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Prevention of ischemia-induced arrhythmias in the rat : a comparative study of ester-linked RSD compounds… Wang, Wei-Qun 1999

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Prevention of ischemia-induced arrhythmias in the rat: A comparative study of ester-linked R S D c o m p o u n d s with their amide-linked analogues. by Wei-Qun Wang  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF PHARMACOLOGY & THERAPEUTICS FACULTY OF MEDICINE  We accept this thesis as conforming to the required standard  University of British Columbia © J a n u a r y 1999 Wei-Qun Wang  In  presenting  degree freely  at  this  the  thesis  in  partial  fulfilment  of  University  of  British  Columbia,  I agree  available for  copying  of  this  department publication  or of  reference  thesis by  this  for  his thesis  and study. scholarly  or for  her  financial  pJwYK&Cpity%)  of  The University of British Vancouver, Canada  Date  DE-6 (2/88)  J*  n  .  2? .  ff  Columbia  purposes  gain  shall  requirements that  agree  may  representatives.  permission.  Department  I further  the  It not  be is  that  the  Library  permission  granted  by  understood be  for  allowed  an  advanced  shall for  the that  without  make  it  extensive  head  of  my  copying  or  my  written  Abstract These  studies  were  intended  to  investigate  the  in  vivo  cardiac  electrophysiological and antiarrhythmic activities of a related series of RSD compounds, which  included  ester-linked  compounds  (RSD973,  RSD1009,  RSD1046) and their amide-linked analogues (RSD996, RSD997, RSD1044). Special emphasis was placed on a comparison of their selectivity for myocardial ischemia-induced arrhythmias. The regional ischemia model used was ligation of a branch of left coronary artery in anesthetized rats. Antiarrhythmic activity was expressed arrhythmias,  as  effects including  on  incidence,  premature  severity  and  ventricular  tachycardia (VT) and ventricular fibrillation (VF). antiarrhythmic activity was constructed  duration  beats  of  (PVBs),  ventricular ventricular  A dose-response curve for  by plotting the percent change in  arrhythmia score, from control value, versus log-m dose.  The effective dose  producing 50% protection (ED ) was calculated from the dose-response curves. 50  The results showed that all compounds studied were protective against ischemiainduced arrhythmias in a dose-dependent manner.  At sufficiently high doses,  these compounds completely abolished VT or VF and markedly reduced PVB. The mortality caused by sustained VT or VF was also significantly reduced. There were no obvious similarities in antiarrhythmic effectiveness among the different pairs of ester and amide compounds with respect to antiarrhythmic dose-response curves. However, antiarrhythmic dose-response curve analyses showed that esters RSD973 and RSD1009 had antiarrhythmic effectiveness similar to that of their corresponding amide analogues RSD996 and RSD997,  II  respectively, in terms of efficacy, potency and curve slope. The antiarrhythmic dose-response curve for the ester RSD1046 was parallel to that for its amide analogue RSD1044 with a right shift (i.e. lower potency). The results suggested that the underlying mechanisms for the antiarrhymic effects of esters and corresponding  amide analogues  might be the same, although the  exact  mechanisms remain unclear. The  effects  of  ester  and  amide  analogues  on  haemodynamic  and  electrocardiographic (ECG) parameters in the absence of myocardial ischemia were investigated. In addition, electrical stimulation was applied to the heart to examine  drug  effects  on  cardiac  tissue  excitability  and  refractoriness.  Cumulative dose-response curves were constructed and the potencies were expressed as D 5 values (i.e. effective dose producing 25% change from predrug 2  value). Both ester and amide compounds decreased blood pressure (BP) and heart rate (HR) in a dose-dependent manner.  The ester-linked compounds were less  potent for effects on blood pressure and heart rate than their amide-linked analogues. In most cases, a maximal response (efficacy) could not be obtained while the slopes of dose-response curves did not differ between ester and amide analogues. According to their effects on the ECG and electrical stimulation variables, the compounds could possess both sodium (Na ) and potassium (K ) channel +  +  blocking activities. Prolongation of PR and QRS intervals of ECG, and increase in iT and VF-VTt of electrical stimulation were considered to be mainly  in  attributable to Na channel blockade, while prolongation of QT interval, increase +  in ERP and decrease in MFF to be mainly attributable to K channel blockade. +  RSD compounds influenced most of these indicators in a dose-dependent fashion. Ester analogues appeared to be less potent for the effects on indicators than their corresponding amide analogues, although it was not possible to establish this conclusion statistically for all the different indicators. Comparison (by ratios) of potencies for indicators of Na channel blockade (PR, iT) to those +  for indicators of K channel blockade (QT, ERP) suggested no preference for Na +  +  or K blockade between ester and amide analogues. +  Finally, in order to evaluate selectivity for antiarrhythmic effects on arrhythmias induced by myocardial ischemia, therapeutic indices were estimated from the ratio of D 5 values for BP, HR, ECG and electrical stimulation to the E D 2  antiarrhythmic activity.  50  for  The morpholino esters RSD973 and RSD1009 had  higher therapeutic indices, especially for those indices related to the ECG, than their amide analogues RSD996 and RSD997. However, for the corresponding dimethoxy pair, RSD1046 failed to show higher therapeutic indices than its amide analogue RSD1044, due to its lower potencies for both antiarrhythmic activity as well as electrophysiological effects on normal cardiac tissue.  Such results  suggest that the ester or amide group on morpholino compounds might be involved in the selectivity for myocardial ischemia-induced arrhythmias through interaction with other molecular moieties. The tentative suggestion drawn from this study, that ester-linked compounds with certain molecular structures provide wider safety margin with less haemodynamic  IV  depression, less bradycardiac effect and less potential proarrhythmic activity than their amide-linked analogues, needs to be confirmed in a larger series (at least twice the size of the present series) of structurally complementary compounds. The underlying mechanisms of action of the esters and amides remain to be elucidated.  v  Contents Abstract  II  Abbreviations  VIII  Acknowledgments  )C  1  Introduction  1  1.1.  Mechanisms causing arrhythmias  2  1.1.1.  Enhanced automaticity  2  1.1.2.  Triggered automaticity  2  1.1.3.  Reentry  3  1.2.  Myocardial ischemia and arrhythmias  4  1.3.  Antiarrhythmic drugs  7  1.3.1.  N a and K channel blocking actions of some antiarrhythmic drugs  9  1.3.2.  Proarrhythmic effects and side effects of antiarrhythmic drugs  10  1.3.3.  State-dependent block by antiarrhythmic agents  12  1.3.4.  Arrhythmia and ischemia selectivity of antiarrhythmic agents  15  1.3.5.  +  +  Structural and physico-chemical properties of ion channel blocking agents  18  1.4.  Objective of this study  22  1.5.  Experimental models for the study of myocardial ischemia and  1.5.1.  Arrhythmias  26  Models of ischemia-induced arrhythmias  26  1.5.2. Models of electrically-induced arrhythmias  2. 2.1.  Methods General  31  33 33  2.2.  Ischemia-induced arrhythmias in anesthetized rats  34  2.3.  Electrically-induced arrhythmias in anesthetized rats  35  2.4.  Materials  36  VI  2.5.  Data analyses  36  3.  Results  38  3.1.  Drug effects on haemodynamics and electrocardiogram  38  3.2.  Drug effects on electrical stimulation variables  46  3.3  Frequency-dependence of electrophysiological effects of RSD Compounds  3.4.  46  Drug effects on ischemia-induced arrhythmias  55  3.5.  Therapeutic indices of RSD compounds  55  4.  Discussion  62  4.1.  Putative N a and K channel blocking activities of RSD +  +  compounds 4.2.  62  Potency for different drug effects on haemodynamics and normal cardiac electrophysiology between ester-linked and amide-linked analogues  4.3.  64  Protective effects of RSD compounds against ischemia-induced Arrhythmias  65  4.4.  Selectivity of RSD compounds for ischemia-induced arrhythmias  66  4.5.  Possible factors contributing to the differences in action between  5.  ester-linked and amide-linked compounds  68  References  73  VII  Abbreviation AA  50  Drug dose producing 50% of a maximum antiarrhythmic response  ANOVA  Analysis of variance  AS  Arrhythmia score  BP  Blood pressure  Ca  Calcium  D5  Drug dose producing a 25% change from pre-drug  2  value DAD  Delayed afterdepolarization  EAD  Early afterdepolarization  ECG  Electrocardiogram  ED  Drug dose producing 50% of a maximum response  50  ERP  Effective refractory period  EtOH  Ethanol  HR  Heart rate  Hz  Hertz  iT  Current threshold  K  Potassium  Log P  Log-10 partition  MFF  Maximum following frequency  pA  Microampere  |jmole/kg/min  Micromoles / kilogram (rat weight) / minutes  coefficient  VIII  min  minutes  ml/hr/300g  Milliliters / hour / 300 gram (rat weight)  msec  Milliseconds  MW  Molecular weight  Na  Sodium  OZ  Occluded zone of heart following occlusion of the left coronary artery  pH  -Log  [H ]  pKa  -Logio dissociation constant  PVB  Premature ventricular beat  VF  Ventricular fibrillation  VF-VTt  Ventricular fibrillo-flutter threshold  VT  Ventricular tachycardia  +  10  IX  Acknowledgements  I gratefully acknowledge  Dr. Pang for her constant support through the  experience of my Master's Degree.  I know an ordinary "thanks" is entirely  inadequate to express how grateful I am for the great care and encouragement she has given to me. To Dr. Walker, my supervisor, I am indebted to him for his excellent guidance and supervision, and his effective motivational techniques, without him this thesis would not have been possible. I also wish to thank Drs. Sutter and McLarnon for agreeing to be part of my committee. Their invaluable advice has been incorporated into this thesis. Also contributing to my personal development as a pharmacologist was Dr. Beatch.  I appreciated  his constructive  recommendations  concerning  my  research. Thanks to  Dr. Zolotoy for  reviewing this thesis  and  providing  valuable  suggestions, Dr. Pugsley, Sandra Yong, Eric Hayes, and Terry Barrett for showing patience in teaching me many of the new techniques that I have come to master, David Dickenson for his technical expertise and protocol development. Thanks to all members of the Nortran Pharmacology laboratory for their kind willingness to help whenever I encountered difficulties. Finally, many thanks to my mother Li Yao, my wife Hong and my son Jing, for their loving support and patience throughout the course of my studies.  x  1. Introduction Highly integrated electrophysiological activities of different ion channels in cardiac cells result in a regular heart beat, which ensures that the heart and circulatory system functions normally and efficiently. When these ion channels are perturbed by factors such as acute myocardial ischemia, sympathetic stimulation and drugs, abnormalities of cardiac rhythm, i.e. arrhythmias, occur. Arrhythmias can be bradyarrhythmias (failure of impulse initiation), heart block (failure of impulse propagation) but the most serious in terms of lethality are tachyarrhythmias.  The serious tachyarrhythmias such as sustained ventricular  tachycardia (VT) and ventricular fibrillation (VF) are major clinical problems as a cause of sudden death.  Currently, various antiarrhythmic drugs are available.  However, none of them is uniformly effective, free of proarrhythmic properties and other adverse effects.  There is a need to develop better antiarrhythmic  agents by targeting arrhythmogens, or arrhythmic mechanisms, so as to provide compounds with greater therapeutic indices.  A rational approach to the  treatment of cardiac arrhythmias is based on an understanding of the origin and the mechanisms underlying electrical abnormalities, as well as the mechanism of action of a proposed antiarrhythmic drug. The following is a brief summary of possible mechanisms underlying arrhythmias.  1  1.1. Mechanisms causing arrhythmias The underlying mechanisms of arrhythmia are complex and not yet fully understood.  Generally accepted mechanisms include enhanced automaticity,  triggered arrhythmia, and reentry (Roden, 1995; Lipka etal., 1995).  1.1.1. Enhanced automaticity Sinus  or  ectopic  automatic  arrhythmias,  which  occur  in the  cells  with  spontaneous pacemaker activity, may be caused by an increase in the slope of diastolic depolarization, or reduction in threshold for generation of an action potential.  