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  • 1. REVIEW Drug Transporters in the Lung—Do They Play a Role in the Biopharmaceutics 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 ( DOI 10.1002/jps.21995 ABSTRACT: The role of transporters in drug absorption, distribution and elimination processes as well as in drug–drug interactions is increasingly being recognised. Although the lungs express high levels of both efflux and uptake drug transporters, little is known of the implications 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 studies that have looked at their impact on pulmonary drug absorption is extensively reviewed. The emphasis is on transporters most likely to affect the disposition of inhaled drugs: (1) the ATPbinding 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 cation transporter (OCT) family, the peptide transporter (PEPT) family, the organic anion transporter (OAT) family and the organic anion transporting polypeptide (OATP) family. Whenever available, expression and localisation in the intact human tissue are compared with those in animal lungs and respiratory epithelial cell models in vitro. The influence of lung diseases or exogenous agents on transporter expression is also mentioned. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:2240–2255, 2010 Keywords: drug inhalation; pulmonary delivery; multidrug resistance transporters; peptide transporters; organic cation transporters; organic anion transporters; Calu-3 cells; isolated perfused lungs; cell culture; permeability INTRODUCTION The implication of membrane transport proteins in the pharmacokinetic, pharmacodynamic (PKPD) and safety profiles of a large range of drugs is now well established, although probably not yet fully appreciated.1,2 Due to their critical role in the successful development of drug candidates,1 the study of drug transporters is currently the topic of intense research. The focus is essentially on transporters in the intestine, liver, kidney, brain and their relevance to drug disposition in those organs. Comparatively, the influence of transporters on the disposition of inhaled drugs has hardly been investigated, although access Additional Supporting Information may be found in the online version of this article. Correspondence to: Cynthia Bosquillon (Telephone: 44-1158466078; Fax: 44-115-9515122; E-mail: Journal of Pharmaceutical Sciences, Vol. 99, 2240–2255 (2010) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association 2240 to drug target sites in the lung tissue might partly depend on their activity. Although less than 40 drugs are currently administered by the pulmonary route, drug absorption, distribution and elimination processes in the lung remain overall poorly understood. Hence, the PKPD profile 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 commonly administered as aerosols in the treatment of respiratory diseases, for example glucocorticoids, and cationic b2-agonists, might interact with those transporters (Tab. 2). An evaluation of the impact of active transport systems on drug absorption from the lungs would help in the interpretation and optimisation of PKPD parameters after drug inhalation. This task is, nevertheless, complicated by the complexity of the organ and the lack of validated models to investigate drug transport mechanisms in the lung. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
  • 2. DRUG TRANSPORTERS IN THE LUNG 2241 Table 1. Summary of Drug Transporter Expression in Human Lungs Protein Name Gene Symbol ABC transporters P-gp ABCB1 Expression in Human Lungs Moderate MRP1 ABCC1 High MRP2 ABCC2 No or low MRP3 MRP4 MRP5 MRP6 MRP7 MRP8 MRP9 BCRP ABCC3 ABCC4 ABCC5 ABCC6 ABCC10 ABCC11 ABCC12 ABCG2 Low or high Moderate High Moderate Moderate to high Low or high Low or high Low or high SLC transporters OCT1 SLC22A1 Contradictory data OCT2 SLC22A2 Contradictory data OCT3 SLC22A3 Contradictory data in airways OCTN1 SLC22A4 Yes OCTN2 SLC22A5 Yes PEPT1 PEPT2 SLC15A1 SLC15A2 Low High OAT1 OAT2 OAT3 OAT4 SLC22A6 SLC22A7 SLC22A8 SLC22A11 No Contradictory data No No SLCO transporters OATP1A2 SLCO1A2 OATP1B1 SLCO1B1 OATP1B3 SLCO1B3 OATP1C1 SLCO1C1 OATP2B1 SLCO2B1 OATP3A1 SLCO3A1 OATP4A1 SLCO4A1 OATP4C1 SLCO4C1 OATP5A1 SLCO5A1 OATP6A1 SLCO6A1 DOI 10.1002/jps No No No No Yes Yes Yes Yes No No Cellular Distribution Bronchial/bronchiolar epithelium Alveolar epithelium (contradictory data) Alveolar macrophages endothelium Bronchial/bronchiolar epithelium Alveolar macrophages Bronchial/bronchiolar epithelium Unknown Unknown Unknown Unknown Unknown Unknown Unknown Airway epithelium Seromucinous glands Small capillaries Tracheal/bronchial ciliated cells Tracheal/bronchial ciliated cells Basal cells Basal cells Airway smooth muscles Endothelium Tracheal epithelium Alveolar macrophages Airway epithelium Alveolar epithelium Bronchial epithelium Airway epithelium Type II pneumocytes Endothelium Unknown Unknown Unknown Unknown Unknown Cellular Localisation Apical Refs. 22–27 Apical Basolateral 24,61,62 Apical 24,63 Apical 21,23,60 21,23,60 21,23,60 21,23,60 21,23,60 21,23,60 21,23,60 23,24,73,74 Apical/cytoplasmic 23,78,79 Apical 23,78,79 Entire membrane Entire membrane 23,78,79 Apical Cytoplasmic Apical Apical Unknown Apical Cytoplasmic Apical? Unknown Unknown Unknown Unknown Unknown 23,79 23,79 23,101 23,102 23,109–111 23,109–111 23,109–111 23,109–111 23,120 23,120,121 23,120,121 23,122 23,120,125 23,120,126 23,120,127 23 23 23,123,124 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
  • 3. 2242 BOSQUILLON Figure 1. Expression and localisation of drug transporters in human upper airway epithelial cells. Ã Indicates the existence of conflicting data in the literature. Human in vitro models of the epithelial barrier of the lungs have been developed recently in response to ethical concerns regarding the use of laboratory animals.3,4 The two bronchial cell lines Calu-3 and 16HBE14o- as well as normal human epithelial bronchial (NHBE) cells, when cultured as monolayers on permeable supports at an air–liquid interface, provide in vitro representations of the absorption barrier of the upper airways morphologically close to the native bronchial epithelium while exhibiting similar permeability properties.5–7 In the absence of an alveolar cell line suitable for permeability studies, modelling the alveolar epithelium must exclusively rely on primary cultured alveolar type-I like epithelial cells.3,4 To date, it is unclear whether those cell culture models express the same range of transporters found in human lungs and hence, whether they are of any utility for the identification of compounds actively transported across the respiratory epithelium. Besides in vitro models, isolated perfused lung (IPL) techniques are gaining popularity as tools to predict pulmonary drug absorption as, in contrast to permeability studies in cell layers, pharmacokinetic data can be obtained following drug delivery to an intact organ.8 Ex