7-hydroxymethotrexate and Leukemia--Lymphoid

Topics: Child; Chromatography, High Pressure Liquid; Humans; Leukemia, Lymphoid; Methotrexate; Spectrophotometry, Ultraviolet; Sulfamethoxazole; Trimethoprim

Cell growth survival studies have revealed that 7-OH methotrexate is two orders of magnitude less cytotoxic to human melanoma and human acute lymphoblastic leukaemia (ALL) cells in vitro than methotrexate. The influence of 7-OH methotrexate on methotrexate toxicity was investigated by studying cell growth in the presence of methotrexate and its 7-OH metabolite and by studying [3H]-methotrexate movement across the plasma membrane of isolated human cells. Transport was followed for net entry of the drug into drug-free cells, net exit of drug into drug-free medium and for unidirectional exit fluxes with drug and/or metabolite in the extracellular medium (exchange exit). Results indicate that 7-OH methotrexate (10(-6) M) interacts with melanoma cells to reduce the initial cellular uptake rate of [3H]-methotrexate but that no such interaction occurs with ALL cells. Efflux measurements revealed that a stimulatory effect of extracellular methotrexate on [3H]-methotrexate exit was apparent and that extracellular 7-OH methotrexate had a less stimulatory effect. Overall, loss of intracellular drug was greater from melanoma cells than from ALL cells. The results suggest that the drug resistance encountered following high dose therapy may be due to reduced cellular uptake and/or increased efflux of methotrexate from cells, both events being enhanced by 7-OH methotrexate. In addition, there is an apparently endogenous resistance of the melanomas to methotrexate as regards time of exposure to this agent which could also contribute to the lack of clinical response when compared to ALL.

Topics: Acute Disease; Cell Division; Cell Line; Cell Survival; Cells, Cultured; Humans; Leukemia, Lymphoid; Melanoma; Methotrexate; Time Factors

7-Hydroxymethotrexate is a major metabolite of methotrexate in patients undergoing chemotherapy with this drug. It has now been demonstrated that when cultures of CCRF-CEM, a human lymphoblastic cell line, are incubated in the presence of 100 microM methotrexate, significant quantities of 7-hydroxymethotrexate are formed. This finding is relevant to clinical use of the drug since it is now clear that during the treatment of acute lymphocytic leukemia conversion of methotrexate to the relatively inactive 7-hydroxy derivative probably occurs not only in the liver but also in the target cells.

Topics: Cell Line; Chromatography, High Pressure Liquid; Humans; Leukemia, Lymphoid; Methotrexate

The intracellular synthesis of polyglutamyl derivatives of both methotrexate (4-amino-N-10-methylpteroylglutamic acid) and 7-hydroxymethotrexate, the primary plasma metabolite of methotrexate in humans, was evaluated in a methotrexate-sensitive, acute lymphoblastic leukemia cell line, MOLT 4. These studies were performed using a highly specific ion-pairing high-pressure liquid chromatography method which permits the simultaneous determination of methotrexate, 7-hydroxymethotrexate, and their corresponding polyglutamyl derivatives. When MOLT 4 cells were exposed to 1 microM methotrexate, the monoglutamate attained a steady state after 30 min, and polyglutamyl derivatives having from one to 4 additional glutamyl residues were observed over 4 hr. Four additional metabolites were also detected upon incubation with 1 microM 7-hydroxymethotrexate. On the basis of the retention times for these compounds relative to methotrexate polyglutamyl standards and since these metabolites reverted to 7-hydroxymethotrexate upon treatment with a preparation of hog kidney conjugase, they were identified as polyglutamyl derivatives of 7-hydroxymethotrexate. The identification of 7-hydroxymethotrexate polyglutamyl derivatives in vitro raises the possibility of an important new dimension in the pharmacological action of methotrexate. We investigated the effect of extracellular 7-hydroxymethotrexate on net methotrexate uptake and metabolism when cells were exposed simultaneously to 1 microM [3H]-methotrexate and unlabeled 7-hydroxymethotrexate. A decrease in the levels of both intracellular methotrexate and the corresponding polyglutamyl derivatives was noted for cells treated with 1 or 10 microM 7-hydroxymethotrexate. However, no appreciable effect of 7-hydroxymethotrexate on the amount of polyglutamyl derivatives formed relative to the total intracellular antifolate was noted. These studies show that in MOLT 4 cells (a) both methotrexate and 7-hydroxymethotrexate are rapidly converted to polyglutamyl derivatives, and (b) 7-hydroxymethotrexate interferes with net methotrexate accumulation and metabolism when present simultaneously in the extracellular medium. These results, moreover, suggest a potential role for 7-hydroxymethotrexate in modulating the biochemical effects of methotrexate in vivo.

