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Transport of Acyclovir Ester Prodrugs Through Rabbit Cornea and SIRC‐Rabbit Corneal Epithelial Cell Line
Transport of Acyclovir Ester Prodrugs Through Rabbit Cornea and SIRC-Rabbit Corneal Epithelial Cell Line RAHUL V. TAK, DHANANJAY PAL, HONGWU GAO, SURAJIT DEY, ASHIM K. MITRA Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri±Kansas City, 5005 Rockhill Road, Kansas City, Missouri 64110 Received 5 May 2000; revised 6 March 2001; accepted 16 March 2001
ABSTRACT: The purpose of this study is to assess the permeability of acyclovir (ACV) prodrugs through the rabbit corneal cell line (SIRC) as well as the cornea, and characterize the SIRC cell line for transport and metabolism studies of ester prodrugs. Prodrug derivatization of an acycloguanosine antiviral agent, acyclovir, was employed to improve its permeability across the cornea. New Zealand albino rabbits were used as an animal model for corneal studies. The SIRC cell line grown on polyester membranes was used for transport of these prodrugs. SIRC cells grown on the membrane support for 10 days developed four to six layers of epithelial cells, and this is comparable to the normal rabbit corneal epithelial layer. Transport experiments were conducted across the rabbit cornea and con¯uent SIRC cells using side-by-side diffusion-cell apparatus. Enzymatic hydrolysis of these compounds was evaluated in SIRC cell lysates. Appropriate reversed phase HPLC method(s) were employed for quantitation of both the prodrug and ACV simultaneously. Corneal permeabilities of some of these prodrugs (Malonyl ACV and Acetyl ACV) were higher relative to ACV. The SIRC cell line permeability values of all the prodrugs were higher compared to that of the intact cornea. The total amount of ACV-prodrugs transported, i.e., unhydrolyzed prodrugs and regenerated ACV, across the SIRC cell line was more relative to ACV. Hydrolytic studies in the SIRC cell line homogenate demonstrated the bioreversion potential of the prodrugs and the presence of enzymes, particularly the cholinesterase in the SIRC cell line. It may be concluded that the SIRC cell line is leakier compared to the cornea. Keeping in mind the limitations, the SIRC cell line after further characterization may be used for transport and metabolism studies of ester prodrugs. ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1505±1515, 2001
Keywords:
rabbit cornea; SIRC; ACV; prodrugs; permeability
INTRODUCTION Herpes simplex keratitis is the leading infectious cause of blindness in the United States.1 Ocular Herpes Simplex Virus (HSV-1) infections are increasing in individuals whose immune system is compromised. Approximately, 12% of human immunode®ciency virus (HIV) infected patients present with opportunistic infection and herpes Correspondence to: A. K. Mitra (Telephone: 816-235-1615; Fax: 816-235-5190; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 90, 1505±1515 (2001) ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association
simplex virus is among the most common pathogens to affect them.2 Primary herpetic keratitis is self-limiting, and leaves no scarring. HSV-1 can remain latent in the trigeminal ganglion, and shed periodically, producing clinical manifestations of secondary herpetic keratitis. The treatment of ocular HSV-1 infection is a major concern of ophthalmologists. ACVÐa synthetic purine nucleoside analog of guanine, is clinically used in the treatment of herpes simplex virus infections because of its af®nity for the viral thymidine kinase.3 However, the treatment of these infections has become problematic because of the inability of the active
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drug to achieve desirable concentrations in ocular tissues requiring frequent administrations of a topical dose. ACV has low ocular bioavailability because of its hydrophilic nature and rapid precorneal drainage, upon topical administration, relative to its transcorneal ¯ux. A lipophilic prodrug approach can be utilized to overcome the low penetration of ACV, thereby achieving optimum therapeutic concentrations in the cornea as well as the internal ocular tissues. Hughes and Mitra4 showed that a series of aliphatic 20 -esters of ACV signi®cantly increased the corneal permeability of the ACV. In this decade signi®cant efforts have been dedicated to establish cell culture as an ef®cient screening tool to assess the transport and metabolism processes of compounds. A number of cell lines have been characterized, and are used routinely in pharmaceutical research and development.5 Development of a cell line model to assess the corneal transport and metabolism characteristics of compounds would be desirable. Such a model will also be economical, as it will minimize the use of animal tissues. The SIRCrabbit corneal epithelial cell line has been used for in vitro studies of corneal physiology, immunology, and toxicology.6±10 SIRC cells form multiple elongated epithelial-like cell layers. Hutak et al.11 established a growth pattern of SIRC in microwell inserts. SIRC cells grown for 10 days on the surface of microwell inserts showed four to six layers of cells. The number of cell layers remained stable of average four-cell layers up to 14 days. This number gradually decreased during 21 days of culture. There was a decrease in the nuclear-tocytoplasmic ratioÐa sign of aging and an exfoliation of surface cells was also evident. Very recently Goskonda et al.12 studied the permeability characteristics of novel mydriatic agents in SIRC rabbit corneal cells. However, the SIRC cell line has not been characterized yet to assess permeability of a wide range of compounds. The objectives of this work are to assess the permeability of ACV prodrugs through the rabbit cornea as well as the SIRC corneal epithelial cell line, and to evaluate the suitability of the SIRC cell line for transport and metabolism of ester prodrugs.
Triangle Park, NC). Heptanesulfonic acid, acid anhydrides, and 4-dimethylaminopyridine (DMAP) were obtained from Aldrich Chemical Company (Milwaukee, WI), 1-octanol and dimethyl formamide (DMF) were provided by Fisher Scienti®c Company (Fairlawn, NJ). Other reagents were of analytical grade, and were used as received. Distilled, deionized water was used in the preparation of all buffers and mobile phases. The SIRC cell line was obtained from American Type Culture Collection (ATCC). Ketamine hydrochloride was obtained from the animal lab, University of Missouri±Kansas City. Sodium pentobarbital was obtained from the school of Pharmacy, UMKC stock, and used under supervision. Methods Synthesis The simple alkyl esters of [9-(2-hydroxyethoxymethyl) guanine] (ACV) were prepared in a single step reaction, using a modi®ed procedure of Shao et al.13 A solution of ACV in dimethyl formamide was treated with acid anhydride in the presence of catalytic amounts of DMAP (Figure 1). Diacyl derivatives of ACV were observed to be the major product using this literature protocol. Reducing the acid anhydride amounts (from 10 to 3 equivalents) and reaction time (from 2±3 days to 1.5 days) produced more satisfactory results. ACV pivalate is an exception because of the steric hindrance effect of the t-butyl group. The diacetyl derivatives were separated as by-products in about 4 to 12% yields. Valcyclovir, 2-[(2-Amino-1, 6-dihydro-6-oxo9H-purin-9-yl) methoxy]ethyl L-valinate hydro-
EXPERIMENTAL SECTION Materials ACV, [9-(2-hydroxyethoxymethyl)guanine] was a gift from Burroughs Wellcome Co. (Research
Figure 1. Scheme for the synthesis of ester prodrugs of ACV.
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Phosphate-Buffer Saline (DPBS) pH 7.4 for transport studies with SIRC cell line. Analytical Procedure Figure 2. Scheme for synthesis of L-valyl ester of ACV.
