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ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

114 AMD: New Drugs, Delivery Systems and Mechanisms

Sunday, May 6, 2012, 8:30 AM - 10:15 AM

Hall B/C

Poster Session

Program #/Board # Range: 433-457/D1110-D1134

Organizing Section: Physiology/Pharmacology

Program Number: 433 Poster Board Number: D1110 Presentation Time: 8:30 AM - 10:15 AM In-vitro Activity Of Transgenic Anti- VEGF Molecules

Tobias Wimmer, Nina Wagner, Eva Senger, Birgit Lorenz, Knut Stieger.

Department of Ophthalmology, Justus-Liebig University Giessen, Giessen, Germany. Purpose: Upregulation of VEGF (vascular endothelial growth factor) in the eye leads to uncontrolled retinal vessel growth in diseases like AMD (age related macula degeneration) or DR (diabetic retinopathy). Repeated injections of anti- angiogenic molecules like Lucentis® (Ranibizumab) or Avastin® (Bevacizumab) are the state of the art treatment for these disorders. The aim of this study was to develop a gene addition therapy, with which anti-VEGF molecules are produced over long time periods in the eye. To achieve this, we expressed the soluble

isoform of the VEGF receptor 1 (sFlt1) under the control of the Tetracyclin- inducible TetOn-promotor and the F(ab)-fragment Ranibizumab (Ra01) under the control of a CMV promoter in two different eukaryotic cell lines and analyzed the biological activity. Methods: The sequences of both chains of Ranibizumab were synthesized and cloned into the same, IRES (internal ribosomal entry site) containing, expression vector. The sFlt1 cDNA was amplified from corneal extracts and cloned into a TetOn expression vector. HEK293 and HeLa cell lines were transfected with these constructs and the expression of sFlt1 was induced with the Tetracyclin-derivate Doxycyclin. The gene-product containing supernatand was collected after 24 hours of expression. HUVEC (human umbilical vein endothelial cells) migration assays and HUVEC tube formation assays were performed to determine the biological activity of the expressed molecules compared to Lucentis® and Avastin®. The concentration of the expressed molecules was determined by ELISA. Results: In the HUVEC tube formation assay, Ra01 showed a reduction in tube formation (inhibition of VEGF) of 67%, the induced sFlt1 57% and the non- induced sFlt1 a reduction of 6%, compared to Lucentis® (100ng) of 45% and Avastin® (100ng) of 51%. The migration assay showed an inhibition of VEGF- induced HUVEC migration of 15% with expressed sFlt1 and a decrease of 40% with Ra01, compared to Lucentis (100ng) with 47%. Conclusions: The expression of sFlt1 can be controlled using the TetOn system, which produces sufficient amounts of sFlt1 to inhibit VEGF in the tube formation assay compared to Lucentis® or Avastin®. Ra01 can be assembled into a heterodimer in eukaryotic cells and shows a VEGF inhibiting activity comparable or slightly better than Lucentis® or Avastin®. These results provide the basis for a gene-addition therapy to inhibit VEGF in pathologies like AMD or DR. Commercial Relationships: Tobias Wimmer, None; Nina Wagner, None; Eva Senger, None; Birgit Lorenz, None; Knut Stieger, None Support: DFG Sti 597/2-1

Program Number: 434 Poster Board Number: D1111 Presentation Time: 8:30 AM - 10:15 AM The Olmesartan Effect in the Choroid-scleral Leukocyte Recruitment in Hypercholesterolemia Model

Rogil J. Torres 1 , Dalton Precoma 1 , Lucia Noronha 1 , Mario Sturzeneker 1 , Regiane Torres 1 , Andrea Luchini 2 , Antonio M. Casella 3 , Caroline Torres 4 , Lucas Torres 4 , Robson Torres 4 . 1 Retina Vitreous, Pontificia Universidade Catolica do Parana,

Curitiba, Brazil; 2 Centro Oftalmologico de Curitiba, Curitiba, Brazil;

  • 3 Ophthalmology, University of East London, Londrina, Brazil; 4 Universidade Positivo, Curitiba, Brazil. Purpose: Demonstrate that the blockade of angiotensin II AT-1 receptors, through the systemic administration of olmesartan, can reduce the MCP-1 expression and the resulting macrophage accumulation in the choroid and sclera of hypercholesterolemic rabbits. Methods: 32 New Zealand rabbits were divided into 3 groups: group 1(NG), was fed a standard rabbit diet; group 2 (HG) was fed a hypercholesterolemic diet; and group 3 (OG) was fed a hypercholesterolemic diet enriched with olmesartan. The rabbits underwent serum dosages of total cholesterol, triglyceride, HDL cholesterol and fasting blood glucose at the beginning of the experiment and at the euthanasia day. Sclera and choroid underwent immunohistochemical analysis with MCP-1 and RAM-11 markers. Results: No abnormality was detected in NG. HG showed a significant increase in immunoreactivity for MCP-1 in relation to NG (p= 0.001) and OG (p=0.004). HG showed a significant increase in immunoreactivity for RAM-11 of the choroid- scleral complex in relation to (p<0.001). A significant decrease in immunoreactivity for RAM-11 was observed in OG in relation to HG (p= 0.034) and a significant increase in immunoreactivity for RAM-11 was observed in OG in relation to NG (p=0.008).

Conclusions: Olmesartan reduced the MCP-1 expression and the resultant macrophage accumulation in the choroid-scleral complex of hypercholesterolemic rabbits. Commercial Relationships: Rogil J. Torres, None; Dalton Precoma, None; Lucia Noronha, None; Mario Sturzeneker, None; Regiane Torres, None; Andrea Luchini, None; Antonio M. Casella, None; Caroline Torres, None; Lucas Torres, None; Robson Torres, None Support: None

Program Number: 435 Poster Board Number: D1112 Presentation Time: 8:30 AM - 10:15 AM Biocompatibility Of A Novel Ocular Drug Delivery System Nathan Gooch 1A , Michael Burr 1 , Bruce Gale 1B , Balamurali Ambati 1C . A Bioengineering, B Mechanical Engineering, C Ophthalmology, 1 University of Utah, South Jordan, UT. Purpose: To determine ocular biocompatibility of a novel, sustained release, refillable intraocular, drug delivery device. Methods: The intraocular drug delivery device is a reservoir and delivery agent which is designed to be placed within the capsular bag during cataract surgery. The capsule drug ring (CDR) prototypes were manufactured by hot melt extrusion of Bionate II® (DSM), a polycarbonate urethane. As the Bionate II® tubing was extruded from the dye, it was wrapped around a 8mm pipe to incorporate the correct inner and outer diameters into the polymer before fully setting. A filter composed of polyether sulfone was fitted to one end of the devices for controlled drug release. The other end was sealed. The devices have been optimized using Avastin® as the drug of interest. In vitro biocompatibility was assessed with human lens epithelial cell (B-3), mouse macrophage (J774A.1), and mouse fibroblast (L- 929) cell lines. Cell migration and proliferation were assessed after in vitro culture. Pro-inflammatory cytokines (i.e. MIP-1β, MIP-1α, MCP-1, IL-1β, TNF, TGF-β1) were quantified using cytometric bead array (CBA). Preliminary in vivo biocompatibility and pharmacokinetics testing has been performed in rabbits. Results: The use of hot melt extrusion for CDR manufacture in place of previous design methodologies has dramatically improved the performance and reproducibility of drug delivery and pharmacokinetics. The devices have been designed to be a circular ring shape so as to fit in the capsular bag without impeding vision and have a drug reservoir of 50µL. In vitro cell migration and proliferation experiments show that the CDR components and devices had no measurable cytotoxic impact on B-3, J774A.1, and L-929 cell lines. Pro- inflammatory cytokine concentrations were also unchanged by the use of CDR device materials when compared to the gold standard tissue culture polystyrene cultured cells. Manufacturing methods were shown to be sterile as LPS was detected at levels below 0.0303 EU/mL. Preliminary in vivo histology shows Avastin® concentrations in the retina out to >90 days with very little host foreign body or inflammatory response. Conclusions: The results show the successful manufacture of the CDR, a potentially refillable drug delivery device. In vitro results show the devices and their individual components to be highly biocompatible with cells showing no difference in migration, proliferation, and pro-inflammatory cytokine generating behaviors. Avastin® was used as the primary drug of interest to test pharmacokinetics in vivo. Histology showed that the CDR devices performed as designed with very good biocompatibility. The CDR shows great potential as an implantable ocular device for drug delivery. Commercial Relationships: Nathan Gooch, None; Michael Burr, None; Bruce Gale, None; Balamurali Ambati, None Support: NIH Grant EY017185-01A2

Program Number: 436 Poster Board Number: D1113 Presentation Time: 8:30 AM - 10:15 AM Hyaluronic Acid-Containing Silicone Hydrogels: Their Use as Extended Drug Delivery Systems of Hydrophobic Ocular Drugs Myrto Korogiannaki, Heather Sheardown. Chemical Engineering, McMaster University, Hamilton, ON, Canada. Purpose: While eyedrops are a well-accepted method of delivering drugs to the eye, they suffer from significant limitations including significant losses, low residence times, pulsatile concentration profiles in the tear fluid and the need for patient compliance. The high oxygen permeability of silicone hydrogel (SH) based materials makes them attractive for extended release in front of the eye applications. Based on the hypothesis that the drug release can be controlled by controlling the hydrophilicity of the materials, we have developed a series of hyaluronic acid (HA) modified SH materials that have utility for extended release of hydrophobic ocular drugs. Methods: The modified SH that were used consist of a hydrophilic monomer, either hydroxyethyl methacrylate (HEMA) or N,N-dimethylacrylamide (DMAA), a hydrophobic silicone monomer of methacryloxypropyltris (trimethylsiloxy) silane (TRIS) and hyaluronic acid (HA) (7.5 kDa). Ethylene glycol dimethacrylate (EGDMA) was used as the cross-linker. The reaction was performed by UV induced free-radical polymerization. Atropine was used as a model hydrophobic

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

drug. HA and atropine were incorporated into the SH during synthesis with direct entrapment. The hydrogels were analyzed for surface hydrophilicity and equilibrium water content (EWC) in order to determine the effect of HA on these materials. Drug release was monitored and quantified by UV spectroscopy technique. Results: The swelling study showed that the incorporation of HA significantly increased the EWC of drug loaded-DMAA/TRIS hydrogels. Based on the results of the captive air bubble study, it was found that the surface of the model SH were more hydrophobic than our previous HA containing materials presumably due to the polyester sheets used for materials casting. Moreover, the presence of HA into the drug loaded-DMAA/TRIS materials improved the surface hydrophilicity as the advancing contact angles were decreased. The contact angles were also examined at the end of the release experiment and it was found that the silicone hydrogel surfaces were significantly more hydrophilic. Atropine was released for more than two weeks and the incorporation of HA during synthesis led to an increase in the amount and the duration of the drug released. Conclusions: The HA-containing SH materials have the potential to be used as extended drug delivery systems for hydrophobic drugs with potential application in the treatment of front of the eye diseases as a replacement of eyedrops. Commercial Relationships: Myrto Korogiannaki, None; Heather Sheardown, None Support: NSERC

Program Number: 437 Poster Board Number: D1114 Presentation Time: 8:30 AM - 10:15 AM The Combination of Bevacizumab and 3,4 Dihydroxyphenyl Ethanol Reduces Angiogenin in Retinal Pigment Epithelial Cells

Tamara J. Granner, Shawn C. Maloney, Sebastian Di Cesare, Tiago Briccoli,

Cristina Miyamoto, Miguel N. Burnier, Jr

..

Ophthalmology, McGill University,

Montreal, QC, Canada. Purpose: Choroidal neovascularization is the hallmark of the wet form of Age- Related Macular Degeneration (AMD) and is currently the target of multiple anti- angiogenic pharmacotherapies. The current study evaluated if 3,4 Dihydroxyphenyl Ethanol (DPE) reduces secretion of pro-angiogenic cytokines from a human retinal pigment epithelial cell line (ARPE-19). Moreover, additional anti-angiogenic effects of DPE in combination with bevacizumab were evaluated. Methods: ARPE-19 cells were cultured for 24 hours under normoxic conditions or with a hypoxia-mimicking agent (100μM cobalt chloride [CoCl2]). After 24 hours, all media was removed and replaced with one of the following serum-free conditions: Control media, DPE (100μM), bevacizumab (0.25mg/ml), or combination of DPE (100μM) and bevacizumab (0.25mg/ml). Media was harvested after 24 hours for sandwich ELISA-based angiogenesis arrays. The secretion of the following ten pro-angiogenic cytokines was measured: Angiogenin, ANG2, EGF, bFGF, HB-EGF, PDGF-BB, Leptin, PIGF, HGF, and VEGF-A. Statistical significance was evaluated using a Student’s t-test with p<0.05 as a cutoff for significance. Results: Of the ten cytokines assayed, three (Angiogenin, ANG2, and VEGF-A) were secreted by ARPE-19 cells under normoxia and all three were significantly increased under CoCl2-simulated hypoxia. HB-EGF and PIGF were not secreted under normoxia, but secretion was induced under simulated hypoxia. Following treatment with DPE, levels of Angiogenin and VEGF-A significantly decreased under normoxia while all five detectable cytokines significantly decreased under simulated hypoxia compared to the control. The combination of bevacizumab with DPE significantly reduced secretion of Angiogenin and ANG2 under normoxia. Angiogenin was significantly down-regulated by the combination under simulated hypoxia compared to bevacizumab alone. Conclusions: This study demonstrated that DPE significantly reduced the secretion of multiple pro-angiogenic cytokines to varying degrees under normoxia and simulated hypoxia. Further, the combination of DPE and bevacizumab proved to be a more effective approach to reduce Angiogenin than bevacizumab alone. Therefore the combination of DPE and bevacizumab may represent a valuable therapeutic option for the wet form of AMD. Commercial Relationships: Tamara J. Granner, None; Shawn C. Maloney, None; Sebastian Di Cesare, None; Tiago Briccoli, None; Cristina Miyamoto, None; Miguel N. Burnier, Jr., None Support: None

Program Number: 438 Poster Board Number: D1115 Presentation Time: 8:30 AM - 10:15 AM Three Year Results of Visual Outcome with Disease-Activity-Guided

Ranibizumab Algorithm for the Treatment of Exudative Age-Related Macular Degeneration

Lala Ceklic 1,2 , Carsten Framme 2 , Ute E. Schnurrbusch-Wolf 2 , Sebastian Wolf 2 .

  • 1 Eye Clinic "Kasindo", Clinical Center of Eastern Sarajevo, Eastern Sarajevo, Bosnia and Herzegovina; 2 University Of Bern, Universitatsklinik fur Augenheilkunde, Bern, Switzerland.

Purpose: To evaluate three year follow up treatment outcomes with ranibizumab (Lucentis®) 0.5 mg administered either monthly or quarterly on a pro re nata (PRN) basis according to a disease-activity-guided monitoring and treatment algorithm. Methods: A total of 316 treatment-naive eyes of 316 patients with exudative age- related macular degeneration met the criteria for inclusion in this retrospective, interventional case series. Patients were treated with ranibizumab 0.5 mg according to a disease-activity-guided algorithm with monthly monitoring following the standard loading phase with 3 injections. Spectral Optical coherence tomography was routinely used to assess disease activity by qualitatively judging subretinal fluid: active lesions showing subretinal fluid were treated with a series of 3 monthly injections, whereas inactive lesions (dry retinal conditions) were treated with quarterly injections. Results: Mean best-corrected ETDRS visual acuity improved from 52 letters at baseline to 59 letters at 12 months, achieved with a mean of 7.4 injections, 60 letters at 24 months with a mean of 12.1 injections administered up to the second year and again 60 letters at 36 months with a total mean number of 16 injections. Conclusions: A morphology driven Pro re nata regimen with ranibizumab administered either monthly or quarterly in long term follow up (up to 36 months) resulted in BCVA gain of 8 letters which is in favorable correlation to the expected visual gain derived from the MARINA- and ANCHOR studies; however, using significant less injections. Commercial Relationships: Lala Ceklic, None; Carsten Framme, None; Ute E. Schnurrbusch-Wolf, None; Sebastian Wolf, None Support: None

Program Number: 439 Poster Board Number: D1116 Presentation Time: 8:30 AM - 10:15 AM Tissue Kallikrein Suppresses Laser-Induced Choroidal Neovascularization in Mice

Junichi Fukuhara 1A,1B , Kousuke Noda 1A,1B , Chikako Yoshizawa 1A , Satoshi Kinoshita 1A,1B , Zhenyu Dong 1A,1B , Ryo Ando 1A,1B , Anton Lennikov 1A,1B , Atsuhiro Kanda 1A,1B , Susumu Ishida 1A,1B . A Department of Ophthalmology, B Laboratory of Ocular Cell Biology and Visual Science, 1 Hokkaido University Graduate School of Medicine, Sapporo, Japan. Purpose: Tissue kallikrein is a serine protease that contributes to a flow-dependent arterial dilation through activation of bradykinin B2 receptors coupled with

endothelial nitric oxide release. Recently, it has been reported that tissue kallikrein also possesses the antiangiogenic effects through the cleavage of vascular endothelial growth factor (VEGF) 165 isoform and thereby reduces the pathological vascular changes in the murine model of oxygen-induced retinopathy.

Here, we investigate the impact of tissue kallikrein in laser-induced choroidal neovascularization (CNV). Methods: Male C57Bl/6 mice (7-8 weeks old) were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. CNV was induced by laser photocoagulation (200mW, 75µm, 100msec). The

animals received daily subcutaneous injections of tissue kallikrein (50μg/kg), or

vehicle control (PBS) for 2 days before laser photocoagulation, and the treatment was continued until the end of the study. Seven days after laser injury, choroidal

flat mounts were prepared and the size of the CNV lesions was quantified. The RPE-choroid complex was harvested 3 days after laser injury and the levels of inflammation-associated molecules, monocyte chemoattractant protein (ΜCP)-1 and intercellular adhesion molecule (ICAM)-1, were assessed by enzyme-linked immunosorbent assay. Immunoblotting was performed using the RPE-choroid complex with anti-VEGF164 antibody. Results: The animals treated with tissue kallikrein showed a significant decrease in CNV size (27168.3±2432.2µm 2 ), compared with vehicle-treated controls (36374.6±3204.1µm 2 , p<0.05). Tissue kallikrein treatment significantly reduced the levels of ΜCP-1 (p<0.05) and ICAM-1 (p<0.05). Furthermore, immunoblotting showed the approximately 16kDa-band, presumably corresponding to fragmented VEGF164 protein, in the RPE-choroid complex samples obtained from the animals treated with tissue kallikrein. Conclusions: The current data indicate an antiangiogenic property of tissue kallikrein through VEGF164 cleavage in CNV.

Commercial Relationships:

Junichi Fukuhara, Sanwa Kagaku Kenkyusho Co.,

Ltd. (F); Kousuke Noda, Sanwa Kagaku Kenkyusho Co., Ltd. (F); Chikako

Yoshizawa, None; Satoshi Kinoshita, None; Zhenyu Dong, None; Ryo Ando, None; Anton Lennikov, None; Atsuhiro Kanda, None; Susumu Ishida, None Support: None

Program Number: 440 Poster Board Number: D1117 Presentation Time: 8:30 AM - 10:15 AM VEGF Trap Exhibits VEGF Binding Stoichiometry Distinct From Antibodies, and Does Not Support Platelet Aggregation Douglas A. MacDonald 1 , Jiann-Kae Luo 1 , Nicholas Papadopoulos 1 , Erica Pyles 1 , Stanley Wiegand 1 , Françoise Bono 2 , Nathalie Delesque 2 , Pierre Savi 2 , John

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Francis 3 , Todd Meyer 3 . 1 Regeneron Pharmaceuticals, Tarrytown, NY; 2 Sanofi,

France, Toulouse, France;

3

Center for Thrombosis Research, Orlando, FL.

Purpose: VEGF Trap (aflibercept) is a novel, soluble decoy receptor that binds VEGF-A, VEGF-B and placental growth factor. Like anti-VEGF antibodies, VEGF Trap contains an Fc domain. However, given its otherwise distinct structure, VEGF Trap may bind VEGF with different stoichiometry than antibodies. The present studies were conducted to compare binding stoichiometries of bevacizumab (Bev, an anti-VEGF antibody) and VEGF Trap to VEGF, as well as FcγRIIA -mediated platelet activating capability of Bev:VEGF and VEGF Trap:VEGF complexes. Methods: Stoichiometry of binding for Bev and VEGF Trap to VEGF 165 was measured using multi-angle laser light scattering. Platelet activation was measured using platelet aggregometry & 14 C-serotonin release assays (SRA). Animal studies used human FcγRIIA transgenic mice. Pre-formed 1:1 molar mixtures of Bev or VEGF Trap with VEGF 165 plus heparin were injected into the tail vein (n=10/group). After 10 min, blood drawn by cardiac puncture was used to measure platelet number. Lungs were dissected en bloc, PBS washed and formalin-fixed. H&E sections were analyzed for evidence of thrombosis. Results: Bev:VEGF 165 complexes were heterogeneous and predominantly multimeric (~400kDa to >2,000 kDa). In contrast, VEGF Trap formed a homogeneous 1:1 binding complex with VEGF 165 , with a M.W. of 155 kDa. Platelet aggregometry and SRA showed that preformed equal molar Bev:VEGF 165 complexes (100-500 nM) plus heparin (0.5-50 ug/ml) activated platelets, while VEGF Trap:VEGF 165 complexes did not. Increasing the molar excess of Bev (≥ 4 fold relative to VEGF) resulted in smaller complexes that did not trigger platelet activation. Injection of pre-formed VEGF Trap:VEGF 165 complexes with heparin into FcγRIIA transgenic mice did not significantly affect platelet count (966±168) vs saline-injected controls (1173±179). Animals receiving Bev+VEGF 165 complexes with heparin exhibited behavior consistent with thrombotic shock and experienced profound thrombocytopenia (platelet count of 331±217; P < 0.001). Lung sections from these animals showed patterns of occlusive thrombi.

Conclusions: Bev when mixed at near equal molar ratios with VEGF 165 , in the presence of heparin, can support platelet aggregation. This is not the case for VEGF Trap. The clinical relevance of these observations remains to be determined.

Commercial Relationships:

Douglas A. MacDonald, Regeneron

Pharmaceuticals (E); Jiann-Kae Luo, Regeneron Pharmaceuticals (E); Nicholas Papadopoulos, Regeneron Pharmaceuticals (E); Erica Pyles, Regeneron Pharmaceuticals (E); Stanley Wiegand, Regeneron Pharmaceuticals (E); Françoise Bono, Sanofi France (E); Nathalie Delesque, Sanofi France (E); Pierre Savi, Sanofi France (E); John Francis, None; Todd Meyer, Funding grant from Regeneron (F) Support: None

Program Number: 441 Poster Board Number: D1118 Presentation Time: 8:30 AM - 10:15 AM Nanostructured Biopolymer Films for Retinal Drug Delivery

Kevin D. Lance, Daniel A. Bernards, Natalie A. Ciaccio, Robert B. Bhisitkul, Tejal

A. Desai. UCSF, San Francisco, CA. Purpose: The goal of this research is to develop a sustained drug delivery device for intraocular release of therapeutics, with a focus on the treatment of age-related macular degeneration (AMD). We investigate the ocular tolerance, structural durability, and functionality of our nanoporous biopolymer devices. Methods: We utilized a modular fabrication approach with polycaprolactone (PCL) that combines template-based fabrication to produce nanopores and a polymer mixture to generate a mechanically robust supporting layer. PCL-based devices were characterized in vitro to assess physical degradation and the characteristic release of model therapeutics. Parallel experiments in microporous PCL devices were used to characterize drug payload stability for a model antibody. New Zealand White rabbits were used for our in vivo studies. Structured poly(caprolactone) (PCL) thin films were implanted in rabbit eyes for survival studies and surveillance of ocular tolerability up to 9 months. Histology of enucleated post-mortem eyes was used to evaluate morphologic abnormalities and adverse reactions; scanning electron microscopy was used to examine the durability and stability of extracted thin films. Complete devices loaded with IgG were implanted for a 6 week feasibility study, and vitreous and device samples were analyzed to gauge device performance and payload stability. Results: Devices utilizing nanoporous thin films for controlled release exhibit constant rates of release in vitro for greater than 6 months. Model protein drug payloads maintained stability at least 10 weeks. Nanostructured thin films lacked an observable immune response through 9 months of implantation. Structural integrity of implanted films was maintained throughout this time course in vivo. Equivalent films tested in vitro became increasingly fragile after 1 year and had noticeable structural breakdown occurring in excess of 1 year. A short duration in

vivo trial with completed devices demonstrated stable activity for the IgG payload over the course of 6 weeks. Conclusions: The fabrication procedures we have developed are capable of generating robust nanoporous biopolymer films, and preliminary studies have established several important benchmarks of device function, including sufficient

drug loading, controlled release of therapeutic over extended times, materials

biocompatibility, and maintenance of drug activity.

