A composition containing hyaluronic acid, amino acids, and Krebs cycle intermediates that are useful for increasing gene expression in the extracellular matrix.

Abstract

The present invention relates to compositions for dermatological and cosmetic applications comprising a mixture of amino acids, hyaluronic acid or a salt thereof, and Krebs cycle acids, particularly malic acid and succinic acid or salts thereof. The compositions of the present invention are useful in suppressing skin aging caused by a decrease in the amount of extracellular matrix deposition.

Description

Technical Field

[0001] The present invention relates to a composition for use in dermatology and cosmetics, comprising hyaluronic acid, an amino acid or its salt, and a mixture of Krebs cycle acids, particularly malic acid and succinic acid or their salts. The composition according to the present invention is useful for combating skin aging caused by a decrease in extracellular matrix deposition.

Background Art

[0002] In the human body, the skin is one of the organs most affected by aging. Nutritional approaches devised to counteract the age-related decline in extracellular matrix (ECM) deposition can be a useful means of reducing the degenerative processes underlying skin aging.

[0003] Skin aging is characterized by continuous and cumulative changes in the structure and function of the skin, mainly due to a decrease in the production of its extracellular matrix (ECM) components (Shin et al., 2019; Sparavigna, 2020). The decline in ECM function causes a decrease in skin elasticity and resilience and the appearance of wrinkles, which are typical features of skin aging (Black et al., 2008; Sparaviga, 2020, Birch, 2018). Fibroblasts mainly produce ECM consisting of proteins and glycosaminoglycans, and its main constituent is hyaluronic acid (HA), which is an important molecule involved in skin moisturization due to its ability to bind and retain water molecules. The age-related decrease in the content and activity of HA in the dermis results from both a decrease in synthesis and an increase in degradation by tissue hyaluronidase (HAse), leading to dry skin and a decrease in moisturizing power (Papakonstantinou et al., 2012; Scarano et al., 2021).

[0004] The protein components of ECM are composed of fibrous proteins such as collagen, elastin, fibronectin, and laminin, and are characterized by a well-defined amino acid (AA) composition. Elastin (ELN) is the main protein responsible for skin elasticity, accounting for approximately 2% of all dermal proteins (Theocharis et al., 2016). ELN is synthesized via tropoelastin, a soluble isoform in which hydrophobic and hydrophilic domains are alternately formed. The hydrophobic domain is rich in nonpolar AAs such as glycine, valine, proline, and alanine, with 3 to 9 AAs repeating in sequence. On the other hand, the hydrophilic domain is rich in alanine and lysine. Fibronectin (Fbn) plays a crucial role in cell binding, migration, and adhesion. Therefore, Fbn exhibits various binding patterns in its protein structure, characterized by defined AA sequences such as Arg-Gly-Asp, Arg-Gly-Asp-Ser, Leu-Asp-Val, and Arg-Glu-Asp-Val, which mediate the adhesion function of Fbn cells (Rosso et al., 2004).

[0005] Collagen (Col) is the most abundant protein in mammals, making up the main component of the extracellular matrix (ECM), reaching 75% in the dermis. Col has a triple helix structure derived from molecular bonding by rigid AA sequences repeating a glycine-XY pattern, where glycine stabilizes the triple helix, and X and Y are often proline or hydroxyproline. Thus, like ELN and Fbn, Col exhibits a distinctive AA composition with high proportions of glycine, hydroxyproline, proline, and alanine. The collagen family consists of 28 members from I to XXVIII, but different isoforms exist even within the same type of collagen, and the use of alternative promoters and cleavage by other proteases gives rise to further diversity in the Col protein family (Vuorio and De Crombrugghe, 1990; Langton et al, 2010; Chu, 2011; Dalton and Lemmon, 2021).

[0006] In addition to a decline in the structural components of the endothelial cell mass (ECM), the overproduction of reactive oxygen species (ROS) is also a significant factor in skin aging. According to Hermann's theory of aging, this process is primarily due to a decline in mitochondrial function, with increased ROS production as a byproduct of aerobic respiration. Therefore, oxidative stress is a major cause of UV-induced premature skin aging, as it damages mitochondrial DNA (mtDNA), leads to dysfunction of oxidative phosphorylation (OXPHOS), and increases ROS production (Ziada et al., 2020).

