HK40108865A - Injectable composition comprising hyaluronic acid and amino acids - Google Patents

Description

Injectable compositions containing hyaluronic acid and amino acids

Technical Field

This invention relates to injectable compositions comprising a specific combination of non-crosslinked low molecular weight hyaluronic acid (HA) and high molecular weight hyaluronic acid (HA), and a specific composition of amino acids (AA), which are capable of resisting the reduction of extracellular matrix (ECM) proteins and skin hydration caused by oxidative stress-induced aging.

Background Technology

The skin is the largest organ in the human body, with an adult having a total surface area of approximately 2 square meters. It performs many essential functions, including thermoregulation, defense against pathogens and UV damage, and acting as a waterproof barrier. It is connected to the brain through a vast network of nerves and cells, serving as an environmental sensory organ (1,3). The skin consists of three layers: the epidermis (outer layer) contains different types of cells, such as squamous cells, basal cells, and melanocytes. The dermis is the middle layer of the skin; besides blood vessels and lymphatic vessels, hair follicles, and sweat glands, it mainly contains collagen fiber bundles and fibroblasts. The hypodermis is the deepest layer, containing adipose tissue, hair follicles, sensory neurons, and blood vessels (3,2,4). In addition to cells, one of the main components of the skin is the extracellular matrix (ECM), a complex macromolecular composition of water, polysaccharides (glycosaminoglycans such as hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratin sulfate), and proteins, with collagen, fibronectin, laminin, proteoglycans, and elastin being the most abundant (5,6,7,8). ECM (extracellular matrix membrane) is mainly produced by fibroblasts and mesenchymal cells, playing a crucial role in tissue development, maintenance, and repair. ECM endows tissues with mechanical and unique properties, while also playing a vital role in regulating cellular function. Cell-ECM interactions are mediated by specific receptors, promoting not only cell adhesion and migration but also regulating cell differentiation and gene expression, and playing a key role in wound healing (6,7). In particular, ECM imparts elasticity, resistance, and compressibility to the skin. The most important protein responsible for skin elasticity is elastin, one of the main structural proteins of the ECM, accounting for approximately 2% of total dermal protein. Structurally, elastin exhibits alternating hydrophobic and hydrophilic domains; it typically consists of short repeating units of three to nine amino acids, rich in glycine, proline, alanine, leucine, and valine.

At the origin of basic elastin production, fibroblasts synthesize and secrete precursor elastin (ELN, a soluble monomer) into the extracellular space, where it forms a highly cross-linked, insoluble polymer composed of covalently bonded elastin molecules, generating elastin (9,10,11) within the ECM. Another prevalent structural protein in the ECM is collagen, also the most common protein in mammals; collagen comprises a family of molecules that differ structurally and functionally, with 28 members numbered in Roman numerals (I-XXVIII) in vertebrates and encoded by 28 different genes, of which type I is most common in humans. Despite the high heterogeneity among the various types, all members of this family possess a characteristic triple helix structure composed of three α chains, which can be formed from the same collagen chains (homotrimer) or from the combination of different collagen homotypes (heterotrimer). All collagen isotypes contain repeating domains of the Gly-X-Y tripeptide, where the X and Y positions are typically occupied by proline and hydroxyproline; these n(Gly-X-Y) repeats are essential for linking the triple helix structure, and depending on the collagen type, proline and lysine residues also play a role because they are important sites for post-translational modifications (12,13).

Skin structure and function are characterized by continuous and cumulative changes, including alterations in the activity and proliferation of cellular components and the expression and production of the extracellular matrix. Decreased ECM function leads to loss of elasticity and resilience, as well as the appearance of wrinkles, a consequence of skin aging (14,15). Furthermore, another major characteristic of skin aging is dryness and loss of skin moisture.

Hyaluronic acid (HA) glycosaminoglycans are key molecules for skin hydration due to their unique and fundamental ability to bind and retain water molecules (16); in addition to hydration, HA also plays important roles in wound healing, fibroblast migration, immune responses, and tumor development (16). The size of HA greatly influences its function; large HA molecules (typically exceeding 1,000 kDa) have anti-angiogenic and immunosuppressive effects, while smaller HA polymers are potent inducers of inflammation and angiogenesis. But most importantly, aging not only leads to a decrease in HA synthesis but also to the production of even smaller, harmful HA molecules (16,17,18).

