RESUMEN
P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are important transporters causing drug-drug interaction (DDI). Here, we investigated the involvement of P-gp and BCRP in the oral absorption of ensitrelvir in non-clinical studies and estimated the DDI risk mediated by P-gp and BCRP inhibition in humans. Although ensitrelvir is an in vitro P-gp and BCRP substrate, it demonstrated high bioavailability in rats and monkeys after oral administration. Plasma exposures of ensitrelvir following oral administration were comparable in wild type (WT) and Bcrp (-/-) mice. On the other hand, the area under the plasma concentration-time curve (AUC) ratio of ensitrelvir in the Mdr1a/1b (-/-) mice to the WT mice was 1.92, indicating that P-gp, but not BCRP, was involved in the oral absorption of ensitrelvir. Based on our previous retrospective analyses, such a low AUC ratio (<3) in the Mdr1a/1b (-/-) mice indicates a minimal impact of P-gp on the oral absorption in humans. In conclusion, our studies demonstrate that the involvement of both P-gp and BCRP in the oral absorption of ensitrelvir is minimal, and suggest that ensitrelvir has a low risk for DDIs mediated by P-gp and BCRP inhibition in humans.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Ratones Noqueados , Animales , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Administración Oral , Humanos , Ratones , Ratas , Masculino , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Disponibilidad Biológica , Interacciones Farmacológicas , Inhibidores de Proteasas/farmacocinética , Inhibidores de Proteasas/administración & dosificación , Inhibidores de Proteasas/farmacología , Células CACO-2 , Ratas Sprague-Dawley , Perros , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Tratamiento Farmacológico de COVID-19 , Macaca fascicularis , Células de Riñón Canino Madin Darby , Indazoles , Triazinas , TriazolesRESUMEN
Coproporphyrin-I (CP-I) has been investigated as an endogenous biomarker of organic anion transporting polypeptide (OATP) 1B. Here, we determined the CP-I concentrations in a cocktail drug-drug interaction (DDI) study of ensitrelvir to evaluate the OATP1B inhibitory potential because ensitrelvir had increased plasma concentrations of rosuvastatin in this study, raising concerns about breast cancer resistance protein and OATP1B inhibition. Furthermore, CP-I concentrations were compared between active and placebo groups in a first-in-human (FIH) study of ensitrelvir to verify whether the OATP1B inhibitory potential could be estimated at an early drug development stage. In the cocktail DDI study, CP-I did not differ between with/without administration of ensitrelvir, indicating that ensitrelvir has no OATP1B inhibitory effect. Although there were some individual variabilities in CP-I concentrations among the treatment groups in the FIH study, the normalization of CP-I concentrations with pre-dose values minimized these variabilities, suggesting that this normalized method would be helpful for comparing the CP-I from different participants. Finally, we concluded that CP-I concentrations were not affected by ensitrelvir in the FIH study. These results suggested that the CP-I determination in an FIH study and its normalized method can be useful for an early evaluation of the OATP1B-mediated DDI potential in humans.
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Antiinfecciosos , COVID-19 , Indazoles , Triazinas , Triazoles , Humanos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , SARS-CoV-2 , Inhibidores de Proteasas , Coproporfirinas/metabolismo , Coproporfirinas/farmacología , Transportador 1 de Anión Orgánico Específico del Hígado/metabolismo , Proteínas de Neoplasias/metabolismo , Inhibidores Enzimáticos , Antivirales/farmacología , Interacciones FarmacológicasRESUMEN
Oral drug absorption involves drug permeation across the apical and basolateral membranes of enterocytes. Although transporters mediating the influx of anionic drugs in the apical membranes have been identified, transporters responsible for efflux in the basolateral membranes remain unclear. Monocarboxylate transporter 6 (MCT6/SLC16A5) has been reported to localize to the apical and basolateral membranes of human enterocytes and to transport organic anions such as bumetanide and nateglinide in the Xenopus oocyte expression system; however, its transport functions have not been elucidated in detail. In this study, we characterized the function of MCT6 expressed in HEK293T cells and explored fluorescent probes to more easily evaluate MCT6 function. The results illustrated that MCT6 interacts with CD147 to localize at the plasma membrane. When the uptake of various fluorescein derivatives was examined in NaCl-free uptake buffer (pH 5.5), the uptake of 5-carboxyfluorescein (5-CF) was significantly greater in MCT6 and CD147-expressing cells. MCT6-mediated 5-CF uptake was saturable with a Km of 1.07 mM and inhibited by several substrates/inhibitors of organic anion transporters and extracellular Cl ion with an IC50 of 53.7 mM. These results suggest that MCT6 is a chloride-sensitive organic anion transporter that can be characterized using 5-CF as a fluorescent probe.
