RESUMO
Isotope dilution is currently the most accurate technique in humans to determine vitamin A status and bioavailability/bioconversion of provitamin A carotenoids such as ß-carotene. However, limits of MS detection, coupled with extensive isolation procedures, have hindered investigations of physiologically-relevant doses of stable isotopes in large intervention trials. Here, a sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) analytical method was developed to study the plasma response from coadministered oral doses of 2 mg [(13)C10]ß-carotene and 1 mg [(13)C10]retinyl acetate in human subjects over a 2 week period. A reverse phase C18 column and binary mobile phase solvent system separated ß-carotene, retinol, retinyl acetate, retinyl linoleate, retinyl palmitate/retinyl oleate, and retinyl stearate within a 7 min run time. Selected reaction monitoring of analytes was performed under atmospheric pressure chemical ionization in positive mode at m/z 537â321 and m/z 269â93 for respective [(12)C]ß-carotene and [(12)C] retinoids; m/z 547â330 and m/z 274â98 for [(13)C10]ß-carotene and [(13)C5] cleavage products; and m/z 279â100 for metabolites of [(13)C10]retinyl acetate. A single one-phase solvent extraction, with no saponification or purification steps, left retinyl esters intact for determination of intestinally-derived retinol in chylomicrons versus retinol from the liver bound to retinol binding protein. Coadministration of [(13)C10]retinyl acetate with [(13)C10]ß-carotene not only acts as a reference dose for inter-individual variations in absorption and chylomicron clearance rates, but also allows for simultaneous determination of an individual's vitamin A status.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Vitamina A/metabolismo , beta Caroteno/farmacocinética , Adolescente , Adulto , Disponibilidade Biológica , Biotransformação , Feminino , Humanos , Isótopos , Masculino , Pessoa de Meia-Idade , Fatores de Tempo , Vitamina A/sangue , Adulto Jovem , beta Caroteno/sangue , beta Caroteno/metabolismoRESUMO
The antitumour effect of thymidylate synthase inhibitors such as raltitrexed (RTX) may be reversed by salvage of thymidine (Thd). Since thymidine phosphorylase (TP) depletes Thd, the potential for tumour-selective depletion of Thd using antibody-mediated delivery of TP to tumours was investigated. In vitro studies demonstrated that 25 x 10(-3) units/ml TP depleted extracellular Thd (3 microM) and restored sensitivity to the growth inhibitory effects of RTX in Lovo and HT29 cell lines. Thymidine concentrations in xenograft tumours were inversely proportional to the activity of TP in the tumour, and the presence of a subcutaneous Lovo xenograft reduced plasma Thd concentrations from 0.92 +/- 0.07 to 0.37 +/- 0.04 microM. Intravenous administration of native TP enzyme depleted plasma Thd to 5 nM, but following rapid elimination of TP, plasma Thd returned to pretreatment values. There was no effect on tumour TP or Thd. Conjugation of TP to the A5B7 F(ab)2 antibody fragment, which targets carcinoembryonic antigen (CEA) expressed on colorectal cell-lines such as Lovo, did result in selective accumulation of TP in the tumour. However, there was no tumour-selective depletion of Thd and there did not appear to be any potential benefit of combining antibody-targeted TP with RTX.
