THEREOF

FIELD OF THE INVENTION

The present invention relates to the artificially designed peptide of the sequence X-Phe- Trp-Ala-His-Lys-Lys-Z, derivatives or complexes with metal ions thereof, a topical composition comprising thereof and topical use thereof. It relates more particularly to use of the peptides for the treatment of the skin of humans or animals for cosmetic or dermo- pharmaceutical purposes.

BACKGROUND OF THE INVENTION

Acne vulgaris

Acne vulgaris (henceforth acne) is a very common chronic skin disease of the pilosebaceous unit with profound negative psychosocial impact on the quality of life of patients. Although it affects individuals of all ages, it usually appears during adolescence and frequently persists into the adulthood (Yentzer et al. 2010).

The pathogenesis of acne vulgaris is multifactorial with four core events: hyperseborrhoea, epithelial hyperproliferation and hyperkeratinization, Cutibacterium acnes colonization, and inflammation.

Hyperseborrhoea

An increased production of sebum is considered one of the key events in the pathogenesis of acne. Various compounds and signaling pathways are involved in the regulation of the skin sebum production from which sex hormones play a key role. Among the most potent stimulators of sebum production are androgens whose upregulation is often associated with acne development (da Cunha, Fonseca, and Machado 2013). Within the skin cells, testosterone, the most common androgen, is quickly reduced to 5α-dihydrotestosterone (DHT) by the 5α- reductase, type I (Fritsch, Orfanos, and Zouboulis 2001). Both testosterone and DHT bind to androgen receptors (ARs), however, DHT is 5-10 times more potent AR agonist and it is considered the main androgenic hormone in the hair follicles (Anderson and Liao 1968). Epithelial hyperproliferation and hyperkeratinization

Another typical event in acne pathogenesis is hyperproliferation of follicular keratinocytes and their abnormal differentiation (Thiboutot 2000). The latter is associated with increased cohesiveness of comeocytes leading to their impaired desquamation (Thiboutot 2000). The accumulated mass of keratinized comeocytes together with sebum partially obstructs follicles resulting in their extension and creating favorable conditions for the growth of C. acnes (Thiboutot 2000).

There are several hypotheses explaining the cause of keratinocytes hyperproliferation and altered differentiation in acne. Androgens, besides stimulation of sebum production, have been shown to induce the above-mentioned processes and also production of the pro-inflammatory interleukin IL-1α (Kumtornrut et al. 2019; Guy and Kealey 1998). Another possible trigger of the hyperkeratinization is low concentration of an essential fatty acid, linoleic acid, which is decreased in the acne-affected skin probably due to its dilution by high amounts of sebum (Downing et al. 1986).

Cutibacterium acnes

C. acnes is a bacterium living in and on the human skin as part of the normal human skin microbiome. It predominantly resides deep within the sebaceous follicle in contact with keratinocytes.

It is well-established that C. acnes plays a significant role in the pathogenesis of acne (McLaughlin et al. 2019). It seems, that for acne development the overall C. acnes number is not as important as the presence of certain C. acnes strains (Dreno et al. 2018). These acne- associated strains were shown to be more virulent and produce a large number of various pro- inflammatory factors (Dreno et al. 2018). Inflammation

Inflammation is regarded as a key part in the pathogenesis of acne. In the past, inflammation was thought to be a secondary event induced by C. acnes colonization. Recently, inflammatory processes are suggested to be involved in all stages of acne development and acne is nowadays considered as genuinely inflammatory disease (Tanghetti 2013). Acne treatment

Because acne is a multifactorial disease, combination therapy is a recommended approach. Typically, a combination of a topical retinoid (tretinoin, isotretinoin, adapalene, tazarotene, retinol, retinaldehyde etc.) and an antimicrobial agent (e.g. benzoyl peroxide) is chosen as a first-line treatment (Moradi Tuchayi et al. 2015). Hormonal, anti-androgen therapy (oral contraceptives, spironolactone) is often given to women to reduce sebum production. Topical or oral antibiotics (erythromycin, clindamycin, doxycycline, minocycline, and sarecycline) are used in treatment of moderate-to-severe acne usually in combination with benzoyl peroxide or retinoids. From other used topical anti-acne agents, salicylic acid, azelaic acid, zinc, sulfur, niacinamide, glycolic acid and antimicrobial peptides can be mentioned (Liu et al. 2020). Zinc in acne treatment

Zinc level in acne patients is often significantly lower and its supplementation (oral or topical) have been shown to improve acne (Yee et al. 2020). Although its exact mechanism of action is not fully understood, current knowledge suggests multiple mechanisms including anti- inflammatory, antioxidant effects, inhibition of C. acnes proliferation and inhibition of 5 a- reductase leading to reduced sebum production in vivo (Abendrot and Kalinowska-Lis 2018; Cervantes et al. 2018).

Peptides in acne treatment

Another promising group of compounds in acne treatment are peptides showing antimicrobial (AMP) (Ma et al. 2021; Melo, Dugourd, and Castanho 2006; Zhang et al. 2013), anti-inflammatory (Zhang et al. 2013) and additional properties such as antioxidant effects (Mills et al. 2016). Some natural-derived or purely synthetic, designed AMP have been reported to have promising anti-acne effects:

• Omiganan pentahydrochloride derived from bovine AMP indolicidine (Melo, Dugourd, and Castanho 2006; Rubinchik et al. 2009)

• GDP -20 (granulysin-derived peptides) (Lim et al. 2015; Ma et al. 2021; Mclnturff, Modlin, and Kim 2005)

• Peptide LZ1 (VKRWKKWWRKWKKWV-NH2) (Zhang et al. 2013)

• Frog skin-derived AMP ([D4k]ascaphin-8, [G4K]XT-7, [T5k]temporin-DRa, brevinin- 2GU, and B2RP-Era (Popovic et al. 2012)

• Peptides (Br-p) isolated from burdock (Arctium lappa L.) roots (Miazga-Karska, Michalak, and Ginalska 2020)

• Temporin-lDra and moronecidin (Wu, Zhang, and Zhou 2020)

• Bombinin-like peptide 7 (BLP-7) from Bombina orientalis (Wu et al. 2020)

