EP0752859A1

EP0752859A1 - Pharmaceutical composition containing a class iii antiarrhythmic agent and a class iv antiarrhythmic agent

Info

Publication number: EP0752859A1
Application number: EP95914321A
Authority: EP
Inventors: SmithKline Beecham Lab.Pharm. BRIL; SmithKline Beecham Lab.Pharm. FAIVRE; B.E.J. SmithKline Beecham Lab. Pharm. GOUT; Marie-Cl. SmithKline Beecham Lab.Pharm. FOREST
Original assignee: SmithKline Beecham Laboratoires Pharmaceutiques
Current assignee: Laboratoire GlaxoSmithKline SAS
Priority date: 1994-03-31
Filing date: 1995-03-28
Publication date: 1997-01-15
Also published as: WO1995026726A1; JPH09510985A

Abstract

A pharmaceutical composition, which composition comprises a class III antiarrhythmic agent and a class IV antiarrhythmic agent, providing that the composition is not a combination of 10νg/kg E4031 and 0.1mg/kg verapamil, and optionally a pharmaceutically acceptable carrier therefor and in particular a pharmaceutical composition which composition comprises a class III and a class IV antiarrhythmic agent, characterised in that the class III reagent is present in an antiarrhythmic effective amount and the class IV antiarrhythmic agent is present in an amount lower than that which provides a substantial calcium blocking effect, and optionally a pharmaceutically acceptable carrier therefor; a process and medical use for such a composition.

Description

Pharmaceuti cal compositi on contai n i ng a cl ass III aπtiarrhytmi c agent and a cl ass IV anti arrhythmi c agent

This invention relates to a novel pharmaceutical composition and in particular the use of a combination of antiarrhythmic agents in the treatment of arrhythmic disorders in mammals, including humans, in order to achieve a synergistic effect.

Antiarrhythmic drugs have been grouped according to the pattern of electrophysiological effects they produce and/or their presumed mechanism of action. A classification in four groups has been originally proposed by Vaughan Williams in 1970. At the molecular level, class I compounds act on Na⁺ currents; class II compounds possess β-adrenoceptor blocking activity; class III drugs prolong cardiac action potential such as potassium channel Mockers; and class IV drugs target Ca⁺⁺ channels.

E-4031 (Eisai), whose chemical name is methanesulfonamide, N-[4-[[l-[2-(6- methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl]-, dihydrochloride is a known potassium channel blocking agent i.e. a class III drug. Verapamil (5-[(3,4- dimethoxyphenethyl)methylamino]-2-(3,4-dimethoxyphenyl)-2- isopropylvaleronitrile) is a known class IV agent having calcium channel blocking activity.

It is also known from Katoh et al (Journal of Mol. Cell Cardiol. 22, (Supplement III) 1990, page s70) that E-4031 and Verapamil in doses which are ineffective against premature ventricular arrhythmia when administered alone (E4031 lOμg/ml and Verapamil 0. Img/kg) act in combination to suppress premature ventricular arrhythmia.

However, it has now been discovered that a combination of a class HI and class IV antiarrhythmic agents provides an effect which is more fundamental than a mere enhancement of the antiarrhythmic effect of each compound: It is now considered that the combined effect of the two anti arrhythmia agents leads to a beneficial modification of the mode of antiarrythmic effect for the combination. In particular the combination provides an improved antiarrhythmic activity in conjunction with a reduced adverse effect profile.

Accordingly, the present invention provides a pharmaceutical composition, which composition comprises a class III antiarrhythmic agent and a class IV antiarrhythmic agent, providing that the composition is not a combination of lOμg/kg E4031 and 0. Img/kg verapamil, and optionally a pharmaceutically acceptable carrier therefor. The present invention also provides the use of a combination of a class III and a class IV antiarrhythmic agent in the manufacture of a medicament for use in treating cardiac arrhythmia, providing that the medicament is not a combination of lOμg/kg E403 and 0. Img/kg verapamil. The class III agent will normally be a potassium channel blocking agent, and any such agent known in the art would be suitable.

Class III agents include the compounds of EP 235752, EP 245997, EP 322390, USP 4544654 and DE 3419067.

A favoured potassium channel blocking agent is an inhibitor of the delayed rectifier potassium current. A particular example of such an agent is the compound E4031. Further examples include sematilide, dofetilide, almokalant and d-sotalol.

The class IV agent will be a compound having calcium channel blocking activity, suitably having an L-type calcium blocking effect.

Class IV agents include the compounds of USP 3261859, DE 2117571, ZA 6801482 and DE 2549568.

A particular example of a class IV agent is verapamil. Further examples include the dihydropyridines such as nitrendipine, nifedipine and nisoldipine.

As stated above, it has been found that the combination of a class III and class IV agent has been found to provide an improved antiarrhythmic activity in conjunction with a reduced adverse effect profile. These benefits are considered to be due to the calcium blocking effect of the class IV compound.

However it is important that the calcium blocking effect in the combination is not too great. It is considered that the upper limit for the calcium blocking effect is suitably met if the class IV agent is present in an amount lower than that which provides a substantial calcium blocking effect. A 'substantial calcium blocking effect' in this context is present when the class IV agent decreases action potential duration, increases PR interval, reduces arterial blood pressure and/or decreases cardiac contractile force. This is to be contrasted with 'a calcium blocking effect' which is considered to be present when the class IV agent decreases action potential duration and/or increases PR interval but has no significant effect upon arterial blood pressure and/or cardiac contractile force.

Accordingly, in a particular aspect of the invention there is provided a pharmaceutical composition which composition comprises a class III and a class IV antiarrhythmic agent, characterised in that the class HI reagent is present in an antiarrhythmic effective amount and the class IV antiarrhythmic agent is present in an amount lower than that which provides a substantial calcium blocking effect, and optionally a pharmaceutically acceptable carrier therefor.

