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Antifibrillatory actions of K+ channel blocking drugs Beatch, Gregory N. 1991

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ANTIFIBRILLATORY ACTIONS OF K  T  CHANNEL BLOCKING DRUGS.  by GREGORY N. BEATCH , B.Sc.  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PHARMACOLOGY & THERAPEUTICS  We accept t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1991 (c) Gregory. N. Beatch, 1991  In presenting this thesis in partial fulfilment  of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis 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 of frjMLMrKDUb&H The University of British Columbia Vancouver, Canada  DE-6 (2/88)  k  tHe^(€0llCS  ii  ABSTRACT Class  III antiarrhythmic drugs share  mechanism of widening  the common  the cardiac action p o t e n t i a l  a f f e c t i n g conduction v e l o c i t y .  without  This thesis reports on the  actions of newly developed putative Class III antiarrhythmic drugs,  tedisamil,  KC  8851,  RP  62719,  r i s o t i l i d e , as well as an ATP-sensitive K Studies  actions  drugs i n acute myocardial  performed  to  possible mechanisms responsible f o r these hypothesis  tested  was  that  drug  68798,  examine  the  ischaemia  and  actions.  treatment  The  prevented  arrhythmias induced by acute myocardial ischaemia. dependent actions of these drugs on ECG  and  channel blocker,  +  glibenclamide. of these  were  UK  Species  and blood pressure  were examined i n rats, guinea pigs, pigs and primates. The  five  putative class  III drugs l i s t e d  assessed  f o r antiarrhythmic a c t i v i t y  model  myocardial  of  ischaemia.  tedisamil and  KC  8851,  the  up  to  ECG  (by  fibrillation decreased  I t was  conscious  found  which widened the Q-T  65%) ,  were  i n t h i s species.  the  in a  incidence  of  above were  effective  c  that  only  interval  at  of  suppressing  None of the drug ventricular  rat  treatments  premature  beats.  Tedisamil, but not glibenclamide, prevented tachycardias i n a r a t model of myocardial ischaemia- and reperfusion-induced arrhythmias.  In  an  anaesthetized  pig  model  of  acute  myocardial ischaemia, tedisamil and UK 68,798 were shown to mildly  prolong  the  Q-T  c  interval  by  less  protection against arrhythmias was equivocal.  than  20%,  but  In  further  studies,  tedisamil  and UK  68,798  were  compared to each other for e f f e c t s on v e n t r i c u l a r e p i c a r d i a l action p o t e n t i a l morphology using i n t r a c e l l u l a r recording in vivo,  and e f f e c t s on v e n t r i c u l a r e f f e c t i v e refractory  using e l e c t r i c a l stimulation in vivo pigs.  Tedisamil  (up to 1 mg/kg, Tedisamil  (4 mg/kg,  ventricular  e p i c a r d i a l potentials increased  induction  ineffective  i.v.) widened  in this  guinea p i g  Action  potential  i n ventricular  refractoriness  of premature beats.  I t was found  that the species s e l e c t i v e actions  of these drugs was most  l i k e l y related to differences i n s e l e c t i v i t y for K which contribute  while  by 80%, while UK 68,798  these by 30%.  widening p a r a l l e l e d increases to e l e c t r i c a l  fourfold in vivo,  i.v.) was  species.  (25 ug/kg, i.v.)  i n both rats and guinea  (4 mg/kg, i.v.) prolonged r a t v e n t r i c u l a r  e p i c a r d i a l action potential duration UK68,798  period  to r e p o l a r i z a t i o n i n myocardium.  +  channels  iv  TABLE OF CONTENTS  ABSTRACT  i i  LIST OF TABLES  X  LIST OF FIGURES  xi  SYMBOLS AND ABBREVIATIONS  xii  ACKNOWLEDGEMENTS  xiii  CHAPTER 1. 1.1  1  INTRODUCTION Ventricular F i b r i l l a t i o n  1  1.1.1  Overview  1  1.1.2  Myocardial Ischaemia  1  1.1.3  Coronary Heart Disease:  3  Magnitude of the Problem 1.1.4  Risk Factors  5  1.1.5  Mechanisms of Arrhythmogenesis: Abnormal Automaticity Mechanisms of Arrhythmogenesis:  9  1.1.6  12  Reentry 1.1.7 1.2  14  Animal Models  Potassium Channels  16  1.2.1  16  1.2.2  K Channels i n Cardiac Tissue: Overview K Currents Underlying the +  +  17  Cardiac Action Potential 1.2.2.1  Inward R e c t i f i e r ,  1.2.2.2  Delayed R e c t i f i e r , Ij<  21  1.2.2.3  Transient Outward Current, I t o  22  IJQ  19  V  1.2.2.4  Plateau  P  24  1.2.2.5  "Pacemaker" Current, I f  24  1.2.2.6  Na Activated Current, iK(Na)  2  5  1.2.2.7  Ca  2  6  1.2.2.8  ATP Sensitive  1.2.2.9  Acetylcholine/Adenosine Activated K Current, I (ACh/Ado)  29  K+ Channel D i s t r i b u t i o n i n  30  K  +  Current, I  K  +  + +  Activated Current, iK(Ca) K  +  Current,  IR(ATP)  2  7  +  K  1.2.3  Mammalian Heart  1.3  1.2.4  K Channel Stimulators  36  1.2.5  K Channel Blockers  38  1.2.5.1  Sulphonylureas  +  +  -  IR(ATP)  Blockers?....39  Pharmacology of K Channel Blockers  40  1.3.1  Pharmacology of New Class I l l ' s  41  1.3.2  Class III Drug Mechanism of Action....42  1.3.3  Cardiovascular E f f e c t s of  +  44  New Class I l l ' s 1.3.4  C l i n i c a l l y Available Class III Drugs..48  1.3.5  Torsade de Pointes  50  1.4  Non Pharmacological  1.5  Rationale  54  1.6  Experimental Plan  55  2  METHODS  57  2.1  Pharmacology  57  2.1.1  Cardiovascular Assessment  58  2.1.1.1  In Rats  58  2.1.1.2  In Guinea Pigs  59  2.1.1.3  In Pigs  60  Interventions  53  vi  2.2  2.3  2.4  2.1.1.4  In Primates  62  2.1.2  Drug and Dose Regimens  63  2.1.3  Data A q u i s i t i o n and Analysis  64  E l e c t r i c a l Stimulation  65  2.2.1  Introduction  . 65  2.2.2  Preparation  2.2.2.1  In Anaesthetized Rats  68  2.2.2.2  In Guinea Pigs  69  2.2.2.3  In Baboons  69  2.2.3  Data A c q u i s i t i o n  70  2.2.3.1  Thresholds  70  2.2.3.2  Maximum Following Frequency  71  2.2.3.3  E f f e c t i v e Refractory Period  72  2.2.3.4  F i b r i l l o f l u t t e r Threshold  72  2.2.4  Drug and Dose Regimens  73  2.2.5  Data Analysis  73  ..66  f o r Capture  E l e c t r o p h y s i o l o g i c a l Analysis  74  2.3.1  Introduction  74  2.3.2  In vivo Preparation  74  2.3.3  Drug, and Dose Regimens  77  2.3.4  Data Analysis...  77  Myocardial Ischaemia-Induced Arrhythmias  78  2.4.1  Introduction  78  2.4.2  Preparation  79  2.4.2.1  Conscious Rats  81  2.4.2.1.1 Infarcted Rats  84  2.4.2.2  Acutely Prepared Anaesthetized Rats... 85  vii  2.4.2.3  Acutely Prepared Anaesthetized  Pigs...86  2.4.3  Data Analysis  87  2.4.3.1  ECG Changes  87  2.4.3.2  Arrhythmia  analysis  89  3  RESULTS  91  3.1  Pharmacology  91  3.1.1  3.2  Species Dependent E f f e c t s on the Cardiovascular System  91  3.1.1.1  E f f e c t s on HR, BP & ECG i n Rats  91  3.1.1.2  E f f e c t s i n anaesthetized G. Pigs  95  3.1.1.3  E f f e c t s i n Pigs  95  3.1.1.4  E f f e c t s i n Primates  97  E l e c t r i c a l Stimulation Studies  102  3.2.1  Tedisamil vs Class l ' s i n Rats  102  3.2.2  Tedisamil vs UK68,798 i n Rats  103  3.2.3  Tedisamil vs UK68,798  110  in Guinea Pigs  3.3  3.4  3.2.4  Tedisamil i n Baboons  110  3.2.5  Summary  116  I n t r a c e l l u l a r Recording Studies in vivo  116  3.3.1  Tedisamil i n Rats  117  3.3.2 •  Tedisamil i n Guinea Pigs  117  3.3.2.1  E f f e c t s of Vagal Stimulation  122  3.3.3  UK68798 i n Rats  122  3.3.4  UK68798 i n Guinea Pigs  129  3.3.5  Summary  129  Myocardial Ischaemia-Induced Arrhythmias  130  viii  3.4.1  Overview  13 0  3.4.2  Tedisamil i n Conscious Rats  131  3.4.2.1  Tedisamil i n Infarcted Rats  14 0  3.4.3  Other K  3.4.4  K  +  Channel Blockers i n Acute Rats....150  3.4.5  K  +  Channel Blockers i n Acute Pigs....155  3.4.6  Summary  +  Channel Blockers i n R a t s — . 1 4 1  157  4  DISCUSSION  158  4.1  Pharmacology  158  4.2  4.1.1  E f f e c t s on HR & BP  158  4.1.2  Overview of ECG Analysis  161  4.1.2.1  E f f e c t s on ECG Intervals  163  4.1.2.2  Proarrhythmic E f f e c t s  167  4.1.2.3  Summary  171  4.1.3  Comparison with Other Class Ill's....171  4.1.3.1  Amiodarone  4.1.3.2  In Search of a Reference Standard.... 172  4.1.3.3  Clof ilium  177  4.1.3.4  D-Sotalol  178  4.1.3.5  Acecainide.  180  4.1.4  Summary: Analysis of ECG Intervals... 182  171  E l e c t r i c a l Stimulation  183  4.2.1  Overview  183  4.2.2  Tedisamil vs. Class I Drugs  184  4.2.3  Tedisamil vs UK68,798  4.2.4  Studies i n Primates  186  4.2.5  E f f e c t s of Other Class I l l ' s  188  ...185  ix  4.3  4.4  4.5  5  Action Potential Morphology  190  4.3.1  Frequency Dependence of APD  190  4.3.2  E f f e c t s of Tedisamil  192  4.3.3  E f f e c t s of UK68,798  195  4.3.4  Comparison to Other Class I l l ' s  196  Myocardial Ischaemia  198  4.4.1  Studies i n Rats  198  4.4.2  Reperfusion-Induced  4.4.3  Studies i n Pigs  Arrhythmias  202 203  General Conclusions  203  4.5.1  Species Dependent Actions  204  4.5.2  Antiarrhythmic Actions  2 04  References  2 06  X  LIST OF TABLES TABLE  PAGE  1  Haemodynamic E f f e c t s of K i n Conscious Rats.  2  ECG E f f e c t s of K Conscious Rats.  3  Cardiovascular E f f e c t s of Tedisamil and UK68,798 i n Anaesthetized Guinea Pigs.  96  4  Cardiovascular Actions of Tedisamil and UK68,798 i n Pigs.  98  5  E f f e c t s on the ECG of Tedisamil and UK68,798 i n Pigs.  99  6  BP and HR Effects of Tedisamil i n Primates.  100  7  ECG E f f e c t s of Tedisamil i n Primates. •  101  8  Arrhythmias Following Occlusion i n Conscious Rats: E f f e c t s of Tedisamil.  13 3  9  Arrhythmias Following Occlusion i n Conscious Rats: E f f e c t s of K Channel Blockers.  143  10  E f f e c t s of Glibenclamide and Tedisamil.  152  11  Arrhythmias Following Occlusion (0-lh) i n Pigs.  156  +  Channel Blockers  Channel Blockers i n  +  92 94  xi  LIST OF FIGURES FIGURE  PAGE  1  E l e c t r i c a l Stimulation: Tedisamil vs. Class I Drugs.  105  2  ECG E f f e c t s of Tedisamil & Class I Drugs.  107  3  E l e c t r i c a l Stimulation Studies i n Rats: Tedisamil vs. UK68,798.  109  4  ECG E f f e c t s of Tedisamil & UK68,798 i n Anaesthetized Rats.  112  5  E l e c t r i c a l Stimulation Studies i n Guinea Pigs: Tedisamil vs. UK68,798.  114  6  E f f e c t s of Tedisamil on MFF i n Baboons.  115  7  Electrophysiological E f f e c t s of Tedisamil i n Rats.  119  8  Electrophysiological E f f e c t s of Tedisamil i n Guinea Pigs.  121  9  E f f e c t s of HR on APD.  124  10  Electrophysiological E f f e c t s of UK68,798 i n Rats.  126  11  Electrophysiological E f f e c t s of UK68,798 i n Guinea Pigs.  128  12  E f f e c t s of Tedisamil on Arrhythmias.  135  13  E f f e c t s of Tedisamil on ST Segment Elevation.  137  14  E f f e c t s of Tedisamil on BP and HR.  139  15  E f f e c t s of K Channel Blockers on Arrhythmias.  145  16  E f f e c t s of K Channel Blockers on ST Segment Elevation.  147  17  E f f e c t s of K Channel Blockers on BP and HR.  149  18  E f f e c t s of Glibenclamide and Tedisamil on Arrhythmia Incidence.  154  +  +  +  xii  SYMBOLS AND ABBREVIATIONS action  potential  AP  action potential duration  APD  arrhythmia score  A.S.  a t r i o v e n t r i c u l a r node  AVN  calcium  Ca  conductance  g  coronary heart disease  CHD  current  I  e f f e c t i v e refractory period  ERP  hour(s)  h  maximum following  frequency  MFF  membrane potential  Em  myocardial  MI  ischaemia  + +  minute(s)  min  occluded zone  O.Z.  potassium  K  second(s)  s  slow inward current  I« -si  sodium  Na  +  n  standard error of the mean  S.E.M.  time constant torsade de pointes  TdeP  v e n t r i c u l a r premature beat  VPB  v e n t r i c u l a r tachycardia  VT  ventricular  VF  voltage  fibrillation  V  xiii  ACKNOWLEDGMENTS I would l i k e t o thank the members of the Department of Pharmacology & Therapeutics, and i n p a r t i c u l a r  Drs. Cathy  Pang and Bernard MacLeod f o r t h e i r help and encouragement. I am also grateful to the members of my committee f o r the time  and e f f o r t  thesis.  spent  i n review  and discussion  of t h i s  Michael Pugsley has been an invaluable help and a  l o t of fun to work with.  To my supervisor, Michael Walker,  I think the best compliment I can give him i s that he was a true  educator.  I  will  never  forget  his spirit  and  generosity. To my family and friends I owe a great deal, e s p e c i a l l y Cindy  who  triumphs.  has stood I would  by me  also  like  through  both  to thank  struggles and  Dale  Hodgins and  members of Bad Medicine f o r keeping me on track. I  am also  indebted  to the B.C. and Canadian  Foundations f o r t h e i r f i n a n c i a l support.  Heart  1  1.  INTRODUCTION  1.1  Ventricular F i b r i l l a t i o n  1.1.1  Overview  Cardiac arrhythmias  can be caused  by either  abnormal  impulse formation or abnormal impulse conduction (Hoffman & Cranefield, either  1960).  altered  Conduction  Abnormal  automaticity  abnormalities  impulse and/or  include  formation  involves  triggered  activity.  unidirectional  block,  b i d i r e c t i o n a l block, functional and anatomical reentry, and reflection.  The  responsible electrolyte dysfunction, accessory  for  underlying  these  mechanisms  imbalances,  alterations.  may  autonomic  cardiomyopathies,  pathways,  pathological  ischaemia,  nervous  system  anatomical v a r i a t i o n s e.g.,  or drug  Differences  be  processes  induced in  electrophysiological  mechanism  often  require  d i f f e r e n t therapeutic approaches.  1.1.2 Myocardial Ischaemia  Cessation of blood flow through the coronary a r t e r i e s , i.e.  myocardial  heart's  ischaemia,  electrical  causes and  rapid  changes  mechanical  i n the activity.  E l e c t r o p h y s i o l o g i c a l studies have shown that acute ischaemia causes  depolarization  of the resting  membrane  potential,  reduction i n r i s e rate and height of the AP upstroke, and  2  b r i e f widening and subsequent abbreviation of the AP plateau (Kardesch et al., 1958;  Prinzmetal et al.,  al.,  al.,  1977;  increases Zipes,  Russel at  1977)  et  first but  1979).  (Holland this  is  &  Downar et  Conduction  Brooks,  soon  1961;  1976;  followed  velocity  Elharrar  by  &  progressive  slowing (Boineau & Cox, 1973; Waldo & Kaiser, 1973; Williams et  al., 1974).  The refractory period i n the ischaemic zone  becomes heterogeneous 1964;  Levites  (Elharrar et al.,  et al.,  1975).  The  1977;  Han  edge of the  &  Moe,  ischaemic  zone, or border zone, has been implicated i n the genesis of "injury  currents"  depolarization"  or  between  areas  "repolarization"  Nahum et al., 1943;  of (Janse  Samson & Scher, 1960).  "out-of-phase et  al.,  1980;  Injury currents  have been reinvestigated by numerous investigators searching for  the mechanisms of arrhythmogenesis  ischaemia  (MI) .  The  role  of  the  i n acute myocardial  metabolic,  ionic  and  neurohumoral events following acute MI has been the focus of intense research.  I n i t i a l studies of the role of K by Harris et al. [K ] +  Q  (1954) who  +  ions were c a r r i e d out  proposed that the elevation i n  secondary to ischaemia was a major arrhythmogen.  were able to show arrhythmias p r e c i p i t a t e d by K in  ischaemic  investigations  hearts have  following  ischaemia;  elevation  to  up  to  (Harris shown  three  initial 15mM,  et  (30  al.,  s  injection  +  1958).  stage  followed by  Subsequent  changes  to  5-15 a  10  They  -  in  [K ] +  0  min)  rapid  15  minute  3  plateau Hill  and  &  finally  Gettes,  a slowly r i s i n g  1980).  This  phase  [K ]  (Kleber,  1983;  accumulation  +  0  might  contribute to the depolarization according to the GoldmanHodgkin-Katz  equation.  relative  permeability  membrane  potential  [K ]i/[K ]o +  ratio  +  This  sodium  velocity. have  to  equation  largely  (Goldman,  1943;  could  channels  in  shown  and  pointed  Hodgkin  that  a  slowing  greater  degree  occurs  in  to  a  possible  of  the  that in  by  the  & Katz,  slow  high  resting  inactivate  thereby  log  1949). voltage  conduction  Clusin's group  depolarization  first  3  and  minutes  f o r by the accumulation  alone (Blake et al., 1988).  Q  governed  turn  occlusion than can be accounted +  results  However, recent observations by  conduction  [K ]  dictates  potassium,  being  depolarization  dependent  The  of of  These investigators have  interaction  with - C a , + +  because  infusions of C a , or pacing, augmented the early ischaemia+ +  induced depolarization. CO2 accumulation  and  A c i d i f i c a t i o n (Couper et al., (Case  et  al.,  1979)  occur  1984) within  seconds of coronary occlusion, and are augmented by pacing. Using  the  Ca  + +  indicator  dye,  Indo 1,  Lee  et al.  (1988)  showed a rapid r i s e i n [ C a ] i n response to ischaemia which ++  may al.,  stimulate an inward 1981;  answer  Kass et al.,  questions  depolarizing current (Colquhoun et 1978).  generated  by  These studies attempt clinical  experience  to of  myocardial ischaemia induced arrhythmias.  1.1.3  Coronary Heart Disease:  Magnitude of the Problem  4  The  mortality  rate  from  coronary heart disease has  decreased by 40% i n the U.S.A. since 1950  (Feinleib,  1984).  t h i s remarkable decline has not occurred i n a l l countries, but  Canada  has  experienced  a  decline  mortality of 20% i n the same period.  in  cardiovascular  Despite the decline,  200,000 - 600,000 persons i n the U.S.A. and approximately 40,000 Canadians succumb annually to cardiac arrest &  Kannel,  1989)  1971;  N.C.H.S. Advance Report,  which makes CHD  America. (i.e.  Goldberg,  the leading cause of death i n North  The term sudden  "resuscitated  1981;  (Gordon  death has been used and  from  sudden  death")  to  abused  describe  unexpected death occurring within one hour a f t e r the onset of  signs  or symptoms  (Rapaport,  1988).  The  term  sudden  cardiac death refers to death from any cardiac cause, but i t i s usually  due  to atherosclerotic  (Roberts, 1986). aortic  Sudden cardiac death can be the r e s u l t of  dissection,  cardiomyopathy,  coronary artery disease  ventricular  vascular  aneurysm  causes,  arrhythmias (Roberts, 1990).  valvular  discussion  will  cardiac death (SACD).  be  rupture,  disease  and/or  As antiarrhythmic drugs would  only be of benefit to the l a t t e r of t h i s further  and  limited  to  incomplete sudden  list,  arrhythmic  Typical patients s u f f e r i n g an episode  of cardiac arrest do not have an acute myocardial i n f a r c t i o n associated with an episode of VF (Greene, 1990). of patients may CKMB)  into  While 38%  show leak of i n t r a c e l l u l a r enzymes (e.g.,  their  circulation,  only  19%  develop  Q-waves  5  associated with transmural  infarction  (Cobb et al.,  1980a;  1980b).  In some cases t h i s i s probably due to the fact that  necrosis  takes  time  to develop  (Weisman & Healy, 1987)  The  rhythm  after  an  ischaemic  insult  and ischaemic zones vary i n s i z e .  seen  early  in  cardiac  arrest  has  been  reported as VF i n 75%, asystole i n 20% and electromechanical dissociation  i n 5%  (Weaver et  appreciated that the f i r s t  al.,  1986).  I t must  rhythm recorded may  not be  i n i t i a t i n g arrhythmia, VT can of course degenerate and  VF  further change to asystole.  Similarly,  be the  into VF  there are  uncertainties regarding the aetiology of SACD.  1.1.4  Risk Factors  The  longitudinal studies of the Framingham population  (Kannel  &  Schatzkin,  1985)  have  provided  most  of  the  information regarding r i s k factors f o r sudden cardiac death. The  r i s k of sudden death  increases disease  with  weight,  i s present.  associated (Dawber, factors  age,  with 1980;  has  causation  and  i f underlying  Interestingly, increased r i s k  Doll  et  al.,  been questioned, must  1  an  i s apparently greater f o r males,  always  be  of  1980).  smoking  'correlation  remembered  may  infarction The  when  coronary  value  not  i n women of  does not one  be  risk imply  looks  at  epidemiological data.  McCormick & Skrabanek (1988) recently  reviewed  of  a  number  population  based  risk  factor  6  intervention preventable  studies by  and  altering  concluded diet,  exercise l e v e l s or body weight. "risk  f a c t o r " be  stated by  replaced  Grundy  by  CHD  was  blood  not  pressure,  They advocate that the term " r i s k marker" as that  the  previously  implied  causal  Risk factor i d e n t i f i c a t i o n has reached  absurd proportions: by 1981, found 246.  smoking,  (1973) i n order  r e l a t i o n be reduced.  that  Eventually we  Hopkins and Williams  (1981) had  a l l die, h a l f of us from CHD,  seems u n l i k e l y that a l i f e untainted  it  by r i s k factors would  escape t h i s destiny.  Sudden cardiac arrhythmic death has some cases overlapping, compared  with  CHD.  These  poor  left  fraction  3 0%,  extensive  incidence angina  include  or  a  previous  v e n t r i c u l a r function coronary  of VPBs, old age, pectoris  artery  therapy  might  conditions However,  hypotension  be  would drugs  encainide  increased programmed  i . e . ejection disease,  and  given. not  that  Drugs  inducibility  be  optimal  reduce  and  mortality electrical  as  which  shown by  flecainide despite  exacerbate  risk  the  reductions  stimulation  for  factors  were  at  These conditions  candidates  these  high  induced  some of those i n which antiarrhythmic  prevent cardiac mortality, which  myocardial  male gender, exercise  e l e c t r o p h y s i o l o g i c a l study (Greene, 1990). are, therefore,  in  r i s k factors associated with i t as  infarction, <  d i f f e r e n t , but  drug these  therapy. may  not  CAST study i n  associated of  VPBs  inducibility  with and (CAST  7  investigators, 1989).  What then can be done?  I t would seem  r a t i o n a l t o prevent or ameliorate the arrhythmias themselves which lead t o compromised pump function and death.  Thus,  a b o l i t i o n of VF plus, at the l e a s t , slowing of VT, would be desirable properties classes 1984)  of any antiarrhythmic.  of antiarrhythmics  classification,  would  seem  reentrant  suitable  (1970,  I I I antiarrhythmics  candidates  for  slowing  VT and possibly eliminating VF (Bacaner et al.,  1986).  A  conduction  selective velocity  treatment with 1988).  i n the Vaughan-Williams  e f f e c t i v e Class  the most  Of the four  gNa  +  Class  I I I agent  or c o n t r a c t i l i t y and g C a  not  impair  as can occur  blockers  ++  Given that antiarrhythmic  under conditions of impaired  would  (Jaillon  with  & Ferry,  drugs are most often given  pump function, t h i s fact alone  makes Class III drugs a more desirable choice.  While associated  sudden with  cardiac  the previously  i n i t i a t e s the event? a  sudden  arrhythmic  occlusive  mentioned  death  has  been  conditions,  what  An a t t r a c t i v e hypothesis proposed that thrombus  forms  i n a coronary  acutely giving r i s e to ischaemia (Epstein et al., this  p r e c i p i t a t e s the arrhythmias.  studies  indicate that  artery  1989) and  However, pathological  75% of the cases of sudden  death involve no coronary thrombosis (DiMaio et al.,  cardiac 1990).  An i n t e r e s t i n g c o r o l l a r y of the above hypothesis i s that an acute occlusion would be more l i k e l y  f a t a l i n a previously  asymptomatic patient due to a lack of c o l l a t e r a l c i r c u l a t i o n  8  (Epstein et al., enlarge  and  occlusive  1989).  mature  disease  resistance  to  in  Human coronary artery  collaterals  response  and  to  i . e . , greater than  flow  i s inversely  chronic 75%  narrowing,  proportional  ( P o i s e u i l l e , 1842, c f : Shepherd & Vanhoutte, 1963;  Schaper,  1989).  1971;  While  Rentrop  the results  et al.,  severe  to  [as  radius , 4  1979)] (Fulton,  1988;  Cohen et  al.,  of an occluding thrombus would  possibly be worse i n a patient with mild stenosis (less than 70%) , the l i k e l i h o o d of an occluding thrombus developing i s much greater i n patients with severe stenosis (Moise et al., 1984;  Proudfit et al., 1980).  In accordance with the above  mentioned hypothesis much e f f o r t i n the l a s t decade has been focused on the phenomenon of s i l e n t myocardial ischaemia and i t s r o l e i n sudden arrhythmic death Amsterdam, 1987). coronary  artery  patients  with  asymptomatic  (Gottleib et al.,  S i l e n t or painless ischaemia i s common i n disease  (Cohn, 198 6) and  angina, patients  previous (Kannel  Holter ambulatory  ECG  &  Abbot,  or during exercise t e s t i n g  This  method  validated with  ischaemia  (Tzivoni et al.,  other  also  in  Silent  segment a l t e r a t i o n s (Cecchi et  (Amsterdam et al.,  detection 1985;  and  often i n  1984).  monitoring  1983)  of  occurs  infarction  ischaemia i s generally detected as S-T during  1986;  has  al.,  1986).  recently  been  Rocco et al. , 1986).  prognostic indicators,  whether  or  not  As  silent  ischaemia plays a causal r o l e or only an association with ventricular limited  arrhythmias  number  of  i s equivocal.  cases  of  sudden  The death  data  from  a  i n patients  9  undergoing ambulatory  monitoring suggest that most cases of  l e t h a l v e n t r i c u l a r tachycardia are not associated with evidence  of myocardial  Lereq et al.,  1987).  ischaemia  (Meissner  et al.,  However, many i s o l a t e d  ECG 1986;  instances of  i n d i v i d u a l cases support a r o l e f o r ischaemia provocation of arrhythmias  (Gradman  et al.,  1977;  Savage et  al.,  1983;  Meissner et al., 1986).  Interventions developed. as  type  percutaneous  plasminogen  and  activator,  t-PA,  transluminal angioplasty have  revascularization  1988).  ischaemia  have been  F i b r i n o l y t i c streptokinase and urokinase as well  tissue  formation  aimed at reversing  in  cases  i n more  of  acute  as  been  coronary  well used  as for  thrombus  chronic stenosis conditions  (Lew,  These procedures o f f e r a less invasive approach than  coronary artery bypass g r a f t techniques.  1.1.5  Mechanisms of Arrhythmogenesis:  Abnormal  Automaticity  Ventricular  fibrillation  been reported to be  caused  i n myocardial by  both  automaticity (Pogwizd & Corr, 1987).  ischaemia  reentry and  has  abnormal  Normal automaticity i s  found i n the sinus node and Purkinje f i b r e s , although i n the l a t t e r automaticity i s not normally seen owing to the faster rate  of the node.  sensitivity  to  This property overdrive  i s responsible f o r the  suppression  of  enhanced  10  automaticity  i n Purkinje  other  abnormal  hand,  ventricular 1976)  and/or  is difficult  works  by  1981).  occuring  fibres  On the  i n depolarized  (Imanishi  & Surawicz,  to suppress by overdrive pacing blockers  +  and i s  (Cranefield, 1977; Dangman &  I t has been suggested that overdrive pacing  hyperpolarizing  effective  (Vassalle, 1977).  automaticity  Purkinje  not s e n s i t i v e to gNa Hoffman, 1983).  fibres  i n severely Abnormal  the  membrane  and  depressed c e l l s  automaticity  agent f o r most tachycardias  induction  of  infarction  (Hoffman  has been  causative  thus  i s not &  Rosen,  implicated as the seen 24 hour  (Wellens et al.,  1974).  after While  altered automaticity may occur under any conditions i n which a loss of membrane p o t e n t i a l i s present, triggered a c t i v i t y appears to r e s u l t  from afterdepolarizations (Rosen, 1986).  These o s c i l l a t i o n s are linked to a preceding be  classified  as  repolarization), phase  III)  early,  or  (occurring  delayed  (Rosen et al.,  after 1973;  beat, and can  during  Phase  depolarizations Ferrier,  1973).  III (post Early  a f t e r depolarizations (EAD) have been demonstrated in under conditions which either reduce outward, inward, currents. catecholamines  &  Cranefield,  (Coraboeuf et al., 1980), low [Ca ] ++  al.,  (Sano and Sawanobori,  1954)  Hoffman,  and  or increase  Thus, EADs have been produced in vitro  (Hoffman  [K ] +  (Schmidt, 1960;  by  acidosis 1961), low  1972), hypoxia (Trautwein  numerous drugs  1981; Strauss  1960),  (Carmeliet,  vitro  et  Dangman &  et al., 1970; Gough et al., 1988).  On the other hand, few reports implicating EADs in vivo have  11  appeared  (Levine  et al., 1985; E l - S h e r i f et al., 1988;  Carlsson et al., 1990).  These groups have proposed that U-  waves on the surface ECG correspond to EADs, and that the occurrence  of torsade  de  pointes  i s initiated  (Brachman et al., 1983) (see section 1.3.5).  by  EAD  While cesium  induced EADs have been reported t o be favored by bradycardia (Brachman et al.,  1983) delayed a f t e r depolarizations (DADs)  appear t o be augmented by increased 1980).  rate  (Rosen & Reder,  DADs can be produced by [ C a ] i overload, + +  reperfusion ATPase  by  (Ferrier  et al., 1985),  digitalis  hypomagnesemia, etc.  inhibition  glycosides,  DADs r e s u l t i n g from sympathetic stimulation + +  0  1988).  A  Ca  + +  +  +  and  Recently,  i n conjunction  and strophanthidin treatment, have  been d i r e c t l y shown using MAP recordings al.,  of Na /K  hypokalemia  (Marriott & Conover, 1989).  with elevation of [ C a ]  such as i n  overload  in vivo  induced  ( P r i o r i et  transient  inward  current may be the actual mediator of DADs (Lederer & Tsien, 1976;  Kass  et al., 1978; Matsuda  et al., 1982).  transient inward current has been reported electrogenic N a / C a +  ++  + +  1986;  induced nonspecific i o n i c current Colquhoun  t o be linked t o  exchange (Lipp & Pott, 1988; Arlock &  Katzung, 1985; Noble, 1984), while others Ca  The  et al. ,  suggest i t i s a  (Cannell  1981; Shimoni  & Lederer,  & Giles,  1987).  Whether or not a DAD t r i g g e r s an extrasystole would depend on both the magnitude of the induced depolarization and the e x c i t a b i l i t y of the l o c a l t i s s u e at the DAD's focus.  12  It  might  (Hoffman  appear  &  Rosen,  arrhythmogenesis because  unlikely 1981)  i n acute  abnormal  that would  be  ischaemia  automaticity i s  extracellular  [K ]  1975), while  [K ]  responsible  (Janse et al.,  suppressed  (Hoffman & Rosen, 1981;  +  rises  +  0  ( H i l l & Gettes, 1980; However, triggered  abnormal automaticity  subsequent  may  1986)  elevated  Katzung  to coronary  Hirsche et al. , 1980;  activity  by  for  et al., occlusion  Kleber, 1983).  play a r o l e  i n ischaemia  induced arrhythmias due to the possible presence of hypoxia (Trautwein et al., 1954), acidosis (Coraboeuf et a l . , 1953), elevated catecholamines possible  myocardial  increased [ C a ]  (Brooks et al., stretch  1955), high  (Pirzada  et  al.,  [K ] , +  Q  1976),  (Clusin et al., 1983), which may contribute  ++  to the generation of o s c i l l a t o r y a f t e r depolarizations.  1.1.6  Mechanisms of Arrhythmogenesis:  Reentry  was  (McWilliams, 1887,  mentioned  over  one  Reentry  hundred  c f : Marriott & Conover, 1989).  years  ago  Proof of  i t s existence i n rings of j e l l y f i s h subumbrella t i s s u e came 20 years l a t e r 1989) and  (Mayer, 1906;  and  the concepts  their  requirements  tissue  by  Mines  (1913  1908,  c f : Marriott & Conover,  of reciprocating rhythms and were  later  demonstrated  &  1914).  The  beats  i n heart  simplest  case  requirements f o r reentry are:  1.  an available c i r c u i t ;  2.  unequal  segments of the c i r c u i t ;  3)  responsiveness i n two  slow conduction.  13  Reentry can be random, as i n f i b r i l l a t i o n , or ordered, and follow a fixed pathway.  Micro-reentry has been used to  describe the small c i r c u i t s such as might occur i n the AV node  or  distal  Purkinje  fibres,  while  longer  c i r c u i t s are referred to as macro-reentry. form  of reentry that  parallel  unbranching  i s produced fibres  Reflection i s a  through  with  pathway  reflection in  depressed  segments  (Antzelevitch et al., 1980; J a l i f e & Moe, 1981).  Since acute ischaemia i s associated with areas of slow conduction, short refractory periods and inhomogeneities i n refractory periods  (see above) reentry has been  for many years as the most important cause induced arrhythmias.  Sasyniuk  & Mendez, 1971)  et al. 1976, 1977). reentry  i n ischaemic  demonstrated  when conduction  was slowed excessively i n Purkinje f i b r e s 1972;  f o r ischaemia-  Circus movement has been  using microelectrodes, in vitro,  indicated  (Wit et al. 1971;  or a t r i a l muscle  E l - S h e r i f et a l . (1977) myocardium  in  velocity  vitro.  (Allessie  demonstrated Epicardial  mapping studies i n i s o l a t e d  dog or p i g hearts  injury  i n the subendocardium flowing  across  currents o r i g i n a t i n g the  border  zone  of  depolarized  implicated  ischaemic  and  polarized perfused myocardium as the substrate f o r reentry in  early  acute  ischaemia  (Janse  Kleber, 1981; Janse et al., 1986).  et al., 1980; Janse  &  Activation mapping has  shown reentry i n humans (de Bakker et al., 1988).  Three  14  dimensional Corr  a c t i v a t i o n mapping in vivo was used by Pogwizd &  (1987) to record  reentrant  and  abnormal  automaticity  mechanisms p a r t i c i p a t i n g i n the induction and maintenance of arrhythmias following coronary occlusion i n the own  r a t studies in vivo  (Inoue et al., shortening This  1984)  mechanisms appears  and  i n rats  that  and in  vitro  of Vmax  the  necessary  conduction subjected  reentry  can  slowing  to  be  heterogeneity for  coronary  the  tachycardias  as  well  as  of  reentrant  occlusion.  underlying  It  sustaining  mechanism i n some cases of v e n t r i c u l a r bigeminy, or  and  following coronary artery occlusion.  provide  refractoriness  1989)  Our  have shown a depression  of the APD  would  (Abraham et al.,  cat.  trigeminy  supraventricular  and  nodal  arrhythmias.  1.1.7  Animal Models  The c l i n i c a l experience  of arrhythmogenic mechanisms i s  clouded by uncontrolled variables. of  c a u s a l i t y to  result  of  lethality  this,  animal  are  at  Therefore, r e l a t i o n s h i p s best  models  equivocal.  have  been  As  used  a to  systematically investigate the various hypotheses.  Numerous animal models of developed chemically-,  (Review  see:  electrical  arrhythmogenesis have been  Winslow, stimulation-,  ischaemia/infarction-induced  1984).  There  mechanically-,  arrhythmia models.  Due  are and  to the  15  variability  of  encountered  conditions  i n patients,  under  been produced  to f a c i l i t a t e  data  et  al.,  of  gauging  stimulus  (e.g.  ischaemia) .  interlaboratory  1988).  the  These  use  zone of  the  in  rat  and  the  arrhythmogenic  models  as  of  conventions  random protocols  size  the  comparisons  Lambeth  strength of  occluded The  are  Recently guidelines have  h i g h l i g h t the need f o r b l i n d and necessity  arrhythmias  there are no single animal models  that can be considered as i d e a l .  (Walker  which  an  utilizing assay  for  antiarrhythmic a c t i v i t y has been recently evaluated (Curtis et al., 1987; Brooks et al., 1989)  and found to be generally  useful because of s i z e , cost and most importantly consistent coronary important  artery  anatomy  (lack  of  collaterals).  