阅读说明：本技术 用于治疗和预防皮肤衰老和光老化的肽 (Peptides for treating and preventing skin aging and photoaging ) 是由 P·佩莱格里尼 于 2019-10-10 设计创作，主要内容包括：本发明涉及包含Seq.ID No.1的肽及其化妆品组合物在治疗和预防皮肤衰老和光老化中的用途。(The present invention relates to peptides comprising seq.ID No.1 and the use of cosmetic compositions thereof for the treatment and prevention of skin ageing and photoaging.)

1. A peptide comprising seq.id No.1 or a sequence having at least 85%, preferably at least 90%, and more preferably at least 95% sequence identity to seq.id No.1, and derivatives or salts thereof, for use in the treatment and prevention of skin ageing and photoaging.

2. The peptide for use according to claim 1, wherein the peptide has a length of up to 16 amino acids, preferably up to 15 amino acids, and more preferably 14 amino acids.

3. The peptide for use according to claim 2, wherein the peptide consists of: seq.id No.1, or a sequence having at least 85%, preferably at least 90%, and more preferably at least 95% sequence identity to seq.id No.1, and derivatives or salts thereof.

4. The peptide for use according to any of the preceding claims 1 to 3, wherein said derivative comprises the N-and/or C-terminus of said peptide chemically modified or protected with an organic compound.

5. The peptide for use according to claim 4, wherein the organic compound is selected from phosphoryl, glycosyl, acyl, alkyl, carboxyl, hydroxyl, biotinyl, ubiquitin and amido.

6. Cosmetic composition comprising a peptide as defined in any one of the preceding claims 1 to 5 and at least one cosmetically acceptable excipient for the treatment and prevention of skin ageing and photoaging.

7. The cosmetic composition for use according to claim 6, wherein the composition comprises the peptide or derivative and/or salt thereof in an amount ranging from 0.00000001% to 20% by weight, preferably from 0.000001% to 15% by weight, more preferably from 0.0001% to 10% by weight, and even more preferably from 0.0001% to 5% by weight.

8. The cosmetic composition for use according to claim 6, wherein the at least one cosmetically acceptable excipient is selected from cosmetically acceptable carriers, volatile and non-volatile solvents, water, surfactants, preservatives, absorbents, chelating agents, lubricants, humectants, water repellents, antioxidants, ultraviolet light absorbers, anti-irritants, vitamins, trace metals, antimicrobials, fragrances, dyes, and coloring ingredients and/or structurants.

9. The cosmetic composition for use according to claim 8, wherein the cosmetically acceptable carrier comprises a solution, an aerosol, an oil-in-water emulsion, a water-in-oil emulsion, a gel, a solid, and a liposome.

10. The cosmetic composition for use according to claim 8, wherein the cosmetic composition is in the form of a lotion, cream, gel, stick, spray, ointment, paste, mousse, and cosmetic.

11. A non-therapeutic method for the treatment and prevention of skin aging and photoaging in a human in need thereof, comprising the topical application of a cosmetic composition comprising a peptide as defined in any one of the preceding claims 1 to 5 and at least one cosmetically acceptable excipient.

12. The non-therapeutic method of claim 11, wherein the method comprises pre-treating skin by Low Frequency Sonophoresis (LFS).

Technical Field

The present invention relates to peptides for cosmetic applications. In particular, the present invention relates to peptides having seq.id 1, which are capable of reducing skin aging and photoaging. More specifically, the invention also relates to a cosmetic composition comprising a peptide having seq.id No.1 and at least one cosmetically acceptable excipient.

Background

Nerve Growth Factor (NGF) is a neurotrophic factor and neuropeptide, primarily involved in the regulation of the growth, maintenance, proliferation and survival of certain target neurons.

NGF was discovered by professor Rita Levi-Montalciini at the Institute of Zoology, St.Louis, University of St.Louis, the Zoolology Institute of the Washington University of St.Louis (Levi-Montalciini R., Harvey Lect.,60:217,1966), and its discovery represents a significant progress in the study of the mechanism of growth and differentiation of neurons, since NGF can affect the development and maintenance of neuronal biological functions and neuronal regeneration. As this molecule was discovered and its biological functions in both the peripheral and central nervous systems were characterized, professor r. Numerous in vitro and in vivo studies have shown the pathophysiological importance of NGF in preventing neuronal damage of surgical, chemical, mechanical and ischemic nature, making it an ideal candidate for the treatment of a variety of conditions of the peripheral and central nervous systems (Hefti f., j.neurobiol.,25:1418,1994; Fricker j., Lancet,349:480,1997). Indeed, since many years ago, clinical trials have been conducted on Parkinson's Disease and Alzheimer's Disease patients by intracerebral administration of murine NGF (see, e.g., Olson L. et al, J. neural trains: Parkinson's Disease and Dementia Section,4:79, 1992). The results of these studies confirm the observations made in animal models and suggest that there are no possible side effects following administration of murine NGF. This feature was subsequently confirmed for human recombinant NGF (Petty B.G. et al, Annals of Neurology,36:244-246, 1994).

