MODIFIED PEPTIDES, COMPOSITION, METHOD FOR INHIBITING MYOCYTE CONTRACTION, METHOD FOR IMPROVING SKIN, AND USE OF A MODIFIED PEPTIDE

Abstract

MODIFIED PEPTIDES, COMPOSITION, METHOD FOR INHIBITING MYOCYTE CONTRACTION, METHOD FOR SKIN IMPROVEMENT, AND USE OF A MODIFIED PEPTIDE. The present invention relates to modified peptides comprising an amino acid sequence of SEQ ID No. 1 to 80 and a modification of the N- and / or C-terminal ends, preferably with a modification of an acetyl and / or carboxyl group. The present invention also relates to a composition comprising said modified peptides, methods for inhibiting myocyte contraction and improving skin, and the use of said modified peptide. Figure for abstract: none

Description

Title of the invention: MODIFIED PEPTIDES, COMPOSITION, METHOD FOR INHIBITING MYOCYTE CONTRACTION, METHOD FOR IMPROVING SKIN AND USE OF A MODIFIED PEPTIDE FIELD OF THE INVENTION

[0001] The present invention relates to modified peptides comprising modifications in the N- and / or C-terminal regions. The present invention also relates to a composition comprising said modified peptides, methods for inhibiting myocyte contraction and improving skin, and the use of said modified peptide. BACKGROUND OF THE INVENTION

[0002] Peptides derived from natural peptide precursors isolated from snake venom, for example, are widely used for various therapeutic or cosmetic purposes. A well-known example is captopril, the natural peptide precursor of which is isolated from the venom of the snake Bothrops jararaca. Captopril is a peptide drug that inhibits angiotensin-converting enzyme, producing an antihypertensive effect. Other useful peptides prepared from natural peptide precursors include natriuretic peptides, bradykinin-potentiating peptides, and sarafotoxins. Low-mass proteins such as crotamine, disintegrins, and three-finger toxins are derived from snake venom.

[0003] Some commercial peptide products contain synthetic peptides derived from natural peptides isolated from snake venom. For example, SYN® -AKE (DSM) is an effective anti-wrinkle compound based on a synthetic tripeptide, i.e., dipeptide diaminobutyroyl benzylamide diacetate, which mimics the activity of Waglerin 1, a polypeptide present in the venom of the temple viper Tropidolaemus wagleri. SYN®-AKE acts at the postsynaptic membrane and is a reversible antagonist of the muscle nicotinic acetylcholine receptor (mnAChR). Upon binding to the mnAChR by SYN®-AKE, Na+ uptake is blocked at the postsynaptic membrane and myocyte contraction is attenuated. SYN®-AKE helps reduce signal transmission between nerves and works in a similar way to Botox to relax muscles.

[0004] Although known peptides are already used in the cosmetic, dermo-cosmetic and dermatological fields, it has been shown that they are less stable than desired in terms of chemical stability in a composition and also in terms of metabolic / biological stability after being applied to a patient.

[0005] One of the main causes of the poor metabolic / biological stability of peptides is that they are susceptible to enzymatic degradation by peptidases, which are proteases capable of cleaving, and therefore often inactivating, small peptides. These enzymes are widely distributed on the surface of many different cell types and, given their wide distribution in the body, several tissues, including the skin, have the capacity to metabolize peptides into amino acid residues.

[0006] There remains a need for bioactive peptides that have better stability, particularly chemical and / or metabolic / biological.

[0007] In view of the above, the objective of the present invention is to provide peptides which have improved metabolic / biological and / or chemical stability in the composition and after being applied to a patient, in particular to the skin.

[0008] Thus, the inventors have succeeded in overcoming the problem of the state of the art and have surprisingly developed new peptides comprising C- and / or N-terminal modifications which have the desired improvement, said modifications preferably being with acetyl and / or carboxyl groups. Summary of the invention

[0009] The present invention relates to modified peptides comprising modifications in the N- and / or C-terminal regions, preferably with acetyl and / or carboxyl modification.

[0010] The present invention also relates to a composition comprising said modified peptides, methods for inhibiting myocyte contraction and improving the skin, and the use of said modified peptide in the cosmetic, dermo-cosmetic and dermatological fields. Brief description of the drawings

[0011] [Fig-1] [Fig. 1] shows the mass spectrum of the unacetylated peptide of SEQ ID NO: 2.

[0012] [Fig.2] [Fig.2] shows the chromatogram and mass spectra of the peptides acetylated SEQ ID NO: 2, 4 and 48 quantified via high-resolution LC / MS.

