Yuichiro Amano

Lipid‐lowering properties of TAK‐475, a squalene synthase inhibitor, in vivo and in vitro

Squalene synthase is the enzyme that converts farnesyl pyrophosphate to squalene in the cholesterol biosynthesis pathway. We examined the lipid‐lowering properties of 1‐[[(3R,5S)‐1‐(3‐acetoxy‐2,2‐dimethylpropyl)‐7‐chloro‐5‐(2,3‐dimethoxyphenyl)‐2‐oxo‐1,2,3,5‐tetrahydro‐4,1‐benzoxazepin‐3‐yl]acetyl]piperidine‐4‐acetic acid (TAK‐475), a novel squalene synthase inhibitor. TAK‐475 inhibited hepatic cholesterol biosynthesis in rats (ED50, 2.9 mg kg−1) and showed lipid‐lowering effects in beagle dogs, marmosets, cynomolgus monkeys and Wistar fatty rats. In marmosets, TAK‐475 (30, 100 mg kg−1, p.o., for 4 days) lowered both plasma non‐high‐density lipoprotein (HDL) cholesterol and triglyceride, but did not affect plasma HDL cholesterol. On the other hand, atorvastatin (10, 30 mg kg−1, p.o., for 4 days) lowered the levels of all these lipids. A correlation between decrease in triglyceride and increase in HDL cholesterol was observed, and TAK‐475 increased HDL cholesterol with a smaller decrease in triglyceride than did atorvastatin. TAK‐475 (60 mg kg−1, p.o., for 15 days) suppressed the rate of triglyceride secretion from the liver in hypertriglyceridemic Wistar fatty rats, which show an enhanced triglyceride secretion rate from the liver compared with their lean littermates. In HepG2 cells, TAK‐475 and its pharmacologically active metabolite, T‐91485, increased the binding of 125I‐low‐density lipoprotein (LDL) to LDL receptors. 6 These results suggest that TAK‐475 has clear hypolipidemic effects in animals via inhibition of hepatic triglyceride secretion and upregulation of LDL receptors, and that TAK‐475 might increase HDL cholesterol by decreasing triglyceride. Thus, TAK‐475 is expected to be useful for the treatment of dyslipidemia.

Lipid-lowering effects of TAK-475, a squalene synthase inhibitor, in animal models of familial hypercholesterolemia.

The lipid-lowering effects of 1-[2-[(3R,5S)-1-(3-acetoxy-2,2-dimethylpropyl)-7-chloro-1,2,3,5-tetrahydro-2-oxo-5-(2,3-dimethoxyphenyl)-4,1-benzoxazepine-3-yl] acetyl] piperidin-4-acetic acid (TAK-475), a novel squalene synthase inhibitor, were examined in two models of familial hypercholesterolemia, low-density lipoprotein (LDL) receptor knockout mice and Watanabe heritable hyperlipidemic (WHHL) rabbits. Two weeks of treatment with TAK-475 in a diet admixture (0.02% and 0.07%; approximately 30 and 110 mg/kg/day, respectively) significantly lowered plasma non-high-density lipoprotein (HDL) cholesterol levels by 19% and 41%, respectively, in homozygous LDL receptor knockout mice. The 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, simvastatin and atorvastatin (in 0.02% and 0.07% admixtures), also reduced plasma levels of non-HDL cholesterol. In homozygous WHHL rabbits, 4 weeks of treatment with TAK-475 (0.27%; approximately 100 mg/kg/day) lowered plasma total cholesterol, triglyceride and phospholipid levels by 17%, 52% and 26%, respectively. In Triton WR-1339-treated rabbits, TAK-475 inhibited to the same extent the rate of secretion from the liver of the cholesterol, triglyceride and phospholipid components of very-low-density lipoprotein (VLDL). These results suggest that the lipid-lowering effects of TAK-475 in WHHL rabbits are based partially on the inhibition of secretion of VLDL from the liver. TAK-475 had no effect on plasma aspartate aminotransferase and alanine aminotransferase activities. Thus, the squalene synthase inhibitor TAK-475 revealed lipid-lowering effects in both LDL receptor knockout mice and WHHL rabbits.

