a comprehensive study demonstrating that p-glycoprotein function is directly affected by changes in...

A Comprehensive Study Demonstrating That P-glycoproteinFunction Is Directly Affected by Changes in pH: Implicationsfor Intestinal pH and Effects on Drug Absorption

PALLABI MITRA,1 KENNETH AUDUS,1 GERVAN WILLIAMS,2 MEHRAN YAZDANIAN,2 DEBORAH GALINIS2

1Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, Kansas

2World Wide Drug Development, Cephalon, Inc., West Chester, Pennsylvania

Received 21 January 2011; revised 11 March 2011; accepted 12 April 2011

Published online 27 April 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22596

ABSTRACT: The purpose of this study was to investigate whether changes in the pH of thegastrointestinal tract can directly affect P-glycoprotein (P-gp) function. The effect of changesin extracellular pH on P-gp functionality was examined by testing colchicine (a nonionizableP-gp substrate) in bidirectional Caco-2 and MDR1–Madine Darby canine kidney (MDCK) cellpermeability assays, in which the pH of the apical and basolateral chambers was varied. Re-duction of the pH from 7.4 to 5.0 and 4.5 markedly increased the apical-to-basolateral flux ofcolchicine and reduced the basolateral-to-apical flux. The efflux ratio for colchicine was reducedto 1.2 at pH 4.5, compared with values greater than 20 that were measured in the pH range of5.5–7.4. A similar result was obtained when MDR1–MDCK cells were used in the bidirectionalpermeability studies. Other nonionizable P-gp substrates (digoxin, dexamethasone, paclitaxel,and etoposide) responded to acidic pH (4.5) in a manner similar to colchicine. Reduced P-gpATPase activity is a reason for the diminished P-gp function observed at pH 4.5. © 2011 WileyLiss, Inc. and the American Pharmacists Association J Pharm Sci 100:4258–4268, 2011Keywords: ABC transporters; absorption; active transport; bioavailability; Caco-2 cells; drugtransport; efflux pumps; gastrointestinal; intestinal absorption; MDCK cells

INTRODUCTION

Efflux transporters play a major role in chemother-apy resistance as well as drug distribution anddisposition.1 One subset of transporters has re-cently been noted as clinically important: the fam-ily of adenosine triphosphate (ATP)-binding cassette(ABC) efflux transporters.2 An important member ofthis family, P-glycoprotein (P-gp, MDR1, or ABCB1),was the first mammalian multidrug-resistant (MDR)-linked transporter protein to be discovered,3 and ithas been studied extensively for over 25 years. P-gpis functional in several tissues such as intestine, liver,kidney, and also at the blood–brain barrier. It is wellknown that its location in tissues affects the dispo-sition of pharmaceutical products by reducing theirabsorption and/or enhancing their elimination.4 TheUS Food and Drug Administration recommends test-ing of investigational drugs as substrates, inhibitors,

Correspondence to: Deborah Galinis (Telephone: 610-883-5898;Fax: 610-738-6305; E-mail: [email protected])Journal of Pharmaceutical Sciences, Vol. 100, 4258–4268 (2011)© 2011 Wiley-Liss, Inc. and the American Pharmacists Association

and inducers of P-gp. Drug–drug interactions, whichare P-gp mediated, are also clinically important, par-ticularly for compounds that have a narrow therapeu-tic index such as digoxin.5,6

In the intestine, P-gp is located on the lumen-facing membrane of the enterocytes, and its ex-pression increases along the length of the intestinefrom stomach to colon.7 It limits the bioavailabil-ity of some drugs that have been determined tobe P-gp substrates in vitro.8 Attempts at overcom-ing P-gp-mediated efflux of investigational drugs aremostly focused on structural modifications to reduceP-gp recognition. However, one of the pharmacolog-ical parameters that modify intestinal drug absorp-tion is pH. The pH varies along the gastrointestinaltract as follows: stomach (1–2), duodenum (4–5.5),jejunum (5.5–7), ileum, colon, and rectum (7–7.5).9

The pH variation along the gastrointestinal tract haslong been exploited by the pharmaceutical industryto design formulations that have improved solubil-ity, stability, and regionally targeted release profiles.Medications prescribed for gastroesophageal refluxdisease, such as proton pump inhibitors (PPIs) and

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EFFECTS OF CHANGES IN pH ON FUNCTIONS OF P-GLYCOPROTEIN 4259

histamine H2 receptor antagonists, elevate intragas-tric pH. Coadministration of these drugs can alter thepharmacokinetics of other medicinal drugs.10,11 Thislikely results from altered physicochemical propertiesas a result of elevated pH and/or the direct inhibitionof the efflux transporters by PPIs.10,11 The effect ofpH on ionization and how it indirectly affects effluxactivity are well recognized.12 Much less is knownabout any direct effects of pH on the function of effluxtransporters.

