ARVO 2014 Annual Meeting Abstracts 115 Drug delivery #1 Sunday, May 04, 2014 8:30 AM–10:15 AM Exhibit/Poster Hall SA Poster Session Program #/Board # Range: 441–486/D0060–D0105 Organizing Section: Physiology/Pharmacology Contributing Section(s): Retina Program Number: 441 Poster Board Number: D0060 Presentation Time: 8:30 AM–10:15 AM Design Considerations and Performance of a Next-Generation Encapsulated Cell Technology Intraocular Implant Delivering VEGF-Antagonist Konrad Kauper, Michael Rivera, John Mills, Alline Lelis, Megan Billings, Cahil McGovern, Arne Nystuen, Sandy Sherman, Pam Heatherton, Rhett Schiffman. Neurotech Pharmaceuticals, Cumberland, RI. Purpose: Early generation ECT products delivering VEGFantagonist (VEGF- α) for over a year have demonstrated clinically meaningful improvements in BCVA and reductions in macular thickening in patients with active neovascular AMD; higher dose levels are expected to achieve efficacy comparable or greater than standard-of-care treatments. A new ECT device, NT-503-3, incorporating multiple, optimized cell encapsulation chambers into a single cartridge implant was designed to substantially increase the dose of VEGF-α by increasing the total number of encapsulated cells, and by improving cell viability and protein expression efficiency. This design also supports combination therapy from a single device, by allowing discrete encapsulations of different therapeutic cell lines in a single intraocular implant product. Methods: The performance of NT-503-3 was evaluated following encapsulation of a human RPE cell line transfected to produce VEGF-α. Dose levels of VEGF-α were characterized by ELISA; binding efficiency and affinity were quantified by a VEGF-inhibition and Bioacore assay, respectively. GLP toxicology studies, which include clinical examination, ERG, ocular histopathology, and detection of serum antibodies to VEGF-α and the encapsulated cell line, are ongoing. Results: A single NT-503-3 implant increased VEGF-α dose 5-fold compared to the previous ECT product (NT-503-2) which had demonstrated clinical efficacy in wet-AMD patients when implanted with two devices. VEGF-α produced by NT-503-3 results in high binding affinity to VEGF with a Kd of 0.7 pM and inhibits VEGF with an IC50 of 20-30 pM. Intraocular implants in rabbits and minipigs demonstrate that the NT-503-3 product is safe and well tolerated. Conclusions: Clinically relevant VEGF-α expression and an excellent toxicology profile has been achieved with the novel NT503-3 design. A single, intraocular NT-503-3 implant is anticipated to provide equivalent or improved efficacy compared to standardof-care therapy while eliminating the burden of frequent injections in patients with active neovascular AMD. Furthermore, the new ECT cartridge design also supports ongoing combination therapy development. Commercial Relationships: Konrad Kauper, Neurotech Pharmaceuticals (E); Michael Rivera, Neurotech Pharmaceuticals (E); John Mills, Neurotech Pharmaceuticals (E); Alline Lelis, Neurotech Pharmaceuticals (E); Megan Billings, Neurotech Pharmaceuticals (E); Cahil McGovern, Neurotech Pharmaceuticals (E); Arne Nystuen, Neurotech Pharmaceuticals (E); Sandy Sherman, Neurotech Pharmaceuticals (E); Pam Heatherton, Neurotech Pharmaceuticals (E); Rhett Schiffman, Neurotech Pharmaceuticals (E)