Commercial Relationships:

Kevin D. Lance, Santen, Inc. (F); Daniel A.

Bernards, Santen, Inc. (F); Natalie A. Ciaccio, Santen, Inc. (F); Robert B.

Bhisitkul, Santen, Inc. (C); Tejal A. Desai, Santen, Inc. (F) Support: NIH Grant 2 T32 GM 8155-27

Program Number: 442 Poster Board Number: D1119 Presentation Time: 8:30 AM - 10:15 AM Resveratrol inhibits Choroidal Endothelial Cell Proliferation through the induction of SAPK/JNK Pathway Vijay Khetpal, S Balaiya, K V. Chalam. Ophthalmology, University of Florida College of Medicine, Jacksonville, FL. Purpose: Exudative AMD results from hypoxia induced choroidal neovascularization. Resveratrol, a common antioxidant present in red wine and natural plants is anti-angiogenic in carcinoma and pro-angiogenic in experimental models of vascular endothelial cells. It decelerate the aging process and modulate vascular endothelial cell function in diverse angiogenic beds. In this study, we investigated the effects of resveratrol on cell viability and its effects on apoptosis in hypoxic choroidal endothelial cells. Methods: Choroidal endothelial cells (RF/6A) where exposed to escalating doses of resveratrol 4 and 12 µg/ml and cell viability was analysed by WST-1 assay. Hypoxia was induced through cobalt chloride (200 µM) and the effect of resveratrol (4 and 12µg/ml) was evaluated by assessing Stress Activated ProteinKinase/c-Jun N-terminal Kinase (SAPK/JNK) pathway using immunoblot and densitometric analysis. Results: In the presence of hypoxia (200µM of cobalt chloride), cell proliferation increased to 129.3±1.13% whereas it decreased to 125.6±1.02% after the addition of resveratrol at 4µg/ml and 86.1±2.16% at 12µg/ml of concentration. In comparison to basal level (0.3% ODU), phosphorylation state of SAPK/JNK increased after hypoxic insult to 5.3% at 200 µM concentration of cobalt chloride which decreased to 3.8 and 2.5% after the addition of 4 and 12 µg/mL of resveratrol, respectively. (P<0.01) Conclusions: Resveratrol inhibits proliferation of hypoxic choroidal endothelial cells and its apoptotic effects may be relayed through the SAPK/JNK pathway. Commercial Relationships: Vijay Khetpal, None; S. Balaiya, None; K. V. Chalam, None Support: None

Program Number: 443 Poster Board Number: D1120 Presentation Time: 8:30 AM - 10:15 AM Anti-phosphatidylserine Antibodies As A Potential New Therapy Against

Choroidal Neovascularization Rafael Ufret-Vincenty 1 , Bogale Aredo 1 , Kaiyan Zhang 1 , Cynthia X. Wang 1 , Shusheng Wang 1 , Jose Pulido 2 , Philip E. Thorpe 1 . 1 Ophthalmology, UT Southwestern Medical Center, Dallas, TX; 2 Ophthalmology, Mayo Clinic, Rochester, MN. Purpose: Despite the dramatic changes in clinical outcomes brought by anti-VEGF agents, additional drugs directed against different targets in the neovascular process are needed. This may allow for combination therapies that could potentially eradicate choroidal neovascularization (CNV). In normal cells the aminophospholipid phosphatidylserine (PS) is almost exclusively localized to the

inner leaflet of the cell membrane’s lipid bilayer. Endothelial cells of tumor neovasculature lose their capacity to maintain PS asymmetry. Anti-PS antibodies bind to the newly exposed phosphatidylserine in the outer leaflet of the tumor vascular endothelium, mediating antibody-dependent cell-mediated cytotoxicity, and causing the collapse of the tumor neovasculature. Radiotherapy increases the exposure of PS in tumor vasculature, enhancing the antitumor effects of anti-PS antibodies. We propose to evaluate if there is also exposure of PS in CNV, and if anti-PS antibodies can treat CNV. Methods: We induced CNV in B6 mice using the laser model. Immunostaining for PS was done after perfusing mice with an anti-PS antibody and paraffin-embedding the eyes. We tested the effect of intravitreal anti-PS antibodies alone, or in combination with eye radiation, on the CNV size as measured with ICAM-2 staining. Results: Paraffin sections of eyes perfused with an anti-PS antibody stained positive for PS. The staining co-localized with ICAM-2-stained CNV, demonstrating that PS was exposed on the choroidal neovascular membranes. The anti-PS antibody (11.31) led to a 52% reduction in the laser-induced CNV size (p=0.02) when injected intravitreally. We have established a system for irradiation of the eyes. We will show data on the effect of radiation alone or in combination with anti-PS antibodies on the neovascular complex. Conclusions: Anti-phosphatidylserine antibodies may have therapeutic value in wet AMD alone or in combination with radiation or anti-VEGF agents. Commercial Relationships: Rafael Ufret-Vincenty, None; Bogale Aredo, None; Kaiyan Zhang, None; Cynthia X. Wang, None; Shusheng Wang, None; Jose Pulido, None; Philip E. Thorpe, Peregrine (P)

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Support: Unrestricted RPB Department Grant, Core grant to Department of Ophthalmology (EY020799), Disease Oriented Clinical Scholars Grant to RUV

Program Number: 444 Poster Board Number: D1121 Presentation Time: 8:30 AM - 10:15 AM Biological Activity of Different Transgenic Ranibizumab Compositions

Knut Stieger, Tobias Wimmer, Birgit Lorenz. Department of Ophthalmology, Justus-Liebig-University Giessen, Giessen, Germany. Purpose: Ocular neovascularisation due to uncontrolled growth of new vessels into the retina following overexpression of vascular endothelial growth factor (VEGF) is the main cause of visual impairment in retinal neovascular diseases such as age- related macular degeneration (AMD) or diabetic retinopathy (DR). The intraocular expression of anti-VEGF molecules potentially represents a therapeutic strategy to block neovascularization in these pathologies. The aim of this study was to characterize the optimal composition of the expression cassette for the production of these molecules in standard cell lines in vitro. Methods: Both antibody chains of Ranibizumab, the light and part of the heavy chain were designed containing secretory leader sequences or restriction sites for subsequent subcloning. The fragments were either expressed separately using an

IRES containing expression cassette (Ra01), or were cloned together into one reading frame containing either a glycine or glycine-proline anchor in between (Ra02-Ra06). Plasmids were transfected into HEK293 and Hela cell lines and the expression of the molecules verified by Western blot analysis. A Ranibizumab specific ELISA was developed in order to measure the concentration of the anti- VEGF molecules. The biological activity was tested using HUVEC (human umbilical vein endothelial cell) tube formation assays and HUVEC migration assays. Results: All Ra compositions were detected in the supernatant of transfected Hela and HEK293 cells. Generation of long cellular tubes in the HUVEC tube formation assay, which is predominantly due to VEGF activity, was reduced to 50% with Ra01 and similar levels were achieved with Ra02-Ra06. VEGF activity in the HUVEC migration assay was reduced by about 50% for all Ra compositions. Similar VEGF inhibition results were obtained using commercially available recombinant Ranibizumab (Lucentis®) at equal concentrations. Conclusions: Transgenic Ranibizumab, either expressed separately or as one molecule have similar inhibitory effects on VEGF activity as commercially available Ranibizumab in vitro. These results lay the foundation for the development of an alternative treatment strategy for patients with AMD or DR, in which Ranibizumab is produced at low doses directly in retinal cells. Commercial Relationships: Knut Stieger, None; Tobias Wimmer, None; Birgit Lorenz, None Support: DFG STI 597/2-1

Program Number: 445 Poster Board Number: D1122 Presentation Time: 8:30 AM - 10:15 AM RGD-targeted Nanoparticles Expressing Flt23k Inhibit CNV In a Murine CNV Model

Xiaohui Zhang, Ling Luo, Hironori Uehara, Tadashi Miya, Christina Mamalis,

Alex Jones, Bonnie Archer, Balamurali K. Ambati. John Moran Eye Center, University of Utah, Salt Lake City, UT. Purpose: Choroidal neovascularization (CNV) is a leading cause of blindness in age-related macular degeneration (AMD) patients in the developed world. Currently, the most effective therapies are monthly intravitreal injections of anti- VEGF agents such as bevacizumab or ranibizumab. However there are significant risks associated with repeated intravitreal injections. The purpose of this study was to determine whether a single intravenous administration of RGD-coated nanoparticles delivering plasmids expressing Flt23k intraceptors could suppress CNV in a murine model. Methods: We prepared nile-red labeled nanoparticles which were blank, loaded with pCMV.Flt23k, or loaded with pCMV.Flt23k conjugated with RGD oligopeptides (which home to alpha-v-beta-3 integrin). All three nanoparticles were

dissolved in MES buffer. The total volume delivered was 4 μl (plasmid concentration is 0.1μg/μl) in each mouse, and similar volumes of MES buffer

served as blank control. Mice CNV was induced by 532 nm laser or subretinal injecton of adeno-associated virus mediated small hairpin ribonucleic acid (shRNA) sFlt-1. Tail vein injection was performed 2 weeks after induction of CNV. CNV regression was evaluated 2 weeks after tail vein injection in histological sections and CNV volume quantified using newly developed software, Seg3D in vivo image. RGD.Flt23k.NR.NP was detected by immunostaining in CNV. Results: α5 expression in CNV area was confirmed by immunostaining. H&E stained sections show the CNV size was dramatically decreased in

RGD.Flt23k.NR.NP injected mice (treatment group) compare to the other three control groups (Flt23k.NR.NPs, blank NR.NPs or MES buffer). The treatment group mice CNV volume was decreased by 23%, which showed significantly more reduction than observed with unlabeled nanoparticles, blank nanoparticles, or MES control (all p’s <0.05), As a positive control, CNV lesions treated with anti-mouse

VEGF antibody intravitreal injections were decreased by 11% (p=0.1). Conclusions: This study exploits a new method to treat CNV by providing a novel therapeutic, a safer route of delivery, and also a nonviral delivery system for extended gene delivery. Commercial Relationships: Xiaohui Zhang, None; Ling Luo, None; Hironori Uehara, None; Tadashi Miya, None; Christina Mamalis, None; Alex Jones, None; Bonnie Archer, None; Balamurali K. Ambati, None Support: 5R01EY017182-04

Program Number: 446 Poster Board Number: D1123 Presentation Time: 8:30 AM - 10:15 AM Microbial and Fungal Contamination Following A Day Use Of Multiple Use Bottles Of Fluoresceine Sodium 0.25% And Benoxinate Hydrochloride 0.4% In An Outpatient Ophthalmology Clinic Olivier Lasnier, Anne Faucher. Ophthalmology, Sherbrooke University, Sherbrooke, QC, Canada. Purpose: Fluorescein and benoxinate solution in a multiple use bottle is an important clinical tool for diagnostics and tonometry measurement in ophthalmology clinics. However, this solution may also be the source for dissemination of ocular infections. Contamination of ocular solutions may occur due to rapid administration, poor patient cooperation and clinician distraction. We aimed to determine the rate of bacterial and fungal contamination of 5 mL multiple usage bottles of fluorescein 0.25% and benoxinate 0.4% solution after a single day of use in an ophthalmology outpatient clinic. We also calculated the average duration of a 5 mL solution. Methods: This project is a prospective blinded study. One unopened bottle of fluorescein sodium 0.25% and benoxinate hydrochloride 0.4% solution was placed in every exam room of the University of Sherbrooke ophthalmology clinics at the beginning of the day. All staff members working in the clinics were unaware of the ongoing project to prevent any change in their usual practice. At the end of the clinical day, all bottles were collected, left at room temperature for less than 24 hours and sent for culture. Three drops from each bottle were put on each of five culture media: blood agar, chocolate agar, Brucella agar, Sabouraud agar and Thioglycolate broth. All media where immediately stored in an air oven at 35 degrees Celsius until being delivered to the microbiology laboratory for further analysis. All cultures were kept for at least one week of observation before being discarded. Results: A total of 27 bottles were collected from nine ophthalmologists. All of the bottles had negative culture after one week of incubation. All physicians were unaware of the project at the time of sample collection and reported to have used this solution, on average, for 80% of their patients. Considering the average frequency of usage and the average number of patients seen per day, a 5 ml of this solution would last two and a half days. Conclusions: The multiple use of a single bottle of Fluoresceine sodium 0.25% and Benoxinate hydrochloride 0.4% is safe on a 24 hours basis in regard of the bacterial and fungal contamination risk. To our knowledge, this is the largest in-use contamination experimentation for this solution. The safety profile over a longer period of utilization warrants further investigation. Commercial Relationships: Olivier Lasnier, None; Anne Faucher, None Support: None

Program Number: 447 Poster Board Number: D1124 Presentation Time: 8:30 AM - 10:15 AM Treatment For Persistent Uveitic Macular Edema With intravitreal dexamethasone 0.7 mg implant (Ozurdex®)

Alfredo Adan Civera 1 , Laura Pelegrin 1 , Amanda Rey Torrente 1 , Victor Llorens 1 , Marina Mesquida 1 , Blanca Molins 2 . 1 Ophthalmology, Hospital Clinic, Barcelona, Spain; 2 Ophthalmology, IDIBAPS, Barcelona, Spain. Purpose: To evaluate the effects of intravitreal dexamethasone (DEX) 0.7 mg implant (Ozurdex®; Allergan, Inc., Irvine, CA) for the treatment of persistent uveitic macular edema (ME) Methods: Medical records of 16 patients (23 eyes) with persistent (>or= 90 days) uveitic ME treated with intravitreal DEX 0.7 mg implant were reviewed. Complete ophthalmic examination including visual acuity, fundus biomicroscopy, fundus photography, and spectral domain optical coherence tomography (Cirrus HD- OCT,Carl Zeiss Meditec, Dublin, CA) was performed at baseline and follow-up .Main outcome measures were improvement of central retinal thickness (CRT) measured with optical coherence tomography and changes in best corrected visual acuity (BCVA). Tolerability of the implant was assessed. Results: At a mean postoperative follow-up of 5.3 months,central retinal thickness on optical coherence tomography exams decreased significatively (p < 0.001) at one month and three month examination.At day 60, a 10-letter or more BCVA improvement was seen in 60.86% (14/23) of eyes.A 15-letter or more mprovement was achieved in 34,78% (8/23) of eyes.ME relapsed in 21.7% (5/23) and an additional intravitreal injection of DEX 0.7 mg implant was performed. The implant was well tolerated. Throughout the study, an increase in intraocular pressure of 10 mm Hg or more was seen in 26,08 % (6/23) of eyes. There were no

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

cases of endophthalmitis. At the time of intravitreal injection, 17 eyes (73.9%) were vitrectomized and 6 were non-vitrectomized (26.1%) Conclusions: Intravitreal DEX 0.7 mg implant seems to be a safe and effective for treatment of persistent uveitic ME .Our results suggest that efficacy of the implant in vitrectomized eyes with uveitic ME. Commercial Relationships: Alfredo Adan Civera, None; Laura Pelegrin, None; Amanda Rey Torrente, None; Victor Llorens, None; Marina Mesquida, None; Blanca Molins, None Support: None

Program Number: 448 Poster Board Number: D1125 Presentation Time: 8:30 AM - 10:15 AM JUNCTION Study: SD-OCT Shows Shrinking And Disappearing Of Drusen

In Nonexudative AMD Eyes Treated With AREDS 2 Supplements Plus A Complex Containing The Natural Compounds Curcumin, Omega 3 DHA/EPA and Phospholipids Andreas U. Bayer. "The Life100 Concept" Study Group, Weilheim, Germany. Purpose: To evaluate safety and efficacy of Ayurveda´s anti-inflammatory and anti-oxidative natural compound Curcumin in the treatment of nonexudative AMD.

Preliminary results (6-months follow-up) of the Justification for the Use of Natuaral Compounds in the Treatment of Inflammatory, Ophthalmic and Neurodegenerative diseases Study (JUNCTION Study) are presented here. Methods: 126 patients with nonexudative AMD with large drusen in both eyes (AREDS Category 3) were randomized to the AREDS2 supplements 1000mg Omega 3 and 10mg Lutein / 2mg Zeaxanthin alone or to these supplements in addition to 1000mg Curom TM (complex of Curcumin, Omega 3 DHA/EPA and phospholipids). SD-OCT (Heidelberg Spectralis OCT) and fundus photography (Zeiss Visucam NMpro) were performed at baseline and after 6 months. Before the beginning of this study, the bioavailability of different Curcumin products have been studied. Results: The treatment with Curom TM shows very good 24-hours bioavailabilities. In the 66 patients treated with AREDS2 plus Curom TM , there was a highly significant reduction of drusen size between baseline and after 6 months (p<0.0001). In the 132 eyes of these 66 patients, we found a disappearing of one or more drusen in 109 eyes. There was only a non-significant change of drusen size in the eyes of the 60 patients who were treated with AREDS2 supplements alone. Tolerance of AREDS2 supplements was slightly better than of AREDS2 supplements plus Curom TM . There were 5 out of the 66 patients who were treated with AREDS2 supplements plus Curom TM , who complained about diarrhea and/or a dull stomach. Biomarkers of atherosclerosis improved significantly in patients who were treated with AREDS2 supplements plus Curom TM (p<0.05). Conclusions: Curom TM is a powerful natural compound (complex) to treat nonexudative AMD (high-risk dry AMD). The anti-inflammatory agent Curcumin should be part of any treatment of nonexudative AMD. Thereby, bioavailability is of major concern. Follow-up of patients in the JUNCTION Study will test the hypothesis, that (whether) Curom TM is able to reduce the risk of progression of high-risk dry AMD (AREDS Category 3) to exudative AMD and/or Geographic Atrophy (AREDS Category 4). In addition, following the guidelines for assessment of cardiovascular risk in asymptomatic adults of the American College of Cardiology Foundation/American Heart Association, the large number of patients recruited until April 30, 2012, will give insights into a possible prevention of stroke and/or congestive heart failure.

Commercial Relationships:

Andreas U. Bayer, Dr. Andreas Bayer (P)

Support: None Clinical Trial: Bavarian Medical Association, Munich, Germany, 11130

Program Number: 449 Poster Board Number: D1126 Presentation Time: 8:30 AM - 10:15 AM Design and Optimization of a Transscleral Iontophoresis Applicator for Delivering Biologics to the Anterior and Posterior Segments

Michael Manzo 1 , Peyman Moslemy 1 , Begona Ruiz-Perez 1 , Fengqui Fan 1 , Lydia Mbocha 1 , Will Schubert 1 , Phil Isom 1 , Tracy Dowie 1 , Pammy Subramony 1 , Michael A. Patane 2 . 1 Eyegate Pharmaceuticals Inc, Waltham, MA; 2 EyeGate Pharmaceuticals, Inc, Waltham, MA. Purpose: To develop a transscleral iontophoresis applicator capable of delivering biologics to the anterior and posterior segments. Methods: The engineering designs and testing focused on optimizing conductivity, buffering capacity, product stability, and minimizing drug volume requirements. Multiple biocompatible conductive matrices were prepared and evaluated in the ocular iontophoresis applicator drug reservoir. The devices were loaded with Cytochrome C (CytC, 12.4 kDa globular protein) in H 2 O and tested ex vivo via anodal iontophoresis in a Franz cell apparatus with phosphate buffered saline

solution in the receptor chamber. For in vivo testing, New Zealand albino rabbits were dosed with CytC using the test devices impregnated with different biocompatible conductive matrices. Results: Following iontophoretic transport experiments in rabbits, bioanalytical results revealed that CytC was present in all ocular tissues harvested. The levels of

CytC in conjunctiva, sclera, choroid, retina, vitreous, iris and ciliary body, and aqueous humor were quantified by triple-quad MS. The iontophoretic transport efficiency (percent ratio of total delivery to the initial amount of protein in dosing solution) increased from < 0.7% for passive delivery of 40 and 80 mg/mL CytC solutions to > 7% for iontophoresis. The concentration of CytC in the ocular tissues increased directly with the increase in dosing solution concentration from 10 - 40

mg/mL but remained at comparable levels for 80 mg/mL. Initial pH of dosing solution (7.4 vs. 5.0) had no considerable effect on the amount of protein delivered to the ocular tissues. Varying the current intensity (1 - 8 mA) with a fixed 5 min application time enhanced the concentration of CytC in the tested ocular tissues. Likewise, increasing the iontophoretic dose at a fixed current intensity resulted in enhanced delivery of CytC to the ocular tissues. Conclusions: The experimental results demonstrate that significant amounts of CytC can be delivered non-invasively into all rabbit ocular tissues using a novel transscleral iontophoresis ocular applicator.

Commercial Relationships:

Michael Manzo, Eyegate Pharmaceuticals Inc (E);

Peyman Moslemy, Eyegate Pharmaceuticals (E); Begona Ruiz-Perez, Eyegate Pharmaceuticals (E); Fengqui Fan, Eyegate Pharmaceuticals (E); Lydia Mbocha, Eyegate Pharmaceuticals (E); Will Schubert, Eyegate Pharmaceuticals (E); Phil Isom, Eyegate Pharmaceuticals (E); Tracy Dowie, Eyegate Pharmaceuticals (E); Pammy Subramony, Eyegate Pharmaceuticals (E); Michael A. Patane, Eyegate Pharmaceuticals (E) Support: None

Program Number: 450 Poster Board Number: D1127 Presentation Time: 8:30 AM - 10:15 AM Multicenter Phase 1 Clinical Trial Targeting Tissue Factor for the Treatment

of Neovascular AMD John A. Wells, III 1 , Brian B. Berger 2 , Christine Gonzales 3 , Victor H. Gonzales 4 , David L. Johnson 1 , Brian D. Sippy 5 , Manju Soni 6 . 1 Ophthalmology, Palmetto Retina Center, West Columbia, SC; 2 Retina Research Center, Austin, TX; Ophthalmology, Retina and Vitreous Center of Southern Oregon, Ashland, OR; 4 Valley Retina Institute, McAllen, TX; 5 Ophthalmology, Rocky Mountain Eye Ctr, PC, Missoula, MT; 6 Numa LLC, Mystic, CT. Purpose: To evaluate the safety and tolerability of binding tissue factor with hI- con1™, alone or in combination with anti-VEGF therapy, in eyes with active neovascular AMD.

3

Methods: This prospective, multi-center, dose-escalating clinical study evaluated the safety and tolerability of a single, intravitreal injection of 60µg, 150µg and 300µg of hI-con1 in 18 patients (6 per cohort). Ocular inclusion criteria included active CNV with at least a 30% classic component on angiography and VA 20/63 - CF. Eyes with end-stage CNV, eyes on chronic anti-VEGF therapy and treatment naïve eyes were enrolled. Anti-VEGF therapy was allowed 2 weeks after the hI-

con1 injection at the investigator’s discretion. Results: No ocular or systemic dose limiting toxicities were identified. No retinal or choroidal vascular, inflammatory or hemorrhagic toxicities were identified. Patients reported no drug related adverse events. hI-con1, administered alone or adjunctively with an anti-VEGF agent, showed multiple, dose-related biologic

signals across the broad spectrum of treated eyes. An interim analysis of 6 eyes in the high-dose 300μg cohort at Day 57 showed:

• 67% showed reduced OCT thickness, some CNV regression on angiography and ≥ 3 lines improved BCVA. • 100% of the 3 treatment naïve eyes showed reduced OCT thickness, some CNV regression on angiography and ≥ 3 lines improved BCVA.

