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PubMed1997

gamma-Glutamyl hydrolase from human sarcoma HT-1080 cells: characterization and inhibition by glutamine antagonists.

Elevated gamma-glutamyl hydrolase (GGH) activity as a contributing factor in mechanisms of acquired and intrinsic antifolate resistance has been reported for several cultured cell lines. Despite this, little is known about this enzyme, especially the human species. Using the human HT-1080 sarcoma line, we observed the secretion of GGH activity into media during culture (a phenomenon that could be markedly stimulated by exposure to NH4Cl) and an acidic pH optimum for in vitro catalytic activity of the enzyme. These properties are consistent with a lysosomal location for the enzyme. Unlike rodent GGH, preparations of HT-1080 enzyme (purified < or = 2000-fold) displayed exopeptidase activity in cleaving successive end-terminal gamma-glutamyl groups from poly-L-gamma-glutamyl derivatives of folate, methotrexate (MTX), and para-aminobenzoic acid substrates and a marked preference for long-chain polyglutamates (Km values for glu4 versus glu1 derivatives were 17- and 15-fold lower for folate and MTX versions, respectively). Using an in vitro assay screen, several glutamine antagonists [i.e., 6-diazo-5-oxo-norleucine (DON), acivicin, and azaserine] were identified as human GGH inhibitors, with DON being the most potent and displaying time-dependent inhibition. In cell culture experiments, simultaneous exposure of DON (10 microM) and [3H]MTX for 24 hr resulted in modest elevations of the long-chain gamma-glutamyl derivatives of the antifolate for HT-1080 and another human sarcoma line. These compounds may serve as useful lead compounds in the development of specific GGH inhibitors for use in examining the relationship between GGH activity and antifolate action and may potentially be used in clinical combination with antifolates that require polyglutamylation for effective cellular retention.

Azaserine↗
PubMed1977

Abnormal folate polyglutamate ratios in untreated pernicious anaemia corrected by therapy.

The relative amounts of folate compounds having 1--3--, 4--, 5-- and 6--glutamic acid residues in the molecule were measured in red blood cells. The results were expressed in relation to methyltetrahydrofolate pentaglutamate (5-glutamic acid residues) which is the analogue present in highest concentration. When the concentration of pentaglutamate is given a value of 100 the relative concentration of folates with 1--3--, 4--, 5-- and 6--glutamic acid residues in control subjects are 48, 48, 100 and 50 respectively. In folate deficiency there is a fall in the relative amount of the short chain glutamic acid chain compounds in addition to having an overall fall in the amount of folate present, the values for 1--3, 4, 5 and 6 chain folates being 16, 14, 100 and 39. By contrast, in untreated pernicious anaemia there is an accumulation of the short chain folates as well as an overall fall in polyglutamate concentration and the relative concentrations were 72, 44, 100 and 30. Folate polyglutamate appears to be the active coenzyme and the reltive amounts of glutamic acid residues may serve to regulate the rate of enzyme activity. In pernicious anaemia the amount of enzyme is reduced and on this hypothesis the regulatory function impaired.

Anemia, Pernicious↗
PubMed2004

Bioavailability of polyglutamyl folic acid relative to that of monoglutamyl folic acid in subjects with different genotypes of the glutamate carboxypeptidase II gene.

