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Pharmacological and antiarrhythmic properties of quinacainol : a new sodium channel blocker? Howard, Paisley Gail 1990

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PHARMACOLOGICAL AND ANTIARRHYTHMIC PROPERTIES OF QUINACAINOL SODIUM CHANNEL BLOCKER? by PAISLEY GAIL HOWARD B.Sc. Simon Fraser University, British Columbia A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PHARMACOLOGY & THERAPEUTICS We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1990 (c) Paisley Gail Howard, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. 1 further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada DE-6 (2/88) i i ABSTRACT Q u i n a c a i n o l , 1 - [ 2 - ( 1 , 1 - d i m e t h y l e t h y l ) - 4 - q u i n o l y l ] - 3 - ( 4 -p i p e r i d y l ) - 1 - p r o p a n o l i s a c l a s s I a n t i a r r h y t h m i c agent p r o v i s i o n a l l y s u b c l a s s i f i e d as I c . Studies were c a r r i e d out i n order t o (1) determine the a c t i o n s of q u i n a c a i n o l i n acute myocardial ischaemia, (2) a s c e r t a i n the mechanism(s) r e s p o n s i b l e f o r these a c t i o n s , and (3) a s c e r t a i n the appropriateness of i t s s u b c l a s s i f i c a t i o n . T o x i c o l o g i c a l , haemodynamic, and ECG e f f e c t s i n c h r o n i c a l l y prepared conscious r a t s were determined f o l l o w i n g a d m i n i s t r a t i o n of 1, 2, 4, or 8 mg/kg of q u i n a c a i n o l given i . v . over 10 minutes on a l t e r n a t e days. T o x i c i t y r e f e r a b l e t o the hea r t was seen at doses of 8 mg/kg and above. In r a t s given 8 or 16 mgkg, arrhythmias occurred. Q u i n a c a i n o l had no major e f f e c t s on blood pressure, u n l i k e most c l a s s I a n t i a r r h y t h m i c s , but lowered he a r t r a t e (not s t a t i s t i c a l l y s i g n i f i c a n t l y ) and prolonged P-R i n t e r v a l and QRS d u r a t i o n . I n an attempt t o p r o t e c t a g a i n s t ischaemic arrhythmias, doses of 2 mg/kg and 4 mg/kg were given. The high dose gave the best p r o t e c t i o n . I t reduced the in c i d e n c e of v e n t r i c u l a r t a c h y c a r d i a (VT) from a c o n t r o l value of 80% t o 3 0%, and reduced the in c i d e n c e of v e n t r i c u l a r f i b r i l l a t i o n (VF) from a c o n t r o l value of 60% t o 10%. An in c r e a s e i n the in c i d e n c e of premature v e n t r i c u l a r c o n t r a c t i o n s was seen at both doses. Blood pressure was not adversely e f f e c t e d although s l i g h t b r a d y c a r d i c e f f e c t s as w e l l as p r o l o n g a t i o n i i i of the P-R i n t e r v a l were seen at both doses. Both doses reduced S-T segment and delayed onset of e l eva t ion of S-T segment and R-wave which were induced by coronary o c c l u s i o n . S e n s i t i v i t y to e l e c t r i c a l s t imulat ion was tes ted i n pentobarbi ta l anaesthetised rats us ing v e n t r i c u l a r e l ec trodes . Doses of 0.5, 1, 2, and 4 mg/kg were given cumulat ively as a 10 min in fus ion every 25 min. Quinacainol d i d not a f f e c t QRS durat ion or the Q-T c i n t e r v a l but dose-dependently widened P-R i n t e r v a l when compared to p r e -treatment. Quinacainol dose-dependently increased threshold current , thresho ld durat ion , and v e n t r i c u l a r f i b r i l l a t i o n thresho ld . In a d d i t i o n , quinacainol e levated e f f e c t i v e r e f r a c t o r y per iod while decreasing maximum fo l lowing frequency. Open-chest ra t s under pentobarbi ta l anaesthesia were used to record the e f fec t s of quinacainol on e p i c a r d i a l i n t r a c e l l u l a r p o t e n t i a l s . Recordings were made by conventional microelectrode techniques before and a f t e r cumulative doses of 0.5, 1, 2, 4, and 8 mg/kg i . v . Quinacainol dose-dependently reduced phase zero of the a c t i o n p o t e n t i a l (AP) and AP height but d i d not inf luence other phases of the AP (with the exception of prolonging r e p o l a r i z a t i o n at the highest dose); act ions i n d i c a t i v e of c la s s I c . E f f e c t s of quinaca ino l on i s o l a t e d r a t hearts were assessed us ing a modif ied Langendorff heart preparat ion and were compared with those of te trodotoxin (TTX). Quinacainol i v widened the P-R i n t e r v a l and QRS d u r a t i o n without having major e f f e c t on the Q-Tc i n t e r v a l . In a d d i t i o n i t slowed the s i n u s beating r a t e . Quinacainol was more potent than TTX. A l l f i n d i n g s i n d i c a t e d t h a t q u i n a c a i n o l i s a potent a n t i a r r h y t h m i c agent w i t h Na + channel b l o c k i n g p r o p e r t i e s . V TABLE OF CONTENTS CHAPTER Page ABSTRACT i i TABLE OF CONTENTS V LIST OF FIGURES v i i i LIST.OF TABLES X LIST OF ABBREVIATIONS x i ACKNOWLE DGEMENTS x i i i DEDICATION xiv 1 INTRODUCTION 1 1.1 The Need fo r Antiarrhythmic Drugs 1 1.2 Experimental Approach to Myocardial Ischaemia and I n f a r c t i o n 3 1.3 Necessity of Animal Studies and Choice of Species i n the Study of Antiarrhythmic Agents 6 1.4 Experimental Models used to t e s t Antiarrhythmic Agents 10 1.5 Mechanistic Models of Arrhythmogenesis 15 1.5.1 Abnormalities i n Impulse Generation 16 1.5.2 Abnormalities i n Impulse Conduction 2 0 1.5.3 E l e c t r o p h y s i o l o g i c a l Consequences of Myocardial Ischaemia 24 1.6 Mode of Action of Antiarrhythmic Agents 25 1.6.1 C l a s s i f i c a t i o n System for Antiarrhythmic Agents 2 6 1.6.2 Class I Agents 28 1.6.2.1 The Sodium Channel 28 1.6.2.2 S u b c l a s s i f i c a t i o n of Class 1 Agents 35 1.7 Quinacainol 4 6 1.8 Objectives 48 2 METHODS 51 2.1 Haemodynamic studies i n the Conscious Rat 51 2.1.1 Preparation 51 2.1.2 Experimental Design 53 2.1.3 Data Analysis 54 2.2 Ischaemia-induced Arrhythmias 56 2.2.1 Experimental Preparation 56 2.2.2 Experimental Design 59 2.2.3 ECG Changes Pre and Post Occlusion 61 v i 2.2.4 Ischaemia-induced Arrhythmias Commonly seen w i t h O c c l u s i o n 62 2.2.5 R a t i o n a l e and Methods of D e f i b r i l l a t i o n 63 2.2.6 Scoring System f o r Arrhythmia A n a l y s i s 65 2.3 E l e c t r i c a l l y - i n d u c e d Arrhythmias 67 2.3.1 P r e p a r a t i o n 67 2.3.2 Experimental Design 67 2.3.3 Experimental Endpoints 68 2.4 E p i c a r d i a l I n t r a c e l l u l a r Recordings in vivo 69 2.4.1 P r e p a r a t i o n 7 0 2.4.2 Experimental Design 71 2.4.3 Experimental Endpoints and Data A n a l y s i s 72 2.5 E f f e c t s of Quinacainol i n I s o l a t e d Rat Hearts 72 2.5.1 P e r f u s i o n Apparatus 7 3 2.5.2 P r e p a r a t i o n 7 3 2.5.3 Experimental Design 75 2.6 S t a t i s t i c a l Analyses 76 3 RESULTS 7 8 3.1 E f f e c t s of Quinacainol on Blood Pressure, V e n t r i c u l a r Pressure, and Heart Rate 78 3.2 ECG E f f e c t s of Quinacainol 81 3.3 Coronary A r t e r y Occlusion-induced Arrhythmias i n Conscious Rats 89 3.3.1 Occluded Zone and M o r t a l i t y 97 3.3.2 Occlusion-induced ECG E f f e c t s 97 3.3.3 Arrhythmias 101 3.4 E l e c t r i c a l l y - i n d u c e d Arrhythmias i n Anaesthetised Rats 103 3.5 E f f e c t s of Quinacainol on E p i c a r d i a l A c t i o n P o t e n t i a l s i n the Anaesthetised Rat 109 3.6 I s o l a t e d Perfused Rat Hearts 111 4 DISCUSSION 115 4.1 Haemodynamic and ECG E f f e c t s of Quinacainol 115 4.2 A n t i a r r h y t h m i c A c t i o n s of Quinacainol a g a i n s t Ischaemia-induced Arrhythmias 116 4.3 A c t i o n s of Quinacainol a g a i n s t E l e c t r i c a l l y -induced Arrhythmias i n the Anaesthetised Rat 119 v i i 4.4 A c t i o n s of Quinacainol on E p i c a r d i a l I n t r a c e l l u l a r Recordings 120 4.5 E f f e c t s of Quinacainol in vitro 123 5 SUMMARY 12 7 6 REFERENCES 131 LIST OF FIGURES FIGURE Page 1 Model of sodium channel s t a t e s . 34 2 S t r u c t u r e of q u i n a c a i n o l . 4 6 3 I l l u s t r a t i o n of a t y p i c a l ECG and p o i n t s taken f o r i n t e r v a l measurements. 55 4 Occluder placement f o r coronary o c c l u s i o n . 58 5 Examples of arrhythmias and ECG changes induced by coronary o c c l u s i o n . 64 6 Time course of drug and o c c l u s i o n e f f e c t s on blood pressure and heart r a t e i n conscious r a t s . 80 7 Blood pressure and heart r a t e e f f e c t s of q u i n a c a i n o l i n anaesthetised r a t s . 82 8 E f f e c t s of q u i n a c a i n o l on the P-R i n t e r v a l i n conscious r a t s . 8 6 9 Changes i n the P-R i n t e r v a l pre and post o c c l u s i o n . 87 10 E f f e c t s of q u i n a c a i n o l on the QRS d u r a t i o n i n conscious r a t s . 88 11 E f f e c t s of q u i n a c a i n o l on the Q-Tc i n t e r v a l i n conscious r a t s . 9 0 12 P-R i n t e r v a l p r o l o n g a t i o n i n anaesthetised r a t s d u r i n g e l e c t r i c a l s t i m u l a t i o n s t u d i e s . 91 13 In vitro e f f e c t s of q u i n a c a i n o l and TTX on the P-R i n t e r v a l . 93 i x FIGURE Page 14 E f f e c t s of q u i n a c a i n o l and TTX on QRS d u r a t i o n d u r i n g in vitro s t u d i e s . 94 15 E f f e c t s of q u i n a c a i n o l and TTX on Q-Tc i n t e r v a l d u r i n g in vitro s t u d i e s . 95 16 S-T segment e l e v a t i o n f o l l o w i n g coronary a r t e r y o c c l u s i o n . 100 17 E f f e c t s of q u i n a c a i n o l on t h r e s h o l d c u r r e n t and t h r e s h o l d d u r a t i o n . 105 18 E f f e c t s of q u i n a c a i n o l on v e n t r i c u l a r f i b r i l l a t i o n t h r e s h o l d . 107 19 E f f e c t s of q u i n a c a i n o l on the maximum f o l l o w i n g frequency and e f f e c t i v e r e f r a c t o r y p e r i o d . 108 2 0 E f f e c t s of q u i n a c a i n o l on dV/dt m a x and a c t i o n p o t e n t i a l height. 110 21 E f f e c t s of q u i n a c a i n o l on r e s t i n g membrane p o t e n t i a l . 112 22 E f f e c t s of q u i n a c a i n o l on v a r i o u s phases of r e p o l a r i z a t i o n . 113 23 In vitro e f f e c t s of q u i n a c a i n o l and TTX on a measure of c o n t r a c t i l i t y , dP/dt. 114 X LIST OF TABLES TABLE Page 1 Blood pressure and heart rate e f f e c t s of quinacainol i n conscious ra t s . 79 2 Blood pressure and heart rate e f f e c t s of quinacainol during i n t r a c e l l u l a r p o t e n t i a l recordings in vivo. 84 3 Haemodynamic e f f e c t s of quinacainol and TTX in vitro. 8 5 4 ECG e f f e c t s i n anaesthetised r a t s . 9 2 5 Major acute t o x i c e f f e c t s of quinacainol i n conscious r a t s . 96 6 Mor t a l i t y from arrhythmic and non-arrhythmic causes. 98 7 ECG changes produced by coronary occlusion. 99 8 Arrhythmia incidence at 0-0.5 h and 0.5-4 h following occlusion. 102 9 Comparison of experimental r e s u l t s with established d e f i n i t i o n s of class I agents. 129 x i LIST OF ABBREVIATIONS action p o t e n t i a l AP action p o t e n t i a l duration APD action p o t e n t i a l duration at 10, 50 or 90% re p o l a r i z a t i o n APDio,50,90 arrhythmia score AS blood pressure BP current i e f f e c t i v e dose achieving 50% E D 5 0 e f f e c t i v e r e f r a c t o r y period ERP electrocardiogram ECG hertz Hz hour(s) h i n t r a p e r i t o n e a l l y i . p . intravenous i . v . l e f t a n t e r i o r descending LAD l e t h a l dose LD 5 0 l o g 1 0 of the number of PVC's log 1 0 PVC maximum d i a s t o l i c p o t e n t i a l MDP maximum following frequency MFF maximum rate of r i s e of phase 0 of the AP dV/dt maximum rate of r i s e of v e n t r i c u l a r pressure dP/dt mean x milligram per kilogram mg/kg millisecond(s) ms minute(s) min x i i LIST OF ABBREVIATIONS non-spontaneously reverting VT NSVT non-spontaneously reverting VF NSVF occluded zone OZ pacemaker current i f premature v e n t r i c u l a r contraction PVC Q-T i n t e r v a l corrected f o r heart rate Q-Tc rate-dependent block RDB tetrodotoxin TTX second(s) sec sino - a u r i c u l a r node SAN sodium Na + sodium conductance gNa standard error of the mean s.e.mean subcutaneous s.e. threshold current i T threshold duration (threshold pulse width) t T v e n t r i c u l a r f i b r i l l a t i o n VF v e n t r i c u l a r f i b r i l l a t i o n threshold VF T v e n t r i c u l a r tachycardia VT x i i i ACKNOWLEDGEMENTS I would l i k e t o thank Cathy Pang and B e r n i e MacLeod f o r t h e i r a s s i s t a n c e and encouragement. To Greg B e a t c h I owe much o f what I t a k e away from h e r e . He sometimes a c t e d as my mentor, was a s o u r c e o f numerous memorable d i s c u s s i o n s , many good l a u g h s , and a t r u e f r i e n d . Had M i c h a e l Walker been "demoted t o god", perhaps I would have l e a r n e d f a r l e s s . I doubt t h a t he w i l l e v e r f u l l y r e a l i z e t h e l a s t i n g i m p r e s s i o n s he has made on me. Though I ar g u e d w i t h him e n d l e s s l y , more on t h e p h i l o s o p h y o f l i f e t h a n on s c i e n c e , h i s words d i d n o t fade i n t o t h e a i r b u t have sometimes become p a r t o f my own c o n s c i e n c e . He f r u s t r a t e d me, i n f u r i a t e d me, p r o v o k e d me, and c h a l l e n g e d me c o n s t a n t l y . I n s h o r t , he c o n t i n u o u s l y churned t h e t r u e e motions o f l i f e . Many o t h e r p e o p l e have added t o my memories and h e l p e d me i n numerous ways. J i p i n g Huang, L a r r y S e l b y , and D i c k W a l l ( t e c h n i c a l a s s i s t a n c e ) , J a n e l l e Swetnam ( g e n e r a l a d v i s o r ) , Maureen Murphy (computer d i s a s t e r r e l i e f ) , M i c h a e l P u g s l e y and my f e l l o w g r a d u a t e s t u d e n t s ( f r i e n d s and c o l l e a g u e s ) , and o f c o u r s e t h e department as a whole. I am i n d e b t e d t o t h e B.C. & Yukon H e a r t F o u n d a t i o n , The U n i v e r s i t y o f B.C., and Rhone P o u l e n c S a n t e f o r t h e i r f i n a n c i a l s u p p o r t . F i n a l l y , t o my p a r e n t s . I t i s i m p o s s i b l e f o r me t o e x p r e s s t h e d e p t h o f my g r a t i t u d e t o them. F o r now, I can o n l y t h a n k them w i t h a l l my h e a r t and s o u l f o r t h e i r u n d y i n g s u p p o r t t h r o u g h t h e w o r s t o f i t and t h e b e s t o f i t . x i v Dedicated t o the memory of my grandfather, ANDREW COCHRANE HOWARD 1 1 INTRODUCTION 1.1 The Need f o r A n t i a r r h y t h m i c Drugs V e n t r i c u l a r f i b r i l l a t i o n i s r e s p o n s i b l e f o r the m a j o r i t y of deaths (90%) due t o heart a t t a c k s ( O l i v e r , 1982). McCormick and Skrabanek (1988) examined the i d e n t i f i c a t i o n of r i s k f a c t o r s and the b e l i e f t h a t m o d i f i c a t i o n of such f a c t o r s can prevent or reduce the i n c i d e n c e of diseases such as coronary heart disease. Analyses of data from numerous s t u d i e s such as the WHO, H e l s i n k i , and Framingham s t u d i e s found t h a t l i f e s t y l e i n t e r v e n t i o n s ( i . e . d i e t a r y , smoking, blood pressure, etc.) f a i l e d t o demonstrate any s i g n i f i c a n t b e n e f i t (McCormick and Skrabanek, 1988). E p i d e m i o l o g i c a l s t u d i e s can i d e n t i f y r i s k f a c t o r s as c a u s a l agents, but experiment alone provides evidence of cause and e f f e c t . Regardless of the above, the need f o r aggressive a n t i a r r h y t h m i c drug research and development i s obvious s i n c e sudden death w i t h myocardial ischaemia/infarction i s p r i m a r i l y a t t r i b u t a b l e t o v e n t r i c u l a r f i b r i l l a t i o n ( O l i v e r , 1982; Hoffman and Dangman, 1986). Despite the a v a i l a b i l i t y of a l a r g e number of a n t i a r r h y t h m i c agents, there i s a c o n t i n u i n g need f o r s a f e r and more e f f i c a c i o u s drugs (Podrid, 1989). However, the route t o new drugs i s d i f f i c u l t due t o the l a c k of exact knowledge concerning the mechanisms u n d e r l y i n g the genesis of VF i n the c l i n i c a l p o p u l a t i o n and the r e l a t i v e l a c k of 2 knowledge regarding pharmacological p r o p e r t i e s which confer a n t i f i b r i l l a t o r y a c t i v i t y ( B o t t i n g et al. , 1986). A compounding problem i n the c l i n i c a l s i t u a t i o n r e g a r d i n g a p p r o p r i a t e choice of a p a r t i c u l a r a n t i a r r h y t h m i c agent i s t h a t t h i s d e c i s i o n depends not only on i t s demonstrable e f f e c t i v e n e s s a g a i n s t arrhythmias, but a l s o on a knowledge of i t s pharmacokinetics, haemodynamic a c t i o n s , and adverse e f f e c t s (Muhiddin and Turner, 1985). With r e s p e c t t o the l a c k of knowledge regarding the pharmacological p r o p e r t i e s which confer a n t i a r r h y t h m i c a c t i o n s , i t appears t h a t u s e f u l drugs might be developed from two routes: (1) agents which prevent the p r o d u c t i o n or a c t i o n of an e s s e n t i a l arrhythmogen or (2) c l a s s i c a l a n t i a r r h y t h m i c agents. The f i r s t route has not proven t o be s u c c e s s f u l although i t has been suggested on a number of occasions, t h a t e s s e n t i a l endogenous arrhythmogens are i n v o l v e d i n ischaemic arrhythmogenesis. P o s s i b l e arrhythmogens i n c l u d e e i c o s a n o i d s , catecholamines, f r e e r a d i c a l s , p l a t e l e t a c t i v a t i n g f a c t o r , e t c . (Braquet et al. , 1987). Overwhelming evidence f o r the o b l i g a t o r y involvement of such arrhythmogens has not been provided v i a s t u d i e s of blockade of p r o d u c t i o n or a c t i o n of p u t a t i v e arrhythmogens. In f a c t , i t has been recognized t h a t a c t i v a t i o n of the ATP-dependent K + channel may be s u f f i c i e n t alone t o induce the arrhythmogenic changes seen dur i n g the e a r l y phase of ischaemia (Wilde e t al., 1989). 3 The second route; development of c l a s s i c a l a n t i -arrhythmic agents, has more pot e n t i a l although i t i s tedious and protracted. I t has been con s i s t e n t l y reported that, despite the use of a v a r i e t y of models and species, the c l a s s i c a l ion channel blocking antiarrhythmics have a n t i f i b r i l l a t o r y and antiarrhythmic actions against ischaemia-induced arrhythmias (Botting et al. , 1986). 1.2 Experimental Approach to Myocardial Ischaemia and I n f a r c t i o n Sudden cardiac death i s considered the foremost challenge of modern cardiology (Bayes de Luna et a l . , 1988). The most frequent cause i s VF although VT and asystole can also r e s u l t i n sudden cardiac death (Cobb et a l . , 1980; Janse, 1986). The underlying cause of these arrhythmias i s a t h e r o s c l e r o t i c coronary artery disease which narrows or occludes the artery causing cardiac ischaemia and i n f a r c t i o n (Davies, 1981; Janse and Wit, 1989). In only a few of those patients resuscitated and reaching h o s p i t a l a l i v e , are signs of myocardial necrosis found, suggesting that a b r i e f period of ischaemia not even long enough to cause i r r e v e r s i b l e myocardial damage, can induce f a t a l arrhythmias (Janse, 1986) . Experimental evidence providing a l i n k between obstruction of a coronary artery, arrhythmias, VF, and sudden death can be traced back to the 19th century (Lazzara 4 et al., 1978). E r i c h s e n , a pioneer i n t h i s f i e l d , d e s c r i b e d a ' s l i g h t tremulous motion' a f t e r c e s s a t i o n of a r e g u l a r heart beat f o l l o w i n g coronary a r t e r y o c c l u s i o n i n the dog i n 1842 (reviewed i n L a z a r r a et al., 1978). Some 10 years l a t e r P o r t e r determined t h a t disturbance of c a r d i a c rhythm was the r e s u l t of coronary o c c l u s i o n (reviewed i n L a z a r r a et al., 1978). In 1909, Thomas Lewis demonstrated a c o r r e l a t i o n between coronary o c c l u s i o n i n experimental animals and the appearance of paroxysmal VT (reviewed i n Lazzara et al., 1978). C l i n i c i a n s observed a m u l t i t u d e of arrhythmias r e s u l t i n g from ischaemia and i n f a r c t i o n . Wiggers and coworkers (1941) noted t h a t a r e g i o n of ischaemia f a c i l i t a t e d i n d u c t i o n of VF by applying a s t r o n g s t i m u l u s dur i n g the T wave. They concluded t h a t ischaemia lowered the f i b r i l l a t i o n t h r e s h o l d (current requirement) and broadened the v u l n e r a b l e p e r i o d f o r i n d u c t i o n of VF (Wiggers e t al. , 1941). In the e a r l y 1950's, H a r r i s and a s s o c i a t e s (1950) developed a technique t o determine the mechanisms u n d e r l y i n g d i s o r d e r s of rhythm by d i r e c t l y r e c o r d i n g from ischaemic t i s s u e s i n dogs w i t h occluded coronary a r t e r i e s . H a r r i s ' group drew a number of c o n c l u s i o n s . They concluded t h a t e c t o p i c beats were generated near the border between i n f a r c t e d and normal t i s s u e by automatic f o c i induced by potassium r e l e a s e d from ischaemic c e l l s . They discounted the importance of the i n t e r i o r of the ischaemic zone i n the gen e r a t i o n of arrhythmias. They a l s o discounted r e e n t r y 5 because they were not able t o record continuous a c t i v a t i o n ( i . e . a c t i v a t i o n p o t e n t i a l s detected throughout the i n t e r v a l between 2 beats, d u r i n g t a c h y c a r d i a ) . Furthermore, they concluded t h a t e c t o p i c v e n t r i c u l a r rhythms occur i n 2 phases f o l l o w i n g coronary o c c l u s i o n , and t h a t these are separated by a quiescent p e r i o d of sinus rhythm. The f i r s t phase occurs d u r i n g the f i r s t 30 min f o l l o w i n g o c c l u s i o n w i t h frequent occurrences of v e n t r i c u l a r tachyarrhythmias w h i l e the second, or delayed phase, begins 4-8 h p o s t - o c c l u s i o n and can l a s t f o r 2-4 days ( H a r r i s , 1950). During t h i s time p e r i o d , v e n t r i c u l a r tachyarrhythmias again occur, although episodes of VF are r a r e . H a r r i s (1950) speculated t h a t the e l e c t r o p h y s i o l o g i c a l mechanisms i n v o l v e d d u r i n g the 2 phases were d i f f e r e n t , as would be concluded i n l a t e r s t u d i e s . The term "acute phase of myocardial ischaemia" r e f e r s t o events o c c u r r i n g w i t h i n the f i r s t 2-4 h a f t e r sudden r e d u c t i o n of blood flow through a coronary a r t e r y (Janse and Wit, 1989) . In p a t i e n t s r e s u s c i t a t e d from VF, only a few subsequently develop a myocardial i n f a r c t i o n (Cobb e t al. , 1980) which suggests t h a t , i f myocardial ischaemia i s i n v o l v e d , i t i s t r a n s i e n t . Ischaemia need only t o e x i s t very b r i e f l y (< 1 min) i n order t o induce arrhythmias (Janse and Wit, 1989) . I t has a l s o been shown t h a t arrhythmias can r e s u l t both from t r a n s i e n t ischaemia (where S-T segment e l e v a t i o n has a l r e a d y reached maximal l e v e l s ) or from r e p e r f u s i o n ( a f t e r the S-T segment changes have returned to normal) (Janse and Wit, 1989). 6 I t i s g e n e r a l l y accepted t h a t the occurrence of l e t h a l arrhythmias i n humans i s the r e s u l t of the i n t e r p l a y between s e v e r a l f a c t o r s . F i r s t , acute ischaemia produces e l e c t r o p h y s i o l o g i c a l changes thereby c r e a t i n g the s e t t i n g f o r r e e n t r y c i r c u i t s w i t h i n the ischaemic myocardium (Janse and Wit, 1989). Second, the anatomical arrangement of s u r v i v i n g myocardial f i b e r s w i t h i n a healed i n f a r c t provides a n a t o m i c a l l y d e f i n e d r e e n t r a n t c i r c u i t s (Janse and Wit, 1989). S p e c i f i c t r i g g e r s can manifest themselves as arrhythmias i n the s e t t i n g of acute ischaemia or i n f a r c t i o n (Szekeres, 1986). PVC's, e s p e c i a l l y i f they occur a f t e r a long pause or a f t e r increases i n heart r a t e , may be considered a t r i g g e r t o more severe arrhythmias (Janse and Wit, 1989). Modulating f a c t o r s i n c l u d e the sympathetic nervous system, e l e c t r o l y t e d i s t u r b a n c e s (e.g. hyper/hypokalemia), or impaired l e f t v e n t r i c u l a r f u n c t i o n which may modify a t r i g g e r or f u n c t i o n a l change brought about by acute ischaemia (Szekeres, 1986) . The numerous experimental models used to study arrhythmias r e s u l t i n g from ischaemia-infarction vary w i t h respect t o the above f a c t o r s . 