RESUMEN
The cause of nonlinear pharmacokinetics (PK) (more than dose-proportional increase in exposure) of a urea derivative under development (compound A: anionic compound [pKa: 4.4]; LogP: 6.5; and plasma protein binding: 99.95%) observed in a clinical trial was investigated. Compound A was metabolized by CYP3A4, UGT1A1, and UGT1A3 with unbound Km of 3.3-17.8 µmol/L. OATP1B3-mediated uptake of compound A determined in the presence of human serum albumin (HSA) showed that unbound Km and Vmax decreased with increased HSA concentration. A greater decrease in unbound Km than in Vmax resulted in increased uptake clearance (Vmax/unbound Km) with increased HSA concentration, the so-called albumin-mediated uptake. At 2% HSA concentration, unbound Km was 0.00657 µmol/L. A physiologically based PK model assuming saturable hepatic uptake nearly replicated clinical PK of compound A. Unbound Km for hepatic uptake estimated from the model was 0.000767 µmol/L, lower than the in vitro unbound Km at 2% HSA concentration, whereas decreased Km with increased concentration of HSA in vitro indicated lower Km at physiological HSA concentration (4%-5%). In addition, unbound Km values for metabolizing enzymes were much higher than unbound Km for OATP1B3, indicating that the nonlinear PK of compound A is primarily attributed to saturated OATP1B3-mediated hepatic uptake of compound A.
Asunto(s)
Hígado/metabolismo , Albúmina Sérica Humana/metabolismo , Urea/análogos & derivados , Urea/farmacocinética , Adulto , Disponibilidad Biológica , Transporte Biológico , Simulación por Computador , Citocromo P-450 CYP3A/metabolismo , Femenino , Glucuronosiltransferasa/metabolismo , Humanos , Masculino , Modelos Biológicos , Unión Proteica , Urea/metabolismoRESUMEN
The expression of C/EBPalpha, which may govern transcription of mature hepatocyte marker genes, was suppressed in periportal hepatoblasts in mouse liver development, leading to biliary cell differentiation. This study was undertaken to analyze how inactivation of the Cebpa gene affects biliary cell differentiation and gene expression of the regulatory genes for that differentiation, including Hnf1b and Hnf6. In the knockout mouse liver at midgestation stages, pseudoglandular structures were abundantly induced in the parenchyma with elevated expression of Hnf6 and Hnf1b mRNAs. The wild-type liver parenchyma expressed mRNAs of these transcription factors at low levels, though periportal biliary progenitors had strong expression of them. These results suggest that expression of Hnf6 and Hnf1b is downstream of C/EBPalpha action in fetal liver development, and that the suppression of C/EBPalpha expression in periportal hepatoblasts may lead to expression of Hnf6 and Hnf1b mRNAs. Immunohistochemical studies with biliary cell markers in knockout livers demonstrated that differentiated biliary epithelial cells were confined to around the portal veins. The suppression of C/EBPalpha expression may result in upregulation of Hnf6 and Hnf1b gene expression, but be insufficient for biliary cell differentiation. When liver fragments of Cebpa-knockout fetuses, in which hepatoblasts were contained as an endodermal component, were transplanted in the testis of Scid (Prkdc) male mice, almost all hepatoblasts gave rise to biliary epithelial cells. Wild-type hepatoblasts constructed mature hepatic tissue accompanied by biliary cell differentiation. These results also demonstrate that the suppression of C/EBPalpha expression may stimulate biliary cell differentiation.
Asunto(s)
Conductos Biliares/citología , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Factor Nuclear 1-beta del Hepatocito/metabolismo , Factor Nuclear 6 del Hepatocito/metabolismo , Hepatocitos/fisiología , Animales , Conductos Biliares/crecimiento & desarrollo , Biomarcadores/metabolismo , Proteína alfa Potenciadora de Unión a CCAAT/genética , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Moléculas de Adhesión Celular/metabolismo , Trasplante de Células , Factor Nuclear 1-alfa del Hepatocito/genética , Factor Nuclear 1-beta del Hepatocito/genética , Factor Nuclear 6 del Hepatocito/genética , Hepatocitos/citología , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Hígado/citología , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Ratones SCID , ARN Mensajero/metabolismo , Receptor Notch2/genética , Receptor Notch2/metabolismo , Proteínas Serrate-Jagged , Testículo/citología , Testículo/embriología , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
BACKGROUND/AIMS: Intrahepatic biliary cell differentiation takes place in periportal hepatoblasts under the influence of the subjacent mesenchyme, which leads to the suppression of mature hepatocyte marker expression. This study was undertaken to analyze C/EBP alpha and beta expression, which may govern transcription of mature hepatocyte marker genes, during mouse liver development with special attention given to biliary differentiation. METHODS: Expression of C/EBP alpha and beta was immunohistochemically examined. Expression of alpha-fetoprotein, albumin and urea cycle enzymes, the genes of which have CCAAT motifs in their upstream regulatory sequences, was examined immunohistochemically or by using in situ hybridization. RESULTS: C/EBP alpha started to be expressed in endodermal cells of 9.5-day liver primordium, and continued to be expressed in hepatoblasts and hepatocytes throughout development. Although biliary cell progenitors transiently expressed mature hepatocyte markers, their expression of C/EBP alpha was weak or totally absent. The signals of C/EBP beta in hepatocytes were weak in fetal liver, but became stronger with postnatal development. Differentiated epithelial cells of intrahepatic biliary structures did not express C/EBP alpha. CONCLUSIONS: These data suggest that the suppression of C/EBP alpha expression may be prerequisite to biliary cell differentiation in the hepatoblast population and one of its earliest signs.
