Kinase experiment:

The inhibitory potency (IC50) of Dalcetrapib, Torcetrapib, and Anacetrapib to decrease CE transfer from HDL to LDL by rhCETP and C13S CETP is measured using a scintillation proximity assay kit. Briefly, [3H]CE-labeled HDL donor particles are incubated in the presence of purified CETP proteins (final concentration 0.5 μg/mL) and biotinylated LDL acceptor particles for 3 h at 37℃. Subsequently, streptavidin-coupled polyvinyltoluene beads containing liquid scintillation cocktail binding selectively to biotinylated LDL are added, and the amount of [3H]CE molecules transferred to LDL is measured by β counting[1].

Cell experiment:

Cells are seeded in a 96 well plate overnight prior to the treatment by different concentrations of CETP inhibitors (e.g., Anacetrapib) for 24 h. Cell viability is measured using the CellTiter-Glo Luminescent Cell Viability Assay kit. Four wells are evaluated under each experimental condition[2].

Animal experiment:

Mice[2] Eight-week old male C57BL/6J mice are housed (4 animals/cage) under controlled temperature (72°F) and lighting (12 h light/dark cycle). Animals have free access to autoclaved water and food. In the first in vivo study, after an acclimatization period of 7 days, mice are fed a high-fat high-cholesterol diet (HFHC) containing 35% calories from fat and 1.25% cholesterol for two weeks. Mice are then divided into two groups (n=8 per group) and are given a daily dose of Anacetrapib at 50 mg/kg by oral gavage. The control group receive vehicle (0.5% methyl cellulose). The drug treatment lasts 7 days. In the second in vivo study, mice fed a normal chow diet are treated with Anacetrapib (50 mg/kg, n=8) or vehicle (n=8) for 10 days. Serum samples are collected after a 4 h fasting before and after the drug treatment. After the last dosing, all animals are sacrificed for collection of serum and liver tissues. Livers are immediately removed, cut into small pieces, and stored at –80℃ for RNA and protein isolations and cholesterol measurement. Rats[3] Adult male Sprague-Dawley rats, weighing 280 to 330 g, are fasted overnight before the study. The animals are allowed access to food 4 h after dosing, but water is provided ad libitum. For intravenous administration, four rats received a dose of Anacetrapib (dosing volume 2.5 mL/kg) via bolus injection through a catheter previously implanted into the femoral vein, followed by a 300 μL saline flush. The intravenous dose of Anacetrapib is formulated in polyethylene glycol 300-water (7:3, v/v) at a concentration of 0.2 mg/mL. For oral administration, four rats received a dose via oral gavage (dosing volume 2 mL/kg). These oral doses of 5, 50, 100, and 500 mg/kg are formulated in Imwitor 742-Tween 80 (1:1, w/w) at concentrations of 2.5, 25, 50, and 250 mg/mL, respectively. Blood samples (0.3 mL) are collected into EDTA-containing tubes at the following time points: predose and 0.083 (intravenous only), 0.25, 0.5, 1, 2, 4, 6, 8, 24, and 48 h postdose. Blood samples are stored on ice, and plasma is prepared by centrifugation. Plasma is transferred to a 96-well plate and stored at –70℃ until analysis.

Anacetrapib, also known as MK-0859, is a potent and selective inhibitor of cholesteryl ester transfer protein (CETP) that exhibits strong inhibition against CETP-mediated transfer of cholesteryl esters and triglycerides with values of inhibition constant IC₅₀of 16 nM and 29 nM respectively. Anacetrapib is mainly metabolized by CYP3A4 through O-demethylation, hydroxylation on the biphenyl moiety and hydroxylation on the isopropyl side chain resulting in three oxidative metabolites M1, M2 and M3 respectively. Anacetrapib is being investigated for the treatment of primary hypercholesterolemia and mixed hyperlipidemia due to its impressive effects to lower low-density lipoprotein (LDL) cholesterol levels and increase high-density lipoprotein (HDL) cholesterol levels without any associated major adverse events.

Reference

[1].Tan EY, Hartmann G, Chen Q, Pereira A, Bradley S, Doss G, Zhang AS, Ho JZ, Braun MP, Dean DC, Tang W, Kumar S. Pharmacokinetics, metabolism, and excretion of anacetrapib, a novel inhibitor of the cholesteryl ester transfer protein, in rats and rhesus monkeys. Drug Metab Dispos. 2010;38(3):459-473.
[2].Kumar S, Tan EY, Hartmann G, Biddle Z, Bergman AJ, Dru J, Ho JZ, Jones AN, Staskiewicz SJ, Braun MP, Karanam B, Dean DC, Gendrano IN, Graves MW, Wagner JA, Krishna R. Metabolism and excretion of anacetrapib, a novel inhibitor of the cholesteryl ester transfer protein, in humans. Drug Metab Dispos. 2010;38(3):474-483