Meeting future challenges in topical ocular drug delivery:

Journal of Controlled Release 65 (2000) 1–11 www.elsevier.com / locate / jconrel

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 a

a

Johnny J. Yang , Hideo Ueda , Kwang-Jin Kim a

b,c,d,e,g

, Vincent H.L. Lee

a,f ,

*

Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, 1985 Zonal Avenue, PSC 704, Los Angeles, CA 90033, USA b Department of Medicine, University of Southern California, Los Angeles, CA 90033, USA c Department of Physiology and Biophysics, University of Southern California, Los Angeles, CA 90033, USA d Department of Molecular Pharmacology and Toxicology, University of Southern California, Los Angeles, CA 90033, USA e Department of Biomedical Engineering, University of Southern California, Los Angeles, CA 90033, USA f Department of Ophthalmology, University of Southern California, Los Angeles, CA 90033, USA g Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles, CA 90033, USA Received 7 July 1999; accepted 4 September 1999

Abstract The purpose of this study was to develop and characterize a functional air-interfaced primary culture of rabbit conjunctival epithelial cells grown on a permeable support for drug transport studies. Conjunctival epithelial cells from the pigmented rabbit were isolated, seeded at 1.2310 6 cells cm 22 on permeable Transwell filters, and cultured at the air interface using a modified PC-1 medium. Conjunctival epithelial cell layers showed a transepithelial resistance of 1.160.1 kV cm 2 , a potential difference of 17.060.5 mV, and an equivalent short-circuit current (Ieq ) of 16.160.4 mA cm 22 . The Ieq was reduced by 35% using 0.01 mM bumetanide, 66% using 0.1 mM ouabain, 46% using 2 mM barium chloride (all three in the basolateral fluid), and 63% using 0.3 mM NPAA in the apical fluid, consistent with active Cl 2 -secretion across the conjunctival epithelial barrier. Amiloride-sensitive Na 1 channels were absent. The permeability of the cell layers to polar solutes decreased with increased solute size, and the calculated equivalent pore size was about 8.0 nm. The Papp of b-blockers varied with lipophilicity in a sigmoidal fashion. Uridine transport showed temperature sensitivity and directionality, favoring transport in the apical-to-basolateral direction. Apical L-carnosine uptake was reduced by 46% in the absence of an inwardly directed proton gradient, and lowering the temperature to 48C abolished direction-dependent L-carnosine uptake. Furthermore, uptake was inhibited by 73% using apical 10 mM glycyl sarcosine (a dipeptide transporter substrate) and by 60% using 1 mM L-valacyclovir (a dipeptide prodrug). In conclusion, a functional air-interfaced primary culture of rabbit conjunctival epithelial cell layers was established. This air-interfaced primary culture model may be useful for studying passive and active transport processes for ion and solute translocation in the mammalian conjunctival epithelial barrier in a defined experimental setting.  2000 Elsevier Science B.V. All rights reserved.

*Corresponding author. Tel.: 11-323-442-1368; fax: 11-323-442-1390. E-mail address: [email protected] (V.H.L. Lee) 0168-3659 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0168-3659( 99 )00226-6

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J. J. Yang et al. / Journal of Controlled Release 65 (2000) 1 – 11

Keywords: Conjunctival epithelial cell; Air-interfaced culture; Drug transport; Ion transport; Transporter; Topical ocular drug delivery

1. Introduction The conjunctiva is a thin, transparent mucous membrane covering the inner surface of the eyelids and exterior part of the sclera. It plays an important role in both ocular [1] and systemic absorption [2,3] of topically applied ophthalmic drugs. The bulbar conjunctiva is the first tissue across which a topically applied drug must pass in order to reach the underlying tissues in the uveal tract via the non-corneal route [1]. Previous work with excised tissues in our laboratory has revealed that the conjunctiva actively secretes Cl 2 [4], is commensurately permeable to hydrophilic molecules of up to |40 kDa [5], and is capable of active drug transport mediated by nucleoside [6], monocarboxylate [7], and dipeptide transporters [8,9]. Cell cultures have proven to be a useful tool for evaluating transport mechanisms as well as drug, formulation, and physiological factors influencing drug transport [10,11]. In 1996, Saha et al. [12] reported the development of functional primary cultures of rabbit conjunctival epithelial cells (RCEC) under the conventional liquid-covered condition (LCC). Since air-interfaced cultures (AIC) of tracheal epithelial cells [13] seem to mimic the tissue better when compared with liquid-covered cultures, we undertook the present study to determine whether this would also be the case in the conjunctiva on the basis of passive and active transport characteristics for ions and drugs.

