Our long-standing objective has been to develop the chemistry of glucuronide and glutathione conjugates of therapeutic agents in support of their clinical use. Historically our experiments have been designed to exploit mass spectrometry and the catalytic capabilities of UDP-glucuronyl and glutathione transferases immobilized in microsomal protein. The current proposal addresses both the inactivation and the activation of drugs by conjugation. Previously we have demonstrated that several classes of therapeutic agents that contain carboxylic acid groups can be activated for covalent bonding of biopolymers by conjugation with glucuronic acid or sulfate. Acyl-linked glucuronides also undergo intramolecular isomerization to ensembles of polar isomers that complicate structure identification and pharmacokinetic analyses. We propose to continue studies of the rates and range of the transacylating reactivities of 1-0-acyl glucuronides, intermolecularly with albumin and intramolecularly to form B-glucuronidase resistant isomers, working with conjugates of clofibric acid, indomethacin, retinoic acid and thyroxine. We will also evaluate the reactivity of acyl-linked glutathiones, working first with the conjugate of clofibric acid, the necrotic hypolipidaemic agent. With support from the expiring grant we have collaborated in studies of the mechanisms by which patients acquire resistance to chemotherapeutic alkylating agents. My research group has demonstrated that melphalan, chlorambucil and cis platin are substrates for glutathione transferases in vitro. This is significant in the context of elevated levels of glutathione and glutathione transferase reported to accompany acquired resistance to these drugs in vivo. We propose to extend these studies to cyclophosphamide and its alkylating metabolite phosphoramide mustard, to confirm that these drugs are conjugated with glutathione, and to provide reference material and assay methods. We will also evaluate the conjugation of glutathione with thyroxine. Plasma levels of glutathione-Stransferase are elevated and intracellular glutathione is decreased when thyroxine is administered therapeutically. We will develop laser desorption electron impact mass spectrometry as a new technique to provide ion radical directed fragmentation in involatile samples.
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