Asunto(s)
Transportadores de Anión Orgánico , Animales , Humanos , Transportadores de Anión Orgánico/metabolismo , Cloruros/metabolismo , Células HEK293 , Transporte Biológico , Fluoresceínas , Mamíferos/metabolismoRESUMEN
OBJECTIVE: An in vitro relative activity factor (RAF) technique combined with mechanistic static modeling was examined to predict drug-drug interaction (DDI) magnitude and analyze contributions of different clearance pathways in complex DDIs involving transporter substrates. Atorvastatin and rifampicin were used as a model substrate and inhibitor pair. METHODS: In vitro studies were conducted with transfected HEK293 cells, hepatocytes and human liver microsomes. Prediction success was defined as predictions being within twofold of observations. RESULTS: The RAF method successfully translated atorvastatin uptake from transfected cells to hepatocytes, demonstrating its ability to quantify transporter contributions to uptake. Successful translation of atorvastatin's in vivo intrinsic hepatic clearance (CLint,h,in vivo) from hepatocytes to liver was only achieved through consideration of albumin facilitated uptake or through application of empirical scaling factors to transporter-mediated clearances. Transporter protein expression differences between hepatocytes and liver did not affect CLint,h,in vivo predictions. By integrating cis and trans inhibition of OATP1B1/OATP1B3, atorvastatin-rifampicin (single dose) DDI magnitude could be accurately predicted (predictions within 0.77-1.0 fold of observations). Simulations indicated that concurrent inhibition of both OATP1B1 and OATP1B3 caused approximately 80% of atorvastatin exposure increases (AUCR) in the presence of rifampicin. Inhibiting biliary elimination, hepatic metabolism, OATP2B1, NTCP, and basolateral efflux are predicted to have minimal to no effect on AUCR. CONCLUSIONS: This study demonstrates the effective application of a RAF-based translation method combined with mechanistic static modeling for transporter substrate DDI predictions and subsequent mechanistic interpretation.
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Transportadores de Anión Orgánico , Rifampin , Humanos , Atorvastatina/metabolismo , Rifampin/farmacología , Rifampin/metabolismo , Células HEK293 , Transportador 1 de Anión Orgánico Específico del Hígado/metabolismo , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Interacciones Farmacológicas , Transportadores de Anión Orgánico/metabolismoRESUMEN
In hepatic dysfunction, renal pharmacokinetic adaptation can be observed, although information on the changes in drug exposure and the interorgan regulation of membrane transporters in kidney in liver diseases is limited. This study aimed to clarify the effects of renal exposure to nephrotoxic drugs during cholestasis induced by bile duct ligation (BDL). Among the 11 nephrotoxic drugs examined, the tissue accumulation of imatinib and cisplatin in kidney slices obtained from mice 2 weeks after BDL operation was higher than that in sham-operated mice. The uptake of imatinib in the kidney slices of BDL mice was slightly higher, whereas its efflux from the slices was largely decreased compared to that in sham-operated mice. Proteomic analysis revealed a reduction in renal expression of the efflux transporter multidrug resistance-associated protein 6 (Mrp6/Abcc6) in BDL mice, and both imatinib and cisplatin were identified as Mrp6 substrates. Survival probability after cisplatin administration was reduced in BDL mice. In conclusion, the present study demonstrated that BDL-induced cholestasis leads to the downregulation of the renal basolateral efflux transporter Mrp6, resulting in drug accumulation in renal cells and promoting drug-induced renal injury.
