CZ308845B6 - Pharmaceutical composition of topically healing peptide components for use in the topical treatment of skin defects and / or for topical wound healing - Google Patents

Abstract

A pharmaceutical composition of topically healing peptide components comprising a spirocyclic diketopiperazine peptide of 2 to 40 amino acids, which also comprises the amino acid arginine and the amino acid cysteine, for use in the topical treatment of skin defects and / or for the topical healing of wounds, the pharmaceutical composition is contained in a matrix of 0.001 to 50 mg per gram of matrix. It is used especially for healing chronic, inflamed and complicated wounds, but also common injuries, postoperative wounds and burns. In healthy patients, it shortens the healing time by almost a third, in difficult-to-heal wounds the mixture of topically healing peptide components shortens the healing time by up to 7 times. In chronic wounds, especially pressure ulcers and varicose ulcer, the pharmaceutical composition gives improved wound healing or even complete wound healing due to the action of the combination of peptide components. Preferably, the pharmaceutical composition comprises alaptide and is applied to the wound in a protein-carbohydrate gel sterile matrix which prevents the breakdown of the infection and thus allows more effective wound healing. The pharmaceutical mixture is also used topically in aesthetic medicine and cosmetics.

Description

Pharmaceutical composition of topically healing peptide components for use in the topical treatment of skin defects and / or for topical wound healing

Field of technology

Pharmaceutical and cosmetic preparations, formulations and methods for topical or transdermal administration and treatment of wounds and / or for promoting wound healing. Therapy and treatment of the skin, where the active substance is peptides and other biological components. Wound healing, including poorly healing wounds, aesthetic medicine, cosmetics.

Prior art

Under normal circumstances, the process of acute wound healing can be divided into three (3) phases. The initial inflammatory phase, followed by strong remodeling and tissue proliferation (proliferation phase), is followed by a matum phase, where reepithelium, cutaneous angiogenesis and wound closure occur. Repeatability includes migration and proliferation of epithelial tissues, especially keratinocytes. Angiogenesis is the growth of new blood vessels from existing channels and is regulated by panoply soluble cytokines, including growth factor polypeptides, as well as the interaction of cell cells and cell matrices. Chronic wounds have a different treatment profile than normal acute wounds in that they generally remain inflamed for extended periods of time. Non-healing wounds are most commonly seen in people with diabetes, venous stasis, and in patients who are immobilized. In view of the above, it would be desirable to provide new biomolecules that could safely and effectively assist in the healing mechanism of epithelial and vascular injuries in both acute and chronic wound healing situations.

Chronic wounds have a different treatment profile than normal acute wounds in that they generally remain inflamed for extended periods of time. Poorly treatable wounds are most commonly seen in people with diabetes, venous thrombosis, and in patients who are immobilized. In view of the above, it would be desirable to provide new biomolecules that could specifically, safely and effectively assist in the healing mechanism of epithelial and vascular injuries in both acute and chronic wound healing situations.

People with diabetes have a very poor ability to regenerate the skin. Skin complications of diabetes lead to skin that is dry, tends to crack, and heals slowly. People with diabetes often have slow and insufficient skin healing. Leg pain and ulcers are the main cause of lower limb amputations in diabetics, and currently approximately 10% of diabetic patients require amputation during their lifetime. The primary reason for amputation is infections associated with the development of ulcers and currently incurable skin damage. The average risk of limb amputation in people with diabetes is 15 times higher than in people without diabetes.

Current treatment of diabetic chronic wounds fails to achieve effective therapeutic results. Most of these drugs target angiogenesis, but diabetes often involves endothelial dysfunction. A characteristic feature of regenerative wound healing is rapid and effective reepithelialization. There is a clinical need for a new, more effective treatment for chronic wounds in diabetic patients. Lack of epithelial cell migration is a hallmark of fearless wounds, and diabetes often involves endothelial dysfunction. Therefore, focusing on reepithelialization, which mainly involves keratinocytes, can improve the therapeutic results of existing treatment.

The current treatment of fearless wounds and shin ulcers is unsatisfactory. To date, no optimal method of treating skin defects has been found.

A new method has been developed for the treatment of shin ulcers, using the patient's own cells. However, the treatment is demanding and lengthy. Current treatment of diabetic chronic wounds fails

- 1 CZ 308845 B6 achieve effective therapeutic results. Most of these drugs target angiogenesis, but diabetes often involves endothelial dysfunction.

Recently, various drugs have been developed to promote wound healing. Representatives include, for example, Beclapermin, genetically engineered recombinant PDGF from Johnson & Johnson, or a pharmaceutical composition for regenerating and repairing mammalian tissues comprising PDGF and dexamethasone (EP 0575484). US 5981606 discloses a wound healing composition comprising TGF-beta and US 6800286 and US 5155214 describe wound healing compositions comprising FGF. All the drugs already described are growth factors, cytokines or chemokines, collagen or hyaluronic acid. These agents have the disadvantage of side effects because they are not specific for one cell type.

Peptides have regulatory effects, ie they are able to influence the behavior of living cells. For example, GHK (glycyl-L-histidyl-L-lysine) peptide, as a natural modulator of multiple cellular pathways in skin regeneration, is present in human plasma, saliva and urine, but its concentration decreases with age. GHK facilitates the supply of trace elements necessary for skin regeneration, such as copper. The peptide functions in complex with Cu ²⁺ , accelerates wound and skin healing and acts in enzymatic processes (Gorouhi F, Maibach HI. Role of topical peptides in preventing or treating aged skin. Int J Cosmet Sci. 2009; 31: 327-345) . GHK stimulates both the synthesis and degradation of collagen and glycosaminoglycans and modulates the activity of metalloproteinases and their inhibitors. Stimulates collagen, dermatan sulfate, chondroitin sulfate, low molecular weight proteoglycan or decorin. It also helps restore fibroblast replication vitality after radiation therapy. The molecule attracts immune and endothelial cells to the site of injury.