[0013] [Fig.3A] and [Fig.3B] Figures 3A and 3B show the results of a comparative study of skin metabolism, where [Fig.3A] represents the residual percentage of non-acetylated peptide of SEQ ID NO: 2 over time and [Fig.3B] represents the residual percentage of acetylated peptide of SEQ ID NO: 2 over time in a 2-hour experiment. The dotted lines represent the controls and the solid lines represent the data points with metabolic activity (Skin S9). DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention relates to modified peptides comprising an amino acid sequence of SEQ ID NO: 1 to 80, with a modification of the N- and / or C-terminals. In a preferred embodiment, the modification of the N- and / or C-terminal ends is selected from acetyl and / or carboxyl groups. In a preferred embodiment, the modified peptide comprises an amino acid sequence selected from SEQ ID NOS: 1 to 10, 48 and 51, with modification of the N- and / or C-terminal ends. In a more preferred embodiment, the modified peptide comprises an amino acid sequence of SEQ ID NO: 2 with modification of the N- and / or C-terminal ends.

[0015] The term "peptide" as used herein refers to a compound having two or more amino acids linked in a chain by peptide bonds, with or without a branched chain. Any amino acid in the peptide may have one or more post-translational modifications.

[0016] In a preferred embodiment, the modified peptide is a low mass peptide containing no more than 5, 10, 50 or 100 amino acids. In a more preferred embodiment, the modified peptide is a tetrapeptide having four amino acids linked in a straight chain without a branched chain comprising a terminal modification.

[0017] The modified peptides of the present invention are particularly suitable for skin improvement and may be applied topically and / or combined with certain medical / aesthetic procedures / devices (such as laser, radiofrequency, microneedling, among other techniques). By "skin improvement" one may understand one or more beneficial characteristics such as improvement of elasticity, skin quality (acne scars, pores, inflammation) and firmness, appearance of pores, as well as prevention of acne scars and improvement of the appearance of such scars, improvement / prevention of the appearance and appearance of wrinkles, among others.

[0018] The modified peptide can be used in the cosmetic, dermo-cosmetic and / or dermatological fields, such as a ready-to-use product intended to be applied by the end consumer, or a product intended to be applied by a health professional such as a dermatologist or a beautician for aesthetic purposes.

[0019] The present invention also relates to a composition comprising an effective amount of said modified peptide and a cosmetically, dermo-cosmetically and / or dermatologically acceptable vehicle, diluent or carrier.

[0020] The composition may comprise the modified peptide at a concentration of about 0.01 to 500 qM, about 0.1 to 100 qM, about 1 to 50 qM or about 1 to 10 qM, for example, 1 or 10 qM.

[0021] The composition may be a cosmetic composition and further comprise a cosmetically acceptable vehicle, diluent or carrier. The composition may be a dermo-cosmetic composition and further comprise a vehicle, a dermocosmetically acceptable diluent or carrier. The composition may be a dermatological composition and further comprise a dermatologically acceptable vehicle, diluent, or carrier. Cosmetically, dermocosmetically, and dermatologically acceptable vehicles, diluents, or carriers are known in the art and may be selected by the judgment of a technician.

[0022] The composition may be in the form of a lotion, gel, milk, serum, cream, emulsion, liquid, lyophilized powder, aerosol or spray. The composition may be incorporated into microneedle systems, such as patches or hydrogel devices. The composition may further contain other compounds commonly used in the art, such as surfactants, thickeners or gelling agents, preservatives, basifying or acidifying agents well known in the art, and in sufficient amounts to obtain the desired presentation form, in particular a more or less thick lotion, gel, emulsion or cream. The composition may be in the form of a pressurized aerosol or spray from a pump bottle.

[0023] The term "effective amount" means an amount of a modified peptide or composition comprising the modified peptide according to the present invention that is necessary to achieve a stated objective. The effective amount may vary depending on the nature of the modified peptide or composition, the type of target cells, the duration of treatment and the stated objective. A specific effective amount for a given peptide or composition can generally be determined by the judgment of a technician.

[0024] The present invention also relates to a method of inhibiting myocyte contraction, in which these cells are in contact with an effective amount of the modified peptide or the composition containing it, said contact resulting in the inhibition of myocyte contraction. The inhibition may be irreversible.

[0025] Depending on the method of inhibition, the myocytes may be treated for at least about 0.5, 1, 2, 5, 10, 12, 18, 24 or 48 hours, for example, at least about 5 or 24 hours. The contraction of the myocytes may be inhibited by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% relative to that observed before treatment.