species dependent differences  There  i n coronary  anatomy (Johns & Olsen, 1954; Maxwell et al., 1987).  are  artery Hearts  from r a t s , cats, rabbits, pigs and f e r r e t s have less than 5% 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 of t h e i r coronary a r t e r i e s , while dog  hearts  have  variable  and  extensive c o l l a t e r a l i z a t i o n . are  important  occlusion.  produced  pig  hearts  These anatomical  determinants  ischaemia/infarction  guinea  of  the  have  differences extent  by permanent coronary  of artery  Blood flow i n c o l l a t e r a l s may prevent i n f a r c t i o n  of t i s s u e a f t e r l i g a t i o n of a single coronary artery.  Since  the s i z e of ischaemic zone has been shown to be an important determinant  of  subsequent  arrhythmias,  consistently  ischaemic zones are necessary i n order to make  sized  comparisons  between groups of treated animals (Curtis et al. , 1987).  16  While models, of  there  when  (1.2)  activated  Class  are  in  the  al., the  K  to  K  1978).  subside  techniques  K  the  discussed  differences  of  of  single with  voltage  channel K  in  of  different  in  aware  channels  AP.  K  rat  section  the  the  in led  definable with to  the  different  the  cells  Thus  channels  +  as has  has  +  of  channels.  area.  in  +  channels  et  some  of  preparations, of  particular  depletion space  and/or  (Kline &  in  knowledge  the  number o f  This  Previous  (Hamill  alleviated  and,  since  an i n c r e a s e d number o f  growth  introduction  K  technique  extracellular  a growth  Overview  developed  multicellular  a result  area, to  K  clamp  has  clamp c o n t r o l  limited  the also  channels  electrophysiology,  in  +  +  patch  Perhaps as  has  separately  +  of  investigators  studies  of  of  associated  accumulation  not  As  of  one must be  Channels i n Cardiac T i s s u e :  +  inadequate  channels  drugs.  blockade  The use  problems  Morad,  III  understanding  1981).  interest  usefulness  efficacy.  introduction  s u c h as  the  Potassium Channels  1.2.1  The  to  r e p o l a r i z a t i o n phase  i n our studies,  1.2  point  marked s p e c i e s  s p e c i e s dependent  the  studies  are important considerations  testing  there  shown  these  of  of  K  +  reputed  trend probably  will  molecular  biological  genetic  sequencing  to were  characterized  by  17  q u a l i t i e s such as t h e i r current/voltage relationships, time dependent k i n e t i c s , as well as responses to pharmacological manipulations.  With  regard  to  the  latter,  the  pharmacological tools used i n t h i s area have u n t i l now been rather l i m i t e d . define  I to  a  n  standard drugs, T E A and 4AP,  The *K  d  n  a  v  e  i n the  ECSQ'S  mM  used to  range.  The  inorganic ions which are also used to characterize channels are  not  themselves  entirely  specific.  Despite  these  possible l i m i t a t i o n s , recent reviews have given what may  be  a deceptively clear description of the currently proposed K channels 1988; K  present  i n cardiac  Carmeliet, 1989).  cells  (Irisawa,  1987;  +  Cook,  There are at l e a s t eight separate  channels found i n myocardium and t h e i r occurrence i s both  +  tissue  and  species dependent.  summarizes K Those Inward  +  with  primarily (IRI)/  list  briefly  voltage  dependent  delayed r e c t i f i e r  (IR)/  kinetics; transient  (I-to) ' "pacemaker" (If or 1^) , and plateau current and those which  (IRP),  following  channel taxonomy i n cardiac t i s s u e :  rectifier  outward  The  ( K(Na))/  ( K(Ca))'  I  I  are primarily chemically dependent;  ( K(Ach/Ado)) I  a  n  ( K(ATP)) (Carmeliet,  d  X  1989) .  1.2.2  K  +  Currents Underlying the Cardiac AP.  O r i g i n a l l y a single K was  invoked  1964) .  As  to explain apparent  channel, which only activated,  +  the  from  cardiac  the  AP  above,  (Johnson this  view  & was  Tille, soon  18  demonstrated  to be inadequate.  s e l e c t i v e l y permeable to K , close  to  the  K  equilibrium  +  at rest are  so that the membrane p o t e n t i a l  +  is  Cardiac c e l l s  potential.  Wiedemann's  (1951) pioneering work, involving resistance measurements i n cardiac pacemaking c e l l s , led to the proposal that a slowly decaying  outward  responsible Trautwein,  conductance  i n part 1958).  after  repolarization  f o r pacemaker depolarization Noble's  was  (Dudel &  (1962) model incorporated  this  idea, even before the f i r s t description of t h i s delayed conductance  (Hall  et al.,  1963).  Application  clamp techniques to the heart by (19 64)  led  to  a  of previous currents.  inward  currents  carried  (1967)  after  evidence  (Niedergerke, Shanes,  1963)  1962).  recently  by was  and  flux  reported by  transient  from  current  ++  <3  AP  through  (T)  the  Reuter  experiments (Winegrad  i n the  conducted and  the  an  existence of  measurements  Ca -dependent  the  The  was  + +  provided  been shown to be  channels;  Ca  & Trautwein  i . e . I^o  reinterpretation  +  of voltage  Deck, Kern  greater complexity,  K  two  heart  & has  separate  dihydropyridine  s e n s i t i v e (L) channels (Hess et al., 1984).  With  regard to K  +  channels  a  schism  developed  over  i n t e r p r e t a t i o n of pacemaking mechanisms when DiFrancesco & Noble  reinterpreted  Tsien,  1968)  dependent called,  as  inward If  the  "pacemaker" current  consisting rectifier  (Brown  et  of  both  (IJQ)  al.,  a n c  *  1979;  the a  n  e  1^2  time w  (Noble & and  current  DiFrancesco,  [K ] +  Q  they 1981;  19  DiFrancesco & Noble, and  1982;  This nonspecific, inward  1984).  hyperpolarization-activated "funny"  current  has been  rejected as a major contributor to pacemaker depolarization i n S.A. Node by other investigators (Noma, Morad & Irisawa, 1983) the  The gK original  modulatory  +  of I f although  they  maintain  a  effects  1 9 8 4 ; DiFrancesco,  (Noble  A detailed description of these and  1987).  channels found i n cardiac tissue follows:  Inward R e c t i f i e r ,  1.2.2.1  The  proponents  chronotropic  Irisawa  other K  "decay" hypothesis has again been adopted by  r o l e of I f e s p e c i a l l y with regard t o adrenaline's  positive 1985;  +  most  thoroughly  IRI  studied K  +  channel  which  helps  maintain the resting potential i s the inward r e c t i f i e r ( I R I ) originally 1963;  thought  to be time-independent  McAlister & Noble,  channel  has rapid  (Carmeliet, characteristic  activation  1982; area  Sakmann  calf: Dog:  Hutter & Noble,  &  Trube, slope  et  The  kinetics  1984)  and  conductance  at plateau potentials  al.,  1960).  and i n a c t i v a t i o n  of negative  makes i t s conductance (Guinea  1966;  (Hall  almost  a  which zero.  p i g : Hume and Uehara, 1 9 8 5 , Sheep: Isenberg, 1 9 7 6 DiFrancesco et al., 1 9 8 4 , Cat: Tseng et al. ,  Shah et al.,  1987).  lyj. i s present i n a t r i a l  1987,  (Sackman  et al., 1 9 8 3 ) and more so i n v e n t r i c u l a r t i s s u e , but i s very sparse i n nodal c e l l s ,  which may explain the lower  K /Na +  +  permeability r a t i o and thus the low "resting" p o t e n t i a l of  20  nodal t i s s u e resistance changes  (Pelzer & Trautwein, 1987) .  of nodal c e l l s  i n membrane  membrane potential single  channel conductance  square  root  studies  (12-15 KOhms cm ) allows small 2  current  (Bean,  The high membrane  t o cause  large  changes i n  1985; Noma et al., 1984). of I J Q i s proportional  of [ K ] (Carmeliet, 1982) and thus +  Q  i n multicellular  preparations were  accumulation/depletion of K  to the original  hampered  i n extracellular  +  The  by  compartments  (Baumgarten & Isenberg, 1977; Kline & Morad, 1978; Cohen & Kline, IKI, the  1982).  Although the single channel conductance of  i n v e n t r i c l e and a t r i a i s similar, i n the l a t t e r tissue gating k i n e t i c s are much faster  (Pelzer and Trautwein,  1987) .  The inward r e c t i f i e r channel can conduct more than one K  +  ion at a time (Shen et al., 1990; H i l l et al., 1989) and  can be activated by hyperpolarization (Kurachi, 1985).  The  activation  that  gate  of I  K  1  could  be a charged  moves due t o changes i n e l e c t r i c due  t o the change i n l o c a l  field  particle  of the membrane or  [K ] ion (subsequent t o f i e l d +  changes) at a c r i t i c a l s i t e (Carmeliet 1982; 1989). in  [ K ] also +  Q  guinea  shifted  pig ventricle  inward r e c t i f i c a t i o n which  blocks  the voltage range (Kurachi,  1985).  of a c t i v a t i o n i n The mechanism of  was found to be dependent  outward  current  through  Changes  on [ M g ] i  the channel  ++  at  physiological concentrations (Matsuda et al., 1987; Horie et al.,  1987; Vandenberg, 1987).  21  1.2.2.2  The  delayed  current, was T  Xi  Delayed R e c t i f i e r ,  rectifier,  f i r s t described  (Noble & Tsien  called I  or  time  1969).  In v e n t r i c l e ,  the The  the  reversal p o t e n t i a l of 1^ between the two  was  again  found  to  be  related to  the  adopted.  Before the current was  f i n d i n g of a threshold  (Vassalle,  1966)  consequence  of  and  that  was  difference i n t i s s u e types K  +  Thus the term  described, however,  repolarization  voltage  I R has  membrane conductance.  current  for all-or-none r e p o l a r i z a t i o n  indicated time  called  extracellular cleft  accumulation i n m u l t i c e l l u l a r preparations. I R was  outward  i n Purkinje f i b r e s and  (McDonald & Trautwein, 1978).  K  dependent  dependent  was  a  changes  in  slow a c t i v a t i o n k i n e t i c s i n  response to depolarization which can be modulated by protein kinase A (Walsh et al., al.,  1987).  (Bennett  et  Beta al.,  1988)  adrenergic  1986).  (Scamps & Carmeliet,  and protein kinase C (Tohse et stimulation  Internal  1989)  and  Ca  + +  enhances  concentrations  of  (Tohse et a l . ,  Na  K  +  1987)  also modulated a c t i v a t i o n , but  [K ]  single channel conductance was  found to be proportional to  the  square root of  rectification  [K ] +  D  properties  DiFrancesco et al.,  1979;  +  did not.  I  (Shibasaki, (Noma  and  Daytner et al  1987).  However, the  I t s inward  Irisawa, 1984)  1976;  i n mammalian  t i s s u e have been a t t r i b u t e d to fast i n a c t i v a t i o n (Shibasaki, 1987) .  Compared to I R of mammalian cardiocytes, the 1^ of  22  frog  atria  i s simpler,  relationship  and exhibits  (Hume & G i l e s , 1983).  a single  ohmic I/V  Recently, the c l a s s i c a l  Ij/r has been subdivided into a rapidly a c t i v a t i n g component (iKr)  a n c  *  a  slowly a c t i v a t i n g component (IRS)  Jurkiewicz,  (Sanguinetti &  1990) on the basis of s p e c i f i c block of I j ^ by r  the new class III antiarrhythmic E4031 (see l a t e r ) .  1.2.2.3  Transient  Early studies  Outward Current,  of a transient  I^  0  outward current  believed  to play a r o l e i n action potential r e p o l a r i z a t i o n were done using cardiac Purkinje c e l l s from sheep (Peper & Trautwein, 1968;  Kenyon & Gibbons, 1979), and cow (Siegelbaum & Tsien,  1980,  Siegelbaum et al., 1977).  Josephson and colleagues  (1984) were the f i r s t group to quantitatively in  r a t single  ventricular  myocytes.  describe  Canine  epicardial  Action Potentials have a c h a r a c t e r i s t i c notch which has been attributed  t o I^o (Litovsky  transient outward current to  exhibit  result  negative  & Antzelevitch,  1988).  of human atrium has been reported  frequency  dependence which may  be a  of i t s time and voltage dependence of r e a c t i v a t i o n  (Escande et al., 1985; Shibata et al., 1989).  Inactivation  of I-to develops quickly and decays r e l a t i v e l y slowly et  The  al., 1988; Litovsky  & Antzelevitch,  (Clark  1989; Hiraoka  &  Kawano, 1986; 1987) thus the current apparently decreases i n a  frequency  dependent  manner,  delayed r e c t i f i e r (Ruiz-Petrich  i . e . the reverse & Leblanc, 1989).  of the  23  Various  authors  have  pointed  to  two  separate Ito  currents; one that i s voltage dependent and s e n s i t i v e to 4AP blockade and another that i s C a 1987;  Benndorf  al.,  et  dependent (Escande et al. ,  + +  1987;  Tseng  Coraboeuf and Carmeliet, 1982). is  one  current  inactivation divalent  that  has  curves which  cations  depolarizing s h i f t  Ba  + +  steady  et  Hoffman,  1989;  Others contend that there state  are s h i f t e d  (Agus  al.,  activation  to the r i g h t  1989),  similar  and by to  i n 1^ a c t i v a t i o n and i n a c t i v a t i o n gates  (Mayer & Sugiyama, 1988). by  &  distinguishes  A lack of s e n s i t i v i t y to blockade  Ito i n cardiac  tissue  from the 1^  current i n neurons (Yellen, 1987), otherwise they are very similar.  O r i g i n a l l y Ito  w  a  s  attributed to an inward current  c a r r i e d by C l ~ (Carmeliet, 1961; Dudel et al.,  1967; Fozzard  & Hiraoka, 1973) on the basis of an apparent s e n s i t i v i t y to [Cl~] . 0  This C l ~ s e n s i t i v i t y was l a t e r shown to r e s u l t from  the chelation of C a Gibbons, 1979). background (Inward  + +  by the C l ~ substitutes used (Kenyon &  I t i s i r o n i c to note that a cAMP dependent  C l ~ conductance  C l ~ movement)  v e n t r i c u l a r myocytes, Harvey et al.,  1990).  has  with been  lacking  outward reported  rectification i n guinea p i g  Ito (Harvey and Hume, 1989;  A previous unconfirmed report  also  described a TTX sensitive C l ~ conductance i n r a t v e n t r i c u l a r myocytes  (Pidoplichko  & Verkhratsky,  1987).  A  chloride  channel from SR reconstituted into l i p i d b i l a y e r s has been  24  studied  at single channel l e v e l  human cardiac SR  (Rousseau, 1989)  (Holmberg & Williams,  and  from  1989).  Inactivation of I^o develops quickly and decays slowly (Clark et al., &  Kawano,  1988;  1986;  Litovsky & Antzelevitch, 1989;  1987).  r e p o l a r i z a t i o n due  Thus,  to I^o  i t has  been  Hiraoka  argued  decreases, whereas I R  that  increases,  with increasing frequency of stimulation, because the former current  recovers  too  slowly  inactivates too slowly  1.2.2.4  Plateau K  Another K  +  while  the  later  current  (Ruiz-Petrich & Leblanc, 1989).  Current,  +  current  has  I  K P  been recently described  which  appears to carry current throughout the duration of the  AP  (Yue & Marban, 1988).  I  show r e c t i f i c a t i o n .  Yue and Marban, (1988) suggested that  i s apparently  K P  Ohmic and does not  the current which i s activated by n i c o r a n d i l and (Kakei et al.,  1986;  Iijima and Taira, 1987)  pinacidil  resembles iRp*  More work needs to be done to confirm the existence of t h i s depolarization activated K  1.2.2.5  Na  +  proposed  and  K, +  to  channel.  "Pacemaker" Current,  A hyperpolarization by  +  activated,  denoted variously contribute  I f or  to  I  N  inward current c a r r i e d as  I f or  spontaneous  1^,  has  been  ("pacemaker")  25  depolarization Seyama,  (Brown et al.,  cells  1976; Yanagihara  Ojeda, 1980; do  i n nodal  & Irisawa,  1980; DiFrancesco  DiFrancesco 1981b; 1982b).  not conform  to Hodgkin-Huxley  1977; 1979; &  The k i n e t i c s of I f  type  of models  (Hart,  1983), but have slow sigmoidal a c t i v a t i o n and deactivation time courses which can be represented by a complicated model with  3 "closed" and 5 "open" states  (DiFrancesco,  1984).  This current slowly activates at membrane p o t e n t i a l s more negative  than  approximately 1984;  -80  mV,  with  Earm et al.,  would  tend  to negate  resistance).  occurring  (DiFrancesco,  1983; Callewaert et al.,  pacemaker depolarization (even input  current  2 seconds a f t e r a voltage step  characteristics  high  peak  1984).  IfS contribution to  i n nodal  Pacemaker  cells  which have  activity  has  recorded i n SA node c e l l s even when I f was f u l l y (Kreitner, 1981; Brown et al., role  on  pacemaking  investigators  1981).  for  If  (DiFrancesco,  is  1985;  These  been  inhibited  However, a modulatory maintained Noble,  by  1984)  adrenaline's augmentation of I f (Brown et al.,  some  due to  1979a), the  observation that increased [ C a ] i increases I f (Hagiwara & + +  Irisawa,  1989),  and  i t s dependence  on  external  [K ] +  (DiFrancesco & Ojeda, 1980).  1.2.2.6  This Na  Na Activated K +  +  activated K  +  +  Current,  channel  mammalian v e n t r i c l e by Kameyama et al.  IjK(Na)  was f i r s t  reported i n  (1984).  For [ N a ] i > +  26  20mM t h i s channel has a large single channel conductance of 207pS  (Kameyama  al.,  et  1984).  It  shows  outward  r e c t i f i c a t i o n f o r [ K ] i > [ K ] (Carmeliet, 1990, Lux, 1990) +  +  0  while  the o r i g i n a l  [K ]i  <  +  [K ]  Q  to  [Ca ]  Carmeliet, The  al.,  et  and  ++  physiologically  showed inward r e c t i f i c a t i o n f o r  (Kameyama  +  insensitive  report  1984).  ^K(Na) i -  i s not voltage  relevant voltages  dependent at et al.,  (Kameyama  1984;  1990).  channel may be activated i n conditions of Na /K +  ATPase  inhibition  and  thus  may  contribute  to  the  shortening induced by d i g i t a l i s glycosides (Kameyama et 1984; with  s  Carmeliet,  1990; Lux 1990).  +  AP al.,  In single channel studies  guinea p i g v e n t r i c u l a r myocytes  (Sanguinetti,  1990),  T-K(Na) had a high slope conductance of approximately 150pS (compared to I ^ i = 30pS) with many sub conductance l e v e l s at multiples of 1/12 x 150pS or 12-13pS. mM  f o r Na  +  iR(Na)  h  a  d  a  K  d  o  f  6 6  a c t i v a t i o n i n inside out patches and therefore  would not be active i n normal r e s t i n g cardiac c e l l s . open  probability  increase  increases  i n [Na *]^. 4  sigmoidally  with  Open p r o b a b i l i t y varies  The  logarithmic sigmoidally  from -200 mV to -80 mV but shows no voltage dependence above -80 mV and  (Sanguinetti, 1990) .  Very few (3%) c e l l s had iR(Na)  these had low r e s t i n g Em  (-20 to -lOmV) , but appeared  healthy according to t h i s report.  1.2.2.7  Ca  + +  Activated K  +  Current, iR(Ca)  27  A  Ca  activated  + +  Purkinje f i b r e s  K  current  +  was f i r s t  reported i n  (Isenberg 1975; 1977; Siegelbaum, 1977) and  l a t e r i n patch clamped bovine Purkinje c e l l s (Callewaert, et al.,  1986)  and guinea p i g a t r i a l myocytes  (Baro & Escande,  The channel showed both a l i n e a r I/V relationship  1989).  from +10 t o HOmV, and C a  dependence,  + +  exhibiting a rapid  a c t i v a t i o n followed by a slower i n a c t i v a t i o n (time constant, t  = 30 - 100 ms) .  was  120pS  In 10.8 mM [ K ] +  (Callewaert  Q  the slope conductance  et al., 1986).  This  designated t h i s channel as d i f f e r e n t from large  conductance  fibres is  (18pS)  tenuous  conductance  (Coraboeuf & Carmeliet, 1982). not useful  i n numerous  Trautwein,  because of i t s  to I-^Q i n sheep  as compared  and perhaps  1987).  group has  tissues  Purkinje  This d i v i s i o n  as  has varying  (Review:  The s u b c l a s s i f i c a t i o n  Pelzer  &  of an iR(Ca)  channel i s a seductive endeavor because such a channel would provide  an elegant  negative  feedback mechanism  entry by shortening AP duration i n C a (Eisner & Vaughan-Jones,  1.2.2.8  K  +  intracellular and  guinea  Hescheler,  levels  which  +  Current,  overloaded states  were  IR(ATP)  inhibited  of ATP were f i r s t  p i g cardiac i983).  + +  1983) .  ATP Sensitive K  channels  + +  for Ca  cells  (Noma,  by  normal  recorded i n rabbit 1983; Trube and  When the [ATP]^ f a l l s  below  cardiac myocytes these channels are activated  0.2mM i n  (K^ = 100 uM)  28  (Noma, 1983).  Other nucleotide blockers of the cardiac ATP  channel include non-hydrolysable analogues of ATP; AMP-PNP, ADP  (partial  agonist)  Kakei et al.,  1983;  et al.,  (Kakei  1986) and GTP (Noma,  1985) although i n pancreatic  c e l l s the  l a t t e r nucleotide has been reported t o have no e f f e c t s (Cook &  Hales,  Petersen,  1984) or t o activate 1986).  As w i l l be discussed  drugs i n h i b i t the channel.  patch  current  clamp  induced  studies,  +  sulfonylurea  channels has been derived  early  shortening  hypoxia (Trautwein et al.,  later,  (Dunne and  While almost a l l of our d e t a i l e d  information about ATP s e n s i t i v e K from  the channel  studies  of the AP  of an outward  i n conditions  1954) and/or metabolic  of  inhibition  (Carmeliet and Boulpaep, 1957) pointed towards the existence of such a channel.  When both sides of the membrane are exposed t o equal [K ]  of  +  150  mM,  the channel's  K  +  selective  unitary  conductance i n guinea p i g cardiac myocytes i s 80pS while i n [K ]  of 5mM i t i s 20pS (outward at Em > -80 mV)  +  Q  Kakei et al.,  1983;  Noma, 1984). inward  block  For large depolarizations the channel shows  al. ,  (Noma,  +  currents  > 0 mV) and Na 1987).  physiological  1983; Cook  &  Hales,  1984;  1984) which r e s u l t s from voltage dependent  of outward K  efficient et  1985; Trube & Hescheler, 1984; Kakei &  rectification  Ashcroft et al.,  (Noma,  +  For  by i n t r a c e l l u l a r  moderate  Q  (only  ++  cations, and deactivation  [ K ] outward currents +  Mg  (Horie  depolarizations are larger  in  (Findlay,  29  1987) .  Typical recordings of the single channel show bursts  separated  by  quiescence  which has  led to a simplest  case  model with one open state and one short and one long closed state (Kakei & Noma, 1984; al.,  1985;  Trube & Hescheler,  1984;  Spruce et al.,  Rorsman & Trube 1985;  Kakei et  1987).  ATP  reduces mean open time and the number of openings per burst (Kakei et al., single  1985;  channel  Spruce et al.,  current  1987)  amplitude.  without  Recently  affecting open  and  "short" closed times have also been found to depend on  the  electromotive d r i v i n g force, V-V^, 1988) , i . e . , with  increased  [K ]  for K  +  Q  Q  was  (Zilbeter et  +  the frequency  increases within a burst of openings. reversal p o t e n t i a l , - t  the  al.,  of c l o s i n g  In the region of the  maximal and X  was  c  minimal, and  open p r o b a b i l i t y decreased with hyperpolarization.  1.2.2.9  Acetylcholine/Adenosine  Activated K  +  Current,  ACh/Ado  K  Early  indications  acetylcholine's observations atrium and 1951;  1958) .  that ACh  &  the  mechanism  Later i t was I  K 1  of  bradycardic e f f e c t s were derived from increased flux of K  Hutter,  in fibres  inward r e c t i f i e r gating  to  from guinea p i g  +  frog and t o r t o i s e sinus venosus (Holland et  Harris  resistance  (ACh)  as  1956)  from dog  al.,  and  decreased  membrane  atrium  (Trautwein  & Dudel,  shown that iR(Ach) ^ (Gamier et al.,  (shorter open times than IJQ)  s  s i m i l a r to the  1978), but and  that i t s  conductance  (13pS  30  i n 5.4 mM [ K ] +  (Sakmann  v. 6pS f o r T-KI) properties were d i f f e r e n t  et al.,  Trautwein al.,  Q  1983; Soejima  and Noma, 1984; Noma &  1978; Carmeliet & Mubagwa, 1986; Heidbuchel et  1990).  Intracellular Mg  i s responsible f o r inward  ++  r e c t i f i c a t i o n of both channels. Also, iR(Ach) doesn't show sensitivity to B a shown  that  blockade as I  + +  adenosine  K  1  does.  Recently, i t was  receptors and ACh receptors couple  through a G protein to the same K and Isenberg, 1983; Kurachi et al.,  channels  +  (Belardinelli  1986; Pfaffinger et al.,  1985; Breitwieser and Szabo, 1985; Logothetis et al., Kirsch et al.,  1988).  1987;  ATP (K^ 9.5 mM) has also been shown  to activate an inward r e c t i f y i n g current which was proposed to  be conducted  1988;  through  iR(ACh)  channels  (Friel  & Bean,  1990).  1.2.3  Inward  K  +  Channel D i s t r i b u t i o n i n Mammalian Heart  rectifier,  IKI  :  T  current has been characterized guinea p i g (Sakmann & Trube, (Payet et a l . , al., 1989),  1983),  1985;  n  i n ventricular  1984;  Josephson,  channel underlying t h i s  e  Hume & Uehara,  1988),  cat (Harvey & Ten Eick  and dog (Tseng et al.,  cells  rabbit  1988;  1987).  from:  1985),  rat  (Kameyama, et  Kleiman & Houser,  S i m i l a r l y ubiquitous  i n Purkinje f i b r e s I J Q has been recorded from canine, sheep and bovine Purkinje c e l l s  (Callewaert et al.,  1984).  IJQ  has been reported i n a t r i a l c e l l s of rabbit (Sakmann et al., 1983;  Noma et a l . ,  1984;  Giles & Imaizumi,  1988),  guinea p i g  31  (Hume and Uehara, 1985), and human (Heidbuchel et al., 1990) .  However, others have concluded  present i n a t r i a of rabbit discussed  earlier,  that  1987;  I ^ i was not  (Soejima & Noma, 1984),  and as  I J Q does not appear  the a t r i a l  equivalent to the v e n t r i c u l a r I ^ i  (Hume et al.,  to be  1990).  reviews of Irisawa (1987) and Pelzer & Trautwein  The  (1987) of  currents i n cardiac tissue conclude that there are few I ^ i channels i n nodal c e l l s ; t h i s agrees with previous reviews (Noble, 1984).  Delayed r e c t i f i e r , I  K 2  i n Purkinje  1^:  fibres  This current o r i g i n a l l y c a l l e d  (Hall  et al.,  1963) was  analyzed i n sheep Purkinje f i b r e s by Noble  & Tsien  first (1969)  and has been i d e n t i f i e d i n the v e n t r i c l e s of cat (McDonald & Trautwein, 1978) as well as i n a number of amphibian preparations  (Rougier et al.,  1969b), SA node  1968; Brown & Noble,  (DiFrancesco et al.,  node (Kokubun et al.,  1982).  1969a &  1979) and rabbit AV  The K accumulation/depletion +  phenomenon  seen  preparations  (Baumgarten & Isenberg,  1978)  in  atrial  voltage  clamped  multicellular  1977; Kline  & Morad,  l e d to a r e a l i z a t i o n of the need f o r single c e l l and  single channel analyses of I . K  difficulties  This approach has had i t s  due to the low single  channel  conductance  (llpS) found i n c e l l attached patches of rabbit nodal c e l l s (Shibasaki,  1987).  Whole c e l l  recordings have been made  from canine Purkinje c e l l s (Datyner et al., ventricular  cells  (Matsuura  et  al.,  1984) guinea p i g 1987),  feline  32  ventricular  cells  ventricular  cells  Purkinje  (Bennett  ventricular cells cells  (Kleiman  &  (Clapham  &  al.,  et  Houser,  1989) ,  Logothetis,  1986),  (Giles & Imaizumi,  rabbit  1988) ,  1987).  insignificant  i n rat ventricle  et al.,  Yatani  1984).  analysis of the K mention  1^ appears  of  +  calf  atrial  and  1988) rabbit Purkinje  (Scamps & Carmeliet, 1989) canine v e n t r i c u l a r  (Tseng et a l . ,  chick  cells  to be either absent or  (Josephson  et al.,  et al.,  Heidbuchel  (1990)  1984; i n an  channels i n human a t r i a l c e l l s , made no  i n these  cells.  Shibata  et al.  (1989)  suggested the contribution of I R to r e p o l a r i z a t i o n may be small, i . e . , suggest  that  < 2 0% Ito* the delayed  v e n t r i c l e , because actions  Pharmacological evidence would rectifier  i s present  i n human  an I R blocker, has class I I I  clofilium,  (Arena & Kass, 1988).  However, one study of human  v e n t r i c u l a r c e l l s f a i l e d to find I R although the short (100 ms) t e s t pulses used may have precluded i t (Mitchell et  al.,  1986) .  Transient this  outward, I t o  :  T  n  e  channel  responsible f o r  current has been reported to be present i n Purkinje  fibres  of cow (Siegelbaum  1987;  Callewaert et al.,  1987)  sheep,  & Tsien,  1986), rabbit,  (Deck & Trautwein,  1968; Coraboeuf  1980; Kenyon & Sutko, (Carmeliet et a l . ,  1964; Peper  & Trautwein,  & Carmeliet, 1982; Carmeliet et al.,  1987),  dog single Purkinje c e l l s (Nakayama & Fozzard, 1988), rabbit a t r i a l and v e n t r i c u l a r c e l l s  (Hiraoka & Kawano, 1986; 1989;  33  Imaizumi  and  Imaizumi,  Giles  1988)  1987;  Crista  Clark  et al.,  Terminalis  1988;  (Giles  Giles  and  & van Ginneken,  1985) and AV node (Nakayama and Irisawa, 1985) , guinea p i g atrial  epicardium  reported  to  endocardium  be  present  (Litovsky  anatomically ventricle  (Wang & Nattel, in  studies  (Tseng and Hoffman, 1987).  1985; Escande et al.,  canine  have  I^o  1988;  shown  i  I-to has been reported  1984)  (McDonald  (Josephson et  and  although t h i s  Trautwein,  latter  questioned by Ten Eick and Robertson, (1983).  1985;  However, as  and guinea p i g v e n t r i c l e s et al.,  MacDonald m a  even  Y  b  e  elephant  (Schoutten  i n mouse v e n t r i c l e  1987), r a t v e n t r i c l e  cat v e n t r i c l e  1985)  al.,  1989; Heidbuchel  1984).  Shibata,  1989;  1987; Shibata et al.,  al.,  et al.,  canine  (Escande et  et  McDonald  not  Human atrium has  1990), and i t may be i n human v e n t r i c l e  and  n  been  Less  1989; Nakayama et al.,  al.,  1984)  but  1989).  et  (Benndorf et al. ,  has  epicardium  & Antzelevitch,  specific  Tseng et al.,  1989).  1978;  finding  was  Frog (Giles &  (Hume & Uehara,  1984) appear not to have an I to*  common to most mammalian a t r i a l seal  al.,  atrial  muscle  has  tissues  i t (Maylie  and  Morad, 1984) .  Plateau, iRp^  This current has only been described i n  one publication using guinea p i g v e n t r i c u l a r myocytes and Marban, 1988) current  was  ventricle  (Yue  although these authors contend that the  reported,  but not recognized  (Apkon and Nerbonne,  as  lRp>  in rat  1988) and i n chick embryonic  34  cells  (Mazzanti and DeFelice, 1990).  I t remains to be seen  i f channels responsible for Ij^p,  where, and  are present i n  myocardium.  Hyperpolarization-activated, I f : supporters  and  detractors.  t i s s u e / c e l l s i s supported al.,  I t s existence i n rabbit  Maylie  & Morad,  Ginneken & G i l e s , 1985;  & Irisawa,  1984;  Nathan  Noma et a l . ,  1980;  & Roberts,  DiFrancesco et a l . ,  are negative reports (Kokubun et al.,  1982;  1985;  1986)  et al.,  1983), cow  supported al.,  1987)  and sheep Purkinje c e l l s  Studies  i n human a t r i a l  rejected  it  (Shibata  mentioned i t at a l l (Heidbuchel single  channel  study  may  not  et al., have  have  1987;  al.,  et  The  able  either  Escande et  1989),  1990). been  (Earm  (Callewaert et  cells  a r o l e f o r I f (Heibuchel et al.,  al.,  Nakayama et  I f has been reported to exist i n sheep atrium  1984).  van  but, there  1984) .  al.,  nodal  by a number of studies (Brown et  1979a & 1979b; Yanigara  1980;  This current has both  to  or  not  latter see  If  because of the short time course of the voltage steps used.  Sodium dependent, iR(Na)  This current has  :  reported i n a few preparations. pig  ventricular  Sanguinetti, 1990;  myocytes  I t has been shown i n guinea (Kameyama  Carmeliet, 1990;  Calcium dependent, i K ( C a )  :  only been  Luk,  et 1990).  al.,  1984;  35  This current has been suggested to be present preparations  (Carmeliet, 1989).  proposed that a s p e c i f i c C a  However, i t has  activated K  + +  has not been found, but that only C a K  +  channels occurs  these  al.,  iR(Ca)  fibres/cells  1977;  modulation of other  + +  been  reported  (Isenberg,  1975;  1977;  Despite  in  bovine  Siegelbaum et  Callewaert et a l . , 1986), sheep Purkinje  & Escande,  al.,  1990)  al.,  1989).  1982), guinea p i g a t r i a l  1989), rat T-tubule  membrane  and human a t r i a (Escande et al.,  Acetylcholine/adenosine current  s e l e c t i v e channel  has  (Coraboeuf & Carmeliet, (Baro  also been  (Eisner & Vaughan-Jones, 1983).  reservations,  Purkinje  +  i n most  is  responsible  acetylcholine (Ach)  myocytes  (Cecchi  1987;  the  bradycardic  and adenosine (Ado).  et  Shibata et  activated, lR(ACh/Ado)  for  fibres  :  This  effects  of  The receptors for  ACh and Ado are linked to the same G protein which activates this  current  (Kurachi  pharmacological atrial  tissue,  evidence  al.  1986).  Thus  there  is  for t h i s current i n both nodal  and  i . e . t i s s u e s innervated by the vagus nerve.  Since muscarinic t h i s current may  et  receptors  are  also present  also be found there.  in ventricle,  Patch-clamp/voltage-  clamp studies have shown lK(ACh/Ado) i - c e l l s  from guinea  n  pig  atrium  (Iijima et al.  al.,  1983;  Sakmann et al.,  ,  1985)  AV  node ( B e l a r d i n e l l i et  1983), rabbit atrium  Noma, 1984), SA node (Noma et a l . , 1981; 1978), AV  node (Nishimura  et al.,  (Soejima  &  Noma & Trautwein,  1988), and  human atrium  36  (Escande  et al. ,  Heidbuchel  1987;  et al. ,  1987;  1990).  ^(ACh/Ado) has also been shown i n sheep Purkinje (Carmeliet &  Ramon,  Carmeliet,  1980),  and rabbit  1983),  although  Purkinje  fibres  i t s functional  (Mubagwa &  significance i n  t h i s t i s s u e i s less clear.  ATP-sensitive,  channel  which  IR(ATP)  responds  This current r e s u l t i n g from a  :  to metabolic  inhibition,  mechanisms f o r A T P depletion, was f i r s t  or other  shown i n i s o l a t e d  c e l l s from guinea p i g and rabbit a t r i a and v e n t r i c l e (Noma, 1983;  Trube,  Along with numerous confirmations i n  1983).  these tissues the channel has also been shown t o e x i s t i n rabbit AV node (Kakei & Noma,  1984),  et a l . ,  1983;  An ischaemia-induced outward  current  i n cat v e n t r i c l e  Findlay,  proposed t o have been  1987).  rat ventricle  (Vleugels et al.,  IK(ATP)  investigators proposed that  1 9 8 0 ) has been  (Stern et al.,  IR(ATP)  (Conrad  1988).  These  channels were activated  i n ischaemic c e l l s despite bulk i n t r a c e l l u l a r l e v e l s of A T P high enough t o suppress the channel, on the premise compartmentalization  of A T P was occurring  or that  that other  nucleotides were a c t i v a t i n g the channel.  1.2.4  K  While  there  adenosine  +  Channel Stimulators  are numerous  muscarinic  receptor and  receptor agonists which would activate K^ch/Ado  channels i n the heart, these receptor linked  channels are  37  mainly  present  i n supraventricular t i s s u e .  t h i s thesis i s on K  ACh/Ado i l l w  of the Kjj from  n  digitalis  shown in  Thus pharmacological activators of  ° t be discussed.  channel  a  S i m i l a r i l y , the a c t i v a t i o n  by high l e v e l s of  glycoside treatment,  vivo  and  discussed here.  focus of  channels which play putative roles i n  +  v e n t r i c u l a r arrhythmias. K  The  Na /K +  ATPase  +  [ N a ] i might  result  +  but t h i s has inhibitors  not been  will  not  be  On the other hand, a newly developed group  of vasodilator drugs has recently been shown to activate ATP sensitive K beta  channels i n heart, s k e l e t a l muscle, pancreatic  +  cells  and  Escande, 1989;  of  course  Cole & Leblanc, 1990;  prototypes of t h i s new et  a l . , 1979)  Bray et al.,  is  analogues  in  Taira, 1980; 1986) .  A number of compounds with  similar  1983; 1988;  has  pinacidil  (P1134)  (main metabolite i n a c t i v e ) , SG114; RP49,356; EMD  drugs  produce  an  (Yanagisawa et al.,  been  shown to  Yanagisawa  cromakalim Mestre  including  et  and  52,962.  