vivo systems offer the opportunity to quantify the actual contribution of active transport mechanisms on pulmonary drug absorption and consequently, rat IPL models have recently been used to evaluate the influence of P-glycoprotein (P-gp) on the transport of model substrates across the respiratory barrier.9,10 Although models employing isolated and perfused human lung lobes have been described,11,12 the majority of IPL systems are based on rodent lungs.8 This entails inter-species variations in drug permeability and renders any extrapolation hypothetical with respect to the situation in humans. The large majority of inhaled drugs are delivered locally to treat respiratory conditions and are administered to an inflamed or infected tissue. As either origins or consequences of the pathology, the expression and activity of drug transporters might be altered in diseased lungs, which could potentially affect drug PKPD profiles. Similarly, the progression or remission of the disease state and chronic pharmacotherapy might also modify the transporter expression pattern in the lungs. However, with the exception of ATP-binding cassette (ABC) transporters in chronic obstructive pulmonary disease (COPD),13,14 how transporters are regulated in common pulmonary affections has essentially not been considered so far. Table 2. Inhaled Compounds That Interfere With Drug Transporters Drug Name Drug Class Transporter(s) Refs. Beclomethasone dipropionate Corticosteroid Budesonide Corticosteroid Ciclesonide Flunisolide Fluticasone propionate Mometasone furoate Triamcinolone acetonide Albuterol/salbutamol Corticosteroid Corticosteroid Corticosteroid Corticosteroid Corticosteroid b2-agonist Formoterol Ipratropium b2-agonist Antimuscarinic N-acetylcysteine Tobramycin Ciprofloxacin Mucolytic Antibiotic Antibiotic Pentamidine Antiprotozoal BCRP, P-gp OCT1, OCT2 P-gp MRP OCT1, OCT2, OCT3 BCRP, P-gp P-gp OCT2, OCT3 BCRP, P-gp P-gp P-gp? OCT, OCTN2 OCT3, OCTN2 MRP1 OCTN2 MRP1 P-gp? BCRP MRP4 OCTN2 OCT1, OCT2, OCT3 129 79 130 70 78,80 129 39 78,80 129 131 132 79,84 84 70 86 70,133 134 135 136 137 138 ? Indicates inconclusive evidence. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010 DOI 10.1002/jps
  • 4. DRUG TRANSPORTERS IN THE LUNG This review aims to give an insight into the current knowledge of the expression, localisation and activity of drug transporters in the lungs from a drug delivery perspective. Each family of transporters is reviewed in terms of their expression in normal human lungs and when data are available, expression in healthy lungs is contrasted with that in diseased lungs, excluding lung tumours which are not included in the discussion. The few studies that have evaluated the activity of transporters at the pulmonary absorption barrier or their interaction with inhaled drugs are summarised and the suitability of animal and cell culture absorption models for the investigation of active transport systems in the lungs is analysed. Transporters considered herein are those known to affect drug absorption, reabsorption or elimination in the intestine, kidney, liver or at the blood–brain barrier. Those encompass (1) the efflux pumps belonging 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 the solute-linked carrier (SLC or SLCO) superfamilies, that is the organic cation transporters (OCTs), the peptide transporters (PEPT1 and PEPT2), the organic anion transporters (OATs) and the organic anion transporting polypeptide (OATPs). Although the lung resistance-related protein (LRP) is highly expressed in the lung and its critical role in multidrug resistance against chemotherapeutic agents is well documented,15 little information is available on its involvement in the transport of conventional molecules across the respiratory epithelium. Hence, that transporter is not discussed in this review. ATP-BINDING CASSETTE (ABC) TRANSPORTERS ABC transporters are a large family of transmembrane proteins which function as ATP-dependent efflux pumps capable of exporting a broad range of chemically diverse substances from the cell cytoplasm to the external environment. Approximately 50 members of the ABC family have been identified in humans. Those transporters are classified into seven subfamilies designated from A to G. Amongst those, P-gp, MRPs and BCRP are well known for their role in multidrug resistance (MDR), a phenomenon which results from the expulsion of chemotherapeutic agents from cancerous cells that overexpress efflux pumps.16 ABC transporters are also present in normal tissues where they prevent the accumulation of xenobiotics and therefore, they actively contribute to the tissue defense mechanisms.17 In addition, their involvement in the poor oral bioavailability and/or tissue distribution of a large series of drugs as well as in their hepatobiliary and DOI 10.1002/jps 2243 renal excretion has been demonstrated.18 Due to their potential role in limiting the transport of inhaled therapeutic molecules across the respiratory epithelium and in the pathophysiology of airway diseases, the expression and functions of ABC transporters in normal and diseased lungs have been granted great attention. Those topics were first covered in an excellent review published a few years ago.19 Several subsequent publications have largely contributed to the evaluation of the actual role of ABC transporters in the pharmacokinetic profile of aerosolised drugs and in the development of COPD. Those recent studies together with older ones exploring the expression of ABC transporters in respiratory cell culture models in vitro are summarised below. P-gp/MDR1 P-gp, also called MDR1 or ABCB1, is a 170 kDa transporter mainly expressed in the apical membrane of the enterocytes, hepatocytes, proximal renal tubules and at the blood–brain barrier. The protein is encoded by the MDR1 gene in humans and both the mdr1a and mdr1b genes in rodents. Since it is well established that P-gp limits the oral absorption of drugs, prevents their entry into the central nervous system and is responsible for many drug–drug interactions,20 that transporter has been the most extensively studied in the lungs. P-gp in the Lung Tissue P-gp mRNA was detected in normal human lung tissue by RT-PCR21,22 and microarray analyses.23 The intensity of expression was lower than in other major organs involved in drug absorption, distribution and elimination such as the small intestine, liver, kidney and brain. Immunohistochemistry techniques localised the transporter on the apical membrane of the bronchial and bronchiolar epithelia,22,24–26 in the endothelial cells of the bronchial capillaries18 and in alveolar macrophages.24,25 Contradictory data has been published regarding P-gp expression in the alveolar region. No staining of the alveolar epithelium was observed in three studies24–26 while type I pneumocytes stained positive at their apical side in the study by Campbell et al.27 Both mdr1a and mdr1b mRNA are present in the lungs of mice28 and rats,29 with highest levels of the mdr1b messenger in both species. The cellular distribution of P-gp in rodent lungs was nevertheless shown to be similar to that in humans.24 P-gp expression in diseased lungs or in the lungs of smokers compared to normal lungs has not or hardly been quantified. mRNA levels in the lungs of smokers, nonsmokers or ex-smokers were reported not to be statistically different.22 However, the pulmonary JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
