RESUMEN
As organoids and organ-on-chip (OoC) systems move toward preclinical and clinical applications, there is an increased need for method validation. Using a liquid chromatography-mass spectrometry (LC-MS)-based approach, we developed a method for measuring small-molecule drugs and metabolites in the cell medium directly sampled from liver organoids/OoC systems. The LC-MS setup was coupled to an automatic filtration and filter flush system with online solid-phase extraction (SPE), allowing for robust and automated sample cleanup/analysis. For the matrix, rich in, e.g., protein, salts, and amino acids, no preinjection sample preparation steps (protein precipitation, SPE, etc.) were necessary. The approach was demonstrated with tolbutamide and its liver metabolite, 4-hydroxytolbutamide (4HT). The method was validated for analysis of cell media of human stem cell-derived liver organoids cultured in static conditions and on a microfluidic platform according to Food and Drug Administration (FDA) guidelines with regards to selectivity, matrix effects, accuracy, precision, etc. The system allows for hundreds of injections without replacing chromatography hardware. In summary, drug/metabolite analysis of organoids/OoCs can be performed robustly with minimal sample preparation.
Asunto(s)
Hígado , Organoides , Humanos , Organoides/metabolismo , Organoides/citología , Cromatografía Liquida/métodos , Hígado/metabolismo , Espectrometría de Masas/métodos , Tolbutamida/metabolismo , Tolbutamida/análisis , Dispositivos Laboratorio en un Chip , Preparaciones Farmacéuticas/metabolismo , Preparaciones Farmacéuticas/análisis , Extracción en Fase Sólida , Bibliotecas de Moléculas Pequeñas/análisis , Bibliotecas de Moléculas Pequeñas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Cromatografía Líquida con Espectrometría de MasasRESUMEN
Arsenite is an important heavy metal. Some Chinese traditional medicines contain significant amounts of arsenite. The aim of this study was to investigate subacute exposure of arsenite on activities of cytochrome P450 enzymes and pharmacokinetic behaviors of drugs in rats. Midazolam, tolbutamide, metoprolol, omeprazole, caffeine, and chlorzoxazone, the probe substrates for cytochrome P450 (CYP) s3A, 2C6, 2D, 2C11, 1A, and 2E, were selected as probe drugs for the pharmacokinetic study. Significant decreases in areas under the curves of probe substrates were observed in rats after consecutive 30-day exposure to As at 12 mg/kg. Microsomal incubation study showed that the subacute exposure to arsenite resulted in little change in effects on the activities of P450 enzymes examined. However, everted gut sac study demonstrated that such exposure induced significant decreases in intestinal absorption of these drugs by both passive diffusion and carrier-mediated transport. In addition, in vivo study showed that the arsenite exposure decreased the rate of peristaltic propulsion. The decreases in intestinal permeability of the probe drugs and peristaltic propulsion rate most likely resulted in the observed decreases in the internal exposure of the probe drugs. Exposure to arsenite may lead to the reduction of the efficiencies of pharmaceutical agents coadministered resulting from the observed drug-drug interactions. SIGNIFICANCE STATEMENT: Exposure to arsenite may lead to the reduction of the efficiencies of pharmaceutical agents coadministered resulting from the observed drug-drug interactions. The present study, we found that P450 enzyme probe drug exposure was reduced in arsenic-exposed animals (areas under the curve) and the intestinal absorption of the drug was reduced in the animals. Subacute arsenic exposure tends to cause damage to intestinal function, which leads to reduced drug absorption.
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Arsenitos , Sistema Enzimático del Citocromo P-450 , Interacciones Farmacológicas , Ratas Sprague-Dawley , Animales , Arsenitos/toxicidad , Arsenitos/farmacocinética , Masculino , Ratas , Sistema Enzimático del Citocromo P-450/metabolismo , Absorción Intestinal/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Omeprazol/farmacología , Omeprazol/farmacocinética , Midazolam/farmacocinética , Cafeína/farmacocinética , Clorzoxazona/farmacocinética , Metoprolol/farmacocinética , Metoprolol/farmacología , Tolbutamida/farmacocinética , Compuestos de Sodio/toxicidad , Compuestos de Sodio/farmacocinéticaRESUMEN
Since glucose stimulates protein biosynthesis in beta cells concomitantly with the stimulation of insulin release, the possible interaction of both processes was explored. The protein biosynthesis was inhibited by 10 µM cycloheximide (CHX) 60 min prior to the stimulation of perifused, freshly isolated or 22 h-cultured NMRI mouse islets. CHX reduced the insulinotropic effect of 25 mM glucose or 500 µM tolbutamide in fresh but not in cultured islets. In cultured islets the second phase of glucose stimulation was even enhanced. In fresh and in cultured islets CHX strongly reduced the content of proinsulin, but not of insulin, and moderately diminished the [Ca2+]i increase during stimulation. The oxygen consumption rate (OCR) of fresh islets was about 50% higher than that of cultured islets at basal glucose and was significantly increased by glucose but not tolbutamide. In fresh, but not in cultured, islets CHX diminished the glucose-induced OCR increase and changes in the NAD(P)H- and FAD-autofluorescence. It is concluded that short-term CHX exposure interferes with the signal function of the mitochondria, which have different working conditions in fresh and in cultured islets. The interference may not be an off-target effect but may result from the inhibited cytosolic synthesis of mitochondrial proteins.
