lometrexol and Leukemia--Lymphoid

5,10-dideaza-5,6,7,8-terrahydrofolic acid (DDATHF) is a potent antiproliferative agent in cell culture systems and in vivo in a number of murine and human xenograft tumors. In contrast to classical antifolates, which are dihydrofolate reductase inhibitors, DDATHF primarily inhibits GAR transformylase, the first folate-dependent enzyme along the pathway of de novo purine biosynthesis. The (6R) diastereomer of DDATHF (Lometrexol), currently undergoing clinical investigation, was used to develop CCRF-CEM human leukemia sublines resistant to increasing concentrations of the drug. Three cell lines were selected for ability to grow in medium containing 0.1 microM, 1.0 microM, and 10 microM of (6R)DDATHF, respectively. Impaired polyglutamylation was identified as a common mechanism of resistance in all three cell lines. A progressive decrease in the level of polyglutamylation was associated with diminished folylpolyglutamate synthetase activity and paralleled increasing levels of resistance to the drug. However, the expression of folylpolyglutamate synthetase RNA was not altered in the resistant cell lines compared to the parent cells. The most resistant cell subline also displayed an increased activity of gamma-glutamyl hydrolase. The sublines were scrutinized for other possible mechanisms of resistance. No alterations in drug transport or in purine economy were found. Modest increases were found in the activity of methylene tetrahydrofolate dehydrogenase but no alterations of other folate-dependent enzymes were observed. Increases in accumulation and conversion of folic acid to reduced forms, particularly 10-formyltetrahydrofolate, was also seen. The resistant cell lines were sensitive to dihydrofolate reductase inhibitors, methotrexate and trimetrexate, for a 72-h exposure period but showed cross-resistance to methotrexate for 4 and 24 h exposures. Cross-resistance was also shown toward other deazafolate analogues for both short- and long-term exposures.

Topics: 5,10-Methylenetetrahydrofolate Reductase (FADH2); Acyltransferases; Antineoplastic Agents; Clone Cells; Drug Resistance; Folic Acid Antagonists; Formate-Tetrahydrofolate Ligase; gamma-Glutamyl Hydrolase; Glycine Hydroxymethyltransferase; Humans; Hydroxymethyl and Formyl Transferases; Leukemia, Lymphoid; Methylenetetrahydrofolate Dehydrogenase (NADP); Methylenetetrahydrofolate Reductase (NADPH2); Oxidoreductases; Peptide Synthases; Phosphoribosylaminoimidazolecarboxamide Formyltransferase; Phosphoribosylglycinamide Formyltransferase; Stereoisomerism; Tetrahydrofolate Dehydrogenase; Tetrahydrofolates; Thymidylate Synthase; Tumor Cells, Cultured

We previously reported (Matherly et al., J Biol Chem 267: 23253-23260, 1992) that impaired methotrexate transport in a drug-resistant CCRF-CEM variant (CEM/MTX) involved the synthesis of a structurally altered isoform of the "classical" carrier for methotrexate and related derivatives. Although CEM/MTX cells were highly resistant (162- to 300-fold) to assorted antifolate substrates for the classical transporter, including methotrexate, aminopterin, 10-ethyl-10-deazaaminopterin, ICI D1694, and 1843U89, they were only 3.6-fold resistant to (6R)-5,10-dideaza-5,6,7,8-tetrahydrofolate (DDATHF). These divergent antifolate sensitivities were not associated with appreciable differences in the levels of dihydrofolate reductase, thymidylate synthase, and 5'-phosphoribosylglycinamide (GAR) transformylase, or the expression of a high affinity membrane folate binding protein receptor in either line. The initial rate of [14C]DDATHF influx was increased 2.9-fold over that for [3H]methotrexate in parental cells (at 2 microM). Whereas [14C]DDATHF initial uptake was, likewise, increased over [3H]methotrexate in CEM/MTX cells (5.3-fold), influx of both compounds was impaired substantially (95-97%). For the parent, influx of [14C]DDATHF was inhibited by substrates for the classical transporter including unlabeled DDATHF, methotrexate, (6R,S)-5-formyl tetrahydrofolate, 10-ethyl-10-deazaaminopterin, ICI D1694, 1843U89, and folic acid. The synthesis of a modified transporter in CEM/MTX cells was accompanied by significant changes in the binding of all these transport substrates. In spite of its impaired transport, [14C]DDATHF (at 2 microM), unlike methotrexate, continued to accumulate in CEM/MTX cells, eventually reaching 62% of the parental drug levels after 4 hr. At this time, 53% (parent) and 71% (CEM/MTX) of the intracellular radioactivity from [14C]DDATHF was identified as polyglutamates. DDATHF polyglutamates in CEM/MTX cells after 4 hr reached 90% of the levels measured in parental cells. While significant levels of methotrexate polyglutamates were detected in the parental line, methotrexate polyglutamylation was negligible in intact CEM/MTX cells. The specific activity of folylpolyglutamate synthetase was measured in cell-free extracts from parental and CEM/MTX cells using aminopterin, methotrexate, and DDATHF as substrates; in each case, CEM/MTX cells showed 2-fold higher enzyme activity than parental cells. These data show that even for tumor cells with severely impai

