Cholesteryl ester transfer protein (CETP); recombinant human (rh) CETP (IC50 = 204.6 nM)[1]; human plasma CETP (IC50 = 6 μM)[2]

Dalcetrapib (JTT-705) (0.1-10 μM; 21 h) enhances pre-β-HDL production in a dose-dependent manner[1]. In HepG2, Dalcetrapib (0-30 μM; 24 h) dose-dependently suppresses the media's CETP activity[3].

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The mechanism by which cholesteryl ester transfer protein (CETP) activity affects HDL metabolism was investigated using agents that selectively target CETP (dalcetrapib, torcetrapib, anacetrapib). In contrast with torcetrapib and anacetrapib, dalcetrapib requires cysteine 13 to decrease CETP activity, measured as transfer of cholesteryl ester (CE) from HDL to LDL, and does not affect transfer of CE from HDL3 to HDL2. Only dalcetrapib induced a conformational change in CETP, when added to human plasma in vitro, also observed in vivo and correlated with CETP activity. CETP-induced pre-β-HDL formation in vitro in human plasma was unchanged by dalcetrapib ≤3 µM and increased at 10 µM. A dose-dependent inhibition of pre-β-HDL formation by torcetrapib and anacetrapib (0.1 to 10 µM) suggested that dalcetrapib modulates CETP activity. [1]

In rabbits, dacletrapib (JTT-705) (30 or 100 mg/kg; po; once daily for three days) considerably raises plasma HDL cholesterol[2]. Neutral sterol, bile acids, and plasma HDL-cholesterol are considerably increased in the feces upon administration of dacletrapib (100 mg/kg; ir; twice daily for 7 days)[1].

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In hamsters injected with [3H]cholesterol-labeled autologous macrophages, and given dalcetrapib (100 mg twice daily), torcetrapib [30 mg once daily (QD)], or anacetrapib (30 mg QD), only dalcetrapib significantly increased fecal elimination of both [3H]neutral sterols and [3H]bile acids, whereas all compounds increased plasma HDL-[3H]cholesterol. These data suggest that modulation of CETP activity by dalcetrapib does not inhibit CETP-induced pre-β-HDL formation, which may be required to increase reverse cholesterol transport.[1]

Selective binding of dalcetrapib to Cys13.[1]
CETP containing a serine residue instead of Cys13 (C13S CETP) was constructed by site-directed mutagenesis. The protein was expressed in HEK293EBNA cells from large-scale transient transfection, and purified as described below for recombinant human (rh)CETP.

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Inhibition of rhCETP and C13S CETP-mediated transfer of CE from HDL to LDL.[1]
The inhibitory potency (IC50) of dalcetrapib, torcetrapib, and anacetrapib to decrease CE transfer from HDL to LDL by rhCETP and C13S CETP was measured using a scintillation proximity assay kit. Briefly, [3H]CE-labeled HDL donor particles were incubated in the presence of purified CETP proteins (final concentration 0.5 µg/ml) and biotinylated LDL acceptor particles for 3 h at 37°C. Subsequently, streptavidin-coupled polyvinyltoluene beads containing liquid scintillation cocktail binding selectively to biotinylated LDL were added, and the amount of [3H]CE molecules transferred to LDL was measured by β counting.

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Inhibition of transfer of CE from HDL3 to HDL2.[1]
Assessment of lipid movement among HDL subfractions was performed using radiolabeled lipid transfer assays as previously described. Lipoprotein subfraction (d > 1.063 g/ml) was labeled with [3H]CE. [3H]CE-labeled HDL3 (1.125 < d < 1.210 g/ml) was prepared by sequential ultracentrifugation. [3H]CE-labeled HDL3 and nonradiolabeled HDL2 (1.063 < d < 1.125 g/ml) were added on an equal phospholipid basis (2.3 μg/tube). The lipoprotein mixture was incubated in the presence of 1% BSA, 21 mM tris-HCl (pH 7.4), 0.5% NaCl, and 0.006% EDTA with or without rhCETP (0.5 μg/tube). Dalcetrapib, torcetrapib, and anacetrapib were tested at concentrations of 0.001, 0.01, 0.1, 1, and 10 µM in a total volume of 0.715 ml and incubated for 4 h at 37°C. After incubation, HDL2 and HDL3 fractions were separated by ultracentrifugation (d = 1.125 g/ml) for 19 h at 4°C. Total radioactivity in the HDL2 (upper layer) and HDL3 (lower layer) subfractions was measured by scintillation counting. CETP activity was expressed as the percentage of total radioactivity recovered in the HDL2 fraction.

