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Title:
SKIN ANTI-AGING COMPOSITION
Document Type and Number:
WIPO Patent Application WO/2014/037561
Kind Code:
A2
Abstract:
It relates to a combination comprising a compound able to activate endogenous synthesis of SIRT3 protein in skin cells and a peptide comprising the amino acid sequence of formula (I) wherein AA is an amino acid or a derivative thereof, n is 0, 1, 2 or 3, and at least one of the lysine residues is acetylated. It also relates to a microcapsule containing it, to a pharmaceutical or cosmetic composition containing it, to its use in the prevention and/or treatment of cutaneous signs of aging, and to its use as skin care agent.

Inventors:
GARRE CONTRERAS AURORA DEL CARMEN (ES)
GUTIERREZ REYES CARMEN (ES)
RAMOS RODRIGUEZ ISABEL (ES)
ELIZARI GALAR MAIALEN (ES)
Application Number:
PCT/EP2013/068615
Publication Date:
March 13, 2014
Filing Date:
September 09, 2013
Export Citation:
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Assignee:
CINFA S A LAB (ES)
International Classes:
A61K8/64; A61Q19/08
Domestic Patent References:
WO2011113785A22011-09-22
Other References:
SAHOO S K ET AL: "Residual polyvinyl alcohol associated with poly (d,l-lactide-co-glycolide) nanoparticles affects their physical properties and cellular uptake", JOURNAL OF CONTROLLED RELEASE, ELSEVIER, AMSTERDAM, NL, vol. 82, no. 1, 18 July 2002 (2002-07-18), pages 105-114, XP004369794, ISSN: 0168-3659, DOI: 10.1016/S0168-3659(02)00127-X
Attorney, Agent or Firm:
DAVIU FOLGUERA, Noemi (Barlocci & MarkvardsenPl. Cataluny, 1 Barcelona, ES)
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Claims:
CLAIMS

1 . A combination comprising a compound able to activate endogenous synthesis of SIRT3 protein in skin cells and a peptide comprising the amino acid sequence of formula (I)

(AA)n-Arg-His-Lys1-Lys2-Gln-(AA)n

(I) wherein AA is an amino acid or a derivative thereof, n is 0, 1 , 2 or 3, and at least one of the lysine residues is acetylated.

2. The combination according to claim 1 , wherein the amino acid sequence of formula (I) is Arg-His-Lys-Lys(Ac)-Gln (SEQ ID NO.1 ).

3. The combination according to any of the claims 1 -2, wherein the compound able to activate endogenous synthesis of SIRT3 protein in skin cells is hydrolyzed soy protein. 4. The combination according to any of the claims 1 -3, which is in the form of a microcapsule.

5. The combination according to claim 4, wherein the microcapsule comprises a core polymer and an outer shell polymer.

6. The combination according to claim 5, wherein the core polymer is poly(D,L-lactide-co-glycolide) (PLGA) and the outer shell polymer is made of polyvinyl alcohol (PVA). 7. The combination according to any of the claims 5-6, wherein the compound able to activate endogenous synthesis of SIRT3 protein is encapsulated within the core polymer, and the peptide comprising the amino acid sequence of formula (I) is attached to the outer shell polymer on the surface of the microcapsule.

8. The combination according to claim 7, wherein the peptide comprising the amino acid sequence of formula (I) is attached to the outer shell polymer an by amide bond between the amino group of the N-terminal amino acid and the carboxyl group of the outer shell polymer.

9. A pharmaceutical or cosmetic composition comprising the combination as defined in any of the claims 1 -8 and pharmaceutically or cosmetically acceptable excipients or carriers.

10. The composition according to claim 9, which is a cosmetic composition. 1 1 . The composition according to any of the claims 9-10, wherein the excipients or carriers are adapted for external topical administration.

12. A combination as defined in any of the claims 1 -8 for use in the prevention and/or treatment of cutaneous signs of aging, wherein aging comprises chronological aging and/or photoaging.

13. The combination for use according to claim 12, for use in reducing the cutaneous signs of chronological aging or for use in the prevention of cutaneous signs of photoaging.

14. Use of the combination as defined in any of the claims 1 -8, as a skin care agent.

15. Use according to claim 14, wherein the skin care comprises ameliorating at least one of the following symptoms: roughness, flakiness, dehydration, tightness, chapping, lack of elasticity, lines, fine lines, wrinkles,

telangiectasia, skin sagging, excess sebum, enlarged pores, loss of skin firmness, brown spot, dull skin, disturbance of sebum production, loss of skin comfort, skin devitalization, dark eye circles, bags under eyes, blemishes, and spots.

Description:
Skin anti-aging composition

The present invention relates to the field of pharmacy and cosmetics, in particular, it relates to a combination of active ingredients, to compositions containing them, to their pharmaceutical use for the prevention and/or treatment of cutaneous signs of aging, including photoaging caused by solar radiation, as well as to their cosmetical use as a skin care agent.

BACKGROUND ART

The normal process of energy production in cells, which is essential for maintaining vital functions, takes place within the mitochondria. Collaterally, during this process free radicals are also generated. Free radicals, in particular, Reactive Oxygen Species (ROS) are highly reactive, unstable molecular compounds, which by reaction with organic substances alter all components of the cell (nucleic acids, proteins, carbohydrates and lipids) and and lead to cellular degeneration.

Substantial evidence in the literature suggests that organismic aging is correlated with increasing damage to mitochondria and decay of

mitochondrial function has been invoked as a major determinant of aging. The mitochondrial theory of aging predicts that, over the lifespan of an organism, increasing damage (predominantly oxidative damage) occurs in the mitochondrial genome and/or mitochondrial proteins, and ROS

(predominantly superoxide) are produced in the mitochondria (either chronically during normal mitochondrial function or increased production as a result of mitochondrial damage).

Mitochondrial DNA (mtDNA) is particularly vulnerable because it is devoid of histone-type proteins, and physically close to the main source of ROS. It has been calculated that each molecule of DNA is subject to 10000 of free radicals attacks per day. As a consequence of repeated damage in

mithochondrial DNA, the mitochondria become increasingly unable to produce energy, the ATP production is decreased and the production of oxygen radicals is increased leading to a vicious cycle of respiratory impairment. DNA has a repair intrinsic function to compensate the damage caused by these toxic agents. However, when the repair activity becomes inefficient, the ability to respond to toxic agents is decreased. As a consequence, a series of gradual deterioration processes of cells takes place, which leads to deterioration of organs (including skin) and their associated functions. Thus, when in a tissue, such as skin, a sufficient number of cells reach such state, the tissue is compromised and signs of aging begin to show. Accumulation of ROS may, in turn, damage neighboring mitochondrial complexes, membranes and mtDNA and further accelerate the aging process in a kind of feedback loop. Once the damage of macromolecules has reached the level of mtDNA, leading to mutations, the energetic age of a mitochondrial, and thus of a cell, is carved in stone.

A change in the composition of the macromolecules causes a progressive loss of physiological capacity and adaptation to environmental changes. Therefore, to obtain an optimal therapeutic response to pharmacological or cosmetic ingredient, it is necessary to regulate, balance and/or improve the energy status of the cell delaying or limiting these changes in biomolecules caused by free radicals.

