Composition for use in cosmetics

Abstract

FIELD: cosmetic composition.SUBSTANCE: injectable cosmetics for the skin. An injection composition for treating deterioration and / or aging of the skin, elastosis and dermoepidermal atrophy caused by oxidative stress comprises non-crosslinked sodium hyaluronate with a molecular weight of 100 to 400 kDa at a concentration of 7 to 20 mg / ml, non-crosslinked sodium hyaluronate with a molecular weight of at least 2000 kDa at a concentration of 10 to 25 mg / ml, a mixture of amino acids consisting of glycine at a concentration of 6 to 12.5 mg / ml, L-proline and / or L-hydroxyproline at a concentration of 5 to 8 mg / ml, L-alanine at a concentration of 1 to 5 mg / ml, L-valine at a concentration of 1 to 5 mg / ml, L-leucine at a concentration of 1 to 5 mg / ml, L-lysine HCl at a concentration of 1 to 5 mg / ml, L-arginine HCl at a concentration of 1 to 5 mg / ml. The use of a composition and kit for treating a cosmetic condition of the skin and for stimulating collagen synthesis, a non-therapeutic method for treating deterioration and / or aging of the skin, elastosis and dermoepidermal atrophy caused by oxidative stress in a subject.EFFECT: counteraction to the reduction in the amount of extracellular matrix proteins and skin hydration due to aging, especially that caused by oxidative stress.17 cl, 4 dwg, 4 tbl, 1 ex

Description

[0001] Field of engineering

[0002] The present invention relates to injectable compositions comprising specific combinations of low- and high-molecular-weight hyaluronic acid (HA), non-crosslinked, in combination with a specific composition of amino acids (AA), capable of counteracting the decrease in the level of extracellular matrix (ECM) proteins and skin hydration due to aging caused by oxidative stress.

[0003] Level of technique

[0004] The skin is the largest organ in the body, with a total surface area of ​​approximately 2 square meters in adults. It performs many fundamental functions, including thermoregulation, protection against pathogens and damage caused by ultraviolet rays, and a waterproof barrier. It is connected to the brain through a wide network of nerves and cells and acts as an environmental sensory system (1, 3). The skin is composed of three layers: the epidermis, the outer layer, is made up of various cell types, such as squamous epithelial cells, basal cells, and melanocytes. The dermis is the intermediate layer of the skin; in addition to blood and lymphatic vessels, hair follicles, and sweat glands, it mainly contains collagen fiber bundles and fibroblasts. The hypodermis is the deepest layer and contains adipose tissue, hair follicles, sensory neurons, and blood vessels (3, 2, 4).In addition to the cellular matrix, one of the most important components of the skin is the extracellular matrix (ECM), a complex composition of macromolecules consisting of water, polysaccharides (glycosaminoglycans such as hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate), and proteins, the most common of which are collagen, fibronectin, laminin, proteoglycans, and elastin (5,6,7,8). The ECM is primarily produced by fibroblasts, mesenchymal cells that also play a vital role in tissue development, maintenance, and repair. The ECM gives tissues their mechanical and ECM-specific properties and also plays an important role in the regulation of cellular functions; Cell-ECM interactions are mediated by specific receptors and not only promote cell adhesion and migration but also regulate cell differentiation and gene expression and play a key role in wound healing (6, 7).In particular, the ECM provides skin with characteristics such as elasticity, strength, and compressibility. The most important protein responsible for skin elasticity is elastin, one of the main structural proteins of the ECM, which accounts for approximately 2% of the total dermal protein content. Structurally, elastin alternates between hydrophobic and hydrophilic domains; it often consists of repeating short units of three to nine amino acids, enriched in glycine, proline, alanine, leucine, and valine.

[0005] The fundamental process of elastogenesis involves fibroblasts synthesizing and secreting tropoelastin precursor (ELN), a soluble monomer, into the extracellular space, which then forms a highly cross-linked insoluble polymer composed of covalently linked tropoelastin molecules to produce elastin in the ECM (9,10,11). The other most abundant structural protein in the ECM is collagen, which is also the most abundant protein in mammals; collagen comprises a family of molecules, diverse both structurally and functionally, numbering 28 members in vertebrates, numbered with Roman numerals (I-XXVIII), encoded by 28 different genes, of which type I is the most abundant in humans.Despite the high heterogeneity of the various types, all members of the family share a characteristic triple-helix structure consisting of three α-chains, which can be formed by identical collagen chains (homotrimers) or by combining different collagen isoforms (heterotrimers). All collagen isoforms contain repeating Gly-XY tripeptide domains, where the X and Y positions are often occupied by proline and hydroxyproline; these n(Gly-XY) repeats are necessary for holding the triple-helix structure together and, depending on the collagen type, proline and lysine residues are also beneficial, as they are important sites of post-translational modifications (12, 13).

[0006] Skin structure and function are characterized by sequential and cumulative changes and include alterations in both the viability and proliferation of cellular components, as well as the expression and production of the extracellular matrix. Decreased ECM function leads to both loss of elasticity and firmness and the appearance of wrinkles, which are disadvantages of skin aging (14, 15). Furthermore, another key sign of skin aging is dryness and loss of skin hydration.

[0007] Hyaluronic acid (HA), a glycosaminoglycan, is a key molecule for skin hydration because it has a unique and fundamental ability to bind and retain water molecules (16). In addition to hydration, HA also plays an important role in wound healing, fibroblast migration, immune response, and tumor development (16). The size of HA greatly influences its functions: large-molecular HA, typically over 1000 kDa, has antiangiogenic and immunosuppressive effects, while smaller HA polymers are potent inducers of inflammation and angiogenesis. But above all, aging leads not only to a decrease in HA synthesis but also to the production of harmful smaller HA molecules (16,17,18).

[0008] The world is still far from fully understanding the numerous causes of aging; the main ones include, among others, glycation, telomere shortening, secondary reactions, mutations and protein aggregation. However, one of the major and well-known factors in the aging of organic substances is the formation of reactive oxygen species (ROS); in fact, the mitochondrial free radical theory of aging was put forward as early as 1956, which is mainly based on the generation of ROS as a by-product of mitochondrial respiration (19,20). Due to constant exposure to UV rays, the production of ROS in this organ, in addition to the endogenous (internal) mitochondrial origin, also occurs due to an exogenous (external) source; this makes the amount of free radicals in the skin particularly large, triggering the process of photoaging of the skin (21,22).One of the main consequences of UV exposure on the skin is accelerated ECM turnover, which is a normal and fundamental process in maintaining tissue health, during which old proteins are broken down and replaced by newly synthesized proteins (14,23). However, UV irradiation also produces intracellular ROS, such as superoxide anion (O2). - ) and hydrogen peroxide (H2O2), which leads to the synthesis of matrix metalloproteinase (MMP) (24,25). Thus, the cleavage of the ECM component MMP without a simultaneous increase in the production of new ECM proteins alters the balance of exchange between synthesis and degradation in favor of the latter. This results in the loss of collagen, elastin, and fibrillary fibers, leading to decreased skin elasticity and the appearance of wrinkles (21,22).

[0009] Another important effect of oxidative stress is the increase in the percentage of cellular senescence, which also plays a fundamental role in skin aging; senescent fibroblasts no longer divide and acquire a senescence-associated secretory phenotype (SASP) (26), with increased secretion of proinflammatory cytokines and chemokines, ECM remodeling proteases; moreover, SASP increases the amount of ROS, thereby creating a vicious cycle of free radical formation, which ultimately causes premature aging (26,27,28).

[0010] To prevent and / or counteract the harmful effects of oxidative stress on skin photoaging, various therapeutic and dietary approaches have been proposed. An important strategy is to prevent oxidative stress-induced ECM remodeling by stimulating the synthesis of new ECM proteins, especially collagen and elastin, to prevent the loss of skin elasticity (29, 30, 31). Furthermore, improving ECM protein translation by stimulating fibroblast viability also blocks oxidative stress-induced aging, thereby delaying the onset of skin aging.

[0011] Amino acids, the building blocks of proteins, are known to stimulate protein translation when used as substrates. However, specific amino acid compositions, only when properly identified and administered, can promote the synthesis of a specific subset of proteins. Several compositions currently exist that are used to counteract oxidative stress and skin aging; these compositions are specifically designed for injection. Currently, numerous compositions are available for intradermal injection, either for volume creation (e.g., cross-linked hyaluronic acid (HA) fillers) or for long-term effects by inducing neocollagenesis.

