Journal of Pharmaceutical Investigation (2012) 42:147153 DOI 10.

1007/s40005-012-0020-9

RESEARCH ARTICLE

Application of biopharmaceutics classication system (BCS) in drug transport studies across human respiratory epithelial cell monolayers
Hyun-Jong Cho Prabagar Balakrishnan Hongxia Lin Min-Koo Choi Dae-Duk Kim

Received: 23 April 2012 / Accepted: 11 May 2012 / Published online: 24 May 2012 The Korean Society of Pharmaceutical Sciences and Technology 2012

Abstract Transport studies of model drugs were conducted across the human nasal epithelial (HNE) and normal human bronchial epithelial (NHBE) cell monolayers cultured by airliquid interface method. Physicochemical properties (e.g., molecular weight, calculated partition coefcient, dose number) of model drugs were quoted from literatures and apparent permeability coefcients (Papp) across the HNE and NHBE cell monolayers were directly measured. A linear relationship was observed between the Papp values of model drugs in the HNE and NHBE cell monolayers. As the molecular weight of model drugs increased, the Papp showed a decreasing pattern while the increase of partition coefcients resulted in the increment of Papp. These results indicated that the transport of model drugs across both cell monolayers followed mainly the passive diffusion mechanism, although substrates mediated by drug transporters showed a deviating pattern. It was also interesting to note that almost all model drugs could be grouped into the same biopharmaceutics classication system as that classied by the human intestinal permeability when the Papp was plotted as a function of dose number (D0) of each drug.

Keywords HNE cell monolayers NHBE cell monolayers Physicochemical properties Papp value Dose number BCS

Introduction Drug delivery via respiratory (i.e., nasal, bronchial and pulmonary) routes has acquired interests as an alternative administration route due to the avoidance of rst pass metabolism and the rapid absorption and onset of action. In spite of its limitations including proteolytic enzymes, volume of administration and ciliary movement for the absorption of drugs across respiratory mucosa, a variety of peptides/proteins and hormones have been successfully delivered, especially through nasal route (Abe et al. 1995; Hinchcliffe and Illum 1999; Laursen et al. 1996; Wang et al. 2006; Hermens et al. 1990). Pulmonary route has also been considered as an ideal administration route for gene and vaccine delivery (Densmore et al. 2000; Lu and Hickey 2007). To predict pharmacokinetic patterns of drugs in animal models, in vitro permeation study has been performed previously with several kinds of respiratory epithelium-derived cell culture systems, including human bronchial epithelial cell line (16HBE14o-) (Forbes et al. 2003), human broncho-tracheal epithelial cell line (Calu-3) (Grainger et al. 2006), human nasal RPMI 2650 cells (Bai et al. 2008), ATI-like human alveolar epithelial cell (hAEpC) monolayers (Fuchs et al. 2003), A549 lung cancer cells (Wang and Zhang 2004), normal human bronchial epithelial (NHBE) cells (Lin et al. 2007) and human nasal epithelial (HNE) cells (Lee et al. 2005; Cho et al. 2011). Among these cell culture systems, the characteristics of HNE and NHBE cell monolayers cultured by airliquid interface (ALI) method have been investigated in our

H.-J. Cho College of Pharmacy, Sunchon National University, Sunchon 540-950, Republic of Korea P. Balakrishnan H. Lin D.-D. Kim (&) College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Republic of Korea e-mail: ddkim@snu.ac.kr M.-K. Choi College of Pharmacy, Dankook University, Cheon-an 330-714, Republic of Korea