[0026] In some embodiments, the myocytes are located in a muscle of a patient. Upon treatment of the myocytes with the modified peptide or composition containing it, the muscle may be relaxed.

[0027] The invention also relates to a method for improving the skin, comprising applying an effective amount of the modified peptide or the composition containing it. The skin can be treated at least once, twice or three times per day and / or for at least 7, 14, 21, 28, 60 or 90 days.

[0028] The present invention also relates to the use of the modified peptide for the manufacture of a composition or a product for cosmetic, dermo-cosmetic and / or dermatological treatments. In a preferred embodiment, said use is carried out using an aesthetic device for laser, radiofrequency and / or for microneedling.

[0029] A person skilled in the art can easily determine which field of application is of interest depending on the type of composition desired, for example, a treatment based on a ready-to-use cream, a dermatological composition to be applied by a health professional to the consumer. TERMS

[0030] The term "about," as used herein, when referring to a measurable value such as an amount, percentage, etc., is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the stated value, as the case may be.

[0031] Although the invention is illustrated and described herein with reference to specific embodiments, the invention is not intended to be limited to the details shown. Rather, various modifications may be made to the details within the scope and range of equivalents of the claims without departing from the invention. PEPTIDE MODIFICATIONS

[0032] As already mentioned, the term "peptide" as used herein refers to a compound having two or more amino acids linked in a chain by peptide bonds, with or without a branched chain. Any amino acid in the peptide may have one or more post-translational modifications.

[0033] In a peptide, the carboxyl group of one amino acid is linked to the amine group of another. One end of the peptide has a free amine group, while the other end has a free carboxyl group, forming the amino-terminal (N-terminal - [NH2]) and carboxy-terminal (C-terminal - [COOH]) ends, respectively. In one embodiment, the modified peptide is a tetrapeptide having four amino acids linked in a straight chain without a branched chain comprising a terminal modification.

[0034] "Terminal modification" may occur at the N- and / or C-terminal ends of the modified peptide and may include, but is not limited to, modifications, substitutions and / or conjugations of the amino- and carboxy-terminal ends. Terminal modifications may occur with an acetyl group, a methyl group, a phosphate group, a carboxyl group, a fatty acid including capryloyl groups, capryl and lauryl, an amide, an N-alkylamide, an aldehyde, a urea, an alkylamine, etc. In a preferred embodiment, the terminal modification comprises modification of the N- and / or C-termini with an acetyl and / or carboxyl group. In a preferred embodiment, the peptide is N-acetylated. In another preferred embodiment, the peptide is carboxylated.

[0035] Acetylation describes a reaction in which the positive charge on the N-terminus of peptides is removed. Carboxylation describes a reaction in which a carboxyl group is incorporated into the terminal end of the peptide. Amidation describes a reaction in which the C-terminus of the peptide is synthesized as an amide to neutralize the negative charge of the COOH C-terminus.

[0036] Terminal modifications may be synthesized via techniques customary in the art. The modified peptide may be synthesized chemically and / or enzymatically, and in a preferred embodiment, the peptide is synthesized enzymatically. Suitable enzymes may include transferases, which are enzymes that transfer a group from one compound to another and are classified according to their donor / acceptor group pattern. In another preferred embodiment, the peptide is synthesized chemically.

[0037] Terminal modifications may increase the cosmetic, metabolic / biological and / or chemical stability of the modified peptides by preventing degradation of the N- and / or C-termini by peptidases and / or by modifying the physicochemical properties of the peptides which may include the length, molecular weight, hydrophobicity, etc. of the peptides. EXAMPLES Example 1 - Modified Peptide

[0038] The modified peptide of the present invention comprises an amino acid sequence selected from SEQ ID NOS: 1 to 80, as shown in Table 1.

