Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Triazinate (TZT), a potent inhibitor of dihydrofolate reductase, was selected for detailed investigation to determine its mechanism of selective action as well as its metabolic fate in mice, rats, dogs, and monkeys. The serum disappearance of TZT in normal and tumor-bearing mice was similar, with a rapid tissue equilibration phase and a slower elimination phase. Serum disappearance in normal and tumor-bearing rats was 1.5 to 2.2 hr. Serum disappearance in dogs and monkeys was similar, with half-lives of 3 to 4 and 2 to 4 hr, respectively. Urinary excretion of TZT at 24 hr was only 5 to 6% of the injected dose in mice and rats; in contrast, the dogs excreted 60% of the injected dose in 8 hr. TZT accumulated to comparable degrees in the organs of rats and mice, with progressively lesser concentrations in liver, kidney, spleen, and brain. Dihydrofolate reductase activity became almost undectectable in all tissues studied within 15 min after drug adminsitration. An important difference in drug accumulation was in the ascites cells of tumor-bearing animals: in mice, the drug level was consistently lower in the L1210 cells than in the ascites fluid; in contrast, by 30 min after treatment with TZT the drug level in Walker 256 cells was 10-fold higher than the level in the ascites fluid. No evidence for drug metabolism was found in extracts of urine, feces, or organ tissues from either mice or rats. TZT and two related triazines were studied for their ability to accumulate in the cerbrospinal fluid of dogs after i.v. administration. TZT achieved a cerebrospinal fluid level of approximately 15% of the serum concentration at 1 hr; in contrast, the other two triazines reached maximum cerebrospinal fluid values of 1% at 1 hr.
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PMID:Pharmacology of a new triazine antifolate in mice, rats, dogs, and monkeys. 80 54

Triazinate (TZT), a triazine folate antagonist, is a potent inhibitor of dihydrofolate reductase from mammalian cells. Because antitumor activity of triazinate in experimental tumors correlated closely with the in vitro inhibition of DNA synthesis in tumor cells derived from these tumors, we studied cells from patients with leukemia, solid tumor effusions, and cells from normal marrow to determine their in vitro sensitivity to TZT. DNA synthesis in cells from patients with acute leukemia was less sensitive to TZT than it was to methotrexate (MTX) at 2 X 10(-6) M concentration of the inhibitor, whereas the sensitivity was similar at 10(-5) M. This could be accounted for by the known greater sensitivity of dihydrofolate reductase to MTX than to TZT, and the observation that, whereas intracellular drug levels were similar at low (2 X 10(-6) M) extracellular concentrations of TZT or MTX, at the higher (10(-5) M) extracellular drug concentration intracellular TZT was greater than 3 times intracellular MTX. In vitro inhibition of DNA synthesis in cells obtained after patients were treated with TZT was correlated with drug serum concentration and with leukemia cell kill. The sensitivity of cells from solid tumor effusions to TZT was similar to the sensitivity to MTX. Since patients can tolerate doses of TZT five times higher than MTX with less toxicity, there may be advantage to the clinical use of TZT in some tumor cell types.
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PMID:Inhibition of DNA synthesis in normal and malignant human cells by triazinate (Baker's antifol) and methotrexate. 95 90

Roswell Park Memorial Institute 4265 human lymphoblasts were grown with three dihydrofolate reductase inhibitors: a 2,4-diaminopteridine, methotrexate; a 2,4-diaminoquinazoline, chlorasquin; and, a 2,4-diaminotriazine, triazinate. In the absence of inhibitor, dihydrofolate reductase activity increased to a peak at mid-log growth and then declined during the later growth stages. When cells were grown with 10(-8) M antifolate, cell growth was not affected, but dihydrofolate reductase activity (assayed at pH 7.0) remained at approximately initial levels throughout the growth cycle. This represented 60 to 70% less activity at the mid-log stage of growth, as compared to control cells. Dihydrofolate reductase activity in cells grown with 10(-8) M methotrexate, when assayed at pH 8.5, reached levels twice those in control cells. Enzyme activity in cells grown with 10(-8) M chlorasquin, when assayed at pH 8.5, was also higher than at pH 7.0, but it was not as high as that observed in methotrexate-treated cells. Activity in cells grown with 10(-8) M triazinate was approximately the same when assayed at either pH 7.0 or 8.5. At 10(-8) M, the three antifolates had no effect on the activities of thymidylate synthetase, thymidine kinase, serine trans-hydroxymethylase, 5,10-methylenetetrahydrofolate dehydrogenase, 10-formyltetrahydrofolate synthetase, and thymidylate kinase. However, when concentrations were used which completely inhibited growth (10(-7) to 10(-5) M methotrexate or chlorasuin; 10(-6) to 10(-5) M triazinate), dihydrofolate reductase was progressively inhibited, and there was a two- and a threefold elevation of thymidylate synthetase and thymidine kinase activity, respectively. Quantitatively, the elevation of either enzyme was similar over the range of growth-inhibitory concentrations studied. The activities of the other enzymes were unaffected. Methotrexate and chlorasquin inhibited thymidylate synthetase in a noncompetitive manner (with respect to 5,10-methylenetetrahydrofolate) with approximate Ki values of 4.5 X 10(-5) M and 4.9 X 10(-6) M, respectively. Triazinate, at 10(-3) M, had no significant effect on thymidylate synthetase activity. At 10(-3) M, the antifolates produced a negligible inhibition of thymidine kinase. Deoxyuridine 5'-monophosphate (10(-5) M) effectively protected thymidylate synthetase from heat inactivation in vitro. Dihydrofolate or 5,10-methylenetetrahydrofolate, at 10(-3) M, only partially protected thymidylate synthetase. Concentrations of methotrexate (10(-7) to 10(-6) M), chlorasquin (10(-7) M), and triazinate (10(-6) to 10(-5) M), which produced thymidylate synthetase elevation in vivo, did not protect the enzyme from heat inactivation in vitro. Methotrexate at 10(-5) M and chlorasquin at 10(-6) M gave slight protection. Thymidine kinase was stabilized only by thymidine.
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PMID:Elevation of dihydrofolate reductase, thymidylate synthetase, and thymidine kinase in cultured mammalian cells after exposure to folate antagonists. 127 51