BACKGROUND: Before dietary folate is absorbed, polyglutamate folates are deconjugated to monoglutamates by folylpoly-gamma-glutamyl carboxypeptidase in the small intestine. The 1561T allele of the glutamate carboxypeptidase II gene (GCPII), which codes for folylpoly-gamma-glutamyl carboxypeptidase, may impair intestinal absorption of dietary folates. OBJECTIVE: Our aim was to study the bioavailability of polyglutamyl folic acid relative to that of monoglutamyl folic acid across GCPII 1561 genotypes. DESIGN: In a randomized study, 180 healthy adults aged 50-75 y received 323 nmol monoglutamyl folic acid/d (n = 59), 262 nmol heptaglutamyl folic acid/d (n = 61), or placebo (n = 60) for 12 wk. Genotypes were assessed after the intervention. The bioavailability of heptaglutamyl folic acid relative to that of monoglutamyl folic acid was calculated by using the changes in serum folate concentration in the treatment groups, after correction for changes in the placebo group and for the administered dose. RESULTS: No subjects with the TT genotype were encountered. At baseline, serum and erythrocyte folate concentrations were higher (P < 0.05) in subjects with the CT genotype [16.3 nmol/L (geometric x; 95% CI: 13.7, 19.3 nmol/L) and 863 nmol/L (735, 1012 nmol/L), respectively; n = 19] than in subjects with the CC genotype [13.7 (13.1, 14.3) and 685 (652, 721) nmol/L, respectively; n = 161]. Baseline homocysteine concentrations were not significantly different between genotypes. The bioavailability of heptaglutamyl folic acid relative to that of monoglutamyl folic acid was not significantly different between subjects with the CC (64%; 52%, 76%) and CT genotypes (70%; 49%, 91%). CONCLUSIONS: The 1561T allele of the GCPII gene does not impair the bioavailability of polyglutamyl folic acid. However, the allele is associated with higher folate status. This association may be explained by yet unidentified factors controlling the expression of the GCPII gene.

Administration, Oral↗
PubMed2000

The mechanism of transport of the multitargeted antifolate (MTA) and its cross-resistance pattern in cells with markedly impaired transport of methotrexate.

MTA (LY231514) is an antifolate that targets multiple folate-dependent enzymes. In this report, MTA transport was characterized in wild-type L1210 cells and variants with impaired membrane transport or polyglutamation. MTA influx via the reduced folate carrier was somewhat faster (approximately 30%) than that for methotrexate (MTX). Unlike MTX, MTA was rapidly polyglutamated in L1210 cells; hence, a folylpoly-gamma-glutamate synthetase-deficient L1210 variant was used to assess net transport and efflux properties. The MTA transmembrane gradient for exchangeable drug was 2.5 times greater than the MTX gradient, attributable primarily to an efflux rate constant 40% that of MTX. No MTA was bound to dihydrofolate reductase. When grown with folic acid, MTX-resistant L1210 variants with mutations in the reduced folate carrier demonstrated cross-resistance to MTA, markedly reduced MTA accumulation, and only a slightly decreased intracellular folate cofactor pool as compared to L1210 cells. However, when 5-formyltetrahydrofolate was the growth substrate, these MTX-resistant cells were less resistant or negligibly resistant to MTA, accumulated more MTA, and had a lower folate pool as compared to L1210 cells. MTA activity and the intracellular folate pool in L1210 cells were inversely related. These data indicate that MTA polyglutamation in L1210 cells is favored by both the generation of high intracellular drug levels and high MTA affinity for FPGS relative to MTX. Cells resistant to MTX because of impaired transport may retain appreciable sensitivity to MTA because of a concurrent reduction in tetrahydrofolate cofactor transport resulting in cellular folate depletion, which diminishes endogenous folate suppression of MTA polyglutamation.

Animals↗
PubMed2004

Folate requirements of the 2-keto-L-gulonic acid-producing strain Ketogulonigenium vulgare LMP P-20356 in L-sorbose/CSL medium.

In this study, the requirements for growth factors of Ketogulonigenium vulgare LMP P-20356, a 2-keto-L-gulonic acid-producing strain of particular interest for the manufacture of vitamin C, were assessed. Various growth factors were studied in order to obtain improved growth of the strain when cultured in an L-sorbose/corn steep liquor medium. Cultures grown in the presence of reduced mono- and polyglutamated folate derivatives showed a 15- to 20-fold higher biomass content than control cultures lacking these supplements, indicating that the strain has a requirement for folate. Although most folate derivatives used in this study promoted growth, the amplitude of the response varied depending on the compound used. Dihydrofolic acid was found to be the most active form, followed by 5-formyltetrahydrofolic acid, 5-methyltetrahydrofolic acid and tetrahydrofolic acid. Folic acid had no effect. The effectiveness of polyglutamated derivatives was inversely proportional to the polyglutamated chain-length of the derivative used. Our results suggest that the rate-limiting step in the utilisation of monoglutamated folates is most probably related to their transport and/or their intracellular interconversion rather than their polymerisation into polyglutamated forms (physiological forms). The industrial production of 2-keto-L-gulonic acid by K. vulgare LMP P-20356 could be improved by using media in which low-molecular-weight reduced folates are present.

