Increased accumulation of methotrexate by murine tumor cells in vitro in the presence of probenecid which is mediated by a preferential inhibition of efflux.

Carrier-mediated influx and efflux of [3H]methotrexate by L1210 leukemia cells was inhibited by probenecid. The concentration of probenecid required for inhibition of influx was markedly greater than that required to inhibit efflux. The concentration determined for 50% inhibition of influx was 1.35 +/- 0.15 (S.D.) mM. Inhibition of this flux was competitive (Ki = 1.23 +/- 0.2 mM) and was reversed after removal of probenecid. In contrast, the concentration determined for 50% inhibition of efflux was only 0.12 +/- 0.016 mM, and inhibition was also reversed after removal of probenecid. As a consequence of the different extent of inhibition of each flux by probenecid, the level of intracellular [3H]methotrexate at steady state was markedly increased. At 0.1 and 1 mM probenecid, the steady state level was increased 2- and 2.6-fold, respectively. These observed increases are in close agreement with that expected from the effect on each flux at these concentrations. From other data on the inhibition of each flux at higher concentrations of probenecid, a maximum effect (3- to 4-fold increase) in steady-state level would be expected at a probenecid concentration between 2 and 3 mM. A similar relationship between the inhibition by probenecid of influx and efflux of [3H]methotrexate was also shown for Sarcoma 180 and Ehrlich carcinoma cells. These results have pharmacological implications with respect to the adjuvant use of probenecid or related organic ions during folate analog therapy of human cancer.