kinetic characterization of p-glycoprotein...

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KINETIC CHARACTERIZATION OF P-GLYCOPROTEIN

MEDIATED EFFLUX OF RHODAMINE 6G IN THE INTACT

RABBIT LUNG

DAVID L. ROERIG, SAID H. AUDI, SUSAN B. AHLF

Research Service, V.A. Medical Center, Milwaukee, Wisconsin (DLR, SBA)

Department of Anesthesiology (DLR) and

Department of Pulmonary and Critical Care Medicine (SHA), Medical College of Wisconsin

Department of Biomedical Engineering, Marquette University, Milwaukee, Wisconsin (SHA).

DMD Fast Forward. Published on June 9, 2004 as doi:10.1124/dmd.104.000042

Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on June 9, 2004 as DOI: 10.1124/dmd.104.000042

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Running Title

P-gp MEDIATED EFFLUX OF RHODAMINE 6G IN THE RABBIT LUNG

Corresponding Author

Dr. David L. Roerig

Research Service 151

V.A. Medical Center

5000 W. National Ave.

Milwaukee, WI 53295

Telephone (414) 384-2000 X-41541

Fax (414) 382-5374

e-mail [email protected]

Text pages 19

Table 1

Figures 6

References 36

Number of Words

Abstract 249

Introduction 603

Discussion 1487


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Abbreviations

P-gp, P-glycoprotien; R6G, rhodamine 6G; BSA, bovine serum albumin; 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; MID, multiple indicator dilution.


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ABSTRACT

P-glycoprotein (P-gp) is an ATP dependent drug efflux transporter involved in multidrug

resistance and drug disposition in many organ systems. A majority of P-gp substrates are

lipophilic amine drugs which also exhibit rapid extensive accumulation in lung tissue. P-gp is

expressed in lung tissue and the very nature of this drug efflux mechanism suggests a moderating

role in pulmonary drug disposition. Little is known about P-gp mediated efflux out of lung

tissue or its kinetic characteristics as they may relate to P-gp impact on pulmonary drug

accumulation. The present study develops an experimental and kinetic model to characterize the

kinetics of P-gp mediated efflux of rhodamine 6G dye (R6G) out of the intact rabbit lung. The

perfusate concentration of R6G with time during recirculation through an isolated perfused rabbit

lung was measured and 66.6±2.6% (SE) of the perfusate R6G was taken up by the lung. In the

presence of P-gp inhibitors, R6G uptake increased significantly to 87.5±1.1% (P<0.002)

indicating a functional pulmonary P-gp efflux transporter. Fractional lung accumulation of R6G

increased with increasing R6G perfusate concentration, a result consistent with saturation of an

efflux transporter. A parsimonious three compartment kinetic model of R6G pulmonary

disposition was used to interpret data sets from experiments with different perfusion variables

and to estimate parameters descriptive of the dominant kinetic processes involved in R6G

pulmonary accumulation. The estimated value of the kinetic parameter, kpgp, rate constant for P-

gp mediated R6G efflux, indicates this transporter plays a significant role in moderating R6G

pulmonary disposition.


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INTRODUCTION

P-glycoprotein (P-gp) is a 170,000Da transmembrane phosphoglycoprotein that was first

correlated with decreased drug accumulation and increased drug resistance in certain cell lines

(Gottesman and Pasten,1993; Ambudkar et al., 1999; Ford and Hait, 1990). P-gp is a member of

the ATP binding cassette (ABC) transporter family and is encoded by the MDR1 and MDR2

genes in humans and the mdr1a, mdr1b and mdr2 genes in rodents (Borst et al., 1993; Thiebaut

et al., 1987). Over-expression of P-gp is recognized as an important mechanism in the

development of multidrug resistance in cancer chemotherapy. P-gp is also present in normal

tissue (Thiebaut et al., 1987; Endicott and Ling, 1989; Sharom, 1997). In humans, P-gp is

located in the plasma membrane in specific cell types of different organs including intestine,

kidney, liver and in capillary endothelial cells of the brain wherein roles for P-gp in drug uptake,

metabolism, excretion and limiting CNS concentrations, respectively, have been reported