1.3 Necessity of Animal Studies and Choice of Species i n the Study of Antiarrhythmic Agents The d i v e r s e number of experimental animal models e x e m p l i f i e s the inadequacy of any one p a r t i c u l a r model t o e f f e c t i v e l y mimic the mechanisms or causes of sudden c a r d i a c 7 death i n man (Davies, 1981) as w e l l as the v a r i o u s types of questions asked. Models of acute or chr o n i c ischaemia do not n e c e s s a r i l y take i n t o account f a c t o r s which may i n f l u e n c e s u s c e p t i b i l i t y f o r v e n t r i c u l a r arrhythmias. Such f a c t o r s i n c l u d e neuro-humoral i n f l u e n c e s , emotional responses, anatomical l o c a t i o n s of the diseased v e s s e l s , regions of i n f a r c t i o n , c o l l a t e r a l c i r c u l a t i o n , metabolic derangements, coronary spasm, and unstable coronary a r t e r y plaques, i . e . p r i o r coronary or c a r d i a c disease (David e t al. , 1986; Janse and Wit, 1989; Skereres, 1988). Experimental methods f o r inducing myocardial ischaemia and i n f a r c t i o n have been developed out of n e c e s s i t y from l a c k of adequate human data. U n f o r t u n a t e l y , experimental p r e p a r a t i o n s do not n e c e s s a r i l y mimic those seen i n man, due i n p a r t t o the f a c t t h a t the u n d e r l y i n g mechanisms of c l i n i c a l arrhythmias are not f u l l y understood (Winslow, 1984). C r i t e r i a were described by C u r t i s e t a l . (1987) f o r a s s e s s i n g animal models of myocardial ischaemia and i n f a r c t i o n . The c r i t e r i a e s t a b l i s h e d t h a t an i d e a l model would t h e o r e t i c a l l y : (1) completely mimic one or more of the v a r i o u s c l i n i c a l c o n d i t i o n s ; (2) respond t o drugs i n a manner which corresponded w i t h the c l i n i c a l response e x a c t l y ; (3) have s u f f i c i e n t p r e c i s i o n and accuracy t o f u n c t i o n as a bioassay; (4) permit a v a r i e t y of responses t o be measured; and (5) be p r a c t i c a l i n terms of c o s t , time, and demand ( C u r t i s e t al., 1987). As no one model meets a l l 8 these requirements, compromises have been made. A number of techniques and models must be used t o provide pharmacological and e l e c t r o p h y s i o l o g i c a l i n f o r m a t i o n as t o p r o p e r t i e s which confer a n t i f i b r i l l a t o r y a c t i v i t y i n myocardial ischaemia. In order t o perform such e x t e n s i v e s t u d i e s , a r e a d i l y a v a i l a b l e , e a s i l y used, and c o s t -e f f i c i e n t s p e c ies has t o be chosen. Such e x t e n s i v e s t u d i e s i n dogs or any other l a r g e species make the choice of the r a t a l o g i c a l a l t e r n a t i v e . While "man i s not a r a t " , d i s c o v e r i e s i n r a t s have been proven t o have t h e i r c l i n i c a l c o unterpart. For example, r a t s t u d i e s have shown t h a t blockade of the endogenous systems b e l i e v e d t o p l a y a p a r t i n ischaemia-induced arrhythmogenesis, does not r e s u l t i n a n t i f i b r i l l a t o r y a c t i o n s (Beatch et al., 1989). Thus many of the lessons l e a r n t i n the r a t can be d i r e c t l y t r a n s f e r a b l e t o other s p e c i e s . While the r a t has d i s t i n c t d i f f e r e n c e s from man i n terms of c a r d i a c anatomy and e l e c t r o p h y s i o l o g y , i t s advantages i n the study of myocardial ischaemia and arrhythmias seem t o outweigh i t s disadvantages. A notable advantage i s the uniform l a c k of e f f e c t i v e coronary c o l l a t e r a l s which r e s u l t s i n r e p r o d u c i b l e occluded (ischaemic) zones ( C u r t i s et al., 1987). This i s of prime importance s i n c e both ischaemia-induced arrhythmias and i n f a r c t s i z e depend upon the extent of c o l l a t e r a l anastamoses ( C u r t i s , 1986). 9 The primary disadvantage of producing ischaemia-induced arrhythmias i n r a t s v i a coronary a r t e r y l i g a t i o n i s shared w i t h a l l animal s p e c i e s , t h a t i s the l a c k of c l a r i t y r e g a r d i n g a b s o l u t e c l i n i c a l relevance. As w e l l , some i n v e s t i g a t o r s c o n s i d e r the h i g h r e s t i n g h e a r t r a t e i n the r a t (approximately 400 beats/min) t o be a disadvantage. However, others have concluded t h a t heart r a t e does not c o r r e l a t e w i t h ischaemia-induced arrhythmia s e v e r i t y i n conscious r a t s (Johnston et al., 1983). C e r t a i n e l e c t r o p h y s i o l o g i c a l p r o p e r t i e s of the r a t heart complicate analyses. V e n t r i c u l a r APD i s b r i e f i n r a t s but i t i s u n c l e a r how, and i f , the narrow AP j e o p a r d i s e s r a t p r e p a r a t i o n s (Payet et al. , 1978; c i t e d i n Inoue e t al., 1984) . The narrow AP may reduce the l i k e l i h o o d of r e e n t r y d u r i n g acute myocardial ischaemia although the f a s t r e s t i n g h eart r a t e would be expected t o compensate f o r t h i s ( B o t t i n g et al., 1986). I t i s unavoidable t h a t p a r t of the v e n t r i c u l a r muscle and coronary v e i n s must be t i e d w i t h the a r t e r y upon o c c l u s i o n . However, i t has been shown t h a t the t i g h t e n i n g of the o c c l u d e r produces no s i g n i f i c a n t sequelae (e.g. t r i g g e r i n g of r e f l e x e s or receptor a c t i v a t i o n c o n t r i b u t i n g t o arrhythmogenic e f f e c t s of ischaemia) u n l e s s the a r t e r y i s a l s o occluded (Hirche et al., 1980). 10 1.4 Experimental Models used to Test A n t i a r r h y t h m i c Agents A number of problems are a s s o c i a t e d w i t h the treatment of c a r d i a c arrhythmias. Treatment remains l a r g e l y e m p i r i c a l i n nature l i k e l y due t o the f a c t t h a t myocardial ischaemia and i n f a r c t i o n i n man i s a disease of m u l t i p l e a e t i o l o g y and v a r i a b l e outcome ( B r e i t h a r d t et a l . , 1989). Numerous models have been developed to help i d e n t i f y the o r i g i n and i d e a l i s t i c a l l y the a c t u a l u n d e r l y i n g e l e c t r o p h y s i o l o g i c a l b a s i s of the v a r i o u s disturbances i n c a r d i a c rhythm (Winslow, 1984). A more r a t i o n a l approach t o treatment w i l l r e q u i r e c h a r a c t e r i z a t i o n of the v a r i o u s types of arrhythmias w i t h r e s p e c t t o the s i t e ( s ) of o r i g i n , u n d e r l y i n g e l e c t r i c a l a b n o r m a l i t i e s , and the p r e c i s e mechanism of a c t i o n of each a n t i a r r h y t h m i c agent ( i n both normal and ischaemic areas) (Winslow, 1984). One p a r t i c u l a r problem w i t h some models of c a r d i a c arrhythmias i s t h a t w h i l e some deal w i t h model methodology and c h a r a c t e r i z a t i o n , few a c t u a l l y c o n s i d e r the model's a b i l i t y t o d i s c r i m i n a t e among a n t i a r r h y t h m i c agents of d i f f e r e n t c l a s s e s or whether a l l agents i n one c l a s s are e q u a l l y e f f e c t i v e i n a p a r t i c u l a r model (Brooks e t al. , 1989). Animal models have p r i m a r i l y attempted t o mimic v e n t r i c u l a r arrhythmias as these are most o f t e n p r e c i p i t a t e d by acute myocardial i n f a r c t i o n and are known t o occur i n out of h o s p i t a l p a t i e n t s dying of sudden c a r d i a c death (David et a l . , 1986; Winslow, 1984). 11 Numerous methods are a v a i l a b l e f o r the i n d u c t i o n of arrhythmias i n order t o assess the e f f e c t i v e n e s of p u t a t i v e a n t i a r r h y t h m i c agents or t o explore mechanisms a s s o c i a t e d w i t h the v a r i o u s arrhythmias. Arrhythmogenic s t i m u l i can be d i v i d e d i n t o 3 groups: mechanical, e l e c t r i c a l , and chemical (Winslow, 1984). One experimental procedure u s i n g a mechanical arrhythmogenic s t i m u l i c r e a t e s a region of acute myocardial ischaemia by the acute o c c l u s i o n of a major coronary a r t e r y ( H a r r i s , 1950; C l a r k e t al., 1980; Johnston et al. , 1983). This can be done e i t h e r open-chest under anaesthesia, or c l o s e d chest i n a c h r o n i c conscious model u s i n g a p r e v i o u s l y implanted occluder or c a t h e t e r technique. A v a r i e t y of species have been used i n t h i s technique i n c l u d i n g guinea-p i g , r a t , c a t , dog, p i g , and baboon (Johnston e t al., 1983). Each species has i t s unique anatomic and e l e c t r o p h y s i o l o g i c a l c h a r a c t e r i s t i c s t o c o n s i d e r w i t h respect t o methodology, i n t e r p r e t a t i o n of r e s u l t s and c r o s s -species comparison. C u r t i s e t a l . (1987) p o i n t out t h a t the major source of v a r i a b i l i t y i n the extent, s e v e r i t y , and outcome of myocardial ischaemia i s coronary a r t e r y anatomy (with r e spect t o the extent of c o l l a t e r a l v a s c u l a r i z a t i o n ) . However, the r e s u l t of an acute ischaemic event can be compared q u a n t i t a t i v e l y and q u a l i t a t i v e l y w i t h the e f f e c t s of treatment upon the ensuing v e n t r i c u l a r arrhythmias (Johnston e t al. , 1983). Drugs can be administered e i t h e r p r o p h y l a c t i c a l l y or subsequent t o the onset of ischaemia. 12 Acute myocardial ischaemia i n r a t s (the animal model we have chosen) produces v e n t r i c u l a r arrhythmias i n a p r e d i c t a b l e and r e p r o d u c i b l e manner ( C u r t i s e t al. , 1987). Rats e x h i b i t 3 phases of arrhythmias i n response t o coronary o c c l u s i o n ( C u r t i s e t al., 1987). In both conscious and pentobarbitone anaesthetised r a t s , an e a r l y phase of ischaemia-induced arrhythmias begins 4 t o 8 min a f t e r o c c l u s i o n , and l a s t s f o r 5 t o 10 min ( C u r t i s e t al., 1987). A second phase of severe v e n t r i c u l a r arrhythmias s t a r t s approximately 1.5 - 2.5 h f o l l o w i n g o c c l u s i o n and l a s t s f o r s e v e r a l hours ( C u r t i s et al., 1987). A t h i r d phase of arrhythmias i s present i n r a t s . In at l e a s t 90% of animals s t i l l a l i v e 24 h p o s t - o c c l u s i o n , m u l t i - f o c a l PVC 1s occur ( C u r t i s e t al., 1987). VT i s r a r e i n r a t s a t 24 h f o l l o w i n g permanent coronary o c c l u s i o n ( u n l i k e i n dogs) ( C u r t i s et al., 1987). Experimental evidence suggests t h a t these d i f f e r e n t phases of arrhythmias may represent d i f f e r e n t mechanisms of arrhythmia i n i t i a t i o n (David e t al., 1986). O c c l u s i o n induced arrhythmias i n c l u d e PVC's, VT, VF, and bradyarrhythmias ( C u r t i s , 1986). Sinus b r a d y c a r d i a and a t r i a l and a t r i o v e n t r i c u l a r nodal arrhythmias are much l e s s common than v e n t r i c u l a r arrhythmias ( C u r t i s , 1986). Spontaneous r e v e r s i o n of these arrhythmias can occur and i s not unique t o r a t s and indeed may be a f u n c t i o n of heart s i z e ( C u r t i s et a l . , 1987). Numerous v a r i a t i o n s i n a second technique which i n v o l v e s i n d u c t i o n of arrhythmias by e l e c t r i c a l s t i m u l a t i o n 13 of the heart have a r i s e n s i n c e i t s i n d u c t i o n over a century ago (Winslow, 1984). A l l e l e c t r i c a l methods used t o induce f i b r i l l a t i o n i n the heart are dependent upon the b a s i c p h y s i o l o g i c a l concept t h a t not a l l c a r d i a c f i b e r s (even those i n c l o s e p r o x i m i t y w i t h one another) w i l l r e p o l a r i z e ( i . e . recover t h e i r e x c i t a b i l i t y ) simultaneously (Mines, 1913; Moe e t al., 1964; i n Winslow, 1984). Thus a v u l n e r a b l e p e r i o d i s e s t a b l i s h e d during the c a r d i a c c y c l e at the end of s y s t o l e a t which time some f i b e r s w i l l be completely r e p o l a r i z e d w h i l e others are s t i l l r e f r a c t o r y (Winslow, 1984) . A p p l y i n g an e x t r a e l e c t r i c a l s t i m u l u s of s u f f i c i e n t i n t e n s i t y i n t h i s time p e r i o d w i l l induce f i b r i l l a t i o n (Winslow, 1984). A l l e s s i e et a l . (1973, 1976) have mapped the spread of a c t i v a t i o n of a s i n g l e premature stim u l u s and measured r e f r a c t o r y periods a t m u l t i p l e s i t e s i n r a b b i t atrium. They suggested t h a t the e x i s t e n c e of nonuniform recovery of e x c i t a b i l i t y i s of primary importance i n the genesis of tachyarrhythmias r e s u l t i n g from r e e n t r y . I f the i n t e n s i t y of the e l e c t r i c a l s t i m u l u s i s f u r t h e r i n c r e a s e d , e x c i t a t i o n w i l l spread t o become d i s o r g a n i z e d , thereby i n s t i g a t i n g f i b r i l l a t i o n (a measure of the v e n t r i c u l a r f i b r i l l a t i o n threshold) ( A l l e s s i e et a l . , 1976). In view of the f a c t t h a t c l a s s I and I I I a n t i a r r h y t h m i c agents are expected t o increase the i n t e n s i t y of the e l e c t r i c a l s t i m u l u s (current) r e q u i r e d t o evoke f i b r i l l a t i o n , t h i s method i s o f t e n used t o a s c e r t a i n the e f f e c t i v e n e s s of p u t a t i v e a n t i a r r h y t h m i c agents (Winslow, 14 1984). The a n t i a r r h y t h m i c i s t e s t e d a g a i n s t v a r i o u s e l e c t r i c a l s t i m u l a t i o n v a r i a b l e s and i t s a b i l i t y t o i n f l u e n c e arrhythmias induced by e l e c t r i c a l s t i m u l a t i o n of the h e a r t . The advantages of the e l e c t r i c a l s t i m u l a t i o n model i n c l u d e (Winslow, 1984): (1) good r e p r o d u c a b i l i t y , (2) small c o s t , (3) d u r a t i o n of drug a c t i o n can be f o l l o w e d , (4) minimal surgery i s r e q u i r e d , (5) each animal e f f e c t i v e l y serves as i t s own c o n t r o l such t h a t l a r g e sample s i z e s are not r e q u i r e d f o r s t a t i s t i c a l a n a l y s i s , (6) e l e c t r i c a l l y induced arrhythmias i n animals do have p r e d i c t e d value f o r man (Horowitz et a l . , 1980). Two f u r t h e r experimental models used t o t e s t a n t i a r r h y t h m i c agents i n c l u d e chemical agents and re p e r f u s i o n - i n d u c e d arrhythmias. A l a r g e number of chemical agents, alone or i n combination, have been found t o induce arrhythmias (Winslow, 1984) . I t has been found t h a t some chemical agents are s p e c i e s - s p e c i f i c (Brooks e t a l . , 1989; Winslow, 1984) and capable of d i s c r i m i n a t i n g among a n t i a r r h y t h m i c agents of d i f f e r e n t mechanistic c l a s s e s (Brooks e t a l . , 1989). Chloroform, a c o n i t i n e , and oubain are the chemical agents most commonly used (Winslow, 1984). Reperfusion of blood flow t o myocardium p r e v i o u s l y made ischaemic by a p e r i o d of coronary o c c l u s i o n , has been a s s o c i a t e d w i t h arrhythmias and high m o r t a l i t y i n animal experiments (Janse and Wit, 1989; Winslow, 1984). In f a c t , the occurrence of VF may be g r e a t e r a f t e r r e p e r f u s i o n than 15 a f t e r coronary a r t e r y l i g a t i o n (Stephenson et a l . , 1960; c i t e d i n Janse and Wit, 1989) . Both in vivo and in vitro models are used t o study reperfusion-induced arrhythmias. There i s a r e l a t i o n s h i p between the l e n g t h of the ischemic p e r i o d d u r i n g the o c c l u s i o n and the occurrence of r e p e r f u s i o n arrhythmias (reviewed i n Janse and Wit, 1989). The i n c i d e n c e of reperfusion-induced VF increas e s when o c c l u s i o n periods are lengthened from 5 min t o 2 0 or 3 0 min but decrease when r e p e r f u s i o n i s delayed beyond 3 0-60 min (Janse and Wit, 1989). O c c l u s i o n can be repeated ( u s u a l l y at 30-90 min i n t e r v a l s ) so t h a t animals may serve as t h e i r own c o n t r o l s f o r drug assessment (Winslow, 1984). 1 . 5 M e c h a n i s t i c M o d e l s o f A r r h y t h m o g e n e s i s An arrhythmia i s an i r r e g u l a r i t y i n the rhythm of the hea r t ' s b e a t i n g , due t o an abnormality of impulse i n i t i a t i o n , conduction, or both (Hoffman and Rosen, 1981). This abnormality i n the r a t e , r e g u l a r i t y , s i t e of o r i g i n of the c a r d i a c impulse, or disturbance i n conduction, r e s u l t s i n the normal sequence of a c t i v a t i o n of a t r i a then v e n t r i c l e s being a l t e r e d (Wit et al., 1974). In c o n s i d e r i n g the mechanisms f o r c a r d i a c arrhythmias, i t i s necessary t o f i r s t i d e n t i f y the a b n o r m a l i t i e s of c e l l u l a r e l e c t r i c a l f u n c t i o n or s t r u c t u r e t h a t can induce arrhythmic a c t i v i t y and second, t o determine which of these p o s s i b l e mechanisms are a c t u a l l y r e s p o n s i b l e f o r s p e c i f i c arrhythmias i n the in 16 situ h e a r t (Hoffman and Dangman, 1987). This i s a c r i t i c a l p o i n t because, i f i t i s p o s s i b l e t o make a c e r t a i n and e x p l i c i t i d e n t i f i c a t i o n of the c e l l u l a r e l e c t r o p h y s i o l o g i c a l mechanism t h a t i s i n v o l v e d i n the genesis of the arrhythmia, then perhaps i t would a l s o be p o s s i b l e t h a t the response of s p e c i f i c arrhythmias t o drug i n t e r v e n t i o n would be dependent on the i d e n t i f i e d arrhythmogenic mechanism. 1.5.1 Abnormalities i n Impulse Generation C e r t a i n c a r d i a c c e l l s have the property of a u t o m a t i c i t y which means t h a t they are capable of spontaneously i n i t i a t i n g t h e i r own impulses which r e s u l t s from d e p o l a r i z a t i o n of f i b e r s during e l e c t r i c a l d i a s t o l e - phase 4 d e p o l a r i z a t i o n (Rosen, 1988). T h i s slow spontaneous d e p o l a r i z a t i o n d u r i n g d i a s t o l e lowers the membrane p o t e n t i a l t o t h r e s h o l d p o t e n t i a l and a spontaneous AP occurs (Vera and Mason, 1981). These f i b e r s are t h e r e f o r e considered automatic (Wit e t a l . , 1974). Normally automatic c e l l s i n c l u d e the pacemaker c e l l s of the SAN, subsidary a t r i a l f i b e r s , f i b e r s i n and around the coronary sinus ostium, c a r d i a c f i b e r s i n the t r i c u s p i d and m i t r a l v a l v e l e a f l e t s , the NH r e g i o n of the a t r i o v e n t r i c u l a r j u n c t i o n , and the His bundle and P u r k i n j e f i b e r r a m i f i c a t i o n s i n the v e n t r i c l e (Hoffman and Dangman, 1987; Wit e t al., 1974). The i n t r i n s i c r a t e of impulse i n i t i a t i o n i n the SAN i s f a s t e r when compared w i t h the r e s t of the s p e c i a l i z e d conducting 17 system, a l l o w i n g i t t o f u n c t i o n as the primary pacemaker (Rosen, 1988). However, when the si n u s r a t e i s absent, other s i t e s i n the s p e c i a l i z e d conducting system take over the pacemaker f u n c t i o n (Rosen, 1988). Thus arrhythmias t h a t are caused by abnormal impulse generation can be regarded as r e s u l t i n g from f o c a l mechanisms (Hoffman and Dangman, 1987). The o r i g i n or focus of these arrhythmias i s i n a s i n g l e f i b e r or a small group of w e l l - c o u p l e d f i b e r s w i t h e c t o p i c impulses spreading r a d i a l l y from t h i s focus (Hoffman and Dangman, 1987). I t i s assumed t h a t the rhythms are automatic i n the sense t h a t generation of one impulse does not depend on a p r i o r impulse (Hoffman and Rosen, 1981). Two major c l a s s e s of arrhythmias caused by a b n o r m a l i t i e s i n impulse generation can be i d e n t i f i e d ; those r e s u l t i n g from t r u l y spontaneous impulse generation or a u t o m a t i c i t y (and t h e r e f o r e not r e l i a n t on a p r i o r impulse) and second, those from t r i g g e r e d a c t i v i t y ( i . e . the gen e r a t i o n of one or more impulses as a consequence of a p r i o r impulse) (Hoffman and Rosen, 1981). An important c h a r a c t e r i s t i c of automatic pacemakers i s t h e i r a b i l i t y t o be o v e r d r i v e suppressed (Rosen, 1988). I f a p r e p a r a t i o n i s s t i m u l a t e d a t a f a s t e r r a t e than t h a t of the i n t r i n s i c pacemaker, the pacemaker r a t e w i l l be t r a n s i e n t l y reduced (Rosen, 1988). This i s r e f e r r e d t o as o v e r d r i v e suppression (Rosen, 1988). In f i b e r s w i t h high l e v e l s of membrane p o t e n t i a l , the normal pacemaker mechanism tends t o be r e a d i l y suppressed by o v e r d r i v e pacing (Rosen, 18 1988) . This i s contingent upon the en t r y of sodium ions d u r i n g phase 0 of each AP (Rosen, 1988). Enhanced pacemaker a c t i v i t y i n normal s p e c i a l i z e d conducting t i s s u e s can r e s u l t i n automatic arrhythmias (Hoffman and Dangman, 1987). However, many of the p r o p e r t i e s of normal automatic c e l l s suggest t h a t a s i g n i f i c a n t f r a c t i o n of the arrhythmias a t t r i b u t e d to abnormal impulse generation are not caused by normal a u t o m a t i c i t y (Hoffman and Dangman, 1987). Abnormal a u t o m a t i c i t y can be the occurrence of phase 4 d e p o l a r i z a t i o n ( i . e . spontaneous impulse i n i t i a t i o n ) , at l e v e l s of transmembrane p o t e n t i a l c o n s i d e r a b l y l e s s negative than the normal maximum d i a s t o l i c p o t e n t i a l or normal r e s t i n g p o t e n t i a l of the f i b e r s i n v o l v e d (Hoffman and Rosen, 1981; Janse and Wit, 1989). I f there i s a decrease i n background potassium conductance or an inc r e a s e i n inward Na + c u r r e n t , the r e s t i n g transmembrane p o t e n t i a l i s reduced to -60 mV or l e s s , i n i t i a t i n g a spontaneous impulse (Hoffman and Dangman, 1987). When the transmembrane p o t e n t i a l i s reduced, slow d i a s t o l i c d e p o l a r i z a t i o n seems not t o r e s u l t from the pacemaker c u r r e n t , i f but from time- and v o l t a g e -dependent changes i n K + and Ca 2 + c u r r e n t s t h a t occur at the p l a t e a u p o t e n t i a l (Hoffman and Dangman, 1987). U n l i k e normal automatic impulses, s i n g l e premature impulses do not per t u r b rhythms caused by abnormal automatic impulses (Hoffman and Dangman, 1987). In a d d i t i o n , the marked o v e r d r i v e suppression t h a t can occur i n normal automatic 19 c e l l s i s reduced or even l o s t i n abnormal automatic c e l l s (Hoffman and Dangman, 1987; Rosen, 1988). This may r e f l e c t the f a c t t h a t the Na + channels are l a r g e l y i n a c t i v a t e d by the low maximum d i a s t o l i c p o t e n t i a l (Hodgkin and Huxley, 1952; Hoffman and Dangman, 1987). A t h i r d proposed arrhythmogenic mechanism i s t r i g g e r e d a c t i v i t y . I t i s r e p e t i t i v e impulse formation i n i t i a t e d by a propagated or automatic AP (Janse and Wit, 1989). T r i g g e r e d a c t i v i t y i s dependent on o s c i l l a t i o n s i n membrane p o t e n t i a l t h a t f o l l o w the AP upstroke, i . e . a f t e r d e p o l a r i z a t i o n s (Janse and Wit, 1989) , and may be e i t h e r e a r l y or delayed (Rosen, 1988). When such an o s c i l l a t i o n occurs d u r i n g r e p o l a r i z a t i o n i t i s c a l l e d an e a r l y a f t e r d e p o l a r i z a t i o n ; when i t occurs a f t e r the membrane has r e p o l a r i z e d t o i t s maximum d i a s t o l i c p o t e n t i a l (Rosen, 1988), or n e a r l y so, i t i s c a l l e d a delayed a f t e r d e p o l a r i z a t i o n (Janse and Wit, 1989). When a f t e r d e p o l a r i z a t i o n s are l a r g e enough t o reach t h r e s h o l d , the r e s u l t a n t AP, or r e p e t i t i v e AP's, i s s a i d t o be " t r i g g e r e d " and may r e s u l t i n a t r i a l or v e n t r i c u l a r arrhythmias (Janse and Wit, 1989). Impulses r e s u l t i n g from a f t e r d e p o l a r i z a t i o n s are by d e f i n i t i o n t r i g g e r e d by a p r i o r impulse and thus are not automatic (Hoffman and Dangman, 1987). Arrhythmias induced by delayed a f t e r d e p o l a r i z a t i o n s occur more r e a d i l y when the preceding s t i m u l a t i o n r a t e i s r a p i d and w i l l tend t o in c r e a s e i n r a t e as the preceding d r i v e r a t e i s incre a s e d (Monk and Rosen, 1984). I t has been suggested t h a t delayed a f t e r d e p o l a r i z a t i o n s r e f l e c t 20 o s c i l l a t i o n s of calcium-loaded sarcoplasmic r e t i c u l u m (Lazzara and Scherlag, 1988) . In summary, the mechanisms i n v o l v i n g arrhythmias caused by abnormal impulse generation may be e i t h e r automatic or t r i g g e r e d : the automatic rhythms can be due t o e i t h e r normal or abnormal a u t o m a t i c i t y , and t r i g g e r e d rhythms by e i t h e r e a r l y or delayed a f t e r d e p o l a r i z a t i o n s . 