RESUMEN
Determination of plasma uracil was reported as a method for evaluation of Dihydropyrimidine dehydrogenase (DPD) activity that is highly demanded to ensure the safe administration of 5-fluorouracil (5-FU)-based therapies to cancer patients. This work reports the development of a simple electroanalytical method based on adsorptive stripping square wave voltammetry (AdSWV) at mercury film-coated glassy carbon electrode (MF/GCE) for the highly sensitive determination of uracil in biological fluids that can be used for diagnosis of decreased DPD activity. Due to the formation of the HgII-Uracil complex at the electrode surface, the accuracy of the measurement was not affected by the complicated matrices in biological fluids including human serum, plasma, and urine. The high sensitivity of the developed method results in a low limit of detection (≈1.3 nM) in human plasma samples, falling below the practical cut-off level of 15 ng mL-1 (≈0.14 µM). This threshold concentration is crucial for predicting 5-FU toxicity, as reported in buffer, and ≤1.15% in biological samples), and accuracy (recovery percentage close to 100%).
Asunto(s)
Técnicas Biosensibles , Deficiencia de Dihidropirimidina Deshidrogenasa , Electrodos , Fluorouracilo , Mercurio , Uracilo , Humanos , Uracilo/sangre , Mercurio/sangre , Límite de Detección , Técnicas Electroquímicas/métodos , Dihidrouracilo Deshidrogenasa (NADP)/metabolismoRESUMEN
Fluorouracil is among the most used antimetabolite drugs for the chemotherapeutic treatment of various types of gastrointestinal malignancies. Dihydropyrimidine dehydrogenase (DPYD) genotyping prior to fluorouracil treatment is considered standard practice in most European countries. Yet, current pre-therapeutic DPYD genotyping procedures do not identify all dihydropyrimidine dehydrogenase (DPD)-deficient patients. Alternatively, DPD activity can be estimated by determining the DPD phenotype by quantification of plasma concentrations of the endogenous uracil and thymine concentrations and their respective metabolites dihydrouracil (DHU) and dihydrothymine (DHT). Liquid chromatography - mass spectrometry (LC-MS) detection is currently considered as the most adequate method for quantification of low-molecular weight molecules, although the sample preparation method is highly critical for analytical outcome. It was hypothesized that during protein precipitation, the recovery of the molecule of interest highly depends on the choice of precipitation agent and the extent of protein binding in plasma. In this work, the effect of protein precipitation using acetonitrile (ACN) compared to strong acid perchloric acid (PCA) on the recovery of uracil, thymine, DHU and DHT is demonstrated. Upon the analysis of plasma samples, PCA precipitation showed higher concentrations of uracil and thymine as compared to ACN precipitation. Using ultrafiltration, it was shown that uracil and thymine are significantly (60-65â¯%) bound to proteins compared to DHU and DHT. This shows that before harmonized cut-off levels of DPD phenotyping can be applied in clinical practice, the analytical methodology requires extensive further optimization.
Asunto(s)
Dihidrouracilo Deshidrogenasa (NADP) , Fenotipo , Unión Proteica , Timina , Uracilo , Timina/metabolismo , Uracilo/análogos & derivados , Uracilo/metabolismo , Uracilo/sangre , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Dihidrouracilo Deshidrogenasa (NADP)/genética , Humanos , Cromatografía Liquida/métodos , Fluorouracilo/metabolismo , Fluorouracilo/sangre , Genotipo , Deficiencia de Dihidropirimidina Deshidrogenasa/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
In clinical practice, 25-30% of the patients treated with fluoropyrimidines experience severe fluoropyrimidine-related toxicity. Extensively clinically validated DPYD genotyping tests are available to identify patients at risk of severe toxicity due to decreased activity of dihydropyrimidine dehydrogenase (DPD), the rate limiting enzyme in fluoropyrimidine metabolism. In April 2020, the European Medicines Agency recommended that, as an alternative for DPYD genotype-based testing for DPD deficiency, also phenotype testing based on pretreatment plasma uracil levels is a suitable method to identify patients with DPD deficiency. Although the evidence for genotype-directed dosing of fluoropyrimidines is substantial, the level of evidence supporting plasma uracil levels to predict DPD activity in clinical practice is limited. Notwithstanding this, uracil-based phenotyping is now used in clinical practice in various countries in Europe. We aimed to determine the value of pretreatment uracil levels in predicting DPD deficiency and severe treatment-related toxicity. To this end, we determined pretreatment uracil levels in 955 patients with cancer, and assessed the correlation with DPD activity in peripheral blood mononuclear cells (PBMCs) and fluoropyrimidine-related severe toxicity. We identified substantial issues concerning the use of pretreatment uracil in clinical practice, including large between-center study differences in measured pretreatment uracil levels, most likely as a result of pre-analytical factors. Importantly, we were not able to correlate pretreatment uracil levels with DPD activity nor were uracil levels predictive of severe treatment-related toxicity. We urge that robust clinical validation should first be performed before pretreatment plasma uracil levels are used in clinical practice as part of a dosing strategy for fluoropyrimidines.