Topics: Antineoplastic Agents; Biological Transport; Blotting, Western; Catalysis; Cell Membrane; Drug Resistance; Folic Acid Antagonists; Humans; Kinetics; Leukemia, Lymphoid; Methotrexate; Peptide Synthases; Polyglutamic Acid; Tetrahydrofolates; Tumor Cells, Cultured

5,10-Dideazatetrahydrolic acid (DDATHF) is representative of a new class of antifolates acting through inhibition of de novo purine synthesis. We report here the transport characteristics of the diastereomers of DDATHF, which differ in configuration at C6, and comparison studies with other folate and antifolate analogs. (6R)-DDATHF showed high affinity for the influx system of CCRF-CEM cells with a Km of 1.07 microM and an influx Vmax of 4.04 pmol/min/10(7) cells. Comparative studies with methotrexate yielded an influx Km of 4.98 microM and a Vmax of 6.64 pmol/min/10(7) cells, and with 5-formyltetrahydrofolate an influx Km of 2.18 microM and a Vmax of 6.84 pmol/min/10(7) cells. Uptake of (6R)-DDATHF was competitively inhibited by (6S)-DDATHF, methotrexate (MTX), and 5-formyltetrahydrofolate, all with Ki values similar to their influx Km. The (6S)-DDATHF diastereomer had an influx Km of 1.04 microM, similar to that of (6R)-DDATHF; however, the Vmax of 1.72 pmol/min/10(7) cells was 2.3-fold lower than for (6R)-DDATHF. The transport properties of DDATHF were also studied in a mutant cell line (CEM/MTX), resistant to MTX based on impaired drug transport. In this system (6R)-DDATHF showed an influx Km of 1.49 microM and a decreased influx Vmax of 0.60 pmol/min/10(7) cells. A similar effect was shown for MTX (Km of 7.48 microM, Vmax of 1.02 pmol/min/10(7) cells). The number of binding sites in CCRF-CEM cells was similar for (6R)-DDATHF, (6S)-DDATHF, and MTX, 0.74, 0.71, and 0.76 pmol/10(7) cells, respectively. These values were slightly higher in the CEM/MTX cell line (1.07 and 1.09 pmol/10(7) cells for (6R)-DDATHF and MTX, respectively). Treatment of CCRF-CEM cells with either the N-hydroxysuccinimide ester of MTX or the corresponding N-hydroxysuccinimide ester of (6R)-DDATHF caused substantial inhibition (> 90%) of the influx of (6R)-[3H]DDATHF and [3H]MTX, respectively. These results suggest strongly that DDATHF and MTX share a common influx mechanism through the reduced folate transport system. The internalization of DDATHF by monkey kidney epithelial MA104 cells, which express a high affinity folate receptor, was also studied. Competitive binding studies using purified folate receptor and radiolabeled 5-methyltetrahydrofolate showed that (6S)- and (6R)-DDATHF both had I50 values lower than 5-methyltetrahydrofolate (12 nM). Further studies indicate that both DDATHF isomers are actively intracellularly concentrated through this route and are also rapidly con

Topics: Biological Transport; Carrier Proteins; Drug Resistance; Folate Receptors, GPI-Anchored; Folic Acid Antagonists; Humans; Kinetics; Leukemia, Lymphoid; Methotrexate; Receptors, Cell Surface; Stereoisomerism; Substrate Specificity; Tetrahydrofolates; Tritium; Tumor Cells, Cultured