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Binding sites of compounds on CETP: competition for binding site on sepharose-immobilized rhCETP.[1]
Binding studies were performed according to Connolly et al. using rhCETP expressed by a cell line described by Weinberg et al and purified by hydrophobic interaction chromatography and size exclusion chromatography (SEC). BSA and rhCETP were immobilized on CNBr-activated sepharoseTM 4 Fast Flow. Both competition (co-incubation experiments) and displacement after preincubation [the latter with or without the reducing agent tris(2-carboxyethyl)phosphine (TCEP)] with radioactive compound were determined for 300 pmol immobilized rhCETP (3 μM) or the same mass of BSA mixed with 0.25 μM [14C]torcetrapib or 2.5 μM [14C]dalcetrapib, respectively, and unlabeled CETP inhibitors in a total volume of 100 μl. [14C]dalcetrapib was treated with pancreatic lipase to produce [14C]dalcetrapib-thiol prior to incubation with CETP. Radioactivity bound to sepharose was measured by scintillation counting.

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In Vitro CETP Activity. [2]
In vitro CETP activity was determined form the rate of [3H]cholesteryl ester transfer from HDL to apoprotein B-containing lipoproteins.13,14 Blood was collected from normolipidemic volunteers into tubes containing heparin. Plasma was isolated by centrifugation at 1500g for 15−30 min at 4 °C. An appropriate amount of each test compound, dissolved in a 1:1 mixture of N-methyl-2-pyrrolidinone and poly(ethylene glycol) (with an average molecular weight of 400), was added to the plasma. The final concentration of the organic solvent was 2%. After the reaction mixture with HDL containing [3H]CE was incubated at 37 °C for 4 h, apoprotein B-containing lipoproteins were precipitated using dextran sulfate and magnesium chloride (at final concentrations of 0.075% and 37.5 mM, respectively), and 100 μL of the supernatant containing HDL was used for counting the radioactivity. CETP-mediated transfer was determined by the decrease in radioactivity of the supernatant, and the concentration achieving 50% inhibition of CETP activity was estimated from the dose−response curves obtained with five doses.

CETP ODNs and CETP chemical inhibitor Dalcetrapib (JTT-705) delivery.[3]
The HepG2 cells were plated in 6-well plates for transfection. ODNs were delivered into cultured HepG2 cells by means of cationic liposome Tfx-20. Sense or antisense ODNs and liposomes (12 μM) were mixed in Opti-MEM I. The cultured cells at 70–80% confluence were rinsed twice with PBS before the addition of the ODNs-liposome mixture. The cells were exposed to the mixture for 12 h at 37°C, 5% CO2, and 100% humidity and were returned to the growth medium. After 8 h of incubation, the cells were rinsed twice with PBS before total RNA and cellular protein preparation. After 24 h of incubation, the growth medium and cells were collected for CETP mass determination and protein quantitation. Different concentrations of ODNs (0–8 μM) were determined.
The HepG2 cells were plated in 6-well plates and cultured to 70–80% confluence. After being washed with PBS, the cells were incubated with growth medium and a different concentration (0–30 μM) of chemical inhibitor Dalcetrapib (JTT-705) was dissolved in 2% DMSO for 24 h. Total RNA was used for RT-PCR.

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These ELISAs were applied to plasma samples from 22 healthy subjects receiving a single oral dose of 600 mg dalcetrapib. Plasma samples were collected before, and 2, 4, 6, 8, 12, and 24 h after dalcetrapib intake.Dalcetrapib (JTT-705) level was measured as described elsewhere and CETP immunoreactivity was determined using the ELISA described above with JHC-1 or 6/2 as capture antibody and 6/6 or 6/17 for detection. CETP activity was determined by an ex vivo CETP activity assay kit.[1]

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Quantification of pre-beta-HDL by ELISA.:Samples with or without added rhCETP were incubated for 21 h in the presence of torcetrapib, anacetrapib, and Dalcetrapib (JTT-705) (0.10 µM to 10 µM). Pre-β-HDL concentration was measured by ELISA as previously described[1].

