European Journal of Pharmaceutical Sciences 26 (2005) 203–210

Transport of anti-allergic drugs across the passage culturedhuman nasal epithelial cell monolayer

Hongxia Lina, Jin-Wook Yooa, Hwan-Jung Rohb, Min-Ki Leeb, Suk-Jae Chungc,Chang-Koo Shimc, Dae-Duk Kimc,∗

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 Korea

Received 22 September 2004; received in revised form 3 May 2005; accepted 2 June 2005Available online 8 August 2005

Abstract

The purpose of this study was to investigate the nasal absorption characteristics of a series of anti-allergic drugs across the human nasale ® isc xhibited theT M andS s, albuterolh mpounds fort in the log( in thea sale oncentration-d rugs and nos l may be au©

K

1

whDmaah

eof

s for

for

eds of

, nasaldels,on ofninghance

0d

pithelial cell monolayer, which was passage cultured by the liquid-covered culture (LCC) method on Transwell. Characterization of thell culture model was achieved by bioelectric measurements and morphological studies. The passages 2–4 of cell monolayers eEER value of 1731± 635� cm2 after 2 days of seeding and maintained high TEER value for 4–6 days. Morphological study by TEEM showed the existence of the tight junctions, and the cuboidal shaped epithelial cells monolayer. A series of anti-allergic drugemisulfate, albuterol, fexofenadine HCl, dexamethasone, triamcinolon acetonide, and budesonide were selected as model co

ransport studies. All the drugs were assayed using reversed-phase HPLC under isocratic conditions. Results indicated that withPapparent 1-octanol/water partition coefficient) range from−1.58 (albuterol) to 3.21 (budesonide), there existed 100-fold differencepparent permeability coefficients (Papp). A log-linear relationship was shown between the drug logP and thePappacross passaged human napithelial monolayers. The amount of fexofenadine HCl and dexamethasone across passaged human nasal cell monolayers was cependent in the direction of apical to basolateral. The direction dependent transport studies were investigated among all these dignificant difference in the two directions was observed. In conclusion, this LCC passaged human nasal epithelial culture modeseful in vitro model for studying the passive transport processes in nasal drug delivery.2005 Elsevier B.V. All rights reserved.

eywords: Passaged human nasal epithelial cell culture; Drug transport; Permeability; Anti-allergic drugs

. Introduction

The drug delivery via the nasal route has recently receivedidespread attention due to several advantages includingigh systemic bioavailability and rapid onset of action.rug candidates ranging from small metal ions to largeacromolecular proteins have been investigated in variousnimal models by nasal drug delivery (Krishnamoorthynd Mitra, 1998). Nearly, complete absorption of certainormones 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 thpotential value of the nasal route for administrationsystemic medications as well as for local effects, such anasal allergy, nasal congestion, and nasal infections.

Although in vivo animal models have been widely usedthe investigation of nasal drug transport studies (Morimotoet al., 1991), in vitro nasal models are recently employin the studies of the transport and metabolic propertiethe nasal mucosa using the excised nasal tissue modelhomogenates, and cell culture model. Among these mothe nasal cell culture models have attracted the attentipharmaceutical researchers as promising tools for defitransport mechanisms and testing novel strategies to en

928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.ejps.2005.06.003

204 H. Lin et al. / European Journal of Pharmaceutical Sciences 26 (2005) 203–210

drug transport and absorption. Furthermore, primary cultureof nasal epithelial cells from a variety of species includ-ing human, 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 fibro-sis, 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 path-ways and 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 useful-ness of in vitro cell culture model especially if human tissueis preferred. In order to overcome the shortage of humannasal tissue, researchers have shifted from primary cultureso for-m irus( e int than2 ures( ab-l od fortm werei Thec n oft edu tilityo ithe-l ctedf drugl ers.T rosst rcinen ibilityo

2

2

hem-i fate,a SS),

h-EGF, and d-(+)-glucose were purchased from SigmaChemical Co. (St. Louis, MO). Triamcinolone acetonide andfexofenadine HCl were gifts from Handok-Aventis Pharma-ceutical Co. (Seoul, Korea). Other cell culture reagents andsupplies were obtained from GIBCO Invitrogen Co. (GrandIsland, NY). BEGM Bulletkit was obtained from Cam-brex Bio 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 cellmethod

The 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 sinusi-tis, using a protocol approved by the Institutional ReviewBoard (IRB) at Pusan National University Hospital, SouthKorea. Briefly, the nasal specimens were dissociated enzy-matically using 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/mLs disha stsb ithe-l forf nd cul-t %C gede

nflu-e andwT era mLp n-e alc inb main-t ays.R lturedu y was5 ay.

