Biomechanical and wound healing characteristics of corneas after excimer laser keratorefractive surgery: is there a difference between advanced surface ablation and sub-Bowman's keratomileusis?

PURPOSE: To describe the biomechanical and wound healing characteristics of corneas after excimer laser keratorefractive surgery. METHODS: Histologic, ultrastructural, and cohesive tensile strength evaluations were performed on 25 normal human corneal specimens, 206 uncomplicated LASIK specimens, 17 uncomplicated sub-Bowman's keratomileusis (SBK) specimens, 4 uncomplicated photorefractive keratectomy (PRK) specimens, 2 uncomplicated advanced surface ablation (ASA) specimens, 5 keratoconus specimens, 12 postoperative LASIK ectasia specimens, and 1 postoperative PRK ectasia specimen and compared to previously published studies. RESULTS: Histologic and ultrastructural studies of normal corneas showed significant differences in the direction of collagen fibrils and/or the degree of lamellar interweaving in Bowman's layer, the anterior third of the corneal stroma, the posterior two-thirds of the corneal stroma, and Descemet's membrane. Cohesive tensile strength testing directly supported these morphologic findings as the stronger, more rigid regions of the cornea were located anteriorly and peripherally. This suggests that PRK and ASA, and secondarily SBK, should be biomechanically safer than conventional LASIK with regard to risk for causing keratectasia after surgery. Because adult human corneal stromal wounds heal slowly and incompletely, all excimer laser keratorefractive surgical techniques still have some distinct disadvantages due to inadequate reparative wound healing. Despite reducing some of the risk for corneal haze compared to conventional PRK, ASA cases still can develop corneal haze or breakthrough haze from the hypercellular fibrotic stromal scarring. In contrast, similar to conventional LASIK, SBK still has the short- and long-term potential for interface wound complications from the hypocellular primitive stromal scar. CONCLUSIONS: Ophthalmic pathology and basic science research show that SBK and ASA are improvements in excimer laser keratorefractive surgery compared to conventional LASIK or PRK, particularly with regard to maintaining corneal biomechanics and perhaps moderately reducing the risk of corneal haze. However, most of the disadvantages caused by wound healing issues remain.

Depth-dependent cohesive tensile strength in human donor corneas: implications for refractive surgery.

PURPOSE: To determine the cohesive tensile strength throughout the stroma of normal human donor corneas and evaluate the relevance of these findings within the context of current excimer laser surgical techniques. METHODS: Twenty normal corneoscleral buttons from 11 donors were obtained from the Georgia Eye Bank. The corneas were cut into 3-mm strips, dissected at varying stromal depths, mechanically separated through the dissection plane using a motorized extensometer, and measured for cohesive tensile strength. Central corneal thickness and dissection depth were measured by routine light microscopy and correlated with cohesive tensile strength measurements. RESULTS: A strong negative correlation was noted between stromal depth and cohesive tensile strength (r = -0.93). The anterior corneal stroma directly adjacent to Bowman's layer followed by the underlying anterior 40% of the corneal stroma had the highest cohesive tensile strength. Cohesive tensile strength plateaued from 40% to 90% corneal stromal depth and then declined rapidly from the posterior 10% of the stroma to Descemet's membrane. The anterior 40% of the corneal stroma had significantly higher cohesive tensile strength than the posterior 60% (33.3 g/mm vs 19.6 g/mm, P < .00001). Within the central 40% to 60% depth, a positive correlation was found between increased age and increased tensile strength (r = 0.67), with corneal tensile strength increasing 38% from ages 20 to 78 years. CONCLUSIONS: The anterior 40% of the central corneal stroma is the strongest region of the cornea, whereas the posterior 60% of the stroma is at least 50% weaker. The risk for ectasia may therefore be greater with ablations into the posterior stroma. Increasing age is associated with increased corneal cohesive tensile strength.

Targeting tissue factor-expressing tumor angiogenesis and tumors with EF24 conjugated to factor VIIa.

