Cell/extracellular matrix interactions: application to skin repair – SKINHEALING

First, the peptide properties will be optimized according to in vivo constraints and uses in human patients (production according to good manufacturing practices, sterilisation, assessment of peptide stability after sterilization and time, evaluation and optimisation of peptide stability in proteolytic situations such as in normal and chronic wound fluids and evaluation of biocompatibility, tolerance, and toxicology studies according to pharmaceutical standards). Secondarily, pharmaceutical forms will be developed and adapted according to the wound type and severity. A pharmaceutical form peptide delivery will be developed for simple acute wounds and a formulation or device containing the peptide will be adapted for hard-to-heal wounds. The in vivo proof of concept of the healing effect of the peptide will be assessed in models of acute partial- and full-thickness wounds in pigs. This model was chosen because of skin similarities between pig and human in which wounds heal by a combination of both granulation tissue formation and contraction. Moreover, additional preclinical studies will consist in evaluating whether the peptide has a beneficial effect in hard-to-heal wounds. The rat model will be used here to assess large wounds mimicking 3rd degree burns and chronic wounds. In the later situation, diabetic rats will be produced. Basic research aimed at understanding the mechanisms underlying the peptide-induced dependent signalling cascades in keratinocytes will be conducted. In addition, in vitro re-epithelialisation models will be developed to assess the peptide activity and analyze its mechanism of action.

The work conducted during the last 18 months allowed us to define the cGMP production conditions of the peptide and identify the sterilisation protocol compatible with maintaining the peptide activity. We have characterized the peptide degradome with time by the use of proteases commonly found in the wound environment. The nano-LC/MSMS analysis of the peptide revealed the exo- and endo-peptidic activities of serum enzymes and elastase or auréolysine respectively toward the peptide, showing its high sensitivity to degradation. Different strategies of peptide modification were assessed to improve the stability of the original peptide (termini-protection, various cyclisation approaches, replacement of amino-acids, N-methylation, size reduction..). Syndecan-1 mediated cell assays were used to assess the modified peptides biological activity and digestion assays were applied to all bioactive peptides. While resistance to proteolytic degradation to the enzymes described above was improved in all cases, two peptides displayed an extremely interesting behaviour because their sequence was 80 to 100 % maintained after 20 h incubation. In the mean time, cell migration assays (time LAPS videomicroscopy and wound assays) performed with the modified peptides revealed a pro-migratory activity similar to the original peptide. Several peptide associated pharmaceutical forms (gel and compress) were produced for their future in vivo evaluation using the pig wound model. Preliminary assays of skin wounding in the pig model allowed us to define the most reproducible conditions for efficacy studies. During this time frame, fundamental research was conducted. To analyse the dynamic of syndecan-1 during keratinocyte migration we produced a syndecan-1 combined with the Green Fluorescent Protein. Our preliminary experiments revealed that the GFP-syndecan-1 receptor is functional.

The aspects we will develop are the outcome of the recent significant progress we have made and will consist in determining whether the peptide can be considered as a potential bio-therapeutic agent for clinical uses in both acute and chronic wounds. Superficial and superficial partial-thickness wounds will be performed in a porcine model to analyse the peptide efficacy on the epithelialisation steps of wound closure. A clinical examination of the wounds will be done at different time points. To study whether the peptide has a beneficial effect in hard-to-heal wounds, the rat model will be used as it has been widely used in the study of skin wound healing and of efficacy of different treatment modalities. In a first set of experiments, large wounds mimicking third degree burns will be generated to evaluate whether the peptide is efficient in promoting epithelial closure. The millimetric patches autograft technique, widely used for the healing of extensive burns, will be applied. In a second set of experiments, peptide efficacy will be evaluated in chronic wounds generated in a diabetic rat model. Meanwhile, toxicological, pharmacological, stability and bioavailability studies of the chosen peptide will be assessed with a view to its use in the human clinical setting studies. Strategies to understand its mechanism of action at the molecular and cellular levels will also be undertaken. More precisely the peptide interaction with the syndecan-1 receptor and its associated signalling cascade leading to keratinocyte migration and wound closure will be examined at the molecular level. In addition, in vitro skin epithelialisation models will be used as three-dimensional models to analyze the GFP-syndecan-1 function in keratinocyte migration.

