Human scleral diffusion of anticancer drugs from solution and nanoparticle formulation.

PURPOSE: To determine the transscleral permeability of chemotherapeutic drugs vinblastine and doxorubicin for treatment of intraocular tumors, and to compare the use of doxorubicin encapsulated in PLGA and liposome nanoparticles. METHODS: Human sclera was isolated and mounted in a Lucite chamber. Fluorescently tagged vinblastine (VIN), innately fluorescent free doxorubicin (DOX), PLGA doxorubicin (PLGA-DOX), or Doxil (Tibotec Therapeutics) were added to the episcleral donor chamber. The choroidal side was perfused with Balanced Salt Solution. Perfusate fractions were collected over 24 h and measured for fluorescence. Following the experiment, tissue sections were imaged, underwent a drug wash out procedure, and tissue drug content was analyzed using an LC-MS/MS method. RESULTS: Within 24 h, a total of 68%, 74%, 29%, and 1.9% of the drug dose from VIN, DOX, PLGA-DOX, and Doxil, respectively, diffused across the sclera. VIN and DOX scleral tissue showed strong fluorescence after 24 h. PLGA-DOX displayed scattered fluorescence, and Doxil indicated minimal fluorescence. LC-MS/MS revealed strong tissue binding of DOX. CONCLUSIONS: This study suggests both vinblastine and doxorubicin are able to diffuse across human sclera. In addition, PLGA nanoparticles delivered doxorubicin at a slower rate across the sclera, and the liposome preparation resulted in the slowest delivery of drug.

Trans-scleral permeability of Oregon green 488.

PURPOSE: The aim of this study was to determine the scleral permeability of a commercially available version of 2',7'-difluorofluorescein (OG) and compare it to that of sodium fluorescein (NaF). METHODS: Both in vitro and in vivo experiments were performed. For the ex vivo experiment, a Lucite block perfusion chamber with human donor sclera was used. Two hundred microliters (200 microL) of 2.5 mg/ml OG or NaF was placed in the donor chamber. The OG and NaF concentration that diffused across the sclera was measured every 2 h for 24 h by fluorometry, and the fluorescence in the sclera was examined by fluorescent microscopy. In vivo experiments consisted of live rabbits treated with a 0.2-mL subtenon injection of 7.5 mg/ml solution of either OG or NaF in the right eye. Intraocular fluorescence was measured by ocular fluorophotometry. RESULTS: The scleral permeability coefficient (K(trans)) of OG was 3.93 +/- 1.01 x 10(-7) cm/sec and that of NaF was 4.41 +/- 1.32 x 10(-7) cm/s. Both OG and NaF were visible throughout the sclera after 24 hours. Peak vitreous concentration after subtenon injection in rabbits was 6.48 +/- 2.65 ng/mL of OG at 2 min and 47.15 +/- 13.3 ng/mL of NaF at 10 min. CONCLUSIONS: OG was able to diffuse across the sclera and thus could be potentially useful as a fluorescent tag for intraocular drug delivery studies. However, its permeability was substantially less than that of NaF.

Nonpeptide somatostatin receptor agonists specifically target ocular neovascularization via the somatostatin type 2 receptor.

PURPOSE: To define the molecular pharmacology underlying the antiangiogenic effects of nonpeptide imidazolidine-2,4-dione somatostatin receptor agonists (NISAs) and evaluate the efficacy of NISA in ocular versus systemic delivery routes in ocular disease models. METHODS: Functional inhibitory effects of the NISAs and the somatostatin peptide analogue octreotide were evaluated in vitro by chemotaxis, proliferation, and tube-formation assays. The oxygen-induced retinopathy (OIR) model and the laser model of choroidal neovascularization (CNV) were used to test the in vivo efficacy of NISAs. Transscleral permeability of a candidate NISA was also measured. RESULTS: NISAs inhibited growth factor-induced HREC proliferation, migration and tube formation with submicromolar potencies (IC(50), 0.1-1.0 microM) comparable to octreotide. In the OIR model, systemic administration of the NISAs RFE-007 and RFE-011 inhibited retinal neovascularization in a dose-dependent manner, comparable to octreotide. In the CNV model, intravitreal RFE-011 resulted in a 56% reduction (P < 0.01) in CNV lesion area, whereas systemic administration resulted in a 35% reduction (P < 0.05) in lesion area. RFE-011 demonstrated transscleral penetration. CONCLUSIONS: Micromolar concentrations of octreotide and NISAs are necessary for antiangiogenic effects, whereas nanomolar concentrations are effective for endocrine inhibition. This suggests that the antiangiogenic activity of NISAs and octreotide is mediated by an overall much less efficient downstream coupling mechanism than is growth hormone release. As a result, the intravitreal or transscleral route of administration should be seriously considered for future clinical studies of SSTR2 agonists used for treatment of ocular neovascularization to ensure efficacious concentrations in the target retinal and choroidal tissue.