Tissue factor (TF) is aberrantly expressed on tumor vascular endothelial cells (VECs) and on cancer cells in many malignant tumors, but not on normal VECs, making it a promising target for cancer therapy. As a transmembrane receptor for coagulation factor VIIa (fVIIa), TF forms a high-affinity complex with its cognate ligand, which is subsequently internalized through receptor-mediated endocytosis. Accordingly, we developed a method for selectively delivering EF24, a potent synthetic curcumin analog, to TF-expressing tumor vasculature and tumors using fVIIa as a drug carrier. EF24 was chemically conjugated to fVIIa through a tripeptide-chloromethyl ketone. After binding to TF-expressing targets by fVIIa, EF24 will be endocytosed along with the drug carrier and will exert its cytotoxicity. Our results showed that the conjugate inhibits vascular endothelial growth factor-induced angiogenesis in a rabbit cornea model and in a Matrigel model in athymic nude mice. The conjugate-induced apoptosis in tumor cells and significantly reduced tumor size in human breast cancer xenografts in athymic nude mice as compared with the unconjugated EF24. By conjugating potent drugs to fVIIa, this targeted drug delivery system has the potential to enhance therapeutic efficacy, while reducing toxic side effects. It may also prove to be useful for treating drug-resistant tumors and micro-metastases in addition to primary tumors.

Review of corneal endothelial specular microscopy for FDA clinical trials of refractive procedures, surgical devices, and new intraocular drugs and solutions.

Specular microscopy can provide a noninvasive morphologic analysis of the corneal endothelial cell layer from subjects enrolled in clinical trials. The analysis provides a measure of the endothelial cell physiologic reserve from aging, ocular surgical procedures, pharmaceutical exposure, and general health of the corneal endothelium. The purpose of this review is to discuss normal and stressed endothelial cell morphology, the techniques for determining the morphology parameters, and clinical trial applications.

Pharmacokinetics of intraocular drug delivery by periocular injections using ocular fluorophotometry.

PURPOSE: To evaluate the pharmacokinetics of the periocular injections: posterior subtenon (PST), retrobulbar (RB), and subconjunctival (SC) injection. METHODS: Two sodium fluorescein (NaF) concentrations, 2.5 mg in 0.1 mL (NaF1) and 2.5 mg in 0.5 mL (NaF2) were injected into live rabbits by the PST (NaF1 n = 4, NaF2 n = 3), RB (NaF1 n = 10), SC (NaF1 n = 6), and intravenous (IV, NaF1 n = 6) routes and into euthanatized rabbits by the RB (NaF1 n = 8) route. NaF concentrations in the choroid/retina, vitreous, and anterior segment were measured by ocular fluorophotometry. The NaF level in the contralateral choroid/retina was used as a measure of the systemic drug levels. RESULTS: The maximum NaF concentrations (nanograms per milliliter) in the choroid/retina after PST, RB, SC, and IV were 757 +/- 549 at 2 hours, 906 +/- 1014 at 1 hour, 320 +/- 462 at 2 hours, and 865 +/- 363 at 5 to 10 minutes, respectively. The PST had the highest and most prolonged vitreous NaF1 concentration (maximum: 270 +/- 226 ng/mL at 3.5 hours). The contralateral peak choroid/retina NaF levels after the RB, SC, and IV injections were 7, 4, and 21 times greater than after the PST injection. The SC injection had the highest anterior segment NaF concentration (5364 +/- 2840 ng/mL at 2 hours). PST with NaF2 resulted in intraocular NaF levels higher than with NaF1. CONCLUSIONS: NaF reaches the choroid/retina by transscleral diffusion from the periocular depot. The orbital and conjunctival vasculature and lymphatics have a larger role in NaF clearance than does the choroid. NaF diffuses into the vitreous from the choroid and the anterior segment; the periocular depot location determines the predominant diffusion pathway. The duration of high NaF levels in the choroid/retina or the anterior segment determines vitreous NaF levels. PST is the best periocular route for vitreous NaF delivery with minimal systemic levels. Increasing the volume of NaF PST depot enhances transscleral drug delivery.

Coated microneedles for drug delivery to the eye.

