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

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

European Journal of Pharmaceutical Sciences 26 (2005) 203–210 Transport of anti-allergic drugs across the passage cultured human nasal epithelial cel...

379KB Sizes 0 Downloads 86 Views

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

Transport of anti-allergic drugs across the passage cultured human nasal epithelial cell monolayer Hongxia 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 Korea College of Medicine, Pusan National University, Pusan 602-739, South Korea c College of Pharmacy, Seoul National University, Seoul 151-742, South Korea

b

Received 22 September 2004; received in revised form 3 May 2005; accepted 2 June 2005 Available 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 nasal epithelial cell monolayer, which was passage cultured by the liquid-covered culture (LCC) method on Transwell® . Characterization of this cell culture model was achieved by bioelectric measurements and morphological studies. The passages 2–4 of cell monolayers exhibited the TEER value of 1731 ± 635  cm2 after 2 days of seeding and maintained high TEER value for 4–6 days. Morphological study by TEM and SEM showed the existence of the tight junctions, and the cuboidal shaped epithelial cells monolayer. A series of anti-allergic drugs, albuterol hemisulfate, albuterol, fexofenadine HCl, dexamethasone, triamcinolon acetonide, and budesonide were selected as model compounds for transport 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 the apparent permeability coefficients (Papp ). A log-linear relationship was shown between the drug log P and the Papp across passaged human nasal epithelial monolayers. The amount of fexofenadine HCl and dexamethasone across passaged human nasal cell monolayers was concentrationdependent in the direction of apical to basolateral. The direction dependent transport studies were investigated among all these drugs and no significant difference in the two directions was observed. In conclusion, this LCC passaged human nasal epithelial culture model may be a useful 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 drugs

1. Introduction The drug delivery via the nasal route has recently received widespread attention due to several advantages including high systemic bioavailability and rapid onset of action. Drug candidates ranging from small metal ions to large macromolecular proteins have been investigated in various animal models by nasal drug delivery (Krishnamoorthy and Mitra, 1998). Nearly, complete absorption of certain hormones and steroids by nasal administration (Hussain ∗

Corresponding author. Tel.: +82 2 880 7870; fax: +82 2 888 5969. E-mail address: [email protected] (D.-D. Kim).

0928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2005.06.003

et al., 1981; Lipworth and Jackson, 2000) revealed the potential value of the nasal route for administration of systemic medications as well as for local effects, such as for nasal allergy, nasal congestion, and nasal infections. Although in vivo animal models have been widely used for the investigation of nasal drug transport studies (Morimoto et al., 1991), in vitro nasal models are recently employed in the studies of the transport and metabolic properties of the nasal mucosa using the excised nasal tissue model, nasal homogenates, and cell culture model. Among these models, the nasal cell culture models have attracted the attention of pharmaceutical researchers as promising tools for defining transport mechanisms and testing novel strategies to enhance