We are far from fully understanding the many causes of aging; key causes include glycation, telomere shortening, secondary reactions, mutations, and protein aggregation. However, one of the well-known major factors contributing to organic aging is the production of reactive oxygen species (ROS); in fact, the mitochondrial free radical aging theory was proposed as early as 1956, based primarily on the production of ROS as a byproduct of mitochondrial respiration (19,20). Due to continuous exposure to ultraviolet radiation, the production of ROS in this organ, in addition to endogenous (intrinsic) mitochondrial origin, also has exogenous (external) sources; this results in a particularly high number of free radicals in the skin, triggering the photoaging process (21,22). One of the main consequences of skin exposure to ultraviolet radiation is the accelerated renewal of the ECM, a normal and essential process for maintaining healthy tissue, in which aged proteins are degraded and replaced by newly synthesized proteins (14,23). However, ultraviolet radiation generates intracellular ROS, such as superoxide anions ( _O2- ) and hydrogen _peroxide ( _H2O2 ), leading to the synthesis of matrix metalloproteinases (MMPs) (24,25). Therefore, without simultaneously increasing the production of new ECM proteins, the digestion of ECM components by MMPs alters the turnover balance between synthesis and degradation, thus favoring degradation. The result is the loss of collagen, elastin, and fibrillary fibers, leading to decreased skin elasticity and the appearance of wrinkles (21,22).

Another important effect of oxidative stress is the increased percentage of senescent cells, which also plays a fundamental role in skin aging. Senescent fibroblasts no longer divide and acquire the aging-associated secretory phenotype (SASP) (26), while increasing the secretion of pro-inflammatory cytokines and chemokines (ECM remodeling proteases); in addition, SASP increases the amount of ROS, thereby forming a vicious cycle of free radical production, ultimately leading to premature aging (26,27,28).

To prevent and/or counteract the harmful effects of oxidative stress on photoaging of the skin, various treatment and nutritional 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 loss of skin elasticity (29,30,31). Furthermore, stimulating fibroblast activity to improve ECM protein translation can also block oxidative stress-induced aging, thereby delaying the onset of skin aging.

As is well known, amino acids are the building blocks of proteins and stimulate protein translation when placed as substrates. However, only by correctly identifying and administering specific amino acid compositions can they promote the synthesis of specific protein subsets. To date, several compositions have been developed to combat oxidative stress and skin aging; these compositions are specifically designed for injection. Currently, many compositions are available for intradermal injection with the aim of increasing volume (e.g., fillers containing cross-linked hyaluronic acid (HA)) or providing long-term effects by inducing the production of new collagen.

In the human body, injected hyaluronic acid remains in place for several days in its natural state because the polymer chains are easily degraded by enzymes and free radicals present in the body. To overcome this problem, most commercially available compositions are prepared through a cross-linking process using the hydroxyl groups of HA via chemical cross-linking agents. The final effect of such compositions on tissues can be controlled by varying the cross-linking density using various cross-linking agents; however, this method has a significant drawback: HA-based cross-linked fillers are actually very dense and difficult to inject.

Therefore, the object of the present invention is to provide alternative and improved injectable compositions to resist damage caused by photoaging and improve collagen production in the dermis.

Summary of the Invention

The object of the present invention is to provide an injectable composition comprising a specific combination of non-crosslinked low molecular weight hyaluronic acid (HA) and high molecular weight hyaluronic acid (HA), and a specific composition of amino acids (AA).

Specifically, the composition according to the present invention comprises:

Non-crosslinked sodium hyaluronate, with a molecular weight of 100-400 kDa and a concentration of 7 to 20 mg/ml;

Non-crosslinked sodium hyaluronate with a molecular weight of 2000 kDa or higher and a concentration of 10 to 25 mg/ml;

An amino acid mixture, comprising:

Glycine 6-12.5 mg/ml;

L-proline and/or L-hydroxyproline 5-8 mg/ml;

L-alanine 1-5 mg/ml;

L-valine 1-5 mg/ml;

L-Leucine 1-5 mg/ml;

L-Lysine hydrochloride (HCl) 1-5 mg/ml;

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

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

Glycine 9mg/ml;

Proline 6.5 mg/ml;

Alanine 2mg/ml;

Valine 2.5 mg/ml;

Leucine 1mg/ml;

Lysine hydrochloride 2.5 mg/ml;

Arginine hydrochloride 1.5 mg/ml.