Folic Acid↗
PubMed1980

Transport, binding, and polyglutamation of methotrexate in freshly isolated rat hepatocytes.

Influx of [3H]methotrexate into freshly isolated hepatocytes in suspension is mediated by two routes, one with a high affinity (Km = 5.9 microM) and another with a low affinity for methotrexate. Both transport routes are equally sensitive to the sulfhydryl group inhibitor, p-chloromercuriphenylsulfonic acid, alterations in temperature, substitution of extracellular Na+ with choline, and inhibition by ouabain or azide. The high-affinity pathway for methotrexate shows specificity for the 4-amino group of the pteridine moiety as methotrexate and aminopterin similarly inhibit influx of the labeled drug. On the other hand, 100 microM concentrations of the naturally occurring folates, folic acid, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate, are not inhibitory to influx of 1 microM methotrexate. Once in the cell, methotrexate rapidly reaches molar equivalence with dihydrofolate reductase following which both exchangeable and nonexchangeable intracellular methotrexate accumulates. The exchangeable component reaches steady state within 0.5 hr while the nonexchangeable component increases for at least 1 hr. The nonexchangeable component represents both bound methotrexate and methotrexate polyglutamates. Polyglutamates of methotrexate are a trivial component of total 3H within the cell until about 15 min, but thereafter, their rate of accumulation is constant so that by 1 hr they represent approximately 30% of total intracellular 3H. At steady state, there is a transmembrane chemical gradient for exchangeable methotrexate of 2.4:1; this is 24 times greater than the chemical gradient predicted for equilibrium when the transcellular membrane potential is considered. These results indicate that there are multiple routes for methotrexate transport in the rat hepatocyte that appear to be, at least in part, distinct from the routes for folic acid and the tetrahydrofolate cofactors. The data suggest that transport is energy and Na+ dependent and that the transport carrier requires intact sulfhydryl groups. Net association of methotrexate with the cells is a complex process determined by transport and binding to multiple sites within the cell and metabolism to polyglutamate derivatives that are retained within the cell.

4-Chloromercuribenzenesulfonate↗
PubMed1988

Determination of native folates in milk and other dairy products by high-performance liquid chromatography.

Folates were measured in dairy products by high-performance liquid chromatography without prior sample clean-up. Detection limits for individual folates range from 0.3 to 7.3 ng/g. The folates were extracted from the sample matrix by adjusting the pH to 4.5 with acetic acid, centrifuging to remove precipitated proteins, and treating with conjugase to remove multiple polyglutamate residues. Folates were separated from other sample components using a reversed-phase column with a methanol-phosphate buffer (pH 6.8), and ion-pairing with tetrabutylammonium ion. Fluorescence was found to be the most useful detection technique. Fluorescence detection of reduced forms of the vitamin was achieved by post-column pH adjustment of the eluent with phosphoric acid, while the parent folic acid molecule required chemical oxidation with hypochlorite in order to obtain a fluorescent response.

Animals↗
PubMed1984

Effect of phenobarbitone on folic acid metabolism in the rat.

Anticonvulsant drugs have well-known relationships with folic acid, often leading, in epileptic patients on long term treatment to signs of folic acid deficiency and to haematological complications. The findings of this work, carried out on rats, show strong interference of phenobarbitone with folic acid metabolism, particularly with pteroylpolyglutamate synthesis in liver. This is proved by lower polyglutamate content, lower 3H folate incorporation in polyglutamate forms and in protein-bound folates, and also by lower blood radioactivity, higher urinary excretion of 3H folate metabolites and by higher 3H folate concentration in kidneys.

Animals↗
PubMed1991

Folate analogues. 34. Synthesis and antitumor activity of non-polyglutamylatable inhibitors of dihydrofolate reductase.

