November 21, 2024 /
Age-related macular degeneration (AMD), a leading cause of vision loss in older adults, casts a long shadow over millions of lives worldwide. Wet AMD, a subtype characterized by abnormal blood vessel growth beneath the retina, presents a particularly treacherous threat, stealing central vision with chilling swiftness. While treatments exist, they require frequent injections and don’t address the root cause of the disease. Apart from antibody drugs with intravitreal injection already in the market, other modalities and dosing routes are increasingly being tested in preclinical and clinical stage to replace or supplement current treatment. This new trend requires careful selection and tailored modification of the animal study designs to suit less or non-invasive deliveries, long lasting formulation or implant, and anti-fibrotic features.
Targeted pathways in wet AMD therapies (Source: RETINA TODAY)
Changing Landscape of R&D of Wet AMD Therapies:
- Gene therapy: more than 10 in clinical trials worldwide
- A lot of biosimilars to ranibizumab or aflibercept lining up
- Multi-targets going mainstream
- VEGF signaling has always been the first choice
- Subretinal fibrosis targeted: PLGF, integrin, Ang2, PDGF, complement, etc…
- Tyrosine Kinase Inhibition gaining momentum with improved bioavailability and various dosing strategies
- Supplementary treatment to reduce current injection frequencies
Table 1: Animal models for wet AMD at PharmaLegacy
Choosing the Right Model
The choice of an animal model hinges on specific research questions and desired aspects of medical needs to be studied. Each model comes with its own set of advantages and limitations, especially with all the various modalities and dosing strategies that are in development. It is always good to know the cross reactivity of the test drug with the targets among different animal species before moving into animal study. Furthermore, the correlation of drug’s working mechanism with pathogenic lesions in animal model, as well as clinical manifestations, need to be carefully considered. To better examine the efficacy in preclinical stage, at least two models from different species are recommended.
PharmaLegacy has the longest list of animal models for wet AMD. Aflibercept and faricimab are currently the most used reference drug. Apart from the usual targets in the VEGF signaling and TIE2 signaling, PharmaLegacy also has extensive experience with novel targets such as WNT signaling and JAGGED1.
DL-AAA-induced Retinal Neovascularization and Leakage in Rabbits
DL-alpha-aminoadipic acid (AAA) is a glial (Müller) cell toxin that results in disintegration of the blood-retinal barrier when used in the eye, causing retinal neovascularization (RNV) and chronic leakage. After 8~11 weeks, the RNV and chronic leakage becomes stable and can be maintained for years, making it a perfect model for long term evaluation. In efficacy studies, the RNV and leakage will return to baseline after drug washout, ideal for assessing the test article’s long-term efficacy and estimation of dosing frequency. Given the size of the eye, it is also a good model for all types of administration methods: intravitreal, suprachoroidal, eye drops, systemic, etc.. PharmaLegacy maintains a large cohort of induced DL-AAA model animals in-house, which greatly shortens your experimental time.
Figure 1: DL-AAA induced retinal neovascularization and leakage in DB rabbit
JR5558 Mice (Spontaneous CNV)
JR5558 mice strain is the Crb1rd8 and Jak3m1J double homozygous that develops a more severe phenotype than the single mutated strains, with early-onset multifocal depigmented retinal lesions and photoreceptor damage, leading to retinal neovascular phenotype (retinal vascularization 3 model). Spontaneous CNV appears gradually from week 3, without laser treatment. The window of therapeutic intervention is longer than the laser CNV model in mice. Both aflibercept and faricimab have shown good efficacy in this model which has become a very useful tool of efficiently study for drug targeting wAMD, DME, and subretinal fibrosis. PharmaLegacy offers this valuable strain for efficacy studies with an internally bred colony.
Figure 2: Progression of spontaneous CNV over time in JR5558 mice
VEGFA or ANG2 Humanized Mice
The first issue facing large molecule drugs before initiating animal studies is the cross-reactivity of drugs with target antigens across different species. Limited cross-reactivity will greatly reduce the availability of animal models. We have been tackling this by adopting target humanization, such as fully humanized VEGFA or ANG2 in mice. For the first time, we verified laser CNV model in hVEGFA mice and hANG2 mice, as well as the efficacy of Faricimab in these strains. These mice show normal reproductive and vascular systems. Bevacizumab and faricimab have shown significant efficacy in different disease models using hVEGFA mice and have a much lower cost than non-human primates. Double humanization of VEGFA/ANG2 will be available by the end of 2024.
