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ARVO 2015 Annual Meeting Abstracts 416 Drug delivery Wednesday, May 06, 2015 8:30 AM–10:15 AM Exhibit Hall Poster Session Program #/Board # Range: 4135–4167/C0018–C0050 Organizing Section: Physiology/Pharmacology Contributing Section(s): Clinical/Epidemiologic Research, Cornea, Retinal Cell Biology, Retina Program Number: 4135 Poster Board Number: C0018 Presentation Time: 8:30 AM–10:15 AM Vancomycin incorporated collagen hydrogel implant for the prevention of ocular infections DEBASISH MONDAL1, Andri K. Riau1, 3, Elavazhagan Murugan1, Thet Tun Aung2, Bo Liedberg3, 4, Subbu S. Venkatraman3, Jodhbir S. Mehta1, 5. 1Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore, Singapore; 2Anti-Infectives Research Group, Singapore Eye Research Institute, Singapore, Singapore; 3School of Materials Science and Engineering, Nanyang Technological University, Singapore, Singapore; 4Interdisciplinary Graduate School, Nanyang Technological University, Singapore, Singapore; 5Cornea and External Eye Disease, Singapore National Eye Center, Singapore, Singapore. Purpose: To develop a sustained delivery system to improve the release kinetics of vancomycin as well as its therapeutic effectiveness to prevent postoperative corneal infection. Methods: Vancomycin was incorporated into an engineered collagen hydrogel (10 and 15 wt%) scaffold through N-ethyl-N’[3-dimethylaminopropyl] carbodiimide/N-hydroxy succinimide (EDC/NHS) crosslinking technique. Vancomycin incorporation into the collagen hydrogel was examined by fourier transform infrared spectroscopy (FTIR) and quantified spectrophotometrically after collagen digestion with collagenase. Mechanical stability of hydrogel was measured by Instron instrument. In vitro release profile of vancomycin was measured in PBS at 37°C. Structural integrity of released vancomycin was assessed by circular dichroism (CD) spectroscopy. The minimum inhibitory concentration (MIC) of control and released vancomycin against S.aureus (SA) was determined by inoculating with bacterial suspension (106 colonyforming unit/ml). The wells were examined spectrophotometrically for visible bacterial growth as evidenced by turbidity after 24h incubation at 37°C. Results: FTIR spectrum showed the incorporation of vancomycin in the collagen hydrogel as evidenced by presence of characteristic peaks of vancomycin (3075 cm-1, 1640 cm-1, and 1545 cm-1and 1423 cm-1, the bands were attributed to phenolic O-H, C=O stretching, C=C and C-C mode of vibration, respectively). Mechanical test result showed 15% collagen hydrogel yielded significantly higher tensile strength compared to the 10% hydrogel (0.317 ± 0.021 and 0.259 ± 0.007 MPa for 15% and 10% respectively, p=0.03). The addition of vancomycin in the hydrogel slightly reduced the tensile strength and Young’s modulus, but the difference was not significant (p=0.3). Vancomycin release pattern and amount was same for the different percentage (10% and 15%) hydrogel (p>0.05). The release was in a sustained manner for 7 days period with concentrations above the theoretical MIC of vancomycin against SA (2 mg/ml). Antimicrobial assay showed that the MIC of released vancomycin up to day 5 was the same as control (1 mg/ml), but MIC on day 7 was increased to 4 mg/ml. CD result revealed significant perturbation of vancomycin structure on day 7. Conclusions: Our study suggests that collagen hydrogel implant can be used as a delivery system of vancomycin for 7 days above MIC, to prevent the postoperative ocular infection. Commercial Relationships: DEBASISH MONDAL, None; Andri K. Riau, None; Elavazhagan Murugan, None; Thet Tun Aung, None; Bo Liedberg, None; Subbu S. Venkatraman, None; Jodhbir S. Mehta, None Program Number: 4136 Poster Board Number: C0019 Presentation Time: 8:30 AM–10:15 AM Comparative ex-vivo trans-scleral diffusion permeability of dexamethasone sodium phosphate in equine, porcine and rabbit scleras Pamela P. Ko1, 2, Jonathan Moreno2, Robert Brito2, Monica Vargas Dougherty2, Ruebuen Merideth1, Ricardo Carvalho2. 1Ophthalmology, Eye Care for Animals, Irvine, CA; 2Ophthalmology, 3T Ophthalmics Inc., Irvine, CA. Purpose: To characterize the ex vivo trans-scleral diffusion of dexamethasome sodium phosphate and compare its diffusion and permeability profile across the equine, porcine and rabbit scleras. Methods: The scleras were harvested from fresh equine, porcine and rabbit globes. The samples were mounted between the donor and the receptor compartments onto the curved surface of water-jacketed Franz cells, comprising a 5-mm diffusion window and maintained at 37° C. Balanced salt solution was the media utilized in the receptor chamber. Solutions containing 10 and 100 mg/mL of dexamethasone sodium phosphate (Dex. SP.) (n=10 per concentration) were dispensed into the donor compartments and samples were collected from the receptor sites at 30,60,90,120,180,240,300,360 minutes. The sclera and residual solutions in the donor chambers were collected at the end of the run. The collected samples were analyzed by an UV-Vis spectrophotometer. The trans-scleral flux (F), permeability coefficient (Papp) and effective diffusion coefficient (Deff) of Dex. SP. were calculated. Permeability parameters were determined and compared with ANOVA statistical analysis (p<0.05). Results: Preliminary spectrophotometery analysis of dexamethasone SP. was carried out to calculate the cumulative amount recovery (microgram) over time and permeability parameters of three species were calculated. The equine flux at 100 mg/mL concentration had a higher average recovery rate than the 10 mg/mL concentration. It was the opposite in the porcine flux of the two concentration study. Rabbit sclera had reached drug diffusion saturation time fastest and equine was the slowest. It suggested a potential scleral anatomical compositional difference among species. The permeability coefficient values of two concentrations were similar within the same species (i.e. 100 mg/mL vs. 10 mg/mL in porcine). Conclusions: We have shown that a model of water-soluble steroid anti-inflammatory drug is able to permeate the sclera in other species such as equine, porcine and rabbit animal models. The data obtained from this ex-vivo experiment demonstrated interspecies difference pertaining to the drug diffusion across the scleras. This suggests a possible use of trans-scleral route as an alternative drug delivery method for translational research and development, but also for the treatment of retinal diseases in veterinary ophthalmology. Commercial Relationships: Pamela P. Ko, None; Jonathan Moreno, None; Robert Brito, None; Monica Vargas Dougherty, None; Ruebuen Merideth, None; Ricardo Carvalho, None ©2015, 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 2015 Annual Meeting Abstracts Program Number: 4137 Poster Board Number: C0020 Presentation Time: 8:30 AM–10:15 AM Sustained Release from Biodegradable Microparticles of Bioactive Cysteinyl Leukotriene Receptor Antagonists for the Treatment of Ocular Neovascularisation Claire Kilty1, Adolfo Lopez-Noriega2, Camille Hurley1, Neil O’Conner3, Alison Reynolds1, Cormac Murphy3, Fergal O’Brien2, 4 , Breandan N. Kennedy1. 1UCD Conway Institute & UCD School of Biomolecular and Biomedical Science, University College Dublin, Dublin, Ireland; 2Tissue Engineering Research Group, The Department of Anatomy, Royal College of Surgeons in Ireland, Dublin, Ireland; 3UCD School of Biomolecular & Biomedial Science, Ardmore House, University College Dublin, Dublin, Ireland; 4Trinity Centre for Bioengineering, Trinity College Dublin, Dublin, Ireland. Purpose: There is an unmet need in ocular neovascular-based diseases to improve current delivery modalities to the eye. This is highlighted by the requirement of current gold-standard anti-VEGF drugs to be administered bi-/monthly by intravitreal injection. Our objective is to enhance the delivery of bespoke novel anti-angiogenic drugs using biodegradable sustained-release devices. Methods: Two novel VEGF-independent, cysteinyl leukotriene receptor antagonists (Quininib [QB] & Quininib-12 [QB12]) previously identified as anti-angiogenic in zebrafish, cells and mice were formulated into biodegradable PLGA and alginate microparticles. Drug-loaded microparticles were characterised in terms of shape, size and loading efficiency. Drug release from microparticle subtypes was determined by in vitro release studies using HPLC. The Efficacy of released drug was evaluated in vitro with tubule formation assays, ex vivo with rodent aortic rings and in vivo using larval zebrafish angiogenesis assays. Results: QB & QB12 were successfully formulated into PLGA and alginate microspheres of ~1-2 mm. Notably, in vitro release studies demonstrate high concentrations of QB drug released from PLGA and alginate microparticles for up to one month. Importantly, QB released from microparticles retained anti-angiogenic efficacy in vivo. We are currently evaluating if these microparticles inhibit HMEC1 tubule formation and reduce sprouting angiogenesis from rodent aortic rings. Future directions will test the safety, pharmacokinetics and efficacy of these Quininib-loaded microparticle formulations in rodent models. Conclusions: We successfully encapsulated small molecule cysteinyl leukotriene receptor antagonists into slow release preparations. The ultimate goal is to determine if these encapsulated anti-angiogenic drugs offer an improved sustained and effective treatment for ocular neovascularisation. Commercial Relationships: Claire Kilty, ‚ÄúAnti-angiogenic compounds‚Äù, priority date 18/7/2012; published as WO 2014/012889; entered PCT 15/7/2013 (P); Adolfo Lopez-Noriega, None; Camille Hurley, None; Neil O’Conner, None; Alison Reynolds, ‚ÄúAnti-angiogenic compounds‚Äù, priority date 18/7/2012; published as WO 2014/012889; entered PCT 15/7/2013 (P); Cormac Murphy, None; Fergal O’Brien, None; Breandan N. Kennedy, ‚ÄúAnti-angiogenic compounds‚Äù, priority date 18/7/2012; published as WO 2014/012889; entered PCT 15/7/2013 (P), ‚ÄúAnti-angiogenic compound‚Äù, priority date 14/1/2011; published as WO 2012/095836; progressed to National Regional Phase 14/7/2013 (P) Support: Irish Research Council Goverment of Ireland Postdoctoral Fellowship, SFI TIDA, Enterprise Ireland Program Number: 4138 Poster Board Number: C0021 Presentation Time: 8:30 AM–10:15 AM Penetration of Polar Sulforhodamine B into the Cornea Sangly P. Srinivas1, Wanachat Chaiyasan2, Pattravee Niamprem2, Katelyn Keefer1, Waree Tiyaboonchai2, Uday B. Kompella3. 