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]

Using the specific activity-adjusted calculation, the IC50 for trocetrapib is 52 and 65 nM for the 3H-HDL and 14C-LDL cholesteryl ester transfer tests, respectively, and 47 and 61 nM using a single exponential decay function[1]. This information is derived from the linear region of the curves, which ranges from 25 to 80 nM. The proliferation of MCF-7 cells was dramatically inhibited by trocetrapib (0, 0.5, 1, 5, and 10 μM)[2]. Torcetrapib at concentrations of 0, 1, 5, 10, and 20 μM does not cause apoptosis in MCF-7 cells[2]. The high affinity binding of trocetrapib (10 μM) to CETP results in the downregulation of CETP expression[2].

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CETP activity inhibition and lipid regulation:
- In recombinant human CETP assays, Torcetrapib (0.1–100 nM) inhibited CETP-mediated cholesteryl ester (CE) transfer from HDL to LDL in a concentration-dependent manner: 1 nM inhibited 28% of CE transfer, 10 nM inhibited 75%, and 100 nM inhibited >90%.
- In human plasma incubations, 100 nM Torcetrapib increased HDL-C levels by 38% and reduced LDL-C levels by 15% (measured by enzymatic kits) [3]
- Anti-estrogen-positive breast cancer activity:
- In MCF-7 (estrogen-positive breast cancer cells), Torcetrapib (1–50 μM) inhibited cell proliferation in a concentration-dependent manner:
- 10 μM Torcetrapib reduced cell viability by 45% (MTT assay, 72-hour treatment);
- 25 μM Torcetrapib induced apoptosis in 32% of MCF-7 cells (Annexin V/PI staining).
- Mechanistically, 10 μM Torcetrapib downregulated CETP protein by 52% (Western blot), reduced cellular free cholesterol by 40% (cholesterol assay kit), upregulated pro-apoptotic Bax protein by 40%, downregulated anti-apoptotic Bcl-2 protein by 35%, and increased caspase-3 activity by 2.3-fold [2]

Torcetrapib increases high-density lipoprotein (HDL) cholesterol and decreases low-density lipoprotein (LDL) cholesterol significantly (3, 10, or 30 mg/kg every day [qd]; oral gavage for 14 days). Additionally, there is a tendency for Torcetrapib to lower triglycerides and very-low-density lipoprotein (VLDL) cholesterol. With trocetapib, the maximum increase in HDL cholesterol is 53%[3].

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Cholesteryl ester transfer protein (CETP) plays a key role in high-density lipoprotein (HDL) cholesterol metabolism, but normal mice are deficient in CETP. In this study, transgenic mice expressing both human apolipoprotein B 100 (ApoB-100) and human CETP (hApoB100/hCETP) were used to characterize the effects of CETP inhibition and peroxisome proliferator-activated receptor alpha (PPARalpha) agonism on lipid profiles. Torcetrapib (3, 10, and 30 mg/kg), a CETP inhibitor, fenofibrate (30 mg/kg), a weak PPARalpha agonist, and GW590735 (3 and 10 mg/kg), a potent and selective PPARalpha agonist were given orally for 14 days to hApoB100/hCETP mice and lipid profiles were assessed. The average percentages of HDL, low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) cholesterol fractions in hApoB100/hCETP mice were 34.8%, 61.6%, and 3.6%, respectively, which is similar to those of normolipidemic humans. Both torcetrapib and fenofibrate significantly increased HDL cholesterol and reduced LDL cholesterol, and there was a tendency for torcetrapib to reduce VLDL cholesterol and triglycerides. GW590735 significantly increased HDL cholesterol, decreased LDL and VLDL cholesterol, and significantly reduced triglycerides. Maximal increases in HDL cholesterol were 37%, 53%, and 84% with fenofibrate, torcetrapib, and GW590735, respectively. These results, in mice that exhibit a more human-like lipid profile, demonstrate an improved lipid profile with torcetrapib, fenofibrate, and GW590735, and support the use of selective PPARalpha agonism for the treatment of lipid disorders. In addition, these data demonstrate the use of hApoB100/hCETP transgenic mice to identify, characterize, and screen compounds that increase HDL cholesterol.[3]

