RESUMEN
Ketone ester ((R)-3-hydroxybutyl (R)-3-hydroxybutyrate) has gained popularity as an exogenous means to achieve ketosis. Regarding its potential as a therapeutic prodrug, it will be necessary to study its pharmacokinetic profile and its proximal metabolites (beta-hydroxybutyrate, 1,3-butanediol, and acetoacetate) in humans. Here we develop and validate two LC-MS methods for quantifying KE and its metabolites in human plasma. The first assay uses a C18 column to quantitate ketone ester, beta-hydroxybutyrate, and 1,3-butanediol, and the second assay uses a hydrophilic interaction liquid chromatography (HILIC) column for the quantitation of acetoacetate. The method was partially validated for intra- and inter-day accuracy and precision based on the ICH M10 guidelines. For both the assays, the intra- and inter-run accuracy was ±15% of the nominal concentration, and the precision (%CV) was <15% for all 4 molecules being quantified. The matrix effect for all molecules was evaluated and ranged from -62.1 to 44.4% (combined for all molecules), while the extraction recovery ranged from 65.1 to 119% (combined for all molecules). Furthermore, the metabolism of ketone ester in human plasma and human serum albumin was studied using the method. Non-saturable metabolism of ketone ester was seen in human plasma at concentrations as high as 5 mM, and human serum albumin contributed to the metabolism of ketone ester. Together, these assays can be used to track the entire kinetics of ketone ester and its proximal metabolites. The reverse-phase method was used to study the metabolic profile of KE in human plasma and the plasma protein binding of 1,3-BD.
Asunto(s)
Acetoacetatos , Cetonas , Humanos , Ácido 3-Hidroxibutírico , Cromatografía Liquida , Espectrometría de Masas en Tándem , Butileno Glicoles , ÉsteresRESUMEN
Ursolic acid (UA) is a naturally occurring pentacyclic triterpenoid that exhibits extensive pre-systemic metabolism from in vitro studies. However, there are no available authentic metabolite standards or validated analytical methods to quantitate UA metabolites. We have identified ursolic acid sulfate (UAS) as one of the major metabolites. We were able to identify and characterize its structure via comparison to the chemically synthesized UAS. A cyano (CN, 150 × 4.6 mm, 5 µm) column along with a gradient elution of acetonitrile and 0.08% (v/v) acetic acid, pH 3.0 were employed for chromatographic separation. Negative single ion recording mode (SIR) with electron-spray ionization (ESI) source at mass-to-charge ratios of 455.3 and 535.3 were monitored for UA and UAS, respectively. UAS linearity range was 0.010-2.500 µM. The absolute values of intra-day and inter-day precision (CV, %) and accuracy (DFN, %) were all below 15%. Thus, the analytical method has been validated in the human subcellular fractions to facilitate in vitro/ in vivo DMPK and future clinical disposition studies on UA.
Asunto(s)
Sulfatos , Triterpenos , Humanos , Espectrometría de Masas en Tándem/métodos , Ácido Acético , Hígado , Reproducibilidad de los Resultados , Ácido UrsólicoRESUMEN
The Caco-2 cell model has been widely used to assess the permeability of drug candidates. It has provided a high throughput in vitro platform, functionally resembling the enterocytes. Since the oral route is the most preferred for drug administration, the Caco-2 cell model acts as a very important tool to elucidate the oral "druggability" of a molecule by providing a fairly reliable estimate of its permeability through the intestinal membrane. Despite its shortcomings (the lack of a mucus layer, long cultivation period, inter-lab variability, and differences in expression of enzymes, transporters, and tight junction complexes) it remains heavily used due to its reliability, predictive performance, and wide acceptance. Various modifications have been made: co-culturing with other intestinal cells, applying biosimilar mucus, reducing culturing time, combining Caco-2 monolayer with the dissolution apparatus, enhancing protein expression, and redesigning the sampling apparatus. These modifications are intended to overcome some of the shortcomings of the Caco-2 model in order to make its use easier, quicker, economical, and more representative of the intestine. The aim of this review is to discuss such modifications to enhance this model's utility, predictive performance, and reproducibility.
