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1.
J Vet Intern Med ; 37(6): 2429-2442, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37670479

RESUMO

BACKGROUND: Cytosine arabinoside (Ara-C) is a nucleoside analog prodrug utilized for immunomodulatory effects mediated by its active metabolite Ara-CTP. Optimal dosing protocols for immunomodulation in dogs have not been defined. Cytarabine ocfosfate (CO) is a lipophilic prodrug of Ara-C that can be administered PO and provides prolonged serum concentrations of Ara-C. OBJECTIVES: Provide pharmacokinetic data for orally administered CO and determine accumulation and functional consequences of Ara-CTP within peripheral blood leukocytes. ANIMALS: Three healthy female hound dogs and 1 healthy male Beagle. METHODS: Prospective study. Dogs received 200 mg/m2 of CO PO q24h for 7 doses. Serum and cerebrospinal fluid (CSF) CO and Ara-C concentrations were measured by liquid chromatography-tandem mass spectroscopy (LC-MS/MS). Complete blood counts, flow cytometry, and leukocyte activation assays were done up to 21 days. Incorporation of Ara-CTP within leukocyte DNA was determined by LC-MS/MS. RESULTS: Maximum serum concentration (Cmax ) for Ara-C was 456.1-724.0 ng/mL (1.88-2.98 µM) and terminal half-life was 23.3 to 29.4 hours. Cerebrospinal fluid: serum Ara-C ratios ranged from 0.54 to 1.2. Peripheral blood lymphocyte concentrations remained within the reference range, but proliferation rates poststimulation were decreased at 6 days. Incorporation of Ara-CTP was not saturated and remained >25% of peak concentration at 13 days. CONCLUSIONS AND CLINICAL IMPORTANCE: Oral CO may produce prolonged serum Ara-C half-lives at concentrations sufficient to induce functional changes in peripheral leukocytes and is associated with prolonged retention of DNA-incorporated Ara-CTP. Application of functional and active metabolite assessment is feasible and may provide more relevant data to determine optimal dosing regimens for Ara-C-based treatments.


Assuntos
Arabinofuranosilcitosina Trifosfato , Pró-Fármacos , Feminino , Masculino , Cães , Animais , Cromatografia Líquida/veterinária , Estudos Prospectivos , Espectrometria de Massas em Tandem/veterinária , Leucócitos , Biomarcadores , Citarabina , DNA
2.
Blood Adv ; 7(11): 2538-2550, 2023 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-36689724

RESUMO

Cytarabine arabinoside (Ara-C) has been the cornerstone of acute myeloid leukemia (AML) chemotherapy for decades. After cellular uptake, it is phosphorylated into its active triphosphate form (Ara-CTP), which primarily exerts its cytotoxic effects by inhibiting DNA synthesis in proliferating cells. Interpatient variation in the enzymes involved in the Ara-C metabolic pathway has been shown to affect intracellular abundance of Ara-CTP and, thus, its therapeutic benefit. Recently, SAMHD1 (SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1) has emerged to play a role in Ara-CTP inactivation, development of drug resistance, and, consequently, clinical response in AML. Despite this, the impact of genetic variations in SAMHD1 on outcome in AML has not been investigated in depth. In this study, we evaluated 25 single nucleotide polymorphisms (SNPs) within the SAMHD1 gene for association with clinical outcome in 400 pediatric patients with newly diagnosed AML from 2 clinical trials, AML02 and AML08. Three SNPs, rs1291128, rs1291141, and rs7265241 located in the 3' region of SAMHD1 were significantly associated with at least 1 clinical outcome: minimal residual disease after induction I, event-free survival (EFS), or overall survival (OS) in the 2 cohorts. In an independent cohort of patients from the COG-AAML1031 trial (n = 854), rs7265241 A>G remained significantly associated with EFS and OS. In multivariable analysis, all the SNPs remained independent predictors of clinical outcome. These results highlight the relevance of the SAMHD1 pharmacogenomics in context of response to Ara-C in AML and warrants the need for further validation in expanded patient cohorts.


Assuntos
Leucemia Mieloide Aguda , Proteína 1 com Domínio SAM e Domínio HD , Criança , Humanos , Arabinofuranosilcitosina Trifosfato/metabolismo , Arabinofuranosilcitosina Trifosfato/uso terapêutico , Citarabina/uso terapêutico , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Polimorfismo de Nucleotídeo Único , Proteína 1 com Domínio SAM e Domínio HD/genética
3.
J Intern Med ; 292(6): 925-940, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35934913