Favourably, the class IV antiarrhythmic agent is present in the composition in an amount which exhibits a threshold calcium blocking effect, that is an amount which only just provides an electrophysiological effect due to a calcium antagonist effect.

A lower limit for the amount of class IV antiarrhythmic agent in the composition is considered to be an amount which is below the threshold calcium blocking effect in that it is ineffective on action potential duration and on the PR interval.

'Action potential duration' and 'increase the PR interval' are art terms for effects which are measured using conventional electrocardiographic methods such as those used herein.

The class III and IV agents can be used in a wide variety of weight ratios to achieve the above mentioned synergistic effect. For example, the ratio of class III to class IV agent may be from 1:1 to 10:1, preferably from 1:1 to 5:1, with a particular ratio of around 3:1.

In particular, the dosage of class III agent may be within wide ranges, for example from 0.1 to 2.0 mg/kg, and the class IV agent may be within 0.01 to 1.0 mg/kg.

The invention also provides a method for the treatment and/or prophylaxis of cardiac arrhythmias in mammals, particularly humans, which comprises administering to the mammal in need of such treatment and/or prophylaxis an effective amount of a combination of a class III antiarrhythmic agent and class IV antiarrhythmic agent, providing that the combination is not a combination of lOμg/kg E4031 and 0.1 mg/kg verapamil.

The invention further provides a method for the treatment and/or prophylaxis of cardiac arrhythmias in mammals, particularly humans, which comprises administering to the mammal in need of such treatment and/or prophylaxis an amount of a combination of a class III antiarrhythmic agent and class IV antiarrhythmic agent, wherein the class III antiarrhythmic agent is administered in an antiarrhythmic effective amount and the class IV antiarrhythmic agent is administered in an amount lower than that which provides a substantial calcium blocking action.

The invention also provides the use of a combination of a class IH and a class IV antiarrhythmic agent in the manufacture of a medicament for use in treating cardiac arrhythmia, wherein the class III antiarrhythmic agent is present in the medicament in an antiarrhythmic effective amount and the class IV antiarrhythmic agent is present in an amount lower than that which provides a substantial calcium blocking action

Suitably, the class III antiarrhythmic agent and class IV antiarrhythmic agent are administered together, preferably in a pharmaceutical composition.

The class III antiaιτhythmic agent and class IV antiarrhythmic agents are prepared according to conventional procedures depending upon the nature of the agent. For example the above mentioned agents are prepared according to methods disclosed in the following publications: Class III: E-4031 (EP235752 ), sematilide (USP4544654 ), dofetilide

(EP245997), almokalant (EP322390 ), and d-sotalol (DE3419067 ).

Class IV: Verapamil (USP3261859 ), nitrendipine (DE2117571 ), nifedipine (ZA6801482 ), and nisoldipine (DE2549568 ).

The composition of the invention may be prepared using known techniques. In a further aspect the invention provides a process for the preparation of a composition, the composition comprising a class III antiarrhythmic agent, a class IV antiarrhythmic agent and, optionally, a pharmaceutically acceptable carrier, which process comprises the admixture of a class III antiarrhythmic agent, a class IV antiarrhythmic agent and, optionally, a pharmaceutically acceptable carrier, providing that said admixture does not give a combination of lOμg/kg E4031 and 0. Img/kg verapamil.

The invention also particularly provides a process for the preparation of a pharmaceutical composition, the composition comprising a class III and a class IV antiarrhythmic agent, in which the class III reagent is present in an antiarrhythmic effective amount and the class IV antiarrhythmic agent is present in an amount lower than that which provides a substantial calcium blocking effect, and optionally a pharmaceutically acceptable carrier therefor, which process comprises admixing the antiarrhythmic effective amount of the class III antiarrhythmic agent, the class IV antiarrhythmic agent in an amount which is lower than that which provides a substantial calcium blocking effect and, optionally, the pharmaceutically acceptable carrier.

It will be appreciated that an antiarrhythmic agent and includes the pharmaceutically acceptable salts and solvates of any particular agent including the class HI antiarrhythmic agent and class IV antiarrhythmic agents specifically mentioned herein. Such compounds are prepared according to known methods. An antiarrhythmic effective amount of a class III antiarrhythmic agent is that amount which would be considered by those skilled in the art to provide such as effect depending upon such factors as the efficacy of the particular agent, the nature of the pharmaceutically acceptable salt or pharmaceutically acceptable solvate chosen and the nature and severity of the disorders being treated and the weight of the mammal. However, a unit dose will normally contain 0.1 to 500 mg for example 2 to 50 mg, of the compound of the invention. Unit doses will normally be administered once or more than once a day, for example 2,3,4,5 or 6 times a day, more usually 2 to 4 times a day, such that the total daily dose is normally in the range, for a 70 kg adult of 0.1 to 2500 mg, more usually 50 to 2000 mg, for example 10 to 75mg, that is in the range of approximately 0.002 to 35 mg/kg/day, more usually 1 to 30 mg/kg/day, for example 0.15 to 1 mg/kg/day.

The above mentioned calcium blocking activity of a class IV antiarrhythmic agent can be determined using the techniques disclosed herein. In the present treatment, the active compounds may be administered by any suitable route, e.g. by the oral, parenteral or topical routes. As indicated, for such use, the compounds will normally be employed in the form of a pharmaceutical composition in association with a human or veterinary pharmaceutical carrier, diluent and/or excipient, although the exact form of the composition will naturally depend on the mode of administration.

Active compounds or a pharmaceutically acceptable salt thereof and/or a pharmaceutically acceptable solvate thereof is normally administered in unit dosage form.

Compositions are prepared by admixture and are suitably adapted for oral, parenteral or topical administration, and as such may be in the form of tablets, capsules, oral liquid preparations, powders, granules, lozenges, pastilles, reconstitutable powders, injectable and infusable solutions or suspensions, suppositories and transdermal devices. Orally administrable compositions are preferred, in particular shaped oral compositions, since they are more convenient for general use.

