[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-H4PteGlun) and tetrahydrofolates (H4PteGlun), 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 (tv2 = 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-H4PteGlun and H4PteGlun 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-H4PteGlun and H4PteGlun, suggesting saturation in the cellular metabolism of [6RS]leucovorin.

In HxGC3 tumors, CH2-H4PteGlu4–5 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-H4PteGlu3–5 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-H4PteGlun and H4PteGlun 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-H4PteGlun and H4PteGlun, the inhibition of thymidylate synthase by FUra, and FUra cytotoxicity in colon tumors under the in situ conditions of tumor growth.


Supported by National Cancer Institute Awards CA32613, CA23099, and Cancer Center Support (CORE) Grant CA21765; Center of Excellence Award, University of Tennessee; and American Lebanese Syrian Associated Charities.

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