Asunto(s)
Absorción Intestinal , Mucosa Intestinal , Células CACO-2 , Técnicas de Cocultivo , Células HT29 , Humanos , Mucosa Intestinal/metabolismo , Permeabilidad , Reproducibilidad de los ResultadosRESUMEN
Every year, complications during pregnancy affect more than 26 million women. Some of those diseases are associated with significant morbidity and mortality, as is the case of preeclampsia, the main cause of maternal deaths globally. The ability to improve the delivery of drugs to the placenta upon administration to the mother may offer new opportunities in the treatment of diseases of pregnancy. The objective of this study was to develop megalin-targeting liposome nanocarriers for placental drug delivery. Megalin is a transmembrane protein involved in clathrin-mediated endocytic processes, and is expressed in the syncytiotrophoblast (SynT), an epithelial layer at maternal-fetal interface. Targeting megalin thus offers an opportunity for the liposomes to hitchhike into the SynT, thus enriching the concentration of any associated therapeutic cargo in the placental tissue. PEGylated (2 KDa) lipids were modified with gentamicin (GM), a substrate to megalin receptors as we have shown in earlier studies, and used to prepare placental-targeting liposomes. The ability of the targeting liposomes to enhance accumulation of a fluorescence probe was assessed in an in vivo placental model - timed-pregnant Balb/c mice at gestational day (GD) 18.5. The targeting liposomes containing 10 mol% GM-modified lipids increased the accumulation of the conjugated fluorescence probe in the placenta with a total accumulation of 2.8% of the initial dose, which corresponds to a 94 fold increase in accumulation compared to the free probe (p < .0001), and 2-4 fold accumulation compared to the non-targeting control liposomes (p < .0001), as measured by both tissue extraction assay and ex vivo imaging. Furthermore, confocal images of placental SynT cross-sections show a 3-fold increase of the targeting liposomes compared with the non-targeting liposomes. The rate and extent of uptake of a fluorescent probe encapsulated within targeting liposomes was also probed in an in vitro model of the human placental barrier (polarized BeWo monolayers) using flow cytometry. Targeting liposomes containing 5 mol% GM-modified lipids enhanced the uptake of the probe by 1.5 fold compared to the non-targeting control. An increase to 10 mol% of the modified lipid resulted in further enhancement in uptake, which was 2 fold greater compared to control. In a competition assay, inhibition of the megalin receptors resulted in a significant reduction in uptake of the fluorescence probe encapsulated in GM-modified liposomes compared to the uptake without free inhibitor (p < .0001), implicating the involvement of megalin receptor in the internalization of the liposomes. Taken together, these results demonstrate that megalin-targeted liposomes may offer an opportunity to enhance the delivery of therapeutics to the placenta for the treatment of diseases of pregnancy.
Asunto(s)
Liposomas , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad , Animales , Sistemas de Liberación de Medicamentos , Femenino , Gentamicinas , Placenta , EmbarazoRESUMEN
Five pulchinenosides (pulchinenoside B3, pulchinenoside BD, pulchinenoside B7, pulchinenoside B10, and pulchinenoside B11) isolated from Pulsatilla chinensis (Bge) Regel saponins extract exhibited strong antitumor activities but poor gastrointestinal absorption properties. The enteric induction of P-glycoprotein (P-gp) is understood to restrict the oral bioavailability of some pharmaceutical compounds and lead to adverse drug reactions. Therefore, the present investigation was intended to delineate the impacts of pulchinenosides on cellular P-gp function and expression using Sf9 membrane vesicles and LS180 cells as a surrogate of human intestinal epithelial cells. Preliminary cytotoxic studies showed that 10 µM was an acceptable concentration for cytotoxicity and antiproliferation studies for all pulchinenosides using the alamarBlue assay. The cell cycle of LS180 cells detected by flow cytometry was not significantly influenced after 48 hours of coincubation with 10 µM of pulchinenosides. In the presence of pulchinenosides, the ATP-dependent transport of N-methyl-quinidine mediated by P-glycoprotein was stimulated significantly. The upregulation of P-glycoprotein and mRNA levels was found by Western blot and real-time PCR analysis in LS180 cells. Parallel changes indicate that all pulchinenosides are exposed to pulchinenosides-mediated transcriptional regulation. In conclusion, pulchinenosides could induce P-glycoprotein expression and directly increase its functional activity.