Tablets and capsules for oral administration are usually presented in a unit dose, and contain conventional excipients such as binding agents, fillers, diluents, tabletting agents, lubricants, disintegrants, colourants, flavourings, and wetting agents. The tablets may be coated according to well known methods in the art. Suitable fillers for use include cellulose, mannitol, lactose and other similar agents. Suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycollate. Suitable lubricants include, for example, magnesium stearate. Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulphate.

These solid oral compositions may be prepared by conventional methods of blending, filling, tabletting or the like. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of fillers. Such operations are, of course, conventional in the art.

Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats, emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example, almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl rj-hydroxybenzoate or sorbic acid, and if desired conventional flavouring or colouring agents.

For parenteral administration, fluid unit dose forms are prepared containing a compound of the present invention and a sterile vehicle. The compound, depending on the vehicle and the concentration, can be either suspended or dissolved. Parenteral solutions are normally prepared by dissolving the compound in a vehicle and filter sterilising before filling into a suitable vial or ampoule and sealing. Advantageously, adjuvants such as a local anaesthetic, preservatives and buffering agents are also dissolved in the vehicle. To enhance the stability, the composition can be frozen after filling into the vial and the water removed under vacuum.

Parenteral suspensions are prepared in substantially the same manner except that the compound is suspended in the vehicle instead of being dissolved and sterilised by exposure to ethylene oxide before suspending in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the active compound.

For topical administration, the composition may be in the form of a transdermal ointment or patch for systemic delivery of the active compound and may be prepared in a conventional manner, for example, as described in the standard textbooks such as 'Dermatological Formulations' - B.W. Barry (Drugs and the

Pharmaceutical Sciences - Dekker) or Harrys Cosmeticology (Leonard Hill Books). In addition such compositions may contain further active agents such as anti-hypertensive agents and diuretics.

As is common practice, the compositions will usually be accompanied by written or printed directions for use in the medical treatment concerned. No toxicological effects are indicated when an active compound is administered in the above mentioned dosage ranges.

The invention is described in more detail below.

Summary

The effects of E-4031 (a class III antiarrhythmic agent), verapamil (a calcium channel blocking drug) and a combination of both E-4031 and verapamil on ventricular fibrillation were evaluated in anesthetized dogs 3 days following myocardial infarction. Ventricular arrhythmias were induced by programmed electrical stimulation protocols. In dogs with myocardial infarction E-4031 (0.1 to 1.0 mg/kg) significantly increased QT intervals and ventricular effective refractory periods in both the normal in the infarcted areas. Verapamil reduced heart rate, mean arterial pressure, pressure-rate product and to a lesser extent dP/dtmax. In contrast to E-4031, verapamil did not change QT intervals and ventricular effective refractory periods but increased PR interval. In combination with E-4031, high doses of verapamil induced more pronounced cardiodepressive effects than given alone. The incidence of ventricular arrhythmias was changed neither by cumulative administration of vehicle nor by increasing doses of verapamil. E-4031 (0.1 to 3.0 mg/kg) reduced the severity of ventricular arrhythmias from ventricular fibrillation to non sustained ventricular tachycardia. A combination of verapamil with E-4031 was able to suppress completely the occurrence of arrhythmias induced by programmed electrical stimulation (i.e. rendered these non-inducible) in several dogs, an effect which was never observed with E-4031 alone. However, when the dose of verapamil was further increased, the occurrence of conduction block led to adverse effects. These results clearly established that a correct combination of a compound with a potassium channel blocking activity and a compound with a calcium channel activity ayu represent a new and novel mechanism for the treatment of cardiac arrhythmias.

Introduction The blockade of cardiac outward potassium channels by class III antiarrhythmic agents represents the most recent therapeutic approach developed for the suppression of re¬ entrant ventricular tachyarrhythmias and sudden death (1,2).

Some newly developed class III antiarrhythmic agents, such as dofetilide (UK-68,798), E-4031 and sematilide, that selectively prolong action potential duration and refractoriness in ventricular preparations, have been shown to reduce the incidence of ventricular fibrillation in canine models of sudden coronary death (3-6). Experiments were performed in anesthetized dogs with a previous myocardial infarction to determine the effects of the pure class III agent E-4031 on the ventricular effective refractory period (ERP) and on the inducibility of ventricular tachycardia or ventricular fibrillatio n. Using the same protocol, verapamil and a combination verapamil with E-4031 were tested in order to evaluate the benefit of a combination of the two mechanisms, potassium and calcium blocking activities.

Methods

Surgical preparation for chronic infarction Mongrel dogs of either sex (13-23 kg) were used. All experimental procedures were in compliance with NIH guidelines (Publication n°85-23) for the use and care of experimental animals.

Dogs were anesthetized using 30 mg/kg i.v. sodium pentobarbital. Subcutaneous needle electrodes were installed to monitor a lead II electrocardiogram (ECG). After tracheal intubation for artificial respiration (Harvard 613 ventilator, South Natick, MA, U.S.A.) a left thoracotomy was performed at the fourth intercostal space and the heart was exposed in a pericardial cradle. The left anterior descending (LAD) coronary artery was isolated close to its origin, a silk suture was placed around the artery and a two stage coronary artery occlusion was performed. When the extent of area of underperfusion appeared too small under visual inspection several epicardial collaterals were also ligated for two hours. Reperfusion was performed in the presence of a critical stenosis for 30 min then complete reflow of the occluded zone was achieved. The pericardium was closed and the surgical incision was repaired. Postoperatively, animals received amoxicillin (1.0 g, i.m., SmithKline-Beecham, Paris, France). Buprenorphine (0.3 mg, i.m., Schering-Plough, Kenilworth, NJ, U.S.A.) and droperidol (1.5 mg/kg, i.m., Janssen Pharmaceutica, Beerse, Belgium) were given as required.