In one embodiment, the composition according to the invention comprises sodium hyaluronate with a molecular weight of 100-400 kDa and a concentration of 16 mg/ml, and sodium hyaluronate with a molecular weight of at least 2000 kDa and a concentration of 16 mg/ml.

In one embodiment, the concentration of sodium hyaluronate with a molecular weight of 100-400 kDa is 12 mg/ml, and the concentration of sodium hyaluronate with a molecular weight of 2000 kDa is 20 mg/ml.

In a preferred embodiment, the composition according to the invention comprises a total sodium hyaluronate concentration of greater than 25 mg/ml.

In a preferred embodiment, the pH of the composition is preferably 6.8 to 7.5, more preferably 7 to 7.3.

In a preferred embodiment, the composition according to the invention comprises at least one pharmaceutically acceptable excipient or adjuvant, a buffer (preferably a phosphate buffer), and an anesthetic (preferably a local anesthetic).

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

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

The present invention also relates to the use of the compositions described above in treating skin deterioration and/or aging caused by oxidative stress, degeneration of elastic tissue and dermal-epidermal atrophy or other pathologies caused by oxidative stress, and for cosmetic purposes, preferably for treating photoaging, skin depressions, scars, facial defects and asymmetry, wrinkles and skin lines, preferably facial wrinkles and skin lines, more preferably frown lines, nasolabial folds, chin wrinkles, marionette lines, cheek lines, perioral wrinkles, and crow's feet.

Finally, the object of the present invention is to provide the use of the composition or kit according to the invention to stimulate collagen synthesis, and to provide a non-therapeutic method for skin treatment of a subject, including intradermal injection of the composition.

Further objectives will become apparent from the detailed description below.

Attached Figure Description

Figure 1: Expression of catalase (CAT) and Ink4 mRNA in BJ fibroblasts pretreated with or untreated (culture medium only, CT) with three compositions (AA1, AA2 or _AA3 ) and then treated with hydrogen peroxide ( _H2O2 ) or completely untreated (NT).

Figure 2: mRNA expression of elastin (ELN), fibrin ( _FBN ), and collagen isotype IV (Col4a1) in BJ fibroblasts pretreated with or untreated (culture medium only, CT) with three compositions (AA1, AA2, or _AA3 ) and then treated with hydrogen peroxide (H2O2) or completely untreated (NT).

Figure 3: The proliferation of BJ fibroblasts was analyzed by MTT assay before treatment (0 hours) and after incubation for 24, 48, and 72 hours with two mixtures of AA4 and AA3 at different final concentrations (0.1%, 0.3%, and 0.5%).

Figure 4: mRNA gene expression of two collagen isoforms (col1a1 and col4a1) after 72 hours of incubation in BJ fibroblasts pretreated with the two compositions (AA4 or AA3).

The values 0.1%, 0.3%, and 0.5% refer to the different final concentrations of the two mixtures.

Detailed Implementation

Therefore, the inventors have developed a novel composition to combat the skin aging process and stimulate collagen production. This composition comprises a specific combination of non-crosslinked low-molecular-weight hyaluronic acid (HA) and high-molecular-weight hyaluronic acid (HA), and a specific composition of amino acids (AA), which are able to combat the aging-related reduction in extracellular matrix (ECM) proteins and skin hydration caused by oxidative stress.

In one embodiment, the composition according to the invention further comprises a biomimetic peptide selected according to its effects.

Therefore, the subject of this invention is a novel composition comprising an amino acid composition and non-crosslinked hyaluronic acid of different molecular weights, which can both stimulate the synthesis of elastin and collagen and exert a protective function against ROS and oxidative stress-induced aging.

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

The composition (AA3) according to the present invention comprises:

Non-crosslinked hyaluronic acid with a molecular weight of 100-400 kDa.

Non-crosslinked hyaluronic acid with a molecular weight of 2000 kDa or higher.

The total amino acid concentration of 25 mg/ml is as follows:

Glycine 6-12.5 mg/ml;

L-proline and/or L-hydroxyproline 5-8 mg/ml;

L-alanine 1-5 mg/ml;

L-valine 1-5 mg/ml;

L-Leucine 1-5 mg/ml;

L-Lysine hydrochloride (HCl) 1-5 mg/ml;

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

In a preferred embodiment, the composition comprises

Glycine 9mg/ml;

Proline 6.5 mg/ml;

Alanine 2mg/ml;

Valine 2.5 mg/ml;

Leucine 1mg/ml;

Lysine hydrochloride 2.5 mg/ml;

Arginine hydrochloride 1.5 mg/ml.