Asunto(s)
Deficiencia de Dihidropirimidina Deshidrogenasa , Dihidrouracilo Deshidrogenasa (NADP) , Uracilo , Antimetabolitos Antineoplásicos , Deficiencia de Dihidropirimidina Deshidrogenasa/tratamiento farmacológico , Deficiencia de Dihidropirimidina Deshidrogenasa/genética , Dihidrouracilo Deshidrogenasa (NADP)/genética , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Humanos , Leucocitos Mononucleares/metabolismo , Estudios Prospectivos , Uracilo/sangreRESUMEN
BACKGROUND: Pretherapeutic screening for dihydropyrimidine dehydrogenase (DPD) deficiency is recommended prior to the administration of fluoropyrimidine-based chemotherapy. However, the best strategy to identify DPD deficiency in End Stage Renal Disease (ESRD) patients is unknown. METHODS: We assessed the characteristics of both DPD phenotypes and DPYD genotypes in 20 dialyzed patients before and after dialysis session. The extent to which the concentrations of uracil [U] and dihydrouracil [UH2] were affected by dialysis was evaluated. RESULTS: Mean [U] was 14 ± 3.3 ng/ml before the dialysis session, and 7.9 ± 2.7 ng/ml after. Notably, mean [U] in 119 non-ESRD patients during the same timeline was 8.7 ± 3.9 ng/ml, which is similar to [U] values after dialysis session (p = 0.38). [U] values > 16 ng/ml were measured in 4 ESRD patients (20%), whereas the rate was 3.3% in the non-ESRD cohort. Whole gene sequencing did not reveal DPYD deleterious allelic variants in the 4 ESRD patients with [U] values > 16 ng/ml. The profile of [UH2] values during dialysis was similar to that of [U]: 385 ± 86 ng/ml before, and 185 ± 62 ng/ml after (mean reduction rate 42.5%). Thus, [UH2]:[U] ratio remained unaffected by dialysis, and was similar to the values in non-ESRD patients (22.4 ± 7.1). CONCLUSION: Phenotyping based on measuring plasma [U] before a dialysis sessions in ESRD patients is associated with an unacceptable high rate of false positives. The optimal strategy for the identification of patients with DPD deficiency in this population would be the monitor the [UH2]:[U] ratio, which remains unaffected.
Asunto(s)
Deficiencia de Dihidropirimidina Deshidrogenasa/diagnóstico , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Fallo Renal Crónico/terapia , Tamizaje Masivo/métodos , Diálisis Renal/efectos adversos , Uracilo/sangre , Estudios de Casos y Controles , Deficiencia de Dihidropirimidina Deshidrogenasa/enzimología , Deficiencia de Dihidropirimidina Deshidrogenasa/etiología , Reacciones Falso Positivas , Estudios de Seguimiento , Humanos , Fallo Renal Crónico/patología , Estudios ProspectivosRESUMEN
Our previously reported carboxyl-containing DPP-4 inhibitors were highly potent but were poorly bioavailable. Esters of the carboxyl analogs exhibited a significant DPP-4 potency loss albeit with enhanced oral absorption. Herein, we described identification and structure-activity relationship (SAR) exploration of a novel series of benzoic acid and ester derivatives as low single-digit nanomolar DPP-4 inhibitors. Importantly, the esters displayed comparable activities to the acids counterparts. Molecular simulation revealed that ester adopts a similar binding mode to acid. Moreover, the selected esters and acids demonstrated high selectivity and low cytotoxicity, as well as good metabolic stability. And more importantly, the esters possessed excellent pharmacokinetic profiles for oral administration. The best compound ester 19b demonstrated long DPP-4 inhibition in vivo, and robustly improved the glucose tolerance in normal and db/db mice while ensuring glucose-lowering potency in chronic treatment. Our results supported that the compound 19b can be served as a potential candidate for the treatment of type 2 diabetes.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Dipeptidil Peptidasa 4/metabolismo , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Hipoglucemiantes/farmacología , Administración Oral , Animales , Ácido Benzoico/administración & dosificación , Ácido Benzoico/sangre , Ácido Benzoico/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/metabolismo , Inhibidores de la Dipeptidil-Peptidasa IV/administración & dosificación , Inhibidores de la Dipeptidil-Peptidasa IV/sangre , Relación Dosis-Respuesta a Droga , Ésteres/administración & dosificación , Ésteres/sangre , Ésteres/farmacología , Humanos , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/sangre , Inyecciones Intravenosas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Modelos Moleculares , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Uracilo/administración & dosificación , Uracilo/sangre , Uracilo/farmacologíaRESUMEN
