International consortium for innovation and quality: An industry perspective on the nonclinical and early clinical development of intravitreal drugs.

The eye, which is under constant exposure to environmental pathogens, has evolved various anatomic and immunological barriers critical to the protection of tissues lacking regenerative capacity, and the maintenance of a clear optic pathway essential to vision. By bypassing the ocular barriers, intravitreal (IVT) injection has become the mainstay for the delivery of drugs to treat conditions that affect the back of the eye. Both small molecules and biotherapeutics have been successfully administered intravitreally, and several drugs have been approved for the treatment of (wet) age-related macular degeneration and diabetic macular edema. However, IVT injection is an invasive procedure, which requires sufficient technical expertise from the healthcare professional administering the drug. Potential side effects include bleeding, retinal tear, cataracts, infection, uveitis, loss of vision, and increased ocular pressure. Pharmaceutical companies often differ in their drug development plan, including drug administration techniques, collection of ocular tissues and fluids, ophthalmology monitoring, and overall conduct of nonclinical and clinical studies. The present effort, under the aegis of the Innovation & Quality Ophthalmic Working Group, aims at understanding these differences, identifying pros and cons of the various approaches, determining the gaps in knowledge, and suggesting feasible good practices for nonclinical and early clinical IVT drug development.

Direct Tie2 Agonists Stabilize Vasculature for the Treatment of Diabetic Macular Edema.

Purpose: 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. Methods: 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. Results: 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. Conclusions: Tie2.1-hexamer directly activates the Tie2 pathway, reduces vascular leak, and is persistent within the vitreal humor. Translational Relevance: Our study presents a promising potential therapeutic for the treatment of DME.

Nonclinical Toxicology and Biocompatibility Program Supporting Clinical Development and Registration of the Port Delivery System With Ranibizumab for Neovascular Age-Related Macular Degeneration.

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.

EVALUATION OF SURGICAL FACTORS AFFECTING VITREOUS HEMORRHAGE FOLLOWING PORT DELIVERY SYSTEM WITH RANIBIZUMAB IMPLANT INSERTION IN A MINIPIG MODEL.

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.

Rapid Development of Glaucoma Via ITV Nonselective ANGPT 1/2 Antibody: A Potential Role for ANGPT/TIE2 Signaling in Primate Aqueous Humor Outflow.

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.

The Port Delivery System with Ranibizumab for Neovascular Age-Related Macular Degeneration: Results from the Randomized Phase 2 Ladder Clinical Trial.

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.

Evaluation of the Toxicity of Intravitreally Injected PLGA Microspheres and Rods in Monkeys and Rabbits: Effects of Depot Size on Inflammatory Response.

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.

Menadione degrades the optical quality and mitochondrial integrity of bovine crystalline lenses.

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<0.0001), and 1,000 µM- (p<0.0001) treated lenses. No change in optical quality was observed for the 50 µM-treated lenses. Analysis of confocal micrographs indicated a trend of shorter mitochondria for 200 µM- and 600 µM-treated lenses with time and analysis of the distributions of mitochondrial lengths indicated a relative increase in the number of shorter mitochondria with higher doses of, and longer exposures to, menadione. CONCLUSIONS: The data show that menadione has a detrimental effect on mitochondrial integrity and this change is associated with degradation of optical quality, suggesting a possible link between mitochondrial function and optical function.

Corneal, limbal, and conjunctival epithelium of bovine eyes imaged in vitro by using a confocal laser scanning microscope.

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.

In vitro assays for evaluating the ultraviolet B-induced damage in cultured human retinal pigment epithelial cells.

