WO2010013190A2

WO2010013190A2 - Composition for hypertrophic tissue regeneration, products and uses thereof

Info

Publication number: WO2010013190A2
Application number: PCT/IB2009/053261
Authority: WO
Inventors: Luisa Gennero; Chiara Cesano; Antonio Ponzetto
Original assignee: Luisa Gennero; Chiara Cesano; Antonio Ponzetto
Priority date: 2008-07-28
Filing date: 2009-07-27
Publication date: 2010-02-04
Also published as: ITTO20080579A1; WO2010013190A3

Abstract

A composition for the cosmetic-aesthetic anti- hypertrophic and/or repairing-regenerative treatment of the skin and/or of the integumentary system comprising at least three among: at least salicylic acid, a salt and/or derivative thereof, at least one proteolytic enzyme, at least zinc oxide or a zinc salt.

Description

"Composition for hypertrophic tissue regeneration, products and uses thereof" ***

TEXT OF THE DESCRIPTION

FIELD OF THE INVENTION

The present invention relates to a composition useful for hypertrophic tissue regeneration, in particular for the aesthetic treatment of hypertrophic cutaneous tissues and adnexae, mucosa and connective tissues. The above-said composition is suitable for being prepared in various physical forms and formulations specifically adapted to their envisioned use as cosmetic compositions, cell culture media, pharmaceutical compositions, medical devices, for human or veterinary use.

The composition of the invention is efficacious in the regeneration and eutrophic recovery of hypertrophic cutaneous tissues and adnexae, mucosa and connective tissues both in vitro and in vivo, in particular as aesthetic treatment, creating the microenvironmental conditions suitable for regeneration of native stem cells and their differentiation with physiological synthesis and secretion of collagen.

SUMMARY OF THE INVENTION The object of the present invention is to provide a valid and efficacious solution for inducing the repair of hypertrophic tissues such as cutaneous and mucosal tissues and the regeneration of connective tissues in vitro and in vivo, in human and veterinary fields, through (re) creation of the physiological biochemical conditions and the optimal microenvironment for simulating the vitality and improving the trophism of compromised tissues, obtaining an overall aesthetic improvement of the tissues.

According to the present invention, such object is achieved by means of the composition defined in the annexed claims . The composition of the invention is based on the combination of at least three among: salicylic acid, a salt and/or derivative thereof, at least one proteolytic enzyme, preferably papain, zinc oxide, and at least one zinc salt. Depending on the applications and uses envisioned, the composition of the invention may be provided as a cosmetic composition (in different tissue-specific formulations), medical device, pharmaceutical composition, or cell culture medium for in vitro use. As will be described in greater detail below, the composition of the invention, based on the combination of salicylic acid, a salt and/or derivative thereof, at least one proteolytic enzyme, zinc oxide, and/or at least one zinc salt, was tested both in vitro and in vivo. The results obtained confirm the aesthetic repair, regeneration and re-trophisation of hypertrophic tissues such as skin, mucosa, hypoderm and of connective tissue in general.

Particularly significant are the histological results obtained in vitro after six months of treatment of hypertrophic scar biopsies. The repair and re-trophisation of tissues induced by the composition of the invention is morphologically comparable to the trophic condition of the intact tissues in vivo, with optimal histo-functional characteristics. Note that with normal in vitro culturing media, tissues from cutaneous biopsies generally do not survive more than one month.

For a general definition of the characteristics of hypertrophic tissues and keloids see bibliographic references Costa AM, et al., Am J Pathol. 1999 Nov; 155(5) : 1671-9; and Ehrlich PH, et al., Am J Pathol. 1994; 145:105-113.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in detail, by way of non-limiting example only, with reference to some preferred embodiments. The present invention provides a valid and efficacious solution for inducing a restoring and regenerating activity of cells and tissues so to allow recovery of the somatosensory picture through the correct synthesis, metabolism and catabolism of collagen and so to accelerate tissue repair and, finally, to restore the correct tissue aesthetic. In particular, the present invention provides a valid and efficacious solution to improve the vitality and trophism of hypertrophy-compromised, epidermal, dermal, mucosal and connective tissues in general, through the creation of conditions favourable to repair, differentiation of pluripotent stem cells resident in the involved tissues, native and non-native, resulting in a complete aesthetic regeneration . The invention is based on the surprising observation made by the present inventors of a particular pro- proliferative stimulus exerted by the combination of at least salicylic acid, a salt and/or derivative thereof and at least one proteolytic agent, such as, for example, papain, in combination with zinc oxide or, alternatively, with a zinc salt, on cutaneous and mucosal tissue, hypoderm and connective tissue in general.

In fact, the present inventors surprisingly verified that salicylic acid, a salt and/or derivative thereof participate in several enzymatic processes and have a regulatory action on the metabolism and catabolism of collagen, inducing the control and proliferation of fibroblasts at the level of cutaneous and connective tissues.

In performing the experiments the present inventors could verify that parallel to the action induced by salicylic acid, a salt and/or derivative thereof, the proteolytic enzyme (preferably papain) in synergy with salicylic acid, a salt and/or derivative thereof promotes two fundamental functions, which are: (i) activation of the growth factors physiologically present in the microenvironment of damaged or senescent tissues, and (ii) activation of the same growth factors attracted by the salicylic acid, a salt and/or derivative thereof, with consequent chemotactic induction of the resident stem cell compartment and a rapid repairing differentiation. Chemotaxis is a process through which a cell is able to detect gradients of extracellular chemical signals and to move in the direction of the concentration gradient. Chemotaxis plays a fundamental role in the development and aesthetic regeneration of covering tissues.