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

Table 1

The compositions according to the invention optionally comprise 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:

Acetyl decapeptide 3, SEQ ID NO:1, Ac-Tyr-Arg-Ser-Arg-Lys-Tyr-Thr-Ser-Trp-Tyr-NH ₂ ,

Oligopeptide 24, SEQ ID NO:2, H-RGDGCMYIEGGGG-OH,

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

Velos pentapeptide 3, SEQ ID NO:4, Gly-Pro-Arg-Pro-Ala-NH ₂ ,

Acetyl hexapeptide 8 (hexapeptide), SEQ ID NO:5, Ac-Glu-Glu-Met-Gln-Arg-Arg-NH ₂ ,

Myristoyl pentapeptide 8, SEQ ID NO:6, Myr-RGDGK-NH ₂ ,

GHK-Cu peptide, with the unreported sequence Gly-His-Lys-Cu (containing only 3 amino acids),

Tripeptide 29 (collagen tripeptide), with the sequence H-Gly-Pro-Hyp-OH (containing only 3 amino acids), which is not reported in the sequence listing.

The composition according to the present invention may further comprise at least one of the following:

Octapeptide 3, SEQ ID NO:7, Ac-Glu-Glu-Met-Gln-Arg-Arg-Ala-Asp-NH ₂ ,

Pentapeptide, SEQ ID NO:8, Lys-Thr-Thr-Lys-Ser,

Hexapeptide, SEQ ID NO:9, Val-Gly-Val-Ala-Pro-Gly.

The above concentrations apply to each peptide.

In a preferred embodiment, the composition according to the invention comprises a total sodium hyaluronate concentration of greater than 25 mg/ml.

The compositions according to the invention may also contain pharmaceutically acceptable excipients or adjuvants.

The compositions according to the invention may also contain a buffer, such as a phosphate buffer, to adjust the pH.

The pH of the composition is preferably 6.8 to 7.5, more preferably 7 to 7.3.

The compositions according to the invention may also contain an anesthetic, particularly a local anesthetic, preferably lidocaine, at a concentration of 0.1% to 0.4%, preferably 0.2% to 0.3%.

To verify the improvement effect of the compositions according to the invention on collagen production compared with compositions currently available on the market, comparative tests were conducted, in which the compositions according to the invention were compared with other products currently available on the market, and their compositions are shown in Tables 2 and 4: one (AA1) contains 33 mg/ml total amino acids (as shown in Table 2) + 10 mg/ml hyaluronic acid (100 kDa), and one (AA2) contains only 16 mg/ml cross-linked hyaluronic acid (80-100 kDa) + 16 mg/ml cross-linked hyaluronic acid (1100-1400 kDa). AA4 contains 32 mg/ml low molecular weight hyaluronic acid (100-400 kDa) and an amino acid mixture identical to the compositions according to the invention but lacking lysine.

Table 2

To assess the induction of oxidative stress, the mRNA expression levels of catalase (CAT, a known ROS scavenger and antioxidant enzyme) in the BJ8 human fibroblast cell line (ATCCCRL-2522) were first analyzed and compared. _CAT mRNA levels increase in response to _H₂O₂ , thus constituting a marker of intracellular ROS levels. In Figure 1, as expected, a 48% increase in CAT mRNA induced by _{H₂O₂ treatment} was observed compared to untreated cells (NT). Cells were pretreated with compositions AA1, AA2, and AA3 for comparison.

The inventors were surprised to find that pretreatment of BJ fibroblasts with AA3 significantly reduced CAT mRNA levels to levels similar to those of control cells without induced oxidative stress, indicating that the composition has the property of counteracting the effects of oxidative stress in cells (a 51% reduction compared to CT cells treated with _{H2O2 alone} ). Conversely, pretreatment with composition AA1 and composition AA2 did not significantly alter CAT levels (reducing them by 8% and 4% respectively relative to CT).

Therefore, compared with the formulations described in the prior art, the compositions according to the present invention are extremely effective in counteracting the effects of oxidative stress in fibroblasts.

