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Diabetic macular edema (DME) is the leading cause of vision loss and blindness among working-age adults. Although current intravitreal anti-vascular endothelial growth factor (VEGF) therapies improve vision for many patients with DME, approximately half do not achieve the visual acuity required to drive. We therefore sought additional approaches to resolve edema and improve vision for these patients.We explored direct agonists of Tie2, a receptor known to stabilize vasculature and prevent leakage. We identified a multivalent PEG-Fab conjugate, Tie2.1-hexamer, that oligomerizes Tie2 and drives receptor activation and characterized its activities in vitro and in vivo.Tie2.1-hexamer normalized and stabilized intercellular junctions of stressed endothelial cell monolayers in vitro, suppressed vascular leak in mice under conditions where anti-VEGF alone was ineffective, and demonstrated extended ocular exposure and robust pharmacodynamic responses in non-human primates.Tie2.1-hexamer directly activates the Tie2 pathway, reduces vascular leak, and is persistent within the vitreal humor.Our study presents a promising potential therapeutic for the treatment of DME.

Sustained drug delivery formulations are developed to reduce dose frequency while maintaining efficacy of intravitreal (ITV) administered therapeutics. Available safety data for components novel to the eye’s posterior segment may be limited, requiring preclinical assessments to identify potential toxicities. We evaluated the in vivo and in vitro safety of two solvents, acetyl triethyl citrate (ATEC) and benzyl benzoate (BB), as novel sustained delivery formulations for ITV administration. In vivo tolerability was assessed following ITV administration of ATEC and BB to rabbits and cynomolgus monkeys. In rabbits, ITV solvent administration resulted in moderate to severe retinal toxicity characterized by focal retinal necrosis and/or degeneration, sometimes accompanied by inflammation, with a clear association between the physical presence of the solvent and areas of retinal damage. In contrast, solvent administration in monkeys appeared well tolerated, producing no histologic abnormalities. Toxicity in primary human retinal pigment epithelial cells, characterized by cellular toxicity and mitochondrial injury, corroborated the retinal toxicity in rabbits. In conclusion, ITV solvent depots of ATEC or BB result in chemical and focal retinal toxicity in rabbits, but not monkeys. Additional investigation is needed to demonstrate a sufficient margin of safety prior to use of ATEC or BB in ITV drug products.

Purpose To evaluate the safety and efficacy of the Port Delivery System with ranibizumab (PDS) for neovascular age-related macular degeneration (nAMD) treatment. Design Phase 2, multicenter, randomized, active treatment–controlled clinical trial. Participants Patients diagnosed with nAMD within 9 months who had received 2 or more prior anti–vascular endothelial growth factor intravitreal injections and were responsive to treatment. Methods Patients were randomized 3:3:3:2 to receive the PDS filled with ranibizumab 10 mg/ml, 40 mg/ml, 100 mg/ml, or monthly intravitreal ranibizumab 0.5-mg injections. Main Outcome Measures Time to first implant refill assessed when the last enrolled patient completed the month 9 visit (primary efficacy end point), improvement in best-corrected visual acuity (BCVA) and central foveal thickness (CFT), and safety. Results The primary analysis population was 220 patients, with 58, 62, 59, and 41 patients in the PDS 10-mg/ml, PDS 40-mg/ml, PDS 100-mg/ml, and monthly intravitreal ranibizumab 0.5-mg arms, respectively. Median time to first implant refill was 8.7, 13.0, and 15.0 months in the PDS 10-mg/ml, PDS 40-mg/ml, and PDS 100-mg/ml arms, respectively. At month 9, the adjusted mean BCVA change from baseline was ‒3.2 Early Treatment Diabetic Retinopathy Study (ETDRS) letters, ‒0.5 ETDRS letters, +5.0 ETDRS letters, and +3.9 ETDRS letters in the PDS 10-mg/ml, PDS 40-mg/ml, PDS 100-mg/ml, and monthly intravitreal ranibizumab 0.5-mg arms, respectively. At month 9, the adjusted mean CFT change from baseline was similar in the PDS 100-mg/ml and monthly intravitreal ranibizumab 0.5-mg arms. The optimized PDS implant insertion and refill procedures were generally well tolerated. After surgical procedure optimization, postoperative vitreous hemorrhage rate was 4.5% (7/157; 1 event classified as serious). There was no evidence of implant clogging. Conclusions In the phase 2 Ladder trial, the PDS was generally well tolerated and demonstrated a dose response across multiple end points in patients with nAMD. The PDS 100-mg/ml arm showed visual and anatomic outcomes comparable with monthly intravitreal ranibizumab 0.5-mg injections but with a reduced total number of ranibizumab treatments. The PDS has the potential to reduce treatment burden in nAMD while maintaining vision.