Asunto(s)
Colestasis , Hepatopatías , Ratones , Animales , Hígado/metabolismo , Regulación hacia Abajo , Mesilato de Imatinib , Cisplatino , Proteómica , Colestasis/metabolismo , Conductos Biliares/metabolismo , Conductos Biliares/cirugía , Hepatopatías/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Riñón/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismoRESUMEN
Metformin is an antidiabetic drug, increasingly prescribed in pregnancy and has been shown to cross the human placenta. The mechanisms underlying placental metformin transfer remain unclear. This study investigated the roles of drug transporters and paracellular diffusion in the bidirectional transfer of metformin across the human placental syncytiotrophoblast using placental perfusion experiments and computational modelling. 14C-metformin transfer was observed in the maternal to fetal and fetal to maternal directions and was not competitively inhibited by 5 mM unlabelled metformin. Computational modelling of the data was consistent with overall placental transfer via paracellular diffusion. Interestingly, the model also predicted a transient peak in fetal 14C-metformin release due to trans-stimulation of OCT3 by unlabelled metformin at the basal membrane. To test this hypothesis a second experiment was designed. OCT3 substrates (5 mM metformin, 5 mM verapamil and 10 mM decynium-22) added to the fetal artery trans-stimulated release of 14C-metformin from the placenta into the fetal circulation, while 5 mM corticosterone did not. This study demonstrated activity of OCT3 transporters on the basal membrane of the human syncytiotrophoblast. However, we did not detect a contribution of either OCT3 or apical membrane transporters to overall materno-fetal transfer, which could be represented adequately by paracellular diffusion in our system.
Asunto(s)
Metformina , Placenta , Humanos , Embarazo , Femenino , Intercambio Materno-Fetal/fisiología , Hipoglucemiantes/farmacología , Proteínas de Transporte de Membrana , Simulación por ComputadorRESUMEN
The study of transporter proteins is key to understanding the mechanism behind multi-drug resistance and drug-drug interactions causing severe side effects. While ATP-binding transporters are well-studied, solute carriers illustrate an understudied family with a high number of orphan proteins. To study these transporters, in silico methods can be used to shed light on the basic molecular machinery by studying protein-ligand interactions. Nowadays, computational methods are an integral part of the drug discovery and development process. In this short review, computational approaches, such as machine learning, are discussed, which try to tackle interactions between transport proteins and certain compounds to locate target proteins. Furthermore, a few cases of selected members of the ATP binding transporter and solute carrier family are covered, which are of high interest in clinical drug interaction studies, especially for regulatory agencies. The strengths and limitations of ligand-based and structure-based methods are discussed to highlight their applicability for different studies. Furthermore, the combination of multiple approaches can improve the information obtained to find crucial amino acids that explain important interactions of protein-ligand complexes in more detail. This allows the design of drug candidates with increased activity towards a target protein, which further helps to support future synthetic efforts.
RESUMEN
Pregabalin is an anti-neuropathic pain drug inhibiting the α2δ subunit of the voltage-dependent calcium channel in the spinal cord. The aim of this study is to characterize the transport mechanism of pregabalin at the blood-spinal cord barrier (BSCB) by means of in vivo experiments in rats and in vitro studies using primary-cultured rat spinal cord endothelial cells. We isolated endothelial cells by culturing rat spinal cord tissue in the presence of puromycin, and confirmed the expression of BSCB markers such as Cd31, Mdr1a, and Claudin-5. The uptake of pregabalin by primary-cultured rat spinal cord endothelial cells was sodium-independent and was significantly inhibited by L-leucine, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, and JPH203. These results suggest the involvement of L-type amino acid transporter (LAT) 1. LAT1 mRNA and protein was expressed in primary-cultured rat spinal cord endothelial cells, which is consistent with LAT1 expression at the BSCB. In the in vivo study, the transfer of pregabalin to rat spinal cord and brain was significantly decreased by the pre-administration of branched chain amino acids (BCAAs), which are endogenous substrates of LAT1. Our results indicate that pregabalin transport across the BSCB is mediated at least in part by LAT1 and is inhibited by plasma BCAAs.
