Novel non-systemic inhibitor of ileal apical Na⁺-dependent bile acid transporter reduces serum cholesterol levels in hamsters and monkeys

Abstract

1-{7-[(1-(3,5-Diethoxyphenyl)-3-{[(3,5-difluorophenyl)(ethyl)amino]carbonyl}-4-oxo-1,4-dihydroquinolin-7-yl)oxy]heptyl}-1-methylpiperidinium bromide, R-146224, is a potent, specific ileum apical sodium-dependent bile acid transporter (ASBT) inhibitor; concentrations required for 50% inhibition of [³H]taurocholate uptake in human ASBT-expressing HEK-293 cells and hamster ileum tissues were 0.023 and 0.73 μM, respectively. In bile-fistula rats, biliary and urinary excretion 48 h after 10 mg/kg [¹⁴C]R-146224, were 1.49 ± 1.75% and 0.14 ± 0.05%, respectively, demonstrating extremely low absorption. In hamsters, R-146224 dose-dependently reduced gallbladder bile [³H]taurocholate uptake (ED₅₀: 2.8 mg/kg). In basal diet-fed hamsters, 14-day 30–100 mg/kg R-146224 dose-dependently reduced serum total cholesterol (∼ 40%), high density lipoprotein (HDL) cholesterol (∼ 37%), non-HDL cholesterols (∼ 20%), and phospholipids (∼ 20%), without affecting serum triglycerides, associated with reduced free and esterified liver cholesterol contents. In normocholesterolemic cynomolgus monkeys, R-146224 specifically reduced non-HDL cholesterol. In human ileum specimens, R-146224 dose-dependently inhibited [³H]taurocholate uptake. Potent non-systemic ASBT inhibitor R-146224 decreases bile acid reabsorption by inhibiting the ileal bile acid active transport system, resulting in hypolipidemic activity.

Introduction

Atherosclerosis is a disease of the vascular wall leading to myocardial infarction, heart failure, peripheral vascular disease, and stroke (Glass and Witztum, 2001). Although multiple risk factors have been identified contributing to atherosclerotic lesion formation, its growth is initiated and sustained by increased levels of low density lipoprotein (LDL), and low and/or dysfunctional high density lipoprotein (HDL). Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (statins) are currently the mainstay of dyslipidemia management for prevention of cardiovascular disease (Adult Treatment Panel III, 2001) with recent data indicating additional benefits of further lipid lowering at higher dosage (Cannon et al., 2004, Grundy et al., 2004, Heart Protection Study Collaborative Group, 2002, LaRosa et al., 2005, Mosca et al., 2004). Occasionally serious side effects occur, such as myopathy (rhabdomyolysis), which can cause acute renal failure. Statins also elevate serum transaminases in a small percentage of patients. Although little evidence suggests progression to liver disease (Tolman, 2002), persistent transaminase elevation is implicated. To limit such potential side effects, some clinicians may combine a lower dose of statin with a cholesterol absorption inhibitor such as ezetimibe (McKenney, 2005). The relative safety and efficacy of this and other strategies to lower LDL and increase HDL cholesterol levels, including fibrates, HDL cholesterol mimetics, nicotinic acid derivatives, and inhibitors of cholesterol ester transfer protein in combination with a low-dose statin, need further evaluation. Much effort is directed to developing agents acting via other hypocholesterolemic mechanisms to use with statins.

One area of focus is the bile acid system. Bile acids play a critical role in the intestinal absorption of fat and cholesterol. After fulfilling their function as detergent agents in the intestinal lumen, they are absorbed by active ileal uptake and recycled in the enterohepatic circulation. Bile acid sequestrants such as anion exchanging resins have been used to treat hypercholesterolemia and hyperlipidemia with a good safety record for over two decades (Hoeg, 1991, Melian and Plosker, 2001), and in combination with statins have achieved synergistic reduction in blood cholesterol levels (Leren et al., 1988, Vega and Grundy, 1987). Limitations of bile acid sequestrant therapy include discomfort owing to their bulkiness as well as constipation, which contribute to poor patient compliance. Therefore, it is desirable to develop hypocholesterolemic agents that interrupt the reabsorption of bile acids at more tolerable therapeutic doses.

Ileal apical Na⁺-dependent bile acid transporter (ASBT), which plays a critical role in the reabsorption of bile acids in the ileum in humans and animals (Dietschy et al., 1993, Hofmann, 1999, Mitropoulos et al., 1973, Shneider, 2001), is considered an attractive target for a new class of cholesterol-lowering drugs (Zhang et al., 2002). ASBT inhibitors would increase excretion of bile acids, causing increased catabolism of hepatic cholesterol to bile acids, and subsequently reducing hepatic and eventually serum cholesterol levels. Moreover, ASBT inhibitors would not be absorbed since bile acid binding to ASBT occurs within the lumen of the most distal part of the ileum. Thus, a non-systemic ASBT inhibitor would carry low risk of potential systemic toxicity and drug–drug interaction (Huang et al., 2005, Tremont et al., 2005). Human ASBT is expressed at the highest levels in the distal half of the ileum and in the kidney (Craddock et al., 1998). Gene cloning and expression techniques have facilitated rapid screening of potential drugs and several compounds have been identified (Hara et al., 1997, Lewis et al., 1995, Root et al., 2002, West et al., 2002).

Absorbed bile acids are transferred to the hepatic portal circulation where they are taken up into hepatocytes by another bile acid transporter, Na⁺-taurocholate co-transporting polypeptide (NTCP) (Meier et al., 1997). Recently, we synthesized a novel series of ASBT inhibitors having potent cholesterol-lowering activity in vivo by screening with hamsters and human ASBT (hASBT)- or human NCTP (hNCTP)-expressing HEK-293 cells. Some derivatives, designed with poor absorbability and greater molecular mass than that of “Lipinski's Rule of Five”, were previously reported to reduce serum cholesterol levels in hamsters fed a chow diet (Kurata et al., 2004).

Herein, we report the in vitro and in vivo pharmacological profiles of R-146224, a new ASBT inhibitor synthesized in our laboratory, and its effects on bile acid metabolism. We also present pharmacokinetic analysis using [¹⁴C]labeled R-146224 and demonstrate the potential of R-146224 as a hypocholesterolemic drug in vivo.