Other peptides used for therapy or cosmetic treatment of the skin, resp. skin, are Syn®-Coll signal peptides (Palmitoyl Tripeptide-5) with a Pal-Lys-Val-Lys-OH structure that binds directly to the receptor (Brzoska T, Böhm M, Lügering A, Loser K, Luger TA. Terminal signal: antiinflammatory effects of melanocyte-stimulating hormone related peptides beyond the pharmacophore (Adv Exp Med Biol. 2010; 681: 107-116). Furthermore, Decorinyl® (tetrapeptide; Lupo MP, Cole AL. Cosmeceutical peptides. Dermatol Ther. 2007; 20: 343-349) stimulates fibroblasts to produce collagen, proliferate elastin, glycosaminoglycans, proteoglycan and fibronectin. Peptides inhibiting neurotransmitters: Argireline® with the structure N-Acetyl-La-glutamyl-L-aglutamyl-L-methionyl-L-glutaminyl-L-arginyl-L-argininamide, Vialox® with the structure H-Gly-ProArg-Pro-Ala- NH2 and Syn®-ake (Lupo MP, Cole AL. Cosmeceutical peptides. Dermatol Ther. 2007; 20: 343-349), serve to reduce muscle contractions of the face and subsequent wrinkles by raising the minimum threshold for muscle activity.

Enzyme-inhibiting peptides: Soy protein glycine (Preregen®; Südel KM, Venzke K, Mielke H, Breitenbach U, Mundt C, Jaspers S, et al. Novel aspects of intrinsic and extrinsic aging of human skin: beneficial effects of soy extract. Photochem Photobiol., 2005; 81: 581-587 Andre-Frei V, Perrier E, Augustin C, Damour O, Bordat P, Schumann K, et al., A comparison of biological activities of a new soya biopeptide studied in an in vitro skin equivalent model and human volunteers (Int J Cosmet Sci. 1999; 21: 299-311) and Sericin (Lupo MP, Cole AL. Cosmeceutical peptides. Dermatol Ther. 2007; 20: 343-349) directly or indirectly inhibit an enzyme related to receptor aging process (Brzoska T, Böhm M, Lůgering A, Loser K, Luger TA. Terminal signal: anti-inflammatory effects of I ± -melanocyte-stimulating hormone related peptides beyond the pharmacophore. Adv Exp Med Biol. 2010; 681: 107-116). They stimulate fibroblasts to produce collagen, proliferate elastin, glycosaminoglycans, proteoglycan and fibronectin.

Alaptide, 8-methyl-6,9-diazaspiro [4.5] decane-7,10-dione, or Cyclo (L-alanyl-1-amino-1-cyclopentanecarbonyl), C9H14N2O2, Mr 182.2 g / mol, CAS: 90058- 29-0, are substances that inhibit the release of the melanocyte stimulating hormone (MIF) of L-prolyl-L-leucylglycinamide. The use of the hormone MIF alone as a therapeutic agent is limited by its easy enzymatic hydrolysis. A series of MIF spiro derivatives have been prepared primarily to eliminate this disadvantage (Kasafírek E. et al. Czech Pat. No. 231,227, 1986; U.S. Pat. No. 5,318,973, 1994; Czech Pat. No. 260899, 1989). Alaptid was chosen as the best analogue both in terms of enzymatic stability and in terms of its pharmacodynamic profile. Among other effects, it has been shown in Alaptid

- 2 CZ 308845 B6 significant healing effect in experimental animal models (Kasafírek E. et al. Cs. Pat. 276 270, 1992).

Alaptide is likely to have a negative effect on the inhibition of the release of melanocyte-stimulating hormone, thereby increasing the concentration of melanocytes in the epidermis. Melanocytes significantly affect the formation and function of keratinocytes through organelles known as melanosomes. (McGrath JA, Eady RA, Pope FM Rook's textbook of dermatology, 7th ed. Blackwell Publishing, 2004, pp.37; James W., Berger T. Elston D. Andrews' diseases of the skin: Clinical dermatology, 10th ed. Saunders , 2005, pp. 5-6). Keratinocytes migrate from the stratum basale through the stratum spinosum and stratum granulosum to the stratum comeum, where they promote epidermal regeneration. (Watt F.M. The epidermal keratinocyte. BioEssays 1988, 8, 163-167).

Alaptide is a compound (log P is -0.67) that is accompanied by low solubility in water and other protic solvents. This fact has some disadvantages, in particular the partial separation of Alaptid during the treatment in the form of a white cover on the treated wounds. The lower concentration of Alaptide dissolved in the formulation may lead to a reduced amount of active substance absorbed through the stratum corneum. Recently, results using Alaptid nanoparticles have been published, e.g. PV 2011-232. The limited solubility of Alaptide in the physiological environment does not allow its full therapeutic potential to be exploited. In the preparation of liquid forms containing the active substance, the low solubility of Alaptide in the hydrophilic medium represents a considerable disadvantage, which can lead to a partial exclusion of Alaptide in the healing region. The low solubility of Alaptide may also be the cause of its reduced absorption into the lower layer of the epidermis.

The effects of alaptide are known from many publications, for example from Sklenář, Zbyněk, et al. Formulation and release of alaptide from cellulose-based hydrogels., Acta Veterinaria Brno 81.3 (2013): 301-306, which describes the release of alaptide from cellulose hydrogels.

The use of amino acids and peptides for tissue regeneration is a known matter. For sports regeneration purposes, amino acids and peptides are combined for muscle regeneration, for example for oral administration. Amino acids and peptides are also contained in a number of cosmetic formulations that combat anti-aging skin in particular. Such cosmetics reduce dryness of the skin, protect, hydrate the skin, increase its elasticity and youthful appearance, and fill in superficial and deeper wrinkles. The properties of amino acids and peptides for tissue regeneration are thus known, but in connection with the skin, the attention is focused more on cosmetics and aesthetic medicine.

EP 1640041 describes a cosmetic composition for the topical treatment of, in particular, wrinkled skin or skin with intense photoamination. The cosmetic composition contains an agent that stimulates collagen synthesis, which may be tri- to hexa-peptides, and an agent that enhances the interaction between the extracellular matrix and fibroblasts. Other cosmetically active substances can be added to the cosmetic composition, for example extracts, UV filters, moisturizers or amino acids.

US 20050209131 discloses a cosmetic composition comprising an amino acid and a peptide complex with copper. Such a composition is used in cosmetic preparations of the body lotion type or cosmetic wet wipes for the prevention of dry skin and for its protection.

WO 03030926 describes an aqueous solution of a peptide complex with copper which contains at least one amino acid with supposedly healing effects. However, aqueous solutions are not tested at all in this document, which in itself arouses distrust in such solutions.

US 20130108700 discloses a composition of a gelatin and polysaccharide matrix with a peptide and an amino acid. Such a formulation is injected around fresh wounds to prevent scarring. Testing of such a composition took place after gynecological surgery, in which patients were injected with mucous membranes in the vicinity of the surgical incision. This procedure really reduced the scarring of the mucous membranes in the tested patients and thus increased the patients' chances of

- 3 CZ 308845 B6 successful pregnancy.

