PRECLINICAL PHARMACOLOGY OF THE ANTITUMOR AGENT O-6-METHYLGUANINE IN CDF1 MICE

0-6-methylguanine (O6-mG), a guanine analog recently shown to be a pot ent inhibitor of alkylguanine-DNA alkyltransferase, has been found to potentiate the antitumor activity of nitrosoureas, in particular, carm ustine (BCNU), in resistant cell lines (HT-29 mer+) and is targeted fo r development as a modulating agent with chloroethyl nitrosoureas. A h igh-performance liquid chromatography (HPLC) assay of O6-mG in plasma has been developed using a muC18 reverse-phase column. O6-mG and the i nternal standard deoxyguanosine (dGuo) were eluted with a linear gradi ent of from 15% to 35% methanol in 0.5 M ammonium acetate (pH 6.5) at a flow rate of 1 ml/min. The assay was linear over a 4-log concentrati on range with a detection limit of 0.1 mug/ml. The within-run and betw een-run coefficients of variation (CV) were found to be 8.1% and 9.3%, respectively. The pharmacokinetics (PK) of O6-mG were investigated in healthy CDF1 mice following separate i.v. and i.p. administrations. A t 20 mg/kg i.v., plasma O6-mG gave a biexponential profile with a term inal half-life (t1/2) of 24 min and a total clearance (CLT) of 23.7 ml min-I kg-1. Higher doses (40 - 80 mg/kg) revealed a fluctuating third phase, probably due to enterohepatic cycling. Dose-dependent kinetics as measured by CLT and area under the plasma-concentration curve (AUC ) values were also seen. Following i.p. dosing, O6-mG was completely a bsorbed and available to the circulation. No acute toxicity was observ ed in the animals, except for mild sedation, a possible side effect of the 10% ethanol used in the formulation. Studies on the cellular meta bolism of highly purified [H-3]-O6-mG have shown that the compound is not anabolized by a human lymphoblastoid cell line (CEM). Biochemistry studies have shown that the parent molecule is inactivating the alkyl guanine-DNA alkyltransferase (AGT), thus exerting its pharmacological effect.