Antibody to thrombin receptor inhibits neointimal smooth muscle cell accumulation without causing inhibition of platelet aggregation or altering hemostatic parameters after angioplasty in rat.

An antibody was raised in rabbits against SFFLRNPSEDTFEQF peptide, which is an NH2-terminal peptide of the thrombin-cleaved rat thrombin receptor. In vitro, the antibody inhibited rat smooth muscle cell proliferation but had no effect on rat platelet aggregation or clotting time. These data indicate that the antibody is a specific blocker of the thrombin receptor-signaling pathway in rat smooth muscle cells but does not work as a blocker in rat platelets, suggesting the existence of a second thrombin receptor in the platelets. Using an in vivo balloon catheter-induced injury model in rats, we examined the effect of the anti-rat thrombin receptor IgG on intimal smooth muscle cell accumulation 2 weeks after angioplasty. Analysis of the ratio of intimal to medial cross-sectional areas showed that injection of immune IgG resulted in 43.7% and 53.1% reduction (P<0.01) of neointimal smooth muscle cell accumulation compared with saline and nonimmune IgG treatment, respectively. Moreover, the injection of immune IgG caused a significant decrease of thrombin receptor mRNA expression and also 40.5% and 43.0% decreases (P<0.01) of the proliferating cell nuclear antigen (PCNA) index in the intima compared with the PCNA index after saline and nonimmune IgG treatment, respectively. The suppression of the PCNA index was also observed in the immune IgG-treated group at an early stage after angioplasty. These results suggest that thrombin receptor activation is involved in the proliferation and accumulation of neointimal smooth muscle cells induced by balloon injury.

Anti-atherosclerotic effect of E5324, an inhibitor of acyl-CoA:cholesterol acyltransferase, in Watanabe heritable hyperlipidemic rabbits.

E5324, n-butyl-N'-[2-[3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxy]-6- methylphenyl]urea, a novel and potent inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), was evaluated for its anti-atherosclerotic and lipid-lowering effects in Watanabe heritable hyperlipidemic (WHHL) rabbits. At 3 months of age, 40 male WHHL rabbits were divided into 4 groups. The rabbits were fed a standard rabbit chow (control group), or standard rabbit chow containing E5324 (0.1% or 0.02%) or 1% probucol for 16 weeks. Even the high dose of E5324 did not lower the plasma total cholesterol levels throughout the experiment. Probucol slightly reduced the plasma cholesterol levels, and showed anti-atherosclerotic activity, i.e., reductions of atherosclerotic plaque formation and cholesterol content in the aorta. Although E5324 did not lower plasma cholesterol, atherosclerotic plaque formation in the aortic arch and thoracic aorta was reduced (by about 34% and 41%, respectively, at the high dose; P < 0.05). Cholesterol content in the aortic arch and thoracic aorta was also reduced (by about 59% and 62% at the high dose, respectively) compared with the control. These results suggest that E5324 acts directly on the arterial wall through ACAT inhibition, and prevents the progression of atherosclerosis in WHHL rabbits.

Effect of E5324, a novel inhibitor of acyl-CoA:cholesterol acyltransferase, on cholesteryl ester synthesis and accumulation in macrophages.

The in vitro potencies of a novel inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), E5324 (n-butyl-N'-[2-[3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxy]-6- methylphenyl]urea), were studied. E5324 was found to be a potent ACAT inhibitor in microsomes from a various tissues and in cultured cell homogenate, with IC50 values in the range of 0.044 to 0.19 microM. The kinetic study on E5324 showed that the inhibition of rat intestine ACAT was competitive with respect to oleoyl CoA. E5324 inhibited [3H]olate incorporation into cholesteryl [3H]oleate in phorbol ester-treated THP-1 cell lines (IC50 = 0.44 microM). The rate of [3H]oleate incorporation into phospholipids and triglycerides was not affected by E5324. In an experiment with [3H]cholesterol as the substrate for ACAT, E5324 also inhibited [3H]cholesteryl ester synthesis (IC50 = 0.41 microM). Furthermore, E5324 prevented accumulation of both esterified and total cholesterol in acetyl low density lipoprotein-loaded THP-1 cells. These results indicate that E5324 is a potent and selective ACAT inhibitor and prevents cholesteryl ester accumulation in macrophages.

