Patent Application
20250136641
The present invention relates to the field of molecular biology, more precisely to molecular biology applied to cosmetics, nutraceuticals and medicine, even more precisely to peptides and compositions comprising said peptides, able, among others, to protect telomers and to protect from inflammation and oxidation, therefore, said peptides provide for the prevention and/or treatment of aging (preferably, skin aging), including inflammation.
In the last decades life expectancy of the population has increased significantly. In addition, there is also an increased concern in the population regarding personal aesthetics and to try to delay or minimize the appearance of signs related to aging.
The skin is the largest organ in humans and due to its location, in the body interface, is subject to intrinsic (chronologic) aging and extrinsic aging (mainly, photoaging). Intrinsic aging is accompanied by cell loss, thinning of epidermis, flattening of DEJ (dermal-epidermal junction) and fine lines of wrinkles. On the other hand, photoaged skin is coarsely wrinkled and associated with dyspigmentation. Inflammation and accumulation of ROS (reactive oxygen species) are now believed to be the causative factors in both types of skin aging.
One of the main functions of the skin is to protect the body from external factors and it does so by employing physical barriers, biomolecules, and an intricate network of resident immune and non-immune cells and skin structures. Concretely, the most external layer of the skin, named the epidermis, provides a barrier that confers the protection from environmental factors, pathogens, heat, ultraviolet and moisture loss. In addition, keratinocytes are keratin-producing epidermal cells and account for approximately 90% of epidermal cells (Nguyen A V, Soulika A M (2019). The Dynamics of the Skin's Immune System. International journal of molecular sciences, 20(8), 1811; Lee H S, et al. (2019). Anti-inflammatory effects of ethanol extract from the leaves and shoots of Cedrela odorata L. in cytokine-stimulated keratinocytes. Experimental and therapeutic medicine, 18(1), 833-840).
Skin aging is a complex biological process influenced by combination of endogenous and exogenous factors (endogenous: genetics, cellular metabolism, hormone and metabolic processes; exogenous: chronic light exposure, pollution, ionizing radiation, chemicals, toxins) and, hence, as noted above, lead to changes in the skin and to the appearance of imperfections therein (for example, loss of firmness, wrinkling, roughness and/or sagginess).
These factors induce cumulative structural and physiological alterations at the different layers of the skin as well as changes in overall skin appearance (Ganceviciene, R, et al. (2012). Skin Anti-Aging Strategies. Dermato-Endocrinology, 4, 308-319).
As the aging process occurs, the ability of the human body to resolve inflammation becomes significantly reduced, resulting in an imbalance between proinflammation and anti-inflammation. “Inflammaging” refers to a continuous, low-grade inflammation associated with aging. Such chronic inflammatory response could build up with time and gradually causes tissue damage (Zhuang Y and Lyga J (2014) Inflammaging in skin and other tissues—the roles of complement system and macrophage. Inflamm Allergy Drug Targets. 13, 153-61). Inflammaging is driven by a systemic increase in multiple pro-inflammatory cytokines and is significantly influenced by several extrinsic factors, including UV radiation (UVR), air particulate matter (PM), and the microbiome (Xia S, et al. (2016) An Update on Inflamm-Aging: Mechanisms, Prevention, and Treatment. J Immunol Res. 2016, 8426874).
In addition, or in parallel, UV irradiation, xenobiotics, and thermal stress disturb skin cell metabolism and lead to an increase in reactive oxygen species (ROS) generation and to redox imbalance contributing to oxidative stress which exposes skin cells to the formation and accumulation of irreversibly damaged proteins, lipids, nucleic acids and carbohydrates and, hence to skin aging. Key elements or mechanisms of skin cells against oxidative stress are Nrf2 (able to modulate anti-inflammatory and antioxidant response through multiple mechanisms, such as the regulation of redox homeostasis and the suppression of pro-inflammatory genes, either directly or through the interaction with NF-κB) and catalase (crucial antioxidant enzymes that mitigates oxidative stress to a considerable extent by destroying cellular hydrogen peroxide to produce water and oxygen).
Senescence typically occurs in response to damaging stimuli, such as telomere shortening or dysfunction (replicative senescence) or persistent activation of the DNA-damage response (DDR). The adoption of a pro-inflammatory phenotype known as the senescence-associated secretory phenotype (SASP) may promote tissue inflammation and deterioration. Growing evidence suggests that senescent cells accumulate in tissues and organs with advancing age, thereby impairing physiological regeneration processes and contributing to organismal aging (Rube, C E, et al. (2021). Human skin aging is associated with increased expression of the histone variant H2A.J in the epidermis. Aging Mech Dis 7, 7). Moreover, as noted above, ROS production increases with age, causing also double strand breaks (DSBs) in DNA while skin cells ability to repair this damage decreases over the years, contributing to chronological aging. It has been shown that during RS (replicative senescence), fibroblasts accumulate proteins involved in the DDR at telomeric regions, including γH2A.X, 53BP1, MDC1 and NBS1 (d'Adda di Fagagna et al., (2003). A DNA damage checkpoint response in telomere-initiated senescence, Nature 426, 194-198). DDR activation can result in the activation of transcription factor p53 which is involved in a variety of processes including DNA repair and apoptosis, but most notably results in expression of cyclin-dependent kinase inhibitor p21, which together with activation of p16, are thought to be the major pathways in the induction of senescence (de Magalhães J P and Passos J F (2018). Stress, cell senescence and organismal ageing; Mechanisms of Ageing and Development, Volume 170, 2-9). Antioxidant substances have the ability to bind free radicals, which are caused by oxidative stress, reducing DNA DSBs, and therefore, the apparition of 53BP1 foci, preventing and slowing the skin aging process.
On its side, Interleukin-8 (IL-8) is a potent chemotactic and proinflammatory cytokine, produced in the skin by a variety of cells in response to inflammatory stimuli. Recent studies suggest that in addition to its potent actions on leukocytes, IL-8 exerts a direct influence on several functions of human epidermal cells such as chemotaxis, Candida albicans killing activity or proliferation (Kemény L et al. (1994). Role of interleukin-8 receptor in skin. International archives of allergy and immunology, 104(4), 317-322).
In addition, one of the main players in innate immune response is toll-like receptor2 (TLR2). This protein is a membrane receptor involved in recognizing pathogen-associated molecular patterns (PAMPs) or exogenous ligands, and also recognizes danger associated molecular patterns (DAMPs), also known as alarmins or endogenous ligands. This recognition leads to the activation of the proinflammatory response in order to protect the organisms against external insults (Chen L and DiPietro L A (2017). Toll-Like Receptor Function in Acute Wounds. Adv Wound Care (New Rochelle) 6, 344-355). Focusing on the skin, which main function is to protect the body from external factors, TLR2 is expressed in many of the cell types that compose this organ, including keratinocytes (Nguyen A V and Soulika A M (2019). The Dynamics of the Skin's Immune System. International journal of molecular sciences, 20(8), 1811; Pivarcsi A et al. (2003). Expression and function of Toll-like receptors 2 and 4 in human keratinocytes. Int Immunol. 15, 721-30). Misfunction or overactivation of TLR2 in skin, can lead to an excessive pro-inflammatory response, causing inflammation-related diseases like acne and contributing to maintain a sustained inflammatory state, that can lead to inflammaging (Bailey K L et al. (2019). Aging leads to dysfunctional innate immune responses to TLR2 and TLR4 agonists. Aging Clin Exp Res. 31, 1185-1193.; Zhang B, et al. (2019) Toll-like receptor 2 plays a critical role in pathogenesis of acne vulgaris. Biomed Dermatol 3, 4).
Moreover, it is important to highlight that telomeres form the ends of human chromosomes. These structures consist of simple tandem DNA repeats (TTAGGG), that do not encode for any gene product and their main function is to cap the chromosome ends. Telomere capping is necessary to distinguish the chromosome ends, that should not be repaired, from DNA breaks within the genome, which lead to cell cycle arrest and DNA repair or to induction of apoptosis when the damage is too severe or non-repairable. Telomeres prevent the induction of such responses at the chromosome termini and this capping is a prerequisite to maintain chromosome stability, being necessary a minimum telomere length to fulfil this capping function (Buckingham E M and Klingelhutz A J (2011). The role of telomeres in the ageing of human skin. Exp Dermatol. 20, 297-302; Jiang H et al. (2007). Telomere shortening and ageing. Z Gerontol Geriatr. 40, 314-24).
Telomers shorten with each round of cell division, due to the “end replication problem” of DNA-polymerase, being one of the most relevant features of aging. Additional factors (processing of telomeres during the cell cycle, reactive oxygen species) can contribute to telomere shortening and different cellular mechanisms are involved in telomere preservation. Among these mechanisms, the sheltering complex is essential for telomere function, telomere maintenance, and connections with intracellular signalling pathways. Said sheltering complex is a multi-protein structure that protects telomere endings, and it is formed by different proteins, including TRF1, TRF2, POT1, RAP1, TIN2, and TPP1 (Imbert I et al. (2012). Modulation of telomere binding proteins: a future area of research for skin protection and anti-aging target. J Cosmet Dermatol. 11, 162-6).