1 Optometry, Indiana University, Bloomington, IN; 2Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand; 3 Pharmaceutical Sciences, University of Colorado, Denver, CO. Purpose: Several fluorescent dyes are employed as tracers to investigate barrier function of the corneal epithelia and as drug analogs to assess pharmacokinetics of topical drugs. Unlike fluorescein and its derivatives, the fluorescence of Sulforhodamine B (SRB) is pH independent and therefore can serve as a better tracer in the presence of changes in pH. This study has examined the transcorneal dynamics of SRB using a custom-built confocal scanning microfluorometer (CSMF; depth resolution ~ 7 μm). Methods: SRB (0.1%) was injected into a/c of pig eyes (n = 3). The epithelium was exposed to a dish containing the dye (n = 3). CSMF with a water-immersion objective (Zeiss 40x; 0.75 NA and wd = 1.2 mm) was employed to quantify the penetration dynamics of SRB. The output of a white LED modulated at 10 kHz was filtered through an interference filter (565 + 10 nm) and led to the excitation port of the CSMF. The SRB fluorescence ( > 585 nm) and scattered light passing through a parfocal exit slit in the eyepiece were detected by 2 photomultiplier tubes coupled to 2 lock-in amplifiers. Measurements were performed with eyeballs held underneath the objective on a motorized linear stage. Results: Exposure of the corneal epithelium to SRB over 3-12 hrs led to significant but variable fluorescence in the stroma (n= 3). The fluorescence distribution showed a marked discontinuity at the interface between epithelium and stroma, with gradient in the stroma itself. Injection of SRB into the anterior chamber also produced increase in fluorescence from the stroma over time with noticeable discontinuity at the interface between endothelium and anterior chamber (n = 3). Unlike a small peak of fluorescence in the epithelium, there was no notable fluorescence from the endothelial layer and stromal gradient was also much smaller. These transcorneal fluorescence profiles of SRB are very much similar to those obtained with hydrophilic carboxyfluorescein administered into the a/c. Conclusions: A significant increase in the stromal fluorescence with a small fluorescence increase corresponding to the epithelium and negligible increase in fluorescence corresponding to the endothelium indicates that SRB crosses barriers of the corneal epithelia via penetration through the tight junctions. Therefore, SRB could be used as a polar tracer to assess barrier function of the ocular epithelia in situations of marked changes in pH. Commercial Relationships: Sangly P. Srinivas, None; Wanachat Chaiyasan, None; Pattravee Niamprem, None; Katelyn Keefer, None; Waree Tiyaboonchai, None; Uday B. Kompella, None Support: R01-EY018940 (UBK), NEI P30EY019008 Program Number: 4139 Poster Board Number: C0022 Presentation Time: 8:30 AM–10:15 AM A Novel Carboxymethylated Hyaluronic Acid Polymer for Sustained Drug Delivery to Ocular Surface Hee-Kyoung Lee1, 2, Michael Onorato3, Isaac Erickson3, MaryJane Rafii1, McKenna M. Drysdale2, Brittany Coats2, Thomas Zarembinski3, Barbara M. Wirostko1, 2. 1Jade Therapeutics Inc., Salt Lake City, UT; 2University of Utah, Salt Lake City, UT; 3BioTime Inc., Alameda, CA. Purpose: Jade Therapeutics uses a biocompatible, biodegradable cross-linked hyaluronic acid-based polymer for sustained drug delivery to enhance the treatment of ophthalmology disorders ©2015, 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 2015 Annual Meeting Abstracts and improve visual outcomes. Jade’s proprietary, thiolated carboxymethylated hyaluronic acid (CMHA) film requires infrequent application, and is easy to install “urgently” on-site by a physician. Films of four different cross-linked CMHA formulations were fabricated and the in vitro characteristics were determined and optimized to establish film drug release properties. The cytocompatibility of four CMHA film formulations was evaluated using two cell lines. Methods: Films were fabricated varying the HA-based polymers (thiolated CMHA alone versus thiolated CMHA with thiolated gelatin). Poly(ethyleneglycol) diacrylate (PEGDA) or glutathione disulfide (GSSG) was used as cross-linkers. The polymerized gel in silicone mold was dried at room temperature overnight to create thin films. Swelling was assessed by analysis of film diameter and mass before and after hydration. Using carbazole assay, the integrity of CMHA-PEGDA film after ethylene oxide (EtO) sterilization was compared to the integrity of pre-sterilized film. Film’s mechanical property was measured under various levels of stress. Film-related effects on bone marrow derived mesenchymal stem cell (MSCs) and NIH 3T3 fibroblast viability and morphology were analyzed using fluorescence imaging and an alamarBlue® metabolic assay. Results: Six mm diameter films were created, and swelled to 8 mm in PBS within 30 min. The mass increased as much as 700% after 24 hrs. The CMHA-PEGDA film was the most favorable in terms of tensile strength, relaxation modulus, durability and flexibility. This film was also the closest to soft contact lens under various levels of stress. Based on the carbazole assay, the integrity of CMHAPEGDA film remained the same after EtO sterilization. Cells readily proliferated over a 7-day period for each film and cell type with no significant reductions in metabolic activity or morphology as compared to the control. Conclusions: Based on their physical characteristics, cytocompatability, comfort, and sterilizability, CMHA-PEGDA film shows the potential for use in sustained-release delivery of a wide range of molecules for treating various ocular surface conditions. Commercial Relationships: Hee-Kyoung Lee, Jade Therapeutics Inc (E), Jade Therapeutics Inc (F), Jade Therapeutics Inc (I), Jade Therapeutics Inc (R); Michael Onorato, BioTime Inc (E), BioTime Inc (F); Isaac Erickson, BioTime Inc (E), BioTime Inc (F); MaryJane Rafii, Jade Therapeutics (F), Jade Therapeutics (P), Jade Therapeutics (S), Jade Therapeutics Inc (I); McKenna M. Drysdale, Jade Therapeutics (F); Brittany Coats, Jade Therapeutics (C); Thomas Zarembinski, BioTime Inc (E), BioTime Inc (F); Barbara M. Wirostko, Jade Therapeutics (F), Jade Therapeutics (P), Jade Therapeutics (S), Jade Therapeutics Inc (I) Support: Department of Defense SBIR Phase 1 W81XWH14-C-0025 Program Number: 4140 Poster Board Number: C0023 Presentation Time: 8:30 AM–10:15 AM A Novel, Non-invasive, Self-Administered, Preservative-Free, Sustained Release Product (EySite-TPTM) for Glaucoma Therapy Shikha P. Barman1, Moli Liu2, Kevin Ward2, Koushik Barman2, Kathryn Crawford3, Thomas Leland2. 1Executive, Integral BioSystems, Bedford, MA; 2Microencapsulations, Integral BioSystems, Bedford, MA; 3PharmaOcu, Andover, MA. Purpose: One of the leading causes for blindness among the elderly is glaucoma. Among leading standards of care for glaucoma treatment are prostaglandin eye-drops, administered once daily. However, treatment is confounded by lack of patient compliance, inefficient placement of drops and losses of ~90% of the dosage to drainage by the tear duct. We present feasibility data of a novel, preservativefree, self-administered, Travoprost-containing sustained release, nanoengineered product that can be placed in the conjunctival cul de sac by the patient, every 30 days. Methods: Appropriately-sized drug-containing nanoengineered matrices were prepared using a combination of proprietary process conditions and blends of PLG/ hydrophilic polymers. Formulations varied in composition, end groups and amount and type of amphiphilic co-excipient. Travoprost-containing matrices were analyzed as followes: content (mg/mg device) by HPLC, integrity of encapsulated drug (HPLC), microstructure (SEM), in-vitro release studies at 37°C, pH 7.4 using a flow-through system and rate of hydration model developed in-house. Results: Travoprost-containing dry nano-matrices contained 39-100 mg of intact drug per device, with >90% encapsulation efficiency. Ester-end group PLGA combined with hydrophilic polymers provided a flexible, biodegradable matrix. SEM of the matrices showed a fine nanostructure, with interconnecting pores, suitable for rapid water uptake into a flexible hydrogel, post-placement. The prototypes released Travoprost in-vitro at 37°C at a rate of 0.8-3.5 mg per day, at 65% released in 2 weeks. The nanomatrix remained flexible and hydrated throughout the study, with its hydrated flexural modulus similiar to that of ocular conjunctiva. The nanomatrix was assessed for the rate of water update and its hydration into a hydrogel. By visual assessment, the dry, flexible nanomatrix absorbed water in approximately 30 seconds to become a tissue-conforming, adherent hydrogel. Conclusions: The data demonstrates the the feasibility of a noninvasive, preservative-free, self-administered Travaprost-containing 30-Day sustained release, biodegradable dosage form. This novel product addresses a vital clinical need and has the potential to transform drug administration for both ocular surface disorders and intraocular diseases. The sterile product is placed in the conjunctival cul de sac with an applicator. Commercial Relationships: Shikha P. Barman, Integral BioSystems (F), Integral BioSystems (P); Moli Liu, Integral BioSystems (E), Integral BioSystems (F); Kevin Ward, Integral BioSystems (E), Integral BioSystems (F); Koushik Barman, Integral BioSystems (E), Integral BioSystems (F); Kathryn Crawford, None; Thomas Leland, Integral BioSystems (E), Integral BioSystems (F) Program Number: 4141 Poster Board Number: C0024 Presentation Time: 8:30 AM–10:15 AM Biodegradable and Injectable Thermosensitive Pentablock Copolymers Hydrogels for Sustained Delivery of Proteins for Posterior Segment Ocular Diseases Ashim K. Mitra1, Sulabh Patel2, 1, Ravi Vaishya1, Vibhuti Agrahari1. 1 School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO; 2Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland. Purpose: To investigate the sustained delivery of protein therapeutics from biodegradable and injectable thermosensitive hydrogels for the treatment of ocular posterior segment diseases including age-related macular degeneration, macular edema and proliferative diabetic retinopathy. The hydrogel matrix protects protein therapeutics from enzymatic degradation and provides sustained release over a longer period of time, eliminating the need for monthly injections. Methods: This study includes synthesis and characterization of triblock (TB) and pentablock (PBC) copolymers with an emphasis on effect of block arrangements of polymers, rheological properties, sol-gel transition, in vitro cytotoxicity/biocompatibility, in vitro release, release kinetics and in vitro degradation study. Purity and molecular weight were analyzed by NMR. Mw, Mn and PDI indices were examined by GPC. Crystallinity of TB and PBC were analyzed by XRD. Rheological properties were estimated with an Ubbelohde ©2015, 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 2015 Annual Meeting Abstracts capillary viscometer. In vitro cell viability and biocompatibility studies were performed on ARPE 19 and RAW 264.7. In vitro release studies of proteins were performed in PBS, pH 7.4 at 340C. The release data was fitted to various kinetic models to investigate release mechanism. In vitro degradation studies were performed at four different incubation conditions, further subjected to GPC, XRD and ESM. Results: NMR, GPC, FTIR and XRD analyses of TB and PBC provided complete characterization of the polymers. Results from sol-gel transition studies demonstrated that aqueous solutions of TB and PBC can immediately transform to hydrogel at 32-34 °C. PBC provide significantly longer sustained release of IgG relative to TB copolymers. Kinematic viscosity of aqueous solution of PBCs was noticeably lower than the TB copolymers suggesting easy syringeability. In Vitro biocompatibility and cell viability assay exhibited negligible release of cytokines. Based on the R2 value, best fit model was identified. Rapid degradation of TB and PBC depends on the amorphous and hydrophilic nature of thermosensitive polymer. Conclusions: TB and PBC were evaluated for their utility as injectable hydrogel forming depot for sustained ocular protein delivery. These outcomes clearly suggest that PBC based controlled drug delivery system may serve as a promising platform for back of the eye complications. Commercial Relationships: Ashim K. Mitra, None; Sulabh Patel, None; Ravi Vaishya, None; Vibhuti Agrahari, None Program Number: 4142 Poster Board Number: C0025 Presentation Time: 8:30 AM–10:15 AM HP-GUAR-LIPID BASED NANOCARRIER FOR TOPICAL OCULAR DELIVERY OF EPA AND DHA Maria G. Saita, Danilo Aleo, Barbara Melilli, Sergio Mangiafico, Melina Cro, Sebastiano Mangiafico. R&D, MEDIVIS, Catania, Italy. Purpose: The greatest difficulties in the development of ophthalmic formulations based on polynsaturated fatty acids (PUFAs), such as EPA and DHA are derived mainly from their water solubility as well as their poor stability to oxidation. The objective of our work was to