1.5.2 A b n o r m a l i t i e s i n impulse Conduction A b n o r m a l i t i e s i n impulse conduction may be the r e s u l t of complete f a i l u r e of propagation or due t o u n i d i r e c t i o n a l b l o c k and r e e n t r y of an impulse (Rosen, 1988) . The term r e e n t r y i s used i n the sense t h a t d u r i n g the arrhythmia the r e i s continuous propagation of the impulse (Rosen, 1988) . Evidence i s accumulating i n support of r e e n t r y as a mechanism f o r generation of arrhythmias by ischaemic t i s s u e s i n animal models (Lazzara and Scherlag, 1988). C e r t a i n c o n d i t i o n s are necessary f o r the i n i t i a t i o n and maintenance of r e e n t r y and i n c l u d e : (1) u n i d i r e c t i o n a l b l ock of the impulse i n a region(s) of the h e a r t ; (2) s t a b l e propagation at a s u f f i c i e n t l y low v e l o c i t y ; (3) delayed e x c i t a t i o n of the t i s s u e j u s t d i s t a l t o the blocked s i t e , and (4) s u f f i c e n t r e p o l a r i z a t i o n of the t i s s u e proximal t o the s i t e of b l o c k such t h a t the sodium channels can be opened when 21 the impulse t h a t i s propagating around the b a r r i e r enters the r e g i o n of i n i t i a l b l o c k (Sasyniuk and Mendez, 1971; Spach and Dolber, 1985) . Thus conduction time over the a l t e r n a t i v e route must be longer than the r e f r a c t o r y p e r i o d of the path t o be reentered (Sasyniuk and Mendez, 1971) . D i f f i c u l t y a r i s e s when c o n s i d e r a t i o n i s given t o the long r e f r a c t o r y periods ( p a r t i c u l a r l y i n the v e n t r i c l e s ) and b r i n g s about the q u e s t i o n whether propagation over an a l t e r n a t i v e route i n v e n t r i c u l a r t i s s u e could be slow enough t o a l l o w t i s s u e w i t h extended r e f r a c t o r y p e r i o d s t o be r e e x c i t e d (Sasyniuk and Mendez, 1971). M o d i f i c a t i o n s have been made regarding these c o n d i t i o n s and the mechanisms t h a t cause the slow conduction and conduction block. A l l e s i e e t al. (1977) demonstrated i n a t r i a l t i s s u e t h a t s t a b l e r e e n t r y c i r c u i t s need not form around a hole or f i x e d b a r r i e r but can form around a t h i n i n t e r f a c e between regions of t i s s u e t h a t are a l t e r n a t e l y r e f r a c t o r y and r e c e p t i v e d u r i n g a s i n g l e c i r c u i t . In A l l e s i e ' s model, absolute r e f r a c t o r i n e s s serves as the f u n c t i o n a l b a r r i e r t o the c i r c u l a t i n g impulse w h i l e r e l a t i v e r e f r a c t o r i n e s s meets the requirements of slow conduction (Lazzara and S c h e r l a g , 1988). I t has been suggested i n past t h a t f i b r i l l a t i o n might represent c h a o t i c r e e n t r a n t e x c i t a t i o n or m u l t i p l e c o n t i n u a l l y m i g r a t i n g a c t i v a t i o n wavefronts (Mines, 1913; Moe et al., 1964). This type of r e e n t r y has been termed random r e e n t r y (as opposed t o s t a b l e r e e n t r y based on a 22 f i x e d anatomical path as discussed above) where the path of e x c i t a t i o n c o n t i n u o u s l y changes such t h a t i n d i v i d u a l groups of f i b e r s may be repeatedly e x c i t e d (Hoffman and Rosen, 1981) . Myocardial ischaemia can produce a l l of the c o n d i t i o n s necessary f o r r e e n t r y t o occur: holes (scars or f i x e d b a r r i e r s ) , slow conduction, and abnormal r e f r a c t o r i n e s s , e i t h e r abbreviated or prolonged (Lazzara and S c h e r l a g , 1988). Consequently, str o n g support i s given t o the important r o l e r e e n t r y may have as a mechanism f o r arrhythmias i n ischaemia. Mapping has been used e x t e n s i v e l y t o study r e e n t r a n t c i r c u i t s and t o o f f e r m o d i f i c a t i o n s t o the c o n d i t i o n s f o r r e e n t r y . Kramer et al. (1985) used a computerized system capable of d e t e c t i n g , s t o r i n g and a s s e s s i n g i n f o r m a t i o n from 232 s i t e s i n dog hearts a f t e r permanent or t r a n s i e n t coronary o c c l u s i o n . They determined t h a t i n t r a m u r a l r e e n t r y (microreentry) was a mechanism f o r VT whereby small e p i c a r d i a l conduction loops e x i t e d i n t o n o n - r e f r a c t o r y subendocardium i n i t i a t i n g succeeding beats. Kramer e t al. (1985) a l s o determined t h a t the s i t e of conduction delay neccessary f o r r e e n t r y was the t h i n s u r v i v i n g e p i c a r d i a l t i s s u e rim, i . e . a f i x e d hole or b a r r i e r was not needed f o r a r e e n t r y c i r c u i t (Lazzara and Scherlag, 1988). Furthermore, the " p r e f e r r e d pathways" of e x i t i n t o the subendocardium occurred a t the "border zone" of the i n f a r c t and were of v a r i a b l e c o n f i g u r a t i o n (Kramer et al., 1985). 23 Reentry can a l s o r e s u l t from r e f l e c t i o n of an impulse from an i n e x c i t a b l e segment ( A n t z e l e r i t c h et a l . , 1985; C r a n f i e l d , 1975; c i t e d i n Hoffman and Dangman, 1987). I f a delayed AP i s caused by the e l e c t r o t o n i c d e p o l a r i z a t i o n of a blocked impulse and i s d i s t a l t o an i n e x c i t a b l e segment, then r e f l e c t i o n occurs (Hoffman and Dangman, 1987) . The delayed AP then r e e x c i t e s the t i s s u e proximal t o the s i t e of blo c k (Hoffman and Dangman, 1987). These r e f l e c t e d impulses can be modulated by changes i n r a t e and rhythm (Hoffman and Dangman, 1987). Spach and Dolber (1985) proposed t h a t the spread of e x c i t a t i o n i n c a r d i a c muscle occurs by a p r e v i o u s l y unrecognized type of propagation t h a t i s d i s c o n t i n u o u s i n nature (proceeding i n steps) due t o r e c u r r e n t d i s c o n t i n u i t i e s of a x i a l r e s i s t i v i t y t h a t a f f e c t the k i n e t i c s of the f a s t Na + channels ( i . e . membrane c u r r e n t s ) . They proposed t h a t t h i s propagation i n c a r d i a c muscle p l a y s a primary r o l e i n c a r d i a c conduction disturbances l e a d i n g t o re e n t r y . T h is hypothesis does not meet the r e q u i s i t e s f o r re e n t r y ( d e t a i l e d e a r l i e r ) due t o s p a t i a l nonuniformity of r e f r a c t o r y p e r i o d s (Spach and Dolber, 1985) . F o l l o w i n g the d i r e c t i o n of Spach and co-workers i t has been suggested t h a t the alignment of f i b e r s and d i r e c t i o n a l d i f f e r e n c e s i n the d e n s i t y of t i g h t j u n c t i o n s may determine the l o c i of b l o c k and the c o n f i g u r a t i o n of r e e n t r a n t c i r c u i t s (Lazzara and Sche r l a g , 1988). 24 1.5.3 El e c t r o p h y s i o l o g i c a l Consequences of Myocardial Ischaemia Acute ischaemia causes myocardial c e l l s t o r e l e a s e K + (Hirche e t al., 1980). This i n c r e a s e i n e x t r a c e l l u l a r K + i s accompanied by a decrease i n the r e s t i n g membrane p o t e n t i a l . This causes a decrease i n AP amplitude, maximum r a t e of d e p o l a r i z a t i o n , and APD (Janse and Wit, 1989) . A combination of f a c t o r s produce these e f f e c t s and i n c l u d e i n c r e a s e d e x t r a c e l l u l a r K +, a c i d o s i s , and the combined l a c k of oxygen and s u b s t r a t e i n the ischaemic t i s s u e (Janse and Wit, 1989) . Both f a s t and slow c u r r e n t s are e q u a l l y depressed by ischaemia ( C a r d i n a l et al. , 1981). Due t o the r e d u c t i o n i n APD, ERP i s a l s o expected t o be abbreviated (Lazzara et al., 1978). However t h i s i s not the case. ERP i s i n f a c t prolonged i n a c u t e l y ischaemic c e l l s s i n c e i t continues beyond f u l l r e p o l a r i z a t i o n (Lazzara et al., 1978). This i s known as p o s t - r e p o l a r i z a t i o n - r e f r a c t o r i n e s s (Lazzara et al., 1978). The above e l e c t r o p h y s i o l o g i c a l changes occur i n d i f f e r e n t ischaemic c e l l s t o d i f f e r e n t degrees (Janse et al., 1979). Thus delayed conduction and conduction b l o c k ; necessary f o r r e e n t r y and subsequent r e e n t r a n t arrhythmias, are present during acute ischaemia ( B o t t i n g et al., 1986). 25 1.6 Mode of A c t i o n of Ant i a r r h y t h m i c Agents While the c a r d i a c e l e c t r o p h y s i o l o g i c a l a c t i o n s of an t i a r r h y t h m i c agents have been e x t e n s i v e l y d e s c r i b e d and indeed are the b a s i s f o r the Vaughan W i l l i a m s c l a s s i f i c a t i o n system, the mechanisms of the a n t i a r r h y t h m i c a c t i o n s of these drugs i s f a r l e s s def i n e d (Davy et al., 1988). A n t i a r r h y t h m i c agents can a f f e c t the arrhythmogenic s u b s t r a t e (whatever i t s e l e c t r o p h y s i o l o g i c a l o r i g i n ) , or the i n i t i a t i n g events such as e c t o p i c beats or changes i n the sinu s c y c l e l e n g t h (Davy et al., 1988). Both diseased c e l l s and normal c e l l s are i n v o l v e d , and can be profoundly a f f e c t e d by such drugs (Davy et al., 1988). Consequently, the choice of an a n t i a r r h y t h m i c agent o f t e n remains e m p i r i c a l . An e m p i r i c a l b a s i s of therapy f i r s t may r e s u l t i n f a i l u r e but, more imp o r t a n t l y , the arrhythmia being t r e a t e d may be aggravated or a new arrhythmia generated (Zipes, 1987). Mechanisms of a n t i a r r h y t h m i c a c t i o n s have been demonstrated in v i t r o and in vivo both e x p e r i m e n t a l l y and c l i n i c a l l y . Based on the arrhythmogenic mechanisms di s c u s s e d e a r l i e r ( a u t o m a t i c i t y , t r i g g e r e d a c t i v i t y , and r e e n t r y ) , the a n t i a r r h y t h m i c a c t i o n of drugs on automatic rhythms could i n v o l v e an i n h i b i t i o n of i f , the pacemaker c u r r e n t , a s h i f t i n g of the maximum d i a s t o l i c p o t e n t i a l t o more negative v a l u e s , or an increase i n APD, these a c t i o n s 26 a l t o g e t h e r lowering the automatic focus f i r i n g r a t e (Davy et a l . , 1988). Triggered rhythms, as opposed t o automatic rhythms, are more commonly r e s p o n s i b l e f o r c l i n i c a l arrhythmias (Davy et al., 1988). A n t i a r r h y t h m i c a c t i o n on t r i g g e r e d a c t i v i t y c o u l d i n v o l v e suppression of the a f t e r d e p o l a r i z a t i o n (Davy et al., 1988). A n t i a r r h y t h m i c agents can decrease Ca 2 + or Na + inward c u r r e n t s (Thale et al., 1987). F i n a l l y , e a r l y a f t e r d e p o l a r i z a t i o n s can be e f f e c t i v e l y suppressed by decreasing the b a s i c c y c l e length w h i l e delayed a f t e r d e p o l a r i z a t i o n s can be suppressed by i n c r e a s i n g i t (Davy et al., 1988). A n t i a r r h y t h m i c a c t i o n of drugs on r e e n t r a n t rhythms c o u l d i n c l u d e an i n c r e a s e i n r e f r a c t o r i n e s s i n one p a r t of the c i r c u i t or secondly, a conduction block changing a u n i d i r e c t i o n a l b l o c k , i n t o a b i d i r e c t i o n a l block (Hoffman, 1985) . I t i s important t o r e a l i z e t h a t the u n d e r l y i n g mechanisms are unknown f o r some arrhythmias and t h a t the same drug can act i n d i f f e r e n t ways. 1.6.1 C l a s s i f i c a t i o n System f o r A n t i a r r h y t h m i c Agents The c l a s s i f i c a t i o n of a n t i a r r h y t h m i c agents was i n i t i a l l y based on drug e f f e c t s on AP morphology (Vaughan W i l l i a m s , 1970). This c l a s s i f i c a t i o n has become the most w i d e l y accepted. A n t i a r r h y t h m i c agents are grouped i n t o c l a s s I , those t h a t b l o c k sodium channels; c l a s s I I , those 27 agents which b l o c k sympathetic a c t i v i t y - the B b l o c k e r s ; c l a s s I I I , potassium channel b l o c k e r s ; and c l a s s IV, calc i u m channel b l o c k e r s (Singh and Vaughan W i l l i a m s , 1972; Vaughan W i l l i a m s , 1970, 1984a). Vaughan W i l l i a m s emphasized t h a t h i s c l a s s i f i c a t i o n system was not so much a c a t e g o r i z a t i o n of drugs according t o chemical s t r u c t u r e s or p h y s i c a l p r o p e r t i e s (as e x e m p l i f i e d by the d i v e r s i t y of agents i n any given c l a s s ) , but merely described four ways i n which abnormal c a r d i a c rhythms c o u l d be prevented or c o r r e c t e d (Vaughan W i l l i a m s , 1984a). Some b a s i c problems arose when attempts were made t o t r a n s f e r Vaughan W i l l i a m s c l a s s i f i c a t i o n t o the c l i n i c . F i r s t , the Vaughan W i l l i a m s c l a s s i f i c a t i o n of a n t i a r r h y t h m i c agents was based on observations made on the c a r d i a c AP under normal c o n d i t i o n s (Brugada, 1990). E x t r a p o l a t i o n of such i n f o r m a t i o n t o the c l i n i c a l \ s e l e c t i o n of an a n t i a r r h y t h m i c agent has proven d i f f i c u l t (Zipes, 1987). The same a n t i a r r h y t h m i c agent can have d i f f e r e n t e f f e c t s on the same c e l l depending upon a multitude of f a c t o r s such as the presence o r absence of ischaemia, e l e c t r o l y t e d i s t u r b a n c e s , drug c o n c e n t r a t i o n at the t a r g e t s i t e , r a t e of s t i m u l a t i o n , e t c . (Brugada, 1990; David et a l . , 1986; Szekeres, 1986). The behaviour of normal and abnormal c e l l s i n the presence of a drug depends not only on the i n t r i n s i c c h a r a c t e r i s t i c s of the c e l l s but a l s o upon the autonomic balance (Brugada, 1990). Arrhythmias seen c l i n i c a l l y 28 i n v o l v e areas (as opposed t o s i n g l e c e l l s ) of myocardium and r e l a t i v e l y long a c t i v a t i o n pathways (Brugada, 1990). Further a n a l y s i s w i t h i n each c l a s s both e x p e r i m e n t a l l y and c l i n i c a l l y has r e s u l t e d i n f u r t h e r c l a r i f i c a t i o n and s u b c l a s s i f i c a t i o n of the Vaughan W i l l i a m s c l a s s i f i c a t i o n system perhaps g i v i n g i t more c l i n i c a l relevance. The c l i n i c a l d i f f e r e n c e s between v a r i o u s drugs w i t h c l a s s I a c t i o n has n e c e s s i t a t e d t h e i r s u b d i v i s i o n (Harrison e t al., 1980) . 1.6.2 Cl a s s I Agents 1.6.2.1 The Sodium Channel Attempts t o e l u c i d a t e the nature and p r o p e r t i e s of the c a r d i a c sodium channel are r e l e v a n t t o hypotheses concerning the a c t i o n of c l a s s I a n t i a r r h y t h m i c agents on these channels. Ion channels are molecules t h a t form pores i n the membrane t o a l l o w i o n flow ( H i l l e , 1984; Jan and Jan, 1989). The e l e c t r i c p o t e n t i a l across the c e l l membrane i s determined l a r g e l y by i o n channels opening and c l o s i n g (Jan and Jan, 1989). Channel s p e c i f i c i t y t o a p a r t i c u l a r i o n spe c i e s i s con f e r r e d by a " s e l e c t i v i t y f i l t e r " t h a t r e s i d e s i n the outer p o r t i o n of the channel (Rosen and S p i n e l l i , 1988). Voltage or channel gates are s t r u c t u r e s c o n t r o l l i n g the passage of ions so t h a t ions do not enter the channel ad l i b i t u m u n t i l the c o n c e n t r a t i o n and charge g r a d i e n t s are 29 balanced (as t h i s would not maintain the transmembrane p o t e n t i a l ) (Rosen and S p i n e l l i , 1988). These gates are on the i n n e r p o r t i o n of the channel and depending on whether they are i n an open or c l o s e d p o s i t i o n , passage of ions w i l l be p e r m i t t e d or prevented (Rosen and S p i n e l l i , 1988). From Hodgkin and Huxley's (1952) c l a s s i c s t u d i e s on squi d axon, these gates were designated m and h. Hodgkin and Huxley (1952) introduced the concept of channel gates i n an attempt to mathematically model the time and v o l t a g e dependent stages of the inward Na + c u r r e n t and d e p o l a r i z a t i o n . I n the c l o s e d s t a t e of the channel, the m gate i s c l o s e d and the h gate open (Sheldon e t a l . , 1989). In the open s t a t e , the m gate opens i n response t o an e l e c t r i c a l s t i m u l u s (voltage s e n s i t i v e ) and Na + ions enter causing the AP upstroke (Rosen and S p i n e l l i , 1988). As the c e l l d e p o l a r i z e s , the v o l t a g e -s e n s i t i v e h gate c l o s e s , i n a c t i v a t i n g the channel (Rosen and S p i n e l l i , 1988). Both gates are time-dependent such t h a t d u r i n g r e p o l a r i z a t i o n , the m gate c l o s e s and the h gate opens (Rosen and S p i n e l l i , 1988; Sheldon e t a l . , 1989). These v o l t a g e sensors or gates act independently of the s e l e c t i v i t y f i l t e r (Rosen and S p i n e l l i , 1988). E l e c t r o p h y s i o l o g i c a l s t u d i e s have examined the movements of charges i n t r i n s i c t o the c a t i o n channels (known as the g a t i n g c u r r e n t ) as p r e d i c t e d by Hodgkin and Huxley (1952) (Jan and Jan, 1989). These s t u d i e s have l e a d t o the p r e d i c t i o n of a s t r i n g of p o s i t i v e charges a c t i n g as a v o l t a g e sensor which may p a i r w i t h neighbouring negative 30 charges i n the c e l l membrane (Armstrong, 1981). Subsequent s t u d i e s (reviewed by H i l l e , 1984) i n d i c a t e d t h a t components i n t r i n s i c t o these channels f u n c t i o n as the v o l t a g e sensor. The d i f f e r e n c e s between the i n a c t i v a t e d s t a t e and the c l o s e d s t a t e i s c l e a r l y demonstrated by measuring the movement of charges i n t r i n s i c t o the channel (the g a t i n g current) under v a r i o u s c o n d i t i o n s which a f f e c t i n a c t i v a t i o n of Na + channels (Jan and Jan, 1989). I n a c t i v a t i o n immobilizes the g a t i n g charges i n the open s t a t e (Armstrong, 1981), i m p l y i n g t h a t a channel has t o be open before i t can be i n a c t i v a t e d (Jan and Jan, 1989). Na + channels i n a c t i v a t e more r e a d i l y when they are open, although i t i s p o s s i b l e t h a t some channels may be i n a c t i v a t e d before reaching the open s t a t e (Jan and Jan, 1989). The sodium channel i s composed of 1820 amino a c i d r e s i d u e s and con t a i n s f o u r homologous i n t e r n a l domains, each of which has s i x p u t a t i v e transmembrane segments (Stuhmer et al., 1989) of 19 or more predominantly hydrophobic r e s i d u e s (Jan and Jan, 1989). One of these segments, S4, c o n t a i n s s e v e r a l a r g i n i n e o r l y s i n e r e s i d u e s at every t h i r d p o s i t i o n i n t e r p o s e d w i t h mostly nonpolar residues (Stuhmer et a l . , 1989) . This S4 sequence has been p o s t u l a t e d t o form a transmembrane h e l i x , so t h a t the p o s i t i v e l y charged r e s i d u e s r e s i d e w i t h i n the membrane and f u n c t i o n as the v o l t a g e sensors discussed above ( C a t t e r a l l , 1986). In the S4 hypothesis, the f o u r S4 sequences may correspond t o the g a t i n g p a r t i c l e s r e s p o n s i b l e f o r channel a c t i v a t i o n and the 31 steep voltage-dependence as proposed by Hodgkin and Huxley (Jan and Jan, 1989; Stuhmer et al., 1989). From measurements of g a t i n g charge, i t has been estimated t h a t f o u r t o s i x charges w i t h i n the channel molecule move from one s i d e of the membrane t o the other as the channel opens (Jan and Jan, 1989) . The S4 model accounts f o r t h i s i f d e p o l a r i z a t i o n causes each of the S4 sequences t o move by roughly one h e l i c a l t u r n (Jan and Jan, 1989). Stuhmer et a l . (1989) i d e n t i f i e d f u n c t i o n a l regions of the Na + channels by i n v e s t i g a t i n g the e f f e c t s of s i t e d i r e c t e d mutations on r a t sodium channels. They provided evidence t h a t the p o s i t i v e charges i n segment S4 were i n v o l v e d i n the v o l t a g e - s e n s i n g mechanism f o r a c t i v a t i o n of the channel, and t h a t the region between repeats I I I and IV were important f o r channel i n a c t i v a t i o n . Na + channels i n squid g i a n t axon are s a i d t o e x i s t i n 3 s t a t e s , as are Na + channels of P u r k i n j e c e l l s : (1) c l o s e d , at p o t e n t i a l s near the r e s t i n g p o t e n t i a l , but a v a i l a b l e t o be opened by d e p o l a r i z a t i o n ; (2) open, s e l e c t i v e l y p e r m i t t i n g passage of Na + i o n s ; and (3) c l o s e d , and not a v a i l a b l e t o be opened, i . e . i n a c t i v a t e d (Fozzard e t al., 1985). A f t e r the c e l l membrane d e p o l a r i z e s , Na + p e r m e a b i l i t y markedly increases and then a f t e r 1 msec decreases t o the b a s e - l i n e l e v e l ( C a t t e r a l l , 1987). This b i p h a s i c behaviour represents a c t i v a t i o n (opening of Na + channels) and i n a c t i v a t i o n ( c l o s i n g of Na + channels) ( C a t t e r a l l , 1987). These events can be d e s c r i b e d as 32 separate voltage-dependent processes t h a t change t h e i r r a t e s i n s t a n t l y as a f u n c t i o n of v o l t a g e (Hodgkin and Huxley, 1952). Both r e s t i n g and i n a c t i v a t e d s t a t e s are non-conducting ( C a t t e r a l l , 1987) . However, evidence has been accumulating over the years t h a t the c a r d i a c Na + c u r r e n t s are not a c c u r a t e l y d e s c r i b e d by the o r i g i n a l Hodgkin-Huxley f o r m u l a t i o n s . Armstrong (1981) and others (reviewed i n Fozzard et a l . , 1985) have shown t h a t a c t i v a t i o n occurs w i t h a l a g which i s thought t o r e f l e c t the intermediate s t a t e t h a t the channel must pass through p r i o r t o opening. I n a c t i v a t i o n a l s o occurs w i t h a l a g , thus suggesting t h a t channels must open before they can i n a c t i v a t e (Armstrong, 1981). I n a c t i v a t i o n and recovery from i n a c t i v a t i o n o f t e n occurs w i t h more than one time constant (Chiu, 1977) suggesting t h a t the channel may have more than one i n a c t i v a t e d s t a t e (Fozzard e t a l . , 1985). Meves and Nagy (1989) di s c u s s e d m u l t i p l e conductance l e v e l s i n the sodium channel of mouse neuroblastoma c e l l s . They po i n t e d out t h a t these subconductance s t a t e s were ra r e events and t h a t i t d i d not seem l i k e l y t h a t they c o n t r i b u t e d s i g n i f i c a n t l y t o the macroscopic inward Na + c u r r e n t r e s p o n s i b l e f o r the r i s i n g phase of the AP (Meves and Nagy, 1989) . However, the subconductance s t a t e s may help e x p l a i n f u n c t i o n as r e l a t e d t o s t r u c t u r e of the sodium channel (Meves and Nagy, 1989). In t h i s regard, the f o u r repeated homology u n i t s of the sodium channel undergo conformational changes d u r i n g d e p o l a r i z a t i o n before an " i o n p e r m e a b i l i t y pathway" i s 33 formed (Meves and Nagy, 1989). Meves and Nagy (1989) speculated t h a t the subconductance - l e v e l s of the sodium channel corresponded t o the a c t i v a t i o n of each homology u n i t i n t u r n . The delay i n the onset of i n a c t i v a t i o n c o uld be very important i n heart muscle s i n c e the c u r r e n t i n v o l v e d i n generating the conducted AP upstroke r e q u i r e s 100-200 usee t o develop (Fozzard et al. , 1985). A very slow phase of recovery from i n a c t i v a t i o n i n heart muscle i s i n f l u e n c e d by the d u r a t i o n of d e p o l a r i z a t i o n or the presence of a n t i a r r h y t h m i c agents (Cohen et a l . , 1981). Based on recent experimental r e s u l t s , v a r i a t i o n s t o the Hodgkin-Huxley model have been made. Figure 1 i s an i l l u s t r a t i o n of one such m o d i f i c a t i o n . In a time- and voltage-dependent manner, Na + channels pass between the c l o s e d (C), open (O), and i n a c t i v a t e d (I) s t a t e s (Hondeghem, 1984) . Time and v o l t a g e dependent r a t e constants or p r o b a b i l i t y f u n c t i o n s can d e s c r i b e these t r a n s i t i o n s (Hondeghem, 1984). The c l o s e d s t a t e of the channel i s most p r e v a l e n t i n the r e s t i n g membrane (Hondeghem, 1984). Upon d e p o l a r i z a t i o n of the membrane, the channel may f i r s t go to the open s t a t e and then the i n a c t i v a t e d s t a t e (or go there d i r e c t l y ) then r e t u r n i n g t o the c l o s e d s t a t e before any f u r t h e r channel opening (Fozzard e t al., 1985). The development of the patch clamp technique by Neher and c o l l e a g u e s (Hamill et al., 1981) has allowed the study of s i n g l e membrane channels. Data obtained from patch clamp HH' F i g u r e 1. Diagram i l l u s t r a t i n g the mechanism of a c t i o n of a n t i a r r h y t h m i c drugs i n a sodium channel undergoing t r a n s i t i o n s between t h r e e s t a t e s . R = r e s t i n g ( c l o s e d ) , A = a c t i v a t e d (open), and I = i n a c t i v a t e d (closed) drug-free f r a c t i o n s of the p o p u l a t i o n of sodium channels. R*, A*, and I 1 are the r e s p e c t i v e drug-associated f r a c t i o n s . The t r a n s i t i o n between the s t a t e s f o l l o w s Hodgkin and Huxley f i r s t - o r d e r k i n e t i c s w i t h voltage-dependent r a t e constants (HH) . HH' are the same r a t e constants s h i f t e d on the v o l t a g e a x i s f o r the drug-associated channels. k., , k2 , and k 3 are the a s s o c i a t i o n r a t e constants f o r the a n t i a r r h y t h m i c drug and k.i , k .2 , and k .3 are the d i s s o c i a t i o n r a t e c o n s t a n t s . (From Hondeghem and Katzung, 1977). 35 a n a l y s i s of c a r d i a c Na channels has allowed the f o l l o w i n g c o n c l u s i o n s t o be drawn (Fozzard et al, 1985): (1) Na + channel d e n s i t y i s 2-10/Mm2. Whether channel d i s t r i b u t i o n i s random or l o c a l i z e d i s not yet known (Aimers e t al., 1983; c i t e d i n Fozzard e t al., 1985); (2) Under c e r t a i n d e f i n e d c o n d i t i o n s , a s i n g l e Na + channel has a conductance of approximately 15 pS, which i s about 10 7 ions/sec per channel. With an open time of 1 msec, a s i n g l e channel event i s recorded by the movement of about 10 000 Na + i o n s ; (3) The Na + c u r r e n t changes i t s magnitude upon d e p o l a r i z a t i o n which increases p r o b a b i l i t y of opening and b y a somewhat longer mean open time (Rosen and S p i n e l l i , 1988). Studies have provided data suggesting t h a t t h e r e may be two p o p u l a t i o n s of Na + channels, based on channel c u r r e n t s , k i n e t i c s , and/or response t o TTX (Ten E i k e t al., 1984 ; c i t e d i n Fozzard e t al., 1985). 