Syntheses and pharmacokinetic evaluations of four metabolites of 2-(4-(2-((1H-benzo[d]imidazol-2-yl)thio)ethyl)piperazin-1-yl)-N-(6-methyl-2,4-bis-(methylthio)pyridin-3-yl)acetamide hydrochloride [K-604], an acyl-CoA:cholesterol O-acyltransferase-1 inhibitor.

We synthesized and identified four metabolites of acyl-coenzyme A:cholesterol O-acyltransferase (ACAT)-1 inhibitor, K-604 (1). Two of the metabolites M1 and M2, were prepared from 1 using a combination reagent of hydrogen peroxide and sodium tungstate with either phosphoric acid or trifluoroethanol as the solvent to control the regioselectivity. Upon exposure of 4b to tert-butyl hypochlorite at -78 °C, the monosulfoxidation afforded synthetic intermediate of M3 in excellent yield. The efficient synthesis of M4 was established. The in vitro metabolic study exhibited a high clearance value (720 µL/min/mg protein) of 1 using human liver microsomes. We orally administered a single dose of 10 mg/kg of 1 to monkeys because the in vitro metabolic patterns are quite similar. Fortunately, the drug concentration of 1 was much higher than those of M1, M2, M3 and M4.

Prediction of circulating human metabolites of pemafibrate, a novel antidyslipidemic drug, using chimeric mice with humanized liver.

Pharmacokinetics and metabolism of recently launched antidyslipidemic drug pemafibrate ((2R)-2-[3-({1,3-benzoxazol-2-yl[3-(4-methoxyphenoxy)propyl]amino}methyl)phenoxy]butanoic acid) was investigated in chimeric mice with humanized liver in the present study.The plasma unbound fractions of [14C]pemafibrate in mice (0.0046-0.0048) were higher than those in monkeys and humans (0.0015-0.0022).In chimeric mice with humanized liver intravenously treated with pemafibrate at 1.0 mg/kg body weight, the pharmacokinetic parameters (CLtotal, Vss and AUC0-inf) of unbound pemafibrate in chimeric mice with humanized liver were more similar to those reported in monkeys and humans than those in control mice.High concentrations of N-dealkylated form (M4) and benzoxazole 6-hydroxylated form (M6) of pemafibrate in plasma were observed as the main circulating metabolites in chimeric mice with humanized liver treated with pemafibrate. Moreover, the concentrations of other specified metabolites of pemafibrate were much higher in chimeric mice with humanized liver than in control mice.These results suggest that there are species differences in the pharmacokinetics of pemafibrate in vivo between mice tested and humans reported. Moreover, chimeric mice with humanized liver seem to be a beneficial animal model for further studies to predict the circulating human metabolites of pemafibrate and their pharmacokinetics.

Brain Targeting of Acyl-CoA:Cholesterol O-Acyltransferase-1 Inhibitor K-604 via the Intranasal Route Using a Hydroxycarboxylic Acid Solution.

An acyl-CoA:cholesterol O-acyltransferase-1 (ACAT-1/SOAT-1) inhibitor, K-604 is a promising drug candidate for the treatment of Alzheimer's disease and glioblastoma; however, it exhibits poor solubility in neutral water and low permeability across the blood-brain barrier. In this study, we report the successful delivery of K-604 to the brain via the intranasal route in mice using a hydroxycarboxylic acid solution. In cerebral tissue, the AUC of K-604 after intranasal administration (10 µL; 108 µg of K-604/mouse) was 772 ng·min/g, whereas that after oral administration (166 µg of K-604/mouse) was 8.9 ng·min/g. Thus, the index of brain-targeting efficiency was 133-fold based on the dose conversion. Even with intranasal administration of K-604 once per day for 7 days, the level of cholesteryl esters markedly decreased from 0.70 to 0.04 µmol/g in the mouse brain. Thus, this application will be a crucial therapeutic solution for ACAT-1 overexpressing diseases in the brain.

Pharmacokinetics and metabolism of pemafibrate, a novel selective peroxisome proliferator-activated receptor-alpha modulator, in rats and monkeys.