These events are mediated by NGF binding to its two cell surface receptors, TrkA and p 75. Co-expression of the two genes, the p75 receptor and the Trk NGF receptor, may lead to a greater response to NGF and suggest an additional level of regulation of the neurotrophic factor family (Chao MV, Hempstead BL., "p 75 and Trk: a two-receptor system," Trends neurosci.1995; 18: 321-6). Chick embryo sensory neurons display two distinct types of neurotrophin receptors, p75 and Trk, with dissociation constants of 10^-9M and 10^-11M, suggesting that only a low occupancy of higher affinity receptors is required to mediate The biological effects of neurotrophins (Meakin SO, Shooter EM, "The neural growth factor family of receptors," Trends neurosci.1992; 15: 323-31).

TrkA is a receptor with tyrosine kinase activity that forms a high affinity binding site for NGF.

p75 is a transmembrane protein without the recognizable cytoplasmic catalytic domain belonging to the Tumor Necrosis Factor (TNF) receptor superfamily, which forms the low affinity binding site for NGF.

International patent publications WO2004026329a1, WO2011116090, WO2011049758a1 and WO2005019266a2 relate to peptides or antibodies that interact with or bind to human Nerve Growth Factor (NGF) and neutralize NGF function, and to their use in the prevention and/or treatment of various diseases and disorders in which NGF activity is detrimental, including sunburn and vitiligo.

Contrary to what is suggested by the above-cited patent publications, international patent publication WO2013065078a2 discloses that topical application of a formulation containing NGF to the skin is effective in obtaining an enhancement of the skin colour, i.e. an increase in pigmentation, and an improvement in skin conditions involving a deficiency or hypopigmentation of the skin, such as in the case of vitiligo, in healthy skin not affected by dyschromatosis.

Peptide fragments comprising the 1-14 sequence of human NGF (NGF1-14) are known in the art as NGF mimetics as disclosed in some references (P.Di Pietro et al, "Immobilization of Neurosperm on Gold Nanoparticles by Direct and Lipid-Mediated Interaction: A New Multi cosmetic Nanoplatform for CNS Disorders", ACS Omega (2017),2(8), 4071-42-4079 e C.Satriano et al, "Neurosperm-binding Peptides at the bio tissue with Gold depression to copper stimulation", Physical Chemistry Physics (2016),18 (30595) 30604).

On the other hand, NGF1-14 was shown to interact only with TrkA receptors, in particular activating the PI3/Akt cascade and CREB phosphorylation, but not with p75 receptors and inducing ERK1/2 phosphorylation (A. Travaglia et al, "A small linear peptide peptides employing the NGF N-term polypeptide peptides PC12 cells", ACS Chem neurosci. 2015.8/19 days; 6(8): 1379-92).

Finally, WO99/39728 discloses that p75 signals apoptosis of keratinocytes and melanocytes when activated alone, and cell survival when activated together with Trk family of receptors.

Disclosure of Invention

Starting from the knowledge derived from the prior art (sometimes contradictory), the applicant has studied the potential use of NGF1-14 in cosmetic applications.

NGF1-14 is represented by the following sequence:

Seq.ID No.1：SSSHPIFHRGEFSV

in extensive experiments, applicants have surprisingly found that NGF1-14 is able to promote the production of procollagen I and elastin, as well as activate melanocytes to produce melanin. This finding suggests the use of NGF1-14 in cosmetic compositions for the treatment and prevention of skin aging and photoaging.

In addition, applicants have found that the NGF1-14 peptide retained all of the rh-NGF effect required for dermatological use.

Another advantage discovered by applicants is that, in contrast to intact proteins, NGF1-14 can be readily synthesized by conventional peptide synthesis methods and can be stored under mild conditions for extended periods of time.

These advantages lead to a significant reduction in production costs and make NGF1-14 well suited and attractive for cosmetic applications.