• Cathelicidin-BF has been purified from the snake venoms of Bungarus fasciatus (Wang et al. 2011)

• Head-to-tail cyclized bacteriocin AS-48 (Cebrian et al. 2018)

• CEN1HC-Br isolated from the green sea urchin (Han et al. 2018)

• AMP C12-50 (Nair et al. 2017)

• α-helical cationic peptide, P5 (Sunhyo Ryu et al. 2015)

• Helicobacter pylori-AenveA synthetic antimicrobial peptide HPA3NT3 (S. Ryu et al.2014). • Synthetic epinecidin- 1 (22-42) peptide was derived from positions 22-42 of Epinephelus coioides epinecidin-1 (Pan et al. 2009)

• Enterococcus faecalis-derived AMP SL-5 (Kang et al. 2009)

• Bacteriocin-like inhibitory substance (BLIS-like substance) produced by Streptococcus salivarius (Bowe et al. 2006)

• Synthetic, designed AMP (Woodburn, Jaynes, and Clemens 2020)

Examples of patents describing the use of peptides in acne treatment:

• WO2021175186A1 Use of small molecule short peptide in preparation of product for treating acne (LQLQAEER, derived from bFGF, 100 μg/mL, inhibits growth of sebocytes, reduces sebum production)

• CN111253469A Self-assembly short-chain peptide and application of short-chain peptide in treatment of acne (FFLQLQAEER)

• CN107698662A Peptide combination for treating acne (WKIKIDSEAE and PNMIYSKDY)

• CN112979762A Cyclic peptide PIZ and application thereof (E YP YKHSGYYHRAV)

• KR20200101767A Peptide having anti-microbial activity and compositions for anti- microbial comprising the same (KTTKS)

• W003015809A2 Antimicrobial cationic peptides and formulations thereof

• W02005025598A1 Antibacterial drug for propionibacterium acnes

• EP1853620A2 Antimicrobial hexapeptides

However, there is no patent nor publication describing any peptide of the amino acid sequence according to the present invention which is crucial for the physicochemical properties of the peptide as well as for its biological activity.

Many of the above-mentioned treatment options have negative adverse effects. Retinoids, in particularly, but also zinc or salicylic acid commonly cause skin dryness, peeling, itching, redness and sensitivity to sun (Thielitz and Gollnick 2008; Cervantes et al. 2018; Arif 2015). Of particular concern is also the known teratogenic effect of retinoids observed after oral use (Thielitz and Gollnick 2008). Although topical use of retinoids has not been shown to increase risk of birth defects, they are precautionary not recommended for pregnant women (Panchaud et al. 2012). Use of antibiotics as another common approach in acne therapy is associated with high risk of selection of resistant bacterial strains (Walsh, Efthimiou, and Dreno 2016). Therefore, they cannot be used as a monotherapy and are recommended only for a short-term use (Walsh, Efthimiou, and Dreno 2016). For all these reasons, there is still a need for new, more effective, complex and safer acne treatments.

SUMMARY OF THE INVENTION

Surprisingly novel activities of the Phe-Trp-Ala-His-Lys-Lys hexapeptide on the skin cells and improvement of the skin condition were found.

The subject-matter of the invention concerns a hexapeptide having a general formula I

X- Phe-Trp-Ala-His-Lys-Lys -Z (I) wherein:

X is -NHX ¹ group of phenylalanine at the N-terminal end of the hexapeptide, wherein

X ¹ is selected from a group comprising H, acetyl, octanoyl, decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, elaidoyl, oleoyl, biotinoyl or lipoyl. Preferably, X ¹ is H.

Z is -COZ ¹ group of lysine at the C-terminal end of the hexapeptide, wherein Z ¹ is selected from a group comprising OH, OCH ₃ , OCH ₂ CH ₃ or NH ₂ . Preferably, Z ¹ is OH. The hexapeptide according to the present invention is preferably the enhancer of the skin cells so that it can be applied topically in the skin and it can be called also the topical hexapeptide.

The hexapeptide of the general formula I according to the present invention is preferably in a form of a complex with a metal ion selected from a group comprising zinc ion, copper ion, manganese ion or magnesium ion. The metal ion is bound by a coordination covalent bond in the complex. Preferably, metal ion is zinc ion (Zn ²⁺ ) and the complex is schematically stated below as Zn-FWAHKK complex or simply as Zn-FWAHKK.

The hexapeptide of the general formula I according to the present invention may be optically pure or be composed of L- or D-isomers or a mixture thereof. L-isomers, which are those found in nature, are preferred.

The hexapeptide of the present invention may be in the form of salts, especially salts of hydrochloric acid, formic acid or acetic acid, or any salts commonly used in cosmetics.

Another embodiment according to the present invention is a topical composition comprising at least one hexapeptide of the general formula I as defined above.

According to the preferred embodiment of the topical composition according to the present invention a concentration of the hexapeptide of the general formula I, as stated above, is in the range of 0.0001 to 0.135 % (w/w), preferably in the range of 0.001 to 0.05 % (w/w), more preferably from 0.001 to 0.0135 % (w/w). Amount of the hexapeptide depends on the purpose of the composition and the desired final effect. This invention also includes the topical composition, that contains at least one hexapeptide according to the invention in a form for the topical skin application that may be selected from a group comprising cream, serum, anhydrous gel, paste, dispersion of vesicles, powder, nanofibers, macro-, micro-, or nano-capsules, macro-, micro- or nano-spheres, liposomes, oleosomes or chylomicrons, macro-, micro-, or nanoparticles or macro-, micro- or nano-sponges, which may be adsorbed on organic polymer powders, talcs, bentonites and other inorganic or organic supports.

The topical composition according to the present invention is in the form of the cream selected from a group comprising a water-in-oil or oil-in-water emulsion, a micro- or nano- emulsion.

The topical composition according to the present invention is in the form of the serum selected from a group comprising sterile or nonsterile aqueous or hydro-alcoholic solution or gel.

According to another preferred embodiment of the topical composition according to the present invention the composition is in a form selected from a group comprising cream or serum.

In the case the topical composition according to the present invention is in the form of cream, the concentration of the hexapeptide of the general formula I, as stated above, is in the range of 0.0001 to 0.135 % (w/w), preferably 0.001 % to 0.0135 % (w/w).

