Peptides and compositions for use in cosmetics and medicine

Abstract

The present invention relates to a family of peptides which are able to interfere in the formation of complex Munc18-Syntaxin-1 and, hence, are useful in the prevention and/or treatment of neuronal exocytosis and/or muscle contractility disorders; and to prevent, reduce and/or eliminate skin aging and/or expression signs.

Claims

1. A method of reducing skin aging and/or facial expression lines in a subject in need thereof comprising applying to the subject in need thereof a peptide or cosmetic composition comprising said peptide, wherein the peptide has a sequence in accordance with formula (I):
R.sub.1-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6-R.sub.2   (I) cosmetically and pharmaceutically acceptable isomers, salts, solvates and/or derivatives and mixtures thereof, wherein: AA.sub.1 is His; AA.sub.2 is selected from the group consisting of Gly, Ala, Val, Leu, Met and Ile; AA.sub.3 is selected from the group consisting of Gly, Ala, Val, Leu, Met and Ile; AA.sub.4 is selected from the group consisting of Lys, Arg, His, Asp and Glu; AA.sub.5 is selected from the group consisting of Met, Phe, Tyr and Trp; and AA.sub.6 is Trp; R.sub.1 is selected from the group consisting of H, substituted or unsubstituted non-cyclic aliphatic, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl and R.sub.5—CO— wherein R.sub.5 is selected from the group formed by substituted or unsubstituted C.sub.1-C.sub.24 alkyl radical, substituted or unsubstituted C.sub.2-C.sub.24 alkenyl, substituted or unsubstituted C.sub.2-C.sub.24 alkynyl, substituted or unsubstituted C.sub.3-C.sub.24 cycloalkyl, substituted or unsubstituted C.sub.5-C.sub.24 cycloalkenyl, substituted or unsubstituted C.sub.8-C.sub.24 cycloalkynyl, substituted or unsubstituted C.sub.6-C.sub.30 aryl, substituted or unsubstituted C.sub.7-C.sub.24 aralkyl, substituted or unsubstituted heterocyclyl ring of 3 to 10 members, and substituted or unsubstituted heteroarylalkyl of 2 to 24 carbon atoms and 1 to 3 atoms other than carbon and an alkyl chain of 1 to 6 carbon atoms; and R.sub.2 is selected from the group consisting of H, —NR.sub.3R.sub.4—, —OR.sub.3 and —SR.sub.3, wherein R.sub.3 and R.sub.4 are independently selected from H, substituted or unsubstituted non-cyclic aliphatic group, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted aralkyl (R.sub.1-SEQ ID NO: 15-R.sub.2), wherein the peptide interferes in the Munc18-Syntaxin-1 complex interaction and competes with SEQ ID NO: 3 and/or SEQ ID NO: 4, acceptable isomers, salts, solvates and/or derivatives and/or mixtures thereof.

2. The method in accordance with claim 1, wherein the peptide of formula (I) is: TABLE-US-00011 (Ac-SEQ ID NO: 5-NH.sub.2) Ac-His-Ile-Leu-Asp-Met-Trp-NH.sub.2; (Ac-SEQ ID NO: 6-NH.sub.2) Ac-His-Ile-Met-Asp-Phe-Trp-NH.sub.2; (Ac-SEQ ID NO: 7-NH.sub.2) Ac-His-Ile-Leu-Asp-Trp-Trp-NH.sub.2; (Ac-SEQ ID NO: 8-NH.sub.2) Ac-His-Ala-Leu-Arg-Phe-Trp-NH.sub.2; and/or (Ac-SEQ ID NO: 9-NH.sub.2) Ac-His-Ile-Met-Asp-Trp-Trp-NH.sub.2.

3. A method of reducing skin aging and/or facial expression lines in a subject in need thereof comprising applying to the subject in need thereof a peptide or cosmetic composition comprising said peptide, the peptide having a sequence in accordance with formula (II) R.sub.1-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-R.sub.2, wherein the peptide of formula (II) is: TABLE-US-00012 (Ac-SEQ ID NO: 10-NH.sub.2) Ac-Arg-Arg-Arg-Phe-NH.sub.2; and/or (Ac-SEQ ID NO: 11-NH.sub.2) Ac-Arg-Met-Arg-Phe-NH.sub.2.

4. The method in accordance with claim 1, wherein AA.sub.1 is His; AA.sub.2 is selected from the group of Ala and Ile; AA.sub.3 is selected from the group of Leu and Met; AA.sub.4 is selected from the group of Arg and Asp; AA.sub.5 is selected from the group of Met, Phe and Trp; and AA.sub.6 is Trp.

5. The method in accordance with claim 1, wherein the peptide of formula (I) is TABLE-US-00013 (R.sub.1-SEQ ID NO: 5-R.sub.2) R.sub.1-His-Ile-Leu-Asp-Met-Trp-R.sub.2; (R.sub.1-SEQ ID NO: 6-R.sub.2) R.sub.1-His-Ile-Met-Asp-Phe-Trp-R.sub.2; (R.sub.1-SEQ ID NO: 7-R.sub.2) R.sub.1-His-Ile-Leu-Asp-Trp-Trp-R.sub.2; (R.sub.1-SEQ ID NO: 8-R.sub.2) R.sub.1-His-Ala-Leu-Arg-Phe-Trp-R.sub.2; and/or (R.sub.1-SEQ ID NO: 9-R.sub.2) R.sub.1-His-Ile-Met-Asp-Trp-Trp-R.sub.2.

6. A method of reducing skin aging and/or facial expression lines in a subject in need thereof comprising applying to the subject in need thereof a peptide or cosmetic composition comprising said peptide, wherein the peptide has a sequence in accordance with formula (II) R.sub.1-AA1-AA.sub.2-AA.sub.3-AA.sub.4-R.sub.2, wherein the peptide of formula (II) is R.sub.1-Arg-Arg-Arg-Phe-R.sub.2 (R.sub.1-SEQ ID NO: 10-R.sub.2); or R.sub.1-Arg-Met-Arg-Phe-R.sub.2 (R.sub.1-SEQ ID NO: 11-R.sub.2) and wherein R.sub.1 is selected from the group consisting of H, substituted or unsubstituted non-cyclic aliphatic, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, substituted or unsubstituted aralkyl and R.sub.5—CO— wherein R.sub.5 is selected from the group formed by substituted or unsubstituted C.sub.1-C.sub.24 alkyl radical, substituted or unsubstituted C.sub.2-C.sub.24 alkenyl, substituted or unsubstituted C.sub.2-C.sub.24 alkynyl, substituted or unsubstituted C.sub.3-C.sub.24 cycloalkyl, substituted or unsubstituted Cs-C.sub.24 cycloalkenyl, substituted or unsubstituted Cs-C.sub.24 cycloalkynyl, substituted or unsubstituted C.sub.6-C.sub.3O aryl, substituted or unsubstituted C-C.sub.24 aralkyl, substituted or unsubstituted heterocyclyl ring of 3 to 10 members, and substituted or unsubstituted heteroarylalkyl of 2 to 24 carbon atoms and 1 to 3 atoms other than carbon and an alkyl chain of 1 to 6 carbon atoms; and R.sub.2 is selected from the group consisting of H, —NR.sub.3R.sub.4—, —OR.sub.3 and —SR.sub.3, wherein R.sub.3 and R.sub.4 are independently selected from H, substituted or unsubstituted non-cyclic aliphatic group, substituted or unsubstituted alicyclyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted aralkyl (R.sub.1-SEQ ID NO: 15-R.sub.2).

7. The method in accordance with claim 1, wherein the skin aging and/or facial expression line being reduced are facial wrinkles and facial asymmetry.

8. The method in accordance with claim 1, wherein the cosmetic composition is applied by means of iontophoresis.

9. The method in accordance with claim 1, wherein the cosmetic composition is applied by subcutaneous injection.

10. The method in accordance with claim 1, wherein the cosmetic composition is applied topically.

11. The method in accordance with claim 1, wherein the cosmetic composition comprises the peptide at a concentration of 0.0001%-0.05% (m/v).

