Lipid‐lowering effects of the squalene epoxidase inhibitor FR194738 in dogs,hamsters, and rats

Squalene epoxidase is a microsomal enzyme that catalyzes the conversion of squalene to 2,3‐oxidosqualene and is an important control site in the cholesterol synthetic pathway. FR194738 is a potent inhibitor of hepatic squalene epoxidase from dogs, hamsters, and rats with IC₅₀ values of 49, 14, and 68 nM, respectively. In dogs, FR194738 at 10 and 32 mg/kg/day decreased serum total cholesterol levels by 26% and 40%, and serum triglyceride levels by 47% and 76%, respectively. Pravastatin, an inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase, at 3.2 and 10 mg/kg/day also decreased serum total cholesterol levels by 32% and 36%, and serum triglyceride levels by 56% and 50%, respectively. Both FR194738 and pravastatin preferentially decreased low‐density lipoprotein cholesterol levels among the lipoprotein fractions examined. In hamsters, FR194738 decreased serum total cholesterol levels by 22% and led to a decrease in serum triglyceride levels of 9% at 100 mg/kg/day, whereas pravastatin did not decrease total serum cholesterol levels up to 100 mg/kg/day even though it decreased serum triglyceride levels at doses as low as 3.2 mg/kg/day. In rats, both FR194738 and pravastatin failed to decrease total serum cholesterol levels up to 100 mg/kg/day. FR194738 dose‐dependently decreased serum triglyceride levels by 30%, 41%, and 65% at 10, 32, and 100 mg/kg/day, respectively, whereas pravastatin decreased them only by 19% at 100 mg/kg/day. These results clearly showed a species difference in cholesterol and triglyceride metabolism, and suggest that dogs are the most appropriate animal model for evaluating hypocholesterolemic drugs. FR194738 that induced comparable effects with pravastatin in the dog model may provide another treatment for hypercholesterolemia in men. Drug Dev. Res. 54:202–208, 2001. © 2002 Wiley‐Liss, Inc.     

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.     

Experimental model of escape phenomenon in hamsters and the effectiveness of YM-53601 in the model

1. The aim of this study was to establish an experimental model of the escape phenomenon, in which plasma cholesterol, initially reduced by a 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitor such as pravastatin, increases again on long-term administration. We also evaluated the efficacy of YM-53601 ((E)-2-[2-fluoro-2- (quinuclidin-3-ylidene) ethoxy]-9H-carbazole monohydrochloride), a squalene synthase inhibitor, in this model. 2. Pravastatin inhibited cholesterol biosynthesis in hamster primary hepatocytes (IC(50), 14 nM). After pre-treatment with pravastatin, in contrast, almost no effect on cholesterol biosynthesis was seen. 3. In hamsters fed a high fat diet, 3 mg kg(-1) pravastatin for 9 days decreased plasma non-HDL cholesterol (total cholesterol - high density lipoprotein cholesterol) (P<0.01), but this effect was lost between 17 and 27 days of treatment, accompanied by an increase in HMG-CoA reductase activity. No such increase in plasma non-HDL cholesterol was seen with YM-53601 at 30 mg kg(-1) after 9 (P<0.001), 17 (P<0.01) or 27 (P<0.001) days of treatment. Replacement of pravastatin with YM-53601 caused a decrease in plasma non-HDL cholesterol by 53% (P<0.001) and in HMG-CoA reductase activity. 4. This animal model thus satisfactorily replicates the escape phenomenon observed in humans and may therefore be useful in evaluation of lipid-lowering agents, specifically comparison of HMG-CoA reductase inhibitors. Further, YM-53601 may be useful in the treatment of hypercholesterolemia without induction of the escape phenomenon.     