There are several publications in which the effect ofpH on the function of the P-gp efflux transporter hasbeen probed and the available data are contradictoryas some studies have concluded that pH does not af-fect P-gp functionality, whereas others have deducedthat this function is altered by pH.12–15 For example,Altenberg et al.13 performed efflux studies with the P-gp substrate rhodamine 123 in various drug-resistantcell lines and showed that changes in the intra- orextracellular pH environment did not mediate P-gpactivity in a pH range of 5.5–7.5. Neuhoff et al.12 gen-erated data that were in agreement with this conclu-sion, by demonstrating that the efflux ratio (ER) ofdigoxin across Caco-2 is independent of the apical pH(5.0–8.0). However, Thews et al.15 reported that thereduction of extracellular pH to 6.6 from 7.4 led to

doubling of P-gp-mediated efflux of rhodamine 123.They also reported that the cytotoxicity of daunoru-bicin, a P-gp substrate, was reduced in the acidic en-vironment, which was attributed to higher activity ofthe efflux transporter. Because daunorubicin is a ba-sic drug (pKa of 8.4),16 the lowered activity could beexplained by the pH-partition hypothesis in which thecell membrane favors the permeation of unchargedspecies as opposed to the ionized fraction. Thus, theuptake of the weakly basic daunorubicin is impairedat a low pH. With the exception of the Neuhoff et al.12

study, in all of the previous studies, the P-gp sub-strates and inhibitors have been ionizable in the pHranges studied, which most likely contributed to thecontradictory results. In addition, some compoundschosen in these studies were substrates for other drugtransporters.17

The objective of the present study was to eluci-date the effects, if any, of variable apical (represent-ing luminal) pH (4.5–) on P-gp activity in Caco-2cells. Colchicine and other neutral P-gp substrates(digoxin, taxol, dexamethasone, and etoposide) werechosen for the test. In theory, the change in extracellu-lar pH should not alter the passive permeability of theneutral substrates (Fig. 1). If pH-dependent changesin passive permeability can be ruled out, observed

Figure 1. Structures of the neutral P-gp substrates.

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011

4260 MITRA ET AL.

differences in the ERs can be attributed to the ac-tive efflux being affected by pH. The results obtainedin Caco-2 cells were compared with those obtainedin Madine Darby canine kidney (MDCK) cells trans-fected with the human MDR1 gene (MDR1–MDCKcells), which have been shown to be a useful modelin screening the P-gp substrate activity of drugs.18 IfP-gp function can be manipulated with pH changes,then it may be possible to develop drug formulationsthat transiently modify the intestinal pH to improvedrug absorption.

MATERIALS AND METHODS

Materials

Dulbecco’s modified Eagle’s medium (DMEM)buffered with 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES) was obtained fromGibco Invitrogen (Carlsbad, California). All othercell culture reagents were obtained from Mediat-ech (Herndon, Virginia). Transwell

TMplates were

obtained from Costar Corporation (Acton, Mas-sachusetts). Colchicine and unlabeled digoxin werepurchased from Acros Organics (Geel, Belgium)[3H]-Digoxin (40:Ci/nmol) and Ultima Gold scintil-lation cocktail were obtained from PerkinElmer LifeSciences (Waltham, Massachusetts). [3H]-Paclitaxel(16.1:Ci/nmol) was purchased from Moravek Bio-chemicals, Inc. (Brea, California). Alprenolol anddexamethasone were purchased from MP Biomedi-cals, Inc. (Solon, Ohio). Zosuquidar trihydrochloride(LY3359793) was purchased from API Services Inc.(Westford, Massachusetts). Lucifer yellow (LY),testosterone, etoposide, sodium acetate trihydrate, 2-(N-morpHolino) ethanesulfonic acid (MES), HEPES,and all other chemicals required to measure ATPaseactivity were obtained from Sigma–Aldrich (St. Louis,Missouri). Caco-2 cells originating from a humancolorectal carcinoma were acquired from AmericanTissue Culture Collection (Manassas, Virginia). TheMDR1–MDCK19,20 were obtained from Dr. Piet Borst(The Netherlands Cancer Institute, Amsterdam, theNetherlands). P-gp expressing Spodoptera frugiperda(Sf9) membranes were purchased from GENTEST(Woburn, Massachusetts).

Cell Culture

Caco-2 cells and MDR1–MDCK cells were grownat 37◦C in an atmospHere of 5% CO2 and 90%relative humidity. Caco-2 cells were maintained inDMEM supplemented with 10% heat-inactivated fe-tal bovine serum, 100 IU/mL penicillin, 100:g/mLstreptomycin, and 10 mM nonessential amino acids.The MDR1–MDCK cells were maintained using thesame culture conditions with an exception of the me-dia being supplemented with 20 nM colchicine to pre-

serve selective pressure. Confluent cell monolayerswere subcultured every 7 days by treatment with0.25% trypsin containing 1 mM ethylenediaminete-traacetic acid.