2. Materials and methods

2.1. Materials Fluorescein isothiocyanate (FITC)-labeled dextrans (FD) with average molecular weights of 4400 (FD4), 9400 (FD10), 21 200 (FD20), 71 200 (FD70), a series of b-adrenergic blockers (atenolol, timolol maleate, metoprolol tartrate, and propranolol hydrochloride), amiloride, bumetanide, ouabain, Nphenylathranilic acid (NPAA), barium chloride, uridine, and L-carnosine were purchased from Sigma

Chemical (St. Louis, MO). Sotalol was obtained from Bristol-Myers (Evansville, IN). [ 3 H]-Propranolol (specific activity, 19.3 Ci mmol 21 ) and [ 3 H]betaxolol (specific activity, 51 Ci mmol 21 ) were purchased from Amersham (Arlington Heights, IL). [ 3 H]-Metoprolol (specific activity, 1.05 Ci mmol 21 ) ¨ was a gift from Astra Hassle (Sweden). [ 14 C]-Man21 nitol (56 mCi mmol ), [ 3 H]-L-carnosine (5 Ci mmol 21 ) and [5,6- 3 H]-uridine (42.7 Ci mmol 21 ) were purchased from Moravek Biochemical (Brea, CA). L-Valacyclovir was a gift of Dr. Philip Smith of SmithKline Beecham Pharmaceuticals (Collegeville, PA). Cell culture reagents and supplies were purchased from Life Technologies (Grand Island, NY). PC-1 culture medium was purchased from BioWhittaker (Walkersville, MD). Transwell filters (6.5 mm diameter, 0.4 mm pore size), were obtained from Costar (Cambridge, MA). Male Dutch-belted pigmented rabbits, weighing 2.0–2.5 kg, were obtained from Irish Farm (Los Angeles, CA), and all animals were handled in accordance with the Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 80-23).

2.2. Primary culture of conjunctival epithelial cells Rabbit conjunctival epithelial cells were harvested using a protocol modified from that developed by Saha et al. [12]. Briefly, following excision, the conjunctiva was washed in ice-cold Ca 21 / Mg 21 -free Hanks’ balanced salt solution and treated with 0.2% protease (type XIV) for 60 min at 378C in 95% air / 5% CO 2 to dissociate the cells. The isolated cells were treated with S-MEM containing 10% FBS and 1 mg ml 21 deoxyribonuclease (DNAase I) to stop protease reaction. The cell pellet was washed, centrifuged at 100 g for 10 min at room temperature and filtered through a 40-mm cell strainer. The final cell pellet was resuspended in Dulbecco’s Modified Eagle Medium / Nutrient Mixture F-12 (DMEM / F12) medium supplemented with 100 U ml 21 penicillin–streptomycin, 0.5% gentamicin, 0.4% fungizone, 2 mM L-glutamine, 1% ITS 1 (insulin 6.5 mg ml 21 , transferrin 6.5 mg ml 21 , selenious acid 6.5 ng ml 21 , BSA 1.25 mg ml 21 , and linoleic acid 5.35

J. J. Yang et al. / Journal of Controlled Release 65 (2000) 1 – 11

mg ml 21 ), 30 mg ml 21 bovine pituitary extract (BPE), 1 mM hydrocortisone, and 1 ng ml 21 epidermal growth factor (EGF). These cells were seeded at a density of 1.2310 6 cells cm 22 on Transwell inserts precoated with collagen, and cultured in 5% CO 2 and 95% air at 378C. From Day 2 onwards, the growth medium was changed to PC-1 growth medium supplemented with 2 mM L-glutamine, 100 U ml 21 penicillin–streptomycin, 0.5% gentamicin and 0.4% fungizone. Cells were then switched to an air interface (i.e. nominally fluid-free on the apical surface of the cell layers from that day onwards) on Day 4 onwards, unless otherwise indicated.