Effects of Propionic Acid and Pravastatin on HMG-CoA Reductase Activity in Relation to Forestomach Lesions in the Rat

Abstract: Administration of 4% propionic acid in powdered diet to rats for 12 weeks induces severe hyperplastic lesions in the forestomach mucosa. The mechanisms underlying this damage are not yet clear. Several lipophilic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors such as lovastatin and simvastatin produce forestomach lesions similar to propionic acid after oral administration and the degree of alterations is correlated with their in vitro inhibitory potency (Kloss et al. 1991). Therefore it is possible, that sustained inhibition of HMG-CoA reductase and induction of hyperplasias may be somehow connected. For that reason we investigated, whether or not propionic acid has any influence on HMG-CoA reductase activity in vitro and in vivo, because propionic acid has been suggested to suppress liver cholesterol synthesis, and also whether or not pravastatin, a more polar HMG-CoA reductase inhibitor than lovastatin displays similar effects on forestomach mucosa. In untreated forestomach microsomes in vitro, propionic acid at a concentration of 51 mM (pH 5.7) inhibited HMG-CoA reductase activity by 51±10%, but at pH 7.2 no inhibition of the enzyme could be detected. Furthermore 4% propionic acid-treatment did not lower serum cholesterol. In contrast to lovastatin (Kloss et al. 1991), oral administration of pravastatin (up to 25% in the diet) did not produce any forestomach lesions in the rat. On the other hand, pretreatment with pravastatin revealed that HMG-CoA reductase activity in microsomes exceeded the activity of control forestomach and liver microsomes by 4.9 fold and 6.7 fold respectively, whereas no induction of this enzyme (neither liver nor forestomach) could be observed by pretreatment with 4% propionic acid for 12 weeks. Despite increased hepatic HMG-CoA reductase activity, pravastatin-treatment significantly lowered serum cholesterol levels of rats. These results show that sustained inhibition of HMG-CoA reductase activity in forestomach microsomes is not strongly connected with hyperplasia development.     

Pravastatin prevents arrhythmias induced by coronary artery ischemia/reperfusion in anesthetized normocholesterolemic rats

HMG-CoA reductase inhibitors (statins) have been shown to decrease cardiovascular mortality. Since ventricular tachyarrhythmias are closely related to cardiovascular mortality, we tested effects of the hydrophilic statin pravastatin and the lipophilic statin fluvastatin in a rat arrhythmia model of ischemia/reperfusion and simultaneously measured serum total cholesterol level. Anesthetized rats were subjected to 5-min ischemia and 10-min reperfusion after chronic administration of oral pravastatin (0.02, 0.2, or 2 mg/kg), fluvastatin (0.2, 2, or 4 mg/kg), or vehicle for 22 days, once daily. The acute effect of pravastatin (0.2 or 2 mg/kg, once orally) was also observed. Chronically administrated pravastatin significantly reduced the incidence of ischemia-induced ventricular tachycardia (VT) from 70% (control) to 9% at 2 mg/kg, and it reduced the incidence of reperfusion-induced lethal ventricular fibrillation (VF) from 90% (control) to 20% at 0.2 mg/kg. Acute pravastatin and chronically administrated fluvastatin had no significant effect on these arrhythmias. There were no significant changes in blood pressure, heart rate, QT interval, and serum cholesterol among pravastatin-, fluvastatin-, and vehicle-treated groups. Hydrophilic pravastatin prevented reperfusion-induced lethal VF in anesthetized rats by chronic administration independent of its cholesterol lowering effect. This may be a new beneficial role of pravastatin in decreasing cardiovascular mortality.     

Bicyclol, a synthetic dibenzocyclooctadiene derivative, decreases hepatic lipids but increases serum triglyceride level in normal and hypercholesterolaemic mice

Bicyclol is used for the treatment of chronic hepatitis B in China. In this study, the effects of bicyclol (100 or 300 mg kg(-1), p.o.) on serum and liver lipid contents were investigated in both normal and experimentally induced hypercholesterolaemic mice. Hypercholesterolaemia was induced by either oral administration of cholesterol/bile salt or feeding a diet containing lard/cholesterol. Daily administration of bicyclol for 7 days dose-dependently increased the serum triglyceride level (29-80%) but slightly decreased the hepatic total cholesterol level (12-17%) in normal mice. Co-administration of bicyclol with cholesterol/bile salt decreased the hepatic triglyceride and total cholesterol levels (7-15% and 25-31%, respectively), when compared with the drug-untreated and cholesterol/bile salt-treated group. Bicyclol treatment for 7 days decreased hepatic triglyceride (5-76%) and total cholesterol (5-48%) levels in mice fed with high-fat/cholesterol diet. In contrast, bicyclol treatment increased the serum triglyceride level (18-77%) in mice treated with cholesterol/bile salt or fed with high-fat/cholesterol diet. Bicyclol treatment also caused an increase in hepatic index of normal and hypercholesterolaemic mice (3-32%). The results indicate that bicyclol treatment can invariably decrease hepatic lipid levels and increase serum triglyceride levels in normal and hypercholesterolaemic mice.     

