Vascular Binding, Blood Flow, Transporter, and Enzyme Interactions on the Processing of Digoxin in Rat Liver

Abstract

The roles of vascular binding, flow, transporters, and enzymes as determinants of the clearance of digoxin were examined in the rat liver. Digoxin is metabolized by Cyp3a and utilizes the organic anion transporting polypeptide 2 (Oatp2) and P-glycoprotein (Pgp) for influx and excretion, respectively. Uptake of digoxin was found to be similar among rat periportal (PP) and perivenous (PV) hepatocytes isolated by the digitonin-collagenase method. The K m values for uptake were 180 Ϯ 112 and 390 Ϯ 406 nM, V max values were 13 Ϯ 8 and 18 Ϯ 4.9 pmol/min/mg protein, and nonsaturable components were 9.2 Ϯ 1.3 and 10.7 Ϯ 2.5 l/min/mg for PP and PV, respectively. The evenness of distribution of Oatp2 and Pgp was confirmed by Western blotting and confocal immunofluorescent microscopy. When digoxin was recirculated to the rat liver preparation in Krebs-Henseleit bicarbonate (KHB) for 3 h in absence or presence of 1% bovine serum albumin (BSA) and 20% red blood cell (rbc) at flow rates of 40 and 10 ml/min, respectively, biexponential decays were observed. Fitted results based on compartmental analyses revealed a higher clearance (0.244 Ϯ 0.082 ml/min/g) for KHB-perfused livers over the rbc-albuminperfused livers (0.114 Ϯ 0.057 ml/min/g) (P Ͻ 0.05). We further found that binding of digoxin to 1% BSA was modest (unbound fraction ϭ 0.64), whereas binding to rbc was associated with slow on (0.468 Ϯ 0.021 min Ϫ1) and off (1.81 Ϯ 0.12 min Ϫ1 ) rate constants. We then used a zonal, physiologically based pharmacokinetic model to show that the difference in digoxin clearance was attributed to binding to BSA and rbc and not to the difference in flow rate and that clearance was unaffected by transporter or enzyme heterogeneity.Digoxin, an important cardiotonic drug of narrow therapeutic index, is cleared by both the kidney and liver, and exhibits a long half-life in vivo due to the large volume of distribution (Kramer et al., 1974;Rodin and Johnson, 1988). In the rat liver, digoxin is primarily metabolized by cytochrome P450 3a to the didigitoxoside and monodigitoxoside as well as digitoxigenin (Harrison and Gibaldi, 1976; Sal- ABBREVIATIONS:Pgp, P-glycoprotein; Oatp, rat organic anion transporting polypeptide; OAT, organic anion transporter; PP, periportal; PV, perivenous; rbc, red blood cell; KHB, Krebs-Henseleit bicarbonate buffer; BSA, bovine serum albumin; PBPK, physiologically based pharmacokinetic; Dg3, unlabeled digoxin; Dg2, digitoxigenin bis-digitoxoside; Dg1, digitoxigenin monodigitoxoside; Dg0, digitoxigenin; HPLC, high-performance liquid chromatography; HRP, horseradish peroxidase; Hct, hematocrit; AUC, area under the curve; A 2 , Dg3 amount in peripheral compartment; A bile and A met , amounts of Dg3 excreted into bile and metabolized, respectively; C 1 , Dg3 concentration in central compartment (compartmental modeling); C R (or Dg3 R ), Dg3 concentration in the reservoir; C rbc,R (or Dg3 rbc,R ), Dg3 concentration in rbc in the reservoir; C p,R (or Dg3 p,R ), Dg3 concentration in plasma, in the reservoir; C rbci...

Culture Period-Dependent Changes in the Uptake of Transporter Substrates in Sandwich-Cultured Rat and Human Hepatocytes

“…d Data from Harrison and Gibaldi (1976). e Data from Liu et al (2005). f Data from Watanabe et al (2010).…”

P-Glycoprotein and an Unstirred Water Layer Barring Digoxin Absorption in the Vascularly Perfused Rat Small Intestine Preparation: Induction Studies with Pregnenolone-16α-carbonitrile

“…These developed TMs and SFMs are further expanded to include a bilayer membrane compartment that houses the efflux transporter, Pgp, adjacent to the apical membrane, and an unstirred water layer (UWL) compartment external to the apical membrane that bars the absorption of this lipophilic drug. The slow partitioning of digoxin between the plasma and red blood cells in the vascular compartments is also considered (Liu et al, 2005). The data suggest that intestinal efflux is a major mechanism for digoxin elimination, and digoxin bioavailability and efflux were subject to changes with a Pgp inducer such as PCN.…”

“…Two previously developed, physiologically based pharmacokinetic models for the intestine, the traditional model (TM) and the segregated flow model (SFM) (Cong et al, 2000), were expanded to include the partitioning of Dg3 in red blood cells (rbc), plasma protein, and intestinal tissue binding, as we did for digoxin handling by the liver (Liu et al, 2005). A separate bilayer membrane that housed the Pgp adjacent to the apical membrane and an unstirred water layer (UWL) external to the apical membrane that may retard the diffusion of digoxin were included as additional models to fit the data of digoxin (Fig.…”

“…Digoxin (Dg3) and its metabolites, digoxigenin bis-digitoxoside (Dg2), digoxigenin mono-digitoxoside (Dg1), and digoxigenin (Dg0), were quantified by the modified HPLC procedure (Liu et al, 2005). Briefly, a Shimadzu HPLC system consisting of a C18 reverse-phase column (Alltech Associates, Deerfield, IL; 4.6 ϫ 250 mm, particle size, 10 m) and guard column (Waters Bondapak C18/Corasil 37-55 m) was used for the separation of digoxin and its metabolites at 220 nm.…”

Characterization of Digoxin Uptake in Sandwich-Cultured Human Hepatocytes

“…In rats, hepatic disposition of digoxin has been fairly well characterized. Digoxin has been shown to be a substrate of rat Oatp1a4 (Noé at al., 1997;Kodawara et al, 2002) and mouse Oatp1a4 (Ose et al, 2010), and hepatocyte uptake K m values range from 0.18 to 36 M (Hedman and Meijer, 1998;Lam and Benet, 2004;Liu et al, 2005). In human hepatocytes, digoxin uptake is decreased by 95% at 4°C (Olinga et al, 1998).…”