Animal/Disease Models: Male JW rabbits[2]
Doses: 30 or 100 mg/kg
Route of Administration: Oral administration, one time/day for 3 days
Experimental Results: Increased plasma HDL cholesterol by 27% and 54% at 30 mg/kg and 100 mg/kg, respectively.

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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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Ex Vivo CETP Activity. [2]
Ex vivo CETP activity was measured in whole plasma obtained from male JW rabbits (11−15 weeks old), which were orally administered the test compounds (10 or 30 mg/kg). 15 Plasma was isolated by centrifugation at 11000g for 5 min at 4 °C. Whole plasma (1.8 μL) from control or treated rabbits was diluted to 600 μL with Tris-buffered saline (pH 7.4) containing 0.1 mg/mL of bovine serum albumin and was incubated at 37 °C for 15 h in the presence of HDL containing [3H]CE and VLDL/LDL. After precipitation of VLDL/LDL using dextran sulfate and magnesium chloride, the radioactivity of the supernatant containing HDL was counted. CETP activity was determined by the decrease in radioactivity versus that of the blank sample without plasma.

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In Vivo Plasma HDL Cholesterol Level. HDL cholesterol levels were measured in the plasma after precipitation of apoprotein B-containing lipoproteins with 13% poly(ethylene glycol) (at an average molecular weight of 6000) by an enzyme assay. Plasma was obtained from male JW rabbits (11−15 weeks old, N = 5), which were orally administered compound 27/Dalcetrapib (JTT-705) (30 or 100 mg/kg) once a day for 3 days.[2]

[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]. J Med Chem. bis(2-(Acylamino)phenyl) disulfides, 2-(acylamino)benzenethiols, and S-(2-(acylamino)phenyl) alkanethioates as novel inhibitors of cholesteryl ester transfer protein. 2000, 43(19), 3566-3572.

[3]. Dual effects on HDL metabolism by cholesteryl ester transfer protein inhibition in HepG2 cells. Am J Physiol Endocrinol Metab. 2003, 284(6), E1210-E1219.

2-methylpropanethioic acid S-[2-[[[1-(2-ethylbutyl)cyclohexyl]-oxomethyl]amino]phenyl] ester is an anilide.
Dalcetrapib is under investigation for the treatment of Acute Coronary Syndrome.
Dalcetrapib is a cholesteryl ester transfer protein (CETP) inhibitor that can produce an increase in serum HDL-cholesterol levels and a decrease in serum LDL-cholesterol levels. Dalcetrapib is structurally dissimilar from other CETP inhibitors, including torcetrapib, and raises functional HDL by a different mechanism.

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Drug Indication
Lipoprotein deficiency

C23H35NO2S

389.59

389.239

C, 70.91; H, 9.06; N, 3.60; O, 8.21; S, 8.23

211513-37-0

6918540

White to off-white solid powder

1.066 g/cm3

528.912ºC at 760 mmHg

273.676ºC

6.749

1

3

9

27

481

0

YZQLWPMZQVHJED-UHFFFAOYSA-N

InChI=1S/C23H35NO2S/c1-5-18(6-2)16-23(14-10-7-11-15-23)22(26)24-19-12-8-9-13-20(19)27-21(25)17(3)4/h8-9,12-13,17-18H,5-7,10-11,14-16H2,1-4H3,(H,24,26)

S-[2-[[1-(2-ethylbutyl)cyclohexanecarbonyl]amino]phenyl] 2-methylpropanethioate

Dalcetrapib; JTT-705; RO-4607381; RO4607381; 211513-37-0; Dalcetrapib (JTT-705, RO4607381); S-[2-[[1-(2-ethylbutyl)cyclohexanecarbonyl]amino]phenyl] 2-methylpropanethioate; JTT705;RO 4607381; JTT705; JTT 705

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 (6.42 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (6.42 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 3: ≥ 2.5 mg/mL (6.42 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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Solubility in Formulation 4: 0.5% methylcellulose: 30 mg/mL

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