2m

ea-s Mv d bys sertsa

f epithelial cells to passage cultured cell lines. Transation of human nasal polyp cells using a chimaeric v

Ad5/SV40 1613 ori-) is one of the examples of the changrends. The extended lifespans ranged from 20 to more50 population doublings beyond that of the primary cultBuchanan et al., 1990). In a previous report, we have estished the passaged human nasal epithelial culture methhe drug transport studies (Yoo et al., 2003), where the LCCethod was utilized and properties of nasal epithelium

nvestigated by bioelectric and morphological studies.haracteristic high TEER value, which was an indicatiohe formation of a tight junction formation, was maintainp to the monolayers of passage 4. In order to verify the uf this passaged culture model for actual nasal transep

ial drug delivery, a series of anti-allergic drugs were seleor transport studies and evaluated for the influence ofipophilicity on solute permeability across the monolayhe permeability characteristics of the model drugs ac

he monolayers was compared with those of excised poasal mucosa in the literatures to demonstrate the feasf the in vitro model for nasal drug transport studies.

. Materials and methods

.1. Materials

Dexamethasone was purchased from Wako Pure Ccal Industries Ltd. (Osaka, Japan). Albuterol hemisullbuterol, budesonide, Hank’s balanced salt solution (HB

treptomycin. Cell suspension was pre-plated on a plastict 37◦C for 1 h in order to eliminate fibroblasts and myoblay differential attachment to plastic dish. Suspended ep

ial cells were frozen and stored in liquid nitrogen tankuture usages. Frozen passage-1 stocks were thawed aured in T-flask using BEGM at 37◦C in an atmosphere of 5O2 and 95% relative humidity. The medium was chanvery 2 days.

When cultures reached approximately 70–80% concy, the cells were detached with 0.1% trypsin–EDTA,ere seeded at densities of 2× 105 to 3× 105 cells/cm2 onranswell® insert with 0.5 mL BEGM in the apical chambnd 1.5 mL DMEM (supplemented with 10% FBS, 100 U/enicillin, 100�g/mL streptomycin, 1 ng/mL EGF, 1% nossential amino acid, and 1%l-glutamine) in the basolaterhamber. After 24 h, media were changed with DMEMoth the apical and basolateral sides and the cells were

ained in the medium by changing the medium every 2 demaining cells after seeding were subsequently subcusing BEGM for the next passage. The subculture densit00 cells/cm2 and the media were changed every other d

.3. Measurement of bioelectric parameters andorphological studies

The transepithelial electrical resistance (TEER) was mured daily by directly reading the values of an EVOoltohmmeter device (WPI, Sarasota, FL), and correcteubtracting the background due to the blank Transwell innd medium.

H. Lin et al. / European Journal of Pharmaceutical Sciences 26 (2005) 203–210 205

Scanning (SEM) and transmission (TEM) electronmicroscopy were processed for morphological studies on day5. For SEM, each monolayer was fixed in 2.5% glutaralde-hyde in 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 alco-hol series 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 micro-scope (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-allergicdrugs

The solubility of model drugs in transport medium wasmeasured at room temperature. Excess amount of each com-pound was added to 2 mL of transport medium, and themixtures were stirred for 24 h. After filtering through Minis-art RC 4 filter (0.45�m, Satorius, Germany), solutions werea nol.

o , asr1 withg d. Th1 L)w , andt vial.A eres con-c ed byH

2

eed-i portet ith ap mMd thei

ans-p ousc l sidea aso-l als,a qualv f the

basolateral 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 vol-ume of fresh transport medium to maintain the sink condition.To monitor integrity of the cultured epithelial cell monolay-ers, the TEER value was measured at the beginning and endof each transport experiment.

