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International Journal of Cancer Feb 2003We have studied the molecular basis of drug resistance in human CCRF-CEM leukemia cells exposed to high dose intermittent pulses of novel polyglutamatable antifolates...
Loss of folylpoly-gamma-glutamate synthetase activity is a dominant mechanism of resistance to polyglutamylation-dependent novel antifolates in multiple human leukemia sublines.
We have studied the molecular basis of drug resistance in human CCRF-CEM leukemia cells exposed to high dose intermittent pulses of novel polyglutamatable antifolates that target various folate-dependent enzymes. These include the dihydrofolate reductase (DHFR) inhibitors edatrexate, methotrexate and aminopterin, the thymidylate synthase (TS) inhibitors ZD1694 and GW1843, the glycinamide ribonucleotide formyltransferase (GARTF) inhibitor DDATHF as well as the multitargeted antifolate LY231514 inhibiting both TS, DHFR and GARTF. Fourteen antifolate-resistant sublines were isolated, 11 of which displayed a drug resistance phenotype that was based on impaired folylpoly-gamma-glutamate synthetase (FPGS) activity as these cell lines: 1) typically lost 90-99% of parental FPGS activity; 2) expressed 1.4-3.3-fold less FPGS mRNA (only 4 cell lines); 3) displayed up to 10(5)-fold resistance to polyglutamylation-dependent antifolates including ZD1694 and MTA; 4) retained sensitivity to polyglutamylation-independent antifolates including ZD9331 and PT523; 5) were up to 19-fold hypersensitive to the lipid-soluble antifolates trimetrexate and AG377; 6) had a normal or a small decrease in [(3)H]MTX transport; and 7) had a 2.1-8.3-fold decreased cellular folate pools and a consequently increased folate growth requirement. The remaining 3 antifolate-resistant sublines lost 94-97% of parental [(3)H]MTX transport and thus displayed a high level resistance to all hydrophilic antifolates. To screen for mutations in the hFPGS gene, we devised an RT-PCR single strand conformational polymorphism (SSCP) assay. RT-PCR-SSCP analysis and DNA sequencing showed that only a single FPGS-deficient subline harbored an FPGS mutation (Cys346Phe). Three-dimensional modeling of the human FPGS based on the crystal structure of Lactobacillus casei FPGS suggested that this mutation maps to the active site and interferes with the catalytic activity of the enzyme due to a putative bulky clash between the mutant Phe346 and a native Phe350 within alpha-helix A10 in a highly conserved C-terminal hydrophobic core. This was consistent with a 23-fold decreased affinity of the mutant Cys346Phe FPGS for L-glutamate. We conclude that decreased FPGS activity is a dominant mechanism of resistance to polyglutamylation-dependent novel antifolates upon a high-dose intermittent exposure schedule. The finding that cells may exhibit 5 orders of magnitude of resistance to polyglutamylation-dependent antifolates but in the same time retain parental sensitivity or hypersensitivity to polyglutamylation-independent antifolates or lipophilic antifolates offers a potentially promising treatment strategy in the overcoming of FPGS-based anticancer drug resistance.
Topics: Base Sequence; Binding Sites; Biological Transport; Blotting, Northern; Blotting, Western; Cell Division; DNA Primers; Down-Regulation; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Enzyme Inhibitors; Folic Acid; Folic Acid Antagonists; Genes, Dominant; Humans; Leukemia; Mutation; Peptide Synthases; Polyglutamic Acid; Polymorphism, Single-Stranded Conformational; Protein Conformation; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured
PubMed: 12494465
DOI: 10.1002/ijc.10829 -
American Journal of Clinical Oncology Apr 2010The primary objective of this trial was to evaluate the response rate for trimetrexate in conjunction with 5-FU and leucovorin (LV) (= TFL) in the treatment of advanced...
OBJECTIVE
The primary objective of this trial was to evaluate the response rate for trimetrexate in conjunction with 5-FU and leucovorin (LV) (= TFL) in the treatment of advanced gastric cancer in a phase II, cooperative group setting.
METHODS
Patients with locally advanced, unresectable, or metastatic adenocarcinoma of the stomach received trimetrexate 110 mg/m IV over 60 minutes day 1, followed by 5-FU 500 mg/m IV bolus and LV 200 mg/m IV over 60 minutes day 2, followed by oral LV 15 mg every 6 hours x 7 doses, all weekly for 6 weeks followed by 2 weeks of rest, continued until progression.
