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International Journal of Toxicology 2014Decitabine (5-aza-2'-deoxycytidine; DAC) in combination with tetrahydrouridine (THU) is a potential oral therapy for sickle cell disease and β-thalassemia. A study was...
Decitabine (5-aza-2'-deoxycytidine; DAC) in combination with tetrahydrouridine (THU) is a potential oral therapy for sickle cell disease and β-thalassemia. A study was conducted in mice to assess safety of this combination therapy using oral gavage of DAC and THU administered 1 hour prior to DAC on 2 consecutive days/week for up to 9 weeks followed by a 28-day recovery to support its clinical trials up to 9-week duration. Tetrahydrouridine, a competitive inhibitor of cytidine deaminase, was used in the combination to improve oral bioavailability of DAC. Doses were 167 mg/kg THU followed by 0, 0.2, 0.4, or 1.0 mg/kg DAC; THU vehicle followed by 1.0 mg/kg DAC; or vehicle alone. End points evaluated were clinical observations, body weights, food consumption, clinical pathology, gross/histopathology, bone marrow micronuclei, and toxicokinetics. There were no treatment-related effects noticed on body weight, food consumption, serum chemistry, or urinalysis parameters. Dose- and gender-dependent changes in plasma DAC levels were observed with a Cmax within 1 hour. At the 1 mg/kg dose tested, THU increased DAC plasma concentration (∼ 10-fold) as compared to DAC alone. Severe toxicity occurred in females receiving high-dose 1 mg/kg DAC + THU, requiring treatment discontinuation at week 5. Severity and incidence of microscopic findings increased in a dose-dependent fashion; findings included bone marrow hypocellularity (with corresponding hematologic changes and decreases in white blood cells, red blood cells, hemoglobin, hematocrit, reticulocytes, neutrophils, and lymphocytes), thymic/lymphoid depletion, intestinal epithelial apoptosis, and testicular degeneration. Bone marrow micronucleus analysis confirmed bone marrow cytotoxicity, suppression of erythropoiesis, and genotoxicity. Following the recovery period, a complete or trend toward resolution of these effects was observed. In conclusion, the combination therapy resulted in an increased sensitivity to DAC toxicity correlating with DAC plasma levels, and females are more sensitive compared to their male counterparts.
Topics: Animals; Antimetabolites; Antimetabolites, Antineoplastic; Azacitidine; Blood Cell Count; Body Weight; Bone Marrow Cells; Decitabine; Eating; Female; Male; Mice; Micronucleus Tests; Pharmacokinetics; Tetrahydrouridine
PubMed: 24639139
DOI: 10.1177/1091581814524994 -
Therapeutic Drug Monitoring Jun 2017Gemcitabine (2',2'-difluoro-2'-deoxycytidine) is a nucleoside analog used as a single agent and in combination regimens for the treatment of a variety of solid tumors....
BACKGROUND
Gemcitabine (2',2'-difluoro-2'-deoxycytidine) is a nucleoside analog used as a single agent and in combination regimens for the treatment of a variety of solid tumors. Several studies have shown a relationship between gemcitabine peak plasma concentration (Cmax) and hematological toxicity. An immunoassay for gemcitabine in plasma was developed and validated to facilitate therapeutic drug monitoring (TDM) by providing an economical, robust method for automated chemistry analyzers.
METHODS
A monoclonal antibody was coated on nanoparticles to develop a homogenous agglutination inhibition assay. To prevent ex vivo degradation of gemcitabine in blood, tetrahydrouridine was used as a sample stabilizer. Validation was conducted for precision, recovery, cross-reactivity, and linearity on a Beckman Coulter AU480. Verification was performed on an AU5800 in a hospital laboratory. A method comparison was performed with (LC-MS/MS) liquid chromatography tandem mass spectrometry using clinical samples. Selectivity was demonstrated by testing cross-reactivity of the major metabolite, 2',2'-difluorodeoxyuridine.
