IT202300002910A1 - SKIN PATCH COMPRISING A BIO-ABSORBABLE SKIN SUBSTITUTE AND RELATED PRODUCTION PROCESS - Google Patents

Description

TITLE: ?Skin patch comprising a bioabsorbable skin substitute and related manufacturing process?

DESCRIPTION

FIELD OF INVENTION

The field of the present invention is that of skin substitutes. In particular, the present invention relates to a skin patch comprising a bioabsorbable skin substitute and a related manufacturing process.

STATE OF THE ART

Some lesions of the epithelial tissue are defined with the expression "difficult wound" which represents a multiple concept that aims to highlight the characteristics of chronicity, absence of spontaneous closure and presence of concomitant systemic biological factors that hinder normal tissue repair. "Difficult wound" means a lesion that, regardless of the triggering cause, is outside the normal processes and normal resolution times.

In the context of epithelial lesions, a chronic skin ulcer (English acronym CCU or CU for chronic ulcer) is defined as a lesion characterized by a loss of substance, which persists in the absence of a tendency to spontaneous healing for more than 60 days (Westerhof W. Leg ulcers: diagnosis and treatment. 1993 Elsevier). It can be assumed that the current prevalence of ulcers, including those of the foot, is between 0.18% and 0.32%, while in a percentage between 0.06 and 1.0% for venous ulcers. Studies that consider both open and closed ulcers estimate a global prevalence of the order of 1-1.3 (The Alexander House Group -1992-Consensus Paper on Venous Leg Ulcer. Phlebology 7:48-58). The prevalence data for vascular lesions range from 1.8 to 3.05 per thousand, with an increasing prevalence as age increases. In Western countries, it has been calculated that 10 per thousand of the adult population has been affected at least once in their life by an ulcer on the lower limbs.

Venous leg ulcer: Mar;46(3):381-6.). The incidence is significantly higher in females, with a ratio of 3 to 1. Phlebostatic ulcers are among the most frequent, with a prevalence of 70-80%, followed by arterial ulcers (15-20%). An epidemiological survey conducted by Canonico et al. for the Geriatric Observatory of the Campania Region Canonico et al. Prevalence of veins in an Italian elderly population (Angiology 1998; 49: 129 - 3135), has shown that 42.5% of males and 57.5% of women over 65 years of age suffer from varicose disease of the lower limbs, and that 3% suffer from skin ulcers. Even more detailed analyses show that the incidence of venous leg ulcers is higher in females. Recent studies have confirmed the epidemiological relevance of venous ulcers (Agale SV Chronic Leg Ulcers: Epidemiology, Aetiopathogenesis, and Management 2013 Ulcers 5 Volume 2013 Article ID 413604, http://dx.doi.org/10.1155/2013/413604).

While pressure ulcers develop in hospitalized patients with a prevalence ranging from 18% to 29% (A.I.S.Le.C. 2010), it is difficult to find large-scale and well-conducted studies in the home-based population. The World Health Organization forecasts have estimated that the number of diabetics in 2025 will be over 300 million compared to the 120 million calculated in 1996; from this, one can easily understand the size of this problem. Estimates of this pathology in fact report that approximately 15% of diabetics will experience a foot ulcer in their lifetime that will require medical care. Although the diabetic population represents approximately 3% of the general population, more than 50% of all major amputations involve diabetics. (http://www.iwgdf.org/).

The socio-economic impact of UC is significant. In assessing the costs of this pathology, expenses related to dressing materials, transport times and medical and nursing staff were taken into account; other expenses directly borne by patients (private healthcare, adjuvant care, loss of working days, etc.) were not taken into consideration, as is the case for indirect expenses borne by the National Health System (hospitalization for surgical operations, infectious complications, haemorrhagic episodes, neoplastic degeneration, etc.) which would further increase the costs of this pathology (Rith-Najaran SJ et al. Identifying diabetic patients at high risk for lower extremity, amputation in a primary care setting. Diabetes Care 1992; 15:1386-9). Anglo-Saxon studies on the costs of dressing materials for the treatment of chronic leg ulcers indicate that the cost in the United Kingdom is around £1,300 to £2,500 per patient, which corresponds to an annual cost of between £2 and £3 billion (Rippon et al The economic 30 impact of hard to heal leg ulcers Wounds 2007; Vol 3, N2; (2008) The burden of chronic wounds in the UK. Nursing Times; 104: 3, 44?45). The impact of this pathology on healthcare costs has been known for some time. For example, in Sweden (

(1991). Epidemiology of chronic venous ulcers. British Journal of Surgery, 78(7), 864-867) were spent more than 120 million pounds annually, corresponding to about one percent of the national health budget. In the United States, the authors

(A study of the impact of leg ulcers on quality of life: financial, social and psychological implications. J Am Acad Dermatol 1994, 31: 49-53) reported the loss of 2 million working days due to leg ulcers (about 2.5 million people are affected). These considerable figures can be understood in the light of the fact that 45% of skin ulcers cause immobility or marked reduction of individual autonomy. This is without taking into account the worsening of the quality of life. More recently, the estimated cost per patient with venous leg ulcer in Germany was about ? 9,500

Cost-of Illness of chronic leg ulcers in Germany Int Wound 2010; 7: 97-102), as well as extremely high costs to the healthcare budget continue to be reported (van Gent WB et al Management of venous ulcer disease; BMJ 2010; 341: c6045).

This picture is associated with the prospective involvement of an ever-increasing number of patients, both due to the increase in average life expectancy with the consequent increase in the age groups most exposed, and due to the increase in pathologies associated with aging and/or peripheral vascular disease (for example diabetes), with a consequent increasingly large share of lesions that will become chronic and/or that will prove refractory to available therapies.

