Metabolite Kinetics: The Segregated Flow Model for Intestinal and Whole Body Physiologically Based Pharmacokinetic Modeling to Describe Intestinal and Hepatic Glucuronidation of Morphine in Rats In Vivo

ABSTRACT We used the intestinal segregated flow model (SFM) versus the traditional model (TM), nested within physiologically based pharmacokinetic (PBPK) models, to describe the biliary and urinary excretion of morphine 3b-glucuronide (MG) after intravenous and intraduodenal dosing of morphine in rats in vivo. The SFM model describes a partial (5%-30%) intestinal blood flow perfusing the transporter-and enzyme-rich enterocyte region, whereas the TM describes 100% flow perfusing the intestine as a whole. For the SFM, drugs entering from the circulation are expected to be metabolized to lesser extents by the intestine due to the segregated flow, reflecting the phenomenon of shunting and route-dependent intestinal metabolism. The poor permeability of MG crossing the liver or intestinal basolateral membranes mandates that most of MG that is excreted into bile is hepatically formed, whereas MG that is excreted into urine originates from both intestine and liver metabolism, since MG is effluxed back to blood. was better predicted by the SFM-PBPK (2.59 at 4 hours) and not the TM-PBPK (1.0), supporting the view that the SFM is superior for the description of intestinal-liver metabolism of morphine to MG. The SFM-PBPK model predicts an appreciable contribution of the intestine to first pass M metabolism

Disrupted murine gut-to-human liver signaling alters bile acid homeostasis in humanized mouse liver models

The humanized liver mouse model is being exploited increasingly for human drug metabolism studies. However, its model stability, intercommunication between human hepatocytes and mouse nonparenchymal cells in liver and murine intestine, and changes in extrahepatic transporter and enzyme expressions have not been investigated. We examined these issues in FRGN [fumarylacetoacetate hydrolase (Fah2/2), recombination activating gene 2 (Rag22/2), and interleukin 2 receptor subunit gamma (IL-2rg 2/2) triple knockout] on nonobese diabetic (NOD) background] and chimeric mice: mFRGN and hFRGN (repopulated withmouse or human hepatocytes, respectively). hFRGN mice showed markedly higher levels of liver cholesterol, biliary bilirubin, and bile acids (liver, bile, and plasma; mainly human forms, but also murine bile acids) but lower transforming growth factor beta receptor 2 (TGFBR2) mRNA expression levels (10%) in human hepatocytes and other proliferative markers in mouse nonparenchymal cells (Tgf-1) and cholangiocytes [plasma membrane-bound, G protein-coupled receptor for bile acids (Tgr5)], suggestive of irregular regeneration processes in hFRGN livers. Changes in gene expression in murine intestine, kidney, and brain of hFRGN mice, in particular, induction of intestinal farnesoid X receptor (Fxr) genes: fibroblast growth factor 15 (Fgf15), mouse ileal bile acid binding protein (Ibabp), small heterodimer partner (Shp), and the organic solute transporter alpha (Osta), were observed. Proteomics revealed persistence of remnant murine proteins (cyotchrome P450 7α-hydroxylase (Cyp7a1) and other enzymes and transporters) in hFRGN livers and suggest the likelihood ofmouse activity.When comparedwith normal human liver tissue, hFRGN livers showed lower SHP mRNA and higher CYP7A1 (300%) protein expression, consequences of tb- and ta-muricholic acid-mediated inhibition of the FXR-SHP cascade and miscommunication between intestinal Fgf15 and human liver fibroblast growth factor receptor 4 (FGFR4), as confirmed by the unchanged hepatic pERK/total ERK ratio. Dysregulation of hepatocyte proliferation and bile acid homeostasis in hFRGN livers led to hepatotoxicity, gallbladder distension, liver deformity, and other extrahepatic changes, making questionable the use of the preparation for drug metabolism studies

Up-regulation of transporters and enzymes by the vitamin D receptor ligands

ABSTRACT The effects of 1␣,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] on gene expression and function were studied in Caco-2 cells. Microarray analyses, real-time quantitative polymerase chain reactions, and Western blotting were used to determine the mRNA and protein expression of transporters and enzymes after 1,25(OH) 2 D 3 or vehicle (0.1% ethanol) treatment for 1, 3, 6, and 10 days. The mRNA and protein expressions of the apical sodium-dependent bile acid transporter, oligopeptide transporter 1, multidrug resistance-associated protein (MRP) 3, and sulfotransferase 1E1 remained unchanged with 1,25(OH) 2 D 3 treatment, whereas those for CYP3A4, multidrug resistance protein 1, and MRP2 were significantly increased (P Ͻ 0.05). 1,25(OH) 2 D 3 treatment significantly enhanced MRP4 protein expression by increasing protein stability without affecting mRNA expression, as confirmed in cycloheximide experiments. Marked increase in 6␤-hydroxylation of testosterone by CYP3A4 was also observed in the 6-day 1,25(OH) 2 D 3 -treated (100 nM) cell lysate. The transport of [ 3 H]digoxin, the P-glycoprotein (P-gp) substrate, after treatment with 100 nM 1,25(OH) 2 D 3 for 3 days revealed a higher apparent permeability (P app ) value in the basal (B)-to-apical (A) direction over that of vehicle treatment (15.1 Ϯ 0.53 ϫ 10 Ϫ6 versus 11.8 Ϯ 0.58 ϫ 10 Ϫ6 cm/s; P Ͻ 0.05), whereas the P app in the A-to-B direction was unchanged; the efflux ratio was increased (from 5.8 to 8.0). Reduced cellular retention of 5-(and-6)-carboxy-2Ј,7Ј-dichlorofluorescein, suggestive of higher MRP2 activity, was observed in the 3-day 100 nM 1,25(OH) 2 D 3 -treated cells over controls. Higher protein expression of CYP3A4, MRP2, P-gp, and MRP4 was also observed after a 6-day treatment with other vitamin D analogs (100 nM 1␣-hydroxyvitamin D 3 , 1␣-hydroxyvitamin D 2 or Hectorol, and 25-hydroxyvitamin D 3 ) in Caco-2 cells, suggesting a role of 1,25(OH) 2 D 3 and analogs in the activation of enzymes and transporters via the vitamin D receptor. The intestine plays an important role in the absorption of orally administered drugs. The expression and proximity of metabolic enzymes and efflux transporters in the enterocyte contribute to intestinal first-pass removal and delimit the tissue accumulation of endo-and xenobiotics. In the small intestine, cytochrome P450 3A4 (CYP3A4) accounts for approximately 70% of total cytochrome P450 content and is responsible for the metabolism of approximately 50% of drugs currently in us