O6-Benzylguanine is known to inactivate the DNA repair protein, 06- alkylguanine-DNA alkyltransferase (AGT) and increase the therapeutic index of BCNU in animals carrying human tumor xenografts. The overall goal of this proposal is to provide companion biochemical and metabolic studies to the Phase l clinical trial of O6-benzylguanine/BCNU. This goal will be accomplished by the development of a highly sensitive assay to determine AGT activity in human lymphocytes and tumor biopsy samples. The sensitive assay utilizes oligodeoxynucleotides containing 06- methylguanine labeled with 35S as a substrate for the AGT protein. Upon reaction with AGT, the methyl group is removed and the synthetic oligomer is cleaved by a restriction enzyme. The parental oligomer and its products are separated by reverse phase HPLC and analyzed using a radioactive detector. This assay will be used to determine the dose of o6-benzylguanine required to deplete human lymphocytes of AGT and to analyze the repletion kinetics of AGT in human lymphocytes after administration of drug. Whenever possible, tumor biopsy samples will be analyzed for AGT activity before and after O6-benzylguanine treatment. A second major objective is to identify and quantify the urinary and plasma metabolites of 06-benzylguanine and the enzymes responsible for 06-benzylguanine metabolism in humans. In vitro studies will include the determination of the Km and Vmax upon incubation of 06-benzylguanine with various isozymes of cytochrome P450 and N-acetyltransferase. Invivo studies will include a simple, non-invasive method to measure variations of cytP450 1A2 and acetylation by quantitating urinary caffeine metabolites after administration of caffeine-containing beverages. The N-acetylating and 1A2 oxidizing capacity of patients will be measured in an effort to correlate these levels with the metabolism and biological effects of 06-benzylguanine.
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| Dolan, M E; Pegg, A E (1997) O6-benzylguanine and its role in chemotherapy. Clin Cancer Res 3:837-47 |