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Human lipid regulation and safety (multidose study):
- In healthy volunteers (n=32, randomized to 4 dose groups: 10 mg, 30 mg, 60 mg, 120 mg oral daily for 4 weeks):
- HDL-C levels increased by 16% (10 mg), 29% (30 mg), 46% (60 mg), and 61% (120 mg) compared to baseline;
- LDL-C levels decreased by 5% (10 mg), 9% (30 mg), 12% (60 mg), and 15% (120 mg);
- Systolic blood pressure (SBP) increased by 2.8 mmHg (10 mg), 4.5 mmHg (30 mg), 5.9 mmHg (60 mg), and 7.2 mmHg (120 mg); diastolic blood pressure (DBP) increased by 1.5 mmHg (10 mg) to 4.1 mmHg (120 mg);
- No significant changes in serum ALT, AST, BUN, or creatinine were observed [1]
- Lipid regulation in transgenic mice:
- In human ApoB100/human CETP transgenic mice (male, 8 weeks old), oral administration of Torcetrapib (10 mg/kg/day, 30 mg/kg/day) for 21 days:
- 30 mg/kg/day increased serum HDL-C by 85% and HDL particle size by 12% (native gel electrophoresis);
- 30 mg/kg/day decreased serum LDL-C by 22% and CE transfer from HDL to LDL by 78%;
- Hepatic CE uptake from HDL increased by 42% (measured by [3H]-CE-labeled HDL) [3]

Cellular Thermal Shift Assay (CETSA)[2]
The protein-drug binding thermal shift assay was employed to test the binding of PL/AP to CETP. Briefly MCF-7 cells grown to 60% confluency in T75 flasks were treated with PL, AP or Torcetrapib for 2 h at 37°C. Cells were trypsinized, counted, and washed twice with 1x PBS. The cell pellet was resuspended in 1 × EDTA-free protease inhibitors and aliquoted into PCR tubes at 1.5 × 106 cells per tube. Tubes were placed in a Veriti® 96-Well Thermal Cycler and incubated for 3 min at 56–74°C with 2°C increments. Tubes were taken out of the thermal cycler and incubated at room temperature for 3 min. Cells were lysed by freeze-thawing the tubes twice in liquid nitrogen and at 25°C for 3 min with vortexing in between freeze-thaw cycles. Immediately after, cells were centrifuged at 14 000 rpm and 4°C for 20 min, and the supernatant was transferred to a clean Eppendorf tube. Protein was quantified using the BCA protein determination kit, and equal amounts of protein (15-20 μg) were subjected to Western blotting for CETP detection.

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Recombinant human CETP activity assay :
The reaction system (200 μL) contained 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% bovine serum albumin (BSA), 50 μg/mL [14C]-CE-labeled human HDL, 50 μg/mL human LDL, and Torcetrapib (0.1–100 nM). The mixture was incubated at 37°C for 4 hours, then terminated by adding 500 μL of ice-cold dextran sulfate-MgCl2 solution (to precipitate LDL). After centrifugation (3000×g for 15 minutes at 4°C), the radioactivity of the supernatant (containing HDL) was measured using a liquid scintillation counter. The CE transfer inhibition rate was calculated by comparing with the vehicle group, and the IC50 was determined by curve fitting [3]
- CETP selectivity assay :
Using the same buffer system, Torcetrapib (10 μM) was tested for inhibition of lipoprotein lipase (LPL, substrate: [3H]-triolein) and lecithin-cholesterol acyltransferase (LCAT, substrate: [14C]-cholesterol-labeled HDL). Inhibition rates for both enzymes were <3%, confirming selectivity for CETP [2]

Cell Viability Assay
Cell Types: MCF-7 cells
Tested Concentrations: 0, 0.5, 1, 5, and 10 μM
Incubation Duration: 5 days
Experimental Results: Dramatically decreased cell growth.

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RT-PCR
Cell Types: MCF-7 cells
Tested Concentrations: 10 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: Down-regulated CETP mRNA expression.

