Kazuhiko Tamura

The anti-ischemic effects of CP-060S during pacing-induced ischemia in anesthetized dogs

CP-060 S, (-)-( S)-2-[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]-3-[3-[N-methyl-N-[2-(3 ,4-methylenedioxyphenoxy)ethyl]-amino]propyl]-1,3-thiazolidin++ +-4-one hydrogen fumarate, is a novel cardioprotective drug which prevents Na+-, Ca2+-overload and has Ca2+ channel blocking activity. We compared the anti-ischemic effects of CP-060S with those of diltiazem, a Ca2+ channel blocker, and R56865, N-[1-[4-(4-fluorophenoxy)butyl]-4-piperidinyl]-N-methyl-2-benzothiazo lamine, a Na+-, Ca2+-overload inhibitor, in a canine pacing-induced ischemia model. CP-060S 100 microg kg(-1) significantly suppressed the pacing-induced ischemic epicardial ST-segment elevation by maximally 75%, while diltiazem 100 microg kg(-1) suppressed it by maximally 35%. R56865 100 microg kg(-1) significantly suppressed the ST-segment elevation by maximally 30%. In addition, diltiazem 100 microg kg(-1) caused synergistic suppression of ST-segment elevation by 70% when administered simultaneously with R56865 100 microg kg(-1). These results suggest that a Na+-, Ca2+-overload preventive action and a Ca2+ channel blocking action independently contribute to the suppression of the ST-segment elevation. Therefore, CP-060S may suppress pacing-induced ST-segment elevation by a dual action by preventing Na+-, Ca2+-overload and the Ca2+ channel blockade.

Actions of CP-060S on veratridine-induced Ca2+ overload in cardiomyocytes and mechanical activities in vascular strips.

CP-060S, (-)-(S)-2-[3,5-bis(1, 1-dimethylethyl)-4-hydroxyphenyl]-3-[3-[N-methyl-N-[2-(3, 4-methylenedioxyphenoxy)ethyl]amino]propyl]-1,3-thiazolidin- 4-one hydrogen fumarate, is a novel cardioprotective drug which is designed to prevent Ca2+ overload and cause vasorelaxation. The effects of this compound were evaluated and compared with those of CP-060R (enantiomer of CP-060S,) and diltiazem (Ca2+ channel antagonist) in a veratridine-induced model of Ca2+ overload and vasorelaxation. After 5-min superfusion of veratridine (74 microM), intracellular free calcium concentrations ([Ca2+]i) of rat single cardiomyocytes, as measured with the fura-2 procedure, were greatly elevated, from 44 +/- 5 nM to 3705 +/- 942 nM, and subsequently generated cell contracture. Pretreatment of cardiomyocytes with more than 300 nM of CP-060S or CP-060R for 30 min provided almost complete protection against the veratridine-induced cell contracture; in CP-060S(1 microM)-treated myocytes, [Ca2+]i were minimal and partially elevated from 42 +/- 5 nM to 72 +/- 14 nM after 5 min of veratridine superfusion. In comparison, diltiazem showed no protection below 1 microM and only partial protection at 10 microM. CP-060S, CP-060R and diltiazem all shifted the concentration-response curve for CaCl2 to the right in a competitive manner in depolarized rat thoracic aorta. The pA2 values of CP-060S, CP-060R and diltiazem were 9.16 +/- 0.18, 8.24 +/- 0.14 and 7.66 +/- 0.09, respectively. Our results indicate that CP-060 behaves stereoselectively as a Ca2+ channel antagonist and non-stereo-selectively to protect against veratridine-induced contracture. The latter effect suggests that Ca2+ entry blockade is not the mechanism by which CP-060S exerts cardioprotection.

