Evaluation of Animal Models for Retinal Degenerative

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International Broadcasting Convention(IBC)'s 5th International Conference Ocular Angiogenesis & Retinal Degeneration. New Targets and Treatments, March 30 – 31, 2009, Las Vegas, NV, USA
Evaluation of Animal Models for Retinal Degenerative Treatments
Epidemiology of Blindness (Cont.)
161 million people with visual impairment worldwide,
37 million people blind worldwide,
3.8 million people blind in the western
PURPOSE: To develop and compare different animal models for neovascularization to evaluate
potential anti-angiogenic drugs for treatment of age-related macular degeneration and diabetic
retinopathy.
CONCLUSIONS: The accomplishment of the neovascularization and corneal neovascularization
rodent model and Laser-induced choroidal neovascularization rodent model are effective, fast and
efficient models for evaluating new drugs for the treatment of age-related macular degeneration
and diabetic retinopathy.
# of Patients
15 million
17%
Ocular NV:
Choroidal NV
(Subretinal NV)
Retinal NV
5 million
Alkali cauterization mouse/rat models (NaOH, 30”)
Epithelial wound healing mouse/rat models
Suture rat model
Model Creation
D0
Laser photocoagulation mouse model (Argon laser
photocoagulation, 50mum, 400mW, 0.05s)
Dry AMD (Atrophic)
D20
90.00
80.00
Prevention Model
Retinopathy of prematurity (ROP) mouse model (low oxygen
environment)
Lipid hydroperoxide (LHP) rabbit model (Intravitreous injection)
Streptozotocin (STZ) rat model (Diabetic retinopathy, i.m.)
> 85% mature vessels
Regression Model
Corneal NV (CoNV) Mouse Model
Age-related macular degeneration (AMD)
Normal
Hematoxylin-eosin (HE) staining of a representative
area of CNV at the site of laser-induced CNV in mice
new vessels
Cataracts are curable. AMD, glaucoma & DR are treatable.
Toxikon Corporation
A multidisciplinary contract research organization (CRO) laboratory focused on evaluating the
safety toxicity of biologics, pharmaceuticals & medical devices.
D10
Results:
Wet AMD (Neovascular)
Normal
Angiogenesis

Relevant Species
 Mice/rats
 Rabbits: New Zealand White, Dutch-belted (Pigmented)
 Beagle Dogs
 Mini-Pigs
 Cynomolgus Monkeys



Laser-Induced Choroidal NV Mouse/Rat Model
Advanced Age-Related Macular
Degeneration (AMD)
(With Associated Vision Loss)
Glaucoma
Diabetic Retinopathy (DR)
Cataract


2020 Projections
(in millions)
1.8*
2.9
2.2
4.1
20.5
3.3
7.2
30.1
Transparent
Epithelial injury
Drug Screening
Choroidal NV and Leakage
7 days after Laser shots
CoNV epithelial wound healing mouse model procedure:
Retinal agiogenesis by diabetes
Diabetic control
Anti-angiogenic drugs
Types
- Corneal Chemical or mechanical insult
- Retinal Retinopathy of prematurity (ROP)
- Choroidal Laser-induced choroidal neovascularization
Remove the corneal and limbal epithelia

Animal model validation for 4 weeks

Screening anti-angiogenic compounds for 4 texts

Histology-pathology, immunostaining and images for 2 weeks
Procedure of Laser Photocoagulation:
Wound healing and angiogenesis of the cornea
PECAM-1 immunostaining and CoNV measurement:
Animals
- Mice/Rats
- Rabbits (Dutch Belted Rabbits and New Zealand White Rabbits)
Endpoint
- Ocular examination (cornea and retina), photography
- Fluorescein angiography
- Histology with paraffin embedding and whole mount retinas or choroids
Choroidal NV and Leakage
14 days after Laser shots
Positive
Control
n=3
0 Day
10 Days
100
90
80
70
60
50
40
30
20
10
0
0
5
7
10
14
21
Days after microsurgical process
Neovascularization (NV) Rodent Animal Models
28

Anesthesia with Ketamine/Xylazine

Pupil dilation with 1% Tropicamide

Compound-1 Compound-2 Compound-3
n=3
Group
n=3
n=3
yellow area – pink area
NV is a key factor of retinal degeneration in AMD and DR
% of CoNV =

Treatment of AMD and DR is to find new drugs to inhibit NV
yellow area
NV animal models are useful for screening anti-angiogenic compounds
30.00
25.20
20.00
10.16
10.00
0.00
Positive
Control
n=3
Compound-1 Compound-2 Compound-3
n=3
Group
n=3
n=3
The Laser-induced choroidal neovascularization mouse/rat model was effective, quantitative,
accurate, middle- and long-term for screening anti-angiogenic compounds.
Summary

Toxikon is contract research and testing company that primarily conducts compliant
GLP/non-GLP studies for product safety.
Fundus photography

The Ocular Science Department at Toxikon is driven by Dr. Zhong, who specializes in creating
ophthalmic disease models, and offers efficacy screening and safety testing of drugs.

