BIOACTIVE PEPTIDES AND COMPOSITIONS COMPRISING THEM

Abstract

The present invention relates to bioactive peptides and to the cosmeceutical compositions containing them, useful for the prevention and treatment of the signs of skin aging, such as wrinkles, fine lines and loss of firmness and elasticity of the skin, thanks to their efficacy in the protection of the dermal collagen.

Claims

1. A peptide derivative having formula (I) $\begin{array}{cc}\text{X-DAsn-DVal-DVal-DLys-Y}& \left(I\right)\end{array}$ wherein: X is an acyl group —CO—(CH.sub.2).sub.nCH.sub.3 linked to the Asparagine N-terminus, wherein n is an integer comprised between 0 and 20, and Y is the —OH at the Lysine C-terminus, or it is an amino group —NR.sub.1R.sub.2 linked to the Lysine C-terminus, wherein R.sub.1 and R.sub.2, equal or different from each other, are selected from between H and an alkyl group —(CH.sub.2).sub.mCH.sub.3 wherein m is an integer comprised between 0 and 2.

2. The peptide according to claim 1, having general formula (I) wherein, when Y is —NR.sub.1R.sub.2, R.sub.1 and R.sub.2 are both H.

3. The peptide of claim 1, having general formula (I) wherein n is 0.

4. The peptide of claim 1, having general formula (I) wherein n is 14.

5. The peptide of claim 1, which is selected from the group consisting of H.sub.3C—CO-DAsn-DVal-DVal-DLys-NH.sub.2 (SEQ ID NO:1) and H.sub.3C—(CH.sub.2).sub.14—CO-DAsn-DVal-DVal-DLys-NH.sub.2 (SEQ ID NO:2).

6. A process for the preparation of the peptide derivative as defined in claim 1, comprising a step of immobilizing a first amino acid on a solid support, followed by a step of adding each of the amino acids from the second to the fourth in the same order of general formula (I), and by a step of acylation at the Asn N-terminus, optionally followed by a step of amidation at the Lys C-terminus.

7. A cosmetic composition comprising a peptide derivative of general formula (I) as defined in claim 1, and one or more cosmetically acceptable excipients, adjuvants or carriers.

8. The cosmetic composition of claim 7, wherein said cosmetically acceptable excipients, adjuvants or carriers are selected from the group consisting of perfumes, colouring agents, wetting/moisturising agents, preservatives, functional ingredients and/or claim ingredients, solvents, and mixtures thereof.

9. The cosmetic composition of claim 7, which is in the form of serum, lotion, cream, gel, ointment or mousse for topical application.

10. A cosmetic method for preventing and treating the signs of skin ageing comprising the application on the skin of the cosmetic composition of claim 7.

11. A pharmaceutical composition comprising a peptide derivative of general formula (I) as defined in claim 1, and one or more pharmaceutically acceptable excipients, adjuvants or carriers.

12. A method of treating the skin following cosmetic surgery, comprising applying a pharmaceutical composition of claim 11 to an area of skin after cosmetic surgery.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. 1 shows a histogram representing the concentration of Type I procollagen detected on cellular samples of fibroblasts treated with different products and in parallel with a peptide of this invention, as described in detail in the following examples.

DETAILED DESCRIPTION OF THE INVENTION

[0015] In the present invention, the terms “cosmetic composition” and “cosmeceutical composition” are indifferently used to mean a composition that has been formulated and manufactured by using the methods typical of the pharmaceutical preparations.

[0016] The bioactive peptides derivatives of the present invention are tetrapeptides, consisting of amino acids of the D series only, having the following general formula (I):

[00001]$\begin{array}{cc}\text{X-DAsn-DVal-DVal-DLys-Y}& \left(I\right)\end{array}$

[0017] wherein:

[0018] X is an acyl group —CO—(CH.sub.2).sub.nCH.sub.3 linked to the N-terminus of asparagine, wherein n is an integer ranging between 0 and 20, and

[0019] Y represents a —OH group at the C-terminus of lysine, or it is an amine group —NR.sub.1R.sub.2 linked to the C-terminus of lysine, wherein R.sub.1 and R.sub.2, equal or different to each other, are selected from between H and an alkyl group —(CH.sub.2).sub.mCH.sub.3 wherein m is an integer ranging between 0 and 2.

