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BACKGROUND: Tenofovir alafenamide fumarate (TAF) co-formulated with elvitegravir (EVG; E), cobicistat (C), and emtricitabine (F), a recommended antiretroviral regimen, was evaluated for distribution and antiviral activity in cerebrospinal fluid (CSF) as well as neurocognitive (NC) performance change in participants switching from E/C/F/tenofovir disoproxil fumarate (TDF) to E/C/F/TAF. METHODS: This was a 24-week, single-arm, open-label study in treatment-experienced adults living with human immunodeficiency virus (HIV). Nine participants switched from E/C/F/TDF (150/150/200/300 mg once daily) to E/C/F/TAF (150/150/200/10 mg once daily) at week 12. CSF and total plasma concentrations of EVG, TDF, TAF, tenofovir (TFV), and HIV RNA levels were measured at baseline and week 24. NC performance was estimated by the Montreal Cognitive Assessment. RESULTS: EVG concentrations in CSF and the CSF:plasma ratio remained stable (P = .203) over time. Following the switch, TFV concentrations in CSF and plasma declined (P = .004), although the TFV CSF:plasma ratio increased (P = .004). At week 24, median TAF plasma concentration was 11.05 ng/mL (range, 2.84-147.1 ng/mL) 2 hours postdose but was below assay sensitivity 6 hours after dosing. TAF was below assay sensitivity in all CSF specimens. HIV RNA was ≤40 copies/mL in all CSF and plasma specimens. Three participants (33%) had NC impairment at baseline and 2 (22%) remained impaired at week 24. CONCLUSIONS: Switch to E/C/F/TAF was associated with reductions in TFV concentrations in CSF but stable EVG concentrations that exceeded the 50% inhibitory concentration for wild-type HIV, suggesting that EVG achieves therapeutic concentrations in the central nervous system. No virologic failure or significant NC changes were detected following the switch. CLINICAL TRIALS REGISTRATION: NCT02251236.
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Fármacos Anti-VIH , Infecciones por VIH , Quinolonas , Adenina/análogos & derivados , Adenina/uso terapéutico , Adulto , Alanina , Fármacos Anti-VIH/uso terapéutico , Emtricitabina/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Humanos , Quinolonas/uso terapéutico , Tenofovir/análogos & derivadosRESUMEN
The liver is crucial to pharmacology, yet substantial knowledge gaps exist in the understanding of its basic pharmacologic processes. An improved understanding for humans requires reliable and reproducible liver sampling methods. We compared liver concentrations of paritaprevir and ritonavir in rats by using samples collected by fine-needle aspiration (FNA), core needle biopsy (CNB), and surgical resection. Thirteen Sprague-Dawley rats were evaluated, nine of which received paritaprevir/ritonavir at 30/20 mg/kg of body weight by oral gavage daily for 4 or 5 days. Drug concentrations were measured using liquid chromatography-tandem mass spectrometry on samples collected via FNA (21G needle) with 1, 3, or 5 passes (FNA1, FNA3, and FNA5); via CNB (16G needle); and via surgical resection. Drug concentrations in plasma were also assessed. Analyses included noncompartmental pharmacokinetic analysis and use of Bland-Altman techniques. All liver tissue samples had higher paritaprevir and ritonavir concentrations than those in plasma. Resected samples, considered the benchmark measure, resulted in estimations of the highest values for the pharmacokinetic parameters of exposure (maximum concentration of drug in serum [Cmax] and area under the concentration-time curve from 0 to 24 h [AUC0-24]) for paritaprevir and ritonavir. Bland-Altman analyses showed that the best agreement occurred between tissue resection and CNB, with 15% bias, followed by FNA3 and FNA5, with 18% bias, and FNA1 and FNA3, with a 22% bias for paritaprevir. Paritaprevir and ritonavir are highly concentrated in rat liver. Further research is needed to validate FNA sampling for humans, with the possible derivation and application of correction factors for drug concentration measurements.
