Disturbance of Ca homeostasis, especially intracellular Ca overload, represents one of the critical alterations responsible for the development of cell injury as well as arrhythmias observed in the myocardium during ischemia and subsequent reperfusion (1,2). Increased cellular Ca can damage the sarcolemmal membrane by activating phospholipases and proteases, which in turn induce further abrupt Ca overload. This vicious cycle finally leads to loss of cell viability.
Many Ca channel blockers, which are potent hypotensive and vasodilating agents, have been shown to protect the myocardium against ischemia/reperfusion injuries in animal models (3-5). These effects are presumed to result from coronary vessel dilation and the energy-sparing effect due to suppression of myocardial oxygen consumption after direct cardiodepression and/or afterload reduction. On the other hand, the cardioprotection by α1-adrenoceptor blocking is not widely recognized, although α1-adrenoceptor blocking action can be expected to cause both coronary vasodilation and afterload reduction by the hypotensive effect. However, α1-adrenoceptor blockers were recently reported to inhibit Ca gain in ischemic/reperfused cat heart (6) and to prevent changes in energy metabolism in ischemic dog heart (7) in addition to their inhibitory effects on reperfusion-induced ventricular fibrillation (VF) (8).
S-2150, (+)-cis-3-acetoxy-8-chloro-2,3-dihydro-2-(4-methoxyphenyl)-5-[3-[4-(2-methoxy-phenyl)-1-piperazinyl]propyl]-1,5-benzothiazepin-4(5H)-one citrate, is a new synthetic analogue of the Ca channel blocker diltiazem and has a vasodilatory effect on the vascular smooth muscle (VSM) of rat aorta precontracted with KCl. S-2150 also has a relaxing effect on phenylephrine (PE)-induced contraction of rat aorta (9).
To study the usefulness of S-2150 against ischemic/reperfused myocardial injury, we investigated its cardioprotective effects with an anesthetized rat model by measuring creatine phosphokinase (CPK) loss from the heart and reperfusion-induced fatal arrhythmias. We also compared the direct effects of S-2150 on the contractile function and on the coronary perfusion flow of isolated rat heart with those of diltiazem. S-2150 exhibited greater cardiac antiischemic efficacy than diltiazem, and combination studies of diltiazem and prazosin suggested that the dual blocking action against both the Ca channel and α1-adrenoceptor is effective for myocardial protection against ischemia/reperfusion injury.
The cardiac ventricle and cerebral cortex were excised from male Wistar rats (Slc: 11-13 weeks old) after decapitation. The tissue was homogenized in ice-cold 0.25 M sucrose containing 5 mM Tris-HCl (pH 7.4) with a Polytron homogenizer. The homogenate was centrifuged at 1,000 g for 10 min; the supernatant was then centrifuged at 48,000 g for 20 min at 4°C. The resultant pellet suspended in 50 mM Tris-HCl (pH 7.4) was used as the membrane fraction. cis (+) [3H]diltiazem, (+) [3H]PN200-110, and [3H]WB4101 were used as radioligands of the 1,5-benzothiazepine-type Ca channel binding site, 1,4-dihydropyridine-type Ca channel binding site, and α1-adrenoceptor binding site, respectively, for the binding assay with this membrane fraction.
The membrane fraction (≈0.3 mg protein) was incubated in 0.5 ml 50 mM Tris-MCl (pH 7.4) containing 5 nM cis (+) [3H]diltiazem with or without test compounds for 60 min at 30°C and 0.025 nM (+) [3H]PN200-110 with or without test compounds for 120 min at 25°C, respectively. The reaction was terminated by filtration through a glass filter (Whatman GF/C) under vacuum and dried. The radioactivity of the filter was counted with a liquid scintillation counter. The amounts of total and nonspecific bindings of cis (+) [3H]diltiazem were determined from the radioactivities without and with 10 μM diltiazem in the reaction mixture, respectively. The amounts of total and nonspecific bindings of (+) [3H]PN200-110 were determined in the same way with 1 μM nifedipine. The difference between the total and nonspecific bindings were defined as the specific binding. [3H]WB4101 binding to rat cerebral cortical membrane fraction was assessed according to the same procedure. In the binding assay of 1 nM [3H]WB4101, incubation was performed for 15 min at 25°C and nonspecific binding was determined from the radioactivity with 10 μM phenoxybenzamine in the reaction mixture; the Ki value was obtained. Protein was measured by the method of Lowry and colleagues (10) with crystalline bovine serum albumin (BSA) as the standard.