(Ambudkar et al., 1999; Ford and Hait, 1990; Endicott and Ling, 1989). In brain, P-gp appears

to be a functional aspect of the blood-brain barrier (Cordon-Cardo et al., 1989; Shinkel et al.,

1996; Regina et al., 1998). Using various methods, P-gp expression has been reported in other

organs including lung (Demeule et al., 1999; Jette et al., 1996). Brady et al. (2002) found that

mdr1b mRNA expression in the lung was second only to the gastrointestinal tract. Other studies

have located P-gp in bronchial and alveolar epithelial cells and in pulmonary and bronchial

capillary endothelial cells (Campbell et al., 2003; Lechapt-Zalcman et al., 1997; Demeule et al.,

2001).

P-glycoprotein has been referred to as an atypical ATP dependent transporter that is highly

promiscuous based on the hundreds of compounds that have been identified as substrates

(Sharom, 1997). The physical chemical properties of P-gp substrates are diverse and most


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commonly include some degree of lipophilicity and a positive charge and/or an amine nitrogen

(Frezard et al., 2001). Seelig (1998) reviewed 100 compounds that interact with P-gp and over

60 were basic lipophilic amines, a property shared with compounds that readily accumulate in

lung tissue. The lung is well known for its extensive accumulation of lipophilic amines and this

similarity to the substrate specificity of P-gp plus the very nature of this drug efflux transporter

strongly suggests P-gp mediated drug efflux could play a moderating role in the pulmonary

accumulation of amine drugs (Junod, 1976; Wilson et al., 1979; Bend et al., 1985; Roerig et al.,

1983; 1984). However, being a substrate for P-gp does not insure that this efflux transporter will

significantly moderate drug disposition in lung tissue. For an intact organ, tissue accumulation is

dependent on multiple, and often competing, vascular and intracellular dispositional processes.

These include rate of delivery to the organ (blood flow), diffusion into or out of specific cell

types, interactions such as binding to intracellular sites or accumulation in subcellular organelles.

An additional factor is saturation of one or more of these kinetic processes, which could reduce

the impact of the saturable process on overall tissue accumulation of the drug.

This work is aimed at developing a kinetic model of pulmonary drug disposition to identify

the contribution of the different kinetic processes, including P-gp mediated drug efflux, in the

lung tissue accumulation of lipophilic amines. Because of its ease of ex vivo perfusion, the

ability to manipulate perfusion variables and expression of P-gp in normal lung tissue, the lung

may also serve as an organ model in general. For this study, we selected the lipophilic amine

dye, rhodamine 6G (R6G) as the test substrate based on its reported uptake in cells (Loetchutinat

et al., 2003).


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MATERIAL AND METHODS

Chemicals and Reagents: Rhodamine 6G (R6G) was obtained from Sigma-Aldrich (St.

Louis, MO). Sodium Pentobarbital (50mg/ml), heparin sodium (1000 units/ml), and verapamil

HCl were obtained from Abbott Laboratories (N. Chicago, IL), Elkins-Sinn INC. (Cherry Hill,

NJ) and Knoll Pharmaceutical Co. (Whippany, NJ) respectively. Bovine Serum Albumin

(Standard Powder, BSA) was purchased from Serologicals Corp. (Norcross, GA). The P-

glycoprotein inhibitor, N (4-[2-(1,2,3,4-tetrahydro-6, 7 dimethyl-2 isoquinolinyl)-ethyl]-phenyl)

9,10-dihydro-5-methoxy-9-oxo-4 acridine carboxamide (GF120918), was generously supplied

by Glaxo Smith Wellcome Inc. (Research Triangle Park, NC) and dissolved in dimethylsulfoxide

(1mg/ml). All other chemicals were of reagent grade.

