Sclera-Choroid-RPE Transport of Eight β-Blockers in Human, Bovine, Porcine, Rabbit, and Rat Models

Abstract

Purpose.: To determine the influence of drug lipophilicity, ocular pigmentation, and species differences on transscleral solute transport.

Methods.: The transport of eight β-blockers across excised sclera/sclera-choroid-RPE (SCRPE) of albino rabbit, pigmented rabbit, human, porcine, and bovine eyes was determined over 6 hours. The ex vivo transscleral β-blocker transport to the vitreous at the end of 6 hours was determined in euthanatized, pigmented Brown Norway rats. The thicknesses of the sclera and SCRPE and the melanin content in choroid-RPE (CRPE) were measured to determine whether species differences in drug transport can be explained on this basis.

Results.: Solute lipophilicity inversely correlated with the SCRPE cumulative percentage of transport in all species (R ² ≥ 0.80). The CRPE impeded the SCRPE transport of all β-blockers (51%–64% resistance in the rabbits; 84%–99.8% in the bovine and porcine eyes) more than the sclera, with the impedance increasing with lipophilicity. SCRPE transport followed the trend albino rabbit > pigmented rabbit > human > porcine > bovine, and a cross-species comparison showed good Spearman's rho correlation (R ² ≥ 0.85). Bovine (R ² = 0.84), porcine (R ² = 0.84), and human (R ² = 0.71) SCRPE transport was more predictive than that in the rabbit models (R ² = 0.60–0.61) of transscleral solute transport to the vitreous in rats. The CRPE concentrations were higher in pigmented rabbits than in albino rabbits. The melanin content of the CRPE exhibited the trend albino rabbit ≪ pigmented rabbit < porcine ∼ bovine < rat. Normalization to scleral thickness abolished the species differences in scleral transport. Normalization to SCRPE thickness and melanin content significantly reduced species differences in SCRPE transport.

Conclusions.: Owing to the presence of pigment and drug binding, choroid-RPE is the principal barrier to transscleral β-blocker transport, with the barrier being more significant for lipophilic β-blockers. Although different in magnitude between species, sclera/SCRPE transport can be correlated between species. Tissue thickness accounts for the species differences in scleral transport. Differences in tissue thickness and melanin content largely account for the species differences in SCRPE transport.

Drug delivery to the back of the eye has received considerable attention during the past decade because of advances in understanding the pathophysiology of the various diseases that afflict the posterior segment of the eye and the successful development of various intravitreally administered pharmacologic agents, including pegaptanib (Macugen; Pfizer, New York, NY), ranibizumab (Lucentis; Genentech, South San Francisco, CA), triamcinolone acetonide (Triesence, Alcon, Fort Worth, TX; Trivaris, Allergan, Irvine, CA), and dexamethasone (Ozurdex, Allergan, Irvine, CA), for treating posterior eye diseases. The intravitreal route of drug administration, although approved and effective for several drug molecules, is associated with many complications, such as retinal detachment, endophthalmitis, vitreous hemorrhage, and cataract formation. ^1,2 Transscleral drug delivery or drug delivery across the sclera to the back of the eye is expected to minimize the complications associated with intravitreal injection. ³ Our earlier studies demonstrated the suitability of sustained transscleral delivery of celecoxib in treating diabetic retinopathy. ⁴ Although several reports have been published regarding transscleral drug delivery, there is a dearth of knowledge with respect to species differences and drug properties that are critical for enhanced delivery to the retina. Probably due to the lack of such fundamental knowledge, some clinical transscleral delivery efforts have failed to result in approval of drug products (e.g., posterior juxta-scleral administration of an anecortave acetate suspension). Inadequate efficacy due to poor drug delivery is believed to be one of the reasons for the failure of the products. Therefore, we investigated species differences and the influence of drug properties on transscleral drug delivery. 

A thorough understanding of the role of various factors influencing the absorption of drugs across a membrane is beneficial during drug development. The physicochemical properties of the drug molecule such as lipophilicity, molecular weight, and acid/base properties, along with anatomic, physiological, and pathologic factors associated with various organs and tissues play an important role in drug delivery to target sites. Previously, we have shown that the pigmented choroid-Bruch's layer underlying the sclera, in the absence of retinal pigment epithelium (RPE), can significantly reduce the transport of drugs across the sclera. ⁵ In addition, data from our previous studies indicated that lipophilic solutes preferentially bind to the choroid-RPE (CRPE). ⁶ Further, using celecoxib, a lipophilic solute, we demonstrated that transscleral delivery to the retina is impeded in pigmented Brown Norway (BN) rats when compared to delivery in albino Sprague-Dawley rats. The sclera, choroid, and RPE collectively regulate the transscleral solute transport to the retina. We have investigated the influence of drug lipophilicity on sclera–choroid transport without the RPE. However, the influence of drug lipophilicity on transport across the sclera-choroid-RPE (SCRPE) has not been investigated. 