Asunto(s)
Aminoácidos de Cadena Ramificada , Transportador de Aminoácidos Neutros Grandes 1 , Ratas , Animales , Pregabalina , Transportador de Aminoácidos Neutros Grandes 1/genética , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Aminoácidos de Cadena Ramificada/metabolismo , Células Endoteliales/metabolismo , Médula Espinal/metabolismoRESUMEN
The inert gas xenon (Xe) is increasingly used in medicine as a universal anesthetic, a regulator of cellular metabolism, and a broad-spectrum organoprotector. Commonly utilized Xe inhalation requires expensive equipment that is not universally available. Here we describe the production process and physical characteristics of a solid, highly stable xenon carrier based on α-cyclodextrin (α-CD), developed for oral administration. It was found, that the interaction of α-CD with Xe in an aqueous solution and elevated pressure leads to precipitation of the α-CD-Xe complex. We have discovered three new properties of the resulting complex that promote long-term storage and oral delivery of Xe. (i) At temperatures below 0 °C, the precipitated α-CD-Xe complex containing water is so stable that it allows the removal of water by vacuum freeze-drying (lyophilization). (ii). Lyophilized α-CD-Xe remains stable for months at room temperature. (iii) Upon contact with water, α-CD-Xe rapidly releases gaseous Xe. As revealed in the forced swim test, after oral administration of lyophilized α-CD-Xe to rats, the duration of swimming was significantly increased. The obtained data open up prospects for the development of drugs based on the lyophilized α-CD-Xe complex suitable for storage, transportation, and medical use, including outside the hospital.
Asunto(s)
Xenón , alfa-Ciclodextrinas , Ratas , Animales , Administración Oral , Excipientes , AguaRESUMEN
Breast cancer resistance protein (BCRP/Abcg2 in human, Bcrp/Abcg2 in rat), a member of the ATP-binding cassette (ABC) transporter family, acts as an efflux pump for xenobiotics, with ability to transport various drugs out of cells. Capsaicin may have the potential to modulate the function of Bcrp transport. This study was to evaluate the effects of capsaicin on the pharmacokinetics of sulfasalazine, a Bcrp substrate, in rats and investigate the mechanism of this food-drug interaction.The rats were pre-treated with 5% carboxymethylcellulose sodium (vehicle), capsaicin (3, 8, 25 mg/kg) and cyclosporine A (10 mg/kg) by gastric gavage for 7 days. On day 7, blood, liver and intestine samples were collected after sulfasalazine administered. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to study the effects of capsaicin on the pharmacokinetics of sulfasalazine in rats. RT-PCR and western blotting were used to study the mechanism in biomolecules in rats, respectively.Compared with vehicle group, AUC0-∞ of sulfasalazine in rats were increased by 1.5-folds, 1.6-folds and 1.7-folds in 3, 8 and 25 mg/kg/d capsaicin pre-treated groups. At the same time, the CL/F in rats were decreased by 33%, 38% and 42% in the three groups. In addition, we found Bcrp mRNA levels and protein expressions in rat livers and intestines were decreased in 3, 8 and 25 mg/kg/d capsaicin-treated groups.Our study demonstrated that long-term ingestion of capsaicin significantly enhanced the AUC of sulfasalazine involved down-regulate Bcrp gene and protein expression in rat liver and intestine.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Capsaicina , Sulfasalazina , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Capsaicina/farmacología , Cromatografía Liquida , Femenino , Ratas , Sulfasalazina/farmacocinética , Espectrometría de Masas en TándemRESUMEN
Pravastatin is currently under evaluation for prevention of preeclampsia. Factors contributing to placental disposition of pravastatin are important in assessment of potential undesirable fetal effects. The purpose of this study was to identify the uptake transporters that contribute to the placental disposition of pravastatin. Our data revealed the expression of organic anion transporting polypeptide 1A2 (OATP1A2) and OATP2A1 in the apical, and OATP2B1 and OATP5A1 in the basolateral membranes of the placenta, while organic anion transporter 4 (OAT4) exhibited higher expression in basolateral membrane but was detected in both membranes. Preloading placental membrane vesicles with glutarate increased the uptake of pravastatin suggesting involvement of glutarate-dependent transporters such as OAT4. In the HEK293 cells overexpressing individual uptake transporters, OATP2A1, OATP1A2 and OAT4 were determined to accept pravastatin as a substrate at physiological pH, while the uptake of pravastatin by OATP2B1 (known to interact with pravastatin at acidic pH) and OATP5A1 was not detected at pH 7.4. These findings led us to propose that OATP1A2 and OATP2A1 are responsible for the placental uptake of pravastatin from the maternal circulation, while OAT4 mediates the passage of the drug across placental basolateral membrane in the fetal-to-maternal direction.
