E-5324

N-[2-[N'-pentyl-(6,6-dimethyl-2,4-heptadiynyl)amino]ethyl]- (2-methyl-1-naphthylthio)acetamide (FY-087). A new acyl coenzyme a:cholesterol acyltransferase (ACAT) inhibitor of diet-induced atherosclerosis formation in mice

FY-087 (N-[2-[N'-pentyl-(6,6-dimethyl-2,4-heptadiynyl)amino]ethyl]- (2-methyl-1-naphthylthio)acetamide) was found to be a competitive inhibitor of human microsomal acyl coenzyme A:cholesterol acyltransferase (ACAT) with an IC50 value of 0.11 microM. Under our assay conditions, other ACAT inhibitors tested, specifically YM-750, E-5324, and melinamide, all of which are now in phase I clinical trials or in clinical use in Japan, inhibited this enzyme with IC50 values of 0.18, 0.14, and 3.2 microM, respectively. FY-087 also inhibited ACAT in acetyl-low density lipoprotein loaded human macrophages (THP-1 cells) with an IC50 of 0.17 microM. Following the oral administration of FY-087 (30 mg/kg) to rats, the plasma concentration of FY-087 reached 0.42 microgram/mL after 2 hr. This concentration of FY-087 was enough to inhibit blood vessel ACAT activity. Cholesterol-lowering and anti-atherogenic effects of FY-087 were examined using C57BL/6J mice fed an atherogenic diet. In this mouse model, treatment with FY-087 (28 mg/kg) inhibited the increase in plasma cholesterol levels by about 20% and decreased the hepatic accumulation of free and esterified cholesterol by 61 and 67%, respectively. FY-087 also significantly inhibited the atherogenic diet-induced increase in the fatty-streak lesion area of the proximal aorta by 57% in C57BL/6J mice. These results indicate that FY-087 is not only a therapeutically bioavailable ACAT inhibitor that lowers plasma cholesterol levels, but also an effective anti-atherogenic agent in mice fed an atherogenic diet.

Biochemical evidence for oligomerization of rat adrenal acyl-coenzyme A:cholesterol acyltransferase

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) in rat adrenal was compared with that in rat liver. Immunoblot analyses of the microsomal fractions from adrenal with an anti-human ACAT antibody detected a 45 kDa protein. Upon pretreatment of these microsomal fractions with chemical cross-linkers such as BS3 and Sulfo-EGS, the 45 kDa band decreased with a concomitant increase in high molecular weight proteins (55, approximately 100, and approximately 230 kDa), suggesting that ACAT constitutes oligomers of 45 kDa monomers associated with a 10 kDa protein. In sharp contrast, the same immunoblot analysis of rat liver microsomal fractions identified a 50 kDa protein which was not cross-linked by these cross-linkers. Moreover, when four ACAT inhibitors were tested for their effects on adrenal and liver enzymes, NTE-122, CI-976, and E5324 were more effective for the liver enzyme, whereas 58-035 was much more effective for adrenal ACAT. These biochemical and pharmacological observations support the notion that the rat liver ACAT protein is distinct from the adrenal counterpart.

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.

Inhibition of cholesterol esterification in macrophages and vascular smooth muscle foam cells: evaluation of E5324, an acyl-CoA cholesterol acyltransferase inhibitor

Cholesteryl esters (CE) comprise the principal lipid class that accumulates within macrophages and smooth muscle cells of the atherosclerotic lesion. Acyl-CoA cholesterol acyl-transferase (ACAT) is the major enzyme responsible for esterification of intracellular cholesterol. We evaluated the ability of E5324 (n-butyl-N'-[-2-[3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxy]-6- methyl-phenyl]urea), a novel, orally absorbable ACAT inhibitor, to inhibit esterification of fatty acids to cholesterol and CE accumulation in macrophages and in smooth muscle cells. E5324 significantly inhibited cholesterol esterification in rat aortic smooth muscle cells and in macrophages. In addition, E5324 reduced the cellular mass of CE, the significant measure of the efficacy of drugs designed to modulate cholesterol metabolism. E5324 treatment of macrophages exposed to acetylated low-density lipoprotein reduced CE mass by 97%, and treatment of lipid-loaded smooth muscle cells reduced CE mass by 29%. Although free cholesterol increased approximately twofold, this free cholesterol would presumably be accessible to the membrane for efflux in vivo (reverse cholesterol transport). These results demonstrate that E5324 can inhibit cholesterol esterification and CE mass in atherosclerotic foam cells, derived from either macrophages or arterial smooth muscle cells.