Bidirectional Transport Studies

Caco-2 cells were seeded at a density of 80,000 cells/cm2 in 12-well plates on polycarbonate filters (CostarTranswellTM) cell culture inserts (purchased fromCorning Life Sciences, Lowell, MA), 12.0 mm diam-eter, 3.0:m pore size). Caco-2 cells were grown onfilters for 21–25 days. The MDR1–MDCK cells wereseeded at a density of 50,000 cells/cm2 in 12-wellplates on polycarbonate filters (Costar Transwell

TM

cell culture inserts, 12.0 mm diameter, 0.4:m poresize). The MDR1–MDCK cells were grown for 6–8 days. Caco-2 cells of passage numbers 41–60 andMDR1–MDCK cells of passage numbers 15–37 wereused for all experiments in this paper.

Prior to the transport studies, the culture mediumwas replaced with Hank’s Buffered Salt Solution(HBSS) at pH 7.4 and equilibrated for 30 min at 37◦C.For experiments that included a standard P-gp in-hibitor, zosuquidar trihydrochloride21 (concentration1 :M in HBSS) was added 30 min prior to the start ofthe transport experiment. HBSS was buffered with10 mM sodium acetate for pH 4.5 and 5.0; 10 mMMES for pH 5.5 and 6.0; and 25 mM HEPES for pH7.4. Transport studies that involved a pH gradientacross the monolayer (apical chamber contained adrug/buffer solution at pH values varying from 4.5 to7.4, whereas the basolateral chamber contained drug/HEPES-buffered HBSS at pH 7.4) were attempted.Measurement of the pH of the apical and basolat-eral chambers at the end of the experiment indicatedthat the chambers were equilibrating (Table 1); there-fore, transport experiments were performed withboth chambers containing drug/buffer solutions at thesame pH. Drug solutions were prepared in HBSS (atthe adjusted pH value for the experiment) at a concen-tration of 100:M for LY and 20:M for digoxin (3:Ci/mL of [3H]-digoxin), colchicine, etoposide, dexametha-sone, and testosterone and then added to either theapical (A) or basolateral (B) sides of the cell monolay-ers. A concentration of 0.6:M paclitaxel (3:Ci/mL of

Table 1. Buffer pH Values at the Beginning and End ofthe Bidirectional Caco-2 Permeability Studies

Apical (A) pHt = 60 min

Basolateral (B) pHt = 60 min

pH (A/B) t = 0 HBSS HEPES HBSS HEPES

4.5/7.4 5.5 6.0 7.2 7.45.0/7.4 5.9 6.1 7.0 7.35.5/7.4 5.7 5.7 6.9 7.56.0/7.4 6.1 6.1 7.5 7.4

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011 DOI 10.1002/jps


[3H]-paclitaxel) was used due to its low aqueous solu-bility. The volume of the solution added to the apicalchamber was 0.5 mL, whereas that added to the baso-lateral chamber was 1.0 mL. Transport experimentswere carried out in an oven maintained at 37◦C. Theplates were shaken at 100 rpm using an orbital shaker(VWR, West Chester, Pennsylvania) for the durationof the experiment (1 h). At discrete time intervals,200:L of the receiver chamber solution was with-drawn and replaced with fresh buffer solution (sinkcondition).

Colchicine, dexamethasone, etoposide, and testos-terone samples were analyzed by liquid chromatog-raphy–mass spectrometry (LC–MS) [an integratedCohesive Technologies LX-2 series liquid chromatog-raphy system coupled with an Applied BiosystemsMDS-SCIEX 4000 Q Trap triple quadrupole massspectrometer (Foster City, California). The radiola-beled digoxin and paclitaxel were measured using ascintillation counter (Wallac Microbeta TriLux 1450LSC & Luminescence Counter; PerkinElmer Analyt-ical Sciences). The LY samples were measured usinga Wallac 1420 Multilabel Reader at 485 nm excitationand 535 nm emission (PerkinElmer Life Sciences).

Drug Uptake Studies

All drug uptake experiments were performed usingMDR1–MDCK cells seeded in six-well tissue cultureplates at a density of 25,000 cells/cm2 and grown for5 days. Prior to the uptake studies, the cells werewashed twice with prewarmed (37◦C) HBSS buffer so-lution. The buffer solutions at various pH values wereprepared as previously described. The cell monolay-ers were then incubated with 4 mL of test compoundsolution (1:M) at 37◦C on an orbital shaker (rota-tion speed 100 rpm) for up to 1 h. At discrete timeintervals, the drug solution was removed from thewells and the cells were washed twice with ice-coldHBSS and lysed in 1 mL of freshly prepared lysisbuffer, which consisted of 20 mM ammonium acetate/methanol (4:1) and alprenolol (20 ng/mL) as an in-ternal LC–MS standard. Contents of each well werecentrifuged at 24104 g for 15 min and 200:L of thesupernatant was then transferred for quantitation byLC–MS. The concentration of the test drug was thenmeasured using a standard curve prepared by spik-ing known concentrations of the drug into the lysisbuffer. The percent uptake was calculated by dividingthe amount of drug recovered from the lysed cells bythe amount of drug applied to the cells. Uptake datawere normalized for protein content. For the uptakestudies with a P-gp inhibitor, the cells were preincu-bated with a 1:M solution of zosuquidar21 for 30 min.The cells were then washed with fresh HBSS and thedrug/inhibitor solution (1:M for both) was applied,and the experiment was completed as outlined above.

