EA044414B1 - PEPTIDES WITH INHIBITORY ACTIVITY AGAINST NEURONAL EXOCYTOSIS - Google Patents

Description

The invention relates to peptides capable of inhibiting neuronal exocytosis, and to products containing such peptides, in particular to pharmaceutical and cosmetic products useful for improving conditions, disorders and/or diseases of the skin mediated by neuronal exocytosis.

State of the art

Acetylcholine (ACh) is a fast-acting positional neurotransmitter at the neuromuscular junction, autonomic ganglia, glandular innervation, and various parts of the central and peripheral nervous system. ACh is contained in the secretory vesicles of presynaptic cholinergic fibers and is released into the synaptic cleft, acting on cholinergic receptors on postsynaptic membranes. Neuronal exocytosis is a C ²⁺ -dependent process triggered by action potentials and controlled by proteins of the soluble N-ethylmaleimide-sensing factor attachment protein receptor (SNARE) family of proteins. NSF (Nethylmaleimide-sensitive factor) is a hexameric ATPase required for all steps of intracellular membrane trafficking. In its role in membrane trafficking, NSF is recruited to membranes by SNAPs (soluble NSF attachment proteins), which in turn are recruited by the SNARE protein complex formed during membrane fusion. This complex is formed by one vesicular SNARE, synaptobrevin or vesicle-associated membrane protein (VAMP), and two target plasma membrane SNAREs called syntaxin and SNAP25, which are involved in vesicle docking and membrane fusion with the plasma membrane (Kasai et al., Physiol Rev. , (2012), 92: 1915-1964).

SNARE protein complex-mediated vesicle docking and fusion are key elements controlling the secretion of any neurotransmitter (Purves et al., Neuroscience, 2nd edition, (2001)), as they have identical molecular mechanisms. Among them, like ACh, serotonin, histamine, GABA, glutamate, aspartate, ATP, epinephrine, norepinephrine, dopamine, epinephrine, norepinephrine or neuropeptides (calcitonin gene-related peptide (CGRP), substance P, neurokinins, VIP) are released through a SNARE-dependent mechanism , neurotrophins, endorphins).

Cleavage of the SNARE protein by botulinum toxins (BoNT) impairs vesicle fusion and neurotransmitter release (Binz et al., Toxins (Basel), (2010), 2 (4): 665-682). At the molecular level, botulinum toxins BoNT/A, /C and /E break down SNAP-25; BoNT/B, /D, /F, and /G cleave VAMP. Only BoNT/C is able to cleave both SNAP25 and syntaxin (Schiavo et al., Physiol Rev. (2000); 80: 717-66). Therefore, SNARE proteins have become targets for therapeutic and/or cosmetic compounds and/or products used for the treatment and/or prevention of conditions caused by neurotransmitter release. Indeed, approved botulinum toxin-based drugs block the release of neurotransmitters from presynaptic vesicles by deactivating SNARE proteins, mainly SNAP-25 or VAMP (Zakin et al., Toxicon (2018), EP 2318033 A2, EP 1856139 A2, EP 1180524 A1 EP 2123673 A1, WO 97/34620).

The skin interacts closely with the peripheral nervous system. Increasing evidence indicates that the neurological system is directly involved in many skin processes. For example, due to the inhibition of ACh release, the main cosmetic use of botulinum toxin-based products is the well-known anti-wrinkle effect of relaxing the facial muscles. However, in the skin, cholinergic fibers provide innervation to several other accessory structures in the integumentary system, including sweat glands, hair follicles, blood vessels, and muscles such as the arrector pili muscles. Indeed, botulinum toxin derivatives are used experimentally for a number of dermatological indications that include hyperhidrosis, scar prevention, skin inflammation, wound healing, facial redness, postherpetic neuralgia, sebum control and itching, with successful results (Kim et al., Toxins (2017) , 9, 403). The general mechanism underlying these new indications involves, in addition to ACh, inhibition of substance P, CGRP, glutamate and histamine release or even mast cell activation.

For example, in addition to classic thermoregulatory hyperhidrosis, improvement can be achieved in skin diseases with excessive sweating, such as dyshidrotic eczema or inflammatory dermatosis. Inhibition of ACh release prevents its direct action on the sweat glands and the smooth muscle surrounding the sweat glands (Swartling et al., J. Am. Acad. Dermatol. (2002), 47, 667-671; Wollina, U., J. Eur. Acad Dermatol Venereol (2002), 16, 40-42). In this regard, botulinum toxin has been shown to have a beneficial effect on dermatitis or psoriasis, skin diseases that are aggravated by excessive sweating, by reducing local sweating (Zanchi et al., J. Eur. Acad. Dermatol. Venereol. (2008), 22, 431-436). In addition, subsequent inhibition of neuropeptide, substance P, and/or CGRP release reduces associated itch and vasodilation (Humm et al., Exp. Neural. (2000), 161, 361-372, Ishikawa. et al., Jpn. J. Opthalmol (2000), 44, 106-109), which cause discomfort and worsen symptoms.

Inhibition of neuronal exocytosis reduces and prevents pruritus in several diseases and through different molecular mechanisms. ACh mediates itch in pruritic skin diseases such as atopic dermatitis (Hallett M., Ann. Neural. (2000), 48, 7-8). Substance P is associated with itching and aggravation due to the release of histamine upon activation of mast cells, and CGRP due to vasodilation. Therefore, corresponding disruption of neurotransmitter release by botulinum toxin reduces pruritus, both histaminergic and non-histaminergic types (Gazerani et al., Br. J. Dermatol. (2009), 161, 737-745). Pruritogenic itch is accompanied by skin inflammation and suppression of neuronal exocytosis, which reduces neurogenic inflammation, so itching in atopic dermatitis and psoriasis is reduced (Han et al., Dermatol. Surg. (2017); Ward et al., J. Investig. Dermatol. (2012 ), 132, 1927-1930; Saber et al., Arch. Dermatol. (2011), 147, 629-630; Gilbert et al., J. Drugs Dermatol. (2014), 13, 1407-1408; Gazerani et al. ., Br. J. Dermatol. (2009), 161,737-745; Cao et al., Neuroreport. (2017), 28, 518-526; Ramachandran et al., Toxins (2018), 10, pii: E134). Similarly, inflammatory skin dermatosis such as rosacea, characterized by facial redness and erythema, is improved by blocking the release of ACh, substance P and CGRP as cutaneous vasodilation and local skin inflammation are reduced. (Eshghi et al., Acta Med. Iran (2016), 54, 454-457; Bloom et al., Dermatol. Surg. (2015), 41 (Suppl. 1), S9-S16; Geddoa et al., Int. J. Dermatol. (2013), 52, 1547-1550; Odo et al., Dermatol. Surg. 2011, 37, 1579-1583).

In skin appendages such as the sebaceous glands, ACh increases lipid synthesis in sebocytes, and botulinum toxin significantly reduces sebum production in humans (Min et al., Aesthet. Surg. J. (2015), 35, 600-610; Rose et al. al., Dermatol. Surg. (2013), 39, 443-448). Therefore, the arrector pili muscles and local muscarinic receptors on the sebaceous glands are targets for neuromodulatory regulation through inhibition of ACh release.