RESUMO

BACKGROUND: Treatment of newly diagnosed acute myeloid leukaemia (AML) is based on combination chemotherapy with cytarabine (ara-C) and anthracyclines. Five-year overall survival is below 30%, which has partly been attributed to cytarabine resistance. Preclinical data suggest that the addition of hydroxyurea potentiates cytarabine efficacy by increasing ara-C triphosphate (ara-CTP) levels through targeted inhibition of SAMHD1. OBJECTIVES: In this phase 1 trial, we evaluated the feasibility, safety and efficacy of the addition of hydroxyurea to standard chemotherapy with cytarabine/daunorubicin in newly diagnosed AML patients. METHODS: Nine patients were enrolled and received at least two courses of ara-C (1 g/m2 /2 h b.i.d. d1-5, i.e., a total of 10 g/m2 per course), hydroxyurea (1-2 g d1-5) and daunorubicin (60 mg/m2 d1-3). The primary endpoint was safety; secondary endpoints were complete remission rate and measurable residual disease (MRD). Additionally, pharmacokinetic studies of ara-CTP and ex vivo drug sensitivity assays were performed. RESULTS: The most common grade 3-4 toxicity was febrile neutropenia (100%). No unexpected toxicities were observed. Pharmacokinetic analyses showed a significant increase in median ara-CTP levels (1.5-fold; p = 0.04) in patients receiving doses of 1 g hydroxyurea. Ex vivo, diagnostic leukaemic bone marrow blasts from study patients were significantly sensitised to ara-C by a median factor of 2.1 (p = 0.0047). All nine patients (100%) achieved complete remission, and all eight (100%) with validated MRD measurements (flow cytometry or real-time quantitative polymerase chain reaction [RT-qPCR]) had an MRD level <0.1% after two cycles of chemotherapy. Treatment was well-tolerated, and median time to neutrophil recovery >1.0 × 109 /L and to platelet recovery >50 × 109 /L after the start of cycle 1 was 19 days and 22 days, respectively. Six of nine patients underwent allogeneic haematopoietic stem-cell transplantation (allo-HSCT). With a median follow-up of 18.0 (range 14.9-20.5) months, one patient with adverse risk not fit for HSCT experienced a relapse after 11.9 months but is now in second complete remission. CONCLUSION: Targeted inhibition of SAMHD1 by the addition of hydroxyurea to conventional AML therapy is safe and appears efficacious within the limitations of the small phase 1 patient cohort. These results need to be corroborated in a larger study.


Assuntos
Citarabina , Leucemia Mieloide Aguda , Humanos , Citarabina/uso terapêutico , Citarabina/farmacologia , Hidroxiureia/uso terapêutico , Arabinofuranosilcitosina Trifosfato/uso terapêutico , Proteína 1 com Domínio SAM e Domínio HD , Temperatura Alta , Protocolos de Quimioterapia Combinada Antineoplásica , Recidiva Local de Neoplasia , Leucemia Mieloide Aguda/tratamento farmacológico , Daunorrubicina/uso terapêutico
4.
J Pharm Biomed Anal ; 211: 114582, 2022 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-35101802

RESUMO

In this study, a sensitive and rapid ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous analysis of cytarabine (ara-C), cytarabine monophosphate (ara-CMP), cytarabine diphosphate (ara-CDP) and cytarabine triphosphate (ara-CTP) in the cytosol and nucleus. The separation of analytes and endogenous interferents was achieved in 8 min on a hypercarb column (2.1 mm × 100 mm, 3 µm) by using a gradient elution with 95% acetonitrile and aqueous 5 mM hexylamine with 0.4% (v/v) diethylamine adjusted to pH 10. The analytes were detected with both negative and positive electrospray ionization in multiple reaction monitoring (MRM) mode. The calibration curve demonstrated good linearity ranging from 5 to 750 nM for ara-C, 50-7500 nM for ara-CMP, 20-3000 nM for ara-CDP and 1-150 nM for ara-CTP in the cytosol. In the nucleus, good linearity was achieved over a concentration range of 1-100 nM for ara-C, 5-500 nM for ara-CMP, 2.5-250 nM for ara-CDP and 0.5-50 nM for ara-CTP. Intra- and interbatch accuracies and precisions met the standards of validation. The matrix effect, recovery and stability were also within acceptable ranges. After incubation with 10 µM ara-C for 3 h, the levels of ara-C, ara-CMP, ara-CDP and ara-CTP in the cytosol and nucleus of HL-60 cells and HL-60/ara-C cells were determined. Most of the metabolites were found within the quantitation range. The results showed that the nuclear ara-CTP level was significantly different than the intracellular ara-CTP level between HL-60 and HL-60/ara-C cells.


Assuntos
Arabinofuranosilcitosina Trifosfato , Citarabina , Arabinofuranosilcitosina Trifosfato/análise , Arabinofuranosilcitosina Trifosfato/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Citosol/metabolismo , Difosfatos , Humanos , Espectrometria de Massas em Tandem
5.
EMBO Mol Med ; 12(3): e10419, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-31950591

RESUMO

The deoxycytidine analogue cytarabine (ara-C) remains the backbone treatment of acute myeloid leukaemia (AML) as well as other haematological and lymphoid malignancies, but must be combined with other chemotherapeutics to achieve cure. Yet, the underlying mechanism dictating synergistic efficacy of combination chemotherapy remains largely unknown. The dNTPase SAMHD1, which regulates dNTP homoeostasis antagonistically to ribonucleotide reductase (RNR), limits ara-C efficacy by hydrolysing the active triphosphate metabolite ara-CTP. Here, we report that clinically used inhibitors of RNR, such as gemcitabine and hydroxyurea, overcome the SAMHD1-mediated barrier to ara-C efficacy in primary blasts and mouse models of AML, displaying SAMHD1-dependent synergy with ara-C. We present evidence that this is mediated by dNTP pool imbalances leading to allosteric reduction of SAMHD1 ara-CTPase activity. Thus, SAMHD1 constitutes a novel biomarker for combination therapies of ara-C and RNR inhibitors with immediate consequences for clinical practice to improve treatment of AML.


