Development of Pentablock Copolymer Based Formulations for the Sustained Delivery of Protein Therapeutics in the Treatment of Posterior Segment Ocular Diseases
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We have successfully synthesized pentablock (PB) copolymers comprised of various FDA approved polymer blocks such as polyethylene glycol (PEG), polycaprolactone (PCL), polylactic acid (PLA) and polyglycolic acid (PGA). PB copolymers with different composition, molecular weights and block arrangements were utilized to develop protein-embedded thermosensitive gels or nanoparticles (NPs) for sustained delivery in the treatment of posterior segment ocular diseases. In order to eliminate the burst release effect, we have studied PB composite formulation comprised of protein-encapsulated PB NPs dispersed in PB thermosensitive gel. The composite formulation eliminated burst release effect and exhibited nearly zero-order protein release for significantly longer durations. In this research work, we have utilized various model proteins (lysozyme, IgG-Fab, IgG, BSA, and catalase) and therapeutic proteins (octreotide, insulin and bevacizumab) to optimize the formulation. We have synthesized various triblock (TB) (PCL-PEG-PCL, B-A-B) and PB (PLA-PCL-PEG-PCL-PLA (C-B-A-B-C) and PEG-PCL-PLA-PCL-PEG (A-B-C-B-A)) copolymers based thermosensitive gelling polymers. We have observed distinct effect of block arrangement and molecular weights of block copolymers on the sol-gel transition and on the kinematic viscosity of aqueous solutions. PB copolymers with A-B-C-B-A block arrangement exhibited significantly lower viscosity relative to TB copolymers or other types of PB copolymers (C-B-A-B-C). The difference in viscosity and sol-gel transition behavior has been explained by two different processes of micellization for A-B-C-B-A and B-A-B, or C-B-A-B-C types of copolymers. Moreover, a PB copolymer based formulation sustained the release of IgG up to ~20 days, which is significantly longer relative to TB copolymers based formulations. In order to sustain release for longer duration, we have synthesized various PB copolymers (PLA-PCL-PEG-PCL-PLA and PGA-PCL-PEG-PCL-PGA) with high molecular weight and utilized them for the fabrication of protein-encapsulated NPs. We observed a significant effect of the presence of PLA or PGA on entrapment efficiency (EE), drug loading (DL) and in vitro release behavior. This may be due to the fact that PB copolymers exhibited significantly reduced crystallinity relative to TB copolymers. In addition, we have successfully optimized NP preparation methods to achieve maximum possible DL. This achievement allowed the loading of a large amount of drug which can last for ~6 month in a limited injection volume (100 μL). The optimized methods were successfully utilized to encapsulate a wide variety of peptides and proteins with molecular weights ranging from 1 - 237 kDa in PB NPs. PB NPs alone exhibited significant burst release in the first few days of release study. However, a composite formulation comprised of protein-encapsulated PB-NPs prepared with optimized method and optimized PB copolymers (PB copolymers for NPs and thermosensitive gel) exhibited protein release for significantly longer duration of time (~6 months) with nearly zero-order release rate. We have evaluated the structural integrity of released protein at different time intervals by CD spectroscopy. Moreover, biological activity of bevacizumab was evaluated by cell proliferation and cell migration assays. Enzymatic activity of lysozyme and catalase were confirmed with their respective enzymatic assays. Our results indicated that proteins retained their structural integrity and bioactivity during the preparation of formulation and also during the release process. In vitro cell culture studies such as cell viability, cytotoxicity and biocompatibility studies performed on various ocular cell lines confirmed the safety of PB copolymers for ocular applications. Further, we have performed in vivo ocular tolerability studies with optimized PB formulations which demonstrated no inflammation, retinal toxicity, change in intraocular pressure or cataract even after 16 week of exposure. Moreover, in vivo studies further revealed that PB copolymers based formulations were slowly degraded and dissolved in vitreous humor confirming biodegradability of polymers. Our studies indicated that PB copolymer based composite formulation can serve as a platform technology for the development of sustained release therapy in the treatment of posterior segment ocular diseases such as wet age-related macular degeneration (wet-AMD), diabetic macular edema (DME) and diabetic retinopathy (DR). This technology has a scope beyond ocular treatments and can also be used for the treatment of other chronic diseases.
Table of Contents
Literature review -- Introduction -- Novel thermosensitive pentablock (PB) copolymers for sustained delivery of proteins in the treatment of posterior segment diseases -- Sustained delivery of proteins employing novel pentablock copolymer based nanoparticlulate systen for the treatment of posterior segment ocular diseases -- Tailor-made pentablock copolymer based composite formulation for sustained ocular delivery of protein therapeutics -- Optimization of novel pentablock copolymer based composite formulation for sustained delivery of protein therapeutics in the treatment of ocular diseases -- In vivo tolerability studies of various pentablock copolymer based formulations: delivered topically or intravitreally -- Summary and recommendations -- Appendix