Lapaquistat acetate, a squalene synthase inhibitor, changes macrophage/lipid‐rich coronary plaques of hypercholesterolaemic rabbits into fibrous lesions

Inhibition of squalene synthesis could transform unstable, macrophage/lipid‐rich coronary plaques into stable, fibromuscular plaques. We have here treated WHHLMI rabbits, a model for coronary atherosclerosis and myocardial infarction, with a novel squalene synthase inhibitor, lapaquistat acetate (TAK‐475).

Effects of a farnesoid X receptor antagonist on hepatic lipid metabolism in primates.

We aimed to elucidate the mechanism underlying the anti-dyslipidemic effect of compound-T3, a farnesoid X receptor antagonist, by investigating its effects on hepatic lipid metabolism in non-human primates. We administered lipid-lowering drugs for 7 days to cynomolgus monkeys receiving a high-fat diet, and subsequently measured the levels of lipid parameters in plasma, feces, and hepatic tissue fluids. Compound-T3 (0.3 and 3mg/kg p.o.) significantly decreased the plasma levels of non-high-density lipoprotein (non-HDL) cholesterol and apolipoprotein B in a dose-dependent manner. It also decreased the mRNA levels of hepatic small heterodimer partner-1, induced the mRNA expression of hepatic cholesterol 7α-hydroxylase, reduced hepatic cholesterol and triglyceride levels, increased fecal bile acid excretion, and upregulated the expression of hepatic low-density lipoprotein (LDL) receptor. Furthermore, compound-T3 significantly increased plasma HDL cholesterol and apolipoprotein A-I levels. The mRNA expression levels of hepatic apolipoprotein A-I tended to increase after compound-T3 treatment. Compound-T3 also induced accumulation of hepatic bile acids and decreased the mRNA expression levels of the hepatic bile acid export pump. The effects of cholestyramine (300mg/kg p.o.) on the plasma and hepatic lipid parameters were similar to those of compound-T3, and it increased fecal bile acid levels without causing accumulation of hepatic bile acids. These findings suggest that LDL receptor-mediated hepatic LDL incorporation due to cholesterol catabolism catalyzed by cholesterol 7α-hydroxylase decreases plasma non-HDL cholesterol levels. Upregulation of hepatic apolipoprotein A-I mRNA expression may partially contribute to the increase in HDL cholesterol levels mediated by compound-T3.

Antidyslipidemic effects of a farnesoid X receptor antagonist in primates

AIMS We investigated antidyslipidemic effects of a farnesoid X receptor antagonist compound-T3 in non-human primates as a novel treatment approach for dyslipidemia. MAIN METHODS Cynomolgus monkeys were fed a high-fat diet over 3 weeks. Drugs were administered to the monkeys for a week, and their plasma and fecal lipid parameters were measured. KEY FINDINGS Compound-T3 dose-dependently decreased the plasma non-high-density lipoprotein (non-HDL) cholesterol and apolipoprotein B levels in high-fat diet-fed cynomolgus monkeys. The plasma levels of 7α-hydroxy-4-cholesten-3-one, a marker of hepatic cholesterol 7α-hydroxylase activity, and total fecal bile acid levels increased, suggesting that the hypocholesterolemic effects would be dependent on the activation of cholesterol catabolism in the liver. Compound-T3 significantly increased the plasma levels of HDL cholesterol and apolipoprotein A-I. In this condition, the cholesterol absorption inhibitor ezetimibe significantly decreased the plasma non-HDL cholesterol levels and increased the fecal cholesterol levels without affecting plasma HDL cholesterol and triglyceride levels. Bile acid sequestrant cholestyramine tended to decrease plasma non-HDL cholesterol and increase fecal bile acid levels. The cholesteryl ester transfer protein inhibitor torcetrapib significantly increased plasma HDL cholesterol levels without affecting plasma non-HDL cholesterol and fecal cholesterol levels. SIGNIFICANCE The results of ezetimibe, cholestyramine, and torcetrapib treatments indicate that our high-fat diet fed monkey model would be a preferred animal model for studying non-statin type antidyslipidemic drugs. Compound-T3 significantly decreased non-HDL cholesterol levels and increased HDL cholesterol levels in the monkey model, suggesting that a farnesoid X receptor antagonist could be a therapeutic option in human dyslipidemia.

