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We investigated the hepatotoxicity induced by AQ using a glutathione (GSH)-depleted mice model. Although sole administration of either AQ or L-buthionine-S,R-sulfoxinine (BSO), a well-known GSH synthesis inhibitor, produced no significant hepatotoxicity, combined administration of AQ with BSO induced hepatotoxicity characterized by centrilobular necrosis of the hepatocytes and an elevation of plasma alanine aminotransferase activity. Pretreatment of aminobenzotriazole, a nonspecific inhibitor for P450s, completely suppressed the above hepatotoxicity caused by AQ co-treatment with BSO. Administration of radiolabeled AQ in combination with BSO exhibited significantly higher covalent binding to mice liver proteins than that observed after sole dosing of radiolabeled AQ. The results obtained in this GSH-depleted animal model suggest that the reactive metabolite of AQ formed by hepatic P450 binds to liver proteins, and then finally leads to hepatotoxicity. These observations may help to understand the risk factors and the mechanism for idiosyncratic hepatotoxicity of AQ in humans.

Nearly all bodily processes exhibit circadian rhythmicity. As a consequence, the pharmacokinetic and pharmacodynamic properties of a drug may also vary with time of day. The objective of this study was to investigate diurnal variation in processes that regulate drug concentrations in the brain, focusing on P-glycoprotein (P-gp). This efflux transporter limits the distribution of many drugs in the brain. To this end, the exposure to the P-gp substrate quinidine was determined in the plasma and brain tissue after intravenous administration in rats at six different time points over the 24-h period. Our results indicate that time of administration significantly affects the exposure to quinidine in the brain. Upon inhibition of P-gp, exposure to quinidine in brain tissue is constant over the 24-h period. To gain more insight into processes regulating brain concentrations, we used intracerebral microdialysis to determine the concentration of quinidine in brain extracellular fluid (ECF) and cerebrospinal fluid (CSF) after intravenous administration at two different time points. The data were analyzed by physiologically based pharmacokinetic modeling using NONMEM. The model shows that the variation is due to higher activity of P-gp-mediated transport from the deep brain compartment to the plasma compartment during the active period. Furthermore, the analysis reveals that CSF flux is higher in the resting period compared to the active period. In conclusion, we show that the exposure to a P-gp substrate in the brain depends on time of administration, thereby providing a new strategy for drug targeting to the brain.Electronic supplementary materialThe online version of this article (doi:10.1208/s12248-014-9625-4) contains supplementary material, which is available to authorized users.

ABSTRACT:We have identified several novel metabolites of ticlopidine, a well known antiplatelet agent and have revealed its metabolic route in rats. The main biliary metabolite of ticlopidine was characterized as a glutathione (GSH) conjugate of ticlopidine S-oxide, in which conjugation had occurred at carbon 7a in the thienopyridine moiety. Quantitative analysis revealed that 29% of the dose was subjected to the formation of reactive intermediates followed by conjugation with GSH after oral administration of ticlopidine (22 mg/ kg) to rats. In vitro incubation of ticlopidine with rat liver 9000g supernatant fraction (S9) fractions led to the formation of multiple metabolites, including 2-oxo-ticlopidine, the precursor for the pharmacologically active ticlopidine metabolite, [1-(2-chlorobenzyl)-4-mercaptopiperidin-(3Z)-ylidene] acetic acid. A novel thiophene ring-opened metabolite with a thioketone group and a carboxylic acid moiety has also been detected after incubation of 2-oxo-ticlopidine with rat liver microsomes or upon incubation of ticlopidine with rat liver S9 fractions.Ticlopidine is a well known antiplatelet agent (Quinn and Fitzgerald, 1999) and has been widely used for the secondary prevention of atherothrombosis (Jacobson, 2004). The metabolism of ticlopidine is complex because of extensive oxidation in the liver (Saltiel and Ward, 1987). For example, less than 1% of the parent compound was detected in urine, whereas approximately 60 and 25% of radioactivity was recovered in urine after a single oral administration of [ 14 C]ticlopidine to humans (Noble and Goa, 1996) and rats (Tuong et al., 1981), respectively. Furthermore, a large difference was observed between the total radioactivity and unchanged ticlopidine level in the plasma (Panak et al., 1983). In the 1980s, metabolic studies suggested that N-dealkylation, N-oxidation, and oxidation of the thiophene ring followed by ring opening appeared to be the main routes, but numerous highly polar urinary and biliary metabolites in both humans and animals remained unidentified (Tuong et al., 1981;Panak et al., 1983). More recently, novel ticlopidine metabolites such as the S-oxide form (Ha-Duong et al., 2001) and the pharmacologically active metabolite (Yoneda et al., 2004) have been detected. However, the whole metabolism of ticlopidine has remained unclarified.In the present study, we have identified several novel metabolites of ticlopidine and revealed the metabolic pathways of ticlopidine quantitatively. Moreover, an in vitro experiment was conducted to elucidate the whole metabolic route of ticlopidine including the biotransformation to the pharmacologically active metabolite.