RESUMEN
Fluorouracil (5FU) acts by RNA-incorporation and inhibition of thymidylate synthase; the first action is counteracted by uridine, and the second is enhanced by leucovorin (LV). Growth inhibition of C26-10 colon cancer cells by 5FU was enhanced by LV and rescued by uridine, but 5FU-LV was only partially rescued by uridine. In WiDr cells, 5FU sensitivity was not enhanced by LV, while both 5FU and 5FU-LV were rescued by uridine. Intermediate trends were found in SW948 and HT29 cells. Uridine rescue in mice allowed 1.5-fold increase in 5FU dose, leading to 2-fold increase in the antitumor effect and thymidylate synthase inhibition in resistant Colon-26 tumors. In the sensitive Colon-26-10 tumor, uridine rescue decreased 5FU-RNA incorporation > 10-fold, without affecting the antitumor activity. The use of LV and uridine can differentiate between two mechanisms of action of 5FU.
Asunto(s)
Fluorouracilo/metabolismo , Fluorouracilo/farmacología , Leucovorina/farmacología , ARN/metabolismo , Timidilato Sintasa/antagonistas & inhibidores , Timidilato Sintasa/metabolismo , Uridina/farmacología , Animales , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Bovinos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , ARN/genética , Interferencia de ARN/efectos de los fármacos , Sensibilidad y EspecificidadRESUMEN
Gemcitabine (dFdC) was tested in a Phase I trial at 14 doses (40-5700 mg/m(2)), administered every 2 weeks as a (1/2) -h infusion to 52 patients with refractory solid cancer. Gemcitabine and its deaminated metabolite difluorodeoxyuridine (dFdU), measured with HPLC, reached plasma peak levels of 2-3 microM at 40 mg/m(2) which increased to 512 microM at 5700 mg/m(2). Gemcitabine was eliminated rapidly with a t(1/2) beta of 2.3-15.8 min in the 40-5700 mg/m(2) dose range, with one exception of 38 min at 4500 mg/m(2) . dFdU was still present at a plateau of +/- 20 microM from 4-24 h at doses >960 mg/m(2). Up to 3650 mg/m(2) linear pharmacokinetics were observed for gemcitabine, while those for dFdU were linear over the whole range. Gemcitabine clearance varied between 1.5-12.6 l/min and was 1.5-fold higher in males than in females (p= 0.024); its volume of distribution was 45.2-248 l. In lymphocytes peak levels of the active metabolite dFdCTP were 100-380 pmol/10( 6 )cells in the first course. Apparently a plateau was reached which was confirmed by incubation of white blood cells with increasing gemcitabine concentrations up to 500 microM, reaching a plateau of about 350 pmol/10(6 )cells; in contrast in cancer cells this concentration dependence did not exist and accumulation reached about 1590 pmol/10( 6 )cells. In tumors isolated from patients treated with gemcitabine dFdCTP reached about 70 pmol/g wet weight. Gemcitabine itself was eliminated only to a limited extent in the urine, but dFdU was eliminated almost completely in the urine in the first 24 h (51-92%). In conclusion, dFdC was rapidly eliminated in contrast to dFdU, which was present for at least 18 h, as well as dFdCTP in lymphocytes.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacocinética , Desoxicitidina/análogos & derivados , Neoplasias/tratamiento farmacológico , Adulto , Anciano , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/farmacología , Desoxicitidina/administración & dosificación , Desoxicitidina/farmacocinética , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Humanos , Infusiones Intravenosas , Masculino , Dosis Máxima Tolerada , Tasa de Depuración Metabólica , Persona de Mediana Edad , GemcitabinaRESUMEN
Thymidine phosphorylase (TP) and uridine phosphorylase (UP) catalyze the (in)activation of several fluoropyrimidines, depending on their catalytic activity and substrate specificity. Blood cells are the first compartment exposed to most anticancer agents. The role of white blood cells in causing toxic side effects and catalyzing drug metabolism is generally underestimated. Therefore we determined the contribution of the white blood cell compartment to drug metabolism, and we investigated the activity and substrate specificity of TP and UP for the (fluoro)pyrimidines thymidine (dThd), uridine (Urd), 5'-deoxy-5-fluorouridine (5' dFUrd) and 5-fluorouracil (5FU) in peripheral blood mononuclear cells (PBMC) and undifferentiated monocytes and differentiated monocytes: macrophages and dendritic cells. PBMC had an IC50 of 742 microM exposed to 5'dFUrd, increasing to > 2000 microM when both TP and UP activities were inhibited. Total phosphorolytic activity was higher with dThd than with Urd, 5'dFUrd or 5FU. Using a specific TP inhibitor (TPI) and UP inhibitor (BAU) we concluded that dThd and Urd were preferentially converted by TP and UP, respectively, while 5'dFUrd and 5FU were mainly converted by TP (about 80%) into 5FU and FUrd, respectively. 5FU was effectively incorporated into RNA. dThd conversion into thymine was highest in dendritic cells (52.6 nmol thymine/h/10(6) cells), followed by macrophages (two-fold) and undifferentiated monocytes (eight-fold). TPI prevented dThd conversion almost completely. In conclusion, PBMC were relatively insensitive to 5'dFUrd, and the natural substrates dThd and Urd were preferentially converted by TP and UP, respectively. TP and UP were both responsible for converting 5'dFUrd/5FU into 5FU/FUrd, respectively.
