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Siponimod as a novel therapeutic for ocular neovascular diseases: biological characterization and development of controlled release systems
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Date
2024
Authors
Alshaikh, Rasha A.
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Publisher
University College Cork
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Abstract
Background: Neovascular ocular diseases, including age-related macular degeneration, diabetic retinopathy, and retinal vein occlusion, are characterized by pathological processes such as angiogenesis, oedema, inflammation, cell death, and fibrosis. Current therapeutic approaches predominantly target vascular endothelial growth factor (VEGF), one of the primary mediators of pathological angiogenesis. However, while effective, VEGF blockers are costly macromolecules requiring frequent intravitreal administration and are associated with various adverse effects and high treatment resistance rates. Consequently, identifying small molecular weight angiogenesis inhibitors with alternative mechanisms of action and developing sustained, less invasive delivery systems for intravitreal administration are needed.
In this thesis, I explored the potential of siponimod (a sphingosine 1-phosphate receptor modulator approved for multiple sclerosis) as an inhibitor of ocular angiogenesis in vitro and in vivo. Subsequently, upon demonstrating its potential efficacy, I characterized siponimod's ocular pharmacokinetics (PK) and toxicity in vivo, along with its solubility and stability profiles. Leveraging siponimod's small molecular weight and lipophilic nature, we investigated the ability of electrospinning to sustain the release of siponimod and improve the current standard of monthly intravitreal injection. Then, we explored the potential of improving the current treatment standard of diabetic retinopathy and diabetic macula oedema by cocrystallisation of siponimod and fluocinolone acetonide. If successful, these cocrystals can concomitantly address the components of angiogenesis and inflammation in these diseases.
Methods: The effect of siponimod on angiogenesis in vitro was confirmed using endothelial cells and various functional assays, including growth factor-induced cell proliferation and migration. Siponimod’s impact on the integrity of the retinal endothelial barrier under stress conditions was also investigated using a combination of functional assays and immunofluorescence. Furthermore, the effect of siponimod on ocular neovascularisation in vivo was assessed using a suture-induced corneal neovascularisation model in albino rabbits. Then, siponimod’s ocular PK and toxicity were investigated after intravitreal injection in albino rabbits and the PK parameters of the drug were computed. This was followed by a preformulation study to determine the drug's solubility and stability under stress conditions.
A microfibrous PLGA implant carrying siponimod was developed using electrospinning. The implant was characterised using solid-state characterisation techniques and drug-polymer interaction, and the drug release from the implant in vitro was investigated. The implant stability under stress conditions and the ability of the released drug to produce a therapeutic effect was also confirmed.
Finally, novel siponimod fluocinolone acetonide cocrystals were produced for dual drug delivery in diabetic retinopathy and macular oedema. Cocrystals were characterised using solid-state characterisation techniques. Their dissolution rate, saturation solubility, and stability under stress conditions were also evaluated. Then, PLGA microparticles loaded with cocrystals were produced using electrospraying. Their drug loading, size, morphology, drug release and ability to produce dual angiogenesis and inflammation inhibition over 100 days were investigated.
Results: In vitro data showed that siponimod is a potent angiogenesis inhibitor and protects endothelial barrier function. The drug inhibited endothelial cell migration towards different growth factors, increased retinal endothelial barrier integrity, and reduced TNF-α-induced barrier disruption. These actions were mediated by sphingosine 1-phosphate receptor-1 modulation. Furthermore, siponimod, tested in two different doses, completely abolished the progression of suture-induced corneal neovascularisation in albino rabbits and reduced corneal epithelial thinning.
Siponimod’s half-life after intravitreal injection of low and high doses was 2.8 h and 3.9 h, respectively. No signs of retinal toxicity were observed after intravitreal administration. Siponimod showed a significant increase in solubility in porcine vitreous compared to aqueous saline and exhibited temperature-dependent degradation in aqueous solution.
An electrospun bead-free microfibrous PLGA implant carrying siponimod was successfully produced under optimised conditions. Siponimod showed a uniform distribution within the electrospun fibres as a stabilised, amorphous, solid dispersion with a significant drug-polymer interaction. Siponimod dispersion and drug-polymer interactions formed smooth fibres devoid of porous structures. This inherent lack of porosity, coupled with the drug’s hydrophobic dispersion, afforded resistance to water penetration. This results in a slow Higuchi-type diffusion of siponimod, with approximately 30% of the drug load being released over 90 days. The released drug maintained a pharmacological effect in vitro comparable to that of a freshly prepared drug solution, indicating the stability of the drug cargo. Moreover, the implant retained physical and chemical stabilities under stress conditions for 3 months.
Finally, siponimod and fluocinolone acetonide cocrystals exhibited higher physical stability than the parent siponimod, maintaining their crystalline pattern under stress conditions. The cocrystals also showed a lower dissolution rate under sink conditions compared to siponimod. Electrosprayed cocrystal-loaded PLGA microparticles released ~ 70% of the drug load over 100 days. The released drugs successfully and simultaneously inhibited retinal endothelial cell migration and IL-6 production. This indicates the superior efficacy of the microparticles in the dual inhibition of angiogenesis and inflammation, crucial aspects in managing diabetic retinopathy and macular oedema.
Conclusion: The efficacy of siponimod in vitro and in vivo supports its therapeutic potential in ocular neovascular diseases. The drug has a small molecular weight, lipophilic nature, short ocular half-life and is prone to thermal degradation in solution. Siponimod was formulated in a stabilising, sustained-release microfibrous electrospun PLGA implant. The drug interaction with PLGA provided a unique opportunity to sustain drug release from the electrospun fibres, thereby reducing the frequency of intravitreal injection, improving patient adherence, and representing a potential alternative to anti-VEGF treatments in AMD and DR. Such drug-polymer interactions can provide a low-cost, effective method for sustaining small molecules for ocular applications. Finally, siponimod-fluocinolone acetonide cocrystals in PLGA microparticles offer a promising new approach to treating AMD and DR. By providing sustained drug release and targeting both angiogenesis and inflammation; this formulation has the potential to reduce the frequency of intravitreal injections, improve patient adherence, and offer a viable alternative to single treatments.
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Keywords
BAF 312 , Neovascularization , Diabetic retinopathy , Age-related macular degeneration , Sphingosine 1 phosphate , Corneal neovascularization , Diabetes mellitus , Age-related macular degeneration , Intravitreal administration , Neovascularisation , Siponimod degradation , Siponimod stability , Ocular half-life , Sustained release , Controlled release , Drug-polymer interaction , PLGA
Citation
Alshaikh, R. A. 2024. Siponimod as a novel therapeutic for ocular neovascular diseases: biological characterization and development of controlled release systems. PhD Thesis, University College Cork.