RESUMO
BACKGROUND: Hypoxia is a driving force in pancreatic-ductal-adenocarcinoma (PDAC) growth, metastasis and chemoresistance. The muscle-isoform of lactate dehydrogenase (LDH-A) constitutes a major checkpoint for the switch to anaerobic glycolysis, ensuring supply of energy and anabolites in hypoxic-environments. Therefore, we investigated the molecular mechanisms underlying the pharmacological interaction of novel LDH-A inhibitors in combination with gemcitabine in PDAC cells. METHODS: Lactate dehydrogenase A levels were studied by quantitative RT-PCR, western blot, immunofluorescence and activity assays in 14 PDAC cells, including primary-cell-cultures and spheroids, in normoxic and hypoxic conditions. Cell proliferation, migration and key determinants of drug activity were evaluated by sulforhodamine-B-assay, wound-healing assay, PCR and LC-MS/MS. RESULTS: Lactate dehydrogenase A was significantly increased under hypoxic conditions (1% O2), where the novel LDH-A inhibitors proved to be particularly effective (e.g., with IC50 values of 0.9 vs 16.3 µM for NHI-1 in LPC006 in hypoxia vs normoxia, respectively). These compounds induced apoptosis, affected invasiveness and spheroid-growth, reducing expression of metalloproteinases and cancer-stem-like-cells markers (CD133+). Their synergistic interaction with gemcitabine, with combination index values <0.4 in hypoxia, might also be attributed to modulation of gemcitabine metabolism, overcoming the reduced synthesis of phosphorylated metabolites. CONCLUSION: Lactate dehydrogenase A is a viable target in PDAC, and novel LDH-A inhibitors display synergistic cytotoxic activity with gemcitabine, offering an innovative tool in hypoxic tumours.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Desoxicitidina/análogos & derivados , Inibidores Enzimáticos/farmacologia , L-Lactato Desidrogenase/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Antígeno AC133 , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Carcinoma Ductal Pancreático , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Desoxicitidina/administração & dosagem , Desoxicitidina/farmacologia , Regulação para Baixo , Sinergismo Farmacológico , Proteína Potenciadora do Homólogo 2 de Zeste , Inibidores Enzimáticos/administração & dosagem , Glicoproteínas/biossíntese , Glicoproteínas/genética , Isoenzimas/antagonistas & inibidores , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , L-Lactato Desidrogenase/biossíntese , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5 , Metaloproteases/biossíntese , Metaloproteases/genética , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Peptídeos/genética , Complexo Repressor Polycomb 2/biossíntese , Complexo Repressor Polycomb 2/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esferoides Celulares , Células Tumorais Cultivadas , GencitabinaRESUMO
INTRODUCTION: Glioblastoma (GBM) is an incurable cancer type. New therapeutic options are investigated, including targeting the mitogen-activated protein kinase (MAPK) pathway using MEK inhibitors as radio-sensitizers. In this study, we investigated whether MEK inhibition via PD0325901 leads to radio-sensitization in experimental in vitro and in vivo models of GBM. MATERIALS AND METHODS: In vitro, GBM8 multicellular spheroids were irradiated with 3 fractions of 2 Gy, during 5 consecutive days of incubation with either PD0325901 or MEK-162. In vivo, we combined PD0325901 with radiotherapy in the GBM8 orthotopic mouse model, tumor growth was measured weekly by bioluminescence imaging and overall survival and toxicity were assessed. RESULTS: Regrowth and viability of spheroids monitored until day 18, showed that both MEK inhibitors had an in vitro radio-sensitizing effect. In vivo, PD0325901 concentrations were relatively constant throughout multiple brain areas and temporal PD0325901-related adverse events such as dermatitis were observed in 4 out of 14 mice (29%). Mice that were treated with radiation alone or combined with PD0325901 had significantly better survival compared to vehicle (both P < 0.005), however, no significant interaction between PD0325901 MEK inhibition and irradiation was observed. CONCLUSION: The difference between the radiotherapy-enhancing effect of PD0325901 in vitro and in vivo urges further pharmacodynamic/pharmacokinetic investigation of PD0325901 and possibly other candidate MEK inhibitors.