When telomeres reach a critically short length, they lose the capping function at the chromosome termini. These dysfunctional telomeres are then recognized as DNA damage and induce DNA damage checkpoints, leading to senescence or even cell death (Imbert I et al. (2012). Modulation of telomere binding proteins: a future area of research for skin protection and anti-aging target. J Cosmet Dermatol. 11, 162-6; Maleki M et al. (2020) Stabilization of telomere by the antioxidant property of polyphenols: Anti-aging potential. Life Sci. 259, 118341). Focusing in skin aging, accumulation of senescent cells with short telomeres in skin tissues, is a hallmark of the ageing process (Buckingham E M and Klingelhutz A J (2011). The role of telomeres in the ageing of human skin. Exp Dermatol. 20, 297-302; Jiang H et al. (2007). Telomere shortening and ageing. Z Gerontol Geriatr. 40, 314-24).
Telomerase is an enzyme that elongates telomeres and compensates for telomere attrition through de novo addition of TTAGGG repeats onto chromosome ends in those cells where it is normally expressed, such as pluripotent stem cells and adult stem cell compartments (Blackbum E H (1991). Structure and function of telomeres. Nature 350, 569-573; Liu L, et al. (2007). Telomere lengthening early in development. Nat. Cell Biol. 9:1436-1441; Flores I et al. (2005). Effects of telomerase and telomere length on epidermal stem cell behavior. Science. 309: 1253-1256; Marion R M et al. (2009). Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells. Cell Stem Cell 4:141-54). Although telomerase is expressed in adult stem cell compartments, this is not sufficient to counteract telomere attrition associated with cell division throughout life, and therefore, as noted above, telomeres shorten with age in vitro and in vivo. This progressive telomere shortening can impair the regenerative capacity of tissues and has been proposed as one of the molecular hallmarks of aging (López-Otín C et al. (2013). The hallmarks of aging. Cell. 153:1194-1217; Martínez P, Blasco M A (2017). Telomere-driven diseases and telomere-targeting therapies. J Cell Biol. 216(4):875-887). Telomere attrition in stem cell compartments impairs their tissue and self-renewal capacity and is considered to be one of the primary molecular causes of aging and the onset of aging-associated diseases (Flores I et al. (2005). Effects of telomerase and telomere length on epidermal stem cell behavior. Science 309: 1253-1256; Sharpless N E and DePinho R A (2007). How stem cells age and why this makes us grow old. Nat. Rev. Mol. Cell Biol. 8:703-713). Specifically, for skin aging, telomerase, has been implicated in having a key role in the maintenance of skin cell function and proliferation (Buckingham E M, Klingelhutz A J (2011) The role of telomeres in the ageing of human skin. Exp Dermatol. 20, 297-302).
In recent years, the number of active ingredients to improve signs of skin aging, such as loss of firmness, skin texture and wrinkling, has increased considerably. Examples of such active ingredients are retinoids, vitamins or botanical extracts (Bradley E J et al. (2015). Over-the-counter anti-ageing topical agents and their ability to protect and repair photoaged skin. Maturitas, 80, 265-272).
Antioxidants are used in order to reduce the concentration of free radicals in the skin and, therefore, counteract collagen degradation. An example of said antioxidants is ascorbic acid (also known as Vitamin C). Ascorbic acid, in addition to its antioxidant effect, induces the synthesis of proteins from the ECM (collagen I and Ill and elastin) while promoting epidermal differentiation and inhibiting Matrix metalloproteinase-1 (MMP1), among others. Unfortunately, ascorbic acid, is extremely unstable and undergoes oxidation especially at high temperatures, aerobic conditions, high pH and/or when exposed to light (Manela-Azulay M et al. (2017). Vitamins and other Antioxidants. Daily Routine in Cosmetic Dermatology, 1-13).
In addition, a wide range of botanical extracts and plant derived compounds are found in the market with multiple applications, such as, for example, grape extracts which comprise resveratrol (an antioxidant); green tea which comprises polyphenols; or soy which comprises isoflavones. Another example is turmeric (Curcuma longa), which was used in Chinese and Ayurvedic medicine (as a treatment for inflammatory conditions) and heralded for its use in cooking and beauty rituals around the world. Turmeric comprises turmerin, which has shown antioxidant and anti-inflammatory activities.
However, the in vivo efficacy and composition of these ingredients is not sufficiently scientifically validated.
On the other hand, peptides can also be incorporated in cosmetic formulas to improve the signs of skin aging. Bioactive peptides can imitate body's own molecules and influence processes such as collagen synthesis, with the advantage that they have much better tolerability and stability. In addition, a wide range of activities, chemistries and indications can be developed for them (Zhang L and Falla T J (2009). Cosmeceuticals and peptides. Clinics in dermatology, 27, 485-494).
Despite the extensive variety of compounds and/or extracts in the field, there is still the need to find new molecules (preferably, peptides) which can provide for the prevention and/or treatment of aging (preferably, skin aging) and which show a broad spectrum of activities against said aging as well as diseases related to aging, inflammation and oxidative stress.
The inventors of the present invention, after extensive and exhaustive research, have surprisingly found peptides with a broad spectrum of activities which, among others, include protection of telomeres and antioxidative and anti-inflammatory activities. These peptides of the present invention are, therefore, useful for the treatment of skin aging and signs related to said skin aging as well as inflammatory diseases and diseases related with oxidative stress and, hence, solve the problem present in the state of the art mentioned above.
To allow a better understanding, the present invention is described in more detail below with reference to the enclosed drawings, which are presented by way of example, and with reference to illustrative and non-limitative examples. In the figures: *means p<0.05; **means p<0.01; ***means p<0.001.
In a first aspect, the present invention refers to a peptide with a broad spectrum of activities against skin aging which include, protection of telomeres and anti-inflammatory and antioxidant activities.
In a second aspect, the present invention refers to a composition comprising a peptide of the present invention.
In an additional aspect, the present invention refers to the use as a cosmetic of a peptide of the present invention or of a composition of the present invention, in a subject in need of the treatment.
In a fourth aspect, the present invention refers to the cosmetic use of a peptide of the present invention or a composition of the present invention in a subject in need of the treatment.
In a fifth aspect, the present invention refers to a method for the prevention and/or treatment of skin aging in a subject comprising the administration of a peptide of the present invention or a composition of the present invention to the subject.
In a further aspect, the present invention refers to a composition or a peptide of the present invention for use as a medicament.
In a seventh aspect, the present invention refers to the use of the composition or a peptide of the present invention for the manufacture of a medicament for the prevention, amelioration and/or treatment of an age-related disease, an inflammatory disease or a disease related to oxidative stress.
In an eighth aspect, the present invention refers to a method for the prevention, amelioration and/or treatment of an age-related disease, an inflammatory disease or a disease related to oxidative stress, comprising administering a peptide or a composition of the present invention to a subject in need thereof.
In a ninth aspect, the present invention refers to a nutraceutical composition comprising a peptide of the present invention.
In a final aspect, the present invention refers to the use as a nutraceutical of a peptide of the present invention or of a composition of the present invention.
The term “non-cyclic aliphatic group” and its plural, as used herein, have the common meaning given in the state of the art to said terms. Therefore, these terms refer to, for example and not restricted to, linear or branched alkyl, alkenyl and alkynyl groups.
The term “alkyl group” and its plural, as used herein, refer to a saturated, linear or branched group, which has between 1 and 24, preferably between 1 and 16, more preferably between 1 and 14, even more preferably between 1 and 12, and even more preferably still between 1, 2, 3, 4, 5 or 6 carbon atoms and which is bound to the rest of the molecule by a simple bond, including, for example and not restricted to, methyl, ethyl, isopropyl, n-propyl, i-propyl, isobutyl, tert-butyl, n-butyl, sec-butyl, n-pentyl, n-hexyl, heptyl, octyl, decyl, dodecyl, lauryl, hexadecyl, octadecyl, amyl, 2-ethylhexyl, 2-methylbutyl, 5-methylhexyl and similar. The alkyl groups can be optionally substituted by one or more substituents, such as, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto and alkoxythio.
The term “alkenyl group” and its plural, as used herein, refer to a linear or branched group which has between 2 and 24, preferably between 2 and 16, more preferably between 2 and 14, even more preferably between 2 and 12, even more preferably still 2, 3, 4, 5 or 6 carbon atoms, with one or more carbon-carbon double bonds, preferably with 1, 2 or 3 carbon-carbon double bonds, conjugated or unconjugated, which is bound to the rest of the molecule through a single bond, including, for example and not restricted to, the vinyl, oleyl, linoleyl and similar groups. The alkenyl groups can be optionally substituted by one or more substituents, such as, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto and alkoxythio.
The term “alkynyl group” and its plural, as used herein, refer to a linear or branched group which has between 2 and 24, preferably between 2 and 16, more preferably between 2 and 14, even more preferably between 2 and 12, even more preferably still 2, 3, 4, 5 or 6 carbon atoms, with one or more carbon-carbon triple bonds, preferably with 1, 2 or 3 carbon-carbon triple bonds, conjugated or unconjugated, which is bound to the rest of the molecule through a single bond, including, for example and not restricted to, the ethinyl group, 1-propinyl, 2-propinyl, 1-butinyl, 2-butinyl, 3-butinyl, pentinyl, such as 1-pentinyl and similar groups. The alkynyl groups can be optionally substituted by one or more substituents, such as, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto and alkoxythio.