The synthesis and biological evaluation of a number of analogues of N-[4-[4-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidyl) butyl]benzoyl]-L-glutamic acid (2) (7-DM-DDATHF), an acyclic modification of the novel folate antimetabolite 5,10-dideazatetrahydrofolic acid (DDATHF), are described. The synthetic procedure utilized previously for the synthesis of 2, 15, and 16 was extended to the preparation of analogues modified in the benzoyl region with thiophene and methylene groups replacing the benzene ring (compounds 27a-c) and in the glutamate region with aspartic acid and phenylalanine replacing L-glutamic acid (compounds 36, 37). The 2-amino-4,6-dioxo derivative 33 was obtained from intermediate 30 via a palladium-catalyzed carbon-carbon coupling reaction with diethyl (4-iodobenzoyl)-L-glutamate, followed by reduction and removal of protecting groups with base. Cell culture cytotoxicity studies of all of the above acyclic analogues of DDATHF against CCRF-CEM human lymphoblastic leukemic cells gave IC50s ranging from 0.042 greater than 48 microM. Inhibition and cell culture reversal studies against isolated enzymes suggest the mode of action of these compounds. Compound 2 was only 3-fold less inhibitory toward glycinamide ribonucleotide formyltransferase (GARFT, isolated from L1210 leukemic cells) than DDATHF itself. These acyclic analogues were less efficient substrates for the enzyme folylpolyglutamate synthetase (FPGS) compared with their bicyclic counterparts. Moderate antitumor activity was observed for compound 2 against 6C3HED lymphosarcoma and C3H mammary adenocarcinoma in vivo.

Topics: Adenocarcinoma; Animals; Antineoplastic Agents; Cell Line; Cells, Cultured; Humans; Leukemia, Lymphoid; Mammary Neoplasms, Experimental; Mice; Mice, Inbred C3H; Structure-Activity Relationship; Tetrahydrofolates

5,10-Dideazatetrahydrofolic acid (DDATHF) is a new potent antitumor agent that specifically inhibits purine biosynthesis, primarily through inhibition of glycinamide ribonucleotide transformylase, the first of the tetrahydrofolate-requiring enzymes in the de novo synthesis pathway. DDATHF has been shown to be an excellent substrate for mouse liver folylpolyglutamate synthetase in vitro, suggesting that intracellular conversion to polyglutamates could play an important role in the action of this antifolate. In this report, metabolic studies of the 6R-diastereomer of DDATHF in the cultured human leukemia cell lines CCRF-CEM and HL-60 are presented. At both 1 and 10 microM (6R)-DDATHF was rapidly converted to polyglutamates in both cell lines. DDATHF(Glu)5 and DDATHF(Glu)6 were the main intracellular metabolites. After incubation in drug-free medium, (6R)-DDATHF polyglutamates were better retained intracellularly with increasing glutamate chain length. (6R)-DDATHF showed reduced cytotoxicity toward a folylpolyglutamate synthetase-deficient cell line, CCRF-CEM30/6 related to a dramatically diminished accumulation of polyglutamates. The activity of (6R)-DDATHF in CCRF-CEM30/6 cells was decreased after both short and prolonged exposures. These results suggest that polyglutamylation of (6R)-DDATHF not only represents a mechanism for trapping the drug inside the cells but also produces a more potent inhibitor of the target enzyme.

Topics: Cell Division; gamma-Glutamyl Hydrolase; Humans; Leukemia, Lymphoid; Polyglutamic Acid; Tetrahydrofolates; Tumor Cells, Cultured

The synthesis of the antifolate 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF) has been modified. It is prepared from 2-acetamido-6-formyl-4(3H)-pyrido[2,3-b]pyrimidone and [P-(N-[1,3-bis(ethoxycarbonyl)propan-1-yl]aminocarbonyl)] phenylmethyl]-triphenylphosphonium bromide. The synthesis proceeds via a sodium hydride promoted Wittig condensation in 1-methyl-2-pyrrolidone followed by catalytic reduction, mild base hydrolysis, and acid precipitation of the product. Synthesis of DDATHF is achieved in a total of seven steps from commercially available reagents. DDATHF is transported effectively into CCRF-CEM cells and inhibits growth of both human (CEM) and murine (L1210) cells in culture. Studies reported here support the view that methotrexate and DDATHF are transported via a shared transport mechanism.

Topics: Animals; Antineoplastic Agents; Binding, Competitive; Cell Division; Cell Line; Folic Acid Antagonists; Humans; Leukemia L1210; Leukemia, Lymphoid; Methotrexate; Mice; Tetrahydrofolates