204

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

drug transport and absorption. Furthermore, primary culture of nasal epithelial cells from a variety of species including human, bovine, rat, and rabbit can provide valuable in vitro models in the study of nasal physiology. The culture of cells and tissue derived from differentiated human nasal epithelium is an established tool for the studies of fibrosis, electrolyte transport, ciliogenesis, and ciliary movement (Ruckes et al., 1997; Jorissen and Bessems, 1995). In vitro cell culture models offer many advantages, including: (a) a controlled environment for the study of epithelial cell growth and differentiation; (b) the elucidation of drug transport pathways and mechanisms; (c) rapid and convenient means of evaluating drug permeability; (d) opportunity to minimize the expensive and limited controversial use of animals (Schmidt et al., 1998). It has been reported that human nasal primary cultures as in vitro models show the potentials for the study of nasal drug absorption, especially for the peptide transport studies (Werner and Kissel, 1995; Kissel and Werner, 1998; Agu et al., 2001). However, efforts to develop and characterize nasal cell culture systems for drug metabolism and permeation are still in their infancy. The main limitation is the difficulty in obtaining a reliable tissue source, which hinders the usefulness of in vitro cell culture model especially if human tissue is preferred. In order to overcome the shortage of human nasal tissue, researchers have shifted from primary cultures of epithelial cells to passage cultured cell lines. Transformation of human nasal polyp cells using a chimaeric virus (Ad5/SV40 1613 ori-) is one of the examples of the change in trends. The extended lifespans ranged from 20 to more than 250 population doublings beyond that of the primary cultures (Buchanan et al., 1990). In a previous report, we have established the passaged human nasal epithelial culture method for the drug transport studies (Yoo et al., 2003), where the LCC method was utilized and properties of nasal epithelium were investigated by bioelectric and morphological studies. The characteristic high TEER value, which was an indication of the formation of a tight junction formation, was maintained up to the monolayers of passage 4. In order to verify the utility of this passaged culture model for actual nasal transepithelial drug delivery, a series of anti-allergic drugs were selected for transport studies and evaluated for the influence of drug lipophilicity on solute permeability across the monolayers. The permeability characteristics of the model drugs across the monolayers was compared with those of excised porcine nasal mucosa in the literatures to demonstrate the feasibility of the in vitro model for nasal drug transport studies.

2. Materials and methods 2.1. Materials Dexamethasone was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Albuterol hemisulfate, albuterol, budesonide, Hank’s balanced salt solution (HBSS),

h-EGF, and d-(+)-glucose were purchased from Sigma Chemical Co. (St. Louis, MO). Triamcinolone acetonide and fexofenadine HCl were gifts from Handok-Aventis Pharmaceutical Co. (Seoul, Korea). Other cell culture reagents and supplies were obtained from GIBCO Invitrogen Co. (Grand Island, NY). BEGM Bulletkit was obtained from Cambrex Bio Science Inc. (Walkersville, MD), and Transwells® (0.4 ␮m, 12 mm diameter, polyester) were obtained from Costar Co. (Cambridge, MA). All other materials were of analytical grade or better. 2.2. Passage cultured human nasal epithelial cell method The primary human nasal cell culture method used in this study has been described in detail previously (Roh et al., 1999). Human nasal specimens were obtained from patients undergoing surgery due to septal deviation or chronic sinusitis, using a protocol approved by the Institutional Review Board (IRB) at Pusan National University Hospital, South Korea. Briefly, the nasal specimens were dissociated enzymatically using 1.0% protease XIV (Sigma, St. Louis, MO) overnight at 4 ◦ C. Dissociated epithelial cells were washed three times in DMEM containing 10% fetal bovine serum supplemented with 100 U/mL penicillin and 100 ␮g/mL streptomycin. Cell suspension was pre-plated on a plastic dish at 37 ◦ C for 1 h in order to eliminate fibroblasts and myoblasts by differential attachment to plastic dish. Suspended epithelial cells were frozen and stored in liquid nitrogen tank for future usages. Frozen passage-1 stocks were thawed and cultured in T-flask using BEGM at 37 ◦ C in an atmosphere of 5% CO2 and 95% relative humidity. The medium was changed every 2 days. When cultures reached approximately 70–80% confluency, the cells were detached with 0.1% trypsin–EDTA, and were seeded at densities of 2 × 105 to 3 × 105 cells/cm2 on Transwell® insert with 0.5 mL BEGM in the apical chamber and 1.5 mL DMEM (supplemented with 10% FBS, 100 U/mL penicillin, 100 ␮g/mL streptomycin, 1 ng/mL EGF, 1% nonessential amino acid, and 1% l-glutamine) in the basolateral chamber. After 24 h, media were changed with DMEM in both the apical and basolateral sides and the cells were maintained in the medium by changing the medium every 2 days. Remaining cells after seeding were subsequently subcultured using BEGM for the next passage. The subculture density was 500 cells/cm2 and the media were changed every other day. 2.3. Measurement of bioelectric parameters and morphological studies The transepithelial electrical resistance (TEER) was measured daily by directly reading the values of an EVOM voltohmmeter device (WPI, Sarasota, FL), and corrected by subtracting the background due to the blank Transwell inserts and medium.