Applicants have further discovered that some modifications can be made to the number and type of amino acids of NGF1-14 without substantially altering the activity of NGF 1-14.

Accordingly, a first object of the present invention relates to a peptide comprising seq.id No.1, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity with seq.id No.1, and derivatives or salts thereof, for use in the treatment and prevention of skin aging and photoaging.

Furthermore, a second object of the present invention relates to a cosmetic composition comprising a peptide comprising seq.id No.1, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity to seq id No.1, and derivatives or salts thereof, together with at least one cosmetically acceptable excipient for use in the treatment and prevention of skin ageing and photoaging.

A third object of the present invention relates to a non-therapeutic method for treating and preventing skin aging and photoaging in a human in need thereof, comprising the topical application of a cosmetic composition comprising a peptide comprising seq.id No.1, or a sequence having at least 85%, preferably at least 90%, more preferably at least 95% sequence identity to seq.id No.1, and derivatives or salts thereof, together with at least one cosmetically acceptable excipient.

Advantageously, the non-therapeutic method of the invention comprises pre-treatment of the skin by Low-Frequency Sonophoresis (LFS).

Drawings

FIG. 1 shows NGF1-14 at various concentrations and reference Negermin^TMActivity profile of compound (fig. 1A) and EC50 curve (fig. 1B), as described in example 1.

Figure 2 shows the treatment protocol for a skin region of interest by the experiment described in example 2.

Figure 3 shows the levels of NGF1-14 on the epidermis (figure 3A) and dermis (figure 3B) measured after the treatment described in example 2.

Figure 4 shows the detected phosphorylation levels of TrkA receptor after the treatment described in example 2.

Figure 5 shows the levels of procollagen I (figure 5A) and elastin (figure 5B) in the dermis of rats measured after a single administration test as described in example 3.

Figure 6 shows the levels of procollagen I (figure 6A) and elastin (figure 6B) in the dermis of rats measured after the two-week administration test described in example 3.

Detailed Description

A first object of the present invention relates to a peptide comprising seq.ID No.1 for use in the treatment and prevention of skin aging and photoaging.

The seq.id No.1 according to the invention is represented by the sequence SSSHPIFHRGEFSV.

The abbreviations for the amino acid sequences used herein correspond to the IUPAC-IUB nomenclature reported in Table A below.

TABLE A

When optimally aligned, the peptide according to the invention may have at least 85%, at least 90% and at least 95% sequence identity with seq.id No. 1. Optimal alignment of sequences can be performed by various known methods and computerized implementations of known algorithms (e.g., BLAST, TFASTA, BESTFIT, see, e.g., Wisconsin Genetics Software Package, Release 7.0, Genetics Computer Group, Madison, Wis.). The BLAST algorithm, software available through the national center for Biotechnology information (www.ncbi.nlm.nih.gov /), Altschul et al, mol. biol. (1990),215, 403-.

"percent sequence identity" with respect to a peptide sequence refers to the percentage of residues in the two sequences that are identical. Percent sequence identity (% SI) is calculated by the following formula:

％SI＝(nt-nd)x 100/nt

where nt is the number of residues in the base sequence and nd is when aligned toThe total number of residues that are not identical in the sequences that face the maximum number of amino acids. Thus, sequence SSSHPIFHRGDFSV has about 92.8% sequence identity with seq.id No.1 (nd 1, nt 14).

The peptide according to the invention may have the same length as seq.ID No.1 or a length of up to 16 amino acids.

Changes in the amino acid sequence of the peptide comprising seq.ID No.1 of the present invention include conservative substitutions of amino acids that do not affect the activity of the peptide. Substitutions capable of maintaining peptide activity were selected according to the following conditions: (a) efficacy in maintaining the backbone structure of the peptide (e.g., a sheet-like or helical three-dimensional structure) in the substitution region, (b) efficacy in maintaining the charge or hydrophobicity of the molecule in the target region, or (c) efficacy in maintaining the majority of the side chains.

Examples of conservative substitutions belong to the group consisting of: basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid addition), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, valine and methionine), aromatic amino acids (phenylalanine, tryptophan and tyrosine) and small amino acids (glycine, alanine, serine and threonine).

Amino acid substitutions that do not generally alter specific activity are known in the art of the present invention.

The most common changes are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly, and vice versa. Additional examples of conservative substitutions are shown in table C below.