FHTR2163 is an antigen-binding fragment of a humanized immunoglobulin G1 monoclonal antibody directed against high-temperature requirement A serine peptidase 1 (HTRA1) that is being developed as a potential intravitreal (ITV) treatment for patients with geographic atrophy (GA), an advanced form of dry age-related macular degeneration. The nonclinical toxicology program was designed to assess the safety and tolerability of HTRA1 inhibition following ITV administration of FHTR2163 to support ITV administration in patients with GA. FHTR2163 was well tolerated in a single-dose ITV-administered 8-day toxicity study in cynomolgus monkeys following a 50 µL high (>700 mOsm/kg) osmolality formulation up to 12.5 mg/eye; however, 100 µL (2× 50 µL injections) of a high-osmolality formulation resulted in transient retinal detachment. Repeat-dose ITV administration every 2 weeks of FHTR2163 was well tolerated in 8- and 26-week studies with ITV injection of 100 µL (2× 50 μL) of iso-osmolar formulation up to 15 mg/eye, or 50 µL of the high-osmolality formulation up to 12.5 mg/eye. Observed transient and reversible ocular effects included inflammation and perivascular infiltrates, consistent with an immune response attributed to the administration of heterologous (humanized) protein. Overall, FHTR2163 was well tolerated, and the nonclinical package supported the continued clinical development of FHTR2163 in patients with GA.

The Port Delivery System with ranibizumab (PDS) is an investigational drug delivery system designed to provide continuous intravitreal release of ranibizumab for extended durations. The PDS consists of a permanent, surgically placed, refillable intraocular implant; a customized formulation of ranibizumab; and ancillary devices to support surgery and refill procedures. A toxicology program was conducted to evaluate the ocular toxicology and biocompatibility of the PDS to support its clinical development program and product registrational activities. PDS safety studies included a 6-month chronic toxicology evaluation in minipigs as well as evaluation of nonfunctional surrogate implants (comprised of the same implant materials but without ranibizumab) in rabbits. Biocompatibility of the implant and ancillary devices was evaluated in both in vitro and in vivo studies. Implants and extracts from implants and ancillary devices were nongenotoxic, noncytotoxic, nonsensitizing, and nonirritating. Ocular findings were comparable between implanted and sham-operated eyes, and no systemic toxicity was observed. The results of this nonclinical toxicology program demonstrated that the PDS was biocompatible and that intravitreal delivery of ranibizumab via the PDS did not introduce any new toxicology-related safety concerns relative to intravitreal injections, supporting ongoing PDS clinical development and product registrational evaluation.

Supplemental Digital Content is Available in the Text. This minipig model of vitreous hemorrhage demonstrated that scleral dissection followed by thorough laser ablation of the pars plana before the pars plana incision could mitigate vitreous hemorrhage during Port Delivery System with ranibizumab implant insertion surgery., Purpose: To develop an animal model of vitreous hemorrhage (VH) to explore the impact of surgical parameters on VH associated with insertion of the Port Delivery System with ranibizumab (PDS) implant. Methods: Ninety eyes from 45 treatment-naive male Yucatan minipigs received PDS implant insertion or a sham procedure. The effect of prophylactic pars plana hemostasis, scleral incision length, scleral cauterization, surgical blade type/size, and viscoelastic usage on postsurgical VH was investigated. Results: Postsurgical VH was detected in 60.0% (54/90) of implanted eyes. A systematic effect on VH was only detected for pars plana hemostasis before the pars plana incision. The percentage of eyes with VH was 96.6% (28/29) among eyes that did not receive prophylactic pars plana hemostasis and 42.4% (24/58) among eyes that did. There was no VH in eyes that received laser ablation of the pars plana using overlapping 1,000-ms spots; pars plana cautery or diathermy was less effective. The majority of all VH cases (83.3% [45/54]) were of mild to moderate severity (involving ≤25% of the fundus). Conclusion: In this minipig surgical model of VH, scleral dissection followed by pars plana laser ablation before pars plana incision most effectively mitigated VH secondary to PDS implant insertion.