An interim analysis of all dose cohorts additionally showed:

• The mean BCVA of 7 eyes on chronic anti-VEGF therapy (mean number of previous anti-VEGF injections = 6; all with persistent sub-retinal fluid; VA = 20/80

or worse) at Day 29 was +9 letters compared to baseline; at Day 57, +7 letters compared to baseline. • The mean BCVA of 9 patients receiving one hI-con1 plus one anti-VEGF injection was +11 letters 29 days after the anti-VEGF injection. • 67% of all 18 eyes went 57 days without resuming anti-VEGF therapy; 50% of all eyes went 85 days or longer without additional anti-VEGF therapy. Conclusions: A single injection of hI-con1, alone or in combination with anti- VEGF agents, showed no ocular or systemic safety signals. Evidence of biologic activity with reduced OCT thickness, evidence of CNV regression, and gains in BCVA was observed in many of the treated eyes. Further studies are planned.

Commercial Relationships:

John A. Wells, III, Eyetech (C, R), Genentech (F),

LPath Inc (F), Novartis (F), Ophthotech (F), Pfizer Inc (F), Steba (F); Brian B.

Berger, Genentech (F), Iconic Therapeutics (F), Lpath Inc (F), NeoVista Pharmaceuticals (F), Pfizer Inc (F); Christine Gonzales, Eyetech (C), Iconic Therapeutics (F), Ophthotech (F); Victor H. Gonzales, Eyetech (C, R), Genentech (F, C), Iconic Therapeutics (F), Ophthotech (F), Pfizer (C, R), Pfizer Inc (F), Regeneron (F), Steba (F); David L. Johnson, Genentech (F), Iconic Therapeutics (F), Lpath Inc (F), Ophthotech (F), Regeneron (F); Brian D. Sippy, Iconic Therapeutics (F), Regeneron (F); Manju Soni, Iconic Therapeutics (C) Support: Iconic Therapeutics, Inc Clinical Trial: http://www.clinicaltrials.gov, NCT01485588

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Program Number: 451 Poster Board Number: D1128 Presentation Time: 8:30 AM - 10:15 AM Trasferrin-functionalized PLGA Nanopartilces Sustain Diclofenac Delivery to Choroid-RPE Uday B. Kompella 1A , Arun K. Upadhyay 1B . A Pharmaceutical Sciences, Ophthlamology and Bioengineering, B Pharmaceutical Sciences, 1 University of Colorado Denver, Aurora, CO. Purpose: To develop topically applied transferrin-functionalized, biodegradable, polymeric nanoparticles to enhance and sustain diclofenac delivery for treating choroidal neovascularization. Methods: Diclofenac and Nile red loaded poly(lactide-co-glycolide) (PLGA) nanoparticles were prepared using double emulsion and solvent evaporation method. Nanoparticles were chemically conjugated to amino groups on transferrin through 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl (EDAC) activation of carboxyl group present on PLGA. In vitro release of diclofenac from nanoparticles was assessed under sink condition at 37 °C. Diclofenac and Nile red were quantified using UV absorbance at 276 nm and 520 nm (Nile red absorbance maxima in 1:1 acetonitrile:water mixture), respectively. In vitro uptake of nanoparticles was evaluated in ARPE-19 and MDCK1 cells. Briefly, cells at 80 % confluence were incubated with 100 μg/ml nanoparticles in serum free medium for

5 min, the medium was removed, cell monolayer was washed - 2 times each with physiological PBS (7.4) and acidic PBS (pH 5.2), cells were lysed using 2% Triton X-100 and Nile red was extracted from particles using dichloromethane, and Nile red content was estimated using UV absorbance at 554 nm (absorption maxima in

dichloromethane). Following single topical application of two drops of 10 μl

nanoparticle suspension (0.35% w/v diclofenac acid) NP or equivalent dose of plain diclofenac sodium solution in Brown Norway rats, drug delivery was determined by LC-MS method. Nanoparticles were assessed for size using Malvern nanosizer. Results: Transferrin-functionalized diclofenac PLGA nanoparticles had a mean diameter of 230-260 nm and a negative zeta-potential. Drug release was sustained during the one week in vitro study. ARPE-19 and MDCK1 cells showed 3-4 times higher uptake of transferrin-functionalized diclofenac-PLGA nanoparticles when compared to non-functionalized nanoparticles. At the end of one week following single dose, diclofenac levels in rat choroid-RPE were significantly higher with functionalized PLGA nanoparticles when compared to non-functionalized nanoparticles and plain drug solution.

Conclusions: Transferrin functionalization enhances cellular delivery of nanoparticles and allows sustained and enhanced delivery of diclofenac to choroid- RPE for one week following eye drop administration.

Commercial Relationships:

Uday B. Kompella, International Patent

Application. 12/09/2007. WO/2008/033924 (P); Arun K. Upadhyay, None

Support: NIH Grant R41 EY020097

Program Number: 452 Poster Board Number: D1129 Presentation Time: 8:30 AM - 10:15 AM

Planar SU-8/PEGDMA Microdevices for Retinal Drug Delivery of Lucentis

Jennifer S. Wade, Tejal A. Desai Ph.D

..

Bioengineering and Therapeutic Sciences,

University of California - San Francisco, San Francisco, CA. Purpose: Due to the complications associated with intravitreal injection novel drug delivery technologies are desired. We have developed planar SU-8/PEGDMA microdevices, coated with the permeation enhancer Chitosan. These devices maximize contact surface area and provide consistent drug volume. Using the retinal epithelial cell line ARPE19, the influence of microdevice geometry and surface coating on paracellular drug delivery has been investigated. Methods: Device fabrication was achieved by a three-mask photolithography process. SU-8 and PEGDMA hydrogel solutions of FITC-Dextran (FD) or Lucentis were spun onto a silicon wafer. UV-light was used to crosslink the hydrogel in the device reservoir. Surface modification was conducted by deposition of a 1.6% w/v Chitosan solution onto a drug-loaded wafer until dry film formation. Devices were placed in the apical chamber of an ARPE19 coated transwell insert. Aliquots were removed from the basolateral chamber and analyzed for released drug concentration using a fluorimeter or spectrophotometer. Results: Microdevices with payloads of FD and Lucentis were succesfully fabricated. Consistent elution of FD and Lucentis from devices was achieved. The quantity of FD transported across the ARPE19 monolayer of cells using a microdevice was greater than the amount transported using a bolus administration. The effect Chitosan coating has on the amount of drug transported is still being investigated and further optimization of the coating process will clarify if the mucoadhesive inhibits drug elution. Conclusions: A planar microdevice capable of housing therapeutics of varying molecular weight was developed. Preliminary data suggests this device enhances

the transport of large molecules across an ARPE19 in vitro retina model in comparison to bolus administration alone.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology (PH) Program Number: 451 Poster Board

Commercial Relationships:

Jennifer S. Wade, Genentech Inc (F); Tejal A.

Desai Ph.D., Genentech Inc (F) Support: Genentech Grant 71868-01

Program Number: 453 Poster Board Number: D1130 Presentation Time: 8:30 AM - 10:15 AM Therapeutic Effect of Fenofibrate Eyedrops on Diabetic Retinopathy Ying Chen 1 , Boyu Lu 1,2 , Qingjiong Zhang 2 , Jianxing Ma 1 . 1 Physiology, OUHSC, Oklahoma City, OK; 2 Sun Yat-sen University, Zhongshan Ophthalmic Center, Guangzhou, China. Purpose: The new studies from The Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) reported that oral administration of fenofibrate, a relativlye safe and low cost lipid-lowering drug, prevented the progression of diabetic retinopathy (DR) in type 2 diabetic patients. The purpose of this study is to evaluate if fenofibrate has therapeutic effects on DR in type 1 diabetes and if topical administration of fenofibrate is able to prevent DR in type 1 diabetes. Methods: Human retinal endothelial cells (HRECs) were cultured on Matrigel or Transwell inserts in the presence or absence of various concentrations of fenofibrate, the effects of fenofibrate on tube formation and cell migration were evaluated. Oxygen-induced retinopathy (OIR) was generated by exposing newborn rats to 75% oxygen. Diabetes was induced in adult rats by injection of streptozotocin (STZ). Eyedrop containing 3% fenofibrate or control vehicle were topically administered to the cornea of OIR rats or diabetic rats 5 and 3 times a day, respectively. Levels of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule 1 (ICAM-1) were measured by Western blot and enzyme-linked immunosorbent assay (ELISA). Retinal vascular leakage was evaluated by retinal vascular permeability assay, and retinal neovascularization (NV) was evaluated by fluorescein angiography. Results: Fenofibrate inhibited HREC tube formation and cell migration. Topical application of fenofibrate significantly decreased retinal vascular permeability and reduced levels of VEGF and ICAM-1 in the retina from OIR rats and from STZ- induced diabetic rats, and arrested pre-retinal NV in the OIR rat model. Conclusions: Fenofibrate attenuates retinal angiogenesis and inflammation, and topical administration of fenofibrate has therapeutic effects on DR in type 1 diabetes. Commercial Relationships: Ying Chen, None; Boyu Lu, None; Qingjiong Zhang, None; Jianxing Ma, None Support: 711JF10-Junior Faculty Award from ADA , P20RR024215 from the National Center for Research Resources.

Program Number: 454 Poster Board Number: D1131 Presentation Time: 8:30 AM - 10:15 AM Development of an Optimized Culture Media to Improve VEGF Antagonist Secretion and Stability by Encapsulated Cell Technology Implants

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Alline M. Lelis 1A , Michael Rivera 1A , Sue Elliott 1A , Brenda Dean 1A , Pam Heatherton 1A , Bruce Bouchard 1A , Melissa Stiles 1A , Vicent Ling 1A , Konrad Kauper 1B ,

Weng Tao 1 . A Research and Development, B Core Technology Development,

  • 1 Neurotech USA, Lincoln, RI. Purpose: Encapsulated Cell Technology (ECT) is designed to deliver therapeutic factors directly to the retina through a semi-permeable, hollow fiber membrane implant that encapsulates genetically modified cells. The goal of this study was to optimize ECT pre-implant culture conditions to maximize and stabilize secretion of a VEGF-antagonist targeting microgram per day daily production levels and to remove all animal and human components from the culture media. Effects of culture media and additional supplementation on encapsulated cell protein secretion levels and encapsulated cell health were evaluated. Methods: ECT devices were manufactured and encapsulated using a platform cell line engineered to secrete a VEGF antagonist molecule. Media supplements, including lipids, vitamins, amino acids and growth factors were added to the platform culture media in an effort to increase protein expression and optimize encapsulated cell line performance. Multiple commercial media designed for enhanced recombinant protein expression, differentiation, and viability were compared to the current platform media. Protein production of the encapsulated cells was evaluated by ELISA. Device health and cell viability was analyzed qualitatively by the preparation of histology slides. Media analyte and metabolite concentration were evaluated using a chemistry analyzer. Results: The addition of GlutaMAX TM and removal of L-glutamine from all media led to decreased ammonia accumulation and improved encapsulated cell stability. One animal and human component free media formulation increased protein production of the encapsulated cell line three to four fold, maintaining steady-state expression levels in micrograms per day. Most media supplements evaluated did not improve protein expression, with the exception of cholesterol. However, long- term exposure to high concentrations of cholesterol was detrimental to cell health. While select stem cell factors improved protein production, changes to cell morphology and growth rate occurred as well. Conclusions: Optimization of media used to support ECT function resulted in identification of an improved formulation capable of achieving sustained ECT production of a VEGF antagonist at a rate of micrograms daily secretion. In addition, the absence of animal and human components enables this media to be used for clinical development and commercialization.

Commercial Relationships:

Alline M. Lelis, Neurotech USA (E); Michael

Rivera, Neurotech USA (E); Sue Elliott, Neurotech USA (E); Brenda Dean, Neurotech USA (E); Pam Heatherton, Neurotech USA (E); Bruce Bouchard, Neurotech USA (E); Melissa Stiles, Neurotech USA (E); Vicent Ling, Neurotech USA (E); Konrad Kauper, Neurotech USA (E); Weng Tao, Neurotech USA (E) Support: None

Program Number: 455 Poster Board Number: D1132 Presentation Time: 8:30 AM - 10:15 AM Long-Term, Sustained Intraocular Delivery of a VEGF Antagonist Using Encapsulated Cell Technology Implant for the Treatment of Choroidal Neovascular Diseases

Konrad Kauper, Vincent Ling, Sue Elliot, Cahil McGovern, Sandy Sherman,

Brenda Dean, Lisa Orecchio, Mike Rivera, Pam Heatherton, Weng Tao. Core Technology Development, Neurotech USA, Lincoln, RI. Purpose: To develop Encapsulated Cell Technology (ECT) intraocular implants capable of microgram daily intraocular delivery of a VEGF-antagonist over a sustained period. Long-term delivery of a VEGF antagonist will potentially improve the standard-of-care treatment modality of monthly injections by providing a consistent dose, eliminating patient compliance issues and minimizing safety concerns associated with repeated injections in the eye. Methods: A human derived cell line with a clinically tested history of safety and long-term implant viability was genetically engineered to produce a VEGF- antagonist in doses ranging from nanogram to microgram daily sustained delivery by ECT intraocular implants. The ability to neutralize VEGF and to block VEGF- induced endothelial cell proliferation was evaluated for the VEGF-antagonist. Studies were conducted to determine the steady state concentrations of VEGF- antagonist in the rabbit eye over a one year period. Maximum vitreous concentrations from several encapsulated cell lines producing escalating doses of VEGF-antagonist were quantified. Sustained intraocular levels of VEGF-antagonist delivered by ECT implants were compared to modeled data of standard-of-care injections for Lucentis TM , Avastin TM and Eylea TM to determine the projected efficacy requirements of steady-state concentration of a VEGF antagonist. Results: All doses of VEGF-antagonist produced by ECT implants were determined to be potent and bioactive. Controlled delivery by ECT ranged from nanogram per day to greater than 5 microgram per day sustained delivery in the

rabbit eye. While improvements to the production rate of the ECT implant continue, the current steady state concentration of VEGF antagonist exceeds 25 micrograms in the rabbit eye. Potential levels of VEGF-antagonist in the human eye, adjusted for the half-life of the ECT produced protein, conservatively extrapolate to concentrations greater than 50 ug, exceed the modeled levels for steady-state, standard-of-care treatments by monthly injections.

Conclusions: ECT intraocular implant is capable of sustained delivery of a VEGF antagonist for periods greater than one year in the rabbit. The ability of ECT implant to deliver an efficacious dose over sustained periods, suggest that the ECT implant would have unique advantages compared to traditional standard-of-care treatment for choroidal neovascular diseases, including improved patient compliance and safety.

Commercial Relationships:

Konrad Kauper, Neurotech USA (E); Vincent

Ling, Neurotech USA (E); Sue Elliot, Neurotech USA (E); Cahil McGovern, Neurotech USA (E); Sandy Sherman, Neurotech USA (E); Brenda Dean, Neurotech USA (E); Lisa Orecchio, Neurotech USA (E); Mike Rivera, Neurotech USA (E); Pam Heatherton, Neurotech USA (E); Weng Tao, Neurotech USA (E) Support: None

Program Number: 456 Poster Board Number: D1133 Presentation Time: 8:30 AM - 10:15 AM Nanostructured Porous Silicon Dioxide Microparticles as an Intravitreal Injectable Drug Delivery System for Avastin (Bevacizumab) Lasting Six Months William R. Freeman 1 , Michael Sailor 2 , Michelle Chen 3 , Lingyun Cheng 1 . 1 Ophthalmology, UCSD Jacobs Retina Center, La Jolla, CA; 2 Chemistry and Biochemistry, UCSD, La Jolla, CA; 3 Spinnaker Biosciences, La Jolla, CA. Purpose: Intravitreal anti-VEGF therapy has become the standard of care in treatment of CNV, diabetic macular edema and other conditions. Most studies suggest that injections of Avastin (bevacizumab) every 4 weeks may be the optimal treatment although some interruption in therapy may be possible. The goal of our study was to develop a long-acting intravitreally injectable form of Avastin bound in a nanostructured porous silicon dioxide microparticles and to show the ability of these Avastin-loaded microparticles to release drug over many months. Methods: Porous silicon dioxide was prepared by electrochemical etch of a single crystal silicon wafer in hydrofluoric acid and then oxidized. Microparticles were prepared by ultrasonic fracture. Commercial Avastin was loaded into the nanopores, which had mean diameters of ~ 100 nm. The porous silicon dioxide carrier prepared in this fashion had previosuly been shown to be non-toxic after intravitreal injection. Elution of drug into phosphate buffered saline (PBS) solution at pH 7.4 was determined by placing 5 mg of drug loaded microparticles into tightly capped glass vials containing 1.5 mL PBS and gently reciprocating the vials at 37°C. Free Avastin released from the microparticles was measured using a micro BCA protein test (Pierce). Results: The mass loading efficiency of Avastin in the porous silicon dioxide microparticles was 137 ug Avastin/mg porous silicon dioxide. Elution experiments were initially performed with a drug load of 700 ug. Avastin release was nearly linear with a steady-state free (released) drug concentration between 10 and 30 ug/mL (therapeutic is >> 0.06 ug/mL). The free drug concentration remained > the therapeutic concentration for 5.5 months. Conclusions: Commercial Avastin can be loaded into nanoporous silicon dioxide.

We loaded a total of 1 mg of Avastin into a 0.1 cc injection volume (50% particles/ 50% dextrose). Drug releases in a linear manner maintaining a therapeutic concentration for 5.5 months (165 days). Optimized Avastin loading can increase the load to 4.7 mg per 0.1 cc injection and would result in a therapeutic effect for over six months. A clinical trial with an Avastin-loaded porous silicon dioxide formulation is anticipated with in the next 12 months.

Commercial Relationships:

William R. Freeman, Spinnaker Biosciences (F, I,

C); Michael Sailor, Spinnaker Biosciences (F, I, C); Michelle Chen, Spinnaker

Biosciences (E); Lingyun Cheng, Spinnaker Biosciences (F, I, C) Support: NIH EY020617-01A1 and Research to Prevent Blindness, Inc.

Program Number: 457 Poster Board Number: D1134 Presentation Time: 8:30 AM - 10:15 AM A Novel Eye Drop Formulation of Squalamine For Exudative AMD:

Evaluation Of Ocular Distribution And Ocular Safety In Rabbits Irach B. Taraporewala 1 , Michael J. Elman 2 , Shalom Z. Hirschman 1 , Samuel I. Backenroth 1 . 1 Ohr Pharmaceutical Inc, New York, NY; 2 Elman Retina Group, Baltimore, MD. Purpose: To evaluate the ocular safety and ocular tissue distribution of a novel eye drop formulation of Squalamine, a potent antiangiogenic small molecule inhibitor of multiple growth factors (VEGF, PDGF, bFGF) with previously demonstrated systemic activity in vivo in ocular pathologies and in clinical trials for exudative macular degeneration. Methods: Male Dutch belted rabbits (n=24) were administered Squalamine eye drops bilaterally, either QD (every 24 hours) or BID (every 12 hours) for 1, 7, and 14 days (n=4/group/dose). Ocular tissues were harvested 24 (±2) or 12(±1) hours post last dosing in the QD or BID groups, respectively. Posterior sclera/choroid,

aqueous and vitreous humors, and plasma were assayed for Squalamine concentrations using a validated LC-MS/MS method with a lower limit of quantification (LLOQ) of 10ng/g of tissue. Ocular toxicity and irritation were also evaluated. Results: Squalamine eye drops, given QD or BID were well tolerated with no

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

adverse clinical effects. Given QD, mean concentrations of Squalamine in posterior sclera/choroid were 9.5, 21.9, and 39.8ng/g in the 1, 7, and 14 day groups, respectively. Given BID, mean concentrations of Squalamine in posterior sclera/choroid were 21.7, 62.6, and 68ng/g in the 1, 7, and 14 day groups, respectively. Values represent levels at one full dosing interval after last administration (QD 24(+2) hours, BID 12(+1) hours). Squalamine concentrations in aqueous and vitreous humors were <LLOQ in all animals and <LLOQ in plasma in 23/24 animals. Conclusions: Squalamine eye drops were well tolerated, consistent with previous longer term preclinical studies in which there were no adverse clinical findings or changes in ocular histopathology. Mean posterior segment tissue concentrations of Squalamine given QD and BID exceeded the threshold at which Squalamine is known to inhibit neovascularization in a cell-based model. Importantly, as evidenced by concentrations in posterior sclera/choroid above the anti-angiogenic threshold level, sustained therapeutically relevant posterior ocular exposure levels were maintained for the duration of a full dosing interval (QD 24h, BID 12h). Squalamine has prolonged residence time and slow tissue clearance when administered QD and BID up to 14 days. Minimal systemic uptake reduces potential systemic safety concerns. The absence of Squalamine concentrations in aqueous humor suggests a passive diffusion mechanism from anterior to posterior sclera and subsequently into the choroid. These results, consonant with previous preclinical topical data and intravenous clinical studies, warrant further clinical investigation of Squalamine eye drops to treat neovascular ophthalmic disorders.

Program Number: 459 Poster Board Number: D1136 Presentation Time: 8:30 AM - 10:15 AM Preparation And Characterization Of Fk-506 Micelles For Potential Application In The Treatment Of Uveoretinitis li xingyi, chen hao. Institute of Biomedical Engineering, wenzhou medical college, wenzhou, China. Purpose: To develop a novel injectable FK-506 micelles and the potential application in the treatment of uveoretinitis was primarily evaluated in rat model. Methods: Briefly, PEG-PCL diblock copolymer was prepared by ring-opening copolymerization of e-CL initiated by MPEG at 130 o C using Sn(Oct)2 as catalyst.To improve the water solubility of FK506, thin-film method was employed to develop a novel FK506 nanoformulation based on MPEG-PCL copolymer.Briefly, 0.2g of FK506 and 0.8g of MPEG-PCL copolymer were co- dissolved into 10ml acetone solution, and then evaporated by a rotary evaporation at 37 o C. Finally,the product was re-suspended into 40ml water solution at 55 o C to obtain FK506 nanoformulation. The obtained FK 506 nanoformulation was lyophilized and storage at 4 o C for further usage. In the rate model, rats were intravitreally injected with saline, FK506, FK506 nanoformulation and its efficiency on the inflammation was evaluated. Results: Due to the solubilization of MPEG-PCL co-polymer, the water solubility of FK506 was greatly improved,and the obtained FK506 nanoformulation could be freeze-dried into a power state while could re-dissolved into the water solution. As presented in Fig.1, the obtained FK506 nanoformulation were uniform particle size

Commercial Relationships:

Irach B. Taraporewala, Ohr Pharmaceutical (I, E,

(about 50nm) and monodisperse with no aggregation. Meanwhile, the preliminary

P); Michael J. Elman, Genentech (C), Ohr Pharmaceutical (C); Shalom Z.

study on the rate model revealed that the application of FK506 nanoformulation

Hirschman, Ohr Pharmaceutical (C); Samuel I. Backenroth, Ohr Pharmaceutical (I, E, P) Support: None

could significantly reduce the intraocular inflammation and markedly inhibite the development of uveoretinitis . Conclusions: In the presented paper, a novel FK 506 micelles was developed and

115 Drug Delivery I

 

its potential application in the treatment of uveoretinitis was investigated. The results suggested that the application of FK506 nanoformulation could significantly

Sunday, May 6, 2012, 8:30 AM - 10:15 AM

reduce the intraocular inflammation and markedly inhibite the development of

Hall B/C

Poster Session

uveoretinitis .