Watch C

Depending on the length of the peptide, the peptides of the invention may be synthesized by methods well known in the art, for example by automated peptide synthesizers, or produced by genetic engineering techniques. For example, a fusion gene encoding a fusion protein comprising a fusion partner and the peptide of the present invention is prepared by genetic engineering, and then transformed into a host cell to express the fusion protein. Thereafter, the peptide of the present invention is cleaved and isolated from the fusion protein using a protease or a compound, thereby producing the desired peptide. To this end, a DNA sequence encoding an amino acid residue which can be cleaved by a protease (such as factor Xa or enterokinase) or a compound (such as CNBr or hydroxylamine) may be inserted between the polynucleotide encoding the fusion partner and the peptide of the invention.

The cosmetic composition of the invention comprises a peptide comprising seq.ID No.1 and at least one cosmetically acceptable excipient.

The cosmetic composition of the present invention may comprise an amount of peptide or derivative and/or salt thereof in the range of from 0.00000001% to 20% by weight, preferably from 0.000001% to 15% by weight, more preferably from 0.0001% to 10% by weight, even more preferably from 0.0001% to 5% by weight.

The cosmetic compositions of the present invention may contain a variety of other optional components suitable for making such compositions more cosmetically or aesthetically acceptable, or for providing them with additional use benefits. Such conventional optional ingredients are well known to those skilled in the art. These include any cosmetically acceptable ingredients such as those found in "CTFA International Cosmetic Ingredient Dictionary and Handbook", edited by Wenninger and McEwen (7 th edition) (The Cosmetic, Toiletry, and france Association, inc., Washington, d.c., 1997). As used herein, "cosmetically acceptable" refers to a material (e.g., a compound or composition) suitable for use in contact with skin, hair, or other suitable substrate as defined below.

Cosmetically acceptable ingredients useful in the present invention include cosmetically acceptable carriers, volatile and non-volatile solvents, water, and other additional ingredients such as surfactants, preservatives, absorbents, chelating agents, lubricants, humectants, water repellents, antioxidants, ultraviolet absorbers, anti-irritants, vitamins, trace metals, antimicrobials, fragrances, dyes, and coloring and/or structuring agents.

As used herein, the expression "cosmetically acceptable carrier" refers to one or more compatible solid or liquid fillers, diluents, extenders and the like, which are cosmetically acceptable as defined above. As used herein, the term "compatible" means that the components of the compositions of the present invention are capable of being combined with the primary active of the present invention and with each other in a manner that does not interact with each other in a manner that would substantially reduce the efficacy of the composition under normal use conditions.

The type of carrier used in the present invention depends on the type of product desired. The compositions useful in the present invention may be in a variety of product forms. These include, but are not limited to, lotions, creams, gels, sticks, sprays, ointments, pastes, mousses, and cosmetics (e.g., solid, semi-solid, or liquid toiletries, including foundations).

These product forms can contain several types of carriers including, but not limited to, solutions, aerosols, emulsions (including oil-in-water or water-in-oil), gels, solids, and liposomes.

Depending on the form of the composition, the composition of the invention may comprise varying amounts of water. The amount of water, if present, may range from less than 1% to greater than 99% by weight, compared to the total composition weight. The aqueous compositions of the invention are especially formulated as aqueous lotions or as water-in-oil or oil-in-water emulsions or built emulsions (oil-in-water-in-oil or water-in-oil triple emulsions). Such emulsions are known, for example, from C.FOX, described in "Cosmetics and Toiletries", 11.1986, Vol.101, p.101-112.

Solid compositions, spray compositions and water-in-oil creams generally comprise less than 10% by weight, more preferably less than 5% by weight, of water relative to the total weight of the composition. The roll-on compositions, aqueous compositions, and deodorants typically comprise from about 15% to about 99%, more preferably from about 30% to about 90%, even more preferably from about 50% to about 80%, by weight, of water, relative to the total weight of the composition.

The compositions of the present invention may comprise one or more volatile solvents. If present, the volatile solvent or solvent mixture is generally present in an amount of from about 10% to about 90%, more preferably from about 25% to about 75%, even more preferably from about 35% to about 65% by weight, relative to the total weight of the composition. The solvent which can be used in the present invention is preferably an organic volatile solvent.

As understood by one of skill in the art, "volatile" as used herein refers to a substance having a significant amount of vapor pressure at ambient conditions.