RESULTS
Coefficients of variation for repeatability and within-laboratory precision were <8%. The deviation between measured and assigned values was <3%. Linear range was from 0.40 to 33.02 μ/mL (1.5-125.5 μM). Correlation with validated LC-MS/MS methods was R = 0.977. The assay was specific for gemcitabine: there was no cross-reactivity to 2',2'-difluorodeoxyuridine, chemotherapeutics, concomitant, or common medications tested. Tetrahydrouridine was packaged in single-use syringes. Gemcitabine stability in whole blood was extended to 8 hours (at room temperature) and in plasma to 8 days (2-8°C).
CONCLUSIONS
The assay demonstrated the selectivity, test range, precision, and linearity to perform reliable measurements of gemcitabine in plasma. The addition of stabilizer improved the sample handling. Using general clinical chemistry analyzers, gemcitabine could be measured for TDM.
Topics: Antibodies, Monoclonal; Chromatography, High Pressure Liquid; Deoxycytidine; Drug Monitoring; Humans; Immunoassay; Limit of Detection; Nanoparticles; Plasma; Reproducibility of Results; Tandem Mass Spectrometry; Gemcitabine
PubMed: 28490046
DOI: 10.1097/FTD.0000000000000402 -
International Journal of Cancer Jul 2012Apoptosis genes, such as TP53 and p16/CDKN2A, that mediate responses to cytotoxic chemotherapy, are frequently nonfunctional in melanoma. Differentiation may be an...
Apoptosis genes, such as TP53 and p16/CDKN2A, that mediate responses to cytotoxic chemotherapy, are frequently nonfunctional in melanoma. Differentiation may be an alternative to apoptosis for inducing melanoma cell cycle exit. Epigenetic mechanisms regulate differentiation, and DNA methylation alterations are associated with the abnormal differentiation of melanoma cells. The effects of the deoxycytidine analogue decitabine (5-aza-2'-deoxycytidine), which depletes DNA methyl transferase 1 (DNMT1), on melanoma differentiation were examined. Treatment of human and murine melanoma cells in vitro with concentrations of decitabine that did not cause apoptosis inhibited proliferation accompanied by cellular differentiation. A decrease in promoter methylation, and increase in expression of the melanocyte late-differentiation driver SOX9, was followed by increases in cyclin-dependent kinase inhibitors (CDKN) p27/CDKN1B and p21/CDKN1A that mediate cell cycle exit with differentiation. Effects were independent of the TP53, p16/CDKN2A and also the BRAF status of the melanoma cells. Resistance, when observed, was pharmacologic, characterized by diminished ability of decitabine to deplete DNMT1. Treatment of murine melanoma models in vivo with intermittent, low-dose decitabine, administered sub-cutaneously to limit high peak drug levels that cause cytotoxicity and increase exposure time for DNMT1 depletion, and with tetrahydrouridine to decrease decitabine metabolism and further increase exposure time, inhibited tumor growth and increased molecular and tumor stromal factors implicated in melanocyte differentiation. Modification of decitabine dose, schedule and formulation for differentiation rather than cytotoxic objectives inhibits the growth of melanoma cells in vitro and in vivo.
Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Azacitidine; Base Sequence; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor Proteins; Cyclin-Dependent Kinase Inhibitor p16; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Decitabine; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Nude; Promoter Regions, Genetic; Proto-Oncogene Proteins B-raf; SOX9 Transcription Factor; Sequence Analysis, DNA; Tetrahydrouridine; Tumor Suppressor Protein p53; Up-Regulation
PubMed: 21796622
DOI: 10.1002/ijc.26320 -
PloS One 2012Tetrahydrouridine (THU) is a well characterized and potent inhibitor of cytidine deaminase (CDA). Highly expressed CDA catalyzes and inactivates cytidine analogues,...
Tetrahydrouridine (THU) is a well characterized and potent inhibitor of cytidine deaminase (CDA). Highly expressed CDA catalyzes and inactivates cytidine analogues, ultimately contributing to increased gemcitabine resistance. Therefore, a combination therapy of THU and gemcitabine is considered to be a potential and promising treatment for tumors with highly expressed CDA. In this study, we found that THU has an alternative mechanism for inhibiting cell growth which is independent of CDA expression. Three different carcinoma cell lines (MIAPaCa-2, H441, and H1299) exhibited decreased cell proliferation after sole administration of THU, while being unaffected by knocking down CDA. To investigate the mechanism of THU-induced cell growth inhibition, cell cycle analysis using flow cytometry was performed. This analysis revealed that THU caused an increased rate of G1-phase occurrence while S-phase occurrence was diminished. Similarly, Ki-67 staining further supported that THU reduces cell proliferation. We also found that THU regulates cell cycle progression at the G1/S checkpoint by suppressing E2F1. As a result, a combination regimen of THU and gemcitabine might be a more effective therapy than previously believed for pancreatic carcinoma since THU works as a CDA inhibitor, as well as an inhibitor of cell growth in some types of pancreatic carcinoma cells.