2.3. Periodic Acid Schiff ( PAS) staining of rabbit conjunctival epithelial cell layers RCECs were fixed for PAS staining to estimate the proportion of secretory cell population on Day 0 and Day 6 of culture. Day-0 cells in suspension were fixed with 3.7% formaldehyde in PBS, incubated with 0.5% periodic acid for 10 min and then with a Schiff reagent for 15 min. The cells were subsequently rinsed with sodium carbonate working solution for 5 min and Light Green SF Yellowish for 10 s, and finally dehydrated in absolute alcohol and toluene [12]. Day-6 cell layers were incubated with 0.5% trypsin–EDTA for 15 min at 378C to detach cells from the Transwell filter, and the resultant cell suspension was fixed and stained in the same manner as for Day-0 cells. The percentage of positively stained cells in the entire population was estimated under a light microscope accordingly. Day-6 cell layers were also directly stained on Transwell filter in the same way and examined under light microscopy.

2.4. Evaluation of ion transport properties of conjunctival epithelial cell layers Confluent cell layers (TEER.1 kV cm 2 ) were equilibrated in a pH 7.4 bicarbonated Ringer’s solution (BRS) containing 1.8 mM CaCl 2 , 5.6 mM KCl, 0.8 mM MgSO 4 , 0.8 mM NaH 2 PO 4 , 116 mM NaCl, 25 mM NaHCO 3 , 15 mM HEPES, and 5.5 mM D-glucose (378C) for 30 min. Ion transport inhibitors were then added separately to either the

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apical or basolateral fluid. These inhibitors were: basolateral 100 mM ouabain, a Na 1 / K 1 -ATPase inhibitor; basolateral 10 mM bumetanide, a Na 1 -K 1 -2Cl 2 cotransport blocker; basolateral 2 mM barium chloride, a potassium channel blocker; apical 300 mM NPAA, a chloride channel blocker; and 1 apical 10 mM amiloride, a Na channel blocker. Potential difference (PD) and transepithelial electrical resistance (TEER) were measured at predetermined time points throughout the experiment using EVOM (World Precision Instruments, Sarasota, FL). The equivalent short-circuit current (Ieq ) was calculated using Ohm’s law, i.e. Ieq 5 PD/ TEER.

2.5. Solute transport studies in air-interface cultured cell layers Confluent RCEC layers grown on Transwell inserts were washed once with BRS buffer. After equilibration for 30 min, transport studies were initiated by adding the dosing solution to the apical compartment. The final concentrations used were: 1 mg ml 21 for FITC, FD4, FD10, FD20 and FD70; 1 mM for atenolol and sotalol; 100 mM for timolol; and 10 mM for metoprolol, propranolol and betaxolol. Uridine transport was measured with 1 mCi ml 21 [ 3 H]-uridine and 10 mM unlabelled uridine in the donor fluid. In the case of sotalol, atenolol, metoprolol, timolol, propranolol and betaxolol, their transport in freshly excised conjunctiva was also measured. We have previously reported the detailed procedure for preparing the excised rabbit conjunctiva for transport studies in the modified Ussing chamber [4]. Briefly, rabbits were euthanized with an injection of 85 mg kg 21 sodium pentobarbital solution into a marginal ear vein, and the entire eye ball was removed from the orbit. After trimming, the excised conjunctiva was mounted in the tissue adapter with a circular aperture of 1.0 cm 2 , which was then placed in a modified Ussing chamber. The bathing solutions (6 ml each) were bubbled with 95% air–5% CO 2 to maintain the pH at 7.4 and to provide adequate agitation. The Ussing chamber assembly was maintained at 36618C with a circulating water bath. At predetermined times for up to 240 min, a 200-ml aliquot was removed from the basolateral side and was replaced with same volume of fresh