Asunto(s)
Leucocitos Mononucleares/metabolismo , Pirimidinas/metabolismo , Timidina Fosforilasa/fisiología , Uridina Fosforilasa/fisiología , Células Cultivadas , Floxuridina/metabolismo , Floxuridina/farmacología , Fluorouracilo/análisis , Fluorouracilo/metabolismo , Fluorouracilo/farmacología , Humanos , Concentración 50 Inhibidora , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/enzimología , Pirimidinas/farmacología , ARN/química , ARN/metabolismo , Especificidad por Sustrato , Timidina/metabolismo , Timidina/farmacologíaRESUMEN
BACKGROUND: Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are essential enzymes for 5-fluorouracil (5-FU) metabolism. In patients with advanced colorectal cancer (ACRC), retrospective studies have shown that low expression levels of TS and DPD correlated with response to 5-FU. We performed a prospective study in which the choice of first-line chemotherapy with either 5-FU or a non-5-FU containing regimen was based on TS and DPD expression. PATIENTS AND METHODS: Fresh-frozen samples of metastases were obtained from 58 previously untreated patients with ACRC. mRNA expression of TS and DPD was quantified using an RT-PCR assay. Patients with low tumor expression of both TS and DPD received weekly bolus 5-FU/leucovorin (LV) 500 mg/m2 (group A); patients with high TS and/or DPD received 3-weekly oxaliplatin 85 mg/m2 and irinotecan 200 mg/m2 (group B). After progression, cross-over to the alternative regimen was attempted. RESULTS: Of 53 eligible patients, 31 had tumors with both low TS and low DPD, and were treated in group A. A response was observed in 11 patients [35%; 95% confidence interval (CI) 19% to 54%]. Cross-over to second-line oxaliplatin/irinotecan resulted in a partial response in two out of 16 patients (13%; 95% CI 1% to 38%). In group B, four out of 22 patients responded (18%; 95% CI 5% to 40%), while no responses were observed in 12 patients after cross-over to 5-FU/LV (0%; 95% CI 0% to 28%). CONCLUSIONS: Prospective selection of 5-FU/LV chemotherapy based on low TS and DPD expression in patients with ACRC did not confirm the high response rates reported in retrospective studies. The procedure of obtaining metastatic tissue and quantitation of enzymes appeared feasible but cumbersome. Before assessing the clinical utility of a predictive marker in a randomized trial, future studies should focus on prospective validation of the assay in a large and well defined population.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Timidilato Sintasa/metabolismo , Adulto , Anciano , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Neoplasias Colorrectales/enzimología , Neoplasias Colorrectales/genética , Dihidrouracilo Deshidrogenasa (NADP)/genética , Femenino , Fluorouracilo/administración & dosificación , Humanos , Irinotecán , Leucovorina/administración & dosificación , Masculino , Persona de Mediana Edad , Compuestos Organoplatinos/administración & dosificación , Oxaliplatino , Pronóstico , Estudios Prospectivos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tasa de Supervivencia , Timidilato Sintasa/genéticaRESUMEN
UNLABELLED: Gemcitabine is a deoxycytidine analog, which can be inactivated by deamination catalyzed by deoxycytidine deaminase (dCDA). Altered transport over the cell membrane is a mechanism of resistance to gemcitabine. To facilitate accumulation, the fatty acid derivative CP-4125 was synthesized. Since, the fatty acid is acylated at the site of action of dCDA, a decreased deamination was expected. CP-4125 was equally active as gemcitabine in a panel of rodent and human cell lines and in human melanoma xenografts bearing mice. In contrast to gemcitabine, CP-4125 was not deaminated but inhibited deamination of deoxycytidine and gemcitabine. Pools of the active triphosphate of gemcitabine increased for over 20 hr after CP-4125 exposure, while these pools decreased directly after removal of gemcitabine. IN CONCLUSION: CP-4125 is an interesting new gemcitabine derivative.
Asunto(s)
Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Ácidos Grasos/metabolismo , Leucemia/tratamiento farmacológico , Animales , Antimetabolitos Antineoplásicos/farmacología , Carbono/química , Línea Celular Tumoral , Membrana Celular/metabolismo , Proliferación Celular , Citidina Desaminasa , ADN/química , Daño del ADN , Humanos , Concentración 50 Inhibidora , Melanoma/patología , Ratones , Ratones Desnudos , Modelos Químicos , Trasplante de Neoplasias , Nucleósido Desaminasas/metabolismo , Fosforilación , Ratas , Factores de Tiempo , GemcitabinaRESUMEN
Two methods for measurement of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) mRNA expression were compared. Although the relative mRNA levels compared to beta-actin measured with competitive template RT-PCR were different from the data obtained with a TaqMan based PCR, a significant correlation between the two assays was found.