Electrophysiologic study and programmed electrical stimulation

Three days after induction of the anterior infarction, animals were reanesthetized, as described previously, for the programmed electrical stimulation (PES) protocol. Systemic arterial pressure was measured from the right femoral artery (P23 ID transducer, Gould Electronics, Cleveland, OH, U.S.A.). A 5F microtip catheter pressure transducer (Millar Instruments Inc., Houston, TA, U.S.A.) was inserted via the left carotid artery to measure the left ventricular pressure (LVP) and its first derivative dP/dt. A peripheral venous catheter was used for administration of fluids. Electrocardiographic parameters were measured via a standard lead II ECG. For determination of the QT interval a CV5RL precordial lead of ECG was measured as previously described (7). Under artificial respiration the heart was re-exposed by thoracotomy through the left fifth intercostal space. One pair of stainless steel electrodes was attached to the left atrium for subsequent pacing. Two pairs of teflon coated needle electrodes were sutured respectively in the normal zone (NZ) and the infarcted zone (IZ) of the left ventricle to measure the effective refractory period (ERP) and for induction of trains of stimulation by PES testing. The ventricular ERP was determined in both NZ and IZ during atrial pacing at 300 msec. Every 8 beats a single premature stimulus S2 (3 msec duration, 1.5 x threshold) was introduced at progressive shorter delay from the atrial stimulus S 1 until no ventricular response was obtained. The ERP was calculated from the longest (S1-S2) interval which did not induce a propagated response in the tissue. To determine the actual ERP duration, the ventricular conduction time (time from the S 1 stimulus to the ventricular response) was substracted from the (S 1-S2) duration.

The pacing protocol for programmed ventricular stimulation was adapted from Katoh et al. (4). During atrial pacing at 286 msec, animals were subjected to ventricular stimulation by delivering a triple-pulse train (S2-S3-S4) in the ventricular IZ using a Grass S8800 stimulator and SIU-5 isolation unit (Grass Instruments, Quincy, MA, USA). The (S1-S2) coupling interval of the train of stimuli was 10 msec longer than the measured refractoriness. The (S2-S3) and (S3-S4) train intervals were tested in the range of 125 to 200 msec to determine the susceptibility of the ventricle. Inducibility of animals was classified into four groups according to their responsiveness to stimulation : non-inducible animals (NI) which elicited less than 4 ventricular premature beats in response to the PES ; non-sustained ventricular tachycardia (nsVT), animals responding with 4 or more ventricular premature beats lasting for less than 15 sec ; sustained ventricular tachycardia (sVT), ventricular tachycardia persisting for more than 15 sec and less than 30 sec ; ventricular tachycardia-ventricular fibrillation (VT/VF), ventricular tachycardia lasting for more than 30 sec or ventricular fibrillation. The latter response to PES required a DC cardioversion (Defi 2, S&W Medico Teknik A/S, Albertslund, Denmark).

Experimental time protocol

After stabilization, baseline hemodynamic and ECG parameters were measured. The ventricular ERP was determined in both NZ and IZ. A control PES test was then performed in the absence of drug to determine the pretreatment response of dogs. Hemodynamic and ECG parameters were measured again. Inducible animals (n=8 in all groups) were randomly assigned to receive either vehicle or increasing doses of drugs over a 10 min period 15 min before determination of ERP and subsequent PES test. Each dose was infused at 50 min intervals to allow recovery of animals from PES.

Drugs and measurements

All drugs (hydrochloride salt) were dissolved in bidistilled water. Drug solutions were prepared every day. E-4031 was given at 0.1, 0.3 and 1.0 mg/kg, i.v. Verapamil was administered at 0.03, 0.1 and 0.3 mg/kg. For the combination E-4031 + Verapamil, E- 4031 (0.1 mg/kg) was given with increasing doses of verapamil.

Heart rate was integrated from the ECG. Intervals of ECG were the mean of at least five determinations. A corrected QT interval [QTc = (QT interval in msec)/(RR interval in sec)^] was determined according to Bazett (8). Parameters were recorded on a thermal recorder (8800, Gould). At every time point of measurement, data were analyzed on 80286 IBM computer using Clodia interactive software (Clod, Saint- Maur, France). Stored data were transferred to a VAX 4000-200 computer (DEC, Maynard, MA, U.S.A.) for further analysis. Statistical analysis

Values were expressed as mean±SE and were compared using analysis of variance followed by Duncan's new multiple range test. Durations of VT not found Gaussian were compared using the Mann-Withney test. The incidence of inducibility by PES in individual groups was compared with vehicle group using a Fisher's Exact test. All statistical comparisons were performed using Crunch 4 statistical package (Crunch

Software Corporation, Oakland, CA, U.S.A.). A p value less than 0.05 was considered statistically significant.

RESULTS

Fifty-one dogs have been prepared for this study, forty of which were included into the protocol. Five animals died between the coronary artery occlusion and the PES protocol and six other dogs were excluded because they responded as noninducible to the control PES test.

Hemodynamic responses

In the vehicle treated group, cumulative administration of saline did not significantly change the hemodynamic parameters (table 1). A reduction in heart rate was observed in the presence of E-4031, but this reduction was not sufficient to produce a decrease in pressure-rate product. Verapamil reduced heart rate, mean arterial blood pressure and pressure-rate product in a dose dependent manner. In addition, a slight but not significant reduction in left ventricular pressure and (+)dP/dtmax was also noted at the high dose (0.3 mg/kg). In the presence of E-4031 the effects of verapamil were more pronounced since the reduction in hemodynamic parameters were significantly different from the other treated groups (analyse of variance followed by Tukey test).