chloride, was synthesized by Beauchamp et al.14 method (Figure 2). Step I: Acyclovir (2.0 g, 8.88 mM) was dissolved in dry DMF (150 mL) by warming on a steam bath. Successively, DMAP (0.154 g, 1.25 mM), N-cbz-L-valine (3.01 g, 12.0 mM), and DCC (3.0 g, 14.4 mM) were added to the cooled solution. The solution was stirred under nitrogen atmosphere at ambient temperature for 18 h. The mixture was recharged with same amounts of DMAP, N-cbz-L-valine, and DCC, and stirring was continued at ambient temperature for 2 days. The mixture was ®ltered and the DMF was removed by treating the mixture with water and ethyl acetate under acidic conditions. The intermediate partitions into the ethyl acetate layer and the DMF is miscible in the water layer. The ethyl acetate fraction was separated and rotavaped. The residue was chromatographed on silica gel, using 1:4 methanol (MeOH), CH2Cl2 as the eluant, to generate 3.5 g of the desired intermediate, 2-[(2-amino-1, 6-dihydro-6-oxo-9H-purin-9-yl) methoxy] ethyl N-CbzL-valinate, as a white solid. The 1H spectra was consistent with the desired structure. Step II: solution of the intermediate in MeOH (130 mL), THF (tetrahydrofuran, 65 mL), and H2O (25 mL) was added to 0.5 N aqueous HCl (18 mL) and 377 mg of 5% palladium on charcoal. The mixture was shaken in a Parr apparatus under an initial pressure of 50-psi hydrogen at ambient temperature for 18 h. The mixture was ®ltered, the catalyst was washed with MeOH, and the combined washings and ®ltrate were evaporated in vacuo at a bath (< 608C) temperature. The 1HNMR spectra was satisfactory for the desired structure. Preparation of Drug Solutions A fresh, 1±2 mM aqueous solution of the ester prodrugs (butyryl-ACV, iso-butyryl-ACV, valylACV, 1 mM; malonyl-ACV, acetyl-ACV, 2 mM; pivaloyl-ACV, 0.5 mM) was prepared in isotonic phosphate buffer pH 7.4 for corneal transport studies. A 1-mM aqueous solution of the ester prodrugs was prepared in Dulbecco's Modi®ed
A high-pressure liquid chromatographic (HPLC) method was developed for the analyses of ACV and its monoesters (Table 1). The system comprised of a Waters 515 HPLC pump, a Rheodyne TM injector, and a Waters 486 tunable absorbance detector. For the esters of acyclovir, a dualcolumn isocratic HPLC method was used. A 250 4.6 mm i.d. reversed phase C18, 5 mm particles, connected in series with a cation exchange column, Partisil 10 SCX, 5 mm, 250 4.6 mm i.d. was employed. A mobile phase consisting of 0.005 M ammonium phosphate buffer pH 2.2 (60%) and organic phase (40%) comprising of methanol was employed. In case of ACV±acetate and ACV±malonate, a 250 4.6 mm i.d. reversedTM Ê (Varian) conphase C18 Microsorb-MV 100 A nected to Beckman System Gold1 128 solvent module with ¯uorescence detection (Schoeffel Instr.-Corp). A mobile phase comprising of ammonium phosphate 0.0025 M pH 2.2 (90%) and an organic phase (10%) comprising of MeOH and acetonitrile (1:1) was used. For guanosine and guanosine monophosphate, a similar reversephase C18 column connected to a 515 Waters pump with a UV detection was used. A mobile phase consisting of ammonium phosphate 0.002M pH 5.6, and a varying proportion of methanol was used. This method was capable of detecting guanosine, guanosine monophosphate, guanosine diphosphate, and guanosine triphosphate in a single run. Data acquisition and chromatographic analysis was carried out using a Hewlett Packard integrator (HP 3396 series III). For all analyses, a ¯ow rate of 1 mL/min was maintained. Transport Through Cornea A typical side-by-side diffusion apparatus (typeVSC-1, Crown Glass Company Inc.) was used for performing transport experiments. Cornea obtained from New Zealand albino rabbits weighing 2.0 to 3.0 kg was used for transport study. The animals were euthanized by an overdose of pentobarbital through a marginal ear vein. Eyes were proptosed and carefully enucleated, and washed with ice-cold isotonic phosphate buffer (IPB) pH 7.4 to remove any traces of blood. After a small incision to the sclera, vitreous humor was aspirated by using a 1-mL. syringe. Cornea was carefully excised, leaving some scleral portion JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 10, OCTOBER 2001
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Table 1. HPLC Conditions for Ester Prodrugs of ACV, Guanosine, and GMP Compounds Butyryl-ACV, isobutyryl-ACV, pivaloyl-ACV Acetyl-ACV, malonyl-ACV L Valyl-ACV Guanosine GMP
Mobile Phase
Detection
Ammonium phosphate (0.005 M, pH 2.5): methanol 60:40 Ammonium phosphate (0.0025 M, pH 2.2): methanol: acetonitrile 90:5:5 Ammonium phosphate (0.0025 M, pH 2.2) Ammonium phosphate (0.002 M, pH 5.6): methanol 95:5 Ammonium phosphate (0.002 M, pH 5.6)
attached to the cornea. The scleral part attached to the cornea helps to hold the cornea in place in between the half-cells during the transport experiment. The lens was removed, and the irisciliary body was separated from the cornea. The cornea was washed immediately with ice-cold IPB pH 7.4 and mounted on a side-by-side diffusion half-chamber. The temperature was maintained at 348C by circulating water through the jacketed chambers of the diffusion apparatus. Aqueous drug solutions (3 mL) were added on the epithelial side of the cornea (donor chamber). In the other half-chamber (receiver chamber), 3.4 mL of IPB pH 7.4 was added. Both the chambers were stirred continuously by using magnetic stirrer bars. Receiver chamber volume of IPB added was little more than that of the donor chamber to maintain the curvature of the cornea throughout the experiment. Sink conditions were maintained during the experiment. One hundred microliter aliquots were removed from the receiver chamber at appropriate intervals, and were replaced with an equal volume of IPB pH 7.4. The samples were either immediately analyzed by HPLC or stored at ÿ808C until further analysis. All experiments were done in triplicate. Transport Through SIRC Rabbit Corneal Epithelial Cell Line The SIRC cells grown on a polyester membrane support (Whatman) were used for transport studies. The rabbit corneal epithelial cell line (SIRC) was received from the American Type Culture Collection1 (ATCC). The cells were cultured according to the guidelines by ATCC with Minimum Essential Medium (pH 7.4) supplemented with 10% calf serum, lactalbumin (1.76 mg/mL), HEPES (1.3 mg/mL), and Penicillin-streptomycin
UV lmax 249 nm Fluorescence lex 249 nm lem 370 nm Fluorescence lex 249 nm lem 370 nm UV lmax 254 nm UV lmax 254 nm
(100 mg/mL). The cells were incubated at 378C in humidi®ed atmosphere of 5% CO2/95% air. The cells were plated at a density of 33,000 cells/cm2 either on tissue culture-treated plastic dishes or collagen±®bronectin-coated polyester clear membrane (pore size 0.4 mm). The cells used for the transport study were grown for 10 days, and the medium was changed every alternate day. Before the transport study the cells were rinsed with Dulbecco's modi®ed phosphate-buffer saline (DPBS) containing 1 mM CaCl2, 0.74 mM MgSO4, 5.3-mM glucose, and pH 7.4. The cells grown on the membrane support were mounted on side-byside diffusion chambers, and the drug solution was added on the apical side of the membrane (donor chamber). DPBS was added to the other half-chamber (receiver chamber). One hundred microliters of aliquots were removed from the receiver chamber at predetermined time points and analyzed by HPLC. The receiver was replaced with equal volume of buffer after removing the sample. Transport of the hydrophilic marker 14C mannitol was carried out in triplicate for each compound used for the transport study to assess the integrity of cells during the length of the experiments. A similar sampling procedure was used for 14C mannitol transport studies. The samples were analyzed by a scintillation counter (Beckman, LS 6500). The temperature was maintained at 348C through circulated water from a water bath, and the solutions were stirred by using magnetic stirrer bars throughout the length of the experiment. Data was obtained by performing experiments in triplicate or quadruplicate. Transmission Electron Microscopy Transmission electron microscopy (TEM) was used to determine the number of layers in the
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SIRC cell line. The cells grown for 10 days were washed two times with DPBS at 48C. The cells were then washed with cacodylate buffer (48C) for 5 min. Then the cells were ®xed with 2% glutaraldehyde in 0.15 M Na-cacodylate buffer for 60 min. The cells were washed several times with cacodylate buffer and post®xed with osmium tetraoxide for 30 min. Then the cells were processed for thin sectioning using a routine TEM procedure and photographed using a JEOL Electron Microscope (Model JEM 1200 EX II) at 100 kV. Preparation of Ocular Tissues for Enzymatic Hydrolysis Studies New Zealand albino rabbits were used as an animal model for metabolism studies. Animals were euthanized, and eyes were enucleated as described earlier. The eyes were washed in icecold IPB pH 7.4 to remove any traces of blood. Aqueous humor was removed by inserting a 27-1/ 2 gauge needle through the sclera into the anterior chamber. Vitreous humor was collected by incision in the sclera. Part of the sclera was removed to expose the lens. The lens was separated followed by the iris-ciliary body. The cornea was cut carefully to leave no traces of scleral tissue attached to it. All the tissues were immediately washed with ice-cold IPB pH 7.4 and stored in ice. The procedure takes approximately 10±15 min. The tissues were homogenized immediately using a homogenizer (Tissue Tearer Model 985-370 Type 2, Biospace Products, Inc.) in 2 mL ice-cold IPB pH 7.4 and the supernate was stored at ÿ808C for further studies. The homogenate was centrifuged at 50,000 rpm for 30 min using an ultracentrifuge (Beckman TL-100). The supernate obtained was used for hydrolysis studies. Reactions were initiated by adding 100±200 mL (10±50 mg/mL) of drug solution made in IPB pH 7.4 to 1.0 to 1.2 mL of supernate. The reactions were carried out in a water bath at 348C. Samples were taken at appropriate intervals and analyzed by HPLC. Equal volume of methanol was added to stop the reaction. Experiments were done in triplicate. Protein concentration was estimated by using BioRad1 assay method for protein estimation. Preparation of SIRC Cell Homogenate for Enzymatic Hydrolysis Studies The SIRC cells were grown in a 75-cm2 ¯ask for 10 days, the medium was removed, and the cells
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were washed three times with DPBS (pH 7.4). Ten milliliters of buffer was added to a ¯ask and the cells were scraped with the help of a rubber policeman (cell scraper). The cell suspension was centrifuged at 1500 rpm for 10 min. The pellet was resuspended in 2 mL ice cold DPBS, and homogenized by a tissue homogenizer (Tissue Tearer Model 985-370 Type 2, Biospace Products, Inc.) for 1 min. The homogenate was centrifuged (Beckman TL-100 Ultracentrifuge) at 10,000rpm, at 48C for 30 min. The supernate obtained after centrifugation was the cytosol fraction and used for hydrolysis studies. Protein content of the cytosol was estimated by BioRad1 Protein Assay method. Hydrolysis experiments were carried out as mentioned before. All experiments were carried out in triplicate. Cholinesterase Assay in SIRC Cell Homogenate Calibration was done according to the method given in Sigma Diagnostics procedure1 No. 420 (catalog No. 420-MC) cholinesterase kit. Cholinesterase catalyzes the hydrolysis of cholinesters of various short-chain organic acids, including acetylcholine, which is the substrate used in this Sigma Diagnostic procedure. The reaction was conducted in the presence of an acid-base indicator, such as m-nitrophenol. The acetic acid produced lowers the pH, causing a loss of color. This color change is proportional to the cholinesterase activity present. A serum ``blank'' was prepared by inactivation at 608C for 10 min to compensate for background absorbance contributed by the sample. Absorbance of both ``blank'' and ``test'' was read at 420 nm, and the ``difference'' was used to estimate the cholinesterase level. Calf serum was used instead of pooled serum. Sodium chloride solution (0.2 mL) and SIRC cell homogenate were taken in two separate tubes and then mixed. One tube (blank) was placed in a 608C water bath for 10 min to inactivate the enzymes. The tube was heated thoroughly to ensure that all enzymes have been inactivated. It was removed and cooled in tap water. To both ``blank'' and ``test'' tubes, 3.0 mL water, 2.0 mL nitrophenol solution, and 0.2 mL acetylcholine chloride solution was added. All these reagents were brought to room temperature before making the additions. Exact time of addition of acetylcholine chloride solution was recorded, and both the ``blank'' and ``test'' tubes were placed in water bath at 258C. Exactly 30 min later, absorbance (A) was recorded by using a Beckman UV at 420 nm and water as a reference. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 10, OCTOBER 2001
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Calculations (DA DBLANK ÿ DTEST) Using DA, the cholinesterase activity of the SIRC homogenate was determined in Rappaport Units from the calibration curve. One Rappaport unit is the amount of cholinesterase that liberates 1 mm of acetic acid from acetylcholine in 30 min at 258C at pH 7.4 under test conditions. Data Analysis Flux values for ACV and ester prodrugs were calculated by dividing the slope (obtained by plotting a cumulative amount of the compound permeated versus time) with the area available for diffusion, i.e., 0.636 cm2. The permeability values were calculated by dividing the ¯ux values with the concentration of the prodrug used in the donor. Statistical tests were performed to compare the differences between the corneal and the SIRC permeability values using one-way ANOVA. A p-value less than 0.05 was considered as signi®cant. Apparent ®rst-order rate constants for enzymatic hydrolysis was calculated by linear regression of the line obtained by plotting log percent remaining versus time pro®les. Triplicate samples were analyzed, and a mean rate constant was calculated. The rate constants were expressed as per milligram of protein.
