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PubMed1993

The role of the reduced-folate carrier and metabolism to intracellular polyglutamates for the activity of ICI D1694.

The uptake of ICI D1694 into L1210 cells is very rapid and evidence strongly suggests that transport is via the reduced-folate/MTX cell membrane carrier (RFC); for example a cell line with a greatly impaired RFC is highly resistant to ICI D1694. Polyglutamates can be found intracellularly within a few minutes, so that experiments initially designed to measure transport were actually measuring transport and polyglutamation. After 30mins, in normal serum-containing tissue culture medium, the concentration of polyglutamates (di, tri and tetra) exceeded that of the parent drug 6-fold. Studies where cells were resuspended in drug-free medium demonstrated that the parent drug and its diglutamate could readily leave the cell. Folinic acid could markedly decrease the polyglutamation of ICI D1694, but had to be given simultaneously with the drug as a 4hr delayed rescue was less effective because substantial polyglutamation had already occurred. This effect was translated into considerable antagonism for cell growth inhibition by simultaneous folinic acid. The importance of the metabolism of ICI D1694 to polyglutamates to its potent cytotoxic activity is demonstrated by compounds related in structure to ICI D1694 but with different properties for the RFC and FPGS. For example, 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolate (ICI 198583) owes its less potent cytotoxic activity to its poorer FPGS substrate activity (Km 40 microM compared with 1.3 microM for ICI D1694). Replacing the 2-methyl of either compound with amino, which appears to prevent use of the RFC, has a deleterious effect on growth inhibitory activity presumably by limiting the transport of the parent compounds into the cells, thereby slowing the rate of polyglutamate formation. Again a single change to another part of the molecule, that is methylation of the 7-position can have serious consequences on cytotoxic potency, particularly for the ICI D1694 molecule. The 7-methylated compounds are apparently poor or non-substrates for FPGS and therefore retain activity against a cell line unable to polyglutamate antifolates. These same compounds are only slightly affected by coincubation with folinic acid in L1210 tissue culture, consistent with the failure of these compounds to form intracellular polyglutamates. The results of short-exposure assays and in situ TS assays confirms that 7-methylation largely prevents the formation of a retained drug-form (polyglutamates), continuous exposure being necessary to maintain TS inhibition and cause a cytotoxic effect after removal of extracellular drug.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

The in vivo metabolic stability of dipeptide analogues of the quinazoline antifolate, ICI 198583, in mice.

In the search for quinazoline thymidylate synthase inhibitors that are not subject to intracellular polyglutamation, a class of dipeptide analogues of the diglutamate of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583-gamma-L-glu) has been evaluated for their stability to in vivo hydrolysis. Replacement of the second glutamate with another amino acid, e.g. alanine, prevented polyglutamation in vitro but such compounds were subject to hydrolysis when injected into mice. The extent of hydrolysis was measured in plasma, liver and kidney by HPLC analysis of tissue removed from mice 1 hr after i.p. injection. The enzyme responsible for this hydrolysis is thought to be a gamma-glutamyl hydrolase which hydrolyses the amide bond, releasing ICI 198583 which may then be polyglutamated. Development of stable dipeptide compounds was achieved by structural modification in two principal ways: either by replacement of the second amino acid (e.g. glutamate or alanine) with its D-enantiomer or removal of the carboxyl on the alpha-carbon of the second amino acid (alpha'-COOH). In this second approach two series of compounds were investigated. Monocarboxylate-derived dipeptides, e.g. ICI 198583-gamma-L-phenylalanine or ICI 198583-gamma-phenylglycine, resulted in stable compounds after removal of the alpha'-COOH (to give -ethylamide and -benzylamide derivatives, respectively). However, for the dicarboxylic amino acids a less clear picture emerged. Although removal of the alpha'-COOH from ICI198583-gamma-L-glutamate to give ICI 198583-gamma-gamma-aminobutyric acid resulted in a stable compound, the corresponding aspartate analogue (-beta-alanine) was subject to hydrolysis.

Animals↗
PubMed1982

Synthesis, retention, and biological activity of methotrexate polyglutamates in cultured human breast cancer cells.

