Human liver microsomes reactive metabolites

In a more thorough and balanced evaluation of the in vitro reactive metabolite tests in both human liver microsomes and S9 fractions, Obach et al. found that a panel of safe drugs in the clinic can form as much reactive metabolites as those hepatotoxic drugs, even after corrections for drug clearance parameters.92-93 Obviously, a more balanced and system-based approach to reactive metabolite formation and its clinical implications is needed hence forward. 

In the first case study, a lead candidate 12 (Fig. 6) was assessed for its potential to form reactive metabolites in LC-MS-based trapping assays using a variety of trapping agents. There were no indications of the formation of any thiol or cyanide adducts when GSH, N-acetylcysteine, or cyanide were included in incubations of 12 with human liver microsomes. However, when a tritium-labeled derivative of 12 was incubated with human liver microsomes under standard conditions... 

Zhang Z, Chen Q, Li Y, Doss GA, Dean BJ, Ngui JS, Silva EM, Kim S, Wu JY, Dininno F, Hammond ML, Steams RA, Evans DC, Baillie TA, Tang W (2005) In vitro bioactivation of dihydrobenzoxathiin selective estrogen receptor modulators by cytochrome P450 3A4 in human liver microsomes formation of reactive iminium and quinone type metabolites. Chem Res Toxicol 18 675-685... 

FIGURE 10.10 Analysis of [ H]GSH trapped reactive metabolites by HPLC with TopCount (Zhu et al., 2005b). A nonlabeled drug (50 mM) was incubated with a mixture of GSH (1 mM) and trace [ H]GSH (1-2 p,Ci/mL) in human liver microsomes. After precipitating proteins, the samples were analyzed by HPLC (1 mL/min) with TopCount (4wlls/min, 10-min counting time) (a) Radioactivity profile of the incubation. Ml and M2 were GSH-trapped reactive metabolites and (b) Radioactivity profile of a control incubation sample (without NADPH). 

Scheme 19) (Sun et al., 2008). The characterization of a hydroxycarboxylic acid metabolite of trovafloxacin in preclinical species (Dalvie et al., 1996) lends further support for the metabolism of the cyclopropylamine ring in trovafloxacin to a reactive intermediate. The formation of the hydroxycarboxylic acid can occur from the addition of water to the a,(3-unsaturated aldehyde via Michael addition followed by oxidation as depicted for the model compound (Scheme 19). However, the proposal for reactive metabolite formation with trovafloxacin remains a speculation since the bioactivation studies did not involve the parent fluoroquinolone and no a,(3-unsaturated aldehyde or the corresponding glutathione conjugate has been detected in trovafloxacin incubations in human liver microsomes (Sun et al., 2008). Furthermore, the primary pathways of trovafloxacin clearance in humans include phase II metabolism (iV-acetylation, acyl glucuronidation, and iV-sulfation) (Scheme 19) with very minor contributions from phase I oxidative pathways (Dalvie et al., 1997). 

In a general sense, the kinetics and metabolism of toluene in humans, rats and mice are very similar the hippurate is in all cases by far the major metabolite, while in all species the ortho- and / ora-cresols are minor metabolites. To what extent formation of a potentially reactive sulfate conjugate of benzyl alcohol occurs (van Doom et al., 1980 Chidgley et al., 1986) is uncertain, mainly because mercapturates formed from toluene have not been characterized. Similarly, whether the covalent binding observed in rat liver microsomes has any toxicological relevance is uncertain. 

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