeghem and Katzung, 1977; Hondeghem and Katzung, 1980; Courtney, 1980;Carmeleit et al., 1989; Hondeghem, 1991). During diastole, or the R state of the channel,drugs tend to unbind from the binding site and the level of block is reduced. However,drugs such as lidocaine and quinidine show a differential high affinity (lidocaine>quinidine)for the open state of the channel. This has implications for the tonic and use-dependentblocking properties of each drug. High affinity, open (active) channel state blockingantiarrhythmic drugs would, consequently, have the ability to increase the degree of blockattained with each action potential (or evoked sodium current), while high affinity inactivestate blockers would continuously develop block throughout the duration of the actionpotential until a steady-state is reached (Hondeghem, 1990). Since each drug selectivelydepresses various aspects of the action potential they may have differential effectiveness139against arrhythmias. However, as Hondeghem (1987) has proposed it is irrelevant whetherblock develops during the upstroke (active) or plateau (inactive) phases of the actionpotential but rather the amount of block which develops during each evoked actionpotential be quantified as it is this property which is most important in determining drugeffectiveness.According to this hypothesis, the arylacetamide compounds possess similar actionsto drugs such as quinidine or even lidocaine. This is revealed through examination ofsodium channel activation/inactivation properties. Studies by Lee et al. (1981) withquinidine and lidocaine in isolated rat myocytes showed that neither of these drugsproduced a change in the I-V relationship. The arylacetamides, at concentrations whichmarkedly reduce peak available sodium current, had no effect either.More interesting results were observed when inactivation kinetics were examined inthe presence of the arylacetamides. Drug associated (non-conducting) channels produceshifts in the voltage-dependence of inactivation (Hille, 1984; Hondeghem and Katzung,1984). Spiradoline and related compounds produced such a shift of the inactivation curve(hc,) for the sodium current in a hyperpolarizing direction indicating that the drug maypreferentially bind to the inactive state of the sodium channel (Hille, 1984). Unlike otherclass I agents, which produce a dose-dependent shift and reduction in peak current at verynegative pre-pulse potentials and low rates of stimulation (Bean et aL, 1983), spiradolineproduced a large block and shift in the inactivation curve which was not dependent uponconcentration (since both 50 and 150 pM concentrations produced similar changes in halfmaximal inactivation). This action is indicative of a tonic component of block, and apotential affinity for the resting state of the channel.Estimates of the resting channel affinity for antiarrhythmic drugs are determined forthe MRH by using global fifting equations (which consider the rate of kinetics of drugactions simultaneously for all three states of the sodium channel) (Hondeghem andKatzung, 1984). However these estimates are not accurate since the calculated values for140channel state affinities must be obtained from drug effects which occur in the pMconcentration range (Hondeghem and Katzung, 1977, 1984). As a result, the values forresting channel affinity for antiarrhythmic drugs are large (Kdr>O.l M) reflecting theinaccuracy of the calculated values (Hondeghem and Katzung, 1984). No attempt wasmade to characterize the rate constants for arylacetamide interaction using global fittingequations for the various channel states but rather qualitatively describe the observedeffects of these compounds.In a qualitatively similar manner to that seen with quinidine (Lee et al., 1981;Snyders and Hondeghem, 1990) and lidocaine (Bennett et al., 1988) our results showedthat the arylacetamide compounds, spiradoline (Figure 10, 11, 1 2A, B) and U-50,488H(Figure 5a,b,c, Appendix 3) block sodium channels in both a tonic and use-dependentmanner. Use-dependence of spiradoline was slow to develop and reminiscent of thatexhibited by quinidine (Hondeghem and Matsubara, 1988). Spiradoline (Figure 12A, B)and U-50,488H (Figure 5 a,b,c, Appendix 3), at concentrations producing class Ia effects,produced only a small tonic reduction in sodium current. Since this is only a small fractionof the total block produced at these concentrations, this indicates a low affinity for this stateof the channel. If the block were similar to that observed with the higher doses (150 pM)this may lead one to assume that the drug has a high affinity for the resting state of thesodium channel. This form of tonic block, according to Clarkson et al. (1988), may not bean accurate reflection of closed or rest-state drug binding. These studies suggest that withstrong depolarizing pulses, and when the drug is at a high concentration, tonic block mayresult and that this is due to open channel block. Even when a small fraction of channelsare inactivated the drug will be trapped in these channels in the ID (inactivated drugassociated) state and produce tonic block (Hondeghem, 1987). Tonic block is composedof two components, an early fast open channel phase following membrane depolarizationand a later slow phase which is distinctly associated with sodium channel inactivation(Matsubara et al., 1989; Clarkson et al., 1988). These components in turn depend upon141drug concentration and action potential duration (Courtney, 1975; Mason et al., 1984).With an increase in drug concentration an increase in tonic block results. Both spiradoline(Figure 12A, B) and U-50,488H (Figure 5a, Appendix 3) produce tonic block in aconcentration-dependent manner similar to lidocaine (Matsubara et al., 1987) and quinidine(Snyders and Hondeghem, 1990). When drug effects were examined on the cells atseveral concentrations which span the range of antiarrhythmic doses (low therapeuticconcentrations) the increase in tonic