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Investigating Small Molecule Therapeutics to Improve Regeneration and Functional Recovery Following Peripheral Nerve Damage

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LONDON’S GLOBAL UNIVERSITY Investigating small molecule therapeutics to improve regeneration and functional recovery following peripheral nerve damage. Melissa Lucy Doreen Rayner Thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy. School of Pharmacy University College London September, 2018 1 Declaration I, Melissa Rayner, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. Signed: ________________________ Date: ________________________ 2 Acknowledgements I would like to express my sincere gratitude to my supervisors Dr Jess Healy and Dr James Phillips for their continuous support and guidance throughout the PhD. Thank you for all the time and encouragement you have given during the project but also towards the additional projects and activities I had the opportunity to get involved with. I would particularly like to thank the biological services unit staff for all of their generous support during in vivo experiments and especially to Francesca Busuttil for her willingness to give her time so generously to assist during long surgery days. In addition, special thanks also go to Rachael Evans for her input and hard-work during the development of the in vitro screening tool, Alessandra Grillo for her great effort and contribution to the biomaterials work and to Essam Tawfik for providing the PLGA nanofibres. I am grateful to all of my colleagues and friends in the Healy and Phillips labs for all of their inspiration and making the lab a friendly and enjoyable place to work. Furthermore, I am thankful to Professor Steve Brocchini and all of the CDT management team for giving me the opportunity to complete my PhD as a part of the CDT. Special thanks go to my CDT cohort for sharing this journey with me and all of our wonderful adventures. Finally, and most importantly, I would like to thank all of my family and friends who have been there for me throughout this challenging journey. A special mention goes to Ahmed Jamel for his continuous encouragement and belief in me. 3 Publications Papers MLD Rayner, S Laranjeira, RE Evans, RJ Shipley, J Healy, JB Phillips (2018). Developing an in vitro model to screen drugs for nerve regeneration. Anatomical record. (In press). MLD Rayner, JB Phillips, J Healy (2018) Repurposing small molecules to target the Rho/ROCK pathway as new therapies for peripheral nerve injuries. (Submitted). MLD Rayner, T Quick, JB Phillips (2018) Quantifying regeneration in humans following peripheral nerve injury. (Submitted). MLD Rayner, A Grillo, J Healy, JB Phillips (2018) Ibuprofen loaded ethylene vinyl acetate for controlled local delivery to treat peripheral nerve injuries. (Paper in preparation). MLD Rayner, E Tawfik, C Vasiliki, D Craig , J Healy, JB Phillips (2018) Ibuprofen loaded electrospun PLGA nanofibres for controlled local delivery to treat peripheral nerve injuries. (Paper in preparation). AGE Day, C Murray-Dunning, C Schuh, MLD Rayner, L Thanabalasundaram, L Stevanato, N Grace, G Cameron, RAL Drake, J Sinden, JB Phillips (2018) Optimisation of peripheral nerve repair constructs containing Engineered Neural Tissue made using CTX0E03 cells. (Paper in preparation). MLD Rayner, A Grillo, J Healy, JB Phillips (2017) Developing biomaterials for the local delivery of drugs following peripheral nerve injury Tissue and Cell Engineering Society, Manchester (UK). C O‘Rourke, MLD Rayner, PJ Kingham, JB Phillips (2017) Tuning collagen hydrogel stiffness to enhance the regenerative phenotype of adipose stem cells for peripheral nerve repair European Cells and Materials Meeting abstracts, Collection 2, P277 MLD Rayner, R Evans, J Healy, JB Phillips (2016) Development of an advanced 3D neuronal cell culture as a screening tool for drugs promoting nerve regeneration. European Cells and Materials Meeting abstracts, Collection 1, page 177. MLD Rayner, J Healy, JB Phillips (2016) Using advanced 3D engineered cell cultures to analyse the effect of drugs on peripheral nerve regeneration in vitro. European Cells and Materials Meeting abstracts, Collection 5, page 145. A Finch, K Akpokavie, S Grieb, C Da Costa Mathews, and M Rayner (2015). Characterisation of a haze observed in reconstituted lyophile solutions. Analytical Matters, Pfizer. 