The consequence of all this is that chronic ulcers of the lower limbs (English acronym CLLU) constitute and will continue to constitute in the coming years one of the most arduous challenges of current medical research, both for their significant epidemiological relevance and because there is a large share of lesions that do not heal, and indeed recur, despite the patient being subjected to a correct therapeutic approach, with a consequent impact on the costs of therapy and management (hospitalization, surgical interventions, etc.) and on social costs.

Traditional therapy for the treatment of ulcers includes, for example, compression bandaging, which allows healing only in the long term; however, a complete and faster healing is desirable, in order to avoid complications, often capable of threatening the life of the patient. In the last 30 years, tissue engineering techniques have allowed the large-scale production of skin substitutes (acronym English SS) (Funct Biomaterials 2015; 6: 547-563).

Skin substitutes are classified based on the Technology Assessment Program ? Agency for Healthcare ? Meriland USA ? report Dec 12: into the following product types:

1. products containing autologous or allogeneic human cells;

2. products made with human tissue from donors;

3. products derived from decellularized animal tissues;

4. biosynthetic products.

Although these new treatment options represent significant improvements over more traditional approaches, there is still a need to find a definitive cure for chronic ulcers.

For example, the typology of products 1-4 reported above has allowed an improvement in the treatment capacity, without however satisfactorily resolving the healthcare demand. It should be noted in this regard that UC and consequently CLLU are included in the pathologies that have "unmet medical need": in this category the European Medicines Agency (EMA) includes those pathologies whose current therapeutic approach is not able to ensure correct treatment for patients.

Due to the characteristics of the wound bed of chronic ulcers, the most suitable skin substitute for this type of injury is product 1, containing autologous or allogenic human cells processed in pharmaceutical factories authorised for GMP production, called Cell Factories for the production of ATMPs (Advanced Therapies Medicinal Products), which fall under European regulation 1394/2007.

This type of product also presents a series of drawbacks. The causes that lead to a failure in the healing of UC and in particular of CLLU are of various types such as: the size of the ulcers; the presence of bacterial species inside the wound; the prolongation of such ulcers over time (Phlebological rev 2015; 2:29-38).

In addition to this, the skin substitutes currently in use present a series of limitations of use, mostly due to the fact that they are not able to ensure sufficient revascularization and have low adhesion to the wound bed, hindering the regeneration of the skin tissue and therefore the healing of the wound.

As regards chronic skin lesions, autologous skin substitutes, i.e. those made from skin samples from the patient to be treated, require relatively long production times and are consequently not generally available for the emergency treatment of lesions over a large area of the body surface (English acronym BSA). Autologous skin substitutes are, moreover, of little use in the treatment of patients affected by dystrophic epidermolysis bullosa (DEB); furthermore, the cells of patients with infectious diseases present serious difficulties in their processing in pharmaceutical factories authorised to produce ATMPs and therefore these patients are effectively precluded from accessing this type of treatment (this occurs in over 70% of cases).

Allogeneic skin substitutes, i.e. those made from skin samples from different subjects (living donors or cadavers), processed in Cell Factories, are instead very useful for the treatment of patients with extensive burns, with large skin losses and can be used on infected lesions after careful surgical cleaning of the lesion bed.

Recently, several procedures have been proposed for the production of skin substitutes based on both fibroblasts and keratinocytes and other human cells (peripheral blood monocyte cells - PBMNC), such as those developed by:

1. Organogenesis ? Apligraft: ? product consisting of a skin graft containing live keratinocyte and fibroblast cells, grafted onto a type 1 bovine collagen support.

2. S&N ? HP802-247: The product consists of expanded keratinocyte and fibroblast cells grafted onto a fibrin gel. The cells are irradiated and then subsequently frozen to stop their growth process.

3. CureXcell ? Macrocure: ? a product based on PBMNC cells concentrated by centrifugation and activated through a hypo-osmotic shock process (suspension of the cells in a low saline solution). The product is injected directly into the lesion site: the proposed mechanism of action is to stimulate tissue regeneration through the action of growth factors contained in the cells.

However, these procedures still present several disadvantages such as:

1. Organogenesis ? Apligraft. ? is authorized on the US market as a medical device, but has not obtained approval on the European market due to the incorrect definition of the mechanism of action of the cellular components. Furthermore, the clinical evidence is judged by the American regulatory authority (FDA) not to meet the needs of treatment.

2. S&N ? HP802-247: after expansion, the cells are subjected to an irradiation process that destroys the alarmin proteins and growth factors within the fibroblasts. This results in a safe product, but without the regenerative capacity necessary for the treatment of LCC. During the execution of the phase 3 clinical trial phase in the USA, it was found that the product did not reach the pre-established efficacy objectives: it was published on the website of the manufacturing company that the trial was suspended as the product did not show a significant clinical advantage compared to the placebo treatment (http://www.smith-nephew.com/news-andmedia/news/top-line-results-of-hp802-247-phase-3-study/).

3. CureXcell is Macrocure. The company conducted the phase 3 study for CureXcell for registration as an advanced therapy medicinal product (EMA study code is MC-105) for the treatment of CLL: the company admitted that the product did not meet the clinical efficacy endpoints and that therefore the registration process is suspended.

The skin is the fundamental organ that separates the organism from the outside, and for this reason it has some peculiarities at the molecular level that allow it to act as a physical barrier, to produce antimicrobial substances (at the epidermis level) and to perform immunomodulatory functions (at the dermis level).

In the skin, repair processes require the cooperation of several factors, including hyaluronic acid, which is the most abundant molecule in both the dermis and the epidermis. The presence of hyaluronic acid is a necessary marker for the initial processes of healing and neoangiogenesis, as well as being accompanied by inflammatory phenomena necessary for repair processes. In the epidermis, the role of hyaluronic acid is critical as it allows the differentiation of keratinocytes and is particularly abundant in the spinous and granular layer of the epidermis. The absence of hyaluronic acid is not compatible with life, while its hyperproduction in the dermis is present in the Shar-Pei dog breed, while the longevity of the African hairless rat (

High-molecular-mass hyaluronan mediates the cancer resistance of the naked mole rat. Nature. 2013 Jul 18;499(7458):346-9). From what has been reported, the topical use on damaged epidermis of preparations containing both high molecular weight HA with hydrating and protective action appears scientifically sustainable.

