e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 4 ( 2 0 0 8 ) 345–350
avai lab le at www.sc iencedi rec t .com
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Kinetic analysis of saturable hepatic uptake of digoxin andits inhibition by rifampicin
Michael Weissa,∗, Daniel Y. Hungb, Klaus Poenickea, Michael S. Robertsb
a Section of Pharmacokinetics, Department of Pharmacology, Martin Luther University Halle-Wittenberg, D-06097 Halle, Saale, Germanyb Department of Medicine, Princess Alexandra Hospital, University of Queensland, Woolloongabba, Queensland, Australia
a r t i c l e i n f o
Article history:
Received 4 February 2008
Received in revised form
14 April 2008
Accepted 22 May 2008
Published on line 3 June 2008
Keywords:
Hepatic uptake
a b s t r a c t
Although the organic anion transporter Oatp2 plays a critical role in determining the hepatic
clearance of some drugs, little quantitative information exists about its functional character-
istics in relation to inhibition of sinusoidal drug uptake. We investigated the uptake kinetics
of the Oatp2 substrate digoxin in the isolated perfused rat liver. In the single-pass perfused
liver three consecutive digoxin doses of 15, 30 and 45 �g were administered in the pres-
ence or absence of rifampicin (100 �M), an inhibitor of Oatp2. Digoxin was determined in
the outflow samples by HPLC and all data were analyzed by simultaneous nonlinear regres-
sion assuming a Michaelis–Menten uptake mechanism. Hepatocellular uptake of digoxin
was concentration-dependent with a Michaelis constant (KM) of 577.8 ng/ml. Rifampicin
Oatp2
Digoxin
Rifampicin
R
significantly reduced uptake (KM increased 2.5-fold) without affecting other parameters.
in the perfused rat liver is still lacking. Since digoxin is also
0d
at liver
. Introduction
he liver represents the major organ for drug metabolism.embrane transporters as organic anion transporting
olypeptides (Oatp 1, 2 and 4), sodium/taurocholate-otransporting polypeptide (Ntcp), organic cation transportersOct 1) and organic anion transporters (Oat 3) facilitate theptake of drugs from the blood into rat hepatocytes wherehey undergo metabolism and excretion into bile (e.g., Fabert al., 2003). Oatp-mediated uptake is a prerequisite to hepaticlimination of some poorly membrane-permeable drugs,uch as, for example, digoxin (Kodawara et al., 2002; Lau etl., 2004), cerivastatin (Shitara et al., 2003), bosentan (Treibert al., 2004) and erythromycin (Lam et al., 2006; Frasetto et al.,
007). Drug clearance then depends on sinusoidal membraneransport and it has been pointed out that inhibition ofptake transporters may reduce hepatic drug clearance and
represents a potential source of drug–drug interactions. Theinterplay of transporters and enzymes in defining net hepaticelimination has been discussed in detail recently (Lau etal., 2004; Lam and Benet, 2004; Wu and Benet, 2005; Liu andPang, 2006). Digoxin is a high affinity substrate of OATP2 (ratOatp2), located in the hepatic sinusoidal membrane, predom-inantly in perivenous hepatocytes (Noe et al., 1997; Reichelet al., 1999; Liu et al., 2005) and rifampicin was reported tobe a selective inhibitor of Oatp2 (Fattinger et al., 2000). Itwas shown that rifampicin reduces digoxin clearance andmetabolite formation in the perfused rat liver (Lau et al., 2004)and metabolism in isolated rat hepatocytes (Lam and Benet,2004), but a quantitative kinetic analysis of saturable uptake
secreted into bile via the P-glycoprotein (P-gp) efflux pump,it is important to note that rifampicin, in the concentrationused here, does not inhibit P-gp (Lau et al., 2004). Furthermore,
346 e u r o p e a n j o u r n a l o f p h a r m a c e
the main route of elimination in the rat for digoxin is throughmetabolism by cyp3a (Salphati and Benet, 1999).
The goal of the present study was to describe hepatocellu-lar uptake kinetics of digoxin at various doses in single-passperfused rat livers in the presence and absence of rifampicin.Parameters describing the kinetics of digoxin uptake in theperfused rat liver were estimated using the Michaelis–Mentenmodel. These model parameters may then be used in thequantitative prediction of the effect of changes in uptakekinetics on hepatic drug clearance.