Effects of CP-060S, a novel cardioprotective drug, on cardiac function and myocardial oxygen consumption

1. We examined the effects of CP-060S on cardiac function and myocardial oxygen consumption (MVO2) in anesthetized dogs. 2. CP-060S (10-300 microg/kg i.v.) decreased heart rate, increased aortic flow and decreased mean blood pressure in a dose-dependent manner. The PR interval was significantly prolonged by administration of CP-060S (300 microg/kg i.v.). 3. CP-060S (10-300 microg/kg i.v.) increased coronary blood flow in a dose-dependent manner. Left ventricular end-diastolic pressure and maximal first derivative of left ventricular pressure were not significantly affected. 4. CP-060S (10-300 microg/kg i.v.) increased coronary sinus blood flow and decreased arteriovenous oxygen difference and MVO2 in a dose-dependent manner. 5. The effects of CP-060S on cardiac function and MVO2 are qualitatively similar to those of diltiazem, a typical Ca2+ antagonist.

CP-060S, a novel cardioprotective drug, limits myocardial infarct size in anesthetized dogs.

The myocardial infarct size (IS)-limiting effect of CP-060S, a novel cardioprotective drug that prevents Na+-, Ca2+-overload and has Ca2+ channel-blocking activity, was compared with that of diltiazem, a pure Ca2+ antagonist, to determine whether the prevention of Na+-, Ca2+-overload contributes to this IS-limiting effect. Dogs were subjected to 90 min of left circumflex coronary artery (LCx) occlusion followed by 5 h of reperfusion. Either CP-060S (300 microg/kg) or diltiazem (600 microg/kg) was administered intravenously 20 min before the occlusion. CP-060S significantly limited IS compared with that of vehicle (percentage of the area at risk: vehicle, 50.64 +/- 6.08%; CP-060S, 21.13 +/- 3.75%; p < 0.01 vs. vehicle). Although diltiazem exerted a significant decrease in rate-pressure product (RPP; an index of myocardial oxygen consumption) during occlusion equal to that of CP-060S, diltiazem did not significantly reduce IS (33.90 +/- 4.30%). Regional myocardial blood flow (RBF) was not significantly different between any of the groups. Therefore the IS-limiting effect of CP-060S cannot be explained in terms of changes in RPP or RBF. Thus the IS limitation induced by CP-060S is probably the consequence of a direct cardioprotective effect on myocytes. The prevention of Na+-, Ca2+-overload may be the primary reason for this IS-limiting effect.

The protective effects of CP-060S on ischaemia- and reperfusion-induced arrhythmias in anaesthetized rats

CP‐060S is a novel sodium and calcium overload inhibitor, and is also characterized as a calcium channel blocker. As these activities have each been shown independently to ameliorate ischaemia damage in the myocardium, the combination may synergistically exert cardioprotection. In this study, therefore, the protective effect of CP‐060S against ischaemia‐ and reperfusion‐induced arrhythmia was evaluated in anesthetized rats. Rats were anaesthetized with pentobarbitone, and the left anterior descending coronary artery was occluded for either 5 min with subsequent reperfusion (a reperfusion‐induced arrhythmia model) or 30 min without (an ischaemia‐induced arrhythmia model). All drugs were intravenously administered 1 min before the onset of occlusion. In the reperfusion‐induced arrhythmia model, the animals in the vehicle‐treated group exhibited ventricular tachycardia (VT) in 100%, ventricular fibrillation (VF) in 89%, and death caused by sustained VF in 56%. CP‐060S (30–300 μg kg−1) dose‐dependently suppressed the incidences of arrhythmias. Significant decreases occurred at 100 μg kg−1 in VF (incidence: 42%) and mortality (8%), and at 300 μg kg−1 in VT (50%), VF (33%) and mortality (8%). This protective effect of CP‐060S was 10 times more potent than that of a pure calcium channel blocker, diltiazem (30–1000 μg kg−1) we tested, in terms of effective dose ranges. As both drugs decreased myocardial oxygen consumption estimated by rate‐pressure product to a similar extent, the calcium channel blocking activity of CP‐060S would not seem to be sufficient to explain its potency. In the same model, co‐administration of ineffective doses of diltiazem (300 μg kg−1) and a sodium and calcium overload inhibitor, R56865 (100 μg kg−1), produced significant suppression of VT (incidence: 62%), VF (46%) and mortality (8%). By contrast, co‐administration of R56865 at the same dose with CP‐060S (300 μg kg−1) did not add to the effect of a single treatment of CP‐060S. In the ischaemia‐induced arrhythmia model, CP‐060S (300 μg kg−1) significantly decreased the incidence of VF from 75% to 29%, whereas diltiazem (1 mg kg−1) was ineffective. These results suggest that CP‐060S inhibits both ischaemia‐ and reperfusion‐induced arrhythmia. The combination of the calcium channel blocking effect and the calcium overload inhibition was hypothesized to contribute to these potently protective effects.