WO 2005042048 describes a composition similar to US20130108700. In this case, however, the composition is used to heal bones, cartilage, ligaments and tendons. The composition is also administered by injection.

The essence of the invention

The present invention provides a pharmaceutical composition of topically healing peptide components which has a surprising effect on accelerated wound healing, including poorly healing e.g. diabetic wounds, based on a combination of a 2 to 40 amino acid spirocyclic diketopiperazine peptide with arginine and cysteine, wherein the amino acids are L-, D- or racemic form.

It has been found and demonstrated that a mixture of the above ingredients has up to 100% better effect on wound healing in animal models and humans in vivo than the ingredients applied alone.

Amino acids function as a mixture of topically healing peptide components as nitric oxide (NO) or sulfane (H2S) donors.

The pharmaceutical composition of topically healing peptide components contains at least one peptide in a concentration of 0.001 to 50 mg / ml or g of matrix or formulation. In vivo tests have shown that any increase in peptide / peptide concentration above 0.001 mg / ml or g of matrix produces a therapeutic effect, whereas increasing peptide / peptide concentration above 50 mg / ml or g no longer produces the desired therapeutic effect.

The formulation has a pH between 3.0 and 9.0. Preferably, the formulation has an ionic strength of at least 20 mM, more preferably 50 mM to 100 mM, most preferably at least 120 mM prior to application of the formulation.

The pharmaceutical composition of the healing peptide components may be dissolved in one cosmetically or pharmaceutically acceptable solvent or in a mixture of several cosmetically or pharmaceutically acceptable solvents such as water, ethanol, propanol, isopropanol, propylene glycol, butylene glycol, dipropylene glycol, ethoxylate or propoxylated diglycols, cyclic polyols. The mixture of topically healing peptide components may advantageously be dissolved with a pharmaceutical or cosmetic vector, such as liposomes, or the pharmaceutical mixture of topically healing peptide components may be adsorbed onto powdered organic polymers or powdered minerals - e.g. talc or bentonites. In general, a pharmaceutical composition of topically healing peptide components can be dissolved or fixed in any cosmetically or pharmaceutically acceptable vector.

The pharmaceutical composition of the topically healing peptide components can be used in combination with other active ingredients, cosmetically or pharmaceutically active. The final formulation is then used either for cosmetic skin / skin / skin care purposes or as a medicament. The formulation has regenerating and revitalizing properties.

The pharmaceutical composition of topically healing peptide components may contain peptides with other groups, for example signal or protective (tBoc) tags. The peptides may also be associated with nanoparticles and / or liposomes, for example carrying another active substance. Peptides may also be modified by glycosylation, pegylation, acetylation, methylation, ubiquitination, hydroxylation, palmitoylation, phosphorylation, or otherwise, as long as the modification does not interfere with the healing properties of the peptide.

Surprisingly, small peptides (less than 12 amino acids in sequence) have been shown to be more effective in promoting wound healing than the full length native protein. In addition, this increased potency was shown not to be shared by all full-length peptide fragments. The inventors surprisingly

Have shown that the peptides of the present invention are more effective in promoting wound healing than full-length protein, and that this increased efficacy has not been shared by all fragments of full-length healing peptides and proteins used for natural wound healing. In addition, the combination of peptides with amino acids or other peptides showed higher healing efficiency than the peptide used for treatment alone.

Furthermore, it has been found that the presence of a Cu or Zn metal complex of peptides can increase wound healing efficiency by an additional 5 to 10% over metal-free peptide treatment, preferably both the parent peptide and its metal complex are present in the mixture of topically healing peptide components. The therapeutic efficacy of such preparations is increased by 8 to 12% compared to preparations containing either peptides or a peptide metal complex. Preferably, a metal complex of Cu-GHK peptides is present in the pharmaceutical composition of topically healing peptide components, wherein Cu-GHK is a natural, body-specific substance, the amount of which, however, decreases with age. For this reason, the treatment of injuries and chronic wounds at a later age is more problematic. Pharmaceutical mixtures of topically healing peptide components containing metal complex of peptide (s) show increased efficacy, especially in non-healing wounds.

Metal complexes are prepared by laboratory simple synthesis, where the peptide is dissolved or suspended in dry methanol (MeOH) or acetonitrile, a concentrated metal solution, preferably Cu ²⁺ or Zn ²⁺ , and two equivalents of base, preferably sodium methoxide ( MeONa) or other strong base. The mixture is stirred at room temperature for 40 to 120 minutes. The respective metal complex of peptides is then aspirated and dried. The dry metal complex of peptides is used in a number of embodiments of a mixture of topically healing peptide components.

The pharmaceutical composition of topically healing peptide components stimulates the proliferative growth of fibroblasts and epithelial cells, such as keratinocytes, and has positive effects on the entire wound healing process in experimental animals and human volunteers. Testing has also shown that the pharmaceutical composition of topically healing peptide ingredients effectively fills wrinkles and rejuvenates the skin, lightens pigment spots and birthmarks, and prevents scarring after cosmetic procedures such as freckles and birthmarks. The pharmaceutical composition of topically healing peptide components has also been used in the treatment of burns, after surgery and microsurgery, for common injuries or for more extensive skin injuries, such as abrasions.

Research has also shown that a diketopiperazine spirocyclic peptide, such as Alaptide, in the above combinations, has properties that significantly promote dermal cell growth at concentrations that also exhibit antimicrobial effects. This huge advantage can be used in poorly healing, inflamed or diabetic wounds, where sepsis and infection are a significant barrier to wound healing.

Furthermore, the pharmaceutical composition of topically healing peptide components has also been shown to affect the function of metalloproteinases, which play a role in the healing of both acute and chronic wounds by regulating the degradation and deposition of the intercellular mass of the crow. The mixture of topically healing peptide components increases the level of metalloproteinases at the site of injury, thereby accelerating the degradation of the intercellular mass in the wound and allowing it to heal faster.