Zinc is an essential trace mineral present in the organism in quantities greater than any other oligo element apart from iron. It is involved in the normal uptake and action of vitamins, in particular those of the B complex. It is a constitutive element of more than 2,000 enzymes involved in digestion and metabolism. Furthermore, it was experimentally verified by the present inventors that, in addition to acting as an emollient, zinc oxide or alternatively a zinc salt is able to synergise with salicylic acid, a salt and/or derivative thereof and the at least one proteolytic enzyme to induce an increase in local vascularisation important both in the cosmetic treatment of hypertrophic scars and keloids to correct their aesthetic defects.

Furthermore, it was discovered that salicylic acid, papain and zinc oxide act synergistically, determining: (i) a micro-desiccation and a superficial exfoliation of the wounds, eliminating the severely damaged cells and correcting the peripheral disemia (imbalance of mineral salts in the blood arriving to the dermal capillaries consequent to a lesion) which appears in the dermis after trauma, (ii) acceleration of the uptake of the nutrients present in the preparation and, finally (iii) induction of tissue repair, of protein synthesis, hormonal functions and the process of cutaneous regeneration, inhibiting the lipases of bacteria, yeast and various skin saprophytes. This treatment has the purpose of actuating the esthetical improvement of the treated tissues. Furthermore, the present inventors discovered that with papain the Langerhans cells (stratum spinosum) are stimulated to take up foreign substances (excess collagen) and digest them. Such mechanism was particularly useful in the aesthetic treatment of keloids.

Such absolutely surprising and unexpected observations led the present inventors to the creation of a composition based on the combination of salicylic acid, a salt and/or derivative thereof (introduced in the composition in the form of F. U. salts - official pharmacopoeia), at least one proteolytic enzyme, preferably papain, and zinc oxide, or alternatively, at least one zinc salt.

The histological results obtained in vitro after cosmetic treatment with the composition of the invention confirm the induction of aesthetic regeneration and re- trophisation of cutaneous tissues and adnexae, hypodermal, mucosal and connective tissues, morphologically comparable to the intact tissues in vivo with optimal histo-functional characteristics (see Example 1) and with the maximum correction of aesthetic defects.

The cosmetic treatment of hypertrophic scar biopsy tissue in vitro with the composition of the invention results in a surprising repair of the aesthetic damage and in a total recovery of the original trophism. All tissues represented in the treated biopsies are subjected to complete functional and morphological regeneration.

The histological results obtained in vivo after 30 - 60 days of treatment with the composition object of the present invention and its tissue-specific formulations (see the section relative to Clinical Studies) , confirm at histological examination the formation of regeneration and re- trophisation of cutaneous and subcutaneous tissues, mucosa and connective tissues in general with optimal histo- functional characteristics, morphologically comparable to intact cutaneous, subcutaneous, mucosal and connective tissues in vivo. In detail, the composition object of the present invention comprises at least three among: i) salicylic acid, a salt and/or derivative thereof, ii) at least one proteolytic agent, preferably papain, iϋ) zinc oxide, and iv) at least one zinc salt.

The zinc salt is preferably selected from zinc chloride, zinc sulphate, zinc phosphate, zinc nitrate.

The salicylic acid, a salt and/or derivative thereof is preferably present in the composition at a concentration comprised in the interval from 1 to 10 g/kg, preferably from 4 to 8 g/kg.

The proteolytic enzyme is preferably present in the composition at a concentration comprised in the interval from 0.1 μg/L to 100 mg/L, preferably from 10 mg/L to 80 mg/L.

The zinc salt and/or the zinc oxide is preferably present in the composition at a concentration comprised in the interval from 0.1 to 30 g/kg, preferably from 1 to 10 g/kg. Furthermore, in an even more preferred embodiment of the invention, the composition comprises additional active ingredients, selected for their enhancing and synergistic activity with the two (three) active principles defined above, specifically for each tissue type to be treated. Such active ingredients, for example, are selected from an amino acid, a sugar, a vitamin, ventilated green clay.

The composition is advantageously formulated as a lyophilised preparation, cream, gel, foam, or injectable solution. Examples of tissue-specific compositions

In order to obtain a deep tissue disinfection before treatment, the use of topical prophylaxis with physiological solution supplemented with Gentamicin 640 mg/kg is recommended . Table 1. Composition CHELl-MD GEL (medical device) for the treatment of cutaneous and subcutaneous tissue

Table 2. Composition CHEL1-CREMA for treatment of cutaneous and subcutaneous tissues.

BASE in the form of a cream or emulsion (O/A or A/0) (such as: water, white vaseline, cetostearyl alcohol, q.s. per kg of product mineral oil, ceteth-20, sodium phosphate, p-chloro-m-cresol, phosphoric acid)

Table 3. Composition CHELl-INFUS (liquid infusion) for the treatment of cutaneous and subcutaneous tissues.

