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

The transfer of CE from HDL3 to HDL2 is considerably and dose-dependently reduced by anacetrapib (P<0.001 for doses up to and including 0.1 µM). The amount that [14C]Torcetrapib (0.25 µM) binds to immobilized rhCETP is reduced by 82% and 60%, respectively, by excess anacetrapib (25 µM). Pre-β-HDL production is reduced by over 46% (P<0.001) by anacetrapib at all investigated concentrations (0.1, 1, 3, and 10 µM)[1]. Anacetrapib (ANA) significantly reduces PCSK9 promoter activity; this is seen at 3 µM concentration (−22%, p<0.01), and at 10 µM, it is even lower, at 68% of control. Similarly, Anacetrapib reduces the luciferase activity of B11 cells starting at a concentration of 3 µM and reaches a maximum reduction of 38% of control at 10 µM. Anacetrapib reduces PCSK9 mRNA to 60% of control and LDLR mRNA to 67% of control at 10 µM concentration[2].

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]

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Anacetrapib is administered to hamsters for seven days prior to the injection of [3H]cholesterol-labeled macrophages (day 0). Day 0 HDL-C values are significantly elevated following anacetrapib treatment. Day 3 [3H]cholesterol radioactivity in the HDL fraction is substantially higher than Anacetrapib control values[1]. When compared to a vehicle control, anacetrapib (ANA) medication slightly raises serum levels of total serum cholesterol by around 10% (p<0.05) and serum levels of LDL-C by 26% (p<0.05)[2]. The mean values for terminal half-life, steady-state volume of distribution, and systemic plasma clearance following an intravenous dosage of 0.5 mg/kg are 12 hours, 1.1 L/kg, and 2.3 mL/min/kg, respectively. Anacetrapib has a 38% bioavailability after oral dosage at 5 mg/kg. Exposures (AUC) rise from 23 μM·h at 5 mg/kg to 362 μM·h at 500 mg/kg in a manner that is not dose-proportional. The time to attain peak plasma level (Tmax) ranged from 3 to 4.5 hours, and the peak plasma level (Cmax) ranged from 5 to 26 μM in this dosing range[3].

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.

LDL uptake assay[2]
HepG2 cells in 6-well culture plates were treated with anacetrapib for 24 h. The fluorescent DiI-LDL at a concentration of 2 µg/ml was added to the cells at the end of treatment for 4 h and cells were trypsinized. The mean red fluorescence of 1×104 cells was measured using FACScan. [2]

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Small interference RNA (siRNA) transfection[2]
A pool of four pre-designed siRNAs targeted to human CETP mRNA were obtained from Dharmacon. The silencer negative control siRNA was obtained from Applied Biosystem. 4×10 cells were mixed with 50 nM siRNA using siPORT NeoFX siRNA transfection reagent and plated in 6-well plates. Next day, fresh medium was added to the transfected cells and then cells were treated with anacetrapib for 24 h prior to isolation of total RNA.[2]

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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].

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

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)

Solubility in Formulation 1: ≥ 2.75 mg/mL (4.31 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 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 30% PEG400+0.5% Tween80+5% propylene glycol:10 mg/mL

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