ARVO 2016 Annual Meeting Abstracts
112 Ocular Toxicology/New drug
Sunday, May 01, 2016 8:30 AM–10:15 AM
Exhibit/Poster Hall Poster Session
Program #/Board # Range: 269–301/B0302–B0334
Organizing Section: Physiology/Pharmacology
Contributing Section(s): Low Vision
Program Number: 269 Poster Board Number: B0302
Presentation Time: 8:30 AM–10:15 AM
Corneal Toxicity of ABT-414 in Glioblastoma (GBM): Clinical
Manifestations, Ophthalmological Findings and Management
Marian Macsai1, Ashiyana Nariani2, Hui Gan3, Andrew Lassman4,
Ryan Merrell1, Erica Gomez5, Rajendar Mittapalli5, Hao Xiong5,
Christopher Ocampo5, Kyle Holen5, Golnaz Moazami6. 1NorthShore
University Health System, Evanston, IL; 2Duke University Eye
Center, Durham, NC; 3Austin Health and Olivia Newton-John
Cancer Research Institute, Melbourne, VIC, Australia; 4Department
of Neurology and Herbert Irving Comprehensive Cancer Center,
Columbia University Medical Center, New York, NY; 5AbbVie
Inc., North Chicago, IL; 6Harkness Eye Institute Columbia Medical
Center, New York, NY.
Purpose: ABT-414 is an antibody-drug conjugate consisting of a
toxin, MMAF (monomethyl auristatin F), linked to an anti-EGFR
antibody (ABT-806). Although there is promising antitumor activity
of ABT-414 in GBM, it has been associated with corneal toxicity
(CT). The pathophysiology of these toxicities is poorly understood.
Herein we report the manifestations and management of CTs
associated with ABT-414 in GBM.
Methods: M12-356 (NCT01800695) is an open-label, phase 1, 3-arm
study: Arm A (ABT-414+radiation (RT)/ temozolomide (TMZ) in
newly diagnosed GBM (nGBM)), Arm B (ABT-414+TMZ either in
nGBM as adjuvant therapy, or in recurrent GBM (rGBM)) and Arm
C (ABT-414 monotherapy in rGBM). Severity of each ocular adverse
event (AE) was rated according to NCI CTCAE Version 4.1. The
dose-toxicity relationship was analyzed by logistic regression and the
onset of CTs was determined by Kaplan-Meier analysis.
Results: As of September 29, 2015, total number of patients reported
with one or more ocular treatment-emergent AEs (TEAEs) were
40/45 (89%, Arm A), 42/48 (88%, Arm B) and 59/74 (80%, Arm
C). Most common TEAEs (≥25% in at least one arm) for Arms
A/B/C respectively, were blurred vision (64%/52%/53%), dry
eye (36%/17%/24%), keratitis (33%/25%/20%) and photophobia
(33%/38%/24%). Most common Grade (G) 3/4 TEAEs (≥10%
in at least one arm) were keratitis (13%/15%/14%) and blurred
vision (11%/6%/4%). Overall percent ocular G3/4 TEAEs were
27%/25%/28%. An apparent dose-toxicity relationship was
observed in each arm. Median onset times of G2 or higher CTs were
comparable among all arms at recommended phase 2 doses, ranging
from 49-65 days. Manifestations included microcystic keratopathy,
corneal crystals, stromal opacities, limbal stem cell deficiency,
punctate epithelial erosion, and filament/corneal abrasions.
Management included prophylactic dexamethasone or prednisolone
acetate (PA) eye drops, and treatment with bandage contact lenses,
punctal plugs, topical preservative-free artificial tears, antibiotics,
PA, and/or cyclosporine eye drops. For G3/4 toxicities, ABT-414 was
held until toxicity resolved and re-treatment began at a lower dose.
Conclusions: Frequent CTs were observed with ABT-414 treatment
in GBM. However, the promising antitumor activity of ABT-414 in
GBM warrants further evaluation into strategies to help manage or
prevent these toxicities.
Commercial Relationships: Marian Macsai, None;
Ashiyana Nariani, None; Hui Gan, Merck Serono (R), Pfizer
(R), AbbVie, Inc. (R), Ludwig Institute for Cancer Research (S),
AbbVie, Inc. (F); Andrew Lassman, Celldex (F), Novartis (F),
Medimmune (F), Boehringer Ingelheim (F), Heron (R), Karyopharm
(F), Amgen (F), Genentech (R), Agenus (F), AbbVie (F), AbbVie,
Inc. (R), Novocure, Stemline, E-Therapeutics, Millennium (F),
Genentech (F), Plexxicon (F), Pfizer (F), Regeneron (R), Novartis
(R), NW Biotherapeutics (F), Angiochem (F), Foundation Medicine
(R); Ryan Merrell, None; Erica Gomez, AbbVie, Inc. (I),
AbbVie, Inc.; Rajendar Mittapalli, AbbVie, Inc. (I), AbbVie, Inc.;
Hao Xiong, AbbVie, Inc. (I), AbbVie, Inc.; Christopher Ocampo,
AbbVie, Inc. (I), AbbVie, Inc.; Kyle Holen, AbbVie, Inc. (I), AbbVie,
Inc.; Golnaz Moazami, None
Clinical Trial: NCT01800695
Program Number: 270 Poster Board Number: B0303
Presentation Time: 8:30 AM–10:15 AM
Volumetric Single-Layer Inner Retinal Analysis in Patients with
Hydroxychloroquine Toxicity
Vishal Parikh1, Yasha Modi1, Adrian Au2, Justis P. Ehlers1,
Andrew Schachat1, Rishi P. Singh1. 1Ophthalmalogy, Cole Eye
Institute - Cleveland Clinic Foundation, Cleveland, OH; 2Case
Western Reserve School of Medicine, Cleveland, OH.
Purpose: This is a retrospective, observational study to evaluate
single layer retinal volumes using spectral-domain optical coherence
tomography (SD-OCT) between eyes with hydroxychloroquine
(HCQ) toxicity and controls to better understand changes in the
various retinal layers in
Methods: Using a previously validated algorithm, automated
volumetric analysis of the ganglion cell layer (GC), inner plexiform
layer (IPL), inner nuclear layer (INL), and outer retina [outer
plexiform layer to retinal pigment epithelium (OPL-RPE)] layers
from the SD-OCT macular cube were compared in three sets of 14
patients: patients with a clinical diagnosis of HCQ toxicity, agematched patients taking HCQ but not manifesting overt toxicity, and
age-matched controls.
Results: The GC (p=0.01), IPL (p=0.004), INL (p<0.001), and
OPL-RPE (p<0.001) volumes were significantly reduced in HCQ
toxicity eyes relative to the HCQ exposure eyes. There were no
significant inner and outer retinal volume differences between the
HCQ exposure group and group with no HCQ use (p > 0.05 for all
layers). Increasing disease severity correlated with increasing volume
loss in the inner (2.27 mm3 in early disease vs. 1.78 mm3 in advanced
retinopathy, p = 0.02) retina.
Conclusions: HCQ toxicity appears to result in both outer and inner
retinal volumetric thinning compared to age-matched controls and
patients taking HCQ but not manifesting toxicity.
Commercial Relationships: Vishal Parikh, None; Yasha Modi,
None; Adrian Au, None; Justis P. Ehlers, None; Andrew Schachat,
None; Rishi P. Singh, None
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Program Number: 271 Poster Board Number: B0304
Presentation Time: 8:30 AM–10:15 AM
Efficacy of Rat Primary Cells transfected with Pigment
Epithelium-Derived Factor using the Sleeping Beauty Transposon
system in choroidal neovascularization
Maria Hernandez1, 2, Laura Garcia-Garcia1, 2,
Sergio Recalde1, 2, Patricia Fernandez1, 2, Juan R. Rodriguez3, 2,
Jaione Bezunartea-Bezunartea1, 2, Daniel Scherman4,
Sandra Johnen6, Gabriele Thumann5, Alfredo Garcia-Layana1, 2.
1
Experimental Ophthalmology laboratory, Clinica Universidad de
Navarra, Pamplona, Spain; 2Idisna, Navarra Institute for Health
Research, Pamplona, Spain; 3Cell Therapy Area. Division of Cancer,
Center of Aplied Medical Research (CIMA), Pamplona, Spain;
4
Unité de Technologies Chimiques et Biologiques pour la Santé,
INSERM U1022 – CNRS UMR8258, Paris, France; 5Département
des neurosciences cliniques, Service d’ophtalmologie, Hôpitaux
universitaires de Genève, Genève, Switzerland; 6Department of
Ophthalmology, University Hospital RWTH Aachen, Aachen,
Germany.
Purpose: The objective was to analyze the effect of PEDF
release using the non-viral Sleeping Beauty (SB100X) transposon
system (pFAR4-ITRs CMV PEDF BGH/pFAR4-CMV SB100x
SV40) in rat primary cells in a model of laser-induced choroidal
neovascularization (CNV).
Methods: Retinal pigment epithelia cells (RPEs) and iris pigment
epithelia cells (IPEs) were transfected with pFAR4-pigment
epithelium-derived factor (PEDF) plasmid. Laser-induced CNV was
performed and 48h later the transfected cells were injected in the
subretinal space of Brown Norway rats. Follow up of the lesions was
assessed by fluorescein angiographies (FA) at 7 and 14 days post
laser. The animals were sacrified at 5 and 12 days post injection.
Flat mounts were labeled with isolectin, retinal cross sections
were subjected to immunofluorescence technique with microglial/
macrophage markers (isolectin, Iba1, CD68) and visualized under a
confocal microscope.
Results: Transplantation of PEDF-transfected cells subretinally
showed feasibility of the procedure. A significant reduction of FA
leakage was observed in cell injected groups mainly 14 days after
laser. Microglial and macrophage cell activation was localized in
the angiogenesis area after subretinal injection. Finally, a significant
reduction of neovascularization was observed in the CNV area
labelled with isolectin marker.
Conclusions: The non-viral Sleeping Beauty (SB100X) transposon
system for PEDF secretion was effective in reducing the area of
leakage and CNV volume and area. This is an important step for
the establishment of a safe, specific and effective gene therapeutic
treatment for angiogenic retinal diseases such as exudative agerelated macular degeneration.
Commercial Relationships: Maria Hernandez, None;
Laura Garcia-Garcia, None; Sergio Recalde, None;
Patricia Fernandez, None; Juan R. Rodriguez, None;
Jaione Bezunartea-Bezunartea, None; Daniel Scherman,
None; Sandra Johnen, None; Gabriele Thumann, None;
Alfredo Garcia-Layana, Bayer (C), Allergan (C), Novartis (C), Thea
(C), Alcon (C)
Support: FP7 HEALTH 2012-305134. LGG received ADA
predoctoral grant from University of Navarra
Program Number: 272 Poster Board Number: B0305
Presentation Time: 8:30 AM–10:15 AM
Autoimmune Optic Neuropathy Transiently Reversed with
Expanded Visual Fields Following Rituximab in a Patient with
Monoclonal B Lymphocytosis
April Marquardt, Steven K. Lundy, John R. Heckenlively.
Ophthalmology, University of Michigan, Ann Arbor, MI.
Purpose: To perform a within subject prospective study of an optic
neuropathy (ON) patient who was treated with rituximab.
Methods: A 56-year-old woman with night blindness, peripheral
vision loss, and no family history of retinal degeneration. Clinical
exam: Relatively normal fundus and ERG, narrow Goldmann visual
fields (GVF), and thickening of the nerve fiber layer at the optic nerve
head. Blood tests revealed anti-CCP antibodies and a high level of B
lymphocytes. Diagnosis of optic neuropathy secondary to monoclonal
B lymphocytosis and rheumatoid arthritis was made. Patient was
treated with 4 doses of rituximab (375 mg/m2) to deplete B cells.