In addition, under conditions such as myocardial ischemia, an  abnormal automaticity may be invoked in cells which normally lack spontaneous diastolic depolarization, by virtue of partial depolarization of resting membrane potential (Roden, 1995; Lipka, 1995).  1.1.2. Triggered automaticity Two  major  forms  afterdepolarization  of  triggered  (DAD)  and  arrhythmias  early  are  mediated  afterdepolarization  (EAD).  by  delayed If  these  depolarizations reach threshold they may give arise to a second upstroke which can create abnormal rhythms. Delayed afterdepolarizations occur as a result of intracellular calcium (Ca ) overload under pathological conditions such as 2+  myocardial ischemia and digitalis intoxication.  Delayed afterdepolarizations  might mediate triggered arrhythmias when the underlying cardiac rate is rapid, whereas EAD mediated triggering is most common when the underlying heart  2  rate is slow and action potential prolonged (Roden & Hoffman, 1985; Jackman, 1988). Early afterdepolarization is thought to contribute to a polymorphic ventricular tachycardia (torsades de pointes) associated with long QT syndrome. The mechanism underlying EADs is not fully understood.  It may be due to  disturbance of the balance of currents by increasing inward C a  2+  or N a currents +  and/or decreasing outward K currents (Moore, 1993). There is a report that the +  recovery and reactivation of L-type C a  2 +  current ( l ) during the action potential Ca  plateau might be a primary ionic mechanism in EAD formation. currents such as prolonged l  and N a - C a +  N a  2+  Other plateau  exchange may modulate EAD  formation by changing the balance of currents. The delayed rectifier K current +  with its very long activation time constant might play an important role in determining the rate dependence (bradycardia-related) of EADs (Zeng & Rudy, 1995).  1.1.3. Reentry Reentry can occur when impulses propagate via more than one anatomical or functional  pathway  associated  with  dispersion  of the  electrophysiological  properties (e.g. refractoriness and conduction) of action potentials.  When the  impulse fails in one pathway (unidirectional block) it continues to conduct through the alternative pathway. If conduction along this pathway is slow enough to allow the retrograde impulse to reach the tissue proximal to the unidirectional block with a delay so that cells are no longer refractory, a recirculating rhythm may occur. Therefore reentry is determined by (1) the presence of an anatomical or  functional circuit, (2) heterogeneity in refractoriness among regions in the circuit, (3) slow conduction in one part of the circuit. A reentrant arrhythmia may be initiated not only by itself, but also by automatic or triggered beats in situations where there are potential reentrant circuits.  1.2. Myocardial ischemia and arrhythmias The underlying pathological conditions most often responsible for lethal ventricular arrhythmias are myocardial ischemia and infarction. An inadequate blood supply to meet the energy needs of cells leads to biochemical alterations that lead to altered ionic homeostasis and result in accumulation of metabolic byproducts. During ischemia, extracellular K , as well as intracellular Na and Ca , +  increase.  +  2+  In addition, myocardial tissue pH decreases, amphipathic lipid  metabolites (such as the long-chain acylcarnitine and lysophosphatidylcholine, LPC) accumulate and catecholamine release increases. Cellular geometry and gap junctions are also altered in the late stage of myocardial ischemia and infarction. Ionic and metabolic changes may be heterogeneous throughout the ischemic zone, at the ischemic margins, between Purkinje and ventricular fibers, as well as between epicardial, mid-myocardial and endocardial cells.  Such changes  influence a host of other factors such as cardiac ion channels, transmembrane ion exchangers, pumps and cell-to-cell coupling. The above changes lead to alterations in resting membrane potential, excitability, automaticity, refractoriness and conduction, all of which contribute substrates and triggers for ventricular  4  arrhythmias through abnormal impulse formation and reentry (Haverkamp,1991; Cascio, 1995; Corr & Yamada, 1995). The substrates and mechanisms for arrhythmias differ during the different phases of myocardial ischemia and infarction (Janse & Wit, 1989). In this study we focussed on the acute phase of myocardial ischemia due to its clinically important role in producing lethal ventricular arrhythmias (Roelandt, 1984; Bayes, 1989). During acute ischemia, the resting membrane potential is decreased (partial depolarization) mainly due to alteration in K gradient across the membrane. The +  underlying mechanisms for the increase in extracellular K concentration are not +  well established.  It might result from a net K efflux by (1) activation of a K +  channel (such as K T P ) by substantial reduction in intracellular ATP, (2) partial A  inhibition of Na -K pump, (3) K efflux secondary to loss of intracellular anions. +  +  +  The accumulation of extracellular K is accentuated by the lack of blood flow. +  The increase in extracellular K concentration is not homogeneous as a result of +  the diffusion of K from the ischemic zone to the normal zone. In addition, other +  ischemic components such as increases in intracellular C a  2+  and the effects of  LPC on the membrane may also contribute to the depolarization of resting membrane potential (Janse & Wit, 1989). Depolarization of resting membrane potential, in combination with other factors such as hypoxia and acidosis, causes reductions in the amplitude, upstroke velocity and duration of action potentials.  Such electrophysiological changes  show a marked spatial and temporal heterogeneity, particularly at the boundary  5  between the ischemic and normal zone, which is probably important for the generation of arrhythmias.  Such differences in membrane potential between  closely adjacent regions either during diastole, or during the action potential, will generate injury currents which may be involved in initiation of some arrhythmias (Janse & Wit, 1989; Haverkamp, 1991; Arnar et. al., 1997). As a result of reduction in resting membrane potential and amplitude of action potentials, as well as the changes in cellular coupling in the late phase of ischemia, conduction velocity is decreased.  Also, this conduction change shows heterogeneous  dispersion with more delay in epicardial than endocardial myocardium. The latter may be nourished by ventricular cavity blood (Janse & Wit, 1989). Refractoriness  is  lengthened  in  the  ischemia  zone  and  so-called  postrepolarization-refractoriness is postulated to relate to the depolarization of resting or maximum diastolic potential, whereas in the border zone refractoriness is shortened associated with reduction of action potential (Janse & Wit, 1989). The spatial and temporal inhomogeneity in conduction, and in recovery of excitability facilitates unidirectional block, and therefore facilitates initiation and maintenance of reentry.  Reentry is responsible for the majority of ventricular  arrhythmias during acute ischemia (Janse, 1980; Janse & Wit, 1989; Pogwizd & Corr, 1990; Haverkamp, 1991). Ventricular tachycardia might be caused by a single unstable reentry circuit.  Three dimensional activation  mapping studies show that in areas of block, excitability may recover and cells in conducting pathways may temporarily become inexcitable. Therefore, size and location of reentrant circuits change from beat to beat, and tachycardias are  6  irregular.  Fibrillation ensues when a single circuit is broken up into many  independent reentry wavelets (Janse & Wit, 1989; Janse & Kleber, 1992). The first beat of a VT or VF may itself be caused by reentry but also may be caused by a non-reentrant mechanism. The sites of origin of the arrhythmias are always close  to  the  ischemic  border,  possibly  in  the  subendocardium  and  midmyocardium (Pogwizd & Corr, 1990). Arnar (Arnar, 1997) and others (Janse, 1986) demonstrated that ventricular tachycardia with focal Purkinje origin is common in the early ischemic period, suggesting that microreentry and triggered activity in Purkinje fibers close to ischemic myocardial cells may play an important role in development of early ischemic VT. However, our understanding of the electrical  macromechanisms  or micromechanisms  as well  as the  biochemical events of ischemia-induced arrhythmias remains incomplete, a fact which hampers the development of ideal antiarrhythmic drugs.  1.3. Antiarrhythmic drugs An ideal antiarrhythmic drug for ischemic arrhythmias might be expected to target arrhythmogens, should such mediators cause ischemia-induced arrhythmias. However, such targeting is impossible until the essential mediator molecules involved have been identified.  Nevertheless, such substances must ultimately  produce their arrhythmogenic effect by altering cardiac cell membrane ion channels, thereby changing intracellular potentials and causing the arrhythmias. By interacting with cardiac ion channels, antiarrhythmic drugs modify the ion currents  which  are  most  likely  to  modulate  vulnerable  parameters  7  (electrophysiological properties most susceptible to alteration while manifesting minimal undesirable effect on the heart), thereby suppressing abnormal impulse propagation and reentry. At concentrations sufficient for ion channel blockade, most current antiarrhythmic drugs have little or no effect on cellular metabolism (Rabinowitz, 1997). Drugs may slow automatic rhythms by depressing the slope of spontaneous diastolic depolarization (phase 4), by shifting the threshold voltage toward a more positive  level,  or  by  hyperpolarizing  the  resting  membrane  potential.  Antiarrhythmic drugs may block DAD- or EAD-induced arrhythmias by inhibition of the development of afterdepolarizations, or by interference with the inward current, usually through N a o r Ca channels (Roden, 1995; Lipka, 1995). +  2+  Reentry depends critically on timing and spatial interrelations for refractoriness, conduction and excitability. Whether a given drug exacerbates or suppresses a reentrant  arrhythmia  depends  on  the  balance  between  its  refractoriness and on conduction in a particular reentrant circuit.  effects  on  If conduction  velocity is slowed or refractoriness prolonged so that the wavelength of tachycardia is greater than the pathlength, reentry can not be maintained (Lipka, 1995).  Usually, slow conduction generally promotes the development of  reentrant arrhythmias while prolongation of refractoriness is most likely to terminate functionally determined reentry, probably by reducing dispersion of refractoriness (Roden, 1994). Acceleration of conduction in the area of slow conduction, and shortening of refractoriness under certain circumstances also could be antiarrhythmic in  8  reentry. However, these properties do not characterize most currently available drugs (Task force, 1991). Since heterogeneity of refractory periods is thought to be the most important determinant for the initiation and propagation of ischemia-induced arrhythmias, most studies give emphasis to electrophysiological effects of drugs that prolong refractoriness (a vulnerable parameter for reentrant arrhythmias) either by increasing action potential duration, or by decreasing sodium channel availability. In the following we will discuss antiarrhythmic drugs according to their actions on cardiac electrophysiology, mainly K channel and N a channel blockade, with +  +  less attention being paid to classifications according to conventional classification systems.  1.3.1. N a and K channel blocking actions of some antiarrhythmic drugs +  +  Drugs with N a channel blocking activity decrease automaticity by increasing the +  threshold for excitability and decreasing phase 4 slope.  They also inhibit  triggered activity arising from DADs and EADs by interfering with N a currents. +  Most importantly, block of N a channels can shift the voltage dependence of +  recovery from inactivation to more negative potentials and/or slow the time course of recovery from inactivation, thereby tending to increase refractoriness and extinguish propagating reentrant wavefronts (Roden, 1996). conduction  slowing with  Na  +  channel  blockade  is a desirable  Whether effect or  proarrhythmic effect is questionable.  