Purpose: To characterize the inflammatory response and determine the no-observable-effect level (NOEL) in cynomolgus monkey eyes after intravitreal (ITV) injection of endotoxin. Methods: The inflammatory response to endotoxin was assessed in a single-dose study in monkeys at doses of 0.01 to 0.51 endotoxin units (EU)/eye. Tolerability was assessed by clinical ophthalmic examinations, intraocular pressure measurements, fundus color photography, optical coherence tomography, and anatomic pathology. Results: ITV injection of endotoxin at ≥0.04 EU/eye resulted in a dose-related anterior segment inflammatory response. No aqueous flare or cell was noted in the 0.01 EU/eye dose group. A more delayed posterior segment response characterized by vitreous cell was observed beginning on day 5, peaking on day 15, and decreasing in some groups. Microscopic findings of mononuclear cell infiltrates in the vitreous were observed in eyes given ≥0.21 EU/eye. Conclusion: The NOEL for ITV endotoxin in cynomolgus monkeys was 0.01 EU/eye, suggesting that this species is as sensitive as rabbits to the effects of endotoxin. The vitreous cavity also appears more sensitive to endotoxin than the anterior segment/aqueous chamber. Overall, the magnitude of the inflammatory response at ≥0.04 EU/eye suggests that dose-response curve in monkeys is steeper than in rabbits. These data highlight the importance of assessing endotoxin level in ITV formulations, as levels as low as 0.04 EU/eye may confound the safety evaluations of ITV therapeutics in cynomolgus monkeys.

To evaluate the safety and efficacy of the Port Delivery System with ranibizumab (PDS) for neovascular age-related macular degeneration (nAMD) treatment.Phase 2, multicenter, randomized, active treatment-controlled clinical trial.Patients diagnosed with nAMD within 9 months who had received 2 or more prior anti-vascular endothelial growth factor intravitreal injections and were responsive to treatment.Patients were randomized 3:3:3:2 to receive the PDS filled with ranibizumab 10 mg/ml, 40 mg/ml, 100 mg/ml, or monthly intravitreal ranibizumab 0.5-mg injections.Time to first implant refill assessed when the last enrolled patient completed the month 9 visit (primary efficacy end point), improvement in best-corrected visual acuity (BCVA) and central foveal thickness (CFT), and safety.The primary analysis population was 220 patients, with 58, 62, 59, and 41 patients in the PDS 10-mg/ml, PDS 40-mg/ml, PDS 100-mg/ml, and monthly intravitreal ranibizumab 0.5-mg arms, respectively. Median time to first implant refill was 8.7, 13.0, and 15.0 months in the PDS 10-mg/ml, PDS 40-mg/ml, and PDS 100-mg/ml arms, respectively. At month 9, the adjusted mean BCVA change from baseline was ‒3.2 Early Treatment Diabetic Retinopathy Study (ETDRS) letters, ‒0.5 ETDRS letters, +5.0 ETDRS letters, and +3.9 ETDRS letters in the PDS 10-mg/ml, PDS 40-mg/ml, PDS 100-mg/ml, and monthly intravitreal ranibizumab 0.5-mg arms, respectively. At month 9, the adjusted mean CFT change from baseline was similar in the PDS 100-mg/ml and monthly intravitreal ranibizumab 0.5-mg arms. The optimized PDS implant insertion and refill procedures were generally well tolerated. After surgical procedure optimization, postoperative vitreous hemorrhage rate was 4.5% (7/157; 1 event classified as serious). There was no evidence of implant clogging.In the phase 2 Ladder trial, the PDS was generally well tolerated and demonstrated a dose response across multiple end points in patients with nAMD. The PDS 100-mg/ml arm showed visual and anatomic outcomes comparable with monthly intravitreal ranibizumab 0.5-mg injections but with a reduced total number of ranibizumab treatments. The PDS has the potential to reduce treatment burden in nAMD while maintaining vision.

