Physiology Pharmacology

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

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 [email protected].

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



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

Torres1, Andrea Luchini

2, Antonio M. Casella

3, Caroline Torres

4, Lucas Torres

4,

Robson Torres4.

1Retina Vitreous, Pontificia Universidade Catolica do Parana,

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

3Ophthalmology, University of East London, Londrina, Brazil;

4Universidade

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 (p90 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



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



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



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-

all media was removed and replaced with one of the following serum-free

conditions: Control media, DPE (1

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

-test with p



Francis3, Todd Meyer

3.

1Regeneron Pharmaceuticals, Tarrytown, NY;

2Sanofi,

France, Toulouse, France; 3Center 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 -mediated platelet activating capability of Bev:VEGF and VEGF Trap:VEGF complexes.

Methods: Stoichiometry of binding for Bev and VEGF Trap to VEGF165 was

measured using multi-angle laser light scattering. Platelet activation was measured

using platelet aggregometry & 14

C-serotonin release assays (SRA). Animal studies

-formed 1:1 molar mixtures of Bev or

VEGF Trap with VEGF165 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:VEGF165 complexes were heterogeneous and predominantly

multimeric (~400kDa to >2,000 kDa). In contrast, VEGF Trap formed a

homogeneous 1:1 binding complex with VEGF165, with a M.W. of 155 kDa.

Platelet aggregometry and SRA showed that preformed equal molar Bev:VEGF165 complexes (100-500 nM) plus heparin (0.5-50 ug/ml) activated platelets, while

VEGF Trap:VEGF165

fold relative to VEGF) resulted in smaller complexes that did not trigger platelet activation. Injection of pre-formed VEGF Trap:VEGF165 complexes with heparin

vs saline-injected controls (1173179). Animals receiving Bev+VEGF165

complexes with heparin exhibited behavior consistent with thrombotic shock and

experienced profound thrombocytopenia (platelet count of 331217; P < 0.001).

Lung sections from these animals showed patterns of occlusive thrombi.

Conclusions: Bev when mixed at near equal molar ratios with VEGF165, 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);

Franoise 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



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



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 g/ml and cell viability was analysed by WST-1 assay.

Hypoxia was induced through cobalt chloride (200 M) and the effect of

resveratrol (4 and 12g/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 (200M of cobalt chloride), cell proliferation

increased to 129.31.13% whereas it decreased to 125.61.02% after the addition

of resveratrol at 4g/ml and 86.12.16% at 12g/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 M concentration of cobalt chloride

which decreased to 3.8 and 2.5% after the addition of 4 and 12 g/mL of resveratrol, respectively. (P



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



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



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

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: 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

ti-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



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



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 Mesquida1, Blanca Molins

2.

1Ophthalmology, Hospital Clinic, Barcelona,

Spain; 2Ophthalmology, 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



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



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 Ayurvedas 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 CuromTM

shows very good 24-hours bioavailabilities.

In the 66 patients treated with AREDS2 plus CuromTM

, there was a highly significant reduction of drusen size between baseline and after 6 months

(p



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.

APharmaceutical Sciences,

Ophthlamology and Bioengineering, BPharmaceutical Sciences,

1University 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 %

es 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

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



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.

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

Desai Ph.D., Genentech Inc (F)

Support: Genentech Grant 71868-01



Therapeutic Effect of Fenofibrate Eyedrops on Diabetic Retinopathy Ying Chen

1, Boyu Lu

1,2, Qingjiong Zhang

2, Jianxing Ma

1.

1Physiology, OUHSC,

Oklahoma City, OK; 2Sun 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.



Development of an Optimized Culture Media to Improve VEGF Antagonist

Secretion and Stability by Encapsulated Cell Technology Implants



Alline M. Lelis1A

, Michael Rivera1A

, Sue Elliott1A

, Brenda Dean1A

, Pam Heatherton

1A, Bruce Bouchard

1A, Melissa Stiles

1A, Vicent Ling

1A, Konrad Kauper

1B,

Weng Tao1.

