Amino acid composition for hair care

Abstract

Amino acid composition for hair care The present invention provides an oral composition for hair care comprising L-arginine, L-alanine, L-glutamine, L-glycine, L-glutamic acid, and L-lysine, wherein the weight ratio of L-glycine / L-glutamic acid is from 4.0:1 to 0.05:1; the weight ratio of L-glycine / L-glutamine is from 1.4:1 to 0.1:1; the weight ratio of L-glycine / L-alanine is from 2.6:1 to 0.05:1; the weight ratio of L-glycine / L-arginine is from 4.2:1 to 0.05:1; and the weight ratio of L- glycine / L-lysine is from 5.6:1 to 1.1:1. The invention also provides a cosmetic use of the composition for improving hair growth, brightness of hair, hydration, elasticity, firmness, hair shine and / or prevention of hair loss, wherein the use comprises administering an effective amount of the composition for the intended use.

Description

[0001] Amino acid composition for hair care

[0002] This application claims the benefit of European Patent Application EP24383244.1 filed on 15 November 2024.

[0003] Technical Field

[0004] The present invention belongs to the field of compositions for hair care. In particular, the invention relates to amino acid compositions and the use of the compositions for improving hair growth, brightness of hair, hydration, elasticity, firmness, hair shine and / or prevention of hair loss.

[0005] Background Art

[0006] Collagen is a structural protein of hair which provides elasticity to the skin and scalp, strengthens the hair follicle, accelerates healing, and nourishes the hair bulb.

[0007] Consequently, this protein improves hair growth, prevents loss of hair density through different modes of action. In humans, at least 28 different types of collagens have been identified.

[0008] In addition to collagen, keratin is an essential protein for hair health, as this fibrous protein is primarily responsible for hair structure and strength. Maintaining adequate levels of keratin in the body is essential to promote hair growth, prevent hair loss and keep hair in optimal condition. There are 54 functional keratin genes in the human genome, which are classified into three major groups, i.e. , epithelial keratins, hair follicle cell-specific epithelial keratins and hair keratins.

[0009] In recent years, it has become increasingly accepted that keratin and collagen-based products have beneficial effects on hair care. These products, such as collagen and keratin supplements, are extensively used among people already showing early signs of hair loss such as: gradual loss on the top of the head, circular or irregular bald patches or sudden loosening. These products help in maintaining the structure and strength of the hair, thus contributing to reinforcing it structurally, promoting hair growth and / or preventing hair loss. These products, which are mainly based on hydrolyzed collagen or keratin peptides, have gained special attention in the field of hair care.

[0010] WO2012065782A2 discloses a collagen hydrolysate used to improve the health of human skin, hair and / or nails, wherein least 90 wt. % of said collagen hydrolysate has a molecular weight of less than 3500 Da, and the collagen hydrolysate has at least four characteristic peptides with a molecular weight of between 600 and 1200 Da.

[0011] WO2021224310A1 discloses a keratin hydrolysate comprising at least 88%, preferably at least 90%, by weight of free amino acids relative to the total weight of the amino acids of the hydrolysate and comprising a content of free tyrosine between 2% and 4% by weight relative to the total weight of the free amino acids of the hydrolysate.

[0012] These peptide compositions are the result of enzymatic digestion of collagen and keratin. However, the actual composition in these hydrolysates is not clearly defined and batch-to-batch variability is subject to the action of the enzymes. Therefore, in view of all the above there is a clear need for amino acid-based compositions precisely designed for targeting and promoting the formation of its main structural elements, thus resulting in strengthening of the hair structure, hair growth and / or prevention of hair loss.

[0013] Summary of Invention

[0014] The inventors have developed new amino acid compositions comprising free amino acids in specific ratios, which have been shown to have beneficial effects on hair care. The compositions according to the invention not only promote hair reinforcement, growth and prevention of hair loss but also the brightness of hair, hydration, elasticity, firmness and hair shine.

[0015] Contrary to the products known in the art, and thanks to the specific combination of free amino acids and the specific ratios between them, the amino acid-based compositions of the present invention target the formation of the most important structural proteins in the human hair, such as collagens and keratins, and are precisely designed for reinforcing the structure and strength of the hair and / or prevention of hair loss. Furthermore, they are easier to absorb and be used by the body. Unlike protein hydrolysates, the composition according to the invention comprises free amino acids in a defined molar ratio, enabling industrial reproducibility, traceability, and modification of the amino acid content within the claimed ranges.

[0016] With the aim of promoting the formation of collagen and keratin, and on the basis of the aminogram, i.e., the amino acid profile, of the most relevant structural proteins in the human hair and scalp, and through extensive experimentation, the present inventors surprisingly found out amino acid compositions particularly beneficial for hair care. The ratios of amino acids of two of the more relevant collagens involved in the formation of hair, collagen IV and XVII, were selectively modified for obtaining compositions according to the invention. Surprisingly, the testing of these compositions in volunteers having Female Pattern Hair Loss (FPHL) led to an improvement in hair density and hair volume, brightness of the hair, general appearance and hair quality, patient satisfaction with the treatment and the time to positive response and reduction of hair loss. Likewise, the ratios of amino acids in respect to other three proteins, keratin types 35 and 85, and trichohyalin, a protein that participates in keratin intermediate filaments inter-filamentous cross-linking, were selectively modified in the compositions according to the invention. Advantageously, as it is illustrated in the examples, an improvement in hair growth, hair density and hair volume, brightness of the hair, general appearance and hair quality, patient satisfaction with the treatment and the time to positive response and reduction of hair loss in volunteers administered with these compositions was obtained.

[0017] Therefore, in a first aspect, the invention provides composition for hair care comprising L-arginine, L-alanine, L-glutamine, L-glycine, L-glutamic acid, and L-lysine, wherein the weight ratio of L-glycine / L-glutamic acid is from 4.0:1 to 0.05:1; the weight ratio of L-glycine / L-glutamine is from 1.4:1 to 0.1:1; the weight ratio of L-glycine / L-alanine is from 2.6:1 to 0.05:1; the weight ratio of L-glycine / L-arginine is from 4.2:1 to 0.05:1; and the weight ratio of L-glycine / L-lysine is from 5.6:1 to 1.1:1.

[0018] It has been shown that the compositions according to the invention are able to promote rapid and sustained hair growth by stimulating HGF and the Wnt / p-catenin pathway while reducing its inhibitor SFRP1, particularly in Dermal Papilla (DP) fibroblasts. This mechanism supports the activation of key molecular signals responsible for initiating and maintaining the hair growth phase.

[0019] The inventors have also found that the use of these compositions contribute to improved follicular morphogenesis, since they increase the inductive activity of Dermal Papilla (DP) fibroblasts through the upregulation of alkaline phosphatase (ALPL) and versican (VCAN).

[0020] In addition, the compositions strengthen hair follicles by stimulating keratin 6A (KRT6A) and collagen 17A1 expression in Outer Root Sheath (ORS) keratinocytes (KCs). This response supports the maintenance of the stem cell niche and reinforces the structural integrity of the follicle.

[0021] Furthermore, the compositions also enhance mitochondrial activity (MTCO1), providing increased cellular energy availability for hair shaft production. This boost in metabolic efficiency supports more robust and continuous hair formation, helping sustain visible hair growth over time.

[0022] In the longer term, the inventors have found that the formulations favor prolonged follicle survival and reduced regression, as they stimulate IGF-1 while inhibiting TGF-P2. By promoting growth-supporting signals and counteracting inhibitory factors, this mechanism contributes to more durable hair.