To determine the pharmacologic importance of methotrexate (MTX) polyglutamates, we examined the formation, retention, and effect of these metabolites in cultured human breast cancer cells. Two cell lines (MCF-7 and ZR-75-B) converted the drug to gamma-polyglutamate derivatives in a dose- and time-dependent reaction. After 24-h incubations with 2 muM MTX, polyglutamates of two to five amino acids in length accounted for 55.4% (51.9 nmol/g) of intracellular drug in the MCF-7 cells and 87.6% (62.4 nmol/g) of drug in ZR-75-B cells. In contrast, MDA-231 cells showed lesser accumulation of MTX, and only 32% (4.06 nmol/g) of the intracellular drug was in the form of polyglutamates, a difference that could only partially be explained by decreased ability of these cells to take up free drug from the medium. When MCF-7 and ZR-75-B cells containing polyglutamates were transferred to drug-free medium for 24 h, 22 and 51% of the total intracellular drug were, respectively, retained in each cell line. The loss of intracellular drug was primarily accounted for by disappearance of parent compound and polyglutamates containing 1-3 additional glutamyl residues. The rates of disappearance from cells decreased with increasing glutamyl chain length. All of the 4-NH(2)-10-CH(3)-PteGlu(5) and 47 and 38% of the 4-NH(2)-10-CH(3)-PteGlu(4) remained in the MCF-7 and ZR-75-B cells, respectively, and could be identified in the cytosol after 24 h in drug-free medium. The retention of MTX polyglutamates in these two cell lines in excess of dihydrofolate reductase binding capacity led to prolonged inhibition of thymidylate synthesis and loss of cell viability after removal of extracellular MTX. After 24-h incubation with 2 muM MTX and an additional 24 h in drug-free medium, [(3)H]deoxyuridine incorporation was still inhibited to 30% of control in the MCF-7 cells and 34.7% of control in ZR-75-B cells; this persistent inhibition was associated with a 30% reduction in cell numbers in each cell line during the 24-h period in drug-free medium. In contrast, [(3)H]deoxyuridine incorporation and cell growth quickly recovered to normal in the MDA-231 cells following removal of 2 muM MTX from the medium after a 24-h incubation. Prolonged inhibition of both thymidylate synthesis and cell growth was observed in this cell line in drug-free medium only after a 24-h incubation with 10 muM MTX, a condition that leads to the synthesis of 11.3 nmol/g of MTX polyglutamates. These studies demonstrate that polyglutamate formation allows a prolonged retention of drug in a noneffluxable form and prolonged inhibition of both thymidylate synthesis and cell growth following removal of extracellular drug.

Breast Neoplasms↗
PubMed1993

Decreased expression of the human folate receptor mediates transport-defective methotrexate resistance in KB cells.

The human membrane-associated folate binding protein, or folate receptor (hFR), is a necessary component of folate and methotrexate (MTX) transport in some cell lines. To investigate the role of hFR in acquired MTX resistance in human cells, we characterized nine MTX-resistant clones selected from human nasopharyngeal epidermoid carcinoma (KB) cells (cells which transport folates/anti-folates via the hFR) cultured in media containing low folate concentrations. Compared with wild type KB cells, the level of resistance of the clones ranges from 2- to 80-fold higher and the resistant phenotypes of the clones are characterized as follows. 1) DHFR levels are increased (3-13-fold) in four of nine clones; 2) MTX polyglutamation is not detectably different; 3) the extents of MTX efflux are similar; 4) initial rates of MTX efflux are similar except for two clones which exhibit slightly faster efflux rates (approximately 2-fold); and 5) the Vmax for specific MTX and 5-methyltetrahydrofolate transport are decreased (2-18-fold) in all mutants. The Kt values for MTX transport of each mutant are similar to the Kt of KB cells. These results indicate that all nine MTX-resistant clones exhibit defective MTX transport and that four clones also have increased DHFR levels. Based on folic acid binding assays, the hFR is reduced by 1.8-24-fold in these clones relative to KB cell hFR expression. Western, Northern, and Southern analyses are consistent with decreased hFR expression in these clones rather than mutations, resulting in alterations in the size or ligand binding affinities of the hFR. The decrement in hFR expression correlates closely with the degree of reduction in MTX transport Vmax for each clone. Since folate and MTX influx proceed via hFR in KB cells and in these mutants, the correlation (R2 = 0.90) between hFR expression and the MTX transport Vmax of each clone indicates that hFR expression is an important determinant of acquired MTX resistance in this human tumor cell line. These studies demonstrate that defective transport (manifested by decreased Vmax) resulting from decreased expression of the hFR is frequent in KB cells cultured under these conditions and suggest that modulation of hFR may be relevant to MTX cytotoxicity or resistance in tissues or cells expressing functionally significant levels of hFR.

Biological Transport↗
PubMed1979

Polyglutamate forms of folate in liver of folic acid-deficient rats.