To measure the level of oxidative stress-induced senescence, the mRNA expression level of p16(INK4a)(INK4), a well-known biomarker of senescence, was measured, and its mRNA level is significantly elevated in senescent cells (36). In the same case, treatment with _H₂O₂ resulted in a significant increase _in INK4 mRNA, confirming senescence of BJ fibroblasts due to oxidative stress; comparative cells were also pretreated with compositions AA1, AA2, and AA3, respectively, in this case.

Similar to what was observed at the CAT level, a significant decrease in INK4 mRNA expression levels was observed after AA3-based pretreatment (65% reduction compared to CT), indicating lower senescence in fibroblasts treated with composition AA3. Although reductions in INK4 mRNA were also recorded in cells pretreated with compositions AA1 (33% reduction compared to CT) and AA2 (28% reduction compared to CT), composition AA3 proved to be significantly more effective. Table 3 below summarizes the data discussed above.

Table 3

The percentage reduction in mRNA levels in cells treated with _the indicated composition and hydrogen peroxide ( _H₂O₂ ) compared to the control treated with hydrogen peroxide alone.

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

To verify the effect of the composition according to the present invention on reducing the mRNA level in ECM proteins induced by oxidative stress, the inventors analyzed the mRNA expression of three ECM proteins, namely elastin (ELN), fibrinogen (FBN), and Col4a1 collagen.

The expression of these mRNAs is strongly affected by oxidative stress. As shown in Figure ₂ , after treatment with _H2O2 , the mRNA of all ECM genes was significantly reduced, confirming the ECM deterioration caused by oxidative stress.

In this case, the cells were also treated with the three compositions reported in Table 2; after treatment, it was observed how the mRNA expression level of ELN was not affected by treatment with composition AA1 or composition AA2 (reduced by 0.9% and 4% compared to CT).

Conversely, ELN mRNA expression levels were restored by treatment with AA3 (37% increase compared to CT).

Similarly, the FBN mRNA depletion caused by oxidative stress (31% lower than NT) was almost completely blocked by composition AA3 (32% higher than CT), and partially blocked by AA1 (26% higher than CT), but not blocked by AA2 (3% higher).

Finally, _{H₂O₂ -} induced col₄a₁ consumption (reduced by 55% compared to NT) was slightly improved by using the composition of AA₁ and AA₂ (increased by 13% and 24%, respectively), and was slightly more significantly improved by using the composition of AA₃ (increased by 28% compared to CT).

Composition AA3 not only significantly reduced oxidative stress and senescence in _{H₂O₂ -} treated BJ fibroblasts, but also restored the resulting reduction in ECM mRNA. Specifically, only composition AA3 was shown to be effective against oxidative stress and senescence, while AA1 or AA2 were not; similarly, only AA3 restored ELN expression. Furthermore, the composition according to the invention has been shown to be more effective in increasing FBN mRNA compared to the control composition, and was able to restore Col4a1 levels along with the other two compositions. ELN is one of the most important proteins in the ECM, responsible for skin elasticity (9,10); FBN is also another essential protein in the formation of ECM elastic fibers, and its levels are known to decrease with aging, especially under exogenous oxidative stress (37). Therefore, the ability of the AA3 composition to restore mRNA levels by counteracting the _{H₂O₂ -} induced reduction suggests that the composition plays an effective role in counteracting age-related ECM deterioration. Among skin collagen isotypes, type IV (Col4a1) is one of the most severely affected by aging; its deterioration also highlights skin aging and photoaging caused by oxidative stress (38). The compositions according to the invention have been shown to partially restore col4a1 mRNA levels reduced after _{H2O2 treatment} .

Of all the compositions tested, the composition according to the invention proved to be the most effective. The greater efficacy of the composition according to the invention in counteracting oxidative stress, aging, and ECM reduction compared to AA1 and AA2 is attributed to its specific composition; indeed, although the composition according to the invention contains fewer total amino acids than AA1, the specific ratio between them and the presence of arginine, in addition to the selected molecular weight of HA, allows for a greater beneficial effect on fibroblast aging and stronger antioxidant capacity than the control composition.

By further comparison, the effect of composition AA3 according to the present invention was tested compared with composition (AA4) lacking high molecular weight hyaluronic acid and lysine. Table 4 illustrates these compositions.

Table 4

The effects of the composition were evaluated by analyzing fibroblast proliferation and gene expression of collagen (Col4a1, Col1a1).