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Hígado/metabolismo , Compuestos Macrocíclicos/farmacocinética , Ritonavir/farmacocinética , Animales , Biopsia con Aguja Fina , Cromatografía Liquida , Ciclopropanos , Hepatocitos/metabolismo , Inactivación Metabólica/fisiología , Lactamas Macrocíclicas , Hígado/cirugía , Masculino , Prolina/análogos & derivados , Ratas , Ratas Sprague-Dawley , Sulfonamidas , Espectrometría de Masas en TándemRESUMEN
Chronic kidney disease (CKD) affects the nonrenal clearance of drugs by modulating the functional expression of hepatic drug-metabolizing enzymes and transporters. The impact of CKD on oxidative and conjugative metabolism has been extensively studied. However, its effect on hepatic drug reduction, an important phase I drug-metabolism pathway, has not been investigated. We aimed to assess the effect of experimental CKD on hepatic reduction using warfarin as a pharmacological probe substrate. Cytosolic and microsomal cellular fractions were isolated from liver tissue harvested from five-sixths-nephrectomized and control rats (n = 10 per group). The enzyme kinetics for warfarin reduction were evaluated in both fractions, and formation of warfarin alcohols was used as an indicator of hepatic reductase activity. Selective inhibitors were employed to identify reductases involved in warfarin reduction. Gene and protein expression of reductases were determined using quantitative real-time polymerase chain reaction and Western blotting, respectively. Formation of RS/SR-warfarin alcohol was decreased by 39% (P < 0.001) and 43% (P < 0.01) in cytosol and microsomes, respectively, in CKD rats versus controls. However, RR/SS-warfarin alcohol formation was unchanged in the cytosol, and a trend toward its decreased production was observed in microsomes. Gene and protein expression of cytosolic carbonyl reductase 1 and aldo-keto reductase 1C3/18, and microsomal 11ß-hydroxysteroid dehydrogenase type 1 were significantly reduced by >30% (P < 0.05) in CKD rats compared with controls. Collectively, these results suggest that the functional expression of hepatic reductases is selectively decreased in kidney disease. Our findings may explain one mechanism for altered nonrenal clearance, exposure, and response of drugs in CKD patients.
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Enfermedades Renales/enzimología , Enfermedades Renales/metabolismo , Hígado/enzimología , Hígado/metabolismo , Oxidorreductasas/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Oxidorreductasas de Alcohol/metabolismo , Aldehído Reductasa/metabolismo , Aldo-Ceto Reductasas , Animales , Citosol/enzimología , Citosol/metabolismo , Modelos Animales de Enfermedad , Masculino , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Ratas , Ratas Sprague-Dawley , Warfarina/metabolismoRESUMEN
An international HIV pharmacology specialty laboratory (PSL) was established at the University of Zimbabwe to increase bioanalytical and investigator capacities. Quantitation of plasma nevirapine in samples from the AIDS Clinical Trials Group protocol 5279 was compared between the University of Nebraska Medical Center PSL and the University of Zimbabwe PSL. Both PSLs employed internally developed methods utilising reverse-phase high-performance liquid chromatography with ultraviolet detection. Eighty-seven percent of the cross-validation results exhibited ± 20% difference.
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PURPOSE: To investigate whether follicular fluid lipid-soluble micronutrients are associated with embryo morphology parameters during IVF. METHODS: Follicle fluid and oocytes were obtained prospectively from 81 women. Embryo morphology parameters were used as surrogate markers of oocyte health. HDL lipids and lipid-soluble micronutrients were analyzed by high-pressure liquid chromatography. Non-parametric bi-variate analysis and multivariable ordinal logistic regression models were employed to examine associations between biochemical and embryo morphology parameters. RESULTS: Follicular fluid HDL cholesterol (r = -0.47, p < 0.01), alpha-tocopherol (r = -0.41, p < 0.01), delta-tocopherol (r = -0.38, p < 0.05) and beta-cryptoxanthine (r = -0.42, p < 0.01) are negatively correlated with embryo fragmentation. Ordinal logistic regression models indicate that a 0.1 mumol/L increase in beta-cryptoxanthine, adjusted for gamma-tocopherol, is associated with a 75% decrease in the cumulative odds of higher embryo fragmentation (p = 0.010). CONCLUSION: Follicular fluid HDL micronutrients may play an important role in the development of the human oocyte as evident by embryo fragmentation during IVF.