1.6.2.2 S u b c l a s s i f i c a t i o n of Class I Agents Despite the complexity of the c l a s s i f i c a t i o n of a n t i a r r h y t h m i c agents based on the c l a s s i c a l i o n channel b l o c k i n g p r o p e r t i e s , such agents have c o n s i s t e n t l y demonstrated a n t i f i b r i l l a t o r y and a n t i a r r h y t h m i c a c t i o n s a g a i n s t ischaemia-induced arrhythmias. The c l a s s I agents have a long h i s t o r y , both i n t h e i r use i n the treatment of a v a r i e t y of arrhythmias, and i n t h e i r development by m o d i f i c a t i o n of b a s i c chemical s t r u c t u r e s (Courtney, 1988). 36 However, endeavours t o improve on p r o t o t y p i c a l c l a s s I agents have not met w i t h much success d e s p i t e the i n t r o d u c t i o n of numerous new agents (Schlepper, 1989). Agents d i f f e r i n t h e i r mode of a c t i o n on Na + channels and i n other p r o p e r t i e s (Borchard e t al., 1989) such t h a t s u b c l a s s i f i c a t i o n w i t h i n t h i s group has s i m p l i f i e d matters and provided a d d i t i o n a l r e l e v a n t i n f o r m a t i o n f o r experimental and c l i n i c a l purposes. In reviewing the complex e l e c t r o p h y s i o l o g i c a l a c t i o n s of v a r i o u s new c l a s s I a n t i a r r h y t h m i c agents, Vaughan W i l l i a m s (1984b) proposed a s u b c l a s s i f i c a t i o n of t h i s group of drugs. Vaughan W i l l i a m s (1984b) suggested p l a c i n g l i d o c a i n e and f l e c a i n i d e i n one c l a s s , and q u i n i d i n e , procainamide and disopyramide i n another sub-class on the b a s i s of t h e i r e f f e c t s on APD. In 1979, H a r r i s o n et al. proposed a f u r t h e r d i v i s i o n ( i . e . the i n t r o d u c t i o n of the I c s u b c l a s s i f i c a t i o n ) of c l a s s I i n t o subgroups l a , l b , and l c ( H a r r i s o n et al., 1979). H a r r i s o n (1985) s u b c l a s s i f i e d c l a s s I agents p r i m a r i l y on the d i f f e r e n t e f f e c t s of c l i n i c a l c o n c e n t r a t i o n s of these drugs, on conduction i n the s p e c i a l i z e d conducting t i s s u e , and on v e n t r i c u l a r r e f r a c t o r i n e s s and r e p o l a r i z a t i o n . According t o H a r r i s o n (1985) ; c l a s s l a agents widen the QRS d u r a t i o n , slow conduction a t h i g h c o n c e n t r a t i o n s , prolong the Q-T i n t e r v a l and widen the AP. P r o t o t y p i c a l l a agents i n c l u d e q u i n i d i n e , procainamide, and disopyramide. C l a s s l b agents demonstrate l i m i t e d ( i f any) e f f e c t on QRS d u r a t i o n and conduction w h i l e 37 s h o r t e n i n g the Q-T i n t e r v a l and APD and e l e v a t i n g the f i b r i l l a t i o n t h r e s h o l d (Harrison, 1985). L i d o c a i n e , m e x i l e t i n e , t o c a i n i d e , and ethmozine are a l l l b agents. Class l c agents widen the QRS complex, slow conduction at low c o n c e n t r a t i o n s and have l i t t l e e f f e c t on r e p o l a r i z a t i o n and d u r a t i o n of the AP. Class l c agents produce small changes i n r e f r a c t o r i n e s s , e.g. f l e c a i n i d e , e n c a i n i d e , l o r c a i n i d e , propafenone, and i n d e c a i n i d e ( H a r r i s o n , 1985). These c h a r a c t e r i s t i c e l e c t r o p h y s i o l o g i c d i f f e r e n c e s can a l s o be seen c l i n i c a l l y (Milne et a l . , 1984). C r i t i c i s m c ould be made again s t H a r r i s o n ' s s u b c l a s s i f -i c a t i o n not only f o r compounds w i t h m u l t i p l e e l e c t r o p h y s i o l o g i c a c t i o n s such as amiodarone, but a l s o f o r drugs such as q u i n i d i n e and procainamide, which prolong r e p o l a r i z a t i o n of the AP at higher c o n c e n t r a t i o n s by mechanisms u n r e l a t e d t o sodium channel blockade (Harrison, 1985). Since a l l c l a s s 1 agents b l o c k f a s t sodium channels, i t has been p o s t u l a t e d t h a t the s u b c l a s s i f i c a t i o n i s not a fundamental one but one based on pharmacodynamics i n t h a t the o n l y d i f f e r e n c e between the subclasses i s i n the c o n c e n t r a t i o n necessary t o bl o c k Na + channels (Ha r r i s o n , 1985) . In 1983, Campbell subdivided c l a s s 1 agents based on t h e i r k i n e t i c s of onset and r a t e dependent depression of the maximum r a t e of d e p o l a r i z a t i o n (V m a x or MRD) in vitro (Campbell, 1983a, 1983b). Campbell's l a b o r a t o r y i n v e s t i g a t e d 3 new c l a s s I agents: e n c a i n i d e , f l e c a i n i d e , 38 and l o r c a i n i d e . These agents a l l markedly depressed conduction i n the H i s - P u r k i n j e system and v e n t r i c l e i n v i v o , not s u r p r i s i n g l y based on t h e i r e f f e c t s on V m a x in vitro (Campbell, 1983b). At t h e r a p e u t i c c o n c e n t r a t i o n s these agents d i d not prolong r e f r a c t o r i n e s s i n the atrium and v e n t r i c l e t o the extent expected (Campbell, 1983b). In a d d i t i o n , and common t o other c l a s s I agents, increased frequency of s t i m u l a t i o n p r o g r e s s i v e l y enhanced the depression of V m a x ( i . e . rate-dependent block) (Campbell, 1983b) . However, the r a t e at which V m a x was depressed f o l l o w i n g a sudden increase i n frequency was much slower than r e p o r t e d f o r other c l a s s I agents i n cu r r e n t c l i n i c a l use (Campbell, 1983b). Campbell (1983b) s t u d i e d nine c l a s s I a n t i a r r h y t h m i c agents and found t h a t they f e l l i n t o 3 well-demarcated subgroups based on onset k i n e t i c s of r a t e dependent block (RDB). While a l l agents produced comparable amounts of RDB, i t was p o s s i b l e t o separate them i n t o l b , l a , and l c subgroups based on the marked d i f f e r e n c e s observed i n the speed at which V m a x f e l l t o the new p l a t e a u l e v e l w i t h these d i f f e r e n c e s p e r s i s t i n g at a l l c o n c e n t r a t i o n s and d r i v i n g r a t e s s t u d i e d (Campbell, 1983b). l b agents ( l i d o c a i n e , t o c a i n i d e , and m e x i l e t i n e ) demonstrated f a s t onset k i n e t i c s ; l a ( q u i n i d i n e , disopyramide, and procainamide), i n t e r m e d i a t e ; and l c (enc a i n i d e , f l e c a i n i d e , and l o r c a i n i d e ) , slow onset k i n e t i c s (Campbell, 1983b). 39 A major f a c t o r i n determining the a b i l i t y of a c l a s s I drug t o prolong the ERP r e l a t i v e t o the APD i s the speed w i t h which the drug can f u r t h e r depress i n response t o a sudden i n c r e a s e i n s t i m u l a t i o n frequency ( i . e . the speed of onset of RDB) (Campbell, 1983b) . Reasons f o r these k i n e t i c d i f f e r e n c e s among c l a s s I drugs i n v o l v e s a number of aspects. The depression of alone i s i n s u f f i c i e n t t o b l o c k conduction of a propagated AP in v i t r o at a b a s i c d i a s t o l i c i n t e r v a l of 1000 ms (Campbell, 1983b). An a d d i t i o n a l f a c t o r must r e s u l t i n f u r t h e r depression of the number of Na + channels a v a i l a b l e at 300 ms ( i n a d d i t i o n to the depression a l r e a d y present at the time of the preceding b a s i c d r i v e beat) , such t h a t an AP w i l l not propagate and the t i s s u e would be, by d e f i n i t i o n , r e f r a c t o r y (Campbell, 1983b) . T h i s f a c t o r may be the a b i l i t y of a given drug t o f u r t h e r depress i n response t o a step i n c r e a s e i n r a t e which p e r s i s t s f o r one i n t e r s t i m u l u s i n t e r v a l (Campbell, 1983b). When the k i n e t i c d i f f e r e n c e s between subgroups are taken i n t o account, then drugs w i t h the f a s t e s t onset of RDB ( l b agents) w i l l produce the g r e a t e s t i n c r e a s e s i n ERP-APD90 (Campbell, 1983b). Agents w i t h f a s t k i n e t i c s have r e l a t i v e l y g r e a t e r e f f e c t s on r e f r a c t o r i n e s s than on APD thus c o n f e r r i n g s e l e c t i v i t y of l b agents f o r hig h frequency arrhythmias ( N a t t e l and Zeng, 1984; Varro e t al., 1985). Thus the d i f f e r e n c e s between drugs i n t h e i r onset k i n e t i c s at c o n c e n t r a t i o n s producing equal depression of V m a x , and 40 t h e i r a b i l i t y t o prolong ERP r e l a t i v e t o APD, can be seen to be a d i r e c t consequence of t h e i r d i f f e r i n g a b i l i t i e s t o prolong recovery from i n a c t i v a t i o n (Campbell, 1983b). The c l a s s I c agents may then be considered more potent s i n c e t h e i r longer o f f s e t k i n e t i c s w i l l produce a s t e a d i l y accumulating depression of without s i g n i f i c a n t i n t e r f e r e n c e from d i a s t o l i c recovery (Varro et a l . , 1985). However, they are a l s o more t o x i c s i n c e they can e x e r t t h e i r e f f e c t s at normal s i n u s rhythm r e s u l t i n g i n adverse s i d e e f f e c t s such as myocardial depression (Schlepper, 1989). C l a s s l b agents on the other hand, can be g i v e n at c o n c e n t r a t i o n s t h a t have l i t t l e e f f e c t on V m a x and hence conduction v e l o c i t y at normal heart r a t e s , but s e l e c t i v e l y depress conduction of premature AP's ( P a l l a n d i and Campbell, 1988; Varro et a l . , 1985). Class l a agents are l e s s able t o respond t o sudden changes of r a t e and thus depress conduction of normal beats at co n c e n t r a t i o n s t h a t depress premature beats ( P a l l a n d i and Campbell, 1988). This i s seen t o an even g r e a t e r extent w i t h I c agents ( P a l l a n d i and Campbell, 1988). Experimental s t u d i e s on c a r d i a c and nerve t i s s u e t h a t focus on molecular mechanisms of a c t i o n of c l a s s I agents have provided f u r t h e r i n s i g h t on the k i n e t i c d i f f e r e n c e s of c l a s s I agents (Campbell, 1983b). I t has been e s t a b l i s h e d t h a t the primary a c t i o n of c l a s s I drugs i s t o prolong recovery from i n a c t i v a t i o n , p o s s i b l y by b i n d i n g d i r e c t l y t o the i n a c t i v a t i o n g a t i n g mechanism (Hondeghem and Katzung, 41 1977; Courtney, 1980). In a d d i t i o n , before " l a r g e - s c a l e " drug b i n d i n g can occur, i t appears t h a t the channels must be i n the i n a c t i v a t e d s t a t e (Campbell, 1983b). Therefore, r e p e t i t i v e s t i m u l a t i o n enhances the b i n d i n g of c l a s s I drugs to the sodium channel by producing r e p e t i t i v e i n a c t i v a t i o n and i s known as the rate-dependent e f f e c t (Campbell, 1983b). Kodama's group (1986, 1987) have shown channel s t a t e s e l e c t i v i t y of blockade. They determined t h a t l a agents b l o c k the Na + channel mainly d u r i n g the upstroke of the AP ( i . e . a c t i v a t e d channel block) w h i l e l b agents do so mainly d u r i n g the p l a t e a u phase ( i n a c t i v a t e d channel block) (Kodama and Toyama, 1988). The importance of physico-chemical p r o p e r t i e s i n determining the k i n e t i c s of the e f f e c t s of the c l a s s I agents on maximum r a t e of d e p o l a r i z a t i o n have been examined (Campbell, 1983c). P r o p e r t i e s taken i n t o account i n c l u d e molecular weight, l i p o p h i l i c i t y , and the s p a t i a l dimensions of the molecule. Numerous s t u d i e s of s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s of c l a s s I agents have been p u b l i s h e d (Courtney, 1980). These v a r i o u s groups found t h a t the degree t o which c l a s s I agents could produce r e s t i n g b l ock ( i . e . depress i n unstimulated myocardium) c o r r e l a t e d w e l l w i t h the agent's l i p o p h i l i c i t y (from logP, the p a r t i t i o n c o e f f i c i e n t , and pKa). Poor c o r r e l a t i o n s have been found w i t h some other physico-chemical p r o p e r t i e s such as molecular weight (Campbell, 1983c). However as molecular weight i n c r e a s e d , the a f f i n i t y f o r the r e c e p t o r and the 42 p e r s i s t e n c e of drug a c t i o n a l s o increased (Campbell, 1983c). This i s normally a t t r i b u t e d t o g r e a t e r Van Der Waal's (and other) f o r c e s between drug and b i n d i n g s i t e (Campbell, 1983c) . The r a p i d i t y of onset of r a t e dependent b l o c k c o r r e l a t e d w i t h molecular weight; agents w i t h lower molecular weights being f a s t e r (Campbell, 1983c). The time constant of recovery from RDB on c e s s a t i o n of s t i m u l a t i o n a l s o c o r r e l a t e d w e l l w i t h molecular weight (Campbell, 1983c), t h i s c o r r e l a t i o n being improved by t a k i n g l i p o p h i l i c i t y ( l o g P and pKa) i n t o account (Campbell, 1983c). Permanently charged molecules were a s s o c i a t e d w i t h very slow recovery r e g a r d l e s s of molecular weight (Campbell, 1983c). V o i g t et a l . (1988) demonstrated t h a t the b i n d i n g of c l a s s I a n t i a r r h y t h m i c s t o p h o s p h a t i d y l c h o l i n e membranes was determined mainly by t h e i r l i p o p h i l i c i t y i r r e s p e c t i v e of pKa. Based on previous concepts and Campbell's (1983c) data on the physico-chemical p r o p e r t i e s of c l a s s I agents, Campbell proposed a model of the sodium channel and i t s i n t e r a c t i o n w i t h c l a s s I a n t i a r r h y t h m i c agents. Campbell (1983c) f e l t t h a t the c l a s s I agents act on a s i t e , p o s s i b l y w i t h i n , or f u n c t i o n a l l y a s s o c i a t e d w i t h the Na + channel, t h i s s i t e being a c c e s s i b l e from the c y t o s o l or hydrophobic membrane, but not d i r e c t l y from the e x t r a c e l l u l a r s i d e of the membrane. Furthermore, d e p o l a r i z a t i o n g r e a t l y enhances drug b i n d i n g t o the b i n d i n g s i t e (Campbell, 1983c). I f the d e p o l a r i z a t i o n i s r a p i d and r e p e t i t i v e , then the incre a s e d 43 b i n d i n g i s manifest as RDB, but i f a steady d e p o l a r i z a t i o n i s a p p l i e d , i t w i l l appear as an i n c r e a s e i n r e s t i n g b l ock (Campbell, 1983c). These are e s s e n t i a l l y two d i f f e r e n t aspects of a s i n g l e voltage-dependent process (Campbell, 1983c). D e s t r u c t i o n of the i n a c t i v a t i o n g a t i n g process by some technique which leaves the a c t i v a t i o n i n t a c t l a r g e l y e l i m i n a t e s t h i s v o l t a g e dependence, thus s t r o n g l y suggesting t h a t the b i n d i n g s i t e i s the i n a c t i v a t i o n gate or a s i m i l a r s t r u c t u r e (Cahalan, 1980; Shapiro and Aimers, 1980; c i t e d i n Campbell, 1983c). D e p o l a r i z a t i o n presumably changes the i n a c t i v a t i o n gate which exposes the r e c e p t o r s i t e t o the c y t o s o l so t h a t b i n d i n g i s f a c i l i t a t e d (Campbell, 1983b). R e a c t i v a t i o n i n response t o r e p o l a r i z a t i o n i s only p o s s i b l e i f the drug d i s s o c i a t e s from the b i n d i n g s i t e (Campbell, 1983b; Courtney, 1987). Another important f a c t o r i n determining the k i n e t i c s of o f f s e t of RDB of v a r i o u s c l a s s I agents aside from i o n i c charge (permanently charged agents have very long o f f s e t k i n e t i c s r e g a r d l e s s of molecular weight), and molecular weight i s s t e r e o s p e c i f i c i t y ( H i l l e t a l . , 1988). The o r i e n t a t i o n s of aromatic and amine groups on c l a s s I agents are important i n drug b i n d i n g t o the c a r d i a c Na + channel ( H i l l e t a l . , 1988). S t r u c t u r a l hypotheses have been proposed f o r c l a s s I agents w i t h respect t o t h e i r w i d e l y d i f f e r i n g a b i l i t i e s t o b l o c k myocardial Na + channels d u r i n g i n d i v i d u a l AP's and t h e i r a s s o c i a t e d r e p r i m i n g k i n e t i c s . The s i z e - s o l u b i l i t y 44 hypothesis j u s t d i s c u s s e d provides support f o r s m a l l e r a n t i a r r h y t h m i c drugs w i t h good l i p i d d i s t r i b u t i o n c a p a b i l i t i e s (e.g. Ib agents) e x h i b i t i n g r a p i d r e p r i m i n g k i n e t i c s (Courtney, 1990) . While the hypothesis seemed t o s u c c e s s f u l l y p r e d i c t the unblocking k i n e t i c s , i t was not c l e a r from a group of drugs t e s t e d by Courtney (1983), whether a high l i p i d s o l u b i l i t y would speed up recovery k i n e t i c s f o r a l a r g e r drug, or whether j u s t a low l i p i d s o l u b i l i t y would slow recovery k i n e t i c s f o r a s m a l l e r drug. Courtney (1983) suggested t h a t h y d r o p h i l i c drugs but not l i p o p h i l i c drugs show modulation of t h e i r size-dependent k i n e t i c s . Furthermore, the more t i g h t l y bound waters of h y d r a t i o n could be e f f e c t i v e l y adding t o the s i z e of such h y d r o p h i l i c drug molecules (Courtney, 1983). With the i n t r o d u c t i o n of new drugs, p r e d i c t i o n s based on a q u a n t i t a t i v e s t r u c t u r e - a c t i v i t y r e l a t i o n proposed by Courtney (1987) have been i n e r r o r . Such was the case w i t h a p u t a t i v e c l a s s I agent BW A256C which has an u n u s u a l l y high pKa (11.2) (Donoghue et al. , 1987). As a r e s u l t of t h i s f a i l u r e t o make c o r r e c t p r e d i c t i o n s r e g a r d i n g new drugs, Courtney (1990) looked at agents s t r u c t u r a l l y and t h r e e - d i m e n s i o n a l l y . He found t h a t the end-on view of the molecule, provided a b e t t e r e x p l a n a t i o n f o r the s i z e dependence of r e p r i m i n g k i n e t i c s than molecular weight thus o f f e r i n g a m o d i f i c a t i o n t o the s i z e - s o l u b i l i t y hypothesis. Courtney (1990) d e s c r i b e d a q u a n t i t a t i v e model f o r recovery time which coupled proton exchange r a t e s w i t h a d r u g - s i z e 45 dependent process r e l a t e d t o recovery from Na channel i n a c t i v a t i o n . Thus drugs having a wider span a t t h e i r aromatic recovery k i n e t i c s from i n a c t i v a t i o n (Courtney, 1990). C l i n i c a l l y , the main i n t e r e s t of s u b d i v i s i o n of c l a s s I agents, based on p u r e l y o b j e c t i v e e l e c t r o p h y s i o l o g i c a l measurements in vitro, i s t h a t the q u a n t i t a t i v e d i s p a r i t i e s i n such c h a r a c t e r i s t i c s as frequency-(or rate) dependency, voltage-dependency, and time-dependency to the channels may account f o r observed d i f f e r e n c e s i n the c l i n i c a l p r o f i l e s of c l a s s I agents (Vaughan W i l l i a m s , 1984b). Subgrouping e x p e r i m e n t a l l y then may p o s s i b l y c o r r e l a t e w i t h t h a t determined c l i n i c a l l y (Vaughan W i l l i a m s , 1984a). 1.7 Quinacainol Quinacainol ( l - [ 2 - ( 1 , 1 - d i m e t h y l e t h y l ) - 4 - q u i n o l y l ] - 3 - ( 4 -p i p e r i d y l ) - 1 - p r o p a n o l ) (Figure 2) was developed by Rhone Poulenc Sante i n an e f f o r t t o d i s c o v e r a more e f f e c t i v e and l e s s t o x i c drug f o r the treatment of c a r d i a c arrhythmias. A p r e l i m i n a r y r e p o r t produced by Rhone Poulenc Sante (PK 10139, 1984) summarized a number of pharmacological s t u d i e s w i t h q u i n a c a i n o l . These s t u d i e s assessed q u i n a c a i n o l 1 s pharmacokinetics, i t s e f f e c t s i n conscious and a n a e s t h e t i s e d animals, i t s t o x i c o l o g i c a l p r o p e r t i e s , and i t s c l i n i c a l e f f e c t i v e n e s s . 46 Figure 2. The structure of quinacainol, ( l - [ 2 - ( l , l -dimethylethyl)-4-quinolyl]-3-(4-piperidyl)-1-propanol). It has a pKa of 10.8, the empirical formula is C 2 1 H 3 0 N 2o / and i t has a molecular weight of 326.4 g/mole. 47 Pharmacokinetic r e s u l t s showed t h a t the u r i n a r y and faecal routes were e q u a l l y important f o r e l i m i n a t i o n ; unchanged q u i n a c a i n o l was the l a r g e s t u r i n a r y and b i l i a r y component w i t h only s m a l l amounts of conjugated d e r i v a t i v e s found. At l e a s t 4 metabolites were found i n plasma and u r i n e although t h e i r s t r u c t u r e s s t i l l need t o be determined. In conscious and anaesthetised dogs as w e l l as i n anaesthetised r a t and r a b b i t , q u i n a c a i n o l was w e l l t o l e r a t e d . Q u i n a c a i n o l 1 s e f f e c t s on P-R i n t e r v a l and QRS d u r a t i o n were seen from the lowest doses up to approximately t w i c e the 100% a c t i v e dose. Quinacainol d i d not compromise blood pressure but reduced c o n t r a c t i l i t y s l i g h t l y . The a n t i a r r h y t h m i c a c t i v i t y of q u i n a c a i n o l was assessed i n mice, r a t s , or dogs on arrhythmias induced by v a r i o u s methods. Quinacainol was potent and lacked d e l e t e r i o u s s i d e e f f e c t s i n comparison w i t h other c l a s s I a n t i a r r h y t h m i c agents. The e l e c t r o p h y s i o l o g i c a l a c t i o n s of q u i n a c a i n o l were assessed i n a number of d i f f e r e n t models. Concentrations of q u i n a c a i n o l above 10"6 M decreased amplitude and dV/dt i n sheep P u r k i n j e f i b r e s , a depression of p l a t e a u phase (60% r e p o l a r i z a t i o n ) , s hortening of the APD and a s l i g h t decrease i n RMP. Quinidine was t e s t e d i n a s i m i l a r manner f o r comparative purposes and i t s e f f e c t s were completely reversed w h i l e p a r t i a l e f f e c t s of q u i n a c a i n o l could s t i l l be seen 1 h l a t e r . S i m i l a r e f f e c t s were seen i n sheep v e n t r i c u l a r f i b e r s and canine a t r i a l f i b e r s f o r 48 q u i n i d i n e and q u i n a c a i n o l although both produced a s l i g h t i n c r e a s e i n APD i n these p r e p a r a t i o n s . The acute t o x i c e f f e c t s of q u i n a c a i n o l (LD 5 0 ) were s i m i l a r t o other c l a s s I (la) agents. Quinacainol was devoid of e f f e c t s on the CNS and autonomic nervous system. 1.8 O b j e c t i v e s H i s t o r i c a l l y , c l a s s I agents have proven t o have a n t i a r r h y t h m i c and a n t i f i b r i l l a t o r y p r o p e r t i e s a g a i n s t ischaemia-induced arrhythmias. Recent endeavours t o improve upon p r o t o t y p i c a l agents have not met w i t h much success. Drugs have been produced w i t h the app r o p r i a t e l b p r o f i l e , f a s t k i n e t i c s of blockade, e t c . , but these advantages have not t r a n s l a t e d i n t o i d e a l drugs (Impact Research Group, 1984). C l a s s l b agents penetrate the b r a i n and produce CNS-r e l a t e d s i d e - e f f e c t s (Davy et al., 1988). In an analogous manner, there have been developments i n c l a s s I c agents, but r e c e n t l y newer c l a s s I c agents have come under a cl o u d of s u s p i c i o n as a r e s u l t of the much discussed CAST t r i a l (1989) . C l i n i c a l s t u d i e s w i t h c l a s s I c agents have been terminated and researc h on t h i s subgroup has been reduced (Woosley, 1990). A recent meta-analysis by Hine e t al. (1989) supported the c o n c l u s i o n t h a t the e n t i r e c l a s s of c l a s s I a n t i a r r h y t h m i c agents cause increased m o r t a l i t y . In view of such d i f f i c u l t i e s , i t i s not s u r p r i s i n g t h a t a t t e n t i o n has been d i r e c t e d t o developing b e t t e r 49 a n t i a r r h y t h m i c s from other c l a s s e s . Most notable are the c l a s s I I I a n t i a r r h y t h m i c agents. I n t e r e s t i n g l y , many of the newer drugs t h a t have been developed as c l a s s I I I a n t i a r r h y t h m i c s were developed by chemical manipulations which change sodium channel b l o c k e r s i n t o potassium channel b l o c k e r s (Lumma e t al., 1990). However, the question remains as t o whether the i d e a l a n t i f i b r i l l a t o r y drug can be developed from c l a s s I I I agents. Studies w i t h i n our l a b o r a t o r y have shown t h a t a n t i - f i b r i l l a t o r y a c t i o n s occur only a f t e r massive i n c r e a s e s i n APD (Beatch et a l . , 1990). This b r i n g s us back then t o the c l a s s I agents. Perhaps a r e a l p o s s i b i l i t y e x i s t s of developing a c l a s s I a n t i a r r h y t h m i c agent which a f f o r d s e x c e l l e n t p r o t e c t i o n a g a i n s t arrhythmias w i t h i n the s e t t i n g of myocardial ischaemia without i n c u r r i n g s i g n i f i c a n t c a r d i o v a s c u l a r t o x i c i t y w h i l e demonstrating s e l e c t i v i t y f o r the ischaemic myocardium. The most r e c e n t l y marketed c l a s s I c agents ( q u i n a c a i n o l 1 s p u t a t i v e s u b c l a s s i f i c a t i o n ) have been noted t o have potent e f f e c t s i n the treatment of v e n t r i c u l a r arrhythmias but are l i m i t e d by t h e i r c a r d i a c and non-cardiac t o x i c e f f e c t s (Kreeger and Hammill, 1987; Nathan et al., 1985) . Our experiments were designed t o r e v e a l the u n d e r l y i n g e l e c t r o p h y s i o l o g i c a l a c t i o n s of q u i n a c a i n o l and the appropriateness of i t s p u t a t i v e a n t i a r r h y t h m i c c l a s s i f i c a t i o n , i . e . I c (PK 10139, 1984). The e l e c t r o p h y s i o l o g i c a l s t u d i e s were chosen t o show how these 50 a c t i o n s may be t r a n s f e r r e d t o e f f e c t s on responses t o e l e c t r i c a l s t i m u l a t i o n . The r e l a t i v e t h e r a p e u t i c u s e f u l n e s s of q u i n a c a i n o l was assessed by comparing i t s e f f i c a c y a g a i n s t ischaemia-induced arrhythmias t o i t s t o x i c e f f e c t s . E f f i c a c y a g a i n s t e l e c t r i c a l l y - i n d u c e d arrhythmias c o u l d be compared w i t h e f f i c a c y a g a i n s t ischaemia-induced arrhythmias. 