Transport of anti-allergic drugs across the passage cultured human ...

European Journal of Pharmaceutical Sciences 26 (2005) 203–210Transport of anti-allergic drugs across the passage culturedhuman nasal epithelial cell monolayerHongxia Lin a , Jin-Wook Yoo a , Hwan-Jung Roh b , Min-Ki Lee b , Suk-Jae Chung c ,Chang-Koo Shim c , Dae-Duk Kim c,∗a College of Pharmacy, Pusan National University, Pusan 609-735, South Koreab College of Medicine, Pusan National University, Pusan 602-739, South Koreac College of Pharmacy, Seoul National University, Seoul 151-742, South KoreaReceived 22 September 2004; received in revised form 3 May 2005; accepted 2 June 2005Available online 8 August 2005AbstractThe purpose of this study was to investigate the nasal absorption characteristics of a series of anti-allergic drugs across the human nasalepithelial cell monolayer, which was passage cultured by the liquid-covered culture (LCC) method on Transwell ® . Characterization of thiscell culture model was achieved by bioelectric measurements and morphological studies. The passages 2–4 of cell monolayers exhibited theTEER value of 1731 ± 635 cm 2 after 2 days of seeding and maintained high TEER value for 4–6 days. Morphological study by TEM andSEM showed the existence of the tight junctions, and the cuboidal shaped epithelial cells monolayer. A series of anti-allergic drugs, albuterolhemisulfate, albuterol, fexofenadine HCl, dexamethasone, triamcinolon acetonide, and budesonide were selected as model compounds fortransport studies. All the drugs were assayed using reversed-phase HPLC under isocratic conditions. Results indicated that within the log P(apparent 1-octanol/water partition coefficient) range from −1.58 (albuterol) to 3.21 (budesonide), there existed 100-fold difference in theapparent permeability coefficients (P app ). A log-linear relationship was shown between the drug log P and the P app across passaged human nasalepithelial monolayers. The amount of fexofenadine HCl and dexamethasone across passaged human nasal cell monolayers was concentrationdependentin the direction of apical to basolateral. The direction dependent transport studies were investigated among all these drugs and nosignificant difference in the two directions was observed. In conclusion, this LCC passaged human nasal epithelial culture model may be auseful in vitro model for studying the passive transport processes in nasal drug delivery.© 2005 Elsevier B.V. All rights reserved.Keywords: Passaged human nasal epithelial cell culture; Drug transport; Permeability; Anti-allergic drugs1. IntroductionThe drug delivery via the nasal route has recently receivedwidespread attention due to several advantages includinghigh systemic bioavailability and rapid onset of action.Drug candidates ranging from small metal ions to largemacromolecular proteins have been investigated in variousanimal models by nasal drug delivery (Krishnamoorthyand Mitra, 1998). Nearly, complete absorption of certainhormones and steroids by nasal administration (Hussain∗ Corresponding author. Tel.: +82 2 880 7870; fax: +82 2 888 5969.E-mail address: ddkim@snu.ac.kr (D.-D. Kim).et al., 1981; Lipworth and Jackson, 2000) revealed thepotential value of the nasal route for administration ofsystemic medications as well as for local effects, such as fornasal allergy, nasal congestion, and nasal infections.Although in vivo animal models have been widely used forthe investigation of nasal drug transport studies (Morimotoet al., 1991), in vitro nasal models are recently employedin the studies of the transport and metabolic properties ofthe nasal mucosa using the excised nasal tissue model, nasalhomogenates, and cell culture model. Among these models,the nasal cell culture models have attracted the attention ofpharmaceutical researchers as promising tools for definingtransport mechanisms and testing novel strategies to enhance0928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.ejps.2005.06.003

204 H. Lin et al. / European Journal of Pharmaceutical Sciences 26 (2005) 203–210drug transport and absorption. Furthermore, primary cultureof nasal epithelial cells from a variety of species includinghuman, bovine, rat, and rabbit can provide valuable invitro models in the study of nasal physiology. The cultureof cells and tissue derived from differentiated human nasalepithelium is an established tool for the studies of fibrosis,electrolyte transport, ciliogenesis, and ciliary movement(Ruckes et al., 1997; Jorissen and Bessems, 1995). In vitrocell culture models offer many advantages, including: (a) acontrolled environment for the study of epithelial cell growthand differentiation; (b) the elucidation of drug transport pathwaysand mechanisms; (c) rapid and convenient means ofevaluating drug permeability; (d) opportunity to minimize