RESUMEN
Caco-2 cells are widely used as an in vitro intestinal epithelial cell model because they can form a monolayer and predict drug absorption with high accuracy. However, Caco-2 cells hardly express cytochrome P450 (CYP), a drug-metabolizing enzyme. It is known that CYP3A4 is the dominant drug-metabolizing enzyme in human small intestine. In this study, we generated CYP3A4-expressing Caco-2 (CYP3A4-Caco-2) cells and attempted to establish a model that can simultaneously evaluate drug absorption and metabolism. CYP3A4-Caco-2 cells were generated by piggyBac transposon vectors. A tetracycline-controllable CYP3A4 expression cassette (tet-on system) was stably transduced into Caco-2 cells, thus regulating the levels of CYP3A4 expression depending on the doxycycline concentration. The CYP3A4 expression levels in CYP3A4-Caco-2 cells cultured in the presence of doxycycline were similar to or higher than those of adult small intestine. The CYP3A4-Caco-2 cells had enough ability to metabolize midazolam, a substrate of CYP3A4. CYP3A4 overexpression had no negative effects on cell proliferation, barrier function, and P-glycoprotein activity in Caco-2 cells. Thus, we succeeded in establishing Caco-2 cells with CYP3A4 metabolizing activity comparable to in vivo human intestinal tissue. This cell line would be useful in pharmaceutical studies as a model that can simultaneously evaluate drug absorption and metabolism.
Asunto(s)
Citocromo P-450 CYP3A/genética , Elementos Transponibles de ADN , Regulación de la Expresión Génica/efectos de los fármacos , Vectores Genéticos/genética , Regiones Promotoras Genéticas , Tetraciclina/farmacología , Células CACO-2 , Células Cultivadas , Citocromo P-450 CYP3A/metabolismo , Descubrimiento de Drogas/métodos , Citometría de Flujo , HumanosRESUMEN
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.
Asunto(s)
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
BACKGROUND & AIMS: To develop an effective and safe orally administered drug, it is important to predict its intestinal absorption rate, intestinal first-pass effect, and drug-drug interactions of orally administered drugs. However, there is no existing model to comprehensively predict the intestinal pharmacokinetics and drug-response of orally administered drugs. In this study, we attempted to generate homogenous and functional intestinal epithelial cells from human induced pluripotent stem (iPS) cells for pharmaceutical research. METHODS: We generated almost-homogenous Villin- and zonula occludens-1 (ZO1)-positive intestinal epithelial cells by caudal-related homeobox transcription factor 2 (CDX2) transduction into human iPS cell-derived intestinal progenitor cells. RESULTS: The drug absorption rates in human iPS cell-derived intestinal epithelial cell monolayers (iPS-IECM) were highly correlated with those in humans (R2=0.91). The expression levels of cytochrome P450 (CYP) 3A4, a dominant drug-metabolizing enzyme in the small intestine, in human iPS-IECM were similar to those in human small intestine in vivo. In addition, intestinal availability in human iPS-IECM (the fraction passing the gut wall: Fg=0.73) was more similar to that in the human small intestine in vivo (Fg=0.57) than to that in Caco-2 cells (Fg=0.99), a human colorectal adenocarcinoma cell line. Moreover, the drug-drug interaction and drug-food interaction could be observed by using our human iPS-IECM in the presence of an inducer and inhibitor of CYP3A4, i.e., rifampicin and grape fruit juice, respectively. CONCLUSION: Taking these results together, we succeeded in generating the human iPS-IECM that can be applied to various intestinal pharmacokinetics and drug-response tests of orally administered drugs.