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previous studies and these cell monolayers have several advantages over the other cell culture systems as follows: optimal transepithelial electrical resistance (TEER) values to perform in vitro drug permeation study, the formation of tight junction and primary cells originated from human respiratory tract (Lin et al. 2007; You et al. 2003). Since the introduction of biopharmaceutics classication system (BCS) in 1995, it has become an increasingly crucial standard in the classication of drug substances. According to BCS guideline, drug substances can be classied into class I*IV based on their solubility and permeability. High solubility means dose/solubility (D:S) ratio is under 1 with a volume of 250 ml over the pH range 17.5 and high permeability means that fraction absorbed is over 90 % by mass balance studies and absolute bioavailability studies in human or in vitro/in situ permeation models. According to US Food and Drug Administration (FDA) guidance, permeability has been determined in the respect of oral administration with oral dosage forms. However, since various drug delivery systems via respiratory route have been developed and received increasing attention in recent years, the necessity of classication of drugs in nasal and bronchial epithelial cell monolayers has become more important. The objective of this study was to investigate the correlation between physicochemical properties of model drugs selected from all four classications of BCS and their permeability coefcients in HNE and NHBE cell monolayers cultured by ALI method. Based on their permeation behavior and physicochemical properties, it was determined whether the selected model drugs t with the same classication of BCS.

were obtained from Invitrogen Co. (Grand Island, NY, USA). Solubility and dose number calculations Based on BCS, 10 model drugs were selected. Solubility (mg/ml) of model drugs were obtained from the following website: http://www.drugbank.ca, quoted from reference (Ross and Riley 1990) and some of them were measured directly. Estimated values of Log P were obtained using ChemDraw Ultra 10.0 software (CambridgeSoft Corp., Cambridge, MA, USA). Dose number (D0) was calculated according to the following equation: D0 M=V0 Cs 1

where M is maximum dose strength (mg), V0 is the volume of water consumed with drug, 250 ml, and Cs is the aqueous solubility (mg/ml). ALI method culture of HNE and NHBE cell monolayers Passaged HNE cell monolayers were cultured by previously reported method (Lee et al. 2005). When human nasal epithelial cells of passage 1 and 2 attained about 7080 % conuency, the cells were trypsinized and seeded on Transwell insert (12-well) at a density of 1.5 9 105 cells per well. NHBE cells were also cultured on Transwell insert as previously described (Lin et al. 2007). The apical side (0.5 ml) and basolateral side (1.5 ml) were lled with BEGM:DME/F12 (50:50, v/v) supplemented with epinephrine (0.5 lg/ml), gentamycin (50 lg/ml), insulin (5 lg/ml), hydrocortisone (0.5 lg/ml), transferrin (10 lg/ml), triiodothyronine (6.5 lg/ml), epidermal growth factor(0.5 ng/ml human recombinant), amphotericin-B (50 lg/ml) and retinoic acid (0.1 ng/ml) (all supplied by Cambrex Bio Science Inc., Walkersville, MD, USA). The culture media in both sides were changed after 1 day and then the apical side was exposed to the air on day 3 for ALI culture, after which the culture medium in the basolateral side was changed every other day. The incubator for culturing cells was maintained at 37 C in atmosphere 5 % CO2 and 95 % relative humidity. Transport studies across HNE and NHBE cell monolayers cultured by ALI method HNE and NHBE cell monolayers cultured by ALI method which have at least 500 X cm2 of TEER value were selected for drug transport study. The TEER was measured by an EVOM voltohmmeter device (WPI, Sarasota, FL, USA)

Materials and methods Materials

L-phenylalanine, caffeine, theophylline, metoprolol tartrate, piroxicam, naproxen, atenolol, ooxacin, furosemide, Hanks balanced salt solution (HBSS), 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) and D-(?)-glucose were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fexofenadine hydrochloride (HCl) was gifted from Chong Kun Dang Pharmaceutical Co. (Seoul, Korea). HNE cells were obtained from human nasal tissue according to the procedure described in the reference (Lin et al. 2005). Normal human bronchial epithelial (NHBE) cells (CloneticsTM, 1st passage) and BEGM bullet kit were purchased from Cambrex Bio Science, Inc. (Walkersville, MD, USA). Transwell (0.4 lm pore size, 12 mm diameter, polyester) was obtained from Costar Co. (Cambridge, MA, USA). Other reagents for cell culture and supplies