Many long-acting delivery strategies for ocular indications rely on pH- and/or temperature-driven release of the therapeutic agent and degradation of the drug carrier. Yet, these physiological parameters are poorly characterized in ocular animal models. These strategies aim at reducing the frequency of dosing, which is of particular interest for the treatment of chronic disorders affecting the posterior segment of the eye, such as macular degeneration that warrants monthly or every other month intravitreal injections. We used anesthetized white New Zealand rabbits, Yucatan mini pigs, and cynomolgus monkeys to characterize pH and temperature in several vitreous locations and the central aqueous location. We also established post mortem pH changes in the vitreous. Our data showed regional and species differences, which need to be factored into strategies for developing biodegradable long-acting delivery systems.

Historically, it was thought that lens protein was sequestered, and injury to the lens capsule causing release of lens material into the eye would always result in ocular inflammation. Currently, it is believed that lens antigens are recognized as self, subject to normal T-cell tolerance. Three different single-dose intravitreal injection/implantation studies of 4 different test materials, ranging from 4 to 6 weeks in length, were performed in New Zealand White rabbits. The test materials included polymer microspheres, polymer rods, a solvent, and a hydrogel. Intravitreal injection/implantation procedures were performed on day 1, and indirect ophthalmoscopy and slit-lamp biomicroscopy examinations were performed by board-certified veterinary ophthalmologists periodically throughout the course of each study. None of the affected animals received corticosteroids or other immunomodulatory agents during the course of the studies. Four rabbits had perforation of the posterior lens capsule during the injection/implantation procedure on day 1, visible on clinical ophthalmic examination as lens capsule alterations described as "lens hits" and/or incipient posterior cataracts. Findings on slit-lamp biomicroscopy examination were limited to vitreous cells in 2 of the animals, although not centered on the area of lens capsule disturbance. Histologically, there was no evidence of inflammation in association with extruded lens protein material in any of the affected eyes. These results indicate that iatrogenic damage to the lens capsule during aseptically performed intravitreal injections/implantations does not appear to induce inflammation in rabbits.

Purpose Investigate a significant, dose-related increase in IOP, leading to glaucomatous damage to the neuroretina and optic nerve following intravitreal (ITV) administration of a bispecific F(ab')2 [anti-VEGF/Angiopoietins [ANGPT]F(ab')2] molecule in adult monkeys. Methods ITV ocular tolerability and investigation of anti-VEGF/ANGPT F(ab')2 (blocking both ANGPT1 and ANGPT2) was done in monkeys; mechanistic studies were done in neonatal mice. Results Following the second ITV dose of anti-VEGF/ANGPT F(ab')2, all 1.5- and 4-mg/eye treated monkeys developed elevated IOP, which eventually was associated with optic disc cupping and thinning of the neuroretinal rim. Histopathologic examination showed nonreversible axonal degeneration in the optic nerves of animals administered 1.5 mg/eye and higher that was considered secondary to high IOP. Anti-ANGPT Fab also caused elevated IOP in monkeys, but anti-VEGF Fab did not contribute to the IOP increase. In addition, an anti-ANGPT2-selective antibody did not change IOP. In mice simultaneous blockade of ANGPT1 and ANGPT2 impaired the expansion and formation of Schlemm's canal (SC) vessels, similar to genetic ablation of Angpt1/Angpt2 and their receptor TIE2. As previously reported, blocking ANGPT2 alone did not affect SC formation in mice. Conclusions Dual inhibition of ANGPT1/ANGPT2, but not ANGPT2 alone, leads to increased IOP and glaucomatous damage in monkeys. This confirms a role for TIE2/ANGPT signaling in the control of IOP in adults, a finding initially identified in transgenic mice. Dual pharmacologic inhibition of ANGPT1/ANGPT2 may affect aqueous drainage and homeostasis in adult monkeys and may be useful in developing novel models of glaucoma.

