Celulas

REVIEW

Drug Transporters in the Lung—Do They Play a Role in theBiopharmaceutics of Inhaled Drugs?

CYNTHIA BOSQUILLON

Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, University Park, Nottingham NG7 2RD,United Kingdom

Received 8 July 2009; accepted 25 September 2009

Published online 30 November 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21995

Additional Sversion of this a

Corresponde8466078; Fax: 4E-mail: cynthia

Journal of Pharm

� 2009 Wiley-Liss

2240 JOURN

ABSTRACT: The role of transporters in drug absorption, distribution and elimination processesas well as in drug–drug interactions is increasingly being recognised. Although the lungsexpress high levels of both efflux and uptake drug transporters, little is known of the implica-tions for the biopharmaceutics of inhaled drugs. The current knowledge of the expression,localisation and functionality of drug transporters in the pulmonary tissue and the few studiesthat have looked at their impact on pulmonary drug absorption is extensively reviewed. Theemphasis is on transporters most likely to affect the disposition of inhaled drugs: (1) the ATP-binding cassette (ABC) superfamily which includes the efflux pumps P-glycoprotein (P-gp),multidrug resistance associated proteins (MRPs), breast cancer resistance protein (BCRP) and(2) the solute-linked carrier (SLC and SLCO) superfamily to which belong the organic cationtransporter (OCT) family, the peptide transporter (PEPT) family, the organic anion transporter(OAT) family and the organic anion transporting polypeptide (OATP) family. Whenever avail-able, expression and localisation in the intact human tissue are compared with those in animallungs and respiratory epithelial cell models in vitro. The influence of lung diseases or exogenousagents on transporter expression is also mentioned. � 2009 Wiley-Liss, Inc. and the American

Pharmacists Association J Pharm Sci 99:2240–2255, 2010

Keywords: drug inhalation; pulmonary del
ivery; multidrug resistance transporters; peptidetransporters; organic cation transporters; organic anion transporters; Calu-3 cells; isolatedperfused lungs; cell culture; permeability
INTRODUCTION

The implication of membrane transport proteins inthe pharmacokinetic, pharmacodynamic (PKPD) andsafety profiles of a large range of drugs is now wellestablished, although probably not yet fully appre-ciated.1,2 Due to their critical role in the successfuldevelopment of drug candidates,1 the study of drugtransporters is currently the topic of intense research.The focus is essentially on transporters in theintestine, liver, kidney, brain and their relevance todrug disposition in those organs. Comparatively, theinfluence of transporters on the disposition of inhaleddrugs has hardly been investigated, although access

upporting Information may be found in the onlinerticle.nce to: Cynthia Bosquillon (Telephone: 44-115-4-115-9515122;[email protected])

aceutical Sciences, Vol. 99, 2240–2255 (2010)

, Inc. and the American Pharmacists Association

AL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 201

to drug target sites in the lung tissue might partlydepend on their activity.

Although less than 40 drugs are currently admi-nistered by the pulmonary route, drug absorption,distribution and elimination processes in the lungremain overall poorly understood. Hence, the PKPDprofile of inhaled drugs is suboptimal in most cases.Many transporters expressed in the intestine, liver,kidney or brain are also present in the lung (Tab. 1;Fig 1) and evidence indicates drugs commonlyadministered as aerosols in the treatment of respira-tory diseases, for example glucocorticoids, andcationic b2-agonists, might interact with thosetransporters (Tab. 2). An evaluation of the impactof active transport systems on drug absorption fromthe lungs would help in the interpretation andoptimisation of PKPD parameters after drug inhala-tion. This task is, nevertheless, complicated by thecomplexity of the organ and the lack of validatedmodels to investigate drug transport mechanisms inthe lung.

0

Table 1. Summary of Drug Transporter Expression in Human Lungs

ProteinName

GeneSymbol

Expression inHuman Lungs

CellularDistribution

CellularLocalisation Refs.

ABC transportersP-gp ABCB1 Moderate Bronchial/bronchiolar

epitheliumApical 22–27

Alveolar epithelium(contradictory data)

Apical

Alveolar macrophagesendothelium

MRP1 ABCC1 High Bronchial/bronchiolarepithelium

Basolateral 24,61,62

Alveolar macrophagesMRP2 ABCC2 No or low Bronchial/bronchiolar

epitheliumApical 24,63

MRP3 ABCC3 Low or high Unknown 21,23,60MRP4 ABCC4 Moderate Unknown 21,23,60MRP5 ABCC5 High Unknown 21,23,60MRP6 ABCC6 Moderate Unknown 21,23,60MRP7 ABCC10 Moderate to high Unknown 21,23,60MRP8 ABCC11 Low or high Unknown 21,23,60MRP9 ABCC12 Low or high Unknown 21,23,60BCRP ABCG2 Low or high Airway epithelium Apical 23,24,73,74

Seromucinous glandsSmall capillaries

SLC transportersOCT1 SLC22A1 Contradictory data Tracheal/bronchial

ciliated cellsApical/cytoplasmic 23,78,79

OCT2 SLC22A2 Contradictory data Tracheal/bronchialciliated cells

Apical 23,78,79

Basal cells Entire membraneOCT3 SLC22A3 Contradictory Basal cells Entire membrane 23,78,79

data in airways Airway smooth musclesEndothelium

OCTN1 SLC22A4 Yes Tracheal epithelium Apical 23,79Alveolar macrophages Cytoplasmic

OCTN2 SLC22A5 Yes Airway epithelium Apical 23,79Alveolar epithelium Apical

PEPT1 SLC15A1 Low Bronchial epithelium Unknown 23,101PEPT2 SLC15A2 High Airway epithelium Apical 23,102

Type II pneumocytes CytoplasmicEndothelium Apical?

OAT1 SLC22A6 No 23,109–111OAT2 SLC22A7 Contradictory data Unknown Unknown 23,109–111OAT3 SLC22A8 No 23,109–111OAT4 SLC22A11 No 23,109–111

SLCO transportersOATP1A2 SLCO1A2 No 23,120OATP1B1 SLCO1B1 No 23,120,121OATP1B3 SLCO1B3 No 23,120,121OATP1C1 SLCO1C1 No 23,122OATP2B1 SLCO2B1 Yes Unknown Unknown 23,120,125OATP3A1 SLCO3A1 Yes Unknown Unknown 23,120,126OATP4A1 SLCO4A1 Yes Unknown Unknown 23,120,127OATP4C1 SLCO4C1 Yes Unknown Unknown 23OATP5A1 SLCO5A1 No 23OATP6A1 SLCO6A1 No 23,123,124

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010

DRUG TRANSPORTERS IN THE LUNG 2241

Figure 1. Expression and localisation of drug transpor-ters in human upper airway epithelial cells. � Indicates theexistence of conflicting data in the literature.