[0007] Currently, methods devised to slow skin aging are based on preventative approaches such as avoiding excessive sun exposure and / or using anti-UV creams (Mohiuddin, 2019). Simultaneously, other therapeutic strategies involve supplementing with natural compounds such as polyphenols, plant extracts, or HA to suppress age-related skin dryness. However, it has been demonstrated that adding a mixture of six amino acids (glycine, L-proline, L-leucine, L-lysine, L-valine, and L-alanine) to fibroblasts increases the expression of ECM components. Furthermore, these six amino acids induce the expression of antioxidant genes (Tedesco et al., 2022). De novo protein synthesis and the reconstruction and regeneration of ECM components, which underlie the mechanotransduction and ability to maintain the tensile properties of the ECM, are both energy-intensive and require increased mitochondrial ATP production (Romani et al., 2021). Therefore, stimulating mitochondrial activity may promote ECM deposition in fibroblasts and improve aerobic respiration in the skin, potentially combating skin aging caused by oxidative stress. [Overview of the Initiative]

[0008] We have found that a mixture of six amino acids, consisting of glycine, L-proline, L-leucine, L-lysine, L-valine, and L-alanine, combined with hyaluronic acid, malic acid, succinic acid, or pharmaceutically acceptable salts thereof, exhibits a synergistic effect on stimulating ECM gene expression. Therefore, the object of the present invention is a composition containing the above amino acids in combination with hyaluronic acid, malic acid, and succinic acid, which is useful for combating skin aging. For this purpose, the composition can be suitably administered by injection in the form of a readily available sterile solution or as a lyophilized powder that is redissolved in sterile water for injection. The composition may also be in gel form for use as an intradermal filler. Alternatively, the formulation according to the present invention may be used for topical or transdermal administration in the form of, for example, an ointment, gel, or cream.

[0009] The weight ratios of amino acids substantially correspond to the ratios present in collagen and elastin. The relative ratios, based on glycine, are as follows: - Glycine: 1. - L-proline: 0.7-0.8%, preferably 0.75%. - L-alanine: 0.47-0.76, preferably 0.48-0.51, more preferably 0.75-0.76. - L-valine: 0.35-0.56, preferably 0.35-0.37 or 0.54-0.56. - L-leucine 0.13-0.27, preferably 0.13-0.15. - L-lysine hydrochloride: 0.10-0.12, preferably 0.10-0.11. The amino acid mixture in the above ratio is present in the composition according to the present invention at a concentration ranging from 5 to 30% by weight, preferably 5 to 15% by weight.

[0010] The weight ratio of malic acid to succinic acid is preferably about 1:1. Malic acid and succinic acid or their salts are preferably present at a concentration of 1 to 5% by weight. Hyaluronic acid or its salts, particularly sodium hyaluronate, preferably have an average molecular weight (Mn) in the range of 50,000 to 2,500,000 Da. The proportion of hyaluronic acid or sodium hyaluronate is in the range of 1 to 5% by weight of the total composition.

[0011] For anticipated dermatological, cosmetic, or therapeutic uses, the compositions according to the present invention are administered subcutaneously or intradermally in amounts ranging from 0.1 to 10 ml, depending on the application site and the condition being treated or prevented. For topical administration, the formulation can be applied in amounts ranging from 0.1 to 10 g. [Examples]

[0012] The present invention will be described in detail by the following examples. Example 1 [Table 1]

[0013] Example 2 [Table 2]

[0014] Example 3 [Table 3]

[0015] Example 4: Pharmacological Experiment The most important experiment conducted will be explained as an example. In particular, the effects of adding succinate (6AAHS), malate (6AAHM), or succinate / malate (6AAHSM) to a HA-enriched 6-amino acid mixture (6AAH) on ECM gene expression in human fibroblasts were investigated under both basal conditions and in response to oxidative stress (hydrogen peroxide).

[0016] 6AAH, 6AAHS, 6AAHM, and 6AAHSM were added to BJ human fibroblasts, and the mRNA levels of various ECM markers were evaluated. 6AAHSM significantly increased the expression of all ECM markers compared to 6AAH alone or 6AAH supplemented with only succinic acid or malic acid. Furthermore, in an in vitro oxidative stress model, 6AAHSM suppressed the decrease in ECM gene expression induced by hydrogen peroxide. These data suggest that dietary supplementation with 6AAH, which is rich in malic acid and succinic acid, is useful as a non-pharmacological approach to combat skin aging.

[0017] Materials and Methods Cell Culture and Treatment BJ human skin fibroblasts (ATCC® CRL-2522™) were cultured under standard conditions in EMEM medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) and antibiotics. Cell culture was performed at 37 °C, 95% humidity, and 5% CO2. Cells were treated with a 0.1% (w / v) mixture (composition shown in Table 1), + / - malic acid and succinic acid (5 mM each) for 3 days. Untreated cells were seeded as controls. Every 24 hours, the medium was replaced with fresh medium or a mixture of amino acids in both the control and treated flasks. Oxidative stress with hydrogen peroxide was performed in cells pretreated for 24 hours with only the culture medium or the selected mixture (Figure 3). Then, H2O2 (500 μM) was added for 2 hours, and at the end of the experimental treatment, cells were harvested for mRNA extraction.