Protein content of MDR1–MDCK cells grown in in-dividual wells of the six-well plates was determinedseparately by Quant-iT

TMProtein Assay Kit with a

QubitTM

fluorometer (Invitrogen). The extraction so-lution containing ammonium acetate and methanolwas not used to prevent damage to the extracted pro-teins. Instead, lysis buffer containing Triton X-100was used as reported in the literature.22 Cells weredissolved in 1 mL of freshly prepared lysis buffer con-sisting of 1% Triton X-100 (v/v), 20 mM Tris–HCl, and150 mM NaCl.

P-gp ATPase Assay

Drug- and pH-stimulated P-gp ATPase activity wasestimated by measuring the inorganic phosphate re-leased from ATP, according to the Gentest protocolwith modification in buffer pH. The reaction was per-formed by preincubating 20:L of P-gp membranes(2 mg/ml) at 37◦C for 10 min in 20:L of Tris–MESbuffer at varying pH values ranging from 4.5 to 6.8(50 mM Tris–MES for pH values 5.0–6.8 and 25 mMsodium acetate for pH 4.5) with or without 300:Msodium orthovanadate. The reaction was started bythe addition of 20:L of 12 mM MgATP (magnesiumsalt) and was stopped 20 min later by the additionof 30:L of 10% sodium dodecyl sulfate containingantifoam A. Detection reagent (200:L) was addedto the reaction and incubated at 37◦C for an ad-ditional 20 min. The absorbance was measured at650-nm wavelength in a SpectraMax 190 microplatespectrophotometer (Molecular Devices, Sunnyvale,California). The drug- or pH-stimulated, vanadate-sensitive ATPase activity [nmol/(min mg of pro-tein)] was determined as the difference between theamounts of inorganic phosphate released from ATP inthe presence and absence of orthovanadate phosphatestandards were prepared in each plate, and verapamil(20:M) served as a positive control.

Permeability Calculations

The following equation was used to calculate appar-ent permeability23 in either the apical-to-basolateraldirection (Papp, A–B) or basolateral-to-apical direction(Papp, B–A):

Papp = dC/dt × Vr/A × Cd

where dC/dt is the change in concentration in the re-ceiver compartment with respect to time, Vr is thevolume of the receiver compartment, A is the growthsurface area, and Cd is the initial concentration of thedrug in the donor compartment. Mass balance wascalculated for each experiment. In all transport ex-periments, the mass balance was greater than 70%.

An ER was calculated as follows:

ER = Papp, B−A/Papp, A−B


4262 MITRA ET AL.

Table 2. Apparent Permeability Coefficients (Papp) of Lucifer Yellow and Testosterone Across Caco-2 Cell Monolayers as a Function ofpH

Compound pH (A/B) Papp, A–B × 106 (cm/s) Papp, B–A × 106 (cm/s) ER

Lucifer yellow (LY) 4.5/7.4 0.09 ± 0.01 – –LY 5.0/7.4 0.12 ± 0.06 – –LY 7.4/7.4 0.07 ± 0.01 – –LY 4.5/4.5 0.08 ± 0.01 – –LY and colchicine (20 :M) 4.5/7.4 0.18 ± 0.04Testosterone 4.5/4.5 28.0 ± 2.8 27.9 ± 1.3 1.0 ± 0.1Testosterone 7.4/7.4 28.0 ± 2.6 25.2 ± 1.2 0.9 ± 0.1

Efflux ratio (ER) = Papp,B–A/Papp,A–B.Data are plotted as mean ± SD (n = 3).

Statistical Analysis

Statistical significance was calculated with one-wayanalysis of variance followed by Dunnet’s post-hoccomparison test (GraphPad Prism Software, version5, La Jolla, California). A p value of less than 0.05was considered to be statistically significant.

RESULTS

Caco-2 Cell Monolayer Integrity

The Caco-2 cell monolayer integrity for each set ofpassaged cells was routinely tested at pH 7.4 withthe paracellular permeability marker LY. Monolay-ers with LY Papp values greater than 0.3 × 10–6 cm/s were considered to be unsuitable for the transportstudies. The permeability coefficients of LY remainedconstant over the pH range 4.5–7.4 for up to 1 h(Table 2), indicating that pH did not affect the para-cellular route. This was in agreement with the previ-ous reports, wherein LY and/or mannitol were usedas paracellular permeability markers in this pHrange.23,24

To monitor potential pH-dependent changes in themembrane composition of the Caco-2 cell monolay-ers, the bidirectional transport of testosterone wasmeasured at pH 4.5 and 7.4 (Table 2). Testosteroneis a neutral compound that undergoes transcellulartransport. It is known to be a substrate of CYP3A4,but it is not a P-gp substrate.25,26 Testosterone perme-ability coefficients were similar at pH 4.5 and 7.4 (Ta-ble 2), providing additional evidence that the Caco-2cell monolayers remain intact during the transportexperiment. Furthermore, the testosterone Papp,A–Bvalue measured at pH 7.4 is in agreement with thepublished value (Papp, A–B = 28.0 ± 2.6 × 10–6 cm/s).27

pH-Dependent Bidirectional Transport of Colchicineand Digoxin Across Caco-2 Cell Monolayers