Asunto(s)
Factor de Transcripción CDX2/genética , Células Madre Pluripotentes Inducidas/citología , Intestinos/citología , Transducción Genética/métodos , Factor de Transcripción CDX2/metabolismo , Células CACO-2 , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Cultivadas , Citocromo P-450 CYP3A/metabolismo , Interacciones Farmacológicas , Células Epiteliales/citología , Células Epiteliales/metabolismo , Interacciones Alimento-Droga , Jugos de Frutas y Vegetales , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Absorción Intestinal , Rifampin/farmacocinéticaRESUMEN
The purpose of this study was to elucidate the involvement of Mate1 in the tubular secretion of trimethoprim and saturation of Mate1-mediated efflux to address the mechanisms underlying the pharmacokinetic drug interactions with trimethoprim. Trimethoprim is a more potent inhibitor of MATE2-K than MATE1 with Ki values (µM) of 0.030-0.28 and 2.4-5.9, respectively. Trimethoprim is a substrate of human MATE1 and MATE2-K with Km values of 2.3 ± 0.9 and 0.018 ± 0.004 µM, and mouse Mate1, but not human OCT2, mouse Oct1 and Oct2. Pyrimethamine significantly reduced the renal clearance (CLR) of trimethoprim (mL/min/kg) from 40.0 ± 5.1 to 20.1 ± 3.7 (p < 0.05). Trimethoprim was given to mice at three infusion rates (150, 500, and 1500 nmol/min/kg). Together with an increase in the plasma concentrations of trimethoprim, the CLR (mL/min/kg) of trimethoprim decreased to 25.9 ± 3.2, 13.5 ± 5.7, and 8.92 ± 1.50 at the respective rates. Trimethoprim decreased the CLR of rhodamine 123 in an infusion rate-dependent manner: 11.5 ± 1.3 (control), 5.17 ± 1.55, 1.31 ± 0.50, and 0.532 ± 0.180. These results suggest that Mate1 mediates the tubular secretion of trimethoprim, and at therapeutic doses, MATEs-mediated efflux can be saturated, and thereby, cause drug interactions with other MATE substrates.
Asunto(s)
Inhibidores del Citocromo P-450 CYP2C8/metabolismo , Riñón/metabolismo , Dinámicas no Lineales , Proteínas de Transporte de Catión Orgánico/metabolismo , Trimetoprim/metabolismo , Animales , Inhibidores del Citocromo P-450 CYP2C8/farmacología , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas/fisiología , Células HEK293 , Humanos , Riñón/efectos de los fármacos , Masculino , Ratones , Trimetoprim/farmacologíaRESUMEN
We synthesized [11C]sulpiride as a positron emission tomography probe for investigating the drug distribution in the human body. [11C]Sulpiride was injected to healthy male subjects in either tracer dose of [11C]sulpiride (approximately 222 MBq) or with therapeutic dose of sulpiride (500 mg, peroral) 3 h before the injection in a crossover fashion. Whole-body positron emission tomography imaging demonstrated that [11C]sulpiride accumulated exceedingly in the bladder, followed by liver, gall bladder, and kidney, respectively, at 30 min after the injection, whereas scarcely in the brain. Oral dose of sulpiride decreased the hepatic accumulation of the radioactivity by 60%. From in vitro experiments, we found that sulpiride is a substrate of hOCT1 (Km 2.6 µM), hOCT2 (Km 68 µM), hMATE1 (Km 40 µM), and hMATE2-K (Km 60 µM). Moreover, the uptake of sulpiride by human hepatocytes was diminished by tetraethylammonium, and saturable with Km of 18 µM. Oct1/2 double knockout mice and wild-type mice received Mate1 inhibitors (pyrimethamine/cimetidine) manifested reduced renal clearance of sulpiride, accompanied with its accumulation in the plasma. In conclusion, we found that sulpiride is a substrate of OCT1, OCT2, MATE1, and MATE2-K, and this suggests that [11C]sulpiride would be a useful radioligand to investigate the organic cation transporters in humans.