In view of role played by pteroylpolyglutamates as actual folate coenzymes in single-carbon unit metabolism, the effect of folic acid deficiency on the distribution of these compounds in rat liver has been studied. In deficient rats the liver levels of pteroylpolyglutamates and in particular of those containing a higher number of glutamyl residues are more decreased as compared with monoglutamates. In fact penta- and other higher polyglutamates: mono-, di- and tri-glutamates ratio is lower in deficient rats than that in control rats. The decreased amount of these coenzymic forms observed in this condition may be considered as the consequence of the severe alterations of process which adds glutamate moieties to monoglutamate forms.

Animals↗
PubMed1983

An electrophoretic method for the identification of poly-gamma-glutamyl chain lengths in folates and related compounds.

An electrophoretic method for the identification and separation of folyl polyglutamates of different chain lengths and of the corresponding p-aminobenzoyl polyglutamate compounds has been developed. These compounds have been separated using electrophoresis in a 40% polyacrylamide gel using a higher voltage and other modifications of the standard polyacrylamide gel electrophoresis procedures used to separate larger polypeptides. Good separation has been obtained on folates containing up to 12 glutamyl residues. Further, this method has been used to investigate the nature of the products formed by the gamma-glutamyl carboxypeptidases from hog kidney and bovine liver.

Animals↗
PubMed1987

Characterization of structural component of cell walls of alkalophilic strain of Bacillus sp. C-125. Preparation of poly(gamma-L-glutamate) from cell wall component.

The cell wall of an alkalophilic strain of Bacillus sp. C-125 is composed of A1 gamma-peptidoglycan, a teichuronic acid and an unknown acidic polymer composed of glutamic acid and glucuronic acid, of which the molar ratio is approx. 4-5:1. Poly(gamma-L-glutamate) was prepared from the acidic polymer by removal of almost all of the glucuronic residues with trifluoromethanesulphonic acid treatment and purified chromatographically. The Mr of the polyglutamate preparation was estimated to be 14,000 by gel chromatography, or 43,000 on the basis of the content of N-terminal acid residues. The acidic polymer found in the cell wall of the organism was concluded to be a polyglutamate substituted with (oligo)glucuronic acid residues or a complex composed of two kinds of polymers (polyglutamate and polyglucuronate).

Bacillus↗
PubMed1979

The effect of age in culture on the uptake and metabolism of methotrexate by primary cultures of hepatocytes.

Primary monolayer cultures of rat hepatocytes show an initial transient azide-sensitive capacity to concentrate methotrexate several-fold from the medium. However, after 48 h in culture they lose the azide-sensitive ability to accumulate methotrexate. The uptake of (+) 5-methyltetrahydrofolate and folate is not altered greatly with time in culture and is unaffected by azide. Once inside the cell, methotrexate is converted predominantly to di- and triglutamate derivatives. After a short exposure to methotrexate (up to 4 h) most of the intracellular methotrexate is unaltered, but by 24 h nearly 80% is present as polyglutamates. Unlike the capacity of the cells to accumulate methotrexate, the ability of the cells to form polyglutamates does not appear to diminish with the age of the culture.

Animals↗
PubMed1986

Regulation of methotrexate polyglutaminate formation in Ehrlich ascites carcinoma cells by endogenous folate pool.

The conversion of methotrexate to poly-gamma-glutamyl derivatives in Ehrlich ascites carcinoma cells which are characterized by different pools of endogenous folates is described. The cells in which folate pool was high (the 5-fluorodeoxy-uridine-resistant cell line) the ability to convert methotrexate to its polyglutamate derivatives was much lower than in the cells in which folate pool was smaller (the parental cell line). When the cellular folate pool was reduced by treatment of the cells with lysolecithin, a similar methotrexate polyglutamate concentration in both cell lines was observed. These data suggest that cellular folate pool has a regulatory effect on methotrexate polyglutamate synthesis.

Animals↗
PubMed1972

Observations on folate absorption with particular reference to folate polyglutamate and possible inhibitors to its absorption.

A test system for detection of inhibitors to the enzyme which removes the glutamic acid peptide chain from folate polyglutamates (folate conjugase) is described. This utilizes the reaction between plasma conjugase and the folate polyglutamate in red blood cell haemolysate. Diphenylhydantoin and yeast extracts did not inhibit plasma conjugase. The better absorption of monoglutamate forms of folate as compared to polyglutamate forms was confirmed. Polyglutamates were absorbed normally by patients with pernicious anaemia, suggesting that conjugase enzymes (optimally active at pH 4.5) did not normally function to a significant extent in the gut lumen. Diphenylhydantoin and bicarbonate did not interfere with absorption of either mono- or polyglutamate forms of folate.