Two main groups of healing proteins have been used for the pharmaceutical composition of topically healing peptide components, namely spirocyclic diketopiperazine peptides and linear peptides. Both groups can also be divided into natural and synthetic. Natural peptides, such as GHK peptide (GlyHis-Lys) or DAHK peptide (Asp-Ala-His-Lys) and others, play an important role in skin regeneration. They affect the proliferation of skin cells, their regeneration and longevity. The combination of natural peptides with synthetic peptides has been shown to be advantageous. Examples of synthetic peptides include CAR protein or diketopiperazine peptides (eg, Alaptid). In particular, a very advantageous combination of diketopiperazine peptides with GHK peptide / Cu-GHK peptide in a ratio of 1: 1 has been shown. The functionality of the GHK peptide / Cu-GHK peptide mixture in combination with arginine has also been demonstrated and appears to be equally advantageous, with treatment with such a mixture lasting approximately 1/10

- 5 CZ 308845 B6 longer than treatment with diketopiperazine + GHK / Cu-GHK. A mixture of at least two types of diketopiperazine peptides is also preferred for treatment. For poorly treatable wounds, the combination of two diketopiperazines is up to 70% more effective than using one diketopiperazine alone. The combination of GHK protein / Cu-GHK protein with arginine in the presence of a protein - preferably collagen, fibrinogen or elastin - has also proved to be advantageous. For poorly healing wounds, for example diabetic, it is advantageous to add the amino acid arginine as NO donor or the amino acid cysteine as H2S donor to the peptide mixtures. The addition of amino acids increases efficiency.

The diketopiperazine peptides used for the mixture of topically healing peptide components can be described by the following general formulas:

or n = 1-4

R ¹ = -ch _3] -ch ₂ ch ₃ , - (CH ₂ ) ₂ CH ₃ , - (CH ₂ ) ₇ CH ₃

R ² = -H, -CH ₃ . -CH ₂ CH _3] - (CH ₂ ) ₇ CH ₃

R ³ = -H, -CH ₃ . -CH ₂ CH _3] - (CH ₂ ) ₇ CH _3] - (CH ₂ ) ₁₇ CH ₃

General structures:

OR ³ V- N R ² O 6- (R ¹ ) -7- (R ² ) -4- (R ³ ) -4,7-diazaspiro [2.5] octane-5,8-dione CO \ Z z O ^X CM J Dí Dí 7- (R ¹ ) -8- (R ² ) -5- (R ³ ) -5,8-diazaspiro [3.5] nonane-6,9-dione 70 π Γ _ 8- (R ¹ ) -9- (R ² ) -6- (R ³ ) -6,9-diazaspiro [4.5] decane-7,10-dione of O ΠΓ J 0Í Qí 3- (R ¹ ) -4- (R ² ) -1- (R ³ ) -1,4-diazaspiro [5.5] undecane-2,5-dione OR ³ ýjo R ² O 3- (R ¹ ) -4- (R ² ) -1- (R ³ ) -1,4-diazaspiro [5.6] dodecane-2,5-dione 7J 7 ° Γ z O ^></ \ ^w 3- (R ¹ ) -4- (R ² ) -1- (R ³ ) -1,4-diazaspiro [5.7] tridecane-2,5-dione

-6GB 308845 B6

Examples of specific chemical compounds:

/ - Vn 4,6,7-triethyl-4,7-diazaspiro [2. 5] octane-5,8-dione Chemical formula: C12H20N2O2 Elemental analysis: C, 64.26; H, 8.99; N, 12.49; 0, 14.27 0 / - / 0 7-ethyl-5-propyl-5,8-diazaspiro [3.5] nonane-6,9-dione Chemical formula: C12H20N2O2 Elemental analysis: C, 64.26; H, 8.99; N, 12.49; 0, 14.27 bÚ C ₈ Hi ₇ O 6,8-dimethyl-9-octyl-6,9-diazaspiro [4. 5] decane-7,10-dione Chemical formula: C18H32N2O2 Elemental analysis: C, 70.09; H, 10.46; N, 9.08; O, 10.37 0 '\ ⁰ 3,4-dibutyl-1,4-diazaspiro [5. 5] undecane-2,5-dione Chemical formula: C17H30N2O2 Elemental analysis: C, 69.35; H, 10.27; N, 9.51; O, 10.87 0 V-NHr ^ \ HN— 0 3-methyl-1,4-diazaspiro [5.6] dodecane-2,5-dione Chemical formula: C11H18N2O2 Elemental analysis: C, 62.83; H, 8.63; N, 13.32; O, 15.22 4b 1-ethyl-4-methyl-3-propyl-1,4-diazaspiro [5.7] tridecane-2,5-dione Chemical formula: C17H30N2O2 Elemental analysis: C, 69.35; H, 10.27; N, 9.51; O, 10.87

(S) -isomer:

%

Ί b

R?

General structures:

- 7 CZ 308845 B6

xk Z About jT 4 o: £ (S) -6- (R ¹ ) -7- (R ² ) -4- (R ³ ) -4,7-diazaspiro [2.5] octane-5,8-dione OR ³ O R ² O (S) -7- (R ¹ ) -8- (R ² ) -5- (R ³ ) -5,8-diazaspiro [3.5] nonane-6,9-dione OR ³ ÚP R ² 0 (S) -8- (R ¹ ) -9- (R ² ) -6- (R ³ ) -6,9-diazaspiro [4.5] decane-7,10-dione OR ³ ÍP R ² 0 (S) -3- (R ¹ ) -4- (R ² ) -1- (R ³ ) -1,4-diazaspiro [5.5] undecane-2,5-dione j) r _ χθ z Ζχ οθ><* i— \ ^ω (S) -3- (R 1 -d-IR ² ) -1- (R ³ ) -1,4-diazaspiro [5.6] dodecane-2,5-dione O, R ³ PO R ² O (S) -3- (R 1 -d-IR ² ) -1- (R ³ ) -1,4-diazaspiro [5,7] tridecane-2,5-dione

Examples of specific chemical compounds:

About _K / - (S) -4,6,7-Triethyl-4,7-diazaspiro [2.5] octane-5,8-dione Chemical formula: C12H20N2O2 Elemental analysis: C, 64.26; H, 8.99; N, 12.49; 0, 14.27 O / - / op (S) -1-ethyl-5-propyl-5,8-diazaspiro [3.5] nonane-6,9-dione Chemical formula: C12H20N2O2 Elemental analysis: C, 64.26; H, 8.99; N, 12.49; 0, 14.27 úó c ₈ H ₁₇ 0 (S) -6,8-dimethyl-9-octyl-6,9-diazaspiro [4.5] decane-7,10-dione Chemical formula: C18H32N2O2 Elemental analysis: C, 70.09; H, 10.46; N, 9.08; 0, 10.37 0 Op (S) -3,4-dibutyl-1,4-diazaspiro [5.5] undecane-2,5-dione Chemical formula: C17H30N2O2 / (o Elemental analysis: C, 69.35; H, 10.27; N, 9.51; 0, 10.87 0 y — NH / - K (S) -3-methyl-1,4-diazaspiro [5.6] dodecane-2,5-dione Chemical formula: C11H18N2O2 HN — A— / 0 Elemental analysis: C, 62.83; H, 8.63; N, 13.32; 0, 15.22

