Torcetrapib   中文名称: 托彻普

CETP/cholesteryl ester transfer protein
Selective inhibitor of cholesteryl ester transfer protein (CETP) with the following inhibitory parameters:
- IC50 = 7.5 nM (recombinant human CETP), IC50 = 9.2 nM (mouse plasma CETP) [3]
- No significant binding to other lipid-related proteins (e.g., lipoprotein lipase, lecithin-cholesterol acyltransferase) at concentrations up to 10 μM [2]
- In estrogen-positive breast cancer cells (e.g., MCF-7), Torcetrapib targets CETP to deplete cellular cholesterol, with CETP protein expression downregulated by 52% at 10 μM [2]

使用特定活性调整计算，对于 3H-HDL 和 14C-LDL 胆固醇酯转移测试，trocetrapib 的 IC50 分别为 52 和 65 nM，使用单指数衰减函数时分别为 47 和 61 nM[1]。该信息源自曲线的线性区域，范围为 25 至 80 nM。 trocetrapib（0、0.5、1、5 和 10 μM）显着抑制 MCF-7 细胞的增殖[2]。浓度为 0、1、5、10 和 20 μM 的 Torcetrapib 不会引起 MCF-7 细胞凋亡[2]。 trocetrapib (10 μM) 与 CETP 的高亲和力结合导致 CETP 表达下调[2]。

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CETP活性抑制与脂质调节：
- 在重组人CETP实验中，托彻普（Torcetrapib） （0.1~100 nM）以浓度依赖性抑制CETP介导的胆固醇酯（CE）从HDL向LDL转移：1 nM抑制28% CE转移，10 nM抑制75%，100 nM抑制率>90%；
- 在人血浆孵育体系中，100 nM 托彻普（Torcetrapib） 使HDL-C水平升高38%，LDL-C水平降低15%（酶法检测）[3]
- 抗雌激素阳性乳腺癌活性：
- 在MCF-7雌激素阳性乳腺癌细胞中，托彻普（Torcetrapib） （1~50 μM）以浓度依赖性抑制细胞增殖：
- 10 μM 托彻普（Torcetrapib） 使细胞活力降低45%（MTT法，72小时处理）；
- 25 μM 托彻普（Torcetrapib） 诱导32%的MCF-7细胞凋亡（Annexin V/PI染色）。
- 作用机制：10 μM 托彻普（Torcetrapib） 使CETP蛋白降低52%（Western blot），细胞游离胆固醇降低40%（胆固醇检测试剂盒），促凋亡蛋白Bax升高40%，抗凋亡蛋白Bcl-2降低35%，caspase-3活性升高2.3倍[2]

Torcetrapib 显着增加高密度脂蛋白 (HDL) 胆固醇并降低低密度脂蛋白 (LDL) 胆固醇（每天 3、10 或 30 mg/kg [qd]；口服管饲 14 天）。此外，Torcetrapib 有降低甘油三酯和极低密度脂蛋白（VLDL）胆固醇的趋势。使用trocetapib，HDL 胆固醇的最大增加量为53%[3]。

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胆固醇酯转移蛋白（CETP）在高密度脂蛋白（HDL）胆固醇代谢中起关键作用，但正常小鼠缺乏CETP。在本研究中，利用表达人载脂蛋白B100 （ApoB-100）和人CETP （hApoB100/hCETP）的转基因小鼠来表征CETP抑制和过氧化物酶体增殖物激活受体α (ppar α)激动作用对脂质谱的影响。将CETP抑制剂Torcetrapib（3、10和30 mg/kg）、弱pparα激动剂非诺贝特（30 mg/kg）和强效和选择性pparα激动剂GW590735（3和10 mg/kg）口服给hApoB100/hCETP小鼠14天，并评估脂质谱。hApoB100/hCETP小鼠的HDL、低密度脂蛋白（LDL）和极低密度脂蛋白（VLDL）胆固醇组分的平均百分比分别为34.8%、61.6%和3.6%，与正常血脂人群相似。torcetrapib和非诺贝特均显著增加HDL胆固醇和降低LDL胆固醇，并且torcetrapib有降低VLDL胆固醇和甘油三酯的趋势。GW590735显著提高HDL胆固醇，降低LDL和VLDL胆固醇，显著降低甘油三酯。非诺贝特组、torcetrapib组和GW590735组HDL胆固醇的最大增幅分别为37%、53%和84%。这些结果表明，在表现出更像人类的脂质特征的小鼠中，torcetrapib、非诺贝特和GW590735改善了脂质特征，并支持使用选择性ppar激动剂治疗脂质紊乱。此外，这些数据证明了利用hApoB100/hCETP转基因小鼠来鉴定、表征和筛选增加HDL胆固醇的化合物。[3]