12. The method in accordance with claim 1, wherein the subject in need thereof is a mammal.

13. The method in accordance with claim 12, wherein the mammal is a human.

14. The method in accordance with claim 3, wherein the cosmetic composition is applied by means of iontophoresis.

15. The method in accordance with claim 3, wherein the cosmetic composition is applied by subcutaneous injection.

16. The method in accordance with claim 3, wherein the cosmetic composition is applied topically.

17. The method in accordance with claim 3, wherein the cosmetic composition comprises the peptide at a concentration of 0.0001%-0.05% (m/v).

18. The method in accordance with claim 3, wherein the subject in need thereof is a mammal.

19. The method in accordance with claim 18, wherein the mammal is a human.

20. The method in accordance with claim 6, wherein the cosmetic composition is applied by means of iontophoresis.

21. The method in accordance with claim 6, wherein the cosmetic composition is applied by subcutaneous injection.

22. The method in accordance with claim 6, wherein the cosmetic composition is applied topically.

23. The method in accordance with claim 6, wherein the cosmetic composition comprises the peptide at a concentration of 0.0001%-0.05% (m/v).

24. The method in accordance with claim 6, wherein the subject in need thereof is a mammal.

Description

(1) To allow a better understanding, the present invention is described in more detail below with reference to the enclosed drawings, which are presented by way of example, and with reference to illustrative and non-limitative examples.

(2) FIG. 1 shows the percentage acetylcholine release in vitro by LAN cells treated with the analyzed peptides in comparison with the positive control (this is, establishing the percentage of acetylcholine release of the positive control sample as 100% and then performing the comparison with the rest of the samples). FIG. 1 shows the results obtained for peptides: Ac-SEQ ID NO: 5-NH.sub.2 (A), Ac-SEQ ID NO: 6-NH.sub.2 (B), Ac-SEQ ID NO: 7-NH.sub.2 (C), Ac-SEQ ID NO: 8-NH.sub.2 (D), Ac-SEQ ID NO: 9-NH.sub.2 (E), Ac-SEQ ID NO: 10-NH.sub.2 (F), Ac-SEQ ID NO: 11-NH.sub.2 (G), Ac-SEQ ID NO: 13-NH.sub.2 (H) and Ac-SEQ ID NO:14-NH.sub.2 (I). All peptides, except Ac-SEQ ID NO: 8-NH.sub.2 (D), were tested at concentrations of 0.001 mg/mL, 0.005 mg/mL and 0.01 mg/mL; and peptide Ac-SEQ ID NO: 8-NH.sub.2 (D) was tested at 0.005 mg/mL and 0.05 mg/mL. For FIGS. 1(D) columns from left to right in the x-axis correspond to: basal state (cells without treatment), positive control (cells treated with 50 mM of KCl), cells treated with 100 nM of toxin (Botulinum neurotoxin A light chain (BoNT A LC) produced in accordance with Ibañez C., Blanes-Mira C., Fernández-Ballester G., Planells-Cases R., and Ferrer-Montiel A., (2004) Modulation of botulinum neurotoxin A catalytic domain stability by tyrosine phosphorylation, FEBS Letters 578, 121-127), cells treated with 0.1 μM Palmitoyl-Argireline® (palmitoyl-acetyl hexapeptide-8®) and cells treated with 0.005 mg/mL and 0.05 mg/mL of the corresponding peptide. On its side, for FIGS. 1(A) to 1(C) and 1(E) to 1(I), columns from left to right in the x-axis correspond to: basal state (cells without treatment), positive control (cells treated with 50 mM of KCl), cells treated with 100 nM of toxin (BoNT A LC) and cells treated with 0.001 mg/mL, 0.005 mg/mL and 0.01 mg/mL of the corresponding peptide. For FIGS. 1(A) to 1(I) the y axis shows the percentage of acetylcholine release (with regard to the positive control).

(3) FIG. 2 shows the percentage of binding of the complex Munc18-Syntaxin-1 with respect to the control (no treatment), which represents 100% of binding. In FIG. 2 the capacity of peptides Ac-SEQ ID NO:8-NH.sub.2 (A) and Ac-SEQ ID NO: 5-NH.sub.2 (B) to inhibit the formation of the complex Munc18-Syntaxin-1 can be observed. Columns from left to right in the x-axis of both, FIGS. 2(A) and 2(B), correspond to: control and 0.1, 0.5 and 1 mM concentration of the corresponding peptide at a ratio Munc18 100 nM:Syntaxin-1 nM for the first group of columns (left group of columns) and, for the second group of columns, control and 0.1, 0.5 and 1 mM concentration of the corresponding peptide at a ratio Munc18 100 nM:Syntaxin-1 5 nM (right group of columns). For both figures the y axis shows the percentage of formation of the complex, being 100% the signal obtained without any treatment.

(4) FIG. 3 shows the modulation in gene expression profile of human skeletal myocytes induced by the treatment with the peptides of the present invention. FIG. 3(A) shows the results obtained for the treatment with Ac-SEQ ID NO: 8-NH.sub.2 (at a concentration of 0.05 and 0.5 mg/mL, during 6 hours), wherein bars, from top to bottom refer to the following genes: SCN3A (Sodium Voltage-Gated Channel Alpha Subunit 3), UTRN (Utrophin), ACTA1 (Actin alpha 1), TNNC1 (Troponin C1), CALM3 (Calmodulina 3), CAV1 (Caveolin 1), CACNB1 (Calcium Voltage-Gated Channel Auxiliary Subunit Beta 1), LRP4 (LDL Receptor Related Protein 4) and MYH1 (Myosin Heavy Chain 1). In FIG. 3(A) the results shown for genes MHY1, LRP4, CACNB1 and UTRN correspond to a treatment with 0.05 mg/mL, while the rest correspond to a 0.5 mg/mL treatment. FIG. 3(B) shows the results obtained for the treatment with Ac-SEQ ID NO: 8-NH.sub.2 at a concentration of 0.5 mg/mL during 24 hours, wherein bars, from top to bottom refer to the following genes: RAPSN (Receptor Associated Protein of The Synapse) and ATP2A (ATPase Sarcoplasmic/Endoplasmic Reticulum Ca.sup.2′ Transporting). FIG. 3(C) shows the results obtained for the treatment with Ac-SEQ ID NO: 5-NH.sub.2 (at a concentration of 0.05 mg/mL and during 24 hours), wherein bars, from top to bottom refer to the following genes: UTRN (Utrophin), ACTA1 (Actin alpha 1), TNNC1 (Troponin C1), RAPSN (Receptor Associated Protein of The Synapse), SCN3A (Sodium Voltage-Gated Channel Alpha Subunit 3) and MYH1. FIG. 3(D) shows the results obtained for the treatment with Ac-SEQ ID NO: 10-NH.sub.2 (at a concentration of 0.1 mg/mL and during 24 hours), wherein bars, from top to bottom refer to the following genes: UTRN (Utrophin), TNNC1 (Troponin C1), SCN3A (Sodium Voltage-Gated Channel Alpha Subunit 3), CHRNA1 (Cholinergic Receptor Nicotinic Alpha 1 Subunit) and CACNB1 (Calcium Voltage-Gated Channel Auxiliary Subunit Beta 1). In the four cases, x-axis refers to the fold change with regard to the basal state. A negative fold change refers to downregulation of gene expression while a positive fold change refers to upregulation.

(5) FIG. 4 shows the decrease in calcium influx on human skeletal muscle myocytes after treatment with peptide Ac-SEQ ID NO: 8-NH.sub.2 and stimulation with 60 mM KCl with regard to the control (untreated sample stimulated with 60 mM KCl) which is established as 100%. Columns from left to right in the x-axis correspond to: control and 0.01, 0.05 and 0.1 mg/mL of peptide concentration. The y axis shows the percentage of decrease in calcium signal with regard to the control, established as 100% signal.

(6) FIG. 5 shows representative images of the expression levels of myosin heavy chain protein, on non-treated primary human skeletal muscle cells (FIG. 5(A)) and on primary human skeletal muscle cells treated with 0.05 mg/mL of peptide Ac-SEQ ID NO: 8-NH.sub.2 (FIG. 5(B)). A reduction of myosin heavy chain protein levels is observed in the primary human skeletal muscle cells treated with 0.05 mg/mL of peptide with regard to the non-treated cells, which can be seen as a loss of staining or signal in FIG. 5B in comparison with FIG. 5(A).