AResearch and Development,

BCore Technology Development,

1Neurotech 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 GlutaMAXTM

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



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 LucentisTM

, AvastinTM

and EyleaTM

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



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.

1Ophthalmology, UCSD Jacobs Retina Center, La Jolla, CA;

2Chemistry and

Biochemistry, UCSD, La Jolla, CA; 3Spinnaker 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 37C. 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.



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.

Backenroth1.

1Ohr Pharmaceutical Inc, New York, NY;

2Elman 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



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



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.

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


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. Salmon1, Sydney Cartiff

1, Skip Ballou

2, Corey Ballou

2, Brian C. Gilger

1.

1Clinical Sciences, North Carolina State University, Raleigh, NC;

2Corinthian

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




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



Transscleral Iontophoretic Delivery of a Macromolecule into the Rabbit Eye John Higuchi

1, Kongnara Papangkorn

1,2, Sarah Molokhia

2, Donald Mix

1,

Charlotte Butler1, Prasoona Karra

1, Balbir Brar

1, S. Kevin Li

1,3, William I.

Higuchi1,2

. 1Aciont Inc, Salt Lake City, UT;

2University of Utah, Salt Lake City,

UT; 3University 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/cm2), the total amount IgG delivered

was 438 63 and 364 27 g with 400 L and 200 L IgG solution volumes in

Visulex system, respectively. At 8 mA (current density = 3.6 mA/cm2), the total

amount IgG delivered was 727 38 and 457 94 g with 400 L and 200 L

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 g 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



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 Stiles1B

, Karen Ahola1A

, Alice Lee1B

, Konrad Kauper1C

, Bruce Bouchard1D

,

Weng Tao1E

. AProcess Development,

BQuality Control,

CEngineering,

DHistology,

EReasearch and Development,

1Neurotech 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 37C 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


Ternary Complex With Hp --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--cyclodextrin (HP--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. 1H- and

13C- NMR experiments

were undertaken to verify the real inclusion of ACZ in the

ACZ-HP--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--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--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--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



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



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



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 -80C. 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 100L plasma aliquot and a 50L 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



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. Robinson1A

, Jie Shen1.

AOphthalmology,

1Allergan, Irvine, CA;

2ANSYS,

Evanston, IL; 3ANSYS, 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.

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



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

Katsukura1A

, Machiko Sato1A

, Yumi Ishikawa1A

, Toru Nakazawa1A

, Matsuhiko



Nishizawa1B

, Toshiaki Abe1A

. AGraduate School of Medicine,

BGraduate School of

Engineering, 1Tohoku 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



Delivery Of Dexamethasone To The Posterior Segment Of The Eye Using An

Eye Gel Formulation Vincenzo Papa

1A, Elena Solfato

1B, Claudine Civiale

1B.

AMedical Marketing-BU

Pharma, BPharma Tech & Anal Chem-BU Pharma,

1SIFI 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 l 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. CMAX and AUCALL were calculated for

each tissue. Data obtained from control eyes were considered expression of systemic absorption.

Results: CMAX (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. AUCALL (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 AUCALL in

study eyes was statistically significant higher (p



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



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

Ling1A

, Konrad Kauper1B

, Weng Tao1.

ADevelopment,

BCore Technology

Development, 1Neurotech 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

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 g/day, maintaining a vitreous

steady state concentration of approximately 25 g/eye. Bevacizumab injections

have been shown to maintain vitreous steady state concentrations of > 5 g/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



Suppression of Rat Choroidal Neovascularization by Transscleral Vasohibin-1

Delivery Device Hideyuki Onami

1A, Nobuhiro Nagai

1A, Ryosuke Wakusawa

1A, Hirokazu Kaji

1B,

Takuya Yamada1B

, Yumi Ishikawa1A

, Matauhiko Nishizawa1B

, Yasufumi Sato1A

, Toru Nakazawa

1A, Toshiaki Abe

1A.

AGraduate school of medicine,

BGraduate school

of engineering, 1Tohoku univercity, Sendai, Japan.

Pur

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