Asunto(s)
Antagonistas de los Receptores de Dopamina D2/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Sulpirida/metabolismo , Animales , Transporte Biológico , Isótopos de Carbono , Cimetidina/química , Cimetidina/metabolismo , Antagonistas de los Receptores de Dopamina D2/administración & dosificación , Relación Dosis-Respuesta a Droga , Células HEK293 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Factores de Transcripción de Octámeros/metabolismo , Tomografía de Emisión de Positrones , Sulpirida/administración & dosificación , Tetraetilamonio/química , Tetraetilamonio/metabolismo , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/metabolismoRESUMEN
This study aimed to investigate the interactions of 3 anticoagulants, rivaroxaban, apixaban, and dabigatran, with 5 human solute carrier transporters, hOAT1, hOAT3, hOCT2, hOATP1B1, and hOATP1B3. Apixaban inhibited hOAT3, hOATP1B1, and hOATP1B3, and rivaroxaban inhibited hOAT3 and hOATP1B3, with IC50 values of >20 and >5 µM, respectively. The effect of dabigatran was negligible or very weak, so significant drug interactions at therapeutic doses are unlikely. Specific uptake of rivaroxaban was observed only in human and mouse OAT3-expressing cells. The Km for mouse Oat3 (mOat3) was 1.01 ± 0.70 µM. A defect in mOat3 reduced the kidney-to-plasma concentration ratio of rivaroxaban by 38% in mice. Probenecid treatment also reduced the kidney-to-plasma concentration ratio of rivaroxaban in rats by 73%. Neither mOat3 defect nor probenecid administration in rats reduced the renal clearance of rivaroxaban. The uptake of rivaroxaban by monkey kidney slices was temperature dependent and inhibited by probenecid but not by tetraethylammonium. Taken together, organic anion transporters, mainly OAT3, may mediate basolateral uptake of rivaroxaban in kidneys. hOAT3 could be an additional factor that differentiates the potential drug-drug interactions of the 3 anticoagulants in the urinary excretion process in clinical settings.
Asunto(s)
Anticoagulantes/farmacocinética , Dabigatrán/farmacocinética , Riñón/metabolismo , Transportadores de Anión Orgánico/metabolismo , Pirazoles/farmacocinética , Piridonas/farmacocinética , Rivaroxabán/farmacocinética , Animales , Anticoagulantes/metabolismo , Anticoagulantes/farmacología , Transporte Biológico/efectos de los fármacos , Dabigatrán/metabolismo , Dabigatrán/farmacología , Interacciones Farmacológicas , Femenino , Células HEK293 , Haplorrinos , Humanos , Riñón/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Transportadores de Anión Orgánico/antagonistas & inhibidores , Pirazoles/metabolismo , Pirazoles/farmacología , Piridonas/metabolismo , Piridonas/farmacología , Ratas , Ratas Sprague-Dawley , Rivaroxabán/metabolismo , Rivaroxabán/farmacologíaRESUMEN
Organic anion-transporting polypeptide (OATP) 1A2 has the potential to be a target for central nervous system drug delivery due to its luminal localization at the human blood-brain barrier and broad substrate specificity. We found OATP1A2 mRNA expression in the human brain to be comparable to breast cancer resistance protein and OATP2B1 and much higher than P-glycoprotein (P-gp), and confirmed greater expression in the brain relative to other tissues. The goal of this study was to establish a model system to explore OATP1A2-mediated transcellular transport of substrate drugs and the interplay with P-gp. In vitro (human embryonic kidney 293 cells stably expressing Oatp1a4, the closest murine isoform) and in vivo (naïve and Oatp1a4 knock-out mice) studies with OATP1A2 substrate triptan drugs demonstrated that these drugs were not Oatp1a4 substrates. This species difference demonstrates that the rodent is not a good model to investigate the active brain uptake of potential OATP1A2 substrates. Thus, we constructed a novel OATP1A2 expressing Madin-Darby canine kidney (MDCK) II wild type and an MDCKII-multidrug resistance protein 1 (MDR1) system using BacMam virus transduction. The spatial expression pattern of OATP1A2 after transduction in MDCKII-MDR1 cells was superimposed to P-gp, confirming apical membrane localization. OATP1A2-mediated uptake of zolmitriptan, rosuvastatin, and fexofenadine across monolayers increased with increasing OATP1A2 protein expression. OATP1A2 counteracted P-gp efflux for cosubstrates zolmitriptan and fexofenadine. A three-compartment model incorporating OATP1A2-mediated influx was used to quantitatively describe the time- and concentration-dependent apical-to-basolateral transcellular transport of rosuvastatin across OATP1A2 expressing the MDCKII monolayer. This novel, simple and versatile experimental system is useful for understanding the contribution of OATP1A2-mediated transcellular transport across barriers, such as the blood-brain barrier.