Recently, it has been observed that the biological action of HA fragments is strongly dependent on the size of the oligosaccharides. In fact, the functions of HA change radically depending on its size, sometimes producing opposite effects. Recently, it has been noted that the role of HA fragments is critical in repair processes such as neoangiogenesis (

Hyaluronan oligosaccharides are potential stimulators to angiogenesis via RHAMM mediated signal pathway in wound healing. Clin Invest Med.2008;31(3): E106-16.) and the repair of the epidermis

Low molecular weight hyaluronic acid increases the self-defense of skin 15 epithelium by induction of betadefensin 2 via TLR2 and TLR4. J Immunol.2008 Aug 1;181(3):2103-10.), as well as the presence of oligosaccharides in tissues during tissue repair processes is a natural phenomenon. HA oligosaccharides are produced by the tissue itself through mechanisms that are still poorly understood but which involve both enzymatic (endogenous hyaluronidases) and physical (free radicals) digestion.

Despite this, dermal substitutes based on hyaluronic acid derivatives do not allow for an appropriate treatment of LCC.

Furthermore, there are known processes for the production of skin substitutes, which may also include fibroblast cells, which involve a cryopreservation phase. The latter, however, seems to be merely functional to the conservation of the skin substitute, in addition to the purpose of obtaining reserves to be stored. The temperature of the cryopreservation phase for the known processes is very low and ranges between -180?C and -80?C. These low temperatures require a greater degree of complexity in the production process, as well as higher management costs. In fact, low temperatures are often obtained by using liquid nitrogen.

Therefore, there is a need for epithelial tissue substitutes, in particular allogenic skin substitutes, that overcome the drawbacks of those known from the state of the art and/or that present a better clinical efficacy. In addition to this, there is a need to use leaner or less complex production processes, and that favor an increase in the production of molecules capable of promoting the partial or total regeneration of the damaged/wounded or ulcerated epithelium.

SUMMARY OF THE INVENTION

The Applicant has developed, as the first object of the invention, a bioabsorbable skin substitute A) for ulcers and lesions, which is obtained by subjecting

a composition A1) comprising

a) a cellular component comprising fibroblasts,

b) a physical mixture comprising hyaluronic acid, fibrin and/or platelet-rich plasma gel;

to a cryogenic process comprising the following phases:

- cooling to 4°C and maintaining composition A1) at this temperature for a period of between 1 and 2 hours;

- freezing of composition A1) at -20°C and maintaining it at this temperature for a period of at least 18 hours.

A second object of the invention is a skin patch C) comprising

- a layer comprising or consisting of the bioabsorbable skin substitute A);

- an external protective layer B) comprising an adhesive layer B1 placed directly in contact with the skin substitute A) and an external support B2) placed on the adhesive layer B1.

A third object of the invention is a process for preparing the skin patch C) comprising the following stages:

i) Preparation of the cellular component a) including fibroblasts;

ii) Addition of the cellular component a) of the previous stage i) to a physical mixture b?) comprising hyaluronic acid, fibrinogen and/or platelet-rich plasma and obtaining a composition A2);

iii) Addition of a fibrinogen activating agent comprising or consisting of thrombin and an inorganic salt preferably chosen from: Trisodium citrate, Sodium bicarbonate Sodium chloride to obtain fibrin, and/or a platelet-rich plasma activator preferably chosen from batroxobin and/or a calcium salt with an organic or mineral acid to obtain PRP gel, to the mixture b?) from step ii) and obtaining composition A1).

iv) Transfer of the composition A1) coming from stage iii) onto the protective layer B) in contact with the adhesive layer B1) and obtaining a patch C1);

v) Cryogenic treatment of the patch C1) with a process comprising the following sequential phases:

or cooling to 4?C and maintaining the patch C1) at that temperature for a period of between 1 and 2 hours;

or freezing the patch C1) at -20?C and maintaining it at this temperature for at least 18 hours; to obtain the patch C); or packaging the patch in a sterile atmosphere.

Overall, the invention developed by the Applicant is advantageous because:

- The cryogenic treatment that the cellular component a) undergoes, induces an increase in the productive activity including fibroblasts, of substances produced by the cells themselves caused by the thermal stress to which the aforementioned cells have been subjected with the aforementioned cryogenic treatment.

Such substances are, for example, growth factors and alarmins; which promote the regeneration of the epithelium and have anti-inflammatory properties;

At the same time, the reduction in temperature of the cryogenic treatment reduces or eliminates the proliferation of fibroblast cultures, decreasing or eliminating the problem relating to a possible immune response by the subject receiving treatment with the allogenic skin substitute of the invention;

- induces a significant clinical efficacy due both to the increase in the controlled release of the aforementioned substances produced by the cells of the cellular component following the heat treatment, but is also associated with the presence of component b), i.e. a physical mixture comprising hyaluronic acid, fibrin and/or platelet-rich plasma gel.

- the production process is streamlined and less complex than those known in the state of the art; since it does not require particular or expensive technological measures, for example those that are possibly linked to reaching relatively low temperatures.

Further advantages will become apparent as the detailed description of the invention proceeds.

DESCRIPTION OF THE FIGURES

Figure 1? Graph showing the relative expression of pre-freezing vs. post-freezing alarmins from the Example 2 study, relative to the First trial.

Figure 2? Graph showing the relative expression of pre-freeze vs. post-freeze alarmins from the Example 2 study, relative to the Second trial.