theexpensive and limited controversial use of animals (Schmidtet al., 1998). It has been reported that human nasal primarycultures as in vitro models show the potentials for the studyof nasal drug absorption, especially for the peptide transportstudies (Werner and Kissel, 1995; Kissel and Werner, 1998;Agu et al., 2001).However, efforts to develop and characterize nasal cellculture systems for drug metabolism and permeation arestill in their infancy. The main limitation is the difficulty inobtaining a reliable tissue source, which hinders the usefulnessof in vitro cell culture model especially if human tissueis preferred. In order to overcome the shortage of humannasal tissue, researchers have shifted from primary culturesof epithelial cells to passage cultured cell lines. Transformationof human nasal polyp cells using a chimaeric virus(Ad5/SV40 1613 ori-) is one of the examples of the change intrends. The extended lifespans ranged from 20 to more than250 population doublings beyond that of the primary cultures(Buchanan et al., 1990). In a previous report, we have establishedthe passaged human nasal epithelial culture method forthe drug transport studies (Yoo et al., 2003), where the LCCmethod was utilized and properties of nasal epithelium wereinvestigated by bioelectric and morphological studies. Thecharacteristic high TEER value, which was an indication ofthe formation of a tight junction formation, was maintainedup to the monolayers of passage 4. In order to verify the utilityof this passaged culture model for actual nasal transepithelialdrug delivery, a series of anti-allergic drugs were selectedfor transport studies and evaluated for the influence of druglipophilicity on solute permeability across the monolayers.The permeability characteristics of the model drugs acrossthe monolayers was compared with those of excised porcinenasal mucosa in the literatures to demonstrate the feasibilityof the in vitro model for nasal drug transport studies.2. Materials and methods2.1. MaterialsDexamethasone was purchased from Wako Pure ChemicalIndustries Ltd. (Osaka, Japan). Albuterol hemisulfate,albuterol, budesonide, Hank’s balanced salt solution (HBSS),h-EGF, and d-(+)-glucose were purchased from SigmaChemical Co. (St. Louis, MO). Triamcinolone acetonide andfexofenadine HCl were gifts from Handok-Aventis PharmaceuticalCo. (Seoul, Korea). Other cell culture reagents andsupplies were obtained from GIBCO Invitrogen Co. (GrandIsland, NY). BEGM Bulletkit was obtained from CambrexBio Science Inc. (Walkersville, MD), and Transwells ®(0.4 m, 12 mm diameter, polyester) were obtained fromCostar Co. (Cambridge, MA). All other materials were ofanalytical grade or better.2.2. Passage cultured human nasal epithelial cellmethodThe primary human nasal cell culture method used in thisstudy has been described in detail previously (Roh et al.,1999). Human nasal specimens were obtained from patientsundergoing surgery due to septal deviation or chronic sinusitis,using a protocol approved by the Institutional ReviewBoard (IRB) at Pusan National University Hospital, SouthKorea. Briefly, the nasal specimens were dissociated enzymaticallyusing 1.0% protease XIV (Sigma, St. Louis, MO)overnight at 4 ◦ C. Dissociated epithelial cells were washedthree times in DMEM containing 10% fetal bovine serumsupplemented with 100 U/mL penicillin and 100 g/mLstreptomycin. Cell suspension was pre-plated on a plastic dishat 37 ◦ C for 1 h in order to eliminate fibroblasts and myoblastsby differential attachment to plastic dish. Suspended epithelialcells were frozen and stored in liquid nitrogen tank forfuture usages. Frozen passage-1 stocks were thawed and culturedin T-flask using BEGM at 37 ◦ C in an atmosphere of 5%CO 2 and 95% relative humidity. The medium was changedevery 2 days.When cultures reached approximately 70–80% confluency,the cells were detached with 0.1% trypsin–EDTA, andwere seeded at densities of 2 × 10 5 to 3 × 10 5 cells/cm 2 onTranswell ® insert with 0.5 mL BEGM in the apical chamberand 1.5 mL DMEM (supplemented with 10% FBS, 100 U/mLpenicillin, 100 g/mL streptomycin, 1 ng/mL EGF, 1% nonessentialamino acid, and 1% l-glutamine) in the basolateralchamber. After 24 h, media were changed with DMEM inboth the apical and basolateral sides and the cells were maintainedin the medium by changing the medium every 2 days.Remaining cells after seeding were subsequently subculturedusing BEGM for the next passage. The subculture density was500 cells/cm 2 and the media were changed every other day.2.3. Measurement of bioelectric parameters andmorphological studiesThe transepithelial electrical resistance (TEER) was measureddaily by directly reading the values of an EVOMvoltohmmeter device (WPI, Sarasota, FL), and corrected bysubtracting the background due to the blank Transwell insertsand medium.