[0012] In humans, the shelf life of injected hyaluronic acid in its natural state is several days, as the polymer chains are easily broken down by enzymes and free radicals present in the body. To overcome this problem, most commercially available compositions are produced by cross-linking the hydroxyl groups of HA using a chemical cross-linking agent. The final tissue effect of such compositions can be controlled by varying the cross-link density using various cross-linking agents. However, this approach has a significant drawback: cross-linked HA-based fillers are actually very dense and difficult to inject.

[0013] Thus, the object of the present invention is to provide alternative and improved injectable compositions for counteracting photoaging damage and improving collagenogenesis in the dermis.

[0014] The essence of the invention

[0015] The object of the present invention is an injection composition comprising a specific combination of low- and high-molecular hyaluronic acid (HA), non-crosslinked, in combination with a specific composition of amino acids (AA).

[0016] In particular, the composition of the present invention includes:

[0017] - Sodium hyaluronate with a molecular weight of 100-400 kDa, non-crosslinked, in a concentration of 7 to 20 mg / ml

[0018] - Non-cross-linked sodium hyaluronate with a molecular weight of 2000 kDa or higher, at a concentration of 10 to 25 mg / ml

[0019] - a mixture of amino acids, including:

[0020] Glycine 6-12.5 mg / ml

[0021] L-proline and / or L-hydroxyproline 5-8 mg / ml

[0022] L-alanine 1-5 mg / ml

[0023] L-valine 1-5 mg / ml

[0024] L-leucine 1-5 mg / ml

[0025] L-lysine HCl 1-5 mg / ml (hydrochloride)

[0026] L-arginine HCl 1-5 mg / ml (hydrochloride)

[0027] In a preferred embodiment, the composition according to the invention comprises:

[0028] 9 mg / ml glycine

[0029] 6.5 mg / ml proline

[0030] 2 mg / ml alanine

[0031] 2.5 mg / ml valine

[0032] 1 mg / ml leucine

[0033] 2.5 mg / ml lysine HCl

[0034] 1.5 mg / ml arginine HCl

[0035] In one embodiment, the composition according to the invention comprises sodium hyaluronate with a molecular weight of 100-400 kDa, which is present at a concentration of 16 mg / ml, and sodium hyaluronate with a molecular weight of less than 2000 kDa, which is present at a concentration of 16 mg / ml.

[0036] In one embodiment, sodium hyaluronate with a molecular weight of 100-400 kDa is present at a concentration of 12 mg / mL and sodium hyaluronate with a molecular weight of 2000 kDa is present at a concentration of 20 mg / mL.

[0037] In a preferred embodiment, the composition according to the invention contains a total concentration of sodium hyaluronate per ml greater than 25 mg / ml.

[0038] In a preferred embodiment, the composition has a pH value of preferably from 6.8 to 7.5, even more preferably from 7 to 7.3.

[0039] In a preferred embodiment, the composition according to the invention comprises at least one of pharmaceutically acceptable excipients or adjuvants, a buffer, preferably a phosphate buffer, an anesthetic, preferably a local anesthetic.

[0040] In a preferred embodiment, the composition according to the invention comprises at least one biomimetic peptide selected from acetyl decapeptide 3 SEQ ID NO: 1, oligopeptide 24 SEQ ID NO: 2, acetyl tetrapeptide 5 SEQ ID NO: 3, vialox pentapeptide-3 SEQ ID NO: 4, acetyl hexapeptide 8 SEQ ID NO: 5, myristoyl pentapeptide-8 SEQ ID NO: 6, peptide GHK-Cu of the sequence Gly-His-Lys-Cu, tripeptide-29 of the sequence H-Gly-Pro-Hyp-OH, octapeptide-3 SEQ ID NO: 7, matrixyl SEQ ID NO: 8, hexapeptide SEQ ID NO: 9.

[0041] The present invention also relates to a kit comprising the injection composition described above, preferably in the form of a gel, contained in a pre-filled syringe, and optionally including instructions for use.

[0042] The present invention also relates to the use of the compositions described above for the treatment of deterioration and / or aging of the skin, elastosis and dermoepidermal atrophy or other pathologies caused by oxidative stress, and for cosmetic applications, preferably for the treatment of photoaging, skin depressions, scars, facial imperfections and asymmetries, wrinkles and skin folds, preferably of the face, more preferably glabellar wrinkles, nasolabial folds, chin folds, marionette lines, wrinkles in the cheek area, perioral wrinkles, crow's feet.

[0043] Finally, an object of the present invention is the use of a composition or kit of the invention for stimulating collagen synthesis and a non-therapeutic method for treating the skin of a subject, comprising intradermal injection of the composition.

[0044] Additional objects will become apparent from the following detailed description.

[0045] Figure Description

[0046] Fig. 1 - Expression of catalase (CAT) and Ink4 mRNA in BJ fibroblasts pretreated with three compositions (AA1, AA2, or AA3) or without pretreatment (culture medium only) (CT), and then treated with hydrogen peroxide (H2O2) or not treated at all (NT).

[0047] Fig. 2 - Expression of tropoelastin (ELN), fibrillin (FBN), and collagen isoform IV (Col4a1) mRNA in BJ fibroblasts pretreated with three compositions (AA1, AA2, or AA3) or without pretreatment (culture medium only) (CT), and then treated with hydrogen peroxide (H2O2) or not treated at all (NT).

[0048] Fig. 3 - Analysis of BJ fibroblast proliferation using MTT colorimetric assay before treatment (0 h) and after 24, 48 and 72 h of incubation with two mixtures of AA4 and AA3 at different final concentrations (0.1%, 0.3%, 0.5%).

[0049] Fig. 4 - Expression of mRNA genes of two collagen isoforms (col1a1 and col4a1) after 72 hours of incubation in BJ fibroblasts pre-treated with two compositions (AA4 or AA3).

[0050] The values ​​0.1%, 0.3%, 0.5% refer to the different final concentrations of the two mixtures.

[0051] Detailed description of the invention:

[0052] Based on the above, the inventors developed a new composition to counteract skin aging and stimulate collagenogenesis. This composition includes a specific combination of non-cross-linked medium and high molecular weight hyaluronic acid (HA) in conjunction with a specific amino acid (AA) composition to counteract the loss of ECM proteins and skin hydration due to aging caused by oxidative stress.

[0053] In one embodiment, the composition of the invention also further comprises biomimetic peptides selected for their action.

[0054] Thus, the subject of the present invention is a new composition comprising a composition of amino acids and hyaluronic acid of various molecular weights, non-crosslinked, capable of both stimulating the synthesis of elastin and collagen and performing a protective function against ROS and aging caused by oxidative stress.

[0055] The present invention also relates to a composition further comprising biomimetic peptides.

[0056] The composition according to the invention (AA3) is composed as follows:

[0057] - non-cross-linked hyaluronic acid with a molecular weight of 100-400 kDa

[0058] - Non-cross-linked hyaluronic acid with a molecular weight of 2000 kDa or higher

[0059] - 25 mg / ml of the following amino acids in total:

[0060] Glycine 6-12.5 mg / ml

[0061] L-proline and / or L-hydroxyproline 5-8 mg / ml

[0062] L-alanine 1-5 mg / ml

[0063] L-valine 1-5 mg / ml

[0064] L-leucine 1-5 mg / ml

[0065] L-lysine HCl (hydrochloride) 1-5 mg / ml

[0066] L-arginine HCl (hydrochloride) 1-5 mg / ml.

[0067] In a preferred embodiment, the composition comprises

[0068] 9 mg / ml glycine

[0069] 6.5 mg / ml proline

[0070] 2 mg / ml alanine

[0071] 2.5 mg / ml valine

[0072] 1 mg / ml leucine

[0073] 2.5 mg / ml lysine HCl

[0074] 1.5 mg / ml arginine HCl

[0075] In a preferred embodiment, the composition is shown in Table 1.