The present invention is based on the observation of a particular repairing and aesthetic regenerative stimulus exerted on cutaneous, subcutaneous, mucosal and connective tissues by the combination of salicylic acid, a salt and/or derivative thereof, with proteolytic agents (preferably papain) , zinc oxide and possibly zinc salts optionally supplemented with further active components such as:

- mixtures of amino acids, preferably L-Glycme, L- Leucme, L-Lysme, L-Proline;

- vitamins A, B, C, D, E, H and PP; - Ventilated green clay. The salicylic acid is preferably introduced in the compositions of the invention in the form of a salt and/or derivative thereof. In addition to the salicylic acid mentioned in the above tables, it is possible to obtain the same results using acetyl-salicylic acid, compositions containing salicylate, salix extracts, their combinations and derivatives, am'ong others.

As proteolytic enzymes, in addition to the already cited papain, for example, collagenases may be used (preferably of type Ia, type II, type IV), serratiopeptidases, elastases, bromelain, bradykinase, Clostridium peptidases, enzymes expressed by Lactobacillus acidophilus , enzymes expressed by the genus Aspergillus, proteases, alliinases, fibrinolysin. Mixtures of amino acids, preferably containing L- Glycine, L-Leucine, L-Lysine, L-Proline, constitute pro- eutrophic elements that function as important growth factors for cutaneous (basal layer) and subcutaneous tissues, regulating cellular and tissue turnover. Among the amino acids that may be present in the compositions of the invention we cite by way of example methionine, cystine, N-acetylcysteine, cysteine, glycine, leucine, isoleucine, proline, glutamine, arginine, glutamic acid, histidine, histidine-HCl, lysine, lysine-HCl, phenylalanine, serine, threonine, tryptophan, tyrosine, tyrosine-disodium salt, valine, hydroxyproline . Such amino acids are often used in mixtures comprising a large number of different amino acids.

In addition to amino acids, the compositions of the invention may also comprise peptides and proteins, such as, glutathione, glycocolic acid, soy lecithin, collagen, elastin, wheat extract, and the like.

Among the vitamins that may be present in the compositions of the invention we cite, by way of example, retinoic acid, retinaldehyde, retinol, alpha-tocopherol, beta-carotene, cholecalciferol, ascorbic acid, pantothenic acid, dexpanthenol, D-calcium pantothenate, cocarboxylase tetrahydrate, pyridoxine, pyridoxine-HCl, folic acid, niacinamide, nicotinamide, riboflavin, riboflavin dihydrate sodium phosphate, cyanocobalamin, para-aminobenzoic acid, biotin.

It was discovered by the present inventors that vitamin A, used at doses that are free of any pharmacological effect, potentiates correct tissue nutrition, bringing about the gradual physiological restoration of the cutaneous and subcutaneous microenvironments (restoration of the correct pH and antioxidant potential) .

Always based on the experiments of the present inventors, it was verified that in the composition object of the present invention the B vitamins, preferably Bl, B2, B5, B6, B9 and B12 contribute to the normalisation of the local- regional microenvironment, promoting an ideal exchange of albumin and of the cell nuclei favouring correction of the protein and electrolyte concentration altered during hypertrophic tissue reactions. Always based on the experiments of the present inventors, it was verified that in the composition object of the present invention vitamin C promotes the recovery of physiological collagen synthesis in the damaged dermis and basal membrane, while vitamin D at physiological values plays an auxiliary role in the consolidation of an ideal sodium- calcium ionic exchange, favouring adjustment to physiological values of altered electrolyte concentration.

The present inventors have also demonstrated that vitamin E in the composition object of the present invention, used at doses free of any pharmacologic effect, brings about the gradual physiological recovery of the microenvironment, mainly of the subcutaneous microenvironment, actively contrasting the aesthetic imperfections deriving from hypertrophic tissue alterations, and acts loco-regionally as a co-adjuvant for physiological collagen biosynthesis by fibroblasts . Concerning the activity of vitamin H (Biotin) in the composition object of the present invention it was discovered that it induces an anti-inflammatory and loco-regional anti oedemic action, restoring reduced permeability of the physiological microenvironment and favouring the activation in the organism of two repair mechanism: the attraction of stem cells from the circulatory stream and an accelerated specialisation of resident stem cells in the skin towards a rapid differentiation into mature cells destined to restore correct aesthetics of the damaged tissues.

Furthermore, the present inventors have discovered that vitamin PP (Nictinamide) , in association with the various components of the composition object of the present invention, local-regionally induces several mechanisms of action:

1) it functions as a constituent of enzymes involved in redox reactions;

2) it participates in energy metabolism of carbohydrates and proteins; 3) it takes part in stimulating capillary turnover.

4) participates as an active cofactor for the skin, in the synthesis of sex hormones.

Regarding the Ventilated Green Clay, the present inventor verified that it has an excellent absorbing and thickening power when present in gel or cream formulations; furthermore, it has good re-mineralising capacity and it activates tissue cationic exchange.

The cosmetic, pharmaceutical and medical device type compositions object of the present invention may also comprise further accessory elements such as excipients and vehicles whose choice and employment fall within the capacity of one skilled in the art with no need to exert inventive activity.