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Islotes Pancreáticos , Ratones , Femenino , Animales , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Cicloheximida/farmacología , Insulina/metabolismo , Glucosa/metabolismo , Tolbutamida/farmacología , Tolbutamida/metabolismo , NAD/metabolismo , Mitocondrias/metabolismo , Calcio/metabolismoRESUMEN
Accumulating evidence supports the use of higher doses of rifampicin for tuberculosis (TB) treatment. Rifampicin is a potent inducer of metabolic enzymes and drug transporters, resulting in clinically relevant drug interactions. To assess the drug interaction potential of higher doses of rifampicin, we compared the effect of high-dose rifampicin (40 mg/kg daily, RIF40) and standard-dose rifampicin (10 mg/kg daily, RIF10) on the activities of major cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp). In this open-label, single-arm, two-period, fixed-order phenotyping cocktail study, adult participants with pulmonary TB received RIF10 (days 1-15), followed by RIF40 (days 16-30). A single dose of selective substrates (probe drugs) was administered orally on days 15 and 30: caffeine (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and digoxin (P-gp). Intensive pharmacokinetic blood sampling was performed over 24 hours after probe drug intake. In all, 25 participants completed the study. Geometric mean ratios (90% confidence interval) of the total exposure (area under the concentration versus time curve, RIF40 versus RIF10) for each of the probe drugs were as follows: caffeine, 105% (96%-115%); tolbutamide, 80% (74%-86%); omeprazole, 55% (47%-65%); dextromethorphan, 77% (68%-86%); midazolam, 62% (49%-78%), and 117% (105%-130%) for digoxin. In summary, high-dose rifampicin resulted in no additional effect on CYP1A2, mild additional induction of CYP2C9, CYP2C19, CYP2D6, and CYP3A, and marginal inhibition of P-gp. Existing recommendations on managing drug interactions with rifampicin can remain unchanged for the majority of co-administered drugs when using high-dose rifampicin. Clinical Trials registration number NCT04525235.
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Citocromo P-450 CYP1A2 , Tuberculosis Pulmonar , Adulto , Humanos , Midazolam/uso terapéutico , Citocromo P-450 CYP2D6/metabolismo , Cafeína , Rifampin/uso terapéutico , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP3A/metabolismo , Dextrometorfano/uso terapéutico , Tolbutamida , Citocromo P-450 CYP2C9/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Omeprazol , Interacciones Farmacológicas , Tuberculosis Pulmonar/tratamiento farmacológico , Digoxina/uso terapéuticoRESUMEN
BACKGROUND: Hypoxia-ischemia predisposes to atrial arrhythmia. Atrial ATP-sensitive potassium channel (KATP) modulation during hypoxia has not been explored. We investigated the effects of hypoxia on atrial electrophysiology in mice with global deletion of KATP pore-forming subunits. METHODS: Whole heart KATP RNA expression was probed. Whole-cell KATP current and action potentials were recorded in isolated wild-type (WT), Kir6.1 global knockout (6.1-gKO), and Kir6.2 global knockout (6.2-gKO) murine atrial myocytes. Langendorff-perfused hearts were assessed for atrial effective refractory period (ERP), conduction velocity, wavefront path length (WFPL), and arrhymogenicity under normoxia/hypoxia using a microelectrode array and programmed electrical stimulation. Heart histology was assessed. RESULTS: Expression patterns were essentially identical for all KATP subunit RNA across human heart, whereas in mouse, Kir6.1 and SUR2 (sulphonylurea receptor subunit) were higher in ventricle than atrium, and Kir6.2 and SUR1 were higher in atrium. Compared with WT, 6.2-gKO atrial myocytes had reduced tolbutamide-sensitive current and action potentials were more depolarized with slower upstroke and reduced peak amplitude. Action potential duration was prolonged in 6.1-gKO atrial myocytes, absent of changes in other ion channel gene expression or atrial myocyte hypertrophy. In Langendorff-perfused hearts, baseline atrial ERP was prolonged and conduction velocity reduced in both KATP knockout mice compared with WT, without histological fibrosis. Compared with baseline, hypoxia led to conduction velocity slowing, stable ERP, and WFPL shortening in WT and 6.1-gKO hearts, whereas WFPL was stable in 6.2-gKO hearts due to ERP prolongation with conduction velocity slowing. Tolbutamide reversed hypoxia-induced WFPL shortening in WT and 6.1-gKO hearts through ERP prolongation. Atrial tachyarrhythmias inducible with programmed electrical stimulation during hypoxia in WT and 6.1-gKO mice correlated with WFPL shortening. Spontaneous arrhythmia was not seen. CONCLUSIONS: KATP block/absence leads to cellular and tissue level atrial electrophysiological modification. Kir6.2 global knockout prevents hypoxia-induced atrial WFPL shortening and atrial arrhythmogenicity to programmed electrical stimulation. This mechanism could be explored translationally to treat ischemically driven atrial arrhythmia.