Animals: All studies were carried out with New Zealand White Rabbits of either gender with

a mean ± SE weight of 2.65 ± 0.2 kg. The rabbits were housed one per cage with free access to

food and water and maintained on a 12 hr light/dark cycle. All rabbits were purchased from New

Franken Research Rabbits (New Franken, WI). All animal care and treatment procedures were

approved by the V.A. Medical Center Research Institutional Animal Care and Use Committee

prior to initiating these studies.

Isolated Lung Perfusion System: The isolated perfused rabbit lung (IPL) system was similar

to that previously described (Roerig et al., 1992; Audi et al., 1995; 1996). Briefly rabbits were

anesthetized with sodium pentobarbital (25-40mg/kg. iv, ear vein) and the carotid artery

cannulated. The rabbits were heparinized (1000 units/kg) and exsanguinated via the carotid

cannula. The chest was opened and the pulmonary artery, pulmonary vein and trachea were


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cannulated. The lung was removed from the chest, suspended in the perfusion apparatus by the

cannulas and the perfusion system primed with a perfusate consisting of a physiological salt

solution containing 4.5% BSA. Perfusate was initially pumped through the IPL at 200 ml/min

(Master Flex roller pump Cole-Palmer Inc.) and the uniformity of blanching after the residual

blood was washed out of the lung was used to estimate the extent of perfusion of the IPL. Only

lungs greater than 90% perfused were used for these studies. The venous outflow was then

directed to the reservoir supplying the pump. Additional perfusate was added to the reservoir to

give a recirculating perfusate volume of 134 ± 1 ml. The pulmonary artery pressure, referenced

to the level of the left atrium, was 8.1± 0.3 cm H2O at end expiration and IPLs that exhibited an

arterial perfusion pressure greater than 10 cm H2O were not studied. The perfused lung was

maintained at 37°C. The lungs were continuously ventilated (10 breaths/min) with a gas mixture

containing 15% O2, 5% CO2, balance N2 with end inspiratory and expiratory airway pressures of

7.7± 0.1 and 2.1± 0.1 cm H2O, respectively. At the end of each experiment the lungs were

weighed and lyophilized to a constant weight. The wet/dry weight ratio of all lungs was 6.11±

0.06.

Experimental Protocols: The uptake of R6G with time was determined by adding known

amounts of R6G to the recirculating reservoir and then removing 1.7ml perfusate samples from

the reservoir at fixed time intervals out to 120 min depending on the particular experiment. The

sampling interval after R6G addition to the reservoir was every 5 min for the first 30 min, every

10 min between 30 and 60 min and every 15 min thereafter. Depending on the experiment the P-

gp inhibitor, GF120918, was added to the perfusate reservoir 5 min prior to the R6G or at

various times after R6G addition. A standard concentration curve for R6G was constructed using


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perfusate that had recirculated through the lung prior to R6G addition and a portion of the stock

R6G solution that was added to the recirculating reservoir. All samples and standards were

centrifuged to remove trace red blood cells and the absorbance at 540 nm used to determine the

R6G perfusate concentration. The amount of R6G taken up by the lung was determined by its

disappearance from the recirculating reservoir corrected for the amount of R6G removed from

the perfusion system during sampling. Each lung was used only once and the number (n) of

individual rabbit lungs used in the different experiments is given in the figure legends.


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RESULTS

Uptake of R6G in The Lung: Figure 1 shows the nmol of R6G in the isolated perfused rabbit

lung with time after 2.5µM R6G was added to the recirculating reservoir in control lungs (open

circles) and in lungs to which the P-gp efflux inhibitor verapamil (closed triangles, Ford and

Hait, 1990) or GF120918 (closed circles, Hyafil et al., 1993) were added to the perfusate

reservoir 5 min prior to addition and recirculation of the R6G. With either inhibitor, the R6G

accumulation increased. At all time points, R6G accumulation was significantly greater in the

presence of GF120918 (P<0.002). At 90 min after the start of R6G recirulcation 87.5±1.1% (SE)

of the R6G had accumulated in GF120918 treated lungs compared to 66.6±2.6% for control

lungs.