Assuntos
Citarabina/farmacologia , Leucemia Mieloide Aguda , Pirofosfatases/metabolismo , Ribonucleotídeo Redutases/antagonistas & inibidores , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Animais , Arabinofuranosilcitosina Trifosfato/metabolismo , Camundongos
6.
Exp Hematol ; 74: 13-18.e3, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31054867

RESUMO

Cytarabine remains the backbone of therapy in acute myeloid leukemia (AML). The ability to assess intracellular cytarabine triphosphate (ara-CTP) levels in patients receiving cytarabine represents a major goal in the prediction of treatment response. This study, conducted within a clinical setting, aimed to assess ara-CTP levels in circulating peripheral blasts from non-M3 AML patients receiving cytarabine at one of three dosing levels, using a novel biosensor assay. Results from the initial 72 hours post-commencement were correlated with day 28 remission status, with feasibility parameters concurrently assessed. Intracellular ara-CTP was detectable in ex vivo blasts post-treatment for standard-dose (SD) and high-dose (HD) patients (p < 0.05), and quantification revealed a 27-fold increase in intracellular steady-state concentration between the two dosing levels. For low-dose cytarabine, high rates of patient discharge and low intracellular concentrations limited analysis; however, assessment of intracellular ara-CTP concentration was achievable in a dwindling population of blasts for SD and HD treatment cohorts, with 4 hours post-treatment commencement potentially being most predictive of clinical response (r = -0.912, p = 0.0113). Concurrent assessment of peripheral leukemia-associated immunophenotype (LAIP)-positive cells revealed a decline in burden (0-72 hours), which correlated with remission status (p < 0.05). Unexpectedly high rates of night sampling led to challenges associated with sampling rates, but did not have an impact on patient compliance. Additional training of night staff improved feasibility substantially. Multiple peripheral sampling during the initial 72 hours of treatment is feasible in newly diagnosed patients, and ara-CTP is detectable over the initial 24 hours, facilitating prediction of chemosensitivity of leukemic blasts to cytarabine.


Assuntos
Arabinofuranosilcitosina Trifosfato , Crise Blástica , Leucemia Mieloide Aguda , Indução de Remissão , Idoso , Arabinofuranosilcitosina Trifosfato/administração & dosagem , Arabinofuranosilcitosina Trifosfato/farmacocinética , Crise Blástica/sangue , Crise Blástica/tratamento farmacológico , Crise Blástica/mortalidade , Intervalo Livre de Doença , Feminino , Humanos , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Taxa de Sobrevida , Fatores de Tempo
7.
Gan To Kagaku Ryoho ; 44(5): 413-415, 2017 May.
Artigo em Japonês | MEDLINE | ID: mdl-28536338

RESUMO

A 69-year-old woman underwent total gastrectomy for advanced gastric cancer with pyloric stenosis. She had a good postoperative course and was discharged 2 weeks after surgery. She received adjuvant chemotherapy with S-1 after discharge. One month after the initiation of the adjuvant chemotherapy, she complained of wobbling and weakness of her limbs. She stopped intake of S-1, but the symptoms did not improve. She was admitted to the hospital, but she became unconscious and had headache and blurred vision. We conducted a cerebrospinal fluid examination and made a diagnosis of meningeal carcinomatosis. After we started intrathecal infusion of methotrexate and Ara-C, referring to case reports clinical symptoms, including unconsciousness, headache, and left upper limb paralysis, improved and the CEA level in cerebrospinal fluid decreased.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Meningite/etiologia , Neoplasias Gástricas/tratamento farmacológico , Idoso , Arabinofuranosilcitosina Trifosfato/administração & dosagem , Biópsia , Quimioterapia Adjuvante , Feminino , Gastrectomia , Humanos , Meningite/patologia , Metotrexato/administração & dosagem , Recidiva , Neoplasias Gástricas/complicações , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgia
8.
PLoS One ; 12(1): e0169052, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28046007