Figure 3? Graph showing the relative expression of pre-freeze vs. post-freeze alarmins from Example 2 study, relative to the Third Trial.

Figures 4A and 4B? Example of skin patch C) of the invention, seen from above (Figure 4A) and in perspective (Figure 4B).

DETAILED DESCRIPTION OF THE INVENTION

Below, Applicant describes the invention in more detail.

For the purposes of this invention, the definition "includes" does not exclude the presence of additional components/stages not explicitly listed immediately after such definition. On the contrary, the definition "consisting of" or "consisting of" excludes the presence of such additional components/stages.

For the purposes of the present invention, a "physical mixture" is understood to be a set of several substances that are not linked together by covalent or ionic bonds but at most form labile bonds of the hydrogen bridge type or Van der Walls forces.

For the purposes of this invention, the definition of platelet-rich plasma or PRP refers to the supernatant obtained after centrifuging whole blood to separate red and white blood cells.

The definition of PRP gel refers to a gel obtained from PRP placed in contact with an activating agent such as batroxobin, possibly in the presence of an organic or inorganic calcium salt, more preferably calcium chloride or calcium gluconate.

The first object of the invention is the bioabsorbable skin substitute A), which is obtained by subjecting a composition A1) comprising

a) a cellular component comprising fibroblasts,

b) a physical mixture comprising hyaluronic acid, fibrin and/or Platelet Rich Plasma,

to a cryogenic process comprising the following phases:

- cooling to 4°C and maintaining composition A1) at this temperature for a period of between 1 and 2 hours;

- freezing composition A1) at -20°C and maintaining it at this temperature for at least 18 hours.

Preferably, the cellular component a) is allogeneic. In surgery, the term ?allogeneic? indicates a tissue coming from another organism, that is, from a donor; it is contrasted with the term ?autologous?.

Preferably, the cellular component a) is non-proliferating; this means that, although active and/or capable of producing exogenous substances, the cellular component a) is not, at the same time, capable of proliferating or reproducing or initiating mitosis processes. The cellular component a) comprising fibroblasts is, therefore, preferably exploited as a natural source of growth factors and alarmins, protagonists of the tissue repair or regeneration process.

Preferably, the cryogenic process comprises the following steps:

- cooling to 4°C and maintaining composition A1) at this temperature for 1.5 hours;

- freezing of composition A1) at -20?C and maintaining it at this temperature for a period of 18 hours.

The Applicant believes that the cryogenic process, which coincides with the cryogenic treatment of stage v) of patch C1) according to the process of the invention, is functional to an increase in the production of exogenous substances or cellular products by the cellular component a) comprising fibroblasts, compared to when the cryogenic process is absent, in other words when the skin substitute coincides with A1).

Preferably, such cellular products are chosen from the group consisting of: growth factors, alarmins, interleukins, and mixtures of the previous ones.

Preferably, alarmins (also called Damage-associated molecular patterns or DAMPs) have the ability to trigger the cellular response following damage and promote the regeneration/repair of the lesion/ulcer. Their antibiotic and immunomodulatory properties allow for faster healing of lesions, wounds or skin ulcers.

Preferably, the cellular products are chosen from the group consisting of: High-Mobility Group Box1 (HMGB-1), biglycan, tenascin-C (TNC), Fibroblast Growth Factor 2 (FGF-2), IL-6, IL-8, and mixtures of the above.

Preferably, the bioabsorbable skin substitute A) is of the allogenic type; furthermore, it preferably constitutes a vector for the release of proteins with regenerative capacity.

Please note that the cellular component a) comprising fibroblasts of the skin substitute A) is obtained from the skin substitute A1) by cryogenic treatment comprising the above-mentioned phases:

- cooling to 4°C and maintaining composition A1) at this temperature for a period of between 1 and 2 hours;

- freezing composition A1) at -20°C and maintaining it at this temperature for at least 18 hours.

This cryogenic treatment, as anticipated above, coincides with stage v) of the preparation process of the patch C) further object of the invention.

Skin substitute A) differs substantially from intermediate A1) in the cellular component a) which, due to freezing, contains substances

Component b) of the intermediate skin substitute A1) is made up of a physical mixture comprising hyaluronic acid, fibrin and/or Platelet Rich Plasma gel? b).

Component b) of both the intermediate composition A1) and the skin substitute or the physical mixture comprising hyaluronic acid, fibrin and/or Platelet Rich Plasma gel? b) is obtained starting from a physical mixture comprising hyaluronic acid, fibrinogen and/or Platelet Rich Plasma, in the presence of at least one fibrinogen activator, and/or in the presence of at least one platelet rich plasma activator, as described in stage iii) of the process object of the invention to prepare the skin patch further object of the invention.

The bioabsorbable skin substitute A) is particularly suitable for the treatment of ulcers and lesions and preferably chronic ulcers and lesions as it allows the regeneration of damaged or ulcerated skin tissue.

Skin patch C)

A second object of the invention is the skin patch C) comprising

- an inner layer comprising or consisting of the bioabsorbable skin substitute A);

- an external layer B) placed on the bioabsorbable skin substitute A) comprising an adhesive material B1) placed in contact with the skin substitute A) and an external layer B2) placed above the adhesive layer B2).

The support B2) is a sterile support, preferably transparent, more preferably a highly permeable polyurethane film, the adhesive layer B2 is a hypoallergenic PSA (Pressure sensitive adhesive) type adhesive, free of latex. This allows for optimal exchange of oxygen and water vapor, such as to maintain the correct level of humidity in contact with the wound/ulcer. Furthermore, it is impermeable to liquids, bacteria and viruses and therefore protects the insertion site from external contamination.

Preferably, the skin patch C) can also contain active ingredients useful for the purposes of the invention, not produced by the cellular component a) comprising fibroblasts; these, which can be contained for example in the adhesive layer B1.

