-
British Journal of Pharmacology Oct 2009The metabolism and efficacy of 5-fluorouracil (FUra) and other fluorinated pyrimidine (FP) derivatives have been intensively investigated for over fifty years. FUra and... (Review)
Review
The metabolism and efficacy of 5-fluorouracil (FUra) and other fluorinated pyrimidine (FP) derivatives have been intensively investigated for over fifty years. FUra and its antimetabolites can be incorporated at RNA- and DNA-levels, with RNA level incorporation provoking toxic responses in human normal tissue, and DNA-level antimetabolite formation and incorporation believed primarily responsible for tumour-selective responses. Attempts to direct FUra into DNA-level antimetabolites, based on mechanism-of-action studies, have led to gradual improvements in tumour therapy. These include the use of leukovorin to stabilize the inhibitory thymidylate synthase-5-fluoro-2'-deoxyuridine 5' monophoshate (FdUMP)-5,10-methylene tetrahydrofolate (5,10-CH(2)FH(4)) trimeric complex. FUra incorporated into DNA also contributes to antitumour activity in preclinical and clinical studies. This review examines our current state of knowledge regarding the mechanistic aspects of FUra:Gua lesion detection by DNA mismatch repair (MMR) machinery that ultimately results in lethality. MMR-dependent direct cell death signalling or futile cycle responses will be discussed. As 10-30% of sporadic colon and endometrial tumours display MMR defects as a result of human MutL homologue-1 (hMLH1) promoter hypermethylation, we discuss the use and manipulation of the hypomethylating agent, 5-fluorodeoxycytidine (FdCyd), and our ability to manipulate its metabolism using the cytidine or deoxycytidylate (dCMP) deaminase inhibitors, tetrahydrouridine or deoxytetrahydrouridine, respectively, as a method for re-expression of hMLH1 and re-sensitization of tumours to FP therapy.
Topics: Antimetabolites, Antineoplastic; Cell Line, Tumor; DNA Mismatch Repair; Fluorouracil; Humans; Models, Biological; Neoplasms; Signal Transduction
PubMed: 19775280
DOI: 10.1111/j.1476-5381.2009.00423.x -
Asian Journal of Pharmaceutical Sciences Sep 2017A simple and rapid UPLC-MS/MS method to simultaneously determine gemcitabine and its L-carnitine ester derivative (2'-deoxy-2', 2'-difluoro-N-((4-amino-4-oxobutanoyl)...
Development and validation of a UPLC-MS/MS assay for the determination of gemcitabine and its L-carnitine ester derivative in rat plasma and its application in oral pharmacokinetics.
A simple and rapid UPLC-MS/MS method to simultaneously determine gemcitabine and its L-carnitine ester derivative (2'-deoxy-2', 2'-difluoro-N-((4-amino-4-oxobutanoyl) oxy)-4-(trimethyl amm-onio) butanoate-cytidine, JDR) in rat plasma was developed and validated. The conventional plasma sample preparation method of nucleoside analogues is solid-phase extraction (SPE) which is time-consuming and cost-expensive. In this study, gradient elution with small particles size solid phase was applied to effectively separate gemcitabine and JDR, and protein precipitation pretreatment was adopted to remove plasma protein and extract the analytes with high recovery(>81%). Method validation was performed as per the FDA guidelines, and the standard curves were found to be linear in the range of 5-4000 ng/ml for JDR and 4-4000 ng/ml for gemcitabine, respectively. The lower limit of quantitation (LLOQ) of gemcitabine and JDR was 4 and 5 ng/ml, respectively. The intra-day and inter-day precision and accuracy results were within the acceptable limits. Finally, the developed method was successfully applied to investigate the pharmacokinetic studies of JDR and gemcitabine after oral administration to rats.
PubMed: 32104361
DOI: 10.1016/j.ajps.2017.01.001 -
Cancer Chemotherapy and Pharmacology Aug 2008Cytidine analogues such as cytosine arabinoside, gemcitabine, decitabine, 5-azacytidine, 5-fluoro-2'-deoxycytidine and 5-chloro-2'-deoxycytidine undergo rapid catabolism...