Simultaneous measurement of cytosolic free calcium concentration and cell circumference during contraction, both in a single rat cardiomuscular cell, by digital imaging microscopy with indo-1

We have developed a novel system using digital imaging microscopy with indo-1 to measure the cytosolic free calcium concentration [( Ca2+]i). The method is particularly suitable for measuring the rapid change in [Ca2+]i in relation to the cell motion. With this system, we made the first successful simultaneous measurement of [Ca2+]i and cell circumference during contraction in an electrically stimulated single rat ventricular myocyte. It was found that the level of [Ca2+]i was elevated during contraction, and that the onset and peak time of the calcium transient preceded that of the decrease in circumference.

CP-060S interacts with three principal binding sites on the L-type Ca2+ channel

CP-060S, (-)-(S)-2-[3,5-bis(1,1-dimethylethyl)-4-hydroxypheny1]-3-[3-[N-met hyl-N-[2-(3,4-methylenedioxyphenoxy)ethyl]amino]propyl]-1,3-thi azolidin-4-one hydrogen fumarate is a novel cardioprotective drug, which is able to prevent Na+-, Ca2+-overload and also has Ca2+ channel blocking activity. The latter action of CP-060S was characterized by radioligand binding experiments with rat cardiac membranes in terms of the interaction with the three principal binding sites on the L-type Ca2+ channel, which bind such drugs as the 1,4-dihydropyridines, phenylalkylamines and benzothiazepines. CP-060S exhibited complete and concentration-dependent inhibition of [3H](+)-PN200-110, [3H](-)-desmethoxyverapamil and [3H]cis-(+)-diltiazem binding to their specific binding sites. Saturation studies showed that CP-060S increased the Kd of [3H](+)-PN200-110 and [3H](-)-desmethoxyverapamil without causing a significant change in the maximum binding density. The dissociation kinetics of the three radioligands were accelerated by CP-060S. These results suggest that CP-060S interacts with a novel binding site on the L-type Ca2+ channel and has a negative allosteric interaction with the three principal binding sites for the 1,4-dihydropyridines, phenylalkylamines and benzothiazepines.

Action of KC-399, a newly synthesized potassium channel opener, on mechanical activity and 86Rb efflux in rat aorta.

To clarify the characteristics of KC-399, a newly synthesized potassium channel opener, we investigated the effects of KC-399 and lemakalim on the contractions induced by norepinephrine (NE 1 μM) and K + (30 and 90 mM) and on 86Rb efflux in rat thoracic aorta. KC-399 (0.01–10 nM) and lemakalim (0.001–10 μM) induced relaxation in aortic rings precontracted with 30 mM K+ or NE, but not with 90 mM K +. The vasorelaxant effect of KC-399 was almost 500 times more potent than that of lemakalim. The vasorelaxation with KC-399 developed more slowly and was more resistant to washout than that induced by lemakalim. Glibenclamide (0.1–1 μM), a blocker of ATP-sensitive K-channels, produced concentration-dependent inhibition of the relaxant action of KC-399 in aorta treated with 30 mM K +. KC-399 (3–100 nM) and lemakalim (0.3–10 μM) stimulated 86Rb efflux in rat aorta; the potency of KC-399 was 100 times greater than that of lemakalim. The effects of KC-399 on 86Rb efflux persisted after an 18-min washout period, but those of lemakalim did not. The stimulatory effects of KC-399 (10 and 30 nM) and lemakalim (1 and 3 μM) on 86Rb efflux were also significantly reduced by glibenclamide (1 μM). These results suggest that KC-399 is a potent and long-lasting vasodilator in vitro and that opening of the ATP-sensitive K+ channel may be involved in its mechanism of action.