[0023] In a second aspect, the invention provides a cosmetic use of the composition according to the first aspect, for improving brightness of hair, hydration, elasticity, firmness, hair shine, hair growth and / or prevention of hair loss, wherein the use comprises administering an effective amount of the composition for the intended use.

[0024] In view of the above, the amino acid compositions described herein provide a great advance in the field of hair care, in particular in hair strengthening, hair growth and prevention of hair loss.

[0025] Brief description of the figures

[0026] FIG. 1 shows the gene expression of SFRP1 , HGF, ALPL, VCAN after 6 hours treatment of DP fibroblasts spheroids with the composition of Examples 1 and 2, at two different concentrations according to the protocol described in Example 9. The fold change in mRNA expression relative to GAPDH was truncated to improve visualization of the data.

[0027] FIG. 2 shows the gene expression of SFRP1, HGF, ALPL, VCAN and VEGFA after 72 hours treatment of DP fibroblasts spheroids with the composition of Examples 1 and 2, at two different concentrations according to the protocol described in Example 9. The fold change in mRNA expression relative to GAPDH was truncated to improve visualization of the data.

[0028] FIG. 3 shows the protein SFRP1 expression after treatment of DP fibroblasts spheroids with the composition of Examples 1 and 2, at two different concentrations according to the protocol described in Example 9.

[0029] FIG. 4 shows the gene expression of TGF 2, KRT6A, LEF1, AXIN2, MTCO1, COL17A1 after 6 hours treatment of ORS KCs with the composition of Examples 1 and 2, at two different concentrations according to the protocol described in Example 10.

[0030] FIG. 5 shows the protein SFRP1 expression after treatment of ORS KCs with the composition of Examples 1 and 2, at two different concentrations according to the protocol described in Example 10.

[0031] FIG. 6 shows the protein TGFP2 expression after treatment of ORS KCs with the composition of Examples 1 and 2, at two different concentrations according to the protocol described in Example 10.

[0032] Detailed description of the invention

[0033] All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly through-out the specification and claims unless an otherwise expressly set out definition provides a broader definition.

[0034] For the purposes of the invention, any ranges given include both the lower and the upper endpoints of the range. Ranges given, such as temperatures, quantities, times, sizes, and the like, should be considered approximate, unless specifically stated.

[0035] The term “free amino acid” refers to amino acids which are not covalently linked to other amino acids or molecules (e.g., not forming peptides or proteins). In other words, it refers to the amino acid in free form.

[0036] The term "effective amount” when used for cosmetic purposes, refers to the amount of a compound that, when administered, is sufficient to provide cosmetic benefits such as improves brightness, strength, hydration, elasticity, firmness, hair shine, hair growth and / or prevention of hair loss. 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.

[0037] The term “weight ratio” refers to the relation of weights of the molecules or compounds indicated. For instance, a weight ratio (weight / weight) of 3.50 between L-glycine and glutamic acid refers to 3.50 units of L-glycine per 1 unit of glutamic acid. As used herein, "% by weight" or “% w / w” of a component refers to the amount of the single component relative to the total weight of the composition or, if specifically mentioned, of other component. The term “non-proteinogenic amino acid” refers to amino acids which are naturally nonencoded in the genome of organisms for the assembly of proteins. Examples of non-proteinogenic amino acids are citrulline, taurine, ornithine, gamma-aminobutyric acid, carnosine and creatine.

[0038] The term “nucleoside” refers to molecules consisting of a nitrogenous base (purine or pyrimidine) and a five-carbon sugar (ribose or 2'-deoxyribose).

[0039] The term “solvents” refers to a liquid in which a solute is dissolved or suspended to form a solution or suspension, respectively.

[0040] The term “emulsifier” refers to a surface-active agent promoting the formation and stabilization of an emulsion.

[0041] The term “viscosifier” refers to a substance that when added to the composition increases its viscosity.

[0042] The term “sweetener” refers to a substance to sweeten the composition.

[0043] The term “flavouring” refers to a substance that when added to the composition gives it a particular taste.

[0044] The term “fragrance” refers to a substance that when added to the composition gives it a particular odour or smell.

[0045] The term “for human consumption” refers to an edible substance that has been prepared and processed correctly for people to safely consume.

[0046] The term “cellulose derivative” refers to cellulose whose reactive hydroxyl groups have been chemically modified by reactions such esterification or etherification.

[0047] The term “Female Pattern Hair Loss” (FPHL) refers to a pattern of hair loss (alopecia) occurring in women caused by hormones, aging and genetics.

[0048] The term “orally acceptable excipients” refers to excipients that are suitable for oral human consumption. The term “topically acceptable excipients” refers to excipients that are suitable for application onto the skin or scalp of a user and which no cause irritation or toxicity.

[0049] SFRP1 (Secreted Frizzled-Related Protein 1) refers to a Wnt pathway inhibitor; its reduction activates Wnt / p-catenin signalling and promotes hair follicle growth.

[0050] HGF (Hepatocyte Growth Factor) refers to a growth factor that stimulates hair follicle growth partly by decreasing SFRP1 and enhancing Wnt activity.

[0051] LEF1 (Lymphoid Enhancer-Binding Factor 1) refers to a transcription factor mediating Wnt / p-catenin signalling; elevated expression indicates active follicle regeneration.

[0052] AXIN2 refers to transcriptional target of Wnt signalling used as a molecular marker of pathway activation.

[0053] ALPL (Alkaline Phosphatase) refers to an enzyme marker of dermal papilla inductive capacity and hair growth potential.

[0054] VCAN (Versican) refers to an extracellular matrix proteoglycan in the dermal papilla linked to hair-inductive and regenerative functions.

[0055] VEGFA (Vascular Endothelial Growth Factor A) refers to a key angiogenic factor promoting perifollicular blood vessel formation and follicle nourishment.

[0056] TGF-P2 (Transforming Growth Factor Beta 2) refers to a cytokine that induces catagen (regression phase); its inhibition prolongs anagen (growth phase).

[0057] MT-CO1 (Mitochondrially Encoded Cytochrome c Oxidase Subunit I) refers to a mitochondrial enzyme indicating oxidative phosphorylation efficiency and energy production in follicular cells.

[0058] COL17A1 (Collagen Type XVII Alpha 1) refers to a structural protein maintaining the hair follicle stem cell niche; its presence supports stem cell adhesion and regenerative capacity.

[0059] KRT6A refers to keratin 6A, a structural keratin that supports epithelial activation and is positively associated with hair follicle growth and regeneration.

[0060] As mentioned above, the first aspect of the invention provides composition for hair care comprising L-arginine, L-alanine, L-glutamine, L-glycine, L-glutamic acid, and L-lysine, wherein the weight ratio of L-glycine / L-glutamic acid is from 4.0:1 to 0.05:1; the weight ratio of L-glycine / L-glutamine is from 1.4:1 to 0.1:1; the weight ratio of L-glycine / L-alanine is from 2.6:1 to 0.05:1; the weight ratio of L-glycine / L-arginine is from 4.2:1 to 0.05:1; and the weight ratio of L-glycine / L-lysine is from 5.6:1 to 1.1:1. This composition has been shown to improve hair growth, hair density and hair volume, brightness of the hair, general appearance and hair quality and thus, since collagens and keratins are two of the most important types of proteins for hair care, this composition is considered suitable for promoting keratin or collagen formation.

