Several organ systems are involved in the elimination of xenobiotics, including the kidney, intestine, and liver. Common to these organs is the polarized nature of the cells that mediate vectorial transport of endogenous and exogenous compounds. In the liver, P-gp is localized to the bile canalicular membrane of the hepatocyte where several other transport proteins, including the multidrug resistance-associated protein 2 (Mrp2), reside (Thiebaut et al., 1987; Buchler et al., 1996). These canalicular proteins mediate active transport of many substrates from the liver into bile for excretion from the body. Coupled with active transport mechanisms at the basolateral membrane of the hepatocyte, the overall process of hepatobiliary transport is complex, potentially consisting of multiple parallel, sequential, and/or opposing transport pathways.

The concept of multiplicity of drug transport mechanisms responsible for the vectorial movement of drugs into and out of the liver has gained considerable attention; the identification of these processes has been a major research focus over the past decade (Sathirakul et al., 1994; Yamazaki et al., 1996; Zamek-Gliszczynski et al., 2003). Recent advances in molecular biology have enhanced the ability to clone and express transport proteins from multiple species into a number of cellular-based systems (Cvetkovic et al., 1999; Sasaki et al., 2002; Xiong et al., 2002). These systems permit direct identification of substrates for individual transport proteins and represent valuable tools for characterization and evaluation of transport mechanisms that may be important in vivo. However, the ability to extrapolate these observations to the whole organ, especially when compounds are substrates for multiple transporters, is challenging. Furthermore, the attempt to predict the in vivo pharmacokinetic behavior of a compound from expression systems may be even more difficult.

One approach to understanding the impact of individual drug transport pathways on the pharmacokinetics of compounds has been to use chemical inhibition of specific transport proteins in complex systems such as the intact organ or the whole animal. For example, probenecid was shown to inhibit the biliary excretion of the organic anion 5-(and 6)-carboxy-2′7′-dichlorofluorescein (Zamek-Gliszczynski et al., 2003) and the anticancer agent methotrexate (Ueda et al., 2001), implicating the involvement of organic anion transport pathways for these compounds. These studies illustrate clearly the pharmacokinetic consequences of transport inhibition for a given substrate. However, inhibitors often interact with multiple transport pathways, as is the case for probenecid (Bakos et al., 2000; Sugiyama et al., 2001), which may limit or confound data interpretation. Moreover, inhibition may not be competitive, or the specificity of inhibitors may not be understood completely (Kouzuki et al., 2000). Nevertheless, a few inhibitors have been considered selective and have been used to inhibit isolated transport mechanisms. An example is the potent acridonecarboxamide GF120918, which has been shown to inhibit P-gp (Hyafil et al., 1993), and more recently Bcrp (Allen et al., 1999), both at nanomolar potencies. As a selective inhibitor, GF120918 is useful in determining the impact of P-gp and/or Bcrp in the disposition of some compounds (Allen et al., 2003).

P-gp typically transports lipophilic cationic compounds (Horio et al., 1989), whereas substrates of Mrp2 are usually anionic and often possess multiple negative charges (Keppler et al., 1997). However, due to the zwitterionic nature of many peptides, the classification of transport protein specificity based solely upon chemical structure is difficult for these compounds. For example, the cyclooctapeptide somatostatin analog octreotide is transported primarily by P-gp (Yamada et al., 1998), whereas the cyclopentapeptide endothelin antagonist BQ-123 demonstrates specificity for Mrp2 (Shin et al., 1997). Several prodrugs of the cyclic opioid peptide DADLE (H-Tyr-d-Ala-Gly-Phe-d-Leu-OH) have been synthesized; the acyloxyalkoxy-based derivatives show preference for P-gp-mediated transport, whereas the coumarinic acid derivatives have affinity for both Mrp2 and P-gp (Tang and Borchardt, 2002a,b).