develop a new drug delivery system able to solubilize and stabilize EPA and DHA both chemically and physically. Methods: EPA and DHA in oil solution were mixed with an Oxygen Blocker Substance (OBS) and nanodispersed in a carbopol / hyaluronic acid / hydroxypropilguar hydrogel to form a lipid based nanocarrier for PUFAs delivery to the ocular surface (FH0114). Osmolality, pH, droplet size as well as ocular biocompatibility (Corneal Epithelim Cells-HCE test) were evaluated. The chemical stability of EPA and DHA in the final formulation (FH0114) was evaluated with HPLC and compared with that of FH0114 without the OBS (FH0214) under different ICH recommended conditions. Results: FH0114 nanoparticles size was stable and showed a narrow range of distribution (size: 400-600 nm with an average of 440+20nm). Osmolality ranged from 300-310 mOsm/Kg and pH=6.8-7.2. EPA and DHA have maintained a concentration higher than 90% after 24 months at room temperature (25°+/- 2° and 60%+/- 5% R.H.). Concentrations of EPA and DHA at the same ICH conditions in the FH0214 were lower than 25% after 3 months. Ocular biocompatibility (ocular irritation and cytotoxicity tests) evaluated on Human Corneal Epithelium Cells, was good. Conclusions: The new HP-Guar-Lipid-Based vehicle maintains EPA and DHA chemically and physically stable. The new FH0114 formulation was showed biocompatible with HCE cells and may represents a potentially new therapeutic tool in corneal wound healing as well as in dry eye patients. Commercial Relationships: Maria G. Saita, MEDIVIS (E); Danilo Aleo, MEDIVIS (E); Barbara Melilli, MEDIVIS (E); Sergio Mangiafico, MEDIVIS (E); Melina Cro, MEDIVIS (E); Sebastiano Mangiafico, MEDIVIS (E) Program Number: 4143 Poster Board Number: C0026 Presentation Time: 8:30 AM–10:15 AM Formulation and Release of sd-rxRNA® from a Cross-linked, Thiolated CMHA-based Film for Topical Delivery to Reduce the Formation of Corneal Scarring Michael Byrne1, Hee-Kyoung Lee2, James Cardia1, Lakshmipathi Pandarinathan1, Katherine Holton1, Karen Bulock1, Pamela A. Pavco1, Barbara M. Wirostko2. 1Pharmacology, RXi Pharmaceuticals, Marlborough, MA; 2Jade Therapeutics, Salt Lake City, UT. Purpose: Injury to the front of the eye can lead to scarring and negatively impact the transparency of the cornea and vision. Connective tissue growth factor (CTGF) is expressed in cornea after injury and is believed to play a key role in development of ocular fibrosis. RXi has developed a new class of stable, self-delivering RNAi compounds (sdrxRNA) that incorporate features of both RNAi and antisense and are spontaneously taken up by cells. RXI109 is a CTGF-targeting sdrxRNA that is currently in Phase 2 clinical trials for the reduction of dermal scarring and in pre-clinical development for reduction of retinal scarring. Intravitreal administration of RXI109 to monkey eyes resulted in dose dependent reduction of CTGF protein levels in the retina and in the cornea. In order to treat injuries of the front of the eye to prevent corneal scarring, a topical formulation would be ideal. Here, the formulation and release of a control sdrxRNA from thiolated carboxymethyl hyaluronic acid (CMHA)/poly ethylene glycol diacrylate (PEGDA) hydrogel films from Jade was evaluated to investigate the possibility of sustained topical release. Methods: sd-rxRNA was formulated in 1.0% and 1.2 % CMHA with a 1.5:1 thiol:acrylate ratio, molded and dried to create thin flexible 6 mm diameter films. Fifty to four-hundred micrograms of sd-rxRNA was incorporated during formulation. The release was monitored for 16 days by measuring UV absorption at 260 nm and was calculated from a standard curve. Results: sdrxRNA was successfully formulated in CMHA films. The majority (50-80%) was released within 24 hours; however, the remaining release continued over approximately 6 days. For the films formulated with 400 mg, release was still detectable at day 16. The accumulated release was between 65% and 95% depending on the dose. The release profile was similar between 1.0% and 1.2% CMHA. Conclusions: sd-rxRNAs were successfully formulated in CMHA films and show a favorable release profile over a period of approximately two weeks. Future studies will be focused on optimizing the release profile and producing in vivo grade films formulated with RXI109 for studies focused on anterior segment uptake of RXI109. Sustained topical delivery of RXI109 could serve as a potential therapeutic to be administered at the time of corneal injury and lead to reduced scarring and improved vision. Commercial Relationships: Michael Byrne, RXi Pharmaceuticals (E); Hee-Kyoung Lee, Jade Therapeutics (E); James Cardia, RXi (E); Lakshmipathi Pandarinathan, RXi (E); Katherine Holton, RXi (E); Karen Bulock, RXi (E); Pamela A. Pavco, RXi (E); Barbara M. Wirostko, Jade (E) ©2015, 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 2015 Annual Meeting Abstracts Program Number: 4144 Poster Board Number: C0027 Presentation Time: 8:30 AM–10:15 AM Development and optimization of clear, aqueous triamcinolone acetonide eye drop Hoang Trinh, Kishore Cholkar, Ashim K. Mitra. University of Missouri kansas city, Kansas city, MO. Purpose: There are almost 18.8 million people suffering from diabetes in U.S and this population develops diabetic macular edema (DME) or retinopathy during their life time. Triamcinolone acetonide (TA) is an anti-inflammatory and anti-angiogenic drug indicated for treat back of the eye diseases to reduce the central macular thickness. The purpose of this study is to develop, optimize, characterize and evaluate toxicity of triamcinolone acetonide loaded clear aqueous mixed nanomicellar formulation Methods: A full factorial design was selected from screening design to predict response variables. Amount of Polymer 1 and Polymer 2 were selected as independent variables. Percent triamcinolone acetonide entrapment, loading and critical micellar concentration (CMC) were evaluated as dependent variables. Formulations were prepared by solvent casting/film hydration method. Response data was analyzed with standard least square fit analysis. Based on t-test, variables which had significant effect were determined. For optimization process, prediction profiler was generated to predict the levels of independent variables allowing maximum entrapment efficiency, loading efficiency while minimizing CMC Results: The formulation was prepared with predicted polymer ratio, Polymer 1 and Polymer 2 (5:1.5). The results were in agreement with predicted profile. Furthermore, all formulations were characterized for micelle size, polydispersity index, zeta potential, and viscosity. Qualitative 1H NMR studies confirmed the absence of free triamcinolone acetonide in aqueous solution. In vitro biocompatibility of formulations studies with WST-1 reagent assay produced no toxicity on human cornea epithelical cells (HCEC) Conclusions: In conclusion, an aqueous, clear mixed nanomicellar triamcinolone acetomide loaded formulation is successfully prepared with the aid of Polymer 1 and Polymer 2. Fig 1. a. Real-time scanning transmission electron microscope (STEM) image of triamcinolone acetonide-loaded nanomicelles (x147,000). Scale bar 100 nm. b. Image showing visual appearance of 0.1% triamcinolone acetonide-loaded nanomicelles on the left side in comparison to water on the right side Commercial Relationships: Hoang Trinh, None; Kishore Cholkar, None; Ashim K. Mitra, None Support: NIH R01EY09171-16 and R01EY010659-14 Program Number: 4145 Poster Board Number: C0028 Presentation Time: 8:30 AM–10:15 AM Controlled vancomycin release from a biodegradable hydrogel ocular drug delivery system Emily Dosmar1, William F. Mieler2, Jennifer J. Kang Mieler1. 1 Biomedical Engineering, Illinois Institute of Technology, Chicago, IL; 22Department of Ophthalmology and Visual Sciences, University of Illinois, Chicago, IL. Purpose: The purpose of this study was to investigate the use of a biodegradable hydrogel to deliver prophylactic vancomycin (VAN) for two weeks following ocular surgery. Methods: VAN was encapsulated in hydrolytically degradable poly(ethylene glycol)-co-(L-lactic acid) diacrylate (PEG-PLLA-DA) and poly(ethylene glycol) diacrylate (PEG-DA) hydrogels. Polymer concentration, polymer PEG-PLLA-DA:PEG-DA ratio, PEG-DA molecular weight (575 MW or 700 MW), and polymerization time were varied to assess the degradation rate and time of hydrogel. Polymer composition was varied to determine swelling ratios. The mesh size of the hydrogel network was estimated using the Flory-Rehner equations. VAN release profiles were conducted at 37°C; at predetermined intervals, samples were analyzed via highperformance liquid chromatography to quantify VAN concentration. Results: Hydrogel swelling ratios decreased significantly with increased PEG-DA concentrations (p=7×10-4), demonstrating the dependency of hydrogel swelling ratio on polymer concentration. Hydrogel mesh size estimations ranged from 6.79-7.76 nm regardless of investigated polymer composition. Hydrogels degraded slower as polymer concentration increased and hydrogels with a higher degradable to non-degradable polymer ratio showed a faster overall degradation time. Hydrogels with the same polymer ratio degraded more quickly with lower PEG-DA molecular weight: hydrogels rendered with PEG-DA 575MW degraded 5 days faster than those with 700MW. Hydrogels composed of 20:0 PEG-PLLA-DA:PEG-DA exhibited ~30% cumulative release (of theoretically encapsulated VAN) after 24 hours, 40% release by 1 week, and degraded completely in 9 days. Hydrogels with 17:3 PEG-PLLA-DA:PEGDA showed ~30% release at 24 hours, 33% at 1 week and degraded completely in 16 days. Conclusions: This study demonstrated that by modifying polymer concentration and ratio, the degradation and release time of VAN can be controlled. Biodegradable hydrogels may have promise for application as prophylactic antibiotic ocular drug delivery devices. Commercial Relationships: Emily Dosmar, None; William F. Mieler, Genentic (C), Thrombogenics (C); Jennifer J. Kang Mieler, None Program Number: 4146 Poster Board Number: C0029 Presentation Time: 8:30 AM–10:15 AM Ranibizumab Delivery Device Using Polyethyleneglycol Dimethacrylates Toshiaki Abe, Aya Katsuyama, Hideyuki Onami, Toru Nakazawa, Nobuhiro Nagai. Graduate School of Medicine, Tohoku University, Sendai, Japan. Purpose: To test the extended release of ranibizumab using a polymeric system made of photopolymeized poly(ethyleneglycol) dimethacrylates. Methods: The device consists of a reservoir, controlled-release cover, and drug formulation, which were made of photopolymeized triethyleneglycol dimethacrylate (TEGDM) and polyethyleneglycol dimethacrylate (PEGDM). Ranibizumab (10 mg/ml) was mixed with PEGDM prepolymer at the ratio of 4 to 1 (volume), and the mixture (12.5 μl) was loaded in a TEGDM reservoir (12 mm × 4.4 mm × 1.2 mm), then photopolymerized for 40 seconds with UV light. After loading the ranibizumab, a prepolymer mixture of 20% PEGDM in water was applied to the reservoir, and a glass slide was placed on the prepolymer mixture, followed by UV curing for 4 min to provide a reservoir cover. The device was incubated in 1 ml of phosphate-buffered saline (PBS) at 370C. The released ranibizumab in the collected PBS was seperated by electrophoresis and the release amount was estimated from the band intensity using a standard curve. ©2015, 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 2015 Annual Meeting Abstracts To study the bioactivity of release ranibizumab, endothelial tube formation was assessed using anti-CD31 immunostaining. Results: In vitro release results show the extended release of ranibizumab from the device over 150 days. The release rate estimated from the gradient curve was 0.245 ng per day. The capiliary formation was inhibited by the medium