Cytidine analogues such as cytosine arabinoside, gemcitabine, decitabine, 5-azacytidine, 5-fluoro-2'-deoxycytidine and 5-chloro-2'-deoxycytidine undergo rapid catabolism by cytidine deaminase (CD). 3,4,5,6-tetrahydrouridine (THU) is a potent CD inhibitor that has been applied preclinically and clinically as a modulator of cytidine analogue metabolism. However, THU pharmacokinetics has not been fully characterized, which has impaired the optimal preclinical evaluation and clinical use of THU. Therefore, we characterized the THU pharmacokinetics and bioavailability in mice. Mice were dosed with THU iv (100 mg/kg) or po (30, 100, or 300 mg/kg). Plasma and urine THU concentrations were quantitated with a validated LC-MS/MS assay. Plasma pharmacokinetic parameters were calculated compartmentally and non-compartmentally. THU, at 100 mg/kg iv had a 73 min terminal half-life and produced plasma THU concentrations >1 microg/ml, the concentration shown to effectively block deamination, for 4 h. Clearance was 9.1 ml/min/kg, and the distribution volume was 0.95 l/kg. Renal excretion accounted for 36-55% of the THU dose. A three-compartment model fit the iv THU data best. THU, at 100 mg/kg po, produced a concentration versus time profile with a plateau of approximately 10 mug/ml from 0.5-3 h, followed by a decline with an 85 min half-life. The oral bioavailability of THU was approximately 20%. The 20% oral bioavailability of THU is sufficient to produce and sustain, for several hours, plasma concentrations that inhibit CD. This suggests the feasibility of using THU to decrease elimination and first-pass metabolism of cytidine analogues by CD. THU pharmacokinetics are now being evaluated in humans.
Topics: Administration, Oral; Animals; Biological Availability; Chromatography, Liquid; Cytidine Deaminase; Enzyme Inhibitors; Half-Life; Injections, Intravenous; Male; Mice; Mice, Inbred Strains; Protein Binding; Tandem Mass Spectrometry; Tetrahydrouridine
PubMed: 18008070
DOI: 10.1007/s00280-007-0625-2 -
The Journal of Clinical Investigation Apr 2024Cell and antibody therapies directed against surface molecules on B cells, e.g., CD19-targeting chimeric antigen receptor T cells (CD19 CAR-T), are now standard for...
Cell and antibody therapies directed against surface molecules on B cells, e.g., CD19-targeting chimeric antigen receptor T cells (CD19 CAR-T), are now standard for patients with chemorefractory B cell acute lymphoblastic leukemias and other B cell malignancies. However, early relapse rates remain high. In this issue of the JCI, Aminov, Giricz, and colleagues revealed that leukemia cells resisting CD19-targeted therapy had reduced CD19 but also low CD22 expression and were sensitive to Bruton's tyrosine kinase and/or MEK inhibition. Overall, their observations support the evolution of resistance following a Lamarckian model: the oncotherapy directly elicits adaptive, resistance-conferring reconfigurations, which are then inherited by daughter cells as epigenetic changes. The findings prompt reflection also on the broader role of epigenetics in decoupling of replication from lineage differentiation activation by the B cell lineage master transcription factor hub. Such oncogenesis and resistance mechanisms, being predictable and epigenetic, offer practical opportunities for intervention, potentially non-cross-resistant and safe vis-à-vis present cytotoxic and CAR-T treatments.
Topics: Humans; Receptors, Chimeric Antigen; Adaptor Proteins, Signal Transducing; Agammaglobulinaemia Tyrosine Kinase; Antigens, CD19; B-Lymphocytes
PubMed: 38618954
DOI: 10.1172/JCI179788 -
Annals of Neurology May 2017Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the...
OBJECTIVE
Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the nuclear gene, TK2, cause TK2 deficiency, which manifests predominantly in children as myopathy with mtDNA depletion. Molecular bypass therapy with the TK2 products, deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP), prolongs the life span of Tk2-deficient (Tk2 ) mice by 2- to 3-fold. Because we observed rapid catabolism of the deoxynucleoside monophosphates to deoxythymidine (dT) and deoxycytidine (dC), we hypothesized that: (1) deoxynucleosides might be the major active agents and (2) inhibition of deoxycytidine deamination might enhance dTMP+dCMP therapy.
METHODS
To test these hypotheses, we assessed two therapies in Tk2 mice: (1) dT+dC and (2) coadministration of the deaminase inhibitor, tetrahydrouridine (THU), with dTMP+dCMP.