Five analogues of methotrextate (MTX), 10-deazaaminopterin (10-DAM), and 10-ethyl-10-deazaaminopterin (10-EDAM) in which the glutamate moiety was replaced by either a gamma-methyleneglutamate or beta-hydroxyglutamate were synthesized and evaluated for their antifolate activity. These analogous are 4-amino-4-deoxy-N10-methylpteroyl-beta-hydroxyglutamic acid (1), 4-amino-4-deoxy-10-deazapteroyl-beta-hydroxyglutamic acid (2), 4-amino-4-deoxy-N10-methylpteroyl-gamma-methyleneglutamic acid (3, MMTX), 4-amino-4-deoxy-10-deazapteroyl-gamma-methyleneglutamic acid (4, MDAM), and 4-amino-4-deoxy-10-ethyl-10-deazapteroyl-gamma-methyleneglutamic acid (5, MEDAM). None of these compounds were metabolized to the respective polyglutamate derivative as judged by their inability to serve as substrates for CCRF-CEM human leukemia cell folylpolyglutamate synthetase (FPGS) in vitro. All compounds inhibited recombinant human-dihydrofolate reductase (DHFR) at nearly equivalent magnitude as MTX. Growth-inhibition studies with H35 hepatoma, Manca human lymphoma, and CCRF-CEM human leukemia cells established greater cytotoxic effects with compounds 3-5 than with compounds 1 and 2. gamma-Methyleneglutamate derivatives 3-5 were transported to H35 hepatoma cells better than MTX or beta-hydroxyglutamate derivatives 1 and 2. Compound 3 was 2.5 times better than MTX in competing with folinic acid transport in H35 hepatoma cells. Compound 1 did not have a significant inhibitory effect on folinic acid transport even at 50 microM under identical conditions. The IC50 for compound 1 against H35-hepatoma cell growth was 8.5-fold higher than MTX. Compounds with the gamma-methyleneglutamate moiety (3-5) exhibited almost equal or lower IC50 values than MTX against the growth of CCRF-CEM human leukemia cells. These studies show that on continuous exposure, the non-polyglutamylatable inhibitors DHFR (3-5) can exhibit superior antifolate activity compared to the polyglutamylatable methotrexate, presumably due to their enhanced transport to these cell lines. Compounds 3-5 appear to be excellent models to study the role of polyglutamylation of antifolates in antitumor activity and host toxicity.

Animals↗
PubMed1995

Quinazoline antifolate thymidylate synthase inhibitors: replacement of glutamic acid in the C2-methyl series.

The synthesis of a series of analogues of the potent thymidylate synthase (TS) inhibitor N-[4-[N-[(3,4-dihydro-2-methyl-4-oxo-6- quinazolinyl)methyl]-N-prop-2-ynylamino]benzoyl]-L-glutamic acid (ICI 198583, 1) is described in which the glutamic acid residue has been replaced by other alpha-amino acids. Most of these analogues were prepared by coupling of tert-butyl-4-(prop-2-ynylamino)benzoate (37) with 6-(bromomethyl)-3,4-dihydro-2-methyl-4-oxoquinazoline (34) followed by deprotection of the tert-butyl ester to the acid and azide-mediated coupling to the appropriate amino acid or amino acid ester. In cases where the amino acid ester was unreactive with the acid azide, a modification was used in which the quinazolinone moiety was protected as its 3-(pivaloyloxy)methyl derivative. This permitted the generation of the more reactive acid chloride of the p-aminobenzoate unit. In general these modifications result in compounds that have equivalent potency to 1 as inhibitors of isolated TS except where the amino acid lacks a lipophilic alpha-substituent. These compounds appear to require the reduced folate carrier (RFC) for transport into cells, but since they are not converted intracellularly into polyglutamated forms, they have a lower level of cytotoxicity compared to 1. The removal of the alpha-carboxylic acid has given a second set of analogues of 1 which contain simple alkyl amide, benzyl, substituted benzyl, and heterocyclic benzyl amide derivatives. These are considerably less potent than 1 as TS inhibitors but display 1-10 microM cytotoxicities due to the fact that they do not require RFC transport and can presumably readily enter cells by passive diffusion through the cell membrane. Molecular modeling and NMR studies indicated that the incorporation of, respectively, 7-methyl and 2'-fluoro substituents would favor the optimum conformation of these molecules for interaction with the TS enzyme. Accordingly, these substituents were incorporated into selected examples to give the series of analogues 47-55. These all show enhanced (approximately 10-fold) inhibition of TS compared to their unsubstituted counterparts. In the substituted benzylamides (51, 52) and heterocyclic benzyl amides (53-55) the ability to enter cells by passive diffusion results in highly potent (< 1 microM) cytotoxic agents.