H. Lin et al. / European Journal of Pharmaceutical Sciences 26 (2005) 203–210 205Scanning (SEM) and transmission (TEM) electronmicroscopy were processed for morphological studies on day5. For SEM, each monolayer was fixed in 2.5% glutaraldehydein PBS at 4 ◦ C for 1 h, rinsed in ice-cold PBS, and thenfixed in 1.0% osmium tetroxide in PBS at room temperaturefor 1 h. The specimens were dehydrated through an alcoholseries and allowed to air-dry overnight. The specimenswere mounted on stubs with adhesive tape, sputter coated,and viewed in a Hitachi S-4200 scanning electron microscope(Hitachi, Japan). For TEM, the specimens were fixedas SEM. After dehydration, the specimens were embedded inEpon 812 semi-thin sections (80 nm), stained with toluidinblue and observed by light microscopy. Appropriate areaswere selected for ultra-thin sections, which were treated withuranyl acetate for 6 min and treated with lead citrate for 3 min.These sections were viewed under a JEM 1200 EXII electronmicroscope (Jeol, Japan).2.4. Physicochemical characteristics of anti-allergicdrugsThe solubility of model drugs in transport medium wasmeasured at room temperature. Excess amount of each compoundwas added to 2 mL of transport medium, and themixtures were stirred for 24 h. After filtering through MinisartRC 4 filter (0.45 m, Satorius, Germany), solutions wereanalyzed by HPLC after appropriate dilution with methanol.Apparent 1-octanol/water partition coefficient (log P)of model drugs was determined at room temperature, asreported in the literature (Dearden and Bresnen, 1988). An1-octanol/water mutual saturation was prepared for 24 h withgentle mechanical stirring, and each phase was separated. The100 L methanolic solution of each model drug (1 mg/mL)was placed in a glass vial and completely evaporated, andthen 1.0 mL of each saturated solvent was added to the vial.After shaking the vial for 24 h at 150 rpm, the phases wereseparated by centrifugation at 4000 rpm for 20 min. The concentrationof the compound in each phase was determined byHPLC after appropriate dilution with methanol.2.5. Transport studiesNasal epithelial cell monolayers after 4–8 days of seedingwere used for drug transport studies. All the transportexperiments were performed in an incubator at 37 ◦ C. Priorto each experiment, the monolayers were washed with apre-equilibrated transport medium (10 mM HEPES, 10 mMd-(+)-glucose), and allowed to equilibrate for 20 min in theincubator.For measurement of the apical to basolateral (A–B) transport,0.4 mL of the transport medium containing variousconcentrations of model drugs was added on the apical sideand 1.0 mL of the transport medium was added on the basolateralside of the insert. At pre-determined time intervals,all the receiver fluid was immediately replaced with an equalvolume of fresh transport medium. For measurement of thebasolateral to apical (B–A) transport, 1.0 mL of the transportmedium containing various concentrations of model drugswas added on the basolateral side and 0.4 mL of the transportmedium was added on the apical side of the insert. In orderto avoid damaging the monolayers, an aliquot of 0.3 mL wassampled from the apical side and replaced with the same volumeof fresh transport medium to maintain the sink condition.To monitor integrity of the cultured epithelial cell monolayers,the TEER value was measured at the beginning and endof each transport experiment.2.6. HPLC assayThe samples collected from transport studies were directlydetermined by HPLC. A reversed-phase C-18 column(Lichrospher ® 100, RP −18, 125 mm × 4 mm, 5 m, MerckDarmstadt, Germany) was used, except for albuterol, whichwas determined on a C-8 column (Lichrospher ® 100, RP-8,25 mm × 4 mm, 5 m, Merck Darmstadt, Germany). GilsonHPLC system equipped with a pump (Gilson Model 306,Gilson Inc., France), an automatic