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Human in vitro models of the epithelial barrier ofthe lungs have been developed recently in response toethical concerns regarding the use of laboratoryanimals.3,4 The two bronchial cell lines Calu-3 and16HBE14o- as well as normal human epithelialbronchial (NHBE) cells, when cultured as monolayerson permeable supports at an air–liquid interface,provide in vitro representations of the absorptionbarrier of the upper airways morphologically close tothe native bronchial epithelium while exhibitingsimilar permeability properties.5–7 In the absence ofan alveolar cell line suitable for permeability studies,modelling the alveolar epithelium must exclusivelyrely on primary cultured alveolar type-I like epithe-lial cells.3,4 To date, it is unclear whether those cellculture models express the same range of transpor-ters found in human lungs and hence, whether theyare of any utility for the identification of compoundsactively transported across the respiratory epithe-lium.

Table 2. Inhaled Compounds That Interfere With Drug Tran

Drug Name Drug Class

Beclomethasone dipropionate Corticosteroid

Budesonide Corticosteroid

Ciclesonide CorticosteroidFlunisolide CorticosteroidFluticasone propionate CorticosteroidMometasone furoate CorticosteroidTriamcinolone acetonide CorticosteroidAlbuterol/salbutamol b2-agonist

Formoterol b2-agonistIpratropium Antimuscarinic

N-acetylcysteine MucolyticTobramycin AntibioticCiprofloxacin Antibiotic

Pentamidine Antiprotozoal

? Indicates inconclusive evidence.

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010

Besides in vitro models, isolated perfused lung(IPL) techniques are gaining popularity as toolsto predict pulmonary drug absorption as, in contrastto permeability studies in cell layers, pharmacoki-netic data can be obtained following drug delivery toan intact organ.8 Ex vivo systems offer the opportu-nity to quantify the actual contribution of activetransport mechanisms on pulmonary drug absorptionand consequently, rat IPL models have recently beenused to evaluate the influence of P-glycoprotein (P-gp)on the transport of model substrates acrossthe respiratory barrier.9,10 Although models employ-ing isolated and perfused human lung lobes have beendescribed,11,12 the majority of IPL systems are basedon rodent lungs.8 This entails inter-species variationsin drug permeability and renders any extrapolationhypothetical with respect to the situation in humans.

The large majority of inhaled drugs are deliveredlocally to treat respiratory conditions and areadministered to an inflamed or infected tissue. Aseither origins or consequences of the pathology, theexpression and activity of drug transporters might bealtered in diseased lungs, which could potentiallyaffect drug PKPD profiles. Similarly, the progressionor remission of the disease state and chronicpharmacotherapy might also modify the transporterexpression pattern in the lungs. However, withthe exception of ATP-binding cassette (ABC) trans-porters in chronic obstructive pulmonary disease(COPD),13,14 how transporters are regulated incommon pulmonary affections has essentially notbeen considered so far.

sporters

Transporter(s) Refs.

BCRP, P-gp 129OCT1, OCT2 79P-gp 130MRP 70OCT1, OCT2, OCT3 78,80BCRP, P-gp 129P-gp 39OCT2, OCT3 78,80BCRP, P-gp 129P-gp 131P-gp? 132OCT, OCTN2 79,84OCT3, OCTN2 84MRP1 70OCTN2 86MRP1 70,133P-gp? 134BCRP 135MRP4 136OCTN2 137OCT1, OCT2, OCT3 138

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This review aims to give an insight into the currentknowledge of the expression, localisation and activityof drug transporters in the lungs from a drug deliveryperspective. Each family of transporters is reviewedin terms of their expression in normal human lungsand when data are available, expression in healthylungs is contrasted with that in diseased lungs,excluding lung tumours which are not included in thediscussion. The few studies that have evaluated theactivity of transporters at the pulmonary absorptionbarrier or their interaction with inhaled drugs aresummarised and the suitability of animal and cellculture absorption models for the investigation ofactive transport systems in the lungs is analysed.Transporters considered herein are those known toaffect drug absorption, reabsorption or elimination inthe intestine, kidney, liver or at the blood–brainbarrier. Those encompass (1) the efflux pumpsbelonging to the ABC superfamily of transporters:P-gp, the multidrug resistance associated proteins(MRPs) and breast cancer resistance protein (BCRP)as well as (2) the uptake transporters members of thesolute-linked carrier (SLC or SLCO) superfamilies,that is the organic cation transporters (OCTs), thepeptide transporters (PEPT1 and PEPT2), theorganic anion transporters (OATs) and the organicanion transporting polypeptide (OATPs). Althoughthe lung resistance-related protein (LRP) is highlyexpressed in the lung and its critical role in multidrugresistance against chemotherapeutic agents is welldocumented,15 little information is available on itsinvolvement in the transport of conventional mole-cules across the respiratory epithelium. Hence, thattransporter is not discussed in this review.

ATP-BINDING CASSETTE (ABC) TRANSPORTERS

ABC transporters are a large family of transmem-brane proteins which function as ATP-dependentefflux pumps capable of exporting a broad rangeof chemically diverse substances from the cellcytoplasm to the external environment. Approxi-mately 50 members of the ABC family have beenidentified in humans. Those transporters are classi-fied into seven subfamilies designated from A to G.Amongst those, P-gp, MRPs and BCRP are wellknown for their role in multidrug resistance (MDR), aphenomenon which results from the expulsion ofchemotherapeutic agents from cancerous cells thatoverexpress efflux pumps.16 ABC transporters arealso present in normal tissues where they prevent theaccumulation of xenobiotics and therefore, theyactively contribute to the tissue defense mechan-isms.17 In addition, their involvement in the poor oralbioavailability and/or tissue distribution of a largeseries of drugs as well as in their hepatobiliary and

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renal excretion has been demonstrated.18 Due to theirpotential role in limiting the transport of inhaledtherapeutic molecules across the respiratory epithe-lium and in the pathophysiology of airway diseases,the expression and functions of ABC transporters innormal and diseased lungs have been granted greatattention. Those topics were first covered in anexcellent review published a few years ago.19 Severalsubsequent publications have largely contributed tothe evaluation of the actual role of ABC transportersin the pharmacokinetic profile of aerosolised drugsand in the development of COPD. Those recentstudies together with older ones exploring theexpression of ABC transporters in respiratory cellculture models in vitro are summarised below.