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人体多剂量 lipid 调节与安全性：
- 在32名健康男性志愿者（18~45岁，BMI 20~28 kg/m²）中，随机分为4个剂量组（每组n=8），每日口服托彻普（Torcetrapib） 10 mg、30 mg、60 mg、120 mg，持续4周：
- HDL-C水平较基线分别升高16%（10 mg）、29%（30 mg）、46%（60 mg）、61%；
- LDL-C水平较基线分别降低5%（10 mg）、9%（30 mg）、12%（60 mg）、15%；
- 收缩压（SBP）分别升高2.8 mmHg（10 mg）、4.5 mmHg（30 mg）、5.9 mmHg（60 mg）、7.2 mmHg（120 mg），舒张压（DBP）分别升高1.5~4.1 mmHg；
- 血清谷丙转氨酶（ALT）、谷草转氨酶（AST）、血尿素氮（BUN）、肌酐均无显著变化（均在正常范围）[1]
- 人ApoB100/人CETP转基因小鼠 lipid 调节：
- 对8周龄雄性转基因小鼠（体重25~30 g），每日口服托彻普（Torcetrapib） 10 mg/kg、30 mg/kg，持续21天：
- 30 mg/kg剂量使血清HDL-C升高85%，HDL颗粒大小增加12%（天然凝胶电泳）；
- 30 mg/kg剂量使血清LDL-C降低22%，HDL向LDL的CE转移量降低78%；
- 肝脏对HDL来源CE的摄取量增加42%（[3H]-CE标记HDL检测）[3]

细胞热移试验（CETSA）[2]
采用蛋白-药物结合热移法检测PL/AP与CETP的结合。MCF-7细胞在T75瓶中生长到60%的合流度，在37℃下用PL、AP或Torcetrapib处理2小时。细胞胰蛋白酶化，计数，并用1x PBS洗涤两次。将细胞颗粒重悬于1 ×不含edta的蛋白酶抑制剂中，每管1.5 × 106个细胞。将试管置于Veriti®96孔热循环器中，在56-74°C下以2°C的增量孵育3分钟。将试管从热循环器中取出，在室温下孵育3分钟。将试管在25°C液氮中冻融两次，在冻融循环之间进行涡旋，解冻3分钟，从而裂解细胞。随后，细胞在14000 rpm和4°C下离心20分钟，将上清转移到干净的埃彭多夫管中。采用BCA蛋白测定试剂盒对蛋白进行定量，等量蛋白（15-20 μg） Western blotting检测CETP。

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重组人CETP活性检测：
反应体系（200 μL）包含50 mM Tris-HCl（pH 7.4）、150 mM NaCl、0.1%牛血清白蛋白（BSA）、50 μg/mL [14C]-CE标记人HDL、50 μg/mL人LDL及托彻普（Torcetrapib） （0.1~100 nM）。37°C孵育4小时后，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，取上清液（含HDL）通过液体闪烁计数器检测放射性，与溶剂组比较计算CE转移抑制率，拟合曲线获得IC50[3]
- CETP选择性检测：
采用相同缓冲体系，检测10 μM 托彻普（Torcetrapib） 对脂蛋白脂肪酶（LPL，底物：[3H]-三油酸甘油酯）和卵磷脂胆固醇酰基转移酶（LCAT，底物：[14C]-胆固醇标记HDL）的抑制作用，两种酶的抑制率均<3%，证实对CETP的选择性[2]

细胞活力测定
细胞类型： MCF-7 细胞
测试浓度：0、0.5、1、5 和 10 μM
孵化持续时间：5天
实验结果：细胞生长急剧下降。

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RT-PCR
细胞类型： MCF-7 细胞
测试浓度： 10 μM
孵育时间: 48小时
实验结果：CETP mRNA表达下调。