2. Materials and methods
2.1. Chemicals
Digoxin and rifampicin were purchased from Sigma–Aldrich(Germany), [U-14C]sucrose was purchased from Amersham,Buckinghamshire, UK. Digoxin was dissolved in 70% ethanolsolution and diluted with Krebs–Henseleit buffer. The finalconcentration of ethanol in vehicle was 0.7%. Rifampicin wasdissolved with DMSO (0.075% (v/v) final concentration). Allother chemicals and solvents were of the highest grade avail-able.
2.2. In situ perfusion of the isolated rat liver
Perfusion of the isolated rat liver used in this study was per-formed as described elsewhere (Cheung et al., 1996). Briefly,male Wistar rats, weighing 200–250 g were anaesthetisedusing an intraperitoneal injection of xylazine/ketamine(10/80 mg kg−1). The laparatomised rats were heparinized with200 units heparin injected into the inferior vena cava. Thebile duct and the portal vein were cannulated (PE-10, ClayAdams, Franklin Lakes, NJ) and using an intravenous 16-gauge catheter, respectively. The liver was then perfused withMOPS [3-(N-morpholino-)propanesulfonic acid]-buffer con-taining 2% BSA adjusted to pH 7.40 and oxygenated via asilastic tubing lung, ventilated with an atmosphere of 100%pure oxygen. A peristaltic pump was used as non-circulatingperfusion system. Perfusions were adjusted to a flow rate of25 ml/min. The animals were sacrificed by thoracotomy onceperfusion was established and the inferior vena cava was can-nulated for collection of samples. The animals were placed ina temperature-controlled environment at 37 ◦C. Assessmentof liver viability was by macroscopic appearance, measure-ment of bile flow, oxygen consumption and portal resistancepressure.
2.3. Experimental protocol
After a 10 min perfusion-stabilization period [U-14C]sucrose(1.5 × 106 dpm) was injected into each liver (n = 5) with outletsamples collected via a fraction collector over 4 min (1 s × 20,4 s × 5, 10 s × 5, 30 s × 5). Samples were centrifuged at 1500 × g(25 ◦C) for 3 min, and aliquots (75 �l) of the supernatant were
taken for scintillation counting to determine [U-14C]sucroseusing a MINAXI beta TRI-CARB 4000 series liquid scintillationcounter (Packard Instruments; Meriden, CT) and “Emulsifier-Safe” scintillation fluid (Packard BioScience; Groningen, The
a l s c i e n c e s 3 4 ( 2 0 0 8 ) 345–350
Netherlands). Each liver was then first perfused for 30 minin the absence and after 10 min for 30 min in the presenceof rifampicin (100 �M). At t = 0, 10 and 20 min in each 30 minperiod, digoxin doses of 15, 30 and 45 �g were infused within1 min and 24 samples of outflow concentration were collectedup to 7 min after start of infusion. Digoxin outflow sampleswere kept frozen at −20 ◦C until assayed for digoxin by high-pressure liquid chromatography (HPLC). Note that the digoxindoses were selected on the basis of previous experiments inthe perfused rat heart (Kang and Weiss, 2002) to ensure thatthe doses are not cardiotoxic and the results remain relevantfor studies in the rat in vivo (in view of the free digoxin concen-tration). The rifampicin concentration was the same as usedby Lau et al. (2004) in similar experiments performed with arecirculating liver perfusion system.