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

Scanning (SEM) and transmission (TEM) electron microscopy were processed for morphological studies on day 5. For SEM, each monolayer was fixed in 2.5% glutaraldehyde in PBS at 4 ◦ C for 1 h, rinsed in ice-cold PBS, and then fixed in 1.0% osmium tetroxide in PBS at room temperature for 1 h. The specimens were dehydrated through an alcohol series and allowed to air-dry overnight. The specimens were 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 fixed as SEM. After dehydration, the specimens were embedded in Epon 812 semi-thin sections (80 nm), stained with toluidin blue and observed by light microscopy. Appropriate areas were selected for ultra-thin sections, which were treated with uranyl acetate for 6 min and treated with lead citrate for 3 min. These sections were viewed under a JEM 1200 EXII electron microscope (Jeol, Japan). 2.4. Physicochemical characteristics of anti-allergic drugs The solubility of model drugs in transport medium was measured at room temperature. Excess amount of each compound was added to 2 mL of transport medium, and the mixtures were stirred for 24 h. After filtering through Minisart RC 4 filter (0.45 ␮m, Satorius, Germany), solutions were analyzed by HPLC after appropriate dilution with methanol. Apparent 1-octanol/water partition coefficient (log P) of model drugs was determined at room temperature, as reported in the literature (Dearden and Bresnen, 1988). An 1-octanol/water mutual saturation was prepared for 24 h with gentle mechanical stirring, and each phase was separated. The 100 ␮L methanolic solution of each model drug (1 mg/mL) was placed in a glass vial and completely evaporated, and then 1.0 mL of each saturated solvent was added to the vial. After shaking the vial for 24 h at 150 rpm, the phases were separated by centrifugation at 4000 rpm for 20 min. The concentration of the compound in each phase was determined by HPLC after appropriate dilution with methanol.

205

basolateral to apical (B–A) transport, 1.0 mL of the transport medium containing various concentrations of model drugs was added on the basolateral side and 0.4 mL of the transport medium was added on the apical side of the insert. In order to avoid damaging the monolayers, an aliquot of 0.3 mL was sampled from the apical side and replaced with the same volume of 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 end of each transport experiment. 2.6. HPLC assay The samples collected from transport studies were directly determined by HPLC. A reversed-phase C-18 column (Lichrospher® 100, RP −18, 125 mm × 4 mm, 5 ␮m, Merck Darmstadt, Germany) was used, except for albuterol, which was determined on a C-8 column (Lichrospher® 100, RP-8, 25 mm × 4 mm, 5 ␮m, Merck Darmstadt, Germany). Gilson HPLC system equipped with a pump (Gilson Model 306, Gilson Inc., France), an automatic injector (Gilson Model 234, Gilson Inc., France) and UV–vis detector (Gilson Model 118, Gilson Inc., France) was used. 2.7. Data analysis The apparent permeability coefficients, Papp (cm/s) were calculated using the following equation: Papp =

dQ 1 dt AC0

(1)

where dQ/dt is the solute flux obtained from linear regression, A the surface area across which transport studies were measured (1.0 cm2 ), and C0 is the initial drug concentration. All the data were expressed as the mean ± S.D. (n > 3). The statistical significance of differences between treatments was evaluated using the unpaired Student’s t-test.