injector (Gilson Model234, Gilson Inc., France) and UV–vis detector (Gilson Model118, Gilson Inc., France) was used.2.7. Data analysisThe apparent permeability coefficients, P app (cm/s) werecalculated using the following equation:P app = dQ 1(1)dt AC 0where dQ/dt is the solute flux obtained from linear regression,A the surface area across which transport studies weremeasured (1.0 cm 2 ), and C 0 is the initial drug concentration.All the data were expressed as the mean ± S.D. (n > 3). Thestatistical significance of differences between treatments wasevaluated using the unpaired Student’s t-test.3. Results and discussion3.1. Measurement of bioelectric parametersWhen seeded at a density of 2 ∼ 3 × 10 5 cells/cm 2 , theLCC cell monolayers of passages 2–4 appeared to reachconfluence and began to exhibit a measurable transepithelialresistance from day 2. As shown in Fig. 1, a similar pattern ofTEER time-course profile was observed in passages 2–4 cultures.The mean maximum TEER value (1731 ± 635 cm 2 )of all the three passaged cultures appeared on 2 days afterseeding, and then rapidly decreased thereafter. The highTEER value implies the formation of tight junction that is animpermeable junction located at the apical side. The promptformation of tight monolayers in LCC condition can beadvantageous for the short culture duration for in vitro nasaltransport studies. Based on our results from the previous

206 H. Lin et al. / European Journal of Pharmaceutical Sciences 26 (2005) 203–210Fig. 1. Time-course of TEER across passaged human nasal epithelial cellmonolayer during 2 weeks; () mean value of passages 2–4 (n = 9).study (Yoo et al., 2003), transport studies were conductedafter 4–8 days of seeding when the TEER value maintained800–1200 cm 2 .The characteristics of maximum TEER appearing in 2days after seeding was different from that of the rabbittracheal epithelial cell monolayer, primary human alveolarepithelial cells or air-interfaced primary rabbit conjunctivalepithelial culture (Mathias et al., 1995; Elbert et al., 1999;Yang et al., 2000). Relatively high TEER value was alsoreported on the collagen matrix-based human nasal primaryculture using air-liquid interface culture (Agu et al., 2001).High TEER value at the early phase of the culture could bedue to the influence of culture media (DMEM containing 10%FBS), since most of respiratory epithelial cells are culturedin serum-free media, such as PC-1 medium for the primaryculture of rabbit tracheal and conjunctival epithelial cells andDME-F12 for human primary nasal culture (Mathias et al.,1995; Yang et al., 2000; Agu et al., 2001). It could be speculatedthat the serum, an important source of extracellularmatrix (e.g., laminin), promoted cell proliferation, adhesionfactors, and/or anti-trypsin activity (Freshney, 1994).3.2. Morphological studies of passaged human nasalmonolayer cultureIn order to estimate the barrier properties of epithelialcells, morphological studies, bioelectric determination, andnon-electrolyte solute permeability are commonly investigated.In addition to TEER value, SEM, and TEM wereobserved for passage 2 culture on day 5 after seeding. Asshown in Fig. 2(A), tight junctions were clearly observedin TEM (indicated by a white arrowhead). Microvilli andincomplete cilia were also observed at the apical side ofepithelial cells. However, SEM results showed less prominentcilia or denuded ciliated cells of passage 2 in LCCmethod on day 5, which was consistent with the report onrabbit tracheal epithelial cell culture using the same method(Mathias et al., 1995). The epithelial monolayers developed aFig. 2. Morphological appearance of passage 2 human nasal epithelialmonolayer after 5 days of seeding under the transmission and scanning electronmicroscopy. Plate A shows the tight junction (indicated by a white arrow)and the