including ranibizumab released from the device, compared with the medium including PBS released from a PBS-loaded device or non-treated medium. The results indicate that ranibizumab retained its activity when released from the device. Conclusions: We established a sustained ranibizumab release device by using a photocurable polymers. The device may be promissing for ocular drug delivery system for the treatment of age-related macular disease. Commercial Relationships: Toshiaki Abe, None; Aya Katsuyama, None; Hideyuki Onami, None; Toru Nakazawa, None; Nobuhiro Nagai, None Support: JSPS KAKENHI Grant Number 26861435 Program Number: 4147 Poster Board Number: C0030 Presentation Time: 8:30 AM–10:15 AM Cell-penetrating peptide constructs as non-invasive drug delivery vehicles for ranibizumab and bevacizumab Felicity De Cogan, Peter Morgan-Warren, Lisa J. Hill, Robert A H Scott, Ann Logan. Clinical and Experimental Medicine, University of Birmingham, Birmingham, United Kingdom. Purpose: To investigate cell penetrating peptide constructs (CPPCs), with a protein transduction domain, as novel topical ocular drug delivery vehicles that transport macromolecule biopharmaceutical agents into the posterior segment of the eye. Methods: CPPCs were synthesised using standard solid phase peptide synthesis. Synthesised CPPCs were mixed with OvalbuminTexas Red (OVA-TR), a macromolecule used as a drug model for ranibizumab, in PBS to create macromolecule carrying eye drops. The loaded and unloaded CPPC eye drops were topically administered to the adult rat cornea in vivo. The concentration of CPPC and of macromolecule loaded CPPCs that passed into retinal tissue was quantified. Results: When delivered as an eye drop formulation, fluorescently labelled unloaded CPPCs were capable of penetrating the retina from topical application to the cornea. CPPC levels were determined using fluorescence spectroscopy, with significantly higher fluorescence measured in the retina of unloaded CPPC treated eyes (23.76 ± 4.4 %) when compared to the background signal (12.99 ± 2.9 %), (p=0.045) obtained from the retina of untreated eyes. OVA-TR loaded CPPCs were used to deliver OVA-TR to the retina following topical administration to the front of the eye. The measured macromolecule concentration of OVA-TR in the retina, determined by absorbance of the conjugated tag, was 0.5 ± 0.1 mg at 120 minutes following topical application, significantly higher than levels in control untreated eyes (p=0.036). Conclusions: CPPCs: (1), are non-toxic to ocular cells in vitro and in vivo; (2), are capable of decorating large macromolecules ; (3), enabled macromolecules to penetrate ocular tissue in vivo; (4), delivered mg quantities of macromolecules to the anterior and posterior segments in vivo. In summary, topically administered CPPC eye drops effectively deliver physiologically relevant concentrations of macromolecules to the anterior and posterior segments of rat eyes in vivo. Commercial Relationships: Felicity De Cogan, None; Peter Morgan-Warren, None; Lisa J. Hill, None; Robert A H Scott, None; Ann Logan, None Support: NIHR Program Number: 4148 Poster Board Number: C0031 Presentation Time: 8:30 AM–10:15 AM Cataracts as an adverse event in the drug development process: findings from a systematic review and economic model Andrew F. Smith1, 2, Alex Klotz1, Michael Wormstone3. 1Medmetrics Inc, Ottawa, ON, Canada; 2Department of Ophthalmology, King’s College London, London, United Kingdom; 3School of Biological Sciences, University of East Anglia, Norwich, United Kingdom. Purpose: Limited information exists on the degree to which cataract formation impacts the drug development process of compounds across a number of therapeutic areas. As such, we conducted a systematic literature search on cataract formation as an adverse event in the overall drug development process and developed an economic model to estimate its impact. Methods: A systematic literature search was conducted using Google scholar, which incorporates a number of literature database search engines. The key search criteria used were the terms “phase I trial” cataract. The word trial was replaced with study and clinical and the phase was varied from pre-clinical to phase I,II,III, and IV, respectively. The economic model was developed in Visual Basic for Applications (VBA). The key input parameters included: the number of patients in each of the clinical phases, the probability of the trial phase being successful, the costs of treating cataract, the cost of other adverse events and the length of time spent in each of the various phases. Costs were discounted over the length of the trial using a discount rate of 5%. Results: Data from the systematic review indicated that in those cases where cataract occurred in drugs for a life-threatening condition, it did not impede the trial and the costs of treating cataract were out-weighed by those costs attributable to the life-threatening condition of interest. The economic model forecasted that the mean break even time for a drug with possible cataractogenic adverse events in the drug development process ranged from a low of 1.26 years to a high of 6.36 years assuming that the drug would be marketed to a patient population of 100,000 at a cost of US$ 25 per unit. Such break even measures may facilitate the comparison between the costs and adverse events in the selection of potential compounds for development. Conclusions: Our model provides an understanding of the relative costs of cataract in the context of drug trials over a range of lifethreatening, non-life-threating and sight-threatening indications. Such information is of particular value to a wide audience of industrial R & D organizations, clinical trial scientists, clinicians and healthcare funding administrators. Utilization of the model may help to guide and refine the drug development process and improve candidate selection. Commercial Relationships: Andrew F. Smith, University of East Anglia (F); Alex Klotz, Medmetrics Inc (F); Michael Wormstone, University of East Anglia (E) Support: School of Biological Sciences, Unitersity of East Anglia, Norwich, UK ©2015, 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 2015 Annual Meeting Abstracts Program Number: 4149 Poster Board Number: C0032 Presentation Time: 8:30 AM–10:15 AM Biointegration of collagen hydrogel, as donor corneal substitute, on PMMA for Boston keratoprosthesis Andri K. Riau1, 2, DEBASISH MONDAL1, Gary Hin-Fai Yam1, Bo Liedberg2, 3, Subbu S. Venkatraman2, Jodhbir S. Mehta1, 4. 1Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore, Singapore; 2School of Materials Science and Engineering, Nanyang Technological University, Singapore, Singapore; 3 Interdisciplinary Graduate School, Nanyang Technological University, Singapore, Singapore; 4Singapore National Eye Centre, Singapore, Singapore. Purpose: To improve biointegration and adhesion strength of collagen hydrogel, as human donor corneal substitute, on PMMA for Boston keratoprosthesis. Methods: PMMA sheets of 25 x 25 x 0.5 mm were surface modified by: oxygen plasma for 5 minutes (referred as plasma group); oxygen plasma followed by hydroxyapatite (HAp) coating (plasma+HAp group); L-3,4-dihydroxyphenylalanine and 11-mercaptoundecanoic acid followed by HAp coating (DOPA+HAp group); or oxygen plasma followed by 5% (3-aminopropyl)triethoxysilane coating (3-APTES group). The modified surfaces were characterized by water contact angle changes, ATR/FTIR and AFM. Bovine collagen type I hydrogel was casted and allowed to gel overnight on the modified surfaces and then subjected to shear adhesion strength tests. Morphology of primary human corneal fibroblasts seeded on the modified PMMA surfaces was assessed by SEM. Results: ATR/FTIR showed the presence of respective coating on the PMMA surface. Oxygen plasma, plasma+HAp and DOPA+HAp treatment significantly enhanced the surface hydrophilicity of the PMMA (all p<0.05 compared to untreated PMMA). Water contact angle produced by 3-APTES treatment (71.99±0.90o) was slightly increased relative to that of untreated PMMA (68.43±3.00o; p=0.09). AFM revealed relatively rough surface after plasma+HAp (RMS = 294.33±38.43 nm) and DOPA+HAp treatment (337.77±109.89 nm), but they are not significantly different (p=0.499). There was a decrease in surface roughness after 3-APTES coating (8.25±1.10 nm) relative to after plasma treatment (18.58±1.19 nm; p<0.001). On SEM, human corneal fibroblasts were able to maintain their normal morphology on all treated surfaces, comparable to cells seeded on cover slips. Plasma+HAp yielded the best shear adhesion strength with collagen hydrogel (179.50 ± 29.99 mN/cm2), followed by DOPA+HAp (159.50 ± 26.56 mN/cm2), 3-APTES (133.25 ± 20.23 mN/cm2) and plasma treatment (114.50±11.68 mN/cm2). All treatments produced significantly better adhesion strength with collagen hydrogel than untreated PMMA (67.25±10.09 mN/cm2; p<0.05). Conclusions: HAp coating can potentially be used to enhance the interfacial seal between PMMA optical cylinder and collagen hydrogel. Our study also suggests that collagen hydrogel can be used as donor corneal substitute for Boston keratoprosthesis and thereby, reducing our heavy dependence on transplant-grade donor corneas in the future. Commercial Relationships: Andri K. Riau, None; DEBASISH MONDAL, None; Gary Hin-Fai Yam, None; Bo Liedberg, None; Subbu S. Venkatraman, None; Jodhbir S. Mehta, None Support: Singapore National Research Foundation Translational and Clinical Research Fund Program Number: 4150 Poster Board Number: C0033 Presentation Time: 8:30 AM–10:15 AM An In Vitro Reconstructed Normal Human Corneal Tissue Model for Corneal Drug Delivery Studies of Ophthalmic Formulation Yulia Kaluzhny, Miriam Kinuthia, Viktor Karetsky, Laurence d’Argembeau-Thornton, Patrick Hayden, Mitchell Klausner. MatTek Corporation, Ashland, MA. Purpose: Permeation of topically applied ocular drugs occurs predominantly through the cornea and therefore absorption studies using corneal tissues play a critical role in ocular drug formulation. Currently, most ocular absorption studies use in vivo or ex vivo animal tissues that have many disadvantages including poor standardization, species extrapolation, high cost, and ethical concerns. Methods: A reconstructed corneal tissue model (EpiCornealTM) was recently developed. The model consists of normal human corneal epithelial cells that have been cultured using serum free medium to form a highly differentiated organotypic corneal epithelial tissue. The multilayered cultures contain tight junctions and develop barrier properties comparable to the in vivo human cornea. Real time qPCR confirmed that the reconstructed tissues express ALDH-A1 and TXNRD1 (corneal epithelium specific enzymes that confer resistance to UV light damage), MUC4 (corneal glycoprotein mucin), and ABCC1 and ABCB1 (efflux transporters with important roles in corneal drug distribution). The permeability of the model was evaluated using compounds with a wide range of properties: a) the hydrophilic dyes sodium fluorescein (NaFl), fluorescein isothiocyanate-labeled dextran (FD-4, MW=4000), and lucifer yellow (LY); b) the hydrophobic dye rhodamine B (RdB), and c) ophthalmic related antibiotics, ofloxacin (OFL) and voriconazole (VCZ). The effect of permeation enhancing agents, 0.01% Benzalkonium Chloride (BAK), 0.05-0.5% EDTA, 0.005% Cetylpyridinium chloride (CPC), and 0.02% Polyoxyethelen-20 stearyl ether (PSE), was investigated. Results: The permeation coefficient (Kp) for NaFl/ FD-4/ LY/ RdB/ OFL/ and VCZ was 6.0±0.1x10-7/ 5.7±3.8x10-8/ 6.8±0.7x10-7/ 3.5±0.4x10-5/ 1.0±0.2x10-6/ and 3.9±1.1x10-5 cm/s. The Kp’s agree with literature values. 0.25% EDTA increased permeability of NaFl/ FD-4/ LY/ and VCZ by 2.2/ 3.9/ 7.1/ and 4.0 fold, respectively. 