Finally, inhibition of ACh release can be used to prevent and manage healing, and/or control the symptoms of hypertrophic scars. Decreasing local ACh release immobilizes the muscles surrounding the healing tissue and reduces skin tension. This process releases blocked nerve fibers in the keloid, counteracting the associated itching (Uyesugi et al., Am J Phys Med Rehabil. (2010), 89 (2): 153-155). In this regard, botulinum toxin has been shown to inhibit fibroblast proliferation, transforming growth factor-beta, collagen I and III, myosin II and α-smooth muscle actin in keloid fibroblasts (Xiao et al., Aesthet. Plast. Surg. (2011) , 35, 802-807; Chen et al., Ann. Plast. Surg. 2016, 77, e46-e49; Jeong al., Plast. Reconstr. Surg. (2015), 136, 171 e-178e; Wang, X . et al., Aesthet. Surg. J. (2014), 34, 154-159. Based on this, the potential effects of blockade of neuronal exocytosis on scars surrounding muscle and fibroblasts suggest its use for wound healing and scar prevention.

Thus, in general, inhibition and/or modulation of neurotransmitter release is useful not only in the case of facial wrinkles and motor muscle dysfunction, but also for the prevention, treatment or care of new skin-related pathological conditions such as excessive sweating, itching, skin inflammation, dermatitis, atopy, psoriasis, vascular hyperreactivity, rosacea, acne, hair growth, wound healing, calluses, warts or sensitive skin conditions such as ulcers and skin lesions.

Botulinum toxin-based treatments require repeated injections and may cause loss of effectiveness due to an immune reaction. Other side effects include cephalalgias, nausea, paralysis or muscle weakness, and respiratory failure. In addition, the lability and instability of pharmaceutical drugs make their treatment expensive. Thus, the development of simpler and more stable molecular structures to replace them is required. To this end, peptides derived from the primary structure of the core SNARE complex proteins are capable of disrupting the release of neurotransmitters.

A synthetic hexapeptide derived from the primary structure of the amino-terminal fragment of SNAP25 is widely used for the treatment and prevention of facial wrinkles, as described in EP1180524A1 and EP2123673A1. This peptide crosses the membrane and specifically interacts with SNAP25, thereby disrupting the assembly of the SNARE complex and the exocytosis of neurotransmitters.

Similarly, peptides derived from the carboxy-terminal region of SNAP25, or from synaptobrevin, or syntaxin, were also selected to inhibit neuronal exocytosis, as described in WO97/34620. However, for optimal activity they must have a minimum length of 20 amino acids and a maximum length of 28 amino acids. And their large length increases production costs and makes it difficult for further development as cosmetics and/or therapeutics.

Other peptides not directly derived from core SNARE complex proteins, with an unknown mechanism, have also been published to reduce neuronal exocytosis, as described in WO2013153192A1 and W02013070808A1. Similarly, peptides derived from the membrane component C subunit of V-ATPase are described in WO2011/048443 as inhibitors of neuronal exocytosis, targeting synaptobrevin and exhibiting potential anti-wrinkle effects.

In US patent No. 6169074 issued to Montal et al. (Montal, et al.), combinations of peptides are disclosed that interfere with the functioning of the SNARE complex at the synaptic cleft of the neuromuscular junction.

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In US Patent No. 6866856 issued to Liu et al. (Lu, et al.) describe limonoids (citrus alkaloid extracts) that inhibit the release of acetylcholine at the neuromuscular junction of skeletal muscles.

US Patent No. 7,566,464 to Belfer discloses a skin care composition that reduces fine lines and wrinkles on the human face. This product contains Acmella oleracea extract, which quickly relaxes the contractile muscle elements and suppresses the activity of the facial muscles based on the synergy of strengthening the dermis and suppressing muscle tissues associated with expression lines.

In US Patent No. 7015192 issued to Blaines et al. (Blanes, et al.), it was shown that peptides from the N-terminus of the SNAP-25 protein, which is part of the SNARE complex, inhibit the release of acetylcholine. The base molecule, acetylhexapeptide-8 (also formally called acetylhexapeptide-3 or ARGIRELINE®), is said to rival the effectiveness of botulinum toxin but reduce the risks associated with the latter's administration as well as the cost of production.

There are additional molecules that regulate the SNARE complex that are also important for neuronal exocytosis. For example, snapin is a SNAP25-binding protein that stabilizes the association between synaptotagmin 1 and the SNARE complex during Ca ^{2+ -triggered} exocytosis (Ilardi et al., Nat Neurosci. (1999) 2:119-124; Buxton et al., Biochem J. (2003) 375, 433-440). Removal of snapin does not completely eliminate neurotransmitter release, but rather reduces excitatory postsynaptic currents by 70% (Pan et al., Neuron. (2009) 61:412-424), indicating that snapin is an auxiliary modulator of neuronal exocytosis. The recombinant carboxyl terminus of snapin blocked the association of the SNARE complex with synaptotagmin. Indeed, peptides derived from the carboxyl terminus sequence of snapin disrupted the assembly of the SNARE complex, thereby impairing neuronal exocytosis (Ilardi et al., Nat Neurosci. (1999), 2:119-124). Only four 20-mer peptide fragments corresponding to the C-terminal coiled-coil domain, only from positions 117-136, inhibited exocytosis. Thus, snapin is a potential target for the development of exocytosis regulators rather than complete inhibitors, perhaps reducing the undesirable effects of more potent compounds.

In conclusion, this innovation represents an alternative to current needs and involves the identification of novel peptide sequences that are capable of reducing neuronal exocytosis.

The essence of the invention

The Applicant has unexpectedly discovered peptides capable of inhibiting or at least reducing the release of neurotransmitters, in particular acetylcholine and the neuropeptide CGRP, from neurons.

Even if the exact molecular mechanism has not yet been fully elucidated and confirmed, and without being bound by any theory, we believe that inhibition or reduction of exocytosis may be due to indirect modulation of SNARE complex formation through disruption of SNAP25 interaction.

The Applicant has discovered that peptides having the following SEQ ID NOs: 1-5 have the effect of blocking neuronal exocytosis, and therefore such peptides are capable of inhibiting or at least reducing the release of acetylcholine from peripheral nerve terminals.

Seq. ID No. 1 HYWRELQYR

Seq. ID No. 2 MQVWLRMWIDYRAT

Seq. ID No. 3 RRVVLVNNIL

Seq. ID No. 4 LRVQMVNMFL

Seq. ID No. 5 WEQEFLRR

The Applicant has also discovered that the effect of blocking neuronal exocytosis is also achieved with sequences having a length of no more than 20 amino acids and containing the above-described SEQ ID NO: 1-5 or a sequence having at least 70%, preferably at least 80% or more preferably at least 90% sequence identity to any one of SEQ ID NOs: 1-5.

The Applicant has also discovered that the effect of blocking neuronal exocytosis can be modulated by binding to the N-terminus of the above SEQ ID NOs: 1-5 an alkylcarbonyl group, such as, for example, an acetyl group, a palmitoyl group or a myristoyl group, as well as by forming a salt of the above SEQ IDs NO: 1-5 with a suitable anion such as, for example, chloride, acetate or trifluoroacetate.