block evident with an increase in drug concentrationwas small at low therapeutic doses, but approached equal blocking activity at highconcentrations when compared to the use-dependent block which quickly ensued. Theantiarrhythmic doses which were effective against ventricular fibrillation in occlusion studieswould approximately be equivalent to 25 pM for spiradoline and 60 pM for U-50,488H inour patch-clamp myocyte studies. If we examine the concentration-response curves(Figures 9A and B) for spiradoline and Figure 2, Appendix 3 for U-50,488H thisconcentration corresponds to an approximate reduction in sodium current by 20 and 75%,respectively. Thus, at therapeutic doses of the drug tonic block is minimal and hence thedrugs would have a low affinity for the resting state of the channels.The use-dependent block of the cardiac sodium channel consists of an additionalreduction in 1Na which develops in a frequency-dependent manner with trains ofdepolarizing pulses (Strichartz, 1977; Courtney et al., 1978). The predicted recovery-timeconstant (t112) for channel block was calculated using the molecular weight, PKa andlipophilicity of spiradoline according to an equation developed by Courtney (1980). Thepredicted ‘r112 for spiradoline was 2.3 sec based on lipophilicity (ethanol:water partitioncoefficient = 1.3 at pH = 7.4), pKa = 7.8 and molecular weight (411 Da). The predictedvalue for spiradoline is similar to a range of values calculated by Courtney forantiarrhythmic agents with slow kinetics (such as procainamide, quinidine and imipramine)using the octanol:water partition coefficient at pH = 7.3 (Courtney, 1980).142The patch-clamp results qualitatively suggest that spiradoline may have a greateraffinity for the active (open) or inactive (closed) form of the channel than for rested (closed)channels. Since recovery from block is calculated to be slow (2.3secs for spiradoline and2.4 sec for U-50,488H) it is predicted that if these compounds interact primarily with eitherthe open or inactive state of the channel, then according to the Modulated ReceptorHypothesis, recovery from the drug-inactivated (ID) channel state will be slow (Hondeghemand Katzung, 1984). Studies for both spiradoline and U-50,488H suggest that these drugsinteract with the sodium channel slowly since it takes between 7 and 30 pulses before asteady-state level of block is produced by either compound. Quinidine displays theseproperties (Hondeghem & Katzung, 1980). In vivo confirmation of the patch-clamp resultsshow that, at the high heart rates associated with ventricular fibrillation and tachycardia,spiradoline and U-50,488H effectively reduce these arrhythmias. Thus, the predicted slowkinetics of drug block and low affinity for the resting state of the sodium channel suggestthat the antiarrhythmic properties of these drugs may be due to accumulated channel blockat high rates. This use-dependent action is an event similar to that seen with quinidine,lidocaine and propafenone (Kohlhardt and Siefert, 1983; Hondeghem & Matsubara, 1988;Snyders and Hondeghem, 1990). These experiments cannot confirm whether open vs.inactive channel block develops with each action potential only that arylacetamidecompounds block cardiac ion channels. The accumulation of block occurs at high rateswhich may be important for antiarrhythmic activity (Hondeghem, 1987).Hille (1977) postulated that hydrophilic molecules which interact with the bindingsite on the sodium channel are confined to a hydrophilic pore, formed by the proteincomprising the sodium channel, pathway. A second pathway which was suggested wasthat hydrophobic molecules would diffuse across the lipid bilayer cell membrane to thebinding site. It is thought that local anaesthetics and class I antiarrhythmic drugs interact ata site on the cytoplasmic side of the sodium channel in close proximity to the inactivationgate (Hille, 1977; Hondeghem and Katzung, 1977; Courtney, 1980; Hondeghem 1987;143Narahashi et al., 1970). however, studies conducted by Alpert et al. (1989), Baumgartenet al. (1991) and Sheldon and Thakore (1993) suggest that in addition to this intracellularbinding site, there may be an extracellular binding site.Studies with lidocaine and its permanently charged analogue, QX-314, suggest thatthe charged lidocaine analogue blocks sodium current when applied outside as well asinside in cardiac myocytes (Alpert et al., 1989). The permanently charged quaternaryamine, QX-222, was also shown to bind to an external site associated with the cardiacsodium channel (Sheldon and Thakore, 1993). These studies, in addition to highlightingthe differences between local anaesthetic and antiarrhythmic drug actions on cardiac andneuronal sodium channels, provide the basis for the putative site of cardiac ion channelblockade by the arylacetamides we examined.The studies conducted with (±)PDII7,302 and its inactive enantiomer(+)PD123,497 suggest that these compounds block the cardiac sodium channel from anextracellular site. Our studies showed that the compounds did not effectively block theevoked sodium current when applied inside the pipette. The low leak current and high sealresistence attained with each whole-cell patch-clamp, would suggest that the drug, whenplaced in the pipette, could not leak out through this interface. The size of the patch-pipette is of some concern when examining drug effects applied by this route. However,the pore size of our patch-clamp electrodes (approximately 2-4 pm) and resistences ofbetween 5-10 M2 should provide sufficient intracellular perfusion of the pipette contentswith the compounds examined. Thus the observed results are probably not a result ofexperimental artifacts.The most convincing evidence of an external site of block by the arylacetamides isthe time to onset of and recovery from block when cells were exposed to the drug. Thedrug perfusion cannula used in our experiments ensured that there was a minimal delay intime to drug exposure (Saint et al., 1991). Figure 15A shows that half-maximal blockoccured in less than 6 sec, and that this time was not concentration dependent. The144lipophilicity of these arylacetamide compounds is less than that of quinidine (log P=3.6)and imipramine (4.7) (Courtney, 1980) and share similar off-rate kinetics. Patch-clampstudies suggest that these compounds readily interact with the A or I state of the sodiumchannel using either a hydrophilic pathway to gain access to an intracellular binding site orby simply binding to an external site. It follows that hydrophilic agents gain access to thecell interior when the channel opens during depolarization. However, we propose that therapid rate of binding which occurs was due to block at an external site, eliminating time-dependent diffusion of the drug to the binding site. The external binding site by whichthese compounds exert their channel blocking effects is pH-dependent and may beimportant when we examine changes during ischaemia.In the acidic conditions associated with ischaemia, arylacetamide compounds withpredominantly PKa values >7.0 become charged and hence are confined to the hydrophilicpathway for their drug channel blocking action. Hondeghem et al. (1974) and Grant et al.(1980) showed that quinidine, procainamide and other class I drugs effectively suppressischaemic arrhythmias under acidic conditions due to a slowed diastolic recovery fromblock. Acidic conditions have significant effects of sodium channel properties such asslowing the onset of and reducing peak sodium current amplitude, producing ahyperpolarizing shift in inactivation and slow depolarizing shifts in activation kinetics(Woodhull, 1973 ; Watson and Gold, 1995). Barber et al. (1991) examined the effects ofpH changes on amitryptaline and diphenylhydantoin. Their studies suggested two use-dependent binding sites for these drugs, whereby one site was sensitive to external pH andthe other was insensitive to external pH. The pH insensitive site was postulated to belocated inside the cell since changing the intracellular pH does not change the channelblocking properties of the drug. The pH-sensitive site was located at a more “superficial”site which is accessible to external protons (Barber et al., 1991).Preliminary studies conducted with (±)PD1 17,302 and its inactive enantiomer,(+)PD123,497, suggest that a pH-dependent blockade of ion channels occurs and this may145apply to arylacetamides in general. Under acidic conditions (pH = 6.4) there is a reductionin block of the evoked cardiac sodium channel with these compounds. This agrees withthe data for the existence of an external binding site (Alpert et al., 1989; Barber et al.,1991). It also agrees well with the proposal by Grant et al. (1980) whereby a reduction inpH (acidification) adversely affects drug hydrophilicity (which is dependent upon the pKa ofthe drug) such that when the drug is in the charged form it has a reduced affinity for theextracellular binding site. Drug block could be relieved by such a mechanism. However, itmay be that drug block is not relieved under acidic conditions, but only that the availabilityof sodium channels for block is reduced. Many models have been proposed for thereduction in peak sodium conductance under acidic conditions. The most plausible waspostulated by Drouin and Neumcke (1974) and later elaborated upon by Zhang andSiegelbaum (1991). This theory suggests that protons titrate the negative external surfacecharge of the sodium channel protein reducing the local sodium concentration near thepore entrance and reduce the magnitude of the resulting current. This paradoxically mayresult in a reduction in channel block or a removal of block by protons.We do not know whether the proposed binding site is the same extracellular pH-sensitive binding site as discussed by Barber et al. (1991) or for TTX (Narahashi, 1970;Duff et al., 1988). These preliminary studies with arylacetamides do not address this issue,but merely suggest that perhaps these compounds bind to an extracellular locus and mayprovide the basis for a fundamentally novel mechanism for sodium channel blockade andpossibly antiarrhythmic properties.Spiradoline (Figure 13) and U-50,488H (Figure 6, Appendix 3) reduced at least tworepolarizing potassium currents found in the rat heart. The dominant transient outward (ito)current responsible for the early phase of repolarization of the rat action potential(Josephson et al., 1984), and the sustained outward plateau current (Wang et al., 1993)which we ascribe to a delayed rectifier plateau potassium current (iKSUS) are similarlyreduced by arylacetamides. Spiradoline (Figure 13) and U-50,488H (Figure 6, Appendix 3)146increased the rate of decay of to and markedly reduced the sustained plateau componentin a concentration-dependent manner. When the actions of U-50,488H were examined indetail with respect to their effects on on‘to and currents neither the voltage-dependence of activation or inactivation of ito (Figure 7, 8a, Appendix 3) nor voltage-dependence of1Ksus activation (Figure 8b, Appendix 3) was changed. Thus, depression ofthese potassium currents prolongs refractoriness and in conjunction with the predicted slowrate of drug-bound inactivated (ID) sodium channel recovery should provide antiarrhythmicactivity. Snyders and Hondeghem (1990) and Balser et al. (1991) showed that quinidineand amiodarone block transient sodium and potassium currents in myocardial tissue. Noother studies have examined the effects of arylacetamide compounds on except ourlaboratory (Pugsley et al., 1993a). However, Roden et al. (1988) examined the voltage-dependence of quinidine on the delayed rectifier current (iK), of which is a member(Wang et al., 1993), and suggest that the‘K channel proceeds from a closed state throughtwo open channel states at which time quinidine associates with the latter open state. Thesecond open state is presumed to occur at membrane potentials greater than +20 mV(Roden et al., 1988). Perhaps the arylacetamides act in a similar manner.Thus spiradoline is another arylacetamide which possesses both sodium andpotassium channel blocking properties in rat cardiac tissue. It is suggested, and has beenshown, that the combination of a class lb and class Ill antiarrhythmic agent may be ofpotential therapeutic benefit (Hondeghem & Katzung, 1980; Carlsson et al., 1993). The icagonists and related compounds may possess the structural properties which confer theseactions to ventricular ion channels.4.4 Are the ion channel blocking actions of the arylacetamides responsible for theantiarrhythmic actions of these compounds?4.4.1 Electrical Stimulation studies147Both the in vivo and in vitro results suggest that spiradoline and the arylacetamidecompounds depress excitability by virtue of sodium channel blockade and increaserefractoriness by blocking potassium channels. However, since prolongation of the P-Rinterval is not considered a good index of sodium channel blockade, even in the rat, weused increases in QRS width and RSh as additional indices of blockade (Penz et al., 1992;Pugsley et al., 1995). Electrical stimulation studies were also used to examine theantiarrhythmic properties of spiradoline and the other arylacetamides, and to verify and/ordelineate ion channel involvement in drug action.Spiradoline (Figure 7,8), U-50,488H (Figure 4b, Appendix 1), (+)PD 129,289 and (-)PD129,290 (Figure 3a, Appendix 4) and in Table 2 of Appendix 5 for (±)PDI 17,302 at therelatively high doses and concentrations used in these studies, produced significantactions on electrical stimulation variables. These responses included dose-dependentincreases in threshold currents for capture (it) and ventricular fibrillation (VFt) and thresholdduration (tt), responses characteristic of those produced by blockade of sodium channels inrat cardiac muscle and reflecting putative antiarrhythmic actions (Spear et al., 1972;Winslow, 1984; Abraham et al., 1989; Howard et al., 1991).Electrical stimulation studies take advantage of the properties of inhomogeneity ofcardiac muscle repolarization. After a wavefront of excitability occurs in the cardiac musclethere exists a certain degree of heterogeneity of recovery in excitability amongst cardiaccells (Moe et al., 1964; Winslow, 1984). The resulting disparity which occurs amongst cellsduring this period of ventricular vulnerability occurs at the end of the cardiac cycle (Wiggersand Wegria, 1940). This period corresponds to the phase of the T-wave waveform on theECG. Therefore, during this period the degree of instability in the ventricle may be suchthat an extra-stimulus of adequate strength and duration applied at this critical momentmay precipitate VF (Han, 1969). This has been termed the “R-on-T” phenomenon since itinvolves a depolarizing R-S complex appropriately activating a portion of the activatable148ventricle (i.e., cells which are no longer refractory) during the vulnerable period (see reviewby Moore and Spear, 1975). Thus, electrical stimulation is thought to, via the delivery ofhigh currents (approximately 100 pA), produce VF in this manner (Han, 1969; Sugimato etal., 1967).Principally, circus movement re-entry is reduced by prolongation of refractoriness.This is because extended refraction of ventricular myocyte action potentials reduces orlimits the potential re-entrant loop required to sustain the propagation of the impulse(Adaikan et al., 1992). That portion of the re-entrant loop which is no longer refractory andis therefore susceptible to excitation is known as an excitable gap (Janse 1992; Niwano etal., 1994). If a plot of it is made against tt, the boundary conditions described by the i vs tcurve define sodium channel availability, and may also represent a path length for circusmovement (McC. Brooks et al., 1951; Antoni, 1971). Thus, any conditions whereby it and ttfall below the defined exponentially-decaying curve provides cause for enhancedventricular excitability and possible induction of circus movement re-entrant arrhythmias(Antoni, 1971). Blockade of sodium channels by antiarrhythmic drugs, and arylacetamides,produces a shift in the curve by reducing sodium channel availability (through drug-boundchannel inactivation) and ventricular excitability such that induction of re-entrant circuits isless probable.However, it was shown that low doses of U-50,488H (Figure 4a, Appendix 1), (-)PD129,290, (Figure 3c, Appendix 4) and, although not to the same degree, spiradoline(Figure 7) which are associated with selective actions on K receptors, reduced VFt (and toa less marked extent, it and tt) in a manner that was blocked by naloxone. Thus, as seenby Brasch with naloxone (Kaschube and Brasch, 1991), the K agonist arylacetamidesappeared to have two actions: a low dose and possible arrhythmogenic effect mediatedthrough opiold receptors, and a higher dose antiarrhythmic effect independent of opioidreceptors (Pugsley et al., 1992a, 1993).149It is generally regarded that two possible mechanisms may produce or exacerbatearrhythmias associated with class I antiarrhythmic drug administration or ischaemia (Han,1969; Hope et al., 1974; Velbeit et al., 1982). The first occurs if conduction is depressed inventricular tissue without an alteration in refractoriness. This depression in conductionestablishes the conditions necessary for potential re-entrant circuit development in thevulnerable period of the myocardium, as discussed above (Binah and Rosen, 1982). Thesecond mechanism by which arrhythmias may occur is due to prolongation of the Q-aTinterval or delayed repolarization (Janse, 1992). Development of EADs and DADs, mostcommonly associated with the torsades de pointes arrhythmia, is characteristic of agentswhich prolong repolarization, most notably drugs possessing class Ill properties.Electrical stimulation studies did not allow us to determine whether one or all of thepossible mechanisms of arrhythmogenesis