Pharmacokinetics of intraocular drug delivery of Oregon green 488-labeled triamcinolone by subtenon injection using ocular fluorophotometry in rabbit eyes.

PURPOSE: To evaluate the transscleral delivery of Oregon Green-labeled triamcinolone acetonide (OGTA) into the eye. METHODS: Ex vivo experiments were performed on rabbit sclera in a Lucite block perfusion chamber. Two hundred microliters OGTA (5 mg/mL) was placed on the outer surface of the sclera for 24 hours. The exposed sclera was divided into two pieces; one half for a washout of OGTA and the other for histology. The concentration of OGTA that diffused through the sclera (n = 6) was measured by fluorometry. Two hundred microliters of OGTA (5 mg/mL) was also injected subtenon into live (n = 6) and euthanatized rabbits (n = 3). Intraocular OGTA concentrations were measured by ocular fluorophotometry. RESULTS: The permeability constant for the transscleral diffusion (K(trans)) of OGTA was 1.12 x 10(-7) +/- 0.08 cm/s (n = 8) during the steady state perfusion. Washout tests showed higher OGTA concentration in the sclera exposed to OGTA for 4 hours than in that exposed for 1 hour. Fluorescent microscopy showed OGTA fluorescence throughout the exposed sclera, as evidence of scleral penetration of OGTA. The maximum OGTA concentration in the retina/choroid after subtenon injection was 25.77 +/- 10.26 ng/mL in the live rabbit at 3 hours and 84.68 +/- 21.04 ng/mL in the euthanatized rabbits at 8 hours. CONCLUSIONS: OGTA is capable of diffusing across isolated rabbit sclera ex vivo and into the retina/choroid via transscleral diffusion from a subtenon depot in vivo. Conjunctival and choroidal circulation decreased the drug delivery of OGTA.

Measurement and prediction of lateral diffusion within human sclera.

PURPOSE: Drug delivery via the sclera is a promising approach to retinal disorder treatments that require access to the posterior segment of the eye. To complement existing studies of transverse diffusion across the sclera, this study examined lateral diffusion within the sclera parallel to the scleral surface. METHODS: Using sulforhodamine as a model hydrophilic drug, rates of diffusion were measured in strips of human cadaveric sclera for up to 1 week. Data were analyzed with a mathematical model based on theoretical expressions for one-dimensional diffusion. RESULTS: Measurable amounts of sulforhodamine were detected at distances of 5 and 10 mm from the sulforhodamine donor reservoir at 4 hours and 3 days, respectively. The effective lateral diffusivity of sulforhodamine was determined to be 3.82 x 10(-6) cm2/s, which is similar in magnitude to the transverse diffusivity. The theoretical model agreed with experimental values with an average error of 39%. CONCLUSIONS: This study shows that the lateral diffusion of sulforhodamine in human sclera is slow and localizes to the site of administration.

The influence of intraocular pressure on the transscleral diffusion of high-molecular-weight compounds.

PURPOSE: To evaluate the effects of intraocular pressure on the permeability of the human sclera to high-molecular-weight compounds. METHODS: Human transscleral permeability to FITC-albumin (70 kDa) and 70-kDa and 150-kDa FITC-dextran was determined at transscleral pressures from 0 to 60 mm Hg. For each compound at each pressure, six to eight experiments were performed. Scleral sections were mounted in a two-compartment perfusion chamber. Temperature was maintained at 37 degrees C. Fractions of choroidal perfusate were collected, and fluorescence was measured with a spectrofluorometer. From these data, scleral permeability K(trans) (in centimeters per second) was calculated. RESULTS: Permeability to FITC-albumin was decreased by approximately one half when pressure was elevated from 0 to 30 mm Hg (P < 0.05). No significant differences in permeability to 70-kDa FITC-dextran were observed at pressures from 0 to 60 mm Hg. Permeability to 150-kDa FITC-dextran decreased by a little more than one half when transscleral pressure was raised from 0 to 15 mm Hg and was approximately 10 times lower at 60 mm Hg than at 0 mm Hg (P < 0.01). CONCLUSIONS: Human sclera was permeable to compounds with a molecular weight of up to 150 kDa at transscleral pressures ranging from 0 to 60 mm Hg. Transscleral diffusion was relatively unaffected by the pressure gradient, although for 150-kDa FITC-dextran at 60 mm Hg a 10-fold decrease was observed compared with that at 0 mm Hg. These experiments suggest that high-molecular-weight compounds (e.g., immunoglobulins and oligonucleotides) could be effectively delivered transsclerally to the intraocular tissues under circumstances of physiological or elevated intraocular pressure.