Preferably the composition in the form of cream additionally comprises at least one cosmetic or dermo-pharmaceutical auxiliary ingredient selected from a group comprising oil, wax, butter, emulsifier, auxiliary active ingredient and preservative.

Preferably the amount of oil, wax, butter or a mixture thereof in the composition according to the present invention is in the range of 20 to 55 % (w/w), preferably 20 to 40 % (w/w), more preferably 20 to 30 % (w/w). Preferably the amount of emulsifier in the composition according to the present invention is in the range of 1 to 20 % (w/w), preferably 2-15 % (w/w), more preferably 2-10 % (w/w). Preferably the amount of auxiliary active ingredient in the composition according to the present invention is in the range of 0.001 to 20 % (w/w), preferably 0.001 to 10 % (w/w), more preferably 0.001 to 5 % (w/w). Preferably the amount of preservative in the composition according to the present invention is in the range of 0.1 to 2.5 % (w/w), preferably 0.1 to 1.5 % (w/w), more preferably 0.8 to 1.2 % (w/w).

Preferably the oil is selected from a group comprising argan, coconut, avocado, almond, sesame, olive, sunflower, hemp, jojoba, macadamia, wheat germ, marula, meadowfoam, rice, poppy, rosehip, apricot, castor oil, caprylic/capric triglyceride; the butter is selected from a group comprising cocoa butter, shea butter, illipe butter, kokum butter, murumuru butter, mango butter, cupuacu butter, avocado butter; and the wax is selected from a group comprising lanolin, beeswax, carnauba, candelilla wax and petrolatum.

Preferably the emulsifier is selected from a group comprising glyceryl stearate, glyceryl caprylate, behenyl alcohol, glyceryl behenate, cetearyl glucoside, methyl glucose sequistearate, glyceryl stearate citrate, polyglyceryl-3 stearate, cetearyl olivate, lecithin, stearyl alcohol, sorbitan oleate, polysorbate, stearic acid, cetyl alcohol and cetearyl alcohol, sodium acrylate, sodium acryloyldimethyl taurate copolymer, isohexadecane, polysorbate 80 or mixtures thereof.

The topical composition according to the present invention in the form of cream containing at least one hexapeptide according to the invention may also be combined with other auxiliary active ingredients that can have synergistic or additional effect to enhance or extend the desired activity described in the invention. The auxiliary active ingredient is selected from a group comprising following agents: anti-acne, anti-aging, anti- wrinkle, lightening, whitening, anti-spots, pro-pigmenting, hydrating, moisturizing, humectants, slimming, exfoliating, anti- redness, anti-inflammatory, antioxidants, radical scavengers, anti-glycation, volumizing, restructuring, rejuvenating, regenerating, anti-carbonylation, dermo-relaxing, improving stratum corneum, dermo-epidermal junction, firmness, elasticity, collagen boosters, eye contours (dark circles and under eye bags), promoting blood circulation etc.

The above-mentioned auxiliary active ingredients may be synthetic or obtained from plant, bacteria, fungi, cell cultures or their products.

Preferably the auxiliary active ingredient is selected from a group comprising vitamins A, D, E, K, C, B-group vitamins, coenzyme Q10, allantoin, bisabolol, bakuchiol, resveratrol, lactic acid, amino acids, benzoyl peroxide, sulfur, azelaic acid, peptides preferably acetyl hexapeptide-8, palmitoyl tripeptide- 1, palmitoyl tetrapeptide-7, copper tripeptide- 1, hexapeptide- 1, palmitoyl pentapeptide-4, saccharomyces peptides, and proteins, preferably rice, soy, quinoa or wheat protein; polysaccharides preferably hyaluronic acid or its pharmaceutically and cosmetically acceptable salt or derivative preferably sodium oleoyl hyaluronate, sodium retinoyl hyaluronate, sodium hyaluronate crosspolymer-3; carboxymethyl glucan, schizophyllan, glucomannan; panthenol; urea; glycerin; plant extracts, preferably aloe vera extract, cammomile extract, acai extract, green tea extract, algae extract, oat extract, cannabis extract, cranberry extract; extracts, ferments, lysates or filtrates from bacteria preferably from Lactobacillus spp., Thalassospira spp., Bifidobacterium spp., Halobacterium spp., or from fungi preferably from Saccharomyces spp., Agaricus subrufescens, Choiromyces maeandriformis, Cordyceps sinensis, Ganoderma lucidum, Grifola frondosa, Hypsizygus ulmarium, Inonotus obliquus, Lentinula edodes, Polyporus spp., Trametes versicolor, Tremella fuciformis, Tuber spp., Schizophyllum commune.

Preferably the preservative is selected from the group comprising aromatic acids and derivatives thereof preferably benzoic acid, sodium benzoate, salicylic acid, dehydroacetic acid, potassium sorbate, parabens; alcohols preferably ethanol, isopropanol, benzyl alcohol, phenoxyethanol, phenethyl alcohol; imidazole derivatives preferably hydantoin, imidazolidinyl urea; cationic surfactants preferably benzalkonium chloride.

In the case the topical composition according to the present invention is in the form of serum, the concentration of the hexapeptide of the general formula I, as stated above, is in the range of 0.001 to 0.135 % (w/w), preferably 0.001 % to 0.0135 % (w/w).

Preferably the composition in the form of serum additionally comprises at least one cosmetic or dermo-pharmaceutical auxiliary ingredient selected from a group comprising thickener, auxiliary active ingredient and preservative as defined above.

Preferably the amount of thickener in the composition according to the present invention is in the range of 0.1 to 20 % (w/w), preferably 0.1 to 5 % (w/w), more preferably 0.2 to 0.5 % (w/w). Preferably the amount of auxiliary active ingredient in the composition according to the present invention is in the range of 0.001 to 20 % (w/w), preferably 0.001 to 10 % (w/w), more preferably 0.001 to 5 % (w/w). Preferably the amount of preservative in the composition according to the present invention is in the range of 0.1 to 2.5 % (w/w), preferably 0.1 to 1.5 % (w/w), more preferably 0.8 to 1.2 % (w/w).