[0061] In a particular embodiment, the weight ratio of L-glycine / L-glutamic acid is from 3.8:1 to 3.0:1, from 3.5:1 to 2.8:1, from 3.5:1 to 2.5:1, from 3.5:1 to 2.0:1, from 3.3:1 to 2.0:1, from 3.0:1 to 1.5:1, from 3.3:1 to 0.2:1, from 3.0:1 to 0.3:1, from 2.8:1 to 0.4:1, from 2.5:1 to 0.5:1, from 2.2:1 to 0.6:1, from 2.0:1 to 0.7:1, from 1.7:1 to 0.8:1, from 1.4:1 to 0.9:1, 1.1:1 to 0.9:1, from 0.9:1 to 0.7:1, from 0.7:1 to 0.5:1, from 0.5:1 to 0.3:1 or from 0.3:1 to 0.1:1. In a particular embodiment, the composition comprises from 5 to 15% w / w of L-glutamic acid of the total weight of the composition, preferably from 8 to 12 % w / w of the total weight of the composition.

[0062] In a particular embodiment, the weight ratio of L-glycine / L-glutamine is from 1.4:1 to 0.3:1, from 1.4:1 to 1.0:1, from 1.2:1 to 0.3:1, from 1.1:1 to 0.3:1, from 1.0:1 to 0.5:1, from 1.0:1 to 0.6:1, from 1.0:1 to 0.7:1, from 1.0:1 to 0.8:1, from 0.5:1 to 0.2:1 or from 0.5:1 to 0.3:1. In particular embodiment, the composition comprises from 5 to 18 % w / w of L-glutamine of the total weight of the composition, preferably from 10 to 14 % w / w of the total weight of the composition.

[0063] In a particular embodiment, the weight ratio L-glycine / L-alanine is from 2.5:1 to 0.1:1, from 2.3:1 to 0.3:1, from 2.3:1 to 0.85:1, from 0.85:1 to 0.13:1, from 0.9:1 to 0.7:1, from 2.1:1 to 0.4:1, from 1.9:1 to 0.5:1, from 1.7:1 to 0.6:1, from 1.5:1 to 0.7:1, from 1.2:1 to 0.7:1, from 0.9:1 to 0.7:1, from 2.5:1 to 2.0:1, from 2.4:1 to 2.2:1 or from 0.2:1 to 0.1:1. In particular embodiment, the composition comprises from 2 to 20 % w / w of L-alanine of the total weight of the composition, preferably from 10 to 14 % w / w of the total weight of the composition.

[0064] In particular embodiment, the weight ratio of L-glycine / L-arginine is from 4.2:1 to 0.05:1, from 3.9:1 to 0.1:1, from 3.9:1 to 0.3:1, from 3.7:1 to 0.5:1, from 3.5:1 to 0.7:1, from 2.9:1 to 0.08:1, from 0.63:1 to 0.08:1, from 2.9:1 to 0.63:1, from 3.3:1 to 0.9:1, from 3.0:1 to 1.2:1, from 2.8:1 to 1.5:1, from 2.6:1 to 1.7:1, from 2.4:1 to 1.5:1, from 2.2:1 to 1.8:1, from 4.0:1 to 2.6:1, from 0.2:1 to 0.05:1 or from 0.63:1 to 0.5:1. In particular embodiment, the composition comprises from 2 to 30% w / w of L-arginine of the total weight of the composition, preferably from 14 to 18 % w / w of the total weight of the composition. It has been found by the inventors that fine tuning the weight ratio of L-glycine / L-arginine is important for obtaining the effects associated with the composition according to the invention. These effects are specially obtained when the weight ratio of L-glycine / L-arginine is from 0.63:1 to 0.5:1.

[0065] In a particular embodiment, the weight ratio L-glycine / L-lysine is from 5.5:1 to 1.1:1, from 5.5:1 to 1.3:1, from 5.3:1 to 1.1:1, from 5.3:1 to 1.3:1, from 5.2:1 to 1.7:1, from 4.9:1 to 2.0:1, from 4.7:1 to 2.3:1, from 4.5:1 to 2.6:1, from 4.2:1 to 2.9:1, from 4.1:1 to 3.2:1. In a particular embodiment, the composition comprises from 1 to 5 % w / w of L-lysine of the total weight of the composition, preferably from 2 to 4 % w / w of the total weight of the composition. The presence of lysine in the composition in combination with the other amino acids has particularly been found to promote hair density, brightness and reduce hair loss. Additionally, the inventors have found that is highly relevant that the weight ratio L-glycine / L-lysine is higher than 1:1.

[0066] In a particular embodiment, the weight ratio of L-glycine / L-glutamic acid is from 4.0:1 to 0.05:1; the weight ratio of L-glycine / L-glutamine is from 1.4:1 to 0.1:1; the weight ratio of L-glycine / L-alanine is from 2.6:1 to 0.05:1; the weight ratio of L-glycine / L-arginine is from 0.63:1 to 0.5:1; and the weight ratio of L-glycine / L-lysine is from 4.1:1 to 3.2:1. This composition has been shown to specially improve hair density, brightness and reduce hair loss.

[0067] In a particular embodiment, the composition further comprises L-leucine, and L-serine. The inventors found that the presence of these two amino acids improved the hair growing properties of the composition. In a particular embodiment, the weight ratio of L-glycine / L-leucine is from 2.5:1 to 0.2:1 and the weight ratio of L-glycine / L-serine is from 2.9:1 to 0.3:1. In another particular embodiment, the weight ratio of L-glycine / L-leucine is from 1.1:1 to 0.7:1 and the weight ratio of L-glycine / L-serine is from 2.0:1 to 1.6:1.

[0068] In a particular embodiment, the weight ratio of L-glycine / L-leucine is from 2.3:1 to 0.3:1, from 2.2:1 to 0.4:1, from 2.1:1 to 0.5:1 from 2.0:1 to 0.6:1, from 1.9:1 to 0.8:1, from 1.8:1 to 1.0:1 or from 1.7:1 to 1.2:1. In a particular embodiment, the composition comprises from 5 to 20 % w / w of L-leucine of the total weight of the composition, preferably from 8 to 12 % w / w of the total weight of the composition. In particular embodiment, the weight ratio of L-glycine / L-serine is from 2.9:1 to 0.4:1, from 2.8:1 to 0.5:1, from 2.6:1 to 0.7:1, from 2.4:1 to 0.9:1, from 2.4:1 to 0.9:1, from 2.2:1 to 1.1:1, from 2.0:1 to 1.3:1 or from 1.9:1 to 1.5:1. In a particular embodiment, the composition comprises from 3 to 10 % w / w of L-serine of the total weight of the composition, preferably from 5 to 8 % w / w of the total weight of the composition.

[0069] In a particular embodiment, the composition further comprises L-proline. In a particular embodiment, the weight ratio of L-glycine / L-proline is from 1.3:1 to 0.7:1. In a particular embodiment, the weight ratio of L-glycine / L-proline is from 1.1:1 to 0.9:1. In another particular embodiment, the composition is free from proline. In a particular embodiment, the composition comprises from 5 to 20 % w / w of L-proline of the total weight of the composition, preferably from 8 to 12 % w / w of the total weight of the composition.