RESUMO

SAMHD1 hydrolyzes 2'-deoxynucleoside-5'-triphosphates (dNTPs) into 2'-deoxynucleosides and inorganic triphosphate products. In this paper, we evaluated the impact of 2' sugar moiety substitution for different nucleotides on being substrates for SAMHD1 and mechanisms of actions for the results. We found that dNTPs ((2'R)-2'-H) are only permissive in the catalytic site of SAMHD1 due to L150 exclusion of (2'R)-2'-F and (2'R)-2'-OH nucleotides. However, arabinose ((2'S)-2'-OH) nucleoside-5'-triphosphates analogs are permissive to bind in the catalytic site and be hydrolyzed by SAMHD1. Moreover, when the (2'S)-2' sugar moiety is increased to a (2'S)-2'-methyl as with the SMDU-TP analog, we detect inhibition of SAMHD1's dNTPase activity. Our computational modeling suggests that (2'S)-2'-methyl sugar moiety clashing with the Y374 of SAMHD1. We speculate that SMDU-TP mechanism of action requires that the analog first docks in the catalytic pocket of SAMHD1 but prevents the A351-V378 helix conformational change from being completed, which is needed before hydrolysis can occur. Collectively we have identified stereoselective 2' substitutions that reveal nucleotide substrate specificity for SAMHD1, and a novel inhibitory mechanism for the dNTPase activity of SAMHD1. Importantly, our data is beneficial for understanding if FDA-approved antiviral and anticancer nucleosides are hydrolyzed by SAMHD1 in vivo.


Assuntos
Inibidores Enzimáticos/farmacologia , Proteínas Monoméricas de Ligação ao GTP/antagonistas & inibidores , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Animais , Arabinofuranosilcitosina Trifosfato , Carboidratos/química , Galinhas , Humanos , Hidrólise , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Modelos Moleculares , Monócitos/citologia , Nucleotídeos/metabolismo , Multimerização Proteica/efeitos dos fármacos , Proteína 1 com Domínio SAM e Domínio HD , Especificidade por Substrato/efeitos dos fármacos
9.
Nat Med ; 23(2): 256-263, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28067901

RESUMO

The cytostatic deoxycytidine analog cytarabine (ara-C) is the most active agent available against acute myelogenous leukemia (AML). Together with anthracyclines, ara-C forms the backbone of AML treatment for children and adults. In AML, both the cytotoxicity of ara-C in vitro and the clinical response to ara-C therapy are correlated with the ability of AML blasts to accumulate the active metabolite ara-C triphosphate (ara-CTP), which causes DNA damage through perturbation of DNA synthesis. Differences in expression levels of known transporters or metabolic enzymes relevant to ara-C only partially account for patient-specific differential ara-CTP accumulation in AML blasts and response to ara-C treatment. Here we demonstrate that the deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) promotes the detoxification of intracellular ara-CTP pools. Recombinant SAMHD1 exhibited ara-CTPase activity in vitro, and cells in which SAMHD1 expression was transiently reduced by treatment with the simian immunodeficiency virus (SIV) protein Vpx were dramatically more sensitive to ara-C-induced cytotoxicity. CRISPR-Cas9-mediated disruption of the gene encoding SAMHD1 sensitized cells to ara-C, and this sensitivity could be abrogated by ectopic expression of wild-type (WT), but not dNTPase-deficient, SAMHD1. Mouse models of AML lacking SAMHD1 were hypersensitive to ara-C, and treatment ex vivo with Vpx sensitized primary patient-derived AML blasts to ara-C. Finally, we identified SAMHD1 as a risk factor in cohorts of both pediatric and adult patients with de novo AML who received ara-C treatment. Thus, SAMHD1 expression levels dictate patient sensitivity to ara-C, providing proof-of-concept that the targeting of SAMHD1 by Vpx could be an attractive therapeutic strategy for potentiating ara-C efficacy in hematological malignancies.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Citarabina/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Proteínas Monoméricas de Ligação ao GTP/efeitos dos fármacos , Proteínas Virais Reguladoras e Acessórias/farmacologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Arabinofuranosilcitosina Trifosfato/metabolismo , Criança , Pré-Escolar , Citarabina/uso terapêutico , Modelos Animais de Doenças , Feminino , Humanos , Técnicas In Vitro , Lactente , Leucemia Mieloide Aguda/metabolismo , Masculino , Camundongos , Terapia de Alvo Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Prognóstico , Proteína 1 com Domínio SAM e Domínio HD
10.
DNA Repair (Amst) ; 49: 51-59, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27989484

RESUMO

Human PrimPol is a recently discovered bifunctional enzyme that displays DNA template-directed primase and polymerase activities. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn2+-dependent enzyme as it shows significantly improved primase and polymerase activities when binding Mn2+, rather than Mg2+, as a divalent metal ion cofactor. Consistently, our fluorescence anisotropy assays determined that PrimPol binds to a primer/template DNA substrate with affinities of 29 and 979nM in the presence of Mn2+ and Mg2+, respectively. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct dNTPs with 100-fold higher efficiency with Mn2+ than with Mg2+. Notably, the substitution fidelity of PrimPol in the presence of Mn2+ was determined to be in the range of 3.4×10-2 to 3.8×10-1, indicating that PrimPol is an error-prone polymerase. Furthermore, we kinetically determined the sugar selectivity of PrimPol to be 57-1800 with Mn2+ and 150-4500 with Mg2+, and found that PrimPol was able to incorporate the triphosphates of two anticancer drugs (cytarabine and gemcitabine), but not two antiviral drugs (emtricitabine and lamivudine).


