The overall objective of this research proposal is to enhance the safety of volatile anesthetics by diminishing or preventing their toxicity. Halothane, the most widely used anesthetic in the world, causes a rare but fatal fulminant hepatic necrosis. Moreover, it commonly causes altered postoperative mixed function oxidase activity. The toxicity of halothane, and other fluorinated anesthetics, is intrinsically linked to their metabolism by cytochrome P450. For example, the immunologic cascade culminating in fulminant hepatic necrosis (""""""""halothane hepatitis"""""""") is initiated by P450-mediated halothane oxidation to products which bind to liver proteins, rendering them antigenic. P450 also activates halothane to free radicals which initiate lipid peroxidation, and catalyzes nephrotoxic fluoride ion release from fluorinated ethers. Specific human P450 isoforms responsible for anesthetic metabolism have been identified. P4502E1 catalyzes fluorinated ether defluorination and halothane oxidation to protein antigens, while P4503A4 catalyzes halothane reduction to free radicals. Certain volatile anesthetics, alone among the myriad protoxins and procarcinogens activated by P4502E1, are metabolized stereoselectively. Furthermore, P4502E1 metabolizes halothane aerobically but not anaerobically, while the converse occurs for P4503A4. One central hypothesis to be tested is that selective inactivation of the specific human cytochrome P450 isoforms responsible for anesthetic metabolism can be accomplished clinically, and that such inactivation will prevent anesthetic toxification. Disulfiram is an effective clinical inhibitor of P4502E1 and P4502E1-mediated anesthetic metabolism. However, clinical disulfiram P450 specificity and efficacy in preventing metabolism-based toxicity are unknown. Human studies will establish the specificity, safety and optimal disulfiram dosing regimen for clinical inhibition of P4502E1-mediated anesthetic toxicity. The ability of clinical P450 inhibitors, developed to diminish anesthetic metabolism, to prevent metabolism-based anesthetic toxicity will be investigated in vitro and in vivo in animals and humans. If the hypothesis is correct, then volatile anesthetic metabolism will be diminished, toxicity will be reduced or prevented, and the therapeutic index of volatile anesthetics dramatically enhanced. The second major hypothesis to be tested is that unique structural dynamic features of the P4502E1 and P4503A4 active sites restricts substrate binding and active site mobility, and that oxygen-dependent conformational changes have a critical modulatory role in these processes. Cloned, expressed human P450s 2E1 and 3A4 will be used in catalytic structure-activity studies with anesthetic stereoisomers and analogues, in concert with NMR studies of substrate-hemoprotein interactions, to test this hypothesis. If the hypothesis is correct, then unique insights into molecular mechanisms of substrate-protein interactions will be obtained. More importantly, potentially bioactivated anesthetics or protoxins may be rationally designed to avoid this metabolic hazard.
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