Pharmacological evaluation of pioglitazone and candesartan cilexetil in a novel mouse model of non-alcoholic steatohepatitis, modified choline-deficient, amino acid-defined diet fed low-density lipoprotein receptor knockout mice

Low‐density lipoprotein receptor knockout (LDLR‐KO) mice fed a modified choline‐deficient and amino acid‐defined (mCDAA) diet show non‐alcoholic steatohepatitis (NASH)‐like pathophysiology. In order to pharmacologically benchmark this model, effects of pioglitazone (a thiazolidinedione) and candesartan cilexetil (an angiotensin II type 1 receptor blocker) on steatosis and liver fibrosis were examined.

Non-alcoholic steatohepatitis-associated hepatic fibrosis and hepatocellular carcinoma in a combined mouse model of genetic modification and dietary challenge

Experimental models of non‐alcoholic steatohepatitis (NASH) are still required for understanding the pathophysiology of this disease. This study aimed to examine whether disease progression is accelerated by combining dyslipidemic genetic modification and dietary challenges and develop NASH‐associated hepatic fibrosis, cirrhosis, and carcinoma in a short period.

Amelioration of sexual behavior and motor activity deficits in a castrated rodent model with a selective androgen receptor modulator SARM-2f

Sarcopenia and cachexia present characteristic features of a decrease in skeletal muscle mass and strength, anorexia, and lack of motivation. Treatments for these diseases have not yet been established, although selective androgen receptor modulators (SARMs) are considered as therapeutic targets. We previously reported that a novel SARM compound, SARM-2f, exhibits anabolic effect on muscles, with less stimulatory effect on prostate weight compared with testosterone, in rat Hershberger assays and cancer cachexia models. In this study, we studied the mechanism of action for SARM-2f selectivity and also assessed whether the muscle increase by this compound might lead to improvement of muscle function and physical activity. First, we examined the tissue distribution of SARM-2f. Tissue concentration was 1.2-, 1.6-, and 1.9-fold as high as the plasma concentration in the levator ani muscle, brain, and prostate, respectively. This result showed that the tissue-selective pharmacological effect did not depend on SARM-2f concentration in the tissues. The ability of SARM-2f to influence androgen receptor (AR)-mediated transcriptional activation was examined by reporter assays using human normal prostate epithelial cells (PrEC) and skeletal muscle cells (SKMC). SARM-2f exerted higher activity against AR in SKMC than in PrEC. Mammalian two hybrid assays showed different co-factor recruitment patterns between SARM-2f and dihydrotestosterone. Next, we studied the effect of SARM-2f on motivation and physical functions such as sexual behavior and motor activities in castrated rat or mouse models. SARM-2f restored the sexual behavior that was lost by castration in male rats. SARM-2f also increased voluntary running distance and locomotor activities. These results suggest that tissue-specific AR regulation by SARM-2f, but not tissue distribution, might account for its tissue specific androgenic effect, and that the muscle mass increase by SARM-2f leads to improvement of physical function. Together, these findings suggest that SARM-2f might represent an effective treatment for sarcopenia and cachexia.

Combinational effects of farnesoid X receptor antagonist and statin on plasma lipid levels and low-density lipoprotein clearance in guinea pigs.