occurs with arylacetamides, however, mostprobably it is K receptor mediated. If we compare the doses of class I antiarrhythmic drugswhich induce arrhythmias to the potential proarrhythmic doses for the arylacetamides, it ismost likely that the class I agent produce arrhythmias at higher than therapeutic doses(Janse, 1992; Nattel, 1991).ERP (and MFF) was dose-dependently affected by the arylacetamides. This canindicate an action to block sodium channels and/or prolong the duration of the actionpotential. Similar effects are seen with class Ia antiarrhythmics (Vaughan-Williams,1984b). MFF is related to ERP by the equation MFF(Hz) = 1000/ERP (msec) (Pugsley etal., 1992a). Although these measures are similar they may be differentially susceptible toantiarrhythmic drug action. ERP is a measure of the effective refractory period while MFFmore accurately describes the relative refractory period (RRP) of the ventricle. Themanner in which MFF is obtained may be associated with an increase in extracellularpotassium which may locally alter drug actions in the vicinity of the depolarized cells.Studies with spiradoline (Figure 8), U-50,488H (Figure 5, Appendix 1) and (-)PD129,290150and its inactive enantiomer (+)PD129,289 (Figure 3b, Appendix 4) suggest that thesecompounds produce dose-dependent, but differential, actions on these measures.4.4.2 lschaemic Arrhythmia StudiesSpiradoline and related arylacetamides demonstrated sodium channel blockadeand possible antiarrhythmic properties and these studies showed, for the first time, a novelclass of potential antiarrhythmic drugs with an unexplored antiarrhythmic profile. Whencompared to sodium channel blocking antiarrhythmic drugs, such as quinidine, lidocaineand flecainide, the arylacetamides protect against acute ischaemia-induced arrhythmias atdoses which moderately reduce blood pressure and heart rate. In similar rat modelsquinidine and flecainide produced only moderate antiarrhythmic protection. Lidocainecompletely abolished arrhythmias but only at doses which produced convulsions, limiting itsusefulness against ischaemia-induced arrhythmias (Barrett et al., in press). Thus, apotentially new class of antiarrhythmic drugs exist with which to explore arrhythmogenesisand antiarrhythmic mechanisms.The low dose, potentially proarrhythmic actions seen with some of the K agonists inthe electrical stimulation studies did not exacerbate arrhythmias in the ischaemicarrhythmia model. The low dose, like naloxone, was without effect on the incidence ofventricular arrhythmias.The proarrhythmic actions of class I antiarrhythmic drug actions are thought toinvolve at least two components related to pharmacodynamic and pharmacokinetic factors.The pharmacodynamic component of conduction delay relates to a synergism betweenischaemic tissue and drug action (Hope et al., 1974). Antiarrhythmic drugs which increaseconduction delay in ischaemic tissue may or may not suppress arrhythmias (Cranefield,1975; Gettes et al., 1982). Antiarrhythmic drugs may suppress conduction and producearrhythmias by creating an area of myocardium with inconsistent activation, i.e. an area151whereby no uniform conduction can occur (El Harrar et al., 1977; Hoffman et al., 1981).Class I drugs, through perhaps such a mechanism, may facilitate local re-entry andincrease ectopic pacemaker site activity (Patterson et al., 1995).Since coronary artery occlusion can profoundly affect drug distribution in themyocardium, pharmacokinetic consideration may also play a role in causing arrhythmias.Nattel et al. (1981) examined the antiarrhythmic properties of aprindine in dogs subject tocoronary artery occlusion. It was found that regional distribution of drug influencedarrhythmia incidence. Although the clinical importance of the cardiovascular actions of Kreceptor agonists are likely to be limited, they may be of theoretical interest in view of theproarrhythmic findings with class Ic antiarrhythmic agents in the CAST-I and CAST-Il trials(CAST Investigators, 1989; CAST-Il Investigators, 1992).The antiarrhythmic activity of spiradoline (Table 1), U-50,488H (Table 3, Appendix2), (-)PD129,290 and its (+) enantiomer PD129,289 (Pugsley et al., 1993) could not beascribed to either an increase in extracellular potassium or occluded zone size. Anincrease in extracellular potassium occurs in ischaemic tissue shortly after the onset ofischaemia and is responsible for limited depolarization of cells within the ischaemic zone(Harris et al., 1954; Hill and Gettes, 1980). The increase in potassium is most probablydue to a decrease in cellular ATP levels which cause the ATP-dependent potassiumchannels (iTp) to open and enhance potassium efflux from cells (Wilde and Kieber,1986). As well, inhibition of the sodium/potassium ATPase membrane exchanger results inan increase in extracellular potassium and intracellular sodium concentrations;consequently, an increase in intracellular calcium usually occurs (Carmeleit, 1984). Lastly,the simple lack of blood flow preventing washout of potassium from the ischaemic areafurther adds to the elevation in potassium (Janse and Kleber, 1981). However, the exactmechanism by which potassium loss occurs is not defined, only that critical environmentalchanges occur in the acutely ischaemic myocardium.152In ischaemic tissue, action potential amplitude is reduced as well as the rise rate(Vm) of phase 0. This is accompanied by an initial increase, and then decrease, inaction potential duration (Downar et al., 1977). Partially depolarized ischaemic fibres showa reduced recovery from excitability compared to normal tissue (post-repolarizationrefractoriness) (Gettes and Reuter, 1974) These factors, in addition to acidification in theextracellular milieu (Yan and Kleber, 1992), may increase dispersion of refractoriness andprecipitate arrhythmias (Janse and Kleber, 1981). The ic receptor agonists and relatedcompounds may be antiarrhythmic by virtue of their ability to differentially