Search

The cell-kinetics and biochemical pharmacology of simultaneous and sequential combination treatment with 0.02 microM methotrexate (MTX) and 2 microM 6-mercaptopurine (6MP) were studied in MOLT-4 malignant T-lymphoblasts. The results were compared with our data from earlier studies of separate treatment with these antimetabolites. A synergistic effect of combination treatment could be demonstrated, based on the inhibition of purine de novo synthesis by both agents, on DNA and RNA synthesis, on the incorporation of 6-thioguanine nucleotides into DNA and RNA, and on inhibition of cell growth and clonal growth. The synergistic effects of combination treatment with MTX and 6MP will only be available in malignant lymphoblasts, and will be absent in normal bone marrow cells and normal lymphocytes, because the activity of purine de novo synthesis in these cells is absent or low. Based on the synergistic effects of MTX and 6MP and the good penetration of both agents in the cerebrospinal fluid, the Dutch Childhood Leukemia Study Group presently performs a randomized study during protocol M of the BFM/DCLSG-ALL-90 protocol comparing the results of 4 times each two weeks 24 hr intravenous administration of MTX (5 g/m2) versus intravenous MTX, immediately followed by 24 hr intravenous administration of 6MP (1.3 g/m2). The pharmacokinetics and intracellular biochemical pharmacology of 6MP in lymphocytes will be studied, comparing intravenous administration and low dose oral administration.

Methotrexate (MTX) causes an inhibition of purine de novo synthesis (PDNS), resulting in increased intracellular availability of 5-phosphoribosyl-1-pyrophosphate (PRPP) in human malignant lymphoblasts with an active PDNS. Normal bone marrow cells and peripheral blood lymphocytes lack this capacity. The increased levels of PRPP can be used for enhanced incorporation of 6-mercaptopurine (6MP), indicating a potential time-, sequence- and dose-dependent synergism of both drugs. The effects of 0.02 microM and 0.2 microM MTX on the PDNS of MOLT-4 (T-), RAJI (B-) and KM-3 (non-B-non-T-) human malignant lymphoblasts were studied with respect to PRPP levels, aminoimidazolecarboxamide ribonucleosidemonophosphate (AICAR) levels and the incorporation of labeled glycine into purine metabolites. These results were correlated with the activity of the PDNS (labeled glycine incorporation) and the purine salvage pathway (labeled hypoxanthine incorporation) in untreated cells. Inhibition of PDNS by 0.02 microM MTX was complete in KM-3 cells with a moderately active PDNS and salvage pathway. RAJI cells, with a relatively low PDNS and high salvage pathway, demonstrated an incomplete, but increasing inhibition of PDNS, whereas inhibition of PDNS in MOLT-4 cells with both pathways active was minimal and recovered in time. Treatment with 0.2 microM MTX resulted in a complete inhibition of PDNS in all cell lines. After treatment with MTX an enhanced incorporation of labeled hypoxanthine and 6MP was noticed, confirming the potential rescue from MTX cytotoxicity by hypoxanthine and a potential synergism of MTX and 6MP on cytotoxicity. The enhanced incorporation of 6MP was more obvious in RAJI and KM-3 cells in comparison with MOLT-4 cells. These data demonstrate the important role of both the activities of the PDNS and the purine salvage pathway in malignant lymphoblasts of different subclasses with respect to the synergism of MTX and 6MP.

A survey is given of our present investigations of the pharmacokinetics of 6MP and the effects of the combination of MTX and 6MP on purine and pyrimidine metabolism. Both drugs are used in the oral maintenance therapy of ALL in children. The very low serum-concentrations after oral administration of 6MP and the short serum-half-life-times after intravenous administration point to more efficacy after prolonged intravenous infusion. The penetration of 6MP in the cerebrospinal fluid after intravenous administration could account for (prophylactic) treatment of the CNS in ALL. Pretreatment of leukemic lymphoblasts with MTX results in an increase of intracellular PRPP due to inhibition of purine de novo synthesis. This can be used for an increased incorporation and conversion of 6MP. Thus, increased incorporation and conversion of 6MP can be obtained when 6MP is added to MTX-pretreated leukemic lymphoblasts at that point of time where MTX causes maximal PRPP accumulation. This will result in increased incorporation of 6MP into nucleic acids and thus in enhanced cytotoxicity. Pharmacokinetic and metabolic investigations of 6MP and MTX could lead to a more rational and more effective use of both agents in patients with ALL.

The effects of methotrexate (MTX) on cytotoxicity (trypan blue exclusion and soft agar clonal growth), cell cycle perturbation, and purine and pyrimidine ribonucleotide and deoxyribonucleotide pools have been studied in MOLT-4 malignant T-lymphoblasts. Two concentrations of MTX, 0.02 microM and 0.2 microM have been utilized, which can be maintained in vivo during many hours in the maintenance therapy of acute lymphoblastic leukemia (ALL). The results are correlated with the effects of MTX on the inhibition of purine de novo synthesis. Treatment with 0.02 microM MTX results in an accumulation of cells in early S phase after 20 hr, as measured by DNA flow cytometry and by a significant increase of dCTP levels, followed by a slow progression of a cohort of cells through the cell cycle. Cytotoxicity also becomes evident starting from this point of time. The effects on deoxyribonucleotide pools are discussed in correlation with the inhibition of DNA synthesis. The changes in ribonucleotide pools are associated with the partial inhibition of purine de novo synthesis at 20-28 hr and suggest an inhibition of RNA synthesis. After 48 hr a reutilization of nucleotide precursors due to nucleic acid breakdown and a recovery of purine de novo synthesis is shown, associated with a recovery of RNA synthesis, whereas cytotoxicity increases. Treatment of MOLT-4 cells with 0.2 microM MTX results in a rapid complete cessation of cell progression through all parts of the cell cycle after 8 hr, associated with a depletion of all deoxyribonucleotide pools, complete inhibition of purine de novo synthesis, inhibition of RNA synthesis and a marked cytotoxicity. Ribonucleotide pools demonstrate a reutilization of nucleotide precursors after 12 hr of incubation without a recovery of purine de novo synthesis and RNA synthesis. These data show a close dose- and time-dependent correlation of the effects of MTX on purine de novo synthesis, UMP levels and other (deoxy)ribonucleotide pools, and on RNA and DNA synthesis in MOLT-4 cells having an active purine de novo synthesis. This correlation is absent in normal bone marrow cells and peripheral blood lymphocytes. These data can be used in order to elucidate the synergistic effects of sequential administration of MTX and 6-mercaptopurine.