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Transportadores de Anión Orgánico/metabolismo , Preparaciones Farmacéuticas/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico Activo , Perros , Células HEK293 , Humanos , Células de Riñón Canino Madin Darby , Ratones , Ratones Noqueados , Modelos Biológicos , Transportadores de Anión Orgánico/biosíntesis , Transportadores de Anión Orgánico/genética , Rosuvastatina Cálcica/farmacocinética , Especificidad de la Especie , Distribución Tisular , Triptaminas/metabolismoRESUMEN
BACKGROUND: Abnormal behaviors of young patients after taking the anti-influenza agent oseltamivir (Tamiflu®, F. Hoffmann-La Roche, Ltd., Basel, Switzerland) have been suspected as neuropsychiatric adverse events (NPAEs). Immune response to viral infection is suspected to cause elevation of drug concentration in the brain of adolescents. In the present study, the effect of innate immune activation on the brain uptake of [(11)C]oseltamivir was quantitatively evaluated in juvenile monkeys. METHODS: Three 2-year-old monkeys underwent positron emission tomography (PET) scans at baseline and immune-activated conditions. Both scans were conducted under pre-dosing of clinically relevant oseltamivir. The immune activation condition was induced by the intravenous administration of polyinosine-polycytidylic acid (poly I:C). Dynamic [(11)C]oseltamivir PET scan and serial arterial blood sampling were performed to obtain [(11)C]oseltamivir kinetics. Brain uptake of [(11)C]oseltamivr was evaluated by its normalized brain concentration, brain-to-plasma concentration ratio, and plasma-to-brain transfer rate. Plasma pro-inflammatory cytokine levels were also measured. RESULTS: Plasma interleukin-6 was elevated after intravenous administration of poly I:C in all monkeys. Brain radioactivity was uniform both at baseline and under poly I:C treatment. The mean brain concentrations of [(11)C]oseltamivir were 0.0033 and 0.0035% ID/cm(3) × kg, the mean brain-to-plasma concentration ratios were 0.58 and 0.65, and the plasma-to-brain transfer rates were 0.0047 and 0.0051 mL/min/cm(3) for baseline and poly I:C treatment, respectively. Although these parameters were slightly changed by immune activation, the change was not notable. CONCLUSIONS: The brain uptake of [(11)C]oseltamivir was unchanged by poly I:C treatment in juvenile monkeys. This study demonstrated that the innate immune response similar to the immune activation of influenza would not notably change the brain concentration of oseltamivir in juvenile monkeys.
RESUMEN
PURPOSE: Multidrug and toxin extrusion proteins (MATEs) are multispecific organic cation transporters mediating the efflux of various cationic drugs into the urine. The present study aimed at identifying endogenous compounds in human plasma and urine specimens as biomarkers to evaluate drug interactions involving MATEs in the kidney without administration of their exogenous probe drugs. METHODS: An untargeted metabolomic analysis was performed using urine and plasma samples from healthy volunteers and mice treated with or without the potent MATE inhibitor, pyrimethamine. Plasma and urinary concentrations of candidate markers were measured using liquid chromatography-mass spectrometry. Transport activities were determined in MATE- or OCT2-expressing HEK293 cells. The deuterium-labeled compounds of candidates were administered to mice for pharmacokinetics study. RESULTS: Urinary excretion of eleven compounds including thiamine and carnitine was significantly lower in the pyrimethamine-treatment group in humans and mice, whereas no endogenous compound was noticeably accumulated in the plasma. The renal clearance of thiamine and carnitine was decreased by 70%-84% and 90%-94% (p < 0.05), respectively, in human. The specific uptake of thiamine was observed in MATE1-, MATE2-K- or OCT2-expressing HEK293 cells with Km of 3.5 ± 1.0, 3.9 ± 0.8 and 59.9 ± 6.7 µM, respectively. The renal clearance of carnitine-d 3 was decreased by 62% in mice treated with pyrimethamine. CONCLUSIONS: Our findings indicate that MATEs account for the efflux of thiamine and perhaps carnitine as well as drugs into the urine. The urinary excretion of thiamine is useful to detect drug interaction involving MATEs in the kidney.