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BRS. For FITC-dextran experiments, the fluorescence intensity of the samples was measured in a spectrofluorometer F-2000 (Hitachi, Tokyo, Japan) at an excitation wavelength of 490 nm and an emission wavelength of 515 nm. In the case of atenolol, sotalol and timolol, 200 ml was sampled from the receiver compartment, mixed with 200 ml of acetonitrile to precipitate the proteins, and centrifuged at 3000 g for 10 min. The supernatant was evaporated and redissolved in 200 ml water containing an appropriate internal standard. One hundred ml of these reconstituted samples was then injected into a HPLC equipped with a reversed-phase C18 Microsorb column (4.63250 mm, particle size 5 mm, Rainin, Woburn, MA). The HPLC conditions for each compound are listed in Table 1. For metoprolol, propranolol, betaxolol and uridine, the samples was mixed with 5 ml of EconoSafe scintillation cocktail (Research Products International, Mount Prospect, IL) for assay of radioactivity in a liquid scintillation spectrometer (Beckman, Fullerton, CA).

2.6. Dipeptide uptake studies All the uptake experiments were performed in a humidified atmosphere of 5% CO 2 and 95% air at 378C. An inwardly directed proton gradient (apical pH 6.0, basolateral pH 7.4) from the apical fluid to the cell interior was imposed [8]. Prior to each experiment, the cell layers were washed with BRS and pre-equilibrated for 30 min. Uptake was initiated by spiking the apical or basolateral fluid with 20 mCi ml 21 [ 3 H]-L-carnosine and an appropriate amount of unlabeled L-carnosine. After 10 min, uptake was terminated by suctioning off the dosing solution and washing the cell layers with ice-cold BRS (pH 7.4). The cell layers were then solubilized

in 0.5 ml of 0.5% Triton X-100 solution. Twenty ml of the cell lysate were taken for protein assay using the method of Bradford [14] with bovine serum albumin as a standard. The rest of the cell lysate sample was mixed with 5 ml of EconoSafe scintillation cocktail for measurement of radioactivity in a liquid scintillation counter. Drug uptake was expressed as amount of drug accumulated per mg of cellular protein over the duration of measurement.

2.7. Data analysis Data were presented as mean6S.E.M. Flux was derived from a plot of the steady-state slope of a plot of the cumulative amount appearing in the receiver fluid vs. time. The Papp of unidirectional fluxes for solutes was estimated by normalizing the flux dQ / dt (mol s 21 ), against the nominal surface area (A50.33 cm 2 ) and initial solute concentration in the donor fluid C0 (mol ml 21 ), or Papp 5 (dQ / dt) /(A 3 C0 ). The FD transport data was subjected to equivalent pore analysis. The permeability-to-diffusion coefficient ratio at 378C of a test solute with a Stokes– Einstein radius (r e ) for a single homogeneous population of equivalent pores (with a radius of r p ) is shown as follows [5]: Papp /D 5 (Ap /dx)(1 2 m )2 (1 2 2.104m 1 2.809m 3 1 0.948m 5 2 1.372m 6 ) where Ap is the total pore area, dx is the mean thickness of the barrier, and m 5 r e /r p . The diffusivity of FD was estimated from the relationship: D(MW )1 / 3 5 constant, where the constant was calculated using bovine serum albumin as a reference (MW567 000 Daltons, D50.88310 26 cm 2 s 21 ) [15]. From the experimental Papp’s and the calcu-

Table 1 HPLC conditions for assaying b-blockers: detection wavelength, internal standard, composition of the mobile phase, and retention time Drug

Wavelength (nm)

Internal standard

Mobile phase a

RT b (min) Drug

RT (min) IS c

Sotalol Atenolol Timolol

224 224 280

Atenolol Sotalol Metoprolol

3.5% AcN 3.5% AcN 18% AcN

16.2 19.1 14.2

19.1 16.2 17.2

a

The aqueous portion of the mobile phase was 0.2% triethylamine at pH 3.0. Key: AcN, acetonitrile. Retention time. c Internal standard at 5 mg ml 21 . b

