Metabolic Stability and Cytochrome P450 Phenotyping of Triptolide in Human and Rat Liver Microsomes

Triptolide, the primary active component of Tripterygium wilfondii Hook F (WHF), which has anti-inflammatory, anti-fertility, anti-neoplastic, and immunosuppressive activities, has been widely used in Asian countries. Although the pharmacokinetic profiles of triptolide indicated triptolide was extensively metabolized after i.v. administration, its metabolic profile and the enzymes responsible for its metabolism have not been fully elucidated. The aim of the present study was to characterize the metabolites of triptolide and identify the cytochrome P450 (CYP) isoforms involved in the metabolism in human and rat liver microsomes in vitro. We found that the biotransformation rate of triptolide was low in both RLM and HLM, but the rate was elevated in phenobarbital induced RLMs, where triptolide was converted to four metabolites (M1, M2, M3 and M4). In RLMs incubating system, all these four metabolites were identified by LC-MS to be mono-hydroxylated products. 1-Aminobenzotriazole (ABT), a non-specific cytochrome P450 inhibitor, inhibited the generation of all these four metabolites. Ketoconazole (KTZ), a specific inhibitor of CYP3A4, was found to inhibit the production of M2, M3, and M4. CYP2C19 inhibitor omeprazole inhibited the production of M1. Three metabolites (M2, M3, and M4) were found in both HLM and cDNA-expressed human CYP3A4 incubating system, whereas M1 and M4 were found in cDNA-expressed human CYP2C19 incubating system. In HLMs, the production of the three metabolites could be significantly inhibited by ABT and KTZ but not by omeprazole. The kinetics parameters were determined in the phenobarbital induced RLMs with Kms of 277 ±76, 267 ± 64, 30 ± 2 and 28 ± 4 μM and Vmaxs of 2.99 ± 0.65, 0.44 ± 0.08, 0.64 ± 0.02 and 0.98 ± 0.06 nmol/min/ mg protein for M1, M2, M3, and M4, receptivity. Our results indicated that the metabolic activity of triptolide was low in HLM and RLM, and CYP3A4 was the primary CYP isoform contributed to the metabolism of triptolide in human. This work was supported by the National Natural Science Foundation of China (30630075) and Dalian Institute of Chemical Physics Innovation and Ph.D. Exploration Fund of Chinese Academy of Sciences.