Accordingly, the first aspect of the present invention relates to peptides having a length equal to or less than 20 amino acids, preferably equal to or less than 15 amino acids, and containing any one of SEQ ID NOs: 1-5, or a sequence having at least 70%, preferably at least 80% and more preferably at least 90% sequence identity with any one of SEQ ID NOs: 1-5, and a derivative or salt thereof.

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A second aspect of the present invention relates to a pharmaceutical or cosmetic composition comprising (i) a peptide having a length equal to or less than 20 amino acids, preferably equal to or less than 15 amino acids, and containing any one of SEQ ID NO: 1-5, or a sequence, having at least 70%, preferably at least 80%, and more preferably at least 90% sequence identity with any one of SEQ ID NOs: 1-5, and a derivative or salt thereof, and (ii) according to at least one pharmaceutically or cosmetically acceptable ingredient.

A third aspect of the present invention relates to the use of a peptide having a length equal to or less than 20 amino acids, preferably equal to or less than 15 amino acids, and containing any one of SEQ ID NOs: 1-5, or a sequence having at least 70%, preferably at least 80% and more preferably at least 90% sequence identity with any one of SEQ ID NOs: 1-5, and a derivative or salt thereof for ameliorating neuronal exocytosis-mediated conditions, disorders and/or diseases of the skin.

A fourth aspect of the present invention relates to a therapeutic or non-therapeutic method for improving conditions, disorders and/or diseases of the skin mediated by neuronal exocytosis, comprising topical application of a pharmaceutical or cosmetic composition containing (i) a peptide having a length equal to or less than 20 amino acids, preferably equal to or less than 15 amino acids, and containing any one of SEQ ID NO: 1-5 or a sequence having at least 70%, preferably at least 80%, and more preferably at least 90% sequence identity with any one of SEQ ID NOs: 1-5, and a derivative or salt thereof, and (ii) at least one pharmaceutically or cosmetically acceptable ingredient.

In particular, skin conditions, disorders and/or diseases mediated by neuronal exocytosis include wrinkles, excessive sweating, itching, skin inflammation, dermatitis, atopy, psoriasis, vascular hyperreactivity, rosacea, acne, hair growth, wound healing, calluses, warts or sensitive skin conditions such as ulcers and skin lesions.

A further aspect of the present invention relates to a polynucleotide that encodes a peptide having a length equal to or less than 20 amino acids, preferably equal to or less than 15 amino acids, and containing any one of SEQ ID NOs: 1-5, or a sequence having at least 70 %, preferably at least 80% and more preferably at least 90% sequence identity with any one of SEQ ID NOs: 1-5.

Detailed Description of the Invention

According to a first aspect, the present invention provides peptides having a length equal to or less than 20 amino acids, preferably equal to or less than 15 amino acids, and containing any one of SEQ ID NOs: 1-5, or a sequence having at least 70%, preferably , at least 80% and more preferably at least 90% sequence identity with any one of SEQ ID NOs: 1-5, and a derivative or salt thereof.

Preferably, the present invention relates to a peptide having a length equal to or less than 20 amino acids, preferably equal to or less than 15 amino acids, more preferably equal to or less than 10 amino acids, and containing SEQ ID NO: 1, or a sequence having at least 70 %, preferably at least 80% and more preferably at least 90% sequence identity to SEQ ID NO: 1, and a derivative or salt thereof.

Preferably, the present invention relates to a peptide having a length equal to or less than 20 amino acids, preferably equal to or less than 15 amino acids, and comprising SEQ ID NO: 2 or a sequence having at least 70%, preferably at least 80% and more preferably at least 90% sequence identity to SEQ ID NO: 2, and a derivative or salt thereof.

Preferably, the present invention provides a peptide having a length equal to or less than 20 amino acids, preferably equal to or less than 15 amino acids, more preferably equal to or less than 10 amino acids, and comprising SEQ ID NO: 3, or a sequence having at least 70 %, preferably at least 80% and more preferably at least 90% sequence identity to SEQ ID NO: 3, and to a derivative or salt thereof.

Preferably, the present invention provides a peptide having a length equal to or less than 20 amino acids, preferably equal to or less than 15 amino acids, more preferably equal to or less than 10 amino acids, and comprising SEQ ID NO: 4, or a sequence having at least 70 %, preferably at least 80% and more preferably at least 90% sequence identity to SEQ ID NO: 4, and a derivative or salt thereof.

Preferably, the present invention provides a peptide having a length equal to or less than 20 amino acids, preferably equal to or less than 15 amino acids, more preferably equal to or less than 10 amino acids, and comprising the sequence SEQ ID NO: 5, or the sequence having at least 70%, preferably at least 80%, and more preferably at least 90% sequence identity to SEQ ID NO: 5, and a derivative or salt thereof.

To the best of the knowledge of the listed inventors and the applicant of this application, none of the peptides described herein are known in the art prior to the priority date of this application. However, any peptide known in the art prior to the priority date of this application that falls within the scope of the present invention is accordingly excluded from this document.

The amino acid sequence abbreviations used herein correspond to the IUPAC-IUB nomenclature given in the following table. A.

Table A

Alanin Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G G istidine His H Isoleucine lie I Leucine Leu L Lysine Lys TO Methionine Met M Phenylalanine Phe F Proline Pro P Serin Ser S Threonine Thr T Tryptophan Trp w Tyrosine Tyr Y Valin Val V

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

where nt is the number of residues in the base sequence and nd is the total number of non-identical residues in the opposing sequence when aligned so that the maximum number of amino acids is identical. Accordingly, the sequence RKVVLVNQJL will have 80% sequence identity with SEQ ID NO: 3 RRVVLVNNIL (nd=2 and nt=10).

A peptide of the invention may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% sequence identity to a reference sequence when optimally aligned. Optimal sequence alignment can be accomplished by various known methods and by computerized implementation of known algorithms (eg, BLAST, TFASTA, BESTFIT, for example, in the Wisconsin Genetics software package, release 7.0, Genetics Computer Group, Madison, Wis.). The BLAST algorithm (Altschul et al., Mol. Biol. (1990), 215, 403-410) can also be used, software for which is available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).

The amino acid sequence variation in the peptides comprising SEQ ID NO: 1-5 of the present invention involves conservative amino acid substitutions that do not affect the activity of the peptide. Substitutions capable of preserving peptide activity are selected based on (a) effectiveness in maintaining the structure of the peptide backbone in the region of substitution, such as sheet or helical three-dimensional structures, (b) effectiveness in maintaining the electrical charge or hydrophobicity of the molecule in the target region, or (c) efficiency of maintaining the main part of the side chain.

Amino acids are classified according to their general side chain properties, as described in the following table. IN.

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Table B

hydrophobicity Norleucine, Met, Ala, Val, Leu, lie; neutral hydrophobicity Cys, Ser, Thr; acidity Asp, Glu; basicity Asn, Gln, His, Lys, Arg; residues that affect chain orientation Gly, Pro; aromaticity Trp, Tyr, Phe.

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), 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 are known in the art that generally do not change the specific activity.

The most common substitutions 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 opposite changes. Another example of conservative substitutions is shown in the following table. WITH.