P-gp/MDR1

P-gp, also called MDR1 or ABCB1, is a 170 kDatransporter mainly expressed in the apical membraneof the enterocytes, hepatocytes, proximal renaltubules and at the blood–brain barrier. The proteinis encoded by the MDR1 gene in humans and both themdr1a and mdr1b genes in rodents. Since it is wellestablished that P-gp limits the oral absorption ofdrugs, prevents their entry into the central nervoussystem and is responsible for many drug–druginteractions,20 that transporter has been the mostextensively studied in the lungs.

P-gp in the Lung Tissue

P-gp mRNA was detected in normal human lungtissue by RT-PCR21,22 and microarray analyses.23

The intensity of expression was lower than in othermajor organs involved in drug absorption, distribu-tion and elimination such as the small intestine, liver,kidney and brain. Immunohistochemistry techniqueslocalised the transporter on the apical membrane ofthe bronchial and bronchiolar epithelia,22,24–26 in theendothelial cells of the bronchial capillaries18 and inalveolar macrophages.24,25 Contradictory data hasbeen published regarding P-gp expression in thealveolar region. No staining of the alveolar epithe-lium was observed in three studies24–26 while type Ipneumocytes stained positive at their apical side inthe study by Campbell et al.27

Both mdr1a and mdr1b mRNA are present in thelungs of mice28 and rats,29 with highest levels of themdr1b messenger in both species. The cellulardistribution of P-gp in rodent lungs was neverthelessshown to be similar to that in humans.24

P-gp expression in diseased lungs or in the lungs ofsmokers compared to normal lungs has not or hardlybeen quantified. mRNA levels in the lungs of smokers,nonsmokers or ex-smokers were reported not to bestatistically different.22 However, the pulmonary


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clearance rate of 99mTc-sestamibi, a P-gp substrate,after aerosol delivery was reduced in healthy smokersas compared to nonsmokers.30 This was assumed toresult from an upregulation of P-gp in the lungs ofsmokers. As 99mTc-sestamibi is also a substrate forMRP1,31 the delayed elimination of the tracer fromthe lungs of smokers might also be due toa downregulation of the basolaterally located MRP1caused by cigarette smoke.14 While decreased P-gplevels in the inflamed intestinal tissue of patientssuffering from gastrointestinal inflammatory disor-ders have been reported,32 no statistical differencewas found between the immunostaining intensity ofbronchial biopsies of COPD patients versus healthycontrols or of patients suffering from severe COPDversus patients with a milder form of the disease.13 Incontrast, whether P-gp expression is altered inthe bronchial epithelium of asthmatic patients hasnot been evaluated. Similarly, the effect of chronicadministration of inhaled glucocorticoids on P-gpexpression in the lungs has not been investigatedwhereas related studies suggest they might upregu-late the transporter in the pulmonary tissue. Forexample, a stronger immunohistochemical stainingfor P-gp was observed in nasal polyps of patientstreated with local doses of budesonide,33 a commoncorticoid used in the prophylaxis of asthma. Also, P-gpexpression increased about twofold in the lungs ofrats following oral34 or intraperitoneal35 administra-tion of dexamethasone.

Cystic fibrosis (CF) is a congenital disorder causedby a mutation in the CF transmembrane conductanceregulator (CFTR) gene. CFTR is a member of the ABCfamily of transporters which regulates the transportof ions. Due to a structurally close similitude with P-gp, it has been hypothesised that some alteredfunctions of CFTR might be compensated by anoverexpression of P-gp in CF patients.36,37 On theother hand, it has been reported that Cif, a toxinproduced by Pseudomonas aeruginosa whose infec-tions are frequent in the lungs of CF patients,inhibited P-gp as well as CFTR.38

P-gp in Respiratory Cell Culture Models In Vitro

P-gp has been detected in all in vitro humanrespiratory cell models currently available for drugpermeability studies, that is the Calu-339–42 and16HBE14o-14,41,43 bronchial cell lines, NHBE7,41,44

and alveolar type-I like cells41,45 with however,contradictory data regarding its expression andlocalisation on the cell membrane.9 For instance, novectorial transport of ciprofloxacin, digoxin andvinblastine, all P-gp substrates, has been observedin Calu-3 cell layers, suggesting those cells did notexpress a functional transporter.46 Western blotanalysis however revealed that cell line expresses


P-gp.39,40,42,47 Functional studies in Calu-3 layersusing rhodamine 123,40 cyclosporine47 and digoxin9

as P-gp substrates showed a polarised transport inthe basolateral to apical (B-A) direction, indicating anapical localisation of the efflux pump on the cellmembrane. By contrast, P-gp was localised inimmunofluorescence on the basolateral side of thecell layer and flunisolide transport was enhanced inthe absorptive direction in the study by Florea et al.39

Those conflicting results can likely be explained bydifferences in cell culture conditions and by the useof nonspecific P-gp substrates and inhibitors. Byusing GF120918a, a highly potent and more selectiveP-gp inhibitor,48 Madlova et al.9 measured a P-gpmediated polarised digoxin transport in the B-Adirection only in Calu-3 cell layers at passages over 50grown for three weeks on cell culture inserts. It wastherefore suggested that layers of Calu-3 cells mightnot express functional P-gp at low passages and whencultured for a shorter period of time on permeablefilters. This hypothesis was however not confirmed byBrillault et al.42 who detected P-gp by Westernblotting in Calu-3 layers at passages 22–30 grownfor 15 days on cell culture filters. In addition, theymeasured a B-A polarised transport of the fluoroqui-nolone antibiotic moxifloxacin that was inhibited byPSC-833, another P-gp potent inhibitor, while pro-benicid, an MRP inhibitor, had no effect.

A dependence of the time in culture on P-gp activitywas observed in NHBE cells with no vectorialtransport of digoxin after 14 days in culture and anet absorptive transport reversed by GF120918Aafter 21 days.9 This was in agreement with theincreased mRNA levels quantified by RT-PCR inNHBE cells after 14 and 21 days on cell cultureinserts as compared to after 7 days on those inserts.7

Functional studies in NHBE using digoxin as thesubstrate suggested a modest P-gp activity waspresent at the basolateral side of the cells.9 However,that assumption was not confirmed by localisationstudies.

