RESUMEN
Organic anion transporting polypeptide 2B1 (OATP2B1) is a widely expressed membrane transporter with diverse substrate specificity. In vitro and clinical studies suggest a role for intestinal OATP2B1 in the oral absorption of medications. Moreover, OATP2B1 is highly expressed in hepatocytes where it is thought to promote liver drug clearance. However, until now, a shortcoming of studies implicating OATP2B1 in drug disposition has been a lack of in vivo models. Here, we report the development of a knockout (KO) mouse model with targeted, global disruption of the Slco2b1 gene to examine the disposition of two confirmed mOATP2B1 substrates, namely, fexofenadine and rosuvastatin. The plasma pharmacokinetics of intravenously administered fexofenadine was not different between KO and wild-type (WT) mice. However, after oral fexofenadine administration, KO mice had 70% and 41% lower maximal plasma concentration (C max) and area under the plasma concentration-time curve (AUC0-last) than WT mice, respectively. In WT mice, coadministration of fexofenadine with grapefruit juice (GFJ) or apple juice (AJ) was associated with reduced C max by 80% and 88%, respectively, while the AUC0-last values were lower by 35% and 70%, respectively. In KO mice, AJ coadministration reduced oral fexofenadine C max and AUC0-last values by 67% and 59%, respectively, while GFJ had no effects. Intravenous and oral rosuvastatin pharmacokinetics were similar among WT and KO mice. We conclude that intestinal OATP2B1 is a determinant of oral fexofenadine absorption, as well as a target for fruit juice interactions. OATP2B1 does not significantly influence rosuvastatin disposition in mice. SIGNIFICANCE STATEMENT: A novel mouse model with targeted disruption of the Slco2b1 gene revealed that OATP2B1 is a determinant of oral absorption but not systemic disposition of fexofenadine, as well as a target of fruit juice interactions. Rosuvastatin oral and intravenous pharmacokinetics were not dependent on OATP2B1. These findings support the utility of the Slco2b1 KO mouse model for defining mechanisms of drug disposition at the intersection of in vitro and clinical pharmacology.
Asunto(s)
Mucosa Intestinal/metabolismo , Transportadores de Anión Orgánico/metabolismo , Rosuvastatina Cálcica/farmacocinética , Terfenadina/análogos & derivados , Administración Intravenosa , Administración Oral , Animales , Área Bajo la Curva , Interacciones Alimento-Droga , Jugos de Frutas y Vegetales , Células HEK293 , Células HeLa , Humanos , Absorción Intestinal , Masculino , Ratones , Ratones Noqueados , Transportadores de Anión Orgánico/genética , Rosuvastatina Cálcica/administración & dosificación , Terfenadina/administración & dosificación , Terfenadina/farmacocinéticaRESUMEN
Trimethylamine-N-oxide (TMAO) is a recently identified predictor of cardiovascular and chronic kidney disease. TMAO is primarily generated through gut-microbiome mediated conversion of dietary choline and carnitine to TMA, which is converted to TMAO by hepatic flavin monooxygenase 3 (FMO3) and subsequently undergoes renal elimination. We investigated the role of uptake and efflux drug transporters in TMAO disposition in vitro and in vivo. After screening a large array of uptake transporters, we show organic cation transporter 2 (OCT2) is the key transporter for TMAO cellular uptake. In Oct1/2 knockout mice, we observed increased plasma TMAO levels with reduced renal retention, suggesting the importance of Oct2 in facilitating the uptake of TMAO into renal tubular cells in vivo. Multiple transporters of the ATP-binding cassette (ABC) family, including ABCG2 (BCRP) and ABCB1 (MDR1), were capable of TMAO efflux. In human subjects, clinical, dietary, and pharmacogenetic covariates were evaluated for contribution to TMAO levels in a cohort of dyslipidemic patients (n = 405). Interestingly, genetic variation in ABCG2, but not other transporters, appeared to play a role in modulating TMAO exposure.
Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Metilaminas/metabolismo , Óxidos/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico , Carnitina/metabolismo , Humanos , Masculino , Ratones , Ratones Noqueados , Proteínas de Transporte de Catión Orgánico/metabolismo , Oxigenasas/metabolismoRESUMEN
Nonalcoholic fatty liver disease (NAFLD) alters drug response. We previously reported that NAFLD is associated with reduced in vivo CYP3A drug-metabolism activity and hepatic CYP3A4 expression in humans as well as mouse and human hepatoma models of the disease. Here, we investigated the role of the lipid- and glucose-modulating hormone fibroblast growth factor 21 (FGF21) in the molecular mechanism regulating CYP3A4 expression in NAFLD. In human subjects, mouse and cellular NAFLD models with lower CYP3A4 expression, circulating FGF21, or hepatic FGF21 mRNA levels were elevated. Administration of recombinant FGF21 or transient hepatic overexpression of FGF21 resulted in reduced liver CYP3A4 luciferase reporter activity in mice and decreased CYP3A4 mRNA expression and activity in cultured Huh7 hepatoma cells. Blocking canonical FGF21 signaling by pharmacological inhibition of MEK1 kinase in Huh7 cells caused de-repression of CYP3A4 mRNA expression with FGF21 treatment. Mice with high-fat diet-induced simple hepatic steatosis and lipid-loaded Huh7 cells had reduced nuclear localization of the pregnane X receptor (PXR), a key transcriptional regulator of CYP3A4 Furthermore, decreased nuclear PXR was observed in mouse liver and Huh7 cells after FGF21 treatment or FGF21 overexpression. Decreased PXR binding to the CYP3A4 proximal promoter was found in FGF21-treated Huh7 cells. An FGF21-PXR signaling pathway may be involved in decreased hepatic CYP3A4 metabolic activity in NAFLD.
Asunto(s)
Citocromo P-450 CYP3A/genética , Regulación hacia Abajo , Factores de Crecimiento de Fibroblastos/metabolismo , Enfermedad del Hígado Graso no Alcohólico/enzimología , Enfermedad del Hígado Graso no Alcohólico/genética , Receptores de Esteroides/metabolismo , Transducción de Señal , Animales , Línea Celular Tumoral , Citocromo P-450 CYP3A/metabolismo , Modelos Animales de Enfermedad , Femenino , Factores de Crecimiento de Fibroblastos/administración & dosificación , Factores de Crecimiento de Fibroblastos/sangre , Factores de Crecimiento de Fibroblastos/genética , Glucuronidasa/metabolismo , Humanos , Proteínas Klotho , Hígado , Sistema de Señalización de MAP Quinasas , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/sangre , Receptor X de Pregnano , Regiones Promotoras Genéticas/genética , Unión Proteica , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Fracciones Subcelulares/metabolismo , Transcripción GenéticaRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is the leading cause of liver disease in the Western world, given its association with obesity, type 2 diabetes, and dyslipidemia. Medications are widely used in NAFLD to manage comorbid conditions, and there is significant interest in developing new drug therapies to treat the disease. Despite this, little is known about the effects of NAFLD on drug metabolism. We examined the activity and expression of the major drug-metabolizing enzyme subfamily, CYP3A, in subjects with NAFLD as well as in mouse and cellular models. CYP3A activity was determined in healthy volunteers and subjects with biopsy-proven NAFLD by oral midazolam phenotyping and measurement of plasma 4ß-hydroxycholesterol, an endogenous metabolic biomarker. CYP3A4 transcriptional activity, metabolic activity, and expression were also assessed in a mouse and cellular model of NAFLD. Subjects with nonalcoholic steatohepatitis (NASH) had 2.4-fold higher plasma midazolam levels compared with controls. Plasma 4ß-hydroxycholesterol was 51% and 37% lower than controls in subjects with simple steatosis and NASH, respectively. Fibrosis was associated with 57% lower plasma 4ß-hydroxycholesterol levels than controls. Furthermore, hepatic CYP3A4 mRNA expression in NASH was 69% lower than control livers. CYP3A4 gene luciferase activity in the livers of NAFLD mice was 38% lower than that of controls. Lipid-loaded Huh7 human hepatoma cells had a 38% reduction in CYP3A4 activity and 80% lower CYP3A4 mRNA expression compared with the control. CYP3A activity is reduced in human NAFLD in addition to mouse and in vitro cell models of the disease.