Asunto(s)
Fluorouracilo/farmacología , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Actinas/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Neoplasias Colorrectales/metabolismo , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Regulación de la Expresión Génica , Humanos , Timidilato Sintasa/metabolismoRESUMEN
A non-radioactive method to determine 5-Fluorouracil (5FU) incorporation into DNA has been developed. Isolated DNA was enzymatically degraded to bases and the resulting 5FU was measured with standard gas-chromatography coupled to mass spectrometry (GC-MS) and compared with that of radioactive 5FU in a cell line. Incorporation into DNA of the murine Colon 26-B tumor treated with maximal tolerated doses of 5FU and fluorodeoxyuridine (FUdR) was maximal after 2 hour and was 15.4 and 71.0 fmol/microg DNA, respectively. After a plateau for about 3 days a decrease was observed to +/- 2 fmol/microg DNA after 10 days. The assay is very sensitive and reproducible and can be used in a clinical setting.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacocinética , ADN/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Floxuridina/farmacología , Fluorouracilo/farmacocinética , Cromatografía de Gases y Espectrometría de Masas/métodos , Neoplasias/tratamiento farmacológico , Animales , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , ADN/metabolismo , Ratones , Sensibilidad y Especificidad , Factores de TiempoRESUMEN
BACKGROUND: The mechanism of action of 5-fluorouracil (5-FU) has been associated with inhibition of thymidylate synthase (TS) and incorporation of 5-FU into RNA and DNA, but limited data are available in human tumor tissue for the latter. We therefore measured incorporation in human tumor biopsy specimens after administration of a test dose of 5-FU alone or with leucovorin. PATIENTS AND METHODS: Patients received 5-FU (500 mg/m(2)) with or without high-dose leucovorin, low-dose leucovorin or l-leucovorin, and biopsy specimens were taken after approximately 2, 24 or 48 h. Tissues were pulverized and extracted for nucleic acids. 5-FU incorporation was measured using gas chromatography/mass spectrometry after complete degradation to bases of isolated RNA and DNA. RESULTS: Maximal incorporation into RNA (1.0 pmol/micrograms RNA) and DNA (127 fmol/micrograms DNA) of 59 and 46 biopsy specimens, respectively, was found at 24 h after 5-FU administration. Incorporation into RNA but not DNA was significantly correlated with intratumoral 5-FU levels. However, DNA incorporation was significantly correlated with the RNA incorporation. Primary tumor tissue, liver metastasis and normal mucosa did not show significant differences, while leucovorin had no effect. Neither for RNA (30 patients) nor DNA (24 patients) incorporation was a significant correlation with response to 5-FU therapy found. However, in the same group of patients, response was significantly correlated to TS inhibition (mean TS in responding and non-responding groups 45 and 231 pmol/h/mg protein, respectively; P=0.001). CONCLUSIONS: 5-FU is incorporated at detectable levels into RNA and DNA of human tumor tissue, but no relation between the efficacy of 5-FU treatment and incorporation was found, in contrast to TS.
Asunto(s)
Neoplasias Colorrectales/tratamiento farmacológico , ADN de Neoplasias/metabolismo , Fluorouracilo/uso terapéutico , ARN Neoplásico/metabolismo , Timidilato Sintasa/antagonistas & inhibidores , Adulto , Anciano , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/metabolismo , Antimetabolitos Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/metabolismo , Arteria Hepática/lesiones , Humanos , Infusiones Intraarteriales/métodos , Leucovorina/administración & dosificación , Masculino , Persona de Mediana Edad , Timidilato Sintasa/metabolismo , Factores de Tiempo , Resultado del TratamientoRESUMEN
S-1 is an oral formulation of ftorafur (FT), oxonic acid and 5-chloro-2,4-dihydroxypyridine (CDHP) at a molar ratio of 1:0.4:1. FT is a 5-fluorouracil (5-FU) prodrug, CDHP is a dihydropyrimidine dehydrogenase (DPD) inhibitor and oxonic acid is an inhibitor of 5-FU phosphoribosylation in the gastrointestinal mucosa and was included to prevent gastrointestinal toxicity. We determined the pharmacokinetics of S-1 in 28 patients at doses of 25, 35, 40 and 45 mg/m(2). The plasma C(max) values of FT, 5-FU, oxonic acid and CDHP increased dose-dependently and after 1-2 h were in the ranges 5.8-13 microM, 0.4-2.4 microM, 0.026-1.337 microM, and 1.1-3.6 microM, respectively. Uracil levels, indicative of DPD inhibition, also increased dose-dependently from basal levels of 0.03-0.25 microM to 3.6-9.4 microM after 2-4 h, and 0.09-0.9 microM was still present after 24 h. The pharmacokinetics of CDHP and uracil were linear over the dose range. The areas under the plasma concentration curves (AUC) for CDHP and uracil were in the ranges 418-1735 