4 Conference proceedings MLD Rayner, A Grillo, J Healy, JB Phillips (2018) Ibuprofen-loaded biomaterials for controlled local delivery to treat peripheral nerve injuries. TERMIS 5th World Congress, Kyoto (Japan). AGE Day, MLD Rayner, JB Phillips (2018) Development methods for delivering engineered neural tissue within peripheral nerve repair conduit. TERMIS 5th World Congress, Kyoto (Japan). MLD Rayner, J Healy, JB Phillips (2018). Using advanced 3D engineered cell cultures to investigate drug synergy in promoting peripheral nerve regeneration. Tissue and Cell Engineering Society. Keele (UK) MLD Rayner, J Healy, JB Phillips (2017). Investigating the effects of ibuprofen on axon regeneration and functional recovery following peripheral nerve injury ISPNR, Barcelona (Spain). MLD Rayner, A Grillo, J Healy, JB Phillips (2017). Developing biomaterials for the local delivery of drugs following peripheral nerve injury Tissue and Cell Engineering Society, Manchester (UK). MLD Rayner, J Healy, JB Phillips (2017). Investigating the effects Ibuprofen on axonal regeneration and functional recovery following peripheral nerve injury 6th Vienna Symposium on Surgery of Peripheral Nerves, Vienna (Austria). MLD Rayner, J Healy, JB Phillips (2017). Investigating the effects Ibuprofen on axonal regeneration and functional recovery following peripheral nerve injury Neuroscience Symposium, UCL, London (UK). M Rayner, J Healy, JB Phillips (2016). Using advanced 3D engineered cell cultures to analyse the regenerating effect of drugs on neural cells in isolation. Eastman Dental Institute PhD Research Symposium. M Rayner M, J Healy, JB Phillips (2016). Using advanced 3D engineered cell cultures to analyse the effect of drugs on peripheral nerve regeneration in vitro. Tissue and Cell Engineering Society, University College London, London (UK). M Rayner, R Evans, J Healy, JB Phillips (2016). Development of an advanced 3D neuronal cell culture as a screening tool for drugs promoting nerve regeneration. Tissue Engineering and Regenerative Medicine International Society, Uppsala (Sweden). M Rayner, C Da Costa Matthews C, B Cooper (2015). Dynamic Light Scattering as a tool to benchmark the submicron region in parenteral formulations. Global Research & Development, Drug Product Design, Pfizer, Sandwich (UK). M Rayner, R Kendall, S Orubu, C Tuleu (2015) The milky way: Towards development of a formulation and process design space to ensure the development of innovative, safe, effective and natural drug delivery to young children. UKICRS, University of Nottingham, Nottingham (UK). 5 Abstract Peripheral nerve injury (PNI) can be debilitating and results in loss of function, coupled with slow neuron regeneration. Microsurgical treatments remain the gold standard therapy, with no drug therapies currently available. Effective pharmacological treatments could potentially maintain neuronal viability, encourage axonal growth, improve axonal specificity to targets and reduce neuropathic pain. Some drugs and targets have been identified but challenges remain with clinical translation. Advancements in understanding the molecular and cellular events occurring following PNI identifies signalling pathways that could be targeted with drug therapies. The failure in drug therapies reaching PNI clinical trials may be due to the lack of effective in vitro and in vivo pre-clinical models. This study developed and applied models to be used as effective screening tools to address this need. Many compounds demonstrated positive effects on neurite growth when screened in a 3D-engineered co-culture model. NSAIDs (ibuprofen and sulindac sulfide) demonstrated beneficial effects and were studied further in two injury models demonstrating increased axonal growth and improved function. Local controlled-release drug delivery systems have become more attractive because of the drawbacks in conventional drug treatments. This study investigated drug release from various biomaterials in order to obtain an optimal material for implantation and sustained drug delivery. Suitable biomaterials were implanted in vivo to deliver ibuprofen or sulindac sulfide. Both drugs demonstrated beneficial effects on axonal regeneration and functional recovery. Embedding drugs into biocompatible and bio-degradable materials provides effective delivery systems for future translation. Studying NSAIDs revealed a previously unreported relationship between PPAR-γ affinity and regeneration. A NSAID derivative demonstrated the greatest effects on neurite growth in vitro at lower doses than other compounds tested. In summary, this work has 6 identified therapeutic targets to aid the development of novel compounds, as well as, drug repurposing, and effective tools for the pre-clinical screening of these drugs. 7 Impact statement This thesis contributes to translational research in peripheral nerve injury (PNI), specifically the identification of small molecule therapies to improve regeneration and functional recovery. The findings from aspects of this work have already started to enhance the clinical translation of drug therapies in this area through being used to underpin the development of a clinical trial in repurposing a drug
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