RESUMEN
For thousands of years opioids have been the first-line treatment option for pain management. However, the tolerance and addiction potential of opioids limit their applications in clinic. NFP, a MOR/KOR dual-selective opioid antagonist, was identified as a ligand that significantly antagonized the antinociceptive effects of morphine with lesser withdrawal effects than naloxone at similar doses. To validate the potential application of NFP in opioid addiction treatment, a series of in vitro and in vivo assays were conducted to further characterize its pharmacological profile. In calcium mobilization assays and MOR internalization studies, NFP showed the apparent capacity to antagonize DAMGO-induced calcium flux and etorphine-induced MOR internalization. In contrast to the opioid agonists DAMGO and morphine, cells pretreated with NFP did not show apparent desensitization and down regulation of the MOR. Though in vitro bidirectional transport studies showed that NFP might be a P-gp substrate, in warm-water tail-withdrawal assays it was able to antagonize the antinociceptive effects of morphine indicating its potential central nervous system activity. Overall these results suggest that NFP is a promising dual selective opioid antagonist that may have the potential to be used therapeutically in opioid use disorder treatment.
Asunto(s)
Morfinanos/farmacología , Trastornos Relacionados con Opioides/tratamiento farmacológico , Receptores Opioides kappa/antagonistas & inhibidores , Receptores Opioides mu/antagonistas & inhibidores , Analgésicos Opioides/farmacología , Animales , Transporte Biológico , Células CHO , Células CACO-2 , Calcio/metabolismo , Línea Celular Tumoral , Cricetulus , Encefalina Ala(2)-MeFe(4)-Gli(5)/farmacología , Humanos , Ligandos , Masculino , Ratones Endogámicos C57BL , Antagonistas de Narcóticos/farmacología , Receptores Opioides kappa/agonistas , Receptores Opioides mu/agonistas , Receptores Opioides mu/genética , Receptores Opioides mu/metabolismoRESUMEN
Oxidative stress may arise from a variety of pathologies and results in the formation of toxic and reactive chemical species. Extensive research has been done to establish mechanisms of formation and cytotoxic effects of a number of different products of oxidation stress including peroxynitrite (PN) and 4-hydroxynonenal (4HNE). However, relatively few studies have investigated their effects on ATP-binding cassette (ABC) transporters. The objective of this investigation was to determine the effects of PN and 4HNE on BCRP/ABCG2. To eliminate the effect of metabolic enzymes, the experiments were carried out with inside-out Sf9 membrane vesicles overexpressing BCRP/ABCG2 using riboflavin as a substrate. The experiments revealed that PN produced IC50 of about 31.2 ± 2.7 µM, based upon initial concentrations. The IC50 for 4HNE was estimated to be 92 ± 1.4 µM. Preincubation of membrane vesicles with either PN or 4HNE caused the maximal rate of transport (Vmax) to drop drastically, up to 19 times, with no or much smaller effect on Km. Thus, PN and 4NE can inhibit BCRP transport activity.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Aldehídos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Proteínas de Neoplasias/metabolismo , Ácido Peroxinitroso/farmacología , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Femenino , Humanos , Células Sf9RESUMEN
Monoamine oxidases (MAOs) regulate local levels of neurotransmitters such as dopamine, norepinephrine, and serotonin and thus have been targeted by drugs for the treatment of certain CNS disorders. However, recent studies have shown that these enzymes are upregulated with age in nervous and cardiac tissues and may be involved in degeneration of these tissues, since their metabolic mechanism releases hydrogen peroxide leading to oxidative stress. Thus, targeting these enzymes may be a potential anti-aging strategy. The purpose of this study was to compare the MAO inhibition and selectivity of selected dietary phenolic compounds, using a previously validated assay that would avoid interference from the compounds. Kynuramine metabolism by human recombinant MAO-A and MAO-B leads to formation of 4-hydroxyquinoline, with Vmax values of 10.2±0.2 and 7.35±0.69 nmol/mg/min, respectively, and Km values of 23.1±0.8 µM and 18.0±2.3 µM, respectively. For oral dosing and interactions with the gastrointestinal tract, curcumin, guaiacol, isoeugenol, pterostilbene, resveratrol, and zingerone were tested at their highest expected luminal concentrations from an oral dose. Each of these significantly inhibited both enzymes except for zingerone, which only inhibited MAO-A. The IC50 values were determined, and selectivity indices (MAO-A/MAO-B IC50 ratios) were calculated. Resveratrol and isoeugenol were selective for MAO-A, with IC50 values of 0.313±0.008 and 3.72±0.20 µM and selectivity indices of 50.5 and 27.4, respectively. Pterostilbene was selective for MAO-B, with IC50 of 0.138±0.013 µM and selectivity index of 0.0103. The inhibition of resveratrol (MAO-A) and pterostilbene (MAO-B) was consistent with competitive time-independent mechanisms. Resveratrol 4'-glucoside was the only compound which inhibited MAO-A, but itself, resveratrol 3-glucoside, and pterostilbene 4'-glucoside failed to inhibit MAO-B. Additional studies are needed to establish the effects of these compounds on MAO-A and/or MAO-B in humans.