suppressexcitability and conduction in the ischaemic and non-ischaemic zones in a uniform mannerand thereby convert any uni-directional block to bi-directional block preventing re-entrantcircuit development.At higher doses the arylacetamides had potassium channel blocking properties. Assuggested by Hondeghem and Katzung (1980), Hondeghem and Snyders (1990) andCarlsson et al. (1993) such blockade may be of therapeutic benefit. Hondeghem andKatzung (1980) examined the antiarrhythmic effectiveness of combinations of drugs suchas quinidine and lidocaine and argue that selective depression of abnormal impulsegeneration could be obtained by combining drugs with slow and fast offset kinetics forsodium channel blockade. Spiradoline and the other arylacetamides may have thesedesired properties providing a novel structure with which to explore the relationship andinterdependence of block between sodium and potassium channels.In these studies, the anaesthetized rat model of ischaemia was used to assess theonset of ventricular arrhythmias which usually occur within 10 mm after the onset ofocclusion (Walker et al., 1991). Like many other species a later phase occurs 1-2 hrs afterocclusion. Arrhythmias include PVC, VT and VF. In rats VF often spontaneously reverts,which is almost unknown in larger species including man (Janse and Kleber, 1981). Manymechanisms for the induction of VT and VF have been proposed (see review by Binah andRosen, 1992). This discussion will concentrate upon re-entry first conceptualized by Mines153(1913) as circulating excitation. Many mapping experiments have been conducted whichreveal that the movement of excitatory wavefronts are responsible for premature excitationwithin the heart (Janse and Kleber, 1981). In addition, Harris (1950) and Hoffman (1981)suggested that at the boundary between normal and ischaemic tissue arrhythmogenic“injury currents” occur. This may be due to the variable potential differences which existsbetween the two tissues.The abnormal electrical activity and distinct differences in cellular electrophysiologyin ischaemic tissue provide an ideal “target” for antiarrhythmic drug development.Consequently, for a drug to effectively suppress the re-entry circuits involving ischaemictissue it should ideally be activated within the ischaemic zone, i.e. at low pH and highpotassium concentrations. Since cells are partially depolarized a useful drug should bindto the channel when it is activated (open block), or inactivated (closed block), and unbindwhen rested (Hondeghem and Katzung, 1977; Janse, 1992). This would ensurefrequency-dependence in a manner similar to class lb antiarrhythmic drugs. Suchfrequency-dependence eliminates high-frequency arrhythmias such as VT and VF. Inaddition, sodium channel inactivation is greatest at the end of the cardiac cycle, maximalblock should theoretically occur at this point thereby suppressing ectopic pacemakeractivity (Hondeghem, 1990).When the arylacetamide compounds were examined for antiarrhythmiceffectiveness a degree of selective protection against VT and VF was found whereas theincidence of PVC occurrence was not changed. The frequency-dependent actions of thedrugs shown in the patch-clamp studies indicate enhanced effectiveness of thesecompounds at high rates of stimulation. However, in addition to suppressing conduction byblockade of sodium channels, the arylacetamides also prolonged refractoriness. Increasedrefractoriness can occur by two mechanisms, inhibition of repolarizing potassium currents,and prolongation of sodium channel inactivation (Janse, 1992). As mentioned above, theexcitable gap concept of re-entry suggests that drugs which both inhibit sodium channels154and prolong refractoriness may, in addition to abolishing arrhythmias, decrease thepossible pro-arrhythmic effects associated with sodium channel blockade alone (Hope etal., 1974; El Harrar et al., 1977). Pro-arrhythmic actions were not seen with thearylacetamides at any of the doses administered. If, according to the long excitable gaptheory, excitability and conduction are the requirements by which a portion of the re-entrantloop may be susceptible to arrhythmia induction (Niwano et al., 1994) blockade of sodiumchannels would selectively suppress conduction along the re-entrant path rendering thetissue inexcitable (Derakhchan et al., 1994). The proposed antiarrhythmic mechanism ofthe arylacetamides in this study is suggested to be due multiple ion channel block incardiac ventricular tissue.In contrast, some studies have shown that excitation may follow rapidly behindrefractoriness resulting in a short excitable gap (Janse, 1992). In this case prolongation ofrepolarization by inhibition of potassium currents may increase refractoriness and abolisharrhythmias (Spinelli and Hoffman, 1989). A large number of repolarizing potassiumcurrents are found in the myocardium (Carmeleit, 1993). It is suggested that the delayedrectifier current,‘K’ is an important target for block by antiarrhythmic drugs (Colatsky et al.,1990). In animal species in which‘K plays a dominant, or at least major role inrepolarization, block by antiarrhythmic drugs should effectively abolish arrhythmias.However, in the rat model used to conduct the experiments outlined in this thesis,‘K playsonly a minor role in contributing to ventricular repolarization.In the rat, the transient outward potassium current, ito, is the dominant repolarizingpotassium current in the ventricle (Josephson et al., 1984). Blockade of this current isantiarrhythmic in the rat and primates (Beatch et al., 1991; Adaikan et al., 1992). Actionpotentials in atrial tissue are predominantly repolarized by ito currents which, wheneffectively blocked, abolishes SVT (Wang et al., 1991). A suggested mechanism by whichito blockers effectively abolish high frequency ventricular arrhythmias such as VF has beenexplained in detail by Adaikan et al. (1992). Essentially these drugs prolong ERP to the155point