Title: Development of Hypoxia-Mimicking materials for Soft Tissue ( Wound healing ) : Regenerative Medicine Product Development
Funder: Department of Health (England)

Abstract: Research Plan An international search report did not highlight any conflicting documents, but did highlight seven potentially overlapping documents which the examiner felt were of relevance to the inventive step of the patent, when considered in combination. Further discussions with our patent lawyers, Kilburn and Strode (London) will shortly provide assistance to refine the claims to ensure novelty, inventiveness and ability for reduction to practice. The International phase of this patent application is now complete. RepRegen has nominated regional/national filings to be made in Europe, UK, US and Japan, and such instructions were issued in 2010. Selection of these territories has been based upon an understanding of market size for device sales with the US (2010 figures suggest this market is worth $94.9 billion), Japan, Germany and France constituting the top 4 markets, with the UK following closely behind its European counterparts, with a market worth $8.4 billion in 2010. Aims Inventive steps and technical challenges: The project is highly innovative by using the first synthetic hypoxia mimicking material for wound regeneration. The inventive steps are reflected in patented technology from Imperial College London and licensed to RepRegen. The concentration and duration of hypoxia ion release is vital for initiating favourable biological responses. The technical challenges are to successfully develop formulations and scalable manufacturing. Cutting edge spectroscopic, diffraction and microscopy characterisation techniques will be used to identify how the addition of cobalt affects the atomic structure of the glasses, including solid-state magic angle spinning nuclear magnetic resonance (SS MAS-NMR) spectroscopy will analyse the silica network of the glasses. These results will be correlated with synchrotron X-ray diffraction (XRD) results and complemented with X-ray Adsorption Near Edge Structure (XANES), which will provide a fingerprint of local cobalt environment. This data will enable the optimisation and correlation of the atomic glass structure with respect to human skin cell response. The Project PlanThe project will be run as a highly collaborative venture, with close working relations between Imperial College, The Blond McIndoe Research Foundation and RepRegen. The project plan comprises 10 work packages to enable regulatory approval and market launch of the HyHeal product and deliverables for each WP and 4 major milestones identified. Outputs of the project will include the following: delivering scalable product production validated using RepRegen's quality management system (ISO13485) for the manufacture of bioactive glasses. A detailed technical file containing plans for testing to gain regulatory approval, technical data, literature reviews, competitor analysis, patents, design reviews and risk analysis. Progress product development towards human clinical trials - ensuring by the end of the project a detailed plan has been approved and signed off to obtain regulatory approval. Project work packages: WP1) We will conduct a stakeholder analysis to determine the relevant end users. The analysis will form the basis of the product specification and user needs. By speaking to surgeons and nurses at Queen Victoria Hospital and St Mary's Hospital, the correct inputs will be defined for the product design review process and risk analysis.Deliverables: Draft product specification and design inputs. Design review and risk analysis. Report and recommendations (Milestone 1).Completion Date: Q2WP2) Formulation Optimisation: Development of glass compositions for manufacturing and optimisation for incorporation in wound dressings in preparation for scale-up. Glass compositions (%cobalt) and particle size distributions will be chosen based on input from WP1 and dissolution kinetics studies. Product format (gel, cream, gauze, bandage) chosen to suit given indication.Deliverables: Technical reports. Design review and risk analysis.Completion Date: Q6WP3) Materials Properties Testing: glass structure, metrology of materials, antibacterial testing, formulation optimisation.Deliverables: Technical reports. Design review and risk analysis. Validation of materials properties of HyHeal (Milestone 2)Completion Date: Q7WP4) In Vitro Cell Testing: Candidate formulations will be screened using in vitro assays with an ex vivo skin explant model and primary human skin cells (keratinocyte and fibroblast with assays for: MTT, HIF, VEGF, proliferation, migration, differentiation, HIF