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after 5 min incubation with BEGM : DME/F12 (50 : 50, v/v) medium for stabilizing prior to the measurement of TEER value. The selected model drugs classied by BCS were solubilized in transport medium (HBSS buffer containing 10 mM HEPES and 10 mM D-(?)-glucose). Piroxicam, naproxen, ooxacin, which have low solubility in the transport medium, were solubilized in transport medium containing 1 % (v/v) dimethyl sulfoxide (DMSO). The concentration of drug solution added in the apical side was 200 lM in the case of piroxicam, naproxen, ooxacin and furosemide, but that for other drugs was 500 lM. The cell culture medium was aspirated off and transport medium was added to equilibrate the cell monolayers for 30 min. Transport study was carried out in a shaking water bath at 50 rpm at 37 C. The transport study was performed only from apical to basolateral (AB) direction after applying 0.4 ml of each drug solution in the donor compartment, while 1.0 ml of transport medium was added to the receptor compartment. Aliquot (1.0 ml) of samples from the basolateral side was collected at pre-determined times (30, 60, 90 and 120 min), and the same volume of fresh transport medium was applied immediately into the basolateral side. Analytical method and data analysis The samples from transport study were analyzed by a Waters HPLC system (Waters Co., Milford, MA, USA) composed of a pump (Waters 515), an automatic injector (Waters 717plus) and UV detector (Waters 2487) or uorescence detector (Series 200, PerkinElmer Instrument, Norwalk, CT, USA). Two reverse phase C-18 columns (Lichrospher 100, RP-18, 125 9 4 mm, 5 lm, Merck Darmstadt, Germany and Xterra, RP-18, 250 9 4.6 mm, 5 lm, Waters Co., Milford, MA, USA) were used for the analysis. The wavelength and mobile phase composition

for the drug substances studied are shown in Table 1. Flow rate was 1.0 ml/min and the injection volume was 30 ll. The apparent permeability coefcients (Papp, cm/s) were acquired according to the following equation: Papp dQ 1 dt A C0 2

where, dQ/dt is the transported amounts of drug in the receptor compartment, A is the surface area of Transwell insert (1.12 cm2) and C0 is the drug concentration added in the donor side. Each datum was represented as the mean standard deviation (SD).

Results and discussion Relationship of Papp between HNE and NHBE cell monolayers Transport studies of 10 model drugs across the HNE and NHBE cell monolayers were carried out, and Papp (cm/s) values were calculated. The relationship of Papp values between the HNE and NHBE cell monolayers was plotted in Fig. 1. It was noticeable that a good linearity was shown between Papp values of model drugs in HNE and NHBE cell monolayers (r2 = 0.94). In our previous report, a good loglinear relationship (r2 = 0.92) was observed in the permeability of anti-allergic drugs between HNE cell monolayers cultured by the liquid-covered culture (LCC) and ALI method despite the difference in the culturing method (Lin et al. 2007). The good relationship observed in this study could be due to the similar anatomical structure between human nasal and bronchial epithelium. They are sections of airway epithelium and both cell shapes are pseudostratied epithelium. In addition, the morphology of these two cells is

Table 1 HPLC analysis conditions of all model drugs Drug

L-phenylalanine

The composition of mobile phase (v:v, %) Water: acetonitrile = 95:5 Methanol: 0.05 M phosphate buffer (pH 6.0) = 30:70 Methanol: 0.05 M phosphate buffer (pH 6.0) = 30:70 0.05 M KH2PO4 (pH 3.0 with phosphoric acid): acetonitrile = 60:40 0.3 % (v/v) phosphoric acid : acetonitrile = 80:20 0.1 M sodium acetate:acetonitrile:triethylamine = 61:39:0.05 Acetonitrile:water (pH 3.2 with phosphoric acid) = 55:45 Phosphate buffer (pH 2.5 with phosphoric acid):acetonitrile = 60:40 0.05 M KH2PO4 (pH 3.0 with phosphoric acid):acetonitrile = 60:40 Acetonitrile:water (pH 5.0 with phosphoric acid) = 40:60

Wavelength (nm) Ex: 210/Em: 303 270 270 Ex : 230/Em : 300 306 330 270 220 Ex: 230/Em: 300 280

