Mechanism-based approaches to inhibition of the synthesis and degradation of folate and antifolate polyglutamates.
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Biological Transport↗
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Synthesis of folate polyglutamates in human cells.
1. Synthesis of polyglutamate derivatives from radioactively labelled folic acid, folinic acid and methotrexate has been studied in human phytohaemagglutinin-transformed lymphocytes. 2. With labelled folic acid as the precursor, approximately 20% of the labelled cell folate was found in each of the mono-, tetra-, penta- and hexaglutamate peaks after 72 h of incubation. At earlier times, the amounts of labelled folate polyglutamates were proportionately greater and the amount of labelled higher folate polyglutamates was lower. After only 4 h incubation over 80% of the intracellular labelled folate was still in the monoglutamate form. 3. With labelled folinic acid used as precursor, approximately 30% of labelled cell folate after 72 h incubation was in the form of folate pentaglutamate, with tri- and tetra-glutamate forming the other major peaks. The speed of formation of polyglutamate derivatives was substantially more rapid than from folic acid. 4. Methotrexate was found to decrease considerably the amount of folate polyglutamate formation from labelled folic acid in lymphocytes byt nevertheless some di-, tri- and traces of tetra-glutamate were formed. On the other hand, methotrexate had no effect on folate polyglutamate formation with labelled folinic acid used as the precusor. 5. Polyglutamate derivatives of labelled methotrexate were formed by human lymphocytes with mean amounts of 4-5% of di-, 1-5% of tri-, 1-0% of tetra- and 0-7% of penta-glutamate derivatives being formed over the period 48-72h of culture. 6. Pentaglutamate derivatives probably constitute the largest group of intracellular folates in human cells but a complex mixture exists. 7. The enzyme synthesizing polyglutamate derivatives of folate in human cells prefers a reduced folate as substrate but the requirement for a reduced form is not absolute. The nature of the reduced folate is uncertain but it is suggested on the basis of previous work that tetrahydrofolate rather than methylhydropfolate or any other reduced folate monoglutamate is the preferred substrate.
Adult↗
Leucovorin rescue of human cancer and bone marrow cells following edatrexate or methotrexate.
We have examined the cytotoxic activities of edatrexate (EDX) and methotrexate (MTX) and their reversal by leucovorin in nine human cancer cell lines and in human bone marrow CFU-GM cells. EDX was 3.7- to 123-fold more toxic than MTX against the cancer cell lines and 25-fold against the bone marrow cells. Lower EDX concentrates generally were needed to inhibit cancer cell growth relative to bone marrow cells, however, whereas bone marrow and cancer cell growth were more often susceptible to the same MTX concentrations. The new antifolate was metabolized to long-chain polyglutamates to a greater extent than MTX in seven cell lines. Leucovorin at 0.2 microM rescued two breast cancer and two non-small cell lung cancer cell lines to a lesser extent following EDX than MTX, but significant rescue was observed in two head and neck cancer cell lines that formed large amounts of polyglutamates. These cell lines also accumulated reduced folates to a greater extent than the other cell lines following leucovorin exposure. Leucovorin rescued bone marrow cells following MTX but only partially following the highest EDX concentrations. EDX may enjoy a better therapeutic index than MTX against some cancer cell lines relative to bone marrow precursor cells, especially after leucovorin rescue.
Aminopterin↗
Differential effects of folinic acid and glycine, adenosine, and thymidine as rescue agents in methotrexate-treated human cells in relation to the accumulation of methotrexate polyglutamates.