Use of skin patch C)

Skin patch C) is preferably used in tissue regeneration, preferably of epithelial or skin tissue and in particular in the treatment of wounds, ulcers or skin lesions.

Preferably, these ulcers are of the chronic type.

Preferably, the skin patch C) is applied to the wound or ulcer lesion on the side of the skin substitute A) and the protective layer is removed after a period of time ranging from 15 to 23 days, preferably between 17 and 23 days, preferably between 17 and 21 days, preferably equal to 21 days.

Skin patch preparation process C)

A further object of the invention is the process of preparing the skin patch C) comprising the following stages:

i) Preparation of the cellular component a) including fibroblasts;

ii) Addition of the cellular component a) of the previous stage i) to a physical mixture b?) comprising hyaluronic acid, fibrinogen and/or Platelet Rich Plasma

and obtaining an A2 composition);

iii) Addition of a crosslinking agent comprising or consisting of thrombin to composition A2) from step ii) and obtaining composition A1);

iv) Transfer of the composition A1) from stage iii) onto the external adhesive layer B) and obtaining a patch C1);

v) Cryogenic treatment of the patch C1) with a process comprising the following phases:

or cooling to 4?C and maintaining the patch C1) at that temperature for a period of between 1 and 2 hours;

or freezing the patch C1) at -20?C and maintaining it at this temperature for at least 18 hours.

Stage i) - Preparation of the cellular component a) comprising fibroblasts Preferably, stage i) of preparation of the cellular component a) comprising fibroblasts comprises the following steps:

i-1) prepare at least one fragment of human skin obtained by biopsy from a skin donor;

i-2) separate the epidermis from the dermis using conventional methods;

i-3) enzymatically treat (or with enzymes) the dermis to isolate fibroblast cells;

i-4) grow fibroblast cells in a suitable culture medium until confluence to obtain fibroblast cell cultures;

i-5) freeze the fibroblast cell cultures obtained in the previous stage.

Phase i-1) - prepare at least one fragment of human skin obtained by biopsy from a skin donor

Phase i-1) consists of preparing at least one fragment of human skin obtained by biopsy from a skin donor.

Preferably, a skin donor is initially selected. For example, such skin may be waste skin obtained from a weight reduction operation. The selection of the skin donor occurs in accordance with the European Directives on the donation, procurement, testing, processing, preservation, storage and distribution of human tissues and cells (2004/23/EC, 2006/17/EC and subsequent updates and national implementations).

Preferably, once the skin donor has been identified, a biopsy is performed to obtain at least one fragment of human skin.

Preferably, the at least one skin fragment is preferably stored in a suitable transport medium, then it is subsequently sent, via a qualified courier, and with controlled transport at a temperature between 2?C and 8?C, to a pharmaceutical plant authorized for the production of cellular products.

Preferably, human skin waste of approximately 36 cm<2> is collected; however, by maintaining the same geometry, it is possible to reduce or increase the number of flasks to be seeded after the digestion step.

Phase i-2) - separate the epidermis from the dermis

The separation of the epidermis from the dermis is preferably done by incubation in the presence of Neutral Protease. The operating conditions are preferably the following: 6 DMCU/cm2 of tissue in 7 ml of HBSS 14?l of a solution of CaCl21M, preferably for 18 h at 4?C. After enzymatic digestion with Neutral Protease, the skin fragment is washed with DPBS (or Dulbecco`s Phosphate Buffered Saline); then, transferred to a Petri dish and using sterile forceps the epidermis is separated from the dermis. The dermis fragment is then minced into smaller pieces with a scalpel and transferred to a 15 mL tube. DMCU is the following compound: (3-(3,4-dichlorophenyl)-1,1-dimethylurea). HBSS is Hanks' Balanced Salt Solution.

Phase i-3) - enzymatically treat the dermis and isolate the fibroblasts

The dermis is enzymatically treated preferably with collagenase. Preferably, the operating conditions are as follows: 0.3 PZU/ml in 10 ml of HBSS 20ul of CaCl21M, preferably for 2 h at 37?C. At the end of the digestion, the solution is filtered to eliminate the larger pieces and is subjected to centrifugation to obtain a pellet of cells extracted from the tissue.

Step i-4) - grow fibroblasts in a suitable culture medium until confluence to obtain fibroblast cell cultures

Preferably, fibroblasts are cultured and expanded, preferably flask-expanded, in a suitable medium until confluence, preferably to 70-80% confluence.

Preferably, a suitable culture medium or culture medium is composed of: Dulbecco's Modified Eagle Medium (DMEM), Glutamine, Sodium Pyruvate and Fetal Bovine Serum (FBS) (irradiated and with European Directorate for the Quality of Medicines (EDQM) certificate); the formula of the complete culture medium or Complete Medium (CM) is as follows: 500 mL DMEM, 10 mL Glutamine 200 mM, 50 mL FBS, 5 mL Sodium Piruvate 1M.

Preferably, the cultivation and expansion process scheme according to phase i-4) is as follows:

- Sow in 6 T25 flasks

- Expansion in 6 T150 flasks

- Sow in 1 5-tier cellstack

- Perform further splits into 5-tier cellstacks depending on the required batch size while maintaining the seeding density at a value of approximately 5000 cells/cm<2>.

All components of the medium must be sterile.

Preferably, cultivation and expansion are performed under a laminar flow hood (grade A) with a grade B environment, while the components are added sterilely into the bottle containing DMEM.

Preferably, the medium is changed every 2-4 days, checking cell growth under the microscope at each medium change.

At each split, the medium is removed from the flask, the flask is then washed with Dulbecco's Phosphate-Buffered Saline (DPBS) and recombinant trypsin is added, cells are then seeded into the flask or cell stack.

Step i-5) - freeze the fibroblast cell cultures obtained in the previous stage. Preferably, at the end of the expansion of step i-4) above, the fibroblast cell cultures are collected and divided into containers or vials or cryovials, preferably each vial or cryovial contains 10x10<6 >cells.