Laser shots: 4-6 photocoagulation lesions between the retinal vessels using an Coherent’s 900Z
Argon laser (100-um spot size, 0.1-second duration, 120 ~160 mW, wavelength 514 nm)

NV animal models, including CoNV and CNV mouse/rat models, are effective, quick and quality
methods for screening potential compounds to find new drugs to treat AMD and DR

Post-laser photocoagulation: 0.5% Erythromycin applied

Fluorescein angiography at the designed time-points Day 7, 14, 21 and 28 after laser shots
References


Histology-pathology, immunostaining and whole-mount retinas or choroids at endpoint





50.00
40.00
Negative
Control
n=3


48.78
50.00
0.00
Study Design:
Male C57BL/6 mice
Ocular Angiogenesis Animal Models
Eye Disease Prevalence and Projections
(Number of Adults 40 Years and Older in the U.S.)
Current Estimates
(in millions)
Vascular endothelial growth factor (VEGF)
VEGF inhibitors/antibodies: Macugen™, Avastin and Lucentis
Eye is under microscopy

Epidemiology of Blindness
20.00
60.00
Diabetic Retinopathy (DR)
Capabilities
 Slit-Lamp Exams and Draize and McDonald-Shadduck Scoring
 Direct ophthalmoscopy and indirect ophthalmoscopy
 Tonometry (IOP), Pupilometry and ERG
 Anterior/Posterior Segment Photography
 Ocular Tissue Dissections and PK/PD Process
 Retrogradely labeling RGCs, wholemount retina/choroid, and images
 Ocular disease model development (Dry eye, OHT, CoNV, CNV, etc.)
30.00
Vessel staining
% Neovascularization of cornea

40.00
Reduced % of CNV Area in Group


Picture Courtesy: National Eye Institute, NIH
50.00
Negative
Control
n=3
Ophthalmic Science
Ocular Dose Routes
 Topical
 Subconjunctival, Subtenon and Intracameral
 Intravitreal and Anterior chamber injection
 Subretinal
60.00
0.00
Ocular Study Director
Lichun Zhong, M.D., M.S., Ph.D., was trained in ophthalmology at Beijing University in China and
has led the Ocular Science Department at Toxikon for 2 years.

70.00
10.00
The corneal neovascularization mouse/rat model was effective, quantitative, accurate, economic, and
short-term for screening anti-angiogenic compounds
To prevent blindness, a critical strategy on AMD, glaucoma and DR
Introduction
CoNV mouse model for studying anti-angiogenic agents with the advantages of consistency,
scale, visualization and regression:
2.5 million
12%
Laser-Induced Choroidal NV Mouse/Rat Model (Cont.)
Redured % of CNV Area
METHODS and RESULTS: Several studies were conducted to create neovascularization animal
models in mice and rats. Successful surgical procedures exhibited that the corneal
neovascularization in rodents was reproducible and effective. These models will be effective,
quantitative, accurate and cost effective for screening anti-angiogenic compounds in short term
studies. Laser-induced choroidal neovascularization rodent models were also effective, accurate,
quantitative models for middle- and long-term studies for evaluation of anti-angiogenic drugs.
Leading causes of blindness in the United States:
Age-related macular degeneration (AMD)
30%
Cataract
13%
Congenital Cataract
4%
Primary Open-angle Glaucoma (POAG)
6%
Other Glaucoma
6%
Corneal Opacity
7%
Diabetic Retinopathy (DR)
6%
Macular Scar
7%
Other
22%
Corneal NV
Corneal NV (CoNV) Mouse Model (Cont.)
Area of CNV (mm2)
Abstract
Lichun Zhong and Laxman S. Desai
Ocular Science Department, Toxikon Corporation
15 Wiggins Avenue, Bedford, Massachusetts, 01730 U.S.A.
[email protected],
www.toxikon.com
Retinal
Degeneration Animal Model
Development
Draize, J. H. “Appraisal of the Safety of Chemicals in Foods, Drugs, and Cosmetics.” Association of Food and Drug
Officials of the United States, Austin, Texas, 1965. 36-45.
McDonald, T.O. and Shadduck J.A. 1983. Eye irritation in Dermatotoxicology (2nd Ed.). Edited by Marzulli F.N.
Hemishpere Publishing Corp., New York, NY.
Gelatt, K.N. 2007, Veterinary Ophthalmology, 4th Ed. p169-170, p447 and p468-471. Blackwell Publishing, Gainesville,
Florida.
Ju M, et al. Simultaneous but not prior inhibition of VEGF165 enhances the efficacy of photodynamic therapy in
multiple models of ocular neovascularization. Invest Ophthalmol Vis Sci. 2008 Feb;49(2):662-70.
Jo N, et al. Inhibitory effect of an antibody to cryptic collagen type IV epitopes on choroidal neovascularization. Mol
Vis. 2006 Oct 26;12:1243-9.
Jo N. Inhibition of platelet-derived growth factor B signaling enhances the efficacy of anti-vascular endothelial growth
factor therapy in multiple models of ocular neovascularization. Am J Pathol. 2006 Jun;168(6):2036-53.
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