[0070] In a particular embodiment, the composition comprises L-arginine, L-alanine, L-glutamine, L-glycine, L-glutamic acid, L-lysine, L-leucine, L-serine, and L-proline, wherein the weight ratio of L-glycine / L-glutamic acid is from 1.1:1 to 0.9:1; the weight ratio of L-glycine / L-glutamine is from 1.0:1 to 0.8: 1 ; the weight ratio of L-glycine / L-alanine is from 0.9: 1 to 0.7:1; the weight ratio of L-glycine / L-arginine is from 0.63:1 to 0.5:1; the weight ratio of L-glycine / L-lysine is from 4.1:1 to 3.5: 1 ; the weight ratio of L-glycine / L-leucine is from 1.1:1 to 0.8:1; the weight ratio of L-glycine / L-serine is from 1.9:1 to 1.5:1; and the weight ratio of L-glycine / L-proline is from 1.1:1 to 0.9:1.

[0071] In a particular embodiment, the composition further comprises one or more non-proteinogenic amino acids. In another embodiment the non-proteinogenic amino acid is selected from the group consisting of L-ornithine, L-carnitine, taurine, and mixtures thereof. In vitro studies using the inventive compositions containing one or more non-proteinogenic amino acids demonstrated a positive and selective enhancement in the expression of markers associated with hair growth.

[0072] In a particular embodiment, the weight ratio of L-glycine / non-proteinogenic amino acid is from 15:1 to 0.2:1. In a particular embodiment, the weight ratio of L-glycine / non-proteinogenic amino acid is from 10:1 to 0.2:1. In another embodiment, the weight ratio of L-glycine / non-proteinogenic amino acid is from 13:1 to 1.5:1, from 11:1 to 2:1, from 9:1 to 2.6:1, from 7:1 to 2.8:1, from 3.35:1 to 0.25:1 or from 5:1 to 0.25:1. In an embodiment, the composition comprises from 1 to 15% w / w of the non-proteinogenic amino acid of the total weight of the composition, preferably from 5 to 9 % w / w of the total weight of the composition. In a particular embodiment, the composition further comprises one or more nucleosides. In another particular embodiment the nucleoside is selected from the group consisting of adenosine, cytidine, guanosine, 5-methyluridine, uridine, and mixtures thereof.

[0073] In a particular embodiment, the weight ratio of L-glycine / nucleoside is from 250:1 to 5:1. In another particular embodiment, the weight ratio of L-glycine / nucleoside is from 240:1 to 9:1, from 161:1 to 9:1, from 85:1 to 9:1, from 47:1 to 9:1, from 75:1 to 35:1, from 70:1 to 40:1, from 65:1 to 35:1 or from 60:1 to 40:1. In a particular embodiment, the composition comprises from 0.1 to 1 % w / w of the nucleoside of the total weight of the composition, preferably from 0.1 to 0.5 % w / w of the total weight of the composition.

[0074] In a particular embodiment, the weight ratio of L-glycine / L-glutamic acid is from 1.1:1 to 0.9:1; the weight ratio of L-glycine / L-glutamine is from 1.0:1 to 0.8:1; the weight ratio of L-glycine / L-alanine is from 0.9:1 to 0.6:1; the weight ratio of L-glycine / L-arginine is from 0.63:1 to 0.5:1; and the weight ratio of L-glycine / L-lysine is from 4.1:1 to 3.2:1.

[0075] In a particular embodiment, the weight ratio of L-glycine / L-glutamic acid is from 4.0:1 to 3.0:1; the weight ratio of L-glycine / L-glutamine is from 1.4:1 to 1.0:1; the weight ratio of L-glycine / L-alanine is from 2.6:1 to 2.0:1; the weight ratio of L-glycine / L-arginine is from 4.2: 1 to 2.6: 1 ; and the weight ratio of L-glycine / L-lysine is from 5.6: 1 to 5.0: 1.

[0076] In a particular embodiment, the weight ratio of L-glycine / L-glutamic acid is from 0.4 to 0.05; the weight ratio of L-glycine / L-glutamine is from 0.5 to 0.1; the weight ratio of L-glycine / L-alanine is from 0.2 to 0.05; the weight ratio of L-glycine / L-arginine is from 0.2 to 0.05; and the weight ratio of L-glycine / L-lysine is from 1.3 to 1.1.

[0077] In a particular embodiment, the weight ratio of L-glycine / L-glutamic acid is from 4.0:1 to 0.05:1; the weight ratio of L-glycine / L-glutamine is from 1.4:1 to 0.1:1; the weight ratio of L-glycine / L-alanine is from 2.6:1 to 0.05:1; the weight ratio of L-glycine / L-arginine is from 0.63:1 to 0.5:1; and the weight ratio of L-glycine / L-lysine is from 4.1:1 to 3.2:1.

[0078] In a particular embodiment, the composition is free from cysteine.

[0079] The composition according to the first aspect of the invention can be administered orally or topically. However, the inventors surprisingly found that the hair appearance in users in whom the composition was administered orally works particularly well, observing that hair loss was even less than in patients where the composition was administered topically. Therefore, in an embodiment, optionally in combination with any of the embodiments provided above or below, the composition is an oral composition.

[0080] In a particular embodiment, the composition is a liquid, solid or semisolid dosage form. In a particular embodiment the dosage form is selected from the group consisting of pill, tablet, capsule, powder, solution, suspension, emulsion, syrup, gel, hydrogel, cream, lotion, serum or shampoo. In a particular embodiment, the composition is liquid. In a particular embodiment, the composition is a liquid suspension.

[0081] In a particular embodiment, the composition further comprises one or more orally acceptable excipients.

[0082] In a particular embodiment, the composition further comprises one or more solvents, viscosifiers, emulsifiers, sweeteners, flavourings or fragrances.

[0083] In a particular embodiment comprises one or more solvents. In a particular embodiment comprises one or more solvents selected from the group consisting of water, ethanol, propylene glycol, glycerine, polyethylene glycol (PEG), castor oil derivatives, oils, and dimethyl sulfoxide (DMSO). In a particular embodiment, the composition comprises water. In another particular embodiment, the composition comprises from 40 to 80 % w / w of water of the total weight of the composition.

[0084] In a particular embodiment, the composition comprises one or more emulsifiers. In a particular embodiment, the composition comprises one or more emulsifiers selected from the group consisting of polysorbates, polyoxylglycerides derivatives, polyoxyethylene, and isopropyl miristate. In another particular embodiment, the composition comprises from 0.1 to 1 % w / w of the one or more emulsifiers of the total weight of the composition.

[0085] In a particular embodiment, the composition comprises one or more viscosifiers. In a particular embodiment, the composition comprises one or more viscosifiers selected from the group consisting of cellulose derivatives, xanthan gum, alginic acids, polysaccharides, and starch. In a particular embodiment, the composition comprises xanthan gum and / or a cellulose derivative. In another particular embodiment, the composition comprises from 0.2 to 2 % w / w of xanthan gum and / or a cellulose derivative of the total weight of the composition.

[0086] In a particular embodiment, the composition comprises one or more sweeteners. In a particular embodiment, the composition comprises one or more sweeteners selected from the group consisting of saccharin, sucralose, fructose, acesulfame k, aspartame, erythritol, sorbitol, mannitol, xylitol, and steviol glycosides. In a particular embodiment, the composition comprises fructose. In another particular embodiment, the composition comprises from 5 to 20 % w / w of the one or more sweeteners of the total weight of the composition.

[0087] In a particular embodiment, the composition comprises one or more flavourings. In a particular embodiment, the composition comprises one or more flavourings selected from the group consisting of mango, chocolate, pineapple, methyl salicylate, lemon oil, orange oil, peppermint oil, glycyrrhiza syrup, maple, butterscotch, peach, Wintergreen, apricot, cinnamon, raspberry, and vanillin. In another embodiment, the composition comprises from 0.01 to 0.05 % w / w of the one or more flavourings of the total weight of the composition.