2.6. HPLC assay

The 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 analysis

c

P

w es-s werem n.As wase

3

3

L reachc helialr fT 4 cul-to afters highT ani mptf bea asalt ious

nalyzed by HPLC after appropriate dilution with methaApparent 1-octanol/water partition coefficient (logP)

f model drugs was determined at room temperatureeported in the literature (Dearden and Bresnen, 1988). An-octanol/water mutual saturation was prepared for 24 hentle mechanical stirring, and each phase was separate00�L methanolic solution of each model drug (1 mg/mas placed in a glass vial and completely evaporated

hen 1.0 mL of each saturated solvent was added to thefter shaking the vial for 24 h at 150 rpm, the phases weparated by centrifugation at 4000 rpm for 20 min. Theentration of the compound in each phase was determinPLC after appropriate dilution with methanol.

.5. Transport studies

Nasal epithelial cell monolayers after 4–8 days of sng were used for drug transport studies. All the transxperiments were performed in an incubator at 37◦C. Prioro each experiment, the monolayers were washed wre-equilibrated transport medium (10 mM HEPES, 10-(+)-glucose), and allowed to equilibrate for 20 min in

ncubator.For measurement of the apical to basolateral (A–B) tr

ort, 0.4 mL of the transport medium containing varioncentrations of model drugs was added on the apicand 1.0 mL of the transport medium was added on the b

ateral side of the insert. At pre-determined time intervll the receiver fluid was immediately replaced with an eolume of fresh transport medium. For measurement o

e

The apparent permeability coefficients,Papp (cm/s) werealculated using the following equation:

app = dQ

dt

1

AC0(1)

here dQ/dt is the solute flux obtained from linear regrion,A the surface area across which transport studieseasured (1.0 cm2), andC0 is the initial drug concentratioll the data were expressed as the mean± S.D. (n > 3). Thetatistical significance of differences between treatmentsvaluated using the unpaired Student’st-test.

. Results and discussion

.1. Measurement of bioelectric parameters

When seeded at a density of 2∼ 3× 105 cells/cm2, theCC cell monolayers of passages 2–4 appeared toonfluence and began to exhibit a measurable transepitesistance from day 2. As shown inFig. 1, a similar pattern oEER time-course profile was observed in passages 2–

ures. The mean maximum TEER value (1731± 635� cm2)f all the three passaged cultures appeared on 2 dayseeding, and then rapidly decreased thereafter. TheEER value implies the formation of tight junction that is

mpermeable junction located at the apical side. The proormation of tight monolayers in LCC condition candvantageous for the short culture duration for in vitro n

ransport studies. Based on our results from the prev

206 H. Lin et al. / European Journal of Pharmaceutical Sciences 26 (2005) 203–210

Fig. 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� cm2.

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 spec-ulated that 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 culture

elialc andn sti-g ereo g. Ass edi ndi e ofe mi-n CCm t onr thod( d a

Fig. 2. Morphological appearance of passage 2 human nasal epithelialmonolayer after 5 days of seeding under the transmission and scanning elec-tron microscopy. 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 mono-layers (600× magnification). The insert shows a SEM of passage 2 humannasal epithelial monolayer exhibiting denuded cilia (4500× magnification).

cuboidal shape, as shown inFig. 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 exper-iment time and cost.

3.3. The effect of lipophilicity on the permeability acrossthe human nasal cell monolayer

In 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 trans-port studies (data not shown).