Topics: Cell Line; Chromatography, High Pressure Liquid; Humans; Leukemia, Lymphoid; Methotrexate; Peptides; Polyglutamic Acid

Methotrexate (MTX), a widely used antineoplastic agent, is metabolized to a non-active derivative, 7-OH-MTX, and to some active poly-gamma-glutamyl derivatives (MTX-Gn) which are retained within cells. Pharmacokinetic studies in humans indicate (i) a higher concentration of 7-OH-MTX than of MTX in plasma after a 24-h infusion and (ii) a time-dependent relationship for MTX and 7-OH-MTX kinetics in plasma and urine which might be explained by the variation of MTX metabolism. The intracellular metabolism of MTX and 7-OH-MTX has been investigated using a specific ion-paired method of high-performance liquid chromatography (HPLC) which permits the simultaneous determination of DAMPA, MTX, 7-OH-MTX and their respective polyglutamate derivatives. The formation of 7-OH-MTX polyglutamates and the possible effect of 7-OH-MTX on the transport and/or metabolism of the unchanged MTX in a human acute lymphoblastic leukaemia cell line (Molt 4) have been studied. After incubation of the cells to 1 microM (3H) 7-OH-MTX, four radiolabelled peaks, representing 75% of the intracellular 3H, were converted to 7-OH-MTX upon exposure to hog kidney conjugase indicating the formation of 7-OH-MTX polyglutamyl derivatives. The effects of 7-OH-MTX on MTX-PG formation were analysed after simultaneous incubation of the cells to 1 microM (3H) MTX and 10 microM 7-OH-MTX. The formation of the higher glutamyl derivatives, MTX-G3 and MTX-G4 was completely inhibited and the total intracellular accumulation of the MTX-PG's decreased by 35% compared to control. These data suggest that the 7-OH-MTX and the 7-OH-MTX-PG might modify the chemotherapeutic activity of this agent in vivo.

Topics: Cell Line; Folic Acid Antagonists; Glutamates; Humans; Kinetics; Leukemia, Lymphoid; Methotrexate; Models, Biological

Methotrexate (MTX) shows consistent cytotoxicity for melanoma cells in vitro but it is ineffective in clinical use at equivalent concentrations in vivo. This apparent paradox has been investigated by cell culture techniques and results quantified by cell number. In an in vitro model of high dose MTX therapy followed by leucovorin rescue (HD-MTX-LCR) there was survival of both melanoma and choriocarcinoma cell lines but not of an acute lymphocytic leukaemia cell line. The 70H metabolite of MTX was identified by HPLC in plasma samples of melanoma patients treated by HD-MTX-LCR, in which MTX concentrations approximately 10(-5) M were maintained for 24 h. However, metabolism per se is unlikely to account for the lack of response to MTX clinically. In vitro 70H MTX (10(-7) - 10(-6) M) was two orders of magnitude less cytotoxic for melanoma than MTX (10(-9) - 10(-8) M). The cellular accumulation of [3H]-MTX, using a rapid gradient centrifuge technique for separation of melanoma cells from medium, was reduced in the presence of 70H-MTX. The results suggest that reduced cellular uptake of MTX combined with biochemical rescue of tumour cells may partially explain the paradoxical lack of clinical response of melanoma to the drug.

Topics: Animals; Cell Count; Cell Line; Cell Survival; Choriocarcinoma; Humans; Leucovorin; Leukemia, Lymphoid; Male; Melanoma; Methotrexate; Mice

Topics: Child; Chromatography, High Pressure Liquid; Drug Therapy, Combination; Humans; Leucovorin; Leukemia, Lymphoid; Methotrexate