9  /  Drugs  with  K  +  channel  blocking  activity  prolong  repolarization,  increase  refractoriness and hence should prevent reentrant arrhythmias. A homogeneous ventricular repolarization process would decrease the probability of arrhythmias. An ideal antiarrhythmic drug should prolong action potentials without increasing dispersion between the shortest and longest action potential, whereas increased dispersion will be arrhythmogenic (Moore, 1993). However, most assumptions concerning mechanisms of antiarrhythmic activity are mainly based on the investigation of electrophysiological effects on normal cardiac tissue. The effects of these agents on the arrhythmogenic substrate may be quite different under pathological conditions such as ischemia (Haverkamp, 1991). Furthermore, disturbing normal electrophysiological function raises the  serious  problem  of  proarrhythmia, especially  when  drugs  are  used  prophylactically after myocardial ischemia. Aggravation of arrhythmias by some antiarrhythmic drugs have been demonstrated during experimental myocardial ischemia (Haverkamp, 1991).  Clinical evidence for the proarrhythmic  effects of current antiarrhymic drugs was obtained from analyses of clinical trials such as Cardiac Arrhythmia Suppression Trial (CAST) (Echt, 1991) and trial of Survival With Oral d-Sotalol (SWORD) (Waldo, 1996).  1.3.2 Proarrhythmic effects and side effects of antiarrhythmic drugs The results of the CAST and SWORD trials were an excess of deaths due to arrhythmias and cardiac causes after acute recurrent myocardial infarction in patients  treated  with  encainide, flecainide  and  d-sotalol.  Although  the  10  mechanism underlying the excess mortality remains unclear, the adverse outcome of the trials has highlighted the proarrhythmic properties of these antiarrhythmics (Echt, 1991; Waldo, 1996).  Case reports have  appeared implicating almost all classes of antiarrhythmic drug as having the potential to exacerbate arrhythmias either by induction of new arrhythmias and/or increasing the frequency of sustained ventricular tachycardia (Creamer, 1987). The occurrence of drug-induced arrhythmias is not easy to predict while such arrhythmias usually are very difficult to manage and have possibly fatal consequences (Creamer, 1987). Drugs with K channel blocking activity prolong cardiac action potentials (and QT +  interval) which, in turn, results in EADs and torsades de pointes (Mortensen, 1993).  Prolongation of repolarization may be associated with temporal  dispersion of refractoriness and nonuniform recovery of excitability, leading to reentry circuits.  Conduction slowing associated with N a channel block may +  facilitate the occurrence and maintenance of reentry circuits by increasing regional slowing in conduction and thereby increasing heterogeneity in local activation times (Creamer, 1987; Haverkamp, 1991).  Besides  tachyarrhythmias, most antiarrhythmic agents depress sinoatrial node function and atrioventricular conduction, thereby causing bradyarrhythmias or heart block (Roden, 1995).  Torsades  de pointes occur most often in the presence of  underlying bradycardia. Most class I agents have negative inotropic effects.  This deleterious cardiac  action will exacerbate cardiac failure and prejudice recovery from ventricular  n  arrhythmias (Roden, 1995; Ito, 1996).  In the presence of an already  compromised myocardium, further drug-induced cardiovascular depression is unacceptable. On the other hand, class III agents usually do not produce a negative inotropic effect or may even show moderate positive inotropy. This may be advantageous under the condition of myocardial ischemia (Mortensen et. al., 1993).  However, prolongation of action potential and shortening of diastolic  interval will augment calcium influx and reduce calcium extrusion. This may have undesired consequences, especially in the patients in whom cardiac calcium handling is already disturbed (Hondeghem, 1994). Therefore whether a class III drug may reduce or accelerate heart failure is not entirely clear. Drugs that modify cardiac electrophysiology often have a very narrow safety margin between the dose required to produce a desired (antiarrhythmic) effect and that associated with adverse (proarrhythmic) effect. Therefore selectivity of antiarrhythmic drugs for arrhythmias is more important than potency.  In the  present study, we compared the selectivity and safety of some novel compounds with similar chemical structures in an attempt to provide useful structure-activity information for further development of new agents with higher selectivity. .  1.3.3. State-dependent block by antiarrhythmic agents The conformation of ion channel proteins is modulated by membrane voltage and time. Conformational changes may be associated with changes not only in ion conduction, but also in drug affinity since drugs bind to ion channel proteins. The  12  affinity for binding can vary with the different ion channel states (Hille, 1977; Hondeghem & Katzung, 1984; Snyders, 1991). Drugs with sodium channel blocking activity can have high affinity for activated, inactivated, or both, states (i.e. block channels during the action potential), but a low affinity for the resting state (i.e. dissociate from binding sites during diastole). For open or inactivated state block, drug effects could develop in a frequencydependent fashion whenever the resting interval is too short for complete recovery from block.  This effect would increase from a basic value at normal  cycle length, to steady-state values characteristic for shorter cycle lengths, in an exponential manner (Hondeghem, 1991).  The kinetics of block, e.g. rate of  frequency-dependent block onset and recovery of block, which is thought to be dependent on the time constant of recovery from block  (irecovery)  (Starmer &  Grant, 1985), is very important for heart rate selectivity of drug action.  Use-  dependence is determined by the ratio of binding and unbinding rates. If a drug dissociates from its binding site too fast, there will be no cumulative effect even at very high heart rates.  On the other hand, drugs with very long  xrecovery  will  block ion channels during both systolic and diastolic period even at normal heart rate.  An ideal antiarhythmic drug would dissociate from its binding site  completely during diastole at normal heart rates so allowing for no cumulative effect.  However, should tachycardia develop, there would be more cardiac  action potentials per unit time and hence more drug binding.  In addition, the  diastolic interval would shorten, therefore less dissociation would occur.  As a  13  result, drug effect would accumulate in a frequency-dependent fashion until it precluded continuation of the tachycardia. Electrophysiological alterations induced by drugs (e.g. conduction decrease, refractoriness increase) are not perfectly uniform throughout the heart. could trigger serious arrhythmias as the effects of the drug develop.  This  However,  the effects of drugs with fast kinetics (fast onset and fast recovery from block) increases reasonably quickly upon acceleration of heart rate so terminating the tachyarrhythmia promptly, and most channels become unoccupied with a few normal heartbeats upon termination of the arrhythmia. provide less chance for proarrhythmias.  Therefore those drugs  Conversely, drugs with slow kinetics  lack selectivity for high heart rate and increase the likelihood of proarrhythmias (Hondeghem, 1991). Blockers of inactivated channels may be more effective during ischemia, because the rate of recovery from blockade becomes slower when cells are partially depolarized and more channels are in an inactivated state (Roden, 1995). In contrast to frequency-dependent block, when channel blockade with drugs develops at negative potential and declines during depolarizations, such as occurs in blocking of outward current (Ik) with quinidine, reverse frequencydependent block occurs.  Many antiarrhythmic agents that lengthen action  potential duration (e.g. sotalol, N-acetyl procainamide and melperone) cause virtually no prolongation of action potential duration during tachycardia (when they should) but markedly modify the normal beat instead. Following long cycle  14  lengths, the action potential can be lengthened so dramatically that repolarization disturbances may deteriorate into torsades de pointes.  Reverse frequency-  dependent block of ion channels can be associated with a reverse frequencydependent drug effect. versa.  However this does not need to be the case, and vice  For instance, many agents (such as almokalant) that show reverse  frequency-dependent effects block open potassium channels in a frequency dependent manner (Carmeliet, 1992, 1993a,b; Hondeghem, 1994; Roden, 1996). The outcome of drug effect on cardiac electrophysiological behavior, which is dependent on the integrated interaction of different ion channels or ion channel subtypes, may be different from its effect on one specific ion channel.  1.3.4. Arrhythmia and ischemia selectivity of antiarrhythmic agents Selectivity of action is very important for antiarrhythmic drugs. Selectivity means not only selectivity for cardiac tissue over other tissues so as to avoid neuronal or vascular adverse effects, but also within cardiac tissue. Antiarrhythmics should have minimal effects upon ion channels in normal tissue under normal voltagetime profiles to avoid proarrhythmic effects, but be maximally effective when arrhythmias occur under pathological conditions, i.e. the drugs should be selective for pathological conditions {Hondeghem, 1991). Drugs, which bind to ion channels at relatively positive potentials (e.g. blockers of  inactivated  channels), may exhibit cardioselectivity because of the longer duration of the cardiac action potential, and thus have few side effects in the central nervous system. Furthermore, they may also be selective for ischemic tissue. Drugs with  15  high frequency-dependence may be selective for high heart rate, and hence provide high antiarrhythmic and low pro-arrhythmic activities. It is an reasonable assumption that, regardless of whether an arrhythmia originates from ischemic or non-ischemic zones, confining a drug's action to ischemic tissue may ensure maximum refractoriness in ischemic tissue before the tissue become electrically silent.  This would prevent arrhythmias at doses  producing less adverse electrophysiological effects (proarrhythmic effects) due to less drug action on the normal tissue. It is proposed that this may be achieved by a drug which is activated or potentiated only under conditions associated with ischemia such as acid pH and high K  +  concentration.  Drugs (weak base  compounds) with low pKa might be confined to, and more potent in, ischemic myocardial tissue with its acid pH, if such drugs block an ion channel at an extracellular site and are active in their charged state (Bain, 1998). Unfortunately, current drugs with N a channel blocking activity are less effective +  in preventing sustained reentrant tachyarrhythmia occurring in the setting of myocardial ischemia (Haverkamp, 1991).  Previous studies in our laboratory  (Barret, 1995), and by others (Campell & Hemsworth, 1990; Campell, 1991; Ye, 1993), found that class I drug flecainide and quinidine lacked frequency dependence and ischemic selectivity.  Lidocaine had selectivity for  high frequency and for ischemia probably related directly to its greater affinity for inactivated  channels  (Dumaine  &  Kirsch,  1998).  Its  usefulness  as  an  antiarrhythmic drug was limited by its central nervous system and cardiovascular toxicity.  There are reports of effects of the class I antiarrhythmic drugs  16  disopyramide (la), lidocaine (lb) and flecainide (Ic) on vulnerability to filbrillation , evaluated by electrical fibrillation threshold being suppressed, and even inverted by ischemia.  This effect depends on the oxygen debt which varies with the  severity and duration of ischemia ( Bui-Xuan,1996; Aupetit, et. al., 1997). The activities of current class III agents are significantly attenuated and even lost at high heart rates and under the conditions of ischemia (Culling, 1984; Cobbe, 1988; Hondeghem, 1991). Amiodarone, which acts on many types of molecular targets including Na , K and C a +  +  2 +  channels (Kodama, 1996),  is considered to be one of most efficacious antiarrhythmic drugs available due to its lack of reverse frequency-dependence in lengthening the action potential (Hondeghem & Snyders, 1990; Hondeghem, 1994). torsades  de pointes.  It is not likely to induce  Both the European Myocardial Infarction Amiodarone Trial  (EMIAT) and Canadian Amiodarone Myocardial Infarction Arrhythmia (CAMIA) showed a reduction in arrhythmic deaths with amiodarone treatment (Julian, 1997; Cairns, 1997). This suggests that combination of activities of Na  +  channel  blockade  and  action  potential  prolongation  could  increase  antiarrhythmic activity and reduce toxicity. It has been suggested (Bain, 1998) that a better antiarrhythmic to prevent ischemia-induced  arrhythmias would require the following  pharmacological  profile: (1) cardiac selective ion channel blockade; (2) increased potency in, or selectivity for, ischemic myocardium; (3) sodium and potassium ion channel blocking action; (4) a positive frequency dependent blocking action, (5) an  17  extracellular site of ion channel blockade. The compounds in this study have, in part, some of this profile.  