The present study demonstrates broadband UV-B-induced damage of cultured human retinal pigment epithelial cells as an effort to develop an in vitro model that can be used, along with in vivo research and other in vitro efforts, to evaluate the need for retinal UV protection in humans after cataract removal. The human retinal pigment epithelial cell line, ARPE-19, was cultured in two groups: control and treated. Treated cells were irradiated with three broadband UVB radiations at energy levels of 0.05, 0.1 and 0.2J/cm(2). After irradiation, cells were incubated for 48h while cellular viability, morphology, and phagocytotic activity were analyzed using the Alamar blue assay, confocal microscopy, and fluorescent microspheres. Confocal analysis concentrated on the study of the cell nuclei and mitochondria. The Alamar blue assay of UV-B-exposed cells showed dose and time-dependent decreases in cellular viability in comparison to control cells. Loss of cell viability was measured at the two higher energy levels (0.2 and 0.1J/cm(2)), but the cell group exposed to 0.05J/cm(2) showed no significant viability change at 1-h time point. Morphological evaluation also showed dose and time-dependent degradation of mitochondria and nucleic acids. Cells exposed with 0.05J/cm(2) UVB did not show significant degradation of mitochondria and nucleic acids during the entire culture period. Phagocytotic activity assay data for UVB-exposed cells showed dose-dependent decreases in phagocytotic activity in comparison with the control cells. The control cells have significantly greater capacities for uptake than the 0.1 and 0.2J/cm(2) UV-B-exposed cells, while the 0.05J/cm(2) UV-B-exposed cell group showed no significant difference from the control cell group. The findings suggest that UVB radiation-induced cultured RPE cell damage can be evaluated by assays that probe cellular viability, morphological change, and phagocytotic activity, and that these assay methods together provide a valuable in vitro model for ultraviolet radiation-induced retinal toxicology research.

Effect of hyperbaric oxygen on guinea pig lens optical quality and on the refractive state of the eye.

The main objective of this study was to investigate the effect of in vivo hyperbaric oxygen (HBO) treatment of albino guinea pigs on ocular refractive state and optical properties of the lens in vitro, as well as on the integrity of the mitochondria of the lens. The animals were treated 30-35 times (2.5-3 months) or 70 times (6 months) with HBO. An increased level of lens nuclear light scattering was evident by slit-lamp at 30 treatments, and this increased at 70 treatments. After 30-35 HBO treatments a myopic shift in refractive state of the eye was seen in two separate studies with two different refractionists. Also, the average back vertex distance of the lens was significantly shorter after 35 HBO treatments while spherical aberration (focal variability) increased after 70 treatments. No difference in refractive state was noted after 70 HBO treatments (a reversal of the initial myopic effect). The mitochondrial distribution and morphology of the lens epithelium and the superficial cortical fibre cells were normal after both 35 and 70 HBO treatments, highlighting that HBO treatment does not affect the superficial cortex of the lens. The results of the in vitro lens optical analysis carried out in this study correlate with the myopia observed after 30-35 HBO in vivo treatments. A similar reversible myopia and increase in lens nuclear light scattering is known to occur in humans treated with HBO for extended periods and the results suggest that the myopia was caused by a change in the refractive index of the lens. The significant loss of sharp focus after 70 HBO treatments can be correlated with previous reports of biochemical and morphological changes associated with HBO-induced loss of lens nuclear transparency in mature guinea pigs. The guinea pig HBO model may be a useful approach for the study of lens development and refractive error.

Mechanisms of ocular toxicity using the in vitro bovine lens and sodium dodecyl sulfate as a chemical model.

Previous work using the in vitro bovine lens as a model has shown a correlation between toxicity and lens optical function and showed much higher sensitivity in detecting irritancy of several surfactants at much lower concentrations than the Draize score. In the current study, cultured bovine lenses were used to study the effects of the surfactant sodium dodecyl sulfate (SDS) on lens optical properties and mitochondrial integrity. Bovine lenses were exposed to SDS (0.1 to 0.00625%) for 30 min and cultured for 24 h. Compared to controls (n = 17), loss of sharp focus was evident immediately following exposure to 0.1% SDS (n = 14, p < 0.0001). At 24 h loss of sharp focus became evident in all groups. Loss of lens transparency, significant increase in lens wet weight, and axial length were seen 24 h postexposure in lenses treated with 0.1 to 0.025% SDS. Confocal analysis 24 h postexposure showed SDS concentration-dependent decrease in number and length of the mitochondria in lens epithelial and superficial cortical fiber cells. The results of this study show a correlation between lens optical properties and metabolic function and together provide a sensitive in vitro model of ocular chemical toxicity. Results of confocal analysis suggest that the mitochondrial integrity of the superficial cortical fiber cells is most sensitive to damage caused by SDS. The results further suggest that recovery of lens metabolic function is necessary for the recovery of lens optical properties.