3. Results and discussion 2.5. Transport studies 3.1. Measurement of bioelectric parameters Nasal epithelial cell monolayers after 4–8 days of seeding were used for drug transport studies. All the transport experiments were performed in an incubator at 37 ◦ C. Prior to each experiment, the monolayers were washed with a pre-equilibrated transport medium (10 mM HEPES, 10 mM d-(+)-glucose), and allowed to equilibrate for 20 min in the incubator. For measurement of the apical to basolateral (A–B) transport, 0.4 mL of the transport medium containing various concentrations of model drugs was added on the apical side and 1.0 mL of the transport medium was added on the basolateral side of the insert. At pre-determined time intervals, all the receiver fluid was immediately replaced with an equal volume of fresh transport medium. For measurement of the

When seeded at a density of 2 ∼ 3 × 105 cells/cm2 , the LCC cell monolayers of passages 2–4 appeared to reach confluence and began to exhibit a measurable transepithelial resistance from day 2. As shown in Fig. 1, a similar pattern of TEER time-course profile was observed in passages 2–4 cultures. The mean maximum TEER value (1731 ± 635  cm2 ) of all the three passaged cultures appeared on 2 days after seeding, and then rapidly decreased thereafter. The high TEER value implies the formation of tight junction that is an impermeable junction located at the apical side. The prompt formation of tight monolayers in LCC condition can be advantageous for the short culture duration for in vitro nasal transport studies. Based on our results from the previous

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 cell monolayer during 2 weeks; () mean value of passages 2–4 (n = 9).

study (Yoo et al., 2003), transport studies were conducted after 4–8 days of seeding when the TEER value maintained 800–1200  cm2 . The characteristics of maximum TEER appearing in 2 days after seeding was different from that of the rabbit tracheal epithelial cell monolayer, primary human alveolar epithelial cells or air-interfaced primary rabbit conjunctival epithelial culture (Mathias et al., 1995; Elbert et al., 1999; Yang et al., 2000). Relatively high TEER value was also reported on the collagen matrix-based human nasal primary culture using air-liquid interface culture (Agu et al., 2001). High TEER value at the early phase of the culture could be due to the influence of culture media (DMEM containing 10% FBS), since most of respiratory epithelial cells are cultured in serum-free media, such as PC-1 medium for the primary culture of rabbit tracheal and conjunctival epithelial cells and DME-F12 for human primary nasal culture (Mathias et al., 1995; Yang et al., 2000; Agu et al., 2001). It could be speculated that the serum, an important source of extracellular matrix (e.g., laminin), promoted cell proliferation, adhesion factors, and/or anti-trypsin activity (Freshney, 1994). 3.2. Morphological studies of passaged human nasal monolayer culture In order to estimate the barrier properties of epithelial cells, morphological studies, bioelectric determination, and non-electrolyte solute permeability are commonly investigated. In addition to TEER value, SEM, and TEM were observed for passage 2 culture on day 5 after seeding. As shown in Fig. 2(A), tight junctions were clearly observed in TEM (indicated by a white arrowhead). Microvilli and incomplete cilia were also observed at the apical side of epithelial cells. However, SEM results showed less prominent cilia or denuded ciliated cells of passage 2 in LCC method on day 5, which was consistent with the report on rabbit tracheal epithelial cell culture using the same method (Mathias et al., 1995). The epithelial monolayers developed a

Fig. 2. Morphological appearance of passage 2 human nasal epithelial monolayer after 5 days of seeding under the transmission and scanning electron microscopy. Plate A shows the tight junction (indicated by a white arrow) and the cilia (indicated by a black arrow) (8000× magnification). Plate B displays a cuboidal appearance of passage 2 human nasal epithelial monolayers (600× magnification). The insert shows a SEM of passage 2 human nasal epithelial monolayer exhibiting denuded cilia (4500× magnification).

cuboidal shape, as shown in Fig. 2(B), which were observed to be broader and shorter than the “native” pseudo-stratified columnar epithelial cells. Even though the differentiation of the cell monolayers was incomplete, this passaged culture model seems to be suitable for drug transport studies since the development of the tight junction was completed within 2–3 days, which can be advantageous due to reduced experiment time and cost. 3.3. The effect of lipophilicity on the permeability across the human nasal cell monolayer In preliminary studies, all the model drugs were stable in transport medium and the integrity of the monolayer checked by the change of TEER was maintained for 60 min of transport studies (data not shown).