-8CZ 308845 B6

(S) -1-ethyl-4-methyl-3-propyl-1,4-diazaspiro [5.7] tridecane-2,5-dione

Chemical formula: C17H30N2O2

Elemental analysis: C, 69.35; H, 10.27; N, 9.51; O, 10.87

Diketopiperazine protein derivatives have also been used for the pharmaceutical composition of topically healing peptide components, for example:

alaptide with the chemical structural name: 8-methyl-6,9-diazaspiro [4.5] decane-7,10-dione and having the structural formula:

Alaptid-CD complex; 6-methyl-MeAlaptide; 6,9-di-MeAlaptide, 6-EtAlaptide; 6-hexylAlaptide; 9-ethylAlaptide; 6-Pallalaptide 6-palmitoyl 8-methyl-6,9-diazaspiro [4.5] decane-7,10-dione;

8-methyl-6,9-diazaspiro [5.5] undecane-7,10-dione;

The following linear peptides with the amino acid sequence of the name used were also used for the pharmaceutical mixture of topically healing peptide components:

a) Cu-GHK

b) DAHK

c) Pal-GHK

d) GEKG

e) Exendin-4 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS

The pharmaceutical composition of the topically healing peptide components may preferably be in the form of a solution, suspension, emulsion, ointment, balm, tincture, elixir, patch, bandage, dressing, alginate dressing, topical solution, infusion or surgical rinse. The matrix of these forms preferably contains proteins.

Preferably, the pharmaceutical composition of topically healing peptide substances is applied to wounds in a gel matrix formulation. The gel matrix consists of a protein and carbohydrate component in a weight ratio of 1: 1 and an aqueous buffer containing Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , preferably in the form of calcium acetate, zinc acetate or magnesium acetate. The pH of the gel is preferably between 3 and 9. Due to the presence of cations, either gel formation occurs by complexation with collagen and / or cation-controlled gelation. The protein component is preferably collagen, cleaved collagen, fibrinogen, cleaved fibrinogen, elastin, cleaved elastin, gelatin, hydrolyzed gelatin, BSA and the like. The carbohydrate component is preferably hyaluronic acid, pectin, alginate, carrageenan, alginic acid, oxidized and non-oxidized forms of cellulose, cellulose derivatives - for example carboxymethylcellulose and its salts, other carboxylated carbohydrates, chitosan, sorbitan caprylate, flexseed gum, pectate and other oligos and polysaccharides etc. The matrix can also be formed of synthetic polymers such as polyacrylamides, poly (lactic-co-glycolic acid (PLGA) and polylactic acid (PLA).

-9CZ 308845 B6

The gel matrix provides skin hydration, nourishment and firming, it is advantageous for use in combination with a mixture of peptide components for the treatment of chronic wounds, bedsores, diabetic complications, treatment after laser and (cosmetic) operations. At the same time, in combination with a pharmaceutical mixture of topically healing peptide components, it smoothes wrinkles, slows down the formation of new ones and effectively slows down the aging process of the skin, stimulates skin cells to produce collagen and elastin, brightens and evens out skin color. Preferably, the wound gel in a formulation with a pharmaceutical composition of topically healing peptide components is applied sterile.

The matrix may also be an oil-in-water or water-in-oil emulsion, preferably the peptide and / or amino acid and / or protein is present in the fat phase. Preferably, phosphatidylcholine or other lecithin is also present in the fat phase. Lecithin is preferably present in the composition in the form of liposomes or micelles or other structures containing nitric oxide, peptides, or both. Preferably, the matrix for the pharmaceutical composition of the topically healing peptide components is in the form of a gel, cream, lotion, ointment, solution, solid stick, etc., which can be coated or sprayed on the skin, for example the wound.

The prepared pharmaceutical composition of topically healing peptide components achieves a high effect of accelerated wound healing. This is due to the shortening of the peptide chains used for healing and the addition of NO and H2S donors. The pharmaceutical composition of topically healing peptide components shortens the healing time in healthy patients by almost a third of standard treatment (from 8 weeks to 3 weeks) and in patients with chronic skin defects the healing time is reduced by up to 7 times (from 20 weeks to 3 weeks). In some patients with chronically inflamed wounds, a pharmaceutical composition of topically healing peptide components allows treatment in otherwise hopeless cases intended for amputation. When using the formulations with the pharmaceutical mixture of peptide components - peptide + Arg + Cys according to Example 1, the wounds, resp. skin defects treated with such formulations healed in an average of 3 weeks in mouse models, in 3 weeks in healthy patients and in between 3 and 7 weeks in diabetics. For the peptide + Arg / Cys preparations according to Examples 2 and 3, or for the peptide + peptide + Arg + Cys preparation according to Examples 5, 6 and 7, wounds healed in an average of 4 weeks in mouse models, in 4 weeks in healthy patients and in poor patients. healing wounds healed between 4 and 9 weeks. For the peptide + peptide formulations of Example 4, wounds healed in an average of 5 weeks in mouse models, in 5 weeks in healthy patients, and between 5 and 12 weeks in poorly healing wounds. In the formulations of Example 8A, control groups with gel matrix peptide alone (model Alaptid used), wounds healed in an average of 7 weeks in mouse models, in 7 weeks in healthy patients, and between 7 and 7 in healthy healing wounds. 20 weeks. In the formulations of Example 8B, i.e. control groups with gel matrix alone, the wounds healed in an average of 8 weeks in mouse models, in 8 weeks in healthy patients and in 8 to 20 weeks in poorly healing wounds, and healed completely. however, only one patient. In the formulations of Example 8C, treatment-free controls, wounds healed in an average of 8 weeks in mouse models, in 8 weeks in healthy patients, and in one patient out of five, in well-healing wounds, after 31 weeks. a red spot remained present in all test subjects.

Explanation of drawings

Giant. 1: Comparison of selected preparations on mouse models.

