Inhibition of Hepatobiliary Transport as a Predictive Method for Clinical Hepatotoxicity of Nefazodone

Abstract

Treatment with the antidepressant nefazodone has been associated with clinical idiosyncratic hepatotoxicty. Using membranes expressing human bile salt export pump (BSEP), human sandwich hepatocytes, and intact rats, we compared nefazodone and its marketed analogs, buspirone and trazodone. We found that nefazodone caused a strong inhibition of BSEP (IC(50) = 9 microM), inhibition of taurocholate efflux in human hepatocytes (IC(50) = 14 microM), and a transient increase in rat serum bile acids 1 h after oral drug administration. Buspirone or trazodone had no effect on biliary transport system. Nefazodone produced time- and concentration-dependent toxicity in human hepatocytes with IC(50) = 18 microM and 30 microM measured by inhibition of protein synthesis after 6 h and 24 h incubation, respectively. Toxicity was correlated with the amount of unmetabolized nefazodone. Partial recovery in toxicity by 24 h has been associated with metabolism of nefazodone to sulfate and glucuronide conjugates. The saturation of nefazodone metabolism resulted in sustained decrease in protein synthesis and cell death at 50 microM. The toxicity was not observed with buspirone or trazodone. Addition of 1-aminobenzotriazole (ABT), an inhibitor of CYP450, resulted in enhancement of nefazodone toxicity at 10 microM and was associated with accumulation of unmetabolized nefazodone. In human liver microsomes, ABT also prevented metabolism of nefazodone and formation of glutathione conjugates. We suggest that inhibition of bile acid transport by nefazodone is an indicator of potential hepatotoxicity. Our findings are consistent with the clinical experience and suggest that described methodology can be applied in the selection of nonhepatotoxic drug candidates.

In Vitro Investigation of the Hepatobiliary Disposition Mechanisms of the Antifungal Agent Micafungin in Humans and Rats

“…Our in vitro biliary excretion data with micafungin in SCRH and SCHH support the primary involvement of BSEP/Bsep in micafungin biliary excretion. In SCHH, inhibition of micafungin biliary excretion by the BSEP inhibitor nefazodone (Kostrubsky et al, 2006) provided further evidence for the role of BSEP. The inhibitory effects observed with the mixed P-gp/BCRP inhibitor GF120918 and the selective BCRP inhibitor fumitremorgin C in SCHH suggests that BCRP, but not P-gp, plays some role in micafungin biliary excretion.…”

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“…7,8 In turn, malfunctioning mitochondria produce less adenosine triphosphate, which in turn further impairs adenosine triphosphate-dependent BSEP functions. 9 Therefore, drug-induced and/or bile acid-induced dual inhibition of mitochondrial and BSEP functions might result in prolonged and severe drug-induced cholestasis, as in the case presented here. 10 Although rapid reversal of liver cholestasis after cessation of the insulting drug is expected in the simple form of drugrelated BSEP malfunction, severe and prolonged cholestasis requiring medical intervention (as presented in our case) can be theoretically explained by this dual-inhibition mechanism.…”

Involvement of mitochondrial dysfunction in nefazodone-induced hepatotoxicity

“…Moreover, drugs are frequently accumulated in the liver, becoming several times more concentrated in the hepatic tissue compared to the plasma (Chu et al, 2013). Although data on plasma-to-liver ratio of NEF is not available in the literature, if NEF does accumulate in the liver it could potentially inhibit its own elimination (Dykens et al, 2008;Kostrubsky et al, 2006), contributing to further increases in tissue drug concentration. As our objective was to use NEF as a model hepatotoxic compound withdrawn from the market, to help predict the toxicity of future drug candidates, we had to find an experimental setting where drug toxicity was manifested.…”

“…As a consequence of NEF treatment, hepatotoxic symptoms were common and included jaundice, dark urine, clay colored stool and increased plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin and prothrombin time (Aranda-Michel et al, 1999;Lucena et al, 1999). NEF hepatotoxic effects have been previously associated with the inhibition of cytochrome P450 3A4 (CYP3A4), since NEF treatment in human hepatocytes and human liver microsomes in the presence of the non-selective inhibitor of P450, 1-aminobenzotriazole (ABT), resulted in a significant decrease in protein synthesis together with a decrease in NEF metabolism (Kostrubsky et al, 2006). The same work showed that NEF treatment resulted in inhibition of both bile acid transport, via the bile salt export pump (BSEP) present in membrane vesicles, and the canalicular transport in cultured human hepatocytes (Kostrubsky et al, 2006).…”

“…NEF hepatotoxic effects have been previously associated with the inhibition of cytochrome P450 3A4 (CYP3A4), since NEF treatment in human hepatocytes and human liver microsomes in the presence of the non-selective inhibitor of P450, 1-aminobenzotriazole (ABT), resulted in a significant decrease in protein synthesis together with a decrease in NEF metabolism (Kostrubsky et al, 2006). The same work showed that NEF treatment resulted in inhibition of both bile acid transport, via the bile salt export pump (BSEP) present in membrane vesicles, and the canalicular transport in cultured human hepatocytes (Kostrubsky et al, 2006). NEF was also shown to directly impair hepatic mitochondrial function through inhibition of OXPHOS complexes, particularly complex I (Dykens et al, 2008;Nadanaciva et al, 2007).…”