Troglitazone-induced intrahepatic cholestasis by an interference with the hepatobiliary export of bile acids in male and female rats. Correlation with the gender difference in troglitazone sulfate formation and the inhibition of the canalicular bile salt export pump (Bsep) by troglitazone and troglitazone sulfate
Introduction
Troglitazone is an insulin sensitizer of the thiazolidinedione class for the treatment of type 2 non-insulin-dependent diabetes mellitus (Chen, 1998). Elevations in liver enzyme levels were observed during clinical trials, and since its market introduction in early 1997 several cases of fulminant hepatic failure were reported, leading to the withdrawal of this compound from the market in March 2000 (Gitlin et al., 1998, Neuschwander-Tetri et al., 1998, Shibuya et al., 1998, Herrine and Choudhary, 1999, Fukano et al., 2000). The mechanism(s) underlying the troglitazone-associated hepatotoxicity are at present unclear. Several reports suggested a cholestatic mechanism to be involved in this liver injury (Gitlin et al., 1998, Fukano et al., 2000) and a strong reduction of the bile flow has been observed in isolated perfused rat liver (Preininger et al., 1999). Several cases of liver injury following concurrent troglitazone and glibenclamide treatment lead to the speculation that this latter drug, known to induce cholestasis in some patients (Krivoy et al., 1996) might enhance the probability for troglitazone-induced liver injury (Shibuya et al., 1998, Fukano et al., 2000). The cholestatic potential of troglitazone has been studied previously in vitro and in vivo in male rats (Funk et al., 2001).
An intrahepatic cholestasis can be induced by an interference with the vectorial transport of biliary constituents from blood to bile resulting in an intracellular accumulation of bile salts (Meier-Abt, 1990, Erlinger, 1997, Trauner et al., 1998). High intracellular bile salt levels have been reported to induce cellular necrosis and mitochondrial dysfunction due to their intrinsic detergent activity (Delzenne et al., 1992, Desmet, 1995, Gores et al., 1998). The vectorial transport of both, endobiotics (bile acids, steroids, bilirubin glucuronide and other metabolic products) and xenobiotics and their metabolites from plasma to bile is facilitated by several transport systems (Zimniak et al., 1999). The export across the canalicular membrane, where the greatest uphill concentration gradient has to be overcome, often represents the rate-limiting step in this excretion process (Kadmon et al., 1993, Arrese et al., 1998). For bile acids this step is catalyzed by a primary active, ATP-dependent transporter of the ABC protein family, the canalicular bile salt export pump (Bsep) localized in the canalicular liver plasma membrane (Gerloff et al., 1997). For several cholestatic compounds interactions with the export of bile acids were found at the level of the canalicular ATP-dependent Bsep (Stieger et al., 2000). The immunosuppressive agent cyclosporin A was found to inhibit the canalicular Bsep in vitro (Kadmon et al., 1993). A similar mechanism was recently postulated for the NSAID sulindac (Bolder et al., 1999), for rifamycin, rifampicin and glibenclamide (Stieger et al., 2000). Such interactions at the level of hepatobiliary export processes have to be considered in addition to metabolic drug–drug interactions, mostly induced by cytochrome-P450 inhibition. The importance of a close interplay of drug-metabolizing systems and active hepatobiliary export processes for the overall drug elimination process has been pointed out recently (Schuetz and Schinkel, 1999, Zimniak et al., 1999).
Troglitazone is extensively metabolized in the liver mainly by sulfation, glucuronidation and oxidation (Loi et al., 1999). The main metabolite, troglitazone sulfate, undergoes biliary excretion, and accounted for up to 60% of the dose in rats (Kawai et al., 1997). In patients with hepatic impairment, troglitazone sulfate was found to accumulate approximately fourfold in plasma, with a threefold increased half-life (Ott et al., 1998). Therefore the cholestatic potential of troglitazone and troglitazone sulfate has been studied previously in vitro and in vivo in rats (Funk et al., 2001). The formation of troglitazone sulfate as main metabolite, the accumulation of this metabolite in rat liver tissue and the high affinity of troglitazone sulfate for the canalicular bile salt export pump (Bsep) were hypothesized to lead to an intrahepatic cholestasis in the rat (Funk et al., 2001). Based on these results, the formation of troglitazone sulfate was further studied as a key step in the formation of an intrahepatic cholestasis in the rat. Several sulfotransferase isoenzymes exhibit a sex-specific expression pattern, with phenol sulfotransferases being expressed mainly in male rats, while the estrogen sulfotransferases are predominant in female rats (Iwasaki et al., 1994, Dunn and Klaassen, 1998). A polymorphic expression pattern has been described for several human phenol-sulfotransferase isoenzymes (Jones et al., 1993, Ozawa et al., 1998).
The dependence of the cholestatic effect of troglitazone on the formation of troglitazone sulfate has been studied using both, an in vivo cholestasis model in male and female rats and isolated perfused rat livers of both genders. In addition, mechanistic in vitro studies were performed to address the formation rates of troglitazone sulfate and the interference of troglitazone sulfate with the hepatobiliary export of bile acids in male and female rats.
Section snippets
Substances
All buffer salts (Hepes, Tris, NaHCO3, KNO3, Mg(NO3)2, CaCl2, MgSO4), taurocholic acid, ATP, 3′-phospho-adenosine 5′-phosphosulfate (PAPS), creatine phosphate and creatine phosphokinase were from Fluka AG, Buchs, Switzerland. Sucrose was obtained from E. Merck (Darmstadt, Germany). Radiolabeled [3H]-taurocholic acid was purchased from DuPont NEN (NET322) at a specific activity of 128.4 GBq mmol−1. Troglitazone was synthesized at Roche Diagnostics, Mannheim, Germany and glibenclamide (Ro
Results
The cholestatic potential of troglitazone has been studied in two rat models, the isolated perfused rat liver and an acute in vivo model, using male and female rats. Mechanistic studies have been performed in cell-free in vitro systems to elucidate differences in troglitazone sulfate formation and Bsep inhibition between male and female rats.
Discussion
Interference of troglitazone with the hepatobiliary export of bile acids in male isolated perfused rat livers (IPRL) was shown previously (Preininger et al., 1999). Upon addition of troglitazone (3.15 μM) to the perfusion medium, a strong decline in bile flow by 67% within 1 h was noted, accompanied by a slight increase in portal pressure (Preininger et al., 1999). Based on these results and our own, previously reported studies on the cholestatic effect of troglitazone (Funk et al., 2001), we
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