RESUMEN
Thiopurine (TP) treatment is discontinued in up to 30% of patients suffering from inflammatory bowel diseases (IBD) due to various adverse effects. Therapeutic drug monitoring of biologically active TP metabolites, i.e. thiopurine nucleotides (TPN), can help to optimize the efficacy and safety of the TP treatment. In our work, a novel strategy for TPN profiling, based on a porous graphitic carbon (PGC) chromatography, was developed. The validated PGC-MS method was compared with ion-exchange LC-MS, a currently leading analytical approach established for the determination of TPN. The innovative approach enabled an enhancement of several key performance parameters demanded in a clinical routine use, namely (i) selectivity (time- and mass-recognition of all 12 TPN in one run), (ii) sensitivity (2-5-fold increase in intensities of the analytical signals), (iii) sample throughput (25% shorter analysis time). Application of the novel TPN profiling strategy to a pilot clinical study (12 patients) revealed significantly higher levels of 6-methylthioguanine 5'-diphosphate (MeTGDP) in non-responsive IDB patients treated with azathioprine. Some other TPN are very close to the critical level (p = 0.05) and they will need larger groups of IBD patients to confirm definitively their relevance. In conclusion, the developed PGC-MS method represents a significant improvement to currently available methods for detailed profiling of TPN and its use in bigger clinical studies should lead to a better understanding of the relationship between TPN profiles and therapeutic outcome.
Asunto(s)
Grafito , Enfermedades Inflamatorias del Intestino , Carbono , Humanos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Espectrometría de Masas , Nucleótidos , PorosidadRESUMEN
The compositions of leaf infusions of three genotypes of Lycopus europaeus L. with origins in central Europe, namely L. europaeus A (LeuA), L. europaeus B (LeuB), and L. europaeus C (LeuC), and one genotype of L. exaltatus (Lex), were examined by LC-MS-DAD (Liquid Chromatography Mass Spectrometry and Diode Array Detection) analysis. This revealed the presence of thirteen compounds belonging to the groups of phenolic acids and flavonoids, with a predominance of rosmarinic acid (RA) and luteolin-7-O-glucuronide (LGlr). The antimicrobial activity of leaf infusions was tested on the collection strains of Gram-positive and Gram-negative bacteria, and on the clinical Staphylococcus aureus strains. We detected higher activity against Gram-positive bacteria, of which the most susceptible strains were those of Staphylococcus aureus, including methicillin-resistant and poly-resistant strains. Furthermore, we examined the antioxidant activity of leaf infusions using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) methods, and on NIH/3T3 cell lines using dichlorodihydrofluorescein diacetate (DCFH-DA). We also studied the mutual interactions between selected infusions, namely RA and/or LGlr. In the mixtures of leaf infusion and RA or LGlr, we observed slight synergism and a high dose reduction index in most cases. This leads to the beneficial dose reduction at a given antioxidant effect level in mixtures compared to the doses of the parts used alone. Therefore, our study draws attention to further applications of the Lycopus leaves as a valuable alternative source of natural antioxidants and as a promising topical antibacterial agent for medicinal use.
Asunto(s)
Antiinfecciosos/química , Antiinfecciosos/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Lycopus/química , Extractos Vegetales/química , Extractos Vegetales/farmacología , Antibacterianos/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Fitoquímicos/químicaRESUMEN
Thiopurines (TP) represent an important therapeutic tool for the treatment of inflammatory bowel diseases (IBD) in the current situation of rising incidence and health care costs. The results of multiple clinical studies aimed at finding correlations between levels of TP metabolites and response of IBD patients to the treatment are, however, often controversial due to variability in analytical and sample preparation procedures among these studies. In this work, therefore, an updated analytical and sample preparation procedure for therapeutic drug monitoring (TDM) of TP metabolites in blood samples obtained from patients with IBD was proposed to establish a unified protocol. An advanced analytical method based on ion-exchange liquid chromatography hyphenated with tandem mass spectrometry (IEC-ESI-MS/MS) was used for the determination of the profiles of 12 individual TP metabolites in the particular steps of sample preparation procedure including blood collection, red blood cells (RBC) isolation, lysis, and storage. Favorable performance parameters of the IEC-ESI-MS/MS method (LLOQs 1â»10 nmol/L, accuracy 95â»105%, intra-day and inter-day precision < 10%, selectivity demonstrated via no sample matrix interferences) and acceptable stability (peak area fluctuations < 15%) of clinical samples under the proposed sample preparation conditions {(i) EDTA anticoagulant tube for the blood collection; (ii) 4 °C and 4 h between the sample collection and RBC isolation; (iii) phosphate-buffered saline for RBC washing and re-suspendation; (iv) -20 °C for RBC lysis and short-term storage; (v) 50 mmol/L phosphate buffer, pH 7.4, 10 mmol/L DTT as a stabilizing medium for TPN in RBC lysates} demonstrated the suitability of such protocol for a well-defined and reliable routine use in studies on thiopurines TDM.
