5-10-methylenetetrahydrofolic-acid and Adenocarcinoma

Modufolin® ([6R]-5,10-methylene tetrahydrofolate; [6R]-MTHF) is an endogenous biomodulator that is being developed as an alternative to leucovorin, a folate prodrug used in the treatment of colorectal cancer. The objective of this phase 1 dose de-escalation trial was to estimate the minimum tolerated dose of [6R]-MTHF to be used in combination with pemetrexed 500 mg/m(2) in the neoadjuvant treatment of patients with rectal cancer.. Adult patients (≥18 years) with resectable rectal adenocarcinoma were allocated to [6R]-MTHF doses of 500, 100, 50, and 10 mg/m(2) in combination with pemetrexed 500 mg/m(2). [6R]-MTHF was administered as an intravenous (i.v.) bolus injection 1 week prior to the first dose of pemetrexed and then once weekly for 9 weeks; pemetrexed was administered by i.v. infusion once every 21 days for three cycles.. Twenty-four patients (mean [SD] age, 63.1 [12.9] years) were enrolled in the study. A total of 72 treatment-related adverse events (AEs) were reported, of which the most common were fatigue (n = 17; 23.6 %), nausea (n = 10; 13.9 %), and diarrhea (n = 5; 6.9 %). The incidence of treatment-related AEs by [6R]-MTHF dose level (500, 100, 50, 10 mg/m(2)) was 11.1 % (n = 8), 13.9 % (n = 10), 45.8 % (n = 33), and 29.2 % (n = 21), respectively. There were no dose-limiting toxicities, and only two (2.8 %) treatment-related AEs were grade 3 in severity. Of the 11 serious AEs reported, none were considered to be related to [6R]-MTHF treatment.. The results of this phase 1 study indicate that the estimated minimum tolerated dose of [6R]-MTHF was 100 mg/m(2) once weekly in combination with pemetrexed 500 mg/m(2). The low toxicity profile of [6R]-MTHF supports its further evaluation as a component of systemic chemotherapy in the management of colon and rectal cancer.

Topics: Adenocarcinoma; Aged; Antineoplastic Combined Chemotherapy Protocols; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Humans; Male; Middle Aged; Neoadjuvant Therapy; Pemetrexed; Rectal Neoplasms; Tetrahydrofolates

Aberrant DNA methylation occurs in a subset of colorectal cancers and is characterized by regional areas of hypermethylation at CpG islands. The aims of this study were firstly to evaluate the levels of folate intermediates (FIs) in tumors with aberrant DNA methylation and secondly to determine whether these levels are affected by polymorphisms in key genes involved in folate metabolism.. The concentrations of two major intracellular FIs, 5,10-methylenetetrahydrofolate and tetrahydrofolate (FH4), were measured in 103 surgically resected colorectal cancers. DNA hypermethylation at seven different CpG islands was measured using the MethylLight assay. Genotyping for polymorphisms in the thymidylate synthase, cystathionine beta-synthase, methionine synthase, and methylenetetrahydrofolate reductase (MTHFR) genes was carried out using PCR and PCR-RFLP.. Significantly higher levels of FH4 were found in tumors from the proximal colon compared with those originating in the distal colon and rectum. Tumors with aberrant DNA methylation of CpG islands within promoter regions of the hMLH1, TIMP3, and ARF genes also contained higher levels of both 5,10-methylenetetrahydrofolate and FH4. In contrast, patients who were homozygous for the C667T polymorphism of the MTHFR gene had significantly lower concentrations of both these FIs in their tumor tissue.. The concentrations of FIs in colorectal tumors are directly related to the presence of frequent DNA hypermethylation and inversely related to the presence of a common polymorphism in the MTHFR gene. FIs could serve as biochemical markers for the risk of developing this disease, as well as for the prediction of toxicity and efficacy of fluorouracil-based treatments.