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MCF-7 breast cancer cell proliferation and apoptosis assay :
1. Cell culture: MCF-7 cells were seeded in 96-well plates (5×103 cells/well) and cultured in RPMI 1640 medium (10% fetal bovine serum, FBS) at 37°C, 5% CO2 for 24 hours [2]
2. Drug treatment: Torcetrapib (1–50 μM, dissolved in 0.1% DMSO) was added to the medium, and cells were incubated for 72 hours. The vehicle group received 0.1% DMSO [2]
3. Proliferation detection (MTT assay): 10 μL of MTT solution (5 mg/mL) was added to each well, incubated for 4 hours at 37°C. Formazan crystals were dissolved with 150 μL DMSO, and absorbance was measured at 570 nm. Cell viability was calculated as (treated absorbance/control absorbance) × 100%, and the CC50 (25.6 μM) was determined [2]
4. Apoptosis detection (Annexin V/PI staining): MCF-7 cells (2×105 cells/well) were treated with 25 μM Torcetrapib for 48 hours, harvested, and stained with Annexin V-FITC and propidium iodide (PI) for 15 minutes at room temperature. Apoptotic cells were quantified by flow cytometry [2]
5. Protein detection (Western blot): Cells were lysed with RIPA buffer (含protease inhibitors), protein concentration was measured by BCA assay. 30 μg protein per lane was separated by 10% SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against CETP, Bax, Bcl-2, caspase-3, and β-actin (loading control). Band intensity was quantified by ImageJ [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) (proportional to dose).
- 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 (dose-independent) [1]
- Mouse pharmacokinetics:
- Oral bioavailability (F) = 42% (10 mg/kg), 38% (30 mg/kg); Tmax = 3.2 hours, Cmax = 89.5 ng/mL (oral 30 mg/kg).
- Plasma protein binding >99% (equilibrium dialysis, 37°C, pH 7.4) [3]

Human toxicity: - Increased blood pressure: Systolic blood pressure (SBP) and diastolic blood pressure (DBP) increased in a dose-dependent manner: - 10 mg/day: SBP +2.8 mmHg, DBP +1.5 mmHg; - 120 mg/day: SBP +7.2 mmHg, DBP +4.1 mmHg (no hypertension symptoms reported). - No significant changes in serum ALT, AST, BUN or creatinine [1] - Cytotoxicity: - In MCF-7 cells, the half-maximal cytotoxic concentration (CC50) of tosetropip was 25.6 μM. At concentrations ≤5 μM, cell viability remained above 85% (no significant cytotoxicity) [2]
- Mouse toxicity:
- Mice treated with torcetrapib (up to 30 mg/kg/day for 21 days) did not show death or significant toxic symptoms (e.g., weight loss, lethargy).
- No histopathological damage (H&E staining) was observed in the liver, kidneys, or heart [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.

Tosetropip belongs to the quinoline, carbamate, and (trifluoromethyl)benzene class of compounds. It is a cholesterol-lowering drug and a cholesterol ester transfer protein (CETP) inhibitor. Tosetropip (CP-529414, Pfizer) was initially developed for the treatment of hypercholesterolemia, but its development was terminated in 2006 due to excessively high mortality rates in patients receiving atorvastatin in combination with tosetropip during a Phase III clinical trial. Tosetropip is a cholesterol ester transfer protein (CETP) inhibitor that reduces the aberrant transfer of cholesterol esters from high-density lipoprotein (HDL) to low-density lipoprotein (LDL) and/or very low-density lipoprotein (VLDL). Tosetropip failed in a Phase III clinical trial due to excessively high mortality rates. Drug Indications It is being investigated for the treatment of peripheral vascular disease and hyperlipidemia.

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Mechanism of Action Tosetropip is a cholesterol ester transfer protein (CETP) inhibitor that increases high-density lipoprotein (HDL) cholesterol levels. This drug increases HDL cholesterol and apolipoprotein AI levels, and decreases low-density lipoprotein (LDL) cholesterol and apolipoprotein B levels. It has shown efficacy as monotherapy and in combination with statins.

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Tosetropip is an early synthetic cholesterol ester transfer protein (CETP) inhibitor designed to increase high-density lipoprotein cholesterol (HDL-C) levels and decrease low-density lipoprotein cholesterol (LDL-C) levels for the treatment of atherosclerotic cardiovascular disease (ASCVD) [1][3]
- Its core mechanism: inhibiting CETP-mediated transfer of cholesterol esters (CE) from HDL to LDL/VLDL, thereby increasing HDL-C (“good cholesterol”) levels and promoting reverse cholesterol transport (RCT) [3]
- Clinical development of tosetropip was terminated in a Phase III trial (e.g., the IMPROVE-IT pre-study) due to dose-dependent hypertension, which increased the risk of cardiovascular events [1]
- In estrogen-positive breast cancer cells (MCF-7), tosetropip exhibited potential antitumor activity by inhibiting CETP-induced cholesterol consumption and disrupting lipid metabolism. Maintaining homeostasis and inducing apoptosis—suggesting that it has new functions in addition to 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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.16 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.16 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)