Anemia, Pernicious↗
PubMed1984

Studies on the polyglutamate specificity of thymidylate synthase from fetal pig liver.

Thymidylate synthase has been purified 1700-fold from fetal pig livers by using chromatography on Affigel-Blue, DEAE-52, and hydroxylapatite. Steady-state kinetic measurements indicate that catalysis proceeds via an ordered sequential mechanism. When 5,10-methylenetetrahydro-pteroylmonoglutamate (CH2-H4PteGlu1) is used as the substrate, dUMP is bound prior to CH2-H4PTeGlu1, and 7,8-dihydropteroylmonoglutamate (H2PteGlu1) is released prior to dTMP. Pteroylpolyglutamates (PteGlun) are inhibitors of thymidylate synthase activity and are competitive with respect to CH2-H4PteGlu1 and uncompetitive with respect to dUMP. Inhibition constants (Ki values), which correspond to dissociation constants for the dissociation of PteGlun from the enzyme-dUMP-PteGlun ternary complex, have been determined for PteGlun derivatives with one to seven glutamyl residues: PteGlu1, 10 microM; PteGlu2, 0.3 microM; PteGlu3, 0.2 microM; PteGlu4, 0.06 microM; PteGlu5, 0.10 microM; PteGlu6, 0.12 microM; PteGlu7, 0.15 microM. Thus, thymidylate synthase from fetal pig liver preferentially binds pteroylpolyglutamates with four glutamyl residues, but derivatives with two to seven glutamyl residues all bind at least 30-fold more tightly than the monoglutamate. When CH2-H4PteGlu4 is used as the one carbon donor for thymidylate biosynthesis, the order of substrate binding and product release is reversed, with binding of CH2-H4PteGlu4 preceding that of dUMP and release of dTMP preceding release of H2PteGlu4. Vmax and Km values for dUMP and CH2-H4PteGlun show relatively little change as the polyglutamate chain length of the substrate is varied.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Inhibition of gamma-glutamyl hydrolases in human cells by 2-mercaptomethylglutaric acid.

Cultured human lymphocytes and fibroblasts accumulate methotrexate during 24 hours and synthesize methotrexate polyglutamates up to the hexaglutamate, with the triglutamate predominating. In the interval after incubation with methotrexate, drug is lost, metabolites are converted to longer chain-lengths, and methotrexate pentaglutamate predominates. 2-Mercaptomethylglutaric acid, an inhibitor of neutral pH gamma-glutamyl hydrolases in vitro, had little effect on polyglutamate synthesis during incubation of the cells with methotrexate, but maintained for 48 hours almost all the methotrexate as the pentaglutamate when added after the removal of the drug. These findings demonstrate that inhibition of gamma-glutamyl hydrolases is an effective approach to alter the distribution of polyglutamate forms of methotrexate in vivo and indicate that enzymatic hydrolysis may contribute to regulation of polyglutamate chain lengths in human cells.

Carboxypeptidases↗
PubMed1990

Interaction of methotrexate polyglutamates and dihydrofolate during leucovorin rescue in a human breast cancer cell line (MCF-7).

Previous investigations have suggested that high-dose methotrexate with leucovorin rescue is a potentially useful strategy for overcoming antifolate resistance. Interactions between methotrexate (MTX) and leucovorin and their respective metabolites appear to occur at multiple intracellular sites, including dihydrofolate reductase (MTX/MTX polyglutamates versus dihydrofolate) and other folate-dependent enzymes (MTX polyglutamates versus reduced folate substrates). The present studies were designed to test the ability of dihydrofolate to compete with methotrexate and methotrexate polyglutamates for dihydrofolate reductase activity using an intact human breast carcinoma cell line (MCF-7) as the model system. Exposure of the breast cells to methotrexate for 24 h resulted in a concentration-dependent formation of methotrexate polyglutamates that markedly exceeded the dihydrofolate reductase-binding capacity for up to 24 h after the removal of drug from the growth media. Under these conditions of dihydrofolate reductase inhibition, we found that tritium-labeled dihydrofolate was capable of competing with methotrexate and its metabolites for dihydrofolate reductase activity as evidenced by the appearance of tritium-labeled reduced folates in the treated cells. We found the interaction between dihydrofolate and methotrexate to be dependent on the exposure concentrations of both methotrexate and dihydrofolate. These studies provide direct evidence that competition during leucovorin rescue occurs at the level of dihydrofolate reductase between methotrexate polyglutamates and dihydrofolate polyglutamates in intact human cells.