Several studies performed in Edelhauser's laboratory ^{7 –9} have documented the permeability of excised human sclera. However, there are no reports of studies of SCRPE permeability in human tissues, possibly because of the difficulty in obtaining the tissues with an intact CRPE layer. As an alternative to human tissues, excised tissues from a variety of animal sources (e.g., rabbit, pig, and bovine) have been studied. ^10,11 In the present study, we used tissues from rabbit (albino and pigmented), human, porcine, and bovine sources in modified Ussing chambers, to understand SCRPE permeability as a function of drug lipophilicity. In addition, since rodent models (e.g., BN rat) are widely used as disease models for diabetic retinopathy ¹⁰ and choroidal neovascularization, ¹² we assessed the transscleral transport of β-blockers in BN rats. We assessed the solute delivery in intact eyes in the rat model after euthanatization and periocular injection, to mimic the transport with excised tissues from other species, in the absence of circulatory clearance mechanisms. Rat tissues are not large enough to allow excised tissue-mounting in Ussing chambers. Thus, we assessed solute transport across SCRPE in six models. In addition, we determined whether species differences in transscleral solute transport can be explained on the basis of differences in tissue thickness and melanin content. 

Materials and Methods

Atenolol (99.0%), sotalol hydrochloride (∼98%), nadolol (∼98%), pindolol (98%), timolol maleate (98%), metoprolol tartrate (99%), betaxolol (∼98%), labetalol hydrochloride (99%), and propranolol hydrochloride (99%) were purchased from Sigma-Aldrich (St. Louis, MO). HPLC-grade acetonitrile and methanol were purchased from Fisher Scientific (Fair Lawn, NJ); triethyl amine (99.5%), sodium hydroxide, and formic acid (88%) from Sigma-Aldrich; and ammonium formate (99.9%) from Fluka BioChemika (Buchs, Switzerland). All other chemicals and reagents were of analytical reagent grade. 

Tissue Isolation

Freshly excised eyes of New Zealand White rabbits (age, 2–3 months; Harlan Sprague-Dawley, Indianapolis, IN), Dutch Belted rabbits (age, 2–3 months; Myrtle's Rabbitry, Thompson's Station, TN), pigs (age unknown; Decker & Son, Colorado Springs, CO), and cows (age, 1–1.5 years; G & C Packing Co., Colorado Springs, CO) were used as models. Human cadaveric eyes were obtained from anonymous donors (San Diego Eye Bank, San Diego, CA) within 48 hours of death, in accordance with the protocol approved by the Institutional Review Board. The procedures are in compliance with the Declaration of Helsinki for research involving human tissue. The age of donor patients ranged from 55 to 83 years, and the primary causes of death were renal failure and melanoma. Human eyes were received overnight in moist chambers maintained at 4°C. After the adherent muscle tissue was removed, the anterior and posterior segments of each eye were separated by a circumferential cut behind the limbus. The neural retina was separated from the SCRPE layer by exposing the eye cup to isotonic assay buffer at pH 7.4. Rectangular pieces (∼1.5 × 1.5 cm) of the tissue containing the CRPE layer along with the sclera were dissected from the equatorial region of the sclera. For solute transport studies across the sclera, the CRPE layer was gently scraped off and removed, and the sclera was washed with assay buffer. Because of the limited availability of human tissue samples, the transport study was performed only across human SCRPE, not across the sclera, since data are abundant in the literature for solute transport across human sclera. ^{7 –9}  

In Vitro Transport Study

All transport studies were performed using a modified Ussing chamber assembly (Navicyte, Sparks, NV) in isotonic assay buffer (pH 7.4). The excised tissues were mounted in the chambers such that the episcleral side faced the donor chamber and the retinal side faced the receiver chamber. The chambers were filled with equal volumes (1.5 mL) of assay buffer, with (donor side) or without (receiver side) a cocktail of eight β-blockers (100 μg/mL of each drug). The temperature of the buffer was maintained at 37°C in a circulating water bath. The pH of the bathing fluid was maintained at 7.4 by aeration in 95% air–5% CO₂. At predetermined time points (1, 2, 3, 4, 5, and 6 hours), 200-μL of sample was collected from the receiver side and replaced with fresh assay buffer pre-equilibrated at 37°C. The drug levels were analyzed with an LC-MS/MS assay. The apparent permeability coefficient (P _app) was calculated by equation 1.  

 

where dM/dt is the slope of the linear region of the cumulative amount of the solute transported versus time plot, A is the area of the tissue surface (0.64 cm²) available for transport, and C _d is the initial donor drug concentration. Permeation data were corrected for dilution of the receiver solution with sample volume replenishment. All permeability values in this study refer to apparent permeability coefficients. 

Tissue Thickness Measurement

The thicknesses of the SCRPE and sclera after removal of the CRPE were measured with an outside micrometer (0–25-mm range with an accuracy of 0.001 mm; Mitutoyo USA., Aurora, IL). For measurement of tissue thickness, the SCRPE was dissected from the equator regions of the eye with a 10-mm skin biopsy punch (Acu Punch; Acuderm Inc., Fort Lauderdale, FL). Caution was taken not to compress the tissues during the measurements. 

Ex Vivo Tissue Distribution Study in BN Rats

Animals were treated according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and guidelines by the Animal Care Committee of the University of Colorado Denver. Male BN (pigmented) rats (200–250 g) were obtained from Charles River Laboratories (Wilmington, DE). The tissue distribution study was performed in euthanatized rats to eliminate the effect of blood and lymphatic circulations and associated drug clearance. The cocktail mixture of eight β-blockers (250 μg/mL) was prepared in phosphate-buffered saline (pH 7.4). The rats were euthanatized with an intraperitoneal injection of pentobarbital sodium (250 mg/kg). After euthanatization, the β-blocker solution (25 μL) was injected into the posterior subconjunctival space of one eye with a 30-gauge needle. The animals were maintained at room temperature for 6 hours after injection. The eyes were enucleated and immediately frozen in a mixture of isopentane and dry ice. Drug levels were measured in the sclera, CRPE, retina, and vitreous by LC-MS/MS. 