RESULTS AND DISCUSSION Electron Microscopy The TEM photomicrograph depicts six layers of SIRC cells (Figure 3). This observation is in agreement with Hutak et al.11 that the SIRC cells grown on the membrane support for 10 days developed four to six layer of cells and comparable to the normal corneal epithelial layer. The TEM photomicrograph shows an elongated epithelial structure and has a ®broblast like appearance. There are no microvilli and microplicae on the surface. There is a sparse distribution of the intercellular space and the cells appear to be fairly tight. However, the cellular tight-junction components such as desmosomes are lacking. Our TEM results of SIRC cells are similar with the previous reports that SIRC cells lack desmosomes, cytoplasmic ®laments, and cytokeratinÐ the structures that are characteristic of corneal epithelial cells.6 The SIRC cells grown on six-well Transwell1 ®lters did not develop enough transe-
Figure 3. Transmission electron microscopic photomicrograph of SIRC cells exhibiting six layers after 10 days of culture.
lectrical epithelial resistance (TEER value < 100 O cm2) indicative of the leaky nature of the cells. During the 10 days of our culture, no exfoliation of SIRC cells was observed. These studies also con®rmed that the SIRC cell line grown for 10 days differentiated to a multicell layer epithelial membrane. Enzymatic Hydrolysis in Ocular Tissues and SIRC Cell Line Homogenate The apparent ®rst-order rate constants for the hydrolysis of the ACV esters in SIRC cells have been reported in Table 5. Loss of ACV ester was accompanied by the formation of ACV. A mass balance with respect to total ACV concentration was maintained throughout the experiment. Controls with no homogenate and containing only the DPBS (pH 7.4) and the respective esters showed no quanti®able degradation over the time course of the study. Cytosolic fraction of the SIRC cell line was used for the hydrolysis studies. The amount of cholinesterase in the SIRC cell line homogenate was found to be 38 Rappaport Units/ mL. The esterase activity in the epithelium of the
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rabbit cornea is higher than that of stroma± endothelium, and this esterase activity varies with rabbits's age and strain.15 The hydrolysis of ACV prodrugs and formation of ACV demonstrated the presence of esterase activity. The hydrolytic rate constant for butyryl± ACV in SIRC homogenate studies was found to be the highest ( p < 0.05). The concentration versus time pro®le for isobutyryl±ACV is shown in Figure 4. Lee et al.16 demonstrated that there exists a chain length at which a prodrug would be hydrolyzed equally well by both cholinesterases. This could be attributed to the higher hydrolytic rate for the butyryl±ACV. A lipophilic pocket at the active site of the ocular esterase has been suggested.17 The isobutyryl and pivaloyl esters showed low enzymatic hydrolytic rate constants in the SIRC cell homogenate, most likely due to a steric hindrance at the enzyme active site. The hydrolysis studies of acetyl ester in SIRC cell line homogenate also showed regeneration of ACV (Figure 5). The SIRC cell line enzymatic data con®rms the presence of esterase activity and thereby bioreversibility of the ACV prodrugs. The apparent ®rst-order rate constants for acetyl± ACV and malonyl±ACV in ocular tissue homogenates is illustrated in Table 6. The rate of hydrolysis of acetyl±ACV in ocular tissues was highest in the iris±ciliary body, followed by in the cornea and in the aqueous humor ( p < 0.05). For malonyl ester, the highest rate constant was also found in the iris±ciliary body ( p < 0.05). The amount of esterases present in the iris±ciliary body was signi®cantly higher (almost twice) than in the cornea. Transport of Acyclovir Esters Across SIRC Cell Line and Rabbit Cornea Corneal and SIRC cell line permeability values for the prodrugs and ACV are reported in Table 2. Integrity of the SIRC cell line was determined by
Figure 4. Concentration versus time pro®le for isobutyryl±ACV in the SIRC cell lysate.
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Figure 5. Percent log versus time pro®le for acetyl± ACV in the SIRC cell lysate.
the transport of a paracellular marker 14C mannitol for each set of experiments. All experiments were conducted in triplicate, with 14C mannitol throughout the length of the experiments for each prodrug used. Mannitol transport was found to be between 2±4% per hour. Permeability values for mannitol for each prodrug are summarized in Table 3. To monitor transcellular permeability, transport of 14C diazepum was conducted across SIRC and the freshly excised cornea (Figure 7). The permeability of 14C diazepum across SIRC was more than double (8.37 10ÿ5 cm/s) compared to the cornea (3.43 10ÿ5 cm/s). All the ACV prodrugs, except ACV±acetate and ACV±malonate, were partially hydrolyzed to ACV during transport across the SIRC cell layers. Enzymatic activity of the SIRC cell line is evident from the regeneration of the parent compound ACV during transport (Figure 6). Permeability values for the prodrugs through SIRC were found to be higher than that of across the intact cornea ( p < 0.05). From the electron microscopic data it was observed that SIRC cells grown for 10 days developed four to six cell layers thick. Normal rabbit corneal tissue consists of four to seven layers of epithelial cells, followed by stroma and endothelium. The top two super®cial layers offer the primary resistance to transport of drugs. Although SIRC cells grown for 10 days were four to six layers thick, but they did not produce enough resistance, having a poor TEER value (< 100 O cm2). The TEER value for rabbit cornea is > 1500 O cm2. Thus, SIRC cell layers are leakier than the cornea, and hence, the higher permeability values result for the ACV prodrugs. The amounts of ACV generated during transport across the SIRC cell line are reported in Table 4. Hughes and Mitra18 reported 50% hydrolysis of isobutyryl±ACV and about 80% hydrolysis of other esters in the homologous series for transport experiment in the cornea. However, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 10, OCTOBER 2001
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Table 2. Permeability Values of ACV Prodrugs, Guanosine, and GMP in Cornea and SIRC Cells Prodrugs ACV Malonyl-ACV Acetyl-ACV Butyryl-ACV Isobutyryl-ACV Pivaloyl-ACV L-Valyl-ACV Guanosine GMP
Cornea P cm/s
SIRC P cm/s
ÿ6 a
Ratio: SIRC/Cornea ÿ5
3.65 10 2.33 0.42 10ÿ5 1.83 1.2 10ÿ5 5.12 10ÿ6 a 3.92 10ÿ6 a n/d 5.8 0.95 10ÿ6 7.95 0.21 10ÿ6 2.36 0.14 10ÿ6
2.6 0.34 10 4.0 1.4 10ÿ5 2.5 0.21 10ÿ5 1.25 0.11 10ÿ5 1.36 0.12 10ÿ5 1.67 0.10 10ÿ5 1.20 0.33 10ÿ5 9.1 0.24 10ÿ6 5.06 0.13 10ÿ6
7.12 1.72 1.37 2.44 3.47 2.07 1.14 2.14
a
Data obtained from ref. 18. n/d not detected.
b
in SIRC transport studies, isobutyryl±ACV ester hydrolyzed to about 5%. Other ACV esters except acetyl±ACV and malonyl±ACV were hydrolyzed to about 10±13% during transport. In the SIRC homogenate study the apparent ®rst-order rate constant for the hydrolysis of isobutyryl±ACV was also low compared to other ACV esters in the study. Moreover, hydrolysis of acetyl±ACV and malonyl±ACV was also observed in the SIRC cell line homogenate study. The epithelial layer contributes approximately 70% of the enzymatic activity of the cornea. The SIRC cell line consists of only epithelial layers, and these cells might not be expressing the esterases at the same level, which are responsible for hydrolysis of ester prodrugs. Hence, a lower percentage of hydrolysis for the ACV±esters in the SIRC cell line was observed during transport. Higher permeability for acetyl±ACV might be attributed to its inability to undergo hydrolysis during transport across the SIRC cell line. However, the relationship observed by Hughes and Mitra12 for log partition coef®cient and permeability may not be predicted for these compounds. Similarly, a higher permeability value was observed for malonyl±ACV ( p < 0.05). The permeability values for the unhydrolyzed
prodrugs except malonyl±ACV and acetyl±ACV were lower compared to that of ACV across the SIRC cell line ( p < 0.05). These prodrugs were hydrolyzed during transport, and the total amount of ACV transported was more than that of ACV, resulting in increased amounts of ACV transported across the SIRC cell line. It is well known that the optimum log partition coef®cient values for the compounds to diffuse across cornea are between 1 and 2. However, higher log partition coef®cient values would compromise aqueous solubility of prodrugs. Thus, watersoluble, solution stable, L-valyl ester of ACV was synthesized. The permeability value for valacyclovir was comparable to other simple ester prodrugs. Higher corneal permeability values for Malonyl±ACV and Acetyl±ACV were observed compared to corneal permeability of ACV ( p < 0.05). ACV was regenerated from acetyl prodrug in a consistent manner during transport through the cornea. Hydrolysis of the malonyl prodrug was observed during transport across the cornea to a lesser extent and in an inconsistent manner compared to other prodrugs. The permeability value for valacyclovir across the cornea was also more than that of ACV ( p < 0.05). Its mechanism
Table 3. 14C Mannitol Permeability Values Across SIRC Cells for Each Compound Compounds Butyryl, iso-butyryl, pivaloyl ACV Valyl ACV Guanosine GMP
Permeability (cm/s) 10ÿ6 1.12 0.10 2.02 0.50 1.41 0.34 1.20 0.047
Figure 6. Receptor cell concentrations of ACV, total ACV, and the unhydrolyzed butyryl±ACF. (a) Each point is a mean of n 3.
