Biochemical and pharmacokinetic effects of leucovorin after high-dose methotrexate in a murine leukemia model.

The administration of calcium leucovorin, either concurrently or after high dosages of methotrexate in L1210 leukemic mice, has both pharmacokinetic and biochemical effects in tumor cells and drug-limiting proliferative normal tissue in small intestine. A reduction in the maximal level of accumulation and retention of exchangeable drug (unbound to dihydrofolate reductase) in tissue could be demonstrated when calcium leucovorin was given simultaneously with methotrexate at equal or greater dosages than the latter. The dose dependence for calcium leucovorin-introduced drug loss is similar in both tissues and showed the expected variation when the time interval between methotrexate and calcium leucovorin doses was increased. With 400 mg methotrexate per kg, greater than 96 mg calcium leucovorin per kg were required maximally to affect overall drug retention in tissue 2 hr after drug, whereas only 24 mg calcium leucovorin per kg were required 16 hr after drug. Calcium leucovorin, given after methotrexate, induced synchronous recovery of DNA synthesis (measured by labeled deoxyruridine incorporation) in both small intestine and L1210 cells. An initial cycle of synthesis was induced in the presence of exchangeable levels of drug. Two hr after methotrexate, 12 mg/kg, calcium leucovorin induced an immediate but only partial (20 to 25% of control rate) recovery of synthesis with dose dependence from 3 to 12 mg calcium leucovorin per kg. Less synthesis was induced after 96 mg/kg and almost none after methotrexate, 400 mg/kg. With calcium leucovorin, 24 mg/kg, given 2 hr after methotrexate, 12 or 96 mg/kg, a major cycle of synthesis occurred when total drug levels approached the equivalence of the dihydrofolate reductase content. The magnitude of this cycle of synthesis in both L1210 cells and small intestine was the same as that seen in animals recovering from methotrexate alone. However, this is based on the assumption that an approximately equivalent relationship between DNA synthesis and labeled doexyuridine incorporation in each tissue during the period of maximal incorporation within the cycle. The major effect of calcium leucovorin, then, was to induce an earlier resumption of DNA synthesis as a consequence of the pharmacokinetic effect in each tissue. With calcium leucovorin, 24 or 400 mg/kg, given 16 hr after methotrexate, an identical effect on drug retention was observed in both L1210 cells and small intestine. Although there was a difference in the time course for recovery in small intestine at each dosage of calcium leucovorin, the recovery of DNA synthesis as drug levels approached the dihydrofolate reductase content was similar in magnitude. In L1210 cells, however, substantial recovery of synthesis to a comparable level and with a similar time-course occurred only after leucovorin, 400 mg/kg. Little or no recovery of DNA synthesis was observed after calcium leucovorin, 24 mg/kg, during the same time period. This dosage schedule (methotrexate, 400 mg/kg, s.c...

Tissue pharmacokinetics, inhibition of DNA synthesis and tumor cell kill after high-dose methotrexate in murine tumor models.

In Sarcoma 180 and L1210 ascites tumor models, the initial rate of methotrexate accumulation in tumor cells in the peritoneal cavity and in small intestine (intracellularly) after s.c. doses up to 800 mg/kg, showed saturation kinetics. These results and the fact that initial uptake in these tissues within this dosage range was inhibited to the expected relative extent by the simultaneous administration of leucovorin suggest that carrier mediation and not passive diffusion is the major route of drug entry at these extremely high doses. Maximum accumulation of intracellular drug occurred within 2 hr and reached much higher levels in small intestine than in tumor cells at the higher dosages. At a 3-mg/kg dose of methotrexate s.c., intracellular exchangeable drug levels persisted more than four times longer in L1210 cells than in small intestine, but differences in persistence (L1210 cell versus gut) diminished markedly with increasing dosage. At 96 mg/kg, the difference in persistence was less than 2-fold. In small intestine and L1210 cells, theduration of inhibition of DNA synthesis at different dosages correlated with the extent to which exchangeable drug was retained. Toxic deaths occurred when inhibition in small intestine lasted longer than 25 to 30 hr. Recovery of synthesis in small intestine and L1210 cells occurred synchronously and only below dosages of 400 mg/kg. Within 24 hr after dosages of greater than 24 mg/kg, the rate of tumor cell loss increased to a point characterized by a single exponential (t1/2=8.5 hr). The total cell loss, but not the rate of cell loss, was dose dependent.

Kinetic correlates of methotrexate transport and therapeutic responsiveness in murine tumors.

Net accumulation of methotrexate by carrier-mediated transport in different murine tumor cells in vitro exhibits a positive correlation with the relative drug pharmacokinetics and therapeutic responsiveness in these tumors in vivo. The transport of methotrexate by Sarcoma 180, Ehrlich carcinoma, P388, P288, and L1210 leukemia cells is qualitatively similar. Influx of drug exhibits saturation kinetics and is highly temperature dependent (Q10, 6.1 to 9.4). Efflux of exchangeable methotrexate from all of the different tumor cells exhibited first-order kinetics and the same high temperature dependence seen for influx (Q10, 6.1 to 8.0). The major kinetic determinant of responsiveness is the Km for influx. Values vary from 3.1 to 11.2 X 10(-6) M and are highest in cells from a nonresponsive Sarcoma 180 tumor, somewhat lower in the poorly responsive Ehrlich tumor, lower in moderately responsive P388 and P288 leukemias, and lowest in the highly responsive L1210 leukemia. Values for the influx Vmax differ to some extent, but in a manner not correlatable with responsiveness. The level of responsiveness of the P388 leukemia in vivo can also be partially attributed to an efflux rate that is lower than that measured for the other tumor cells. Steady-state levels of drug accumulation in vitro reflected influx and efflux rates and were consistently correlatable with therapeutic responsiveness. There was no significant difference in the extent to which folate and reduced 5-substituted folate derivatives compete with methotrexate for uptake in cells from all five tumors. The average value for Ki measured with folate for each tumor cell type was 50- and 80-fold higher than for 5-formyltetrahydrofolate and 5-methyltetrahydrofolate.