Estimation of Melanin

The melanin content of bovine, porcine, and albino and pigmented rabbit sclera and CRPE was measured according to a published method. ¹³ Briefly, the weighed amount of tissues (20 mg) were placed in microcentrifuge tubes containing 100 μL of 10% DMSO solution in 1 M NaOH in water. The samples were heated at 70°C for 45 minutes to solubilize the melanin. The samples were then diluted in distilled water to 500 μL and neutralized using 500 μL of dilute acetic acid (1 M), and absorbance was measured at 475 nm. The calibration curve for melanin estimation was prepared with synthetic melanin. 

LC-MS/MS for Estimating β-Blockers

The concentration of β-blockers in the transport study samples was measured according to a published method. ¹⁴ Briefly, the transport study samples were diluted 5- to 10-fold with acetonitrile to reduce the salt concentration. The concentrations in transport study tissue samples and rat ocular tissue samples were measured after liquid–liquid extraction using dichloromethane and ethyl acetate (1:1 vol/vol) mixture. The mobile phase was a mixture of 5 mM ammonium formate adjusted to pH 3.5 (A) and acetonitrile:methanol (75:25) containing 0.02% triethylamine HCl, pH adjusted to 3.5 (B). The linear gradient elution at a flow rate of 0.4 mL/min with total run time of 13 minutes was as follows: 90% A (0–1.0min), 10% A (6–9 minutes), and 90% A (10–13 minutes). The following transitions were monitored: 267/145 (atenolol), 273/255 (sotalol), 310/254 (nadolol), 249/116 (pindolol), 317/261 (timolol), 268/133 (metoprolol), 308/116 (betaxolol), 260/116 (propranolol), and 327/162 (labetalol). 

Statistical Analysis

The data are represented as the mean ± SD. For statistical comparisons between the two experimental groups, an independent-samples Student's t-test was used. The comparisons of the mean between multiple experimental groups were performed with one-way ANOVA followed by Tukey's post hoc analysis (SPSS, ver.11.5; SPSS, Chicago, IL). The differences were considered statistically significant at P < 0.05. The correlation of the permeability coefficient or cumulative percentage of transport between the species was performed by using Spearman's rank correlation with statistical significance set at P < 0.05. 

Results

Scleral Transport: Species Differences

The physicochemical properties of the β-blockers are shown in Table 1. The cumulative percentages of transport and the permeability coefficients for the β-blockers across rabbit, porcine, and bovine sclera are shown in Figure 1 and Table 2, respectively. Because of the limited availability of human tissue, transport study was not performed across sclera alone. The rank of the cumulative percentage of transport of β-blockers across the sclera was rabbit > porcine > bovine. The cumulative percentage of transport of β-blockers across the sclera showed an inverse relation with scleral thickness, with the percentage of transport being the highest in albino rabbit and the lowest in bovine tissue. Normalization of the cumulative percentage of transport and P _app by multiplying with scleral thickness diminished the species differences (Fig. 2). The rank of the normalized cumulative percentage of transport of the β-blockers across the sclera was generally sotalol ∼ atenolol > metoprolol ∼ timolol ∼ betaxolol ∼ pindolol > propranolol (Table 3). The hydrophilic β-blockers (sotalol and atenolol) exhibited the highest percentage of transport in all species. 

Table 1.

 

Physicochemical Properties of the β-Blockers

 

 Solubility data were obtained from the website: http://www.drugbank.ca (provided by the Department of Biological Sciences, University of Alberta, Calgary, Alberta, Canada). Molecular weight and pKa values were obtained from SciFinder Scholar 2007 (provided by Chemical Abstracts Science, The American Chemical Society, Columbus, OH; http://www.cas.org). Octanol-PBS (pH 7.4) distribution coefficients (log D) of the drug forms listed in the Materials and Methods section were previously determined by us at 37°C using the shake flask method by our group. ⁶ Partition coefficients (log P) were obtained from the literature. ¹⁵

Figure 1.

 

Scleral transport of β-blockers in various species. The data show cumulative percentages of transport of β-blockers across excised bovine, porcine, pigmented rabbit, and albino rabbit sclera devoid of CRPE. Solid line: transport before normalization for scleral thickness; dashed line: transport after normalization by multiplying with scleral thickness in mm. The data are expressed as the mean ± SD for n = 6.

Table 2.