Electrocardiographic parameters

Electrocardiographic parameters, PR interval, QT interval and QTc were not changed by cumulative administration of vehicle. E-4031 induced a dose dependent increase in QT and QTc intervals, with no change in PR interval. Verapamil given alone, only increased PR interval without any effect on QT intervals. However, in presence of E- 4031 (0.1 mg/kg) verapamil markedly increased QT intervals, with limited effect on PR interval suggesting a potentiation of the action of the class III antiarrhythmic agent. In addition, verapamil, given at 0.3 mg/kg alone or in presence of 0.1 mg/kg of E-4031 produced frequent atrio-ventricular blocks. Such conduction disturbances were never observed in presence of saline, or E-4031.

Ventricular effective refractory periods (VERP) were measured in both the normal and the infarcted areas. The vehicle did not significantly change VERP in both zones. E- 4031 increased effective refractory periods in both the normal and the infarcted zones (Table 2). In presence of E-4031, increasing doses of verapamil (0.03, 0.1 and 0.3 mg/kg) induced a marked increase in VERP. Effective refractory periods which were 158±7 and 152±6 msec in the normal and infarcted zones before the combination were increased to 202±9 and 200±11 msec respectively in presence of 0.1 mg kg (p < 0.001) and to 219±16 and 223±13 msec with 0.3 mg/kg (p < 0.001).

Programmed electrical stimulation

In the vehicle group, pretreatment responses to PES was represented by VT/VF in 6 out of 8 dogs (figure 1). A steady response was obtained during the whole experiment in 5 dogs while one dog dropped toward nsVT during the third PES test. Two dogs responding with nsVT to pretreatment PES did not change throughout the experimental period.

E-4031 administered at 0.1 mg/kg suppressed the incidence of VT/VF and sVT in 5 out of 7 dogs. At higher doses, a significant protection was obtained with respect to the induction of VT/VF by PES (p = 0.013 vs vehicle) When compared to pretreatment inducibility, a significant reduction of the incidence of VT/VF and sVT by E-4031 was observed from the first administration (p = 0.02, by Fisher's Exact test at 0.1 mg/kg). However, none of the dogs were converted into non-inducible at either dose of E-4031 (figure 1).

The effects of verapamil administered alone on the inducibility of dogs to PES are shown in figure 1. Although some instability of the response to PES was observed in 3 dogs during successive testing, there was no reduction of the baseline inducible tachyarrhythmias in presence of verapamil. Inducibility to PES was not modified by either dose of verapamil in 4 out of 5 animals responding with VT/VF. Verapamil converted only one animal from VT/VF to nsVT at the higher dose (0.3 mg/kg).

In contrast, using the same dose-range of verapamil in combination with a single dose of E-4031 (0.1 mg/kg), a significant reduction of the incidence of VT/VF was observed in response to the PES test. This protective effect occurred from the first combined dosing (0.03 mg/kg verapamil + 0.1 mg/kg E-4031, p = 0.020 by Fisher's Exact test). At higher doses of verapamil, 3 dogs were converted from nsVT to non- inducible in response to PES (figure 1). At 0.3 mg/kg verapamil, the number of dogs rendered NI was significantly higher than in the vehicle group (p = 0.013).

CONCLUSION Ventricular arrhythmias induced by PES are known to be generated by a re-entrant mechanism (9). In experimental studies class III antiarrhythmic agents may convert PES induced ventricular fibrillation to non-sustained ventricular tachycardia (4, 10). A similar effect is observed in the present study with a typical class III antiarrhythmic compound (E-4031). However, it appeared that the combination of E-4031 and verapamil was able to cause a greater protection of canine hearts since several animals given 0.01 or 0.03 mg/kg of verapamil (in the presence of 0.1 mg/kg of E-4031) were in a non-inducible state. Although verapamil alone did not change the incidence of ventricular arrhythmias induced by PES, an adjusted combination of a potassium and calcium blocking effect may represent a potent and safe therapeutic approach for cardiac arrhythmias.

The antiarrhythmic effects observed with E-4031 and the combination of E-4031 and verapamil are associated with a dose dependent increase in ventricular effective refractory period. This suggest that the protection of PES induced arrhythmias may be related to a prolongation of the cardiac action potential. The observation that verapamil neither prevented PES -induced arrhythmias nor prolonged ventricular effective refractory period reinforces the hypothesis that action potential prolongation may represent an effective antifibrillatory mechanism.

E-4031 did not change left ventricular pressure or (+)dP/dtmax but reduced heart rate. This effect was essentially responsible for the slight reduction in pressure-rate product, an index of cardiac work. Except at the high dose of 0.3 mg/kg, verapamil either in the absence or in the presence of E-4031 (0.1 mg/kg) did not markedly change cardiac function. However, the higher dose used in this study, induced a marked cardiodepressive effect with the occurrence of atrio-ventricular conduction disturbances suggesting a limitation in the beneficial effect of this combination.

REFERENCES

1. Singh BN, Nademanee K. Control of cardiac arrhythmias by selective lengthening of repolarization: Theoretic considerations and clinical observations. Am Heart J 1985;109:421-430. 2. Colatsky TJ, Follmer CH, Starmer CF. Channel specificity in antiarrhythmic drug action. Mechanism of potassium channel block and its role in suppressing and aggravating cardiac arrhythmias. Circulation 1990;82:2235-2242.

3. Black SC, Chi L, Mu DX, Lucchesi BR. The antifibrillatory actions of UK-68,798, a class III antiarrhythmic agent. J Pharmacol Exp Ther 1991;258:416-423. 4. Katoh H, Ogawa S, Furuno I, Sato Y, et al. Electrophysiologic effects of E-4031, a class III antiarrhythmic agent, on re-entrant ventricular arrhythmias in a canine 7- day-old myocardial infarction model. J Pharmacol Exp Ther 1990;253:1077-1082.

5. Chi L, Mu DX, Lucchesi BR. Electrophysiology and antiarrhythmic actions of E- 4031 in the experimental animal model of sudden coronary death. J Cardiovasc Pharmacol 1991;17:285-295.

6. Chi L, Mu DX, Driscoll EM, Lucchesi BR. Antiarrhythmic and electrophysiologic actions of CK-3579 and sematilide in a conscious canine model of sudden coronary death. J Cardiovasc Pharmacol 1990;16:312-324.