Table C

Parent amino acid Possible replacement Preferred replacement Ala (A) Val; Leu; lie Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly (G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg He(I) Leu; Val; Met; Ala; Ph; norleucine Leu Leu (L) norleucine; He; Val; Met; Ala; Phe lie Lys (K) Arg; Gln; Asn Arg Met(M) Leu; Ph; lie Leu Phe (F) Leu; Val; lie; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Ph; Thr; Ser Phe Val(V) lie; Leu; Met; Ph; Ala; norleucine Leu

The peptide of the present invention may be in the form of a modified peptide, the N or/and C terminus of which is chemically modified or protected with organic compounds.

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The term derivative or derivative thereof, as used herein in relation to a peptide of the present invention, means a peptide in which its N- and/or C-terminus is chemically modified or protected with an organic compound.

Examples of modification include phosphorylation, glycosylation, acylation (including acetylation, lauroylation, myristorylation, palmitoylation), alkylation, carboxylation, hydroxylation, glycation, biotinylation, ubiquitinylation and amidation.

Preferably, the peptide of the present invention may be modified at its N-terminus, more preferably by acylation, including acetylation, lauroylation, myristorylation and palmitoylation. N-terminal acetyl and palmitoyl derivatives of the peptide are a preferred aspect of the present invention.

The term salt or a salt thereof, as used herein in relation to a peptide of the present invention, means a salt of the peptide or a derivative thereof with a suitable acid or base.

Typical examples of acids include, for example, hydrochloric acid, acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, methanesulfonic acid, p-toluenesulfonic acid, succinic acid, citric acid, tartaric acid and lactic acid.

Typical examples of bases include, for example, mono-, di- and trialkylamines, for example methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, propylamine, dipropylamine, tripropylamine, ethylenediamine, mono-, di- and trialkanolamines, for example monoethanolamine, diethanolamine and triethanolamine ; guanidine, morpholine, piperidine, pyrrolidine, piperazine, 1-butylpiperidine, 1-ethyl-2-methylpiperidine, N-methylpiperazine, 1,4-dimethylpiperazine, N-benzylphenylethylamine, Nmethylglucosamine and tris-(hydroxymethyl)aminomethane.

According to the present invention, the acetate or trifluoroacetate salt of the peptide or its derivative is preferably used.

Depending on its length, the peptide of the present invention can be synthesized by a method well known in the art, for example, using an automatic peptide synthesizer, or obtained using genetic engineering technology. For example, a fusion gene encoding a fusion protein comprising a fusion partner and a peptide of the present invention is genetically engineered and then transformed into a host cell to express the fusion protein. Thereafter, the peptide of the present invention is cleaved and separated from the fusion protein using a protease or compound to obtain the desired peptide. To this end, a DNA sequence encoding amino acid residues that can be cleaved by a protease, such as factor Xa or enterokinase, or a compound, such as CNBr or hydroxylamine, can be inserted between the polynucleotides encoding the fusion partner and the peptide of the present invention.

The peptides of the present invention may exist as stereoisomers or mixtures of stereoisomers; for example, the amino acids of which they are composed may be L-configured, D-configured, or may be racemic independently of each other. Therefore, it is possible to obtain isomeric mixtures, as well as racemates or diastereomeric mixtures, or pure diastereomers or enantiomers, depending on the number of asymmetric carbon atoms and which isomers or isomeric mixtures are present. Preferred structures of the peptides of the present invention are pure isomers, i.e. enantiomers or diastereomers. The most preferred structures of the peptides of the present invention include amino acids having the L configuration. Unless otherwise indicated, it is understood that if one amino acid is stated to be Ala, it is intended to be selected from L-Ala-, D-Ala- or racemic or non-racemic mixtures of both.

The cosmetic composition of the present invention contains at least one of the above-described peptides together with at least one cosmetically acceptable ingredient.

The pharmaceutical composition of the present invention contains at least one of the above-described peptides together with at least one pharmaceutically acceptable ingredient.

The pharmaceutical or cosmetic composition of the present invention may contain an amount of peptide or derivative and/or salt thereof 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.

The cosmetic composition of the present invention may contain a variety of other optional components suitable to make such compositions more cosmetically or aesthetically acceptable or to provide them with additional benefits in use. Such common optional ingredients are well known to those skilled in the art. This includes any cosmetically acceptable ingredients such as those contained in the CTFA International Dictionary and Reference of Cosmetic Ingredients, 7th ed., ed. Wenninger and McEwen

- 7 044414 (The Cosmetic, Toiletry and Fragrance Association, Inc., Washington, DC, 1997). As used herein, the term cosmetically acceptable means a substance (eg, compound or composition) that is suitable for use in contact with skin, hair, or other suitable substrate, as defined below.

Cosmetically acceptable ingredients used 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, UV absorbers radiation, anti-irritants, vitamins, rare earth metals, antimicrobial agents, fragrances, dyes and coloring ingredients and/or structuring agents.

The expression cosmetically acceptable carrier as used herein means one or more compatible solid or liquid fillers, diluents, bulking agents, and the like that are cosmetically acceptable as defined above. The term compatible as used herein means that the components of the compositions of the present invention can be combined with the parent active substances of the present invention and with each other in such a way that there is no interaction that would significantly reduce the effectiveness of the composition in normal use.

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

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

The compositions of the present invention may contain water in varying amounts depending on the form of the composition. The amount of water, if present, may range from less than 1% to more than 99% by weight, based on the weight of the entire composition. The aqueous composition of the present invention is specifically formulated as aqueous lotions or emulsions of water in oil or oil in water, or as multiemulsions (triple emulsions: oil in water in oil or water in oil in water). Such emulsions are known and described, for example, by C. FOX in Cosmetics and Toiletries. - November 1986. - t. 101. - p. 101-112.

Solid compositions, sprays and water-in-oil creams typically contain an amount of water of less than 10%, more preferably less than 5% by weight, based on the total weight of the composition. Roll-on compositions, aqueous compositions, and deodorant typically contain an amount of water 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, based on the total weight of the composition .

The compositions of the present invention may also contain silicones. If present, silicones will generally be present at a level of from about 30% to about 85%, more preferably from about 40% to about 75%, even more preferably from about 50% to about 65% by weight, based on the total weight of the composition .

The silicones used in the present invention are preferably linear or cyclic silicones containing from 2 to 7 silicon atoms, these silicones being optionally substituted with alkyl or alkoxy groups containing from 1 to 10 carbon atoms. Suitable silicones include dodecamethylcyclohexasiloxane, cyclopentasiloxane, decamethylcyclopentasiloxane, cyclotetrasiloxane, heptamethyloctyltrisiloxane, hexamethyldisiloxane, decamethyltetrasiloxane, dodecamethylpentasiloxane, octamethyltetrasiloxane, and mixtures thereof.

The compositions of the present invention may contain one or more volatile solvents. If present, the volatile solvent or solvent mixture will typically be present at a level 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 solvents used herein are preferably volatile organic solvents.

As used herein, the term volatile refers to substances with significant vapor pressure at ambient conditions, as understood by those skilled in the art.