The metabolically stable cyclopentapeptide DPDPE is excreted rapidly and extensively into the bile of rats as unchanged peptide (Chen and Pollack, 1997). Although DPDPE is transported by P-gp, the absence of functional P-gp due to genetic manipulation or inhibition with GF120918 does not seem to alter the systemic disposition of [³H]DPDPE in vivo (Chen and Pollack, 1998, 1999). The in vitro biliary clearance of [³H]DPDPE in polarized sandwich-cultured rat hepatocytes from Mrp2-deficient (TR^-) rats was impaired significantly relative to hepatocytes obtained from Wistar control animals (1.48 ± 0.36 versus 0.23 ± 0.17 μl/min/mg protein in control versus TR^-, respectively), indicating that Mrp2 plays some role in the biliary excretion of the opioid peptide (Hoffmaster et al., 2005). However, the contribution of Mrp2 to the overall hepatic disposition of DPDPE is unknown.

The purpose of this study was to examine the mechanisms of [³H]DPDPE hepatobiliary transport and to determine the contributions of these pathways to the disposition of [³H]DPDPE in the intact liver. These studies used isolated perfused livers (IPLs) from Wistar control and Mrp2-deficient (TR^-) rats in the presence or absence of GF120918 to identify the primary mechanisms of [³H]DPDPE biliary excretion and to determine the individual contributions of P-gp and Mrp2 to the canalicular transport of [³H]DPDPE. Stepwise pharmacokinetic modeling was applied to gain insight into the complex process of [³H]DPDPE biliary excretion and to assess the interplay between multiple transport systems in determining the hepatobiliary disposition of this opioid peptide.

Materials and Methods

Chemicals and Reagents. Unlabeled DPDPE was obtained from American Peptide Company (Sunnyvale, CA); [³H]DPDPE (44 Ci/mmol, >95% purity) was from PerkinElmer Life and Analytical Sciences (Boston, MA). Sodium taurocholate was purchased from Sigma-Aldrich (St. Louis, MO). GF120918 [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide] was a generous gift from GlaxoSmithKline (Research Triangle Park, NC). All other chemicals and reagents were of analytical grade and were available from commercial sources.

Animals. Male Wistar rats (250–300 g) from Charles River Laboratories, Inc. (Raleigh, NC), or male TR^- rats bred in our facility (275–300 g; breeding stock obtained from Dr. Mary Vore, University of Kentucky, Lexington, KY), were used as liver donors for perfused liver studies. Retired male Wistar breeder rats (400–500 g; Charles River Laboratories, Inc.) were used as blood donors. Rats were housed in an alternating 12-h light/dark cycle with rat chow and water provided ad libitum. All animals were allowed to acclimate for at least 1 week before experimentation. Rats were anesthetized with ketamine/xylazine (60:12 mg/kg i.p.). The Institutional Animal Care and Use Committee at the University of North Carolina approved all animal procedures.

IPL Studies. Recirculating IPL studies were conducted with control and TR^- rat livers as described previously (Brouwer and Thurman, 1996). Briefly, after portal vein and bile duct cannulation, livers were perfused in situ with oxygenated Krebs-Henseleit bicarbonate buffer (pH 7.4). Livers were removed from the body cavity and placed into a humidified perfusion chamber heated to maintain liver temperature at 37°C. Perfusion was continued with oxygenated buffer containing 20% (v/v) heparinized male rat blood at a flow rate of 20 ml/min. Livers were allowed to acclimate for 10 min before [³H]DPDPE infusion. Liver viability was assessed by monitoring portal pressure (<15 cm of H₂O), observing gross morphology, and measuring initial bile flow in control and TR^- IPLs (>0.8 and >0.3 μl/min/g liver, respectively). Taurocholate (0.5 μmol/min, in saline) was infused into the perfusate reservoir to maintain bile flow. [³H]DPDPE was infused (40 nmol/min; 4 μCi) over 30 min. For P-gp inhibition studies, GF120918 (480 μg in DMSO) was administered as a bolus 5 min before the start of [³H]DPDPE infusion. Previous studies in isolated hepatocytes demonstrated that GF120918 achieved distributional equilibrium within 5 min (Booth et al., 1998). In agreement with prior studies in IPLs, DMSO (0.3%) or GF120918 did not affect liver viability (Booth et al., 1998; Zamek-Gliszczynski et al., 2003). Bile was collected in toto every 10 min, and the volume was determined gravimetrically (specific gravity 1.0). Perfusate (0.5 ml) was sampled every 10 min and immediately centrifuged to obtain supernatant for analysis. After perfusion, livers were blotted dry and weighed. To avoid photodegradation of GF120918, liver perfusions were conducted under minimal lighting, and all samples were protected from light during experimentation, collection, and storage at -80°C until analysis.