Asunto(s)
Transportadores de Anión Orgánico/metabolismo , Pravastatina , Transporte Biológico , Femenino , Células HEK293 , Humanos , Placenta/metabolismo , Pravastatina/metabolismo , EmbarazoRESUMEN
The aim of the present study was to investigate changes in plasma concentrations and tissue distribution of endogenous substrates of organic anion transporting polypeptide (OATP) 1B, hexadecanedioate (HDA), octadecanedioate (ODA), tetradecanedioate (TDA), and coproporphyrin-III, induced by its weak inhibitor, probenecid (PBD), in rats. PBD increased the plasma concentrations of these four compounds regardless of bile duct cannulation, whereas liver-to-plasma (Kp,liver) and kidney-to-plasma concentration ratios of HDA and TDA were reduced. Similar effects of PBD on plasma concentrations and Kp,liver of HDA, ODA, and TDA were observed in kidney-ligated rats, suggesting a minor contribution of renal disposition to the overall distribution of these three compounds. Tissue uptake clearance of deuterium-labeled HDA (d-HDA) in liver was 16-fold higher than that in kidney, and was reduced by 80% by PBD. This was compatible with inhibition by PBD of d-HDA uptake in isolated rat hepatocytes. Such inhibitory effects of PBD were also observed in the human OATP1B1-mediated uptake of d-HDA. Overall, the disposition of HDA is mainly determined by hepatic OATP-mediated uptake, which is inhibited by PBD. HDA might, thus, be a biomarker for OATPs minimally affected by urinary and biliary elimination in rats.
Asunto(s)
Transportadores de Anión Orgánico , Probenecid , Animales , Transporte Biológico , Hepatocitos/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Transportador 1 de Anión Orgánico Específico del Hígado/metabolismo , Transportadores de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Probenecid/farmacología , RatasRESUMEN
The Breast Cancer Resistance Protein (BCRP) is a key transporter in drug efflux and drug-drug interactions. However, endogenous expression of Multidrug Resistance Protein 1 (MDR1) confounds the interpretation of BCRP-mediated transport in in vitro models. Here we used a CRISPR-Cas9 edited Madin-Darby canine kidney (MDCK) II cell line (MDCKcMDR1-KO) for stable expression of human BCRP (hBCRP) with no endogenous canine MDR1 (cMDR1) expression (MDCK-hBCRPcMDR1-KO). Targeted quantitative proteomics verified expression of hBCRP, and global analysis of the entire proteome corroborated no or very low background expression of other drug transport proteins or metabolizing enzymes. This new cell line, had similar proteome like MDCKcMDR1-KO and a previously established, corresponding cell line overexpressing human MDR1 (hMDR1), MDCK-hMDR1cMDR1-KO. Functional studies with MDCK-hBCRPcMDR1-KO confirmed high hBCRP activity. The MDCK-hBCRPcMDR1-KO cell line together with the MDCK-hMDR1cMDR1-KO easily and accurately identified shared or specific substrates of the hBCRP and the hMDR1 transporters. These cell lines offer new, improved in vitro tools for the assessment of drug efflux and drug-drug interactions in drug development.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Sistemas CRISPR-Cas , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Animales , Transporte Biológico , Línea Celular , Perros , Humanos , Células de Riñón Canino Madin Darby , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMEN
Recent studies have focused on coproporphyrin (CP)-I and CP-III (CPs) as endogenous biomarkers for organic anion transporting polypeptides (OATPs). Previous data showed that CPs are also substrates of multidrug resistance-associated protein (MRP/Mrp) 2 and 3. This study was designed to examine the impact of loss of Mrp2 function on the routes of excretion of endogenous CPs in wild-type (WT) Wistar compared to Mrp2-deficient TR- rats. To exclude possible confounding effects of rat Oatps, the transport of CPs was investigated in Oatp-overexpressing HeLa cells. Results indicated that CPs are substrates of rodent Oatp1b2, and that CP-III is a substrate of Oatp2b1. Quantitative targeted absolute proteomic (QTAP) analysis revealed no differences in Oatps, but an expected significant increase in Mrp3 protein levels in TR- compared to WT rat livers. CP-I and CP-III concentrations measured by LC-MS/MS were elevated in TR- compared to WT rat liver, while CP-I and CP-III estimated biliary clearance was decreased 75- and 840-fold in TR- compared to WT rats, respectively. CP-III concentrations were decreased 14-fold in the feces of TR- compared to WT rats, but differences in CP-I were not significant. In summary, the disposition of CPs was markedly altered by loss of Mrp2 and increased Mrp3 function as measured in TR- rats.