Figure 2 depicts a family of curves showing the increase in the total nmol of R6G in the rat

lungs versus time with initial perfusate R6G concentrations ranging from 1.25 µM to 12.6 µM.

This experiment provides one of the discriminating data sets necessary to estimate values of the

kinetic model parameters (see Kinetic Model and Data Analysis). This experiment also provides

insight into possible saturation of dispositional processes involved in R6G lung accumulation.

For example, in the absence of any saturation, the fraction of the initial amount of R6G in the

perfusate in the lung at the end of the perfusion should be constant over the R6G perfusate

concentration range studied. However, as shown in Figure 3, the fraction of total R6G in the

lung at 90 min increased as a function of total R6G initially present in the reservoir.

The effect of GF120918 concentration on R6G accumulation in the lung was also determined.

Lungs were perfused for 5 min with perfusate concentrations of GF120918 of 0, 2, 10, 20, and

70 µM prior to recirculation of 2.5µM R6G. Accumulation of R6G in the lung was then

determined at each inhibitor concentration and is shown in Figure 4. The accumulation of R6G


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in the lung increased with increasing GF120918 perfusate concentration. This experiment also

provides a data set necessary to estimate values of the kinetic model parameters as explained

under Kinetic Model and Data Analysis described below. It is apparent in Figure 4 that

inhibition of P-gp is approaching a maximum and the 20µM GF120918 concentration (open

triangles) used to inhibit P-gp mediated R6G efflux out of the lung in Figures 1 and 5 results in

near maximal P-gp inhibition.

In an additional study, 20µM GF120918 was added to the recirculating perfusate at various

times after the start of R6G recirculation through the lung. Figure 5 shows the increase in the

total R6G in the lung versus time with GF120918 added at the times indicated by the arrows.

Addition of the P-gp inhibitor caused a sudden increase in R6G accumulation which was greater

the earlier the GF120918 was added to the recirculating perfusate.

Kinetic Model and Data Analysis: To evaluate the effect of P-gp and other cellular

processes on R6G accumulation in the lung (Figures 2-5), we developed a parsimonious kinetic

model for the pulmonary disposition of R6G. The model (Figure 6) consists of three

compartments, a vascular compartment and two extravascular compartments. The vascular

compartment (compartment 1) represents the lung vascular region, reservoir, and tubing. The

two extravascular compartments (compartments 2 and 3) represent distribution of R6G within

the lung tissue. The model allows for R6G diffusion between compartments 1 and 2 with the

rate of diffusion represented by the permeability-surface area product, PS (ml/min); P-gp

mediated efflux of R6G from compartment 2 into compartment 1 described by a Michaelis-

Menten type process with an apparent maximum efflux rate Vmax21 (nmols/min) and a

Michaelis constant Km21 (nmol); accumulation of R6G in compartment 3 is also described by


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Michaelis-Menten type process with an apparent maximum uptake rate Vmax23 (nmols/min) and

a Michaelis constant Km23 (µM); and a passive intercompartmental transfer from compartment

3 to compartment 2 described by the first order rate constant k32 (min−1). Compartment 3 could

represent intracellular binding sites or sequestration in organelles. The extensive pulmonary

uptake of R6G suggests this is a high capacity, high affinity intracellular pool for R6G. Thus,

in equations (1-3) below, Vmax23 and Km23 are replaced by the first order rate constant k23 =

Vmax23 /(Km23V2) (min−1). The inhibitor GF120918 is assumed not only to inhibit P-gp

mediated R6G efflux with an inhibition constant Ki1 (µM), but also to inhibit R6G

accumulation in compartment 3 with an inhibition constant Ki2 (µM). The kinetic model

makes no assumption of the anatomical location of the P-gp protein but does assume that P-gp

transports R6G from the lung tissue into the vascular space.