[0088] In an embodiment, the one or more binders, lubricants, glidants, solvents, viscosifers, emulsifiers, sweeteners, flavourings or fragrances are suitable for human consumption.

[0089] In a particular embodiment, the composition is a topical composition, particularly a cosmetic topical composition. In a particular embodiment, the composition further comprises one or more permeation enhancers. Examples of permeation enhancers include isopropyl myristate, polysorbate 80, limonene, phosphatidylcholine, propylene glycol, ethanol, lauric acid, dimethyl sulfoxide, sodium cholate, and hydroxypropyl-p-cyclodextrin, or mixtures thereof.

[0090] The composition of the invention may be a cosmetic oral composition comprising an effective amount of the composition for hair care as described above, together with one or more orally acceptable excipients.

[0091] In a particular embodiment, the cosmetic oral composition comprising the composition for hair care as described above, further comprises one or more orally acceptable excipients selected from the group consisting of solvents, binders, lubricants, glidants, viscosifers, emulsifiers, sweeteners or flavourings.

[0092] A cosmetic topical composition comprising the composition as described above, further comprising one or more topically acceptable excipients selected from the group consisting of solvents, binders, lubricants, glidants, viscosifers, emulsifiers or fragrances is also part of the invention. The sum of the % w / w of each of the components in the amino acid compositions according to the invention must not add-up to more than 100%. Therefore, when different particular embodiments are combined, the sum of the % w / w of the components indicated in the present application will be 100% or less of the total weight of the composition.

[0093] In a second aspect, the invention relates to the cosmetic use of the composition according the first aspect, for improving hair growth, brightness of hair, strength, hydration, elasticity, firmness, hair shine and / or prevention of hair loss, wherein the use comprises administering an effective amount of the composition for the intended use.

[0094] This aspect can also be reformulated as a cosmetic method of improving hair growth, brightness of hair, hydration, elasticity, firmness, hair shine and / or prevention of hair loss by use of the composition according to the first aspect of the invention, wherein the use comprises administering an effective amount of the composition for the intended use.

[0095] In a particular embodiment, the cosmetic use comprises administering orally or topically an effective amount of the composition for the intended use. As it has been discussed above, the composition being administered orally improves hair growth, the brightness of hair, strength, hydration, elasticity, firmness, shine and / or prevention of hair loss.

[0096] Therefore, in a particular embodiment, the cosmetic use comprises administering orally an effective amount of the composition for the intended use.

[0097] In a particular embodiment, the effective amount of the composition is from 300 to 2000 mg. In another embodiment, the effective amount of the composition is from 350 to 1800 mg, from 500 to 1400 mg, from 700 to 1250 mg or from 800 to 1200 mg. In a particular embodiment, the composition is administered two or three times a day.

[0098] In a particular embodiment, the composition is administered one or more times a day, particularly once a day. In a particular embodiment, the effective amount of the composition is from 300 to 2000 mg and is administered once a day.

[0099] 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.

[0100] 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 and drawings 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 embodiments described herein.

[0101] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0102] Clause 1. A composition for hair care comprising L-arginine, L-alanine, L-glutamine, L-glycine, L-glutamic acid, and L-lysine, wherein

[0103] - the weight ratio of L-glycine / L-glutamic acid is from 4.0:1 to 0.05:1;

[0104] - the weight ratio of L-glycine / L-glutamine is from 1.4:1 to 0.1:1;

[0105] - the weight ratio of L-glycine / L-alanine is from 2.6:1 to 0.05:1;

[0106] - the weight ratio of L-glycine / L-arginine is from 4.2:1 to 0.05:1; and

[0107] - the weight ratio of L-glycine / L-lysine is from 5.6:1 to 1.1:1.

[0108] Clause 2. The composition according to clause 1, further comprising L-leucine, and L-serine.

[0109] Clause 3. The composition according to clause 2, wherein the weight ratio of L-glycine / L-leucine is from 2.5:1 to 0.1:1 and the weight ratio of L-glycine / L-serine is from 2.9:1 to 0.3:1.

[0110] Clause 4. The composition according to any of the clauses 1-3, further comprising L-proline.

[0111] Clause 5. The composition according to clause 4, wherein the weight ratio of L-glycine / L-proline is from 3.1:1 to 0.7:1.

[0112] Clause 6. The composition according to any of the clauses 1-5, further comprising a non-proteinogenic amino acid selected from the group consisting of L-ornithine, L-carnitine, taurine, and mixtures thereof.

[0113] Clause 7. The composition according to clause 6, wherein the weight ratio of L-glycine / non-proteinogenic amino acid is from 10:1 to 0.2:1.

[0114] Clause 8. The composition according to any of the clauses 1-7, further comprising a nucleoside selected from the group consisting of adenosine, cytidine, guanosine, uridine, and mixtures thereof.

[0115] Clause 9. The composition according to clause 8, wherein the weight ratio of L-glycine / nucleoside is from 250:1 to 5:1. Clause 10. The composition according to any of the clauses 1-9, wherein

[0116] - the weight ratio of L-glycine / L-glutamic acid is from 1.1:1 to 0.9: 1 ;

[0117] - the weight ratio of L-glycine / L-glutamine is from 1.0:1 to 0.8:1;

[0118] - the weight ratio of L-glycine / L-alanine is from 0.9:1 to 0.6:1;

[0119] - the weight ratio of L-glycine / L-arginine is from 0.63:1 to 0.5:1; and

[0120] - the weight ratio of L-glycine / L-lysine is from 4.1:1 to 3.2:1.

[0121] Clause 11. The composition according to any of the clauses 1-9, wherein

[0122] - the weight ratio of L-glycine / L-glutamic acid is from 4.0:1 to 3.0:1;

[0123] - the weight ratio of L-glycine / L-glutamine is from 1.4:1 to 1.0:1;

[0124] - the weight ratio of L-glycine / L-alanine is from 2.6:1 to 2.0:1;

[0125] - the weight ratio of L-glycine / L-arginine is from 4.2:1 to 2.6:1; and

[0126] - the weight ratio of L-glycine / L-lysine is from 5.6:1 to 5.0:1.

[0127] Clause 12. The composition according to any of the clauses 1-9, wherein

[0128] - the weight ratio of L-glycine / L-glutamic acid is from 0.4 to 0.05;

[0129] - the weight ratio of L-glycine / L-glutamine is from 0.5 to 0.1 ;

[0130] - the weight ratio of L-glycine / L-alanine is from 0.2 to 0.05;

[0131] - the weight ratio of L-glycine / L-arginine is from 0.2 to 0.05; and

[0132] - the weight ratio of L-glycine / L-lysine is from 1.3 to 1.1.

[0133] Clause 13. A cosmetic oral or, alternatively, topical composition comprising an effective amount of the composition of any of the clauses 1-12, together with one or more either orally or topically acceptable excipients, respectively.

[0134] Clause 14. Cosmetic use of the composition according to any of the clauses 1-12, for improving hair growth, brightness of hair, hydration, elasticity, firmness, hair shine and / or prevention of hair loss, wherein the use comprises administering an effective amount of the composition for the intended use.

[0135] Clause 15. The cosmetic use of the composition according to clause 14, wherein the composition is administered orally.