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lated Stokes–Einstein radii of molecules, the equivalent pore radius (r p ) and the ratio (Ap /dx) between the total pore area and pore length were estimated on the basis of a non-linear curve fitting software TableCurve (Jandel Scientific, San Rafael, CA), assuming a dx of 10 mm for the conjunctival epithelial cell layers [12]. The Papp value of b-blockers was plotted against the logarithm of the n-octanol–water partition coefficient (log P), and fitted to the sigmoidal model as defined by the following equation using TableCurve [16]: Papp 5 m 1 n /(1 1 exp((o 2 log P) /q)) where m is the minimal Papp observed, n is the maximal Papp observed, o is the half-maximal log P (where Papp 5 (m 1 n) / 2), and q is the slope of the linear increase in Papp vs. log P. One-way analysis of variance (ANOVA) was used to determine the significant difference among group means. p,0.05 was considered as statistically significant.

3. Results

3.1. Air-interface culture conditions and measurement of bioelectric parameters Approximately 15–20 million conjunctival epithelial cells were obtained from each animal, with a viability.90% as assessed by trypan blue exclusion. We found that a density of 1.2310 6 cm 22 was the best condition for forming a functional epithelial barrier. When air-interface conditions were instituted from Day 4 of culture, a peak TEER of 1.0660.06 kV cm 2 and PD of 17.060.5 mV were observed (Fig. 1 and Table 2). In contrast, when air-interface conditions were imposed on Day 2 or 3, a lower peak TEER of 0.5860.03 and 0.6760.03 kV cm 2 and a lower PD of 7.660.6 and 11.060.2 mV, respectively, were found. Therefore, we used airinterface cultures (AIC) starting at Day 4 for the remaining studies. Compared with LCC, air-interface Day-4 culture developed a higher Ieq (181%) and PD (130%) but a lower TEER (73%), mimicking the tissue better [4].

Fig. 1. Mean transepithelial electrical resistance (TEER), potential difference (PD), and equivalent short-circuit current (Ieq ) of cultures grown on Transwell filter in LCC (d) and AIC [AIC Day 2 (D), 3 (h) or 4 (s) onwards]. Key: ←, values observed in the excised tissue [12]. Each data point represents mean6S.E.M. for n512.

3.2. Periodic Acid Schiff ( PAS) staining of RCEC A sporadic PAS positive staining pattern (3–4%)

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Table 2 Peak transepithelial electrical resistance (TEER), potential difference (PD), and equivalent short-circuit current (Ieq ) of the excised tissue, liquid covered culture, and air-interface culture Conditions

TEER (kV cm 2 )

PD (mV)

Isc or Ieq (mA cm 22 )

Excised tissue a Liquid-covered culture b Liquid-covered culture c Air-interfaced culture c

1.360.1 1.960.2 1.560.1 1.160.1

17.760.8 14.261.6 13.160.7 17.060.5

14.560.7 8.060.4 8.960.6 16.160.4

a

From Ref. [4]. From Ref. [12]. c From this study. Mean6S.E.M. (n512). b

Fig. 2. Periodic Acid Schiff (PAS) staining of Day-6 AIC (A) and LCC cells (B). The black dots correspond to PAS positive cells (magnification 903).

was seen in AIC cells, suggesting the presence of mucin-secreting goblet cells [Fig. 2(A)]. By contrast, PAS positive cells in LCC cells were fewer (|1%) [Fig. 2(B)].

3.3. Ion transport properties Fig. 3 summarizes the effect of ion transport inhibitors on Ieq as percent inhibition compared to control Ieq of AIC cells on Days 6–8. Ieq was reduced by 66% using 0.1 mM ouabain (basolateral), 35% using 0.01 mM bumetanide (basolateral), 63% using 0.3 mM NPAA (apical), and 46% using 2 mM BaCl 2 (basolateral). By contrast, 0.01 mM amiloride (apical) did not affect the Ieq .