The effect of etofibrate on cholesterol and bile acid metabolism in the hamster

Hamsters were given etofibrate at a dose of 300 mg/kg body wt, by gavage for 5 days, while being fed a chow diet. After treatment, serum cholesterol levels were 27% lower compared to those of the control animals. A similar trend was observed for triglyceride levels. Hepatic lipid levels were unchanged by the treatment. HMG-CoA reductase and acylCoA cholesterol acyltransferase were decreased while cholesterol 7 alpha-hydroxylase was not significantly modified by etofibrate. A choleretic effect and an increase of cholesterol excretion into hepatic bile was observed in treated animals. Nevertheless, composition and cholesterol saturation index of gallbladder bile were similar in control and treated animals. With respect to controls, hepatic bile of treated hamsters contained a lesser amount of cholic and deoxycholic acid and a greater amount of ursodeoxycholic acid.     

Hypolipidemic effect of NK-104 and other 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors in guinea pigs

Hypolipidemic effects of various HMG-CoA reductase inhibitors (statins) were examined in guinea pigs. After 2-week administration of NK-104 ((+)-monocalcium bis((3R,5S,6E)-7-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl]-3, 5-dihydroxy-6-heptenoate), CAS 147526-32-7), simvastatin (SV), pravastatin (PV), fluvastatin (FV), cerivastatin (CV) and atorvastatin (AV) to guinea pigs at various doses, which did not affect body weight, plasma and liver lipids were measured. Liver endoplasmic reticulum (ER) was isolated and measured for lipid content and activity of ER enzymes (NK-104: 3 mg/kg, SV: 30 mg/kg). Each statin except CV dose-dependently lowered total cholesterol (TC), with NK-104 showing the greatest effect. Only NK-104 and AV, long-acting statins, significantly lowered triglycerides (TG). A significant reduction of the liver cholesterol content was observed for NK-104 and AV. The liver TG content increased with dose for SV or PV, but not for NK-104 or AV. The TG content in ER and activity of diacylglycerol acyltransferase of ER, increased with SV and slightly with NK-104. Activity of microsomal triglyceride transfer protein, which is necessary for both apoB particle formation at rough ER and TG droplet production at smooth ER, increased by SV but not by NK-104. It is suggested that statins with prolonged action lower TG by decreasing cholesterol ester supply necessary for very low density lipoprotein formation and by attenuating an increase in TG production at ER after HMG-CoA reductase inhibition.     

RPR 101821, a new potent cholesterol-lowering agent: inhibition of squalene synthase and 7-dehydrocholesterol reductase

RPR 101821 (trans-2-[4-(benzoxazol-2-yl)phenylmethoxy] amino cyclohexane hydrochloride) is a potent cholesterol-lowering agent in rodents and marmoset. The compound inhibited rat liver microsomal squalene synthase (IC₅₀ = 1 nM) and 7-dehydrocholesterol (7DHC) reductase (IC₅₀ = 1 M; Lewis et al. 1995). When RPR 101821 (10 mg/kg), the 7DHC reductase inhibitor BM 15.766 (4[2-[4-(4-chlorocinnamyl)piperazine-1-yl]ethyl] benzoic acid; 10 mg/kg) or the HMG-CoA reductase inhibitor lovastatin (30 mg/kg) was given orally to rats at –29 h, – 21 h and – 5 h, serum cholesterol was reduced by 56%, 46% or 15%, respectively. The reduction in cholesterol with RPR 101821 was associated with an accumulation of 7DHC in serum, suggesting an inhibition of 7DHC reductase. In the presence of BM 15.766, RPR 101821 reduced the serum accumulation of 7DHC in a dose-dependent manner, with complete inhibition at 30 mg/kg, p.o. In Balb-cJ mice, RPR 101821 and lovastatin (50 mg/kg, b.i.d., p.o., for 14 days) lowered serum cholesterol by 67% and 2%, respectively. In marmosets, RPR 101821 and lovastatin (both at a dose of 10 mg/kg, p.o., b.i.d., for 7 days) reduced cholesterol by 28% and 19%, respectively.In summary, RPR 101821 is an orally effective potent cholesterol-lowering agent in rodents and a small primate species. The suggested mechanism of hypocholesterolemic effect is the inhibition of squalene synthase and 7DHC reductase.     