Visual exams and immunological tests were performed bimonthly
over six months. Flow cytometry tracked B and T lymphocyte
subsets, and cell activation markers. Western blots measured antiretinal (ARA) and anti-optic nerve (AONA) antibodies. Plasma
anti-recoverin antibody titers were determined by ELISA. Blood
mRNA was analyzed for immune response genes using NanoString
technology. T cell responses toward recoverin were measured by
cytokine output in cell cultures.
Results: Before treatment, the patient had 77.2% CD19+ B cells in
blood (normal: 12.0±9.1%), which were 67.8% CD5+CD43+CD27+
cells (normal: 3.1±1.7%). There were six ARA bands, one AONA
band (~19kDal), a high titer of anti-recoverin IgG, and a very strong
IFNg+ TH1-type immune response toward recoverin. Immediately
after depletion (B cells <1% PBL), GVF improved and the recoverinspecific IFNg response decreased 19-fold. ARA were modestly
reduced but the AONA band increased in intensity. Two months
after treatment, GVF expanded 4-fold versus pretreatment and B
cells remained low (1.1%). However, a spike in TH1 response was
observed, accompanied by an increase in circulating effector memory
TH cells, activated CD69+CD8+ T cells, and CD56+CD3neg natural
killer (NK) cells. Four months after treatment, the monoclonal B cell
subset began to repopulate, NK cells remained elevated and GVF
declined, returning to the pretreatment levels.
Conclusions: This study demonstrates the utility of performing
immunologic tests on patients with non-classical presentations of
retinal degeneration, and the potential of B cell depletion for therapy
in cases in which autoimmunity and B lymphocyte abnormalities are
present.
Commercial Relationships: April Marquardt, None;
Steven K. Lundy; John R. Heckenlively, AGTC (F)
Program Number: 273 Poster Board Number: B0306
Presentation Time: 8:30 AM–10:15 AM
Age-related choroidal and retinal changes in Sprague-Dawley
and Fischer 344 Rats
Malinda E. Fitzgerald1, 2, Chunyan Li1, Nobel Del Mar1,
Ryan W. Piche3, Anton Reiner1, 4. 1Anatomy and Neurobiology,
UTHSC, Memphis, TN; 2Biology, Christian Brothers University,
Memphis, TN; 3Southern College of Optometry, Memphis, TN;
4
Ophthalmology, UTHSC, Memphis, TN.
Purpose: Choroidal blood flow (ChBF) is adaptively controlled to
maintain stable flow despite variations in systemic blood pressure,
a phenomenon called baroregulation. We examined if ChBF
baroregulation changes with age in both Sprague-Dawley (SD)
and Fischer-344 (F344) rats, and whether any such changes were
associable with changes in retinal structure and function.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Methods: The following assessments were made in male SD rats
from 120-500 days of age and male F344 rats from 140-750 days of
age: 1) the flash-evoked scotopic electroretinogram (ERG); 2) ChBF
as measured by Laser Doppler Flowmetry while also monitoring
systemic arterial blood pressure (ABP); 3) visual acuity and contrast
sensitivity as measured using optomotry (F344 only); and 4) the
thickness of the retina and its individual layers in plastic-embedded
sections as measured using Neurolucida morphometric analysis.
Results: ChBF was uncorrelated with ABP (i.e. showed
baroregulation) during its fluctuations above and below basal ABP in
young (120-200 day old) SD rats, but became increasingly correlated
with ABP as SD rats aged, so that by about a year of age ChBF
tended to change linearly with ABP. Associated with the age-related
progressive loss in ChBF baroregulation in SD rats was a progressive
age-related decline in both the scotopic ERG b-wave amplitude
and a-wave amplitude, and a thinning of the retina, particularly the
inner plexiform layer and outer nuclear layer. For F344 rats, ChBF
was highly correlated with ABP already in 170 day-old rats – i.e.
baroregulation was already failing in young F344 rats. Acuity,
contrast sensitivity and a-wave deficits and ONL thinning were
evident by 400 days, and ERG b-wave deficits and additional acuity,
contrast sensitivity and a-wave loss and ONL thinning were noted by
660 days.
Conclusions: These studies indicate that ChBF compensatory
baroregulation declines with age in SD rats, and its loss is correlated
with functional and morphological decline in the retina. By contrast,
impaired baroregulation is already evident in young F344 rats,
before loss in vision and ERG is seen and before retinal thinning.
This early loss of baroregulation in F344 rats may be a factor in their
accelerated and severe subsequent functional decline and loss of
photoreceptors. Our studies highlight the important role age-related
failure in ChBF baroregulation may play in age-related disruption in
retinal health and function.
Commercial Relationships: Malinda E. Fitzgerald, None;
Chunyan Li, None; Nobel Del Mar, None; Ryan W. Piche, None;
Anton Reiner, None
Support: The Methodist Hospitals Professor of Neuroscience,
RO1EY05298 (AR), 5P30EY13080 (D. Johnson), Research to
Prevent Blindness (MECF), NSF DUE 9850780, Southern College
of Optometry Summer Research Fellow (RP), and The University of
Tennessee Neuroscience Institute (CL).
Program Number: 274 Poster Board Number: B0307
Presentation Time: 8:30 AM–10:15 AM
Anti-VEGF Drug Interference in the R&D Systems Quantikine
VEGF-A ELISA Kit
Albert F. Torri, Camille Georgaros, Ashique Rafique, Giane Sumner.
Bioanalytical Sciences, Regeneron Pharmaceuticals, Tarrytown, NY.
Purpose: To examine the impact of anti-VEGF drugs on the
quantitation of VEGF by the R&D Systems Quantikine VEGF-A
ELISA kit.
Methods: Surface Plasmon Resonance (SPR) experiments were
performed using a Biacore 3000 instrument to determine the relative
binding affinities of the anti-VEGF drugs and the Quantikine
VEGF-A capture antibody. Aflibercept and bevacizumab were
captured on a coupled Protein A chip surface, while ranibizumab
was captured on a coupled anti-human Fab polyclonal antibody chip
surface. The Quantikine VEGF-A capture antibody was captured on
a coupled anti-mouse Fcγ chip surface. Following the capture step,
varying concentrations of test ligand were individually injected over
the surfaces. The Quantikine VEGF-A ELISA was performed as per
manufacturer’s instructions. Separate samples containing 1.3 pM (50
pg/mL) of VEGF were prepared with increasing concentrations of
each of the 3 anti-VEGF inhibitors.
Results: The binding constant (KD) of the Quantikine VEGF-A
capture antibody for VEGF was 1.41 pM, which fell between the KD
of aflibercept (0.490 pM) and the KDs of ranibizumab (46 pM) and
bevacizumab (58 pM). Aflibercept, ranibizumab, and bevacizumab all
interfered with the measurement of VEGF concentrations using the
Quantikine VEGF-A ELISA. However, the IC50 for aflibercept (0.45
pM) was much lower than the IC50 values of either ranibizumab (24
pM) or bevacizumab (138 pM). The observed degree of interference
correlated with the relative binding affinities of the 3 anti-VEGF
inhibitors for VEGF.
Conclusions: Aflibercept, ranibizumab and bevacizumab all caused
assay interference in the Quantikine VEGF-A ELISA. The level
of assay interference was proportional to the binding affinities of
each anti-VEGF inhibitor for VEGF. Aflibercept, with the highest
binding affinity for VEGF A165, interfered with the detection of
VEGF by the Quantikine VEGF-A ELISA to a greater degree than
either ranibizumab or bevacizumab. However, serum concentrations
of each of these drugs following IVT administration all reach
circulating levels that have the potential to negatively affect accurate
measurement of VEGF levels.
Commercial Relationships: Albert F. Torri; Camille Georgaros,
Regeneron Parmaceuticals; Ashique Rafique, Regeneron
Parmaceuticals; Giane Sumner, Regeneron Parmaceuticals
Program Number: 275 Poster Board Number: B0308
Presentation Time: 8:30 AM–10:15 AM
Increased uptake of lipophilic immunosuppressive compounds
in cornea and retina based on solubilization in an aqueous
formulation
Sabine Nakowitsch, Cornelia Kaintz, Philipp Graf,
Marielle Koenig-Schuster, Eva Prieschl-Grassauer,
Angelika Bodenteich, Andreas Grassauer. Marinomed Biotechnology,
Vienna, Austria.
Purpose: Second generation corticosteroids and many macrolide
immunosuppressants show very poor solubility. Hence, these
substances are formulated as dispersions for application to ocular
tissues. However, dissolved drugs permeate faster into different
ocular compartments and are less likely washed out before reaching
therapeutic levels than solid disperse drug particles. Here, we show
that the novel biocompatible aqueous formulation Marinosolv allows
solubilizing of lipophilic drugs like Fluticasone propionate and
Tacrolimus. For visualization of the increased uptake into cornea
and retina of solubilized lipophilic compounds in comparison to
dispersions, fluorescently labeled estradiol was used in porcine exvivo models.
Methods: Fluticasone propionate, Tacrolimus, and fluorescently
labeled estradiol were solubilized in the Marinosolv formulation. The
concentrations of dissolved compounds were determined by standard
HPLC methods. For visualization of the permeation, fluorescently
labeled estradiol was applied as Marinosolv solution or dispersion
onto porcine eyes ex-vivo and the amount of compound was
determined by light scattering microscopy (LSM).
Results: Fluticasone propionate, Tacrolimus and fluorescently labeled
estradiol can be dissolved in Marinosolv, a formulation suitable for
ocular application. An increase of solubility of approximately 200fold was observed for all three compounds. The ex-vivo visualization
experiment with fluorescently labeled estradiol clearly showed that
dispersed estradiol hardly penetrates into eye compartments. In
contrast, Marinolsov solubilized estradiol was detected within the
cornea and the sclera/retina tissues in remarkable amounts.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Conclusions: Utilization of Marinosolv enables the solubilization of
otherwise insoluble compounds for ocular application. The increased
penetration of these compounds into ocular tissues, visualized with
solubilized labeled estradiol, suggests an enhanced tissue availability
of dissolved drugs in comparison to dispersions. The application of
Marinosolv may enable the development of otherwise insoluble drugs
for the treatment of ocular diseases.
Commercial Relationships: Sabine Nakowitsch,
Marinomed Biotechnology; Cornelia Kaintz,
Marinomed Biotechnlogy; Philipp Graf, Marinomed
Biotechnology; Marielle Koenig-Schuster, Marinomed
Biotechnology; Eva Prieschl-Grassauer, Marinomed
Biotechnology; Angelika Bodenteich, Marinomed Biotechnology;
Andreas Grassauer, Marinomed Biotechnology
Program Number: 276 Poster Board Number: B0309
Presentation Time: 8:30 AM–10:15 AM
Functional and Morphological Changes Following Macular
Pucker Surgery After Topical Administration of Indomethacin
0.5%, Bromfenac 0.09%, Nepafenac 0.1%, or Placebo
Raffaele Turano1, Andrea Russo1, Elena Gambicorti1,
Anna Cancarini1, Sarah Duse1, Francesco Morescalchi1,
Ciro Costagliola2, Francesco Semeraro1. 1University of Brescia,
Brescia, Italy; 2Eye Clinic, University of Molise, Campobasso, Italy.
Purpose: Nonsteroidal antiinflammatory drugs (NSAIDs) are
reported to penetrate the vitreous and lower basal prostaglandin E2
level. We investigated the functional and morphological changes in
patients treated with NSAIDs before vitrectomy for macular pucker.
Methods: A prospective, investigator-masked, randomized study was
performed in 64 patients scheduled to undergo 25-gauge vitrectomy.
The patients were randomized 1:1:1:1 to receive indomethacin 0.5%,
bromfenac 0.09%, nepafenac 0.1%, or placebo three times a day one
week before surgery. Main outcome measures were best-corrected
visual acuity (BCVA) and central macular thickness (CMT) findings.
