دانلود رایگان مقاله هیدرولیز کتوکونازول داستیلاز آریل استامید انسانی به دنبال سمیت کبدی

abstract

Ketoconazole (KC), an antifungal agent, rarely causes severe liver injury when orally administered. It has been reported that KC is mainly hydrolyzed to N-deacetyl ketoconazole (DAK), followed by the N-hydroxylation of DAK by flavin-containing monooxygenase (FMO). Although the metabolism of KC has been considered to be associated with hepatotoxicity, the responsible enzyme(s) remain unknown. The purpose of this study was to identify the responsible enzyme(s) for KC hydrolysis in humans and to clarify their relevance to KC-induced toxicity. Kinetic analysis and inhibition studies using human liver microsomes (HLM) and recombinant enzymes revealed that human arylacetamide deacetylase (AADAC) is responsible for KC hydrolysis to form DAK, and confirmed that FMO3 is the enzyme responsible for DAK N-hydroxylation. In HLM, the clearance of KC hydrolysis occurred to the same extent as DAK N-hydroxylation, which indicates that both processes are not rate-limiting pathways. Cytotoxicity of KC and DAK was evaluated using HepaRG cells and human primary hepatocytes. Treatment of HepaRG cells with DAK for 24 h showed cytotoxicity in a dose-dependent manner, whereas treatment with KC did not show due to the low expression of AADAC. Overexpression of AADAC in HepaRG cells with an adenovirus expression system elicited the cytotoxicity of KC. Cytotoxicity of KC in human primary hepatocytes was attenuated by diisopropylfluorophosphate, an AADAC inhibitor. In conclusion, the present study demonstrated that human AADAC hydrolyzes KC to trigger hepatocellular toxicity

4. Discussion

KC is widely used for treatment of fungal infections. Because hypoadrenalism and hepatotoxicity are adverse reactions of KC, a number of countries recommended that KC not be administered orally. Although the mechanism of hypoadrenalism is suggested to be blocked adrenal steroidogenesis through inhibition of cytochrome P450s, the mechanism of hepatotoxicity has yet to be determined. Drug-induced hepatotoxicity is often caused via the production of a reactive metabolite(s). Identifying the enzymes involved in the metabolic activation of drugs would help to understand this mechanism of drug toxicity and may help researchers develop better processes for drug development. In this study, we sought to identify the enzyme(s) responsible for the metabolism of KC in humans to obtain some insight into the KC hepatotoxicity. First, we found that high KC hydrolase activity was detected in HLM and HIM (Fig. 2A). KC hydrolase activity was detected with recombinant AADAC but not with recombinant CES1 and CES2 (Fig. 2A). The Km values of KC hydrolysis by recombinant AADAC were similar to those in HLM and HIM (Fig. 2B–D, Table 1). These results suggest that AADAC is responsible for KC hydrolysis. The responsibility of human AADAC in KC hydrolysis in human liver was confirmed with an inhibition study (Fig. 3). We previously found that AADAC prefers compounds with quite small acyl moiety [13]. Because KC also possesses an acetyl moiety, it is conceivable that AADAC can hydrolyze KC. Other potential enzyme candidates were examined, and there were no contributions of BCHE and PON to KC hydrolysis because human plasma expressing BCHE and PON did not show any KC hydrolase activity (data not shown). The responsibility of AADAC for KC hydrolysis in human liver was also confirmed by the fact that KC hydrolase activity in a panel of 24 individual HLM samples was significantly correlated with the hydrolase activities of flutamide, indiplon, phenacetin, and rifampicin, which are specifically hydrolyzed by human AADAC [15,21–23]. It should be noted that although flutamide is hydrolyzed by CES2 at relatively low substrate concentration, it is hydrolyzed primarily by AADAC [30] at a concentration of 500 lM, which was used in this study. HLM samples showed a 27-fold interindividual variability in KC hydrolase activity. Interestingly, a sample showing the lowest KC hydrolase activity was from a liver genotyped as the homozygote of AADAC*3 (data not shown), which produces inactive AADAC enzyme [31]. Thus, the variability in KC hydrolysis could partly be due to genetic polymorphisms of AADAC.