2.4. Analytical procedure
The outflow concentration of digoxin was determined using ahigh-performance liquid chromatography (HPLC) system withpost-column derivatization and fluorescence detection; sim-ilar to the method of Embree and McErlane (1989). 75 �l ofthe collected perfusate were transferred into a 500 �l safe-locktube, spiked with 10% trichloroacetic acid (TCA, 75 �l) and werevigorously vortexed. After centrifugation at 2000 × g for 10 minat 4 ◦C, aliquots of the upper phase were stored at −80 ◦C untilanalysis. Frozen samples were thawed at 15 ◦C and centrifugedat 200 × g for 10 min at 4 ◦C. 10 �l of the supernatant weredirectly injected onto the analytical column. The HPLC sys-tem consisted of a Degasser ECR-3114 (JCN9), a HPLC pumpL-7100 and autosampler AS-200S (Merck-Hitachi, Germany), areaction pump 655A-13 (Merck-Hitachi, Germany) for the post-column derivatization and a fluorescence spectrophotometerF-1050 (Merck-Hitachi, Germany). The analysis was carriedout on Eurospher 100-C8 column (250 mm × 4 mm i.d., 5 �m,Knauer, Germany). The mobile phase consisting of 250 ml ace-tonitrile and 750 ml dehydroascorbic acid solution (preparedfrom 406.2 mg ascorbic acid and 105 �l 30% hydrogen perox-ide as previously described (Embree and McErlane, 1989). Theflow rate was 0.6 ml/min. By means of the reaction pump, theHPLC-column effluent was mixed with 32% hydrochloric acid,used as post-column derivatization reagent (0.25 ml/min), andpassed into a 15 m knitted reactor (PTFE capillary) at 78.5 ◦C.Fluorescence detector was adjusted at excitation and emis-sion wavelengths; 360 and 485 nm, respectively. We used 1 �gdigoxin per ml perfusion buffer solution as external standard.Aliquots of that were diluted with buffer solution to producethe other standard concentrations. The detector response waslinear at least over the range from the limit of quantification10 ng to 1.0 �g digoxin per ml perfusate. The detection limitwas 2 ng digoxin/ml (signal twice the noise level). The base lineof the blank perfusion solution was free of interference. Thestandard curve was prepared daily with digoxin in final con-centrations of 0.1, 0.4 and 1.0 �g/ml perfusion buffer solutionby using the peak height. The representative linear equa-tion was y = 16460x − 10 with a correlation coefficient r = 0.9998
(n = 6). Intra-day variability showed a mean standard devia-tion of 3.1% (0.1 �g/ml), 2.2% (0.4 �g/ml) and 1.8% (1.0 �g/ml)by analyzing each solution six times. The inter-day precisionwas determined to 2.5% (0.1 �g/ml), 3.31% (0.4 �g/ml) as well
t i c a
arma
2
ImTcbt
k
wrdfedCst
f(M(a
FHdVsNm
e u r o p e a n j o u r n a l o f p h a r m a c e u
s 3.76% (1.0 �g/ml) on five different days. After the analyticalun, samples with concentration more than 1.0 �g digoxin per
l perfusate were diluted with perfusion buffer solution andnalyzed once again.
.5. Model
n order to deal with nonlinearity, a compartmental liverodel (e.g., Sirianni and Pang, 1997) was used (Fig. 1).
ransporter-mediated uptake of digoxin from the extracellularompartment, V1 (sucrose space) across the sinusoidal mem-rane into hepatocytes was described by a Michaelis–Mentenype process with an apparent fractional uptake rate:
12(t) = Vmax
KM + C(t)(1)
here Vmax and KM, denote the apparent maximal transportate and Michaelis constant, respectively. The rate constantue to hepatic outflow is given by Q/V1, where Q denotes per-usate flow. The first-order rate constants describing cellularfflux and elimination (metabolism and biliary excretion) areenoted by k21 and ke, respectively. The outflow concentration(t) = x1(t)/V1 (where x1 denotes the amount of digoxin in theucrose space) after a dosing rate R(t) is obtained by solvinghe set of differential equations:
dx1(t)dt
= − Vmax
KM + x1/V1x1(t) − Q
V1x1(t) + k21x2(t) + R(t) (2)
dx2(t)dt
= Vmax
KM + x1/V1x1(t) − k21x2(t) − kex2(t) (3)
In order to calculate the hepatic extraction E at steady-staterom the model parameters, the set of nonlinear equations
Eqs. (2) and (3)) was solved numerically for dxi(t)/dt = 0 using
APLE 8 (Waterloo Maple Inc., 2002). For the linear caseC � KM) where Eq. (1) can be approximated by k12 = Vmax/KM,
closed form solution is obtained for E and the total hepatic
ig. 1 – Model of digoxin kinetics in the perfused rat liver.epatocellular uptake from the sucrose space (V1) isescribed by a Michaelis–Menten process with parameters
max and KM. The rate constants k21 and ke denoteinusoidal efflux and cellular elimination, respectively.ote that the latter is the sum of biliary excretion andetabolism (ke = kb + km).
l s c i e n c e s 3 4 ( 2 0 0 8 ) 345–350 347
clearance CL = QE:
CL = Q CLint,app
Q + CLint,app(4)
where the apparent intrinsic clearance is defined as
CLint,app = CLuptakeke
k21 + ke(5)
accounts for the effect of sinusoidal influx on metabolism andbiliary excretion (e.g., Shitara et al., 2005).
