Clentiazem, [2(+) (2S,3S)-3-acetoxy-8-chloro-5-(2-(dimethylamino)ethyl)-2,3-dihydro-2(4-methoxyphenyl)-1,5-benzothiazepin-4-(5H?)-one maleate], is a new synthetic analogue of the Ca2+ channel antagonist diltiazem (Fig. 1). It has been shown to have a stronger and longer-lasting antihypertensive action than that of diltiazem in various hypertensive animal models (1). Clentiazem also caused a more potent increase in vertebral blood flow than in peripheral blood flow in anesthetized dogs (2). The spasmolytic activity of clentiazem observed in various dog blood vessels shows a selectivity for basilar arteries and is stronger than that of diltiazem (3). In accord with these observations, the Ca2+ antagonistic effect of clentiazem has been reported in various vascular preparations (3-8). These pharmacological profiles suggest that clentiazem is a potent and a long-acting Ca2+ antagonist, which is expected to exert an antihypertensive action and to be beneficial for the treatment of spastic disorders of cerebral and coronary arteries.
Although clentiazem exhibited negative inotropic effects on isolated rabbit (6) and human (7) myocardial preparations, electrophysiological effects of clentiazem have not yet been examined. Therefore, we undertook the present studies to clarify the membrane action of clentiazem on cardiac muscles. We examined the effects of clentiazem as compared with those of diltiazem on electrical activity of both Ca2+ channel-dependent and normal cardiac cells.
MATERIALS AND METHODS
All experiments were performed in albino rabbits weighing 2.0-3.0 kg. After animals were anesthetized with an intravenous injection of 30-50 mg/kg sodium pentobarbital and 0.2 mg/kg heparin, the chest was opened and a cannula was inserted into the aorta under artificial respiration. The heart was then quickly removed and placed under the Langendorff perfusion system. The isolated heart was mounted in a tissue bath on a thermostatic chamber maintained at 36 ° ± 0.5 °C and was perfused through the cannula with Tyrode's solution gassed with 95% O2/5% CO2. The tissue bath (≈40-ml vol), was also perfused with oxygenated Tyrode's solution. The aorta was retrogradely perfused by Langendorff perfusion at a constant pressure equivalent to 80 cm H2O by a peristaltic pump (MP 3, Tokyo Rikakikai, Tokyo, Japan). The right atrial appendage was cut open to expose its endocardial surface. Four spots at the cut ends of the atrial tissue were fixed by threads to the tissue bath under slight tension.
Local electrograms were obtained with bipolar, Teflon-coated silver electrodes (diameter 0.2 mm, interelectrode distance 0.6 mm). They were placed on the right branch of the crista terminalis near the sinus node (HRA) and right ventricle (V). The His bundle electrogram (HBE) was also obtained with a bipolar electrode catheter placed across the tricuspid valve. Preparations were stimulated through bipolar silver electrodes placed on the right atrium close to the sinus node region. Stimuli were supplied from a pulse generator and isolation transformer (SEN-7103, Nihon Kohden, Tokyo, Japan). Pulses for stimulation were of 1-ms duration and twice the diastolic threshold in intensity. The basic frequency of stimulation was set at a value 10-20% above the spontaneous rate.
Atrioventricular (A-V) node preparations
Atrioventricular (A-V) node preparations were obtained from the excised heart in a Tyrode's solution. The preparation, consisting of interatrial septum, coronary sinus, A-V node, and His bundle, was transferred to a tissue bath (8 ml) in which a Tyrode's solution was perfused by a peristaltic pump (MP 3, Tokyo Rikakikai, Tokyo, Japan) at a constant rate of 6 ml/min. Solution was bubbled with 95% O2/5% CO2 at 36 ° ± 0.5 °C. Preparations were stimulated by bipolar silver electrodes with a square pulse of 3-ms duration and stimulus intensity of twice diastolic threshold delivered from a stimulator (SEN-7103, Nihon Kohden). Preparations were allowed to equilibrate with electrical stimulation at 400- to 500-ms intervals for 30 min before the electrical recordings were started.
Sinoatrial (S-A) node preparations
The right atrium, with the sinoatrial (S-A) node region intact, was dissected from other parts of the heart in a bath (8 ml) containing Tyrode's solution. The S-A node preparation was prepared by repetitive dissections to achieve a final dimension of ≈1.0 × 1.0 mm. The preparations were then superfused with Tyrode's solution oxygenated with 95% O2/5% CO2 at 36 ° ± 0.5 °C; they were spontaneously beating.
Ventricular muscle preparations
Thin endocardial layers (<1.5 mm) of right ventricular free wall were dissected from the excised heart in a Tyrode's solution. The preparation was fixed in a tissue bath (8 ml) and superfused continuously with Tyrode's solution equilibrated with a gas mixture of 95% O2/5% CO2 at 36 ° ± 0.5 °C. Preparations were stimulated by bipolar silver electrodes with a square pulse of 3-ms duration and stimulus intensity of twice diastolic threshold. Preparations were allowed to equilibrate in Tyrode's solution for 1 h and were stimulated at 1,000-ms intervals before the electrical recordings were started.