Figure 3: Progression of spontaneous CNV over time in JR5558 mice
VEGF-induced Retinal Leakage in Rabbits
VEGF is up-regulated in wet AMD and diabetic retinopathy; it has always been the main target in drug development for wAMD and DR/DME. This model involves the injection of VEGF or a VEGF-like substance into the vitreous cavity of the eye, which leads to increased vascular permeability and retinal edema. It has been used extensively to investigate the mechanisms underlying retinal vascular leakage and to test potential therapeutic interventions. This induction can also be implemented by combining VEGF with other target proteins. With a short timeline and being relatively inexpensive, this model is also suitable for large scale screening at early stages.
Figure 4: Efficay of Eylea in VEGF-induced retinal leakage model in rabbit
Two-stage Laser-induced Subretinal Fibrosis Secondary to CNV
Subretinal fibrosis is a condition where scar tissue forms beneath the retina. It often occurs as a complication of other eye conditions, including choroidal neovascularization (CNV). As CNV progresses, the leaking blood and fluid can irritate the surrounding tissues. This irritation can trigger the body’s healing response, leading to the growth of scar tissue. Poor prognosis of wAMD treatment is often caused by subretinal fibrosis. While there’s no cure for it, there is a tremendous need for the development of a treatment.
First developed at Prof. Heping Xu’s lab at QUB, the model sets itself apart from traditional single laser models, with closer features to human lesions (such as vascularized fibrotic membrane), and stronger and persistent subretinal fibrosis. The model can be used to evaluate anti-angiogenic and anti-fibrosic efficacy simultaneously. With close collaboration with Prof. Xu, PharmaLegacy is one of the earliest to adopt this model for anti-fibrosis efficacy studies. We have tested several drug candidates ranging from AAVs, antibodies to small molecules.
Oxygen-Induced Retinopathy (OIR)
Oxygen-Induced Retinopathy (OIR) in mice is a well-established animal model for studying the pathophysiology of retinopathy of prematurity (ROP), a leading cause of childhood blindness. ROP is a retinal vascular disease that occurs in premature infants due to exposure to high levels of oxygen. As an in vivo screening tool, it can also be used for other indications such as wAMD, DME, RVO.
The model involves exposing newborn mice to hyperoxia (high oxygen levels) for a period of time, typically 5 days. This mimics the oxygen therapy often required for premature infants. After the hyperoxia period, the mice are returned to room air (normoxia) for a recovery phase. This transition from high to low oxygen can induce retinal vascular abnormalities, including neovascularization (growth of new, fragile blood vessels), hemorrhages, and retinal detachment. The model recapitulates many of the key pathological processes observed in ROP, such as inflammation, oxidative stress, and dysregulation of angiogenic factors.
Figure 5: Isolectin-B4 staining of retinal wholemount of OIR mice
Neonatal Retinal Vascular Outgrowth in Mice
Neonatal retinal vascular outgrowth is a critical developmental process, where blood vessels grow and extend in the retina after birth. This period of outgrowth gives a perfect window for pharmacological intervention. This model’s relatively short timelines and its affordability make it very suitable for large scale candidates screening aimed for ocular indications such as ROP, nMD, DME and RVO. Humanized mice such as hVEGFA or hANG2 can also be utilized. Additionally, test article’s safety characteristics can be measured simultaneously.
Figure 6: Isolectin-B4 staining of retinal wholemount of neonatal mice after treatment
Exciting Times Ahead
Since the first anti-VEGF medication for wet AMD was approved by the FDA in 2004, there has been great improvement of the standard of care for AMD patients. As new unmet medical needs emerged since then, animal models have also evolved along the way. Preclinical study designs will always have room for improvement to accommodate new modalities, less invasive delivery routes, long term treatment, novel targets, or managing subretinal fibrosis. Fortunately, with numerous models in house and consistent results, PharmaLegacy is up to the challenge.
References and Additional Resources
- https://retinatoday.com/resource/therapeutic-pipeline-for-amd
- Li, Y., et al. (2018). A novel model of persistent retinal neovascularization for the development of sustained anti-VEGF therapies. Exp Eye Res 174, 98-106.
- Regula, J.T., et al. (2016). Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases. EMBO molecular medicine 8, 1265-1288.
- Chidiac, R., et al. (2021). A Norrin/Wnt surrogate antibody stimulates endothelial cell barrier function and rescues retinopathy. EMBO molecular medicine 13, e13977.
- Nagai, N., et al. (2014). Spontaneous CNV in a novel mutant mouse is associated with early VEGF-A-driven angiogenesis and late-stage focal edema, neural cell loss, and dysfunction. Investigative ophthalmology & visual science 55, 3709-3719.