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Fibrilación Atrial , Canales KATP , Humanos , Animales , Ratones , Canales KATP/genética , Fibrilación Atrial/genética , Tolbutamida , Taquicardia , Atrios Cardíacos , Hipoxia/complicaciones , Hipoxia/genética , Adenosina TrifosfatoRESUMEN
The drug interaction potential of enarodustat (doses: 25, 50 mg) on the activity of cytochrome P450 (CYP) 1A2, 2C9, 2C19, 2D6, and 3A4 was evaluated after once-daily administration for 15 days in a phase 1 multiple-ascending-dose study in healthy subjects. Probe substrates specific for the enzymes, i.e., caffeine (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and midazolam (CYP3A4), were administered orally as a cocktail with (day 15) and without (day -3) enarodustat. Drug interaction was based on geometric mean maximum plasma concentration (Cmax ) and area under the plasma concentration-time curve from the time of dosing to infinity (AUCinf ) ratios (day 15/day -3) for CYP1A2, 2C9, 2C19, 2D6, 3A4, and urinary excretion of dextromethorphan metabolite dextrorphan for CYP2D6. At the 2 enarodustat doses, for caffeine, the geometric mean ratios (range) for Cmax and AUCinf were 0.99-1.06 and 1.61-1.63, respectively. The ratios for peak concentrations and total exposures were 0.98-1.07 and 0.71-1.78 for tolbutamide and omeprazole, respectively. For dextrorphan the Cmax and AUCinf ratios were 0.83-0.90 and 1.02-1.04, respectively. The mean dextrorphan cumulative amount excreted into the urine from the time of dosing to 24 hours values on day -3 and day 15 were 8.25 mg and 8.20 mg at the lower dose, and 9.40 mg and 9.51 mg at the higher dose. The ratios for midazolam Cmax and AUCinf were 1.42-1.63. Overall, there was a lack of enarodustat dose dependency regarding the geometric mean ratios and 90% confidence intervals and urinary excretion of dextrorphan. There were some cases where the 90% confidence intervals at the 2 enarodustat doses were outside the 0.80-1.25 range, but changes in the geometric mean ratios were all <2-fold.
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Citocromo P-450 CYP1A2 , Citocromo P-450 CYP2D6 , Humanos , Citocromo P-450 CYP1A2/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Cafeína , Midazolam , Tolbutamida , Dextrometorfano , Dextrorfano , Sistema Enzimático del Citocromo P-450/metabolismo , Interacciones Farmacológicas , OmeprazolRESUMEN
In this study, we investigated the effect of type 1 diabetes mellitus on the modulation of the activities of CYP450s in dynamics by a UHPLC-MS/MS method. The diabetic rat model was constructed by an intraperitoneal single injection of streptozotocin. Fasting blood glucose levels > 16.7 mmol/L were considered as diabetic. The rats were given a cocktail of four probe drugs (10 mg/kg phenacetin, 1 mg/kg tolbutamide, 10 mg/kg metoprolol, and 10 mg/kg midazolam) by oral administration for the pharmacokinetic study. Thereafter, the metabolic ratio (MR) of the metabolites to probe substrates were determined. The results indicated that two weeks after diabetes was induced, diabetes increased the MRs of acetaminophen/phenacetin (CYP1A2) and 4-hydroxyl tolbutamide/tolbutamide (CYP2C9); however, it decreased the MRs of α-hydroxy metoprolol/metoprolol (CYP2D6) and 1-hydroxy midazolam/midazolam (CYP3A4). Two months after diabetes was induced, diabetes increased the MRs of acetaminophen/phenacetin and 4-hydroxyl tolbutamide/tolbutamide. The MR of α-hydroxy metoprolol/metoprolol was decreased and the MR of 1-hydroxy midazolam/midazolam was increased but the difference was not significant. According to the results, CYP1A2 and CYP2C9 activities were enhanced in the diabetic rats. and CYP2D6 activity was inhibited in a short period of diabetes; however, the decrease in CYP2D6 activity was not significant in the long period. CYP3A4 activity was decreased in a short period of diabetes and increased in a long period of diabetes but was not significant in the two periods. This study suggests the activity change rule of the CYP450 enzyme system in diabetes mellitus, which can provide a reference for precise clinical medication.