Biochemical changes and morphological alterations of the liver in guinea-pigs after administration of simvastatin (HMG CoA reductase-inhibitor)

Simvastatin is a potent competitive inhibitor of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) which is the rate-limiting enzyme of cholesterol synthesis. In guinea-pigs, administration of a high oral dose of simvastatin (125 mg/kg/day at the beginning of the study) during 18 days had a major hepatotoxic effect whereas a lower oral dose (30 mg/kg/day) did not seem to cause any liver damage. A significant reduction in microsomal Cyt P 450 content was only observed on a high dose of simvastatin whereas HMG CoA reductase activity was reduced in the group with the low simvastatin dose. The hepatic microsomal aminopyrine N-demethylase activity remained unchanged in all groups. The liver lesion was hepatocellular necrosis accompanied in some animals by a biliary duct proliferation. It was associated with a 10-fold elevation in serum aspartate and alanine aminotransferase activities, as well as a great reduction in daily food intake and body weight (28%). The hepatotoxicity of simvastatin could result from the low basal content of HMG-CoA reductase in guinea-pig liver, the prolonged inhibition of mevalonate synthesis and probably, from the absence of HMG-CoA reductase enzyme de novo synthesis.     

Effect of the hypocholesterolemic agent YM-16638 on cholesterol biosynthesis activity and apolipoprotein B secretion in HepG2 and monkey liver

YM-16638 ([[5-[[3-(4-acetyl-3-hydroxy-2-propylphenoxy)propyl]thio]-1,3,4-++ +thiadiazol-2-yl] thio] acetic acid) showed a strong hypocholesterolemic effect in humans and monkeys. To clarify the mechanism of this hypocholesterolemic effect, the action of YM-16638 on cholesterol biosynthesis in the cultured human hepatoma cell line HepG2 and cynomolgus monkey liver was examined. Cholesterol biosynthesis activity derived from [14C]acetic acid, [3H/14C]mevalonic acid or [14C]isopentenyl pyrophosphate substrates was significantly decreased, but not that from [3H]farnesyl pyrophosphate or [3H]squalene substrates in HepG2 cells treated with YM-16638. Simultaneously, treatment of these cells with YM-16638 changed neither the rate of apolipoprotein B synthesis from [35S]methionine nor its secretion. In addition, the activities of hepatic cholesterol biosynthesis enzymes HMG-CoA reductase, mevalonate kinase (MK), isopentenyl pyrophosphate isomerase (IPPI), farnesyl pyrophosphate synthase (FPPS), squalene synthase and squalene epoxidase were measured in monkeys fed a diet supplemented with YM-16638. Among these enzymes, MK, IPPI and FPPS activities in the YM-16638-treated group significantly decreased by 38%, 56% and 30%, respectively, when compared to those from control animals receiving no drug treatment. These results indicate that YM-16638 has the characteristics of a cholesterol biosynthesis inhibitor.     

Lifibrol increases hepatic cholesterol 7α-hydroxylase activity in sprague-dawley rats

The hypocholesterolemic drug lifibrol was administered orally to Sprague-Dawley rats to determine its effects on lipoprotein cholesterol distribution and hepatic HMG-CoA reductase and cholesterol 7α-hydroxylase activities. The effects of lifibrol on those endpoints were compared with the effects of gemfibrozil and lovastatin. When administered to either chow-fed or cholesterol-fed rats, lifibrol (25 or 50 mg/kg/day) caused a redistribution of lipoprotein cholesterol such that HDL increased and VLDL + LDL decreased significantly. Gemfibrozil (30 or 50 mg/kg/day) caused similar changes in lipoprotein cholesterol distribution. In contrast, lovastatin (10 mg/kg/day) decreased both HDL and VLDL + LDL in chow-fed animals, but had no effect in cholesterol-fed animals. Hepatic HMG-CoA reductase activity was increased in rats treated with lifibrol (50 mg/kg/day), gemfibrozil (50 mg/kg/day), and lovastatin (10 mg/kg/day). The most significant finding is that lifibrol was the only drug that increased hepatic cholesterol 7α-hydroxylase activity. This observation in rats separates the mechanism of action of lifibrol from those of the HMG-CoA reductase inhibitors and the fibrates, and suggests that one mechanism of action of lifibrol is to enhance cholesterol elimination through conversion to bile acids. © 1994 Wiley-Liss, Inc.     