 

Apparent Permeability Coefficients (×10⁻⁶ cm/s) of the β-Blockers across Albino and Pigmented Rabbit, Porcine, and Bovine Ocular Tissues

β-Blocker	Albino Rabbit			Pigmented Rabbit			Porcine			Bovine
	Sclera	Sclera-Choroid-RPE	Choroid-RPE (% Resistance)*	Sclera	Sclera-Choroid-RPE	Choroid-RPE (% Resistance)*	Sclera	Sclera-Choroid-RPE	Choroid-RPE (% Resistance)*	Sclera	Sclera-Choroid-RPE	Choroid-RPE (% Resistance)*
Atenolol	23.7 ± 4.74 (53.6)	12.7 ± 0.87†	27.4 (46.4)	22.1 ± 3.97 (46.6)	10.3 ± 1.05	19.3 (53.4)	14.4 ± 2.02 (15.9)	2.28 ± 0.34	2.71 (84.1)	10.1 ± 1.04 (12.7)	1.28 ± 0.57	1.47 (87.3)
Sotalol	30.5 ± 7.25 (46.9)	14.3 ± 1.01†	26.9 (53.1)	24.2 ± 1.28 (49.5)	12.0 ± 1.7	23.8 (50.5)	16.7 ± 2.41 (11.8)	1.96 ± 0.38	2.24 (88.2)	10.7 ± 2.81 (10.9)	1.17 ± 0.32	1.31 (89.1)
Nadolol	24.5 ± 6.02 (50.2)	12.3 ± 1.57†	24.7 (49.8)	21.1 ± 3.1 (48.0)	10.1 ± 1.8	19.4 (52.0)	12.9 ± 5.53 (14.9)	1.91 ± 0.20	2.25 (85.1)	9.24 ± 2.37 (11.4)	1.05 ± 0.21	1.18 (88.6)
Pindolol	22.7 ± 7.03 (45.4)	10.3 ± 0.71†	18.9 (54.6)	19.0 ± 1.93 (41.3)	7.84 ± 0.95	13.4 (58.7)	10.1 ± 0.43 (1.1)	0.11 ± 0.024	0.11 (98.9)	8.44 ± 0.27 (1.5)	0.13 ± 0.09	0.13 (98.5)
Metoprolol	24.0 ± 4.55 (42.9)	10.3 ± 1.33†	18.0 (57.1)	18.5 ± 3.97 (43.7)	8.1 ± 1.45	14.4 (56.3)	11.3 ± 0.06 (5.5)	0.62 ± 0.098	0.65 (94.5)	8.70 ± 0.29 (4.0)	0.38 ± 0.26	0.40 (96.0)
Timolol	22.5 ± 5.68 (49.8)	11.2 ± 0.68†	22.3 (50.2)	19.2 ± 1.38 (46.0)	8.83 ± 1.09	16.4 (54.0)	10.7 ± 0.32 (3.3)	0.36 ± 0.025	0.37 (96.7)	9.49 ± 0.39 (4.1)	0.36 ± 0.20	0.37 (95.9)
Betaxolol	20.8 ± 3.84 (41.4)	8.61 ± 0.74†	14.7 (58.6)	15.4 ± 2.01 (40.8)	6.27 ± 1.3	10.6 (59.2)	9.05 ± 0.52 (3.2)	0.29 ± 0.060	0.30 (96.8)	8.60 ± 0.26 (2.2)	0.19 ± 0.14	0.19 (97.8)
Propranolol	18.8 ± 3.82 (34.4)	6.46 ± 1.38†	9.84 (65.6)	13.0 ± 1.30 (36.1)	4.7 ± 1.28	7.36 (63.9)	6.13 ± 0.48 (1.6)	0.096 ± 0.022	0.10 (98.4)	6.25 ± 0.38 (0.2)	0.013 ± 0.009	0.01 (99.8)

 Data are expressed as the mean ± SD for n = 6.

*  Values in the parentheses indicate the relative percentage of resistance offered by the tissue (sclera or choroid-RPE) to the permeability of the β-blocker across the SCRPE combination.

†  Significant (P < 0.05) difference compared with the corresponding pigmented rabbit tissues. P _app values for CRPE were calculated from the corresponding sclera and SCRPE permeability values.

Figure 2.

 

The species differences in β-blocker transport across the sclera were abolished after normalization for scleral thickness. The cumulative percentage of transport at the end of 6 hours (A) before and (B) after normalization for tissue thickness. The apparent permeability coefficients (C) before and (D) after normalization for tissue thickness. The results are expressed as the mean ± SD for n = 6.

Table 3.

 

P _app Coefficients (×10⁻⁶ cm/s) and Cumulative Percentages of Transport of the β-Blockers across Human SCRPE at the End of 6 Hours

β-Blocker	P app	Cumulative Transport (%)
Atenolol	5.67 ± 1.03	4.71 ± 0.88
Sotalol	6.03 ± 1.02	4.92 ± 0.95
Nadolol	4.62 ± 0.76	3.71 ± 0.70
Pindolol	3.32 ± 0.98	2.57 ± 0.72
Metoprolol	4.53 ± 1.04	3.48 ± 0.86
Timolol	3.99 ± 0.92	3.12 ± 0.69
Betaxolol	3.49 ± 0.87	2.68 ± 0.59
Propranolol	1.34 ± 0.59	1.04 ± 0.43

 Data are expressed as the mean ± SD for n = 6.

Scleral Transport Strain Differences: Albino versus Pigmented Rabbit

The cumulative percentage of transport and the apparent permeability coefficients for the β-blockers across the pigmented and albino rabbit sclera are shown in Figure 1 and Table 2, respectively. Between the strains, the apparent permeability coefficient, as well as the cumulative percentage of transport for all β-blockers except propranolol, were not significantly different. For propranolol, the most lipophilic β-blocker assessed, there was lower transport across the pigmented rabbit sclera than across the albino rabbit sclera. For propranolol, the P _app and cumulative percentages of transport across the pigmented rabbit sclera were 31% and 14% lower, respectively, than across the albino rabbit sclera. 