[0020] They are therefore tetrapeptides having the sequence of D amino acids shown above in the general formula (I), acylated at the N-terminus of the amino acid asparagine and optionally amidated at the C-terminus of the lysine.

[0021] According to a preferred embodiment of the invention, when Y is —NR.sub.1R.sub.2, then R.sub.1 and R.sub.2 are both H.

[0022] In a particular embodiment of the present invention, the present peptide has the above said formula (I) wherein n is an integer selected from 0 and 14, therefore X is respectively acetyl (indicated below also with the symbol “Ac”) or palmitoyl.

[0023] Preferred according to the invention are the following tetrapeptide derivatives:

TABLE-US-00001 (SEQ ID NO: 1) H.sub.3C-CO-DAsn-DVal-DVal-DLys-NH.sub.2, and (SEQ ID NO: 2) H3C-(CH.sub.2).sub.14-CO-DAsn-DVal-DVal-DLys-NH.sub.2.

[0024] The peptide derivatives subject of this invention can be prepared by synthesis. For instance they can be prepared by peptide synthesis on solid phase, wherein the single amino acids of the peptide, in the same order of their position in formula (I), are added and attached to a first amino acid immobilised on a solid support, for example on a suitably functionalised resin. Then they are suitably acylated at the N-terminus before detachment from the resin. The peptides of the present invention can be advantageously prepared by using a strategy 9-fluorenyl-methoxycarbonyl/tert-butyl (Fmoc/tBu), such as that described in detail in the following experimental part.

[0025] Inventors have found that the peptides of the invention, and in particular Ac-DAsn-DVal-DVal-DLys-NH.sub.2 (SEQ ID NO:1), have a surprising activity of protection against degradation of the dermal collagen. Furthermore, they have higher stability in vivo, thus being able to guarantee their protective effect on collagen for a prolonged period. They are therefore particularly useful as active principles in cosmetic or cosmeceutical compositions. Subject of this invention are therefore also those compositions comprising at least a peptide of formula (I) as defined above, together with one or more cosmetically acceptable excipients, adjuvants or carriers. The amount of peptide of formula (I) present in the composition can be for example comprised between 0.01 and 1% by weight with respect to the total weight of the composition, and it can be accompanied by one or more further cosmetically active principles. Examples of cosmetically acceptable excipients, adjuvants or carriers can be selected amongst perfumes, colouring agents, wetting/moisturizing agents, preservatives, functional and/or claim ingredients, solvents, and mixtures thereof. In an aspect of the present invention, the present compositions are formulated for the topical administration, for example in the form of serum, gel, lotion, ointment, cream or mousse.

[0026] Any person with ordinary technical skills in the field could easily and without efforts select the excipients, adjuvants and/or carriers most suitable to formulate the peptide composition of the invention to obtain the desired final form. The optimal conditions of preparation of uniform and stable compositions could be established as well. The thus prepared, present compositions can be packed in jar containers or can be distributed by means of pump dispensers, typically used for cosmetic lotions and gels.

[0027] Subject of this invention is moreover a cosmetic method for the prevention and treatment of the cutaneous ageing of skin, such as wrinkles, fine lines, loss of firmness and elasticity of the skin, comprising applying on the skin of a subject in need thereof the present cosmetic composition.

[0028] The peptides of this invention, for their properties towards dermal collagen, can furthermore find application also as active principles in pharmaceutical compositions useful in treatments following aesthetic surgery, where it is required a stimulation of the collagen production or a protection against degradation of the dermal collagen, for example after thread lifting treatments, or similar. Subject of this invention are therefore also such pharmaceutical compositions comprising at least a peptide of formula (I) as defined above, together with one or more pharmaceutically acceptable excipients, adjuvants or carriers; and the same compositions for use in the stimulation of the production of collagen or in the protection against collagen degradation in treatments following aesthetic surgery.

