Anacetrapib (MK-0859)   中文名称: 安塞曲匹

Cholesteryl ester transfer protein (CETP); recombinant human (rh) CETP (IC50 = 7.9 nM)[1]; CETPC13S (IC50 = 11.8 nM)[2]

anacetrapib 可显着且剂量依赖性地减少 CE 从 HDL3 到 HDL2 的转移（对于高达并包括 0.1 µM 的剂量，P<0.001）。过量的 anacetrapib (25 µM) 使 [14C]Torcetrapib (0.25 µM) 与固定化 rhCETP 结合的量分别减少 82% 和 60%。 anacetrapib 在所有研究浓度（0.1、1、3 和 10 µM）下，前 β-HDL 产量减少了 46% 以上（P<0.001）[1]。 Anacetrapib (ANA) 显着降低 PCSK9 启动子活性；这是在 3 µM 浓度下观察到的（-22%，p<0.01），在 10 µM 浓度下甚至更低，为对照的 68%。同样，Anacetrapib 从 3 µM 浓度开始降低 B11 细胞的荧光素酶活性，在 10 µM 浓度时最大程度降低 38%。 Anacetrapib 在 10 µM 浓度下将 PCSK9 mRNA 降低至对照的 60%，将 LDLR mRNA 降低至对照的 67%[2]。

在兔子身上，dalcetrapib（JTT-705）（30或100mg/kg；口服；每天一次，持续三天）显著提高了血浆HDL胆固醇[2]。服用达克雷替布（100mg/kg；ir；每天两次，持续7天）后，粪便中的中性甾醇、胆汁酸和血浆高密度脂蛋白胆固醇显著增加[1]

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在注射[3H]胆固醇标记的自体巨噬细胞的仓鼠中，并给予dalcetrapib（100 mg每日两次）、torcetrapib[30 mg每日一次（QD）]或anacetrapib（30 mg QD），只有dalcetrapib显著增加了[3H]中性甾醇和[3H]胆汁酸的粪便清除，而所有化合物都增加了血浆HDL-[3H]胆固醇。这些数据表明，dalcetrapib对CETP活性的调节不会抑制CETP诱导的前β-HDL形成，这可能是增加胆固醇逆向转运所必需的。1.

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在注射 [3H]胆固醇标记的巨噬细胞（第 0 天）之前，将anacetrapib 给予仓鼠 7 天。 Anacetrapib 治疗后第 0 天 HDL-C 值显着升高。第 3 天 HDL 部分中的 [3H] 胆固醇放射性显着高于 anacetrapib 对照值[1]。与媒介对照相比，anacetrapib(ANA) 药物使血清总胆固醇的血清水平轻微升高约 10% (p<0.05)，使 LDL-C 的血清水平轻微升高 26% (p<0.05)[2]。静脉注射0.5 mg/kg剂量后的终末半衰期、稳态分布容积和全身血浆清除率的平均值分别为12小时、1.1 L/kg和2.3 mL/min/kg。 anacetrapib 口服 5 mg/kg 后生物利用度为 38%。暴露量 (AUC) 从 5 mg/kg 时的 23 μM·h 上升至 500 mg/kg 时的 362 μM·h，其方式与剂量不成比例。在此剂量范围内，达到峰值血浆水平（Tmax）的时间为3至4.5小时，峰值血浆水平（Cmax）为5至26μM[3]。

dalcetrapib与Cys13的选择性结合。[1]
通过定点突变构建了含有丝氨酸残基而不是Cys13的CETP（C13S CETP）。该蛋白在大规模瞬时转染的HEK293EBNA细胞中表达，并按照下文所述的重组人（rh）CETP纯化。

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抑制rhCETP和C13S CETP介导的CE从HDL向LDL的转移。[1]
使用闪烁邻近分析试剂盒测量了达昔珠单抗、托昔珠单抗和阿曲匹布减少rhCETP和C13S CETP从HDL到LDL的CE转移的抑制效力（IC50）。简而言之，在37°C下，将[3H]CE标记的HDL供体颗粒与纯化的CETP蛋白（终浓度0.5µg/ml）和生物素化的LDL受体颗粒一起孵育3小时。随后，加入含有选择性结合生物素化LDL的液体闪烁鸡尾酒的链霉抗生物素蛋白偶联聚乙烯甲苯珠，并通过β计数测量转移到LDL的[3H]CE分子的量。