AIMS We previously reported anti-dyslipidemic effects of a farnesoid X receptor antagonist in monkeys. In this study, we compared the cholesterol-lowering effects of single and combined administration of a farnesoid X receptor antagonist, compound-T8, and the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor atorvastatin in a guinea pig model. MAIN METHODS Plasma levels of 7α-hydroxy-4-cholesten-3-one, a marker of hepatic cholesterol 7α-hydroxylase activity, were measured after a single administration of compound-T8. The effects of compound-T8 or atorvastatin on plasma cholesterol levels and low-density lipoprotein (LDL) clearance were investigated after 14 or 16 days of repeated dosing, respectively. Fractional catabolic rate of plasma LDL was estimated by intravenous injection of DiI-labeled human LDL. The cholesterol-lowering effects of combination therapy were investigated after 7 days of repeated treatment. KEY FINDINGS Compound-T8 (10 and 30 mg/kg) increased plasma 7α-hydroxy-4-cholesten-3-one levels in a dose-dependent manner. Single administration of compound-T8 (30 mg/kg) and atorvastatin (30 mg/kg) reduced plasma non-high-density lipoprotein (non-HDL) cholesterol levels by 48% and 46%, respectively, and increased clearance of plasma DiI-labeled LDL by 29% and 35%, respectively. Compound-T8 (10mg/kg) or atorvastatin (10mg/kg) reduced non-HDL cholesterol levels by 19% and 25%, respectively, and combination therapy showed an additive effect and lowered cholesterol levels by 48%. SIGNIFICANCE Similar to atorvastatin, compound-T8 reduced plasma non-HDL cholesterol levels accompanied with accelerated LDL clearance in guinea pigs. Combination therapy additively decreased plasma non-HDL cholesterol levels. Therefore, monotherapy with a farnesoid X receptor antagonist and combination therapy of a farnesoid X receptor antagonist with atorvastatin would be attractive dyslipidemia treatment options.

Antidyslipidemic potential of a novel farnesoid X receptor antagonist in a hamster model of dyslipidemia: Comparative studies of other nonstatin agents

We attempted to clarify the therapeutic capability of antagonists of the farnesoid X receptor (FXR), a nuclear receptor that regulates lipid and bile acid metabolism. Herein, we report the antidyslipidemic effects of a novel synthesized FXR antagonist, compound‐T1, utilizing a dyslipidemic hamster model. Compound‐T1 selectively inhibited chenodeoxycholic acid‐induced FXR activation (IC50, 2.1 nmol·L−1). A hamster model of diet‐induced hyperlipidemia was prepared to investigate the antidyslipidemic effects of compound‐T1 through comparative studies of the nonstatin lipid‐modulating agents ezetimibe, cholestyramine, and torcetrapib. In the hamster model, compound‐T1 (6 mg·kg−1·day−1, p.o.) increased the level of plasma high‐density lipoprotein (HDL)‐cholesterol (+22.2%) and decreased the levels of plasma non‐HDL‐cholesterol (−43.6%) and triglycerides (−31.1%). Compound‐T1 also increased hepatic cholesterol 7α‐hydroxylase expression and fecal bile acid excretion, and decreased hepatic cholesterol content. Moreover, the hamster model could reflect clinical results of other nonstatin agents. Torcetrapib especially increased large HDL particles compared with compound‐T1. Additionally, in the human hepatoma Huh‐7 cells, compound‐T1 enhanced apolipoprotein A‐I secretion at a concentration close to its IC50 value for FXR. Our results indicated the usefulness of the hamster model in evaluating FXR antagonists and nonstatin agents. Notably, compound‐T1 exhibited beneficial effects on both blood non‐HDL‐cholesterol and HDL‐cholesterol, which are thought to involve enhancement of cholesterol catabolism and apolipoprotein A‐I production. These findings aid the understanding of the antidyslipidemic potential of FXR antagonists with a unique lipid and bile acid modulation.