(7) FIG. 6 shows the decrease of myosin heavy chain protein levels on primary human skeletal muscle cells after treatment with the peptide Ac-SEQ ID NO: 8-NH.sub.2 with regard to the basal control (non-treated cells) which is established as 100%. Columns from left to right in the X-axis correspond to non-treated cells and 0.05 and 0.5 mg/ml of peptide concentration. The Y-axis shows the levels of myosin heavy chain protein with regard to the basal control, established as 100% signal (on the basis of the fluorescence signal observed for myosin heavy chain protein in each of the treatment groups, as measured by means of mean cell fluorescence).

(8) FIG. 7 shows the modulation in the contraction frequency observed on human motor neurons and human skeletal myocytes co-cultures after treatment with either peptide Ac-SEQ ID NO: 8-NH.sub.2, acetyl hexapeptide-8, as a benchmark control, or α-bungarotoxin, as a positive control of inhibition, with regard to the basal control (non-treated cells) which is established as 100%. The line with squares represents the contraction frequency of the non-treated cells (basal control). The line with crosses represents the contraction frequency by the positive control of contraction inhibition, α-bungarotoxin. The line with diamonds represents the contraction frequency by the benchmark acetyl hexapeptide-8 at 0.5 mg/ml. The line with circles represents the contraction frequency by peptide Ac-SEQ ID NO: 8-NH.sub.2 at 0.1 mg/ml; and the line with triangles represents the contraction frequency by peptide Ac-SEQ ID NO: 8-NH.sub.2 at 0.05 mg/ml. The X-axis corresponds to the length of the treatment by the different actives and it points out different time points: TO, T30 min, T2 h, T24 h and recovery. TO corresponds to the contractions before the treatment with the above-mentioned compounds; T30 min corresponds to the 30 min-treatment; T2 h corresponds to the 2 h-treatment; T24 h corresponds to the 24 h-treatment and Recovery corresponds to the 24 h incubation after removal of all compounds. The Y-axis shows the percentage of contraction frequency with regard to the basal control (non-treated cells), established as 100% signal.

(9) FIG. 8 shows the decrease of exocytosis on a human neuroblastoma cell line after treatment with the peptide Ac-SEQ ID NO: 8-NH.sub.2 or the benchmark acetyl hexapeptide-8 with regard to the basal control of non-treated cells which is established as 100%. Columns from left to right in the X-axis correspond to non-treated cells, cells treated with 1 mg/ml of acetyl hexapeptide-8 and cells treated with 0.01 mg/ml of peptide Ac-SEQ ID NO: 8-NH.sub.2. The Y-axis shows the percentage of the fluorescence signal corresponding to the level of exocytosis with regard to the basal control, established as 100% level.

(10) FIG. 9 shows the delay in time of exocytosis on a human neuroblastoma cell line after treatment with the peptide Ac-SEQ ID NO: 8-NH.sub.2 or the benchmark acetyl hexapeptide-8 with regard to the basal control of non-treated cells which is established as 100%. Columns from left to right in the X-axis correspond to non-treated cells, 1 mg/ml of acetyl hexapeptide-8 and 0.01 mg/ml of peptide Ac-SEQ ID NO: 8-NH.sub.2. The Y-axis shows the percentage of the response time corresponding to the delay of exocytosis with regard to the basal control, established at 100% level.

(11) FIG. 10 shows the collagen type I production by primary human dermal fibroblasts after treatment with Ac-SEQ ID NO: 8-NH.sub.2 with regard to the basal control (non-treated cells) which is established as 100%, at 24 h after the beginning of the treatment (FIG. 10(A)) and at 48 h after the beginning of the treatment (FIG. 10(B)). Columns from left to right in the X-axis correspond to: basal control (non-treated cells) and cells treated with 0.01, 0.05 or 0.1 mg/ml of peptide Ac-SEQ ID NO: 8-NH.sub.2, respectively. The Y-axis shows the percentage of increase of collagen type I protein synthesis with regard to the control, established at 100% level.

EXAMPLES

Abbreviations

(12) The abbreviations used for amino acids follow the 1983 IUPAC-IUB Joint Commission on Biochemical Nomenclature recommendations outlined in Eur. J. Biochem. (1984) 138:937.

(13) Ac, acetyl; Ala, alanine; Arg, arginine; Asn, Asparagine; Asp, Aspartic acid; Boc, tert-butyloxycarbonyl; C-terminal, carboxy-terminal; DCM, dichloromethane; DIEA, N,N′-diisopropylethylamine; DIPCDI, N,N′-diisopropylcarbodiimide; DMF, N,N-dimethylformamide; equiv, equivalent; ESI-MS, electrospray ionization mass spectrometry; Fmoc, 9-fluorenylmethyloxycarbonyl; Glu, Glutamic acid; hiPSC, human induced pluripotent stem cells; His, histidine; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; HRP, Horseradish peroxidase; Ile, Isoleucine; INCI, International Nomenclature of Cosmetic Ingredients; MBHA, p-methylbenzhydrylamine; Leu, leucine; Lys, lysine; Me, methyl; MeCN, acetonitrile; MeOH, methanol; Met, Methionine; N-terminal, amino-terminal; Palm, palmitoyl; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; PFA, paraformaldehyde; PMA, phorbol 12-myristate 13-acetate; Phe, Phenylalanine; PMSF, Phenylmethanesulfonyl; RT, room temperature; tBu, tert-butyl; Thr, Threonine; TFA, trifluoroacetic acid; TIS, triisopropylsilane; TMB, Tetramethylbenzidine; Trt, triphenylmethyl or trityl; Trp, Tryptophan; Tyr, Tyrosine.

(14) Regarding the chemical synthesis procedures included in the examples, it is noted that all synthetic processes were carried out in polypropylene syringes fitted with porous polyethylene discs or Pyrex® reactors fitted with porous plates. All the reagents and solvents were synthesis quality and were used without any additional treatment. The solvents and soluble reagents were removed by suction. The Fmoc group was removed with piperidine-DMF (2:8, v/v) (at least 1×1 min, 2×10 min, 5 mL/g resin) (Lloyd Williams P. et al., Chemical Approaches to the Synthesis of Peptides and Proteins, C R C, 1997, Boca Raton (Fla., USA)). Washes between stages of deprotection, coupling, and, again, deprotection, were carried out with DMF (3×1 min) and DCM (3×1 min) each time using 10 ml solvent/g resin. Coupling reactions were performed with 3 ml solvent/g resin. The control of the couplings was performed by carrying out the ninhydrin test (Kaiser E. et al., Anal. Biochem., 1970, 34: 595598). All synthetic reactions and washes were carried out at RT.

Example 1. In Silico Determination of Peptides Interfering in the Interaction of Mync18-Syntaxin-1

(15) In this experiment, the objective was to generate in silico peptides with affinity and/or specificity for the interaction regions of Munc18 and Syntaxin-1 and, hence, that could be able to compete, interfere and disrupt the interaction and/or binding between said two proteins.

(16) Since the structure of Munc18-Syntaxin-1 complex was known and available at 2.6 A resolution (Protein Data Bank reference number 3C98), a three-dimensional structure model of this interaction was generated and the interaction fragments of Munc18 included in table 1 were selected.

(17) TABLE-US-00005 TABLE 1 Interaction fragments of Munc18 with Syntaxin-1 selected by means of the in silico study. Length Protein Sequence of the interaction fragment (amino acids) Munc18 46-Lys-Met-Thr-Asp-Ile-Met-51 (SEQ ID NO: 3) 6 Munc18 59-Glu-Asp-Ile-Asn-Lys-Arg-Arg-Glu-66 (SEQ ID NO: 12) 8 Munc18 63-Lys-Arg-Arg-Glu-66 (SEQ ID NO: 4) 4

(18) On the basis of said target fragments, as a first step of design, the wild type peptide (considered as the 100% binder peptide) was mutated to poly-Ala (considered as the non-binder peptide 0%), and all the positions were treated as independent. Each individual position was mutated to the 20 natural amino acids, while the other positions remained as Ala. The theoretical binding energy between the fragment and the rest of the complex was determined to assess the improvement of the interaction with the mutagenesis in the different positions. The tabulation and the normalization of the e binding energies produced scoring matrices which showed how well an amino acid residue fitted in a given position of the peptide. These matrices were used to propose and build a ranked list of peptides that putatively inhibit the Munc8-Syntaxin-1 complex formation.