2.6. Data analysis
A mixture of two inverse Gaussian density functions withcorrection for catheter effects was fitted to the [14C] sucroseoutflow data to estimate the extracellular sucrose space (V1)(Weiss et al., 2000). The parameter V1 was than used as fixedparameter in fitting the digoxin data.
Differential equations describing the mass transportamong compartments (Eqs. (2) and (3)) were solved numeri-cally and C(t) was fitted to the data using ADAPT II, release4 (D’Argenio and Schumitzky, 1997). Due to its low hep-atic extraction, the determination of digoxin clearance wouldnormally require a recirculating design (Liu et al., 2005),but the analysis of uptake kinetics appears only possiblein single-pass experiments. One way to solve this prob-lem is to incorporate prior information on the uptake rateand elimination rate constants, k12 = Vmax/KM = 40 min−1 andke = 0.02 min−1, respectively, estimated in experiments withtracer doses (Liu et al., 2005) by using maximum a posteri-ori Bayesian (MAP) estimation implemented in ADAPT II. Inorder to improve the accuracy and reliability of parameter esti-mation, the two outflow data sets measured for three dosesas consecutive 1-min infusions in each liver in the absenceand presence of rifampicin were simultaneously fitted by asingle set of parameter values, allowing only a change in KM
or Vmax in the presence of rifampicin under the assump-tion of competitive or noncompetitive inhibition, respectively.This method, also known as global or simultaneous nonlinearregression, has recently become practical for fitting nonlin-ear enzyme-kinetic models to data (Kakkar et al., 2000). UsingMAP estimation, we assumed that the measurement error hasa standard deviation which is a linear function of the mea-sured quantity. The ‘goodness of fit’ was judged by visualexamination of the distribution of residuals, the generalizedinformation criterion (GEN-IC) (D’Argenio and Schumitzky,1997) and the R2 value of the fits. The GEN-IC index was appliedto discriminate between alternative models (competitive vs.noncompetitive inhibition of uptake). The paired t-test wasused to compare KM estimates in the presence and absence ofrifampicin. A P-value of <0.05 was considered significant.
3. Results
The model shown in Fig. 1 was capable to fit the digoxin out-flow data. The data measured in the presence of rifampicinwere successfully described by the model assuming compet-itive inhibition. The outflow concentration data after three
348 e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 3 4 ( 2 0 0 8 ) 345–350
Fig. 2 – Simultaneous fit of the model to outflowconcentration data for three consecutive digoxin doses (15,
Table 1 – Parameters estimated by simultaneous fittingof digoxin outflow concentration data for threeconsecutive digoxin doses (15, 30 and 45 �g as 1-mininfusions) in the absence and presence of 100 �Mrifampicin in perfusate, where only a change in KMaccounted for the effect of rifampicin (KM,rif)
Mean and coefficient of variation, n = 5.a P < 0.05 compared to KM.
Fig. 3 – The interplay between cellular elimination (rateconstant ke) and hepatocellular uptake in determininghepatic extraction (E) under conditions where uptake isunsaturated (C < 500 ng/ml) and the uptake rate constant isgiven by Vmax/KM. Uptake inhibition by rifampicin (100 �M)leads to a 2.5-fold increase in KM leaving ke unchanged.This reduces E (and hepatic clearance CL = QE) to 43% of the
30 and 45 �g as 1-min infusions) in the absence (A) andpresence (B) of 100 �M rifampicin in perfusate.