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Citocromo P-450 CYP1A2 , Diabetes Mellitus Experimental , Animales , Ratas , Acetaminofén , Cromatografía Líquida de Alta Presión/métodos , Citocromo P-450 CYP2C9 , Citocromo P-450 CYP2D6 , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Metoprolol , Midazolam/metabolismo , Fenacetina , Espectrometría de Masas en Tándem/métodos , TolbutamidaRESUMEN
Licorice, the roots and rhizomes of Glycyrrhiza glabra L., has been used as a medicinal herb, herbal adjuvant, and flavoring agent since ancient times. Recently, licorice extracts have become popular as dietary supplements used by females to alleviate menopausal symptoms. Exposure to licorice products containing high levels of glycyrrhizic acid can cause hypokalemia, but independent from this effect, preclinical data indicate that licorice can inhibit certain cytochrome P450 (P450) enzymes. To evaluate whether clinically relevant pharmacokinetic interactions of licorice with P450 enzymes exist, a phase 1 clinical investigation was carried out using a licorice extract depleted in glycyrrhizic acid (content <1%) and a cocktail containing caffeine, tolbutamide, alprazolam, and dextromethorphan, which are probe substrates for the enzymes CYP1A2, CYP2C9, CYP3A4/5, and CYP2D6, respectively. The botanically authenticated and chemically standardized extract of roots from G. glabra was consumed by 14 healthy menopausal and postmenopausal female participants twice daily for 2 weeks. The pharmacokinetics of each probe drug were evaluated immediately before and after supplementation with the licorice extract. Comparison of the average areas under the time-concentration curves (AUCs) for each probe substrate in serum showed no significant changes from licorice consumption, whereas time to reach peak concentration for caffeine and elimination half-life for tolbutamide showed small changes. According to the US Food and Drug Administration guidance, which is based on changes in the AUC of each probe substrate drug, the investigated licorice extract should not cause any clinically relevant pharmacokinetic interactions with respect to CYP3A4/5, CYP2C9, CYP2D6, or CYP1A2. SIGNIFICANCE STATEMENT: Despite generally-recognized-as-safe status, the licorice species Glycyrrhiza glabra has been associated with some toxicity. Preclinical studies suggest that G. glabra might cause pharmacokinetic drug interactions by inhibiting several cytochrome P450 enzymes. This phase 1 clinical study addressed these concerns by evaluating clinically relevant effects with respect to CYP3A4/5, CYP2C9, CYP2D6, and CYP1A2. These results showed that a standardized G. glabra extract did not cause any clinically relevant pharmacokinetic drug interactions with four major cytochrome P450 enzymes.
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Citocromo P-450 CYP1A2 , Glycyrrhiza , Humanos , Femenino , Citocromo P-450 CYP2D6 , Cafeína/farmacocinética , Citocromo P-450 CYP3A , Tolbutamida , Ácido Glicirrínico , Citocromo P-450 CYP2C9 , Sistema Enzimático del Citocromo P-450 , Glycyrrhiza/química , Suplementos DietéticosRESUMEN
PURPOSE: In this study, the drug-drug interaction potential of vatiquinone with cytochrome P450 (CYP) substrates was investigated in both in vitro and clinical studies. METHODS: The inhibitory potential of vatiquinone on the activity of CYPs 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4/5 was assessed in vitro. In an open-label, drug-drug interaction study in 18 healthy human subjects, a single oral dose of 500 mg tolbutamide and 40 mg omeprazole was administered on day 1, followed by a washout of 7 days. Multiple oral doses of 400 mg vatiquinone (three times a day [TID]) were administered from day 8 to day 13 with coadministration of a single oral dose of 500 mg tolbutamide and 40 mg omeprazole on day 12. RESULTS: In vitro, vatiquinone inhibited CYP2C9 (IC50 = 3.7 µM) and CYP2C19 (IC50 = 5.4 µM). In the clinical study, coadministration of vatiquinone did not affect the pharmacokinetic (PK) profile of tolbutamide and omeprazole. The 90% confidence intervals (CIs) of geometric least-square mean ratios for maximum plasma concentration (Cmax), areas under the plasma concentration-time curve (AUC0-t), and AUC0-inf of tolbutamide and omeprazole were entirely contained within the 80 to 125% no effect limit, except a minor excursion observed for Cmax of omeprazole (geometric mean ratio [GMR], 94.09; 90% CI, 78.70-112.50). Vatiquinone was generally well tolerated, and no clinically significant findings were reported. CONCLUSION: The in vitro and clinical studies demonstrated vatiquinone has a low potential to affect the pharmacokinetics of concomitantly administered medications that are metabolized by CYP enzymes.
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Omeprazol , Tolbutamida , Área Bajo la Curva , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Interacciones Farmacológicas , Voluntarios Sanos , Humanos , Omeprazol/farmacología , Tolbutamida/farmacocinéticaRESUMEN
Specific noncovalent drug-polymer interactions were analytically identified using Raman and Fourier transform infrared spectroscopy for amorphous solid dispersions (ASD) formed between either chlorpropamide or tolbutamide and polyvinylpyrrolidone vinyl acetate random copolymer (PVPVA). Spectral changes in the C-Cl stretching vibrations due to changes in the electronic environment of the Cl atom confirmed halogen bond formation in chlorpropamide-PVPVA ASDs, the extent of which was established to be inversely related to the concentration of the drug using 2D correlation spectroscopy analysis. Hydrogen bonding between the secondary amide of each drug and the pyrrolidone carbonyl of the copolymer was also confirmed in all dispersions. Implications of coexistent interactions were investigated for drug-polymer solubility, mixing free energy, and molecular mobility relative to tolbutamide, which only formed hydrogen bonds with PVPVA. Chlorpropamide had a higher solubility, a larger negative mixing free energy, and lower mobility in PVPVA relative to tolbutamide. These thermodynamic and kinetic differences demonstrate the significance of halogen bond formation even when hydrogen bonding is present.