and 2281-8627 micromol x min/l, respectively. The t(1/2) values were in the ranges 213-692 and 216-354 min, respectively. Cumulative urinary excretion of FT was predominantly as 5-FU and was 2.2-11.9%; the urinary excretion of both fluoro-beta-alanine and uracil was generally maximal between 6 and 18 h. During 28-day courses with twice-daily S-1 administration, 5-FU and uracil generally increased. Before each intake of S-1, 5-FU varied between 0.5 and 1 microM and uracil was in the micromolar range (up to 7 microM), indicating that effective DPD inhibition was maintained during the course. In a biopsy of an esophageal adenocarcinoma metastasis that had regressed, thymidylate synthase, the target of 5-FU, was inhibited 50%, but increased four- to tenfold after relapse in subsequent biopsies. In conclusion, oral S-1 administration resulted in prolonged exposure to micromolar 5-FU concentrations due to DPD inhibition, and the decrease in uracil levels after 6 h followed the pattern of CDHP and indicates reversible DPD inhibition.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacocinética , Neoplasias/metabolismo , Ácido Oxónico/farmacocinética , Piridinas/farmacocinética , Tegafur/farmacocinética , Adulto , Anciano , Antimetabolitos Antineoplásicos/sangre , Antimetabolitos Antineoplásicos/uso terapéutico , Área Bajo la Curva , Disponibilidad Biológica , Relación Dosis-Respuesta a Droga , Combinación de Medicamentos , Femenino , Fluorouracilo/sangre , Fluorouracilo/farmacocinética , Fluorouracilo/orina , Semivida , Humanos , Masculino , Tasa de Depuración Metabólica , Persona de Mediana Edad , Neoplasias/tratamiento farmacológico , Ácido Oxónico/sangre , Ácido Oxónico/uso terapéutico , Piridinas/análisis , Piridinas/sangre , Piridinas/uso terapéutico , Tegafur/análisis , Tegafur/sangre , Tegafur/uso terapéutico , Distribución Tisular , Uracilo/farmacocinéticaRESUMEN
Platelet derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) catalyzes the phosphorolysis of thymidine (TdR) to thymine and deoxyribose-1-phosphate (dR-1-P) and has a pro-angiogenic effect for which dR-1-P may be responsible. Using a purine nucleoside phosphorylase based assay it was found that TdR incubation did not increase dR-1-P accumulation in colon cancer cell line Colo320 and its PD-ECGF/TP transfected variant Colo320TP1. The assay was linear up to 25,000pmol dR-1-P with complete recovery of dR-1-P from cellular extracts. There was a huge discrepancy between thymine production and the measured dR-1-P level, 0.05% of the expected value for dR-1-P was found, indicating that there was a rapid disappearance of dR-1-P. However, in cellular extracts, TdR incubation increased dR-1-P, measurable by trapping, which was inhibited by a thymidine phosphorylase inhibitor. dR-1-P directly added to cellular extracts disappeared within 5-10min. In conclusion, large amounts of dR-1-P are produced by Colo320TP1 cells, which rapidly disappear thus not resulting in a net accumulation of dR-1-P in these cells.
Asunto(s)
Ribosamonofosfatos/metabolismo , Timidina Fosforilasa/metabolismo , Línea Celular , Neoplasias del Colon/metabolismo , Humanos , Cinética , Purina-Nucleósido Fosforilasa/metabolismo , Timidina/metabolismo , Timidina Fosforilasa/genética , Transfección , Células Tumorales CultivadasAsunto(s)
Metotrexato/farmacología , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Antimetabolitos Antineoplásicos/análisis , Antimetabolitos Antineoplásicos/farmacocinética , Antimetabolitos Antineoplásicos/farmacología , Células Sanguíneas/metabolismo , Células Sanguíneas/patología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Resistencia a Antineoplásicos , Inhibidores Enzimáticos/análisis , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Humanos , Lactante , Recién Nacido , Metotrexato/análisis , Metotrexato/farmacocinética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologíaRESUMEN
Non-invasive magnetic resonance spectroscopy (MRS) can be used in the clinic to monitor the pharmacokinetics of the chemotherapeutic drug 5-fluorouracil (5-FU) and the effects of modifiers. We report two studies of 5-FU toxicity in normal tissue--one with patients and the other an animal study. 1) 19F MRS signals from fluoronucleotides, cytotoxic anabolites of 5-FU metabolism, were observed in the livers of two patients treated with 5-FU for colorectal cancer, shown by computed tomography (CT) and ultrasound (US) to have no liver metastases. This is the first report of non-invasive monitoring of toxic 5-FU metabolites in normal human tissues. 2) In animals, carbogen-breathing enhances tumour uptake and the efficacy of 5-FU, and the method is under trial in patients. This study demonstrates that there were no significant effects of carbogen breathing on the levels of 5-FU and its metabolites in normal rat tissues, or on the histology of the tissues assessed after treatment.