Program #/Board # Range: 458-509/D1135-D1186

Organizing Section: Physiology/Pharmacology

Program Number: 458 Poster Board Number: D1135 Presentation Time: 8:30 AM - 10:15 AM Molecularly Imprinted Hydrogels with Hyaluronic Acid for Ocular Drug Delivery

Giuliano Guidi, Andrea K. Weeks, Heather Sheardown. Chemical Engineering, McMaster University, Hamilton, ON, Canada. Purpose: The use of eye drops for ocular drug delivery while convenient is inefficient and poses a risk of systemic side effects. Contact lenses with a well- documented history of use in the eye represent a promising alternative vehicle. The potential of a contact lens delivery system for the prostaglandin analogue, timolol maleate was investigated and the effect of factors such as material polarity, molecular imprinting and HA were analyzed to further understand the drug- hydrogel interactions which govern the release kinetics. Methods: Two model lens materials were created based on combinations of dimethylacrylamide (DMAA), hydroxyethyl methacrylate (HEMA) and methacryloxypropyltris(trimethylsiloxy)silane (TRIS). The materials were prepared with and without the direct addition of 7.5 kDa hyaluronic acid at 0.1 wt% and timolol maleate at 0.3 wt% and cured under UV light. Molecular imprinting was accomplished and a subsequent uptake study was performed for two days using two solutions, one containing timolol in PBS at a concentration of 0.2mg/mL and another containing both timolol and hyaluronic acid in PBS at 0.2mg/mL. Release studies were then performed on the molecular imprinted materials using UV- spectroscopy to quantify release. Materials were also characterized by swelling and contact angles. Results: Release studies show that 90% of the release for both pHEMA/TRIS and DMAA/TRIS occurs within approximately 3 days and 2 days respectively. The pHEMA/TRIS materials show an average 25% increase in total drug release over the duration of the study in comparison to the DMAA/TRIS materials. The effect of imprinting yields a consistent trend with both timolol and HA imprinted materials independently showing increasing release and a compounded effect when materials are imprinted with both compounds simultaneously. The increase in uptake affinity associated with timolol imprinted materials is consistent with established literature, however, the effect of HA increasing drug release is a novel result. Conclusions: It is evident the polarity of monomers, the ratio of their contribution and the nature of the therapeutic are all determining factor in uptake and release for these systems. Hyaluronic acid imprinting appears to affect the uptake and release of therapeutics regardless of its similarity to the target compound and this

interaction requires further characterization. Commercial Relationships: Giuliano Guidi, None; Andrea K. Weeks, None; Heather Sheardown, None Support: NSERC 20/20 Ophthalmic Network

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology (PH) adverse clinical effects. Given QD,

Commercial Relationships: li xingyi, None; chen hao, None Support: Zhejiang Provincial Program for the Cultivation of High-level Innovative Health talents

Program Number: 460 Poster Board Number: D1137 Presentation Time: 8:30 AM - 10:15 AM Corticosteroid Delivery from a Punctum Plug in a Canine Model Over 21- Days Ankita Desai, Michael Bassett, Art Driscoll, Peter Jarrett, Amar Sawhney, Leslie Jost, Abbe Miller, Charles Blizzard. R&D, Ocular Therapeutix, Inc., Bedford, MA. Purpose: To examine the influence of aqueous solubility on the in vivo release rate of three corticosteroids from a hydrogel punctum plug in a canine model. Methods: Micronized prednisolone (Pred), prednisolone acetate (PA) and dexamethasone (Dex) having a water solubility of 223, 89 and 17 µg/mL, respectively were suspended in a multi-arm PEG solution and injected into small bore tubing prior to cross-linking. The steroid in hydrogel matrix was subsequently dried and cut into punctum plugs. The plugs were inserted into the inferior canaliculus of beagles and a subset was removed each week for photographic imaging and to analyze drug content following extraction. Percent drug release was plotted over time relative to content prior to insertion. Results: As shown in Figure 1, the steroids release in vivo from the plug at a rate

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

relative to their aqueous solubility. The most soluble Pred is > 95% released in 14 days, whereas the lesser soluble Dex and PA has released 54 and 41%, respectively over 21 days. Images of a steroid loaded plug demonstrating weekly drug release are seen in the bottom of Figure 1. Conclusions: Topical corticosteroids, such as Dex, Pred and PA are used to treat inflammation in a variety of ophthalmic conditions. Many therapies require multiple daily administrations (hourly for severe conditions) for a prolonged period making a single dose therapy a more convenient option that may help ensure patient compliance and better provide necessary treatment. Aside from drug potency and ocular penetration, aqueous solubility must be considered when developing an ophthalmic sustained drug delivery system, as it can greatly influence the in vivo release rate as demonstrated in the canine model. A more water soluble drug may be preferred if short term therapy is required, whereas a less soluble drug may be preferred if long term therapy is needed.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology (PH) relative to their aqueous solubility.

Commercial Relationships:

Ankita Desai, Ocular Therapeutix, Inc. (E);

Michael Bassett, Ocular Therapeutix, Inc. (E); Art Driscoll, Ocular Therapeutix, Inc. (E); Peter Jarrett, Ocular Therapeutix, Inc. (E); Amar Sawhney, Ocular Therapeutix, Inc. (E); Leslie Jost, Ocular Therapeutix, Inc. (E); Abbe Miller,

Ocular Thjerapeutix, Inc. (E); Charles Blizzard, Ocular Therapeutix, Inc. (E) Support: None

Program Number: 461 Poster Board Number: D1138 Presentation Time: 8:30 AM 10:15 AM Comparison Of Ocular Effects After Use Of Topical Eye Drops Versus Use Of The WhisperTM Topical Drug Applicator

Jacklyn H. Salmon 1 , Sydney Cartiff 1 , Skip Ballou 2 , Corey Ballou 2 , Brian C. Gilger 1 .

  • 1 Clinical Sciences, North Carolina State University, Raleigh, NC; 2 Corinthian Ophthalmic, Inc., Raleigh, NC. Purpose: To compare the mydriatic, mitotic, and intraocular pressure (IOP) effects of topical tropicamide and latanoprost when delivered as an eye drop versus delivery of the same medication via the WhisperTM device. Methods: Six adult, female beagles were used in this study. With at least 1 week washout between treatments, each dog received topical 1% tropicamide HCl (Bausch & Lomb Inc., Tampa, FL) or 0.005% latanoprost (Greenstone LLC, Peapack, NJ) delivered as a single eye drop (via the commercial bottle) or in a separate study, via a single application using the WhisperTM device (Corinthian Ophthalmic, Inc.) in the right eye while receiving balanced salt solution (BSS, Alcon Laboratories, Fort Worth, TX) in the left eye using the same application method. Pupil diameter (PD) (tropicamide and latanoprost) using a digital pupilometer (VIPTM 200, Neuroptics, Irvine, CA) and IOP (latanoprost only) using a TonoVet tonometer (Icare, Espoo, Finland) were measured in both eyes at - 1, 0, 0.5, 1, 2, 3, 4, 6, and 8 hours after topical application. Results: Eyes receiving topical tropicamide via eye drops or WhisperTM device had significantly greater PD (P<0.01) than those receiving BSS from 30 minutes to

  • 6 hours after application. Eyes receiving topical latanoprost via eye drops or

WhisperTM device had significantly smaller (P<0.0001) PD from 30 minutes to 8 hours after application and significantly lower (P<0.001) IOP from 1 to 6 hours after application compared to eyes receiving BSS. There were no significant

differences in PD in eyes treated with tropicamide by eye drops or by the WhisperTM device through 6 hours after treatment. Eyes treated with latanoprost via the WhisperTM device had significantly lower IOP 1 hour (P=0.049) after treatment compared to IOP of eye drop latanoprost. IOP was not significantly different in eyes treated with latanoprost by either method at times 2, 3, and 4 hours after treatment, but IOP was significantly lower (P=0.048) in eyes treated with latanoprost eye drops compared to the WhisperTM latanoprost at 6 and 8 hours after treatment. Conclusions: Drugs delivered via the WhisperTM device, an innovative approach to application of topical ocular medications, induce similar ocular effects compared to traditional eye drops when using 2 common ocular medications in the dog. Furthermore, the latanoprost IOP data suggests that the WhisperTM device may induce a more rapid onset of effect compared to the same drug administered via an eye drop. These results strongly support the further development of this innovative device for application of topical ocular medications. Commercial Relationships: Jacklyn H. Salmon, Corinthian Ophthalmic, Inc.; Sydney Cartiff, Corinthian Ophthalmic, Inc.; Skip Ballou, Corinthian Ophthalmic, Inc.; Corey Ballou, Corinthian Ophthalmic, Inc., Brian C. Gilger, Corinthian Ophthalmic, Inc. Support: None

Program Number: 462 Poster Board Number: D1139 Presentation Time: 8:30 AM - 10:15 AM Effect Of Melatonin On Prednisolone Eye Disposition In Cats Maria J. Del Sole 1 , Paula Schaiquevich 2 , Marcelo A. Aba 1 , Carlos E. Lanusse 1 , Laura Moreno 1 . 1 Fisiopatologia, Facultad Ciencias Veterinarias, UNCPBA, Tandil, Argentina; 2 Unit of Clinical Pharmacokinetics, CONICET-Hosp de Ped JP Garrahan, Ciudad de Buenos Aires, Argentina. Purpose: To characterize prednisolone (PRED) ocular and systemic pharmacokinetics in cats after oral administration and to study the effect of concomitant administration of melatonin (MEL) on PRED disposition. Methods: Six (6) castrated young physically and ophthalmologically healthy male European Short Hair cats were orally administrated with a single dose (10 mg) of PRED or a single dose of PRED (10 mg) and MEL (3 mg) in tablet. In anesthetized cats 2 mL of blood and 450 µL of aqueous humor (AH) were obtained from preplaced cephalic antebrachial intravenous catheters and from one eye by direct puncture, respectively, at: 0.25, 0.5, 1, 1.5, 2, 3, 4 and 5 h after administration. Plasma and AH samples were assayed for PRED by HPLC. A two-compartment model was used to simultaneously fit PRED plasma and aqueous concentration vs. time data using ADAPT 5. The estimated pharmacokinetic (PK) parameters included the absorption rate constant (ka), elimination rate constant from the central compartment (kc), intercompartment rate constants between plasma and aqueous humor (kpa), apparent volume of distribution of the central compartment (Vc/F). PK parameters were compared between groups (PRED vs. PRED+MEL) by means of Wilcoxon matched pairs test (p<0.05). Results: The model adequately fitted the data and the estimated median (interval) PK parameters are shown in Table 1. No significant difference was observed in the PK parameters when comparing between groups of treatments (p>0.05). Conclusions: These results indicated that MEL does not modify PRED systemic or ocular disposition in cats when both are administrated simultaneously. A possible synergic pharmacological effect may be account for different mechanisms of action, but not for pharmacokinetic synergism and will be further studied.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology (PH) relative to their aqueous solubility.

Commercial Relationships: Maria J. Del Sole, None; Paula Schaiquevich, None; Marcelo A. Aba, None; Carlos E. Lanusse, None; Laura Moreno, None Support: None

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Program Number: 463 Poster Board Number: D1140 Presentation Time: 8:30 AM - 10:15 AM Vitreous Humor Buffering Capacity Of Rabbit, Bovine, And Porcine

Mohannad Shawer, Martin J. Coffey, Eric Phillips. Bausch and Lomb, Rochester, NY. Purpose: To compare the vitreous buffering capacity of three species: rabbit, bovine, and porcine. This study examines the ability of the vitreous to accommodate formulations with wide ranges of pH and its effect on the local and whole vitreous pH, and clarity of the vitreous as result of such changes in its pH. Methods: The vitreous from three species (bovine, porcine, and rabbit) were used for pH titration. For each measurement, a 5-mL sample of vitreous was placed in a scintillation vial and stirred. After the initial pH stabilization, the pH was recorded and aliquots of either 0.1M HCl or 0.1M NaOH were added every minute for 30 minutes. Prior to the performing the titrations, vitreous samples were equilibrated with a 5% CO2 in air mixture and the pH was adjusted to 6.5 - 7.5. Equilibration with the 5% CO2 in air mixture was maintained during the HCl titrations, but not during the NaOH titrations because doing so was found to artificially increase the observed buffer capacity as the CO2 solubility increased at higher pH. Vitreous clarity was evaluated by optical density measurements at low pH after incubation with HCl aliquots for 20 minutes.

Results: Although the titration curves for the three species had some notable differences, all three species exhibited similar buffering capacity toward both acidic and basic titration. Titration toward both acidic and basic sides (over about 3 pH units) showed a buffer capacity of 6.3-8 mmol L-1 pH-1. Using published information about the composition of the vitreous in various species, we can identify which vitreous components are contributing to the observed buffer capacity. The clarity of the vitreous was found to diminish at low pH. The vitreous

maintained its clarity at pH 3 and 2, but significant turbidity was observed at pH 1. Conclusions: All three animal species are predicted to react similarly with regard to vitreous pH when injected with a non-neutral pH formulation. The observed buffer capacity for the vitreous in all three species examined here is in good agreement with what would be predicted based on published information on the vitreous composition.

Commercial Relationships:

Mohannad Shawer, Bausch and Lomb (E); Martin

J. Coffey, Bausch and Lomb (E); Eric Phillips, Bausch and Lomb (E)

Support: None

Program Number: 464 Poster Board Number: D1141 Presentation Time: 8:30 AM - 10:15 AM Transscleral Iontophoretic Delivery of a Macromolecule into the Rabbit Eye

John Higuchi 1 , Kongnara Papangkorn 1,2 , Sarah Molokhia 2 , Donald Mix 1 , Charlotte Butler 1 , Prasoona Karra 1 , Balbir Brar 1 , S. Kevin Li 1,3 , William I. Higuchi 1,2 . 1 Aciont Inc, Salt Lake City, UT; 2 University of Utah, Salt Lake City, UT; 3 University of Cincinnati, Cincinnati, OH. Purpose: To study the in vivo transscleral anodal iontophoresis (AI) delivery of Immunoglobulin G (IgG, MW~150 kDa and a surrogate for bevacizumab) and to evaluate key iontophoretic conditions (i.e., current, current density, and drug solution volume) on the amount and distribution of IgG delivered into the eye. Methods: AI was performed on New Zealand White rabbits using a Visulex system containing IgG (2.5%). There were 4 groups of rabbits (n=3 for each group) under 4 different conditions of AI. After 20 min of AI delivery, the rabbits were sacrificed and the eyes were enucleated. The eyes were dissected and the cornea, aqueous humor, vitreous, conjunctiva, sclera, choroid and retina analyzed for IgG content by ELISA. Results: At 4 mA (current density = 1.8 mA/cm 2 ), the total amount IgG delivered was 438 ± 63 and 364 ± 27 µg with 400 µL and 200 µL IgG solution volumes in Visulex system, respectively. At 8 mA (current density = 3.6 mA/cm 2 ), the total amount IgG delivered was 727 ± 38 and 457 ± 94 µg with 400 µL and 200 µL solution volumes, respectively. With regard to tissue distributions, at both current densities, most of the IgG was found in the conjunctiva and sclera at amounts roughly in proportion to their total amounts at the two current densities. There were significant amounts of IgG found in the deeper tissues, including the retina/choroid. It was interesting to find that the average amounts in the deeper tissues (e.g., retina/choroid and vitreous) were much higher at the higher current density relative to the total amounts (e.g., 8 vs 42 µg for the retina/choroid). Conclusions: This study might be the first to demonstrate successful noninvasive macromolecule delivery in vivo to the posterior eye tissues with the amounts of IgG delivered being of the same order of magnitude of that used in the intravitreal injection of Avastin. It was estimated that iontophoretic delivery of IgG is approximately 1000-fold superior over passive. The reproducibility was found to be quite satisfactory (5-20%). The volume of drug solution used as well as current density appears to be significant in AI drug delivery efficiency for both amounts

and depth of penetration.

Commercial Relationships:

John Higuchi, Aciont Inc (E); Kongnara

Papangkorn, Aciont Inc (E); Sarah Molokhia, Aciont Inc (C); Donald Mix, Aciont Inc (E); Charlotte Butler, Aciont Inc (E); Prasoona Karra, Aciont Inc (E); Balbir Brar, Aciont Inc (E); S. Kevin Li, Aciont Inc (C); William I. Higuchi, Aciont Inc (E)

Support: 1R43EY020791-01

Program Number: 465 Poster Board Number: D1142 Presentation Time: 8:30 AM - 10:15 AM Evaluation of a Candidate Cell Line Employed to Deliver an Antiangiogenic Factor Using Encapsulated Cell Technology

Cahil McGovern 1A , Sandy Sherman 1A , Crystal Cortellessa 1A , Suzanna Borges 1A , Melissa Stiles 1B , Karen Ahola 1A , Alice Lee 1B , Konrad Kauper 1C , Bruce Bouchard 1D , Weng Tao 1E . A Process Development, B Quality Control, C Engineering, D Histology, E Reasearch and Development, 1 Neurotech USA, Lincoln, RI. Purpose: To investigate the cell stability and in vitro performance of Encapsulated Cell Technology (ECT) devices using a cell line to deliver an antiangiogenic factor. Methods: The cell line secreting the antiangiogenic factor was constructed using NTC-200 cells. The candidate cell line was cultured for 40 passages. At predetermined passages, cells were seeded at a defined density in 24 well plates and allowed to attach. Cells were washed twice with a balanced salt solution and incubated with 1 ml of Endo-SFM for 2 hours. The pulsate was then analyzed for antiangiogenic factor release. Once cell stability had been established out to 40 passages, the cell line was encapsulated in a hollow fiber membrane. Each device

was manufactured using standard protocols and held at 37°C in closed packages containing 37 mls of Endo-SFM. ECT devices were pulsed for factor production in 1 ml of Endo-SFM for 24 hours at days 2, 7, and 14 post manufacture. The devices were subsequently analyzed for metabolic activity and then subjected to either total DNA or histological analysis. Unencapsulated cells and device performance were quantified by Elisa. Device metabolic activity was determined using the CCK-8 assay (Dojindo). Total DNA was determined using the Hoefer DyNA Quant 200 fluorometer (Pharmacia). Histological examination of the devices was performed using standard hematoxylin and eosin staining techniques. Results: Cell stability: The candidate cell line released the desired factor for 40 passages which is favorable for manufacturing purposes. During this time, the cells maintained a typical and consistent morphology as well as exhibited a consistent doubling rate. Device stability: The ECT devices released the desired factors and maintained viable cells during the evaluation period. Total DNA analysis of devices showed a consistent number of cells was maintained between 1 and 2 weeks and histological analysis of device sections showed a high density of healthy cells distributed throughout the device. Conclusions: The data suggested that the Encapsulated Cell Technology platform was able to achieve sustained delivery of antiangiogenic factors under in vitro conditions. This technology can deliver other factors for a broad range of indications where long-term treatment is required.

Commercial Relationships:

Cahil McGovern, Neurotech (E); Sandy Sherman,

Neurotech (E); Crystal Cortellessa, Neurotech (E); Suzanna Borges, Neurotech (E); Melissa Stiles, Neurotech (E); Karen Ahola, Neurotech (E); Alice Lee, Neurotech (E); Konrad Kauper, Neurotech (E); Bruce Bouchard, Neurotech (E); Weng Tao, Neurotech (E) Support: None

Program Number: 466 Poster Board Number: D1143 Presentation Time: 8:30 AM - 10:15 AM

Rediscovering An “Old Drug”: Topical Application Of Acetazolamide Using A

Ternary Complex With Hp-ß-cd And Tea

Luis I. Tartara, Santiago D. Palma, Daniela A. Quinteros, Marcela R. Longhi,

Daniel A. Allemandi, Gladys E. Granero. Departamento de Farmacia, Universidad Nacional de Cordoba, Cordoba, Argentina. Purpose: In order to enhance the ocular bioavailability of acetazolamide (ACZ), a novel liquid formulation based on a multicomponent complex with hydroxypropyl- ß-cyclodextrin (HP-ß-CD) and triethanolamine (TEA) was prepared. In vitro e In vivo performance of this formulation was assayed in rabbits. Methods: The background of the design of this formulation was the interaction between the components of the ternary complex. 1 H- and 13 C- NMR experiments were undertaken to verify the real inclusion of ACZ in the ACZ-HP-ß-CD-TEA complex. The biopharmaceutical performance of the formulation was evaluated by mean of In vitro corneal permeation and the In vivo effect on the intraocular pressure (IOP). The marketed ophthalmic solution AZOPT® (1% w/v brinzolamide) was also included in the assays for comparison. Results: The ternary system ACZ-HP-ß-CD-TEA seemed to be able to reduce IOP in about 30% after two hours. This effect was sustained for four hours after instillation. In vitro corneal permeation studies demonstrated that the ACZ permeation was increased as consequence of the multicomponent complex formation. RMN experiments indicated that TEA can weaken the association between ACZ and HP-ß-CD increasing the drug ocular hypotensive effect by increasing rate and extent of drug dissolution; due to the relative stability of the ternary ACZ-HP-ß-CD-TEA system. All formulations, including the commercial product, were considered practically non-irritant. Conclusions: These results indicate that this new strategy for ACZ formulation could improve the treatment of IOP. The new formulation thus obtained was to be

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

able to facilitate ACZ corneal permeation and showed appreciable pharmacological activity. Commercial Relationships: Luis I. Tartara, None; Santiago D. Palma, None; Daniela A. Quinteros, None; Marcela R. Longhi, None; Daniel A. Allemandi, None; Gladys E. Granero, None Support: FONCYT PICT 2010-0380

Program Number: 467 Poster Board Number: D1144 Presentation Time: 8:30 AM - 10:15 AM Moxifloxacin: A Valuable New Addition To Chemotherapeutic Armamentarium For The Treatment Of Retinoblstoma Megha Barot, Mitan R. Gokulgandhi, Dhananjay Pal, Ashim K. Mitra. Pharmaceutical Science, University of Missouri, Kansas City, Kansas City, MO. Purpose: Multidrug resistance (MDR) proteins (P-gp and MRPs) mediated chemoresistance have been considered as a major cause of treatment failure in the management of retinoblastoma (RB) with systemic chemotherapy. Here, we have examined an interaction of fluoroquinolone moxifloxacin (MFX) with three anticancer drugs (Topotecan, Etoposide, Vinblastine) for the treatment of RB. We hypothesized that in such interactions, MFX will not only modulate the bioavailability of anticancer agent at the ocular site such as retina (due to competitive inhibition at efflux sites) but it will also synergize antiproliferative activity of anticancer agent. Methods: Time dependent uptake and transport of three anticancer drugs was performed in presence of MFX across model cell lines (MDCK-MDR1 and MDCK-MRP2). Modulation of time dependent cell cytotoxicity and caspase-3 enzyme activity of anticancer drugs in presence of MFX was evaluated using retinoblastoma (Y-79) cells. IC-50 value of anticancer drugs alone and in presence of MFX was also determined. Results: 2-2.5 fold increased uptake of all three anticancer drugs was observed in presence of MFX across MDCK-MDR1 and MDCK-MRP2 cells suggesting MFX mediated evasion of efflux pumps. Significant reduction in efflux ratio (B-A/A-B permeability) of three anticancer drugs was also observed in presence of MFX indicating MFX mediated improved transport. Following cytotoxicity study, tenfold reduction in IC-50 value of Topotecan and Etoposide and twofold reduction in IC-50 value of Vinblastine was observed in combination with MFX. Significant enhancement in caspase-3 enzyme activity of three anticancer drugs was observed in combination with MFX on Y-79 cells. Conclusions: Strategy of utilizing efflux pump inhibitors (which is yet not clinically approved) for improving ocular bioavailability of anticancer agent has its own limitations in terms of little or no improvement in toxicity of chemotoxic agents. However, ocular cells have shown good tolerability against MFX, which is a clinically well accepted drug even at higher dose level. Our results suggest that MFX may be a valuable new addition to chemotherapeutic armamentarium, concurrently improving cytotoxic activity while evading MDR mediated chemoresistance of various anticancer drugs currently used for the treatment of RB. These novel drug interactions will provide dual benefit in terms of overcoming chemoresistance and synergistic cytotoxic effect will help reducing chemotherapeutic dose which eventually reduces probability of dose-limiting toxicity. Commercial Relationships: Megha Barot, None; Mitan R. Gokulgandhi, None; Dhananjay Pal, None; Ashim K. Mitra, None Support: NIH grants RO1 EY 09171-16 and RO1 EY 10659-14

Program Number: 468 Poster Board Number: D1145 Presentation Time: 8:30 AM - 10:15 AM Lc-ms/ms Quantification Of Melphalan Plasma Levels In Children Undergoing Selective Intra-arterial Infusion Of Chemotherapy For Retinoblastoma