PURPOSE: Standard dosages of fluoropyrimidine chemotherapy result in severe toxicity in a substantial proportion of patients, however, routine pre-therapeutic toxicity prediction remains uncommon. A thymine (THY) challenge test can discriminate risk of severe gastrointestinal toxicity in patients receiving fluoropyrimidine monotherapy. We aimed to measure endogenous plasma uracil (U) and its ratio to dihydrouracil (DHU), and assess the performance of these parameters compared with the THY challenge test to evaluate risk of severe toxicity. METHODS: Plasma samples, previously collected from 37 patients receiving 5-fluorouracil (5-FU) or capecitabine monotherapy for a THY challenge test (ACTRN12615000586516; retrospectively registered), were assessed for endogenous plasma concentrations of U and DHU using a validated LC-MS/MS method. Renal function was estimated from blood creatinine, and patients with ≥ grade 3 toxicity (CTCAE v4.0) were classified as cases. RESULTS: There were no differences in median endogenous U plasma concentrations or U/DHU ratios between severe toxicity cases and non-cases. Significant differences between cases and non-cases were noted when these measures were normalised to the estimated renal function (CrCL), Unorm p = 0.0004; U/DHUnorm p = 0.0083. These two parameters had a sensitivity of 29%, compared with 57% for the THY challenge test in the same patients. Genotyping for clinically relevant DPYD variants was inferior to either of these pyrimidine phenotyping tests (sensitivity of 14%). CONCLUSIONS: The endogenous uracil-based parameters, adjusted to CrCL, were more predictive of increased risk of severe fluoropyrimidine toxicity than DPYD genotyping. However, endogenous U measurement detected fewer cases of severe toxicity than the THY challenge test.
Asunto(s)
Capecitabina/efectos adversos , Fluorouracilo/efectos adversos , Timina/farmacología , Uracilo/análogos & derivados , Uracilo/sangre , Dihidrouracilo Deshidrogenasa (NADP)/genética , Genotipo , HumanosRESUMEN
Combination therapy of tegafur/uracil (UFT) and leucovorin (LV) is widely used to treat colorectal cancers. Although this therapy has a significant therapeutic effect, severe adverse effects occur frequently. Therapeutic drug monitoring (TDM) may help to prevent adverse effects. A useful assay that can quantitate plasma levels of 5-FU, uracil, and tegafur simultaneously for TDM has been desired, but such a method is not currently available. In this study, we aimed to develop a sensitive method for simultaneous quantification of 5-FU, uracil, and tegafur in human plasma using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). After preparing plasma samples by protein precipitation and liquid extraction, 5-FU, uracil, and tegafur were analyzed by UPLC-MS/MS in negative electrospray ionization mode. Validation was performed according to US Food and Drugs Administration guidance. The calibration curves were linear over concentration ranges of 2-500 ng/mL for 5-FU, 20-5000 ng/mL for uracil, and 200-50,000 ng/mL for tegafur. The corresponding average recovery rates were 79.9, 80.9, and 87.8%. The method provides accuracy within 11.6% and precision below 13.3% for all three analytes. Matrix effects of 5-FU, uracil, and tegafur were higher than 43.5, 84.9, and 100.2%, respectively. This assay was successfully applied to assess the time courses of plasma 5-FU, uracil, and tegafur concentrations in two patients with colorectal liver metastasis who received UFT/LV therapy after hepatectomy. In conclusion, we succeeded to develop a sensitive and robust UPLC-MS/MS method for simultaneous quantification of 5-FU, uracil, and tegafur in human plasma. This method is potentially useful for TDM in patients receiving UFT/LV combination therapy.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Cromatografía Líquida de Alta Presión/normas , Monitoreo de Drogas/métodos , Fluorouracilo/sangre , Espectrometría de Masas en Tándem/normas , Tegafur/sangre , Uracilo/sangre , Antimetabolitos Antineoplásicos/sangre , Calibración , Cromatografía Líquida de Alta Presión/métodos , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/patología , Hepatectomía/métodos , Humanos , Leucovorina/uso terapéutico , Hígado/patología , Hígado/cirugía , Neoplasias Hepáticas/sangre , Neoplasias Hepáticas/secundario , Neoplasias Hepáticas/cirugía , Sensibilidad y Especificidad , Espectrometría de Masas en Tándem/métodosRESUMEN