Preferably the thickener is selected from a group comprising xantham gum, sclerotium gum, guar gum, cellulose gum, carbomer, hydroxy ethylcellulose, Amorphophallus konjac root extract, karagenan, pullulan, lecithin, lysolecithin, sodium acrylate, sodium acryloyldimethyl taurate copolymer, isohexadecane, polysorbate 80 or mixtures thereof.

The topical composition according to the present invention containing at least one hexapeptide according to the invention may also be combined with other auxiliary active ingredients that can have synergistic or additional effect to enhance or extend the desired activity described in the invention. The auxiliary active ingredient is selected from a group comprising following agents: anti-acne, anti-aging, anti-wrinkle, lightening, whitening, anti- spots, pro-pigmenting, hydrating, moisturizing, humectants, slimming, exfoliating, anti- redness, anti-inflammatory, antioxidants, radical scavengers, anti-glycation, volumizing, restructuring, rejuvenating, regenerating, anti-carbonylation, dermo-relaxing, improving stratum comeum, dermo-epidermal junction, firmness, elasticity, collagen boosters, eye contours (dark circles and under eye bags), promoting blood circulation etc.

The above-mentioned auxiliary active ingredients may be synthetic or obtained from plant, bacteria, fungi, cell cultures or their products.

Preferably the auxiliary active ingredient is selected from a group comprising vitamin C and B-group vitamins; amino acids, peptides preferably acetyl hexapeptide-8, palmitoyl tripeptide- 1, copper tripeptide- 1, Saccharomyces peptides, hexapeptide- 1, and proteins preferably rice protein, soy protein, quinoa or wheat protein; further polysaccharides preferably hyaluronic acid or its pharmaceutically and cosmetically acceptable salt or derivative preferably sodium oleoyl hyaluronate, sodium retinoyl hyaluronate, sodium hyaluronate crosspolymer-3; carboxymethyl glucan, schizophyllan, glucomannan; panthenol; urea; glycerin; plant extracts preferably aloe vera extract, cammomile extract, acai extract, green tea extract, algae extract, oat extract, cannabis extract, cranberry extract; extracts, ferments, lysates or filtrates from bacteria preferably from Lactobacillus spp., Thalassospira spp., Bifidobacterium spp., Halobacterium spp., or from fungi preferably from Saccharomyces spp., Agaricus subrufescens, Choiromyces maeandriformis, Cordyceps sinensis, Ganoderma lucidum, Grifola frondosa, Hypsizygus ulmarium, Inonotus obliquus, Lentinula edodes, Polyporus spp., Trametes versicolor, Tremella fuciformis, Tuber spp., Schizophyllum commune.

Preferably the preservative is selected from the group comprising aromatic acids and derivatives thereof preferably benzoic acid, sodium benzoate, salicylic acid, dehydroacetic acid; potassium sorbate, parabens; alcohols preferably ethanol, isopropanol, benzyl alcohol, phenoxyethanol, phenethyl alcohol; imidazole derivatives preferably hydantoin, imidazolidinyl urea; cationic surfactants preferably benzalkonium chloride.

All % stated below are (w/w%) unless otherwise stated and are related to the total weight of the composition if % concerns to the topical composition according to the invention.

Generally, the amount of water in the composition according to the present invention in the form of cream or serum corresponds to the amount of water added up to 100 % (w/w) of the composition.

Cosmetically and pharmaceutically acceptable salts of hyaluronic acid are said to be those formed from acids which form non-toxic acid anions selected from a group comprising sodium, potassium, calcium, magnesium, zinc.

New is the hexapeptide of the general formula I according to the present invention and use thereof or use of the cosmetic or dermo-pharmaceutical topical compositions according to the invention containing said hexapeptide to improve oily and acne-prone skin condition and appearance. More specifically, the hexapeptide according to the invention inhibits epidermal keratinization, reduces sebum production by inhibition of a key enzyme 5α-reductase, possesses an anti-inflammatory activity by inhibiting pro-inflammatory interleukins, has an antimicrobial effect towards Cutibacterium acnes, and reduces number of acne lesions in vivo. Furthermore, the said hexapeptide according to the present invention also has an anti-ageing effect by stimulating collagen synthesis and reducing wrinkles in vivo.

The said hexapeptide of the topical composition according to the invention is dedicated for oily and acne-prone skin as well as for normal skin.

According to the other preferred embodiment of the hexapeptide of the general formula I according to the present invention and the topical composition according to the present invention are dedicated for use for treatment of the skin. Preferably for use for medical treatment of skin diseases selected from a group comprising acne, atopic dermatitis, psoriasis, peeling skin disease, ichthyosis. Or preferably for use for cosmetic treatment of the skin.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Hexapeptide FWAHKK forms a complex with zinc. Spectrophotometric determination of the ability of zinc to form a complex with FWAHKK hexapeptide using dithizone as a free zinc indicator.

Fig. 2. Zinc penetrates more effectively into the cells when in Zn-FWAHKK complex. HaCaT keratinocytes were incubated with Zn-FWAHKK or ZnSO ₄ *7H ₂ O (Zn) of corresponding concentration for 3 h. Intracellular zinc was visualized using fluorescence microscopy.

Fig. 3. Hexapeptide FWAHKK increases cell viability and prevents cytotoxic effect of zinc in Zn-FWAHKK complex. NIH-3T3 fibroblasts were incubated with FWAHKK, Zn-FWAHKK or ZnSO4*7H ₂ O (Zn) of corresponding concentrations for 48 h. Cell viability was determined by MTT assay. * p< 0.05; ** p<0.01; *** p0.001 in comparison to control if not stated otherwise

Fig. 4. Hexapeptide FWAHKK and ZnSO ₄ *7H ₂ O reduced 5α-reductase gene expression in contrast to retinol. Zn-FWAHKK complex was even more effective than its individual components. HaCaT keratinocytes were treated with FWAHKK, Zn-FWAHKK, ZnSO4*7H ₂ O (Zn) and retinol for 72 h. Relative gene expression of 5α-reductase (gene SRD5A1) was determined by qRT-PCR. * pO.05; ** p<0.01; *** p0.001 in comparison to the respective controls