Topics: 5-Methyltetrahydrofolate-Homocysteine S-Methyltransferase; Adenocarcinoma; Adenoma; Aged; Colorectal Neoplasms; CpG Islands; Cystathionine beta-Synthase; DNA Methylation; Female; Genotype; Humans; Male; Methylenetetrahydrofolate Reductase (NADPH2); Polymerase Chain Reaction; Polymorphism, Genetic; Polymorphism, Restriction Fragment Length; Tetrahydrofolates; Thymidylate Synthase

Various factors, including thymidylate synthase, thymidine kinase, 5-fluorouracil phosphorylation and degradation pathways, folate concentrations, and the stability of ternary complex, which influence thymidylate synthase inhibition rate of fluoropyrimidines, were studied in 87 human adenocarcinoma tissues.. The activity of the 5-fluorouracil degradation pathway was not significantly lower than the activity of the 5-fluorouracil phosphorylation pathway. The activity of the catabolism pathway of 5-fluorouracil should be considered in human adenocarcinoma tissue during chemotherapy. On the other hand, the means plus or minus standard deviations (means +/- SD) of the concentration of 5,10-methylenetetrahydrofolate and tetrahydrofolate were 0.69 +/- 0.54 and 1.25 +/- 0.69 nM, respectively, for the adenocarcinoma tissues without previous chemotherapy.. Because the half-life of tritium-labeled ternary complex and folate concentration in cytosol were correlated well, the differences in folate concentration among tumors must influence the dynamic equilibrium of ternary complex formation. Moreover, these results show that the ratio of 5,10-methylenetetrahydrofolate concentration to thymidylate synthase concentration influences the thymidylate synthase inhibition rate in tumor, and that the new synthesis of 5,10-methylenetetrahydrofolate and tetrahydrofolate from other endogenous reduced folates is also important in tumors with high thymidylate synthase concentrations.

Topics: Adenocarcinoma; Colorectal Neoplasms; Culture Techniques; Fluorouracil; Folic Acid; Humans; Leucovorin; Lymph Nodes; Phosphorylation; Stomach Neoplasms; Tetrahydrofolates; Thymidine Kinase; Thymidylate Synthase

Leucovorin (LV) increases the cytotoxic effect of fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd) by enhancing the formation of the fluorodeoxyuridine monophosphate (FdUMP) thymidylate synthase (TS) 5,10-methylenetetrahydrofolate (mTHF) ternary complex. To study the difference in the efficacy of this combination against different tumors, we compared the effect of LV (20 microM) on the cytotoxicity of FUra, FdUrd, and 5-fluorouridine (FUrd) in vitro against cell lines of five colorectal carcinomas (CC), five gastric carcinomas (GC), and four non-small-cell lung carcinomas (NSCLC) using the colony-forming assay. At the concentration used in the experiments, LV alone failed to inhibit colony formation in any of the cell lines tested. The NSCLC cell lines were more resistant to FdUrd than were the CC and GC lines. LV modulated the cytotoxicity of FdUrd in all five CC lines and in three of the five GC lines but failed to do so in any of the NSCLC lines. In addition, following 20 h treatment with 1 microM [3H]-FdUrd, formation of the FdUMP/TS/mTHF ternary complex was enhanced by LV in the LV-sensitized CC and GC cell lines but not in the LV-refractory NSCLC lines. These in vitro data corresponded well to the results of clinical trials. Therefore, the colony-forming assay may be useful for the identification of the sensitivity of tumors according to phenotype.

Topics: Adenocarcinoma; Carcinoma, Non-Small-Cell Lung; Cell Survival; Colony-Forming Units Assay; Colorectal Neoplasms; Drug Synergism; Floxuridine; Fluorodeoxyuridylate; Fluorouracil; Leucovorin; Lung Neoplasms; Stomach Neoplasms; Tetrahydrofolates; Thymidylate Synthase; Tumor Cells, Cultured

[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 (t1/2 = 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

Topics: Adenocarcinoma; Animals; Colonic Neoplasms; Female; Floxuridine; Half-Life; Humans; Injections, Intravenous; Leucovorin; Mice; Mice, Inbred CBA; Tetrahydrofolates; Thymidylate Synthase; Time Factors

