Avasimibe (PD-148515) is an orally bioavailable inhibitor of acyl-Coenzyme A: cholesterol acyltransferase (ACAT, IC50: 3.3 μM) that prevents cholesterol deposition in the arterial wall. It also inhibits human P450 isoenzymes CYP2C9/1A2/2C19 (IC50: 2.9/13.9/26.5 μM).

CYP2C9:2.9 μM, CYP1A2:13.9 μM, CYP2C19:26.5 μM, ACAT:3.3 μM

Avasimibe通过降低低密度脂蛋而降低总胆固醇.在9只健康雄性猴子体内,Avasimibe明显降低脂蛋白(a)和总胆固醇水平,Avasimibe（30 mg/kg/day,p.o.）饲喂3周,可使总胆固醇和脂蛋白(a)水平分别降低至对照水平的73和68%.

在HepG2 细胞中温育24 h,Avasimibe（0.01/1/10 μM）可使分泌到培养基中的ApoB分别降低 25%,27%和43%。在人类单核细胞衍生的巨噬细胞中,通过抑制泡沫细胞形成期低密度脂蛋结合和降低清除剂受体数,Avasimibe（1μg/ml）可降低酯化胆固醇和总胆固醇。Avasimibe（2μg/ml）与低密度脂蛋白（10μg/ml）预温育,可使胆固醇从HMM泡沫细胞中的外排增强。Avasimibe是通过增强细胞内ApoB降解来降低ApoB分泌,但不影响其ApoB合成。 在IC-21巨噬细胞中,Avasimibe抑制 ACTC（IC50：3.3 μM）。 在胶质瘤细胞中,Avasimibe对胆固醇酯的合成和ACAT-1 表达有抑制作用。通过诱导细胞周期停滞和caspase-8/3激活引起的凋亡,Avasimibe可抑制胶质瘤细胞生长。Avasimibe剂量依赖性抑制原代猴肝脏细胞培养基中的脂蛋白(a)累积（11.9% -31.3%）,这与 ApoA降低有关。

P450 Inhibition Studies: Pooled human liver microsomes (HLM) from at least 15 donors are used for all inhibition assays. For IC50 determinations, the substrate probes are used at their approximate in vitro Km values. All incubations are performed with 100 mM potassium phosphate buffer (pH 7.4) and 1 mM NADPH. For CYP1A2 inhibition study, incubations are performed in a total volume of 0.5 ml, in duplicates with 0.1 mg/ml HLM, 30 μM phenacetin, 1 mM NADPH, and in the presence of avasimibe (0, 0.3, 0.75, 1.5, 3, 7.5, 15, 30, and 40 μM in 50 mM) in a potassium phosphate buffer at pH 7.4. After preincubation at 37 °C for 7 min, NADPH is added to initiate the enzyme reaction. The reaction mixture is quenched with 500 μl of ice-cold 100 ng/ml paracetamol-D4/CH3CN after 25 min. The standards (4-acetamidophenol, singlet) and quality controls (triplicates for low, medium, and high) are prepared at room temperature. After mixing, 0.2 ml of the samples is transferred to another plate and submitted for LC/MS/MS analysis after centrifugation at 3000 rpm for 10 min. A Supelco Discovery Amide C16, 100 × 2.1 mm (5-μm particle size) column (Supelco, Bellefonte, PA) is used. The mobile phase is isocratic, 40:60 [acetonitrile/formic acid, 0.1% (v/v)] at 0.2 ml/min.

For foam cell formation, the growth medium (RPMI medium containing 10% human serum) is aspirated and the BMMs are rinsed four times with RPMI medium, and then HMMs are exposed to RPMI medium containing bovine serum albumin (BSA, 0.2%) and dimethylsulfoxide (DMSO, 0.2%, vehicle for CI-1011) (control medium) with and without agacLDL (100 μg protein/ml) and CI-1011 (1 μg/ml) for 48 hours. For cholesterol efflux experiments, HMMs are preincubated with ag-acLDL (100 μg protein/ml) for 24h, and then exposed to control RPMI medium with and without HDL (100 μg protein/ml), CI-1011 (2 μg/ml) or HDL plus CI-1011 (2 μg/ml) for 24–48 hours. Additionally, the appearance of [14C]FC in the medium is monitored by first preincubating HMMs with RPMI medium containing ag-acLDL (100 μg protein/ml) radiolabeled with [4-14C]FC (0.5 μCi/ml) in an ethanolic spritz (final concentration, 0.1%) for 24 h. The medium is removed, cells rinsed three times with RPMI medium, and then cells are exposed to control RPMI medium with and without CI-1011 (1–10 μg/ml) for 4–48 h. At each time point, the medium is aspirated and centrifuged to pellet nonadherent cells. The appearance of [14C]FC in the medium is measured by liquid scintillation spectroscopy. Cellular lipids are extracted using hexane:isopropanol (3:2, v/v) for 1 h. The distribution of cellular radiolabeled cholesterol is measured by subjecting an aliquot of the cell extract and FC and EC standards to thin layer chromatography using petroleum ether:hexane:glacial acetic acid solvent system (85:15:2, v/v). The percent FC efflux is calculated as: medium [14C]FC dpm/ cell [14C] dpm×100. FC and TC mass are quantified by gas liquid chromatography using stigmasterol (1 mg/ml) as an internal standard. EC mass is calculated as the difference between TC and FC, and all values are normalized to cell protein. The MBC is de?ned as the lowest concentration that exhibited 99.9% or more reduction of the numbers of colonies compared with the cfu in the initial inoculum. (Only for Reference)