The human bronchial CF epithelial cell lineCFBE41o- was shown to form tight monolayersexpressing P-gp when cultured under submergedconditions on permeable supports and hence, wasdeemed to be a suitable in vitro model for studying thedisease at the cellular level.49

Due to the unsuitability of the A549 alveolar cellline to represent the absorption barrier of the deeplung in vitro3 and the scarcity of human lung tissue,animal primary cell culture models of the alveolarepithelium have been developed as alternatives tohuman systems. The expression and functionality ofP-gp was confirmed in monolayers of rat type-1 likepneumocytes by Western blot and vinblastine bi-directional transport studies, respectively.27

Although monolayers of porcine alveolar epithelial

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cells stained positive for P-gp under a confocalmicroscope, no asymmetric transport of several P-gp substrates was measured, indicating a lack offunctionality of the transporter in that model.50

Disposition Studies of P-gp Substrates in Ex Vivoand In Vivo Animal Models

Uptake studies in isolated perfused animal lungsdemonstrated the contribution of efflux pumpsin drug accumulation from the perfusate into thelungs and therefore a significant activity of ABCtransporters in that organ. Idarubicin concentrationsin rat perfused lungs were enhanced after its infusionthrough the pulmonary circulation together with theP-gp modulators cinchonine and rutin.51 Similarly, inrabbit lungs, the disappearance of the P-gp substraterhodamine 6G from the perfusate was increased inpresence of the inhibitors verapamil and GF120918.52

In both studies, inhibitors were added in theperfusion solution and could therefore modulate P-gp present on both the endothelial cells of thepulmonary capillaries and the epithelial cells of theairways. The enhanced pulmonary accumulation ofthe substrates in presence of inhibitors suggests ahigher P-gp activity at the endothelium site than atthe airway epithelium since inhibition of the epithe-lial transporter would rather slow down the diffusionof the substrates from the perfusate. This hypothesisis in agreement with two absorption studies of inhaledP-gp substrates that have failed to highlight anysignificant influence of an efflux mechanism on theirdisposition from the airspaces. The percentage oflosartan transferred from the airways to the perfu-sate in 120 min reached 94� 2% after aerosolisationto a rat IPL model, indicating a negligible P-gp-mediated efflux at the apical membrane of the airwayepithelium.53 Nevertheless, relatively high drugconcentrations were used which might potentiallyhave saturated the transporter. In the second study,the coinstillation of GF120918a with digoxin to a ratIPL did not modify the pulmonary absorption profileof that model P-gp substrate.9 In contrast, therecovery of rhodamine123 in the perfusion solutionafter intratracheal delivery to a rat IPL was enhancedin presence of GF120918a both in the instillate andperfusate,10 which indicates an efflux mechanismrestricted the pulmonary absorption of the dye.However, GF120918a possesses some inhibitoryactivity against BCRP48 and rhodamine123 is asubstrate for that transporter.54 The actual contribu-tion of P-gp in the restricted absorption of rhoda-mine123 is therefore unclear.

The only in vivo study so far that aimed atevaluating P-gp impact on drug disposition fromintact lungs showed that the pharmacokinetic profileof digoxin after intratracheal instillation was similar

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in mdr1a (�/�) deficient and mdr1a (þ/þ) wild-typemice.55 However, mdr1b P-gp is still expressed in thelungs of mdr1a (�/�) mice and an upregulation of thepulmonary mdr1b P-gp as a compensation mechan-ism in mdr1a knockout animals is conceivable.Digoxin was used at concentrations below P-gpsaturation but, as in the study by Madlova et al.,9

the test solution was administered to the lungs ofanimals by intratracheal instillation and an over-saturation of the transporter locally in the pulmonarytissue cannot be excluded. Digoxin was shown to bewell and rapidly absorbed from the lungs.9,55 Hence,an epithelial efflux mechanism might not impacton its permeation profile to a significant extentwhereas it might hinder the absorption of a compoundwith a prolonged retention in the airspaces. Digoxin isalso a substrate for members of the organic aniontransporting polypeptide (OATP) family of transpor-ters;56,57 the presence of some of which has beenconfirmed in the lungs (see below). The contributionof an active uptake mechanism in digoxin pulmonaryabsorption might therefore have counterbalanced apotential P-gp-mediated efflux.

MRPs

The MRPs are nine organic anion efflux pumpsidentified as MRP1–9. MRP1, 4, 5, 7, 8, 9 are presentin many tissues while MRP2, 3, 6 are mainlyexpressed in the liver and kidneys.58 MRP1, 3, 4are basolateral transporters while MRP2 and 5 arelocated in the apical membrane of the cells.58 MRP2 isknown to play an important role in the biliaryexcretion of drug conjugates, especially those withglutathione.59

MRPs in the Lung Tissue

Using RT-PCR techniques,21,60 MRP1 and MRP5were shown to be highly expressed in normal humanlung tissue while MRP6 and 7 were moderatelyexpressed and MRP2, MRP3, MRP4, MRP8 andMRP9 levels were either low or undetectable.Subsequent gene microarray analyses confirmedthe high expression of MRP1 and MRP5 and theabsence of MRP2 in the lungs.23 However, theintensity of expression was found to be very highfor MRP7, high for MRP3, MRP8, MRP9 andmoderate for MRP4 and MRP6.23

The high expression of MRP1 in the lungs wasfurther corroborated by Western blotting61 andimmunohistochemistry.24,61,62 Bronchial/bronchiolarepithelial cells were stained but while labelling waslocalised in the cytoplasm of ciliated cells just belowthe cilia in the study by Flens et al.,61 MRP1 wasfound on the basolateral membrane of ciliated,mucous-producing and basal cells in two other


2246 BOSQUILLON

studies.24,62 Alveolar macrophages exhibited stainingin their cytoplasm whereas no staining was observedin pneumocytes.24,61 In concordance with geneexpression data, weak63 or absent staining24 wasobserved in bronchial/bronchiolar epithelial cells forMRP2. MRP3 could not be detected in any area of thelung.24

A similar expression pattern with high levelsof MRP1 mRNA and low levels of MRP2 was reportedin rats and mice23,64,65 although MRP1 expressionseemed to be lower in mice than in rats andhumans.23,65 Gene microarrays indicated the lungsof rodents might express lower levels of MRP3 andMRP6 but higher levels of MRP4 than humanlungs.23

MRP1 mRNA levels were not statistically differentin healthy smokers, ex-smokers and nonsmokers.62

However, MRP1 expression, as assessed by immu-nostaining, was lower in bronchial biopsies of COPDpatients as compared to that in healthy patients aswell as in patients affected by severe COPD versusthose with a mild to moderate form of the disease.13

Consequently, a role of that transporter in thepathophysiology for COPD might be postulated.13

MRPs in Respiratory Cell Culture Models In Vitro

In accordance with gene microarray data in humanlungs, high levels of MRP1, MRP3, MRP5 and MRP7mRNA were measured by RT-PCR in human epithe-lial respiratory cell culture models.41 MRP4 andMRP8 transcripts were detected in normal humanbronchial and alveolar cells but were absent in theCalu-3 and 16HBE14o- cell lines.41 MRP6 wasmoderately expressed in bronchial models but highlyexpressed in alveolar type-I like cells.41 The moststriking discrepancy with human lungs was thepresence of MRP2 transcripts in all in vitro modelswhile that transporter seems not to be expressedin vivo.23