[0136] Examples

[0137] Examples 1 to 7. Preparation of compositions The following compositions according to the first aspect of the invention were prepared by mixing the indicated substances (amino acids selected from the group consisting of L-arginine, L-glutamine, L-glycine, L-glutamic acid, L-lysine, L-leucine, L-serine, L-proline, L-alanine, non-proteinogenic amino acids and / or nucleosides):

[0138]

[0139]

[0140] Example 8. Evaluation of a product according to the present invention for hair treatment

[0141] Subjects:

[0142] 7 Volunteers (V1-V7).

[0143] Women and men aged 45-65.

[0144] Volunteers with Initial Female Pattern Hair Loss (FPHL).

[0145] Commitment not to use systemic, topical, or oral products with an effect similar to that of the product to be evaluated throughout the study period.

[0146] - Do not change the daily routine regarding the use of cosmetic products and eating habits.

[0147] Availability to comply with all study visits.

[0148] Product: Oral composition of examples 1-7, one for each of the volunteers.

[0149] Mode of Ingestion: Oral

[0150] Duration: 6 months.

[0151]

[0152]

[0153] Results of the treatment after 6 months

[0154] Visual inspection

[0155] In the visual inspection test, the response is classified qualitatively in five categories: 5-points Likert scale (1 = very little; 2 = little; 3 = neutral; 4 = much; and 5 very much). Comb test: results are shown as a percentage of reduction of hair loss.

[0156]

[0157] Evaluation of side effects

[0158] The patients have not reported any side effects or discomfort either during or after the use of the compositions throughout the entire clinical study.

[0159] IN VITRO DATA

[0160] Example 9. Dermal papilla (DP) fibroblast spheroids

[0161] DP Spheroids culture

[0162] Primary DP fibroblasts (from 2 male donors, 19-21y-o) were extracted and expanded in culture until passage 2 to obtain enough cells to perform DP spheroids using DMEM / F12 (1:1) medium (Gibco-ThermoFisher Scientific) supplemented with 1% penicillin / streptomycin (Gibco-ThermoFisher Scientific) and 10% fetal bovine serum (FBS; Gibco-ThermoFisher Scientific). 48 hours before passaging DP fibroblasts to perform spheroids, the medium was changed and replaced with DMEM / F12 (1:1) medium supplemented with 10% fetal bovine serum without penicillin / streptomycin, since the use of antibiotics prevents the formation of spheroids (Higgins et al., 2013). DP fibroblasts were trypsinized with 0.25% Trypsin / EDTA (Gibco-ThermoFisher Scientific), and cells were diluted to obtain a cell suspension of 300,000 cells / ml in DMEM / F12 (1:1) medium supplemented with 10% FBS. A 10 pL drop (containing 3,000 cells) of the cell suspension was deposited on the internal surface of the lid of the plastic petri dish. A total of 40 drops were deposited on each petri dish lid and left to hang for 48 hours. After incubation, the DP fibroblasts spheroids were formed (one spheroid per drop). Each spheroid was collected and added to a well of a V-shape 96-well plate coated with 20 mg / ml poly-HEMA (Sigma Aldrich) solution containing either vehicle (medium alone), 5pM 6AA Blend, 50pM 6AA Blend, 5pM 8AA Blend, or 50pM 8AA Blend in modified DMEM / F12 medium (Gibco-ThermoFisher Scientific). 6AA Blend corresponds to Example 1 and 8AA Blend corresponds to Example 2.

[0163] After 6 or 72hrs of culture, the DP spheroids were collected for quantitative immunohistomorphometry analyses (spheroids were collected and embedded in cryomatrix (Epredia), and snap frozen), or qRT-PCR. Furthermore, culture medium was collected for LDH analyses.

[0164] LDH assay

[0165] As an indication of HF cytotoxicity, lactate dehydrogenase activity (LDH) was quantified through a colorimetric assay after 6 and 72hrs of culture in the medium collected from each well of DP spheroid. The Cytotoxicity Detection kit Plus (Roche), which measures the conversion of tetrazolium salt to formazan, a water-soluble dye with a broad absorption maximum at approximately 490 nm, was used to evaluate the LDH levels in the different conditions at both time points.

[0166] qRT-PCR

[0167] Total RNA was isolated from DP fibroblast spheroids using the PicoPure™ RNA Isolation Kit (Applied Biosystems-Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. RNA purity and concentrations were determined using the Nanodrop ND-1000 assay (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription of the RNA into cDNA was performed using the SuperScript™ IV VILO™ Master Mix Kit (Applied Biosystems-Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. RNA concentrations were adjusted to a range of 50 to 500 nM to ensure the same amount of RNA was present among samples from the same donor, allowing for further quantification and comparison between the samples after quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Given the low expression level of some genes (e.g. LEF1 and AXIN2), a pre-amplification step was performed using the TaqMan™ PreAmp Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. Normalization was performed using the housekeeping gene GAPDH. qRT-PCR was run in duplicate using TaqMan Fast Advanced Master Mix and Gene Expression Assay probes (Id:

[0168] Hs02786624_g1 for GAPDH, Hs01029144_m1 forALPL, Hs00300159_m1 for HGF, Hs00610060_m1 forSFRPI, Hs00171642_m1 forVCAN, Hs00900055_m1 forVEGFA, Thermo Fisher Scientific, Waltham, MA, USA) on a QuantStudio 1 Real-Time-PCR-System (Thermo Fisher Scientific, Waltham, MA, USA). The QuantStudio™ Design & Analysis Software collected and stored real-time quantification plots and Ct values. The amount of the transcripts was normalized to the housekeeping gene (GAPDH) using the AACT method.

[0169] Immunofluorescence staining and quantitative immunohistomorphometry (qIHM)

[0170] OCT-embedded DP fibroblasts spheroids were sectioned at 6 pm thickness. Briefly, after fixation (see table below), cryosections were pre-incubated for 30 min at room temperature (RT) (see table below), followed by incubation with the primary antibody (see table below overnight at +40C. After three times 5 min-long washes, slides were incubated with the corresponding fluorescent-labelled secondary antibody for 45 min at RT. Finally, cryosections were counterstained with 4Z,6Z-diamidino-2-phenylindole dihydrochloride (DAPI). Negative controls were performed by demonstrating negative immunoreactivity by omitting primary antibodies.

[0171] For alkaline phosphatase activity, DP spheroids cryosections were fixed in acetone at -20°C for 10 mins. The sections were then washed twice for 5min each in TBS, followed by an incubation of 30 min at 37°C with Vector Blue AP Substrate (Vector labs), following the manufacturer’s instructions (a negative control solution was also made with Levamisole). After a wash of 5min in TBS and another in distilled water, the sections were dehydrated in isopropanol twice 5min and embedded in a non-aqueous mounting medium, VectaMount Express Mounting Medium (Vector labs).

[0172] Quantitative immunohistomorphometry (qIHM) data for each donor were normalized to the vehicle (set as 1) to stratify inter-individual variability between donors.

[0173] List of primary and secondary antibodies used for DP fibroblasts spheroids qIHM.

[0174]

[0175] Results

[0176] In DP fibroblasts spheroids, both blends tend to have a rapid effect on stimulating the expression of the key hair growth-promoting pathway, the Wnt / 3 -catenin pathway, by reducing the gene expression of the canonical Wnt / 3 -catenin signalling inhibitor, SFRP1 (FIG. 1 and 2). The 6AA blend at a 50 pM concentration is also increasing the gene expression of the known SFRP1 inhibitor and hair growth promoter, hepatocyte growth factor (HGF). This data indicates that the AA blends tend to stimulate hair growth by inhibiting the inhibitor of the canonical Wnt / 3 -catenin signalling. Furthermore, both blends tend to rapidly stimulate DP fibroblasts' inductivity by increasing the gene expression of alkaline phosphatase (ALPL) and versican (VCAN) (FIG. 1). These results indicate that both blends have the capacity to stimulate DP fibroblasts to induce hair follicle growth by sending hair growth-promoting signals to epithelial cells.