BACKGROUND: Pretherapeutic screening for dihydropyrimidine dehydrogenase (DPD) deficiency is recommended or required prior to the administration of fluoropyrimidine-based chemotherapy. However, the best strategy to identify DPD-deficient patients remains elusive. METHODS: Among a nationwide cohort of 5886 phenotyped patients with cancer who were screened for DPD deficiency over a 3 years period, we assessed the characteristics of both DPD phenotypes and DPYD genotypes in a subgroup of 3680 patients who had completed the two tests. The extent to which defective allelic variants of DPYD predict DPD activity as estimated by the plasma concentrations of uracil [U] and its product dihydrouracil [UH2] was evaluated. RESULTS: When [U] was used to monitor DPD activity, 6.8% of the patients were classified as having DPD deficiency ([U] > 16 ng/ml), while the [UH2]:[U] ratio identified 11.5% of the patients as having DPD deficiency (UH2]:[U] < 10). [U] classified two patients (0.05%) with complete DPD deficiency (> 150 ng/ml), and [UH2]:[U] < 1 identified three patients (0.08%) with a complete DPD deficiency. A defective DPYD variant was present in 4.5% of the patients, and two patients (0.05%) carrying 2 defective variants of DPYD were predicted to have low metabolism. The mutation status of DPYD displayed a very low positive predictive value in identifying individuals with DPD deficiency, although a higher predictive value was observed when [UH2]:[U] was used to measure DPD activity. Whole exon sequencing of the DPYD gene in 111 patients with DPD deficiency and a "wild-type" genotype (based on the four most common variants) identified seven heterozygous carriers of a defective allelic variant. CONCLUSIONS: Frequent genetic DPYD variants have low performances in predicting partial DPD deficiency when evaluated by [U] alone, and [UH2]:[U] might better reflect the impact of genetic variants on DPD activity. A clinical trial comparing toxicity rates after dose adjustment according to the results of genotyping or phenotyping testing to detect DPD deficiency will provide critical information on the best strategy to identify DPD deficiency.
Asunto(s)
Deficiencia de Dihidropirimidina Deshidrogenasa/diagnóstico , Anciano , Estudios de Cohortes , Estudios Transversales , Deficiencia de Dihidropirimidina Deshidrogenasa/epidemiología , Deficiencia de Dihidropirimidina Deshidrogenasa/genética , Dihidrouracilo Deshidrogenasa (NADP)/genética , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Femenino , Francia/epidemiología , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Fenotipo , Prevalencia , Estudios Retrospectivos , Uracilo/análogos & derivados , Uracilo/sangre , Uracilo/metabolismoRESUMEN
PURPOSE: Several clinical guidelines recommend genetic screening of DPYD, including coverage of the variants c.1905 + 1G>A(DPYD*2A), c.1679T>G(DPYD*13), c.2846A>T, and c.1129-5923C>G, before initiating treatment with fluoropyrimidines. However, this screening is often inadequate at predicting the occurrence of severe fluoropyrimidine-induced toxicity in patients. METHODS: Using a complementary approach combining whole DPYD exome sequencing and in silico and structural analysis, as well as phenotyping of DPD by measuring uracilemia (U), dihydrouracilemia (UH2), and the UH2/U ratio in plasma, we were able to characterize and interpret DPYD variants in 28 patients with severe fluoropyrimidine-induced toxicity after negative screening. RESULTS: Twenty-five out of 28 patients (90%) had at least 1 variant in the DPYD coding sequence, and 42% of the variants (6/14) were classified as potentially deleterious by at least 2 of the following algorithms: SIFT, Poly-Phen-2, and DPYD varifier. We identified two very rare deleterious mutations, namely, c.2087G>A (p.R696H) and c.2324T>G (p.L775W). We were able to demonstrate partial DPD deficiency, as measured by the UH2/U ratio in a patient carrying the variant p.L775W for the first time. CONCLUSION: Whole exon sequencing of DPYD in patients with suspicion of partial DPD deficiency can help to identify rare or new variants that lead to enzyme inactivation. Combining different techniques can yield abundant information without increasing workload and cost burden, thus making it a useful approach for implementation in patient care.