Asunto(s)
Enfermedades del Sistema Nervioso Central/dietoterapia , Monoaminooxidasa/química , Estilbenos/farmacología , Envejecimiento/efectos de los fármacos , Envejecimiento/patología , Envejecimiento/fisiología , Enfermedades del Sistema Nervioso Central/metabolismo , Enfermedades del Sistema Nervioso Central/patología , Cromatografía Líquida de Alta Presión , Curcumina/farmacología , Eugenol/análogos & derivados , Eugenol/farmacología , Guayacol/administración & dosificación , Guayacol/análogos & derivados , Guayacol/farmacología , Humanos , Hidroxiquinolinas/farmacología , Concentración 50 Inhibidora , Kinuramina/química , Kinuramina/farmacología , Monoaminooxidasa/efectos de los fármacos , Monoaminooxidasa/metabolismo , Inhibidores de la Monoaminooxidasa/química , Inhibidores de la Monoaminooxidasa/farmacología , Estrés Oxidativo/efectos de los fármacos , Fenoles/química , Fenoles/farmacología , Resveratrol/química , Resveratrol/farmacologíaRESUMEN
INTRODUCTION: The in vivo disposition and metabolism of phenylephrine have not been establishedby previous analytical methods and there is a lack of available standards for quantitating the metabolites. METHODS: We pursued and compared the preparation of sulfation metabolites of phenylephrine and its ethyl analog etilefrine via chemical and bio-synthesis. RESULTS: Both sulfates were obtained in higher yield and purity through chemical syntheses compared to biosynthesis. DISCUSSION: A facile method for the production of phenylephrine 3-O-sulfate and etilefrine 3-O-sulfate was established. These compounds will be useful in the development of analytical assays for studying the pharmacokinetics of phenylephrine and its main route of metabolism in the presence of formulation changes and pharmacogenetic variation.
Asunto(s)
Etilefrina/síntesis química , Descongestionantes Nasales/síntesis química , Fenilefrina/síntesis química , Sulfatos/síntesis química , Evaluación Preclínica de Medicamentos/métodos , Etilefrina/metabolismo , Etilefrina/farmacocinética , Descongestionantes Nasales/metabolismo , Descongestionantes Nasales/farmacocinética , Variantes Farmacogenómicas , Fenilefrina/metabolismo , Fenilefrina/farmacocinética , Sulfatos/metabolismo , Sulfatos/farmacocinéticaRESUMEN
The present study evaluated the potential of five generally recognized as safe (GRAS) or dietary compounds (α-mangostin, chrysin, ginger extract, pterostilbene and silybin) to inhibit oxidative (CYP) and conjugative (UGT) metabolism using pooled human intestinal and liver microsomes. Buprenorphine was chosen as the model substrate as it is extensively metabolized by CYPs to norbuprenorphine and by UGTs to buprenorphine glucuronide. Chrysin, ginger extract, α-mangostin, pterostilbene and silybin were tested for their inhibition of the formation of norbuprenorphine or buprenorphine glucuronide in both intestinal and liver microsomes. Pterostilbene was the most potent inhibitor of norbuprenorphine formation in both intestinal and liver microsomes, with IC50 values of 1.3 and 0.8 µM, respectively, while α-mangostin and silybin most potently inhibited buprenorphine glucuronide formation. The equipotent combination of pterostilbene and ginger extract additively inhibited both pathways in intestinal microsomes. Since pterostilbene and ginger extract showed potent CYP and/or UGT inhibition of buprenorphine metabolism, their equipotent combination was tested to assess the presence of synergistic inhibition. However, because the combination showed additive inhibition, it was not used while performing IVIVE analysis. Based on quantitative in vitro-in vivo extrapolation, pterostilbene (21 mg oral dose) appeared to be most effective in improving the mean predicted Foral and AUC∞ PO of buprenorphine from 3 ± 2% and 340 ± 330 ng*min/ml to 75 ± 8% and 36,000 ± 25,000 ng*min/ml, respectively. At a 10-fold lower dose of pterostilbene, the predicted buprenorphine Foral approximated sublingual bioavailability (~35%) and showed a 2-4 fold reduction in the variability around the predicted AUC∞ PO of buprenorphine. These results demonstrate the feasibility of using various GRAS/dietary compounds to inhibit substantially the metabolism by CYP and UGT enzymes to achieve higher and less variable oral bioavailability. This inhibitor strategy may be useful for drugs suffering from low and variable oral bioavailability due to extensive presystemic oxidative and/or conjugative metabolism.
