levoleucovorin and lometrexol

Lometrexol is an antifolate which inhibits glycinamide ribonucleotide formyltransferase (GARFT), an enzyme essential for de novo purine synthesis. Extensive experimental and limited clinical data have shown that lometrexol has activity against tumours which are refractory to other drugs, notably methotrexate. However, the initial clinical development of lometrexol was curtailed because of severe and cumulative antiproliferative toxicities. Preclinical murine studies demonstrated that the toxicity of lometrexol can be prevented by low dose folic acid administration, i.e. for 7 days prior to and 7 days following a single bolus dose. This observation prompted a Phase I clinical study of lometrexol given with folic acid supplementation which has confirmed that the toxicity of lometrexol can be markedly reduced by folic acid supplementation. Thrombocytopenia and mucositis were the major toxicities. There was no clear relationship between clinical toxicity and the extent of plasma folate elevation. Associated studies demonstrated that lometrexol plasma pharmacokinetics were not altered by folic acid administration indicating that supplementation is unlikely to reduce toxicity by enhancing lometrexol plasma clearance. The work described in this report has identified for the first time a clinically acceptable schedule for the administration of a GARFT inhibitor. This information will facilitate the future evaluation of this class of compounds in cancer therapy.

Topics: Acyltransferases; Administration, Oral; Adult; Aged; Antidotes; Antineoplastic Combined Chemotherapy Protocols; Bone Marrow Diseases; Dose-Response Relationship, Drug; Drugs, Investigational; Enzyme Inhibitors; Female; Folic Acid; Folic Acid Antagonists; Gastrointestinal Diseases; Humans; Hydroxymethyl and Formyl Transferases; Kidney Diseases; Leucovorin; Male; Middle Aged; Phosphoribosylglycinamide Formyltransferase; Tetrahydrofolates

Lometrexol (5,10-dideazatetrahydrofolic acid) is a new antifolate that is highly selective in inhibiting the key enzyme of purine synthesis glycinamide ribonucleotide formyltransferase. The most promising preclinical features of lometrexol in animal models were its significant activity against a broad panel of solid tumors, the schedule dependency of its antitumor activity, and the availability of a rescue regimen with folinic or folic acid. In the present study, lometrexol was first given daily for 3 consecutive days, repeated every 4 weeks (part I). The occurrence of delayed myelotoxicity prompted the development of a rescue regimen with lometrexol given in a single dose on day 1, followed by oral folinic acid, 15 mg four times a day, from day 3 to day 5 (part II). Longer time intervals between administration of lometrexol and start of rescue were then evaluated (part III), and in the last part of the study (part IV), the maximum tolerated dose of single intermittent doses of lometrexol with folinic acid given from day 7 to day 9 was established. Sixty adult patients entered the study. In part I, the highest daily dose that could be safely given was 4 mg/m2, for a total dose of 12 mg/m2. Cumulative early stomatitis and delayed thrombocytopenia were dose limiting. The use of oral folinic acid made it possible to escalate the dose up to 60 mg/m2, and the maximum tolerated dose was reached at this dose when folinic was given from day 7 to day 9, with anemia being the dose-limiting toxicity. A shorter time interval between lometrexol and folinic acid administrations (from day 5 to day 7) is recommended for Phase II evaluations to optimize the antitumor effect. Anemia was normochromic and macrocytic, possibly due to a deficiency of folic acid. One partial response of 8 months' duration was reported in a patient with epithelial cancer of the ovary, relapsing after cisplatin and alkylating agents. The use of folic acid as rescue, proposed on the basis of experimental data and pharmacological considerations, has also allowed the repeated administration of lometrexol at doses higher than in the previous studies. The advantages of rescue with folinic acid over supplementation with folic acid, however, are difficult to define.

Topics: Adult; Bone Marrow; Folic Acid Antagonists; Humans; Leucovorin; Neoplasms; Tetrahydrofolates