The term “alicyclic group” and its plural, as used herein, have the common meaning given in the state of the art to said terms. Hence, these terms are used to refer to, for example and not restricted to, cycloalkyl or cycloalkenyl or cycloalkynyl groups.
The term “cycloalkyl” and its plural, as used herein, refer to a saturated mono- or polycyclic aliphatic group which has between 3 and 24, preferably between 3 and 16, more preferably between 3 and 14, even more preferably between 3 and 12, even more preferably still 3, 4, or 6 carbon atoms and which is bound to the rest of the molecule through a single bond, including, for example and not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methyl cyclohexyl, dimethyl cyclohexyl, octahydroindene, decahydronaphthalene, dodecahydro-phenalene, adamantyl and similar, and that can optionally be substituted by one or more groups, such as, alkyl, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto and alkoxythio.
The term “cycloalkenyl” and its plural, as used herein, refer to a non-aromatic mono- or polycyclic aliphatic group which has between 5 and 24, preferably between 5 and 16, more preferably between 5 and 14, even more preferably between 5 and 12, even more preferably still 5 or 6 carbon atoms, with one or more carbon-carbon double bonds, preferably with 1, 2 or 3 carbon-carbon double bonds, conjugated or unconjugated, which is bound to the rest of the molecule through a single bond, including, for example and not restricted to, the cyclopent-1-en-1-yl group and similar groups, and that can optionally be substituted by one or more groups, such as, alkyl, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto and alkoxythio.
The term “cycloalkynyl” and its plural, as used herein, refer to a non-aromatic mono- or polycyclic aliphatic group which has between 8 and 24, preferably between 8 and 16, more preferably between 8 and 14, even more preferably between 8 and 12, even more preferably still 8 or 9 carbon atoms, with one or more carbon-carbon triple bonds, preferably with 1, 2 or 3 carbon-carbon triple bonds, conjugated or unconjugated, which is bound to the rest of the molecule through a single bond, including, for example and not restricted to, the cyclooct-2-yn-1-yl group and similar, and that can optionally be substituted by one or more groups, such as, alkyl, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto and alkoxythio.
The term “aryl group” and its plural, as used herein, refer to an aromatic group which has between 6 and 30, preferably between 6 and 18, more preferably between 6 and 10, even more preferably 6 or 10 carbon atoms, which comprises 1, 2, 3 or 4 aromatic rings, bound by a carbon-carbon bond or fused, and which is bound to the rest of the molecule through a single bond, including, for example and not restricted to, phenyl, naphthyl, diphenyl, indenyl, phenanthryl or anthranyl among others. The aryl group can be optionally substituted by one or more substituents, such as, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto and alkoxythio.
The term “aralkyl group” and its plural, as used herein, refer to an alkyl group substituted by an aromatic group, with between 7 and 24 carbon atoms and including, for example and not restricted to, —(CH2)1-6-phenyl, —(CH2)1-6-(1-naphtyl), —(CH2)1-6-(2-naphtyl), —(CH2)1-6-CH(phenyl)2 and similar. The aralkyl groups can be optionally substituted by one or more substituents, such as, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto and alkoxythio.
The term “heterocyclic group” and its plural, as used herein, refer to a 3-10-member heterocyclyl or hydrocarbon ring, in which one or more of the ring atoms, preferably 1, 2 or 3 of the ring atoms, is a different element to carbon, such as nitrogen, oxygen or sulfur and may be saturated or unsaturated. For the purposes of this invention, the heterocyclyl can be a cyclic, monocyclic, bicyclic or tricyclic system which may include fused ring systems; and the nitrogen, carbon or sulfur atoms can be optionally oxidized in the heterocyclyl radical; the nitrogen atom can optionally be quaternized; and the heterocyclyl radical may be partially or completely saturated or may be aromatic. With increasing preference, the term heterocyclic relates to a 5 or 6-member ring. The heterocyclic groups can be optionally substituted by one or more substituents, such as, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto and alkoxythio.
The term “heteroarylalkyl group” and its plural, as used herein, refer to an alkyl group substituted with a substituted or unsubstituted aromatic heterocyclyl group, the alkyl group having from 1 to 6 carbon atoms and the aromatic heterocyclyl group between 2 and 24 carbon atoms and from 1 to 3 atoms other than carbon and including, for example and not restricted to, —(CH2)1-6-imidazolyl, —(CH2)1-6-triazolyl, —(CH2)1-6-thienyl, —(CH2)1-6-furyl, —(CH2)1-6-pyrrolidinyl and similar. The heteroarylalkyl groups can be optionally substituted by one or more substituents, such as, halo, hydroxy, alkoxy, carboxy, carbonyl, cyano, acyl, alkoxy-carbonyl, amino, nitro, mercapto and alkoxythio.
The terms “halo” or “halogen”, as used in the present document, refer to fluorine, chlorine, bromine or iodine, and its anions are referred to as halides.
As used herein, the term “derivative” and its plural, refer to cosmetically, pharmaceutically and/or food acceptable compounds, this is, derived from the compound of interest that can be used in the preparation of a cosmetic, pharmaceutical and/or food composition, and to cosmetically, pharmaceutically and/or food unacceptable derivatives since these may be useful in the preparation of cosmetically, pharmaceutically and/or food acceptable derivatives.
As used in the present document, the term “salt” and its plurals refer to any type of salt from among those known in the state of the art, for example, halide salts, hydroxy acid salts (such as oxyacid salts, acid salts, basic salts and double salts), hydroxy salts, mixed salts, oxy salts or other hydrated salts. This term comprises cosmetically, pharmaceutically and/or food acceptable salts; and cosmetically, pharmaceutically and/or food unacceptable salts, since the latter may be useful in the preparation of cosmetically, pharmaceutically and/or food acceptable salts.
As used in the present document, the term “isomer” and its plural refer to optical isomers, enantiomers, stereoisomers or diastereoisomers. The individual enantiomers or diastereoisomers, as well as their mixtures, may be separated by conventional techniques known in the state of the art.
As used herein, the term “solvate” and its plural refer to any solvate known in the state of the art, such as polar, apolar or amphiphilic solvates, and include any cosmetically, pharmaceutically and/or food acceptable solvate which, when administered or applied to the interested subject (directly or indirectly) provides the compound of interest (the peptide or peptides of the present invention). Preferably, the solvate is a hydrate, a solvate with an alcohol such as methanol, ethanol, propanol or isopropanol, a solvate with an ester such as ethyl acetate, a solvate with an ether such as methyl ether, ethyl ether or THF (tetrahydrofuran) or a solvate with DMF (dimethylformamide), and more preferably a hydrate or a solvate with an alcohol such as ethanol.
In addition, as used herein, the term “amino acid” and its plural include the amino acids codified by the genetic code as well as uncodified amino acids, whether they are natural or not and whether they are D- and L-amino acids. Examples of uncodified amino acids are, without restriction, citrulline, ornithine, sarcosine, desmosine, norvaline, 4-aminobutyric acid, 2-aminobutyric acid, 2-aminoisobutyric acid, 6-aminohexanoic acid, 1-naphthylalanine, 2-naphthylalanine, 2-aminobenzoic acid, 4 aminobenzoic acid, 4-chlorophenylalanine, 2,3-diaminopropionic acid, 2,4 diaminobutyric acid, cycloserine, carnitine, cysteine, penicillamine, pyroglutamic acid, thienylalanine, hydroxyproline, allo-isoleucine, allo-threonine, isonipecotic acid, isoserine, phenylglycine, statin, β-alanine, norleucine, N-methylamino acids, α-amino acids and β-amino acids, among others, as well as their derivatives. Nevertheless, further unnatural amino acids are known in the state of the art (see, for example, “Unusual amino acids in peptide synthesis” by Roberts D C and Vellaccio F (1983) The Peptides, Vol. 5, Chapter VI. Gross E and Meienhofer J, Eds., Academic Press, New York, USA). The abbreviations used in the present text for amino acids follow the 1983 IUPAC-IUB Joint Commission on Biochemical Nomenclature recommendations outlined in Eur. J. Biochem. (1984) 138:937.
The “percentage of identity” regarding peptides, polypeptides and proteins, as used herein, has the meaning commonly attributed in the state of the art and, hence, relates to the percentage of amino acids which are identical between two amino acid sequences which are compared after an optimal alignment of these sequences, where said percentage is merely statistical and the differences between the two amino acid sequences are randomly distributed throughout the sequence. “Optimal alignment” is understood as that alignment of amino acid sequences giving rise to a greater percentage of identity. The percentage of identity is calculated by determining the number of identical positions in which an amino acid is identical in the two compared sequences, dividing the number of identical positions by the number of compared positions and multiplying the result obtained by 100 to obtain the percentage of identity between the two sequences. The sequence comparisons between two amino acid sequences can be carried out manually or by means of computer programs known in the state of the art, such as the BLAST (Basic Local Alignment Search Tool) algorithm.
The terms “inflammaging” and “inflamm-aging” are used herein interchangeably and acquire the meaning they commonly have in the state of the art, this is, they refer to the chronic, low-grade inflammation that characterizes aging.
Therefore, in a first aspect, the present invention refers to a peptide of formula (I):
R1 is linked to the N-terminal of the peptide, this is, to the amino group of AA1 (it substitutes or replaces an H from the amino group of AA1).