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Halógenos , Tolbutamida , Solubilidad , Enlace de Hidrógeno , Clorpropamida , Polímeros/química , Composición de Medicamentos/métodosRESUMEN
BACKGROUND AND OBJECTIVE: Tucatinib is approved for treatment of human epidermal growth factor receptor 2-positive metastatic breast cancer. Understanding potential drug-drug interactions (DDIs) informs proper dosing when co-administering tucatinib with other therapies. The aim of this study was to evaluate DDIs between tucatinib and metabolizing enzymes and transporters in healthy volunteers. METHODS: Parts A-C assessed the impact of itraconazole (cytochrome P450 [CYP] 3A4 inhibitor), rifampin (CYP3A4/CYP2C8 inducer), or gemfibrozil (CYP2C8 inhibitor) on the pharmacokinetics of a single 300 mg dose of tucatinib administered orally and its primary metabolite, ONT-993. Parts D and E assessed the effect of steady-state tucatinib on the pharmacokinetics of repaglinide (CYP2C8 substrate), tolbutamide (CYP2C9 substrate), midazolam (CYP3A4 substrate), and digoxin (P-glycoprotein substrate). RESULTS: Tucatinib area under the concentration-time curve from time 0 extrapolated to infinity (AUC0-inf) increased by ~ 1.3- and 3.0-fold with itraconazole and gemfibrozil, respectively, and decreased by 48% with rifampin, indicating that tucatinib is metabolized primarily by CYP2C8, and to a lesser extent via CYP3A. Tucatinib was a strong inhibitor of CYP3A (midazolam AUC0-inf increased 5.7-fold), a weak inhibitor of CYP2C8 and P-glycoprotein, and had no impact on CYP2C9-mediated metabolism in humans. Tucatinib was well tolerated, alone and with co-administered drugs. CONCLUSION: The potential DDIs identified here may be mitigated by avoiding concomitant use of tucatinib with strong CYP3A inducers, moderate CYP2C8 inducers, CYP3A substrates with a narrow therapeutic window (modifying substrate dose where concomitant use is unavoidable), and strong CYP2C8 inhibitors (decreasing tucatinib dose where concomitant use is unavoidable), or by reducing the dose of P-glycoprotein substrates with a narrow therapeutic window. TRIAL REGISTRATION: This trial (NCT03723395) was registered on October 29, 2018.
Asunto(s)
Inductores del Citocromo P-450 CYP2C8 , Inductores del Citocromo P-450 CYP3A , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Área Bajo la Curva , Citocromo P-450 CYP2C8/metabolismo , Inhibidores del Citocromo P-450 CYP2C8 , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Inductores del Citocromo P-450 CYP3A/farmacocinética , Digoxina , Interacciones Farmacológicas , Gemfibrozilo , Voluntarios Sanos , Humanos , Itraconazol/farmacología , Midazolam/farmacocinética , Oxazoles , Piridinas , Quinazolinas , Rifampin/farmacología , TolbutamidaRESUMEN
INTRODUCTION: Pharmacokinetic variability in disease state is common in clinical practice, but its underlying mechanism remains unclear. Recently, gut microbiota has been considered to be pharmacokinetically equivalent to the host liver. Although some studies have explored the roles of gut microbiota and host Cyp450s in drug pharmacokinetics, few have explored their effects on pharmacokinetic variability, especially in disease states. OBJECTIVES: In this study, we aim to investigate the effects of gut microbiota and host Cyp450s on pharmacokinetic variability in mice with non-alcoholic steatohepatitis (NASH), and to elucidate the contribution of gut microbiota and host Cyp450s to pharmacokinetic variability in this setting. METHODS: The pharmacokinetic variability of mice with NASH was explored under intragastric and intravenous administrations of a cocktail mixture of omeprazole, phenacetin, midazolam, tolbutamide, chlorzoxazone, and metoprolol, after which the results were compared with those obtained from the control group. Thereafter, the pharmacokinetic variabilities of all drugs and their relations to the changes in gut microbiota and host Cyp450s were compared and analyzed. RESULTS: The exposures of all drugs, except metoprolol, significantly increased in the NASH group under intragastric administration. However, no significant increase in the exposure of all drugs, except tolbutamide, was observed in the NASH group under intravenous administration. The pharmacokinetic variabilities of phenacetin, midazolam, omeprazole, and chlorzoxazone were mainly associated with decreased elimination activity in the gut microbiota. By contrast, the pharmacokinetic variability of tolbutamide was mainly related to the change in the host Cyp2c65. Notably, gut microbiota and host Cyp450s exerted minimal effects on the pharmacokinetic variability of metoprolol. CONCLUSION: Gut microbiota and host Cyp450s co-contribute to the pharmacokinetic variability in mice with NASH, and the degree of contribution varies from drug to drug. The present findings provide new insights into the explanation of pharmacokinetic variability in disease states.