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抑制CE从HDL3转移到HDL2。[1]
如前所述，使用放射性标记的脂质转移试验评估HDL亚组分之间的脂质运动。脂蛋白亚组分（d>1.063 g/ml）用[3H]CE标记。通过连续超速离心制备[3H]CE标记的HDL3（1.125 CETP上化合物的结合位点：琼脂糖固定化rhCETP上结合位点的竞争。[1]
根据Connolly等人的研究，使用Weinberg等人描述的细胞系表达的rhCETP进行结合研究，并通过疏水相互作用色谱和尺寸排阻色谱（SEC）纯化。BSA和rhCETP被固定在溴化氰活化的sepharoseTM 4 Fast Flow上。测定了300 pmol固定的rhCETP（3μM）或相同质量的BSA分别与0.25μM[14C]torcetrapib或2.5μM[114C]dalcetrapib混合，以及总体积为100μl的未标记CETP抑制剂与放射性化合物预孵育后的竞争（共孵育实验）和置换（后者有或没有还原剂三（2-羧乙基）膦（TCEP））。在与CETP孵育之前，用胰脂肪酶处理[14C]达昔珠单抗以产生[14C]达昔珠单抗硫醇。通过闪烁计数测量与琼脂糖结合的放射性。

LDL摄取测定[2]
将6孔培养板中的HepG2细胞用anacetrapib处理24小时。处理结束时，将浓度为2µg/ml的荧光DiI-LDL加入细胞中4小时，并对细胞进行胰蛋白酶处理。使用FACScan测量1×104个细胞的平均红色荧光。2.

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小干扰RNA（siRNA）转染[2]
从Dharmacon 获得了四种针对人类CETP mRNA的预先设计的siRNA。消音器阴性对照siRNA购自Applied Biosystem。使用siPORT NeoFX siRNA转染试剂将4×10个细胞与50 nM siRNA混合，并放置在6孔板中。第二天，将新鲜培养基加入转染的细胞中，然后在分离总RNA之前用anacetapib处理细胞24小时。[2]

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ELISA定量前β-HDL：在torcetrapib、anacetrapib和Dalcetrapib（JTT-705）（0.10µM至10µM）的存在下，将添加或不添加rhCETP的样品孵育21小时。如前所述，通过ELISA测量前β-HDL浓度[1]。

In vivo RCT study.[1]
To investigate the effect of dalcetrapib, torcetrapib, and anacetrapib on macrophage-to-feces RCT, radiolabeled macrophages from the peritoneal cavity of donor Golden Syrian hamsters preinjected with [3H]cholesterol were prepared as previously described. Male recipient Golden Syrian hamsters, 8 weeks old, on a standard chow diet were preadministered dalcetrapib [100 mg/kg twice daily (BID)], torcetrapib [30 mg/kg once daily (QD)], anacetrapib (30 mg/kg QD), or vehicle (0.5% methylcellulose BID) for 7 days by oral gavage before intraperitoneal injection of [3H]cholesterol-labeled macrophages (3.8 × 106 cells/90.6 kBq/0.5 ml per animal) at day 0. The percentage of esterified cholesterol in injected macrophages was 21% (mass) and 16% (labeled). Animals continued to receive vehicle or test compounds daily for 10 days. Samples for plasma lipid analysis were obtained on days −7, 0, 3, 7, and 10 and for radioactivity levels on days 3, 7, and 10. Total cholesterol and HDL-C were measured by enzymatic methods. HDL-C was measured as the cholesterol concentration in the HDL fraction separated by polyethylene glycol 6000 solution. The area under the plasma HDL-C concentration-time curve (HDL-C·AUC) during the RCT study period (day 0 to day 10) was calculated from plasma HDL-C levels (at day 0, 3, 7, and 10) by the trapezoidal method.