Fig. 5. Hexapeptide FWAHKK and Zn-FWAHKK significantly reduced UVB-induced ILIA gene expression in contrast to zinc or retinol. HaCaT keratinocytes were irradiated with 10 mJ/cm ² UVB and treated with FWAHKK, Zn-FWAHKK, ZnSO ₄ *7H ₂ O (Zn) and retinol for 24 h. Relative gene expression of IL-la (gene IL1A) was determined by qRT-PCR. *** p<0.001 in comparison to the respective UVB controls

Fig. 6. Hexapeptide FWAHKK and Zn-FWAHKK reduced expression of genes associated with keratinocyte differentiation similarly to retinol whereas zinc had no such effect. HaCaT keratinocytes were treated with FWAHKK, Zn-FWAHKK, ZnSO ₄ *7H ₂ O (Zn) and retinol for 72 h. Relative gene expression of 5α-reductase (gene SRD5AI) was determined by qRT-PCR. * p<0.05; ** p0.01; *** p0.001 in comparison to the respective controls

Fig. 7. Hexapeptide FWAHKK and Zn-FWAHKK significantly reduced UVB-induced IL6 and IL8 gene expression in contrast to zinc or retinol. HaCaT keratinocytes were irradiated with 10 mJ/cm ² UVB and treated with FWAHKK, Zn-FWAHKK, ZnSO ₄ *7H2O (Zn) and retinol for 24 h. Relative gene expression of the pro-inflammatory interleukins IL-6 and IL-8 (genes IL6 and IL8) was determined by qRT-PCR. * p<0.05; ** p<0.01; *** p0.001 in comparison to the respective UVB controls

Fig. 8. Hexapeptide FWAHKK and zinc have an antimicrobial activity against C. acnes growing in suspension. Zn-FWAHKK has higher activity than individual compounds. C. acnes growing in suspension was cultivated with FWAHKK, Zn-FWAHKK and ZnSO4*7H ₂ O (Zn) for 72 h. OD590 was then measured. * p<0.05; ** p0.01; *** p0.001 in comparison to control if not stated otherwise

Fig. 9. Hexapeptide FWAHKK and Zn-FWAHKK stimulated collagen 1 gene expression in contrast to zinc or retinol. HaCaT keratinocytes were treated with FWAHKK, Zn-FWAHKK, ZnSO4*7H ₂ O (Zn), and retinol for 72 h. Relative gene expression of 5α-reductase (gene SRD5A1) was determined by qRT-PCR. * pO.05; ** p0.01; *** p0.001 in comparison to the respective controls Fig. 10. Zn-FWAHKK complex has anti-acne and anti-ageing activity and is more effective than retinol. In vivo study on 40 volunteers with acne-prone skin who applied two emulsions with 13.5 μg/mL Zn-FWAHKK and placebo (30 subjects) or 0.2 % retinol and placebo (10 subjects) on the two respective halves of the face once daily for 6 weeks. The number of acne lesions (A) was determined by an image analysis of the whole-face images obtained by VisiaCR. Crow's feet wrinkle depth (B) was determined by a 3D camera. * p<0.05; ** p<0.01, *** p0.001 in comparison to placebo

EXAMPLES OF THE EMBODIMENTS OF THE INVENTION

Example 1, Preparation of FWAHKK, Zn-FWAHKK, acetyl-FWAHKK and palmitoyl- FWAHKK peptide

Firstly, FWAHKK peptide was synthetized by standard solid phase peptide synthesis using Fmoc/tBu strategy on Wang resin (Amblard et al. 2006).

The attachment of the first amino acid to Wang resin (Sunresin, China) was performed in a syringe reactor in dimethylformamide (DMF) using diisopropylcarbodiimide (DIC) and OxymaPure® (Iris Biotech, Germany) in 3 molar excess (meq) of Fmoc-Lys(Boc)-OH overnight. After coupling, residual hydroxyl groups on the resin were capped by acetic anhydride/pyridine solution (1/1, v/v).

The FWAHKK peptide was synthetized at 0.1 mmol scale using the CEM Liberty Blue automated micro wave peptide synthesizer (CEM, USA) on Wang resin preloaded with the first Fmoc-AA. Fmoc deprotections were performed with 20% piperidine in DMF. Coupling reactions were performed using 5 equivalents of Fmoc-AA/DIC/Oxyma Pure® in DMF.

For the preparation of acetyl-FWAHKK and palmitoyl-FWAHKK, the V-terminus of the synthetized peptide FWAHKK on the resin was acetylated or palmitoylated using 2 meq of acetic or palmitic anhydride/pyridine (1/1, v/v) in DMF for 30 min at room temperature.

Cleavage of the peptides (FWAHKK, acetyl-FWAHKK or palmitoyl-FWAHKK) from the resin was performed for 30 min at elevated temperature (38 °C) using the CEM Razor® high-throughput peptide cleavage system (CEM, USA) with trifluoracetic acid/anisole/thioanisole/H ₂ O/dichloromethane (85/2,5/2,5/5/5) mixture. The peptides were then precipitated in cold diethyl ether.

Crude FWAHKK and acetyl-FWAHKK peptides were dissolved in 50% acetonitrile, crude palmitoyl-FWAHKK was dissolved in 100 % acetonitrile. Then, they were purified by Shimadzu preparative HPLC (Prominence series). XBridge OBD Prep Column Reversed-Phase 5 pm Spherical Hybrid, 50 mm x 50 mm was used for the separation with flow rate 30 ml/min. Mobile phase consisted of A 0.1% HCOOH in water and B acetonitrile. The purity of the peptides was verified by PDA and MS detector. The purified peptides were then lyophilized.

For the preparation of FWAHKK hexapeptide in complex with zinc (Zn-FWAHKK), water solution of 10 % FWAHKK hexapeptide and 3.52 % ZnSO ₄ *7H ₂ O (zinc sulfate, heptahydrate, Lachner, Czech Republic) corresponding to a molar ratio 1 :1 was prepared, mixed overnight at room temperature and then the solution was lyophilized.