Asunto(s)
Dióxido de Carbono/farmacología , Radioisótopos de Flúor/farmacocinética , Fluorouracilo/efectos adversos , Oxígeno/farmacología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/radioterapia , Adenocarcinoma/secundario , Adenocarcinoma/cirugía , Animales , Antimetabolitos Antineoplásicos/uso terapéutico , Disponibilidad Biológica , Médula Ósea/química , Médula Ósea/ultraestructura , Carcinoma de Células Transicionales , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/cirugía , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Terapia Combinada , Resultado Fatal , Femenino , Fluorodesoxiuridilato/análisis , Fluorouracilo/farmacocinética , Fluorouracilo/toxicidad , Humanos , Intestino Delgado/química , Intestino Delgado/ultraestructura , Leucovorina/uso terapéutico , Hígado/química , Hígado/diagnóstico por imagen , Hígado/ultraestructura , Neoplasias Hepáticas/secundario , Imagen por Resonancia Magnética , Persona de Mediana Edad , Neoplasias Primarias Múltiples , Ratas , Ratas Endogámicas WF , Neoplasias del Colon Sigmoide/tratamiento farmacológico , Neoplasias del Colon Sigmoide/radioterapia , Neoplasias del Colon Sigmoide/cirugía , Tomografía Computarizada por Rayos X , Ultrasonografía , Neoplasias de la Vejiga UrinariaRESUMEN
The main mechanism of action of the anticancer drug gemcitabine is assumed to be incorporation of its triphosphate (dFdCTP) into DNA, resulting in inhibition of DNA polymerization, inhibition of DNA synthesis and repair. Another mechanism is inhibition of ribonucleotide reductase leading to imbalance in the deoxyribonucleotide (dNTP) pools. One assay to measure dNTP pools is based on oligonucleotide elongation mediated by DNA polymerase. Since the latter may be affected by dFdCTP, we studied the effect of 0.1-600 pmol dFdCTP on this assay; 10 pmol and more dFdCTP significantly increased the average dpm of the blank (absence of other dNTP) and that of the calibration line of dATP (1.4-1.6-fold); 0.1 pmol and more increased that of the standard dGTP curve significantly (1.1-1.8-fold); 10-75 pmol decreased that of dCTP while 75 and 100 pmol significantly increased that of dCTP (1.3-fold); 50 pmol significantly increased that of dTTP (1.3-1.5-fold). For dATP, dGTP and dTTP, a saturation was reached at 100 pmol dFdCTP, but not yet for dCTP. To minimize these effects, we added an excess of 200 pmol dFdCTP to all samples and calibration lines when measuring dNTP levels of gemcitabine treated samples. In this way the effects of gemcitabine on dNTP levels were studied in human A2780 ovarian, HT29 colon, K562 myelogenous leukemia, H322 non-small cell lung cancer cell lines and the murine lung cancer cell line Lewis Lung. In all cell lines, intrinsic dTTP pools (3-77 pmol/106 cells) were the highest, followed by dATP (1.5-31), dCTP (0.7-27) and (nd-14) dGTP. Exposure to 1 and 10 microM gemcitabine for 4-h concentration dependently decreased dATP 3-10-fold and dGTP to undetectable levels, but dCTP at most 3-fold, while dTTP increased. In conclusion, dFdCTP affects dNTP measurements with the DNA polymerase elongation assay, but its effect could be controlled by addition of similar amounts of dFdCTP to each assay.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxicitidina/farmacología , Desoxirribonucleótidos/metabolismo , Células Tumorales Cultivadas/efectos de los fármacos , ADN de Neoplasias/biosíntesis , ADN de Neoplasias/metabolismo , Desoxicitidina/análogos & derivados , Humanos , Inhibidores de la Síntesis del Ácido Nucleico , GemcitabinaRESUMEN
Acute myeloid leukaemia (AML) is thought to be methotrexate (MTX)-resistant. However, a small study suggested that acute monocytic leukemia (AML-M5) is sensitive to MTX. We measured MTX accumulation/polyglutamylation in 20 AML-nonM5, 37 AML-M5 and 83 common/preB-acute lymphoblastic leukaemia (c/preB-ALL) samples. Membrane transport was determined in 11 childhood AMLs (including 3 AML-M5) and in 25 c/preB-ALL samples. MTX sensitivity was determined in 23 AML-nonM5, 15 AML-M5 and 63 common/preB-ALL samples using the thymidylate synthase (TS) inhibition assay. MTX transport was higher in AML samples compared with c/preB-ALL precluding a transport defect in AML. Accumulation of long-chain polyglutamates MTX-Glu(4-6) was 3-fold lower for AML-nonM5 compared with c/preB-ALL cells (median 268 versus 889 pmol MTX-Glu(4-6)/10(9) cells; P < or = 0.001); for AML-M5 samples, median accumulation of MTX-Glu(4-6) was 0 pmol/10(9) cells (P < or = 0.001). After short-term MTX exposure, AML-nonM5 was 6-fold more resistant to MTX compared with c/preB-ALL cells (2.16 versus 0.39 microM; P < 0.001), while AML-M5 was 2-fold more resistant (P = 0.02). In both AML-nonM5 and AML-M5 cells, MTX resistance was circumvented by continuous MTX exposure (median TSI(50) values: 0.052 and 0.041 microM, respectively) compared with a c/preB-ALL value of 0.066 microM. In conclusion, AML-M5 is relatively sensitive to MTX compared with other AML-subtypes even though polyglutamylation of MTX is poor. Using continuous exposure, AML-nonM5 and AML-M5 cells were at least as sensitive to MTX as c/preB-ALL cells. This report suggests that MTX might be an overlooked drug in the treatment of childhood AML.
Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Leucemia Monocítica Aguda/tratamiento farmacológico , Metotrexato/uso terapéutico , Antimetabolitos Antineoplásicos/farmacocinética , Transporte Biológico , Niño , Resistencia a Antineoplásicos , Humanos , Leucemia Monocítica Aguda/enzimología , Metotrexato/farmacocinética , Timidilato Sintasa/antagonistas & inhibidores , Células Tumorales CultivadasRESUMEN
BACKGROUND: A method that provides standardized data and is relatively inexpensive and capable of high throughput is a prerequisite to the development of a meaningful gene expression database suitable for conducting multi-institutional clinical studies based on expression measurement. Standardized RT (StaRT)-PCR has all these characteristics. In addition, the method must be reproducible. StaRT-PCR has high intralaboratory reproducibility. The purpose of this study is to determine whether StaRT-PCR provides similar interlaboratory reproducibility. METHODS AND RESULTS: In a blinded interlaboratory study, expression of ten genes was measured by StaRT-PCR in a complementary DNA sample provided to each of four laboratories. The average coefficient of variation for interlaboratory comparison of the nine quantifiable genes was 0.48. In all laboratories, expression of one of the genes was too low to be measured. CONCLUSION: Because StaRT-PCR data are standardized and numerical and the method is reproducible among multiple laboratories, it will allow development of a meaningful gene expression database.
Asunto(s)
Perfilación de la Expresión Génica/normas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Unión Competitiva/genética , Línea Celular , ADN Complementario/genética , Bases de Datos Genéticas , Método Doble Ciego , Expresión Génica , Perfilación de la Expresión Génica/clasificación , Perfilación de la Expresión Génica/estadística & datos numéricos , Humanos , Pulmón/química , Pulmón/citología , Pulmón/metabolismo , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/estadística & datos numéricos , Moldes Genéticos , Terminología como AsuntoRESUMEN
Gemcitabine (2'-2'-difluorodeoxycytidine; dFdC) is a deoxycytidine analogue which is effective against solid tumours, including lung cancer and ovarian cancer. dFdC requires phosphorylation by deoxycytidine kinase (dCK) for activation. In the human ovarian cancer cell line A2780 and its 30,000-fold dFdC-resistant variant AG6000 (P<0.001), we investigated the cross-resistance profile to several drugs. AG6000, which has a complete dCK deficiency, was approximately 1000-10,000-fold resistant to other deoxynucleoside analogues such as 1-beta-D-arabinofuranosyl cytosine, 2-chloro-deoxyadenosine, aza-deoxycytidine and 2', 2'-difluorodeoxyguanosine (dFdG) (P<0.001). dFdG can be activated by dCK and deoxyguanosine kinase (dGK), but the latter enzyme was not altered in AG6000 cells. Thus dFdG resistance was only due to dCK deficiency. AG6000 was 1.6- and 46.7-fold resistant to 5-fluorouracil (5-FU) and ZD1694, respectively (the latter was significant; P<0.01), which may be due to the 1.7-fold higher thymidylate synthase (TS) activity, but AG6000 cells were also 2. 7-fold resistant to the lipophilic TS inhibitor AG337 (P<0.05). Remarkably, AG6000 cells were 2.5-fold more sensitive to methotrexate (MTX) (P<0.01) than A2780 cells, but 1.6-fold more resistant to trimetrexate (TMQ) (P<0.10). However, no differences in reduced folate carrier activity, folylpolyglutamate synthetase (FPGS) activity and polyglutamation of MTX were found between the cell lines. AG6000 cells were approximately 2 to 7.5-fold more resistant to doxorubicin (DOX), daunorubicin (DAU), epirubicin and vincristine (VCR) (the latter was significant; P<0.02) and approximately 4-fold more resistant to the microtubule inhibitors paclitaxel and docetaxel (P<0.001). Fluorescent activated cell sorter (FACS) analysis revealed no P-glycoprotein (Pgp) or multidrug resistance-associated protein (MRP) expression, but less fluorescence of intercalated DAU in AG6000 cells. An approximately 2-fold resistance to the topoisomerase I and II inhibitors etoposide, CPT-11 and SN38 was found in AG6000 cells. Topoisomerase I and IIalpha RNA expression was decreased in AG6000 cells. AG6000 was 2.4, 2.4, 2.3 and 3.7-fold more resistant to EO9 (P<0.02), mitomycin-C (MMC) (P<0.05), cisplatin (CDDP) (P<0.10) and maphosphamide (MAPH), respectively. DT-diaphorase (DTD), which activates EO9, was 2.2-fold lower in AG6000 cells. CDDP resistance might be related to a reduced retention of DNA adducts in AG6000. However, glutathione levels were equal in A2780 and AG6000 cells. A 24 h exposure to DOX, VCR and paclitaxel at equimolar and equitoxic concentrations, resulted in more double-strand breaks (1.5- to 2-fold) in A2780 than in AG6000 cells. MAPH at 1120 nM and 17 nM of EO9 did not cause DNA damage in either cell line. In conclusion, AG6000 is a cell line highly cross-resistant to a wide variety of drugs. This cross-resistance might be related to altered enzyme activities and/or increased DNA repair.