The aging process is the result of the accumulated damage on the

macromolecules such as nucleic acids, proteins and lipids, which causes a loss of their function. Sirtuins (silent information regulators) are a family of enzymes involved in the regulation of gene expression, cellular apoptosis, fatty acid metabolism, and the regulation of cell longevity. There are seven human Sirtuins (SIRT1 -7) that display diversity in cellular localization and function hree of which are localized to mitochondria (SIRT3, SIRT4, and SIRT5). In human skin, sirtuins are found both in the fibroblasts and keratinocytes.

SIRT3 gene plays an important role in the energy metabolism. It decreases the membrane potential and ROS production and stimulates the production and regeneration of mitochondrial antioxidants, thereby preserving cellular and skin vitality and longevity. In addition to changes associated to chronological aging, the skin is damaged by various environmental stressors, especially by solar ultraviolet radiation (UVR). Stress responses induced by UVR elicit premature skin aging

(photoaging), resulting in extensive damage to dermal connective tissue. Disruption of the normal dermal structure of skin connective tissue, primarily collagen, impairs a variety of skin functions and is considered to be the main cause of wrinkle formation.

Thus, for instance, UV radiation, in particular UVB, plays a central role in photodamage, such as clinical sunburn, hyperpigmentation, erythema, plaque-like thickening, loss of skin tone, deep furrowing, and fine wrinkle formation. Furthermore, it induces the development of skin cancer (photo- carcinogenesis). UV-induced photodamage and photocarcinogenesis both involve epidermal damage (such as induction of apoptosis),

immunosuppression, inflammation (activation of pro-inflammatory cytokines and chemokines), and DNA damage. Since most UVB radiation is absorbed at the epidermis, keratinocytes become a major target of its deleterious effects. When cells are exposed to stressors, a number of so-called stress proteins are induced to confer protection against such stressors. Heat shock proteins (HSPs) are representative of these stress proteins, and their cellular up- regulation of expression, especially that of HSP70, provides resistance given that HSPs re-fold or degrade denatured proteins produced by stressors such as ROS. It has been demonstrated that HSP70 expression suppresses UV- induced epidermal damage via anti-inflammatory and anti-apoptotic effects and suppression of DNA damage. Thus, HSP70 expression is considered an early repair mechanism which limits the appearance of irreversible cell injury. An anti-aging composition acting at the level of mitochondrial DNA has been described in WO 201 1/1 13785. This document discloses a complex comprising at least one active ingredient showing activity at the level of mitochondrial DNA, such as a pentapeptide including serine, cysteine, isoleucine, asparagine, threonine amino acids; and hydrolized soy protein; and at least one active ingredient showing activity at the level of nuclear DNA, such as teprenone and Myrtus communis extract. However, the pentapeptide referred to as pentapeptide-28 in the examples, is solely defined by amino acid content and not by an amino acid sequence, so that it is not clear what is the structure of the pentapetide.

From what is known in the art it is derived that there is still the need of providing effective anti-aging agents, in particular to delay chronological aging and to prevent photoaging.

SUMMARY OF THE INVENTION The inventors have developed a peptide comprising an amino acid sequence of formula (I) which can be useful in the prevention and/or treatment of cutaneous signs of aging, wherein aging comprises chronological aging and/or photoaging. The peptide of the invention is capable of reducing stress levels, e.g, caused by UV radiation, (HSP70 assay) and also reducing the DNA degradation under stress conditions, e.g, caused by hydrogen peroxide or UV radiation.

The peptide comprising an amino acid sequence of formula (I) can be conveniently combined with a compound able to activate the synthesis of SIRT3 ptotein. As it will be demonstrated in the examples this combination of actives increases the ATP production in human dermal fibroblasts and prevents cell overoxidation (especially in mitochondria). In addition, it decreases the levels of protein oxidation induced either directly by the effect of radicals (ROS) or indirectly by the action of secondary products resulting from oxidative stress. It also reduces mitochondrial membrane potential, demonstrating its ability to reduce cellular oxidative stress.

Therefore, an aspect of the invention relates to a combination comprising a compound able to activate endogenous synthesis of SIRT3 protein in skin cells and a peptide comprising the amino acid sequence of formula (I)

(AA) n -Arg-His-Lys 1 -Lys 2 -Gln-(AA)n

(I) wherein AA is an amino acid or a derivative thereof, n is 0, 1 , 2 or 3, and at least one of the lysine residues is acetylated. The combination of actives of the invention can be administered in the form of a microcapsule. Thus, it also forms part of the invention a microcapsule comprising the combination comprising a compound able to activate endogenous synthesis of SIRT3 protein in skin cells and a peptide comprising the amino acid sequence of formula (I) as defined above.

The use of the microcapsules allows to decrease the amounts nedded of the active ingredients with the consequence that potential side-effects are also reduced. Further, the microcapsules have also the advantage that are capable of penetrating skin and show a uniformly distribution on the entire epidermis.

The combination of actives of the invention, preferably in the form of the microcapsules as disclosed above, may be formulated as pharmaceutical or cosmetic compositions. Thus, another aspect of the invention relates to a pharmaceutical or cosmetic composition comprising the combination comprising a compound able to activate endogenous synthesis of SIRT3 protein in skin cells and a peptide comprising the amino acid sequence of formula (I) as defined above and pharmaceutically or cosmetically acceptable excipients or carriers.

The combination comprising a SIRT3 activating compound and a peptide of formula (I) as defined above is useful as skin anti-aging agent. Thus, the invention also relates to the combination comprising a SIRT3 activating compound and a peptide of formula (I) as defined above for use in the prevention and/or treatment of cutaneous signs of aging, wherein aging comprises chronological aging and/or photoaging.

This aspect can also be formulated as the use of the combination comprising a SIRT3 activating compound and a peptide of formula (I) as defined above for the preparation of a medicament for the prevention and/or treatment of cutaneous signs of aging, wherein aging comprises chronological aging and/or photoaging. It also relates to a method for the prevention and/or treatment of cutaneous signs of aging in a mammal, including a human, wherein aging comprises chronological aging and/or photoaging, the method comprising administering to said mammal an effective amount of the combination as defined above together with pharmaceutically acceptable carriers or excipients.

Finally, another aspect of the present invention refers to the use of the combination of actives as defined above, as a skin care agent. It also forms part of the invention a cosmetic method for skin care of a mammal, including a human, said method comprising the administration to said mammal, including a human, of an effective amount of the combination comprising a SIRT3 activating compound and a peptide of formula (I) as defined above, together with acceptable excipients or carriers.

DETAILED DESCRIPTION OF THE INVENTION

All the percentages mentioned herein are in weight.

As mentioned above, the present invention relates to a combination comprising a compound able to activate endogenous synthesis of SIRT3 protein in skin cells (herein also referred to as SIRT3 activating compound) and a peptide comprising the amino acid sequence of formula (I) (herein also referred to as peptide of formula (I)).

For the purposes of the invention, the aminoacid sequence is written from N-terminus (N(t)) to C-terminus (C(t)), being the he N-terminus placed on the left. Thus, the peptide of formula (I) refers the peptide comprising the amino acid sequence: N(t)-(AA) n -Arg-His-Lys 1 -Lys 2 -Gln-(AA) n -C(t).