Asunto(s)
Coproporfirinas , Proteómica , Animales , Cromatografía Liquida , Células HeLa , Humanos , Hígado , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Ratas , Ratas Wistar , Espectrometría de Masas en TándemRESUMEN
Although an abundance of drug candidates exists which are aimed at the remediation of central nervous system (CNS) disorders, the utility of some are severely limited by their inability to cross the blood brain barrier. Potential drug delivery systems such as the Angiopep family of peptides have shown modest potential; however, there is a need for novel drug delivery candidates that incorporate peptidomimetics to enhance the efficiency of transcytosis, specificity, and biocompatibility. Here, we report on the first in vitro cellular uptake and cytotoxicity study of a peptidomimetic, cationic peptide, L57. It binds to cluster 4 of the low-density lipoprotein receptor-related protein 1 (LRP1) receptor which is expressed in numerous cell types, such as brain endothelial cells. We used early-passage-number brain microvascular endothelial cells and astrocytes harvested from rat pup brains that highly express LRP1, to study the uptake of L57 versus Angiopep-7 (A7). Uptake of L57 and A7 showed a concentration-dependent increase, with L57 being taken up to a greater degree than A7 at the same concentration. Additionally, peptide uptake in LRP1-deficient PEA 10 cells had greatly reduced uptake. Furthermore, L57 demonstrated excellent cell viability versus A7, showing promise as a potential drug delivery vector for CNS therapeutics.
Asunto(s)
Preparaciones Farmacéuticas , Receptores de Lipoproteína , Animales , Barrera Hematoencefálica , Encéfalo , Sistemas de Liberación de Medicamentos , Células Endoteliales , Péptidos , RatasRESUMEN
In predicting the hepatic elimination of compounds, the extended clearance concept has proven useful. Yet, its experimental proof was scarce partly due to the lack of models with the controlled expression of transporters. Here, the uptake and efflux transporters [NTCP (SLC10A1) and BSEP (ABCB11), respectively] were doubly and transiently expressed in MDCKII cells by electroporation-based transfection (with the BSEP plasmid amount varied and with the NTCP plasmid fixed), achieving the activity levels of NTCP and BSEP comparable to those of sandwich cultured human hepatocytes. The biliary excretion clearance for taurocholate increased proportionally to the BSEP expression level. Under the same conditions, the basal-to-apical transcellular clearance of taurocholate displayed an initial increase, and a subsequent plateau, indicating that the basolateral uptake of taurocholate became rate-limiting. The doubly transfected MDCKII cells were also used to kinetically analyze the inhibitory effects of rifampicin on BSEP and NTCP. The obtained results showed a bell-shaped profile for cell-to-medium concentration ratios over a range of rifampicin concentrations, which were quantitatively captured by kinetic modeling based on the extended clearance concept. The present study highlights the utility of the transient, tunable transporter expression system in delineating the rate-determining process and providing mechanistic insights into intracellular substrate accumulation.
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Simportadores , Ácido Taurocólico , Transportadoras de Casetes de Unión a ATP , Ácidos y Sales Biliares , Hepatocitos , Humanos , Hígado , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Rifampin/farmacología , Simportadores/genéticaRESUMEN
Determination of abundances of proteins involved in uptake, distribution, metabolism and excretion of xenobiotics is a prerequisite to understand and predict elimination mechanisms in tissue. Mass spectrometry promises simple and accurate measurements of individual proteins in complex mixtures using isotopically labeled peptide standards. However, comparisons of measurements performed in different laboratories have shown considerable discrepancies in the data generated. Even when very similar approaches are compared, the results differ significantly. An alternative method of measuring protein titers is global proteomics. Depending on sample type, this allows quantification of hundreds to thousands of proteins in a single analysis. It enables system-wide insights by providing protein copy numbers and cell sizes. Regardless of differences, the workflows of both the labeled standard-based and the proteomic approach share several steps. Each can be critical. Selection of optimal techniques is the prerequisite for accurate and reproducible protein quantification.