[0039]

[0040]

[0041] [Tables 1] SEQ ID N° Peptide SEQ ID N° Peptide SEQ ID N° Peptide SEQ ID N° Peptide 1 WQHE 21 KYWFY 41 KALA 61 KKKY 2 KWKK 22 IPSP 42 KFFCK 62 KYKK 3 NFKD 23 KVFL 43 RNYL 63 YKKK 4 RADH 24 YWFYP 44 YLKP 64 FKKK 5 CSEI 25 WKTYW 45 QLGK 65 KKKI 6 IIHK 26 NRNY 46 LKPF 66 KKFK 7 LWEY 27 FANL 47 KNYK 67 KKIK 8 IWDH 28 FCKKP 48 WKKK 68 KKKF 9 EPPA 29 GKIP 49 KYWL 69 KKLK 10 ACDN 30 YLQK 50 KKWK 70 KLKK 11 FYPAK 31 WFYG 51 KYWF 71 KKKS 12 WFYP 32 CYKK 52 YPAK 72 LKKK 13 YNKK 33 YSSY 53 NKKY 73 IKKK 14 KALAI 34 FYPA 54 KYKI 74 KIKK 15 YGCY 35 TYNK 55 NYKI 75 KSKK 16 CCYKK 36 KKLT 56 FCKK 76 KKSK 17 WFYPA 37 CKKP 57 KKKW 77 KKPK 18 GKVF 38 FFCK 58 YWFY 78 KKKL 19 NKYWF 39 YGAK 59 KKYK 79 KPKK 20 KFFC 40 KNNY 60 KFKK 80 PKKK Séquences de peptides Example 2 - Quantification of peptides via LC / MS with high resolution Acetylated peptides were quantified using a Vanquish UPLC chromatography system (Thermo™) coupled to a Q-Exactive mass spectrometer (Thermo™). Peptides were ionized using the positive electrospray ionization mode and detected using the full scan mode with a resolving power of 35,000 amu. Mass spectra showed doubly charged ions, and quantification was performed using the m / z ratio of 295.195 amu (with z=2, [Fig. 2]) for peptides KWKK (SEQ ID NO: 2) and WKKK (SEQ ID NO: 48) and the m / z ratio of ions of 322,166 (with z=2, [Fig.2]) for the tetrapeptide KYWF (SEQ ID NO: 51). The data were processed with Excalibur software and are illustrated in [Fig.2].

[0042] Example 3 - Comparative study of skin metabolism

[0043] The metabolic / biological stability of the acetylated and non-acetylated peptides of SEQ ID NO: 2 was studied with S9 subcellular fractions (S9) obtained from Episkin™ reconstructed human skin models, and liver. The half-life of the peptides was quantitatively determined and the qualitative appearance of the potential metabolites was studied. The protocol for the metabolism study with S9 skin is presented below.

[0044] [Tables2] Liver and skin S9 [Biological material] (mg prot. / ml) 2 [peptides] (pM) 5 Buffer Phosphate buffer (100 mM, pH 7.4) [Cofactors] (pM): None Kinetics at 37°C: 0 to 120 min Analytical method: - Quantification of the substrate - Identification of metabolites LC / MS LC / MS

[0045] Protocol for the study of skin metabolism with skin S9

[0046] A control carried out under the same experimental conditions without S9 was also performed in order to verify whether the disappearance of the parent compound is linked to the enzymatic activities. At different time points (0, 5, 10, 15, 30, 60 and 120 min), 100 μl of medium were inactivated with 200 μl of methanol with 0.1% formic acid. Samples were then analyzed by LC / MS to quantify the peptides. Potential metabolites (loss of one or two amino acids on the C-terminus) were identified by LC / MS.

[0047] The half-life of the disappearance of the test peptide results from an adaptation of the curve using data of residual concentration of the parent peptide versus time using GraphPad software (V. 5.02). The percentage of residual peptides over time is shown in FIG. 3.

[0048] It is clear from [Fig.3A] and 3B that this non-acetylated peptide shows a very rapid decrease over time with a half-life of less than 5 min, while the acetylated peptide shows the same behavior with or without the metabolically active fraction.

Claims

Claims

1. A modified peptide comprising an amino acid sequence of SEQ ID NO: 2 and a modification of the N- and / or C-terminal ends.

2. Peptide according to claim 1, wherein the modification of the N- and / or C-terminal ends is carried out with an acetyl and / or carboxyl group.

3. Modified peptide comprising an amino acid sequence of SEQ ID NO: 2 and an N- and / or C- terminal modification, for use as a cosmetic, dermo-cosmetic and / or dermatological product.

4. A composition comprising a modified peptide comprising an amino acid sequence of SEQ ID NO: 2 and an N- and / or C-terminal modification, as well as a cosmetically, dermo-cosmetically and / or dermatologically acceptable vehicle, diluent or support.

5. A composition according to claim 4, the composition being in the form of a lotion, gel, milk, serum, cream, emulsion, liquid, lyophilized powder, aerosol or spray or being incorporated into microneedle systems, such as patches or hydrogel devices.