pathway upregulation).Deliverables: Reports. Validation of hypoxia bio-activation in human skin cells (Milestone 3).Completion Date: Q7.WP5) Scale Up and Manufacture: The scale-up of HyHeal processing will be optimised, demonstrating the robustness of the established processes and/or troubleshooting until robust processes are achieved and validated by testing against manufacturing specifications. Each batch of the scaled-up material will be packaged and sterilised, using validated configurations. Shelf life testing, sterilisation validation, bioactivity, accelerated aging will be preformed for the technical file. Deliverables: Finalised product specification (Milestone 4). SOPs. Design review and risk analysis. Finalised sterilised product (Milestone 5).Completion Date: Q8WP6) Clinical Trial Design and Planning: We will complete a proposed clinical study plan to deliver HyHeal to the marketplace and identify suitable study sites and patient numbers for statistical significance in compliance with ISO14155. This WP includes a risk and gap analysis identifying the ISO10993 biocompatibility tests and animal model to be undertaken for product approval (these studies are outside of the scope of this study and will be run in parallel by RepRegen).Deliverables: Clinical study plan (Milestone 6).Completion Date: Q7WP7) Patent activity: we will generate a freedom to operate and other supporting patent activity for the chosen product, to ensure appropriate protection. Deliverables: FTO report (Milestone 7).Completion Date: Q12WP8) Patient engagement: patients, nurses and surgeons will be engaged to feed input into the design control process by organising workshops and visits with prototype devices.Deliverables: Product feedback forms for design inputs.Completion Date: Q12WP9) Regulatory: a regulatory plan and strategy will be formulated to identify the necessary steps to obtain approval for this novel device,Deliverables: Completed Regulatory plan (Milestone 8).Completion Date: Q12.WP10) Project management: project management activities.Deliverables: Project plan, cost estimates, risk matrix and consortium management. Final project report (Milestone 9)Completion Date: Throughout the project and re-evaluated at quarterly meetings.A full description of each work package including resources and costs is contained within annex 2 and project Gantt chart annex 3.Research methodology (Further Details):The work packages above will ensure regulatory approval is obtained quickly after performing the additional ISO10993 and in vivo (animal testing required for regulatory approval) tests funded separately by RepRegen outside of the scope of this project.Ethically approved access to discarded human skin tissue will allow us to utilise an ex vivo skin explant model and primary human keratinocytes and fibroblasts at the Blond McIndoe Research Foundation. These well established in vitro models will be used to assess the effect of HyHeal materials on the behaviour of skin cells which will indicate any stimulatory effect on wound repair. Initial work will focus on cytotoxicity and the rate of epithelial outgrowth from ex vivo skin will also be measured to provide a measure of the wound healing effect of HyHeal in tissue with an intact epithelial architecture. Proliferation in response to HyHeal of keratinocyte and fibroblast cells will be measured by the use of anti-BrdU and phenotypic changes in keratinocytes will be measured by real-time PCR for cytokeratins K1,10, 5, 14, 6 and 16 which are markers of a differentiated, basal and wound repair phenotype respectively. Myofibroblast differentiation and expression of collagen type 1 and type 3 (vital constituents of the dermis) will also be measured. We will measure both the activity of HIF1 by Transam HIF1 activation assay and the expression of Selenbp1 and VEGFa by real-time PCR in keratinocytes. In addition VEGF protein levels will be assessed by ELISA. The findings from these in vitro studies will inform further pre-clinical studies in a well established pig model of wound repair for which additional funding will be supplied by RepRegen. Findings from this pre-clinical work will inform the design of preliminary clinical studies. The work will follow a clear translational pathway through BMRF and our collaborating clinicians Dr Hutan Ashrafian (IC) and consultant plastic surgeon Mr Baljit Dheansa (both included in this grant application) at QVH will ensure that patients quickly derive benefit from this novel material. The project will take HyHeal upto the point of clinical delivery. MHRA clinical trials licencing and ethical approval will be sought with Queen Victoria Hospital as the sponsoring organisation such