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Desoxicitidina/uso terapéutico , Resistencia a Antineoplásicos , Neoplasias Ováricas/tratamiento farmacológico , División Celular , Daño del ADN/efectos de los fármacos , ADN de Neoplasias/efectos de los fármacos , Desoxicitidina/análogos & derivados , Ensayos de Selección de Medicamentos Antitumorales , Drogas en Investigación/uso terapéutico , Femenino , Citometría de Flujo , Humanos , Neoplasias Ováricas/patología , Células Tumorales Cultivadas/efectos de los fármacos , GemcitabinaRESUMEN
PURPOSE: To investigate the side effects, determine the maximum-tolerated dose (MTD), and study the pharmacokinetics of S-1, an oral fluoropyrimidine-based antineoplastic agent consisting of the fluorouracil (5-FU) prodrug tegafur combined with two modulators, 5-chloro-2,4-dihydroxypyridine and potassium oxonate. PATIENTS AND METHODS: Patients with advanced solid tumors received S-1 bid for 28 days, followed by 1 week of rest. 5-FU pharmacokinetics were investigated after a single initial dose of S-1 during the first 24 hours and weekly thereafter. RESULTS: Twenty-eight patients received S-1 at the four consecutive dose levels of 25, 45, 35, and 40 mg/m(2). The MTD was initially found at 45 mg/m(2), with diarrhea as the dose-limiting toxicity (DLT). Diarrhea was also the DLT at the dose of 40 mg/m(2), which was the MTD for patients exposed to extensive prior chemotherapy. Other toxicities were generally mild. Two patients had a reduction of more than 50% in tumor dimension. Plasma pharmacokinetics of 5-FU were linear; at the highest S-1 dose level, 5-FU plasma peak concentrations reached 1 to 2 micromol/L, and the half-life of 5-FU was 3 to 4 hours. A statistically significant relationship was observed between the severity of diarrhea and pharmacokinetic parameters of 5-FU. CONCLUSION: The recommended dose of S-1 in chemotherapy-naive or minimally chemotherapy-exposed patients is 40 mg/m(2) bid on 28 consecutive days, every 5 weeks. In heavily pretreated patients, the recommended dose is 35 mg/m(2) bid. Phase II trials are warranted in tumors known to be responsive to 5-FU treatment.
Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Neoplasias/tratamiento farmacológico , Ácido Oxónico/uso terapéutico , Piridinas/uso terapéutico , Tegafur/uso terapéutico , Administración Oral , Adulto , Anciano , Antimetabolitos Antineoplásicos/administración & dosificación , Antimetabolitos Antineoplásicos/farmacocinética , Combinación de Medicamentos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Ácido Oxónico/administración & dosificación , Ácido Oxónico/farmacocinética , Piridinas/administración & dosificación , Piridinas/farmacocinética , Tegafur/administración & dosificación , Tegafur/farmacocinéticaRESUMEN
Treatment failure in childhood acute lymphoblastic leukaemia (ALL) might be associated with methotrexate (MTX) resistance. Little is known about MTX resistance in relapsed ALL. In this study, we determined ex vivo MTX resistance in precursor-B ALL at relapse (rALL) and determined possible defects in MTX membrane transport and polyglutamylation. Using the in situ thymidylate synthase inhibition assay, 21 rALL samples were threefold more MTX resistant than 63 initial precursor-B ALL samples, both after short-term and after continuous MTX exposure (P < or = 0.01). [3H]-MTX membrane transport did not differ between eight rALL and 25 precursor-B ALL samples. Incubation for 24 h with 1 microM [3H]-MTX resulted in a trend towards a lower accumulation of MTX in 20 relapsed than in 83 initial samples of precursor-B ALL samples (906 vs. 1364 pmol/109 cells; P = 0.07). Accumulation of long-chain MTX polyglutamates (MTX-Glu4-6) did not differ between relapsed and newly diagnosed samples (746 and 889 pmol/109 cells; P = 0.1). Activities of the enzymes involved in polyglutamylation (folylpolyglutamate synthetase and folylpolyglutamate hydrolase) did not differ between rALL and untreated c/pre-B-ALL. This study demonstrates that leukaemic cells of children with relapsed precursor-B ALL are relatively MTX resistant, but that this MTX resistance is not associated with major impairments in MTX uptake or polyglutamylation.