Caffeine Theophylline Metoprolol tartrate Ooxacin Piroxicam Naproxen Fexofenadine HCl Atenolol Furosemide

Ex and Em indicate excitation and emission wavelength, respectively

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30

25

20

15

10

10

20

30

Papp values in HNE cell monolayers (x 10-6 cm/s)

Fig. 1 Correlationship between the apparent permeability coefcient (Papp) of drugs across the HNE and NHBE cell monolayers

known to be similar when observed by light microscopy (Lin et al. 2007; Lin et al. 2004). When metoprolol was used as a reference drug, the permeability coefcients of drugs that belonged to the conventional BCS class I and II (high permeability) were also higher than that of metoprolol in HNE and NHBE cell monolayers, except for ooxacin. On the contrary, the permeability coefcients of drugs classied as BCS class III and IV (low permeability) were also lower than that of metoprolol (Table 2). Among the model drugs studied, the highest and lowest Papp values across the HNE

and NHBE cell monolayers were observed for caffeine (BCS class I) and fexofenadine HCl (BCS class III), respectively (Table 3). This seems to be related with their aqueous solubility and transport mechanism. Caffeine showed higher permeability than expected, which could be due to its relatively high aqueous solubility, and it is expected to be transported by aqueous channel in the mucosal membrane by paracellular route. Though the coordinates of phenylalanine and naproxen in the correlation plot (Fig. 1) were slightly apart from linear line, all of model drugs, including those two drugs, seemed to be permeated by common transport mechanism across HNE and NHBE cell monolayers irrespective of BCS class. These results indicated that the model drugs were transported by identical routes (transcellular or paracellular) and they were also able to be inuenced by commonly expressed drug transporters in both HNE and NHBE cell monolayers, such as P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), organic anion-transporting polypeptide (OATP), and organic cation transporter (OCT) (Cho et al. 2011; Lin et al. 2007; Horvath et al. 2007; Wioland et al. 2000; Kaler et al. 2006). The effect of the molecular weight (MW) of drugs on Papp Relationship between MW of the model drug candidates and their permeability across HNE and NHBE cell monolayers was investigated. In general, compounds with high

Table 2 Molecular weight (MW), partition coefcient (Log P), solubility in H2O, and dose number (D0) of model drugs BCS class I Drug
L-Phenylalanine

MW (g/mol) 165.19 194.19 180.16 684.80 361.37 331.35 230.26 538.13 266.34 330.75

Log P -1.49 -0.80 -1.19 2.18 1.35 0.29 2.97 0.49 0.22 0.74

Solubility (mg/ml) 29.0a 22.0b 8.00c 1,000 3.23d 0.02b 0.02 1.00 26.5 0.01
b

Maximum dose strength (mg) 500 300e 300 100 400 20 500 180 100 80

D0 0.069 0.0545 0.15 0.004 0.496 4.00 125 0.72 0.015 32.0

Caffeine Theophylline Metoprolol tartrate Ooxacin II III IV

a b

Piroxicam Naproxen Fexofenadine HCl Atenolol Furosemide

All Log P values of drugs were calculated by ChemDraw Ultra 10.0 software except fexofenadine HCl (Lin et al. 2005) Solubility was measured directly Solubility data was found and quoted from following website: http://redpoll.pharmacy.ualberta.ca/drugbank/cgi-bin/getCard.cgi?CARD= APRD01011.txt Solubility was quoted from reference (Paruta et al. 1965) Solubility was quoted from reference (Ross and Riley 1990) If the dose of caffeine per day is over 300 mg, pregnant women may have a possibility of miscarriage

c d e

Solubility, maximum dose strength, and dose number values of the others were quoted from the reference (Kasim et al. 2004)

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Respiratory drug transport studies and BCS Table 3 Apparent permeability coefcient (Papp) of model drugs in ALI HNE and NHBE cell monolayers BCS class I Drug Papp in HNE cell monolayers (910-6 cm/s) 8.22 0.86 25.9 0.98 9.93 0.22 4.02 0.13 1.95 0.12 11.0 0.33 10.3 0.60 0.51 0.03 2.66 0.24 0.67 0.05 Papp in NHBE cell monolayers (910-6 cm/s) 4.60 0.32 24.2 1.1 7.31 0.50 4.15 0.02 1.66 0.22 8.29 0.15 6.68 0.99 0.28 0.07 1.98 0.43 3.58 0.22