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Microbioma Gastrointestinal , Enfermedad del Hígado Graso no Alcohólico , Animales , Clorzoxazona/farmacología , Metoprolol/farmacología , Ratones , Midazolam/farmacología , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Omeprazol/farmacología , Preparaciones Farmacéuticas , Fenacetina/farmacología , Tolbutamida/farmacologíaRESUMEN
BACKGROUND: Gnaphalium affine D. Don extract (GAD) enhanced efficacy and reduced toxicity of benzbromarone (BBR) in combination use. However, little is known about effects of GAD on the pharmacokinetics (PKs) and metabolic enzymes of BBR. PURPOSE: To investigate the pharmacokinetic (PK) and pharmacodynamic (PD) mechanism of the herb-drug interactions (HDIs) between GAD and BBR. STUDY DESIGN AND METHODS: Intragastric single BBR (4.5 or 50 mg/kg), single BBR (4.5 or 50 mg/kg) + single GAD (450 mg/kg, 2 h after BBR-administration), or single BBR (4.5 or 50 mg/kg) + multiple GAD (450 mg/kg/day, once daily for 7 days) were administered to both sexes for BBR PK studies in normal rats. Intragastric multiple BBR (4.5 mg/kg/day), or multiple BBR (4.5 mg/kg/day) + multiple GAD (450 mg/kg/day, 2 h after BBR-administration) were administered for BBR PK and PD studies in male rats with hyperuricemic nephropathy (HN). The cumulative anti-hyperuricemic effects of BBR and BBR+GAD were determined by plasma uric acid (UA) concentration-time curve and area under curve (AUCUA). An in vivo cocktail approach was employed to determine the effects of GAD on cytochrome P450 (CYP) 2C11(9) and 1A2 - mediated drug metabolism. RESULTS: In normal rats, the repeated dose administration of GAD induced a significant increase of BBR AUC and prolonged the mean residence time (MRT) (p < 0.05). systemic exposure to BBR and metabolically derived hydroxybenzbromarones was significantly greater in female compared with male rats (p < 0.05). In HN rats, post-administration of GAD resulted in significantly higher bioavailability and enterohepatic recycling (ER) of BBR relative to the BBR alone administrated group from the prolongation of terminal elimination half-life (T1/2) and MRT of BBR (p < 0.05). Significantly higher urate-lowering effect of BBR+GAD compared with BBR alone was generally observed at post-dosing most time points with a maximal effect of 84.3% (acute treatment), 71.4% (7-day subchronic treatment) and 82.5% (14-day subchronic treatment) reduction in UA levels. Additionally, GAD showed a significant inhibitory effect on CYP2C11(9)-mediated tolbutamide (probe substrate) metabolism with ≥ 1.25 but < 2-fold increase in AUCtolbutamide. CONCLUSIONS: PD synergism demonstrated with the BBR+GAD combination could be explained by the PK interaction observed partially from CYP2C11(9)-mediation and enterohepatic recycling.
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Gnaphalium , Interacciones de Hierba-Droga , Animales , Benzbromarona/farmacología , Femenino , Masculino , Extractos Vegetales/farmacología , Ratas , Tolbutamida/farmacocinéticaRESUMEN
The primary objectives of this study were to investigate the suitability of a 6-probe cocktail (caffeine, tolbutamide, omeprazole, dextromethorphan, midazolam, and digoxin) to be used as a tool for assessing the activity of drug metabolizing enzymes and transporters, and examine differences in the way drugs are handled among groups with different genetic regulation of these processes. This was a single-center, open-label, phase I clinical study involving 20 young, healthy Chinese volunteers (equal gender distribution). The subjects were administered a single, oral dose of the 6-probe cocktail and serum samples were collected to assess the disposition of the different probe substrates and produced metabolites. The serum samples were analyzed using ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry technology. The DNA samples were subjected to whole exome sequencing. Nineteen healthy volunteers completed the study. The 6-probe cocktail was safe and well-tolerated by all the subjects. The parent substrates and metabolites-caffeine (paraxanthine), dextromethorphan (dextrorphan), digoxin, midazolam (1-hydroxy-midazolam), omeprazole (5-hydroxy-omeprazole), and tolbutamide (4-hydroxy-tolbutamide)-were within the detectable window. Genetic variations known to alter drug metabolism (CYP2D6*10, CYP2C19*2, CYP2C19*3, and CYP2C9*3) were identified and generally correlated with phenotypic status. The 6-probe cocktail appeared to be suitable for assessing drug metabolizing activities. This, in conjunction with individual genetics, will pave the way for the implementation of personalized medicine in clinical practice. This will hopefully improve efficacy and reduce the incidence of adverse drug reactions.