Assuntos
Coenzimas/metabolismo , DNA Primase/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Magnésio/metabolismo , Manganês/metabolismo , Enzimas Multifuncionais/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/uso terapêutico , Antivirais/metabolismo , Antivirais/uso terapêutico , Arabinofuranosilcitosina Trifosfato/metabolismo , Arabinofuranosilcitosina Trifosfato/uso terapêutico , Cátions Bivalentes/metabolismo , Citidina Trifosfato/análogos & derivados , Citidina Trifosfato/metabolismo , Citidina Trifosfato/uso terapêutico , Desoxirribonucleotídeos/metabolismo , Didesoxinucleotídeos/metabolismo , Didesoxinucleotídeos/uso terapêutico , Emtricitabina/análogos & derivados , Emtricitabina/metabolismo , Emtricitabina/uso terapêutico , Humanos , Cinética , Lamivudina/análogos & derivados , Lamivudina/metabolismo , Lamivudina/uso terapêutico
11.
Adv Biochem Eng Biotechnol ; 145: 189-214, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25216956

RESUMO

Bioluminescent bacterial biosensors can be used in a rapid in vitro assay to predict sensitivity to commonly used chemotherapy drugs in acute myeloid leukemia (AML). The nucleoside analog cytarabine (ara-C) is the key agent for treating AML; however, up to 30 % of patients fail to respond to treatment. Screening of patient blood samples to determine drug response before commencement of treatment is needed. To achieve this aim, a self-bioluminescent reporter strain of Escherichia coli has been constructed and evaluated for use as an ara-C biosensor and an in vitro assay has been designed to predict ara-C response in clinical samples. Transposition mutagenesis was used to create a cytidine deaminase (cdd)-deficient mutant of E. coli MG1655 that responded to ara-C. The strain was transformed with the luxCDABE operon and used as a whole-cell biosensor for development an 8-h assay to determine ara-C uptake and phosphorylation by leukemic cells. Intracellular concentrations of 0.025 µmol/L phosphorylated ara-C were detected by significantly increased light output (P < 0.05) from the bacterial biosensor. Results using AML cell lines with known response to ara-C showed close correlation between the 8-h assay and a 3-day cytotoxicity test for ara-C cell killing. In retrospective tests with 24 clinical samples of bone marrow or peripheral blood, the biosensor-based assay predicted leukemic cell response to ara-C within 8 h. The biosensor-based assay may offer a predictor for evaluating the sensitivity of leukemic cells to ara-C before patients undergo chemotherapy and allow customized treatment of drug-sensitive patients with reduced ara-C dose levels. The 8-h assay monitors intracellular ara-CTP (cytosine arabinoside triphosphate) levels and, if fully validated, may be suitable for use in clinical settings.


Assuntos
Antimetabólitos Antineoplásicos/metabolismo , Arabinofuranosilcitosina Trifosfato/análise , Bioensaio , Citarabina/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Antimetabólitos Antineoplásicos/farmacologia , Arabinofuranosilcitosina Trifosfato/metabolismo , Técnicas Biossensoriais , Linhagem Celular Tumoral , Citarabina/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Reporter , Engenharia Genética , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Limite de Detecção , Luciferases Bacterianas/genética , Luciferases Bacterianas/metabolismo , Medições Luminescentes , Óperon , Fosforilação
12.
Artigo em Inglês | MEDLINE | ID: mdl-24880220

RESUMO

A specific and reliable HPLC-MS/MS method was developed and validated for the simultaneous determination of 1-ß-d-Arabinofuranosylcytosine (ara-C), 1-ß-d-Arabinofuranosyluracil (ara-U) and 1-ß-d-Arabinofuranosylcytosine triphosphate (ara-CTP) in the leukemic cells for the first time. The analytes were separated on a C18 column (100mm×2.1mm, 1.8µm) and a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source was used for detection. The ion-pairing reagent, NFPA, was added to the mobile phase to retain the analytes in the column. The cell homogenates sample was prepared by the simple protein precipitation. The calibration curves were linear over a concentration range of 3.45-3450.0ng/mL for ara-C, 1.12-1120.0ng/mL for ara-U and 4.13-4130.0ng/mL for ara-CTP. The intra-day and inter-day precision was less than 15% and the relative error (RE) were all within ±15%. The validated method was successfully applied to assess the disposition characteristics of ara-C and support cell pharmacokinetics after the patients with leukemia were intravenously infused with SDAC and HiDAC. The result of the present study would provide the valuable information for the ara-C therapy.


