Analysis of the Metabolic Pathway of Bosentan and of the Cytotoxicity of Bosentan Metabolites Based on a Quantitative Modeling of Metabolism and Transport in Sandwich-Cultured Human Hepatocytes.
To quantitatively understand the events in the human liver, we modeled a hepatic disposition of bosentan and its three known metabolites (Ro 48-5033, Ro 47-8634, and Ro 64-1056) in sandwich-cultured human hepatocytes based on the known metabolic pathway. In addition, the hepatotoxicity of Ro 47-8634 and Ro 64-1056 was investigated because bosentan is well known as a hepatotoxic drug. A model illustrating the hepatic disposition of bosentan and its three metabolites suggested the presence of a novel metabolic pathway(s) from the three metabolites. By performing in vitro metabolism studies on human liver microsomes, a novel metabolite (M4) was identified in Ro 47-8634 metabolism, and its structure was determined. Moreover, by incorporating the metabolic pathway of Ro 47-8634 to M4 into the model, the hepatic disposition of bosentan and its three metabolites was successfully estimated. In hepatocyte toxicity studies, the cell viability of human hepatocytes decreased after exposure to Ro 47-8634, and the observed hepatotoxicity was diminished by pretreatment with tienilic acid (CYP2C9-specific inactivator). Pretreatment with 1-aminobenzotriazole (broad cytochrome P450 inactivator) also tended to maintain the cell viability. Furthermore, Ro 64-1056 showed hepatotoxicity in a concentration-dependent manner. These results suggest that Ro 64-1056 is directly involved in bosentan-induced liver injury partly because CYP2C9 specifically mediates hydroxylation of the t-butyl group of Ro 47-8634. Our findings demonstrate the usefulness of a quantitative modeling of hepatic disposition of drugs and metabolites in sandwich-cultured hepatocytes. In addition, the newly identified metabolic pathway may be an alternative route that can avoid Ro 64-1056-induced liver injury.
Department of Membrane Transport and Biopharmaceutics, Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan (N.M., A.Y.S., T.N., I.T.); and Pharmacokinetic Research Laboratories, Ono Pharmaceutical Co., Ltd., Tsukuba, Japan (N.M., N.K., K.N., H.I.).
Norikazu
N
Matsunaga
Department of Membrane Transport and Biopharmaceutics, Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan (N.M., A.Y.S., T.N., I.T.); and Pharmacokinetic Research Laboratories, Ono Pharmaceutical Co., Ltd., Tsukuba, Japan (N.M., N.K., K.N., H.I.).
Naomi
N
Kaneko
Department of Membrane Transport and Biopharmaceutics, Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan (N.M., A.Y.S., T.N., I.T.); and Pharmacokinetic Research Laboratories, Ono Pharmaceutical Co., Ltd., Tsukuba, Japan (N.M., N.K., K.N., H.I.).
Angelina Yukiko
AY
Staub
Department of Membrane Transport and Biopharmaceutics, Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan (N.M., A.Y.S., T.N., I.T.); and Pharmacokinetic Research Laboratories, Ono Pharmaceutical Co., Ltd., Tsukuba, Japan (N.M., N.K., K.N., H.I.).
Takeo
T
Nakanishi
Department of Membrane Transport and Biopharmaceutics, Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan (N.M., A.Y.S., T.N., I.T.); and Pharmacokinetic Research Laboratories, Ono Pharmaceutical Co., Ltd., Tsukuba, Japan (N.M., N.K., K.N., H.I.).
Ken-ichi
K
Nunoya
Department of Membrane Transport and Biopharmaceutics, Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan (N.M., A.Y.S., T.N., I.T.); and Pharmacokinetic Research Laboratories, Ono Pharmaceutical Co., Ltd., Tsukuba, Japan (N.M., N.K., K.N., H.I.).
Haruo
H
Imawaka
Department of Membrane Transport and Biopharmaceutics, Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan (N.M., A.Y.S., T.N., I.T.); and Pharmacokinetic Research Laboratories, Ono Pharmaceutical Co., Ltd., Tsukuba, Japan (N.M., N.K., K.N., H.I.) tamai@p.kanazawa-u.ac.jp.
Ikumi
I
Tamai
D001692:Biological Transport; D001711:Biotransformation; D000077300:Bosentan; D018929:Cell Culture Techniques; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D056486:Chemical and Drug Induced Liver Injury / Q000201:enzymology / Q000209:etiology* / Q000473:pathology; D065729:Cytochrome P-450 CYP2C9 / Q000378:metabolism; D065688:Cytochrome P-450 CYP2C9 Inhibitors / Q000494:pharmacology; D065128:Endothelin Receptor Antagonists / Q000378:metabolism* / Q000633:toxicity*; D005260:Female; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology* / Q000473:pathology; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D008954:Models, Biological; D011743:Pyrimidines / Q000378:metabolism / Q000633:toxicity; D013449:Sulfonamides / Q000378:metabolism* / Q000633:toxicity*; D000096926:Benzenesulfonamides
D000077300:Bosentan; D065729:Cytochrome P-450 CYP2C9; D065688:Cytochrome P-450 CYP2C9 Inhibitors; D065128:Endothelin Receptor Antagonists; D011743:Pyrimidines; D013449:Sulfonamides; C120596:Ro 47-8634; D000096926:Benzenesulfonamides; C120597:Ro 48-5033; C450260:CYP2C9 protein, human