7. Koerner JE, Dage RC. Antiarrhythmic and electrophysiologic effects of MDL 11,939, a novel class III antiarrhythmic agent in anesthetized dogs. J Cardiovasc

Pharmacol 1990;16:383-393. 8. Bazett HC. An analysis of the time-relations of electrocardiograms. Heart 1920;7:353-370.

9. Wit AL, Janse MJ. The ventricular arrhythmias of ischemia and infarction. Electrophysiological mechanisms. Futura Publishing Inc, New York, 1993.

10. Zuanetti G, Corr PB. Antiarrhythmic efficacy of a new class III agent, UK-68,798, during chronic myocardial infarction: Evaluation using three dimensional mapping. J Pharmacol Exp Ther 1991;256:325-334.

Table 1 : Effects ofE-4031 and verapamil on hemodynamic parameters in dogs with

3 days of myocardial infarction.

Dose HeartRate MAP LVP LVPx HR (+)dP/dtmax

(mg/kg) (beat/min) (rnmHg) (mmH ) (mmHg.b/min) (mmHg/sec)

Vehicle

173±6 114±10 123111 2160512508 23321239

172±6 117±7 12518 2172411911 22791153

178±7 116±9 - 12719 2276312388 23331230

179±7 112±8 12418 2241012211 22131194

E-4031

0 171±11 106±6 11717 20011+1692 20001141

0.1 148±10 10815 12316 1815111172 22261204

0.3 145+9* 10016 11417 1633411177 19941123

1.0 14519** 102+4 11615 1688011199 21381167

Verapamil

0 170±11 10316 11618 20090+2354 21981175

0.03 16119 10015 11216 1838111805 20101148

0.1 155±8 10315 11716 1822011503 19501144

0.3 119±9*** 7919* 9718 1192811770** 15131203

Verapamil in presenceofE-4031 (0.1 mg/kg)

0 163±6 10612 11914 194741998 2075156

0.03 12717* 10614 12415 1577611314 22821116

0.1 11116*** 10214 12015 1347211234** 20461124

0.3 7516*** 5716*** 83+9* 6480+1183*** 12531196*

Values represent mean±SEM for 8 experiments in each group. Analysis of variance followed by Tukey test was used for comparison, * p < 0.05, ** p < 0.01, *** p <

0.001.

Table 2 : Effects ofBRL-32872, E-4031 and verapamil on electrocardiographic parameters in dogs with 3 days of myocardial infarction.

Dose PR interval QT interval QTc interval (msec ERP-NZ ERP-IZ

(mg kg) (msec) (msec) .sec¹^) (msec) (msec)

Vehicle

9713 22616 38116 151 17 15216

9812 22615 38217 149 15 149 15

9712 22215 38116 149 15 146 + 5

9712 21814 37614 149 15 146 + 5

E-4031

0 9715 234113 390+9 153+6 15616

0.1 9613 291118* 448115** 17316 17919*

0.3 10115 299120** 454120* 183110* 186110*

1.0 96+4 306120** 470117** 192+13 195114*

Verapamil

0 94+2 216+8 359110 15513 15115

0.03 10113 21917 35516 15214 14516

0.1 11514* 22118 352110 15215 14614

0.3 148+11*** 250113 342110 169110 15718

Verapamil in presence of E-4031 (0.1 mg/kg)

0 10213 23519 385111 15817 15216

0.03 10515 324121*** 464120** 19017* 194+7***

0.1 10916 342120*** 473123** 202+9*** 200111***

0.3 11010 36515** 442147 219116*** 223113***

0.001.

VEHICLE

Figure 1 : Programmed electrical stimulation (PES) in anesthetized dogs, 3 days after anterior wall myocardial infarction. Effects of PES before drug dosing (0) and after cumulative administration of each drug. Each line represents one animal with the nature of the response to PES indicated on the ordinate (see text for inducibility criteria of animals to the stimulation).

Further Studies

Inhibition of the calcium current has been suggested to be involved in the rate- dependent alterations of action potential duration (Jurkiewicz and Sanguinetti, 1993). The purpose of the present study was to assess the reverse frequency dependence of a specific class III agent E-4031 in the absence and in the presence of the calcium antagonist, verapamil. Experiments were conducted in guinea-pig papillary muscle using the standard microelectrode technique. Similar methods were also repeated replacing verapamil with nitrendipine (0.03 μmol).

METHODS

Male Hartley guinea-pigs (250-300g) were anaesthetised with intraperitoneal injection of sodium pentobarbital (120 mg/kg). The heart was quickly excised and dissected in an oxygenated Tyrode solution (95% 02 / 5% C02). A thin papillary muscle was removed from either right or left ventricle and pinned onto the silastic floor of a tissue bath superfused with oxygenated Tyrode solution maintained at 37°C and at pH 7.35. The modified Tyrode solution contained (mM) : NaCl 125, KC1 4.0, CaCl₂ 1.8, MgCl₂ 1.0, NaHC0₃ 24, NaH₂P0₄ 0.9, glucose 11. The solution was equilibrated with a gas mixture (95% O₂/ 5% CO₂). E-4031 and verapamil solutions were prepared daily as stock solutions in water (2 mM). After a stabilisation period of at least 1 hour, transmembrane action potentials were recorded with conventional microelectrodes (10-30 Mohms) connected to a high input impedance amplifier (BIOLOGIC VF180). External stimuli (1Hz, 2msec, twice threshold) were delivered by a stimulator (PULSAR 6i) through bipolar platinum electrodes placed at the base of the preparation. Signals were monitored on a storage 20 MHz oscilloscope (GOULD 1604) and simultaneously recorded on a digital tape recorder (BIOLOGIC DTR1204). Action potentials were digitalized for subsequent analysis (Software Clovis, CLOD sari) on a personal microcomputer equipped with a 12 bit analog-digit DAS50 converter.

