High-dose edatrexate with oral leucovorin rescue: a phase I and clinical pharmacological study in adults with advanced cancer.

Our objective was to determine the maximum tolerated dose and toxicity of i.v. edatrexate with p.o. leucovorin. Thirty-one adults with advanced solid tumors received edatrexate as a 2-h infusion, once a week for 3 weeks, recycled every 28 days. p.o. leucovorin (10 mg/m2, every 6 h for 10 doses) began 24 h later. All had urinary alkalinization and p.o. hydration. Nine dosage levels ranging from 120 to 3750 mg/m2 were explored. Fatigue, epistaxis, nausea/emesis, mucositis, rash, myalgias, leukopenia, thrombocytopenia, and transient elevations of serum aspartate transferase were observed. Leukoencephalopathy with clinical manifestations occurred in two patients (one had prior cranial irradiation). Pharmacokinetic studies carried out at the 120- and 1080-mg/m2 dose levels revealed no significant difference in the elimination half-life at the two dose levels studied and no significant intrapatient variability between day 1 and day 8 edatrexate administration. Serum edatrexate levels measured using a dihydrofolate reductase inhibition assay correlated with those by high-performance liquid chromatography. Three major and two minor antitumor responses occurred. The maximum tolerated dose was 3750 mg/m2, with grade 3 or 4 leukopenia (one patient), stomatitis (one patient), and leukoencephalopathy (one patient). Because of the occurrence of leukoencephalopathy, further study of high-dose edatrexate with leucovorin rescue is not recommended.

Functional aspects of membrane folate receptors in human breast cancer cells with transport-related resistance to methotrexate.

Two methotrexate (MTX)-resistant human breast-cancer cell lines with impaired transport via the reduced folate carrier (RFC), one established in vitro (MTX(R)-ZR-75-1) and another inherently resistant (MDA-231), were adapted to grow in medium containing 2 nM folic acid. This induced the expression of previously undetectable membrane folate receptors (MFR) to levels of 8.2 and 2.3 pmol/10(7) cells, respectively. Polymerase chain reaction (PCR) quantitation revealed that MFR messenger-RNA levels of the isoform first described in human nasopharyngeal carcinoma KB cells (MFR-alpha) were increased in low-folate-adapted MTX(R)-ZR-75-1 cells, whereas placental transcripts (MFR-beta) coincided with MFR-alpha expression in low-folate (LF)-adapted MDA-231 cells. These cell lines were used to study the role of MFR in the uptake and growth-inhibitory effects of five different antifolates with varying affinities for MFR: N10-propargyl-5, 8-dideazafolic acid (CB3717) > 5,10-dideazatetra-hydrofolic acid (DDATHF) > N-5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-methyl) -N-methyl-amino]-2-theonyl}-glutamic acid (ZD1694) >> MTX > edatrexate (EDX). Expression of MFR only slightly decreased the resistant phenotype for MTX, EDX, and ZD1694, suggesting that these drugs are not transported intracellularly to cytotoxic concentrations at these levels of MFR expression. On the other hand, both cell lines became from at least 180- to 400-fold more sensitive to growth inhibition by CB3717 and DDATHF, which may be correlated with their high affinity for MFR. These sensitivity/resistance profiles were largely similar following cell culture in medium containing 1 nM L-leucovorin, a folate with an affinity for MFR 10-fold lower than that of folic acid, the one exception being the increased sensitivity for ZD1694 seen in the LF-adapted cells with the highest level of MFR expression (MTX(R)-ZR-75-1). These results illustrate that the efficacy of MFR in mediating antifolate transport and cytotoxicity depends on their affinity for the folate antagonist, their degree of expression, and the levels of competing folates.

Antifolates in rheumatoid arthritis: a hypothetical mechanism of action.

The antifolates, methotrexate, aminopterin, 10-deazaaminopterin and sulfasalazine are clinically useful in the treatment of rheumatoid arthritis. Toxicity, rather than efficacy, appears to the the major factor limiting the usefulness of the classical antifolates (i.e., methotrexate and 10-deazaaminopterin). The fact that folate supplementation of methotrexate-treated rheumatoid arthritis patients reduces toxicity without altering efficacy also suggests that inhibition of the drug's target enzyme, dihydrofolate reductase, is not complete and not essential for efficacy. Since polyglutamates of methotrexate are direct inhibitors of thymidylate synthase and folate dependent enzymes of purine biosynthesis, the efficacy of this agent may involve blockade of these pathways. We hypothesize that blockage of aminoimidazole carboxamide ribotide transformylase, the folate dependent enzyme responsible for the insertion of carbon 2 into the purine ring, produces an immunosuppression mediated by secondary inhibition of adenosine deaminase, and S-adenosyl homocystein hydrolase by aminoimidazolecarboxamide metabolites. This mechanism of immunosuppression may explain the clinical effect of methotrexate, 10-deazaaminopterin, and possibly sulfasalazine. Since purine biosynthesis is a fundamental process, blockading this pathway may also decrease leukotriene production and interleukin-1 expression, which also could contribute to the efficacy of methotrexate.