Results: Mean BCVA improvement was 0.24 ± 0.15 LogMAR for
indomethacin, 0.26 ± 0.13 LogMAR for bromfenac, 0.23 ± 0.1
LogMAR for nepafenac, and 0.23 ± 0.1 LogMAR for placebo
(P = 0.87). Mean CMT reduction was 50.2 ± 85 µm for indomethacin,
122.4 ± 63 µm for bromfenac, 65.6 ± 100 µm for nepafenac, and
117.2 ± 164.3 for placebo (P = 0.3). No statistical differences among
the different NSAID groups were noticed in either BCVA or CMT.
Conclusions: Despite the antiinflammatory activity of the NSAIDs
in the vitreous, no functional or morphological improvements were
observed after macular pucker surgery.
Representative OCT scan depicting the macula before (A) and after
(B) macular pucker surgery in a patient in bromfenac group.
Commercial Relationships: Raffaele Turano, None;
Andrea Russo, None; Elena Gambicorti, None; Anna Cancarini,
None; Sarah Duse, None; Francesco Morescalchi, None;
Ciro Costagliola, None; Francesco Semeraro, None
Clinical Trial: NCT02361645
Program Number: 277 Poster Board Number: B0310
Presentation Time: 8:30 AM–10:15 AM
Pharmacological normalization of the complement system
dysregulation in the animal model of enhanced lipofuscin
formation
Konstantin Petrukhin, Boglarka Racz. Department of Ophthalmology,
Columbia University, New York, NY.
Purpose: Complement system dysregulation in the retina plays
a critical role in pathogenesis of dry AMD. Normalization of the
complement system dysregulation may represent a treatment strategy
for dry AMD and, potentially, Stargardt disease. It has been recently
reported that enhanced accumulation of lipofuscin in the eyes of
abca4-/- mice is associated with complement activation. We developed
a novel cyclopentyl fused pyrrolidine Retinol-Binding Protein 4
(RBP4) antagonist as a potential drug candidate for Stargardt disease
and dry AMD. The compound partially restricts retinol supply to
the RPE and induces drastic inhibition of bisretinoid synthesis in
the abca4-/- model. Here we report the data on normalization of the
complement system dysregulation by this drug candidate in the
abca4-/- mouse model of enhanced lipofuscin formation
Methods: Abca4-/- mice were treated with the compound formulated
into a chow at the dose that induces ~80% of serum RBP4 reduction.
Two control groups of vehicle-treated abca4-/- and abca4+/+ animals
were included in the study. Compound dosing was conducted
for 3 months. Immunoblot analysis of retinal extracts as well as
immunofluorescence analysis of retinal sections were conducted in
the treatment and control groups to determine levels of expression for
C3/C3b, CFH, CFD, MCP-1 as well as for C-reactive protein
Results: Significant dysregulation of C3/C3b, CFH, CFD and
C-reactive protein was observed in eyes of the vehicle-treated abca4/mice in comparison to the untreated abca4+/+ controls. Expression
of C3/C3b, CFD and C-reactive protein was significantly increased
in the abca4-/- mice while expression of CFH was significantly
decreased. There was no difference in the MCP-1 expression
between untreated abca4-/- and abca4+/+ animals. Compound dosing
normalized the expression of all dysregulated complement system
components in the treatment group
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Conclusions: Administration of the optimized RPB4 antagonist
normalized complement system dysregulation in eyes of the abca4-/mice. The effect seems to relate to the ability of the drug candidate
to drastically inhibit bisretinoid synthesis in this animal model. In
addition, the drug candidate may directly interfere with the retinolindependent pro-inflammatory signaling function of RBP4 that was
ascribed to this adipokine in alternative experimental systems
Commercial Relationships: Konstantin Petrukhin, The Trustees
of Columbia University in the City of New York (P), iCura Vision (I);
Boglarka Racz, None
Support: This study was supported by NIH Grants U01 NS074476
(to K.P.), P30 EY019007 (Core Support for Vision Research), and
unrestricted funds from Research to Prevent Blindness (New York,
NY) to the Department of Ophthalmology, Columbia University.
Program Number: 278 Poster Board Number: B0311
Presentation Time: 8:30 AM–10:15 AM
Thin Outer Nuclear Layer and Retinal Volume Measurements
with Automated Segmentation on Optical Coherence
Tomography Supports Hydroxychloroquine Toxicity
Akshay Jain1, 3, Zach Dupureur2, Alexandra Almasov4,
Michael S. Tsipursky2, Leanne Labriola2, 5. 1School of Molecular
and Cellular Biology, The University of Illinois Urbana Champaign,
Urbana, IL; 2Ophthalmology, Carle Foundation Hospital, Urbana,
IL; 3The Department of Chemistry, The University of Illinois Urbana
Champaign, Urbana, IL; 4Research, Carle Foundation Hospital,
Urbana, IL; 5Interdisciplinary Health Science Initiative, The
University of Illinois Urbana Champaign, Urbana, IL.
Purpose: Hydroxycloroquine (HCQ) is a frequently used medication
to treat conditions including rheumatoid arthritis, lupus, and
inflammatory joint disease. HCQ toxicity is rare but devastating due
to retinal atrophy. Screening guidelines for HCQ toxicity identify
optical coherence tomography (OCT) as an important tool with
atrophic changes present in the parafoveal macula in confirmed cases.
We hypothesize patients with HCQ toxcicity will show low retinal
volumes (RV) and thickness measurements of the outer nuclear layer
(ONL) on automated segmentation OCT as compared to healthy
controls.
Methods: We performed a retrospective chart review of patients
evaluated at Carle Foundation Hospital with OCT from 20112015 (Spectralis HRA+OCT, Heidelberg Engineering, Germany).
Subjects had a confirmed diagnosis of HCQ toxicity made by a
retina specialist. The mean age of affected subjects was 68 and the
control group was 65. Diagnosis was based on clinical exams and
ancillary testing (including automated visual field tests, fundus
autofluorescence, and OCT). OCT was used for further analysis
by dividing the retina into foveal, parafoveal, and perifoveal
zones with an EDTRS overlay grid and measurements of retinal
thickness and volumes were recorded from each region (Figure 1a).
The segmentation function in Heidelberg Eye Explorer (1.9.10.0)
automatically isolated the ONL (Figure 1b). Cases with image quality
less then 20 were excluded. Age-matched controls were used for
comparison. The two groups were compared using a two-sample
T-Test. Carle Foundation Hospital Institutional Review Board
approved the study.
Results: Thirteen eyes from 7 subjects had confirmed HCQ
retinopathy. Fourteen eyes from 14 subjects with normal retinas
served as controls. Statistical significance between the two groups
was observed in all regions for thickness and volume measurements,
with 95% confidence interval (Table 1).
Conclusions: This study confirms our hypothesis that the ONL is
thinner in patients with HCQ toxicity. ONL thickness and overall RV
serve as a significant comparison tool for patients with HCQ toxicity
as compared to healthy controls. These traits may be a valuable
marker for identifying patients with toxicity. Further investigation
is needed to explore whether a threshold thickness using automatic
segmentation could serve as a screening tool for HCQ retinopathy.
Commercial Relationships: Akshay Jain, None; Zach Dupureur,
None; Alexandra Almasov, None; Michael S. Tsipursky, None;
Leanne Labriola
Support: Neal Fund provided through Carle Foundation Hospital
Program Number: 279 Poster Board Number: B0312
Presentation Time: 8:30 AM–10:15 AM
Laser Guided Catheter for Sub-retinal Injection
M. Ali Nasseri, Mathias M. Maier, Chris P. Lohmann.
Ophthalmology, Klinikum rechts der Isar, Munich, Germany.
Purpose: The rise of Intraoperative optical coherence tomography
(iOCT) as well as interventional intra- and sub-retinal therapies
such as stemcell injection and gene therapy methods have increased
the demand for tools to facilitate precise intraocular operations in
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ARVO 2016 Annual Meeting Abstracts
different retinal layers. In this regard, this work introduces a method
and a tool that allows ophthalmic surgeons to perform augmented
sub-retinal injection.
Methods: Our method consists of a 23G cannula for inserting a
catheter in the region of interest. The cannula comprises two parallel
channels: the first channel is for receiving and guiding the catheter
(Channel: 200μm in diameter – Catheter: 40μm in diameter) to
provide intuitive access this channel has a bent tip; the second
channel or the pointing channel (200μm in diameter) has an optical
fiber that emits a visible laser beam (Safety class I, 40μW) to indicate
the catheter insertion point on the retina. The surgeon uses this tool
manually or with the help of a micromanipulator to precisely inject
drugs, cells or other substances into the sub-retinal regions. This
method is introduced mainly for aged-related macular degeneration
(AMD) treatment but also is applicable to other sub-retinal
interventions.
Results: We prototyped an up-scaled (20G) cannula and we used
a guiding laser pointer (Class II, 120μW, 560nm). We mounted the
cannula on a micromanipulator with 10μm precision. To prove the
concept we performed the vein cannulation procedure, which is
more complicated than sub-retinal injection. In second experiment
we used image-processing techniques to detect the aiming point and
autonomously guide the cannula. With this method we successfully
injected a visible test liquid (blue dye) into the region of interest. The
limiting factor on the precision in these experiments was the size of
the aiming point (100μm). This limiting factor will be mitigated in
the next [23G] prototype.
Conclusions: We proved our concept, “instrumental sub-retinal
injection using guiding channel” on pig eyes. The authors believe
that in order to reduce hardware complications, one can use intraoperative optical and OCT image data to detect the tool direction and
simulate the laser beam. This needs augmented reality to overlay the
simulated aiming point on the surface of the retina in the field view of
the microscope.
Program Number: 280 Poster Board Number: B0313
Presentation Time: 8:30 AM–10:15 AM
Analysis of choroidal effusion fluid using Light’s criteria in acute
systemic lupus erythematous (SLE)
James A. Stefater, Dean Eliott, Leo A. Kim. Ophthalmology,
Massachusetts Eye and Ear Infirmary, Boston, MA.
Purpose: While choroidal effusions are known to be associated with
SLE, the cause is unknown. Some have postulated a transudative
process, either related to hypoalbunemia/hypoproteinema or from
abnormal anatomy. Others have suggested an exudative etiology
from an inflammatory process. In the work here, we investigated the
etiology of an SLE-related choroidal effusion by analyzing the fluid
after surgical drainage using Light’s criteria. Understanding the cause
of fluid accumulation will help guide development of therapies.
Methods: In a patient with bilateral choroidal effusions secondary to
acute SLE (Fig 1), a partial-thickness scleral flap was made under the
effusion site. A small full-thickness scleral hole was then created and
the choroidal fluid was allowed to percolate onto the everted flap. As
the fluid slowly effluxed, the fluid was aspirated from the flap surface
and sent to the lab for analysis using Light’s criteria.
Results: The choroidal fluid from the patient’s right eye contained
3.4 g/dL protein and 187 U/L LDH. The fluid from the left eye was
similar in composition. Using Light’s criteria, the fluid is exudative if
at least one of the following criteria is met: the fluid-to-serum protein
ratio is greater that 0.5, the fluid-to-serum LDH ratio is greater than
0.6, or the fluid LDH level is at least 2/3 the serum LDH upper-limitof-normal. Applying Light’s criteria, the choroidal fluid from both
eyes was exudative (Fig 2).
Conclusions: Light’s criteria was originally developed to help
internists distinguish transudative from exudative pleural effusions.