RESULTS
We observed that dC+dT delayed disease onset, prolonged life span of Tk2-deficient mice and restored mtDNA copy number as well as respiratory chain enzyme activities and levels. In contrast, dCMP+dTMP+THU therapy decreased life span of Tk2 animals compared to dCMP+dTMP.
INTERPRETATION
Our studies demonstrate that deoxynucleoside substrate enhancement is a novel therapy, which may ameliorate TK2 deficiency in patients. Ann Neurol 2017;81:641-652.
Topics: Animals; Antimetabolites; DNA, Mitochondrial; Deoxycytidine Monophosphate; Disease Models, Animal; Drug Therapy, Combination; Metabolism, Inborn Errors; Mice; Mice, Transgenic; Mitochondrial Diseases; Tetrahydrouridine; Thymidine; Thymidine Kinase
PubMed: 28318037
DOI: 10.1002/ana.24922 -
Drug Metabolism and Disposition: the... Mar 2020Cytidine deaminase (CDA) is a determinant of in vivo gemcitabine elimination kinetics and cellular toxicity. The impact of CDA activity in pancreatic ductal...
Cytidine deaminase (CDA) is a determinant of in vivo gemcitabine elimination kinetics and cellular toxicity. The impact of CDA activity in pancreatic ductal adenocarcinoma (PDAC) cell lines has not been elucidated. We hypothesized that CDA regulates gemcitabine flux through its inactivation and activation pathways in PDAC cell lines. Three PDAC cell lines (BxPC-3, MIA PaCa-2, and PANC-1) were incubated with 10 or 100 µM gemcitabine for 60 minutes or 24 hours, with or without tetrahydrouridine, a CDA inhibitor. Extracellular inactive gemcitabine metabolite (dFdU) and intracellular active metabolite (dFdCTP) were quantified with liquid chromatography tandem mass spectrometry. Cellular expression of CDA was assessed with real-time PCR and Western blot. Gemcitabine conversion to dFdU was extensive in BxPC-3 and low in MIA PaCa-2 and PANC-1, in accordance with their respective CDA expression levels. CDA inhibition was associated with low or undetectable dFdU in all three cell lines. After 24 hours gemcitabine incubation, dFdCTP was highest in MIA PaCa-2 and lowest in BxPC-3. CDA inhibition resulted in a profound dFdCTP increase in BxPC-3 but not in MIA PaCa-2 or PANC-1. dFdCTP concentrations were not higher after exposure to 100 versus 10 µM gemcitabine when CDA activities were low (MIA PaCa-2 and PANC-1) or inhibited (BxPC-3). The results suggest a regulatory role of CDA for gemcitabine activation in PDAC cells but within limits related to the capacity in the activation pathway in the cell lines. SIGNIFICANCE STATEMENT: The importance of cytidine deaminase (CDA) for cellular gemcitabine toxicity, linking a lower activity to higher toxicity, is well described. An underlying assumption is that CDA, by inactivating gemcitabine, limits the amount available for the intracellular activation pathway. Our study is the first to illustrate this regulatory role of CDA in pancreatic ductal adenocarcinoma cell lines by quantifying intracellular and extracellular gemcitabine metabolite concentrations.
Topics: Adenocarcinoma; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cytidine Deaminase; Deoxycytidine; Humans; Pancreatic Neoplasms; Gemcitabine
PubMed: 31871136
DOI: 10.1124/dmd.119.089334 -
Cancer Chemotherapy and Pharmacology May 2020Following promising responses to the DNA methyltransferase (DNMT) inhibitor 5-fluoro-2'-deoxycytidine (FdCyd) combined with tetrahydrouridine (THU) in phase 1 testing,...
Intravenous 5-fluoro-2'-deoxycytidine administered with tetrahydrouridine increases the proportion of p16-expressing circulating tumor cells in patients with advanced solid tumors.
PURPOSE
Following promising responses to the DNA methyltransferase (DNMT) inhibitor 5-fluoro-2'-deoxycytidine (FdCyd) combined with tetrahydrouridine (THU) in phase 1 testing, we initiated a non-randomized phase 2 study to assess response to this combination in patients with advanced solid tumor types for which tumor suppressor gene methylation is potentially prognostic. To obtain pharmacodynamic evidence for DNMT inhibition by FdCyd, we developed a novel method for detecting expression of tumor suppressor protein p16/INK4A in circulating tumor cells (CTCs).