cilia (indicated by a black arrow) (8000× magnification). Plate Bdisplays a cuboidal appearance of passage 2 human nasal epithelial monolayers(600× magnification). The insert shows a SEM of passage 2 humannasal epithelial monolayer exhibiting denuded cilia (4500× magnification).cuboidal shape, as shown in Fig. 2(B), which were observedto be broader and shorter than the “native” pseudo-stratifiedcolumnar epithelial cells. Even though the differentiation ofthe cell monolayers was incomplete, this passaged culturemodel seems to be suitable for drug transport studies sincethe development of the tight junction was completed within2–3 days, which can be advantageous due to reduced experimenttime and cost.3.3. The effect of lipophilicity on the permeability acrossthe human nasal cell monolayerIn preliminary studies, all the model drugs were stable intransport medium and the integrity of the monolayer checkedby the change of TEER was maintained for 60 min of transportstudies (data not shown).

H. Lin et al. / European Journal of Pharmaceutical Sciences 26 (2005) 203–210 207Table 1Apparent 1-octanol/water partition coefficient (log P) and solubility of model drugsModel drug Chemical structure (M.W.) log P Solubility a (g/mL)Albuterol hemisulfate −1.58 ± 0.02 bAlbuterol −0.79 ± 0.03 3222.37 ± 50.23Fexofenadine HCl 0.49 ± 0.01 1518.95 ± 22.10 cDexamethasone 1.92 ± 0.01 84.16 ± 0.78Triamcinolone acetonide 2.40 ± 0.01 19.96 ± 0.02Budesonide 3.21 ± 0.01 15.75 ± 0.37a Solubility was determined in transport medium at room temperature.b Solubility is above 5 mg/mL.c Solubility of fexofenadine HCl was determined in water at room temperature.

208 H. Lin et al. / European Journal of Pharmaceutical Sciences 26 (2005) 203–210In order to investigate the influence of lipophilicity ondrug permeation across the passaged human nasal epithelialcell monolayers, a series of anti-allergic drugs withvarious 1-octanol/H 2 O partition coefficient (log P) wereselected as model drugs. Their log P values and solubilityin transport medium at room temperature are listed inTable 1. The permeation profiles of model drugs are shownin Fig. 3. Interestingly, the P app values of the most hydrophiliccompound studied, albuterol hemisulfate (log P = −1.58;P app 0.20 × 10 −6 cm/s), was close to that of a paracellularmarker mannitol (log P = −3.10; P app , 0.16 × 10 −6 cm/s).Highly lipophilic budesonide showed the P app value of 21.92(±0.78) × 10 −6 cm/s. Within the log P range from −1.58to 3.21, there was a 100-fold difference in the P app . TheP app value significantly increased with the enhancement oflipophilicity, which indicated that log P is the most importantfactor affecting the permeability of nasal transepithelialroute.Various anti-allergic drugs have been usually selected asmodel drugs to study the effect of lipophilicity on the transepithelial(e.g., conjunctival and alveolar) permeability due totheir wide distribution in log P value. However, there has beenno report on the transport of these drugs across the nasalcell monolayers or animal excised tissue, thus, it was notpossible to estimate the intrinsic nasal epithelial cells permeabilityof these drugs in vivo. It was exciting though tofind a good log-linear relationship between the lipophilicity(log P) of anti-allergic drugs and the permeability coefficient(P app ) across the nasal epithelial monolayers (r 2 = 0.92, A–Bdirection; r 2 = 0.94, B–A direction), as shown in Fig. 4 andTable 2. A sigmoid relationship was previously reported inthe transport studies of -blockers across the primary conjunctivaland the alveolar epithelial cell monolayers (Yang etal., 2000; Saha et al., 1994). This sigmoid relationship wasFig. 