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Table 5. Apparent First-Order Rate Constants for ACV Prodrugs in SIRC Cell Lysate Prodrugs Malonyl ACV Acetyl ACV Butyryl ACV Iso-butyryl ACV Pivaloyl ACV Figure 7. Transport of 14C diazepam (a transcellular marker) across the SIRC cell line and freshly excised rabbit cornea. (a) Each point is a mean of n 4.
of transport across the cornea is not clear, and needs to be studied further in detail. In a recent study Goskonda et al.19 have shown good correlation between permeability coef®cients and lipophilicities of drugs. The ester prodrugs, such as phenylacetyl ester, isovaleryl ester, and pivalyl ester, having higher lipophilicities, showed a fourto sixfold increase in corneal permeability compared to phenylephrine HCl. The permeability of ester prodrugs varied, depending on the pH of the transport medium, because the degree of ionization is dependent on the pH of the medium. Permeability values for transport of guanosine and guanosine monophosphate (GMP) are reported in Table 2. Metabolism of guanosine was found to occur during diffusion through the cornea. These metabolites remain to be identi®ed. However, no metabolism was observed during transport through the SIRC cell line. GMP was used as a model for antiviral nucleoside monophosphate to investigate whether it gets converted to higher phosphates, namely guanosine diphosphate, and guanosine triphosphate, by the cellular kinases during transport through the cornea. These studies show that there was no such conversion of GMP to higher phosphates. However, three metabolites, which are yet to be identi®ed, were found during the transport of Table 4. Amount of ACV Transport Through SIRC Cells Prodrugs Butyryl ACV Iso-butyryl ACV Pivaloyl ACV Valyl ACV
Generated
During
Amount of ACV (mg/mL) 0.823 0.020 0.426 0.049 0.300 0.064 0.410 0.021
K minÿ1/mg of Protein 10ÿ4 2.41 0.5 2.9 0.13 14.4 0.83 1.6 0.13 3.12 0.17
GMP through the cornea. No phosphate metabolites were found during transport through the SIRC cell line. The permeability value for guanosine across the SIRC was higher than that of across the cornea ( p < 0.05). A possible reason might be the lower resistance offered by SIRC epithelial cell layers to diffusion of guanosine. Moreover, absence of hydrolysis might have resulted in higher transport. Guanosine is poorly water soluble (less than 1 mg/mL) at pH 7.4, although it has relatively lower log partition coef®cient. ACV shares some of the physicochemical characteristics of guanosine; hence, it was selected as a model for antiviral nucleosides. Similar results were obtained for GMP. GMP is fairly hydrophilic compared to guanosine, and diffuses through the cornea as well as the SIRC cell line very poorly. The permeability values for GMP were found to be lower compared to that of guanosine ( p < 0.05). This also suggests that the epithelial layer offers resistance to transport of hydrophilic compounds. It can also be concluded that the topmost two layers of cornea present a considerable barrier to permeation of hydrophilic compounds. The SIRC permeability values for guanosine and GMP were 10 and 25% higher than that of guanosine and GMP through the cornea, respectively. Although SIRC expresses cholinesterase, it may not be expressing the wide variety of kinases present in the cornea as no metabolism (phosphorylation) of guanosine and GMP was observed during transport across the SIRC. Characterization of the SIRC cell line for other enzyme systems needs to be performed. The permeability values obtained for these compounds across the SIRC cell line were suf®ciently close to predict their permeabilities across the cornea. However, caution must be exercised while interpreting transport data for compounds, considering the limitations of the SIRC cell line. From the results of in vitro transport and metabolism studies in the cornea and SIRC cells, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 10, OCTOBER 2001
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Table 6. Apparent First-Order Rate Constants for ACV Prodrugs in Ocular Tissue Homogenates ICB Prodrugs Acetyl-ACV Malonyl-ACV
K minÿ1 per mg of Protein
Cornea 4.61 0.16 10ÿ5 4.61 0.14 10ÿ5
some encouraging observations were obtained. However, a better correlation between the corneal and the SIRC permeability values could be obtained by further characterization of the SIRC cell line in detail. Optimizing the growth conditions using additional growth-promoting agents [pituitary extracts, insulin-transferin, epidermal growth factor (EGF)] in the medium, air interface culture,20,21 etc., would obtain a cell line with consistent characteristics and thus minimize the variability in the data. This would also yield four to seven layers with tight junctions with suf®cient TEER value to offer resistance to transport of compounds similar to the cornea. The ACV prodrugs were hydrolyzed to ACV in the cornea and the SIRC cell line. However, hydrolysis of the malonyl and acetyl prodrug was not observed during transport through the SIRC cell line although these prodrugs were hydrolyzed to ACV in the SIRC cell lysates. Further study of the enzyme systems present in the SIRC cell line would enhance the understanding of hydrolysis of these prodrugs. Moreover, qualitative and quantitative characterization of the SIRC cell line for the enzyme systems would facilitate the evaluation of a wide variety of prodrugs. Transport of valacyclovir was increased signi®cantly relative to ACV in the cornea and the SIRC cell line. Its mechanism of transport could be studied in detail by investigating the carrier systems present in the cornea as well as the SIRC cell line, which can be achieved by evaluating the transport of valacyclovir and amino acid prodrugs.
CONCLUSIONS An ideal cell line will have the similar anatomical and physiological characteristics as that of the tissue of interest. Such a cell line will be an ef®cient tool in screening the compounds for assessment of transport characteristics of compounds. Based on our studies, it can be concluded that SIRC cells form multilayers, but are leaky in nature compared to the cornea. Moreover, one of
AQ
9.5 0.20 10ÿ5 1.26 0.11 10ÿ4
0.150 0.08 10ÿ5 1.00 0.02 10ÿ4
the desirable characteristics of a cell line is to express the enzymes present in the original animal tissues. Ester prodrugs must also display the appropriate hydrolysis kinetics by the esterases present in the cell line model similar to that of the corneal tissues obtained from animals. The prodrug must be metabolized to the parent compound in the ocular tissues including the cornea. Based on the experimental data obtained in this study, it is concluded that the SIRC cell line expresses the enzyme cholinesterases, which are responsible for hydrolysis of ester prodrugs. The bioreversion of ACV prodrugs and the esterase determination con®rms the presence of esterases in the SIRC cell line. From the SIRC transport and metabolism data butyryl±ACV may be chosen as the best prodrug. Based on the corneal data for acetyl±ACV, it can be considered as a prodrug with favorable transport and metabolism characteristics in addition to butyryl±ACV. The water-soluble amino acid prodrug valacyclovir can also be considered as a potential prodrug for topical delivery of ACV, although it needs further evaluation. In conclusion, the SIRC cell, upon further characterization, may be used for assessing the permeabilities and metabolism of ester prodrugs, thereby eliminating the need of animal tissues. Further characterization of the SIRC cell line using different growth supplements (pituitary extracts, EGF, insulin-transferin, etc.) and air interface culture is in progress in this laboratory. However, considering the limitations of in vitro cell culture model, caution must be exercised to avoid over interpretation of results.
ACKNOWLEDGMENTS We would like to thank Dr. Paul Brown for his help in Mass Spectrometry, and Greg Williams for electron microcopy. This work was supported by NIH Grants RO1 EY 09171-05 and RO1 EY 10659-04.
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REFERENCES 1. Green MT, Dunkel E. 1985. Herpes simplex virus infections. In: Darell RW, editor. Viral diseases of the eye. Philadelphia: PA: Lea and Febiger. pp 30± 35. 2. Cotran RS, Kumar V, Robbins S. 1994. Diseases of immunity. In: Cotran K, Kumar V, Robbins S, editors. Robins pathologic basis of disease, 5th ed. Saint Louis: W.B. Saunders Co. pp 229. 3. Douglas RG Jr. 1986. Anti-microbial agents. In: Gilman AG, Rall TW, Nies AS, Taylor P, editors. The pharmacological basis of therapeutics, 8th ed. New York: MacMillan. pp 1182. 4. Hughes PM, Krishnamoorthy R, Mitra AK. 1993. Effect of acylation on the ocular disposition of acyclovir. I: Synthesis, physicochemical properties, and antiviral activity of 20 -esters. J Ocul Pharmacol 9:287±297. 5. Audus KL, Bartel RL, Hidalgo IJ, Borchardt RT. 1990. The use of cultural epithelial and endothelial cells for drug transport and metabolism. Pharm Res 7:435±451. 6. Niederkorn JY, Meyer DR, Ubelaker JE, Martin JH. 1990. Ultrastural and immunological characterization of the SIRC corneal cell line. In Vitro Cell Dev Biol 26:923±930. 7. North-Root H, Yackovich F, Demetrulias J, Gacula M, Heinz JE. 1982. Evaluation of an in vitro cell toxicity test using rabbit corneal cells to predict the eye irritation potential of surfactants. Toxicol Lett 14:207±212. 8. Jacaruso RB, Barletta MA, Carson S, Hardig W. 1985. An in vitro method for assessing corneal opaci®cation potential using a rabbit corneal cell line. J Toxicol Cut Ocular Toxicol 4:49±58. 9. Korbmacher C, Helbig H, Forster C, Wiederholt M. 1988. Characterization of Na/Hexchange in a rabbit corneal epithelial cell line (SIRC). Biochem Biophys Acta 943:405±410. 10. Jacaruso RB, Carson S, Barletta MA. 1986. The use of cell lysis as an index of ocular irritation potential. J Toxicol Cut Ocular Toxicol 5:143±161.