Assuntos
Glioblastoma , Camundongos , Animais , Glioblastoma/tratamento farmacológico , Glioblastoma/radioterapia , Glioblastoma/patologia , Proteínas Quinases Ativadas por Mitógeno , Benzamidas/farmacologia , Difenilamina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinases de Proteína Quinase Ativadas por Mitógeno/uso terapêutico , Linhagem Celular TumoralRESUMO
BACKGROUND: The aim of this study was to evaluate whether cytidine deaminase (CDA) polymorphisms 79A>C and 435C>T and/or CDA enzymatic activity influenced clinical outcome in 126 advanced non-small-cell lung cancer patients treated with gemcitabine-platinum-regimens. PATIENTS AND METHODS: CDA polymorphisms and activity were analysed by PCR and high-performance liquid chromatography, respectively. Univariate and multivariate analyses compared biological/clinical parameters with response, clinical benefit, time to progression (TtP) and overall survival (OS) using Pearson's χ(2) test, log-rank test and Cox proportional hazards model. RESULTS: Patients with CDA A79A/A79C genotypes had significantly longer TtP (6.0 versus 3.0 months; P = 0.001) and OS (11.0 versus 5.0 months; P = 0.001) than patients with C79C genotype. Patients harbouring CDA C435C/C435T genotypes also had a longer OS (P = 0.025), but no correlations were observed with TtP. Conversely, patients with low-CDA activity had a significantly higher response rate (37.7% versus 13.8%; P = 0.006), clinical benefit (91.8% versus 51.7%; P < 0.001), as well as longer TtP (8.0 versus 3.0 months; P < 0.001) and OS (19.0 versus 6.0 months; P < 0.001). Furthermore, enzymatic activity emerged as an independent predictor for death/progression risk at multivariate analysis. CONCLUSIONS: CDA enzymatic activity appears to be the strongest candidate biomarker of activity and efficacy of platinum-gemcitabine-based chemotherapy and should be validated in a prospective study.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Citidina Desaminase/genética , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/genética , Polimorfismo Genético , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Área Sob a Curva , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Cromatografia Líquida de Alta Pressão , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Intervalo Livre de Doença , Genótipo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/enzimologia , Estadiamento de Neoplasias , Compostos de Platina/administração & dosagem , Modelos de Riscos Proporcionais , Curva ROC , Reação em Cadeia da Polimerase Via Transcriptase Reversa , GencitabinaRESUMO
BACKGROUND: (Pre)clinical evidence is accumulating that intermittent exposure to increased doses of protein kinase inhibitors may improve their treatment benefit. In this phase I trial, the safety of high-dose, pulsatile sorafenib was studied. PATIENTS AND METHODS: High-dose sorafenib was administered once weekly in exposure escalation cohorts according to a 3 + 3 design. Drug monitoring was performed in weeks 1-3 and doses were adjusted to achieve a predefined target plasma area under the curve (AUC)(0-12 h). The effect of low gastric pH on improving sorafenib exposure was investigated by intake of the acidic beverage cola. RESULTS: Seventeen patients with advanced malignancies without standard treatment options were included. Once weekly, high-dose sorafenib exposure was escalated up to a target AUC(0-12 h) of 125-150 mg/L/h, achieving a twofold higher Cmax compared to standard continuous dosing. Dose-limiting toxicity was observed in three patients: grade 3 duodenal perforation (2800 mg sorafenib), grade 5 multiorgan failure (2800 mg sorafenib) and grade 5 biliary tract perforation (3600 mg sorafenib). The mean difference between observed and target AUC(0-12 h) was 45% (SD ± 56%) in week 1 using a fixed starting dose of sorafenib compared to 2% (SD ± 32%) in week 3 as a result of drug monitoring (P = 0.06). Dissolving sorafenib in cola, instead of water, did not improve sorafenib exposure. Clinical benefit with stable disease as the best response was observed in two patients. CONCLUSION: Treatment with high-dose, once weekly sorafenib administration resulted in dose-limiting toxicity precluding dose escalation above the exposure cohort of 125-150 mg/L/h. Drug monitoring was a successful strategy to pursue a target exposure.
Assuntos
Neoplasias/tratamento farmacológico , Pulsoterapia/métodos , Sorafenibe , Área Sob a Curva , Relação Dose-Resposta a Droga , Esquema de Medicação , Monitoramento de Medicamentos/métodos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/prevenção & controle , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacocinética , Sorafenibe/administração & dosagem , Sorafenibe/efeitos adversos , Sorafenibe/farmacocinética , Resultado do TratamentoRESUMO
Glioblastoma multiforme (GBM), the most frequent malignant brain tumor, has a poor prognosis, but is relatively sensitive to radiation. Both gemcitabine and its metabolite difluorodeoxyuridine (dFdU) are potent radiosensitizers. The aim of this phase 0 study was to investigate whether gemcitabine passes the blood-tumor barrier, and is phosphorylated in the tumor by deoxycytidine kinase (dCK) to gemcitabine nucleotides in order to enable radiosensitization, and whether it is deaminated by deoxycytidine deaminase (dCDA) to dFdU. Gemcitabine was administered at 500 or 1000 mg/m(2) just before surgery to 10 GBM patients, who were biopsied after 1-4 h. Plasma gemcitabine and dFdU levels varied between 0.9 and 9.2 microM and 24.9 and 72.6 microM, respectively. Tumor gemcitabine and dFdU levels varied from 60 to 3580 pmol/g tissue and from 29 to 72 nmol/g tissue, respectively. The gene expression of dCK (beta-actin ratio) varied between 0.44 and 2.56. The dCK and dCDA activities varied from 1.06 to 2.32 nmol/h/mg protein and from 1.51 to 5.50 nmol/h/mg protein, respectively. These enzyme levels were sufficient to enable gemcitabine phosphorylation, leading to 130-3083 pmol gemcitabine nucleotides/g tissue. These data demonstrate for the first time that gemcitabine passes the blood-tumor barrier in GBM patients. In tumor samples, both gemcitabine and dFdU concentrations are high enough to enable radiosensitization, which warrants clinical studies using gemcitabine in combination with radiation.