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HDL-Colesterol/metabolismo , Embrión de Mamíferos/metabolismo , Líquido Folicular/metabolismo , Micronutrientes/metabolismo , Adulto , Embrión de Mamíferos/anatomía & histología , Femenino , Fertilización In Vitro/métodos , Humanos , Modelos Logísticos , Masculino , Análisis Multivariante , Embarazo , Estudios Prospectivos , Adulto JovenRESUMEN
The therapeutic use of cannabinoids has increased with providers often recommending cannabinoid-containing products with limited pre-clinical and clinical pharmacokinetic studies. An ultra-performance liquid chromatography with triple quadrupole mass spectrometry method was developed and validated for the determination of cannabidiol and Δ9-tetrahydrocannabinol in human ethylenediaminetetraacetic acid (EDTA) plasma. The cannabinoids are extracted from plasma with a liquid-liquid procedure utilizing methyl tert-butyl ether. UHPLC Separation was achieved with a Waters Acquity HSS T3 column (100â¯×â¯2.1â¯mm, 1.8⯵m) under isocratic conditions (18:82:0.02 water:methanol:formic acid v/v/v). The run time was 8.5â¯min. Detection of analytes was achieved using electrospray ionization and triple quadrupole selected reaction monitoring. Standard curve concentrations ranged from 0.5 to 250â¯ng/mL for cannabidiol and Δ9-tetrahydrocannabinol. The intra- and inter-day accuracy (% bias) and precision (relative standard deviation) were <9.20% in low, medium, and high quality control samples. The validated method was applied to the analysis of donated human EDTA plasma. The assay provides an important patient monitoring capability to determine variability in clinical pharmacokinetics during use of cannabinoid-containing products.
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Cannabidiol/sangre , Cromatografía Líquida de Alta Presión/métodos , Dronabinol/sangre , Espectrometría de Masas en Tándem/métodos , Ácido Edético , Humanos , Modelos Lineales , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: There are several instances where nevirapine pharmacokinetic monitoring may be useful, such as in special populations or pharmacokinetic drug interaction studies that require the ascertainment of nevirapine pharmacokinetics in the sub-Saharan region. OBJECTIVES: The main aim of this study was to produce a validated, sustainable and relevant nevirapine assay method that meets bio-analytical regulatory requirements. METHODS: The developed method utilised a Waters 2795 Alliance high performance liquid chromatography system with a 2996 photo diode array detector, an Atlantis dC18 5 micron, 3.9 mm × 150 mm analytical column and a gradient flow rate of 1 mL/min. Ultraviolet detection data were collected from 210 nm to 400 nm, extracted at 260 nm, and processed for nevirapine and internal standard peak height responses. RESULTS: The method proved to be linear (R2 0.995), precise (+1.92% - +9.69%) and accurate (-9.70% - 12.0%). Recovery for the analyte and internal standard was between 98.8% and 114%. The method showed good specificity as no interferences were caused by common African traditional medicines, anti-tuberculosis medications or other concomitant antiretrovirals nor endogenous components. CONCLUSION: The method is reproducible, relevant to our setting and uses considerably low plasma volumes with preservation of some consumables, a desirable key factor in a resource-limited setting.
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A liquid chromatography with triple quadrupole mass spectrometry method was developed and validated for the determination of tenofovir and tenofovir alafenamide concentrations in human plasma and cerebrospinal fluid. Tenofovir and tenofovir alafenamide were extracted from matrix by solid phase extraction. The dried extraction eluents were dissolved in water for LC-MS/MS analysis. Separation was achieved with a Phenomenex Synergi 4⯵m Polar-RP 80A column (50â¯×â¯2â¯mm) with a gradient elution of 0.1% formic acid in water and acetonitrile. The total run time was 5â¯min. Detection of analytes was achieved using electrospray ionization (positive mode) and triple quadrupole selected reaction monitoring. Standard curve concentrations ranged from 0.5 to 500â¯ng/mL for the plasma assay and 0.1-50â¯ng/mL for the cerebrospinal fluid assay. The intra- and inter-day accuracy and precision were less than 12% in low, medium, and high quality control samples for both matrices. The validated methods were applied to the analysis of plasma and cerebrospinal fluid samples of a patient undergoing tenofovir therapy which involved the switch from Stribild® (elvitegravir 150â¯mg/cobicistat 150â¯mg/emtricitabine 200â¯mg/tenofovir disoproxil fumarate 300â¯mg) to Genvoya® (elvitegravir 150â¯mg/cobicistat 150â¯mg/emtricitabine 200â¯mg/tenofovir alafenamide 10â¯mg).