R2 is linked to the C-terminal of the peptide, this is, to the carboxy group of AA8 or in case AA8 is absent to the carboxy group of AA7 (it substitutes or replaces the OH of the carboxy group).
It is contemplated that the amino acids used or present in the peptides of the present invention are L-amino acids, D-amino acids or combinations thereof. In a preferred embodiment, the amino acids used or present in the peptides of the present invention are L-amino acids.
Preferably, the isomers mentioned above are stereoisomers. It is contemplated that said stereoisomers are enantiomers or diastereoisomers. Hence, in a preferred embodiment of the present invention, the peptide is a racemic mixture, a diastereomeric mixture, a pure enantiomer or a pure diastereoisomer.
In addition, isomers, salts, solvates, derivatives and/or mixtures thereof are, preferably, cosmetically, pharmaceutically and/or food acceptable isomers, salts, solvates, derivatives and/or mixtures thereof.
Preferably, AA1 is selected from Cys, Val, Ala, Pro, Ser, Asn, Ala, Gly or Thr, more preferably, AA1 is selected from Ser, Cys, Ala or Gly; even more preferably, AA1 is Gly.
Also preferably, AA2 is selected from Tyr, Phe, Trp, Ser, Thr, Gln, Asn, Cys or Gly, more preferably AA2 is selected from Tyr, Trp, Ser, Asn or Gln, more preferably AA2 is selected from Tyr or Asn.
Preferably, AA3 is absent or selected from Tyr, Phe or Trp, more preferably AA3 is absent or Tyr.
In the present invention, AA4 is, preferably, selected from Asp or Glu, more preferably AA4 is Glu.
Also preferably, AA5 is selected from Ala, Val, lie, Leu, Met, Pro, Phe or Trp, more preferably AA5 is selected from Ala, lie, Leu or Val, even more preferably AA5 is Leu.
In the present invention, AA7 is, preferably, selected from Asp or Glu, more preferably AA7 is Asp.
Preferably, AA8 is absent or selected from Tyr, Phe or Trp, more preferably AA8 is absent or Tyr.
Therefore, in a preferred embodiment, in formula (I):
In any of the above embodiments, in case AA3 is absent, preferably AA8 is not absent and vice versa. This is, in a preferred embodiment, only one of AA3 and AA8 is absent.
Therefore, in an embodiment, AA3 and AA8 are both present. In another embodiment, AA3 and AA8 are both absent. However, in a most preferred embodiment, AA3 is absent and AA8 is present; and in another most preferred embodiment AA3 is present and AA8 absent.
In a preferred embodiment, the peptide of the present invention is:
being R1 and R2 as disclosed above.
More preferably, the peptide of the present invention is:
In any of the above embodiments, preferably, R1 is H or acetyl, more preferably R1 is H.
Also, in any of the above embodiments, R2 is NH2 or OH, more preferably R2 is NH2.
Hence, in a most preferred embodiment, R1 is H and R2 is NH2.
In the most preferred embodiment, the peptide of the present invention is:
even more preferably, the peptide of the present invention is:
Peptide SEQ ID NO: 2 is a biomimetic peptide derived or generated from Turmerin protein (Curcuma longa)—UniProt ref P85278 (TURM_CURLO).
Peptide SEQ ID NO: 3 is a biomimetic peptide derived or generated from Ricin protein (Ricinus communis (Castor bean))—UniProt ref P02879 (RICI_RICCO).
In a preferred embodiment, combinable with any of the above embodiments, the peptide of the present invention provides for a protection of the telomeres, preferably by increasing the expression and/or levels of TRF1.
In a further preferred embodiment, combinable with any of the above embodiments, the peptide of the present invention provides for an increase or a recovery in the length in telomeres.
In a further preferred embodiment, combinable with any of the above embodiments, the peptide of the present invention provides for antioxidant activity, preferably by means of: increasing the levels of catalase, translocation of Nfr2 into the nuclei of cells or combinations thereof, more preferably, by means of increasing the levels of catalase and translocation of Nfr2 into the nuclei of cells.
In a further preferred embodiment, combinable with any of the above embodiments, the peptide of the present invention provides for anti-inflammatory activity, preferably by means of modulation of inflammation-related genes and/or miRNAs, decreasing the levels of IL-8 and/or reducing the levels of TLR2 or combinations thereof, most preferably at least by decreasing the levels of IL-8.
Modulation of inflammation-related genes is preferably upregulation of genes TNFAIP3, TOLLP, HMOX1, IRAK3, NQO1 and downregulation of genes NFKB1, IL1B, TNF, CXCL8, IL6, MMP3 and TLR2.
Modulation of inflammation-related miRNAs is preferably upregulation of miR-146a-5p and miR-21-5p.
Within the scope of the present invention are also included peptides derived or obtained from any of the peptides disclosed above, preferably incorporating one or more conservative substitutions, as long as they retain or show or improve at least one of the activities mentioned above (and, preferably, all of the activities mentioned above for the peptides of the present invention).
In addition, also included within the scope of the present invention are peptides with a 70% percentage identity, preferably 80%, more preferably 90%, more preferably 95%, even more preferably 99% percentage identity with any of the specific sequences mentioned above as long as they retain or show or improve at least one of the activities mentioned above (and, preferably, all of the activities mentioned above for the peptides of the present invention).
As it is directly derivable from the above, it is contemplated that the peptide of the present invention is a linear peptide or a cyclic peptide.
In an embodiment, which can be combined with any of the above embodiments, the peptide is cyclic. In this case, the first and the last amino acid of the peptide of the present invention (in accordance with the above explanations) are bound. Therefore, appropriate modification to any of the above embodiments has to be done (for example, R1 and R2 may be absent as the amino acids to which said moieties are bound, are now bound to each other).
In a preferred embodiment, which can be combined with any of the above embodiments, the peptide is linear.
The peptides of the present invention may be synthesized and produced by any means known in the state of the art. For example, they may be synthesized and produced by chemical synthesis (preferably, by means of solid phase peptide synthesis), expressing said peptides in cell cultures or by means of transgenic production of the peptide in plants or animals. In addition, the peptides of the present invention may be purified by any means known in the state of the art.
In a preferred embodiment, which can be combined with any of the embodiments disclosed above, the peptide of the present invention is suited or adapted to be applied by means of iontophoresis, more preferably, in the face of a subject (preferably, a human).
In a further preferred embodiment, which can be combined with any of the embodiments disclosed above, the peptide of the present invention is suited or adapted to be applied subcutaneously, more preferably, in the face of a subject (preferably, a human).
In another preferred embodiment, which can be combined with any of the embodiments disclosed above, the peptide of the present invention is suited or adapted to be applied topically (preferably, in the form of a cream), more preferably, in the face of a subject (preferably, a human).
As it can be directly derived from the examples included below, the peptides of the present invention provide for a broad spectrum of activities which make them useful in cosmetics for the prevention and/or treatment of skin aging and inflammaging:
Therefore, the peptides of the present invention provide for a broad-spectrum activity for the prevention and/or treatment of inflammaging and skin aging and solve the problems present in the state of the art and mentioned above.
In addition, the peptides of the present invention are also useful in medicine due to the above-mentioned activities (especially anti-inflammatory and antioxidative activities). Said activities demonstrate that the peptides of the present invention are useful, for example, for the treatment of age-related diseases, inflammatory diseases and/or diseases related with oxidative stress.
Moreover, due to the above activities (especially anti-inflammatory and antioxidative activities), which appear demonstrated in the examples included below, the peptides of the present invention are also useful in the food industry, preferably as nutraceuticals.
In a second aspect, the present invention refers to a composition comprising a peptide of the present invention.
The peptide of the present invention is as disclosed above in the first aspect of the present invention.
The composition of the present invention is preferably a cosmetic composition. Hence, in this embodiment, the cosmetic composition of the present invention comprises a cosmetically effective amount of the at least one peptide of the present invention, more preferably the cosmetic composition of the present invention comprises from 0.0001% (weight/volume in g/100 mL, hereinafter, w/v) to 0.05% (w/v) of a peptide of the present invention. In a most preferred embodiment, the cosmetic composition of the present invention comprises from 0.0001% (w/v) to 0.005% (w/v) of a peptide of the present invention, more preferably, from 0.0005% (w/v) y 0.0015% (w/v).
It is contemplated that the cosmetic composition of the present invention also comprises at least one additional cosmetic ingredient. Said additional cosmetic ingredient can be at least one excipient and/or at least one additional cosmetic active ingredient. Said additional cosmetic ingredient can be any cosmetic ingredient known in the state of the art as long as it does not affect, or it does not affect in an unacceptable manner the activity of the peptides of the present invention.
In another embodiment, the composition of the present invention is a pharmaceutical composition.
It is contemplated that the pharmaceutical composition of the present invention also comprises at least one additional pharmaceutical ingredient. Said additional pharmaceutical ingredient can be at least one excipient and/or at least one additional pharmaceutical active ingredient. Said additional pharmaceutical ingredient can be any cosmetic ingredient known in the state of the art as long as it does not affect, or it does not affect in an unacceptable manner the activity of the peptides of the present invention.
In a further embodiment, the composition of the present invention is a food composition.