The temporal variations in the concentrations of the various species in the three

compartments are described by equations (1-3).

d[C1](t)dt

=C2(t)V1

PSV2

+Vmax21

Km21V2 1 +λ [i]Ki1

+ C2(t)

– PS[C1](t)

V1 (1)

dC2(t)dt

= PS [C1](t) + k32C3(t)

– C2(t) PSV2

+Vmax21

Km21V2 1 +λ [i]Ki1

+ C2(t)

+k 23

1 +λ [i]Ki2

(2)

dC3(t)

dt=

k 23 C2(t)

1 +λ [i]Ki1

– k32C3(t) (3)

where [C1](t) is the vascular (perfusate) concentration (µM) of R6G; C2(t) and C3(t) are the

amounts (nmols) of R6G in compartments 2 and 3, respectively; [i] is the concentration (µM)


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of the inhibitor GF120918 in the extravascular region; λ is the inhibitor’s extravascular-to-

vascular partition coefficient; k23 (min−1) is an approximation to the Vmax23/(Km23V2) ratio,

and V1 and V2 (ml) are the volumes of compartments 1 and 2, respectively.

The identifiable model parameters are the following parameter groups: PS (ml/min); kout =

PS/V2 (min−1), a measure of the rate constant for diffusion mediated lung efflux of R6G;

(Km21V2) (nmols), measure of the affinity of P-gp for R6G; Vmax21 (nmols/min), measure of

the capacity of P-gp for R6G; k23 and k32 (min−1), first order rate constants for the transfer of

R6G in and out of the subcellular pool (compartment 3), respectively; Ki1/λ and Ki2/λ,

respective measures of the inhibition constants for P-gp mediated R6G efflux and R6G

accumulation in compartment 3. The volume of compartment 1, V1, was set at 134 ml.

The above model parameters are not estimable from a single set of data or experimental

condition. In other words, the information content of a single set of data or experimental

condition does not provide sufficiently discriminating information about all dominant

processes to separately estimate all the model parameters. Thus, the approach that we have

taken to estimate values for the kinetic model parameters was to carry out simultaneous

nonlinear regression fits of the solution of equations (1-3) to all the mean data in Figures 2 and

4 obtained by varying the perfusion conditions as indicated above. The model solution was

corrected for the change in reservoir volume due to sampling. The estimated values of the

model parameters are given in table 1. The solid lines imposed on the data in Figures 2 and 4

are the model fit to the data. The solid lines superimposed on the data in Figures 3 and 5 are

model predictions obtained by solving equations (1-3) with the model parameters set at the

values estimated from the data in Figures 2 and 4.


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To examine the contribution of P-gp mediated efflux to the pulmonary uptake of R6G, we

compared the estimated values of the kinetic parameters kpgp = Vmax21/(Km21V2), kout =PS/V2,

kin = PS/V1, and k23 = Vmax23/(Km23V2) which represent rate constants (min−1) for the competing

dispositional processes, namely P-gp mediated efflux, non-P-gp mediated efflux, diffusion into

the lung and intracellular transfer into the subcellular pool (see Table 1).

A Fortran based computer program (Fortran PowerStation, Version 4.0) was developed for

evaluating equations (1-3) (kinetic model) and for estimating the model parameters descriptive of

the various processes (Audi, et al., 1998). As described above, for the appropriate initial

conditions, the model equations were solved numerically (Gear’s method) using International

Mathematics and Statistics Library (IMSL) subroutine DIPAG (IMSL Math/Library). The

optimization for fitting the model to the data and parameter estimation was carried out using

IMSL subroutine DBCLSF, which finds the best nonlinear least squares solution using a

modified Levenberg-Marquardt algorithm with finite difference Jacobian. The 95% asymptotic

parameter confidence intervals (Table 1) were calculated from the sensitivity matrix as

previously described (Audi, et al., 1998).