Using fluid protein and LDH levels, and normalizing to serum levels,
one can predict the cause of fluid accumulation. Here, this technique
was applied to SLE choroidal effusions which revealed a frankly
exudative process. This suggests the effusions in SLE are most
likely related to localized inflammation. It is therefore likely that
the choroid is a primary target of immune complex deposition and
not just responding to the low oncotic pressure. This suggests that
treatment of the eye with an anti-inflammatory medication may be an
effective therapy for SLE-induced choroidal effusions. This technique
could be applied to a wide range of diseases with chroidal fluid
retention, help elucidate disease mechanisms, and guide development
of new therapies.
This image shows the cannula performing injection into the retinal
vessels. The catheter, guiding channel and the laser beam can be seen
here.
Commercial Relationships: M. Ali Nasseri, None;
Mathias M. Maier, None; Chris P. lohmann, None
Fundus images showing 360-degree choroidal effusions
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ARVO 2016 Annual Meeting Abstracts
Commercial Relationships: James A. Stefater; Dean Eliott, None;
Leo A. Kim, None
Program Number: 281 Poster Board Number: B0314
Presentation Time: 8:30 AM–10:15 AM
RT-qPCR quantification of mRNAs specific of Pigment
Epithelium-Derived Factor (PEDF) and Vascular Endothelial
Growth Factor (VEGF) in Rat eyes injected in the subretinal
space with Primary Cells transfected with hPEDF
Sergio Recalde1, 5, Laurence Jeanson-LEH4, Patricia Fernandez1, 5,
Maria Hernandez1, 5, Laura Garcia-Garcia1, 5,
Jaione Bezunartea-Bezunartea1, 5, Daniel Scherman3,
Séverine Pouillot4, Gabriele Thumann2, Alfredo Garcia-Layana1, 5.
1
Experimental Ophthalmology Laboratory, Clinica Universidad
de Navarra, PAMPLONA, Spain; 2Département des neurosciences
cliniques, Service d’ophtalmologie, Hôpitaux universitaires de
Genève, Geneve, Switzerland; 33Unité de Technologies Chimiques
et Biologiques pour la Santé, INSERM U1022 – CNRS UMR8258,
Paris, France; 4Genosafe, Evry, France; 5IdiSNA, Navarra Institute for
Health Research, Pamplona, Spain.
Purpose: The aim of this study was to quantify human and rat
PEDF, rat VEGF mRNAs in a model of laser-induced choroidal
neovascularization (CNV) transfected with SB100X transposase
with PEDF transposon encoding pFAR4 plasmids using real-time
quantitative RT-PCR (RT-qPCR).
Methods: Retinal pigment epithelia cells (RPEs) and iris pigment
epithelia cells (IPEs) were transfected with pFAR4-pigment
epithelium-derived factor (PEDF) plasmid using the non-viral
Sleeping Beauty (SB100X) transposon system (pFAR4-ITRs CMV
PEDF BGH-pFAR4-CMV SB100x SV40) in the subretinal space
of Brown Norway rats. The treatment groups were as follows: not
injected, injected with PBS, injected with the Venus plasmid (GFP),
injected with the human PEDF plasmid or injected with the human
PEDF plasmid + the SB plasmid. Total RNA was extracted from the
injected eyes and purified for reverse transcription. Human PEDF, rat
VEGF and rat PEDF mRNAs were quantified in eyes of rats using
real-time quantitative RTqPCR. Relative quantity from each RNA
was calculated for human and rat PEDF or rVEGF Ct normalized
to Ct of RPL30 and have been determined with the Relative
Quantification software from Applied Biosystems v1.2 after analysis
of each qPCR plate with the SDS v2.3 software.
Results: There were no significant differences in the mRNA
expression in rat VEGF and PEDF for any group analyzed. Human
PEDF was detected only in eyes injected with the hPEDF plasmid,
with or without SB.
Conclusions: Human PEDF was detected in rat eyes injected with
the hPEDF plasmid demonstrating the efficacy of this SB system to
release the PEDF. This system could be useful as an antiangiogenic
therapy for retinal diseases.
Commercial Relationships: Sergio Recalde, None;
Laurence Jeanson-LEH, Genosafe; Patricia Fernandez,
None; Maria Hernandez, None; Laura Garcia-Garcia, None;
Jaione Bezunartea-Bezunartea, None; Daniel Scherman,
None; Séverine Pouillot, Genosafe; Gabriele Thumann, None;
Alfredo Garcia-Layana
Support: FP7 HEALTH 2012-305134. LGG received ADA
predoctoral grant from University of Navarra
Program Number: 282 Poster Board Number: B0315
Presentation Time: 8:30 AM–10:15 AM
Lymphocytic Microparticles Modulate Angiogenic Properties of
Macrophages in Laser-induced Choroidal Neovascularization
Houda Tahiri5, 2, Samy Omri3, 4, CHUN YANG4, 2, Sylvain Chemtob1, 2,
Pierre Hardy1, 2. 1Pediatrics/ Pharmacology, Université de Montréal,
Montreal, QC, Canada; 2Ste-Justine Hospital Research Center,
Montreal, QC, Canada; 3Centre de recherche Maisonneuve
Rosemont, Montreal, QC, Canada; 4Université de Montréal,
Montreal, QC, Canada; 5Pharmacology, Université de Montréal,
Montreal, QC, Canada.
Purpose: Choroidal neovascularization (CNV) is the major
cause of severe vision loss in which choroidal vessels penetrate
retinal pigment epithelium (RPE). The scavenger receptor CD36
mediates antiangiogenic effect, which is highly expressed in RPE,
microvascular endothelial cells and macrophages. Previously, we
have demonstrated that human T-lymphocyte-derived microparticles
(LMPs) are capable of modulating macrophages activities. This study
is aimed to determine whether CD36 is involved in LMPs-induced
antiangiogenic effect in choroidal angiogenesis.
Methods: LMPs were produced from apoptotic human T
lymphocytes after treated with actinomycin D. Gene expression in
LMPs-treated macrophages was evaluated by quantitative RTPCR, RNA array and FACS analysis. The uptake experiment was
performed to determine the involvement of CD36 receptor. In vivo,
the antiangiogenic effect of LMPs was investigated in laser-induced
CNV model. Immunohistostaining was performed to reveal the
angiogenesis-related factors expression by macrophages in CNV
areas. Laser capture micro-dissection using to collect tissues in the
CNV regions followed by PCR
Results: LMPs dose-dependently inhibited macrophages cell growth
without inducing cell death. LMPs pretreated macrophages increased
IL-12, decreased CD206. LMPs-pretreated macrophages exhibited
strong inhibitory effect on endothelial cell growth and this effect was
associated with decreased expression of proangiogenic factor VEGF
and increased antiangiogenic factors TSP-1. LMPs co-localized
with Dil-LMPs, and LMPs uptake by macrophages is reduced by
60% after CD36 antibody treatment. This inhibition consequently
abrogated the effects of LMPs VEGFa and TSP-1 in macrophages.
The role of CD36 in mediating the antiangiogenic effect of LMPs
was demonstrated in mice and human choroidal explants. In vivo,
intravitreal injection of LMPs significantly suppressed laser-induced
CNV this antiangiogenic effect was less effective in CD36 KO mice.
In CNV region LMPs significantly induced expression of IL-12 but
decreased the expression of VEGF and IL-10. Moreover, we found
that JNK-1 expression was increased in LMPs-treated macrophages
Conclusions: The expression of critical angiogenesis-related
genes in macrophages was effectively modulated by LMPs, this
antiangiogenic effect of LMPs-treated macrophages was largely
dependent on CD36.
Commercial Relationships: Houda Tahiri, None; Samy Omri,
None; CHUN YANG, None; Sylvain Chemtob, None;
Pierre Hardy, None
Support: Fonds de recherche du Québec-Santé
Program Number: 283 Poster Board Number: B0316
Presentation Time: 8:30 AM–10:15 AM
ELU Study: Aflibercept decreases the number of follow-up and
intravitreal injection visits compared with ranibizumab, in
patients with exudative (wet) age-related macular degeneration
Frederic Queguiner, Kristina Bezirganyan. Ophtalmology, Hopital
saint joseph, Marseille, France.
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ARVO 2016 Annual Meeting Abstracts
Purpose: The efficacy and safety of Ranibizumab and Aflibercept
are comparable and well known in (wet) age related macular
degeneration (AMD) treatment. Considering their same level of
efficacy, we rather chose one molecule as first line, because of its
incidence in decreasing the number of follow-up (FU) and intravitreal
injection (IVI).
The main objective of this study was to compare the number FU
and IVI visits in patients with AMD, successively treated with
Ranibizumab and Aflibercept.
Methods: We retrospectively enrolled 33 patients (38 eyes) with
AMD (mean age 77±7.7) treated first with Ranibizumab (group1),
and switched to Aflibercept (group 2).
For each patient, we compared the number of FU and IVI visits for
each treatment period. Visual Acuity (VA) evolution was analyzed as
a secondary criteria as well as the reason for “switching”.
Results: The median (min; max) numbers of FU and IVI visits were
respectively 16 (10; 30) and 11 (5; 20) in group 1 (Ranibizumab), and
16 (13;18) and 11 (6; 14) in group 2 (Aflibercept).
The median numbers of monthly FU were respectively 1.0 (0.81;
1.49) versus 0.79 (0.67; 0.86) in group 1 and group 2. This number
was significantly lower with Aflibercept (p(W)=0.0005 and
p(BM)=0.0002).
The median number of IVI by treatment period was 0.67 (0.55; 0.90)
in group 1 and 0.55 (0.45; 0.67) in group 2. This number of IVI
visits also significantly decreased during the Aflibercept treatment
(group 2) (p(W)=0.0049 and p(BM)=0.0041). The VA delta, between
initial and final visits, was calculated for each group and compared.
The mean (+/- SD) evolution of VA was 0.0066±0.2377 in group 1
versus 0.0305±0.1792 in group 2, VA evolution was similar in both
groups (p(W)=0.8113 and p(BM)=0.7886). Whatever the reason
for “switching” from Ranibizumab to Aflibercept (loss of efficacy,
tachyphylaxis, tolerance problems), there was no incidence on the VA
evolution in time.
Conclusions: Our results showed that, Aflibercept could significantly
reduce the number of FU and IVI visits, with the same efficacy than
Ranibizumab. This decrease in visit number could improve patients’
quality of life and reduce surgical risk by reducing the number of
injections. Randomized prospective studies on larger scales are
necessary to confirm these results
Commercial Relationships: frederic queguiner;
Kristina Bezirganyan, None
Support: Partial Grant from Novartis
Program Number: 284 Poster Board Number: B0317
Presentation Time: 8:30 AM–10:15 AM
Degradation pathway and biological activity of heat-stressed
brolucizumab (RTH258)
Rodney Bannwart, Robert Ritter, Ted Chu, Charles Blaylock. Alcon
Labs, a Novartis Company, Fort Worth, TX.
Purpose: Brolucizumab (RTH258) is a humanized single-chain
antibody fragment expressed recombinantly in Escherichia coli and
in development for the treatment of neovascular (wet) age-related
macular degeneration. By targeting human vascular endothelial
growth factor (VEGF), brolucizumab inhibits binding to and
activation of the VEGF receptors, VEGFR1 and VEGFR2, critical
activators of angiogenesis. Brolucizumab is formulated at high
concentration for delivery of up to 6 mg in a 50 μL intravitreal
injection. An implantable micro-volume pump is in development to
deliver the drug to the eye, potentially reducing patient visits and
possibly increasing compliance. The effect of prolonged exposure of
brolucizumab in the device at physiologically relevant temperatures
is unknown; therefore a stability study was conducted to characterize
the molecular profile of brolucizumab after exposure to elevated
temperatures.
Methods: Chemical and biological profiles were generated after heat
treatment of brolucizumab at 40°C for 5 months by ion-exchange
high-performance liquid chromatography (IE-HPLC), size-exclusion
high-performance liquid chromatography (SE-HPLC), sodium
dodecyl sulfate-capillary gel electrophoresis (SDS-CGE), and
VEGF-induced human umbilical vein endothelial cells (HUVEC)
proliferation assay.