[0177] Both blends at a 5 pM concentration also exhibit a long-term hair growth-promoting effect by reducing SFRP1 expression and stimulating gene expression of HGF, as observed with the 6AA blend at 5 pM and the 8AA blend at 5 and 50 pM concentrations (FIG.2). Thus, an increase in HGF expression was detected, which is of particular interest; this mechanistically links and supports the observed downregulation of SFRP1. Furthermore, 6AA blend at 5pM and 8AA blend at 5 and 50pM concentrations tend to also stimulate long-term DP fibroblasts' inductivity by increasing the gene expression of alkaline phosphatase (ALPL). Versican (VCAN) expression increases with the 8AA blend at both concentrations (FIG. 2). Interestingly, 8AA blend at both concentrations tends to stimulate the gene expression of the rejuvenation and angiogenic factor, VEGFA. This means that the 8AA blend can reduce hair aging and also increase the amount of blood vessels within the DP to provide HFs with nutrients needed for their long-term growth. VEGFA levels were increased at 72 hours but not at 6 hours post-treatment, indicating a delayed yet sustained pro-angiogenic response, which is considered unexpected and supportive of long-lasting follicular activation.

[0178] At the protein level, both blends at 50 M concentration tend to decrease the expression of the inhibitor of the Wnt / p-catenin pathway, SFRP1 , indicating that they can stimulate hair growth long-term through activation of the Wnt / -catenin pathway (FIG. 3). Thus, a markedly positive and consistent reduction of SFRP1 was observed at both the gene expression level in dermal papilla fibroblast spheroids and at the protein level, confirming effective inhibition of this Wnt antagonist.

[0179] LDH assay indicates that neither blend shows significant cytotoxicity in DP fibroblasts spheroids, both at short and long term.

[0180] The dermal papilla (DP) fibroblast spheroid assay provides a biologically relevant framework for evaluating the effects of compounds that reach the follicle through circulation. Given that free amino acids are efficiently absorbed and achieve measurable systemic levels following oral intake, their direct exposure in culture media can effectively reproduce a similar in vivo environment encountered by follicular cells, as well as the ORS KCs assays described below.

[0181] Example 10. Outer root sheath keratinocytes

[0182] Primary ORS KCs (from 2 male donors, 32-55y-o) were extracted and expanded in culture on top of a feeder layer of Mitomycin C-treated dermal fibroblasts until passage 2 to obtain enough cells to perform the culture with the test compounds using DMEM / F12 (1:1) medium (Gibco-ThermoFisher Scientific) supplemented with 1% penicillin / streptomycin (Gibco-ThermoFisher Scientific) and 10% FBS. (Gibco-ThermoFisher Scientific). ORS KCs were trypsinized with 0.25% Trypsin / EDTA, (Gibco-ThermoFisher Scientific), and cells were diluted to obtain a cell suspension of 20,000 cells / ml of DMEM / F12 (1:1) medium supplemented with 1% penicillin / streptomycin and 10% FBS in a 12-wells plate. After 48hrs of culture, the medium was removed and either vehicle (medium alone), 5pM 6AA Blend, 50pM 6AA Blend, 5pM 8AA Blend, or 50pM 8AA Blend in modified DMEM / F12 medium (Gibco-ThermoFisher Scientific) was added. 6AA Blend corresponds to Example 1 and 8AA Blend corresponds to Example 2. After 6hrs or 72 hrs of culture, ORS KCs were collected for MTT, quantitative immunohistomorphometry analyses (these were cultured on top of glass coverslips), or qRT-PCR. Furthermore, culture medium was collected for LDH analyses. MTT assay

[0183] After 6 and 72hrs of culture, the medium was removed from the well of ORS KC and replaced by 200ul of 500 ug / ml of MTT solution (Sigma-Aldrich) in OptiMEM (Gibco-ThermoFisher Scientific), the cells were then incubated for 3hrs at 37° C. After 3hrs, 200ul of DMSO (Sigma-Aldrich) was added per well to dissolve the insoluble purple formazan crystal and the absorbance was measured at 540nm.

[0184] LDH assay

[0185] As an indication of HF cytotoxicity, lactate dehydrogenase activity (LDH) was quantified through a colorimetric assay after 6 and 72hrs of culture in the medium collected from each well of ORS KC. The Cytotoxicity Detection kit Plus (Roche), which measures the conversion of tetrazolium salt to formazan, a water-soluble dye with a broad absorption maximum at approximately 490 nm, was used to evaluate the LDH levels in the different conditions at both time points.

[0186] qRT-PCR

[0187] Total RNA was isolated from DP fibroblast spheroids or ORS KCs using the PicoPure™ RNA Isolation Kit (Applied Biosystems-Thermo FisherThermoFisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. RNA purity and concentrations were determined using the Nanodrop ND-1000 assay (Thermo FisherThermoFisher Scientific, Waltham, MA, USA). Reverse transcription of the RNA into cDNA was performed using the SuperScript™ IV VI LO™ Master Mix Kit (Applied Biosystems-Thermo FisherThermoFisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. RNA concentrations were adjusted to a range of 50 to 500 nM to ensure the same amount of RNA was present among samples from the same donor, allowing for further quantification and comparison between the samples after quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Given the low expression level of some genes (e.g. LEF1 and AXIN2), a pre-amplification step was performed using the TaqMan™ PreAmp Master Mix (Thermo FisherThermoFisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. Normalization was performed using the housekeeping gene GAPDH. qRT-PCR was run in duplicate using TaqMan Fast Advanced Master Mix and Gene Expression Assay probes (Id: Hs02786624_g1 for GAPDH, Hs00610344_m1 forAXIN2, Hs00990036_m1 forCOL17A1, Hs01699178_g1 for KRT6A, Hs01547250_m1 for LEF1, Hs02596864_g1 for MT-CO1, Hs00234244_m1 forTGFB2, Thermo FisherThermoFisher Scientific, Waltham, MA, USA) on a QuantStudio 1 Real-Time-PCR-System (Thermo FisherThermoFisher Scientific, Waltham, MA, USA). The QuantStudio™ Design & Analysis Software collected and stored real-time quantification plots and Ct values. The amount of the transcripts was normalized to the housekeeping gene (GAPDH) using the AACT method.

[0188] Immunofluorescence staining and quantitative immunohistomorphometry (qIHM) Briefly, after fixation (see Table 2), coverslips containing ORS KCs were pre-incubated for 30 min at room temperature (RT) (see Table 2), followed by incubation with the primary antibody (see Table 2) overnight at +4 °C. After three times 5 min-long washes, coverslips were incubated with the corresponding fluorescent-labelled secondary antibody for 45 min at room temperature (RT). Finally, coverslips were counterstained with 4Z,6Z-diamidino-2-phenylindole dihydrochloride (DAPI). Negative controls were performed by demonstrating negative immunoreactivity by omitting primary antibodies.

[0189] Quantitative immunohistomorphometry (qIHM) data for each donor were normalized to the vehicle (set as 1) to stratify inter-individual variability between donors.