increase  1979;  in  et al.  Nakajuna ,  1988;  & Taira, &  shorten 1980;  Kurachi,  the  cardiac  Imanishi 1985;  Scholtysik, 1987)  K  +  Yanagisawa &  Weir & Weston, 1986a & 1986b; Hamilton et al.  This  (nicorandil:  (Taira  1986;  cromakalim  development,  cells  The  (Hamilton et al.,  and  vasodilator  from  Ashcroft, 1988).  1980;  the n i c o r a n d i l analogues:  efflux  see  (Arrigoni-Martelli  P1060, and P13 68  These  review  al.,  pinacidil 1987)  (for  class of drug are n i c o r a n d i l ,  Weir & Weston, 1986). actions  vasculature  et  , AP al.,  Osterrieder, by decreasing  38  the  time  constant  amplitude of outward  for activation  and  increasing  the  currents at potentials more p o s i t i v e  than -40 mV, i n part through removal of inward r e c t i f i c a t i o n as shown by L i u et al. sensitive K rapid  al.,  upon depolarization  current  1987)  Cromakalim or  channel shows inward r e c t i f i c a t i o n due to both  +  closing  outward  similar  o  to  I  K  (Matsuda  1  both, but since  r  al.,  1988)  al.,  1988;  Osterrieder et al. , does  not  (an  1985; Trube et al.,  reverses  cromakalim's  IR(ATP)  1986; Fossett effects,  IR(ATP)  these (Escande al.,  1986; Sanguinetti et  1988).  reduce  1987).  of either I J Q  glibenclamide  1988; Hamilton et al.,  cromakalim  block of  al.,  et  vasodilator drugs are thought to activate et  ++  (Noma, 1983; Cook & Hales, 1984; Horie et  blocker) (Sturgess et al., et  and also M g  could reduce inward r e c t i f i c a t i o n  iK(ACh)  The A T P  (1990) with cromakalim.  I t has been shown that  slow  inward  I  current,  s  i /  d i r e c t l y , although shortening of the A P would decrease the time available f o r C a 1990; Steinberg et al.,  1.2.5  K  +  entry i n a beat (Liu et al.,  + +  1988;  1988; Morad & Trautwein, 1968).  Channel Blockers  There are a number of inorganic ions and a few ammonium compounds that are routinely used to block K cardiac t i s s u e or c e l l s in vitro. being  used  analysis,  to  dissect  out  +  channels i n  These ions have, and are currents  but are not generally  used  for in vivo  quantitative because of  39  their  toxicity.  myocardium  has  For been  example  used  to  guinea  show  pig  the  effects  blockade of I R and I J Q on slow response AP 1980) .  Similarly,  effects  of  blockade  (Isenberg, 1976). observe  CsCl has been used  the  of  of  morphology i n r a t and  Ito  has  Gibbons,  been  Ba  (Ehara et  to investigate  I J Q in  and  of  Purkinje  + +  al., the  fibres  Tetraethylammonium, TEA, has been used to  effects  Vassort, 1968).  If  ventricular  reduction of  K  currents on  +  guinea p i g v e n t r i c l e  (Coraboeuf  AP and  The 4-aminopyridine s e n s i t i v e component of  studied  1979) .  i n sheep Purkinje f i b r e s  While  useful  as tools,  (Kenyon &  these K  channel  +  blockers are not cardiac s e l e c t i v e , they were a c t u a l l y f i r s t used  i n squid giant  axons,  and  their  actions  on  neural  t i s s u e l i m i t s t h e i r usefulness as antiarrhythmic drugs. N  one  drug  that  has  been  derived from  quaternary  The  ammonium  compounds (QA) i s c l o f i l i u m , but i t i s much more potent than the simple "channel plugger" QAs  1.2.5.1  Sulphonylureas -  (Arena & Kass,  IR(ATP)  1988).  Blockers?  Sulphonylurea drugs have been widely used to t r e a t type II  diabetes.  questioned  by  In  the  findings  early of  seventies  the  University  their Group  use  was  Diabetes  Program which suggested tolbutamide treatment increased the risk  of cardiovascular death  (UGDP, 1970).  This sparked  i n t e r e s t into t h e i r cardiac e f f e c t s and as reviewed by Levey et a l . (1974) these hypoglycemic  agents were found to have  40  direct noted  positive cAMP  inhibition  inotropic  accumulation  actions.  Studies at the time  and suggested  as a mechanism of action  phosphodiesterase  (Roth et al.,  Stimulation of adenylate cyclase was also invoked & Fichman, & Broth (1983)  1977;  observed  Leichter et al.,  on  Kramer et al.  1981).  tolbutamide to increase glycogenolysis and  glucose u t i l i z a t i o n . explained  (Brooker  Others questioned these hypothesis (Brown  1971).  al.,  1971).  These early observations can now be  the basis  of  the observation  that the  sulphonylureas block (Sturgess et a l . , 1 9 8 5 ; Trube et  al.,  1 9 8 6 ; Fosset et a l . , 1 9 8 5 ; Sanguinetti et a l . , 1 9 8 8 ) the ATP sensitive K  +  channel,  (Noma,  IR(ATP)'  Blockade of  1983).  t h i s channel prevents shortening of the AP i n the presence of  ATP depletion,  a complication which may have  been a  factor i n the o r i g i n a l c i t e d work on i s o l a t e d t i s s u e .  Patch  clamp studies have shown tolbutamide and glibenclamide t o be specific al.,  IR(ATP)  1986;  blockers (Sturgess et a l . ,  Belles et al.,  no e f f e c t s of I R or C a I  K(Ca) (Trube et a l . ,  Pancreatic  beta  cell  1987; + +  magnitude more sensitive  l . ,  A T P  or  IJQ  channels  (Belles et al.,  1986) 1987).  are two orders of  t o tolbutamide  v e n t r i c u l a r myocytes (Trube et al.,  with  1986),  currents (Rorsman & Trube,  1986),  K  Ashcroft et a  Trube et  1985;  1986).  than  channels i n  Glibenclamide i s  the most potent sulphonylurea, being 1 0 0 x more potent than tolbutamide (Sturgess et al.,  1.3  1985;  Pharmacology of K  +  Trube et al.,  Channel Blockers  1986).  41  1.3.1  Pharmacology of New Class I l l ' s  Since Vaughan-Williams' (1970; 1975) c l a s s i f i c a t i o n of antiarrhythmic drugs, development of class I I I agents, which prolong r e f r a c t o r i n e s s without has gNa  progressed +  This may be related to a perceived  p o t e n t i a l of Class I I I drugs.  Cardiac Arrhythmia Suppression increased mortality r i s k and  velocity,  slowly r e l a t i v e to development of c l a s s I,  blocking, drugs.  proarrhythmic  slowing conduction  flecainide,  investigators,. 1989) .  T r i a l , CAST, pointed to a an  associated with  despite  However, the  use of encainide  suppression  of  These r e s u l t s with  VPB  Class  (CAST  l c , gNa  +  blocking drugs, may provide a more receptive environment f o r the introduction of agents with Class I I I a c t i v i t y (Woosley, 199 0)  and currently a number of pharmaceutical  companies are  a c t i v e l y persuing t h i s avenue (Colatsky and Follmer, 1989). New  Class  sematilide  I I I drugs  currently  i n development  (Lumma et al., 1987) and CK-3579  include:  (Chi et al.,  1990)  from Berlex-Schering; E4031 (Katoh et al., 1988; 1990)  from  Esai;  UK68,798  (Gwilt  et al. ,  1989a  &  1989b) and  UK66,914 (Gwilt, 1988) from P f i z e r , (the l a t t e r compound has been  withdrawn);  MDL-11,939  M e r r i l Dow; r i s o t i l i d e  (Koerner  & Dage,  1989)  from  (Colatsky et al., 1989) from Wyeth-  Ayerst; RP58,866 (Mestre et al., 1989; Escande et a l . , 1990) and RP62,719 from Rhone Poulenc-Sante, LY190147 from L i l l y ; MS-551 (Kamiya et al., 1990) from M i t s u i . KC8851 (Beatch  Tedisamil and  et al., 1990) are presently being  developed  42  as  antianginal  1989a).  drugs  by  KaliChemie  (Buschmann  et  al.,  While most of these compounds have been reported to  have s e l e c t i v e Class III actions, the data supporting claims will  i s limited.  be  safer to  A f t e r t e s t i n g on ascribe  these  a broader scale, i t  selectivity  of  action  to  these  compounds.  The sotalol  4-[methylsulfonylamino] is  the  basis  of  benzamide  sematilide,  UK68,798, UK66,914, MDL-11,939 and the N-acetyl  group of acecainide  CK3579,  E4031.  has  nucleus  In  of  risotilide, sematilide,  been replaced  with a  methanesulfonamide group, which i s a more stable amide than N-acetyl,  because  transformation  of  the  associated with a lupus l i k e t o x i c i t y Notable  exceptions  to  this  latter  has  (Lumma et al.,  structural  Buschmann,  derivative  1987),  RP58,866,  (Escande et al.,  substituted pyrimidinedione  1.3.2  of outward K  +  reduction  inward Na  1990), and  is  a  are (Kuhl  benzopyran  MS-551, which i s a  (Kamiya et al.,  1990).  Class III Drug Mechanisms of Action  The cardiac APD  ii)  which  1989).  similarity  tedisamil and KC8851, which are sparteine derivatives &  been  +  currents which contribute to r e p o l a r i z a t i o n , of  or C a  electrogenic  can be prolonged by either, i) blockade  i n a c t i v a t i o n or + +  conductance  currents, or i i i ) increased  Na /Ca +  increased  ++  exchange.  Prolonging  activity APD  of of  delays  43  recovery of voltage dependent Na refractory  periods.  "selectively"  block  Class K  +  channels,  thus increasing  I I I antiarrhythmics  channels  are  being  that  developed  (Colatsky and Follmer, 1989; 1990).  The  exact  structural  requirements f o r synthesis of a  compound producing s p e c i f i c K at present.  +  channel blockade i s not c l e a r  The p-aromatic methylsulfonamides l i s t e d above  have been reported to block the cardiac delayed r e c t i f i e r K current ( r i s o t i l i d e :  Follmer et al., 1989; UK66,914:  +  Gwilt  et al., 1988; UK68,798: Gwilt et al. , 1989; E4031: Sawada, 1989;  Follmer and Colatsky, 1990).  However, the presence of  t h i s group alone does not confer Class I I I actions (Lumma et al., 1987).  Within a group of p-aromatic methylsulfonamides  structure a c t i v i t y substitutions  r e l a t i o n s h i p (SAR) analysis showed that  f o r the methyl  group  or  removal  of the  sulfonamide moiety i t s e l f renders the compound i n a c t i v e ( L i s et al., 1987).  The s u b s t i t u t i o n of an electron withdrawing  group i n place of an electron-donating group on the aromatic portion of the basic l o c a l anaesthetic backbone appears to confer s e l e c t i v e Class I I I actions. 1990) .  This  trend  can be seen with  (Colatsky & Follmer, the electronegative  methylsulfonamide s u b s t i t u t i o n above and with the para-Cl~ or NO2 i n the s e l e c t i v e Class I I I agent c l o f i l i u m and i t s t e r t i a r y ammonium derivatives (Arena & Kass, 1988).  44  The  sulfonamides  bacteriostatic  familiar  to a pharmacologist as the  agents used u n t i l  the 1940's are  different  from these Class III drugs i n that the amide-N l i n k s to the aromatic ring  i n the antiarrhythmic compounds, whereas the  sulfonamide group i s linked to a paraamino benzene r i n g v i a the s u l f u r atom i n the a n t i b i o t i c s .  Clofilium rectifier,  has  while  been  reported  i t s tertiary  to  block  the  derivatives,  LY97119  LY97241 also block the inward r e c t i f i e r i n i s o l a t e d pig v e n t r i c u l a r  cells  (Arena & Kass, 1988).  been  block  I-^o  shown  to  *K  a n d  a  ^  room  in  respectively  1990).  Cardiovascular E f f e c t s of New  A  with  reported that 3.5  sematilide mM  in  ventricular  Class I l l ' s  isolated  canine  widened the APD95 by 20%  f i b r e s and 17 mM prolonged the functional in  guinea  temperature  1.3.3  study  and  Tedisamil has  i s o l a t e d r a t and guinea p i g v e n t r i c u l a r c e l l s , (Dukes et al.,  delayed  muscle  by  20%  (Lumma  i n Purkinje  refractory et  tissue  period  al.,  1987).  Sematilide (CK-1752A) and CK-3579 have both been reported to have Class III e f f e c t s i n dogs (Chi et al., 1990).  In vivo  there was  P-R  was  while  Q-T  widened  no  after  effect  by  CK-3579  either due  drug  to  i n t e r v a l s were widened by only 5% refractory  period was  beta  on  QRS,  blockade  but  (Chi et al., 1990).  The  widened only 5 - 15% by these drugs  45  whether measured i n normal  or infarcted t i s s u e and yet 80%  protection from mortality following i n f a r c t i o n was seen (Chi et al.,  1990).  In s i m i l a r models, E4031 was tested f o r antiarrhythmic effects  (Lynch et al.,  1990).  without e f f e c t s on QRS and R-R Q-T  c  i n t e r v a l by 11%.  In t h i s  study,  E4031  was  i n t e r v a l s , while i t prolonged  S i m i l a r l y v e n t r i c u l a r conduction time  was unaltered by E4031 treatment but refractory periods were increased  by  a  maximum  infarcted tissue protection  from  of  25%  (Lynch et al., mortality  in  normal,  1990).  (70%)  was  as  Again, seen  well  as  remarkable  when  a  second  ischaemic i n s u l t was  applied i n the presence of a previous  infarct.  group  A  second  of  investigators  found  similar  s e l e c t i v e Class I I I actions of E4031 i n a canine model of programmed  electrical  myocardial i n f a r c t i o n  stimulation  (PES),  (Katoh et al., 1990).  in  7  day  old  In t h i s study  e f f e c t i v e refractory periods were increased by  18-22% (in  both normal and infarcted zones) with plasma l e v e l s of 330 ± 150  ng/ml  mapping  to  E4031. suggest  This  study  that  ERP  also  employed  epicardial  prolongation prevented  the  induction of reentrant VT.  UK68,798 and UK66,914 have been shown to prolong and  ERP  i n dog  ventricular  increase v e n t r i c u l a r ERP dog in vivo  and  Purkinje f i b r e s  and  (VERP) i n guinea p i g in vitro  (Gwilt et al., 1988;  1989).  APD to and  These e f f e c t s were  46  found to be correlated with widening  of the Q-T  interval.  In addition, both drugs were found to s e l e c t i v e l y block I R and  I ^ i i n whole  not  ventricular cells were seen on QRS  in  5 0  patch  (Gwilt et al., or P-Q  conduction times. EC  cell  clamp  1988;  of  guinea  1989).  No  pig  effects  i n t e r v a l s of the surface ECG, or on  These drugs were remarkably potent with  (for Class I I I effects) values for UK68,798 of 20-50 nM  vitro  and  10-25  ug/kg in vivo  1989b; Dalrymple et a l . ,  Risotilide  (Gwilt et a l . , 1989a &  1989).  (WY48,986)  has  been  reported  to  have  s e l e c t i v e Class I I I antiarrhythmic actions i n dogs and pigs subjected  to  coronary  artery  ligation  (Colatsky et  al. ,  1989).  As with the drugs reported above, the APD  and ERP  prolonging e f f e c t s of r i s o t i l i d e were suggested to  result  from  s e l e c t i v e 1^ blockade  Preliminary  studies  demonstrated  antifibrillatory  or reperfusion 1989; 1990).  i n rats  and  MDL  (Follmer et al. , 1989).  11,939  (10  mg/kg)  e f f e c t s with acute PES  i n dogs  have  ischaemia  (Koerner  & Dage,  These e f f e c t s were suggested to r e s u l t from AP  widening seen in vitro  RP58,866 has  ( L i & Dage, 1989).  been reported to  (Escande et al., 1989; prolong APD  with  widening  1990)  selectively  block I J Q  i n guinea p i g myocytes and to  and refractory periods i n guinea p i g v e n t r i c l e  and Purkinje f i b r e s (Mestre et al., 1989).  These Class I I I  actions were found to protect against ischaemia/reperfusion  47  induced VF i n halothane  anaesthetized dogs, and micropigs,  at a dose of 0.3 mg/kg (Mestre et al., 1990). risotilide, a  marked  As opposed to  UK68798, sematilide and E4031, RP58866 produced reduction  Unfortunately,  i n HR  (31%) (Mestre  et al., 1990).  the assumption of antiarrhythmic actions of  RP58,866, was based on a single dose, open study, with only 5 animals  per group i n which the occluded  zone sizes were  not reported (Mestre et al., 1990).  A single abstract has been recently published on the Class I I I protective e f f e c t s of MS-551 against  arrhythmias  induced by PES i n 3-5 day old infarcted dogs (Kamiya et al., 1990).  In t h i s study, MS-551 was found to increase a t r i a l  ERP by 56% and v e n t r i c u l a r ERP by only 21% while increasing Q-T i n t e r v a l  by 14% at a maximal  10 mg/kg dose.  Up to  lOmg/kg MS-551 d i d not have e f f e c t s on QRS, P-Q i n t e r v a l s , conduction time, blood pressure, a o r t i c flow nor myocardial c o n t r a c t i l i t y , while only a s l i g h t bradycardia was seen.  Although bradycardic  tedisamil has been tested actions  for i t s specific  and applications to angina  pectoris  (Buschmann et al., 1989; Grohs et al., 1989) there has been only one report of i t s antiarrhythmic other than ourselves  actions by a group  (Curtis et al., 1990 i n press).  This  group found tedisamil to reduce the duration or "turn-off" VF i n i s o l a t e d r a t hearts subjected to ischaemia. work which i s presented  Our own  i n t h i s t h e s i s has been published  48  previously 1990;  (Walker  1988,  Beatch  et al.,  1988;  1991).  1.3.4  It  C l i n i c a l l y Available Class III Drugs  i s i r o n i c that the f i r s t  i . e . amiodarone, any  & Beaten,  class III antiarrhythmic,  can no longer be considered as belonging to  single class i n the c l a s s i f i c a t i o n  Williams  (1970;  1975).  antiarrhythmic by originally al.,  1962).  1970)  as  Initial  et al.  an  first  (1969) although i t was  antianginal  reports  a selective  (Singh  drug &  widening  (Charlier et  Vaughan-Williams, i n rabbit  a t r i a and v e n t r i c l e , with minimal Vmax reduction.  Recently,  APD prolonging e f f e c t s of amiodarone have been shown to  result  from blockade of both  1987; Sato et al., 1987). the Ca  AP  shown to be  effect  the  suggested  Amiodarone was  Charlier  developed  scheme of Vaughan-  I J Q (Balser et al.,  and  The h i s t o r i c a l events leading to  discovery of amiodarone's + +  1^  blocking e f f e c t s on Na  +  and  channels, interactions with thyroid function and beta  adrenoceptors, been well 1983;  chronicled  Singh,  1989).  unusual  1983;  While  apparent, unsuitable  the  toxicity  i n a number of reviews  Nattel  i t s lack for  pharmacokinetics and  & Talajic,  clinical of  (Broekhuysen,  1988;  Singh et al.,  usefulness of  amiodarone i s  selectivity  examinations  have  of  of  action  specific  antiarrhythmic mechanisms i n basic research.  renders i t Class  III  49  The  other  prototypical  compound, d , l - s o t a l o l beta  blocker  with  (Singh & Vaughan-Williams, 1 9 7 0 )  APD widening  Singh & Vaughan-Williams,  1967;  While  both  I I I antiarrhythmic  Class  the dextro-  effects  1970;  (Schmid  & Hanna,  Strauss et al.,  and levo-rotary  is a  1970).  enantiomers of  s o t a l o l have s i m i l a r APD widening e f f e c t s and antiarrhythmic effects  (Somani  1968)  & Watson,  potent as a beta blocker  d-sotalol  (Manley et a l . ,  i s only  7%  as  Sotalol's  1986).  Class I I I actions are thought t o r e s u l t from i t s a b i l i t y t o block I R and to a lesser extent —6  concentrations of 1 0 and  Purkinje  reduce  - 10  M i n both v e n t r i c u l a r muscle  High  Although  +  1 9 8 5 ) at  —4.  fibres.  gNa .  I J Q (Carmeliet,  doses  there  (10~  M) were  4  are species  seen to  differences i n  s e n s i t i v i t y t o s o t a l o l , t y p i c a l l y the APD i s widened for  a plasma concentration of 2 x 1 0 ~ M s o t a l o l 5  (Edvardsson  et al.,  drug  has been  which  currently  under  i n man  The uses and mechanisms of t h i s  1980).  seminal  development  recently reviewed (Singh,  15-30%  i n the design (see  section  of compounds  1.3.1)  has been  1987).  There are a number of drugs which prolong APD as an adjunct  to their  other  actions,  such  drugs  include the  adrenergic neuron blocking drug bretylium (Waxman & Wallace, 1972) and  i t s analogues bethanidine meobentine  neuroleptic  (Bacaner and Benditt, 1 9 8 2 )  (Wastilla et al.,  melperone  Vaughan-Williams, 1 9 8 2 )  (Arlock  1981);  the butyrophenone  et al., 1 9 7 8 ; M i l l a r  and some t r i c y c l i c  &  antidepressants  50  e.g.  amoxapine  (Kinugawa  et  al.,  1988).  Clofilium,  a  d e r i v a t i v e of bretylium, on the other hand appears to be a relatively Molloy,  specific  1979)  Class  which  III antiarrhythmic  blocks I  Arena & Kass, 1988).  K  (Snyders  (Steinberg &  & Katzung,  1985;  Acecainide (N-acetylprocainamide) also  has r e l a t i v e l y s e l e c t i v e Class III actions  (Drayer et al.,  1974; J a i l l o n & Winkle, 1979).  The prompted  historical  absence  of s e l e c t i v e  us to assess the antiarrhythmic  class  III drugs  efficacy  of  new  p u t a t i v e l y s e l e c t i v e class III compounds (tedisamil, KC8851, UK68,798,  RP62,719,  and  risotilide).  We  performed  experiments designed to determine the mechanism(s) of action of  these  compounds  at  equivalent  dose  levels  used  to  determine antiarrhythmic e f f i c a c y .  Section 1.3.5  Any  Torsade de Pointes  discussion  of antiarrhythmic drugs  i s incomplete  without an examination of t h e i r potential f o r proarrhythmia. A p a r t i c u l a r form of polymorphic VT which i s preceded by a prolonged  diastolic  interval  was  first  described  by  Dessertenne (1966) who coined the term, ''torsade de pointes" (TdeP).  Agents  which  prolong  repolarization  have  been  reported to cause t h i s arrhythmia, which can be suppressed by overdrive pacing and i s favoured by hypokalemia. see:  Strattmann  & Kennedy,  1987;  Jackman et al.,  (Review 1988;  51  Surawicz,  1987;  1989;  induction  of  important  factor influencing the  this  Fish  arrhythmia  &  Roden,  1989).  i s perhaps  the  The  unclear,  investigators  some  afterdepolarizations al.,  1988).  single most  development of Class III  antiarrhythmics. but  mechanisms  underlying  have  (Brachmann et al.,  1983;  to  are early  E l - S h e r i f et  Other factors such as bradycardia, hypokalemia  1984;  assessing  TdeP  pointed  and/or hypomagnesemia have also been considered al.,  Possible  Surawicz,  1987).  One  of  the  (McKibbin et  difficulties  a drug's p o t e n t i a l to cause TdeP i s a  in  lack  of  appropriate animal models for TdeP (Nayebpour et al., 1989).  Recently, (Carlsson  et  adrenoceptor  a rabbit model of TdeP has al.,  1990).  agonist  In  methoxamine  this (15  been developed  model ug/kg/min  infused together with various Class III agents. speculated  that  the  increased  concomitant  infusions of  mediated  increases  phosphatidylinosital greater  to  The  of  (Berridge, EADS.  was  authors  TdeP  with  from  via  + +  turnover,  i.v.)  resulted  [Ca ]i,  alpha^  alpha  increased  1984)  and  However,  thus recent  of alpha^ adrenoceptor mediated i n h i b i t i o n  I-to (Tohse, 1988; provide  in  susceptibility  observations  incidence  methoxamine  the  an  Tohse et al.,  alternate  1990,  mechanism.  Ravens et al. ,  The  largest  time  of  1989) and  voltage dependent outward current i n rabbit v e n t r i c l e i s I^o (Imaizumi & G i l e s , 1987; also  have  a  greater  Hiraoka  than  & Kawano, 1986).  average  density  of  Rabbits myocardial  52  alpha-adrenoceptors  (Mukherjee  et  al. , 1983).  Thus, i f  methoxamine were to i n h i b i t Ito» t h i s would cause additional widening of the action potential to that attributed to class III actions. needed  This would explain why 50 x less c l o f i l i u m was  to  produce  TdeP  during  concomitant  methoxamine  infusion (Carlsson et a l . ,  1990).  Our own r a t studies have  shown that the proarrhythmic actions of tedisamil depend on an i n t a c t  autonomic nervous system  Elevations  of  augment K  currents,  Hiraoka  +  &  intracellular  Kawano,  (Callewaert  [Ca ] ++  such as I to 1989;  et al.,  et I  and  have  been  1989).  reported  (Tseng & Hoffman,  Albitz  1986),  (Howard et al.,  K  to  1989;  a l . , 1988),  iR(Ca)  (Tohse et al.,  1987).  Thus, i f methoxamine i n fact d i d cause a marked increase i n [ C a ] i , the e f f e c t s on K + +  of  the  III  Class  +  channels would oppose the e f f e c t s  agents.  Furthermore,  the  dose  of  methoxamine used would not appear to have been high enough to disrupt  Ca  handling  + +  (arterial  pressure was  minimally  affected) to the extent necessary f o r production of EADS as suggested by Carlsson et al., (1990).  Phenylephrine (10~ -  10~ M)  acetylstrophanthidin  5  has  induction  been of  found  transient  to  inhibit  inward  7  current  reputed  to  be  responsible f o r o s c i l l a t o r y a f t e r potentials (OAP) i n rabbit Purkinje f i b r e s (Ferrier & Carmeliet, 1990).  However, these  authors d i d f i n d alpha stimulation to increase OAPs induced by 8mM is  [Ca ] « ++  0  In the absence of [ C a ] i measurements i t  hard to reach a  induction  of torsade  + +  firm  conclusion  de pointes  on  the mechanism of  by methoxamine/Class  III  53  combination. research.  I t would be an i n t e r e s t i n g area f o r further  Since drug treatment may  be associated with side  e f f e c t s , other therapies have been developed.  1.4  In  Non Pharmacological Interventions  the  last  30 years c a r d i o l o g i s t s  have come from  a  p o s i t i o n of recommending bed rest a f t e r a cardiac arrest to an aggressive approach to the treatment of rhythm disorders. In  1958,  complete  the f i r s t pacemaker was AV  block, and  Z o l l ' s demonstration by e l e c t r i c  implanted f o r treatment of  f o r years Mirowski,  (Zoll et al.,  shock i n man,  1956)  spurred on  by  of d e f i b r i l l a t i o n  struggled to develop a r e l i a b l e  automatic implantable d e f i b r i l l a t o r (Mirowski et al., 1980). He  succeeded  1980  i n implanting the f i r s t  (Mirowski  approve  these  et al. , devices  devices  offer  telemetry,  extended  one  1980b), although until  1985.  i n a patient i n the  Presently  multi-programability, battery l i f e ,  FDA  data  did not available  microcircuitry,  storage  capability  and more physiological control of the heart (Dreifus, 1989). Transvenous electrodes are simplifying the surgery necessary for  implantation  limitations their  cost;  (Campbell,  to the use some  1990).  One  of pacemaker and  $40,000.00  Downar, personal communication).  in total  of  the  major  cardioverters i s  i n Canada  (Eugene  These units are apparently  e f f e c t i v e , with sudden death rates of 1% and t o t a l mortality of  4% i n one year (Winkle et al., 1989)  while the r i s k f o r  54  cardiac death i n the year following MI i s from 8-15% (Bigger et al., 1984).  Perhaps effectiveness has been i n f l a t e d by  counting every non f a t a l shock as successful d e f i b r i l l a t i o n , as devices have been known t o f i r e inappropriately.  Progress has also been made i n suppressing arrhythmias with  catheter ablative  techniques  (Fontaine, 1987).  The  methods of ablation can be chosen from DC shock fulguration, radio  frequency  radiation.  fulguration,  cryomodification and  A common use of t h i s  technique  laser  i s t o ablate  accessory pathways responsible f o r AVN reentrant tachycardia i n Wolff-Parkinson-White  Syndrome.  The obvious benefit of  catheter ablation i s that a cure i s possible f o r an a l b e i t l i m i t e d number of arrhythmias.  Surgical ablation whether by  catheter route or more c l a s s i c a l  techniques when combined  with endocardial/epicardial mapping have been  successfully  used i n patients (Camm & Davies, 1989).  1.5 Rationale  The  o v e r a l l goal underlying studies i n the laboratory  i s t o define the pharmacological c h a r a c t e r i s t i c s required of the ideal a n t i f i b r i l l a t o r y drug i n the setting of myocardial ischaemia. class  In keeping  I I I drugs  with t h i s ,  we tested  for antifibrillatory  new putative  activity  against  arrhythmias induced by myocardial ischaemia.  In addition we  sought,  determine  at  a  whole  animal  level,  to  the  55  antiarrhythmic  mechanisms involved by means of e l e c t r i c a l  stimulation protocols and i n t r a c e l l u l a r recording in ECG  and  haemodynamic  data  were  characterize the pharmacological  collected  profile  to  vivo.  further  of each drug.  A  number of d i f f e r e n t species were used since the occurrence of  different  (Carmeliet,  K  channels  +  1977;  Pelzer  i s highly &  species  dependent  1987).  For the  Trautwein,  purposes of the study, Class I I I antiarrhythmic agents were classified  as  those  drugs  which  selectively  widen  the  v e n t r i c u l a r action p o t e n t i a l (Vaughan-Williams, 1970) and as a  result  increase  Q-T  c  interval  of the surface  ECG and  prolong v e n t r i c u l a r refractory periods.  1.6  Experimental  In keeping with  Plan  our rationale, the following studies  were performed with a s e l e c t i o n of new K  +  channel blockers  as well as with reference drugs:  1) Assays of antiarrhythmic e f f i c a c y against induced  arrhythmias  i n rats  ischaemia-  (tedisamil, KC8851,  RP62,719, r i s o t i l i d e and glibenclamide)  UK68,798,  and pigs (tedisamil  and UK68,798).  2a) Determination KC8851,  UK68,798,  mexiletine,  and  of ECG e f f e c t s RP62,719,  propafenone),  i n rats (tedisamil,  risotilide, guinea  pigs  quinidine, (tedisamil,  56  UK68,798), and baboons  (tedisamil,  UK68,798,  RP62,719  and  risotilide).  2b)  Determination of possible mechanisms of underlying  antiarrhythmic e f f i c a c y by means of e l e c t r i c a l induction of fibrilloflutter propafenone,  i n rats, UK68,798),  (tedisamil, quinidine, and  guinea  pigs,  mexiletine, (tedisamil,  UK68,798).  2c)  Determination  through analysis by  use  of  (tedisamil, guinea pigs,  2d)  of possible  mechanism  of  action,  of e f f e c t s on refractory periods assessed  electrical quinidine,  stimulation mexiletine,  protocols  in  rats,  propafenone, UK68,798),  (tedisamil, UK68,798) and baboons, (tedisamil).  Determination  of  possible  mechanism  of  action  through analysis of e f f e c t s on v e n t r i c u l a r e p i c a r d i a l action p o t e n t i a l morphology i n rats guinea pigs  (tedisamil  and UK68,798) and  (tedisamil and UK68,798).  Attempts were made to explain these drug e f f e c t s on the basis of underlying electrophysiological actions on membrane currents,  as reported i n the l i t e r a t u r e .  57  2 2.1  The  METHODS Pharmacology  structural  formulae of the new compounds used i n  our studies were: tedisamil,  3,7-di-(cyclopropylmethyl)-9,9-  tetramethylene-3,7-diazabicyclo[3,3,1]nonane dihydrochloride;  KC8851,  3,7-di(isobutyl)-9,9-  pentamethylene-3,7-diazabicyclo  [3,3,1]nonane  dihydrochloride;  risotilide,  N-(1-methylethyl)-N-(2-(Cl-  methylethyl) amino) ethyl)-4-((methylsulfonyl)amino)benzenesulfonamide  hydrochloride;  UK68,798,  l-(4-  methanesulfonamidophenoxy)-2-(N-(4methanesulfonamidophenethyl)-N-methylamino)ethane; RP62719  is a  benzopyran  formula  not a v a i l a b l e ) .  derivative  (complete  The structures  KC8851 are based upon sparteine, while are  derived  from  sotalol,  and structural  of tedisamil and  the other  as discussed  earlier  compounds (Section  1.3.1.)•  Tedisamil  Risotilide:  UK68,798:  H3C-S02-fH  oo  N -  58  2.1.1  Cardiovascular Assessment  Dose response studies were performed to evaluate the blood pressure, heart rate, and ECG responses to the various drugs. to  In some instances data from these studies was used  establish  the dose  range  to be  used  i n subsequent  antiarrhythmic and/or electrophysiological assays.  Section 2:1:1:1 Cardiovascular Assessment i n Rats  The t o x i c i t y and e f f e c t i v e dose range of tedisamil i n rats was investigated previously using a continuous infusion protocol autonomic  (Howard  et a l . , 1989).  nervous  system  In t h i s  interventions  study were  various used  to  elucidate the nature of adverse e f f e c t s associated with high doses of tedisamil. doses  Proarrhythmic e f f e c t s were also seen at  above 15 mg/kg.  From these studies we decided to  perform the remainder of investigations with tedisamil i n the 0 - 8 mg/kg range. from  conscious  studies.  rats  ECG, HR and BP data were obtained  used  f o r coronary  artery  ligation  The i n s e r t i o n of ECG leads and cannulae was done  under halothane (1%) anaesthesia one week before the t e s t as previously abdominal floating  described aorta  (Curtis,  and vena  cannulae.  polyethylene tubing  These  1986).  Basically,  the  cava were cannulated with  free  cannulae  were  fashioned  from  (PE10 and PE50) as described o r i g i n a l l y  59  by Weeks (Weeks and Jones, 1960; Weeks, 1981) and i n replete d e t a i l by C u r t i s (1986).  The d i s t a l  ends of the cannulae  were e x t e r i o r i z e d at the mid-scapular monitoring  of conscious  freely  region,  facilitating  moving animals.  The ECG  leads were made from Teflon coated s t a i n l e s s s t e e l wire and inserted  in a  (Johnston also  V3  configuration as  et al., 1983; Curtis,  exteriorized  i n the  pressure was recorded  mid  described  1986).  previously  These leads were  scapular  region.  on a Grass Polygraph  Blood  chart recorder  (Series VII) u t i l i z i n g a Statham pressure transducer. ECG was recorded on the Grass Polygraph on an o s c i l l o s c o p e (Honeywell,  All  speed  and also displayed  Model E f o r M).  drugs used were infused over  vena caval cannula.  The  Recordings  10 minutes v i a the  were measured at a chart  of 100 mm/sec 4 minutes a f t e r  the infusion  ended.  Tedisamil was tested at three separate doses of 1,2 and 4 mg/kg.  KC8851 was tested at 4 mg/kg.  were tested at 1 mg/kg, and f i n a l l y , at  5 and 10 mg/kg.  RP62719 and UK68798 r i s o t i l i d e was tested  These doses were chosen to represent  supramaximally e f f e c t i v e doses i n other al.,  1989;  Colatsky  et  al. ,  1989;  species Cavero  (Gwilt et personal  communication).  2.1.1.2  Cardiovascular Assessment i n Guinea Pigs  60  Guinea pigs were anaesthetized with urethane and  their  jugular vein and l e f t  with polyethylene tubing  c a r o t i d artery  (PE50).  Blood  (1.5 g/kg) cannulated  pressure  and ECG  (lead II configuration) were recorded on the Grass Polygraph as  for rats.  Only  tedisamil  examined i n t h i s species.  and UK68798  (n =  6) were  The doses of tedisamil were 0.5,  0.5, 1, 2, 4 mg/kg i . v . given consecutively every 15 minutes as a bolus, i . e . cumulative doses of 0.5, 1.0, 2.0, 4.0, and 8.0 mg/kg.  ECG and blood pressure responses were recorded  10 minutes a f t e r dosing. 10,  The doses of UK68,798 tested were  10, 20, 40 and 80 ug/kg i . v . given consecutively as  above.  2.1.1.3  Cardiovascular Assessment i n Pigs  Yorkshire  weanling  pigs  determine the ECG, BP and l e f t ventricular  pressure  dP/dt) i n response  (17 - 25 kg) were used  to  (and r i g h t f o r UK68,798 only)  (plus rate of pressure  to tedisamil  development,  and UK68,798.  Pigs were  anaesthetized with 30 mg/kg i . p . pentobarbitone.  A marginal  ear vein was cannulated with a 23 gauge (Jelco , butterfly) needle  and additional  surgical  pentobarbitone  anaesthesia, then  infusion was commenced.  was given to achieve  a 30 mg/kg/hour  pentobarbitone  The femoral vein and artery were  then cannulated with a polyethylene tubing Swan-Ganz catheter, respectively.  (PE 160) and a  The a r t e r i a l catheter was  advanced u n t i l i t was positioned i n the thoracic aorta.  A  61  tracheotomy was was  performed  inserted.  