Assuntos
Neoplasias Encefálicas/metabolismo , Desoxicitidina/análogos & derivados , Glioblastoma/metabolismo , Radiossensibilizantes/farmacocinética , Adulto , Idoso , Antimetabólitos Antineoplásicos/sangue , Antimetabólitos Antineoplásicos/farmacocinética , Antimetabólitos Antineoplásicos/uso terapêutico , Disponibilidade Biológica , Biópsia , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/cirurgia , Citidina Desaminase , Desoxicitidina/sangue , Desoxicitidina/farmacocinética , Desoxicitidina/uso terapêutico , Desoxicitidina Quinase/metabolismo , Feminino , Floxuridina/sangue , Floxuridina/farmacocinética , Floxuridina/uso terapêutico , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Nucleosídeo Desaminases/metabolismo , Radiossensibilizantes/uso terapêutico , GencitabinaRESUMO
PURPOSE: Preclinical research and prior clinical observations demonstrated reduced toxicity and suggested enhanced efficacy of cisplatin due to folic acid and vitamin B12 suppletion. In this randomized phase 2 trial, we evaluated the addition of folic acid and vitamin B12 to first-line palliative cisplatin and gemcitabine in patients with advanced esophagogastric cancer (AEGC). METHODS: Patients with AEGC were randomized to gemcitabine 1250 mg/m2 (i.v. days 1, 8) and cisplatin 80 mg/m2 (i.v. day 1) q 3 weeks with or without folic acid (450 µg/day p.o.) and vitamin B12 (1000 µg i.m. q 9 weeks). The primary endpoint was response rate (RR). Secondary endpoints included overall survival (OS), time to progression (TTP), toxicity, and exploratory biomarker analyses. Cisplatin sensitivity and intracellular platinum levels were determined in adenocarcinoma cell lines cultured under high and low folate conditions in vitro. RESULTS: Adenocarcinoma cells cultured in medium with high folate levels were more sensitive to cisplatin and this was associated with increased intracellular platinum levels. In the randomized phase 2 clinical trial, which ran from October 2004 to September 2013, treatment was initiated in 78 of 82 randomized pts, 39 in each study arm. The RR was similar; 42.1% for supplemented patients vs. 32.4% for unsupplemented patients; p = 0.4. Median OS and TTP were 10.0 and 5.9 months for supplemented vs. 7.7 and 5.4 months for unsupplemented patients (OS, p = 0.9; TTP, p = 0.9). Plasma homocysteine was lower in the supplemented group [n = 20, 6.9 ± 1.6 (mean ± standard error of mean, SEM) µM; vs. 12.5 ± 4.0 µM; p < 0.001]. There was no significant difference in the Cmax of gemcitabine and cisplatin in the two treatment groups. CONCLUSION: Folic acid and vitamin B12 supplementation do not improve the RR, PFS, or OS of cisplatin and gemcitabine in patients with AEGC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Esofágicas/tratamento farmacológico , Ácido Fólico/administração & dosagem , Neoplasias Gástricas/tratamento farmacológico , Vitamina B 12/administração & dosagem , Adenocarcinoma/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Cisplatino/efeitos adversos , Cisplatino/farmacocinética , Desoxicitidina/administração & dosagem , Desoxicitidina/efeitos adversos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacocinética , Suplementos Nutricionais , Sinergismo Farmacológico , Neoplasias Esofágicas/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Gástricas/metabolismo , GencitabinaRESUMO
A fast, sensitive and accurate method for the determination of gemcitabine (difluorodeoxycytidine; dFdC) and deoxycytidine (CdR) in human plasma/tissue was developed using LC-MS/MS techniques. Effectiveness of the method is illustrated with the analysis of plasma from a phase I trial of dFdC administered as a 24h infusion. The method was developed using (15)N(3) CdR as an internal standard across the concentration range of 1-500ng/ml, using a cold alcohol-protein precipitation followed by desorption with freeze drying. Sample clean-up for LC-MS/MS analysis was performed by an innovative liquid/liquid back extraction with ethyl acetate and water. Chromatography was performed using a Chrompak-spherisorb-phenyl-column (3.1mmx200mm, 5microm) with a 50mM formic acid: acetonitrile (9:1) mobile phase eluted at 1ml/min. Extracted samples were observed to be stable for a minimum of 48h after extraction when kept at 4 degrees C. Detection was performed using an atmospheric pressure chemical ionization (APCI) source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) for dFdC (264 m/z; 112 m/z), CdR (228 m/z; 112 m/z), and (15)N(3) CdR (231 m/z; 115 m/z) at an ion voltage of +3500V. The accuracy, precision and limit-of-quantitation (LOQ) were as follows: dFdC: 99.8%, +/-7.9%, 19nM; CdR: 100.0%, +/-5.3%, 22nM, linear range LOQ to 2microM. During 24h infusion dFdC levels were detected with no interference from either CdR or difluorodeoxyuridine (dFdU). CdR co-eluted with dFdC but selectivity demonstrated no "crosstalk" between the compounds. In conclusion the analytical assay was very sensitive, reliable and robust for the determination of plasma and tissue concentrations of dFdC and CdR.