Volatile solvents for use in the present invention will preferably have a vapor pressure of about 2 kPa or more, more preferably about 6 kPa or more at 25°C. Volatile solvents for use in the present invention will preferably have a boiling point at normal atmosphere (1 atm) of less than about 150°C, more preferably less than about 100°C, even more preferably less than about 90°C, even more preferably less than about 80°C.

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Preferably, volatile solvents for use herein 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 herein is ethanol.

The compositions of the present invention may also contain one or more non-volatile solvents. If present, the non-volatile solvent or solvent mixture will typically be present at a level 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, caprylyl methicone, and mixtures thereof.

Several other additional ingredients may be present in the compositions of the present invention. These include, but are not limited to, hydrophilic polymers selected from polyethylene glycols (PEG), polyvinylpyrrolidones (PVP), hydroxypropyl methylcellulose (HPMC) and poloxamers; UV stabilizers such as benzophenone-3; antioxidants such as tocopheryl acetate; preservatives such as phenoxyethanol, benzyl alcohol, methylparaben, propylparaben; pH adjusting agents 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 tribegenin, glycerin; skin conditioning agents such as allantoin; cooling agents such as trimethylisopropylbutanamide and menthol; hair conditioning ingredients such as panthenol, pantethine, pantotheine, panthenyl ethyl ether and combinations thereof; propellants such as propane, isopropane, butane and isobutene; common salts such as potassium acetate and sodium chloride and mixtures thereof; fragrances and dyes.

If present, these additional ingredients will preferably be present at a level of less than 10%, more preferably less than 5% by weight, based on the total weight of the composition.

Preferably, the pharmaceutical composition of the present invention is formulated in suitable dosage forms containing an effective amount of at least one of the above-described peptides together with at least one pharmaceutically acceptable ingredient.

Examples of suitable dosage forms are tablets, capsules, coated tablets, granules, solutions and syrups for oral administration; solutions, lipsticks and ointments for topical use; medicinal patches for transdermal administration; suppositories for rectal administration and sterile injection solutions. Other suitable dosage forms are sustained release and liposome based forms for oral, injectable or transdermal administration.

As described herein, the pharmaceutical composition of the present invention contains at least one of the above-described peptides together with a pharmaceutically acceptable excipient, which in the context of the present invention includes any solvents, diluents or other excipients, dispersion or suspension auxiliaries, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, depending on the particular dosage form desired.

Some examples of substances that can serve as a pharmaceutically acceptable excipient include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline solution; Ringer's solution; ethyl alcohol and phosphate buffer solutions, other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, dyes, release agents, coating agents, sweeteners, flavors and fragrances, preservatives and antioxidants.

The terms pharmaceutically acceptable and physiologically acceptable are intended to refer, without any particular limitation, to any substance suitable for the manufacture of a pharmaceutical composition for administration to a living being.

Dosage forms may also contain other conventional ingredients such as preservatives, stabilizers, surfactants, buffers, osmotic salts, emulsifiers, sweeteners, colors, flavors, and the like.

The pharmaceutical compositions of the present invention can be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or through

- 9 044414 implanted reservoir. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

The pharmaceutical compositions of the present invention can also be administered via nasal aerosol or inhalation, or delivered by implantation (eg, surgery), for example, using an implantable or permanent device such as a stent.

Dosage forms of the pharmaceutical composition of the present invention can be prepared by methods known to the pharmaceutical chemist and include mixing, granulating, compressing, dissolving, sterilizing and the like.

The peptides of the present invention are capable of inhibiting or at least reducing the release of neurotransmitters, in particular acetylcholine and the neuropeptide CGRP, from neurons.

Accordingly, a further aspect of the present invention relates to the use of at least one of the above-described peptides and a derivative or salt thereof for the improvement of neuronal exocytosis-mediated conditions, disorders and/or diseases of the skin.

As discussed above, this property can be used not only to treat facial wrinkles and motor muscle dysfunction, but also to reduce, prevent, or treat several skin-related conditions where the release of neuronal vesicles is involved, such as excessive sweating, itching, inflammation skin, dermatitis, atopy, psoriasis, vascular hyperreactivity, rosacea, acne, hair growth, wound healing, calluses, warts or sensitive skin conditions such as ulcers and skin lesions.

In other words, conditions, disorders and/or diseases of the skin mediated by neuronal exocytosis include facial wrinkles, motor muscle dysfunction, excessive sweating, itching, skin inflammation, dermatitis, atopy, psoriasis, vascular hyperreactivity, rosacea, acne, hair growth, healing wounds, calluses, warts or sensitive skin conditions such as ulcers and lesions on the skin, etc.

Accordingly, the present invention also relates to a therapeutic or non-therapeutic method for improving skin conditions, disorders and/or diseases mediated by neuronal exocytosis, in particular for reducing wrinkles, excessive sweating, itching, skin inflammation, dermatitis, atopy, psoriasis, vascular hyperreactivity, rosacea , acne, hair growth, wound healing, calluses, warts or sensitive skin conditions such as ulcers and skin lesions, including topical application of a pharmaceutical or cosmetic composition containing (i) at least one of the above-described peptides and their derivatives or salts, and (ii) at least one pharmaceutically or cosmetically acceptable ingredient.

An additional aspect of the present invention relates to a polynucleotide that encodes at least one of the above-described peptides.

The above polynucleotide allows the peptides of the present invention to be produced in large quantities. For example, the cultivation of vectors that include polynucleotides encoding peptides allows the production of peptides in large quantities.

A polynucleotide is a nucleic acid molecule, which may be natural or artificial DNA or RNA molecules, single-stranded or double-stranded. A nucleic acid molecule may be one or more nucleic acids of the same type (eg, having the same nucleotide sequence) or nucleic acids of different types. Nucleic acid molecules include, but are not limited to, one or more DNA, cDNA, decoy DNA, RNA, siRNA, siRNA, shRNA, mvRNA, snoRNA, snRNA, PNA, antisense oligomer, plasmid, and other modified nucleic acids.

The following examples are intended to better illustrate the present invention, but do not limit it.

Examples

Example 1. Chemical synthesis.

All peptides were synthesized with C-terminus amidation using a standard solid-phase Fmoc method (Perez de la Vega et al., Molecules (2010), 15:4924-4933; Behrendt et al., J. Pept. Sci. (2016) , 22(1):4-27; Made et al., Beilstein J Org Chem (2014), 10: 1197-1212). Synthesis of the inventive peptides, mixtures and/or cosmetically acceptable salts thereof can be carried out by conventional methods known in the art, such as solid-phase peptide synthesis methods, enzymatic synthesis or any combination (Bondazky et al., Int. J. Pept. Protein Res. (1993), 42(1): 103).

All synthesis methods were carried out with Kromasil-C18-HPLC (5 μm, 4.6x250 mm). The peptides were then eluted with linear gradients of acetonitrile (CH3CN) with trifluoroacetic acid (TFA) (gradient: 5-55% B over 2 min, flow: 1 ml/min, eluent A: 100% H ₂ O + 0.1% TFA; eluent B: 100% CH ₃ CN+0.l% TFA). Peptide detection was performed by measuring absorbance at 220 nm. The Fmoc group was removed with 20% piperidine/DMF solution during a 30-min reaction. Washings between stages were carried out with DMF (5 times). All synthesis and washing reactions were carried out at 25°C. HPLC analysis of the resulting peptides showed purity exceeding 80% in all cases. The identity of the resulting peptides was confirmed using ESI-MS.