[0029] The cosmetic compositions and the pharmaceutical compositions of this invention ca comprise, besides at least an active peptide of formula (I), also one or more further ingredients having respectively cosmetic and/or pharmaceutical/dermatological activity.

[0030] As shown below in the experimental part, the peptides of this invention are endowed with an inhibitory activity in the degradation of dermal collagen that is surprisingly higher also with respect to Serpin A1 and fragments thereof previously identified by the inventors. Furthermore, they have been proved without undesired effects of cellular proliferation, they only have a protective action on the dermal collagen. This action was proved to be efficacious both on cells coming from old subjects, and also on cells coming from young subjects, thus showing a usefulness of the present peptides towards the physiological skin ageing conditions, but also promising good protective results also in the case of an excessive degradation rate of the dermal collagen with respect to the capacities of the subject of synthesising new collagen because of pathological conditions.

[0031] The present peptides are moreover tetrapeptides, therefore they are short-chain peptides, easier to synthesise, also on a large scale in industry.

[0032] The experimental part that follows is provided as an illustrative, non-limiting, example of the present invention.

[0033] Experimental Part

[0034] Synthesis of the Peptide Ac-DAsn-DVal-DVal-DLys-NH.sub.2 (SEQ ID NO. 1)

[0035] The peptide of the title according to the invention, also indicated in the following and in FIG. 1 with the code AAT11RI, has been prepared by Solid-Phase Peptide Synthesis (SPPS) in a manual way, using a resin Rink-Amide AM and the 9-fluorenyl-methoxycarbonyl/tert-butyl strategy (Fmoc/tBu). After the attachment of the final amino acid to the resin, the peptide was acetylated at the N-terminus with acetic anhydride, then detached from the solid support with a mixture of TFA:H.sub.2O:TIS:EDT (94:2.5:1:2.5).

[0036] The so obtained raw peptide was purified by semi-preparative RP-HPLC chromatography, to obtain a chromatographic purity of 98%, and then freeze-dried. Finally, the so obtained purified peptide of the title was characterised by analytical HPLC chromatography and by electrospray ionization mass spectrometry (ESI-MS).

[0037] Cell Cultures

[0038] Neonatal human dermal fibroblasts (NHDF) have been obtained from Lonza. Cultures of adult dermal fibroblasts have been prepared from cutaneous skin biopsies of healthy donors having different ages, as described in Pani et al. J. Alzheimer's Dis. (2009) 18:829-41, C6 (36-year-old men), C3 (57-year-old woman) and C12 (84-year-old woman). A written informed consent to use these dermal cells was obtained from all the donor subjects involved, according to the Guidelines indicated by the Ethics Committee of the Careggi University Hospital (Florence, Italy) and by the Helsinki Declaration of 1975, as reviewed in the 1983. All cell cultures have been subjected to a similar number of steps, from 6 to 9, at the beginning of the experiments, and were preserved in the modified Dulbecco Eagle culture medium (DMEM, Lonza) added with FBS 10% (Gibco), 100 units/ml of penicillin G, 0.05 mg/ml of streptomycin and glutamine 2 mM (PAN-Biotech GmbH), at 37° C. in a humidified incubator containing 5% of CO.sub.2.

[0039] The cells were seeded in 12-wells plates (30000 cells per well) and treated the day after with the peptide AAT11RI (20 mM). In parallel the same cells were treated with TGF-β1 (10 ng/mL) and with the peptide SA1III (SEQ ID No: 3), which is the peptide previously described in Cipriani C. et al., Cell. Biol. International (2018) 42:1340-1348, as positive controls. The same cells were furthermore treated also with comparison peptides, not part of the invention, Peptide 1 (Ac-Lys-Val-Val-Asn-NH.sub.2-SEQ ID No: 4), and Peptide 2 (Ac-DLys-DVal-DVal-DAsn-NH.sub.2— SEQ ID No: 5).

