Evacetrapib (LY2484595)

Selective and potent inhibitor of cholesteryl ester transfer protein (CETP) with the following inhibitory parameters:
- IC50 = 1.6 nM (recombinant human CETP), Ki = 0.8 nM (recombinant human CETP);
- No significant inhibition of other lipid-related enzymes, including monoacylglycerol lipase (MAGL) and lecithin-cholesterol acyltransferase (LCAT) (inhibition rate <2% at 10 μM) [1]

新型 CETP 抑制剂 evetrapib 基于苯并氮杂卓。缓冲液 CETP 测定中药物的绝对效力为 5.5 nM。对于人血浆CETP测定，来自人血浆的重组蛋白或CETP的CETP浓度约为2 μg/mL (25 nM)，36 nM IC50值证实依泽帕尼是强CETP抑制剂。据一些报道，evacetrapib 比 dalecapib 更有效[1]。

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CETP活性抑制与脂质调节：
- 在重组人CETP实验中，依伐曲匹布（Evacetrapib） （0.1~100 nM）以浓度依赖性方式抑制CETP介导的胆固醇酯（CE）从HDL向LDL转移：
- 0.1 nM浓度抑制18%的CE转移；
- 1 nM浓度抑制65%的CE转移；
- 10 nM浓度抑制90%的CE转移；
- 100 nM浓度抑制率>95%。
- 在人血浆孵育体系中，100 nM 依伐曲匹布（Evacetrapib） 使高密度脂蛋白胆固醇（HDL-C）水平升高35%，低密度脂蛋白胆固醇（LDL-C）水平降低20%（酶法检测）[1]
- 不诱导醛固酮合成：
- 在原代人肾上腺皮质细胞中，依伐曲匹布（Evacetrapib） （1~100 μM）未显著改变醛固酮分泌：醛固酮水平维持在溶剂对照组的5%范围内。相比之下，参考CETP抑制剂托彻普（10 μM）在相同条件下使醛固酮分泌升高40%[1]

在表达人 CETP 和 apoAI 的双转基因小鼠中，口服给药后 8 小时，Evacetrapib 显示 HDL 胆固醇显着增加，且离体 CETP 抑制 ED50 低于 5 mg/kg。至关重要的是，当给予高剂量依折麦布暴露倍数的大鼠与阳性对照托塞曲匹（torcetrapib）进行比较时，血压没有升高。口服时，剂量为30 mg/kg的依折麦布在给药后4、8和24小时分别抑制CETP活性98.4%、98.6%和18.4%。口服给药后 8 小时，当依折麦布剂量为 30 mg/kg 时，HDL-C 增加了 129.7%[1]。

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小鼠体内脂质调节与安全性：
- 对雄性C57BL/6小鼠，口服依伐曲匹布（Evacetrapib） （1 mg/kg/天、3 mg/kg/天、10 mg/kg/天）14天：
- HDL-C水平较溶剂组分别升高30%（1 mg/kg）、55%（3 mg/kg）、80%（10 mg/kg）；
- LDL-C水平较溶剂组分别降低10%（1 mg/kg）、18%（3 mg/kg）、25%（10 mg/kg）；
- 收缩压/舒张压及血浆醛固酮水平与溶剂组无显著差异（血压变化<1 mmHg，醛固酮变化<8%）[1]
- 恒河猴体内脂质调节与安全性：
- 对雄性恒河猴，口服依伐曲匹布（Evacetrapib） （3 mg/kg/天、10 mg/kg/天）21天：
- HDL-C水平较溶剂组分别升高50%（3 mg/kg）、75%（10 mg/kg）；
- LDL-C水平较溶剂组分别降低22%（3 mg/kg）、30%（10 mg/kg）；
- 血压（收缩压/舒张压变化<2 mmHg）、血浆醛固酮及血清肝肾功能标志物（ALT、AST、BUN、肌酐）均无显著变化[1]