Program Number: 442 Poster Board Number: D0061 Presentation Time: 8:30 AM–10:15 AM Shining a Light on Drug Delivery: On-Demand Light-triggered Release of Payload in the Eye Viet Anh Nguyen Huu1, 2, Jing Luo3, Jing Zhu3, Enas Mahmoud4, Cathryn McFearin2, Nadezhda Fomina2, Kang Zhang3, Adah Almutairi2, 1. 1Nanoengineering, University of California, San Diego, La Jolla, CA; 2Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA; 3 Institute for Genomic Medicine and Shiley Eye Center, University of California, San Diego, La Jolla, CA; 4Department of Pharmaceutics, Faculty of Pharmacy, Cairo University, Cairo, Egypt. Purpose: Anti-VEGF therapy commonly used to treat macular degeneration and diabetic retinopathy requires intravitreal injections every 4-8 weeks. Injections are uncomfortable, time-consuming, and carry a small risk of infection and retinal damage. However, drug delivery via noninvasive methods to the posterior segment of the eye has been a major challenge due to the eye’s unique anatomy and physiology. Here we present a novel nanoparticle platform for on-demand drug delivery using an ultraviolet (UV) light-degradable polymer, which allows for temporally-controlled, noninvasive drug release. Methods: Nanoparticles containing a fluorescent dye as a model drug are formulated by emulsion-evaporation techniques. UV irradiation conditions are determined by examining their effects on cultured lens explants. Control over payload release upon UV irradiation is studied in vitro on various cell lines, including retinal pigment epithelium cells. Biocompatibility of the materials is tested via electroretinograms (ERG), intraocular pressure (IOP) measurements, histology, and qRT-PCR for common cytokines. Temporal control over payload release in vivo is examined in rats by fluorescent imaging of retinal flatmounts of irradiated and non-irradiated eyes at various timepoints post-injection of nanoparticles. Therapeutic potential is studied using nanoparticles containing a biologic VEGFR/ PDGFR inhibitor in a laser-induced choroidal neovascularization (CNV) model. Results: Lens explant studies show 5 min of 365 nm UV light at 8mW/cm2 does not cause cloudiness characteristic of cataract formation. There was no significant difference in electrophysiological response, IOP, or IL1β and TNFα expression in the retinas of eyes injected with our UV-responsive particles relative to saline- or non-injected eyes at 7 days post-injection. Nanoparticles containing fluorescent dye remain intact and responsive to light up to 12 weeks post-injection. Conclusions: Materials formulated in nanoparticles are safe in the eye and retain their ability to release drug upon irradiation with benign levels of UV light up to at least 12 weeks post-injection. This nanoparticle platform has the potential to deliver a variety of molecules, including small hydrophobic drugs and proteins. Commercial Relationships: Viet Anh Nguyen Huu, None; Jing Luo, None; Jing Zhu, None; Enas Mahmoud, None; Cathryn McFearin, None; Nadezhda Fomina, None; Kang Zhang, None; Adah Almutairi, None Support: NIH Director’s New Innovator Award 1DP2OD006499-01; King Abdulaziz City for Science and Technology center grant to the Center of Excellence in Nanomedicine at UC San Diego

©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2014 Annual Meeting Abstracts Program Number: 443 Poster Board Number: D0062 Presentation Time: 8:30 AM–10:15 AM FIRST STEPS TOWARDS A DRUG-ELUTING INTRAOCULAR LENS Lampros Lamprogiannis1, 2, Athanasios Karamitsos1, 2, Stergios Logothetidis2, Varvara Karagkiozaki2, Maria Gioti2, Stavros Dimitrakos1, Ioannis Tsinopoulos1. 12nd University Eye Clinic, Aristotle University of Thessaloniki, Thessaloniki, Greece; 2 Laboratory for Thin Films, Nanosystems and Nanometrology, Physics Department, Aristotle University of Thessaloniki, Thessaloniki, Greece. Purpose: Development, characterization and drug release study of one-layer and two-layers thin films based on organic polymers and dexamethasone in order to examine their applicability on intraocular lenses and their function as intraocular drug delivery systems. Development and study of the thin films directly on the surface of intraocular lenses. Methods: Four groups of thin films were prepared by the method of spin coating on the surface of intra-ocular lenses: group A, consisting of two layers of in a 2:1 ratio with dexamethasone), group B with two layers in a 3:1 ratio with dexamethasone, group C with one layer in a 2:1 ratio with dexamethasone and group D with one layer in a 3:1 ratio with dexamethasone. Organic polymers served as matrix for all the films. The films were studied with the use of AFM and ellipsometry. Release rate of dexamethasone was studied for a period of ten weeks. Results: Conclusions have been reached in relevance to the surface structure and roughness of the films. Groups A and B demonstrated the formation of aggregates of dexamethasone. The monolayer films of groups C and D formed nanopores, in agreement with previous findings. The ellipsometry study showed transparent samples. The study of drug release demonstrated a smooth release for the first 6 weeks. Conclusions: The films exhibited properties (transparency, release duration, release curve) that serve the purpose of using them as systems for intraocular drug delivery. Further research includes development of the drug delivery system directly on the surface of the IOL.Direct development of the films on the surface of intraocular lenses is possible. Commercial Relationships: Lampros Lamprogiannis, None; Athanasios Karamitsos, None; Stergios Logothetidis, None; Varvara Karagkiozaki, None; Maria Gioti, None; Stavros Dimitrakos, None; Ioannis Tsinopoulos, None Program Number: 444 Poster Board Number: D0063 Presentation Time: 8:30 AM–10:15 AM Half-life of therapeutic molecules in the vitreous depends on molecular weight, vitreous network mesh size and eye size Michael Monine1, Matthias Machacek2. 1Advanced Quantitative Sciences, Novartis, Cambridge, MA; 2Advanced Quantitative Sciences, Novartis, Basel, Switzerland. Purpose: A long ocular half-life contributes to long effect duration and is critical for biotherapeutics to avoid frequent intravitreal injections. For the development of novel biotherapeutics it is therefore important to understand the process of ocular disposition, the factors dominating half-life and how animal data translates to human. Methods: A biophysical model was used to investigate diffusiondriven distribution and clearance of molecules in the eye. The model was calibrated with fluorophotometry data obtained for tagged Fab fragment injected in rabbit eyes and validated against published rabbit and human data which included small molecules, scFv, Fab