Topics: Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Cytidine Deaminase; Deoxycytidine; Enzyme Inhibitors; Humans; Lung Neoplasms; Pancreatic Neoplasms; Tetrahydrouridine; Gemcitabine
PubMed: 22616006
DOI: 10.1371/journal.pone.0037424 -
RNA (New York, N.Y.) Jul 2011Dihydrouridine (DHU) positions within tRNAs have long been used as sites to covalently attach fluorophores, by virtue of their unique chemical reactivity toward...
Dihydrouridine (DHU) positions within tRNAs have long been used as sites to covalently attach fluorophores, by virtue of their unique chemical reactivity toward reduction by NaBH(4), their abundance within prokaryotic and eukaryotic tRNAs, and the biochemical functionality of the labeled tRNAs so produced. Interpretation of experiments employing labeled tRNAs can depend on knowing the distribution of dye among the DHU positions present in a labeled tRNA. Here we combine matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS) analysis of oligonucleotide fragments and thin layer chromatography to resolve and quantify sites of DHU labeling by the fluorophores Cy3, Cy5, and proflavin in Escherichia coli tRNA(Phe) and E. coli tRNA(Arg). The MALDI-MS results led us to re-examine the precise chemistry of the reactions that result in fluorophore introduction into tRNA. We demonstrate that, in contrast to an earlier suggestion that has long been unchallenged in the literature, such introduction proceeds via a substitution reaction on tetrahydrouridine, the product of NaBH(4) reduction of DHU, resulting in formation of substituted tetrahydrocytidines within tRNA.
Topics: Cytidine; Escherichia coli; Fluorescent Dyes; Models, Biological; Organisms, Genetically Modified; RNA, Transfer; Ribonucleases; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staining and Labeling; Tissue Distribution; Uridine
PubMed: 21628433
DOI: 10.1261/rna.2670811 -
The Journal of Biological Chemistry Mar 2020Pentatricopeptide repeat (PPR) proteins with C-terminal DYW domains are present in organisms that undergo C-to-U editing of organelle RNA transcripts. PPR domains act as...
Pentatricopeptide repeat (PPR) proteins with C-terminal DYW domains are present in organisms that undergo C-to-U editing of organelle RNA transcripts. PPR domains act as specificity factors through electrostatic interactions between a pair of polar residues and the nitrogenous bases of an RNA target. DYW-deaminase domains act as the editing enzyme. Two moss () PPR proteins containing DYW-deaminase domains, PPR65 and PPR56, can convert Cs to Us in cognate, exogenous RNA targets co-expressed in We show here that purified, recombinant PPR65 exhibits robust editase activity on synthetic RNAs containing cognate, mitochondrial sequences , indicating that a PPR protein with a DYW domain is solely sufficient for catalyzing C-to-U RNA editing Monomeric fractions possessed the highest conversion efficiency, and oligomeric fractions had reduced activity. Inductively coupled plasma (ICP)-MS analysis indicated a stoichiometry of two zinc ions per highly active PPR65 monomer. Editing activity was sensitive to addition of zinc acetate or the zinc chelators 1,10--phenanthroline and EDTA. Addition of ATP or nonhydrolyzable nucleotide analogs stimulated PPR65-catalyzed RNA-editing activity on substrates, indicating potential allosteric regulation of PPR65 by ATP. Unlike for bacterial cytidine deaminase, addition of two putative transition-state analogs, zebularine and tetrahydrouridine, failed to disrupt RNA-editing activity. RNA oligonucleotides with a single incorporated zebularine also did not disrupt editing , suggesting that PPR65 cannot bind modified bases due to differences in the structure of the active site compared with other zinc-dependent nucleotide deaminases.