Jonathan W. Kim, Ludmila Alexandrova, Emilia DeMarchis, Diana Lee, Allis

Chien. Ophthalmology, Stanford University, Palo Alto, CA. Purpose: Melphalan is an alkylating agent with effective tumoricidal properties but also severe systemic side effects. A recent clinical trial has demonstrated promising results in retinoblastoma patients when melphalan is infused selectively into the ophthalmic artery. A minority of subjects developed neutropenia, which suggests that systemic diffusion of the drug does occur. Developing a reliable systemic assay to determine plasma levels of melphalan following ophthalmic artery infusion is critical in optimizing the benefits of this treatment. Methods: We utilized high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) to determine the melphalan levels in human plasma. Melphalan and the internal standard (N-phenyldiethanolamine (N-PEA)) were purchased from Sigma (Steinhein, Germany) and drug-free normal human plasma was obtained from a regional blood bank. Stock solutions of Melphalan 2 mg/mL solution in dimethyl sulfoxide (DMSO) and the internal standard (IS) 1 mg/mL in ethanol were both stored at -80°C. Two concentration ranges (range 1: 2 - 400 ng/mL and range 2: 20 - 4000 ng/mL) were tested in order to develop a calibration curve. The extraction procedure for the concentration ranges 1 and 2 utilized 100 uL and 50 uL plasma respectively. Blank human plasma was spiked with

appropriate calibration solutions of melphalan and internal standard. A methanol/acetonitrile protein precipitation was performed and the samples were analyzed with an LC-MS/MS assay. Results: Mass chromatograms for the range 1 provided eight calibration points:

2ng/mL; 4; 10; 20; 50; 100; 200; 400 ng/mL. A calculated ratio of the peak intensities for both melphalan and the internal standard were then used to generate a calibration curve. Similarly, mass chromatograms for range 2 generated eight calibration points: 20 ng/mL; 40; 80; 200; 400; 1000; 2000; 4000 ng/mL. A calculated ratio of the peak intensities for both melphalan and the internal standard were then used to generate a second calibration curve (figure 2). Weighting of 1/x 2 was used to fit the data to a linear least-squared regression curve, where x represents concentration (ng/mL). The linear detection response was defined for concentrations within the range of 2 to 400 ng/mL and within the range of 20 to 4000 ng/mL. Conclusions: A LC-MS/MS method for determination of melphalan levels in human plasma has been developed, and calibration curves for range 1 and range 2 were generated by using a 100µL plasma aliquot and a 50µL plasma aliquot, respectively. Ongoing aspects of the project include assay validation to demonstrate accuracy, reproducibility and stability of melphalan in human plasma. Commercial Relationships: Jonathan W. Kim, None; Ludmila Alexandrova, None; Emilia DeMarchis, None; Diana Lee, None; Allis Chien, None Support: None

Program Number: 469 Poster Board Number: D1146 Presentation Time: 8:30 AM - 10:15 AM 3D Computational Fluid Dynamic Model Comparing Rabbit and Human Intravitreal Pharmacokinetics Using Fluorescein Dyes Julie E. Whitcomb 1 , Susan S. Lee 1 , Marc Horner 2 , Mohammad R. Kazemi 3 , Michael R. Robinson 1A , Jie Shen 1 . A Ophthalmology, 1 Allergan, Irvine, CA; 2 ANSYS, Evanston, IL; 3 ANSYS, San Jose, CA. Purpose: Ocular pharmacokinetic experiments are commonly performed in rabbits to predict the intraocular distribution of drug in humans. However, results may not translate directly to humans because of anatomical and physiological differences. Computational fluid dynamic (CFD) models were developed in an effort to discern the intravitreal drug distribution differences between the two species. Methods: 3D half-globe symmetric CFD models of rabbit and human eyes were developed to simulate diffusion and convection of drug in the vitreous. The geometries were constructed using SpaceClaim Direct Modeler and anatomical dimensions were based on average values for each species. ANSYS Fluent was used to simulate the flow of vitreous humor and drug delivery from a centrally- placed bolus of fluorescent dye. Fluorescein and fluorescein glucuronide were selected as model compounds based on available experimental data from Araie and Maurice, 1991. Results: Anatomical differences, such as a larger lens and smaller vitreous volume in the rabbit, altered the aqueous humor flow in the rabbit relative to the human. The average drug concentration in the rabbit eye was found to be higher than the human due to the smaller vitreous volume. Conclusions: While the rabbit is an excellent animal model for studying ocular pharmacokinetics, anatomical and physiological differences should be considered when extrapolating rabbit data to human. CFD modeling can be effective to use rabbit ocular pharmacokinetic data to simulate human drug distribution.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology (PH) able to facilitate ACZ corneal

Commercial Relationships:

Julie E. Whitcomb, Allergan (E); Susan S. Lee,

Allergan (E); Marc Horner, ANSYS (E); Mohammad R. Kazemi, ANSYS (E);

Michael R. Robinson, Allergan (E); Jie Shen, Allergan (E) Support: None

Program Number: 470 Poster Board Number: D1147 Presentation Time: 8:30 AM - 10:15 AM Protective Effects of Transscleral Drug Delivery Device Against Light-induced Retinal Damage in Rats Nobuhiro Nagai 1A , Hideyuki Onami 1A , Hirokazu Kaji 1B , Takuya Yamada 1B , Yuki Katsukura 1A , Machiko Sato 1A , Yumi Ishikawa 1A , Toru Nakazawa 1A , Matsuhiko

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Nishizawa 1B , Toshiaki Abe 1A . A Graduate School of Medicine, B Graduate School of Engineering, 1 Tohoku University, Sendai, Japan. Purpose: To evaluate the protective effects of a transscleral drug delivery device that can release geranylgeranylacetone (GGA) in a controlled release manner against light-induced retinal damage in rats. Methods: The device consists of a reservoir, controlled-release cover, and drug formulations, which were made of photopolymeized poly(ethyleneglycol) dimethacrylate that partially contains tri(ethyleneglycol) dimethacrylate. These parts were fabricated via a microfabrication technique that used an AutoCAD design. GGA, a heat shock protein (HSP) inducer, was loaded in the device. High- performance liquid chromatography was used to evaluate the release amount of GGA. After the devices were placed onto the sclera of rat eyes, HSP inductions of retinal tissues were evaluated by real-time RT-PCR and western blot analyses. Flash electroretinograms were recorded 4 days after white light exposure (8000 lux for 18h). Histological examinations were perfomred to evaluate the thickness of the outer nuclear layer. Results: GGA was released with zero-ordered kinetics from the device. One or four weeks after implanation, gene and protein expression of HSP70 were upregulated in the sclera-choroid-retinal pigment epithelium fraction of the eyes treated with GGA-loaded devices compared with those treated with saline-loaded devices or non-treated rats. Electroretinographic amplitudes of the a- and b-waves increased significantly in rats treated with GGA-loaded devices compared with saline-loaded devices. The outer nuclear layer thickness was thinned in the group treated with saline-loaded devices, but the group treated with GGA-loaded devices suppressed the photic damage. Conclusions: Transscleral GGA delivery device protected against light-induced retinal damage in rats. The device may offer a less-invasive method of drug delivery to achieve sustained release of medications for intravitreal drug delivery and the treatment of various retinal diseases. Commercial Relationships: Nobuhiro Nagai, None; Hideyuki Onami, None; Hirokazu Kaji, None; Takuya Yamada, None; Yuki Katsukura, None; Machiko Sato, None; Yumi Ishikawa, None; Toru Nakazawa, None; Matsuhiko Nishizawa, None; Toshiaki Abe, None Support: Grant-in-Aid for Young Scientists (A) from the MEXT, Japan, Health Labour Sciences Research Grant from the MHLW, Japan

Program Number: 471 Poster Board Number: D1148 Presentation Time: 8:30 AM - 10:15 AM Delivery Of Dexamethasone To The Posterior Segment Of The Eye Using An Eye Gel Formulation

Vincenzo Papa 1A , Elena Solfato 1B , Claudine Civiale 1B . A Medical Marketing-BU Pharma, B Pharma Tech & Anal Chem-BU Pharma, 1 SIFI SPA, Lavinaio, Catania, Italy. Purpose: Corticosteroids are valuable drugs in the management of macular edema (ME) but therapeutic concentrations in the retina can be usually achieved only after invasive injections, making the use of topical corticosteroids not suitable for the treatment of ME. We describe herein the PK results obtained in rabbits after topical administration of a gel-based delivery system containing 1% xanthan gum and 0.15% dexamethasone phosphate (DP), a water soluble prodrug of dexamethasone (D). Methods: A single dose of 50 µl of 0.15% DP gel (Etacortilen Gel) was applied to one eye of pigmented rabbits (n=24). D levels were measured by LC/MS-MS in aqueous humour (AH), vitreous humour (VH) and retina/choroid (R/C) of both

eyes after 15, 30, 60, 120, 180 and 240 min. C MAX and AUC ALL were calculated for each tissue. Data obtained from control eyes were considered expression of systemic absorption. Results: C MAX (mean ±SE): 83.72 ± 10.64 (study eye) vs. 0 (control eye) ng/ml in the AH, 1.76 ± 0.59 vs. 4.24 ± 1.89 ng/g in the VH and 67.79 ± 22.73 vs. 39.98 ± 9.97 ng/g in the R/C. AUC ALL (mean ±SE): 178.92 ± 17.08 (study eye) vs. 0 (control eye) hr*ng/ml in the AH, 1.88 ± 0.38 vs. 6.19 ± 1.25 hr*ng/g in the VH and 129.92 ± 16.81 vs. 77.14± 9.02 hr*ng/g in the R/C. In the R/C the AUC ALL in study eyes was statistically significant higher (p<0.01, Student t test) than that in control eyes. Conclusions: A single topical administration of DP gel is able to deliver effective concentrations of D to the posterior segment of the eye through both systemic and topical absorption. Topical absorption across the conjunctiva and the sclera accounted for about 40% of the D reaching the posterior part of the eye. Since D levels found in the R/C are sufficient to reduce central macular thickness in patients with diabetic ME (Tanito IOVS 2011), this eye gel formulation may be useful as adjuvant topical treatment of ME, obviating any concerns associated with more invasive routes.

Commercial Relationships:

Vincenzo Papa, SIFI SpA (E); Elena Solfato, SIFI

SpA (E); Claudine Civiale, SIFI SpA (E) Support: None

Program Number: 472 Poster Board Number: D1149 Presentation Time: 8:30 AM - 10:15 AM

SMVT Targeted Lipid Prodrug Of Cidofovir: Novel Treatment Strategy For CMV Retinitis

Mitan R. Gokulgandhi, Megha Barot, Mahuya Bagui, Dhananjay Pal, Ashim K.

Mitra. Pharmaceutical Science, University of Missouri Kansas City, Kansas City, MO. Purpose: Cidofovir (CDF) has shown potential antiviral activity and currently indicated for the treatment of AIDS-related cytomegalovirus (CMV) retinitis. The major drawback with CDF is its low bioavailability (attributable to low lipid solubility) and hence poor passive transport into virus infected tissue such as retina which leads to limited therapeutic efficacy. Therefore, with a strategy to simultaneously enhancing lipid mediated and transporter targeted cellular uptake of CDF, we have evaluated novel transporter targeted lipid prodrugs of CDF for the treatment of CMV retinitis. Methods: We have successfully synthesized sodium dependent multivitamin transporter (SMVT/Biotin) targeted lipid prodrugs with lipid moiety of carbon chain length C6 or C12 as a linker between biotin (B) and CDF (B-C6-CDF and B- C12-CDF). All synthesized prodrugs were characterized and evaluated for their physicochemical properties and cytotoxicity. We have also performed in vitro uptake and transport of [3H]Biotin in presence of these prodrugs on a model cell line MDCK-MDR1 and human retinal cell line ARPE-19. To elucidate the affinity

interaction of these prodrugs with SMVT transporter, uptake of [3H]Biotin in presence of different concentrations of prodrugs has been performed and IC50 value of individual prodrugs has been calculated. Results: Inhibition of [3H]Biotin uptake in presence of all prodrugs shows strong interaction with SMVT transporter. B-C12-CDF (0.90±0.07µM, IC50) shows higher affinity towards SMVT transporter than B-C6-CDF (11.25±1.48 µM, IC50) and B-CDF (31.42± 3.05µM, IC50) suggesting that increasing in lipid chain length will improve cellular uptake via SMVT transporter. Cellular uptake of B-C12-CDF (18 fold), B-C6-CDF (5 fold) and B-CDF (3.5 fold) prodrugs was found to be higher relative to CDF, shows our novel conjugates have higher cellular accumulation in retina. Conclusions: Above studies demonstrated that transporter targeted lipid prodrugs of CDF pose superior affinity for SMVT transporter and hence will have higher bioavailability into human retinal cells owing to higher expression of SMVT on the human retina. This strategy will augment antiviral and therapeutic efficacy of CDF into retina due to synergistic effect of lipid and transporter targeting moiety. Finally, these novel prodrugs appear to be potential clinical candidates for the treatment of CMV retinitis. Commercial Relationships: Mitan R. Gokulgandhi, None; Megha Barot, None; Mahuya Bagui, None; Dhananjay Pal, None; Ashim K. Mitra, None Support: NIH grants RO1 EY 09171-16 and RO1 EY 10659-14

Program Number: 473 Poster Board Number: D1150 Presentation Time: 8:30 AM - 10:15 AM Three Methods of Topical Lidocaine-Based Anesthesia for Intravitreal Injections

Anthony F. Kokx, Gary J. Miller. Ophthalmology, West Virginia University, Morgantown, WV. Purpose: The purpose of this study is to compare 3 different methods of topical anesthesia for intravitreal injections. This study will compare effectiveness of each method at achieving pre-injection anesthesia and each method's cost effectiveness. Methods: Patients already requiring an intravitreal (0.05mL) injection with either Bevacizumab or Ranibizumab were enrolled in this study. Three different methods of topical anesthesia were utilized; 3.5% lidocaine gel (LG), 4% lidocaine soaked pledget (LP), and 4% lidocaine soaked cotton tipped applicator (CTA). Twenty- four patients were enrolled in the study (power of 80%), with all patients scheduled to receive each treatment arm. After each injection, a pain survey (100 point Visual Analog Scale) was administered, recording pain associated with both numbing and injection. The following day, the patients used the same scale to retrospectively rate their pain from the day of the injection as well as rate their residual pain (RP). Patients had no reference to the prior survey. Results: The average number of injections prior to enrollment in the study was 4.7. Average post-injection IOP was 30mm Hg. Subconjunctival hemorrhage (SCH) was noted in 1 out of 7 patients. The average pain score for each patient (all methods same day) associated with the numbing method was 6.5/100 (n=7); for the injection was 5.43/100 (n=7). The average pain score for each patient (all methods next day) associated with the numbing method was 6.8/100 (n=6); for the injection was 8.4/100 (n=6). The pain score in the LP group (n=5) was 5.8/100 for numbing and 3.5/100 injection (same day). The pain score in the LP group was 5.3/100 for numbing and 7.9/100 for injection (next day). The RP was 1.0/100. There was one SCH in this group that did not have higher retrospective or residual pain scores. The pain score in the LG group (n=1) was 0 for both numbing and injection (same

and next day) without any RP. The pain score in the CTA group (n=1) was 16.5/100 for numbing and 18.5/100 injection (same day). The pain score in the CTA group was 7.5/100 for numbing and 6.0/100 injection (next day). RP was 1.5/100. There were no cases of endophthalmitis. The average unit cost for each method is as follows: LG $1.13 per injection; LP $0.80 per injection; CTA $0.24 per injection. Average staffing time for each numbing protocol; LG group - 10

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

seconds (for technician); LP group - 30 seconds (for technician); CTA group - 60 seconds (for physician). Data collection will finish March 2012. Conclusions: All three anesthetic methods appear to be capable of achieving an effective level of anesthesia. Currently, there is not a link between SCH and higher RP or retrospective pain scores. There is a range of cost and time of staffing required with each method. Commercial Relationships: Anthony F. Kokx, None; Gary J. Miller, None Support: WVU Research to Prevent Blindness

Program Number: 474 Poster Board Number: D1151 Presentation Time: 8:30 AM - 10:15 AM Pharmacokinetics of a VEGF Antagonist Delivered by an Intraocular

Encapsulated Cell Technology Implant Michael Rivera 1A , Alline Lelis 1A , Bruce Bouchard 1A , Melissa Stiles 1A , Vincent Ling 1A , Konrad Kauper 1B , Weng Tao 1 . A Development, B Core Technology Development, 1 Neurotech USA, Lincoln, RI. Purpose: Agents that neutralize VEGF isoforms have been shown to be effective for treating the wet form of Age-Related Macular Degeneration (AMD), a leading

cause of blindness. Neurotech’s encapsulated cell technology (ECT) platform

utilizes genetically engineered cells to secrete therapeutic proteins from an implantable hollow fiber capsule. The purpose of the current study was to investigate the pharmacokinetics (PK) of a VEGF antagonist delivered by an intraocular ECT implant in rabbits over a 3 months implantation period. Additionally, an arm evaluating the PK of Bevacizumab injection was included for comparison. Methods: ECT implants were manufactured using polymer membrane encapsulated cells genetically engineered to continuously secrete a VEGF antagonist molecule, designated NT-503. Standard-of-care injections of 1.25 mg Bevacizumab were given at Day 0 and Day 42. Plasma and vitreous were harvested from 4 eyes/two animals per time point following implantation or injection. Implant secretion rates, vitreous and serum concentrations of the VEGF antagonist were quantified by ELISA. Samples were collected at days 1, 3, 7, 14, 28, 40, 56 and 84 for both ECT implanted and injected eyes. Results: The NT-503 implants have demonstrated stable expression of the VEGF antagonist molecule through the time period evaluated. Data suggests that the levels of VEGF antagonist are delivered at up to 5 µg/day, maintaining a vitreous

steady state concentration of approximately 25 µg/eye. Bevacizumab injections have been shown to maintain vitreous steady state concentrations of > 5 µg/mL for up to 30 days. Conclusions: Pharmacokinetics data demonstrates that ECT delivery of a VEGF antagonist can maintain microgram per eye concentrations over an extended period and the steady state level established by the ECT implant exceeds the maintenance dose established by Bevacizumab monthly intraocular injections.

Commercial Relationships:

Michael Rivera, Neurotech USA (E); Alline Lelis,

Neurotech USA (E); Bruce Bouchard, Neurotech USA (E); Melissa Stiles, Neurotech USA (E); Vincent Ling, Neurotech USA (E); Konrad Kauper, Neurotech USA (E); Weng Tao, Neurotech USA (E) Support: None

Program Number: 475 Poster Board Number: D1152 Presentation Time: 8:30 AM - 10:15 AM

Suppression of Rat Choroidal Neovascularization by Transscleral Vasohibin-1 Delivery Device Hideyuki Onami 1A , Nobuhiro Nagai 1A , Ryosuke Wakusawa 1A , Hirokazu Kaji 1B , Takuya Yamada 1B , Yumi Ishikawa 1A , Matauhiko Nishizawa 1B , Yasufumi Sato 1A , Toru Nakazawa 1A , Toshiaki Abe 1A . A Graduate school of medicine, B Graduate school of engineering, 1 Tohoku univercity, Sendai, Japan. Purpose: To evaluate the effects of transscleral sustained vasohibin-1(VASH) delivery for rat laser-induced choroidal neovascularization (CNV) by a novel drug delivery device. Methods: Transscleral VASH delivery device (VASH-DD) consists of a drug reservoir covered with a controlled-release membrane. The controlled release membrane is made of photopolymerised polyethylene glycol dimethacrylate (PEGDM) that contains interconnected collagen microparticle. VASH is pelletised with PEGDM and loaded in the reservoir. The amount of released VASH from VASH-DD was measured by ELISA. The release was also confirmed in vivo by immunostaining 2 weeks after the device transplantation. Rat laser-induced CNV model was used to evaluate the effect of VASH-DD. Fluorescein angiography and choroidal flat mounts were used to evaluate the CNV. The results of not only

transplantation different amount of VASH loaded device (0, 1, and 10μM, respectively) and just pelletised VASH (VASH-pellet) but also VASH and vehicle itravitreal injection were compared each other. Results: The sustained release of VASH from VASH-DD was confirmed by ELISA. VASH was detected in the retina, especially RPE and ganglion cells aroud the transplanted region and optic nerve. Statisitically significant less fluorescein leakage was observed in eyes with VASH-DD than eyes with 0 μM VASH-DD 2

weeks after transplantation (p=0.038). Statistically significant less CNV size was also observed in eyes transplanted with VASH-DD or intravitreal VASH injection than those of others by choroidal flat mounts. Conclusions: Our device showed sustained protein release and might offer a less- invasive method than those previously reported for treatment, such as age-related macular degenelation. Commercial Relationships: Hideyuki Onami, None; Nobuhiro Nagai, None; Ryosuke Wakusawa, None; Hirokazu Kaji, None; Takuya Yamada, None; Yumi Ishikawa, None; Matauhiko Nishizawa, None; Yasufumi Sato, None; Toru Nakazawa, None; Toshiaki Abe, None Support: Health Labour Sciences Research Grant from the MHLW,Japan

Program Number: 476 Poster Board Number: D1153 Presentation Time: 8:30 AM - 10:15 AM

Polyesteramide Microparticles For Ophthalmic Drug Delivery Vanessa Andres-Guerrero 1A , Beatriz de las Heras 1B , George Mihov 2 , Aylvin Dias 2 , Rocío Herrero-Vanrell 1A . A Pharmaceutical Technology, B Pharmacology, 1 Faculty

of Pharmacy/Complutense University, Madrid, Spain;

2

DSM Ahead & DSM

Biomedical, Geleen, The Netherlands. Purpose: Polyesteramides (PEAs) are a new family of polymeric materials. PEAs combine good mechanical, thermal and processing properties and are also biodegradable. The purpose of the current study was to evaluate PEA-II microparticles to be used as carriers for controlled drug delivery in the eye. Methods: Microparticles (MPs) were prepared using an emulsion-solvent evaporation technique. Particle size and morphology of MPs were characterized by dynamic light scattering and scanning electron microscopy (SEM), respectively. To study the in vitro degradation behavior, MPs were incubated in a phosphate buffered solution isotonized with NaCl (PBS, pH 7.4, 37ºC) at a constant agitation speed of 100 rpm. At different time points (1 hour, 24 hours, 48 hours and 5 days) MPs morphology was studied by SEM. In vitro tolerance studies were performed by the MTT technique in human corneal limbal epithelial cells and macrophage cells. Cells were exposed to MPs suspensions (5mg and 10mg MPs/ml in PBS) for 15 minutes (short term exposure), 1 hour and 4 hours (long term exposure). Dexamethasone (DX) was used as a lipophilic drug model to determine the encapsulation efficiency of PEA-II MPs (0.5:10 DX:PEAII). Results: Unloaded PEA-II MPs were spherical and had smooth surface. MPs size ranged between 10-30µm (mean particle size 21.3±0.2 µm). MPs started to lose their shape after being incubated in PBS for 1 hour. 5 days later, MPS had turned into an unshaped depot of polymer. The cytotoxicity assays demonstrated good tolerance in the two cell lines in all cases after short- and long-term exposures (cell viability>90%) at the assayed concentrations. DX-loaded MPs (9.62±0.56 µg DX/mg MPs and mean particle size 21.3±1.8 µm) did not show any morphological difference with unloaded MPs. Conclusions: Biodegradable PEA-II MPs are potentially useful to develop new controlled drug delivery systems for treating ophthalmic diseases. Commercial Relationships: Vanessa Andres-Guerrero, None; Beatriz de las Heras, None; George Mihov, DSM Biomedical (E); Aylvin Dias, DSM Biomedical (E); Rocío Herrero-Vanrell, None Support: PANOPTES (project number 246180) under the 7th Research Framework Programme of the European Union and Spanish Ministry of Health (RETICS RD07/0062).

Program Number: 477 Poster Board Number: D1154 Presentation Time: 8:30 AM - 10:15 AM Sustained Back Of The Eye Delivery Following Sub-tenon Administration Of

Dexamethasone-loaded PLGA Microspheres In Rabbits Rocio Herrero-Vanrell 1 , Deyanira Barbosa-Alfaro 1 , Irene Bravo-Osuna 1 , RS Kadam 2 , I. Fernandez-Bueno 3 , MT García-Gutierrez 3 , Jose-Carlos Pastor 3 , Uday B. Kompella 2 , Irene Teresa Molina Martínez 1 . 1 Pharmaceutical Techn Sch of Pharm, Complutense University, Madrid, Spain; 2 Pharmaceutical Sci & Ophthal, University of Colorado Denver, Aurora, CO; 3 IOBA-Campus Miguel Delibes, University of Valladolid, Valladolid, Spain. Purpose: Quantification and pharmacokinetics evaluation of dexamethasone in ocular tissues after sub-tenon administration of dexamethasone-loaded PLGA microspheres in rabbits. Dexamethasone is widely used in ophthalmology to treat several ocular diseases affecting the posterior segment. Due to short half-live of dexamethasone, repeated intraocular injections are employed in the treatment of most of these diseases. Controlled drug delivery systems, such as as microspheres (MPh) are being designed to avoid frequent injections. MPhs based on a biodegradable polymer (PLGA) have been developed to release dexamethasone in vitro for 65 days. MPhs can be periocularly administered as a conventional injection with less risk of adverse effects than intravitreal administration.