Innovative protein engineering and chemical conjugation technologies have yielded an impressive number of drug candidates in clinical development including >80 antibody drug conjugates, >60 bispecific antibodies, >35 Fc-fusion proteins and >10 immuno-cytokines. Despite these innovations, technological advances are needed to address unmet medical needs with new pharmacological mechanisms. Age-related eye diseases are among the most common causes of blindness and poor vision in the world. Many such diseases affect the back of the eye, where the inaccessibility of the site of action necessitates therapeutic delivery via intravitreal (IVT) injection. Treatments administered via this route typically have vitreal half-lives

Pycnogenol was used (a) to study its antioxidant activity, (b) to study its effects on lens integrity in organ culture and (c) in vivo to determine whether it could reduce the damage in model diabetic cataract.For (a) our luminescent antioxidant assay was used, (b) lenses were incubated in medium 199, with 55.6 mM glucose. Lenses were stained with 0.014 mM rhodamine 123 for 15 min to stain mitochondria, immobilized in 1% agarose in M199, and the equatorial region examined by a Zeiss confocal microscope. For (c) cataract grades of streptozotocin diabetic rats fed 1% pycnogenol were followed for 12 weeks.(a) Pycnogenol in vitro was an antioxidant when challenged with peroxide. (b) In vitro, when [570 mg/L] pycnogenol in dimethyl sulfoxide (DMSO) was used, lenses turned opaque after 3 d of incubation, in both pycnogenol controls and glucose + pycnogenol. Normal controls (DMSO, n = 4) and controls (n = 4) remained clear after 8 d of incubation. After 3 d of incubation with pycnogenol, cumulative protein leakage was greater than 0.28 mg/mL versus 8 d controls (0.018 mg/mL). Similar damage occurred at pycnogenol concentrations as low as 20 mg/L. The 20 mg/L pycnogenol control showed mitochondrial death, and calcium concentration in the lens equatorial differentiating fiber cells increased. (c) In vivo feeding pycnogenol resulted in similar growth and body condition for diabetic rats, and lower cataract grades at 9 and 11 weeks: final serum glucose levels were not significantly different, but glycohemoglobin A1 levels were significantly lower (83.9% of normal, p 0.05) in pycnogenol-fed diabetic rats.Although it appears that pycnogenol has a potential toxic effect on incubated lenses, it appears in vivo to have a marginal protective effect, and also significantly reduces glycation of proteins. Supported by Cognis US (formerly Henkel Chemical Co.) and Horphag Research.

PURPOSE To provide a description of the distribution and cell morphology, by using fluorescent markers and confocal laser scanning microscopy, of the corneal, limbal, and conjunctival epithelium of bovine eyes in vitro. METHODS Fresh enucleated bovine eyes were dissected within 2 hours postmortem. Central cornea, limbus, and bulbar conjunctiva were imaged with confocal microscopy after staining with acridine orange (AO) or calcein-acetoxymethyl and ethidium homodimer-1. Epithelial thickness, cell density, cell lamination, and cell morphology were evaluated at these 3 locations. RESULTS Corneal epithelium was the thickest, and the conjunctival epithelium was the thinnest. The cell morphology was similar to that found in previous histologic studies, and the cell density gradually decreased from the basal to superficial layers. Nuclear AO staining particles increased from basal to superficial cells. Limbal superficial epithelial cells showed less AO staining than corneal and conjunctival superficial cells. CONCLUSIONS Confocal results of the corneal central, limbal, and conjunctival morphology are similar to those found in traditional microscopic observations. Bovine central corneal epithelium is thicker than limbal epithelium. However, the nuclear AO staining pattern of unfixed ocular surface epithelium of bovine eyes in vitro might represent the cell differentiation status. With the aid of the fluorescence dye, confocal laser scanning microscopy can provide unique morphometric information about corneal, limbal, and conjunctival epithelial cells.