Fibroblast cell cultures are then frozen, preferably in a suitable medium and in the presence of a cryopreservative or cryoprotectant. Preferably, the suitable medium is Fetal Bovine Serum (FBS or PBS); preferably, the cryopreservative is dimethyl sulfoxide (DMSO), preferably the latter is equal to 10% of the total content.

Preferably, before step ii) of adding component a) of previous step i) to a physical mixture b?) comprising hyaluronic acid, fibrinogen and Platelet Rich Plasma, cellular component a) is thawed. Preferably, for the purposes of the invention, the volume of thawed cellular component a) is 1 ml. Preferably, the thawed cellular component a) is placed in a 15 ml tube.

Preferably, after thawing, a suitable medium, physiological saline or PBS (Phosphate-buffered saline), preferably 9 ml of PBS, is added to the cellular component a). After that, the cellular component a) added with the suitable medium is centrifuged, then the supernatant is removed and the resulting solid (or pellet) is resuspended with physiological NaCl solution, preferably 200 ?l of NaCl.

Stage ii) - Addition of the cellular component a) of the previous stage i) to a physical mixture comprising hyaluronic acid, fibrinogen and/or Platelet Rich Plasma

The physical mixture b?) comprising hyaluronic acid, fibrinogen and/or Platelet Rich Plasma is initially prepared.

The hyaluronic acid used in this stage is previously sterilized by filtration through 0.2 micron filters; then, aseptically added in stage ii) of the invention process to the fibrinogen or PRP in sterile solution, in an ISO 7 clean room inside an ISO 5 biological safety cabinet to obtain the physical mixture b?).

Preferably, the amount of hyaluronic acid added in step ii) is between 0.5 and 5 mg/ml, preferably between 1 and 4 mg/ml by weight on the total volume of the physical mixture and shows a weight average molecular weight between 1000 and 3000kDa.

The physical mixture b? is then added to the cellular component suspended in a suitable medium and coming from the previous stage.

Step iii) - Addition of a crosslinking agent comprising or consisting of thrombin to the primary mixture from step ii) and obtaining composition A1)

Stage iii) involves the addition of an activating agent to activate the fibrinogen to transform it into fibrin and/or comprising or consisting of thrombin and at least one inorganic salt, batroxobin and/or calcium salt of an organic or inorganic acid to precipitate the PRP gel to the mixture b) from stage ii) resulting in composition A1).

The organic or inorganic salt present in the aqueous solution of thrombin is preferably chosen from trisodium citrate, sodium bicarbonate, sodium chloride, calcium chloride.

Thrombin is added in the form of a diluted aqueous solution, preferably the dilution ? 1: 40 thrombin solution/calcium chloride solution; this is obtained by adding 10 ?l of thrombin 1000 U/ml to 390 ?l of CaCl21M.

Preferably, step iii) involves the addition of 134 ?l of thrombin to the primary mixture obtained from step ii) to initiate polymerization with formation of fibrin glue. Preferably, during this step iii), gentle mixing is performed to avoid the formation of bubbles.

Preferably, sodium bicarbonate is at a concentration of 1 to 3 mg/ml, preferably 2 mg/ml.

Preferably, sodium chloride is at a concentration of 7 to 10 mg/ml, preferably 8 to 9 mg/ml.

Preferably, the formation of the skin substitute A1) is carried out at a temperature between 35?C and 40?C, preferably between 36?C and 38?C, preferably equal to 37?C.

The organic or inorganic calcium salt to be added to batroxobin to precipitate the PRP gel is chosen between calcium chloride and calcium gluconate.

This combination of hyaluronic acid, fibrin and/or PRP gel obtained in stage iii) and contained in the skin substitute A. object of the invention and in turn also contained in the patch C., allows to obtain a safe and stable product. The mixture of hyaluronic acid, fibrin and/or PRP gel in fact combines the viscoelastic, protective and anti-inflammatory properties typical of hyaluronic acid with those of a matrix based on fibrin and/or PRP gel, to generate a unique biocompatible environment, which stimulates the physiological regenerative processes of the skin.

Stage iv) - Transfer of the composition A1) from stage iii) onto the external adhesive layer B) and obtaining a patch C1)

Preferably, a protective layer B) is initially prepared comprising a support B1 based on transparent polyurethane film, on which an adhesive layer B2 is applied comprising a hypoallergenic and therefore latex-free pressure-sensitive adhesive (PSA).

Preferably, such a protective layer B) comprising both the support B2 and the adhesive layer B1 is commercially available. Preferably, the dimensions of the adhesive protective layer B) are 10x12 cm.

Preferably, the package of the adhesive protective layer B) is opened in the laminar flow hood, then the adhesive protective layer B) is placed in a dish or Petri dish, preferably rectangular and of adequate dimensions.

Composition A1) is preferably transferred immediately onto the surface of the external protective layer B) from the side of the adhesive layer B1, arranged inside a Petri dish, to obtain a patch C1). Preferably, the Petri dish is covered with a suitable lid.

Preferably, the C1) patch is left at room temperature (RT) for 15 to 25 minutes, preferably 20 minutes. Room temperature is defined as a temperature between 18?C and 25?C, preferably not exceeding 25?C.

Stage v) - Cryogenic treatment of the patch C1).

Then, after the required time has elapsed, a suitable freezing solution is prepared and placed over the patch C1), preferably covering the latter with at least one layer (veil) of freezing solution.

Preferably, the freezing solution consists of 90% NaCl saline and 10% dimethyl sulfoxide (DMSO). Preferably, the freezing solution to be placed on the patch (C1) is equal to 1 ml.