The volatile solvent used in the present invention preferably has a vapor pressure of about 2kPa or higher, more preferably about 6kPa or higher, at 25 ℃. The volatile solvents used in the present invention preferably have a boiling point of less than about 150 deg.C, more preferably less than about 100 deg.C, even more preferably less than about 90 deg.C, and even more preferably still less than about 80 deg.C under normal atmosphere (1 atm). Preferably, the volatile solvents used in the present invention will be relatively odorless and safe for use on human skin. Suitable volatile solvents include, but are not limited to, C1-C4 alcohols, volatile silicones, and mixtures thereof. Preferred volatile solvents are C1-C4 alcohols and mixtures thereof. More preferred for use in the present invention is ethanol.

The compositions of the present invention may also comprise one or more non-volatile solvents. If present, the non-volatile solvent or solvent mixture is generally present in an amount of from about 1% to about 20%, more preferably from about 2% to about 10%, even more preferably from about 3% to about 5% by weight, relative to the total weight of the composition. Suitable non-volatile solvents include, but are not limited to, benzyl benzoate, cetearyl alcohol, cetyl alcohol, diethyl phthalate, isopropyl myristate, dimethicone, octyl methicone (captylmethicone), and mixtures thereof.

Several other additional ingredients may be present in the compositions of the present invention. These include, but are not limited to: a hydrophilic polymer selected from the group consisting of polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), Hydroxypropylmethylcellulose (HPMC), and poloxamer; UV stabilizers, such as benzophenone-3; antioxidants, such as tocopheryl acetate; preservatives, such as phenoxyethanol, benzyl alcohol, methyl paraben, propyl paraben; pH adjusters such as lactic acid, citric acid, sodium citrate, succinic acid, phosphoric acid, sodium hydroxide, sodium carbonate; deodorants and antimicrobials such as farnesol, zinc phenolsulfonate and ethylhexylglycerin; humectants, such as tribehenamine (tribehenin), glycerin; skin conditioning agents, such as allantoin; coolants such as trimethyl isopropyl butanamide and menthol; hair conditioning ingredients such as panthenol, pantethine (pantethine), pantetheine (pantetheine), panthenol ethyl ether, and combinations thereof; propellants such as propane, isopropane, butane and isobutylene; general salts, such as potassium acetate and sodium chloride and mixtures thereof; fragrances and dyes.

If present, these additional ingredients are preferably present in an amount of less than 10%, more preferably less than 5% by weight relative to the total weight of the composition.

The peptide of the present invention can promote the production of procollagen I and elastin, and can activate melanocytes to produce melanin. The cosmetic compositions of the invention are therefore particularly useful for the treatment and prevention of skin ageing and photoaging.

The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1

In vitro Activity

NGF treatment of the rat pheochromocytoma cell line (PC12) resulted in c-fos mRNA and protein expression (J.Milbrandt, "New growth factor Rapid indeces c-fos mRNA in PC12 rate phytochromacytocells", Proc Natl Acad Sci U S A.1986, 7 months; 83(13): 4789-93).

Starting from the work of milbradt, applicants monitored activation of the NFG receptor by using a stably transfected clone (B9) with a luciferase reporter gene (PC12-Luci) driven with the human c-fos promoter. This clone was used to establish functional and quantitative assays to characterize the activity of NGF 1-14.

The experiments were performed in PC12-Luci cells, which endogenously express the NGF receptors TrkA and p 75. Maintaining the cells in a complete medium comprising:

DMEM-F12 mixture (DMEM-F12 Glutamax; Life Technologies, GIBCO 10565-

5% fetal bovine serum (FBS, Sigma Aldrich, F7524)

10% horse serum (HS, Sigma Aldrich, H1138)

1% penicillin-streptomycin (Life Technologies, GIBCO 15140-

For passage, cells were gently washed with PBS and then incubated with trypsin for 5 minutes at room temperature or 37 ℃. Media was added and cells were resuspended (6-8 times).

Experiments were performed in seven replicates (sestupplicate) in black 96-well plates pre-coated with 50 μ g/ml type I collagen.

PC-12-Luci cells were seeded at 25000 cells/well in 100. mu.l of complete growth medium. After 24 hours, the cell culture medium was carefully removed. The cells were then incubated with five concentrations (ranging from 30ng/ml to 300. mu.g/ml, 1:10 serial dilutions) of NGF1-14 in standard Tyrode buffer containing 0.1% BSA (50. mu.l/well) for 4 hours at 37 ℃. Reference Negermin was added in an amount of 30. mu.g/ml^TMThe compound (supplied by MimeTech Srl) was used as reference. Stock solutions of NGF1-14 were resuspended in sterile water, aliquoted and stored at-20 ℃. Mixing Negermin^TMStock solutions of compounds were resuspended in formulation buffer and stored at-20 ℃.