Animals↗
PubMed2004

The role of alpha-folate receptor-mediated transport in the antitumor activity of antifolate drugs.

PURPOSE: Raltitrexed, pemetrexed, lometrexol, and ZD9331 are antifolate drugs transported into cells via the ubiquitously expressed reduced-folate carrier. They display also high affinity for the alpha-folate receptor (alpha-FR), a low capacity folate transporter that is highly overexpressed in some epithelial tumors. The role of alpha-FR in the activity of the antifolates has been evaluated in two alpha-FR-overexpressing cell lines grown in a physiological concentration of folate (20 nM R,S-Leucovorin). EXPERIMENTAL DESIGN AND RESULTS: A431-FBP cells (transfected with the alpha-FR) were 3-5-fold more sensitive to the antifolates than A431 cells. KB cells (constitutive alpha-FR overexpression) were less sensitive to the drugs when coexposed to 1 microM folic acid to competitively inhibit binding to the alpha-FR. Raltitrexed, pemetrexed, and lometrexol are polyglutamated in cells leading to drug retention, e.g., the raltitrexed 4- and 24-h IC(50)s in A431 cells were approximately 0.6 and 0.008 microM, respectively, compared with 0.003 microM for 72-h continuous exposure. A431-FBP cells were approximately 3-fold more sensitive to raltitrexed and pemetrexed at all exposure times. ZD9331 is not polyglutamated, and the 4- and 24-h IC(50)s in A431 cells were >100 and approximately 100 microM, respectively, reducing to 2 and 0.1 microM, respectively, in A431-FBP cells. The ZD9331 4- and 24-h IC(50)s in KB cells were 20 and 1 microM, respectively, and reversible by coaddition of 1 microM folic acid. An in situ thymidylate synthase assay demonstrated continued thymidylate synthase inhibition after ZD9331-treated A431-FBP and KB, but not A431, cells were placed in drug-free medium for 16 h. A model is proposed in which the antifolates accumulate in the alpha-FR/endosomal apparatus, leading to slow release into the cytoplasm. In particular, this leads to cellular retention of the nonpolyglutamatable ZD9331. CONCLUSIONS: Antifolate drugs, particularly ZD9331, have the potential for increased efficacy in tumors that highly overexpress the alpha-FR.

Antimetabolites, Antineoplastic↗
PubMed1987

The effects on 4-aminoantifolates on 5-formyltetrahydrofolate metabolism in L1210 cells. A biochemical basis of the selectivity of leucovorin rescue.

This report describes studies designed to evaluate possible inhibitory effects of diaminoantifolates on folate-dependent biosynthetic enzymes in intact L1210 leukemia cells. A novel approach is described which involves an assessment of the metabolism of and biosynthetic flux of the one-carbon moiety from (6S)5-formyltetrahydrofolate in folate-depleted cells. Pretreatment with methotrexate (10 microM), resulting in the formation of methotrexate polyglutamates, or continuous incubation with trimetrexate (1 microM) inhibited growth of folate-depleted L1210 cells in the presence of folic acid or 5-formyltetrahydrolate. In both control and drug-treated cells, double-labeled (6S)-5-[14C]formyl[3H]tetrahydrofolate was rapidly metabolized with the loss of the [14C]formyl group. Under all conditions, the predominant metabolite was 10-formyl[3H]tetrahydrofolate, detectable both intracellularly and extracellularly. In drug-treated cells, there was a remarkably small decrease in the level of 10-formyl[3H]tetrahydrofolate (approximately 30%) and a 10-fold rise in the level of [3H]dihydrofolate to less than 20% of the total folate pool. The incorporation of [14C]formyl group from 5-[14C]formyltetrahydrofolate into thymidylate, serine, and methionine was unaffected by the presence of 1 microM trimetrexate, consistent with the generation of sufficient 5,10-[14C]methylenetetrahydrofolate to drive these reactions. Similarly, the presence of methotrexate polyglutamates had no effect at the level of amino acid synthesis; however, carbon transfer into thymidylate was markedly inhibited. Even though 10-formyltetrahydrofolate was readily formed from 5-formyltetrahydrofolate in this model, the net incorporation of 14C from 5-[14C]formyltetrahydrofolate into purine nucleotides was inhibited by both methotrexate and trimetrexate treatments. Similar findings were obtained when [14C]glycine incorporation into purine nucleotides was monitored in cells incubated with unlabeled 5-formyltetrahydrofolate. Finally, in antifolate-treated cells incubated with unlabeled 5-formyl-tetrahydrofolate, transfer of 14C from [14C]formate or [14C]serine into biosynthetic products or incorporation of [3H]deoxyuridine into nucleic acids was potently inhibited. These results suggest that insufficient levels of tetrahydrofolate and 5, 10-methylenetetrahydrofolate were formed to drive these reactions despite the presence of high levels of 10-formyltetrahydrofolate.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine↗
PubMed1997