The resulting impurities, as well as the parent, were isolated
in parallel by SE-HPLC and IE-HPLC semi-preparative
chromatographies, and further characterized by analytical IE-HPLC
and SE-HPLC, SDS-CGE, and VEGF-induced HUVEC proliferation
assay.
Results: No fragmentation of the brolucizumab molecule was
observed after the heat treatment. Seven charge variants in addition
to the parent were identified by IE-HPLC, and seven higherorder oligomers were partly resolved from parent by SE-HPLC.
The reversibility of the oligomerization was assessed by dilution
of isolated oligomers with vehicle followed by 3-day ambient
temperature treatment and analysis by SE-HPLC. No reversibility in
the soluble and insoluble oligomerization was found. The heat-treated
sample, as well as all isolated charge variants, were efficacious at
inhibiting VEGF-induced HUVEC proliferation.
Conclusions: The results indicate the major degradation pathways
of brolucizumab are both chemical (monomeric) and physical
(aggregation), but brolucizumab remains biologically active after
prolonged exposure to physiologically relevant temperatures.
Commercial Relationships: Rodney Bannwart, Alcon Labs, a
Novartis Company; Robert Ritter, Alcon Labs, a Novartis Company;
Ted Chu, Alcon Labs, a Novartis Company; Charles Blaylock,
Alcon Labs, a Novartis Company
Program Number: 285 Poster Board Number: B0318
Presentation Time: 8:30 AM–10:15 AM
Cumulative dosing enhances the beneficial effects of antisense
oligonucleotide treatment on visual function in a mouse model of
Usher Syndrome
Russell Amato1, Robert F. Rosencrans1, Francine M. Jodelka2,
Frederic Depreux2, Nicolas G. Bazan1, Frank Rigo3,
Michelle Hastings2, Jennifer J. Lentz1. 1Neuroscience, Louisiana State
University Health Sciences Center, New Orleans, LA; 2Rosalind
Franklin University, North Chicago, IL; 3ISIS Pharmaceuticals,
Carlsbad, CA.
Purpose: Usher syndrome (USH) is characterized by concurrent
hearing and vision impairment. There are three clinical types (USH13) depending on the severity and age of onset of clinical symptoms.
6-8% of USH1 cases are caused by mutations in the USH1C gene,
which encodes the protein harmonin. Mice containing the human
USH1C c.216G>A splicing mutation (216A) have a loss of visual
function and slow retinal degeneration similar to human Usher
syndrome. Antisense oligonucleotides (ASOs) targeting the 216A
mutation are known to correct Ush1c splicing and translation, and
be efficacious in rescuing hearing and vestibular function when
administered systemically. Treatment of ASOs in the eye improves
visual function and retinal structures for several months after a single
injection. The purpose of this study was to test the effect of multiple
ASO treatments on Ush1c expression, visual function and retinal
structure in 216AA mice.
Methods: Various of doses of 216A-targeted ASOs were delivered
locally to the eye by intravitreal injection (IVI) in 216AA mutant
and control-treated littermate mice. The expression of Ush1c splice
variants in the retina was determined by reverse transcription-
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ARVO 2016 Annual Meeting Abstracts
polymerase chain reaction (RT-PCR). Visual function and retinal
structures were evaluated by electroretinogram (ERG) and optical
coherence tomography (OCT) imaging analyses, respectively.
Results: RT-PCR analysis of retinal RNA isolated from 216AA mice
treated with various doses of ASOs demonstrated a dose-responsive
correction in Ush1c splicing. ERG and OCT analysis showed
significant improvements in photoreceptor function and structure,
respectively, after a single IVI treatment of ASO in neonatal and adult
216AA mutant mice compared to control mice. These effects were
sustained for 3 months. Additional ASO treatments increased the
duration of efficacy for visual function.
Conclusions: Our results show that ASOs delivered locally to the
eye can effectively target Ush1c mutations in the retina. These
results suggest the therapeutic potential of early ASO intervention
to improve gene expression, photoreceptor structure and function in
Usher syndrome.
Commercial Relationships: Russell Amato, None;
Robert F. Rosencrans; Francine M. Jodelka, None;
Frederic Depreux, None; Nicolas G. Bazan, None; Frank Rigo,
Isis Pharmaceuticals; Michelle Hastings, None; Jennifer J. Lentz,
None
Support: TA-NMT-0613-0609-LSU-WG
Program Number: 286 Poster Board Number: B0319
Presentation Time: 8:30 AM–10:15 AM
Efficacy of Suprachoroidal Aflibercept in a Laser Induced
Choroidal Neovascularization Model
Samirkumar R. Patel1, Jennifer Kissner1, Rafal Farjo2,
Vladimir Zarnitsyn1, Glenn Noronha1. 1Clearside Biomedical Inc,
Alpharetta, GA; 2EyeCRO, Oklahoma City, OK.
Purpose: The purpose of this study is to determine the efficacy
of suprachoroidal administration of aflibercept in reducing
neovascular area in a rat laser induced choroidal neovascularization
model. Reduction in neovascular area in this model could provide
evidence for treating retinal disease such as wet age related macular
degeneration.
Methods: Brown Norway rats (4/group) of approximately 8
weeks were used for this study. Both eyes for each rat were used
and 3 laser spots/eye were applied on day 1. On day 3 rats were
treated using a microneedle (Clearside Biomedical) to perform a
suprachoroidal injection. The microneedle was inserted 1-2 mm
posterior to the limbus and 5 microliters of test article was injected
into the suprachoroidal space. Rats were treated with either saline
or aflibercept (Eylea 40 mg/mL, Regeneron Pharmaceuticals). Eyes
were examined using fluorescein angiography 3 weeks after laser
treatment and images were obtained for quantitative analysis. Area of
neovascularization was quantified for each laser spot using computer
software. Statistical analysis was performed using a Mann Whitney
t-test.
Results: Saline treated animals exhibited approximately 4862 ± 192
pixels2 while aflibercept treated animals showed approximately
3318 ± 353 pixels2 based on evaluation of neovascular leak area.
The difference between these measurements represents a statistically
significant (p<0.001) reduction in neovascularization on comparing
the aflibercept treated group to the saline treated group.
Conclusions: Suprachoroidal injection of aflibercept lead to a
significant reduction in neovascular area in this 21 day model of laser
induced choroidal neovascularization model in rats. This 3-week
treatment effect with a favorable reduction in neovascular area is the
first reported evidence provided for duration of treatment following
suprachoroidal dosing with a soluble biological agent (Eylea).
Further, these results indicate that suprachoroidal injection of an anti-
VEGF agent may provide another treatment option for diseases such
as wet age related macular degeneration.
Commercial Relationships: Samirkumar R. Patel, Clearside
Biomedical (I), Clearside Biomedical, Clearside Biomedical (P);
Jennifer Kissner, Clearside Biomedical; Rafal Farjo, None;
Vladimir Zarnitsyn; Glenn Noronha, Clearside Biomedical (P),
Clearside Biomedical
Program Number: 287 Poster Board Number: B0320
Presentation Time: 8:30 AM–10:15 AM
Effectiveness of AR-13154 monotherapy and combination
therapy in animal models of wet age-related macular
degeneration and proliferative diabetic retinopathy
Cheng-Wen Lin, Jill M. Sturdivant, Mitchell A. deLong,
Casey Kopczynski. Aerie Pharmaceuticals, Inc., Research Triangle
Park, NC.
Purpose: Rho-associated protein kinase (ROCK), Janus
kinase (JAK), and platelet-derived growth factor receptor beta
(PDGFR-b) have been implicated in the development of choroidal
neovascularization (NV) and vascular leakage in wet age-related
macular degeneration (AMD) and retinal NV in proliferative diabetic
retinopathy (PDR). This study evaluates AR-13154, a selective
ROCK/JAK/PDGFR-b kinase inhibitor for its ability to inhibit NV
in relevant animal models, either as monotherapy or in combination
with an anti-VEGF agent.
Methods: 184 compounds from a library of ROCK inhibitors were
screened at 500nM for activity against a panel of 456 human kinases.
Subsequently, AR-13154 was selected for further investigation in
rat laser-induced choroidal neovascularization (CNV) and mouse
oxygen-induced ischemic retinopathy (OIR) models. In the rat
CNV model, after retinal laser treatment on Day 0, AR-13154
(6mcg/mL; estimated vitreous concentration) or vehicle was
administered by intravitreal (IVT) injection on Days 1, 4, and 10.
Aflibercept (800mcg/mL) was administered by IVT injection on
Day 1 as a positive control. In the OIR model, mice were treated
from postnatal day P12 to P17 with either: 1) 0.06% AR-13154(S)
administered topically to both eyes t.i.d.; 2) aflibercept (1mg/kg)
administered intraperitoneally q.d.; 3) co-administration of 0.06%
AR-13154(S) and aflibercept (1mg/kg); or 4) vehicle control. Retinal
flat-mounts were stained with isolectin and areas of NV in both
models were quantified using imaging software.
Results: AR-13154 (500nM) inhibited ROCK2, JAK2, JAK3, and
PDGFR-b by >99%, 72%, 97%, and 89%, respectively. In the rat
CNV model, mean CNV lesion size was reduced by 35% (p<0.001)
and 23% (p<0.05) following IVT administration of AR-13154
(6mcg/mL) or aflibercept (800mcg/mL), respectively. In the mouse
OIR model, topical 0.06% AR-13154(S) reduced NV by 37%
and aflibercept (1mg/kg) reduced NV by 34% (both p<0.0001).
The combination of 0.06% AR-13154(S) and aflibercept (1mg/
kg) reduced NV by 57%, and was statistically superior to either
monotherapy (p<0.005).
Conclusions: AR-13154, a selective multi-kinase inhibitor of
ROCK/JAK/PDGFR-b, significantly inhibited NV in rat CNV
and mouse OIR models. AR-13154 has therapeutic potential as a
treatment option for wet AMD and PDR, either as monotherapy or in
combination with anti-VEGF agents.
Commercial Relationships: Cheng-Wen Lin; Jill M. Sturdivant,
Aerie Pharmaceuticals, Inc.; Mitchell A. deLong, Aerie
Pharmaceuticals, Inc.; Casey Kopczynski, Aerie Pharmaceuticals,
Inc.
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ARVO 2016 Annual Meeting Abstracts
Program Number: 288 Poster Board Number: B0321
Presentation Time: 8:30 AM–10:15 AM
Development of a hydrogel flowable dressing for the prevention
of corneal scarring
Gurpreet Chouhan, Felicity De Cogan, Lisa J. Hill, Saaeha Rauz,
Ann Logan, Liam M. Grover. University of Birmingham,
Birmingham, United Kingdom.
Purpose: Blindness caused by corneal opacity is often a result
of corneal scarring and vascularisation from infectious diseases,
inflammation and corneal trauma. The ‘gold standard’ amniotic
membrane (AM) is often applied to the ocular surface as a treatment
to induce the healing process and reduce scarring, as it is capable of
releasing anti-fibrotic/anti-inflammatory factors. The reproducibility
and repeatability however of the clinical effects of the AM are
limited due to their biological variability. Consequently a plethora
of treatment strategies have previously been investigated however
very few have replaced the AM. We have developed a transparent gel
dressing that can be applied as an eye drop to the ocular surface that
is capable of occluding the wound with sufficient lubrication as well
as delivering the anti-scarring agent, decorin, in a sustained manner
to the cornea.
