Extended-release formulations of somatostatin for ocular application and their effect on vascular endothelial growth factor as a biomarker of diabetic retinopathy

Do Jai Prakash Rai, Uma Devi 2018, Extended-release formulations of somatostatin for ocular application and their effect on vascular endothelial growth factor as a biomarker of diabetic retinopathy, Doctor of Philosophy (PhD), Health and Biomedical Sciences, RMIT University.

Document type: Thesis
Collection: Theses

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Title Extended-release formulations of somatostatin for ocular application and their effect on vascular endothelial growth factor as a biomarker of diabetic retinopathy
Author(s) Do Jai Prakash Rai, Uma Devi
Year 2018
Abstract The International Diabetes Federation reports 642 million adults globally will be living with diabetes by 2040. Approximately one third will go on to develop some extent of diabetic retinopathy (DR), with ca. 93 million current sufferers.

Current therapeutic options for DR target the late stages of the disease when vision is already significantly degraded. These treatments aim to prevent haemorrhaging using laser therapy or intraocular injections of corticosteroids and therapeutic anti-vascular endothelial growth factor (VEGF) antibodies. However, efficacy is low or variable with frequent injections required and
associated with serious ocular side-effects. Moreover, all result in some degree of tissue destruction. New therapies to prevent the onset or arrest the progression of the disease before overt microvascular pathologies are therefore essential.

Somatostatin (SST) has been reported to have neuroprotective, antiangiogenic effects in addition to the regulation of water and ion transport in the retina. It is postulated that SST could attenuate key pathological changes in DR. However, studies with SST analogues such as octreotide demonstrated that systemic administration resulted in the drug reaching the retina only when there is a breakdown of the blood retinal barrier.

SST is produced by the retina and there is downregulation of SST levels in the retina of diabetic patients, correlated to retinal neurodegeneration. SST eye drops show some promise (EUROCONDOR). The purpose of this thesis was to develop a modified-release formulation of SST enhancing the treatment of diabetic retinopathy.

The physicochemical characterization of SST self-assembled hydrogels was performed to investigate the rheological and morphological properties of the self-assembled hydrogels and their influence on the mechanism and rate of SST release in vitro. This study demonstrated that the SST hydrogels were shear-thinning, did not exhibit thermogelation and exhibited a higher degree of structure with higher somatostatin concentration and in the presence of electrolytes. Transmission electron micrographs demonstrated the presence of nanofibrils that were narrower and denser in appearance in the presence of electrolytes. This corresponds to the higher G' (storage modulus) of the SST hydrogels in electrolytes and at a higher SST concentration. In the presence of electrolytes SST hydrogels released a lower cumulative percentage of SST monomers at 48 hours compared to those in water. The release kinetics of the SST monomers demonstrated concurrent first-order and zero-order mechanisms. However the limited release at 48 hours in vitro precludes these selfassembled hydrogels as depots for SST delivery.

Two formulation candidates of SST were produced and characterized. The first candidate was SST 2.5% w/w microparticles encapsulated in a triblock polymer of polycaprolactone-polyethylene glycol-polycaprolactone (PCL-PEG-PCL) prepared by the double emulsion method. Encapsulation efficiency of SST ranged from 19% to 43% of which 1.4% -4% was released over 16-24 days. Scanning electron microscopy of the microparticles showed spherical aggregates which averaged 32 µm in size. The release kinetics of somatostatin from the microparticles demonstrated first-order decay to a plateau with good mean R2 value of 0.979.

The second formulation candidate consisted of somatostatin 5% w/w entrapped in hydroxypropyl methylcellulose (HPMC) 3% w/w or 6% w/w hydrogel. The SST in HPMC hydrogels were subjected to rheological studies and FTIR characterization, transmission electron microscopy and release studies.

Rheological tests demonstrated that inclusion of SST at a concentration of 5% w/w did not alter the characteristic properties of HPMC matrices. FTIR data showed transition of SST structure from random conformations to structured organisations upon exposure to simulated lachrymal fluid (SLF). These transitions were hindered in the HPMC hydrogels compared to SST 5% w/w in water, consistent with the hypothesis that the entanglement network of HPMC acts a barrier to the interaction of the electrolytes in SLF with SST. This was supported by the TEM images. Release studies demonstrated an extended release of SST monomers from the SST/HPMC hydrogels compared to SST 5% w/w in water. The SST 5% w/w solution reached a 100% release at 6 hours. The SST 5% w/w in HPMC 3% w/w gels released a total of 45.6% incorporated SST at 96 hours whereas the SST 5% w/w in HPMC 6% w/w gels continued to release a total of 37.6% at 10 days. The release kinetics of SST 5% w/w in HPMC 3% w/w and HPMC 6% w/w hydrogels were fitted to a hybrid model of release with R2 values of 0.998 and 0.997 respectively. Mass spectrometry confirmed the structural integrity of the SST released from hydrogels. SST 5% w/w in HPMC 6% w/w hydrogels are the most promising candidate in terms of SST delivery, with delayed SST nanofibril formation and extended duration of release in comparison to SST 5% w/w aqueous dispersions.

The safety and efficacy of SST solutions at different concentrations and that from hydrogel formulations were tested on immortalized human retinal pigment epithelium cells (ARPE-19). MTT cell viability assay was conducted to assess the effect of the release samples from SST/HPMC hydrogels and SST 10-7M and 10-6 M on the viability of the ARPE-19 cells. The results of the MTT assay demonstrated a significant decrease in cell viability only by the Release Day 1 samples compared to control. It is postulated that the combination of the viscosity of the Release Day 1 sample with the higher concentration of SST present at 24 hours may have additive effects on the viability of the cells. The markers for assessment for efficacy were expression of vascular endothelial growth factor receptor-2 (VEGF-R2) and secretion of VEGF by the ARPE-19 cells.

Immunofluorescence was used to assess the effect of SST concentrations (10-8 M to 10-5 M) on the expression of VEGF-R2 levels. The results of the first study showed that SST 10-7 M produced significant downregulation of VEGF-R2 expression in high glucose (25 mM) conditions compared to normal glucose (5 mM) conditions. The quantitative analysis of VEGF secretion in the same conditions performed by ELISA did not show any significant effect by the treatments applied in the same conditions.
In conclusion, two formulations of somatostatin for ocular delivery were developed and characterised. The microparticle formulation showed reasonable encapsulation and duration of release, however, the more promising candidate consisted of somatostatin entrapped in a HPMC network and demonstrated modified release of somatostatin over 10 days.

This work has introduced HPMC as a suitable candidate for modifying the release rate of peptides. The current work also evidenced the downregulation of the VEGF receptor 2 (VEGF-R2) by somatostatin at a concentration of 10-7 M in high glucose (25 mM) conditions. Although further studies are warranted on other retinal cell lines and animal models with additional markers, the identification of a new polymeric carrier for the delivery of somatostatin adds to the current body of work in the search for a noninvasive treatment for diabetic retinopathy.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Health and Biomedical Sciences
Subjects Pharmaceutical Sciences
Keyword(s) Somatostatin
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