The organic
acid probenecid has been shown to interfere with the active extrusion of
methotrexate (MTX) from L1210
tumor cells in vitro leading to enhanced MTX accumulation and increased formation of MTX
polyglutamate derivatives. In the presence of
serum albumin (4 g/100 ml), to which
probenecid is bound, the inhibition by
probenecid of [3H]MTX efflux from the
Ehrlich ascites tumor cell was reduced markedly. While half-maximal inhibition of MTX efflux occurred with 0.12 mM
probenecid in the absence of
albumin, 1.45 mM
probenecid was required in its presence. The presence of
albumin also modified the
probenecid-induced elevation of steady-state MTX levels in the
tumor cell. Maximal elevation of cellular MTX levels occurred with 0.5 mM
probenecid in the absence of
albumin, and 3 mM
probenecid in its presence.
Serum albumin further reversed the effects of
probenecid on MTX influx. While
probenecid inhibited influx of 1 microM [3H]MTX in the absence of
albumin (half-maximal inhibition at approximately 1 mM
probenecid),
probenecid stimulated MTX influx in its presence (half-maximal effect at 0.5 to 1 mM). Equilibrium dialysis studies demonstrated that
probenecid displaced MTX from
albumin, increasing the effective free concentration of MTX in the incubation medium, and hence the rate of MTX influx. Therefore,
probenecid may enhance the accumulation of MTX in the
tumor cells by increasing the level of free (as opposed to
albumin bound) MTX in the extracellular medium as well as by direct inhibition of MTX efflux. These observations may provide an additional explanation for
probenecid enhancement of the therapeutic efficacy of MTX in
tumor bearing mice and highlight the importance of assessing
drug-
protein interactions in an in vitro experimental model.