Methods: Gel dressings were produced by heating the hydrocolloid
to melting point and by controlled temperature processing, reduced
the temperature to form a gel. In vitro decorin release studies from
the gel dressing were carried out using an ELISA assay. The gel was
applied to the corneal surface of rat eyes and thickness was measured
using OCT for 2h. The decorin containing gel was applied as a single
dose to a 2mm ex-vivo corneal ulcer to observe wound healing and
re-epithelialisation.
Results: The gels containing the anti-scarring molecule released a
sustained dose of decorin over 4h. Thickening of the gel occurred
immediately in vivo when dropped on the surface of a rat eye. The
gel evenly covered the ocular surface and remained for upto 2h
whereas a PBS drop was not visible after 30min. Re-epithelialisation
occurred within 48h in the presence the decorin gel in the ex-vivo
models when compared to a decorin-PBS drop which showed limited
wound closure over the same time. Collagen type IV deposition was
significantly reduced in 14 days in the presence of the decorin gel,
indicative of reduced fibrosis on the corneal surface.
Conclusions: We have successfully demonstrated a gel ‘eye drop’
therapy for the attenuation of corneal scarring. The properties of the
gel allow a sustained, effective dose of decorin to be released onto
the corneal surface over 4h after application and the formation of a
gel on the ocular surface provides a protective transparent dressing
promoting re-epithelialisation and reducing fibrosis in ocular surface
disease.
Commercial Relationships: Gurpreet Chouhan, None; Felicity De
Cogan, None; Lisa J. Hill, None; Saaeha Rauz, None; Ann Logan,
None; Liam M. Grover, None
Support: MRC
Program Number: 289 Poster Board Number: B0322
Presentation Time: 8:30 AM–10:15 AM
Biocompatibility Assessment of End-Thiolated Hyaluronate
Coated Gold Nanoparticles on Retinal Pigment Epithelial
(ARPE-19) cells
Bedia B. Karakocak1, 2, Joshua T. Davis2, 3, Pratim Biswas1,
Nathan Ravi2, 3. 1Energy&Environmental and Chemical Engineering,
Washington University, Saint Louis, MO; 2Department of
Ophthalmology and Visual Sciences, Washington University, Saint
Louis, MO; 3Veterans Affairs Medical Center, Saint Louis, MO.
Purpose: Gold nanoparticles (Au NPs) are promising targeting
agents in drug and gene delivery for various cell types, including
ocular cells due to their unique optical, magnetic properties, and ease
in manipulating the surface characteristics. However, nascent Au NPs
have been reported to have significant toxicity. The goal of this study
is to create biocompatible Au NPs, which can be used as delivery
agents for ocular cells.
Methods: In this study, an established retinal pigmented epithelial
(ARPE-19) cell line was used to assess toxicity. Au NPs were
synthesized via citrate reduction method and further coated with
end thiolized hyaluronate (HA). Total organic carbon (TOC) and
thermal gravimetric analysis (TGA) measurements were performed
to quantify the amount of HA coating on Au NPs. HA conjugated Au
NPs were tracked inside the cell via confocal microscopy (Figure
1A). The cell proliferation behavior was monitored continuously
via electrical cell-substrate impedance sensing (ECIS; Applied
Biophysics, NY) for 96 hours (Figure 1B). The biocompatibility of
resultant HA-Au NPs was also tested with MTT and Apo Tox-GloTM
(Promega, CA) assays (Figure 2).
Results: The presence of hyaluronate coating on Au NPs was
confirmed and quantified. ECIS, MTT and Apo Tox-GloTM results
show that the ARPE-19 cells were able to proliferate and maintain a
monolayer in the presence of the HA-Au NPs at concentrations where
nascent Au NPs were toxic (Figures 1&2). Evidence is also provided
to show that the HA Au NP’s crossed the cell membrane and were
observed within the cells.
Conclusions: Our results indicate that hyaluronate coated Au NPs
have potential as delivery agents for ocular cells as they are shown
to have enhanced biocompatibility compared to the nascent Au NPs
with ARPE-19 cells.
Figure 1. (A) The 3-D confocal images of HA coated Au NPs
within the ARPE-19 cells. (B) Comparison of cell attachment
impedance measurements at 4000Hz as a function of time for
nascent and Hyaluronate (HA) coated Gold (Au) NPs. The exposure
concentrations used for comparison are the critical concentrations for
nascent Au NPs (0.05 mg/ml (5nm), 0.09 mg/ml (10nm), and 0.15
mg/ml (20nm).
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ARVO 2016 Annual Meeting Abstracts
induce autophagy in ocular cells in vitro and in vivo. Autophagy
is an evolutionarily conserved process, of which cells catabolize
damaged proteins and organelles in lysosome dependent manner
during nutrient deprivation and stress. Autophagy is highly associated
with development and degeneration of eye. However, the role of
autophagy in ocular cells in response to the vital dyes is completely
unknown. The purpose of this study is to investigate the differential
effects of vital dyes in retinal pigment epithelial and photoreceptor
cells.
Methods: Human retinal pigment epithelial ARPE-19 cells and
mouse photoreceptor 661W cells were treated with ICG or BBG
(0.05 mg/ml) for 30 mins and recovered for 24h to examine cell
viability with CellTiter-Glo® Luminescent Cell Viability Assay. The
plasmid expressing GFP-LC3 was transfected into cells and treated
with ICG or BBG to examine the GFP-LC3 puncta and fluorescence
intensity with fluorescence microscopy and flow cytometry,
respectively. Autophagic flux in the treated cells was further
determined with immunoblotting using antibody against LC3.
Results: We found that ICG and BBG reduced cell viability in
both ARPE19 and 661W cells. Moreover, the conversion of LC31 to LC3-II and GFP-LC3 puncta were increased in the vital dyes
treated-ARPE19 and 661W cells, indicating the vital dyes modulate
autophagy in ocular cells. We further combine the treatment with
autophagy inhibitor chloroquine (CQ) to inspect the role ICG and
BBG on autophagic flux in ocular cells. Interestingly, ICG and
BBG inhibited autophagic flux in ARPE-19 cells, whereas the vital
dyes induced autophagic flux in 661W cells. Ablation of autophagy
with inhibitor CQ or shRNA against ATG7 diminished cell viability
in ARPE-19, but elevated cell viability in 661W cell, suggesting
autophagy play protective and detrimental role in vital dyes treatedARPE-19 and 661W cells, respectively.
Conclusions: Our results imply autophagy modulation could prevent
the damage caused by vital dyes during ocular therapy.
Commercial Relationships: Shwu-Jiuan Sheu, None; Yi-An Chen,
None; Chih-Wen Shu, None
Support: VGHKS104-088
Figure 2. Biocompatibility results for hyaluronate (HA) coated vs
nascent Au NPs with (A) MTT (B); Apo Tox-GloTM assays.
Commercial Relationships: Bedia B. Karakocak, None;
Joshua T. Davis, None; Pratim Biswas, None; Nathan Ravi, None
Support: NIH Grant EY021620; Core Grant: P30EY02687;
Research to Prevent Blindness
Program Number: 290 Poster Board Number: B0323
Presentation Time: 8:30 AM–10:15 AM
Differential autophagic effects of vital dyes in retinal pigment
epithelial and photoreceptor cells
Shwu-Jiuan Sheu1, 2, Yi-An Chen1, Chih-Wen Shu3. 1Ophthalmology,
Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan;
2
Ophthalmology, National Yang Ming University, Taipei, Taiwan;
3
Research and Education, Kaohsiung Veterans General Hospital,
Kaohsiung, Taiwan.
Purpose: Indocyanine green (ICG) and Brilliant blue G (BBG) are
commonly used vital dyes for removal of internal limiting membrane
(ILM). Recent reports showed the dyes impair mitochondria and
Program Number: 291 Poster Board Number: B0324
Presentation Time: 8:30 AM–10:15 AM
Computational Prediction of Intravitreal Pharmacokinetics of
Macromolecules: Tool for Ocular Drug Development
Eva M. del Amo Páez1, Arto Urtti1, 2. 1Biopharmacy, University
of Eastern Finland, Kuopio, Finland; 2Center of Drug Research,
University of Helsinki, Helsinki, Finland.
Purpose: Intravitreal pharmacokinetics is important in the
development of ocular medications, but computational tools for
intravitreal pharmacokinetic prediction are missing. We aimed to
develop pharmacokinetic simulation models to predict the intravitreal
concentrations of biologicals after intravitreal administration. Such
models are needed in ocular drug development to estimate the drug
doses during chronic treatments with injections or long acting drug
delivery systems.
Methods: The primary pharmacokinetic parameter values of a
universal collection of intravitreal macromolecule drugs (7.1-149
kDa) in rabbit eye were used: 80% of the molecules presented
intravitreal volume of distribution of 1.2-2.3 ml and clearance from
the vitreous of 0.011 – 0.025 ml/h. The values were implemented
into pharmacokinetic simulation models for drugs in solution or in
controlled delivery systems using STELLA® Modelling & Simulation
software.
Results: The pharmacokinetic simulation models with
macromolecules yielded good estimates of vitreous drug
concentrations. The models could be used for dosage form design to
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ARVO 2016 Annual Meeting Abstracts
estimate the required release rates and doses that are needed to reach
the target concentration profile. The translation from intravitreal
rabbit data into intravitreal human prediction is possible.
Conclusions: The present work offers useful in silico predictions
of vitreal concentration profiles of macromolecule drugs after
intravitreal injection, either in solution or incorporated in implants.
Such in silico models are expected to advance ocular drug
development.
Commercial Relationships: Eva M. del Amo Páez, None;
Arto Urtti
Program Number: 292 Poster Board Number: B0325
Presentation Time: 8:30 AM–10:15 AM
The influence of drop size of tropicamide 0,5% eye drops on pupil
dilation
Hans Van Der Heiden2, 1, Nynke A.M. Troelstra2, Janneke V. Lith3,
Jacques Verzijl2. 1mu-Drop, Apeldoorn, Netherlands; 2ZAMB,
Tilburg, Netherlands; 3Ophthalmology, St Elisabeth Hospita, Tilburg,
Netherlands.
Purpose: Ocular and systemic side-effects are an important reason
to discontinue eye medication. Side-effects are dose-dependent to a
great extend, so establishing the minimal effective dose is of great
importance. This study was performed to assess whether clinical
equivalent mydriasis can be achieved with reduced size tropicamide
eye drops compared to regular tropicamide eye drops.
Methods: Randomised single-blind cross-over trial in 30 healthy
volunteers. On day 1, one intervention group received a micro
drop (2,4 microliter) in both eyes, the other group a regular eye
drop (38 microliter). Pupil size measurements were performed 10
times during a 2 hour timeframe. Side effects were assessed using a
questionnaire. The measurements were repeated with the other eye
drop after 7 days.
Results: After 40 minutes maximum pupil dilation is reached. The
mean difference in pupil size (within subject analysis) between the
micro drop and the regular drop is 0,41 mm (statistically significant:
t=8,43; df=28; p<0.001). The non-inferiority margin of 0,5 mm lies
above the confidence interval (0,39-0,49 mm).
All 30 subjects report to prefer the micro drop. Less discomfort is
experienced and less complaints of impaired vision are reported with
the micro drop.
Conclusions: Non-inferior mydriasis can be achieved with a micro
eye drop that is 15 times smaller compared to a regular size eye
drop tropicamide and less side-effects are experienced. All subjects
prefer micro drops. Development of ocular micro drops offers new
possibilities in improving treatments with eye drops.
Commercial Relationships: Hans Van Der Heiden, mu-Drop (P);
Nynke A.M. Troelstra; Janneke V. Lith, None; Jacques Verzijl,
None
Clinical Trial: EudraCT 2012-005219-18
Program Number: 293 Poster Board Number: B0326
Presentation Time: 8:30 AM–10:15 AM
Evaluation of cytotoxicity of palomid 529 on human retinal
pigment epithelial cells and choroidal vascular endothelial cells
Bharani Krishna Mynampati Arunadithya, Kumar Sambhav,
KV Chalam. Dept of Ophthalmology, University of Florida,
JACKSONVILLE, FL.
