Identification of glutathione conjugates of troglitazone in human hepatocytes

doi:10.1016/S0009-2797(02)00056-X

Chemico-Biological Interactions

Volume 142, Issues 1–2, 10 November 2002, Pages 83-97

https://doi.org/10.1016/S0009-2797(02)00056-X Get rights and content

Abstract

Troglitazone (TGZ) is an orally active antihyperglycemic agent used in the treatment of noninsulin-dependent diabetes mellitus. Several cases of liver failure following TGZ administration led to its withdrawal from the market. The mechanism of toxicity is still not understood. The formation of toxic metabolites is believed to play an important role. Herein, we report the biotransformation of TGZ in human hepatocytes. TGZ at 50 μM concentration was incubated with cryopreserved human hepatocytes. Four metabolites were found—glucuronide, sulfate, and two glutathione (GSH) conjugates of TGZ. The two GSH metabolites could be conjugation at the 6-hydroxychromane nucleus and the thiazolidinedione ring. Alternatively, the conjugation could be one of the two rings, with the two GSH metabolites are diastereomers. The sulfate conjugate was the major metabolite found. The cytochrome P450 (CYP) inhibitors furafylline (CYP1A1/2), omeprazole (CYP2C19), ketoconazole (CYP3A4), and sulfaphenazole (CYP2C9) had no inhibitory effect on the TGZ metabolism suggesting that several P450s may play a role in the TGZ metabolic pathway. Previous studies in our laboratory have shown a large interindividual variation between different donors in cytotoxicity after dosing with TGZ. Based on EC₅₀ values, donors were classified as sensitive or resistant. The sensitive human donors were found to form significantly less troglitazone GSH conjugates and glucuronides than the resistant donors.

Introduction

Troglitazone (TGZ; Rezulin), a relatively new drug for the treatment of type II diabetes or noninsulin-dependent diabetes, is an oral antihyperglycemic agent which acts primarily by decreasing the insulin resistance by acting as an insulinomimetic agent [1], [2], [3]. It improves sensitivity to insulin in muscle and adipose tissue and inhibits hepatic gluconeogenesis. Chemically, TGZ (5-[4-(6-hydroxy-2,5,7,8-tetramethyl-chroman-2-yl)methoxy)-benzyl]-2,4-thiazolidinedione) belongs to the thiazolidinedione class of compounds, and is chemically not related to either the sulfonylureas, the biguanides, or the γ-glucosidase inhibitors. The molecule contains two chiral centers, with each of the four stereoisomers having similar pharmacological effects. The thiazolidinediones bind to peroxisome proliferator-activated receptor-gamma, a nuclear receptor which regulates the growth and differentiation of cells at the transcriptional level [4], [5]. It also inhibits the expression of phosphoenolpyruvate carboxykinase gene, which is the rate-limiting enzyme in the hepatic gluconeogenesis [6]. However, the mechanism of action of TGZ is not completely understood. It decreases plasma glucose levels as monotherapy but is more effective when used in combination with insulin, metformin, or sulfonylureas [7].

The biotransformation of TGZ has been studied in rodents, dogs, monkeys, pigs, and humans. In humans, it is rapidly absorbed with an absolute bioavailability of 40–50% [8]. The mean elimination half-life ranges from 7.6 to 24 h. Age, race, gender, existence of type II diabetes, or renal impairment has no effect on the pharmacokinetics of TGZ. However, in subjects with hepatic impairment, there is a 30%, four and two times increase in the plasma concentration of TGZ, sulfate conjugate of TGZ (TGZS), and quinone derivative of TGZ (TGZQ), respectively. About 85% of the drug is recovered in the feces, suggesting that the major route of excretion is bile. The major metabolites of TGZ in human plasma are TGZS and TGZQ, accounting for about 70 and 10%, respectively of the plasma metabolites. TGZ glucuronide (TGZG) is also found in the plasma, albeit in very low amounts [9], [10], [11], [12]. CYP3A4 and CYP2C8 are the main P450 enzymes involved in the TGZQ formation. TGZ is also a potent inducer of CYP3A4, and is similar to rifampin in inducing CYP3A4 in human hepatocytes. It also induces CYP2C8 [13], [14], [15], [16].

The use of TGZ has been associated with severe hepatotoxicity since its introduction in March, 1997. In 3 years of marketing, about 90 cases of liver failure, believed to be related to TGZ, were reported. It finally resulted in the voluntarily withdrawal of TGZ from the market [17]. The exact mechanism of liver toxicity is still not clear. An interpretation of the currently available data suggests that TGZ can operate through several mechanisms—formation of reactive metabolites such as the quinone or the epoxide metabolite [18], [19], [20], decreased conjugation of the parent resulting in toxicity due to the accumulated parent [21], induction of apoptosis (as observed in cultured rat hepatocytes, [22]), and induction of intrahepatic cholestasis [23], [24]. Although the TGZ metabolites have been implicated in human liver toxicity, there is lack of direct evidence supporting this hypothesis. The objectives of the present study were: 1) to determine the formation and identification of TGZ metabolites in human hepatocytes, 2) to identify the metabolic pathway for TGZ activation, and 3) to differentiate between the metabolic profile of sensitive and resistant donors.

Section snippets

Materials

TGZ was purchased from Biomol Research Labs, Inc. (Plymouth Meeting, PA). Verapamil, poly-alanine, omeprazole, sulfaphenazole, Percoll, and Krebs Henseleit Buffer (KHB) were obtained from Sigma Chemical Co. (St. Louis, MO). Furafylline was purchased from Ultra Fine (Manchester, UK) and ketoconazole from Janssen Biotech (Berse, Belgium). HPLC grade solvents were from Fisher Scientific (Fair Lawn, NJ). The suspension media was Dulbecco's modified Eagle's medium fortified with fetal bovine serum

Metabolism of TGZ in human hepatocytes

Cryopreserved human hepatocytes were used to study the metabolism of TGZ. Human hepatocytes, containing a full complement of drug-metabolizing enzymes, are valuable in vitro physiological-based models to evaluate the metabolism of xenobiotics [27]. Hepatocytes from three donors were pooled together. The demographics of the three donors are given in Table 1. Comparing the controls (media control and vehicle control) with the sample incubated with TGZ indicated the formation of four metabolites (

Acknowledgements

The authors wish to sincerely thank Katrina Smith and Elizabeth Duble for helping with the hepatocyte incubations, Presely Mozone for assistance with hepatocyte handling and cryostorage, and Dan Dryden for assistance with DME characterization data for cryopreserved human hepatocytes.

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