Combined Effect Of Methotrexate Research Articles

Combined effect of methotrexate and bifidobacteria metabolites on TNFα AND IFNγ production by human peripheral blood mononuclears

Methotrexate (Mtx) is a first-line drug for the treatment of numerous rheumatic and non-rheumatic disorders, including oncological disdiseases. However, therapeutic efficacy of Mtx is limited by severe toxicity to many organs (myelo-, hepato-, nephrotoxicity, mucositis, enteritis, dysbiosis at various human biotopes, etc.). Recently, a number of studies showed that some metabolites of Bifidobacteria and Lactobacilli are able to enhance effect of chemotherapeutic drugs and limit their toxic properties. The aim of the present work was to study the possible potentiating action of Bifidobacteria cell-free supernatants and methotrexate upon secretion of pro-inflammatory TNF and IFN cytokines by human peripheral blood mononuclear cells (PBMCs). The immunoregulatory effects upon production of TNF and IFNg was evaluated in the in vitro model of cultured PBMC supplemented with Bifidobacteria metabolites, methotrexate, or their combination. Analysis of the combined effect of Bifidobacteria metabolites and Mtx on the cytokine production revealed their synergism towards the key pro-inflammatory cytokines (TNF and IFN). We found an increase against the control cultures (with Mtx only), inhibition of the early pro-inflammatory cytokine TNF production. On the contrary, we revealed an increased secretion of IFN which regulates the effector cells. The results obtained with these cytokines suggest the presence of a potentiating effect of Bifidobacteria metabolites upon anti-inflammatory and immunoregulatory properties of methotrexate. Thus, Bifidobacteria metabolites can be considered a promising agent which potentiates the therapeutic action of methotrexate by suppressing TNF secretion and stimulating IFN by immunocompetent cells. Further studies of the combined effects of Mtx and metabolites from the intestinal microbiota upon the cytokine production by effector cells could be recommended, aiming to enhance therapeutic effect of methotrexate and limit its toxic properties using the Bifidobacteria metabolites.

Histone deacetylase inhibition enhances the therapeutic effects of methotrexate on primary central nervous system lymphoma.

BackgroundPolyglutamylation is a reversible protein modification that commonly occurs in tumor cells. Methotrexate (MTX) in tumor cells is polyglutamylated and strongly binds to dihydrofolate reductase (DHFR) without competitive inhibition by leucovorin. Therefore, tumor cells with high polyglutamylation levels are supposed to be selectively killed, whereas normal cells with lower polyglutamylation are rescued by leucovorin. This study investigated the combined effects of MTX plus histone deacetylase inhibitors (HDACIs), which upregulate MTX polyglutamylation, in primary central nervous system lymphoma (PCNSL).MethodsWe evaluated cell viability after MTX treatment and leucovorin rescue and compared the expression of folylpolyglutamate synthetase (FPGS), γ-glutamyl hydrolase (GGH), and DHFR in 2 human PCNSL-derived cell lines (HKBML and TK) and a human Burkitt lymphoma cell line (TL-1). Combination treatments were created using 4 HDACIs: panobinostat, vorinostat, sodium butyrate, and valproic acid. The expression of DHFR was examined as well as ratios of FPGS/GGH expression. The combined effects of MTX plus HDACIs were evaluated using a cell viability assay, mass spectroscopy imaging, and subcutaneous and intracranial xenograft models.ResultsHDACIs upregulated the ratio of FPGS/GGH expression resulting in increased polyglutamylation of MTX, but also downregulated expression of the target molecule of MTX: DHFR. The combination of MTX and vorinostat decreased cell viability in vitro (P < .05) and tumor volumes in a subcutaneous model (P < .0001), and prolonged survival in an intracranial model (P < .01), relative to controls.ConclusionHDACIs enhanced the therapeutic effect of MTX through increased polyglutamylation of MTX and concomitant downregulation of DHFR expression.

CBMT-09. THERAPEUTIC EFFECT OF INDUCTION OF POLYGLUTAMYLATION IN HIGH-DOSE METHOTREXATE THERAPY TO PRIMARY CENTRAL NERVOUS SYSTEM LYMPHOMA

Abstract BACKGROUND Polyglutamylation is a reversible protein modification with a high occurrence rate in tumor cells. Methotrexate (MTX) incorporated into cells is polyglutamylated and strongly binds to dihydrofolate reductase without competitive inhibition by leucovorin (LV). Tumor cells with high polyglutamylation levels are selectively killed, whereas normal cells with lower polyglutamylation are rescued by LV. In this study, we investigated the therapeutic response of PCNSL to HD-MTX therapy with LV rescue based on polyglutamylation status and evaluated the combined effect of MTX and the drugs which upregulated polyglutamylation of MTX. METHODS Among 113 consecutive PCNSL patients who underwent HD-MTX therapy in our department between 2001 and 2014, polyglutamylation was evaluated by immunostaining in 82 cases, with relationships between polyglutamylation and therapeutic response retrospectively examined. Human malignant lymphoma cell lines were used for in vitro and in vivo experiments. The association of polyglutamylation and the response to MTX with LV rescue was assessed in these cell lines. Histone-deacetylase inhibitor (HDACI) has been reported to induce polyglutamylation by elevating folpolyglutamate synthetase (FPGS) expression, so the effects of HDACIs on polyglutamylation were evaluated. Combined effects of MTX and HDACI were evaluated by cell viability assay and xenograft mouse models. RESULTS The complete response rate was significantly higher in the group with polyglutamylation than in the non-polyglutamylation group [58.1% (25/43) and 33.3% (13/39), respectively] (p &lt; 0.05), and progression-free survival was also significantly increased in the group with polyglutamylation (p &lt; 0.01). The combined effect of MTX and HDACI was significantly enhanced in cell viability assay in vitro (p&lt; 0.05), in subcutaneous (p&lt; 0.001) and intracranial tumor xenografts (p&lt; 0.001). CONCLUSION These findings suggested that polyglutamylation could be a predictor of therapeutic response to HD-MTX therapy with LV rescue in PCNSL. Combined therapy with HD-MTX and HDACIs might represent a promising treatment for HD-MTX resistant intractable PCNSL.