Methods: Sterilized dexamethasone-loaded PLGA microspheres (5 mg of microspheres, dose: 828 µg of Dexamethasone) were administered by sub-tenon injection to rabbits. Dexamethasone concentrations in rabbit ocular tissues (choroid-RPE, retina and vitreous) were evaluated at different time points (1, 2, 4 and 6 weeks after injection). Samples were treated and the drug was quantified by

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

means of electrospray ionization MS/MS analysis after separation by HPLC. Results: After administration in rabbits, dexamethasone reached measurable concentrations in choroid-RPE, retina and vitreous during the six-weeks study. The tmax was observed at 2 weeks post-administration in the three tissues evaluated, with Cmax values of 579.02 ng/g, 1,749.65 ng/g and 69.19 ng/mL for choroid- RPE, retina and vitreous respectively. Calculations of area under the concentration- time curve (AUC0,42d) in the target tissue (retina) were performed as measurement of drug bioavailability, with a value of 21,337.52 ng day/g. Conclusions: According to the results obtained in this work, the microsphere formulation developed is suitable for sustained transscleral delivery of dexamethasone. Periocular microspheres can be considered as a potential alternative to dexamethasone intravitreal administrations indicated for the treatment of diseases affecting the posterior segment of the eye. Commercial Relationships: Rocio Herrero-Vanrell, None; Deyanira Barbosa- Alfaro, None; Irene Bravo-Osuna, None; RS Kadam, None; I. Fernandez- Bueno, None; MT García-Gutierrez, None; Jose-Carlos Pastor, None; Uday B. Kompella, None; Irene Teresa Molina Martínez, None Support: Research Group UCM 920415 (GR35/10-A). MAT-2010-18242.

Program Number: 478 Poster Board Number: D1155 Presentation Time: 8:30 AM - 10:15 AM A Topical Ocular Toxicity Study of Dexamethasone Sodium Phosphate

Formulations Administered with Visulex-p in Rabbits Kongnara Papangkorn 1,2 , Donald Mix 1 , Charlotte Butler 1 , David J. Knauss 2 , John

W. Higuchi 1 , Balbir Brar 1 , William I. Higuchi 1,2 .

  • 1 Aciont Inc, Salt Lake City, UT;

  • 2 Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT. Purpose: To assess the ocular tolerability of Visulex-p (Vp) containing dexamethasone sodium phosphate (DSP) following daily dosing for 7 days and weekly dosing for 12 weeks in New Zealand white rabbits. Methods: Twenty-one 5-6 months old rabbits were assigned to seven groups according to Table 1. Vp loaded with 250 ul of the test formulation was applied to the right eye. Vp was then removed at the end of the application. The ocular examinations, with an indirect ophthalmoscope, modified McDonald-Shadduck scoring, and fluorescein staining were completed at pre-, post-, and at selected time-points post-dose. The body weight was recorded at baseline and during the study. After the final observation, animals were sacrificed and the eyes were collected for histological examination for any signs of inflammation, including edema/congestion (conjunctiva, ciliary body, cornea), inflammatory cell infiltration (conjunctiva, cornea, anterior chamber, trabecular meshwork, iris, ciliary body), and neovascularization of cornea. Results: Ocular findings are summarized in Table 1. Conjunctival injection and chemosis were observed right after dosing in all groups, with severity increasing with dosing concentrations. Resolution was observed within the 1-7 days after each application. At doses of 8% and higher, there was a longer time to resolution with continued weekly treatment. Weight loss occurred with 15% and 25% DSP. No corneal damage was seen at any of the dose groups. No ocular findings were observed in histopathology. Conclusions: Vp containing 4% and 8% DSP formulation is well tolerated in rabbit for 7 days of daily dosing and over a 3 month period of weekly dosing. Although Vp containing 8% and 15% DSP formulation treatments showed an accumulation of irritation scores over 3 months, there were no signs of irreversible ocular tissue damage in any of the rabbits.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology (PH) means of electrospray ionization MS/MS

Commercial Relationships:

Kongnara Papangkorn, Aciont Inc (E); Donald

Mix, Aciont Inc (E); Charlotte Butler, Aciont Inc (E); David J. Knauss, Aciont

Inc (F); John W. Higuchi, Aciont Inc (E); Balbir Brar, Aciont Inc (E); William I. Higuchi, Aciont Inc (E) Support: NEI Grant 2R44EY014772-02A1

Program Number: 479 Poster Board Number: D1156 Presentation Time: 8:30 AM - 10:15 AM

Drug Eluting Contact Lenses For The Treatment Of Glaucoma Joseph B. Ciolino 1 , Cristina F. Stefanescu 2,3 , Katherine A. Wymbs 2 , Sarah L. Spraque 2 , Daniel R. Mascoop 2 , Shireen S. Rudina 2 , Fabiano Cade 1 , Daniel S. Kohane 3,2 . 1 Ophthalmology, Massachusetts Eye and Ear Infirmary/ Harvard Medical School, Boston, MA; 2 Massachusetts Institute of Technology, Cambridge, MA; 3 Anesthesiology, Children's Hospital Boston/Harvard Medical School, Boston, MA. Purpose: To report the in vivo and in vitro testing of a latanoprost-eluting contact lens designed for the treatment of glaucoma. Methods: Drug-eluting therapeutic contact lenses (TCL) were created by encapsulating latanoprost-Poly(lactic-co-glycolic acid) films in methafilcon A by ultraviolet light polymerization. TCLs (n=4) were placed in 5 mL of phosphate buffered saline at 37°C with continuous rotation (64 RPM). The release media was sampled and changed daily. Drug concentrations were measured with an enzyme- linked immunosorbent assay (ELISA). TCLs were inserted on the left eye of New Zealand white rabbits (n=3) for 10 days. The eyes were examined under an operating microscope and the anterior chamber (AC) fluid was collected during 1,

3, 5, 7, 10, and 14 days of continuous wear. Latanoprost 0.005% solution (drops)

was topically applied to rabbit eyes and the AC fluid was sampled at the following times after drop administration (1, 3, 6, 12, and 24 hrs). Each sample was collected on a different day and the AC drug concentration was measured by ELISA. The 24- hr area under the curve (AUC) for latanoprost drops was calculated and compared to the AC concentration measured during 14 days of TCL wear. Results: In vitro, TCLs demonstrated an initial burst and then eluted a sustained and therapeutic daily amount of latanoprost for 4 weeks. In vivo, the TCLs demonstrated no signs of toxicity. Through 14 days of continuous wear, TCLs delivered more drug to the anterior chamber each day than latanoprost drops. Conclusions: This contact lens design can potentially be used as a treatment for glaucoma and as a platform for ocular drug delivery with widespread applications.

Commercial Relationships:

Joseph B. Ciolino, M0925.70250US00

(P); Cristina F. Stefanescu, None; Katherine A. Wymbs, None; Sarah L.

Spraque, None; Daniel R. Mascoop, None; Shireen S. Rudina, None; Fabiano Cade, None; Daniel S. Kohane, M0925.70250US00 (P) Support: 1K08EY019686

Program Number: 480 Poster Board Number: D1157 Presentation Time: 8:30 AM - 10:15 AM Thermoresponsive and Biodegradable Star-Branched Dendritic Polymers for Drug Delivery across the Blood-Ocular Barriers

Xiaoxun Li, Sibo Jiang, Tao L. Lowe. Department of Pharmaceutical Sciences, University of Tennessee Health Science Center, Memphis, TN. Purpose: The objective of this study was to develop thermoresponsive and biodegradable star-branched dendritic polymers as drug carriers for drug delivery across the blood-ocular barriers. Methods: The thermo-responsive and biodegradable star-branched dendritic polymers were synthesized using a combination of solid phase peptide synthesis, ring-opening polymerization and atom transfer radical polymerization techniques. These dendritic polymers contained biodegradable poly-L-lactic acid (PLLA) branches, thermoresponsive poly (N-isopropyl acrylamide) segmants, and poly-L- lysine (PLL) dendrons. Their chemical structure was characterized using 1 H-NMR and FT-IR. Their molecule weights were determined using MALDI-TOF. Their thermoresponsive property was measured using both UV-vis spectroscopy (to measure the transmittance) and dynamic light scattering (to measure the hydrodynamic size of the dendritic polymers). In the degradation studies, the viscosity, molecular weight, and chemical structure of the dendritic polymers were monitored using rheometer, MALDI-TOF and FTIR, respectively. Their cytotoxicity to retinal pigment epithelium (RPE) cells was evaluated using the MTT assay. The permeabilities of dendritic polymers across the porcine sclera- choroid-RPE, sclera, and cornea were determined using side-by-side diffusion cells. The DTAF-labeled nanoparticle solutions were added to the donor cell while equal volume of transport buffer was put in the receiver cell. The fluorescence intensity in the receiver cell was assayed for 4 h. Results: 1 H-NMR, FT-IR and MALDI-TOF confirmed the successful synthesis of the star-branched dendritic polymers. UV-vis spectroscopy and dynamic light scattering measurements showed that they were thermoresponsive with LCSTs around of 30-40 °C at different concentrations (0.05-1mg·mL). Degradation studies showed that both viscosity and molecular weight decreased with time during the degradation course. MTT data indicated that the dendritic polymers were not toxic the retinal pigment epithelium (RPE) cells. The ex-vivo data demonstrated that these dendritic polymers were more permeable to the porcine sclera-choroid-RPE than the control, dextran with a molecular weight of 70,000.

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

Conclusions: The developed thermo-responsive and biodegradable star-branched dendritic polymers showed great potential to be used as drug carriers for deliver drug across the blood-ocular barriers. Commercial Relationships: Xiaoxun Li, None; Sibo Jiang, None; Tao L. Lowe, None Support: NIH Grant

Program Number: 481 Poster Board Number: D1158 Presentation Time: 8:30 AM - 10:15 AM Molecular Parameters Associated With Uptake Of Multi-functional Antioxidants For The Lens, Retina, And Brain Hiroyoshi Kawada, Zifeng Zhang, Karen A. Blessing, Peter F. Kador. Pharmaceutical Sciences, College of Pharmacy, Univ of Nebraska Medical Center, Omaha, NE. Purpose: Our laboratory has reported the synthesis of multi-functional antioxidant analogs of N,N-dimethylsulfamoyl-4-(2-pyrimidyl)piperazine, which can independently chelate redox-active metals and scavenge reactive oxygen species (ROS). While they readily accumulate in the lens and retina after oral administration, they fail to significantly reach the brain. To increase the ability of these multi-functionals to cross the blood brain barrier (BBB), a new series of analogs retaining the 2-amino-4-hydroxypyrimidine ring system have been synthesized (HK 1-16). Here, the molecular parameters required to target these analogs to the lens, retina, or brain are defined. Methods: Molecular parameters such as log P and Clog P, that are associated with lipophilicity; dipole moment, which implies an electric moment apart from the total net charge on the molecule; kappa shape index, which is associated with steric bulk of the molecule; molar refractivity, which is related to polarizability of the molecule; and hydrophilic volume of the molecule were calculated by MOE. The new descriptor fMF, which is associated with topology, was calculated as described in J. Med. Chem. 1996, 39, 2887-289. All compounds examined were administered to C57BL/6 mice for 14 days by individually incorporating each compound into chow (0.05%). After 14 days of feeding, all mice were perfused with PBS and drug levels in the brain, lens, and retina were quantified by HPLC-ESI-MS. Results: Significant negative correlations between the accumulation of drugs in the brain and either the kappa shape index, dipole moment, molar refractivity, or hydrophilic volume were observed (linear fit analysis, R2 = 0.66-0.95, P< 0.05). In contrast, significant positive correlations were observed between the lenticular accumulation of drugs and either the kappa shape index or dipole moment (linear fit analysis, R2 = 0.63-0.87, P< 0.05). No significant correlations between any molecular parameter and the uptake of drugs into the neural retina were observed. Conclusions: Inverse correlations between the uptake of drugs and their molecule parameters related to steric bulk, refractivity, polarity, and hydrophilic volume, were observed in the brain versus the lens. Although the retina is considered an extention of the brain, no correlations between the examined molecular parameters and accumulation of compound into the retina were observed. Commercial Relationships: Hiroyoshi Kawada, None; Zifeng Zhang, None; Karen A. Blessing, None; Peter F. Kador, None Support: R21EY016460

Program Number: 482 Poster Board Number: D1159 Presentation Time: 8:30 AM - 10:15 AM Fluid Infusion Into the Corneal Stroma Using a Microneedle

Samirkumar R. Patel 1 , Eric Powers 1 , Mark R. Prausnitz 1 , Henry F. Edelhauser 2 .

  • 1 Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA; 2 Ophthalmology, Emory Univ Eye Center, Atlanta, GA. Purpose: Corneal infections, especially deep stromal infections, can require an aggressive therapy regimen using topically applied agents. It would be ideal to have a delivery method that can administer drug directly to the stroma to stop spread of the infection quickly without relying on patient compliance. Here, we investigate the capability of a microneedle to infuse a fluid into the corneal stroma and study the volume that can be delivered, the pressures needed for injection and the spread of injected material. Methods: Enucleated porcine eyes were placed in a holder and microneedles (700- 800 µm in length) were inserted perpendicularly into the cornea. The needles were attached to tubing with an in-line pressure transducer connected to a computer for continuous pressure monitoring. The tubing was attached to a 1 mL syringe that was mounted onto a syringe pump. The needle was inserted near the central cornea and the pump was used to inject a target volume of sulforhodamine B into the stroma at a flow rate of 100 µL/min. Images of the cornea were taken to measure the spread of the colored dye. Results: Intrastromal injections of up to 300 µL were achieved in all attempts using a microneedle. The spread of the injection was radial from the site of injection and the injection of 50, 80, 100, 200 and 300 µL resulted in a spread diameter of 6.3±0.5, 6.9±1.0, 8.2±0.1, 10.0±0.5 and 11.4±0.7 mm, respectively. The pressure profile during injection reached a peak within 25-35 s after the injection was started and then dropped off. The peak pressure during all injections was between 27-32 psi. During injection of volumes between 100-300 µL, the pressure leveled off

before injection was stopped. In these cases the pressure averaged between 15-20 psi. All volumes caused the cornea to expand and injection of 200-300 µL resulted in a 3-4 mm increase in thickness. Conclusions: This study demonstrated that a microneedle can inject up to 300 µL of fluid directly into the corneal stroma of porcine eyes. The injected fluid spread

radially and a volume as small as 50 µL was able to spread significantly. Large volumes, however, caused significant corneal thickening. As a result, if minimal corneal thickening is desired, small volumes, < 50 µL should be used. This approach could be used to deliver drugs to the cornea for the treatment of corneal infections.

Commercial Relationships:

Samirkumar R. Patel, 12/767,768 (P), Clearside

Biomedical (E); Eric Powers, None; Mark R. Prausnitz, 12/767,768 (P),

Clearside Biomedical (I); Henry F. Edelhauser, 12/767,768 (P), Clearside Biomedical (I) Support: R24 EYE017404, P30 06360, RPB

Program Number: 483 Poster Board Number: D1160 Presentation Time: 8:30 AM - 10:15 AM Pharmacokinetics of intravitreal bevacizumab (avastin) In vitrectomized eyes Se Joon Woo 1 , Jeeyun Ahn 2 , Ji Hyun Park 1 , Sunyoung Park 3 , Kyu Hyung Park 1 , Hyuncheol Kim 3 . 1 Ophthalmology, Seoul National University Bundang Hospital, Seongnam, Republic of Korea; 2 Department of Ophthalmology, Seoul Metropolitan Boramae Medical Center, Seoul, Republic of Korea; 3 Chemical & Biomolecular Engineering, Sogang University, Seoul, Republic of Korea. Purpose: To perform comparative analysis of pharmacokinetics of intravitreally injected bevacizumab in vitrectomized versus non-vitrectomized rabbit eyes. Methods: Among the 35 eyes of 35 rabbits included in the study, 25-gauge pars plana vitrectomy was performed in 18 right eyes (vitrectomized eyes), and the remaining 17 right eyes served as control (non-vitrectomized eyes). Both groups received 1.25mg/0.05mL bevacizumab intravitreally. Eyes were enucleated at 1 hour, 1, 2, 5, 14 and 30 days after the intravitreal injection and were immediately frozen at -70°C. Bevacizumab concentrations were determined after separation of frozen vitreous and aqueous humor compartments using direct enzyme-linked immunosorbent assay. Bevacizumab concentration-time data were fit by two- compartmental analysis to determine half-life. Results: The vitreous concentration of bevacizumab in both groups showed two phases of clearance which are the fast distribution phase and the slow elimination phase. Bevacizumab vitreous concentration in vitrectomized eye showed 94.7% (1 hour), 70.5% (1 day), 89.2% (2 days), 94.2% (5 days), 99.2% (14 days), and 79.1% (30 days) of that of non-vitrectomized eyes. The calculated overall half-lives of intravitreal bevacizumab were 6.99 days for vitrectomized eyes and 7.06 days for non-vitrectomized eyes (1.6 hours difference). The clearance of intravitreal bevacizumab in vitrectomized eyes was accelerated only in the first phase but not in the second phase. Conclusions: The increase of intravitreal bevacizumab clearance after vitrectomy is not substantial in rabbit eyes. This experimental data suggests that the therapeutic efficacy and duration of intravitreal bevacizumab in patients who previously underwent vitrectomy may be comparable to those without vitrectomy history. Commercial Relationships: Se Joon Woo, None; Jeeyun Ahn, None; Ji Hyun Park, None; Sunyoung Park, None; Kyu Hyung Park, None; Hyuncheol Kim, None Support: SNUBH research grant 11-2011-016

Program Number: 484 Poster Board Number: D1161 Presentation Time: 8:30 AM - 10:15 AM In Vivo Verification of the Bioresponsive Potential of an Intelligent Intraocular Implant Lisa C. du Toit 1A , Viness Pillay 1A , Trevor R. Carmichael 1B , Thirumala Govender 2 , Yahya E. Choonara 1A . A Pharmacy and Pharmacology, B Ophthalmology, 1 University of the Witwatersrand, Johannesburg, South Africa; 2 Pharmaceutics, University of KwaZulu-Natal, Kwazulu-Natal, South Africa. Purpose: An autofeedback polymeric platform was used in the design of an intelligent intraocular implant - the I 3 - using stimuli-responsive polymers, producing a smart release system capable of delivering therapeutic levels of anti- inflammatory agent and antibiotic for posterior segment disorders of the eye in response to inflammation. Here the I 3 was assessed for its ability to respond to the inflammatory state created in a suitable animal model. Methods: In vivo design and surgical technique in the healthy rabbit eye: Fifty New Zealand Albino rabbits were used, randomly assigned to the experimental (25 rabbits) and control (25 rabbits) groups (n=5). A placebo device was implanted into one eye of the control group at sub-Tenon’s space and the drug-loaded implant (containing indomethacin and ciprofloxacin) into one eye of the experimental group. All procedures were undertaken in accordance with ARVO. For each study, one animal in each group was euthanized at each sampling point (3, 7, 14, 21, 28 days) with consequent device removal, enucleation and vitreous humor aspiration. In vivo design and surgical technique in the inflamed rabbit eye: Rabbits were assigned to experimental (5 rabbits) and control (5 rabbits) groups (n=5 at the

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

sampling point on day 7). Device implantation in the control and experimental groups was on day 0. Intraocular inflammation was induced via intravitreal injection of Salmonella typhimurium lipopolysaccharide (LPS) (100-200 ng/10μL of phosphate-buffered saline) into the I 3 -implanted eye of each rabbit. One animal in each group was euthanized on day 7 with device removal, enucleation and vitreous aspiration. Analysis of implant, tissue and fluid samples: Device biocompatibility and the degree of inflammation present were histologically established. The disparity in drug release behaviour in the healthy versus the inflamed rabbit eye was ascertained via analysis of vitreous samples. Results: Histological evaluation indicated that sub-Tenon device implantation did not induce a notable degree of inflammation. In vivo results demonstrated good penetration of drug from sub-Tenon’s space, through the ocular membranes, into the vitreous humour. There was enhanced release of both drugs in the inflamed rabbit eye even after 7 days with indomethacin levels of 0.749±0.126µg/mL and 1.168±0.186µg/mL, and ciprofloxacin levels of 1.181±0.150µg/mL and 6.653±0.605µg/mL being attained in the normal and inflamed eye, respectively. Conclusions: The I 3 displayed exemplary inflammation-responsive behaviour following assessment in a rabbit eye model. Commercial Relationships: Lisa C. du Toit, None; Viness Pillay, None; Trevor R. Carmichael, None; Thirumala Govender, None; Yahya E. Choonara, None Support: None

Program Number: 485 Poster Board Number: D1162 Presentation Time: 8:30 AM - 10:15 AM Generation Of Dual Secreting CNTF / VEGF-antagonist Cell Lines And ECT Devices

Vincent Ling, Susan Elliott, Brenda Dean, Lisa Orecchio, Konrad Kauper, Michael

Rivera, Weng Tao. Biological Sciences, Neurotech USA, Lincoln, RI. Purpose: Anti-angiogenic molecules have been successfully used in the treatment of wet AMD. However, only about 30% of treated patients gain 3 lines of vision. The presence of 70% afflicted patients suggests a separate mechanism in wet AMD pathology not addressed by standard anti-VEGF therapies. The current study was carried out to explore the concept of a combination therapy - the simultaneous delivery of two biotherapeutics, each with different bioactivities, from one device. An example of a clinically relevant dual-secreting biologic device would be one that produces an anti-angiogenic VEGF antagonist and also the neuroprotective cytokine, CNTF, as dual mode therapy for Wet AMD with underlying

photoreceptor lesions. Methods: An iterative transfection strategy was used to create combination VEGF antagonist / CNTF cell lines. VEGF antagonist expression vectors were cloned, then transfected into NTC-200 cells followed by screening for highest productivity. Top producing VEGF antagonist cell lines were subsequently expanded, transfected with CNTF expression vectors and screened for top CNTF producers. Dual producing VEGF antagonist / CNTF cell lines were obtained and subsequently encapsulated to create Neurotech ocular ECT implants. The rate of

protein secretion was determined by ELISA. Results: Dual biologic producing clonal cell lines exhibited robust recombinant protein secretion, with levels of some cell lines approaching 15 pcd for VEGF antagonist, and 0.5 pcd for CNTF cytokine. Cell lines were then encapsulated, and production of recombinant proteins from individual ECT devices were detected. Conclusions: Proof-of-concept, dual-secreting cell line studies suggest that Neurotech ECT devices may be an effective method of delivering biologics in a combinatorial approach where such therapies may be preferable, such as a case for Wet / Dry AMD.

Commercial Relationships:

Vincent Ling, Neurotech (E); Susan Elliott,

Neurotech (E); Brenda Dean, Neurotech (E); Lisa Orecchio, Neurotech (E); Konrad Kauper, Neurotech (E); Michael Rivera, Neurotech (E); Weng Tao, Neurotech (E) Support: None

Program Number: 486 Poster Board Number: D1163 Presentation Time: 8:30 AM - 10:15 AM

Microneedle-Based Suprachoroidal Injections in Rabbits: Histological Study Using Triamcinolone Acetonide Damian E. Berezovsky 1 , Samirkumar R. Patel 2 , Hans E. Grossniklaus 1 , Henry F. Edelhauser 1 . 1 Ophthalmology, Emory University, Atlanta, GA; 2 Chem & Biomolecular Eng, Georgia Institute of Technology, Atlanta, GA.