SCRPE Transport: Species Differences

The cumulative percentages of transport and the apparent permeability coefficients for the β-blockers across bovine, porcine, human, and rabbit SCRPE are shown in Figure 3, Table 2, and Supplementary Table S1. The cumulative percentage of transport and apparent permeability coefficients for human SCRPE are summarized in Table 3. The cumulative percentage of transport of the β-blockers in the presence of CRPE was significantly lower than that in the sclera alone (Supplementary Table S1). The cumulative percentage of transport and permeability coefficient of each β-blocker across the SCRPE were ranked as rabbit > human > porcine > bovine. However, the order of the β-blockers for cumulative percentage of transport and permeability followed different trends between the species. In the cases of bovine and porcine SCRPE, the order was sotalol ∼ atenolol ∼ nadolol > metoprolol ∼ timolol > betaxolol > pindolol > propranolol. The order in human SCRPE was sotalol ∼ atenolol > nadolol > metoprolol ∼ timolol > betaxolol ∼ pindolol > propranolol (Table 3). The order in pigmented rabbit SCRPE was sotalol ∼ atenolol ∼ nadolol > pindolol ∼ metoprolol ∼ timolol > betaxolol > propranolol. Normalization of the cumulative percentages of transport and the apparent permeability coefficients with SCRPE thickness and pigment content in CRPE (by multiplying with the latter two parameters) reduced species differences but significant differences remained, especially for the more lipophilic solutes (Fig. 4; Supplementary Fig. S1). 

Figure 3.

 

SCRPE transport of β-blockers in various species. Data show the cumulative percentages of transport of β-blockers across bovine, porcine, human, pigmented rabbit, and albino rabbit SCRPE. Solid line: the transport before normalization for SCRPE thickness. Dashed line: transport after normalization by multiplying with SCRPE thickness in mm. The data are expressed as the mean ± SD for n = 6.

Figure 4.

 

The species differences in β-blocker transport across the SCRPE was reduced but remained after normalization for SCRPE thickness and pigment content in the CRPE. The cumulative percentage of transport at the end of 6 hours (A) before normalization, (B) after normalization for SCRPE thickness, and (C) after normalization for SCRPE thickness and melanin content in the CRPE. The results are expressed as the mean ± SD for n = 6.

SCRPE Transport Strain Differences: Albino versus Pigmented Rabbit

The cumulative percentages of transport and the apparent permeability coefficients for the β-blockers across albino and pigmented rabbit SCRPE are shown in Figure 3 and Table 2, respectively. The cumulative percentage of transport and apparent permeability coefficients were significantly lower across pigmented rabbit SCRPE than across albino rabbit SCRPE (Table 2; Supplementary Table S1). The difference in transport is more significant with lipophilic β-blockers than with the hydrophilic ones. For the highly lipophilic β-blocker propranolol, the P _app across pigmented rabbit SCRPE was 27% lower than that of albino SCRPE. The cumulative percentage of transport of propranolol was 29% lower in the pigmented rabbit. 

Estimation of CRPE Permeability of β-Blockers

The apparent permeability coefficients for the CRPE layer are derived with the following equation:  

 

where P _app (AB) is the apparent permeability coefficient across the SCRPE combination, P_app (A) is the apparent permeability coefficient across the sclera, P _app (B) is the apparent permeability coefficient across CRPE, and 1/P _app represents the corresponding resistance offered by each tissue layer. The derived apparent permeability of CRPE is summarized along with the experimental values of the sclera and SCRPE in Table 2. The resistance offered by the sclera and CRPE to β-blocker permeability across the SCRPE is summarized in Table 2 (values in parentheses). The CRPE offers a significantly greater barrier than does the sclera for the transport of lipophilic β-blockers across the SCRPE. For hydrophilic molecules, the resistance for rabbit SCRPE transport offered by the sclera and CRPE were similar. In bovine and porcine tissues, the CRPE offered a greater barrier than the sclera for both hydrophilic and lipophilic molecules. In the bovine and porcine models, the CRPE offered a >84% barrier for the transport of hydrophilic β-blockers. The barriers were most resistant (>95%) to lipophilic molecules. 

Influence of Solute Lipophilicity on Transport across the Sclera and SCRPE

The correlation of solute lipophilicity (log P) with the cumulative percentages of transport across the sclera and SCRPE is shown in Figure 5. Solute lipophilicity correlated negatively with both apparent permeability (P_app) (Supplementary Figs. S2, S3) and cumulative percentage of transport across the sclera and SCRPE in all species (Fig. 5 and Supplementary Fig. S4). A better correlation was observed with log P (Fig. 5; Supplementary Fig. S2) than log D (Supplementary Figs. S3, S4) in all species for both the sclera and SCRPE. 

Figure 5.

 

The cumulative percentage of transport across the sclera and SCRPE declined with the increase in drug lipophilicity in the various species. Correlation of the cumulative percentages of β-blocker transport at the end of 6 hours with the log P in (A) albino rabbit, (B) pigmented rabbit, (C) human, (D) porcine, and (E) bovine tissues. The results are expressed as the mean ± SD for n = 6.

Tissue Thickness

The measured tissue thickness data for the sclera and SCRPE from the equator region of bovine, porcine, and pigmented and albino rabbit eyes are summarized in Table 4. The reported literature values are also summarized for comparison purposes. 

Table 4.

