Kohei Nishikawa

CV-3988 - a specific antagonist of platelet activating factor (PAF).

CV-3988, rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl 2-thiazolioethyl phosphate was shown to be a specific inhibitor of platelet activating factor (PAF). This compound in concentrations of 3 x 10(-6) to 3 x 10(-5)M inhibited aggregation of rabbit platelets induced by PAF (3 x 10(-8)M), while it had no effect on the aggregation induced by arachidonic acid, ADP, collagen or A-23187. CV-3988 alone even at a concentration of 10(-3)M had no effect on platelet aggregation. The inhibitory action of CV-3988 on the PAF-induced aggregation was independent of the formation of micelles. The PAF (0.1 to 1.0 micrograms/kg, i.v.)-induced hypotension in anesthetized rats was also inhibited dose-dependently by the i.v. administration of CV-3988 (1 and 10 mg/kg), while the hypotensive actions induced by the i.v. administration of acetylcholine (1 micrograms/kg), arachidonic acid (1 mg/kg), bradykinin (10 micrograms/kg), isoproterenol (1 microgram/kg) and histamine (100 micrograms/kg) were not altered by CV-3988 (10 mg/kg, i.v.). All these findings indicate that CV-3988 specifically inhibits the action of PAF in vitro and in vivo. This is the first report of a PAF antagonist which can specifically inhibit the PAF-induced hypotension as well as the PAF-induced platelet aggregation.

Is platelet activating factor (PAF) a mediator of endotoxin shock

To determine whether endogenous PAF contributes to the pathogenesis of endotoxin shock, CV-3988, a specific PAF antagonist, was injected i.v. to rats before, simultaneously with or after endotoxin. CV-3988 (5 mg/kg i.v.) injected 5 min before the endotoxin completely inhibited endotoxin (15 mg/kg i.v.)-induced hypotension, and CV-3988 (0.05-1 mg/kg i.v.) injected 7-10 min after the endotoxin rapidly reversed endotoxin-induced hypotension. A combination of CV-3988 (10 mg/kg) with endotoxin (5 mg/kg) administered i.v. improved the survival rate for 20 h or more. CV-3988 (0.05-1 mg/kg i.v.) rapidly reversed the PAF (1 microgram/kg i.v.)-induced hypotension. These findings strongly suggest that endogenous PAF may play a pivotal role in the pathogenesis of endotoxin shock.

Pathophysiological role of endothelin in acute renal failure

In conscious rats, the i.v. injection of endothelin (ET) caused an increase in blood urea nitrogen (BUN), an index of renal dysfunction. In the model of acute renal failure which was induced by occlusion of the bilateral renal arteries of rats followed by reperfusion, ET-monoclonal antibody improved the renal function. In this model, ET-antibody also protected the kidneys from renal proximal tubular necrosis and suppressed Ca++-accumulation in necrotic tissues. Plasma ET level increased 5 min and 5 hr and renal ET content did 5 and 20 hr after reperfusion. BUN level increased 5 and 20 hr after reperfusion. These results strongly suggest that the endogenously increased ET may be one of the important deleterious mediators in the pathogenesis of ischemic acute renal failure.

Protective effects of candesartan cilexetil (TCV-116) against stroke, kidney dysfunction and cardiac hypertrophy in stroke-prone spontaneously hypertensive rats.

The effects of chronic treatment with an angiotensin II receptor antagonist, candesartan cilexetil (TCV-116, 0.1, 1, 10 mg/kg), and an angiotensin converting enzyme inhibitor, enalapril maleate (enalapril, 10 mg/kg), on the development of end-organ damage were examined in stroke-prone spontaneously hypertensive rats (SHRSP). The control SHRSP developed severe hypertension with stroke signs and increased urinary protein excretion. TCV-116 (0.1 mg/kg) reduced the stroke incidence and urinary protein excretion without affecting the blood pressure. TCV-116 (1 and 10 mg/kg) and enalapril reduced blood pressure, the stroke incidence, the urinary indices and left ventricular weight. Circulating renin-angiotensin system (RAS) and renal renin mRNA expression were significantly accelerated or tended to be accelerated in the control SHRSP with end-organ damages. A low dose of TCV-116 tended to reduce the RAS indices in plasma by improving the damages, whereas a high dose (10 mg/kg) increased them by the reflexes with blocking RAS. The present results indicate that chronic All blockade reduces the increase in blood pressure, end-organ damages and RAS related to the damages in SHRSP.