(19) In a second step of the experiment, the list of peptides was modelled on the complex, but mutating all positions in the peptide at the same time. In this way it was ensured that interactions or incompatibilities between neighbouring positions in the peptides were taken into account. The bonding energy of peptide-complex interaction was evaluated again, and the peptides were re-ranked according to this energy. The binding energies, which could be higher than 100% or lower than 0%, were then compared to the 100% binder (wild type) and the 0% binder (poly Ala peptide). The best peptides were selected and proposed for further study and verification (see table 2).

(20) TABLE-US-00006 TABLE 2 peptides of the in silico study selected for further study and verification. Sequence of the ID of ΔG interaction fragment Sequence of the peptide the peptide (Joules) 46-Lys-Met-Thr-Asp-Ile- His-Ile-Leu-Asp-Met-Trp SEQ ID NO: 5 −11.02 Met-51 46-Lys-Met-Thr-Asp-Ile- His-Ile-Met-Asp-Phe-Trp SEQ ID NO: 6 −10.99 Met-51 46-Lys-Met-Thr-Asp-Ile- His-Ile-Leu-Asp-Trp-Trp SEQ ID NO: 7 −10.76 Met-51 46-Lys-Met-Thr-Asp-Ile- His-Ala-Leu-Arg-Phe-Trp SEQ ID NO: 8 −8.68 Met-51 46-Lys-Met-Thr-Asp-Ile- His-Ile-Met-Asp-Trp-Trp SEQ ID NO: 9 −11.21 Met-51 63-Lys-Arg-Arg-Glu-66 Arg-Arg-Arg-Phe SEQ ID NO: 10 −5.71 63-Lys-Arg-Arg-Glu-66 Arg-Met-Arg-Phe SEQ ID NO: 11 −5.26 59-Glu-Asp-Ile-Asn-Lys- Glu-Arg-Ile-Asn-Lys-Arg- SEQ ID NO: 13 −9.23 Arg-Arg-Glu-66 Arg-Trp 59-Glu-Asp-Ile-Asn-Lys- Glu-Arg-Ile-Asn-Lys-Met SEQ ID NO: 14 −8.12 Arg-Arg-Glu-66 Arg-Tyr

Example 2. Synthesis and Preparation of the Peptides

(21) Obtaining Fmoc-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6-Rink-MBHA-resin, wherein AA.sub.1 is L-His; AA.sub.2 is L-Ile or L-Ala; AA.sub.3 is L-Leu or L-Met; AA.sub.4 is L-Asp or L-Arg; AA.sub.5 is L-Met, L-Trp or L-Phe; and AA.sub.6 is L-Trp.

(22) Weights were normalized. 4.8 g (2.5 mmol) of the Fmoc-Rink-MBHA resin with a functionalization of 0.52 mmol/g were treated with piperidine-DMF according to the described general protocol known in the state of the art in order to remove the Fmoc group. 3.94 g of Fmoc-L-Trp(Boc)-OH (7.5 mmol; 3 equiv) were incorporated onto the deprotected resin in the presence of DIPCDI (1.17 mL; 7.5 mmol; 3 equiv) and HOBt (1.01 g; 7.5 mmol; 3 equiv) using DMF as a solvent for one hour.

(23) The resin was then washed as described in the general methods known in the state of the art and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. Following the previously described protocols 2.90 g of Fmoc-Phe-OH, 2.78 g of Fmoc-L-Met-OH or 3.94 g of Fmoc-L-Trp(Boc)-OH (7.5 mmol; 3 equiv); subsequently 4.86 g of Fmoc-L-Arg(Pbf)-OH or 3.08 g of Fmoc-L-Asp(tBu)-OH (7.5 mmol; 3 equiv); subsequently 2.65 g of Fmoc-L-Leu-OH or 2.78 g of Fmoc-L-Met-OH (7.5 mmol; 3 equiv); subsequently 2.33 g Fmoc-L-Ala-OH or 2.65 g Fmoc-L-Ile-OH (7.5 mmol; 3 equiv) and subsequently 4.64 g of Fmoc-L-His(Trt)-OH (7.5 mmol; 3 equiv) were coupled, sequentially, each coupling in the presence of 1.01 g of HOBt (7.5 mmol; 3 equiv) and 1.17 mL of DIPCDI (7.5 mmol; 3 equiv). As already noted above, between each amino acid addition step, a deprotection treatment of the Fmoc group was performed.

(24) After the synthesis, the peptide resins were washed with DCM (5 times for 3 minutes each one) and dried under vacuum.

(25) Obtaining Fmoc-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-Rink-MBHA-Resin, Wherein AA.sub.1 is L-Arg; AA.sub.2 is L-Arg or L-Met; AA.sub.3 is L-Arg and AA.sub.4 is L-Phe.

(26) Weights were normalized. 4.8 g (2.5 mmol) of the Fmoc-Rink-MBHA resin with a functionalization of 0.52 mmol/g were treated with piperidine-DMF according to the described general protocol of the state of the art in order to remove the Fmoc group. 2.90 g of Fmoc-L-Phe-OH (7.5 mmol; 3 equiv) were incorporated onto the deprotected resin in the presence of DIPCDI (1.17 mL; 7.5 mmol; 3 equiv) and HOBt (1.01 g; 7.5 mmol; 3 equiv) using DMF as a solvent for one hour.

(27) The resin was then washed as described in the general methods and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. Following the previously described protocols, 4.86 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equiv); subsequently 4.86 g of Fmoc-L-Arg(Pbf)-OH or 2.78 g of Fmoc-L-Met-OH (7.5 mmol; 3 equiv) and subsequently 4.86 g of Fmoc-L-Arg(Pbf)-OH (7.5 mmol; 3 equiv) were coupled, sequentially, each coupling in the presence of 1.01 g of HOBt (7.5 mmol; 3 equiv) and 1.17 mL of DIPCDI (7.5 mmol; 3 equiv). As already noted above, between each amino acid addition step, a deprotection treatment of the Fmoc group was performed.

(28) After the synthesis, the peptide resins were washed with DCM (5 times for 3 minutes each one) and dried under vacuum.

(29) Obtaining Fmoc-AA.sub.1-AA.sub.2-AA.sub.3-AA.sub.4-AA.sub.5-AA.sub.6-AA.sub.7-AA.sub.8-Rink-MBHA-Resin, Wherein AA.sub.1 is L-Glu; AA.sub.2 is L-Arg; AA.sub.3 is L-Ile; AA.sub.4 is L-Asn; AA.sub.5 is L-Lys; AA.sub.6 is L-Arg or L-Met; AA.sub.7 is L-Arg and AA.sub.8 is L-Trp or L-Tyr.

(30) Weights were normalized. 4.8 g (2.5 mmol) of the Fmoc-Rink-MBHA resin with a functionalization of 0.52 mmol/g were treated with piperidine-DMF according to the described general protocol known in the state of the art in order to remove the Fmoc group. 3.94 g of Fmoc-L-Trp(Boc)-OH or 3.44 g of Fmoc-L-Tyr(tBu)-OH (7.5 mmol; 3 equiv) were incorporated onto the deprotected resin in the presence of DIPCDI (1.17 mL; 7.5 mmol; 3 equiv) and HOBt (1.01 g; 7.5 mmol; 3 equiv) using DMF as a solvent for one hour.

(31) The resin was then washed as described in the general methods and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. Following the previously described protocols, 4.86 g of Fmoc-Arg(Pbf)-OH (7.5 mmol; 3 equiv); subsequently 4.86 g of Fmoc-L-Arg(Pbf)-OH or 2.78 g of Fmoc-L-Met-OH (7.5 mmol; 3 equiv); subsequently 3.51 g of Fmoc-L-Lys(Boc)-OH (7.5 mmol; 3 equiv); subsequently 4.45 g Fmoc-L-Asn(Trt)-OH (7.5 mmol; 3 equiv); subsequently 2.65 g Fmoc-L-Ile-OH; subsequently 4.86 g Fmoc-L-Arg(Pbf)-OH and subsequently 3.19 g of Fmoc-L-Glu(OtBu)-OH (7.5 mmol; 3 equiv) were coupled, sequentially, each coupling in the presence of 1.01 g of HOBt (7.5 mmol; 3 equiv) and 1.17 mL of DIPCDI (7.5 mmol; 3 equiv). As already noted above, between each amino acid addition step, a deprotection treatment of the Fmoc group was performed.