Binding of biotinylated thrombin receptor peptide to cloned human thrombin receptor overexpressed in baby hamster kidney cells.

Baby hamster kidney (BHK) cells transfected with an expression vector for the human thrombin receptor, and then treated with basic fibroblast growth factor, were found to express specific and saturable binding sites for biotinylated thrombin receptor peptide (SFLLRNPNDKYEPF). Analysis of the binding to live BHK cells yielded an equilibrium dissociation constant (Kd) of 3.0 +/- 0.3 mu mol/l and a maximal binding capacity (Bmax) of 31.0 +/- 0.5 nmol/mg of protein. In competitive binding experiments, the thrombin receptor agonist peptide (SFLLRN), which is a strong inducer of human platelet aggregation, was the most potent competitor. In contrast, position 1 to 2 inverted peptides such as FSLLRNPNDKYEPF and FSLLRNP, which fail to induce for the platelet aggregation, were less potent. This simple and convenient binding assay system using the biotinylated thrombin receptor peptide as a labeled ligand and the cloned thrombin receptor overexpressed in BHK cells may be useful for exploring specific antagonists of the receptor.

Effect of the acyl-CoA:cholesterol acyltransferase inhibitor, E5324, on experimental atherosclerosis in rabbits.

E5324, n-butyl-N'-[2-[3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxy]-6- methylphenyl]urea, a novel and orally absorbable acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, was evaluated for its antiatherosclerotic and antihyperlipidemic effects in cholesterol-fed hypercholesterolemic rabbits. When administered concurrently with a high-cholesterol (0.5% cholesterol) diet for 12 weeks, E5324 (0.0025%, 0.005% and 0.01% in diet) lowered plasma total cholesterol levels dose-dependently (by about 55%-87% at the end of the experiment compared with the control) and also reduced atherosclerotic plaque formation (about 90% reduction at the highest dose; P < 0.01). In pre-established hypercholesterolemic rabbits, which had been pre-fed a high-cholesterol diet for 8 weeks, E5324 administered in the same diet at a dose of 0.005%, 0.01% or 0.02% for 4 weeks significantly reduced plasma cholesterol levels dose-dependently. Cholesterol content and ACAT activity in the aortic arch were also decreased (by about 72% and 58% at the highest dose, respectively) compared with the control. Another ACAT inhibitor, CI-976, had a similar action, but cholestyramine and probucol (2% and 1% in diet, respectively) lacked anti-atherosclerotic activity in this model. Furthermore, when pre-established hypercholesterolemic rabbits were fed normal rabbit chow diet with or without 0.02% E5324 for 4 weeks, changes in plasma cholesterol levels were similar in both E5324-treated and control groups. On the other hand, E5324 significantly reduced cholesterol content and ACAT activity in the aortic arch (by about 52% and 50%, respectively) compared with the control group. These results indicate that E5324 not only has hypocholesterolemic activity, but also may have a direct effect on the arterial wall in experimental atherosclerosis.

Structure-activity relationship of N-[2-(dimethylamino)-6-[3-(5-methyl-4-phenyl-1H-imidazol-1-yl)propoxy] phenyl]-N'-pentylurea and analogues. Novel potent inhibitors of acyl-CoA:cholesterol O-acyltransferase with antiatherosclerotic activity.

