Takaki Koga

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.

Factor VIIa inhibitors: target hopping in the serine protease family using X-ray structure determination.

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. Compound 1 was discovered from focused screening of serine protease-directed compounds from our internal collection. Using parallel synthesis supported by structure-based drug design, we identified peptidemimetic FVIIa/TF inhibitors (compounds 4-11) containing L-Gln or L-Met as the P2 moiety. However, these compounds lacked the selectivity of other serine proteases in the coagulation cascade, especially thrombin. Further optimization of these compounds was carried out with a focus on the P4 moiety. Among the optimized compounds, 12b-f showed improved selectivity.

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.

In Vitro and In Vivo Studies of AT-1362, a Newly Synthesized and Orally Active Inhibitor of Thrombin

AT-1362 was found to be a potent, selective, and competitive inhibitor of thrombin, with a Ki value of 6.7 nM. In a rat model of venous thrombosis induced by partial stasis and endothelial disruption, the ID(50) values (a dose required to obtain 50% inhibition of thrombus formation over each vehicle group) of AT-1362 and argatroban were 0.03 mg/kg i.v. plus 0.5 microg/kg/minute and 0. 13 mg/kg i.v. plus 8.7 microg/kg/minute, respectively, and the antithrombotic effect of AT-1362 without prolongation of bleeding time lasted for 2 hours and disappeared 4 hours after oral administration of 30 mg/kg. In the rat tail transection model, the BT(2) values (a dose causing two-fold prolongation of the bleeding time over each vehicle group) of AT-1362 and argatroban were 0.56 mg/kg i.v. plus 9.3 microg/kg/minute and 1.1 mg/kg i.v. plus 73.3 microg/kg/minute, respectively. The reduction of thrombus formation and the prolongation of bleeding time were correlated with an ex vivo activated partial thromboplastin time (APTT) for both drugs. AT-1362 at 0.3 mg/kg i.v. plus 5 microg/kg/minute and argatroban at 0.6 mg/kg i.v. plus 40 microg/kg/minute significantly (p<0.05 and p<0.01, respectively) improved the vessel patency in a FeCl(2)-induced carotid artery thrombosis model in rats. These results suggest that AT-1362 may be a potent antithrombotic agent for the treatment of thrombotic diseases.

The effect of a novel benzopyran derivative, KC 399, on the isolated guinea-pig trachealis and human bronchi.

1. In isolated guinea-pig trachealis, KC 399, BRL 38227 and salbutamol suppressed the spontaneously generated tone in a concentration-dependent manner with pD2 values of 8.89 +/- 0.09 (n = 14), 6.18 +/- 0.07 (n = 11) and 7.72 +/- 0.12 (n = 8), respectively. 2. The bronchodilator effects of KC 399 and BRL 38227 were antagonized by glibenclamide but not by charybdotoxin or apamin. The effect of salbutamol was antagonized by charybdotoxin but not by glibenclamide or apamin. 3. KC 399 and BRL 38227 failed to inhibit the tone evoked by 90 mM K+ in guinea-pig trachealis, whereas salbutamol did inhibit it, in a concentration-dependent manner. 4. These bronchodilators also relaxed the tone of isolated guinea-pig trachealis supported by histamine, carbachol, U46619 or leukotriene D4. Their order of potency was always KC 399 > salbutamol > BRL 38227. 5. KC 399 and BRL 38227 relaxed isolated human bronchi contracted with histamine or carbachol. 6. We conclude that KC 399 is a potent relaxant of isolated guinea-pig trachealis and human bronchi in vitro. The relaxant action of KC 399 could be due to the opening of glibenclamide-sensitive K+ channels.

Structure of human factor VIIa/tissue factor in complex with a peptide-mimetic inhibitor: high selectivity against thrombin by introducing two charged groups in P2 and P4.