Evaluation of antinecrotic effect against coronary artery occlusion/reperfusion in rats
Male rats [Slc; Wistar, 9-11 weeks old; Crj; spontaneously hypertensive rats (SHR), 10 weeks old] were anesthetized with urethane (1 g/kg intraperitoneally, i.p.), intubated, and placed in a positive-pressure respirator. Left thoracotomy was performed, and the left coronary artery was ligated 3-4 mm from this origin with a needled suture (no. 4-0, Akiyama Medical MFG, Tokyo, Japan). A polyethylene tube was placed between the ventricular wall and the suture to make it easy to cut the ligature after 20-min occlusion. Body temperature was maintained at 37°C with a heated operating pad throughout the experiment. Heart rate (HR) was counted from R-R intervals of the ECG recorded through the standard limb lead.
At 3 h after reperfusion, the animals were killed by decapitation and their hearts were excised for the determination of CPK activity. According to the method described by Bernauer (11), we homogenized the left ventricular free wall in ice-cold 0.1 M Tris-HCl (pH 7.5) containing 1 mM mercaptoethanol using a Polytron homogenizer. The homogenate was centrifuged at 20,000 g for 10 min at 4°C, and the supernatant was obtained. CPK activity of the supernatant was determined by spectrophotometric assay with a kit (CPK-Test Wako, Wako Chemicals, Osaka, Japan). Protein was measured by the method of Lowry and colleagues (10) with crystalline BSA as the standard.
The percent antinecrotic effect was estimated as follows: 100 × [(CPKd - CPKv)/(CPKs - CPKv)], where CPKd is the CPK value of drug-treated animals, CPKv is the CPK value of vehicle-treated control animals, and CPKs is the CPK value of sham-operated (i.e., without ligation) animals.
Evaluation of antiarrhythmic effect against reperfusion in coronary artery-occluded rats
Male rats (Slc; Wistar, 9-11 weeks old) were used for study of antiarrhythmic effect against reperfusion. Anesthesia was induced with sodium pentobarbital (50 mg/kg i.p.). The coronary artery was occluded and reperfused as described before except that the coronary artery was occluded for 4 min. The ECG was recorded through the standard limb lead (lead I) after anesthesia until 5 min of reperfusion, and the incidences and duration of ventricular tachycardia (VT) and VF were monitored. VF was defined as the total irregularity of morphology of the repetitive rapid ventricular beats, and cardiac death was defined as a flattened ECG. The percent incidence of VT and VF for each animal was estimated as follows: 100 × [duration of incidence]/[duration of survival].
Evaluation of direct cardiac effect in isolated rat heart preparation
Male rats (Slc; Wistar, 13-14 weeks old) were anesthetized with ether and heparinized (100 U intravenously, i.v.). The chest was opened, and the heart with the cannulated aorta was excised. The isolated heart was mounted on a Langendorff apparatus, and retrogradely perfused with Krebs-Henseleit buffer (in mM): NaCl 118, KCl 4.7, KH2PO4 1.2, MgCl2 1.2, CaCl2 2.5, and NaHCO3 25, pH 7.4) supplemented with 11 mM glucose and 0.1% defibrinated rat blood. The heart was placed in a warm box at 37°C to maintain its temperature. The perfusate was continuously gassed with a 95% O2/5% CO2 mixture at 37°C. Buffer was allowed to flow freely into the aortic root at a hydrostatic pressure of 60 cm, and the heart was left to stabilize for 25 min before the test drugs were injected.
To measure the contractile tension (CT), a silk suture was tied to the apex of the heart and connected to a force-displacement transducer (TB-611T, Nihon Kohden, Tokyo, Japan) and a recorder of the polygraph system (RM-6000, Nihon Kohden). The HR was counted from the contraction signal observed with an HR counter (AT-601G, Nihon Kohden). Coronary perfusion flow (CPF) was recorded with a drop counter made in our laboratory. Test agents were injected into the aorta just proximal to the entrance of the perfusion medium in the myocardium.
S-2150 (molecular weight 802) and nifedipine were synthesized at Shionogi Research Laboratories, Osaka, Japan. Diltiazem HCl, prazosin HCl, and verapamil HCl were obtained from Sigma Chemical, St. Louis, MO, U.S.A. These drugs were suspended in 0.5% methylcellulose solution for oral administration. For intravenous and intraduodenal administrations, they were dissolved in a small amount of dimethyl sulfoxide:polyoxyethylene 50 hydrogenated castor oil (9:1) and then diluted with 0.25 M sucrose solution before use. All other chemicals were purchased from standard commercial sources.
Ki values for the displacement response of the binding assay and IC20 and EC20 values for the parameters of isolated hearts were calculated by the least-squares method. Values are mean ± SEM. The significance of the differences between groups was determined by Student's t test and standard Chi-square method. A p-value <0.05 was regarded as statistically significant.