In one embodiment, the peptide of the combination above consists of the amino acid sequence (AA) n -Arg-His-Lys 1 -Lys 2 -Gln-(AA) n . In the amino acid sequence of formula (I), each AA is independently an amino acid or a derivative thereof.

For the purposes of the invention, an "amino acid derivative" refers to a non- natural or modified amino acid which contains one or more modifications to backbone and/or side chain(s). These modifications can be introduced by incorporation of amino acid mimetics that show similarity to the natural amino acids. In the context of this invention, mimetics of amino acids include, but are not limited to, [beta]2- and [beta]3-amino acids, [beta]2,2-, [beta]2,3, and [beta]3,3-disubstituted amino acids, [alpha], [alpha]-disubstituted amino acids, statine derivatives of amino acids, D-amino acids, [alpha]- hydroxyacids, [alpha]-aminonitriles, N-alkylamino acids and the like.

Furthermore, derivatives include amino acids with a modified amino acid side chain (referring to the side chain of the corresponding natural amino acid), such as 4-fluorophenylalanine, 4-hydroxylysine, 3-aminoproline, 2- nitrotyrosine, N-alkylhistidine or [beta]-branched amino acids or [beta]- branched amino acid mimetics with chirality at the [beta]-side chain carbon atom opposed to the natural chirality (e.g. allo-threonine, allo-isoleucine and derivatives). Furthermore, derivatives also include those amino acids wherein the amino group present in the amino acid side chain is modified, e.g. is acetylated. Examples of modified and unusual amino acids include without limitation: 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, beta- aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, piperidinic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3- aminoisobutyric acid, 2-aminopimelic acid, 2,4 diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N- ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3- hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N- methylglycine, sarcosine, N-methylisoleucine, 6-N-methyllysine, N- methylvaline, norvaline, norleucine, and ornithine.

In a particular embodiment, the amino acid derivative is selected from a) a natural amino acid in which one or more hydrogen atoms of its side chain is replaced by a substituent selected from the group consisting of halogen, -NO 2 , -NH 2 , -NH(CrC 4 )alkyl, -OH, -SH and -NHCOCH 3 ; and/or one or more methylene groups (-CH 2 -) of its side chain is replaced by a substituent selected from the group consisting of a bond or (Ci-C 4 )alkyl, and b) a natural amino acid with chirality at the [beta]-side chain carbon atom opposed to the natural chirality. More particularly, the amino acid derivative is selected from 4-fluorophenylalanine, 4-hydroxylysine, 3-aminoproline, 2-nitrotyrosine, N- alkylhistidine, allo-threonine, allo-isoleucine, 2-aminoadipic acid, 2- aminobutyric acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 2- aminopimelic acid, 2,4 diaminobutyric acid, 2,3-diaminopropionic acid, N- ethylglycine, N-ethylasparagine, allo-hydroxylysine, 3-hydroxyproline, 4- hydroxyproline, allo-isoleucine, N-methylglycine, N-methylisoleucine, 6-N- methyllysine, N-methylvaline, norvaline, norleucine, and ornithine.

In anotherembodiment, each AA is independently selected from natural L- amino acids, such as Alanine (Ala), Arginine (Arg), Asparagine (Asn), Aspartic Acid (Asp), Cysteine (Cys), Glutamic Acid (Glu), Glutamine (Gin), Glycine (Gly), Histidine (His), Isoleucine (lie), Leucine (Leu), Lysine (Lys), Methionine (Met), Phenylalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr) and Valine (Val).

In one other embodiment, above mentioned group of peptides may contain close structural analogues of amino acid or amino acids mimetics, for instance ornithine instead of lysine, homophenylalanine or phenylglycine instead of phenylalanine, [beta]-alanine instead of glycine, pyroglutamic acid instead of glutamic acid, norleucine instead of leucine or the sulfur-oxidized versions of methionine and/or cysteine.

Moreover, variants of peptides of formula (I) subject to additions or substitutions also form part of the present invention. In particular, a "variant" of the peptide of formula (I) refers herein to a peptide of formula (I) wherein from one to three amino acids, preferably one amino acid, of the amino acid sequence of formula (I) is substituted with any other natural amino acid or derivative thereof as defined above. The amino acid substitution may be conservative (i.e. one or more amino acids are are replaced by one or more amino acids with similar chemical properties; e.g. between Ala, Val, Leu and lie; between Ser and Thr; between the acid residues Asn and Gin; and between the basic residues Lys and Arg, or between the aromatic residues Phe and Tyr) or non-conservative (i.e. one or more amino acids are substituted by one or more amino acids with different physicochemical properties). In a variant of the peptide of formula (I), the C-terminus may be carboxamide, or other resulting from incorporation of one of the above mentioned amino acid mimetics. Furthermore, in variants of the peptide of formula (I), the peptide may contain one or more replacements of native peptide bonds with groups including, but not limited to, sulfonamide, retroamide, aminooxy-containing bond, ester, alkylketone, [alpha], [alpha]- difluoroketone, [alpha]-fluoroketone, peptoid bond (N-alkylated glycyl amide bond). The linear and cyclized forms of the peptides mentioned above are covered by this invention, as well as their retro, inverso and/or retroinverso analogues. Such variants should not affect the properties of the peptides described herein. In one embodiment, a peptide or peptidomimetic according to the present invention is at most 1 1 amino acids in length, preferably 7, 6, 5 amino acids in length.

In another embodiment, all amino acids constituting the peptide of the combination above are L-amino acids.

In another embodiment, only Lys 2 is acetylated. In a preferred embodiment, the peptide of formula (I) comprises or consists of the amino acid sequence: Arg-His-Lys-Lys(Ac)-Gln, more preferably, the peptide of formula (I) comprises or consists of the amino acid sequence SEQ ID NO. 1 :

L-Arg-L-His-L-Lys-L-Lys(Ac)-L-Gln.

The peptides described herein can be prepared by any known methods including, without limitation, solid phase synthesis, solution synthesis, expression of protein by a transformed host, cleavage from a synthetic or semisynthetic polypeptide, or a combination of these techniques.

The peptides according to the present invention can also be obtained by methods known in the art in the form of pharmaceutically or cosmetically acceptable salts thereof, such as the sodium salt, potassium salt, calcium salt, magnesium salt and addition salts with acids. Examples of salts include salts of inorganic acids (e.g. hydrochloric acid, sulfuric acid and phosphoric acid) and organic acids (e.g. acetic acid, propionic acid, citric acid, tartaric acid, malic acid and methanesulfonic acid).

As it is shown in the examples the peptide of the combination is capable to decrease the DNA degradation in skin cells such as fibroblasts and

keratinocytes when exposed to exogenous stressors such as UV radiation and H 2 O 2 .

The combination of the present invention apart from the peptide of formula (I) also comprises a compound able to activate endogenous synthesis of SIRT3 protein in skin cells or SIRT3 activating compound. For the purposes of the invention, the term "SIRT3 activating compound" refers to an agent that increases the level of SIRT3 protein. In one embodiment, a SIRT3 activating compound may increase the production of SIRT3 protein by at least 5%, 10%, 15%, 25% or 30% with respect to control (untreated cells). The identification of SIRT3 activating compounds may be generally carried out by cellular assay which comprises treating the cells with the test compound, lysing them and separating SIRT3 protein by electrophoresis as described in more detail in the examples.