151

Relationship between Papp and Log P of drugs The relationship between Papp in HNE and NHBE cell monolayers and Log P values of 10 model drugs was investigated, as also reported in the literature (Kasim et al. 2004). Metoprolol tartrate has been used as a reference drug for its high absorption ([90 %) in the gastrointestinal tract (Kim et al. 2006). Drugs exhibiting higher partition coefcients and Papp values in HNE and NHBE cell monolayers than the corresponding value for metoprolol were considered as high-permeability drugs. On the other hand, drugs with lower partition coefcients and Papp values in HNE and NHBE cell monolayers than that of metoprolol were classied as low-permeability drugs. False negatives were drugs exhibiting higher Papp values in HNE and NHBE cell monolayers than metoprolol but with lower partition coefcients than that of metoprolol. On the contrary, drugs with higher partition coefcients and lower Papp values in HNE and NHBE cell monolayers than the corresponding values of metoprolol were considered as false positives (Kasim et al. 2004). Experimentally determined Papp values in HNE and NHBE cell monolayers were presented in Table 3 and Log P values of model drugs were also shown in Table 2. A correlation plot of the Log P and Papp in HNE and NHBE cell monolayers indicated that the permeability classication was correct for 6 drugs (including metoprolol) out of 10 drugs when metoprolol was used as a reference drug (Fig. 3). Among the studied model drugs, 4 drugs (caffeine, theophylline, piroxicam, phenylalanine) that were classied as low-permeability drugs are regarded as false negatives due to their higher permeability as determined by the experiment. About 60 % accordance in the classication of drug candidates based on the Papp values in HNE and NHBE cell monolayers and Log P values seemed to be induced by the biological characteristics of human nasal and bronchial epithelial cells (e.g., expression of transporters, the difference of epithelial cell shape, mucin secreting degree) and the variation of Log P according to the used calculation method. It is interesting to note that there is no drug belonged to false positives in this study. Relationship between Papp and D0 of drugs Model drugs selected for this study were classied into provisional BCS classes based on the D0 and Papp values in the HNE and NHBE cell monolayers. In BCS classication, if D0 of a drug is over 1, that drug can be regarded as low solubility drugs and classied as BCS class II or IV. On the contrary, in the case of D0 \ 1, it can be regarded as high solubility drugs (BCS class I or III). As there is limited or no availability of D0 for the intranasal application from reference sources, we used D0 and V0 (250 ml),

L-phenylalanine

Caffeine Theophylline Metoprolol tartrate Ooxacin II III IV Piroxicam Naproxen Fexofenadine HCl Atenolol Furosemide

Data represents the mean SD (n = 3)

MW ([ 1 kDa) are known to be hardly absorbed across nasal mucosa without absorption enhancers. The intranasal bioavailability of drugs with high MW ([ 1 kDa), such as peptides and proteins, could be improved only with the presence of absorption enhancers (Costantino et al. 2007). No model drug with MW over 1 kDa was selected in this investigation. In Fig. 2, it was observed that Papp of model drug was decreased as the MW was increased in both HNE and NHBE cell monolayers. The regression lines of Papp values in HNE (line A) and NHNE (line B) are y = -0.0231 (0.014) x ? 15.0862 (5.0877) and y = -0.0168 (0.0131) x ? 11.7733 (4.7755), respectively. The absolute value of slope of regression line plotting MW and Papp values in HNE cell monolayers was higher than that of NHBE cell monolayers.