Enhanced thromboxane A2 biosynthesis in the kidney of spontaneously hypertensive rats during development of hypertension.

Arachidonic acid (AA)-induced pressor response and production of thromboxane YXB2, the stable metabolite of TXA2, prostaglandin (PG)-like substance (PLS) and 6-keto-PGF1 alpha the stable metabolite of prostacyclin (PGIs), were studied using isolated, perfused kidneys of 6- and 18-week old spontaneously hypertensive rats (SHR), Wistar-Kyoto rats (WKY), two-kidney, one clip hypertensive rats (RHR) and DOCA/salt hypertensive rats (DOCA/salt HR). The AA-induced pressor response and release of TXB2 were highest in the 6-week old SHR, whereas, the release of PLS and 6-keto-PGF 1 alpha was marked in the 18-wek old SHR and the established hypertensive stages of both RHR and DOCA/salt HR. In the kidneys of SHR and WKy, exogenous TXA2 induced a severe vasoconstriction and there was a positive correlation between the AA-induced pressor response and the release of TXB2 or PLS. Thus, the initiation of hypertension in SHR may follow an accelerated synthesis of TXA2 against PGI2 in response to stimuli which induce a release of AA.

Coronary vasospastic action of thromboxane A2 in isolated, working guinea pig hearts.

The effects of thromboxane A2 (TXA2) on isolated, working guinea pig heart and left ventricular papillary muscle preparations were investigated. TXA2 was generated by the enzymatic conversion of prostaglandin H2 (PGH2) with indomethacin-treated horse platelet microsomes (IPM). TXA2 caused dose-dependently a considerable decrease in the coronary flow, left ventricular systolic pressure and left ventricular dp/dt in the perfused heart, while the similar effects of PGH2 were transient and weaker than those of TXA2. These effects of TXA2 disappeared after the TXA2-generating system was left standing at 37 degree C for 3 min. When IPM in the TXA2-generating system was pretreated with a TXA2 synthetase inhibitor, L-8027, the enhanced cardiac responses induced by the mixture of IPM and PGH2 disappeared. TXA2 had no direct effect on the contractile force of the papillary muscle. These results suggest that TXA2 has a specific coronary vasopastic action without a direct inotropic effect.

Angiotensin-II-stimulated release of thromboxane a2 and prostacyclin (PGI2 ..) in isolated, perfused kidneys of spontaneously hypertensive rats

Abstract Angiotensin-II(A-II)-stimulated release of rabbit aorta-contracting substance (RCS), prostaglandin-like substance (PLS) and dog coronary-relaxing substance (DRS) was studied in isolated, perfused kidneys of spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY). When the perfusion pressure was set at 60–70 mmHg, the dose-related pressor response of the kidney to A-II did not differ significantly between the strains, or between the ages of 6, 12 and 18 weeks. The A-II-induced release of RCS from the kidney was most remarkable in 6-week SHR, slight in 12- and 18-week SHR, but negligible for all ages of WKY. The DRS release was remarkable in 12- and 18-week SHR, but was hardly detectable in 6-week SHR and all ages of WKY. The PLS release was much more remarkable in 18-week SHR than in 6- and 12-week SHR and all ages of WKY. The RCS was confirmed to be thromboxane A2 (TXA2) by: (1) its half-life of 38 sec at 37°, (2) a concomitant release of radio-immunoassayable TXB2: (a stable metabolite of TXA2) into the perfusate and (3) specific inhibition of its release by L-8027 (a TXA2 synthetase inhibitor). The DRS was considered to be prostacyclin (PGI2) by: (1) its half-life of 5.2 min at 37° and (2) its anti-platelet aggregation activity. It is speculated that an enhanced TXA2 synthesis in the 6-week SHR kidney may be involved in increasing the renal vascular resistance and in initiating the process of hypertension.