The  and a p e d i a t r i c endotracheal tube  pig was  then  artificially  ventilated  (Palmer  V e n t i l a t o r Pump) with O 2 at 20 cycles/min, stroke  volume  10  ml/kg.  A  saline  infusion  i n i t i a t e d v i a the femoral cannula. also performed  (0.5  1/hour)  was  A midline sternotomy  was  to permit placement of a loose LAD  occluder f o r subsequent antiarrhythmic assay.  coronary  Leads I, I I ,  and III were recorded using subcutaneous needle electrodes. In pigs treated with tedisamil, a 14 gauge J e l c o  R  catheter  heart  was  inserted  thoracotomy.  into  the  apex  (0 s i l k ) .  the l e f t v e n t r i c l e was  In l a t e r studies with UK  was  after  difference  68,798,  catheterized v i a the c a r o t i d  with the a i d of a J wire locator.  placement.  the  The catheter was held i n p o s i t i o n with a purse  s t r i n g suture  pressure  of  atraumatic  was  taken  The  as  artery  diastolic/systolic  evidence  of  correct  In experiments with UK68,798 the r i g h t v e n t r i c l e  also catheterized v i a the jugular vein - superior vena  cava  - auricle  Grass  route.  Polygraph  (Honeywell visual  (8 channel,  Model  assessment  ventricular  E  f o r M) of  pressures  Polygraph.  A l l ECG  ECG were  Ventricular  leads were recorded  Series VII); an was  also  effects. also  used Blood  recorded  pressures  were  on  a  oscilloscope  for  continuous  pressure on  the  and Grass  electronically  d i f f e r e n t i a t e d (dP/dt) and recorded on the chart recorder.  Tedisamil was 0.5,  0.5,  1,  given i n a cumulative manner i n doses of  2, and  4 mg/kg i . v . at 15 minute  intervals.  62  UK68798 was given i n consecutive doses of 2.5, 5, 10, 20, 40 and 80 ug/kg i . v . at a dosing i n t e r v a l of 5 minutes.  2.1.1.4  The  Cardiovascular Assessment i n Primates  effects  of  tedisamil  (and  to  a  lesser  extent  UK68,798, RP62719, and r i s o t i l i d e ) were investigated i n male and female baboons (Papio anubis) and male and female p i g tailed  monkeys  (Macaque  University of Singapore. the  performance  fascicularis)  at  the  National  I did not p a r t i c i p a t e d i r e c t l y i n  of these  experiments.  These  experiments  were c a r r i e d out by my supervisor, Dr. Michael J . A. Walker and  Dr.  Ganesan  planning Since  and  the  data  results  Adiakan.  However,  analysis of  of  these  the  I  participated  experimental  studies are  records.  useful,  j u s t i f i e d i n including some of the relevant data.  in  I  feel  The data  add considerably to the o v e r a l l pharmacological p r o f i l e f o r the new compounds we have investigated.  Preparation of primates  Animals were sedated with ketamine (15 mg/kg, i.m.) anaesthetized with pentobarbitone (15 mg/kg i.m.) h fast.  a f t e r a 12  The brachial vein was cannulated and femoral artery  catheterized  for  respectively. as  and  needed  in  intravenous  access  and  BP  recording  The animals were given extra pentobarbitone order  to  maintain  a  "light"  degree  of  63  anaesthesia, and were allowed to recover a f t e r completion of each dose response  study.  A full  12 lead ECG was recorded  along with blood pressure, heart rate and respiratory rate. The  data  reported  here  were recorded  14 min  after  each  consecutively administered dose; the dosing i n t e r v a l was 15 min.  There were two d i f f e r e n t studies performed i n baboons.  In the f i r s t  study,  the cumulative  doses were 0.05, 0.1,  0.2, 0.4, 0.8, and 1.6 mg/kg tedisamil. given t h i s dose regimen.  Monkeys were also  In the second study the cumulative  doses were 0.5, 1.0, and 2.0 mg/kg, e l e c t r i c a l stimulation of the r i g h t atrium and v e n t r i c l e was also performed i n t h i s group (see Section 2.2.2.3). (0-0.4  mg/kg,  risotilide The  n=2),  In p i l o t experiments UK68,798  RP62,719  (0-0.4  mg/kg,  n=2)  (0-0.3 mg/kg, n=2) were also tested i n baboons.  effects  on the ECG  (QRS,  P-R,  Q-U,  and Q-Uc)  analyzed from recordings made at chart speeds of 25  2.1.2  in  dissolution. dissolved studies  KC8851  and  risotilide  i n a l l experiments.  dissolved  were  mm/s.  Drug and Dose Regimens  Tedisamil, saline  and  acidified  were  UK68798  and  and  warmed  saline  dissolved i n RP62719 to  were  promote  In primate studies, UK68,798 and RP62,719 were  i n ethanol  i n rats  and d i l u t e d  with  saline.  the drugs were administered  b l i n d randomized design.  For the  in a  double  64  2.1.3  Data A c q u i s i t i o n and Analysis  As described above the Grass Polygraph (Series VII) used throughout (except f o r primate studies).  was  ECG i n t e r v a l s  were measured by hand with a micrometer from recordings made at  100  mm/sec  Cardiograph  chart  speed.  In  primate  (Model M1700A Page Writer  traces were recorded at 25 mm/s  XLi)  studies,  was  used,  a and  chart speed, with 3 beats of  each lead configuration for each time period printed.  ECG  i n t e r v a l s were defined as follows: P-R  i n t e r v a l - taken from the foot of the P wave to the  upstroke of the R wave. QRS  -  from  the  first  d e f l e c t i o n of  the  QRS  to i t s  return to the i s o e l e c t r i c . Q-T - from the s t a r t of the Q wave to the f i n a l return to the  isoelectric.  Q-T  =  C  Q-T/(R-R) / 1  2  from  Bazett's  formula  (Bazett,  1920) . Q-U  (where appropriate)  from the s t a r t of the Q wave to  the f i n a l r e p o l a r i z a t i o n to i s o e l e c t r i c .  This was  because  often  the  shape  of  tedisamil treatment.  the  T-wave  was  necessary  altered  by  In some cases a U-wave developed, i n  others the T-wave had a biphasic morphology.  Therefore,  the  i n t e r v a l to the f i n a l return to i s o e l e c t r i c , whether from a positive  or  arbitrarily  negative decided,  d e f l e c t i o n was recognizing  the  measured.  This  was  previously  described  65  difficulty  in  accurately  determining  Q-T  intervals  (Surawicz, 1987; 1989).  A Differences  in  L  effects  on  ECG  intervals  between  treatments were examined by analysis of variance, unweighted means ANOVA, and i f s i g n i f i c a n t (p < 0.05), were followed by Duncan's multiple range t e s t (Hintze,  1987).  significantly  As  different  using NCSS computer packages  pre-infusion from  values  each other,  were  not  comparisons were  made between s a l i n e and drug treatment post-infusion data i n rat studies. baboons)  data  For cumulative dosing studies (guinea pigs and were  compared  between  pre-drug  and dosage  level.  2.2  E l e c t r i c a l Stimulation  2.2.1 Introduction  Class Williams,  I I I drugs 1970) as  were agents  initially which  classified  increased  a d d i t i o n a l l y decreasing conduction v e l o c i t y . assess  whether  electrical  drugs  increased  (Vaughan-  APD  without  In order t o  refractoriness,  simple  stimulation protocols were c a r r i e d out in vivo.  66  It has been well established that drugs which decrease gNa , +  increase  thresholds  fibrillation  f o r capture  threshold  of  single  (Wiggers & Wegria,  beats  1940).  and  On the  other hand, pure Class III drugs might not a f f e c t thresholds for  capture,  heart  yet might suppress VF induction by making the  r e f r a c t o r y to the f r a c t i o n a t i n g wavefront  Winslow, 1984).  However, a pure Class  expected t o prolong  the e f f e c t i v e  (Review:  I I I drug would be  r e f r a c t o r y period (ERP)  and decrease the maximum following frequency (MFF) to square wave  stimuli  (Vaughan-Williams,  1970; 1975).  Thus, by  t e s t i n g the drugs f o r t h e i r influence on VF^, ERP, and MFF we hoped to quickly e s t a b l i s h an index of t h e i r Class I vs. Class III actions. the  effects  on  Simple comparisons could also be made of electrical  stimulation  variables  with  antiarrhythmic e f f e c t s for a given l e v e l of ECG change (e.g. increase i n Q-T ). c  2.2.2  Preparation  I n i t i a l l y we attempted t o develop a conscious rat model for  electrical  Beatch,  stimulation  1988).  of the v e n t r i c l e  B a s i c a l l y , rats  were  (Walker and  anaesthetized  with  halothane (1%) and free f l o a t i n g cannulae were implanted i n the  inferior  previously. that  a  vena  cava  and abdominal  aorta  as described  The chest was opened and the heart exposed so  specially  loosely sutured  designed  electrode  carriage  could  be  to the anterior a p i c a l region of the l e f t  67  ventricle.  The leads from the carriage assembly was routed  subcutaneously and  and e x t e r i o r i z e d i n the mid scapular region,  the chest  occluders  closed  as i n placement  (section 2.4.2.1).  recover from surgery. three p a r a l l e l  of coronary  artery  Animals were given a week to  The electrode carriage consisted of  11cm lengths  of polyethylene  joined together at one end by melting.  tubing  (PE10)  A p r o l i n e suture was  inserted i n the central tube and s t a i n l e s s s t e e l or s i l v e r wire  electrodes were inserted i n the tubes on e i t h e r side  (see diagram).  Electrode assembly:  Thus, a f t e r anchoring the assembly to the heart, gentle traction  on  the  central  guide  tube  could  electrodes into apposition with the v e n t r i c l e .  bring  the  Stimulation  protocols are described below.  We used an acutely prepared for  a l l other  electrical  anaesthetized animal model  stimulation  experiments.  The  v a r i a b l e s measured were current and pulse width thresholds for  capture  of the v e n t r i c l e  following frequency  on  a  1:1  basis,  maximum  (MFF), E f f e c t i v e r e f r a c t o r y period (ERP)  68  and f i b r i l l o f l u t t e r threshold (VF^)•  These variables  will  be described i n d e t a i l below.  2.2.2.1  Male  Electrical  rats  (250  Stimulation i n Anaesthetized Rats  -  400  g)  anaesthetized with pentobarbitone  Sprague-Dawley,  (45 - 60 mg/kg i.p.) and  intubated with a 14 gauge Teflon catheter. artery was  were  The l e f t c a r o t i d  cannulated with PE50 polythene tubing f o r blood  pressure recording.  The r i g h t jugular vein was  cannulated  i n the same manner f o r the purpose  of drug administration.  The  cut away to  skin  overlying  placement  of  the  the thorax was pacing  electrodes.  The  facilitate stimulating  electrodes were made from Teflon coated s i l v e r wire. mm  segment of i n s u l a t i o n was  removed from the end  A  1-2  of the  wire and the wire was passed through the lumen of a 27 gauge needle.  The  desheathed  t i p of the wire was  bent back to  form a barb and the needle plunged through the chest wall, and the wire deposited into the l e f t v e n t r i c u l a r free wall towards the apex. wire,  2-3  mm  This process was  from the f i r s t one.  repeated with a second This technique  rapid i n s e r t i o n of stimulating electrodes 2-3 mm minimal  trauma  to the  absence of arrhythmias  animal.  allowed  apart with  Stable threshold values,  and/or hypotension  and  post mortem  examination confirmed a correct placement of the electrodes. A lead II or I I I configuration of the ECG was used.  The  ECG  and BP were recorded on a standard Grass Polygraph, and a  69  delayed loop oscilloscope (Honeywell Model E f o r M) was used for continuous  2.2.2.2  Guinea activity  assessment of the ECG.  E l e c t r i c a l Stimulation i n Guinea Pigs  pigs  because  were  also  there  result  potential  to t e s t  are species  response to Class I I I drugs. the  used  of v a r i a t i o n repolarization.  f o r Class I I I  differences  i n the  These species differences are  i n currents While  underlying  the r a t has  action a  large  transient outward current, t h i s current has been reported to be absent i n guinea pigs (Josephson et al., 1984; MacDonald et al., 1984).  Guinea pigs, male Hartley using  the same  prepared was  techniques  (450 - 600g) were  and apparatus  rats with one exception; urethane  used  f o r anaesthesia.  This  i n acutely  (1.5 g/kg i.p.)  anaesthetic  because i t produces a smooth CNS depression without  as  prepared  was  chosen  i n guinea pigs  influencing sympathetic tone.  2.2.2.3  E l e c t r i c a l Stimulation i n baboons  Baboons monitoring sections protocols.  which  were  prepared  for  cardiovascular  (study two, above) as described i n the previous were  also  subjected  to  electrical  stimulation  These experiments were performed i n "recovery"  70  animals and thus f o r v e n t r i c u l a r stimulation an atraumatic electrode was positioned  i n the lower r i g h t v e n t r i c l e v i a  the femoral vein, and the r i g h t atrium was stimulated with an intra-oesophageal electrode inserted o r a l l y .  Threshold  pulse width and threshold voltage were estimated at f i f t e e n minute  intervals  determine  MFF.  and  twice  these  thresholds  used  to  Doses of 0.5, 1.0 and 2.0 mg/kg tedisamil  were assessed.  2.2.3  For  Data A c q u i s i t i o n  rats  and guinea  protocols were used. to  establish  pigs  equivalent  equipment and  A constant current stimulator was used  thresholds and estimate  Refractory periods were determined  refractory  periods.  by a single extra pulse  technique at a basic stimulation frequency of 7.5 Hz (rats) or  6.5 Hz  using  (guinea pigs).  square  waves.  A l l determinations were made  Each end point  (MFF, ERP, VF-^) was  determined i n t r i p l i c a t e 10 minutes a f t e r each dose of drug. The ECG and BP were recorded on a polygraph chart-recorder (Grass, Series VII).  2.2.3.1  Thresholds f o r capture  The current necessary f o r capture of the v e n t r i c l e on a 1:1 basis was determined at the stimulation frequency of 7.5 Hz f o r rats, and 6.5 Hz f o r guinea pigs, and a pulse width  71  of  1 ms.  The minimum  current  necessary  capture was taken as the threshold current.  to obtain  1:1  The pulse width  threshold f o r capture was determined at the same frequency of  stimulation  (rats 7.5 Hz, guinea pigs  twice the current threshold.  6.5 Hz) and at  Average threshold pulse widths  were 0.3 ms using t h i s method.  2.2.3.2  Maximum Following Frequency  This v a r i a b l e i s a measure of the heart's  a b i l i t y to  follow an increasing rate of stimulation on a 1:1 basis. Interventions which increase v e n t r i c u l a r r e f r a c t o r y periods could  be  expected  frequency (MFF). effectiveness. setting their  to  decrease  maximum  following  The MFF i s thus another t e s t for class I I I In normal myocardium, MFF was determined by  the stimulation current respective  increasing  the  (3 Hzsec  threshold  and pulse  values  and  width  at twice  then  smoothly  ) the stimulation frequency from 6 Hz  u n t i l the heart f a i l e d to follow on a 1:1 basis.  This end  point was determined by noting the point at which the blood pressure  (which s t e a d i l y decreases at increasing rates of  stimulation) suddenly r e f l e c t e d a compensatory pause from a non-captured pulse. a  sudden  missed  oscilloscope. (reciprocal  The end point was also e a s i l y seen (as beat)  The estimate of the MFF)  on  the  ECG  displayed  on the  of e f f e c t i v e r e f r a c t o r y periods obtained  using  this  method  may  72  reflect  underlying drug binding k i n e t i c s to sodium channels  (Hondeghem & Katzung, 1984).  2.2.3.3  E f f e c t i v e Refractory Period  Another  method  used  to  gain  an  estimate  of  the  e f f e c t i v e r e f r a c t o r y period i n the v e n t r i c l e was the paired pulse method.  In t h i s method the v e n t r i c l e i s stimulated at  a baseline frequency  (7.5 Hz r a t s ; 6.5 Hz guinea pigs) and a  single extra stimulus width)  i s added  stimuli. beat  (of same current strength and pulse  at a v a r i a b l e delay  from  the entrained  The minimum delay which resulted i n a conducted  was  taken  Comparisons  as  between  method allowed  the  effective  1/MFF  and  ERP  refractory determined  us to ascertain the frequency  period.  with  this  dependency of  r e f r a c t o r i n e s s increase i n the upper range of frequencies.  2.2.3.4  Fibrilloflutter  Threshold  This threshold i s generally accepted vulnerability drugs with  of the v e n t r i c l e  c l a s s I or gNa  Wegria, 1940).  +  as a measure of  and i s normally  reducing  actions  r a i s e d by  (Wiggers and  The end point was determined by increasing  the current strength (at 50 Hz and 0.8 ms duration) u n t i l a fibrilloflutter  was  elicited.  generally non-sustained.  The  tachycardias  were  The high rate of stimulation was  used i n order to increase the p r o b a b i l i t y of an R on T type  73  initiation  of the tachyarrhythmia  (see sections 2.2.1 &  4.2.1).  2.2.4  Drug and Dose Regimens  Tedisamil dissolved baseline either  was  dissolved  in acidified control  values  i n saline.  saline. rats  After  or guinea  UK68798  was  establishment pigs  were  of  given  0.5 mg/kg tedisamil or 5 ug/kg UK68798 as a bolus  i.v. injection.  At 10 minutes a f t e r i n j e c t i o n ,  electrical  stimulation protocols were i n i t i a t e d and t r i p l i c a t e readings obtained.  Fifteen  was given.  minutes a f t e r  i n j e c t i o n the next dose  The cycle was repeated u n t i l the maximum dose  l e v e l was achieved.  For tedisamil the doses were 0.5, 0.5,  1, 2, and 4 mg/kg i . v . given consecutively as indicated; f o r UK68798 the doses were 5, 5, 10, 20, 40, 80 ug/kg i . v . given as above.  The exact same protocol was used  guinea pigs.  f o r rats and  Six animals per group were used f o r each drug.  Primates however received tedisamil 0.5, 0.5, & 1.0 mg/kg, i.v.  given  every  15 minutes  as described above  (section  2.1.1.4).  2.2.5  ECG  Data Analysis  and  blood  pressure  records  were  examined  at  baseline and 10 minutes a f t e r each dose f o r r a t and guinea pig studies.  ECG intervals were measured manually using the  74  same  conventions  as  described  previously.  Electrical  stimulation end points determined between 10 and 15 minutes a f t e r dosing were compared to baseline values using analysis of variance, ANOVA followed by Duncan's multiple range t e s t (NCSS package, Hintze, 1987).  Repeated measures ANOVA was  also done to show trends.  2.3  E l e c t r o p h y s i o l o g i c a l Analysis  2.3.1  Introduction  In order  to examine drug e f f e c t s on action p o t e n t i a l  morphology and upstroke were made from intact  velocity,  intracellular  the v e n t r i c u l a r e p i c a r d i a l  rats and guinea pigs.  With t h i s  recordings  cell  layer i n  technique  i t was  possible to show the e f f e c t s of the drugs at s i m i l a r doses used i n other i n vivo technique  with  studies.  An i n t r a c e l l u l a r  free f l o a t i n g microelectrodes,  order to obtain accurate measurements in vivo.  recording  was used i n The f l e x i b l e  t i p method we used was modified from the method of Woodbury & Brady, (1956) .  2.3.2  Tn vivo  preparation  The technique consists of the following: A  rat  pentobarbitone  or  guinea  pig  was  anaesthetized  45 mg/kg i . p . or urethane  1.5  with  g/kg i . p . ,  respectively, and intubated with a 14 gauge Teflon Jelco .  75  The  animal  was  artificially  ventilated  with  cycles/minute, 10 ml/kg stroke volume).  O2  100%  (60  The r i g h t jugular  vein and l e f t c a r o t i d artery were cannulated with polythene tubing (PE50) as described previously (Section 2.2.2.1). bipolar oesophageal oesophagus  or  heating  lamp  ECG lead was appropriately placed i n the  alternatively  configuration,  was  used.  was  A  inserted  a  surface  into  the  the thorax was  The l e f t  over-lying  muscles  retracting  the  retractors.  A  electrode was  delicately  heart  was  moistened  to the  Grass  +  4; lactate 28).  left  continuously  Polygraph  and  The ECG  was  loop  ventricular  a simple s i l v e r wire wick  l a c t a t e solution (composition i n mM: 109; K  rectus)  reference  electrode was positioned against the v e n t r i c l e . the  to  s p e c i a l l y tooled  chloride  sutured  a  removed and  A pericardial sling  surface or, i n l a t e r experiments,  of  to  order  abdominus  r i b cage with  silver/silver  II  The skin covering  and  positioned away from the opening. made a f t e r  in  4th r i b was  {pectoralis  lead  connected  rectum  at 37 ± 1° C.  cut away.  ECG,  thermocouple  maintain body temperature  the  A  Na  +  The surface  with  Ringer's  130; C a  1.5; C l ~  + +  and BP were recorded on a  on video tape  (NEC  VCR  DX1000C) a f t e r  analogue to d i g i t a l conversion with a pulse code modulator (initially  Sony PCM,  Intracellular  electrodes  borate glass (WPI, electrode p u l l e r resistance  l a t e r Medical Systems Corp. PCM  10-20  were  pulled  1BBL W/FIL 1.0 mm,  from  fibre  4/8) . filled  4 i n ) , using a pipette  (Narashige PaOl, set at magnet 7, heat 9), MOhms,  filled  with  filtered  (0.45  um,  76  Millex)  3 M KC1 and a coated tungsten wire  (0.002 i n Am  systems Inc.) back inserted down the barrel of the pipette. The t i p (1 - 1.5 cm) of the pipette was broken o f f leaving the wire attached.  The wire was bent at r i g h t angles 2 cm  from the pipette t i p and then cut 2 cm d i s t a l to t h i s bend. The wire was clipped to a connector which was inserted into the amplifier The  (WPI instruments Model 750 Dual micro-probe).  connector  was  positioned  using  micromanipulators  (Narashige #2749), allowing the electrode to be dropped onto the v e n t r i c u l a r surface.  The i n t r a c e l l u l a r potentials were  d i g i t a l l y recorded on video tape a f t e r pulse code modulation of  the amplified  signals.  Transmembrane potentials  were  also d i f f e r e n t i a t e d e l e c t r o n i c a l l y and d i g i t a l l y recorded on video  tape.  The quality  of the impalement  was  easily  monitored by an on-line oscilloscope (Gould Advance OS250B) which displayed both the DC potential and i t s d i f f e r e n t i a l . Stable recordings could often be maintained f o r 15 minutes using t h i s technique. the  quality  potential  of  I t was r e l a t i v e l y easy to determine  impalement  (Em), the r i s e  morphology.  by rate  (dV/dt),  the  and  a  resting "clean"  Unsatisfactory impalements (dV/dt < 100 V/s, Em  < -70 mV) were not analyzed.  Resting potentials could be  ascertained by the voltage s h i f t electrode  determining  passed  from  i n t r a c e l l u l a r milieu.  the extra  of the baseline when the cellular  space  to the  A continuous spoken record was also  made d i r e c t l y on the video tape to l o g the progress of the experiment.  77  2.3.3  Drugs and Dose regimens  Tedisamil  (dissolved  i n saline)  was given  as bolus  i n j e c t i o n s i . v . at doses of 0.5, 0.5, 1, 2, and 4 mg/kg to rats or guinea pigs, n = 4-6 per group. in  acidified  saline was given  UK68798 dissolved  as i . v . bolus at doses of  12.5, 12.5, 25, 50, 250, 1000 ug/kg t o rats and guinea pigs. The dosing i n t e r v a l was 15 minutes between each dose given consecutively.  Multiple  impalements  were made with the  microelectrode throughout the experiment, but when possible, a  single  cell  was  held  following 2 minutes.  during  drug  injection  and the  Recordings were made from the lower  (apical) h a l f of the l e f t v e n t r i c u l a r surface.  2.3.4  Data Analysis  The DC recordings were analyzed from the video tape by feeding the signal  back through the A/D converter  storage oscilloscope (Tektronix type 549). was  calibrated  into a  The oscilloscope  from a 200 V/s sawtooth c a l i b r a t i o n  signal  recorded at the beginning of each tape using a saw tooth wave generator.  Measurements were made at 10, 25, 50 and  75% r e p o l a r i z a t i o n of the AP. were also measured.  The AP height and r i s e rate  In addition the slope of the plateau  (phase 2) and l a t e r e p o l a r i z a t i o n phase calculated  manually.  The d i g i t a l l y  (phase 3) were also recorded  ECG  & BP  78  records  were  played  back  through  the Grass  Polygraph  allowing f a s t trace records to be replayed and made at any time.  Values were measured at 1, 2, 5, 10 and 15 minutes  a f t e r each dose. Data followed  were by  analyzed  Duncan's  by analysis of variance Multiple  Range  test  (ANOVA)  f o r means.  Repeated measures ANOVA was used to detect trends.  2.4  Myocardial Ischaemia - Induced Arrhythmias:  2.4.1  The  Introduction  use of animal models f o r assessing  antiarrhythmic  drug e f f i c a c y has been a major contributor t o the s e l e c t i o n of p o t e n t i a l l y useful drugs. ischaemia  The production  of myocardial  by l i g a t i o n of one or more coronary  arteries i n  numerous species has been used by numerous investigators. No s i n g l e species or model of acute ischaemia has proven to be i d e a l . aim  Recently, guidelines have been published with the  of standardizing  comparisons  procedures  can be made  so that interlaboratory  (Walker  et al., 1988).  These  "Lambeth Conventions" were followed i n our studies using the rat. model  Recently the rat has been endorsed as an appropriate for  independent  testing  antiarrhythmic  laboratories  Curtis et al.,  (Winslow,  1987; Brooks et al.,  drugs 1984,  1989).  by Curtis,  several 1986;  One of the main  advantages to the use of the rat i s that the species has few c o l l a t e r a l a r t e r i e s i n i t s coronary  vasculature  (Johns and  79  Olsen,  1954;  Maxwell  consistently  sized  ligations  the  of  artery.  The  et  al.,  occluded  left  1984:  zones  anterior  1987)  such  be  produced  can  descending  (LAD)  that by  coronary  s i z e of the occluded zone corresponds well  the i n t e n s i t y of the arrhythmogenic stimulus (Austin et 1982;  Bernauer, 1982;  Curtis et al.,  1987), thus low  to al.,  inter-  animal v a r i a b i l i t y i s desirable for multi treatment t r i a l s . As  discussed  in  the  Introduction,  fundamental electrophysiological of other mammalian species, negligible outward  1^  and  repolarization  differences  i n that  a uniquely large  current,  Ito*  which  the  ventricular  1984) .  Thus the  rat  has  feature  renders  the  which i n h i b i t Ito inhibit  I R or  heavily  on  ideal  to  short  more  component of  AP  I R I than the  (Josephson  sensitive  to  currents  for AP  has  transient to  ventricular  guinea pig  has  rat v e n t r i c l e  rapid et  al. ,  AP.  This  interventions  less s e n s i t i v e to interventions  these l a t t e r  its ventricle. it  a n d  rat  the  rat  from a number  contributes  of  a  the  which  which r e l i e s more repolarization  in  However, t h i s feature of the r a t also makes study  class  III  antiarrhythmic  mechanisms  because i t s v e n t r i c u l a r AP can be widened to such an extent (5 fold) by Ito blockade (Dukes et al., 1990; 1990).  Therefore,  the  degree  of  widening  Beatch et necessary  al., to  produce antiarrhythmic a c t i v i t y i n t h i s model can be r e a d i l y obtained empirically.  2.4.2  Preparation  80  The  preparation  commonly  used  for  production  ischaemia consists of simple l i g a t i o n of the l e f t descending  coronary artery  of  anterior  (Review: Botting et a l . , 1986).  There are a number of variations to t h i s basic theme.  For  instance the c l a s s i c Harris (1950) two stage l i g a t i o n model in  dogs  is  ischaemia  used  to  produced  test  by  for  ligation  arrhythmias of  arising  from  a previously p a r t i a l l y  l i g a t e d artery (done to prevent f a t a l VF).  Acutely prepared  rats have been widely used to t e s t the e f f e c t s of drugs on permanent occlusions of the LAD et al., 1960; is  briefly  Curtis et a l . 1987).  exteriorized  s i l k l i g a t u r e may of  the  Although  Selye  In t h i s model the heart  a f t e r thoracotomy i n order that a  be placed around the LAD.  stabilization,  monitored.  (Johns & Olson, 1954;  ligature  i s tied,  A f t e r a period and  the  animal  there are numerous ways of occluding  the artery i . e . , glass bead i n j e c t i o n (Wilkerson and Downey, 1978), sutures (Johnston et a l . , 1935), e l e c t r i c a l l y induced thrombogenesis "ameroid  (Salazar,  1961;  Patterson  et  downstream from been  resulted  the occlusion.  studied  pharmacological has  and  using  The  or  sequelae  biochemical,  electrophysiological  in identification  ischaemia,  of occlusion biophysical,  techniques,  of potential  Sobel et al., Janse  1978; et  al.,  H i l l and Gettes, 1980; 1981;  Coker  et  which  mediators  arrhythmias and cardiac dysfunction (Dennis and Moore,  1980;  1981),  c u f f " occluders etc. the basic outcome desired i s  the production of an area of hypoperfusion,  have  al.,  of  1938;  Hirche et al.,  a l . , 1981).  These  81  p o t e n t i a l mediators have then been used to mimic ischaemia i n non-ischaemic tissue i n order to determine  arrhythmogenic  mechanisms or to assay pharmacological interventions (Harris et a l . , 1958; Ettinger et a l . , 1973; F e r r i e r et a l . , There  1985).  i s no ideal model f o r predicting the e f f i c a c y of an  intervention  f o r transfer to c l i n i c a l  al.,  Recently, a paradigm  1988).  practice  (Walker et  f o r examining  arrhythmia  mechanisms has evolved out of chaos theory (Guevara et a l . , 1981;  Winfree,  1987;  Chialvo and  Jalife,  1987).  These  studies suggest that long range predictions of the e f f i c a c y of  antiarrhythmic  arrhythmia  interventions  occurrence  conforms  nonlinear dynamic systems (May,  2.4.2.1  The  may  be  to  impossible, i f  the  rules  1976).  Conscious rats  conscious  r a t model  used  i n these  in this  laboratory  from  a  developed  governing  useful drug assay  (Johnston et al.,  desire  1983).  studies to  was  create a  Over the past  decade our laboratory has produced a large data base of the effects  of  interventions  on  arrhythmias using t h i s model. has  recently  been reviewed  myocardial  ischaemia  induced  The examination of t h i s model (Curtis,  et al.,  1986).  The  model as described below was used to evaluate the e f f e c t s of compounds with reported Class III antiarrythmic actions.  82  Basically,  as described  previously  (Johnston et al.,  1983, Curtis, 1986), male Sprague-Dawley rats were anaesthetized with halothane Teflon 14 gauge J e l c o  R  (250 - 450 g)  (1%), intubated with a  catheter and v e n t i l a t e d on 100% O 2 ,  (10 ml/kg, 60 cycles/min) to keep blood gases i n the normal range (MacLean & Hiley, 1988). to  obtain  a  lead  II  ECG  Needle electrodes were used which  was  displayed  on  an  oscilloscope (Honeywell, Model E f o r M) f o r the purposes of monitoring the animal during surgery. were cleaned and soaked  i n 70%  floating  disinfected  ethanol.  with A35  After  Surgical instruments detergent and  then  midline laparotomy,  free  cannulae, made from polyethylene tubing (Weeks &  Jones, 1960; Weeks, 1981; Curtis, 1986) were inserted i n the abdominal aorta and i n f e r i o r vena cava i n close proximity to the renal artery branches.  The d i s t a l ends of the cannulae  were e x t e r i o r i z e d i n the mid-scapular region with the a i d of a trocar.  The  cannulae were flushed with s a l i n e  ends heat sealed.  and  the  The abdomen was closed with s i l k sutures.  In some cases the blood pressure l i n e was  connected to the  transducer and a recording was made on the Grass Polygraph during surgery.  The chest  was  then opened at the fourth  i n t e r c o s t a l space, a p e r i c a r d i a l s l i n g was made, and a loose coronary closed  occluder placed around  with  stainless intercostal  a silk  steel space  purse  electrode  string wire  (approximate V3  the occluder guide and  ECG  lead  the LAD.  The  chest  suture, a Teflon implanted  over  configuration), exteriorized  was  coated  the  third  and both  at the  mid-  83  scapular region. negative  The chest was closed with s i l k sutures and  pressure  applied t o prevent  wound s i t e s were i n f i l t r a t e d  with Marcaine  with C i c a t r i n .  Reference  were  subcutaneously  implanted  forelimbs  and  exteriorized then  on  hindlimb,  t o recover  All  and sprinkled  R  s t a i n l e s s s t e e l wire ECG leads  at the mid-scapular  allowed  tube.  left  pneumothorax.  after  the  left  connected region. removal  and  right  together  and  The animal was  of the intubation  Total surgical preparation took about 40 minutes i n  most cases. before  The animal was allowed to recover f o r one week  use.  During  this  time  they  had access  (Purina Rat Chow) and water ad libitum.  to food  On the day of the  experiment the conscious animal was weighed and i t s cannulae and leads connected to the Grass polygraph, oscilloscope and drug infusion pump as appropriate. to s t a b i l i z e drug  f o r at least  15 minutes before a ten minute  infusion was i n i t i a t e d .  drawn j u s t with K  +  prior  sensitive  The animal was allowed  A blood sample  to infusion  and serum  electrodes (Ionetics  (0.2 ml) was  [K ] was measured +  Instruments).  The  LAD l i g a t u r e was tightened 5 minutes a f t e r the infusion was complete. blind  Drug treatments  were administered  randomized design as described l a t e r .  ligation  were  monitored  disconnecting the animal.  for a  four  hour  in a  double  Responses to period  before  A repeat drug dose was given as a  30 minute infusion, commencing 1.5 hours a f t e r occlusion i n order drugs.  to compensate Arrhythmias  f o r metabolism during  and excretion of the  the monitoring  period  were  84  diagnosed from the oscilloscope screen and noted d i r e c t l y on the chart recording f o r l a t e r analysis, as described below. Twenty-four  hours  and monitored  after  occlusion the r a t was  f o r a further  30 minutes.  reconnected  In the case of  tedisamil, fourteen of these rats with one day o l d i n f a r c t s were  further  monitoring  analyzed  period  (section  (or as  soon  2.4.2.2.). as  the  After  animal  this  died) ,  the  animal was s a c r i f i c e d and the heart removed and retrogradely perfused (Langendorff mode) i n order to assess the occluded zone (O.Z.), s i z e by cardiac green dye exclusion.  The  O.Z.  was defined as weight of non-perfused v e n t r i c l e / weight of perfused v e n t r i c l e x 100%.  The animals were examined post  mortem f o r any obvious signs of pathology e.g. scarring and adhesion  of  the  myocardium,  pulmonary oedema etc. outlined by Curtis  infarction  of  the  kidneys,  A r i g i d set of exclusion c r i t e r i a as  (Curtis,  1986)  was  adhered to.  In the  event of exclusion of a r a t by these c r i t e r i a the treatment was immediately repeated i n another r a t before continuation. This conscious r a t model of myocardial ischaemia was used to t e s t the e f f i c a c y of tedisamil, KC8851, UK68798, RP62719 and risotilide.  2.4.2.1.1 Infarcted Rats  The proarrhythmic potential of tedisamil was i n rats with previous myocardial i n f a r c t i o n .  