It is contemplated that the food composition of the present invention also comprises at least one food ingredient. Said additional food ingredient can be any food ingredient known in the state of the art as long as it does not affect, or it does not affect in an unacceptable manner the activity of the peptides of the present invention.
Preferably, the food composition of the present invention is a nutraceutical.
In an embodiment, the composition of the present invention consists essentially of peptides of the present invention.
In another embodiment, the composition of the present invention consists of peptides of the present invention.
In a third aspect, the present invention refers to the use as a cosmetic of a peptide of the present invention or of a composition of the present invention, in a subject in need of the treatment.
The peptide of the present invention is as disclosed above in the first aspect of the present invention.
The composition of the present invention is as disclosed above in the second aspect of the present invention.
The peptide or the composition of the present invention are used in a cosmetically effective amount or quantity.
The composition of the present invention is, preferably, a cosmetic composition as disclosed above in the second aspect of the present invention.
In a preferred embodiment, the use as a cosmetic is to prevent, treat and/or reduce skin aging, more preferably, reduce, prevent and/or eliminate signs of skin aging.
Preferably, the signs of skin aging are wrinkles, skin roughness (preferably, wrinkle roughness), looser skin or sagging skin, more preferably, wrinkles, skin roughness, non-inflammatory looser skin or non-inflammatory sagging skin, more preferably, wrinkles, even more preferably facial wrinkles. Said wrinkles can be chronological wrinkles or expression (muscle-derived) wrinkles.
Non-inflammatory looser skin is equivalent to cosmetic looser skin, this is, non-pathological (non-medical condition).
Non-inflammatory sagging skin is equivalent to cosmetic sagging skin, this is, non-pathological (non-medical condition).
In another preferred embodiment, the use as a cosmetic is to prevent, treat and/or reduce inflammaging, more preferably skin inflammaging.
In this third aspect of the present invention, the subject in need of the treatment is preferably a mammal, more preferably, a human.
In this third aspect of the present invention, it is also contemplated that the use as a cosmetic is for the improvement of skin firmness and/or elasticity.
In a fourth aspect, as noted above, the present invention refers to the cosmetic use of a peptide of the present invention or a composition of the present invention in a subject in need of the treatment.
Preferably, in this fourth aspect of the present invention, the cosmetic use is to prevent, treat and/or reduce skin aging, more preferably, reduce, prevent and/or eliminate signs of skin aging.
In another preferred embodiment, the cosmetic use is to prevent, treat and/or reduce inflammaging, more preferably skin inflammaging. Inflammaging is cosmetic inflammaging, preferably cosmetic skin inflammaging.
In this fourth aspect of the present invention, it is also contemplated that the cosmetic use is for the improvement of skin firmness and/or elasticity.
The peptide of the present invention is as disclosed above in the first aspect of the present invention.
The composition of the present invention is as disclosed above in the second aspect of the present invention.
The composition of the present invention is, preferably, a cosmetic composition as disclosed above in the second aspect of the present invention.
The signs of skin aging and the subject in need of the treatment are as explained above in the third aspect of the present invention.
In addition, all the embodiments explained above in the third aspect of the present invention are directly applicable to this fourth aspect of the present invention, with the appropriate adaptations.
In a fifth aspect, the present invention refers to a method for the prevention and/or treatment of skin aging or of inflammaging in a subject comprising the administration of a peptide of the present invention or a composition of the present invention to the subject.
The peptide of the present invention is as disclosed above in the first aspect of the present invention.
The composition of the present invention is as disclosed above in the second aspect of the present invention.
The composition of the present invention is, preferably, a cosmetic composition as disclosed above in the second aspect of the present invention.
The signs of skin aging, inflammaging and the subject are as explained above in the third aspect of the present invention.
In addition, all the embodiments explained above in the third and fourth aspects of the present invention are directly applicable to this fifth aspect of the present invention, with the appropriate adaptations.
In a sixth aspect, the present invention refers to a composition or a peptide of the present invention for use as a medicament.
The peptide of the present invention is as disclosed above in the first aspect of the present invention.
The composition of the present invention is as disclosed above in the second aspect of the present invention.
The peptide or the composition of the present invention are used in a pharmaceutically effective amount or quantity.
The composition of the present invention is, preferably, a pharmaceutical composition as disclosed above in the second aspect of the present invention.
In this sixth aspect of the present invention, the composition or peptide of the present invention is administered to a subject in need of the treatment. Preferably, the subject in need of the treatment is a mammal, more preferably, a human.
In a preferred embodiment, the use as a medicament is for use in the prevention, amelioration and/or treatment of an age-related disease, an inflammatory disease and/or a disease related to oxidative stress.
In a preferred embodiment, the use as a medicament is for the prevention, amelioration and/or treatment of an inflammatory disease.
Preferably, the inflammatory disease is a chronic inflammatory disease, more preferably rheumatoid arthritis, inflammatory bowel disease, psoriasis or palmoplantar pustulosis.
Also preferably, the inflammatory disease is rosacea, atopic skin, psoriasis or acne.
It is also contemplated that the inflammatory disease is inflammaging, more preferably, non-cosmetic inflammaging, this is, pathological inflammaging.
It is also contemplated that the inflammatory disease is inflammatory looser skin or inflammatory sagging skin (this is, pathological looser skin or pathological sagging skin).
In a further preferred embodiment, the use as a medicament is for the prevention, amelioration and/or treatment of a disease related with oxidative stress.
Preferably, the disease related with oxidative stress is selected from cardiovascular diseases (CVDs) (preferably, atherosclerosis), chronic obstructive pulmonary disease, acute or chronic kidney disease, clinical frailty, neurodegenerative diseases (preferably, AD, Parkinson's disease (PD), Sarcopenia, Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and vascular dementia), macular degeneration (MD), biliary diseases cancer, biliary cirrhosis, cholangitis, osteoarthritis and diabetes.
In a seventh aspect, the present invention refers to the use of the composition or a peptide of the present invention for the manufacture of a medicament for the prevention, amelioration and/or treatment of an age-related disease, an inflammatory disease or a disease related to oxidative stress.
The peptide of the present invention is as disclosed above in the first aspect of the present invention.
The composition of the present invention is as disclosed above in the second aspect of the present invention.
The peptide or the composition of the present invention are used in a pharmaceutically effective amount or quantity.
The composition of the present invention is, preferably, a pharmaceutical composition as disclosed above in the second aspect of the present invention.
The age-related disease, the inflammatory disease and the disease related to oxidative stress are as disclosed above in the sixth aspect of the present invention.
In this seventh aspect of the present invention, the composition or peptide of the present invention is administered to a subject in need of the treatment. Preferably, the subject in need of the treatment is a mammal, more preferably, a human.
In an eighth aspect, the present invention refers to a method for the prevention, amelioration and/or treatment of an age-related disease, an inflammatory disease or a disease related to oxidative stress, comprising administering a peptide or a composition of the present invention to a subject in need thereof.
The peptide of the present invention is as disclosed above in the first aspect of the present invention.
The composition of the present invention is as disclosed above in the second aspect of the present invention.
The peptide or the composition of the present invention are used in a pharmaceutically effective amount or quantity.
The composition of the present invention is, preferably, a pharmaceutical composition as disclosed above in the second aspect of the present invention.
The age-related disease, the inflammatory disease and the disease related to oxidative stress are as disclosed above in the sixth aspect of the present invention.
Preferably, the subject in need of the treatment is a mammal, more preferably, a human.
In a ninth aspect, the present invention refers to a nutraceutical composition comprising a peptide of the present invention.
The peptide of the present invention is as disclosed above in the first aspect of the present invention.
In a final aspect, the present invention refers to the use as a nutraceutical of a peptide of the present invention or of a composition of the present invention.
The peptide of the present invention is as disclosed above in the first aspect of the present invention.
The composition of the present invention is as disclosed above in the second aspect of the present invention. More preferably, the composition of the present invention is a food composition as explained in the second aspect of the present invention.
The activities mentioned above and demonstrated in the experiments included below, especially the anti-inflammatory and antioxidative activities, demonstrate the usefulness of the peptides and compositions of the present invention as nutraceuticals.
The abbreviations used in the present text for amino acids follow the 1983 IUPAC-IUB Joint Commission on Biochemical Nomenclature recommendations outlined in Eur. J. Biochem. (1984) 138:937.
Asn, Asparagine; Asp, aspartic acid; BSA, Bovine Serum Albumin; cDNA, Complementary DNA; C-terminal, carboxy-terminal; DAPI, 4′,6-diamidino-2-phenylindole; DCM, dichloromethane; DIPCDI, N,N′-diisopropylcarbodiimide; DMEM, Dulbecco's Modified Eagle Medium; DMF, N,N-dimethylformamide; DMSO, Dimethyl sulphoxide; DNA, Deoxyribonucleic acid; equiv, equivalent; ESI-MS, electrospray ionization mass spectrometry; FBS, Fetal Bovine Serum; Fmoc, 9-fluorenylmethyloxycarbonyl; Glu, Glutamic acid; Gly, Glycine; HOBt, 1-hydroxybenzotriazole; HPLC, high performance liquid chromatography; IFN-γ, Interferon gamma; Leu, Leucine; LSGS, Low Serum Growth Supplement; MBHA, p-methylbenzhydrylamine; Me, methyl; MeCN, acetonitrile; MeOH, methanol; N-terminal, amino-terminal; PBS, Phosphate Borate Saline; qPCR, real-time polymerase chain reaction; RH, Relative Humidity; RIPA, Radioimmunoprecipitation assay buffer; RNA, Ribonucleic acid; RT, room temperature; RT-qPCR, Quantitative reverse transcription Polymerase Chain Reaction; tBu, tert-butyl; TFA, trifluoroacetic acid; TIS, triisopropylsilane; Trt, triphenylmethyl or trityl; Tyr, tyrosine.