Assuntos
Desoxicitidina/análogos & derivados , Desoxicitidina/análise , Espectrometria de Massas em Tandem/métodos , Cromatografia Líquida de Alta Pressão , Ensaios Clínicos Fase I como Assunto , Desoxicitidina/sangue , Desoxicitidina/farmacocinética , Humanos , Reprodutibilidade dos Testes , Distribuição Tecidual , GencitabinaRESUMO
Deoxycytidine (CdR) analogs are increasingly popular as chemotherapeutic agents and their effectiveness can be linked to the direct competition with active forms of endogenous CdR. A tandem mass spectrometric assay was developed to determine the plasma concentrations of CdR. Plasma extracts were prepared by protein precipitation and an ethyl acetate/water back extraction, and then separated chromatographically. Detection parameters were optimized for multi-reaction monitoring (MRM) tandem mass spectrometry and assay efficiency was improved using 15N3 CdR as an isotopic internal standard. Preliminary results from a gemcitabine trial are shown which indicate that CdR concentrations increase systemically during infusion, from about 5 nM to 78 nM after hepatic artery infusion and to 102 nM after systemic infusion for 24 hours. The developed assay demonstrated good sensitivity and selectivity for CdR.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/biossíntese , Desoxicitidina/farmacocinética , Antineoplásicos/farmacologia , Química Clínica/métodos , Cromatografia/métodos , Desoxicitidina/sangue , Desoxicitidina/farmacologia , Humanos , Espectrometria de Massas , Modelos Biológicos , Modelos Químicos , Sensibilidade e Especificidade , Fatores de Tempo , GencitabinaRESUMO
The pulmonary deposition and pharmacokinetics of fine and coarse radioactive aerosols of nedocromil sodium, of mass median aerodynamic diameters 16 microns and 24 microns respectively, delivered by metered dose inhaler (MDI) have been investigated. The corresponding geometric standard deviations of the particle size distributions were 5.32 and 3.93. Pulmonary deposition was assessed by both planar radionuclide scintigraphy and multi-modality three dimensional imaging using single photon emission computed tomography (SPECT) and x-ray computed tomography (CT). The three dimensional data were analysed by transformation to a hemispherical shape based on the fractional radial distance of each point in the lung from the centre to the corresponding extrapolated point on the periphery. This enabled parameters on the variation of both concentration of deposition and total amount deposited with penetration distance to be calculated. For both planar and SPECT data the central to peripheral concentration ratio (C/P ratio) was calculated. The three dimensional C/P ratio showed a median value (3.21) which was significantly higher than for the planar imaging (2.03) (p < 0.001). The parameter used to express the variation of total amount deposited was the median dose position. This showed that for both aerosols 50% of the dose was deposited at sites with a percentage central to peripheral distance of greater than 68%. There was a trend for total percentage of the fine aerosol in the lungs to be higher than for the coarse and for its deposition to be more peripheral. In addition the mean concentrations in blood were measured to be greater for the fine aerosol. However these differences were relatively small and none were individually statistically significant. The technique of combined SPECT and CT imaging was shown to be valuable in obtaining more accurate information on pulmonary distribution of inhaled aerosol deposition. The merits, limitations and potential applications of the technique are discussed.
Assuntos
Aerossóis/administração & dosagem , Antiasmáticos/administração & dosagem , Pulmão/diagnóstico por imagem , Nedocromil/administração & dosagem , Tecnécio , Tomografia Computadorizada de Emissão de Fóton Único , Administração por Inalação , Adulto , Aerossóis/farmacocinética , Antiasmáticos/farmacocinética , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Nebulizadores e Vaporizadores , Nedocromil/farmacocinética , Tamanho da Partícula , Tomografia Computadorizada por Raios X/métodosRESUMO
Consumption of caffeine containing products is very popular in African children, particularly during ill health in the belief that caffeine promotes good health. This study aims to define the metabolism of caffeine, which takes place in the liver in a group of healthy Nigerian children. About 100 mg of caffeine was ingested after an overnight fast. Urine was collected before caffeine ingestion and over 12-hour periods for 36 hours in 13 healthy Nigerian children. The percentage of caffeine and metabolites recovered in urine was determined by high performance liquid chromatography. The total urinary caffeine and metabolites recovered over the 36-hour sampling period was 63.6%, with only 0.4% of the caffeine dose ingested recovered as unchanged caffeine during the same period. Insignificant amounts of 3,7-dimethyluric acid (0.2%), 3-methyluric acid (0.3%) and 1,3,7-dimethyluric acid (0.4) were recovered in the 36hour urine sample. This study also found that the N3-demethylation pathway was the principal pathway of caffeine metabolism accounting for 83.3% of the total metabolites recovered while C8-hydroxylation accounted for only 0.6% of metabolites recovered. The pattern of urinary metabolites recovered suggested that N3-demethylation is the principal pathway of caffeine metabolism in healthy African children and that small amounts of unchanged caffeine, as well as 3,7-dimethyluric acid, 3-methyluric acid and 1,3,7-dimethyluric acid were recovered during the sampling period.