MRP1 was reported to be expressed both atthe mRNA and protein level in undifferentiatedNHBE and normal alveolar lung cells grown on cellculture dishes, with nevertheless high intra-indivi-dual variations.44,66 The functionality ofthe transporter in normal lung cells was demon-strated by the decreased efflux of the MRP substratecarboxydichlorofluorescein (CDF) in presence of theMRP inhibitor MK-571.44 Immunodetection ofMRP1–5 in undifferentiated NHBE and alveolar cellsshowed that MRP1 and MRP3 were localised in thecell membrane while MRP2, MRP4 and MRP5 wereintracellular proteins.67 However, when cells werecultured on inserts at an air–liquid interface, MRP1and MRP2 were detected on the basolateral mem-brane of the cells or on both the apical and basolateralmembranes, respectively.67 This illustrates the


necessity of investigating transporter expressionand localisation in differentiated cells grown underphysiologically relevant conditions.

Torky et al.68 investigated the influence of pro-inflammatory mediators on MRP1 functions innormal human respiratory epithelial cells. Theuptake of CDF by undifferentiated NHBE cellsexposed to arachidonic acid and prostaglandin E2for 3 days was decreased as compared to that inuntreated cells while prostaglandin F2a had no effecton MRP1 activity.68 In alveolar cells, only arachidonicacid modified CDF efflux.68

In Calu-3 cells, MRP1 was localised on thebasolateral side of monolayers cultured on permeablefilters.69 Efflux and transport studies were carried outusing calcein as the substrate and indomethacin,probenicid or MK-571 as inhibitors. However,appraising the actual contribution of MRP1 in calceintranslocation across Calu-3 cells was rendered com-plex due to the dye interacting with P-gp.69

In 16HBE14o- cells, a strong immunohistochemicalstaining was obtained for MRP1 while this was weakfor MRP4 and negative for MRP2, MRP3 andMRP5.14 Because MRP1 was shown to be down-regulated in the lungs of COPD patients,13 that cellline was used to evaluate the effect of cigarette smokeand drugs commonly used in the treatment of COPDon MRP1 activity in the bronchial epithelium. Theefflux of the fluorescent dye CDF out of cells exposedto cigarette smoke extracts was diminished whencompared to that in untreated cells.14 This inhibitoryeffect was caused by a direct interaction of cigarettesmoke components with the transporter.14 In addi-tion, in the cytotoxicity MTT assay, the reduction ofthe metabolic activity of 16HBE14o- cells provoked bycigarette smoke extracts was enhanced in presence ofthe MRP inhibitor MK-571,14 confirming the protec-tive role of MRP1 against cell damage induced bycigarette smoke and hence, the probable role playedby the transporter in the development of COPD. Theintracellular accumulation of CDF by 16HBE14o-cells was enhanced in presence of budesonide but thisinhibitory effect on MRP1 transport was reduced byformoterol whereas formoterol on its own had littleeffect on MRP1 activity.70 By contrast, ipratropiumand N-acetylcysteine decreased the accumulation ofthe dye, suggesting they both stimulated MRP1-mediated efflux.70 Whether those modulations ofMRP1 activity caused by drugs used in COPD arebeneficial or detrimental for the treatment of thedisease is currently unknown.

Both type I and type II rat pneumocytes in primaryculture stained positive for MRP1 at their basolateralsurface when grown on permeable filters andindomethacin increased the basolateral to apicaltransport of fluorescein across type IIcell monolayers as well as the intracytoplasmic

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accumulation of the dye by those cells.71 Similarfunctionality studies could not be carried out usingtype I cell monolayers as the monolayer integrity wascompromised in presence of indomethacin. Never-theless, in absence of apical to basolateral perme-ability data and control experiments demonstratingthe involvement of an active mechanism in fluor-escein translocation, transport data in type IImonolayers must be interpreted with caution. Fluor-escein permeability data are indeed commonly used toverify the integrity of epithelial cell monolayers as thedye is assumed to be exclusively transported by apassive paracellular route.3,4

BCRP/ABCG2

BCRP is a 72 kDa transporter encoded by the ABCG2gene which was first isolated from a breast cancer cellline. Besides being overexpressed in many cancercells, it is also highly expressed in the placenta, thegastrointestinal tract, the brain, the liver and thebreast tissue where it regulates the transfer andaccumulation of xenobiotics.72

Data regarding BCRP expression in the lung aresparse and contradictory. In early studies, BCRPmRNA was either not present73 or detected only ata low level74 in normal human lung and a weakbut detectable immunostaining was observed inthe epithelium, in seromucinous glands and smallcapillaries.24 Gene microarrays recently showedBCRP was relatively highly expressed in humanlungs whereas its expression was comparatively lowin the lungs of rats and mice.23

Transcripts for BCRP were found in all humanlung epithelial cell culture models with howeverunderexpression in Calu-3 and overexpression in16HBE14o-.41 A strong immunostaining for BCRPwas obtained in 16HBE14o- cells,14 which confirmedthe transporter is highly expressed in that cell line.

ORGANIC CATION TRANSPORTERS

OCTs are members of the SLC22A family oftransporters which belongs to the major facilitatorsuperfamily (MFS). They comprise five main sub-types of carriers; the electrogenic OCT1, OCT2, OCT3and the electroneutral OCTN1, OCTN2. In addition,OCT6 and octn3 have been cloned from humans orrodents, respectively, where they are found mainly intestis.75 They all have the capacity to translocatevarious endogenous and exogenous molecules acrossthe plasma membrane in both directions. Althoughneutral molecules can be transported, the majority oftheir substrates are positively charged at physiolo-gical pH and include hormones, neurotransmitters,

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metabolites, xenobiotics and drugs.75 Their involve-ment in the intestinal absorption and renal excretionof cationic drugs has been demonstrated and recentstudies indicate they play a crucial role in theregulation of brain functions as well as those ofbasophils.75

As the structure, tissue distribution, physiologicalfunctions of OCTs and their roles in drug absorptionand elimination was reviewed recently,75–77 the focushere is on their expression, localisation and functionsin the lung with an emphasis on their interaction withinhaled drugs.