After incubation, the ground state luminescence level was monitored for 30 seconds by using an ensspire multimode plate reader (PerkinElmer). Then, the Triton-Luciferin mixture was mixed at a ratio of 1: a ratio of 1 was added to the cells (50. mu.l/well), resulting in cell lysis. The luminescence signal obtained was monitored for an additional 60 seconds.

The compositions of the above solutions and buffers were as follows:

preparation of buffer: 50mM NaH₂PO₄、100mM NaCl,pH 7.2，

Tyrode buffer: 130mM NaCl, 5mM KCl, 2mM CaCl in water₂、1mM MgCl₂、5mM NaHCO₃20mM HEPES, pH 7.4; sterile filtered and stored at 4 c,

bovine Serum Albumin (BSA), fatty acid free (Sigma, a 8806); liquid storage: 10% in Tyrode buffer; the mixture was aliquoted and stored at-20 c,

triton lysis stock: 25mM TRIS, 25mM Na₂HPO₄2mM DTT, 10% glycerol, 2% TRITON X-100; the mixture was aliquoted and stored at-20 c,

fluorescein stock solution: 20mM Tricine, 2.67mM MgSO₄0.1mM EDTA, 33.3mM DTT, 270. mu.M coenzyme A, 470. mu.M fluorescein, 530. mu.M ATP; aliquots were aliquoted and stored at-80 ℃.

All luminescence values are normalized with respect to the ground state luminescence level. The response values were calculated as the average of the recorded kinetic luminescence.

The data were further analyzed (e.g., dose response curves and EC50 values calculated) and graphed using GraphPad Prism (GraphPad Software, Inc.). The results are shown in FIG. 1, showing different concentrations of NGF1-14 and reference Negermin^TMActivity profile of compound (fig. 1A) and EC50 curve (fig. 1B). The results show that NGF1-14 increased the ground state luminescence level in a dose-dependent manner (Ec 50-290.95 ng/ml).

Taken together, these results indicate that NGF1-14 is functionally active, showing in vitro potency above the reference standard Negermin^TMCompound, but less than rhNGF (Ec50 rhNGF 1.4 ng/ml). The reduced efficacy is consistent with the proposed cosmetic use.

Example 2

Preclinical study

Profiling experiments were also performed to assess the ability of NGF1-14 gel formulations to penetrate the stratum corneum (cornual stratum) and reach the dermis by promoting skin penetration using strategies similar to sonophoresis.

Tape stripping and sonication were performed on shaved back areas of adult female Sprague Dawley (SD) rats (10-12 months of age).

Since prolonged use of sonophoresis is known to increase skin permeability, but also causes considerable thermal damage, it is not suitable (see International Journal of Health Sciences & Research 477 Vol.5; Issue: 5; 5. 2015. A Review on Ultrasonic Parameters and Methods of Application in Transdermal Drug Delivery), and therefore, according to conventional clinical practice, Low Frequency Sonophoresis (LFS) was applied using sonic Ultrasonic Processor Model VCX 400 operating at 20KHz (tip displacement of about 2cm, continuous mode) for 6 or 10 minutes.

Vehicle control and NGF1-14 gel formulations were applied to lfs(s) -pretreated skin portions or shaved/tape-peeled skin (NS).

Table 1 below reports the composition of the vehicle control and NGF1-14 gel formulations.

TABLE 1

The sodium hyaluronate-based hydrogel was prepared by dissolving sodium hyaluronate (manufactured by Contipro) having a molecular weight ranging from 50 to 150KDa in water.

Treatment was repeated two hours later and rats were sacrificed to dissect skin samples 2 hours after the second gel/NGF mock administration.

The treatment protocol is shown in FIG. 2, where N and G represent regions not treated with LFS with NGF1-14 and vehicle gel, respectively, and NS and GS represent regions treated with LFS plus NGF1-14 and vehicle gel, respectively. NL and GL denote skin areas adjacent to the LFS area without overlap.

The epidermis and dermis of the sonicated (S) and non-sonicated (NS) skin sections were separated using a lancet, washed with PBS and cut into two parts. The former were stored in sterile tubes at-80 ℃ for processing for total protein extraction and biochemical analysis.

One skin section was immersed in 4% PFA for treatment for subsequent histological and immunohistochemical evaluation.

Epidermal and dermal protein extracts were used to assess the effect of LFS application on the ability of NGF1-14 gel formulations to penetrate the stratum corneum and reach the dermis.