Using preclinical models of human colon adenocarcinomas in immune-deprived mice, the influence of dose of [6RS]leucovorin ([6RS]LV, 20 to 1000 mg/m2) administered by 24-h i.v. infusion was determined on the following parameters: (a) plasma concentrations of the active [6S] and inactive [6R] isomers of [6RS]LV and the biologically active diastereoisomer of 5-methyltetrahydrolate (5-CH3-H4PteGlu); (b) expansion of intratumor pools of 5,10-methylenetetrahydrofolates (CH2-H4PteGlun) and tetrahydrofolates (H4PteGlun), that may influence the binding of 5-fluorodeoxyuridylate to thymidylate synthase; (c) the distribution of polyglutamate forms of CH2-H4PteGlun and H4PteGlun; and (d) (5-fluorouracil (FUra)-mediated thymidylate synthase inhibition in Hx-ELC2, HxGC3, HxVRC5, and HxHC1 tumors. Folypolyglutamate synthetase activities were also determined in each line. Linear increases in plasma concentrations of [6R]LV, [6S]LV, and 5-CH3-H4-PteGlu were determined over the complete range of [6RS]LV doses examined. However, in neoplastic tissues three patterns of biochemical modulation by [6RS]LV were evident. (a) In HxELC2 and HxVRC5 tumors, pools of CH2-H4PteGlun and H4PteGlun were elevated in proportion to the dose of [6RS]LV between dose levels of 50 and 200 mg/m2. Subsequent expansion of these pools continued that was disproportionate to the dose of [6RS]LV until no further increase was observed beyond 800 mg/m2 [6RS]LV, at which point pools were maximally expanded by 4- to 4.5-fold. The extent of retardation of recovery of thymidylate synthase activity increased as the dose of [6RS]LV was increased in both tumors, when FUra (15 or 50 mg/kg), was administered by i.v. bolus injection 3 h into the 24-h infusion of [6RS]LV. This was related to the increase in predominance of CH2-H4PteGlu2-5 with increasing dose of [6RS]LV. (b) For HxHC1 tumors, little expansion of CH2-H4PteGlun and H4PteGlun pools (maximum, 137% of control) was detected at the highest dose levels of [6RS]LV, and no significant modulation of FUra-inhibited thymidylate synthase activity was detected, even at 1000 mg/m2 [6RS] LV. CH2-H4PteGlu5 remained similar or decreased as the dose of [6RS] LV was increased. (c) For line HxGC3, pools of CH2-H4PteGlun and H4PteGlun increased gradually from 169% of control at 20 mg/m2 [6RS] LV to 233% of control at 1000 mg/m2 [6RS]LV, and were intermediate between the expansion observed in HxHC1 in comparison to HxELC2 and HxVRC5 tumors. CH2-H4PteGlu3-5 were elevated a

Topics: Adenocarcinoma; Animals; Colonic Neoplasms; Dose-Response Relationship, Drug; Female; Fluorouracil; Humans; Leucovorin; Mice; Mice, Inbred CBA; Peptide Synthases; Tetrahydrofolates; Thymidylate Synthase; Time Factors

Four hr infusions i.v. of [6RS]5-formyltetrahydrofolate ([6RS]5-CHO-H4PteGlu; 500 mg/m2) and [6RS]5-methyltetrahydrofolate ([6RS]5-CH3-H4PteGlu; 500 mg/m2) were compared for their relative effects on expansion of pools of 5,10-methylenetetrahydrofolates (CH2-H4PteGlun) and tetrahydrofolates (H4PteGlun) in two human colon adenocarcinoma xenografts in mice. Expansion of these pools by 253-661% of control and increase in predominance of di-, tri-, and tetra-glutamate species were observed during [6RS]5-CHO-H4PteGlu infusion. In contrast, only modest pool size expansion (148-164% of control) and limited modulation of polyglutamate species were detected in four tumor lines during infusion with [6RS]5-CH3-H4PteGlu. The data suggest that [6RS]5-CH3-H4PteGlu is less effective than [6RS]5-CHO-H4PteGlu as a precursor for pools of CH2-H4PteGlun and H4PteGlun in colon tumors.