0.01% BAC increased the permeability of OFL and VCZ by 3.4 and 18.5 fold, respectively. 0.02% PSE increased the permeability of NaFl and FD-4 by 4.1 and 3.0 fold, respectively. Conclusions: The reconstructed in vitro EpiCorneal tissue morphology, barrier properties, and permeability resemble those of the in vivo human cornea. This model is anticipated to be a useful tool to evaluate new corneal drug formulations. Commercial Relationships: Yulia Kaluzhny, MatTek Corporation (E); Miriam Kinuthia, MatTek Corporation (E); Viktor Karetsky, MatTek Corporation (E); Laurence d’Argembeau-Thornton, MatTek Corporation (E); Patrick Hayden, MatTek Corporation (E); Mitchell Klausner, MatTek Corporation (E) Program Number: 4151 Poster Board Number: C0034 Presentation Time: 8:30 AM–10:15 AM Nanofiber-assembled biomatrix for corneal tissue engineering: Enhanced drug delivery by integration of specific surface linkers Piotr Stafiej2, 1, Sahar Salehi1, Jochen Gutmann1, Dirk W. Schubert3, Friedrich E. Kruse2, Thomas Bahners1, Thomas A. Fuchsluger2. 1 Deutsches Textilforschungszentrum Nord-West e.V., Duisburg, Germany; 2Department of Ophtalmology, University of ErlangenNurnberg, Erlangen, Germany; 3Institute of Polymer Materials, University of Erlangen-Nurnberg, Erlangen, Germany. ©2015, 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 2015 Annual Meeting Abstracts Purpose: We have previously demonstrated that Polycaprolactone (PCL) / Poly(glycerol sebacate) (PGS) nanofiber-biomatrix shows properties for ocular surface reconstruction (biodegradability, extracellular matrix (ECM) attributes). Manufactured by electrospinning, this biocompatible scaffold promotes growth of corneal cells. To further optimize the biomatrix for drug delivery we now assembled specific surface groups for immobilization of proteins, like growth-factors or specific cell-binding proteins. Methods: The biomatrix was electrospun in different PCL:PGS blend ratios (1:1, 1:2, 1:3, 1:4) and cut into a sample size of 1 cm2. Fiber surfaces were functionalized by a wet-chemical treatment of the scaffolds. In a first step, amino-functional groups were introduced to existing hydroxyl groups, after which thio-functional groups were added. The amount of thiol groups was analyzed by Ellman’s reagent and subsequently measured. MTT apoptosis tests were performed to determine negative effects following this modification. Fiber morphology was examined by Scanning Electron Microscopy (SEM). Results: Thiol groups could be introduced by the described wetchemical process in each PGS containing sample. Interestingly, the amount of introduced thiol groups decreased with increasing concentration of PGS (PGS:PCL 110±3,74 nM [1:1], 51±33 nM [2:1], 23±14 nM [3:1], 8±2 nM [4:1]). Separate samples made of unblended PGS, PCL, and of untreated cotton served as controls. Here, thiol groups could not be established on PCL fibers, while significant amounts were detected on PGS and cotton, both materials having hydroxyl groups. SEM images did not show major changes in fiber morphology. No significant increase of apoptosis could be measured. Conclusions: Thio-functional groups could be established on the surfaces of a PGS:PCL nanofiber biomatrix by a wet-chemical process. The treatment did not affect fiber morphology and did not significantly increase apoptosis. Hence, the fiber modification did not reactively affect biomatrix properties. Further research will reveal, how binding of specific proteins to these surface groups will increase proliferation and differentiation of corneal cells. Commercial Relationships: Piotr Stafiej, None; Sahar Salehi, None; Jochen Gutmann, None; Dirk W. Schubert, None; Friedrich E. Kruse, None; Thomas Bahners, None; Thomas A. Fuchsluger, None Program Number: 4152 Poster Board Number: C0035 Presentation Time: 8:30 AM–10:15 AM Development of triamcionolone acetonide based lipid nanocapsules as platforms for ocular drug delivery María L. Formica1, 2, Gabriela V. Ullio Gamboa1, 2, Jean P. Benoit4, Daniel A. Allemandi1, 2, Jose D. Luna Pinto3, Santiago D. Palma1, 2. 1 Faculty of Chemical Sciences - Pharmacy, University of Córdoba, Córdoba, Argentina; 2Unidad de Investigación y Desarrollo en Tecnología Farmacéutica (UNITEFA) - CONICET, Córdoba, Argentina; 3Ctr Privado de Ojos Romagosa-Fndtn VER, Córdoba, Argentina; 4Laboratoire INSERM U1066-IBS-CHU Angers (France), Angers, France. Purpose: Triamcinolone acetonide (TAA) is considered a first-line drug by itself or as a combined treatment of several intraocular diseases such as macular edema, retinal vein thrombosis, uveitis and age-related macular degeneration. The development of TAA dosage forms is limited due to its poor solubility in water and physiologically acceptable solvents. Lipid nanocapsules (LNCs) are biocompatible systems that allow loading both hydrophobic and hydrophilic drugs. LNCs present a versatile composition and application suitable for different routes of administration. The aim of this work was to develop and characterize a novel lipid LNCs formulation containing TAA as drug delivery system. Methods: LNCs were prepared in triplicate using an optimized phase inversion-based method described by Heurtault et al., 2002. Due to the poor solubility of TAA in the oily phase of the original formulation, two co-surfactants (captex® 500p -Glyceryl triacetate and oleic acid) in three proportions (20, 30 and 50%) were tested. The average particle size (APS), polydispersity index (PI), zeta potential (ZP) and entrapment efficacy (EE) were measured. Results: Acceptable results were obtained with a 20% of both cosurfactants. LNCs with captex® 500p leads to about (40±1) nm size nanoparticles with a narrow size distribution (PI less than 0.2), a negative ZP (-1.2±0.7) mV and EE (85.8±0.8) % while LNCs with oleic acid showed an APS of (35.9± 0.6) nm and a PI below 0.1 with a negative ZP (-3.6±0.6) mV and EE (87±2) %. Moreover, both systems were stable for two months. Conclusions: LNCs allow encapsulation of TAA and their properties remain constant over long periods of time. Thus, LNCs are promising systems than may be a potential strategy to improve efficacy and decrease side effects of this drug so used in the treatment of intraocular diseases. Commercial Relationships: María L. Formica, None; Gabriela V. Ullio Gamboa, None; Jean P. Benoit, None; Daniel A. Allemandi, None; Jose D. Luna Pinto, None; Santiago D. Palma, None Program Number: 4153 Poster Board Number: C0036 Presentation Time: 8:30 AM–10:15 AM Pharmacokinetic and Safety Evaluation of a Transscleral Sustained Unoprostone Release Device Nobuhiro Nagai1, Yasuko Izumida1, Eri Koyanagi1, Hirokazu Kaji2, Matsuhiko Nishizawa2, Takahito Imagawa3, Akiko Morikawa3, Toru Nakazawa1, Yukihiko Mashima3, Toshiaki Abe1. 1Graduate School of Medicine, Tohoku University, Sendai, Japan; 2Graduate School of Engineering, Tohoku University, Sendai, Japan; 3R-tech Ueno Ltd., Tokyo, Japan. Purpose: To evaluate the ocular tissue distribution of unoprostone isopropyl (UNO) and retinal toxicity after transscleral administration of UNO by a drug delivery device in rabbits. Methods: The device consists of a reservoir, controlled-release cover, and drug formulation, which were made of photopolymeized poly(ethyleneglycol) dimethacrylates. UNO, a prostone and BK channel activator for antiglaucoma eyedrops marketed in Japan, was loaded in the device (length, 10 mm; width, 3.6 mm; height, 0.7 mm) at a content of 2.85 mg UNO. High-performance liquid chromatography was used to evaluate the release amount of UNO in vitro. The UNO metabolite, unoprostone-free acid (M1), ©2015, 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 2015 Annual Meeting Abstracts concentrations in the retina, choroid, and plasma were determined by liquid chromatography-tandem mass spectrometry at 4, 12, and 24 weeks after implantation in rabbits. Retinal toxicity was evaluated by electroretinogram and optical coherence tomography. Results: The UNO released from the device in vitro showed zeroordered kinetics for 12 weeks, then the release gradually decreased to 24 weeks. The area under the M1 concentration curve (AUC) of the retina during 24-week device implantation was higher than the simulated AUC of the retina after topical administration of 0.12% UNO eye-drop (once-a-day for 24-week). No substantial toxic reactions were observed by electroretinogram and optical coherence tomography. Conclusions: The device could be a useful carrier for intraocular sustained delivery of UNO without producing severe retinal toxicity. Commercial Relationships: Nobuhiro Nagai, Tohoku University (P); Yasuko Izumida, None; Eri Koyanagi, None; Hirokazu Kaji, Tohoku University (P); Matsuhiko Nishizawa, Tohoku University (P); Takahito Imagawa, R-tech Ueno Ltd. (E); Akiko Morikawa, R-tech Ueno Ltd. (E); Toru Nakazawa, None; Yukihiko Mashima, R-tech Ueno Ltd. (E); Toshiaki Abe, Tohoku University (P) Support: JSPS KAKENHI Grant Number 26560232, Health Labour Sciences Research Grant from the MHLW (H24-nanchitohippan-067), Japan Program Number: 4154 Poster Board Number: C0037 Presentation Time: 8:30 AM–10:15 AM Sustained Release Dexamethasone Implants for In Vitro – In Vivo Correlations YUNPENG BAI1, David Bourne1, yan wang2, Stephanie Choi2, Uday B. Kompella1. 1Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO; 2U. S. Food and Drug Administration, Silver Spring, MD. Purpose: To prepare qualitatively (Q1) and quantitatively (Q2) identical dexamethasone-poly(lactic-co-glycolic acid) (PLGA) implants with variable diameters and mechanical properties in order to identify implants suitable for developing in vitro-in vivo correlations. Methods: Powders of dexamethasone and PLGA-503H were mixed at a ratio of 1:4 and implants with different diameters were manufactured with Telfon molds and warm compression. Blade cutting mode of the Texture Analyzer was used to measure the firmness and toughness of implants. Dexamethasone content in implants was quantified using a QTRAP 4500 LC-MS instrument. Results: Warm compression followed by cooling of the molds allowed easy removal of the implants from the mold using a stylus. Dexamethasone loaded implants of diameters about 1 and 0.8 mm with a drug content of about 20 and 17%, respectively, were made. Implants with the larger diameter exhibited higher hardness (1074.6 vs 344.1 g) and toughness (108.7 vs 31.4 g•sec). Conclusions: Using Teflon molds and warm compression, dexamethasone-PLGA implants of various diameters could be prepared. Future studies will involve preparation of implants with low, medium, and high in vitro release rates for in vivo studies and the development of in vitro-in vivo correlations. Commercial Relationships: YUNPENG BAI, None; David Bourne, None; yan wang, None; Stephanie Choi, None; Uday B. Kompella, None Support: Supported by the FDA grant U01 FD0004929-01. Program Number: 4155 Poster Board Number: C0038 Presentation Time: 8:30 AM–10:15 AM Micelles based on tri-block copolymer with positive charges enhance its corneal penetration Junjie Zhang, Jingguo Li, Tianyang Zhou, Siyu He, Zhanrong Li, Hongmin Zhang, Liya Wang. Henan Eye Institute, Henan Eye Hospital, Zhengzhou, China. Purpose: Cornea is a main barrier for drug penetration after topical application. The aim of this study was to evaluate the ability of corneal penetration by newly generated micelles based on tri-block copolymer with a positive charge. Methods: The tri-block copolymer poly (ethylene glycol)-poly (ε-caprolactone)-g-polyethylenimine (mPEG–PCL-g–PEI) was synthesized. The physicochemical properties of the self-assembled polymeric micelles were investigated including hydrodynamic sizes, zeta potentials, the morphology, the drug loading content (DLC), drug loading efficiency (DLE) and in vitro drug release. The behavior of polymeric micelles penetration was in vivo monitored using a