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Papp values in HNE cell monolayer

25 20 15
A

Papp values in NHBE cell monolayers

Papp (x 10 cm/s)

-6

10
B

5 0 -5 100

200

300

400

500

600

700

800

M. W.

Fig. 2 Relationship between molecular weight (MW) and Papp of model drugs across the HNE and NHBE cell monolayers. A and B line indicate the regression line of Papp values across the HNE and NHBE cell monolayers against MW of drugs, respectively

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A
Papp in HNE cell monolayers (10 cm/s)
-6

30
B

Papp across ALI HNE cell monolayers ( X10-6cm/s)

false negatives

A
100
BCS class 1 B E F BCS class 2

25

20

10
D H

15
C E F

10

1
G

5
H I G J D false positives

0 -2 -1 0 1 2 3 4

LogP

0.1 0.001

BCS class 3

BCS class 4

0.01

0.1

10

100

1000

B
Papp in NHBE cell monolayers (10 cm/s)
-6

30
B

Dose number (D0 )

false negatives

B
Papp across ALI NHBE cell monolayers ( X10 cm/s)
100
BCS class 1 B BCS class 2
-6

25

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D H A

10
C A

1
G

H G

false positives

0 -2 -1 0 1 2 3 4

LogP

0.1 0.001

BCS class 3

BCS class 4

0.01

0.1

10

100

1000

Fig. 3 Relationship between Log P of model drugs and Papp across the a HNE and b NHBE cell monolayers. Each point represents the mean SD (n = 3). (A L-phenylalanine, B caffeine, C theophylline, D metoprolol tartrate, E piroxicam, F naproxen, G fexofenadine HCl, H atenolol, I ooxacin, J furosemide)

Dose number (D0)

values of oral administration from reference sources. D0 values, based on the intranasal application, should be further determined for more accurate drug classication in the drug delivery via respiratory routes. Papp of metoprolol in HNE and NHBE cell monolayers was used as a reference value in the permeability scale. In both cell monolayers, 9 drugs (including metoprolol) were classied identically with classication patterns based on D0 and the human intestinal permeability (Fig. 4). Ooxacin, which is classied as BCS class I, was only moved to BCS class III due to its low permeability in both kinds of respiratory epithelial cell monolayers. Thus, 90 % of the model drugs was classied in provisional BCS as identical to oral BCS class. This indicated that the absorption patterns of tested drugs with various physicochemical characteristics in both human intestinal mucosa and human nasal and bronchial epithelium might be similar in spite of the differences of anatomical location and their function. Furthermore, it is

Fig. 4 Relationship between dose number (D0) and Papp of drugs across the a HNE and b NHBE cell monolayers. Each point represents the mean SD (n = 3). (A L-phenylalanine, B caffeine, C theophylline, D metoprolol tartrate, E piroxicam, F naproxen, G fexofenadine HCl, H atenolol, I ooxacin, J furosemide)

expected that in vivo absorption patterns of various drugs via respiratory routes could be predicted on the basis of provisional BCS with D0 and Papp values in HNE and NHBE cell monolayers.

Conclusion Although nasal and pulmonary drug deliveries of numerous compounds have been tried, there is no drug classication by BCS in human nasal and bronchial epithelial cell monolayers which enables to predict in vivo absorption patterns in human respiratory epithelium. In this study, the relationship between physicochemical properties of model drugs and their Papp values across human nasal and bronchial epithelial cell monolayers was investigated. Inverse

