European Journal of Pharmacology, 229 (1992) 179 - 187

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© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

EJP 52803

Ro 40-5967, a novel calcium channel antagonist, protects against ventricular fibrillation G e o r g e E. B i l l m a n The Department of Physiology, The Ohio State Uni~:ersity, Columbus, Ohio, USA Received 15 June 1992, revised MS received 4 September 1992, accepted 15 September 1992

Ro 40-5967 is a new calcium channel antagonist that binds at the same membrane sites as verapamil, yet has minimal negative inotropic effects. The effects of Ro 40-5967 on the susceptibility to ventricular fibrillation were investigated and compared to diltiazem. Ventricular fibrillation (VF) was induced in 40 mongrel dogs with healed myocardial infarctions by a 2-rain coronary occlusion during exercise. Twenty-four animals were found to be susceptible to VF and were given the treatments described below. Pretreatment with Ro 40-5967 (n = 17, 1000 p~g/kg i.v.) significantly (P < 0.001) reduced the incidence of VF (13 of 17 protected) during the exercise plus ischemia test. Diltiazem (n = 8, 1000 /~g/kg) completely suppressed VF. Lower doses of diltiazem and Ro 40-5967 did not prevent VF. The hemodynamic effects of Ro 40-5967 were also compared to diltiazem and verapamil. Diltiazem and verapamil, but not Ro 40-5967, increased P-R interval in a dose-dependent manner. Even when reflex tachycardia was controlled by /3-adrenoceptor blockade, Ro 40-5967 still exerted only minimal effects on P-R interval. Verapamil, but neither Ro 40-5967 nor diltiazem, provoked a dose-dependent negative inotropic response. All three drugs elicited large increases in coronary blood flow. These data support the hypothesis that calcium entry may play a critical role in the development of malignant arrhythmias during ischemia. Further, Ro 40-5967 can protect against ventricular fibrillation without significant negative inotropic or dromotropic effects. Ca 2+ channel antagonists; Ventricular fibrillation; Myocardial ischemia; Myocardial infarction; Cardiac arrhythmias

I. Introduction

A growing body of evidence suggests that an accumulation of myocardial cytosolic calcium, particularly during myocardial ischemia (Steenbergen et al., 1987; Lee et al., 1988; Marban et al., 1989; Mohabir et al., 1991), plays a critical role in the genesis of malignant arrhythmias (Opie et al., 1979; Levy, 1989; Billman, 1991). For example, Merillat et al. (1990) recently demonstrated that a slow inward calcium current was required for the initiation and maintenance of ventricular fibrillation (VF). In a similar manner, calcium channel antagonists have been shown to reduce cardiac mortality in patients (Danish Study G r o u p on Verapamil in Myocardial Infarction, 1990; Boden et al., 1991) and prevent VF in animals (Kaumann and Aramendia, 1968; Fondacaro et al., 1978; Clusin et al., 1982; Billman, 1989). However, since myocardial contractile function is critically dependent on cellular calcium (Fabiato, 1985),

Correspondence to: G.E. Billman, Department of Physiology, The Ohio State University, 302 Hamilton Hall, 1645 Nell Avenue, Columbus, OH 43210, USA. Tel. 1 (614) 292-5189, fax 1 (614) 292-4888.

these interventions could also adversely affect cardiac mechanical performance. In fact, diltiazem has been shown to decrease cardiac mortality in post-myocardial infarction patients with well preserved left ventricular function, but to increase mortality in patients with radiographic evidence of pulmonary congestion (Boden et al., 1991). Calcium antagonists could, therefore, have deleterious effects in patients with compromised cardiac function - the very patients at the greatest risk for malignant arrhythmias (Biggers et al., 1984). The challenge, therefore, is to develop calcium antagonists that can selectively modulate myocardial calcium without compromising cardiac mechanical function. Recently, a novel calcium channel antagonist, Ro 40-5967 ((1S,2S)-2(2-((3-(benzimidazolyl)propyl) methyl-amino)ethyl)-6-fluoro- 1,2,3,4-tetrahydro-l-isopropyl-2-naphthyl methoyacetate dihydrochloride) has been developed (Osterrieder and Holck, 1989). This drug binds at or near the same m e m b r a n e sites as verapamil (Clozel et al., 1989; Osterrieder and Holck, 1989), yet does not reduce myocardial force in either normal (Osterrieder and Holck, 1989; Clozel et al., 1990) or diseased hearts (Clozel et al., 1989; Ezzaher et al., 1991; Vdniant et al., 1991). It was therefore the purpose of this series of studies to investigate the

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effects of Ro 40-5967 on the susceptibility to ventricular fibrillation. Specifically, the hypothesis that Ro 40-5967 could protect against ventricular fibrillation without exerting significant negative inotropic or dromotropic effects was tested. In addition, the hemodynamic effects of Ro 40-5967 were compared with two well characterized calcium antagonists, diltiazem and verapamil, using cumulative d o s e - r e s p o n s e studies.