In vitro sustained human transscleral drug delivery of fluorescein-labeled dexamethasone and methotrexate with fibrin sealant.

PURPOSE: To study the use of fibrin sealant as a drug delivery system for the sustained transscleral delivery of dexamethasone and methotrexate. METHODS: Scleral sections excised from moist-chamber-stored human globes were mounted in a perfusion chamber. Dexamethasone-fluorescein or methotrexate-fluorescein in either fibrin sealant or balanced salt solution (BSS) was applied to the episcleral surface. BSS was perfused to the choroidal side, fluorescence was measured in perfusate fractions, and an apparent scleral permeability P(A) was calculated for each solute-vehicle combination. RESULTS: P(A) for both compounds was significantly lower with fibrin sealant delivery compared to delivery in BSS (p < 0.001). However, the fibrin sealant vehicle provided a more sustained release of both drugs through 24 hr. CONCLUSIONS: Incorporating dexamethasone and methotrexate into a fibrin sealant provided a more gradual drug delivery and a more uniform delivery compared to dissolving these drugs in BSS. Fibrin sealant could be useful for transscleral delivery for posterior segment disease.

Ex vivo confocal microscopy of human LASIK corneas with histologic and ultrastructural correlation.

OBJECTIVE: To perform confocal microscopy on postmortem human LASIK corneas and correlate these findings to histologic and ultrastructure evaluations. DESIGN: Prospective, consecutive, observational case series. PARTICIPANTS: Ninety postmortem LASIK corneas (47 patients) were evaluated for histopathology, of which 22 consecutive corneas (12 patients) were also evaluated by confocal microscopy. Six normal corneas (3 patients) served as controls. METHODS: This observational case series involving 22 corneas from 12 patients with postoperative intervals from 1 month to 6.5 years after LASIK surgery were collected. The corneas were mounted in an artificial anterior chamber and perfused with balanced salt solution before confocal microscopy was performed on the center of the cornea. The corneas were then bisected and processed for light and transmission electron microscopy. RESULTS: Confocal microscopy, along with histologic and ultrastructural correlations, demonstrated that the most prevalent alterations in the centers of LASIK corneas were a slightly thickened epithelium caused by focal basal epithelial cell hypertrophic modifications, random undulations in Bowman's layer over the flap surface, and a variably thick hypocellular primitive stromal interface scar. By using confocal microscopy, the interface wound was easily identified in 100% of the cases because numerous brightly reflective interface particles were always present in the hypocellular primitive stromal scar. These particles were found primarily to consist of organic cellular constituents, some of which were transient in nature. CONCLUSION: After LASIK, active stromal wound healing in the central cornea results in the production of a hypocellular primitive stromal scar, whereas secondary tissue adjustments seem to cause the Bowman's layer undulations and the subsequent epithelial cell modifications. Most of the interface particles revealed by confocal microscopy in the region of the stromal scar are organic in nature and presumably innocuous to the cornea.

Transscleral permeability of fluorescent-labeled antibiotics.

PURPOSE: To determine the in vitro transscleral permeability of antibiotics for posterior segment infection. METHODS: Scleral sections from moist chamber-stored human globes were mounted in a 2-compartment perfusion chamber. Fluorescent-labeled vancomycin, polymyxin B, or penicillin G was added to the episcleral surface, while the choroidal side was slowly perfused with balanced salt solution (Alcon Laboratories, Inc., Ft. Worth, TX). The perfusate was collected and fluorescence was measured using a fluorometer. From the measurements, permeability (Ktrans) was calculated. Photomicrographs were taken with a fluorescent microscope to evaluate tissue absorption. RESULTS: The Ktrans values (cm/s, mean +/- standard error) were 6.66 +/- 1.46 x 10(-7) for vancomycin, 3.90 +/- 0.59 x 10(-7) for polymyxin B, and 1.89 +/- 0.21 x 10(-6) for penicillin G. The percent of antibiotic that diffused across the sclera from the donor chamber in 24 hours was 20.6 +/- 4.2 for vancomycin, 12.6 +/- 2.0% for polymyxin B, and 50.8 +/- 4.8% for penicillin G. CONCLUSIONS: This study shows that human sclera is more permeable to lower molecular weight, water-soluble penicillin G than to vancomycin or polymyxin B. The data suggests that a local, noninvasive, transscleral drug-delivery method may be reasonable for treating intraocular infections.

