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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.

To study the body distribution of 6-mercaptopurine (6MP), [8-14C]6MP was given by infusion to 2 marmoset monkeys at a dose rate of 5 mg/kg body weight/h for 1 and 4 h, respectively. Both experimental animals were sacrificed 2 h after the end of the drug infusion and instantly frozen at -70 degrees C. Whole-body sagittal sections were made later. Blood samples were obtained regularly during the experiments to quantitate 6MP, 6MP riboside (6MPR), 6-thioxanthine, and 6-thiouric acid in plasma. The autoradiograms revealed extensive distribution of the 14C label. High levels of radioactivity were seen in liver, bile, and intestinal contents. Labeling of the central nervous system and bone marrow was obvious. The plasma concentration-time curves of 6MP and 6MPR attained steady-state concentrations of 30-40 microM and 6-12 microM, respectively. After stopping the infusion of the drug, the concentrations of 6MP and 6MPR became equal. 6MPR contributes to the biological effect of 6MP, as degradation of 6MPR results in 6MP. In studies on the pharmacokinetics and dynamics one should try to include all relevant metabolites of 6MP, both in plasma and in the cells.

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.

In vitro investigations have indicated the need for both prolonged exposure to 6-mercaptopurine (6MP) and the use of high concentrations to achieve maximal cell kill. After the customary oral administration the bioavailability of 6MP appeared to be low, and i.v. bolus injections resulted in short-lived high concentrations of 6MP, so prolonged infusions seemed rational. To test the feasibility of this approach 24-h infusions were given to goats. We used our improved HPLC method to quantitate 6MP and 6MP riboside (6MPR) in plasma, CSF, and urine. The concentrations of 6MPR were in excess of those of 6MP. Since 6MPR can easily be converted to 6MP, 6MPR acts as a depot for 6MP. Penetration of both 6MP and 6MPR into CSF was excellent. Of the total dose administered, 38% to 68% could be accounted for in the urine, with about equal amounts of 6MP and 6MPR. At doses of 20 and 10 mg kg-1 h-1 total concentrations of 6MP and 6MPR in excess of 100 microM were reached during 24-h infusions. However, all three experimental animals died due to toxicity. A dose of 2 mg kg-1 h-1 was tolerated; the total steady state concentration of 6MP and 6MPR in two experiments was about 10 microM. We conclude that the prolonged infusion of 6MP is feasible, and in view of the excellent penetration of 6MP and 6MPR into CSF, studies using prolonged infusions of thiopurines are warranted in man.