S-2150 concentration-dependently inhibited specific cis (+) [3H]diltiazem binding to rat cardiac ventricular membranes with a mean Ki of 0.20 μM, a value 1.7 times higher than that of diltiazem, but the difference between the two drugs was not statistically significant. Neither S-2150 nor diltiazem had an effect on the binding of (+) [3H]PN200-110 with a mean Ki >10 μM (data not shown). On the other hand, S-2150 also concentration-dependently inhibited [3H]WB4101 binding to rat cerebral cortical membrane with a mean Ki value of 0.021 μM, which was 21 times higher than that of prazosin. Diltiazem had little effect on the binding of [3H]WB4101. The mean Hill coefficients and their SE for S-2150 in bindings with cis (+) [3H]diltiazem and [3H]WB4101 were 0.94 ± 0.09 (n = 3) and 1.03 ± 0.01 (n = 3), respectively (Table 1).
Effects on ischemia/reperfusion-induced necrosis
The effects of intraduodenal pretreatments with S-2150 and diltiazem on ischemia/reperfusion-induced myocardial necrosis are shown in Table 2. In the untreated control group, myocardial CPK activity decreased markedly after 20-min ischemia and subsequent 3-h reperfusion. S-2150 (30 and 60 mg/kg) administered 30 min before coronary artery occlusion suppressed the loss of myocardial CPK activity. The estimated antinecrotic effects were 40% at 30 mg/kg and 48% at 60 mg/kg. On the other hand, pretreatment with diltiazem (30 and 100 mg/kg) showed only a tendency to suppress it.
The effects of orally administered S-2150 on ischemia/reperfusion-induced myocardial necrosis was determined in hypertensive rats (Table 3). S-2150 (30 and 60 mg/kg) was given as pretreatment to conscious SHR; the temporal coronary artery was occluded 2 h later. Under urethane anesthesia, the mean arterial blood pressure (MAP) measured from the left carotid artery became ≈100 mm Hg, and S-2150 further dose-dependently decreased it by 30 mm Hg. There were no changes in HR with S-2150. Under this condition, S-2150 suppressed the loss of myocardial CPK activity in SHR with temporarily occluded coronary artery. The antinecrotic activity of S-2150 in SHR was ≈50%.
Next, the antinecrotic effects of S-2150 and related drugs on ischemia/reperfusion-induced necrosis were determined by intravenous infusion (Table 4). Drugs were infused during the 10 min before the start of temporal occlusion. S-2150 and diltiazem suppressed the loss of myocardial CPK activity at 5 mg/kg. Another type of Ca channel blocker, verapamil (3 mg/kg), was also effective. However, a third type of Ca channel blocker, nifedipine (1 mg/kg), did not preserve the myocardial CPK activity. Preservation did occur with a combination of noneffective dosages of diltiazem (1 mg/kg) and prazosin (0.1 mg/kg). No effect was noted with a high dose of prazosin (0.5 mg/kg) or a low dose of nifedipine (0.3 mg/kg).
Effects of reperfusion-induced arrhythmias
The effects of S-2150 and reference drugs on coronary artery reperfusion-induced arrhythmias are shown in Table 5. After coronary reflow after temporal occlusion for 4 min, all vehicle-treated control animals exhibited arrhythmias such as VT and VF. Arrhythmias developed immediately after coronary artery reperfusion and disappeared in 5 min. In the control group, the calculated incidences of ventricular arrhythmias were ≈80% and the survival rate was 32% because of the irreversible incidence of VF. Test agents were administered orally to conscious animals; temporal coronary artery occlusion was performed 2 h later under anesthesia. S-2150 (10 and 60 mg/kg) produced dose-related reduction of the incidence of reperfusion-induced ventricular arrhythmias. At 60 mg/kg, the incidence was almost completely suppressed. All animals treated with S-2150 survived. Diltiazem at 10 and 60 mg/kg did not significantly modify the incidence of reperfusion-induced arrhythmias, but the higher dose of 100 mg/kg produced an antiarrhythmic effect and prevented cardiac death. On the other hand, combined administrations of ineffective doses of diltiazem (60 mg/kg) and prazosin (1 mg/kg) also reduced the incidence of arrhythmias and mortality.