Retinal function after subconjunctival injection of carboplatin in fibrin sealant.

PURPOSE: To evaluate retinal function in a rabbit model after subconjunctival delivery of carboplatin in balanced saline solution (BSS) or fibrin sealant to determine possible retinal toxicity. METHODS: One group of rabbits (n = 5) received a unilateral subconjunctival injection of 12.2 +/- 1.0 mg/mL of carboplatin (Paraplatin) in BSS. Another group of rabbits (n = 5) received 25.1 +/- 7.7 mg/mL of carboplatin in fibrin sealant (Hemaseel APR). Rabbits injected with fibrin sealant only (n = 2) and BSS only (n = 2) were used as control groups. Electroretinographic recordings consisted of a series of intensities presented under dark- and light-adapted conditions. Electroretinograms were recorded before the injection (baseline) and 2 days, 1, 2, and 3 weeks after the injection. After 3 weeks, all rabbit eyes were obtained for histopathologic examination. RESULTS: Transient reductions in the dark-adapted b-wave amplitudes were noted 2 days after treatment for eyes injected with carboplatin compared with the vehicle-only treatment groups. Other treatment groups and postinjection time points showed no significant changes from baseline. Retinal structure and thickness were normal 3 weeks after treatment for all treatment groups. CONCLUSION: Subconjunctival delivery of carboplatin in fibrin sealant or BSS does not have a toxic effect on retinal function or structure in a non-tumor-bearing rabbit model at the doses used in this study at 3 weeks' follow-up.

Drug delivery through the sclera: effects of thickness, hydration, and sustained release systems.

The purpose of this study was to determine whether trans-scleral pressure affects scleral solute permeability by altering scleral thickness or hydration, and to investigate the sustained release delivery of dexamethasone. Scleral sections from donor human globes were mounted for in vitro flux studies. Scleral thickness and hydration were measured as functions of trans-scleral pressure. For the sustained release studies, 3H-dexamethasone in pluronic F-127 gel or in fibrin sealant was added to the episcleral side of the tissue and flux studies were performed. While scleral thickness showed a tendency to decrease with increasing pressure, a significant decrease in thickness was measured only at a trans-scleral pressure of 60 mmHg. No significant changes in scleral hydration were measured over the range of trans-scleral pressures studied. The apparent permeability constants (Ktrans) of human sclera for 3H-dexamethasone in BSS plus, fibrin sealant and F-127 gel were 11.5 x 10(-6), 7.3 x 10(-6), and 1.5 x 10(-6) cm sec(-1), respectively. Human scleral permeability to dexamethasone differed significantly among the three vehicles (p < 0.0001). Cumulative delivery of dexamethasone from BSS plus, F-127 gel, and fibrin sealant were 85.0, 29.3, and 67.9% at 20 hr, respectively. Scleral hydration was unaffected by trans-scleral pressures. Scleral thinning was only observed at 60 mmHg. Trans-scleral pressures below 60 mmHg would not be expected to significantly affect the permeability of the tissue to solutes in the size range of conventional drugs. F-127 gel and fibrin sealant provided a slow, relatively uniform sustained release through a 24 hr period. These systems might be employed to achieve sustained therapeutic levels of drugs to the posterior segment of eye.

Transscleral permeability and intraocular concentrations of cisplatin from a collagen matrix.

This study determined the in vitro permeability of cisplatin through isolated human sclera as delivered by a collagen matrix vehicle. Short-term and long-term intraocular levels of cisplatin were also measured in the rabbit eye after a subconjunctival injection. Cisplatin in either a collagen matrix vehicle or a control balanced salt solution (BSS) vehicle was applied to human sclera mounted in a specially designed in vitro perfusion chamber. The amount of cisplatin that diffused across the sclera was measured in hourly samples for 24 h using atomic absorption spectrometry. In vivo studies were also performed in Dutch Belted rabbits given subconjunctival injections of cisplatin in collagen matrix or in BSS. Eyes were enucleated at 1.5 h and 2 weeks after injection, frozen, and dissected to determine the intraocular cisplatin concentrations. Cisplatin had a peak in vitro scleral permeability constant of 8.3+/-1.2 x 10(-6) and 20.1+/-1.8 x 10(-6) cm/s, delivered in collagen matrix and in BSS, respectively (mean+/-S.D.). At the end of the in vitro experiments, 35.9+/-4.6% of the cisplatin remained in the collagen matrix, while 0.8+/-0.2% remained in the BSS vehicle. Subconjunctival injection of cisplatin in the collagen matrix vehicle achieved 3.3+/-0.1 microg/ml in the vitreous humor at 1.5 h and 0.1+/-0.1 microg/ml at 2 weeks. This vehicle also achieved a cisplatin concentration of 73.5+/-23.9 microg/mg in the choroid and retina at 1.5 h and 3.2+/-1.3 microg/mg at 2 weeks. Compared to BSS, the collagen matrix vehicle provided a more controlled release of cisplatin, and after subconjunctival injection into rabbits, attained higher drug levels in several ocular tissues.