Asunto(s)
Azatioprina , Monitoreo de Drogas/métodos , Enfermedades Inflamatorias del Intestino/sangre , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Mercaptopurina , Tioguanina , Adulto , Azatioprina/administración & dosificación , Azatioprina/farmacocinética , Eritrocitos/metabolismo , Femenino , Humanos , Masculino , Mercaptopurina/administración & dosificación , Mercaptopurina/farmacocinética , Persona de Mediana Edad , Tioguanina/administración & dosificación , Tioguanina/farmacocinéticaRESUMEN
It is supposed that plant functional foods, rich in phytochemicals, may potentially have preventive effects in carcinogenesis. In this study, the anticancer effects of cloves in the in vivo and in vitro mammary carcinoma model were assessed. Dried flower buds of cloves (CLOs) were used at two concentrations of 0.1% and 1% through diet during 13 weeks after the application of chemocarcinogen. After autopsy, histopathological and immunohistochemical analyses of rat mammary carcinomas were performed. Moreover, in vitro evaluation using MCF-7 cells was carried out. Dietary administered CLO caused the dose-dependent decrease in tumour frequency by 47.5% and 58.5% when compared to control. Analysis of carcinoma cells in animals showed bcl-2, Ki67, VEGFA, CD24 and CD44 expression decrease and Bax, caspase-3 and ALDH1 expression increase after high-dose CLO administration. MDA levels were substantially decreased in rat carcinomas in both CLO groups. The evaluation of histone modifications revealed increase in lysine trimethylations and acetylations (H4K20me3, H4K16ac) in carcinomas after CLO administration. TIMP3 promoter methylation levels of CpG3, CpG4, CpG5 islands were altered in treated cancer cells. An increase in total RASSF1A promoter methylation (three CpG sites) in CLO 1 group was found. In vitro studies showed antiproliferative and pro-apoptotic effects of CLO extract in MCF-7 cells (analyses of cytotoxicity, Brdu, cell cycle, annexin V/PI, caspase-7, Bcl-2 and mitochondrial membrane potential). This study showed a significant anticancer effect of clove buds in the mammary carcinoma model in vivo and in vitro.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Epigénesis Genética/efectos de los fármacos , Neoplasias Mamarias Experimentales/dietoterapia , Syzygium/química , Adenocarcinoma/dietoterapia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Familia de Aldehído Deshidrogenasa 1 , Animales , Antineoplásicos Fitogénicos/aislamiento & purificación , Neoplasias de la Mama/dietoterapia , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Caspasa 3/genética , Caspasa 3/metabolismo , Metilación de ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Flores/química , Histonas/genética , Histonas/metabolismo , Humanos , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Células MCF-7 , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Extractos Vegetales/química , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Ratas Sprague-Dawley , Retinal-Deshidrogenasa/genética , Retinal-Deshidrogenasa/metabolismo , Transducción de Señal , Carga Tumoral/efectos de los fármacos , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteína X Asociada a bcl-2/genética , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Thiopurine S-methyltransferase (TPMT) plays an important role in the metabolism of thiopurines used in the therapy of inflammatory bowel diseases (IBD). In this work a new progressive method for the determination of TPMT activity in red blood cells lysates was developed. Analysis was carried out by means of hydrophilic interaction liquid chromatography (HILIC) hyphenated with mass spectrometry (MS). In comparison with reversed-phase high-performance liquid chromatography (RP-HPLC), that has been typically applied in determination of TPMT activity, the HILIC significantly improved the analytical signal provided by MS, shortened analysis time, and improved chromatographic resolution. The HILIC-HPLC-MS method was optimized and validated, providing favorable parameters of detection and quantitation limits (5.5 and 16.5pmol/mL, respectively), linearity (coefficient of determination 0.9999 in the range of 0.01-1.0nmol/mL), recovery and precision (93.25-100.37% with RSD 1.06-1.32% in the whole concentration range of QC samples). Moreover, in contrast to the conventional RP-HPLC-UV approach, the complex phenotype TPMT profiles can be reliably and without interferences monitored using the HILIC-HPLC-MS method. Such advanced monitoring can provide valuable detail information on the thiopurines (e.g. evaluating ratio of methylated and non-methylated 6-mercaptopurine) and, by that, TPMT action in biological systems before and during the therapy of IBD.