51 2 METHODS 2. l Haemodynamic Studies i n Conscious Rats The dose-response r e l a t i o n s h i p i s e s s e n t i a l i n studying drug a c t i o n s . T h i s experiment was designed t o study the haemodynamic e f f e c t s of q u i n a c a i n o l , u n d e r l y i n g t o x i c e f f e c t s , and t o a s c e r t a i n t h e r a p e u t i c r a t i o s and ED 5 0 's i n the r a t . As a l l s t u d i e s were performed i n the same species under only two c o n d i t i o n s , conscious or p e n t o b a r b i t a l anaesthetised, e x t r a p o l a t i o n w i t h respect t o the doses used i n the v a r i o u s s t u d i e s was p o s s i b l e due t o s i m i l a r i t y of c o n d i t i o n s i n d i f f e r e n t models. 2.1.1 Preparation Blood pressure and drug i n f u s i o n cannulae f o r permanent i m p l a n t a t i o n were modeled a f t e r Weeks (1981) and t h e i r manufacture has been described elsewhere (Johnston et al. , 1981, 1983; C u r t i s , 1986; Igwemezie, 1990). F o l l o w i n g i n d u c t i o n of anaesthesia w i t h 5% halothane i n oxygen d e l i v e r e d by a vap o u r i z e r t o a g l a s s chamber, male Sprague Dawley r a t s (245-34 0 g) were in t u b a t e d w i t h a 14 gauge human intravenous c a t h e t e r w i t h the a i d of a modified p a e d i a t r i c laryngoscope. The i n t u b a t i o n tube was secured and the anaesthetic maintained w i t h 1-1.5% halothane throughout the remainder of the surgery. A m i d l i n e 52 laparotomy was performed and the i n f e r i o r vena cava and the abdominal a o r t a cannulated f o r drug a d m i n i s t r a t i o n and blood pressure r e c o r d i n g , r e s p e c t i v e l y . These v e s s e l s were used (as opposed t o c a r o t i d , j u g u l a r , or femoral blood v e s s e l s ) because they are l a r g e , e a s i l y a c c e s s i b l e , and do not tend to become ob s t r u c t e d by blood or muscle t i s s u e ( C u r t i s , 1986). The cannulae were pushed through the back muscles and routed subcutaneously u s i n g a t r o c a r t o the mid-scapular r e g i o n and e x t e r i o r i s e d . The abdomen was dusted w i t h C i c a t r i n " a n t i b i o t i c powder and the abdominal muscle was c l o s e d u s i n g a continuous suture (4-0 s i l k ) . The l o c a l anaesthetic, Marcaine" was l i b e r a l l y a p p l i e d p r i o r t o s u t u r i n g the s k i n w i t h discontinuous sutures. The cannulae were heat-sealed f o l l o w i n g i n j e c t i o n of 0.5-1.0 ml of s a l i n e to ensure patency. ECG leads (prepared from t e f l o n coated s t a i n l e s s s t e e l w i re, 0.0001 cm diameter, w i t h the i n s u l a t i o n removed from e i t h e r end) were next placed i n a lead I I c o n f i g u r a t i o n u s i n g a 21 guage needle. These were t w i s t e d together and secured t o the s k i n . A chest lead was i n s e r t e d through the p e c t o r a l i s major muscle and e x t e r i o r i s e d i n the mid-scapular r e g i o n and secured t o the s k i n . The anaesthetic was d i s c o n t i n u e d . Animals were l e f t u n t i l i t was evident the anaesthesia was wearing o f f a t which time the t r a c h e a l tube was removed. Animals were p l a c e d i n separate cages i n a temperature and l i g h t - c o n t r o l l e d animal 53 room, given r a t chow and water ad l i b i t u m and allowed t o recover from surgery f o r 5 t o 7 days. 2.1.2 Experimental Design Animals (n=6) re c e i v e d a randomly chosen dose (or v e h i c l e ) every 48 h over a p e r i o d of 10 days. Using a b l i n d , random, cross-over design 1, 2, 4, or 8 mg/kg i . v . doses of q u i n a c a i n o l or v e h i c l e alone, were each given as an i n f u s i o n over 10 min. Dosing was performed on a l t e r n a t e days as a pre c a u t i o n a r y measure because q u i n a c a i n o l produces at l e a s t 4 met a b o l i t e s which disappear w i t h i n 36 h (PK 10139, 1984). In some animals a f i n a l dose of 16 mg/kg was given. The drug was d i s s o l v e d i n 26% ethanol i n s a l i n e . This v e h i c l e acted as the c o n t r o l . The v e h i c l e f o r the 16 mg/kg dose of q u i n a c a i n o l was 40% ethanol i n s a l i n e . Rats were brought i n t o the l a b o r a t o r y 1 h p r i o r t o beginning the experiment. The a o r t i c cannula was connected t o a Grass Polygraph (model 79D) and kept open by a t t a c h i n g a le a k pump i n s e r i e s w i t h the l i n e according t o Weeks' method (1981). ECG leads were a l s o connected t o the Grass Polygraph. Animals were allowed t o s t a b i l i s e f o r 3 0 min p r i o r t o s t a r t i n g the experiment. Blood pressure and ECG were recorded d u r i n g t h i s time and f o r 20 minutes f o l l o w i n g dosing. In a d d i t i o n t o r e c o r d i n g c a r d i o v a s c u l a r responses to q u i n a c a i n o l , t o x i c e f f e c t s were a l s o noted throughout the experiment and du r i n g post-mortem a n a l y s i s of i n t e r n a l 54 organs. D r i s c o l l (1981) s t r e s s e d t h a t the use or the non-use of anaesthesia (e.g. telemetry, r e s t r a i n t , etc.) i s perhaps the most important f a c t o r having a d i r e c t b e a r i n g on t o x i c o l o g i c a l t e s t i n g as i t p e r t a i n s t o the r a t ECG. 2.1.3 Data Analysis The P, QRS, and T d e f l e c t i o n s i n the r a t ECG u s u a l l y do not share a common b a s e l i n e ; any given wave may terminate at a l e v e l d i f f e r e n t from t h a t of i t s o r i g i n ( D r i s c o l l , 1981). In order t o measure the magnitudes of the v a r i o u s d e f l e c t i o n s i t i s necessary t o choose a refe r e n c e p o i n t f o r determination of the i s o e l e c t r i c b a s e l i n e ( D r i s c o l l , 1981). The best r e f e r e n c e p o i n t i s t h a t p o i n t a t which the P-R i n t e r v a l terminates and the QRS complex begins ( D r i s c o l l , 1981). This p o i n t i s i n f l u e n c e d the l e a s t by other e l e c t r i c a l events which may occur during the c a r d i a c c y c l e ( B e i n f i e l d and Lehr, 1968; c i t e d i n D r i s c o l l , 1981). As i s common w i t h most leads of the r a t ECG, the Q wave i s absent i n most cases. T h i s p e c u l i a r i t y , along w i t h the l a c k of a d e f i n i t e S-T segment and the occurrence of an asymmetric T wave has been d i s c u s s e d e x t e n s i v e l y i n the l i t e r a t u r e (reviewed i n D r i s c o l l , 1981) and has been w i d e l y s t u d i e d . A composite of a normal r a t ECG i s given i n F i g u r e 3 which notes the p o i n t s taken t o measure ECG v a r i a b l e s . The Q-T i n t e r v a l was measured from the Q wave t o the mid-peak of the T wave as the T wave was o f t e n i l l - d e f i n e d w i t h respect t o 55 R ' R interval Q T i n t e r v a l Figure 3. A composite drawing of a- t y p i c a l ECG trace from the r a t . Two normal cycles are shown. Dashed l i n e s represent i s o e l e c t r i c baselines f o r each. Also shown are the points used f o r measurement of the i n t e r v a l s indicated. (Reproduced from D r i s c o l l , 1981). The Q-T i n t e r v a l measurement was modified; instead of using the end-point of the T wave ( D r i s c o l l , 1981), the mid-point was used as shown above. 56 an obvious upstroke or downstroke. The Q-T i n t e r v a l was corrected for heart rate (QT C), QTC = Q-T i n t e r v a l (sec)/yR-R i n t e r v a l (sec) due to the consistent differences i n the heart rate seen between anaesthetised and non-anaesthetised rats ( D r i s c o l l , 1981). In a l l experiments with quinacainol the peak of the s y s t o l i c wave was measured and the lowest point of the d i a s t o l i c was measured. For the haemodynamic study mean blood pressure was calculated from the following formula: Mean BP = s y s t o l i c BP + V 3 ( s y s t o l i c BP - d i a s t o l i c BP) . For a l l other experiments the average of s y s t o l i c and d i a s t o l i c pressures was taken to be the mean blood pressure. 2.2 Ischaemia-induced Arrhythmias 2.2.1 Experimental Preparation Male Sprague Dawley rats (270-350 g) were prepared as described i n 2.1.1 except for the addit i o n a l s u r g i c a l preparation needed for implantation of the occluder. Cannulae were implanted f i r s t followed by an occluder. We routinely study the e f f e c t s of myocardial ischaemia i n chronic, conscious rats and consequently have designed a snare device which can be implanted and l e f t i n t a c t for up to 7 days. The occluder, manufactured from polyethylene, was f i r s t described by Au et al. (1979) and Johnston et al., (1983) . I t s design and manufacture have been extensively 57 d e s c r i b e d elsewhere ( C u r t i s , 1986; Igewemezie, 1990). A 5.0 gauge atraumatic polypropylene suture (Ethicon) was threaded through the p o l y e t h y l e n e guide such t h a t the needle end of the suture appeared at the f l a r e d end of the guide. The i m p l a n t a t i o n of the occluder has a l s o been de s c r i b e d i n d e t a i l elsewhere (Johnston e t a l . , 1983; C u r t i s , 1986). B r i e f l y , an o c c l u s i o n s i t e was chosen to produce an ischaemic zone 35-40% of t o t a l v e n t r i c u l a r mass. The polypropylene suture of the occluder was looped under the l e f t a n t e r i o r descending coronary a r t e r y . T h i s was done b l i n d l y s i n c e the v e s s e l could not be seen w i t h the naked eye, thus we r e l i e d upon knowledge of the anatomy of the r a t heart and when v i s i b l e , we were able t o use the coronary v e i n s as landmarks (see Figure 4) . A wide loop (approximately 4 mm) was made i n the v e n t r i c u l a r muscle to ensure t h a t the a r t e r y was occluded. Minor b l e e d i n g was apparent i n l e s s than 15% of cases. Any b l e e d i n g was stopped by a l l o w i n g the blood t o c l o t and the t h o r a c i c c a v i t y was c l e a r e d of excess blood. A f t e r i m p l a n t a t i o n of the coronary a r t e r y occluder the lungs were h y p e r i n f l a t e d d u r i n g c l o s u r e of the chest by b l o c k i n g the e x h a l a t i o n tube f o r 3 consecutive breaths i n order t o avoid a pneumothorax. F o l l o w i n g completion of surgery, the r a t s were placed i n i n d i v i d u a l r a t cages and given tap water ad l i b i t u m . The r a t s remained i n the l a b o r a t o r y u n t i l the anaesthesia had worn o f f . They were placed i n a separate room used only t o house r a t s and g i v e n r a t chow and water ad l i b i t u m . The Figure 4. Diagram of the r a t heart showing approximate placement of the occluder around the l e f t a n t e r i o r descending coronary a r t e r y (reproduced from Igwemezie, 1990). 59 e n t i r e s u r g i c a l procedure took 40-60 min t o complete from i n d u c t i o n of anaesthesia t o recovery. 2.2.2 Experimental Design S i x t o e i g h t days f o l l o w i n g surgery, r a t s were brought i n t o the l a b o r a t o r y i n t h e i r home cages 2 h before l i g a t i o n and allowed f r e e access t o food and water. ECG and cannulae were connected. Blood samples (1 ml) were taken t o measure serum potassium l e v e l s ( I o n e t i c s Potassium Analyzer) p r i o r t o drug i n f u s i o n and 4 h p o s t - o c c l u s i o n i n those animals s u r v i v i n g t o t h i s time. Recordings at f a s t paper speed (100 mm/sec) were taken 15 min and 5 min p r i o r t o drug i n f u s i o n once c a l i b r a t i o n was completed. These served as c o n t r o l p e r i o d s f o r data analyses. The b a s i c design was a 3x10 randomized block design w i t h replacement of excluded animals. Our c r i t e r i a f o r e x c l u s i o n were based on the Lambeth Conventions (1988) and have been d e t a i l e d by C u r t i s (1986). In a d o u b l e - b l i n d and random manner, a p p r o p r i a t e s o l u t i o n s were given by i . v . i n f u s i o n over a 10 min p e r i o d i n a t o t a l volume of 2 ml or l e s s . T h e r e a f t e r , a f u r t h e r 5 min elapsed before o c c l u s i o n . O c c l u s i o n was performed as de s c r i b e d by C u r t i s (1986). U s u a l l y , o c c l u s i o n causes a sudden change i n the ECG but t h i s c ould not be used as a c r i t e r i o n f o r o c c l u s i o n s i n c e i t was p o s s i b l e t h a t drug treatment could mask or at l e a s t delay the ECG changes caused by o c c l u s i o n . Rats were monitored c o n t i n u o u s l y f o r 4 60 h p o s t - o c c l u s i o n or u n t i l death. Recordings a t f a s t paper speed were taken every min f o r the f i r s t 10 min, every 5 min f o r the f o l l o w i n g 20 min, then every 15 min. A l l r a t s s u r v i v i n g 4 h were s a c r i f i c e d by concussion and exsanguination and occluded zone determined. The s i z e of the occluded zones i n the hearts were a l s o determined i n animals dying p r i o r t o 4 h. The heart was e x c i s e d t a k i n g care not t o cut through the occluder and the occluded zone was determined as f o l l o w s . The heart was perfused v i a the a o r t a according t o Langendorff (1895) w i t h 0.9% s a l i n e f o l l o w e d by s a l i n e c o n t a i n i n g indocyanine (Fast Green dye, BDH) 0.5 g/1. Approximately 30-60 s was necessary t o a l l o w the dye t o pass through the coronary c i r c u l a t i o n t o s t a i n the perfused t i s s u e a dark green c o l o u r and leave the ischaemic area (occluded zone) an opaque c o l o u r . The occluded zone was cut away from the normal or non-occluded v e n t r i c u l a r t i s s u e and each was weighed a f t e r b l o t t i n g t o remove excess p e r f u s a t e ; the occluded zone could be expressed as a percent of v e n t r i c u l a r weight. Arrhythmias, arrhythmias score (discussed below), and m o r t a l i t y were a l s o recorded along w i t h the blood pressure and ECG. A l l responses (to drug and coronary occlusion) were monitored w i t h the a i d of permanent re c o r d i n g s of the ECG. For c l a s s i f i c a t i o n and q u a n t i f i c a t i o n of arrhythmias dur i n g the experiment, a delayed loop o s c i l l i s c o p e (Honeywell type E f o r M) w i t h a 4 sec delay and a 4 sec r e a l time d i s p l a y was used. 61 2 . 2 . 3 E C G C h a n g e s P r e a n d P o s t O c c l u s i o n The ECG was recorded p r i o r t o drug i n f u s i o n , f o l l o w i n g i n f u s i o n and 1 min p r i o r t o o c c l u s i o n so t h a t the f u l l e l e c t r o p h y s i o l o g i c a l e f f e c t s of the drug could be assessed. C h a r a c t e r i s t i c ECG changes produced by o c c l u s i o n i n c l u d e an immediate and r a p i d r i s e i n the he i g h t of the R wave and a slower but c o n s i s t e n t e l e v a t i o n of S-T segment. A Q wave occurs i n the chest l e a d approximately 2 h f o l l o w i n g o c c l u s i o n . A s i g n i f i c a n t Q wave has been d e f i n e d as an i n i t i a l downward d e f l e c t i o n from the i s o e l e c t r i c p o t e n t i a l approximately 10% of the R wave amplitude ( C u r t i s , 1986) . The time a t which t h i s was noted was recorded. R wave amplitude was e a s i l y measured (mV) from the i s o l e c t r i c t o the peak of the R wave. Maximum R wave amplitude was a l s o recorded as was the time at which t h i s occurred. Before o c c l u s i o n the S wave i s negative t o the i s o e l e c t r i c p o t e n t i a l so t h a t values of S-T segment e l e v a t i o n are negative as w e l l . A l l negative values were assigned a value of zero. The e l e v a t i o n of the S-T segment was expressed as a percent of R wave amplitude. Maximum value of the S-T segment e l e v a t i o n were a l s o determined. 62 2.2.4 Ischaemia-induced Arrhythmias Commonly Seen with Occlusion V a r i a t i o n s e x i s t w i t h r e s p e c t t o d e f i n i n g t h e v a r i o u s a r r h y t h m i a s seen w i t h o c c l u s i o n o f t h e LAD c o r o n a r y a r t e r y . A v a r y i n g amount o f s u b j e c t i v i t y i s i n e v i t a b l e i n d e t e r m i n i n g what a r r h y t h m i a s a r e seen. However, b l i n d and random d e s i g n s r e s u l t i n random d i s t r i b u t i o n o f t h e i n c o n s i s t e n c i e s i n d e f i n i n g t h e a r r h y t h m i a s . The f o l l o w i n g d e f i n i t i o n s were used i n t h e d i a g n o s i s o f a r r h y t h m i a s f o l l o w i n g o c c l u s i o n o f t h e LAD i n r a t s . A premature v e n t r i c u l a r c o n t r a c t i o n (PVC) was d e f i n e d as a premature QRS complex o c c u r r i n g i n d e p e n d e n t o f a P wave, n o r m a l l y accompanied by a t r a n s i e n t drop i n b l o o d p r e s s u r e . S i n g l e t s , d o u b l e t s ( b i g e m i n y ) , and t r i p l e t s were c o u n t e d as PVC's w h i l e l o n g e r r u n s (4 o r more) were r e c o r d e d as v e n t r i c u l a r t a c h y c a r d i a . T h i s p o o l i n g o f s i n g l e t s , d o u b l e t s and t r i p l e t s i m p l i e s t h a t t h e y were one and t h e same a r r h y t h m i a . However, t h e s e a r r h y t h m i a s were a s s o c i a t e d o n l y f o r c o n v e n i e n c e i n a n a l y s i s . I n f a c t , t h e i n c i d e n c e o f b i g e m i n y and t r i p l e t s i s v a r i a b l e and l o w e r t h a n t h e i n c i d e n c e o f PVC's. However t h e s t u d y group s i z e would have t o be e n l a r g e d t o i n v e s t i g a t e t h e d r u g e f f e c t s on each a r r h y t h m i a j u s t mentioned. I n t h e absence o f c l e a r e v i d e n c e o f a common e l e c t r o p h y s i o l o g i c a l mechanism amongst PVC's, d o u b l e t s , and t r i p l e t s and knowledge o f t h a t p o i n t i n w h i c h 63 a r u n o f PVC's s h o u l d a c t u a l l y be c o n s i d e r e d a s h o r t r u n o f VT, we a r b i t r a r i l y d e f i n e d PVC and VT. VT was d e f i n e d as 4 o r more v e n t r i c u l a r e c t o p i c b e a t s (PVC's) i n s u c c e s s i o n . No r e s t r i c t i o n was made on a s s o c i a t e d r a t e . VT was s u b d i v i d e d based upon d u r a t i o n , i n t o s p o n t a n e o u s l y r e v e r t i n g VT (SVT), w h i c h l a s t e d l e s s t h a n 10 s, and n o n - s p o n t a n e o u s l y r e v e r t i n g VT (NSVT) i n w h i c h , a f t e r 10 s, c a r d i o v e r s i o n was a t t e m p t e d by f l i c k i n g t h e c h e s t w i t h a f o r e f i n g e r t o a t t e m p t t o c o n v e r t t h e h e a r t back t o s i n u s rhythm. VF was d e f i n e d as c h a o t i c d i s o r d e r i n t h e ECG ( i . e . no i d e n t i f i a b l e QRS complex) accompanied by a p r e c i p i t o u s f a l l i n BP ( e s s e n t i a l l y , z e r o c a r d i a c o u t p u t ) . As i n VT, d e f i b r i l l a t i o n was a t t e m p t e d i n a l l r a t s e x p e r i e n c i n g g r e a t e r t h a n 10 s o f VF, and c o u l d t h e r e f o r e be d i f f e r e n t i a t e d i n t o s p o n t a n e o u s l y r e v e r t i n g VF (SVF, <10 s and n o t r e q u i r i n g d e f i b r i l l a t i o n ) , and n o n - s p o n t a n e o u s l y r e v e r t i n g VF (NSVF, >10 s, r e q u i r i n g d e f i b r i l l a t i o n ) . VT sometimes d e g e n e r a t e d i n t o VF; b o t h e p i s o d e s were c o u n t e d . F i g u r e 5 i l l u s t r a t e s a t y p i c a l ECG p a t t e r n i n d u c e d by c o r o n a r y o c c l u s i o n and examples o f t h e a r r h y t h m i a s d i s c u s s e d above. 2.2.5 Rationale and Methods of D e f i b r i l l a t i o n Manual d e f i b r i l l a t i o n was a t t e m p t e d t o c o n v e r t a l l e p i s o d e s o f VT o r VF l a s t i n g l o n g e r t h a n 10 s t o a v o i d 64 Examples of Arrhythmias and ECG Changes A ECC ST'elevation T . note R ~av« 1 \ WOr BP Occlusion Xx^l KOI ^^Cg^BQgonMi v/x > y \ >A ^ ^ Control R wave increase with »2 +5_ * 3 0 + 2 h , 4 f c occlusion Change postfigation B Typical PVC A V Dissociation ECG til * Bradycardia BP C Ventricular Tachycardia • E C C > . . Flutter D . Ventricular Fibrillatii V Torsade de pointes Fibrillation Figure 5. T y p i c a l ECG pattern induced by l i g a t i o n of the l e f t a n t e r i o r descending (LAD) coronary artery and appearance of arrhythmias. Panel A shows ECG and blood pressure traces with R-wave increase, S-T segment changes, and Q-wave appearance. Typical arrhythmias of PVC and A-V d i s s o c i a t i o n (bradycardia) are shown i n panel B, VT i n C, and VF i n D. The horizontal bars i n d i c a t e 1 s at various chart speeds. (Reproduced from Johnston et a l , 1983). 65 e x c e s s i v e c e n s o r i n g due t o d e a t h f o l l o w i n g VF. I t i s e x p e c t e d t h a t d e a t h due t o VF w i l l be h i g h e r i n c o n t r o l g r oups when t e s t i n g a n t i f i b r i l l a t o r y d r u g s , such t h a t t h e group s i z e may become so s m a l l as t o have t o f o r e g o m e a n i n g f u l c o m p a r i s o n s . A l s o , t h e e x p e r i m e n t was s e t up t o examine t h e e f f e c t s o f a n t i f i b r i l l a t o r y d r u gs o v e r t i m e . Much i n f o r m a t i o n c o n c e r n i n g t h e t i m e c o u r s e and i n t e r r e l a t i o n s h i p s o f v a r i a b l e s i s l o s t as a n i m a l s d i e d u r i n g t h e c o u r s e o f t h e s t u d y . F o r a l l t h e s e r e a s o n s , d e f i b r i l l a t i o n was a t t e m p t e d i n a l l e p i s o d e s o f any VT o r VF l a s t i n g l o n g e r t h a n 10 s. D e f i b r i l l a t i o n was a c c o m p l i s h e d by l i f t i n g t h e r a t and f l i c k i n g t h e c h e s t d i r e c t l y o v e r t h e h e a r t w i t h a f o r e - f i n g e r u n t i l s i n u s rhythm r e t u r n e d . N o r m a l l y , l e s s t h a n 10 f l i c k s were r e q u i r e d ; however, i n t h o s e r a t s where " t h u m p - v e r s i o n " was n o t s u c c e s s f u l , t h e c h e s t was compressed w i t h t h e thumb and 2 f o r e f i n g e r s t o d e f i b r i l l a t e . R e v e r s i o n was d i a g n o s e d by a sudden i n c r e a s e i n a o r t i c BP and r e t u r n t o s i n u s rhythm. VF n o r m a l l y p r o d u c e d c o n v u l s i o n s w i t h i n a few seconds o f i t s i n i t i a t i o n , and syncope q u i c k l y f o l l o w e d . T h i s was n o t a p p a r e n t w i t h e p i s o d e s o f VT. 2.2.6 Scoring System for Arrhythmia Analysis I n o r d e r t o f a c i l i t a t e a n a l y s i s o f a r r h y t h m i a s , p a r t i c u l a r l y w i t h m y o c a r d i a l ischaemia, a number o f a r r h y t h m i a s c o r e s have been d e v e l o p e d ( C u r t i s and Walker, 66 1988). I t i s p o s s i b l e t o design many arrhythmia scores t h a t w i l l show changes i n s e v e r i t y of arrhythmias when more conv e n t i o n a l analyses show only n o n - s t a t i s t i c a l l y s i g n i f i c a n t trends ( C u r t i s and Walker, 1988). The Lambeth Conventions (1988) recommends a cautionary use of arrhythmia scores. Arrhythmia scores should be considered only i n r e l a t i o n t o dose-response data and i n co n j u n c t i o n w i t h the raw arrhythmia data i n order t o avoid f a l s e c laims f o r a drug's e f f e c t i v e n e s s ( C u r t i s and Walker, 1988). C u r t i s and Walker (1988) examined seven s c o r i n g systems. The f o l l o w i n g s c o r i n g system was used. S c o r i n g system f o r q u a n t i f i c a t i o n of arrhythmias: 0 = 0-4 9 PVCs 1 = 50-499 PVCs 2 = >499 PVCs and/or 1 episode of spontaneously r e v e r t i n g VT or VF 3 = >1 episode of VT or VF or both (< 60 s t o t a l combined duration) 4 = VT or VF or both (60-119 s t o t a l combined duration) 5 = VT or VF or both (>119 s t o t a l combined duration) 6 = f a t a l VF s t a r t i n g a t >15 min a f t e r o c c l u s i o n 7 = f a t a l VF s t a r t i n g at between 4 min and 14 min 59 s a f t e r o c c l u s i o n 8 = f a t a l VF s t a r t i n g a t between 1 min and 3 min 59 s a f t e r o c c l u s i o n 67 9 = f a t a l VF s t a r t i n g <1 min a f t e r o c c l u s i o n 2.3 E l e c t r i c a l l y - I n d u c e d Arrhythmias 2.3.1 Preparation Male Sprague Dawley r a t s (250-350 g) were anaesthetised w i t h sodium p e n t o b a r b i t a l (50 mg/kg i . p . ) . The l e f t c a r o t i d a r t e r y and r i g h t j u g u l a r v e i n were cannulated f o r blood pressure monitoring and drug i n f u s i o n , r e s p e c t i v e l y . A tracheotomy was performed and a l l r a t s were allowed t o breathe spontaneously throughout the experiment. Using a 2 7 gauge needle, t e f l o n coated s i l v e r w i r e e l e c t r o d e s (with exposed ends) were passed through the t h o r a c i c c a v i t y and lodged 2-5 mm apart i n the upper l e f t v e n t r i c l e . T h i s was confirmed by d i s s e c t i o n at the end of the experiment. Subcutaneous ECG e l e c t r o d e s i n a Lead I I c o n f i g u r a t i o n were used and a l l 4 limbs were grounded t o reduce noise. 