[0076] Table 1

[0077] Composition AA3 mg / ml (MW) Sodium Hyaluronate 7-20(100-400) 10-25 (≥ 2000) Glycine 9 Proline 6,5 Alanine 2 Valin 2,5 Leucine 1 Lysine 2,5 Arginine 1,5 Total AA 25

[0078] The composition according to the invention optionally includes a concentration of from 0.005 mg / ml to 0.080 mg / ml, preferably from 0.005 mg / ml to 0.05 mg / ml, more preferably from 0.005 mg / ml to 0.02 mg / ml of at least one of the following peptides:

[0079] Acetyl decapeptide 3 SEQ ID NO: 1 Ac-Tyr-Arg-Ser-Arg-Lys-Tyr-Thr-Ser-Trp-Tyr-NH2,

[0080] oligopeptide 24 SEQ ID NO: 2 H-RGDGCMYIEGGGG-OH,

[0081] Acetyltetrapeptide 5 SEQ ID NO: 3-Ac-β-Ala-His-Ser-His-OH,

[0082] Vialox pentapeptide-3 SEQ ID NO: 4 Gly-Pro-Arg-Pro-Ala-NH2,

[0083] acetyl hexapeptide - 8 (argireline) SEQ ID NO: 5 Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2,

[0084] Myristoyl pentapeptide-8 SEQ ID NO: 6 Myr-RGDGK-NH2,

[0085] GHK-Cu peptide with the sequence Gly-His-Lys-Cu, not listed in the sequence listing (having only 3 amino acids),

[0086] Tripeptide-29 (Collagen tripeptide) with the sequence H-Gly-Pro-Hyp-OH, not listed in the sequence listing (having only 3 amino acids).

[0087] The composition of the present invention may further comprise at least one of:

[0088] Octapeptide-3 SEQ ID NO: 7 Ac-Glu-Glu-Met-Gln-Arg-Arg-Ala-Asp-NH2,

[0089] matrixyl SEQ ID NO: 8 Lys-Thr-Thr-Lys-Ser,

[0090] hexapeptide SEQ ID NO: 9 Val-Gly-Val-Ala-Pro-Gly,

[0091] The concentration above is for each peptide.

[0092] In a preferred embodiment, the composition of the invention comprises a total concentration of sodium hyaluronate per ml greater than 25 mg / ml.

[0093] The composition of the present invention may further include pharmaceutically acceptable excipients or adjuvants.

[0094] The composition of the present invention may further include a buffer, such as a phosphate buffer, to adjust the pH.

[0095] The pH value of the composition is preferably from 6.8 to 7.5, even more preferably from 7 to 7.3.

[0096] The composition of the present invention may further comprise an anesthetic, in particular a local anesthetic, preferably lidocaine, at a concentration of 0.1% to 0.4%, preferably 0.2% to 0.3%.

[0097] In order to test the improved effect on collagenogenesis of the composition of the invention compared with the compositions currently on the market, comparative tests were conducted in which the composition of the invention was compared with other products currently on the market, the compositions of which are shown in Tables 2 and 4: one (AA1) containing 33 mg / ml of total amino acids (according to Table 2) plus 10 mg / ml of hyaluronic acid (100 kDa), and one (AA2) containing only 16 mg / ml of cross-linked hyaluronic acid (80-100 kDa) +16 mg / ml of cross-linked hyaluronic acid (1100-1400 kDa). AA4 contains 32 mg / ml of low molecular weight hyaluronic acid (100-400 kDa) and the same amino acid mixture as the composition according to the invention, but does not contain lysine.

[0098] Table 2

[0099] Composition AA1 mg / ml (MW) AA2 mg / ml (MW) AA3 mg / ml (MW) Sodium hyaluronate (MW) 10 (200) 16 (80-100) 16 (1100-1400) 16 (100-400) 16 (2000) Glycine 10 9 Proline 8 6,5 Alanine 7 2 Valin 5 2,5 Leucine 2 1 Lysine 1 2,5 Arginine 1,5 Total AA 33 25

[0100] To evaluate the induction of oxidative stress, the mRNA expression levels of catalase (CAT), a known ROS scavenger and antioxidant enzyme, were first analyzed and compared in the human fibroblast cell line BJ 8 (ATCC CRL-2522). CAT mRNA levels increase in response to H2O2 and, therefore, are a marker of intracellular ROS levels. In Fig. 1, as expected, CAT mRNA induced by H2O2 treatment (+48% compared to untreated NT cells) can be observed. The comparison cells were pretreated with compositions AA1, AA2, and AA3, respectively.

[0101] The inventors unexpectedly found that pretreatment of BJ fibroblasts with AA3 was able to significantly reduce CAT mRNA levels to levels similar to those in control cells where oxidative stress was not induced, indicating the ability of the composition to counteract the effect of oxidative stress in cells (-51% compared to cells treated with H2O2-CT alone). In contrast, neither pretreatment with composition AA1 nor composition AA2 significantly altered CAT levels in (-8% and -4%, respectively, relative to CT).

[0102] Thus, the composition according to the invention, compared with the preparations described in the prior art, is extremely more effective in counteracting the effects of oxidative stress in fibroblasts.

[0103] To measure the level of aging caused by oxidative stress, the mRNA expression level of p16 (INK4a) (INK4), a well-known aging marker whose mRNA levels are very high in senescent cells (36), was determined. In this case, INK4 mRNA also significantly increased upon H2O2 treatment, confirming the onset of aging in BJ fibroblasts due to oxidative stress; in this case, the comparison cells were also pretreated with compositions AA1, AA2, and AA3, respectively.

[0104] Similar to what was observed with CAT levels, a significant decrease in INK4 mRNA expression levels was noted with AA3-based pretreatment (-65% compared to CT), indicating a lower degree of senescence in fibroblasts treated with the AA3 formulation. Although a decrease in INK4 mRNA was also recorded in cells pretreated with AA1 (-33% compared to CT) and AA2 (-28% compared to CT), the AA3 formulation proved to be significantly more effective. Table 3 below presents the data discussed above.

[0105] Table 3

[0106] Percentage reduction in mRNA levels in cells treated with the indicated compositions and hydrogen peroxide (H2O2) compared to the control treated with hydrogen peroxide alone.

[0107] AA1 AA2 AA3 CAT mRNA -8% -4% -51% INK4 mRNA -33% -28% -65%

[0108] To test the effect of the composition of the invention on the oxidative stress-induced decrease in mRNA levels of ECM proteins, the inventors analyzed the mRNA expression of three ECM proteins, namely, elastin (ELN), fibrillin (FBN), and collagen Col4a1.

[0109] The expression of these mRNAs is greatly affected by oxidative stress, and as shown in Fig. 2, after H2O2 treatment, the mRNA of all ECM genes was greatly reduced, confirming the deterioration of ECM due to oxidative stress.

[0110] In this case, the cells were also treated with the 3 compositions shown in Table 2; after the treatments, it could be observed that the ELN mRNA expression levels were not affected by either the AA1 treatment or the AA2 treatment (-0.9 and -4% compared with CT).

[0111] In contrast, AA3 treatment could achieve restoration (+37% compared with CT) of ELN mRNA expression level.

[0112] Similarly, FBN mRNA depletion due to oxidative stress (-31% compared with NT) was almost completely blocked by AA3 (+32% compared with CT) and partially by AA1 (+26% compared with CT), but not by AA2 (+3%).

[0113] Finally, the depletion of col4a1 induced by H2O2(-55% compared with NT) was slightly improved by the formulations with AA1, AA2 (+13%, +24%) and somewhat more significantly by the formulation with AA3 +28% compared with CT.

[0114] The AA3 composition not only was extremely effective in reducing oxidative stress and aging in H2O2-treated BJ fibroblasts, but also in restoring the decrease in ECM mRNA induced by it. Specifically, only the AA3 composition was effective against oxidative stress and aging, but not AA1 or AA2; similarly, only AA3 restored ELN expression. Furthermore, the inventive composition was more effective in increasing FBN mRNA than the comparative compositions and was able to restore Col4a1 levels along with the other two compositions. ELN is one of the most important ECM proteins and is responsible for skin elasticity (9,10); also, FBN is another fundamental protein for the formation of ECM elastic fibers, the levels of which are known to decrease with age, especially in the case of external oxidative stress (37).Thus, the ability of the AA3 composition to restore mRNA levels, counteracting the decrease caused by H2O2, demonstrates that this composition effectively counteracts the destruction of the ECM caused by aging. Among the skin collagen isoforms, type IV (Col4a1) is one of the most susceptible to aging; its deterioration also accentuates skin aging and photoaging caused by oxidative stress (38). The composition according to the invention was shown to be able to partially restore col4a1 mRNA levels reduced after H2O2 treatment.