Example 2. The ability of FWAHKK peptide to form a complex with zinc

The ability of FWAHKK peptide to form a complex with zinc was evaluated as described previously with slight modifications (Catapano etal. 2018). Briefly, 20 μL of solution of FWAHKK (0-600 μM (0-490 μg/mL; 0-0.049 w/w %), prepared as described in Example 1) and 60 μM (17.3 μg/mL; 0.00173 %) ZnSO ₄ *7H ₂ O in 15 mM HEPES, pH 6.8 was mixed with 20 μL of 250 μM dithizone in DMSO and 80 μL of 15 mM HEPES, pH 6.8. Dithizone was used as an indicator of free zinc. After 30 min incubation at room temperature (RT), the absorbance was measured at 520 nm and percentage of zinc in complex with FWAHKK peptide was calculated.

The results (Fig. 1) confirmed the ability of peptide FWAHKK to form a complex with zinc. In the complex Zn-FWAHKK prepared as described in Example 1, in which the molar ratio of FWAHKK and zinc is 1 : 1 , approx. 50 % of zinc is in the complex with the hexapeptide.

Example 3. Enhanced cell penetration of zinc when in Zn-FWAHKK complex

HaCaT keratinocytes (CLS collection, Germany) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal bovine serum, 0.3 mg/mL glutamine, lOO U/mL penicillin and 0.1 mg/mL streptomycin (all Sigma- Aldrich, USA) at 37 °C, in a humidified atmosphere with 5 % CO2.

The cells seeded in appropriate density into 96- well plates were treated with 1352 μg/mL (0.1352 %) Zn-FWAHKK complex (prepared as described in Example 1) or ZnSO ₄ *7H ₂ O of corresponding concentration (352 μg/mL (0.0352 %)) for 3 h. Then, the cells were washed by the cell culture medium and incubated with 1 μM FluoZin™-3 AM (Invitrogen, USA), a cell-permeant, Anorogenic Zn ²⁺ -selective indicator, and 20 μM Hoechst (Abeam, UK), a fluorescent DNA stain in the cell culture medium for 30 min at 37 °C in the dark. Then, the cells were washed in phosphate-buffered saline (PBS). The fluorescence was observed using an Eclipse 50i fluorescence microscope equipped with a DS-Fil camera (both Nikon, Japan).

The results (Fig. 2) showed that penetration of zinc into the skin cells was more effective when the ion was in the Zn-FWAHKK complex.

Example 4. FWAHKK increases cell viability and prevents cytotoxic effect of zinc in Zn- FWAHKK complex

NIH-3T3 mouse embryonic fibroblasts (ATCC collection, USA) were cultured in the same manner as HaCaT keratinocytes as described in Example 3. The cells seeded in appropriate density into 96-well plates were treated with FWAHKK (2.5 - 80 μg/mL (0.00025 - 0.008 %)), Zn-FWAHKK (3.4 - 108 μg/mL (0.00034 - 0.0108 %)) (both prepared as described in Example 1) and ZnSO ₄ *7H ₂ O (0.9 - 28.2 μg/mL (0.00009 - 0.00282 %)) for 48 h. Then, cell viability was determined by MTT assay (Riss et al. 2016). Briefly, the cells were incubated with 0.5 mg/mL MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) in the cell culture medium for 2.5 h at 37 °C. After MTT removal, cell lysis and solubilization of formazan was carried out by incubation of the cells with a solubilizing solution (45 % isopropanol, 45 % DMSO, 10 % Triton-X, 0.3 M HC1) for 30 min at RT, shaking. The absorbance was then measured at 570 run by a spectrophotometer (EnVision® 2105 Multimode Plate Reader, PerkinElmer, USA). T-test was used for the statistical evaluation of the results.

The results (Fig.3) showed that ZnSO ₄ *7H ₂ O in concentrations starting from 10.6 μg/mL significantly decreased cell viability. On the other hand, FWAHKK peptide enhanced cell viability and creation of Zn-FWAHKK complex prevented the cytotoxic effect of zinc.

Example 5. Inhibition of 5α-reductase gene expression by FWAHKK and Zn-FWAHKK

HaCaT keratinocytes (CLS collection, Germany) were cultured as described in Example 3. The cells seeded in appropriate density into 6-well plates were treated with FWAHKK (1 μg/mL (0.0001 %) and 10 μg/mL (0.001 %)), Zn-FWAHKK (1.35 μg/mL (0.000135 %) and 13.5 μg/mL (0.00135 %)) (both prepared as described in Example 1), ZnSO ₄ *7H ₂ O (Zn, 0.35 μg/mL (0.000035 %) and 3.5 μg/mL (0.00035 %)), and 10 μM retinol (in DMSO, final concentration of DMSO was 0.1 %) for 72 h. Control cells were untreated or treated with 0.1 % DMSO in the case of retinol control.

Then, 5α-reductase gene expression (gene SRD5A1) was determined by quantitative, real-time, reverse-transcription PCR (qRT-PCR) as follows. The total RNA was isolated from the cells by the acid guanidine thiocyanate-phenol extraction method using TRI Reagent (Sigma Aldrich, USA) according to the instructions provided by the supplier. Reverse transcription was performed using High capacity cDNA reverse transcription kit (Thermo Fisher Scientific, MA, USA) in GenePro thermal cycler (Bioer Technology, China) according to the manufacturer’s instructions. Subsequent qPCR was performed with specific TaqMan gene expression assays for SRD5A1 (qHsaCEP0052536, BioRad, CA, USA), and RPL13A (Hs04194366_gl, Thermo Fisher Scientific, MA, USA) as a reference gene; and TaqMan Fast Advanced Master mix (all Thermo Fisher Scientific, MA, USA) according to the supplier’s recommendations in a StepOne real-time PCR cycler (Thermo Fisher Scientific, MA, USA). The data were analyzed using the 2 ^-ΔΔCt method. The data were normalized to the negative controls or in the case of retinol to DMSO controls. T-test was used for the statistical evaluation of the data.

5α-reductase is a key enzyme involved in the androgens-induced sebum production. The enzyme converts testosterone, the most common androgen, into DHT which is several times more potent in stimulation of sebum production. The results (Fig. 4) showed that the hexapeptide FWAHKK, Zn-FWAHKK as well as ZnSO4*7H2O significantly reduced gene expression of 5α-reductase (gene SRD5AP). The complex Zn-FWAHKK was more effective than its individual components of corresponding concentrations. On the other hand, retinol did not have any significant effect on 5α-reductase gene expression.