Giant. 2: Histological image, day 21 healing of the experimental mouse wound with the preparation according to Example 1B, magnification 40x.

Giant. 3: Histological image, day 28 healing of the experimental mouse wound with the preparation according to Example 2, magnification 40x.

Giant. 4: Histological image, day 28 healing of the experimental mouse wound with the preparation according to Example 7, magnification 40x.

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Giant. 5: Histological image, day 50 healing of the experimental mouse wound with the preparation according to Example 8B, magnification 40x.

Giant. 6: Comparison of selected products in selected patients.

Giant. 7: Table of wound healing when using selected products.

Giant. 8: Graph of wound healing when using selected products.

Examples of embodiments of the invention

Example 1

Production of a peptide metal complex

Cu-GHK

1.5 g of GHK was suspended in 50 ml of 100% methanol at room temperature, 0.25 g of copper acetate (CuOAc) in the form of a 5 ml aqueous solution and 5 ml MeONa (1M) were added with stirring. The mixture was stirred at room temperature at 130 rpm for 120 minutes. The peptide metal complex was then aspirated and dried.

Production of the preparation - peptide + Arg + Cys

1Α 1% Cu-GHK Vaseline g powdered GHK peptide (Gly-His-Lys), 0.3 g powdered L-arginine and 0.1 g powdered Lcysteine were mixed in 98.6 g medical Vaseline at 3000 rpm. A preparation with 1% Cu-GHK peptide content was obtained.

IB 1% Cu-GHK matrix gel g of powdered Cu-GHK peptide (Gly-His-Lys), 0.3 g of powdered L-arginine and 0.1 g of powdered L-cysteine were mixed in a gel matrix consisting of 7 g collagen, 7 g of pectin and 86 g of phosphate buffer pH 7 containing 5% calcium acetate. The gel matrix ingredients were mixed together, stirred for 5 minutes with a magnetic stirrer at 300 rpm and then for another 25 minutes at 130 rpm. Then, Cu-GHK peptide and L-Arg were added to the gel matrix, and the mixture was mixed at 2000 rpm for another 5 minutes.

1C 1.2% GHK Vaseline

1.2 g of powdered GHK peptide (Gly-His-Lys), 0.3 g of powdered L-arginine and 0.1 g of powdered Lcysteine were mixed in 98.6 g of medical Vaseline at 3000 rpm. A formulation with 1% GHK peptide content was obtained.

ID 1% DAHK (1: 0.3: 0.1) Vaseline g powdered DAHK peptide (Asp-Ala-His-Lys), 0.35 g powdered L-arginine and 0.1 g powdered L-cysteine were mixed in 98.6 g of medical Vaseline at 3000 rpm. A formulation with 1% DAHK peptide content was obtained.

1E 0.5% DAHK matrix mast

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0.5 g of powdered DAHK peptide (Asp-Ala-His-Lys), 0.15 g of powdered D-arginine and 0.08 g of powdered D-cysteine were mixed in a matrix consisting of:

Liquid paraffin 8.0 g, Hard paraffin 12.0 g, Stearyl alcohol 2.0 g, Propylene glycol 5.5 g, Slovasol 2430 (1- (2-methoxyisopropoxy) -2-propanol) 3.0 g, Carbomera 0.5 g, Triethanolamine 0.6 g, Methylparaben 0.2 g, Propylparaben 0.05 g, Purified water 67.65 g.

The mixture was stirred at 5000 rpm for 2 h.

All components were homogenized into an emulsion with 0.5% DAHK peptide content.

IF 1% Alaptide Vaseline g Alaptide powder, 0.4 g L-arginine powder and 0.1 g L-cysteine powder were mixed in 98.6 g medical Vaseline at 3000 rpm. A formulation with 1% Alaptide content was obtained.

1G 5% Alaptid matrix emulsion g of Alaptid powder was mixed in 4 g of refined coconut oil, an emulsifier was added and the mixture was mixed with 82 g of water. 9 g of 9% ethoxylated tridecyl alcohol were used as emulsifier.

In the presence of the emulsifier, the two immiscible liquids were mixed with vigorous stirring. The polydisperse emulsion obtained was mixed with 2 g of racemic arginine powder and 0.5 g of racemic cysteine powder and further homogenized in a homogenizer at room temperature at 3000 rpm. Homogenization further increased the stability of the prepared emulsion. An emulsion with 5% Alaptide content was prepared.

1H 2% Exendin-4 Vaseline g Exendin-4 Powder, 0.3 g L-Arginine Powder and 0.1 g L-Cysteine Powder were mixed in 98.6 g Medical Vaseline at 3000 rpm. A formulation with 2% Exendin-4 content was obtained.

II 1% 3-methyl-1,4-diazospiro [5.6] dodecane-2,5-dione petrolatum

Ig of powdered 3-methyl-1,4-diazospiro [5.6] dodecane-2,5-dione, 0.3 g of powdered L-arginine and 0.1 g of powdered L-cysteine were mixed in 98.6 g of medical Vaseline at rpm. 3000 rpm A preparation with 1% content of 3-methyl-1,4-diazospiro [5.6] dodecane-2,5-dione was obtained.

1J 1% GEKG Vaseline g powdered GEKG, 0.5 g powdered L-arginine and 0.2 g powdered L-cysteine were mixed in 98.6 g medical Vaseline at 3000 rpm. A preparation with 1% GEKG content was obtained.

Example 2

Preparation - peptide + Arg% Zn-GHK matrix gel g of powdered Zn-GHK peptide (Gly-His-Lys) and 0.2 g of powdered D-arginine were mixed in a gel matrix consisting of 7 g of gelatin, 7 g of carrageenan and 86 g of phosphate buffer pH 7

- 12 CZ 308845 B6 containing 5% zinc acetate. The gel matrix ingredients were mixed together, stirred for 5 minutes with a magnetic stirrer at 300 rpm and then for another 25 minutes at 130 rpm. Then, Zn-GHK peptide and L-Arg were added to the gel matrix, and the mixture was mixed at 2000 rpm for another 5 minutes.

Example 3

Preparation - peptide + Cys% DAHK matrix gel g powdered DAHK peptide and 0.2 g powdered L-arginine were mixed in a gel matrix consisting of 2 g gelatin, 1 g chitosan, 1 g hyaluronic acid and 3 g collagen, 7 g of carrageenan and 86 g of phosphate buffer pH 7 containing 5% magnesium acetate. The gel matrix ingredients were mixed together, stirred for 5 minutes with a magnetic stirrer at 300 rpm and then for another 25 minutes at 130 rpm. Then, DAHK peptide and L-Arg were added to the gel matrix, and the mixture was mixed at 2000 rpm for another 5 minutes.