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

207

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

Solubilitya (␮g/mL)

Albuterol hemisulfate

−1.58 ± 0.02

b

Albuterol

−0.79 ± 0.03

3222.37 ± 50.23

Model drug

Chemical structure (M.W.)

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 b c

Solubility was determined in transport medium at room temperature. Solubility is above 5 mg/mL. 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 on drug permeation across the passaged human nasal epithelial cell monolayers, a series of anti-allergic drugs with various 1-octanol/H2 O partition coefficient (log P) were selected as model drugs. Their log P values and solubility in transport medium at room temperature are listed in Table 1. The permeation profiles of model drugs are shown in Fig. 3. Interestingly, the Papp values of the most hydrophilic compound studied, albuterol hemisulfate (log P = −1.58; Papp 0.20 × 10−6 cm/s), was close to that of a paracellular marker mannitol (log P = −3.10; Papp , 0.16 × 10−6 cm/s). Highly lipophilic budesonide showed the Papp value of 21.92 (±0.78) × 10−6 cm/s. Within the log P range from −1.58 to 3.21, there was a 100-fold difference in the Papp . The Papp value significantly increased with the enhancement of lipophilicity, which indicated that log P is the most important factor affecting the permeability of nasal transepithelial route. Various anti-allergic drugs have been usually selected as model drugs to study the effect of lipophilicity on the transepithelial (e.g., conjunctival and alveolar) permeability due to their wide distribution in log P value. However, there has been no report on the transport of these drugs across the nasal cell monolayers or animal excised tissue, thus, it was not possible to estimate the intrinsic nasal epithelial cells permeability of these drugs in vivo. It was exciting though to find a good log-linear relationship between the lipophilicity (log P) of anti-allergic drugs and the permeability coefficient (Papp ) across the nasal epithelial monolayers (r2 = 0.92, A–B direction; r2 = 0.94, B–A direction), as shown in Fig. 4 and Table 2. A sigmoid relationship was previously reported in the transport studies of ␤-blockers across the primary conjunctival and the alveolar epithelial cell monolayers (Yang et al., 2000; Saha et al., 1994). This sigmoid relationship was

Fig. 4. Relationship between Papp and log P in different directions across passaged 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 nasal epithelial monolayer, apical to basolateral; (䊉) porcine nasal mucosa (Ussing chamber) (Osth et al., 2002; Wadell et al., 2003).

also reported in the permeability studies of ␤-blockers across the excised rabbit conjunctiva and cornea (Wang et al., 1991), and in the studies of barbiturates across the excised rat nasal mucosa (Huang et al., 1985). In order to demonstrate the feasibility of the passage cultured human nasal cell monolayer model in estimating the “intrinsic” nasal permeability of drugs, the Papp of model antiallergic drugs need to be compared with that across the human nasal mucosa in vivo. However, since there was no report on the nasal transport of these in human, the Papp values of various compounds in the literatures across the excised porcine nasal mucosa were compared with the results of this study. As shown in Fig. 4, a similar log-linear relationship was reported in transport studies of various compounds across the excised porcine nasal mucosa (r2 = 0.81, when lidocaine was deleted) (Osth et al., 2002; Wadell et al., 2003). The Papp values of antiallergic drugs across the passage cultured human nasal cell monolayer were significantly lower than those various compounds across the excised porcine nasal mucosa. For example, the Papp values of mannitol across the passage cultured human nasal cell monolayer was 0.90 ± 0.30 × 10−6 cm/s Table 2 Permeability coefficient of model drugs in the direction of apical to basolaterol and reverse direction across passaged human nasal epithelial monolayers (mean ± S.D., n > 3) Model drug

Fig. 3. Transport profiles of all the model drugs across the passaged cultured human nasal cell monolayers with TEER value higher than 800  cm2 using LCC 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; () triamcinolone acetonide, 20 ␮g/mL; (♦) budesonide, 15 ␮g/mL.