[0115] Of all the tested compositions, the composition according to the invention proved to be the most effective. The higher efficacy of the composition according to the invention, compared to AA1 and AA2, in counteracting oxidative stress, aging, and ECM reduction is due to its specific composition; in fact, although the composition according to the invention contains fewer total amino acids than AA1, the specific ratios between them and the presence of arginine, in addition to the selected molecular weights of HA, allow for a greater beneficial effect on fibroblast aging and greater antioxidant power than the comparative compositions.

[0116] For further comparison, the effect of the AA3 composition according to the invention was tested against a composition (AA4) lacking both high-molecular-weight hyaluronic acid and lysine. Examples of the compositions are shown in Table 4.

[0117] Table 4

[0118] Composition AA3 (mg / ml) AA4 (mg / ml) sodium hyaluronate 16 (100-400 kDa) 16 (2000 kDa) 32 (100-400 kDa) -- Glycine 9 9 Proline 6,5 6,5 Alanine 2 2 Valin 2,5 2,5 Leucine 1 1 Lysine 2,5 -- Arginine 1,5 1,5 Total AA 25 22,5

[0119] The effects of the compositions were assessed both by analyzing fibroblast proliferation and by the expression of collagen genes (Col4a1, Cola1a1).

[0120] Human BJ fibroblasts were cultured at 70%-80% confluence in F12 medium plus 10% (v / v) fetal bovine serum (FBS) and 2 mM L-glutamine in a 5% CO2 / 95% air atmosphere. The cells were then treated with AA3 and AA4 formulations at the final concentrations indicated in Figs. 3 and 4 (0.1%-0.3%-0.5%). Proliferation was determined by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. 8x10 3cells / well were seeded in a 96-well plate in 100 μl of medium. Purple formazan crystals were dissolved overnight at 37°C in 5% SDS / 0.1 M HCl (100 μl / well) and the absorbance was recorded on a microplate reader at a dual wavelength of 570 nm / 655 nm at time zero (as a control), 24, 48 and 72 hours. The results are shown in Fig. 3 and demonstrate how the composition of the invention at each tested concentration and depending on the dose is more effective than the comparative composition in stimulating the proliferation of BJ fibroblasts. Furthermore, the composition of the invention increases the proliferation of BJ fibroblasts and is more effective than the comparative composition even at the lowest dose.

[0121] Alternatively, at the end of the experimental treatment (72 hours), the cells were used for mRNA extraction. RNA was isolated using the RNeasy Mini Kit (Qiagen), and cDNA (1 μg) was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories). The relative gene expression level was calculated as 2-ΔΔCT, where ΔΔCT corresponded to the difference between the ΔCT of both treatments and the ΔCT of the untreated group. GAPDH was used as a reference housekeeping gene. The results are shown in Fig. 4 and demonstrate how the composition of the invention is more effective than the reference composition in stimulating the expression of both analyzed collagen genes; moreover, the composition of the invention increases the expression of both genes in a dose-dependent manner and at each concentration, proving to be more effective than the reference composition even at relatively low doses.

[0122] Thus, the inventors have developed a composition that, compared to the compositions currently described in the known art, is capable of blocking the harmful effects of oxidative stress, aging and the decrease in the amount of ECM proteins in fibroblasts in an extremely more effective manner.

[0123] According to the present invention, "injectable" means delivered from syringes under normal conditions at normal pressure and refers to injection into the skin, dermis or other tissues to deliver the composition to the desired destination.

[0124] Thus, the object of the present invention is an injection composition comprising:

[0125] - from 7 to 20 mg / ml of non-cross-linked sodium hyaluronate with a molecular weight of 100-400 kDa,

[0126] - from 10 to 25 mg / ml of non-cross-linked sodium hyaluronate with a molecular weight of 2000 kDa,

[0127] - a mixture of amino acids, including:

[0128] Glycine 6-12.5 mg / ml

[0129] L-proline and / or L-hydroxyproline 5-8 mg / ml

[0130] L-alanine 1-5 mg / ml

[0131] L-valine 1-5 mg / ml

[0132] L-leucine 1-5 mg / ml

[0133] L-lysine HCl 1-5 mg / ml

[0134] L-arginine HCl 1-5 mg / ml

[0135] In a preferred embodiment, the composition comprises:

[0136] 9 mg / ml glycine

[0137] 6.5 mg / ml L-proline

[0138] 2 mg / ml L-alanine

[0139] 2.5 mg / ml L-valine

[0140] 1 mg / ml L-leucine

[0141] 2.5 mg / ml L-lysine HCl

[0142] 1.5 mg / ml L-arginine HCl

[0143] In a preferred embodiment, the composition comprises 16 mg / ml of non-cross-linked sodium hyaluronate with a molecular weight of 100-400 kDa and 16 mg / ml of non-cross-linked sodium hyaluronate with a molecular weight of 2000 kDa or higher.

[0144] In another preferred embodiment, the composition according to the invention comprises 12 mg / ml of non-cross-linked sodium hyaluronate with a molecular weight of 100-400 kDa and 20 mg / ml of non-cross-linked sodium hyaluronate with a molecular weight of 2000 kDa.

[0145] In a preferred embodiment, the composition comprises a total concentration of sodium hyaluronate per ml greater than 25 mg / ml.

[0146] In one embodiment, the composition of the invention optionally comprises at least one of the following peptides at a concentration of 0.005 mg / ml to 0.080 mg / ml, preferably 0.005 mg / ml to 0.05 mg / ml, more preferably 0.005 mg / ml to 0.02 mg / ml:

[0147] Acetyl decapeptide 3 SEQ ID NO: 1 Ac-Tyr-Arg-Ser-Arg-Lys-Tyr-Thr-Ser-Trp-Tyr- NH2,

[0148] oligopeptide 24 SEQ ID NO: 2 H-RGDGCMYIEGGGG-OH,

[0149] Acetyl tetrapeptide 5 SEQ ID NO: 3 Ac-β-Ala-His-Ser-His-OH,

[0150] Vialox pentapeptide-3 SEQ ID NO: 4 Gly-Pro-Arg-Pro-Ala-NH2,

[0151] acetyl hexapeptide - 8 (argireline) SEQ ID NO: 5 Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2,

[0152] Myristoyl pentapeptide-8 SEQ ID NO: 6 Myr-RGDGK-NH2,

[0153] GHK-Cu peptide with the sequence Gly-His-Lys-Cu, not listed in the sequence listing (having only 3 amino acids),

[0154] Tripeptide-29 (Collagen tripeptide) with the sequence H-Gly-Pro-Hyp-OH, not listed in the sequence listing (having only 3 amino acids).

[0155] The composition of the present invention may further comprise at least one of:

[0156] Octapeptide-3 SEQ ID NO: 7 Ac-Glu-Glu-Met-Gln-Arg-Arg-Ala-Asp-NH2,

[0157] matrixyl SEQ ID NO: 8 Lys-Thr-Thr-Lys-Ser,

[0158] hexapeptide SEQ ID NO: 9 Val-Gly-Val-Ala-Pro-Gly,

[0159] The composition of the present invention may further include at least one pharmaceutically acceptable excipient or adjuvant.

[0160] The composition of the present invention may further comprise a buffer, such as a phosphate buffer, to adjust the pH. Said pH is preferably from 6.8 to 7.5, and even more preferably from 7 to 7.3.

[0161] The composition of the present invention may further comprise an anesthetic, in particular a local anesthetic, preferably lidocaine, at a concentration of 0.1% to 0.4%, preferably 0.2% to 0.3%.

[0162] The present invention also relates to a kit comprising an injection composition of the invention in the form of a gel in a pre-filled syringe and, optionally, instructions for use.

[0163] Thus, the object of the present invention is a composition or kit as defined above for use in the treatment of deterioration and / or aging of the skin, elastosis and dermoepidermal atrophy caused by oxidative stress.

[0164] The composition and kit according to the invention are also used for the treatment of photoaging, skin depressions, scars, defects and asymmetries of the nose, lips, cheeks, perioral region, infraorbital region, facial asymmetry, jaw and cheekbone lines, wrinkles and skin folds, for example on the face, as a non-limiting example of glabellar wrinkles, nasolabial folds, chin folds, marionette lines, wrinkles in the cheek area, perioral wrinkles, crow's feet.

