J.A.R. Mead

The effect of reduced derivatives of folic acid on toxicity and antileukemic effect of methotrexate in mice

Abstract Mice inoculated with leukemia L1210 were treated with combinations of methotrexate (amethopterin) and one of the following folic acid derivatives: (a) folic acid, (b) dihydrofolic acid, (c) tetrahydrofolic acid, (d) 10-formyltetrahydrofolic acid, (e) 5-formyltetrahydrofolic acid (citrovorum factor), and (f) prefolic A (5-methyltetrahydrofolic acid). Daily treatment was started 3 days after leukemic inoculation. Treatment with methotrexate (MTX) alone, or in combination with folic acid, resulted in a considerable prolongation of lifetime. All other treatments resulted in leukemic death of the animals at the same time as the untreated controls; i.e. the antileukemic effect of methotrexate was blocked by the metabolite. Delayed administration of ctirovorum factor and prefolic A, after large doses of MTX, showed that both compounds were effective in reducing toxicity but had little or no effect on the antileukemic activity when given 12 to 24 hr after MTX. At 48 hr after administration of MTX neither compound protected against the toxicity of the drug. In view of the extreme sensitivity of dihydrofolic reductase to inhibition by MTX in vitro , it was of particular interest that dihydrofolic acid was able to bring about extensive reversal of the antileukemic effect and toxicity of this substance. A study of folic and dihydrofolic reductase activity in the livers of mice which had received combinations of MTX and folic acid derivatives showed that there was no difference in the extent of inhibition, regardless of whether the combination was toxic or nontoxic to the animals.

Effect of pretreatment with methotrexate on the reduction of dihydrohomofolic acid in mice

Abstract Reduction of dihydrohomofolate and dihydrofolate was studied in normal and leukemic mice. Liver, kidney and spleen from mice treated with the compounds were analyzed by column chromatography on DEAE cellulose columns and the isolated compounds were characterized by ultraviolet absorption spectrum and Chromatographic behavior. Both dihydrohomofolic and dihydrofolic acid were found to be reduced ( in vivo ) to the corresponding tetrahydro derivatives which were isolated from liver, kidney and spleen. The reduction was found to be blocked by pretreatment with methotrexate and the blocking effect was found to be time and dose dependent. The results of these studies strongly suggest that dihydrofolate reductase is responsible for the conversion of the dihydro derivatives to the corresponding tetrahydro forms. While it is likely that the conversion occurs in all tissues containing the enzyme, the possibility exists that distributional factors could account for the presence of tetrahydro derivatives in some tissues.

The pharmacological and biochemical activity of 4-amino-4-deoxy-10-methyl-pteroylaspartic acid

4-Amino-4-deoxy-10-methylpteroylaspartic acid (AspMTX), the aspartate analog of methotrexate (MTX), was synthesized, and its pharmacological and bio-chemical effects were studied. AspMTX, even at comparatively large doses, was less effective against mouse leukemia L1210 than MTX. Pretreatment with MTX, but not with AspMTX, abolished the protection afforded by folate against a lethal dose of aminopterin in mice. Dihydrofolic reductase activity in the liver of normal mice, as well as incorporation of formate-14C into acid-soluble adenine in the spleen of leukemic mice, was inhibited much less after administration of AspMTX than of MTX. Asp MTX was a competitive inhibitor (KiKm = 1.8 × 10−3at pH 7.4) of dihydrofolic reductase prepared from an antifolic-resistant subline of leukemia L1210. The findings are explained by a decreased affinity of the inhibitor for the enzyme after substitution of aspartate for glutamate.

A method for assessing dihydrofolate reductase inhibition in vivo

Abstract A method for studying inhibition of dihydrofolate reductase activity in vivo has been described. Mice were given 500 μCi (25 mg) 3 H-H 2 F/kg intraperitoneally, the tetrahydrofolate (H 4 F) formed in liver and gut were separated by DEAE-cellulose column chromatography of tissue homogenates, and the radioactivity in peaks corresponding to H 4 F and H 2 F was determined. A linear relationship was observed in the inhibition of H 2 F-reductase by methotrexate in both liver and intestine when the mice were treated with methotrexate 1 hr before H 2 F injection and the tissues were assayed 2 hr later. The recovery of enzyme activity from inhibition by methotrexate in gut and liver was biphasic: a rapid recovery (45% in gut and 72% in liver) within 6 hr was followed by a slower recovery (92% in gut and 98% in liver) in 2 to 3 days. The data presented indicate greater inhibition and slower recovery of H 2 F-reductase in intestine than in liver and illustrate the feasibility of assessing H 2 F reductase activity in vivo .

The effect of folate and folate analogs upon dihydrofolate reductase and DNA synthesis in kidneys of normal mice.