  • 5. 2244 BOSQUILLON clearance rate of 99mTc-sestamibi, a P-gp substrate, after aerosol delivery was reduced in healthy smokers as compared to nonsmokers.30 This was assumed to result from an upregulation of P-gp in the lungs of smokers. As 99mTc-sestamibi is also a substrate for MRP1,31 the delayed elimination of the tracer from the lungs of smokers might also be due to a downregulation of the basolaterally located MRP1 caused by cigarette smoke.14 While decreased P-gp levels in the inflamed intestinal tissue of patients suffering from gastrointestinal inflammatory disorders have been reported,32 no statistical difference was found between the immunostaining intensity of bronchial biopsies of COPD patients versus healthy controls or of patients suffering from severe COPD versus patients with a milder form of the disease.13 In contrast, whether P-gp expression is altered in the bronchial epithelium of asthmatic patients has not been evaluated. Similarly, the effect of chronic administration of inhaled glucocorticoids on P-gp expression in the lungs has not been investigated whereas related studies suggest they might upregulate the transporter in the pulmonary tissue. For example, a stronger immunohistochemical staining for P-gp was observed in nasal polyps of patients treated with local doses of budesonide,33 a common corticoid used in the prophylaxis of asthma. Also, P-gp expression increased about twofold in the lungs of rats following oral34 or intraperitoneal35 administration of dexamethasone. Cystic fibrosis (CF) is a congenital disorder caused by a mutation in the CF transmembrane conductance regulator (CFTR) gene. CFTR is a member of the ABC family of transporters which regulates the transport of ions. Due to a structurally close similitude with Pgp, it has been hypothesised that some altered functions of CFTR might be compensated by an overexpression of P-gp in CF patients.36,37 On the other hand, it has been reported that Cif, a toxin produced by Pseudomonas aeruginosa whose infections 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 human respiratory cell models currently available for drug permeability studies, that is the Calu-339–42 and 16HBE14o-14,41,43 bronchial cell lines, NHBE7,41,44 and alveolar type-I like cells41,45 with however, contradictory data regarding its expression and localisation on the cell membrane.9 For instance, no vectorial transport of ciprofloxacin, digoxin and vinblastine, all P-gp substrates, has been observed in Calu-3 cell layers, suggesting those cells did not express a functional transporter.46 Western blot analysis however revealed that cell line expresses JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010 P-gp.39,40,42,47 Functional studies in Calu-3 layers using rhodamine 123,40 cyclosporine47 and digoxin9 as P-gp substrates showed a polarised transport in the basolateral to apical (B-A) direction, indicating an apical localisation of the efflux pump on the cell membrane. By contrast, P-gp was localised in immunofluorescence on the basolateral side of the cell layer and flunisolide transport was enhanced in the absorptive direction in the study by Florea et al.39 Those conflicting results can likely be explained by differences in cell culture conditions and by the use of nonspecific P-gp substrates and inhibitors. By using GF120918a, a highly potent and more selective P-gp inhibitor,48 Madlova et al.9 measured a P-gp mediated polarised digoxin transport in the B-A direction only in Calu-3 cell layers at passages over 50 grown for three weeks on cell culture inserts. It was therefore suggested that layers of Calu-3 cells might not express functional P-gp at low passages and when cultured for a shorter period of time on permeable filters. This hypothesis was however not confirmed by Brillault et al.42 who detected P-gp by Western blotting in Calu-3 layers at passages 22–30 grown for 15 days on cell culture filters. In addition, they measured a B-A polarised transport of the fluoroquinolone antibiotic moxifloxacin that was inhibited by PSC-833, another P-gp potent inhibitor, while probenicid, an MRP inhibitor, had no effect. A dependence of the time in culture on P-gp activity was observed in NHBE cells with no vectorial transport of digoxin after 14 days in culture and a net absorptive transport reversed by GF120918A after 21 days.9 This was in agreement with the increased mRNA levels quantified by RT-PCR in NHBE cells after 14 and 21 days on cell culture inserts as compared to after 7 days on those inserts.7 Functional studies in NHBE using digoxin as the substrate suggested a modest P-gp activity was present at the basolateral side of the cells.9 However, that assumption was not confirmed by localisation studies. The human bronchial CF epithelial cell line CFBE41o- was shown to form tight monolayers expressing P-gp when cultured under submerged conditions on permeable supports and hence, was deemed to be a suitable in vitro model for studying the disease at the cellular level.49 Due to the unsuitability of the A549 alveolar cell line to represent the absorption barrier of the deep lung in vitro3 and the scarcity of human lung tissue, animal primary cell culture models of the alveolar epithelium have been developed as alternatives to human systems. The expression and functionality of P-gp was confirmed in monolayers of rat type-1 like pneumocytes by Western blot and vinblastine bidirectional transport studies, respectively.27 Although monolayers of porcine alveolar epithelial DOI 10.1002/jps