1.3.5. Structural and physico-chemical properties of ion channel blocking agents Physico-chemical properties, structure and steric configuration of classic sodium channel blockers will influence their interaction with ion channels (Courtney & Strichartz, 1985; Courtney, 1987).  Three physico-chemical properties often  related to N a channel blockade are molecular weight (MW), hydrophobicity and +  pKa.  A size/solubility hypothesis was described by Courtney such that the  smaller antiarrhythmic drugs with higher lipid distribution capabilities provide more rapid repriming kinetics of N a channel blocking. +  In addition, very lipid  soluble drugs are more potent at blocking myocardial N a channels during an +  action potential.  The pKa will have important effects on drug distribution  processes in the membrane. Moreover, the affinity for the receptor site may well depend on the degree of protonation (Courtney & Strichartz, 1985; Courtney, 1987). Many N a channel blocking agents (such as current class I antiarrhythmics and +  local anesthetics) appeared to have a common basic chemical structure. That is a hydrophilic domain linked to a hydrophobic domain by short alkyl chain containing an electronegative functional group such as amide, ester, ether or alcohol groups. These structures are amphipathic with lipophilic and hydrophilic characteristics, generally, at opposite ends of the molecule (Colatsky & Follmer, 1990; Catterall & Mackie, 1995). The structure-activity relationship, i.e. the role  18  that each of these different portions of the molecule plays in determining the activities, were studied mostly based on interaction of local anesthetics agents with N a channels.  However most local anesthetic agents have demonstrated  +  antiarrhythmic effects, in that they reduce N a currents in cardiac tissue (Gintant +  & Hoffman, 1987). Regarding the contribution of local anesthetic effects to the action of antiarrhythmic agents, the information provided by these studies should be helpful in designing antiarrhythmic agents. Furthermore, it has been reported that some local anaesthetics, antiarrhythmics and anticonvulsants interact with a common receptor site in N a channels in a overlapping manner (Ragsdale, +  1996). However, the differences between ion channels in heart and in nerve (Qu, 1995) should be considered as important factors in the comparative pharmacology of local anesthetics and antiarrhythmics. The hydrophilic group usually is a tertiary amine, but it also may be a secondary amine, which is important for affinity to its binding site. Once the molecule is at its binding site, its affinity for that site depends on the terminal N being protonated. Charge also may affect drug distribution processes in the membrane (Courtney & Strichartz, 1987). interaction  with  the  Na  +  In addition a charged group is involved in  selectivity filter which  normally  limits  a  drug's  extracellular access to, and escape from its binding site (Sunami, 1997). The  hydrophobic  domain  must  be  an  aromatic  moiety  to  provide  the  hydrophobicity to the molecule structure. This group may interact closely with a structural hydrophobic zone of the binding site, or may determine unstructured absorption in the cell membrane. The intermediate alkyl chain may influence the  19  hydrophobicity of structure or influence the ability of the terminal amine group to add a proton (pKa) (Courtney & Strichartz, 1987). Courtney found that several anticonvulsant drugs, which are cyclic amides, are less potent for tonic block of N a channels in nerve, while several non-amides +  including the ethers, mexiletine, alprenolol and propranolol, and the ester procaine, are much potent than linear amides with corresponding hydrophobicity (Courtney, 1980). Ether-linked antiarrhythmic drugs appeared to be more potent than amide-linked drugs of the same solubility in producing N a channel block in +  guinea-pig papillary muscle (Courtney, 1983). Similar results were reported byothers (Wildsmith, 1985; Wildsmith,1987; Strichartz, 1990) using in vitro nerve preparations. Potencies for tonic blockade of N a currents with ester+  linked anesthetic agents exceeded those with their amide-linked counterparts. These results suggest the nature of the linking group e.g. ester or amide bond, determines some of the pharmacological properties of a molecule (Catterall & Mackie, 1995). The amide bond might provide, relative to ester or ether bond, some steric hindrance for interaction with the effector site. The rigid amide-linked rings in anticonvulsant drug molecules may present even greater steric hindrance than with linear amides. As a result, their potency may be reduced compared to that of equivalent linear amides (Courtney & Strichartz, 1985). It is generally accepted that there are differences in the pharmacokinetic processes acting on esters and amides. The majority of amide compounds are metabolized  by dealkylation  in liver, while ester compounds  are  usually  20  hydrolyzed readily by plasma esterases, such as pseudocholinesterase (Arthur, 1987). The influence of physico-chemical properties, structural and steric configuration on K channel blockade are less clear at the present time. Potassium channel +  blockers appear to be chemically diverse. However, many local anesthetics and class I antiarhythmic agents have been shown to possess K channel blocking +  activity, to a greater or lesser extent. The basic pharmacophore for K channel blockers seems to be similar to that for +  local anesthetics, and other N a channel blockers, in which an electronegative +  aryl substituent is important to limit sodium channel interaction and to convert Na  +  channel blocking activity to K  Follmer,  1989,  1990).  electrophysiological  +  channel blocking activity (Colatsky and  This assumption  was tested  by comparing  properties of procainamide with N-acetyl  the  procainamide  (NAPA). The former depresses cardiac conduction and prolongs refractoriness, while NAPA is almost entirely devoid of effects on conduction, but retains the ability to prolong repolarization (Bagwell, 1976). Furthermore, the replacement of N-acetyl group of NAPA with a methysulfonamide group, like those in sotalol, produced a pure class III compound, sematilide (Lumma, 1987).  Another  example is that replacing the 4-NH aryl group in Wy-47804 molecule with 4 - N 0 2  2  produced a compound Wy-47792 with K blocking action which contrasts with the +  class I profile of its parent compound (Colatsky and Follmer, 1989,1990). The above suggests that the electronic changes induced by substitution of an electron-withdrawing for an electron-donating group on the aromatic portion of  21  basic local anesthetic pharmacophore can convert a class I to class III electrophysiological profile, and thus the blocking site in the sodium channel for drugs of this type is a sterically restricted, electron-deficient structure. The structure-activity relationship of antiarrhythmic agents, which are diverse in structure, has not been extensively studied as that of local anesthetics. We are still not clear as to how antiarrhythmic drugs interact with ion channels at a molecular level. Furthermore, almost all the structure-activity studies were based on investigation of the interaction of compounds with ion currents. The effects of antiarrhythmic  agents  on  antiarrhythmic  activities.  ion channels Therefore,  the  may  not  be  equivalent  structure-activity  to  relationship  their of  antiarrhythmic agents remained unclear.  1.4. Objective of this study The intent of our laboratory is to develop better antiarrhythmic compounds with improved selectivity and wider safety margins than existing drugs, aimed at preventing lethal ventricular arrhythmias due to myocardial ischemia.  The  compound, RSD921, provided a lead compound with better therapeutic index than existing drugs and had sufficiently novel pharmacological properties, and chemical structure, to form a rational basis for a drug discovery program. Thereafter, several series of compounds, including amides and esters, were produced by modifying the molecular structure of RSD921. These compounds usually contain a hydrophilic domain (usually tertiary nitrogen) and a hydrophobic domain (aromatic group) linked by ester or amide group.  Differences exist  22  between amides and esters with respect to pharmacological properties and chemical characteristics, such as the hydrophobicity, dynamics of structural configuration, action on different ion channel or different channel states. However, these differences remained to be systematically evaluated. Three pairs of compounds were studied, RSD996 and RSD973, RSD997 and RSD1009, RSD1044 and RSD1046. Each pair had the same chemical structure except that the amide group in the first of each pair was replaced by ester group in the second (Figure 1).  Physico-chemical properties of compound were not  altered by such structural change as is shown in Table 1.  We intended to  investigate the functional and gross cardiac electrophsiological effects, and antiarrhythmic activities, of these compounds with special emphasis being given to the comparison of their selectivity for ischemia-induced arrhythmias. As mentioned before, changes in normal electrophysiology such as conduction velocity and refractoriness induced by a drug may in fact create a new re-entrant circuit capable of causing a more serious arrhythmia. potent  Compounds which are  in their depression of normal electrophysiology  ineffective  against  (Hondeghem, 1987).  arrhythmias,  or  even  hazardous  may be via  relatively  proarrhythmia  With respect to antiarrhythmic drugs, selectivity for  arrhythmias or pathological factors initiating arrhythmias (such as ischemia) is most important.  In this study, the selectivity was examined by comparing the  potency for antiarrhythmic activity with potency for effects on haemodynamic and electrophysiology under normal condition. It was hoped that the study would  23  RSD996  RSD973  RSD997  RSD1009  RSD1044  RSD1046  H  Figure 1. The molecular structures of the RSD compounds studied. RSD921.  On the top is  On the left are amide-linked compounds RSD996, RSD997 and RSD1044,  while on the right are structurally complementary ester-linked compounds RSD973, RSD1009and RSD1046.  24  Table 1. The physico-chemical properties of RSD compounds related to their electrophysiological effects  Compounds  MW  pKa  Log P  RSD996 RSD973  (A) (E)  359 360  6.2 6.0  3.1 3.2  RSD997 RSD1009  (A) (E)  353 353  6.3 6.2  3.2 3.3  RSD 1044 RSD1046  (A) (E)  400 399  7.0 7.0  3.8 3.8  A = amide; E = ester. MW is molecular weight, pKa is -Log-in dissociation constant and Log P is Log-| partition coefficient between octanol and water. 0  25  provide structure-activity clues for the development of better antiarrhythmic compounds. Discovery and improvement of drug aimed at selectively preventing ischemiainduced arrhythmias requires the use of models in which such arrhythmias are produced.  Thus,-a pathological model of acute ischemia, due to coronary  occlusion, in anaesthetized rats was chosen as the primary model.  Electrical  stimulation experiments were also performed to observe the effects of the compounds on eletrophysiological properties of normal tissue.  1.5. Experimental models for the study of myocardial ischemia and arrhythmias Animal studies have played an important role in our understanding of the mechanisms  of  ischemia-induced  ventricular  arrhythmias,  and  in  the  development of new and better antiarrhythmic drugs. Arrhythmias in animals can be induced by mechanical, electrical and chemical methods.  Our study  concentrated  myocardial  on protective effect of RSD compounds against  ischemia-induced  arrhythmias.  Electrically-induced  arrhythmias  were  also  studied to allow comparison of selectivity of the compounds for ischemia- versus electrically-induced arrhythmias.  1.5.1. Models of ischemia-induced arrhythmias Methods for producing myocardial ischemia-induced arrhythmia by ligation of coronary artery were first developed in early 1950's. There appear to be different phases of arrhythmias in response to coronary artery occlusion in most animal  26  models. In conscious rats, for example, the first burst of arrhythmias occurs 4-8 minute after occlusion and lasts for 5-10 minutes.  The second phase of  arrhythmias starts approximately 1-2 hour after occlusion and lasts for several hours (Curtis, 1987). Evidence suggests that different mechanism may be involved in the genesis of arrhythmias in different phases (Winslow, 1984).  