Yoshimi Imura

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.

Lipid‐lowering properties of TAK‐475, a squalene synthase inhibitor, in vivo and in vitro

Squalene synthase is the enzyme that converts farnesyl pyrophosphate to squalene in the cholesterol biosynthesis pathway. We examined the lipid‐lowering properties of 1‐[[(3R,5S)‐1‐(3‐acetoxy‐2,2‐dimethylpropyl)‐7‐chloro‐5‐(2,3‐dimethoxyphenyl)‐2‐oxo‐1,2,3,5‐tetrahydro‐4,1‐benzoxazepin‐3‐yl]acetyl]piperidine‐4‐acetic acid (TAK‐475), a novel squalene synthase inhibitor. TAK‐475 inhibited hepatic cholesterol biosynthesis in rats (ED50, 2.9 mg kg−1) and showed lipid‐lowering effects in beagle dogs, marmosets, cynomolgus monkeys and Wistar fatty rats. In marmosets, TAK‐475 (30, 100 mg kg−1, p.o., for 4 days) lowered both plasma non‐high‐density lipoprotein (HDL) cholesterol and triglyceride, but did not affect plasma HDL cholesterol. On the other hand, atorvastatin (10, 30 mg kg−1, p.o., for 4 days) lowered the levels of all these lipids. A correlation between decrease in triglyceride and increase in HDL cholesterol was observed, and TAK‐475 increased HDL cholesterol with a smaller decrease in triglyceride than did atorvastatin. TAK‐475 (60 mg kg−1, p.o., for 15 days) suppressed the rate of triglyceride secretion from the liver in hypertriglyceridemic Wistar fatty rats, which show an enhanced triglyceride secretion rate from the liver compared with their lean littermates. In HepG2 cells, TAK‐475 and its pharmacologically active metabolite, T‐91485, increased the binding of 125I‐low‐density lipoprotein (LDL) to LDL receptors. 6 These results suggest that TAK‐475 has clear hypolipidemic effects in animals via inhibition of hepatic triglyceride secretion and upregulation of LDL receptors, and that TAK‐475 might increase HDL cholesterol by decreasing triglyceride. Thus, TAK‐475 is expected to be useful for the treatment of dyslipidemia.

Identification of Benzoxazin-3-one Derivatives as Novel, Potent, and Selective Nonsteroidal Mineralocorticoid Receptor Antagonists

Mineralocorticoid receptor (MR) blockade has come into focus as a promising approach for the treatment of cardiovascular diseases such as hypertension and congestive heart failure. In order to identify a novel class of nonsteroidal MR antagonists that exhibit significant potency and good selectivity over other steroidal hormone receptors, we designed a novel series of benzoxazin-3-one derivatives and synthesized them from 6-(7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazin-6-yl)-2H-1,4-benzoxazin-3(4H)-one (1a), high-throughput screening (HTS) hit compound. Our design was based on a crystal structure of an MR/compound complex and a docking model. In the course of lead generation from 1a, a 1,2-diaryl framework was characterized as a key structure with high binding affinity. On the basis of scaffold hopping and optimization studies, benzoxazin-3-one derivatives possessing 1-phenyl-3-trifluoromethylpyrazol-5-yl moiety at the 6-position were identified as a novel series of potent and selective MR antagonists. Among these compounds, 6-[1-(4-fluoro-2-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl]-2H-1,4-benzoxazin-3(4H)-one (14n) showed highly potent activity and good selectivity and also exhibited a significant antihypertensive effect in deoxycorticosterone acetate-salt hypertensive rats. On the basis of these results, compound 14n was progressed for further pharmacological evaluation.

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.

Lipid-lowering effects of TAK-475, a squalene synthase inhibitor, in animal models of familial hypercholesterolemia.

The lipid-lowering effects of 1-[2-[(3R,5S)-1-(3-acetoxy-2,2-dimethylpropyl)-7-chloro-1,2,3,5-tetrahydro-2-oxo-5-(2,3-dimethoxyphenyl)-4,1-benzoxazepine-3-yl] acetyl] piperidin-4-acetic acid (TAK-475), a novel squalene synthase inhibitor, were examined in two models of familial hypercholesterolemia, low-density lipoprotein (LDL) receptor knockout mice and Watanabe heritable hyperlipidemic (WHHL) rabbits. Two weeks of treatment with TAK-475 in a diet admixture (0.02% and 0.07%; approximately 30 and 110 mg/kg/day, respectively) significantly lowered plasma non-high-density lipoprotein (HDL) cholesterol levels by 19% and 41%, respectively, in homozygous LDL receptor knockout mice. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, simvastatin and atorvastatin (in 0.02% and 0.07% admixtures), also reduced plasma levels of non-HDL cholesterol. In homozygous WHHL rabbits, 4 weeks of treatment with TAK-475 (0.27%; approximately 100 mg/kg/day) lowered plasma total cholesterol, triglyceride and phospholipid levels by 17%, 52% and 26%, respectively. In Triton WR-1339-treated rabbits, TAK-475 inhibited to the same extent the rate of secretion from the liver of the cholesterol, triglyceride and phospholipid components of very-low-density lipoprotein (VLDL). These results suggest that the lipid-lowering effects of TAK-475 in WHHL rabbits are based partially on the inhibition of secretion of VLDL from the liver. TAK-475 had no effect on plasma aspartate aminotransferase and alanine aminotransferase activities. Thus, the squalene synthase inhibitor TAK-475 revealed lipid-lowering effects in both LDL receptor knockout mice and WHHL rabbits.

