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

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.