In  this study, the focus was on the early phase of arrhythmias due to its potentially fatal consequences. Curtis and Walker (Curtis, 1987) suggested that an ideal model would (1) completely mimic one or more of the various clinical conditions; (2) respond to drugs in a manner which corresponds with the clinical response; (3) have sufficient precision and accuracy to function as a bioassay; (4) permit a variety of responses to be measured; and (5) be undemanding in terms of cost, time and expertise.  However, the clinical relevance of any animal model, e.g. exactly  mimicking clinical conditions and providing same response to a given drug, is difficult to assess because of the complexity of human myocardial ischemia and lack of well established effective drugs. The animals that are most often used for producing ischemia dependent arrhythmias are dog, cat, rabbit, guinea pig and rat.  All species have their  perceived advantages and disadvantages in addition to clinical relevance.  The  severity and duration of arrhythmias may vary markedly with animal species and with the coronary artery ligated because of the considerable variability in collaterals.  For instance, the coronary circulation of pig or rat is devoid of  collaterals. However, this is not the case for canine heart. Therefore, events in  27  pig or rat most likely mimic the events occurring in the human heart without longstanding ischemic heart disease (no collaterals), whereas the dog heart is similar to human hearts in which collaterals have developed because of coronary artery disease (Wit & Janse, 1992). The rat was chosen in this study due to its reproducibility (uniform lack of effective coronary collateral results in reproducible occluded zone and ischemiainduced arrhythmias), predictability (the precision of measurement of arrhythmias and other variables has been defined quantitatively) and convenience (ready availability and low cost). Like other animal models, the clinical relevance of rat model is unclear and the electrophysiological composition of rat heart is different from that of humans. The rat has a high heart rate, its ventricular action potential duration is brief and the  transient  repolarization.  outward  current  l  to  is  the  major  current  responsible  for  Although it is unclear to what extent these disadvantages  invalidate results from the rat preparation, the usefulness of rat model in the study of myocardial ischemia cannot be said to be less valid than that of any other species (Curtis, 1987). Winslow (Winslow, 1984) considered that the stretch of cardiac tissue, causing by unavoidably tying off part of ventricular muscle with artery, perhaps could trigger reflexes or activate receptors, which might contribute to the arrhythmogenic effects of ischemia alone. However, the tightening of an occluder has been shown to produce no significant sequelae unless the artery is occluded (Curtis, 1987).  28  Since acute ischemia is carried out in anesthetized rats, it is necessary to consider the possible influence of anesthesia on the outcome of coronary occlusion. Pentobarbitone, which is used in our laboratory, does not influence arrhythmias to any measurable extent (Au, 1983).  Extracellular K and  ischemic zone size are important determinants of arrhythmias.  +  Studies in our  laboratory revealed that there is a strong correlation between the arrhythmias and occluded zone (Curtis, 1987), while manipulation of serum K had a +  profound influence on arrhythmia severity following coronary occlusion (Curtis, 1986; Saint et. al.,1992). In order to ensure equivalence among different groups, these sources of variance are eliminated by setting exclusion criteria. Antiarrhythmia data are usually expressed as incidence and severity of arrhythmias.  However, untransformed raw data is often not suitable for  parametric statistical analysis. Arrhythmia scores (Table 2) were established, which are an arbitrary numerical grading of ventricular arrhythmias in terms of their perceived severity.  They are also Gaussian-distributed so that graded  dose-response curve may be generated. When used in combination with raw arrhythmia data, comprehensive dose ranges, and appropriate parametric statistical tests, arrhythmia scores facilitate the quantification of arrhythmias and the response to drug interventions (Curtis and Walker, 1988).  29  Table 2 Arrhythmia score for the quantification of ischemia-induced arrhythmias  0 = 0-49 PVBs 1 = 50-499 PVBs 2 => 499 PVBs and/or 1 episode of spontaneously reverting VT or VF 3 => 1 episode of spontaneously reverting VT or VF of both (< 60 seconds total combined duration) 4 = VT or VF of both (60-119 seconds total combined duration) 5 = VT or VF of both (>119 seconds total combined duration) 6 = fatal VF starting at > 15 minute after occlusion 7 = fatal VF starting between 4 minute and 4 minute 59 second after occlusion 8 = fatal VF starting between 1 minute and 3 minute 59 second after occlusion 9 = fatal VF starting <1 minute after occlusion  PVB = premature ventricular beat, VT = ventricular tachycardia, VF = ventricular fibrillation.  30  1.5.2. Models of electrically-induced arrhythmias Induction of arrhythmias by electrical stimulation has been extensively used to assay the antiarrhythmic activity of drugs. Allessie (Allessie, 1977) pointed out that cardiac fibers naturally exhibit nonuniform recovery of excitability and there exists a vulnerable period during the cardiac cycle, at the end of systole, when some cells have recovered from excitability while others are still refractory. This vulnerable period is an important source of re-entry.  When an extra  stimulus of sufficient intensity is applied to the heart during this period, spread of excitation becomes sufficiently disorganized to precipitate fibrillation. The ventricular fibrillation threshold (the minimum current intensity required to produce fibrillation), is used to assess antiarrhythmic activity.  It can be  determined either by applying a single shock, or by a train of stimuli during the vulnerable period.  The method is simplified by applying a continuous train of  high frequency impulses whence the vulnerable period does not have to be located. The multiple (or repetitive) extrasystole threshold (MRET), which is the minimum current intensity required to evoke two or more repetitive extra beats, is an alternative to avoid the problem of defibrillation and has good predictive value for VFT (Winslow, 1984). Our laboratory uses such electrical methods in anesthetized rat to evaluate the effects of agents on cardiac eletrophysiological properties.  Continuous square  waves are applied to left ventricle via bipolar electrodes.  The thresholds of  current intensity to pace the heart at 7.5 Hz and to evoke sustained VT or VF are used as an estimate of cardiac cellular excitability.  Effective refractory period  31  (ERP) and maximum following frequency (MFF) are also measured to estimate refractoriness (details in Methods).  Values for these variables are highly  reproducible and BP, HR and ECG parameters are not significantly changed after stimulation. Thus, each animal can serve as its own control and used to cover a large range of doses.  Results are open to statistical analysis without  having to use large numbers of animals.  However, to exclude the influence of  solvents and to avoid the subjective error, blind and randomized experimental design is used.  Comparison of the results of studies on different species is necessary to formulate an overall concept of the mechanisms of ischemic arrhythmias in human.  However, this is sometimes difficult due to the differences  in  electrophysiological properties and differences in cardiac and coronary artery anatomy (Wit & Janse. 1992).  We believe the promising results obtained from  our rat models may provide useful information for future research.  32  2. Methods 2.1. General The experimental procedures were approved by the Animal Care Committee of the University of British Columbia. A general surgical preparation was performed in both ischemia and electrical stimulation experiments. Male Sprague-Dawley rats weighing between 250-350g were used. They were randomly selected from a single group and anesthetized with pentobarbitone (65mg/kg, ip.) with additional anesthetic given if necessary. The trachea was cannulated and rat was artificial ventilated at a stroke volume of 10 ml/kg, 60 strokes/minute. The right external jugular vein and the left carotid artery were cannulated for intravenous injections of compounds and blood pressure (BP) recording, respectively. Needle electrodes were subcutaneously inserted along the suspected anatomical axis (right atrium to apex) of the heart for ECG measurement.  The superior  electrode was placed at the level of the right clavicle about 0.5 cm from the midline, while the inferior electrode was placed on the left side of the thorax, 0.5 cm from the midline and at the level of the ninth rib. BP and ECG signals were recorded and analyzed using a computer program LabView  (National  Instruments)  with  a customized  autoanalysis  software  (Nortran Pharmaceuticals Inc./Stopper Computer Solutions) to calculate mean BP (mmHg): 2/3 diastolic + 1/3 systolic blood pressure; HR (bpm): 6000/R-R interval (msec); PR (msec): the interval from the beginning of the P-wave to the peak of the R-wave; QRS (msec): the interval from the beginning of the R-wave  3 3  (lack of Q wave in rat ECG) to the peak of the S-wave, QT (msec): the interval from the beginning of the R-wave to the peak of the T-wave. In order to limit the influence of pharmacokinetic factors, an infusion regimen was used to obtain relatively slowly rising plasma levels during the time periods when drug effects were measured (i. e. 5-15 minutes of drug administration).  2. 2. Ischemia-induced arrhythmias in anesthetized rats Experiments with each compound involved a vehicle and 5 dose groups. Each group contained 5 animals. A coronary artery occluder was implanted loosely around a branch of left coronary artery exposed by means of a thoracotomy at the level of the 5  th  left intercostal space. The chest was loosely closed after the  operation, and the rat was allowed to recover for 15-20 minutes.  Body  temperature was monitored via rectal thermometer and maintained at 34-35°C by means of a heating lamp or ice in order to keep the rat's HR below 400 beats per minute (bmp). A sample of arterial blood (0.5-1 ml) was taken and its potassium concentration measured before drug administration.  In a random and blind  manner, drug or vehicle (30% ethanol or saline) was infused intravenously at a rate of 1 ml/hr/300g throughout the whole period of the experiment. The occluder was pulled to ligate the coronary artery 5 minutes after commencing drug infusion. All arrhythmias were recorded and summarized as arrhythmia score. After 15 minutes of occlusion, another blood sample was taken.  The chest was then  reopened and the heart was excised to determine the occluded zone.  The  34  occluded zone was visualized by perfusing the heart with Kreb's buffer containing cardiac green, and calculated as a percentage of total ventricular mass. The rats with serum K concentrations outside the normal range of 2.9-3.9 mM, +  or OZ outside the range of 25-50% or with pre-drug arrhythmias were excluded. To ensure parity of ischemia, all rats had to show the expected ischemia-induced ECG changes of increased R wave size and S-T segment elevation.  2.3. Electrically-induced arrhythmias in anesthetized rats Two Teflon-coated silver electrodes were implanted in the left ventricle (1-2 mm apart) passed directly through the chest wall. Square pulse stimulation provided by a Grass model SD9 stimulator was used to assess: threshold current (iT, uA) and minimal pulse width (tT, msec) for induction of extra systoles, maximum following frequency (MFF, Hz), effective refractory period (ERP, msec) and ventricular fibrillo-flutter threshold (VF-VTt, uA). Briefly, iT was measured as the minimal current to capture the heart at a frequency of 7.5 Hz and a pulse width of 1msec; tT was measured as the minimal pulse width to capture the heart with pulse current of twice iT and frequency of 7.5 Hz; ERP was the minimal delay for a second manually triggered pulse required to cause an extra systole with heart entrained at a frequency of 7.5 Hz, twice iT and tT; MFF was the maximum stimulation frequency which captured the heart at twice iT and twice tT; VF-VTt was the minimal pulse current to evoke sustained VT or VF at twice tT and 50 Hz.  35  After measures had stabilized, drug or vehicle (30% ethanol) was infused intravenously at a rate which started at 0.5 ml/hr/300g and was doubled every 5 minutes.  Electrical stimulation was applied to the heart during the last two  minutes of each infusion. Each experiment was terminated when the dose was too high to be tolerated (e.g. rats died of suspected cardiac output failure).  