Concentrated (␮g/mL)

Albuterol hemisulfate 500 Albuterol 500 Fexofenadine HCl 500 Dexamethasone 50 Triamcinolone acetonide 20 Budesonide 15

Papp (A–B) (10−6 cm/s) 0.20 0.78 0.54 4.88 10.31 21.92

± ± ± ± ± ±

0.02 0.23 0.12 0.30 0.25 0.78

Papp (B–A) (10−6 cm/s) 0.23 0.61 0.60 4.10 10.90 19.81

± ± ± ± ± ±

0.05 0.04 0.16 0.64 0.58 1.39

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

(Yoo et al., 2003), while that across the excised porcine nasal mucosa was 3.9 ± 2.2 × 10−6 cm/s (Wadell et al., 2003). This discrepancy could be due to the species difference (human versus porcine) and/or to the difference in the tight junction of passage cultured monolayer and excised nasal mucosa. The TEER value of 40–120  cm2 was reported in the excised nasal mucosa from animals (sheep, rabbit, and cattle) and human (Schmidt et al., 1998), which was considerably lower than that obtained in this study (>1000  cm2 ). However, a good log-linear relationship of the both models indicates that lipophilicity (log P) of the compounds is the most important factor that determine the nasal permeability and that the passage cultured human nasal cell monolayer model is useful to predict the nasal permeability of small molecular drugs.

209

Table 3 The effect of drug concentration on transport in the direction of apical to basolaterol 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 1000

0.54 ± 0.12 0.55 ± 0.14

0.97 ± 0.22 1.96 ± 0.51

50 80

4.88 ± 0.30 4.66 ± 0.46

0.88 ± 0.05 1.34 ± 0.13

Dexamethasone

constant permeability coefficients. This implies that transcellular transport of dexamethasone was non-polarized and suggests that passive diffusion is predominant. Similar results were reported in passive diffusion of budesonide transport across Calu-3 cell line (Borchard et al., 2002).

3.4. The effect of transport direction on Papp The possibility of the expression of active transporter(s) in the passaged human nasal cell monolayers and their involvement in the permeability of anti-allergic drugs was investigated by comparing the Papp 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 in Papp values between two directions was observed in all the model drugs. The Papp value of A-to-B direction was insignificantly higher than that of B-to-A direction, probably, due to the gravity and/or to the sampling condition. Thus, the transporters known to be expressed in human respiratory epithelium, which include members of the super family of ABC transporter, MDR1, MRP, and amino transporters (Brechot et al., 1998; Bremer et al., 1992), are absent or incompletely expressed in the human nasal epithelial cell monolayers in LCC condition used in this study. However, several transporters, such as MDR1 and MRP1 have been reported to be expressed in the epithelium and glands of the excised human nasal respiratory mucosa in immunohistochemistry study (Wioland et al., 2000; Henriksson et al., 1997). A large variety of hydrophobic and amphiphilic compounds as well as organic cations are known to be the substrates for MDR1 in superficial respiratory mucosa (Bremer et al., 1992). Fexofenadine HCl was reported to be a probe compound for the P-gp transporter in Caco-2 cell study (Perloff et al., 2002), yet there was no significant difference in the Papp values of A–B and B–A direction in this study. Thus, the culture condition needs to be further improved to express transporters and to study their effect on drug transport in in vitro nasal epithelial cell culture system. 3.5. Concentration dependency of the transport of fexofenadine HCl and dexamethasone The transport studies of different concentration of fexofenadine HCl (500 and 1000 ␮g/mL) and dexamethasone (50 and 80 ␮g/mL) were performed over a period of 60 min. As shown in Table 3, the transport rate of drugs across the monolayer increased in a concentration-dependent manner with