Effects on mechanical function and coronary perfusion flow in isolated heart
The direct effects of various concentrations (4, 12, 40, and 120 nmol) of S-2150 and diltiazem on cardiac mechanical function and CPF were evaluated with Langen-dorff perfused rat heart preparations. Figure 1 shows the representative effect of both drugs (12 and 40 nmol) on CT, HR, and CPF. S-2150 produced slight but sustained decrease in CT, whereas diltiazem produced a marked but transient decrease. S-2150 barely changed the HR, whereas diltiazem markedly decreased it. Unlike the marked but transient negative inotropism, the negative chronotropism of diltiazem was long-lasting. S-2150 and diltiazem produced the increase of CPF. The effect of diltiazem was transient and weak as compared with the effect of S-2150, and diltiazem subsequently decreased the CPF below the initial value.
Both S-2150 and diltiazem concentration-dependently decreased cardiac function assessed by the product of CT and HR and increased CPF. The IC20 value of cardiodepression and the EC20 value of coronary vasodilation were calculated from their concentration-response curves at the timepoint showing maximal effect; results are summarized in Table 6. S-2150 was 7.5 times less cardiodepressive than diltiazem, whereas S-2150 was ≈3.3 times more potently coronary vasodilatory than diltiazem. S-2150 and diltiazem at high concentrations induced arrhythmias by themselves, such as atrioventricular (AV) block, and the arrhythmogenesity was stronger with diltiazem. The concentration that induced AV block in 50% of heart preparations was 40 nmol for diltiazem and >120 nmol for S-2150 (Table 6).
Most of the antiischemic effects of Ca channel blockers have been explained by their hemodynamic and cardiac effects, which include coronary vasodilation and reduced peripheral resistance and cardiac work output. Particularly important as potential mechanisms are the direct negative inotropic and chronotropic actions in the case of nondihydropyridines such as diltiazem and verapamil (3,12). Their protective effects during ischemia secondarily modify the reperfusion injury.
S-2150 is a Ca channel blocker with a structure similar to that of diltiazem, with an additional blocking effect on the α1-adrenoceptor, as indicated by an earlier study (9) and the binding assay of the present study. As for the Ca channels, S-2150 had a binding affinity to the 1,5-benzothiazepine binding site but not to the 1,4-dihydropyridine binding site. On the other hand, S-2150 had a binding affinity to the α1-adrenoceptor even when the Ki value was much higher than that of prazosin. Diltiazem did not have a marked effect. Hill coefficients of ≈1.0 indicate that the inhibitory activities of S-2150 to both binding sites are competitive. Therefore, it is of interest to determine whether the dual blocking character of the new compound S-2150 has useful cardioprotective properties as compared with the representative 1,5-benzothiazepine compound diltiazem. We performed experiments to assess the potential of S-2150 for protection of the reperfused ischemic myocardium against necrosis and arrhythmias. We also attempted to determine the direct cardiac effects of this compound and observe any differences from those of diltiazem to assess its therapeutic advantages.
Pretreatment with S-2150 significantly reduced the infarct size (IS) assessed by the preservation of myocardial CPK activity. The significant finding in the antinecrotic study was that S-2150, in contrast to diltiazem, manifested its effect intraduodenally. This method of administration is valuable if the test agent can act through the gastrointestinal tract in anesthetized animals. S-2150 was effective at 30 and 60 mg/kg, which are hypotensive dosages in normotensive rats and experimental hypertensive rats, as indicated in an earlier report (9). On the other hand, diltiazem did not show significant antinecrotic action even at the hypotensive dosage of 100 mg/kg in hypertensive rat models, which suggests that S-2150 has both antinecrotic and hypotensive characteristics within the same dose range of oral administration. To ascertain these advantageous characteristics using hypertensive rats, we performed experiments to determine whether oral pretreatment with an acute hypotensive dose of S-2150 would offer protection in SHR. S-2150 produced significant antinecrosis at ≈30-60 mg/kg orally in this model.
We attempted to determine the efficacy differences between representative Ca channel blockers and the role of α1-adrenoceptor against ischemia/reperfusion-induced necrosis under continuous intravenous infusion. Prazosin (0.1 and 0.5 mg/kg) alone showed no significant antinecrotic effect; however, combining it with a noneffective dose of diltiazem (1 mg/kg) produced a significant result. Diltiazem alone decreased the HR from 420 to ≈330 beats/min at the end of the infusion, and the combination with prazosin did not modify the decreased HR. Although antinecrotic effects of S-2150, diltiazem, and verapamil were observed at 5, 5, and 3 mg/kg, respectively, no effect was observed with nifedipine 0.2 or 1 mg/kg. Doses of nifedipine >1 mg/kg cannot be applied because of its marked hypotensive activity. In this experiment, the HR decreased from 440 to ≈270 beats/min with diltiazem (5 mg/kg) and verapamil (3 mg/kg) at the end of the infusion. S-2150 (5 mg/kg) slightly decreased the HR from 440 to 390 beats/min. Nifedipine (1 mg/kg) did not change the HR. Therefore, the protective mechanism of diltiazem or verapamil might derive mainly from the energy-sparing effects due to marked negative chronotropism. On the other hand, in the case of S-2150, Ca channel and α1-adrenoceptor dual blocking activities appear to act cooperatively against the injury, in addition to the negative chronotropism.