The crystal structure of human factor VIIa/soluble tissue factor (FVIIa/sTF) in complex with a highly selective peptide-mimetic FVIIa inhibitor which shows 1670-fold selectivity against thrombin inhibition has been solved at 2.6 A resolution. The inhibitor is bound to FVIIa/sTF at the S1, S2 and S3 sites and at the additional S1 subsite. Two charged groups, the amidino group in P2 and the carboxylate group in P4, form ionic interactions with Asp60 and Lys192 of FVIIa, respectively. Structural comparisons between factor VIIa and thrombin show that thrombin has oppositely charged residues, Lys60F and Glu192, in the S2 site and the S1 subsites, respectively. These data suggest that the utilization of the differences of charge distribution in the S2 site and the S1 subsites between FVIIa and thrombin is critical for achieving high selectivity against thrombin inhibition. These results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.

Contribution of the tissue angiotensin converting enzyme to the antihypertensive effect of altiopril calcium (MC‐838) in spontaneously hypertensive rats

Abstract— The effect of a new orally active angiotensin converting enzyme (ACE) inhibitor, calcium (—)‐N‐[(S)‐3–[(N‐cyclohexylcarbonyl‐D ‐alanyl)thio]‐2‐methylpropionyl]‐L ‐prolinate (MC‐838, altiopril calcium), on systemic blood pressure (SBP) and tissue ACE activity has been examined in conscious spontaneously hypertensive rats (SHRs). MC‐838 (3 mg kg−1) given orally to SHRs elicited a long‐lasting hypotension lasting over 24 h. With the development of the hypotension, MC‐838 significantly reduced ACE activity in the lung, kidney and aorta, but not in the brain and heart. Suppression of plasma ACE and rise of plasma renin activity occurred only transiently at an earlier stage.

Comparative effects of ouabain on isolated papillary muscle from tree shrews, guinea‐pigs and rats

Abstract— Inotropic effects of ouabain were investigated in the isolated papillary muscle preparations of the tree shrew (Tupaiaglis), guinea‐pigs and rats. In the guinea‐pig and shrew papillary muscles, ouabain at concentrations of 10−8 to 3 × 10−7 M caused a concentration‐dependent positive inotropic response in a similar magnitude, while in the rat, ouabain at concentrations of 10−7 to 3 × 10−6 M elicited negative inotropic one. Either phentolamine or pindolol in a concentration sufficient to block the α‐ and β‐adrenoceptors did not antagonize the positive inotropic effect of ouabain in the papillary muscle preparation of the shrews.

Demonstration in Tupaia papillary muscle preparations of α‐adrenoceptors mediating positive inotropic effects: comparison with guinea‐pigs

1 Positive inotropic responses to α‐ and β‐adrenoceptor agonists of isolated papillary muscles from the tree shrew (Tupaia), were compared with those from guinea‐pigs. 2 In Tupaia, the concentration‐response curve for phenylephrine, unlike that for isoprenaline, was not affected by pindolol in a concentration (10−8m) sufficient to block β‐adrenoceptor‐mediated responses, but it was significantly shifted to the right by phentolamine (10−6m). In guinea‐pig papillary muscles, however, the concentration‐response curve for phenylephrine, like that for isoprenaline, was shifted to the right by pindolol (10−8m) but was unaltered by phentolamine (10−6m). Furthermore, when the mean concentrations of agonists inducing maximal positive inotropic responses were compared (relative to that of isoprenaline = 1.0), phenylephrine was found to be only slightly less potent (0.84 ± 0.04; n = 5) in Tupaia and much less potent (0.33 ± 0.06; n = 5) in the guinea‐pig. 3 Although in Tupaia papillary muscles the increase in developed tension induced by a combination of phenylephrine and isoprenaline did not significantly differ from that by phenylephrine alone, it was approximately 3 times larger than that produced by phenylephrine alone in guinea‐pigs. 4 These results indicate that in papillary muscles from Tupaia, unlike the guinea‐pig, the positive inotropic effects of phenylephrine can be mediated by α‐adrenoceptors.