4. Conclusion Passaged human nasal epithelial cell monolayer using the LCC method was established up to passage-4, and its utility as an in vitro model for evaluating drug permeability was successfully investigated. Each passage culture formed a tight monolayer with high TEER value for drug transport studies, although the differentiation of cilia was not complete. A good log-linear relationship was observed between the lipophilicity (log P) of a series of anti-allergic drugs and their permeability coefficients (Papp ). Non-polarized transport across the human nasal cell monolayers was observed among all the model drugs, which indicated the absence or incomplete expression of transporter in this culture system. However, this in vitro model seems to be useful since it can offer constant supply of human nasal epithelial cells for transport and mechanism studies, and also is feasible to predict in vivo nasal permeability of small molecular drugs.

Acknowledgement This work was support by the research grant from the Ministry of Health and Welfare in Korea (02-PJ2-PG1-CH120002).

References Agu, 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 nasal primary 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. Transport and local metabolism of budesonide and fluticasone propionate in a human bronchial epithelial cell line (Calu-3). J. Pharm. Sci. 91, 1561–1567. Brechot, J.M., Hurbain, I., Fajac, A., Daty, N., Bernaudin, J.F., 1998. Different pattern of MRP localization in ciliated and basal cells from human bronchial epithelium. J. Histochem. Cytochem. 46, 513–517.

210

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

Bremer, S., Hoof, T., Wilke, M., Busche, R., Scholte, B., Riordan, J.R., Maass, G., Tummler, B., 1992. Quantitative expression patterns of multidrug-resistance P-glycoprotein (MDR1) and differentially spliced cystic-fibrosis transmembrane-conductance regulator mRNA transcripts in human epithelia. Eur. J. Biochem. 206, 137–149. Buchanan, J.A., Yeger, H., Tabcharani, J.A., Jensen, T.J., Auerbach, W., Hanrahan, J.W., Riodan, J.R., Buchwald, M., 1990. Transformed sweat gland and nasal epithelial cell lines from control and cystic fibrosis individuals. J. Cell Sci. 95 (Pt. 1), 109–123. Dearden, J.C., Bresnen, G.M., 1988. The measurement of partition coefficients. Quant. Struct. Act. Relat. 7, 133–144. Elbert, K.J., Schafer, U.F., Schafers, H.J., Kim, K.J., Lee, V.H., Lehr, C.M., 1999. Monolayers of human alveolar epithelial cells in primary culture for pulmonary absorption and transport studies. Pharm. Res. 16, 601–608. Freshney, R.I., 1994. Chapter of Media. Culture of Animal Cells: A Manual of Basic Technique, fourth Wiley-Liss Inc, pp. 99. Henriksson, G., Norlander, T., Zheng, X., Stierna, P., Westrin, K.M., 1997. Expression of p-glycoprotein 170 in nasal mucosa may be increased with topical steroids. Am. J. Rhinol. 11, 317–321. Huang, C.H., Kimura, R., Nassar, R.B., Hussain, A., 1985. Mechanism of nasal absorption of drugs. Part I: physicochemical parameters influencing the rate of in situ nasal absorption of drugs in rats. J. Pharm. Sci. 74, 608–611. Hussain, A.A., Hirai, S., Bawarshi, R., 1981. Nasal absorption of natural contraceptive steroids in rats: progesterone absorption. J. Pharm. Sci. 70, 466–467. Jorissen, M., Bessems, A., 1995. Influence of culture duration and ciliogenesis on the relationship between ciliary beat frequency and temperature in nasal epithelial cells. Eur. Arch. Otorhinolaryngol. 252, 451–454. Kissel, T., Werner, U., 1998. Nasal delivery of peptides: an in vitro cell culture model for the investigation of transport and metabolism in human nasal epithelium. J. Control. Release 53, 195–203. Krishnamoorthy, R., Mitra, A.K., 1998. Prodrugs for nasal drug delivery. Adv. Drug Deliv. Rev. 29, 135–146. Lipworth, B.J., Jackson, C.M., 2000. Safety of inhaled and intranasal corticosteroids: lessons for the new millennium. Drug Saf. 23, 11–33. Mathias, N.R., Kim, K.J., Robison, T.W., Lee, V.H., 1995. Development and characterization of rabbit tracheal epithelial cell monolayer models for drug transport studies. Pharm. Res. 12, 1499–1505. Morimoto, K., Yamaguchi, H., Iwakura, Y., Miyazaki, M., Nakatani, E., Iwamoto, T., Ohashi, Y., Nakai, Y., 1991. Effects of proteolytic

