Relationship between Dose Rate of [6RS]Leucovorin Administration, Plasma Concentrations of Reduced Folates, and Pools of 5,10-Methylenetetrahydrofolates and Tetrahydrofolates in Human Colon Adenocarcinoma Xenografts¹

[6RS]Leucovorin (5-formyltetrahydrofolate; 5-CHO-H₄PteGlu) administered in different regimens in combination with 5-fluorouracil (FUra) has increased the response rates to FUra in patients with colon adenocarcinoma. Using preclinical models of human colon adenocarcinomas as xenografts in immune-deprived mice, the effect of the rate of administration of racemic [6RS]leucovorin on the concentration-time profile of reduced folates in plasma, size of intratumor pools of 5,10-methylenetetrahydrofolates (CH₂-H₄PteGlu_n) and tetrahydrofolates (H₄PteGlu_n), and the distribution of their polyglutamate species have been examined.

Bolus injection i.v., or 4-h or 24-h infusion of [6RS]leucovorin (500 mg/m²) yielded similar concentration profiles of the biologically active [6S] and inactive [6R] isomers of 5-CHO-H₄-PteGlu and 5-methyltetrahydrofolate (5-CH₃-H₄PteGlu) in mouse plasma to those previously reported in humans, but with more rapid elimination half-lives (t_v2 = 11 to 16 min, 23 to 41 min, and 30 to 35 min, respectively). Thus, reduced folates remained elevated in plasma during the period of [6RS]leucovorin administration. In HxELC₂ and HxGC₃ tumors, pools of CH₂-H₄PteGlu_n and H₄PteGlu_n were increased from 350% to 700% of control, but only during [6RS]leucovorin infusion. Intracellular levels subsequently declined rapidly, similar to the loss of reduced folates from plasma. Increasing the rate of [6RS]leucovorin delivery by decreasing the time for administration from a 24-h to a 4-h infusion did not further increase the intratumor pools of CH₂-H₄PteGlu_n and H₄PteGlu_n, suggesting saturation in the cellular metabolism of [6RS]leucovorin.

In HxGC₃ tumors, CH₂-H₄PteGlu_4–5 were elevated more rapidly than in line HxELC₂, which accumulated predominantly a shorter chain length species following i.v. bolus injection. During the 4-h infusion schedule, di- and triglutamate species in particular accumulated in both tumors with no elevation in CH₂-H₄PteGlu₅ until the infusion was discontinued, when this species increased as the shorter chain length forms were declining. However, during the 24-h infusion of [6RS]leucovorin, CH₂-H₄PteGlu_3–5 were elevated in tumors. Since these species have been reported to increase the binding affinity of [6-³H]5-fluorodeoxyuridine monophosphate ([6-³H]FdUMP) to thymidylate synthase, and intratumor pools of CH₂-H₄PteGlu_n and H₄PteGlu_n were elevated during the 24-h infusion of [6RS]leucovorin, this was considered to be the preferred schedule for administration. When FUra (6.25 to 25 mg/kg) was administered 3 h into a 24-h infusion of [6RS]leucovorin (500 mg/m²) in tumor-bearing mice, potentiation of thymidylate synthase inhibition in comparison with FUra administered alone was observed. These studies raise important questions regarding the effect of (a) dose of [6RS]-leucovorin, (b) frequency of administration, (c) utility of 5-CH₃-H₄PteGlu, and (d) [6R]5-CHO-H₄PteGlu on influencing intratumor pools of CH₂-H₄PteGlu_n and H₄PteGlu_n, the inhibition of thymidylate synthase by FUra, and FUra cytotoxicity in colon tumors under the in situ conditions of tumor growth.