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11. Hutak CM, Barletta MA. 1997. Growth pattern of SIRC rabbit corneal cells in microwell inserts. J Toxicol Cut Ocular Toxicol 16:145±156. 12. Goskonda VR, Khan MA, Hutak CM. 1999. Permeability characteristics of novel mydriatic agents using an in vitro cell culture model that utilizes SIRC rabbit corneal cells. J Pharm Sci 88:180±184. 13. Shao Z, Park GB, Krishnamoorthy R, Mitra AK. 1994. The physicochemical properties, plasma enzymatic hydrolysis, and nasal absorption of acyclovir and its 20 -ester prodrugs. Pharm Res 11:237±242. 14. Beauchamp LMO, de Miranda GF, Burnette P, Krenitsky TA. 1992. Amino acid ester prodrugs of acyclovir. Antiviral Chem Chemother 3:157±164. 15. Lee VHL, Morimoto KW, Stratford RE. 1982. Esterase distribution in the rabbit cornea and its implications in the ocular drug bioavailablity. Biopharmacol Drug Dispos 3:291±300. 16. Lee VH, Robinson JR. 1986. Topical ocular drug delivery: Recent developments and future challenges. J Ocul Pharmacol 2:67±108. 17. Dixon M. 1979. In: Boyer PD, editor. Enzymes 3rd ed. New York: Academic Press. p 45. 18. Hughes PM, Mitra AK. 1993. Effect of acylation on the ocular disposition of acyclovir. II: Corneal permeability and anti-HSV 1 activity of 20 -esters in rabbit epithelial keratitis. J Ocul Pharmacol 9:299±309. 19. Goskonda VR, Hill RA, Khan MA, Reddy IK. 2000. Permeability of chemical delivery systems across rabbit corneal (SIRC) cell line and isolated corneas: A comparative study. Pharm Dev Technol 5:409± 416. 20. Chang J, Basu SK, Lee VHL. 2000. Air±interface condition promotes the formation of tight corneal epithelial cell layer for drug transport studies. Pharm Res 17:670±676. 21. Lentz KA, Hayashi J, Lucisano LJ, Polli JE. 2000. Development of a more rapid, reduced serum culture system for Caco-2 monolayers and application to the biopharmaceutics classi®cation system. Int J Pharm 200:41±51.
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ABSTRACT: The purpose of this study is to assess the permeability of acyclovir (ACV) prodrugs through the rabbit corneal cell line (SIRC) as well as the cornea, and characterize the SIRC cell line for transport and metabolism studies of ester prodrugs. Prodrug derivatization of an acycloguanosine antiviral agent, acyclovir, was employed to improve its permeability across the cornea. New Zealand albino rabbits were used as an animal model for corneal studies. The SIRC cell line grown on polyester membranes was used for transport of these prodrugs. SIRC cells grown on the membrane support for 10 days developed four to six layers of epithelial cells, and this is comparable to the normal rabbit corneal epithelial layer. Transport experiments were conducted across the rabbit cornea and con¯uent SIRC cells using side-by-side diffusion-cell apparatus. Enzymatic hydrolysis of these compounds was evaluated in SIRC cell lysates. Appropriate reversed phase HPLC method(s) were employed for quantitation of both the prodrug and ACV simultaneously. Corneal permeabilities of some of these prodrugs (Malonyl ACV and Acetyl ACV) were higher relative to ACV. The SIRC cell line permeability values of all the prodrugs were higher compared to that of the intact cornea. The total amount of ACV-prodrugs transported, i.e., unhydrolyzed prodrugs and regenerated ACV, across the SIRC cell line was more relative to ACV. Hydrolytic studies in the SIRC cell line homogenate demonstrated the bioreversion potential of the prodrugs and the presence of enzymes, particularly the cholinesterase in the SIRC cell line. It may be concluded that the SIRC cell line is leakier compared to the cornea. Keeping in mind the limitations, the SIRC cell line after further characterization may be used for transport and metabolism studies of ester prodrugs. ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:1505±1515, 2001
Keywords:
rabbit cornea; SIRC; ACV; prodrugs; permeability
INTRODUCTION Herpes simplex keratitis is the leading infectious cause of blindness in the United States.1 Ocular Herpes Simplex Virus (HSV-1) infections are increasing in individuals whose immune system is compromised. Approximately, 12% of human immunode®ciency virus (HIV) infected patients present with opportunistic infection and herpes Correspondence to: A. K. Mitra (Telephone: 816-235-1615; Fax: 816-235-5190; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 90, 1505±1515 (2001) ß 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association
simplex virus is among the most common pathogens to affect them.2 Primary herpetic keratitis is self-limiting, and leaves no scarring. HSV-1 can remain latent in the trigeminal ganglion, and shed periodically, producing clinical manifestations of secondary herpetic keratitis. The treatment of ocular HSV-1 infection is a major concern of ophthalmologists. ACVÐa synthetic purine nucleoside analog of guanine, is clinically used in the treatment of herpes simplex virus infections because of its af®nity for the viral thymidine kinase.3 However, the treatment of these infections has become problematic because of the inability of the active
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drug to achieve desirable concentrations in ocular tissues requiring frequent administrations of a topical dose. ACV has low ocular bioavailability because of its hydrophilic nature and rapid precorneal drainage, upon topical administration, relative to its transcorneal ¯ux. A lipophilic prodrug approach can be utilized to overcome the low penetration of ACV, thereby achieving optimum therapeutic concentrations in the cornea as well as the internal ocular tissues. Hughes and Mitra4 showed that a series of aliphatic 20 -esters of ACV signi®cantly increased the corneal permeability of the ACV. In this decade signi®cant efforts have been dedicated to establish cell culture as an ef®cient screening tool to assess the transport and metabolism processes of compounds. A number of cell lines have been characterized, and are used routinely in pharmaceutical research and development.5 Development of a cell line model to assess the corneal transport and metabolism characteristics of compounds would be desirable. Such a model will also be economical, as it will minimize the use of animal tissues. The SIRCrabbit corneal epithelial cell line has been used for in vitro studies of corneal physiology, immunology, and toxicology.6±10 SIRC cells form multiple elongated epithelial-like cell layers. Hutak et al.11 established a growth pattern of SIRC in microwell inserts. SIRC cells grown for 10 days on the surface of microwell inserts showed four to six layers of cells. The number of cell layers remained stable of average four-cell layers up to 14 days. This number gradually decreased during 21 days of culture. There was a decrease in the nuclear-tocytoplasmic ratioÐa sign of aging and an exfoliation of surface cells was also evident. Very recently Goskonda et al.12 studied the permeability characteristics of novel mydriatic agents in SIRC rabbit corneal cells. However, the SIRC cell line has not been characterized yet to assess permeability of a wide range of compounds. The objectives of this work are to assess the permeability of ACV prodrugs through the rabbit cornea as well as the SIRC corneal epithelial cell line, and to evaluate the suitability of the SIRC cell line for transport and metabolism of ester prodrugs.
Triangle Park, NC). Heptanesulfonic acid, acid anhydrides, and 4-dimethylaminopyridine (DMAP) were obtained from Aldrich Chemical Company (Milwaukee, WI), 1-octanol and dimethyl formamide (DMF) were provided by Fisher Scienti®c Company (Fairlawn, NJ). Other reagents were of analytical grade, and were used as received. Distilled, deionized water was used in the preparation of all buffers and mobile phases. The SIRC cell line was obtained from American Type Culture Collection (ATCC). Ketamine hydrochloride was obtained from the animal lab, University of Missouri±Kansas City. Sodium pentobarbital was obtained from the school of Pharmacy, UMKC stock, and used under supervision. Methods Synthesis The simple alkyl esters of [9-(2-hydroxyethoxymethyl) guanine] (ACV) were prepared in a single step reaction, using a modi®ed procedure of Shao et al.13 A solution of ACV in dimethyl formamide was treated with acid anhydride in the presence of catalytic amounts of DMAP (Figure 1). Diacyl derivatives of ACV were observed to be the major product using this literature protocol. Reducing the acid anhydride amounts (from 10 to 3 equivalents) and reaction time (from 2±3 days to 1.5 days) produced more satisfactory results. ACV pivalate is an exception because of the steric hindrance effect of the t-butyl group. The diacetyl derivatives were separated as by-products in about 4 to 12% yields. Valcyclovir, 2-[(2-Amino-1, 6-dihydro-6-oxo9H-purin-9-yl) methoxy]ethyl L-valinate hydro-
EXPERIMENTAL SECTION Materials ACV, [9-(2-hydroxyethoxymethyl)guanine] was a gift from Burroughs Wellcome Co. (Research
Figure 1. Scheme for the synthesis of ester prodrugs of ACV.