that at completion of the project Phase 1 clinical studies can commence. It is anticipated that our investigations will indicate that HyHeal is suitable for clinical application. The project will result in test data, reports, SOPs, records, risk analysis (ISO14971) and specifications used to populate the Design History File (DHF) of the product over the course of the project. Selected data from the DHF will then be used to compile the Technical File (TF) for EU Regulatory Approval and will be submitted to the notified body who grant approval based on compliance to the EU Medical Device Directive (MDD) and ISO13485.Risk management: RepRegen will use its existing SOPs to ensure that all product related risks are managed proactively (ISO14971). Risks will be identified and a probability of occurrence and severity will be assigned to each individual risk. If the risk is considered too high a risk control measure will be put in place and the probability and severity assessed again. One notable risk is that there could be manufacturing variability in batches during scale-up of the product. This risk will be mitigated by considerable expertise of the consortium and the design control and risk analysis procedures which will be followed using the SOPs in RepRegen's and their supply chain sub-contractors quality management systems. Risk is further reduced by the fact that HyHeal has a similar bioceramic structure to RepRegen's bone-repair strontium containing bioceramic (StronBone) for which RepRegen successfully achieved CE marking in 2010. Bioactivity of product will be assessed using in vitro human skin cell tests and the in vitro SBF (ISO23317) test. Benefit The patient healthcare benefit provided by HyHeal over existing technologies is that the stimulation of the natural healing response and vascularisation induced by the hypoxia pathways, a first of its kind in a medical device. This bioactive approach should result in significant patient benefit due to a faster healing time and antibacterial action. Enhanced healing will result in reduced pain during the course of an ulcer, speedier recovery and the associated discomfort of these conditions. This will lead to the benefit of reduced hospital bed days, lower risks of infection and follow-up operations. Furthermore this project could spur a platform of HyHeal products, based on the HyHeal research prototype material, greatly accelerating their commercialisation and readiness for the clinic where further healthcare benefits could manifest. The indications which will benefit from this innovation are described below.1) The escalating prevalence of obesity and Type II Diabetes is resulting in 7 million new patients each year worldwide which reflects the dramatic rise in overweight individuals by 70 million cases each year (Ashrafian et al. Obes Rev 2010). In the UK 4.9% of 20-79 year old have diabetes mellitus (www.diabetesatlas.org), this translates to 4.4m individuals. There were also 18,707 deaths in the UK attributed to diabetes, some of which could be avoidable with better treatments. For people with healed diabetic foot ulcers, the 5-year cumulative rate of ulcer recurrence is 66% and amputation 12%. The most effective preventive measure for major amputation seems to be screening and referral to a foot-care clinic if high-risk features are present. Other interventions include therapeutic footware, topical growth factors, systemic hyperbaric oxygen, debridement, wound dressings, debridement with hydrogel and dimethyl sulfoxide wound dressings. However, these therapies produce variable results.2) Peripheral vascular disease prevalence increases with age: when measured by non-invasive tests, is about 3% in people under the age of 60 years but rises to over 20% in people over 75 years. A quarter of those affected have symptoms (Fowkes et al. Int J Epidemiol 1991). 3) In the UK each year around 250,000 people attend hospital A&E departments for burns (source - Enoch et al BMJ 2009). An estimated 58% of all severe injuries involve victims being admitted for 5 or more days as in-patients at hospitals or specialist burns units. Many of these victims suffer extensive full thickness burns and require plastic surgery, often for many years following the accident with associated physical pain and often suffer acute psychological distress for many years. 4) Approximately 412,000 individuals develop a new pressure ulcer each year in the UK with long stays in intensive care. (Thomas J Wound Care 2006). Combined with diabetic ulcers and active venous ulcers results in around 650,000 wounds which could benefit from this innovation.


Research activity codes: 5.2 CELLULAR AND GENE THERAPIES
Health categories: SKIN