Effects of CP‐060S, a novel cardioprotective drug, in the methacholine‐induced ECG change model in rats

We compared the antianginal effect of CP‐060S, a novel cardioprotective drug with Na+ and Ca2+ overload‐preventing activity as well as Ca2+ channel antagonistic activity, with that of diltiazem, in an experimental model of vasospastic angina induced by methacholine in anaesthetized rats. Intra‐aortic injection of methacholine at the coronary ostium provoked the ST‐segment elevation of the electrocardiogram (ECG), indicating myocardial ischemia. CP‐060S (3, 5 and 10 mg/kg, i.d.) significantly and dose‐dependently suppressed the methacholine‐induced ST‐elevation, with the duration of action being at least 3 h at the highest dose. In addition, CP‐060S at 3 mg/kg could inhibit the ST‐elevation without producing significant changes in blood pressure, heart rate or rate‐pressure product (RPP). In contrast, diltiazem (10 and 30 mg/kg, i.d.) significantly decreased the RPP, a significant suppression of the ST‐elevation could only be achieved at the highest dose and its duration of action was about 2 h. Similar results were obtained with i.v. administration of the drugs, i.e. CP‐060S given i.v. could inhibit the ST‐elevation with less haemodynamic changes than diltiazem. In conclusion, CP‐060S exerted a more potent and sustained protection against myocardial ischemia evoked by methacholine than diltiazem. The characteristics of the effects of CP‐060S observed here suggest that this drug may be a desirable drug for the treatment of vasospastic angina.

The long-lasting anti-anginal effects of CP-0605 in a rat model of arginine vasopressin-induced myocardial ischaemia

The anti‐anginal effect of CP‐060S, a new cardioprotective agent that prevents myocardial Na+‐, Ca2+‐overload and has Ca2+‐channel blocking activity, was evaluated in a rat model of arginine8‐vasopressin (AVP)‐induced cardiac ischaemia. Infusion of AVP (0.2 IU kg−1) depressed the electrocardiogram (ECG) ST segment, an index of myocardial ischaemia. Vehicle, CP‐060S and diltiazem were given orally 1, 2, 4, 8, 12 and 24 h before the administration of AVP. CP‐060S, at 3 mg kg−1 and 10 mg kg−1, suppressed AVP‐induced ST‐segment depression for 2 h and 12 h, respectively. In contrast, diltiazem, at 10 and 30 mg kg−1, suppressed AVP‐induced ST‐segment depression for only 1 h. The persistent suppression of the AVP‐induced ST‐segment depression by CP‐060S correlated with the time course of changes in its plasma concentration. The minimum effective concentration of CP‐060S was estimated to be 30 ng mL−1 (≅ 50 nwi), consistent with its vasorelaxant potency in rat isolated aortic strips (concentration producing 50% relaxation of KCl contraction, IC50 = 32.6 ± 8.3 nM). Intravenously administered CP‐060S, at 300 μg kg−1 and diltiazem at 500 μg kg−1 showed similar haemodynamic changes, whereas CP‐060S, at 300 μg kg−1, significantly suppressed AVP‐induced ST‐segment depression and diltiazem, at 500 μg kg−1, had no effect on AVP‐induced ST‐segment depression. In summary, orally administered CP‐060S exerted a long‐lasting anti‐anginal effect proportionate to the time course of changes in its plasma concentration in a rat model of AVP‐induced ischaemia.