Human BJ fibroblasts were cultured in F12 medium at 70%–80% confluence with 10% (v/v) fetal bovine serum (FBS) and 2 mM L-glutamine under 5% _CO2 /95% air atmosphere. Cells were then treated with compositions AA3 and AA4 at final concentrations (0.1%–0.3%–0.5%) as shown in Figures 3 and 4. Proliferation was assessed using the MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide]. ⁸ x 10³ cells/well were seeded into 100 μL of medium in 96-well plates. Purple formazan crystals were dissolved overnight at 37°C in 5% SDS/0.1 M HCl (100 μL/well), and absorbance at 570 nm/655 nm dual wavelengths was recorded at zero time (as a control), 24, 48, and 72 hours using a microplate reader. The results are shown in Figure 3, demonstrating how the composition according to the invention stimulates the proliferation of BJ fibroblasts more effectively than the control composition in a dose-dependent manner at each tested concentration. Furthermore, the composition according to the invention increases the proliferation of BJ fibroblasts, even at the lowest dose, and is more effective than the control composition.

Alternatively, at the end of the experimental treatment (72 hours), cells were harvested for mRNA extraction. RNA was isolated using the RNeasy Mini Kit (Qiagen), and cDNA (1 μg) was synthesized using the iScript (Bio-Rad Laboratories) cDNA Synthesis Kit. The relative levels of gene expression were calculated as 2 - ΔΔCT, where ΔΔCT corresponds to the difference between the ΔCT of the two treatment groups and the ΔCT of the untreated group. GAPDH was used as a reference housekeeping gene. The results are shown in Figure 4 and demonstrate how the composition according to the invention promotes the expression of the analyzed collagen genes more effectively than the control composition; furthermore, the composition according to the invention increases the expression of both genes in a dose-dependent manner and at each concentration, demonstrating greater effectiveness than the control composition even at relatively low doses.

Therefore, the inventors have developed a composition that, compared with compositions currently described in the prior art, is able to block the harmful effects of oxidative stress, aging, and ECM protein reduction in fibroblasts in a much more effective manner.

According to the present invention, "injectable" means that it can be delivered from a syringe under normal conditions and atmospheric pressure, and means that it can be injected into the skin, dermis or other tissues to deliver the composition to the desired target site.

Therefore, the object of the present invention is to provide an injectable composition comprising:

Non-crosslinked sodium hyaluronate, 7-20 mg/ml, molecular weight 100-400 kDa.

Non-crosslinked sodium hyaluronate, 10-25 mg/ml, molecular weight 2000 kDa.

An amino acid mixture, comprising:

Glycine 6-12.5 mg/ml;

L-proline and/or L-hydroxyproline 5-8 mg/ml;

L-alanine 1-5 mg/ml;

L-valine 1-5 mg/ml;

L-Leucine 1-5 mg/ml;

L-Lysine hydrochloride 1-5 mg/ml;

L-arginine hydrochloride 1-5 mg/ml.

In a preferred embodiment, the composition comprises:

Glycine 9mg/ml;

L-proline 6.5 mg/ml;

L-alanine 2 mg/ml;

L-valine 2.5 mg/ml;

L-Leucine 1 mg/ml;

L-Lysine hydrochloride 2.5 mg/ml;

L-arginine hydrochloride 1.5 mg/ml.

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

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

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

In one embodiment, the composition according to 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:

Acetyl decapeptide 3, SEQ ID NO:1, Ac-Tyr-Arg-Ser-Arg-Lys-Tyr-Thr-Ser-Trp-Tyr-NH ₂ ,

Oligopeptide 24, SEQ ID NO:2, H-RGDGCMYIEGGGG-OH,

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

Velos pentapeptide 3, SEQ ID NO:4, Gly-Pro-Arg-Pro-Ala-NH ₂ ,

Acetyl hexapeptide 8 (hexapeptide), SEQ ID NO:5, Ac-Glu-Glu-Met-Gln-Arg-Arg-NH ₂ ,

Myristoyl pentapeptide 8, SEQ ID NO:6, Myr-RGDGK-NH ₂ ,

GHK-Cu peptide, with the unreported sequence Gly-His-Lys-Cu (containing only 3 amino acids),

Tripeptide 29 (collagen tripeptide), with the sequence H-Gly-Pro-Hyp-OH (containing only 3 amino acids), which is not reported in the sequence listing.