  • 6. DRUG TRANSPORTERS IN THE LUNG cells stained positive for P-gp under a confocal microscope, no asymmetric transport of several Pgp substrates was measured, indicating a lack of functionality of the transporter in that model.50 Disposition Studies of P-gp Substrates in Ex Vivo and In Vivo Animal Models Uptake studies in isolated perfused animal lungs demonstrated the contribution of efflux pumps in drug accumulation from the perfusate into the lungs and therefore a significant activity of ABC transporters in that organ. Idarubicin concentrations in rat perfused lungs were enhanced after its infusion through the pulmonary circulation together with the P-gp modulators cinchonine and rutin.51 Similarly, in rabbit lungs, the disappearance of the P-gp substrate rhodamine 6G from the perfusate was increased in presence of the inhibitors verapamil and GF120918.52 In both studies, inhibitors were added in the perfusion solution and could therefore modulate Pgp present on both the endothelial cells of the pulmonary capillaries and the epithelial cells of the airways. The enhanced pulmonary accumulation of the substrates in presence of inhibitors suggests a higher P-gp activity at the endothelium site than at the airway epithelium since inhibition of the epithelial transporter would rather slow down the diffusion of the substrates from the perfusate. This hypothesis is in agreement with two absorption studies of inhaled P-gp substrates that have failed to highlight any significant influence of an efflux mechanism on their disposition from the airspaces. The percentage of losartan transferred from the airways to the perfusate in 120 min reached 94 Æ 2% after aerosolisation to a rat IPL model, indicating a negligible P-gpmediated efflux at the apical membrane of the airway epithelium.53 Nevertheless, relatively high drug concentrations were used which might potentially have saturated the transporter. In the second study, the coinstillation of GF120918a with digoxin to a rat IPL did not modify the pulmonary absorption profile of that model P-gp substrate.9 In contrast, the recovery of rhodamine123 in the perfusion solution after intratracheal delivery to a rat IPL was enhanced in presence of GF120918a both in the instillate and perfusate,10 which indicates an efflux mechanism restricted the pulmonary absorption of the dye. However, GF120918a possesses some inhibitory activity against BCRP48 and rhodamine123 is a substrate for that transporter.54 The actual contribution of P-gp in the restricted absorption of rhodamine123 is therefore unclear. The only in vivo study so far that aimed at evaluating P-gp impact on drug disposition from intact lungs showed that the pharmacokinetic profile of digoxin after intratracheal instillation was similar DOI 10.1002/jps 2245 in mdr1a (À/À) deficient and mdr1a (þ/þ) wild-type mice.55 However, mdr1b P-gp is still expressed in the lungs of mdr1a (À/À) mice and an upregulation of the pulmonary mdr1b P-gp as a compensation mechanism in mdr1a knockout animals is conceivable. Digoxin was used at concentrations below P-gp saturation but, as in the study by Madlova et al.,9 the test solution was administered to the lungs of animals by intratracheal instillation and an oversaturation of the transporter locally in the pulmonary tissue cannot be excluded. Digoxin was shown to be well and rapidly absorbed from the lungs.9,55 Hence, an epithelial efflux mechanism might not impact on its permeation profile to a significant extent whereas it might hinder the absorption of a compound with a prolonged retention in the airspaces. Digoxin is also a substrate for members of the organic anion transporting polypeptide (OATP) family of transporters;56,57 the presence of some of which has been confirmed in the lungs (see below). The contribution of an active uptake mechanism in digoxin pulmonary absorption might therefore have counterbalanced a potential P-gp-mediated efflux. MRPs The MRPs are nine organic anion efflux pumps identified as MRP1–9. MRP1, 4, 5, 7, 8, 9 are present in many tissues while MRP2, 3, 6 are mainly expressed in the liver and kidneys.58 MRP1, 3, 4 are basolateral transporters while MRP2 and 5 are located in the apical membrane of the cells.58 MRP2 is known to play an important role in the biliary excretion of drug conjugates, especially those with glutathione.59 MRPs in the Lung Tissue Using RT-PCR techniques,21,60 MRP1 and MRP5 were shown to be highly expressed in normal human lung tissue while MRP6 and 7 were moderately expressed and MRP2, MRP3, MRP4, MRP8 and MRP9 levels were either low or undetectable. Subsequent gene microarray analyses confirmed the high expression of MRP1 and MRP5 and the absence of MRP2 in the lungs.23 However, the intensity of expression was found to be very high for MRP7, high for MRP3, MRP8, MRP9 and moderate for MRP4 and MRP6.23 The high expression of MRP1 in the lungs was further corroborated by Western blotting61 and immunohistochemistry.24,61,62 Bronchial/bronchiolar epithelial cells were stained but while labelling was localised in the cytoplasm of ciliated cells just below the cilia in the study by Flens et al.,61 MRP1 was found on the basolateral membrane of ciliated, mucous-producing and basal cells in two other JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
  • 7. 2246 BOSQUILLON studies.24,62 Alveolar macrophages exhibited staining in their cytoplasm whereas no staining was observed in pneumocytes.24,61 In concordance with gene expression data, weak63 or absent staining24 was observed in bronchial/bronchiolar epithelial cells for MRP2. MRP3 could not be detected in any area of the lung.24 A similar expression pattern with high levels of MRP1 mRNA and low levels of MRP2 was reported in rats and mice23,64,65 although MRP1 expression seemed to be lower in mice than in rats and humans.23,65 Gene microarrays indicated the lungs of rodents might express lower levels of MRP3 and MRP6 but higher levels of MRP4 than human lungs.23 MRP1 mRNA levels were not statistically different in healthy smokers, ex-smokers and nonsmokers.62 However, MRP1 expression, as assessed by immunostaining, was lower in bronchial biopsies of COPD patients as compared to that in healthy patients as well as in patients affected by severe COPD versus those with a mild to moderate form of the disease.13 Consequently, a role of that transporter in the pathophysiology for COPD might be postulated.13 MRPs in Respiratory Cell Culture Models In Vitro In accordance with gene microarray data in human lungs, high levels of MRP1, MRP3, MRP5 and MRP7 mRNA were measured by RT-PCR in human epithelial respiratory cell culture models.41 MRP4 and MRP8 transcripts were detected in normal human bronchial and alveolar cells but were absent in the Calu-3 and 16HBE14o- cell lines.41 MRP6 was moderately expressed in bronchial models but highly expressed in alveolar type-I like cells.41 The most striking discrepancy with human lungs was the presence of MRP2 transcripts in all in vitro models while that transporter seems not to be expressed in vivo.23 MRP1 was reported to be expressed both at the mRNA and protein level in undifferentiated NHBE and normal alveolar lung cells grown on cell culture dishes, with nevertheless high intra-individual variations.44,66 The functionality of the transporter in normal lung cells was demonstrated by the decreased efflux of the MRP substrate carboxydichlorofluorescein (CDF) in presence of the MRP inhibitor MK-571.44 Immunodetection of MRP1–5 in undifferentiated NHBE and alveolar cells showed that MRP1 and MRP3 were localised in the cell membrane while MRP2, MRP4 and MRP5 were intracellular proteins.67 However, when cells were cultured on inserts at an air–liquid interface, MRP1 and MRP2 were detected on the basolateral membrane of the cells or on both the apical and basolateral membranes, respectively.67 This illustrates