Example 6. Inhibition of IL-1α gene expression by FWAHKK and Zn-FWAHKK

HaCaT keratinocytes (CLS collection, Germany) were cultured as described in Example 3. The cells seeded in appropriate density into 6-well plates were washed in PBS and irradiated with 10 mJ/cm ² UVB using 300 W xenon arc lamp (Oriel Arc Lamp Housing, Newport, CA, USA) and 300±10 nm optical filter (Newport, CA, USA). Then, they were treated with FWAHKK (1 μg/mL (0.0001 %) and 10 μg/mL (0.001 %)), Zn-FWAHKK (1.35 μg/mL (0.0001 %) and 13.5 μg/mL (0.001 %)) (both prepared as described in Example 1), ZnSO4*7H2O (Zn; 0.35 μg/mL (0.000035 %) and 3.5 μg/mL (0.00035 %)) and 10 μM retinol (in DMSO, final concentration of DMSO was 0.1 %) for 24 h. Control cells were untreated or treated with 0.1 % DMSO in the case of retinol control.

ILIA gene expression was determined by real-time qRT-PCR as described in Example 5 using specific TaqMan gene expression assays for ILIA (qHsaCIP0030471, BioRad, CA, USA), and RPL13A (Hs04194366_gl, Thermo Fisher Scientific, MA, USA) as a reference gene. The data were analyzed using the 2' ^ΔΔCt method. The data were normalized to the UVB untreated controls or in the case of retinol to UVB DMSO controls. T-test was used for the statistical evaluation of the results. IL-1α is a pro-inflammatory interleukin stimulating hyperkeratinization of follicular keratinocytes (Guy and Kealey 1998) in the early phases of acne pathogenesis. In our experiments, we induced IL-1α gene expression in HaCaT keratinocytes using UVB irradiation (Fig. 5). Then, we observed significant inhibition of UVB-induced ILIA gene expression after FWAHKK and Zn-FWAHKK treatment whereas zinc and retinol did not show any effect (Fig. 5).

Example 7. Inhibition of keratinocyte differentiation by FWAHKK and Zn-FWAHKK

HaCaT keratinocytes (CLS collection, Germany) were cultured as described in Example 3. The cells seeded in appropriate density into 6-well plates were treated with FWAHKK (1 μg/mL (0.0001 %) and 10 μg/mL (0.001 %), Zn-FWAHKK (1.35 μg/mL (0.000135 %) and 13.5 μg/mL (0.00135 %)) (both prepared as described in Example 1), ZnSO ₄ *7H ₂ O (Zn; 0.35 μg/mL (0.000035 %) and 3.5 μg/mL (0.00035 %)) and 10 μM retinol (in DMSO, final concentration of DMSO was 0.1 %) for 72 h. Control cells were untreated or treated with 0.1 % DMSO in the case of retinol control.

Expression of the selected genes involved in keratinocyte differentiation was determined by real-time qRT-PCR as described in Example 5 using specific TaqMan gene expression assays for FLG (Hs00856927_gl), OCLN (Hs00170162_ml), LCE2C (Hs02390636_sl), SPRR2E (qHsaCEP0055677, BioRad, CA, USA), KRT10 (Hs01043114_gl) and RPL13A (Hs04194366_gl) as a reference gene (all except SPRR2E Thermo Fisher Scientific, MA, USA). The data were analyzed using the 2' ^ΔΔCt method. The data were normalized to the untreated controls or in the case of retinol to DMSO controls. T-test was used for the statistical evaluation of the results.

The hexapeptide FWAHKK as well as Zn-FWAHKK were shown to downregulate genes associated with keratinocyte differentiation in the same manner as 10 μM retinol whereas zinc of corresponding concentrations had no such effect (Fig. 6).

Example 8. Anti-inflammatory effect of FWAHKK and Zn-FWAHKK

HaCaT keratinocytes (CLS collection, Germany) were cultured as described in Example 3. The cells seeded in appropriate density into 6-well plates were washed in PBS and irradiated with 10 ml/cm ² UVB using 300 W xenon arc lamp (Oriel Arc Lamp Housing, Newport, CA, USA) and 300±10 nm optical filter (Newport, CA, USA). Then, they were treated with FWAHKK (1 μg/mL (0.0001 %) and 10 μg/mL (0.001 %)), Zn-FWAHKK (1.35 μg/mL (0.000135 %) and 13.5 μg/mL (0.00135 %)) (both prepared as described in Example 1), ZnSO ₄ *7H ₂ O (Zn, 0.35 μg/mL (0.000035 %) and 3.5 μg/mL (0.00035 %)), and 10 μM retinol (in DMSO, final concentration of DMSO was 0.1 %) for 24 h. Control cells were untreated or treated with 0.1 % DMSO in the case of retinol control.

IL1A gene expression was determined by real-time qRT-PCR as described in Example 5 using specific TaqMan gene expression assays for IL6 (Hs00174131 ml), IL8 (Hs00174103_ml), and RPL13A (Hs04194366_gl) as a reference gene (all Thermo Fisher Scientific, MA, USA). The data were analyzed using the 2 ^-ΔΔCt method. The data were normalized to the UVB untreated controls or in the case of retinol to UVB DMSO controls. T- test was used for the statistical evaluation of the results.

Inflammation is a key event in acne pathogenesis. We induced inflammatory processes such as the overexpression of the pro-inflammatory interleukins IL-6 and IL-8 in HaCaT keratinocytes using UVB irradiation (Fig. 7). We observed significant inhibition of UVB- induced IL6 and IL8 gene expression after FWAHKK and Zn-FWAHKK treatment similar to 10 μM retinol whereas zinc of corresponding concentrations did not show any effect (Fig. 7).