The composition according to the present invention may further comprise at least one of the following:

Octapeptide 3, SEQ ID NO:7, Ac-Glu-Glu-Met-Gln-Arg-Arg-Ala-Asp-NH ₂ ,

Pentapeptide, SEQ ID NO:8, Lys-Thr-Thr-Lys-Ser,

Hexapeptide, SEQ ID NO:9, Val-Gly-Val-Ala-Pro-Gly.

The compositions according to the invention may also contain at least one pharmaceutically acceptable excipient or adjuvant.

The compositions according to the invention may further comprise a buffer, such as a phosphate buffer, to adjust the pH. The pH is preferably 6.8 to 7.5, more preferably 7 to 7.3.

The compositions according to the invention may also contain an anesthetic, particularly a local anesthetic, preferably lidocaine, at a concentration of 0.1% to 0.4%, preferably 0.2% to 0.3%.

The present invention also relates to a kit comprising an injectable composition according to the present invention, contained in a prefilled syringe in gel form, optionally including instructions for use.

Therefore, the object of the present invention is to provide a composition or kit as defined above for treating skin deterioration and/or aging, elastic tissue degeneration and dermal-epidermal atrophy caused by oxidative stress.

The compositions and kits according to the invention are also used to treat photoaging, skin depressions, scars, defects and asymmetry of the nose, lips, cheeks, perioral region, and infraorbital region, facial asymmetry, jaw and chin lines, wrinkles and skin lines, such as facial lines, with non-limiting examples including frown lines, nasolabial folds, chin wrinkles, marionette lines, cheek lines, perioral wrinkles, and crow's feet.

The compositions and kits according to the present invention are also used to stimulate collagen synthesis and for cosmetic improvement of soft tissues.

The use described in this invention is preferably for treating cosmetic conditions; however, the composition can also be applied for treating therapeutic indications.

Example

Cells and processing.

Human BJ fibroblasts were purchased from the American Type Culture Library (ATCC-CRL-2522) and grown to 70%–80% confluence in F12 medium (ATCC) supplemented with 10% (v/v) fetal bovine serum (FBS) and 2 mM L-glutamine, under a humidified atmosphere containing 5% _CO2 /95% air. Cells were pretreated for 24 hours with the 1% composition shown in Table 2.

Then, to induce oxidative stress, _cells were treated with 200 μM _H₂O₂ for 2 hours, followed by another 48 hours. Untreated cells were plated as a control. At the end of the experimental treatment, mRNA was extracted from the cells.

Total RNA extraction and gene expression analysis.

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 levels of gene expression at 2-ΔΔCT were calculated, where ΔΔCT corresponds to the difference between the ΔCT of the treatment group and the untreated group. GAPDH was used as a constitutive gene reference.

The gene expression levels of CAT, INK4, ELN, COL4a1, and FBN were analyzed.

References:

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

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

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

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

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

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.

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

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

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

10. Vindin H, Mithieux SM, Weiss AS.

11. CU, Heinz A, Majovsky P, Karaman Mayack B, Brinckmann J, SipplW, Schmelzer CEH. Elastin is heterogeneously cross-linked. J Biol Chem. 2018 Sep28; 293(39):15107-15119.

12.Ricard-Blum S.The collagen family.Cold Spring Harb PerspectBiol.2011 Jan 1;3(1).

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.

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

15.Black LD,Allen PG,Morris SM,Stone PJ,Suki B.ECMhanical and failureproperties of extracellular matrix sheets as a func tion of structural proteincomposition.Biophys J.2008 Mar 1;94(5):1916-29.doi:10.1529/biophysj.107.107144.Epub 2007 Nov 9.

16. Dicker KT, Gurski LA, Pradhan-Bhatt S, Witt RL, Farach-Carson MC, JiaX. Hyaluronan: a simple polysaccharide with diverse biological functions. ActaBiomater. 2014 Apr; 10(4):1558-70.

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

18.Papakonstantinou E,Roth M,Karakiulakis G.Hyaluronic acid:A keymolecule in skin ageing.De rmatoendocrinol.2012 Jul 1;4(3):253-8.doi:10.4161/derm.21923.PMID:23467280; PMCID:PMC3583886.

19. da Costa JP, Vitorino R, Silva GM, Vogel C, Duarte AC, Rocha-Santos T. Asynopsis on ageing-Theories, ECMhanisms and future prospects. Ageing ResRev. 2016 Aug; 29:90-112.