Assuntos
Cafeína/farmacocinética , Cafeína/urina , Estimulantes do Sistema Nervoso Central/farmacocinética , Estimulantes do Sistema Nervoso Central/urina , Administração Oral , África , Fatores Etários , Cafeína/administração & dosagem , Estimulantes do Sistema Nervoso Central/administração & dosagem , Criança , Cromatografia Líquida de Alta Pressão , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Valores de Referência , Fatores de TempoRESUMO
Combination of drugs with different targets is a logical approach to overcome multilevel cross-stimulation among key pathways in NSCLC progression such as EGFR, K-Ras and VEGFR. The sorafenib-erlotinib combination showed clinical activity and acceptable safety. Therefore, we evaluated mechanisms underlying sorafenib-erlotinib interaction in seven NSCLC cell lines selected for their heterogeneous pattern of EGFR and Raf-kinase-inhibitor protein (RKIP) expression, and EGFR/K-Ras mutations. Pharmacologic interaction was studied using MTT/SRB assays and the combination index (CI) method, while effects on EGFR, Erk1/2 and Akt phosphorylation, cell cycle and apoptosis were studied with western-blot, ELISA, and flow cytometry. Intracellular drug concentrations were measured with LC-MS/MS, whereas kinase activity profiles were generated on tyrosine kinase peptide substrate arrays. Synergism was detected in all cell lines, with CIs < 0.6 in K-Ras mutated A549, SW1573 and H460, as well as in H1975 (EGFR-T790M) cells. Sorafenib slowed cell cycle progression and induced apoptosis, which was significantly increased in the combination. Moreover, sorafenib reduced Akt/ERK phosphorylation in erlotinib-resistant cells, associated with significant RKIP up-regulation. No direct drug interaction was detected by LC-MS/MS measurement, while lysates from A549 and H1975 cells exposed to erlotinib+sorafenib showed a significant inhibition in the phosphorylation of 16 overlapping peptides, including sites from RAF, VEGFR2, PDGFR, CDK2 and SRC, suggesting new markers to identify NSCLC patients who are likely to respond to this treatment. In conclusion, several mechanisms, including apoptosis-induction, modulation of expression/phosphorylation of RKIP and crucial kinases contribute to erlotinib-sorafenib synergistic interaction and should be evaluated in future trials for the rational development of this combination in NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Quinazolinas/farmacologia , Apoptose/efeitos dos fármacos , Western Blotting , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cromatografia Líquida , Sinergismo Farmacológico , Ensaio de Imunoadsorção Enzimática , Cloridrato de Erlotinib , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Terapia de Alvo Molecular , Niacinamida/administração & dosagem , Niacinamida/farmacologia , Compostos de Fenilureia/administração & dosagem , Proteína de Ligação a Fosfatidiletanolamina , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/administração & dosagem , Sorafenibe , Espectrometria de Massas em TandemRESUMO
A fast, sensitive, universal and accurate method for the determination of four different tyrosine kinase inhibitors from biological material was developed using LC-MS/MS techniques. Utilizing a simple protein precipitation with acetonitrile a 20 microl sample volume of biological matrixes can be extracted at 4 degrees C with minimal effort. After centrifugation the sample extract is introduced directly onto the LC-MS/MS system without further clean-up and assayed across a linear range of 1-4000 ng/ml. Chromatography was performed using a Dionex Ultimate 3000 with a Phenomenex prodigy ODS3 (2.0 mm x 100 mm, 3 microm) column and eluted at 200 microl/min with a tertiary mobile phase consisting of 20mM ammonium acetate:acetonitrile:methanol (2.5:6.7:8.3%). Injection volume varied from 0.1 microl to 1 microl depending on the concentration of the drug observed. Samples were observed to be stable for a maximum of 48 h after extraction when kept at 4 degrees C. Detection was performed using a turbo-spray ionization source and mass spectrometric positive multi-reaction-monitoring-mode (+MRM) for Gefitinib (447.1 m/z; 127.9 m/z), Erlotinib (393.9 m/z; 278.2 m/z), Sunitinib (399.1 m/z; 283.1 m/z) and Sorafenib (465.0 m/z; 251.9 m/z) at an ion voltage of +3500 V. The accuracy, precision and limit-of-quantification (LOQ) from cell culture medium were as follows: Gefitinib: 100.2+/-3.8%, 11.2 nM; Erlotinib: 101.6+/-3.7%, 12.7 nM; Sunitinib: 100.8+/-4.3%, 12.6 nM; Sorafenib: 93.9+/-3.0%, 10.8 nM, respectively. This was reproducible for plasma, whole blood, and serum. The method was observed to be linear between the LOQ and 4000 ng/ml for each analyte. Effectiveness of the method is illustrated with the analysis of samples from a cellular accumulation investigation and from determination of steady state concentrations in clinically treated patients.