2. Materials and methods

2.1. Preparation Seventy mongrel dogs, weighing 13.4-20.4 kg, were used in this study. The animals were anesthetized and instrumented to measure left circumflex coronary blood flow and left ventricular pressure, as previously described (Billman et al., 1982, 1991; Schwartz et al., 1984; Billman, 1989). Briefly, the animals were given lnnovar-vet (0.02 m g / k g of fentanyl citrate and 1 m g / k g of droperidol i.v.) ( P i t t m a n - M o o r e Inc., Mundelein, IL), as a pre-anesthetic. Anesthesia was induced with sodium pentobarbital (10 m g / k g i.v., A.J. Buck and Son, Hunt Valley, MD). A left thoracotomy was made in the fourth intercostal space and the heart was exposed. A 20 MHz pulsed Doppler flow transducer and a hydraulic occluder were placed around the left circumflex coronary artery. A pre-calibrated solid state pressure transducer (Konigsberg Instruments, P 4.5-5.0. Pasadena, CA) was placed into the left ventricle via a stab wound in the apical dimple. Insulated silver-coated copper wires were sutured to the epicardial surface of the left and right ventricles and later were used to record the ventricular electrogram. An experimental myocardial infarction (n = 55) was then induced by a two-stage ligation of the left anterior descending coronary artery distal to its first large diagonal branch (Billman et al., 1982, 1991; Schwartz et aI., 1984; Billman, 1989). Sham procedures (i.e., artery dissected but not ligated) were performed in 15 animals. All leads to the cardiovascular instrumentation were tunneled under the skin to exit on the back of the animal's neck. Pentazocine lactate (30 mg i.m., Talwin, Winthrop Breon Laboratories, New York, NY) was given to minimize post-operative pain. In addition, the long acting local anesthetic bupivacaine HCI (Marcaine, Winthrop Breon Laboratories) was used to block the intercostal nerves (i.e., pain fibers) in the area of the incision to minimize discomfort to the animal. Each animal was placed on antibiotic therapy (ampicillin, 500 mg per os, I D E Laboratories, Amityville, NY) twice daily for 5 - 7 days. The animals with myocardial infarction were placed in an 'intensive care' setting for the first 24 h and given tocainide HCI (600 mg Per Os, Tonocard, Merck Sharp

and Dohme, West Point, PA) every 12 h, beginning the day before surgery. A total of 17 animals (approximately 31%) died acutely or within the first 72 h. Three additional animals could not bc classified due to rupture of the coronary occluder (see below), and five animals were not successfully resuscitated after experimentally induced ventricular fibrillation (see below). Thus, of the original 70 animals, studies were completed in 45 animals (including 15 animals without myocardial infarction). The principles governing the care and use of animals as expressed by the Declaration of Helsinki and in the Guidc for the Care and Use of Laboratory Animals, as adopted by the National Institutes of Health, were followed at all times during this study. In addition, all procedures were approved by the Ohio State University Institutional Animal Care and Use Committee.

2.2. Exercise plus ischemia test: classtfication o]" susceptibifity to sudden death Three to four weeks after surgery the studies began. The animals were walked on a motor driven treadmill and adapted to the laboratory environment during this period. The susceptibility to ventricular fibrillation was tested as previously described (Billman et al., 1982, 1984, 1991; Billman, 1989). Briefly, the animals ran on a motor driven treadmill while work load increased every 3 min. During the last minute of exercise, the left circumflex coronary artery was occluded (inflation of a hydraulic occluder); the treadmill was then stopped and the occlusion was maintained for an additional minute. The occlusion therefore lasted a total of 2 min. Large metal plates (11 cm d i a m e t e r ) w e r e placed across the animal's chest so that electrical defibrillation could be achieved with a minimal delay, but only after the animal was unconscious (10-20 s after VF began). The occlusion was released immediately if ventricular fibrillation occurred. Twenty six (including five dogs without infarction) developed VF (susceptible) during the exercise plus ischemia test, while the remaining 19 (10 without myocardial infarction, resistant) did not. As noted above, five susceptible animals were not successfully resuscitated and were eliminated from the study.