Example 9. Antimicrobial activity of FWAHKK and Zn-FWAHKK against C. acnes

Cutibacterium acnes (strain DSM 1897, DSZM collection, Germany) was maintained in suspension in tryptic soy broth (TSB) at 37 °C. The bacteria were then inoculated in appropriate density into TSB containing FWAHKK (10 - 80 μg/mL (0.001 - 0.008 %)), Zn- FWAHKK (13.5 - 108.2 μg/mL (0.00135 - 0.01082 %)) (both prepared as described in Example 1) and ZnSO4*7H2O (Zn; 3.5 - 28.2 μg/mL (0.00035 - 0.00282 %)) in 96-well plates. The bacteria were cultivated for 72 h at 37 °C and then, optical density was measured at 590 nm (OD590) by a spectrophotometer (EnVision® 2105 Multimode Plate Reader, PerkinElmer, USA). T-test was used for the statistical evaluation of the results.

Both zinc and the hexapeptide FWAHKK have weak antimicrobial activity against C. acnes (Fig. 8). The complex Zn-FWAHKK was more effective than the individual compounds of corresponding concentrations (Fig. 8).

Example 10. Stimulation of collagen production by FWAHKK and Zn-FWAHKK

NIH-3T3 mouse embryonic fibroblasts (ATCC collection, USA) were cultured in the same manner as HaCaT keratinocytes as described in Example 3. The cells seeded in appropriate density into 6-well plates were treated with FWAHKK (1 μg/mL (0.0001 %) and 10 μg/mL (0.001 %)), Zn-FWAHKK (1.35 μg/mL (0.000135 %) and 13.5 μg/mL (0.00135 %)) (both prepared as described in Example 1), ZnSO4*7H2O (Zn, 0.35 μg/mL (0.000035 %) and 3.5 μg/mL (0.00035 %)), and 10 μM retinol (in DMSO, final concentration of DMSO was 0.1 %) for 72 h. Control cells were untreated or treated with 0.1 % DMSO in the case of retinol control.

COL1A1 gene expression was determined by real-time qRT-PCR as described in Example 5 using specific TaqMan gene expression assays for COL1A1 (Mm00801666_gl), and RPL13A (Mm05910660_gl) as a reference gene (both Thermo Fisher Scientific, MA, USA). The data were analyzed using the 2 ^-ΔΔCt method. The data were normalized to the untreated controls or in the case of retinol to DMSO controls. T-test was used for the statistical evaluation of the results.

The results showed a significant stimulation of collagen 1 gene expression after FWAHKK and Zn-FWAHKK treatment whereas zinc of corresponding concentrations and 10 μM retinol did not show any effect (Fig. 9).

Example 11 Zn-FWAHKK improves appearance of acne-prone skin and also has an anti- ageing effect in vivo

We performed a double-blind, placebo-controlled, split-face in vivo study on 40 human volunteers (Caucasians, Fitzpatrick skin type I-III, women/men 36/4, 18-49 years, average 30.5 years) with oily, problematic, acne-prone skin. The study was conducted in accordance with the principles of the Declaration of Helsinki of World Medical Association. The study was approved by Contipro a.s. ethical committee and informed consent was obtained from all the volunteers. Only heathy subjects with no acute skin disorders on the face were included in the study. Other exclusion criteria were pregnancy, breast-feeding, invasive cosmetic facial procedures (face lift, botox etc.) and heavy smoking (> 10 cigarettes per day). Application of any facial skincare products as well as face washing was not allowed during the day of the measurement.

All the volunteers were given two emulsions: 30 volunteers (women/men 27/3, 18-48 years, average 31.0 years) obtained emulsions with 0.00135 % Zn-FWAHKK (13.5 μg/mL; prepared as described in Example 1) and placebo, and 10 volunteers (women/men 9/1, 24-49 years, average 28.9 years) obtained emulsions with 0.2 % retinol and placebo. Composition of the emulsions is described in Table 1. The two emulsions were applied on the two respective halves of the face once daily in the evening for 6 weeks. The measurement of the skin parameters was performed at the beginning of the study, and then after 2, 4 and 6 weeks.

The measurements were performed after 30 min of acclimation of the volunteers in a room with controlled conditions (T: 20-22 °C; RH 40-45 %). The number of acne lesions was determined by an image analysis of the whole-face images obtained by VisiaCR high-resolution camera (Canfield Scientific, USA). The quantification was performed using an Image-Pro 10 analysis software (Media Cybernetics, USA). Crow's feet wrinkle depth was determined by a Primos Lite 3D camera (Canfield Scientific, USA). The results showed an improvement of the appearance of the acne-prone skin represented by the reduced number of acne lesions (Fig. 10A). Zn-FWAHKK was also shown to possess an anti-ageing activity by reducing wrinkles (Fig 10B). In both cases, Zn-FWAHKK was more effective than 0.2 % retinol. Table 1. Compositions of the emulsions used in the in vivo study

Example 12. Compositions of the emulsions with FWAHKK, Zn-FWAHKK, acetyl- FWAHKK, or palmitoyl-FWAHKK

Example of creams (oil in water emulsions) containing the hexapeptides of the present invention FWAHKK, Zn-FWAHKK, acetyl-FWAHKK or palmitoyl-FWAHKK prepared as described in Example 1.

Table 2.

Table 3

Table 4

Table 5

Oil and water phases were prepared and heated to 70 °C. Next, both phases were mixed and emulsified while stirring (290 rpm/min). Then, the emulsion was cooled to 40 °C and the water-soluble ingredient phase and/or oil-soluble active ingredient phase and/or vitamin E and preservative were added. Finally, pH was measured and adjusted to values between 5.2 and 6.5 with 1-50% citric acid or 1-50 % KOH or NaOH.

Example 13. Compositions of the serums with FWAHKK, Zn-FWAHKK and acetyl- FWAHKK Example of serums containing the hexapeptides of the present invention FWAHKK and

Zn-FWAHKK prepared as described in Example 1.

Table 6

Table 7

Table 8

Table 9

First, the hexapeptide FWAHKK or Zn-FWAHKK or acetyl-FWAHKK and active ingredients except sodium hyaluronate and Crosslinked ¹¹⁴ were added to water and stirred till complete dissolution. Next, thickeners and/or sodium hyaluronate and Crosslinked ¹¹⁴ were added and mixed till complete dissolution. Then, a preservative was added. Finally, pH was measured and adjusted to values between 5.2 and 6.5 with 1-50% citric acid or 1-50 % KOH or NaOH.

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