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Phosphate-Buffer Saline (DPBS) pH 7.4 for transport studies with SIRC cell line. Analytical Procedure Figure 2. Scheme for synthesis of L-valyl ester of ACV.
chloride, was synthesized by Beauchamp et al.14 method (Figure 2). Step I: Acyclovir (2.0 g, 8.88 mM) was dissolved in dry DMF (150 mL) by warming on a steam bath. Successively, DMAP (0.154 g, 1.25 mM), N-cbz-L-valine (3.01 g, 12.0 mM), and DCC (3.0 g, 14.4 mM) were added to the cooled solution. The solution was stirred under nitrogen atmosphere at ambient temperature for 18 h. The mixture was recharged with same amounts of DMAP, N-cbz-L-valine, and DCC, and stirring was continued at ambient temperature for 2 days. The mixture was ®ltered and the DMF was removed by treating the mixture with water and ethyl acetate under acidic conditions. The intermediate partitions into the ethyl acetate layer and the DMF is miscible in the water layer. The ethyl acetate fraction was separated and rotavaped. The residue was chromatographed on silica gel, using 1:4 methanol (MeOH), CH2Cl2 as the eluant, to generate 3.5 g of the desired intermediate, 2-[(2-amino-1, 6-dihydro-6-oxo-9H-purin-9-yl) methoxy] ethyl N-CbzL-valinate, as a white solid. The 1H spectra was consistent with the desired structure. Step II: solution of the intermediate in MeOH (130 mL), THF (tetrahydrofuran, 65 mL), and H2O (25 mL) was added to 0.5 N aqueous HCl (18 mL) and 377 mg of 5% palladium on charcoal. The mixture was shaken in a Parr apparatus under an initial pressure of 50-psi hydrogen at ambient temperature for 18 h. The mixture was ®ltered, the catalyst was washed with MeOH, and the combined washings and ®ltrate were evaporated in vacuo at a bath (< 608C) temperature. The 1HNMR spectra was satisfactory for the desired structure. Preparation of Drug Solutions A fresh, 1±2 mM aqueous solution of the ester prodrugs (butyryl-ACV, iso-butyryl-ACV, valylACV, 1 mM; malonyl-ACV, acetyl-ACV, 2 mM; pivaloyl-ACV, 0.5 mM) was prepared in isotonic phosphate buffer pH 7.4 for corneal transport studies. A 1-mM aqueous solution of the ester prodrugs was prepared in Dulbecco's Modi®ed
A high-pressure liquid chromatographic (HPLC) method was developed for the analyses of ACV and its monoesters (Table 1). The system comprised of a Waters 515 HPLC pump, a Rheodyne TM injector, and a Waters 486 tunable absorbance detector. For the esters of acyclovir, a dualcolumn isocratic HPLC method was used. A 250 4.6 mm i.d. reversed phase C18, 5 mm particles, connected in series with a cation exchange column, Partisil 10 SCX, 5 mm, 250 4.6 mm i.d. was employed. A mobile phase consisting of 0.005 M ammonium phosphate buffer pH 2.2 (60%) and organic phase (40%) comprising of methanol was employed. In case of ACV±acetate and ACV±malonate, a 250 4.6 mm i.d. reversedTM Ê (Varian) conphase C18 Microsorb-MV 100 A nected to Beckman System Gold1 128 solvent module with ¯uorescence detection (Schoeffel Instr.-Corp). A mobile phase comprising of ammonium phosphate 0.0025 M pH 2.2 (90%) and an organic phase (10%) comprising of MeOH and acetonitrile (1:1) was used. For guanosine and guanosine monophosphate, a similar reversephase C18 column connected to a 515 Waters pump with a UV detection was used. A mobile phase consisting of ammonium phosphate 0.002M pH 5.6, and a varying proportion of methanol was used. This method was capable of detecting guanosine, guanosine monophosphate, guanosine diphosphate, and guanosine triphosphate in a single run. Data acquisition and chromatographic analysis was carried out using a Hewlett Packard integrator (HP 3396 series III). For all analyses, a ¯ow rate of 1 mL/min was maintained. Transport Through Cornea A typical side-by-side diffusion apparatus (typeVSC-1, Crown Glass Company Inc.) was used for performing transport experiments. Cornea obtained from New Zealand albino rabbits weighing 2.0 to 3.0 kg was used for transport study. The animals were euthanized by an overdose of pentobarbital through a marginal ear vein. Eyes were proptosed and carefully enucleated, and washed with ice-cold isotonic phosphate buffer (IPB) pH 7.4 to remove any traces of blood. After a small incision to the sclera, vitreous humor was aspirated by using a 1-mL. syringe. Cornea was carefully excised, leaving some scleral portion JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 10, OCTOBER 2001
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Table 1. HPLC Conditions for Ester Prodrugs of ACV, Guanosine, and GMP Compounds Butyryl-ACV, isobutyryl-ACV, pivaloyl-ACV Acetyl-ACV, malonyl-ACV L Valyl-ACV Guanosine GMP
Mobile Phase
Detection
Ammonium phosphate (0.005 M, pH 2.5): methanol 60:40 Ammonium phosphate (0.0025 M, pH 2.2): methanol: acetonitrile 90:5:5 Ammonium phosphate (0.0025 M, pH 2.2) Ammonium phosphate (0.002 M, pH 5.6): methanol 95:5 Ammonium phosphate (0.002 M, pH 5.6)
attached to the cornea. The scleral part attached to the cornea helps to hold the cornea in place in between the half-cells during the transport experiment. The lens was removed, and the irisciliary body was separated from the cornea. The cornea was washed immediately with ice-cold IPB pH 7.4 and mounted on a side-by-side diffusion half-chamber. The temperature was maintained at 348C by circulating water through the jacketed chambers of the diffusion apparatus. Aqueous drug solutions (3 mL) were added on the epithelial side of the cornea (donor chamber). In the other half-chamber (receiver chamber), 3.4 mL of IPB pH 7.4 was added. Both the chambers were stirred continuously by using magnetic stirrer bars. Receiver chamber volume of IPB added was little more than that of the donor chamber to maintain the curvature of the cornea throughout the experiment. Sink conditions were maintained during the experiment. One hundred microliter aliquots were removed from the receiver chamber at appropriate intervals, and were replaced with an equal volume of IPB pH 7.4. The samples were either immediately analyzed by HPLC or stored at ÿ808C until further analysis. All experiments were done in triplicate. Transport Through SIRC Rabbit Corneal Epithelial Cell Line The SIRC cells grown on a polyester membrane support (Whatman) were used for transport studies. The rabbit corneal epithelial cell line (SIRC) was received from the American Type Culture Collection1 (ATCC). The cells were cultured according to the guidelines by ATCC with Minimum Essential Medium (pH 7.4) supplemented with 10% calf serum, lactalbumin (1.76 mg/mL), HEPES (1.3 mg/mL), and Penicillin-streptomycin
UV lmax 249 nm Fluorescence lex 249 nm lem 370 nm Fluorescence lex 249 nm lem 370 nm UV lmax 254 nm UV lmax 254 nm
(100 mg/mL). The cells were incubated at 378C in humidi®ed atmosphere of 5% CO2/95% air. The cells were plated at a density of 33,000 cells/cm2 either on tissue culture-treated plastic dishes or collagen±®bronectin-coated polyester clear membrane (pore size 0.4 mm). The cells used for the transport study were grown for 10 days, and the medium was changed every alternate day. Before the transport study the cells were rinsed with Dulbecco's modi®ed phosphate-buffer saline (DPBS) containing 1 mM CaCl2, 0.74 mM MgSO4, 5.3-mM glucose, and pH 7.4. The cells grown on the membrane support were mounted on side-byside diffusion chambers, and the drug solution was added on the apical side of the membrane (donor chamber). DPBS was added to the other half-chamber (receiver chamber). One hundred microliters of aliquots were removed from the receiver chamber at predetermined time points and analyzed by HPLC. The receiver was replaced with equal volume of buffer after removing the sample. Transport of the hydrophilic marker 14C mannitol was carried out in triplicate for each compound used for the transport study to assess the integrity of cells during the length of the experiments. A similar sampling procedure was used for 14C mannitol transport studies. The samples were analyzed by a scintillation counter (Beckman, LS 6500). The temperature was maintained at 348C through circulated water from a water bath, and the solutions were stirred by using magnetic stirrer bars throughout the length of the experiment. Data was obtained by performing experiments in triplicate or quadruplicate. Transmission Electron Microscopy Transmission electron microscopy (TEM) was used to determine the number of layers in the
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SIRC cell line. The cells grown for 10 days were washed two times with DPBS at 48C. The cells were then washed with cacodylate buffer (48C) for 5 min. Then the cells were ®xed with 2% glutaraldehyde in 0.15 M Na-cacodylate buffer for 60 min. The cells were washed several times with cacodylate buffer and post®xed with osmium tetraoxide for 30 min. Then the cells were processed for thin sectioning using a routine TEM procedure and photographed using a JEOL Electron Microscope (Model JEM 1200 EX II) at 100 kV. Preparation of Ocular Tissues for Enzymatic Hydrolysis Studies New Zealand albino rabbits were used as an animal model for metabolism studies. Animals were euthanized, and eyes were enucleated as described earlier. The eyes were washed in icecold IPB pH 7.4 to remove any traces of blood. Aqueous humor was removed by inserting a 27-1/ 2 gauge needle through the sclera into the anterior chamber. Vitreous humor was collected by incision in the sclera. Part of the sclera was removed to expose the lens. The lens was separated followed by the iris-ciliary body. The cornea was cut carefully to leave no traces of scleral tissue attached to it. All the tissues were immediately washed with ice-cold IPB pH 7.4 and stored in ice. The procedure takes approximately 10±15 min. The tissues were homogenized immediately using a homogenizer (Tissue Tearer Model 985-370 Type 2, Biospace Products, Inc.) in 2 mL ice-cold IPB pH 7.4 and the supernate was stored at ÿ808C for further studies. The homogenate was centrifuged at 50,000 rpm for 30 min using an ultracentrifuge (Beckman TL-100). The supernate obtained was used for hydrolysis studies. Reactions were initiated by adding 100±200 mL (10±50 mg/mL) of drug solution made in IPB pH 7.4 to 1.0 to 1.2 mL of supernate. The reactions were carried out in a water bath at 348C. Samples were taken at appropriate intervals and analyzed by HPLC. Equal volume of methanol was added to stop the reaction. Experiments were done in triplicate. Protein concentration was estimated by using BioRad1 assay method for protein estimation. Preparation of SIRC Cell Homogenate for Enzymatic Hydrolysis Studies The SIRC cells were grown in a 75-cm2 ¯ask for 10 days, the medium was removed, and the cells