Fig. 3. Influence of pharmacological modulators of ion transport on the equivalent short-circuit current (Ieq ) of air-interfaced conjunctival epithelial cell layers (h) and the excised tissue (j) [4]. Key: a, apical; b, basolateral; mean6S.E.M. (n54); * and 1 , p,0.05 compared to corresponding AIC and tissue controls, respectively. Tissue data for amiloride treatment are not available.

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3.4. FITC-dextran transport The cumulative appearance of fluorescent label in the receiver fluid was linear in all FDs for the duration of the transport experiment. The Papp for FDs decreased sharply from 3.26310 28 cm s 21 for FD4 to 0.68310 28 cm s 21 for FD70 (Table 3 and Fig. 4). The relationship shown in Fig. 4 is consistent with a single pore population with an equivalent pore radius of about 8.0 nm and a pore density of 5310 7 pores per cm 2 . Fig. 4. Relationship between the apparent permeability coefficient (Papp) and molecular weight (MW ) of mannitol, FITC, FD4, FD10, FD20 and FD70 in AIC (n54–5). Key: s, AIC; d, tissue [5].

3.5. b -Blocker transport The PD and TEER of both the cell layers and excised tissue remained relatively constant throughout these experiments. The lag time was brief (,10 min) for all solutes studied. Papp of b-blockers appeared to follow an apparent sigmoidal relationship (Table 4 and Fig. 5), with a half-maximal log P of 1.2 for AIC and 1.0 for the excised tissue.

3.6. Uridine transport The Papp of uridine transport at 10 mM in AIC exhibited 1.7760.03310 25 cm s 21 for the apical-tobasolateral direction and 0.03660.000310 25 cm s 21 for the basolateral-to-apical direction at 378C. At

48C, it was 0.02260.002310 25 cm s 21 for the apical-to-basolateral direction (Fig. 6).

3.7. L-Carnosine uptake L-Carnosine uptake was direction-dependent, being 4.4-times higher from the apical than from the basolateral fluid. Lowering the temperature to 48C and abolishing the pH gradient reduced apical Lcarnosine uptake by factors of 5 and 2, respectively. Furthermore, uptake was inhibited by 73% in the presence of apical 10 mM glycyl sarcosine (a known substrate for dipeptide transporter) and by 60% in the

Table 3 Molecular weight (MW ), molecular radius, and apparent permeability coefficient (Papp) of FITC-dextrans at 1 mg ml 21 donor concentration in the apical-to-basolateral direction in air-interface cultures of pigmented rabbit conjunctival epithelial cells and excised tissues Solutes

Mannitol 6-CF FITC FD4 FD10 FD20 FD70 a

Mean6S.E.M. (n54). From Ref. [5]. c N.D., not determined. b

MW

182 376 389 4400 9400 21 200 71 200

Radius (nm)

0.43 0.68 0.68 1.44 1.91 2.44 3.81

Papp (310 28 cm s 21 )a AIC

Excised tissue b

21.7861.46 N.D.c 15.2062.80 3.2660.13 2.4060.14 1.2260.03 0.6860.05

27.7064.33 15.7063.30 N.D. 3.4160.79 1.9160.26 0.7160.06 0.3060.02

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Table 4 Logarithm of n-octanol / pH 7.4 buffer partition coefficient (log PC) and apparent permeability coefficient (Papp) of b-blockers in the apical-to-basolateral direction in air-interface cultured pigmented rabbit conjunctival epithelial cell layers and excised tissue Solutes

Sotalol Atenolol Metoprolol Timolol Propranolol Betaxolol a b

log PC

20.62 0.16 1.88 1.91 3.21 3.44

Papp (310 25 cm s 21 )a Air-interface culture

Liquid-covered culture b

Excised tissue

0.0360.01 0.0160.00 1.3460.05 0.5360.03 0.9860.02 1.0660.03

0.0260.01 0.0360.01 3.0960.49 1.6860.17 2.3660.16 3.1660.40

0.1160.04 0.2160.07 0.9460.15 0.9560.06 0.7960.08 0.6960.14

Mean6S.E.M. (n54–7). From Ref. [16].