Mechanisms of natural resistance to antifolates in human soft tissue sarcomas.

Efforts to use fresh human sarcoma cells for evaluating antifolate resistance with an in situ thymidylate synthesis assay using 5-[3H] deoxyuridine were unsuccessful because of low thymidylate synthesis activity in enzymatically disaggregated tumors. By incubating tumor cell suspensions in supplemented RPMI-1640 medium with 10% fetal bovine serum for 3 days, activity of the in situ thymidylate synthesis assay markedly increased (1.42 versus 0.03 pmol/h/10(7) cells), thus allowing 75% of samples to be evaluated for antifolate sensitivity. By criteria developed with a methotrexate-resistant and -sensitive cell line, this assay indicated that most sarcomas are naturally resistant to methotrexate (12 of 15). Natural resistance to 10-ethyl-10-deazaaminopterin and trimetrexate was also observed in 60% of the samples (nine of 15, respectively). The results from the 3-day in situ assay were confirmed by specific tests for resistance mechanisms in most sarcoma samples. The resistance mechanisms detected were impaired polyglutamylation, an increased level of dihydrofolate reductase, and amplification of this gene. These results encourage further exploration of this assay to predict response to antifolates in individual patients and to evaluate efficacy of new antifolates as candidates for clinical trial.

Conformational energy calculations and electrostatic potentials of dihydrofolate reductase ligands: relevance to mode of binding and species specificity.

Classical potential energy calculations are reported for a series of 11 structurally diverse substrates, products, and inhibitors of dihydrofolate reductase. In almost every case, the calculations reveal a range of potential biologically active conformations accessible to the molecule, and geometry optimization with molecular mechanics and molecular orbital calculations further expands the range of accessible conformations. The energy calculations are supplemented with electrostatic potential energy surfaces for the heterocyclic components of each molecule. These data are used in conjunction with the energy calculations and the crystallographically determined enzyme structures to compare two alternative proposed binding modes of folates known to bind with their pteridine rings inverted relative to that of methotrexate. It is shown that the conformational flexibility of the connecting chain between the benzoyl glutamate and pteridine moieties in the folates actually allows the pteridine ring to shift between these alternative binding modes, a combination of which may offer the best explanation for the observed activity. The electrostatic potentials and conformational energy data are also used in an attempt to account for the species specificity of inhibitors of mammalian, bacterial, and protozoal dihydrofolate reductases. The results show that while these techniques can be used to explain many of the observed results, others require recourse to the observed crystal structures to provide a satisfactory explanation.

Folate analogues as substrates of mammalian folylpolyglutamate synthetase.

The antifolate drugs methotrexate (MTX) and aminopterin (AM) have been tested as substrates for folylpolyglutamate synthetase (FPGS) partially purified from beef liver. The Km for MTX is 100 microM, and that for AM is 25 microM. These values are considerably higher than those for either tetrahydrofolate or folinic acid. Based on their ratios of Vmax to Km, AM is a better substrate than is MTX for the beef liver FPGS. Both are poorer substrates than tetrahydrofolate. The 7-hydroxy metabolites of MTX and AM also are substrates for FPGS. The reactivity of 7-hydroxymethotrexate is similar to that of MTX, but 7-hydroxyaminopterin is a poorer substrate than AM. Folinic acid, often used as the rescue agent in high-dose MTX therapy, has a low Km with mammalian FPGS (7 microM). Its activity is comparable to that of the best substrate, tetrahydrofolate. Low concentrations of folinic acid prevent the formation of polyglutamates of MTX. This inhibition is competitive, presumably because folinic acid and MTX are competing substrates for FPGS. The activities of folate and antifolate substrates also have been determined with rat liver FPGS. With near-saturating concentrations of AM, MTX, or 7-hydroxymethotrexate, the reaction velocity exceeds that with an optimal concentration of tetrahydrofolate. However, the Km values of the folate analogues all are greater than those of the tetrahydrofolate coenzymes. In contrast to the formation of long-chain polyglutamates observed when tetrahydrofolate or folinic acid was the substrate, beef liver FPGS, under our reaction conditions, cannot catalyze the formation from MTX monoglutamate of polyglutamates longer than the triglutamate. MTX di- and triglutamates are poorer substrates than is MTX itself. Longer polyglutamates of MTX, while having no activity as substrates, must bind to the enzyme, because they are inhibitors. Our observations using MTX and AM with the enzymatic FPGS system help to rationalize the therapeutic use of antifolates.