The compositions of the invention prepared as culture media were also tested to verify their efficacy in conserving the vitality of hypertrophic biopsy samples. All biopsy samples of hypertrophic scar tissues tested responded positively to the use of the culturing media of the invention, remaining vital, reorganising the three dimensional tissue structure and physiologically depositing collagen with an ordered and three dimensional redistribution during at least six months of culture in vitro.

The results obtained with the culture media object of the present invention demonstrate that the state of cutaneous hypertrophy occurring in degenerative processes can be recovered, restoring the correct eu-aesthetics of the tissue.

According to the present invention, the culture media may comprise further ingredients, such as, for example, the usual inorganic salts, sugars, peptides, amino acids and vitamins required for the maintenance and/or growth in culture of mammalian cells, as well as possible antibiotic and/or antimicrobial agents required to avoid culture contamination.

Examples of cell culture solutions to which the composition of the present invention can be added are, for example, RPMI1640 (cell culture medium) , DMEM-LG (cell culture medium), AIM-V (cell culture medium), high glucose concentration modified D-MEM (cell culture medium) , EBM (cell culture medium), human albumin, FBS (foetal bovine serum for cell culture), F12 (solution for cell culture containing a complete amino acid source), HANK' s solution (solution for cell culture containing sodium bicarbonate).

Finally, in the category of antibiotics and antimicrobials we cite, by way of example, gentamicin, penicillin, streptomycin, ciprofloxacin, levofloxacin, metronidazole, clorhexidine, amphotericin B, fluconazole, itraconazole, antimycotics, triazoles, silver (bacteriostatic activity) .

EXAMPLE 1. Biopsies and prototype-solutions Biopsies The animal biopsy samples under study are constituted of hypertrophic scars surgically removed for the purpose of correcting serious cutaneous and subcutaneous functional retractions .

All samples were washed three times with physiological solution containing antibiotics (100 Units/ml penicillin + 100 μg/ml streptomycin + 160 mg/L gentamicin + fluconazole

0.2 mg/ml) for ten minutes at room temperature.

The biopsies were then sectioned into three parts (two controls, 1 and 2, and one sample 3 to be treated for each patient) and suspended in a final volume equal to 25 ml of the corresponding culture solution in 10 cm plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark) .

Two types of controls were prepared, an untreated negative control (1), that is, treated with physiological solution and antibiotics only (as described above) , and a positive control (2) treated with cell culture medium commonly used for cutaneous biopsies.

1. Negative control: the control 1 biopsy specimens were suspended in physiological solution in 10 cm plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark) . 2. Positive controls: the control 2 biopsy specimens were placed in 10 cm plates (Lab-Tek chamber slides, Nunc,

Kamstrup, Denmark) in D-MEM medium supplemented with:

10% FBS (Celbio, Milano, Italy)

160 mg/L gentamicin (Schering-Plough, Milan, Italy) 2 mM L-glutamine (Life Technologies; growth medium)

50 ng/mL EGF (Sigma Aldrich, Milan, Italy) .

3. Samples: the sample biopsy specimens 3 were placed in

10 cm plates (Lab-Tek chamber slides, Nunc, Kamstrup,

Denmark) in Culture media CHELl-INFUS. All samples were incubated for 15 days in a Heraeus incubator thermostatically controlled at the temperature of

37 °C with a constantly supplied atmosphere containing 5% CO₂

(v/v in air) . Two-thirds of the culture medium were replaced every 7 days. All biopsy tissues used in culture constitute a possible co-conditioning optional support for the three dimensional growth of the cell samples under study .

Staining protocol

After three 10 minute washes at room temperature in PBS

(pH 7,4), the samples were resuspended in a 4% paraformaldehyde fixing solution in D-MEM (Gibco) pH 7.4 for one hour at room temperature. All biopsies object of study were treated with^"'Alcian blue. This stain is made of a group of water soluble polyvalent basic dyes. The colour blue is due to the present of copper in the molecule. Alcian blue at a final concentration of 1% w/v in PBS (pH 7.4) is added to a 3% acetic acid solution (pH 2.5). After a two hour incubation at room temperature this composition permanently stains acidic mucopolysaccharides and glycoproteins both sulfonated and carboxylated. Specific controls were set up for each sample. All samples were washed three times with PBS (pH 7,4) at room temperature for five minutes and then observed with light microscopy. A net increase in type 1 collagen and type 4 collagen that stain in blue is noted in the samples treated with the solution Culture medium CHELl-INFUS with respect to control 1 and to control 2 [2] . RESULTS Staining with the Alcian blue colorimetric method

- Negative controls 1 treated with physiological solution: diffuse blue staining (score = ++++) alternating with cytolytic and necrotic areas.

- Positive controls 2 treated with common biopsy culturing medium, as described above. A very strong diffuse Alcian blue staining (score = +++++) .

- Sample treated with the solution Culture medium CHELl- INFUS. It is noted that the cells, in which a re-deposition of mucopolysaccharides and glycoproteins, stain well with Alcian blue growing in superimposed and ordered layers with physiological distribution of mucopolysaccharides and GAG (Score = +++) . Western blot

Samples were subjected to phenotypic analysis with Western Blot for the markers (Santa Cruz Biotechnology, America, California) anti-collagen type I, anti collagen type IV, anti cytokeratms 1, 5, 10, 14. After five washes, the membranes were incubated with the corresponding secondary antibodies (1:1000) conjugated with horseradish peroxidase (HRP, Santa Cruz, Calif. USA) for Ih at room temperature as reported in table 3 below.