the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010 necessity of investigating transporter expression and localisation in differentiated cells grown under physiologically relevant conditions. Torky et al.68 investigated the influence of proinflammatory mediators on MRP1 functions in normal human respiratory epithelial cells. The uptake of CDF by undifferentiated NHBE cells exposed to arachidonic acid and prostaglandin E2 for 3 days was decreased as compared to that in untreated cells while prostaglandin F2a had no effect on MRP1 activity.68 In alveolar cells, only arachidonic acid modified CDF efflux.68 In Calu-3 cells, MRP1 was localised on the basolateral side of monolayers cultured on permeable filters.69 Efflux and transport studies were carried out using calcein as the substrate and indomethacin, probenicid or MK-571 as inhibitors. However, appraising the actual contribution of MRP1 in calcein translocation across Calu-3 cells was rendered complex due to the dye interacting with P-gp.69 In 16HBE14o- cells, a strong immunohistochemical staining was obtained for MRP1 while this was weak for MRP4 and negative for MRP2, MRP3 and MRP5.14 Because MRP1 was shown to be downregulated in the lungs of COPD patients,13 that cell line was used to evaluate the effect of cigarette smoke and drugs commonly used in the treatment of COPD on MRP1 activity in the bronchial epithelium. The efflux of the fluorescent dye CDF out of cells exposed to cigarette smoke extracts was diminished when compared to that in untreated cells.14 This inhibitory effect was caused by a direct interaction of cigarette smoke components with the transporter.14 In addition, in the cytotoxicity MTT assay, the reduction of the metabolic activity of 16HBE14o- cells provoked by cigarette smoke extracts was enhanced in presence of the MRP inhibitor MK-571,14 confirming the protective role of MRP1 against cell damage induced by cigarette smoke and hence, the probable role played by the transporter in the development of COPD. The intracellular accumulation of CDF by 16HBE14ocells was enhanced in presence of budesonide but this inhibitory effect on MRP1 transport was reduced by formoterol whereas formoterol on its own had little effect on MRP1 activity.70 By contrast, ipratropium and N-acetylcysteine decreased the accumulation of the dye, suggesting they both stimulated MRP1mediated efflux.70 Whether those modulations of MRP1 activity caused by drugs used in COPD are beneficial or detrimental for the treatment of the disease is currently unknown. Both type I and type II rat pneumocytes in primary culture stained positive for MRP1 at their basolateral surface when grown on permeable filters and indomethacin increased the basolateral to apical transport of fluorescein across type II cell monolayers as well as the intracytoplasmic DOI 10.1002/jps
  • 8. DRUG TRANSPORTERS IN THE LUNG accumulation of the dye by those cells.71 Similar functionality studies could not be carried out using type I cell monolayers as the monolayer integrity was compromised in presence of indomethacin. Nevertheless, in absence of apical to basolateral permeability data and control experiments demonstrating the involvement of an active mechanism in fluorescein translocation, transport data in type II monolayers must be interpreted with caution. Fluorescein permeability data are indeed commonly used to verify the integrity of epithelial cell monolayers as the dye is assumed to be exclusively transported by a passive paracellular route.3,4 2247 metabolites, xenobiotics and drugs.75 Their involvement in the intestinal absorption and renal excretion of cationic drugs has been demonstrated and recent studies indicate they play a crucial role in the regulation of brain functions as well as those of basophils.75 As the structure, tissue distribution, physiological functions of OCTs and their roles in drug absorption and elimination was reviewed recently,75–77 the focus here is on their expression, localisation and functions in the lung with an emphasis on their interaction with inhaled drugs. OCTs in the Lung Tissue BCRP/ABCG2 BCRP is a 72 kDa transporter encoded by the ABCG2 gene which was first isolated from a breast cancer cell line. Besides being overexpressed in many cancer cells, it is also highly expressed in the placenta, the gastrointestinal tract, the brain, the liver and the breast tissue where it regulates the transfer and accumulation of xenobiotics.72 Data regarding BCRP expression in the lung are sparse and contradictory. In early studies, BCRP mRNA was either not present73 or detected only at a low level74 in normal human lung and a weak but detectable immunostaining was observed in the epithelium, in seromucinous glands and small capillaries.24 Gene microarrays recently showed BCRP was relatively highly expressed in human lungs whereas its expression was comparatively low in the lungs of rats and mice.23 Transcripts for BCRP were found in all human lung epithelial cell culture models with however underexpression in Calu-3 and overexpression in 16HBE14o-.41 A strong immunostaining for BCRP was obtained in 16HBE14o- cells,14 which confirmed the transporter is highly expressed in that cell line. ORGANIC CATION TRANSPORTERS OCTs are members of the SLC22A family of transporters which belongs to the major facilitator superfamily (MFS). They comprise five main subtypes of carriers; the electrogenic OCT1, OCT2, OCT3 and the electroneutral OCTN1, OCTN2. In addition, OCT6 and octn3 have been cloned from humans or rodents, respectively, where they are found mainly in testis.75 They all have the capacity to translocate various endogenous and exogenous molecules across the plasma membrane in both directions. Although neutral molecules can be transported, the majority of their substrates are positively charged at physiological pH and include hormones, neurotransmitters, DOI 10.1002/jps The five main subtypes of OCTs have been detected in healthy human lungs. However, conflicting observations have been made. Lips et al.78 found high levels of OCT1, OCT2 and OCT3 mRNA in human lung tissue but they did not quantify OCTN1 and OCTN2 levels. Using immunohistochemistry techniques, OCT1 and OCT2 were localised on the apical membrane of ciliated cells of the trachea and bronchi. In addition, OCT1 was detected in the cytoplasm of ciliated cells and OCT2 in the plasma membrane of basal cells. Ciliated cells stained weakly for OCT3, by contrast to the entire membrane of basal cells and the basolateral membrane of intermediate cells which was intensively labelled. In the gene microarray analysis by Bleasby et al.,23 the intensity of expression in human lungs was weak for OCT2, moderate for OCT1 and OCT3 and high for OCTN1 and OCTN2. Horvath et al.79 measured high levels of OCTN1 and OCTN2 mRNA and very low levels of OCT1-3 mRNA in the airway tissue of both healthy and CF patients. The same group also found high levels of OCT3 mRNA in airway smooth muscle cells and using immunohistochemistry, they visualised the transporter in bronchial and pulmonary blood vessels.80 The expression of the other OCT subtypes was low or undetectable in muscular and endothelial cells.80 OCTN1 positive staining was observed on the luminal side of the trachea epithelium and less intensively in alveolar macrophages while OCTN2 staining was positive on the apical membrane of the airway and alveolar epithelia.79 The intensity of OCT1, OCT2 and OCTN2 gene expression was shown to be similar in human and rodent lungs whereas that of OCT3 and OCTN1 appeared, respectively, higher or lower in rodents compared to humans.23 OCT1, OCT2 and OCT3 were identified in rat lungs at the protein level78,81 but although the mRNA of all three transporters was found in murine lungs, only OCT1 and OCT3 were visualised by immunofluorescence in that species.82 In rats, the apical membrane of ciliated cells of the trachea and bronchi stained positive for the three JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