Asunto(s)
Metiltransferasas/análisis , Cromatografía Liquida , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de MasasRESUMEN
The present work illustrates potentialities of CE hyphenated with MS/MS for the simultaneous determination and identification of a mixture of simultaneously acting drugs in pharmaceutical and biological matrices. Here, the hyphenation was provided by ESI interface, while the MS/MS technique was based on the triple quadrupole configuration. Three drugs, namely pheniramine, phenylephrine, and paracetamol were determined and identified with high reliability due to their characterization in three different dimensions, i.e. electrophoresis and MS/MS, that prevented practically any interference. Appropriately selected transitions of the analytes (parent ion-quantifier product ion-qualifier product ion) provided their selective determination at maximum S/N. The proposed CE-MS/MS method was validated (LOD/LOQ, linearity, precision, recovery, accuracy) and applied for (i) the multidrug composition pharmaceuticals, namely Theraflu®, and (ii) human urine taken after per-oral administration of the same pharmaceutical preparation. The method was applied also for the investigation of potential weak associates of the drugs and monitoring of predicted (bio)degradation products of the drugs. Successful validation and application of the proposed method suggest its routine use in highly effective and reliable advanced drug control and biomedical research.
Asunto(s)
Acetaminofén/orina , Electroforesis Capilar/métodos , Espectrometría de Masas/métodos , Feniramina/orina , Fenilefrina/orina , Electroforesis Capilar/instrumentación , HumanosRESUMEN
The idea of utilization of one hydraulic line of a common commercial HPLC pump for direct on-column sample pumping injection of large sample volumes, 20 mL, was further investigated with the aim to develop multicomponent pesticides trace residues HPLC method in gram soil samples. Target pesticides group involve asulam, atrazine, 2,4-D, PCA, propazine, simazine, 4-chloro-2-methylphenoxyacetic acid, 2-(4-chloro-2-tolyloxy) propionic acid, chlortoluron, metoxuron, epoxiconazole. The results proved the applicability of this approach in experiments with mixtures of analytes at low ng/mL levels. Analysis of 20 mL of soil leachates and extracts of fortified soil samples containing these pesticides at the 10-50 ng/g level (in dry soil) revealed good figures of merit, also in the presence of large excess of humics. LODs achieved by detection at 220 nm evaluated from calibration runs of spiked soil extracts by Hubaux et al. method ranged from 5-12 ng per injected volume. For 20 mL large volume injection it represents 0.25-0.6 ng/mL of diluted soil extract, or 2.5-6 ng/mL of crude extract, or 6-5 ng/g dry soil. Recoveries of pesticides at concentration levels approaching half of maximum allowable concentration of pesticides in soil (100 ng/g) ranged from 85 to 98% with acceptable reproducibility, except asulam and metoxuron.
Asunto(s)
Cromatografía Liquida/métodos , Análisis de Inyección de Flujo/métodos , Plaguicidas/análisis , Contaminantes del Suelo/análisis , Suelo/análisis , Animales , Calibración , Cromatografía Liquida/instrumentación , Cromatografía Liquida/normas , Análisis de Inyección de Flujo/instrumentación , Tecnología Química Verde/instrumentación , Tecnología Química Verde/métodos , Tecnología Química Verde/normas , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
A well established method of direct injection of larger than conventional sample volumes ranging from 0.1 mL to 10 mL in HPLC is the injection valve method in which a loop of tubing is totally or partially filled with sample. Recent HPLC pumps have a flow-rate setting accuracy of +/- 1-2% over a flow-rate range from 0.1 mL/min to 10 mL/min and the flow stability is 0.2% or less. Quarternary low pressure gradient pumps are widely available and used, but all their hydraulic lines are seldom utilised. The idea of using one line of a common commercial HPLC quaternary low-pressure pump for direct on-column injection (pumping) of large sample volumes ranging from 1 mL to 100 mL was tested. This approach was evaluated during practical work on the development of an RP-HPLC method of determination of residual atrazine and hydroxyatrazine. In lysimetric environmental experiments hydroxyatrazine was formed in situ in a soil column by hydrolysis of atrazine. The results proved the applicability of this approach not only in experiments with model mixtures of analytes at microg/L levels in solutions. Analysis of 20 mL of soil leachates and extracts of soil samples containing atrazine and hydroxyatrazine at the 10 microg/kg level (in dry soil) revealed that good figures-of-merit were preserved, even in the presence of a large excess of humic substances.