4. Relationship between P app and log P in different directions acrosspassaged human nasal cell monolayers: (1) albuterol hemisulfate; (2)albuterol; (3) fexofenadine HCl; (4) dexamethasone; (5) triamcinolone acetonide;(6) budesonide; (a) mannitol; (b) melagatran; (c) sumatriptan; (d)propranolol; (e) nicotine; (f) lidocaine; (g) testosterone; (○) human nasalepithelial monolayer, apical to basolateral; () porcine nasal mucosa (Ussingchamber) (Osth et al., 2002; Wadell et al., 2003).also reported in the permeability studies of -blockers acrossthe excised rabbit conjunctiva and cornea (Wang et al., 1991),and in the studies of barbiturates across the excised rat nasalmucosa (Huang et al., 1985).In order to demonstrate the feasibility of the passage culturedhuman nasal cell monolayer model in estimating the“intrinsic” nasal permeability of drugs, the P app of model antiallergicdrugs need to be compared with that across the humannasal mucosa in vivo. However, since there was no report onthe nasal transport of these in human, the P app values of variouscompounds in the literatures across the excised porcinenasal mucosa were compared with the results of this study. Asshown in Fig. 4, a similar log-linear relationship was reportedin transport studies of various compounds across the excisedporcine nasal mucosa (r 2 = 0.81, when lidocaine was deleted)(Osth et al., 2002; Wadell et al., 2003). The P app values of antiallergicdrugs across the passage cultured human nasal cellmonolayer were significantly lower than those various compoundsacross the excised porcine nasal mucosa. For example,the P app values of mannitol across the passage culturedhuman nasal cell monolayer was 0.90 ± 0.30 × 10 −6 cm/sFig. 3. Transport profiles of all the model drugs across the passaged culturedhuman nasal cell monolayers with TEER value higher than 800 cm 2 usingLCC culture method. Each point represents the mean ± S.D.; n ≥ 3 experiments;() albuterol hemisulfate, 500 g/mL; (□) albuterol, 500 g/mL;() fexofenadine HCl, 500 g/mL; () dexamethasone, 50 g/mL; (△) triamcinoloneacetonide, 20 g/mL; (♦) budesonide, 15 g/mL.Table 2Permeability coefficient of model drugs in the direction of apical to basolateroland reverse direction across passaged human nasal epithelial monolayers(mean ± S.D., n >3)Model drugConcentrated(g/mL)P app (A–B)(10 −6 cm/s)P app (B–A)(10 −6 cm/s)Albuterol hemisulfate 500 0.20 ± 0.02 0.23 ± 0.05Albuterol 500 0.78 ± 0.23 0.61 ± 0.04Fexofenadine HCl 500 0.54 ± 0.12 0.60 ± 0.16Dexamethasone 50 4.88 ± 0.30 4.10 ± 0.64Triamcinolone acetonide 20 10.31 ± 0.25 10.90 ± 0.58Budesonide 15 21.92 ± 0.78 19.81 ± 1.39

H. Lin et al. / European Journal of Pharmaceutical Sciences 26 (2005) 203–210 209(Yoo et al., 2003), while that across the excised porcine nasalmucosa was 3.9 ± 2.2 × 10 −6 cm/s (Wadell et al., 2003). Thisdiscrepancy could be due to the species difference (humanversus porcine) and/or to the difference in the tight junction ofpassage cultured monolayer and excised nasal mucosa. TheTEER value of 40–120 cm 2 was reported in the excisednasal mucosa from animals (sheep, rabbit, and cattle) andhuman (Schmidt et al., 1998), which was considerably lowerthan that obtained in this study (>1000 cm 2 ). However, agood log-linear relationship of the both models indicates thatlipophilicity (log P) of the compounds is the most importantfactor that determine the nasal permeability and that the passagecultured human nasal cell monolayer model is useful topredict the nasal permeability of small molecular drugs.3.4. The effect of transport direction on P appThe possibility of the expression of active transporter(s) inthe passaged human nasal cell monolayers and their involvementin the permeability of anti-allergic drugs was investigatedby comparing the P app values of “apical-to-basolateral(A–B)” direction with those of “basolateral-to-apical (B–A)”direction. As shown in Table 2, no significant difference inP app values between two directions was observed