Asunto(s)
Biomarcadores/sangre , Biomarcadores/orina , Interacciones Farmacológicas/fisiología , Proteínas de Transporte de Catión Orgánico/metabolismo , Adulto , Animales , Transporte Biológico/fisiología , Línea Celular , Células HEK293 , Humanos , Riñón/metabolismo , Riñón/fisiología , Masculino , Ratones , Adulto JovenRESUMEN
The purpose of this study was to clarify the relationship between the physicochemical properties of drugs and their urinary excretion mechanisms. Three hundred twenty-five drugs were classified into the reabsorption, intermediate, and secretion types based on their ratio of renal clearance to protein-unbound fraction glomerular filtration rate. Fifty percent of ionized and neutral drugs were the secretion and reabsorption types, respectively. The mean molecular weight of the neutral drugs was slightly smaller than those of the ionized drugs (296 vs. 330-368 g/mol). The reabsorption-type anionic drugs were characterized by their low molecular weights (mean value 269 g/mol) and the logarithmic measure of the acid dissociation constants (pKa s) greater than 4.5, whereas the secretion-type anionic drugs all had pKa s below 4.5. Cationic drugs with pKa s lower than 8.0 tended to be the reabsorption type. Some cationic drugs were classified as the secretion type, despite their high molecular weights (734-811 g/mol) and high log P values (3.1-5.3). The organic anion transporter (OAT)1 and OAT3 substrates were all secretion-type drugs. The same trend was observed for the substrates of organic cation transporter 2, multidrug and toxin extrusion, multidrug resistance-associated protein 4, and multidrug resistance 1/breast cancer resistance protein, but substantial fractions of the substrates were categorized as the intermediate or reabsorption types (9%-38%). This work provides a clue to the renal elimination mechanism of new chemical entities during drug development.
Asunto(s)
Preparaciones Farmacéuticas/metabolismo , Preparaciones Farmacéuticas/orina , Proteínas de Transporte de Catión/metabolismo , Tasa de Filtración Glomerular , Humanos , Riñón/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Preparaciones Farmacéuticas/químicaRESUMEN
INTRODUCTION: Renal uptake of Tc-99m-MG3 involves organic anion transporter (OAT). Treatment with drugs showing OAT affinity might interfere with renal uptake of Tc-99m-MAG3, leading to misinterpretation in Tc-99m-MAG3. This study was conducted to discuss a possible drug interference with Tc-99m-MAG3 diagnosis on OAT sites. METHODS: Renal uptake and plasma clearance of Tc-99m-MAG3 were analyzed in healthy volunteers under control and OAT1 and OAT3 related drug treatment conditions. An in vitro uptake study using OAT1 or OAT3 expressing cells was also conducted. RESULTS: Both PAH and probenecid treatment induced delays in Tc-99m-MAG3 clearance from blood, and reductions in the renal uptake clearance. As a result, the normalized effective renal plasma flow estimated from Tc-99m-MAG3 clearance was significantly underestimated, whereas the glomerular filtration rate estimated from plasma creatinine levels was unchanged. The transport activity of Tc-99m-MAG3 was higher in OAT1-expressing cells than in OAT3-expressing cells. CONCLUSION: Drugs with OAT1 affinity affect the renal uptake of Tc-99m-MAG3 and blood clearance. This might cause misinterpretation of functional diagnosis of the kidney using Tc-99m-MAG3.
Asunto(s)
Pruebas de Función Renal , Riñón/efectos de los fármacos , Riñón/fisiología , Proteína 1 de Transporte de Anión Orgánico/metabolismo , Probenecid/farmacología , Tecnecio Tc 99m Mertiatida/metabolismo , Ácido p-Aminohipúrico/farmacología , Adulto , Transporte Biológico/efectos de los fármacos , Estudios Cruzados , Descubrimiento de Drogas , Tasa de Filtración Glomerular/efectos de los fármacos , Voluntarios Sanos , Humanos , Riñón/metabolismo , Masculino , Proteína 1 de Transporte de Anión Orgánico/antagonistas & inhibidores , Transportadores de Anión Orgánico Sodio-Independiente/antagonistas & inhibidores , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Probenecid/metabolismo , Probenecid/uso terapéutico , Unión Proteica , Adulto Joven , Ácido p-Aminohipúrico/metabolismo , Ácido p-Aminohipúrico/uso terapéuticoRESUMEN