Topics: Adenosine Triphosphate; Biocatalysis; Bryopsida; Cytidine; Cytosine; Ions; Magnesium; Mutation; Plant Extracts; Plant Proteins; Protein Aggregates; Protein Domains; Protein Multimerization; RNA Editing; Recombinant Proteins; Repetitive Sequences, Amino Acid; Substrate Specificity; Temperature; Tetrahydrouridine; Uracil; Zea mays; Zinc
PubMed: 31996373
DOI: 10.1074/jbc.RA119.011790 -
International Journal of Radiation... Jul 1995To extend our findings in previous radiation and biochemical studies with five rodent tumors, in which we used one and occasionally two or three irradiations. The extent...
PURPOSE
To extend our findings in previous radiation and biochemical studies with five rodent tumors, in which we used one and occasionally two or three irradiations. The extent of control of the EMT-6 mammary adenocarcinoma was determined using fractionated radiation (12 irradiations) over a 3-week period using the radiosensitizer 5-chloro-2'-deoxycytidine (CldC) and biomodulators of its metabolism: N-(Phosphonacetyl)-L-aspartate (PALA), tetrahydrouridine and 5-fluoro-2'-deoxycytidine (FdC).
METHODS AND MATERIALS
Mammary adenocarcinoma EMT-6 tumors implanted 1 week prior to therapy in BALB/c mice were subjected to single daily doses of focused radiation, not exceeding a total of 60 Gy, on days 2-5 of each week. N-(Phosphonacetyl)-L-aspartate (PALA) was administered on the first day of therapy. Five-fluoro-2'-deoxycytidine and CldC were administered in the morning and afternoon, respectively, of the next 2 days, and CldC was administered on the fourth day. Tetrahydrouridine was always coadministered with FdC or CldC. Drug and radiation treatments overlapped for 3 weeks.
RESULTS
Fifty to 80% cures (usually 70%) were obtained with no apparent morbidity and the same moderate weight loss that occurs with radiation alone. Neither tumor regrowth delay nor cures were obtained with drugs or radiation alone. An apparent threefold dose increase effect was obtained with the end point: "days to reach 4 times initial tumor volume." Increasing the radiation dose threefold (without drugs) resulted in four out of five deaths; increasing the dose twofold (without drugs) resulted in extensive weight loss and hair loss in the entire ventral area and no cures. Increasing the dose of drugs or radiation 1.5-fold, in the complete protocol, did not result in increased morbidity. Comparative studies with Iododeoxyuridine demonstrate the heightened efficacy of CldC.
CONCLUSIONS
One cannot achieve the same results obtained with CldC and the modulators by merely increasing the dose of radiation. There is a significant window of safety in this approach. The evidence we have obtained with EMT-6, the fifth rodent tumor we have studied with CldC, as well as the demonstrated and proposed reasons for its superior efficacy over 5-Iododeoxyuridine (and 5-Bromodeoxyuridine), drugs in current use, indicate that CldC will allow more aggressive treatment of human tumors with radiation than is now feasible.
Topics: Adenocarcinoma; Animals; Aspartic Acid; Body Weight; Deoxycytidine; Idoxuridine; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Phosphonoacetic Acid; Radiation-Sensitizing Agents; Radiotherapy Dosage; Tetrahydrouridine
PubMed: 7607927
DOI: 10.1016/0360-3016(94)00596-d -
Proceedings of the National Academy of... Nov 19825-Fluoro-2'-deoxycytidine (FdCyd) is a potent inhibitor of growth of tissue culture cells. The major cytotoxic event appears to be inhibition of thymidylate synthetase...
