Modified peptide, composition, method for inhibiting muscle cell construction, method for improving skin, and

Abstract

The present invention relates to modified peptides comprising the amino acid sequence of SEQ ID NO: 1 to 80 and N and / or C-terminal modifications, preferably with acetyl and / or carboxyl modifications. The invention also relates to a composition comprising said modified peptide, to a method for inhibiting muscle cell contraction and improving skin, and to the use of said modified peptide.

Description

Technical Field

[0001] The present invention relates to modified peptides, which comprise modifications in the N- and / or C-terminal regions. The present invention also relates to compositions comprising said modified peptides, to methods for inhibiting muscle cell contraction and improving the skin, and to the use of said modified peptides. Background Art

[0002] Peptides derived from natural peptide precursors are widely used for various therapeutic or cosmetic purposes, said natural peptide precursors being isolated from, for example, snake venoms. Well-known examples are captopril, the natural peptide precursor of which is isolated from the venom of the Bothrops jararaca snake. Captopril is a polypeptide-based drug that inhibits angiotensin-converting enzyme and produces an antihypertensive effect. Other useful peptides prepared from natural peptide precursors include natriuretic peptides, bradykinin potentiating peptides, and ceratoxin. Low molecular weight proteins such as crotamine, disintegrins, and three-finger toxins are derived from snake venoms.

[0003] Some commercial peptide products contain synthetic peptides derived from natural peptides, said natural peptides being isolated from snake venoms. For example, SYN®-AKE (DSM) is an effective wrinkle-smoothing compound based on a synthetic tripeptide (i.e., dipeptide diamino butyryl benzamide diacetate), which mimics the activity of Waglerin 1, a polypeptide found in the venom of the Temple Viper (Tropidolaemus wagleri). SYN®-AKE acts on the postsynaptic membrane and is a reversible antagonist of the muscular nicotinic acetylcholine receptor (mnAChR). After SYN®-AKE binds to mnAChR, Na + uptake is blocked at the postsynaptic membrane and muscle cell contraction is attenuated. SYN®-AKE is able to reduce signal transmission between nerves and acts in a manner similar to Botox in relaxing muscles.

[0004] Although known peptides have been used in the fields of cosmetics, skin cosmetics, and dermatology, they have shown less than desired stability in terms of their chemical stability in compositions and in terms of their metabolic / biological stability after being applied to a subject.

[0005] One of the main reasons for the poor metabolic / biological stability of peptides is their susceptibility to enzymatic cleavage by peptidases, which are proteases capable of cleaving small peptides and thus often inactivating them. These enzymes are widely distributed on the surfaces of many different cell types, and due to their widespread distribution in the body, some tissues, including the skin, have the ability to metabolize peptides into amino acid residues.

[0006] There is still a need for bioactive peptides with improved stability, especially chemical and / or metabolic / biological stability.

[0007] In view of the above, an object of the present invention is to provide peptides that have improved metabolic / biological and / or chemical stability in a composition and after administration to a subject, especially after administration to the skin.

[0008] Thus, the inventors have successfully overcome the problems of the prior art and unexpectedly developed new peptides that contain C- and / or N-terminal modifications with the desired improvements, preferably modified with an acetyl group and / or a carboxyl group. Summary of the Invention

[0009] The present invention relates to modified peptides that contain modifications in the N- and / or C-terminal regions, preferably modified with an acetyl group and / or a carboxyl group.

[0010] The present invention also relates to a composition containing the modified peptide, a method for inhibiting muscle cell contraction and improving the skin, and the use of the modified peptide in the fields of cosmetics, skin cosmetics, and dermatology. Brief Description of the Drawings

[0011] Figure 1 The mass spectrum of the non-acetylated peptide of SEQ ID NO:2 is shown.

[0012] Figure 2 The chromatograms and mass spectra of the acetylated peptides of SEQ ID NO:2, 4, and 48 quantified by high-resolution LC / MS are shown.