OBJECTIVES: Multidrug resistance-associated protein 2 (MRP2; ABCC2) is an ATP-binding cassette transporter that mediates the efflux of anionic drugs and phase II metabolites. Our aim was to elucidate the impact of a single nucleotide polymorphism, rs12762549 (G>C), on the in-vivo activity of MRP2. METHODS: Plasma specimens collected from 18 healthy volunteers were subjected to an untargeted metabolomic analysis using liquid chromatography-mass spectrometry. The role of MRP2 in the disposition of substances of interest was then examined in vivo in Mrp2-deficient mutant rats (Eisai hyperbilirubinemic rats; EHBRs) and in vitro in human MRP2-expressing membrane vesicles. RESULTS: A multivariate analysis model using liquid chromatography-mass spectrometry data sets successfully differentiated the GG and the CC genotypes of rs12762549. The C allele is associated with the basal plasma levels of the five phase II metabolites of genistein and dihydrogenistein. Such phase II metabolites were also accumulated in EHBR, compared with normal rats, partly because of the reduced biliary excretion. Following oral administration of genistein and daidzein, the plasma concentrations of total genistein and total daidzein, which were mainly accounted for by sulfoglucuronide conjugates, were markedly higher in EHBR than in normal rats. ATP-dependent uptake of sulfoglucuronides and glucuronides of the isoflavonoids was observed only in MRP2-expressing membrane vesicles. CONCLUSION: MRP2 plays a significant role in preventing the accumulation of phase II metabolites of isoflavonoids. The rs12762549 is associated with an interindividual difference in the plasma levels of MRP2 substrates, phase II metabolites of isoflavonoids, suggesting that this single nucleotide polymorphism is associated with variations in in-vivo MRP2 activity.
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Estudios de Asociación Genética , Genisteína/administración & dosificación , Isoflavonas/administración & dosificación , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/sangre , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Adulto , Alelos , Animales , Genisteína/farmacocinética , Humanos , Isoflavonas/farmacocinética , Japón , Masculino , Fase II de la Desintoxicación Metabólica/genética , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Polimorfismo de Nucleótido Simple , Ratas , Ratas Sprague-Dawley , Adulto JovenRESUMEN
Cimetidine, an H2 receptor antagonist, has been used to investigate the tubular secretion of organic cations in human kidney. We report a systematic comprehensive analysis of the inhibition potency of cimetidine for the influx and efflux transporters of organic cations [human organic cation transporter 1 (hOCT1) and hOCT2 and human multidrug and toxin extrusion 1 (hMATE1) and hMATE2-K, respectively]. Inhibition constants (K(i)) of cimetidine were determined by using five substrates [tetraethylammonium (TEA), metformin, 1-methyl-4-phenylpyridinium, 4-(4-(dimethylamino)styryl)-N-methylpyridinium, and m-iodobenzylguanidine]. They were 95 to 146 µM for hOCT2, providing at most 10% inhibition based on its clinically reported plasma unbound concentrations (3.6-7.8 µM). In contrast, cimetidine is a potent inhibitor of MATE1 and MATE2-K with K(i) values (µM) of 1.1 to 3.8 and 2.1 to 6.9, respectively. The same tendency was observed for mouse Oct1 (mOct1), mOct2, and mouse Mate1. Cimetidine showed a negligible effect on the uptake of metformin by mouse kidney slices at 20 µM. Cimetidine was administered to mice by a constant infusion to achieve a plasma unbound concentration of 21.6 µM to examine its effect on the renal disposition of Mate1 probes (metformin, TEA, and cephalexin) in vivo. The kidney- and liver-to-plasma ratios of metformin both were increased 2.4-fold by cimetidine, whereas the renal clearance was not changed. Cimetidine also increased the kidney-to-plasma ratio of TEA and cephalexin 8.0- and 3.3-fold compared with a control and decreased the renal clearance from 49 to 23 and 11 to 6.6 ml/min/kg, respectively. These results suggest that the inhibition of MATEs, but not OCT2, is a likely mechanism underlying the drug-drug interactions with cimetidine in renal elimination.