(32) After the synthesis, the peptide resins were washed with DCM (5 times for 3 minutes each one) and dried under vacuum.

(33) Using the synthesis procedures mentioned above, with the required selection of amino acids, the following sequences were synthesized:

(34) TABLE-US-00007 (SEQ ID NO: 5) His-Ile-Leu-Asp-Met-Trp; (SEQ ID NO: 6) His-Ile-Met-Asp-Phe-Trp; (SEQ ID NO: 7) His-Ile-Leu-Asp-Trp-Trp; (SEQ ID NO: 8) His-Ala-Leu-Arg-Phe-Trp; (SEQ ID NO: 9) His-Ile-Met-Asp-Trp-Trp; (SEQ ID NO: 10) Arg-Arg-Arg-Phe; (SEQ ID NO: 11) Arg-Met-Arg-Phe; (SEQ ID NO: 13) Glu-Arg-Ile-Asn-Lys-Arg-Arg-Trp; and (SEQ ID NO: 14) Glu-Arg-Ile-Asn-Lys-Met-Arg-Tyr.

Example 3. Removal of Fmoc N-Terminal Protective Group of the Peptides Synthesized in Accordance with Example 2

(35) The N-terminal Fmoc group of the peptidyl resins was deprotected with 20% piperidine in DMF (1×1 min+2×10 min) (Lloyd Williams P. et al. (1997) “Chemical Approaches to the Synthesis of Peptides and Proteins” CRC, Boca Raton (Fla., USA)). The peptidyl resins were washed with DMF (5×1 min), DCM (4×1 min), and dried under vacuum.

Example 4. Process for Introducing the R.SUB.1 .Acetyl Group onto the Peptidyl Resins Obtained in Accordance with Example 3

(36) 1 mmol (1 equiv) of the peptidyl resins obtained in accordance with Example 2 was treated with 25 equivalents of acetic anhydride in the presence of 25 equivalents of DIEA using 5 mL of DMF as a solvent. They were left to react for 30 minutes, after which the peptidyl resins were washed with DMF (5×1 min), DCM (4×1 min), and were dried under vacuum.

Example 5. Cleavage Process from the Polymeric Support of the Peptidyl Resins Obtained in Accordance with Example 3 and 4

(37) Weights were normalized. 200 mg of the dried peptidyl resin obtained in any of Examples 2, 3 or 4 were treated with 5 mL of TFA/TIS/H.sub.2O (90:5:5) for 2 hours at room temperature under stirring. The filtrates were collected and precipitated using 50 mL (8 to 10-fold) of cold diethyl ether. The ethereal solutions were evaporated to dryness at reduced pressure and room temperature, the precipitates were redissolved in 50% MeCN in H.sub.2O and lyophilized.

Example 6. Characterization of the Peptides Synthesized and Prepared in Accordance with Example 5

(38) HPLC analysis of the peptides obtained in accordance with example 5 was carried out with a Shimadzu equipment (Kyoto, Japan) using a reverse-phase column (150×4.6 mm, XBridge Peptide BEH C18, 3.5 μm, Waters, USA) in gradients of MeCN (+0.036% TFA) in H.sub.2O (+0.045% TFA) at a flow rate of 1.25 mL/min and detection was carried out at 220 nm. All peptides showed a purity exceeding 80%. The identity of the peptides obtained was confirmed by ESI-MS in a Water ZQ 4000 detector using MeOH as the mobile phase and a flow rate of 0.2 mL/min. Results obtained demonstrated that the peptides included in table 3 were effectively synthesized.

(39) TABLE-US-00008 TABLE 3 Final peptides synthesized. Peptide ID of the peptide Ac-His-Ile-Leu-Asp-Met-Trp-NH.sub.2 Ac-SEQ ID NO: 5-NH.sub.2 Ac-His-Ile-Met-Asp-Phe-Trp-NH.sub.2 Ac-SEQ ID NO: 6-NH.sub.2 Ac-His-Ile-Leu-Asp-Trp-Trp-NH.sub.2 Ac-SEQ ID NO: 7-NH.sub.2 Ac-His-Ala-Leu-Arg-Phe-Trp-NH.sub.2 Ac-SEQ ID NO: 8-NH.sub.2 Ac-His-Ile-Met-Asp-Trp-Trp-NH.sub.2 Ac-SEQ ID NO: 9-NH.sub.2 Ac-Glu-Arg-Ile-Asn-Lys-Arg- Ac-SEQ ID NO: 13-NH.sub.2 Arg-Trp-NH.sub.2 Ac-Glu-Arg-Ile-Asn-Lys-Met- Ac-SEQ ID NO: 14-NH.sub.2 Arg-Tyr-NH.sub.2 Ac-Arg-Arg-Arg-Phe-NH.sub.2 Ac-SEQ ID NO: 10-NH.sub.2 Ac-Arg-Met-Arg-Phe-NH.sub.2 Ac-SEQ ID NO: 11-NH.sub.2

Example 7. Measurement of Acetylcholine Release

(40) The peptides included in the above table 3 were synthesized in accordance with examples 2 to 6.

(41) Said peptides were tested for their ability to modulate acetylcholine release in vitro. To that end, LAN cells were seeded on 48-well culture plates and allowed to reach the appropriate confluence under controlled conditions (37° C., 5% CO.sub.2) before inducing differentiation by replacing the culture media with Neurobasal® A media, supplemented with N-2 Supplement, GlutaMAX™, choline chloride and Leukemia Inhibition factor (GIBCO, Life technologies, MA, USA). Once the cells acquired its differentiated morphology, they were treated with the peptides mentioned above at the following concentrations for 1 hour: all peptides, except Ac-SEQ ID NO: 8-NH.sub.2, at 0.001 mg/mL, 0.005 mg/mL and 0.01 mg/mL; and peptide Ac-SEQ ID NO: 8-NH.sub.2 at 0.005 mg/mL and 0.05 mg/mL. Cells were then washed with HEPES, before stimulation of acetylcholine release by depolarization with external 50 mM KCl solution. Non-stimulated cells were incubated with non-depolarizing 4 mM KCl solution. After 30 minutes of incubation with the corresponding depolarizing or non-depolarizing KCl solution, cell supernatants were collected and used to measure acetylcholine levels with Amplex Red Acetylcholine Assay Kit (ThermoFisher Scientific, MA, USA). Cell pellets were used to determine protein content by BCA Protein Test Assay (Pierce BCA Protein Assay kit, ThermoFisher Scientific, MA, USA) (for data normalization purposes).

(42) Percentage of acetylcholine release inhibition was calculated considering 100% release for the positive control (non-treated stimulated with KCl cells).

(43) The results obtained appear summarized in table 4:

(44) TABLE-US-00009 TABLE 4 Summary of the results obtained in Example 7. Peptide Result Ac-SEQ ID NO: 5-NH.sub.2 Active in the modulation of acetylcholine release. Ac-SEQ ID NO: 6-NH.sub.2 Active in the modulation of acetylcholine release. Ac-SEQ ID NO: 7-NH.sub.2 Active in the modulation of acetylcholine release. Ac-SEQ ID NO: 8-NH.sub.2 Active in the modulation of acetylcholine release. Ac-SEQ ID NO: 9-NH.sub.2 Active in the modulation of acetylcholine release. Ac-SEQ ID NO: 10-NH.sub.2 Active in the modulation of acetylcholine release. Ac-SEQ ID NO: 11-NH.sub.2 Active in the modulation of acetylcholine release. Ac-SEQ ID NO: 13-NH.sub.2 Non-active in the modulation of acetylcholine release. Ac-SEQ ID NO: 14-NH.sub.2 Non-active in the modulation of acetylcholine release.

(45) The results of this experiment also appear summarized in FIG. 1 (A) to (I). As can be directly derived from said figures, the peptides marked as active in the modulation of acetylcholine in table 4, show a significant decrease in the release of acetylcholine in all the concentrations tested. On the other side, peptides marked as non-active in table 4 show a moderate-to-low inhibition at the lowest concentrations and complete loss of inhibition at the highest concentration. Said peptides did not show a statistical decrease in the release of acetylcholine in any of the concentrations tested. The fact that at low concentrations a slight inhibition was observed was expected due to the technical complication of this type of cells and the inherent variability of the test, but the complete loss of activity at the highest concentration clearly shows these peptides (Ac-SEQ ID NO: 13-NH.sub.2 and Ac-SEQ ID NO: 14-NH.sub.2) were not able to inhibit the release of acetylcholine.