[0040] All treatments have been carried out in the absence of FBS to avoid interferences in the electrophoretic migration. After 72 hours of incubation, the culture media have been collected, centrifuged at 250×g for 5 minutes; the supernatants were aliquoted and stored at −20° C. for analysis of collagen and zymography. The cellular proteins have been extracted in a RIPA buffer containing 1% of a mixture of inhibitors of proteases and phosphatases (Sigma-Aldrich Chemicals) with the help of a cell scraper. Cell lysates were then sonicated, purified by centrifugation and supernatants collected and stored at −20° C. The protein content in the lysates was measured using the Bio-Rad DC protein assay kit.

[0041] Test of Cells Viability

[0042] Cell viability assays were performed with the MTS cell proliferation tests in 96-well microplates (8,000 cells per well) after 72 hours of the treatments described above, following the instructions of the test manufacturer (Promega). All cell cultures have maintained their initial viability after the treatments.

[0043] Analisi Western Blot

[0044] 30-40 μg of protein lysates obtained as described above for each cell sample have been subjected to electrophoretic separation on polyacrylamide gel with 4-12% of sodium dodecyl sulphate (Bis-Tris Plus BOLT, Invitrogen) under standard denaturation and reduction conditions, then they have been transferred onto polyvinylidene fluoride membranes (PVDF, Millipore). Collagen I and GAPDH proteins (the latter used as load control) have been determined by immunohistochemical method with primary polyclonal rabbit antibodies: anti-collagen type I, ab34710, Abcam; anti-GAPDH, 14C10 (Cell Signaling Technology) and with suitable secondary antibodies conjugated to peroxidase (Sigma-Aldrich Chemicals). The protein bands were visualized by using an enhanced chemiluminescence procedure with the Immobilon™ substrate of horseradish peroxidase (Millipore) and the immuno-reactive bands were quantified by densitometric analysis with Quantity-One software (Bio-Rad). Each density measurement was normalized by using the corresponding GAPDH level as an internal control.

[0045] For the measurement of type I soluble collagen in the culture media, 500 μl of each sample have been concentrated for 10 times with centrifuge filters having a cut-off from 3 to 30 K (Amicon Ultra-0.5 ml, Millipore). Approximately 20 μl of each sample have been then used for the Western Blot analysis. Each density measurement was normalised by the protein content of the cells in the corresponding well. As a reference control, in some experiments, type I human collagen (BD Bioscience) has been used together with the experimental samples under investigation. The markers used were Magic Mark (Invitrogen, visible in chemiluminescence) and Page Ruler (Thermo Scientific, visible at light).

[0046] FIG. 1 is a histogram showing the average of the type I procollagen concentrations detected in cell cultures with the various treatments described above and with a control that is a sample of untreated fibroblasts, according to the procedure described above.

[0047] It can be observed from these data that the tetrapeptide of the invention AAT11RI has an ability surprisingly higher than all the other products tested to increase the concentration of procollagen in vitro, also compared to the products used as a positive control as they are already known for their activity of collagen modulation. Moreover, the ability of the present peptides has revealed itself at relatively low concentrations, thus reflecting the high efficacy of the peptide.

[0048] Furthermore, the inventors have detected the same positive effect of the peptide of the invention for the cell cultures of neonatal fibroblasts as well as for the cultures of cells obtained from both adults and old subjects. Therefore the tested peptide has shown its usefulness in the treatment of different conditions of skin ageing, both physiological conditions due to the age of subject and pathological conditions caused by diseases having the skin ageing as a secondary effect because of the reduced capacity of the cells to produce collagen.

[0049] All values measured and reported in the graph of FIG. 1 are expressed as average±standard error. Statistical analyses have been carried out by using the one-way ANOVA test with Bonferroni post-hoc. The Student's t test was used for analysis of paired data. p≤0.05 was considered as a statistically significant difference.

[0050] The present invention was described above with reference to preferred embodiments thereof. It is to be understood that other embodiments may exist which belong to the same inventive core, as defined by the scope of the protection of the claims set forth below.