The metabolic profiles and pharmacokinetics of pemafibrate, a novel selective peroxisome proliferator activated receptor-alpha modulator currently launched as an antidyslipidemic drug, were investigated in vitro using hepatocytes from rats, monkeys and humans and in vivo in rats and monkeys. Hepatocytes from rats, monkeys and humans all biotransformed pemafibrate to its demethylated form (M1). The bioavailabilities of pemafibrate in Sprague-Dawley rats and cynomolgus monkeys were 15% and 87%, respectively, after a single oral administration of pemafibrate (1 mg/kg). In rat plasma, unmetabolized pemafibrate was the major form, accounting for 29% of the area under the curve (AUC) of total radioactivity. In monkey plasma, in contrast, the major circulating metabolites were M2/3 (dearylated/dicarboxylic acid forms, 15%), M4 (N-dealkylated form, 21%) and M5 (benzylic oxidative form, 9%), but pemafibrate was the notable minor form (3%). These results, in combination with the reported findings in humans, suggest that the metabolite profile of pemafibrate in plasma was different for rats and monkeys, and that monkeys could be a suitable animal model for further pharmacokinetic studies of pemafibrate in humans.

Discovery of Clinical Candidate 2-(4-(2-((1 H-Benzo[ d]imidazol-2-yl)thio)ethyl)piperazin-1-yl)- N-(6-methyl-2,4-bis(methylthio)pyridin-3-yl)acetamide Hydrochloride [K-604], an Aqueous-Soluble Acyl-CoA:Cholesterol O-Acyltransferase-1 Inhibitor.

2-(4-(2-((1 H-Benzo[ d]imidazol-2-yl)thio)ethyl)piperazin-1-yl)- N-(6-methyl-2,4-bis(methylthio)pyridin-3-yl)acetamide hydrochloride (K-604, 2) has been identified as an aqueous-soluble potent inhibitor of human acyl-coenzyme A:cholesterol O-acyltransferase (ACAT, also known as SOAT)-1 that exhibits 229-fold selectivity for human ACAT-1 over human ACAT-2. In our molecular design, the insertion of a piperazine unit in place of a 6-methylene chain in the linker between the head (pyridylacetamide) and tail (benzimidazole) moieties led to a marked enhancement of the aqueous solubility (up to 19 mg/mL at pH 1.2) and a significant improvement of the oral absorption (the Cmax of 2 was 1100-fold higher than that of 1 in fasted dogs) compared with those of the previously selected compound, 1. After ensuring the pharmacological effects and safety, we designated 2 as a clinical candidate, named K-604. Considering the therapeutic results of ACAT inhibitors in past clinical trials, we believe that K-604 will be useful for the treatment of incurable diseases involving ACAT-1 overexpression.

Design, synthesis and pharmacology of aortic-selective acyl-CoA: Cholesterol O-acyltransferase (ACAT/SOAT) inhibitors.

We describe our molecular design of aortic-selective acyl-coenzyme A:cholesterol O-acyltransferase (ACAT, also abbreviated as SOAT) inhibitors, their structure-activity relationships (SARs) and their pharmacokinetic (PK) and pharmacological profiles. The connection of two weak ligands-N-(2,6-diisopropylphenyl)acetamide (50% inhibitory concentration [IC50] = 8.6 µM) and 2-(methylthio)benzo[d]oxazole (IC50 = 31 µM)-via a linker comprising a 6 methylene group chains yielded a highly potent molecule, 9-(benzo[d]oxazol-2-ylthio)-N-(2,6-diisopropylphenyl)nonanamide (3h) that exhibited high potency (IC50 = 0.004 µM) toward aortic ACAT. This head-to-tail design made it possible to markedly enhance the activity to 2150- to 7750-fold and to discriminate the isoform-selectivity based on the double-induced fit mechanism. At doses of 1 and 3 mg/kg, 3h significantly decreased the lipid-accumulation areas in the aortic arch to 74 and 69%, respectively without reducing the plasma total cholesterol level in high fat- and cholesterol-fed F1B hamsters. Here, we demonstrate the antiatherosclerotic effect of 3hin vivo via its direct action on aortic ACAT and its powerful modulator of cholesterol level. This molecule is a potential therapeutic agent for the treatment of diseases involving ACAT-1 overexpression.