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.

Endothelin-induced sudden death and the possible involvement of platelet activating factor (PAF)

Endothelin (5 nmol/kg, i.v.) caused a transient hypotension followed by a lasting hypertension in rats. However, an abrupt fall in the blood pressure was observed in most rats 6 to 30 min after the injection of endothelin and sudden death followed with lethality noted over 60 min. An abnormal electrocardiogram (ECG) (ventricular arrhythmias) was observed in rats injected with endothelin. Endothelin (i.v.) also caused sudden death in mice. Pretreatment (5 or 60 min) with specific PAF antagonists, CV-6209 (0.1-3 mg/kg, i.v.) and WEB 2086 (30 mg/kg, p.o.), and a calcium channel blocker, diltiazem (60 mg/kg, p.o.) prevented death and attenuated the ECG changes induced by endothelin, but CV-6209 did not prevent the blood pressure changes induced by endothelin. CV-6209 (0.5-3 mg/kg, i.v.), WEB 2086, diltiazem and dexamethasone (5 mg/kg, i.v.) protected mice against the death induced by endothelin. On the other hand, aspirin (cyclooxygenase inhibitor, 100 mg/kg, p.o.) did not protect mice from the death. Thus, endothelin is a highly toxic peptide with cardiotoxic effects, and PAF may be involved in the pathogenesis of the sudden death.

The thromboxane A2/prostaglandin endoperoxide receptor antagonist activity of CV-4151, a thromboxane A2 synthetase inhibitor.

The thromboxane A2/prostaglandin endoperoxide (TXA2/PGH2) receptor antagonist activity of CV-4151, a potent TXA2 synthetase inhibitor, was examined. CV-4151 inhibited guinea pig and human platelet aggregation induced by U-44069 with IC50 values of 1.2 +/- 0.3 X 10(-5) and 1.9 +/- 0.4 X 10(-5) M, respectively, and inhibited the specific binding of [3H]U-46619 to washed guinea pig and human platelets with IC50 values of 1.2 +/- 0.3 X 10(-6) and 5.1 +/- 1.0 X 10(-6) M, respectively. CV-4151 competitively inhibited the contraction of rabbit aortic strips induced by U-44069 with a pA2 value of 5.90. In experiments in mice in vivo, CV-4151 (1 and 10 mg/kg i.v.) significantly inhibited the thrombocytopenia induced by U-44069 in a dose-dependent manner. These results show that CV-4151 has a distinct TXA2/PGH2 receptor antagonist effect, and that this effect together with its inhibition of TXA2 synthetase could be important for the pharmacological action of this compound.

The role of thromboxane (TX) A2 in rabbit arterial thrombosis induced by endothelial damage

To clarify the role of thromboxane (TX) A2 in arterial thrombus formation, we examined the antithrombotic effects of both a TXA2 synthetase inhibitor (CV-4151) and a TXA2 receptor antagonist (AA-2414) on the rabbit common carotid artery thrombosis which was induced by injury of the endothelium by treatment with 0.25% pronase solution. CV-4151 (1,10 mg/kg, p.o.) and AA-2414 (10 mg/kg, p.o.) significantly inhibited thrombus formation. Furthermore, the combined use of CV-4151 and AA-2414 (0.1 mg/kg, p.o. each) significantly inhibited thrombus formation, though these drugs at the same doses had no effect when administered singly. The plasma level of 11-dehydro TXB2 increased significantly during thrombus formation, and CV-4151 (10 mg/kg) markedly inhibited this increase. There was a significant correlation between the in vivo antithrombotic effects of these drugs and their ex vivo inhibitory effects on arachidonic acid-induced platelet aggregation. The antithrombotic effect of CV-4151 also correlated significantly with its ability to inhibit the production of serum TXA2. These results show that TXA2 may play an important role in the thrombus formation in arterial thrombosis.

Inhibitory effect of TCV-309, a novel platelet activating factor (PAF) antagonist, on endotoxin-induced disseminated intravascular coagulation in rats: possible role of PAF in tissue factor generation.

The possible involvement of platelet activating factor (PAF) in the pathogenesis of endotoxin-induced disseminated intravascular coagulation (DIC) was investigated in rats using a novel potent PAF antagonist, TCV-309. TCV-309 (> 1 mg/kg, i.v.) showed beneficial effects in rats with experimental DIC induced by a 4-hour sustained infusion of endotoxin (1 mg/kg) in a dose-dependent manner. TCV-309 (1 mg/kg) significantly ameliorated the decrease in platelet count and plasma fibrinogen, the prolongation of prothrombin time (PT) and activated partial thromboplastin time (APTT) and the increase in fibrin and fibrinogen degradation products (FDP) and inhibited glomerular fibrin deposition. Furthermore, plasma tissue factor (TF) activity was greatly increased in the DIC rats, and this was also significantly decreased by TCV-309 (1 mg/kg). TCV-309 (1 mg/kg) did not affect these parameters in normal rats. A 4-hour sustained infusion of PAF (60 micrograms/kg) caused mild but significant changes in some DIC parameters such as PT, fibrinogen and FDP concentration and increased the plasma TF activity. TCV-309 (1 mg/kg) inhibited all these PAF-induced changes. TCV-309 (0.1 mM) itself had no direct in vitro effects on the blood coagulation system including TF activity. These results strongly suggest that PAF plays a role in the pathogenesis of endotoxin-induced DIC via the generation of TF. Prophylactic use of PAF antagonists may therefore be useful for the treatment of DIC with sepsis.