Characterisation of samples treated with Culture medium CHELl-INFUS compared to controls

The results relative to the expression of collagen type I, collagen type IV and of the cytokeratms 1, 5, 10 and 14 are expressed on a quantitive scale as follows:

Table 4

Legend band absent

-/+ light presence of band

+ band present thin

++ band present medium +++ band present extensive ++++ band present high

+++++ = band present effuse

EXAMPLE 2. IN VIVO CLINICAL STUDIES Formulation CHEL1 -CREMA.

IN VIVO CLINICAL STUDY 1.

A clinical study was performed apt to evaluate the tolerability and therapeutic efficacy of the product denominated CHEL1-CREMA (composition reported in table 2) in the regeneration and repair of cutaneous and subcutaneous tissues.

The present study was performed on a sample constituted of four dogs of different races and sizes and four humans, these also of different races and ages, presenting cutaneous lesions attributable to scar hypertrophy (canine : laceration contusion wounds, surgical wounds, licking wounds, burns) .

At the enrolment visit subjects affected by the above indicated lesions were selected.

The animal was brought to weekly clinical follow-up visits for cytological examination and evaluation of the extension and depth of the cutaneous lesion, until healing. Case 1 A 9-year-old female Boxer dog was presented at the clinical visit for a lesion of the peroneal nerve and consequent dorsal placement of the foot and auto-traumatic ^"lesions from licking of the skin. After corrective surgery with transposition of the flexor tendon of the toe anchored to the extensor tendon of the toe, the posture allowed for correct placement of the distal extremity of the limb. In spite of resolving the motor problem, the cutaneous lesions from reactive hypertrophic fibrosis consequent to chronic licking persisted. Clinical Examination . Erithema, ulcerations and cutaneous thickening consequent to fibrosis of the dorsal portion of the metacarpal and of the phalanges.

Diagnosis . Clinical picture attributable to chronic auto-traumatic dermatitis complicated by secondary superficial infections. Cytological examination showed a bacterial infection of the ulcerative lesion.

Therapy. Systemic antibiotic therapy with enrofloxacin 5mg/kg per os for three weeks was administered. Wound cleaning with povidone-iodine was performed on the first day, proceeding then to the cleaning of the wound with physiological solution and application of the preparation CHEL1-CREMA twice a day for 30 days.

Follow-up visits. After one week an improvement of the infection and 50% regeneration of the tissue were detected. After two weeks the disappearance of the infection and further reduction of the lesions were observed. Clinical outcome. Four weeks after the beginning of the cosmetic treatment complete disappearance of the dermal fibrosis with recovery of a correct cutaneous aesthetic was observed . Case 2

A 12-year-old female German Shepherd dog brought to the clinical visit for multiple dorsal-caudal burns.

Clinical Examination . Geographic map lesions with liquid exudate and formation of hypertrophic-fibrous scars including fur.

Diagnosis . The cytological examination indicated bacterial superinfection of the burn wounds with hypertrophic scarring.

Therapy. Marbofloxacin was administered per os at 2.5 mg/Kg for 21 days. The fur was shaved and wounds cleaned with povidone-iodine followed by application of the preparation CHEL1-CREMA twice a day for 60 days.

Follow-up visits. After one week granulation tissue was observed, with a reduction of about 20% in the dimensions of the lesions. At the end of the second week complete healing of the wounds was observed with the formation of stable regenerated tissue and a 30% reduction of the hypertrophic scars.

Clinical outcome. At the successive follow-up, 60 days after the initiation of the cosmetic treatment, all scars had a whitish colour and were of normal resistance and consistency, free of hypertrophic components, restoring a normal somatosensory and aesthetic picture.

Case 3 An 8-month-old female Golden retriever dog.

Anamnesis . Spontaneous cutaneous laceration problems from birth resulting in secondary hypertrophic scars. Following various attempts of surgical revision the patient was subjected to various therapies: tetracyclines in association with cortisone deposit, local disinfection of the lesion areas with paromomycin sulfate + prednisolone and clostebol acetate: no appreciable effect and induced cutaneous hypersensitivity. Multiple cutaneous biopsies were performed indicating an alteration of collagen fibres consistent with Ehlers-Danlos syndrome. Day 0. The wounds and cutaneous lacerations were treated twice a day with polivinil-pyrrolidone and the preparation CHELl-CREMA with occlusive bandages on the limbs and free application, without bandages on the ischiatic area. Systemic antibiotic therapy with amoxicillin and clavulanic acid 12.5 mg/kg Bid was associated to control secondary infections.

Day 30. After 30 days, re-epithelisation of all wounds treated with the preparation CHEL1-CREMA was observed, such scars were not hypertrophic.

Within only 30 days, use of the cosmetic preparation CHEL1-CREMA promoted a eutrophic scarring and an efficacious aesthetic correction of extensive cutaneous wounds that had been complicated by superinfections and hypertrophic scarring for months, due to the genetic collagen disorder. Case 4

Description . A 12-year-old male WHWT dog. Anamnesis . For about one year the dog had presented nodular cutaneous areas of inter-digital fibroadnexal dysplasia in the posterior limbs, which had progressively increased in diameter over the previous two months, reaching a size of approximately 4 cm in diameter. Surgical removal and antibiotic therapy with cefalexin 20 mg/kg/bid per 15 days were performed.