By converting methotrexate (MTX) into poly-gamma-glutamyl derivatives, cultured human fibroblasts accumulated high intracellular levels of drug. Once polyglutamates had been formed, DNA synthesis and cell growth remained suppressed even after MTX had been removed from the culture medium. Co-cultivation of cells with MTX and folinic acid reversed the effect of MTX on polyglutamate formation, DNA synthesis, and cell growth. However, if folinic acid was added to the culture medium following a preincubation in methotrexate, DNA synthesis initially remained inhibited and cell growth was only gradually restored. Co-cultivation of cells with 0.67 mM glycine, 37.5 micrometers adenosine, and 41.3 micrometers thymidine (GAT) and MTX did not prevent polyglutamate formation but allowed cells to grow. If GAT was removed from the culture medium along with MTX, cell growth and DNA synthesis were inhibited. If GAT was added to the culture medium following growth in MTX, cell growth recovered. These studies differentiate the effects of GAT and folinic acid treatment. Folinic acid prevented MTX polyglutamate accumulation and reversed the effects of MTX on cell growth when present along with MTX in the cultures. Folinic acid was only partially effective in circumventing the MTX-induced block in folate metabolism when added after pretreatment with MTX. In contrast, GAT allowed growth of cells both in the presence of MTX and after a preincubation in MTX. In contrast, GAT allowed growth of cells both in the presence of MTX and after a preincubation in MTX. However, co-incubation in MTX plus GAT resulted in the accumulation of polyglutamates and a sustained inhibition of cell growth and DNA synthesis upon removal of both MTX and GAT from the culture medium.
Adenosine↗
Inhibition of methotrexate polyglutamate accumulation in cultured human cells.
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Biotransformation↗
PT523 and other aminopterin analogs with a hemiphthaloyl-L-ornithine side chain: exceptionally tight-binding inhibitors of dihydrofolate reductase which are transported by the reduced folate carrier but cannot form polyglutamates.
Nonpolyglutamatable antifolates are potentially of therapeutic interest for the treatment of tumors that are inherently refractory, or have become resistant, to classical antifolates as a result of decreased expression of the enzyme folylpolyglutamate synthetase. An interesting class of water-soluble nonpolyglutamatable analogs of aminopterin (AMT) have been developed, which are much more cytotoxic because they bind more tightly to dihydrofolate reductase (DHFR) and also utilize the reduced folate carrier (RFC) pathway more efficiently for influx into the cell. This review summarizes the in vitro and in vivo preclinical data on the initial lead compound, Nalpha-(4-amino-4-deoxypteroyl)-Ndelta- hemiphthaloyl-L-ornithine (PT523). In addition, the synthesis and in vitro biochemical and biological properties of several types of second-generation analogs are discussed. Analogs modified in the B-ring of the pteridine moiety have been found to be of particular interest because their affinity for DHFR and their influx rate into cells via the RFC pathway are even greater than those of PT523. The hemiphthaloylornithine moiety, which is larger and more hydrophobic than the glutamate side chain of classical antifolates, appears to be chiefly responsible for the exceptionally high biological potency of PT523 and its B-ring analogs.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Hepatic parenchymal cell glutamylation of methotrexate studied in monolayer culture.
A detailed analysis of the gamma-glutamylation of methotrexate has been conducted in primary cultures of rat hepatic parenchymal cells in monolayer culture. The rates of glutamylation are concentration dependent and saturable when measured over a 6-h period at concentrations between 2 and 50 microM. The removal of folate and inclusion of insulin, dexamethasone, and tocopherol enhance glutamylation. Omission of methionine from the medium increases glutamylation, whereas the 6-h period of syntheses, methotrexate the reaction. During the 6-h period of syntheses, methotrexate diglutamate is the primary product, whereas the di- and triglutamates are the major cellular species when the incubation is extended to 24 h. Lower extracellular methotrexate concentrations result in the formation of relatively greater amounts of longer chain length derivatives. The accumulation of methotrexate polyglutamates at steady state is saturable and occurs by 24 h. The predominant species contain two to four glutamate residues, and the distribution depends upon the culture conditions and the extracellular methotrexate concentration. The turnover of cellular polyglutamates at saturation occurs at 30 to 40% of the total cell pool over a 6-h period. Placement of hepatocytes with saturating levels of methotrexate polyglutamates in medium lacking methotrexate results in the slow loss of all derivatives, and the rate of loss is inversely related to polyglutamate chain length. Following a pulse dose of methotrexate, hepatocytes continue to increase the chain length of the cellular pool of polyglutamates, and this process is impaired by addition of folinic acid to the medium. In the pulse experiments as in the longer term incubations, the primary species are the di- and triglutamates. The results demonstrate limited capacity of the hepatocytes to glutamylate longer chain length polyglutamate derivatives.