Cancer chemotherapy with folate antimetabolites has been traditionally targeted at the enzyme dihydrofolate reductase and is based on the requirement of dividing tumor cells for a supply of thymidylate and purines. However, a new compound, 5,10-dideazatetrahydrofolate (DDATHF, whose 6R diastereomer is also known as Lometrexol), has become available that prevents tumor cell growth by inhibiting the first of the folate-dependent enzymes involved in de novo purine synthesis, glycinamide ribonucleotide formyltransferase.. We investigated the toxicity and therapeutic activity of DDATHF in a phase I clinical trial.. DDATHF was given at one of the following dose levels to 33 patients (16 females and 17 males) with malignant solid tumors: 3.0 mg/m2 per week (level A) to 10 patients, 4.5 mg/m2 per week (level B) to 13 patients, or 6.0 mg/m2 per week (level C) to 10 patients. Each drug cycle consisted of three weekly injections of DDATHF followed by a 2-week rest prior to redosing in the next cycle.. Of 33 patients, 27 received at least one full cycle of DDATHF. Thrombocytopenia was the major dose-limiting toxicity, and it was severe in one of 10 patients during the first cycle and in two of four patients during the second cycle. Because of cumulative toxicity at 6.0 mg/m2, second or later cycles were abbreviated to two weekly doses. Stomatitis was generally mild, but it was dose-limiting in one patient. Neutropenia was infrequent and mild, and normocytic anemia requiring blood transfusion was common with repeat dosing. Leucovorin was given for grade 2 or greater thrombocytopenia and resulted in hematologic recovery within 1 week in all eight patients so treated. Without leucovorin, the thrombocytopenia lasted from 7 to 49 days in three patients. A partial response was noted in one patient with non-small-cell lung cancer and a minor response in one patient with breast cancer. Three patients with colorectal cancer achieved stable disease for greater than 3 months with improvement in carcinoembryonic antigen levels in one patient.. DDATHF has an unusual pattern of toxicity with repetitive dosing, and humans with advanced cancer are considerably more sensitive than would be predicted from previous animal studies. Although doses of 6.0 mg/m2 per week on our schedule have been determined to be safe, repeated cycles require careful monitoring because of cumulative toxic effects.. Additional phase I studies of DDATHF that relate toxicity to folate intake and tissue folate pools appear warranted.

Topics: Adult; Aged; Drug Administration Schedule; Female; Folic Acid Antagonists; Hematologic Diseases; Humans; Leucovorin; Male; Middle Aged; Neoplasms; Purines; Tetrahydrofolates; Treatment Outcome

A murine leukemia cell line was identified that is highly resistant to methotrexate (MTX), due to impaired transport, but fully sensitive to 5,10-dideazatetrahydrofolate (DDATHF). A valine-to-methionine substitution at amino acid 104 in the reduced folate carrier (RFC1) explains this disparity in drug resistance. Transfection of the V104M cDNA into an RFC1-deficient cell line markedly increased DDATHF influx (32x) but only modestly increased influx of MTX and 5-formyltetrahydrofolate (4- and 6-fold, respectively). The growth inhibition or growth requirements for these folates fell by factors of 18, 2, and 4, respectively, in the transfectant. Preservation of DDATHF influx in cells with V104M RFC1 resulted in even greater preservation (60%) of the exchangeable drug level. Another major element in the preservation of DDATHF activity was the impact of the mutated carrier on cellular folate pools. For folic acid, folate pools were essentially unchanged but DDATHF polyglutamate levels decreased in lines that express the V104M carrier. However, with 5-formyltetrahydrofolate as the growth source, there was a marked decrease in folate pools in the lines carrying the mutated carrier, and DDATHF polyglutamate levels were unchanged. Hence, DDATHF activity was preserved in cells with V104M RFC1 due to (a) relative conservation of DDATHF transport, and (b) depletion of cellular THF cofactors with diminishing folate cofactor competition at folylpolyglutamate synthetase and possibly glycinamide ribonucleotide formyltransferase. Hence, resistance to one antifolate, in this case MTX, because of a loss of RFC1 transport activity need not exclude the subsequent utility of another antifolate that uses the same carrier.

Topics: Amino Acids; Animals; Antimetabolites, Antineoplastic; Biological Transport; Carrier Proteins; DNA, Complementary; Drug Resistance, Neoplasm; Folic Acid Antagonists; Leucovorin; Leukemia L1210; Membrane Proteins; Membrane Transport Proteins; Methotrexate; Mice; Tetrahydrofolates; Transfection

L1210/D3 mouse leukemia cells are resistant to 5, 10-dideazatetrahydrofolate due to expansion of cellular folate pools which block polyglutamation of the drug (Tse, A., and Moran, R. G. (1998) J. Biol. Chem. 273, 25944-25952). These cells were found to have two point mutations in the reduced folate carrier (RFC), resulting in a replacement of isoleucine 48 by phenylalanine and of tryptophan 105 by glycine. Each mutation contributes to the resistance phenotype. Genomic DNA from resistant cells contained both the wild-type and mutant alleles, but wild-type message was not detected. Folic acid was a much better substrate, and 5-formyltetrahydrofolate was a poorer substrate for transport in L1210/D3 cells relative to L1210 cells. Enhanced transport of folic acid was due to a marked, approximately 20-fold, decrease in the influx Km. Influx of methotrexate and 5,10-dideazatetrahydrofolate were minimally altered. Transfection of mutated rfc cDNA into RFC-null L1210/A cells produced the substrate specificity and 5, 10-dideazatetrahydrofolate resistance observed in the L1210/D3 line. Transfection of the mutant cDNA into wild-type cells also conferred resistance to 5,10-dideazatetrahydrofolate. We conclude that the I48F and W105G mutations in RFC caused resistance to 5, 10-dideazatetrahydrofolate, that the region of the RFC protein near these two positions defines the substrate-binding site, that the wild-type allele was silenced during the multistep development of resistance, and that this mutant phenotype represents a genetically dominant trait.