Purpose:

To assess the histopathological appearance of retina, choroid and sclera following

suprachoroidal injection of Triamcinolone Acetonide (TA, Triesence, Alcon Laboratories) and balanced salt solution in live pigmented rabbits.

Methods:

Ten Dutch-belted rabbits weighing 4-6 lbs. were used in accordance with ARVO’s Statement for the Use of Animals in Ophthalmic and Visual Research. Eyes received single 100uL injections of 2mg TA (n=9) or balanced salt solution (n=9)

using a single hollow metal microneedle in the superior temporal quadrant. Injection location was 5mm posterior to the limbus. Eyes were enucleated

immediately (n=6), after 1 day (n=6), or after 7 days (n=6). Two eyes were used as negative controls. After enucleation, eyes were fixed in 2.5% Glutaraldehyde. Circular sections (4mm in diameter) were cut from the area between injection site and optic nerve, embedded in resin and stained using toluidine blue.

Results:

Sections from all eyes were examined for inflammation, atrophy, necrosis, edema,

enlargement of the suprachoroidal space, choroidal hemorrhage or any other abnormality. No inflammation, atrophy, necrosis, edema, hemorrhage or scarring was found in any of the injected eyes, or in the normal controls. In about one-third of tissue samples, an abnormal separation was observed in the area of the suprachoroidal space. Normal-appearing parallel thin septae were visible within this enlarged suprachoroidal space, without cellular infiltration or evidence of hemorrhage. Two TA-injected samples contained an amorphous, acellular, nonstaining material within the enlarged suprachoroidal space, believed to represent triamcinolone collections. The retina retained normal appearance in all tissue samples.

Conclusions:

This histological study of in vivo suprachoroidal injections shows that triamcinolone acetonide or balanced salt solution can be injected into living rabbit

eyes without causing an immediate or short-term inflammatory response or choroidal hemorrhage. These two materials, injected using a hollow metal microneedle, did not cause any significant disruptions in tissue organization or appearance through seven days post-injection. Commercial Relationships: Damian E. Berezovsky, None; Samirkumar R. Patel, 12/767,768 (P), Clearside Biomedical Inc (I, E); Hans E. Grossniklaus, None; Henry F. Edelhauser, 12/767,768 (P), Clearside Biomedical Inc (I) Support: NIH grants R24 EY017404, P30 EY06360, and RPB

Program Number: 487 Poster Board Number: D1164 Presentation Time: 8:30 AM - 10:15 AM Superior Delivery to Choroid-Retina following Suprachoroidal Injections in Rats: Assessment using Fluorophotometry

Puneet Tyagi, Rajendra S. Kadam, Uday B. Kompella. Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, CO. Purpose: The objective of this study was to evaluate the delivery and pharmacokinetics of sodium fluorescein after suprachoroidal, intravitreal, and posterior subconjunctival injections in Sprague Dawley rats using noninvasive

fluorophotometry. Methods: Delivery and pharmacokinetics of sodium fluorescein was evaluated in Sprague Dawley rats (n=4) after suprachoroidal (500 ng in 5µl), intravitreal (50 ng in 5µl), or posterior subconjunctival injection (500 ng in 5µl). Sodium fluorescein

levels were monitored noninvasively using Fluorotron Master™ up to 6 hours.

Pharmacokinetic parameters were estimated by noncompartmental analysis using WinNonlin. Results: Sodium fluorescein delivery to choroid-retina was in the order:

suprachoroidal > intravitreal > posterior subconjunctival injection. Peak concentration (C max ; ng/ml) in the choroid-retina was the highest after suprachoroidal (1673 ± 363) injection when compared to intravitreal (103 ± 44) and

posterior subconjunctival (76 ± 6) injections. The extent of delivery (AUC 0-t ) to the choroid-retina after suprachoroidal injection was 2- and 6- fold higher than intravitreal and posterior subconjunctival injections, respectively. Conclusions: Suprachoroidal injections resulted in the highest delivery of sodium fluorescein to choroid-retina when compared to intravitreal and posterior subconjunctival injections. Noninvasive monitoring of sodium fluorescein is

possible in rats using Fluorotron Master™ ocular fluorophotometry.

Commercial Relationships: Puneet Tyagi, None; Rajendra S. Kadam, None; Uday B. Kompella, None Support: This work was supported by the NIH grants EY017533, EY018940, and

EY017045.

Program Number: 488 Poster Board Number: D1165 Presentation Time: 8:30 AM - 10:15 AM Nanoemulsion As A Vehicle To Enhance The Ocular Absorption After Topically Applied Cyclosporine A In The Rabbit Eye Junjie Zhang, Tianyang Zhou, Liya Wang, Jijun He, Huiyun Xia. Dpt of Pharmaceutical Science, Henan Eye Institute, Henan Key Laboratory of Keratopathy, Zhengzhou, China. Purpose: To investigate the effect of vehicles on ocular absorption of topically applied Cyclosporin A (CSA) in the rabbit eye. Methods: 0.05% Cyclosporine nanoemulsion (CSA-NE) was prepared. A single dose of 50μl CSA was applied using CSA-NE or oil dissolved drug (CSA-OD), the loading dose (50μl×5 times with an interval of 5min) of CSA-NE or CSA-OD was also performed. CSA concentrations were measured at intervals of 5, 15, 30, 6, 120, 180, 240min, 6, 8, 24 and 48 hours for the single dose and 30, 120, 240min, 6, 24, 48 and 72 hours for the loading dose by high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Results: The size of nanoemulsion was 29.0±0.5nm and zeta potential was -

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

0.036±0.135mv. After single application of CSA-NE, the highest concentrations of CSA in cornea and conjunctiva were 1433.1±340.1ng/g and 1445.5±410.2ng/g, respectively at 30min and remained 236.15±95.03ng/g in cornea at 48h while CSA in cornea and conjunctiva could not be detected after 8 hours for CSA-OD group. The concentrations in cornea at all the time points were significantly higher than the CSA-OD group at the corresponding time points (p<0.01), respectively. The concentrations in conjunctiva were at all the time points except 3h, 4h and 8h were significantly higher than the CSA-OD group at the corresponding time points (p<0.05) , respectively. Relatively low concentrations were measured in the aqueous for both CSA-NE and CSA-OD group 7.60±9.81 and 6.22±5.68ng/ml) and no significant difference were found between the two groups (p>0.05). After loading dose application of CSA-NE, The highest concentrations in cornea and conjunctiva were 2790.8±409.2 and 3365.0±806.1ng/g at 30min, respectively. CSA levels in cornea and conjunctiva were significantly higher than the corresponding time points (p<0.0001 and <0.05),respectively while the drug in both cornea and conjunctiva could not be detected after 8 hours for CSA-OD group. Relatively low concentrations were measured in the aqueous for both CSA-NE and CSA-OD group (7.02±4.36 ng/ml and 4.29±5.35 ng/ml) and no significant difference were found between the two groups (p>0.05). The concentrations in blood for single and loading dose all groups were below the detection limit (5ng/ml). Conclusions: These findings indicate that nanoemulsion may be a promising delivery system for topical use of CSA in ocular immune-mediated diseases. Commercial Relationships: Junjie Zhang, None; Tianyang Zhou, None; Liya Wang, None; Jijun He, None; Huiyun Xia, None Support: 072103810606

Program Number: 489 Poster Board Number: D1166 Presentation Time: 8:30 AM - 10:15 AM

Ex Vivo Permeation of Cystine across Bovine Corneas - A Step towards the Development of an Antioxidant Eye Drop for Cataract Prevention Ilva D. Rupenthal 1A , Simon Raesch 1A,2 , Bo Li 1B , Ulrich F. Schaefer 2 , Claus-Michael

Lehr 2,3 , Julie C. Lim 1B . A Department of Ophthalmology, B Department of Optometry

and Vision Science, 1 The University of Auckland, Auckland, New Zealand;

  • 2 Department of Biopharmaceutics and Pharmaceutical Technology, Saarland University, Saarbruecken, Germany; 3 Department of Drug Delivery, Helmholtz- Institute for Pharmaceutical Research Saarland, Saarbruecken, Germany. Purpose: Age-related nuclear (ARN) cataract is the leading cause of blindness worldwide and is estimated to affect more than 40 million people by 2020. Even though surgical removal of the clouded lens is highly effective, the current demand exceeds the supply. Since ARN cataract is associated with a depletion of antioxidants, mainly glutathione, in the lens core, our research has concentrated on restoring antioxidant levels in this region in order to prevent or delay the onset of cataract formation. This study investigated the permeation of cystine, the limiting factor for glutathione synthesis, across bovine corneas. Methods: Freshly excised bovine corneas were placed between donor and acceptor chambers of standard Franz diffusion cells. A 0.15 mg/ml cystine solution (1 ml) was placed in the donor compartment, while modified Ringer solution, kept at 37°C, served as acceptor medium. Samples were taken at predetermined time points over 24 h and cystine concentrations were analyzed by derivatization with monobromobimane and subsequent fluorescence HPLC analysis. In addition to a simple cystine solution, various penetration enhancers were tested for their ability to improve the cystine permeation across bovine corneas. Results: The established HPLC method allowed cystine quantification to a lower limit of <0.1 nmol/ml with high linearity (R 2 =0.9994) and a reduced sample run time (17 min). Over the 24 h period 45 nmol of cystine permeated across the bovine cornea using the simple solution as donor. This amount was almost tripled (118 nmol over 24 h) when incorporating 0.5% EDTA, a tight junction opener, while 0.01% BAC only exhibited a slight improvement. Immunolabelling of frozen tissue sections revealed the presence of the cystine/glutamate (Xc - ) exchanger in the corneal epithelium, which may be responsible for partial active transport of the antioxidant across the tissue. Conclusions: Franz diffusion cell experiments combined with HPLC analysis offer an accurate way to study the permeation of various cystine eye drops across bovine corneas, with the addition of 0.5% EDTA showing the most promising results so far. Further penetration enhancers in combination with other delivery approaches, such as in situ gelling eye drops, will further be evaluated before testing the most promising formulations in vivo. Commercial Relationships: Ilva D. Rupenthal, None; Simon Raesch, None; Bo Li, None; Ulrich F. Schaefer, None; Claus-Michael Lehr, None; Julie C. Lim, None Support: NZPERF Grant 219

Program Number: 490 Poster Board Number: D1167 Presentation Time: 8:30 AM - 10:15 AM Differential Flow Rate of Triamcinolone and Preservative-Free Triamcinolone through Small-Gauge Needles

Jorge A. Fortun 1 , Alex Gonzalez 2 , Cornelis Rowaan 2 , Mariela Aguilar 2 , William Lee 2 , Andrew A. Moshfeghi 1 , Thomas A. Albini 1 , Jean-Marie A. Parel 2 . 1 Bascom Palmer Eye Institute, Univ of Miami Miller Sch of Med, Miami, FL; 2 Ophthalmic Biophysics Center, Bascom Palmer Eye Institute, Miami, FL. Purpose: The most commonly used commercially available steroid preparations used for intravitreal injections are triamcinolone acetonide (TA) (Kenalog 40, Bristol-Myers Squibb, Princeton, NJ) and the preservative-free triamcinolone acetonide (PFTA) (TRIESENCE, Alcon, Inc., Fort Worth, TX) both in the 40- mg/mL formulation. The most commonly used needle for the injection of TA is a 27-gauge needle. Smaller gauge needles are avoided because of the possibility of clogging, presumably caused by obstructive accumulation of triamcinolone aggregates in these narrow needles. PFTA has been shown to form more aggregates of smaller size than TA. We evaluated the differential flow of TA and PFTA through small gauge needles. Methods: A hydraulic piston mechanism was utilized to transfer a constant vertical 1kg gravitational force to the horizontally positioned 1cc syringe plunger (see figure). The 1 kg force was measured during experimental intravitreal injections given by three clinicians (JAF, AAM, TAA). A piezo electric pressure transducer was placed between the syringe and needle. Specialized software developed with Labview, National Instrument platform, was used to digitize and record the pressure signal over time during an injection of 1cc of TA or PFTA through a 27, 30 or 32 gauge needle. All needles were commercially available and 0.5 inches in length. Measurements were performed in triplicate. From each set of measurements we deduced the injection rate (ml/sec). Injections during which flow stopped prior to complete injection of 1cc, were recorded as having a flow rate of 0. Results: Using a 27-gague needle the mean flow rate of TA was found to be 0.513 cc/sec (±0.064 95% Confidence interval [CI]) and of PFTA 0.620 cc/sec (±0.055 95% CI). Using a 30-gague needle the mean flow rate of TA was found to be 0.06 cc/sec (±0.1 95% CI) and of PFTA 0.180 cc/sec (±0.017 95% CI). Using a 32- gague needle no flow was sustained by TA on any of three attempts and the mean flow rate of PFTA was 0.117 cc/sec (±0.020 95% CI). Conclusions: PFTA can be injected through a 32 gauge needle but TA cannot. The flow rate of PFTA through a 32-gague is adequate for intravitreal injection, i.e. about 0.1 cc/ sec. This is consistent with the finding of smaller trimacinolone aggregates in PFTA than

TA.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology (PH) 0.036±0.135mv. After single application of

Commercial Relationships:

Jorge A. Fortun, Alcon (C); Alex Gonzalez,

None; Cornelis Rowaan, None; Mariela Aguilar, None; William Lee, None; Andrew A. Moshfeghi, Alcon (C); Thomas A. Albini, Alcon (C); Jean-Marie A.

Parel, None Support: The Morgenstern Foundation (TAA); Florida Lions Eye Bank; NIH Center Grant P30EY14801; Research to Prevent Blindness; Henri and Flore Lesieur Foundation (JMP).

Program Number: 491 Poster Board Number: D1168 Presentation Time: 8:30 AM - 10:15 AM Transscleral Iontophoretic Delivery of Avastin® In Vivo: Drug Distribution

and Safety Aspects Sarah A. Molokhia 1 , Kongnara Papangkorn 1,2 , Donald Mix 2 , Charlotte Butler 2 , Prasoona Karra 1 , John Higuchi 2 , Balbir Brar 2 , S. Kevin Li 3 , William I. Higuchi 1,2 . 1 University of Utah, Salt Lake City, UT; 2 Aciont Inc, Salt Lake City, UT; 3 University of Cincinnati, Cincinnati, OH. Purpose: To study anodal iontophoretic delivery of the commercial formulation and a new iontophoretic formulation of Avastin® in vivo using the Visulex iontophoresis system. Methods: All studies were performed under anodal iontophoresis (AI) on New Zealand white rabbits using a Visulex device system with 2.5% bevacizumab (Avastin®). For pharmacokinetics (PK) studies (n=6) the commercial Avastin® formulation and an enhanced (A0-01) iontophoretic formulation were used in Group 1 and 2, respectively. After 20 min of AI delivery, the rabbits were

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

sacrificed and the eyes enucleated. The eyes were dissected and the cornea, aqueous humor, vitreous, conjunctiva, sclera, choroid and retina were assayed for drug concentrations with ELISA. For the safety study Group 3 (n=6), the eyes were examined with an indirect ophthalmoscope before and immediately after iontophoretic dosing, then again on days 2, 4, and 6. The eyes were graded according to a modified McDonald-Shaddock scale for conjunctival injection, chemosis, corneal haze, iritis and other ocular irritation signs. At day 6, the animals were sacrificed and all eyes were processed for histopathological examination. Results: The total amount of Avastin® delivered into the eye after 20 min of AI (1.8 mA/cm2) was 223 ± 71 µg and 315 ± 123 µg for the commercial formulation and (A0-01) iontophoretic formulation, respectively. High concentrations of Avastin were distributed in the conjunctiva and sclera tissues. However, approximately 4 µg was delivered to the retina/choroid tissues. For the safety study, the only adverse effect noted was very minor conjunctival injection, present in four of the six eyes treated on Day 2 which resolved by Day 6. Histopathological evaluation showed no effects due to the iontophoretic treatment. Conclusions: This study demonstrated promising results toward higher Avastin® delivery with the new iontophoretic formulation (AO-01), although this is not statistically significant. Further modifications are required to the new formulation to achieve significant enhanced delivery. The amount delivered to the retina/choroid using the Visulex iontophoretic system is within same order of magnitude reported in literature using intravitreal injection. This study is among the first to demonstrate safe delivery of Avastin® in vivo to the posterior eye tissues within the therapeutic range using iontophoresis.

Commercial Relationships:

Sarah A. Molokhia, Aciont Inc (C); Kongnara

Papangkorn, Aciont Inc (E); Donald Mix, Aciont Inc (E); Charlotte Butler, Aciont Inc (E); Prasoona Karra, None; John Higuchi, Aciont Inc (E); Balbir

Brar, Aciont Inc (E); S. Kevin Li, Aciont Inc (C); William I. Higuchi, Aciont Inc

(E)

Support: 1R43EY020791-01

Program Number: 492 Poster Board Number: D1169 Presentation Time: 8:30 AM - 10:15 AM Ocular Drug Delivery Using A Thermo-responsive Lacritin Fusion Protein

John A. MacKay, Wan Wang, Benjamin Droese, Mihir Shah, Pang-Yu Hsueh,

Guoyong Sun, Sarah F. Hamm-Alvarez. Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA. Purpose: A fusion protein between recombinant human lacritin and an elastin-like- polypeptide (ELP) was designed as a therapeutic for dry eye disease(DED). Fusion of these domains imparts thermo-sensitive assembly to lacritin, which is a candidate biopharmaceutical. Affecting over 3.2 million Americans, predominantly women, DED is characterized by decreased tear production, which leads to discomfort and visual impairment. To develop biopharmaceuticals for DED, better approaches are required to retain proteins on the eye. Protein polymers, such as ELPs, are an emerging solution to this problem composed of an amino acid repeat (VPGXG). ELPs have characteristic inverse phase transition temperatures (Tt), below which they are soluble and above which they self-associate. By adjusting Tt between room and body temperature ELP fusion proteins may act as a therapeutic

depot. The hypothesis tested here is that the fusion protein of ELP and lacritin will undergo thermally-induced phase separation at ocular temperatures without compromising the mitogenic and secretagogue signaling functions of lacritin. Methods: Multiple libraries of synthetic ELP genes were engineered with a range of assembly properties. Genetic engineering enables precise control over their molecular weight, orientation, and Tt. ELP phase behavior was used to purify these biopolymers from bacteria. Fusion protein, free lacritin, and free ELP were characterized for assembly, biodegradation, and induction of secretion from primary rabbit lacrimal gland acinar cells (LGACs). Results: ELP lacritin fusion proteins were developed with three properties: 1) a control that is soluble at body temperature; 2) a thermo-responsive fusion that forms viscous microparticles at body temperature; and 3) a polypeptide nanoparticle that assembles ~200 nm diameter particles at body temperature. Phase behavior and self-assembly was observed over a range of concentrations (5-100 uM) using UV-Vis spectrophotometry and dynamic light scattering. Conclusions: ELP lacritin fusion proteins were developed that assemble between 25 and 30 °C. All fusion constructs induce significant secretion of beta- hexosaminidase from primary LGACs. These results suggest that ELP-mediated assembly may be a useful platform to control ocular drug delivery and efficacy.

Commercial Relationships:

John A. MacKay, Patent disclosure filed on ELP

fusion proteins for ocular drug delivery (P); Wan Wang, None; Benjamin Droese,

None; Mihir Shah, None; Pang-Yu Hsueh, None; Guoyong Sun, None; Sarah F. Hamm-Alvarez, None Support: R21EB012281 to JAM, RO1EY017293 and RO1EY017293S1 to SHA

Program Number: 493 Poster Board Number: D1170 Presentation Time: 8:30 AM - 10:15 AM Ultrasound-enhanced Penetration Of Topical Fluorescein into the Vitreous in Rabbit Eyes

Jay M. Stewart 1A , Chris J. Diederich 1B , Elliot Chan 1A , Vasant Salgaonkar 1B , Ricardo Lamy 1A . A Ophthalmology, B Radiation Oncology, 1 Univ of California-San Francisco, San Francisco, CA. Purpose: To determine whether ultrasound treatment can promote the permeation of topical fluorescein into the vitreous. Methods: Fresh cadaveric rabbit eyes were placed in saline solution at 37C. An eye cup containing 0.1% fluorescein solution was centered on the sclera approximately 5 mm posterior to the limbus. Continuous wave ultrasound at 1 W/cm2 was applied to the sclera within the eye cup for 10 minutes, and the eyes were then left in solution for an additional 50 minutes. Control eyes received the same exposure to fluorescein solution in the eye cup without ultrasound treatment. Eyes were then washed, and vitreous was collected using an automated vitrectomy system. Vitreous samples were analyzed using fluorescence spectophotometry. Fluorescence intensity levels were compared to a standard curve representing known fluorescein concentrations. Results: The vitreous concentration of fluorescein was increased in treated eyes (mean 4.34 mcg/mL, n=10) compared to control eyes (mean 2.88 mcg/mL, n=8), P < 0.05. Conclusions: Ultrasound treatment facilitated the entry of topical fluorescein into the vitreous of intact rabbit eyes. These results suggest that ultrasound-enhanced drug delivery may offer a non-invasive means of treating posterior segment diseases. Commercial Relationships: Jay M. Stewart, None; Chris J. Diederich, None; Elliot Chan, None; Vasant Salgaonkar, None; Ricardo Lamy, None Support: None

Program Number: 494 Poster Board Number: D1171 Presentation Time: 8:30 AM - 10:15 AM Drug Loaded Microparticles For Long-term Sustained Release Of Anti-VEGF Therapies In Age-related Macular Degeneration Leilei Zhang 1A,1B , Cynthia J. Roberts 1A,1B , Alan D. Letson 1B , Ronald X. Xu 1A,1B . A Biomedical Engineering, B Ophthalmology, 1 The Ohio State University, Columbus, OH. Purpose: To investigate an alternative microfabrication technique to enhance the drug encapsulation efficiency and reduce the antibody damage for long-term sustained release of anti-VEGF drug loaded microparticles in age-related macular degeneration. Methods: The co-axial electrohydrodynamic atomization process was used to overcome the limitations of the traditional double emulsion method for microfabrication of multifunctional drug-loaded microparticles. The process setup included a co-axial needle, a ground plate, a droplet collector, and an inline video camera. The inner and outer channels of the co-axial needle were connected to two syringe pumps and infused with a phosphate buffered saline (PBS) solution of the anti-VEGF drugs, and an organic solution of poly (lactic-co-glycolic acid) (PLGA), respectively. Various fluorescent dyes, such as Nile Red, were added to the PLGA solution to visualize the shell structure. Different drugs, such as Lucentis, were encapsulated. A total voltage of 14kV was applied between the co-axial needle and the ground plate so that a stable cone-jet was formed at the co-axial needle, broken into droplets, and collected by the droplet collector filled with distilled water. The core-shell ratio of the fabricated microparticles was controlled by adjusting several process parameters, such as the ratio of the inner and outer flow rates, the concentration of PLGA, and the applied voltage. Results: With the co-axial electrohydrodynamic atomization process, we were able to fabricate drug-loaded microparticles with the size ranging from 1 to 5um. The morphology of these microparticles was characterized by scanning electron microscopy, and the cone-shell structure was confirmed by confocal microscopy. Conclusions: The co-axial electrohydrodynamic atomization process was successful in producing drug-loaded mircoparticles. Future studies will investigate protection of anti-VEGF drugs from process-induced damage with a high encapsulation efficiency. The core-shell ratio can be easily controlled for programmed long-term drug release. Commercial Relationships: Leilei Zhang, None; Cynthia J. Roberts, None; Alan D. Letson, None; Ronald X. Xu, Genentech, Inc. (R) Support: The Ohio Lions Eye Research Foundation Fellowship and a kindly donation through the Muskingum County Community Foundation

Program Number: 495 Poster Board Number: D1172 Presentation Time: 8:30 AM - 10:15 AM Wound Healing Of Human Corneal Epithelial Cells Impacted By Nanoparticles Enhua H. Zhou 1 , Richard Pizzo 2 , Christa Watson 1 , Joel Cohen 1 , Quynh Dang 1 , Georgios Pyrgiotakis 1 , Joseph D. Brain 1 , Jeffrey J. Fredberg 1 , Philip Demokritou 1 . 1 Department of Environmental Health, Harvard School of Public Health, Boston, MA; 2 Department of Physics, Boston Colleague, Boston, MA. Purpose: As engineered nanoparticles (ENPs) enter products, workers and consumers are increasingly exposed. Our body’s first line of defense against ENPs is the epithelium. However, there is a lack of understanding of the effects of ENPs