Purpose To analyze the interactions of benzalkonium chloride (BAK) with silicone-containing (lotrafilcon A and galyfilcon A) and p-HEMA-containing (etafilcon A and vifilcon A) hydrogel contact lenses and to examine the possibility of using sodium fluorescein permeability assay (SFPA), 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the bovine lens assay in conjunction with confocal laser scanning microscopy (CLSM) as a battery of in vitro alternatives to evaluate the potential toxicity of soft contact lenses. Methods Four soft contact lens types (Focus Monthly [vifilcon A], Focus NIGHT & DAY [lotrafilcon A], ACUVUE Advance With Hydraclear [galyfilcon A], and SUREVUE [etafilcon A]) were soaked for 24 hours in various concentrations of BAK (1%, 0.1%, 0.01%, and 0.001%) in 20-mL glass vials. After 24 hours, the lenses were gently washed in Hanks' Balanced Salt Solution (HBSS), placed in 5 mL of HBSS, and incubated for a total of 7 days at 37 degrees C, 5% CO2. BAK released into HBSS (i.e., the extract) was recovered from the vials and used as the test chemical in the SFPA (epithelium integrity), MTT assay (cellular viability), and the bovine lens assay with CLSM (mitochondrial metabolism and optical properties). The amount of BAK extracted from the various contact lenses was measured using an Abbe refractometer. Negative controls consisted of HBSS and contact lenses subjected to the same conditions as the treated contact lenses, but without BAK. Results Extracts obtained from soaking Focus Monthly lenses in BAK caused the most damage to the epithelium and mitochondrial metabolism. However, at 0.1% BAK extraction, all lens extracts showed increased levels of back vertex distance variability of the cultured bovine lens. Conclusions Unexpectedly, lenses extracted with HBSS showed SFPA and MTT assay responses and an observed effect on the bovine lens epithelium visualized by CLSM, indicating that unknown chemical agents may be leached from contact lens polymers.

Purpose Poly(lactic-co-glycolic) acid (PLGA) inserts have been successfully developed for the treatment of posterior eye disease as a means of reducing injection frequency of intravitreally administered therapeutics. PLGA microspheres are also of interest for the delivery of intravitreal drugs, since they offer the advantage of being easily injected without surgical procedures or large injectors. Methods In the current study, the toxicity of PLGA microspheres and rods was investigated in nonhuman primates (NHPs) and rabbits. An in vitro assessment of cytokine responses to PLGA in peripheral blood mononuclear cells (PBMCs) and macrophages was also performed. Results Intravitreal administration of 3, 10, or 12.5 mg/eye of PLGA microspheres in NHPs resulted in a severe immune response characterized by a foreign body response. Follow-up studies in the rabbit confirmed this finding for PLGA microspheres ranging in size from 20 to 100 μm. In contrast, administration of PLGA rod implants with a similar PLGA mass did not elicit a significant immune response. In vitro assays in PBMCs and macrophages confirmed proinflammatory cytokine release upon treatment with PLGA microspheres but not PLGA rods. Conclusions These data demonstrate a lack of tolerability of PLGA microspheres upon intravitreal injection, and suggest that the size, shape, and/or surface area of PLGA depots are critical attributes in determining ocular toxicity.

Intravitreal (ITV) dosing has become a clinically important route of administration for the treatment of uveitis, endophthalmitis, retinal vein occlusion, diabetic macular edema and age-related macular degeneration. Despite this, there are no validated non-clinical models of phototoxicity for ITV products.The objective of this study was to develop an ITV rabbit model of phototoxicity for use in assessing the photosafety of small molecules therapeutics.Dutch Belted rabbits were intravitreally injected bilaterally with four known phototoxicants: 8-methoxypsoralen, lomefloxacin, doxycycline and stannsoporfrin. Triescence(®), a non-phototoxic triamcinolone acetonide steroid formulation designed for ITV administration, was used as a negative control. One eye was then irradiated with solar-simulated ultraviolet radiation for 30 min, 1 h after dosing, while the other eye was occluded, serving as a non-irradiated control.Despite the direct administration of known phototoxicants into the vitreous, no evidence of ocular phototoxicity was observed in any dose group. Direct (non-phototoxic) retinal toxicity was observed in the doxycycline dose group only.These data suggest that the posterior segment of the rabbit eye is protected against phototoxicity by anatomical and/or physiological mechanisms, and is not a useful model for the assessment of phototoxicity of intravitreally administered molecules.