Regarding the chemical synthesis procedures included in the examples, it is noted that all synthetic processes were carried out in polypropylene syringes fitted with porous polyethylene discs or Pyrex® reactors fitted with porous plates. All the reagents and solvents were synthesis quality and were used without any additional treatment. The solvents and soluble reagents were removed by suction. The Fmoc group was removed with piperidine-DMF (2:8, volume/volume, v/v) (at least 1×1 min, 2×10 min, 5 mL/g resin) (Lloyd Williams P. et al., Chemical Approaches to the Synthesis of Peptides and Proteins, CRC, 1997, Boca Raton (Fla., USA)). Washes between stages of deprotection, coupling, and, again, deprotection, were carried out with DMF (3×1 min) and DCM (3×1 min) each time using 10 mL solvent/g resin. Coupling reactions were performed with 3 mL solvent/g resin. The control of the couplings was performed by carrying out the ninhydrin test (Kaiser E. et al., Anal. Biochem., 1970, 34: 595598). All synthetic reactions and washes were carried out at RT.
a) Obtaining Fmoc-AA1-AA2-AA3-AA4-AA5-AA6-AA7-Rink-MBHA-resin, wherein AA1 is L-Gly; AA2 is L-Tyr; AA3 is L-Tyr; AA4 is L-Glu; AA5 is L-Leu; AA6 is L-Asn and AA7 is L-Asp
Weights were normalized. 4.8 g (2.5 mmol) of the Fmoc-Rink-MBHA resin with a functionalization of 0.52 mmol/g were treated with piperidine-DMF according to the described general protocol known in the state of the art in order to remove the Fmoc group. 3.08 g of Fmoc-L-Asp(tBu)-OH (7.5 mmol; 3 equiv) were incorporated onto the deprotected resin in the presence of DIPCDI (1.17 mL; 7.5 mmol; 3 equiv) and HOBt (1.01 g; 7.5 mmol; 3 equiv) using DMF as a solvent for one hour.
The resin was then washed as described in the general methods known in the state of the art and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. Following the previously described protocols 4.45 g of Fmoc-L-Asn(Trt)-OH (7.5 mmol; 3 equiv); subsequently 2.65 g of Fmoc-L-Leu-OH (7.5 mmol; 3 equiv); subsequently 3.19 g of Fmoc-L-Glu(OtBu)-OH (7.5 mmol; 3 equiv); subsequently 3.44 g Fmoc-L-Tyr(tBu)-OH (7.5 mmol; 3 equiv), subsequently 3.44 g Fmoc-L-Tyr(tBu)-OH (7.5 mmol; 3 equiv) and subsequently 2.23 g of Fmoc-L-Gly-OH (7.5 mmol; 3 equiv) were coupled, sequentially, each coupling in the presence of 1.01 g of HOBt (7.5 mmol; 3 equiv) and 1.17 mL of DIPCDI (7.5 mmol; 3 equiv). As already noted above, between each amino acid addition step, a deprotection treatment of the Fmoc group was performed.
After the synthesis, the peptide resins were washed with DCM (5 times for 3 minutes each one) and dried under vacuum.
b) Obtaining Fmoc-AA1-AA2-AA3-AA4-AA5-AA6-AA7-Rink-MBHA-resin, wherein AA1 is L-Gly; AA2 is L-Asn; AA3 is L-Glu; AA4 is L-Leu; AA5 is L-Leu; AA6 is L-Asp and AA7 is L-Tyr
Weights were normalized. 4.8 g (2.5 mmol) of the Fmoc-Rink-MBHA resin with a functionalization of 0.52 mmol/g were treated with piperidine-DMF according to the described general protocol known in the state of the art in order to remove the Fmoc group. 3.44 g of Fmoc-L-Tyr(tBu)-OH (7.5 mmol; 3 equiv) were incorporated onto the deprotected resin in the presence of DIPCDI (1.17 mL; 7.5 mmol; 3 equiv) and HOBt (1.01 g; 7.5 mmol; 3 equiv) using DMF as a solvent for one hour.
The resin was then washed as described in the general methods known in the state of the art and the deprotection treatment of the Fmoc group was repeated to couple the next amino acid. Following the previously described protocols 3.08 g of Fmoc-L-Asp(tBu)-OH (7.5 mmol; 3 equiv); subsequently 2.65 g of Fmoc-L-Leu-OH (7.5 mmol; 3 equiv); subsequently 2.65 g of Fmoc-L-Leu-OH (7.5 mmol; 3 equiv): subsequently 3.19 g of Fmoc-L-Glu(OtBu)-OH (7.5 mmol; 3 equiv); subsequently 4.45 g of Fmoc-L-Asn(Trt)-OH (7.5 mmol; 3 equiv) and subsequently 2.23 g of Fmoc-L-Gly-OH (7.5 mmol; 3 equiv) were coupled, sequentially, each coupling in the presence of 1.01 g of HOBt (7.5 mmol; 3 equiv) and 1.17 mL of DIPCDI (7.5 mmol; 3 equiv). As already noted above, between each amino acid addition step, a deprotection treatment of the Fmoc group was performed.
After the synthesis, the peptide resins were washed with DCM (5 times for 3 minutes each one) and dried under vacuum.
Using the synthesis procedures mentioned above, with the required selection of amino acids, the following sequences were synthesized:
c) Removal of Fmoc N-Terminal Protective Group of the Peptides Synthesized in Accordance with a) or b)
The N-terminal Fmoc group of the peptidyl resins obtained in a) or b) above, was deprotected with 20% (volume/volume, hereinafter v/v) piperidine in DMF (1×1 min+2×10 min) (Lloyd Williams P. et al. (1997) Chemical Approaches to the Synthesis of Peptides and Proteins. CRC, Boca Raton (Fla., USA)). The peptidyl resins were washed with DMF (5×1 min), DCM (4×1 min), and dried under vacuum.
d) Cleavage Process from the Polymeric Support of the Peptidyl Resins Obtained in Accordance with c)
Weights were normalized. 200 mg of the dried peptidyl resin obtained in c) were treated with 5 mL of TFA/TIS/H2O (90:5:5, in volume) for 2 hours at room temperature under stirring. The filtrates were collected and precipitated using 50 mL (8 to 10-fold) of cold diethyl ether. The ethereal solutions were evaporated to dryness at reduced pressure and room temperature, the precipitates were redissolved in 50% (v/v) MeCN in H2O and lyophilized.
e) Characterization of the Peptides Synthesized and Prepared in Accordance with d)
HPLC analysis of the peptides obtained in accordance with d) was carried out with a Shimadzu equipment (Kyoto, Japan) using a reverse-phase column (150×4.6 mm, XBridge Peptide BEH C18, 3.5 μm, Waters, USA) in gradients of MeCN (+0.036% (v/v) TFA) in H2O (+0.045% (v/v) TFA) at a flow rate of 1.25 mL/min and detection was carried out at 220 nm. All peptides showed a purity exceeding 80%. The identity of the peptides obtained was confirmed by ESI-MS in a Water ZQ 4000 detector using MeOH as the mobile phase and a flow rate of 0.2 mL/min. Results obtained demonstrated that peptides SEQ ID NO: 2-NH2 and SEQ ID NO: 3-NH2, were correctly and effectively synthesized.
For this example, peptide SEQ ID NO: 2-NH2 was prepared in accordance with example 1.
A stock solution of the peptide was prepared in DMSO at 50 mg/mL. This stock was further diluted to obtain the final working concentrations of 0.01 mg/mL in complete medium (Dermal Cell Basal Medium (DCBM) supplemented with keratinocyte Growth Kit and 0.1% Penicillin/Streptomycin). Untreated cells were used as negative control sample (basal sample).
The relevant genes for which the expression was measured were: TOLLIP (toll interacting protein), IRAK3 (interleukin 1 receptor associated kinase 3), TNFAIP3 (TNF alpha induced protein 3), HMOX1 (heme oxygenase 1), NQO1 (NAD(P)H quinone dehydrogenase 1), NFKB1 (nuclear factor kappa B subunit 1), TNF (Tumoral necrosis factor), IL1B (interleukin 1 beta), IL6 (Interleukin 6), CXCL8 (C—X—C motif chemokine ligand 8), MMP3 (matrix metallopeptidase 3), TLR2 (toll like receptor 2). The primers used for each of the genes were commercially available TaqMan Gene Expression Assay probes. The experiment was performed in vitro in HEKa cells, one concentration of the peptide was tested (0.01 mg/mL) at 6 h and 24 h.
Briefly, human epidermal keratinocytes from adult (HEKa) were seeded in 6-well plates at a density of 300,000 cells/well and maintained at standard culture conditions (DCBM medium supplemented with keratinocyte Growth Kit and 0.1% Penicillin/Streptomycin; 37° C., 95% Relative Humidity, 5% CO2) for 24 hours. Next, HEKa were incubated in the presence of 0.01 mg/mL of the peptide for 6 or 24 h.