Protocol : Papillary muscles were superfused with increasing doses of verapamil (from 0.3 to 10 μM) or E-4031 (from 0.01 to 0.3 μM). E-4031 was tested either in the absence or in the presence of 0.3 μM of verapamil. In the latter case, verapamil was added to the superfusion medium 30 minutes prior administration of the first dose of E-4031 and was present during the whole experiment. Each concentration was applied for 30 minutes which can be considered as the time necessary to reach steady state. Experiments were conducted at two cycle lengths (300 (CL300) and 1000 (CL1000) msec). Only fibres in which the same impalement was maintained throughout the whole experiment were used for analysis. Statistical analysis :

Results are presented as mean ± sem. Statistical analysis was performed using repeated measures analysis of variance followed by multiple comparisons using the Sidak procedure. For comparisons with equal group sizes the Greenhouse- Geisser adjustment was used to calculate p value, and for comparisons with unequal group sizes, the Geisser-Greenhouse conservative adjustment was taken into consideration (Ludbrook, 1994). All statistical analyses were performed by means of the microcomputer statistical program, CRUNCH 4.0 (Crunch Software Corporation, Oakland, CA). A p value of less than 0.05 was considered as statistically significant.

Effect ofE-4031 at CL 1000 and 300 msec on ventricular action potential.

100 msec

100 msec Effect ofE4031 in the presence of 0.3 μM verapamil at CL 1000 and 300 msec on ventricular action potential.

CL300msec CL 1000msec

DRUGS DOSE n RM APA Vmax APD3 APD9 n RM APA Vmax APD3 APD9

S P 0 0 P 0 0

(mV (mV (V/sec (msec) (msec) (mV (mV (V/sec (msec) (msec)

) ) ) ) ) )

contro 5 -921121+ 23714 8314 13714 6 -921126122211 10515 176±9

1 0 1 5 0 1 7

0.3μM 5 -92+ 118+ 239+4 7414 131+4 6 -921125+ 223+1 102+5 17519 1 1 4 1 1 7

VERAPA l.OμM 5 -92+ 116+238+4 64+3 127+4 6 -92+ 124+ 229+1 9314 17618

MIL 1 1 4 0 1 9

3.0μM 5 -91+ 114+ 235+4 51+3 11913 6 -921122+ 229+1 8112 17117 0 1 3 1 1 8 lO.Oμ 5 -91+ lll± 22714 39+1 10612 6 -92+ 1191221+1 59+3 15113

M 1 2 6 1 1 7

contro 6 -92+ 122125813 8114 136+6 6 -9311251240+4 10716 171±8

1 1 1 0 1 1 8

O.Olμ 6 -921122125612 8613 15216 6 -941125122514 11616 19419

M 1 1 7 1 2 9

E-4031 0.03μ 6 -921121+ 25912 9013 16516 6 -931125123813 1211621419 M 1 1 6 1 2 9

O.lμM 6 -92+ 121+ 24612 9214 179+6 6 -9311251238+3 1241523411 1 1 8 1 1 9 0

0.3μM 6 -93+ 121+ 250+2 9414 186+6 6 -94+ 125+ 244+4 126+524611 1 1 8 1 1 3 2

E-4031 contro 6 -91+ 117+24712 7513 13912 6 -921123+ 230+2 11413 18314 I 1 1 8 1 1 2 inpresence O.Olμ 6 -911116126213 7315 15212 6 -921123122612 11813 19814 M 1 1 2 1 1 0 of 0.03μ 6 -91+ 114± 256+3 75+4 164+2 6 -93+ 122+ 21812 1211321414 M 1 1 5 1 1 2

VERAPA O.lμM 6 -911115125613 74+5 17712 6 -9311221228+2 1211422714

MIL 1 1 5 1 1 0

(0.3μM) 0.3μM 6 -911113+ 229+2 74+4 18313 6 -9311221224+1 1201423115 1 2 0 1 2 9 Effect ofE-4031, verapamil and E-4031 in presence of verapamil on action potential parameters recorded from guinea-pig papillary muscle.

300 msec XWmsec 0.01 0.03 0.1 0.3

Cycle Length E-4031 Concentratio n (μ )

Analysis of variance for repeated measures followed by multiple comparison using the Sidak test was used. - : p<0.001 versus control. -■-< and ->-<-> : p<0.01 and 0.001 respectively versus C LI 000.

300 msec 1000 msec 0.01 0.03 0.1 03

Cycle Length E-4031 Co nee nt ratio n (μM )

Analysis of variance for repeated measures followed by multiple comparison using the Sidak test was used. - : p<0.001 versus control. No statistical difference was obtained between CL300 and CL1000.

RESULTS

JT Action potential amplitude, resting membrane potential and Vmax were not significantly modified by E-4031 whereas this compound induced a dose- dependent increase in action potential duration either at CL 1000 or 300 msec.

^■y However, the effect of E-4031 on APD was significantly reduced as stimulation frequency increased, characterising the reverse rate-dependent effect of class III antiarrhythmic agents. "^ Reverse rate-dependence was not observed in the presence of verapamil (0.3 μM) where effect of E-4031 was not significantly different at CL 300 and 1000 msec.

CONCLUSIONS

The present results show that E-4031 prolonged ventricular action potential more in fibres stimulated at CL1000 than in fibres stimulated at CL300, characterising the reverse rate dependent effect of class III agents. In the presence of verapamil, the increase in APD induced by E-4031 remained pronounced but was similar at both stimulation rates (CL300 and CL1000), and approximated to that seen at the faster stimulation rate (CL300) in fibres treated with E-4031 alone

These results suggest that the reverse-frequency dependent effect of class III agent can be reduced by calcium channel antagonist agents such as verapamil and nitrendipine.