Asunto(s)
Analgésicos Opioides/farmacocinética , Buprenorfina/farmacocinética , Administración Oral , Disponibilidad Biológica , Dieta , Flavonoides/farmacología , Zingiber officinale , Glucurónidos/metabolismo , Intestinos , Hígado , Microsomas/metabolismo , Extractos Vegetales/farmacología , Silibina/farmacología , Estilbenos/farmacología , Xantonas/farmacologíaRESUMEN
Phenylephrine (PE) has low and variable oral bioavailability in humans, due in part to presystemic metabolism by sulfation. LS180 cells were used as a model of the human intestinal epithelium to examine phenylephrine metabolism and its inhibition by generally recognized as safe (GRAS) and dietary compounds. Curcumin, zingerone, resveratrol, guaiacol, pterostilbene and isoeugenol significantly inhibited phenylephrine disappearance, while vanillin, propylparaben and eugenol did not. However, when propylparaben was combined with either vanillin or eugenol, the phenylephrine disappearance was significantly inhibited. These data suggest that these compounds or combinations thereof may have potential to improve phenylephrine oral bioavailability.
Asunto(s)
Interacciones Alimento-Droga , Mucosa Intestinal/metabolismo , Fenilefrina/farmacocinética , Administración Oral , Disponibilidad Biológica , Línea Celular , Suplementos Dietéticos , Humanos , Fenilefrina/administración & dosificaciónRESUMEN
A rapid and sensitive LC-MS/MS method was developed and validated for the simultaneous determination of buprenorphine and its three metabolites (buprenorphine glucuronide, norbuprenorphine and norbuprenorphine glucuronide) as well as naloxone and its metabolite naloxone glucuronide in the rat plasma. A hydrophilic interaction chromatography column and a mobile phase containing acetonitrile and ammonium formate buffer (pH 3.5) were used for the chromatographic separation. Mass spectrometric detection was achieved by an electrospray ionization source in the positive mode coupled to a triple quadrupole mass analyzer. The calibration curves for the six analytes displayed good linearity over the concentration range 1.0 or 5.0-1000 ng/mL. The intra and inter-day precision (CV) ranged from 2.68 to 16.4% and from 9.02 to 14.5%, respectively. The intra- and inter-day accuracy (bias) ranged from -14.2 to 15.2% and from -9.00 to 4.80%, respectively. The extraction recoveries for all the analytes ranged from 55 to 86.9%. The LC-MS/MS method was successfully applied to a pharmacokinetic study of buprenorphine-naloxone combination in rats.
Asunto(s)
Buprenorfina/análogos & derivados , Cromatografía Líquida de Alta Presión/métodos , Glucurónidos/sangre , Naloxona/sangre , Antagonistas de Narcóticos/sangre , Espectrometría de Masas en Tándem/métodos , Animales , Buprenorfina/sangre , Buprenorfina/metabolismo , Línea Celular Tumoral , Glucurónidos/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Límite de Detección , Naloxona/metabolismo , Antagonistas de Narcóticos/metabolismo , RatasRESUMEN
The present study investigated the potential of generally recognized as safe (GRAS) compounds or dietary substances to inhibit the presystemic metabolism of buprenorphine and to increase its oral bioavailability. Using IVIVE, buprenorphine extraction ratios in intestine and liver were predicted as 96% and 71%, respectively. In addition, the relative fraction of buprenorphine metabolized by oxidation and glucuronidation in these two organs was estimated using pooled human intestinal and liver microsomes. In both organs, oxidation appeared to be the major metabolic pathway with a 6 and 4 fold higher intrinsic clearance than glucuronidation in intestine and liver, respectively. The oral bioavailability of buprenorphine was predicted to be 1.16%. Inhibition of 75% and 50% of intestinal and hepatic presystemic metabolism would result in an Foral of 49%, which is comparable to the bioavailability of sublingual buprenorphine. In human liver microsomes, chrysin, curcumin, ginger extract, hesperitin, magnolol, quercetin and silybin inhibited ≥50% glucuronidation, whereas chrysin, curcumin, ginger extract, 6-gingerol, pterostilbene, resveratrol and silybin exhibited ≥30% inhibition of oxidation. In human intestinal microsomes, curcumin, ginger extract, α-mangostin, quercetin and silybin inhibited ≥50% glucuronidation while chrysin, ginger extract, α-mangostin, pterostilbene and resveratrol exhibited ≥30% inhibition of oxidation. These results demonstrate the feasibility of our proposed approach of using GRAS or dietary compounds to inhibit the presystemic metabolism of buprenorphine and thus improve its oral bioavailability. An oral buprenorphine formulation containing these inhibitors or their combinations has promising potential to replace sublingual buprenorphine. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Buprenorfina/metabolismo , Suplementos Dietéticos , Glucurónidos/metabolismo , Interacciones de Hierba-Droga , Intestinos/efectos de los fármacos , Hígado/efectos de los fármacos , Fitoquímicos/toxicidad , Administración Oral , Disponibilidad Biológica , Biotransformación , Buprenorfina/administración & dosificación , Buprenorfina/sangre , Buprenorfina/farmacocinética , Células CACO-2 , Suplementos Dietéticos/efectos adversos , Humanos , Absorción Intestinal , Mucosa Intestinal/metabolismo , Hígado/metabolismo , Modelos Biológicos , Oxidación-Reducción , Unión ProteicaRESUMEN
OBJECTIVES: The aim of this study was to improve the oral bioavailability of buprenorphine by inhibiting presystemic metabolism via the oral co-administration of 'Generally Recognized as Safe' compounds, thus providing an orally administered drug product with less variability and comparable or higher exposure compared with the sublingual route. METHODS: The present studies were performed in Sprague Dawley rats following either intravenous or oral administration of buprenorphine/naloxone and oral co-administration of 'Generally Recognized as Safe' compounds referred to as 'adjuvants'. Plasma samples were collected up to 22 h postdosing followed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. KEY FINDINGS: The adjuvants increased Cmax (21 ± 16 ng/ml vs 75 ± 33 ng/ml; 3.6-fold) and AUC(0-22 h) (10.6 ± 8.11 µg min/ml vs 22.9 ± 11.7 µg min/ml; 2.2-fold) values of buprenorphine (control vs adjuvant-treated, respectively). The absolute oral bioavailability of buprenorphine doubled (from 1.24% to 2.68%) in the presence of the adjuvants. CONCLUSIONS: One may suggest that the adjuvant treatment most likely inhibited the presystemic metabolic enzymes, thus decreasing the intestinal 'first-pass effect' on buprenorphine. Additional studies are now required to further explore the concept of inhibiting presystemic metabolism of buprenorphine by adjuvants to potentially increase the oral bioavailability of buprenorphine.
Asunto(s)
Adyuvantes Farmacéuticos/farmacología , Buprenorfina/farmacocinética , Absorción Intestinal/efectos de los fármacos , Magnoliopsida/química , Extractos Vegetales/farmacología , Administración Oral , Animales , Área Bajo la Curva , Disponibilidad Biológica , Buprenorfina/sangre , Línea Celular , Cromatografía Liquida , Humanos , Inactivación Metabólica/efectos de los fármacos , Masculino , Naloxona/sangre , Naloxona/farmacocinética , Ratas Sprague-Dawley , Espectrometría de Masas en TándemRESUMEN
The efficacy of phenylephrine (PE) is controversial due to its extensive pre-systemic metabolism through sulfation to form phenylephrine-3-O-sulfate (PES). Hence quantitation of PES is important in order to study the metabolism of PE. There are no published methods available for direction detection of PES. We have developed and validated a hydrophilic interaction liquid chromatography (HILIC) method for the direct detection of PES and simultaneous detection of PE to study the enzyme kinetics and metabolism of PE to enable approaches to reduce the presystemic metabolism of PE. This is the first method which facilitates direct detection of PES and simultaneous detection of PE using a zwitterionic HILIC column with improved sensitivity in a single short run. The observed quantitative ranges of our method for PE and PES were 0.39-200µM and 0.0625-32µM (respectively) with a run time of 6.0min. The method was applied to the determination of PE and PES in LS180 human intestinal cell line, recombinant enzymes and human intestinal cytosol (HIC).