Topics: Animals; Biological Transport; Carrier Proteins; Cell Division; DNA Mutational Analysis; Drug Resistance; Folate Receptors, GPI-Anchored; Folic Acid; Kinetics; Leucovorin; Leukemia L1210; Methotrexate; Mice; Phenotype; Point Mutation; Pteroylpolyglutamic Acids; Receptors, Cell Surface; RNA, Messenger; Tetrahydrofolates; Transcription, Genetic; Transfection; Tumor Cells, Cultured

The role of a membrane-associated folate binding protein (mFBP) in transport of folate analogues was investigated in three epithelial cell lines that were grown in high folate medium and folate-conditioned medium and express different levels of mFBP: human nasopharyngeal KB cells, monkey kidney MA104 cells, and IGROV-I ovarian carcinoma cells. Folate analogues were selected for which mFBP exhibits a low affinity, i.e., methotrexate (MTX) and 10-ethyl-10-deazaaminopterin (10-EdAM) or a (moderately) high affinity as compared to folic acid, i.e., N-(5[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl(-N-m ethylamino]-2-theonyl)-L-glutamic acid (ZD1694), N10-propargyl-5,8-dideazafolic acid (CB3717), and 5,10-dideazatetrahydrofolic acid. Regardless of the medium folate status, growth inhibition studies with IGROV-I and MA104 cells demonstrated a lack of correlation between the affinity of mFBP for the antifolate drugs and their sensitivity profile; both cell lines were highly sensitive to growth inhibition by MTX, 10-EdAM, ZD1694 and 5,10-dideazatetrahydrofolic acid, but were insensitive for CB3717. The same drug sensitivity profile was observed for KB cells, with the exception that these cells were also sensitive to growth inhibition by CB3717 but only in folate-conditioned medium. This overall drug sensitivity profile appeared to correlate with the differential efficiency of drug transport via the "classical" reduced folate/MTX carrier (RFC), rather than by mFBP. Characteristics that further supported functional RFC activity in KB, IGROV-I, and MA104 cells included: (a) the growth inhibitory effects of the drugs could be prevented by the reduced folate leucovorin rather than by folic acid; (b) rates for uptake of [3H]10-EdAM were 2-4-fold higher than for [3H]MTX at 1 microM extracellular concentrations and coincided with the affinity of the RFC for these drugs, rather than those of the mFBP; (c) uptake of [3H]10-EdAM and [3H]leucovorin was markedly inhibited by leucovorin and 10-EdAM, respectively, or by an N-hydroxysuccinimide ester of MTX (irreversibly labeling RFC) but only to a minor extent by folic acid or an N-hydroxysuccinimide ester of folic acid (irreversibly labeling mFBP); and, finally, (d) labeling with an N-hydroxysuccinimide ester of [3H]MTX identified a protein with a molecular weight within the range of that reported for the RFC in human leukemic cells. Altogether, these results indicate that both RFC and mFBP are coexpressed in three epithe

Topics: Aminopterin; Animals; Carrier Proteins; Cell Division; Female; Folate Receptors, GPI-Anchored; Folic Acid; Folic Acid Antagonists; Glutamates; Haplorhini; Humans; Kidney; Leucovorin; Methotrexate; Nasopharyngeal Neoplasms; Ovarian Neoplasms; Quinazolines; Receptors, Cell Surface; Tetrahydrofolates; Thiophenes; Tumor Cells, Cultured

Inhibition of clonogenic potential by the glycinamideribonucleosyl transformylase inhibitor 5,10-dideazatetrahydrofolic acid (DDATHF, Lometrexol) was evaluated in vitro in a human ovarian carcinoma cell line, SW626. Drug-induced inhibition of clonogenic potential is a function of the dose and time of exposure and is independent of the formation of DNA single-strand breaks or de novo synthesis of protein. Simultaneous treatment with 100 microM hypoxanthine completely prevented the inhibition of clonogenic potential caused by 0.5 microM DDATHF. DDATHF blocked cells in the early-middle S-phases of the cell cycle, and there was a corresponding marked reduction in the rate of DNA synthesis after drug withdrawal. The cytotoxic potential of DDATHF was modulated by the folic acid concentration present in the medium. In a medium containing 0.22 microM folic acid, DDATHF cytotoxicity was at least 100 times that in a regular medium containing 2.22 microM folic acid, levels which, however, are about 100 times those found in human plasma. DDATHF cytotoxicity differed moderately when folic acid concentrations varied between 0.22 and 0 microM, suggesting that folic acid does not necessarily antagonise DDATHF anti-tumour activity. Folinic acid at a concentration as low as 0.1 microM can completely rescue cells when given simultaneously with 0.5 microM DDATHF. When folinic acid was given 24 h after DDATHF, a reversal of cytotoxicity was observed at 0.5 and 1 microM, but to a much lesser extent than simultaneous treatment. When folinic acid was added after 48 or 72 h of DDATHF washout, even at a high concentration and for a long time, no reduction in DDATHF cytotoxicity was found. In conclusion, the study highlights the modulation of DDATHF cytotoxicity by folic acid or by folinic acid and provides further rationale for in vivo clinical investigation with these combinations.