 

Thickness of Bovine, Porcine, Rabbit, and Human Sclera and SCRPE from the Equator Region

Species	Sclera Thickness (μm)	SCRPE Thickness (μm)	Reported Sclera Thickness (μm)
Albino rabbit	255 ± 28	317 ± 23	200 16
Pigmented rabbit	250 ± 21	295 ± 11	200 16
Porcine	546 ± 16	650 ± 27	560 17
Bovine	654 ± 33	769 ± 41	646 ± 43 5
Human	—	495	390 18

 Data are expressed as the mean ± SD for n = 6.

Melanin Content in the Sclera and CRPE

Measured melanin content of the sclera and CRPE of various species is shown in Figure 6. The melanin content in the CRPE was significantly higher than in the sclera in the BN rat, pigmented rabbit, porcine, and bovine models. There was no difference in the melanin content of these tissues in the albino rabbit. The melanin content in the CRPE was on the order of BN rat > porcine ∼ bovine > pigmented rabbit ≫ albino rabbit. The reported melanin content in human (brown eyes) CRPE (53.6 ± 1.1 μg/g of tissue) ¹⁹ falls between that in the pigmented and albino rabbits. 

Figure 6.

 

Melanin content in the sclera and CRPE of albino rabbit, pigmented rabbit, porcine, bovine, and BN rat eyes. All measures except those in the rat tissues were made in the present study. The rat tissue measurements are reported elsewhere by our group. ¹⁰ The data are expressed as the mean ± SD for n = 4. †Significantly different from the CRPE in the same species. Significantly different from *albino rabbit CRPE and ‡pigmented rabbit CRPE.

Cross-Species Comparison

For the cross-species comparison, the cumulative percentage of transport of β-blockers across the sclera and SCRPE after normalization for tissue thickness and melanin content in the CRPE were compared by using linear regression (Figs. 7). The cross-species correlation for the cumulative percentage of transport before normalization for tissue thickness and melanin content are given in Supplementary Figures S5A and S5B. A one-to-one correlation (slope ≈ 1) was observed for scleral transport normalized with tissue thickness between the bovine and rabbit model (Fig. 7A). For SCRPE, the bovine model showed close correlation (slope = 0.75–1.5) with porcine, human, and pigmented rabbit for SCRPE transport normalized with tissue thickness and melanin content. There was a large difference between the albino rabbit and the bovine, porcine, human, and pigmented rabbit (slope = 9.0–19.1) models for SCRPE transport (Fig. 7B). The Spearman's rho correlation for cumulative percentage of transport and apparent permeability coefficients for β-blockers showed good correlation across the species, with the correlation coefficient ranging from 0.76 to 1.00 (Supplementary Table S2). The cumulative percentage of transport and apparent permeability coefficients for human SCRPE showed very good cross-species correlation across all species with the correlation coefficient being ≥0.90. The cross-species comparison between preclinical models and human tissue data indicated that the preclinical models can predict the delivery across human tissues. 

Figure 7.

 

The cross-species correlation of β-blocker transport in the (A) sclera and (B) SCRPE. Data are expressed as the mean for n = 6. Rho (ρ), Spearman's rho correlation coefficient. Scleral data were normalized to the tissue thickness and the SCRPE data were normalized to the tissue thickness and the CRPE melanin content.

Tissue Accumulation of β-Blockers at the End of the 6-Hour Transport Study

The tissue concentration of β-blockers (micrograms per gram of tissue) in the sclera and CRPE at the end of 6 hours of transport is shown in Figure 8. In all species, the scleral and CRPE drug accumulation was the highest for propranolol followed by pindolol, whereas atenolol, sotalol, and nadolol exhibited the lowest accumulation. The sclera and CRPE concentrations of β-blockers for all species was in the order of propranolol > pindolol > betaxolol > metoprolol ∼ timolol > nadololol ∼ sotalol ∼ atenolol. The tissue concentrations were 1.9- to 3.1-fold higher in the pigmented CRPE compared with the albino CRPE. 

Figure 8.

 

β-Blocker accumulation in the (A) sclera and (B) CRPE at the end of a 6-hour transport study in various species. The β-blockers are arranged from low to high log P. The results are expressed as the mean ± SD for n = 6.

Ex Vivo Tissue Distribution of β-Blockers in a Euthanatized Rat Model

The concentrations of β-blockers in posterior segment eye tissues in euthanatized BN rats at 6 hours after subconjunctival injection are shown in Figure 9. As is evident from the figure, the highest drug accumulation was observed in the CRPE. The drug accumulation of the lipophilic β-blockers was higher than that of the hydrophilic ones. Drug levels in the vitreous of the lipophilic β-blockers were lower than those of the hydrophilic β-blockers. 

Figure 9.

 

An increase in lipophilicity did not result in enhanced drug transport to the vitreous in euthanatized BN rats. Shown are rat ocular tissue concentrations of β-blockers at the end of 6 hours after periocular administration of a solution of eight β-blockers in phosphate-buffered saline. The β-blockers are arranged from low to high log P. The data are expressed as the mean ± SD for n = 4.

We compared the in vitro percentage of transport of β-blockers across SCRPE in rabbit, human, porcine, and bovine models with the vitreous levels in the ex vivo rat model (Fig. 10). It is interesting to note that bovine and porcine models offered the best positive correlations with R ² ≥ 0.84. The human model offered intermediate positive correlation (R ² = 0.71). The pigmented (R ² = 0.61) and albino (R ² = 0.60) rabbit also showed positive correlation, with the R ² values being lower. 