(46) As seen from prior art (Blanes-Mira C., Clemente J., Jodas G., et.al. (2002), A synthetic hexapeptide (Argireline) with antiwrinkle activity, International Journal of Cosmetic Science, 24, 303-310), the inhibition of exocytosis from permeabilized chromaffin cells reached maximum values of 50% when using BoNT A or 40% when using Argireline®. Therefore, the results obtained in this example prove the high activity of the peptides of the present invention as when testing the inhibition of acetylcholine release directly on LAN cells, not even permeabilized, values of 20-30% of inhibition were obtained

Example 8. Binding Assay

(47) The following peptides were synthesized based on the in silico study of example 1 and in accordance with examples 2 to 6:

(48) Ac-SEQ ID NO: 5-NH.sub.2

(49) Ac-SEQ ID NO: 8-NH.sub.2

(50) The capacity of the peptides to inhibit Munc18 protein-protein interaction with Syntaxin-1, a SNARE complex protein, was functionally evaluated by means of an ELISA-based in vitro Munc18/Syntaxin-1 binding assay. Briefly, the above-mentioned peptides at 0.1, 0.5 and 1 mM were preincubated with their target protein Syntaxin-1 (GST-tagged) at 10 and 5 nM in buffer (20 mM Hepes, 150 mM NaCl, 2 mM MgC.sub.2, 2 mM DTT, 0.5% Triton-X100 and 0.5% non-fat milk) for 3 hours at RT. Meanwhile, His-tagged Munc18 was bound to a Nickel-coated 96-well plate at 100 nM and incubated at RT for 1.5 hours. Non-bound protein was subsequently washed away before blocking wells with non-fat milk in wash buffer (PBS+0.02% Triton-X100) for additional 30 minutes. GST-tagged Syntaxin-1 preincubated with the peptides was then added at 10 and 5 nM to the plate and incubated for 2 h at RT. Wells were washed three times with wash buffer before incubation with the primary antibody (GST tag polyclonal antibody—Thermofisher Scientific, Massachusetts, USA) for 45 minutes. Finally, after washing again, HRP-conjugated secondary antibody (Anti-Rabbit-IgG-HRP—Sigma, Missouri, USA) was added and incubated at RT. TMB substrate (3,3′,5,5′-Tetramethylbenzidine) was used to develop signal. Reaction was stopped by addition of Stop Reagent. Amount of binding of Syntaxin-1 protein to Munc18 protein was analysed by measuring absorbance at 450 nm.

(51) The results obtained in this experiment appear summarized in FIG. 2. FIG. 2 evidences that both peptides tested (Ac-SEQ ID NO: 5-NH.sub.2 to an even surprisingly higher degree), were able to significantly block protein-protein interactions between Munc18 and Syntaxin-1, showing even a dose-response manner. Maximum inhibition reached 37% and 67% for Ac-SEQ ID NO: 8-NH.sub.2 and Ac-SEQ ID NO: 5-NH.sub.2 peptides, respectively. Based in these results, it can be seen that the peptides of the present invention are able to disrupt the protein-protein interaction required for regulated exocytosis and consequent neurotransmitter release.

Example 9. Gene Expression Modulation in Skeletal Muscle Human Myocytes

(52) The following peptides were synthesized based on the in silico study of example 1 and in accordance with examples 2 to 6:

(53) Ac-SEQ ID NO: 5-NH.sub.2

(54) Ac-SEQ ID NO: 8-NH.sub.2

(55) Ac-SEQ ID NO: 10-NH.sub.2

(56) Said peptides were tested to evaluate their capacity to modulate the expression of several genes related with muscle contraction and relaxation in human skeletal muscle myocytes (Lodish H, Berk A, Zipursky S L, et al. (2000), Molecular Cell Biology, 4th edition; Section 18.3 Myosin: The Actin Motor Protein, Nueva York, W. H. Freeman; Kuo, I Y, & Ehrlich, B E (2015), Signaling in Muscle Contraction, Cold Spring Harbor Perspectives in Biology, 7(2); RAPSN receptor associated protein of the synapse [Homo sapiens (human)], Gene ID in NCBI 5913; Blake D J, Tinsley J M, Davies K E (1996), Utrophin: a structural and functional comparison to dystrophin, Brain Pathol., 6(1):37-47; Barik A, Lu Y, Sathyamurthy A, et.al. (2014), LRP4Is Critical for Neuromuscular Junction Maintenance, The Journal of Neuroscience, 34(42), 13892-13905; Kim N, Stiegler A L, Cameron T O, Hallock P T, et.al. (2008), Lrp4 is a Receptor for Agrin and Forms a Complex with MuSK, Cell, 135(2), 334-342; Mahavadi S, Nalli A, Kumar D, et.al. (2014), Increased expression of caveolin-1 is associated with upregulation of the RhoA/Rho kinase pathway and smooth muscle contraction in diabetes (1110.11), The FASEB Journal, 28:1_supplement)). To that end, a contractility smart data gene panel was designed. Said contractility smart data gene panel analyses the expression of the genes related with muscular contraction and relaxation included in table 5.

(57) TABLE-US-00010 TABLE 5 Genes analyzed in example 9. Symbol Gene name MYH1 Myosin Heavy Chain SCN3A Sodium Voltage-Gated Channel Alpha Subunit 3 RAPSN Receptor Associated protein of the Synapse TNNC 1 Troponin C1 ACTA 1 Actin UTRN Utrophin CACNB1 Calcium Voltage-Gated channel Auxiliary Subunit Beta 1 CHRNA1 Nicotinic Acetylcholine Receptor Alpha Subunit 1 CALM3 Calmodulin 3 CAV1 Caveolin 1 LRP4 LDL Receptor Related Protein 4

(58) Briefly, human skeletal muscle myoblasts were seeded in duplicate in 12-well culture plates at a density of 1×10.sup.5 cells/well and maintained at standard culture conditions (37° C., 95% humidity, 5% CO.sub.2) for 48-72h. Myoblast differentiation to myocyte was then induced with specific differentiation media (SKM-D medium+1% Antibiotic-Antimycotic) and monitored for 7 more days. Differentiated cells were treated with 0.05 mg/mL of peptide Ac-SEQ ID NO: 5-NH.sub.2; or with 0.1 mg/mL of peptide Ac-SEQ ID NO: 10-NH.sub.2 for 24 hours; or with 0.05 and 0.5 mg/mL of peptide Ac-SEQ ID NO: 8-NH.sub.2 for 6h; or with 0.5 mg/mL of peptide Ac-SEQ ID NO: 8-NH.sub.2 for 24 h. Untreated cells were used as basal control. Cells were then lysed for RNA extraction with a RNA purification commercial kit following manufacturer instructions (RNeasy mini kit, Qiagen, Netherlands). RNA was then quantified by nanodrop, adjusted in concentration and processed for retrotranscription to cDNA using a commercially available kit (High-Capacity cDNA Reverse Transcription kit, Thermofisher Scientific, USA). Resulting cDNA was used to perform a RTqPCR (Real Time Quantitative Polymerase Chain Reaction) using taqman technology and a panel of probes designed to target the specific genes related to muscle contractility mentioned in table 4.

(59) The results of this experiment appear summarized in FIG. 3(A) to (D). In said figures it can be seen that when human myocytes were treated with peptide Ac-SEQ ID NO: 5-NH.sub.2 a downregulation of UTRN, ACTA1, TNNC1, RAPSN, SCN3A and MYH1 was observed. Treatment of myocytes with peptide Ac-SEQ ID NO: 8-NH.sub.2 resulted in a downregulation of SCN3A, UTRN, ACTA1, TNNC1, CALM3, CAV1, CACNB1, LRP4, and MYH1 for the 6-hour treatment, together with a downregulation of RAPSN and an upregulation of ATP2A for the 24-hour treatment. Finally, treatment of myocytes with peptide Ac-SEQ ID NO: 10-NH.sub.2 resulted in a downregulation of UTRN, TNNC1, SCN3A CHRNA1 and CACNB1.