Generally, compounds exhibiting asymmetrical trans-port and ER values of 2 or more are considered to besubstrates of efflux transporters.23 Colchicine has apKa of 12.428 and is unionized within the pH range4.5–7.4. Hence, the passive transport of colchicine

should not be affected by pH. Thus, any variances inthe ER values due to pH change would indicate thatactive transport of colchicine is being affected by pH.Testosterone is a neutral compound that is not subjectto efflux in Caco-2 cells.29 Its apparent permeabilitycoefficient was not affected by low pH, confirming thatpassive diffusion (transcellular) is unchanged at lowpH. It has also previously been demonstrated withanother neutral compound caffeine that pH as low as4.0 does not affect passive diffusion.23

The colchicine bidirectional transport experimentswere performed at several pH values (Fig. 2), andoverall an increasing trend in Papp, A–B and a decreas-ing trend in Papp, B–A was observed with reduction ofpH, with the most dramatic changes being noted atpH values 4.5 and 5.0. The ER values were 97% and88% lower at pH values of 4.5 and 5.0 from that at 7.4.The ER was not significantly different in the pH in-terval 5.5–7.4. The apparent permeability coefficientof LY in the presence of 20:M colchicine at an apicalpH of 4.5 (Table 2) indicated that the altered ER wasnot a result of the colchicine affecting the monolayerintegrity at lower pH values.

The markedly lower ER observed at pH 4.5 and 5.0indicated that active transport of colchicine is reducedat acidic pH. It has been demonstrated previouslythat the polarized transport of colchicine across Caco-2 cell monolayers is mediated by P-gp,30 supportingthe theory that P-gp-mediated colchicine transportis reduced at pH 4.5 and 5.0. As confirmation, thecolchicine transport was examined in the presence ofzosuquidar, a specific P-gp inhibitor.21 At pH 6.0 and7.4, the ER of colchicine was reduced to less than 2in the presence of zosuquidar (Fig. 3). This suggestedthat P-gp, and not other efflux transporters expressedin Caco-2 cells, is mediating colchicine transport. TheERs at pH 4.5 and 5.0 in the absence of zosuquidarwere similar to the values observed in the presence ofzosuquidar at pH 7.4. This confirmed that at pH 4.5and 5.0, the reduced active transport is due to reducedP-gp-mediated colchicine transport.

Neuhoff et al.12 have reported that the ER ofdigoxin across Caco-2 cell monolayers is independentof apical pH in the range 5.0–8.0. To test whether



Figure 2. Apparent permeability of colchicine across Caco-2 monolayers as a function of pH.Data are plotted as mean ± standard deviation (SD) (n = 3). * indicates p < 0.05 and suggestsstatistical significance at a particular pH with respect to the corresponding value at pH 7.4.

the pH-dependant changes observed are specific tocolchicine, the permeability of digoxin was examinedat pH values 4.5, 5.0, and 7.4 (Table 3). Similar to theobservations with colchicine, Papp, A–B increased and

Figure 3. Efflux ratio (ER) of colchicine in a Caco-2 cellpermeability assay in the presence and absence of the P-gp inhibitor zosuquidar at different pH values. Data areplotted as mean ± SD (n = 3).

Papp, B–A decreased at low pH. The changes, however,were less pronounced than colchicine, with statisti-cally significant changes observed only at pH 4.5. Ofnote, ERs of digoxin were comparable to the averageER of digoxin over the pH range 5.0–8.0 reported byNeuhoff et al.12 (8.9 ± 0.8).

pH-Dependent Bidirectional Transport of Colchicineand Other Neutral P-gp Substrates AcrossMDR1–MDCK Cell Monolayers

As further proof that the pH-dependent effluxof colchicine observed in Caco-2 cells is indeeddue to P-gp, transport studies were conducted inMDR1–MDCK cells. MDR1–MDCK cells are fre-quently used to screen for P-gp substrates as they lacksignificant levels of other transporters.18 In agree-ment with the Caco-2 cell data, the ER of colchicinechanged significantly upon lowering the apical sidepH from 7.4 to 4.5 (Table 4). Similar to what was ob-served in Caco-2 cells, there was a greater change inPapp, A–B as compared with Papp, B–A.