RetinoStat(®) is an equine infectious anemia virus-based lentiviral gene therapy vector that expresses the angiostatic proteins endostatin and angiostatin that is delivered via a subretinal injection for the treatment of the wet form of age-related macular degeneration. We initiated 6-month safety and biodistribution studies in two species; rhesus macaques and Dutch belted rabbits. After subretinal administration of RetinoStat the level of human endostatin and angiostatin proteins in the vitreous of treated rabbit eyes peaked at ∼1 month after dosing and remained elevated for the duration of the study. Regular ocular examinations revealed a mild to moderate transient ocular inflammation that resolved within 1 month of dosing in both species. There were no significant long-term changes in the electroretinograms or intraocular pressure measurements in either rabbits or macaques postdosing compared with the baseline reading in RetinoStat-treated eyes. Histological evaluation did not reveal any structural changes in the eye although there was an infiltration of mononuclear cells in the vitreous, retina, and choroid. No antibodies to any of the RetinoStat vector components or the transgenes could be detected in the serum from either species, and biodistribution analysis demonstrated that the RetinoStat vector was maintained within the ocular compartment. In summary, these studies found RetinoStat to be well tolerated, localized, and capable of persistent expression after subretinal delivery.

Purpose The crystalline lens is a unique cellular organ that performs metabolic processes while maintaining transparency for optical functionality. Mitochondria play a role in providing cells with aerobic respiration necessary for these metabolic processes. Using menadione, a mitochondria-specific inhibitor of the quinone family, and bovine lenses in vitro, this study was undertaken to determine whether a relationship exists between mitochondrial function and optical function. Methods Bovine lenses were treated with 50 μM, 200 μM, 600 μM, and 1,000 μM menadione and lens optical function, assessed as optical quality, was observed over 9 days. Confocal micrographs of mitochondria in superficial secondary fiber cells were also analyzed in 50 μM, 200 μM, and 600 μM menadione-treated lenses over 48 h. Results A decrease in lens optical quality was observed in a dose-dependent manner within 24 h for the 200 µM- (p=0.0422), 600 µM- (p

Purpose. To develop a novel in vitro method to detect the depth of penetration of the tear film protein albumin into contact lens materials using confocal laser scanning microscopy (CLSM). Methods. A poly-HEMA-based hydrogel (etafilcon A) and a silicone hydrogel material (lotrafilcon B) were examined. In vitro, bovine serum albumin (BSA) was labeled with 5-(4,6-dichloro-s-triazin-2-ylamino) fluorescein hydrochloride (DTAF). The lenses were incubated in this protein solution (0.5 mg/ml) at 37°C. After 1 and 7 days incubation, the lenses were examined using CLSM (Zeiss 510, config. META 18) and the location of the fluorescently labeled BSA was identified. Results. BSA adsorption on the surface and penetration into the lens matrix occurred at a higher concentration for etafilcon compared to lotrafilcon (p 0.001). For both materials, BSA was detected on the surface after 1 day of incubation. Significant levels of BSA were detected within the matrix of etafilcon after as little as 1 day (p 0.001), but no BSA was detected in the matrix of lotrafilcon at any time (p 0.05). Conclusion. CLSM can be successfully used to examine the depth of penetration of fluorescently labeled proteins into various hydrogel polymers. Our results show that etafilcon lenses both adsorb BSA on the surface and absorb BSA within the matrix, whereas lotrafilcon B adsorbs small amounts of BSA on the surface only. (Optom Vis Sci 2007;84:839–847)