twophoton scanning fluorescence microscopy on murine corneas after topical application. Results: The polymeric micelles had a particle size of 28 nm and a zeta potential of approximately +12 mV, with spherical in morphology. DLE and DLC were 75.37% and 3.47%, respectively. The release of polymeric micelles showed a control-release behavior in vitro. The polymeric micelles with positive charge penetrated significantly across the cornea compared with the control in vivo. Conclusions: Positively charged micelles based on triblock copolymer is a promising vehicle for the topical delivery of hydrophobic agents in ocular applications. Commercial Relationships: Junjie Zhang, None; Jingguo Li, None; Tianyang Zhou, None; Siyu He, None; Zhanrong Li, None; Hongmin Zhang, None; Liya Wang, None Program Number: 4156 Poster Board Number: C0039 Presentation Time: 8:30 AM–10:15 AM Eyedrop Formulation and Evaluation of Quercetin - a component of Ginkgo biloba James Brodie, Ben Davis, Lisa Turner, Shereen Nizari, M Francesca Cordeiro. Glaucoma and Retinal Degeneration Research Group, UCL Institute of Ophthalmology, London, United Kingdom. Purpose: Quercetin, a component of Ginkgo biloba, has been shown to possess a wide range of antioxidant and neuroprotective properties which may have therapeutic potential in ophthalmic disorders such as glaucoma and diabetic retinopathy. Due to its relative insolubility in water, in vivo studies investigating the neuroprotective effects of Quercetin have so far been limited to administering it as an oral preparation. The objective of this study was to formulate Quercetin to enable topical drug delivery to the eye, thereby increasing the stability and bioavailability of the drug, and to evaluate its characteristics and suitability for future in vivo studies. Methods: A polymeric micellar formulation was developed which encapsulated Quercetin using a lipid-film hydration method. The particle size and encapsulation efficiency of the prepared micelles were measured by dynamic light scattering and UV absorption spectroscopy, respectively. The addition of a lyoprotectant to the formulation allowed the micellar preparations to be freezedried in order to maintain stability over the course of the study. Quercetin-loaded micelles were prepared and administered topically to normal and glaucomatous rat eyes to assess corneal and ocular toxicity. Results: The Quercetin-loaded micelles had an average particle size of 18.10 nm and encapsulation efficiency of 77.03%. Stability ©2015, 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 2015 Annual Meeting Abstracts was maintained in the freeze-dried formulations and once reconstituted the preparation was stable for an additional 72 hours at 25°C. The formulation had a pH of 7.0 that is within the ideal range of pH for ophthalmic solutions. Preliminary studies showed that the quercitin formulation was well-tolerated in vivo with no evidence of corneal or ocular toxicity during the study. Conclusions: Quercetin has proven potential as a neuroprotective agent; however the in vivo studies have been limited by the challenges to formulation it poses. The utilisation of polymeric nanoparticles as vehicles for drug delivery appears to be a promising method of incorporating otherwise insoluble compounds with therapeutic potential. This study demonstrates that topical ophthalmic delivery of Quercetin can be achieved by encapsulation of the drug in a pluronic-based polymeric micelle vehicle, and future studies will look to investigate the efficacy of this formulation in vivo. Commercial Relationships: James Brodie, None; Ben Davis, None; Lisa Turner, None; Shereen Nizari, None; M Francesca Cordeiro, None Program Number: 4157 Poster Board Number: C0040 Presentation Time: 8:30 AM–10:15 AM SUSTAINED RELEASE DRUG DELIVERY FOR TREATING OCULAR ANGIOGENESIS Devi Kalyan Karumanchi, Elizabeth R. Gaillard. Department of Chemistry and Biochemistry, Northern Illinois University, Dekalb, IL. Purpose: To develop liposomal formulations for the sustained and controlled release of anti-VEGF antibodies to treat neovascularization associated with diabetic retinopathy and wet AMD. Methods: Stable liposomal formulations were made using a modified lipid hydration and extrusion method. A model fluorescent protein was encapsulated in the liposomes for preliminary studies and evaluation of the vehicle. The liposomes were evaluated based on particle size, surface morphology, percentage drug encapsulation and time of release using dynamic light scattering, transmission electron microscopy, fluorescence spectroscopy and USP4 SOTAX dissolution apparatus, respectively. Results: The liposomal formulations after extrusion exhibited a narrow size distribution of approximately 100-150 nm in diameter with around 85-92% encapsulation efficiency. From the in vitro drug release studies, we observed a timed release over a period of 6-8 months depending on the composition of the formulation. Conclusions: Liposomes are non-toxic, biodegradable artificial vesicles composed of phospholipids and cholesterol. Abrishami et al have been able to obtain a sustained release of the anti-VEGF drugs up to a period of 42 days. Currently, we have been successful in encapsulating a model protein into our stable liposomal formulations and attain a controlled release over a period of 6 months in vitro. In the future, we are interested in encapsulating the protein drugsAvastin and Lucentis to show the efficiency of our drug delivery vehicle. With this study, our efforts would be to decrease the frequency of intravitreal injections from 12 to 2 per year, thereby effectively making the treatment more economical. TEM image of the liposomes In vitro drug release profiles Commercial Relationships: Devi Kalyan Karumanchi, Northern Illinois University (P); Elizabeth R. Gaillard, Northern Illinois University (P) Program Number: 4158 Poster Board Number: C0041 Presentation Time: 8:30 AM–10:15 AM The Aseptic Fabrication of ENV515 (Travoprost) Intracameral Implants Leo Trevino1, Michael Hunter2, Sanjib Das2, Tyler Pegoraro3, Tyler Massey3, Andres Garcia2, Janet Tully2, Kristie Hamby2, Tomas Navratil2, Benjamin Yerxa2. 1Pharmaceutical Development, Envisia Therapeutics Inc., Morrisville, NC; 2Envisia Therapeutics Inc., Morrisville, NC; 3Liquidia Technologies, Inc., Morrisville, NC. Purpose: The ENV515 (travoprost) Intracameral Implant is a biodegradable, rod shaped implant using an extended release formulation of travoprost. These dosage forms do not lend themselves to typical sterilization techniques used for liquid dosage forms, such as filtration, and the drug substance travoprost is prone to degradation via ionizing radiation utilized during terminal gamma sterilization. Therefore, alternate approaches must be utilized to produce a sterile product. We describe a method for producing sterile ENV515 implants using an aseptic cGMP process and evaluate ENV515 implants produced in this process for sterility and other key attributes to support further clinical development. ©2015, 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 2015 Annual Meeting Abstracts Methods: ENV515 implants were produced utilizing PRINT microparticle engineering technology. An aseptic manufacturing process was developed, utilizing a combination of sterile filtration, aseptic processing, and gamma sterilization of consumables, including the mold used to form the implants. The aseptic process was validated using three consecutive process simulations (3 batches, 2,300 implants/batch) and testing 100% of each batch for sterility. The validated aseptic cGMP process was used to fabricate clinical trial material for use in first-time-in-human Phase 2a clinical study. Batch samples were also tested for assay, dose content uniformity, and in vitro drug release. Results: Three consecutive process simulation batches (2,300 implants/batch) were tested for microbial growth and were found to be 100% sterile in a 14 day sterility test. ENV515 clinical trial material was manufactured in this aseptic cGMP process for the initial Phase 2a study, with dose, dose content uniformity, and sterility meeting specifications generally acceptable for US commercial product (90.0 to 110.0% of label claim, content uniformity per USP905). Conclusions: An aseptic cGMP manufacturing process was developed and validated for ENV515 (travoprost) Intracameral Implants. ENV515 clinical trial material was manufactured and tested in support of planned Phase 2a clinical study in glaucoma patients. Figure 1. Aseptic batch process flow for ENV515 Commercial Relationships: Leo Trevino, Envisia Therapeutics Inc. (E); Michael Hunter, Envisia Therapeutics Inc. (E); Sanjib Das, Envisia Therapeutics Inc. (E); Tyler Pegoraro, Liquidia Technologies, Inc. (E); Tyler Massey, Liquidia Technologies, Inc. (E); Andres Garcia, Envisia Therapeutics Inc. (E); Janet Tully, Envisia Therapeutics Inc. (E); Kristie Hamby, Envisia Therapeutics Inc. (E); Tomas Navratil, Envisia Therapeutics Inc. (E); Benjamin Yerxa, Envisia Therapeutics Inc. (E) Program Number: 4159 Poster Board Number: C0042 Presentation Time: 8:30 AM–10:15 AM Long-term cone and ganglion cell survival in organotypic culture of the human fovea and central retina Arnold Szabo1, Akos Lukats1, Akos Kusnyerik2, Katalin Laczko1, Anna Enzsoly2, Klaudia Szabo1, Bulcsu Dekany1, Janos Nemeth2, Agoston Szel1. 1Dept. of Human Morphology & Dev. Biol., Semmelweis University, Budapest, Hungary; 2Dept. Ophthalmolgy, Semmelweis University, Budapest, Hungary. Purpose: Animal models provide useful research tool for investigation of retinal function and diseases, yet the results of these experiments are often difficult to extrapolate to the human where central photopic vision is dependent on the functional integrity of the central retinal regions and the fovea. Previously, we have shown that in appropriate culture system the human retina can be kept alive with near normal morphology for several weeks. In this study we examine the long-term survival of the cone and ganglion cell populations in human organotypic retinal cultures prepared from the fovea and other central retinal regions. Methods: Adult human eyes with very short (2-4 h) post mortem intervals were used in this study. The fovea and parts of the central retina were dissected free from the pigment epithelium and were cut into approximately 5x5 mm pieces. The pieces were placed on polycarbonate membranes and were cultured in serum-free medium for up to 7 weeks. The cultures were fixed in different time points and were analyzed by immunohistochemistry. The number and staining characteristics of cone- and ganglion cell types were studied in detail. Results: The overall retinal morphology was well preserved even after seven weeks. All layers were maintained, all retinal cell types survived and their morphology was comparable to in vivo controls. In the central retina the number of M- and L-cones was near normal. Many M- and L-cones retained their outer segments even after six weeks. The morphology of S-cones was clearly inferior to M- or L-cones, but also they survived in decreased number for seven weeks. To our surprise, in contrast to other literature data, ganglion cells were also detectable in fairly high numbers during the entire culturing period. A significant percentage of the ganglion cells that survived for seven weeks were located in the IPL. Foveas were cultured for two weeks. The general architecture of the fovea was intact, with cone/rod ratios close to the normal values. Ganglion cells were also preserved in high numbers. Conclusions: Even the most sensitive cell types of the human retina can be maintained in culture at least for 7 weeks. Our model allows the long-term investigation of the retina with preserved cytoarchitecture and therefore could be a preferable model in a wide variety of experiments. Commercial Relationships: Arnold Szabo, None; Akos Lukats, None; Akos Kusnyerik, None; Katalin Laczko, None; Anna Enzsoly, None; Klaudia Szabo, None; Bulcsu Dekany, None; Janos Nemeth, None; Agoston Szel, None Support: #OTKA 7300 Program Number: 4160 Poster Board Number: C0043 Presentation Time: 8:30 AM–10:15 AM Nanoparticle-medicated delivery of hydrophobic compounds to retinal microvascular endothelial cells Wenbo Zhang4, 1, Shuang Zhu4, Shariq Ali2, Alexandra N. Martirossian4, Xiaobing Hu4, Sanaalarab Al-Enazy2, Norah Albekairi2, Massoud Motamedi4, 3, Erik Rytting2, 3. 