2.4. Materials RSD996, RSD973, RSD997, RSD1009, RSD1044 and RSD1046 were from Nortran Pharmaceuticals Inc.. The molecular structures and some physicochemical properties of these compounds are shown in Figure 1 and Table 1. RSD996, RSD973, RSD997 and RSD1009 were dissolved in 30% ethanol in saline. RSD1044 and RSD1046 were dissolved in saline (0.9% NaCl).  2.5. Data analyses Responses to compounds were calculated as percent changes from pre-drug (control) values.  This normalization was used to reduce individual variation.  Single dose-response curves, or cumulative dose-response curves, were drawn according to a logistic function y= E  • x / ( E D o x ) , where x is the dose, E D n  m a x  n +  n  5  50  the dose producing 50% of maximum response, n the slope factor (Hill coefficient) and E  m a x  the maximum response (100% for the antiarrhythmic  curves). The SlideWrite program (Advanced Graphics Software, Inc.) was used to obtain lines of best fit with minimum residual sum of squares (least squares).  36  For BP, HR, ECG and electrical stimulation variables, the D25S (effective dose producing 25% change from pre-drug value) were interpolated from individual cumulative dose-response curves and used as indicators for potencies, instead of ED s. 50  The reason is that maximal effect could not be determined and  estimation of E D  50  would require too much extrapolation to be a precise  measure. All data were expressed as mean ± s.e.m. for group of size "n". ANOVA was performed for parametric pairwise comparison. Fisher exact test was applied for proportion data (Zar, 1996).  P<0.05 was chosen as the criterion for statistical  significance.  37  3.  Results  3.1. Drug effects on haemodynamics and electrocardiogram Blood pressure and electrocardiogram were recorded to document a compound's effects  on  conditions.  haemodynamics  and  cardiac  electrophysiology  under  normal  The values of mean BP and ECG parameters immediately before  coronary artery occlusion (5min after drug infusion) or electric stimulation applied (3min after drug infusion) were used to construct dose-response, or cumulative dose-response curves. The two kinds of curves showed similar results although D 5 values were not exactly the same due to differences in dosing regimen, 2  infusion period, influences of surgical operation and experimental procedure. Only cumulative dose-response curves obtained from electrical  stimulation  experiments, which covered a larger dose range, are presented. Ethanol (30%) served as a vehicle control for some compounds. Results showed that 30% ethanol did not significantly affect BP, HR, ECG and electrical stimulation variables in an infusion rate-dependent manner over the same infusion rate range as drug administration (0.5-32 ml/hr/300g). With the exception of RSD1009, all compounds reduced mean BP in a dose dependent manner (Figure 2). RSD1009 did not reduce BP over the dose range of 0.5-16 umole/kg/min and slightly reduced BP at 32 umole/kg/min. Therefore, RSD1009 was less potent for effects on BP than its amide analogue RSD997 (Figure 2, B), although the D 2 5 obtained from individual dose-response curves showed no significant difference between these two analogues (Table 3). The  38  ester, RSD973 and its amide, RSD996 were equi-potent for effects on BP. Ester RSD 1046 was less potent for effects on BP than its amide analogue RSD1044 (Figure 2, Table 3) All these compounds decreased heart rate in a dose dependent fashion. Esters RSD973 and RSD1009 were less potent for effects on heart rate than their amide analogues RSD996 and RSD997, respectively, while the ester RSD1046, and its amide analogue RSD1044, were equi-potent as is shown in Figure 3 and Table 3. All RSD compounds in this study prolonged PR and QT intervals in a doserelated manner. QRS interval was also prolonged by these compounds except for RSD973 which did not significantly change QRS. Ester analogues RSD973 and RSD1009 were less potent for the effects on all ECG parameters than their corresponding amide analogue RSD996 and RSD997, respectively.  Ester  RSD1046 was also less potent in its effects on PR interval than its amide analogue RSD1044, but there were no significant differences in potencies for QRS and QT widening between the two compounds (Figures 4, 5, 6 and Table3). Maximal responses of the compounds could not be obtained in this study while the slopes of dose-response curves, except for the QRS dose-response curves for RSD973 and RSD996, did not differ between ester and amide analogues, The ratio of potency for PR prolongation to potency for QT prolongation was calculated to compare the possible preference for effects on PR or QT interval with either ester or amide analogue. The results showed there were no significant difference in these ratios between ester and amide analogues (Table 5).  39  B 10  0  0  -10  -10  -20  1  -20  -30  o. a  E 1  -30  •40  -40  -50  u SP  -50  -60  3 o  -60  -70  -70  -80  c  -80  -90  -90  -100  0.3  1  10  40  0.3  1  M i s ion rate (ml/hr/300g)  o  0  -10 -20  1 0  -30  -10  AO  -20  -50  If  -60  o  -80  40  Dose (umole/kg/min)  • 10  a,  10  -30 -40  -70  -50 -60  s  -90  -70  -100  0.3  10  Dose (uirttle/kg/min)  Figure 2.  40  -80 0.3 -90  1  10  40  Dose (jirrole/kg/iriin)  -100  Effects of compounds on blood pressure: A = Vehicle (30% EtOH), B =  RSD996(«) and RSD973(A), C = RSD997 (•) and RSD1009(A), D = RSD1044(.) and RSD1046(A). Each point is mean±s.e.m. (n = 5) for change from pre-drug. Filled circles represent amides and filled triangles, ester analogues.  40  B  A  infusion rate (m]/hr/300g)  -  0  -10 -  -10  -20 -  -20  o  •§ CL. B  E  £  60  -30 -  I  -60 -  o  -80 -90 -  •N?  -100  I  L  0.3  s  e  (urrole/kg/rran)  AO  -50 -70 '  o  -30  -40 -  e  - C3 C  D  -50 -60 -70 -80 -90  1  10  40  -100  Dose (urr»le/kg/rnrn)  0.3  1  10  40  Dose (jmiole/kg/min)  Figure 3. Effects of compounds on heart rate: A = Vehicle (30% EtOH), B = RSD996(») and  RSD973(A), C = RSD997  (•)  and RSD1009(A), D = RSD1044(.) and  RSD1046(A). Each point is meanis.e.m. (n = 5) for change from pre-drug. Filled circles represent amides and filled triangles, ester analogues.  41  A  Dose (urrole/kg/iriri)  B  Dose (umole/kg/rnin)  Figure 4. Effects of compounds on PR interval of ECG: A = Vehicle (30% EtOH), B = RSD996(.) and RSD973(A), C = RSD997 (•) and RSD1009(A), D = RSD1044(.) and RSD1046(A). Each point is meanis.e.m. (n = 5) for change from pre-drug. Filled circles represent amides and filled triangles, ester analogues.  42  A  B  Dose (umole/kg/min)  Dose (umole/kg/min)  Figure 5. Effects of compounds on QRS interval of ECG: A = Vehicle (30% EtOH), B = RSD996(») and RSD973(A), C = RSD997 (•) and RSD1009(A), D = RSD1044(.) and RSD1046(A). Each point is mean±s.e.m. (n = 5) for change from pre-drug. Filled circles represent amides and filled triangles, ester analogues.  43  Figure 6. Effects of compounds on QT interval of ECG: A = Vehicle (30% EtOH), B = RSD996(.) and RSD973(A), C = RSD997 (•) and RSD1009(A), D = RSD1044(.) and RSD1046(A). Each point is meanls.e.m. (n = 5) for change from pre-drug. Filled circles represent amides and filled triangles, ester analogues.  44  Table 3. D25 (in umole/kg/min) for effects of compounds on blood pressure (BP), heart rate (HR) and ECG parameters.  Compounds RSD996 RSD973  BP(mmHg) HR(bpm)  PR(msec) QRS(msec)  QT(msec)  (A1) (E1)  9.9±3.2 11 ±3.0  5.5±1.4 18±3.8*  8.4±1.6 38±6.1*  8.4±3.0 >64*  4.7±1.5 19±2.1*  RSD997 (A2) RSD1009 (E2)  42±11.5 55±16.5  6.1+1.6 14±2.1*  14±1.4 44±9.3*  9.8±1.1 48±6.9*  3.5±0.2 7.2±0.4*  RSD1044 (A3) RSD1046 (E3)  3.6±1.3 7.9±1.2*  2.0±0.2 2.610.3  5.0±0.4 13±1.6*  26±9.8 14±4.8  2.2±0.3 4.2±1.1  D 5 was calculated from individual cumulative dose-response curves and expressed as 2  mean ± s.e.m (n=5). Compound A1-3 refers to amides RSD996, RSD997 and RSD1044 while E1-3 refers to esters RSD973, RSD1009 and RSD1046, respectively. Asterisk * indicate statistical significance at P<0.05 for the difference of ester from its amide analogue.  45  3.2. Drug effects on electrical stimulation variables The RSD compounds dose-relatedly increased the resistance to the electrical stimulation i.e. increased iT and VF-VTt, as well as prolonged refractoriness, i.e. increased ERP and decreased MFF. Esters RSD973 and RSD1046 were less potent for effects on all of the above stimulation variables than their amide analogues RSD996 and RSD1044, respectively (Figures 7, 8, 9, 10 and Table 4). The ester RSD1009 and its amide analogue RSD997 were equi-potent for the effects on the electrical stimulation variables iT, VF-VTt and MFF but not ERP, which was more potently increased by its amide analogue RSD997. Maximal effects of the compounds could not be obtained in this study.  The  slopes of dose-response curves were not significant different between ester and amide analogues. Comparison of the ratio of D 5 for iT versus D25 for ERP revealed that there was 2  no obviously preference for increase in iT or ERP with either ester or amide analogues (Table 5).  3.3. Frequency-dependence of electrophysiological effects of RSD compounds The difference in the frequency-dependence of electrophysiological effects of compounds was estimated by comparing the ratio of potency for effects on iT to that on VF-VTt which were measured at low frequency of 7.5 Hz and at high frequency of 50 Hz, respectively, as well as the ratio of potency for effects on ERP to that on MFF which were measured under different condition with ERP being measured at frequency of 7.5 Hz. These potency ratios may reflect the  46  relative frequency-dependence of a compound's action. The higher the ratio, the more frequency-dependence. The results showed that there was no statistically significant differences in the ratios between the ester and amide analogues. However, the potency ratios of ERP versus MFF appeared to be higher for esters RSD 973 and RSD1009 compared to that for amide analogues RSD996 and RSD997, respectively (Table 6).  47  B  A  1  10  Dose (jjirole/kg/min)  l  10  40  Dose (uircle/kg/min)  Figure 7. Effects of compounds on iT: A = Vehicle (30% EtOH), B = RSD996(») and RSD973(A), C = RSD997 (•) and RSD1009(A), D = RSD1044(.) and RSD1046(A). Each point is meanis.e.m. (n = 5) for change from pre-drug. Filled circles represent amides and filled triangles, ester analogues.  48  B -  160  -  140  -  120  -  100  -  80  -  hang  1  180  60  -  40  -  o  20  -  o -20  L  0.3  1  10  40  1  M i s ion rate (ml/hr/300g)  10  40  Dose (urr»le/kg/min)  200 180 ap  160 140  I 1o  120 100 80 60 40 20 0  1  10  40  Dose (urrole/kg/rrrin)  0.3  40  Dose (urrole/kg/rrdn)  Figure 8. Effects of compounds on ERP: A = Vehicle (30% EtOH), B = RSD996(«) and RSD973(A), C = RSD997 (•) and RSD 1009(A), D = RSD1044(«) and RSD1046(A). Each point is mean±s.e.m. (n = 5) for change from pre-drug. Filled circles represent amides and filled triangles, ester analogues.  49  B 20  0  10  -10  0  •1  cx  1  -20  a  -30  ii 00  -40  han  -10  -50  o  -60  oo  -20  ct  -40  -30 -50  u c*  -60 -70 -80  -70  -90  -80  -100 0.3  10  0.3  40  1  a  a.  han  00 o  o  -  0  -10  -  -10  -20  -  -30  -  -40  -  -50  "  -60  -  -70  "  -80  -  -90  -  O O  -20 -30  PH  ^0 -50  If  HS CJ  \?  -loo 0.3  40  Dose (umole/kg/min)  M i s ion rate (ml/hr/300g)  •§  10  1  10  40  Dose (urrcle/kg/rrrin)  -60 -70 -80 -90 -100 0.3  1  10  40  Dose (umole/kg/min)  Figure 9 Effects of compounds on MFF: A = Vehicle (30% EtOH), B = RSD996(«) and RSD973(A), C = RSD997 (•) and RSD1009(A), D = RSD1044(.) and RSD1046(A). Each point is meants.e.m. (n = 5) for change from pre-drug. Filled circles represent amides and filled triangles, ester analogues.  50  B  A  0.3  1  10  40  1  Infusion rate (ml/hr/300g)  l  10  Dose (urrole4cg/mrn)  10  1  Dose (urrole/kg/rnin)  10  Dose (umole/kg/rnin)  Figure 10. Effects of compounds on VF-VTt: A = Vehicle (30% EtOH), B = RSD996(«) and  RSD973(A),  C = RSD997  (•)  and RSD1009(A),  D = RSD1044(«) and  RSD1046(A). Each point is meanis.e.m. (n = 5) for change from pre-drug. Filled circles represent amides and filled triangles, ester analogues.  51  Table 4. D 5 (in umole/kg/min) for the effects of compounds on electrical 2  stimulation.  Compounds  iT (uA)  ERP (msec)  MFF (Hz)  VF-VTt(uA)  RSD996 RSD973  (A1) (E1)  2.7±0.7 14±3.8*  1.4±0.3 7.4±0.5*  3.9±0.5 1.1 ±0.9*  1.1±0.3 8.3±2.1*  RSD997 (A2) RSD1009 (E2)  4.4±1.2 8.1±2.4  3.0±0.4 5.9±0.7*  8.7±1.7 9.2±1.6  4.3±1.0 2.9±1.0  RSD 1044 (A3) RSD1046 (E3)  1.2±0.2 2.8±0.7*  0.7±0.1 1.5±0.3*  1.7±0.1 2.8±0.3*  0.7±0.1 2.0±0.4*  D 5 was calculated from individual cumulative dose-response curves and expressed as 2  mean ± s.e.m (n=5). Compound A1-3 refers to amides RSD996, RSD997 and RSD1044 while E1-3 refers to esters RSD973, RSD1009 and RSD1046, respectively. Asterisk * indicated statistical significance at P<0.05 for the difference of ester from its amide analogue.  