METHODS
Patients in histology-specific strata (breast, head and neck [H&N], or non-small cell lung cancers [NSCLC] or urothelial transitional cell carcinoma) were administered FdCyd (100 mg/m) and THU (350 mg/m) intravenously 5 days/week for 2 weeks, in 28-day cycles, and progression-free survival (PFS) rate and objective response rate (ORR) were evaluated. Blood specimens were collected for CTC analysis.
RESULTS
Ninety-three eligible patients were enrolled (29 breast, 21 H&N, 25 NSCLC, and 18 urothelial). There were three partial responses. All strata were terminated early due to insufficient responses (H&N, NSCLC) or slow accrual (breast, urothelial). However, the preliminary 4-month PFS rate (42%) in the urothelial stratum exceeded the predefined goal-though the ORR (5.6%) did not. An increase in the proportion of p16-expressing cytokeratin-positive CTCs was detected in 69% of patients evaluable for clinical and CTC response, but was not significantly associated with clinical response.
CONCLUSION
Further study of FdCyd + THU is potentially warranted in urothelial carcinoma but not NSCLC or breast or H&N cancer. Increase in the proportion of p16-expressing cytokeratin-positive CTCs is a pharmacodynamic marker of FdCyd target engagement.
Topics: Administration, Intravenous; Antineoplastic Agents; Carcinoma, Transitional Cell; Cell Count; Cyclin-Dependent Kinase Inhibitor p16; DNA (Cytosine-5-)-Methyltransferase 1; Deoxycytidine; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Middle Aged; Neoplastic Cells, Circulating; Pharmacogenetics; Tetrahydrouridine; Urologic Neoplasms
PubMed: 32314030
DOI: 10.1007/s00280-020-04073-5 -
Cancer Chemotherapy and Pharmacology Mar 2015Inhibitors of DNA (cytosine-5)-methyltransferases (DNMT) are active antineoplastic agents. We conducted the first-in-human phase I trial of 5-fluoro-2'-deoxycytidine...
PURPOSE
Inhibitors of DNA (cytosine-5)-methyltransferases (DNMT) are active antineoplastic agents. We conducted the first-in-human phase I trial of 5-fluoro-2'-deoxycytidine (FdCyd), a DNMT inhibitor stable in aqueous solution, in patients with advanced solid tumors. Objectives were to establish the safety, maximum tolerated dose (MTD), pharmacokinetics, and pharmacodynamics of FdCyd + tetrahydrouridine (THU).
METHODS
FdCyd + THU were administered by 3 h IV infusion on days 1-5 every 3 weeks, or days 1-5 and 8-12 every 4 weeks. FdCyd was administered IV with a fixed 350 mg/m(2)/day dose of THU to inhibit deamination of FdCyd. Pharmacokinetics of FdCyd, downstream metabolites and THU were assessed by LC-MS/MS. RBC γ-globin expression was evaluated as a pharmacodynamics biomarker.
RESULTS
Patients were enrolled on the 3-week schedule at doses up to 80 mg/m(2)/day without dose-limiting toxicity (DLT) prior to transitioning to the 4-week schedule, which resulted in an MTD of 134 mg/m(2)/day; one of six patients had a first-cycle DLT (grade 3 colitis). FdCyd ≥40 mg/m(2)/day produced peak plasma concentrations >1 µM. Although there was inter-patient variability, γ-globin mRNA increased during the first two treatment cycles. One refractory breast cancer patient experienced a partial response (PR) of >90 % decrease in tumor size, lasting over a year.
CONCLUSIONS
The MTD was established at 134 mg/m(2) FdCyd + 350 mg/m(2) THU days 1-5 and 8-12 every 4 weeks. Based on toxicities observed over multiple cycles, good plasma exposures, and the sustained PR observed at 67 mg/m(2)/day, the phase II dose for our ongoing multi-histology trial is 100 mg/m(2)/day FdCyd with 350 mg/m(2)/day THU.
Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Chromatography, Liquid; DNA (Cytosine-5-)-Methyltransferases; Deoxycytidine; Dose-Response Relationship, Drug; Female; Humans; Infusions, Intravenous; Male; Maximum Tolerated Dose; Middle Aged; Neoplasms; Tandem Mass Spectrometry; Tetrahydrouridine; Treatment Outcome; gamma-Globulins
PubMed: 25567350
DOI: 10.1007/s00280-014-2674-7 -
Leukemia Apr 2021Mechanisms-of-resistance to decitabine and 5-azacytidine, mainstay treatments for myeloid malignancies, require investigation and countermeasures. Both are nucleoside...
Mechanisms-of-resistance to decitabine and 5-azacytidine, mainstay treatments for myeloid malignancies, require investigation and countermeasures. Both are nucleoside analog pro-drugs processed by pyrimidine metabolism into a deoxynucleotide analog that depletes the key epigenetic regulator DNA methyltranseferase 1 (DNMT1). Here, upon serial analyses of DNMT1 levels in patients' bone marrows on-therapy, we found DNMT1 was not depleted at relapse. Showing why, bone marrows at relapse exhibited shifts in expression of key pyrimidine metabolism enzymes in directions adverse to pro-drug activation. Further investigation revealed the origin of these shifts. Pyrimidine metabolism is a network that senses and regulates deoxynucleotide amounts. Deoxynucleotide amounts were disturbed by single exposures to decitabine or 5-azacytidine, via off-target depletion of thymidylate synthase and ribonucleotide reductase respectively. Compensating pyrimidine metabolism shifts peaked 72-96 h later. Continuous pro-drug exposures stabilized these adaptive metabolic responses to thereby prevent DNMT1-depletion and permit exponential leukemia out-growth as soon as day 40. The consistency of the acute metabolic responses enabled exploitation: simple treatment modifications in xenotransplant models of chemorefractory leukemia extended noncytotoxic DNMT1-depletion and leukemia control by several months. In sum, resistance to decitabine and 5-azacytidine originates from adaptive responses of the pyrimidine metabolism network; these responses can be anticipated and thus exploited.
Topics: Animals; Antimetabolites, Antineoplastic; Azacitidine; Cell Line, Tumor; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; Decitabine; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Humans; Metabolic Networks and Pathways; Mice; Pyrimidines; Uridine Kinase
PubMed: 32770088
DOI: 10.1038/s41375-020-1003-x -
Journal of Pharmaceutical and... Sep 2016The hypomethylating agent 5-fluoro-2'-deoxycytidine (FdCyd, NSC 48006) is being evaluated clinically both via the intravenous route and via the oral route in combination...
The hypomethylating agent 5-fluoro-2'-deoxycytidine (FdCyd, NSC 48006) is being evaluated clinically both via the intravenous route and via the oral route in combination with 3,4,5,6-tetrahydrouridine (THU), a potent inhibitor of FdCyd catabolism. To determine the pharmacokinetics of FdCyd and downstream metabolites, we developed and validated an LC-MS/MS assay for the quantitation of FdCyd, 5-fluoro-2'-deoxyuridine (FdUrd), and 5-fluorouracil (FU) in 0.2mL human plasma. After acetonitrile protein precipitation, the sample was split and separate chromatography was achieved for FdCyd with a Synergi Polar-RP column and for FdUrd and FU with a Shodex Asahipak NH2P-50 2D column. Gradients of 0.1% acetic acid in acetonitrile and water were used. Detection with a Quattromicro quadrupole mass spectrometer with electrospray ionization in positive-ion (FdCyd) or negative-ion (FdUrd and FU) multiple reaction monitoring (MRM) mode. The assay was linear from 5 to 3000ng/mL for all three analytes and proved to be accurate (96.7-105.5%) and precise (<8.1%CV), and fulfilled FDA criteria for bioanalytical method validation. We demonstrated the suitability of this assay for measuring FdCyd and metabolites FdUrd and FU in plasma from a patient who was administered 120mg PO FdCyd 30min after 3000mg THU. Our LC-MS/MS assay will be an essential tool to further define the pharmacology of FdCyd in ongoing and future studies.
Topics: Biological Assay; Chromatography, Liquid; Deoxyuridine; Fluorouracil; Humans; Plasma; Tandem Mass Spectrometry; Tetrahydrouridine
PubMed: 27454087
DOI: 10.1016/j.jpba.2016.07.027