Pharmacokinetic interaction between propranolol and the HMG-CoA reductase inhibitors pravastatin and lovastatin.

1. Single oral 20 mg doses of the HMG-CoA reductase inhibitors pravastatin and lovastatin, with and without concomitant propranolol (40 mg twice daily), were administered to 16 healthy male subjects participating in a randomized, four-way crossover study. 2. Serum concentrations of total and active inhibitors were measured by bioassay and concentrations of pravastatin, two pravastatin metabolites and lovastatin acid were measured by gas chromatography/mass spectrometry. 3. Coadministration of propranolol with pravastatin reduced the mean area under the serum concentration-time curve (AUC) of total inhibitors by 23%, of active inhibitors by 20% and of pravastatin by 16%. 4. Coadministration of propranolol with lovastatin also resulted in decreases in the mean serum AUC of total inhibitors by 18%, of active inhibitors by 12% and of lovastatin acid by 13%. 5. These decreases in systemic drug concentrations may reflect enhanced drug first-pass hepatic clearance in the presence of propranolol. 6. The clinical significance of these changes is likely to be small.     

Comparison of the antiatherosclerotic effects of dihydropyridine calcium channel blocker and HMG-CoA reductase inhibitor on hypercholesterolemic rabbits

The antiatherosclerotic effects of the dihydropyridine-type calcium channel blocker, benidipine hydrochloride (benidipine), and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, pravastatin sodium (pravastatin), were compared in hypercholesterolemic rabbits. Male, New Zealand white rabbits were fed a 0.5% cholesterol diet. Pravastatin (10 mg/kg) or benidipine (10 mg/kg) was orally administered once daily after start of feeding. After 8 weeks of cholesterol feeding, serum cholesterol was increased and endothelial function of thoracic aorta was impaired. Pravastatin prevented elevation of serum cholesterol and aortic tunica intima hyperplasia. Although benidipine had little effect on serum cholesterol, it significantly inhibited aortic tunica intima hyperplasia and impairment of endothelial function. Expression levels of the vascular cell adhesion molecule-1 (VCAM-1) and lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) mRNA in aorta of hypercholesterolemic rabbit were higher than those of normal rabbit. Benidipine significantly prevented upregulation of VCAM-1 mRNA expression and showed a tendency to inhibit elevation of LOX-1 mRNA expression. Pravastatin significantly prevented upregulation of both VCAM-1 and LOX-1 mRNA expression. The results demonstrate that pravastatin inhibits increase of serum cholesterol and vascular dysfunction in hypercholesterolemic rabbit. Benidipine is effective in preventing vascular hyperplasia without altering serum cholesterol levels and this may be due to inhibition of expression of VCAM-1.     

Comparison of the effects of D-003, a mixture of high-molecular-weight aliphatic acids from sugarcane wax, and pravastatin on bones and osteoclast apoptosis of ovariectomized rats

