ABSTRACT:An in vitro screening model was developed to determine the reactivity of acyl glucuronide metabolites from carboxylic drugs. This assay is composed of two phases. The first is a phase of biosynthesis of acyl glucuronides by human liver microsomes (HLM). The second, during which acyl glucuronides are incubated with human serum albumin (HSA), consists of assessing the reactivity of acyl glucuronides toward HSA. Both phases are performed successively in the same experiment. This model was validated using eight carboxylic drugs that were well known for their reactivity, their extent of covalent binding, and their immunological potential. These products were representative of the scale of reactivity. Each compound was incubated with HLM at 400 M and metabolized into acyl glucuronide to different extents, ranging from 5.6% (tolmetin) to 89.4% (diclofenac). The first-order aglycone appearance rate constant and the extent of covalent binding to proteins were assayed during the incubation of acyl glucuronides formed with HSA for 24 h. Extensive isomerization phenomenon was observed for each acyl glucuronide between the two phases. An excellent correlation was observed (r 2 , 0.94) between the extent of drug covalent binding to albumin and the aglycone appearance constant weighted by the percentage of isomerization. This correlation represents an in vitro reactivity scale, which will be helpful in drug discovery support programs to predict the covalent binding potential of new chemical entities. This screening model will also allow the comparison of acyl glucuronide reactivity for related structure compounds.Many acidic drugs with carboxylic acid functions are metabolized to reactive acyl glucuronides. These metabolites are unstable at physiological pH and can result in free aglycone by hydrolysis and lead to positional isomers by acyl migration. Acyl migration involves the transfer of the acyl group from the position 1␤ to the C-2, C-3, or C-4 position of the glucuronic acid ring, which results in the formation of isomeric acyl glucuronides (Faed, 1984;Spahn-Langguth and Benet, 1992).Acyl-migrated glucuronide isomers were shown to bind covalently to proteins in vitro and in vivo causing potential toxicity (SpahnLangguth et al., 1996). The glucuronide-mediated toxicity depends on the covalent binding of acyl glucuronides to specific target proteins located in specific tissues. The toxicological mechanisms are still unknown (Park et al., 1987;Riley and Leeder, 1995;Dansette et al., 1998).However, data literature review provides much information regarding immunologically based and clinically relevant adverse reactions of several drugs that are probably related to the formation of highly reactive acyl glucuronides. These drugs include tolmetin, zomepirac, diclofenac, and diflunisal (Hasegawa et al

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To investigate the localisation of fipronil in dog skin, [14C]-fipronil was topically applied to a male beagle dog (spot-on administration) at the therapeutic dose of 10 mg/kg. By means of autohistoradiography, the radioactivity was precisely detected in the skin and appendages at various intervals after application. Radioactivity was predominantly observed within the stratum corneum, the viable epidermis, and in the pilo-sebaceous units (mainly in the sebaceous glands and epithelial layers). [14C]-fipronil was significantly detected in these structures up to 56 days post-treatment, in the application zone (neck) but also in the lumbar zone, thus indicating the mechanical displacement of fipronil. No radioactivity was detected in either the dermal or the hypodermal layers, confirming the low percutaneous passage of fipronil.

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