Mitochondria provide energy generated by oxidative phosphorylation and at the same time play a central role in apoptosis and aging. As a byproduct of respiration, the electron transport chain is known to be the major intracellular site for the generation of reactive oxygen species (ROS). Exposure to solar and occupational ultraviolet (UV) radiation, and thus production of ROS and subsequent cell death, has been implicated in a large spectrum of skin and ocular pathologies, including cataract. Retinal pigment epithelial cell apoptosis generates photoreceptor dysfunction and ultimately visual impairment. The purpose of this article was to characterize in vitro changes following oxidative stress with UV-B radiation in (a) ocular lens optics and cellular function in terms of mitochondrial dynamics of bovine lens epithelium and superficial cortical fiber cells and (b) human retinal pigment epithelial (ARPE-19) cells. Cultured bovine lenses and confluent cultures of ARPE-19 cells were irradiated with broadband UV-B radiation at energy levels of 0.5 and 1.0 J/cm(2). Lens optical function (spherical aberration) was monitored daily up to 14 days using an automated laser scanning system that was developed at the University of Waterloo. This system consists of a single collimated scanning helium-neon laser source that projects a thin (0.05 mm) laser beam onto a plain mirror mounted at 45 degrees on a carriage assembly. This mirror reflects the laser beam directly up through the scanner table surface and through the lens under examination. A digital camera captures the actual position and slope of the laser beam at each step. When all steps have been made, the captured data for each step position is used to calculate the back vertex distance for each position and the difference in that measurement between beams. To investigate mitochondrial movement, the mitochondria-specific fluorescent dye Rhodamine 123 was used. Time series were acquired with a Zeiss 510 (configuration Meta 18) confocal laser scanning microscope equipped with an inverted Axiovert 200 M microscope and 40-x water-immersion C-Apochromat objective (NA 1.2). The optical analysis showed energy level-dependent increases in back vertex distance variability (loss of sharp focus) from 0.39 +/- 0.04 mm (control, n = 11) to 1.63 +/- 0.33 mm (1.0 J/cm(2), n = 10) and 0.63 +/- 0.13 mm (0.5 J/cm(2), n = 9). Confocal laser scanning microscopy analysis of both bovine lenses and ARPE-19 cells showed that following treatment at 0.5 J/cm(2) the mitochondria stopped moving immediately whereas at 1.0 J/cm(2) not only did the mitochondria stop moving, but fragmentation and swelling was seen. Untreated control tissue exhibited up to 15 microm/min of movement of the mitochondria. This could represent normal morphological change, presumably allowing energy transmission across the cell from regions of low to regions of high ATP demand. Lack of mitochondrial movement, fragmentation, and swelling of mitochondria may represent early morphological changes following oxidative stress that may lead to activation of caspase-mediated apoptotic pathways.

Recent confocal laser scanning microscopy studies of the mitochondria of vertebrate lenses show a striking difference in the distribution and morphology of the mitochondria of lens epithelial and superficial cortical cells. This study, using confocal microscopy, was undertaken to image the movement of the mitochondria specific dye tetramethylrhodamine ethyl ester (TMRE) in the epithelium and superficial cortex of whole live bovine lens.Cultured bovine lenses were loaded with 5 microg/ml TMRE for 15 min at room temperature. TMRE fluorescence was acquired with a Zeiss 510 (configuration META 18) confocal laser scanning microscope for 10 to 15 min using 488 nm Argon laser excitation and 505 nm long pass emission filter settings. The uncoupler of the electron transport chain potential, carbonyl cyanide m-chlorophenylhydrazone (CCCP, 32.5 microM), was used to demonstrate the fluorescent specificity of TMRE.Multidirectional dynamic movement of TMRE was observed in epithelial cells and bidirectional dynamic movement was seen in the superficial cortical fiber cells of live bovine lenses. In the epithelium, the movement of TMRE fluorescence was up to 5 microm/min whereas in the superficial cortex the observed movement was up to 18.5 microm/min. The movement of TMRE fluorescence was abolished with treatment with the uncoupler, CCCP.The observed dynamics of TMRE fluorescence movement may represent actual mitochondrial movement, indicating the dynamic state of the mitochondria in both lens epithelium and superficial cortex. That this activity is found not only in the epithelium but also in the superficial cortex indicates that the superficial cortical fiber cells play a much more active role in lens metabolism than previously suspected. Alternatively, the observed movement of TMRE across a mitochondrial network could represent change in the distribution of potential across the inner membrane, presumably allowing energy transmission across the cell from regions of low to regions of high ATP demand.

The effect of several antioxidants and cysteine-elevating precursor drugs (prodrugs) was tested on lens damage occurring after in vitro exposure to low levels of 60Co-gamma-irradiation, to simulate in vitro the exposure to radiation in vivo of (1) astronauts (2) jet crews (3) military radiation accident personnel. Tocopherol (100 microM), ascorbic acid (1 mM), R-alpha-lipoic acid (1 mM), and taurine (0.5 mM) protected against radiation-associated protein leakage. MTCA and ribocysteine protected lenses against opacification, LDH and protein leakage, indicating that antioxidants and prodrugs of cysteine appear to offer protection against lens damage caused by low level radiation.