subsequent doses (1-min infusions) of digoxin in the absenceand presence of rifampicin and the resulting model fits areexemplified in Fig. 2A and B, respectively. The model fitted thedata well over the whole dose range (15–30–45 �g). The factthat these outflow curves measured in the same liver are fittedby the same parameter set, except of a higher KM in the pres-ence of rifampicin (Table 1) suggests competitive inhibition ofdigoxin uptake, as this model is preferred over the alternativemodel assuming a change in Vmax (GEN-IC difference > 0). Theapparent KM value of 577.8 ng/ml (739.6 nM) observed undercontrol conditions increased 2.5-fold in the presence of 100 �Mrifampicin (P < 0.05). This corresponds to a reduction of theuptake rate constant in the linear range (C � KM), k12 = Vmax/KM
(i.e., of CLuptake = k12V1) to 42 ± 23% of the value under controlconditions and decreases the hepatic extraction ratio E from4.97 to 2.09%. The latter implies a decrease of CL to 43% of thecontrol. This is illustrated in Fig. 3, showing that for unchangedcellular elimination (rate constant ke), the decrease sinusoidaluptake rate constant (due to increase in KM) decreases hepaticextraction of digoxin. The graph in Fig. 4 has been simulated
to illustrate the decrease in extraction due to saturation of thecapacity-limited uptake process for concentration above KM.Both model predictions were made using the mean values ofestimated parameters (Table 1).
control values (The calculation was based on the means ofmodel parameter estimates.).
4. Discussion
In general, it is accepted that Oatp2 is responsible for thesinusoidal uptake of digoxin (Noe et al., 1997; Reichel etal., 1999; Liu et al., 2005). From our estimate an apparentMichaelis–Menten constant KM normalized by the unboundconcentration in perfusate of 473 nM is obtained using the
value of 0.64 for the unbound fraction in the presence of 2%albumin (Liu et al., 2005). This parameter value estimated herein the perfused rat liver is in accordance with the values ofabout 400 nM in isolated rat hepatocytes in vitro (Liu et al.,
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a
Fig. 4 – Dependency of hepatic extraction ratio, E, ondigoxin input concentration as obtained by a numericalsolution of Eqs. (2) and (3) at steady state (dxi(t)/dt = 0). Dueto saturation of Michaelis–Menten uptake kinetics digoxinea
2iiuid(Trdcdea6loniwod(tcbrc
ddkoMam
r
xtraction decreases for concentrations abovepproximately 500 ng/ml.
005; Lam and Benet, 2004; Ito et al., 2007) and Oatp2 expressedn Xenopus oocytes (Kodawara et al., 2002). The most interest-ng result is that rifampicin significantly decreased the hepaticptake of digoxin (due to the 2.5-fold increase in KM), which
s in qualitative accordance with the observed decrease ofigoxin metabolism during Oatp2 inhibition in rat hepatocytes
Lam and Benet, 2004) and perfused rat liver (Lau et al., 2004).he present modeling results suggest that in the presence ofifampicin, the apparent intrinsic clearance CLint,app (Eq. (5))ecreases proportional to the decrease in sinusoidal uptakelearance CLuptake, since k21 and ke remained unchanged. Thisecrease in CLint,app then leads to a reduction in the overallfficiency for hepatic elimination of digoxin (Eq. (4)). Lau etl. (2004) observed a 34% increase in AUC (measured up to0 min) of digoxin in the presence of rifampicin after recircu-ating perfusion. That this change is lower than expected fromur results, may be due to the fact that first, the total AUC wasot measured and second that the rifampicin concentration
n the perfusate decreased from 100 �M to levels below 10 �Mithin the first minutes of perfusion. In general, the effectf an increase in KM on the apparent fractional uptake rateepends both on Vmax and the substrate concentration C (Eq.
1)), but for C � KM, the 2.5-fold increase in KM correspondso a reduction in uptake rate constant Vmax/KM to 40% of theontrol as illustrated in Fig. 3. Although not directly compara-le, in hepatocytes incubated for 2 min with 100 nM digoxin,ifampicin (100 �M) decreased digoxin uptake to 23% of theontrol (Lam and Benet, 2004).