The mevalonate pathway is relevant in bone cells. Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key enzyme of this route, stimulating osteoblast differentiation and activity. Pravastatin increases bone formation markers in postmenopausal women and bone density in diabetics. D-003 is a mixture of high-molecular-weight acids purified from sugarcane wax which inhibits cholesterol biosynthesis through HMG-CoA reductase regulation, preventing bone loss in osteoporosis induced with ovariectomy and prednisolone in rats. We investigated the effects of D-003 (50 mg/kg) and pravastatin (20 mg/kg) orally administered for 12 weeks to ovariectomized rats. Female rats were randomized in four groups (10 rats/group): two control groups treated with the vehicle, one false-operated (sham) and another ovariectomized (positive control), while two other groups received D-003 or pravastatin. Bone resorption and formation was studied through histomorphometry and apoptosis through immunohistochemistry. D-003 and pravastatin significantly (p < 0.001) prevented the changes of trabecular bone versus ovariectomized rats and (p < 0.001) the increase of the surface and number of osteoclasts versus ovariectomized controls. D-003 and pravastatin, however, did not modify osteoblast surfaces, a bone formation marker D-003 and pravastatin increased osteoclast apoptosis and reduced (p < 0.05) osteoblast and osteocyte apoptosis versus ovariectomized controls; D-003 was more effective (p < 0.05) than pravastatin. In conclusion, D-003 (50 mg/kg) orally administered for 12 weeks prevented bone loss and bone resorption in ovariectomized rats, increasing osteoclast apoptosis. The preventive effects of D-003 on bone loss and resorption in ovariectomized rats are comparable to those of pravastatin. Both drugs inhibited osteoblast apoptosis but failed to change osteoblast surface. The effects of D-003 on bone cell apoptosis were greater than those of pravastatin. Therefore, D-003 could be used to prevent or treat bone loss in postmenopausal women, but further animal studies and clinical trials are required to confirm the clinical relevance of this potential effect.     

Schisandrin B from Schisandra chinensis reduces hepatic lipid contents in hypercholesterolaemic mice

The effects of schisandrin B (Sch B) on liver and serum lipid contents were investigated in mice with experimentally-induced hypercholesterolaemia. Hypercholesterolaemia was induced either by oral administration of a cholesterol/bile salts mixture (2/0.5 g kg(-1)) for four days or by feeding a high fat/cholesterol/bile salts (10/1/0.3%, w/w) diet for seven days. Daily co-administration of Sch B (50-200 mg kg(-1), i.g.) for four or six days, respectively, decreased hepatic total cholesterol (TC) and triglyceride (TG) levels (by up to 50% and 52%, respectively) in hypercholesterolaemic mice. Sch B treatment also increased hepatic indices (14-84%) in hypercholesterolaemic mice. The results indicated that Sch B treatment could decrease hepatic TC and TG levels, and increase liver weight, in mouse models of hypercholesterolaemia. Fenofibrate treatment (100 mg kg(-1)) produced effects similar to those of Sch B on the hepatic index and lipid levels of hypercholesterolaemic mice.     

Cholesterol-lowering effect of NK-104, a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, in guinea pig model of hyperlipidemia

Although benefits of statins have been demonstrated even in normolipidemic patients at high risk, the main target of statin therapy is the hypercholesterolemic patient. The aim of this study was to examine the hypocholesterolemic effect of NK-104 ((+)-monocalcium bis((3R,5S,6S)-7-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl]- 3,5-dihydroxy-6-heptenoate), CAS 147526-32-7), a potent 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, and its mechanism of action in hypercholesterolemic animals. In guinea pigs fed a diet containing 15% (w/w) fat rich in laurate for 6 weeks, the liver cholesterol content was markedly increased and plasma total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and LDL-apoB were elevated 4.8, 5.2 and 1.7 times, respectively, compared with normal diet fed animals. These changes were maintained by reduced LDL clearance in the presence of markedly cholesterol-enriched LDL in the plasma. In this model, the LDL-C reduction rates by 0.1, 0.3 and 1 mg/kg of NK-104 orally administered for 2 weeks (from week 4 to week 6), were 11, 27 and 32%, respectively, from controls, being similar in normal guinea pigs previously examined. Those for 3 and 10 mg/kg of atorvastatin (CAS 134523-00-5) were 25 and 39%, respectively. Thus about 10 times higher doses of atorvastatin were required than of NK-104 to cause a similar cholesterol-lowering effect. This reduction of plasma cholesterol was accompanied by an improvement of LDL clearance (24 and 47% increase in fractional catabolic rate by 1 mg/kg of NK-104 and 10 mg/kg of atorvastatin, respectively) and LDL composition. In conclusion, in guinea pig hypercholesterolemia caused by high-laurate diet, NK-104 and atorvastatin lowered plasma cholesterol levels with an improvement of LDL composition and with an increase in LDL clearance, presumably through reduction of the liver cholesterol content, although hepatic cholesterol synthesis might have been markedly suppressed in this model.     