Purpose: Although anti-VEGF therapy is currently an effective
standard treatment for neovascular AMD, exploring the other possible
pathways such as Akt/mTOR pathway may provide an alternative
strategy in the treatment of neovascular AMD. Palomid 529 is a
non-steroidal, synthetic, small molecule drug with a molecular weight
of 406 Daltons that inhibits the AKt/mTOR signaling cascade via
disassociation of both targets of rapamycin complexes, TORC1 and
TORC2 in the immune system. We evaluated the dose dependent
toxicity of palomid 529 on human retinal pigment epithelial cells
(ARPE-19 cells) and choroidal vascular endothelial cells (CVECs)
which has not been well established.
Methods: Human retinal pigment epithelial cells (ARPE-19) and
monkey choroidal vascular endothelial cells (RF6A: CVECs)
were treated with escalating doses of palomid 529 (5,10,15,20,25
µM). Cell proliferation changes were analyzed with water soluble
tetrazolium salts (WST-1) assay. Cytotoxicity in response to palomid
529 was evaluated by trypan blue exclusion assay at different time
intervals i.e 48h, 72h, 1week. Simultaneously, reactive oxygen
species levels were measured using dihydrorhodamine 123 at similar
intervals.
Results: ARPE-19 cells treated with varying doses of palomid
529 (5,10,15,20,25 µM) showed more decline in cell viability than
CVECs. One week after treatment, ARPE-19 cells showed a decrease
in cell proliferation by 16.8% (p<0.0001), 87.6% (p<0.0001), 83.9%
(p<0.0001), 88.6% (p<0.0001), 87.79% (p<0.0001) as compared
to controls respectively by WST-1 assay. Trypan blue exclusion
assay also revealed similar decrease in ARPE-19 proliferation as
15.5%,18.7% (p=0.039), 49.9% (p<0.0001), 53%(p<0.0001), 73.8%
(p<0.0001) compared to controls. Similar results were observed
after one week palomid 529 treatment with CVECs with decrease
in proliferation rates of 27.8% (p=0.002), 33.8% (p=0.0004), 44.5%
(p<0.0001), 44.7% (p<0.0001), 46.2% (p<0.0001). Reactive oxygen
species levels were found to be significantly increased in ARPE19 cells after 48h treatment of palomid 529 compared to control
cells, whereas CVECs showed increased levels of ROS which is not
statistically significant.
Conclusions: Palomid 529 arrests proliferation of choroidal vascular
endothelial cells (CVECs) and human retinal pigment epithelial cells
(ARPE-19) cells in a dose and time dependent fashion
Commercial Relationships: Bharani Krishna Mynampati
Arunadithya, None; Kumar Sambhav, None; KV Chalam, None
Program Number: 294 Poster Board Number: B0327
Presentation Time: 8:30 AM–10:15 AM
Human umbilical tissue-derived cells rescue phagocytosis in
cultured retinal pigment epithelial cells from Royal College of
Surgeons rat through CD36 and integrin αvβ5
Jing Cao1, Christopher Murat2, Weijun An2, Ian Harris1,
George Inana2. 1Janssen Research and Development, LLC., Spring
House, PA; 2University of Miami, Miami, FL.
Purpose: Royal College of Surgeons (RCS) rat exhibits defective
phagocytosis of rod outer segments (ROS) by retinal pigment
epithelium (RPE) cells and photoreceptor degeneration. We showed
that the phagocytosis in the RCS RPE cells was completely rescued
when they were fed with ROS preincubated with human umbilical
tissue-derived cells (hUTC) conditioned medium (CM). We further
demonstrated that hUTC secrete bridge molecules milk-fat-globuleEGF-factor 8 (MFG-E8), thrombospondin (TSP)-1, and TSP-2 which
bound to the isolated ROS in vitro. Knocking down any of the bridge
molecules by siRNA-mediated gene silencing in hUTC significantly
reduced the effect of hUTC CM on the phagocytosis rescue. It has
been reported that the RPE membrane receptors, CD36 and integrin
αvβ5 participate in ROS recognition and internalization through
secreted bridge molecules by the RPE, such as MFG-E8. Therefore,
we aimed to determine the role of αvβ5 and CD36 in hUTC-mediated
phagocytosis rescue in the RCS RPE cells.
Methods: The RCS RPE were preincubated with various doses of
anti-integrin αvβ5 monoclonal antibody P1F6, integrin blocking
peptide GRGDSP, or anti-CD36 monoclonal antibody FA6-152 for 1
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ARVO 2016 Annual Meeting Abstracts
hour at 37oC. The cells were also preincubated with anti-mouse IgG1
isotype control antibody or integrin blocking peptide negative control
peptide GRADSP. The cells were then fed with hUTC CM pretreatedROS and subjected to phagocytosis assay.
Results: The anti-integrin antibody (25, 50 or 100 mg/mL) or
integrin blocking peptide GRGDSP (1 or 2 mg/mL) completely
blocked the phagocytosis of hUTC CM-pretreated ROS. The isotype
control antibody and GRADSP had no effect on phagocytosis when
they were used at 25 mg/mL and 1 mg/mL, respectively. Similar
results were observed when the RCS RPE were preincubated with
anti-CD36 antibody (2.5, 5 or 10 mg/mL), which dose-dependently
blocked the phagocytosis of ROS. The isotype control antibody had
no effect on phagocytosis when applied at 10 mg/mL.
Conclusions: These results suggest that both integrin αvβ5 and
CD36 are involved in hUTC-mediated phagocytosis rescue in
the RCS RPE cells. These phagocytic receptors could recognize
the hUTC-derived bridge molecules that coat the ROS, thereby
facilitating the binding and internalization of ROS by the RCS RPE.
Commercial Relationships: Jing Cao, None; Christopher Murat,
None; Weijun An, None; Ian Harris, None; George Inana, None
Support: The work described was performed under a sponsored
research agreement between University of Miami and Janssen R&D.
Program Number: 295 Poster Board Number: B0328
Presentation Time: 8:30 AM–10:15 AM
Spectral-Domain Optical Coherence Tomography for Early
Detection of Hydroxychloroquine Toxicity
Hemang K. Pandya1, 2, Hunter Porter2, Vinay A. Shah1, 2,
Nawajes A. Mandal2. 1Retina Service, Dean McGee Eye Institute,
Oklahoma City, OK; 2University of Oklahoma Health Sciences
Center, Oklahoma City, OK.
Purpose: Investigate and characterize retinal pathology, using
Optical Coherence Tomography (OCT), in rheumatologic patients
on Hydroxychloroquine (HCQ) for autoimmune disorders, such
as Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus
(SLE). The drug HCQ affects and reduces lysosomal function
and that will affect sphingolipid metabolism and turnover. We
hypothesize sphingolipid metabolite ceramide a known factor for
neuronal cell death will increase in HCQ treated retinas which in turn
induce neuronal cell death throughout the retina, especially, higher
sphingolipid containing retinal ganglion cells (GC) and the nerve
fibers. By imaging the retina of HCQ treated RA and SLE patients we
will determine inner retinal effect of HCQ.
Methods: In a prospective study we recruited 15 subjects with
autoimmune diseases, such as RA and SLE, being treated with HCQ,
and 25 matched healthy control subjects. In a single clinic visit, we
collected patient history to determine HCQ duration and dosage. We
performed OCT to determine macular volume, thickness of macular
GC, inner plexiform layer (IPL) and the optic nerve retinal nerve
fiber layer (RNFL).
Results: We observed a strong negative correlation between
HCQ duration in months and macular volume, average macular
thickness, and macular IPL and GC layer thickness, but a weak
positive correlation with RNFL thickness. HCQ treated subjects
had significantly reduced macular volume, and marked, albeit nonsignificant, reductions in IPL and GC layer thickness and average
macular thickness compared to controls.
Conclusions: HCQ exposure results in reductions of inner retinal
thicknesses in the macula. This identifies the need to reevaluate
clinical practices for detecting retinal toxicity in patients being treated
with HCQ.
Commercial Relationships: Hemang K. Pandya; Hunter Porter,
None; Vinay A. Shah, None; Nawajes A. Mandal, None
Support: NIH Grant EY022071, EY025256; Research to Prevent
Blindness, USA
Program Number: 296 Poster Board Number: B0329
Presentation Time: 8:30 AM–10:15 AM
Ocular Tolerability and Toxicokinetics of Suprachoroidally
Administered CLS-TA, Triamcinolone Acetonide Injectable
Suspension, in Combination with Intravitreal Eylea in Rabbits
Donna Taraborelli, Brian Burke, Glenn Noronha. Clearside
Biomedical, Alpharetta, GA.
Purpose: The ocular tolerability and toxicokinetics of a
suprachoroidal administration of CLS-TA, triamcinolone acetonide
injectable suspension, in combination with an intravitreal injection of
Eylea was evaluated in rabbits.
Methods: Male and female New Zealand White rabbits (4/sex/
group) were randomized according to body weight and assigned
to eight treatment groups. On Day 0, each animal received a single
bilateral intravitreal injection of either vehicle (50 µL) or Eylea
(50 µL) followed 30 minutes later by a single bilateral suprachoroidal
injection of vehicle (100 µL) or CLS-TA (100 µL). Of these
animals, 4/sex/group were each necropsied on Day 1 and on Day
29. Clinical observations, body weight, slit lamp biomicroscopy,
fundus evaluation, intraocular pressure assessment (IOP),
electroretinography (ERG), and systemic exposure were assessed.
Sacrified animals were assessed for macroscopic observations, ocular
toxicokinetics and ocular histopathology.
Results: There were no treatment or administration related effects
on body weight, clinical observations, ophthalmic examinations or
ERG. An increase in IOP was observed in the group treated with
Eylea and CLS-TA, as well as in the groups treated with vehicle, but
not in a pathological range. No treatment or administration related
effects were observed at necropsy and there were no related adverse
effects as assessed by histopathology on Day 1 and Day 29. Findings
observed on Day 1 in individual animals treated with either CLSTA or vehicle included conjunctival inflammatory cells and edema
of the ciliary processes. These findings were not considered to be
pathological since they were transient with minimal severity.
Data from systemic exposure as assessed by concentrations in plasma
suggest that co-administration of CLS-TA and Eylea have no impact
on the plasma pharmacokinetics of either drug.
Conclusions: A single bilateral intravitreal injection of 2 mg Eylea
(50 µL) followed by a single bilateral suprachoridal injection of 4 mg
CLS-TA (100 µL) in albino rabbits was well tolerated. There were no
treatment- or administration-related adverse effects by all evaluation
methods. These findings support development of pharmaceutical
therapies for retinal disease involving suprachoroidal administration
including the use of combination of drugs.
Commercial Relationships: Donna Taraborelli; Brian Burke,
Clearside Biomedical; Glenn Noronha, Clearside Biomedical (S),
Clearside Biomedical, Clearside Biomedical (P)
Program Number: 297 Poster Board Number: B0330
Presentation Time: 8:30 AM–10:15 AM
Combined effects of benzalkonium chloride and UV irradiation
on the bovine lens in vitro
Jordan Rossy, David J. McCanna, Jake Sivak. School of Optometry
and Vision Science, University of Waterloo, Toronto, ON, Canada.
Purpose: Exposure to ocular preservatives and ultraviolet
(UV) irradiation have been associated with cataract formation.
Furthermore, recent evidence shows benzalkonium chloride (BAK)
penetrates into the eye, as deep as the anterior lens capsule. The
purpose of this study was to evaluate the effects produced by
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ARVO 2016 Annual Meeting Abstracts
exposure to BAK and UV irradiation on in vitro lens metabolic
activity.