OCTs in the Lung Tissue

The five main subtypes of OCTs have been detected inhealthy human lungs. However, conflicting observa-tions have been made. Lips et al.78 found high levels ofOCT1, OCT2 and OCT3 mRNA in human lung tissuebut they did not quantify OCTN1 and OCTN2 levels.Using immunohistochemistry techniques, OCT1and OCT2 were localised on the apical membraneof ciliated cells of the trachea and bronchi.In addition, OCT1 was detected in the cytoplasm ofciliated cells and OCT2 in the plasma membrane ofbasal cells. Ciliated cells stained weakly for OCT3, bycontrast to the entire membrane of basal cells and thebasolateral membrane of intermediate cells whichwas intensively labelled. In the gene microarrayanalysis by Bleasby et al.,23 the intensity of expres-sion in human lungs was weak for OCT2, moderatefor OCT1 and OCT3 and high for OCTN1 and OCTN2.Horvath et al.79 measured high levels of OCTN1 andOCTN2 mRNA and very low levels of OCT1-3 mRNAin the airway tissue of both healthy and CF patients.The same group also found high levels of OCT3 mRNAin airway smooth muscle cells and using immuno-histochemistry, they visualised the transporter inbronchial and pulmonary blood vessels.80 The expres-sion of the other OCT subtypes was low or undetect-able in muscular and endothelial cells.80 OCTN1positive staining was observed on the luminal side ofthe trachea epithelium and less intensively inalveolar macrophages while OCTN2 staining waspositive on the apical membrane of the airway andalveolar epithelia.79

The intensity of OCT1, OCT2 and OCTN2 geneexpression was shown to be similar in human androdent lungs whereas that of OCT3 and OCTN1appeared, respectively, higher or lower in rodentscompared to humans.23 OCT1, OCT2 and OCT3 wereidentified in rat lungs at the protein level78,81 butalthough the mRNA of all three transporters wasfound in murine lungs, only OCT1 and OCT3 werevisualised by immunofluorescence in that species.82

In rats, the apical membrane of ciliated cells of thetrachea and bronchi stained positive for the three


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transporters while that of alveolar epithelial cellswas stained for OCT1 and OCT3 only. OCT3 was, inaddition, detected in the plasma membrane ofbronchial basal cells. In mice, OCT1 was observedon the apical side of ciliated cells and OCT3 mainly inbronchial smooth muscles, although a weak stainingwas obtained in the bronchial epithelium as well.82

The protein expression and localisation of OCTN1and OCTN2 in the lungs of rodents have not beeninvestigated to date.

Pulmonary expression of OCTs in human lungdiseases has only been reported for CF, where theexpression pattern was unaltered compared tonormal lungs.79 The expression of OCT1-3 in ratsand mice lungs sensitised with ovalbumin andexposed to the antigen by aerosol delivery wascompared with that in untreated animals in orderto evaluate the effects of acute allergic airwayinflammation on OCTs regulation.81 In allergic rats,OCT1 was upregulated whereas OCT2 and OCT3were downregulated 48 h after antigen challenge.OCT2 expression was similarly reduced in challengedmice but OCT1 and OCT3 levels were identical ininflamed and healthy mice lungs, which suggestsinter-species variations in the regulation of OCTs.Considering those data in animals, it is now para-mount to determine whether OCT expression ismodified in chronic inflammatory respiratory dis-eases, especially since those transporters might haveimplications in asthma/COPD pathophysiology andpharmacotherapy. Indeed, based on uptake andrelease studies in Xenopus laevis transfected withOCT1-3 mRNA78 and the accumulation of acetylcho-line in the bronchial epithelium of OCT1/2 double-knockout mice,82 it has been proposed that OCT1 andOCT2 mediate the release of the nonneuronalacetylcholine produced by bronchial epithelial cellsinto the airway lumen where it controls mucusproduction, cilia beat frequency and epithelial cellproliferation.83

OCTs in Respiratory Cell Culture Models In Vitro

In comparison to the extensive investigation on ABCtransporters in respiratory cell models in vitro, only alimited number of studies have looked at OCTs incultured airway epithelial cells. A recent RT-PCRanalysis of drug transporters in human bronchial cellculture models revealed OCT1 and OCTN2 weremoderately expressed while OCT2 was absent in bothnormal cells and cell lines.41 Various OCT3 andOCTN1 expression were observed amongst models.While high levels of OCT3 mRNA were present innormal bronchial cells and Calu-3 cells, the trans-porter was not detected in 16HBE14o-. Intensesignals were measured for OCTN1 transcripts inCalu-3 and 16HBE14o- cells but not in normal cells.


In a previous study, normal human bronchialepithelial cells were shown to highly express OCTN1and OCTN2 on their apical membrane while expres-sing low amounts of OCT1-3 when grown on perme-able filter at an air–liquid interface for 6–8 weeks.79

OCT expression profile in NHBE layers was similar tothat reported by the same research team in freshlyisolated human bronchial cells. In contrast to thestudy by Endter et al.,41 mRNA for all the five OCTswere detected in the bronchial cell lines Calu-3 and16HBE14o- grown in cell culture flasks.84 Cells were,however, screened for OCTs at passage numbershigher than those commonly used for permeabilitymeasurements in those cell lines.3 Unfortunately, thepassage number at which Endter et al.41 examinedbronchial cell lines was not specified. Therefore, it isunknown whether discrepancies between the twostudies arose from an upregulation of transporterexpression in those in vitro models at high passagenumbers or from differences in culture conditions.

The uptake of the model organic cationguanidine by layers of rabbit alveolar epithelial cellsgrown on Transwell1 inserts was shown to besaturable and was inhibited by a series of positivelycharged molecules.85 This indicated a carrier-mediated transport process for organic cations ispresent in the alveolar epithelium.

Interestingly, the b2-agonist salbutamol, which ispositively charged at physiological pH was activelytransported with a net absorptive flux in layers of thetwo bronchial cell lines Calu-3 and 16HBE14o-.84

Although the transporter involved was not identified,the organic cations TEA and guanidine significantlydecreased the A-B transport of salbutamol, suggest-ing the involvement of one or several member(s) of theOCT family. This assumption was later supported byevidence demonstrating salbutamol (albuterol) andformoterol, another positively charged b2-mimetics,modulate the activity of OCTs. Those two broncho-dilators were shown to inhibit the uptake of the modelcationic fluorophore 4-[4-(demethylamino)-styryl]-N-methylpyridinium (ASPþ) by undifferentiated nor-mal human bronchial cells grown on coverslips.79 Thetransporter involved was identified as OCTN2, basedon the reduction of ASPþ uptake in presence of theOCTN2 inhibitor L-carnitine. However, an interac-tion of formoterol and salbutamol (albuterol) withother members of the OCT family cannot be excludedas cells used in that study did not express OCT1-3.In a related work,80 formoterol was reported tobe internalised into human airway smooth musclecells by an OCT3-mediated process, which indicates itis a substrate or inhibitor for more than one OCTsubtype.