[0165] The composition and kit according to the invention are also used for stimulating collagen synthesis and for cosmetic improvement of soft tissues.

[0166] The use according to the present invention is preferably a use in the treatment of a cosmetic condition, however, the composition can also be administered for treatment according to therapeutic indications.

[0167] EXAMPLES

[0168] Cells and processing methods.

[0169] Human BJ fibroblasts were purchased from the American Type Culture Collection (ATCC-CRL-2522) and grown to 70%–80% confluence in F12 culture medium (ATCC) supplemented with 10% fetal bovine serum (FBS) (vol / vol) and 2 mM L-glutamine and grown in a humidified atmosphere of 5% CO2 / 95% air. Cells were pretreated for 24 hours with 1% of the compositions shown in Table 2.

[0170] Then, to induce oxidative stress, the cells were treated with 200 μM H2O2 for two hours and then for another 48 hours. Untreated cells were plated as a control. After the experimental treatments, the cells were used for mRNA extraction.

[0171] Total RNA isolation and gene expression analysis.

[0172] RNA was isolated from BJ fibroblasts using the RNeasy Mini Kit (Qiagen), and cDNA (1 μg) was synthesized using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories). The relative gene expression level 2-ΔΔCT was calculated, where ΔΔCT corresponds to the difference between the ΔCT of the treated group and the ΔCT of the untreated group. GAPDH was used as a reference for the housekeeping gene.

[0173] The expression levels of CAT, INK4, ELN, COL4a1, and FBN genes were analyzed.

[0174] List of References:

[0175] 1. Kanitakis J. Anatomy, histology and immunohistochemistry of normal human skin. Eur J Dermatol. 2002 Jul-Aug;12(4):390-9; quiz 400-1. PMID: 12095893.

[0176] 2. Urmacher C. Histology of normal skin. Am J Surg Pathol. 1990 Jul;14(7):671-86. doi: 10.1097 / 00000478-199007000-00008. PMID: 1694059.

[0177] 3. Yousef H, Alhajj M, Sharma S. Anatomy, Skin (Integument), Epidermis. In: StatPearls

[0178] 4. Ribeiro, Camila & Leal, Fabiano & Jeunon, Thiago. (2017). Skin Anatomy, Histology, and Physiology. 10.1007 / 978-3-319-20250-1_1-1.

[0179] 5. Theocharis AD, Skandalis SS, Gialeli C, Karamanos NK. Extracellular matrix structure. Adv Drug Deliv Rev. 2016 Feb 1; 97:4-27.

[0180] 6. Manou D, Caon I, Bouris P, Triantaphyllidou IE, Giaroni C, Passi A, Karamanos NK, Vigetti D, Theocharis AD. The Complex Interplay Between Extracellular Matrix and Cells in Tissues. Methods Mol Biol. 2019; 1952:1-20.

[0181] 7. Teti A. Regulation of cellular functions by extracellular matrix. J Am Soc Nephrol. 1992 Apr;2(10 Suppl): S83-7.

[0182] 8. Bosman FT, Stamenkovic I. Functional structure and composition of the extracellular matrix. J Pathol. 2003 Jul;200(4):423-8.

[0183] 9. Mithieux SM, Weiss AS. Elastin. Adv Protein Chem. 2005; 70:437-61.

[0184] 10. Vindin H, Mithieux SM, Weiss AS. Elastin architecture. Matrix Biol. 2019 Nov; 84:4-16. doi: 10.1016 / j.matbio.2019.07.005. Epub 2019 Jul 10.

[0185] 11. Schräder CU, Heinz A, Majovsky P, Karaman Mayack B, Brinckmann J, Sippl W, Schmelzer CEH. Elastin is heterogeneously cross-linked. J Biol Chem. 2018 Sep 28;293(39):15107-15119

[0186] 12. Ricard-Blum S. The collagen family. Cold Spring Harb Perspect Biol. 2011 Jan 1;3(1)

[0187] 13. Sorushanova A, Delgado LM, Wu Z, Shologu N, Kshirsagar A, Raghunath R, Mullen AM, Bayon Y, Pandit A, Raghunath M, Zeugolis DI. The Collagen Suprafamily: From Biosynthesis to Advanced Biomaterial Development. Adv Mater. 2019 Jan;31(1): e1801651.

[0188] 14. Birch HL. Extracellular Matrix and Ageing. Subcell Biochem. 2018; 90:169-190.

[0189] 1 [ PubMed ] Black LD, Allen PG, Morris SM, Stone PJ, Suki B. ECMhanical and failure properties of extracellular matrix sheets as a function of structural protein composition. Biophys J. 2008 Mar 1;94(5):1916-29. doi: 10.1529 / biophysj.107.107144. Epub 2007 Nov 9 .

[0190] 1 [ PubMed ] Dicker KT, Gurski LA, Pradhan-Bhatt S, Witt RL, Farach-Carson MC, Jia X. Hyaluronan: a simple polysaccharide with diverse biological functions. Acta Biomater. 2014 Apr;10(4):1558-70.

[0191] 1 Fallacara A, Baldini E, Manfredini S, Vertuani S. Hyaluronic Acid in the Third Millennium. Polymers (Basel). 2018 Jun 25;10(7):701.

[0192] 1 Papakonstantinou E, Roth M, Karakiulakis G. Hyaluronic acid: A key molecule in skin aging. Dermatoendocrinol. 2012 Jul 1;4(3):253-8. doi: 10.4161 / derm.21923. PMID: 23467280; PMCID: PMC3583886.

[0193] 1 da Costa JP, Vitorino R, Silva GM, Vogel C, Duarte AC, Rocha-Santos T. A synopsis on aging

[0194] -Theories, ECMhanisms and future prospects. Ageing Res Rev. 2016 Aug; 29:90-112.

[0195] 20. Harman D. Free radical theory of ageing. Mutat Res. 1992 Sep;275(3-6):257-66.

[0196] 21. Fisher GJ, Kang S, Varani J, Bata-Csorgo Z, Wan Y, Datta S, Voorhees JJ. ECMhanisms of photoageing and chronological skin ageing. Arch Dermatol. 2002 Nov;138(11):1462-70. doi: 10.1001 / archderm.138.11.1462. PMID: 12437452.

[0197] 22. Battie C, Jitsukawa S, Bernerd F, Del Bino S, Marionnet C, Verschoore M. New insights in photoageing, UVA induced damage and skin types. Exp Dermatol. 2014 Oct;23 Suppl 1:7-12.

[0198] 23. Kehlet SN, Willumsen N, Armbrecht G, Dietzel R, Brix S, Henriksen K, Karsdal MA. Age-related collagen turnover of the interstitial matrix and basement membrane: Implications of age-and sex-dependent remodeling of the extracellular matrix. PLoS One. 2018 Mar 29;13(3): e0194458.

[0199] 24. Huang H, Du W, Brekken RA. Extracellular Matrix Induction of Intracellular Reactive Oxygen Species. Antioxid Redox Signal. 2017 Oct 20;27(12):774-784.

[0200] 25. Herrmann G, Wlaschek M, Lange TS, Prenzel K, Goerz G, Scharffetter-Kochanek K. UVA irradiation stimulates the synthesis of various matrix-metalloproteinases (MMPs) in cultured human fibroblasts. Exp Dermatol. 1993 Mar;2(2):92-7.

[0201] 26. Campisi J. The role of cellular senescence in skin ageing. J Investig Dermatol Symp Proc. 1998 Aug;3(1):1-5.

[0202] 27. Ghosh K, Capell BC. The Senescence-Associated Secretory Phenotype: Critical Effector in Skin Cancer and Ageing. J Invest Dermatol. 2016 Nov;136(11):2133-2139.

[0203] 28. Russell-Goldman E, Murphy GF. The Pathobiology of Skin Ageing: New Insights into an Old Dilemma. Am J Pathol. 2020 Jul;190(7):1356-1369.

[0204] 29. Lupo MP, Cole AL. CosECMeutical peptides. Dermatol Ther. 2007 Sep-Oct;20(5):343-9

[0205] 30. Reddy B, Jow T, Hantash BM. Bioactive oligopeptides in dermatology: Part I. Exp Dermatol. 2012 Aug;21(8):563-8.

