Characteristics of methotrexate polyglutamate formation in cultured hepatic cells

Accumulation of methotrexate polyglutamates in lymphoblasts is a determinant of antileukemic effects in vivo. A rationale for high-dose methotrexate.

“…Although one randomized clinical trial indicated that patients treated initially with high-dose methotrexate (HDMTX) had significantly better event-free survival compared with patients treated with lower-dose methotrexate (LDMTX) (6), questions have been raised about the rationale of using HDMTX in the treatment of childhood ALL (11,12). It has been postulated that higher extracellular MTX concentrations do not necessarily produce higher intracellular concentrations in ALL blasts, due to saturation of receptor-mediated MTX uptake and saturation of metabolism to active MTX polyglutamylated metabolites (13)(14)(15). To address these concerns, we previously conducted a randomized study of HD-MTX versus LDMTX as initial single-agent treatment of childhood ALL and established that significantly higher concentrations of methotrexate's active polyglutamylated metabolites (MTXPG) are achieved in leukemic blasts of patients treated with HDMTX compared with LDMTX (16).…”

gamma-Fluoromethotrexate: synthesis and biological activity of a potent inhibitor of dihydrofolate reductase with greatly diminished ability to form poly-gamma-glutamates.

“…The crucial difference between the glutamylated derivatives and the parent drug was later shown to reside in the prolonged cellular retention of polyglutamates (2)(3)(4)(5)(6)(7)(8)(9)(10). Thus, when cells containing pools of MTX and MTX polyglutamates are placed in medium lacking MTX, they exhibit elevated levels of MTX polyglutamates for prolonged periods of time, during which unmetabolized MTX rapidly leaves the cell (10,11).…”

“…In particular, short-term exposure to FMTX causes growth inhibition that is several orders of magnitude weaker than that obtained with MTX. To establish that limited glutamylation is a major reason for these results, it is necessary to demonstrate (i) that both agents exert similar inhibitory effects toward the target enzyme DHFR, (ii) that FMTX and MTX are accumulated to a similar extent in H35 cells, and (iii) that FMTX is poorly glutamylated by H35 (9) and the concentrations of their solutions were determined by UV spectroscopy (16). [3',5',7-3H]MTX was adjusted to a final specific activity of 2-6 x 105 dpm/nmol.…”

“…Thus, when cells containing pools of MTX and MTX polyglutamates are placed in medium lacking MTX, they exhibit elevated levels of MTX polyglutamates for prolonged periods of time, during which unmetabolized MTX rapidly leaves the cell (10,11). This property, coupled with an equivalent (5,7,8,12) or possibly greater affinity (10) for the target enzyme dihydrofolate reductase (DHFR; EC 1.5.1.3), has led to the speculation that the MTX polyglutamates are the active agents in cells treated with MTX in vitro and in vivo (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14).…”

“…Cell Culture and Drug Interaction Studies. H-11-EC3 cells (referred to as H35 cells) derived from the Reuber rat hepatoma were cultured as described (3,9). The accumulation of [3H]MTX was determined by the use of procedures previously described (3,5,11).…”