(60) Downregulation of the above-mentioned genes may affect normal muscle contractility function in different ways: RAPSN, CHRNA1, UTRN and LRP4, required for the binding of acetylcholine on the surface of the muscle cell, may affect neurotransmitter-induced membrane depolarization and cytoskeleton stability; SCN3A and CACNB1, as voltage-gated channels, may affect membrane potential an excitation transmission; SLN, involved in Ca.sup.2+ transportation, may affect intracellular calcium accumulation; and MYH1, TNNC1 and ACTA1 may affect cytoskeleton integrity and the power strike that drives contraction. Therefore, the results obtained in this example and shown in FIG. 3, demonstrate the ability of the peptides of the present invention to modulate muscular contraction-relaxation, contributing to an increase in the relaxation of muscles.

Example 10. Calcium Mobilization Assay

(61) Peptide Ac-SEQ ID NO: 8-NH.sub.2 was synthesized in accordance with examples 2 to 6.

(62) The potential of said peptide Ac-SEQ ID NO: 8-NH.sub.2 to reduce calcium mobilization on human skeletal muscle cells was evaluated in vitro with Fluo-4 NW Calcium Assay Kit (ThermoFisher Scientific, MA USA). Briefly, human skeletal muscle myoblasts were seeded in quintuplicates in a black 96-well plate, clear bottom, at a density of 1×10.sup.4 cells/well and maintained at standard culture conditions (37° C., 95% humidity, 5% CO.sub.2) for 48-72h. Myoblast differentiation to myocyte was then induced with specific differentiation medium (SKM-D medium+1% a/a) and monitored for 7 more days. Differentiated cells were treated with non-cytotoxic concentrations of peptide Ac-SEQ ID NO: 8-NH.sub.2 (0.01 mg/mL, 0.05 mg/mL and 0.1 mg/mL) for additional 48 hours.

(63) Once incubation was finished, cell culture medium was replaced by 100 μl of Dye loading solution and the plate was kept on the incubator for 30 minutes. Dye solution was then replaced by assay buffer prior to calcium measurement using FLUOstar Omega instrument (BMG Labtech, Germany). For appropriate kinetic measurement, a pre-stimulus phase of 10 seconds was set to determine the baseline signal before induction of calcium influx by addition of 60 mM KCl. Post-stimulus phase was set at 90 seconds, with readings every 0.1 seconds at 494/516 nm (excitation/emission).

(64) The following steps were performed for data analysis: 1) Calculation of a mean kinetic curve for each condition; 2) determination of maximum fluorescent signal for each curve after stimulation with KCl; 3) calculation of the fold change increase versus baseline; 4) normalization of results obtained for each condition (each peptide concentration) versus the one obtained for the control (untreated cells).

(65) The results obtained in this experiment appear summarized in FIG. 4 and reflect the potential of the peptides of the present invention (as exemplified by Ac-SEQ ID NO: 8-NH.sub.2) to reduce calcium influx in a dose dependent manner (−6%, −20% and −30% at 0.01 mg/mL, 0.05 mg/mL and 0.1 mg/mL respectively). This reduction in calcium influx has an impact on cell contractility favoring muscle relaxation.

(66) As can be directly derived from the above examples, the peptides of the present invention effectively interfere or inhibit the formation of the complex Munc18-Syntaxin-1, hence, allowing a regulation (inhibition) of neuronal exocytosis. In addition, said peptides provide a direct effect on muscle cells inducing or contributing to their relaxation (muscle relaxation). Therefore, the peptides of the present invention solve the above-mentioned problems present in the state of the art.

Example 11. Myosin Heavy Chain Protein Decrease

(67) The potential of said peptide Ac-SEQ ID NO: 8-NH.sub.2 to decrease the expression of the protein myosin heavy chain in human skeletal muscle cells was evaluated in vitro by immunofluorescence using a specific antibody against this protein (Biotechne, USA) followed by a secondary fluorescent antibody (Thermofisher, USA). Briefly, human skeletal muscle myoblasts were seeded onto coverslips in SKM-M medium (Tebu-Bio, France) at a density of 3×10.sup.4 cells/cm.sup.2 and incubated overnight at standard culture conditions (37° C., 95% humidity, 5% CO.sub.2). Myoblast differentiation to myocyte was then induced with specific differentiation medium (Skeletal Muscle Cell Differentiation Medium, SKM-D medium) and monitored for 7 more days. Differentiated cells were treated with non-cytotoxic concentrations of peptide Ac-SEQ ID NO: 8-NH.sub.2 (0.05 mg/mL and 0.5 mg/mL) for additional 48 hours.

(68) Once incubation was finished, cells were fixed with 4% PFA and permeabilized using 0.1% (v/v) Triton (Sigma, USA). Myosin was then stained with 0.5 mg/ml myosin heavy chain antibody during 2 h at room temperature. After proper washing, actin protein was labelled with 50 μg/ml Phalloidin (red) (Sigma, USA) for 1 h and, after washing, cells were stained with 4 μg/ml of the secondary antibody Goat anti-Mouse IgG for 1 h. Finally, nuclei were stained with 3.5 μg/ml Hoescht marker for 10 min (Sigma, USA).

(69) Microscopic images were acquired using the 5× and 10× objectives. Three replicates were used for each condition and images of three to four fields of each coverslip were acquired using the same settings.

(70) Images were analyzed using Image J software. Shortly, threshold was adjusted to select myosin and mean fluorescent was measured. The number of positive cells was counted using DAPI staining. Myosin mean fluorescent intensity was divided by the number of myosin-positive cells. Finally, all data was normalized as follows to obtain the % of myosin compared to non-treated cells: % vs. control=(fluorescence per cell in treated wells/fluorescence per cell in non-treated wells)×100.

(71) The results obtained in this experiment appear summarized in FIGS. 5(A), 5(B) and 6 and reflect the potential of the peptides of the present invention (as exemplified by Ac-SEQ ID NO: 8-NH.sub.2) to reduce myosin heavy chain protein levels in a dose dependent manner (−26% and −38% at 0.05 mg/mL and 0.5 mg/mL in FIG. 6, respectively). This reduction in myosin heavy chain protein levels has an impact on cell contractility favoring muscle relaxation.

(72) As can be directly derived from the above example, the peptides of the present invention effectively provide a direct effect on muscle cells inducing or contributing to their relaxation (muscle relaxation).

Example 12. Contraction Frequency

(73) The potential of said peptide Ac-SEQ ID NO: 8-NH.sub.2 to modulate the contraction frequency was evaluated in vitro using human muscle cells and motor neurons derived from hiPSC co-cultures, by means of live-imaging video of localized contractile muscle fibers recorded with an InCell 2200 automated microscope during 60 seconds, before and after treatment (after treatment, at each of the established timepoints).

(74) Human muscle cells were cultivated at a density of 1×10.sup.6 cells in T75 cm.sup.2 flasks and then transferred to 96 well plates for differentiation. Motor neurons derived from hiPSC were transferred onto the 96 well plates containing the muscle cells in a differentiation medium. Co-cultures were maintained for 10 days in order to generate neurons junctions with muscle fibers. Spontaneous contractions were observed within 5 days.

(75) Co-cultures were then treated with control medium (basal), 1 μM α-bungarotoxin, 0.5 mg/mL of acetyl hexapeptide-8 as a benchmark reference and 0.05 or 0.1 mg/mL of peptide Ac-SEQ ID NO: 8-NH.sub.2. Movies of co-cultures were recorded during 60 seconds before treatment and, after 30 minutes of incubation, movies were recorded again during 60 seconds. The culture plate was incubated again for 1 h 30 min with compounds (2 h of total incubation), and movies of co-cultures were recorded again during 60 seconds. The culture plate was incubated again for a total of 24 h with compounds and at the end of the incubation movies were recorded again during 60 seconds. Finally, compounds were washed out and a last recording after 24 h from said washed out of the compounds was done to assess an eventual recovery of muscle contractions (recovery).

(76) Frequencies of contraction were calculated before and after each incubation. For each culture condition, 6 wells were analyzed.