2.3.2 Experimental Design Blood K+ c o n c e n t r a t i o n s were measured on 1 ml a r t e r i a l blood samples p r i o r t o determination of e l e c t r i c a l s t i m u l a t i o n v a r i a b l e s and at the end of the experiment. Q u i n a c a i n o l was administered as a s o l u t i o n i n a c i d i f i e d (HCl, pH 2.7), d i s t i l l e d water. Doses of 0.5, 1, 2, and 4 mg/kg were each given over a 10 min i n f u s i o n p e r i o d i n a 68 cumulative manner w i t h a d d i t i o n of a new dose every 25 min. The same regimen was used w i t h c o n t r o l animals but e q u i v a l e n t volumes of v e h i c l e were s u b s t i t u t e d f o r drug doses. Thus there were 2 groups, a c o n t r o l group (n=6) given v e h i c l e alone, and a q u i n a c a i n o l t r e a t e d group (n=6). Each animal acted as i t s own c o n t r o l . Each group was t e s t e d randomly w i t h a l l experimental components b l i n d e d t o the experimenter. S t i m u l a t i o n of the l e f t v e n t r i c l e w i t h square wave pulses was accomplished using a Grass S t i m u l a t o r (see a l s o Howard and Walker, 1990a). P r i o r t o drug i n f u s i o n , three sets of c o n t r o l values of e l e c t r i c a l s t i m u l a t i o n v a r i a b l e s were determined f i v e min apart. The l a s t s e t of values was taken as the c o n t r o l . A l l measurements, p l u s ECG, heart r a t e and blood pressure were made 10 min a f t e r the end of i n f u s i o n . 2.3.3 Experimental Endpoints End-points were determined using a delayed loop o s c i l l o s c o p e (Honeywell Type E f o r M) and square wave pu l s e s were used t o determine the f o l l o w i n g e l e c t r i c a l s t i m u l a t i o n v a r i a b l e s : t h r e s h o l d c u r r e n t ( i T ) , t h r e s h o l d d u r a t i o n ( t ^ ) , maximum f o l l o w i n g frequency (MFF), v e n t r i c u l a r f i b r i l l a t i o n t h r e s h o l d (VF T), and e f f e c t i v e r e f r a c t o r y p e r i o d (ERP). Each v a r i a b l e was measured 3 times and a mean value obtained. The procedure f o r the measurements has been de s c r i b e d elsewhere ( C u r t i s et a l . , 1984, 1986). 69 Threshold c u r r e n t f o r capture was determined a t 1 ms d u r a t i o n and 7.5 Hz by un i f o r m l y i n c r e a s i n g c u r r e n t a p p l i e d u n t i l capture. The t h r e s h o l d pulse width was determined at 7.5 Hz and twice the t h r e s h o l d c u r r e n t . MFF was determined at twice the cu r r e n t and pul s e width t h r e s h o l d s . MFF was taken a t t h a t p o i n t when the heart f a i l e d t o f o l l o w , on a 1:1 b a s i s , a s t e a d i l y i n c r e a s i n g frequency of s t i m u l a t i o n from 7 t o 2 0 Hz. This was r e a d i l y seen as a sudden upward sp i k e i n the blood pressure a f t e r an i n i t i a l s u s t a i n e d drop. VF T was determined at 50 Hz and twice t h r e s h o l d d u r a t i o n . The frequency was chosen t o ensure t h a t a pulse was d e l i v e r e d d u r i n g the v u l n e r a b l e p e r i o d , i . e . the t e r m i n a l p o r t i o n of the Q-T i n t e r v a l i n the ECG. The maximum cur r e n t which e l i c i t e d s u s t a i n e d f i b r i l l a t i o n w i t h a p r e c i p i t o u s f a l l i n blood pressure was taken as t h r e s h o l d . ERP was determined by the e x t r a - s t i m u l u s method. The heart was paced at 7.5 Hz and a s i n g l e e x t r a s t i m u l u s was a p p l i e d a t v a r y i n g i n t e r v a l s behind the pacing s t i m u l i . The s h o r t e s t i n t e r v a l between the pacing s t i m u l i and the e x t r a -s t i m u l u s i n which an e x t r a - s y s t o l e was obtained was taken as the ERP. 2 .4 E p i c a r d i a l I n t r a c e l l u l a r Recordings in vivo Sodium channel blockade i n normal v e n t r i c u l a r t i s s u e can be assumed from e f f e c t s on the ECG, e.g., widening of 70 the QRS complex or the P-R i n t e r v a l suggests i n h i b i t i o n of c a r d i a c Na + channels. The technique de s c r i b e d below o f f e r e d two advantages: i t demonstrated q u i n a c a i n o l ' s e f f e c t s on the c a r d i a c AP in vivo, and second, i t allowed us t o ( s u b ) c l a s s i f y q u i n a c a i n o l on the b a s i s of i t s e l e c t r o p h y s i o l o g i c a l a c t i o n s . 2.4.1 P r e p a r a t i o n Male Spraque Dawley r a t s (320-390g) were anaesthetised w i t h p e n t o b a r b i t a l (50 mg/kg i.p.) (n=6). The r i g h t j u g u l a r v e i n and l e f t c a r o t i d a r t e r y were cannulated f o r drug a d m i n i s t r a t i o n and blood pressure r e c o r d i n g r e s p e c t i v e l y . The l e f t j u g u l a r v e i n was cannulated f o r a d m i n i s t r a t i o n of s a l i n e and p e n t o b a r b i t a l as needed during the experiment s i n c e the drug i n f u s i o n l i n e i n the r i g h t j u g u l a r v e i n could not be d i s t u r b e d once drug i n f u s i o n had s t a r t e d . The r a t was mounted on a s t a i n l e s s s t e e l p l a t e r a i s e d t o a 15° angle to help maintain venous r e t u r n . Under a r t i f i c i a l v e n t i l a t i o n w i t h room a i r , a thoracotomy was performed by c u t t i n g through the f o u r t h and f i f t h r i b s t o expose the heart. The upper e p i c a r d i a l surface of the l e f t v e n t r i c l e was immobilized by s u t u r i n g i t t o a s i l v e r / s i l v e r c h l o r i d e metal loop u s i n g a s t e r i l e absorbable s u r g i c a l suture (5-0 Dexon P l u s ; Davis & Geek) . The loop a l s o served as a reference e l e c t r o d e . For ECG re c o r d i n g , an e l e c t r o d e was i n s e r t e d down the oesophagus and a needle e l e c t r o d e i n s e r t e d 71 i n t o the chest w a l l w i t h the loop s t i l l a c t i n g as the r e f e r e n c e e l e c t r o d e . M i c r o e l e c t r o d e s (20 megaohm r e s i s t a n c e ) were prepared u s i n g g l a s s t u b i n g (1BBL w / F i l 1.0 MM, 4 inches; World P r e c i s i o n Instruments, Inc.) and a N a r i s h i g e (Pa-01) e l e c t r o d e p u l l e r . The m i c r o e l e c t r o d e s were f i l l e d w i t h 3 M KC1 t h a t had been f i l t e r e d through a 0.45 /nm F i l t e r u n i t ( M i l l e x - H A ) . Coated tungsten wire (0.003 inches, A-M Systems, Inc.) was i n s e r t e d i n t o the e l e c t r o d e u n t i l i t reached the end of the t i p . This end was then c a r e f u l l y snapped o f f and attached t o the e l e c t r o d e h o l d e r . A n g l i n g the tungsten wire at 90° b u f f e r e d the m i c r o e l e c t r o d e a g a i n s t the b e a t i n g of the heart and a s s i s t e d i n keeping the m i c r o e l e c t r o d e w i t h i n the c e l l . The m i c r o e l e c t r o d e s were connected t o a WPI p r e a m p l i f i e r . The output was passed through a d i f f e r e n t i a t o r and the s i g n a l s recorded on a video tape a f t e r p u l s e code modulation (Sony PCM). An o s c i l l i s c o p e was used t o monitor c e l l p e n e t r a t i o n and t o determine dV/dt m a x , w h i l e ensuring a good r e c o r d had been acquired. 2.4 .2 Experimental Design In a 30 min c o n t r o l p e r i o d p r i o r t o drug i n f u s i o n , APs were recorded u s i n g a m u l t i - s a m p l i n g technique. P e n e t r a t i o n w i t h i n a c e l l was considered good i f the AP c o u l d be h e l d f o r 20 sec or longer and was adequate. 72 Q u i n a c a i n o l , ( a c i d i f i e d , d i s t i l l e d water, pH 2.7) was given i n a cumulative manner at doses of 0.5, 1, 2,4, and 8 mg/kg. Each dose was i n f u s e d over a 3-4 minute p e r i o d u s i n g an i . v . i n f u s i o n pump (Harvard Apparatus) w i t h the next dose beginning 10 min a f t e r the end of the previous i n f u s i o n . A m u l t i p l e impalement technique (Inoue et al. , 1982; Abraham et al., 1989) was used f o r the 10 min p e r i o d between doses. 2.4.3 Experimental Endpoints and Data Analysis Using a storage o s c i l l i s c o p e ( T e t r o n i x ) , acceptable APs were examined at two time p e r i o d s , w i t h i n the f i r s t minutes f o l l o w i n g i n f u s i o n (0-3 min and no gre a t e r than 5 min post-i n f u s i o n ) , and du r i n g the l a s t 5 min of the 10 min p e r i o d between doses. From the AP, the f o l l o w i n g parameters were measured: AP h e i g h t , the maximum value of dV/dt f o r the r i s i n g phase of the a c t i o n p o t e n t i a l (dV/dt m a x ) and AP d u r a t i o n at 10, 50, and 90% of r e p o l a r i z a t i o n . For each r a t p r e p a r a t i o n , v a l u e s obtained w i t h i n the time frames mentioned were averaged and the averages f o r a l l r a t s were measured t o g i v e mean and s.e. mean values w i t h n equal t o the number of r a t s t e s t e d (n=6). 2.5 E f f e c t s of Quinacainol i n Isolated Rat Hearts I n v e s t i g a t i o n of the mechanical ( c o n t r a c t i l e force) and e l e c t r i c a l (ECG) a c t i v i t y of the i s o l a t e d mammalian heart 73 was f i r s t devised by Langendorff i n 189 5. The primary advantage t o u s i n g i s o l a t e d organ preparations i s t h a t they are without nervous and humoral r e g u l a t i o n as w e l l as s u b s t r a t e supply and m o d i f i c a t i o n of blood content by the i n t a c t organism. 2.5.1 P e r f u s i o n Apparatus A p e r f u s i o n apparatus f o r mechanical and e l e c t r o p h y s i o l o g i c a l s t u d i e s i n small animals such as r a t , guinea p i g , and r a b b i t hearts was designed and c o n s t r u c t e d i n our l a b o r a t o r y ( C u r t i s et al., 1986). The apparatus c o n s i s t s of nine i n d i v i d u a l p e r f u s i o n chambers connected to the a o r t i c p e r f u s i o n cannula of the Langendorff perfused h e a r t . The m u l t i p l e chambers a l l o w f o r v a r i o u s drug c o n c e n t r a t i o n s or s o l u t i o n s . The design of our apparatus a l l o w s f o r r a p i d s w i t c h i n g of up t o nine d i f f e r e n t pre-heated p e r f u s a t e s and i n v o l v e s a small dead space ( l e s s than 0.1 ml) w h i l e a o r t i c route p e r f u s i o n pressure may be e a s i l y v a r i e d from 0 t o 2 00 mmHg. 2.5.2 P r e p a r a t i o n While the use of whole blood or s o l u t i o n c o n t a i n i n g haemoglobin may l i m i t hypoxia, m u l t i p l e c o m p l i c a t i o n s are a s s o c i a t e d w i t h t h e i r use (e.g. foaming, c l o t t i n g e r y t h r o c y t e aggregation, etc.) (Doring and Dehnert, 1988). 74 Thus we chose t o use a modified K r e b 1 s - H e n s e l e i t s o l u t i o n . The composition (mM) of the Kreb's s o l u t i o n was: NaCl, 118; KCI, 4.74; C a C l 2 - 2 H 2 0 , 2.5; K H 2 P 0 4 , 0.93; NaHC03 , 25; Glucose, 10; MgS0 4-7H 20, 1.2. Male Sprague Dawley r a t s (290-400g) (n=7) were s a c r i f i c e d by concussion and exsanguination and the heart was immediately e x c i s e d . The heart was r e t r o g r a d e l y perfused w i t h c o l d Kreb's s o l u t i o n u s i n g a 50 cc sy r i n g e t o remove blood. The heart was then t r a n s f e r r e d t o the pe r f u s i o n apparatus and t i e d t o the cannula. W i t h i n seconds of i n i t i t a t i n g p e r f u s i o n , the heart began b e a t i n g i n sinus rhythm. The l e f t a trium was then cut o f f i n order t o i n s e r t a b a l l o o n ( C u r t i s , 1986) f o r v e n t r i c u l a r pressure measurements and the d i a s t o l i c pressure w i t h i n the b a l l o o n was adjusted t o maintain l e f t v e n t r i c u l a r end d i a s t o l i c pressure of 10 (±5) mmHg. S p e c i a l atraumatic e l e c t r o d e s were designed f o r ECG reco r d i n g ( C u r t i s , 1986). One el e c t r o d e was pla c e d on the r i g h t atrium t o a l l o w the re c o r d i n g of a l a r g e P wave, and the second on the l e f t v e n t r i c l e . Using a Grass polygraph f o r ECG r e c o r d i n g , a d i f f e r e n t i a t o r was connected t o the channel measuring l e f t v e n t r i c u l a r pressure t o convert i t t o dP/dt, a measure of c o n t r a c t i l i t y . P e r f u s i o n pressure was kept constant at 115 mmHg (±10 mmHg). Measurement of mean coronary flow was done by c o l l e c t i n g the perfu s a t e d r a i n i n g out of the r i g h t atrium. S h i f t i n g of the e l e c t r o d e s was f a i r l y common and care was taken t o ensure they remained i n approximately the 75 same p o s i t i o n . Some d i f f i c u l t y was seen i n determining the beginning and end of the T-wave so t h a t the apex of the T wave was used f o r a l l measurements ( r e f e r t o Figure 3). 2.5.3 Experimental Design To assess the d i r e c t e f f e c t s of q u i n a c a i n o l i n r a t c a r d i a c t i s s u e , i s o l a t e d r a t hearts were perfused w i t h v a r y i n g c o n c e n t r a t i o n s of q u i n a c a i n o l . Q u i n a c a i n o l was compared w i t h t e t r o d o t o x i n (TTX), an agent whose a c t i o n i s a h i g h l y s p e c i f i c b l o c k i n g a c t i o n on sodium channels (Fuhrman, 1986). The heart was allowed t o e q u i l i b r a t e u s i n g the m o d i f i e d Kreb's s o l u t i o n as the p e r f u s a t e f o r a minimum of 10 min. Each heart (n=7) was f i r s t exposed t o the f o l l o w i n g c o n c e n t r a t i o n s of TTX (M) : 3xl0" 7 , 1x10 6 , 3xl0" 6 , 1x10" 5. Each c o n c e n t r a t i o n of TTX perfused the heart f o r 2 min. A 10 min wash p e r i o d then followed. Hearts were exposed t o TTX f i r s t due t o i t s f a s t onset and o f f s e t of e f f e c t s (Fuhrman, 1986). A f t e r 2 min of p e r f u s i n g the h e a r t w i t h m o d i f i e d Kreb's, most hea r t s had r e v e r t e d back to pre-drug v a l u e s . At the end of the 10 minute wash p e r i o d the f o l l o w i n g c o n c e n t r a t i o n s (M) of q u i n a c a i n o l were t e s t e d : 3xl0' 7 , 1x10 "6 , 3xl0" 6 , lxlO" 5 . Hearts were perfused w i t h each c o n c e n t r a t i o n of q u i n a c a i n o l f o r 10 min. The wash p e r i o d acted as the c o n t r o l values f o r q u i n a c a i n o l t o 76 determine drug-induced changes whereas f o r TTX, c o n t r o l values were taken d u r i n g the e q u i l i b r a t i o n p e r i o d . 2.6 S t a t i s t i c a l Analyses The General L i n e a r Model ( a l l ANOVA models from H i n t z e , NCSS S t a t i s t i c a l Package, 1981) was used t o compare treatment means w i t h v e h i c l e i n the haemodynamic study i n conscious r a t s as c e l l frequencies were balanced. O r i g i n a l data v a l u e s i n the v e h i c l e c o n t r o l group and the q u i n a c a i n o l t r e a t e d group were s e p a r a t e l y t e s t e d i n the e l e c t r i c a l s t i m u l a t i o n study (GLM ANOVA) t o determine i f treatment ( v e h i c l e or drug) and time were s t a t i s t i c a l l y s i g n i f i c a n t . An unweighted means a n a l y s i s (UWM ANOVA) was used because of mi s s i n g c e l l s t o d i s c o v e r s i g n i f i c a n t sources of va r i a n c e (p<0.05) between treatment as compared t o c o n t r o l (pre-drug) values and t o determine i f treatment e f f e c t s v a r i e d w i t h time f o r the e p i c a r d i a l i n t r a c e l l u l a r p o t e n t i a l study. UWM ANOVA (p<0.05) was a l s o used t o t e s t a l l co n c e n t r a t i o n s of q u i n a c a i n o l and TTX i n the i s o l a t e d heart experiment t o determine i f drug e f f e c t s on the v a r i a b l e s measured were s t a t i s t i c a l l y s i g n i f i c a n t . As h e a r t s were perfused w i t h each c o n c e n t r a t i o n of TTX and q u i n a c a i n o l over a given time p e r i o d , time-dependent e f f e c t s were t e s t e d f o r i n an analogous manner t o treatment e f f e c t s . A l l values 77 were compared t o pre-treatment values f o r the given treatment (TTX or q u i n a c a i n o l ) . In the experiment t e s t i n g q u i n a c a i n o l 1 s a c t i o n s a g a i n s t ischasmia-induced arrhythmias, a c o n t r o l group ( r e c e i v i n g drug v e h i c l e ) was compared w i t h two t r e a t e d groups. The v a r i a b l e s compared were both G a u s s i a n - d i s t r i b u t e d and b i n o m i a l l y - d i s t r i b u t e d (Johnston et al., 1983). In accordance w i t h these types of d i s t r i b u t e d data, an ANOVA (UWM model) was c a r r i e d out f o r G a u s s i a n - d i s t r i b u t e d v a r i a b l e s , w h i l e Mainland's contingency t a b l e s of minimum c o n t r a s t s were used f o r chi-squared t e s t i n g (Mainland et al., 1956) f o r b i n o m i a l l y - d i s t r i b u t e d v a r i a b l e s . In a l l cases, i f treatment c o n s t i t u t e d a s i g n i f i c a n t source of v a r i a t i o n as revealed by F t e s t v a l u e s , Duncan's m u l t i p l e range t e s t and Newman Keul's t e s t s were used t o compare means. I f any discrepancy e x i s t e d Duncan's r e s u l t s were accepted. ANOVA r e s u l t s were checked by making f u r t h e r comparisons between v e h i c l e c o n t r o l group and treatment group u s i n g two-sample t - t e s t s where ap p r o p r i a t e . 78 3 RESULTS 3 . 1 E f f e c t s of Quinacainol on Blood Pressure, V e n t r i c u l a r Pressure, and Heart Rate The e f f e c t s of q u i n a c a i n o l on blood pressure and heart r a t e were examined in vivo i n conscious and anaesthetised animals and on v e n t r i c u l a r pressure and heart r a t e in vitro. In conscious animals q u i n a c a i n o l showed more dramatic e f f e c t s i n the presence of coronary a r t e r y o c c l u s i o n . In the dose-response study i n conscious r a t s , q u i n a c a i n o l produced a s l i g h t depression i n blood pressure and a s l i g h t e l e v a t i o n i n heart r a t e w i t h t h i s t rend r e v e r s i n g a t the highest dose (8 mg/kg) (Table 1) when expr e s s i n g the e f f e c t as change from pre-treatment values f o r the i n d i c a t e d dose. None of these e f f e c t s achieved s t a t i s t i c a l s i g n i f i c a n c e . S i g n i f i c a n t e f f e c t s of q u i n a c a i n o l on haemodynamics were seen a f t e r coronary a r t e r y o c c l u s i o n . Treatment d i d not have marked a c t i o n s on blood pressure and only occlusion-dependent e f f e c t s were seen 4 h f o l l o w i n g o c c l u s i o n f o r a l l groups (Figure 6a). The e f f e c t s of q u i n a c a i n o l on heart r a t e were seen much e a r l i e r w i t h s i g n i f i c a n t b r a d y c a r d i a noted 15 min p o s t - o c c l u s i o n as compared t o v e h i c l e c o n t r o l group (Figure 6b). Quina c a i n o l • s haemodynamic e f f e c t s were more predominant i n anaesthetised animals than i n conscious r a t s . 79 T a b l e 1. B l o o d p r e s s u r e and h e a r t r a t e e f f e c t s o f q u i n a c a i n o l i n c o n s c i o u s r a t s . Time and Dose Change i n BP Change i n H e a r t R a t e (mmHg) (beats/min) v e h i c l e +5 min -4±8 23±13 +20 min -13±6 34±19 1.0 mg/kg +5 min -5±3 11±7 +20 min -7+4 23±15 2.0 mg/kg : +5 min -9±6 31±24 +20 min -12±9 21±16 4.0 mg/kg +5 min -11±5 6±17 +20 min -14±6 5±18 8.0 mg/kg +5 min -3±7 -10±20 +20 min 6±4 -24±29 R a t s (n=6) were g i v e n q u i n a c a i n o l , 1, 2, 4, o r 8 mg/kg i . v . as an i n f u s i o n o v e r 10 min. Doses were a d m i n i s t e r e d random and b l i n d from a randomized b l o c k d e s i g n w i t h doses b e i n g a d m i n i s t e r e d on a l t e r n a t e days. V a l u e s a r e g i v e n f o r t h a t dose, t i m e f o l l o w i n g end o f dr u g i n f u s i o n , and v a r i a b l e i n d i c a t e d (n=6). R e s u l t s a r e p r e s e n t e d as changes from p r e -t r e a t m e n t v a l u e s ( f o r t h a t d o s e ) . A l l v a r i a b l e s a r e mean ± s.e. mean. No s i g n i f i c a n t d i f f e r e n c e s were found w i t h r e s p e c t t o t i m e o r dose (GLM ANOVA). 80 A 150 pre-drug pre-occl. +15min + 1h + 4 h Time •M vehicle BiH 2 mg/kg V///A 4 mg/kg control Figure 6. Blood pressure and heart r a t e e f f e c t s of q u i n a c a i n o l before and a f t e r coronary a r t e r y o c c l u s i o n . Each bar graph represents mean ± s.e.mean, n=10. Time peri o d s are .-15 min (pre-drug) and -1 min p r e - o c c l u s i o n ( i n d i c a t e d i n the graphs as pre-occl.) and +15 min, 1 h, and 4 h a f t e r o c c l u s i o n . Blood pressure e f f e c t s ar^e shown i n pa r t A and e f f e c t s on heart r a t e i n p a r t B. I n d i c a t e s p<0.05 versus p r e - o c c l u s i o n values ( i . e . occlusion-dependent e f f e c t s ) . + I n d i c a t e s p<0.05 versus v e h i c l e c o n t r o l group. 81 In the r a t s used i n the e l e c t r i c a l s t i m u l a t i o n study, the v e h i c l e alone had no major e f f e c t on blood pressure or heart r a t e w h i l e q u i n a c a i n o l produced a dose-dependent decrease i n blood pressure ( s t a t i s t i c a l l y s i g n i f i c a n t only f o r the highest dose) and produced a s t a t i s t i c a l l y s i g n i f i c a n t dose-dependent decrease i n heart r a t e f o r doses of 1, 2, and 4 mg/kg (Figure 7a and 7b). As i n conscious animals, q u i n a c a i n o l produced a g r e a t e r e f f e c t on heart r a t e than on blood pressure. In the a n a e s t h e t i s e d r a t s (n=7) used t o i n v e s t i g a t e the e f f e c t s of q u i n a c a i n o l on e p i c a r d i a l transmembrane p o t e n t i a l s , a dose-dependent decrease i n heart r a t e and blood pressure was a l s o seen (Table 2) . The c o n t r o l and drug treatment p e r i o d each occupied approximately 90 min. Since c o n t r o l readings d i d not vary over the 90 min c o n t r o l p e r i o d , they are presented as a s i n g l e value i n the t a b l e . The e f f e c t s of treatment d i d not vary w i t h time. B r a d y c a r d i c e f f e c t s of q u i n a c a i n o l were a l s o seen in vitro and were compared t o TTX (Table 3) . Q u i n a c a i n o l reduced v e n t r i c u l a r pressure and coronary flow though not s t a t i s t i c a l l y s i g n i f i c a n t l y (Table 3). 3.2 ECG e f f e c t s of Q u i n a c a i n o l Q u i n a c a i n o l had marked e f f e c t s on the ECG. In conscious animals i t prolonged both the P-R i n t e r v a l and QRS d u r a t i o n (Figure 8,9 and 10) dose-dependently w h i l e the 82 F i g u r e 7. E f f e c t s of q u i n a c a i n o l and v e h i c l e c o n t r o l on blood pressure (A) and heart r a t e (B) i n p e n t o b a r b i t a l anaesthetised r a t s . A l l r e s u l t s are presented as absolute v a l u e s , each v a l u e i s mean ± s.e.mean, n=6. I n d i c a t e s p<0.05 from v e h i c l e c o n t r o l . No s i g n i f i c a n t d i f f e r e n c e s were seen when comparing groups t o t h e i r own pre-treatment or pre-drug v a l u e s . 84 Table 2. Blood pressure and heart r a t e e f f e c t s of q u i n a c a i n o l d u r i n g e p i c a r d i a l i n t r a c e l l u l a r p o t e n t i a l r e c o r d i n g s i n v i v o . Dose Mean BP Heart Rate (mg/kg) (mmHg) (beats/min) c o n t r o l 116±8 433±11 0.5 112±9 407+18 1.0 105±10 393±19* 2 . 0 110+9 387±11* 4.0 95±15* 3 65±21* 8.0 * 69±2 317* The e f f e c t s of q u i n a c a i n o l on blood pressure and heart r a t e are shown as recorded during e p i c a r d i a l i n t r a c e l l u l a r p o t e n t i a l r e c o r d i n g s . * I n d i c a t e s p<0.01 from c o n t r o l v a l u e s . Treatment was determined t o be a s i g n i f i c a n t source of v a r i a n c e . As time was not a s i g n i f i c a n t source of v a r i a n c e a l l values represent xis.e.mean f o r the averaged values i n the second time p e r i o d ( i . e . 5-10 min post-i n f u s i o n ) . * I n d i c a t e s t h a t data was i n s u f f i c i e n t t o perform s t a t i s t i c a l t e s t s or record s.e.mean as heart r a t e s were recorded f o r only 2 animals at t h i s dose. 85 Table 3. Haemodynamic e f f e c t s of t e t r o d o t o x i n (TTX) and q u i n a c a i n o l i n v i t r o . Dose Heart Rate V e n t r i c u l a r Coronary Flow (beats/min) Pressure (ml/min) (mmHg) a) T e t r o d o t o x i n c o n t r o l 226±7.5 134117 10±1.3 3xl0' 7 228±7.9 135118 9.5+2.5 1x10"^ 21119.5 151116 1111.3 3xl0" 6 193111 150+17 9.311 IxlO' 5 200118 150117 8.912 b) Quin a c a i n o l c o n t r o l 17916.8 + 109116 8.011 3xl0^ 7 19116.6 + 105118 7.210.5 1x10 6 185112 97119 6.610.6 3x10"* 17516.8 89+12 6.2 + 0.7 IxlO" 5 163115 + 86119 6.810.7 A l l v alues (x 1 s.e. mean) were recorded at the end of 2 min of p e r f u s i o n w i t h TTX, and 10 min p e r f u s i o n w i t h q u i n a c a i n o l . I n d i c a t e s p<0.05 from c o n t r o l v a l u e s . + I n d i c a t e s p<0.05 as compared t o r e s p e c t i v e doses of TTX (Unweighted Means ANOVA). 86 o If) £ DC I a c CD c ro U + 10s +5min Time from end of infusion +20min + 0 mg/kg <54 ms) 1 mg/kg (61 ms) 2 mg/kg (56 ms) 4 mg/kg (54 ms) 8 mg/kg (51 ms) F i g u r e 8. E f f e c t s of q u i n a c a i n o l and v e h i c l e on P-R i n t e r v a l i n the conscious r a t . Values are expressed as change i n msec (mean + s.e.mean) from pre-treatment v a l u e s . The v a l u e s i n d i c a t e d a t 10 s are those measured immediately f o l l o w i n g end of i n f u s i o n , and those i n d i c a t e d a t 5 min and 20 min are from t h a t time f o l l o w i n g end of i n f u s i o n . * i n d i c a t e s t h a t treatment was a s i g n i f i c a n t source of va r i a n c e as compared t o v e h i c l e (0 mg/kg) (p<0.001). The e f f e c t of a giv e n dose d i d not change w i t h time. Pre-treatment v a l u e s are i n d i c a t e d i n brackets above. Some s.e.mean v a l u e s are omitted f o r c l a r i t y . 