Assuntos
Cromatografia Líquida/métodos , Inibidores de Proteínas Quinases/análise , Espectrometria de Massas em Tandem/métodos , Acetonitrilas/química , Estabilidade de Medicamentos , Humanos , Modelos Lineares , Neoplasias/sangue , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/sangue , Inibidores de Proteínas Quinases/química , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Temozolomide (TMZ) exerts its cytotoxic effects by methylating guanine in DNA, resulting in a mismatch with thymine. We studied possible enhancement of the cytotoxic activity of several other targeted drugs in four lung cancer cell lines by TMZ. the data are in relation to O(6)-alkylguanine-DNA-alkyltransferase (AGT) expression, gene methylation, cell cycle distribution and adduct formation. Synergism/additivity was found with O(6)-BG), gemcitabine, lonafarnib and paclitaxel, but not with platinum analogs and topoisomerase-inhibitors. O(6)-BG enhanced TMZ-induced accumulation in the G2/m-phase by increasing formation and retention of the O(6)-methyldeoxyguanosine adducts. TMZ combinations with drugs showing a different individual effect on the cell cycle (e.g. gemcitabine-induced S-phase) were most effective. The results show that O(6)-BG enhanced the TMZ effect in all cell lines. TMZ enhanced the cytotoxicity of gemcitabine, paclitaxel and lonafarnib in most cell lines, possibly by affecting the cell cycle, supporting possible application of TMZ in the treatment of lung cancer.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Ciclo Celular/efeitos dos fármacos , Adutos de DNA/metabolismo , Dacarbazina/análogos & derivados , Neoplasias Pulmonares/tratamento farmacológico , Animais , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Metilação de DNA , Dacarbazina/farmacologia , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxiguanosina/análogos & derivados , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/patologia , Camundongos , O(6)-Metilguanina-DNA Metiltransferase/genética , Paclitaxel/farmacologia , Piperidinas/farmacologia , Piridinas/farmacologia , Temozolomida , GencitabinaRESUMO
PURPOSE: This study was performed to assess the toxicities, the maximum-tolerated dose (MTD), the pharmacokinetics and the anti-tumour activity of gemcitabine given by 24-h hepatic arterial infusion (HAI). PATIENTS AND METHODS: Patients with liver malignancies received gemcitabine by 24-h HAI, weekly x 3, every 4 weeks. On day 1 or day 8 of the first cycle, patients received one administration by 24-h intravenous infusion for pharmacokinetic comparison and to determine hepatic extraction. RESULTS: Thirteen patients received gemcitabine at the dose levels of 75, 135 and 180 mg/m(2). The MTD was 180 mg/m(2) with thrombocytopaenia as the dose-limiting toxicity. Pharmacokinetic analysis showed a significantly lower maximum gemcitabine plasma concentration (C(max): HAI, 26, 80 and 128 nM, respectively; IV, 229, 264 and 293 nM, respectively) and area under the plasma-concentration-versus-time curve (AUC(0-24h): HAI, 386, 1247 and 2033 nmol x h/L, respectively; IV, 3526, 4818 and 5363 nmol x h/L, respectively) during HAI, compared with intravenous infusion (both P<0.001). Additionally, the mean hepatic extraction ratios of gemcitabine at the 75, 135 and 180 mg/m(2) dose level were 0.89, 0.75 and 0.55, respectively. Hepatic extraction decreased linearly with increasing dose. The C(max) and AUC(0-24h) of 2',2'-difluoro-2'-deoxyuridine, the deaminated product of gemcitabine, were similar for HAI and intravenous infusion. Seven patients had stable disease for a median duration of 9 months (range: 2-11 months). CONCLUSIONS: Gemcitabine given by 24-h HAI was well tolerated and resulted in significantly lower systemic gemcitabine plasma concentrations than intravenous infusion due to a relatively high hepatic extraction.
Assuntos
Antimetabólitos Antineoplásicos , Desoxicitidina/análogos & derivados , Neoplasias Hepáticas , Adulto , Idoso , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/efeitos adversos , Antimetabólitos Antineoplásicos/farmacocinética , Desoxicitidina/administração & dosagem , Desoxicitidina/efeitos adversos , Desoxicitidina/farmacocinética , Feminino , Artéria Hepática , Humanos , Infusões Intra-Arteriais , Infusões Intravenosas , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Trombocitopenia/etiologia , Resultado do Tratamento , GencitabinaRESUMO
We have investigated the pharmacokinetics and pharmacodynamics of nedocromil sodium administered by inhalation (4 mg), by mouth (80 mg) and by intravenous infusion (6 micrograms/kg) against adenosine-5'-monophosphate (AMP) induced bronchoconstriction in 9 mildly asthmatic subjects. Their mean baseline FEV1 was 83.8% of the predicted value, geometric mean (GM) PC20 FEV1 histamine 0.33 mg/ml (range 0.03-4.0), and GM PC50 baseline sGaw AMP 4.37 mg/ml (range 0.158-26.38). Blood collected at intervals for up to 4 h after drug administration was assayed for nedocromil sodium by radioimmunoassay, to derive the maximal drug concentration (Cmax), concentration at time of AMP challenge (Cchall), area under the plasma concentration-time curve (AUC), and the mean residence time (MRT). Airways responsiveness to inhaled AMP was assessed before and after administration of nedocromil sodium by each of the three routes of administration, and the protective efficacy was expressed as the concentration ratio (CR). AMP challenge was carried out 60 min after inhaled nedocromil sodium, 120 min after drug ingestion and 75 min after the start of the intravenous infusion. The GM PC50 sGaw AMP determined after inhaled, oral and intravenous nedocromil sodium was 28.84, 7.24 and 7.94 mg/ml respectively. For the group as a whole, nedocromil sodium by the oral or intravenous routes produced little overall protection against AMP (CR increasing by 0.75 and 0.88 doubling dilutions respectively). However, with inhaled drug, the concentration response curve with AMP was displaced to the right achieving an increased CR of 2.71 doubling dilutions. No relationship could be established between baseline FEV1, and AUC, Cmax or Cchall, or between these indices and the protective efficacy for the drug.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Monofosfato de Adenosina/antagonistas & inibidores , Anti-Inflamatórios não Esteroides/administração & dosagem , Asma/prevenção & controle , Broncoconstrição/efeitos dos fármacos , Quinolonas/administração & dosagem , Administração por Inalação , Administração Oral , Adulto , Anti-Inflamatórios não Esteroides/farmacocinética , Asma/metabolismo , Testes de Provocação Brônquica , Feminino , Humanos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Nedocromil , Quinolonas/farmacocinética , Testes de Função RespiratóriaRESUMO