The pH-dependent permeability of digoxin andthree other neutral P-gp substrates, dexamethasone,etoposide, and paclitaxel, were also evaluated. TheER of all the substrates substantially decreased in


4264 MITRA ET AL.

Table 3. Comparison of the Bidirectional Transport of Colchicine and Digoxin Across Caco-2 cell Monolayers at Various pH Values

Colchicine Digoxin

pH Papp, A–B × 106 (cm/s) Papp, B–A × 106 (cm/s) ER Papp, A–B ×106 (cm/s) Papp, B–A × 106 (cm/s) ER

7.4 0.17 ± 0.01 4.69 ± 0.10 27.4 ± 1.7 1.57 ± 0.16 12.6 ± 1.3 8.0 ± 1.27.4I 0.79 ± 0.03 1.12 ± 0.08 1.4 ± 0.15.0 0.50 ± 0.11(194%)a 1.49 ± 0.18(–68%)a 3.0 ± 0.8(–88%)a 1.24 ± 0.13 12.4 ± 0.4 10.0 ± 1.04.5I 3.00 ± 0.31 3.20 ± 0.28 1.1 ± 0.14.5 1.54 ± 0.27(806%)a 1.24 ± 0.04(–74%)a 0.8 ± 0.1(–97%)a 4.52 ± 0.22(188%)a 8.00 ± 0.40(–37%)a 1.8 ± 0.1(–78%)a

P-gp inhibitor added at pH 7.4 and 4.5, noted with an I.Data are plotted as mean ± SD (n = 3).aNumbers in parenthesis represent the percentage change in Papp or efflux ratio (ER) over the corresponding value observed at pH 7.4.

the presence of the P-gp-specific inhibitor zosuquidar,validating the presence of a P-gp-mediated efflux. Re-ducing the pH to 4.5 also resulted in a sharp decreasein the ER for all, suggesting loss of P-gp function at pH4.5. Analogous to colchicine, the ER change for digoxinand dexamethasone resulted from bigger changes inPapp, A–B than in Papp, B–A. For paclitaxel and etopo-side, there were equivalent changes in Papp, A–B andPapp, B–A. In MDR1–MDCK cells, the extent of changefor digoxin was equivalent to colchicine. Of note, Pappvalues at pH 4.5 matched the values obtained in thepresence of the P-gp inhibitor.

pH-dependent Uptake of Colchicine in MDR1–MDCKCells

To support the conclusion that lowered pH wasaffecting P-gp function, the uptake of colchicinein MDR1–MDCK cell monolayers was examined.Colchicine uptake in MDR1–MDCK cells was foundto be constant at pH 7.4 for 1 h. In the presence ofzosuquidar, colchicine cell concentrations increasedwith time (Fig. 4), demonstrating that reduced P-gp activity resulted in increased drug accumulation.Similarly, at pH 4.5, there was an increase in the

colchicine uptake with time, suggesting diminished P-gp-mediated colchicine uptake. At pH 4.5, there wasan approximate 500% increase in colchicine accumu-lation over 1 h as compared with pH 7.4. There was noincrease in drug accumulation with time at pH 5.0.

Effect of pH on P-gp Atpase Activity

Transport of substrates by P-gp is coupled with ATPhydrolysis, with two molecules of ATP being hy-drolyzed per transport cycle.31 We hypothesized thatthe effect of pH on P-gp could be due to the pH af-fecting either the transmembrane domains or thenucleotide-binding domains (NBDs). If pH is affect-ing the NBDs, then it should be reflected on the P-gpATPase activity as well.31 The optimum pH recom-mended for the P-gp ATPase assay is 6.8, which wasthe highest pH value used in this assay.32,33 Vera-pamil stimulated ATP hydrolysis by P-gp at pH 6.8,which indicated a valid assay procedure (Fig. 5). Com-pared with pH 6.8, ATPase activity did not change aspH was reduced to 5.0. At pH 4.5, however, there wasa marked reduction in ATP hydrolysis, indicating lossof P-gp ATPase activity at pH 4.5.

Table 4. Comparison of the Apparent Permeability Coefficients of Neutral P-gp Substrates in MDR1–MDCK Cells at pH Values4.5 and 7.4 (in the Presence and Absence of the P-gp Selective Inhibitor Zosuquidar)

Substrate pH Papp, A–B × 106 (cm/s) Papp, B–A × 106 (cm/s) ER

Colchicine 7.4 0.11 ± 0.02 4.79 ± 0.08 44.2 ± 6.87.4 (inhibitor) 0.50 ± 0.06 2.40 ± 0.17 4.82 ± 0.66

4.5 0.64 ± 0.12(482%)a 3.52 ± 0.08(–27%)a 5.5 ± 1.1(–88%)a

Digoxin 7.4 0.36 ± 0.09 19.4 ± 2.8 54.4 ± 16.57.4 (inhibitor) 1.05 ± 0.2 5.03 ± 0.9 4.8 ± 1.1

4.5 1.50 ± 0.2(317%)a 11.6 ± 1.0(–40%)a 8.0 ± 1.6(–85%)a

Dexamethasone 7.4 2.50 ± 0.4 29.6 ± 1.9 11.8 ± 2.07.4 (inhibitor) 5.87 ± 0.55 7.90 ± 0.62 1.35 ± 0.16