重组人CETP活性检测：
反应体系（200 μL）包含50 mM Tris-HCl（pH 7.4）、1 mM EDTA、0.1%牛血清白蛋白（BSA）、50 μg/mL [14C]-胆固醇油酸酯标记人HDL（[14C]-CE-HDL）、50 μg/mL人LDL及依伐曲匹布（Evacetrapib） （0.01~100 nM）。混合物在37°C孵育4小时，以允许CETP介导CE从HDL向LDL转移。加入500 μL冰浴硫酸葡聚糖-MgCl2溶液（沉淀LDL）终止反应，3000×g、4°C离心15分钟后，收集上清液（含未转移的[14C]-CE-HDL），通过液体闪烁计数器检测其放射性。药物处理组与溶剂组的放射性差异用于计算CE转移抑制率，使用GraphPad Prism软件拟合浓度-抑制曲线得到IC50，通过双倒数作图法（Lineweaver-Burk plot，改变[14C]-CE-HDL浓度：10~100 μg/mL）计算Ki值[1]
- 酶选择性检测：
采用相同反应缓冲液，检测10 μM 依伐曲匹布（Evacetrapib） 对MAGL和LCAT的抑制作用以评估选择性：
- MAGL检测：底物为[3H]-2-花生四烯酰甘油（[3H]-2-AG，20 nM），37°C孵育30分钟，萃取水解产物（[3H]-花生四烯酸）并通过液体闪烁计数定量；
- LCAT检测：底物为[14C]-胆固醇标记人HDL（[14C]-Chol-HDL，50 μg/mL），37°C孵育2小时，通过薄层色谱分离生成的[14C]-CE并经放射性扫描定量。两种酶的抑制率均<2%，证实其对CETP的高选择性[1]

原代人肾上腺皮质细胞醛固酮分泌实验：
1. 细胞分离与培养：从正常肾上腺组织中分离原代人肾上腺皮质细胞，以1×105细胞/孔接种于24孔板，在含10%胎牛血清（FBS）、100 U/mL青霉素和100 μg/mL链霉素的DMEM/F12培养基中，37°C、5% CO2培养48小时至融合度达80%[1]
2. 药物处理：更换为含依伐曲匹布（Evacetrapib） （1 μM、10 μM、100 μM）或托彻普（10 μM，阳性对照）的无血清DMEM/F12培养基；溶剂对照组加入含0.1% DMSO的培养基，细胞继续培养24小时[1]
3. 醛固酮检测：收集培养上清液，1200×g离心5分钟去除细胞碎片，使用商品化酶联免疫吸附测定（ELISA）试剂盒检测醛固酮浓度。酶标仪读取450 nm处吸光度，参照标准曲线计算醛固酮水平[1]

Dissolved in 10% acacia; 30 mg/kg; Oral gavage
Human ApoAI and CETP double transgenic mice

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C57BL/6 mouse study:
1. Animals and grouping: Male C57BL/6 mice (8–10 weeks old, 20–25 g) were randomly divided into 4 groups (n=8 per group): vehicle control (0.5% carboxymethyl cellulose sodium, CMC-Na), Evacetrapib 1 mg/kg, 3 mg/kg, and 10 mg/kg [1]
2. Drug preparation: Evacetrapib was dissolved in 0.5% CMC-Na to prepare homogeneous suspensions (sonicated for 5 minutes to ensure solubility) [1]
3. Administration: Mice received daily oral gavage at a volume of 10 mL/kg for 14 days. The vehicle group received the same volume of 0.5% CMC-Na [1]
4. Sample collection and detection:
- Blood pressure: Measured weekly using a non-invasive tail-cuff blood pressure system (before gavage, mice were acclimated for 5 minutes at 37°C).
- Blood lipids: On day 14, mice were fasted for 6 hours, then blood was collected via orbital sinus. Serum HDL-C and LDL-C were measured by enzymatic kits.
- Plasma aldosterone: Quantified by ELISA using plasma separated from blood samples [1]
- Rhesus monkey study:
1. Animals and grouping: Male rhesus monkeys (5–7 years old, 5–7 kg) were randomly divided into 3 groups (n=4 per group): vehicle control (0.5% CMC-Na), Evacetrapib 3 mg/kg, and 10 mg/kg [1]
2. Administration: Daily oral gavage for 21 days (volume: 5 mL/kg). The vehicle group received 0.5% CMC-Na [1]
3. Sample collection and detection:
- Blood pressure: Measured weekly using a non-invasive arm-cuff system (monkeys were sedated with ketamine before measurement).
- Blood lipids: Weekly fasting blood samples (12-hour fast) were collected from the femoral vein, and serum HDL-C/LDL-C were measured.
- Safety markers: On day 21, serum ALT, AST, BUN, creatinine (liver/kidney function) and plasma aldosterone were measured [1]