  • 9. 2248 BOSQUILLON transporters while that of alveolar epithelial cells was stained for OCT1 and OCT3 only. OCT3 was, in addition, detected in the plasma membrane of bronchial basal cells. In mice, OCT1 was observed on the apical side of ciliated cells and OCT3 mainly in bronchial smooth muscles, although a weak staining was obtained in the bronchial epithelium as well.82 The protein expression and localisation of OCTN1 and OCTN2 in the lungs of rodents have not been investigated to date. Pulmonary expression of OCTs in human lung diseases has only been reported for CF, where the expression pattern was unaltered compared to normal lungs.79 The expression of OCT1-3 in rats and mice lungs sensitised with ovalbumin and exposed to the antigen by aerosol delivery was compared with that in untreated animals in order to evaluate the effects of acute allergic airway inflammation on OCTs regulation.81 In allergic rats, OCT1 was upregulated whereas OCT2 and OCT3 were downregulated 48 h after antigen challenge. OCT2 expression was similarly reduced in challenged mice but OCT1 and OCT3 levels were identical in inflamed and healthy mice lungs, which suggests inter-species variations in the regulation of OCTs. Considering those data in animals, it is now paramount to determine whether OCT expression is modified in chronic inflammatory respiratory diseases, especially since those transporters might have implications in asthma/COPD pathophysiology and pharmacotherapy. Indeed, based on uptake and release studies in Xenopus laevis transfected with OCT1-3 mRNA78 and the accumulation of acetylcholine in the bronchial epithelium of OCT1/2 doubleknockout mice,82 it has been proposed that OCT1 and OCT2 mediate the release of the nonneuronal acetylcholine produced by bronchial epithelial cells into the airway lumen where it controls mucus production, cilia beat frequency and epithelial cell proliferation.83 OCTs in Respiratory Cell Culture Models In Vitro In comparison to the extensive investigation on ABC transporters in respiratory cell models in vitro, only a limited number of studies have looked at OCTs in cultured airway epithelial cells. A recent RT-PCR analysis of drug transporters in human bronchial cell culture models revealed OCT1 and OCTN2 were moderately expressed while OCT2 was absent in both normal cells and cell lines.41 Various OCT3 and OCTN1 expression were observed amongst models. While high levels of OCT3 mRNA were present in normal bronchial cells and Calu-3 cells, the transporter was not detected in 16HBE14o-. Intense signals were measured for OCTN1 transcripts in Calu-3 and 16HBE14o- cells but not in normal cells. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010 In a previous study, normal human bronchial epithelial cells were shown to highly express OCTN1 and OCTN2 on their apical membrane while expressing low amounts of OCT1-3 when grown on permeable filter at an air–liquid interface for 6–8 weeks.79 OCT expression profile in NHBE layers was similar to that reported by the same research team in freshly isolated human bronchial cells. In contrast to the study by Endter et al.,41 mRNA for all the five OCTs were detected in the bronchial cell lines Calu-3 and 16HBE14o- grown in cell culture flasks.84 Cells were, however, screened for OCTs at passage numbers higher than those commonly used for permeability measurements in those cell lines.3 Unfortunately, the passage number at which Endter et al.41 examined bronchial cell lines was not specified. Therefore, it is unknown whether discrepancies between the two studies arose from an upregulation of transporter expression in those in vitro models at high passage numbers or from differences in culture conditions. The uptake of the model organic cation guanidine by layers of rabbit alveolar epithelial cells grown on Transwell1 inserts was shown to be saturable and was inhibited by a series of positively charged molecules.85 This indicated a carriermediated transport process for organic cations is present in the alveolar epithelium. Interestingly, the b2-agonist salbutamol, which is positively charged at physiological pH was actively transported with a net absorptive flux in layers of the two bronchial cell lines Calu-3 and 16HBE14o-.84 Although the transporter involved was not identified, the organic cations TEA and guanidine significantly decreased the A-B transport of salbutamol, suggesting the involvement of one or several member(s) of the OCT family. This assumption was later supported by evidence demonstrating salbutamol (albuterol) and formoterol, another positively charged b2-mimetics, modulate the activity of OCTs. Those two bronchodilators were shown to inhibit the uptake of the model cationic fluorophore 4-[4-(demethylamino)-styryl]-Nmethylpyridinium (ASPþ) by undifferentiated normal human bronchial cells grown on coverslips.79 The transporter involved was identified as OCTN2, based on the reduction of ASPþ uptake in presence of the OCTN2 inhibitor L-carnitine. However, an interaction of formoterol and salbutamol (albuterol) with other members of the OCT family cannot be excluded as cells used in that study did not express OCT1-3. In a related work,80 formoterol was reported to be internalised into human airway smooth muscle cells by an OCT3-mediated process, which indicates it is a substrate or inhibitor for more than one OCT subtype. The inhaled antimuscarinic bronchodilators ipratropium and tiotropium bear a permanent positive charge due to their quaternary ammonium structure DOI 10.1002/jps