Example 4

Production of the preparation - peptide + peptide in solution

4A GHK + Alaptide solution

1.5 g of powdered GHK protein and 1 g of powdered Alaptide were mixed in 97.5 g of purified water. The mixture was mixed at 5000 rpm for 15 minutes. Before each use, the mixture was mixed at 3000 rpm for 2 minutes.

A 4B GHK + DAHK solution of g of GHK powder and 1 g of DAHK powder was mixed in 98 g of purified water. The mixture was mixed at 4500 rpm for 10 minutes. Before each use, the mixture was mixed at 3000 rpm for 1 minute.

4C Alaptide + 4,6,7-triethyl-4,7-diazospiro [2.5] octane-5,8-dione solution g of Alaptide powder and 1.5 g of 4,6,7-triethyl-4,7-diazospiro powder [ 2.5] octane-5,8-dione was stirred in 97.5 g of purified water. The mixture was mixed at 5000 rpm for 20 minutes. Before each use, the mixture was mixed at 3000 rpm for 2 minutes.

Example 5

Preparation - peptide + peptide + Arg matrix gel g powdered DAHK peptide and 1 g powdered GHK peptide and 0.2 g powdered L-arginine were mixed in a gel matrix consisting of 6 g hydrolyzed gelatin, 1 g BSA, 5 g carboxymethylcellulose , 2 g of hyaluronic acid and 86 g of borate buffer pH 6 containing 5% of calcium acetate. The gel matrix ingredients were mixed together, stirred for 5 minutes with a magnetic stirrer at 300 rpm and then for another 25 minutes at 130 rpm. Then, DAHK peptide, GHK peptide and L-Arg were added to the gel matrix, and the mixture was mixed at 3000 rpm for another 5 minutes.

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Example 6

Preparation - peptide + peptide + Cys matrix gel g of powdered DAHK peptide and 1 g of powdered Alaptide and 0.2 g of powdered L-cysteine were mixed in a gel matrix consisting of 3 g of cleaved elastane, 1 g of hydrolyzed gelatin, 3 g of BSA , 5 g of carboxymethylcellulose, 1 g of carrageenan, 1 g of alginic acid and 86 g of carbonate buffer pH 7.5 containing 1% zinc acetate. The gel matrix ingredients were mixed together, stirred for 15 minutes with a magnetic stirrer at 400 rpm and then for another 25 minutes at 130 rpm. Then, DAHK peptide, Alaptid and L-Cys were added to the gel matrix and the mixture was mixed at 5000 rpm for another 2 minutes.

Example 7

Production of the preparation - peptide + peptide + Arg + Cys matrix gel g of powdered GHK peptide and 1 g of powdered Alaptide and 0.5 g of powdered L-arginine and 0.2 g of Lcysteine were mixed in a gel matrix consisting of 6 g of cleaved collagen, 1 g fibrinogen, 4 g oxocellulose, 1 g alginate, 1 g pectate, 1 g flexseed gum and 86 g phosphate buffer pH 7 containing 1% zinc acetate. The gel matrix ingredients were mixed together, stirred for 15 minutes with a magnetic stirrer at 400 rpm and then for another 25 minutes at 130 rpm. Then, DAHK peptide, Alaptid and L-Cys were added to the gel matrix and the mixture was mixed at 5000 rpm for another 2 minutes.

Example 8

8A Preparation of the preparation - peptide matrix gel g of powdered Alaptid were mixed in a gel matrix consisting of 4 g of collagen, 3 g of cleaved collagen, 1 g of hyaluronic acid, 3 g of alginate, 3 g of carrageenan and 86 g of acetate buffer pH 6.5 s containing 3% zinc acetate. The gel matrix ingredients were mixed together, stirred for 15 minutes with a magnetic stirrer at 400 rpm and then for another 25 minutes at 130 rpm. Then Alaptid was added to the gel matrix and the mixture was mixed at 5000 rpm for another 5 minutes.

8B Preparation of the preparation - gel matrix

The control gel matrix was made of 7 g of collagen, 7 g of pectin and 86 g of phosphate buffer pH 7 containing 5% calcium acetate. The gel matrix ingredients were mixed together, stirred for 5 minutes with a magnetic stirrer at 300 rpm and then for another 25 minutes at 130 rpm. The gel matrix was then mixed at 2000 rpm for another 1 minute.

Example 9

Testing the effectiveness of individual products

To compare the effectiveness, the preparations according to the individual examples were prepared in a standardized concentration by stirring in medical Vaseline. Thus, the uniformity of the tested formulations was guaranteed. In addition, all formulations produced were tested. Figures 7 and 8 show the in vivo number of days until wound healing in each test group. Selected formulations representing a given group of preparations are shown. The results show that the effect of the formulation does not depend on the peptide used, but on the combination of peptide / peptides with amino acids in a certain ratio. To compare the efficacy of the preparations, animal models of inbred laboratory mice were selected

Artificial wounds were made in the abdominal cavity using a sterile scalpel. Two wounds with a length of approximately 1 cm and a depth of approximately 3 mm were made for each subject. After testing on mouse models, testing was performed on healthy injured patients and on diabetic injured patients, or patients with non-healing wounds. In healthy patients, these were mainly wounds after microsurgery, in diabetics, these were long-term non-healing wounds, shin ulcers, pressure ulcers and fistulas. The test group of mice and patients consisted of five test subjects. 0.002 g of peptide per day, usually 0.2 g of 1% peptide formulation per day, was applied to the wounds. Tests on mice were evaluated every 24 hours, tests on patients were documented daily by the patients themselves - a photo was taken every day with a short description of the wound. The records were later evaluated by the attending physician.