in all themodel drugs. The P app value of A-to-B direction was insignificantlyhigher than that of B-to-A direction, probably, due tothe gravity and/or to the sampling condition. Thus, the transportersknown to be expressed in human respiratory epithelium,which include members of the super family of ABCtransporter, MDR1, MRP, and amino transporters (Brechotet al., 1998; Bremer et al., 1992), are absent or incompletelyexpressed in the human nasal epithelial cell monolayers inLCC condition used in this study. However, several transporters,such as MDR1 and MRP1 have been reported tobe expressed in the epithelium and glands of the excisedhuman nasal respiratory mucosa in immunohistochemistrystudy (Wioland et al., 2000; Henriksson et al., 1997). A largevariety of hydrophobic and amphiphilic compounds as wellas organic cations are known to be the substrates for MDR1in superficial respiratory mucosa (Bremer et al., 1992). FexofenadineHCl was reported to be a probe compound for theP-gp transporter in Caco-2 cell study (Perloff et al., 2002), yetthere was no significant difference in the P app values of A–Band B–A direction in this study. Thus, the culture conditionneeds to be further improved to express transporters and tostudy their effect on drug transport in in vitro nasal epithelialcell culture system.3.5. Concentration dependency of the transport offexofenadine HCl and dexamethasoneThe transport studies of different concentration of fexofenadineHCl (500 and 1000 g/mL) and dexamethasone (50and 80 g/mL) were performed over a period of 60 min. Asshown in Table 3, the transport rate of drugs across the monolayerincreased in a concentration-dependent manner withTable 3The effect of drug concentration on transport in the direction of apical tobasolaterol across the passaged human nasal epithelial monolayers (n ≥ 3)Model drugConcentrated(g/mL)P app(×10 −6 cm/s)Transport rate(g/(cm 2 h))Fexofenadine HCl 500 0.54 ± 0.12 0.97 ± 0.221000 0.55 ± 0.14 1.96 ± 0.51Dexamethasone 50 4.88 ± 0.30 0.88 ± 0.0580 4.66 ± 0.46 1.34 ± 0.13constant permeability coefficients. This implies that transcellulartransport of dexamethasone was non-polarized andsuggests that passive diffusion is predominant. Similar resultswere reported in passive diffusion of budesonide transportacross Calu-3 cell line (Borchard et al., 2002).4. ConclusionPassaged human nasal epithelial cell monolayer using theLCC method was established up to passage-4, and its utilityas an in vitro model for evaluating drug permeabilitywas successfully investigated. Each passage culture formeda tight monolayer with high TEER value for drug transportstudies, although the differentiation of cilia was not complete.A good log-linear relationship was observed betweenthe lipophilicity (log P) of a series of anti-allergic drugs andtheir permeability coefficients (P app ). Non-polarized transportacross the human nasal cell monolayers was observedamong all the model drugs, which indicated the absence orincomplete expression of transporter in this culture system.However, this in vitro model seems to be useful since it canoffer constant supply of human nasal epithelial cells for transportand mechanism studies, and also is feasible to predict invivo nasal permeability of small molecular drugs.AcknowledgementThis work was support by the research grant from theMinistry of Health and Welfare in Korea (02-PJ2-PG1-CH12-0002).ReferencesAgu, R.U., Jorissen, M., Willems, T., Augustijns, P., Kinget, R., Verbeke,N., 2001. In-vitro nasal drug delivery studies: comparison of derivatised,fibrillar and polymerised collagen matrix-based human nasalprimary culture systems for nasal drug delivery studies. J. Pharm.Pharmacol. 53, 1447–1456.Borchard, G., Cassara, M.L., Roemele, P.E., Florea, B.I., Junginger, H.E.,2002. 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