  • 10. DRUG TRANSPORTERS IN THE LUNG and hence, it can intuitively be prophesied that they are very likely to be substrates for OCTs. Ipratropium inhibited the uptake of L-carnitine by the human proximal tubule cell line Caki-1, which demonstrates it is recognised by OCTN2.86 Besides cationic bronchodilators, inhaled glucocorticoids were also shown to interact with OCTs, although they are not actually translocated by those transporters. Budesonide and fluticasone inhibited the uptake of acetylcholine by OCT2 transfected X. laevis78 as well as the OCT3-mediated internalisation of formoterol by human airway smooth muscle cells.80 Although strong evidence has shown cationic bronchodilators interact with OCTs, it is at present not known whether those transporters actually contribute to their absorption across the respiratory epithelium. Similarly, the consequences on the treatment of inflammatory respiratory diseases of inhaled bronchodilators and corticosteroids interacting with OCTs are unclear. PEPTIDE TRANSPORTERS Peptide transporters are members of the SLC15 family which is part of the proton-coupled oligopeptide transporter (POT) superfamily. The structure and physiological functions of the two main transporters of that family, PEPT1 and PEPT2 have been extensively described elsewhere.87 Briefly, PEPT1 and PEPT2 are capable of transporting any di- or tripeptide derived from the 20 L-a-amino acids in association with proton translocation independently of the substrate charge. PEPT1 is essentially expressed in the apical membrane of epithelial cells of the small intestine, renal tubules and bile ducts while PEPT2 is present in many organs, such as the kidneys, brain, lung, pituitary, mammary glands, reproductive organs.88–90 Because of their very broad substrate specificity, PEPT1 and PEPT2 have the capacity to translocate peptidomimetic drugs such as the angiotensin-converting enzyme inhibitors captopril, enalapril and fosinopril91 or the b-lactam antibiotics,92 respectively, and it is now well established that PEPT1 and PEPT2 contribute to the high bioavailability of peptide-like drug molecules. Drug inhalation is an attractive route of delivery for the treatment of pulmonary infections. Several antiinfectious agents active against respiratory pathogens are substrates for PEPT1/PEPT2, for example penicillin and cephalosporin antibiotics and the antiviral drugs valacyclovir and valganciclovir.93 The presence of peptide transporters in the respiratory tract can potentially affect the absorption and distribution of those compounds, with consequences on their anti-infectious efficiency.93 DOI 10.1002/jps 2249 PEPT1/PEPT2 in the Lung Tissue High Pept2 mRNA levels have been measured in the lungs of rabbits,94,95 rats90,96,97 and mice.90 By contrast, weak or no signals have been observed for Pept1 in rabbit,95,98 rat90,96,99 and murine90 lung extracts. Based on expression data in animals, PEPT1 was assumed for years to be absent from the human respiratory tract or at least, not to significantly influence inhaled drug distribution in the lungs.93 However, Western blot analysis and uptake studies using di- and tripeptides showed rat alveolar macrophages express functional Pept1 protein.100 Gene microarrays revealed PEPT1 is expressed in the lungs although to a lower extent than PEPT223 and PEPT1 mRNA was detected very recently in the human bronchi of healthy adults.101 The recent discovery of PEPT1 in human lung implies the early assumption that the transporter does not affect pulmonary drug disposition might need reappraisal. Due to the high expression of PEPT2 in human and animal lungs,23 the regional distribution of that transporter has been examined in rat,96 mice96 and healthy or CF human102 lungs using immunohistochemistry techniques. In all species, a positive staining was obtained on the apical membrane of airway epithelial cells, in the cytoplasm of type II pneumocytes and on the endothelium of small blood vessels. The staining intensity was similar in healthy and CF lung samples.102 The presence of Pept2 in rat type II alveolar cells was later confirmed, although the transporter appeared to be located on the apical plasma membrane.103 The functionality of Pept2/PEPT2 in the lungs was demonstrated by performing ex vivo uptake studies of the fluorophore-conjugated dipeptide D-Ala-LysAMCA by isolated mice96 and human102 lung specimens. The model peptide accumulated in airway epithelial cells and type II pneumocytes, two cell types known to express the transporter. In addition, the intracellular fluorescence was reduced after incubation of the lung samples with high concentrations of the dipeptide glycyl-(L)-glutamine or the synthetic cephalosporin and PEPT2 substrate cefadroxil, while addition of the PEPT1 substrate captopril had no effect. PEPT1/PEPT2 in Respiratory Cell Culture Models In Vitro Consistent with gene expression data in human lungs, high PEPT2 mRNA levels were found in human bronchial epithelial cells and bronchial cell lines in vitro.41 In contradiction with expression levels in vivo, PEPT1 was highly expressed in Calu-3 and 16HBE14o- but was absent in normal cells.41 Primary human airway epithelial cells grown on permeable filters at an air–liquid interface were JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010
  • 11. 2250 BOSQUILLON shown to express PEPT2 on their apical cell membrane.104 The two genetic variants previously identified105 PEPT2Ã1 and PEPT2Ã2 were represented amongst the airway samples but the intensity of the RT-PCR signal was similar in all donors. The apical to basolateral transport of the dipeptide GlySar across cell layers was saturable, unaffected by the genotype and inhibited by b-lactam antibiotics (PEPT2 inhibitors) while ACE inhibitors (PEPT1 inhibitors) had no effect. This indicated the transporter was functional in vitro and its activity was not influenced by genetic variations, at least at the pH of the lung fluid, which is about 6.5. Differences in GlySar translocation were indeed observed previously between the two haplotypes at pH 6.105 Both PEPT1 and the PEPT2Ã1 variant were detected in layers of Calu-3 cells grown at an air– liquid interface.101 PEPT1 expression was confirmed by Western blot and the transporter was localised on the apical cell membrane. In contradiction with previous studies which had concluded PEPT1 was not involved in peptide transport across the airway epithelium,102,104 the uptake and transport of GlySar was shown to be mediated by PEPT1 and not PEPT2 in that cell line. b-Ala-L-His uptake by rabbit tracheocytes cultured as air–liquid interfaced monolayers increased when the apical medium was buffered at pH 6.5 as compared to 7.4 and was inhibited by Gly-L-Phe, but neither by Gly-D-Phe or amino acids.106 Although the transporter involved was not identified, the marked effect of the pH gradient coupled to previous evidence of its presence in the lungs suggested it to be PEPT2. Surprisingly, the transport of Gly-L-Phe across the cell layers was symmetrical at pH 7.4 and in the same order of magnitude as the paracellular marker mannitol. Decreasing the pH of the apical medium to 6.5 reduced the A-B permeability of the peptide, despite an enhanced uptake at that pH, while the B-A transport was unchanged. As paracellular diffusion is hindered by the highest