Diethyl (4baalpha,4calpha,9aalpha,9balpha)-3,6-dichlorocyclobuta[1,2-b:3,4-b']bisbenzofuran-9a,9b(4bH,4cH)-dicarboxylate: the cis,syn photodimer of ethyl 5-chlorobenzofuran-2-carboxylate, an analogue related to the antilipidemic drug clofibrate.

The antilipidemic properties of diethyl (4balpha,4calpha,9aalpha,4balpha)-3,6-dichlorocyclobutal[1,2-b:3,4-b']bisbenzofuran-9a,9b(4bH,4cH)-dicarboxylate, herein termed dimer 8, were studied in sucrose-fed and in Triton-induced hyperlipidemic rats. Whereas clofibrate (0.4 mmol/kg) exhibited both anticholesterolemic and antitriglyceridemic activity, dimer 8 showed only antitriglyceridemic properties at the lower dose (0.2 mmol/kg) in sucrose-fed rats. Dimer 8 only lowered serum triglycerides levels in Triton WR-1339 hyperlipidemic rats, whereas clofibrate lowered both cholesterol and triglyceride levels. In the chronic sucrose-fed model, dimer 8 and clofibrate lowered hepatic HMG-CoA reducatase activity and produced significant elevations in several parameters of hepatic drug metabolism. No positive relationship between serum cholesterol lowering and reduction of hepatic HMG-CoA reductase activity was observed by these agents in sucrose-fed rats.     

Are all HMG-CoA reductase inhibitors protective against ischemic heart disease?

Effects of pravastatin, simvastatin, atorvastatin, fluvastatin and cerivastatin on myocardial contractile dysfunction during reperfusion after brief ischemia were examined in dogs. Pretreatment of the dog with lipophilic HMG-CoA reductase inhibitors for 3 weeks, simvastatin (2 mg/kg/day), atorvastatin (2 mg/kg/day), fluvastatin (4 mg/kg/day), and cerivastatin (40 micrograms/kg/day) worsened recovery of myocardial contraction during reperfusion after brief ischemia in association with reduced myocardial ATP level. A hydrophilic HMG-CoA reductase inhibitor, pravastatin (2 and 4 mg/kg/day), did not affect the recovery of myocardial contractile function and ATP level during reperfusion following ischemia. The lipophilic inhibitors may enter the myocardial cell, inhibit ubiquinone biosynthesis, and depress ATP generation in mitochondria, leading to worsening of the myocardial stunning after reperfusion subsequent to ischemia.     

Antioxidative potential of fluvastatin via the inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity

We previously reported that fluvastatin, a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, a strong lipid lowering drug, exerted an anti-atherosclerotic effect at doses insufficient to lower serum lipids in cholesterol fed rabbits. The evidence demonstrated that the superoxide anions from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays a critical role in several steps in the development of atherosclerosis. This study was designed to determine the effects of HMG-CoA reductase inhibitors on the production of the superoxide anions of NADPH oxidase in isolated rat peritoneal neutrophils. Fluvastatin (1-10 microM) decreased phorbol 12-myristate 13-acetate (PMA, 10 nM)-dependent reactive oxygen species (ROS) generation in a concentration-dependent manner. It also (10 microM) decreased PMA-dependent O(2) consumption of the rat neutrophils. These effects were reversed by the addition of mevalonate, a metabolite in the HMG-CoA reductase pathway. Treatment with pravastatin did not show any significant changes. Fluvastatin (10 microM) decreased ROS, such as hydroxyl radicals and superoxide anions generated by the Fenton reaction, and by the xanthine-xanthine oxidase system. Rats were treated with either fluvastatin (5 mg/kg per day, p.o.) or pravastatin (5 mg/kg per day, p.o.) for 1 week. Treatment with fluvastatin decreased the PMA-dependent ROS generation. The fluvastatin induced effect on the PMA-dependent ROS generation was reversed by the combined administration with 40 mg/kg mevalonate per day. The antioxidative effect of fluvastatin was thought to have caused not only the scavenging action of the radicals but also to have inhibited ROS generation by inhibiting the NADPH oxidase activity. This antioxidative potential of fluvastatin via the inhibition of NADPH oxidase activity may be profitable in preventing atherosclerosis.