PURPOSE: To test the hypothesis that coated microneedles can deliver drugs into the eye via intrascleral and intracorneal routes in a minimally invasive manner. METHODS: Solid metal microneedles measuring 500 to 750 microm in length were coated with model drugs, protein, and DNA; inserted into nonpreserved human cadaveric sclera; and imaged. Microneedles coated with sodium fluorescein were then inserted into rabbit cornea in vivo. After needle removal, fluorescein concentration in the anterior segment of the rabbit eye was measured for 24 hours. Similar experiments were performed using pilocarpine-coated microneedles, and the rabbit pupil size was monitored afterward. RESULTS: In vitro insertion tests showed that microneedles were mechanically strong enough to penetrate into human cadaveric sclera and that the drug coating rapidly dissolved off the needles within the scleral tissue within 30 seconds after insertion. In vivo delivery from fluorescein-coated microneedles showed that fluorescein concentrations in the anterior chamber were 60 times greater than those achieved by topical application without microneedles. Similarly, microneedle delivery of pilocarpine caused rapid and extensive rabbit pupil constriction. There were no measurable inflammatory responses caused by microneedle insertion. CONCLUSIONS: This study demonstrated for the first time that coated microneedles can deliver drugs into the eye via intrascleral and intracorneal routes. This minimally invasive approach may avoid the complications associated with intraocular injection and systemic administration.

Corneal epithelial testing strategies for safety evaluation of ophthalmic formulations.

The toxicity of an ophthalmic formulation was tested both in vivo and in vitro. Initial tests on transformed human corneal epithelial (HCE-T) cells in monolayer cultures resulted in adverse effects on cell morphology. The adverse effects were unexpected since the formulation caused no damage to the cornea in vivo. These results suggested HCE-T monolayers do not adequately model the intact corneal epithelium. Therefore, further in vitro studies were conducted to investigate reversibility of morphologic changes, proliferation, cell viability, and effects on corneal epithelial barrier function. These tests showed that the formulation had no adverse effects on cell viability and proliferation. Multilayered cultures of HCE-T cells at an air interface provide a morphologic and physiologic model more relevant to the in vivo cornea. This study demonstrates the importance of selecting appropriate models when conducting in vitro toxicity studies so that potentially effective ophthalmic formulations are not rejected based on false positive in vitro endpoints.

Cohesive tensile strength of human LASIK wounds with histologic, ultrastructural, and clinical correlations.

PURPOSE: To measure the cohesive tensile strength of human LASIK corneal wounds. METHODS: Twenty-five human eye bank corneas from 13 donors that had LASIK were cut into 4-mm corneoscleral strips and dissected to expose the interface wound. Using a motorized pulling device, the force required to separate the wound was recorded. Intact and separated specimens were processed for light and electron microscopy. Five normal human eye bank corneas from 5 donors served as controls. A retrospective clinical study was done on 144 eyes that had LASIK flap-lift retreatments, providing clinical correlation. RESULTS: The mean tensile strength of the central and paracentral LASIK wounds showed minimal change in strength over time after surgery, averaging 2.4% (0.72 +/- 0.33 g/mm) of controls (30.06 +/- 2.93 g/mm). In contrast, the mean peak tensile strength of the flap wound margin gradually increased over time after surgery, reaching maximum values by 3.5 years when the average was 28.1% (8.46 +/- 4.56 g/mm) of controls. Histologic and ultrastructural correlative studies found that the plane of separation always occurred in the lamellar wound, which consisted of a hypocellular primitive stromal scar centrally and paracentrally and a hypercellular fibrotic stromal scar at the flap wound margin. The pathologic correlations demonstrated that the strongest wound margin scars had no epithelial cell ingrowth-the strongest typically being wider or more peripherally located. In contrast, the weakest wound margin scars had epithelial cell ingrowth. The clinical series demonstrated the ability to lift LASIK flaps without complications during retreatments up to 8.4 years after initial surgery, correlating well with the laboratory results. CONCLUSIONS: The human comeal stroma typically heals after LASIK in a limited and incomplete fashion; this results in a weak, central and paracentral hypocellular primitive stromal scar that averages 2.4% as strong as normal comeal stroma. Conversely, the LASIK flap wound margin heals by producing a 10-fold stronger, peripheral hypercellular fibrotic stromal scar that averages 28.1% as strong as normal comeal stromal, but displays marked variability.

Targeted administration into the suprachoroidal space using a microneedle for drug delivery to the posterior segment of the eye.

