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 Km values for uptake were 180 ± 112 and 390 ± 406 nM, Vmax values were 13 ± 8 and 18 ± 4.9 pmol/min/mg protein, and nonsaturable components were 9.2 ± 1.3 and 10.7 ± 2.5 μl/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 ± 0.082 ml/min/g) for KHB-perfused livers over the rbc-albumin-perfused livers (0.114 ± 0.057 ml/min/g) (P < 0.05). We further found that binding of digoxin to 1% BSA was modest (unbound fraction = 0.64), whereas binding to rbc was associated with slow on (0.468 ± 0.021 min-1) and off (1.81 ± 0.12 min-1) 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.
Footnotes
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This work was supported by Grants MOP36457 and MOP65417 (to K.S.P.) from the Medical Research Council of Canada and Grants DK23026 (to A.W.W.) and CA06576 (to P.M.N.) from the National Institutes of Health.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.105.088039.
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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; A2, Dg3 amount in peripheral compartment; Abile and Amet, amounts of Dg3 excreted into bile and metabolized, respectively; C1, Dg3 concentration in central compartment (compartmental modeling); CR (or Dg3R), Dg3 concentration in the reservoir; Crbc,R (or Dg3rbc,R), Dg3 concentration in rbc in the reservoir; Cp,R (or Dg3p,R), Dg3 concentration in plasma, in the reservoir; Crbci (or Dg3rbci), Dg3 concentration in red blood cell in sinusoid in ith zone (1, 2, or 3); Cpi (or Dg3pi), Dg3 concentration in sinusoidal plasma in ith zone (1, 2, or 3); CLi (or Dg3Li), Dg3 concentration in liver tissue in ith zone (1, 2, or 3); CLliver,tot, total hepatic clearance; CLliver,ex and CLliver,met, biliary clearance and hepatic metabolic clearance, respectively; CLini and CLefi, basolateral influx and efflux clearance of the ith zone (1, 2, or 3), respectively; CLint,seci and CLint,meti, secretory intrinsic clearance and metabolic intrinsic clearance of the ith zone (1, 2, or 3), respectively; MSC, model selection criterion; CLuptake, first-order hepatocyte uptake clearance; Dg2Li, “effective” total Dg2 concentration formed in liver tissue in ith zone (1, 2, or 3); E, hepatic extraction ratio; fp, fb, frbc, and ft, unbound fractions of Dg3 in plasma, blood, red blood cells, and liver tissue, respectively; alb, albumin; k10 and k20, elimination rate constants from central compartment and from peripheral compartment, respectively; k12 and k21, intercompartment rate constants between compartment 1 and compartment 2; ke and km, rate constants for biliary excretion and metabolism formation from central compartment or from peripheral compartment, respectively; kpr and krp, exchange rate constants from plasma to rbc and from rbc to plasma, respectively, whereby
equals the product frbckrp; Km,i, Michaelis-Menten constant for the ith transporter system; PSuptake, nonsaturable uptake clearance at sinusoidal membrane; Q and Qbile, total hepatic blood flow rate and bile flow rate, respectively; V1, volume of central compartment (compartment modeling); VR, VS, or VL, volume of reservoir, sinusoid, or liver tissue, respectively; Vmax,i, maximum velocity of the ith transporter system. -
↵1 Current address: Division of Clinical Pharmacology, Vanderbilt University, Nashville, TN.
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↵2 Current address: Drug Metabolism, Merck, Rahway, NJ.
- Received April 15, 2005.
- Accepted June 23, 2005.
- The American Society for Pharmacology and Experimental Therapeutics
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