Assuntos
Quinazolinas/uso terapêutico , Tiofenos/uso terapêutico , Timidina Fosforilase/metabolismo , Timidina/metabolismo , Timidilato Sintase/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Antígeno Carcinoembrionário/imunologia , Antígeno Carcinoembrionário/metabolismo , Linhagem Celular Tumoral , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Células HT29 , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/metabolismo , Injeções Intravenosas , Camundongos , Camundongos Nus , Quinazolinas/farmacologia , Reprodutibilidade dos Testes , Tiofenos/farmacologia , Timidina Fosforilase/administração & dosagem , Timidina Fosforilase/imunologia , Timidilato Sintase/metabolismoRESUMO
The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), are used in the treatment of leukemia. Incorporation of deoxythioguanosine nucleotides (dG(s)) into the DNA of thiopurine-treated cells causes cell death, but there is also evidence that thiopurine metabolites, particularly the 6-MP metabolite methylthioinosine monophosphate (MeTIMP), inhibit de novo purine synthesis (DNPS). The toxicity of DNPS inhibitors is influenced by methylthioadenosine phosphorylase (MTAP), a gene frequently deleted in cancers. Because the growth of MTAP-deleted tumor cells is dependent on DNPS or hypoxanthine salvage, we would predict such cells to show differential sensitivity to 6-MP and 6-TG. To test this hypothesis, sensitivity to 6-MP and 6-TG was compared in relation to MTAP status using cytotoxicity assays in two MTAP-deficient cell lines transfected to express MTAP: the T-cell acute lymphoblastic leukemic cell line, Jurkat, transfected with MTAP cDNA under the control of a tetracycline-inducible promoter, and a lung cancer cell line (A549-MTAP(-)) transfected to express MTAP constitutively (A549-MTAP(+)). Sensitivity to 6-MP or methyl mercaptopurine riboside, which is converted intracellularly to MeTIMP, was markedly higher in both cell lines under MTAP(-) conditions. Measurement of thiopurine metabolites support the hypothesis that DNPS inhibition is a major cause of cell death with 6-MP, whereas dG(s) incorporation is the main cause of cytotoxicity with 6-TG. These data suggest that thiopurines, particularly 6-MP, may be more effective in patients with deleted MTAP.
Assuntos
Mercaptopurina/farmacologia , Neoplasias/tratamento farmacológico , Purina-Núcleosídeo Fosforilase/metabolismo , Tioguanina/farmacologia , Tioinosina/análogos & derivados , Tionucleotídeos/farmacologia , Antimetabólitos Antineoplásicos/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Deleção de Genes , Humanos , Immunoblotting , Mercaptopurina/metabolismo , Mercaptopurina/uso terapêutico , Neoplasias/genética , Neoplasias/metabolismo , Purina-Núcleosídeo Fosforilase/deficiência , Purina-Núcleosídeo Fosforilase/genética , Purinas/biossíntese , Tioguanina/metabolismo , Tioguanina/uso terapêutico , Tioinosina/farmacologiaRESUMO
BACKGROUND: Some anticancer drugs inhibit thymidylate synthase (TS), a key enzyme for thymidine nucleotide biosynthesis. Cells can compensate for depleted thymidine levels by taking up extracellular thymidine via a salvage pathway. We investigated the use of 2-[11C]thymidine positron emission tomography (PET) to measure thymidine salvage kinetics in vivo in humans. METHODS: Five patients with advanced gastrointestinal cancer were PET scanned both before and 1 hour after oral administration of the TS inhibitor AG337 (THYMITAQ [nolatrexed]); seven control patients were scanned twice but not treated with AG337. Thymidine salvage kinetics were measured in vivo using 2-[11C]thymidine PET and spectral analysis to obtain the standardized uptake values (SUV), the area under the time-activity curve (AUC), and the fractional retention of thymidine (FRT). Changes in PET parameters between scans in the AG337-treated and control groups were compared using the Mann-Whitney U test. The relationship between AG337 exposure and AG337-induced changes in tumor FRT and in plasma deoxyuridine levels (a conventional pharmacodynamic systemic measure of TS inhibition) was examined using Spearman's regression analysis. Statistical tests were two-sided. RESULTS: The between-scan change in FRT in patients treated with AG337 (38% increase, 95% confidence interval [CI] = 8% to 68%) was higher than that in control patients (3% increase, 95% CI = -11% to 17%) (P =.028). The level of AG337-induced increase in both 2-[11C]thymidine FRT and plasma deoxyuridine levels was statistically significantly correlated with AG337 exposure (r = 1.00, P =.01 for both). CONCLUSIONS: AG337 administration was associated with increased tumor tracer retention that was consistent with tumor cell uptake of exogenous 2-[11C]thymidine as a result of TS inhibition. 2-[11C]Thymidine PET can be used to measure thymidine salvage kinetics directly in the tissue of interest.