Decreased folylpolyglutamate synthetase activity in tumors resistant to fluorouracil-folinic acid treatment: clinical data.

Thymidylate synthase (TS) is the main target for fluorouracil (FU). Optimal cellular concentrations of reduced folates in polyglutamated forms [via folylpolyglutamate synthetase (FPGS)] are necessary for achieving maximal TS inhibition. The aim of this multicentric prospective study was to analyze the link between clinical response to FU therapy for liver metastases of colorectal carcinoma and tumoral TS and FPGS activities. Forty-four advanced colorectal cancer patients (15 women and 29 men; median age 63, range, 27-78 years) receiving a standard FU-folinic acid protocol were included. A single hepatic tumoral biopsy was obtained systematically at the time of diagnosis. For 24 patients, a biopsy in the primary colon tumor was available. TS and FPGS activities were measured by radioenzymatic assays. Clinical response on hepatic metastases was 1 complete response, 12 partial responses, 14 stabilizations, and 17 progressions. In hepatic biopsies, TS activity (median, 185; range, <10-3111 fmol/min/mg protein) and FPGS activity (median, 1270; range, <400-3730 fmol/min/mg protein) exhibited a wide variability. TS activity in primary tumors (median, 461; range, 35-2565 fmol/min/mg protein) was significantly higher than in hepatic metastases. No difference was observed between primaries and metastases for FPGS. FPGS activity expressed in liver metastases was significantly correlated to that expressed in primaries. The distribution of TS activity in liver metastases was not significantly different between responsive and nonresponsive patients. However, FPGS activity measured in liver metastases was significantly higher in responsive patients (median, 1550 fmol/min/mg protein) than in nonresponsive patients (median, 1100 fmol/min/mg protein). A discriminant analysis revealed that 24 of the 25 patients exhibiting a liver FPGS activity </=1100 fmol/min/mg protein and/or a liver TS >320 fmol/min/mg protein were nonresponding patients. These data establish for the first time the potential importance of tumoral FPGS activity for assessing FU-folinic acid responsiveness in the clinical setting.

Adult↗
PubMed1996

Immunoreactive dUMP and TTP pools as an index of thymidylate synthase inhibition; effect of tomudex (ZD1694) and a nonpolyglutamated quinazoline antifolate (CB30900) in L1210 mouse leukaemia cells.