Abstract A single subcutaneous injection of folate, homofolate or MTX resulted in the inhibition of the activity of dihydrofolate reductase in homogenates prepared from the kidneys of normal mice. Stimulation of 3H-thymidine uptake occurred in the kidneys of treated animals approximately 30 hr after administration of either folate or homofolate, and reached a peak 72 hr after administration. The effects of folate and MTX on dihydrofolate reductase activity in vivo were also determined. One hr after administration of 15 mg/kg methotrexate (MTX) or 300 mg/kg folate, enzyme activity in vivo was inhibited by 90%. 3H-deoxyuridine uptake was neither stimulated nor depressed after treatment with MTX. After administration of folate, uptake of 3H-deoxyuridine was stimulated at approximately 30 hr after drug-treatment and reached a peak at 72 hr after folate administration. Treatment with xanthopterin had no effect on the activity of dihydrofolate reductase in vitro . Xanthopterin stimulated uptake of both deoxyuridine and thymidine in an identical manner. The increased DNA synthesis that occurs in animals after treatment with agents that cause renal damage is distinct from the effect these agents have upon dihydrofolate reductase. Nucleoside incorporation after treatment with folate, homofolate, MTX or xanthopterin cannot be predicted on the basis of enzyme inhibition. Treatment with MTX, folate or homofolate results in enzyme inhibition which is not correlated with the uptake of deoxyuridine into DNA.

Regeneration of tetrahydrohomofolate in cells: Possible basis for antitumor activity of homofolates

Abstract The purpose of this study was to determine if reduction of homofolates to tetrahydrohomofolate in mice and rat tumors in vivo could form the basis for the antitumor activity of reduced homofolates. A single dose (400 mg/kg) of dihydrohomofolate from the optimal therapeutic range was given to animals bearing advanced tumors, and their spleen or tumor tissues were analyzed for dihydro- and tetrahydrohomofolate content by DEAE cellulose column chromatography. Mice bearing leukemia L1210/FR-8, which is responsive to reduced homofolates, showed a measurable reduction of dihydrohomofolate in their spleen, whereas the mice bearing a less responsive tumor (L 1210) or no tumor showed extensive metabolism and no detectable amount of dihydro- or tetrahydrohomofolate in their spleens. The drug was also extensively metabolized in rats bearing Walker carcinosarcoma 256 tumor, which might also explain the minimal response of the tumor to reduced homofolates. The reduction of the parent compound, homofolate, which is a minimally effective antitumor agent, was not detectable in mouse liver. These studies suggested an apparent correlation between the ability of the tumors to reduce homofolates to the tetrahydro level and their response to treatment with these drugs. Since the tetrahydro form of homofolate appears to be the active moiety, the levels of dihydrofolate reductase and catabolizing enzymes, and determinants of the sustained levels of the tetrahydro derivative in tumors might be the important factors in determining the responsiveness of tumors to homofolates.

Chemical transformations of tetrahydrohomofolic acid in vitro and in vivo.

Abstract Analytical and preparative procedures for complete separation of dihydrohomofolic acid from tetrahydrohomofolic acid were established. Following incubation at 25 and 37°, the drug was oxidized to dihydrohomofolic acid within 30 min in the presence of plasma; however, it was stable for 1 h in the presence of Clelands reagent. In 1 h the drug in 0.6% ascorbate solution showed only slight oxidation, but oxidized and decomposed extensively in 17 h. A study in vivo showed that the drug constituted 90–95% of the Bratton-Marshall-positive material present in the liver for 4 h after administration of the drug, as well as its oxidation product, the dihydrohomofolic acid-like material. At 20 min after administration mostly unchanged drug, and no other transformation product, was found in the plasma. However, at 40 min the material eluted in the fractions corresponding to the drug showed at UV absorption spectrum (λmax 287–292) not characteristic of the drug, indicating the presence of transformation products of the drug. Although urine contained unchanged drug in large amounts, biotransformation of the drug in vivo could not be ruled out. The study suggests that the continued presence of the drug in liver might be due to the intracellular reduction of dihydrohomofolic acid-like material and the antitumor activity of the drug might be dependent on high levels of dihydrofolate reductase activity in tumors.

Dihydrofolate reductase activity and loss of resistance in an antifolate-resistant subline of leukemia 11210 after discontinuation of treatment

Abstract A subline of systemic leukemia L1210 resistant to methotrexate (MTX) and dichloromethotrexate (DCM), which had a 70-fold increased dihydrofolate reductase activity, was carried in the absence of treatment. Enzyme activity reverted to the original level of the parent line after 28 transfer generations, but was again increased 70-fold by daily treatment with 80 mg/kg DCM for one generation. After two further similarly treated transfers, 77 untreated transfer generations were required for return of enzyme activity to the original low level. The tumor remained resistant to therapy for several additional generations but finally reverted to the drug-sensitive state.