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