explored  These studies  were c a r r i e d out i n 14 rats with a one day o l d i n f a r c t i o n , 5  85  rats with a one week o l d i n f a r c t i o n and 5 rats with a one month  old infarction.  The rats  ischaemia-induced  arrhythmias  2.4.2.1).  was  It  antiarrhythmic induced  arrhythmias,  prepared  i n conscious  initially  activity  were  hoped  of tedisamil however  the  rats to  (section  assay  against low  as f o r  the  infarction-  incidence  of  arrhythmias i n the animals with one week and one month o l d i n f a r c t s precluded such investigations. for  Rats were monitored  a 30 min s t a b i l i z a t i o n period, during which arrhythmia  incidence  (VPBs) was tabulated, then  tedisamil  were given  intervals.  bolus  i n j e c t i o n s of  (1.0, 2.0, 4.0 mg/kg) at 15 minute  Increases  or  decreases  i n VPB  or  other  arrhythmias were recorded along with the BP and ECG i n these conscious c h r o n i c a l l y infarcted rats.  2.4.2.2  Acutely Prepared Anaesthetized Rats  Acutely  prepared,  pentobarbitone  (45  mg/kg)  anaesthetized, rats were used to detect the antiarrhythmic e f f i c a c y of tedisamil and glibenclamide against arrhythmias induced  by  a  reperfusion.  10  minute  ischaemic  period  followed  by  These investigations were part of a larger  study reported previously (Beatch et al., 1989).  The rats  were cannulated f o r drug administration and blood pressure recording 2.2.2.1). catheter  as i n e l e c t r i c a l  stimulation  studies  (section  The rats were intubated with a 14 gauge Teflon and a r t i f i c i a l l y  ventilated  with  room a i r .  The  86  occluder was  loosely placed around the LAD  as f o r c h r o n i c a l l y prepared recorded  to  infusion  was  An ECG  (lead II)  along with BP on a Grass Polygraph.  allowed  sample  animals.  coronary artery  stabilize  for  commenced.  (0.2 ml)  occluded  min  before  a  10  rat  drug  a  blood  drawn to measure serum [ K ] .  The  post  because  +  5 min  was  min  Just p r i o r to drug infusion  was  glibenclamide was  15  The  was  infusion.  Alternatively,  LAD  insoluble i n polar solvents, a s l u r r y of  glibenclamide i n water was administered per os 3 0 min before occlusion. loosened  A f t e r 10 and  min  of  reperfusion  reperfusion the heart was  occlusion, the effected.  occluder  Ten  min  was  after  excised and retrogradely perfused  (Langendorff mode) with saline to v e r i f y reperfusion and to measure the occluded Eleven  zone a f t e r retightening the occluder.  rats were occluded  treatment  and  (4mg/kg) and  7  after  reperfused,  glibenclamide  5  a l l 5 reperfused.  treated  with  (10 mg/kg) tedisamil  There were seven control  rats.  2.4.2.3  The  Anaesthetized Pigs  antiarrhythmic e f f i c a c y of tedisamil and UK68,798  was  investigated i n acutely  The  preparation  2.1.1.3.).  of  the  pigs  prepared  i s described  of  pigs.  above (section  The primary aim of the pig studies was to obtain  dose/response r e l a t i o n s h i p s of the effects  anaesthetized  tedisamil  (n=6)  and  cardiovascular and UK68,798  (n=5).  ECG  After  87  carrying ligate  out the  the LAD  cumulative in  these  dose  regimen,  preparations  we  and  decided compare  antiarrhythmic e f f e c t s at the maximal dose with control p i g data.  to the  historical  The number of pigs used i n these studies,  the use of h i s t o r i c a l  controls, the open design and the lack  of antiarrhythmic e f f i c a c y dose/response determinations were recognized  and  thus  determined  with  any  antiarrhythmic e f f i c a c y degree  of  scientific  could not  certainty.  be In  short, these were p i l o t studies.  2.4.3  With  Data Analysis  any  scientific  endeavor,  care must be  both the design and analysis of experiments. Conventions  (Walker  et  al.,  1988)  were  taken i n  The  compiled  Lambeth in  an  attempt to approach these problems i n a systematic manner. In accordance with these recommendations, the antiarrhythmic assays i n conscious rats were carried out i n a double b l i n d and random design, with predetermined and While  exclusion  criteria  (Curtis,  a number of the p i l o t  controlled, we  did attempt  statistical  1986;  Igwemezie,  studies were not  as  to obtain dose/response  analysis 1990). rigidly curves  such that the variance due to drug treatment would become apparent.  The  analysis  described below.  2.4.3.1  ECG changes  we  used  i n these  experiments  is  88  The analysis of standard ECG i n t e r v a l s  (QRS, P-R, Q-T)  has been previously defined i n Section 2.1.4, however the effects  of ischaemia  on the R-wave  height,  S-T  segment  elevation and Q-wave appearance were examined i n addition to these standard variables. that  acute  ischaemia  Numerous investigators have shown  and  infarction  have  dramatic  and  reproducible e f f e c t s on the morphology of the ECG (Review: Holland and Brooks,  1977).  Drug treatments have also been  shown to influence these sequelae of ischaemia a l . , 1984). elevation al.,  Ca  + +  (Clusin et  antagonists delay the onset of S-T segment  i n our model  (Johnston et al., 1983; Curtis et  1984; Walker and Beatch,  1990) while Abraham  et a l  (1989) have shown that TTX reduced both R-wave elevation and S-T segment elevation i n chronically prepared anaesthetized rats.  The analysis of these ECG changes has been described  explicitly  i n previous publications  (Curtis,  1986) and the  following description i s only a b r i e f synopsis.  The R-wave height was measured baseline  to  the peak  expressed i n mV.  of  from  the i s o e l e c t r i c  the p o s i t i v e  deflection  and  The S-T segment elevation was expressed as  % of R-wave height, where the S-T segment was defined as the height  ( i n mV)  above  isoelectric  where  d e f l e c t i o n of the S-wave was interrupted isoelectric.  This  second  peak  after  the  negative  i n i t s return to the QRS  was not  confused with a second peak seen with s p l i t t i n g of the QRS  89  secondary to conduction blockade by measuring peaks at least 30 msec a f t e r the QRS i n i t i a t i o n .  Q-wave onset was measured  as the time a f t e r occlusion when a negative d e f l e c t i o n just p r i o r to the R-wave greater than 10% of R-Wave height was seen.  2.4.3.2  Arrhythmia Analysis  Occlusion of the LAD i n rats and pigs resulted i n the occurrence of v e n t r i c u l a r arrhythmias which were equivalent to the premature beats (VPB), bigeminy, tachycardia (VT) and fibrillation the  (VF) seen i n the c l i n i c a l s e t t i n g .  The s i z e of  O.Z. was determined by dye (Cardiac green) exclusion i n  hearts retrogradely perfused at the end of the monitoring period. (1986)  Arrhythmias were analyzed as described by Curtis i . e . VT was  defined  as four  or more consecutive  extrasystoles with a c l e a r l y distinguishable QRS, as opposed to VF which has a chaotic appearance. Arrhythmias  were  divided  into  time  periods  corresponding to early arrhythmias (0-0.5 hr i n rats,  0-lhr  i n pigs) and l a t e arrhythmias (0.5-4hr rats) i n accordance with the biphasic occurrence with time of ischaemia-induced arrhythmias number group.  (Wit and Bigger, 1977) .  of VPBs per time  period  The mean ± SEM  was  determined  log  1 0  f o r each  The mean ± SEM of log^n number and duration of VT  and VF episodes also were counted time period.  f o r each group  i n each  Log^o transformation normalized these data f o r  90  subsequent  parametric s t a t i s t i c a l analysis by one way ANOVA  (Johnston et al., which  had  at  1983).  least  The number of animals per group  one  episode  of  VT  and  VF  also  was the  recorded.  These nonparametric  data were analyzed by  Chi-square  test  of  tables et al  with  (Mainland et al.,  the  aid  1956).  Mainland's  contingency  Arrhythmia score (Johnston  1983) was used to summarize the arrhythmia p r o f i l e f o r  each animal.  The use of arrhythmia scores has recently been  evaluated (Curtis & Walker, 1988).  91  3 RESULTS 3.1  Pharmacology  3.1.1  Species Dependent E f f e c t s on the Cardiovascular System.  The  effects  quantitatively  of tedisamil have been reported  with  species  (Buschmann  et  al.,  to vary 1989).  Similar species dependent e f f e c t s have been reported amiodarone, d-sotalol Since  little  and c l o f i l i u m  with  (Kopia et al., 1985).  was known about the new drugs used i n these  studies, we i n i t i a l l y sought to demonstrate whether or not they had class I I I e f f e c t s on the ECG and BP. these  Data from  experiments could then be compared to more detailed  assessments  of  class  III  actions,  refractoriness and AP morphology. efficacy  was  determined  i . e . , effects  Finally,  on  antiarrhythmic  and interpreted according  to the  pharmacological p r o f i l e obtained f o r each drug.  3.1.1.1  E f f e c t s on HR, BP & ECG i n Rats  Tedisamil species tested. 6  shows  that  reduced  heart  rate  significantly  in  all  Comparison of Table 1 with Tables 3, 4, and the bradycardic  KC8851) were greatest  i n those  actions species  r e s t i n g heart rate (rats, guinea p i g s ) .  of tedisamil (and with  the highest  In conscious rats a  dose-dependent bradycardia was seen with the 4 mg/kg dose producing a 34% drop i n heart rate 4 minutes a f t e r infusion.  Table 1. Haemodynamic E f f e c t s of K Conscious Rats.  Channel Blockers i n  Group  HEART RATE (b/min) predrug postdrug  BLOOD PRESSURE (mmHg) predrug postdrug  CI TI T2 T4  403±16 420112 395± 7 382117  418115 3051 7 263+ 6* 252110*  1201 1141 121+ 1191  7 3 3 4  1211 1241 145+ 1451  6 6 6 5  C2 KC UK RP R5 RIO  436116 416113 397111 376111 398116 420112  4171 2 260111 3761 7 382112 387116 414114  1111 1101 1021 1051 1041 1041  4 4 4 3 4 3  1111 1221 1051 1091 1051 1061  4 5 4 3 3 5  The e f f e c t s of treatment upon heart rate and mean a r t e r i a l blood pressure i n conscious rats are shown. Predrug values were recorded immediately p r i o r to i n i t i a t i o n of a 10 min infusion. Postdrug values were recorded 4 min a f t e r the infusion was complete. The symbol indicates p < 0.05 versus predrug, by ANOVA and Duncan's range t e s t ( a l l predrug values were N.S. versus appropriate control group). The data are from two separate studies, each with t h e i r own control, and are shown here f o r sake of comparison. The groups i n the f i r s t study are CI = control, TI = tedisamil (1 mg/kg), T2 = tedisamil (2 mg/kg), T4 = tedisamil (4 mg/kg). The groups i n the second study are C2 = control, KC = KC8851 (4 mg/kg), UK = UK68,798 (1 mg/kg), RP = RP62,719 (1 mg/kg), R5 = r i s o t i l i d e (5 mg/kg), R10 = r i s o t i l i d e (10 mg/kg). n = 9 per group.  93  S i m i l a r l y , 4mg/kg KC8851 produced a 37.5% drop i n heart rate at t h i s time period. 10 mg/kg, UK68798  On the other hand, r i s o t i l i d e 5 mg or  (lmg/kg) and RP62719  (1 mg/kg) d i d not  reduce heart rate s i g n i f i c a n t l y compared to control  (Table  1). Effects  mg/kg  on BP in conscious  tedisamil  pressure  was  and  the 4  elevated  by  rats:  mg/kg 20%  At doses of 2 & 4  dose (p  respectively, 4 minutes a f t e r infusion.  <  of  KC8851  0.05)  and  blood 11%  The 1 mg/kg dose of  tedisamil, as well as the doses of UK68798, r i s o t i l i d e , and RP62719, d i d not a l t e r  blood pressure  i n conscious  rats.  The elevation i n blood pressure was maintained f o r the f i r s t hour a f t e r occlusion f o r only the 2mg/kg dose of tedisamil; a l l other treatments d i d not attenuate the decrease i n blood pressure produced  by coronary artery occlusion i n conscious  rats (section 3.4.3, Figure 17).  Effects  treatments  on the ECG in conscious  (tedisamil,  KC8851,  rats:  UK68798,  None of the drug  risotilide  nor  RP62719) prolonged the QRS duration when measured 4 minutes post infusion.  Tedisamil (2 & 4 mg/kg) and KC8851 (4 mg/kg)  infusions prolonged the P-R i n t e r v a l to  predrug  values.  A l l doses  markedly increased the Q-T  c  (p<0.05) when compared  of tedisamil  and KC8851  i n t e r v a l while no s i g n i f i c a n t  e f f e c t s were seen with UK68798, RP62719 nor r i s o t i l i d e i n conscious rats (Table 2) .  Table 2. ECG Conscious Rats.  Group  Effects  of  Q-Tc pre  K  Channel  Blockers  P-R  in  QRS  post  pre  post  pre  post  CI Tl T2 T4  215±10 220±5 205±5 195±5  210±10 280±9 320±7 330±9*  41±1 38±1 38±2 44±2  42±1 44±1 47±1* 53±2*  22±1 22±1 23±1 22±1  22±1 23±1 23±1 23±1  C2 KC UK RP R5 RIO  217±6 211±5 205±5 197±7 204±9 203±4  217±10 332110 213±8 202±6 200±10 206±4  42±1 45±1 41±2 46±1 41±1 45±2  42±1 50±2* 43±1 47±1 44±1 47±2  23±1 23±1 22±1 23±1 22±1 24±1  23±1 25±1 22±1 22±1 23±1 24±1  The e f f e c t s of treatment upon the ECG i n conscious ratsi are shown. Values shown are i n ms. Predrug values were recorded immediately p r i o r to i n i t i a t i o n of a 10 min infusion. Postdrug values were recorded 4 min a f t e r the infusion was complete. The symbol indicates p < 0.05; indicates p < 0.01 versus predrug, by ANOVA and Duncan's range t e s t ( a l l predrug values were N.S. versus appropriate control group) . The data are from two separate studies, each with t h e i r own control, and are shown here f o r sake of comparison. The groups i n the f i r s t study are CI = control, T l = tedisamil (1 mg/kg) , T2 = tedisamil (2 mg/kg) , T4 = tedisamil (4 mg/kg). The groups i n the second study are C2 = control, KC = KC8851 (4 mg/kg), UK = UK68,798 (1 mg/kg), RP = RP62,719 (1 mg/kg), R5 = r i s o t i l i d e (5 mg/kg), R10 = r i s o t i l i d e (10 mg/kg). n = 9 per group.  95  3.1.1.2  In  acutely  E f f e c t s i n Anaesthetized Guinea Pigs  prepared  anaesthetized  guinea  pigs, tedisamil  produced a dose dependent bradycardia and widening of the QT . c  The bradycardic  lengthening  effect  as well  e f f e c t s reached  as the Q-T  statistical  interval  significance  after  the second dose ( i . e . , cumulative 2 mg/kg i.v.) while the QT  c  i n t e r v a l widening reached  a t o t a l of 4 mg/kg i . v .  statistical  significance after  On the other hand there were no  e f f e c t s on the QRS duration, and P-R i n t e r v a l s i n t h i s dose range ( 1 - 8 mg/kg).  UK68,798 i n the dose range of 10 - 160  ug/kg produced no s i g n i f i c a n t  e f f e c t s on any ECG  interval  (Table 3).  Tedisimal pressure  i n acutely  and UK68,798 d i d not a f f e c t  prepared  guinea  pigs.  The  blood  8 mg/kg  tedisamil dose when given rapidly occasionally p r e c i p i t a t e d VPB and VT. Also, arrhythmias alternating  sinus  which were characterized by  brady/tachycardia  i . e . sick  sinus  were  seen at t h i s dose.  3.1.1.3  Tedisamil widening producing  E f f e c t s i n Pigs  was  i n pigs  least  efficacious  compared with  a 25% reduction i n HR  other  with  regard  species  to  Q-T  c  at a dose  (Buschmann et a l . , 1989).  However, i t d i d cause a dose dependent bradycardia and Q-T  c  Table 3. Cardiovascular E f f e c t s of tedisamil and TJK68,798 i n Anaesthetized Guinea Pigs.  Dose  Q-Tc (ms)  P-R (ms)  pre. TI T2 T4 T8  270110 280110 300110 315115* 3301/  4912 5211 5211 521/ 541/  pre. UK10 UK20 UK40 UK80 UK160  260110 265115 265115 260115 260115 265110  5714 5413 5413 5313 5413 5313  QRS (ms)  HR b/min  BP (mmHcr)  3112 3012 2912 2912 271/  300120 220115 200120 190+20** 1601/  5514 6515 6518 6319 651/  3112 3112 3112 3212 3112 3112  300115 260120 265120 270120 260120 270120  4215 4215 4314 41+4 3915 3916  The e f f e c t s of treatment upon ECG i n t e r v a l s , heart rate and mean a r t e r i a l blood pressure i n anaesthetized guinea pigs are shown. Predrug values were recorded immediately p r i o r to i n i t i a t i o n of a cumulative dosing regimen. Doses were given at 15 min i n t e r v a l s and values were recorded 10 min a f t e r each dose. The data are from two separate studies, and are shown here f o r sake of comparison. The treatments indicated are, pre = predrug control, TI = tedisamil (1 mg/kg), T2 = tedisamil (2 mg/kg), T4 = tedisamil (4 mg/kg), T8 = tedisamil (8 mg/kg), / indicates n = < 4 f o r p a r t i c u l a r value. The treatments i n the second study are pre = predrug control, UK10 = UK68,798 (10 mg/kg), UK20 = UK68,798 (20 mg/kg), UK40 = UK68,798 (40 mg/kg), UK80 = UK68,798 (80 mg/kg), UK160 = UK68,798 (160 mg/kg), n = 6 throughout. A l l doses indicated are cumulative amounts given. The symbol indicates p < 0.05; indicates p < 0.01 versus predrug, by ANOVA and Duncan's range t e s t .  97  widening. Blood  On the other hand, P-R,  pressure  was  elevated  cumulative  dose  affected.  Unfortunately,  apex d i d not pressure was data  were  of  8  dose  mg/kg.  faithfully  the  were not affected.  dependently,  However, catheter  record LVP.  excluded in  ventricle  v i a the  from  inserted  Since peak  analysis.  studies with  artery.  ug/kg) was without e f f e c t s on HR, i n t e r v a l s except Q-T  c  LVP  This  UK68,798 by  carotid  up  LVEDP  dampened using t h i s technique,  rectified  ECG  QRS  was  not  into  the  systolic and  E f f e c t s i n Primates  Baboons  and  macaque  catheterizing  the  BP, LVP, RVP,  monkeys  dP/dt was  (0 -  167.5  dp/dt or any  i n t e r v a l which rose by 21%  3.1.1.4  a  problem  UK68,798,  the 20 pg/kg dose (37.5 ug/kg cumulative).  to  after  (Tables 4 & 5).  responded  similarly  to  tedisamil with dose dependent elevations i n d i a s t o l i c  and  s y s t o l i c BP, reductions i n HR and prolongations of the  Q-U  interval. the Q-U  and  above 0.5 drug),  The bradycardic e f f e c t maximized at 1 mg/kg, but  continued to widen.  interval  c  QRS  In baboons and monkeys the  duration also tended  to widen  mg/kg (Tables 6 & 7) . In baboons (n=2,  UK68,798  RP62,719(0.4  (50  mg/kg)  ug/kg), also  risotilide  tended  to  at  P-R  doses  f o r each  (0.3  mg/kg)  and  increase  the  Q-U  c  i n t e r v a l , by a maximum of 3 0%, without a n c i l l a r y e f f e c t s on HR,  BP or other ECG intervals (data not shown).  98 Table 4. in Pigs.  Cardiovascular Actions of Tedisamil and UK68,798  Haemodvnamics (mm/Ha) Dose  MAP  LVP  LVEDP  dP/dt  RVP  pre  93±1  /  1.5±2  /  /  Tl  97±4  /  1.3±1  /  /  T2  103±4  /  1.8±2  /  /  T4  106±6  /  6.0±4  /  /  T8  (112±5  /  4.5±2  /  /)  pre  86±7  119± 9  3±1  2800i300  27i4  UK2.5  85±6  118± 9  3±1  2700i300  27i4  UK7.5  89±8  116± 9  2±1  2700i300  28i4  UK20  87±7  118±10  1±1  2800i300  28i4  UK40  88±9  112±12  Oil  2600i300  28i4  UK80  90±10  112±13  Oil  2700i300  27i4  UK160  93±10  111± 9  Oil  2700i300  28i5  The e f f e c t s of treatment upon haemodynamics i n anaesthetized pigs are shown. Predrug values were recorded p r i o r to i n i t i a t i o n of a cumulative dosing regimen. Doses were given at 15 min i n t e r v a l s f o r tedisamil and 5 min i n t e r v a l s f o r UK68,798. Values were recorded 5 min a f t e r each dose. The data are from separate studies, shown here f o r comparison. The cumulative doses indicated are, pre = predrug control, T l = tedisamil (1 mg/kg), T2 = tedisamil (2 mg/kg), T4 = tedisamil (4 mg/kg), T8 = tedisamil (8 mg/kg), UK2.5 = UK68,798 (2.5 /xg/kg) , UK7.5 = UK68,798 (7.5 jug/kg) , UK20 = UK68,798 (17.5 /zg/kg), UK40 = UK68,798 (37.5 Mg/kg), UK80 = UK68,798 (77.5 Mg/kg), UK160 = UK68,798 (157.5 /ig/kg) . The symbol * indicates p < 0.05 versus predrug, by ANOVA and Duncan's multiple range t e s t . T8 values obtained between episodes of arrhythmias, therefore are approximate. Mean i S.E.M.  Table 5. Pigs  E f f e c t s on the ECG of Tedisamil and UK68798 i n  ECG Intervals (ms) Dose  QRS  HR (b/min)  P-R  O-Tc  pre  46±1  96±4  345± 4  125±9  TI  47±1  9814  361± 6  10414  T2  46+1  98±5  369±14  101±3  T4  47±1  95±6  391±18*  104±3  T8  /  /  pre  39±2  84±5  306± 9  141111  UK2.5  40+3  82±5  332±13  140±10  UK7.5  41±3  82+5  333±14  139111  TJK20  41±2  83+5  341117*  132113  UK40  4112  8315  354117*  128112  UK80  4112  8414  346+17*  127112  UK160  4212  8415  340130  132114  /  sinus arrhythmias  The e f f e c t s of treatment upon ECG i n t e r v a l s and heart rate i n anaesthetized pigs are shown. Predrug values were recorded p r i o r to i n i t i a t i o n of a cumulative dosing regimen. Doses were given at 15 min i n t e r v a l s f o r tedisamil and 5 min i n t e r v a l s f o r UK68,798. Values were recorded 5 min a f t e r each dose. The data are from separate studies, shown here for comparison. The cumulative doses indicated are, pre = predrug control, TI = tedisamil (1 mg/kg), T2 = tedisamil (2 mg/kg), T4 = tedisamil (4 mg/kg), T8 = tedisamil (8 mg/kg), UK2.5 = UK68,798 (2.5 jug/kg) , UK7.5 = UK68,798 (7.5 /xg/kg), UK20 = UK68,798 (17.5 Mg/kg), UK40 = UK68,798 (37.5 /ig/kg) , UK80 = UK68,798 (77.5 Mg/kg), UK160 = UK68,798 (157.5 jug/kg) . The symbol * indicates p < 0.05 versus predrug, by ANOVA and Duncan's multiple range t e s t . Mean 1 S.E.M.  100 Table 6.  BP and HR E f f e c t s of Tedisamil i n Primates. Blood pressure (BP)  (2£iU>  Sy.tolic  ( | B a I g )  Di..tolio  H  - ^  t  ^  n  ,  <  H  R  )  BABOONS Study (i) (n = 3-5) Pre-drug  159±15  99±4  0.05 0.10 0.2 0.4 0.8 1.6  +6±5 +7±2 +13±2 +18±6 +20±11 +25±3  +5±3 +3±2 +3±2 +4±3 +19±13 +19 /  101±6 -6±3 -7±4 -20±11 -20±7 -29 / -30 /  Study ( i i ) (n = 4-5). Pre-drug  160±5  117±4  108±3  0.5 1.0 2.0  +9±2 +10±8 +10±8  +8±1 +6±3 +5±3  -16±3 -27±5 -27±5  MONKEYS (n = 5) Pre-drug  104±5  0.05 0.1 0.2 0.4 0.8 1.6  +2±2 +5±5 +8±6 +13±7 +22±3 +31±4  68±5 +5±2 +5±2 +9±6 +15±8 +17±3 +20±5  144±6 -14±2 -22±4 -32±8 -43±12 -52±9 -59±10  The bradycardic and hypertensive responses to tedisamil are shown. Two d i f f e r e n t studies ( i and i i ) were performed i n baboons. In study (i) the cumulative doses were 0.05, 0.1, 0.2, 0.4, 0.8 and 1.6 mg/kg while i n study ( i i ) the cumulative doses were 0.5, 1.0 and 2.0 mg/kg i . v . Values = Mean ± S.E.M. where n > 3; otherwise, only Mean i s given. In the pre-drug period actual values are given, a l l others are changes from pre-drug. The symbol (-) indicates a decrease from pre-drug whereas (+) indicates an increase. Statistical analyses f o r trends (correlation) showed increasing changes i n variables with increasing doses which were statistically significant at p < 0.01 (diastolic pressures) to p < 0.001 (heart rate) i n both species.  101  Table 7. Dose  ECG E f f e c t s of Tedisamil i n Primates. P-R  QRS  Q-U  (ms)  (ms)  (ms)  BABOONS Study Ti) Pre-drug 0.05 0.1 0.2 0.4 0.8  11617 +2.812.7 -2.312.9 +4.012.7 +3.813.1 -1.711.2  Q-U  c  (ms)  (n= 3-5).  34.410.9  332118  413+19  +2.211.8 +3.4+1.7 +4.511.8 +5.311.1 +3.511.5  +2519 +53113 +86121 +152137 +165141  +2018 +57110 +65120 +108117 +133142  Studv ( i i ) (n = 4-5). Pre-drug  13517  3412  0.5 1.0 2.0  +213 +711 +1111  +0.810.8 +1.412.3 +2.811.5  302119  401116  +991 5 +158147 +263175  +89120 +118125 +181115  MONKEYS (n = 5) Pre-drug 0.05 0.1 0.2 0.4 0.8 1.6  9519  231 2  2621 9  413+19  +814 +914 +915 + 1316 +1517 +2213  -0.311.0 +0.310.8 +1.0+2.2 +2.012.2 +1.811.7 +3.311.7  +36111 +56110 +86+27 +121128 +173170 +271177  +15114 +42114 +52+23 +100+36 +109152 +152172  In order to assess the e f f e c t s of tedisamil on the ECG, th P-R, QRS and Q-U i n t e r v a l s were measured. The Q-U i n t e r v a l was corrected f o r rate (Q-U ) by Bazett's, square root of RR correction. Values = Mean 1 S.E.M. where n > 3 otherwise only Mean i s given. In the pre-drug period actual values are given, a l l others are changes from pre-drug. The symbol (-) indicates a decrease from pre-drug whereas (+) indicates an increase. Time had no consistent e f f e c t . Statistical analyses of trends showed that variables with increasing dose 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 at p < 0.05 f o r P-R changes, and p < 0.001 f o r Q-U and Q-Uc. c  102  3.2  E l e c t r i c a l Stimulation Studies  3.2.1  Tedisamil vs. Class Is i n Rats  In an i n i t i a l rat  model  attempt to develop a reusable  for electrical  stimulation  electrode carriage was t r i e d . crossover  design,  conscious  a method  using  an  The intention was to use a  so eliminating between r a t v a r i a b i l i t y .  This method was successful i n that the electrodes could be reversibly  positioned  against  the v e n t r i c l e .  However i n  order to have stable threshold values, the animal had to be anaesthetized. benefit  This  accrued  preparative  latter  by  the  surgery.  requirement  nullified  time-consuming  However,  this  and  method  the  intricate  was used to  compare tedisamil with representatives of the three Class I subclasses; quinidine (Ic) .  ( l a ) ; mexiletine  Later e l e c t r i c a l  (lb) and propafenone  stimulation studies were performed  in acutely prepared pentobarbitone anaesthetized  The  e f f e c t s of tedisamil were d i f f e r e n t  the Class I drugs.  Tedisamil  animals.  from those of  prolonged ERP and decreased  MFF markedly but d i d not elevate  VF^.  mg/kg tedisamil, the c h a r a c t e r i s t i c  However,  after 4  fibrilloflutter  not be induced by e l e c t r i c a l stimulation.  Instead,  could  a VT at  a maximum heart rate of 7 Hz was a l l that could be achieved by  this  drugs  "burst  pacing"  increased  VF^  propafenone  having  method in a  the  (50 Hz, 0.8 ms) .  dose  lowest  dependent EC50  Class I  fashion  for this  with  effect.  103  Propafenone also had the greatest potency i n prolonging  QRS  duration  lc  which  classification.  is The  consistent class  c  Q-T  Class  intervals  Tedisamil widened the  QRS  i n t e r v a l s i g n i f i c a n t l y i n t h i s preparation  with only minimal e f f e c t s on the P-R was  its  1 drugs widened P-R  with apparent E C 5 0 S of 8 mg/kg. duration and Q-T  with  interval.  Tedisamil  eight times more potent than quinidine at widening the interval  c  (Figures 1 & 2) .  These r e s u l t s  published previously (Walker and Beatch,  1988).  3.2.2  Tedisamil vs. UK68,798 i n Rats  After  abandoning  reusable  model  tedisamil  and  of  attempts  at  electrical  developing  a  stimulation,  UK68798 i n acutely prepared,  ( 4 5 mg/kg i.p.) anaesthetized rats.  have been  conscious we  pentobarbitone  Results with tedisamil  were the same as those i n the chronically prepared anaesthetized consuming and there was  model  less invasive.  3) ,  and  were  halothane less  In acutely prepared  of course no r i s k of a s u r g i c a l l y induced  myocardial  adhesion  few  i f the  cases  which  (Figure  caused  an  influencing the r e s u l t s . stab  electrodes damaged  unsteady  tested  baseline  of  time  animals, infarct  However, i n a the  ventricle,  threshold  values,  (3.2.1)  tedisamil  r e s u l t s from such animals were discarded.  As prolonged  seen  i n • the  the ERP  and  previous rendered  study  the heart r e f r a c t o r y to VF  104  Figure 1 shows the responses t o e l e c t r i c a l  stimulation  of halothane anaesthetized chronically prepared rats treated with tedisamil, quinidine, mexiletine, or propafenone cumulative dosing regimen.  in a  Values shown are mean ± S.E.M.  (n = 6) f o r : change i n threshold voltage f o r induction of fibrillation, frequency,  (MFF);  period, (ERP). Slidewrite  (VFt); and  increase  in  maximum  following  i n effective  refractory  Curves were f i t (second order polynomial) by  software.  a f t e r each dose.  decrease  Determinations were made 10-15 min  Dosing i n t e r v a l was 15 min.  105  Figure 1 . E l e c t r i c a l Stimulation: Tedisamil vs. Class I Drugs. change in V F t (V) I  O  O  |  r  +  decrease in M F F (Hz)  o  change in E R P (ms) I  6  (D  a  O o<n  _x  3  *  CD  (Q — 7T (Q  ->• o  ro o  CO O  o  Ol  o  o  106  Figure 2 shows the ECG responses t o drug treatment i n halothane  anaesthetized chronically  prepared  rats  treated  with tedisamil, quinidine, mexiletine, or propafenone cumulative dosing regimen.  in a  Values shown are mean ± S.E.M.  (n = 6) f o r : change i n QRS duration; P-R i n t e r v a l ; and Q-T i n t e r v a l (xlOOO).  Curves were f i t  by Slidewrite software. drug  infusion.  intervals.  c  (second order polynomial)  Measurements were made 10 min a f t e r  Increasing  doses  were  given  at 15 min  107  Figure 2.  E  so  h  70  h  ECG E f f e c t s of Tedisamil & Class I Drugs.  60 SO ±-  36 32  *  J-  -I——  28  r  2-* 20  3  0  0  R  270  180  1  50  0.5  Q u i n  2 A DOSE (mg/kg) pro  16  ted  32  108  Figure 3 shows the response t o e l e c t r i c a l in  pentobarbitone  anaesthetized  treated with tedisamil or UK68,798.  acutely  stimulation  prepared  rats,  Values shown are mean ±  S.E.M. f o r : v e n t r i c u l a r e f f e c t i v e refractory period, ERP; maximum  following  frequency,  f i b r i l l a t i o n threshold, V F f polynomial)  by Slidewrite  MFF;  and  ventricular  Curves were f i t (second order  software.  made 10-15 min a f t e r each dose.  Determinations were  Dosing i n t e r v a l was 15 min.  109 Figure 3. E l e c t r i c a l  Stimulation  Studies  Tedisamil vs. Uk68,798. ERP  (ms)  o o  o  f-\l>—\ D o w  c  o o  (Q  o o o  VFt  o o  (uA) O O  W O  o  in  Rats:  110  induction. MFF,  ERP,  the ECG  UK68,798 had  s i g n i f i c a n t e f f e c t s on  either  or VF-t (Figure 3) . UK68798 also had no effects' on i n t e r v a l s of QRS,  again prolonged the study  no  the  P-R  P-R,  Q-T  or R-R  c  i n t e r v a l and  c  and  Q-T  QRS  intervals  while tedisamil  slowed HR. were  not  In t h i s prolonged  s i g n i f i c a n t l y by tedisamil treatment (Figure 4).  3.2.3  Tedisamil vs. UK68,798 i n Guinea Pigs  UK68,798  tended  to  be  more  potent,  e f f i c a c i o u s , than tedisamil i n prolonging ERP  i n guinea pigs.  significance other  ECG  but  Q-T  However, these trends did not  or  VFf  Tedisamil  did  a f f e c t i n g QRS,  (Table 3 and Figure 5).  3.2.4  nor VFt  Tedisamil  reach of  however  QT  P-R  and ERP  did any  dependently widen the R-R,  C  less  i n t e r v a l s and  c  (unweighted means ANOVA), nor  effects  much  (p < 0.05)  the dose  while not  i n Baboons  In e l e c t r i c a l stimulation studies i n baboons, tedisamil had  no  significant effects  on  stimulation  threshold  for  capture (of a 25% increase i n rate t r a i n ) , but tedisamil did decrease MFF contrast,  when  oesophagus, decreased  by a maximum of 2 6% a f t e r 1.0  the  by  refractoriness (Figure 6) .  the  right  atrium  ventricular  tedisamil, was  not  was  MFF  which  was  stimulated not  suggested  affected  by  mg/kg i . v . via  In the  significantly  that  tedisamil  A.V.  node  treatment  Ill  Figure  4  shows  the ECG  responses  to tedisamil  or  UK68,798 treatment i n the same pentobarbitone anaesthetized acutely prepared rats as shown i n figure 3. are mean ± S.E.M. f o r : i n t e r v a l ; and Q-T  c  heart rate, HR;  interval.  15 min i n t e r v a l s .  QRS duration; P-R  Curves were f i t (second order  polynomial) by Slidewrite software. 10 min a f t e r drug infusion.  Values shown  Measurements were made  Increasing doses were given at  112  P-R  o  <ms) o  QRS (ms)  o  o —I—  o  o  l-C H  o  o  i-t —i  he ^  o  o o  HJH  I—CV—I  3T  1- >H  K )H  c  o o o  01  o  113  Figure 5 shows the response to e l e c t r i c a l in  urethane  anaesthetized acutely  treated with tedisamil or UK68,798.  prepared  stimulation  guinea  pigs,  Values shown are mean ±  S.E.M. f o r : v e n t r i c u l a r e f f e c t i v e refractory period, ERP; maximum  following  frequency,  f i b r i l l a t i o n threshold, V F f polynomial)  by Slidewrite  MFF;  and  ventricular  Curves were f i t (second order  software.  made 10-15 min a f t e r each dose.  Determinations were  Dosing i n t e r v a l was 15 min.  114  Figure 5. E l e c t r i c a l  Stimulation  Studies  .Pigs: Tedisamil vs. UK68,798. ERP 01 O  N oi  (ms) o o  ro oi  01 O  in  Guinea  115  Figure 6. E f f e c t s of Tedisamil on MFF i n Baboons.  10.00 r  7.50  N  I LL LL  5.00  2.50  0.00 0  1  Dose (mg/kg) A  RV  O  RA  The e f f e c t s of tedisamil on v e n t r i c u l a r responses to e l e c t r i c a l stimulation of the r i g h t atrium (RA) and r i g h t ventricle (RV) i n anaesthetized baboons are shown. Cumulative doses of 0.5, 1.0, and 2.0 mg/kg were given. Means and S.E.M. are shown.  116  3.2.5  In  Summary  non-ischaemic hearts in vivo,  tedisamil  prolonged  v e n t r i c u l a r refractoriness i n rats, guinea pigs and baboons. The  increase  bradycardia  i n refractory  periods were not a r e s u l t of  as MFF was lowered by tedisamil  determined by the extra  and ERP was  stimulus method at a fixed  rate.  When compared with class I drugs, tedisamil had a d i f f e r e n t p r o f i l e of action on e l e c t r i c a l stimulation. without e f f e c t s on the threshold VF, but i n rats tedisamil  Tedisamil was  f o r e l e c t r i c a l induction of  (>4 mg/kg) treatment prevented the  e l e c t r i c a l induction of VF.  UK68,798 was f a r less e f f i c a c i o u s than tedisamil and did not produce any s i g n i f i c a n t e f f e c t s on MFF, ERP and VF^ nor  on any of the ECG i n t e r v a l s i n anaesthetized rats or  guinea pigs.  3.3  I n t r a c e l l u l a r Recording Studies In Vivo.  These investigations were c a r r i e d out as an e f f o r t to explore the mechanism of action of tedisamil and UK68798 i n electrophysiological  terms.  used i n other studies. between  studies,  prolongation  was  a  Doses were s i m i l a r  to those  In order to allow f o r comparisons similar  assumed  to  concentrations f o r each drug.  degree  of  indicate  Q-T  c  similar  interval plasma  117  3.3.1  Tedisamil i n Rats  Tedisamil potentials  dose-dependently  (AP).  was delayed epicardial  such  appeared  mammalian species.  tedisamil.  phase As  that  similar  a  slowed.  was result  of  the  APD  repolarization  was  prolonged  effects,  Concomitant  tedisamil  narrow r a t  seen  The slope  reduced  repolarization  cumulative.  repolarization  the normally to that  action  i n larger  The l a t e r stage of r e p o l a r i z a t i o n  3) was not appreciably repolarization  rat  The early phase of rapid  markedly, AP  widened  at  5  10, 5  of the early  fold  this  by  decreased 25,  fold  4  mg/kg  rate  of  50,  and  75%,  after  8  mg/kg  to these highly  s l i g h t l y elevated  (phase  s i g n i f i c a n t APD  AP height  and only  decreased AP r i s e rate at one dose l e v e l (Figure 7).  3.3.2.  Tedisamil i n Guinea Pigs  In guinea pigs tedisamil although  only  by  a maximum  also tended to widen the APD of 80%.  This  effect  was  s u b s t a n t i a l l y reduced by bolus injections of adrenaline (6.7 ug/kg  and 13 ug/kg)  levels. or  restored  There were no consistent  r i s e rate  (phase  which  2)  a f t e r tedisamil  and l a t e  HR  to  pre-tedisamil  trends i n either AP height  treatment.  repolarization  Both the plateau  (Phase  3)  stages of  r e p o l a r i z a t i o n were decreased by 50% at 4 mg/kg cumulative tedisamil  (Figure 8).  