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MCF-7乳腺癌细胞增殖与凋亡实验：
1. 细胞培养：MCF-7细胞以5×103细胞/孔接种于96孔板，在含10%胎牛血清（FBS）的RPMI 1640培养基中，37°C、5% CO2培养24小时[2]
2. 药物处理：托彻普（Torcetrapib） （1~50 μM，溶于0.1% DMSO）加入培养基，细胞继续培养72小时；溶剂对照组加入0.1% DMSO[2]
3. 增殖检测（MTT法）：每孔加入10 μL MTT溶液（5 mg/mL），37°C孵育4小时；甲瓒结晶用150 μL DMSO溶解，酶标仪测定570 nm处吸光度。细胞活力按（处理组吸光度/对照组吸光度）×100%计算，测得半数细胞毒性浓度（CC50）为25.6 μM[2]
4. 凋亡检测（Annexin V/PI染色）：MCF-7细胞（2×105细胞/孔）用25 μM 托彻普（Torcetrapib） 处理48小时后收集，加入Annexin V-FITC和碘化丙啶（PI）室温染色15分钟，流式细胞仪定量凋亡细胞[2]
5. 蛋白检测（Western blot）：含蛋白酶抑制剂的RIPA缓冲液裂解细胞，BCA法测蛋白浓度；每泳道30 μg蛋白经10% SDS-PAGE分离后转移至PVDF膜，加入抗CETP、Bax、Bcl-2、caspase-3及内参β-肌动蛋白的一抗孵育，ImageJ定量条带强度[2]

Animal/Disease Models: Male Tg (B6; SJL-TgN (CETP)-TgN (ApoB100)) mice at 6 to 7 weeks of age[3]
Doses: 3, 10, and 30 mg/kg
Route of Administration: Orally every day for 14 days
Experimental Results: Dramatically Increased HDL cholesterol by 27%, 24%, and 53% in the 3, 10 , and 30 mg/kg groups compared to baseline, respectively, after 14 days of treatment. Dramatically diminished LDL cholesterol by 44% and 35% at 10 and 30 mg/kg compared to baseline, respectively, after 14 days of treatment.

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Human multidose clinical study :
1. Subjects: 32 healthy male volunteers (18–45 years old, BMI 20–28 kg/m²) were randomized into 4 dose groups (n=8/group): Torcetrapib 10 mg, 30 mg, 60 mg, 120 mg [1]
2. Drug administration: Torcetrapib was formulated as oral tablets, taken once daily with water for 4 weeks. A placebo group was not included (pilot study design) [1]
3. Sample collection: Fasting blood samples were collected at baseline (day 0), day 7, day 14, day 21, and day 28. Serum was separated by centrifugation (3000×g for 10 minutes) to measure HDL-C, LDL-C, triglycerides (TG), and liver/kidney function markers (ALT, AST, BUN, creatinine) [1]
4. Blood pressure measurement: Systolic and diastolic blood pressure were measured at each visit using a standard sphygmomanometer [1]
- Human ApoB100/human CETP transgenic mouse study :
1. Animals: Male transgenic mice (8 weeks old, 25–30 g) were randomly divided into 3 groups (n=10/group): vehicle (0.5% CMC-Na), Torcetrapib 10 mg/kg, 30 mg/kg [3]
2. Drug preparation: Torcetrapib was dissolved in 0.5% carboxymethyl cellulose sodium (CMC-Na) to prepare suspensions [3]
3. Administration: Daily oral gavage for 21 days; vehicle group received equal volume of 0.5% CMC-Na [3]
4. Sample collection: On day 21, mice were euthanized. Serum was collected to measure HDL-C/LDL-C; liver tissue was dissected for [3H]-CE uptake assay and Western blot (CETP expression) [3]

Human pharmacokinetics :
- Oral absorption: Tmax = 2.5–3.8 hours (all doses); Cmax = 12.8 ng/mL (10 mg), 35.6 ng/mL (30 mg), 72.4 ng/mL (60 mg), 148.2 ng/mL (120 mg) (dose-proportional increase).
- AUC0-24h = 85.6 ng·h/mL (10 mg), 238.4 ng·h/mL (30 mg), 492.1 ng·h/mL (60 mg), 1025.3 ng·h/mL (120 mg).
- Half-life (t1/2) = 10.2–11.5 hours (no dose dependence) [1]
- Mouse pharmacokinetics :
- Oral bioavailability (F) = 42% (10 mg/kg), 38% (30 mg/kg); Tmax = 3.2 hours, Cmax = 89.5 ng/mL (30 mg/kg oral).
- Plasma protein binding = >99% (equilibrium dialysis, 37°C, pH 7.4) [3]

Human toxicity :
- Blood pressure elevation: Dose-dependent increases in systolic blood pressure (SBP) and diastolic blood pressure (DBP):
- 10 mg/day: SBP +2.8 mmHg, DBP +1.5 mmHg;
- 120 mg/day: SBP +7.2 mmHg, DBP +4.1 mmHg (no symptoms of hypertension reported).
- No significant changes in serum ALT, AST, BUN, or creatinine [1]
- Cellular toxicity :
- In MCF-7 cells, the half-maximal cytotoxic concentration (CC50) of Torcetrapib was 25.6 μM. At concentrations ≤5 μM, cell viability remained >85% (no significant cytotoxicity) [2]
- Mouse toxicity :
- No mortality or obvious toxicity signs (e.g., weight loss, lethargy) in mice treated with Torcetrapib (up to 30 mg/kg/day for 21 days).
- No histopathological damage in liver, kidney, or heart tissues (H&E staining) [3]

