Only a few years ago, it was generally recognized by pharmaceutical scientists that phase II metabolites of drugs, such acyl glucuronide conjugates, are rapidly excreted following their formation in the body and that these metabolites are not active or reactive. We and others have shown that this is not generally true and that acyl glucuronide conjugates are, in fact, reactive intermediates. This application proposes further studies designed to: (a) elucidate the chemical structures of the products that are formed by reactions of acyl glucuronides with proteins in vivo, and the mechanisms of the reactions, and (b) test the hypothesis that acyl glucuronides, via their reaction products with proteins, may play a role in the toxicities associated with many acidic drugs, particularly NSAIDs.
Specific aims of the proposal are: 1.) characterize the in vitro activity of diclofenac glucuronide and measure formation and degradation of the irreversibly-bound drug in vivo; 2.) evaluate the dual mechanisms (imine intermediate and/or nucleophilic displacement) for in vivo formation of irreversibly bound drug/protein complexes in human volunteers and in patients for tolmetin, fenoprofen, and diclofenac; 3.) measure the relative stabilities of modified proteins formed by the imine and the nucleophilic displacement mechanisms; 4.) investigate the influence of the drug glucuronide:protein ratio on the mechanism of covalent binding; 5.) elucidate the structure and mechanism of formation of delta-bilirubin, the biliprotein formed in humans with cholestatic liver disease; 6.) investigate the reaction of selected NSAID glucuronides with glutathione in both in vitro and in vivo studies; 7.) evaluate, in a mouse model, the immunogenicity of drug protein adducts formed via the imine mechanism, as well as by the nucleophilic displacement mechanism; 8.) assess the factors involved in the observed cross-reactivity among NSAIDs by determining the activity of the antibodies formed in mice against drugs, their glucuronides, the drug-lysine and drug glucuronide-lysine conjugates; 9.) investigate the relationship between irreversible-binding in vivo and immunologic toxicity in humans; and 10.) develop an in vitro model to assess the potential of NSAIDs to cause organ toxicity via an antibody dependent hypersensity mechanism. The proposed investigations will further define the pharmacokinetics and biochemical significance of acyl glucuronide reactivity and covalent binding, and will clarify the chemical mechanisms and immunologic aspects of this binding.
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