Next, we studied the suppressive effects of S-2150 and related agents on the incidence of ventricular arrhythmias on reperfusion after a short period of ischemia. S-2150 dose-dependently reduced the incidence of reperfusion arrhythmias within hypotensive dose ranges and the mortality became 0%. Diltiazem was much less effective than S-2150, and prazosin did not affect the incidence and mortality due to irreversible fibrillation. However, combined treatment with noneffective doses of diltiazem and prazosin led to a significant suppressive effect similar to the antinecrotic effect. Diltiazem was reported to protect the heart against coronary artery occlusion/reperfusion-induced ventricular arrhythmias (13,14). On the other hand, the effectiveness of prazosin on the fibrillation during reperfusion in different species is controversial (8). In the present study, we used oral pretreatment because S-2150 is planned to be used as an internal medicine similar to diltiazem for hypertensive patients with ischemic heart diseases. Under this condition, the antiarrhythmic effect of S-2150 was much stronger than that of diltiazem, which was similar to its antinecrotic evaluation after intraduodenal or oral treatment.
The noteworthy finding of the present study was that cardioprotection occurred with a combination of diltiazem and prazosin, which individually were ineffective. Sympathetic activation and local accumulation of norepinephrine in acute myocardial ischemia are believed to be closely associated with the progression of myocardial injury (8,15,16). Myocardial α1-adrenoceptor number also has been reported to increase during hypoxia and ischemia (8,17). A Ca channel blocker selectively inhibits influx of Ca through L-type Ca channel and thus exerts its vasodilatory effect and negative inotropic and chronotropic effects. On the other hand, an α1-adrenoceptor blocker blocks not only receptor-mediated Ca influx but also Ca release from intracellular storage sites. Moreover, α1-adrenergic stimulation in the myocardium raises the Ca sensitivity of intracellular contractile proteins (18,19), which leads to an increase in myocardial oxygen consumption, followed by aggravation of ischemic injury. These findings suggest that the Ca channel and α1-adrenoceptor dual blcoking action may effectively act to prevent excess intracellular Ca overload. This is part of the mechanism of the prominent cardioprotection of S-2150 as compared with diltiazem. Further studies with blood pressure, coronary circulation and cardiac mechanical function are required to define the mode of drug action, especially that of the combination of diltiazem and prazosin.
In isolated rat heart, S-2150 was less cardiodepressive and less arrhythmogenic than diltiazem. Some of these properties are due to the difference in potency for inhibiting the cardiac Ca channel, as shown by radioligand binding analysis. Indeed, diltiazem was reported to have marked inhibitory action on AV nodal conduction (20). However, S-2150 was more strongly coronary vasodilatory than diltiazem, as shown by the CPF. These results suggest that S-2150 should be a coronary vasodilatory at a lower concentration rather than a cardiodepressive, whereas the effects of diltiazem would occur in the reverse order. In our model, the increase in CPF after treatment with diltiazem was followed by its decrease. Because CPF is influenced by the cardiac mechanical function, its decrease due to diltiazem may be a secondary effect on the severe decrease in HR. Further investigation of the actions of these drugs on coronary microcirculation is needed.
Clinically, diltiazem has been shown to prevent cardiac accidents in patients with previous myocardial infarction without heart failure, but it also increased the death rate of patients with heart failure (21,22). Patients with reduced ejection fraction are considered to be at risk of subsequent congestive heart failure when treated with diltiazem. The absence of strong negative inotropism and chronotropism of S-2150 suggests that this compound may be safe for patients of ischemic heart diseases with heart failure.
Our results show that S-2150, a Ca channel and α1-adrenoceptor dual blocker, should be an excellent cardioprotective agent despite its low cardiodepressant activity. The pharmacological characteristics of S-2150 indicate its strong therapeutic potential as an antihypertensive agent with an antiischemic effect on the heart.
Acknowledgment: We thank Drs. Teruo Yamamori and Hiroshi Harada, Director of Discovery Research Laboratories I, and their associates of Shionogi Research Laboratories for their cooperation in the chemical synthesis and development of S-2150. We also thank Drs. Motohiko Ueda and Yukio Yonetani of our laboratory for their helpful suggestions regarding this work.
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