enzyme inhibitors on the nasal absorption of vasopressin and an analogue. Pharm. Res. 8, 1175–1179. Osth, K., Grasjo, J., Bjork, E., 2002. A new method for drug-transport studies on pig nasal mucosa using a horizontal Ussing chamber. J. Pharm. Sci. 91, 1259–1273. Perloff, M.D., von Moltke, L.L., Greenblatt, D.J., 2002. Fexofenadine transport in Caco-2 cells: inhibition with verapamil and ritonavir. J. Clin. Pharmacol. 42, 1269–1274. Roh, H.J., Goh, E.K., Wang, S.G., Chon, K.M., Yoon, J.H., Kim, Y.S., 1999. Serially passaged normal human nasal epithelial cells: morphology and mucous secretory differentiation. Korean J. Rhinol. 6, 107–112. Ruckes, C., Blank, U., Moller, K., Rieboldt, J., Lindemann, H., Munker, G., Clauss, W., Weber, W.M., 1997. Amiloride-sensitive Na+ channels in human nasal epithelium are different from classical epithelial Na+ channels. Biochem. Biophys. Res. Commun. 237, 488–491. Saha, P., Kim, K.J., Yamahara, H., Crandall, E.D., Lee, V.H.L., 1994. Influence of lipophilicity on ␤-blocker permeation across rat alveolar epithelial cell monolayers. J. Control. Release 32, 191–200. Schmidt, M.C., Peter, H., Lang, S.R., Ditzinger, G., Merkle, H.P., 1998. In vitro cell models to study nasal mucosal permeability and metabolism. Adv. Drug Deliv. Rev. 29, 51–79. Wadell, C., Bjork, E., Camber, O., 2003. Permeability of porcine nasal mucosa correlated with human nasal absorption. Eur. J. Pharm. Sci. 18, 47–53. Wang, W., Sasaki, H., Chien, D.S., Lee, V.H., 1991. Lipophilicity influence on conjunctival drug penetration in the pigmented rabbit: a comparison with corneal penetration. Curr. Eye Res. 10, 571–579. Werner, U., Kissel, T., 1995. Development of a human nasal epithelial cell culture model and its suitability for transport and metabolism studies under in vitro conditions. Pharm. Res. 12, 565–571. Wioland, M.A., Fleury-Feith, J., Corlieu, P., Commo, F., Monceaux, G., Lacau-St-Guily, J., Bernaudin, J.F., 2000. CFTR, MDR1, and MRP1 immunolocalization in normal human nasal respiratory mucosa. J. Histochem. Cytochem. 48, 1215–1222. Yang, J.J., Ueda, H., Kim, K., Lee, V.H., 2000. Meeting future challenges in topical ocular drug delivery: development of an air-interfaced primary culture of rabbit conjunctival epithelial cells on a permeable support for drug transport studies. J. Control. Release 65, 1–11. Yoo, J.W., Kim, Y.S., Lee, S.H., Lee, M.K., Roh, H.J., Jhun, B.H., Lee, C.H., Kim, D.D., 2003. Serially passaged human nasal epithelial cell monolayer for in vitro drug transport studies. Pharm. Res. 20, 1690–1696.