Figure 10.

 

In vitro transport across human, porcine, and bovine SCRPE correlated better with ex vivo vitreal drug delivery in BN rats than that in the rabbit. Shown is the correlation of the ex vivo vitreal concentration of β-blockers at the end of 6 hours in euthanatized BN rats after periocular administration with in vitro cumulative percentages of transport at the end of 6 hours across the SCRPE in the various species. The results are expressed as the mean ± SD (n = 4) for ex vivo vitreal concentration and the mean ± SD (n = 6) for in vitro transport data.

Discussion

In this study, six animal and human models, demonstrated the following results, for the first time: (1) the SCRPE transport of a series of β-blockers exhibited species and strain (pigmented versus albino) differences; (2) when compared with that in the sclera, the SCRPE transport decreased more significantly as a function of β-blocker lipophilicity in multiple species; (3) pigment in the CRPE was a significant barrier for SCRPE transport of all β-blockers assessed; (4) sclera/SCRPE transport could be correlated between species; (5) porcine, bovine, and human SCRPE transport was more predictive of ex vivo rat vitreal delivery of β-blockers than was the rabbit SCRPE transport; (6) the CRPE pigment content in various species has an inverse relationship with the SCRPE transport of β-blockers; (7) species differences in scleral transport can be fully explained on the basis of tissue thickness; and (8) species differences in SCRPE transport can be largely explained on the basis of differences in tissue thickness and melanin content. 

In an earlier report, ⁵ using mannitol, sodium fluorescein, celecoxib, and budesonide, we demonstrated that bovine sclera–choroid transport declines with an increase in lipophilicity. In the present study we assessed the influence of solute lipophilicity on transport across the sclera and SCRPE, using eight β-blockers as permeants in five different preclinical models. Further, we assessed human tissue and a rat ex vivo model for SCRPE transport of β-blockers. β-Blockers are commonly used to study the influence of drug lipophilicity on permeability of various tissues, including those of the eye, ^{20 –23} since they are available in a wide range of lipophilicities, similar molecular weight (∼300), and similar pKa (∼9.2). ²⁴ The properties of the β-blockers used in this study are summarized in Table 1. 

On the basis of experiments with albino and pigmented rabbit eye tissues in this study, it is evident that the presence of melanin pigment reduced β-blocker permeability across the SCRPE. Both the cumulative percentage of transport and apparent permeability coefficients are significantly lower across the pigmented SCRPE compared with the albino rabbit tissue (Fig. 3, Table 2; Supplementary Table S1). Since melanin pigment is negatively charged and hydrophobic, ^25,26 this pigment in the choroid and RPE may have bound the β-blockers, potentially leading to tissue retention of the drug and reduced transport during the course of the study. Positively charged molecules as well as lipophilic molecules have more affinity for melanin ^26,27 than do hydrophilic molecules. β-Blockers are positively charged at pH 7.4 and are known to bind significantly to melanin. ^{27 –29} In our previous study we have shown high tissue accumulation of lipophilic β-blockers in the bovine and rat CRPE. ⁶ Consistent with this previous study, we observed that tissue accumulation of the lipophilic β-blockers is about two times higher in vivo in pigmented rabbit CRPE when compared with that in the albino rabbit (Fig. 8B). The melanin pigment is present mainly in CRPE, with very low quantities present in the sclera (Fig. 6). Our previous studies showed that the CRPE concentration of lipophilic celecoxib is significantly higher in vivo in the BN rat (pigmented) than in the SD rat (albino) after periocular injection. ¹⁰ Thus, melanin-containing CRPE retains the β-blockers, thereby reducing their transscleral transport and, potentially, in vivo delivery to the retina and the vitreous. 

Similar to rabbit studies, the permeability and cumulative percentage of transport of β-blockers across SCRPE decreased with an increase in lipophilicity in bovine, porcine, and human species as well (Figs. 3, 5; Supplementary Figs. S2, S3, S4). Further, the apparent permeabilities across the SCRPE as well as the CRPE were lower in bovine and porcine species, when compared with that in rabbits. This finding may be due to the presence of a greater pigment quantity in bovine and porcine species or to any differences in the chemical composition of the pigment or tissue present in the various species. An estimation of the in vitro melanin content showed that the amount of melanin pigment per gram of tissue weight is significantly higher in bovine and porcine CRPE than in the pigmented rabbit CRPE (Fig. 6). 

The resistance offered by the CRPE for β-blocker transport increased with an increase in lipophilicity (Table 2). The CRPE offered 84.1% to 99.8% resistance to the transport of β-blockers across the bovine and porcine SCRPEs. However, this resistance was lower (51%–64%) in the pigmented rabbits, possibly because of pigment and tissue differences. In bovine and porcine tissues, a clear increase in resistance to CRPE transport is evident with an increase in drug lipophilicity. 