Possible role of platelet activating factor (PAF) in disseminated intravascular coagulation (DIC), evidenced by use of a PAF antagonist, CV-3988

The i.v. infusion of endotoxin (ET) (0.25 mg/kg/hr for 4 hr) induced disseminated intravascular coagulation (DIC) in rats; thrombocytopenia, prolongation of prothrombin time (PT) and partial thromboplastin time (PTT), hypofibrinogenemia and elevated levels of fibrinogen/fibrin degradation products (FDP) were observed. Platelet activating factor (PAF) (8 micrograms/kg/hr for 4 hr) also induced DIC-like changes, except in platelets. A specific PAF antagonist, CV-3988 (2 mg/kg bolus 5 min before ET + 1 or 2 mg/kg/hr for 4 hr of ET infusion) improved all the parameters that had been altered by both ET and PAF. CV-3988 (2 mg/kg bolus 2 hr after ET + 2 mg/kg/hr for 2 hr of ET infusion) also had beneficial effects on DIC. CV-3988 itself had no effects on the parameters of DIC. These results strongly suggest that PAF may play a role in the pathogenesis of DIC and CV-3988 may prove to be useful for the treatment of DIC.

Delay of the initiation of hypertension in spontaneously hypertensive rats by CV-4151, a specific thromboxane A2 synthetase inhibitor

When CV-4151, a specific thromboxane (TX) A2 synthetase inhibitor, was given orally to 4 week old (4w) spontaneously hypertensive rats (SHR) daily for 3 weeks, the initiation of hypertension was delayed by about one week. The agent increased urinary excretion of water, sodium and creatinine, reduced that of TXA2 (as TXB2), increased that of PGI2 (as 6-keto-PGF1 alpha) and enhanced urinary PGI2/TXA2. In 4w Wistar-Kyoto rats (WKY) and 18w SHR was established hypertension, the agent had little effect on blood pressure and renal function. In isolated, perfused kidneys of 6w SHR, CV-4151 markedly inhibited both arachidonic acid-induced pressor action and production of TXA2. TXA2 synthetase activity in renal cortical microsomes of 5w SHR was approximately 1.5 times higher than that in age-matched WKY. CV-4151 inhibited TXA2 synthetase activity of medullary and cortical microsomes more effectively in 5w SHR than in age-matched WKY. Thus, in young SHR, the TXA2 synthetase inhibitor seemed to improve renal function by altering the balance of renal TXA2 and PGI2 biosynthesis and subsequently caused a delay in the initiation of hypertension. The present findings lend support to the idea that an imbalance in the renal TXA2-PGI2 biosynthesis may be involved in the initiation of hypertension in SHR.

Cv-4151 — a potent, selective thromboxane A2synthetase inhibitor

(E)-7-Phenyl-7-(3-pyridyl)-6-heptenoic acid (CV-4151) inhibited horse platelet microsomal thromboxane (TX) A2 synthetase with an IC50 of 2.6 X 10(-8) M, but even at a high concentration of 10(-4) M it had little effect on cyclooxygenase, PGI2 synthetase and 5-lipoxygenase in in vitro enzymatic assays. CV-4151 did not affect PGI2 release from rat and rabbit aortic tissues in in vitro (10(-4) M) and ex vivo (10 and 100 mg/kg, p.o.) experiments, whereas aspirin (10(-4) M or 10 and 100 mg/kg, p.o.) markedly inhibited PGI2 release in these preparations. When given orally to rats and dogs, CV-4151 markedly inhibited blood TXA2 synthetase activity: the ID50 values (mg/kg, 2 hr later) were 0.05 in rats and 0.17 in dogs. The inhibitory effects at an oral dose of 1 mg/kg lasted more than 24 hr in both species; the inhibition was 41% in rats and 32% in dogs 24 hr after the administration. When injected i.v. to rats and dogs, CV-4151 caused inhibitory effects on TXA2 synthetase equipotent to those observed with the oral administration. In both species, CV-4151 given orally increased concentration of serum immunoreactive 6-keto-PGF1 alpha concomitant with a decrease of serum TXB2-8 concentration. CV-4151 was equipotent to OKY-1580 (IC50: 2.3 X 10 M), a well documented TXA2 synthetase inhibitor, in an in vitro TXA2 synthetase assay. However, CV-4151, given orally or i.v. to rats and dogs, was much more potent and longer acting in inhibition of blood TXA2 production than OKY-1580. Dazoxiben was less potent than these compounds in vitro. In rats, serial oral administration of CV-4151 (10 mg/kg) once daily for 14 days produced a constant and marked reduction of serum TXB2 concentration with concomitant increase of serum immunoreactive 6-keto-PGF1 alpha concentration. No rebound phenomenon in inhibition of TXA2 synthetase was observed after the dosing was stopped. These findings indicate that CV-4151 is a potent and long acting selective inhibitor of TXA2 synthetase and may reorient the metabolism of PG endoperoxides to PGI2.