118  Figure 7 shows the e f f e c t s of tedisamil ventricular  epicardial  AP morphology  pentobarbitone anaesthetized rats.  action  potential  height,  potential  r e p o l a r i z a t i o n , APD10 etc.; "plateau", -dV/dtp. shown.  AP; maximum  duration  at  i n acutely  prepared  Composite representative  AP are drawn and mean ± S.E.M. values action  treatment on  10,  (n=6) are given f o r : rise 25,  rate, 50  +dV/dt; and  75%  and r e p o l a r i z a t i o n rate of the  Values obtained 10 min a f t e r dosing are  Cumulative doses were given at 15 min i n t e r v a l s .  *p < 0.05, **p < 0.01 from control.  119  Figure 7.  Electrophysiological  Effects  of Tedisamil i n  Rats. 20 ms 100  V/s  25 mV 20  ms  T E D I S A M I L in R a t s  Dose mg/kg  AP mV  con  97*3  183±6  0.5  103+3  173+8  1.0  105+2  175+7  2.0  110+2  4.0  102+4  1 1 + 1** 26+2** 58+6 * * 95+4** ** ** ** ** 170+15 69+2 37+1 114+3 17+1 ** 115+18* 24+4 100+14 162+22 53+8  8.0  107  165  +dV/dt V/s  APD.10 ms  APD25 ms  APD50 ms  APD75 ms  -dV/dtp V/s  5+1  10±1  19+1  45±3  2.7+0.2  8±1  19+2*  41+3**  81+5  1.0+0.3*  29  67  125  195  0.9+0.2* 0.7+0.2$? 0.5+0.2 * * 0.5  120  Figure 8 ventricular urethane adrenaline Composite  shows the e f f e c t s of tedisamil  epicardial  anaesthetized on tedisamil representative  AP morphology guinea induced  treatment on  i n acutely  pigs. changes  The  prepared  effects  are also  of  shown.  AP are drawn and mean ± S.E.M.  values are given for: action potential height, AP; maximum r i s e rate, 75%  dV/dt; action potential duration  at 25, 50 and  r e p o l a r i z a t i o n , APD25 etc.; r e p o l a r i z a t i o n rate of the  "plateau",  -dV/dtp; and phase 3 r e p o l a r i z a t i o n rate, -dV/dt.  Values obtained 5 min a f t e r dosing are shown. doses were given at 15 min i n t e r v a l s (n=4). < 0.01 from control.  Cumulative  *p < 0.05, **p  121  TEDISAMIL in Guinea pigs Dose mg/kg con 0.5 1.0 2.0 4.0 8.0 Adren ug/kg 6.7 13  dV/dt V/s  APD25 APD50 ms ms  105+6 110+7 105+7 100+7 107+7 1 10  140+20 160+20 145+25 135±20 140+30 130  54+3 78+2 91±2 0.52+.05 72+6 109+9 122+6 0.37+.04** 81+4* 115+5* 130+11* 0.36+.03** 93+8**126+13**146±11 0.3H.05** 105+8**144±11**165±13**0.28±.04** 115 155 180 0.26  106+4 100  140+20 160  AP mV  80+7 60  108+15 100  APD75 ms  126 + 18 118  -dV/dtp V/s  0.42+.04 0.40  -dV/dt V/s 2.6+.3 1.9+.1 1.9±.3^ 1.6+.2 1.3+.2** 1.1  1.6+.2 1.5  122  3.3.2.1  E f f e c t s of Vagal  Stimulation  The vagus nerve was stimulated in  order  to study  ventricular that  AP.  guinea pigs  while  rats  The r e s u l t s  effects  no  such  of bradycardia  on  of these experiments showed  APD widening was rate  showed  demonstrated that not  the d i r e c t  i n rats and guinea pigs  dependent in  sensitivity.  This  vivo study  the bradycardic e f f e c t of tedisamil  was  a factor i n i t s a b i l i t y to widen the APD i n rats, but  that major  bradycardia produced i n guinea pigs contribution  to tedisamil's  would have had a  action  i n the species  (Figure 9).  UK68798 i n Rats In Vivo  3.3.3  In rats UK68798 was without e f f e c t s on r i s e rate, AP height, and  S l i g h t prolongation of  APD25.  APD50  also d i d not reach s t a t i s t i c a l s i g n i f i c a n c e was potent (apparent i n t h i s species.  EC50  25ug/kg) despite  and  APD75  (by ANOVA).  It  i t s low e f f i c a c y  The early phase of r e p o l a r i z a t i o n , which  i s rapid i n the rat, was not affected by UK68798 treatment, while s l i g h t slowing of the (Phase 3) f i n a l was seen  (Figure  10) .  repolarization  Comparison of figures  7 & 10 also  show the s l i g h t v a r i a b i l i t y i n morphology (predrug) between i n d i v i d u a l action potentials recorded from r a t epicardium.  123  Figure ventricular  9 shows the e f f e c t s epicardial  of vagal  action potential  guinea pigs (top) and rats (bottom).  stimulation  on  duration, APD, i n  Animals were prepared  as f o r treatment with tedisamil or UK68,798,  except  their  l e f t vagal nerve was stimulated i n order to slow the heart rate. Each point represents a single recording of APD vs. RR interval.  Curves were f i t (second order polynomial) by  Slidewrite software.  E f f e c t s o f HR  9  Figure  on APD  i n G. P i g s and Rats.  150  125  100 o 75  50  25 0.00  0.20  0.40  R-R  0.60  0.80  interval (sec)  60 v  50 o  M £  40  r  30 20  o  10  -if m  0.00  0.20 R-R  +  APD10  "A  0.60  0.40  0.80  interval (sec)  APD25  O  APD50  V  APD75  125  Figure 10 shows the e f f e c t s of UK68,798 treatment on ventricular  epicardial  AP morphology  pentobarbitone anaesthetized r a t s . AP  are redrawn  action  potential  i n acutely  Composite representative  and mean ± S.E.M. values height,  prepared  AP; maximum  rise  are given f o r : rate,  +dV/dt;  action potential duration at 25, 50 and 75% r e p o l a r i z a t i o n , APD25 etc.;  r e p o l a r i z a t i o n rate of the "plateau", -dV/dtp;  and phase 3 r e p o l a r i z a t i o n rate, -dV/dt. min  a f t e r dosing are shown.  15 min i n t e r v a l s (n=3).  Values obtained 10  Cumulative doses were given at  *p < 0.05 from control.  Figure 10.  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 U K 6 8 , 7  i n Rats.  10 ms  10 ms  UK 6 8 , 7 9 8 in Rats Dose ug/kg con 12.5 25 50 100 1000*  AP mV 105+5 105+8 104±7 102+6 106±9 106  dV/dt V/s 130±20 125+20 140+20 125+25 130+20 140  APD25 ms 10+2 10±1 9+3 10+3 10+2 10  APD50 ms  APD75 ms  -dV/dtp V/s  -dV/dt V/s  18±2 19+2 21 ±6 21+5 23±3 22  43±3 45+3 46+3 50+4 53+4 53  3.0+.2 3.0±.2 2.9+.3 2.9±.2 2.8+.3 2.8  0.66+.03 0.68±.04 0.64+.04 0.60+.05 0.60+03 0.60  127  Figure 11 ventricular  shows the e f f e c t s of UK68,798 treatment on  epicardial  AP morphology  i n acutely  prepared  urethane anaesthetized guinea pigs. Composite representative AP are redrawn and mean ± S.E.M. values (as i n figure 10) obtained  10 min a f t e r dosing are shown.  were given control.  at 15 min i n t e r v a l s ,  (n=6) .  Cumulative *p < 0.05  doses from  128  Figure 11.  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 UK68,798  i n Guinea Pigs.  UK 6 8 , 7 9 8 in Guinea pigs Dose ug/kg con 12.5 25 50 100  AP mV 110+4 116+7 114+5 109+7 110+6  dV/dt V/s  APD50 ms  APD75 ms  -dV/dtp V/s  140+10 140+20 145+15 150+20 140+20  84+2 98+9 106+8* 103+5 103+5  95+2 11 7+6^ 125+9 122+9 121±7  0.47±.04 0.45+.04 0.44+.05 0.42+.03 0.42±.04  -dV/dt V/s 2.3±.2 1.9+.1 1.8+.1 1.7+.2 1.7+.1  129  3.3.4.  In  guinea  height,  UK68798 i n Guinea Pigs In Vivo  pigs,  as i n rats,  nor r i s e  rate.  maximally at 25 ug/kg.  UK68798  UK68798  d i d not a f f e c t  widened  APD75  AP  by 32%  I t s main e f f e c t s were on the l a t e r  stage of r e p o l a r i z a t i o n , slowing phase 3 r e p o l a r i z a t i o n rate by 23% while not a l t e r i n g the rate of r e p o l a r i z a t i o n of the plateau (Phase 2). The remarkable potency (despite limited efficacy)  seen  with  UK68798  has  not  been  seen  with  previously available class III drugs (Figure 11).  3.3.5  The  Summary  Class  I I I actions  of  tedisamil  were  clearly  demonstrated i n rats and guinea pigs, although i n the l a t t e r species  APD  bradycardia.  widening  could  have  been  partly  due  to  This was evidenced by the p a r t i a l reversal of  APD widening by adrenaline boluses i n t h i s species and the well known (Anderson and Johnson, 1976; Payet et al. , 1981) frequency  dependence  of APD  i n this  species.  However,  marked frequency dependence appears not to be operative i n rat epicardium at physiological rates, thus the APD widening seen  i n this  species  could  not be simply discarded  secondary  response  to bradycardia.  tedisamil  appeared  to be  minimal dV/dt e f f e c t s .  of l i t t l e  Class  as a  I actions of  consequence  - i.e.  130  Rabbit SA node experiments suggest that the bradycardia produced by tedisamil i s mainly due to i t s e f f e c t s on Phase (3) r e p o l a r i z a t i o n lack  of e f f e c t  (Oexle et al.,  1987).  on pacemaker potential  We  also noted a  in similar  studies  (unpublished observations).  The new  class III drug, UK68798, was remarkably potent  with an E C 5 0 f o r APD  prolongation of 10 - 25 ug/kg.  This  drug also appeared to be completely free of Class I a c t i v i t y (no e f f e c t s on dV/dt). by  UK68798  was  seen  However, the APD as  an  attenuation  widening produced of  the  phase  3  r e p o l a r i z a t i o n rate, such that the l a t e r stages, i . e . A P D 7 5 , were more affected than the e a r l i e r stages i n A P D 2 5 .  The  implications f o r e f f e c t s on refractoriness w i l l be discussed below.  3.4  Myocardial Ischaemia - Induced Arrhythmias  3.4.1  Overview  In l i g h t of the profound e f f e c t s of tedisamil on ERP  and  Q-Tc  interval  i n rats,  i t was  seen  as  an  APD, ideal  candidate to t e s t the e f f i c a c y of Class I I I antiarrhythmic interventions.  I t was possible to r e l a t e the degree of Q-Tc  widening necessary (by implication APD  widening)  to confer  antiarrhythmic a c t i v i t y i n the setting of acute M.I. i n t h i s species.  The congener, KC8851, was expected to behave i n a  131  similar Class  manner to tedisamil, while I I I drugs,  risotilide,  other  UK68798,  newly  and  developed  RP62719  were  expected to confer l e s s protection because of t h e i r lack of e f f e c t s on the Q-T  c  interval i n rats.  Unfortunately,  guinea  pigs cannot be used f o r acute regional myocardial ischaemia studies due to a large number of coronary artery c o l l a t e r a l s in  this  species.  collaterals, species  However,  were  chosen  pigs,  as  an  which  lack  appropriate  i n which to t e s t the antiarrhythmic  tedisamil  and UK68798.  Antiarrhythmic  r i s o t i l i d e i n pigs (Colatsky et al.,  additional p o t e n t i a l of  e f f i c a c y had been  f o r UK68798 i n dogs (Gwilt et al.,  reported  extensive  1989) and f o r  1989).  In r a t s , tedisamil was tested i n a dose response study, while  maximally  analyses,  effective  were used  doses,  f o r the other  as  assessed  drugs.  from  These  ECG  studies  u t i l i z e d double b l i n d randomized treatment regimens.  3.4.2.  Tedisamil i n Conscious Rats  In the f i r s t series of experiments, tedisamil at 1, 2 and 4 mg/kg was tested against the arrhythmias occurring i n the  first  four  hours following  occlusion.  An additional  group of c h r o n i c a l l y prepared rats was also given  2 mg/kg  tedisamil and t h e i r v e n t r i c l e s paced (acutely prepared stab electrodes) at 6.5Hz during the 4 - 1 0 minute post occlusion period;  a highly vulnerable  period  f o r early arrhythmias.  132  An extra s a l i n e control group was pentobarbitone anaesthesia, and  prepared as a control for  electrode  i n s e r t i o n , i n the  animals.  Tedisamil  dose dependently reduced VF  VPB's i n conscious rats subjected the  electrically  occurrence  stimulated  VT  nor  to coronary occlusion.  In  (paced)  were s i g n i f i c a n t l y  but  rats  reduced.  not  both A.S.  VF  and  was  VT  reduced  dose dependently by tedisamil, i n d i c a t i n g that the  incidence  and  tedisamil  severity of the  arrhythmias were reduced by  treatment (Figure 12 & Table 8).  Serum [K ] +  2%)  were  (3.6 ± 0.2  equivalent  equivalence  in  in  O.Z.  The  and O.Z  all  sizes,  appeared to have a greater (Figure 13).  mM)  the  the  s i z e (33 ± 2% to 37 ±  groups.  tedisamil  degree of S-T  i n i t i a l ECG  Despite treated  the  groups  segment elevation  e f f e c t s of tedisamil infusion  in the absence of ischaemia can be seen i n section 3.1.  The  tedisamil  induced  bradycardia  the  first  minutes  following  30  was  occlusion,  elevation seen a f t e r infusion was occlusion  (Figure  14).  maintained  during  however  the  BP  l o s t immediately following  133 Table 8. Arrhythmias Following Occlusion i n Conscious Rats: E f f e c t s of Tedisamil.  VT (0-•l/2h)  VF (0 -l/2h)  1 dur  C  0.5±0.1  0.9±0.2  0.2±0.1  1.7±0.2  Tl  0.6±0.2  0.9±0.4  0.3±0.2  1.8±0.3  T2  0.5±0.2  0.7±0.3  0.2± /  1.3± /  T4  0.7± /  0.9± /  0.5± /  1.4± /  T2S  0.7± /  1.0± /  0.6± /  1.3± /  LocfiQ VPB 0.5-4 h  1 #  1 dur  1 #  Dose  Arrhythmia Score 0-0.5 h  0.5-4 h  Dose  0-0.5 h  C  1.5±0.1  2.0±0.1  4.1±0.8  2.6±0.7  Tl  1.8±0.2  2.5±0.2  3.7±0.9  2.6±0.6  T2  1.6±0.2  2.1±0.4  2.4±0.4*  1.3±0.4*  T4  . 1.4±0.2  2.1±0.3  1.2±0.5*  1.9±0.6  C2  1.3±0.2  3.8±0.4  T2S  1.2±0.2  1.0±0.4*  The e f f e c t s of treatments on the arrhythmias induced by coronary artery occlusion i n conscious rats are shown. Treatments were C = conscious control, T l = tedisamil (1 mg/kg), T2 = tedisamil (2 mg/kg), T4 = tedisamil (4 mg/kg), C2 = control f o r ventricular "paced" rats, T2S = tedisamil (2 mg/kg) i n "paced" rats, see Methods. The columns shown are 1 # = log^Q number, 1 dur = log^n duration of v e n t r i c u l a r tachyarrhythmias (counted only i n those animals which experienced them) Log^o VPB = log^n v e n t r i c u l a r premature beats i n the time periods indicated, A.S. = arrhythmia score. Values are expressed as Mean ± S.E.M. S.E.M. not shown (/) f o r n < 5. indicates p < 0.05 versus control, by ANOVA followed by Duncan's range t e s t .  134  Figure incidence periods The  12  shows  the e f f e c t s  of VT and VF during following  occlusion  of tedisamil  on the  the 0-1/2 h and 1/2-4 h  i n c h r o n i c a l l y prepared  rats.  groups are indicated by: C = controls, TI = 1 mg/kg, T2  = 2 mg/kg, and T4 = 4 mg/kg tedisamil, CPb = pentobarbitone anaesthetized controls, T2S = 2 mg/kg tedisamil rats (0-1/2 h, only). * p < 0.05.  i n "paced"  Figure  12.  E f f e c t s of Tedisamil on Arrhythmias.  ARRHYTHMIA  INCIDENCE  (1/2-4h)  100 r  136  Figure 13 shows the e f f e c t s of tedisamil mg/kg) on ST segment elevation the  (1, 2, and 4  (as % R wave amplitude) i n  f i r s t hour following occlusion.  Each point  represents  mean ± S.E.M. at the time period indicated. Curves were f i t by Slidewrite software.  *p < 0.05.  Figure 13. Elevation.  Effects  of  Tedisamil  on  ST  Segment  138  Figure pressure  14  and  shows  heart  the e f f e c t s rate  before  of tedisamil on and  after  blood  occlusion i n  conscious r a t s . Means ± S.E.M. shown f o r n=9, * p < 0.05.  Figure 14.  E f f e c t s of Tedisamil on BP and HR.  Effects  on Blood Pressure  200 r  Pre-drug  Pre-occl +30min +1h Treatment period  Effects on Heart Rate 600 500 c  +4h  140  3.4.2.1.  Tedisamil i n Infarcted Rats  Administration week,  of tedisamil to rats with one-day, one-  or one-month  old infarcts  pressure, but slowed heart rate non-infarcted  rats.  d i d not a f f e c t  blood  to the' same extent as i n  For example, control values f o r heart  rate were 440 ± 12, 410 ± 15 and 330 ± 30 beats/min i n oneday,  one-week and one-month infarcted rats respectively.  4 mg/kg tedisamil, 259  heart rate  At  i n the three groups f e l l to  ± 9, 245 ± 16 and 225 ± 18 beats/min, respectively.  As  i n the acute ischaemia study, ST segment elevation was seen i n one-day infarcted rats a f t e r tedisamil treatment.  In  one-day  infarcted  rats,  suppressed VPBs i n 8/14 rats.  tedisamil  (1-4 mg/kg)  The control rate of LoginVPB  incidence was 2.0 ± 0.2 over 15 min, i n these r a t s . with older i n f a r c t s , VPB incidence antiarrhythmic e f f e c t s . VPB,  In rats  was too low to t e s t f o r  Proarrhythraic  effects  (increase i n  bigemini or alternating brady/tachycardia) occurred i n  8/14 one-day infarcted rats at a median cumulative dose of 7 mg/kg.  Proarrhythmic  infarcted tedisamil.  rats  after  effects  were  cumulative  seen  doses  i n 1/5 one-week  of 1 and 4 mg/kg  S i m i l a r l y arrhythmias were provoked i n 1/5 of  the one-month infarcted rats a f t e r 8 mg/kg tedisamil.  Respi  ratory t o x i c i t y (asphyxia) appeared to be the cause of death i n animals dying a f t e r doses ranging from 4-16 mg/kg.  141  3.4.3  Other K  Channel Blockers i n Rats.  The i n i t i a l success with tedisamil prompted us to seek out  and t e s t  with  a number of other  reported  Class  investigational  III activity.  Thus,  we  compounds tested the  analogue of tedisamil, KC8851, as well as the s o t a l o l and Nacetylprocainamide RP62719  derivatives,  i n the conscious  UK68,798,  r a t model  risotilide  of coronary  and  artery  occlusion.  These compounds were not e f f e c t i v e i n prolonging interval Only  i n the r a t except  KC8851 was e f f e c t i v e  arrhythmias doses  despite  (with regard  (section  i n preventing  being to Q-T  f o r KC8851  tested c  c  3.1.1.1).  ischaemia-induced  at maximally  widening) .  Q-T  effective  In the f i r s t  minutes following occlusion, none of the treatments  30  reduced  VT or VF incidence r e l a t i v e to control, although the control incidence  was  somewhat  lower  than  previous  studies have  shown (Figure 15). However, when the durations of VT and VF were  analyzed,  control.  KC8851  reduced  VF  duration  Neither VT duration nor Log^o  reduced by any of the treatments.  V  P  B  relative  to  number were  There was no difference  in the A.S. f o r the 0-30 minute time period i n any of the groups (Table 9). 4  hour  prevented  post VF  When arrhythmias were analyzed i n the 1/2 occlusion time  occurrence.  period,  A l l other  KC8851  treatment  treatments  i n e f f e c t i v e i n reducing VF incidence (Figure 15) .  were  None of  142  the treatments  altered  VT incidence nor VPB number.  In  animals i n which VT and/or VF occurred, drug treatment d i d not  shorten the duration of these arrhythmias  When arrhythmias over the f u l l  (Table 9) .  0-4 hour monitoring period  were analyzed again only KC8851 reduced log^n # (0.1 ± 0.1) and  log^n duration (1.1 ± 0.2) of VF r e l a t i v e  to control  (0.6 ± 0.2) and (2.1 ± 0.2) respectively. Also, only reduced the l o g  1 0  KC8851  duration of VT and VF combined (1.3 ± 0.2)  versus control (2.2 ± 0.2).  There were no differences i n O.Z. s i z e between any of the groups  (range 31 ± 4 to 38 ± 2).  Similarly  the ST  segment elevation was s i m i l a r i n a l l the groups, except f o r the KC8851 group, greater  i n which the ST segment  (maximum 81%) than  control  elevation was  (maximum 50%) as seen  previously with tedisamil (Figure 16).  In addition, as seen  with tedisamil, the second KC8851 infusion,  1.5 hours post  occlusion, produced an increased ST segment elevation.  The blood pressure f a l l  induced by occlusion was not  attenuated by any of the drug treatments fact,  over  risotilide  the  first  (10 mg/kg)  15  minutes  treatment  (Figure 17) .  following  exacerbated  In  occlusion hypotension  (p<0.05), the lower dose (5 mg/kg) also lowered BP r e l a t i v e to  control  at the 2 minute  periods (data not shown).  and 5 minute post  occlusion  143 Table 9. Arrhythmias Following Occlusion i n Conscious Rats: E f f e c t s of K+ Channel Blockers.  VT ro -4h)  VF (0-4h)  1 #  1 dur  C  0.910.2  1.310.2  0.510.2  2.010.2  KC  0.5±0.3  1.1+0.4  0.110.1  1.110.2*  UK  0.7±0.1  1.0+0.2  0.710.2  2.010.2  RP  0.8±0.2  1.310.3  0.510.2  1.910.3  R5  0.7±0.3  1.3+0.3  0.310.2  2.010.2  RIO  0.8±0.2  1.3+0.2  0.510.2  1.910.2  Dose  Locr  1 #  1 0 VPB  1 dur  Arrhythmia Score  0.5-4 h  0-0.5 h  Dose  0-0.5 h  0.5-4 h  C  1.4±0.1  2.310.2  2.810.9  4.610.7  KC  1.5±0.2  2.210.2  2.Oil.0  1.410.4*  UK  1.3±0.1  2.310.2  2.310.9  3.710.4  RP  1.310.3  2.510.2  2.811.0  4.010.5  R5  1.310.2  1.710.3  3.611.2  3.2+0.8  RIO  1.710.2  2.410.2  3.610.8  3.610.7  The e f f e c t s of treatments on the arrhythmias induced by coronary artery occlusion i n conscious rats are shown. Treatments were C = control, KC = KC 8851 (4m/kg) , UK = UK68,798 (1 mg/kg), RP = RP62,719 (1 mg/kg), R5 = r i s o t i l i d e (5 mg/kg), R10 = r i s o t i l i d e (10 mg/kg). The columns shown are 1 # = l o g g number, 1 dur = log^o duration of v e n t r i c u l a r tachyarrhythmias, Log^g 9"io v e n t r i c u l a r premature beats i n the time periods indicated, A.S. = arrhythmia score. Values are expressed as Mean 1 S.E.M. indicates p < 0.05 versus control, by ANOVA followed by Duncan's range t e s t . 1  V  P  B  =  lo  144  Figure  15 shows the e f f e c t s  of putative  class III  antiarrhythmic drugs on VT and VF incidence over the 0-1/2 h and  1/2-4 h time periods  rats.  following occlusion i n conscious  The groups indicated are:  C = controls; KC = 4 mg/kg  KC8851; UK = 1 mg/kg UK68,798; RP = 1 mg/kg RP62,719; R5 = 5 mg/kg r i s o t i l i d e ; RIO = 10 mg/kg r i s o t i l i d e . *p < 0.05.  145  Figure 15.  Effects  of  K  Channel  Blockers  Arrhythmias.  ARRHYTHMIA (0-1  100  INCIDENCE /2h)  r  75 -  Tachycardia  Fibrillation  ARRHYTHMIA  INCIDENCE  (1/2-4h) i oo r  O)  Tachycardia  c  I  I KC  Fibrillation  U K mzm  RP  rrrrtrm  R 5  .r=a  R 1 0  on  146  Figure  16 shows  antiarrhythmic amplitude)  the e f f e c t s  of putative  drugs on ST segment elevation  in  the  conscious r a t s .  first  hour  following  The groups indicated are:  risotilide. indicated.  R5  =  5  mg/kg  risotilide;  (as % R wave occlusion  in  C = controls; KC  = 4 mg/kg KC8851; UK = 1 mg/kg UK68,798; RP62,719;  class III  RIO  RP = 1 mg/kg =  10  mg/kg  Each point represents mean at the time period Curves were f i t by Slidewrite  0.05 versus control.  software.  *p <  147  F i g u r e 16. Segment  Effects  of  K  Channel  Blockers  on  ST  Elevation.  100 r  10  0  10  20  30  40  50  60  70  Time post-occlusion (mins) +  C  A  KC  O  UK  v  RP  •  R5  o  R1Q  148  Figure  17 shows  the e f f e c t s  of putative  class III  antiarrhythmic drugs on blood pressure and heart rate before and a f t e r occlusion i n conscious rats. are:  The groups indicated  C = controls; KC = 4 mg/kg KC8851; UK = l mg/kg  UK68,798; RP = 1 mg/kg RP62,719; R5 = 5 mg/kg RIO = 10 mg/kg r i s o t i l i d e . *p < 0.05.  risotilide;  Means ± S.E.M. shown f o r n=9,  149  Effects on Heart Rate  150  The bradycardia produced by KC8851 was maintained u n t i l 30 minute post occlusion. relative  t o control  The heart rate was also decreased  i n the f i r s t  5  minutes  following  occlusion by 5 mg/kg r i s o t i l i d e (p < 0.05) (data not shown). However, by 30 min post occlusion there were no s i g n i f i c a n t differences i n HR between control and any treatment  (Figure  17) .  3.4.4  K  Acutely assess  +  Channel Blockers i n Acutely Prepared Rats  prepared  rats were used  whether  tedisamil  (4mg/kg)  gave  protection  against  rag/kg)  arrhythmias. followed  i n d e t a i l previously  In  tedisamil  study  (10  reperfusion  induced  to 10 minute  ischaemia  The results  been published this  study to  and glibenclamide  Rats were subjected  by reperfusion.  in a pilot  of t h i s  (Beatch  study have  et al., 1989).  (4 mg/kg) and glibenclamide  (10  mg/kg) were given before occlusion and compared t o a number of  interventions  which  reduced  r a d i c a l s and various eicosanoids.  the influence These l a t t e r  of  free  treatments  were i n e f f e c t i v e and are not relevant to the focus of t h i s thesis  and therefore  interest  t o note  channel blocker,  will  whether  not be discussed. the putative  glibenclamide,  could  I t was of  ATP s e n s i t i v e  offer  K  +  protection i n  t h i s model given the numerous speculations and few studies in  the l i t e r a t u r e  1990).  (Kantor  et al., 1990; Bekheit  et al.,  151  In t h i s study tedisamil (4 mg/kg, n = 5) eliminated VT & VF  i n the 10 minute  reperfusion period. of ischaemia (10  ischaemia  period  and VF  The small group s i z e and short duration  may have biased these r e s u l t s .  Glibenclamide  mg/kg, n = 11 occlusion; n = 7 reperfusion)  protective  effect  i n the  on either  arrhythmias  had no  i n either  time  period, and a c t u a l l y increased the duration of the VF seen i n the 10 minute occlusion period Table  10) .  Neither  occurrence of VPB. O.Z.  drug  (p < 0.05) (Figure 18 &  significantly  saline  +  0.05) .  caused bradycardia  (86 ± 5%) r e l a t i v e  i n the groups.  (190 ± 25 b/min) vs. control  (380 ± 30 b/min) and increased  elevation  the  Serum [K ] (3.6 ± 0.2 to 4.1 ± 0.2) and  (33 ± 1 to 36 ± 2) were equivalent  Tedisamil  decreased  maximum  to control  (45  ST segment ± 8%)  (p <  152 Table 10. Antiarrhythmic Effects of Glibenclamide and Tedisamil. Arrhythmias  Following Occlusion i n Anaesthetized Rats VT  VF 1 dur  Dose C T G  0.9±0.1  1.5±0.1  n/a  n/a  0.910.1  1.510.2  , 1 dur  0.210.1  1.310.3  n/a  n/a  0.2+0.1  1.910.1*  Arrhythmias Following Reperfusion i n Anaesthetized Rats VT  VF  1 #  1 dur  C  0.6+0.2  1.510.2  T  0.31 /  1.31 /  G  0.710.2  1.410.2  Dose  Locf  10  VPB  . 1 dur  0.210.1 n/a 0.410.2  1.810.5 n/a 1.510.4  Time to VT (s)  Dose  Occlusion  Reperfus,  Occlusion  Reperfus,  C  1.910.1  1.810.2  330130  10  T  1.310.3  1.310.1  n/a  10  G  1.510.2  1.510.1  320130  7  The e f f e c t s of treatments on the arrhythmias induced by 10 min coronary artery occlusion followed by reperfusion i n anaesthetized r a t s are shown. Treatments were C = control, T = tedisamil (4m/kg), G = glibenclamide (10 mg/kg). The columns shown are 1 # = log^n number, 1 dur = log^n duration of ventricular tachyarrhythmias, Log^n VPB = log^n v e n t r i c u l a r premature beats i n the time periods indicated. Values are expressed as Mean 1 S.E.M. indicates p < 0.05 versus control, by ANOVA followed by Duncan's range t e s t .  153  Figure  18  shows  the  glibenclamide on incidence ischaemia  and followed  effects  (empty  bars);  tedisamil  of VT and VF during  by reperfusion  anaesthetized acutely prepared r a t s . control  of  T4, indicates  and  10 min of  i n pentobarbitone  Con, indicates s a l i n e tedisamil,  4 mg/kg  ( s o l i d bars); GL, indicates glibenclamide, 10 mg/kg (hashed bars);  * indicates p < 0.05 versus control.  154  Figure 18.  Effects  of Glibenclamide and Tedisamil  on Arrhythmia Incidence.  Occlusion — Arrhythmias (% incidence of V T and V F ) 150  8  100  c  10  £  50  Tachycardia  Fibrillation  Reperfusion  — Arrhythmias  (% incidence of V T and V F ) 150  8  100  (0  c  (0  sp  50  Tachycardia con  Fibrillation  T4  155  3.4.5 K  Channel Blockers i n Acutely Prepared Pigs  Tedisimal tested  against  ischaemia adjunct  (8 mg/kg) and UK68798 arrhythmias  i n anaesthetized  produced pigs.  to the cardiovascular  (167.5 by  The  ug/kg) were  acute  myocardial  studies  were an  assessment of the drugs i n  t h i s species and were conducted i n an open design.  Only 5-6  animals per group were used and the arrhythmia incidence was compared to h i s t o r i c a l controls conducted i n our laboratory.  Neither drug decreased log^o VPB incidence i n the f i r s t hour following occlusion. nor  UK68,798  control pigs  Likewise,  (4/5) decreased  (11/16) .  The incidence  VT  neither tedisamil (4/6) incidence  relative  to  of VF was low i n control  (3/16) as compared to rats and as such detecting an  a n t i f i b r i l l a t o r y e f f e c t would require a large sample s i z e . There were no episodes of VF i n the tedisamil treated group while  VT  UK68,798.  degenerated  into  VF  i n two pigs  treated  with  No primary VF was seen i n the UK68,798 group.  The groups had s i m i l a r O.Z. s i z e .  (Table 11).  156 Table 11.  Arrhythmias Following Occlusion (O-lh) i n Pigs  Dose  Locfio  VF  O.Z.  C  1.8±0.1  11/16  3/16  3312  T  2.410.5  4/6  0/6  2612  UK  2.510.2  4/5  2/5  V  P  B  VT  1  2912  The e f f e c t s of treatments on the arrhythmias induced by coronary artery occlusion i n anaesthetized pigs are shown. Treatments were C = control, T = tedisamil (8 mg/kg) , UK = UK68,798 (157.5 /ug/kg). The columns shown are Log^n VPB = login v e n t r i c u l a r premature beats, O.Z. = occluded zone size as percent v e n t r i c u l a r weight, (Mean 1 S.E.M.), VT = VT incidence per group, VF = VF incidence per group. *VF i n i t i a t e d by VT.  Summary:  3.4.6  Tedisamil  and  antifibrillatory ischaemia  Ischaemia induced Arrhythmias  KC8851  activity  demonstrated  dose  i n rats subjected  dependent  to  myocardial  and i n rats further subjected to reperfusion.  An  additional protection against VT was also seen i n rats whose hearts  were  bradycardia  stimulated was  such  reversed.  that  the tedisamil  Tedisamil  induced  otherwise  d i d not  a f f e c t the incidence of VT nor VPBs i n the r a t . results  were seen  ischaemia,  i n pigs  however,  an  subjected unequivocal  to acute  Similar myocardial  demonstration  of  a n t i f i b r i l l a t o r y e f f e c t i n pigs would have required a larger sample  size.  UK68798,  RP62719  antiarrhythmic i n the r a t at a l l .  and r i s o t i l i d e  were not  This was consistent with  t h e i r lack of e f f e c t s on the ECG i n t h i s species.  UK68798,  was not protective i n pigs, despite a small but s i g n i f i c a n t widening of the Q-T  c  i n t e r v a l i n t h i s species.  Again, due  to the small sample s i z e used, an a n t i f i b r i l l a t o r y e f f e c t of UK68798 However,  would  not have  i t would  been  appear  detectable  safer  to  i n this  speculate  study. that  no  protection was afforded against VPB or VT incidence by t h i s drug i n the p i g model.  158  4  DISCUSSION  4.1  Pharmacology  The  first  cardiovascular  section  of the  discussion  deals  e f f e c t s of class III drugs.  e f f e c t s on heart  rate and  with  I n i t i a l l y , drug  blood pressure w i l l be examined.  Species dependent actions of the drugs on the ECG discussed  in  actions.  Possible  these  drug  effects  relation  of  to  possible  proarrhythmic  effects w i l l tedisamil,  the  be  be  electrophysiological  actions  discussed.  KC8851,  will  associated Finally,  UK68798,  with  the  RP62719  ECG and  r i s o t i l i d e w i l l be compared to other class III drugs.  In subsequent sections, mechanisms of drug e f f e c t s w i l l be  interpreted through analysis of responses to  stimulation  and  Antiarrhythmic  effectiveness w i l l be compared f o r the drugs,  and  evaluated  intracellular  electrical  in  relation  to  other  F i n a l l y , general conclusions w i l l be  4.1.1  Class  III  Drug  recording  class  studies.  III  drugs.  discussed.  Effects  on  Heart  Rate  and  Blood Pressure.  The  most prominent e f f e c t of tedisamil  i n a l l species was  dose dependent bradycardia.  administration At a 4mg/kg  dose, the reduction i n heart rate ranged from 17% i n pigs to 34%  i n conscious  rats.  The  order of  sensitivity  to  the  159  bradycardic  effects  (%  reduction  i n HR)  of  tedisamil  was  guinea p i g = r a t = rabbit > cat > baboon > p i g (Buschmann et al. 1989).  The absolute reduction i n heart rate (among the  d i f f e r e n t species) was  dependent on the i n i t i a l heart rate.  This suggests that tedisamil a l t e r s a current which normally controls  cycle  length  blockade i n cat (Howard action  et of  al.,  in  sinus  nodal  (our unpublished 1989)  had  tedisamil.  no  cells.  Autonomic  observations) effect  Tedisamil  on  did not  and  the  i n rat  bradycardic  affect  pacemaker  depolarization rate or threshold p o t e n t i a l i n i s o l a t e d sinus node  and  tissue  apparently  contributed  observations). pacemaker  An  prolongation to  the  earlier  of the AP  bradycardia study  depolarization rate,  (our  reported  which  was  i n pacemaker unpublished  a reduction  in  insufficient  to  account f o r the bradycardia seen (Oexle et al., 1987).  KC8851, tedisamil's analogue, had  s i m i l a r potency  e f f i c a c y to tedisamil i n rats (Table 1). Class  III agents,  and  The other putative  UK68,798, RP62,719, and  risotilide  had  s l i g h t or no e f f e c t s on heart rate when tested i n rats, pigs and guinea pigs and baboons. drugs had  This would suggest that these  minimal e f f e c t s on the currents contributing to  pacemaker rate i n sinus nodal c e l l s .  Since these drugs have  been reported to s e l e c t i v e l y block I R (see section 1.4.1), i t would appear that I R i s either not present, or makes only a  minor  (Irisawa,  contribution 1987).  to  pacemaker  These r e s u l t s  rate  suggest  in  nodal  cells  that tedisamil's  160  bradycardic are not  related to i t s additional suppression of IR  et al.,  1990).  The  bradycardic  action of tedisamil  i t s p o t e n t i a l use as an antianginal Ulrich  Kuhl;  Grohs  et  a p p l i c a t i o n might be shown tedisamil  al.,  to  O2  reduce  ischaemia-induced  model (Johnston et al.,  In  a l l species  tested,  tedisamil  augment  may  have  elevation of BP rats  treated  tedisamil  was  1977;  IR  with  blockers  i n the  Abraham et al.,  tedisamil  conscious  treatment tended  did  the  they  make Class  mild  the  to  other  This suggests not  cause  contractile  force.  hypertensive e f f e c t s of  I I I drugs a more desirable  seen i n  response  did  apparent lack of e f f e c t s on BP of these new  or possible  Slight  None of  used  augment  and  Alternatively,  hypertensive  doses  to  1988), which i n  vasoconstriction.  The  rat  1989).  Beatch et al.,  i n infarcted r a t s .  at  nor  this  Bradycardia does not  contractility.  KC8851.  lost  for  bradycardic  (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 ) was  vasoconstriction The  basis  studies have  its  Class I I I drugs nor glibenclamide altered BP. that  (Dukes  Although,  demand v i a  caused  *  communication,  1989).  1989).  a n <  possibly secondary to bradycardia  AP widening (Carmeliet, might  (personal  arrhythmias  1983;  increase BP, which was  itself  i s the  of questionable benefit,  action i n dogs (Grohs et al., reduce  I^o  e f f e c t s r e s u l t from i t s i n h i b i t i o n of  compounds,  tedisamil,  choice for  would  patients  161  with an  infarction,  than  Class I and  IV drugs which  may  depress c o n t r a c t i l i t y i f given i n s u f f i c i e n t dosage ( J a i l l o n &  Ferry,  1988) .  fraction,  As  infarction  c l e a r l y drugs without  already  negative  lowers  ejection  inotropic  effects  are desirable i n t h i s population of patients. hypertensive  patients,  tedisamil's  BP  However, i n  elevating  effects  might be undesirable.  4.1.2  Overview of ECG Analysis  Class  III  drugs,  as  discussed  r e p o l a r i z a t i o n of the cardiac AP. surface  ECG  ventricle  reflects  (Einthoven,  should widen the Q-T  c  the  previously,  Since the T-wave of the  repolarization  1912;  delay  Katz,  1928),  phase Class  i n t e r v a l of the ECG.  in  I I I drugs  For drugs with  s e l e c t i v e Class III actions no other e f f e c t s on QRS intervals  should  be  seen  due  to  a  the  lack  of  or  P-R  effect  on  conduction v e l o c i t y i n a t r i a l , nodal, or v e n t r i c u l a r t i s s u e . T y p i c a l l y Class I drugs widen QRS  and P-R  i n t e r v a l s i f given  i n doses s u f f i c i e n t to slow conduction v e l o c i t y . drugs , beta blockers, can widen P-R  Class II  i n t e r v a l s in vivo i f AV  nodal conduction has been enhanced by a s i g n i f i c a n t degree of  sympathetic  tone  before  administration,  i n t e r v a l r e f l e c t s the conduction time through Class IV drugs, C a given  + +  in sufficient  dihydropyridines  antagonists, can widen P-R doses  to  inhibit  I i« s  as  the  the AV  P-R node.  intervals i f I n  vivo  (e.g. nifedipine) are less potent  the  in this  162  regard  than  phenylalkylamines  benzothiazepines approximation,  (e.g.  (e.g. diltiazem).  a drug  which  verapamil)  Thus  selectively  as  widens  or  a  first  the Q-T  c  i n t e r v a l i n a dose dependent manner may be characterized as having  Class I I I e f f e c t s .  However, not a l l drugs  which  widen the Q-T i n t e r v a l can be considered primarily as Class c  III  antiarrhythmics.  For example, prolonged  beta-blocker  therapy has been reported to prolong APD (Raine & VaughanWilliams, 1981) possibly through an action  (independent  sotalol).  of d i r e c t APD prolongation as with d-  Q-T prolongation has also been encountered with c  neuroleptics, insecticides, (Fish  alpha-receptor mediated  tricyclic  antidepressants,  erythromycin  & Roden,  1989).  organophosphate  and numerous non-cardiac  Bradycardia produced  drugs  by s p e c i f i c  bradycardic agents or as a side e f f e c t or by a c t i v a t i o n of reflexes also widens the Q-T i n t e r v a l . of  bradycardia,  normalize  numerous  the Q-T  formulae  interval  Due to t h i s  have  f o r changes  been  effect  devised to  i n HR, the most  widely accepted formula i s that of Bazett (1920), which i s Q-T/R-R / . -1  tends slower  2  This formula has been c r i t i c i z e d however as i t  to overcorrect at fast rates.  Antiarrhythmic  electrophysiological antiarrhythmics, block  both  gNa  rates and undercorrect at  actions,  quinidine, +  and gK  +  drugs  f o r example,  disopyramide  with  mixed  the Class l a  and  procainamide  and therefore widen  both  QRS  duration and Q-T i n t e r v a l s i f given i n s u f f i c i e n t dosage. c  163  There are also pathological conditions i n which long Q-T  i n t e r v a l s are  c  long  Q-T  observed i n the  syndrome occurs  which congenital affliction increased  of  (Jackman et al.,  major conditions,  deafness i s present  (Jackman risk  i n two  drug free state.  al.,  et  1988).  TdeP associated  1988).  and  one  There  with  one  without has  long  The in this  been  Q-T  an  syndrome  This apparent r e l a t i o n s h i p as well  as reports of TdeP associated with Class I I I drugs has added an a i r of caution to the introduction of Class III drugs as a therapy for  arrhythmias.  4.1.2.1  E f f e c t s on ECG i n t e r v a l s  In  only  rats,  interval, effects  while  in this  support  the  tedisamil and  the  to  that  repolarization in  contributes  little  to  ventricle.  None of the putative K  tested  i n the  the  decrease  concentrations 1990).  gNa  +  +  the  at  room  current  +  ventricle is present  or  in rat  channel blocking drugs duration  of  the  QRS +  Tedisamil has been shown temperature  > 50uM (Dukes & Morad, 1989;  Interestingly, t h i s gNa blockade was +  c  without  that at the doses used no signs of gNa  blocking a c t i v i t y were manifest. to  rat  K  r e p o l a r i z a t i o n process  rat prolonged  i n t e r v a l suggesting  major  e i t h e r I R i s not  1984)  and  were  Q-T  These r e s u l t s further  the  (Josephson et al.,  we  blockers  (Table 2) .  observation  contributing  IR  putative  species  KC8851 widened the  in  vitro  at  Dukes et  al.,  e f f e c t i v e from  164  the external surface  i n a manner s i m i l a r to TTX  (Kao,  1966;  1986).  This property allowed for rapid r e v e r s i b i l i t y upon  washout  (Dukes & Morad,  proarrhythmic  effects  administration, slowing, may  1989)  as gNa  coupled  to  precipitate  of  and  may  be  tedisamil  related  seen  to  after  the  bolus  channel blockade-induced conduction  +  possible  reentry  heterogeneous  (Mines,  1913;  refractoriness  1914).  While  no  thorough studies on the t i s s u e s e l e c t i v i t y of tedisamil have been performed, the hint  at  a tissue  appeared  which  species  dependent e f f e c t s of  selectivity  as  shows tissue  well.  selectivity  tedisamil  One  abstract  has  of  tedisamil  in  i s o l a t e d rabbit SA node and guinea pig hearts and p a p i l l a r y muscle I-tOf  (Oexle et a l . , 1987).  Guinea pig v e n t r i c l e lacks  but e f f e c t s on 1^ would be seen i n t h i s t i s s u e (Giles &  Imaizumi, 1988).  None  of  the  drugs  tested  had  effects  on  the  P-R  i n t e r v a l i n rats except for tedisamil and KC8851, which both widened P-R due  to C a  to a s i m i l a r extent.  + +  This e f f e c t was  unlikely  antagonism, as these drugs tended to  increase  blood pressure and  not  impair c o n t r a c t i l i t y .  In  addition,  Dukes et a l (1989) have shown tedisamil  (0-100uM) does not  impair g C a  Alternatively,  ++  i n i s o l a t e d rat myocytes.  P-R  i n t e r v a l i s s e n s i t i v e to gNa blocking drugs and bradycardia +  (Driscoll  et  al.,  c e l l s of the AVN  1981).  C l a s s i c a l l y the  do not use Na  (Hoffman & Cranefield,  1960).  +  currents  central  nodal  for depolarization  However, border c e l l s might  165  be more sensitive to the gNa than  ventricular  muscle,  inactivation  of  AVN  cells.  border  component of increase Thus,  a large  gNa  in  by  +  the  with  owing  to  Further  reduction  tedisamil  would be  greater  number of area  of  conducting t i s s u e to traverse,  Ca  In  primates,  remarkably efficacious  in  widened the  Q-U  pigs and  (apparent Q-T  guinea EC50  or  c  i n guinea pigs.  slower r e s t i n g heart rates and which  would  larger  role  possibly  primates, While  allow  the  delayed  i n c o n t r o l l i n g APD,  risotilide  which  most  efficacious guinea pigs,  had  suggests  efficacious at and  Q-T  c  or  an  cells. slowly  influence  of  recently  pigs  10  the been  Q-U  The  2 0%  in Q-U  c  UK68798  i n primates  larger  species have  1^,  to  in  have  larger  species.  to UK68,798 i n  similar  mechanism  rats,  tedisamil  of  action.  was  more  i n baboons than  least e f f e c t i v e i n pigs.  a  I R would  thus blockade of  widening  and  than guinea pigs,  rectifier,  effects  was  minimally  interval.  c  similar effects a  UK68,798  ug/kg) but  longer APD  have more pronounced  RP62719 and  The  i n t e r v a l maximally by  only 9%  to  nodal  dependent  + +  small  equivalent  sinus node has  and  widening c  their  1987).  pigs  potent  of  the  the wave of e x c i t a t i o n might  surrounding the  studied (Kirchof et al.,  dependent  channels i n  +  central  take longer to reach the v e n t r i c l e s . a t r i a l tissue  tedisamil  voltage  proportion of Na  apparent  a  blocking e f f e c t s of  The  from these primate studies i s that humans might be  in  implication sensitive  166  to the Q-T  widening e f f e c t s of tedisamil.  c  r e s t i n g heart as  initial  rate did not govern s e n s i t i v i t y to tedisamil I R blockers,  f o r the putative  widening  Also the  e f f e c t s of  tedisamil  suggesting  that the  were secondary  Q-Tc  to i t s  blocking e f f e c t s .  The pig's r e l a t i v e lack of s e n s i t i v i t y to  tedisamil  suggest  that  markedly  dependent  on  ventricular repolarization i s or  IR  in  this  species.  not The  currents underlying the pig v e n t r i c u l a r action p o t e n t i a l may be  i n f e r r e d from the  ventricular 1977)  morphology of  subepicardium.  recorded  These studies  from  al.,  (Downar et  i s not a major contributor to  r e p o l a r i z a t i o n i n pig v e n t r i c u l a r subepicardium.  recent  report  using  myocardium i t was notch  the  showed a broad plateau AP without the "spike and dome"  appearance which suggests AP  AP  after  transmembrane  recording  of  In a baboon  shown that e p i c a r d i a l c e l l s had a d i s t i n c t  phase  0  endocardial  cells  (Dangman  observations  might  distribution  of  the  be  and  a  shorter et  explained  channels  canine v e n t r i c l e (Litovsky  duration  al., by  AP  than  1988). a  tissue  responsible  & Antzelevitch,  for  These dependent  I-to»  a s  1988), and  i  n  may  p a r t l y explain tedisamil's tendency to generate U waves i n the ECG of baboons.  None of the putative I R blockers prolonged QRS i n t e r v a l s i n any  species tested.  to widen the QRS  and  e f f e c t s i n pigs and  P-R  Tedisamil  or  P-R  however tended  i n t e r v a l s i n baboons with minimal  guinea pigs.  These e f f e c t s i n baboons  167  most l i k e l y resulted from the Na  channel blocking actions  of tedisamil as discussed above.  4.1.2.2  An  Proarrhythmic E f f e c t s  interesting  prominent i n pigs  observation,  (although  which  was  especially  least s e n s i t i v e to tedisamil's  bradycardic action), was the tendency towards a "sick sinus" syndrome or a l t e r n a t i n g sinus bradycardia/tachycardia high  doses  of  tedisamil.  As  the  course  of  after  membrane  p o t e n t i a l changes i s a dynamic process  i n pacemaker c e l l s ,  alterations  phase  in  the  repolarization  influences over the e n t i r e cycle. that  a  bradycardia  dependent  triggered  a  tachyarrhythmia  "overdrive  suppression."  was  1977)  have  p o s s i b i l i t y exists  early  after  depolarization  which  self  terminated  A l t e r n a t i v e l y , since  c e l l s have slow conduction ( A l l e s s i e et al.,  The  could  by  sinus node  a leading c i r c l e micro reentry  could have been i n i t i a t e d  i f there  a dispersion of refractoriness i n the nodal t i s s u e .  Another anecdotal observation, made during studies with primates, was that tedisamil induced v e n t r i c u l a r arrhythmias which  could  be  converted  RP62,719 or r i s o t i l i d e . the  latter  caused  by  to  sinus  rhythm  by  UK68,798,  I t i s possible to speculate  drugs reduced the tedisamil, thereby  Since a c t i v a t i o n mapping was  that  dispersion of r e f r a c t o r i n e s s terminating  a  reentrant  not done and the  VT.  observations  168  are  anecdotal,  no firm  conclusions  can be drawn.  It i s  i n t e r e s t i n g to note i n t h i s regard that amiodarone has been used s u c c e s s f u l l y to t r e a t patients with previous history of drug induced torsade de pointes (Mattioni et al., 1989).  None of the Class III drugs which have been reported to s e l e c t i v e l y block I R (UK68,798, RP62,719, r i s o t i l i d e ) caused arrhythmias  at the doses used.  Tedisamil,  on the other  hand, with mixed electrophysiological e f f e c t s (Dukes et al., 1990) and  and multiple ECG e f f e c t s d i d t r i g g e r episodes VT.  I t therefore  appears  that  some  of VPB  proarrhythmic  e f f e c t s seen with previous Class I I I and l a agents may be related  to  their  mixed  electrophysiological  Selective AP widening may be necessary provoke arrhythmias  effects.  but i n s u f f i c i e n t to  i n normal myocardium without concomitant  hypokalemia, hypomagnesemia, or other abnormalities.  This  i s not e n t i r e l y surprising as the heart already has AP of d i f f e r i n g durations depending upon the t i s s u e , i . e . Purkinje fibres  have  the  longest  APD,  yet normally  the  heart  associated  with  functions i n a remarkably coordinated manner. As  noted  above,  TdeP  has  been  hypomagnesemia (Loeb et al., 1968) and hypokalemia et al., 1967).  (Tamura  Since hypomagnesemia may be associated with  a loss of the inward r e c t i f i c a t i o n properties, and inward conductance through I J Q i s dependent on [ K ] +  Q  V2 these  conditions impair the membrane p o t e n t i a l  function  of  IRI*  I  n  addition  Na /K +  +  ATPase  b o  t h of  stabilizing i s also  169  i n h i b i t e d by these conditions.  Thus, the net r e s u l t might  be  membrane  a  loss  of  subsequent  over  voltage-mediated  Quinidine 1964).  control  has  been  These  two  suppression  of  polarization  inactivation  associated with separate  of Na  TdeP  conduction  velocity  channels.  +  (Selzer  observations  & Wray,  suggest  is  and  an  that  a  additional  component that may be necessary to reveal the proarrhythmic e f f e c t s of drugs which prolong APD. The other factor which may proarrhythmic effects. pointes  potential  may  be  1983;  due  to  was  i t s bradycardic  1985;  VT  in  refer  vivo,  CsCl  and  to  cesium  which  chloride  and  effects  (Howard  et  of tedisamil  pentobarbitone s i g n i f i c a n t QRS  al.,  showed  occurred at doses  and P-R  latter induced  may  not  be  The f i r s t  two  the  third  In rats,  not proarrhythmic.  1989)  anaesthetized  1985;  to  Clearly these studies are  even further removed from our studies. mg/kg tedisamil or KC8851 was  The  anthopleurin-A  respectively  quinidine-induced EADs in vitro.  studies  (Brachman et  E l - S h e r i f et a l . , 1988). to  de  after  Roden & Hoffman,  related to the tedisamil induced arrhythmias. references  torsade early  (EADs) and triggered a c t i v i t y  studies referred  polymorphic  that  bradycardia-dependent  Damiano & Rosen, 1984;  Levine et al., two  tedisamil  Several recent studies suggest  depolarizations al.,  of  have been important i n the  rats.  At  prolongation was  the of > this seen.  up to 4 Previous  proarrhythmic 15 mg/kg i n dose  level  This study  also showed that the proarrhythmic e f f e c t s of tedisamil were  170  eliminated when the autonomic nervous The  proarrhythmic  doses  (4-7  Numerous  effects  mg/kg)  in  studies have  damaged tissue  system  of tedisamil  blocked.  occurred at  infarcted  rats  shown the  arrhythmic  (review:  was  (Section  lower  3.4.2.1).  potential  Hoffman & Dangman, 1987).  of  Whether  tedisamil caused arrhythmias by the same mechanism i n both normal  and  damaged  hearts  is  a  moot  point  (although  deserving of further research), the fact that tedisamil proarrhythmic against  at  acute  doses  showing  ischaemia  was  antiarrhythmic properties  suggests  the  drug  may  have  s i g n i f i c a n t proarrhythmic potential at therapeutic l e v e l s i n post-infarcted receiving  hearts.  As  antiarrhythmic  the  majority  treatment  have  of  patients  a  previous  had  i n f a r c t i o n , the usefulness of tedisamil as an antiarrhythmic drug  may  be  severely limited,  s e n s i t i v i t y as r a t s .  i f patients show the same  Clearly, more studies i n primates with  infarcted hearts should be c a r r i e d out before consideration i s given to t r e a t human subjects with previous myocardial infarction. the  Although,  proarrhythmic  proarrhythmic  not d i r e c t l y  potential  e f f e c t s may  of  result  shown i n our  tedisamil from  studies,  would  suggest  combination  therapy  using one of the more " s e l e c t i v e " Class III agents (UK68798, RP62719, r i s o t i l i d e ) and a Class I antiarrhythmic, although Class  I  and  beneficial 1990)  III combination  endeavor,  has  theoretically,  and with Class l a drugs.  been  proposed  (Hondeghem  to &  be  a  Snyders,  171  4.1.2.3  Summary  In conclusion, analysis of the ECG showed tedisamil and KC8851 exert mixed actions which are species dependent and d i f f e r e n t from the other putative Class I I I agents tested. Tedisamil with  and KC8851 widened Q-T  dose  (Dukes  dependent  et  i n a manner consistent  c  blockade of myocardial  a l . , 1990).  Instances  prolongation may have resulted  from Na  UK68798  Q-T  selectively  widened  of +  c  P-R  intervals  c  channels  +  interval  channel blockade.  consistent with I R blockade (Gwilt et al., Q-T  K  in  1991).  pigs,  However,  widening was not seen i n guinea pigs, which suggests  UK68798 may be i n e f f e c t i v e at higher HR.  UK68798, RP62719  and r i s o t i l i d e had no ECG e f f e c t s i n rats, at supramaximal doses,  which  suggests  they  have  selective  e l e c t r o p h y s i o l o g i c a l actions, as rats are s e n s i t i v e to gNa and  gCa  ++  blocking  drugs.  These  results  should  now  +  be  considered i n the context of other research with Class I I I drugs.  4.1.3  Comparison with Other Class I I I Drugs  4.1.3.1  Amiodarone  As mentioned i n the introduction, amiodarone i s a drug with complex actions. pharmacokinetics,  Not only does amiodarone have complex  multiple  side  effects  unrelated  to i t s  172  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 , e.g. corneal deposits, thyroid dysfunction and lung f i b r o s i s antiarrhythmic  actions  (Singh et al., 1989), but i t s  f i t a l l four  o r i g i n a l classes (Class I:  of  Vaughan-Williams  Singh & Vaughan-Williams, 1970;  Mason et al., 1983; Class I I : C h a r l i e r et al. 1968; Polster & Broekhuysen, III:  Singh  levels  do  1976; Class IV:  & Vaughan-Williams,  not  actions.  correspond  Thus amiodarone  reference  Gloor et al., 1983;  standard  1970).  well  the t i s s u e  with e l e c t r o p h y s i o l o g i c a l  would  Class  Also  Class  be  III  a poor  choice  drug.  for a  However,  the  effectiveness of amiodarone makes i t an agent of l a s t resort for  life-threatening  toxicity,  unclear  pharmacokinetics  4.1.3.2  v e n t r i c u l a r arrhythmias mechanism  of  despite i t s  action  and  bizarre  (Lazzara, 1989).  In Search of a Reference Standard  Thus, what i s an appropriate reference Class I I I drug? According  to Vaughan-Williams  1  classification  scheme, the  ideal compound would have homogeneous AP widening e f f e c t s i n the absence of any e f f e c t s on conduction v e l o c i t y Williams,  1970),  prolongation. clinical  use  or  other  C l e a r l y there which  f i t this  actions are no  (Vaughan-  unrelated  "Class  category.  to  I I I " drugs i n  Bretylium  i s an  adrenergic neuron blocking agent (Boura & Green, 1959). progeny, bethanidine, blocking gK  +  a l t e r s gNa  +  and g C a  ++  AP  Its  i n addition to  (Bkaily et al., 1988), and meobentine has been  173  shown to block  gNa  (Wang et al.,  +  beta blocking a c t i v i t y although  1977), d , l - S o t a l o l has  d-Sotalol i s 1/50 times as  potent as i t s enantiomer i n t h i s regard (Kato et al., Acecainide chronic block  appears  therapy gNa  neuroleptic  well  drugs  in  (Lumma  have  been  I I I , but i n vivo  and thus  al.,  1987).  Certain  reported  to have  Class I I I  et  actions, e.g. melperone (Arlock et al., (Hoglund et al.,  Class  i t may be deacetylated  as  +  t o be s e l e c t i v e l y  1986).  1978) and amperozide  1986), but, c l e a r l y these, and the numerous  other drugs with coincidental Class III properties, can not be  considered  ammonium  as ideal  derivative,  reference  clofilium,  drugs. has  The  been  quaternary  suggested  i n t e r a c t with alpha adrenoceptors and possibly C a (Arena & Kass, 1988). AP  have  profiles  different  + +  to  channels  S i m i l a r l y , cesium chloride, TEA or 4-  of action  from an ideal  in  vivo  that  are markedly  (cardioselective) Class  I I I drug.  I t would be f o l l y to use one of the other newly developed drugs as a reference Class I I I , as s u f f i c i e n t data are not available t o make such a claim.  Thus, one i s l e f t t o choose the l e s s e r of e v i l s . this  regard,  administration) most  selective  the  effects  of  d - s o t a l o l and c l o f i l i u m Class  I I I drugs  sufficient  data  recognized  that using a reference  will  only  acecainide  f o r purposes  assist  in  (on  acute  appear t o be the  f o r which  of comparison. standard  identifying  In  drugs  there  are  I t must be  even i f i d e a l , with  similar  174  properties  (Brugada,  1987;  1990)  and  that  drugs  p o t e n t i a l l y b e n e f i c i a l , but unrelated actions may It  goes without  saying  that  i f the  reference  with  be missed. standard  is  f a l s e l y chosen to begin with, the l i k e l i h o o d of a r r i v i n g at an  ideal  drug  i s greatly diminished.  The  development of  histamine H2 receptor antagonists demonstrated the value of an  appropriate  versus  screening  properties 1972) .  bioassay  of  The  a  (here,  gastric  acid secretion),  for  compounds  with  false  reference  standard  only way  in  to t e s t whether the  vitro  binding  (Black third  et  al.,  c l a s s of  antiarrythmic a c t i v i t y i s indeed antiarrhythmic i s to assay compounds which f i t the  proposed d e f i n i t i o n .  Thus, i f a  drug possesses s e l e c t i v e AP widening e f f e c t s , and a l l other actions of the drug are accounted for, and i t i s found to be protective against arrhythmias, a  defined  probability)  then i t can be assumed (with  that  Class  III  actions  antiarrhythmic  protection i n the model used.  may  proved  only  be  antiarrhythmic, conditions no  if  that  Class  under  the  relationship  controlled  same  However, i t  actions rigidly  are  not  controlled  protection i s seen when the requirements of  the d e f i n i t i o n are f u l f i l l e d . causal  III  confer  there  conditions.  can proof be attained.  In order to obtain proof of a is  a  necessity  Thus r a r e l y  for  rigidly  in biological  science  Dose response (D/R)  i d e n t i f i c a t i o n of drug induced  curves a i d the  e f f e c t s , but are themselves  l i m i t e d by co-existence of dose dependent a d d i t i o n a l actions acting  as  covariants.  Thus,  even  with  D/R  curves,  175  mechanistic  certainty  pharmacology  to have a complete D/R curve f o r each of the  e f f e c t s of a drug. sigmoidal  D/R  i s equivocal.  It  i s rare  in  Further complications a r i s e when non-  curves  are obtained, e.g. serum  [K ] vs. +  arrhythmia incidence.  With a l l of these conditions i n mind then, the ideal Class I I I drug would t h e o r e t i c a l l y have predictable e f f e c t s on the ECG.  A Class III drug should not a f f e c t QRS duration  nor P-R i n t e r v a l since these r e f l e c t conduction v e l o c i t y i n v e n t r i c l e s and AVN, respectively. III  drug  reflects  should the  prolong  the Q-T  c  repolarization  time  (Einthoven, 1912; Katz, 1928). of  By d e f i n i t i o n , interval of  a Class  since  the  this  ventricle  A homogeneous prolongation  the plateau of the AP, as occurs with hypocalcemia or  hypothermia widens the S-T segment, where as a dispersion of repolarization  times,  or  alterations  in  phase  3  r e p o l a r i z a t i o n , r e s u l t i n Q-T widening secondary to T- wave c  broadening  or  simultaneously.  slurring. Thus,  intervals  Both  mechanisms  i t i s important measured  to  operate  distinguish  between  Q-T  initial  foot of the T (Q-TjJ , the apex of the T (Q-T ) or e  to the  Widening of the Q-T i n t e r v a l can  from QRS prolongation  1987; Jackman et al., 1988). distinguish  the Q wave  a  end of the T wave (Q-T ). also r e s u l t  from  may  Q-T widening  detection of the f i r s t  (Review  see:  Surawicz,  While i t i s r e l a t i v e l y easy to  caused  by the l a t t e r  mechanism,  two mechanisms by ECG analysis i s  176  very  challenging  and  requires  multiple  ECG  leads.  presence of U waves further complicates  analysis.  were  of  initially  delayed  interpreted as  regions  repolarization relative  ventricles  (Einthoven,  1912).  support  (Lazzara et al., alter  Q-T  should  be  from  myocardium  with  main mass of  the  and recently t h i s idea has  el-Sherif  1978).  interval,  the  U waves  U waves were l a t e r ascribed  to a f t e r p o t e n t i a l s (Nahum, 1939) received  to  The  and  Lazzara's  groups  As heart rate has been reported to  as  discussed  made at a constant  previously, measurements  heart  rate.  The  underlying  mechanism responsible for AP widening (or dispersion) cannot be  a r r i v e d at by  analysis of Q-T  intervals,  since i t can  occur e i t h e r as a r e s u l t of an increase of inward currents or  a decrease  introduction,  i n outward currents. numerous  drugs,  As  toxins,  e l e c t r o l y t e imbalances, or metabolic  discussed  in  adrenergic  the  agents,  conditions can i n turn  influence these currents.  The underlying electrophysiological mechanism of the T i n t e r v a l prolongation e f f e c t s of d-sotalol, c l o f i l i u m acecainide  (Carmeliet, 1985;  been investigated and channels.  Snyders & Katzung, 1985)  shown to r e s u l t  and have  from blockade of  K  +  Thus, the ECG e f f e c t s of these drugs might appear  d i f f e r e n t from the toxins, aconitine, ATX-II, and AP-A all  Q-  lengthen  Q-T  and  produce  U  waves  via  r e p o l a r i z a t i o n secondary to increases i n inward Na  +  which delayed  currents  177  (Matsuda et al.,  al.,  1959;  Peper & Trautwein, 1967;  1986; e l - S h e r i f et al.,  4.1.3.3  Platou et  1988).  Clofilium  Clofilium  (l-100uM),  a  bretylium  analogue  lacking  sympatholytic actions, " s e l e c t i v e l y " blocks I R i n guinea p i g myocytes (Snyders & Katzung, 1985; Arena & Kass, 1988)  and  produces  P-R  Q-T  widening  (Kopia et al.,  prolongation  without changes  the  absence  1985;  I R blockade  Theoretically,  ventricular  in  produces  (Arena et al.,  QRS  or  Steinberg et al.,  i n the rate of phase  tissue  of  widening  1979).  of  the  AP  3 repolarization in  1990)  thus S-T  segment  widening without T-wave widening on the surface ECG should r e s u l t unless there i s a tissue s e l e c t i v e vs.  Purkinje) s e n s i t i v i t y .  clofilium  proarrhythmic  (Carlsson et al., prolonged Q-T  (1990)  (1985).  tendency 1990).  in  In man,  widening so  carefully  these  and  other  0.80  studies  c l o f i l i u m (60 - 300 pg/kg) intervals  In the rabbit studies of Carlsson et  clofilium s 1  conscious rabbits used, Q-T and  i n the  However, c l o f i l i u m d i d have  proarrhythmic  effects  potentiated by methoxamine and/or propranolol.  0.36  e f f e c t s ofj  i n t e r v a l without a f f e c t i n g AH or HV  (Greene et a l . , 1983). al.  Q-T  i n dogs were not measured  report by Kopia et al., a  The  (e.g., v e n t r i c l e  umol/kg which  widening of 100% was l e d to VT.  were  In the two seen with  In anaesthetized  rabbits, VPB were seen at 1.8 + 1.3 umol/kg c l o f i l i u m and VT  178  at  6  umol/kg  reported.  Our  although results,  (which blocks both Ito (unpublished al.'s  no  The  prolongation  value  was  i n rabbits, showed that tedisamil J  a n d  K ) produced less Q-T  observations)  study.  Q-T  than  studies  clofilium  of  Arena  in  c  widening  Carlsson  and  Kass  et  (1988)  predicted, and our r e s u l t s i n other species a l l showed, less Q-T  widening with s p e c i f i c I R blockers than with t e d i s a m i l .  c  The marked Q-T  prolongation with c l o f i l i u m may  r e s u l t from  i t s completely  d i f f e r e n t structure (quaternary  amine) which  allows i t to block both the "slow" and " f a s t " components of I  K  (Arena  relative  & Kass, to  other  1988;  Sanguinetti  (methylsulfonamide)  appear only to block the  1^  1990),  blockers,  which  less prominent "rapid" component  (Colatsky et a l . , 1990). the " f a s t " and  & Jurkiewicz,  Tedisamil appears to block both  isoproterenol-sensitive "slow" components of  I R i n guinea p i g v e n t r i c u l a r myocytes (Dukes et a l . , 1990).  4.1.3.4  D-Sotalol  D-sotalol blocks both I R I and blocks gNa beta  +  at 10" M 4  blocking  Nattel  and  (Carmeliet, 1985)  potency  colleagues  (plasma concentrations) sotalol Class  (0.8 III  anaesthetized  mg/1)  was  increases dogs.  I R at 10~  of  1-sotalol  (1989)  have  6  - 10~ M, i t 4  and has < l/50th the (Kato shown  et  al.,  that  1986).  the  EC  5 0  for beta blocking e f f e c t s of racemic ten  fold  in  refractoriness  Apparently,  lower than the  EC  5 0  (6.8mg/l)  for in  both enantiomers have the  179  same potency  f o r APD  widening  (Carmeliet, 1985).  These  studies suggest a narrow margin of Class III s e l e c t i v i t y f o r d-sotalol,  i . e . a potency  r a t i o of f o r Class II:Class I I I  e f f e c t s of only 5 f o l d .  Kato's  study  showed equipotent APD  for d- and 1-sotalol, at 10 seen  f o r d-sotalol  1.6  - 3.2  antagonists.  at  10~ M 4  M no beta blocking action  (Kato et a l . , 1986).  (1986) using dogs showed d-sotalol had sotalol  widening  X the potency  was  Gomoll & Bartek  1/12  - 1/14  and  1-  of the racemate as beta-  However, at plasma concentrations giving equi  e f f e c t i v e beta blockade, racemic s o t a l o l and i t s enantiomers had  similar  indicated  potency  that  i n prolonging VERP.  doses  of  d-sotalol  These studies  producing  maximal  APD  widening would be associated with s i g n i f i c a n t beta blockade. Recently, Reid et al. interaction  of  (1990) showed further evidence f o r an  d-sotalol  concentrations producing  with  delayed  beta  receptors  repolarization.  In  at this  study d-sotalol's E C 5 0 f o r APD widening was 13 umol/1, while the  f o r beta-adrenoceptors  studies.  Theoretically,  umol/1  (by Class III effects) and the  P-R  i n t e r v a l s (by Class II effects) without a l t e r i n g  QRS  interval  duration.  This  anaesthetized  has  dogs.  widening  of  the  duration  (Feld  et  been  shown  D-sotalol  Q-T  would  c  interval  al.,  1986).  be  i n binding to  c  d-sotalol  4  expected  widen the Q-T and R-R  was  experimentally,  (2mg/kg) without Clinical  produced increasing findings  in 15% QRS gave  180  (McComb et al.,  1987; Sahar et al.,  1989).  Results with tedisamil on the ECG, at f i r s t glance  appeared  similar results  s i m i l a r to d - s o t a l o l , however no beta receptor a f f i n i t y has been shown f o r tedisamil autonomic blockade widening  In minimal  (Kalichemie Internal Report) and  does not prevent  tedisamil-induced P-R  (Howard et a l . , 1989).  rats,  d-sotalol  effects  on  (2mg/kg) has been  contractility  1988; Tande & Refsum, 1988).  shown to have  (Hoffmeister  &  Siepel,  In a c l i n i c a l study, d-sotalol  (1.5 to 2.75 mg/kg, i.v.) lowered mean BP by 13 ± 9% (Sahar et  a l . , 1989).  This minor  related to beta blockade.  effect  on BP may  The Class I I I e f f e c t s of s o t a l o l  have been reported to be reduced by ischaemia al.,  have been  (Culling et  1984; Cobbe et a l . , 1985); i f the patients had such  abnormalities  the haemodynamic  response  to d - s o t a l o l  may  have been more dependent on beta blockade.  As  discussed  previously,  racemic  sotalol  has  been  reported to be associated with torsade de pointes (Laakso et al.,  1981; McKibbin  demonstrations been reported.  et al.,  of a l i n k  1984).  So f a r , no  between d-sotalol  and TdeP have  Sotalol and acecainide have been shown to  induce EADS in vitro  (Strauss et a l . , 1970; Singh & Vaughan-  Williams, 1970; Dangman & Hoffman, 1981).  4.1.3.5  clinical  Acecainide  181  Acecainide  (30 mg/kg) widened Q-T  i n t e r v a l 16% without  c  a f f e c t i n g QRS i n anaesthetized dogs (Feld et al.,  1988). In  humans, acecainide, at plasma concentrations of up to 1.8 x 10~ M, has been reported to have no e f f e c t s on QRS or P-R 4  (Winkle et al.,  intervals et  al.,  1983),  dependently  while  1981; J a i l l o n et al., i t increased  by a maximum  of 22%.  Q-T  c  These  1981; Sung  interval  dose  selective  Q-T  c  widening e f f e c t s are s i m i l a r to our r e s u l t s with UK68,798, RP62,719 and r i s o t i l i d e when given to guinea pigs, pigs and baboons.  However, despite s i m i l a r e f f i c a c y , UK68798 appears  to be 1000 f o l d more potent than acecainide.  In  anaesthetized  mg/kg i.v.) and  dogs,  acecainide  (12 mg/kg  and 60  was found to have p o s i t i v e i n o t r o p i c actions  produced  respectively.  a  +12%,  and  +33%  increase  in  force,  However, these actions may have been r e l a t e d  to autonomic as well as d i r e c t e f f e c t s (Letora & King, 1986; Letora et al.,  1986).  The r e s u l t of the d i r e c t and i n d i r e c t  actions of the high dose was a reduction i n heart rate and blood  pressure,  hypotensive  while  effects.  12 mg/kg  had no  chronotropic  or  In the human studies referred to above  only J a i l l o n et a l . (1981) reported haemodynamic e f f e c t s , a slight  decrease  above  15  i n BP was noted  ug/ml.  Previous  acecainide to lack myocardial al.,  1975; Atkinson  et al.,  at plasma  studies  concentrations  i n humans  showed  depressant  actions  (Elson et  1977).  Similar  to  these  182  studies, UK68798, tedisamil, and r i s o t i l i d e lack hypotensive effects  (Gwilt  Grohs et al.,  et al., 1989;  4.1.4  QRS, a  drug's  1989,  and Q-T  effects  c  on  and  Intervals  i n t e r v a l s , give useful information on ventricular  and  AVN  conduction  Purkinje or endocardium versus epicardium are  not  Analysis  HV i n t e r v a l s etc. gives further clues to drug e f f e c t s  on some of these tissues. with  and  Tissue dependent e f f e c t s on,the  so r e a d i l y seen with analysis of the standard ECG. of AH,  1989;  1989).  Analysis of Surface ECG  ventricular repolarization. atria  Dalrymple et a l . ,  Colatsky et al.,  Summary:  P-R  1991;  epicardial  More detailed studies can be done  mapping  and  programmed  electrical  stimulation.  Characteristic i.e. and  Q-T  c  Class  widening, can  III  be  electrocardiographic  shown for drugs which block  I R i n most mammalian species including  man.  these drugs appear to be without e f f e c t s i n r a t s . which produced Q-T was  c  most e f f e c t i v e  v e n t r i c u l a r AP  c  I  K 1  However, Tedisamil  widening i n a species dependent manner, i n rats.  As  plateau which may  component (Josephson et al., T  actions  rats have an be  abbreviated  related to a large I ^ Q  1984), tedisamil might widen Q-  i n t e r v a l s i n rats through blockade of t h i s current (Dukes  et a l . , 1990).  Tedisamil and  r e l a t i o n to both c l i n i c a l l y  KC8851 appear to be unique i n available  Class III drugs  and  183  those under development to  have  similar  (see introduction).  efficacy,  but  up  to  UK68798 appears  1000  fold  potency than c l i n i c a l l y available class III drugs. RP62719, and r i s o t i l i d e had s e l e c t i v e Q-T in  primates,  which  were  of  similar  c  greater UK68798,  widening e f f e c t s  magnitude  to  those  reported f o r acecainide and d-sotalol i n man.  4.2  E l e c t r i c a l Stimulation  4.2.1  Overview  There  are many methods of measuring  VF  threshold i n  animal experiments, such as single pulse stimulation, sequential R on T methods.  and  E s s e n t i a l l y a l l that i s needed  i s a c o r r e c t l y timed stimulus of s u f f i c i e n t strength.  