Asunto(s)
Antimetabolitos Antineoplásicos/efectos adversos , Deficiencia de Dihidropirimidina Deshidrogenasa/inducido químicamente , Deficiencia de Dihidropirimidina Deshidrogenasa/genética , Dihidrouracilo Deshidrogenasa (NADP)/genética , Variantes Farmacogenómicas/genética , Adulto , Anciano , Anciano de 80 o más Años , Capecitabina/efectos adversos , Simulación por Computador , Dihidrouracilo Deshidrogenasa (NADP)/química , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/efectos adversos , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Estudios Retrospectivos , Uracilo/análogos & derivados , Uracilo/sangreRESUMEN
Danggui Buxue Decoction (DBD), a famous traditional Chinese medicine (TCM), is often used to treat anemia in China. However, its underlying therapeutic mechanism is unclear. Through the analysis of body weight, spleen and thymus indexes, peripheral blood routine and pathological section of femur, it was obviously that DBD could significantly improve acetylphenylhydrazine (APH)â¯+â¯cyclophosphamide (CTX) induced anemia mice in the present work. Ultra high performance liquid chromatography coupled with quadrupole - Exactive mass spectrometry (UHPLC Q-Exactive MS) based metabolomics and lipidomics was further utilized to screen out differential spleen metabolites associated with DBD treatment. A total of 26 differential metabolites including 8 polar metabolites and 18 lipids were firstly obtained to relate with anemia mice. 7 polar metabolites and 10 lipids among them were reversed by DBD, which the regulation of pyrimidine metabolism and glycerophospholipid metabolism were mainly associated to the anti-anemia effect of DBD based on MetaboAnalyst analysis. Through random forest analysis (RF), ROC analysis and pearson matrix correlation, three metabolites, cytosine, uracil and PC (o-16:1(9Z)/20:0), were further screened out as the potential pharmacodynamic biomarkers associated with the efficacy of DBD. This study provided a methodological reference for the study of the mechanism of TCM.
Asunto(s)
Anemia/tratamiento farmacológico , Medicamentos Herbarios Chinos/farmacología , Metabolismo de los Lípidos/efectos de los fármacos , Lipidómica/métodos , Bazo/efectos de los fármacos , Anemia/sangre , Animales , Biomarcadores/sangre , Cromatografía Líquida de Alta Presión/métodos , Citosina/sangre , Modelos Animales de Enfermedad , Medicamentos Herbarios Chinos/uso terapéutico , Humanos , Masculino , Espectrometría de Masas/métodos , Ratones , Palmitoilcarnitina/sangre , Bazo/metabolismo , Uracilo/sangreRESUMEN
Fluoropyrimidines-based chemotherapies are the backbone in the treatment of many cancers. However, the use of 5-fluorouracil and its oral pre-prodrug, capecitabine, is associated with an important risk of toxicity. This toxicity is mainly due to a deficiency of dihydropyrimidine dehydrogenase (DPD). This deficiency may be detected by using a phenotypic approach that consists in the measurement of uracilemia or the calculation of dihydrouracil (UH2)/uracil (U) ratio. For uracilemia, a threshold value of 16â¯ng/ml has been proposed for partial deficiency, while a value of 150â¯ng/ml has been proposed for complete deficiency. We have developed a rapid, accurate and fully-automated procedure for the quantification of U and UH2 in plasma. Sample extraction was carried out by a programmable liquid handler directly coupled to a liquid chromatography - tandem mass spectrometry (LC-MS/MS) system. The method was validated according to the EMA guidelines and ISO 15189 requirements and was applied to real patient samples (nâ¯=â¯64). The limit of quantification was 5 and 10â¯ng/ml for U and UH2 respectively. Imprecision and inaccuracy were less than 15% for inter and intra-assay tests. Comparison with dedicated routine method showed excellent correlation. An automated procedure perfectly fulfills the need of low inaccuracy and CVs at the threshold values (less than 5% at 16â¯ng/ml) and is highly suitable for the characterization of DPD deficiency. Automatization should guaranty reliable and robust performances by minimizing the sources of variation such as volume inaccuracies, filtration or manual extraction related errors.