that the excitatory gap is reduced to the point that the re-entrant arrhythmia is eitherabolished or the path-length is sufficiently prolonged to sustain only a slow VT. Thisrelates to the concept of wavelength (ERP multiplied by CV) which, as described above, isan index of drug effectiveness against arrhythmias (Rensma et al., 1988). The K receptoragonists and related compounds may block potassium channels and suppress arrhythmiasin rats by a similar manner.Pure potassium channel blockers, which have been the primary focus of drugcompany antiarrhythmic drug development for a number of years, possess two inherentproblems as antiarrhythmics. Blockade of repolarization can induce arrhythmias due toEAD or DAD-type mechanisms as discussed above (Wit et al., 1972a; Cranefield, 1975).In addition, while sodium channel blockers are characterized by positive frequency-dependence blockers of potassium channels (especially for lK) exhibit reverse use-dependence (Hondeghem and Snyders, 1990; Hondeghem, 1994), The term reverse- use-dependence refers to the observation that ERP is prolonged at low heart rates with thesedrugs and reduced at high heart rates (as occurs with VT or VF), thereby limitingantiarrhythmic drug effectiveness.‘to blockers, like tedisamil, also appear to be reverseuse-dependent (Dukes et al., 1990).The inherent degree to which compounds such as spiradoline and relatedarylacetamides block sodium and potassium channels should be explored in detail. Thedevelopment of analogs of these compounds may result in a class of antiarrhythmic drugswith mixed ion channel blocking properties which could be used to treat variousarrhythmias. The properties of antiarrhythmic drug blockade of more than one cardiac ionchannel is well known for amiodarone (Sirigh, 1983) and even for lidocaine (Josephson,1988). Since our studies show that the predominant ion channel which is blocked byarylacetamides, at doses which abolish or reduce arrhythmias (see RSh vs. Q-aT doses inTable 1, Appendix 5 for comparison), is sodium we could classify these arylacetamide156drugs as class Ia antiarrhythmic drugs according to the Vaughan-Williams classificationscheme (Vaughan-Williams, 1984; Pugsley et al., 1992a, 1993).Spiradoline was examined for effects on ischaemia-induced ECG changes. At adose of 2.5pmol/kg/min spiradoline prolonged the time to maximum S-T segment elevationand additionally prolonged the time taken to reach the maximum amplitude of the attainedR-wave following ischaemia. Arrhythmia studies with TTX and quinacainol, a putative classI antiarrhythmic, show results similar to those with spiradoline (Abraham et al., 1989;Howard et aL, 1991). Consistent ischaemic zone sizes between spiradoline and controlgroups (and the other arylacetamides as well) exclude this as a factor which may contributeto the changes in S-T segment elevation.It should be noted that despite the differences which exist between rats andhumans, ECG responses to occlusion are similar in both species (Walker et al., 1991).Elevation of the S-T segment is the first dramatic change which occurs in the ECG afterocclusion (Samson and Scher, 1960; Normann et al., 1961; Walker et al., 1991). Thischange, an indirect index of the extent of ischaemia occurring in the myocardium, issuggested to be the result of depolarization-induced changes in cardiac cells in theischaemic zone which include a reduction in action potential amplitude, action potentialshortening and delayed activation of these cells (Downer et al., 1977; Kleber et al., 1978;Carmeleit, 1984). A similar delay in onset of S-T segment elevation occurs with otherantiarrhythmic agents including the class II B-blockers and class IV calcium channelblockers (Yusuf et al., 1984; Curtis et al., 1986). As with other class I drugs includingquinidine, disopyramide, lidocaine and mexiletine, spiradoline and the relatedarylacetamides do not reduce maximum S-T segment elevation (Johnston et al., 1983;lgwemezie et al., 1990).Thus, the ion channel blocking actions seen with the arylacetamides examined canaccount for the antiarrhythmic actions of these compounds against ischaemic andelectrically-induced arrhythmias in the rat.1574.5 Projections regarding the value arylacetamides have in the study and/ortreatment of arrhythmias and ion channel blocking drugs.The arylacetamides were developed as structurally novel agonists for the K opioidreceptor. However, these compounds also possess non-opioid properties in cardiac tissueand may be valuable in the study of mechanisms, or treatment, of cardiac arrhythmia’s, orfor use as tools in the study of ion channel function. The lack of structural similarity of thearylacetamides to class I and III antiarrhythmic agents offers the opportunity to develop andunderstand the different structural requirements for drug effectiveness against arrhythmias.These compounds may allow for structure-activity relationship analysis for ischaemiaselectivity ultimately leading to the generation of new antiarrhythmic agents which aresuperior to both arylacetamide as well as the presently available drugs.The arylacetamide compounds examined for inhibitory actions on sodium andpotassium currents demonstrate a comparable, if not better, potency for ion channelblockade in rat ventricular myocytes when compared to other class I drugs. The basicelectronic configurations of these compounds may provide insight into the effectiveness ofion channel blockade and the pharmacophore for recognition and binding on or in ionchannels. If arylacetamides can be used to define the geometry of a “pharmacophore” thiswill provide a model for antiarrhythmic drug development.1585 SummarySpiradoline, and other arylacetamides, can have both opioid and non-opioidactions on the cardiovascular system. Actions which are dependent upon the opioidreceptor are blocked by oploid antagonists such as naloxone or Mr 2266. Theseantagonists block actions at other types of opioid receptors such as p, but