A Novel Inhibitor of Oxidosqualene:Lanosterol Cyclase Inhibits Very Low–Density Lipoprotein Apolipoprotein B100 (ApoB100) Production and Enhances Low-Density Lipoprotein ApoB100 Catabolism Through Marked Reduction in Hepatic Cholesterol Content

Abstract

Objective-Inhibition of 2,3-oxidosqualene:lanosterol cyclase (OSC), an enzyme in the cholesterol synthesis pathway, has the unique ability to inhibit cholesterol synthesis while simultaneously enhancing oxysterol synthesis. Our objectives were to determine, in vivo, if a novel OSC inhibitor reduced low-density lipoprotein (LDL) cholesterol and to define the mechanism(s) involved. Methods and Results-Miniature pigs received the OSC inhibitor RO0717625 or placebo and a diet containing fat (34% of energy) and 400 mg per day of cholesterol. Treatment decreased plasma total cholesterol (Ϫ20%) and LDL cholesterol (Ϫ29%). Apolipoprotein B (apoB) kinetic parameters were determined. Very low-density lipoprotein (VLDL) apoB pool size decreased 22% because of inhibition of VLDL production (Ϫ43%). LDL apoB pool size decreased 22% because of a 1.5-fold increase in fractional catabolic rate (FCR). The increased FCR was associated with a 2-fold increase in hepatic LDL receptor mRNA. Hepatic total and microsomal cholesterol were reduced by 16% and 27%, respectively. Plasma lathosterol concentrations decreased 57%, reflecting inhibition of hepatic cholesterol synthesis. Treatment reduced plasma plant sterols and decreased postprandial cholesterol transport in chylomicrons. Conclusions-A novel OSC inhibitor, RO0717625, decreased VLDL and LDL apoB100 through decreased VLDL production and enhanced LDL clearance. Thus, OSC represents a potential therapeutic target for dyslipidemia. Key Words: oxidosqualene:lanosterol cyclase Ⅲ apoB kinetics Ⅲ LDL receptor Ⅲ ABCG5 Ⅲ ABCG8 L andmark trials using 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) revealed reductions in cardiovascular mortality and morbidity associated with low-density lipoprotein (LDL) cholesterol lowering. 1 Statins are well tolerated, although reductions in nonsterol intermediates in the cholesterol-synthesis pathway, such as isoprenoids and coenzyme Q, may theoretically be associated with adverse clinical events. 2,3 This has stimulated the development of compounds that inhibit cholesterol biosynthesis yet act distal to the synthesis of these nonsterol intermediates. 2,3-oxidosqualene:lanosterol cyclase (OSC; enzyme collection 5.4.99.7, also known as lanosterol synthase), a microsomal enzyme, represents a unique target for cholesterol-lowering drugs. 4,5 OSC is downstream of isoprenoid synthesis. Furthermore, compounds that decrease plasma concentrations of atherogenic lipoproteins by more than 1 mechanism are likely to be more efficacious.OSC catalyzes the highly selective cyclization of 2,3-monoepoxysqualene (MOS) to lanosterol, the first sterol to be formed. 4 OSC also catalyzes cyclization of 2,3;22,23-diepoxysqualene (DOS), which itself is derived from MOS, to 24(S),25-epoxylanosterol, the immediate precursor of 24(S),25-epoxycholesterol. Synthesis of 24(S),25-epoxycholesterol is favored over cholesterol synthesis under conditions of partial OSC inhibition, whereas complete OSC inhibition results in decreased synthesis of choleste...

Hepatic ABCG5/G8 overexpression reduces apoB-lipoproteins and atherosclerosis when cholesterol absorption is inhibited

“…In hamster hepatocytes in vitro, hepatic CE and hepatic apoB secretion were closely correlated after the addition of statin or ACAT inhibitor plus or minus oleate (46). A novel oxidosqualene:lanosterol cyclase (OSC) inhibitor that inhibits cholesterol synthesis but enhances oxysterol synthesis in miniature pigs reduced hepatic CE but not FC and inhibited VLDL-apoB production without altering its composition (47). These combined studies demonstrate that hepatic cholesterol or CE levels can alter apoB-Lp cholesterol secretion without changing the lipid content of the apoB-Lps.…”

The molecular mechanisms underlying the reduction of LDL apoB-100 by ezetimibe plus simvastatin

“…The plasma dual isotope ratio method described by Turley, Herndon, and Dietschy (30) (31). Sequential blood samples were obtained, and VLDL, IDL, LDL, and HDL were separated completely by ultracentrifugation.…”

“…Leucine was derivatized to the oxazolinone, and isotopic enrichment was determined by GC-MS as described previously (29). Kinetic parameters of apoB-100 metabolism were analyzed using SAAM II (SAAM Institute; Seattle, WA) according to the model reported previously (31).…”

“…Gene-specific mRNA quantitation for apoB, HMG-CoA reductase, LDLR, SREBP2, SREBP1c, NPC1L1, ABCG5, ABCG8, ABCA1, LXRa, MTP, and GAPDH was performed by quantitative real-time PCR (qRT-PCR) on an ABI Prism (model 7900 HT) Sequence Detection System (Applied Biosystems) according to the manufacturer's instructions (31). Samples (20-75 ng) of reverse transcribed total RNA were assayed in triplicate in 20 ml reactions using a two-step qRT-PCR protocol and the standard curve method to estimate specific mRNA concentrations.…”

“…Expression levels for each gene were normalized to GAPDH expression levels for each tissue type. For genes with known pig (Sus scrofa) sequences (APOB, HMGCR, LDLR, SREBP2, MTP, LXRa, ABCA1, and GAPDH), primer and probe sets were designed using Primer Express 2.0 software (Applied Biosystems) (31). For genes with unknown pig sequences (NPC1L1, ABCG5, ABCG8, and SREBP1c), PCR primers were designed against the mRNA sequence of the human homolog using PrimerQuest software (Integrated DNA technologies).…”