Measurements of local electrograms and action potentials (APs)
Local electrograms of Langendorff-perfused preparations. On the right atrial and His bundle electrograms, the onset of atrial (A) and ventricular (V) deflections and the His spike (H) were identified to measure A-H and H-V intervals. The A-H interval represents the A-V nodal conduction time from the atrial tissue near the A-V node to the bundle of His, and the H-V interval represents the conduction time from the bundle of His to the ventricular tissue. Control measurements were performed after the 40-min equilibration period. Effects of drugs were then assessed 20 min after the perfusion of the test solution containing either clentiazem or diltiazem at various concentrations (10-8-10-6M).
Action potentials of A-V node, S-A node, and ventricular muscle preparations. APs were recorded from the A-V node, the S-A node, or ventricular muscle preparations by the conventional microelectrode technique. Their first derivatives (Vmax) were electronically differentiated from the recorded membrane potentials. The AP and Vmax were displayed on an oscilloscopic screen (VC-10, Nihon Kohden) and photographed (RLG-6101, Nihon Kohden) for later analysis. The following measurements were made from a photograph: maximum diastolic potential, resting membrane potential, AP amplitude (APA), Vmax, AP duration at 20, 50, and 90% of repolarization (APD20, APD50, APD90), slope of slow diastolic depolarization, take-off potential, and spontaneous cycle length.
After an equilibration period of 1-2 h, a stable impalement of the electrode was obtained and control recordings were made in the A-V node, S-A node, and ventricular muscle preparations. Effects of drugs were examined 20 min after application of clentiazem or diltiazem (10-7-10-5M). Only experiments in which a stable impalement of the electrode was maintained from the control through the application of the drug were used for data analysis.
In some experiments, effective refractory period (ERP) of the A-V node was also measured. ERP was defined as the longest interval at which the premature stimulation failed to generate an active response. A single premature stimulus (S2) was introduced after the eighth basic stimuli (S1) through the same electrode. The S1-S2 interval was decreased from the S1-S2 interval in 10-ms steps. ERP was measured before and 20 min after application of 10-6M clentiazem or diltiazem.
Solutions and drugs
The composition of the Tyrode's solution was (in mM): NaCl 125.0, KCl 4.0, CaCl2 1.8, MgCl2 0.5, NaH2PO4 0.4, NaHCO3 24.6, and glucose 5.5; the pH of the solution was adjusted to 7.3-7.4 when bubbled with 95% O2/5% CO2 gas mixture. The compounds used were clentiazem maleate (Tanabe Seiyaku, Osaka, Japan) and diltiazem hydrochloride (Tanabe Seiyaku). The drugs were first dissolved in distilled water and then diluted in the Tyrode's solution at the final concentrations described in the text. Both drugs were applied to either coronary circulation through Langendorff perfusion or to the tissue bath.
All values are the mean ± SEM. Statistical analysis was performed by a paired t test in two-group comparison or by one-way analysis of variance (ANOVA) in multiple comparison; p < 0.05 was considered significant.
Effects of clentiazem and diltiazem on A-V conductions of Langendorff-perfused preparations
Effects of clentiazem and diltiazem on A-V conduction time were investigated in Langendorff-perfused hearts electrically driven at basic cycle lengths of 400-500 ms. Figure 2 shows typical experimental records demonstrating the effects of clentiazem and diltiazem. Like diltiazem, clentiazem prolonged the A-H interval without affecting the H-V interval.
The results for clentiazem and diltiazem on the A-H and H-V intervals are summarized in Fig. 3. The average control value for the A-H interval was 48.6 ± 4.5 ms and was prolonged to 64.1 ± 6.3 ms at 10-7M and to 108.8 ± 18.6 ms at 10-6M clentiazem. At 10-6M clentiazem, one of the four preparations developed Wenckebach-type A-H block. Diltiazem also significantly prolonged the A-H interval at >10-7M concentrations in a concentration-dependent manner, with development of Wenckebach-type A-H block in one of the four preparations at 10-6M. With increase in the concentration of clentiazem or diltiazem to 3 × 10-6M, Wenckebach-type A-H block was observed in all preparations (n = 3 in both groups; data not shown). Clentiazem and diltiazem had almost no effect on the H-V interval even at 10-6M.
Effects of clentiazem and diltiazem on APs of A-V node preparations
Figure 4 shows representative changes in AP configurations produced by clentiazem and diltiazem in rabbit A-V nodes. Clentiazem decreased APA from 100.0 mV in the control to 87.0 mV after 10-6M clentiazem and decreased Vmax from 39.0 to 33.0 V/s, respectively. After exposure to 10-5M clentiazem, the peak amplitude of AP was further decreased, attaining a negative value. In addition, at >10-6M concentrations, APD20 was also shortened. The effects of diltiazem on AP configurations were almost similar to those of clentiazem (Fig. 4B).