[0013] Figure 3 A and 3B show the results of a comparative skin metabolism study, wherein Figure 3 A represents the percentage remaining over time of the non-acetylated peptide of SEQ ID NO:2 in a 2-hour experiment, Figure 3 B represents the percentage remaining over time of the acetylated peptide of SEQ ID NO:2 in a 2-hour experiment. The circles represent the control, and the solid dots represent the data points with metabolic activity (skin S9). Detailed Description

[0014] The present invention relates to modified peptides that contain the amino acid sequences of SEQ ID NOs: 1 to 80 with N- and / or C-terminal modifications. In a preferred embodiment, the N- and / or C-terminal modifications are selected from an acetyl group and / or a carboxyl group. In a preferred embodiment, the modified peptide contains the amino acid sequence selected from SEQ ID NOs: 1 to 10, 48, and 51 with N- and / or C-terminal modifications. In a more preferred embodiment, the modified peptide contains the amino acid sequence of SEQ ID NO:2 with N- and / or C-terminal modifications.

[0015] As used herein, the term "peptide" refers to a compound having two or more amino acids, in which the two or more amino acids are linked 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 molecular weight peptide having no more than 5, 10, 50 or 100 amino acids. In a further preferred embodiment, the modified peptide is a tetrapeptide having four amino acids, which are linked in a straight chain, without a branched chain, and include terminal modifications.

[0017] The modified peptides of the present invention are particularly suitable for improving the skin and can be applied topically and / or in connection with some medical / cosmetic procedures / devices (such as laser, radiofrequency, microneedle and other techniques). By "improving the skin", one or more beneficial features can be understood, such as improving elasticity, skin quality (acne scars, pores, inflammation) and the appearance of tightened pores, as well as preventing acne scars and improving the appearance of the scars, improving / preventing the appearance and aspect of wrinkles, etc.

[0018] The modified peptides can be used in the fields of cosmetics, skin cosmetics and / or dermatology, such as ready-to-use products applied to the end consumer, or products for cosmetic purposes applied by health professionals, such as dermatologists or beauticians.

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

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

[0021] The composition can be a cosmetic composition and further includes a cosmetically acceptable excipient, diluent or carrier. The composition can be a skin-cosmetic composition and further includes a skin-cosmetically acceptable excipient, diluent or carrier. The composition can be a dermatological composition and further includes a dermatologically acceptable excipient, diluent or carrier. Cosmetically, skin-cosmetically and / or dermatologically acceptable excipients, diluents or carriers are known in the art and can be selected according to the judgment of those skilled in the art.

[0022] The composition can be in the form of a lotion, gel, emulsion, serum, cream, milky suspension, liquid, lyophilized powder, aerosol or spray. The composition can be incorporated into a microneedle system, such as a hydrogel patch or device. The composition can further contain other compounds commonly used in the art, such as surfactants, thickeners or gelling agents, preservatives, alkalizing or acidifying agents well known in the art, and in amounts sufficient to obtain the desired presentation form, in particular more or less thickened lotions, gels, milky suspensions or creams. The composition can be in the form of a pressurized aerosol or spray from a pump bottle.

[0023] The term "effective amount" refers to the amount of the modified peptide or the composition comprising the modified peptide according to the invention required to achieve a specified goal. The effective amount can vary depending on the nature of the modified peptide or composition, the type of target cell, the treatment time and the specified goal. The specific effective amount of a given peptide or a given composition can generally be set according to the judgment of a person skilled in the art.

[0024] The present invention also relates to a method for inhibiting muscle cell contraction, wherein these cells are contacted with an effective amount of a modified peptide or a composition comprising it, and said contact results in the inhibition of muscle cell contraction. The inhibition can be irreversible.

[0025] According to this inhibition method, the muscle cells can be treated for at least about 0.5, 1, 2, 5, 10, 12, 18, 24 or 48 hours, such as at least about 5 or 24 hours. Compared with before treatment, muscle cell contraction can be inhibited by at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%.

[0026] In some embodiments, the muscle cells are in the muscle of a subject. After treating the muscle cells with the modified peptide or the composition comprising it, the muscle can relax.

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

[0028] The present invention also relates to the use of the modified peptide in the preparation of a composition or product for cosmetic, skin cosmetic and / or dermatological treatment. In a preferred embodiment, the use is achieved with the assistance of a cosmetic device such as a laser, radiofrequency and / or microneedle.