In another embodiment, the SIRT3 activating compound comprises one or more peptides that have biomimetic activity to mitochondrial respiratory chain proteins. In one embodiment, the compound able to activate endogenous synthesis of SIRT3 is hydrolyzed soy protein.

For the purposes of the invention, the term "hydrolyzed soy protein" (INCI name, CAS number 68607- 88-5) refers to a soy protein which has been hydrolyzed and typically has a reduced molecular weight in comparison to the protein in its non-hydrolyzed (unhydrolyzed) state. It refers to a protein hydrolyzate of soybean, i.e. to the product obtained by acidic, alkaline, or enzymatic hydrolysis of non-hydrolyzed soy protein, i.e. the soy protein that has not lost any quaternary, tertiary, and/or secondary structures present in the original unaltered protein. The hydrolyzed soy protein is composed primarily of amino acids, peptides, and proteins. It may contain impurities consisting chiefly of carbohydrates and lipids along with smaller quantities of miscellaneous organic substances of biological origin. The hydrolyzed soy protein is typically rich in bio-mimetic peptides against the respiratory chain proteins.

The term "soy protein" (glycine max) refers to any protein that may be derived from soy beans, whether or not the protein is actually derived from soy beans. In another embodiment, the hydrolyzed soy protein is commercially available as Dynachondrine ISR (ISP Personal Care). As mentioned above, it also forms part of the invention a microcapsule comprising the combination comprising a SIRT3 activating compound and a peptide comprising the amino acid sequence of formula (I) as defined above. For the purposes of this invention, particular embodiments of the combination comprising the SIRT3 activating compound and the peptide of formula (I) above are also particular embodiments of the microcapsules.

The microcapsules of the invention are generally made of one or more biodegradable polymers. In one embodiment, the microcapsules of the invention are bilayered microcapsules which comprise a core polymer and an outer shell polymer. In a preferred embodiment the core polymer and the outer shell polymer are different. In a preferred embodiment, the core polymer is poly(D,L-lactide-co-glycolide) (PLGA) and the outer shell polymer is polyvinyl alcohol (PVA). In a more preferred embodiment, PLGA has a lactide/glycolide molar ratio from 40:60 to 60:40, more preferably 50:50.

The bilayered microcapsules of the invention can be used as a highly efficient delivery system with sustained and controlled release of the encapsulated active ingredient at the target cells (fibroblasts and keratinocytes). In particular, the microcapsules escape from the endosomal and lysosomal compartment, so that the encapsulated active ingredient is released directly in the cytoplasm and can reach the target quickly, optimizing the application and the bioavailability of the encapsulated active agents. In addition, the microcapsules of the invention have the advantage that they show higher migration and penetration into the skin when compared with conventional microcapsules. By using these microcapsules lower concentrations of the active ingredient are needed to achieve same performance, and, as a consequence side effects are reduced. In one embodiment, the microcapsule comprises from 15 to 30%, preferably from 20 to 25% of SIRT3 activating compound and from 0.05 to 10%, preferably from 0.05 to 5%, preferably from 0.1 to 3% of the peptide of formula (I) in relation to the total weight of the microcapsule. In another embodiment, the SIRT3 activating compound is encapsulated within the core polymer, and the peptide of formula (I) is attached to the outer shell polymer on the surface of the microcapsule. More particularly, the peptide of fornnula (I) is attached to the outer shell by a covalent bond, preferably an amide bond between the amino group of the peptide N-terminal amino acid and the carboxyl group of the outer shell polymer, preferably PVA. The microcapsules of the invention may be prepared by a single emulsion technique as known in the art. Generally, the core polymer, such as PLGA, is mixed with the SIRT3 activating compound in a suitable solvent, such as acetone, and the resulting mixture is emulsified, for example by using a sonicator, with a mixture of the outer shell polymer, such as PVA, in a suitable solvent, such as water. The microcapsules may be isolated by conventional means, such as evaporation of the solvent and ultracentrifugation. The microcapsules may be lyophilized.

In one embodiment, the microcapsules of the invention have a size

distribution from 180 to 400 nm (average size 220 nm) as determined by

Scanning Electron Microscopy (SEM). This size allows the microcapsule to be uptaken by the cells, and to release the actives into the cytosol.

The microcapsules of the invention have the advantage that they reach deep into the hair follicles, where the barrier possess only a few layers of differentiated corneocytes and can be considered highly permeable.

Additionally the hair follicles can act as long-term reservoir, beneficial condition when transdermal delivery is intended. Follicular penetration of particles appear to be a promising mechanism for drug delivery. The hair follicle delivery has several pharmacokinetic advantages as a reduction or bypass of the tortuous pathway of the transepidermal absorption, decrease of the drug systemic toxicity when the follicle acts as long term delivery reservoir and increasing additionally the therapeutic index of some drugs as well as reducing the applied dose or frequency of administration.

The combination of actives of the invention, preferably in the form of the microcapsules as disclosed above, may be formulated as pharmaceutical or cosmetic compositions. Thus, pharmaceutical or cosmetic compositions comprising an effective amount of the combination comprising the SIRT3 activating compound and the peptide of formula (I) as defined above, together with one or more pharmaceutically or cosmetically acceptable excipients or carriers also form part of the invention. In a preferred embodiment, the invention relates to a cosmetic composition.

For the purposes of the present invention, the term "effective amount" means an amount that is sufficient to obtain the expected effect. In the case of pharmaceutical compositions, the effective amount is the "therapeutically effective amount" and refers to the amount of a compound that, when administered, is sufficient to prevent development of, alleviate to some extent, one or more of the symptoms of the disorder, disease, or condition being treated. The particular dose of compound administered according to this invention will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and the similar considerations. The expression "pharmaceutically acceptable excipients or carriers" refers to pharmaceutically acceptable materials, compositions or vehicles for use in the pharmaceutical technology for preparing compositions with medical use. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of mamals, including humans, without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio. The expression "cosmetically acceptable excipients or carriers" refers to that excipients or carriers suitable for use in contact with animal or human skin without undue toxicity, incompatibility, instability, allergic response, among others. In one embodiment, the pharmaceutical or cosmetic composition is a topical composition. Thus, the topical pharmaceutical or cosmetic composition of the present invention comprises an effective amount of the combination

comprising the SIRT3 activating compound and the peptide of formula (I) as defined above together with one or more appropriate topical pharmaceutically or cosmetically acceptable excipients or carriers. In a preferred embodiment, the invention relates to a topical cosmetic composition. Examples of appropriate excipients or carriers for topical administration include, without limitation, repairing cutaneous barrier function agents, hydrating agents, sunscreens, emollients, emulsifiers, thickeners,

humectants, pH-regulating agents, antioxidants, preservative agents, vehicles, or their mixtures. The excipients or carriers used have affinity for the skin, are well tolerated, stable, and are used in an amount adequate to provide the desired consistency, and ease application.

Additionally, the compositions of the present invention may contain other ingredients, such as fragrances, colorants, and other components known in the state of the art for use in topical formulations.