[0018] [Table 4]

[0019] RNA Extraction and Quantitative RT-PCR For mRNA level analysis, 1 μg of total RNA isolated with the RNeasy kit (Qiagen) was reverse transcribed using the cDNA iScript synthesis kit (Bio-Rad Laboratories, Italy). Triple PCR reactions were performed using the iCycler iQ Real-Time PCR detection system (Bio-Rad Laboratories). Corresponding gene expression was calculated using a comparative method (2-ΔΔCt) with GAPDH as the housekeeping gene. Primer sequences were designed using Beacon Designer 2.6 software (Premier Biosoft International, Palo Alto, CA, USA).

[0020] Protein extraction and Western blotting Total protein was extracted using M-PER Mammalian Protein Extraction Reagent (Pierce; ThermoScientific). Protein content was measured by bicinchoninic acid (BCA) protein assay (Pierce). 30–40 μg of protein was analyzed on a sodium dodecyl sulfate-polyacrylamide TGX gel (SDS-PAGE) (BioRad) with a 4–20% concentration gradient. The gel was transferred to PVDF, blocked with 5% skim milk powder, and incubated with anti-Fbn1 (1:1000, GeneTex, Cat#GTX112794) or anti-vinculin (1:1000, Cat#V9131, Sigma-Aldrich). [Brief explanation of the drawing]

[0021] [Figure 1]The addition of malic acid / succinic acid increases the mRNA expression of ECM markers. RT-PCR quantitative analysis of ECM gene expression in BJ fibroblasts. Data are mean ± SEM of triplicate samples. NT: untreated control, *P<0.05 and **P<0.01 vs. NT, $P<0.05 and $P<0.01 vs. 6AAH, #P<0.05 and ##P<0.01 vs. 6AAHS, §P<0.05 and §§P<0.01 vs. 6AAHM. One-way ANOVA followed by Tukey's post-hoc test. [Figure 2] Addition of 6AAHSM induces Fbn expression. Western blot analysis of Fbn protein in BJ samples (top panel) (triple measurement). Vinculin was used as a loading control. Bottom panel: Quantification of immunoblot data. CTRL: Untreated control, **P<0.01 vs NT, $P<0.05 vs 6AAH. One-way ANOVA was followed by Tukey's post-hoc test. [Figure 3] The addition of 6AAHSM protects the ECM from oxidative stress. RT-PCR analysis of ECM markers in hydrogen peroxide-treated BJ fibroblasts. NT: untreated control, hydrogen peroxide. Data are mean ± SEM of triplicate samples. *P<0.05 and **P<0.01 are compared to NT, ¶P<0.05 is compared to H2O2, ≠P<0.05 is compared to 6AAH+H2O2. One-way ANOVA was followed by Tukey's post-hoc test.

[0022] result Adding malic acid and succinic acid to a mixture of six amino acids significantly increases ECM gene expression compared to using the amino acids alone. The addition of succinic acid (6AAHS), malic acid (6AAHM), or succinic acid + malic acid (6AAHSM) to a 6AAH mixture increased the expression of the ECM markers Fbn, Eln1, and two collagen isoforms Col1a1 and Col4a1 compared to 6AAH, 6AAHM, or 6AAHS alone. Conversely, the addition of 6AAH alone (final concentration 0.1% w / v) did not change Fbn expression compared to control cells (NT) (Figure 1), but the addition of succinic acid to the 6AAH mixture significantly increased Fbn mRNA expression in both NT and 6AAH (+93% and +124%, respectively). Furthermore, the addition of malic acid to 6AAH increased Fbn expression by +152% in 6AAH and +116% in the control. Similarly, the addition of malic acid and succinic acid (6AAHSM) to 6AAH significantly increased Fbn expression compared to the control (+167%) and 6AAH (+211%). Furthermore, 6AAHSM also induced Fbn expression more strongly than 6AAHS alone (+38%). Analysis of Eln1 expression showed that only 6AAHSM significantly increased its expression compared to the control (+101%) and other mixtures, while the other mixtures had little effect. Similarly, Col1A1 and Col4A1 expression were also significantly increased by 6AAHSM. Col1A1, in particular, was significantly increased only by the addition of 6AAHSM (+85% relative to NT). On the other hand, Col1A4 expression was significantly induced by both 6AAHS and 6AAHSM (+106% and +136% relative to NT, respectively). However, the addition of 6AAHSM resulted in a greater increase in Col1A4 relative to NT compared to 6AAHS and 6AAHM (+255%, +72%, and +50% relative to NT, 6AAHS, and 6AAHM, respectively).