Asunto(s)
Linfocitos B/efectos de los fármacos , Metotrexato/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras , Transporte Biológico , Membrana Celular/metabolismo , Resistencia a Antineoplásicos , Humanos , Péptido Sintasas/metabolismo , Recurrencia , Timidilato Sintasa/metabolismo , Células Tumorales Cultivadas , gamma-Glutamil Hidrolasa/metabolismoRESUMEN
Drug resistance of leukemic blasts is correlated to event-free survival and might be predicted by mRNA expression of drug resistance-related proteins. Methotrexate (MTX) is an important component in the treatment of childhood leukemia. Mechanisms of MTX resistance include (1) decreased transport via the reduced folate carrier (RFC), (2) altered levels of target enzymes, eg dihydrofolate reductase (DHFR) and thymidylate synthase (TS), (3) decreased ratio of folylpolyglutamate synthetase (FPGS)/folylpolyglutamate hydrolase (FPGH). We designed competitive templates for each of these genes to measure mRNA expression by quantitative RT-PCR and normalized the expression to that of beta-actin. T-lineage acute lymphoblastic leukemia (T-ALL), relatively MTX resistant compared to common/preB-ALL, displayed higher mRNA levels of DHFR and TS (three- and four-fold higher, respectively; P < 0.001), while FPGS expression was lower (three-fold, P = 0.006) compared to common/preB-ALL. The ratio of (DHFR x FPGH)/(RFC x FPGS) was more discriminating between T-ALL and c/preB-ALL (eight-fold higher; P < 0.001) than either target independently. Acute myeloid leukemia (AML) cells, considered MTX resistant, expressed two-fold lower levels of FPGS mRNA compared to c/preB-ALL (P = 0.04). The ratio of FPGH/FPGS was more discriminating between AML and c/preB-ALL (four-fold higher; P = 0.001) than either target independently. For the total group of 79 leukemic samples, mRNA expression of DHFR varied 549-fold and paralleled TS mRNA expression (r = 0.80; P < 0.001). Although variations in mRNA expression resembled variations in functional activity, no direct correlations were found for RFC (58-fold variation in mRNA expression), FPGS (95-fold) and FPGH (178-fold). In conclusion, differences in mRNA expression of MTX resistance parameters between leukemic subtypes as detected by competitive RT-PCR are in line with known differences in MTX resistance.
Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Linfoma de Burkitt/metabolismo , Leucemia-Linfoma de Células T del Adulto/metabolismo , Metotrexato/uso terapéutico , Proteínas de Neoplasias/genética , ARN Mensajero/genética , Antimetabolitos Antineoplásicos/farmacología , Secuencia de Bases , Linfoma de Burkitt/tratamiento farmacológico , Cartilla de ADN , Leucemia-Linfoma de Células T del Adulto/tratamiento farmacológico , Metotrexato/farmacología , Reacción en Cadena de la Polimerasa , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The aim of the present studies was to elucidate the effects and optimal modulatory conditions of 5-ethyl-2'-deoxyuridine (EtdUrd) on the antitumour efficacy, pharmacokinetics and catabolism of 5-fluorouracil (5-FU) on Colon-26 and Colon-38 murine tumours. HPLC and GC-MS techniques were used to measure the concentrations of 5-FU, dihydro-5-fluorouracil, EtdUrd, 5-ethyluracil and uridine in the plasma and that of 5-FU and 5-fluoro-2'-deoxyuridine monophosphate (FdUMP) in the tumours. It was shown that EtdUrd, given 1 h before 5-FU, selectively enhanced the antitumour action of 5-FU, without significantly increasing its toxic side-effects, thus resulting in an approximately three times higher therapeutic index. Pharmacokinetic studies revealed that 1 h after 400 mg/kg EtdUrd administration - i.e. at the time of 5-FU treatment - the plasma concentration of EtdUrd was 269 microM, and that of 5-ethyluracil, as the major metabolite of EtdUrd, was 421 microM. It is of interest that EtdUrd pretreatment did not change the maximal plasma concentration of 5-FU; however, the half-life of the terminal elimination increased from 114.5 min to 171.2 min and thus the mean residence time of 5-FU rose significantly (P < 0.05). After the combined treatment, the maximal concentration of dihydro-5-fluorouracil in the plasma decreased from 61.06 microM to 29.70 microM (P < 0.01). The intratumoral concentrations of 5-FU were 34%-158% higher 6-96 h after the combined treatment than after the single 5-FU treatment. EtdUrd also caused a moderate increase in the intratumoral level of FdUMP. It is noteworthy, that EtdUrd increased the endogenous uridine concentration in the plasma from 18 microM to a maximum of 249 microM, and the level remained high for longer than 6 h. The present studies indicate that EtdUrd enhances the therapeutic index of 5-FU by reducing the catabolism, prolonging the plasma and intratumoral concentrations of 5-FU and, at the same time, offering protection to normal organs by increasing the endogenous uridine level.