Day 0. Disinfection was performed with povidone-iodine and the preparation CHELl-CREMA applied to the inter- digital area once a day with occlusive bandaging until follow-up . The systemic antibiotic therapy with cefalexin was continued at the same dosage. Day 15. Remission of the lesion was noted at this follow-up .

Day 90. Application of the preparation CHEL1-CREMA allowed attainment of eutrophic re-epithelialisation of the surgically treated cutaneous areas.

Day 180. Application of the cosmetic preparation CHEL1-CREMA allowed the attainment of healing of the surgical wound free of relapses and aesthetic imperfections . Case 5

A 19-year-old Caucasian woman.

Anamnesis and therapeutic protocol

The patient presented for the presence of a 1.8 cm keloid existent for 6 years in the tibiotarsal zone. A topical therapy with applications of the preparation CHELl- CREMA twice a day for 60 days was administered.

Day 0. The lesion of the tibial area was 1 cm in size hyperplastic and had modest peri-lesional erythema.

Day 60. At the follow-up the lesion was diminished by 60% with respect to the beginning of therapy.

Summary of the clinical evolution

This case showed progressive reduction in the thickness of the keloid, resulting in a good aesthetic recovery of the compromised tissues (60%) . Case 6

A 58-year-old African man.

Anamnesis ^'and therapeutic protocol

The patient presented with an excoriation at the level of the left zygomatic bone present for about 96 hours, treated only the 10% povidone-iodine . At the time of inclusion (day 0) the preparation CHELl-CREMA was applied twice a day for 21 days.

Day 0. Presence of erosions, ulcerations and peri- lesional depigmentation associated with an irregular 2 cm diameter area of serocellular crust. Day 7. The serocellular crust present at the time of the visit had regressed and the depth of the lesion was diminished by 70%.

Day 14. Small depigmented superficial linear scars remained.

Day 21. Flattening of the scars and progressive cutaneous repigmention were detected at the final visit.

Summary of the clinical evolution

The patient presented predisposition to development of hypertrophic scars. The application of the cosmetic preparation CHEL1-CREMA avoided this problem.

Case 7

A 39-year-old woman.

Anamnesis and therapeutic protocol The patient presented with secondary hypertrophic results on a caesarean section scar in the pelvic zone. A topical therapy with applications of the preparation CHELl- CREMA was administered twice a day for 60 days.

Day 0. The hyperplastic lesion in the pelvic area was about 5 cm in length with slight peri-lesional erythema.

Day 60. At follow-up the lesion had diminished by 80% with respect to the beginning of therapy.

Summary of the clinical evolution

In this case a progressive reduction (80%) in the thickness of the hypertrophic scar occurred, to the point of obtaining an almost complete flattening of the same, characterised by pigmentation similar to the surrounding eutrophic cutaneous zones. The preparation CHELl-CREMA induced a cosmetic-aesthetic correction of the originally compromised part.

Case 8

A 40-year-old Caucasian woman.

Anamnesis and therapeutic protocol

The patient presented with secondary hypertropic results with onset from a scar from removal of a junction nevus in the popliteal fossa. Topical therapy was administered with the preparation CHEL1-CREMA twice daily for 60 days.

Day 0. The lesion in the involved area was about 1 cm, hyperplastic and depigmented.

Day 60. At follow-up the lesion was reduced by 100% with respect to the beginning of therapy.

Summary of the clinical evolution

In this case a progressive reduction in the thickness of the hypertrophic scar occurred, to the point of obtaining a complete flattening of the same, characterised by a persisting lack of pigmentation in the lesioned area. The partial aesthetic correction relative to the results of lack of pigmentation in the lesioned area, could be linked to the short duration of the cosmetic treatment with CHELl- CREMA.

Formulation CHEL1-CREMA.

IN VIVO CLINICAL STUDY 2.

We have used the composition CHEL1-CREMA described in table 2, in the context of compassionate use, in 40 subjects, affected by [3] :

A) pathological scarring resulting from traumatic lesions;

B) pathological scarring resulting from surgical interventions .

Table 5 below summarises the most significant data. The lesions concerned almost exclusively the limbs, with a strong prevalence for the lower limbs.

Table 5.

We have used the Composition CHEL1-CREMA with two daily applications for a period of not less than 35 days to a maximum of 90 days. In no case was the onset of the phenomenon of allergy or intolerance detected, instead most patients reported attenuation of itching, pain and feeling of local tension a few days after beginning therapy.