1Neuroscience and Cell Biology, The University of Texas Medical Branch, Galveston, TX; 2Obstetrics & Gynecology, The University of Texas Medical Branch, Galveston, TX; 3Center for Biomedical Engineering, The University of Texas Medical Branch, Galveston, TX; 4Ophthalmology and Visual Sciences, The University of Texas Medical Branch, Galveston, TX. Purpose: Ischemic retinopathy (IR), including diabetic retinopathy and retinopathy of prematurity, is the leading cause of blindness in persons under 60 years of age in the United States. A major cause of irreversible vision loss in IR is the presence of abnormal retinal neovascurization resulting from pathological angiogenesis. While intravitreally injected large size proteins such as anti-VEGF antibodies can stay in vitreous for long period and effectively block neovascularization, delivery of small anti-angiogenic compounds is challenged by poor drug solubility and rapid ocular elimination. Nanoparticle-based drug delivery involves the encapsulation or incorporation of drugs into microparticles with size and shape at the nanometer scale. It has many advantages over traditional therapeutic methods, such as improved drug efficacy, increased drug solubility and stability, prolonged drug bioavailability, and reduced nonspecific toxicity. This study is to determine whether biocompatible ©2015, 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 2015 Annual Meeting Abstracts nanoparticles comprised of the FDA-approved biodegradable polymer PLGA (poly(lactic-co-glycolic acid)) or PLA (poly(lactic acid)) can be used to effectively deliver hydrophobic compounds into retinal microvascular endothelial cells (RMECs). Methods: PLGA and PLA based nanoparticles were prepared by a modified solvent displacement method. Nanoparticles were either loaded with coumarin-6, a fluorescent hydrophobic dye, to visualize and track nanoparticles; or loaded with Pazopanib, a FDA-approved angiogenic inhibitor, to test whether this approach can inhibit the angiogenic ability of RMECs. Results: PLGA and PLA nanoparticles generated in these studies were negative changed, quite uniform in size, shape and mass distribution, and had diameter ranging from 48 to 144 nm depending on the component of the polymers. They were rapidly uptaken by RMECs within 5 minutes after cells were incubated with the particles. PLGA and the PLA nanoparticles loaded with pazopanib significantly blocked the angiogenic ability of RMECs, such as cell proliferation and migration. Conclusions: These results indicate that PLGA and PLA nanoparticles can effectively deliver small molecular weight water-insoluble compounds into RMECs and therefore serve as a potential vehicle to deliver anti-angiogenic drug to treat retinal neovascularization. Commercial Relationships: Wenbo Zhang, None; Shuang Zhu, None; Shariq Ali, None; Alexandra N. Martirossian, None; Xiaobing Hu, None; Sanaalarab Al-Enazy, None; Norah Albekairi, None; Massoud Motamedi, None; Erik Rytting, None Support: This work was supported by Retina Research Foundation, NIH EY022694, AHA 11SDG4960005, the John Sealy Memorial Endowment Fund for Biomedical Research, International Retinal Research Foundation (to WZ); and an unrestricted grant from Research to Prevent Blindness (to UTMB). Program Number: 4161 Poster Board Number: C0044 Presentation Time: 8:30 AM–10:15 AM Visual Field Indices as the Primary outcome for New Glaucoma Drug Testing: A Systematic Review Richard Filek1, Zarique Z. Akanda2, Nathan Gorfinkel2, William G. Hodge2. 1Pathology, Western University, London, ON, Canada; 2 Western University, London, ON, Canada. Purpose: To synthesize the literature to evaluate visual field assessments as a primary outcome for new glaucoma drug testing. Methods: A systematic search was conducted to help locate published and unpublished studies. Studies which evaluated visual field as primary outcome for new glaucoma drug assessments were identified. Research databases and conference meeting abstracts were searched for articles and included MEDLINE (OVID and PubMed), Cochrane Library (Wiley), BIOSIS (Thomson-Reuters), CINAHL (EBSCO), Web of Science (Thomson-Reuters), and EMBASE (OVID). The search strategies employed database specific subject headings and keywords for glaucoma drug treatment, visual fields and synonyms. Articles were assessed for inclusion by two independent reviewers. A meta-analysis was conducted. Results: Of 2135 studies reviewed, 21 were included of which only 4 were RCT’s that followed patients for at least 36 months. The mean Humphrey defect difference amongst therapeutic vs control groups was very similar at the end of the study compared to the beginning (-5.09 in therapeutic group, -5.06 in control group, p=0.62). Conclusions: There was only a very small sample of studies that have looked at long term non proxy outcomes such as visual fields for new glaucoma drugs. This small sample demonstrates equivalence in VF indices amongst therapeutic and control groups when followed for at least 36 months. More glaucoma drug studies need to use nonproxy outcomes in evaluating drug efficacy in glaucoma. Commercial Relationships: Richard Filek, None; Zarique Z. Akanda, None; Nathan Gorfinkel, None; William G. Hodge, None Support: AMOSO Innovation Fund INN12-010 Program Number: 4162 Poster Board Number: C0045 Presentation Time: 8:30 AM–10:15 AM Bioengineer Functional 3D Human Retina for Understanding Retinal Degeneration Yangzi I. Tian, Yubing Xie. Nanoscale Engineering, SUNY Polytechnic Institute, Albany, NY. Purpose: Progressive deterioration of retinal pigment epithelial (RPE) cells in elderly individuals promotes a cascade of inflammatory events that damages the retina and result in blindnesscausing diseases such as age-related macular degeneration (AMD). Understanding the development of diseased retinal epithelium is critical for the treatment of degenerative eye diseases. However, much of our understanding of the metabolic and phagocytic activities that underlie a healthy, functional retinal epithelium has been gathered from in vitro 2D cell cultures, which do not faithfully recapitulate the human retina architecture and function. Recent advancement in 3D bioprinting and nanofabrication technologies offer tools to create in vivo-like tissue complex. To accurately capture the physiological behavior of RPE cells in vivo, three-dimensional (3D) extracellular environment is needed to overcome limitations of the conventional cell culture. Methods: To reproduce the groove-like geometry of the retina, a 3D printer (BotFactory Replicator 2X) will be used to fabricate a concave, hollow semi-sphere with a diameter of 24 mm and base curvature of approximately 150 degrees, similar to dimensions of human retina. After sterilizing, the scaffold is coated with a selection of FDA-approved natural polymers to support the adhesion and expansion of human retina pigment epithelium stem cells (hRPESC). The formation of an effective blood-retinal barrier is confirmed by examining its transepithelial permeability (or electrical resistance), polarization of membrane domain and distribution of tight junction proteins on RPE cell surface. Results: The result shows the feasibility of culturing hRPESC on our 3D-printed, concave surface to mimic the porous micro-architecture of the native Bruch’s Membrane. We were able to recreate the continuous monolayer of mature RPE cells after 4 weeks in culture. The cells maintained the expression of their cell type specific and functional markers. Conclusions: Controlling the spatial organization and behavior of RPE cells is an important step in understanding behavior of retinal epithelium and modeling disease processes in vitro. Using the 3D printed platform will advance our understanding of retinal morphogenesis, function and the ability to model of disease processes in vitro. Commercial Relationships: Yangzi I. Tian, None; Yubing Xie, None Program Number: 4163 Poster Board Number: C0046 Presentation Time: 8:30 AM–10:15 AM Antimicrobial Activity Detected in Ocular and Salivary Secretions from Marine Mammals Robin Kelleher Davis, Pablo Argueso. Harvard Med Sch/ Ophthalmology, Schepens Eye Research Inst/MEEI, Boston, MA. Purpose: Marine mammal species’ first ancestors evolved from the land to the sea more than 30 million years before man’s ancestors started walking upright, a mere 3-6 million years ago. We hypothesize that the marine mammal tear film has evolved unique mechanisms ©2015, 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 2015 Annual Meeting Abstracts to cope with the harsh environment of the sea. We undertook this study to determine whether mucosal secretions, specifically tears and saliva, from marine mammals have antimicrobial activity. Methods: Tear and saliva samples from bottlenose dolphins, manatees, and humans were collected using IRB and ACUC approved protocols. Samples were lyophilized and reconstituted in sterile water (vehicle). Antimicrobial activity was tested against strains of E. coli (DH5α, New England Biolabs) and P. aeruginosa 6294 (generous gift of Dr. Mihaela Gadjeva, Brigham and Women’s Hospital, Boston, MA). Bacterial cultures were grown in LB (Luria-Bertani) media overnight at 37oC, diluted to an optical density at 600 nm (OD600) of 0.2, and aliquoted to 96-well plates containing vehicle, gentamicin (0.1 mg/ml), or sample in triplicate, in a total volume of 120 ul per well. Final protein concentrations in the assay were 12-39 ug/ml for dolphin tear (n=3), 115-370 ug/ml for dolphin saliva (n=5), 30-51 ug/ml for manatee tear (n=3), and 108 ug/ml for human tear (n=3), respectively. Plates were incubated with shaking in a water bath at 37oC and bacterial growth was monitored by a standard turbidity assay with OD600 measurements taken at hourly intervals. Results: Within two hours of incubation, growth of E. coli was substantially inhibited (p<0.05) by samples of dolphin tear (OD600 = 0.460 +/- 0.025), dolphin mouth mucus (OD600 = .478 +/- 0.230), manatee tear (OD600 = 0.549 +/- 0.313), and human tear (OD 600 = 0.543 +/- 0.051) as compared to vehicle alone (OD600 = 1.012 +/- 0.141). The antibiotic, gentamicin, also inhibited bacterial growth (OD600 = 0.445 +/- 0.086). P. aeruginosa growth was slower and was not affected significantly at 2 hours by any of the samples tested. At 22.5 hours, 2 of the 5 dolphin saliva samples inhibited P. aeruginosa growth by 76 and 79% respectively, however none of the other agents had an effect. Conclusions: It appears that under the conditions of this study, marine mammal tears and saliva do exhibit antimicrobial activity. Future studies will focus on determining which factors in these secretions are responsible for inhibiting bacterial growth. Commercial Relationships: Robin Kelleher Davis, None; Pablo Argueso, None Support: NIH Grant EY05612 and Arey’s Pond Boat Yard. We thank Balaraj B. Menon and David A. Sullivan for technical advice. Program Number: 4164 Poster Board Number: C0047 Presentation Time: 8:30 AM–10:15 AM Development of an ex-vivo trans-corneal permeation model in horses: Epithelial barrier evaluation Eva M. Abarca1, Rosemary Cuming1, Sue Duran1, Jayachandra Rapamuran2. 