RESULTS
Characteristics for 37 eligible patients: median age 63 (range: 23-83); male/female: 69% of 31%; performance status 0/1/2 15/20/1. The confirmed response rate was 19%, and median overall survival was 6 months. Two patients died as a result of therapy, 1 because of infection without significant neutropenia, and 1 due to perforation of a responding gastric lesion. Seventy-two percent experienced grades 3 and 4 toxicity, most commonly diarrhea, fatigue, and lymphopenia.
CONCLUSIONS
This regimen achieves response rates comparable to other 5-FU-based regimens, when used in treatment of incurable gastric cancer. Toxicity appears manageable.
Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Female; Fluorouracil; Humans; Leucovorin; Male; Middle Aged; Neoplasm Staging; Stomach Neoplasms; Survival Rate; Treatment Outcome; Trimetrexate; Young Adult
PubMed: 19770625
DOI: 10.1097/COC.0b013e318199fb84 -
The Journal of Antimicrobial... Dec 2009First-line therapy for Pneumocystis jirovecii pneumonia (PCP) is trimethoprim/sulfamethoxazole. Few data exist to guide the choice of second-line therapy for patients...
OBJECTIVES
First-line therapy for Pneumocystis jirovecii pneumonia (PCP) is trimethoprim/sulfamethoxazole. Few data exist to guide the choice of second-line therapy for patients failing or developing toxicity to first-line therapy.
METHODS
A case note review of 1122 patients with 1188 episodes of HIV-associated PCP from three observational cohorts in Copenhagen, London and Milan, between 1989 and 2004, was conducted.
RESULTS
Trimethoprim/sulfamethoxazole (962 PCP episodes, 81%) was the most frequently used first-line therapy, followed by intravenous pentamidine (87 episodes, 7%), clindamycin/primaquine (72 episodes, 6%) and 'other' (atovaquone, dapsone/pyrimethamine, trimetrexate or inhaled pentamidine; 67 episodes, 6%). Rates of unchanged therapy were trimethoprim/sulfamethoxazole = 79%, clindamycin/primaquine = 65% and pentamidine = 60% (P < 0.001). First-line therapy was changed because of failure in 82 (7%) episodes and because of toxicity in 198 (17%) episodes. Three month survival rates were trimethoprim/sulfamethoxazole = 85%, clindamycin/primaquine = 81% and pentamidine = 76% (P = 0.09). After adjustment for possible confounders, pentamidine was associated with a significantly greater risk of death at 3 months [hazard ratio (HR) = 2.0, 95% confidence interval (CI) = 1.2-3.4]. Second-line therapy survival rates differed: trimethoprim/sulfamethoxazole = 85%; clindamycin/primaquine = 87%; and pentamidine = 60% (P = 0.01). Multivariable time-updated Cox regression analysis showed a greater risk of death associated with pentamidine (HR = 3.3, 95% CI = 2.2-5.0), but not for clindamycin/primaquine, when both were compared with trimethoprim/sulfamethoxazole.
CONCLUSIONS
Pentamidine was associated with a greater risk of death when used as first- and second-line therapy for HIV-associated PCP, and was associated with more treatment changes. Clindamycin/primaquine appeared superior to pentamidine as second-line therapy for PCP in patients failing or developing toxicity with trimethoprim/sulfamethoxazole. In patients failing first-line treatment with non-trimethoprim/sulfamethoxazole regimens, second-line therapy should be trimethoprim/sulfamethoxazole.
Topics: AIDS-Related Opportunistic Infections; Acquired Immunodeficiency Syndrome; Adolescent; Adult; Aged; Antifungal Agents; Clindamycin; Cohort Studies; Denmark; Female; Humans; Italy; London; Male; Middle Aged; Pentamidine; Pneumocystis carinii; Pneumonia, Pneumocystis; Primaquine; Survival Analysis; Treatment Outcome; Trimethoprim, Sulfamethoxazole Drug Combination; Young Adult
PubMed: 19858161
DOI: 10.1093/jac/dkp372 -
Antimicrobial Agents and Chemotherapy Jul 1991Pneumocystis carinii was obtained in high yield from the lungs of immunosuppressed rats by rupturing mammalian host cells, washing away the soluble mammalian...