2.3. Experimental protocol The exercise plus ischemia test was performed after the following treatments: control (normal saline, n 40); diltiazem HC1 (Sigma Chemical Co., St. Louis, MO), was given to 12 susceptible (250 / , g / k g , n = 4; 1000 /,tg/kg, n = 8) and to seven resistant animals (1000 / , g / k g ) ; and Ro 40-5967 (Hoffmann-LaRochc Ltd., Basel, Switzerland), was given to 18 susceptible (250 p,g/kg, n = 6; 500 p g / k g , n = 4; 1000 /.tg/kg, n - 17) and to 11 resistant animals (250 /,tg/kg, n = 2;

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1000 p~g/kg, n = 11). All drugs were mixed in normal saline and given as bolus intravenous injections 3-5 rain before exercise began. The calcium channel antagonists were given in a random order after the initial classification (saline) exercise plus ischemia test. Additional control (i.e., saline) exercise plus ischemia tests were repeated between treatments or one week after the last treatment for each susceptible animal; a maximum of four control exercise plus ischemia tests were given to a particular animal during the course of the experiments. Each animal received at least two exercise plus ischemia tests.

sures. When the F ratio was found to exceed a critical value (P < 0.05), Scheffe's test was used to compare means. The effects of the Various drug treatments on susceptibility to ventricular fibrillation were determined using a X 2 test with Yate's correction for continuity. All data are reported as the mean _+ S.E.M. Cardiac arrhythmias were evaluated at a paper speed of 25 m m / s e c , while P-R interval was calculated at a paper speed of 100 m m / s e c .

2.4. Dose-response studies

3.1. Dose-response studies

The hemodynamic response to Ro 40-5967, diltiazem and verapamil were also evaluated while the animals were lying quietly and unrestrained on a laboratory table. The same animals were used for the exercise plus ischemia and dose-response studies. The following drug treatments were given in a random order, with at least 24-48 h between different drugs: verapamil (n = 10, 5 susceptible and 5 resistant); diltiazem (n = 11, 5 susceptible and 6 resistant); Ro 40-5967 (n = 10, 5 susceptible and 5 resistant); and Ro 40-5967 after/3-adrenoceptor blockade (1.0 m g / k g i.v.) propranolol He1 (Sigma Chemical Co, St. Louis, MO) (n = 10, 5 susceptible and 5 resistant). Since myocardial infarction (MI) could alter the hemodynamic response to the various calcium antagonists, the dose-response studies were repeated in five sham-operated (no MI) animals. Cumulative dose-response curves employing the following concentrations were obtained: 0, 125, 250, 500 and 1000 txg/kg. Ten minutes (or until a new steady state value had been achieved) elapsed between successive drug doses. (Total time was approximately 50 rain).

The hemodynamic dose-response data for the animals with myocardial infarction are shown in figs. 1-6. There were no differences in the response noted in susceptible and resistant animals; the data were therefore combined. P-R interval (fig. 1) increased significantly (P < 0.01) with increasing drug dose for both verapamil and diltiazem, reaching significance at 125 txg/kg and 250 txg/kg, respectively. In fact, the highest dose of verapamil induced either second degree (n = 6) or third degree (n = 3) A-V nodal conduction block in nine of ten animals. In contrast, Ro 40-5967 did not significantly prolong P-R interval. However, since heart rate (fig. 5) reflexively increased in response to arterial smooth muscle relaxation, this tachycardia could have masked the direct dromotropic effects of Ro 40-5967. The dose-response studies were therefore repeated after propranolol He1 was given to attenuate the reflex tachycardia. After /3-adrenoceptor blockade (BB), Ro

3. Results

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2.5. Data analysis All data were recorded on a Gould model 2800 eight channel recorder and a Teac model MR-30 FM tape recorder. Coronary blood flow was measured using a University of Iowa Bioengineering flow-meter model 545 C-4. T h e rate of change of d ( L V P ) / d t was obtained by passing the left ventricular pressure signal through a Gould differentiator that has a frequency response linear to > 300 Hz. The hemodynamic data were averaged over the last 5 s during the last minute of each exercise level, immediately before the coronary occlusion and the 60 sec time point (or immediately before VF) during the occlusion. Dose-response data were obtained in a similar manner. The time to the onset of VF was averaged across trials for each animal. The data were then analyzed using analysis of variance for repeated mea-