Specific examples of appropriate excipients named according to the

International Nomenclature of Cosmetic Ingredients (INCI) include Aqua, BHT, Bis-PEG/PPG-16/16 PEG/PPG-16/16 Dimethicone, Butyl

Methoxydibenzoylmethane, Butylene Glycol, C12-15 Alkyl Benzoate,

Caprylic/Capric Triglyceride, Caprylyl Glycol, Cetearyl Alcohol, Ceteth-20, Cetyl Alcohol, Cyclopentasiloxane, Decapeptide-22, Diethylhexyl Butamido Triazone, Dimer Tripeptide-43, Dimethicone, Dimethicone Crosspolymer, Dimethicone/Vinyl Dimethicone Crosspolymer, Dimethiconol, Disodium EDTA, Ethylhexyl Triazone, Ethylhexylglycerin, Glycerin, Glycerylstearate,

Hydrolyzed Wheat Protein, Isodecyl Neopentanoate, Isohexadecane, Lanolin Alcohol, Mica, Octadecenedioic Acid, Octocrylene, Oligopeptide-78, Palmitoyl Decapeptide-21 , Palmitoyl Nonapeptide-Zn, Paraffinum Liquidum, Parfum, PEG40 Castor Oil, PEG75 Stearate, Pentaerythrityl Tetra-Di-t-Butyl

Hydroxyhydrocinnamate, Pentylene Glycol, Phenoxyethanol,

Polymethylmethacrylate, Polyacrylate-13, Polyisobutene, Polysilicone-15, Polysorbate 20, Potassium Sorbate, Propylene Glycol Dipelargonate, Sodium Acrylate/Sodium Acryloyldimethyl Taurate Copolymer, Sodium Benzoate, Sodium Cetearyl Sulfate, Sodium Hydroxide, Steareth-20, Stearic Acid,

Tocopherol, Tocopheryl Acetate, and Xanthan Gum.

The cosmetic compositions of the invention may be in a solid, semi-fluid or fluid form, in particular in the form of emulsion, cream, milk, lotion, ointment, solid stick, foam, spray, oil, pomade and fluid, among others. They can be in anhydrous form, in an aqueous solution, in suspension form, or in the water- in-oil or oil-in water emulsion form. The topical composition used is formulated preferably as an emulsion. An emulsion is a dispersed system comprising at least two immiscible phases, one phase dispersed in the other as droplets. When water is the dispersed phase and oil is the dispersion medium, the emulsion is termed a water-in-oil emulsion (w/o). When oil is dispersed as droplets throughout the aqueous phase, the emulsion is termed an oil-in-water emulsion (o/w). The emulsions used are preferably oil-in-water emulsions. Preferably, the emulsions for use in the sense of the present invention are compatible with creams and lotions. In general any composition of the invention may be applied to the skin, in any parts of the skin, in any part of the body.

Topical compositions of the present invention can be prepared according to methods well known in the state of the art. The appropriate excipients and/or carriers, and their amounts, can readily be determined by those skilled in the art according to the type of formulation being prepared.

As mentioned above, the combination comprising a SIRT3 activating compound and a peptide of formula (I) as defined above is useful in the prevention and/or treatment of the cutaneous signs of aging. In a preferred embodiment, the combination of actives forms part of a cosmetic composition. In another preferred embodiment, aging is chronological aging. In another preferred embodiment, aging is photoaging. For the purposes of the present invention, the term "photoaging" refers to the premature skin aging caused by solar radiation. This damage does not result from ultraviolet (UV) radiation alone, but also from longer wavelengths, in particular near-infrared radiation (IRA radiation, 760-1440 nm). Since natural sunlight is polychromatic, its ultimate effects on the human skin are the result of not only the action of each wavelength separately, but also interactions among the many wavelengths, including UV, visible light, and infrared (IR). Thus, the term "photoaging" relates to the effects of ultraviolet UV light exposure on skin and/or IR light exposure on skin associated with the formation of coarse wrinkles, uneven skin pigmentation, loss of skin elasticity, a disturbance of skin barrier functions, or a combination thereof. Cutaneous signs of photoaging include changes in pigmentation (mottled pigmentation), sallowness, deep wrinkling, dryness, telangiectasia, premalignant lesions, laxity, atrophy, leathery appearance, elastosis (a coarse, yellow,

cobblestoned effect of the skin), or actinic purpura (easy bruising related to vascular wall fragility in the dermis). For the purposes of the present disclosure, the term "prevention" means reducing the risk of manifestation of a phenomenon. The term "treatment" means compensating for a physiological dysfunction and more generally reducing or even eliminating an undesirable disorder, the manifestation of which is especially a consequence of this dysfunction. The term "reducing" means lessening, ameliorating, or relieving the deleterious effects.

In a preferred embodiment, the invention relates to the combination comprising a SIRT3 activating compound and a peptide of formula (I) as defined above for use in reducing the cutaneous signs of aging, in particular chronological aging. In another preferred embodiment, the invention relates to the combination comprising a SIRT3 activating compound and a peptide of formula (I) as defined above for use in the prevention of cutaneous signs of photoaging, in particular induced or produced by UVA, UVB and IR

radiations, more particularly, induced or produced by UVA, UVB and IRA radiations.

In another embodiment, the prevention and/or treatment of cutaneous signs of aging comprises regenerating, revitalizing and/or increasing longevity, repair or protection of the skin cell layers.

As mentioned above, the combination comprising a SIRT3 activating compound and a peptide of formula (I) of the invention can also be used for cosmetic purposes for the care of the skin. This means that the combination comprising a SIRT3 activating compound and a peptide of formula (I) as defined above are forming part of a cosmetic composition. In one

embodiment, the skin care comprises ameliorating at least one of the following symptoms: roughness, flakiness, dehydration, tightness,

chapping, lack of elasticity, lines, fine lines, wrinkles, telangiectasia, skin sagging, excess sebum, enlarged pores, loss of skin firmness, brown spot, dull skin, disturbance of sebum production, loss of skin comfort, skin devitalization, dark eye circles, bags under eyes, blemishes, and spots. The term "cosmetic" is intended to denote a use intended, principally, to provide an aesthetic and/or comfort effect, in particular, to ameliorate the appearance of the skin, specifically the properties of the skin. When the combination of the invention is used as a skin care agent, the cosmetic composition of the invention comprises the appropriate effective amount of the combination for this use, i.e. an amount that is sufficient to obtain the expected effect which will be easily determined by the skilled in the art, and which mainly will depend on the other excipients and/or components of the cosmetic composition. In the context of the present invention, when the combination of the invention is used as a skin care agent it does not intend to include any therapeutic use.

In one embodiment, the skin care agent is selected from the group consisting of a skin moisturizing agent and a skin barrier recovery agent. For the purposes of the invention, the term "moisturizing agent" refers to a material which increases the water content of the skin and helps keep it soft and smooth. The term "skin barrier recovery agent" refers to material whose composition and/or structure are similar to the skin barrier allowing the reparation of its deficiencies.

Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise" encompasses the case of "consisting of. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

EXAMPLES Ac 2 O: acetic anhydride

Boc: tert butyloxycarbonyl

CCCP: carbonyl cyamide 3-chlorophenylhydrazone CTC: 2-chlorotrityl resin

DCM: dichloromethane

DIPCDI: N,N-diisopropylcarbodiimide

DIPEA: N,N-diisopropylethylamine

DMEM: Dulbecco's Modified Eagle Medium

DMF: N,N-dimethylformamide

DMSO: Dimethylsulfoxide

FCS: Fetal Bobine Serum

Fmoc: 9-fluorenylmethoxycarbonyl

HOAt: 1 -hydroxy-7-azabenzotriazol

JC-1 : 5,5,6,6-tetrachloro-1 ,1 ,3,3-tetraethylbenzimidazolylcarbocianine iodide MeOH: methanol

MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl

PBS: Phosphate Buffer Saline

SDS: sodium dodecyl sulphate

TFA: trifluoroacetic acid

TIS: triisopropylsilane

Trt: trityl

Example 1 : Peptide synthesis

Fmoc-Gln(Trt)-OH (1 eq) was directly incorporated on the CTC-resin (1 .0 mmol/g) with DIPEA (3 eq) in DCM (aprox. 1 .25 mL/1 g resin). After 10 min more DIPEA was added (7 eq) and the mixture allowed to stir for 40 min.

MeOH (0.8 mL/g resin) was then added and the mixture allowed to stir 30 min. Washings were performed with DCM (5 x 30 s) and DMF (5 x 30 s). Kaiser test was used to verify that the coupling was successful. For the deprotection of the Fmoc group, the resin was solvated with DMF (5 x 30 s), treated with a solution of piperidine/DMF 20% (3 x 5 min) and finally washed with DMF (5 x 30 s) and DCM (5 x 30 s). Then, the resin was solvated with DMF (5 x 30 s), and the coupling with Fmoc-Lys(Ac)-OH (3 eq) was carried out in the presence of HOAt (3 eq) and DIPCDI (3eq) in DMF. After 1 h the resin was washed with DMF (5 x 30 s) and DCM (5 x 30 s). Kaiser test was used to verify that the coupling was successful. The same procedure was successively repeated 3 times with the following amino acids Fmoc-Lys(Boc)- OH, Fmoc-His(Trt)-OH, and Fmoc-Arg(Pbf)-OH. Finally, the cleavage of the peptide from the resin was carried out by treating the resin with TFA:TIS: H 2 O (95:2.5:2.5) for 1 h, yielding the peptide of sequence SEQ ID NO.1 .

HPLC: C18 column; UV 220 nm; flux 1 mL/min; gradient acetonitrile-water 0-50 in 8 min, retention time = 3.0 min; MS: M=739; (M+2)/2=370.

Heat Shock proteins (HSP70) assay

Human Dermal Fibroblasts and Human Dermal Keratinocytes obtained from American Type Culture Collection (ATCC; Rockville, MD, USA in primary culture were used. Untreated cells, and cells treated with resveratrol were the negative and positive controls, respectively. The peptide of sequence SEQ ID NO.1 as obtained in Example 1 was tested at two concentrations: 0.015% and 0.030%. Untreated and treated cells were irradiated with 300 mJ/cm 2 and stored in the dark at room temperature. Cell extracts obtained immediately after irradiation were analized by SDS-PAGE.

SDS-PAGE and Immunoblots - Protein samples for electrophoresis were prepared by lysis buffer extraction and precipitation. The precipitates were dissolved in SDS-PAGE sample buffer and electrophoresed in 4.5-15% polyacrylamide gradient gels. The proteins were electrotransferred to nitrocellulose filters, and the membranes were treated with blocking buffer for 1 h at room temperature. This was followed by incubation at 4 °C overnight with an antibody to HSP70 (Santa Cruz Biotechnology, INC.) diluted 1 :1000 in blot buffer, washing, and incubation for 2 h at room temperature with peroxidase-conjugated goat-anti-rabbit IgG (Sigma, Germany) in blot buffer. Immunoreactivity was revealed by chemiluminescence (ECL-kit, Amersham Biosciences, UK). Table 1 shows the percentage of HSP70 in the different cell lines.

Table 1 As can be seen, the peptide of the invention decreased the HSP70 protein levels which had been dramatically increased because of the radiation effect.

Comet test in fibroblasts in fibroblasts and keratinocytes

The comet assay, or single cell gel electrophoresis assay (SCGE), is a common technique for measurement of DNA damage in individual cells.

Under an electrophoretic field, damaged cellular DNA (containing fragments and strand breaks) is separated from intact DNA, yielding a classic "comet tail" shape under the microscope.

Human Dermal Fibroblasts and Human Dermal Keratinocytes obtained from American Type Culture Collection (ATCC; Rockville, MD, USA) in primary culture were used. Untreated cells, and cells treated with resveratrol were the negative and positive controls, respectively. The peptide of sequence SEQ ID NO.1 as obtained in Example 1 was tested at two concentrations: 0.015% and 0.030%. Untreated and treated cells were irradiated with 300 mJ/cm 2 and stored in the dark at room temperature to study the UV effects. On the other hand, H 2 O 2 100 mM was used for inducing chemical oxidative stress. Cell extracts were obtained immediately after irradiation and Comet Test was carried out following supplier's instructions of the Comet Assay Kit (Cell Biolabs, INC). Table 2 shows the size of the signal into a tail that is formed with DNA degradation in the different cell lines after the different treatments.

Table 2

As can be seen, the size of the signal into a tail that is formed with DNA degradation decreases in all cases after treatment with the peptide of sequence SEQ ID NO.1 . Thus, it can be concluded that the peptide of sequence SEQ ID NO.1 is capable to decrease the DNA degradation under stress conditions. Example 2: Preparation of the microcapsules

1 g of PLGA (Resomer RG 502 H, lactide/glycolide molar ratio 48:52 to 52:48) in 10 mL of acetone containing hydrolyzed soy protein (25 mL)

(Dynachondrine ISR, Maymo batch number M.1261 .10, ISP Pharmaceuticals batch number 0Y900000277) was added dropwise to 100 mL of aqueous 1 % (w/v) polyvinyl alcohol (PVA), and the mixture was emulsified for 3 min using a sonicator. Following overnight evaporation of the solvent at 4 °C, the capsules were collected by ultracentrifugation at 60000 g for 30 min, washed three times with distilled water, and then lyophilized for 3 to 4 days.

The peptide of sequence SEQ ID NO.1 was coupled to the above obtained microcapsule via amide bound. Thus, carboxyl groups on the surface of the capsules were activated by re-suspending the capsules in isotonic 0.1 M 2- (N-morpholino)ethanesulphonic acid (MES) saline buffer pH 5.5, and then reacting them with 1 -ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDAC) (10 equiv.) and N-hydroxysuccinimide (NHS) (10 equiv.) for 1 h. The capsules were then centrifuged (15.000 rpm, 45 min) to remove excess EDAC/NHS and the water-soluble isourea byproduct. Activated capsules (1 g) were re- suspended in phosphate-buffered saline (PBS, 40 mL) and reacted with the N-terminal amine group of the peptide of sequence SEQ ID NO.1 (0.150 g) at room temperature for 2 hours. The coated capsules were centrifuged (15000 rpm, 30 min) and washed with PBS (3 x 10 mL) buffer to remove any unbound peptide. The presence of surface-bound peptide was confirmed by Kaiser ninhydrin tests. Unreacted peptide was separated from the capsules after the corresponding reaction step by analysis of the centrifugation supernatant (step described above) using 2,4,6-trinitrobenzenesulphonic acid (TNBS) as a colorimetric assay: 700 microliters of supernatant were added to 700 microliters of 0.1 M sodium borate buffer (pH 9.2). 350 microliter of TNBS aqueous solution (1 .65 mg/mL) were added and the solution was rapidly mixed. After incubation at 40 °C for 45 min., the reaction was stopped by adding 350 microliter of 0.1 M NaH 2 PO 4 containing 1 .5 mM Na 2 SO 3 and absorption at 420 nm was determined on a UVA/IS spectrometer. The amounts of peptide on the surface of the nanoparticles were calculated by substracting the free amount from the total amount added into the reaction system, being in that case 0.125%. The ratio of encapsulated hydrolyzed soy protein to peptide of sequence SEQ ID NO.1 is 25% to 0.125%. The diameter of the capsules was determined by Scanning Electron

Microscopy (SEM), showing a size distribution between 180 and 400 nm (average size 220 nm).