Asunto(s)
Midazolam , Tolbutamida , Cafeína , China , Citocromo P-450 CYP2C19 , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Dextrometorfano , Digoxina , Interacciones Farmacológicas , Voluntarios Sanos , Humanos , OmeprazolRESUMEN
Among the strategies for bioavailability improvement of poorly soluble drugs, co-amorphous systems have revealed to have a significant impact in the increase of the aqueous solubility of the drug, and at the same time increasing the amorphous state stability and dissolution rate when compared with the neat drug. Tolbutamide (TBM) is an oral hypoglycemic drug largely used in the treatment of type II Mellitus diabetes. TBM is a class II drug according to the Biopharmaceutical Classification System, meaning that it has low solubility and higher permeability. The aim of this study was to synthesize a co-amorphous material of tolbutamide (TBM) with tromethamine (TRIS). Density functional theory (DFT), allowed to study the structural, electronic, and thermodynamic properties, as well as solvation effects. In same theory level, several interactions tests were performed to obtain the most thermodynamically favorable drug-coformer intermolecular interactions. The vibrational spectra (mid infrared and Raman spectroscopy) are in accordance with the theoretical studies, showing that the main molecular interactions are due to the carbonyl, sulfonyl, and amide groups of TMB and the alcohol and amine groups of TRIS. X-ray powder diffraction was used to study the physical stability in dry condition at 25 °C of the co-amorphous system, indicating that the material remained in an amorphous state up to 90 days. Differential scanning calorimetry and thermogravimetric results showed a high increase of the Tg when compared with the amorphous neat drug, from 4.3 °C to 83.7 °C, which generally translated into good physical stability. Solubility studies demonstrated an increase in the solubility of TBM by 2.5 fold when compared with its crystalline counterpart.
Asunto(s)
Diabetes Mellitus , Tolbutamida , Rastreo Diferencial de Calorimetría , Sistemas de Liberación de Medicamentos , Estabilidad de Medicamentos , Humanos , Solubilidad , Espectroscopía Infrarroja por Transformada de Fourier , Difracción de Rayos XRESUMEN
The Cocktail probe drug method was used to evaluate the effect of Laportea bulbifera extract on the different subtypes of CYP450 enzyme activities in rats, and to provide references for its clinical rational drug use. The rats were randomly divided into a high-dose L. bulbifera group(0.45 g·kg~(-1)·d~(-1)) and a low-dose L. bulbifera group(0.09 g·kg~(-1)·d~(-1)). After continuous gavage for 7 and 14 days, the Cocktail probe mixing solution, including caffeine, midazolam, tolbutamide, omeprazole, metoprolol, and chlorzoxazone, was injected into the tail vein, and the blood sample was obtained from the tail vein at different time points. Ultra-high performance liquid chromatography-mass spectrometry(UPLC-MS) was established to determine the concentration of six probe drugs in rat plasma. DAS 2.0 was used to calculate its pharmacokinetic parameters, and the effect of L. bulbifera extract on CYP1 A2, CYP2 C9, CYP2 C19, CYP2 D6, CYP2 E1, and CYP3 A4 in rats was investigated. As compared with the blank control group, under the omeprazole index, the AUC_(0-t) and AUC_(0-∞) of the 7-day administration groups and the 14-day high-dose group were significantly decreased, and the CLz and Vz were significantly increased. Under the midazolam index, the AUC_(0-t) and AUC_(0-∞) of the 7-day low-dose group and the 14-day administration group were significantly decreased, and the CLz was significantly increased. In addition, the AUC_(0-∞) of the 7-day high-dose group was also significantly decreased. Under the index of metoprolol, the AUC_(0-t) and AUC_(0-∞) of each experimental group were decreased significantly, and the CLz and Vz of the 7-day low-dose group and the 14-day low-dose group were increased significantly. Under the caffeine index, the AUC_(0-t) and AUC_(0-∞) of the 7-day administration groups were increased significantly, the CLz was decreased significantly, and the t_(1/2 z) of the 14-day high-dose group was increased significantly. Under the chlorzoxazone index, the AUC_(0-t) and AUC_(0-∞) of the 7-day low-dose group were increased significantly, and the CLz was decreased significantly. Under the tolbutamide index, there was no statistical difference in the pharmacokinetic parameters. In conclusion, L. bulbifera extract induces the activities of CYP2 C19, CYP3 A4, and CYP2 D6, inhibits the activities of CYP1 A2 and CYP2 E1, and does not affect the activity of CYP2 C9.
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Cafeína , Midazolam , Ratas , Animales , Midazolam/farmacocinética , Clorzoxazona , Metoprolol , Tolbutamida , Cromatografía Liquida , Espectrometría de Masas en Tándem/métodos , Sistema Enzimático del Citocromo P-450 , Omeprazol/farmacología , Extractos Vegetales/farmacologíaRESUMEN
Natural products embedded crown ethers were prepared by utilizing bioactive natural products including chrysin, tetrahydroisoquinoline (THIQ), and biochanin-A. The prepared crown ether scaffolds were evaluated and compared with their natural product precursors for insulin secretory activity on isolated mice islets and for their fluorescent properties. All the crown adducts were found more active as compared to their natural product precursors. Bischrysin 32-crown-10 (6d), THIQ 15-Crown-5 (6a) and chrysin 16-crown-5 (6c) showed mild, moderate and strong insulin secretory activity, respectively when compared with the standard drug tolbutamide (TB). Particularly crown derivative 6c showed strong activity (31.10 ng/islet/h) that is almost two (02) fold higher than that of standard drug TB (16.82 ng/islet/h). To the best of our knowledge crown ethers based antidiabetic study is being reported for the first time in literature through this work. Furthermore, fluorescence study showed the significant increase in absorption and emission maximum (hypsochromic effect) in crown structures when compared with their natural product precursors. Present optimistic results obtained from this study may be a guided template for developing new effective insulin secretory agents.