Assuntos
Antimetabólitos Antineoplásicos/farmacocinética , Arabinofuranosilcitosina Trifosfato/farmacocinética , Arabinofuranosiluracila/farmacocinética , Citarabina/farmacocinética , Adulto , Antimetabólitos Antineoplásicos/análise , Antimetabólitos Antineoplásicos/sangue , Arabinofuranosilcitosina Trifosfato/análise , Arabinofuranosilcitosina Trifosfato/sangue , Arabinofuranosiluracila/análise , Arabinofuranosiluracila/sangue , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão/métodos , Citarabina/análise , Citarabina/sangue , Humanos , Limite de Detecção , Pessoa de Meia-Idade , Espectrometria de Massas em Tandem/métodos , Adulto Jovem
13.
Biosens Bioelectron ; 52: 345-53, 2014 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-24080214

RESUMO

A novel whole cell bacterial biosensor, which emits light in response to the active metabolite of cytosine arabinoside (ara-C, cytarabine), ara-CTP, has been investigated and verified. The biosensor has been formulated as an ex vivo assay, designed for peripheral blood or bone marrow cells, which can produce a clinical result within a working day. The nucleoside analogue ara-C is a key agent for treatment of acute myeloid leukaemia (AML); treatment decisions are made rapidly with AML, patients often receiving same-day commencement of chemotherapy. Currently no rapid predictive test is available to select appropriate therapy for patients prior to treatment. Experiments were designed to determine optimal assay conditions using leukaemic cell lines. We observed a significant increase (~15 fold) in bioluminescence signal compared to control after 8-h incubation of the biosensor with ara-C. This corresponded to a >2-log increase in light output per bacterial cell. Interestingly, bioluminescence conferred a survival advantage to the bacteria following ara-C treatment. The assay is sensitive (lower limit of quantitation of 0.05 µM), selective, accurate (≤ 15% RE) and precise (≤ 15% coefficient of variation) over a linear concentration range of ara-CTP (0.05-0.5 µM), and detection is independent of reaction volume. Recovery of added standard was tested using ex vivo patient leukaemic cells (n=5). Stability studies on lyophilized bacterial biosensor were performed to ensure maintenance of performance over 12 months. The biosensor assay could be invaluable to the clinician, assisting with treatment selection, and potentially mitigating the risks of resistance and toxicity observed with this drug.


Assuntos
Arabinofuranosilcitosina Trifosfato/isolamento & purificação , Técnicas Biossensoriais , Citarabina/isolamento & purificação , Leucemia Mieloide Aguda/sangue , Arabinofuranosilcitosina Trifosfato/sangue , Biomarcadores Tumorais/sangue , Humanos , Leucemia Mieloide Aguda/patologia , Limite de Detecção , Medições Luminescentes
14.
Leuk Res ; 37(6): 690-6, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23473919

RESUMO

This study evaluates an in vitro biosensor assay capable of detecting the intracellular levels of the tri-phosphorylated form of cytarabine (Ara-CTP) within one working day. The biosensor predicted the response of seven leukaemic cell lines with varying known sensitivities to cytarabine alone and in combination with fludarabine. High-performance liquid chromatography (HPLC), 3-day assessment of cellular viable mass, and flow cytometric assessment of apoptosis were used to validate biosensor performance. A correlation between the biosensor results and Ara-CTP quantitation by HPLC was confirmed (R=0.972). The biosensor was also capable of detecting enhanced accumulation of Ara-CTP following sequential pre-treatment of leukaemic cells with cytarabine ± fludarabine.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Arabinofuranosilcitosina Trifosfato/análise , Técnicas Biossensoriais/métodos , Citarabina/farmacologia , Leucemia/patologia , Vidarabina/análogos & derivados , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/análise , Linhagem Celular Tumoral , Células HL-60 , Humanos , Células K562 , Leucemia/diagnóstico , Leucemia/tratamento farmacológico , Medições Luminescentes/métodos , Proteínas Luminescentes/análise , Resultado do Tratamento , Vidarabina/farmacologia
15.
Biochimie ; 95(2): 320-8, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23063694

RESUMO

In vitro assay of mammalian DNA replication has been variously approached. Using gapped circular duplex substrates containing a 500-base single-stranded DNA region, we have constructed a mammalian cell-free system in which physiological DNA replication may be reproduced. Reaction of the gapped plasmid substrate with crude extracts of human HeLaS3 cells induces efficient DNA synthesis in vitro. The induced synthesis was strongly inhibited by aphidicolin and completely depended on dNTP added to the system. In cell extracts in which PCNA was depleted step-wise by immunoprecipitation, DNA synthesis was accordingly reduced. These data suggest that replicative DNA polymerases, particularly pol delta, may chiefly function in this system. Furthermore, DNA synthesis is made quantifiable in this system, which enables us to evaluate the efficiency of DNA replication induced. Our system sensitively and quantitatively detected the reduction of the DNA replication efficiency in the DNA substrates damaged by oxidation or UV cross-linking and in the presence of a potent chain terminator, ara-CTP. The quantitative assessment of mammalian DNA replication may provide various advantages not only in basic research but also in drug development.