In order to estimate the barrier properties of epithells, morphological studies, bioelectric determination,on-electrolyte solute permeability are commonly inveated. In addition to TEER value, SEM, and TEM wbserved for passage 2 culture on day 5 after seedinhown inFig. 2(A), tight junctions were clearly observ

n TEM (indicated by a white arrowhead). Microvilli ancomplete cilia were also observed at the apical sidpithelial cells. However, SEM results showed less proent cilia or denuded ciliated cells of passage 2 in Lethod on day 5, which was consistent with the repor

abbit tracheal epithelial cell culture using the same meMathias et al., 1995). The epithelial monolayers develope

H. Lin et al. / European Journal of Pharmaceutical Sciences 26 (2005) 203–210 207

Table 1Apparent 1-octanol/water partition coefficient (logP) and solubility of model drugs

Model drug Chemical structure (M.W.) logP Solubilitya (�g/mL)

Albuterol hemisulfate −1.58± 0.02 b

Albuterol −0.79± 0.03 3222.37± 50.23

Fexofenadine HCl 0.49± 0.01 1518.95± 22.10c

Dexamethasone 1.92± 0.01 84.16± 0.78

Triamcinolone acetonide 2.40± 0.01 19.96± 0.02

Budesonide 3.21± 0.01 15.75± 0.37

a 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) 203–210

In order to investigate the influence of lipophilicity ondrug permeation across the passaged human nasal epithe-lial cell monolayers, a series of anti-allergic drugs withvarious 1-octanol/H2O partition coefficient (logP) wereselected as model drugs. Their logP values and solubil-ity in transport medium at room temperature are listed inTable 1. The permeation profiles of model drugs are shownin Fig. 3. Interestingly, thePappvalues of the most hydrophiliccompound studied, albuterol hemisulfate (logP =−1.58;Papp 0.20× 10−6 cm/s), was close to that of a paracellu-lar marker mannitol (logP =−3.10;Papp, 0.16× 10−6 cm/s).Highly lipophilic budesonide showed thePappvalue of 21.92(±0.78)× 10−6 cm/s. Within the logP range from−1.58to 3.21, there was a 100-fold difference in thePapp. ThePapp value significantly increased with the enhancement oflipophilicity, which indicated that logP is the most impor-tant factor affecting the permeability of nasal transepithelialroute.

Various anti-allergic drugs have been usually selected asmodel drugs to study the effect of lipophilicity on the transep-ithelial (e.g., conjunctival and alveolar) permeability due totheir wide distribution in logP 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 per-m tofi city( ent(dT int n-ja s

F ulturedhLi(a

Fig. 4. Relationship betweenPapp and logP in different directions acrosspassaged human nasal cell monolayers: (1) albuterol hemisulfate; (2)albuterol; (3) fexofenadine HCl; (4) dexamethasone; (5) triamcinolone ace-tonide; (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 (Uss-ing chamber) (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 cul-tured human nasal cell monolayer model in estimating the“intrinsic” nasal permeability of drugs, thePappof model anti-allergic drugs 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, thePappvalues of var-ious compounds in the literatures across the excised porcinenasal mucosa were compared with the results of this study. Asshown inFig. 4, a similar log-linear relationship was reportedin transport studies of various compounds across the excisedporcine nasal mucosa (r2 = 0.81, when lidocaine was deleted)(Osth et al., 2002; Wadell et al., 2003). ThePappvalues of anti-allergic drugs across the passage cultured human nasal cellmonolayer were significantly lower than those various com-pounds across the excised porcine nasal mucosa. For exam-ple, thePapp values of mannitol across the passage culturedhuman nasal cell monolayer was 0.90± 0.30× 10−6 cm/s

Table 2Permeability coefficient of model drugs in the direction of apical to baso-laterol and reverse direction across passaged human nasal epithelial mono-l

M

AAFDTB

eability of these drugs in vivo. It was exciting thoughnd a good log-linear relationship between the lipophililogP) of anti-allergic drugs and the permeability coefficiPapp) across the nasal epithelial monolayers (r2 = 0.92, A–Birection;r2 = 0.94, B–A direction), as shown inFig. 4 andable 2. A sigmoid relationship was previously reportedhe transport studies of�-blockers across the primary counctival and the alveolar epithelial cell monolayers (Yang etl., 2000; Saha et al., 1994). This sigmoid relationship wa

ig. 3. Transport profiles of all the model drugs across the passaged cuman nasal cell monolayers with TEER value higher than 800� cm2 usingCC culture method. Each point represents the mean± S.D.; n ≥ 3 exper-

ments; (�) albuterol hemisulfate, 500�g/mL; (�) albuterol, 500�g/mL;�) fexofenadine HCl, 500�g/mL; (�) dexamethasone, 50�g/mL; (�) tri-mcinolone acetonide, 20�g/mL; (♦) budesonide, 15�g/mL.