Cytotoxicity assay

Human Dermal Fibroblasts and human keratinocytes were used. The isolated cells were cultured in DMEM culture medium supplemented with 10% FCS and were incubated at 37 °C for maintenance treatment. Untreated cells were used as negative control. The positive control was 0.1 % SDS. The

microcapsules as obtained in Example 2 were tested in the DMEM culture medium at two concentrations: 0.0032% and 0.032%. These weight percentages refer to the weight of the microcapsule in relation to the total weight.

Cells were seeded at 20000 cells per well in a 96-well plate. 24 hours after seeding, cells were incubated under the conditions described above. After 48h of incubation at 37 °C, the culture medium was removed and the MTT reagent was added. Cells were incubated 2 hours at 37 °C. DMSO was then added and the absorbance of the formed formazan salt was measured at λ 570 nm. Lower absorbance values correspond to cells with lower metabolic activity, which correlates with an increased damage and, therefore, with an increased cytotoxic effect. Thus, the amount of living cells is proportional to the amount of formazan produced. Table 3 shows the percentage of cell viability (as well as the standard deviation (SD)) in the different cell lines after the different treatments.

Table 3 As can be seen, the microcapsules of Example 2 showed no changes in the cell viability at the different tested concentrations. Thus, is can be concluded that the microcapsules of Example 2 are not cytotoxic in any of the cell lines employed.

SIRT3 assay

Human Dermal Fibroblasts and human keratinocytes were used. The isolated cells were cultured in DMEM culture medium supplemented with 10% FCS and were incubated at 37 °C for maintenance treatment. Untreated cells were used as negative control. The microcapsules as obtained in Example 2 were tested in the DMEM culture medium at two concentrations: 0.0032% and 0.032%. These weight percentages refer to the weight of the microcapsule in relation to the total weight.

Cells were seeded at 20000 cells per well in a 96-well plate. 24 hours after seeding, cells were incubated under the conditions described above. For dilution of the sample, DMEM culture medium supplemented with 10% FCS was used. After 48h of incubation at 37 °C, the extract of the total protein content of the samples was carried out. The steps for carrying out the test of Western-Blotting were the following: After the treatment described above, cells were trypsinized and centrifuged. The pellet obtained was diluted with Buffer Shieh supplemented with protease inhibitors (Roche) and 100 mM orthovanadate. The product obtained was centrifuged at 14000 rpm for 15 minutes and the supernatant was stored at -20 °C.

Sample preparation: Based on the protein content of the sample (determined by the Bradford assay), the volume necessary to add in the different lanes of the gel was calculated. In a 10-well mini gel, a volume (including sample buffer and mercaptoethanol beta) of not more than 50 μΙ in each lane was added. 10-15 μΙ of Laemmli buffer in a microtube was added, beta

mercaptoethanol (5% of total volume) was added. The protein sample containing between 100 to 150 g of protein in the tube with the other reagents was added. Tubes were heated to 70 degrees Celsius for 2 hours, and placed on ice. Electrophoresis (SDS-PAGE): 1 X Western Blot SDS-PAGE Buffer was placed in the tank. Acrylamide gels were placed on the appropriate device and immersed in the tank. The internal compartment (between the two gels) was filled with SDS-PAGE buffer. The samples were loaded into the wells. The tank was connected to the power supply at 100V until the samples passed the first part of the gel. Then the voltage was increased at 160V which allowed the separation of proteins according to their molecular weight. The transfer was carried out on nitrocellulose membranes of 0.2 microns for 2 hours to a fixed voltage of 100V. The determination of the protein SIRT3 was performed with commercial antibodies (Rabbit polyclonal anti sirtuin 3 of mouse monoclonal) from Abeam, UK.

Table 4 shows the percentage of SIRT3 protein in the different cell lines after the different treatments.

Table 4

As can be seen, the microcapsules of Example 2 increased SIRT3 levels in human dermal fibroblasts and keratinocytes.

ATP quantification assay

The amount of ATP synthesized by the cells can be quantified by

bioluminescence. This assay is based on measurement of light emitted by the

Mg 2+

Luciferin+ATP+0 2 ► oxyIuciferin+ AM P+ yrophosphate + C0 2 + light

Luciferase

enzyme luciferase according to the reaction shown in the following Scheme:

The light emitted in the presence of enzyme substrate and ATP is directly proportional to the amount of ATP in the reaction. ATP determination kit ref. A22066 Invitrogen, Molecular probes was used. Human Dermal Fibroblasts coming from biopsies of healthy donors were cultured in DMEM culture medium supplemented with 10% FCS and incubated at 37 °C for maintenance and treatment. Untreated cells were used as negative control. The microcapsules as obtained in Example 2 were tested in the DMEM culture medium at two concentrations: 0.0032% and 0.032%. These weight percentages refer to the weight of the microcapsule in relation to the total weight. Cells were seeded at 100000 cells per well in a 96-well plate. 24 hours after seeding, cells were incubated under the conditions described above. For dilution of the sample, DMEM culture medium supplemented with 10% FCS was used. The measures were carried out at 1 h and 3 h. 10 ml_ of the following reaction solution was added: 8.9 ml_ de H 2 O, 0.5 ml_ 20 X Reaction Buffer, 0.1 ml_ 0.1 M DTT (Dithiothreitol), 0.5 ml_ 10 mM D-luciferin, and 2.5 μΙ_ luciferase 5 mg/mL. To prepare the standard curve, dilutions were performed where ATP concentration ranged from 1 nM to 1 μΜ, so that the following ratio was maintained: 90 μΙ_ of reaction solution previously prepared and 10 ml_ ATP standard solution. The samples to be measured were prepared following the steps of the preparation of the calibration curve but substituting the ATP solutions by the sample analyzed. Table 5 shows the amount of ATP produced by g protein after the different treatments

Table 5

As can be seen, the microcapsules of Example 2 increased ATP production in human dermal fibroblasts. Mitochondrial membrane potential assay

The mitochondrial membrane potential evaluates the integrity of the mitochondrial membranes. This parameter is of great interest in studies of oxidative stress because mitochondria are affected significantly in the presence of ROS. The fluorescent marker JC-1 is able to enter the

mitochondria depending on the difference in membrane potential. Under oxidative stress in the presence of Reactive Oxygen Species (ROS) the mitochondrial membrane permeability increases allowing the marker (which gives a red signal when the cell is intact) bind the mitochondrial DNA and gives a green signal.

