Improved methotrexate therapy of murine tumors obtained by probenecid-mediated pharmacological modulation at the level of membrane transport.

Abstract

Our earlier studies showed that both carrier-mediated influx and efflux of [3H]methotrexate in murine tumor cells was inhibited by probenecid. The concentration of probenecid required for inhibition of influx was 10-fold greater than that required to inhibit efflux. As a consequence, the intracellular exchangeable level of [3]methotrexate at steady state was markedly increased. Since these effects were observed at concentrations of probenecid which were pharmacologically achievable, studies were initiated in mice to evaluate the adjuvant use of this agent during therapy with folate analogs. The simultaneous administration of probenecid (125 mg/kg s.c.) with methotrexate (9 to 18 mg/kg s.c.) on a schedule of every 2 days for five doses to tumor-bearing mice resulted in an increased antitumor effect over that obtained with methotrexate alone. Against the L1210 leukemia, >213% increased life span was obtained compared to 152% increased life span with methotrexate alone. Against the Sarcoma 180 ascites tumor, >179% increased life span was obtained compared to 82% with methotrexate alone. Long-term survivors (10 to 20%) were obtained only with methotrexate plus probenecid. Data on plasma levels of [3H]methotrexate following this schedule of administration showed a 2-fold higher initial level in probenecid-treated mice versus mice given [3H]methotrexate alone, a cross-over in levels in these mice 2 to 3 hr later, and a 2-fold lower level sustained for at least 24 hr, Initial rate and maximum level of accumulation in probenecid-treated mice were reduced 2-fold in liver, kidney, bone marrow, and small intestine. Otherwise, overall persistence of [3H]methotrexate in these tissues was minimally affected. In Sarcoma 180 cells, the initial rate but not the maximum level of [3H]methotrexate accumulation was reduced 2-fold by probenecid. However, the overall persistence of [3H]methotrexate in these cells was increased 2-fold with probenecid. The effect on plasma and tissue levels of [3H]methotrexate was dependent on the dosage of probenecid but did not depend on the dosage of the antifolate itself, at least within the dosage ranged used. These results show that the effects of probenecid on methotrexate transport, documented for tumor cells in in vitro studies, were favorably and selectively expressed in these cells at a pharmacological level and as such may have clinical implications.