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were washed three times with DPBS (pH 7.4). Ten milliliters of buffer was added to a ¯ask and the cells were scraped with the help of a rubber policeman (cell scraper). The cell suspension was centrifuged at 1500 rpm for 10 min. The pellet was resuspended in 2 mL ice cold DPBS, and homogenized by a tissue homogenizer (Tissue Tearer Model 985-370 Type 2, Biospace Products, Inc.) for 1 min. The homogenate was centrifuged (Beckman TL-100 Ultracentrifuge) at 10,000rpm, at 48C for 30 min. The supernate obtained after centrifugation was the cytosol fraction and used for hydrolysis studies. Protein content of the cytosol was estimated by BioRad1 Protein Assay method. Hydrolysis experiments were carried out as mentioned before. All experiments were carried out in triplicate. Cholinesterase Assay in SIRC Cell Homogenate Calibration was done according to the method given in Sigma Diagnostics procedure1 No. 420 (catalog No. 420-MC) cholinesterase kit. Cholinesterase catalyzes the hydrolysis of cholinesters of various short-chain organic acids, including acetylcholine, which is the substrate used in this Sigma Diagnostic procedure. The reaction was conducted in the presence of an acid-base indicator, such as m-nitrophenol. The acetic acid produced lowers the pH, causing a loss of color. This color change is proportional to the cholinesterase activity present. A serum ``blank'' was prepared by inactivation at 608C for 10 min to compensate for background absorbance contributed by the sample. Absorbance of both ``blank'' and ``test'' was read at 420 nm, and the ``difference'' was used to estimate the cholinesterase level. Calf serum was used instead of pooled serum. Sodium chloride solution (0.2 mL) and SIRC cell homogenate were taken in two separate tubes and then mixed. One tube (blank) was placed in a 608C water bath for 10 min to inactivate the enzymes. The tube was heated thoroughly to ensure that all enzymes have been inactivated. It was removed and cooled in tap water. To both ``blank'' and ``test'' tubes, 3.0 mL water, 2.0 mL nitrophenol solution, and 0.2 mL acetylcholine chloride solution was added. All these reagents were brought to room temperature before making the additions. Exact time of addition of acetylcholine chloride solution was recorded, and both the ``blank'' and ``test'' tubes were placed in water bath at 258C. Exactly 30 min later, absorbance (A) was recorded by using a Beckman UV at 420 nm and water as a reference. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 10, OCTOBER 2001
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Calculations (DA DBLANK ÿ DTEST) Using DA, the cholinesterase activity of the SIRC homogenate was determined in Rappaport Units from the calibration curve. One Rappaport unit is the amount of cholinesterase that liberates 1 mm of acetic acid from acetylcholine in 30 min at 258C at pH 7.4 under test conditions. Data Analysis Flux values for ACV and ester prodrugs were calculated by dividing the slope (obtained by plotting a cumulative amount of the compound permeated versus time) with the area available for diffusion, i.e., 0.636 cm2. The permeability values were calculated by dividing the ¯ux values with the concentration of the prodrug used in the donor. Statistical tests were performed to compare the differences between the corneal and the SIRC permeability values using one-way ANOVA. A p-value less than 0.05 was considered as signi®cant. Apparent ®rst-order rate constants for enzymatic hydrolysis was calculated by linear regression of the line obtained by plotting log percent remaining versus time pro®les. Triplicate samples were analyzed, and a mean rate constant was calculated. The rate constants were expressed as per milligram of protein.
RESULTS AND DISCUSSION Electron Microscopy The TEM photomicrograph depicts six layers of SIRC cells (Figure 3). This observation is in agreement with Hutak et al.11 that the SIRC cells grown on the membrane support for 10 days developed four to six layer of cells and comparable to the normal corneal epithelial layer. The TEM photomicrograph shows an elongated epithelial structure and has a ®broblast like appearance. There are no microvilli and microplicae on the surface. There is a sparse distribution of the intercellular space and the cells appear to be fairly tight. However, the cellular tight-junction components such as desmosomes are lacking. Our TEM results of SIRC cells are similar with the previous reports that SIRC cells lack desmosomes, cytoplasmic ®laments, and cytokeratinÐ the structures that are characteristic of corneal epithelial cells.6 The SIRC cells grown on six-well Transwell1 ®lters did not develop enough transe-
Figure 3. Transmission electron microscopic photomicrograph of SIRC cells exhibiting six layers after 10 days of culture.
lectrical epithelial resistance (TEER value < 100 O cm2) indicative of the leaky nature of the cells. During the 10 days of our culture, no exfoliation of SIRC cells was observed. These studies also con®rmed that the SIRC cell line grown for 10 days differentiated to a multicell layer epithelial membrane. Enzymatic Hydrolysis in Ocular Tissues and SIRC Cell Line Homogenate The apparent ®rst-order rate constants for the hydrolysis of the ACV esters in SIRC cells have been reported in Table 5. Loss of ACV ester was accompanied by the formation of ACV. A mass balance with respect to total ACV concentration was maintained throughout the experiment. Controls with no homogenate and containing only the DPBS (pH 7.4) and the respective esters showed no quanti®able degradation over the time course of the study. Cytosolic fraction of the SIRC cell line was used for the hydrolysis studies. The amount of cholinesterase in the SIRC cell line homogenate was found to be 38 Rappaport Units/ mL. The esterase activity in the epithelium of the
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rabbit cornea is higher than that of stroma± endothelium, and this esterase activity varies with rabbits's age and strain.15 The hydrolysis of ACV prodrugs and formation of ACV demonstrated the presence of esterase activity. The hydrolytic rate constant for butyryl± ACV in SIRC homogenate studies was found to be the highest ( p < 0.05). The concentration versus time pro®le for isobutyryl±ACV is shown in Figure 4. Lee et al.16 demonstrated that there exists a chain length at which a prodrug would be hydrolyzed equally well by both cholinesterases. This could be attributed to the higher hydrolytic rate for the butyryl±ACV. A lipophilic pocket at the active site of the ocular esterase has been suggested.17 The isobutyryl and pivaloyl esters showed low enzymatic hydrolytic rate constants in the SIRC cell homogenate, most likely due to a steric hindrance at the enzyme active site. The hydrolysis studies of acetyl ester in SIRC cell line homogenate also showed regeneration of ACV (Figure 5). The SIRC cell line enzymatic data con®rms the presence of esterase activity and thereby bioreversibility of the ACV prodrugs. The apparent ®rst-order rate constants for acetyl± ACV and malonyl±ACV in ocular tissue homogenates is illustrated in Table 6. The rate of hydrolysis of acetyl±ACV in ocular tissues was highest in the iris±ciliary body, followed by in the cornea and in the aqueous humor ( p < 0.05). For malonyl ester, the highest rate constant was also found in the iris±ciliary body ( p < 0.05). The amount of esterases present in the iris±ciliary body was signi®cantly higher (almost twice) than in the cornea. Transport of Acyclovir Esters Across SIRC Cell Line and Rabbit Cornea Corneal and SIRC cell line permeability values for the prodrugs and ACV are reported in Table 2. Integrity of the SIRC cell line was determined by
Figure 4. Concentration versus time pro®le for isobutyryl±ACV in the SIRC cell lysate.
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Figure 5. Percent log versus time pro®le for acetyl± ACV in the SIRC cell lysate.
the transport of a paracellular marker 14C mannitol for each set of experiments. All experiments were conducted in triplicate, with 14C mannitol throughout the length of the experiments for each prodrug used. Mannitol transport was found to be between 2±4% per hour. Permeability values for mannitol for each prodrug are summarized in Table 3. To monitor transcellular permeability, transport of 14C diazepum was conducted across SIRC and the freshly excised cornea (Figure 7). The permeability of 14C diazepum across SIRC was more than double (8.37 10ÿ5 cm/s) compared to the cornea (3.43 10ÿ5 cm/s). All the ACV prodrugs, except ACV±acetate and ACV±malonate, were partially hydrolyzed to ACV during transport across the SIRC cell layers. Enzymatic activity of the SIRC cell line is evident from the regeneration of the parent compound ACV during transport (Figure 6). Permeability values for the prodrugs through SIRC were found to be higher than that of across the intact cornea ( p < 0.05). From the electron microscopic data it was observed that SIRC cells grown for 10 days developed four to six cell layers thick. Normal rabbit corneal tissue consists of four to seven layers of epithelial cells, followed by stroma and endothelium. The top two super®cial layers offer the primary resistance to transport of drugs. Although SIRC cells grown for 10 days were four to six layers thick, but they did not produce enough resistance, having a poor TEER value (< 100 O cm2). The TEER value for rabbit cornea is > 1500 O cm2. Thus, SIRC cell layers are leakier than the cornea, and hence, the higher permeability values result for the ACV prodrugs. The amounts of ACV generated during transport across the SIRC cell line are reported in Table 4. Hughes and Mitra18 reported 50% hydrolysis of isobutyryl±ACV and about 80% hydrolysis of other esters in the homologous series for transport experiment in the cornea. However, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 10, OCTOBER 2001
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Table 2. Permeability Values of ACV Prodrugs, Guanosine, and GMP in Cornea and SIRC Cells Prodrugs ACV Malonyl-ACV Acetyl-ACV Butyryl-ACV Isobutyryl-ACV Pivaloyl-ACV L-Valyl-ACV Guanosine GMP
Cornea P cm/s
SIRC P cm/s
ÿ6 a
Ratio: SIRC/Cornea ÿ5
3.65 10 2.33 0.42 10ÿ5 1.83 1.2 10ÿ5 5.12 10ÿ6 a 3.92 10ÿ6 a n/d 5.8 0.95 10ÿ6 7.95 0.21 10ÿ6 2.36 0.14 10ÿ6
2.6 0.34 10 4.0 1.4 10ÿ5 2.5 0.21 10ÿ5 1.25 0.11 10ÿ5 1.36 0.12 10ÿ5 1.67 0.10 10ÿ5 1.20 0.33 10ÿ5 9.1 0.24 10ÿ6 5.06 0.13 10ÿ6
7.12 1.72 1.37 2.44 3.47 2.07 1.14 2.14
a
Data obtained from ref. 18. n/d not detected.