PURPOSE: This study seeks to determine the intraocular pharmacokinetics of molecules and particles injected into the suprachoroidal space of the rabbit eye in vivo using a hollow microneedle. METHODS: Suprachoroidal injections of fluorescein and fluorescently tagged dextrans (40 and 250 kDa), bevacizumab, and polymeric particles (20 nm to 10 µm in diameter) were performed using microneedles in New Zealand white rabbits. The fluorescence intensity within the eye was monitored in each animal using an ocular fluorophotometer to determine the distribution of the injected material in the eye over time as compared with intravitreal injection of fluorescein. Fundus photography and histology were performed as well. RESULTS: Molecules and particles injected near the limbus using a microneedle flowed circumferentially around the eye within the suprachoroidal space. By targeting the suprachoroidal space, the concentration of injected materials was at least 10-fold higher in the back of the eye tissues than in anterior tissues. In contrast, intravitreal injection of fluorescein targeted the vitreous humor with no significant selectivity for posterior versus anterior segment tissues. Half-lives in the suprachoroidal space for molecules of molecular weight from 0.3 to 250 kDa ranged from 1.2 to 7.9 hours. In contrast, particles ranging in size from 20 nm to 10 µm remained primarily in the suprachoroidal space and choroid for a period of months and did not clear the eye. No adverse effects of injection into the suprachoroidal space were observed. CONCLUSION: Injection into the suprachoroidal space using a microneedle offers a simple and minimally invasive way to target the delivery of drugs to the choroid and retina.

In vivo ocular fluorophotometry: delivery of fluoresceinated dextrans via transscleral diffusion in rabbits.

PURPOSE: To evaluate the transscleral delivery of fluoresceinated dextrans (FITC-D) with molecular mass up to 70 kDa to the rabbit posterior segment using sub-Tenon injections. METHODS: Eighteen NZW rabbits received a unilateral 200-µL injection of 2 mg/mL sodium fluorescein (NaF), 25 mg/mL 40-kDa FITC-D, or 25 mg/mL 70-kDa FITC-D, with (n = 9) or without (n = 9) immediate euthanatization. In live animals, fluorescence was measured in the retina/choroid and mid-vitreous by fluorophotometry, immediately after injection and after 4, 24, 48, and 72 hours. Euthanatized animals were examined hourly through 5 or 6 hours. RESULTS: In live animals, the average peak NaF concentration in the retina/choroid was 310.2 ng/mL, measured 3 hours after injection. Average 40- and 70-kDa FITC-D concentrations in the retina/choroid peaked at 5409.6 and 2375.6 ng/mL, respectively, 24 hours after injection. Fluorescence returned to baseline levels 6 hours after NaF injection, and 48 and 72 hours after 40- and 70-kDa FITC-D injections, respectively. Rabbits that received NaF followed by euthanatization exhibited a continuous increase in retina/choroid and mid-vitreous fluorescence, beginning 1 hour after injection, whereas FITC-D-injected eyes did not show elevated retina/choroid or mid-vitreous fluorescence through 6 hours. CONCLUSIONS: FITC-D weighing up to 70-kDa, as well as NaF, reached the posterior retina/choroid after sub-Tenon injections in live rabbits. NaF and 40-kDa FITC-D reached higher peak concentrations and were cleared from the eye more rapidly than was 70-kDa FITC-D. There was minimal penetration of NaF and FITC-D into the mid-vitreous in the in vivo experiments.

Mucoadhesive microparticles in a rapidly dissolving tablet for sustained drug delivery to the eye.

PURPOSE: To test the hypothesis that mucoadhesive microparticles formulated in a rapidly dissolving tablet can achieve sustained drug delivery to the eye. METHODS: Mucoadhesive microparticles, smaller than 5 µm were fabricated with poly(lactic-co-glycolic acid) and poly(ethylene glycol) as a core material and mucoadhesion promoter, respectively, and encapsulated pilocarpine as a model drug. These microparticles were embedded in a poly(vinyl alcohol) matrix to form a dry tablet designed to reduce rapid clearance of the microparticles on initial application to the eye. RESULTS: This in vitro drug release study exhibited that for all formulations, approximately 90% of pilocarpine was released during the first 10 minutes, and the remaining 10% was released slowly for 3 hours. In vivo mucoadhesion test on the rabbit eye indicated that mucoadhesive microparticles adhered significantly better to the preocular surface than other formulations. To assess the pharmacodynamics, the most prolonged pilocarpine-induced pupil constriction was observed in rabbit eyes in vivo using a tablet with mucoadhesive microparticles; it lasted up to 330 minutes. CONCLUSIONS: The authors conclude that mucoadhesive microparticles formulated into a dry dosage form is a promising system for sustained drug delivery to the eye.