Animals↗
FOLIC ACID DERIVATIVES SYNTHESIZED DURING GROWTH OF DIPLOCOCCUS PNEUMONIA.
Sirotnak, F. M. (Sloan-Kettering Institute for Cancer Research, New York, N.Y.), Gloria J. Donati, and Dorris J. Hutchison. Folic acid derivatives synthesized during growth of Diplococcus pneumoniae. J. Bacteriol. 85:658-665. 1963.-Under cultural conditions permitting synthesis of folic acid in an amount greatly in excess (20- to 30-fold) of that required for maximal growth of Diplococcus pneumoniae, 85 to 90% of the growth factor accumulated as polyglutamates. Approximately equal amounts of mono- and diglutamates made up the remaining 10 to 15% found in culture material. Most of the polyglutamates occurred intracellularly, in a proportion of triglutamates to higher glutamates of about two to one. Only 10 to 15% of all folic acid derivatives (mono-, di-, and polyglutamates) found had folinic acid (5-formylfolate-H(4)) activity for Pediococcus cerevisiae. Practically all synthesis of the glutamyl-peptide moieties of folate seems to occur at an enzymatic step prior to folic acid, since no appreciable peptide formation occurred under conditions blocking folate synthesis. Sulfanilamide inhibition of growth by a block in folate synthesis was reversed by the addition of dihydrofolic acid, but not folic acid. The examination of two genetically distinct amethopterin-resistant mutant strains has revealed no gross differences in folate accumulation during growth when compared with the wild strain.
Antimetabolites↗
[Folic acid absorption in tropical enteropathy (author's transl)].
Folic acid absoprtion was measured in 45 patients before and after folic acid treatment by means of a folate tolerance test in serum and red-blood-cell-folate. 10 healthy men recently returned from the tropics volunteered as controls. There is no significant difference in the folate-tolerance tests in serum. The difference is significant however in the red-blood-cell-folate, which is markedly lower with the patients before treatment and raises after folate-treatment to the normal value of the control group. As the transport of folic acid in the mucosa follows two steps it is taken that the disturbance is localized in the hydrolysis of polyglutamate to monoglutamate-folic acid.
Adult↗
Syntheses and thymidylate synthase inhibitory activity of the poly-gamma-glutamyl conjugates of N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-thenoyl]-L-glutamic acid (ICI D1694) and other quinazoline antifolates.
Thirteen poly-gamma-glutamates derived from several novel antifolates have been synthesized by a convergent route. The syntheses of poly-gamma-glutamyl conjugates of N-[5-[N-(3,4-dihydro-2- methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-theno yl]-L-glutamic acid (8) (ICI D1694), 2-desamino-N10-propargyl-5,8-dideazafolic acid (6), 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (7), 2-desamino-2-methyl-N10-propargyl-2'-fluoro-5,8-dideazafolic acid (9), and 2-desamino-2-methyl-4-chloro-N10-propargyl-2'-fluoro-3,5,8-trideazafo lic acid (11) are described. A key step in the route involves coupling of an alpha-tert-butyl-protected poly-gamma-glutamate of the required chain length to the appropriate 5,8-dideazapteroic acid, obtained by carboxypeptidase G2 cleavage of the parent monoglutamate, if available, or by chemical synthesis. Deprotection with trifluoroacetic acid in the final step gave the desired poly-gamma-glutamyl antifolates as their trifluoroacetate salts. As inhibitors of thymidylate synthase, these polyglutamates were more potent in every case than the corresponding non-polyglutamylated drug.
Antineoplastic Agents↗
Rapid assembly of Alzheimer-like paired helical filaments from microtubule-associated protein tau monitored by fluorescence in solution.