Suspended Rat Hepatocyte Isolation/Uptake Studies. Rat hepatocyte isolation and uptake studies were conducted as described previously (Hoffmaster et al., 2005). Briefly, isolated rat hepatocytes were preincubated for 5 min at 37°C before addition of [³H]DPDPE and GF120918 or vehicle (0.1% DMSO). GF120918 (1 μM) was coincubated with [³H]DPDPE at a concentration ∼50-fold higher than the reported K_i for P-gp inhibition. Aliquots of the suspension were removed every 15 s through 1 min and centrifuged immediately through a mixture of silicone and mineral oil (ρ = 1.03). [³H]DPDPE was determined in the cell pellet and the supernatant; uptake data were normalized for protein concentrations in individual hepatocyte suspensions. Where appropriate, data in suspended hepatocytes were normalized per gram of liver using 1.1 × 10⁶ cells/mg protein and 1.3 × 10⁸ cells/g liver (Seglen, 1973).

Data Analysis. [³H]DPDPE was analyzed by liquid scintillation spectroscopy. Residual [³H]DPDPE in the liver was obtained by mass balance, and [³H]DPDPE concentrations in the liver were calculated assuming cytosolic distribution of the peptide and a cytosolic volume of 0.7 ml/g liver (Tsuji et al., 1983). Area under the [³H]DPDPE concentration-time curve in perfusate (AUC_perfusate) and liver (AUC_liver) were calculated by the linear trapezoidal method. Preliminary experiments in control livers indicated steady state was achieved by ∼20 min; perfusate clearance was determined as where k₀ is the infusion rate and C_ss is the [³H]DPDPE concentration at steady-state as determined at the end of the infusion. Uptake clearance (Cl_uptake) in the IPL was determined as where k_uptake and V_perfusate are the first-order rate constant for uptake and volume of the perfusate compartment, respectively, recovered from nonlinear least-squares regression analysis of the data. Biliary clearance was determined based upon the perfusate and liver AUC according to the following equations: where X_bile is the total amount of [³H]DPDPE excreted into the bile through T_last. All data are presented as mean ± S.D. (n = 3–4 livers/condition). Statistically significant differences were determined by Student's t test with Bonferroni's correction for multiple comparisons, or two-way ANOVA with Tukey's post hoc test, where appropriate (^*p < 0.05).

Pharmacokinetic Modeling. A compartmental modeling approach was used to examine the hepatobiliary disposition of [³H]DPDPE and to determine the effect of GF120918 in IPLs from control and TR^- rats. Due to the physical dimensions of the bile duct cannula coupled with the differences in bile flow rates between control and TR^- IPLs (∼1 versus ∼0.4 μl/min/g liver, respectively), biliary excretion of [³H]DPDPE in TR^- rat livers was analyzed initially to estimate a lag time for measurement of [³H]DPDPE at the end of the bile cannula. All subsequent modeling exercises corrected the TR^- biliary excretion rate data for the calculated lag time (5 min). Several models incorporating both linear and nonlinear processes were fit simultaneously to the [³H]DPDPE perfusate concentration versus time, and biliary excretion rate versus time, data from control rat livers to establish appropriate model structure. Differential equations based on the concentration of [³H]DPDPE in the perfusate, amount of [³H]DPDPE in the liver, and the amount of [³H]DPDPE the bile per unit time were resolved simultaneously by nonlinear least-squares regression with 1/Y weighting and the Gauss-Newton (Levenberg and Hartley) minimization method based upon the model structure shown in Fig. 4 (WinNonlin version 4.1; Pharsight, Mountain View, CA). Goodness of fit for each model was evaluated by visual examination of the distribution of residuals, rank and condition number of the matrix of partial derivatives, and Akaike's Information Criterion. Equations based on the model structure in Fig. 4 are as follows: where variables and parameters are defined as in Fig. 4. Stepwise nonlinear least-squares regression was used subsequently to optimize the precision of parameter estimates in all data sets.