1. Eight healthy subjects inhaled nedocromil sodium from a metered-dose inhaler using a standardised inspiratory technique. Blood samples were taken for up to 270 min after inhalation for radioimmunoassay of plasma nedocromil sodium concentrations. 2. To investigate the possibility that respiratory manoeuvres can alter the absorption of the drug from the lungs, on the first (control) study day at 70 min after dosing, subjects performed nine forced expiratory manoeuvres over a 3 min period. At 110 min after dosing, subjects took a slow, full inspiration with a 30 s breath-hold, and at 150 min after dosing the subjects performed one single forced expiration. 3. On the second study day, subjects inhaled methoxamine, 0.15 mg kg-1 of a 20 mg ml-1 solution at 60 min after dosing, and the study continued as above. On the third day, subjects repeated the sequence of respiratory manoeuvres, after having taken phenoxymethyl penicillin and probenecid by mouth for 48 h. 4. Both multiple forced expirations and the deep inspiration with breath-hold produced significant increases in the absorption of nedocromil sodium. Inhaled methoxamine did not alter airway calibre or the response to the respiratory manoeuvres. Probenecid, but not penicillin, was detected in the subjects' plasma, and had the effect of increasing the rise in plasma nedocromil sodium concentrations after the multiple forced expirations when compared with the control day. 5. These data suggest that disruption of epithelial tight junctions induced by the respiratory manoeuvres leads to enhanced paracellular transport of nedocromil sodium into the draining circulation of the airways and alveoli.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Anti-Inflamatórios não Esteroides/farmacocinética , Quinolonas/farmacocinética , Respiração , Administração por Inalação , Adolescente , Adulto , Feminino , Humanos , Masculino , Metoxamina/farmacologia , Nedocromil , Probenecid/farmacologia , Quinolonas/administração & dosagemRESUMO
The teratogenicity of all-trans-retinoic acid, 13-cis-retinoic acid, and retinol was investigated in pregnant Wistar rats given a single oral dose on Day 10 of gestation. External malformations showed dose-dependent increases and the order of potency was all-trans-retinoic acid > retinol > 13-cis-retinoic acid. The metabolites in maternal plasma were determined following a single oral dose on Day 10 of gestation. Equipotent teratogenic doses of all-trans-retinoic acid and 13-cis-retinoic acid had similar plasma levels of all-trans-retinoic acid; however, retinol teratogenicity could not be accounted for by circulating all-trans-retinoic acid or its metabolites. The teratogenicity and maternal pharmacokinetics of all-trans-retinoic were compared in pregnant Wistar rats when given as a single dose (50 mg/kg) and as three equal doses (16.66 mg/kg) over 6 hr. Divided doses were of slightly greater potency than the single dose but the maximum observed concentration (Cmax) and area under the plasma concentration-time curve (AUC) values for the second and third doses were greatly attenuated compared with the first dose; in consequence, both the total AUC and Cmax were reduced compared with the single dose. The altered profile could not be explained by increased formation of all-trans-retinoic acid glucuronide or increased isomerisation to 13-cis-retinoic acid. The maternal plasma levels of all-trans-retinoic acid in pregnant rabbits were reduced by a dose given 24 hr earlier. These data show that all-trans-retinoic acid in maternal plasma is a poor indicator of fetal exposure to teratogenic risk.
Assuntos
Anormalidades Induzidas por Medicamentos , Prenhez/metabolismo , Tretinoína/toxicidade , Animais , Feminino , Glucuronatos/metabolismo , Masculino , Gravidez , RNA Mensageiro/análise , Coelhos , Ratos , Ratos Wistar , Receptores do Ácido Retinoico/genética , Tretinoína/administração & dosagem , Tretinoína/farmacocinéticaRESUMO
OBJECTIVES: Caffeine-containing beverages are generally consumed by Nigerians suffering from malaria and kwashiorkor in the belief that caffeine aids early recovery from these illnesses, which are common in the tropics. However, there are no studies on the influence of these diseases on the absorption and pharmacokinetics of caffeine in Africans. MATERIALS AND METHODS: A single oral dose of caffeine was given to five healthy children and to five and seven children suffering from malaria and kwashiorkor, respectively. Caffeine and its dimethylxanthine metabolites were measured in plasma using high-performance liquid chromatography. RESULTS: The maximum plasma concentration (Cmax) of caffeine and the time of Cmax were similar (P > 0.05) in the three groups. However, the elimination half-life of caffeine was significantly longer in children with malaria (9.2 +/- 3.5 h) (P < 0.01) and kwashiorkor (13.1 +/- 7.9 h) (P < 0.05) than in the healthy controls (3.7 +/- 1.8 h). The total plasma oral clearance of caffeine of 4.4 +/- 1.9 ml/min/kg in healthy children was significantly higher (P < 0.01) than in those with kwashiorkor (2.0 +/- 0.9 ml/min/kg) and malaria (1.6 +/- 1.0 ml/min/ kg) (P < 0.05). Paraxanthine was the principal metabolite in all the three groups with Cmax significantly higher in healthy children (1.3 +/- 0.3 microg/ml) than in children with malaria (0.8 +/- 0.4 microg/ml) (P < 0.05) and kwashiorkor (0.3 +/- 0.1 microg/ml) (P < 0.0001). CYP1A2 activity, measured by the plasma ratios of paraxanthine: caffeine, was significantly lower in kwashiorkor and malaria. CONCLUSIONS: This study showed that the plasma kinetics of caffeine are significantly altered in malaria and kwashiorkor, and CYP1A2 activity was lower in these two disease groups.