52  Table 5. Ratios of D25 for different ECG and electrical stimulation variables (PR v.QT, iT v. ERP)  Compounds  PR/QT  iT/ERP  RSD996 RSD973  (A1) (E1)  2.3±0.5 1.9±0.3  2.6±0.4 1.7±0.6  RSD997 RSD1009  (A2) (E2)  4.0±0.4 6.1±1.9  1.6±0.6 1.5±0.5  RSD1044 RSD1046  (A3) (E3)  2.6±0.6 4.6±1.6  1.8±0.5 1.9±0.2  D  25  was calculated from individual cumulative dose-response curves and expressed as  mean ± s.e.m (n=5). Compound A1-3 refers to amides RSD996.RSD997 and RSD1044 while E1-3 refers to esters RSD973, RSD1009 and RSD1046, respectively. Asterisk * indicated statistical significance at P<0.05 for the difference of ester from its amide analogue.  53  Table 6. Ratios of D 5 for estimation of frequency-dependence of RSD compounds. 2  Compounds  ERP/MFF  iTA/F-VTt  (A1) (E1)  0.4±0.1 0.9±0.2  2.8±0.6 2.9±1.4  RSD997 RSD1009  (A2) (E2)  0.4±0.1 0.9±0.3  1.1±0.3 2.4±0.9  RSD 1044 RSD1046  (A3) (E3)  0.4±0.1 0.5±0.1  1.6±0.3 1.6±0.4  RSD996 RSD973  D 5 was calculated from individual cumulative dose-response curves and ratios were 2  expressed as mean ± s.e.m (n=5). Compound A1-3 refers to amides RSD996, RSD997 and  RSD1044  while  E1-3  refers to esters  RSD973,  RSD1009  and  RSD1046,  respectively.  54  3.4. Drug effect on ischemia induced arrhythmias The RSD compounds studied had protective effects against ischemia-induced arrhythmias.  They markedly reduced the incidences of PVBs and completely  abolished VT and VF at sufficiently high doses. or/and VF was  also  significantly  reduced  Death due to sustained VT  by the compounds  (Table 7).  Antiarrhythmic dose-response curves were constructed by plotting the percent change in arrhythmia score, from control value, versus log-io dose.  Esters  RSD973 and RSD1009 produced similar antiarrhythmic effectiveness to their corresponding amide analogues RSD996 and RSD997, respectively, in terms of efficacy,  potency  and  slope  of  antiarrhythmic  curve  (Hill  coefficient).  Antiarrhythmic dose-response curve for ester RSD1046 paralleled that for its amide analogue RSD1044 with a rightward shift. There were obvious differences in antiarrhythmic effectiveness among the different pairs of compounds with respect to antiarrhythmic dose-response curves (Figure 11, Table 8).  3.5. Therapeutic indices of RSD compounds Therapeutic indices were calculated as a ratio of potencies (D ss) for effects on 2  BP, HR, ECG and electrical stimulation variables versus antiarrhythmic potency (ED o). The higher the ratio, the more selective is the drug for ischemia-induced 5  arrhythmias with less effect on normal electrophysiology (i.e. less potential proarrhythmic effect) and less haemodynamic depression.  Ester RSD973  provided the highest therapeutic indices except for BP, which was only slightly higher than its amide analogue RSD996.  Ester RSD1009 also provided much  55  higher Therapeutic indices related to PR and QRS and slightly higher indices related to BP, HR, QT, iT and ERP, but slightly lower index related VT-VFt than its amide analogue RSD997.  Therapeutic indices for ester RSD1046 and its  amide analogue RSD1044 were very similar (Table 9). Overall comparison of potencies for effects of these RSD compounds on haemodynamic,  electrophysiology,  ischemia-induced  arrhythmias  and  therapeutic indices showed that the amide RSD1044 was most potent for the effects on almost all aspects, including antiarrhythmic activity.  However, It  appeared not to be the most useful on the basis of therapeutic indices. Amides RSD996, RSD997 and ester RSD1046, which have potent effects on most indicators, were less selective for ischemia-induced arrhythmias than the other compounds, among which RSD996 was the least useful.  Esters RSD973,  RSD1009,  the  which  were  generally  the  least  potent  for  effects  on  haemodynamic and electrophysiology, and therefore were the most useful on the basis of their therapeutic indices (Table 10).  56  Table .7. Effects of compounds on ischemia-induced arrhythmias  Compounds  Dose (umole/kg/min)  Incidence of ventricular arrhythmias PVB VT VF Mortality  control  0  30/30  30/30  24/30  16/30  RSD996 (A1)  1 2 4 6 10  5/5 4/5 4/5 3/5* 2/5*  5/5 4/5 3/5* 1/5* 0/5*  2/5 4/5 0/5* 0/5* 0/5*  2/5 2/5 0/5* 0/5* 0/5*  RSD973 (E1)  1 2 4 8 16  5/5 5/5 5/5 2/5* 0/5*  5/5 4/5 3/5* 0/5* 0/5*  2/5 2/5 1/5* 0/5* 0/5*  2/5 1/5 0/5* 0/5* 0/5*  RSD997 (A2)  1 2 4 8 16  5/5 4/5 5/5 4/5 0/5*  5/5 4/5 4/5 3/5* 0/5*  4/5 2/5 2/5 1/5* 0/5*  1/5 1/5 0/5* 0/5* 0/5*  RSD1009 (E2)  1 2 6 8 16  5/5 5/5 5/5 5/5 3/5*  5/5 5/5 4/5 3/5* 1/5*  .5/5 2/5 2/5 2/5 0/5*  5/5 0/5* 1/5* 0/5* 0/5*  RSD 1044 (A3)  0.25 0.5 1 2 4  5/5 5/5 5/5 4/5 1/5*  5/5 5/5 5/5 0/5* 1/5*  2/5 4/5 2/5 0/5* 0/5*  0/5* 2/5 0/5* 0/5* 0/5*  RSD1046 (E3)  0.5 1 2 4 8  5/5 5/5 5/5 3/5* 0/5*  5/5 5/5 3/5* 0/5* 0/5*  4/5 4/5 2/5 0/5* 0/5*  2/5 2/5 0/5* 0/5* 0/5*  The incidence of arrhythmias was expressed as number of animals in which the defined arrhythmias occurred over the number of animals in the group. Compound A1-3 refers to amides RSD996, RSD997 and RSD1044 while E1-3 refers to esters RSD973, RSD1009 and RSD1046, respectively. * indicates statistical significance at P<0.05 from control.  57  0.1  1  10  20  Dose (jirrole/kg/rrrin)  c  Dose (jjrrole/kg/rnin)  Figure 11.  Protective effects against ischemia-induced arrhythmias (antiarrhythmic  dose-response  curve): A = RSD996(«) and RSD973(A),  RSD1009(A), C = RSD1044(») and RSD1046(A).  B = RSD997 (•) and  Values are percent change in  arrhythmia score from control expressed as meants.e.m. (n = 5).  Filled circles  represent amides and filled triangles, ester analogues.  58  Table 8. E D  5 0  (in umole/kg/min) for antiarrhythmic activity of compounds  Compounds  ED  50  RSD996 RSD973  (A1) (E1)  3.4±0.3 2.7±0.2  RSD997 SD1009  (A2) (E2)  4.1±0.9 4.8±1.0  RSD 1044 RSD 1046  (A3) (E3)  1.1±0.3 2.1±0.2*  ED  50  was calculated from antiarrythmic dose-response curves and expressed as mean ±  s.e.m (n=5). Compound A1-3 refers to amides RSD996, RSD997 and RSD1044 while E1-3 refers to esters RSD973, RSD1009 and RSD1046, respectively.  Asterisk *  indicated statistical significance at P<0.05 for the difference of ester from its amide analogue.  59  Table 9. The therapeutic indices of compounds,  Compounds  D25/AA50  BP  HR  PR  QRS  iT  VT-VFt  QT  ERP  MFF  meants.e.m  2.9 4.1  1.6 6.5  2.5 14.0  2.5 23.6  0.8 5.0  0.3 3.1  1.4 7.0  0.4 2.7  1.1 3.9  1.5±0.3 7.8±2.3  RSD997 (A2) 9.9 RSD1009 (E2) 11.4  1.4 2.9  3.3 9.1  2.3 10.0  1.0 1.7  1.0 0.6  0.8 1.5  0.7 1.2  2.0 1.9  2.5±1.0 4.5±1.4  RSD 1044 (A3) RSD1046 (E3)  1.8 1.2  4.4 5.9  23.2 6.5  1.1 1.3  0.6 0.9  1.9 2.0  0.6 0.7  1.5 1.3  4.3±2.4 2.6±0.7  RSD996 RSD973  (A1) (E1)  3.2 3.7  Therapeutic indices were estimated as ratios of D (ED  50  2 5  (for the defined indicator) to A A  50  for antiarhythmic effect). Compound A1-3 refers to amides RSD996, RSD997 and  RSD1044 while E1-3 refered to esters RSD973, RSD1009 and RSD1046, respectively. Mean ± s.e.m. is the mean and standard error for the 9 indices.  60  Table  10  Overall  comparison  of  potencies  of  haemodynamic(D25),electrophysiology(D 5),ischemia-induced 2  compounds  on  arrhythmias(ED ) 50  and gross therapeutic indices(TI)  Potencies Greatest  Least  BP  A3  E3  A1  E1  A2  E2  HR  A3  E3  A1  A2  E2  E1  PR  A3  A1  E3  A2  E1  E2  QRS  A1  A2  E3  A3  E2  E1  IT  A3  A1  E3  A2  E2  E1  VF-VTt  A3  A1  E3  E2  A2  E1  QT  A3  A2  E3  A1  E2  E1  ERP  A3  A1  E3  A2  E2  E1  MFF  A3  E3  A1  A2  E2  E1  AA  A3  E3  E1  A1  A2  E2  Therapeutic index values Greatest  Gross TI  E1 > E2 *  Least  A3 > E3 *  A2 > A1  Compound A1-3 refers to amides RSD996, RSD997 and RSD1044 while E1-3 refers to esters RSD973, RSD1009 and RSD1046, respectively. activity (AA) was estimated as ED . 50  Potency for antiarrhythmic  Potencies for effects on BP, HR, ECG and  electrical stimulation variables were estimated by D . Gross therapeutic index (TI) was 25  estimated by average of the therapeutic indices related to all haemodynamic and electrophysiological indicators. 61  4.  Discussion  With regard to designing ideal antiarhythmic agents, the chemical requirement for selectivity for pathological conditions (such as ischemia) is of interest. This study compared  the  antiarrhythmic  effectiveness  of three  pairs  of  structurally  complementary amides and esters (i.e. RSD996 and RSD973, RSD1009 and RSD997, RSD1044 and RSD1046) against ischemia-induced arrhythmias.  In  two of the pairs the tertiary nitrogen was part of a morpholino ring whereas in the third pair the tertiary nitrogen was not part of a ring structure but had two methoxy groups attached. The antiarrhythmic actions were then compared with action on the normal heart and cardiovascular system.  4.1. Putative N a and K channel blocking activities of RSD compounds +  +  For the purpose of investigating a compound's effects on normal cardiac electrophysiology, ECG changes induced by compounds were measured.  The  ECG reflects changes in cardiac electrical activity during the cardiac cycle. PR interval represents the duration from the initiation of atrial depolarization to the initiation of ventricular depolarization and is a good measurement of conduction time through the AV node. Sodium currents are assumed to play a considerable role in controlling atrioventricular conduction in rat hearts (Pugsley et. al., 1993). QRS complex reflects cardiac electrical activity due to ventricular depolarization and QRS interval is equivalent to the total time for the entire ventricular depolarization. Therefore, prolongation of PR or QRS intervals was considered to be mainly related to N a  +  channel blockade.  QT interval represents the  62  summation  of  ventricular  depolarization  (QRS  complex)  and  ventricular  repolarization. However, QT prolongation is considered to be primarily related to repolarization since an increase in QRS duration contributes little to QT interval prolongation. Thus, QT prolongation was mainly attributed to K  channel  +  blockade. It is appreciated that changes in ECG parameters cannot directly and exactly represent the changes in particular ion currents but they are the integrated activities of different ion currents. Nevertheless, that both esters and amides dose-relatedly widened the PR, QRS and QT intervals without obvious preference for any particular parameter suggest that these compounds might have both N a and K blocking activities. This suggestion was supported by the +  +  results of the compounds' effects on electrical stimulation. Myocardial excitability, which is mainly attributed to N a channel availability, was +  assessed by threshold current for "capture" of the heart (iT) and for induction of ventricular fibrillo-flutter (VF-VTt). In addition, ERP and MFF were measured to estimate duration of the recovery of cardiac cellular excitability.  Increases in  ERP, and correspondingly decreases in MFF, were thought to be indicators of mainly K channel blockade. However, reduction of N a channel availability in a +  frequency  +  dependent  refractoriness.  manner  may  also  contribute  to  prolongation  of  The compounds changed all of these electrical stimulation  variables in a dose-dependent manner. The results of electrical stimulation are in overall agreement with the ECG observations that suggested these compounds possessed both N a and K +  +  blocking activities.  This suggestion needs to be  confirmed by direct evidence obtained from measurement of N a and K currents +  +  63  in vitro. Furthermore, the possibility that these compounds affected other ion channels (such as calcium channel) cannot be excluded. It is not possible to estimate directly the potency for blockade of N a or K +  +  channels by using ECG parameters and response to electrical stimulation since changes in the latter are qualitatively but not quantitatively equivalent to changes in ion currents. However, relative preference for N a or K channel blockade with +  +  different agents may be compared by using the potency ratios of indicators for N a channel blockade (e.g. PR and iT) versus indicators for K channel blockade +  +  (e.g. QT and ERP).  The results suggested that there was no preference for  effects on N a or K channels with either esters or amide analogues. +  4.2.  +  Potency for different drug effects on haemodynamics and normal cardiac  electrophysiology between ester-linked and amide-linked analogues Ester compounds appeared to be less potent in their effects on BP, HR, ECG and electrical stimulation variables, and none of the esters was more potent on any of the above haemodynamic and electrophysiological characteristics than its corresponding amide. Since the only difference in the molecular structure between ester and amide in each pair is the ester / amide bond, any difference in the effects between these two analogues should be related to those groups.  