The transport of β-blockers across the sclera (devoid of CRPE) decreased with an increase in solute lipophilicity in all three species tested (Figs. 1, 5). The transport was the highest across the rabbit sclera followed by the porcine sclera and was the least across the bovine sclera (Fig. 1, Table 2; Supplementary Table S1). A key contributing factor for the species differences in scleral transport is scleral thickness. In this study we used sclera from the equator region of the eye. In this region, the bovine sclera was the thickest followed by the porcine sclera; scleral thickness was the least in the rabbit (Table 4). The permeability of solutes across the sclera is inversely related to the thickness. For sotalol, the cumulative percentage of transport exhibited a high inverse correlation (R ² = 0.961) with scleral thickness. When cumulative percentage of transport and apparent permeability coefficients were normalized by multiplying by scleral thickness, the species differences for scleral transport were abolished (Fig. 2). Thus, differences in scleral thickness appear to contribute to differences in scleral transport of β-blockers among various species. We did not observe a significant difference in the scleral transport of seven of eight β-blockers between albino and pigmented animals. However, we observed a 31% decrease in scleral transport of propranolol in pigmented rabbits when compared to that in albino rabbits. Differences in pigment or other binding elements may account for a slight reduction in the transport of propranolol across pigmented sclera. 

Although different in relative magnitudes, the β-blocker cumulative percentages of transport as well as apparent permeabilities correlated well between species for a given tissue (sclera or SCRPE) in the bovine, porcine, human, and pigmented rabbit models (Fig. 7; Supplementary Figs. S5A, S5B). The Spearman's rho correlation coefficients for these comparisons were ≥0.76 and ≥0.88 for sclera and SCRPE, respectively (Supplementary Table S2). The apparent permeability and cumulative percentages of transport of β-blockers across the human SCRPE strongly correlated with all four preclinical species (bovine, pig, and pigmented and albino rabbits) with a Spearman's rho correlation coefficient ≥0.905 (Fig. 7B; Supplementary Table S2). The differences in magnitude of transport for the sclera were abolished on normalization of transport data to scleral thickness (Fig. 2). The differences in magnitude of transport were substantially reduced on normalization of SCRPE transport data to SCRPE thickness and melanin content in the CRPE (Figs. 4 and S1). Thus, tissue thickness and pigment content are two major contributing factors for the observed interspecies differences in SCRPE transport. Any remaining species differences in SCRPE transport might be due to a more complex dependence of transport on pigment content, differences in retinal pigment epithelial permeability, and differences in drug binding affinities and capacities. 

To translate the excised tissue studies of rabbit, human, porcine, and bovine species to those of commonly used rodent models in preclinical efficacy studies, we assessed the ex vivo tissue distribution of β-blockers in euthanatized BN rats. Euthanatized rats were used to eliminate the effect of blood circulation and clearances pathways, which were also absent in the excised tissue studies. The tissue levels of β-blockers in the BN rat sclera and CRPE increased with an increase in solute lipophilicity (Fig. 9), similar to drug accumulation in the sclera and CRPE at the end of the transport study in the bovine, porcine, human, and pigmented rabbit models (Fig. 8). In the vitreous humor, on the other hand, the drug delivery tended to decline with an increase in solute lipophilicity. In vitro transport across the bovine, porcine, and human SCRPE correlated well with ex vivo vitreal concentrations in BN rats, while the correlation coefficients were lower for SCRPE transport in the rabbit models (Fig. 10). Thus, on the basis of these results, correlation between species appears feasible in some cases for transscleral drug delivery. Further, the euthanatized rat model appears to be useful in assessing solute transport. 

Conclusions

Drug transport across the sclera and SCRPE is affected by the lipophilicity of the permeant, with the permeability being higher for hydrophilic molecules than for lipophilic ones. This difference occurs because the CRPE layer binds and impedes the transport of lipophilic solutes, especially in pigmented animals. Further, pigmented CRPE tissue is the most rate-limiting barrier for the transport of lipophilic solutes across the SCRPE. Thus, lipophilic solutes do not offer a significant advantage in permeating the SCRPE to reach the vitreous humor, especially when presented at low concentrations for sustained drug therapy. The pigmented rabbit CRPE exhibits greater tissue accumulation than its albino counterpart and results in reduced SCRPE transport for all β-blockers in the pigmented rabbit. Rabbit tissues have low pigment levels and are much more permeable than those of the other pigmented species assessed. Species-dependent differences in tissue pigment levels and transscleral and trans-CRPE transport should be taken into account in studies to explain the differences in transscleral drug delivery and/or effects. We could explain species differences in scleral β-blocker transport completely by normalizing the data to scleral thickness. Species differences in the SCRPE transport of β-blockers could also be explained to a large extent by the differences in tissue thickness and melanin content. We demonstrated that interspecies correlations are feasible between various preclinical models as well as the human model. 

Supplementary Materials

Figure sf01, TIF - Figure sf01, TIF 

Figure sf02, TIF - Figure sf02, TIF 

Figure sf03, TIF - Figure sf03, TIF 

Figure sf04, TIF - Figure sf04, TIF 

Figure sf5a, TIF - Figure sf5a, TIF 

Figure sf5b, TIF - Figure sf5b, TIF 

Table st01, DOC - Table st01, DOC 

Table st02, DOC - Table st02, DOC 

Footnotes

 Presented at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2009.

Footnotes

 Supported by National Institutes of Health Grants EY017533, EY018940, and EY017045 (through Emory University) and a UNMC graduate fellowship (NC).

Footnotes

 Disclosure: R.S. Kadam, None; N.P.S. Cheruvu, None; H.F. Edelhauser, None; U.B. Kompella, None

The authors thank Ashish Thakur and Gajanan Jadhav for their assistance during the rabbit studies and Indushekhar Persaud for assistance with the tissue thickness measurements.