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Fig. 4.

Model scheme depicting the disposition of [³H]DPDPE in IPLs from control and TR^- rats in the presence or absence of GF120918. C_perfusate, concentration of [³H]DPDPE in perfusate; V_perfusate, perfusate volume; X_liver, amount of [³H]DPDPE in the liver; X_bile, amount of [³H]DPDPE in the bile; k_uptake, first-order rate constant for uptake into the liver; K_m,blefflux, Michaelis-Menten constant for basolateral excretion; V_m,blefflux, maximal velocity for basolateral excretion; k_bile, first-order rate constant for biliary excretion; k₀, [³H]DPDPE infusion rate into the perfusate reservoir.

Results

[³H]DPDPE Disposition in IPLs. [³H]DPDPE biliary excretion in IPLs from control rats was rapid and extensive. By the end of the 30-min [³H]DPDPE infusion, more than 50% of the dose was excreted into the bile of livers from control rats (Figs. 1 and 2). However, in livers from TR^- rats, the biliary excretion rate was decreased significantly (Fig. 1); the net biliary excretion of [³H]DPDPE was decreased by ∼30% in TR^- rats. In the presence of GF120918, the biliary excretion rate of [³H]DPDPE was not decreased in livers from control rats; in fact, the biliary excretion rate was slightly increased at early time points (Fig. 1). The total mass of [³H]DPDPE excreted into bile in the presence of GF120918 in control rat livers was consistently higher than in the absence of inhibitor, but the slight increase in total [³H]DPDPE mass excreted due to the presence of GF120918 (1.26 ± 0.09 versus 1.11 ± 0.05 μmol) was not significantly different from controls (Table 1). GF120918 decreased significantly the biliary excretion rate in TR^- rat livers (Fig. 1); the total mass of [³H]DPDPE excreted into bile decreased to <10% of the dose (Fig. 2; Table 1). The decreased biliary excretion rate in TR^- relative to control IPLs was reflective of a decrease in biliary clearance of [³H]DPDPE (Table 1). When GF120918 was added to TR^- IPLs, Cl_liver→bile of [³H]DPDPE was negligible. Interestingly, Cl_{perfusate→bile} was increased significantly in livers from control rats with GF120918 (Table 1). In contrast, GF120918 had no effect on the Cl_liver→bile of [³H]DPDPE in livers from control rats (Table 1).

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Fig. 1.

[³H]DPDPE biliary excretion rate and perfusate concentration versus time data in IPLs from control (diamonds) and TR^- (squares) rats in the presence (open symbols) or absence (closed symbols) of GF120918. Data are presented as mean ± S.D. (n = 3–4 livers). Note that [³H]DPDPE concentrations in IPLs from control rats + GF120918 postinfusion (t ≥ 30 min) were below the limit of quantification.

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Fig. 2.

Cumulative biliary excretion of [³H]DPDPE (top) and amount of [³H]DPDPE in the liver (bottom) in IPLs from control (diamonds) and TR^- (squares) rats in the presence (open symbols) or absence (closed symbols) of GF120918. Data are presented as mean ± S.D. (n = 3–4 livers).