Assuntos
Bioensaio , Sistema Livre de Células/metabolismo , Replicação do DNA/genética , DNA de Cadeia Simples , DNA Polimerase Dirigida por DNA/metabolismo , Animais , Afidicolina/farmacologia , Arabinofuranosilcitosina Trifosfato/farmacologia , Sistema Livre de Células/efeitos dos fármacos , Células HeLa , Humanos , Cinética , Inibidores da Síntese de Ácido Nucleico , Plasmídeos/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo
16.
Front Biosci (Landmark Ed) ; 17(2): 569-74, 2012 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-22201761

RESUMO

Cytarabine (araC) is a highly active antimetabolite against hematological malignancy while the agent shows limited activity for some patients despite maintenance or continued therapy with ara-C-containing regiments. In this study, we focused to elucidate the mechanism of intrinsic resistance to araC. The concentration of intracellular ara-CTP and incorporated ara-CTP were monitored in human leukemia cell line-HL-60 for different passages in parental with its variant HL-60R. The expression of mRNA for deoxycytidine kinase (dCK), cytidine deaminas (CDA), human equilibrative nucleoside transporter 1 (hENT1), and cytosolic 50-nucleotidase II (cN-II) were examined by Real-time PCR in HL-60 and HL-60R for different passages. And activities of two metabolizing enzymes for araC, dCK and CDA were further examined. The results showed that the concentration of intracellular ara-CTP was significantly reduced and the ara-U increased in HL-60 cells for 50 passages compared with the 5 passages, and associated with higher CDA activity. All the factors in HL-60R cells did not change by the incubation of ara-C. In conclusion, the long term cultured cells are intrinsically resistant to ara-C through high CDA activity, but not low DCK activity.


Assuntos
Citarabina/farmacologia , Citidina Desaminase/metabolismo , Resistencia a Medicamentos Antineoplásicos/fisiologia , Arabinofuranosilcitosina Trifosfato/metabolismo , Arabinofuranosiluracila/metabolismo , Citidina Desaminase/genética , Células HL-60 , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo
17.
Pharmacogenomics ; 10(10): 1657-74, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19842938

RESUMO

The mainstay of acute myeloid leukemia chemotherapy is the nucleoside analog cytarabine (ara-C). Numerous studies suggest that the intracellular concentrations of the ara-C active metabolite, ara-CTP, vary widely among patients and, in turn, are associated with variability in clinical response to acute myeloid leukemia treatment. Thus, genetic variation in key genes in the ara-C metabolic pathway--specifically, deoxycytidine kinase (a rate-limiting activating enzyme), 5 nucleotidase, cytidine deaminase and deoxycytidylate deaminase (all three are inactivating enzymes), human equilibrative nucleoside transporter (ara-C uptake transporter) and ribonucleotide reductase (RRM1 and RRM2--enzymes regulating intracellular deoxycytidine triphosphate pools)--form the molecular basis of the interpatient variability observed in intracellular ara-CTP concentrations and response to ara-C. Understanding genetic variants in the key candidate genes involved in the metabolic activation of ara-C, as well as the pharmacodynamic targets of ara-C, will provide an opportunity to identify patients at an increased risk of adverse reactions or decreased likelihood of response, based upon their genetic profile, which in future could help in dose optimization to reduce drug toxicity without compromising efficacy. The pharmacogenetic studies on ara-C would also be equally applicable to other nucleoside analogs, such as gemcitabine, decitabine, clofarabine and so on, which are metabolized by the same pathway.


Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Citarabina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , 5'-Nucleotidase/genética , Arabinofuranosilcitosina Trifosfato/metabolismo , Citidina Desaminase/genética , Desoxicitidina Quinase/genética , Transportador Equilibrativo 1 de Nucleosídeo/genética , Previsões , Variação Genética , Humanos , Leucemia Mieloide Aguda/genética , Nucleosídeo Desaminases/genética , Farmacogenética/métodos , Ribonucleotídeo Redutases/genética
18.
Biochem Pharmacol ; 77(12): 1780-6, 2009 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-19428333

RESUMO

Cytarabine (ara-C) is the key agent for treating acute myeloid leukemia (AML). After being transported into leukemic cells by human equilibrative nucleoside transporter 1 (hENT1), ara-C is phosphorylated to ara-C triphosphate (ara-CTP), an active metabolite, and then incorporated into DNA, thereby inhibiting DNA synthesis. Deoxycytidine kinase (dCK) and cytosolic 5'-nucleotidase II (cN-II) are associated with the production of ara-CTP. Because ara-C's cytotoxicity depends on ara-CTP production, parameters that are most related to ara-CTP formation would predict ara-C sensitivity and the clinical outcome of ara-C therapy. The present study focused on finding any correlation between the capacity to produce ara-CTP and ara-C-metabolizing factors. In vitro ara-CTP production, mRNA levels of hENT1, dCK, and cN-II, and ara-C sensitivity were evaluated in 34 blast samples from 33 leukemic patients including 26 with AML. A large degree of heterogeneity was seen in the capacity to produce ara-CTP and in mRNA levels of hENT1, dCK, and cN-II. Despite the lack of any association between each of the transcript levels and ara-CTP production, the ratio of dCK/cN-II transcript levels correlated significantly with the amount of ara-CTP among AML samples. The HL-60 cultured leukemia cell line and its three ara-C-resistant variants (HL-60/R1, HL-60/R2, HL-60/R3), which were 8-, 10-, and 500-fold more resistant than HL-60, respectively, were evaluated similarly. The dCK/cN-II ratio was again proportional to ara-CTP production and to ara-C sensitivity. The dCK/cN-II ratio may thus predict the capacity for ara-CTP production and ultimately, ara-C sensitivity in AML.