Asunto(s)
Deficiencia de Dihidropirimidina Deshidrogenasa/diagnóstico , Uracilo/sangre , Automatización , Capecitabina/metabolismo , Cromatografía Líquida de Alta Presión , Fluorouracilo/metabolismo , Humanos , Límite de Detección , Reproducción , Sensibilidad y Especificidad , Espectrometría de Masas en Tándem , Uracilo/análogos & derivados , Uracilo/metabolismoRESUMEN
Detecting patients with dihydropyrimidine dehydrogenase (DPD) deficiency is becoming a major concern in clinical oncology. Monitoring physiologic plasma uracil and/or plasma uracil-to-dihydrouracil metabolic ratio is a common surrogate frequently used to determine DPD phenotype without direct measurement of the enzymatic activity. With respect to the increasing number of patients rquiring analysis, it is critical to develop simple, rapid, and affordable methods suitable for routine screening. We have developed and validated a simple and robust ultraperformance liquid chromatography-ultraviolet (UPLC-UV) method with shortened (i.e., 12 minutes) analytical run-times, compatible with the requirements of large-scale upfront screening. The method enables detection of uracil (U) over a range of 5-500 ng/ml (265 nm) and of dihydrouracil (UH2) over a range of 40-500 ng/ml (210 nm) in plasma with no chromatographic interference. When used as part of routine screening for DPD deficiency, this method was fully able to discriminate nondeficient patients (i.e., with U levels < 16 ng/ml) from deficient patients at risk of severe toxicity (i.e., U > 16 ng/ml). Results from 1 month of routine testing are presented and, although no complete deficits were detected, 10.7% of the screened patients presented DPD deficiency and would thus require s decresed dose. Overall, this new method, using a simple preanalytical solid-phase extraction procedure, and based on use of a standard UPLC apparatus, is both cost- and time-effective and can be easily implemented in any laboratory aiming to begin routine DPD testing.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacocinética , Deficiencia de Dihidropirimidina Deshidrogenasa/diagnóstico , Dihidrouracilo Deshidrogenasa (NADP)/metabolismo , Neoplasias/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Antimetabolitos Antineoplásicos/administración & dosificación , Biomarcadores/sangre , Biomarcadores/metabolismo , Capecitabina/administración & dosificación , Capecitabina/farmacocinética , Cromatografía Líquida de Alta Presión/métodos , Deficiencia de Dihidropirimidina Deshidrogenasa/sangre , Deficiencia de Dihidropirimidina Deshidrogenasa/metabolismo , Femenino , Fluorouracilo/administración & dosificación , Fluorouracilo/farmacocinética , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/sangre , Neoplasias/metabolismo , Espectrofotometría Ultravioleta/métodos , Uracilo/análogos & derivados , Uracilo/sangre , Uracilo/metabolismoRESUMEN
The direct determination of alogliptin benzoate (ALO) using fluorescence has not yet been accomplished because ALO cannot fluoresce naturally. Accordingly, it should be derivatized first with a fluorogenic reagent to enhance the sensitivity required for its bioanalysis. This method is the first spectrofluorimetric assay for ALO quantification exploiting the nucleophilic nature of its amino group to react with 4-chloro-7-nitrobenzofurazan (NBD-Cl) in borate buffer at pH 8.5 to produce a strong fluorescent compound that is excited at and emits at wavelengths 470 and 527 nm, respectively. Experimental variables concerning the conditions of reaction and fluorogenic intensity were carefully investigated and optimized. Linearity was from 1-250 ng ml-1 with a lower detection limit of 0.29 ng ml-1 and a lower quantification limit of 0.88 ng ml-1 . Validation of the current study was accomplished with mean per cent recovery of 100.62 ± 1.59 in tablets and 99.86 ± 0.82 in human plasma. Furthermore, the current method has been utilized in the bioanalysis of ALO in real rat plasma after oral administration with a simple specimen preparation. The developed method has proven to be a promising alternative method for ALO analysis in bioequivalence studies.