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Adenina/análogos & derivados , Fármacos Anti-VIH/análisis , Infecciones por VIH/tratamiento farmacológico , Tenofovir/análisis , Adenina/análisis , Adenina/uso terapéutico , Alanina , Cromatografía Líquida de Alta Presión , Cobicistat/análisis , Cobicistat/uso terapéutico , Combinación de Medicamentos , Combinación Elvitegravir, Cobicistat, Emtricitabina y Fumarato de Tenofovir Disoproxil/análisis , Combinación Elvitegravir, Cobicistat, Emtricitabina y Fumarato de Tenofovir Disoproxil/uso terapéutico , Emtricitabina/análisis , Emtricitabina/uso terapéutico , Infecciones por VIH/sangre , Infecciones por VIH/líquido cefalorraquídeo , Humanos , Quinolonas/análisis , Quinolonas/uso terapéutico , Reproducibilidad de los Resultados , Extracción en Fase Sólida , Espectrometría de Masas en Tándem , Tenofovir/análogos & derivados , Tenofovir/uso terapéuticoRESUMEN
An ultra-performance liquid chromatography with triple quadrupole mass spectrometry method was developed and validated for the determination of direct acting antiviral drug concentrations in human liver fine needle aspirates. Liver fine needle aspirate (FNA) biopsy samples were homogenized in acetonitrile to stabilize the analytes and precipitate protein. The acetonitrile supernatants were diluted with internal standards and mobile phase. Separation was achieved with a Waters Acquity BEH C18 column (50×2.1mm, 1.7um) with a gradient elution of 0.1% formic acid in water and acetonitrile. The total run time was 4.25min. Detection of analytes was achieved using electrospray ionization (positive mode) and triple quadrupole selected reaction monitoring. Standard curve concentrations ranged from 12.5 to 5000ng/mL for dasabuvir and the m1 metabolite of dasabuvir, 1.25 to 2500ng/mL for ombitasvir and ritonavir, and 5.00 to 5000ng/mL for paritaprevir. The intra- and inter-day accuracy and precision were less than 13.7% in low, medium, and high quality control samples. The validated method was applied to the analysis of a liver fine needle aspirate of a patient undergoing direct acting antiviral therapy for hepatitis C virus.
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Anilidas/análisis , Antivirales/análisis , Carbamatos/análisis , Cromatografía Líquida de Alta Presión/métodos , Hígado/química , Compuestos Macrocíclicos/análisis , Sulfonamidas/análisis , Espectrometría de Masas en Tándem/métodos , Uracilo/análogos & derivados , 2-Naftilamina , Animales , Ciclopropanos , Humanos , Lactamas Macrocíclicas , Límite de Detección , Agujas , Prolina/análogos & derivados , Reproducibilidad de los Resultados , Uracilo/análisis , ValinaRESUMEN
A simple and rapid liquid chromatographic-tandem mass spectrometric method has been developed and validated for the enantiospecific determination of R- and S-warfarin in human urine. Warfarin enantiomers were extracted from urine using methyl tert-butyl ether. Chromatographic separation of warfarin enantiomers and the internal standard d5-warfarin was achieved using a Astec Chirobiotic V column with gradient mobile phase at a flow rate of 400 µL/min over 10 min. Detection was performed on a TSQ Quantum Ultra triple quadrupole mass spectrometer equipped with a heated electrospray ionization source. Analytes were detected in negative ionization mode using selected reaction monitoring. Calibration curves were linear with a correlation coefficient of ≥0.996 for both enantiomers over a concentration range of 5-500 ng/mL. The intra- and interday accuracy and precision for both analytes were within ±9.0%. Excellent extraction efficiency and negligible matrix effects were observed. The applicability of the method was demonstrated by successful measurement of warfarin enantiomers in urine of patients with kidney disease. The method is simple, accurate and reproducible and is currently being used to support warfarin pharmacokinetic studies.
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Anticoagulantes/orina , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas en Tándem/métodos , Warfarina/orina , Calibración , Humanos , Reproducibilidad de los Resultados , Espectrometría de Masa por Ionización de Electrospray , Estereoisomerismo , Warfarina/químicaRESUMEN
AIM: Determination of paritaprevir and ritonavir in rat liver tissue samples. RESULTS: We successfully validated a UPLC-MS/MS method to measure paritaprevir and ritonavir in rat liver using deuterated internal standards (d8-paritapervir and d6-ritonavir). The method is linear from 20 to 20,000 and 5 to 10,000 pg on column for paritaprevir and ritonavir, respectively, and is normalized per milligram tissue. Interday and intraday variability ranged from 0.591 to 5.33% and accuracy ranged from -6.68 to 10.1% for quality control samples. The method was then applied to the measurement of paritaprevir and ritonavir in rat liver tissue samples from a pilot study. CONCLUSION: The validated method is suitable for measurement of paritaprevir and ritonavir within rat liver tissue samples for PK studies.