[0206] 31. Proksch E, Schunck M, Zague V, Segger D, Degwert J, Oesser S. Oral intake of specific bioactive collagen peptides reduces skin wrinkles and increases dermal matrix synthesis. Skin Pharmacol Physiol. 2014;27(3):113-9.

[0207] 32. Murakami H, Shimbo K, Inoue Y, Takino Y, Kobayashi H. Importance of amino acid composition to improve skin collagen protein synthesis rates in UV-irradiated mice. Amino Acids. 2012 Jun;42(6):2481-9.

[0208] 33. Murakami H, Shimbo K, Takino Y, Kobayashi H. Combination of BCAAs and glutamine enhances dermal collagen protein synthesis in protein-malnourished rats. Amino Acids. 2013 Mar;44(3):969-76.

[0209] 34. Albaugh VL, Mukherjee K, Barbul A. Proline Precursors and Collagen Synthesis: Biochemical Challenges of Nutrient Supplementation and Wound Healing. J Nutr. 2017 Nov;147(11):2011-2017.

[0210] 35. Nenoi M, Ichimura S, Mita K, Yukawa O, Cartwright IL. Regulation of the catalase gene promoter by Sp1, CCAAT-recognizing factors, and a WT1 / Egr-related factor in hydrogen peroxide-resistant HP100 cells. Cancer Res. 2001 Aug 1;61(15):5885-94

[0211] 36. Wang AS, Dreesen O. Biomarkers of Cellular Senescence and Skin Ageing. Front Genet. 2018 Aug 23;9: 247.

[0212] 37. Watson RE, Griffiths CE, Craven NM, Shuttleworth CA, Kielty CM. Fibrillin-rich microfibrils are reduced in photoaged skin. Distribution at the dermal-epidermal junction. J Invest Dermatol. 1999 May;112(5):782-7.

[0213] 38. Feru J, Delobbe E, Ramont L, Brassart B, Terryn C, Dupont-Deshorgue A, Garbar C, Monboisse JC, Maquart FX, Brassart-Pasco S. Ageing decreases collagen IV expression in vivo in the dermo-epidermal junction and in vitro in dermal fibroblasts: possible involvement of TGF-β1. Eur J Dermatol. 2016 Aug 1;26(4):350-60.

[0214] 39. Simmers P, Gishto A, Vyavahare N, Kothapalli CR. Nitric oxide stimulates matrix synthesis and deposition by adult human aortic smooth muscle cells within three-dimensional cocultures. Tissue Eng Part A. 2015 Apr;21(7-8):1455-70.

[0215] 40. Frank S, Kämpfer H, Wetzler C, Pfeilschifter J. Nitric oxide drives skin repair: novel functions of an established mediator. Kidney Int. 2002 Mar;61(3):882-8.

[0216] 41. Hummel SG, Fischer AJ, Martin SM, Schafer FQ, Buettner GR. Nitric oxide as a cellular antioxidant: a little goes a long way. Free Radic Biol Med. 2006 Feb 1;40(3):501-6.

[0217] --->

[0218] <?xml version="1.0" encoding="UTF-8"?>

[0219] <!DOCTYPE ST26SequenceListing PUBLIC "- / / WIPO / / DTD Sequence Listing

[0220] 1.3 / / EN" "ST26SequenceListing_V1_3.dtd">

[0221] <st26sequencelisting dtdversion="V1_3" filename="Professional Derma

[0222] - Cosmetica.xml" softwarename="WIPO Sequence" softwareversion="2.1.2"

[0223] productiondate="2022-09-15">

[0224] <applicationidentification>

[0225] <ipofficecode> IB< / ipofficecode>

[0226] <applicationnumbertext>< / applicationnumbertext>

[0227] <filingdate>< / filingdate>

[0228] < / applicationidentification>

[0229] <applicantfilereference> P3552PC00< / applicantfilereference>

[0230] <earliestpriorityapplicationidentification>

[0231] <ipofficecode> IT< / ipofficecode>

[0232] <applicationnumbertext> 102021000023903< / applicationnumbertext>

[0233] <filingdate> 2021-09-16< / filingdate>

[0234] < / earliestpriorityapplicationidentification>

[0235] <applicantname languagecode="it">Professional Derma

[0236] SA< / applicantname>

[0237] <inventiontitle languagecode="it">Composition for use

[0238] cosmetic< / inventiontitle>

[0239] <inventiontitle languagecode="en">Composition for cosmetic

[0240] use< / inventiontitle>

[0241] <sequencetotalquantity> 9< / sequencetotalquantity>

[0242] <sequencedata sequenceidnumber="1">

[0243] <insdseq>

[0244] <INSDSeq_length>10< / INSDSeq_length>

[0245] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0246] <INSDSeq_division>PAT< / INSDSeq_division>

[0247] <INSDSeq_feature-table>

[0248] <insdfeature>

[0249] <INSDFeature_key>source< / INSDFeature_key>

[0250] <INSDFeature_location>1..10< / INSDFeature_location>

[0251] <INSDFeature_quals>

[0252] <insdqualifier>

[0253] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0254] <INSDQualifier_value>protein< / INSDQualifier_value>

[0255] < / insdqualifier>

[0256] <insdqualifier id="q1">

[0257] <INSDQualifier_name>organism< / INSDQualifier_name>

[0258] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0259] < / insdqualifier>

[0260] < / INSDFeature_quals>

[0261] < / insdfeature>

[0262] <insdfeature>

[0263] <INSDFeature_key> MOD_RES< / INSDFeature_key>

[0264] <INSDFeature_location> 1< / INSDFeature_location>

[0265] <INSDFeature_quals>

[0266] <insdqualifier id="q2">

[0267] <INSDQualifier_name>note< / INSDQualifier_name>

[0268] <INSDQualifier_value>ACETYLATION< / INSDQualifier_value>

[0269] < / insdqualifier>

[0270] < / INSDFeature_quals>

[0271] < / insdfeature>

[0272] <insdfeature>

[0273] <INSDFeature_key> MOD_RES< / INSDFeature_key>

[0274] <INSDFeature_location> 10< / INSDFeature_location>

[0275] <INSDFeature_quals>

[0276] <insdqualifier id="q3">

[0277] <INSDQualifier_name>note< / INSDQualifier_name>

[0278] <INSDQualifier_value>Tyr-NH2< / INSDQualifier_value>

[0279] < / insdqualifier>

[0280] < / INSDFeature_quals>

[0281] < / insdfeature>

[0282] < / INSDSeq_feature-table>

[0283] <INSDSeq_sequence>YRSRKYTSWY< / INSDSeq_sequence>

[0284] < / insdseq>

[0285] < / sequencedata>

[0286] <sequencedata sequenceidnumber="2">

[0287] <insdseq>

[0288] <INSDSeq_length>13< / INSDSeq_length>

[0289] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0290] <INSDSeq_division>PAT< / INSDSeq_division>

[0291] <INSDSeq_feature-table>

[0292] <insdfeature>

[0293] <INSDFeature_key>source< / INSDFeature_key>

[0294] <INSDFeature_location>1..13< / INSDFeature_location>

[0295] <INSDFeature_quals>

[0296] <insdqualifier>

[0297] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0298] <INSDQualifier_value>protein< / INSDQualifier_value>

[0299] < / insdqualifier>

[0300] <insdqualifier id="q4">

[0301] <INSDQualifier_name>organism< / INSDQualifier_name>

[0302] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0303] < / insdqualifier>

[0304] < / INSDFeature_quals>

[0305] < / insdfeature>

[0306] < / INSDSeq_feature-table>

[0307] <INSDSeq_sequence>RGDGCMYIEGGGG< / INSDSeq_sequence>

[0308] < / insdseq>

[0309] < / sequencedata>

[0310] <sequencedata sequenceidnumber="3">

[0311] <insdseq>

[0312] <INSDSeq_length> 4< / INSDSeq_length>

[0313] <INSDSeq_moltype> SECOND< / INSDSeq_moltype>

[0314] <INSDSeq_division> PAT< / INSDSeq_division>

[0315] <INSDSeq_feature-table>

[0316] <insdfeature>

[0317] <INSDFeature_key>source< / INSDFeature_key>

[0318] <INSDFeature_location>1..4< / INSDFeature_location>

[0319] <INSDFeature_quals>

[0320] <insdqualifier>

[0321] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0322] <INSDQualifier_value>protein< / INSDQualifier_value>

