Permeability Characteristics of Novel Mydriatic Agents Using an in Vitro Cell Culture Model That Utilizes Sirc Rabbit Corneal Cells VENKAT R. GOSKONDA, † MANSOOR A. KHAN, † CHRISTINE M. HUTAK, ‡ AND INDRA K. REDDY* ,† Contribution from Division of Basic Pharmaceutical Sciences, School of Pharmacy, Northeast Louisiana University, Monroe, Louisiana 71209, and New York College of Osteopathic Medicine of New York Institute of Technology, Department of Pharmacology, Toxicology, and Experimental Therapeutics, Old Westbury, New York 11568. Received September 2, 1998. Accepted for publication November 19, 1998. Abstract 0 The purpose of this study was to evaluate the permeability characteristics of a previously reported in vitro corneal model that utilizes SIRC rabbbit corneal cells and to investigate the permeability of three novel esters of phenylephrone chemical delivery systems (CDS) under different pH conditions using this in vitro model. The SIRC rabbit corneal cell line was grown on transwell polycarbonate membranes, and the barrier properties were assessed by measuring transepithelial electrical resistance (TEER) using a voltohmmeter. The permeabilities of esters of phenylephrone CDS across the SIRC cell layers were measured over a pH range 4.0-7.4. The esters tested include phenylacetyl (1), isovaleryl (2), and pivalyl (3). The SIRC rabbit corneal cell line, when grown on permeable filters, formed tight monolayers of high electrical resistance with TEER values increasing from 71.6 ± 20.8 Ω‚cm 2 at day 3 in culture to 2233.42 ± 15.2 Ω‚cm 2 at day 8 in culture and remained constant through day 14 in culture. The transepithelial permeability coefficients (P app ) at pH 7.4 ranged from 0.58 × 10 -6 cm/s for the hydrophilic marker, mannitol, to 43.5 × 10 -6 cm/s for the most lipophilic molecule, testosterone. The P app at pH 7.4 for phenylephrine was 4.21 × 10 -6 cm/s. The P app values and the lag times of the three esters of phenylephrone were pH dependent. The P app for 1, 2, and 3 at pH 7.4 were 14.76 × 10 -6 , 13.19 × 10 -6 , and 12.86 × 10 -6 cm/s, respectively and the permeabilities decreased at conditions below pH 7.4. The lag times at pH 7.4 were 0.10, 0.17, and 0.12 h for 1, 2, and 3, respectively, and the values increased at lower pH conditions. The TEER values of SIRC cell line observed at day 8 to day 14 in the present investigation are similar to the resistance value reported for rabbit cornea (2 kΩ‚cm 2 ). All the esters showed significantly (p < 0.05) higher permeabilities than phenylephrine at pH 7.4. The rate and extent of transport of the drugs across the cell layers were influenced by the fraction of ionized and un-ionized species and the intrinsic partition coefficient of the drug. The results indicate that the permeability of ophthalmic drugs through ocular membranes may be predicted by measuring the permeability through the new in vitro cell culture model. Introduction Phenylephrine hydrochloride (HCl) is an R-adrenergic agonist and is commonly used in routine ophthalmic practice as a mydriatic and vasoconstrictor agent. Several cases of adverse systemic reactions after topical ocular application of phenylephrine HCl have been reported and include severe hypertension, subarachnoid hemorrhage, ventricular arrhythmia, and possible myocardial infarc- tion. 1-6 Therefore, it would be most desirable to design a drug that could be delivered to the eye compartments with the least possible systemic absorption and/or no systemic side effects. Previous reports have shown that, after topical application to the eye, esters of adrenalone but not adrena- lone itself can be converted via a reduction-hydrolysis sequence to deliver adrenaline (epinephrine) only at the iris-ciliary body, the site of action. 7,8 This suggested that lipophilic ketones can be reduced in the iris-ciliary body. Accordingly, phenylephrone chemical delivery systems (CDS) (Figure 1) were designed to release the active species phenylephrine, by a “reductive-hydrolytic activation” mech- anism, selectively to iris-ciliary body, thus avoiding the various systemic side effects. We recently reported the physicochemical properties of the novel compounds as a part of preformulation study. 9 Investigation of the absorption properties of a new drug moiety is also an important part in the preformulation process. The rate and extent of intraocular absorption and therapeutic effectiveness of topically applied drugs are dependent on the transport characteristics of ocular mem- branes, especially the cornea. 10-12 Characterization of the ocular penetration of drugs has been performed using hard- to-obtain ocular membranes from many animals. The establishment of a predictive method for the drug perme- ation using a cell culture model would be useful. In vitro cell culture models are of potential utility for some screen- ing studies in which large quantities of corneas are needed. The SIRC rabbit cell line has been used by many research- ers as a cellular model in studies of corneal physiology, immunology, and toxicology. 13-17 Hutak et al. recently developed an in vitro model for corneal permeability and reported that a single inoculation of SIRC rabbit corneal cells resulted in the formation of multiple epithelioid cell layers, with the number of layers increasing with culture time. 18 The objectives of the present study are to (a) evaluate the permeability characteristics of the SIRC rabbit corneal cell line system for potential use as an in vitro model for assessing drug permeability and (b) characterize the transport characteristics of three novel esters of the phenylephrone CDS using this model at varying pH condi- tions. Materials and Methods The SIRC cell line was obtained from American Type Culture Collection (ATCC no. CCL60; Rockville, MD). Earle’s balanced salt solution (EBSS) without sodium bicarbonate, bovine calf serum, * Corresponding author: Tel: (318) 342-1709. Fax: (318) 342-1606. E-mail: pyreddy@alpha.nlu.edu. Figure 1sStructures of phenylephrone CDS. 10.1021/js980362t CCC: $18.00 180 / Journal of Pharmaceutical Sciences © 1999, American Chemical Society and Vol. 88, No. 2, February 1999 American Pharmaceutical Association Published on Web 01/12/1999