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Productos Biológicos/farmacología , Éteres Corona/farmacología , Hipoglucemiantes/farmacología , Secreción de Insulina/efectos de los fármacos , Islotes Pancreáticos/efectos de los fármacos , Animales , Productos Biológicos/aislamiento & purificación , Éteres Corona/aislamiento & purificación , Hipoglucemiantes/aislamiento & purificación , Islotes Pancreáticos/metabolismo , Masculino , Ratones Endogámicos BALB C , Tolbutamida/farmacologíaRESUMEN
Selenium is an essential trace element of interest for its potential role in glucose homeostasis. The present study investigated the impact of selenium supplementation as selenomethionine (SeMet) on insulin secretion in MIN6-K8 cells, a pancreatic ß-cell model. We found that SeMet enhanced percent glucose-induced insulin secretion, while also increasing tolbutamide- and KCl-induced percent insulin secretion. RNA-sequencing showed that SeMet supplementation altered expression of several selenoproteins, including glutathione peroxidase 3 (Gpx3) and selenoprotein P (SelP). Targeted knockdown of Gpx3 increased both percent and total insulin release, while SelP knockdown increased insulin content and insulin release. Collectively, these studies support a putative role for selenium and selenoproteins in the regulation of insulin secretion, glucose homeostasis, and diabetes risk.
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Secreción de Insulina/efectos de los fármacos , Insulinoma/metabolismo , Selenometionina/farmacología , Animales , Calcio/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Insulina/metabolismo , Insulinoma/genética , Insulinoma/patología , Ratones , Potasio/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Selenoproteínas/genética , Selenoproteínas/metabolismo , Tolbutamida/farmacologíaRESUMEN
OBJECTIVE: Glucagon is secreted by pancreatic α-cells in response to hypoglycemia and its hyperglycemic effect helps to restore normal blood glucose. Insulin and somatostatin (SST) secretions from ß- and δ-cells, respectively, are stimulated by glucose by mechanisms involving an inhibition of their ATP-sensitive K+ (KATP) channels, leading to an increase in [Ca2+]c that triggers exocytosis. Drugs that close KATP channels, such as sulfonylureas, are used to stimulate insulin release in type 2 diabetic patients. α-cells also express KATP channels. However, the mechanisms by which sulfonylureas control glucagon secretion are still largely debated and were addressed in the present study. In particular, we studied the effects of KATP channel blockers on α-cell [Ca2+]c and glucagon secretion in the presence of a low (1 mM) or a high (15 mM) glucose concentration and evaluated the role of SST in these effects. METHODS: Using a transgenic mouse model expressing the Ca2+-sensitive fluorescent protein, GCaMP6f, specifically in α-cells, we measured [Ca2+]c in α-cells either dispersed or within whole islets (by confocal microscopy). By measuring [Ca2+]c in α-cells within islets and glucagon secretion using the same perifusion protocols, we tested whether glucagon secretion correlated with changes in [Ca2+]c in response to sulfonylureas. We studied the role of SST in the effects of sulfonylureas using multiple approaches including genetic ablation of SST, or application of SST-14 and SST receptor antagonists. RESULTS: Application of the sulfonylureas, tolbutamide, or gliclazide, to a medium containing 1 mM or 15 mM glucose increased [Ca2+]c in α-cells by a direct effect as in ß-cells. At low glucose, sulfonylureas inhibited glucagon secretion of islets despite the rise in α-cell [Ca2+]c that they triggered. This glucagonostatic effect was indirect and attributed to SST because, in the islets of SST-knockout mice, sulfonylureas induced a stimulation of glucagon secretion which correlated with an increase in α-cell [Ca2+]c. Experiments with exogenous SST-14 and SST receptor antagonists indicated that the glucagonostatic effect of sulfonylureas mainly resulted from an inhibition of the efficacy of cytosolic Ca2+ on exocytosis. Although SST-14 was also able to inhibit glucagon secretion by decreasing α-cell [Ca2+]c, no decrease in [Ca2+]c occurred during sulfonylurea application because it was largely counterbalanced by the direct stimulatory effect of these drugs on α-cell [Ca2+]c. At high glucose, i.e., in conditions where glucagon release was already low, sulfonylureas stimulated glucagon secretion because their direct stimulatory effect on α-cells exceeded the indirect effect by SST. Our results also indicated that, unexpectedly, SST-14 poorly decreased the efficacy of Ca2+ on exocytosis in ß-cells. CONCLUSIONS: Sulfonylureas exert two opposite actions on α-cells: a direct stimulation as in ß-cells and an indirect inhibition by SST. This suggests that any alteration of SST paracrine influence, as described in diabetes, will modify the effect of sulfonylureas on glucagon release. In addition, we suggest that δ-cells inhibit α-cells more efficiently than ß-cells.