Pneumocystis carinii was obtained in high yield from the lungs of immunosuppressed rats by rupturing mammalian host cells, washing away the soluble mammalian dihydrofolate reductase, and harvesting intact organisms in association with the mammalian plasma membranes. P. carinii dihydrofolate reductase, measured in the 100,000 x g supernatant from sonicated organisms, was obtained in yields ranging up to 62 IU per rat. The enzyme prepared in the presence of protease inhibitors was stable when frozen in liquid nitrogen. P. carinii dihydrofolate reductase differed from the mammalian enzyme in that the former was slightly inhibited by 150 mM KCl, whereas the latter was stimulated over twofold by 150 mM KCl. The standard assay for P. carinii dihydrofolate reductase contained 0.12 mM NADPH and 92 microM dihydrofolic acid. Under these conditions, the 50% inhibitory concentrations of the known inhibitors trimethoprim, trimetrexate, and pyrimethamine were 12 microM, 42 nM, and 3.8 microM, respectively. These standard compounds were also tested against dihydrofolate reductase from rat liver to allow an assessment of the selectivity of the drugs. Although it was the least potent, trimethoprim was the most selective. Pyrimethamine was more potent but was nonselective. Trimetrexate was extremely potent but was selective for mammalian dihydrofolate reductase. A series of experimental compounds was obtained from the National Cancer Institute and other sources through the Developmental Therapeutics Branch of the Division of AIDS at the National Institute of Allergy and Infectious Diseases. Among the first 87 compounds tested, 11 had 50% inhibitory concentrations below that of trimetrexate and 3 were more selective than trimethoprim. The most promising compounds in this original group were chemically related to methotrexate.
Topics: Animals; Anti-Infective Agents; Drug Evaluation, Preclinical; Female; Folic Acid Antagonists; Liver; Lung; Methotrexate; Pneumocystis; Pneumonia, Pneumocystis; Rats; Rats, Inbred Strains; Structure-Activity Relationship
PubMed: 1929292
DOI: 10.1128/AAC.35.7.1348 -
Proceedings of the National Academy of... Dec 1993A series of methotrexate (MTX)-resistant L1210 leukemia murine ascites tumors were developed in vivo and analyzed for drug resistance. Three of 20 tumors studied...
A series of methotrexate (MTX)-resistant L1210 leukemia murine ascites tumors were developed in vivo and analyzed for drug resistance. Three of 20 tumors studied expressed an altered dihydrofolate reductase (DHFR) and each was identical, having a C to T base transition at nucleotide 46 in the DHFR gene as demonstrated by PCR and direct sequencing. This transition results in a Gly to Trp substitution at amino acid 15 of the enzyme. Purified altered enzyme displays significantly lower binding affinity for the antifolates MTX, trimetrexate, edatrexate, and trimethoprim with respective Ki values 165-, 76-, 30-, and 28-fold higher than values obtained for enzyme isolated from parental tumor (wild-type enzyme). Substrate (dihydrofolate) and cofactor (NADPH) binding is also diminished for the mutant enzyme, although to a lesser extent (17.3- and 3.6-fold higher Km, respectively). Gly-15 is highly conserved for all vertebrate species of DHFR but has no known interaction(s), either directly or indirectly, with bound cofactor, substrate, or inhibitor. Protein molecular modeling reveals that the affected residue is 9-12 A away from the enzyme active site and located in a region analogous to the mobile Met-20 loop domain characterized for Escherichia coli DHFR.
Topics: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Chromatography, Affinity; DNA Primers; DNA, Neoplasm; Drug Resistance; Enzyme Stability; Escherichia coli; Humans; Kinetics; Leukemia L1210; Methotrexate; Mice; Models, Molecular; Molecular Sequence Data; NADP; Point Mutation; Polymerase Chain Reaction; Protein Conformation; Tetrahydrofolate Dehydrogenase; Time Factors
PubMed: 8265628
DOI: 10.1073/pnas.90.24.11797 -
Journal of Chromatography Nov 1986Trimetrexate is a potent inhibitor of dihydrofolate reductase and has demonstrated significant antitumor activity against murine and human cell lines both in vitro and...