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Dissolved in polyethylene glycol 300-water (7:3, v/v); 2.5 mL/kg (2.5, 25, 50, 250 mg/mL); oral gavage

Adult male Sprague-Dawley rats

The pharmacokinetics and metabolism of anacetrapib (MK-0859), a novel cholesteryl ester transfer protein inhibitor, were examined in rats and rhesus monkeys. Anacetrapib exhibited a low clearance in both species and a moderate oral bioavailability of approximately 38% in rats and approximately 13% in monkeys. The area under the plasma concentration-time curve in both species increased in a less than dose-proportional manner over an oral dose range of 1 to 500 mg/kg. After oral administration of [(14)C]anacetrapib at 10 mg/kg, approximately 80 and 90% of the radioactive dose was recovered over 48 h postdose from rats and monkeys, respectively. The majority of the administered radioactive dose was excreted unchanged in feces in both species. Biliary excretion of radioactivity accounted for approximately 15% and urinary excretion for less than 2% of the dose. Thirteen metabolites, resulting from oxidative and secondary glucuronic acid conjugation, were identified in rat and monkey bile. The main metabolic pathways consisted of O-demethylation (M1) and hydroxylation on the biphenyl moiety (M2) and hydroxylation on the isopropyl side chain (M3); these hydroxylations were followed by O-glucuronidation of these metabolites. A glutathione adduct (M9), an olefin metabolite (M10), and a propionic acid metabolite (M11) also were identified. In addition to parent anacetrapib, M1, M2, and M3 metabolites were detected in rat but not in monkey plasma. Overall, it appears that anacetrapib exhibits a low-to-moderate degree of absorption after oral dosing and majority of the absorbed dose is eliminated via oxidation to a series of hydroxylated metabolites that undergo conjugation with glucuronic acid before excretion into bile.[3]

[1]. Modulating cholesteryl ester transfer protein activity maintains efficient pre-β-HDL formation and increases reverse cholesterol transport. J Lipid Res. 2010, 51(12), 3443-3454.

[2]. CETP inhibitors downregulate hepatic LDL receptor and PCSK9 expression in vitro and in vivo through a SREBP2 dependent mechanism. Atherosclerosis. 2014 Aug;235(2):449-62.

[3]. 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.

Anacetrapib is a cholesteryl ester transfer protein (CETP) inhibitor with hypocholesterolemic properties. Anacetrapib reduces the transfer of cholesteryl ester from HDL to LDL and/or VLDL thereby, producing an increase in serum HDL-cholesterol levels and a decrease in serum LDL-cholesterol levels. This agent has not yet been shown to reduce deaths associated with hypercholesterolemia.

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Drug Indication
Investigated for use/treatment in hyperlipidemia.
Prevention of cardiovascular events in patients with hypercholesterolaemia, Treatment of hypercholesterolaemia

C30H25F10NO3

637.51

637.167

C, 56.52; H, 3.95; F, 29.80; N, 2.20; O, 7.53

875446-37-0

11556427

White to off-white solid powder

1.3±0.1 g/cm3

555.3±50.0 °C at 760 mmHg

289.6±30.1 °C

0.0±1.5 mmHg at 25°C

1.494

8.81

38.77

0

13

6

44

964

2

C[C@H]1[C@H](OC(=O)N1CC2=C(C=CC(=C2)C(F)(F)F)C3=CC(=C(C=C3OC)F)C(C)C)C4=CC(=CC(=C4)C(F)(F)F)C(F)(F)F

MZZLGJHLQGUVPN-HAWMADMCSA-N

InChI=1S/C30H25F10NO3/c1-14(2)22-11-23(25(43-4)12-24(22)31)21-6-5-18(28(32,33)34)9-17(21)13-41-15(3)26(44-27(41)42)16-7-19(29(35,36)37)10-20(8-16)30(38,39)40/h5-12,14-15,26H,13H2,1-4H3/t15-,26-/m0/s1

(4S,5R)-5-[3,5-bis(trifluoromethyl)phenyl]-3-[[2-(4-fluoro-2-methoxy-5-propan-2-ylphenyl)-5-(trifluoromethyl)phenyl]methyl]-4-methyl-1,3-oxazolidin-2-one

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 2.75 mg/mL (4.31 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 27.5 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 2 中的溶解度: 30% PEG400+0.5% Tween80+5% propylene glycol:10 mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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