Asunto(s)
Citocromo P-450 CYP3A/biosíntesis , Regulación Enzimológica de la Expresión Génica , Enfermedad del Hígado Graso no Alcohólico/enzimología , Animales , Línea Celular Tumoral , Citocromo P-450 CYP3A/genética , Activación Enzimática/fisiología , Femenino , Humanos , Hígado/enzimología , Hígado/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/patologíaRESUMEN
Tamoxifen is widely prescribed to patients with estrogen receptor-positive breast cancer, and it is a prodrug that requires bioactivation by cytochrome P450 enzymes CYP2D6 and 3A4 to generate the active metabolite, endoxifen. Large interpatient variability in endoxifen plasma levels has been reported, and polymorphisms in CYP2D6 have been implicated as a major determinant of such variability. However, little is known regarding the role of drug transporters such as P-glycoprotein [multidrug resistance 1 (MDR1), ATP-binding cassette B1 (ABCB1)] to endoxifen disposition and response. Therefore, we determined the ability of P-glycoprotein to transport endoxifen in vitro, using a polarized human P-glycoprotein-overexpressing cell line. Markedly higher transport of endoxifen was observed in the basal-to-apical direction, which was abrogated in the presence of the potent and specific P-glycoprotein inhibitor (2R)-anti-5-{3-[4-(10,11-difluoromethanodibenzo-suber-5-yl)piperazin-1-yl]-2-hydroxypropoxy}quinoline trihydrochloride (LY335979). To validate the in vivo relevance of P-glycoprotein to endoxifen disposition, plasma and tissue concentrations were also determined in Mdr1a-deficient mice after oral administration of endoxifen. Plasma endoxifen levels did not significantly differ between wild-type and Mdr1a-deficient mice. However, brain concentrations of endoxifen were nearly 20-fold higher in Mdr1a-deficient mice compared to wild-type mice. Because P-glycoprotein is highly expressed at the blood-brain barrier and in some breast cancer tumors, variation in expression and function of this transporter may alter central nervous system entry and the attained intracellular concentration in such breast cancer cells and therefore may prove to be of relevance to therapeutic outcome.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Antineoplásicos Hormonales/metabolismo , Moduladores de los Receptores de Estrógeno/farmacocinética , Profármacos/metabolismo , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Transporte Biológico Activo/efectos de los fármacos , Encéfalo/metabolismo , Línea Celular , Polaridad Celular , Dibenzocicloheptenos/farmacología , Moduladores de los Receptores de Estrógeno/sangre , Moduladores de los Receptores de Estrógeno/metabolismo , Humanos , Masculino , Moduladores del Transporte de Membrana/farmacología , Ratones , Ratones Transgénicos , Quinolinas/farmacología , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Sus scrofa , Tamoxifeno/sangre , Tamoxifeno/farmacocinética , Distribución TisularAsunto(s)
Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacocinética , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Rosuvastatina Cálcica/farmacocinética , Simportadores/metabolismo , Animales , Células HeLa , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/administración & dosificación , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Modelos Animales , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Rosuvastatina Cálcica/administración & dosificación , Simportadores/genética , Distribución TisularRESUMEN
Rivaroxaban is a novel factor 10a inhibitor, where hepatic metabolism and renal clearance account for its overall disposition. Renal impairment is known to increase rivaroxaban-associated bleeding risk in patients. As renal rivaroxaban clearance exceeds glomerular filtration rate, we suggested that active secretion by efflux transporters P-glycoprotein (MDR1) and breast cancer resistance protein (BCRP) contributes to rivaroxaban clearance. The ability of MDR1 and BCRP efflux transporters to mediate rivaroxaban transport in vitro was assessed in polarized cell monolayers. A significantly greater vectorial transport of rivaroxaban was observed in the basal to apical direction in Caco-2 cells, which was attenuated in the presence of the selective inhibitors. After oral administration of rivaroxaban (2 mg/kg), plasma concentrations did not significantly differ between wild-type and Mdr1a(def) or Bcrp(-/-) mice (n = 6 per group). However, rivaroxaban clearance was significantly reduced in Mdr1a/Mdr1b(-/-)/Bcrp(-/-) mice. Interestingly, rivaroxaban brain-to-plasma ratio did not differ in mice lacking only Mdr1a or Bcrp, but more than two times higher in the Mdr1a/Mdr1b(-/-)/Bcrp(-/-) mice. Rivaroxaban is a shared substrate of MDR1 and BCRP. In vivo, MDR and BCRP function synergistically to modulate rivaroxaban disposition and appear to be particularly relevant to limiting its central nervous system entry. These data have important implications for safety and efficacy of anticoagulation therapy with rivaroxaban as many drugs in clinical use are known MDR1 inhibitors and loss-of-function polymorphisms in BCRP are common.