Cells were finally lysed and RNA extraction was performed using Qiagen RNeasy Mini kit following manufacturer's instructions. Purified RNAs were used to generate the corresponding cDNAs by reverse transcription which served as templates for amplification. RT-qPCR was performed with the panel of TaqMan assay probes specified above and 2× gene expression Master Mix using StepOne plus Real-Time PCR instrument. Amplification included 40 cycles of: 15 seconds at 95° C. (denaturation) and 1 minute at 60° C. (Annealing and extension).
The obtained data were analysed using the ΔΔCt method, which provides the target gene expression values as fold changes in the treated sample compared with an untreated basal sample. All samples were normalized with the relative expression of a housekeeping gene GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).
Gene modulations with a ratio (fold change vs. basal samples)>1.15 or <(−1.15) are considered of potential biological importance.
The results obtained appear summarized in
For this example, peptide SEQ ID NO: 2-NH2 was prepared in accordance with example 1.
A stock solution of the peptide was prepared in DMSO at 50 mg/mL. This stock was further diluted to obtain the final working concentrations of 0.01 mg/mL in complete medium (Dermal Cell Basal Medium (DCBM) supplemented with keratinocyte Growth Kit and 0.1% Penicillin/Streptomycin). Untreated cells were used as negative control sample (basal sample).
The relevant miRNAs for which the expression was measured were: miR-21-5p and miR-146a-5p. During the last years, microRNAs (miRNAs) has been identified as another key element in aging and inflammaging. miR-21-5p and miR-146a-5p are involved in the modulation of NF-κB and NLRP inflammatory pathways, their levels significantly change both in tissues and in the bloodstream during aging and are associated with a variety of inflammatory conditions. Decline in the expression of this miRNAs has been associated with aging and they have been proposed as biomarkers of aging related diseases.
The primers used for each of the genes were commercially available TaqMan Advanced miRNA Assay probes. The experiment was performed in vitro HEKa cells, one concentration of the peptide was tested (0.01 mg/mL) at 24 h.
Briefly, human epidermal keratinocytes from adult (HEKa) were seeded in 6-well plates at a density of 300,000 cells/well and maintained at standard culture conditions (DCBM medium supplemented with keratinocyte Growth Kit and 0.1% Penicillin/Streptomycin; 37° C., 95% Relative Humidity, 5% CO2) for 24 hours. Next, HEKa were incubated in the presence of 0.01 mg/mL of the peptide for 24 h.
Cells were finally lysed and RNA extraction was performed using miRVana miRNA isolation kit following manufacturer's instructions. Purified miRNAs were used to generate the corresponding cDNAs by reverse transcription which served as templates for amplification. RT-qPCR was performed with the panel of TaqMan Advanced miRNA Assay probes specified above and Taqman Fast Advance Master Mix using StepOne plus Real-Time PCR instrument. Amplification included 40 cycles of: 1 second at 95° C. (denaturation) and 20 seconds at 60° C. (Annealing and extension).
The obtained data were analysed using the ΔΔCt method, which provides the target gene expression values as fold changes in the treated sample compared with an untreated basal sample. All samples were normalized with the relative expression of a housekeeping miRNA: miR-16-5p.
Three independent experiments (N=3) were performed.
Obtained results appear summarized in
For this example, peptides SEQ ID NO: 2-NH2 and SEQ ID NO: 3-NH2 were prepared in accordance with example 1.
A stock solution of the peptide was prepared in DMSO at 50 mg/mL. This stock was further diluted to obtain the final working concentrations of 0.005, 0.01 and 0.05 mg/mL in complete medium (DMEM supplemented with 1% FBS and 0.1% Penicillin/Streptomycin). Cells treated with 10 ng/mL IFNγ alone were used as positive control for inflammation induction. Untreated cells were used as basal condition.
For this example, HaCaT keratinocytes were seeded in a 96-well plate at a density f 75,000 cells/well and maintained at standard culture conditions (DMEM with 10% FBS and 1% Penicillin/Streptomycin; 37° C., 95% RH, 5% CO2) for 24 hours. Then keratinocytes were treated for 48 hours with the peptide of the present invention at the concentrations noted above in the presence of 10 ng/mL IFNγ in DMEM+1% FBS. After the treatment, supernatants were collected and centrifuged in order to eliminate cell debris and were kept at −80° C. until they were used for IL-8 quantification with Human IL-8/CXCL8 DuoSet ELISA kit according to the manufacturer instructions.
In order to determine cell viability, at the end of the experiment, cells were cultured in 0.5 mg/mL MTT in complete medium for 1 hour before removing MTT solution and dissolving formazan crystals formed with 150 μL DMSO/well. Finally, OD at 570 nm was determined with spectrophotometer Multiskan.
Each condition was performed in triplicate (n=3) in three independent experiments (N=3). All the obtained data was normalized in accordance with formulas (II) and (Ill):
The obtained results appear summarized in
A similar experiment was carried out under basal conditions (this is, without the inflammatory stimuli of IFNγ), both for peptides SEQ ID NO: 2-NH2 (concentrations of 0.005, 0.01 and 0.05 mg/mL) and SEQ ID NO: 3-NH2 (concentrations of 0.01 and 0.05 mg/mL). In this case, the basal or untreated cells were stablished as 100% (IL-8 secreted by the basal or untreated cells). Obtained results appear summarized in
All the results obtained in this example, demonstrate a protective effect of the peptides of the present invention as IL-8 is a proinflammatory cytokine and, hence, peptides of the present invention are useful for the prevention, reduction and/or treatment of inflammaging and skin aging. In addition, all these results also demonstrate the usefulness of the peptides of the present invention for the treatment of age-related diseases and inflammatory diseases, as explained above. Also, this anti-inflammatory activity demonstrates the usefulness of the peptides of the present invention as nutraceuticals
For this example, peptide SEQ ID NO: 2-NH2 was prepared in accordance with example 1.
A stock solution of the peptide was prepared in DMSO at 50 mg/mL. This stock was further diluted to obtain the final working concentrations of 0.005 or 0.01 mg/mL in complete medium (Medium 106 supplemented with 2% LSGS). HDFa73 (HDFa from 73 years old adult) untreated were used as negative control and HDFa25 (HDFa from 25 years old adult untreated were considered for comparative purpose.
Briefly, Human Dermal fibroblasts from adult (HDFa) were seeded in 12-well plates at a concentration of 150,000 cells/well and maintained at standard culture conditions (Medium 106 supplemented with 2% LSGS, 37° C., 95% RH, 5% CO2) for 24 hours.
Next, HDFa cells were treated with the above-mentioned peptide at the concentrations noted above, for 48 hours. Then, DNA was extracted using Qiagen DNeasy Mini kit following manufacturer's instructions. Finally, telomere DNA vs nuclear DNA ratio was stablished using Absolute Human Telomere Length Quantification qPCR Assay Kit, according to manufacturer instructions. The obtained data was analysed using the ΔΔCt method, which provides the relative telomere length of the target sample as fold changes of telomere sequence (telDNA) compared with a single copy nuclear reference DNA sequence (SCR).
Three independent experiments (N=3) were performed.
Obtained results appear summarized in
For this example, peptide SEQ ID NO: 2-NH2 was prepared in accordance with example 1.
A stock solution of the peptide was prepared in DMSO at 50 mg/mL. This stock was further diluted to obtain the final working concentrations of 0.01 or 0.05 mg/mL in complete medium (Dermal Basal Medium supplemented with keratinocyte Growth Kit and 0.1% Penicillin/Streptomycin). Untreated cells were used as negative control samples (basal condition).
For this study, HEKa cells were cultured in Dermal Cell Basal Medium (DCBM) at a density of 50,000 cell/well and incubated at 37° C. in a humidified atmosphere with 5% CO2. When cell confluence was around 80% cells were treated with the peptide at the different concentration noted above for 24 hours. After the treatment, samples were washed twice with PBS, fixed with 4% formaldehyde and permeabilized with Triton X-100 0.2%. Next, samples were blocked for 1 hour with PBS/BSA 5% in order to avoid nonspecific antibody binding.
For Nrf2 staining, samples were incubated overnight with 1:100 recombinant Alexa Fluor®488 Anti-NRF2 antibody in PBS/BSA 1% at 4° C. After proper washing with PBS, cells were incubated with 50 μg/mL Phalloidin-TRITC (for staining actin filaments) during 1 hour at room temperature and dark conditions. Finally, samples were washed three times with PBS and coverslips were mounted using Prolong-Gold with DAPI, which strongly binds DNA (nucleic staining), and samples were kept protected from light at 4° C. until microscopic images were acquired. Microscopic images were acquired using 10× objective. Immunofluorescence was performed on thee replicates for each condition and images of at least five different fields from each coverslip were acquired using the same settings.
The obtained images were analyzed with Image J software for their fluorescence intensity in the nuclei (DAPI staining) and in the cytoplasm (Nrf2 staining).
The percentage of nuclear located Nrf2 was obtained by normalizing the nuclear fluorescence signal obtained for Nrf2 in the treated samples with regard to the one compared for the untreated sample.
