生物活性

Avasimibe inhibits human P450 isoenzymes, particularly CYP2C9, CYP1A2 and CYP2C19, with an IC50 of 2.9µM, 13.9 µM, and 26.5 µM, respectively.  Avasimibe (CI-1011), an orally bioavailableinhibitor of ACAT, decreased plasma cholesterol and triglyceride concentrations, and reduced early atherosclerotic lesiondevelopment in a variety of small animal models.

Avasimibeinhibits ACAT with an IC50 ranging from 12 nM to 60nM dependingon ACAT assay conditions.[1]

Thein vitro IC50 of avasimibe for pig microsomal ACAT activity is 4μM[2]

P450 enzymes inhibition of avasimibe in human hepatic microsomes[3]

体外研究

体内研究

2% DMSO+corn oil

激酶实验

Quantitation of ACAT Activity[4]

Cholesterol esterification was measured bydetermining the incorporation of [¹¹⁴C]-oleate (100μmol/L, 0.25μCi)into labeled cholesteryl oleate after the incubation of hepatocytes for 22hours, from 18 to 40 hours of culture, with different concentrations ofavasimibe in the presence or absence of βVLDL (providing200μg cholesterol per mL medium). After 22 hours, [¹¹⁴C]-oleate wasadded and cells were incubated for another 2hours at 37°C. Cells were harvestedat 42 hours of culture age to measure cholesterol esterification.

Pooled human liver microsomes (HLM) from atleast 15 donors were used for all inhibition assays. For IC50 determinations,the substrate probes were used at their approximate in vitro Km values. All incubationswere performed with 100mM potassium phosphate buffer (pH7.4) and 1mM NADPH.Organic solvents were used to prepare stock solutions.

CYP1A2

Incubations were performed in a totalvolume of 0.5ml, in duplicates with 0.1mg/ml HLM, 30μM phenacetin,1mM NADPH, and in the presence of avasimibe (0, 0.3, 0.75, 1.5, 3, 7.5, 15, 30,and 40μM in 50mM) in a potassium phosphate buffer at pH 7.4. Afterpreincubation at 37°C for 7 min, NADPH was added to initiate the enzymereaction. The reaction mixture was quenched with 500μl of ice-cold100ng/ml paracetamol-D4/CH3CN after 25min. The standards (4-acetamidophenol,singlet) and quality controls (triplicates for low, medium, and high) wereprepared at room temperature. After mixing, 0.2ml of the samples wastransferred to another plate and submitted for LC/MS/MS analysis aftercentrifugation at 3000rpm for 10min. A Supelco

Discovery Amide C₁₆, 100 X2.1 mm(5-μm particle size) column was used. The mobile phase was isocratic,40:60 [acetonitrile/formic acid, 0.1% (v/v)] at 0.2ml/min.

CYP2C9

Incubations were performed under conditionssimilar to those above with HLM, 7.5μM diclofenac, NADPH, andavasimibe (0, 0.3, 0.75, 1.5, 3, 7.5, 15, 30, and 40μM). Thereaction was quenched with 500μl of 2.5μg/ml ¹³C₆-4’-hydroxydiclofenac/CH3CN after 7min. The standards (4’-hydroxydiclofenac), quality controls, and samples wereprepared as above for LC/MS/MS analysis. A Phenomenex Synergi Max RP, 50 X2.0mm column was used. The mobile phase was 50:50 [acetonitrile/0.1% formic acidin water (v/v)] at 0.27 ml/min (isocratic).

CYP2C19

Incubations were performed under conditionssimilar to those above with HLM, 50μM (S)-(+)-mephenytoin, NADPH,and avasimibe (0, 0.3, 0.75, 1.5, 3, 7.5, 15, 30, and 40) in potassiumphosphate buffer. The reaction mixture was quenched with ice-cold 250 ng/ml3-acetamidophenol/ CH3CN (500 μl) after 30 min. The standards (4’-hydroxymephenytoin) and qualitycontrols were prepared for LC/MS/MS analysis as above. A Phenomenex Max-RP, 50 X2.0mm (5-μm particle size) columns was used. Two mobile phases were used:mobile phase A, 90:10 [10mM ammonium acetate with0.1% acetic acid/CH3CN (v/v)],and mobile phase B: 10:90 [10mM ammonium acetate with 0.1% acetic acid/CH3CN(v/v)]. The gradient was 030.5 min, 100% A; 0.532.0 min, 100% B; 2.033.0 min,100% A.

细胞实验

Cell cultures and RNA interference[5]

The cells were cultured in Dulbecco’sModified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 5mML-glutamine, streptomycin (100μg/ml) and penicillin (100units/ml) at 37°C in ahumidified atmosphere containing 5% CO₂.