[0023] Fbn has also been shown to play a crucial role in controlling and promoting the deposition of various other components of the ECM, including collagen (Sottile et al., 2007; Dalton and Lemmon, 2021). Therefore, when Fbn protein expression was evaluated in BJ fibroblasts supplemented with medium, 6AAH, or 6AAHSM, Fbn mRNA was significantly increased compared to the other mixtures (Figure 1). As shown in Figure 2, 6AAHSM significantly increased Fbn protein expression compared to untreated or 6AAH-treated BJ fibroblasts, further validating the mRNA expression data and confirming that 6AAHSM controls the deposition of ECM components. Furthermore, 6AAH mixtures supplemented with succinate and malate had a greater ability to induce ECM gene expression than 6AAH supplemented with succinate or malate alone.

[0024] After confirming that 6AAHSM is the most effective combination for inducing ECM gene expression, we evaluated whether this ability also enhances the ability to protect human fibroblasts from oxidative stress compared to the 6AAH mixture alone. For this purpose, BJ cells were pretreated with H2O2, H2O2 + 6AAH, or H2O2 + 6AAHSM, and ECM gene expression was evaluated. As expected, H2O2 treatment reduced the expression of all ECM genes, and the addition of 6AAH did not restore their expression, with Col1A4 being the exception, which was significantly increased by 6AAH compared to H2O2 alone (Figure 3). However, when 6AAHSM was added to fibroblasts in the presence of H2O2, the oxidative stress-induced decrease in expression of all ECM genes was significantly restored, and the expression of FBN and Col1A4 was fully restored to control levels.

[0025] These data suggest that the antioxidant capacity of the 6AAH mixture is enhanced by adding malic acid and succinic acid "as is" or in the form of pharmaceutically acceptable salts. As a result, it was shown that the composition according to the present invention significantly stimulates ECM gene expression in human fibroblasts even at a concentration of 0.1% or less. Furthermore, the composition according to the present invention can restore the downregulation of ECM gene expression induced by oxidative stress.

[0026] References Birch, HL (2018). in Subcellular Biochemistry, 169-190. Black, LD, et al., Biophys. J. 94, 1916-1929. Chu, M. (2011). eLS. Dalton, CJ, and Lemmon, CA (2021). Cells 10. Langton,AKet al,(2010).Int.J.Cosmet.Sci.32,330-339. Mohiuddin, AK(2019).Glob.J.Med.Res.,15-60. Papakonstantinou, E. et al, (2012). Dermatoendocrinol.4.doi:10.4161 / derm.21923. Romani,et al,(2021).Nat.Rev.Mol.Cell Biol.22,22-38. Rosso, F., (2004). J. Cell. Physiol. 199, 174-180. Scarano, A. et al. (2021). J. Cosmet. Dermatol. 20, 2296-2304. doi:10.1111 / jocd.13811. Shin,JW,et al.(2019).Int.J.Mol.Sci.20. Sottile, J. et al. (2007) Am.J.Physiol.Cell Physiol.293.doi:10.1152 / ajpcell.00130.2007. Sparavigna, A. (2020).Plast.Aesthetic Res.2020.doi:10.20517 / 2347-9264.2019.73. Tedesco,L.,et al.(2016).Biosci.Biotechnol.Biochem.86,1255-1261. Theocharis,AD,(2016).Adv.Drug Deliv.Rev.97,4-27. Vuorio,E.,and De Crombrugghe,B.(1990).Annu.Rev.Biochem.59,837-872. Ziada,AS,Smith,MSR,and Cote,HCF(2020).Front.Cell Dev.Biol.8.

Claims

1. A composition, a. A mixture of amino acids consisting of glycine, L-proline, L-leucine, L-lysine, L-valine, and L-alanine; b. Hyaluronic acid or its salts; c. Malic acid, succinic acid, or pharmaceutically acceptable salts thereof A composition containing the following:

2. The composition according to claim 1, wherein the amino acid mixture has the following weight ratio of amino acids. - Glycine: 1; - L-proline: 0.7-0.8; - L-alanine: 0.47-0.76; - L-valine: 0.35-0.56; - L-leucine: 0.13-0.27; - L-lysine hydrochloride: 0.10-0.12

3. The composition according to claim 1 or 2, wherein the weight ratio of malic acid to succinic acid is 1:

1.

4. The composition according to any one of claims 1 to 3, wherein the weight percentage of the amino acid mixture is 5 to 30%.

5. The composition according to any one of claims 1 to 4, wherein the weight percentage of malic acid or a pharmaceutically acceptable salt thereof is 1 to 5%.

6. The composition according to any one of claims 1 to 5, wherein the weight percentage of succinic acid or a pharmaceutically acceptable salt thereof is 1 to 5%.

7. The composition according to any one of claims 1 to 6, wherein the hyaluronic acid or a salt thereof, particularly sodium hyaluronate, has an average molecular weight in the range of 50,000 to 2,500,000 Da, and its proportion is 1 to 5% by weight of the whole composition.

8. A composition according to any one of claims 1 to 7, in a form suitable for injection as a sterile solution or for local administration.

9. A composition according to any one of claims 1 to 8, used for inhibiting and preventing skin aging.

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