87 90 r p r e - d r u g + v e h i c l e c o n t r o l 2 m g / k g • 4 m g / k g F i g u r e 9. E f f e c t s of q u i n a c a i n o l and v e h i c l e c o n t r o l on P-R i n t e r v a l before and a f t e r o c c l u s i o n . The a b s c i s s a i n d i c a t e s time (min and h) w i t h respect t o l i g a t i o n of the coronary a r t e r y . R e s u l t s are expressed as x± s.e.mean, n=10. In d i c a t e s p<0.001 versus pre-drug v a l u e s . + I n d i c a t e s s i g n i f i c a n t d i f f e r e n c e s (p<0.05) between treatment groups and v e h i c l e c o n t r o l group at t h a t time p e r i o d i n d i c a t e d on the a b s c i s s a . 88 o <D w E CO tr o Q) CO c ro .C U 1 2 . 0 0 8 . 8 0 5 . 6 0 2 . 4 0 - 0 . 8 0 - 4 . 0 0 O + 1 0 s +5min T ime af ter end of infusion -20min O mg/kg (34 ms) 1 mg/kg (31 ms> 2 mg/kg (32 ms) 4 mg/kg (32 ms) -X-8 mg/kg (32 ms) F i g u r e 10. The e f f e c t s of q u i n a c a i n o l and v e h i c l e on QRS d u r a t i o n i n the conscious r a t . Values are expressed as change from pre-treatment values (x±s.e. mean). Bracketed v a l u e s i n d i c a t e the mean of c o n t r o l values p r i o r t o i n d i c a t e d treatment. Values i n d i c a t e d a t 10 s were recorded immediately f o l l o w i n g end of i n f u s i o n . Compared t o v e h i c l e , 4 mg/kg and 8 mg/kg were a s i g n i f i c a n t source of v a r i a n c e (p<0.01). Treatment e f f e c t s d i d not vary s i g n i f i c a n t l y w i t h time. 89 v e h i c l e was without e f f e c t . The Q-T i n t e r v a l , c o r r e c t e d f o r heart r a t e (Q-T c), was a l s o prolonged dose-dependently although not as markedly (Figure 11). I t was i n t e r e s t i n g t o note t h a t ECG e f f e c t s were most marked 5 min p o s t - i n f u s i o n i n the dose-response study and began to wane s l i g h t l y 20 min p o s t - i n f u s i o n . In anaesthetised animals q u i n a c a i n o l demonstrated s i m i l a r ECG e f f e c t s compared t o those seen i n conscious animals. In the e l e c t r i c a l s t i m u l a t i o n study c o i n c i d e n t w i t h the lowering of heart r a t e , the P-R i n t e r v a l was prolonged (Figure 12) as seen i n conscious animals although l i t t l e e f f e c t was seen on the QRS d u r a t i o n or Q-Tc i n t e r v a l (Table 4) . The v e h i c l e alone had no major e f f e c t s on the ECG. The P-R p r o l o n g a t i o n was s i g n i f i c a n t s t a t i s t i c a l l y (p<0.05) f o r a l l doses when compared to v e h i c l e c o n t r o l group i n f u s i o n s (Figure 12). In vitro, q u i n a c a i n o l widened the P-R i n t e r v a l and QRS d u r a t i o n (Figures 13 and 14) without having a major e f f e c t on the Q-Tc i n t e r v a l (Figure 15). The major acute t o x i c e f f e c t s of q u i n a c a i n o l i n conscious r a t s o c c u r r i n g w i t h i n 1 h of a d m i n i s t r a t i o n can be seen i n Table 5. Proarrhythmic e f f e c t s occurred a t doses of 8 mg/kg and above. 3 . 3 Coronary Artery Occlusion-Induced Arrhythmias i n Conscious Rats 90 5 0 r 4 0 -0 + 1 0 s +5min +20min Time after end of infusion -*-• 0 mg/kg O 1 mg/kg A 2 mg/kg • 4 mg/kg V 8 mg/kg <108ms) (113ms) (105ms) (104ms) (106ms> F i g u r e 11. The e f f e c t s of qu i n a c a i n o l and v e h i c l e on Q-T i n t e r v a l c o r r e c t e d f o r heart r a t e (Q-Tc) i n the conscious r a t . Values are expressed as change from pre-treatment values (x±s.e. mean). Bracketed values i n d i c a t e the mean of c o n t r o l v a l u e s p r i o r t o i n d i c a t e d treatment. Values i n d i c a t e d a t 10s were recorded immediately f o l l o w i n g end of i n f u s i o n . Compared t o v e h i c l e ( i n d i c a t e d as 0 mg/kg above), the h i g h e s t dose was a s i g n i f i c a n t source of va r i a n c e (p<0.002). E f f e c t s of treatment d i d not vary s i g n i f i c a n t l y w i t h time. 91 Figur e 12. E f f e c t s of q u i n a c a i n o l and v e h i c l e c o n t r o l on P-R i n t e r v a l i n p e n t o b a r b i t a l anaesthetised r a t s . Drug treatment w^ith q u i n a c a i n o l was a s i g n i f i c a n t source of va r i a n c e ( p<0.001) as compared t o i t s own c o n t r o l v a l u e s . I n d i c a t e s p<0.05 as compared t o the v e h i c l e c o n t r o l group f o r the i n d i c a t e d dose of q u i n a c a i n o l . A l l v a l u e s are expressed as xts.e.mean. Table 4. ECG e f f e c t s of q u i n a c a i n o l as compared t o v e h i c l e c o n t r o l i n p e n t o b a r b i t a l anaesthetised r a t s . Dose QRS Duration Q-Tc I n t e r v a l (msec) (msec) a) v e h i c l e c o n t r o l group pre-treatment 32±1.9 113±8.4 i n f u s i o n 1 32±1.4 111±6.8 i n f u s i o n 2 32±1.2 107±6.0 i n f u s i o n 3 32±1.4 114±8.7 i n f u s i o n 4 32±1.8 111±9.4 b) q u i n a c a i n o l t r e a t e d group pre-treatment 32±0.9 107±5 0.5 mg/kg 33±1.1 110±2 1.0 mg/kg 33±1.3 106+5 2.0 mg/kg 33±1.0 107±3 4.0 mg/kg . 33±0.7 113±3 A l l v a r i a b l e s are mean ± s.e.mean, n=6. Pre-treatment values are those obtained p r i o r t o i n f u s i o n of s p e c i f i e d dose of q u i n a c a i n o l or e q u i v a l e n t volume f o r body weight of v e h i c l e c o n t r o l ( i n d i c a t e d as i n f u s i o n 1, 2, 3, or 4) . I n d i c a t e s p<0.05 from pre-treatment val u e s . + I n d i c a t e s p<0.05 as compared t o a p p r o p r i a t e i n f u s i o n s i n the v e h i c l e c o n t r o l group. 150 Concentration (M) T T X • Quinacainol F i g u r e 13. In v i t r o e f f e c t s of t e t r o d o t o x i n (TTX) and q u i n a c a i n o l on the P-R i n t e r v a l . I n d i c a t e s p<0.001 as compared t o each drug's own c o n t r o l v a l u e s . + I n d i c a t e s p<0.05 as compared t o r e s p e c t i v e doses of TTX (Unweighted Means ANOVA). 94 Figur e 14. In v i t r o e f f e c t s of t e t r o d o t o x i n (TTX) and q u i n a c a i n o l on the QRS d u r a t i o n . * I n d i c a t e s p<0.001 as compared t o each drug's own c o n t r o l v a l u e s . + I n d i c a t e s p<0.05 as compared t o r e s p e c t i v e doses of TTX (Unweighted Means ANOVA). 95 F i g u r e 15. In v i t r o e f f e c t s of t e t r o d o t o x i n (TTX) and q u i n a c a i n o l on the Q-Tc i n t e r v a l . No s i g n i f i c a n t e f f e c t s were seen when comparing treatment and v e h i c l e t o t h e i r r e s p e c t i v e c o n t r o l values nor when comparing q u i n a c a i n o l t o TTX (Unweighted Means ANOVA). 96 Table 5. Major acute t o x i c e f f e c t s of q u i n a c a i n o l i n conscious r a t s . T o x i c E f f e c t Dose (mg/kg) V e h i c l e 1.0 2.0 4.0 8.0 16 M o r t a l i t y 0 0 0 0 0 1 Arrhythmias 0 0 0 1 3 4 Convulsions 0 0 0 0 0 0 R e s u l t s given f o r v e h i c l e and doses of 1, 2, 4, and 8 mg/kg are based on a sample s i z e of 6 w h i l e n=4 f o r 16 mg/kg. Exces s i v e grooming and p i l o e r e c t i o n were noted d u r i n g i n f u s i o n . 97 3.3.1 Occluded Zone and Mortality To ensure e q u a l i t y of arrhythmic s t i m u l u s between groups, the OZ s i z e (as a percentage of t o t a l v e n t r i c u l a r weight) was determined f o r a l l groups and e x c l u s i o n c r i t e r i a ( C u r t i s , 1986) were followed throughout the experiment. No s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s were seen. The OZs f o r c o n t r o l , 2 mg/kg, and 4 mg/kg were: 41±4, 33±3 , and 41±6% r e s p e c t i v e l y . Dose-related e f f e c t s of q u i n a c a i n o l on m o r t a l i t y were not seen, but q u i n a c a i n o l reduced m o r t a l i t y due t o VF b u t not t h a t due t o non-arrhythmic causes (Table 6) . The high m o r t a l i t y r a t e i n the c o n t r o l group was due t o the high i n c i d e n c e of NSVF. 3.3.2 Occlusion-Induced ECG E f f e c t s I n t e r e s t i n g e f f e c t s of q u i n a c a i n o l treatment on ECG changes produced by coronary o c c l u s i o n ( i n regard t o S-T segment and R-wave changes) were seen. Q u i n a c a i n o l prolonged the time t o maximum S-T segment e l e v a t i o n and R wave amplitude i n c r e a s e s , and reduced the extent of S-T segment e l e v a t i o n (expressed as %R wave amplitude (ST%) i n the manner used by Bernauer (1982; s i t e d i n C u r t i s , 1986)). These r e s u l t s are summarized i n Table 7 and F i g u r e 16. Maximum R-wave values obtained f o l l o w i n g o c c l u s i o n were s i m i l a r i n a l l groups. 98 Table 6. M o r t a l i t y f o l l o w i n g coronary o c c l u s i o n from arrhythmic and non-arrhythmic causes i n v e h i c l e c o n t r o l and qu i n a c a i n o l t r e a t e d conscious r a t s . Dose Arrhythmic Non-Arrhythmic T o t a l Cause Cause a) 0-0.5 h p o s t - o c c l u s i o n c o n t r o l 5/10 1/10 6/10. 2 mg/kg o/ io* 0/10 o/ io* 4 mg/kg 0/10 0/10 0/10 b) 0.5-4 h p o s t - o c c l u s i o n c o n t r o l 1/4 : 1/4 2/4 2mg/kg 0/10 2/10 2/10 4 mg/kg 0/10 1/10 1/10 A l l v a r i a b l e s are expressed as inc i d e n c e (out of n). Incidence of arrhythmias was separated i n t o two time periods (0-0.5 h and 0.5-4 h) f o l l o w i n g o c c l u s i o n due t o the bimodal d i s t r i b u t i o n of arrhythmias f o l l o w i n g o c c l u s i o n s . I n d i c a t e s a s i g n i f i c a n t source of va r i a n c e as compared t o c o n t r o l s (as determined by c h i 2 a n a l y s i s ) . 99 e 7. ECG changes produced by coronary a r t e r y o c c l u s i o n Max %S-T Time of Max R Time of Time of (% of R) Max %S-T (mV) Max R Q-Wave (min) (min) (min) c o n t r o l 73±8 2 mg/kg 52±6* 4 mg/kg 43±6* 8±1.4 0.7±0.1 20.5±4 0.7±0.1 26.3±2.6 0.7±0.1 2.3±0.5 / 11.7±5.8 98±25 12.2±6.3* 93±24 Time ( i n min) i s the time r e q u i r e d f o r the R-wave and S-T changes t o reach t h e i r maxima w h i l e i t i n d i c a t e s the appearance of a Q-wave. I t was not p o s s i b l e t o determine a value f o r the c o n t r o l group as i n d i c a t e d by /. A l l v a r i a b l e s are expressed as mean ± s.e.mean. I n d i c a t e s p<0.05 versus c o n t r o l . 100 100 r ~ 80 -5min 15min 30min 1h 4h Time post-occlusion + vehicle ° 2 mg/kg * 4 mg/kg control Figure 16. E f f e c t s of quinacainol and v e h i c l e c o n t r o l over time on S-T segment e l e v a t i o n (expressed as a % of R wave amplitude) following occlusion. The abscissa i n d i c a t e s the time ( i n min) following coronary occlusion. Results are expressed as x±s.e.mean, n=10. Indicates p<0.05 versus v e h i c l e c o n t r o l group. + Indicates s i g n i f i c a n t d i f f e r e n c e s (p<0.05) between treatment groups and vehicle c o n t r o l group at that time period i n d i c a t e d on the abscissa. 101 3.3.3 Arrhythmias E x t e n s i v e a n a l y s i s w i t h i n our l a b o r a t o r y of the arrhythmias f o l l o w i n g o c c l u s i o n of the LAD i n conscious r a t s showed a bi-modal d i s t r i b u t i o n w i t h time (Johnston et al. , 1983) . W i t h i n the 4 h time p e r i o d i n which we t e s t a n t i -arrhythmic a c t i v i t y i n our conscious ischaemia model, peak frequency of arrhythmias occurs w i t h i n the f i r s t 15 min and 2 h f o l l o w i n g o c c l u s i o n , w i t h a quiescent p e r i o d between these p e r i o d s (Table 8) ( C u r t i s , 1986). Arrhythmia i n c i d e n c e i n the v a r i o u s groups i s shown i n Table 8 f o r the two time p e r i o d s , 0-0.5 h and 0.5-4 h, a f t e r o c c l u s i o n ; arrhythmia scores are a l s o given. Both doses of q u i n a c a i n o l s t a t i s t i c a l l y s i g n i f i c a n t l y reduced arrhythmia score i n the f i r s t time p e r i o d 0-0.5h a f t e r o c c l u s i o n w h i l e q u i n a c a i n o l 1 s p r o t e c t i v e e f f e c t s over the remaining time p e r i o d , 0.5-4 h, were l e s s . With regard t o v a r i o u s types of arrhythmias, the i n c i d e n c e of PVC's was s i m i l a r i n a l l groups. The l o g 1 0 number of PVC was s l i g h t l y higher i n the high dose of 4 mg/kg q u i n a c a i n o l t r e a t e d group but not t o a s t a t i s t i c a l l y s i g n i f i c a n t degree. Quinacainol dose-dependently reduced the i n c i d e n c e of VT and VF over the 4 h f o l l o w i n g o c c l u s i o n . This was s t a t i s t i c a l l y s i g n i f i c a n t f o r VT and VF i n the 0-0.5 h p e r i o d and f o r VF i n the 0.5-4 h p e r i o d . 102 Table 8. A n t i a r r h y t h m i c e f f e c t s of q u i n a c a i n o l a g a i n s t ischaemia-induced arrhythmias i n conscious r a t s i n e a r l y (0-0.5 h) and l a t e (0.5-4 h) periods f o l l o w i n g coronary a r t e r y o c c l u s i o n . Dose Ant i a r r h y t h m i c E f f e c t s AS Log 1 0 PVC VT VF a) 0-0.5 h p o s t - o c c l u s i o n c o n t r o l 2 mg/kg 4 mg/kg 4.8±1.0 2.5±0.5" 1.5±0.5" 1.2±0.1 1.8±0.2 1.810.4 8/10 8/10 3/10* 6/10 4/10 1/10* b) 0.5-4 h p o s t - o c c l u s i o n c o n t r o l 2 mg/kg 4 mg/kg 2.010.2 1.910.4 2.410.5 1.710.4 1.710.4 2.0+0.5 2/4 4/10 3/10 2/4 3/10 1/10* AS=arrhythmia score. C a l c u l a t i o n s t o determine AS were dis c u s s e d i n the methods and a l l values represent mean 1 s.e.mean. PVC's are expressed as l o g 1 0 PV£ number. For VT and VF, values are i n c i d e n c e (out of n) versus c o n t r o l c o n t r o l . ( v e h i c l e ) w h i l e + I n d i c a t e s p<0.05 i n d i c a t e s p<0.01 versus 103 Serum K + l e v e l s did not increase s t a t i s t i c a l l y s i g n i f i c a n t l y 4 h post-occlusion as compared to pre-occlusion values for quinacainol-treated group. In summary quinacainol provided marked protection against ischaemia-induced arrhythmias and was p a r t i c u l a r l y a n t i f i b r i l l a t o r y at 4 mg/kg. The dose-dependent e f f e c t of quinacainol i n reducing the frequency of arrhythmias i s exemplified not only by the arrhythmia scores but also by the decrease i n the incidence of serious arrhythmias (VT and VF) . Higher doses of 8 mg/kg were tested i n a separate study and i t was found to potentiate rather than reduce arrhythmias. This pro-arrhythmic e f f e c t took the form of p r e c i p i t a t i n g arrhythmias within 1 min of occlusion. This was d i f f e r e n t from the groups tested with 2 and 4 mg/kg, where arrhythmias only occurred at l e a s t 5 min a f t e r occlusion. 3.4 E l e c t r i c a l l y - i n d u c e d Arrhythmias i n Anaesthetised Rats Time was a confounding v a r i a b l e i n the e l e c t r i c a l stimulation studies necessitating the presence of a time con t r o l , i . e . the vehicle control group. For t h i s reason, and the sake of continuity i n data analysis of a l l variables (haemodynamics, ECG, and e l e c t r i c a l stimulation) , vehicle data were not pooled. 104 The v e h i c l e d i d not i n f l u e n c e r e s p o n s i v e n e s s t o e l e c t r i c a l s t i m u l a t i o n whereas q u i n a c a i n o l d o s e - d e p e n d e n t l y i n c r e a s e d i T , t,- , and VFj ( F i g u r e s 17a, 17b, and 1 8 ) . I n a d d i t i o n , q u i n a c a i n o l l e n g t h e n e d t h e e f f e c t i v e r e f r a c t o r y p e r i o d w h i l e r e d u c i n g t h e maximum f o l l o w i n g f r e q u e n c y ( F i g u r e 19b and 1 9 a ) . I n summary, q u i n a c a i n o l produced a r e d u c t i o n i n e x c i t a b i l i t y t o e l e c t r i c a l s t i m u l a t i o n . T h i s was a s s o c i a t e d w i t h a n t i a r r h y t h m i c e f f e c t s a g a i n s t e l e c t r i c a l l y - i n d u c e d v e n t r i c u l a r f i b r i l l a t i o n . Serum K + l e v e l s i n c r e a s e d from a c o n t r o l v a l u e o f 3.7 3±0.2 t o 4.14±0.15 a t t h e end o f t h e e x p e r i m e n t f o r t h e v e h i c l e c o n t r o l group. F o r t h e q u i n a c a i n o l t r e a t e d group, serum K + l e v e l s i n c r e a s e d from a c o n t r o l v a l u e o f 3.65±0.19 t o 3.92±0.2 a t t h e end o f t h e e x p e r i m e n t . No s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s were seen when comparing c o n t r o l v a l u e s w i t h p o s t e x p e r i m e n t v a l u e s w i t h i n each group o r comparing c o n t r o l v a l u e s o r p o s t e x p e r i m e n t v a l u e s between gr o u p s . 3.5 E f f e c t s of Quinacainol on E p i c a r d i a l Action Potentials i n the Anaesthetised Rat I t c an be seen t h a t q u i n a c a i n o l m a r k e d l y and dose-d e p e n d e n t l y d e c r e a s e d t h e r i s e r a t e o f t h e AP (phase 0) , d V / d t m a x ( F i g u r e 20a) . The h e i g h t o f t h e AP was a l s o dose-d e p e n d e n t l y d e p r e s s e d ( F i g u r e 20b) w h i l e t h e r e s t i n g 105 Fi g u r e 17. E f f e c t s of q u i n a c a i n o l as compared t o v e h i c l e c o n t r o l on t h r e s h o l d c u r r e n t , i T (A) and t h r e s h o l d d u r a t i o n , t T (B) f o r i n d u c t i o n of VT i n p e n t o b a r b i t a l anaesthetised r a t s s u b j e c t e d t o e l e c t r i c a l s t i m u l a t i o n of the l e f t v e n t r i c l e . As time was not a s i g n i f i c a n t source of v a r i a n c e , the i n d i c a t e d values were measured 10 min a f t e r drug o r v e h i c l e a d m i n i s t r a t i o n and expressed as mean + s.e.mean. Treatment was a s t a t i s t i c a l l y s i g n i f i c a n t source of v a r i a n c e when compared t o pre-drug v a l u e s (0 mg/kg on the a b s c i s s a ) , i n d i c a t i n g p<0.05. + I n d i c a t e s a s i g n i f i c a n t d i f f e r e n c e (p<0.05) between the v e h i c l e c o n t r o l group and q u i n a c a i n o l t r e a t e d group a t the dose i n d i c a t e d . A 90 75 -control gp treated gp H Vehicle control gp Quinacainol treated gp 107 F i g u r e 18. Comparison of q u i n a c a i n o l versus v e h i c l e c o n t r o l on the i n d u c t i o n of v e n t r i c u l a r f i b r i l l a t i o n t h r e s h o l d (VF t) i n p e n t o b a r b i t a l anaesthetised r a t s i n v i v o by s t i m u l a t i o n of the l e f t v e n t r i c l e . I n d i c a t e s p<0.05 from pre-drug v a l u e s . + p<0.05 as compared t o v e h i c l e c o n t r o l . 108 Figure 19. E f f e c t s of quinacainol as compared to vehicle control on maximum following frequency, MFF (A) and e f f e c t i v e r e f r a c t o r y period, ERP (B) as determined by the extra stimulus method. Indicated values were measured 10 min a f t e r end of drug or vehicle administration and expressed as mean ± s.e.mean. Time was not a s i g n i f i c a n t source of variance while treatment was (p<0.05). p<0.05 as compared to pre-drug values (0 mg/kg). p<0.05 as compared to v e h i c l e c o n t r o l . 20 control gp treated gp B 70 60 -Dose (mg/kg) Hi Vehicle IB Quinacainol control gp treated gp 110 A 2 0 0 Dose (mg/kg) B 100 Dose (mg/kg) Figure 20. Effects of quinacainol on phase 0 of the action potent ial . Intracel lular recordings of the epicardial action potential show that the maximum r ise rate of the action potential (dV/dt^ ) (A) and action potential height (B) are dose-dependently decreased in the presence of cumulative doses of quinacainol. Indicates p<0.001 from pre-drug values. I l l membrane p o t e n t i a l was not s i g n i f i c a n t l y a f f e c t e d although a s l i g h t depression was seen at i n c r e a s i n g doses (Figure 21). AP d u r a t i o n was measured at 10, 50, and 90% r e p o l a r i z a t i o n (Figure 22) . P r e f e r e n t i a l e f f e c t s on d i f f e r e n t phases of r e p o l a r i z a t i o n were never observed w i t h the hi g h e s t dose of 8 mg/kg s i g n i f i c a n t l y (p<0.001) pro l o n g i n g r e p o l a r i z a t i o n at a l l times. An obvious trend of delayed r e p o l a r i z a t i o n ( f o r APD 1 0 5 0 ( 9 0 ) w i t h i n c r e a s i n g doses c o u l d be seen although t h i s was s i g n i f i c a n t f o r a l l phases only at the highest dose (8 mg/kg). 3.6 I s o l a t e d P e r f u s e d Rat H e a r t s In order t o assess the d i r e c t e f f e c t s of q u i n a c a i n o l i n c a r d i a c t i s s u e , i s o l a t e d r a t hea r t s were perfused w i t h v a r y i n g c o n c e n t r a t i o n s of q u i n a c a i n o l (modified Langendorff apparatus, C u r t i s et al., 1986) and compared t o t h a t of the c l a s s i c a l sodium channel b l o c k e r TTX. Fi g u r e 23 d e p i c t s the e f f e c t s of q u i n a c a i n o l and TTX on c o n t r a c t i l i t y as measured by dP/dt. TTX d i d not appear to depress c o n t r a c t i l i t y w h i l e a s l i g h t dose-dependent cardiodepressant e f f e c t was seen w i t h q u i n a c a i n o l . This may have been a s s o c i a t e d w i t h i t ' s b r a d y c a r d i c a c t i o n s . 112 Figure 22. E f f e c t s of quinacainol on d i f f e r e n t phases of r e p o l a r i z a t i o n of the e p i c a r d i a l i n t r a c e l l u l a r action p o t e n t i a l , i . e . APD10 = AP duration following 10% r e p o l a r i z a t i o n . Indicates p<0.00l as compared to pre-drug values (Unweighted Means ANOVA). 114 CO ~o> I E E "O CL 4 0 0 0 3 0 0 0 2 0 0 0 1 0 0 0 -0 - 1 0 0 0 - 2 0 0 0 - 3 0 0 0 control TTX +dP/dt i -•I— + — • 3 x 1 0 1 x 1 0 Concentration (M) 3 x 1 0 1 x 1 0 quinacainol +dP/dt TTX -cP/dt quinacainol - d P / d t Figure 23. The e f f e c t s of tetrodotoxin (TTX) and quinacainol on a measure of c o n t r a c t i l i t y (dP/dt) i n the i s o l a t e d r a t heart. Quinacainol had greater e f f e c t s on c o n t r a c t i l i t y than TTX. Treatment was not a s i g n i f i c a n t source of variance when comparing quinacainol and TTX to t h e i r own control values. S i g n i f i c a n t differences were detected when comparing respective doses of quinacainol with TTX ( + indicates p<0.05) (Unweighted Means ANOVA). No error bars are indicated f o r lxlO' 5 M i n the quinacainol treated group as 6 of 7 hearts were not i n sinus rhythm but rather experienced e i t h e r a t r i o v e n t r i c u l a r block or PVC's. 115 4 DISCUSSION 4.1 Hemodynamic and ECG Eff e c t s of Quinacainol The r e s u l t s o f t h e haemodynamic d o s e - r e s p o n s e s t u d y e n a b l e d t h e s e l e c t i o n o f a p p r o p r i a t e doses f o r f u r t h e r e x p e r i m e n t s . The ED 5 0 f o r BP e f f e c t s was e s t i m a t e d t o be 4 mg/kg from t h i s e x p e r i m e n t . S i g n i f i c a n t t o x i c i t y was o n l y e n c o u n t e r e d a t doses o f 8 mg/kg and above and was r e f e r a b l e t o t h e h e a r t , i . e . s p o n t a n e o u s l y r e v e r t i n g t a c h y a r r h y t h m i a s . When compared w i t h t h e v e h i c l e , q u i n a c a i n o l had no major e f f e c t s on BP u n l i k e many c l a s s I a n t i a r r h y t h m i c a g e n t s (Legrand and C o l l i g n o n , 1985; H o n e r j a g e r e t a l . , 1986). Q u i n a c a i n o l ' s major e f f e c t s were t o l o w e r HR and p r o l o n g t h e P-R i n t e r v a l and QRS d u r a t i o n o f t h e ECG. W i t h r e g a r d t o t h e t o x i c i t y o f c l a s s I a g e n t s and t h e i r d e l e t e r i o u s haemodynamic e f f e c t s , t h e most a p p r o p r i a t e c h o i c e o f an agent must t a k e i n t o a c c o u n t c a r d i o v a s c u l a r d e p r e s s i o n (Legrand and C o l l i g n o n , 1985). L e g r a n d and C o l l i g n o n (1985) n o t e d t h a t w h i l e c l a s s l a a g e n t s have minor haemodynamic e f f e c t s and c l a s s l b drugs a r e e x c e p t i o n a l l y w e l l t o l e r a t e d , t h e c a r d i o d e p r e s s a n t e f f e c t s o f c l a s s I c ag e n t s have p o t e n t i a l l y a d v e r s e e f f e c t s e s p e c i a l l y i n p a t i e n t s w i t h l e f t v e n t r i c u l a r d y s f u n c t i o n . There i s c o n t r o v e r s y however, o v e r t h e a s s o c i a t i o n between N a + c h a n n e l b l o c k a d e and n e g a t i v e i n o t r o p y . H o n e r j a g e r e t al. 116 (1986) s t a t e d t h a t sodium channel b l o c k i n v a r i a b l y has a moderate negative i n o t r o p i c i n f l u e n c e , as demonstrated w i t h TTX, although t h i s may not be seen w i t h l b agents due to t h e i r f a s t o f f s e t k i n e t i c s . Cowan and Vaughan W i l l i a m s (1981) and Courtney (1984) found no a s s o c i a t i o n between the e l e c t r o p h y s i o l o g i c a l e f f e c t s of c l a s s I agents and negative i n o t r o p y . 4.2 Antiarrhythmic Actions of Quinacainol against Ischeemia-induced Arrhythmias Q u i n a c a i n o l demonstrated a r a t h e r a t t r a c t i v e p r o f i l e as compared t o other sodium channel b l o c k e r s t e s t e d f o r a n t i a r r h y t h m i c e f f i c a c y . I t gave p r o t e c t i o n a g a i n s t acute ischaemia-induced arrhythmias without inducing c o n v u l s i o n s , s i g n i f i c a n t l y depressing blood pressure nor reducing heart r a t e . However, as i n d i c a t e d by e l e c t r o p h y s i o l o g i c a l a c t i o n s , a n t i a r r h y t h m i c doses caused d e t e c t a b l e depression of sodium c u r r e n t i n non-ischaemic myocardium. This a c t i o n i n normal myocardium i s t y p i c a l of c l a s s I c agents and, t o a l e s s e r extent, l a agents ( P a l l a n d i and Campbell, 1988). At hig h e r doses (8 mg/kg and above), the drug p r e c i p i t a t e d arrhythmias s h o r t l y a f t e r o c c l u s i o n of the coronary a r t e r y ; a f i n d i n g reminiscent of the pro-arrhythmic f i n d i n g s w i t h other c l a s s I c agents (CAST I n v e s t i g a t o r s , 1989; Horowitz, 1990; Levine et al., 1989; Morganroth and Horowitz, 1984). 