Hiroyuki Hagihara

Smooth muscle cell migration induced by inflammatory cell products and its inhibition by a potent calcium antagonist, nilvadipine

The chemotactic activities of inflammatory cell products for rat aortic smooth muscle cells (SMC) were examined in modified Boyden chambers. A checker board analysis revealed that interleukin-1 (IL-1), leukotriene B4 (LTB4), platelet-derived growth factor (PDGF) and inflammatory exudate from zymosan-activated air pouches stimulated chemotaxis of SMC. The chemotaxis, irrespective of the attractants used, was strongly inhibited by nilvadipine, a potent calcium antagonist, and the IC50 values were around 1 x 10(-10) M. Removal of extracellular calcium abolished the chemotactic activities of the attractants. These results suggest that inflammatory cells such as macrophages and polymorphonuclear leukocytes (PMN) have an important role in the migration of SMC into the intima during atherogenesis, and that nilvadipine might be useful for preventing and treating atherosclerosis.

Role of inflammatory responses in initiation of atherosclerosis: effects of anti-inflammatory drugs on cuff-induced leukocyte accumulation and intimal thickening of rabbit carotid artery

Immediately after a cuff-sheathing of rabbit carotid artery, a large number of leukocytes adhered to injured endothelium then infiltrated into the media. These inflammatory responses were followed by an atherosclerotic change, intimal thickening, of the artery. A simultaneous injection of dexamethasone (10 mg/kg i.m.) inhibited the leukocyte accumulation by 74% when evaluated 18 h thereafter. Similarly, 39% inhibition was obtained with the same dose of FR110302, a potent 5-lipoxygenase inhibitor. On the other hand, the same dose of indomethacin, a cyclooxygenase inhibitor, had little effect on the leukocyte accumulation. The intimal thickening which was evaluated 3 weeks after the cuff-treatment was attenuated by a daily dose (10 mg/kg i.m.) of dexamethasone or FR110302 but not by one of indomethacin. The inhibition by the two former drugs were 91 and 58%, respectively. In vitro, the three drugs in concentrations up to 10 microM hardly affected endothelial adhesion of PMN which was induced by LPS or IL-1. Though 10 microM of FR110302 and indomethacin significantly decreased PMN chemotaxis induced by LTB4, the decreases were less than that at 10 microM dexamethasone. These results confirm a possible linkage between inflammation and atherosclerosis, and suggest that 5-lipoxygenase products contribute to the initiation and development of atherosclerosis.

Effect of FR194738, a potent inhibitor of squalene epoxidase, on cholesterol metabolism in HepG2 cells.

(E)-N-ethyl-N-(6,6-dimethyl-2-hepten-4-ynyl)-3-[2-methyl-2-(3-thienylmethoxy)propyloxy]benzylamine hydrochloride (FR194738) inhibited squalene epoxidase activity in HepG2 cell homogenates with an IC50 value of 9.8 nM. In the study using intact HepG2 cells, FR194738 inhibited cholesterol synthesis from [14C]acetate with an IC50 value of 4.9 nM, and induced intracellular [14C]squalene accumulation. On the other hand, the 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor simvastatin reduced both cholesterol and squalene synthesis from [14C]acetate. Incubation with simvastatin for 18 h produced increases in HMG-CoA reductase activity in HepG2 cells, which was related to the degree of reduction in cholesterol synthesis. The HMG-CoA reductase activity increased by 13- and 19-fold at the concentrations of simvastatin that inhibited cholesterol synthesis by 65% and 82%, respectively. In contrast, FR194738 did not increase HMG-CoA reductase activity at the concentrations that inhibited cholesterol synthesis by 24% and 69%, and moderate increase (4.6-fold) was observed at the concentration that inhibited cholesterol synthesis by 90%. These results suggest that non-sterol metabolite(s) derived from mevalonate prior to the squalene epoxidation step in the cholesterol synthetic cascade have a regulatory role in the suppression of HMG-CoA reductase activity. We speculate that FR194738 inhibits cholesterol synthesis with a minimal change of the regulator(s) and would be highly effective in the treatment of hypercholesterolemia.

Effects of FR145237, an Acyl-CoA:cholesterol acyltransferase inhibitor, on diet-induced hypercholesterolemia in diabetic rats

Recent studies have shown that acyl-CoA:cholesterol acyltransferase (ACAT) plays an important role in the initiation of diabetes-associated hypercholesterolemia. To confirm this hypothesis, effects of a potent ACAT inhibitor, FR145237, on diet-induced hypercholesterolemia were examined in streptozotocin (STZ)-induced diabetic rats. One-week feeding of 1% cholesterol and 0.5% cholic acid to normal rats and STZ-induced diabetic rats increased plasma cholesterol levels in both groups, and the response was more remarkable in the STZ rats than in the normal ones (1266 +/- 476 mg/dl and 146 +/- 7 mg/dl, respectively). FR145237 dose-dependently reduced the rise in plasma cholesterol levels in the STZ rats and the levels were almost normalized by treatment with 1 mg/kg/day of the compound. These results suggest that hyperresponse to dietary cholesterol was induced in the STZ rats and that ACAT is involved in the hyperresponse. The effects of FR145237 on other plasma lipids such as high density lipoprotein (HDL) cholesterol and triglyceride (TG) levels were also examined.

Synthesis, X-ray crystal structure, and biological activity of FR186054, a novel, potent, orally active inhibitor of acyl-CoA:Cholesterol O-acyltransferase (ACAT) bearing a pyrazole ring

The synthesis, single crystal X-ray structural analysis, and biological activity of FR186054 (2c), a new, potent, orally efficacious inhibitor of acyl-CoA:cholesterol O-acyltransferase (ACAT), containing a pyrazole ring are described. This compound displayed excellent in vitro efficacy, irrespective of dosing method, indicating superior characteristics compared to other ACAT inhibitors.