Method for introducing the N-terminal acetyl group into peptidyl resins: 1 mmol (1 eq.) peptidyl resins were treated with 25 eq. pre-dissolved acetic anhydride in the presence of 25 eq. DIEA using 5 ml DMF as solvent. After 30 min of reaction, the peptide resins were washed with DMF (1 min x 5), DCM (1 min x 4) and diethyl ether (1 min x 4). Finally, the peptidyl resins were dried under vacuum.

Method for introducing the N-terminal palmitoyl group into peptidyl resins: 3 mmol (3 equiv.) of pre-dissolved palmitic acid was introduced into peptidyl resins in the presence of 3 equiv. FITBU and 6 eq. NMM. They were allowed to react for 30-60 min using DMF as a reagent. After this, the resins were washed 3 times with DMF.

The process of cleavage of peptidyl resins from the polymer carrier: dried peptidyl resins were treated with TFA:TIS:H ₂ O (95:2.5:2.5) for 2 hours at 25°C with vibration.

Example 2: Inhibition of CGRP release on cultured sensory neurons by peptides of the invention.

Induction of calcitonin gene-related peptide (CGRP) release by capsaicin allows direct measurement of neuronal exocytosis (Meng J. et al., J. Cell. Sci. (2007) 15; 120(Pt 16):2864-74, and Meng J. et al., Mol Neurobiol (2014);50(2):574-8).

The test peptides were evaluated by measuring their ability to inhibit induced CGRP release on peptidergic sensory neurons. Dorsal root ganglia were seeded (50,000 cells per well) in a 96-well plate precoated with poly-L-lysine and laminin. 48 hours after seeding, the cells were incubated with Hanks' Balanced Salt Solution containing the peptides of the invention at a concentration of 20, 50 or 100 mM for 1 hour. The cells were then stimulated for 10 minutes with 1 pM capsaicin at 37°C. CGRP content was then determined in the supernatants using a colorimetric CGRP EIA assay (Spi-Bio Inc) following the manufacturer's instructions. Absorbance measurements (405 nm) were standardized to the maximum signal detected upon capsaicin stimulation of vehicle-treated cells. CGRP release inhibition values were calculated as percentages based on the maximum signal for capsaicin-induced release and the minimum signal for unstimulated cells. In table D shows the M3 activity inhibition values obtained for the peptides of the invention.

Table D

Peptide Subsequence N-terminal derivative % release( 100 µM inhibition >birth of CGRP 20 50 µM µM Seq. ID No. 1 HYWRELQYR acetyl 39.1 - 10.1 Seq. ID No. 2 MQVWLRMWIDYRAT acetyl palmitoyl 55.3 53.9 49.3 35.5 Seq. ID No. 3 RRVVLVNNIL palmitoyl 74.8 34.0 Seq. ID No. 4 LRVQMVNMFL acetyl 77.4 14.2

The peptides inhibited capsaicin-induced CGRP release in the range of 10–50% at 20 μM, 50–75% at 50 μM for N-terminal palmitoylated, and 40–77% at 100 pM for N-terminal acetylated.

Example 3: Inhibition of acetycholine release by peptides of the invention in a neuroblastoma cell line.

A human neuroblastoma cell line (50,000 cells/well) was used to determine the effects of the compound of the invention on acetylcholine release. The cells differentiated into a cholinergic neuron phenotype within 4 days. Cells were then preincubated for 60 min with compounds (from 0.1 to 100 μM) in the form of acetylated or palmitoylated forms and conjugated with trifluoroacetate (TFA) or acetate salt. Thereafter, acetylcholine release was induced by membrane depolarization caused by a 15-min incubation with 50 mM KCl. Acetylcholine levels in the supernatants were quantified using the Amplex® Red Acetylcholine Assay Kit (Thermofisher) following the manufacturer's instructions. Acetylcholine is enzymatically transformed to form H2O2, which gives a quantitative fluorescent signal. Fluorescence (excitation 530 nm/emission 590 nm) was measured using FluorStar equipment. Acetylcholine content was normalized to total protein content using the BCA assay (Pierce) according to the manufacturer's instructions. Fluorescence measurements were normalized to the maximum signal detected upon KCl stimulation in vehicle-treated cells. Impairment of vesicle exocytosis by the compounds of the invention leads to a decrease in the release of acetylcholine (Table E).

Table E

% inhibition of A ch release Peptide N-terminal Sequence µM TFA acetate derivative

acetyl 100 50 10 65.9 33.6 87.9 55.9 1 - 27.9 OD 42.2 Seq. ID No. 1 HYWRELQYR 50 75.9 - 10 49.4 87.5 palmitoyl 5 7.9 - 1 - 81.3 o,1 - 42.9 20 10.9 - 10 78.8 55.8 acetyl 5 76.2 - 1 41.0 Seq. ID No. 2 MQVWLRMWIDYRAT od - 39.6 50 20.6 - palmitoyl 10 -35.1 - 5 -46.1 - 50 82.8 - 10 81.8 86.7 Seq. ID No. 3 RRVVLVNNIL palmitoyl 5 75.4 - 1 - 92.5 0.1 - 57.0 100 6.8 - acetyl 50 58.9 - Seq. ID No. 4 LRVQMVNMFL 10 84.3 - palmitoyl 50 83.0 -

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10 46.0 - 5 26.4 - 100 81.6 - 50 51.5 - acetyl 10 52.8 - 1 22.0 - Seq. ID No. 5 WEQEFLRR ο,ι 19.0 - 50 17.9 - 10 24.7 - palmitoyl 5 25.1 - 1 16.1 - ο,ι 16.4 -

All peptides were functionally validated as inhibitors of regulated neuronal exocytosis in a neuroblastoma cell line. Specifically, the acetylated form of SEQ ID NOs: 1, 2, 4 and 5 significantly reduced ACh release by 50-87% at 10 mM as the TFA salt and by approximately 40% as the acetate salt at 0.1 μM. Palmitoylated SEQ ID NO: 4 significantly reduced acetylcholine release by approximately 50% at 10 μM. The most effective peptide was palmitoylated SEQ ID NO: 3, as it was active at the lowest concentration tested, 0.1 μM. Overall, all effective peptides were in the micromolar range.

Example 4: Cell viability assays on human epidermal keratinocytes and dermal fibroblasts.

In this example, the effects of the peptides of the invention on human epidermal keratinocytes and dermal fibroblasts were assessed. Cell viability was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The MTT assay is a colorimetric reaction based on the ability of the enzyme mitochondrial dehydrogenase to degrade and convert the tetrazole rings of MTT. Epidermal keratinocytes (HEKa) were seeded into a precoated 96-well plate at 50-60% confluency in 100 μl of appropriate supplemented medium. Dermal fibroblasts (HDFa) were seeded in a 96-well plate at 70% confluency in 100 μl of supplemented medium. For both cell lines, 24 h after seeding, the medium was replaced with fresh supplemented medium containing peptides dissolved in a concentration range from 0.1 to 200 mM. All test substances were incubated for 24 hours at 37°C and 5% CO ₂ . After this, the medium was replaced with 0.5 mg/ml MTT solution for 4 h in complete medium. The medium was then carefully removed and 150 μL/well of DMSO was added to dissolve the formazan crystals. The plate was protected from light, shaken for 60 s, and absorbance was measured at 570 nm with a 620 nm reference filter. In table F, the effects of the peptides of the invention on the cell viability of keratinocytes and fibroblasts, expressed as percentage inhibition, are described in detail.