[0323] < / insdqualifier>

[0324] <insdqualifier id="q17">

[0325] <INSDQualifier_name>organism< / INSDQualifier_name>

[0326] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0327] < / insdqualifier>

[0328] < / INSDFeature_quals>

[0329] < / insdfeature>

[0330] <insdfeature>

[0331] <INSDFeature_key> MOD_RES< / INSDFeature_key>

[0332] <INSDFeature_location> 1< / INSDFeature_location>

[0333] <INSDFeature_quals>

[0334] <insdqualifier id="q7">

[0335] <INSDQualifier_name> note< / INSDQualifier_name>

[0336] <INSDQualifier_value> bAla< / INSDQualifier_value>

[0337] < / insdqualifier>

[0338] < / INSDFeature_quals>

[0339] < / insdfeature>

[0340] <insdfeature>

[0341] <INSDFeature_key> MOD_RES< / INSDFeature_key>

[0342] <INSDFeature_location> 1< / INSDFeature_location>

[0343] <INSDFeature_quals>

[0344] <insdqualifier id="q8">

[0345] <INSDQualifier_name>note< / INSDQualifier_name>

[0346] <INSDQualifier_value>acetylation< / INSDQualifier_value>

[0347] < / insdqualifier>

[0348] < / INSDFeature_quals>

[0349] < / insdfeature>

[0350] < / INSDSeq_feature-table>

[0351] <INSDSeq_sequence> AHSH< / INSDSeq_sequence>

[0352] < / insdseq>

[0353] < / sequencedata>

[0354] <sequencedata sequenceidnumber="4">

[0355] <insdseq>

[0356] <INSDSeq_length> 5< / INSDSeq_length>

[0357] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[0358] <INSDSeq_division> PAT< / INSDSeq_division>

[0359] <INSDSeq_feature-table>

[0360] <insdfeature>

[0361] <INSDFeature_key>source< / INSDFeature_key>

[0362] <INSDFeature_location>1..5< / INSDFeature_location>

[0363] <INSDFeature_quals>

[0364] <insdqualifier>

[0365] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0366] <INSDQualifier_value>protein< / INSDQualifier_value>

[0367] < / insdqualifier>

[0368] <insdqualifier id="q18">

[0369] <INSDQualifier_name>organism< / INSDQualifier_name>

[0370] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0371] < / insdqualifier>

[0372] < / INSDFeature_quals>

[0373] < / insdfeature>

[0374] <insdfeature>

[0375] <INSDFeature_key> MOD_RES< / INSDFeature_key>

[0376] <INSDFeature_location> 5< / INSDFeature_location>

[0377] <INSDFeature_quals>

[0378] <insdqualifier id="q10">

[0379] <INSDQualifier_name>note< / INSDQualifier_name>

[0380] <INSDQualifier_value>alaninamide< / INSDQualifier_value>

[0381] < / insdqualifier>

[0382] < / INSDFeature_quals>

[0383] < / insdfeature>

[0384] < / INSDSeq_feature-table>

[0385] <INSDSeq_sequence> GPRPA< / INSDSeq_sequence>

[0386] < / insdseq>

[0387] < / sequencedata>

[0388] <sequencedata sequenceidnumber="5">

[0389] <insdseq>

[0390] <INSDSeq_length> 6< / INSDSeq_length>

[0391] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[0392] <INSDSeq_division> PAT< / INSDSeq_division>

[0393] <INSDSeq_feature-table>

[0394] <insdfeature>

[0395] <INSDFeature_key>source< / INSDFeature_key>

[0396] <INSDFeature_location>1..6< / INSDFeature_location>

[0397] <INSDFeature_quals>

[0398] <insdqualifier>

[0399] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0400] <INSDQualifier_value>protein< / INSDQualifier_value>

[0401] < / insdqualifier>

[0402] <insdqualifier id="q19">

[0403] <INSDQualifier_name>organism< / INSDQualifier_name>

[0404] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0405] < / insdqualifier>

[0406] < / INSDFeature_quals>

[0407] < / insdfeature>

[0408] <insdfeature>

[0409] <INSDFeature_key> MOD_RES< / INSDFeature_key>

[0410] <INSDFeature_location> 1< / INSDFeature_location>

[0411] <INSDFeature_quals>

[0412] <insdqualifier id="q12">

[0413] <INSDQualifier_name>note< / INSDQualifier_name>

[0414] <INSDQualifier_value>Acetylated< / INSDQualifier_value>

[0415] < / insdqualifier>

[0416] < / INSDFeature_quals>

[0417] < / insdfeature>

[0418] <insdfeature>

[0419] <INSDFeature_key> MOD_RES< / INSDFeature_key>

[0420] <INSDFeature_location> 6< / INSDFeature_location>

[0421] <INSDFeature_quals>

[0422] <insdqualifier id="q13">

[0423] <INSDQualifier_name>note< / INSDQualifier_name>

[0424] <INSDQualifier_value>argininamide< / INSDQualifier_value>

[0425] < / insdqualifier>

[0426] < / INSDFeature_quals>

[0427] < / insdfeature>

[0428] < / INSDSeq_feature-table>

[0429] <INSDSeq_sequence> EEMQRR< / INSDSeq_sequence>

[0430] < / insdseq>

[0431] < / sequencedata>

[0432] <sequencedata sequenceidnumber="6">

[0433] <insdseq>

[0434] <INSDSeq_length> 5< / INSDSeq_length>

[0435] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[0436] <INSDSeq_division> PAT< / INSDSeq_division>

[0437] <INSDSeq_feature-table>

[0438] <insdfeature>

[0439] <INSDFeature_key>source< / INSDFeature_key>

[0440] <INSDFeature_location>1..5< / INSDFeature_location>

[0441] <INSDFeature_quals>

[0442] <insdqualifier>

[0443] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0444] <INSDQualifier_value>protein< / INSDQualifier_value>

[0445] < / insdqualifier>

[0446] <insdqualifier id="q20">

[0447] <INSDQualifier_name>organism< / INSDQualifier_name>

[0448] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0449] < / insdqualifier>

[0450] < / INSDFeature_quals>

[0451] < / insdfeature>

[0452] <insdfeature>

[0453] <INSDFeature_key>LIPID< / INSDFeature_key>

[0454] <INSDFeature_location>1< / INSDFeature_location>

[0455] <INSDFeature_quals>

[0456] <insdqualifier id="q15">

[0457] <INSDQualifier_name>note< / INSDQualifier_name>

[0458] <INSDQualifier_value>MYRISTATE< / INSDQualifier_value>

[0459] < / insdqualifier>

[0460] < / INSDFeature_quals>

[0461] < / insdfeature>

[0462] <insdfeature>

[0463] <INSDFeature_key> MOD_RES< / INSDFeature_key>

[0464] <INSDFeature_location> 5< / INSDFeature_location>

[0465] <INSDFeature_quals>

[0466] <insdqualifier id="q16">

[0467] <INSDQualifier_name>note< / INSDQualifier_name>

[0468] <INSDQualifier_value>lysinamide< / INSDQualifier_value>

[0469] < / insdqualifier>

[0470] < / INSDFeature_quals>

[0471] < / insdfeature>

[0472] < / INSDSeq_feature-table>

[0473] <INSDSeq_sequence> RGDGK< / INSDSeq_sequence>

[0474] < / insdseq>

[0475] < / sequencedata>

[0476] <sequencedata sequenceidnumber="7">

[0477] <insdseq>

[0478] <INSDSeq_length> 8< / INSDSeq_length>

[0479] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[0480] <INSDSeq_division> PAT< / INSDSeq_division>

[0481] <INSDSeq_feature-table>

[0482] <insdfeature>

[0483] <INSDFeature_key>source< / INSDFeature_key>

[0484] <INSDFeature_location>1..8< / INSDFeature_location>

[0485] <INSDFeature_quals>

[0486] <insdqualifier>

[0487] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0488] <INSDQualifier_value>protein< / INSDQualifier_value>

[0489] < / insdqualifier>

[0490] <insdqualifier id="q26">

[0491] <INSDQualifier_name>organism< / INSDQualifier_name>

[0492] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0493] < / insdqualifier>