ayers (mean± S.D.,n > 3)

odel drug Concentrated(�g/mL)

Papp(A–B)(10−6 cm/s)

Papp (B–A)(10−6 cm/s)

lbuterol hemisulfate 500 0.20± 0.02 0.23± 0.05lbuterol 500 0.78± 0.23 0.61± 0.04exofenadine HCl 500 0.54± 0.12 0.60± 0.16examethasone 50 4.88± 0.30 4.10± 0.64riamcinolone acetonide 20 10.31± 0.25 10.90± 0.58udesonide 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� cm2 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� cm2). 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 pas-sage cultured human nasal cell monolayer model is useful topredict the nasal permeability of small molecular drugs.

3.4. The effect of transport direction on Papp

The possibility of the expression of active transporter(s) inthe passaged human nasal cell monolayers and their involve-ment in the permeability of anti-allergic drugs was investi-gated by comparing thePappvalues of “apical-to-basolateral(A–B)” direction with those of “basolateral-to-apical (B–A)”direction. As shown inTable 2, no significant difference inPapp values between two directions was observed in all themodel drugs. ThePappvalue of A-to-B direction was insignif-icantly higher than that of B-to-A direction, probably, due tot ans-p ithe-l Cte lye rs inL ns-p ed tob cisedh istrysv wella DR1io r thePta tionn nd tos lialc

3f

ofe-n 50a Ass no-l with

Table 3The effect of drug concentration on transport in the direction of apical tobasolaterol across the passaged human nasal epithelial monolayers (n ≥ 3)

Model drug Concentrated(�g/mL)

Papp

(×10−6 cm/s)Transport rate(�g/(cm2 h))

Fexofenadine HCl 500 0.54± 0.12 0.97± 0.221000 0.55± 0.14 1.96± 0.51

Dexamethasone 50 4.88± 0.30 0.88± 0.0580 4.66± 0.46 1.34± 0.13

constant permeability coefficients. This implies that tran-scellular transport 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. Conclusion

Passaged human nasal epithelial cell monolayer using theLCC method was established up to passage-4, and its util-ity as 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 com-plete. A good log-linear relationship was observed betweenthe lipophilicity (logP) of a series of anti-allergic drugs andtheir permeability coefficients (Papp). Non-polarized trans-port across 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 trans-port and mechanism studies, and also is feasible to predict invivo nasal permeability of small molecular drugs.

A

theM 12-0

R

A eke,riva-nasalarm.

B H.E.,sone

arm.

B 998.rom517.

he gravity and/or to the sampling condition. Thus, the trorters known to be expressed in human respiratory ep

ium, which include members of the super family of ABransporter, MDR1, MRP, and amino transporters (Brechott al., 1998; Bremer et al., 1992), are absent or incompletexpressed in the human nasal epithelial cell monolayeCC condition used in this study. However, several traorters, such as MDR1 and MRP1 have been reporte expressed in the epithelium and glands of the exuman nasal respiratory mucosa in immunohistochemtudy (Wioland et al., 2000; Henriksson et al., 1997). A largeariety of hydrophobic and amphiphilic compounds ass organic cations are known to be the substrates for M

n superficial respiratory mucosa (Bremer et al., 1992). Fex-fenadine HCl was reported to be a probe compound fo-gp transporter in Caco-2 cell study (Perloff et al., 2002), yet

here was no significant difference in thePappvalues of A–Bnd B–A direction in this study. Thus, the culture condieeds to be further improved to express transporters atudy their effect on drug transport in in vitro nasal epitheell culture system.

.5. Concentration dependency of the transport ofexofenadine HCl and dexamethasone

The transport studies of different concentration of fexadine HCl (500 and 1000�g/mL) and dexamethasone (nd 80�g/mL) were performed over a period of 60 min.hown inTable 3, the transport rate of drugs across the moayer increased in a concentration-dependent manner

cknowledgement

This work was support by the research grant froministry of Health and Welfare in Korea (02-PJ2-PG1-CH002).

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