[³H]DPDPE perfusate concentrations from TR^- rat livers were higher compared with control rat livers (∼67% increase in AUC; Table 1). Although GF120918 had little impact on the biliary excretion of [³H]DPDPE in livers from control rats, the presence of GF120918 in the perfusate of control livers decreased [³H]DPDPE perfusate concentrations (Fig. 1). GF120918 also decreased [³H]DPDPE perfusate concentrations in TR^- rat livers (Fig. 1). In both control and TR^- IPLs, GF120918 caused an apparent increase in the perfusate clearance of [³H]DPDPE (Table 1).

[³H]DPDPE did not accumulate extensively in IPLs from control rats. GF120918 caused a slight increase in the initial [³H]DPDPE hepatic content in control rat livers (Fig. 2). [³H]DPDPE accumulated >2-fold in IPLs from TR^- rats relative to control; GF120918 further increased [³H]DPDPE hepatic content in livers from TR^- rats (Fig. 2). In TR^- IPLs in the presence of GF120918, >85% of the [³H]DPDPE dose accumulated in the liver by 30 min and remained in the liver through 90 min. Immediately postinfusion, and even at distribution equilibrium, [³H]DPDPE partitioning into the liver was higher in the presence of GF120918 in livers from both control and TR^- rats. [³H]DPDPE liver-to-perfusate ratios averaged over the postinfusion interval were significantly higher in the presence of GF120918 (Fig. 3).

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Fig. 3.

Effect of GF120918 (open columns) on the postinfusion (30–90 min) time-averaged [³H]DPDPE liver-to-perfusate concentration ratio in IPLs from control and TR^- rats. Data are presented as mean ± S.E.M. (n = 3–4 livers; *, p < 0.05 compared with corresponding control without GF120918).

Suspended Hepatocyte Uptake Studies. The uptake of [³H]DPDPE into suspended hepatocytes was rapid. GF120918 did not affect the initial uptake rate of [³H]DPDPE in suspended primary rat hepatocytes (15.2 ± 2.5 versus 14.9 ± 2.1 pmol/min/mg protein, in the absence or presence of GF120918, respectively).

Pharmacokinetic Modeling. Based upon experiments in suspended and sandwich-cultured hepatocytes (Hoffmaster et al., 2005), a compartmental model was used to evaluate the disposition of [³H]DPDPE in the IPL. The scheme shown in Fig. 4 best described the perfusate concentration and biliary excretion rate versus time data in control rat livers. The apparent volume of the perfusate compartment was estimated from preliminary kinetic modeling to be 110 ± 29 and 89.5 ± 5 ml in control and TR^- IPLs, respectively, similar to the actual volume of perfusate plus the mean residual volume in the liver sinusoids (∼88 ml). Therefore, V_p was fixed at the physiological volume of 88 ml for subsequent stepwise analyses. Consistent with observations in suspended hepatocytes (Hoffmaster et al., 2005), the rate constant describing uptake of [³H]DPDPE into the liver was not different between control and TR^- IPLs (0.14 ± 0.04 versus 0.11 ± 0.02 min^-1, respectively). The lack of effect of GF120918 on [³H]DPDPE uptake in the IPL was consistent with the data from suspended hepatocytes; preliminary modeling indicated the rate constant for [³H]DPDPE uptake in IPLs from control (0.14 ± 0.04 versus 0.18 ± 0.06 min^-1) or TR^- (0.11 ± 0.02 versus 0.17 ± 0.05 min^-1) rats in the absence or presence of GF120918 was not different. Therefore, the rate constant for [³H]DPDPE uptake was fixed at the mean value recovered from control and TR^- IPLs (0.12 min^-1) for subsequent parameter estimation. Final parameter estimates describing [³H]DPDPE disposition in IPLs from control and TR^- rats in the presence or absence of GF120918 are shown in Table 2; the correspondence of the model to the observed data is shown in Fig. 5. In the absence of Mrp2, the rate constant for [³H]DPDPE excretion into bile decreased ∼3 fold; addition of GF120918 to the perfusate of TR^- livers decreased the rate constant k_bile by an additional ∼10-fold (Table 2). Interestingly, in the presence of Mrp2, k_bile was similar between control livers in the absence or presence of GF120918 (Table 2). Based upon model selection criteria, a first-order rate constant better described the excretion of [³H]DPDPE from liver to bile. Basolateral efflux of [³H]DPDPE from the liver into the perfusate was best described as a nonlinear process with a K_m below the hepatic concentrations achieved in these studies. Therefore, the model treated basolateral excretion of [³H]DPDPE as a zero-order process, operating at the V_max for transport.