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Cimetidina/farmacología , Riñón/efectos de los fármacos , Proteínas de Transporte de Catión Orgánico/efectos de los fármacos , 1-Metil-4-fenilpiridinio/metabolismo , 3-Yodobencilguanidina/metabolismo , Animales , Unión Competitiva/fisiología , Transporte Biológico/efectos de los fármacos , Cefalexina/administración & dosificación , Cefalexina/sangre , Cefalexina/metabolismo , Cefalexina/farmacocinética , Cefalexina/orina , Cimetidina/administración & dosificación , Cimetidina/metabolismo , Cimetidina/farmacocinética , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas/fisiología , Células HEK293 , Humanos , Riñón/metabolismo , Cinética , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Metformina/administración & dosificación , Metformina/sangre , Metformina/metabolismo , Metformina/farmacocinética , Metformina/orina , Ratones , Ratones Endogámicos , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Transporte de Catión Orgánico/metabolismo , Transportador 1 de Catión Orgánico/antagonistas & inhibidores , Transportador 1 de Catión Orgánico/efectos de los fármacos , Transportador 1 de Catión Orgánico/genética , Transportador 1 de Catión Orgánico/metabolismo , Transportador 2 de Cátion Orgánico , Piridinas/metabolismo , Tetraetilamonio/administración & dosificación , Tetraetilamonio/sangre , Tetraetilamonio/metabolismo , Tetraetilamonio/farmacocinética , Tetraetilamonio/orina , TransfecciónRESUMEN
OBJECTIVE: To clarify the impact of breast cancer resistance protein 1 (BCRP1)/ATP-binding cassette transporter subfamily G member 2 (ABCG2) expression on cardiac repair after myocardial infarction (MI). METHODS AND RESULTS: The ATP-binding cassette transporter BCRP1/ABCG2 is expressed in various organs, including the heart, and may regulate several tissue defense mechanisms. BCRP1/ABCG2 was mainly expressed in endothelial cells of microvessels in the heart. MI was induced in 8- to 12-week-old wild-type (WT) and Bcrp1/Abcg2 knockout (KO) mice by ligating the left anterior descending artery. At 28 days after MI, the survival rate was significantly lower in KO mice than in WT mice because of cardiac rupture. Echocardiographic, hemodynamic, and histological assessments showed that ventricular remodeling was more deteriorated in KO than in WT mice. Capillary, myofibroblast, and macrophage densities in the peri-infarction area at 5 days after MI were significantly reduced in KO compared with WT mice. In vitro experiments demonstrated that inhibition of BCRP1/ABCG2 resulted in accumulation of intracellular protoporphyrin IX and impaired survival of microvascular endothelial cells under oxidative stress. Moreover, BCRP1/ABCG2 inhibition impaired migration and tube formation of endothelial cells. CONCLUSIONS: BCRP1/ABCG2 plays a pivotal role in cardiac repair after MI via modulation of microvascular endothelial cell survival and function.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/fisiología , Células Endoteliales/fisiología , Microvasos/fisiopatología , Infarto del Miocardio/fisiopatología , Proteínas de Neoplasias/fisiología , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Animales , Supervivencia Celular , Femenino , Ratones , Ratones Noqueados , Recuperación de la Función , Cicatrización de HeridasRESUMEN
This report describes a potent and selective inhibitor of multidrug and toxin extrusion (MATE) protein, pyrimethamine (PYR), and examines its effect on the urinary and biliary excretion of typical Mate1 substrates in mice. In vitro inhibition studies demonstrated that PYR is a potent inhibitor of mouse (m)Mate1 (K(i) = 145 nM) among renal organic cation transporters mOctn1 and mOctn2 (K(i) > 30 microM), mOct1 (K(i) = 3.6 microM), and mOct2 (K(i) = 6.0 microM). PYR inhibited the uptake of metformin by kidney brush-border membrane vesicles (BBMVs) (K(i) = 41 nM) and canalicular membrane vesicles in the presence of outward gradient of H+. PYR treatment significantly increased the kidney-to-plasma ratio of tetraethylammonium, and both the liver- and kidney-to-plasma ratios of metformin in mice, whereas it did not affect their plasma concentrations and urinary excretion rates. Furthermore, the plasma lactate concentration, a biomarker for inhibition of gluconeogenesis by metformin, was significantly higher in the PYR-treated group than in the control group. These results not only suggest the importance of mMate1 in the efflux of organic cations into the urine and bile in mice but also the importance of canalicular efflux mediated by MATE proteins for the therapeutic efficacy of metformin. PYR is a potent inhibitor of human (h)MATE1 and hMATE2-K (K(i) = 77 and 46 nM, respectively) and H+ and organic cation exchanger in human kidney BBMVs (K(i) = 31 nM) in the presence of outward gradient of H+. Taken together, PYR can be used as a potent probe inhibitor of human MATE transporters.