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DISCUSSION

This study provides evidence for the expression of the multidrug efflux transporter P-

glycoprotien (P-gp) in isolated perfused lungs from normal rabbits. This conclusion is based on

the increased accumulation of the rhodamine dye, R6G, in the presence of verapamil or

GF120918 (Figures 1,4 and 5), two known P-gp inhibitors (Ford and Hait, 1990; Hyafil et al.,

1993). Also, consistent with P-gp mediated transport of R6G out of the lung is the increased

fractional accumulation of R6G with increasing perfusate R6G (Figure 3). This was interpreted

as saturation where the P-gp mediated efflux rate of R6G approaches a constant, Vmax21, resulting

in a proportionately decreased effect of P-gp on R6G disposition at the higher R6G perfusate

concentrations.

In normal lung tissue, P-gp is expressed in both airway epithelial cells and lung endothelial

cells (Campbell et al., 2003; Lechapt-Zalcman et al., 1997; Demeule et al., 2001). Campbell et

al. (2003) suggested a protective role of epithelial P-gp as a barrier to xenobiotic transfer from

alveoli to pulmonary interstitium and blood. We have focused on the role of P-gp in pulmonary

drug accumulation from the circulation where a protective role of P-gp from blood borne

xenobiotics is of great potential importance as well as an even larger potential role in limiting

pulmonary accumulation of a large number of systemic therapeutic agents where the lung is the

target organ. The accessibility to this transporter from the vascular space and the reported

presence of P-gp in pulmonary endothelial cells suggests endothelial P-gp moderates pulmonary

R6G accumulation (Demeule et al., 2001). However, pulmonary epithelial P-gp must also be

considered. Epithelial P-gp would transport and retain R6G in the airspace rather than transport

it out of the lung back into the circulation. Inhibition of epithelial P-gp would therefore decrease

net lung R6G accumulation. This is the exact opposite of our results, suggesting epithelial P-gp


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contributes little to the observed kinetics of pulmonary R6G accumulation from the vascular

space.

The promiscuous nature of P-gp substrate specificity parallels the affinity of lipophilic amine

drugs for lung tissue. Depending on the drug, accumulation of lipophilic amines in the intact

lung results from both rapidly and slowly equilibrating interactions with lung tissue (Audi et al.,

1995; 1996; 1998; Roerig et al., 1999). To study this differential amine interaction with

macromolecules and/or partitioning among subcellular organelles, we developed kinetic models

of pulmonary drug disposition to identify and characterize kinetic parameters descriptive of

different drug-tissue interactions in the vascular and extravascular space (Audi et al., 1998; 2002;

Roerig et al., 1999). We used a similar approach to develop a kinetic model for pulmonary R6G

accumulation in the intact lung that includes kinetic parameters descriptive of the role of P-gp

mediated efflux and other drug dispositional mechanisms inferred by the data in Figures 1

through 4. Based on Figure 3, P-gp mediated efflux of R6G is saturable and is assumed to

follow Michaelis-Menton kinetics as proposed by others (Weiss and Kang, 2002). The extensive

net accumulation of R6G over the 90 min of recirculation (Figures 1,2,4 and 5) indicates that at

steady state, distribution is far in favor of the lung, suggesting a large apparent lung tissue

volume of distribution. Since R6G is known and marketed as a mitochondrion selective dye,

compartment 3 (Figure 6) represents, at least in part, sequestration in a subcellular organelle

(Haugland, 2002). Other slowly equilibrating R6G-lung tissue interactions may also be involved

but cannot be separately identified with the present data. Weiss and Kang (2002) developed a

similar compartmental model to describe the role of P-gp in idarubicin disposition in the isolated

perfused heart and proposed a carrier-mediated transport process from compartment 2 to 3.

Again, this cannot be separately identified from the data presented here. The model (Figure 6)


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developed from these considerations was simultaneously fit to the data in Figures 2 and 4 and, as

can be seen, resulted in a reasonably good fit (solid line) to the experimental data points. To

explain (fit) all the data in Figures 2 and 4, it was necessary to include an additional dispositional

kinetic parameter, Ki2, that describes inhibition of R6G accumulation in compartment 3 by the P-

gp inhibitor, GF120918. The model was further tested by using the model parameter estimates

obtained from fitting the data in Figures 2 and 4 to predict the shape of the outflow curves in

Figure 5 where the P-gp inhibitor GF120918 was added at various times after the start of R6G

recirculation. As can be seen from the solid lines in Figure 5, the model faithfully predicts the

increase in R6G accumulation that occurs after GF120918 addition at different times during the

recirculation period.