On the other hand, if no  difference existed, logically we cannot exclude the possible alteration of activities of compounds by displacement of amide group with ester group.  Possibly the  alterations are multiple and interactive, which cause contrary effects.  The  64  difference in potency between ester and amide analogues should depend on how the ester or amide group in a particular molecule is involved in interaction with ion channels.  Any inconsistencies in results might be expected in view of  complicated interactions of molecular moieties, and the precision and accuracy of the different cardiovascular indicators measured. Based on above consideration, we supposed that potency for effects on normal cardiac electrophysiology would be decreased by displacement of amide bond with ester bond in this series of compounds.  4.3. Protective effects of RSD compounds against ischemia-induced arrhythmias In predicting antiarrhythmic efficacy, a comparison of pairs based on data obtained in normal tissue alone are probably not too meaningful, since the drug effects are altered by abnormal conditions initiating arrhythmias. A potent agent on normal electrophysiology may be less selective for arrhythmias (Hondeghem, 1987; Haverkamp, 1991). This seemed to be the case for compounds RSD996 and RSD997 which were more potent in the effects on normal cardiac tissue than their ester analogues RSD973 and RSD1009, respectively, but failed to provide the same pattern for protection against ischemia-induced arrhythmias.  Instead,  they had similar antiarrhythmic activity to their ester analogues RSD973 and RSD1009, respectively. However, the results with two pairs could not simply be extrapolated to other structurally complementary compounds with ester or amide groups. For example, the antiarrhythmic potency for ester RSD1046 was lower than that for its amide analogue, and the dose-response curve was parallel to the  65  amide (RSD1044) with a right shift.  Compared to the other two pairs of  compounds RSD1046 and RSD1044 have relative larger molecular weights, higher pKa and a dimethoxy group instead of morpholino group. Whether and how these factors related to the results that are different from the other pairs remains to be elucidated. Although there were some similarities between the ester and amide analogues, the antiarrhythmic activity among different pairs of ester and amide compounds were obviously different in terms of ED and slopes of antiarrhythmic curves. In 50  an overall sense the results indicate that the mechanism(s) underlying antiarrhythmic activity of ester-linked and corresponding amide-linked analogue are the same. Most likely, other moieties (rather than ester or amide group) in the molecular structure (such as the morpholino group) may play even more important roles in determining antiarrhythmic activity of the compounds.  The  interaction of ester or amide group with other moieties, not ester or amide group alone, might modify the activity of compounds.  4.4. Selectivity of RSD compounds for ischemia-induced arrhythmias The major finding in this study is that the esters in two morpholino pairs had better therapeutic indices, i.e. antiarhythmic effects were produced by esters at doses which had limited effects on the normal heart, nor on blood pressure and heart rate.  This was not the case for the dimethoxy pair (RSD1046 and  RSD 1044). It is suggested that the difference in selectivity between the ester and amide analogue might not be dependent on the ester or amide group alone  66  but on the interaction of these group with other moieties.  In a comprehensive  review detailing the search for compounds selective for ischemia cardiac tissue, Bain et. al. (Bain et. al., 1998) have illustrated the importance of the morpholino group in a chemical series similar to the compound used in this study.  In  agreement with that investigation, our results showed that the morpholino group appeared important in conferring ischemia selectivity on ester compounds. The  overall  comparison  of  potencies for effects  of the  compounds  on  haemodynamic, electrophysiology, ischemia-induced arrhythmias and the gross therapeutic index revealed that a potent compound on normal electrophysiology, which might be potent (such as amide RSD1044), or not very potent for antiarrhythmic effect (such as amide RSD996), might be not useful in terms of the selectivity for ischemia-induced arrhythmias, whereas a compound with low potency for normal cardiac effects (such as ester RSD973) might be selective for ischemia-induced arrhythmias. Such observations suggest that the mechanisms of antiarrhythmic action should not be simply attributed to a compound's electrophysiological effects under normal condition.  The activity of compounds in the absence and presence of  ischemic arrhythmias might be different. Pathological conditions, such as ischemia, alter ion channel functions, channel properties as well as the actions of drug binding, further modifying drug action (Task Force, 1991). Perhaps, morpholino ester-linked compounds preferentially interact with the modified channels or binding sites, or interfere with abnormal channel functions associated with ischemia.  67  4.5.  Possible factors contributing to the differences in action between ester-  linked and amide-linked compounds The explanation for the above findings is not obvious. However, certain factors can be taken into consideration in trying to explain the results of this study. Since all the studies were performed in vivo, it might be assumed that pharmacokinetic differences could somehow explain the findings.  However,  unlike use of an i. v. bolus technique where esters would be particularly liable to immediate hydrolysis, the infusion regimen used was designed to obtain relatively slowly rising plasma level during the time periods when drug effects were measured. A specific pharmacokinetic analyses in similar compounds (Beatch, personal communication), revealed that blood levels rose steadily and predictably in the 515 minute period (when measurements were made in this study) following initiation of infusion.  Therefore the influence of pharmacokinetic processes  should not be a major factor.  Furthermore, the pharmacokinetic factors alone  could not explain the apparent selectivity of compounds for ischemia-induced arrhythmias, i.e. while the morpholino ester were less potent in their actions on normal tissue, they retained their potency against ischemia-induced arrhythmias. It could be argued that metabolism pathways were changed under pathological (ischemic) conditions. Supposedly, the breakdown of esters was retarded in rats subject to ischemia giving rise to a higher potency in ischemia. However, such an argument is implausible since no differences were seen with the dimethoxy pair.  68  According to the size/solubility hypothesis, Courtney (Courtney, 1987) suggested that t h e  physico-chemical  properties of a drug  interaction with ion channels.  molecule will influence  its  Molecular weight and lipid solubility a p p e a r to be  the most important determinants for the kinetics of blocking and unblocking of cardiac s o d i u m channels by the classic antiarrhythmic drugs.  T h e effects of  charged and u n c h a r g e d f o r m of the drug on ion channels should be different. However,  replacement  of amide group with ester group did  not alter  the  molecular weight, partition coefficient and pKa of the c o m p o u n d s w e studied. Therefore, these physico-chemical properties should not be considered as the major factors  contributing to differences  in effects on electrophysiology  or  arrhythmic activity between the ester and amide analogues. There are suggestion that steric factors must be considered to be an important physico-chemical (Courtney,  characteristic  1987).  when  a molecule  Structure-activity  relationship  is relatively studies  on  large  (>350)  series  RSD  c o m p o u n d s in our laboratory (Zolotoy, personal communication) suggested that the  conformational  dynamics  of  probability of target penetration.  molecular  structure  might  influence  the  T h e more flexible the molecular structure, the  less probability of target penetration. The replacement of the ester group in R S D c o m p o u n d s studied with amide group might reduce the flexibility of molecular structure a n d thus channels.  This  influence the interaction of amide c o m p o u n d s  hypothesis  is contrary to t h e finding f r o m  with  Courtney  ion and  VVildsmith's e x p e r i m e n t s (Courtney, 1980, 1983; Wildsmith, 1985, 1987).  69  They found that amide bond provided steric hindrance for the interaction with receptor, and hence reduced the potency for N a channel blockade. +  Frequency-dependence of drug actions may have contributed to selectivity for the arrhythmias.  In this study, we attempted to estimate the difference in  frequency-dependence of ester and amide analogue by comparing the effects on different electrical stimulation variables, which were measured at different stimulation frequencies.  While these methods may not be precise or accurate  they may give useful indicators.  A better method would be to investigate the  changes of drug effect on a same indicator with a series of different frequencies of action potential.  However, this was not done. The results of our imperfect  measurements showed that there were no statistically significant differences in these ratios between ester and amide analogues. However, the potency ratios of ERP versus MFF appeared to be higher for the ester RSD973 and RSD1009 (0.9±0.2, 0.9±0.3, respectively), while they were almost the same (0.4±0.1) for the other compounds. dependence  between  Whether these really reflect differences in frequencyRSD973 and RSD1009, as well as RSD1009  and  RSD997, and whether these differences account for the difference in their selectivites for ischemia-induced arrhythmias remains to be proved. If the arrhythmias induced by ischemia are due primarily to disturbed electrical activity in the ischemic zone of heart, the better therapeutic indices could be provided by drugs retaining potency in ischemic cardiac tissue but losing potency in normal cardiac tissue. Ischemia induces many changes in the state or nature of ion channels thereby altering the potency of ion channel blockers. Lidocaine,  70  for instant, can be expected to be more potent in ischemic tissue by virtue of the fact that it binds to inactive N a  +  channels and that the raised extracellular  potassium in ischemia results in an increased proportion of channels in the inactivated state (Dumaine & Kirsch, 1998). Ischemia can also change the state of drug molecule. For example pH will, in a manner that depends upon the pKa of the molecule, change the proportion of ionized to unionized drug. This does not apply to the current discussion since the esters and amides chosen have similar pKa values. However, ischemia can influence the potency of drugs by a variety of mechanisms. For the compounds studied here it appears that potencies for blockade of morpholino esters were potentiated by ischemia (or in another words, the potencies for morpholino esters were reduced in normal tissue) compared with their amide analogues.  It might  imply that a better "fit" between morpholino esters and their effector site under condition of ischemia versus normal conditions. In classical receptor theory, the affinity for a drug is described by the dissociation constant or the ratio of the "off' and "on" rate constant for binding.  Most particularly a decrease in "off rate  constant for a drug will increase its potency and this is what might be happening for the morpholino esters as a consequence of ischemia-induced changes in channel properties. This assumption did not apply to dimethoxy compounds. A reason has to be sought to explain the difference in findings between morpholino and dimethoxy compounds.  71  It has to be emphasized that there are two complementary approaches to analyzing drug action.  One is mechanistic and reductionist in nature and  concentrates on the exact molecular mechanisms involved in drug action. The other is more functional in nature and analyzes action in terms of body systems and the whole organism. approach  since the  In our study we have chosen to use the functional  molecular  mechanisms  involved  in  ischemia-induced  arrhythnias are not known with any certainty. Of necessity this entailed the use of less precise, less accurate and more ambiguous measures of drug action. Caution has to be exercised in extrapolating such data to the in vitro situation, especially with ion channel blocking drugs which block more than one channel. 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