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

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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. Transport and local metabolism of budesonide and fluticasonepropionate 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 fromhuman bronchial epithelium. J. Histochem. Cytochem. 46, 513–517.
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