In vitro activity: Evacetrapib (LY2484595) inhibits human plasma CETP protein with IC50 of 26 nM. Evacetrapib (LY2484595) (< 10 μM) does not induce aldosterone or cortisol synthesis in H295R cells.

Kinase Assay: Human CETP cDNA is amplified from a human liver cDNA library and the sequence is confirmed to be identical to the published sequence. The cDNA is subcloned into a pcDNA3.1 vector, under the control of CMV promoter. A stable line is established in CV1 cells in which the above-mentioned construct is used to express the recombinant human CETP. The medium contained the secreted recombinant CETP protein and the amount (19 ng/μL) is quantified by an ELISA kit. The medium is then aliquoted in 0.2% BSA and stored at -80°C. The stock CETP protein is diluted 150-fold in CETP buffer (10 mM Tris, 150 mM NaCl, and 2 mM EDTA) before use. The assay is set up in a 96-well plate. Each well received 97.5 μL diluted CETP protein (final concentration 7 nM) and 2.5 μL of compound stock. After a 30 min incubation at 37°C, 5 μL of substrate stock (the same stock used in the human plasma CETP assay), 0.16 μL of VLDL stock (2.5 mg/mL, Intracel) and 145 μL of CETP buffer are added, and the incubation is continued for another 4 h. Signal is read for the human plasma CETP assay.

Cell Assay: As a benzazepine-based inhibitor of CETP, evacetrapib is developed to increase HDL cholesterol for the treatment of coronary artery disease. Evacetrapib is efficacious both in vitro and in vivo. The IC50 values of evacetrapib against CETP are 5.5nM and 26nM, respectively in the buffer assay using human recombinant CETP and in the plasma assay using human plasma CETP. In the animal model of human CETP/ApoAI double transgenic mouse line, oral administration of evacetrapib at 30mg/kg significantly inhibits the CETP activity as well as increases the level of HDL. Besides that, the ED50 value of evacetrapib is less than 5mg/kg. Compared to the previous inhibitors of CETP, evacetrapib has less side effects. It is found no to increase blood pressure Zucker diabetic fatty rats and not to induce aldosterone or cortisol synthesis in H295R cells.