4.5 5.16 ± 1.0(106%)a 9.61 ± 0.6(–68%)a 1.9 ± 0.4(–84%)a

Paclitaxel 7.4 1.50 ± 0.30 38.0 ± 4.5 25.4 ± 5.37.4 (inhibitor) 0.24 ± 0.03 1.32 ± 0.18 5.47 ± 0.95

4.5 2.38 ± 0.40(59%)a 16.8 ± 3.1(–56%)a 7.1 ± 1.8(–72%)a

Etoposide 7.4 0.17 ± 0.05 5.41 ± 0.6 31.1 ± 10.97.4 (inhibitor) 0.35 ± 0.06 1.57 ± 0.1 4.5 ± 0.8

4.5 0.29 ± 0.01(71%)a 1.09 ± 0.05(–80%)a 3.7 ± 0.2(–88%)a

Data are plotted as mean ± SD (n = 3).aNumbers in parenthesis represent the percentage change in Papp or efflux ratio (ER) over the corresponding value observed at pH 7.4 in the

absence of a P-gp inhibitor.



Figure 4. Uptake of colchicine in MDR1–MDCK cells atvarious pH values and in the presence of zosuquidar. Dataare plotted as mean ± SD (n = 3). * indicates p < 0.05and suggests statistical significance at a particular timepoint at pH 4.5 or pH 7.4 (+ inhibitor) with respect to thecorresponding value at pH 7.4.

DISCUSSION

The results of this study, as assessed from the trans-port and uptake of several neutral P-gp substrates,suggest that pH affects P-gp function, diminishing itat acidic pH (4.5 and 5.0), with the greatest change be-ing observed at pH 4.5. Decreased P-gp ATPase activ-ity at pH 4.5 is a reason for the loss of P-gp transportactivity. The extent to which P-gp function decreasedalso depended on the substrate, which indicated thatpH possibly also affects the substrate binding sites ofthe transport protein.

The ER value of colchicine at pH 7.4 in Caco-2 cellswas in agreement with a previously reported value.25

The ER value of colchicine (27.4) suggests that it is

Figure 5. Effect of pH on P-gp ATPase activity. Data areplotted as mean ± SD (n = 3). * indicates p < 0.05 and sug-gests statistical significance at a particular pH with respectto the corresponding value at pH 6.8.

a substrate of an efflux transporter located on theapical membrane. In addition to P-gp, Caco-2 cells ex-press other efflux transporters such as breast cancerresistance protein (BCRP) and multidrug resistanceproteins (MRP2 and MRP3)34,35 of which P-gp, BCRP,and MRP2 are located on the apical membrane.36,37

It is known that colchicine is a substrate of P-gp,17

and it was recently reported that it is a substratefor MRP2 as well;38 however, zosuquidar inhibits P-gp, but not MRP2.39 The reduction of the ER valueto less than 2 in the presence of zosuquidar clearlyindicates that efflux of colchicine in Caco-2 cells ismediated by P-gp. In the MDR1–MDCK cells, ERvalues of colchicine as well as the other substrateswere all reduced by zosuquidar, although not com-pletely less than 2. This indicated the involvementof efflux transporter(s) in addition to P-gp. For ex-ample, in a recent kinetic study, it was discoveredthat novel membrane transporters (both apical andbasolateral) assist in the P-gp-mediated transport ofdigoxin40 in MDR1–MDCK cells. However, the con-tribution of these other transporters is likely smallin comparison with P-gp because the ER values ofall substrates were substantially reduced in the pres-ence of zosuquidar, which is a selective inhibitor ofP-gp.

Exhibiting a slight contrast to the transport data,the uptake studies showed that although P-gp effluxactivity was normal at pH 5.0, it decreased sharplyat pH 4.5. The pH affected the ATPase activity of P-gp in a similar manner, suggesting that loss of P-gpATPase activity is the primary reason for diminishedP-gp efflux activity. Among the several amino acids inthe NBDs of P-gp, which are critical for binding andhydrolyzing ATP, are aspartate and glutamate.31,41

Mutations in these amino acid residues result in di-minished or complete loss of ATP hydrolysis.31,41 ThepKa of aspartate and glutamate side chains are 3.8and 4.3, respectively, and the side chains are likelyto undergo a change in ionization status as the pH isreduced to 4.5 from the normal value of 7.4. The aspar-tate is one of the residues that forms an H-bond witha water molecule and initiates nucleophilic attack onATP.31,41 Change in ionization status can affect theinteraction of these amino acids with ATP and hence,its hydrolysis.

It is interesting to note that the extent to whichP-gp is inactivated at pH 4.5 is substrate de-pendant as well. The magnitude of change wasdifferent for each substrate, with the greatest dif-ference for colchicine. With colchicine, diminishedtransport was observed from pH 5.5, but for digoxin,it was observed only when the pH was reduced to4.5. The effect on Papp, A–B was larger than thaton Papp, B–A for colchicine, digoxin, and dexametha-sone, whereas it was equivalent for etoposide andpaclitaxel.