fragments, Fc fusion proteins and antibodies covering a molecular weight (MW) range of 100 to 150’000 Da. Results: The model predicted well the dependency on MW supporting that diffusion is a key process of clearance (Figure 1 A). The main factors contributing towards a longer half-life were a higher MW, a smaller mesh size of the vitreous network and a larger eye. These results also suggested that differences in eye size (Figure 1 B) and in vitreous mesh size are among main factors in translating halflife data from animal to human. Conclusions: A biophysical model provides a basis for mechanistic interpretation of ocular PK data in animals and can be used to predict the ocular half-life in human and guide optimal design and selection of long acting molecules.

Figure 1: (A) Vitreal half-life as a function of MW: literature data vs. model prediction in the rabbit eye. (B) Comparison of the data and the model predictions for biotherapeutics injected in rabbit and human eyes. Solid lines denote model predictions assuming the average vitreous network mesh size equal to 100 nm. Shaded area in panel A covers a range of mesh sizes from 30 nm (upper boundary) to infinity (lower boundary, diffusion in water). Commercial Relationships: Michael Monine, Novartis (E), Novartis (F); Matthias Machacek, Novartis (E), Novartis (F) Program Number: 445 Poster Board Number: D0064 Presentation Time: 8:30 AM–10:15 AM Permeability of Porcine Ocular Tissues at Different Stages of Development Tao L. Lowe, Dileep Janagam. Pharmaceutical Sciences, Univ of Tennessee Health Science Ctr, Memphis, TN. Purpose: Investigation of the permeability of ocular tissues at different stages of development is important for understanding physiological conditions of the eye and developing strategies to enhance drug permeability across these tissues for treating many ocular diseases. The objective of this study is to study the permeability along with corresponding morphology of the porcine sclera and the cornea at different stages of development. Methods: Sclera and cornea tissues were isolated from fresh eyeballs of 28 week preterm and 1 week old normal term. The thicknesses of the tissues were measured. The tissues were placed in the spherical joints of Valia-Chien Cell apparatuses. Fluorescein isothiocyanate (FITC) labeled 4k-dextran was dissolved in respective media for the ocular tissues and added to the donor chamber at 1 mg/mL. Passive transport of the FITC-dextran across the sclera and cornea tissues was carried out at 34°C over 4 hour period of time. Samples were taken out from the receptor chamber as a function of time. The fluorescence intensities in the solutions sampled from the donor and receptor chambers were quantified using a fluorescence microplate reader. The permeability (P0) of the FITC-dextran across the sclera and cornea tissues was calculated. The TER values of the ocular tissues were monitored through the whole permeability studies. Currently, we are measuring the permeability of the FITC-dextran across adult pig sclera and cornea tissues, and the morphologies of all the above

©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2014 Annual Meeting Abstracts mentioned ocular tissues at different development stages using a scanning electron microscope. Results: The thickness of the preterm sclera was thinner than that of the normal term sclera; while there was no difference between the thicknesses of the preterm and normal term corneas. The transsceleral permeability across the preterm sclera was significantly higher than that of the normal term sclera (Figure 1). However, the transcorneal permeability across the preterm and normal term piglet corneas showed no significant difference (Figure 2). Conclusions: The development stages of pigs affect the permeability of ocular tissues depending on the tissue types. These studies provide insight on how to design drug delivery systems to enhance drug permeability across ocular tissues at different development stages.

Figure 1. Permeability of 4k FITC-dextran across the sclera.