[0029] A person skilled in the art can easily determine the target application area based on the desired type of composition. For example, a treatment with a ready-to-use cream of a dermatological composition is administered by a healthcare professional to a consumer.

[0030] Term

[0031] As used herein, the term "about", when referring to measurable values such as amounts, percentages, etc., means a variation encompassing ±20%, or ±10%, more preferably ±5%, even more preferably ±1% and still more preferably ±0.1% of the specified value, as such variation is appropriate.

[0032] Although the present invention has been illustrated and described herein with reference to specific embodiments, the present invention is not intended to be limited to the details shown. Instead, various modifications may be made to the details within the scope and equivalence of the claims without departing from the present invention.

[0033] Modification of Peptide

[0034] As previously mentioned, the term "peptide" as used herein refers to a compound having two or more amino acids, in which the two or more amino acids are linked by peptide bonds, with or without a branched chain. Any amino acid in the peptide may have one or more post-translational modifications.

[0035] In a peptide, the carboxyl group of one amino acid binds to the amino group of another amino acid. One end of the peptide has a free amino group, while the other end has a free carboxyl group, forming an amino (N-terminal – [NH2]) and a carboxyl (C-terminal – [COOH]) end, respectively. In one embodiment, the modified peptide is a tetrapeptide having four amino acids, the four amino acids being linked in a straight chain, without a branched chain, and comprising terminal modifications.

[0036] "Terminal modification" can occur at the N-terminal and / or C-terminal of the modified peptide and can include, but is not limited to, amino and carboxyl terminal modifications, substitutions, and / or conjugations. Terminal modifications can occur with acetyl, methyl, phosphate, carboxyl, fatty acids including octanoyl, decanoyl, and lauroyl, amide, N-alkylamide, aldehyde, urea, alkylamine, etc. In a preferred embodiment, the terminal modification includes N- and / or C-terminal modification with acetyl and / or carboxyl. In one preferred embodiment, the peptide is N-acetylated. In another preferred embodiment, the peptide is carboxylated.

[0037] Acetylation describes a reaction in which the positive charge on the N-terminal of the peptide is removed. Carboxylation describes a reaction in which a carboxyl group is incorporated into the peptide terminus. Amidation describes a reaction in which the C-terminal of the peptide is synthesized as an amide to neutralize the negative charge of the C-terminal COOH.

[0038] Terminal modifications can be synthesized by conventional techniques in the art. Modified peptides can be synthesized chemically and / or enzymatically, and in a preferred embodiment, the peptides are synthesized enzymatically. Suitable enzymes can include transferases, which are enzymes that transfer groups from one compound to another and are classified according to their donor:acceptor group scheme. In another preferred embodiment, the peptides are synthesized chemically.

[0039] Terminal modifications can increase the cosmetic, metabolic / biological, and / or chemical stability of modified peptides by preventing N-terminal and / or C-terminal degradation by peptidases and / or by modifying the physicochemical properties of the peptides, including peptide length, molecular mass, hydrophobicity, etc.

[0040] Example

[0041] Example 1 - Modified Peptide

[0042] The modified peptides of the present invention comprise an amino acid sequence selected from SEQ ID NO: 1 to 80, as shown in Table 1.

[0043] Table 1: Peptide Sequences

[0044]

[0045]

[0046] Example 2 - Peptide Quantification by High Resolution LC / MS

[0047] The acetylated peptides were quantified by connecting to the UPLC Vanquish (Thermo TM ) chromatographic system of the mass spectrometer Q-exactive (Thermo TM ). The peptide was ionized by electrospray positive ion mode and detected by full scan mode with a resolution of 35000 amu. The mass spectrum showed two charged ions. The quantification of the peptides KWKK (SEQ ID NO: 2) and WKKK (SEQ ID NO: 48) was carried out at the ion m / z 295.195 amu (z = 2, Figure 2 ), and the quantification of the tetrapeptide KYWF (SEQ ID NO: 51) was carried out at the ion m / z 322.166 (z = 2, Figure 2 ). The data was processed by Excalibur software, as Figure 2 shown.