 Forsilencing ACAT-1 expression in human glioma cell lines, cells were transfectedwith1.5 or 3 mg of a manufacturer-optimized mixture of human-specific ACAT-1siRNA using Nucleofactor technology according to the manufacturer’sinstructions. Control cells were transfected with3 mg of a mixture ofmouse-specific ACAT-1 siRNA. Culture media was changed once at 72 h post-transfectionand 24 h conditioned media was collected when the cells were harvested 96 hpost-transfection.

Determinationof cell viability

Cells were seeded in 24-well plates at adensity of 5 x 10⁴cells/well in 500μl of tissue culture medium intriplicates. After 48 h incubation time period to allow cell adhesion, the cellswere treated for 48 h with Avasimibe at varied concentrations, as described inindividual experiments. Cell viability was determined by the Trypan blueexclusion assay. The dye exclusion test was used to determine the number ofviable cells present in a glioma cell suspension. It is based on the principle thatglioma live cells possess intact cell membranes that exclude trypan.

动物实验

Animals and diets[6]

Miniature pigs weighing 26.53±0.43kg wereobtained. After being acclimatized for 1 week, animals were maintained on theexperimental diet for 28 days before the postprandial studies. Pigs werestudied in pairs, with each pair being samesex littermates. Each animalreceived a 590 g ration of diet supplemented with lard, butter, and saffloweroil (1:0.6:0.2) generating a final polyunsaturated: monounsaturated: saturatedfatty acid ratio of 1:1:1. Cholesterol was added to the diet to a final concentrationof 0.1% (0.2 mg/kcal). This diet provided 34% calories from fat, 49% ascarbohydrate and 17% as protein.

Pigs were studied in pairs with each pairbeing same sex litter mates. Five animals received the ACAT inhibitor,avasimibe at a dose of 10 mg/kg/ day and four animals, avasimibe at a dose of25 mg/kg/day for 28 days prior to the postprandial studies. Avasimibe wasplaced in gelatin capsules and to ensure ingestion was administered by handbefore the daily feeding. The nine control animals received a placebo capsule.The avasimibe was given at 9 AM each day after a 24 h fast.

Two weeks prior to the postprandialstudies, an indwelling silicone elastomer catheter (1.96 mm internal diameter)was surgically implanted in an external jugular vein. Isoflurane USP was usedas the anesthetic and ketamine USP as the preanesthetic. Catheters that werekept patent by filling with 7% EDTA-Na2, allowed for blood sampling throughouteach postprandial study in unrestrained, unanesthetized animals.

Oralfat tolerance test

After a 24 h fast, pigs were fed the dietdescribed above, in an amount calculated to provide 2 g of fat/kg body weightand either placebo or avasimibe. This test meal was supplemented with 50,000 IUof retinol and consumed within 10 min. The animals were not fed for the 12-hstudy but had free access to drinking water. venous blood samples (20 mL) weredrawn at intervals and collected into tubes containing EDTA-Na2. Samples werekept on ice (up to 30min) prior to isolation of plasma lipoproteins and protectedfrom light during processing. Plasma was obtained by centrifugation at 1000× gfor 25 min at 4°C. The isolated plasma underwent preparative ultracentrifugation atd=1.006 g/ mL in a Beckman 50.4 Ti rotor at 35,500 rpm at 12 °C for 16 h. TRLfractions (db1.006 g/mL; SfN20) were isolated by tube slicing and each plasmasample and TRL fraction, were analyzed for cholesterol, triglyceride andretinyl palmitate (RP) concentrations.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Lee HT SD, Picard JA, Roth BD, Wierenga W, Hicks JL, Bousley RF, Hamelehle KL, Homan R, Speyer C, Stanfield RL, Krause BR. Inhibitors of acyl-CoA: cholesterol O-acyl transferase (ACAT) as hypocholesterolemic agents. CI-1011: an acyl sulfamate with unique cholesterol-lowering activity in animals fed noncholesterol-supplemented diets. J Med Chem. 1996;39(26):5031-5034.
[2] Burnett JR WL, Telford DE, Kleinstiver SJ, Barrett PH, Newton RS, Huff MW. Inhibition of ACAT by avasimibe decreases both VLDL and LDL apolipoprotein B production in miniature pigs. J Lipid Res. 1999 40(7):1317-1327.
[3] Sahi J, Stern RH, Milad MA, et al. Effects of avasimibe on cytochrome P450 2C9 expression in vitro and in vivo. Drug Metab Dispos. 2004;32(12):1370-1376.
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体内溶解度
5mg/mL

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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