1Clinical Sciences, Auburn University, CVM, Auburn, AL; 2Harrison School of Pharmacy, Auburn University, Auburn, AL. Purpose: Keratomycosis is a major concern in human and veterinary ophthalmology, with horses serving as a natural viable model for human keratomycosis. The aim of this study was firstly to describe an ex-vivo trans-corneal drug permeation model for use in equine corneas, and secondly to present evidence on the integrity of the equine epithelial barrier function for 6 hours. Methods: Fresh equine corneas used in the experiment were obtained from terminal laboratories performed at the J. T. Vaughan Large Animal Teaching Hospital, Auburn University, AL. Corneoscleral buttons (16 mm) were dissected using standard eye bank technique within 2 hours of enucleation. Fluorescent permeation experiments using a Franz diffusion cell method were performed to examine the integrity of the epithelial barrier function during the times studied (2 and 6 hours). Corneas were mounted horizontally between the donor and the receiving compartments of the diffusion cells (exposure window 0.64cm2), which were maintained at 370C. The donor compartment was filled with 1ml of a solution containing 10mM sodium fluorescein. Samples (1ml phosphate buffered saline pH 7.4) were removed from the receiving compartment hourly and replaced with fresh receiving fluid. The fluorescent intensities of the receiving solution samples were analyzed using a spectrofluorometer. Experiments without fluorescein in the donor solution were also performed as negative controls. All the experiments were performed in triplicates. The results were expressed as mean value ±SEM. The differences between values were assessed using a one-way ANOVA (p<0.05). Results: Fluorescent concentrations detected in the receiving compartment of experiments maintained for 2 and 6 hours were not significantly different than those obtained from the negative control experiment at any time point, showing that the epithelial barrier function was maintained. Conclusions: This work presented a simple Franz diffusion cell-type modification for use in ex-vivo ocular drug delivery investigations in the equine eye. The results showed that the diffusion cell was able to maintain the integrity of equine epithelial corneal barrier function throughout the 6 hours of the permeation experiment. Investigation into new treatment modalities for horses with keratomycosis may influence development of parallel human therapies and benefit people with this disease. Commercial Relationships: Eva M. Abarca, None; Rosemary Cuming, None; Sue Duran, None; Jayachandra Rapamuran, None Support: American Quarter Horse Foundation Grant-G00008471 Program Number: 4165 Poster Board Number: C0048 Presentation Time: 8:30 AM–10:15 AM Sustained Release Biodegradable Steroid Formulations for Intravitreal Delivery Sanjib Das, Stuart Williams, Janet Tully, Ayush Verma, Jeremy Hansen, Melissa Hernandez, Tyler Pegoraro, Tomas Navratil, Benjamin Maynor, Benjamin Yerxa. Envisia Therapeutics, Morrisville, NC. Purpose: Intraocular implants for the delivery of steroids are used for treatment of Retinal Vein Occlusion (RVO), uveitis and Diabetic Macular Edema (DME). Current therapies are limited by their ability to design specific shape and size ocular implants or microsuspensions with efficacious loadings of steroid. It is desirable to design implants that are biodegradable, tolerable and release therapeutic levels of steroid six months or longer. We have demonstrated the ability to precisely fabricate intraocular implants and microparticle suspensions for the tunable release of steroids. Methods: Specific size and shape biodegradable dexamethasonepolymer steroids implants and particles were fabricated with controlled loadings of dexamethasone using the proprietary PRINT® technology. Implant and microparticle size, shape and morphology were determined by Scanning Electron Microscopy (SEM). Dexamethasone content was measured by RP-HPLC The mobile phase consisted of a gradient of 0.1% TFA in purified water and 0.1% TFA in acetonitrile over 5 minutes at 1.0 mL/min. UV absorbance of the steroid was measured at 245 nm. Dexamethasone release was characterized in vitro in Triton X-100 in 1X PBS at 370C and measured by HPLC. Results: Predefined size and shape biodegradable dexamethasone implants and microparticles were fabricated using the PRINT technology with a high degree of mass uniformity and dexamethasone content. In vitro release was controlled by tuning of the implant formulations. Syringeable high concentration particle suspension formulations were developed allowing for the delivery of mg quantities of steroids in small gauge needle sizes. ©2015, 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 2015 Annual Meeting Abstracts Conclusions: The PRINT technology uniquely allows for the fabrication of intraocular implants with uniform size, shape and dose. These formulations demonstrate tunable, sustained release of steroid. High concentration particulate suspensions allow for the delivery of mg quantities of drug in small needle gauges with potential for sustained release applications. Commercial Relationships: Sanjib Das, None; Stuart Williams, None; Janet Tully, None; Ayush Verma, None; Jeremy Hansen, None; Melissa Hernandez, None; Tyler Pegoraro, None; Tomas Navratil, None; Benjamin Maynor, None; Benjamin Yerxa, None Program Number: 4166 Poster Board Number: C0049 Presentation Time: 8:30 AM–10:15 AM Intravitreal tolerance and ocular pharmacokinetics in nonhuman primates of a carboxymethylated hyaluronic acid/polyethylene glycol diacrylate polymer useful for sustained drug delivery MaryJane Rafii1, Alex Lewis2, Jordan Attwood2, Vernard Woodley2, Shervin Liddie2, Wenzheng Hu2, Robin J. Goody2, Matthew S. Lawrence2, Barbara M. Wirostko1. 1Ophthalmology, Jade Therapeutics Inc., Salt Lake City, UT; 2Research, RxGen, Hamden, CT. Purpose: Developing drug delivery polymers that are well tolerated in the eye and achieve pharmacokinetic profiles that result in sustained therapeutic effect presents a promising strategy for improving the clinical efficacy and safety of existing and new drug candidates for retinal disease and other ophthalmic disorders. Studies were designed to evaluate a novel, biodegradable, fluorophoreloaded, carboxymethylated hyaluronic acid (CMHA) polymer crosslinked with polyethylene glycol diacrylate (PEGDA) in primates. Methods: The ocular tolerance and fluorophore retention of CMHA loaded with the small molecule fluorophore tag, Alexa Fluor® 488, was evaluated in African green monkeys by fluorophotometry and slit lamp exam following intravitreal (IVT) injection (n=4 eyes) and compared to saline controls (n=2 eyes), allowing minimally invasive, nonterminal quantification of fluorophore abundance and test article safety. CMHA was injected via 30 gauge needle as either a pre-gelled polymer (n=2), or immediately after formulation of constituents but prior to complete gelling (n=2) to evaluate the impact of the time of gel formation relative to intravitreal delivery on tolerance and pharmacokinetics. Results: Both pre-gelled and post-gelled Alexa Fluor® 488 tagged CMHA were well tolerated with detectable ocular response to test article limited to a minor vitreous cell infiltrate in one eye receiving the post-gelled polymer, which fully resolved at 12 weeks. Alexa Fluor® 488 signal was detected at an elevated level until week 8, peaking immediately post-injection, and substantially returned to baseline by week 12. Intraocular pressure and flare photometer measures of anterior chamber protein remained within normal limits and similar to controls. Conclusions: A novel, crosslinked CMHA-based hydrogel is well tolerated in the nonhuman primate vitreous and extends release of an incorporated fluorophore over a 2-month interval presenting a clinically meaningful sustained release profile. Release kinetics and ocular safety appears largely independent of whether hydrogel formation occurs before or after IVT delivery, expanding the flexibility with which formulation and delivery might be pursued. Such a hydrogel presents a formulation strategy to improve sustained delivery of existing and candidate ocular therapeutics. Commercial Relationships: MaryJane Rafii, Jade Therapeutics Inc. (I); Alex Lewis, RXGen (F); Jordan Attwood, RXGen (F); Vernard Woodley, RXGen (F); Shervin Liddie, RXGen (F); Wenzheng Hu, RXGen (F); Robin J. Goody, RXGen (F); Matthew S. Lawrence, RXGen (F); Barbara M. Wirostko, Jade Therapeutics Inc. (I) Program Number: 4167 Poster Board Number: C0050 Presentation Time: 8:30 AM–10:15 AM Mechanical Properties of Four Carboxymethylated Hyaluronic Acid Hydrogel Polymer Formulations Brittany Coats1, McKenna M. Drysdale1, Hee-Kyoung Lee2, 3, Barbara M. Wirostko2, 4. 1Mechanical Engineering, University of Utah, Salt Lake City, UT; 2Jade Therapeutics, Inc., Salt Lake City, UT; 3Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT; 4Ophthalmology, University of Utah, Salt Lake City, UT. Purpose: Bioerodable hydrogel polymers are a promising topical drug delivery system because they are biocompatible, diffuse drug easily, and improve retention time. Toward this end, Jade Therapeutics is developing a proprietary biodegradable crosslinked, thiolated carboxymethylated hyaluronic acid (CMHA)-based hydrogel film for topical ocular applications. To assess the structural integrity of the film and establish baselines for future degradation studies, four different cross-linked CMHA formulations were evaluated in uniaxial stress relaxation and pull-to-failure tests before and after ethylene oxide sterilization. Methods: Films were fabricated by varying the HA-based polymers (thiolated CMHA with or without gelatin) and cross-linkers (Poly(ethyleneglycol) diacrylate (PEGDA) or glutathione disulfide (GSSG)). The polymerized gel was dried at room temperature overnight. Dried strips (6 x 10 mm) were hydrated in phosphate buffered saline (PBS) for 24 hrs and dimensioned using an optical stereomicroscope. Hydrated specimens were mounted in custom clamps located in a PBS chamber attached to a material test system (Model 5943, Instron) and a 500 gram submersible load cell (LSB210, Futek). The stress relaxation protocol was applied at a rate of 1.0 in/min up to 9% strain in increments of 0.5% strain (Fig. 1). Pull-to-failure tests were performed at 1.0 in/min. All measured load and displacement data were converted to stress and strain. Elastic modulus, relaxation modulus, and ultimate stress were extracted. Results: All four films exhibited non-linear stress-strain behavior. The CMHA-PEGDA film had the largest hydrated thickness (0.281±0.092 mm), the lowest relaxation modulus (35.4±3.2 kPa, Fig. 2) and lowest ultimate stress (26.2±19.9 kPa). Sterilization significantly increased the relaxation modulus and ultimate stress in all formulations, but following sterilization CMHA-PEGDA was the only formulation that remained comparable to standard soft contact lens properties. Conclusions: CMHA-PEGDA was the most promising formulation in terms of both its comfort as a topical ocular film (i.e., low relaxation modulus) and structural integrity. ©2015, 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 2015 Annual Meeting Abstracts Fig 1. Sample of the strain protocol applied to the hydrogels (maroon) and a typical load response to that strain protocol (red). Fig 2. Comparison of the low strain (0.5-4.5%) and high strain (5.0-9.0%) relaxation moduli for each formulation before and after sterilization. Commercial Relationships: Brittany Coats, None; McKenna M. Drysdale, None; Hee-Kyoung Lee, Jade Therapeutics (E), Jade Therapeutics (I); Barbara M. Wirostko, Jade Therapeutics (E), Jade Therapeutics (I), Jade Therapeutics (P), Jade Therapeutics (S) Support: USAMRMC #W81XWH-14-C-0025 ©2015, 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].