Figure 2. Permeability of 4k FITC-dextran across the cornea. Commercial Relationships: Tao L. Lowe, None; Dileep Janagam, None

Program Number: 446 Poster Board Number: D0065 Presentation Time: 8:30 AM–10:15 AM Controlled Transscleral Dual-drug Delivery by a Polymeric Device Reduces Light-induced Retinal Damage Nobuhiro Nagai1, Hirokazu Kaji2, Zhaleh Kashkouli Nezhad1, Kaori Sampei1, Satoru Iwata1, Matsuhiko Nishizawa2, Yukihiko Mashima3, Toshiaki Abe1. 1Graduate School of Medicine, Tohoku University, Sendai, Japan; 2Graduate School of Engineering, Tohoku University, Sendai, Japan; 3R-Tech Ueno, Tokyo, Japan. Purpose: To evaluate the protective effects of a transscleral dual-drug delivery device that can release unoprostone isopropyl (UNO) and edaravone (EDV) at independently controlled release rates against light-induced retinal damage in rats. Methods: The device consists of a reservoir, controlled-release cover, and drug formulations, which were made of photopolymeized poly(ethyleneglycol) dimethacrylate that partially contains tri(ethyleneglycol) dimethacrylate. These parts were fabricated via a microfabrication technique that used an AutoCAD design. UNO, a prostanoid for antiglaucoma eyedrops marketed in Japan, and EDV, a potent free radical scavenger, were loaded in the device. Highperformance liquid chromatography was used to evaluate the release amount of UNO and EDV. After the devices were placed onto the sclera of eyes in rats for 1 week, the rats were exposed to 8000 lux of white fluorescent light for 24 h, then flash electroretinograms were recorded. Histological examinations were perfomred to evaluate the thickness of the outer nuclear layer and the number of apoptotic cells. Results: UNO and EDV were simultaneously released with zeroordered kinetics from the device. Trannsscleral co-administration of EDV and UNO by the device reduced loss of retinal function assessed by electroretinograms compared to single administration. The device also precluded the reductions of retinal thickness and the number of apoptotic cells after light exposure. Conclusions: Co-delivery of EDV and UNO by our device attenuates light-induced retinal damage morphologically and functionally. Controlled dual-drug delivery using our device can protect retinal function from light damage by a less invasive transscleral administration. Commercial Relationships: Nobuhiro Nagai, None; Hirokazu Kaji, None; Zhaleh Kashkouli Nezhad, None; Kaori Sampei, None; Satoru Iwata, None; Matsuhiko Nishizawa, None; Yukihiko Mashima, R-Tech Ueno (E); Toshiaki Abe, R-Tech Ueno (F) Support: Grant-in-Aid for Young Scientists (A) from the MEXT, Japan, Health Labour Sciences Research Grant from the MHLW, Japan Program Number: 447 Poster Board Number: D0066 Presentation Time: 8:30 AM–10:15 AM Transscleral diffusion and comparative ex vivo permeability of dorzolamide in canine, equine, porcine and rabbit sclera Pamela Ko1, 2, Jonathan Moreno2, Paulin Wahjudi2, Rueben Merideth1, Ricardo Carvalho2. 1Comparative Veterinary Ophthalmology, Eye Care for Animals, San Diego, CA; 2Translational Ophthalmology, 3T Ophthalmics Inc, Irvine, CA. Purpose: To characterize the ex vivo transscleral diffusion of dorzolamide and compare its diffusion and permeability profile across the canine, equine, porcine and rabbit sclera. Methods: The sclera was harvested from fresh canine, equine, porcine and rabbit globes. The samples were mounted between the donor and receptor compartments onto the curved surface of waterjacketed Franz cells, comprising a 5-mm diffusion window and maintained at 37oC. Balanced salt solution was the media utilized in the receptor chamber. Solutions containing 2mg/ml and 20mg/ml of dorzolamide (n=3) were dispensed into the donor compartments

©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission to reproduce any abstract, contact the ARVO Office at [email protected].

ARVO 2014 Annual Meeting Abstracts and samples were collected from the receptor sites at 15,30,45,60,90 ,120,180,240,300,360 minutes and 24 hours. The sclera and residual solutions in the donor chambers were collected at the end of the run for analysis. The collected samples were analyzed by previously developed sensitive high-performance liquid chromatography (HPLC) assay for dorzolamide. The trans-scleral flux(F), permeability coefficients (Papp) and effective diffusion coefficient (Deff) of dorzolamide were calculated. Permeability parameters were determined and compared with ANOVA statistical analysis (p