only at higherdrug concentrations. However, the non-opioid actions of the K agonists and relatedcompounds are not blocked by any opioid antagonist.The non-opioid cardiovascular actions of K receptor agonists and relatedcompounds were only seen at high drug concentrations, or doses, 10-1000 times greaterthan those mediated by the K opioid receptor. Studies on cardiac tissue presented theclearest evidence of non-opioid actions. Studies in intact and isolated hearts suggestedion channel blocking properties in ventricular myocytes involving sodium, and to a lesserextent potassium, current blockade. These actions were associated with antiarrhythmiceffects during electrical stimulation or in the presence of coronary artery occlusion. Thesodium channel blocking actions of the arylacetamides are confounded, to some degree,by an opposing opioid-receptor dependent action. In addition to blocking the cardiacsodium and potassium channels, these compounds may have other membraneperturbational effects which may involve reduced calcium influx and potassium releasefrom neuronal tissue.The clinical relevance of the K agonist actions on the cardiavascular system arelikely to be limited and may be only of theoretical interest. However, recently manysuggestions have been made regarding the use of drug combinations in the treatment ofclinical arrhythmias. 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Permission from the publisher has been obtained foreach paper included in the Appendix.APPENDIX IThe MacMillan Press Limited, Houndmills, Basingstoke, Hampshire, U.K., have grantedpermission for the reproduction of the following material from the British Journal ofPharmacologyPugsley, M.K., Penz, W.P. Walker, M.J.A. and Wong, T-M. Cardiovascularactions of the kappa receptor agonist, U-50,488H, in the absence and presenceof opioid receptor blockade. Br. J. Pharmacol. 105: 521-526, 1992.APPENDIX 2ELSEVIER SCIENCE B.V., Amsterdam Publishing Division grant permission toreproduce the following material from the European Journal of PharmacologyPugsley, M.K., Penz, W.P. Walker, M.J.A. and Wong, T-M. Antiarrhythmiceffects of U-50,488H in rats subject to coronary artery occlusion. Eur. J.Pharmacol. 212: 15-19, 1992.APPENDIX 3The MacMillan Press Limited, Houndmills, Basingstoke, Hampshire, U.K., have grantedpermission for the reproduction of the following material from the British Journal ofPharmacology.Pugsley, M.K., Saint, D.A., Penz, W.P. and Walker, M.J.A. Electrophysiologicaland antiarrhythmic actions of the kappa agonist PD129290, and its R,R (+)enantiomer, PD 129289. Br. J. Pharmacol. 110: 1579-1585, 1993.APPENDIX 4ELSEVIER SCIENCE B.V., Amsterdam Publishing Division grant permission toreproduce the following material from the European Journal of Pharmacology:Pugsley, M.K., Saint, D.A., Walker, M.J.A. An electrophysiological basis for theantiarrhythmic actions of the K-opioid receptor agonist U-50,488H. Eur. J.Pharmacol. 261: 303-309, 1994.195APPENDIX 5Permission has been granted by the Western Pharmacology Society for thereproduction of the following paper from the 1995 volume of the Proceedings:Pugsley, M.K., Hayes, E.S., Saint, D.A. and Walker, M.J.A. Do related kappaagonists produce similar effects on cardiac ion channels? Proc. West. Pharmacol.Soc. 38: 25-27, 1995.APPENDIX 6ELSEVIER SCIENCE B.V., Amsterdam Publishing Division grant permission toreproduce the following material from the Journal of Pharmacological and ToxicologicalMethods:Penz, W.P., Pugsley, M.K., Hsieh, M.Z. and Walker, M.J.A. A new measure(RSh) for detecting possible sodium channel blockade in vivo in rats. J.Pharmacol. Toxicol. Meth. 27(1): 51-58, 1992.APPENDIX 7ELSEVIER SCIENCE B.V., Amsterdam Publishing Division grant permission toreproduce the following material from the Journal of Pharmacological and ToxicologicalMethods:Hayes, E., Pugsley, M.K., Penz, W.P., Adaikan, G. and Walker, M.J.A.Relationship between Q-aT and RR intervals in rats, guinea pigs, rabbits andprimates J. Pharmacol. Toxicol. Meth. 32(4): 201-207, 1994.APPENDIX 8NEVA PRESS, Brantford, CT, U.S.A., grants permission to use the manuscriptpublished in Cardiovascular Drug Reviews for reference in this Doctoral thesis:Pugsley, M.K., Penz, W.P. and Walker, M.J.A. Cardiovascular actions of U50,488H and related kappa agonists. Cardiovasc. Drug Rev. 11(2): 151-164,1993.Br. J. Pharmacol. (1992), 105, 521—526 APPENDIX I196© Macmillan Press Ltd. 1992Cardiovascular actions of the ic-agonist, U-50,488H, in theabsence and presence of opioid receptor blockadeM.K. Pugsley, W.P. Penz,1M.J.A. Walker & 2T.M. WongDepartment of Pharmacology & Therapeutics, Faculty of Medicine, University of British Columbia, 2176 Health Sciences Mall,Vancouver, B.C., Canada, V6T lZ3I The cardiovascular actions of U-50,488H, a ic-receptor agonist, were studied in rat isolated perfusedhearts, and in anaesthetized rats, over concentrations or doses generally above those required to produceic-receptor-mediated effects.2 U-50,488H dose-dependently decreased left-ventricular peak systolic pressure and beating rate in vitroand reduced blood pressure and heart rate in vivo.3 Over the concentration range of 1—3OpM in vitro, and the dose-range of 0.5—32pmolkg in vivo,U-50,488H prolonged the P—R, QRS and Q—T intervals of the ECG.4 The effects of U-50,488H were not antagonized by an opioid receptor antagonist, naloxone (1 JM or8 pmol kg ‘). Similarly, the opioid receptor antagonist, MR 2266, at 8 pmol kg’ did not significantlyreduce the cardiovascular actions of U-50,488H in vivo.5 The actions of U-50,488H on responses to electrical stimulation were also studied. Over the dose rangeof 0.5—32 pmol kg , U-50,488H altered thresholds and effective refractory period. It had a biphasic actionon thresholds for induction of ventricular fibrillation. Thresholds were decreased at lower doses (0.5—4pmol kg ) but increased at higher doses (8—32pmol kg l). The effects of lower doses were blocked bynaloxone.