Fig. 5. Influence of drug lipophilicity on b-blocker transport across AIC (s), LCC (d) [16], and excised conjunctiva (j). Error bars represent S.E.M. for n54–7.

Fig. 7. Ten-minute uptake of L-carnosine at 10 mM donor concentration in the presence of an inwardly directed proton gradient (apical pH56.0, basolateral pH57.4), under various conditions including abolishing pH gradient, pre-incubating cells at 48C, and in the presence of 10 mM glycyl-sarcosine or 1 mM L-valacyclovir in the donor chamber. Values are mean6S.E.M., n54. Asterisks indicate a statistically significant difference from apical uptake at 378C.

presence of 1 mM L-valacyclovir (an amino acid ester prodrug) (Fig. 7).

3.8. Discussion

Fig. 6. Time course of uridine transport measured with 10 mM donor concentration across the air-interface cultured conjunctival epithelial cells. Each data point represents mean6S.E.M. (n55). Key: s, apical-to-basolateral; d, basolateral-to-apical; D, 48C.

The present study demonstrates that it is possible to culture rabbit conjunctival epithelial cells at an air interface that exhibit electrophysiological characteristics more akin to those of the native tissue than does the conventional liquid-covered culture. The TEER of AIC (1.160.1 kV cm 2 ) was not significantly different from the excised tissue value of 1.360.1 kV cm 2 [4], while that in LCC was higher (1.960.2 kV cm 2 ) [12]. Moreover, the PD

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(17.060.5 mV) and Ieq (16.160.4 mA cm 22 ) of AIC resembled those in the excised tissue, while those in LCC were lower (Table 2). It appears that the presence of excess liquid on the apical surface of the cell layers may lower the capacity of active ion transport. Such a phenomenon has been observed in tracheal epithelial cell cultures of the rabbit [13], guinea pig [17], cow [18], and dog [19]. In addition, air interfaced cultures of human bronchial epithelium [20] and nasal turbinate tissue [21] also exhibit better morphological and electrophysiological traits in vitro compared to immersion cultures. The conjunctival epithelial cell layers cultured on permeable filters maintain their barrier properties for approximately 4 days (Days 6–10) (Fig. 1), a suitable time frame for transepithelial drug and ion transport studies. The PAS staining pattern revealed 3–4% of the superficial cell population of the secretory type (goblet cells). This goblet cell population agrees with the 3–5% value seen in freshly isolated cells, well within the 4–22% value seen in the rabbit conjunctival epithelium [22]. Interestingly, cells in liquidcovered culture exhibited a reduced secretory cell population (about 1%) (Fig. 2). Thus, the excess liquid present on the apical cell surface may suppress secretory cell growth. Although the underlying mechanism is not immediately forthcoming, one possibility may be improved oxygenation of or removal of CO 2 from the AIC cells. The overall ion transport properties of AIC are comparable to those observed in the excised tissue (Fig. 3). Thus, Ieq was reduced 66% using basolateral 0.1 mM ouabain, suggesting that Na 1 / K 1 -ATPase is present basolaterally. The inwardly directed Na 1 gradient thus established provides the chemical driving force for Cl 2 uptake by the basolaterally located Na 1 -K 1 -2Cl 2 cotransporter, whose presence is suggested by the 35% reduction in Ieq using 0.01 mM bumetanide. The 46% inhibition of Ieq by Ba 21 reflects reduced K 1 conductance (i.e. raising intracellular [K 1 ]), thereby reducing apical Cl 2 efflux. NPAA effect in AIC indicates the presence of Cl 2 conductive pathways in the apical membranes. The lack of an amiloride effect on Ieq suggests the absence of amiloride-sensitive Na 1 channels. Thus, Cl 2 enters the conjunctival epithelial cell via the basolaterally located Na 1 -K 1 -2Cl 2 cotransporter and Cl 2 exits the cell via the apically localized Cl 2