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Table F % inhibition of cell viability

Peptide Subsequence N-terminal derivative µM NEKa HDFa 200 9.5 2.9 100 6.2 6.8 acetyl 50 7.4 3.9 (acetate salt) 10 1.7 3.3 5 8.8 9.7 Seq. ID HYWRELQYR 1 -1.4 0.2 No. 1 50 37.5 31.6 10 31.0 29.2 palmitoyl 5 23.6 24.3 (acetate salt) 1 8.8 12.2 0.5 6.4 7.2 0.1 2.9 0.3 200 1.2 24.6 100 -0.7 7.8 acetyl 50 -4.6 1.4 (acetate salt) 10 -0.5 8.5 5 -1.8 12.3 Seq. ID MQVWLRMWIDYRAT 1 -1.2 10.1 No. 2 50 32.5 35.3 10 36.5 47.1 palmitoyl 5 36.9 47.3 (acetate salt) 1 25.6 45.8 0.5 20.9 42.9 0.1 12.1 30.8 Seq. ID RRVVLVNNIL palmitoyl 50 24.8 24.3

- 14044414

No. 3 (acetate salt) 10 22.4 27.2 5 9.9 25.7 1 11.7 8.7 0.5 -0.9 15.5 0.1 -3.8 6.0 Seq. ID LRVQMVNMFL No. 4 200 41.1 22.9 100 31.6 41.5 acetyl 50 19.5 40.6 (acetate salt) 10 6.0 37.1 5 2.6 28.4 1 13.6 14.5 50 34.2 37.7 10 18.0 40.9 palmitoyl 5 7.0 42.6 (TFA salt) 1 -7.6 34.2 0.5 1.0 26.9 0.1 -6.2 15.2 Seq. ID WEQEFLRR No. 5 200 23.7 14.7 100 10.4 15.0 acetyl 50 4.3 10.0 (TFA salt) 10 4.2 12.1 5 0.8 10.0 1 -5.7 9.3 50 -24.3 18.0 palmitoyl 10 -6.3 7.6 (TFA salt) 5 -5.5 10.3 1 -2.9 -0.2

Peptides SEQ ID NO: 1, 2 and 5 with N-terminal acetyl substitution did not alter cell viability in human epidermal keratinocytes and skin fibroblasts up to 200 μM. Acetylated and palmitoylated peptide SEQ ID NO: 4 did not alter keratinocyte viability at concentrations below 50 μM. Palmoylated SEQ ID NO: 3 did not alter the viability of keratinocytes and fibroblasts at 1 μM or lower.

Example 5. Evaluation of the antiperspirant effect after short-term administration in a mouse model of sweat secretion.

In this example, the short-term effects of the peptide of the invention SEQ ID NO: 1 in palmitoylated form were assessed in an in vivo model of pilocarpine-induced sweating. This model was established using pilocarpine, a non-selective muscarinic receptor agonist, in 11-week-old male C57BL6/Rcc mice. The test compound is a peptide of the invention having the following sequence (SEQ ID NO: 1): palmitoyl-HYWRELQYRNH ₂ .

The test compound was administered intraplantar (i.pl.) to the right hind paw (10, 30 and 100 μg) 30 min before sweat stimulation. The carrier was saline solution.

Sweating was monitored by determining amylase activity at the skin surface using the iodine-starch reaction. The number of dark sweat droplets was determined after 5 min by inductive counting of the number of droplets per paw in each condition. The sample consisted of n=5-6 individuals per group. Data are presented as mean ± standard error of the mean (SEM). Raw data were normalized as percentages relative to stimulated individuals that were not injected (control, 100%) and unstimulated individuals that received saline injections (vehicle, 0%). Statistical analysis was one-way ANOVA followed by post hoc Dunnett's multiple comparison test comparing each condition with its corresponding control group, ****p<0.0001; ***p<0.001;

**p<0.01; *p<0.05.

The results are presented in the following table. G.

Table G

Palm-SEQ ID NO: 1 % inhibition S.E.M. Statistics 10 mcg/paw 58.2 ±9.8 *** 30 mcg/paw 51.4 ±9.6 ** 100 mcg/paw 34.5 ±8.4 *

The test compound significantly reduced sweating at 10, 30 and 100 μg.

Example 6. Evaluation of the antiperspirant effect during chronic administration in a mouse model of sweat secretion.

In this example, the long-term effects of the peptide of the invention SEQ ID NO: 1 in pamitoylated form were assessed in an in vivo sweating model. This model was established using pilocarpine, a non-selective muscarinic receptor agonist, in 11-week-old male C57BL6/Rcc mice. The test compound was administered topically three times a week for 4 weeks (treatment). Sweating was induced by intraplantar (i.pl) injection of pilocarpine (3 μg/paw) into the right hind paw (sweating) once at weeks 1, 2, and 4 according to Table 1. N.

Table H

A week Monday Tuesday Wednesday Thursday Friday 1 treatment sweating treatment ___ treatment 2 treatment sweating treatment ___ treatment 3 treatment ___ treatment ___ treatment 4 treatment sweating treatment ___ treatment

The test compound is a peptide of the invention having the following sequence (SEQ ID NO: 1): palmitoyl-HYWRELQYR-NH ₂ .

The test compound was administered i.pl. into the right hind paw (1, 10, and 100 μg), and the vehicle was saline.

Sweating was monitored by determining amylase activity at the skin surface using the iodine-starch reaction. The number of dark sweat droplets was determined after 5 min by inductive counting of the number of droplets per paw in each condition. The sample consisted of n=5-6 individuals per group. Data are presented as mean ± standard error of the mean (SEM). Raw data were normalized as a percentage relative to stimulated individuals that were not injected (control, 100%). Statistical analysis was one-way ANOVA followed by post hoc Dunnett's multiple comparison test comparing each condition with its corresponding control group, ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05.

The results are shown in the table below. I.

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Table I

Palm-SEQ ID NO: 1 % inhibition S.E.M. Statistics Week 1 1 mcg/paw 2.6 ±9.7 ** 10 mcg/paw 11.2 ±9.7 100 mcg/paw 24.2 ±6.2 Week 2 1 mcg/paw 26.7 ±6.7 * 10 mcg/paw 27.3 ±5.4 100 mcg/paw 27.1 ±5.0 Week 4 1 mcg/paw -4.3 ± 11.9 * 10 mcg/paw 11.3 ±3.6 100 mcg/paw 25.2 ±3.2

Palm-SEQ ID NO: 1 showed significant suppression of sweating over 4 weeks of treatment when tested at a dose of 100 μg/paw.

Example 7 In Vitro Functional Assay for Neuronal Sensitization This example assessed the in vitro effects of the palmitoylated form of SEQ ID NO: 1 on nociceptor sensitization in the context of sensitive skin.