Topics: Adenocarcinoma; Animals; Biotransformation; Cell Line; Colonic Neoplasms; Female; Formyltetrahydrofolates; Humans; Kinetics; Mice; Mice, Inbred CBA; Neoplasm Transplantation; Pteroylpolyglutamic Acids; Tetrahydrofolates; Transplantation, Heterologous

CH2-H4PteGlu and H4PteGlu exist in human colon adenocarcinoma xenografts predominantly in the form of polyglutamate species at concentrations of less than 3 microM. The interaction of polyglutamates of [6R]CH2-H4PteGlu in the formation and stability of [6-3H]FdUMP-thymidylate synthase-CH2-H4PteGlun ternary complexes has therefore been examined using enzyme purified from a human colon adenocarcinoma xenograft. Dissociation of these complexes was first-order and was dependent upon the concentration of folate. [6R]CH2-H4PteGlu3-6 (0.9 to 1.6 microM) were greater than 200-fold and [6R]CH2-H4PteGlu2 (18.2 microM) was 18-fold more effective than [6R]CH2-H4PteGlu1 (335 microM) at stabilizing ternary complexes for a T1/2 for dissociation of 100 min. Polyglutamylation of CH2-H4PteGlu also increased the affinity of binding of [6-3H]FdUMP to thymidylate synthase as determined by Scatchard analysis at folate concentrations of 10 microM, where the Kd in the presence of [6R]CH2-H4PteGlu1 was in the order of 4.0 x 10(-8) M, and for [6R]CH2-H4PteGlu3-5 was between 3.7 and 5.5 x 10(-9) M. To examine whether this effect was due to differences in the rates at which [6-3H]FdUMP was bound (kon) or dissociated (koff) from the enzyme, the apparent rate of [6-3H]FdUMP binding was determined in the presence of [6R]CH2H4PteGlu1, [6R]CH2-H4PteGlu3 and [6R]CH2-H4PteGlu4. The kon values were similar and were in the range of 1.7 to 2.3 x 10(6) M-1 min-1 for 10 or 20 microM folate concentrations. Differences in binding affinity determined for [6R]CH2-H4PteGlu1 and longer polyglutamate forms of [6R]CH2-H4PteGlu were thus due to differences in koff. The Vmax for the initial velocity of [6-3H]FdUMP binding was achieved at 10 microM folate. Consequently, at concentrations of CH2-H4PteGlu polyglutamates present in tumors, inhibition of thymidylate synthase by FdUMP in vivo would be expected to be transient, based upon the concentration of [6R]CH2-H4PteGlun required for maximal formation and stability of the covalent ternary complex. It would be advantageous for modulation of CH2-H4PteGlun pools to increase the concentrations of the longer polyglutamate species (n greater than or equal to 3) to maximize the interaction between FdUMP, thymidylate synthase and CH2-H4PteGlu.

Topics: Adenocarcinoma; Colonic Neoplasms; Deoxyuracil Nucleotides; Fluorodeoxyuridylate; Folic Acid; Humans; In Vitro Techniques; Kinetics; Pteroylpolyglutamic Acids; Structure-Activity Relationship; Tetrahydrofolates; Thymidylate Synthase