Trimetrexate is a potent inhibitor of dihydrofolate reductase and has demonstrated significant antitumor activity against murine and human cell lines both in vitro and against several murine transplanted tumors. The importance of antifolate concentration and exposure time in determining toxic and therapeutic effects necessitates an assay of suitable sensitivity, accuracy and specificity for investigation of trimetrexate pharmacokinetics. This paper describes a gas chromatographic-mass spectrometric (GC-MS) procedure using selected-ion monitoring (SIM) for the determination of plasma trimetrexate levels. Using the C18 Bond-Elut extraction columns, the drug and internal standard are removed from plasma, derivatized to their bis(trimethylsilyl) derivatives and analysed by GC-SIM-MS. The reproducibility of the daily standard curves had coefficients of variation ranging from 4.9 to 11.4%. The precision of the assay yielded a coefficient of variation ranging from 5.6 to 10.1%, and the concentration means for the seeded control samples were found to be within -3.7 to +0.7% of the theoretical values for trimetrexate. No interfering peaks have been observed in application of the procedure on patient samples. The minimum detectable level under the conditions described was 0.005-0.014 microM trimetrexate.
Topics: Folic Acid Antagonists; Gas Chromatography-Mass Spectrometry; Humans; Kinetics; Quinazolines; Trimetrexate
PubMed: 2950125
DOI: 10.1016/s0378-4347(00)83442-8 -
Anticancer Research 2007Breast cancer patients are at increased risk of osteoporosis. Contributing factors include age and/or chemotherapy. The selective estrogen modulator, raloxifene (RAL),...
BACKGROUND
Breast cancer patients are at increased risk of osteoporosis. Contributing factors include age and/or chemotherapy. The selective estrogen modulator, raloxifene (RAL), effective in the prevention of breast cancer and approved for the treatment and prevention of osteoporosis, may prove beneficial in current breast cancer treatment modules. The purpose of this study was to evaluate RAL in combination with 5-fluorouracil (5-FU) and trimetrexate (TMX) to determine the most effective sequence in which to administer these cell cycle specific agents while taking into consideration the cellular mechanism of action. The goal was to maintain cytotoxicity to breast cancer cells and capitalize on the selective estrogen receptor modulatory effects of RAL.
MATERIALS AND METHODS
MCF-7 cells were exposed to (i) TMX, 5-FU or RAL alone, or (ii) RAL 24 h prior to 5-FU followed 2 h later by TMX, or (iii) 5-FU 2 h prior to TMX followed 24 h later by RAL. The cell viability was determined using the Quick Cell Proliferation Assay.
RESULTS
The growth rate of MCF- 7 cells exposed to early RAL was 68.25 +/- 4.11% that of the control, however, late RAL exposure produced a growth of 34.75 +/- 4.79% that of the control. Late RAL maintained the cytotoxicity of the regimen. The findings were further supported by cell flow cytometry and Western blot analysis data.
CONCLUSION
RAL given prior to 5-FU/TMX significantly compromised cytotoxicity to breast cancer cells.
Topics: Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Bone Density Conservation Agents; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Female; Fluorouracil; Humans; Raloxifene Hydrochloride; Selective Estrogen Receptor Modulators; Trimetrexate
PubMed: 17595753
DOI: No ID Found -
Molecular Microbiology Jul 2008The phenotypes of single- (SKO) and double-knockout (DKO) lines of dihydrofolate reductase-thymidylate synthase (DHFR-TS) of bloodstream Trypanosoma brucei were...
The phenotypes of single- (SKO) and double-knockout (DKO) lines of dihydrofolate reductase-thymidylate synthase (DHFR-TS) of bloodstream Trypanosoma brucei were evaluated in vitro and in vivo. Growth of SKO in vitro is identical to wild-type (WT) cells, whereas DKO has an absolute requirement for thymidine. Removal of thymidine from the medium triggers growth arrest in S phase, associated with gross morphological changes, followed by cell death after 60 h. DKO is unable to infect mice, whereas the virulence of SKO is similar to WT. Normal growth and virulence could be restored by transfection of DKO with T. brucei DHFR-TS, but not with Escherichia coli TS. As pteridine reductase (PTR1) levels are unchanged in SKO and DKO cells, PTR1 is not able to compensate for loss of DHFR activity. Drugs such as raltitrexed or methotrexate with structural similarity to folic acid are up to 300-fold more potent inhibitors of WT cultured in a novel low-folate medium, unlike hydrophobic antifols such as trimetrexate or pyrimethamine. DKO trypanosomes show reduced sensitivity to these inhibitors ranging from twofold for trimetrexate to >10 000-fold for raltitrexed. These data demonstrate that DHFR-TS is essential for parasite survival and represents a promising target for drug discovery.