Sequential methotrexate and 5-Fluorouracil in advanced ovarian carcinoma

Experimental studies have demonstrated that the combined effects of methotrexate and 5-FU may be optimized in schedules of administration where the antifolate is administered prior to the fluoropyrimidine. Seventeen patients with ovarian carcinoma refractory to standard chemotherapy were treated with a sequence of moderate dose MTX (200 mg/m2) and 5-FU. No responses were seen, although the hematologic and gastrointestinal toxicity which was observed was very mild. Because of prior nephrotoxic chemotherapy and the frequent presence of large amounts of “third space” fluid in the form of malignant ascites, patients with ovarian cancer are at high risk for MTX toxicity. In this study, eight patients with significant ascites had higher plasma MTX concentrations measured at 48 and 72 hr following drug administration than nine similar patients without ascites. This suggested delayed excretion of MTX in patients with ascites although this difference was not statistically significant. MTX concentrations in ascites exceeded those in plasma between 6 and 12 hr and remained near 1 μM for 24 hr. Although the lack of response is disappointing, the modest toxicity encountered with the careful drug level monitoring and the favorable pharmacologic findings suggest that this combination of drugs each with established activity in untreated ovarian cancer deserves further study in primary treatment.

Dipyridamole inhibits reversion by thymidine of methotrexate effect and increases drug uptake in Sarcoma 180 cells.

The combined effect of methotrexate (MTX) with dipyridamole, an inhibitor of nucleoside transport, was studied in ascitic Sarcoma 180 cells. It was determined that 10 microM MTX inhibits by greater than 90% deoxy[3H]uridine incorporation into DNA and that this MTX concentration inhibits DNA synthesis as revealed by deoxy[3H]cytidine but not [3H]thymidine incorporation into DNA. Exogenous thymidine (greater than or equal to 1 microM) in the cell culture medium enhances DNA synthesis in nontreated cells and fully restores it in MTX-treated cells, whereas hypoxanthine has no appreciable effect on DNA synthesis. Dipyridamole inhibits deoxy[3H]cytidine and [3H]thymidine uptake by these cells (IC50 = 0.2 and 3 microM, respectively) and blocks the increase in TTP pool produced by 1 microM thymidine in MTX-treated cells (23.1 +/- 4.7 pmol per 1 X 10(6) cells vs. 80.4 +/- 18.9 pmol per 1 X 10(6) cells). Dipyridamole at 10 microM enhances [3H]MTX accumulation by Sarcoma 180 cells and diminishes the efflux of the drug in previously loaded cells. It is suggested that the combination of inhibitors of the de novo pathway for pyrimidine biosynthesis, such as MTX, with inhibitors of the salvage pathway, such as dipyridamole, may increase the cytotoxic activity of MTX alone.

Enteropathy complicating maintenance therapy in acute lymphoblastic leukaemia.

Seven patients being treated for acute lymphoblastic leukaemia between March 1976 and March 1981, in accordance with Medical Research Council protocols, developed clinical malabsorption while on maintenance chemotherapy. All of them received weekly methotrexate and 6 of the 7 were given co-trimoxazole. Five patients had folate deficiency. Stopping the anti-leukaemic therapy led to a resolution of gastrointestinal symptoms. A combined effect of methotrexate and co-trimoxazole is postulated.

Combined effects of methotrexate and 5-fluoropyrimidine on human breast cancer cells in serum-free culture

The combined effects of methotrexate and 5-fluoropyrimidines on the growth of a human breast cancer cell line in serum-free hormone-supplemented medium, lacking nucleosides, have been studied. All combinations of methotrexate (10–50 nM) and 5-fluorodeoxyuridine (10–100 nM) resulted in at least additive inhibition of cell growth and when analyzed by 50% response isobologram demonstrated synergism. Combinations of methotrexate (10–50 nM) and 5-fluorouracil (0.04–0.4 μM) gave enhanced inhibition as compared to single agents, but 50% response isobologram revealed only additive effects. The combination was most effective when methotrexate was added to the cultures prior to the 5-fluoropyrimidines or when the drugs were added simultaneously. The addition of 5-fluoropyrimidine prior to methotrexate appears to decrease the effect of the antifol on purine synthesis and lessen the cytotoxicity of the combination in this cell line.