[0494] < / INSDFeature_quals>

[0495] < / insdfeature>

[0496] <insdfeature>

[0497] <INSDFeature_key> MOD_RES< / INSDFeature_key>

[0498] <INSDFeature_location> 1< / INSDFeature_location>

[0499] <INSDFeature_quals>

[0500] <insdqualifier id="q22">

[0501] <INSDQualifier_name>note< / INSDQualifier_name>

[0502] <INSDQualifier_value>acetylated< / INSDQualifier_value>

[0503] < / insdqualifier>

[0504] < / INSDFeature_quals>

[0505] < / insdfeature>

[0506] <insdfeature>

[0507] <INSDFeature_key> MOD_RES< / INSDFeature_key>

[0508] <INSDFeature_location> 8< / INSDFeature_location>

[0509] <INSDFeature_quals>

[0510] <insdqualifier id="q23">

[0511] <INSDQualifier_name>note< / INSDQualifier_name>

[0512] <INSDQualifier_value>Asparagine< / INSDQualifier_value>

[0513] < / insdqualifier>

[0514] < / INSDFeature_quals>

[0515] < / insdfeature>

[0516] < / INSDSeq_feature-table>

[0517] <INSDSeq_sequence> EEMQRRAD< / INSDSeq_sequence>

[0518] < / insdseq>

[0519] < / sequencedata>

[0520] <sequencedata sequenceidnumber="8">

[0521] <insdseq>

[0522] <INSDSeq_length> 5< / INSDSeq_length>

[0523] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[0524] <INSDSeq_division> PAT< / INSDSeq_division>

[0525] <INSDSeq_feature-table>

[0526] <insdfeature>

[0527] <INSDFeature_key>source< / INSDFeature_key>

[0528] <INSDFeature_location>1..5< / INSDFeature_location>

[0529] <INSDFeature_quals>

[0530] <insdqualifier>

[0531] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0532] <INSDQualifier_value>protein< / INSDQualifier_value>

[0533] < / insdqualifier>

[0534] <insdqualifier id="q27">

[0535] <INSDQualifier_name>organism< / INSDQualifier_name>

[0536] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0537] < / insdqualifier>

[0538] < / INSDFeature_quals>

[0539] < / insdfeature>

[0540] < / INSDSeq_feature-table>

[0541] <INSDSeq_sequence> KTTKS< / INSDSeq_sequence>

[0542] < / insdseq>

[0543] < / sequencedata>

[0544] <sequencedata sequenceidnumber="9">

[0545] <insdseq>

[0546] <INSDSeq_length>6< / INSDSeq_length>

[0547] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0548] <INSDSeq_division>PAT< / INSDSeq_division>

[0549] <INSDSeq_feature-table>

[0550] <insdfeature>

[0551] <INSDFeature_key>source< / INSDFeature_key>

[0552] <INSDFeature_location>1..6< / INSDFeature_location>

[0553] <INSDFeature_quals>

[0554] <insdqualifier>

[0555] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0556] <INSDQualifier_value>protein< / INSDQualifier_value>

[0557] < / insdqualifier>

[0558] <insdqualifier id="q28">

[0559] <INSDQualifier_name>organism< / INSDQualifier_name>

[0560] <INSDQualifier_value>synthetic construct< / INSDQualifier_value>

[0561] < / insdqualifier>

[0562] < / INSDFeature_quals>

[0563] < / insdfeature>

[0564] < / INSDSeq_feature-table>

[0565] <INSDSeq_sequence>VGVAPG< / INSDSeq_sequence>

[0566] < / insdseq>

[0567] < / sequencedata>

[0568] < / st26sequencelisting>

[0569] <---

Claims

1. An injectable composition for the treatment of deterioration and / or aging of the skin, elastosis and dermoepidermal atrophy caused by oxidative stress, comprising: - non-cross-linked sodium hyaluronate with a molecular weight of 100 to 400 kDa at a concentration of 7 to 20 mg / ml, - non-crosslinked sodium hyaluronate with a molecular weight of at least 2000 kDa at a concentration of 10 to 25 mg / ml, - a mixture of amino acids consisting of glycine in a concentration of 6 to 12.5 mg / ml, L-proline and / or L-hydroxyproline in a concentration of 5 to 8 mg / ml, L-alanine in a concentration of 1 to 5 mg / ml, L-valine in a concentration of 1 to 5 mg / ml, L-leucine in a concentration of 1 to 5 mg / ml, L-lysine HCl in a concentration of 1 to 5 mg / ml, L-arginine HCl in a concentration of 1 to 5 mg / ml.

2. The composition according to the preceding paragraph, wherein the amino acid mixture consists of 9 mg / ml glycine, 6.5 mg / ml L-proline, 2 mg / ml L-alanine, 2.5 mg / ml L-valine, 1 mg / ml L-leucine, 2.5 mg / ml L-lysine HCl, 1.5 mg / ml L-arginine HCl.

3. A composition according to any one of the preceding claims, wherein sodium hyaluronate with a molecular weight of 100 to 400 kDa is present at a concentration of 16 mg / ml and sodium hyaluronate with a molecular weight of at least 2000 kDa is present at a concentration of 16 mg / ml.

4. The composition according to one of claims 1 or 2, wherein sodium hyaluronate with a molecular weight of 100 to 400 kDa is present at a concentration of 12 mg / ml and sodium hyaluronate with a molecular weight of at least 2000 kDa is present at a concentration of 20 mg / ml.

5. The composition according to one of claims 1 or 2, wherein the sodium hyaluronate is present in a total concentration of more than 25 mg / ml.

6. A composition according to any one of the preceding claims, wherein said composition has a pH value preferably from 6.8 to 7.5, even more preferably from 7 to 7.

3.

7. A composition according to any one of the preceding claims, comprising at least one of: a saline solution, pharmaceutically acceptable excipients or adjuvants, a buffer, preferably a phosphate buffer, an anesthetic, preferably a local anesthetic, preferably lidocaine, in a concentration in the range of 0.1 to 0.4%, preferably 0.2 to 0.3%.

8. A composition according to any one of the preceding claims, further comprising at least one of acetyl decapeptide 3 SEQ ID NO: 1, oligopeptide 24 SEQ ID NO: 2, acetyl tetrapeptide 5 SEQ ID NO: 3, vialox pentapeptide-3 SEQ ID NO: 4, acetyl hexapeptide 8 SEQ ID NO: 5, myristoyl pentapeptide-8 SEQ ID NO: 6, peptide GHK-Cu with the sequence Gly-His-Lys-Cu, tripeptide-29 with the sequence H-Gly-Pro-Hyp-OH, octapeptide-3 SEQ ID NO: 7, matrixyl SEQ ID NO: 8, hexapeptide SEQ ID NO: 9, each peptide at a final concentration of 0.005 to 0.080 mg / ml, preferably 0.005 to 0.05 mg / ml, more preferably 0.005 to 0.02 mg / ml.

9. Use of a composition according to any one of claims 1-8 for treating a cosmetic condition of the skin.

10. The use according to claim 9, wherein said cosmetic condition is selected from photoaging, atrophic scars, scars, facial defects and asymmetries, wrinkles and folds of the skin, preferably of the face, more preferably interglabellar wrinkles, nasolabial folds, folds in the chin area, marionette lines, wrinkles in the cheek area, perioral wrinkles, crow's feet.

11. Use of a composition according to any one of claims 1-8 for stimulating collagen synthesis.

12. A kit for the treatment of skin deterioration and / or aging, elastosis and dermoepidermal atrophy caused by oxidative stress, including: a) an injection composition according to any one of claims 1 to 8, and b) instructions for use.

13. The kit according to claim 12, wherein the injection composition is in the form of a gel and is contained in a pre-filled syringe.

14. Use of the kit according to any of paragraphs 12 or 13 for treating a cosmetic condition of the skin.

15. The use according to claim 14, wherein said cosmetic condition is selected from photoaging, atrophic scars, scars, facial defects and asymmetries, wrinkles and folds of the skin, preferably of the face, more preferably interglabellar wrinkles, nasolabial folds, folds in the chin area, marionette lines, wrinkles in the cheek area, perioral wrinkles, crow's feet.

16. Use of the kit according to any of paragraphs 12 or 13 for stimulating collagen synthesis.

17. A non-therapeutic method for treating deterioration and / or aging of the skin, elastosis and dermoepidermal atrophy caused by oxidative stress in a subject, comprising intradermal injection of a composition according to one of claims 1-8.

Images