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

Abstract

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 sandwichcultured 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 concentrationdependent 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 sandwichcultured hepatocytes. In addition, the newly identified metabolic pathway may be an alternative route that can avoid Ro 64-1056-induced liver injury.

Metabolic Activation of Cholestatic Drug-Induced Bile Acid-Dependent Toxicity in Human Sandwich-Cultured Hepatocytes

“…Ro 47-8634 is a stronger inhibitor of rat BSEP activity than bosentan. 19,20 In this study, BA tox of bosentan in hSCH was decreased by 1-ABT treatment, suggesting that the bosentan metabolite (like the tolvaptan metabolite) contributes to BA tox development. Likewise, the metabolites of other compounds associated with decreased BA tox by 1-ABT treatment in this study are assumed to have an inhibitory effect on BSEP and contribute to BA tox development.…”

Prediction of Altered Bile Acid Disposition Due to Inhibition of Multiple Transporters: An Integrated Approach Using Sandwich-Cultured Hepatocytes, Mechanistic Modeling, and Simulation

“…There are no literature reports about inhibitory effects of telmisartan metabolites on human bile acid transporters. Although a bosentan metabolite, Ro 47-8634, was reported to be an inhibitor of rat Bsep (K i 5 8.5 mM) (Fattinger et al, 2001), the intracellular concentration of this metabolite in human SCHH is less than 5% of bosentan (Matsunaga et al, 2016). In addition, the concentration of this metabolite in human plasma (Dingemanse et al, 2002) and feces (Weber et al, 1999) is much lower than bosentan.…”

Evaluation of the metabolic capability of primary human hepatocytes in three‐dimensional cultures on microstructural plates

“…evaluation of chronic toxicity of new compounds, evaluation of P450 induction, identification of metabolites, validation of drug targets and development of disease models (Lauschke et al, ), and further work is underway to develop or optimize culture methods and platforms. For example, sandwich‐cultured hepatocytes are used for the evaluation of post‐metabolic biliary excretion (Matsunaga et al, ), and Organ‐on‐a‐Chip platforms enable us to construct a micropatterned coculture environment for hepatocytes (Khetani & Bhatia, ). Moreover, a multi‐organ chip with microfluid has recently been developed (Vivares et al, ).…”