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Descongestionantes Nasales/farmacocinética , Fenilefrina/farmacocinética , Sulfatos/farmacocinética , Cromatografía Líquida de Alta Presión/economía , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Mucosa Intestinal/metabolismo , Intestinos/citología , Límite de Detección , Descongestionantes Nasales/análisis , Descongestionantes Nasales/metabolismo , Fenilefrina/análisis , Fenilefrina/metabolismo , Sulfatos/análisis , Sulfatos/metabolismoRESUMEN
The human placenta fulfills a variety of essential functions during prenatal life. Several ABC transporters are expressed in the human placenta, where they play a role in the transport of endogenous compounds and may protect the fetus from exogenous compounds such as therapeutic agents, drugs of abuse, and other xenobiotics. To date, considerable progress has been made toward understanding ABC transporters in the placenta. Recent studies on the expression and functional activities are discussed. This review discusses the placental expression and functional roles of several members of ABC transporter subfamilies B, C, and G including MDR1/P-glycoprotein, the MRPs, and BCRP, respectively. Since placental ABC transporters modulate fetal exposure to various compounds, an understanding of their functional and regulatory mechanisms will lead to more optimal medication use when necessary in pregnancy.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Placenta/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Animales , Transporte Biológico , Citocinas/metabolismo , Femenino , Hormonas/metabolismo , Humanos , Intercambio Materno-Fetal , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Preparaciones Farmacéuticas/metabolismo , Placenta/metabolismo , Polimorfismo Genético , Embarazo , Xenobióticos/metabolismoRESUMEN
Aminoglycosides (AG) are known to readily cross the placenta, although the mechanisms responsible for placental transport have not been characterized. Megalin is expressed in human placenta, and it is reasonable to speculate, given its role in renal AG uptake, that it is similarly involved in placental transport. However, the role of megalin in placental AG uptake has not been established. An in vitro model to study megalin-mediated placental transport has also not been previously described. The objectives of this study, therefore, were to evaluate the human choriocarcinoma (BeWo) cell line as a model to study megalin-mediated placental transport and to assess the uptake kinetics of gentamicin, an AG antibiotic, using this in vitro model. BeWo cells were grown on Transwell® plates, and megalin expression and functional activity were assessed. Uptake of (3)H-gentamicin was also evaluated in the presence and absence of megalin inhibitors. Expression of megalin protein and mRNA in BeWo cells were confirmed via immunoblot and qPCR analysis. Uptake of fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) (a megalin substrate) was time-, concentration-, and temperature-dependent consistent with a transporter-mediated process. FITC-BSA uptake was also significantly reduced in the presence of unlabeled gentamicin (a megalin substrate) and sodium maleate (to induce megalin shedding) suggesting that megalin is functionally active in BeWo cells. Gentamicin uptake exhibited time and temperature dependence, saturability and Michaelis-Menten kinetics, all of which suggest a transporter-mediated process. Gentamicin uptake was also significantly reduced in the presence of the megalin inhibitors RAP and EDTA suggesting that megalin is likely involved in gentamicin uptake.
Asunto(s)
Antibacterianos/farmacocinética , Gentamicinas/farmacocinética , Proteína 2 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Placenta/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Fluoresceína-5-Isotiocianato/administración & dosificación , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Humanos , Embarazo , Albúmina Sérica Bovina/administración & dosificación , Albúmina Sérica Bovina/farmacocinética , Temperatura , Factores de TiempoRESUMEN
A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with positive electrospray ionization (ESI) mode has been validated for the simultaneous determination of lopinavir (LPV) and three prodrugs, succinic acid ester of lopinavir (SLPV), glutaric acid ester of lopinavir (GLPV), and diglycolic acid ester of lopinavir (DLPV) in BeWo cell lysate. A methanolic precipitation was employed after addition of an internal standard ritonavir (RTV), followed by centrifugation, separation of the supernatant liquid, evaporation, and reconstitution. A reversed-phase Phenomenex C18 column was used for separation of lopinavir and three prodrugs with a mobile phase which comprises methanol (solvent A), acetonitrile (solvent B), and 50mM ammonium acetate buffer adjusted to pH 5.5 with acetic acid (solvent C), with isocratic elution (A:B:C=34:34:32, v/v). The detection of target compounds was conducted using multiple reaction monitoring (MRM) with the following transitions of m/z 729â447, 745â563, 743â429, 629â447 and 721â296 to measure SLPV, DLPV, GLPV, LPV and RTV (I.S.), respectively. The calibration ranges were determined using quadratic regression over concentration ranges of 7-743 ng/mL (DLPV), 7.5-745 ng/mL (GLPV), 7-729 ng/mL (SLPV) and 6-189 ng/mL (LPV). The reverse calculated residuals for LPV and these prodrugs were all less than 15% different from nominal (range). The method we established was fully validated for accuracy (≤15% different from nominal) and precision (≤15% RSD). The results proved that the method is accurate and specific, and also this method has been successfully applied to test the uptake of lopinavir and prodrugs into BeWo cells.