Effect of gemfibrozil on the metabolism of pitavastatin--determining the best animal model for human CYP and UGT activities.

A series of studies was conducted to determine the best animal model for human CYP and UGT activities. The investigation focused primarily on the interactions occurring in the CYP- or UGT-mediated metabolism of pitavastatin, and involved in vitro and in vivo experiments. We found that the best animal models for human CYP-mediated hydroxylation and UGT-mediated lactonization of pitavastatin were rats and dogs, respectively. In addition, a large difference in the metabolic properties of pitavastatin was found between monkeys and humans. In the presence of gemfibrozil, the CYP- or UGT-mediated metabolism of pitavastatin was inhibited in vitro. However, gemfibrozil treatment had no inhibitory effect on the AUC of pitavastatin and its lactone form in rats and dogs. We conclude that the plasma level of pitavastatin would not be increased by co-administration of gemfibrozil in humans.

Interaction between fibrates and statins--metabolic interactions with gemfibrozil.

An in vitro study was carried out in order to examine the metabolic basis of the interaction between fibrates and statins. Metabolic inhibition of statins was noted in the presence of gemfibrozil. However, increase in the unchanged form was fairly small for pitavastatin, compared with other statins. Several CYP enzymes were shown to be principally responsible for the metabolism of gemfibrozil in contrast to other fibrates. In the presence of gemfibrozil, a focal point was obtained in Dixon plots, demonstrating that there was inhibition of CYP2C8-, CYP2C9- and CYP3A4-mediated metabolism. We propose that the increase of plasma concentration caused by co-administration of gemfibrozil and statins is at least partially due to CYP-mediated inhibition.

Studies on the interaction between fibrates and statins using human hepatic microsomes.

To gain a better understanding of the mechanism of drug-drug interaction between fibrates and statins, several in vitro experiments were performed. On coincubation with several fibrates, pitavastatin (CAS 147526-32-7) did not displace fibrates from their protein binding in human plasma. The presence of gemfibrozil (CAS 25812-30-0) inhibited the metabolism of statins (cerivastatin (CAS 145599-86-6) and atorvastatin (CAS 134523-00-5)) remarkably. However, the increase of the unchanged form was fairly small for pitavastatin. The metabolic profile of gemfibrozil was also investigated. The cytochrome P (CYP) enzyme CYP2C9 plays a major role in the metabolism of gemfibrozil. Gemfibrozil showed a high affinity for CYP enzymes and a relatively high metabolism velocity. Moreover, several inhibitory effects of gemfibrozil on CYP-mediated metabolism were detected--in contrast to other fibrates. Although the mechanism of the drug-drug interaction was not completely clarified, it is suggested that the increase of plasma concentration caused by the co-administration of gemfibrozil and statins is at least partially due to the inhibition of the CYP-mediated metabolism.

Interaction between several medicines and statins.

To gain a better understanding of drug-drug interaction between various medicinal substances and statins, in vitro experiments using human hepatic microsomes were performed. The metabolic clearance of atorvastatin (CAS 134523-00-5) was about 32 microliters/min/mg protein, some 15-fold greater than that of pitavastatin (CAS 147526-32-7). On co-incubation with several medicinal substances, metabolic inhibition of pitavastatin was negligible in human hepatic microsomes. However, a remarkable metabolic inhibition of atorvastatin was noted in the presence of various medicinal substances. The intrinsic clearance of atorvastatin lactone was 20-fold greater than that of its acid form, whereas no marked difference was noted between pitavastatin and its lactone form. Pitavastatin lactone showed no inhibitory effect on CYP3A4-mediated metabolism of testosterone in contrast to atorvastatin lactone. These results suggest that pitavastatin and its lactone form will be highly unlikely to interact with other drugs in clinical practice.