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conductive pathway. A counter ion, Na 1 , is likely to move passively via paracellular routes between conjunctival cells, resulting in a net secretion of NaCl with water following passively from the basolateral to the apical side to maintain osmotic equilibrium [4]. The permeability of the air-interfaced rabbit conjunctival epithelial cell layers to hydrophilic solutes was dependent on molecular size, where the molecular weight cut-off of the cell layers was about 20 000 Daltons (Fig. 4). Therefore, hydrophilic solutes near and above the threshold of 20 000 Daltons may experience severely restricted diffusion when considered for topical conjunctival drug delivery. Molecules larger than FD40 may have to rely on fluidphase and other types of endocytosis for transport [5]. The relationship shown in Fig. 4 is consistent with a single population of equivalent pores 8.0 nm in radius at a density of 5310 7 pores per cm 2 , as compared with a 5.5 nm pore size at a density of 1.9310 8 pores per cm 2 in the excised conjunctiva [5]. The slightly larger pore size in the cultured epithelial cells may partly explain the 2-fold larger Papp seen in FD20 and FD70 when compared with the excised tissue, although a more active endocytotic process in the cultured epithelial cells cannot be ruled out. In the case of b-blockers, the Papp of the lipophilic solutes was about 3-fold lower in AIC than in LCC, but in the same range as in the excised tissue (Table 4), indicating that the permeability of AIC in general better reflects that of the excised tissue than LCC. However, the hydrophilic b-blockers exhibited significantly higher ( p,0.05) permeability in the excised tissue than in AIC and LCC. The reason for this phenomenon is unclear. If sotalol and atenolol permeate the cultured epithelial cells via the paracellular route, their Papp should be in the same range of mannitol, like that in AIC. Since b-blockers are organic cations in physiological pH, this difference could be caused by the participation of other transport processes such as organic cation transporters (OCT), which may exist in the tissue [23] but less might be expressed in the cultured epithelial cells. Since the lipophilic beta-blockers can go through cells by simple passive diffusion, the active transport component in their overall permeability may be minimal. By contrast, for the hydrophilic com-

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pounds, their transcellular permeability could be significantly increased by OCT. Whether OCT plays a role in the conjunctival drug absorption remains to be explored [23]. In any event, the influence of drug lipophilicity on drug transport across AIC cell layers is described by a sigmoidal relationship (Fig. 5). An effective half-maximal Papp is seen at a log P value of 1.2, as compared with 1.3 for LCC and 1.0 for the excised tissue. As in the intact tissue [6,8], the AIC epithelial cells do possess the capacity for active nucleoside and dipeptide transport, as suggested by the directionality- and temperature-dependent transport / uptake of their corresponding substrates (uridine and L-carnosine) (Figs. 6 and 7). Thus, uridine transport at 10 mM in AIC was 49-times higher in the apicalto-basolateral than in the opposite direction (Fig. 6), and was reduced 81 times upon lowering the temperature to 48C, suggesting the possible existence of nucleoside transporters in AIC which was confirmed in the intact tissue [6]. L-Carnosine uptake also showed apical preference, being 4.4-times higher than that from the basolateral side. Moreover, apical uptake was temperature- and pH-dependent. Lowering the temperature to 48C and abolishing the inwardly directed pH gradient reduced apical L-carnosine uptake by factors of 5 and 2 from the corresponding control. Furthermore, uptake was inhibited 73% and 60% by 10 mM glycyl sarcosine (a dipeptide substrate) and 1 mM L-valacyclovir (an amino acid ester prodrug [24,25]), respectively, suggesting the possible presence of H 1 -coupled dipeptide transporters in AIC, as confirmed in the intact tissue [8] (Fig. 7). In conclusion, air-interfaced primary cultures of rabbit conjunctival epithelial cells display ion and drug transport characteristics similar to those of the excised tissue. It may prove to be a suitable model for rapidly screening the transport properties of topical ophthalmic drug candidates as well as formulation factors influencing their transport, a future challenge in topical ocular drug delivery.

Acknowledgements This work was supported in part by National

Institutes of Health Research Grants EY10421 (VHLL), HL38658 (KJK), and HL46943 (KJK).

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