Initially, in almost all subjects a hyperaemia was detected as an effect of the superficial vasodilation induced by the Composition CHEL1-CREMA; definitely a positive effect considering the occluded state of many blood vessels observed both in the hypertrophic scars and in keloids. The initial hyperaemia was often accompanied by an increase in local pigmentation due to the melanin biosynthesis- promoting action of zinc oxide. However, the hyperaemia and hyper-pigmentation were progressively attenuated with continuation of the cosmetic treatment. In the greater part of subjects the total or nearly total regression of lesions (80%) and aesthetic imperfections was obtained. Good results were observed in the majority of hypertrophic scars, with disappearance of local adherences (98%), normalisation of pigmentation, reduction in size and restoration of an overall eu-aesthetic picture. In the keloids a significant reduction (80%) in size was observed, with macroscopic modifications (variations in colour, attenuation of fibrotic cords, increase in elasticity, etc.); such observations seem to indicate a possible shift of the lesions from keloid to hypertrophic scar (90%) with net aesthetic-functional improvement. EXAMPLE III. COMPARATIVE STUDIES: Biopsies treated with complete CHEL-I formulation versus partial formulations

Twenty animal biopsy samples from hypertrophic scars removed surgically for the purpose of correcting severe cutaneous and subcutaneous functional retractions were observed.

All samples were washed three times with physiological solution containing antibiotics (100 units/ml penicillin + 100 ug/ml streptomycin + 160 mg/L gentamicin + fluconazole 0.2 mg/ml) for 10 min at room temperature. Every single biopsy of the twenty specimens collected for the experiment was sectioned into nine part (eight controls, 1 - 2 - 3 - 4 - 5 - 6 - 7 and 8, and one sample, 9, to be treated for each patient) and suspended in a final volume of 25 ml of the respective culture solutions in 10-cm plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark) . ' Eight types of controls were prepared: (1) an untreated negative control-1 resuspended in physiological solution containing antibiotics; (2) a positive control-2 treated with cell culture media commonly used for cutaneous biopsies; (3) a positive control-3 treated with incomplete Chel-1 medium containing only salicylic acid as active principle; (4) a positive control-4 treated with incomplete Chel-1 medium containing only zinc oxide as active principle; (5) a positive control-5 treated with incomplete Chel-1 medium containing only papain as active principle; (6) a positive control-6 treated with incomplete Chel-1 medium containing only salicylic acid and zinc oxide as active principles; (7) positive control-7 treated with incomplete Chel-1 medium containing only papain and zinc oxide as active principles;

(8) a positive control-8 treated with incomplete Chel-1 medium containing only papain and salicylic acid as active principles .

A type (9) sample-9 was prepared, treated with COMPLETE Chel-1 medium containing salicylic acid, papain and zinc oxide . The nine parts of each of the twenty biopsies analysed were treated as follows.

1. Negative Control-1: the control 1 biopsy specimens were suspended in physiological solution in 10-cm plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark) . 2. Positive Control-2: the control 2 biopsy specimens were placed in 10-cm plates (Lab-Tek chamber slides, ^'Nunc, Kamstrup, Denmark) in D-MEM medium supplemented with: 10% FBS (Celbio, Milan, Italy) 160 mg/L gentamicin (Schering-Plough, Milan, Italy) 2 mM L-glutamine (Life Technologies; growth medium) 50 ng/mL EGF (Sigma Aldrich, Milan, Italy) . 3. Positive Control-3: the control 3 biopsy specimens were placed m 10-cm plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark) in the solution whose composition is reported in table 6.

Table 6.

4. Positive Control-4: the control 4 biopsy specimens were placed in 10-cm plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark) in the solution whose composition is reported in table 7.

Table 7.

5. Positive Control-5: the control 5 biopsy specimens were placed in 10-cm plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark) in the solution whose composition is reported in table 8.

Table 8.

6. Positive Control-6: the control 6 biopsy specimens were placed in 10-cm plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark) in the solution whose composition is reported in table 9.

Table 9.

7. Positive Control-7: the control 7 biopsy specimens were placed in 10-cm plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark) in the solution whose composition is reported in table 10.

Table 10.

8. Positive Control-8: the control 8 biopsy specimens were placed in 10-cm plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark) in the solution whose composition is reported in table 11. Table 11 .

9. Samples: the sample 9 biopsy specimens were placed in 10-cm plates (Lab-Tek chamber slides, Nunc, Kamstrup, Denmark) in Culture medium CHELl-INFUS COMPLETE (Table 12) .

Table 12.

All samples were incubated for 15 days in a Heraeus incubator thermostatically controlled at the temperature of 37⁰C with an constantly supplied atmosphere containing 5% CO₂ (v/v in air) . Two-thirds of the culture medium were replaced every 7 days. All biopsy tissues, used in culture constitute a possible co-conditioning optional support for the three dimensional support of the cell samples under study.

Characterisation of samples treated with complete CHELl-INFUS culture medium versus controls treated with incomplete CHELl- INFUS culture medium

The results relative to the expression of type I collagen, type IV collagen and of cytokeratins 1, 5, 10 and 14 are presented with a quantative scale in table 13.

Table 13

Legend band absent

-/+ light presence of band

+ band present thin ++ band present medium band present extensive

++++ band present high +++++ band present effuse

The culturing difference between the use of COMPLETE CHEL-I (Sample-9) and the other culture media tested (Controls 1-8) is clear.

It is specified that the COMPLETE solution (Sample-9) comprises in a single solution: said salicylic acid, and/or a salt and/or derivative thereof ; said zinc oxide, or a derivative thereof; and a proteolytic enzyme, for example, papain. The colorimetric method used to evaluate the entire cutaneous and endodermal matrix produced was described above ("Alcian blue"), the preferred method used to evaluate the extracellular matrix in its entirety [2] .

