P183 Identification of Human CYP Enzymes Involved in the Metabolism of 14C‑SRD005825

Gang Luo , Drug Metabolism and Disposition, Covance Laboratories Inc., Madison, WI
Devin Welty , Shire, Lexington, MA
Sara Leitz , Drug Metabolism and Disposition, Covance Laboratories Inc., Madison, WI
Jody Wanta , Drug Metabolism and Disposition, Covance Laboratories Inc., Madison, WI
Daniel Albaugh , Drug Metabolism and Disposition, Covance Laboratories Inc., Madison, WI
Serene Josiah , Shire, Lexington, MA
Donald McKenzie , Drug Metabolism and Disposition, Covance Laboratories Inc., Madison, WI
Zhen Lou , Shire, Lexington, MA
SRD005825 (also known as SHP630) is a drug candidate developed for the treatment of autosomal dominant retinitis pigmentosa (adRP). In this study human cytochrome P450 (CYP) enzymes involved in metabolism of 14C-SRD005825 were identified. Initially 14C-SRD005825 (1 and 10 µM) was incubated with human hepatic microsomes (0.5 and 1.0 mg/mL) and nicotinamide adenine dinucleotide phosphate (reduced form, NADPH, 1 mM) for 5, 15, 30, and 60 minutes. Incubation samples were analyzed by HPLC with radiochemical detection. 14C‑SRD005825 disappeared with increasing incubation times and microsomal protein concentrations. Formation of metabolites M8 (SRD005824) and M9 was observed. SRD005824 was the major metabolite and M9 appeared to be a secondary metabolite derived from SRD005824. Then 14C‑SRD005825 (1, 3, 10, 20, 40, 80, 120, 160, and 200 µM) was incubated with human hepatic microsomes (0.5 mg protein/mL) and NADPH for 15 minutes and the reaction kinetics of SRD005824 formation was calculated according to Michaelis-Menten analysis. The Km and Vmax values were determined to be 97.8 µM and 3830 pmol/minute/mg, respectively. 14C‑SRD005825 (20 mM) was further incubated with human hepatic microsomes (0.5 mg protein/mL) and NADPH for 15 minutes in the absence and presence of selective CYP-specific chemical inhibitors. Compared to the vehicle control, the metabolic rate of SRD005824 formation in the presence of a selective CYP inhibitor was 60.8% for CYP2B6 inhibitor thiotepa (100 µM), 43.6% for CYP2C8 inhibitor montelukast (0.1 µM), 66.4% for CYP2C9 inhibitor sulfaphenazole (3 µM), 71.9% for CYP2C19 inhibitor nootkatone (30 µM), 23.3% for CYP3A4 inhibitor ketoconazole (0.2 µM) and ≥102% for the remaining. These data suggest that in metabolism of 14C-SRD005825, CYP3A4 plays a major role, CYP2B6, CYP2C8, CYP2C9, and CYP2C19 probably play a minor role, but the remaining CYP enzymes tested have no contribution. Finally 14C-SRD005825 (100 mM) was incubated with recombinant human CYP enzymes (SupersomesTM, 50 pmol/mL) including CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 in the presence NADPH for 15 minutes. The rate of SRD005824 formation was 5230, 454, and 249 pmol/mg protein/minute, respectively, for CYP3A4, CYP2D6, and CYP1A2. No formation of SRD005824 was observed with the rest CYP enzymes. In summary, 14C-SRD005825 was primarily metabolized by CYP3A4 and to a much lesser extent by CYP1A1 and CYP2D6.