Isolated dorsal root ganglia from newborn Wistar rats (3-5 days old) were seeded onto microelectrode array chips in DMEM Glutamax, 10% FBS, 1% P/S supplemented with mouse NGF. All experiments were performed 48 h after cell seeding.

Sensory neurons were sensitized by short-term exposure to a proinflammatory cocktail of bradykinin, histamine, and ATP. TRPV1-mediated excitability was analyzed because tingling, burning and itching sensations are mainly caused by TRPV1. Repeated application of the TRPV1 agonist, capsaicin (15-second application of 500 nM), produced a first activation (P1) followed by desensitization (second application). Desensitized cultures were sensitized with a cocktail to restore excitability using an external solution between the second (P2) and third (P3) pulses of capsaicin (8 min). To confirm cell viability, 40 mM KCl was applied at the end. The test compound was incubated with cells for 1 h before monitoring at concentrations of 0.1, 1 and 10 mM. The test compound is a peptide having the following sequence (SEQ ID NO: 1): palmitoyl-HYWRELQYR-NH ₂ .

Sensitization was calculated as the ratio between capsaicin P3 and P1 pulses (P3/P1 ratio), representing the sensitized response induced by the proinflammatory cocktail. The percentage of inhibition was calculated as 100 - sensitization. Data were presented as mean ± standard error of the mean (SEM). Statistical analysis was one-way ANOVA followed by post hoc Dunnett's multiple comparison test comparing each condition with the corresponding sensitization control group, ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05.

The results are presented in the following table. J.

Table J

Παlm-HYWRELQYR-NH ₂ % inhibition sensitization S.E.M. Statistics 0.1 µM 69.7 2.7 **** 1 µM 51.2 4.7 **** 10 µM 59.7 3.3 ****

The test compound at three concentrations significantly inhibited the sensitization process with similar effectiveness.

Claims (20)

1. A peptide having a length equal to or less than 20 amino acids and containing the sequence SEQ ID NO: 1 or a sequence having at least 70% identity with the sequence SEQ ID NO: 1, and a derivative or salt thereof, wherein said derivative contains N - and/or the C-terminus of said peptide, which is chemically modified or protected with an organic compound. 2. A peptide having a length equal to or less than 20 amino acids and containing the sequence SEQ ID NO: 2 or a sequence having at least 70% identity with the sequence SEQ ID NO: 2, and a derivative or salt thereof, wherein said derivative contains N - and/or the C-terminus of said peptide, which is chemically modified or protected with an organic compound. 3. A peptide having a length equal to or less than 20 amino acids and containing the sequence of SEQ ID NO: 3 or a sequence having at least 70% identity with the sequence of SEQ ID NO: 3, and a derivative or salt thereof, wherein said derivative contains N - and/or the C-terminus of said peptide, which is chemically modified or protected with an organic compound. 4. A peptide having a length equal to or less than 20 amino acids and containing the sequence of SEQ ID NO: 4, or a sequence having at least 70% identity with the sequence of SEQ ID NO: 4, and a derivative or salt thereof, wherein said derivative contains The N- and/or C-terminus of said peptide, which is chemically modified or protected with an organic compound. 5. A peptide having a length equal to or less than 20 amino acids and containing the sequence of SEQ ID NO: 5 or a sequence having at least 70% identity with the sequence of SEQ ID NO: 5, and a derivative or salt thereof, wherein said derivative contains N - and/or the C-terminus of said peptide, which is chemically modified or protected with an organic compound. 6. A peptide according to any one of claims 1 to 5, wherein said peptide has a length equal to or less than 15 amino acids. 7. A peptide according to any one of claims 1 to 5, wherein said organic compound is selected from the group consisting of phosphoryl, glycosyl, acyl, alkyl, carboxyl, hydroxyl, biotinyl, ubiquitinyl and amido groups. 8. The peptide of claim 7, wherein said acyl group is selected from the group consisting of acetyl, lauroyl, myristoryl and palmitoyl groups. 9. The peptide according to any one of claims 1 to 5, where its N-terminus is chemically modified or protected with an acetyl or palmitoyl group. 10. A peptide according to any one of claims 1 to 5, wherein said salt is a salt of said peptide or a derivative thereof with a suitable acid or base. 11. The peptide according to claim 10, wherein said acid is selected from the group consisting of hydrochloric acid, acetic acid, trifluoroacetic acid, oxalic acid, maleic acid, methanesulfonic acid, p-toluenesulfonic acid, succinic acid, citric acid, tartaric acid and lactic acid acids. 12. The peptide according to claim 10, wherein said acid is selected from the group consisting of hydrochloric acid, acetic acid and trifluoroacetic acid. 13. The peptide according to claim 10, wherein said base is selected from the group consisting of mono-, di- and trialkylamines, for example methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, propylamine, dipropylamine, tripropylamine, ethylenediamine, mono-, di - and trialkanolamines, for example monoethanolamine, diethanolamine and triethanolamine; guanidine, morpholine, piperidine, pyrrolidine, piperazine, 1-butylpiperidine, 1-ethyl-2-methylpiperidine, N-methylpiperazine, 1,4dimethylpiperazine, N-benzylphenylethylamine, N-methylglucosamine and tris-(hydroxymethyl)aminomethane. 14. The peptide according to any one of claims 1-5, where at least one amino acid listed in the left column of the table. From the description, in any one of the specified SEQ ID NO: 1-5 is replaced by the amino acid listed in the central column of the table. From the description, provided that the resulting sequence has at least 70% sequence identity with, preferably at least 80% and more preferably at least 90% with any one of SEQ ID NO: 1-5, respectively. 15. Peptide according to any one of claims 1-5, where at least one amino acid listed in the left column of the table. From the description, in any one of the specified SEQ ID NO: 1-5 is replaced by the amino acid indicated in the right column of the table. From the description, provided that the resulting sequence has at least 70% sequence identity with, preferably at least 80% and more preferably at least 90% with any of the specified SEQ ID NO: 1-5, respectively. - 18 044414 16. A cosmetic composition containing: (i) a peptide according to any one of claims 1 to 15 and a derivative or salt thereof and (ii) at least one cosmetically acceptable ingredient. 17. A pharmaceutical composition containing: (i) a peptide according to any one of claims 1 to 15 and a derivative or salt thereof and (ii) at least one pharmaceutically acceptable ingredient. 18. A therapeutic or non-therapeutic method for improving conditions, disorders and/or diseases of the skin mediated by neuronal exocytosis, comprising topical application of a pharmaceutical or cosmetic composition containing: (i) a peptide according to any one of claims 1 to 15 and a derivative or salt thereof and (ii) at least one pharmaceutically or cosmetically acceptable ingredient. 19. The method according to claim 18, wherein said conditions, disorders and/or diseases of the skin mediated by neuronal exocytosis are selected from the group consisting of facial wrinkles, motor muscle dysfunction, excessive sweating, itching, skin inflammation, dermatitis, atopy, psoriasis , vascular hyperreactivity, rosacea, acne, hair growth, wound healing, calluses, warts or sensitive skin conditions such as ulcers and skin lesions. 20. A polynucleotide encoding a peptide according to any one of claims. 1-15.