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

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

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

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

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.

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.

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

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.

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.

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

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

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

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

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

34. Albaugh VL, Mukherjee K, Barbul A. Proline Precursors and CollagenSynthesis: Biochemical Challenges of Nutrient Supplementation and WoundHealing.J Nutr.2017 Nov; 147(11):2011-2017.

35.Nenoi M, Ichimura S, Mita K, Yukawa O, Cartwright IL.Regulation of thecatalase 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.

36. Wang AS, Dreesen O. Biomarkers of Cellular Senescence and SkinAgeing. Front Genet. 2018 Aug 23; 9:247.

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.

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 collagenIV expr Ession in vivo in the dermo-epidermal junction and in vitro in dermalfibroblasts: possible involvement of TGF-β1.Eur J Dermatol.2016 Aug 1;26(4):350-60.

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

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

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.

Claims (13)

1. An injectable composition comprising: Non-crosslinked sodium hyaluronate with a molecular weight of 100 to 400 kDa and 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; The amino acid mixture comprises 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 hydrochloride at a concentration of 1 to 5 mg/ml, and L-arginine hydrochloride at a concentration of 1 to 5 mg/ml. 2. The composition according to the preceding claims, wherein the amino acid mixture comprises 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 hydrochloride, and 1.5 mg/ml L-arginine hydrochloride. 3. The composition according to any one of the preceding claims, wherein the concentration of sodium hyaluronate with a molecular weight of 100 to 400 kDa is 16 mg/ml, and the concentration of sodium hyaluronate with a molecular weight of at least 2000 kDa is 16 mg/ml. 4. The composition according to any one of claims 1 or 2, wherein the concentration of sodium hyaluronate with a molecular weight of 100 to 400 kDa is 12 mg/ml, and the concentration of sodium hyaluronate with a molecular weight of at least 2000 kDa is 20 mg/ml. 5. The composition according to any one of claims 1 or 2, wherein the total concentration of sodium hyaluronate is greater than 25 mg/ml. 6. The composition according to any one of the preceding claims, wherein the pH of the composition is preferably 6.8 to 7.5, more preferably 7 to 7.3. 7. The composition according to any one of the preceding claims, comprising at least one of the following: a physiological solution, a pharmaceutically acceptable excipient or adjuvant, a buffer, preferably a phosphate buffer, an anesthetic, preferably a local anesthetic, preferably lidocaine, in a concentration range of 0.1% to 0.4%, preferably 0.2% to 0.3%. 8. The composition according to any one of the preceding claims, comprising at least one of the following: Acetyl decapeptide 3 (SEQ ID NO:1), oligopeptide 24 (SEQ ID NO:2), acetyl tetrapeptide 5 (SEQ ID NO:3), Velos pentapeptide 3 (SEQ ID NO:4), acetyl hexapeptide 8 (SEQ ID NO:5), myristoyl pentapeptide 8 (SEQ ID NO:6), peptide GHK-Cu (Gly-His-Lys-Cu sequence), tripeptide 29 (H-Gly-Pro-Hyp-OH sequence), octapeptide 3 (SEQ ID NO:7), pentapeptide (SEQ ID NO:8), and hexapeptide (SEQ ID NO:9). The final concentration range for each peptide is from 0.005 mg/ml to 0.080 mg/ml, preferably from 0.005 mg/ml to 0.05 mg/ml, and more preferably from 0.005 mg/ml to 0.02 mg/ml. 9. A kit comprising an injectable composition according to any one of the preceding claims, contained in a pre-filled syringe in gel form, optionally including instructions for use. 10. The composition or kit according to any one of the preceding claims for the treatment of skin deterioration and/or aging, elastic tissue degeneration and dermal-epidermal atrophy caused by oxidative stress. 11. Use of the composition or kit according to any one of claims 1 to 9 for cosmetic applications, preferably for treating photoaging, skin depressions, scars, facial defects and asymmetry, wrinkles and skin lines, preferably facial wrinkles and skin lines, more preferably frown lines, nasolabial folds, chin wrinkles, marionette lines, cheek lines, perioral wrinkles, and crow's feet. 12. Use of the composition or kit according to any one of claims 1 to 9 for stimulating collagen synthesis. 13. A non-therapeutic treatment for skin treatment of a subject, comprising intradermal injection of the composition of any one of claims 1 to 8.