[0048] Example 3 - Comparative Skin Metabolism Study

[0049] The metabolic / biological stability of the acetylated and non-acetylated peptides of SEQ ID NO: 2 was studied using the reconstructed human skin model Epikin TMPerformed with subcellular S9 fraction (S9) obtained from the skin and liver. The half-life of the polypeptide was determined quantitatively, and the qualitative appearance of potential metabolites was investigated. The protocol for the metabolism study with skin S9 is shown below.

[0050] Table 2: Protocol for Skin Metabolism Study with Skin S9

[0051]

[0052] Controls without S9 were performed under the same experimental conditions to check whether the disappearance of the parent compound was related to the enzyme activity. At different time points (0, 5, 10, 15, 30, 60, and 120 min), 100 μL of the culture medium was quenched with 200 μL of methanol 0.1% formic acid. Samples were then analyzed by LC / MS to quantify the peptides. Potential metabolites (deletion of one or two amino acids at the C-terminus) were identified by LC / MS.

[0053] The half-life of the disappearance of the test peptide was obtained by curve fitting of the data of the residual concentration of the parent peptide over time using GraphPad software (V. 5.02). The percentage of the remaining peptide over time is as Figure 3 shown.

[0054] From Figure 3 A and 3B, it can be clearly seen that the non-acetylated peptide showed a very rapid decline over time, with a half-life of less than 5 minutes, while the acetylated peptide showed the same behavior with or without the metabolically active fraction.

Claims

1. A modified peptide comprising an amino acid sequence of SEQ ID NOs: 1 to 80 and an N- and / or C-terminal modification.

2. The peptide according to claim 1, wherein the amino acid sequence is selected from SEQ ID NOs: 1 to 10, 48, and 51.

3. The peptide according to claim 1, wherein the amino acid sequence is SEQ ID NO:

2.

4. The peptide according to claim 1, wherein the N- and / or C-terminal modification is effected with an acetyl and / or carboxyl group.

5. A modified peptide comprising an amino acid sequence of SEQ ID NOs: 1 to 80 and an N- and / or C-terminal modification, for use as a cosmetic, skin cosmetic, and / or dermatological product.

6. The peptide according to claim 5, wherein the amino acid sequence is selected from SEQ ID NOs: 1 to 10, 48, and 51.

7. A composition comprising a modified peptide comprising an amino acid sequence of SEQ ID NOs: 1 to 80 and an N- and / or C-terminal modification, and a cosmetically, skin cosmetically, and / or dermatologically acceptable excipient, diluent, or carrier.

8. The composition according to claim 7, wherein the composition is in the form of a lotion, gel, emulsion, serum, cream, milky lotion, liquid, lyophilized powder, aerosol, or spray, or incorporated into a microneedle system, such as in the form of a hydrogel patch or device.

9. The composition according to claim 7, wherein the modified peptide comprises an amino acid sequence selected from SEQ ID No: 1 to 10, 48, and 51.

10. A method of inhibiting muscle cell contraction, comprising treating muscle cells with an effective amount of a modified peptide or a composition comprising said peptide, wherein the peptide comprises an amino acid sequence of SEQ ID NO: 1 to 80 and an N- and / or C-terminal modification, whereby the contraction of the muscle cells is inhibited.

11. A method of improving skin, comprising administering an effective amount of a modified peptide or a composition comprising said modified peptide, wherein the modified peptide comprises an amino acid sequence of SEQ ID NO: 1 to 80 and an N- and / or C-terminal modification.

12. The method according to claim 11, wherein improving the skin relates to the appearance of wrinkles and skin quality (acne scars, pores, inflammation).

13. The method according to claim 10 or 11, wherein the modified peptide comprises an amino acid sequence selected from SEQ ID No: 1 to 10, 48, and 51.

14. Use of a modified peptide for the preparation of a composition or product for cosmetic, skin cosmetic, and / or dermatological treatment, the modified peptide comprising an amino acid sequence of SEQ ID NO: 1 to 80 and an N- and / or C-terminal modification.

15. The use according to claim 14, wherein the use is effected with the aid of a cosmetic device such as a laser, radiofrequency, and / or microneedle.

16. The use according to claim 14, wherein the modified peptide comprises an amino acid sequence selected from SEQ ID NOs: 1 to 10, 48, and 51.

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