The inhaled antimuscarinic bronchodilators ipra-tropium and tiotropium bear a permanent positivecharge due to their quaternary ammonium structure

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and hence, it can intuitively be prophesied that theyare very likely to be substrates for OCTs. Ipratropiuminhibited the uptake of L-carnitine by the humanproximal tubule cell line Caki-1, which demonstratesit is recognised by OCTN2.86 Besides cationicbronchodilators, inhaled glucocorticoids were alsoshown to interact with OCTs, although they are notactually translocated by those transporters. Budeso-nide and fluticasone inhibited the uptake of acet-ylcholine by OCT2 transfected X. laevis78 as wellas the OCT3-mediated internalisation of formoterolby human airway smooth muscle cells.80 Althoughstrong evidence has shown cationic bronchodilatorsinteract with OCTs, it is at present not knownwhether those transporters actually contribute totheir absorption across the respiratory epithelium.Similarly, the consequences on the treatment ofinflammatory respiratory diseases of inhaled bronch-odilators and corticosteroids interacting with OCTsare unclear.

PEPTIDE TRANSPORTERS

Peptide transporters are members of the SLC15family which is part of the proton-coupled oligopep-tide transporter (POT) superfamily. The structureand physiological functions of the two main trans-porters of that family, PEPT1 and PEPT2 have beenextensively described elsewhere.87 Briefly, PEPT1and PEPT2 are capable of transporting any di- ortripeptide derived from the 20 L-a-amino acids inassociation with proton translocation independentlyof the substrate charge. PEPT1 is essentiallyexpressed in the apical membrane of epithelial cellsof the small intestine, renal tubules and bile ductswhile PEPT2 is present in many organs, such as thekidneys, brain, lung, pituitary, mammary glands,reproductive organs.88–90 Because of their very broadsubstrate specificity, PEPT1 and PEPT2 have thecapacity to translocate peptidomimetic drugs such asthe angiotensin-converting enzyme inhibitors capto-pril, enalapril and fosinopril91 or the b-lactamantibiotics,92 respectively, and it is now well estab-lished that PEPT1 and PEPT2 contribute to the highbioavailability of peptide-like drug molecules.

Drug inhalation is an attractive route of delivery forthe treatment of pulmonary infections. Several anti-infectious agents active against respiratory patho-gens are substrates for PEPT1/PEPT2, for examplepenicillin and cephalosporin antibiotics and theantiviral drugs valacyclovir and valganciclovir.93

The presence of peptide transporters in the respira-tory tract can potentially affect the absorption anddistribution of those compounds, with consequenceson their anti-infectious efficiency.93

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PEPT1/PEPT2 in the Lung Tissue

High Pept2 mRNA levels have been measured inthe lungs of rabbits,94,95 rats90,96,97 and mice.90 Bycontrast, weak or no signals have been observed forPept1 in rabbit,95,98 rat90,96,99 and murine90 lungextracts. Based on expression data in animals, PEPT1was assumed for years to be absent from the humanrespiratory tract or at least, not to significantlyinfluence inhaled drug distribution in the lungs.93

However, Western blot analysis and uptake studiesusing di- and tripeptides showed rat alveolar macro-phages express functional Pept1 protein.100 Genemicroarrays revealed PEPT1 is expressed in thelungs although to a lower extent than PEPT223 andPEPT1 mRNA was detected very recently in thehuman bronchi of healthy adults.101 The recentdiscovery of PEPT1 in human lung implies the earlyassumption that the transporter does not affectpulmonary drug disposition might need reappraisal.

Due to the high expression of PEPT2 in human andanimal lungs,23 the regional distribution of thattransporter has been examined in rat,96 mice96 andhealthy or CF human102 lungs using immunohisto-chemistry techniques. In all species, a positivestaining was obtained on the apical membrane ofairway epithelial cells, in the cytoplasm of type IIpneumocytes and on the endothelium of small bloodvessels. The staining intensity was similar in healthyand CF lung samples.102 The presence of Pept2 in rattype II alveolar cells was later confirmed, althoughthe transporter appeared to be located on the apicalplasma membrane.103

The functionality of Pept2/PEPT2 in the lungs wasdemonstrated by performing ex vivo uptake studies ofthe fluorophore-conjugated dipeptide D-Ala-Lys-AMCA by isolated mice96 and human102 lung speci-mens. The model peptide accumulated in airwayepithelial cells and type II pneumocytes, two celltypes known to express the transporter. In addition,the intracellular fluorescence was reduced afterincubation of the lung samples with high concentra-tions of the dipeptide glycyl-(L)-glutamine or thesynthetic cephalosporin and PEPT2 substrate cefa-droxil, while addition of the PEPT1 substratecaptopril had no effect.

PEPT1/PEPT2 in Respiratory Cell CultureModels In Vitro

Consistent with gene expression data in humanlungs, high PEPT2 mRNA levels were found inhuman bronchial epithelial cells and bronchial celllines in vitro.41 In contradiction with expressionlevels in vivo, PEPT1 was highly expressed in Calu-3and 16HBE14o- but was absent in normal cells.41

Primary human airway epithelial cells grown onpermeable filters at an air–liquid interface were


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shown to express PEPT2 on their apical cellmembrane.104 The two genetic variants previouslyidentified105 PEPT2�1 and PEPT2�2 were repre-sented amongst the airway samples but the intensityof the RT-PCR signal was similar in all donors. Theapical to basolateral transport of the dipeptide GlySaracross cell layers was saturable, unaffected by thegenotype and inhibited by b-lactam antibiotics(PEPT2 inhibitors) while ACE inhibitors (PEPT1inhibitors) had no effect. This indicated the trans-porter was functional in vitro and its activity was notinfluenced by genetic variations, at least at the pH ofthe lung fluid, which is about 6.5. Differences inGlySar translocation were indeed observed pre-viously between the two haplotypes at pH 6.105

Both PEPT1 and the PEPT2�1 variant weredetected in layers of Calu-3 cells grown at an air–liquid interface.101 PEPT1 expression was confirmedby Western blot and the transporter was localised onthe apical cell membrane. In contradiction withprevious studies which had concluded PEPT1 wasnot involved in peptide transport across the airwayepithelium,102,104 the uptake and transport of GlySarwas shown to be mediated by PEPT1 and not PEPT2in that cell line.b-Ala-L-His uptake by rabbit tracheocytes cultured

as air–liquid interfaced monolayers increased whenthe apical medium was buffered at pH 6.5 ascompared to 7.4 and was inhibited by Gly-L-Phe,but neither by Gly-D-Phe or amino acids.106 Althoughthe transporter involved was not identified, themarked effect of the pH gradient coupled to previousevidence of its presence in the lungs suggested it to bePEPT2. Surprisingly, the transport of Gly-L-Pheacross the cell layers was symmetrical at pH 7.4and in the same order of magnitude as the para-cellular marker mannitol. Decreasing the pH of theapical medium to 6.5 reduced the A-B permeabilityof the peptide, despite an enhanced uptake at that pH,while the B-A transport was unchanged. As para-cellular diffusion is hindered by the highest proto-nation of Gly-L-Phe at pH 6.5, a passive mechanismwas therefore assumed to be the unique permeationpathway followed by the peptide across the trachealepithelium.