The  vulnerable period r e s u l t s from inhomogeneity of recovery of excitability et  al.,  following normal  1964),  and  the  use  excitation of  (Mines, 1913;  multiple  shocks  Moe  simply  increases the p r o b a b i l i t y of applying an appropriately timed stimulus  (Han,  1969).  that the current  Wiggers and Wegria  strength  needed  (1940) suggested  to e l i c i t  VF,  could  be  used to estimate the s e n s i t i v i t y of the v e n t r i c l e , and thus determine threshold.  the  influence  of  antiarrhythmic  of  on  VF  Recently, i t has been shown that VF threshold i s  dependent on the method of stimulation (Review: al., 1989).  drugs  Sugimoto et  These observations are based on proposed stages  development  of  VF  from  local  excitation,  through  184  ventricular  excitation,  disorganization  repetitive  into VF.  excitation  These authors  and then  showed that  drug  e f f e c t s on VF thresholds were dependent upon which stage the drug worked. by  Marshall  e f f e c t s on  The methods we used are s i m i l a r to those used et al.  (1983) which detects  I and III  VFf  Electrical  stimulation techniques  Hoffman & Cranefield, 1960). a preceding  impulse  The  and in vivo  recovery  of  i s determined  availability  of N a channels,  and  action  thus  have also been used  in vitro  to measure refractory periods  after  Class  excitability  mainly  which are voltage  +  potential  widening  (see  by  the  dependent  can  prolong  refractoriness.  4.2.2  Tedisamil versus Class I Drugs  In r a t s tedisamil prolonged more potently than the  ventricular refractoriness  representative Class  Tedisamil's e f f e c t s on MFF marked (50% increase) Q-T  c  and ERP  generally p a r a l l e l e d i t s  widening e f f e c t s .  not change the r a t i o of 1/MFF  I subclasses.  Treatments did  to ERP values, suggesting that  frequency dependent e f f e c t s were not apparent at rates above 7Hz.  Unlike  elevated VF+-, rendered  the  Class  tedisamil, at a high  the heart  suggests that  I drugs,  i n the  completely small  which  dose  dependently  enough dose,  resistant  rat heart,  (> 4mg/kg)  t o VF.  This  r e f r a c t o r i n e s s was  185  prolonged  to such an extent that multiple f r a c t i o n a t i o n s of  induced reentrant wave fronts were not possible (Sugimoto et al.,  1989).  (except  Propafenone most c l o s e l y  i n the  latter  effect)  resembled tedisamil  which  supports  Wu  and  Hoffman's (1987) observations that e f f e c t s of APD alone, in vitro a  do not necessarily predict antiarrhythmic e f f i c a c y i n  given  model.  (1989) showed  Using Class  the same reentry  I drugs  could  model Wu  et al.  be distinguished from  Class I I I drugs because the former prolong  cycle length of  the reentry more than Class I l l ' s .  4.2.3  Tedisamil versus UK68,798  Tedisamil stimulation  had s i m i l a r  thresholds  pentobarbitone  parameters. the  i n rats  or halothane  UK68,798 was without  anaesthetized  (above).  with  either  On the other  hand,  e f f e c t s on any e l e c t r i c a l stimulation  UK68,798  and UK66,914 have been  increase VERP i n a frequency  shown to  dependent manner i n guinea p i g  and dogs in vivo which i s consistent with i t s Q-T  widening e f f e c t s i n these species Gwilt et al., 1989a & 1989b). elevated  on r e f r a c t o r i n e s s and  This matched the lack of e f f e c t s of UK68798 on  r a t ECG.  in vitro  effects  VF-t i n t h e i r  UK68,798 i n r a t s .  studies,  (Dalrymple et al., 1989,  UK68,798 and UK66,914 also a  finding  not seen  with  In dogs UK68,798 was reported to increase  the incidence of spontaneous reversion to sinus rhythm a f t e r  186  VF  induction  (Gwilt, 1989) by a 50Hz t r a i n of s t i m u l i i n a  manner s i m i l a r to that used here.  In guinea pigs we  found UK68,798 to only  have minor  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 e f f e c t s on r e f r a c t o r i n e s s and VF-f (see  The difference between our r e s u l t s and those in vitro above) may have been due to the high  resting)  heart  stimulation  rate  in vitro,  of guinea  pigs  in  (stimulated  vivo.  &  At 5Hz  minimal VERP prolongation  was  seen  (Gwilt et al., 1989).  Guinea pigs Uehara,1985), effectively  have no  thus increased  (Section 3.2.3).  i n their  tedisamil's ERP  effects  i n guinea  pigs  than  at 10 minutes  e f f e c t s on QRS, P-R or VFt. more  more  UK68,798  bradycardia  properties contributed to the ERP prolongation,  produced  IR  on  was not a  I t i s also u n l i k e l y that tedisamil's gNa  measurements made  (Hume &  ERP was determined at a f i x e d stimulation  rate of 6.5 Hz, so tedisamil-induced factor.  ventricle  "complete"  after  dosing  +  blocking  because the showed no  I t i s possible that tedisamil  block  than  UK68,798  of I R , as  tedisamil has been shown to block the both the " f a s t " and isoproterenol-induced 1990).  "slow" components of I R (Dukes et al.,  However, UK68,798 appears to only block the " f a s t "  component of I R (Gwilt et al., 1989).  4.2.4  E l e c t r i c a l Stimulation Studies i n Primates  187  In primates, MFF than  i n rats  guinea pigs. T  c  tedisamil was less e f f e c t i v e at reducing although  i t was more e f f e c t i v e  than i n  This p a r a l l e l e d the intermediate e f f e c t s on Q-  i n t h i s species (Figure 7) .  Tedisamil appears to have  been more potent i n primates than i n rats with the maximal response occurring at l mg/kg.  In primates,  the index of e x c i t a b i l i t y ,  threshold f o r  capture, was not affected by tedisamil which suggests blockade was not appreciable at the doses used. bradycardia  and  tedisamil, increased  slight  AVN by  P-R  prolongation  refractoriness  tedisamil  was  not  a  slight  (only  v e n t r i c u l a r response to a t r i a l pacing). negative frequency  gNa  +  Despite the produced  by  significantly decrease  in  This may r e f l e c t a  dependency of tedisamil i n the AVN.  It  might be worthwhile to determine ERP by the extra stimulus method at a constant rate and compare i t to the MFF values obtained i n t h i s study. atrial nodal  pacing tissue.  pacing  could  have  In t h i s  corresponded  The lower resting MFF obtained by  to  reflected  longer  ERP values i n  regard the MFF obtained by the  minimum  MFF  atrial  obtained  from  v e n t r i c u l a r pacing, although d i r e c t comparisons can not be made because stimulus  and mode of capture  observations influence  of the difference i n the strength  MFF  assume  of the v e n t r i c l e .  "supernormal"  determination  of the  excitability  (Childers  et  These d i d not  al.,  1968).  188  Another simpler decrease AVN may  occur  reason why  tedisamil did not  r e f r a c t o r i n e s s i s that recovery of e x c i t a b i l i t y  a f t e r r e p o l a r i z a t i o n i s complete i n nodal  (Hoffman & Cranefield, 1960) factors  significantly  affecting  and  cells  therefore might depend on  availability  of  Ca  + +  channels,  which  tedisamil does not block d i r e c t l y (Dukes & Morad, 1989).  Section 4.2.5  E f f e c t s of Other Class III Drugs on  ERP.  In humans, acecainide has been shown to increase a t r i a l and  v e n t r i c u l a r ERP  but  not AVN  ERP  However, d - s o t a l o l did increase AVN patients, at doses producing  P-R  (Sung et a l . , 1983). ERP  as well as VERP i n  widening, which suggests a  beta blockade contribution (Sahar et al., 1989).  Most studies of Class  III drugs include data  drug e f f e c t s on r e f r a c t o r i n e s s . ERP  prolongation  showing  This i s not s u r p r i s i n g as  i s generally believed to be the mechanism  of action of Class III drugs (Vaughan-Williams, 1970;  1975).  Thus, the three "reference" compounds, d - s o t a l o l , acecainide and  clofilium  have a l l been shown to increase v e n t r i c u l a r  r e f r a c t o r y periods in vitro Strauss et al., et a l . , 1989;  1986;  Singh & Vaughan-Williams, 1970;  Sahar  Sung et al.,  & Hoffman, 1987; studies  (Feld et al.,  Singh et al.,  & Brogden 1990;  In  1970;  Wu  using  et al.,  and in vivo  1986; 1983; 1989;  programmed  Feld et al.,  1988;  J a i l l o n et al.,  Harron 1981;  Wu  Euler & Scanlon, 1988). electrical  stimulation,  189  prolongation with  of v e n t r i c u l a r ERP was  generally  an increase i n cycle length of induced  associated  tachycardias,  which often resulted i n termination of the arrhythmias.  In  one model of reentry, ERP prolongation was e f f e c t i v e whether it  was  achieved  by  APD  prolongation  (Class  acecainide) or was accompanied by conduction la,  e.g. procainamide) , although  length  (relative  I I I , e.g.  showing (Class  i n t h i s study  a long  path  to ERP) was a v a i l a b l e f o r the reentrant  impulse (Wu & Hoffman, 1987).  The s i m i l a r e f f i c a c y of UK68,798 i n dogs compared to ds o t a l o l has also been seen with other new Class I I I agents such as E4031 (Lynch et al., 1990, +23% VERP; Katoh et al., 1990,  +18% VERP), MS-551 (Kamiya et al., 1990, +20% VERP),  RP58,866 (Mestre et  et al. , 1989, +12% VERP), sematilide (Chi  a l . , 1990, +15% VERP) and r i s o t i l i d e  1989,  +23% VERP).  shared  by  effects  The remarkable potency of UK68,798 i s  E4031,  MS-551  at ug/kg doses.  studies  was  ventricular  a  "selectively"  produced the least  block K  of  have  these  of a l l these atrial  than  The v e n t r i c u l a r ERP was  no more than 2 5% by any of the drugs  RP58,866 block  which  feature  prolongation  refractory periods.  above).  1989) .  and RP58,866 A general  greater  maximally prolonged (see  (Colatsky et al.,  I  K  1  which (Escande  has et  been  reported  al.,  1989;  (12%) increase i n VERP (Mestre  to  1990)  et al.,  I t thus appears that drugs reported to " s e l e c t i v e l y " +  channels i n myocardium have a maximal effectiveness  190  at prolonging  VERP l i m i t e d to <25%, at least  guinea pigs.  Due to the d i f f e r e n t K  to  r e p o l a r i z a t i o n of the AP  implication, prolonging  currents contributing  i n rats,  I R blockers)  numerous  ERP while  +  i n dogs and  UK68,798  was  (and by-  ineffective  tedisamil was more e f f e c t i v e .  Under  r e s t i n g conditions the r a t VERP i s much shorter than species, as a r e s u l t of an abbreviated plateau  at  other  (see below).  However, the increase i n rat VERP induced by tedisamil makes t h i s species and drug combination i d e a l l y suited f o r t e s t i n g the  effectiveness  of  Class  I I I mechanisms.  Detailed  analysis of reentry and abnormal automaticity models should be  possible,  aconitine,  and the r e s u l t s A-PA  ATX-II,  applicability  can  i n other  to human  never possible with  cardiac  to models  species. arrhythmia  While  using direct  mechanisms i s  animal models, t h e o r e t i c a l  hypothesis  be tested over a broader range of ERP than with the  other "K  blocker" drugs.  +  4.3  Action Potential Morphology  4.3.1  Since  Frequency Dependence of APD  APD width  influences ERP, and rats and guinea  pigs have morphologically AP  compared  (Weidmann,  techniques effects  1956)  different ventricular epicardial  we  used  intracellular  recording  to elucidate tedisamil and UK68,798's disparate on  AP  morphology  in  rats  and  guinea  pigs.  191  Tedisamil's  marked  APD  widening  consistent with blockade of I-to i Morad,  1989;  Josephson  et al.,  effects n  t  n  in  rats  ventricle  e  1984).  I t has  was  (Dukes & also  been  reported that frequency dependent APD prolongation occurs at slow rates of stimulation in vitro et  al.,  1981).  potential  has  However,  an early  the  phase  (Carmeliet, 1977, rat  ventricular  of r e p o l a r i z a t i o n  Payet action  (up  until  controlled by both decay of I ^ and increasing I ^  APD50)  s  and a l a t e phase attributed to inward current electrogenic  Na /Ca  1984b; Noble, (Colquhoun  +  ++  exchange  1987)  or a  al.,  1981)  et  (Mitchell  [Ca ]i These  two  from either  et al.,  activated  + +  0  1984a &  inward current  phases  respond  in  opposite d i r e c t i o n s to frequency of stimulation changes; at slow rates (0.2Hz) A P D 2 5 shortens and A P D 7 5 lengthens, while the opposite e f f e c t s effects  have been  occur at higher rates  attributed  to changes  (5Hz).  These  i n amplitude and  i n a c t i v a t i o n time constant of I i (Payet et al., 1981). s  A l t e r n a t i v e l y , rate dependent prolongation of APD25 and APD50 may cardiac  be mediated by i n a c t i v a t i o n of I t c since muscle  has  slow  kinetics  of  recovery  of from  i n a c t i v a t i o n (Josephson et a l . , 1984; Coraboeuf & Carmeliet, 1982; Hiraoka & Kawano, 1989). that APD25  I t must be borne i n mind,  i n r a t v e n t r i c l e s the frequency dependent  changes i n  and A P D 7 5 were only a decrease from 8.3 to 5.2 ms and  an increase from 42.7 to 59.1 ms respectively f o r a decrease i n stimulation rate from 5Hz  to 0.2  Hz in vitro  (Nobe et  192  al.,  1990).  We  observed  very  little  change  in  rat  e p i c a r d i a l APD  i n response to vagal stimulation, (Figure 9).  More  studies  thorough  of  tedisamil's  d i f f e r e n t stimulation rates in vitro  4.3.2  Our  e f f e c t s on  APD  at  s t i l l need to be done.  E f f e c t s of Tedisamil on AP Morphology.  observations  tedisamil  was  not  indicate that bradycardia produced by  sufficient  marked AP widening i n rats. been expected  to  shorten  to  account  for  tedisamil's  Indeed bradycardia  the  early  However, blockade of Ito by 4-AP  r e p o l a r i z a t i o n phase.  i n rat v e n t r i c l e does not  produce the same degree of AP widening in vitro in our studies (Josephson et al., Although  under  different  action and  4-AP  1984;  conditions  experiments, a clue to the  would have  as  occurred  Nobe et al., from  our  1990).  in  vivo  difference between tedisamil's  can be gained  from the in vitro  Dukes & Morad (1989) and Dukes et al.  studies of  (1990) i n which  4-AP  reduced peak Ito current while tedisamil increased the rate of i n a c t i v a t i o n of Ito*  Dukes and Morad were further able  to  s e n s i t i v e measure  show  action  that was  the a  most  reduction  i n a c t i v a t i o n of Ito»  a  of  K  efflux.  +  of  tedisamil' s By  speeding  decrease i n permeability to K  +  would  r e s u l t at a time when inward currents were activated. Thus, membrane  p o t e n t i a l would  then  Ca  to  their  increased r e l a t i v e permeability to permeability of K .  Thus  equilibrium  p o t e n t i a l to  reflect a  the  greater  Na  +  extent,  and due  +  + +  193  "window"  current  (Colatsky,  current,  and  may  membrane  potential  permeability  of Na  have had  by  increase i n flux of Na rounding  off  of  the  Ca  of vs  + +  increased  without an  +  in  our  inward  contribution to  This was  + +  spike  the K  or C a .  +  background  a greater  virtue  and  +  1982),  relative antecedent  r e f l e c t e d i n the  studies  and  in  gross  q u a l i t a t i v e terms, tedisamil's transmogrification of r a t APs into guinea p i g AP morphologies. have occurred  Additional C a  + +  flux  may  secondary to the prolonged depolarization and  contributed to the increased c o n t r a c t i l e force reported for tedisamil (Grohs et al., Ca  transient  + +  speculations. might  have  hearts  concentration  Without a knowledge of the  these  observations  are  mere  I t i s i n t e r e s t i n g to speculate that tedisamil  converted  rat  (Bowditch 1871,  Tedisamil's likely  1988).  hearts  to  "positive staircase"  c f : Sheperd & van Houtte, 1979).  e f f e c t s i n guinea pig v e n t r i c l e were most  unrelated  to  blockade  of  Ito  i  n  ventricle,  t h e  because Ito i s n e g l i g i b l e i n t h i s t i s s u e (MacDonald et al., 1984) .  However, Ito  (Wang & Nattel, Blockade  of  bradycardia  Ito and  v e n t r i c u l a r AP 1980).  1989) i  been reported  n a s  n  and t n e  nodal node  i n guinea pig a t r i a  tissue may  be  widening  (Anderson & Johnson, 1976; only  partially  1987).  responsible  bradycardia-dependent  Adrenaline  (Irisawa,  Boyett  reversed  of  for the  & Jewell, the  APD  prolongation, despite normalizing the heart rate, s i m i l a r l y tedisamil  prolonged  ERP  in  "paced"  guinea  pigs.  As  194  discussed  earlier,  adrenaline  enhances  I , but tedisamil K  appears t o block t h i s e f f e c t (Dukes et al., 1990). noted  that vagal stimulation widens guinea  epicardial  potentials  (Figure  We have  pig ventricular  9) but has only  minimal  e f f e c t s on r a t v e n t r i c u l a r e p i c a r d i a l p o t e n t i a l s in vivo, i n agreement with 1976;  Boyett  receptors  the work c i t e d  & Jewell,  in  stimulation  1980).  were  the vagus  and ACh i s rapidly  acetylcholinesterase.  (Anderson & Johnson,  I t i s unlikely  the v e n t r i c l e  because  ventricle,  above  activated  does  that by  K h A C  vagal  not innervate the  hydrolyzed  i n plasma by  Indeed, a c t i v a t i o n of iR(ACh) would  have shortened the v e n t r i c u l a r APD, which i s the opposite to what we saw. The degree of widening f o r a s i m i l a r degree of bradycardia  was  stimulation. to  greater  f o r tedisamil  rectifier  explanation (IR)  a  al., 1990). maintained  s  n  a  s  would  bradycardia.  be a blockade  The  of the delayed  been reported f o r tedisamil (Dukes et  The onset of the delayed r e c t i f i e r  (on top of a  component of I i ) contributes to i n i t i a t i o n of AP s  repolarization Trautwein,  f o r vagal  Thus, additional factors must have contributed  tedisamil's APD prolongation besides  simplest  than  in  guinea  pig ventricle  (MacDonald  &  1978; Lee & Tsien, 1982; Hume & Uehara, 1985).  Tedisamil has been shown to have no e f f e c t 1990) on isoproterenol induced  (Dukes et al.,  C l ~ current, I c i / (Harvey &  Hume, 1989) thus the shortening of the APD a f t e r adrenaline could have been due t o reversal of the bradycardia t i s s u e action) and induction of v e n t r i c u l a r  Ici-  (nodal  195  Tedisamil produced greater e f f e c t s on APD however  their  is  e p i c a r d i a l APD  no  a  priori  why  the  prolongation should equal the  lengthening of the surface  extent of  UK68,798  was  or  without  findings  electrical are  ventricle  effects  consistent  with  (Josephson et al.,  UK68,798 for I  K  rat  in this  both 1984)  (Gwilt et al.,  on  Q-T  ventricular  with i t s lack  stimulation  consistent  of  ECG.  Effects of UK68,798 on AP Morphology.  ECG  Q-Tc,  degree  4.3.3  e p i c a r d i a l potentials, on  reason  than on  of  effects  species.  a  lack  and  the  of  I  These in  K  rat  selectivity  of  1989;1991).  I i i s an important membrane potential generator i n the s  early phase of rat v e n t r i c u l a r AP, the  later  phase which depends on  might be due  to electrogenic  ter  1985;  Kerrs,  the than  in rats.  Na /Ca +  ++  al.,  release  of  Ca  + +  less of a role i n  +  et  et al. , 1987).  release of C a  [Na ]  Mitchell  ryanodine-sensitive (Mitchell  but has  + +  D  and  [Ca ]i  exchange (Schouten and 1984a from  &  1984b)  internal  from internal stores i s less simultaneous AP  and  stores  In guinea pig v e n t r i c u l a r  Attempts at  and  + +  cells  important  recording  and  patch clamping have been done on chick embryonic v e n t r i c u l a r cells,  and  macroscopic  have  shown  current  agreement recording  between  microscopic  techniques  (Mazzanti  and &  196  Defelice,  1988) .  ventricular  Studies  cells  have  comparing  not  been  rat  done  and  guinea  yet,  with  pig this  technique.  In  guinea pigs,  i t s e f f e c t s i n dog  UK68,798 produced s i m i l a r  v e n t r i c l e in vitro  1991).  In both species the  can  most  be  easily  blockade of  I  effects  of  UK68,798  suggest  that  Na  et on  al., rise  as  which i s consistent  resulting  1989b; 1991). rate  and  channels were not  +  with i t s lack  blocked  of  e f f e c t s on QRS  This and  P-R  be a most potent and efficacy (+80%  i s not  i s also  e f f e c t on  consistent  intervals.  plateau its  lack  of  the  AP  of  by  the  drug,  conduction  (Gwilt et  with  to  1989;  from  The  height  time i n guinea pig p a p i l l a r y muscle in vitro 1989a; 1991) .  et al.,  minor prolongation of  interpreted  (Gwilt  K  (Gwilt  effects  al.,  i t s lack  of  Thus, UK68,798 appears to  s e l e c t i v e Class III drug, although i t s  remarkable.  UK68,798 was  more  effective  widening) i n Purkinje f i b r e s which have 3 0% wider  AP  than v e n t r i c u l a r muscle i n control conditions (Gwilt et a l . , 1989;  1991;  Myerburg, 1971).  4.3.4  Comparison to Other Class III Drugs  The  APD  prolonging  concentrations of up atrial  and  (Strauss  et  canine al.,  effects  of  sotalol,  to 10~ M, have been studied i n 3  Purkinje 1970)  as  fibres well  as  and cat  at  rabbit  ventricular  muscle  papillary  muscle  197  (Singh  &  Vaughan-Williams,  greater than 1.6  1970).  x 10~ M, were associated  concentrations,  with reductions i n  4  r i s e rate, unlike with UK68,798. pigs,  High  As with UK68,798 i n guinea  s o t a l o l produces more marked widening of dog  f i b r e s than v e n t r i c u l a r  tissue.  However, up  s o t a l o l did not prolong rabbit a t r i a l APD 1970).  This  tissue  has  a  large  to  Purkinje 5 x  10- M 4  (Strauss et  component  al.,  (Giles  &  Imaizumi, 1988)  and resembles rat v e n t r i c l e more than guinea  pig v e n t r i c l e .  Although canine Purkinje f i b r e s have been I+-  reported to have a large  0  current, s o t a l o l and UK68,798  were more e f f e c t i v e i n t h i s tissue than v e n t r i c l e in I t may  be that Purkinje f i b r e s have greater dependence on I R  for final  r e p o l a r i z a t i o n than v e n t r i c u l a r muscle.  generating  ventricular  greater e f f i c i e n c y of and  [Ca ]i  may  be  has  + +  cells Ca  + +  would  be  Ca  + +  expected  and  to  have  contraction,  v e n t r i c u l a r muscle APD  handling r e l a t i v e to  f i b r e "gating function"  As, work  handling than Purkinje f i b r e s ,  marked e f f e c t s on APD  expected that  dependence on  by  vitro.  (Myerburg, 1971)  has  IR.  a greater  The  might be  a slower decay of inward current and  it  Purkinje subserved  repolarization  may  therefore depend to a greater extent on I R a c t i v a t i o n , which by v i r t u e of i t s slow macroscopic a c t i v a t i o n k i n e t i c s would be expected to contribute longer APD.  more to r e p o l a r i z a t i o n i n cases of  This argument i s b a s i c a l l y the  s i t u a t i o n i n rat v e n t r i c l e , where I the  short  (Mitchell  K  has  l a t e phase i s dependent on et  al.,  inverse of  little  rapid  Ca  1984a & 1984b; Schouten and  effect + +  the and  handling  ter  Kerrs,  198  1985).  The prevalence  suggests poorer C a  + +  of EADs  i n Purkinje  handling i n t h i s t i s s u e .  fibres  also  A priori,  it  would be less c r i t i c a l to control [ C a ] i as e f f i c i e n t l y as + +  i n working muscle.  In  vitro  d - s o t a l o l would  not be expected  to d i f f e r  markedly from d , l - s o t a l o l as both enantiomers have s i m i l a r potency  for  APD  prolongation  (Carmeliet,  sympathetic influences are i r r e l e v a n t .  A report  1985)  and  suggesting  (10 - 1000 umol/1) reduced I^o more than I J Q i n  d-sotalol  i s o l a t e d sheep cardiac Purkinje f i b r e s has recently appeared (Berger  et a l . , 1989).  Similarly,  APD widening has been  shown using i n t r a c e l l u l a r recording with acecainide (Dangman & Hoffman, 1981) and c l o f i l i u m (Steinberg et a l . , 1981).  4.4  Myocardial  4.4.1  Ischaemia  Studies In Rats  Our r e s u l t s i n conscious rats support the concept that drugs which can s i g n i f i c a n t l y intervals  can  suppress  prolong  APD, VERP and Q-T  ischaemia-induced  c  arrhythmias.  However, a marked degree (400% and 100% respectively) of APD and VERP prolongation was needed to suppress f i b r i l l a t i o n i n t h i s species. support  The small sized r a t heart was s t i l l  fibrillo-flutter  despite  produced by 2mg/kg tedisamil.  able to  increases of VERP of 65%  This suggests that Class I I I  199  antiarrhythmic drugs may only be useful f o r microreentry of path  length l i m i t e d  i n size by anatomical  substrates.  In  other words, a degree of protection might be afforded by increasing ERP such that the product  of ERP and conduction  v e l o c i t y gives a longer path-length than can be supported by the anatomical this  point;  conduction  substrate. i f one  A simple c a l c u l a t i o n  assumes a  reentrant  illustrates  circuit  with  a  v e l o c i t y of 0.6 m/s and an ERP of 100 ms (e.g.  a f t e r t e d i s a m i l ) , then the minimum path-length would be 6 cm as  compared  with  conditions).  by  an  et al.,  ERP  tedisamil in  ability  action  blockers  (Bacaner  f o r an  actions  antifibrillatory channel  cm  Although  antif i b r i l l a t o r y accompanied  3  the  been  bretylium,  50ms  and  Tedisamil's  not  Selective f o r the  and and  had  was  VPB.  described  bethanidine  (control  KC8851  rat, this  to suppress  has  1986).  of  K  +  meobentine  KC8851's  S-T  segment elevating properties may have resulted from possible greater prolongation of APD i n normal vs. ischaemic t i s s u e (Janse, 1986).  The  lack  risotilide  in  antiarrhythmic ability  of effectiveness of UK68,798, RP62,719 and rats actions  to prolong  supports i n other  ERP i n those  nonelectrophysiological  effect.  the  concept  species  that  their  r e l a t e s to t h e i r  species  and not to some  Our own  results  i n pigs  suggest the effectiveness of UK68,798 may not be as high as o r i g i n a l l y proposed (Gwilt et al.,  1989).  More studies are  200  needed.  UK68,798  may  suppress  specific  types  of  arrhythmias, such as reentrant tachycardias involving fixed path-lengths i n Purkinje f i b r e s . pigs  have  ischaemia  suggested model  Studies with r i s o t i l i d e i n  remarkable  (Colatsky  et  protection al.,  in  1989).  an  The  acute related  compounds E-4031 and sematilide have also been shown to be effective presence 1990).  in of  canine  models  infarction  The  use  of  (Lynch  acute  ischaemia  et a l . , 1990;  in  Chi  of dogs f o r ischaemia-induced  et  the al.,  arrhythmias  has been questioned due to v a r i a b i l i t y i n c o l l a t e r a l artery anatomy as discussed previously (Section 1.1.7). studies with  clofilium  Earlier  i n a canine model of sudden  death  found t h i s compound to be i n e f f e c t i v e despite elevations of VF-t and prolongations of VERP i n non-ischaemic et al., 1985). III drug  Cobbe's group has documented a loss of class  induced  ERP  (Cobbe et al., 1985, and  El-Sherif  proarrhythmic clofilium,  prolongation i n ischaemic conditions Cobbe, 1988).  (1989)  have  tendency  study  which  experimental  whereas  to  an  antiarrhythmic  increased  doses  with  d-sotalol and bretylium i n "ischaemic" Purkinje  el-Sherif's  superfused  On the other hand, Gough  pointed  at  f i b r e s surviving i n f a r c t i o n .  group,  heart (Kopia  may  was  The marked increase i n APD i n  opposite  have  been  conditions  (0.25  fibres Cobbe's  chosen  from  model  is  to  that  related Hz  to  by  Cobbe's  el-Sherif's  stimulation  survivors an  seen  of  arterially  rate,  infarction) perfused  201  i n t e r v e n t r i c u l a r septum stimulated at > 1 Hz  (Cobbe et al.,  1985).  Studies i n r a t ischaemia shown protective  effects  1989), amiodarone and  the  rats  were  reperfusion models have  for sotalol  (Lamontagne et  desethylamiodarone  1989, Varro et al., 1989). and  and  (Riva &  al.,  Hearse,  As these drugs are not s e l e c t i v e  prepared  acutely,  a  false  positive  protective r o l e f o r Class III antiarrhythmics i n rats might have  been  acutely  seen.  prepared  Similarly  Brooks  et  anaesthetized rats  al.,  and  (1989)  found  used  protection  against ischaemia induced arrhythmias with melperone but not with s o t a l o l . bretylium,  In reperfusion studies in vitro  clofilium  and  melperone  strongly protective, but s o t a l o l was et  al.,  1989).  The  broad  were  and in vivo  moderately  less e f f e c t i v e  sensitivity  claimed  to  (Brooks  by  these  authors f o r rodent models, should be accepted with caution as our r e s u l t s  indicate that the new  drugs may not be e f f e c t i v e i n r a t s . prepared  al.,  surgery and 1987,  "selective"  Paletta  shown  et  +  al., was  induced  (Curtis et  Further  clofilium Curtis  and  the  only  i t was  more  & Hearse  arrhythmias  by  be changed by  measured  1988).  than in vivo.  reperfusion  i s determined  [K ] which may  therefore must be  Class III used  e f f e c t i v e in vitro have  The response of acutely  anaesthetized rats to ischaemia  many factors including serum recent  " s p e c i f i c " class III  to  be  (1989) less  202  sensitive  to  [K ] than  ischaemia  induced  arrhythmias  in  vitro.  4.4.2  Reperfusion-Induced  Arrhythmias  A note should be made regarding glibenclamide s lack of 1  effectiveness arrhythmias reported  against  i n the r a t .  antiarrhythmic  hearts by Opie's group carry  ischaemia/reperfusion  out  an  We  found  effects  no protection despite  i n globally  ischaemic r a t  (Kantor et al., 1990).  i n depth  analysis  dependency of glibenclamide*s  induced  of  the  We d i d not  possible  actions, and thus  we  time  cannot  completely dismiss a role f o r glibenclamide i n the treatment of ischaemia/reperfusion. induced  AP  blockade, with  shortening  by  However, prevention of ischaemiaglibenclamide  might be less e f f e c t i v e  "broad  plateau" AP.  mediated  i n rats than  Although  IR(ATP)  IR(ATP)  i n species  blockade  might  delay loss of i n t r a c e l l u l a r K , eventually the c e l l s would +  die  i f ischaemia  were maintained.  Opie's  group further  speculated that K ^ T P channels are activated a f t e r such b r i e f durations of ischaemia despite maintained  l e v e l s of [ A T P ] i n  the cytosol, by invoking compartmentalization  of A T P or the  influence of other nucleotides (Stern et al., 1988; Opie & Clusin, 1990). prevention  This was complete speculation.  Furthermore,  of the AP shortening which occurs  i n ischaemia  may be deleterious by promoting C a 1990).  + +  entry (Cole & Leblanc,  An alternate hypothesis might explain a protective  203  effect  IK(ATP)  of  necrosis v i a C a rendered  + +  blockade,  are  permitting  overload, the ischaemic  electrically  treatment  by  silent.  obviously  of the c o n t r a c t i l e  reported  prevention  K  by  failure. +  loss  cells  benefits  outweighed  worsening  of  The  more  rapid  could be  of such  a  the  potential  Another  group has  with  ischaemia  by  glibenclamide treatment i n dogs (Bekheit et al., 1990).  4.4.3  Our Studies i n Pigs  Although fibrillated treatment  none of the pigs in  response  (8mg/kg),  conclusively incidence Similarly,  ischaemia  the group  demonstrate  of VF  to  (n = 6) used  size  UK68,798  after  was  was  not  controls shown  to  tedisamil  too  an a n t i f i b r i l l a t o r y  in historical  i n our study  small  effect.  was  only  protect  ischaemia-induced f i b r i l l a t i o n i n the same model. degree of Q-T  c  to The  3/16.  against  A similar  prolongation was produced by either tedisamil  or UK68,798 which may have been due to  blockade.  There  was no VF seen a f t e r tedisamil and the VF which occurred i n the  UK68,798  results  treated  indicated  pigs  that  UK68,798 had limited  was  although  efficacy.  initiated  by VT.  These  free  of t o x i c  effects,  While  tedisamil  has less  s e l e c t i v e actions, i t s usefulness w i l l need further studies.  4.5  General Conclusions  204  4.5.1  Species Dependent Actions  The putative class I I I antiarrhythmic drugs we tested exerted  species  dependent  actions  on  the ECG.  These  differences were most l i k e l y related to species dependent K channel  distribution.  +  Tedisamil was markedly e f f e c t i v e i n  prolonging APD, ERP, and Q-T  c  i n t e r v a l i n rats, which can be  most e a s i l y explained by tedisamil's I ^ blocking actions, Q  as t h i s current contributes to the rapid r e p o l a r i z a t i o n of rat  ventricle  AP  (Josephson et al., 1 9 8 4 ; Dukes et al.,  The lack of effectiveness of UK68,798, RP62,719, and  1990).  r i s o t i l i d e at prolonging Q-T  c  i n t e r v a l or APD  (UK68,798)  in  rats can be explained by the r e l a t i v e unimportance of I R i n determining  APD i n rats (Josephson et al., 1 9 8 4 ; Dukes et  al.,  1990).  with  UK68,798  guinea  However, i t was evident that  IR  from the experiments  contributes to AP r e p o l a r i z a t i o n i n  pigs, pigs, and primates.  These studies h i g h l i g h t  the necessity of assessing the pharmacological  p r o f i l e of a  class I I I drug i n the same species i n which one intends to assay the drug for antiarrhythmic a c t i v i t y .  4.5.2  Antiarrhythmic Actions  Antifibrillatory  action  was  evident  at  doses  of  tedisamil which prolonged Q-Tc i n t e r v a l and ERP twofold and APD f o u r f o l d i n r a t s . to  the  This protection can not be attributed  tedisamil-induced  bradycardia,  as  heart  rate  205  reduction has been shown not to protect against i n t h i s model (Curtis et a l . , 1987, Thus,  c l a s s III action can  provided  Abraham et a l . , 1989).  protect  a s u f f i c i e n t degree of APD  prolongation  might  be  these  antifibrillatory experiments s t i l l  APD  ERP  suggests that the other putative c l a s s III drugs  species, although  of  (and  necessary,  effective  amount  fibrillation,  is  fully.  The  against  increase)  not  achieved.  arrhythmias  prolongation  agents  in  other  need to be done  Our preliminary r e s u l t s i n pigs suggest t h i s lack of  e f f i c a c y f o r UK68,798.  I t appears from our r e s u l t s and  r e s u l t s of others, that drugs which have been reported block I R may 25%. had  only maximally prolong Q-T  Tedisamil, with greater  efficacy  additional I to  c  the to  i n t e r v a l by up to  blocking properties,  i n primates which  suggests  that i t  might be possible to design an e f f e c t i v e c l a s s III agent for use i n  man.  206 5.  REFERENCES  Abraham, S., Beatch, G.N., MacLeod, B.A., and Walker, M.J.A. Antiarrhythmic properties of tetrodotoxin against occlusioninduced arrhythmias i n the r a t : A novel approach to the study of the antiarrhythmic e f f e c t s of v e n t r i c u l a r sodium channel blockade. JPET 251: 1166-1173, 1989. Adaniya, H. and Hiraoka, M. 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