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Fig. 1. The effect of increasing doses of calcium channel antagonists on P-R interval. BB, /3-adrenoceptor blockade, propranolol HCI, 1.0 mg/kg. Note the highest dose of verapamil induced second or third degree A-V block in nine of ten animals. Ro 40-5967 (solid circle), verapamil (solid square), diltiazem (solid triangle) and Ro 40-5967+ BB (open circle). Verapamil and diltiazem elicited significant (P < 0.01) increases at 250, 500 and 1000 txg/kg, while Ro 40-5967 only elicited significant (P < 0.01) increases in P-R interval after/3-adrenoceptor blockade at 1000 Ixg/kg.

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DRUG DOSE (/Jg/kg) Fig. 2. The effect of increasing doses of calcium channel antagonists on left ventricular systolic pressure (LVSP). BB, /3-adrenoceptor blockade, propranolol HCI, 1.0 m g / k g ; Ro 40-5967 (solid circle), verapamil (solid square), diltiazem (solid triangle) and Ro 40-5967 + BB (open circle). Verapamil produced the only significant (P < 0.01) change from control (1000/xg/kg).

40-5967 elicited a small but significant (P < 0.01) increase in P-R interval but only at the highest dose (1000 # g / k g ; P-R interval change Ro 40-5967 6.0 + 1.9 versus Ro 40-5967 + BB 19.7_+ 6.2 ms). /3-Adrenoceptor blockade also elicited a significant increase in the control (i.e., pre-Ro 40-5967 treatment) P-R interval (control 92.7 + 2.6 versus 117.0_+ 2.8 ms). Even with heart rate controlled, Ro 40-5967 elicited a much smaller P-R interval increase (maximal change 19.7 _+ 6.2 msec) than was noted for animals treated with either verapamil (maximal change 87.4 +_ 16.1 msec.) or diltiazem (maximal change 64.9 _+ 11.0 msec). In a similar manner, left ventricular systolic pressure (LVSP) decreased slightly with increasing dose of each drug (fig. 2), but it was only significantly (P < 0.01) reduced with the highest dose of verapamil./3-Adrenoceptor blockade did not significantly alter the Ro 405967 response. The inotropic response to the various drugs was also evaluated. The rate of change in left ventricular pressure, positive d ( L V P ) / d t , was used as an index of inotropic state of the heart. Positive d(LVP)dt was significantly (P < 0.01) reduced by the highest dose of verapamil and increased significantly by the highest Ro 40-5967 and diltiazem doses. If reflex changes in heart rate were prevented by/3-adrenoceptor blockade, the d ( L V P ) / d t increase no longer occurred (fig. 3). Heart rate increased significantly with each drug, even at the lowest dose (fig. 5). This heart rate increase was prevented by ¢3-adrenoceptor blockade. The heart rate decrease noted at the highest dose of verapamil resulted from the second and third degree A-V block induced by this drug. Coronary blood flow increased significantly (P < 0.01) in a dose-dependent fashion with the greatest increases noted for diltiazem (fig. 4). Interestingly, the

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D R U G DOSE (/J.g/kg) Fig. 3. The effect of increasing doses of calcium antagonists on an index of inotropic state (positive d(LVP)/dt). BB, fl-adrenoceptor blockade, propranolol HCI, 1.0 m g / k g ; LVP, left vcntricular pressure. Ro 40-5967 (solid circle), verapamil (solid square), diltiazem (solid triangle) and Ro 40-5967+BB (open circle). Note that dihiazem and Ro 40-5967 (1000 # g / k g ) elicited significant (P < 0.01) increases while verapamil 11000 /xg/kg) elicited a significant (P < 0.01) decrease in this inotropic index. Note further that /3-adrenoceptor blockade eliminated the increase in positive d(LVP)/dt.

coronary blood flow increase elicited by Ro 40-5967 was reduced after/3-adrenoceptor blockade, suggesting that reflex increases in heart rate, and thereby metabolic demand, probably contribute significantly to the coronary blood flow increase. Very similar hemodynamic dose-responses were noted in animals with and without myocardial infarction (MI). In fact, no significant hemodynamic differences were noted between animals with or without M1 for any of the calcium channel antagonists. For exam-

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