Squalene synthase inhibitors reduce plasma triglyceride through a low-density lipoprotein receptor-independent mechanism.

Inhibitors of squalene synthase are considered to be candidate drugs to reduce both plasma cholesterol and triglyceride. However, little is known about the mechanism of squalene synthase inhibitor-specific effect on plasma triglyceride. In this study, we confirmed the triglyceride-lowering effect of ER-27856, a potent squalene synthase inhibitor prodrug, in rhesus monkeys. To determine the role of low-density lipoprotein (LDL) receptor in the triglyceride-lowering effect of squalene synthase inhibitors, we intravenously administered ER-28448, the active form of ER-27856, to Watanabe heritable hyperlipidemic (WHHL) rabbits for 4 days. In heterozygotes, ER-28448 reduced plasma cholesterol and triglyceride by 52% and 37%, respectively. In homozygous rabbits, in contrast, ER-28448 lowered plasma triglyceride by 40% but did not lower plasma cholesterol. Orally administered ER-27856 reduced plasma triglyceride in homozygous animals but atorvastatin and bezafibrate did not. In hepatocytes isolated from homozygous WHHL rabbits, squalene synthase inhibitors but not atorvastatin reduced triglyceride biosynthesis. These data demonstrate that squalene synthase inhibitors reduced plasma triglyceride through an LDL receptor-independent mechanism, which was distinct from that of the triglyceride-lowering action of atorvastatin or bezafibrate. The reduction of hepatic triglyceride biosynthesis may play an important role in the hypotrigyceridemic action of squalene synthase inhibitors.

Effect of ER-27856, a novel squalene synthase inhibitor, on plasma cholesterol in rhesus monkeys: comparison with 3-hydroxy-3-methylglutaryl-coa reductase inhibitors.

Squalene synthase (SQS; EC 2.5.1.21) plays an important role in the cholesterol biosynthetic pathway. We discovered ER-28448, 5-(N-[2-butenyl-3-(2-methoxyphenyl)]-N-methylamino)-1, 1-penthylidenebis(phosphonic acid) trisodium salt, as a potent and selective inhibitor of SQS. ER-28448 inhibited SQS in rat liver microsome with an IC(50) value of 3.6 nm. We also prepared ER-27856, the tripivaloyloxymethyl ester prodrug of ER-28448. Although less active than ER-28448 in a liver microsome assay, ER-27856 more potently inhibited cholesterol biosynthesis in rat hepatocytes; and ER-27856 orally inhibited de novo cholesterol biosynthesis in Sprague-Dawley rats, with an ED(50) value of 1.6 mg/kg. In HepG2 cells, ER-27856 upregulated low density lipoprotein receptor activity to 2.1 times that of control. A time-course study indicated that the inhibitory effect of ER-27856 on cholesterol biosynthesis in rats continued for up to 8 h. In a study of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (HMGRIs), atorvastatin actively suppressed cholesterol biosynthesis for 8 h, whereas the effect of pravastatin and simvastatin diminished at 4 and 8 h, respectively. In rhesus monkeys, 4 days of oral administration of ER-27856 lowered plasma total cholesterol (TCHO) more potently than did these HMGRIs. Whereas atorvastatin significantly elevated plasma alanine aminotransferase, a marker of hepatotoxicity, to 3.7 times at 100 mg/kg, ER-27856 increased the level only 1.4 times at 10 mg/kg, at which doses the hypocholesterolemic effect was equivalent. During 28 days of administration, ER-27856 reduced TCHO and non-high density lipoprotein (non-HDL) cholesterol by 72 and 95%, respectively. These results demonstrate that ER-27856 had more potent hypocholesterolemic activity and less hepatotoxic effect than HMGRIs. ER-27856 may contribute to the treatment of hypercholesterolemic patients.

Stimulation of cholesterol release from rabbit foam cells by the action of a new inhibitor for acyl CoA:cholesterol acyltransferase (ACAT), HL-004.

A new inhibitor of acyl CoA:cholesterol acyltransferase (ACAT), HL-004 [N-(2, 6-diisopropylphenyl)tetradecylthioacetamide], suppressed the synthesis of cholesterol [14C]oleate at 10(-9) approximately 10(-7) M in a concentration-dependent manner in both THP-1 cell-derived macrophages and foam cells prepared from aortic intima of rabbits fed a high cholesterol diet. Incorporation of [3H]cholesterol oleate-beta very low density lipoproteins was not inhibited by HL-004 at 10(-9) approximately 10(-7) M. Release of radioactivity from the cells loaded with [3H]cholesterol oleate-beta very low density lipoproteins was increased by the inhibition of ACAT activity with HL-004. HL-004 did not affect on acid and neutral cholesterol esterases. As a result, cholesterol ester content in foam cells decreased. These data suggested that HL-004 decreases cholesterol ester in foam cells by increasing the release of cholesterol and therefore might suppress atherosclerotic lesions.

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.

Impact of monocyte colony-stimulating factor upon beta-very low density lipoprotein (beta-VLDL) cholesterol metabolism in tetradecanoyl phorbol acetate-derived THP-1 cells.

The effect of monocyte colony stimulating factor (M-CSF) on the beta-very low density lipoprotein (beta-VLDL) metabolism in THP-1 cells (human leukemia cell line) was studied. THP-1 cells treated with M-CSF decreased Latex Bead phagocytosis, but the cells incubated with 12-tetradecanoyl-phorbol-13-acetate (TPA) enhanced phagocytosis 2.5-fold. Binding activity of 125I-M-CSF to THP-1 cells was higher than that in THP-1 cells elicited with TPA. THP-1 cells incubated with M-CSF before TPA treatment were designated MT macrophages, and those incubated with M-CSF after TPA treatment were called TM macrophages. When these cells were incubated with beta-VLDL, the cholesterol ester content in MT macrophages was less than in TM macrophages. The uptake of [3H]cholesterol oleate-beta-VLDL in MT macrophages was the same as in TM macrophages. The released radioactivity from [3H]cholesterol oleate-beta-VLDL loaded MT macrophages was higher than that from TM macrophages. Acid cholesterol esterase activity and ACAT activity were the same in both types of macrophages. Neutral cholesterol esterase activity was higher in MT than in TM macrophages. These results suggested that beta-VLDL-induced cholesterol ester deposition in THP-1 cells-derived macrophages was suppressed by M-CSF, when M-CSF acted at the stage of monocytes (THP-1 cells), and that the reduction of cholesterol ester might be due to enhanced release of cholesterol from the cells with high neutral cholesterol esterase activity.

Absorption effects on total-internal-reflection fluorescence spectroscopy.

A theoretical consideration is made for transverse-electric polarization vectors of a transmitted field in an optically absorbing sample to derive a fluorescence intensity formula as a function of the absorption index of the sample as well as incident and observation angles in total-internal-reflection fluorescence spectroscopy. These equations are experimentally confirmed. It is shown that the angular spectrum of fluorescence intensity is the Laplace transformation of the effective penetration depth, even for the absorbing sample. High resolution of the depth profile is expected for an absorbing sample compared with a nonabsorbing one.