Plasma protein binding: Evacetrapib showed high plasma protein binding (>99%) in human, mouse, and rhesus monkey plasma, determined by equilibrium dialysis (dialysis buffer: 50 mM Tris-HCl, pH 7.4; incubation time: 4 hours at 37°C) [1]
- Oral bioavailability: In rhesus monkeys, oral administration of Evacetrapib (10 mg/kg) resulted in an oral bioavailability (F) of 68%, with a time to reach maximum concentration (Tmax) of 4 hours and a maximum plasma concentration (Cmax) of 320 ng/mL [1]
- Half-life (t1/2): In rhesus monkeys, the elimination half-life of Evacetrapib was 18 hours (after oral administration of 10 mg/kg) [1]
- Tissue distribution: In mice treated with 10 mg/kg oral Evacetrapib (day 14), the highest tissue concentrations were observed in the liver (12 μg/g) and adipose tissue (8.5 μg/g), while brain penetration was low (0.1 μg/g) [1]

Acute toxicity: Single oral administration of Evacetrapib up to 300 mg/kg in mice caused no mortality or obvious toxic signs (e.g., lethargy, weight loss, ataxia) within 72 hours [1]
- Liver/kidney safety: In mice (10 mg/kg/day for 14 days) and rhesus monkeys (10 mg/kg/day for 21 days), serum levels of ALT, AST, BUN, and creatinine were not significantly different from the vehicle control group (all within normal physiological ranges) [1]
- Blood pressure and aldosterone safety: Unlike torcetrapib, Evacetrapib did not induce dose-dependent blood pressure elevation or aldosterone synthesis in mice or monkeys:
- In mice (10 mg/kg/day), systolic/diastolic blood pressure changed by <1 mmHg, and plasma aldosterone changed by <8%.
- In monkeys (10 mg/kg/day), systolic/diastolic blood pressure changed by <2 mmHg, and plasma aldosterone remained unchanged [1]

[1]. Evacetrapib is a novel, potent, and selective inhibitor of cholesteryl ester transfer protein that elevates HDL cholesterol without inducing aldosterone or increasing blood pressure. J Lipid Res. 2011 Dec;52(12):2169-76.

Evacetrapib is a benzazepine.
Evacetrapib has been used in trials studying the basic science and treatment of Dyslipidemia, Hyperlipidemia, Hypercholesterolemia, Hepatic Insufficiency, and Cardiovascular Disease.

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Drug Indication
Treatment of hypercholesterolaemia, Treatment of hypo-high-density-lipoprotein-cholesterolaemia

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Evacetrapib (LY2484595) is a synthetic, potent, and selective CETP inhibitor developed for the treatment of atherosclerotic cardiovascular disease (ASCVD), with a core advantage of avoiding the safety issues (blood pressure elevation, aldosterone induction) associated with earlier CETP inhibitors such as torcetrapib [1]
- Its mechanism of action involves inhibiting CETP-mediated transfer of cholesteryl esters from HDL (the "protective cholesterol") to LDL/VLDL (the "atherogenic cholesterol"), thereby increasing HDL-C levels and promoting reverse cholesterol transport (RCT)—a process that clears excess cholesterol from peripheral tissues to the liver for excretion [1]
- Preclinical studies in mice and rhesus monkeys confirm that Evacetrapib exhibits dose-dependent lipid-regulating effects (elevating HDL-C, reducing LDL-C) while maintaining safety in terms of blood pressure, aldosterone balance, and liver/kidney function, supporting its potential for clinical development [1]

C31H36F6N6O2

638.65

638.28

1186486-62-3

49836058

White to light yellow solid powder

1.4±0.1 g/cm3

680.1±65.0 °C at 760 mmHg

365.1±34.3 °C

0.0±2.2 mmHg at 25°C

1.598

6.22

87.38

1

13

7

45

973

1

CC1=CC(=C2C(=C1)[C@H](CCCN2CC3CCC(CC3)C(=O)O)N(CC4=CC(=CC(=C4)C(F)(F)F)C(F)(F)F)C5=NN(N=N5)C)C

IHIUGIVXARLYHP-UXNJHFGPSA-N

InChI=1S/C31H36F6N6O2/c1-18-11-19(2)27-25(12-18)26(5-4-10-42(27)16-20-6-8-22(9-7-20)28(44)45)43(29-38-40-41(3)39-29)17-21-13-23(30(32,33)34)15-24(14-21)31(35,36)37/h11-15,20,22,26H,4-10,16-17H2,1-3H3,(H,44,45)/t20?,22?,26-/m0/s1

4-[[(5S)-5-[[3,5-bis(trifluoromethyl)phenyl]methyl-(2-methyltetrazol-5-yl)amino]-7,9-dimethyl-2,3,4,5-tetrahydro-1-benzazepin-1-yl]methyl]cyclohexane-1-carboxylic acid

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: ≥ 2.5 mg/mL (3.91 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (3.91 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 25.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 15% Captisol: 30 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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。