[0190] List of primary and secondary antibodies used for ORS KCs qIHM.

[0191]

[0192] Results

[0193] The 8AA blend at both concentrations, as well as the 6AA blend at a 5 pM concentration tend to rapidly increase the expression of the p-catenin target genes LEF1 and AXIN2 in ORS KCs in vitro (FIG. 4). This indicates that these conditions can rapidly stimulate hair growth through stimulation of the Wnt / p-catenin signalling. Furthermore, 8AA blend at both concentrations as well as 6AA blend at 5pM concentration tend to rapidly stimulate the expression of the major ORS keratin, important for cell migration, keratin 6A (KRT6A), the mitochondrial activity / function marker, MTCO1, and the stem cell niche integrity marker, collagen 17A1 (COL17A1) (FIG. 4). These results indicate that the structure of hair follicles can be rapidly strengthened, providing resistance to mechanical stress, and can rapidly stimulate the energy production needed for hair growth, as well as prevent short-term stem cell aging / depletion. Furthermore, 6AA blend at 50pM concentration tends to reduce the gene expression of the catagen-inducing growth factor, TGFP2, in ORS KCs, suggesting that this condition can prevent short-term premature catagen induction (FIG. 4).

[0194] Both AA blends at 5 M and 8AA at 50 M concentration tend to increase short term gene expression of KRT6A and the p-catenin target gene LEF1, suggesting that these conditions can stimulate hair growth and increase KRT6A (FIG. 4). At the protein level, 6AA blend at 5pM concentration and 8AA blends at 50pM concentration significantly reduce the long-term expression of SFRP1, while the 8AA blend at 5pM concentration reduces it, but not significantly (FIG. 5). This suggests that the treatment reduced the expression of the inhibitor of hair growth-promoting signalling pathway, Wnt / p-catenin, in ORS KCs, resulting in hair growth promotion. Furthermore, and consistently with the previous experiment showing inhibition of SFRP1 , the same 6AA blend at 5 u. M concentration and 8AA blends at 50uM concentration, significantly reduce the long-term expression of the catagen-inducing growth factor, TGFP2, while 6AA blend at 50 u. M concentration and 8AA blend at 5 u. M concentration reduce it, but not significantly (FIG.

[0195] 6). This result suggests that the different tested conditions may promote hair growth by reducing the production of the hair growth inhibitor TGFP2 in ORS KCs.

[0196] Neither concentration of the 6AA blend displays any significant increase in LDH activity, indicating an absence of cytotoxicity compared to the vehicle control condition for this blend at the short term. MTT assay shows no significant difference in absorbance at longterm for the 8AA blend 5 and 50pM after 6 and 72hrs of treatment as well as for the 8AA blend 5 and 50pM concentration. This result indicates that these conditions do not significantly affect in the long-term ORS KCs viability in vitro.

[0197] Overall, the inhibition of SFRP1 (the Wnt inhibitor) and the suppression of catagen-inducing factors like TGFB2 are the most consistent findings between mesenchymal cells (DP fibroblasts) and epithelium (ORS keratinocytes), which has not been previously demonstrated for amino acid-based compositions. Additionally, these blends also boost the expression of inductivity markers such as versican and alkaline phosphatase in DP fibroblasts, indicating that they stimulate DP fibroblasts to continuously signal the follicle to extend its growth phase (anagen).

[0198] Non-patent literature Higgins, C. A., Chen, J. C., Cerise, J. E., Jahoda, C. A., & Christiano, A. M. (2013). Microenvironmental reprogramming by three-dimensional culture enables dermal papilla cells to induce de novo human hair-follicle growth. Proceedings of the National Academy of Sciences of the United States of America, 110(49), 19679-19688

Claims

1. Claims1. An oral composition for hair care comprising L-arginine, L-alanine, L-glutamine, L- glycine, L-glutamic acid, and L-lysine, wherein3.- the weight ratio of L-glycine / L-glutamic acid is from 4.0:1 to 0.05:1 ;4.- the weight ratio of L-glycine / L-glutamine is from 1.4:1 to 0.1:1;5.- the weight ratio of L-glycine / L-alanine is from 2.6:1 to 0.05:1;6.- the weight ratio of L-glycine / L-arginine is from 4.2:1 to 0.05:1; and7.- the weight ratio of L-glycine / L-lysine is from 5.6:1 to 1.1:1.

2. The oral composition according to claim 1, further comprising L-leucine, and L-serine.

3. The oral composition according to claim 2, wherein the weight ratio of L-glycine / L- leucine is from 2.5:1 to 0.1:1 and the weight ratio of L-glycine / L-serine is from 2.9:1 to 0.3:1.

4. The oral composition according to any of the claims 1-3, further comprising L-proline.

5. The oral composition according to claim 4, wherein the weight ratio of L-glycine / L- proline is from 3.1:1 to 0.7:1.

6. The oral composition according to any of the claims 1-5, further comprising a non- proteinogenic amino acid selected from the group consisting of L-ornithine, L- carnitine, taurine, and mixtures thereof.

7. The oral composition according to claim 6, wherein the weight ratio of L-glycine / non- proteinogenic amino acid is from 10:1 to 0.2:1.

8. The oral composition according to any of the claims 1-7, further comprising a nucleoside selected from the group consisting of adenosine, cytidine, guanosine, uridine, and mixtures thereof.

9. The oral composition according to claim 8, wherein the weight ratio of L- glycine / nucleoside is from 250:1 to 5:1.

10. The oral composition according to any of the claims 1-9, wherein17.- the weight ratio of L-glycine / L-glutamic acid is from 1.1:1 to 0.9:1;18.- the weight ratio of L-glycine / L-glutamine is from 1.0:1 to 0.8:1;19.- the weight ratio of L-glycine / L-alanine is from 0.9:1 to 0.6:1;20.- the weight ratio of L-glycine / L-arginine is from 0.63:1 to 0.5:1; and - the weight ratio of L-glycine / L-lysine is from 4.1:1 to 3.2:1.

11. The oral composition according to any of the claims 1-9, wherein22.- the weight ratio of L-glycine / L-glutamic acid is from 4.0:1 to 3.0:1;23.- the weight ratio of L-glycine / L-glutamine is from 1.4:1 to 1.0:1;24.- the weight ratio of L-glycine / L-alanine is from 2.6:1 to 2.0:1;25.- the weight ratio of L-glycine / L-arginine is from 4.2:1 to 2.6:1; and26.- the weight ratio of L-glycine / L-lysine is from 5.6:1 to 5.0:1.

12. The oral composition according to any of the claims 1-9, wherein28.- the weight ratio of L-glycine / L-glutamic acid is from 0.4 to 0.05;29.- the weight ratio of L-glycine / L-glutamine is from 0.5 to 0.1 ;30.- the weight ratio of L-glycine / L-alanine is from 0.2 to 0.05;31.- the weight ratio of L-glycine / L-arginine is from 0.2 to 0.05; and32.- the weight ratio of L-glycine / L-lysine is from 1.3 to 1.1.

13. The oral composition according to any of the claims 1-12, further comprising one or more orally acceptable excipients.

14. Cosmetic use of the composition according to any of the claims 1-13, for improving hair growth, brightness of hair, hydration, elasticity, firmness, hair shine and / or prevention of hair loss, wherein the use comprises administering orally an effective amount of the composition for the intended use.

15. The cosmetic use of the composition according to claim 14, wherein the effective amount of the composition is from 300 to 2000 mg, and the composition is administered once a day.

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