b
in SIRC transport studies, isobutyryl±ACV ester hydrolyzed to about 5%. Other ACV esters except acetyl±ACV and malonyl±ACV were hydrolyzed to about 10±13% during transport. In the SIRC homogenate study the apparent ®rst-order rate constant for the hydrolysis of isobutyryl±ACV was also low compared to other ACV esters in the study. Moreover, hydrolysis of acetyl±ACV and malonyl±ACV was also observed in the SIRC cell line homogenate study. The epithelial layer contributes approximately 70% of the enzymatic activity of the cornea. The SIRC cell line consists of only epithelial layers, and these cells might not be expressing the esterases at the same level, which are responsible for hydrolysis of ester prodrugs. Hence, a lower percentage of hydrolysis for the ACV±esters in the SIRC cell line was observed during transport. Higher permeability for acetyl±ACV might be attributed to its inability to undergo hydrolysis during transport across the SIRC cell line. However, the relationship observed by Hughes and Mitra12 for log partition coef®cient and permeability may not be predicted for these compounds. Similarly, a higher permeability value was observed for malonyl±ACV ( p < 0.05). The permeability values for the unhydrolyzed
prodrugs except malonyl±ACV and acetyl±ACV were lower compared to that of ACV across the SIRC cell line ( p < 0.05). These prodrugs were hydrolyzed during transport, and the total amount of ACV transported was more than that of ACV, resulting in increased amounts of ACV transported across the SIRC cell line. It is well known that the optimum log partition coef®cient values for the compounds to diffuse across cornea are between 1 and 2. However, higher log partition coef®cient values would compromise aqueous solubility of prodrugs. Thus, watersoluble, solution stable, L-valyl ester of ACV was synthesized. The permeability value for valacyclovir was comparable to other simple ester prodrugs. Higher corneal permeability values for Malonyl±ACV and Acetyl±ACV were observed compared to corneal permeability of ACV ( p < 0.05). ACV was regenerated from acetyl prodrug in a consistent manner during transport through the cornea. Hydrolysis of the malonyl prodrug was observed during transport across the cornea to a lesser extent and in an inconsistent manner compared to other prodrugs. The permeability value for valacyclovir across the cornea was also more than that of ACV ( p < 0.05). Its mechanism
Table 3. 14C Mannitol Permeability Values Across SIRC Cells for Each Compound Compounds Butyryl, iso-butyryl, pivaloyl ACV Valyl ACV Guanosine GMP
Permeability (cm/s) 10ÿ6 1.12 0.10 2.02 0.50 1.41 0.34 1.20 0.047
Figure 6. Receptor cell concentrations of ACV, total ACV, and the unhydrolyzed butyryl±ACF. (a) Each point is a mean of n 3.
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Table 5. Apparent First-Order Rate Constants for ACV Prodrugs in SIRC Cell Lysate Prodrugs Malonyl ACV Acetyl ACV Butyryl ACV Iso-butyryl ACV Pivaloyl ACV Figure 7. Transport of 14C diazepam (a transcellular marker) across the SIRC cell line and freshly excised rabbit cornea. (a) Each point is a mean of n 4.
of transport across the cornea is not clear, and needs to be studied further in detail. In a recent study Goskonda et al.19 have shown good correlation between permeability coef®cients and lipophilicities of drugs. The ester prodrugs, such as phenylacetyl ester, isovaleryl ester, and pivalyl ester, having higher lipophilicities, showed a fourto sixfold increase in corneal permeability compared to phenylephrine HCl. The permeability of ester prodrugs varied, depending on the pH of the transport medium, because the degree of ionization is dependent on the pH of the medium. Permeability values for transport of guanosine and guanosine monophosphate (GMP) are reported in Table 2. Metabolism of guanosine was found to occur during diffusion through the cornea. These metabolites remain to be identi®ed. However, no metabolism was observed during transport through the SIRC cell line. GMP was used as a model for antiviral nucleoside monophosphate to investigate whether it gets converted to higher phosphates, namely guanosine diphosphate, and guanosine triphosphate, by the cellular kinases during transport through the cornea. These studies show that there was no such conversion of GMP to higher phosphates. However, three metabolites, which are yet to be identi®ed, were found during the transport of Table 4. Amount of ACV Transport Through SIRC Cells Prodrugs Butyryl ACV Iso-butyryl ACV Pivaloyl ACV Valyl ACV
Generated
During
Amount of ACV (mg/mL) 0.823 0.020 0.426 0.049 0.300 0.064 0.410 0.021
K minÿ1/mg of Protein 10ÿ4 2.41 0.5 2.9 0.13 14.4 0.83 1.6 0.13 3.12 0.17
GMP through the cornea. No phosphate metabolites were found during transport through the SIRC cell line. The permeability value for guanosine across the SIRC was higher than that of across the cornea ( p < 0.05). A possible reason might be the lower resistance offered by SIRC epithelial cell layers to diffusion of guanosine. Moreover, absence of hydrolysis might have resulted in higher transport. Guanosine is poorly water soluble (less than 1 mg/mL) at pH 7.4, although it has relatively lower log partition coef®cient. ACV shares some of the physicochemical characteristics of guanosine; hence, it was selected as a model for antiviral nucleosides. Similar results were obtained for GMP. GMP is fairly hydrophilic compared to guanosine, and diffuses through the cornea as well as the SIRC cell line very poorly. The permeability values for GMP were found to be lower compared to that of guanosine ( p < 0.05). This also suggests that the epithelial layer offers resistance to transport of hydrophilic compounds. It can also be concluded that the topmost two layers of cornea present a considerable barrier to permeation of hydrophilic compounds. The SIRC permeability values for guanosine and GMP were 10 and 25% higher than that of guanosine and GMP through the cornea, respectively. Although SIRC expresses cholinesterase, it may not be expressing the wide variety of kinases present in the cornea as no metabolism (phosphorylation) of guanosine and GMP was observed during transport across the SIRC. Characterization of the SIRC cell line for other enzyme systems needs to be performed. The permeability values obtained for these compounds across the SIRC cell line were suf®ciently close to predict their permeabilities across the cornea. However, caution must be exercised while interpreting transport data for compounds, considering the limitations of the SIRC cell line. From the results of in vitro transport and metabolism studies in the cornea and SIRC cells, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 90, NO. 10, OCTOBER 2001
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Table 6. Apparent First-Order Rate Constants for ACV Prodrugs in Ocular Tissue Homogenates ICB Prodrugs Acetyl-ACV Malonyl-ACV
K minÿ1 per mg of Protein
Cornea 4.61 0.16 10ÿ5 4.61 0.14 10ÿ5
some encouraging observations were obtained. However, a better correlation between the corneal and the SIRC permeability values could be obtained by further characterization of the SIRC cell line in detail. Optimizing the growth conditions using additional growth-promoting agents [pituitary extracts, insulin-transferin, epidermal growth factor (EGF)] in the medium, air interface culture,20,21 etc., would obtain a cell line with consistent characteristics and thus minimize the variability in the data. This would also yield four to seven layers with tight junctions with suf®cient TEER value to offer resistance to transport of compounds similar to the cornea. The ACV prodrugs were hydrolyzed to ACV in the cornea and the SIRC cell line. However, hydrolysis of the malonyl and acetyl prodrug was not observed during transport through the SIRC cell line although these prodrugs were hydrolyzed to ACV in the SIRC cell lysates. Further study of the enzyme systems present in the SIRC cell line would enhance the understanding of hydrolysis of these prodrugs. Moreover, qualitative and quantitative characterization of the SIRC cell line for the enzyme systems would facilitate the evaluation of a wide variety of prodrugs. Transport of valacyclovir was increased signi®cantly relative to ACV in the cornea and the SIRC cell line. Its mechanism of transport could be studied in detail by investigating the carrier systems present in the cornea as well as the SIRC cell line, which can be achieved by evaluating the transport of valacyclovir and amino acid prodrugs.
CONCLUSIONS An ideal cell line will have the similar anatomical and physiological characteristics as that of the tissue of interest. Such a cell line will be an ef®cient tool in screening the compounds for assessment of transport characteristics of compounds. Based on our studies, it can be concluded that SIRC cells form multilayers, but are leaky in nature compared to the cornea. Moreover, one of
AQ
9.5 0.20 10ÿ5 1.26 0.11 10ÿ4
0.150 0.08 10ÿ5 1.00 0.02 10ÿ4
the desirable characteristics of a cell line is to express the enzymes present in the original animal tissues. Ester prodrugs must also display the appropriate hydrolysis kinetics by the esterases present in the cell line model similar to that of the corneal tissues obtained from animals. The prodrug must be metabolized to the parent compound in the ocular tissues including the cornea. Based on the experimental data obtained in this study, it is concluded that the SIRC cell line expresses the enzyme cholinesterases, which are responsible for hydrolysis of ester prodrugs. The bioreversion of ACV prodrugs and the esterase determination con®rms the presence of esterases in the SIRC cell line. From the SIRC transport and metabolism data butyryl±ACV may be chosen as the best prodrug. Based on the corneal data for acetyl±ACV, it can be considered as a prodrug with favorable transport and metabolism characteristics in addition to butyryl±ACV. The water-soluble amino acid prodrug valacyclovir can also be considered as a potential prodrug for topical delivery of ACV, although it needs further evaluation. In conclusion, the SIRC cell, upon further characterization, may be used for assessing the permeabilities and metabolism of ester prodrugs, thereby eliminating the need of animal tissues. Further characterization of the SIRC cell line using different growth supplements (pituitary extracts, EGF, insulin-transferin, etc.) and air interface culture is in progress in this laboratory. However, considering the limitations of in vitro cell culture model, caution must be exercised to avoid over interpretation of results.
ACKNOWLEDGMENTS We would like to thank Dr. Paul Brown for his help in Mass Spectrometry, and Greg Williams for electron microcopy. This work was supported by NIH Grants RO1 EY 09171-05 and RO1 EY 10659-04.
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