117 Q u i n a c a i n o l had i n t e r e s t i n g e f f e c t s on ischaemia-induced ECG changes. Both doses (2 mg/kg and 4 mg/kg) reduced S-T segment e l e v a t i o n and prolonged the time t o maximum S-T segment e l e v a t i o n and maximum R-wave amplitude. This f i n d i n g i s s i m i l a r t o t h a t seen i n a previous study done i n our l a b o r a t o r y w i t h TTX (Abraham et al., 1989). In each of these s t u d i e s (TTX and q u i n a c a i n o l ) , the decreased extent of S-T segment e l e v a t i o n could not be a t t r i b u t e d to s i g n i f i c a n t d i f f e r e n c e s i n ischaemic zone s i z e . Greater blockade of sodium channels i n ischaemic t i s s u e c ould e x p l a i n the unexpected e f f e c t s of q u i n a c a i n o l on ischaemia-induced ECG changes as has been p o s t u l a t e d p r e v i o u s l y by Abraham et al. (1989) a f t e r f i n d i n g s i m i l a r e f f e c t s w i t h TTX. In c o n t r o l groups (given v e h i c l e ) , coronary o c c l u s i o n produces time-dependent e l e v a t i o n s of R-wave and S-T segment (Johnston et al., 1981, 1983; C u r t i s e t al. , 1987), i n d i r e c t i n d i c e s of the extent and s e v e r i t y of myocardial ischaemia (Janse, 1979; Janse, 1986). Several agents delay the onset of S-T segment e l e v a t i o n , i n c l u d i n g the adrenergic B b l o c k e r , p r o p r a n o l o l (Berdeaux et al. , 1978). Other agents p r e v i o u s l y s t u d i e d i n our l a b o r a t o r y produced s i m i l a r r e s u l t s , most notab l y , the calcium channel b l o c k e r s f e l o d i p i n e ( C u r t i s et al., 1985), anipamil ( C u r t i s et al., 1986b), and verapamil ( C u r t i s and Walker, 1986). However, w h i l e these agents produce s i m i l a r delayed e f f e c t s on the r a t e of development of S-T e l e v a t i o n , they d i d not reduce the maximum S-T segment e l e v a t i o n ( C u r t i s et a l . , 1987). 118 In i s o l a t e d h e a r t s , a dose-dependent cardiodepressant e f f e c t was seen w i t h q u i n a c a i n o l at concentrations which prolonged the P-R i n t e r v a l and QRS d u r a t i o n (Howard and Walker, 1990b). The depressed c o n t r a c t i l i t y seen in vitro w i t h q u i n a c a i n o l may have been a s s o c i a t e d w i t h i t s b r a d y c a r d i c a c t i o n s . However, t h a t q u i n a c a i n o l reduces c o n t r a c t i l i t y (and minimally at t h a t ) , seems an u n l i k e l y e x p l a n a t i o n of the ECG r e s u l t s f o l l o w i n g o c c l u s i o n inasmuch as c a l c i u m channel b l o c k e r s reduce c o n t r a c t i l i t y but do not reduce maximum S-T segment e l e v a t i o n (as reviewed by C u r t i s et a l . , 1987; C u r t i s and Walker, 1986). In a d d i t i o n , r e d u c t i o n of maximum S-T segment e l e v a t i o n has not been seen w i t h any other c l a s s I a n t i a r r h y t h m i c agent used i n our l a b o r a t o r y which i n c l u d e q u i n i d i n e ( l a ) , disopyramide ( l a ) , l i d o c a i n e (lb) (Johnston et al., 1983), and m e x i l e t i n e (lb) (Igwemezie, 1990). F a c t o r s a l t e r i n g myocardial oxygen requirements cannot be excluded as p o s s i b l e c o n t r i b u t o r s t o q u i n a c a i n o l 1 s a c t i o n s on the ischaemic ECG: The primary determinants of myocardial oxygen demand are c o n t r a c t i l i t y and heart r a t e . Both doses of q u i n a c a i n o l produced s i m i l a r e f f e c t s on heart r a t e , and both doses of q u i n a c a i n o l s i g n i f i c a n t l y decreased S-T segment e l e v a t i o n , suggesting e f f e c t s on heart r a t e (tendency t o bradycardia) may be a f a c t o r . The above observations suggest t h a t q u i n a c a i n o l demonstrated some degree of s e l e c t i v i t y on p a r t i a l l y d e p o l a r i z e d t i s s u e under a c i d i c c o n d i t i o n s as occurs i n 119 ischaemic t i s s u e although blockade of v e n t r i c u l a r sodium channels i n normal t i s s u e was a l s o evident. Quinacainol d i d not demonstrate the degree of s e l e c t i v i t y f o r the ischaemic myocardium t h a t would have been d e s i r e d of an i d e a l a n t i a r r h y t h m i c agent. 4.3 Actions of Quinacainol Against E l e c t r i c a l l y - i n d u c e d Arrhythmias i n the Anaesthetised Rat R e s u l t s from the ECG and e l e c t r i c a l s t i m u l a t i o n study suggested t h a t q u i n a c a i n o l produced sodium channel blockade as demonstrated by the p r o l o n g a t i o n i n the P-R i n t e r v a l without changing the Q-Tc i n t e r v a l . Q-Tc i n t e r v a l p r o l o n g a t i o n suggested c l a s s l a a c t i o n s . However, P-R i n t e r v a l widening cannot be considered c o n c l u s i v e evidence of sodium channel blockade s i n c e p r o l o n g a t i o n of the P-R i n t e r v a l i n r a t s i s seen w i t h both sodium and calcium channel b l o c k e r s ( B o t t i n g et al., 1986). In t h i s study the QRS complex was not markedly widened as might be expected from a c l a s I c compound. In a d d i t i o n t o ECG evidence, the e f f e c t s of q u i n a c a i n o l on i T , tr , and VFf were c o n s i s t e n t w i t h sodium channel blockade (Beatch e t al., 1988; Hodess e t al., 1979; M a r s h a l l et al., 1983; Yoon et al., 1974). Quinacainol was a l s o very e f f e c t i v e i n p r o l o n g i n g the ERP and i n reducing the MFF. These a c t i o n s are c o n s i s t e n t w i t h c l a s s l a and l b agents although the l a t t e r produce few ECG s i g n s of sodium channel 120 blockade at normal sinus b e a t i n g r a t e s due t o t h e i r high frequency dependency (Campbell, 1983b; Courtney, 1987). In the above experiment, c h a r a c t e r i z a t i o n of q u i n a c a i n o l was e s t a b l i s h e d a t 7.5 Hz s t i m u l a t i o n frequency and t h e r e f o r e d i d not take i n t o c o n s i d e r a t i o n the marked frequency dependent p r o p e r t i e s of c l a s s I agents (Courtney, 1980). Quinacainol seemed f r e e of t o x i c i t y i n t h a t i t d i d not produce signs of c a r d i o d e p r e s s i o n or CNS t o x i c i t y at doses which had marked e f f e c t s i n reducing e x c i t a b i l i t y t o e l e c t r i c a l s t i m u l a t i o n (Howard and Walker, 1990a). 4.4 Actions of Quinacainol on E p i c a r d i a l I n t r a c e l l u l a r Recordings The predominant e l e c t r o p h y s i o l o g i c a l a c t i o n of c l a s s I a n t i a r r h y t h m i c agents i s t o bl o c k c a r d i a c Na + channels (Sheldon et al. , 1989). This reduces the r a t e of d e p o l a r i z a t i o n of the AP and slows impulse propagation (Sheldon et al. , 1989). Quinacainol had no e f f e c t on the r e s t i n g membrane p o t e n t i a l although i t had major i n h i b i t o r y e f f e c t s on phase 0 of the AP. I t c l e a r l y demonstrated c l a s s I c a c t i o n s i n t h a t i t reduced the maximum r i s e r a t e of the AP and decreased AP height without i n f l u e n c i n g other phases at doses producing e q u i v a l e n t ECG e f f e c t s i n the other experimental p r e p a r a t i o n s . These observations provided evidence f o r s e l e c t i v e v e n t r i c u l a r Na + channel blockade (Sheldon et al., 1989). 121 The use of maximal upstroke v e l o c i t y (V m a x ) as an estimate of a v a i l a b l e Na + conductance i s a controversial subject (Grant et al., 1984; Cohen et al., 1984). In general, a c u r v i l i n e a r r e l a t i o n s h i p describes the dependence o f vmax o n N a + conductance, r i s i n g more sharply at low Na + conductance and approaching a maximum value at high Na + conductance ( C a t t e r a l l , 1987). Measurements i n excitable cardiac c e l l s t y p i c a l l y span t h i s nonlinear range such that measurements of V m a x are only a semi-quantitative measure of Na + channel a v a i l a b i l i t y ( C a t t e r a l l , 1987). Much of the n o n - l i n e a r i t y could be attributed to a c t i v a t i o n k i n e t i c s of the Na + channel (Cohen et al., 1984). In the i n t r a c e l l u l a r study, quinacainol had neither class III nor class IV properties since the plateau duration (class IV) and shape (class III) i n the r a t v e n t r i c l e were r e l a t i v e l y unaffected. There also was no evidence to suggest that quinacainol possessed class II a c t i v i t y . Quinacainol prolonged APD only at the highest dose tested. This suggested that quinacainol may be acting as a class l a agent at higher doses. A lack of e f f e c t on APD90 has been previously described for other c l a s s I agents, notably f l e c a i n i d e (Campbell, 1983a) and BW A256C a putative c l a s s I agent (Allan et al., 1986). However, these agents also showed lack of e f f e c t on ERP (in guinea-pig v e n t r i c l e ) while quinacainol demonstrated a dose-dependent prolongation of ERP. Class I agents such as lidocaine (lb) have been shown to change both APD90 and ERP (Varro et al. , 1985). 122 Such varied e f f e c t s led i n part to Harrison et a l ' s (1981) and Campbell's (1983b) s u b c l a s s i f i c a t i o n schemes based on e f f e c t s on APD and rate-dependent e f f e c t s (onset-offset k i n e t i c s ) , respectively. Based on the combined r e s u l t s obtained with quinacainol, i . e . lack of e f f e c t on a l l phases of the AP except phase 0 and AP height, and a dose-dependent increase i n ERP, quinacainol demonstrated Ic actions with respect to i t s lack of e f f e c t s on APD but lb actions with respect to i t s lengthening of the ERP independent of changes i n APD. Class I agents a l t e r r e p o l a r i z a t i o n i n a v a r i e t y of ways. Lidocaine induces such e f f e c t s on the sodium "window" current while quinidine a f f e c t s the delayed r e c t i f i e r (K + channel blockade) (Colatsky, 1982). The e f f e c t s of encainide (a Ic agent) are attenuated with increasing frequency (Ebharrar and Zipes, 1982; c i t e d i n Nattel and Zeng, 1984). Studies concerning the frequency-dependent action of quinacainol are needed to provide additional evidence of i t s s u b c l a s s i f i c a t i o n . Such a study would te s t i t s frequency-dependency and d i f f e r e n t i a l e f f e c t s on APD might play a r o l e i n determining the o v e r a l l e f f e c t s of the drug on the r e f r a c t o r y period. Nattel (1987) infe r r e d from h i s data that the use-dependence of c l a s s I e f f e c t s on conduction in vivo were q u a n t i t a t i v e l y predictable from the i n t e r v a l dependence of e f f e c t s ort V m a x in vitro. 123 Thus the c e l l u l a r e l e c t r o p h y s i o l o g i c a l p r o f i l e obtained i s not s t r i c t l y consistent with any of the three current subclasses within class I. This anomaly may indicate short-comings i n the present drug c l a s s i f i c a t i o n scheme and may not be i n d i c a t i v e of a "novel" antiarrhythmic p r o f i l e . S i milar conclusions can also be drawn from other studies which tested putative class I antiarrhythmic compounds (e.g. Colatsky et al., 1987). 4.5 E f f e c t s of Quinacainol in vitro Quinacainol 1s possible s i t e ( s ) of action are s t i l l imprecisely determined. Quinacainol could have produced i t s e f f e c t s i n normal myocardium, ischaemic myocardium, or the i n t e r f a c e between the two; the border zone. We must r e l y on i n d i r e c t evidence i n t h i s regard due to the d i f f i c u l t y i n obtaining d i r e c t evidence of drug e f f e c t s on ischaemic t i s s u e (Abraham et al., 1989; Inoue et al., 1982). Th e o r e t i c a l l y , quinacainol could act by protecting the normal myocardium from invasion by arrhythmic impulses a r i s i n g i n the ischaemic zone, or i t could act i n ischaemic t i s s u e to d i r e c t l y suppress arrhythmogenesis. Patch clamp studies would provide stronger evidence on the voltage-dependency of quinacainol. We know that ischaemia i s associated with a 15 to 20 mV depolarization (Inoue et al., 1982), therefore, i f blockade of Na+ channels i n v e n t r i c u l a r c e l l s by quinacainol i s dependent on steady-state depolarization, blockade of 124 sodium channels by q u i n a c a i n o l would be more pronounced i n ischaemic t i s s u e . Greater blockade of sodium channels i n ischaemic t i s s u e may help e x p l a i n the unexpected e f f e c t of q u i n a c a i n o l on ischaemia-induced ECG changes. By determining q u i n a c a i n o l • s a c t i o n s i n i s o l a t e d v e n t r i c u l a r t i s s u e , an opportunity was provided f o r the d i r e c t assessment of q u i n a c a i n o l 1 s e f f e c t s and t o compare these e f f e c t s w i t h TTX, an agent whose only known a c t i o n i s blockade of sodium channels. This experiment showed t h a t q u i n a c a i n o l had a l l of the expected e f f e c t s of a c l a s s I c agent s i n c e i t widened the P-R i n t e r v a l and QRS d u r a t i o n without having a major e f f e c t on the Q-Tc i n t e r v a l . Q u i n a c a i n o l was more potent than TTX, the c l a s s i c sodium channel b l o c k e r , on a l l ECG v a r i a b l e s measured. Blockade of other channels may a l s o prolong the P-R i n t e r v a l . In the r a t heart both sodium and calcium channels p a r t i c i p a t e i n A-V conduction such t h a t p r o l o n g a t i o n of the P-R i n t e r v a l i s a u s e f u l measure of sodium and calcium channel blockade ( B o t t i n g et al., 1985). The g r e a t e r e f f e c t of q u i n a c a i n o l compared w i t h TTX on the P-R i n t e r v a l , suggested t h a t q u i n a c a i n o l may a l s o have blocked calcium channels t o produce i t s e f f e c t . In any event, calcium channel blockade i s not i n v o l v e d i n P-R i n t e r v a l p r o l o n g a t i o n t o the same extent as Na + channel blockade i n s m a l l e r species ( B o t t i n g et al., 1986) . As a r e s u l t of low doses of TTX ( 1 0 7 M) sho r t e n i n g the p l a t e a u of the c a r d i a c P u r k i n j e f i b e r AP without a f f e c t i n g 125 the upstroke v e l o c i t y , Coraboeuf et al. (1979) suggested two c a r d i a c sodium c u r r e n t s - one r e s p o n s i b l e f o r the AP upstroke and a second responding t o TTX. Thus there could be two popul a t i o n s of c a r d i a c Na + channels (Fozzard et al., 1985) . T T X - s e n s i t i v e Na + channels c o u l d i n a c t i v a t e w i t h time constants of s e v e r a l hundred msec, t h e i r s e n s i t i v i t y t o TTX being supported by s t u d i e s i n r a b b i t P u r k i n j e strands demonstrating a s m a l l , s l o w l y i n a c t i v a t i n g component of Na + c u r r e n t (Carmeliet, 1984). In a d d i t i o n , some TTX- s e n s i t i v e Na + channels could be v o l t a g e and time-independent p r o v i d i n g a background inward c u r r e n t important t o the r e s t i n g p o t e n t i a l as w e l l as the p l a t e a u (Fozzard et a l . , 1985). These s l o w l y decaying or steady Na + c u r r e n t s are very s m a l l , t h e i r s i z e being i n the range of c u r r e n t s t h a t could be generated by Na + dependent membrane t r a n s p o r t processes but are not gated Na + channels (Fozzard e t al. , 1985). These p o s s i b i l i t i e s i n c l u d e the e l e c t r o g e n i c Na +-K + pump and the N a + - C a 2 + exchange system, and p o s s i b l y the Na +-H + or Na-K + t r a n s p o r t systems i f they are e l e c t r o g e n i c (Fozzard et al., 1985) . The ex i s t e n c e of 2 d i f f e r e n t c a r d i a c sodium channels may a l s o help e x p l a i n the d i f f e r e n t e f f e c t s observed w i t h q u i n a c a i n o l and TTX (both of which b l o c k sodium channels) on the v a r i a b l e s measured. In r e l a t i o n t o the haemodynamic e f f e c t s of q u i n a c a i n o l , i t i s w e l l known t h a t negative i n o t r o p i s m i s a common s i d e e f f e c t of treatment w i t h c l a s s I agents (Hoffmeister et al., 1987; Honerjager et al., 1986). Honerjager et al. (1986) 126 compared the negative i n o t r o p i c e f f e c t s of TTX and 7 c l a s s I a n t i a r r h y t h m i c s i n r e l a t i o n t o Na + channel blockade. With the exception of 2 agents ( s p a r t e i n e and AR-LH31) a l l the drugs produced a l a r g e r negative i n o t r o p i c e f f e c t than TTX at c o n c e n t r a t i o n s e q u i e f f e c t i v e i n reducing V m a x , suggesting t h a t blockade of Na + channels can account f o r only p a r t of t h e i r negative i n o t r o p i c e f f e c t . Honerjager (1986) a l s o found TTX t o be s i g n i f i c a n t l y more potent i n reducing V m a x than i n reducing f o r c e of c o n t r a c t i o n as compared t o v a r i o u s c l a s s I agents which exerted stronger negative i n o t r o p i c e f f e c t s . One f u r t h e r p o s s i b i l i t y i n regard t o the d i f f e r e n c e s seen on c o n t r a c t i l i t y between TTX and q u i n a c a i n o l may be t h a t not high enough con c e n t r a t i o n s of TTX were used t o v a l i d l y compare i t s e f f e c t s t o q u i n a c a i n o l 1 s . Winslow e t al (1983), i n a study u s i n g Langendorff i s o l a t e d perfused r a t h e a r t s , found " a n t i a r r h y t h m i c c o n c e n t r a t i o n s " (0.16-1.57 u,M) of TTX t o reduce c o n t r a c t i l i t y up t o approximately 48%. The experimental data from i s o l a t e d hearts supports two co n c l u s i o n s , f i r s t , t h a t q u i n a c a i n o l alone can produce the e f f e c t s seen i n in vivo experiments without need f o r conversion t o a c t i v e m e t a b o l i t e s . Secondly, q u i n a c a i n o l e x e r t s e f f e c t s on the normal myocardium and need not r e l y on the c o n d i t i o n s produced by ischaemia t o p o t e n t i a t e i t s e f f e c t s . 127 5 Summary The s t u d i e s showed t h a t doses o f q u i n a c a i n o l , w h i c h i n normal v e n t r i c u l a r myocardium p r o l o n g e d t h e P-R i n t e r v a l , i n c r e a s e d e l e c t r i c a l s t i m u l a t i o n t h r e s h o l d s , and r e d u c e d d V / d t m a x and AP h e i g h t , p o s s e s s e d a n t i a r r h y t h m i c a c t i v i t y a g a i n s t ischaemia-induced a r r h y t h m i a s . The l a t t e r o c c u r r e d w i t h o u t compromising haemodynamic s t a t u s . I f e f f e c t s on t h e ECG, e l e c t r i c a l s t i m u l a t i o n , and i n t r a c e l l u l a r p o t e n t i a l s s h a r e a common mechanism o f sodium c h a n n e l b l o c k a d e (sodium c u r r e n t i n h i b i t i o n ) t h e n i t must be assumed t h a t t h e a n t i a r r h y t h m i c a c t i o n o f q u i n a c a i n o l c o r r e l a t e s w i t h b l o c k a d e o f v e n t r i c u l a r gNa i n normal myocardium. A l l s t u d i e s were p e r f o r m e d i n one s p e c i e s under one o f two c o n d i t i o n s , e i t h e r c o n s c i o u s o r p e n t o b a r b i t a l a n a e s t h e t i s e d . The s i m i l a r i t y o f c o n d i t i o n s i n t h e d i f f e r e n t e x p e r i m e n t a l models a l l o w e d f o r r e a d y e x t r a p o l a t i o n w i t h r e s p e c t t o t h e doses used i n t h e v a r i o u s s t u d i e s . A t a n t i a r r h y t h m i c d o s e s , t o x i c e f f e c t s r e f e r a b l e t o e i t h e r t h e CNS o r c a r d i o v a s c u l a r system were not seen i n marked c o n t r a s t t o o t h e r c l a s s I a g e n t s t h a t we, and o t h e r s , have t e s t e d under s i m i l a r c o n d i t i o n s . The a n t i a r r h y t h m i c e f f i c a c y o f m e x i l e t i n e and l i d o c a i n e ( l b agents) a r e s e v e r e l y l i m i t e d by t h e o c c u r r e n c e o f c o n v u l s i o n s a t c o m p a r a t i v e l y low doses (Igwemezie e t a l . , 1990) w h i l e w i t h d r u g s s u c h as q u i n i d i n e and p r o c a i n a m i d e ( l a agents) , t h e m a j o r t o x i c i t y i s c a r d i o v a s c u l a r d e p r e s s i o n ( n e g a t i v e 128 i n o t r o p i s m ) and p e r i p h e r a l v a s o d i l a t i o n which i s no t h e l p f u l i n p a t i e n t s w i t h c o n g e s t i v e h e a r t f a i l u r e (Legrand and C o l l i g n o n , 1985) . P r o t o t y p i c a l c l a s s I c agents such as f l e c a i n i d e and e n c a i n i d e a l s o have s p e c i f i c and dangerous t o x i c i t i e s i n t h a t p r o a r r h y t h m i c e f f e c t s may o c c u r i n the s e t t i n g o f m y o c a r d i a l ischaemia and i n f a r c t i o n (CAST I n v e s t i g a t o r s 1989a, 1989b) . W i t h r e g a r d t o q u i n a c a i n o l 1 s s u b c l a s s i f i c a t i o n , T a b l e 9 p r e s e n t s the p o o l e d r e s u l t s from a l l e x p e r i m e n t a l s t u d i e s t h a t a r e used f o r d e f i n i t i v e purposes i n s u b c l a s s i f i c a t i o n o f c l a s s I compounds. U s i n g the c h a r a c t e r i s t i c s g i v e n t o the v a r i o u s s u b c l a s s i f i c a t i o n d e f i n i t i o n s and the r e s u l t s o b t a i n e d w i t h the v a r i o u s e x p e r i m e n t a l p r o t o c o l s used h e r e i t can be a s c e r t a i n e d t h a t q u i n a c a i n o l demons tra ted Ic a c t i o n s a t lower doses and l a a c t i o n s a t h i g h e r d o s e s . H i g h e r doses were no t n e c e s s a r y f o r a n t i a r r h y t h m i c e f f e c t s . In c o n c l u s i o n , a t doses p r o d u c i n g e q u i v a l e n t p r o l o n g a t i o n o f t h e P-R i n t e r v a l o f the ECG, a c o n s i s t e n t s e r i e s o f f i n d i n g s were no ted w i t h q u i n a c a i n o l . These c o n s i s t e d o f : (1) a s e l e c t i v e d e p r e s s i o n o f AP h e i g h t and o f d V / d t m a x o f phase 0 o f the A P ; (2) moderate i n c r e a s e i n QRS d u r a t i o n w i t h o u t e f f e c t s on the Q - T c i n t e r v a l ; (3) r e d u c t i o n i n e x c i t a b i l i t y t o e l e c t r i c a l s t i m u l a t i o n , and (4) l a c k o f c a r d i o v a s c u l a r d e p r e s s a n t e f f e c t s ( i . e . no s i g n i f i c a n t e f f e c t s on b l o o d p r e s s u r e , h e a r t r a t e , o r c o n t r a c t i l i t y ) . A l l o f the above e f f e c t s were a s s o c i a t e d w i t h p r o t e c t i o n a g a i n s t e l e c t r i c a l l y and i schaemia- induced 129 Table 9. Established and Experimental E f f e c t s of Class I Agents. Hmodynamic and ECG E f f e c t s E f f e c t s on Ki n e t i c s & AP Class BP HR P-R QRS Q-T ERP MFF iT VFT MRR* APD (a) Established E f f e c t s (as reviewed i n the l i t e r a t u r e ) l a 4- 4. t tt t t t t u t t 4 t l b ** «• ** «• « t t t 444 t tt 4 4<+ Ic u n t t t t «• t ; t t ; (b) Experimental E f f e c t s of Quinacainol (i) Hmodynamic study i n conscious rats 4- 4- t tt tt t / / / / / / ( i i ) Antiarrhythmic study 4 44 t t t / / / / / / / / ( i i i ) E l e c t r i c a l Stimulation study 4 44 t tt «• ** t 44 t t / / (iv) E p i c a r d i a l i n t r a c e l l u l a r p o t e n t i a l study 4 44 / / / / / / / ^ t (v) Isolated Heart study / * t t •» / / / / / / Experimental arrows indicate trend f o r a l l doses tested. Indicates that drug e f f e c t s are dependent on frequency. Based on Vaughan Williams (1984b) c l a s s i f i c a t i o n system which subdivides c l a s s I compounds based on t h e i r e f f e c t s on the APD, quinacainol exerts Ic actions at low doses and l a actions at higher doses. In consideration of frequency-dependency, quinacainol was very e f f e c t i v e i n prolonging ERP and reducing MFF. These actions are consistent with l a and l b agents although the ECG e f f e c t s at normal sinus rhythm seemed to preclude l b agents. The ECG e f f e c t s indicate that quinacainol i s a Ic agent since i t widened the P-R i n t e r v a l and QRS duration without having a major e f f e c t on the Q-Tc i n t e r v a l . 130 arrhythmias. U n f o r t u n a t e l y , the extensive analyses performed s t i l l d i d not provide c o n c l u s i v e evidence w i t h regard t o q u i n a c a i n o l 1 s s u b c l a s s i f i c a t i o n . "A drug n e a t l y pigeon-holed by the pharmacologist as the most potent of i t s group f o r some a l l e g e d l y d e s i r a b l e p r o p e r t y , may have d i s a s t r o u s s i d e - e f f e c t s . P r e c i s e a n a l y s i s of i n d i v i d u a l pharmacological a c t i o n s i s , however, necessary i f a proper s c i e n t i f i c b a s i s i s t o be found f o r a r a t i o n a l e of treatment." (Vaughan W i l l i a m s , 1970). 131 6 REFERENCES Au, T.L.S., C o l l i n s , G.A., MacLeod, B.A. and Walker, M.J.A. Arrhythmic responses t o coronary o c c l u s i o n i n d i f f e r e n t s p e c i e s . Proc. C.F.B.S. 22., 1979. Abraham, S., Beatch, G.N., MacLeod, B.A., and Walker, M.J.A. An t i a r r h y t h m i c p r o p e r t i e s of t e t r o d o t o x i n a g a i n s t o c c l u s i o n -induced arrhythmias i n the r a t : A novel approach t o the study of the a n t i a r r h y t h m i c e f f e c t s of v e n t r i c u l a r sodium channel blockade. J . 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