Asunto(s)
4-Cloro-7-nitrobenzofurazano/química , Benzoatos/sangre , Colorantes Fluorescentes/química , Piperidinas/sangre , Espectrometría de Fluorescencia , Uracilo/análogos & derivados , Animales , Benzoatos/química , Benzoatos/farmacocinética , Humanos , Masculino , Estructura Molecular , Piperidinas/química , Piperidinas/farmacocinética , Teoría Cuántica , Ratas , Ratas Wistar , Espectrometría de Fluorescencia/instrumentación , Uracilo/sangre , Uracilo/química , Uracilo/farmacocinéticaRESUMEN
BACKGROUND: Inconsistent associations between coffee consumption and bone mineral density (BMD) have been observed in epidemiological studies. Moreover, the relationship of bioactive components in coffee with BMD has not been studied. The aim of the current study is to identify coffee-associated metabolites and evaluate their association with BMD. METHODS: Two independent cohorts totaling 564 healthy community-dwelling adults from the Hong Kong Osteoporosis Study (HKOS) who visited in 2001-2010 (N = 329) and 2015-2016 (N = 235) were included. Coffee consumption was self-reported in an food frequency questionnaire. Untargeted metabolomic profiling on fasting serum samples was performed using liquid chromatography-mass spectrometry platforms. BMD at lumbar spine and femoral neck was measured by dual-energy X-ray absorptiometry. Multivariable linear regression and robust regression were used for the association analyses. RESULTS: 12 serum metabolites were positively correlated with coffee consumption after Bonferroni correction for multiple testing (P < 4.87 × 10-5), with quinate, 3-hydroxypyridine sulfate, and trigonelline (N'-methylnicotinate) showing the strongest association. Among these metabolites, 11 known metabolites were previously identified to be associated with coffee intake and 6 of them were related to caffeine metabolism. Habitual coffee intake was positively and significantly associated with BMD at the lumbar spine and femoral neck. The metabolite 5-acetylamino-6-formylamino-3-methyluracil (AFMU) (ß = 0.012, SE = 0.005; P = 0.013) was significantly associated with BMD at the lumbar spine, whereas 3-hydroxyhippurate (ß = 0.007, SE = 0.003, P = 0.027) and trigonelline (ß = 0.007, SE = 0.004; P = 0.043) were significantly associated with BMD at the femoral neck. CONCLUSIONS: 12 metabolites were significantly associated with coffee intake, including 6 caffeine metabolites. Three of them (AFMU, 3-hydroxyhippurate, and trigonelline) were further associated with BMD. These metabolites could be potential biomarkers of coffee consumption and affect bone health.
Asunto(s)
Densidad Ósea/efectos de los fármacos , Cafeína/sangre , Café/efectos adversos , Ingestión de Líquidos/fisiología , Absorciometría de Fotón , Alcaloides/sangre , Café/metabolismo , Encuestas sobre Dietas , Femenino , Cuello Femoral/diagnóstico por imagen , Hipuratos/sangre , Hong Kong/epidemiología , Humanos , Vida Independiente , Modelos Lineales , Vértebras Lumbares/diagnóstico por imagen , Masculino , Metaboloma , Persona de Mediana Edad , Osteoporosis/epidemiología , Osteoporosis/etiología , Estudios Prospectivos , Uracilo/análogos & derivados , Uracilo/sangreRESUMEN
A simple and enantioselective method was developed and validated for the simultaneous determination of (R)- and (S)-trelagliptin in beagle dog plasma by chiral liquid chromatography tandem mass spectrometry. Trelagliptin enantiomers and (R)-rabeprazole (as internal standard, IS) were extracted from plasma samples by liquid-liquid extraction and separated on a CHIRALCEL OX-3R column using acetonitrile-5 ammonium bicarbonate as the mobile phase in gradient elution mode. The multiple reactions monitoring transitions of m/z 358.1â341.2 and 359.9â150.1 were used to quantify trelagliptin enantiomers and IS, respectively. This method was validated for sensitivity, specificity, linearity, precision, accuracy and stability of specific analytes under various conditions. And it was successfully applied to evaluating the pharmacokinetic profile of trelagliptin enantiomers in beagle dogs after single intravenous administration of (R)-trelagliptin injection (at 1 mg/kg) and oral administration (at 6.7 mg/kg). In this study, no chiral bioconversion of (R)-trelagliptin to (S)-trelagliptin in beagle dog plasma was observed. The absolute bioavailability of (R)-trelagliptin was identified to be 128.2%.