The goals of new antifolate development are: 1) improved selectivity, 2) improved penetration into pharmacologic sanctuaries, and 3) effectiveness vs. tumors either with intrinsic or acquired resistance to methotrexate (MTX). The major target for antifolate development has been dihydrofolate reductase (DHFR), but other critical folate-dependent enzymes, i.e., thymidylate synthase, methionine synthetase, and folylpolyglutamate synthetase are also important targets for new antifolate development. The possibility that DHFR from tumor tissue differs significantly from normal tissue DHFR now seems improbable, and the ideas of the late Bill Baker to design specific inhibitors of the tumor enzyme vs. the normal tissue DHFR are unlikely to succeed. However, the experience with triazinate (Baker's antifol; TZT) indicates that transport of antifols could be exploited to provide selective toxicity, as well as to provide agents effective vs. MTX-resistant cells. This work led to a second generation of "nonclassical" folate antagonists, of which trimetrexate (JB-11; TMQ) is now in clinical trial. Uptake of TMQ is via an MTX-independent membrane system, and extremely high intracellular levels of this drug are achieved in human leukemia cells.
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PMID:Design and rationale for novel antifolates. 343 93

A human T-lymphoblast cell line, CCRF-CEM/R1, resistant to methotrexate by virtue of increased dihydrofolate reductase activity, was grown in stepwise increasing concentrations of methotrexate. This additional selection pressure resulted in a cell line, CCRF-CEM/R2, resistant to methotrexate by virtue of both an elevation of dihydrofolate reductase activity and a marked decrease in methotrexate transport. The R1 and R2 cells were approximately 70- and 350-fold more resistant to methotrexate than were the parent cells. The effects of three folate antagonists were studied on these cell lines and also on CCRF-CEM/R3 cells, characterized by impaired methotrexate transport but normal levels of dihydrofolate reductase. The elevated reductase subline CCRF-CEM/R1 was cross-resistant to triazinate [Baker's antifol, NSC 139105; ethanesulfonic acid compounded with alpha-(2-chloro-4-[4,6-diamino-2,2-dimethyl-S-triazine-1-(2H)-yl] phenoxyl)-N,N-dimethyl-m-toluamide (1:1)] and trimetrexate (NSC 249008, JB-11, TMQ; 2,4-diamino-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline), two nonclassical folate antagonists. In contrast, the transport defective subline, CCRF-CEM/R3 was not cross-resistant to these two compounds. In cells resistant to MTX by virtue of both mechanisms, CCRF-CEM/R2, triazinate, and trimetrexate were partially cross-resistant. All three methotrexate-resistant sublines showed minor cross-resistance to isoaminohydroxyquinazoline (IAHQ, NSC 289517; 5,8-dideazaisopteroylglutamate), a folate antagonist inhibitor of thymidylate synthase. These data demonstrate that methotrexate-resistant tumor cells may be effectively inhibited by antifolates with different route of entry into cells or with different enzyme targets.
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PMID:Cytotoxic effects of folate antagonists against methotrexate-resistant human leukemic lymphoblast CCRF-CEM cell lines. 385 84

The tissue distribution of BAF was compared by intravenous and subcutaneous injections in rats bearing Walker 256 carcinoma. Following a single dose (6 mg/kg containing 30 microCi 4,6-di-14C-BAF), the drug concentrations were determined by radiochemical and dihydrofolate reductase assays. The two methods gave comparable results. No metabolites were found by paper chromatographic separations.
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PMID:Comparative distribution of Baker's antifolate (NSC 139105) in rat tissues after subcutaneous and intravenous injections. 696 27

The properties of the folate transport system in H35 hepatoma cells have been studied by measuring the transport of (+)-5-methyltetrahydrofolate. Using initial rates of uptake, it has been demonstrated that the uptake is saturable, carrier mediated, and shared by methotrexate. The accumulation of (+)-5-methyltetrahydrofolate is concentrative, demonstrating the presence of an active transport process. A previous study suggested that methotrexate-resistant sublines (H35R) acquired methotrexate insensitivity because of an impaired capacity for transport. This postulate was substantiated in the present investigation by several observations. The initial uptake and steady-state level of (+)-5-methyltetrahydrofolate were markedly reduced in the resistant sublines as was the case with methotrexate. Triazinate (2-(chloro-4-[4,6-diamino-2,2-dimethyl-S-triazine-1(2H)-ylphenoxyl])-N,N-dimethyl-m-toluamide . ethanesulfonic acid) an inhibitor of dihydrofolate reductase which enters the cells by a pathway independent of the folate coenzyme, was equally toxic to H35 cells and to an H35 subline resistant to 0.3 microM methotrexate. Resistant sublines that are insensitive to methotrexate up to 1 microM display a transport defect but have normal levels of dihydrofolate reductase. Sublines resistant to higher levels of methotrexate showed not only defective transport but also commensurate increases in dihydrofolate reductase. Attempts to demonstrate carried-dependent transport of (+)-5-methyltetrahydrofolate or methotrexate in resistant sublines were negative, suggesting the lack of a functional carrier. These properties were readily demonstrated in H35 cells and included temperature dependence, competition for uptake with analogs, and transstimulation.
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PMID:5-Methyltetrahydrofolate transport by hepatoma cells and methotrexate-resistant sublines in culture. 697 Nov 49