Assuntos
5'-Nucleotidase/genética , Arabinofuranosilcitosina Trifosfato/metabolismo , Desoxicitidina Quinase/genética , Transportador Equilibrativo 1 de Nucleosídeo/genética , Leucemia Mieloide Aguda/metabolismo , Valor Preditivo dos Testes , Citarabina/metabolismo , Citarabina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Células HL-60 , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , RNA Mensageiro/análise
19.
J Chromatogr B Analyt Technol Biomed Life Sci ; 877(14-15): 1417-25, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19328748

RESUMO

A method was developed for the quantification of araCTP, CTP and dCTP in a human follicular lymphoma cell line. This method involves solid phase extraction (SPE) using a weak anion-exchanger (WAX) cartridge, a porous graphitic carbon high-performance liquid chromatography (HPLC) column separation, and tandem mass spectrometry (MS/MS) detection. By using a triple quadrupole mass spectrometer operating in negative ion multiple reaction monitoring (MRM) mode, the method was able to achieve a lower limit of quantification (LLOQ) of 0.1 microg mL(-1) for araCTP and of 0.01 microg mL(-1) for both CTP and dCTP. The method was validated and used to determine the amount of araCTP, CTP and dCTP formed after incubation of araC and an araCMP prodrug in the human follicular lymphoma cell line RL.


Assuntos
Arabinofuranosilcitosina Trifosfato/química , Cromatografia Líquida de Alta Pressão/métodos , Citidina Trifosfato/química , Nucleotídeos de Desoxicitosina/química , Linfoma Folicular/química , Espectrometria de Massas em Tandem/métodos , Tionucleotídeos/química , Linhagem Celular Tumoral , Humanos , Sensibilidade e Especificidade
20.
Mol Cancer Ther ; 7(10): 3373-80, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18852140

RESUMO

The therapeutic efficiency of anticancer nucleoside analogues (NA) strongly depends on their intracellular accumulation and conversion into 5'-triphosphates. Because active NATP cannot be directly administrated due to instability, we present here a strategy of nanoencapsulation of these active drugs for efficient delivery to tumors. Stable lyophilized formulations of 5'-triphosphates of cytarabine (araCTP), gemcitabine (dFdCTP), and floxuridine (FdUTP) encapsulated in biodegradable PEG-cl-PEI or F127-cl-PEI nanogel networks (NGC and NGM, respectively) were prepared by a self-assembly procedure. Cellular penetration, in vitro cytotoxicity, and drug-induced cell cycle perturbations of these nanoformulations were analyzed in breast and colorectal cancer cell lines. Cellular accumulation and NATP release from nanogel was studied by confocal microscopy and direct high-performance liquid chromatography analysis of cellular lysates. Antiproliferative effect of dFdCTP nanoformulations was evaluated in human breast carcinoma MCF7 xenograft animal model. Nanoencapsulated araCTP, dFdCTP, and FdUTP showed similar to NA cytotoxicity and cell cycle perturbations. Nanogels without drugs showed very low cytotoxicity, although NGM was more toxic than NGC. Treatment by NATP nanoformulations induced fast increase of free intracellular drug concentration. In human breast carcinoma MCF7 xenograft animal model, i.v. dFdCTP-nanogel was equally effective in inhibiting tumor growth at four times lower administered drug dose compared with free gemcitabine. Active triphosphates of NA encapsulated in nanogels exhibit similar cytotoxicity and cell cycle perturbations in vitro and faster cell accumulation and equal tumor growth-inhibitory activity in vivo at much lower dose compared with parental drugs, illustrating their therapeutic potential for cancer chemotherapy.


Assuntos
Neoplasias da Mama/patologia , Neoplasias Colorretais/patologia , Nucleosídeos/farmacologia , Polietilenoglicóis/metabolismo , Polietilenoimina/metabolismo , Polímeros/metabolismo , Polifosfatos/metabolismo , Animais , Antineoplásicos/farmacologia , Arabinofuranosilcitosina Trifosfato/farmacologia , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Química Farmacêutica , Citarabina/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Portadores de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Citometria de Fluxo , Humanos , Camundongos , Nanogéis , Gencitabina
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