Peptide promoting cell adhesion and migration.
Applicants : Symatese; Univ Claude Bernard Lyon; CNRS.
European patent, PCT/EP2009/067597 filed Dec. 18, 2009 - WO2009EP67597
United State Patent, US 2012/0114617, filed on May 10, 2012.
Japon patent, 2011-553501, filed on November 26, 2014 ; issued on September 2, 2015

Michopoulou A. and Rousselle P. How do epidermal matrix metalloproteinases support re-epithelialization during skin healing? Eur J Dermatol. 2015 Apr;25 Suppl 1:33-42.

Senyürek I, Kempf WE, Klein G, Maurer A, Kalbacher H, Schäfer L, Wanke I, Christ C, Stevanovic S, Schaller M, Rousselle P, Garbe C, Biedermann T, Schittek B (2014) Processing of Laminin a Chains Generates Peptides Involved in Wound Healing and Host Defense. J Innate Immun 2014, 6:467-84.

Rousselle P, Michopoulou A and Delso Costa C. Rôle du microenvironnement cellulaire dans la cicatrisation cutanée. Biologie Cutanée, CoBiP 2013. pp 210-229, Eds MatriX 2014.

Rousselle P, Carulli S, Chajra H, Dayan G, Pin D and Herbage B (2013) The syndecan binding sequence KKLRIKSKEK in laminin a3 LG4 domain promotes epidermal repair. Eur J Dermatol 23:57-65.

Interventions that promote and/or improve the healing of skin wounds have been around for millennia and are today a medical priority as five million people need wound dressing each year. Skin wound healing is a complex process in which dermal and epidermal events are closely related involving soluble factors, blood elements, extracellular matrix components and cells. Through well-identified continuous phases the wound healing process allow the concomitant repair of both dermal and epidermal compartments. Major progress has been achieved in the handling and treatment of wounds, mainly with the development of innovative wound dressings. In particular, interactive and biological dressings including collagens have allowed important improvements in wound care management as they interact with the wound bed to favour the formation of the dermal granulation tissue. Among the entire wound process, the re-epithelialisation phase is one of the most earliest and crucial event determining the efficiency of the overall wound repair process. However, it is surprising that therapies aimed at promoting re-epithelialisation are largely lacking from the modern clinical approach to improving healing. The development of dressings designed to incorporate and locally deliver signalling proteins such as growth factors and bio-mimetic peptides with properties to support epidermal repair expectantly announces the next generation products for wound management. Epithelialisation of normal wounds occurs by an orderly series of events whereby keratinocytes migrate, proliferate, and differentiate to restore the barrier function. The migratory behaviour is governed at both the extracellular and intracellular levels and depends on the carefully balanced dynamic interaction of the cells with their environment through adhesion complexes that form at sites of matrix contacts.
The aim of the SKINHEALING project is to develop an innovative decapeptide with a sequence derived from an extracellular matrix protein named laminin 332, a protein involved in adhesion and migration of epidermal keratinocytes. The in vitro biological properties of the peptide have been extensively studied and preliminary experiments in a pig partial-thickness cutaneous wound model reported that the peptide application significantly promoted early-stage wound healing by accelerating re-epithelialisation. The technology was co-patented by CNRS and the SYMATESE pharmaceutical company specialized in tissue repair and regeneration biotechnologies. The SKINHEALING project is dedicated to the development of the peptide up to a form that fit for the treatment of acute and hard-to-heal wounds. In the same time the underlying biological process will be uncovered and the mechanism of action of the peptide determined. Our priorities are (1) development of validated animal models of acute and hard-to-heal wounds to evaluate the efficacy of the peptide (2) industrial development of the peptide with a particular focus on its stability in vivo and its resistance to proteolysis, toxicological analysis and optimization and (3) identification of the signalling pathways following keratinocyte interaction with the peptide. At the end of the project, enough data will be collected to support a clinical investigation protocol in order to check the efficacy of the product in human. The synergistic multidisciplinarity among the partners relies on their complementary expertise; partner 1 (P. Rousselle, Coordinator Lyon) is specialized in biochemical and cellular approaches for cell adhesion and migration studies and partner 3 (D. Pin, Ecole Vétérinaire/UPSP, Lyon), is specialized in animal dermatology and wound models. Partner 2 (B. Herbage, SYMATESE) represents the industrial partner that plans to develop and commercialize the peptide for the wound repair and regeneration market. In addition, the project will benefit from the participation of a plastic surgeon (F. Braye), chief of the Edouard Herriot burn centre.