Methods: The study evaluated and compared the effects of three
treatments on the bovine lens: (1) BAK (2) UV irradiation (3) BAK
and UV irradiation (n=5). Control lenses were exposed to phosphate
buffered saline (PBS) solution. For condition 1, lenses were exposed
to BAK solutions (0.01%, 0.005%, and 0.001%) for 10 min. For
condition 2, lenses were exposed to UV (280-400 nm) irradiation for
1.5 h. The measured irradiance was 1132.41 µW cm-2 nm-1. For lenses
in group 3 receiving both treatments, the lenses were exposed first
to a BAK solution and then UV irradiation. Metabolic activity of the
lenses was evaluated using the alamarBlue assay on day 0, day 2, and
day 7 following exposure.
Results: The metabolic activity for lenses exposed to BAK
concentrations of 0.01% and 0.005% on day 7 were significantly less
than the control lenses (p<0.05). The damage produced by exposure
to 0.01% and 0.005% BAK solutions resulted in metabolic activity
68.8%±7.0% and 85.6±5.0% of the control respectively. Similarly,
lenses exposed to UV irradiation had lower metabolic activity
than control lenses on day 7 (p<0.05), with a metabolic activity of
80.6%±7.7% of the control. For group 3, exposure to BAK and UV
produced significant cellular damage as compared to control on day
7 (p<0.05). However, there was no significant difference between the
damage resulting from exposure to BAK alone (group 1) compared to
BAK and UV exposure (group 3) (p>0.05).
Conclusions: Exposure to BAK, UV, or BAK and UV produces
damage to the bovine lens after 7 days. However, combined exposure
of BAK and UV did not produce significantly more damage than
BAK exposure alone.
Commercial Relationships: Jordan Rossy, None;
David J. McCanna, None; Jake Sivak, None
Support: Natural Sciences and Engineering Research Council of
Canada, Canadian Optometric Education Trust Fund
Program Number: 298 Poster Board Number: B0331
Presentation Time: 8:30 AM–10:15 AM
Chemotherapy with biological agents and ocular side effects
Dimosthenis Mantopoulos1, Elaine M. Binkley1, Anne L. Kunkler1, 3,
Kari Kendra2, Colleen M. Cebulla1. 1Department of Ophthalmology
and Visual Science, The Ohio State University, Columbus, OH;
2
Division of Medical Oncology, The Ohio State University,
Columbus, OH; 3Medical School, The Ohio State University,
Columbus, OH.
Purpose: The biological agents are substances derived from living
organisms that have recently found wide applications in medicine.
However, they are a relatively new category of drugs and our
knowledge about their side effects is limited. This retrospective,
observational study reviews the ocular sequelae of these agents in
patients who presented to the ophthalmology clinic of our tertiary
center.
Methods: Approval was obtained from the Institutional Review
Board. A search of the electronic medical record was conducted to
identify adult patients being treated with biologic agents for various
types of malignancy who presented for ophthalmic evaluation. The
patients were either referred for routine screening or presented with
visual/ocular complaints between 1/1/2010 and 2/2/2015
(61 months). For the current study we analyzed the charts of patients
who were treated with biologic agents including: interferon, leukine,
ipilimumab, mirvetuximab, soravtensine and trastuzumab.
Results: From the 1000 charts that were analyzed, 11 patients had
ocular side effects while on biologic agents and met the inclusion
criteria. Ten of these patients (10/11 or 91%) were symptomatic.
The most frequent visual complaint was mild to moderate vision
loss (8/10 or 80%). The most common medication in this category
was interferon (6/11 or 55%) and the most common finding in
these patients was cotton wool spots (4/6 or 67%). For patients
with adequate follow up, discontinuation of the medication lead to
resolution of the symptoms in the majority of cases (6/8 or 75%).
Conclusions: The current study describes the ocular symptoms
and signs in patients treated systemically with biologic agents at
our university hospital. In the majority of cases, discontinuation of
the medication lead to resolution of symptoms. Further studies are
needed to elucidate the incidence and pathophysiology of these side
effects, something that could lead to optimal screening and treatment
guidelines for these patients.
Commercial Relationships: Dimosthenis Mantopoulos, None;
Elaine M. Binkley, None; Anne L. Kunkler; Kari Kendra, None;
Colleen M. Cebulla, None
Support: Ohio Lions Eye Research Foundation. Research reported
in this publication was supported by the National Eye Institute of the
National Institutes of Health under Award Number K08EY022672.
The content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institutes of
Health
Program Number: 299 Poster Board Number: B0332
Presentation Time: 8:30 AM–10:15 AM
Ocular side effects of traditional chemotherapeutics and small
molecule inhibitors
Anne L. Kunkler, Elaine M. Binkley, Dimosthenis Mantopoulos,
Kari Kendra, Colleen M. Cebulla. The Ohio State University,
Columbus, OH.
Purpose: Traditional chemotherapeutics and small molecule
inhibitors have dramatically changed the prognosis for patients
suffering from a variety of malignancies. However, limited data are
available regarding the ocular side effects of many of these novel
agents. This retrospective, observational study aims to examine
the ocular side effects of traditional chemotherapeutics and small
molecule inhibitors in patients treated for their malignancy at the
Ohio State University.
Methods: Approval was obtained from the institutional review board
at the Ohio State University. A search of the electronic medical record
was performed for patients treated with chemotherapeutic agents who
were seen in the department of ophthalmology between 1/1/2010 and
2/2/2015. This search yielded 3,253 cases and for the current project
we analyzed the first 1,000 cases. Patients known to the investigators
were also included. We identified seven patients who experienced
ocular side effects while being treated with traditional agents and
small molecule inhibitors including ibrutinib, dabrafenib/trametinib,
MEK 162, crizotinib, and FOLFOX (folinic acid, fluorouracil,
oxaliplatin).
Results: Small molecule inhibitors were associated with dry eye,
palinopsia, photophobia, branch retinal artery occlusion, and
posterior vitreous detachment. Ibrutinib and crizotinib were the
most frequent medications used in this category. The most common
presenting symptom was mild to moderate vision loss (4/6 or 67%).
The majority of patients remained on treatment (5/6 or 83%) and
for three of the patients the symptoms resolved completely (3/6 or
50%). The most serious adverse drug event was a branch retinal
artery occlusion in a patient receiving ibrutinib, which resulted
in permanent vision loss. One patient treated with the traditional
regimen FOLFOX developed optic disc hyperemia, which resolved
after discontinuing treatment.
Conclusions: This study identified a number of ocular side effects
in patients treated with both traditional chemotherapeutics and small
molecule inhibitors. The side effects varied from mild to severe,
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
including irreversible vision loss secondary to retinal vascular
occlusion. We hope that this study will aid in the development of
improved screening and treatment protocols for patients receiving
these medications.
Commercial Relationships: Anne L. Kunkler, None;
Elaine M. Binkley, None; Dimosthenis Mantopoulos, None;
Kari Kendra, None; Colleen M. Cebulla, None
Support: Ohio Lions Eye Research Foundation; K08EY022672
from the National Eye Institute of the National Institutes of Health.
The content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institutes of
Health.
Program Number: 300 Poster Board Number: B0333
Presentation Time: 8:30 AM–10:15 AM
Visual alterations in a cohort of young male patients with acute/
subacute intoxication with mercury vapor
Jose-Carlos Pastor, Iraxte Zabalza, Ruben Cuadrado, Jose
Alberto de Lazaro, Angela Morejon, Yrbani Lantigua, Rosa Coco.
IOBA (Eye Institute). University of Valladolid, Valladolid, Spain.
Purpose: Mercury intoxication is a well known condition with
several visual and neurological alterations. But there is still a
controversy about the existence of two level of involvement: visual
pathway and/or retinal structures. By the end of 2012, 47 workers
were affected by mercury exposure for 13 days. Blood levels were
high (600-1000 mcg/L, normal < 10 mcg/L) and patients were
not chelated. The purpose of this work is to describe the visual
disturbances encountered in the most severe affected sub-group,
correlating the structural and functional findings
Methods: A prospective observational study on 23 patients (range
28-56 years) was approved by Ethical Committee. Functional tests
were: visual acuity (ETDRS), color vision (Farnsworth-Munsell,
28 Hue), contrast sensitivity (CSV-1000), visual field (Humphrey
750i), ERG, pERG, mERG and VEP (Metrovision, following ISCEV
standard). Additionally autofluorescence, macular thickness by OCT
(3D OCT2000, Topcon), and nerve fiber layer thickness (CFNR)
(Stratus 3000, Zeiss Meditec) were recorded. Appropriate statistical
tests were performed: Spearman, Mann-Whitney, Wilcoxon and t
student. Values were compared against normal
Results: Two patients were excluded, as they have pathological
unrelated findings: one idiopathic parafoveolar telangiectasias type
2 and one congenital dyscromatopsia. All patients showed loss of
contrast sensitivity in all frequencies, alterations in the discrimination
of colors (Confusion index: 1.64±1.18) and defaults in the visual
field in 14 out of 22 (MD, mean defect: -8.9 dB). Also increase of
latency and decrease of the extent of the b wave (ERG), of the p50
wave of the pERG and an increase of the implicit time of the p100
wave of the VEP. No alterations were registered in the structural tests
(macular thickness: 246.1 ± 20.9 µ; CFNR: 101.89 ±11.6 µ) or in the
mERG.
Conclusions: Most of the found alterations were already described
in other series. Alterations in the pERG not described have been
found. These patients are being followed to analyze the progression
of their pathologic findings. Because of the scarcity in reports of this
poisoning any new clinical data is relevant.
Commercial Relationships: Jose-Carlos Pastor, None;
Iraxte Zabalza, None; Ruben Cuadrado, None; Jose Alberto de
Lazaro, None; Angela Morejon, None; Yrbani Lantigua, None;
Rosa Coco, None
Program Number: 301 Poster Board Number: B0334
Presentation Time: 8:30 AM–10:15 AM
Pilot Study Determining Impact of Best Practices Alerts on
Hydroxychloroquine Screening Practice Patterns
Adrian Au, Vishal Parikh, Yasha Modi, Justis P. Ehlers,
Andrew Schachat, Rishi P. Singh. Cleveland Clinic Foundation,
Cleveland, OH.
Purpose: The purpose of this study was to determine the initial
impact of the best practice alert (BPA) on hydroxychloroquine
(HCQ) retinopathy screening practice patterns at one multispecialty
ophthalmic practice.
Methods: This was an observational, retrospective study and
approval from the institutional review board was obtained.
Responses and screening tests for all patients were analyzed after
the implementation of a BPA in the electronic medical record
(August 2015 to December 2015). Screenings were classified as:
“appropriate” when an objective (SD-OCT, mfERG, FAF) and a
subjective test (HVF) were performed; “under-testing” when either
a 10-2 HVF or objective testing was performed; “inappropriate” if
neither 10-2 HVF or objective testing was performed.
Results: Out of the 84 HCQ screening encounters that occurred
after the implementation of the BPA, 57 (67.9%) were appropriately
screened, 17 (20.2%) were under-screened, and 2 (2.4%) were
inappropriately screened. This is in contrast to the previously
described screening adherence at the same multispecialty ophthalmic
practice where 54.8% were appropriately screened, 25.7% were
under-screened, and 19.5% were inappropriately screened (Au et al.
2015). 10-2 HVF and SD-OCT were the preferred testing modalities
at 77.6% and 93.4%, respectively.
Conclusions: Screening for HCQ retinopathy improved with the
implementation of a best practice alert as appropriate screening
improved from 55% to 67.9% while those inappropriately screened
decreased from 19.5% to 2%.
Commercial Relationships: Adrian Au; Vishal Parikh, None;
Yasha Modi, None; Justis P. Ehlers, None; Andrew Schachat,
None; Rishi P. Singh, None
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.