4266 MITRA ET AL.

The X-ray crystal structure of P-gp with a boundsubstrate has not yet been elucidated, but sev-eral studies indicate multiple binding sites on P-gp. Shapiro and Ling42 proposed at least two bind-ing sites on P-gp: an H-site (binds Hoechst 33342and colchicine) and an R-site (binds rhodamine 123).They found that vinblastine and etoposide have equalaffinity for both these sites. Following this study,as many as seven binding sites on P-gp have beenproposed.43,44 In these studies, it was reported thatvinblastine and Hoechst 33342 bind to distinct sites.Because colchicine and Hoechst 33342 bind to thesame site, we can infer that colchicine and vinblas-tine bind to distinct sites as well. Subsequent studiesutilizing pharmacophore models and QSAR studiessuggest that digoxin and vinblastine bind to the samesite on P-gp.45 From this, we can further infer thatcolchicine and digoxin bind to distinct sites. The ap-parent permeability coefficients of paclitaxel, whichalso has a distinct binding site,43,44 are the least af-fected by the decrease in pH. It is a possibility thatpH is differentially affecting these distinct bindingsites. How pH is differentially affecting the distinctbinding sites can only be speculated upon at thisstage. Several amino acids in the binding sites (as-partate, glutamate, and histidine)46 have side chainsthat are likely to undergo a change in the ioniza-tion status as the pH is reduced to 4.5 from 5.5 to6.0. Change in the ionization status can affect bind-ing and could be a reason for reduced transport atacidic pH.

The absolute values of the ERs were not thesame in the Caco-2 and the MDR1–MDCK cells.This could be due to the changes in the lipidmembrane environment and substrate interaction.47

Dissimilarities between Caco-2 and MDR1–MDCKcells have been noted such as P-gp expression lev-els and different orientations of P-gp in the cellmembranes. It has also been noted that the sub-strates can partition differently into the Caco-2 andMDR1–MDCK cell membrane bilayers.18 This canexplain the variations noted using two differentcell types.

CONCLUSION

Our data indicate for the first time that P-gp func-tion is reduced at acidic pH (4.5–5.5). It is well knownthat P-gp reduces the oral bioavailability of severaldrugs such as paclitaxel (chemotherapeutic), talinolol(antihypertensive), and digoxin (indicated in conges-tive heart failure), to name a few.8,48 Several routesare explored by the pharmaceutical industry to evadeor inhibit P-gp-mediated efflux. These include struc-tural modifications of the compounds such as increas-ing lipophilicity or reducing the number of H-bonddonors/acceptors, preparing prodrugs that prevent

recognition by P-gp and allow recognition by otheruptake transporters, incorporating pharmaceuticalexcipients that increase passive diffusion and thusreduce P-gp-mediated efflux, and incorporating phar-maceutical excipients that directly inhibit P-gp (byATP depletion).49 Gastrointestinal pH is one of thephysiological factors that affects bioavailability of P-gp substrates. Coadministration of the PPI omepra-zole, which elevates gastric pH, increases absorptionof digoxin.11,50,51 If the absorption of the basic drugquinidine is compared between the jejunum and theileum, change in the ionization status in the ileumleads to increased passive diffusion, reduced recogni-tion by P-gp, and increased absorption.52 In the fu-ture, it might be possible to exploit P-gp inactivationat pH 4.5 to develop formulations that can transientlyreduce intestinal pH to improve the oral absorptionof P-gp substrates such as paclitaxel. Transient re-duction of intestinal pH is not a novel strategy. Ithas been employed previously to reduce intestinalproteolytic activity and improve absorption.53–55 Forexample, oral administration of the synthetic pep-tide drug salmon calcitonin is limited by poor in-testinal stability. A novel formulation employing cit-ric acid to reduce duodenal/ileal pH in beagle dogsdemonstrated good correlation between intestinal pHreduction and salmon calcitonin absorption.54 Intesti-nal pH recovered to normal levels within 3 h follow-ing administration.55 Such a formulation path maybe explored to improve the absorption of P-gp sub-strates that exhibit limited bioavailability. This for-mulation strategy, of course, would be subject to moreexperimentation. First, pH alters functional activ-ity of BCRP, another efflux transporter in the lu-minal membrane of the intestinal epithelium.56 Thephysiological role of BCRP in the intestine is notyet known, but it affects the bioavailability of sev-eral small molecules by effluxing them and/or theirconjugates.57,58 Transient reduction of intestinal pHhas a chance of affecting the absorption of coadmin-istered drugs that are BCRP substrates. This sideeffect needs to be ruled out from the low-pH formu-lation. Second, several compounds that are P-gp sub-strates in vitro are not limited by this transporterin their in vivo intestinal permeation.59 Within anin vitro setting, the greater contribution of P-gp toabsorption can be due to the non-physiological natureof the transport medium as components of intestinalfluid such as sodium taurocholate inhibit P-gp.60,61 Itwould be interesting to observe whether low pH andintestinal fluid have an additive inhibitory effect onP-gp.

ACKNOWLEDGMENTS

Partial support for this work was provided by grantnumber HD039878 (to K.L.A.).



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