Assuntos
Cafeína/farmacocinética , Kwashiorkor/metabolismo , Malária/metabolismo , Administração Oral , Cafeína/sangue , Criança , Pré-Escolar , Citocromo P-450 CYP1A2/metabolismo , Feminino , Meia-Vida , Humanos , Lactente , Kwashiorkor/sangue , Malária/sangue , Masculino , Taxa de Depuração Metabólica , Nigéria , Teofilina/metabolismoRESUMO
OBJECTIVES: The pharmacokinetics of caffeine and its dimethylxanthine metabolites were evaluated in Nigerians, for whom it is normal to consume caffeine-containing beverages during ill health and recuperation in the belief that caffeine aids early recovery from illness; however, there are no data defining the kinetics of caffeine in healthy and ill Nigerians. MATERIALS AND METHODS: A single oral dose of 300 mg caffeine was given to ten healthy adult Nigerians and ten adults suffering from acute uncomplicated Plasmodium falciparum malaria infection. Caffeine and its dimethylxanthine metabolites were measured in plasma and saliva of healthy subjects and in plasma of patients suffering from malaria using high-performance liquid chromatography. RESULTS: The plasma pharmacokinetics of caffeine per se in both groups was similar (P > 0.05). The maximum plasma concentration (Cmax) of paraxanthine was significantly lower (P < 0.05) in malaria (0.9 +/- 0.4 microg/ ml) than in healthy controls (1.4 +/- 0.5 microg/ml), and the paraxanthine:caffeine area under the plasma concentration time curve ratio, an index of cytochrome P450 (CYP)IA2 activity was significantly lower (P < 0.05) in malaria patients (0.5 +/- 0.1) than in healthy controls (0.3 +/- 0.2). The elimination half-life of theophylline was longer in malaria, while the area under the plasma concentration time curve of theobromine was significantly higher (P < 0.05) in malaria (7.1 +/- 3.4 microg ml(-1) h) than in healthy adults (4.1 +/- 2.2 microg ml(-1) h). Excellent correlations were found between saliva and plasma concentrations of caffeine (r2 = 0.98) with a mean saliva:plasma concentrations ratio of 0.7 +/- 0.1. The plasma concentrations (Cmax and AUC) were therefore higher than the corresponding salivary levels, so that the apparent oral clearance calculated for saliva exceeded the true oral clearance based on plasma data. CONCLUSIONS: Acute Plasmodium falciparum malaria produced significant changes in the disposition of caffeine metabolites. Analysis of concentrations in saliva is a useful non-invasive method for monitoring the kinetics of caffeine and paraxanthine in Nigerians.
Assuntos
Cafeína/farmacocinética , Malária Falciparum/metabolismo , Saliva/química , Doença Aguda , Administração Oral , Adolescente , Adulto , Animais , Cafeína/sangue , Criança , Citocromo P-450 CYP1A2/metabolismo , Feminino , Humanos , Malária Falciparum/sangue , Masculino , Taxa de Depuração Metabólica , Nigéria , Teofilina/sangue , Teofilina/metabolismoRESUMO
1. The pharmacokinetics of doxazosin, following a single oral dose (1 mg) and chronic oral dosing (doubling doses up to a maximum of 16 mg day-1), were studied in 18 patients with mild to moderate hypertension and stable renal function varying from normal to severely impaired. In addition, the effect of chronic administration of doxazosin on renal haemodynamics was evaluated. 2. Significant accumulation of doxazosin occurred with chronic dosing, but comparison of dose-adjusted AUC after single and chronic dosing suggested that there was no change in clearance or bioavailability during chronic administration. 3. There was no significant relationship between plasma elimination half-life or the AUC for doxazosin and the degree of renal impairment. 4. Symptomatic postural hypotension occurred in six patients following the initial dose of 1 mg doxazosin. Systolic and diastolic blood pressure measured 24 h after the previous dose were significantly reduced during chronic administration of doxazosin by a mean of 12/6 mm Hg supine and 10/7 mm Hg on standing. 5. During chronic administration of doxazosin, there was a significant reduction of 13% in glomerular filtration rate compared with pre-treatment, but no change in effective renal plasma flow.