Alzheimer's disease is characterized by the progressive deposition of two types of fibers in the affected brains, the amyloid fibers (consisting of the Abeta peptide, generating the amyloid plaques) and paired helical filaments (PHFs, made up of tau protein, forming the neurofibrillary tangles). While the principles of amyloid aggregation are known in some detail, the investigation of PHF assembly has been hampered by the low efficiency of tau aggregation, the requirement of high protein concentrations, and the lack of suitable detection methods. Here we report a quantitative assay system that permits monitoring of the assembly of PHFs in real time by the fluorescence of dyes such as thioflavine S or T. Using this assay, we evaluated parameters that influence the efficiency of filament formation. Disulfide-linked dimers of tau constructs representing the repeat domain assemble into PHFs most efficiently, but other tau isoforms or constructs form bona fide PHFs as well. The rate of assembly is greatly enhanced by polyanions such as RNA, heparin, and notably polyglutamate which resembles the acidic tail of tubulin. The assembly is optimal at pH approximately 6 and low ionic strengths (<50 mM) and increases steeply with temperatures above 30 degreesC, indicating that it is an entropy-driven process.
Alzheimer Disease↗
Methotrexate for inflammatory bowel disease: pharmacology and preliminary results.
OBJECTIVE: To review the limited published experience with methotrexate treatment for inflammatory bowel disease, to examine the proposed anti-inflammatory and immune-modifying mechanism of action and pharmacologic properties of methotrexate, and to detail its limiting toxicities. DESIGN: A comprehensive synopsis of methotrexate is presented to aid physicians who treat patients with inflammatory bowel disease. RESULTS: Methotrexate and its polyglutamate metabolites are folic acid analogues with inhibitory activity against many enzymes in the metabolic pathway of folic acid. Long-term low-dose methotrexate therapy (25 mg or less once a week) inhibits production of thymidylate, purines, and methionine and leads to accumulation of adenosine, a potent anti-inflammatory substance. These actions inhibit cellular proliferation, decrease formation of antibodies, and decrease production of mediators of inflammation such as interleukins and eicosanoids. Three uncontrolled trials and two controlled trials have demonstrated efficacy of low-dose methotrexate therapy for induction of remission in Crohn's disease and have also suggested possible benefit for ulcerative colitis and for remission maintenance indications in both diseases. Although methotrexate is generally well tolerated for long-term use at a low dose, several serious toxicities potentially limit its use. CONCLUSION: Methotrexate is a promising new agent for the treatment of inflammatory bowel disease.
Anti-Inflammatory Agents↗
Synthesis of pteroylpolyglutamates in rat liver during regeneration after partial hepatectomy.
The liver content of higher polyglutamate forms of folic acid, which in partially hepatectomized rats is markedly lower as compared with sham operated animals, is normalized by the vitamin administration. The greater availability of folic acid and consequently of the tetrahydrofolates, promotes the synthesis of actual coenzymic forms; so their content, in regenerating liver, is maintained at normal values in spite of a higher requirement caused by the exaltation of biosynthetic processes.
Animals↗
Polyglutamation of the antifolate anticancer drug N10-propargyl-5,8-dideazafolic acid (CB 3717) in the mouse.
The antifolate drug N10-propargyl-5,8-dideazafolic acid was shown to be metabolized to its gamma-glutamyl conjugates in mouse liver and kidneys after administration at a dose of 20 mg/kg. The tissue levels of both CB 3717 and its polyglutamates were time dependent. However, in liver the clearance of polyglutamated conjugates was 10 times slower than that of the parent compound. In liver after 48 hr up to 50% of recovered radioactivity consisted of CB 3717 polyglutamates. In addition to polyglutamate derivatives, in both liver and kidney, 72 hr after CB 3717 administration another minor CB 3717 metabolite(s), unsusceptible to conjugase treatment, was detected. The nature of this metabolite(s) is currently unknown.
Animals↗
5-fluorouracil-methotrexate synergy: enhancement of 5-fluorodeoxyridylate binding to thymidylate synthase by dihydropteroylpolyglutamates.