We have discovered N-butyl-N'-[2-(dimethylamino)-6-[3-(4-phenyl-1H- imidazol-1-yl)propoxy]phenyl]urea (4), a novel, potent, and systemically bioavailable inhibitor of ACAT (acylCoA:cholesterol O-acyltransferase). The structure-activity relationships (SARs) of this lead compound 4 were investigated by systematic modification of four regions in the molecule. The compounds prepared in this study were tested for in vitro inhibitory activity toward both aortic and intestinal ACATs, and selected compounds were further tested for in vivo hypocholesterolemic activity. The studies not only resulted in the discovery of N-[2-(dimethylamino)-6-[3-(5-methyl-4-phenyl-1H-imidazol-1-yl) propoxy]phenyl]-N'-pentylurea (24), with potent activity and moderate plasma level after oral administration, but also revealed the SAR in each modified region. Four compounds (4, 13, 14, 24) were further selected for testing of in vivo antiatherosclerotic activity; 4, 13, and 24 reduced atherosclerotic plaque development to 38-45% of the control value in terms of area, while 14 did not have a significant antiatherosclerotic effect.

Structure-activity relationship of a series of phenylureas linked to 4-phenylimidazole. Novel potent inhibitors of acyl-CoA:cholesterol O-acyltransferase with antiatherosclerotic activity. 2.

In our continuing search to find systemically bioavailable ACAT (acyl-CoA:cholesterol O-acyltransferase) inhibitors with more potent antiatherosclerotic effect than N-[2-(dimethylamino)-6-[3-(5-methyl-4-phenyl-1H-imidazol-1-yl)propoxy] phenyl]-N'-pentylurea (3), a series of phenylureas linked to 4-phenylimidazole were synthesized and evaluated for in vitro inhibitory activity toward both aortic and intestinal ACATs, and for in vivo hypocholesterolemic activity. The structure-activity relationships (SARs) were studied by strategic modification of five regions in the molecule of 3, i.e., by introducing functional groups or exchanging carbon atoms for heteroatoms. The SAR studies allowed us to select optimum substituents in the five regions, as follows. (1) Dimethylamino was convertible into nitro, methyl, ethyl, propyl, isopropyl, and chloro. On the basis of preliminary pharmacokinetic studies, the methyl group in the ortho-position of the phenylurea was selected. (2) Butyl, pentyl, isopentyl, and neopentyl were better substituents in the urea moiety. (3) Propoxy was the optimal moiety in the bridging portion. (4) Proton, methyl, ethyl, isopropyl, hydroxymethyl, and chloro were better substituents at the 5-position of the imidazole moiety. (5) An unsubstituted phenyl ring was selected as the phenyl group of phenylimidazole. The subsequent comparison studies of compounds containing various combinations of the optimum substituents in each region resulted in the selection of two compounds (67, 68) for further pharmacological and toxicological testing. These compounds were orally bioavailable, and possessed potent in vitro aortic ACAT inhibitory activity (IC50 = 0.16 and 0.012 microM, respectively) and in vivo cholesterol lowering effect (46% and 52% at 1 mg/kg po, respectively). In particular, 68 was 10-fold more potent in the in vitro aortic ACAT assay and 5-fold more potent with respect to hypocholesterolemic activity in vivo than 3.

Inhibitory effects of a novel antiplatelet agent, E5510, on collagen-induced platelet-derived growth factor release and aggregation of human platelets in vitro.

E5510, 4-cyano-5,5-bis(4-methoxyphenyl)-4-pentenoic acid, is a new anti-platelet-aggregation agent under development. We examined the inhibitory efficacy of E5510 on PDGF-release from washed human platelets. E5510 concentration-dependently inhibited collagen-induced PDGF release from human platelets. PDGF release was reduced to below the detection limit (0.47 ng/ml) by preincubation of platelets with 0.04 microM or higher concentrations of E5510. Total growth factor release from platelets was also measured by a bioassay with cultured smooth muscle cells. E5510 almost completely abolished the mitogenic effect of collagen-induced platelet releasates at concentrations of 0.04 microM or higher. Our data suggest that the release of PDGF and other growth factors was inhibited by E5510 at the same concentration that inhibited platelet aggregation.

A new anti-platelet drug, E5510, has multiple suppressive sites during receptor-mediated signal transduction in human platelets.