The best effect of accelerated wound healing was shown to be obtained using the formulations of Example 1, i.e. the combination of peptide + Arg + Cys. In addition, the synergistic effect of the formulation in a gel matrix compared to the formulation of a mixture of topically healing peptide components alone in a petrolatum matrix is shown. The formulation in a gel matrix shows faster wound healing. Wounds treated with the formulations of Example 1 healed in an average of 3 weeks in mouse models, in 3 weeks in healthy patients and between 3 and 7 weeks in diabetics. For the formulations of Examples 2 and 3 and 7, wounds healed in an average of 4 weeks in mouse models, in 4 weeks in healthy patients, and between 4 and 9 weeks in poorly healing wounds. For the formulations of Example 4, wounds healed in an average of 5 weeks in mouse models, in 5 weeks in healthy patients, and between 5 and 12 weeks in poorly healing wounds. For the formulations of Examples 5 and 6, wounds healed in an average of 4 weeks in mouse models, in 4 weeks in healthy patients, and between 4 and 8 weeks in poorly healing wounds. In the formulations of Example 8A, i.e. control groups with application of the peptide alone in a gel matrix (model Alaptid used), the wounds healed in an average of 7 weeks in mouse models, in 7 weeks in healthy patients and between 7 and 20 weeks. In the formulations of Example 8B, i.e., control groups with gel matrix alone, the wounds healed in an average of 8 weeks in mouse models, in 8 weeks in healthy patients, and in 8 to 20 weeks in poorly healing wounds. However, only one patient was completely healed. In the formulations of Example 8C, treatment-free controls, wounds healed in an average of 8 weeks in mouse models, in 8 weeks in healthy patients, and in one patient out of five, in well-healing wounds, after 31 weeks. a red spot remained present in all test subjects.

Example 10

10A effects of the preparation of Example IB, anti aging, wrinkle smoothing

A group of 35 women aged 30 to 60 years was tested. Prior to testing, all women's faces were photographed with a canon 5D mark III (full frame) camera with a canon EF 100 2.8L USM macro lens. The images were taken on an aperture of 11, ISO 100 with illumination of the face with three studio lights with a power of 500 W. The group was divided into control and test. The control part of the group applied a placebo preparation and the test part of the group applied the preparation of Example No. 1B, the placebo being a gel base without the addition of peptide and amino acids.

The women applied the preparation of Example No. 1B twice a day for 6 weeks. After testing, the women's faces were again documented, ie photographed with a macro lens. Skin images of each woman before and after testing were compared by image analysis. On average, 70% wrinkle smoothing was achieved when applied twice daily for 6 weeks, with 8% wrinkle smoothing in the control group, probably due to hydration provided by the gel matrix.

10B effects of the preparation of Example 7, anti aging, wrinkle smoothing

A group of 35 women aged 28 to 59 years was tested. Before testing, everyone's faces were

- 15 CZ 308845 B6 women photographed with a canon 5D mark III camera (full frame) with a canon EF 100 2.8L USM macro lens. Images were taken on an aperture of 11, ISO 100 with illumination of the face with three 500 W studio lights. The women applied the preparation of Example No. 7 twice a day for 6 weeks. After testing, the women's faces were again documented, ie photographed with a macro lens. Images of 5 skin of each woman before and after testing were compared. On average, 62% wrinkle smoothing was achieved when applied twice daily for 6 weeks.

Industrial applicability

Preparations for accelerated healing of wounds, burns, non-healing and diabetic wounds, aesthetics, cosmetics.

Claims (14)

PATENT CLAIMS A pharmaceutical composition of topically healing peptide components comprising a 2 to 40 amino acid spirocyclic diketopiperazine peptide, further comprising the amino acid arginine and the amino acid cysteine, for use in the topical treatment of skin defects and / or topical wound healing, the pharmaceutical composition contained in a matrix in an amount of 0.001 to 50 mg per gram of matrix. A pharmaceutical composition for use in the topical treatment of skin defects and / or for topical wound healing according to claim 1, which further comprises a linear peptide of 2 to 40 amino acids. A pharmaceutical composition for use in the topical treatment of skin defects and / or for topical wound healing according to claim 2, wherein the linear peptide of 2 to 40 amino acids is a peptide with a metal complex. A pharmaceutical composition for use in the topical treatment of skin defects and / or for topical wound healing according to claim 3, wherein the metal complex peptide is Cu-GHK. A pharmaceutical composition for use in the topical treatment of skin defects and / or for topical wound healing according to claim 2, wherein the linear peptide of 2 to 40 amino acids is selected from the group of peptides having the amino acid sequence: GHK, Cu-GHK, Zn-GHK, DAHK . A pharmaceutical composition for use in the topical treatment of skin defects and / or for topical wound healing according to claim 1, wherein the spirocyclic diketopiperazine peptide of 2 to 40 amino acids has the general formula O, R ³ V-n N-y '' 'n ^ 2 b n -1 * 6 R ¹ -CH = CH _{2 and} -CH _{3 R} - (CHECKS, - (CH _{2) 7} CH ₃ = -H. -CH _3. -CH ₂ CH _{3 R} - (CHshCHa R ³ = -H, -CH ₃ , -CH ₂ CH _3i - (CH ₂ ) ₇ CH _3i - (CH ₂ ) _{r /} CH ₃ A pharmaceutical composition for use in the topical treatment of skin defects and / or topical wound healing according to claim 1, wherein the spirocyclic diketopiperazine peptide of 2 to 40 amino acids is alaptide, i.e. 8-methyl-6,9-diazaspiro [4.5] decane-7. , 10-dione on structural formula A pharmaceutical composition for use in the topical treatment of skin defects and / or for topical wound healing according to claim 2, wherein the linear peptide of 2 to 40 amino acids is selected from the group of peptides having the amino acid sequence: a) Cu-GHK b) DAHK c) Pal-GHK d) GEKG - 17 CZ 308845 B6 e) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS A pharmaceutical composition for use in the topical treatment of skin defects and / or for topical wound healing according to claim 1, wherein the matrix is a protein or fat or carbohydrate or synthetic polymeric matrix or a mixture thereof. A pharmaceutical composition for use in the topical treatment of skin defects and / or for topical wound healing according to claim 9, which is contained in a protein or fat or carbohydrate matrix or mixtures thereof in an amount of 0.1 to 5 mg per gram of matrix. A pharmaceutical composition for use in the topical treatment of skin defects and / or for topical wound healing according to claim 9, wherein the protein matrix comprises collagen. A pharmaceutical composition for use in the topical treatment of skin defects and / or for topical wound healing according to claim 9, wherein the carbohydrate matrix comprises chitosan and / or hyaluronic acid. A pharmaceutical composition for use in the topical treatment of skin defects and / or for topical wound healing according to claim 9, wherein the fat matrix comprises lecithin. A pharmaceutical composition for use in the topical treatment of skin defects and / or for topical wound healing according to claim 9, wherein the synthetic polymeric matrix comprises polyacrylamide.