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Proteínas , Proteómica , Espectrometría de Masas , Péptidos , Estándares de ReferenciaRESUMEN
Vigabatrin (VGB) is a first-line drug used for treatment of infantile spasms. On therapeutic dose, VGB accumulates in the retina causing permanent peripheral visual field constriction. The mechanism involved in retinal accumulation of VGB is ambiguous. In the present study, mechanism of VGB transport into retina was evaluated. VGB uptake into retina was studied in vitro using human adult retinal pigment epithelial (ARPE-19) cells as a model for outer blood retinal barrier. The VGB cell uptake studies demonstrated saturation kinetics with Km value of 13.1 mM and uptake was significantly increased at pH 7.4 and hyperosmolar conditions indicating involvement of carrier-mediated Na+-Cl--dependent transporter. In the presence of taurine transporter (TauT) substrates (taurine and GABA) and inhibitor guanidinoethyl sulfonate (GES), the uptake of VGB decreased significantly demonstrating contribution of TauT. The VGB retinal levels in rats were decreased by 1.5- and 1.3-folds on chronic administration of GES and taurine, respectively. In conclusion, this study demonstrated the TauT involvement in VGB uptake and accumulation in retina.
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Anticonvulsivantes/farmacocinética , Glicoproteínas de Membrana/fisiología , Proteínas de Transporte de Membrana/fisiología , Retina/metabolismo , Vigabatrin/farmacocinética , Animales , Transporte Biológico , Línea Celular , Humanos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Entecavir (ETV) is a first-line antiviral drug against the hepatitis B virus. This study was designed to investigate whether ETV pharmacokinetics changes during pregnancy and the underlying mechanism. The results showed that ETV exposure in plasma was higher in pregnant rats than in nonpregnant rats, whereas the exposure after delivery was recovered to that in nonpregnant rats. Because 70% of orally dosed ETV is eliminated by kidney, the effects of estradiol (E2) and progesterone (P4), 2 important hormones during pregnancy, on ETV-related renal transporters were investigated. Our results revealed that the activities of the ETV-related renal transporters hOAT1, hOAT3, hMATE1, and hMATE2-K were clearly inhibited by E2 and P4, resulting in reduced ETV accumulation in transporter-transfected cell models. However, the cumulative urinary excretion of ETV in pregnant rats exhibited no significant difference compared to nonpregnant rats, although the endogenous creatinine clearance in pregnant rats was 1.5-fold that of nonpregnant rats. In conclusion, ETV plasma exposure is increased during pregnancy, but ETV renal excretion displays no significant alteration. This may be because, during pregnancy, increased glomerular ETV filtration compensated for the decrease in renal tubular ETV secretion that occurs by E2- and P4-mediated inhibition of related transporters.
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Guanina , Eliminación Renal , Animales , Antivirales/metabolismo , Femenino , Guanina/análogos & derivados , Virus de la Hepatitis B , Riñón/metabolismo , Embarazo , RatasRESUMEN
The aims of this study are to quantify the protein levels of nucleoside transporters in placental microvillous membranes (MVMs) and to clarify the contributions of these transporters to ribavirin uptake at the placental barrier. Placental MVMs of human and rat expressed equilibrative nucleoside transporter (ENT) 1 protein, whereas the expression of ENT2 protein was obscure. Maternal-to-fetal transfer of [3H]ribavirin in rats was much higher than that of [14C]sucrose. The uptake of [3H]ribavirin by rat placental trophoblast TR-TBT 18 d-1 cells, which functionally express both ENT1 and ENT2 proteins, was saturable, and was significantly inhibited by 0.1 µM nitrobenzylthioinosine, which selectively abolishes ENT1-mediated uptake. Dipyridamole at 10 µM is capable of inhibiting ENT2 as well as ENT1, but a degree of inhibition by 10 µM dipyridamole on [3H]ribavirin uptake was not much different from that by 0.1 µM nitrobenzylthioinosine (ENT1-specific inhibitor). Therefore, ENT2 may contribute little to [3H]ribavirin uptake by these cells. Rat ENT1 cRNA-injected oocytes showed increased [3H]ribavirin uptake compared with water-injected oocytes, while rat ENT2 cRNA-injected oocytes did not. In conclusion, ENT1 protein expressed in placental MVMs appears to play a predominant role in the uptake of ribavirin.