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153 dimethyl-beta-cyclodextrin in rabbits and rats. Pharm Res 7:500503 Hinchcliffe M, Illum L (1999) Intranasal insulin delivery and therapy. Adv Drug Deliv Rev 35:199234 Horvath G, Schmid N, Fragoso MA, Schmid A, Conner GE, Salathe M, Wanner A (2007) Epithelial organic cation transporters ensure pH-dependent drug absorption in the airway. Am J Respir Cell Mol Biol 36:5360 Kaler G, Truong DM, Sweeney DE, Logan DW, Nagle M, Wu W, Eraly SA, Nigam SK (2006) Olfactory mucosa-expressed organic anion transporter, Oat6, manifests high afnity interactions with odorant organic anions. Biochem Biophys Res Commun 351:872876 Kasim NA, Whitehouse M, Ramachandran C, Bermejo M, Lennernas H, Hussain AS, Junginger HE, Stavchansky SA, Midha KK, Shah VP, Amidon GL (2004) Molecular properties of WHO essential drugs and provisional biopharmaceutical classication. Mol Pharm 1:8596 Kim JS, Mitchell S, Kijek P, Tsume Y, Hilnger J, Amidon GL (2006) The suitability of an in situ perfusion model for permeability determinations: utility for BCS class I biowaiver requests. Mol Pharm 3:686694 Laursen T, Grandjean B, Jorgensen JO, Christiansen JS (1996) Bioavailability and bioactivity of three different doses of nasal growth hormone (GH) administered to GH-decient patients: comparison with intravenous and subcutaneous administration. Eur J Endocrinol 135:309315 Lee MK, Yoo JW, Lin H, Kim YS, Kim DD, Choi YM, Park SK, Lee CH, Roh HJ (2005) Air-liquid interface culture of serially passaged human nasal epithelial cell monolayer for in vitro drug transport studies. Drug Deliv 12:305311 Lin H, Lee CH, Shim CK, Chung SJ, Kim DD (2004) In vitro transport of fexofenadine HCl in deformable liposomes across the human nasal epithelial cell monolayers. J Kor Pharm Sci 34:483489 Lin H, You JW, Roh HJ, Lee MK, Chung SJ, Shim CK, Kim DD (2005) Transport of anti-allergic drugs across the passage cultured human nasal epithelial cell monolayer. Eur J Pharm Sci 26:203210 Lin H, Li H, Cho HJ, Bian S, Roh HJ, Lee MK, Kim JS, Chung SJ, Shim CK, Kim DD (2007) Air-liquid interface (ALI) culture of human bronchial epithelial cell monolayers as an in vitro model for airway drug transport studies. J Pharm Sci 96:341350 Lu D, Hickey AJ (2007) Pulmonary vaccine delivery. Expert Rev Vaccines 6:213226 Paruta AN, Sciarrone BJ, Lordi NG (1965) Solubility proles for the xanthines in dioxane-water mixtures. J Pharm Sci 54:838841 Ross DL, Riley CM (1990) Aqueous solubilities of variously substituted quinolone antimicrobials. Int J Pharm 63:237250 Wang Z, Zhang Q (2004) Transport of proteins and peptides across human cultured alveolar A549 cell monolayer. Int J Pharm 269:451456 Wang X, He H, Leng W, Tang X (2006) Evaluation of brain-targeting for the nasal delivery of estradiol by the microdialysis method. Int J Pharm 317:4046 Wioland MA, Fleury-Feith J, Corlieu P, Commo F, Monceaux G, Lacau-St-Guily J, Bernaudin JF (2000) CFTR, MDR1, and MRP1 immunolocalization in normal human nasal respiratory mucosa. J Histochem Cytochem 48:12151222 You JW, Kim YS, Lee SH, Lee MK, Roh HJ, Jhun BH, Lee CH, Kim DD (2003) Serially passaged human nasal epithelial cell monolayer for in vitro drug transport studies. Pharm Res 20:16901696

relationship between MW of drugs and permeability in HNE and NHBE cell monolayers was shown and drugs transported by passive diffusion mechanism were classied equally on the basis of partition and permeability coefcients in HNE and NHBE cell monolayers. In addition, a good relationship between drug permeability coefcients in HNE and NHBE cell monolayer systems was presented in this study. Even though D0 calculation method for oral formulations was applied for this study due to the absence of D0 calculating equation for nasal formulations, similar BCS classication aspects of drug candidates were observed compared to the drug classication based on human intestinal permeability. Thus, it would be possible to introduce the BCS classication in vitro system for nasal and pulmonary drug delivery and predict the in vivo absorption pattern in human.
Acknowledgments This work was supported by the Industrial Source Technology Development Program funded by the Ministry of Commerce, Industry and Energy (MOCIE) (No. 10031825) and by the MarineBio Research Program (NRF-C1ABA001-2011-0018561) of the National Research Foundation (NRF) grant funded by the Korean government (MEST).

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