The quantitation of NGF1-14 was determined by HPLC-UV on a reversed phase C4 column eluted with phosphate buffer (pH 7.4) and acetonitrile. The HPLC-UV method was validated for linearity, reproducibility and recovery.

The results are shown in fig. 3A and 3B. Significant increases in NGF1-14 were observed in LFS epidermis and dermis compared to treatment with vehicle. The efficacy of NGF mimetic/LFS use was confirmed by analyzing the expression level and activation of NGF survival/trophic receptor TrkA.

The effect of NGF (1-14) on TrkA phosphorylation was tested by western blot analysis of protein extracts from PC12 cell cultures treated with the peptide using antibodies against P-TrkA according to conventional laboratory practice.

As shown in figure 4, the phosphorylation level of TrkA was elevated in all dermal samples administered with NGF1-14, with or without LFS, when compared to the relevant vehicle control.

Example 3

Skin aging and photoaging

Since proteins of extracellular matrix collagen and elastin are involved in wound repair, skin aging and photoaging, the effects of topical administration of NGF1-14 on the levels of these two proteins were also analyzed.

Procollagen I and elastin were measured by using a commercially available Elisa kit, first in a single administration test, then a two week administration test.

The results of the single administration test are summarized in fig. 5. By analyzing these two proteins, a similar trend was observed: after sonication, an increase in both procollagen I (A) and elastin (B) was detected in the dermis of rats treated with 0.1% dose of NGF 1-14. If no ultrasound is used, the effect is less pronounced. Within experimental error, a slight increase in elastin was detected, while collagen I values were comparable to positive control values.

Single and two week administration test

NGF1-14 at a 0.1% dose (P0.1%) was evaluated for exposure to sub-erythema levels of UV-B (twice daily, MED 60 mJ/cm) first in a single administration test (one day) and then for three weeks prior to treatment²) The protective effect of adult skin. This experimental procedure is also called Photo-AGing model (PAG) because it induces structural and molecular changes that occur during AGing.

Topical treatment was performed on shaved dorsal skin of PAG and non-PAG rats for two weeks with P0.1% or vehicle gel. During the last week of treatment, UV-B exposure was also performed (once daily, MED 60 mJ/cm)²)。

The rats were sacrificed and the skin was dissected with a lancet, washed with PBS and cut into two parts. The former fraction was stored in sterile tubes at-80 ℃ for processing for total protein extraction and biochemical analysis. The latter portion was immersed in 4% PFA and treated for histological and immunohistochemical evaluation.

Consistent with the single administration study, photoaging induced a decrease in procollagen I in the dermis (fig. 6A) and detected elastin expression (fig. 6B).

Although this effect was more pronounced in healthy skin, application of P0.1% resulted in an increase in procollagen I in both non-PAG and PAG dermis (fig. 6A). The change in elastin concentration is less pronounced.

Example 4

Tanning effect

The effect of topical application of NGF1-14 on melanocyte activation to produce melanin has been evaluated.

According to an established protocol, primary cell cultures of normal human melanocytes were seeded in petri dishes and cultured for one week in the presence and absence of various concentrations of NGF 1-14. Melanin content was determined by enzyme immunoassay ELISA. An increase was observed in peptide-rich cultures.

Example 5

Skin irritation test

The skin irritation test is performed by applying an assay protocol tailored for skin cosmetic applications (i.e., MTT colorimetric assay and LDH release assay).

The MTT colorimetric assay detects the activity of the mitochondrial enzyme succinate dehydrogenase, which is active only in living cells and reacts with MTT (yellow) to form a blue salt, whose optical density (ODn) is proportional to the number of living cells and is quantified spectrophotometrically at a wavelength of 570 nm.

The LDH release assay measures the amount of lactate dehydrogenase (a cytoplasmic enzyme) in the medium, the release of which implies a loss of integrity of the cell membrane. The amount was quantified spectrophotometrically at a wavelength of 490nm with the aid of specific reagents.

The test was performed in EPI-100-ASY assay medium at EpiDerm^TMTwo samples (1 and 2) of NGF1-14 gel formulation were performed in triplicate at a dose of 0.3% (three times the active dose (0.1%) on tissues (MatTek Corporation, Bratislava, Slovakia). A1% Triton X-100 solution was used as a positive control. As a negative control, a gel matrix without peptide was used. Positive and negative controls were run in duplicate. The samples were exposed overnight for 18 hours.

The test results are summarized in table 2(MTT) and table 3(LDH) below.