ORGANIC ANION TRANSPORTERS

Two families of structurally unrelated transportersare known to mediate the absorption and eliminationof endogenous and exogenous organic anions: theorganic anion transporter (OAT) family and theorganic anion transporting polypeptide (OATP)family (see below). OATs, like OCTs, belong to theSLC22A family of transporters. The six members ofOATs identified so far (OAT1-4, URAT1 and oat5) are


all expressed in the kidneys.107,108 OAT2, OAT3,OAT4 are additionally found in the liver, brain andplacenta, respectively.107,108 OATs are the mainmechanism by which organic anions are excretedand reabsorbed in the kidneys.108 They also con-tribute to drug–drug interactions and to the nephro-toxicity of some drugs and toxins.107

None of the OAT members could be detectedby Northern blot in the lungs of humans,109–111

rats112–114 or mice.115,116 Gene microarrays confirmedthe absence of OAT1, OAT3 and OAT4 in the lungs ofall species. They nevertheless indicated OAT2 wasrelatively highly expressed in human and murinelungs.23

No OAT1, OAT2 and OAT3 transcripts weredetected in any of the human bronchial epithelialcell culture models. As expected, OAT4 mRNA wasabsent in normal cells, but that transporter washighly expressed in the two bronchial cell lines Calu-3and 16HBE14o-.41

ORGANIC ANION TRANSPORTINGPOLYPEPTIDES

OATPs were originally designated by the SLC21 genesymbol and each transporter was essentially namedby the researchers who had isolated them. Thisresulted in huge confusion as a same transporterisolated by independent groups was given differentnames or a human transporter had a designationsimilar to a rodent transporter whereas they were notequivalent.117 OATPs are now classified under theSLCO gene symbol according to a specific nomen-clature whose details can be found elsewhere.57 The11 human OATPs are divided into 6 families (OATP1–6) further subdivided into subfamilies. Some of themare organ specific while others are ubiquitious.57,117

To date, only four of them, OATP1A2 (widelyexpressed), OATP1B1 (liver specific), OATP1B3 (liverspecific), OATP2B1 (widely expressed) have been wellcharacterised and shown to be responsible for drug–drug interactions.118,119 Many OATP substrates arealso transported by the efflux pumps P-gp, MRP1,MRP2 or BCRP57 and assessing the contribution ofeach of those transporters in the pharmacokineticprofile of organic anions is a major challenge inabsence of specific inhibitors and knockout mice forOATPs. Overall, the actual tissue distribution,physiological functions and substrate specificity ofOATPs remain largely unknown.57

RT-PCR, Northern blot or gene microarray ana-lyses of human tissue samples indicatedOATP1A2,23,120 the two liver specific proteinsOATP1B1 and OATP1B3,23,120,121 OATP1C1,23,122

OATP5A123 and OATP6A123,123,124 were notexpressed in the lungs. By contrast, high levels of

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OATP2B1,23,120,125 OATP3A123,120,126 andOATP4A123,120,127 expression were detected inhuman lungs while OATP4C1 was moderately pre-sent.23 Positive signals for oatp2b1 were also observedin rat23,128 and mice23 lungs and for oatp3a1 in ratlungs.126 Nevertheless, the actual expression ofOATPs at the protein levels and their cellularlocalisation in the pulmonary tissue have not yetbeen investigated.

Significant variations in the expression profiles ofOATPs were noticed amongst human bronchialepithelial in vitro models.41 In accordance with genemicroarray data, normal bronchial cellswere reported to express high levels of OATP3A1and OATP4A1 while not expressing OATP1B1,OATP1B1 and OATP1C1. However, in contradictionwith expression profiles in human lungs, OATP1A2was highly expressed and OATP2B1 was undetect-able in those cells. The bronchial cell line Calu-3appeared to express all OATP transporters exceptOATP1A2 while none of OATP transcripts but thosefor OATP3A1 and OATP4A1 were present in16HBE14o- cells.

CONCLUSIONS

Based on the literature, it is manifest that the lungsexpress elevated levels of several drug transporters.Interestingly, transporter gene expression in thelungs appeared to be the highest after that in the liverand kidneys.23 Because techniques used to quantifytransporter levels in whole organs are incapable ofdetermining the cellular origin of the genes, theextent of transporter expression at the epithelial drugabsorption site remains unknown. Not surprisingly,as observed for the exploration of transporters inother organs, the increasing number of studiespublished in the last 5 years attests of a recentinterest in drug transporters in the lungs, both fromacademic institutions and the pharmaceutical indus-try. Nevertheless, contribution in the area has beenlimited and the physiological functions of drugcarriers in the lung tissue, their potential involve-ment in common respiratory diseases and their rolesin the disposition of inhaled drugs are essentiallyunexplored.

This review discussed more specifically the role ofdrug transporters in the biopharmaceuticsof aerosolised medications. To date, there is no strongevidence to indicate active transport mechanismsaffect the distribution of drugs in the lungs.Considering the large surface area offered by therespiratory epithelium and rapidity of pulmonaryclearance observed for most inhaled drugs, drugabsorption from the lungs is less likely to be asinfluenced by transporters as it is from

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the gastrointestinal tract. Delivery strategies cur-rently under development nevertheless envisageexploiting efflux pumps present in the lung tissueto prolong drug retention in situ. However, it isnoteworthy that the impact of transporters on thedistribution of inhaled drugs might be influenced bytheir regional deposition in the respiratory tract andlocal metabolism.

Appraising the absorptive role played by drugtransporters in the lung tissue relies on simplifiedmodels of the respiratory absorption barrier, none ofwhich has been validated yet for such an application.The development of reliable systems to explore drugtransporters in the lungs is therefore a prerequisite tothe evaluation of their influence on the PKPD profileof inhaled drugs. Understanding the regulation oftransporters in pulmonary pathologies is also para-mount for a realistic appreciation of their contribu-tion in inhaled drug disposition under therapeuticconditions.

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