Topics: Animals; Antiprotozoal Agents; Biosynthetic Pathways; Cell Death; Cell Division; Flow Cytometry; Gene Deletion; Genetic Complementation Test; Genotype; Inhibitory Concentration 50; Mice; Microscopy, Electron, Transmission; Oxidoreductases; Protozoan Proteins; Survival Analysis; Tetrahydrofolate Dehydrogenase; Thymidine; Thymidylate Synthase; Trypanosoma brucei brucei; Trypanosomiasis, African; Virulence
PubMed: 18557814
DOI: 10.1111/j.1365-2958.2008.06305.x -
Annals of Oncology : Official Journal... Jan 2002Trimetrexate (TMTX) biochemically modulates 5-fluorouracil (5-FU) and leucovorin (LCV). Two phase II trials demonstrated promising activity for TMTX/5-FU/LCV in patients... (Clinical Trial)
Clinical Trial Randomized Controlled Trial
A double-blind placebo-controlled randomized phase III trial of 5-fluorouracil and leucovorin, plus or minus trimetrexate, in previously untreated patients with advanced colorectal cancer.
BACKGROUND
Trimetrexate (TMTX) biochemically modulates 5-fluorouracil (5-FU) and leucovorin (LCV). Two phase II trials demonstrated promising activity for TMTX/5-FU/LCV in patients with untreated advanced colorectal cancer (ACC). This trial was designed to demonstrate the safety and efficacy of TMTX/5-FU/LCV as first-line treatment in ACC.
PATIENTS AND METHODS
Eligible patients with ACC were randomized in double-blind fashion to receive placebo or TMTX (110 mg/m2) intravenously (i.v.) followed 24 h later by i.v. LCV 200 mg/m2, and 5-FU 500 mg/m2 plus oral LCV rescue. Both schedules were given weekly for 6 weeks every 8 weeks. Patients were evaluated for progression-free survival (PFS), overall survival (OS), tumor response, quality of life (QoL) and toxicity.
RESULTS
A total of 382 eligible patients were randomized. Significant toxicities were noted more frequently with TMTX/5-FU/LCV. Diarrhea was the most common grade 3 or 4 side-effect (41% and 28% on the TMTX and placebo arms, respectively). QoL scores and response rates did not differ between treatment arms. PFS was 5.3 months and 4.4 months in the TMTX and placebo arms, respectively (P = 0.77; Wilcoxon). OS was 15.8 months and 16.8 months, respectively (P = 0.73; Wilcoxon).
CONCLUSIONS
The addition of TMTX to a weekly regimen of 5-FU/LCV worsened grade 3 or 4 diarrhea. The inclusion of TMTX did not yield any significant improvements in response rate, PFS or OS.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Colorectal Neoplasms; Disease Progression; Double-Blind Method; Female; Fluorouracil; Humans; Leucovorin; Male; Middle Aged; Quality of Life; Survival Rate; Time Factors; Trimetrexate
PubMed: 11863117
DOI: 10.1093/annonc/mdf043 -
Acta Crystallographica. Section D,... Dec 2010Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species. These protozoa cause serious diseases for...
High-resolution structures of Trypanosoma brucei pteridine reductase ligand complexes inform on the placement of new molecular entities in the active site of a potential drug target.
Pteridine reductase (PTR1) is a potential target for drug development against parasitic Trypanosoma and Leishmania species. These protozoa cause serious diseases for which current therapies are inadequate. High-resolution structures have been determined, using data between 1.6 and 1.1 Å resolution, of T. brucei PTR1 in complex with pemetrexed, trimetrexate, cyromazine and a 2,4-diaminopyrimidine derivative. The structures provide insight into the interactions formed by new molecular entities in the enzyme active site with ligands that represent lead compounds for structure-based inhibitor development and to support early-stage drug discovery.
Topics: Catalytic Domain; Crystallography, X-Ray; Glutamates; Guanine; Ligands; Models, Molecular; NADP; Oxidoreductases; Pemetrexed; Protein Binding; Triazines; Trimetrexate; Trypanosoma brucei brucei
PubMed: 21123874
DOI: 10.1107/S0907444910040886