Preferably, the cryogenic treatment of the patch C1) occurs with a cryogenic process comprising (or consisting of) the following sequential sub-phases:

or cooling to 4?C and maintaining the patch C1) at that temperature for a period of between 1 and 2 hours;

or freezing the patch C1) at -20?C and maintaining it at this temperature for a period of at least 18 hours and until final use.

Stage vi) packaging the patch in a sterile atmosphere

The patch C1) is transferred from inside the Petri dish into a suitable container, preferably inside an aluminium bag; then, this suitable container is sealed, preferably heat-sealed, at the end and labelled.

EXAMPLES

Below, the Applicant provides examples for illustrative and non-limiting purposes only.

Example 1? In vitro study of cell viability.

We proceeded with the evaluation of the cellular viability of the fibroblasts inserted inside the skin patch C) of the invention, after freezing. The Alamar Blue reagent and the relative colorimetric assay were used, which provides an indication of the metabolic activity of the cells through the calculation of the percentage of reduction of the Alamar Blue reagent.

1.1 First test (27/4/2021)

The freezing process involved a transition to 40?F for 1.5 hours and one to -20?F for 18 hours.

At each time point, the metabolic activity of fibroblasts was evaluated, which correlates with cell viability.

Table 1

The data in Table 1 showed a residual metabolic activity of 4% at -20?C. The results therefore indicate that freezing leads to a high mortality of fibroblasts. This condition is essential for the cells to release a whole series of signal molecules, including alarmins, capable of stimulating tissue regeneration.

1.2 Second test (24/11/2021)

In this test, the metabolic activity of three different batches of fibroblasts (FB batch 1, FB batch 2 and FB batch 3) was analyzed after the scaffold remained at -20?C for 18 hours, 7 days, 14 days and 28 days.

Table 2

The data in Table 2 showed a residual metabolic activity at -20?C after 18h: 4% for FB batch 3, confirming the data from the first test; 11% for FB batch 1 and 9% FB batch 2.

After 7 days at -20?C: the activity for FB batch 2 is negative, while for FB batch 1 and FB batch 3 it seems to increase slightly, reaching 17% and 11% respectively.

After 14 days at -20?C, the metabolic activity of: FB batch 1 drops to zero; FB batch 2 has a percentage of 4% and FB batch 3 of 12%.

After 28 days at -20?C, the metabolic activity of: FB batch 1 becomes negative, that of FB batch 2 drops to 7% and that of FB batch 3 decreases to 8%.

Considering the results obtained, we conclude that a residual metabolic activity below 12% is not related to cell viability, but to the presence of enzymes among the cellular debris that are still able to reduce the Alamar Blue reagent used for the assay.

It is therefore confirmed that freezing at -20?C leads to a high mortality of fibroblasts after just 18 hours. This condition is essential for the cells to release a whole series of signal molecules, including alarmins, capable of stimulating tissue regeneration.

Example 2? In vitro evaluation of alarmin expression levels following the freezing process.

The analyses focused on the production of alarmins by fibroblasts inserted inside the scaffold.

Real Time-PCR (RT-PCR) assays were performed to evaluate the expression levels of the alarmins HMBG1, Biglycan and Tenascin-C, of the fibroblast growth factor (FGF2) and of the interleukins (IL-6 and IL-8).

Genes observed in Real Time:

? High-Mobility Group Box1 (HMGB1) is a non-histone nuclear protein. In physiological situations, it is ubiquitously present and regulates transcription, replication and DNA repair. In stress situations, it is secreted in a hyperacetylated form and supports tissue regeneration by stimulating cell activation, differentiation and migration (fibroblasts, monocytes/macrophages, dendritic cells and endothelial cells).

? BIGLYCAN (BGN) is a proteoglycan present on the cell membrane, where it is able to interact with various extracellular molecules (TNF-alpha, TGF-beta, cytokines, growth factors, etc.). Following stress, the cell increases the production of biglycan which is released at the extracellular level. In its soluble form, it acts as an alarmin by interacting with the cells of the immune system (macrophages and neutrophils) and activating the immune response.

? TENASCIN-C (TNC) ? is an extracellular matrix glycoprotein with regulatory action on inflammation. Following tissue damage or cellular stress, its expression is up-regulated in the first 24 hours to allow the resolution of the inflammatory state.

? Fibroblast Growth Factor 2 (FGF2) is the growth factor expressed by fibroblasts and will be used as a cellular marker.

? IL-6 and IL-8 are two cytokines that mediate inflammation. In particular, IL-8 has pro-inflammatory activity.

The analyses were performed by comparing the relative expression

- pre-freezing (immediately after polymerization) and

- post-freezing (after 18h at -20?C).

2.1 First test (31/3/2021 and 14/4/2021)

Figure 1 shows that, for this first test, dermal-derived fibroblasts express the alarmins studied. The thermal shock, carried out with the passage to 4?C and the subsequent freezing at -20?C, led to a change in expression of the alarmins taken into consideration, with the exception of HMGB1. It is also observed that the freezing ramp used favors the increase in the expression of Biglycan and Tenascin-C and the decrease of the inflammatory cytokines IL-6 and IL-8.

Since our interest is alarmins, in the two subsequent tests (Second and Third test) only Biglycan, Tenascin C, HMGB1 and the growth factor FGF2 as a fibroblastoid marker were analyzed.

2.2 Second test (8/6/2021)

As shown in Figure 2, the results confirm previous analyses: the freezing process leads to an increase in the expression levels of the alarmins Biglycan and Tenascin C, proving to be an important step for the presence of pro-regenerative factors in the final product.

2.3 Third test (20/10/2022)

As can be seen from Figure 3, the data confirm once again the results obtained with the other two tests: the freezing process leads to an increase in the expression of the alarmins Biglycan and Tenascin C. In addition, an increase in the levels of the alarmin HMGB1 is also observed.

Example 3 - In vitro evaluation of alarmin production and release following the freezing process.