Transscleral diffusion of carboplatin: an in vitro and in vivo study.

OBJECTIVES: To compare the in vitro scleral permeability of carboplatin using either a fibrin sealant or a balanced salt solution (BSS) vehicle and to measure in vivo ocular tissue levels following subconjunctival injection of carboplatin in fibrin sealant or BSS. METHODS: The permeability of carboplatin in fibrin sealant or BSS through human eye bank sclera was tested using an in vitro perfusion apparatus. Levels of carboplatin permeating the sclera were measured every hour for 24 hours using atomic absorption spectrometry. In vivo studies were performed in Dutch Belted rabbits injected subconjunctivally with carboplatin in either fibrin sealant or BSS; eyes were enucleated at 1(1/2) hours, 48 hours, and 2 weeks after injection, and levels of carboplatin were measured in various tissues. RESULTS: In vitro carboplatin in fibrin sealant had a peak permeability constant of 13.7 +/- 2.3 x 10(-6) cm/s; carboplatin in BSS, 27.0 +/- 1.7 x 10(-6) cm/s. After 24 hours, 33.2% +/- 1.8% of the carboplatin was retained in the fibrin sealant, while 5.5% +/- 1.0% was retained in the BSS. In vivo subconjunctival injection of carboplatin in fibrin sealant vehicle achieved 11.83 +/- 5.16 microg/mL in the vitreous at 1(1/2) hours and 0.03 +/- 0.06 microg/mL in the vitreous at 2 weeks. The fibrin sealant also attained 396.59 +/- 177.84 microg/mg in the choroid and retina at 1(1/2) hours and 3.38 +/- 1.97 microg/mg in the choroid and retina at 2 weeks. (Data are given as mean +/- SEM.) CONCLUSION: Fibrin sealant provided a more controlled and localized release of carboplatin and delivered carboplatin to the ocular tissues for up to 2 weeks. CLINICAL RELEVANCE: This study reports the use of fibrin sealant as a subconjunctival delivery vehicle for carboplatin, and quantifies ocular drug levels achieved in an animal model.

In vitro human scleral permeability of fluorescein, dexamethasone-fluorescein, methotrexate-fluorescein and rhodamine 6G and the use of a coated coil as a new drug delivery system.

PURPOSE: To determine the in vitro human scleral permeability of several dyes and drugdye combinations with varying molecular weights (MW) and lipid solubilities (fluorescein, dexamethasone-fluorescein, methotrexate-fluorescein, and rhodamine). Coils coated with rhodamine were also evaluated for scleral permeability and sustained release. METHODS: Scleral sections excised from moist chamber stored human globes were mounted in a 2-compartment perfusion chamber. A small depot of drug/dye (100 microl of 10(-4) M fluorescein, dexamethasone-fluorescein, methotrexate-fluorescein or rhodamine) or a coated coil in 100 microl of BSS was added to the episcleral surface while perfusing BSS to the choroidal side. The perfusate was collected and measured for fluorescence. Permeability was calculated as Ktrans from the flux measurements. RESULTS: Ktrans values (cm/sec, mean +/- SE) for the studied dyes and drug-dye combinations were 5.21 +/- 0.71 x 10(-6) for fluorescein, 1.64 +/- 0.17 x 10(-6) for dexamethasone-fluorescein, 3.36 +/- 0.62 x 10(-6) for methotrexate-fluorescein, 1.86 +/- 0.39 x 10(-6) for rhodamine and 2.18 +/- 0.23 x 10(-6) for the rhodamine from the coils. We found a significant difference between the permeability of the sclera to fluorescein and dexamethasone-fluorescein (P < 0.001), methotrexate-fluorescein (P < 0.05) and rhodamine (P < 0.001). Steady state flux was observed from the rhodamine coil. CONCLUSION: The rank order of scleral permeability to the studied dyes is as follows: fluorescein > methotrexate-fluorescein > rhodamine coil > rhodamine 6G > dexamethasone-fluorescein. Differences in scleral permeability are related to MW and lipid solubility. Prolonged transscleral diffusion of rhodamine delivered by solution and by coil are similar.