The inhibition of thymidylate synthase (TS) as a drug development target has received much attention in recent years, and several compounds have reached clinical evaluation. During drug development, the effectiveness of target inhibition can be assessed by determination of the perturbations of deoxythymidine 5-triphosphate (TTP) and deoxyuridine 5'-monophosphate (dUMP) pools in drug-treated cells. Rapid, sensitive, and reproducible radioimmunoassays for TTP pools and immunoreactive dUMP pools have been developed to meet our requirement for the rapid assessment of TS inhibition by quinazoline antifolates. The assays can be carried out on 1-2 million cells, and require minimal sample preparation. The limit of detection for TTP is 1 pmole/10(6) cells and for immunoreactive dUMP ("dUMP"), 3.0 pmole/10(6) cells, both assays being performed on the same cell extract. TTP and "dUMP" pools have been measured in mouse L1210 leukaemia cells treated with the quinazoline antifolates ZD1694 (N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl )-N-methylamine]-2-thenoyl)-L-glutamic acid) and CB30900 (N-[N-[4-[N-[(3,4-dihydro-2,7-dimethyl-4-oxo-6-quinazolinyl)methyl ]-N-prop-2- ynylamino]-2-fluorobenzoyl]-L-gamma-glutamyl]-D-glutamic acid). Unlike ZD1694, CB30900 is a TS inhibitor that does not rely on polyglutamation for activity. In L1210 cells, both compounds caused a rapid inhibition of TTP pools in a dose- and time-related manner. Greater than 90% TS inhibition was achieved following a 4-hr exposure to each compound at equitoxic doses (up to 100 times the IC50 determine by a 48-hr growth inhibition assay). For both compounds, this was accompanied by a 5-10-fold increase in "dUMP" pools. For ZD1694, neither the TTP pool or "dUMP" levels were normalised when cells were resuspended in a drug-free medium for 4 hr and, at the higher doses studied, TS was still inhibited after a 16-hr period in the absence of drug. This is consistent with the formation and intracellular retention of potent polyglutamated forms of ZD1694. In contrast, TS activity as determined by repletion of the TTP pools and normalisation of "dUMP" levels were demonstrated for CB30900. However, at a high dose (50 microM, equivalent to 250 times the IC50), retention of TS inhibition was observed following 4 hr, but not 16 hr in the absence of drug. The radioimmunoassays described will prove useful to further define the extent and time-course of TS inhibition by novel antifolate compounds, and will also provide valuable in vitro and in vivo pharmacodynamic information on established antimetabolites when used alone or in combination with other drugs and modulators.

Animals↗
PubMed1991

ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that is a potent inhibitor of L1210 tumor cell growth in vitro and in vivo: a new agent for clinical study.

N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2-thenoyl)-L-glutamic acid (ICI D1694) is a water-soluble, folate-based thymidylate synthase (TS) inhibitor designed to be a less toxic and more potent analogue of the clinically tested N10-propargyl-5,8-dideazafolic acid. Inhibition of isolated L1210 TS by ICI D1694 is mixed noncompetitive (although tending toward competitive), with a Ki of 62 nM (Kies = 960 nM). The synthetic gamma-polyglutamates are up to 2 orders of magnitude more potent as inhibitors of TS; e.g., the tetraglutamate (glu4) has a Ki of 1.0 nM (Kies = 15 nM). Although inhibitory activity of ICI D1694 toward rat liver dihydrofolate reductase was similar to that of TS (Ki = 92 nM; competitive inhibition) the polyglutamate derivatives did not show enhanced activity. ICI D1694 was also a very potent inhibitor of L1210 cell growth (50% inhibitory activity = 8 nM). L1210 growth inhibition was not observed in the presence of thymidine, consistent with TS being the locus of action. Folinic acid antagonized L1210 growth inhibition in a competitive fashion such that the highest folinic acid concentration used (25 microM) increased the 50% inhibitory activity 6000-fold. When given as a 4-h delayed "rescue", folinic acid was much less effective in antagonizing growth inhibition. These observations are consistent with folinic acid competing with ICI D1694 for uptake into the cell and/or intracellular polyglutamation. The L1210:1565 cell line, which has greatly impaired reduced-folate/methotrexate transport and thus is resistant to methotrexate, was significantly cross-resistant to ICI D1694 (121-fold), suggesting that ICI D1694 is dependent on this uptake mechanism for good cytotoxic potency in L1210 cells. L1210 cells that were incubated for 4 h with 0.1 microM 3H-ICI D1694 accumulated approximately 1.5 microM intracellular 3H, and the high performance liquid chromatography analysis of the cell extracts demonstrated that 96% of the 3H was associated with the ICI D1694 polyglutamate fractions (principally glu4). Upon resuspension in drug-free medium for 24 h, approximately 75% of the cellular 3H was retained, this being the higher polyglutamate pool (glu4-6). In mice, after a single bolus injection of 10 mg/kg of ICI D1694, TS was inhibited greater than 80% for 24 h in ascitic L1210:NCI cells (as measured by the rate of 3H release from [5-3H]deoxyuridine). ICI D1694 cured the L1210:ICR ascitic tumor in mice at 0.4 mg/kg daily for 5 days (maximum tolerated dose, approximately 50 mg/kg).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMedDate not supplied

[Megalocytic anemia of alcoholics and folic acid (author's transl)].