Ternary complex formation of thymidylate synthase (5,10-methylenetetrahydrofolated:dUMP C-methyltransferase, EC 2.1.1.45), 5-fluorodeoxyuridylate (FdUMP), and poly(gamma-glutamyl) conjugates of pteroate and methotrexate (MTX) has been examined as a basis for the sequence-dependent synergism of the 5-fluorouracil-MTX combination in inhibiting viability of L1210 murine tumor cells. A 1.4-log (25-fold) increase in the inhibition of soft agar colony formation was observed when MTX preceded 5-fluorouracil as compared to the reverse sequence. L1210 cells converted 39% of the total intracellular MTX into MTX poly(gamma-glutamate)s within 4 hr of exposure to 1 microM MTX. MTX and MTX(gamma-glutamate) formed reversible ternary complexes with FdUMP on one site of thymidylate synthase, whereas with 7,8-dihydropteroylpentaglutamate and I-5,10-methylenetetrahydropteroylpentaglutamate stoichiometric binding of FdUMP to two sites on thymidylate synthase was observed. The dissociation constants for FdUMP in the ternary complexes formed in the presence of MTX, MTX(gamma-glutamate), 7,8-dihydropteroylpentaglutamate, and I-5-10-methylenetetrahydropteroylpentaglutamate were estimated to be 370, 27, < 10, and < 10 nM, respectively, by equilibrium dialysis. We propose that the sequence-dependent effect of MTX plus 5-fluorouracil on L1210 cell viability results from MTX and MTX polyglutamate inhibition of dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) and consequently a trapping of intracellular folates as dihydropteroylpolyglutamates, which increase the extent of FdUMP binding to thymidylate synthase.
Animals↗
Biochemical and pharmacologic rationale for high-dose methotrexate.
High-dose methotrexate (HDMTX) regimens were initially designed to overcome methotrexate (MTX) resistance due to defective transport of the drug. At high concentrations, enough MTX diffuses into resistant cells to saturate and inhibit the target enzyme, dihydrofolate reductase (DHFR). The high doses of MTX needed to achieve these high drug concentrations must be administered with the reduced folate antidote, leucovorin (LV; 5-formyltetrahydrofolate), to prevent increased toxicity. To increase MTX therapeutic index, LV rescue must be selective, i.e., more effective in normal than in tumor cells. In experimental models, selective rescue can be achieved if strict LV administration guidelines are respected. Since both MTX and LV use the membrane transport system, it was hypothesized that selective rescue occurred because transport-deficient, MTX-resistant tumor cells also transported LV poorly. The unsatisfactory clinical results frequently obtained with HDMTX regimens suggest a need to re-evaluate this underlying rationale, especially in view of recent findings concerning the mechanisms of MTX resistance and LV rescue. Experimentally, cells resistant to MTX because of an increased or altered DHFR, decreased metabolism to polyglutamates, or decreased thymidylate synthase activity are not always significantly more sensitive to higher concentrations of MTX. Furthermore, recent studies on human small cell lung cancer cell lines suggest that decreased MTX polyglutamate metabolism and thymidylate synthase activity might be prevalent MTX-resistant mechanisms in human tumors. Selective LV rescue could also occur through mechanisms other than selective uptake by normal tissues.(ABSTRACT TRUNCATED AT 250 WORDS)
Biological Transport↗
Regulation of methotrexate polyglutamate accumulation in vitro: effects of cellular folate content.
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Animals↗
Methotrexate polyglutamate synthesis by cultured human breast cancer cells.
We studied the conversion of methotrexate to poly-gamma-glutamyl derivatives by cultured human breast cancer cells. After incubation with 2 micro M [3',5',9-3H]methotrexate, MCF-7 cells were washed free of extracellular drug and were boiled to lyse cells and to release drug bound to dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). The supernatant fraction was chromatographed on Sephadex G-15 to separate parent drug from polyglutamate forms. These cells rapidly and quantitatively converted methotrexate to polyglutamates, such that after 24 hr of incubation, 70 +/- (SEM) 3% of intracellular methotrexate existed as polyglutamates. Examination of that portion of intracellular methotrexate specifically bound to dihydrofolate reductase indicated that, with prolonged incubation, methotrexate polyglutamates become the predominant drug form bound to the enzyme. These studies demonstrate that methotrexate polyglutamates are readily formed in human tumor cells and bind to dihydrofolate reductase. Because these forms of the drug may be selectively retained within the cell, they may be important determinants of the duration of action and, ultimately, the cytotoxicity of methotrexate in human solid tumors.
Breast Neoplasms↗