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Antivirales/farmacocinética , Hígado/metabolismo , Compuestos Macrocíclicos/farmacocinética , Inhibidores de Proteasas/farmacocinética , Ritonavir/farmacocinética , Espectrometría de Masas en Tándem/métodos , Animales , Antivirales/análisis , Cromatografía Líquida de Alta Presión/métodos , Ciclopropanos , Hepacivirus/efectos de los fármacos , Hepacivirus/enzimología , Hepatitis C/tratamiento farmacológico , Lactamas Macrocíclicas , Límite de Detección , Compuestos Macrocíclicos/análisis , Masculino , Proyectos Piloto , Prolina/análogos & derivados , Inhibidores de Proteasas/análisis , Ratas , Ratas Sprague-Dawley , Ritonavir/análisis , SulfonamidasRESUMEN
A simple, sensitive, and precise ultra-high performance liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous determination of trimethylamine N-oxide, choline, and betaine in human plasma and urine. Sample preparation involved protein precipitation with methanol containing internal standards. Chromatographic separation was achieved using an Acquity BEH Amide (2.1mm×50mm, 1.7µm) analytical column with gradient elution of solvent A (10mM ammonium formate, pH 3.5) and solvent B (acetonitrile). The flow rate was 0.4mL/min and the total run time was 5min. Detection of analytes was performed using heated electrospray ionization (positive mode) and selected reaction monitoring. Excellent linearity was observed over the standard curve concentration ranges of 0.010-5.00µg/mL (plasma) and 1.00-150µg/mL (urine) for all analytes. The intra- and inter-day accuracy and precision for all quality controls were within ±10%. Excellent recovery was observed. The method is rapid, accurate and reproducible, and was successfully applied to a pilot study of markers of atherosclerosis in patients with kidney disease who underwent successful kidney transplantation.
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Betaína/sangre , Betaína/orina , Colina/sangre , Colina/orina , Metilaminas/sangre , Metilaminas/orina , Calibración , Cromatografía Líquida de Alta Presión , Humanos , Indicadores y Reactivos , Enfermedades Renales/metabolismo , Espectrometría de Masas , Proyectos Piloto , Control de Calidad , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
A sensitive, accurate, and reproducible ultra-high performance liquid chromatography-tandem mass spectrometry method was developed and validated for determination of warfarin and its alcohol metabolites (RS/SR- and RR/SS-warfarin alcohol) in 10mM Tris-HCl incubation buffer (pH 7.4). Sample preparation involved acidification with 4% formic acid, followed by liquid-liquid extraction using methyl tert-butyl ether. Chromatographic separation was achieved using a Hypersil Gold C18 (2.1mm×100mm, 1.9µm) analytical column with gradient elution of solvent A (water containing 0.01% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). The flow rate was 0.4mL/min and the total run time was 5min. Detection of analytes was performed using heated electrospray ionization (negative mode) and selected reaction monitoring. Excellent linearity was observed for all analytes over the standard curve concentration ranges of 100-10,000ng/mL for warfarin, and 0.5-250ng/mL for warfarin alcohols. The intra- and inter-day accuracy and precision for analytes were within ±10.0%. Excellent recovery and negligible matrix effects were observed. The method is robust, sensitive, accurate and reproducible, and was successfully applied to in vitro enzyme kinetic studies of warfarin.
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Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Warfarina/análogos & derivados , Warfarina/análisis , Warfarina/metabolismo , Animales , Estabilidad de Medicamentos , Cinética , Hígado/química , Ratas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Warfarina/químicaRESUMEN
OBJECTIVE: To measure antioxidant enzyme activities and lipid peroxidation levels within follicular fluid (FF) and evaluate correlations with early embryo quality. DESIGN: Individual FF samples were obtained prospectively on the day of oocyte collection and assessed for lipid peroxidation as specific positional isomers of hydroperoxy and hydroxy fatty acids by high-performance liquid chromatography and antioxidant enzyme activities by automated kinetic enzyme assays. Spearman rank correlation coefficients, adjusted for age and day of transfer, were used to assess associations between antioxidant enzymes and lipid peroxidation products and embryo quality using a 1 follicle-1 oocyte/embryo approach. Post hoc power analysis was conducted to help interpret null results. SETTING: A university clinic. PATIENT(S): Thirty-nine women undergoing IVF. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Embryo cell number and embryo fragmentation score (EFS) at transfer. RESULT(S): No significant correlations between lipid peroxidation derivatives or antioxidant enzyme activities and embryo quality were obtained. Post hoc power analysis indicated possible undetected associations between EFS and 13-hydroxy octadecatrienoic acid and 13-hydroperoxy octadecadieneoic acid. CONCLUSION(S): Our preliminary dataset suggests that most lipid peroxidation products and antioxidant enzyme activities within FF are not associated with the quality of embryos, using EFS and embryo cell number as end points. However, further consideration of associations between EFS and 13-hydroxy octadecatrienoic acid and 13-hydroperoxy octadecadieneoic acid is warranted.