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

on the epithelium of the eye, which is a site of environmental exposure and resulting irritation. Moreover, those effects may be aggravated by preexisting wounds in the epithelium. Here we wanted to explore whether ENPs can impede the healing of a wounded epithelium. Methods: To address this question in human corneal epithelial cells (gift from Dr. Ilene K. Gipson), we developed a novel wound healing assay. Using this assay we studied a panel of well characterized, industrially relevant ENPs, including several commercially available ENPs and metals and metal oxides generated using our recently developed Versatile Engineered Nanomaterial Generation System (VENGES). Results: TiO2 and SiO2 ENPs did not affect wound healing at doses up to 100 micro g/ml. By contrast, copper oxide, zinc oxide, and silver ENPs substantially impede wound healing in a dose-dependent manner. Conclusions: Taken together, these studies provide a novel in-vitro model system for evaluating the physiological impact of nanoparticles in the ocular surface. Commercial Relationships: Enhua H. Zhou, None; Richard Pizzo, None; Christa Watson, None; Joel Cohen, None; Quynh Dang, None; Georgios Pyrgiotakis, None; Joseph D. Brain, None; Jeffrey J. Fredberg, None; Philip Demokritou, None Support: Pilot Project funding from the HSPH-NIEHS Center for Environmental Health (ES000002) and the Center for Nanotechnology and Nanotoxicology

Program Number: 496 Poster Board Number: D1173 Presentation Time: 8:30 AM - 10:15 AM Degradation And The Routes Of Clearance Of Intravitreal Hydrosilylated Porous Silicon Particles Lingyun Cheng 1A , Elizabeth Wu 1B , William R. Freeman 1A , Michael J. Sailor 1B , Jennifer Andrew 1B , Laura Conner 1A , Alejandra Nieto 1B . A Jacobs Retina Ctr at Shiley Eye Ctr, B Department of Chemistry and Biochemistry, 1 Univ of California- San Diego, La Jolla, CA. Purpose: We have reported the degradation and intravitreal safety profiles of porous silicon particles as vehicles for ocular drug delivery. Though porous silicon can be degraded in vitreous without toxicity, the routes of clearence of the degradation product, monomeric silicic acid (Si(OH) 4 ), from the eye environment is not yet understood. Methods: Ten pigmented rabbits were used for this study. The right eye of each animal was intravitreally injected with 3mg of hydrosilylated (1-undecylenic acid) porous silicon particles in 100 µL of 5% dextrose. The particles had an average size of 33 by 46 µM. After the intravitreal injections, two rabbits were sacrificed at each of these time points: 1 week, 2 weeks, 3 weeks, 5 weeks, and 8 weeks. Before sacrifice 1 to 2 ml of blood was sampled and the enucleated eye globes were dissected individually into aqueous, vitreous, and retina for quantitation of dissolved Si (as orthosilicate) using ICP-OES (Inductively Coupled Plasma-Optical Emission Spectroscopy). Normal rabbit vitreous and retina were used as controls. Results: No excess Si was detected in the blood samples. The Si concentration in the vitreous and aqueous compartments gradually decreased over time and Si concentration was consistently higher in vitreous than that in aqueous. The peak concentration in the vitreous was 25.5±5µg/ml and 18.9±0.05µg/ml in aqueous, while the peak concentration in the retina was only 2.4±0.8µg/g which was similar to the Si concentration in normal control retina samples (1.9±0.4µg/g). Si concentration in the vitreous at the last time point was 10.5±1.7µg/ml which was not significantly different from Si concentration in the normal control vitreous (11.85±1.6µg/ml). The concentration-time profile suggested a sustained degradation. Conclusions: The data suggest that the degraded porous silicon in vitreous was mainly cleared through aqueous outflow pathways.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology (PH) on the epithelium of the

Commercial Relationships: Lingyun Cheng, None; Elizabeth Wu, None; William R. Freeman, None; Michael J. Sailor, None; Jennifer Andrew, None; Laura Conner, None; Alejandra Nieto, None Support: NIH EY020617

Program Number: 497 Poster Board Number: D1174 Presentation Time: 8:30 AM - 10:15 AM Development of Microemulsions for Enhanced Topical Delivery to the Eye

Ronald A. Wassel, Didier Nuno, Alexander Quiambao, Fadee Mondalek, Rafal

Farjo. Charlesson LLC, Oklahoma City, OK. Purpose: MSH-1001, a novel highly insoluble small molecule, is an ATP-sensitive K channel opener and has been shown to reduce intraocular pressure. The purpose of this study was to develop a microemulsion based eye drop that can deliver therapeutic drug concentrations to the retina better than a micronized suspension. Methods: Twelve different nano- and micro-emulsions were prepared. There formulations were made up of oils and surfactants that are commonly used in ophthalmic formulations. Quantification of MSH-1001 in rabbit aqueous humor at 1 hour following a 60 ul eye drop of the aqueous humor performed using LC- MS/MS.

Results: Following topical administration of the various eye drops it was observed that a 0.5% MSH-1001 microemulsion delivered the same drug levels to the aqueous humor as a 3% nanoemulsion and the 10% micronized suspension. Conclusions: MSH-1001 can be formulated into optically transparent and thermodynamically stable microemulsion eye drops. These eye drops, while having two orders of magnitude lower concentrations of the active ingredient, have demonstrated the ability to deliver the same concentration to the aqueous humor as more traditional eye drop formulations. These results suggest that this eye drop formulation platform technology could be applied to increase ocular delivery of other lipophilic active pharmaceutical ingredients. Commercial Relationships: Ronald A. Wassel, None; Didier Nuno, None; Alexander Quiambao, None; Fadee Mondalek, None; Rafal Farjo, None Support: NIH SBIR Grant 1R43EY021074-01A1

Program Number: 498 Poster Board Number: D1175 Presentation Time: 8:30 AM - 10:15 AM Pharmacokinetics and Biodistribution of Bevacizumab Following

Suprachoroidal Injection into the Rabbit Eye Using a Microneedle Saffar Mansoor 1 , Samikrumar R. Patel 1 , Cetin Tas 2 , Rashmi Pacha-Ravi 3 , Uday B. Kompella 3 , Henry F. Edelhauser 4 , Mark R. Prausnitz 1 . 1 Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA; 2 Pharmaceutical Sciences, Gulhane Military Academy, Ankara, Turkey; 3 Pharmaceutical Sci & Ophthal, University of Colorado Denver, Aurora, CO; 4 Ophthalmology, Emory Univ Eye Center, Atlanta, GA. Purpose: As an alternative to intravitreal injection, this study presents injection of bevacizumab into the suprachoroidal space (SCS) using a hollow microneedle. This minimally invasive approach deposits bevacizumab between the sclera and choroid, which targets drug delivery to its therapeutic site of action. The purpose of this study is to quantify the pharmacokinetics and biodistribution of bevacizumab in the rabbit eye. This is the first study to assess bevacizumab delivery in vivo after injection into the SCS using a microneedle. Methods: Bevacizumab (Avastin®, 1250 µg/50 µl) was injected into the SCS of pigmented rabbits using a metal microneedle measuring 700-800 µm in length inserted 5 mm posterior to the limbus. Rabbits were euthanatized and eyes were enucleated at 15 min, 1 day, and 2 days following SCS injection. Ocular tissues

(sclera, choroid, retina, aqueous humor, vitreous, anterior tissues, lens and optic nerve) were separated. Bevacizumab was then extracted from these tissues and quantified using ELISA. Results: The percent bevacizumab recovered from the eyes was 88.4±0.9% at 15 min, 4.6±0.5% at 1 day, and 0.2±0.1% at 2 days after injection. The distribution of bevacizum in ocular tissues at 15 min after injection was 76% in choroid, 13% in sclera and 2.9% in retina, 1.0% in vitreous, 0.5% in aqueous humor, 0.9% in anterior chamber, 0.6% in lens and 0.1% in optic nerve. After 1 day, drug levels were 34% in choroid, 27% in sclera and 23% in retina, 11% in vitreous, 0.7% in aqueous humor, 1.6% in anterior chamber, 3.8% in lens and 0.3% in optic nerve. After 2 days, the distribution of bavacizum was 0.5% in choroid, 3.3% in sclera, 0.5% in retina, 55% in vitreous, 3% in aqueous humor, 36% in anterior chamber, 1.1% in lens and 0.6% in optic nerve. Conclusions: A microneedle was able to inject bevacizumab into the SCS of rabbit eyes using a minimally invasive procedure. There was a quick biodistribution of bevacizumab into choroid, sclera, and retina, indicating excellent drug targeting. However, choroidal levels of bevacizumab declined very rapidly. The current formulation of bevacizumab is therefore not optimal and a sustained-release formulation of bevacizumab may be needed to achieve sustained delivery to the posterior segment of the eye. Commercial Relationships: Saffar Mansoor, None; Samikrumar R. Patel, 12/767/768 (P), Clearsdie Biomedical (I); Cetin Tas, None; Rashmi Pacha-Ravi,

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

None; Uday B. Kompella, None; Henry F. Edelhauser, None; Mark R. Prausnitz, 12/767,768 (P), Clearside Biomedical (I) Support: NEI grant R24 EY017404

Program Number: 499 Poster Board Number: D1176 Presentation Time: 8:30 AM - 10:15 AM Prolongation of Proparacaine Corneal Anesthesia by an In Situ Cross Linked Hydrogel of Carboxymethylcellulose

Houman D. Hemmati 1,2 , Miguel Manzano-Garcia 2 , Daniel S. Kohane 3 , Robert Langer 2 . 1 Ophthalmology, Mass. Eye & Ear Infirmary, Harvard Medical School, Boston, MA; 2 Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA; 3 Anesthesiology, Laboratory for Biomaterials and Drug Delivery, Children's Hospital Boston, Harvard Medical School, Boston, MA. Purpose: To formulate and characterize a rapid hydrogel-forming eye drop delivery excipient designed to provided extended release of a topical anesthetic drug to the ocular surface. Methods: Periodate oxidation of carboxymethylcellulose (CMC) was performed to generate aldehyde-modified CMC (CMC-ALD). Modification of CMC with adipic dihydrazide (ADH) was performed to generate ADH-modified CMC (CMC-ADH). Separate 0.5% proparacaine solutions were created in phosphate-buffered saline (PBS) with CMC-ALD, CMC-ADH, unmodified CMC, and without CMC. 1:1 mixtures of CMC-ALD and CMC-ADH solutions were created to test hydrogel formation time, adhesiveness, and optical clarity. 25 uL of each of the aforementioned solutions were applied to the corneas of adult male Sprague Dawley rats, with one group of animals receiving 25 uL of PBS alone as a negative control (n=6 rats per group). Hydrogel eyes received 12.5 uL each of CMC-ALD and CMC-ADH simultaneously. Corneal sensation was measured by Cochet- Bonnet esthesiometry 1-2 times a day for 10 days. Results: CMC immediately cross-linked through hydrazone bonds (Figure 1) to form optically clear, pliable hydrogels on both glass slides and rat eyes. In situ cross-linked CMC hydrogel prolonged the duration of topical anesthesia by at least 9 days, significantly longer than unmodified CMC (p<0.05). Conclusions: In situ cross-linked hydrogels of CMC can potentially be used to dramatically prolong the duration of topically-delivered ophthalmic drugs.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology (PH) None; Uday B. Kompella ,

Commercial Relationships:

Houman D. Hemmati, In-situ crosslinked CMC

Hydrogels (P); Miguel Manzano-Garcia, None; Daniel S. Kohane, In situ- crosslinked CMC Hydrogels (P); Robert Langer, In situ-crosslinked CMC Hydrogels (P)

Support: Boston Keratoprosthesis Funds

Program Number: 500 Poster Board Number: D1177 Presentation Time: 8:30 AM - 10:15 AM Clever Contacts: Using Silicone Hydrogel Materials to Deliver Atropine and Roscovitine for the Treatment of Childhood Ocular Conditions

Vida Rahmani 1A , Frances Lasowski 1B , Heather Sheardown 1A . A Chemical Engineering, B School of Biomedical Engineering, 1 McMaster University, Hamilton, ON, Canada. Purpose: A minimally invasive device is ideal for the delivery of therapeutics to treat childhood ocular conditions, such as roscovitine to prevent retinoblastoma and atropine to retard myopia progression. A polymeric, transscleral delivery vehicle is proposed to reduce side effects by localizing drug delivery, creating a prolonged, controlled delivery profile. Methods: The proposed model materials for these applications are a 90/10 mixture of poly(2-hydroxyethyl methacrylate)/ trimethyl siloxy silane (HEMA-TRIS) and a 50/50 mixture of N,N-dimethylacrylamide/trimethyl siloxy silane (DMAA-TRIS), synthesized by UV. Some materials initially contained drug, which was released prior to uptake studies. Both uptake studies and release studies, done at 37°C, were quantified using UV spectrophotometry. Results: Drug uptake, Figure 1, was dependent on the loading concentration. Initial drug incorporation into the disks did not result in greater uptake, as loading with 0.5 mg/ml in 1:2 Ethanol:PBS solutions yielded 0.027±0.006 and 0.028±0.007 mg roscovitine per mg polymer for 0 mg and 21 mg of initial drug loading respectively. HEMA-TRIS disks showed less uptake than DMAA-TRIS materials, such as 0.1654 mg and 0.2356 mg total uptake respectively after 120 hours in 0.08 mg/ml roscovitine solution. Release profiles for drugs incorporated during synthesis, Figure 2, show sustained release over two weeks with a moderate initial burst. Conclusions: These results show that transscleral delivery via polymeric devices is possible for the delivery of these therapeutics. Sufficient drug loading is likely possible from the initial manufacture of the lenses.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology (PH) None; Uday B. Kompella ,

Commercial Relationships: Vida Rahmani, None; Frances Lasowski, None; Heather Sheardown, None Support: NSERC

Program Number: 501 Poster Board Number: D1178 Presentation Time: 8:30 AM - 10:15 AM Gentamycin Release from Collagen Wafers: A Method to Eliminate the Need for Post Operative Eyedrops

Richard A. Eiferman 1 , Dale P. DeVore 2 , Bruce H. DeWoolfson 3 . 1 Ophthalmology, University of Louisville, Louisville, KY; 2 DV Consulting, Chelmsford, MA; 3 Euclid Systems Corp, Herndon, VA. Purpose: To determine the release kinetics of buffer supernatants collected from collagen wafers containing gentamycin Methods: Collagen wafers were incubated with 1.0 ml of 0.1M PBS, pH 7.0 at 37°C. One milliliter solutions were collected at days 1, 3, 7, 9, 17 and 21 and refreshed with 1.0 ml of PBS. Three individual samples per time period were evaluated for gentamycin concentration utilizing a gentamycin EIA Competitive Enzyme Immunoassay. Results: At 24 hours, there was an initial burst of antibiotic release averaging 240 ug/cc. At day 3, the average was 83.5 ug/cc; at day 7, 30 ug/cc; at day 9, 12.5 ug/cc; and at day 17, 4.5 ug/cc. The wafer completely dissolved at day 21.

Conclusions: The collagen wafer released gentamycin in bacteriocidal levels for up to 17 days. There was a very high release rate of antibiotic in the first 24 hours with sustained levels lasting over two weeks. This in vitro study indicates that subconjunctival placement of an antibiotic infused collagen wafer could supply

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)

sufficient antibiotic in the post operative period eliminating the need for self administation of eyedrops.

Commercial Relationships:

Richard A. Eiferman, euclid (R); Dale P. DeVore,

euclid (C, P); Bruce H. DeWoolfson, euclid (I, E)

Support: None

Program Number: 502 Poster Board Number: D1179 Presentation Time: 8:30 AM - 10:15 AM Cellulose Based Tablet For Sustained Release Of Ilomastat Abeer Mohamed Ahmed 1 , Alastair Lockwood 1,2 , Sahar Awwad 1 , Garima Sharma 1 , Peng T. Khaw 2 , Steve Brocchini 1,2 . 1 UCL School of Pharmacy, University of London, London, United Kingdom; 2 NIHR Biomedical Research Centre for Ophthalmology, Moorfields Eye Hospital & UCL, London, United Kingdom. Purpose: Ilomastat is a matrix metalloproteinase inhibitor (MMPi) that has been shown to inhibit fibrosis after glaucoma filtration surgery in a rabbit model of ocular fibrosis. To reduce scarring and fibrosis following glaucoma surgery and to avoid dose dumping when injected, a sustained dosage form is required that allows a prolonged local concentration of ilomastat to be maintained within the subconjunctival space. Cellulose based excipients such as carboxymethylcellulose (CMC) and hydroxypropylmethylcellulose (HPMC), which have been used in ocular medicine were used to fabricate a small tablet for subconjunctival implantation. Methods: Ilomastat (1.0 mg) was dispersed in water (1.0 mL) and sonicated for 30 min to produce a homogenous suspension. An aqueous solution of CMC (2.0%) or HPMC (2.0%) was prepared by dissolving the desired amount of the polymer in water overnight. The suspension of ilomastat was then added to the aqueous polymer solution (2%, 200.0 µL) and homogenized using Ultra-Turrax homogenizer for 2.0 min. The mixture was freeze dried. The lyophilised powder was pressed into a tablet using 3 mm punch and die. The release of ilomastat from the tablet in phosphate buffered saline (PH 7.4) was determined at flow rate of 2.0 µL at 35.0 °C in a flow rig. The concentration of the released ilomastat was measured using high performance liquid chromatography at 280 nm. Results: Ilomastat was successfully fabricated into a tablet that contains a dispersion of a crystalline form of ilomastat in a cellulose based polymer matrix. The weight of the tablet was in the range of 4.5-6.5 mg. Fabrication of the ilomastat tablet using cellulose derivatives (CMC and HPMC) resulted in a sustained release of ilomastat over 10 days. The tablets were prepared using CMC (2.0%), HPMC (2.0 %) and a mixture of CMC/HPMC (1:1). The release profile of ilomastat was 71.8, 52.9 and 66.3 % respectively after 242.8 h (10 days). The concentration of the released ilomastat was in the range of 44.7-200.2 (CMC), 11.8-193.6 (HPMC) and 48.3-162.3 µM (CMC/HPMC 1:1) over a period of 10 days, which was within the therapeutic range (10-100 µM). Conclusions: Sustained release of ilomastat was achieved by its fabrication into a cellulose based tablet. The release period is prolonged, which may be suitable for a successful sub-conjunctiva implant for improvement of the outcome of glaucoma filtration surgery. Commercial Relationships: Abeer Mohamed Ahmed, None; Alastair Lockwood, None; Sahar Awwad, None; Garima Sharma, None; Peng T. Khaw, WO09/063222 (P); Steve Brocchini, WO09/063222 (I) Support: Medical Research Council G801650, Fight for Sight, Freemasons Grand Charity, NIHR Biomedical Research Centre for Ophthalmology,Helen Hamlyn Trust.

Program Number: 503 Poster Board Number: D1180 Presentation Time: 8:30 AM - 10:15 AM Development Of Drug Loaded Nanofiber Sutures For Ophthalmologic Application Fabiana K. Kashiwabuchi 1A , Murilo W. Rodrigues, Jr. 1A , Shuming Zhang 2A , Himatkumar Patel 1B , Jesus S. Vidaurri-Martinez 1A , Qingguo Xu 1B , Justin Hanes 2B , Jiangxia Wang 1C , Hai-Quan Mao 2A , Peter J. McDonnell, III 1A . A Ophthalmology, B Nanomedicine, C School of Public Health, 1 Johns Hopkins Hospital, Baltimore, MD; A Materials Science & Engineering, B Nanomedicine, 2 Johns Hopkins University, Baltimore, MD. Purpose: To fabricate functional nanofibers containing antibiotic drugs and evaluate the possible application of the fibers as sutures capable of drug delivery after ophthalmologic surgery. Methods: Levofloxacin and Poly (L-lactic acid) (PLLA) were used to create the functional nanofibers, employing electrospinning process. Polymer and drug were dissolved in chloroform and the electrospinning applied. For the drug release test, four milligrams of fibers were weighed, placed in tubes filled with buffered saline and kept in incubator shaker at 37°C for 10 days. The entire drug release test was carried out in triplicate. At selected intervals, supernatant was tested with High Performance Liquid Chromatography to quantify the drug release. The fibers were morphologically evaluated by Scanning electron microscope and the tensile strength measured. Antibacterial efficacy was performed against Staphylococcus epidermidis, by placing a piece of fiber on agar plate and incubating overnight at

37°C.

Results: HPLC demonstrated drug release within the first hour followed by a sustained release (p<.0001) for at least ten days. Inhibition zone testing showed antibacterial efficacy against S. epidermidis. The suture average diameter was 60.2 ±13.6 µm and the tensile strength 0.148 ± 0.036 N. Conclusions: Levofloxacin, a third-generation fluoroquinolone, was released from the nanofibers and showed activity against S. epidermidis, one of the most common bacteria residing on the ocular surface. The PLLA used in these experiments is a bioabsorbable and biodegradable synthetic polymer and has been shown to have no cellular toxicity. This study showed that drug loaded nanofibers sutures have potential application as a new drug delivery system in ophthalmic surgery. Commercial Relationships: Fabiana K. Kashiwabuchi, None; Murilo W. Rodrigues, Jr., None; Shuming Zhang, None; Himatkumar Patel, None; Jesus S. Vidaurri-Martinez, None; Qingguo Xu, None; Justin Hanes, None; Jiangxia Wang, None; Hai-Quan Mao, None; Peter J. McDonnell, III, None Support: None

Program Number: 504 Poster Board Number: D1181 Presentation Time: 8:30 AM - 10:15 AM Electrohydrodynamic Spray Drying Technique for Moxifloxacin

Microencapsulation Delivery Systems Qiongyu Guo 1 , Ahmed Aly 1 , Oliver D. Schein 2 , Jennifer H. Elisseeff 1 . 1 Biomedical Engineering, Johns Hopkins University, Baltimore, MD; 2 Ophthalmology, Johns Hopkins Wilmer Eye Inst, Baltimore, MD. Purpose: To develop an electrohydrodynamic spray drying technique to fabricate moxifloxacin microparticle systems in order to achieve sustained antibiotic release for ocular treatments. Methods: Moxifloxacin-loaded PLGA microparticles were prepared using an electrohydrodynamic spray drying (electrospraying) technique. The antibiotic, Moxifloxacin HCl, was encapsulated in poly (lactic-co-glycolic acid) (PLGA) by dissolving both reagents in different solvents and electrospraying the solution using high voltages ranging from 11-13 kV. The microparticles were collected using distilled water, stored at -80 °C then lyophilized. The microparticles were then encapsulated into bioadhesive hydrogels: chondroitin sulfate-polyethylene glycol (CS-PEG) bioadhesive. The morphologies of the microparticles were examined using scanning electron microscopy (SEM). The release of Moxifloxacin was tested in vitro by submerging the vehicles in PBS buffer solution, taking samples at different time intervals and refreshing the solution. Drug concentration was determined using high performance liquid chromatography (HPLC). Results: In order to achieve an optimal, controlled release of moxifloxacin, we encapsulated the antibiotics in PLGA-based microparticles by carefully selecting solvent systems for electrospraying processing. The release speed of moxifloxacin using the solvent of methanol:dichloromethane (MeOH:DCM)=10:90 was found to be close to the one using the solvent of MeOH:DCM=20:80, while the release speed using the solvent of MeOH:DCM=30:70 was much slower than the other two solvent ratios. All of these conditions showed an effective release over ten days with the release concentration continuously higher than the minimum inhibitory concentration (MIC) (Figure 1). In contrast, the Moxifloxacin loaded in hydrogels was released rapidly within 24 hours. Conclusions: This study fabricated surfactant-free antibiotic-loaded polymeric microparticles using an electrospraying technique and achieved sustained release of Moxifloxacin HCl over more than ten days. A delivery system which incorporates a bioadhesive may potentially integrate antibiotic prophylaxis and wound healing.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Physiology/Pharmacology (PH) sufficient antibiotic in the post

Commercial Relationships: Qiongyu Guo, None; Ahmed Aly, None; Oliver D. Schein, None; Jennifer H. Elisseeff, None

Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group Physiology/Pharmacology (PH)