Pharmacodynamics of prolonged treatment with L,S-buthionine sulfoximine.

Abstract

To develop dosing criteria for the use of L-buthionine-S-sulfoximine (active diastereoisomer) as a glutathione depletor in the clinic, using a pharmacodynamic and pharmacokinetic in vitro-in vivo approach. In vitro: L-buthionine-S-sulfoximine uptake was determined in human glioblastoma cells (T98G) and NIH-3T3 cells using 35S-labeled drug. Dose response relationships were derived for inhibition of glutathione synthesis in CHO cells, and for depletion of glutathione in exponentially growing T98G and CHO cells, as a function of extracellular L-buthionine-S-sulfoximine concentration. Steady-state glutathione levels for CHO and NIH-3T3 cells were measured using an enzymatic assay, while glutathione synthesis rates in CHO cells were determined using a flow cytometric assay. In vivo: L-buthionine-S-sulfoximine biodistribution was determined in male nude mice carrying human glioblastomas (T98G) intracranially, using 35S-labeled drug infused subcutaneously by osmotic pump. Tissue glutathione levels were measured using an enzymatic assay. The observed cellular uptake t1/2 of approximately 55 min, coupled with a previously reported, rapid in vivo clearance of buthionine sulfoximine, suggest that continuous infusion would be preferable to bolus dosing. Effective concentrations of L-buthionine-S-sulfoximine (24 h exposure), required to lower cellular glutathione content to 50% of control (EC50), were under 1 mM for both cell lines. The amount of L-buthionine-S-sulfoximine in tissues (estimated from 35S drug disposition) reached steady state within 8 h and was proportional to the rate of infusion. Brain tumors were depleted to approximately 50% of control glutathione by a infusion rate of 0.25 mumoles/h (25 g mice). At lower infusion rates an increase in glutathione content was noted in certain nude mouse tissues including brain tumor xenografts.