Methotrexate (MTX) and 6-mercaptopurine (6MP) have been used since 30 years in the maintenance treatment of acute lymphoblastic leukemia (ALL) of childhood. A synergistic effect of this combination was demonstrated in mouse and childhood leukemia. In this article an overview is given of our investigations, concerning the biochemical basis of this synergism. This synergism is caused by a selective inhibition of the purine de novo synthesis in malignant lymphoblasts by MTX, associated with an enhanced intracellular uptake of 6MP. Pharmacokinetic studies of MTX in various schemes of prophylactic central nervous system treatment in ALL are discussed. Treatment with 24-hr infusions of MTX in a dosage of 5 g/m2, as recommended in the new BFM-86/SNWLK ALL VII protocol, seems to be optimal. Pharmacokinetic studies of intravenous 6MP infusions demonstrated a good cerebral fluid penetration. Exploiting the synergistic action of the combination of MTX and 6MP may offer an improvement of the prophylactic central nervous systems treatment in ALL in the future, using intravenous administration of both MTX and 6MP.

Methotrexate (MTX) and 6-mercaptopurine (6MP) are common drugs in the oral maintenance therapy of acute lymphoblastic leukemia (ALL). On the basis of their biochemical effects on cell metabolism, a sequence-dependent synergism might be anticipated. In order to investigate this hypothesis, MOLT-4 human malignant T-lymphoblasts were incubated with various concentrations of MTX. The time at which maximal increase of intracellular 5-phosphoribosyl-1-pyrophosphate (PRPP) levels was found correlated with the concentrations of MTX used. Determination of aminoimidazolecarboxamide ribonucleoside monophosphate (AICAR) levels and labeled glycine incorporation into purine metabolites revealed an incomplete inhibition of purine de novo synthesis after incubation with 0.02 microM MTX, and a complete inhibition with 0.2 microM MTX. After prolonged periods of incubation, glutamine exhaustion of the medium caused inhibition of purine de novo synthesis in MTX-untreated cells, with a concomitant increase of PRPP levels. Addition of glutamine to the medium prevented this phenomenon. The increased availability of PRPP after pretreatment with MTX can be used for enhanced intracellular incorporation of hypoxanthine and 6MP in their respective nucleotides. The time- and dose-dependent effects of MTX on PRPP levels correlated with the enhanced incorporation of hypoxanthine and 6MP. The data presented in this study demonstrate that a synergistic action of the combination of MTX and 6MP can be anticipated in malignant lymphoblasts with an active purine de novo synthesis depending on the concentration of MTX and on the time and sequence of administration of both drugs.

MOLT-4 (T-), RAJI (B-), and KM-3 (non-B-non-T-, common ALL) malignant lymphoblasts demonstrated significant differences in their activities of purine de novo synthesis (PDNS) and purine salvage pathway and in their cell-kinetic parameters. Incubations with concentrations of methotrexate (0.02 and 0.2 microM), which can be maintained during many hours in the oral maintenance therapy of acute lymphoblastic leukemia, indicated large differences between the three cell lines with respect to the inhibition of PDNS, depending on the concentration of methotrexate (MTX) and on the activities of the two pathways. These dose- and cell line-dependent differences corresponded to the perturbations of cell-kinetics and purine and pyrimidine (deoxy)ribonucleotide pools in the three cell lines. Exposure of MOLT-4 cells to 0.02 microM MTX resulted in an incomplete inhibition of DNA synthesis in early S phase, as shown by DNA-flow cytometry and increase of dCTP levels, which recovered spontaneously after 48 hr. Almost no impairment of RNA synthesis occurred (unbalanced growth). In RAJI cells, exposed to 0.02 microM MTX, DNA synthesis was delayed in the S phase, not arrested, and RNA synthesis was not impaired, also indicating an unbalanced growth pattern, which, however, did not recover in time. KM-3 cells were arrested in G1 phase and subsequently in early S phase after incubation with 0.02 microM MTX, and perturbations of ribonucleotides indicated a complete inhibition of RNA synthesis, resulting in a balanced growth pattern. Cytotoxicity was more pronounced in KM-3 cells. The reliability of the soft agar colony forming assay after low dose MTX treatment is discussed. Exposure of MOLT-4 and KM-3 cells to 0.2 microM MTX resulted in a complete inhibition of DNA synthesis, with cessation of cell progression through all parts of the cell cycle and arrest in G1 phase. RAJI cells showed an increasing accumulation of cells in G1 phase without complete cessation of cell cycle progression. Perturbations of ribonucleotide pools suggested an inhibition of RNA synthesis in all cell lines, indicating a balanced growth pattern in KM-3 cells and MOLT-4 cells.(ABSTRACT TRUNCATED AT 250 WORDS)