The kinetic model parameters obtained from numerically solving equations (1-3) are shown in

Table 1. The effect of P-gp mediated R6G efflux relative to the other dispositional kinetic

processes can be examined by comparing the rate constants (min−1) for P-gp mediated efflux

(kpgp), non-P-gp mediated efflux (kout), diffusion into the lung (kin), and sequestration into

compartment 3 (k23) using the model parameter values in Table 1. If P-gp were fully inhibited or

not present, sequestration in compartment 3 (k23 = 0.22 min−1) would dominate over back

diffusion (kout = 0.011 min−1) resulting in near unidirectional influx and sequestration in the lung.

This is supported by the fact that after 90 min of R6G recirculation in the presence of an inhibitor

(Figure 1), 87.5±1.1% (SE) of the R6G was in the lung. The value of kpgp of 1.44 min−1 is two

orders of magnitude greater than that of kout, suggesting P-gp mediated efflux is by far the

dominant efflux mechanism. In addition, kpgp is 15 times greater than kin = 0.093 min−1 and 6

times the rate constant for sequestration (k23 = 0.22 min−1) resulting in P-gp mediated efflux


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significantly moderating the amount of R6G available for disposition into compartment 3 and the

lung as a whole.

The inhibition of R6G accumulation into compartment 3 in the presence of GF120918 is

interesting since it poses a mechanism that would offset the effect of P-gp inhibition. GF120918

is a lipophilic amine and inhibition of sequestration of one lipophilic amine by another in the

lung has been reported (Anderson et al., 1974; Wilson et al., 1979; Roerig et al., 1989). This

mechanism may be of some importance since reversal or modulation therapy using inhibitors of

P-gp has been proposed and studied as a method of reversing the development of multidrug

resistance in cancer chemotherapy (Ford and Hait, 1990).

Others have proposed models to estimate kinetic parameters to provide insight into the role of

P-gp mediated drug efflux in drug disposition. Loetchutinat et al. (2003) estimated the rate

constants, ka and k, for P-gp mediated efflux and passive membrane permeability, respectively,

of different rhodamine dyes in a human leukemia cell line. For R6G, the ratio of ka/k was about

29, indicating P-gp also dominated the rate of R6G efflux out of the cell in their studies.

Wielinga et al. (2000) also determined a kinetic parameter for passive permeation and a measure

of P-gp mediated efflux using four anthracycline analogs in an MDR cell line. They concluded

that passive permeation plays a substantial role in determining the drug resistance for these

anthracyclines. Wielinga et al. (2000) also identified a rate constant, ks, for passive transport

between an inner compartment and a shell compartment of the cell. Based on their data, like our

findings with R6G, the rate constant for transport into an intracellular sequestered compartment

was greater than that of the passive permeation parameter. The results of both of the above

studies demonstrated that the difference for passive membrane permeablility between the

different compounds studied did not parallel the differences in the derived rate constants for P-gp


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mediated efflux, with high lipophilicity having more effect on passive efflux. Weiss and Kang

(2002) developed a whole organ model for evaluating the role of P-gp in the uptake of idarubicin

in the isolated perfused rat heart. Their three compartment model contained kinetic parameters

descriptive of different intracellular dispositional processes. They concluded that the rate of

idarubicin uptake in the heart represents a balance between two opposing processes: active P-gp

mediated efflux and uptake into mitochondria. These studies, together with the present work, all

serve to emphasize the importance of competing intracellular processes when defining the impact

of P-gp mediated drug efflux on overall tissue disposition.