protonation of Gly-L-Phe at pH 6.5, a passive mechanism was therefore assumed to be the unique permeation pathway followed by the peptide across the tracheal epithelium. ORGANIC ANION TRANSPORTERS Two families of structurally unrelated transporters are known to mediate the absorption and elimination of endogenous and exogenous organic anions: the organic anion transporter (OAT) family and the organic anion transporting polypeptide (OATP) family (see below). OATs, like OCTs, belong to the SLC22A family of transporters. The six members of OATs identified so far (OAT1-4, URAT1 and oat5) are JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 5, MAY 2010 all expressed in the kidneys.107,108 OAT2, OAT3, OAT4 are additionally found in the liver, brain and placenta, respectively.107,108 OATs are the main mechanism by which organic anions are excreted and reabsorbed in the kidneys.108 They also contribute to drug–drug interactions and to the nephrotoxicity of some drugs and toxins.107 None of the OAT members could be detected by Northern blot in the lungs of humans,109–111 rats112–114 or mice.115,116 Gene microarrays confirmed the absence of OAT1, OAT3 and OAT4 in the lungs of all species. They nevertheless indicated OAT2 was relatively highly expressed in human and murine lungs.23 No OAT1, OAT2 and OAT3 transcripts were detected in any of the human bronchial epithelial cell culture models. As expected, OAT4 mRNA was absent in normal cells, but that transporter was highly expressed in the two bronchial cell lines Calu-3 and 16HBE14o-.41 ORGANIC ANION TRANSPORTING POLYPEPTIDES OATPs were originally designated by the SLC21 gene symbol and each transporter was essentially named by the researchers who had isolated them. This resulted in huge confusion as a same transporter isolated by independent groups was given different names or a human transporter had a designation similar to a rodent transporter whereas they were not equivalent.117 OATPs are now classified under the SLCO gene symbol according to a specific nomenclature whose details can be found elsewhere.57 The 11 human OATPs are divided into 6 families (OATP1– 6) further subdivided into subfamilies. Some of them are organ specific while others are ubiquitious.57,117 To date, only four of them, OATP1A2 (widely expressed), OATP1B1 (liver specific), OATP1B3 (liver specific), OATP2B1 (widely expressed) have been well characterised and shown to be responsible for drug– drug interactions.118,119 Many OATP substrates are also transported by the efflux pumps P-gp, MRP1, MRP2 or BCRP57 and assessing the contribution of each of those transporters in the pharmacokinetic profile of organic anions is a major challenge in absence of specific inhibitors and knockout mice for OATPs. Overall, the actual tissue distribution, physiological functions and substrate specificity of OATPs remain largely unknown.57 RT-PCR, Northern blot or gene microarray analyses of human tissue samples indicated OATP1A2,23,120 the two liver specific proteins OATP1B1 and OATP1B3,23,120,121 OATP1C1,23,122 OATP5A123 and OATP6A123,123,124 were not expressed in the lungs. By contrast, high levels of DOI 10.1002/jps
  • 12. DRUG TRANSPORTERS IN THE LUNG OATP2B1,23,120,125 OATP3A123,120,126 and 23,120,127 OATP4A1 expression were detected in human lungs while OATP4C1 was moderately present.23 Positive signals for oatp2b1 were also observed in rat23,128 and mice23 lungs and for oatp3a1 in rat lungs.126 Nevertheless, the actual expression of OATPs at the protein levels and their cellular localisation in the pulmonary tissue have not yet been investigated. Significant variations in the expression profiles of OATPs were noticed amongst human bronchial epithelial in vitro models.41 In accordance with gene microarray data, normal bronchial cells were reported to express high levels of OATP3A1 and OATP4A1 while not expressing OATP1B1, OATP1B1 and OATP1C1. However, in contradiction with expression profiles in human lungs, OATP1A2 was highly expressed and OATP2B1 was undetectable in those cells. The bronchial cell line Calu-3 appeared to express all OATP transporters except OATP1A2 while none of OATP transcripts but those for OATP3A1 and OATP4A1 were present in 16HBE14o- cells. CONCLUSIONS Based on the literature, it is manifest that the lungs express elevated levels of several drug transporters. Interestingly, transporter gene expression in the lungs appeared to be the highest after that in the liver and kidneys.23 Because techniques used to quantify transporter levels in whole organs are incapable of determining the cellular origin of the genes, the extent of transporter expression at the epithelial drug absorption site remains unknown. Not surprisingly, as observed for the exploration of transporters in other organs, the increasing number of studies published in the last 5 years attests of a recent interest in drug transporters in the lungs, both from academic institutions and the pharmaceutical industry. Nevertheless, contribution in the area has been limited and the physiological functions of drug carriers in the lung tissue, their potential involvement in common respiratory diseases and their roles in the disposition of inhaled drugs are essentially unexplored. This review discussed more specifically the role of drug transporters in the biopharmaceutics of aerosolised medications. To date, there is no strong evidence to indicate active transport mechanisms affect the distribution of drugs in the lungs. Considering the large surface area offered by the respiratory epithelium and rapidity of pulmonary clearance observed for most inhaled drugs, drug absorption from the lungs is less likely to be as influenced by transporters as it is from DOI 10.1002/jps 2251 the gastrointestinal tract. Delivery strategies currently under development nevertheless envisage exploiting efflux pumps present in the lung tissue to prolong drug retention in situ. However, it is noteworthy that the impact of transporters on the distribution of inhaled drugs might be influenced by their regional deposition in the respiratory tract and local metabolism. Appraising the absorptive role played by drug transporters in the lung tissue relies on simplified models of the respiratory absorption barrier, none of which has been validated yet for such an application. The development of reliable systems to explore drug transporters in the lungs is therefore a prerequisite to the evaluation of their influence on the PKPD profile of inhaled drugs. Understanding the regulation of transporters in pulmonary pathologies is also paramount for a realistic appreciation of their contribution in inhaled drug disposition under therapeutic conditions. REFERENCES 1. Ayrton A, Morgan P. 2008. Role of transport proteins in drug discovery and development: A pharmaceutical perspective. Xenobiotica 38:676–708. ´ ´ 2. Szakacs G, Varadi A, Ozvegy-Laczka C, Sarkadi B. 2008. 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