These changes in AP parameters are summarized in Table 1. Clentiazem at 10-6M or at the higher concentrations produced concentration-dependent decreases in APA and Vmax. At 10-5M, these parameters were decreased by 21.6 and 19.8% of the control values, respectively. Clentiazem 10-6-10-5M also shortened APD20 and APD50 in a concentration-dependent manner. At the highest concentration (10-5M), clentiazem produced a significant decrease in maximum diastolic potential (-4.7% of the control). Diltiazem produced similar changes in AP parameters.
The effects of clentiazem and diltiazem on ERP were also studied at a concentration of 10-6M(Fig. 5). ERP was significantly prolonged from the control value of 115.0 ± 5.7 to 123.5 ± 7.7 ms (n = 6, p < 0.05) by clentiazem and from 113.8 ± 4.9 ms in the control to 121.8 ± 6.4 ms (n = 5, p < 0.05) by diltiazem.
Effects of clentiazem and diltiazem on APs of S-A node preparations
Although 10-7M clentiazem did not affect APs of S-A node cells, 10-6M clentiazem caused significant decreases in maximum diastolic potential, APA, Vmax, and the slope of slow diastolic depolarization (Fig. 6A). APA was decreased from 96.7 mV in the control to 71.4 mV after clentiazem, and Vmax was decreased from 7.2 to 5.4 V/s. Changes in AP parameters are summarized in Table 2. APA and Vmax were decreased by 31.1 and 47.2% of the control values, respectively, after application of 10-6M clentiazem. The maximum diastolic potential and the slope of slow diastolic depolarization were also decreased by 10-6M clentiazem. Diltiazem had effects similar to those of clentiazem on AP parameters, except for a significant prolongation of spontaneous cycle length. In the present study, after application of both drugs, the sinus arrest was not observed in any of the preparations tested.
Effects of clentiazem and diltiazem on APs of ventricular muscle preparations
The effects of various concentrations (10-7-10-5M) of clentiazem and diltiazem on ventricular APs are summarized in Table 3. Neither clentiazem nor diltiazem had any effect on AP parameters of rabbit ventricular muscles at various concentrations tested.
Our results demonstrate that clentiazem had actions almost identical to those of diltiazem (9) on the electrophysiological parameters of rabbit heart preparations: prolongation of the A-H interval without significant alteration of the H-V interval and suppression of the A-V and S-A nodal APs. Clentiazem also reduced the slope of slow diastolic depolarization of the S-A node APs.
Diltiazem has been shown to decrease A-V conduction and slow APs of the A-V and S-A node cells due to a selective inhibition of the slow inward Ca2+ current (10-14). In addition to prolonging A-H intervals, clentiazem decreased the Vmax and APA of the A-V and S-A node APs, shortened APD20 and APD50, and prolonged ERP of the A-V node cells without affecting APs of ventricular muscles. All these effects could be explained by selective inhibition of the slow inward Ca2+ current by clentiazem. The reduction in the slow diastolic depolarization in spontaneously beating S-A node cells was also attributed to the block of the slow inward Ca2+ current, since the current was assumed to play a significant role in the pacemaker depolarization of this tissue (14,15). Although clentiazem was reported to block the slow inward Ca2+ current in rabbit mesenteric artery with a potency ≈10 times greater than that of diltiazem (4,5), it had a potency similar to that of diltiazem on cardiac slow APs in the present study. Therefore, clentiazem may be assumed to be a selective inhibitor of the slow inward Ca2+ current for vascular smooth muscles rather than for that of cardiac cells.
In the Langendorff-perfused hearts, both clentiazem and diltiazem showed a prolongation of A-H intervals at ≥10-7M, but both drugs decreased Vmax of APs in the A-V node cells at ≥10-6M. This discrepancy might be explained by the differences in the experimental conditions, since the latter preparations were superfused with the drug-containing solutions, whereas the former was applied to the coronary circulation.
We demonstrated that clentiazem and diltiazem have comparable class IV effects in rabbit heart preparations. Ca2+ antagonists such as diltiazem and verapamil have been reported to be highly effective in the treatment of paroxysmal supraventricular tachycardia, but dihydropyridines do not exhibit such action (16-24). Therefore, clentiazem may be beneficial in the treatment of paroxysmal supraventricular tachycardia, in addition to having a possible spasmolytic action on cerebral and coronary circulatory disorders. Clentiazem in clinical therapeutic concentrations prevented the initiation of reentrant type of supraventricular tachycardia in rabbit right atrial preparations (K. Miyazaki et al., unpublished observations). However, because clentiazem may suppress A-V conduction and therefore aggravate A-V block; caution must be used when the drug is administered to patients with ischemic heart disease associated with latent or manifest A-V conduction disorders. Clentiazem exhibited negative dromotropic and chronotropic effects, similar to those of diltiazem, due to suppression of the cardiac Ca2+ current.
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