Table 14. Colour Matrix

Such data were collected in parallel at days 7, 10, 15, 30, 45, 60, 75, and 90 of culture for the Controls and Samples .

Furthermore, also at days 7, 10, 15, 30, 45, 60, 75, and 90 of culturing, methods such as Western Blot (WB) were used to continuously monitor the ability of fibroblasts present in the tested culture to actively produce collagen fibres, such as type I collagen, present at high concentration in the matrix of healthy covering tissues and also m the sample cells treated with the complete CHEL-I INFUS.

It seems appropriate to point out that the culture treated with complete culturing media supplemented only with growth factors (control-2) or with complete culture media supplemented only with salicylates (control-3) or with complete culture media supplemented with only zinc oxide (control-4) or with complete culture media supplemented only with proteolytic enzymes (control- 5) or with complete culture media with only two active principles such as salicylates + zinc oxide (control- 6) , or with only two active principles such as papain + zinc oxide (control- 7) , or, finally, with only two active principles such as salicylates + papain (control-8) , do not lead to a normal and eutrophic deposition of cutaneous and hypodermal matrix equal or comparable to the results obtained with the use of all three synergistic active principles, such as, salicylates + zinc oxide + papain (Sample-9) of the COMPLETE CHELl-INFUS culture medium. Naturally, the details and the embodiments may vary, even appreciably with respect to what has been described and illustrated without departing from the scope of the present invention as defined by the annexed claims.

BIBLIOGRAFIA

1. Costa AM, Peyrol S, Porto LC, Comparin JP, Foyatier JL, Desmouliere A. Mechanical forces induce scar remodeling. Study in non-pressure-treated versus pressure-treated hypertrophic scars. Am J Pathol. 1999 Nov; 155 (5) : 1671-9.

2. French MM, Smith SE, Akanbi K, Sanford T, Hecht J, Farach-Carson MC, Carson DD. Expression of the heparan sulfate proteoglycan, perlecan, during mouse embryogenesis and perlecan chondrogenic activity in vitro. J Cell Biol. 1999 May 31; 145 (5) : 1103-15.

3. Ehrlich PH, Desmouliere A, Diegelmann RF, Cohen IK, Comp- Ton CC, Garner WL, Kapanci Y and Gabbiani G.

Morphological and immunochemical differences between keloid and hypertrophic scar. Am J Pathol. 1994 ; 145 : 105- 113.

Claims

1. A composition comprising at least three among: i) salicylic acid, a salt and/or derivative thereof, ii) at least one proteolytic enzyme, iii) at least one zinc salt, and iv) zinc oxide.

2. The composition according to claim 1, wherein said salicylic acid, a salt and/or derivative thereof is present at a concentration comprised in the interval from 1 to 10 g/kg, preferably from 4 to 8 g/kg.

3. The composition according to claim 1 or claim 2, wherein said at least one proteolytic enzyme is present at a concentration comprised in the interval from 0.1 μg/L to 100 mg/L, preferably from 10 mg/L to 80 mg/L.

4. The composition according to any of the previous claims, wherein said at least one zinc salt is present at a concentration comprised in the interval from 0.1 to 30 g/Kg, preferably from 1 to 10 g/Kg.

5. The composition according to any of the previous claims, wherein said salicylic acid, a salt and/or derivative thereof is selected from salicylic acid, acetyl- salicylic acid, compositions containing salicylate (s) , salix extract, preferably salicylic acid F. U.

6. The composition according to any of the previous claims, wherein said at least one proteolytic enzyme is selected from papain, collagenases, serratiopeptidases, elastases, bromelain, bradykinase, Clostridium peptidases, proteolytic enzymes expressed by Lactobacillus acidophilus, proteolytic enzymes expressed by the genus Aspergillus, proteases, alliinases and fibrinolysin, preferably wherein the proteolytic enzyme is papain.

7. The composition according to any of the previous claims, wherein said zinc salt is selected from zinc chloride, zinc sulphate, zinc phosphate, zinc nitrate.

8. The composition according to any of the previous claims, wherein said composition also comprises at least one among a physiologically acceptable vehicle, an amino acid, a sugar, a vitamin, ventilated green clay.

9. The composition according to any of the previous claims, wherein said composition is formulated as a lyophilised preparation, cream, gel, foam or injectable solution .

10. A pharmaceutical composition for the regeneration of hypertrophic cutaneous tissues and relative adnexae, of hypertrophic mucosal tissues and of hypertrophic connective tissues comprising a composition according to any of the claims 1 to 9.

11. An in vitro cell and/or tissue culture medium, preferably for cutaneous, subcutaneous or mucosal tissue, comprising a composition according to any of the claims 1 to 9.

12. A cosmetic composition for the regeneration of hypertrophic cutaneous tissues, relative adnexae, of hypertrophic mucosal tissues and of hypertrophic connective tissues comprising a composition according to any of the claims 1 to 9.

13. A method for the cosmetic treatment of skin, in particular for the anti-hypertrophic aesthetic treatment of the skin and/or of the integumentary system, envisioning the use of a composition according to any of the claims 1 to 9 .

14. The use of a composition according to any of the claims 1 to 9, for the cosmetic treatment of skin, in particular for the anti-hypertrophic aesthetic treatment of skin and/or of the integumentary system.