Topics: Antineoplastic Agents; Cell Cycle; DNA, Neoplasm; Drug Screening Assays, Antitumor; Female; Folic Acid; Folic Acid Antagonists; Humans; Hypoxanthine; Hypoxanthines; Leucovorin; Ovarian Neoplasms; Tetrahydrofolates; Thymidine; Time Factors; Tumor Cells, Cultured

We previously identified two membrane-bound folate binding proteins, FBP1 and FBP2, in murine L1210 leukemia cells. We now report on the development of two variant murine erythroleukemia cell lines that were used for direct comparison and biochemical characterization of the two murine folate binding proteins. Based on the results of northern analysis and the mobilities of affinity-labeled proteins on polyacrylamide gels, these cell lines exhibit specific up-regulated expression of FBP1 or FBP2. The affinities of the folate binding proteins for various (anti)folates were determined based upon the ability of the compounds to inhibiting of [3H]folic acid. The two proteins exhibited considerably different affinities and stereospecificities and, in general, FBP2 consistently bound each test compound with lesser affinity than FBP1. Both proteins displayed greatest affinity for folic acid, 5-methyltetrahydrofolate, and the antifolates CB3717 and 5,10-dideazatetrahydrofolate (DDATHF). Conversely, the proteins exhibited poor affinity for the dihydrofolate reductase inhibitors methotrexate and aminopterin. For 5-formyltetrahydrofolate, FBP1 had high affinity for the (6S) diastereoisomer, whereas FBP2 showed preference for the non-physiologic (6R) diasterceoisomer. The binding properties of FBP1 and FBP2 overexpressed in these cell lines closely paralleled those of their respective human homologs. These lines provide a model system in which to examine the biochemical characteristics of the individual folate binding proteins without the potential problems associated with expression of proteins in dissimilar cell lines.

Topics: Amidohydrolases; Animals; Biological Transport; Carrier Proteins; Clone Cells; Folate Receptors, GPI-Anchored; Folic Acid; Gene Expression; Humans; Leucovorin; Leukemia, Erythroblastic, Acute; Mice; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; Receptors, Cell Surface; Stereoisomerism; Tetrahydrofolates; Tumor Cells, Cultured

Two membrane folate receptor (MFR) isoforms are present in human tissues i.e. MFR-1 (e.g. placenta) and MFR-2 (e.g. placenta, KB cells, CaCo-2 cells). MFR-1 was expressed in COS-1 cells and the resulting protein had the same polypeptide molecular weight as the native protein. The affinities of (6S) and (6R) diastereoisomers of N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, and 5,10-dideazatetrahydrofolate as well as folic acid and methotrexate to MFR-1, MFR-2 and placental MFR (MFR-1 plus MFR-2) were determined in terms of the Ki values for their competitive inhibition of the binding of [3H]folic acid to these proteins. The results indicated a striking difference in the stereospecificity of MFR-1 and MFR-2 for reduced folate coenzymes; MFR-2 preferentially bound to the physiological (6S) diastereoisomers and MFR-1 bound preferentially to the unphysiological (6R) diastereoisomers, while dideazatetrahydrofolate did not show significant stereospecificity for MFR-1. Furthermore, MFR-2 displayed significantly (2- to 100-fold) greater affinities for all the compounds tested compared to MFR-1. Purified placental MFR, a natural source of MFR-1 which contains variable amounts of MFR-2, showed intermediate Ki values for the compounds tested compared with MFR-1 and MFR-2 and stereospecificities similar to MFR-1. These observations demonstrate striking differences in the ligand binding sites of MFR-1 and MFR-2 which could potentially be exploited in the design of MFR isoform specific antifolates.

Topics: Animals; Binding, Competitive; Carrier Proteins; DNA; Female; Folate Receptors, GPI-Anchored; Folic Acid; Folic Acid Antagonists; Haplorhini; Humans; KB Cells; Kinetics; Leucovorin; Methotrexate; Placenta; Receptors, Cell Surface; Stereoisomerism; Tetrahydrofolates