The mode of action of E5510, 4-cyano-5,5-bis(4-methoxyphenyl)-4-pentenoic acid, which has very potent anti-platelet activities, was investigated by examining its effects on the biochemical responses in the process of human platelet activation. In a whole-cell system, E5510 inhibited the increased turnover of inositol phospholipids arising from phospholipase C activation, arachidonic acid release from phospholipids by phospholipase A2, mobilization of intracellular free Ca2+, protein kinase C activation, and thromboxane A2 production. In a cell-free system, E5510 inhibited cyclooxygenase activity and cyclic AMP-dependent phosphodiesterase activity in a dose-dependent manner. An elevation of cyclic AMP in platelets was also observed at a relatively high concentration of E5510. It was suggested that receptor-mediated turnover of inositol phospholipids, intracellular Ca2+ increase, arachidonic acid release from phospholipids and protein kinase C activation might be indirectly inhibited by the increased cyclic AMP level in platelets. Thromboxane A2 production in the whole-cell system was very strongly inhibited by E5510, and the IC50 for this effect was 100 times lower than that of direct inhibition of cyclooxygenase in the cell-free system. It was concluded that although the primary mode of action of E5510 is the inhibition of the cyclooxygenase pathway of positive signal transduction in platelets, E5510 has another mode of action by increasing platelet cyclic AMP, which can act as a negative messenger in platelet signal transduction, and these multiple sites of action synergistically antagonize platelet cellular activation.

Pharmacological properties of the novel anti-platelet aggregating agent 4-cyano-5,5-bis(4-methoxyphenyl)-4-pentenoic acid.

Various pharmacological properties of a new antiplatelet aggregating agent, 4-cyano-5,5-bis(4-methoxyphenyl)-4-pentenoic acid (E-5510), were examined in order to elucidate its mode of action, Firstly, the inhibitory effect on in vitro aggregation of platelets from humans and various experimental animals was studied. E-5510 inhibited human platelet aggregation induced by collagen, arachidonic acid, adenosine diphosphate (ADP), platelet activating factor (PAF) and epinephrine. Thrombin-induced platelet aggregation, which was not inhibited by acetylsalicylic acid (ASA) or the thiazole drug, 4,5-bis(4-methoxyphenyl)-2-(trifluoromethyl) thiazole, was inhibited by E-5510. E-5510 inhibited collagen-induced platelet aggregation in platelet-rich plasma (PRP) from guinea pigs, beagle dogs and monkey to the same degree as in human PRP, but its effect was weaker in rat PRP. Human platelet adhesion to a collagen-coated plastic disk and thrombin-induced adenosine triphosphate (ATP) release from human platelets were also inhibited by this compound. Next, the ex vivo anti-platelet effect of E-5510 was examined in guinea pigs and beagle dogs. E-5510 was the most potent among the tested drugs (ticlopidine, ASA, cilostazol and the thiazole drug. The anti-platelet effect of this compound appeared within 1 h and lasted more than 8 h after oral administration. The above results suggest that E-5510 may antagonize platelet activation by inhibiting phospholipase C and/or A2, which results in suppression of both phosphatidylinositol breakdown and arachidonic acid release from phospholipids, as well as by inhibiting cyclooxygenase. E-5510 exerted its anti-platelet action without affecting prostaglandin I2 production in the blood vessels. It is considered that E-5510 has a highly potent anti-platelet aggregating effect and a unique multi-site mode of action. This compound is a promising candidate as an antithrombotic drug for clinical use.

Identification of a dephosphorylated oxidation product of the molybdenum cofactor as 2-(1,2-dihydroxyethyl)thieno[3,2-g]pterin.

A new method was developed for the synthesis of 2-(1,2-dihydroxyethyl)thieno[3,2-g]pterin and related 2-substituted thienopterins. A dephosphorylated fluorescent oxidation product of the molybdenum cofactor isolated from xanthine oxidase (EC 1.2.3.2) was identified as 2-(1,2-dihydroxyethyl)thieno[3,2-g]pterin by comparison of electronic and fluorescence spectra and TLC behaviors with those of the synthetic compound.