A limitation of the present experimental design is thatue to the low extraction and the flow-limited uptake ofigoxin (CLuptake � Q), the parameters ke, k12 (=Vmax/KM) and
21 could not be uniquely determined and prior information
n ke and k12 had to be incorporated (Liu et al., 2005) usingAP estimation as described above. Therefore, the Vmax/KM
nd ke estimates (Table 1) simply reflect the priors. The esti-ate of KM, however, was found largely independent from the
l s c i e n c e s 3 4 ( 2 0 0 8 ) 345–350 349
choice of Vmax/KM and ke values in MAP estimation. Thus,our approach appears to be justified for evaluating the effectof rifampicin on hepatic digoxin uptake. By concluding thatthe present model is a minimal model that can accuratelydescribe our data, we do note exclude a contribution of non-saturable uptake transport (10% in isolated hepatozyte studiesby Liu et al. (2005)) nor the possibility of saturable sinusoidalefflux; those assumptions, however, did not improve the fit ofthe model to our data. Due to the low sensitivity of outflowconcentration to changes in ke mentioned above (i.e., the lowextraction of digoxin), we could not test the involvement ofsaturable elimination (metabolism or biliary excretion). Notethat a 1-min infusion instead of bolus injection of digoxin wasused to achieve (at least approximately) distributional equili-bration (well-mixed conditions) in the sucrose compartment.Although it cannot be completely excluded that also a reduc-tion in Vmax could contribute to the observed inhibition ofdigoxin uptake by rifampicin, our modeling result favors thechange in KM, in accordance with earlier results in the litera-ture. In summary, the dilemma arises that the advantage of therecirculating liver perfusion in case of low extracted drugs isaccompanied by the disadvantage that fast uptake processescannot be evaluated kinetically. While this does not hold forisolated hepatocytes, differences to the perfused liver shouldbe taken into account (Webborn et al., 2007). Microsome stud-ies, in contrast, are unsuitable when membrane transportersaffect hepatic elimination, as pointed out by Lam and Benet(2004) using digoxin as an example.
The validity of a model is always defined in terms of themodeling objectives and the present relatively simple modelat this stage adequately describes the measured digoxin out-flow data. The availability of new data, however, may leadto a further improvement of the model as, for example, theincorporation of a zonal structure (Liu and Pang, 2006).
In conclusion, our result is compatible with the hypoth-esis that rifampicin competitively inhibits hepatic uptake ofdigoxin in the isolated perfused rat liver, thereby reducing itsextraction. The estimate of the apparent KM is in accordancewith previously reported results in isolated rat hepatocytes.The present model may be useful in understanding the inter-play between uptake of digoxin into hepatocytes and itssubsequent elimination by metabolism and biliary excretion.
Acknowledgements
This work was supported by grants from the AustralianNational Health & Medical Research Council and the GermanResearch Association.
e f e r e n c e s
Cheung, K., Hickman, P.E., Potter, J.M., Walker, N., Jericho, M.,Haslam, R., Roberts, M.S., 1996. An optimised model for ratliver perfusion studies. J. Surg. Res. 66, 81–89.
D’Argenio, D.Z., Schumitzky, A., 1997. ADAPT II User’s Guide:Pharmacokinetic/Pharmacodynamic Systems AnalysisSoftware. Biomedical Simulations Resource, Los Angeles.
Embree, L., McErlane, L., 1989. Development of ahigh-performance liquid chromatographic-post-column
u t i c
350 e u r o p e a n j o u r n a l o f p h a r m a c e
fluorogenic assay for digoxin in serum. J. Chromatogr. 496,321–334.
Faber, K.N., Muller, M., Jansen, P.L., 2003. Drug transport proteinsin the liver. Adv. Drug Deliv. 55, 107–124.
Fattinger, K., Cattori, V., Hagenbuch, B., Meier, P.J., Stieger, B.,2000. Rifamycin SV and rifampicin exhibit differentialinhibition of the hepatic rat organic anion transportingpolypeptides, Oatp1 and Oatp2. Hepatology 32,82–86.
Frasetto, L.A., Poon, S., Tsourounis, C., Valera, C., Benet, L.Z., 2007.Effects of uptake and efflux transporter inhibition onerythromycin breath test results. Clin. Pharmacol. Ther. 81,828–832.
Ito, S., Nasu, R., Tsujimoto, M., Murakami, H., Ohtani, H., Sawada,Y., 2007. Effect of macrolide antibiotics on uptake of digoxininto rat liver. Biopharm. Drug Dispos. 28, 113–123.
Kang, W., Weiss, M., 2002. Digoxin uptake, receptor heterogeneity,and inotropic response in the isolated rat heart: Acomprehensive kinetic model. J. Pharmacol. Exp. Ther. 302,577–583.