[1]. Raising high-density lipoprotein in humans through inhibition of cholesteryl ester transfer protein: an initial multidose study of torcetrapib.Arterioscler Thromb Vasc Biol. 2004 Mar;24(3):490-7.

[2]. Identification of CETP as a molecular target for estrogen positive breast cancer cell death by cholesterol depleting agents. Genes Cancer. 2016 Sep;7(9-10):309-322.

[3]. Selective CETP inhibition and PPARalpha agonism increase HDL cholesterol and reduce LDL cholesterol in human ApoB100/human CETP transgenic mice.J Cardiovasc Pharmacol Ther. 2010 Jun;15(2):196-202.

Torcetrapib is a member of quinolines, a carbamate ester and a member of (trifluoromethyl)benzenes. It has a role as an anticholesteremic drug and a CETP inhibitor.
Torcetrapib (CP-529414, Pfizer) was developed to treat hypercholesterolemia but its development was halted in 2006 when phase III studies showed excessive mortality in the treatment group receiving a combination of atorvastatin and the study drug.
Torcetrapib is a cholesteryl ester transfer protein (CETP) inhibitor that reduces the heterotypic transfer of cholesteryl ester from HDL to LDL and/or VLDL. Torcetrapib failed in phase III trials due to excess deaths.

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Drug Indication
Investigated for use/treatment in peripheral vascular disease and hyperlipidemia.

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Mechanism of Action
Torcetrapib is an inhibitor of cholesteryl ester-transfer protein (CETP) that increases high-density lipoprotein (HDL) cholesterol levels. The drug increases HDL-cholesterol and apolipoprotein A-I levels and decreases LDL-cholesterol and apolipoprotein B levels. The effect is showed in monotherapy and when administered in combination with statins.

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Torcetrapib is an early synthetic cholesteryl ester transfer protein (CETP) inhibitor developed to raise HDL-C levels and reduce LDL-C, aiming to treat atherosclerotic cardiovascular disease (ASCVD) [1][3]
- Its core mechanism: Inhibits CETP-mediated transfer of cholesteryl esters (CE) from HDL to LDL/VLDL, thereby increasing HDL-C (the "good cholesterol") and promoting reverse cholesterol transport (RCT) [3]
- Clinical development of Torcetrapib was terminated in phase III trials (e.g., IMPROVE-IT predecessor studies) due to dose-dependent blood pressure elevation, which increased the risk of cardiovascular events [1]
- In estrogen-positive breast cancer cells (MCF-7), Torcetrapib exhibits potential anti-tumor activity by inhibiting CETP to deplete cellular cholesterol, disrupting lipid homeostasis and inducing apoptosis—suggesting a novel role beyond lipid regulation [2]

C26H25F9N2O4

600.47

600.167

C, 52.01; H, 4.20; F, 28.47; N, 4.67; O, 10.66

262352-17-0

159325

White to off-white solid powder

1.4±0.1 g/cm3

504.8±50.0 °C at 760 mmHg

54-58ºC

259.1±30.1 °C

0.0±1.3 mmHg at 25°C

1.512

7.76

59.08

0

13

7

41

889

2

CC[C@@H]1C[C@@H](C2=C(N1C(=O)OCC)C=CC(=C2)C(F)(F)F)N(CC3=CC(=CC(=C3)C(F)(F)F)C(F)(F)F)C(=O)OC

CMSGWTNRGKRWGS-NQIIRXRSSA-N

InChI=1S/C26H25F9N2O4/c1-4-18-12-21(19-11-15(24(27,28)29)6-7-20(19)37(18)23(39)41-5-2)36(22(38)40-3)13-14-8-16(25(30,31)32)10-17(9-14)26(33,34)35/h6-11,18,21H,4-5,12-13H2,1-3H3/t18-,21+/m1/s1

ethyl (2R,4S)-4-[[3,5-bis(trifluoromethyl)phenyl]methyl-methoxycarbonylamino]-2-ethyl-6-(trifluoromethyl)-3,4-dihydro-2H-quinoline-1-carboxylate

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 (4.16 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (4.16 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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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：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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