Asunto(s)
Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Uracilo/análogos & derivados , Animales , Perros , Femenino , Masculino , Reproducibilidad de los Resultados , Uracilo/sangreRESUMEN
A novel, high throughput and sensitive LC-MS/MS assay method was developed and fully validated for quantitative determination of pioglitazone, its hydroxyl metabolite and alogliptin in human plasma. A simple and rapid sample preparation procedure based on protein precipitation technique with acetonitrile was utilized. Chromatographic separation was achieved on C8 (50â¯×â¯4.6â¯mm, 5⯵m) Kinetex® analytical column using methanol and 0.1% formic acid in a gradient elution mode at a flow rate of 0.7â¯mL/min with injection volume of 8⯵L. Detection was performed on a triple quadrupole mass spectrometer accompanied with electrospray ionization (ESI) technique in positive mode, operating in multiple reaction monitoring, with the transitions of 357.2â¯ââ¯119.1, 373.1â¯ââ¯150.1, 340.3â¯ââ¯116.1, 361.1â¯ââ¯138.1 and 343.2â¯ââ¯116.1 m/z for pioglitazone, its hydroxyl metabolite, alogliptin, pioglitazone-d4 (IS-1) and alogliptin-d3 (IS-2), in order. Analysis was achieved within 4â¯min over a linear concentration range of 10-3000â¯ng/mL, 5-2000â¯ng/mL and 3-300â¯ng/mL, for pioglitazone, hydroxyl pioglitazone and alogliptin, in order. The method was fully validated according to FDA guidelines. The developed method was used for estimation of the three studied analytes in human plasma and pharmacokinetic parameters were demonstrated after oral dose administration of Oseni® tablets to Egyptian healthy volunteers.
Asunto(s)
Cromatografía Liquida/métodos , Pioglitazona/sangre , Piperidinas/sangre , Espectrometría de Masas en Tándem/métodos , Uracilo/análogos & derivados , Humanos , Modelos Lineales , Pioglitazona/química , Pioglitazona/metabolismo , Pioglitazona/farmacocinética , Piperidinas/química , Piperidinas/farmacocinética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Uracilo/sangre , Uracilo/química , Uracilo/farmacocinéticaRESUMEN
Aim: A sensitive HPLC-MS/MS approach was established to quantify trelagliptin and explore the pharmacokinetic characteristics in rats for up to 7 days. Meanwhile, the pharmacokinetic differences of trelagliptin were investigated for the first time. Results/methodology: The ion pairs of m/z 358.2â341.2 for trelagliptin and m/z 340.3â116.1 for alogliptin (internal standard) were detected in positive mode. Trelagliptin displayed a good linearity in the range of 4-4000 ng/ml (r2 = 0.9997) with a mean recovery rate of 86.9-94.1%. Discussion/conclusion: Compared with normal groups, the T1/2, apparent volume of distribution, area under the curve and bioavailability in model rats were significantly increased while the apparent plasma clearance decreased. The approach is proved to be straightforward and appropriate for quantitation of trelagliptin and application in pharmacokinetics studies.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Inhibidores de la Dipeptidil-Peptidasa IV/sangre , Hipoglucemiantes/sangre , Piperidinas/sangre , Espectrometría de Masas en Tándem/métodos , Uracilo/análogos & derivados , Animales , Diabetes Mellitus/tratamiento farmacológico , Monitoreo de Drogas/métodos , Femenino , Masculino , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Uracilo/sangreAsunto(s)
Antimetabolitos Antineoplásicos/efectos adversos , Deficiencia de Dihidropirimidina Deshidrogenasa/complicaciones , Fluorouracilo/efectos adversos , Acetatos/uso terapéutico , Antídotos/uso terapéutico , Antimetabolitos Antineoplásicos/uso terapéutico , Deficiencia de Dihidropirimidina Deshidrogenasa/genética , Fluorouracilo/uso terapéutico , Humanos , Neoplasias/tratamiento farmacológico , Guías de Práctica Clínica como Asunto , Pirimidinas/efectos adversos , Pirimidinas/antagonistas & inhibidores , Uracilo/sangre , Uridina/análogos & derivados , Uridina/uso terapéuticoRESUMEN
Capecitabine is a 5-fluorouracil (5-FU) derivative that is used widely in the treatment of colorectal cancer. The plasma ratio of dihydrouracil (UH2 ) to uracil (Ura) is expected to gain relevance as an indirect-response biomarker to estimate the activity of dihydropyrimidine dehydrogenase (DPD). The latter is a rate-limiting enzyme in the catabolism of 5-FU in the capecitabine-based regimen. However, the relationship between the pharmacokinetics of capecitabine and the plasma UH2 /Ura ratio is still unknown. This study evaluated the time-course alterations of the plasma UH2 /Ura ratio in rats treated with 180 mg/kg capecitabine. The molar ratio tended to increase within 1.5 h (1.85 ± 0.76 at 1.5 h after administration of capecitabine) and gradually recovered to its initial level (1.00 ± 0.46). The results of the current study suggest that the plasma UH2 /Ura ratio temporarily increases following administration of capecitabine, possibly related to the DPD activity levels. The plasma UH2 /Ura ratio might assist in monitoring the alteration of DPD activity levels in capecitabine treatments.