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Fig. 5.

Final fit of the pharmacokinetic model after stepwise nonlinear least-squares regression analysis of mean [³H]DPDPE data from control (top) and TR^- (bottom) IPLs, in the presence (open symbols, dashed line) or absence (closed symbols, solid line) of GF120918. Biliary excretion rates (control, circles; TR^-, triangles) and perfusate concentrations (control, diamonds; TR^-, squares) were modeled simultaneously for each data set. Data represent mean ± S.D. (n = 3–4 livers).

Discussion

Consistent with previous in vivo (Chen and Pollack, 1997) and in vitro (Hoffmaster et al., 2005) observations, [³H]DPDPE was taken up and excreted rapidly and extensively into bile in IPLs from control rats. Studies in TR^- sandwich-cultured rat hepatocytes suggested a role for Mrp2 in the biliary clearance of [³H]DPDPE (Hoffmaster et al., 2005). In the absence of functional Mrp2 in TR^- IPLs, biliary excretion was decreased significantly. However, net excretion of [³H]DPDPE was decreased only ∼30% in TR^- IPLs, indicating the presence of multiple pathways for [³H]DPDPE transport across the canalicular membrane. At the blood-brain barrier, P-gp has been shown to restrict significantly the penetration of [³H]DPDPE (Chen and Pollack, 1998). When the P-gp inhibitor GF120918 was coadministered to TR^- IPLs, biliary excretion of [³H]DPDPE was negligible, implicating P-gp as the residual biliary excretion mechanism. Based upon these observations and the fact that P-gp expression in TR^- livers is similar to control (Johnson et al., 2003), inhibition of P-gp in control livers should have decreased the biliary excretion of [³H]DPDPE ∼60% if the two excretion processes were additive. Surprisingly, P-gp inhibition in control IPLs did not decrease biliary excretion; in fact, excretion was enhanced slightly. Therefore, P-gp in the liver facilitates [³H]DPDPE biliary excretion only in the absence of functional Mrp2. Chen and Pollack noted that although GF120918 was able to significantly modulate blood-brain disposition of [³H]DPDPE in mice, no change in the systemic pharmacokinetics of [³H]DPDPE was observed (Chen and Pollack, 1999). Clearly, transport by Mrp2, and the inability of P-gp inhibition to affect the biliary excretion of [³H]DPDPE, explain these in vivo observations. Transport by P-gp when Mrp2 function is absent suggests that Mrp2 is more efficient than P-gp for DPDPE transport into the bile, which is the preferential route of hepatic elimination for DPDPE. The conjugated steroid estradiol-17β-glucuronide (E₂17G) also is transported by Mrp2 and P-gp; the predominant mechanism for biliary excretion of E₂17G in the intact organ is Mrp2 (Vore et al., 1996; Morikawa et al., 2000). Interestingly, in the presence of the P-gp inhibitor PSC833, biliary excretion of E₂17G in Mrp2-competent rats in vivo was enhanced slightly and plasma AUC was decreased, similar to the observations in the present study with [³H]DPDPE (Morikawa et al., 2000). However, the presence of PSC833 in Mrp2-deficient rats failed to inhibit the residual excretion of E₂17G into bile (Morikawa et al., 2000). In contrast, the residual mechanism of [³H]DPDPE excretion in Mrp2-deficient IPLs was significantly decreased by GF120918, suggesting that, unlike E₂17G in the intact organ, P-gp is capable of transporting [³H]DPDPE into the bile with moderate efficiency, albeit much less than Mrp2.