(77) The results obtained in this experiment appear summarized in FIG. 7 and reflect the potential of the peptides of the present invention (as exemplified by Ac-SEQ ID NO: 8-NH.sub.2) to induce and important dose-dependent decrease of muscles contraction frequency after only 30 min (25% and 18% of muscle contraction frequency with regard to the basal non-treated cells, at 0.05 and 0.1 mg/mL, respectively) which is maintained during the 24 h incubation period (23% and 10% at 0.05 and 0.1 mg/mL, respectively). The washout of this compound allows a partial recovery of the muscle contraction frequency which is better at 0.05 mg/mL concentration (65% and 35% at 0.05 and 0.1 mg/mL, respectively).

(78) The benchmark (acetyl hexapeptide-8) used at 0.5 mg/mL induced a partial inhibition of muscle contraction frequency after 30 min (18% muscle contraction frequency) but this effect was attenuated with longer incubations (45% after 24 h) and after a washout, the frequency was totally restored (100%).

(79) As can be directly derived from the above example, the peptides of the present invention effectively provide a direct effect on muscle contraction during neurons junctions formation with muscle fibers, contributing to their relaxation (muscle relaxation).

Example 13. Exocytosis Levels and Delay

(80) The potential of said peptide Ac-SEQ ID NO: 8-NH.sub.2 to modulate the exocytosis of vesicles containing neurotransmitters from a neuroblastoma cell line was evaluated in vitro using SH-SY5Y cells, by fluorescence imaging using a Zeiss axiovert 200 inverted epifluorescence microscope with a 20× objective and a Xenon lamp.

(81) SH-SY5Y cells (Sigma, USA) were cultured in T25 flasks in supplemented medium (DMEM/F12, Gibco, USA). Above 90% confluence, cells were trypsinized with 1 ml of 0.5% (v/v) Trypsin-EDTA. Next, 5 mL of said supplemented medium was added and cell concentration determined. Cells were seeded onto polyLysine-coated 12 mm-treated coverslips in 24 well-plates at 15×10.sup.4 cells/well in supplemented medium (Gibco, USA) and incubated at regular conditions (37° C., 5% CO.sub.2). After 24 h cell seeding, cells were transfected with exocytosis reporter using Lipofectamine™ 3000 (Invitrogen, USA) following manufacturer's instructions. Reporter construct contained fusion protein made up of intraluminal-specific proteins and a pH-sensitive fluorescent protein.

(82) For exocytosis monitorization, after 48 h of protein expression, cells were incubated with 0.01 mg/mL of Ac-SEQ ID NO: 8-NH.sub.2 peptide or 1 mg/mL of the benchmark acetyl hexapeptide-8 for 1 h at 37° C. and 5% CO.sub.2. Non-treated cells were used as basal control. Then, cells were pre-treated with 100 nM PMA for 15 min and stimulated with 12.5 μM ionomycin together with PMA (100 nM) for 5 minutes (total stimulation: 20 min). Fluorescence imaging was done for the last 10 min of the stimulation protocol. Fluorescence signal of exocytosis reporter was monitored employing 483-512 nm excitation-filter and 525-530 nm emission-filter. Images were captured with ORCA-ER CCD camera every 5 seconds for 10 min using Aquacosmos software.

(83) Non-treated cells were assayed in N=4 independent experiments with n=16 coverslips (483 cells measured), acetyl hexapeptide-8-treated cells in N=3 independent experiments with n=10 coverslips (328 cells measured) and Ac-SEQ ID NO: 8-NH.sub.2-treated cells in N=3 independent experiments with n=8 coverslips (240 cells measured). Cells were selected to individually monitor fluorescence intensity and time-course. Fluorescence peak evoked by ionomycin addition was used to quantify exocytosis by calculating the Area under the curve (AUC) using GraphPad software. Two parameters were defined and analyzed: Exocytosis levels: For individual cells, total peak area was obtained from AUC analysis and was normalized by the baseline that was defined as fluorescence intensity 3-cycles before lonomycin injection. Exocytosis delay: For individual cells, response time (min) was obtained from AUC analysis as time frame between lonomycin injection (Y) and initiation of peak (firstX).

(84) Fluorescence values were further normalized as percentage change to non-treated cells on each experiment as: (exocytosis level of treated cells/exocytosis levels non-treated cells)×100 and (response time of treated cells/response time of non-treated cells)×100.

(85) For analysis of exocytosis monitorization parameters: Data represent percentages normalized to non-treated cells for exocytosis levels and exocytosis delay. Data are expressed as mean±SEM; Data was obtained from N=4 independent experiments, n=16 replicates (483 cells measured) for non-treated cells; N=3 independent experiments, n=10 replicates for acetyl-hexapeptide-8 (328 cells measured); N=3 independent experiments, n=8 replicates (240 cells measured) for Ac-SEQ ID NO: 8-NH.sub.2.

(86) The results obtained in this experiment appear summarized in FIGS. 8 and 9 and reflect the potential of the peptides of the present invention (as exemplified by Ac-SEQ ID NO: 8-NH.sub.2) to reduce (FIG. 8) and delay (FIG. 9) the exocytosis of vesicles containing acetylcholine released by neurons that activate muscle contractions through specific acetylcholine receptors.

(87) Ac-SEQ ID NO: 8-NH.sub.2 incubated for 1 hour at 0.01 mg/mL significantly delayed exocytosis response time (24% delay) and decreased exocytosis levels (−14%), while acetyl hexapeptide-8 incubated for 1 hour at 1 mg/mL significantly delayed exocytosis response (67% delay) without altering exocytosis level (7%) in human neuroblastoma cell line SH-SY5Y.

(88) As can be directly derived from the above example, the peptides of the present invention effectively provide an indirect effect on muscle contraction by reducing the level of acetylcholine vesicles released by neurons to the synaptic space for muscle contraction and delaying said exocytosis.

Example 14. Collagen Production

(89) The potential of said peptide Ac-SEQ ID NO: 8-NH.sub.2 to modulate the production of collagen type I as a potential improver of skin firming was evaluated in vitro using human skin fibroblasts.

(90) Cell were seeded in 96-well plates at 1×10.sup.4 cells/well and maintained for 24 h at standard culture conditions (37° C., 95% humidity, 5% CO.sub.2). After 24 h incubation, medium was removed and new medium containing the peptide Ac-SEQ ID NO: 8-NH.sub.2 at 0.01, 0.05 or 0.1 mg/mL was added to the wells concentration. Treatment lasted 24 hours or 48 hours and at the end of the assay cell culture media were collected. Cells treated only with culture medium were used as basal control (non-treated cells).

(91) After 24 and 48 hours of treatment the amount of collagen type I produced and released by the cells (ex-novo collagen type I synthesis) was measured in cell culture medium by means of ELISA assay. The results of the test item were compared to basal control condition (non-treated cells). The treatments were performed in triplicate in three different experimental sessions.

(92) The determination of collagen type I synthesis was carried out by means of a competitive ELISA method. Samples were added to enzyme wells which were pre-coated with antibodies, then the recognition antigen labeled with Horseradish peroxidase (HRP) was added; after being incubated 1 hour at 37° C., both compete with solid phase antigen and form immune complex. After washing with phosphate buffer solution, the combined HRP catalyzes Tetramethylbenzidine (TMB) into blue, and turns into yellow by the action of acid; it has an absorption peak under 450 nm wavelength and its absorbance is negatively correlated with antigen density of sample. The plates were read by microplate reader.

(93) The quantitative determination uses a calibration curve made-up of known and growing concentrations of standard collagen type 1. The results are expressed as collagen type I concentration (μg/ml) in 50 μL cell culture medium.

(94) Three trials were performed for each determination in three different experimental sessions. The % variation in collagen type I content between negative controls and samples was calculated and a direct index of the efficacy of the peptide to increase collagen I synthesis was obtained.

(95) The results obtained in this experiment appear summarized in FIGS. 10(A) and 10(B) and reflect the potential of the peptides of the present invention (as exemplified by Ac-SEQ ID NO: 8-NH.sub.2) to induce the production of collagen type I by human dermal fibroblasts.

(96) Ac-SEQ ID NO: 8-NH.sub.2 peptide significantly boosted collagen type I production by fibroblasts after 24 h by 9%, 31% and 37.5% at 0.01, 0.05 and 0.1 mg/mL, respectively and after 48 h by 16%, 29.5% and 56.5% at 0.01, 0.05 and 0.1 mg/mL, respectively.

(97) As can be directly derived from the above example, the peptides of the present invention effectively provide a strong effect and improving the firmness and the quality of the skin.