We initially proceeded with the total quantification of the proteins released by the fibroblasts inserted into the scaffold after the freezing process.

The samples taken into consideration are

- the scaffold or skin patch with fibroblasts inside (called "gel+cells") and

- the scaffold or skin patch without cells (called ?gel only? or ?blank?), which was used as blank (inside the scaffold there is fibrinogen/fibrin, HA and thrombin),

after the freezing procedure at -20?C and 18h.

The BCA (Micro Bicinchoninic Acid (BCA) protein assay) data are summarized in Table 2 below.

Table 2

Subsequently, using ELISA assays, we wanted to verify and quantify the presence of alarmins produced and released by fibroblasts inserted inside the scaffold following freezing.

The targets studied are HMBG1, Biglycan, Tenascin-C and fibroblast growth factor 2 (FGF2).

Table 3 summarizes the data in pg/ml obtained during the two tests performed. The samples taken into consideration are the scaffold with fibroblasts inside (?gel+cells?) and the scaffold without cells (?gel only?), which was used as a negative control (there must be no alarmins in the gel only), after the controlled freezing ramp and 18h at -20?C.

Table 3 [ND = Not Detected]

The analyses show that fibroblasts release proteins inside the scaffold. The skin patch product of the invention will therefore be rich in these regenerative factors useful for healing ulcers.

The Applicant points out that, in the presence of "gel only", it is possible to observe a certain value, for example, of FGF2 because a phenomenon of cross-reactivity or formation of a non-specific binding of the secondary antibodies of the ELISA assay to the proteins present in the gel only could occur.

Example 4? In vitro evaluation of the amount of pre-freezing and post-freezing alarmins.

Following the results obtained, we wanted to understand whether the freezing process (1h and 30 minutes at 4°C and then at -20°C) could stimulate an increase in the production of the alarmins Biglycan, Tenascin C and HMGB1.

Two gels containing the cells were prepared (?gel+cells?). After polymerization, one was used to extract the proteins before freezing (PRE-FREEZING sample) while the other was first frozen and then used for protein extraction (POST-FREEZING sample).

Total protein quantification by BCA:

Table 4

The values obtained from the Elisa tests carried out are summarised in the following table 5 (pg/ml).

Table 5

Comparing the pre-freezing sample with the post-freezing one, an increase of 40% of HMGB1 and 80% of Biglycan is obtained, but not of Tenascin C which remains of the same order of magnitude. The Applicant points out that cryogenic treatment is essential for an enrichment of pro-regenerative factors such as alarmins.

The Applicant points out that for example 4 in question, an increase in HMGB1 is obtained but not in TNC compared to the tests in example 2. This is due to the type of assay that was performed. In example 2, the expression of messenger RNA in the cell that will go on to encode certain proteins is evaluated, while in example 4, the proteins extracted from the gel+cell are quantified.

Claims (10)

1. Bioabsorbable skin substitute A) for ulcers and lesions obtained by subjecting a composition A1) comprising a) a cellular component comprising fibroblasts, b) a physical mixture comprising hyaluronic acid, fibrin and/or Platelet Rich Plasma gel, to a cryogenic process comprising the following sequential phases: - cooling to 4°C and maintaining composition A1) at this temperature for a period of between 1 and 2 hours; - freezing composition A1) at -20°C and maintaining it at this temperature for at least 18 hours. 2. Skin substitute according to claim 1, wherein the cellular component a) is allogeneic. 3. Skin patch C) comprising - a layer comprising or consisting of the bioabsorbable skin substitute A) according to claim 1 or 2; - an external protective layer B) placed on the skin substitute A), comprising an adhesive layer B1) placed directly in contact with the skin substitute A) and an external support B2) placed on the adhesive layer B1). 4. Skin patch where the adhesive layer B) is a polyurethane film comprising a non-allergenic, latex-free pressure sensitive adhesive (PSA). 5. Process for preparing the skin patch C) according to claim 3 or 4 comprising the following steps: i) Preparation of the cellular component a) comprising fibroblasts; ii) Addition of the cellular component a) of the previous stage i) to a physical mixture b?) comprising hyaluronic acid, fibrinogen and/or Platelet Rich Plasma and obtaining a mixture A2); iii) Addition of a fibrinogen activating agent and/or rich plasma gel to the composition A2 from step ii) and obtaining the composition A1 according to claim 1, said activator comprising or consisting of thrombin and at least one inorganic or organic salt of an alkali or alkaline earth metal to precipitate the fibrin, and/or a calcium salt with an organic or inorganic acid to precipitate the platelet gel; iv) Transfer of the composition A1) coming from stage iii) onto the external layer B) in contact with the adhesive layer B1) and obtaining a patch C1); v) Cryogenic treatment of the patch C1) with a process comprising the following phases: or cooling to 4?C and maintaining the patch C1) at that temperature for a period of between 1 and 2 hours; or freezing the patch C1) at -20?C and maintaining it at this temperature for at least 18 hours; vi) packaging the patch in a sterile atmosphere. 6. Process according to claim 5, wherein step i) comprises the following steps: i-1) prepare at least one fragment of human skin obtained by biopsy from a skin donor, i-2) separate the epidermis from the dermis; i-3) enzymatically treat the dermis to isolate fibroblast cells; i-4) grow the fibroblast cells in a suitable culture medium to confluence to obtain fibroblast cell cultures; i-5) freeze the fibroblast cell cultures obtained in the previous stage. 7. Process according to claim 5 or 6, wherein prior to step ii) the cellular component a) is thawed. 8. Process according to any of claims 5 to 7, wherein step v) is carried out in the presence of a cryopreservative agent, preferably dimethyl sulfoxide. 9. Patch according to claim 3 or 4, for use in the treatment of wounds, ulcers or skin lesions. 10. Patch for use according to claim 9, wherein said ulcers are of the chronic type.