Binding, Competitive↗
PubMed1995

Quinazoline-based thymidylate synthase inhibitors: relationship between structural modifications and polyglutamation.

Quinazoline-based analogues of folic acid are a group of thymidylate synthase (TS) inhibitors that display a wide spectrum of activity for cultured tumour cells, partly due to their differential ability to form polyglutamate metabolites that are (i) more potent TS inhibitors and (ii) not readily effluxed from cells. The rate of cell membrane transport and folylpolyglutamate synthetase substrate activity influence compound polyglutamation. A series of intact-cell assays has been used to determine how specific modifications of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolate (ICI 198583) affect compound polyglutamation. Those containing the 'classical' glutamate structure were usually, but not always, well polyglutamated intracellularly. Replacement of N10 propargyl with smaller aliphatic substituents, particularly when combined with replacement of the benzene ring with thiophene or thiazole heterocycles, was beneficial for antitumour activity through polyglutamate formation. Fluorination of the benzene, particularly if a F was adjacent to the 'bridge region' (3'F or 2',5'diF), also gave compounds with a high dependence on polyglutamation for activity. Those analogues with 2-CH2OH or NH2 substituents were poor substrates for the reduced-folate cell membrane carrier which can account for their reduced polyglutamation rate and hence growth-inhibitory activity. A large decrease or prevention of polyglutamation was achieved by the introduction of CH3, CH2CH3, Br or C1 on C7. The concomitant enhancement in TS inhibition by these modifications gave compounds active under continuous-exposure cell culture conditions. Some ICI 198583 analogues had the glutamate moiety replaced with unnatural amino acids or dipeptides. Only the L-gamma-L-glu analogue (a polyglutamate metabolite of ICI 198583) gave activity entirely attributable to polyglutamate formation.

Animals↗
PubMed1976

Subcellular localization of gamma-glutamyl carboxypeptidase and of folates.

The subcellular distributions of glutamyl carboxypeptidase, folate specific activities, and radioactive metabolites of injected [3H] folic acid were studied in rat liver. The specific activity of glutamyl carboxypeptidase in the lysosomal fraction was near or greater than four times that in the other subcellular fractions. The specific activity of folates was highest in the soluble fraction (102 ng folate/mg protein) and lowest in the microsomal fraction (22 ng folate/mg protein). Nuclear, mitochondrial, and lysosomal folates were 95% folate polyglutamates, and microsomal and soluble folates were 85--90% folate polyglutamates. Injected [3H] folic acid was initially concentrated in the microsomal fraction, as measured by 3h cpm per ng folate. Initially, injected [3H] folic acid was found converted to folate penta- and hexaglutamates in all fractions to a similar extent except in the microsomes where the percentage conversion was much less, as measured by the percentage of total 3H cpm determined to be [3H] folate penta- and hexaglutamates. At 24 h, the conversion of [3H] folates to penta- and hexaglutamates in each fraction was less than that found for the endogenous folates. Injected [3H] folic acid after 2h was found to consist of 94% reduced folates in the soluble fraction, 56% in the mitochondrial, 55% in the nuclear, 20% in the lysosomal, and 15% in the microsomal fraction.

Acid Phosphatase↗
PubMed1995

Nutritional supplementation and prevention of congenital abnormalities.

Recent intervention studies have shown a reduction of occurrence and recurrence of neural tube defects caused by the periconceptional supplementation of folic-acid-containing multivitamins or pharmacological doses of folic acid alone. This new primary preventive method can also reduce the occurrence of other major congenital abnormalities, mainly the reduction of conotruncal cardiovascular malformations, defects of the urinary tract, congenital hypertrophic pyloric stenosis and congenital limb deficiencies. The rate of cleft lip, palate, or both, was not lowered by periconceptional multivitamin supplementation, however. The underlying biologic mechanisms of this protective effect are not still understood, but naturally occurring folates (polyglutamates) or synthetic folic acid (monoglutamate) have a key role. The debate over supplementation concerns which vitamins (folic-acid-containing multivitamins, or folic acid alone); what doses (0.4 mg, 0.8 mg or 4 mg folic acid) and to whom (whether it is worthwhile differentiating between women at high and low risk). At present there are three possibilities: folate- and other vitamin-rich diet, supplementation, and food fortification to provide appropriate multivitamin and folic acid consumption for all women of childbearing age who are capable of becoming pregnant.

Congenital Abnormalities↗