Asunto(s)
Cromatografía Liquida/métodos , Inhibidores de la Proteasa del VIH/análisis , Lopinavir/análisis , Profármacos/análisis , Espectrometría de Masas en Tándem/métodos , Trofoblastos/citología , Línea Celular , Inhibidores de la Proteasa del VIH/farmacocinética , Humanos , Límite de Detección , Lopinavir/farmacocinética , Profármacos/farmacocinética , Trofoblastos/metabolismoRESUMEN
OBJECTIVE: To quantitate 2-methoxyestradiol (2-ME) in human corpus luteum (CL) of different ages and to determine the expression of cytochrome-P450-1A1 (CYP1A1) and catechol-O-methyl transferase (COMT) in CL and the action of 2-ME on P, vascular endothelial growth factor (VEGF) secretion, and luteal angiogenesis. DESIGN: Experimental study. SETTING: University division of reproductive endocrinology. PATIENT(S): Twenty-four women of reproductive age. INTERVENTION(S): CL was collected from 15 women during the minilaparotomy for tubal sterilization. Granulosa lutein cells were harvested 36 hours after hCG administration in patients undergoing IVF. MAIN OUTCOMES MEASURE(S): Levels of 2-ME were determined by high-performance liquid chromatography in CL. CYP1A1 and COMT were assessed by immunohistochemistry and Western blot. P and VEGF were measured by radioimmunoassay and ELISA. The angiogenic potential was analyzed using EA.hy926 cells. RESULT(S): Plasma levels of E2 decreased in the late luteal phase in association with an increase in luteal tissue of 2-ME concentrations. Concomitantly, there was a significant reduction of angiogenic activity in late CL. There was no significant variation in CYP1A1 and COMT expression in all CL. In physiological doses, 2-ME inhibited basal VEGF by granulosa lutein cells and diminished the angiogenic activity in conditioned media but did not prevent P and VEGF production stimulated by hCG. CONCLUSION(S): These data suggest the participation of 2-ME in physiological luteolysis by reducing angiogenesis. However, 2-ME did not prevent in vitro hCG stimulation of P biosynthesis, providing a mechanism for CL rescue in the cycle of conception.
Asunto(s)
Cuerpo Lúteo/irrigación sanguínea , Cuerpo Lúteo/metabolismo , Estradiol/análogos & derivados , Células Lúteas/metabolismo , Fase Luteínica/metabolismo , Neovascularización Fisiológica , Progesterona/biosíntesis , 2-Metoxiestradiol , Adulto , Catecol O-Metiltransferasa/metabolismo , Línea Celular , Medios de Cultivo Condicionados/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Estradiol/sangre , Estradiol/metabolismo , Femenino , Humanos , Fase Luteínica/sangre , Factores de Tiempo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: HIV protease inhibitor (PI)-induced inflammatory response in macrophages is a major risk factor for cardiovascular diseases. We have previously reported that berberine (BBR), a traditional herbal medicine, prevents HIV PI-induced inflammatory response through inhibiting endoplasmic reticulum (ER) stress in macrophages. We also found that HIV PIs significantly increased the intracellular concentrations of BBR in macrophages. However, the underlying mechanisms of HIV PI-induced BBR accumulation are unknown. This study examined the role of P-glycoprotein (P-gp) in HIV PI-mediated accumulation of BBR in macrophages. METHODOLOGY AND PRINCIPAL FINDINGS: Cultured mouse RAW264.7 macrophages, human THP-1-derived macrophages, Wild type MDCK (MDCK/WT) and human P-gp transfected (MDCK/P-gp) cells were used in this study. The intracellular concentration of BBR was determined by HPLC. The activity of P-gp was assessed by measuring digoxin and rhodamine 123 (Rh123) efflux. The interaction between P-gp and BBR or HIV PIs was predicated by Glide docking using Schrodinger program. The results indicate that P-gp contributed to the efflux of BBR in macrophages. HIV PIs significantly increased BBR concentrations in macrophages; however, BBR did not alter cellular HIV PI concentrations. Although HIV PIs did not affect P-gp expression, P-gp transport activities were significantly inhibited in HIV PI-treated macrophages. Furthermore, the molecular docking study suggests that both HIV PIs and BBR fit the binding pocket of P-gp, and HIV PIs may compete with BBR to bind P-gp. CONCLUSION AND SIGNIFICANCE: HIV PIs increase the concentration of BBR by modulating the transport activity of P-gp in macrophages. Understanding the cellular mechanisms of potential drug-drug interactions is critical prior to applying successful combinational therapy in the clinic.