Compartmental modeling was performed to assess the pharmacokinetic consequences of multiple drug transport proteins involved in the canalicular excretion of [³H]DPDPE. A kinetic model was developed to describe the disposition of [³H]DPDPE in the IPL in the absence of GF120918. The uptake of [³H]DPDPE into hepatocytes is mediated by parallel linear and nonlinear processes (Hoffmaster et al., 2005). The dose of [³H]DPDPE used in the present study was chosen to achieve perfusate concentrations below the K_m value (28.9 μM) for the saturable process; kinetic modeling confirmed the first-order uptake of [³H]DPDPE. The lack of Mrp2 and the presence of GF120918 did not affect the uptake rate constant or the apparent volume of distribution for [³H]DPDPE in the perfusate compartment. The biliary excretion rate constant was decreased by >60% in livers from TR^- rats, consistent with the major role of Mrp2 in the biliary excretion of [³H]DPDPE. In TR^- IPLs in the presence of GF120918, the decreased rate constant for movement into bile suggested inhibition of a compensatory transport mechanism.

Kinetic modeling of [³H]DPDPE accumulation in sandwich-cultured hepatocytes supported the presence of a basolateral excretion pathway for this peptide (Hoffmaster et al., 2005). Pharmacokinetic modeling of [³H]DPDPE in control IPLs suggested the presence of an apparently saturable basolateral pathway. Although pharmacokinetic modeling was able to recover good estimates of V_m,blefflux for this process, estimates of K_m,blefflux were consistently below hepatic amounts achieved in these studies. Pharmacokinetic modeling indicated that V_m,blefflux was increased in TR^- IPLs, consistent with higher expression of a basolateral transport protein. Mrp3 has been shown to be up-regulated in liver from TR^- rats and may explain these observations (Xiong et al., 2002). Interestingly, basolateral transport also seems to be sensitive to inhibition by GF120918. In both control and TR^- IPLs, GF120918 actually increased [³H]DPDPE perfusate clearance. Since GF120918 inhibits an excretion pathway into bile, the increase in Cl_perfusate was not anticipated. However, liver/perfusate partitioning in the presence of GF120918 was higher in both control and TR^- IPLs, consistent with extensive hepatic uptake coupled with inhibition of basolateral efflux.

The utility of various in vitro models to predict the disposition of xenobiotics lies in the ability of the data from those systems to scale to the in vivo situation. When scaled per gram liver (Seglen, 1973) and compared across suspended hepatocytes, SC hepatocytes, and the IPL (average liver weight 10 g), the uptake clearance of [³H]DPDPE recovered from kinetic modeling (1.8 versus 1.3 versus 1.1 ml/min/g liver, respectively) is reproducible (present study; Hoffmaster et al., 2005). Since hepatobiliary excretion is the major route of clearance, when perfusate clearance in the IPL (7.34 ± 0.83 ml/min; Table 1) is expressed per kilogram of body weight (average body weight for control IPLs in these studies ∼285 g), the clearance (26 ± 3 ml/min/kg) is in good agreement with systemic clearance determined in vivo [23 ± 6 ml/min/kg; (Chen and Pollack, 1996)]. Moreover, when scaled to body weight, biliary clearance calculated based on AUC_perfusate in control IPLs (18 ± 1 ml/min/kg) correlates well with biliary clearance calculated based on AUC_0-Tlast in SC hepatocytes [12 ± 3 ml/min/kg; 0.2 g protein/g liver, 40 g liver/kg b.wt. (Seglen, 1976; Hoffmaster et al., 2005)].