Kakkar, T., Pak, Y., Mayersohn, M., 2000. Evaluation of a minimalexperimental design for determination of enzyme kineticparameters and inhibition mechanism. J. Pharmacol. Exp.Ther. 293, 861–869.
Kodawara, T., Masuda, S., Wakasugi, H., Uwai, Y., Futami, T.T.,Saito, H., Abe, T., Inu, K., 2002. Organic anion transporteroatp2-mediated interaction between digoxin and amiodaronein the rat liver. Pharm. Res. 19, 738–743.
Lam, J.L., Benet, L.Z., 2004. Hepatic microsome studies areinsufficient to characterize in vivo hepatic metabolicclearance and metabolic drug–drug interactions: studies ofdigoxin metabolism in primary rat hepatocytes versusmicrosomes. Drug Metab. Dispos. 32, 1311–1316.
Lam, J.L., Okochi, H., Huang, Y., Benet, L.Z., 2006. In vitro and invivo correlation of hepatic transporter effects onerythromycin metabolism: characterizing the importance oftransporter-enzyme interplay. Drug Metab. Dispos. 34,
Liu, L., Pang, K.S., 2006. An integrated approach to model hepaticdrug clearance. Eur. J. Pharm. Sci. 29, 215–230.
Liu, L., Mak, E., Tirona, R.G., Tan, E., Novikoff, P.M., Wang, P.,Wolkoff, A.W., Pang, K.S., 2005. Vascular binding, blood flow,transporter, and enzyme interactions on the processing ofdigoxin in rat liver. J. Pharmacol. Exp. Ther. 315, 433–448.
Noe, B., Hagenbuch, B., Stieger, B., Meier, P.J., 1997. Isolation of amultispecific organic anion and cardiac glycoside transporterfrom rat brain. Proc. Natl. Acad. Sci. U.S.A. 94, 10346–10350.
Reichel, C., Gao, B., Van Montfoort, J., Cattori, V., Rahner, C.,Hagenbuch, B., Stieger, B., Kamisako, T., Meier, P.J., 1999.Localization and function of the organic anion-transportingpolypeptide Oatp2 in rat liver. Gastroenterology 117, 688–695.
Salphati, L., Benet, L.Z., 1999. Metabolism of digoxin anddigoxigenin digitoxosides in rat liver microsomes:involvement of cytochrome P4503A. Xenobiotica 29, 171–185.
Shitara, Y., Itoh, T., Sato, H., Li, A.P., Sugiyama, Y., 2003. Inhibitionof transporter-mediated hepatic uptake as a mechanism fordrug–drug interaction between cerivastatin and cyclosporinA. J. Pharmacol. Exp. Ther. 304, 610–616.
Shitara, Y., Sato, H., Sugiyama, Y., 2005. Evaluation of drug–druginteraction in the hepatobiliary and renal transport of drugs.Annu. Rev. Pharmacol. Toxicol. 45, 689–723.
Sirianni, G.L., Pang, K.S., 1997. Organ clearance concepts: newperspectives on old principles. J. Pharmacokinet. Biopharm.25, 449–470.
Treiber, A., Schneiter, R., Delahaye, S., Clozel, M., 2004. Inhibitionof organic anion transporting polypeptide-mediated hepaticuptake is the major determinant in the pharmacokineticinteraction between bosentan and cyclosporin A in the rat. J.Pharmacol. Exp. Ther. 308, 1121–1129.
Webborn, P.J.H., Parker, A.J., Denton, R.L., Riley, R.J., 2007. Invitro–in vivo extrapolation of hepatic clearance involvingactive uptake: Theoretical and experimental aspects.Xenobiotica 37, 1090–1109.
Weiss, M., Kuhlmann, O., Hung, D.Y., Roberts, M.S., 2000.Cytoplasmic binding and disposition kinetics of diclofenac in
the isolated perfused rat liver. Br. J. Pharmacol. 130, 1331–1338.
Wu, C.Y., Benet, L.Z., 2005. Predicting drug disposition viaapplication of BCS: transport/absorption/eliminationinterplay and development of a biopharmaceutics drugdisposition classification system. Pharm. Res. 22, 11–23.