The method for measuring polyglutamate forms of CH2-H4PteGlu and H4PteGlu, by entrapment in ternary complexes with [6-3H]5-fluoro-2'-deoxyuridylate and Lactobacillus casei thymidylate synthase has been characterized. Results demonstrated that (a) the relationship between concentration of CH2-H4PteGlu and complex isolated on nondenaturing gels was dependent upon the number of glutamyl residues, and an alternative method for data analysis has been presented, (b) the relationship was linear over a 100-fold change in concentration, (c) formation of isolatable complex was time dependent, (d) noncovalent complexes formed with PteGlu2-5 could be isolated only at concentrations considerably higher than those required for CH2-H4PteGlu1-6, and (e) endogenous deoxyuridylate would be unlikely to interfere significantly with the assay. The distribution of polyglutamates of CH2-H4PteGlu and the combined pools of CH2-H4PteGlu plus H4PteGlu were subsequently examined in three human colon adenocarcinoma xenografts. In each tumor, the pentaglutamate of CH2-H4PteGlu and H4PteGlu was the most prominent species, followed by the hexaglutamate, constituting 68 to 92% of the CH2-H4PteGlu pool, and greater than 93% of the combined pools. A small percentage of di-, tri-, and tetraglutamates were also detected. Using a catalytic assay, the combined pool of CH2-H4PteGlu and H4PteGlu was estimated in the range of 0.5 to 2.7 microM in cell water, and for CH2-H4PteGlu, from 185 nM to 1.7 microM. Using thymidylate synthase purified from colon adenocarcinoma HxVRC5, CH2-H4PteGlu5 (where the subscript digit attached to the glutamate portion equals the number of glutamate residues) stabilized the covalent ternary complex at greater than 200-fold lower concentration in comparison to CH2-H4PteGlu1. Data indicated that in each colon tumor, the concentrations of CH2-H4PteGlun or CH2-H4PteGlun plus H4PteGlun were suboptimal for the interaction of 5-fluoro-2'-deoxyuridylate with thymidylate synthase, and would predict for relatively transient inhibition of thymidylate synthase after treatment with 5-fluorouracil. These data support therapeutic modulation to increase the concentration of CH2-H4PteGlun in the treatment of colon adenocarcinomas with 5-fluorouracil.

Topics: Adenocarcinoma; Animals; Cell Line; Colonic Neoplasms; Humans; Mice; Neoplasm Transplantation; Protein Binding; Structure-Activity Relationship; Tetrahydrofolates; Thymidylate Synthase; Transplantation, Heterologous

This study was designed to examine the endogenous concentrations of 5,10-methylenetetrahydrofolate (CH2FH4) in human colorectal adenocarcinoma xenografts, and to determine the ability of other folate derivatives to increase the formation of the ternary covalent complex between CH2FH4, [6-3H]-5-fluorodeoxyuridylate (FdUMP) and thymidylate synthetase (TS, EC 2.1.1.45). Levels of CH2FH4 were determined by measuring the release of [3H]2O from [5-3H]-dUMP using TS from Lactobacillus casei. The reaction was linear from 1.9 X 10(-13) to 2.4 X 10(-11) mol of CH2FH4 assayed. Concentrations of CH2FH4 were low, ranging from 66 to 233 nM in cell water. Tetrahydrofolate (FH4) and dihydrofolate (FH2) increased complex formation, while 5-formyltetrahydrofolate (5-CHOFH4) and 5-methyltetrahydrofolate (5-CH3FH4) decreased the covalent binding of [6-3H]-FdUMP in vitro. Administration of FH4 or FH2 to tumor-bearing mice reduced subsequent formation of the covalent complex in vitro. Since 5-CH3FH4 is a major derivative of folate in mammalian tissues, its effect on the covalent binding of [6-3H]-FdUMP was examined further; even in the presence of homocysteine and cyanocobalamin (B12), the formation of the covalent complex was not increased. The fate of [5-14CH3]-FH4 was subsequently examined in vivo. In tumors at 1 hr after injection, 72% of the radiolabel remained as [5-14CH3]-FH4, while 17% had been converted to [14C]-methionine or incorporated into protein. By contrast, however, the incorporation of radiolabel into the protein fraction of liver was almost 30-fold greater at this time. At 4 hr, radioactivity in tumors (dpm/g) and in the fraction associated with [5-14CH3]-FH4 was decreased by over 60%, while metabolism was increased by only 13%. No polyglutamate forms of [5-14CH3]-FH4 were detected in tumors at 4 hr after treatment.

Topics: Adenocarcinoma; Animals; Cell Line; Colonic Neoplasms; Deoxyuracil Nucleotides; Female; Fluorodeoxyuridylate; Folic Acid; Homocysteine; Humans; Methyltransferases; Mice; Mice, Inbred Strains; Neoplasm Transplantation; Rectal Neoplasms; Tetrahydrofolates; Thymidylate Synthase; Transplantation, Heterologous; Vitamin B 12