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.

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.

Mucoadhesive microdiscs engineered for ophthalmic drug delivery: effect of particle geometry and formulation on preocular residence time.

PURPOSE: To test the hypothesis that mucoadhesive microdiscs formulated in a rapidly dissolving tablet can increase preocular residence time. METHODS: Microparticles smaller than 10 mum in diameter were fabricated by emulsification with poly(lactic-co-glycolic acid) as a core material and, in some cases, poly(ethylene glycol) as a mucoadhesion promoter. To examine the effect of particle geometry, microparticles were also cut to have flat surfaces (i.e., microdiscs) and were compared with spherical particles (i.e., microspheres). In vitro mucoadhesion of microparticles was tested on a mucous layer under shear stress, mimicking the human blink. The resultant microparticles were also formulated in two dosage forms, an aqueous suspension and a dry tablet, to test the effect of formulation on the retention capacity of microparticles on the preocular space of rabbits in vivo. RESULTS: Mucoadhesive microdiscs adhered better to the simulated ocular surface than did other types of microparticles. When a dry tablet embedded with mucoadhesive microdiscs was administered in the cul-de-sac of the rabbit eye in vivo, these microdiscs exhibited longer retention than the other formulations tested in this study. More than 40% and 17% of mucoadhesive microdiscs remained on the preocular surface at 10 minutes and 30 minutes after administration, respectively. Fluorescence images from the eye surface showed that mucoadhesive microdiscs remain for at least 1 hour in the lower fornix. CONCLUSIONS: This study demonstrated that mucoadhesive microdiscs formulated in a dry tablet can achieve a prolonged residence time on the preocular surface and thus are a promising drug delivery system for ophthalmic applications.

Low incidence of iris pigmentation and eyelash changes in 2 randomized clinical trials with unoprostone isopropyl 0.15%.

OBJECTIVE: To assess whether iris color and eyelash changes occur with the use of unoprostone for 2 years. DESIGN: The 2 clinical trials described herein were prospective, randomized, double-masked, active-controlled, parallel group, multicenter studies. PARTICIPANTS: A total of 1131 patients with primary open-angle glaucoma or ocular hypertension participated in 2 clinical trials and received either unoprostone isopropyl 0.15% (659), timolol maleate 0.5% (331), or betaxolol hydrochloride 0.5% (141), 1 drop per eye twice daily for up to 24 months. METHODS: Color photographs (1:1 magnification) were taken of the iris and eyelid of each patient at baseline and at regular intervals thereafter through month 24 using a standardized camera system. Photography included 7 views of each eye plus a calibration photograph and a patient identification photograph, for a total of 16 photographs per patient per visit. Two independent (masked) readers subjectively compared baseline iris colors to subsequent visits. Side view photographs of the upper and lower eyelashes were used for the eyelash length analysis, with each having sufficient depth of field and a sufficient number of eyelashes in focus. Similarly, frontal eyelash views were used for the eyelash density analysis. MAIN OUTCOME MEASURES: Changes from baseline in iris color and eyelash length and density within and between treatment groups. RESULTS: Seven cases of iris color change (1.06%) were confirmed in patients treated with unoprostone for up to 24 months; no confirmed cases were reported in the timolol or betaxolol groups. In the unoprostone group, cases of iris color change were confirmed at months 12 (1 case), 18 (2 cases), and 24 (4 cases). No clinically relevant differences were observed among treatment groups for changes from baseline in eyelash length or density. CONCLUSION: Although iris hyperpigmentation and abnormal eyelash changes may occur after treatment with unoprostone, the incidence of these events appears to be low in the 2-year clinical study.