Human Dermal Fibroblasts coming from biopsies of healthy donors were cultured in DMEM culture medium supplemented with 10% FCS and incubated at 37 °C for maintenance and treatment. Untreated cells were used as negative control. Untreated cells irradiated with UV, and non-irradiated cells treated with CCCP (carbonyl 3-chlorophenylhydrazone cyamide) were used as positive controls. The microcapsules as obtained in Example 2 were tested in the DMEM culture medium at two concentrations: 0.0032% and

0.032%. These weight percentages refer to the weight of the microcapsule in relation to the total weight.

Cells were seeded at 100000 cells per well in a 96-well plate. 24 hours after seeding, cells were incubated under the conditions described above. For dilution of the sample, DMEM culture medium supplemented with 10% FCS was used. After 72 hours cells were irradiated with UVB light 100 mJ/cm 2 and the samples were allowed to incubate for 4-6 hours. Then the cells were re-suspended in culture medium at a concentration of 10 6 cells/mL. To prepare the CCCP control, the commercially available disruptor of membrane

CCCP (50 μΜ) was used, which was incubated with untreated cells without irradiation at 37 °C for 5 minutes. Then 10 mL of the cationic marker JC-1 was added to the different samples at a concentration of 200 μΜ (2 μΜ final concentration) and the samples were incubated at 37 °C, 5% CO 2 for 15 minutes. Cells were washed with PBS and centrifuged and the pellet obtained was resuspended in 500 μΙ_ of PBS. Samples were analyzed on a flow cytometer at an excitation wavelength of 488 nm.

Table 6 shows the fluorescence intensity (as well as the standard deviation (SD)) in human dermal fibroblasts after the different treatments.

Tested product % fluorescence SD Negative control 93.58 0.45

Control (irradiated cells) 19.49 0.74

Control (CCCP) 13.43 0.36

Example 2 (0.0032%) 25.18 3.93

Example 2 (0.032%) 67.36 4.35

Table 6

As can be seen, the microcapsules of Example 2 increased mitochondrial membrane potential under the tested conditions, demonstrating their ability to reduce cellular oxidative stress.

Cell longevity assay The cell longevity assay is related to the reduction of the galactosidase activity in senescent cells. X-gal (5-bromo-4-chloro-3-indolyl- -D- galactopyranoside) is an insoluble compound formed by an indole covalently linked to a galactoside, which is hydrolyzed in the presence of galactosidase giving a measurable blue color.

Cellular Senescence Assay kit ref: CBA-230 Cell Biolabs, INC. was used. Human Dermal Fibroblasts coming from biopsies of healthy donors were cultured in DMEM culture medium supplemented with 10% FCS and incubated at 37 °C for maintenance and treatment. Untreated non-senescent cells were used as negative control. Untreated senescent (after 12 cell divisions) cells were used as positive control. The microcapsules as obtained in Example 2 were tested in the DMEM culture medium at two concentrations:

0.0032% and 0.032%. These weight percentages refer to the weight of the microcapsule in relation to the total weight.

Cells were seeded at 100000 cells per well in a 96-well plate of 35 mm. 24 hours after seeding, cells were incubated under the conditions described above for 7 passes. For dilution of the sample, DMEM culture medium supplemented with 10% FCS was used. For the measurement, the following steps were carried out:

1 . Aspiration of the medium of the cells with the different treatments. 2. Washing of cells with 1 X PBS (3 mL) three times and aspiration of the final washing.

3. Addition of 2 mL of 1 X Fixing solution and incubation at room temperature for 5 minutes.

4. Removal of the fixing solution and washing with 3 mL of 1 X PBS three times.

5. Aspiration of the last wash, and completely covering with 2 mL of the working cell solution to stain cells.

6. Incubation of cells overnight at 37 °C by protecting them from light.

7. Removal of the working solution and washing with 1 X PBS.

8. Counting of senescent cells stained blue with an optical microscope.

Table 7 shows the percentage of senescence in human dermal fibroblasts after the different treatments.

Table 7

As can be seen, the microcapsules of Example 2 decreased the beta galactosidase activity under the tested conditions, demonstrating their ability to increase cellular longevity.

Protein carbonylation assay Protein oxidation is defined as the modification of proteins induced either directly by the effect of radicals (ROS) or indirectly by the action of secondary products resulting from oxidative stress. The oxidative modifications of proteins can be induced in vitro by numerous pro-oxidant agents. The present assay consists on the derivatization of the carbonyl groups with

dinitrophenylhydrazine (DNPH), followed by detection using an anti-DNP antibody. When DNPH reacts with the formed carbonyl groups gives rise to the corresponding hydrazine, which can be extracted and determined by measuring the absorbance at 375 nm. Human Dermal Fibroblasts coming from biopsies of healthy donors were cultured in DMEM culture medium supplemented with 10% FCS and incubated at 37 °C for maintenance and treatment. Untreated cells were used as negative control. Cells irradiated with UV were used as positive control. The microcapsules as obtained in Example 2 were tested in the DMEM culture medium at two concentrations: 0.0032% and 0.032%. These weight percentages refer to the weight of the microcapsule in relation to the total weight.

Cells were seeded at 100000 cells per well in a 96-well plate of 35 mm. 24 hours after seeding, cells were incubated under the conditions described above. For dilution of the sample, DMEM culture medium supplemented with 10% FCS was used. After 72 hours cells were irradiated with UVB light 100 mJ/cm 2 and the samples were allowed to incubate for 24 hours. Measuring the carbonization of the proteins was performed at 96 hours. Table 8 shows the percentage of carbonylation in human dermal fibroblasts after the different treatments.

Table 8

As can be seen, the microcapsules of Example 2 decreased levels of protein carbonylation after stimulation with UV radiation.

Skin penetration assay

Fluorescein-labeled microcapsules were prepared following an analogous process as described in Example 2, by additionally adding the marker DQ- BSA to the acetone mixture containing PLGA and hydrolyzed soy protein.

Reconstituted skin (SkinEthic, RHE Reconstructed Human Epidermis) was cultured in DMEM culture medium supplemented with 10% FCS and incubated at 37 °C for maintenance and treatment. Untreated RHE was used as negative control. Formulations containing the labelled microcapsules with DQ-BSA were tested medium at three concentrations: 0.032%, 0.16%, 0.32%. These weight percentages refer to the weight of the microcapsule in relation to the total weight.

The reconstructed skin models of size 0.5 cm 2 , 17 day, were reconstituted in the appropriate means provided by the supplier immediately upon arrival at laboratory and kept in the incubator at 37 °C for complete recovery. After 24 hours, treatment with the marked microcapsules was performed under the conditions detailed in the previous section. Incubation with the different treatments was carried out for 48 hours. Then, the samples were frozen and analyzed by fluorescence microscopy.

Images corresponding to a cross section of skin treated with a formulation containing the microcapsules as obtained in Example 2 at 0.16% were recorded. In an image taken by fluorescence microscopy, the green

fluorescence signal indicated that the capsules penetrated and were uniformly distributed along the whole epidermis. The fluorescence did not appear in the nucleus of skin cells, but only in the cytoplasm where the capsule was degraded, DQ-BSA complex was cleaved and the fluorescent molecule released. In a comparative image obtained by light microscopy after hematoxylin-eosin staining, the nucleus of the skin cells were stained in a darker color. In conclusion, the microcapsules of Example 2 showed a suitable penetration and a uniformly distribution on the entire epidermis.

REFERENCES CITED IN THE APPLICATION

- WO 201 1/1 13785