Obtained results appear summarized in
For this example, peptide SEQ ID NO: 2-NH2 was prepared in accordance with example 1.
A stock solution of the peptide was prepared in DMSO at 50 mg/mL. This stock was further diluted to obtain the final working concentrations of 0.01 or 0.05 mg/mL in complete medium (Dermal Basal Medium supplemented with keratinocyte Growth Kit and 0.1% Penicillin/Streptomycin). Cells treated with 10 ng/mL IFNγ alone were used as positive control samples for inflammation induction. Untreated cells were used as negative control samples (basal condition).
For this study, HEKa cells were seeded in a 24-well plate at a density of 50,000 cell/well and maintained at standard conditions (DCBM supplemented with keratinocytes growth kit and 0.1% Penicillin/Streptomycin; 37° C., 95% RH, 5% CO2) for 24 hours. Then, keratinocytes were treated for 24 hours with 10 ng/mL IFNγ. Next IFNγ was removed and cells were incubated with different concentrations of the peptide of the present invention, as noted above, for 24 hours. Positive control samples (IFNγ alone) were incubated with complete medium without peptide during this time. After the treatment, cells were washed with Dubelcco PBS (PBS also including potassium chloride) and lysed with RIPA and a scrapper. Lysates were collected and centrifuged in order to eliminate cell debris and protein concentrates were kept at −80° C. until they were used for TLR2 quantification with Human TLR2 DuoSet ELISA kit according to manufacturer instructions.
Each condition was performed in triplicate (n=3) in three independent experiments (N=3).
Results appear summarized in
For this example, peptide SEQ ID NO: 2-NH2 was prepared in accordance with example 1.
A stock solution of the peptide was prepared in DMSO at 50 mg/mL. This stock was further diluted to obtain the final working concentrations of 0.005, 0.01 or 0.05 mg/mL in complete medium (Dermal Basal Medium supplemented with keratinocyte Growth Kit and 0.1% Penicillin/Streptomycin). Untreated cells were used as negative control samples (basal condition).
For this study, HEKa cells were seeded in a 24-well plate at a density of 50,000 cell/well and maintained at standard culture conditions (DCBM; 37° C., 95% RH, 5% CO2) for 24 hours. Then, keratinocytes were treated for 24 hours with the peptide of the present invention at the concentrations noted above. After treatment, cells were washed with Dubelcco PBS and lysed with RIPA and a scrapper. Lysates were collected and centrifuged in order to eliminate cell debris and protein concentrates were kept at −80° C. until they were used for catalase quantification with human catalase ELISA Kit, according to manufacturer instructions. Finally, in order to normalize data, total protein content of each sample was quantified through Micro BCA Protein Assay kit, following manufacturer instructions.
Each condition was performed at least in triplicate (n=3) in three independent experiments (N=3).
Obtained results appear summarized in
For this example, peptides SEQ ID NO: 2-NH2 and SEQ ID NO: 3-NH2 were prepared in accordance with example 1.
As it is widely known in the state of the art, in vertebrates, telomeres are composed by a tandem repeat of the TTAGGG sequence, bound by a six-protein complex known as sheltering which encompasses TRF1, TRF2, TIN2, POT1, RAP1 and TPP1. The Telomere Repeat Binding factor 1 (TRF1) was the first sheltering to be discovered (Zhong, Z., et al. (1992) A mammalian factor that binds telomeric TTAGGG repeats in vitro. Mol Cell Biol 12, 4834-4843.) and it is one of the best characterized. TRF1 binds to double-stranded TTAGGG repeats and its abundance at telomeres is proportional to the length of repeats (de Lange, T. (2005) Shelterin: the protein complex that shapes and safeguards human telomeres. Genes. Dev. 19, 2100-2110 and Liu, L., et al. (2007) Telomere lengthening early in development. Nat. Cell Biol. 9:1436-1441) being a good reporter of telomere length in vivo.
10,000 CHA9.3 cells were seeded per well in a sterile 96-well black-clear bottom tissue culture plate using 150 μL of DMEM medium supplemented with 10% fetal bovine serum. Cells were incubated at 37° C., 5% CO2/humidified air for 24 h.
In parallel, peptides to be used in this example were weighed out and diluted in dimethyl sulfoxide (DMSO) or water to generate a “mother” plate. From here, an intermediate dilution plate was prepared. 1.5 μL of each compound solution was dispensed automatically using a Biomek FX Dual Pod automated liquid handling robot from the intermediated plate to the media of plated cells to get the final concentration 0.125 mg/mL for peptide SEQ ID NO: 2-NH2 and 0.063 mg/mL for peptide SEQ ID NO: 3-NH2. Each compound was assayed in duplicate. This final concentration for the screening was decided based on viability assays. Water or DMSO controls were also included in each plate as basal condition.
After a 24-hour incubation at 37° C., 5% CO2 humidified air, cells were processed for immunofluorescence. Briefly, cells were fixed with 4% formaldehyde for 13 minutes in phosphate-buffered saline (PBS) and washed two times again with PBS at room temperature. Cells were next permeabilized with 0.2% of Triton X-100 detergent during 10 minutes, washed again with PBS and permeabilized with 0.1% sodium citrate plus 0.1% of Triton X-100 for 5 minutes, the remaining buffer was removed and a final wash was performed. Cells were blocked with 50 μM of PBS-Tween-20 at 0.05% plus 3% of bovine serum albumin during 60 minutes at room temperature. Immunostaining was performed by incubation with anti-TRF1 primary antibody overnight at 4° C. After that, samples were washed 3 times with 0.05% PBS-Tween-20, and secondary antibody anti-rat-555 was dispensed at 1:500 in PBS-Tween plus 3% BSA during 60 minutes. Then, samples were washed 3 times with 0.05% PBS-Tween-20 plus another wash with PBS. Finally, an incubation with Hoechst staining 1 μg/mL to generate masks matching the cell nuclei was performed. SPSS software was used for statistical analysis. Image acquisition was performed on an Opera™ LX spinning disk confocal automated platform (PerkinElmer). Forty images were acquired with a 40× magnification lens under non-saturating conditions and an average of 1×103 cells were analyzed from each well. Quantification of fluorescence at single cell level was done with the Acapella™ platform using built-in analysis algorithms for the identification of foci and nuclei area and intensity.
Cell viability for each condition was also evaluated by comparing the number of nuclei with regards to controls.
Each condition was performed at least in duplicate (n=2) in three independent experiments (N=3). For all data mean TRF1 intensity values of the different experiments were calculated.
Obtained results appear summarized in
For this example, peptide SEQ ID NO: 2-NH2 was prepared in accordance with example 1.
A stock solution of the peptide prepared in DMSO at 50 mg/mL. This stock was further diluted to obtain the final working concentrations of 0.005 mg/mL, 0.01 mg/mL and 0.05 mg/mL of pure active.
HEKa cells were seeded on coverslips in 24-well plates containing Dermal Cell Basal Medium. Cultures were then kept in the 37° C. incubator to attach and expand. When cell confluence was around 50%, media was replaced by media comprising the peptide at different working concentrations tested (5, 10 or 50 μg/mL of peptide SEQ ID NO: 2-NH2) during 24 h. Afterwards, media was replaced by a new media with the compound (for the wells in the basal condition experiment) or media with 100 μM H2O2 and the compound (for the wells in the oxidative stress condition), for another 24 h.
After treatment, cells were harvested, washed once with PBS, and treated for 30 seconds with Triton X-100 buffer for nuclear extraction and then washed again in PBS, fixed for 10 min in 4% buffered formaldehyde, washed in PBS and kept at 4° C. until processed.
For immunofluorescence (IF), cells were permeabilized with 0.2% PBS-Triton for 10 min, washed and blocked with fetal bovine serum for one hour and 5% BSA in PBS for another 1 h in order to avoid nonspecific antibody binding.
For TRF1 and 53BP1 staining, samples were incubated overnight at 4° C. with the corresponding primary antibody. After proper washing with PBS, cells were incubated with 488-Alexa secondary for 1 h at RT in a humid chamber. Samples in cover slides were mounted in Prolong Gold with DAPI which strongly binds DNA (nuclei staining), and samples were kept protected from light at 4° C. until microscopic images were acquired. Fluorescent signals on slides were visualized in a confocal ultra-spectral microscope SP5-MP (Leica). Microscopic images were acquired using 40× objective. Immunofluorescence was performed on three replicates for each condition and images of at least four different fields from each coverslip were acquired using the same settings.
Signal was quantified using the Definiens Developer XD.2 Software. Image analysis was performed blindly. Blue channel (DAPI staining) was used to count the number of nuclei per image. Red and green colour were used to determine TRF1 and 53BP1 intensity, respectively. Then, number of TRF1 or 53BP1 foci was divided by the number of nuclei in order to obtain the total number of TRF1 or 53BP1 foci/nucleus of every image. Finally, all data was normalized with regard to the negative control: basal sample for TRF1 and sample treated with H2O2 for 53BP1, to obtain the % of TRF1 or 53BP1 foci per nucleus.
The results obtained for TRF1 appear summarized in
The results obtained for 53BP1 appear summarized in
| Number | Date | Country | Kind |
|---|---|---|---|
| 22382267.7 | Mar 2022 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/057138 | 3/21/2023 | WO |