Megalocytic anemia of alcoholics is due to the deficiency of folic acid: the proof is the serum hypofolemia. The mechanism is complex and due to : a dietary deficiency; a malabsorption; an insufficiency of conversion of polyglutamate to monoglutamate; a liver deficiency; a specific effect of alcohol on the folate absorption. Alcohol has a toxic effect on the marrow and inhibits the folic acid.

Adult↗
PubMed1973

The synthesis of pteroylpolyglutamates by sheep liver enzymes in vitro.

1. Sephadex G-15 was used to separate pteroylmonoglutamates from corresponding polyglutamate derivatives. 2. Pteroylpolyglutamates were formed when 5-formyltetrahydro[2-(14)C]pteroylglutamic acid, 5-[methyl-(14)C]tetrahydropteroylglutamic acid or tetrahydro[2-(14)C]pteroylglutamic acid was incubated at pH8.4 with ATP, MgCl(2), KCl, l-glutamic acid and sheep liver cytosol. The gamma-glutamyl side chain appeared to be lengthened by the stepwise addition of single glutamate moieties. 3. The subcellular distribution of pteroylpolyglutamates paralleled that of pteroylpolyglutamate synthetase activity, and followed the order cytosol>;nuclear' fraction>microsomal fraction>mitochondria.

Adenosine Triphosphate↗
PubMed1981

Dual functions of bacteriophage T4D gene 28 product: structural component of the viral tail baseplate central plug and cleavage enzyme for folyl polyglutamates. II. Folate metabolism and polyglutamate cleavage activity of uninfected and infected Escherichia coli cells and bacteriophage.

We investigated the role of the T4D bacteriophage gene 28 product in folate metabolism in infected Escherichia coli cells by using antifolate drugs and a newly devised assay for folyl polyglutamate cleavage activity. Preincubation of host E. coli cells with various sulfa drugs inhibited phage production by decreasing the burst size when the phage particles produced an altered gene 28 product (i.e., after infection under permissive conditions with T4D 28(ts) or T4D am28). In addition, we found that another folate analog, pyrimethamine, also inhibited T4D 28(ts) production and T4D 28am production, but this analog did not inhibit wild-type T4D production. A temperature-resistant revertant of T4D 28(ts) was not sensitive to either sulfa drugs or pyrimethamine. We developed an assay to measure the enzymatic cleavage of folyl polyglutamates. The high-molecular-weight folyl polyglutamate substrate was isolated from E. coli B cells infected with T4D am28 in the presence of labeled glutamic acid and was characterized as a folate compound containing 12 to 14 labeled glutamate residues. Extracts of uninfected bacteria liberated glutamate residues from this substrate with a pH optimum of 8.4 to 8.5. Extracts of bacteriophage T4D-infected E. coli B cells exhibited an additional new folyl polyglutamate cleavage activity with a pH optimum of about 6.4 to 6.5, which was clearly distinguished from the preexisting activity in the uninfected host cells. This new activity was induced in E. coli B cells by infection with wild-type T4D and T4D amber mutants 29(-), 26(-), 27(-), 51(-), and 10(-), but it was not induced under nonpermissive conditions by T4D am28 or by T4D 28(ts). Mutations in gene 28 affected the properties of the induced cleavage enzyme. Wild-type T4D-induced cleavage activity was not inhibited by pyrimethamine, whereas the T4D 28(ts) activity induced at a permissive temperature was inhibited by this folate analog. Folyl polyglutamate cleavage activity characteristic of the activity induced in host cells by wild-type T4D or by T4D gene 28 mutants was also found in highly purified preparations of these phage ghost particles. The T4D-induced cleavage activity could be inhibited by antiserum prepared against highly purified phage baseplates. We concluded that T4D infection induced the formation of a new folyl polyglutamate cleavage enzyme and that this enzyme was coded for by T4D gene 28. Furthermore, since this gene product was a baseplate tail plug component which had both its antigenic sites and its catalytic sites exposed on the phage particle, it was apparent that this enzyme formed part of the distal surface of the phage baseplate central tail plug.

Carboxypeptidases↗