TABLE 2

	Mean value (OD). + -. SD	Normalized value. + -. SD
			Negative control	2.0±0.1	100.0±3.1
Positive control	0.7±0.0	33.3±2.0
			Sample 1	1.8±0.0	91.6±1.8
Sample 2	1.9±0.1	99.3±3.8

TABLE 3

	Mean value (OD). + -. SD	Normalized value. + -. SD
			Negative control	0.13±0.00	1.0±0.0
Positive control	1.04±0.04	8.0±0.3
			Sample 1	0.24±0.04	1.9±0.3
Sample 2	0.14±0.05	1.1±0.4

In the MTT assay, a sample is considered "non-irritating" when the viability is above 70% relative to a negative control. In the LDH assay, a sample is considered "non-irritating" when the LDH release value is less than five times the negative control registration value. Both samples 1 and 2 were then considered "non-irritating".

Example 6

Skin sensitization test

The skin sensitization test was performed by performing the IL-18 release assay using the Elisa kit.

The IL-18 release assay measures the amount of interleukin 18(IL-18) in the medium. IL-18 is a proinflammatory cytokine with a key role in inducing allergic contact sensitization. This amount was quantified by direct ELISA, i.e. the color development was directly proportional to the amount of cytokine in the medium.

The test was performed in EPI-100-ASY assay medium at EpiDerm^TMTwo samples (1 and 2) of NGF1-14 gel formulation were performed in triplicate at a dose of 0.3% (three times the active dose (0.1%) on tissues (MatTek Corporation, Bratislava, Slovakia). A0.15% DNCB (2, 4-dinitrochlorobenzene) solution was used as a positive control. A gel matrix without peptide was used as a negative control. Positive and negative controls were run in duplicate. The samples were exposed overnight for 18 hours.

Table 4 below summarizes the results of this test.

TABLE 4

	Mean value (pg/ml). + -. SD	Normalized value. + -. SD
			Negative control	27.0±3.8	1.0±0.1
Positive control	252.3±16.4	9.3±0.6
			Sample 1	38.3±5.6	1.4±0.2
Sample 2	45.6±8.2	1.7±0.3

In the IL-18 release assay, a sample is considered "desensitized" when the IL-18 release value is less than twice the negative control registration value. Both samples 1 and 2 were then considered "non-sensitizing".

Example 7

NGF1-14pKa and Log P and D characterization

Experimental pKa and partition and distribution coefficients (log P and log D) were determined using an automated potentiometric titrator Sirius T3(Sirius Analytical Instruments ltd., East suslex, UK) equipped with an Ag/AgCl dual junction reference pH electrode, a Sirius D-PAS spectrometer and a turbidity sensing device. The pH electrode was titrated in the pH range 1.8-12.2. An overhead stirrer was used and a temperature probe monitored the temperature during titration. Titration experiments were carried out in 0.15M KCl solution (ISA water) at 25 + -1 deg.C under argon atmosphere. All tests were performed using standard 0.5M KOH and 0.5M HCl as titrants, and saturated octanol in ISA water (5% ISA water) was used as partitioning solvent for partition coefficient tests. pKa was determined potentiometrically by pH-titration. The powder (ca. 0.5mg) was dissolved in 1.5ml of ISA water and titrated in triplicate at a pH ranging from 2.0 to 12.0. LogP was performed in triplicate by dissolving the powder (approximately 0.7mg) in 1ml ISA water, followed by pH metric titration in three different percentages of octanol (typically 50%, 60%, 70%). Predicted pKa and calculated log P for the instrument model were performed using ACD/Percepta.

The results are shown in table 5 below. Since peptides are never neutral molecules, Log P values must be considered as relative values.

TABLE 5

Acidic pKa	4.42±0.05(n＝50)
		Basic pKa	7.41±0.07(n＝50)
Basic pKa	6.43±0.06(n＝50)
		Log P(w/o)	-8.50
Log D(7.4)	-7.90

The present invention has been disclosed with particular reference to certain specific embodiments thereof, but it will be understood that modifications and variations can be effected by those skilled in the art without departing from the scope of the invention as defined by the appended claims.

Sequence listing

<110> C4T llc, abbreviated as roman union chemical centers, llc

<120> peptides for treating and preventing skin aging and photoaging

<130> COL3P1WO

<160> 1

<170> BiSSAP 1.3.6

<210> 1

<211> 14

<212> PRT

<213> Artificial sequence

<220>

<223> none

<400> 1

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val

1 5 10