In conclusion, these studies demonstrate a functional P-gp mediated drug efflux transporter in

the lung that significantly moderates the disposition of R6G between the vascular and

extravascular space in the lung. Most importantly, the experimental protocol presented, together

with the proposed kinetic model and appropriate experimental data sets can provide estimates of

kinetic parameters that are descriptive of the different dispositional mechanisms involved in

pulmonary drug accumulation. Reliable estimates of all of these kinetic parameters will be

essential in assessing the role of P-gp mediated drug efflux in the net pulmonary accumulation of

a large variety of therapeutic agents.


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Footnotes This work was supported by the Department of Veterans Affairs and the National Institutes of

Health grant HL24349. This work was presented, in part, at the Experimental Biology 2002 and

2003 Meetings in New Orleans, LA and San Diego, CA, respectively.


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Legends

Figure 1. Total R6G (nmol) in the rabbit lung vs. recirculation time. Initial R6G concentration

was 2.5µM. Verapamil (0.1mM) or GF120918 (20µM) was recirculated through the lung for 5

min prior to addition of R6G to the perfusate reservoir. Data is a mean ± SE for control (n=6)

and GF120918 inhibited (n=3) and * indicates data points in GF120918 inhibited lungs

significantly greater than in control lungs (t-test, P<0.002). The data with verapamil is from a

single experiment.

Figure 2. Total R6G (nmol) in the lung vs. time during recirculation with different initial R6G

concentrations shown. Data is the mean ± SE. For 1.25, 5.0 and 8.5µM R6G concentrations

n=3, for the 2.5µM concentration n=6 and for the 12.5µM concentration n=4. Solid lines are the

model fits to the data.

Figure 3. Fraction of total R6G in the lung 90 min after the start of recirculation vs. the total

nmol initially added to the perfusate reservoir. Data is the mean ± SE and is calculated from the

90 min time points in Figure 2. Solid line calculated from the model fit to the daa in Figure 2.


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Figure 4. Total R6G (nmol) in the lung vs. recirculation time with different concentrations of

GF120918 in the perfusate. Closed circles represent control data (no GF120918, n=6), closed

triangles are 2µM GF120918 (n=3), open circles are 10µM GF120918 (n=2), open triangles are

20µM GF120918 (n=3) and closed diamonds are 70µM GF120918 (n=2). Data is the mean ± SE

and the solid lines are the model fits to the data.

Figure 5. Total R6G (nmol) in the lung vs. recirculation time. The P-gp inhibitor, GF120918,

was added after the start of R6G recirculation at the times indicated by the arrows. Data in each

panel are the mean ± SE and for panels a, b, c, and e, n=3 and in panels d and f, n=2. The solid

lines are the model predictions using the model parameter values estimated from the data in

Figures 2 and 4.

Figure 6. Compartmental model for R6G disposition in the rabbit lung. The model variables and

parameters are defined in Results. Compartment 1 represents the vascular space while

compartment 2 and 3 are intracellular sites of R6G-tissue interactions.


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Table 1: Estimated Values of Kinetic Model Parameters

The kinetic model parameters Vmax21, Km21, V2, PS, k23, k32, Ki1 and Ki2 were estimated by fitting

the solutions of equations (1-3) to data sets in Figures 2 and 4. kpgp, kin and kout were calculated

from the kinetic model parameters as shown in the table. The parameter values are the mean ±

the 95% confidence intervals.

Model parameters Estimated values 95% confidence interval

Vmax21 (nmol/min) 56.6 ± 14.4

Km21V2 (nmol) 39.4 ± 20.0

kpgp = Vmax21/(Km21V2) (min−1) 1.44 ± 0.72

kin = PS/V1 (min−1) 0.093 ± 0.01

kout = PS/V2 (min−1) 0.011 ± 0.02

k23 (min−1) 0.23 ± 0.15

k32 (min−1) 0.006 ± 0.02

Ki1/λ (µM) 0.36 ± 0.29

Ki2/λ (µM) 1.23 ± 1.3


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