5-Fluoro-2'-deoxycytidine (FdCyd) is a potent inhibitor of growth of tissue culture cells. The major cytotoxic event appears to be inhibition of thymidylate synthetase as evidenced by reversal of the cytotoxicity with thymidine but not deoxycytidine and by the effect of FdCyd on nucleotide pools, which is characteristic of specific inhibition of this enzyme. The metabolism of FdCyd was established by using a method in which its cytotoxicity was compared in several S-49 mutant cell lines having defined single or double deficiencies of enzymes involved in nucleoside and nucleotide metabolism. Our results indicate that FdCyd is metabolized to 5-fluoro-2'-deoxyuridylate, a potent inhibitor of thymidylate synthetase by two pathways: (i) sequential reactions catalyzed by deoxycytidine kinase and deoxycytidylate deaminase and (ii) sequential reactions catalyzed by cytidine deaminase and thymidine kinase. We have shown that metabolism of FdCyd can be directed through the former pathway by inhibition of cytidine deaminase with tetrahydrouridine. Since cytidine deaminase appears to be responsible for catabolism of FdCyd in animals, our results suggest that the antineoplastic effects of FdCyd should be examined in combination with inhibitors of cytidine deaminase.
Topics: Animals; Antimetabolites, Antineoplastic; Cell Line; Deoxycytidine; Dose-Response Relationship, Drug; Fluorodeoxyuridylate; Lymphoma; Mice; Neoplasms, Experimental; Structure-Activity Relationship; Thymidylate Synthase
PubMed: 6959127
DOI: 10.1073/pnas.79.21.6419 -
FEBS Open Bio 2015Mycoplasmas may colonize tumor tissue in patients. The cytostatic activity of gemcitabine was dramatically decreased in Mycoplasma hyorhinis-infected tumor cell cultures...
Mycoplasmas may colonize tumor tissue in patients. The cytostatic activity of gemcitabine was dramatically decreased in Mycoplasma hyorhinis-infected tumor cell cultures compared with non-infected tumor cell cultures. This mycoplasma-driven drug deamination could be prevented by exogenous administration of the cytidine deaminase (CDA) inhibitor tetrahydrouridine, but also by the natural nucleosides or by a purine nucleoside phosphorylase inhibitor. The M. hyorhinis-encoded CDAHyor gene was cloned, expressed as a recombinant protein and purified. CDAHyor was found to be more catalytically active than its human equivalent and efficiently deaminates (inactivates) cytosine-based anticancer drugs. CDAHyor expression at the tumor site may result in selective drug inactivation and suboptimal therapeutic efficiency.
PubMed: 26322268
DOI: 10.1016/j.fob.2015.07.007 -
American Journal of Cancer Research 2020DNA methyltransferase 1 (DNMT1) is scientifically validated as a molecular target to treat chemo-resistant pancreatic ductal adenocarcinoma (PDAC). Results of clinical...
DNA methyltransferase 1 (DNMT1) is scientifically validated as a molecular target to treat chemo-resistant pancreatic ductal adenocarcinoma (PDAC). Results of clinical studies of the pyrimidine nucleoside analog decitabine to target DNMT1 in PDAC have, however, disappointed. One reason is high expression in PDAC of the enzyme cytidine deaminase (CDA), which catabolizes decitabine within minutes. We therefore added tetrahydrouridine (THU) to inhibit CDA with decitabine. In this pilot clinical trial, patients with advanced chemorefractory PDAC ingested oral THU ~10 mg/kg/day combined with oral decitabine ~0.2 mg/kg/day, for 5 consecutive days, then 2X/week. We treated 13 patients with extensively metastatic chemo-resistant PDAC, including 8 patients (62%) with ascites: all had received ≥ 1 prior therapies including gemcitabine/nab-paclitaxel in 9 (69%) and FOLFIRINOX in 12 (92%). Median time on THU/decitabine treatment was 35 days (range 4-63). The most frequent treatment-attributable adverse event was anemia (n=5). No deaths were attributed to THU/decitabine. Five patients had clinical progressive disease (PD) prior to week 8. Eight patients had week 8 evaluation scans: 1 had stable disease and 7 PD. Median overall survival was 3.1 months. Decitabine systemic exposure is expected to decrease neutrophil counts; however, neutropenia was unexpectedly mild. To identify reasons for limited systemic decitabine effect, we measured plasma CDA enzyme activity in PDAC patients, and found a > 10-fold increase in those with metastatic resectable PDAC. We concluded that CDA activity is increased not just locally but also systemically in metastatic PDAC, suggesting a need for even higher CDA-inhibitor doses than used here.
PubMed: 33042633
DOI: No ID Found