Biliary or urinary clearance in vivo is typically calculated from plasma AUC and the amount excreted by the pathway of interest (e.g., Cl_biliary = X_bile/AUC_plasma). However, basolateral excretion mechanisms may confound accurate estimates of biliary clearance when clearance is based on perfusate AUC. This was evident in the determination of [³H]DPDPE biliary clearance in control IPLs in the presence of GF120918. Based upon AUC_perfusate, it seemed that biliary clearance was increased >3-fold. The interpretation becomes clear when clearance is calculated using AUC_liver, indicating that GF120918 did not affect the clearance of [³H]DPDPE from liver to bile in control IPLs. When biliary clearance is modulated by GF120918, as in the case of the TR^- IPLs, there still is an underestimate of the true impact of the inhibitor on the biliary clearance when using AUC_perfusate (GF120918 inhibited biliary clearance ∼2.7-fold); when AUC_liver was used, GF120918 decreased biliary clearance more than 10-fold.

The ability of GF120918 to inhibit a basolateral excretion mechanism has potential implications when interpreting pharmacokinetic data. One possible consequence is the misinterpretation of an in vivo drug interaction. An apparent increase in plasma clearance often is attributed to induction of a clearance mechanism such as excretion or metabolism (Dresser et al., 2003). Recently, GF120918 was shown to decrease the perfusate AUC of tacrolimus in the isolated perfused liver (Wu and Benet, 2003). Although not statistically significant, the perfusate AUC of a tacrolimus primary metabolite also was decreased ∼2-fold. The authors concluded that the decrease in AUC was due to GF120918 based inhibition of P-gp-mediated biliary excretion of tacrolimus and the primary metabolite and subsequent enhanced metabolism of both parent compound and the primary metabolite. An alternative explanation is that GF120918 inhibited the translocation of both tacrolimus and its metabolite across the basolateral membrane of the liver, thereby reducing the AUC in perfusate. Another implication of basolateral efflux inhibition is when this interaction is considered in conjunction with inhibition of other hepatic excretory processes. When GF120918 was administered to Mrp2-deficient IPLs, the combined inhibition of biliary and basolateral excretion resulted in a marked accumulation of [³H]DPDPE in the liver. At the termination of the experiment, almost the entire dose remained in the liver (an increase of 35-fold). These studies used a low dose of [³H]DPDPE; the highest accumulation of DPDPE in the liver was only 88 nmol/g liver (TR^- IPLs in the presence of GF120918). However, inhibition of multiple opposing excretion pathways from the liver combined with efficient rapid uptake of compounds into the liver, could result in acute liver toxicity, as well as reduced efficacy for some compounds at therapeutic doses. Moreover, with multiple dosing, substrate could continue to accumulate in the tissue at each dosing interval without a significant change in apparent systemic pharmacokinetics.

These data bring into question the so-called “specificity” of transport inhibitors. GF120918 interacts with at least three transport mechanisms in the liver; two excretion mechanisms at the canalicular membrane, P-gp and Bcrp (Hyafil et al., 1993; Allen et al., 1999), and an efflux mechanism at the basolateral membrane (this study). Interestingly, subsequent investigation of [³H]DPDPE biliary excretion in TR^- IPLs in the presence of a potent, specific P-gp inhibitor (that does not interact with Bcrp) resulted in similar total mass of [³H]DPDPE excreted into bile, suggesting that Bcrp plays a very minor role, if any, in the hepatic transport of the peptide (our unpublished observations). Nevertheless, as more information becomes available regarding the complexities of hepatic transport, inhibitors that once were considered to interact specifically with a discrete process may be found to interact with multiple processes affecting the disposition of a substrate in the liver.

In conclusion, [³H]DPDPE is excreted into bile primarily by Mrp2. Interestingly, P-gp in the liver serves only a compensatory role for transport of [³H]DPDPE in the absence of functional Mrp2. Clearly, the multiplicity of transport mechanisms that dictate the hepatic disposition of [³H]DPDPE would make the extension of observations in transfected systems that express single transport proteins very difficult, if not impossible. The interaction of GF120918 with a basolateral transport process demonstrates another potential drug interaction mechanism in the liver. Moreover, inhibition of hepatic basolateral excretion may explain observations of enhanced systemic clearance of some substrates in the presence of a purported excretory transport inhibitor. Collectively, these studies emphasize the importance of evaluating simultaneously the impact of all relevant transport proteins on the overall disposition of xenobiotics.