REFERENCES

CARMELIET E.E.: Voltage- and time-dependent block of the delayed K+ current in cardiac myocytes by dofetilide. J. Pharmacol. Exp. Ther. 262: 809-817, 1992.

HONDEGHEM L.M. AND SNYDERS D.J.: Class III antiarrhythmic agents have a lot of potential but a long way to go. Reduced effectiveness and dangers of reverse use dependence. Circulation 81: 686-690, 1990. JURKIEWICZ N.K. AND SANGUINETTI M.C: Rate-dependent prolongation of cardiac action potentials by a methanesulfonanilide class III antiarrhythmic agent. Specific block of rapidly activating delayed rectifier K+ current by dofetilide. Circ Res 72: 75-83, 1993.

LUDBROOK J.: Repeated measurements and multiple comparisons in cardiovascular research. Cardiovasc. Res. 28: 303-311, 1994.

SANGUINETTI M.C. AND JURKIEWICZ N.K.: Two components of cardiac delayed rectifier K+ current. Differential sensitivity to block by class III antiarrhythmic agents. J. Gen. Physiol. 96: 195-215, 1990.

VAUGHAN- WILLIAMS, E. M.: Classification of antiarrhythmic drugs. In Symposium on Cardiac Arrhythmias, ed. by E. Sandoe, E. Flensted- Jensen and K. H. Olsen, pp. 449-472, Astra, Sodertalje, Sweden, 1970.

Claims

Claims:

1. A pharmaceutical composition, which composition comprises a class III antiarrhythmic agent and a class IV antiarrhythmic agent, providing that the composition is not a combination of lOμg/kg E4031 and 0. Img/kg verapamil, and optionally a pharmaceutically acceptable carrier therefor.

2. A composition according to claim 1, wherein the Class III agent is a potassium channel blocking agent.

3. A composition according to claim 1 or claim 2, wherein the Class III agent is an inhibitor of the delayed rectifier potassium current.

4. A composition according to any one of claims 1 to 3, wherein the Class III agent is selected from EP 235752, EP 245997, EP 322390, USP 4544654 and DE 3419067.

5. A composition according to any one of claims 1 to 4, wherein the Class III agent is selected from E4031, sematilide, dofetilide, almokalant and d-sotalol.

6. A composition according to claim 1, wherein the Class IV agent is a compound having calcium channel blocking activity.

7. A composition according to claim 6, wherein the Class IV agent is a compound having L-type calcium blocking effect.

8. A composition according to claim 6 or claim 7, wherein the Class IV agent is a compound selected from USP 3261859, DE 2117571, ZA 6801482 and DE 2549568.

9. A composition according to any one of claims 6 to 8, wherein the Class IV agent is verapamil or a dihydropyridine.

10. A composition according to claim 9, wherein the dihydropyridine is selected from nitrendipine, nifedipine and nisoldipine.

11. A pharmaceutical composition, which composition comprises a class III and a class IV antiarrhythmic agent, characterised in that the class III reagent is present in an antiarrhythmic effective amount and the class IV antiarrhythmic agent is present in an amount lower than that which provides a substantial calcium blocking effect, and optionally a pharmaceutically acceptable carrier therefor.

12. A composition according to claim 11, wherein the Class IV agent is present in the composition in an amount which exhibits a threshold calcium blocking effect, that is an amount which only just provides an electrophysiological effect due to a calcium antagonist effect.

13. A composition according to claim 11 or 12, wherein the lower limit for the amount of class IV antiarrhythmic agent in the composition is an amount which is below the threshold calcium blocking effect so that it is ineffective on action potential duration and on the PR interval.

14. A method for the treatment and/or prophylaxis of cardiac arrhythmias in mammals, particularly humans, which comprises administering to the mammal in need of such treatment and/or prophylaxis an effective amount of a combination of a class III antiarrhythmic agent and class IV antiarrhythmic agent, providing that the combination is not a combination of lOμg/kg E4031 and 0.1 mg/kg verapamil.

15. A method for the treatment and/or prophylaxis of cardiac arrhythmias in mammals, particularly humans, which comprises administering to the mammal in need of such treatment and/or prophylaxis an amount of a combination of a class III antiarrhythmic agent and class IV antiarrhythmic agent, wherein the class III antiarrhythmic agent is administered in an antiarrhythmic effective amount and the class IV antiarrhythmic agent is administered in an amount lower than that which provides a substantial calcium blocking action.

16. The use of a combination of a class III and a class IV antiarrhythmic agent in the manufacture of a medicament for use in the treatment of cardiac arrhythmia, providing that the medicament is not a combination of lOμg/kg E4031 and 0. Img/kg verapamil.

17. The use of a combination of a class III and a class IV antiarrhythmic agent in the manufacture of a medicament for use in the treatment of cardiac arrhythmia, wherein the class III antiarrhythmic agent is present in the medicament in an antiarrhythmic effective amount and the class IV antiarrhythmic agent is present in an amount lower than that which provides a substantial calcium blocking action.

18. A process for the preparation of a composition, the composition comprising a class III antiarrhythmic agent, a class IV antiarrhythmic agent and, optionally, a pharmaceutically acceptable carrier, which process comprises the admixture of a class III antiarrhythmic agent, a class IV antiarrhythmic agent and, optionally, a pharmaceutically acceptable carrier, providing that said admixture does not give a combination of lOμg/kg E4031 and 0.1 mg/kg verapamil.

19. A process for the preparation of a pharmaceutical composition, the composition comprising a class III and a class IV antiarrhythmic agent, in which the class III reagent is present in an antiarrhythmic effective amount and the class IV antiarrhythmic agent is present in an amount lower than that which provides a substantial calcium blocking effect, and optionally a pharmaceutically acceptable carrier therefor, which process comprises admixing the antiarrhythmic effective amount of the class III antiarrhythmic agent, the class IV antiarrhythmic agent in an amount which is lower than that which provides a substantial calcium blocking effect and, optionally, the pharmaceutically acceptable carrier.