WO2019023776A1

WO2019023776A1 - Method for utilizing the bioactive hydration of an active ingredient or mixture of ingredients, cosmetic compositions for the modulation of genes responsible for the bioactive hydration of the skin and method for modulating the expression of genes responsible for the bioactive hydration of the skin

Info

Publication number: WO2019023776A1
Application number: PCT/BR2018/050261
Authority: WO
Inventors: Leonardo RODRIGUES DE PAULA
Original assignee: Natura Cosméticos S.A.
Priority date: 2017-07-31
Filing date: 2018-07-27
Publication date: 2019-02-07
Also published as: AR112771A1; BR102017016427A2

Abstract

The present invention relates to a method for characterizing bioactive (or active or dynamic or biological) hydration by means of the use of techniques for utilizing gene and protein expression which makes it possible to identify new ingredients or a combination of ingredients that efficiently stimulate the five endogenous mechanisms of the skin that are associated with said hydration.

Description

METHOD FOR EVALUATING BIOATIVE HYDRATION OF AN INGREDIENT OR MIXTURE OF INGREDIENTS, COSMETIC COMPOSITIONS FOR THE MODULATION OF GENES RESPONSIBLE FOR BIOATIVE SKIN HYDRATION AND A METHOD FOR MODULATING EXPRESSION OF GENES RESPONSIBLE FOR BIOATIVE SKIN HYDRATION FIELD OF THE INVENTION

The present invention relates to a method for characterizing bioactive (or active or dynamic or biological) hydration by employing techniques for assessing gene and protein expression which allows the identification of novel ingredients or a combination of efficient ingredients in the stimulus of the five endogenous mechanisms of the skin associated with said hydration.

BACKGROUND OF THE INVENTION

The epidermis, the outermost layer of the skin, is always renewing and its last layer, which makes contact with the outer medium is called stratum corneum. EC comprises the final stage of epidermal cell differentiation and is specifically represented by dead and anucleated keratinized cells embedded in a lipid matrix acting as a barrier separating the external environment from internal homostasy.

The natural barrier formed by EC depends critically on its composition, represented by proteins (75-80%), lipids (5-15%) and other constituents (5-10%). The lipid composition of human EC can vary quantitatively between individuals and body parts, but mainly comprises ceramides, fatty acids, cholesterol, esters, triglycerides and phospholipids. When these components are not properly balanced, the skin's ability to maintain water is reduced and the skin becomes more susceptible to of environmental factors, leading to an impaired barrier.

[004] A major role in maintaining the integrity of the EC barrier is represented by water. One of the main aspects is related to its ability to mediate the activity of many hydrolytic enzymes in the skin, including those responsible for proper desquamation of corneocytes, as well as those responsible for the formation of the NMF (Natural Moisturizing Factor) . As a proteolytic product of prophylagins - key molecule in maintaining the epidermal barrier -, NMF is essentially composed of amino acids and derivatives, PCA (pyrrolidone carboxylic acid), minerals, urea, sugars and peptides. This highly hygroscopic blend helps maintain hydration and hence EC function.

[005] The epidermis is also responsible for the regeneration of the skin. Every 28 days a cell walks from the dermo-epidermal junction to the stratum corneum, then detached as a dead cell in the desquamation process. When the skin is dehydrated, this reconstitution and exchange are altered, making natural skin regeneration difficult.

[006] When there are changes in the surface of the stratum corneum (eg, by burning, bruising or breakage) or excessive loss of lipids or natural moisturizers, the skin becomes dry, rough, without elasticity or flexibility. The sun, the pollutants, the relative humidity of the air are also factors that interfere with the hydration of the skin.

[007] Another important molecule for skin hydration is hyaluronic acid present in the dermis and epidermal intercellular spaces. This molecule can bind up to a thousand times its weight with water molecules. In the dermis, along with other extracellular matrix molecules, it is responsible for holding water and maintaining adequate tissue drainage.

In addition, membrane protein channels called aquaporins are responsible for facilitating the transport of water and solutes, including glycerol and urea, between biological membranes. This conduction of water occurs through an osmotic gradient.

[009] It is recommended that adults use moisturizer on the whole skin at least once a day. Such products generally include ingredients which typically act by two well known mechanisms: occlusion and wetting.

Occlusion hydration is promoted by ingredients that form a lipophilic film, which hinders the loss of water, increasing water retention in the stratum corneum.

Moisturizing by wetting, in turn, is promoted by ingredients which retain water from the cosmetic composition, the atmosphere and the skin on the surface of the skin by means of a hydrophilic film formed on the horny layer, increasing the retention of water on the skin surface.

However, in evolution to the classic mechanisms mentioned above, other hydration mechanisms, called in the state of active hydration technique, have already been proposed. In general, they refer to the use of ingredients that have the ability to stimulate the skin to produce its own hydrating substances, ie molecules that are able to retain water in all layers of the skin.

Currently, the proof of the moisturizing properties of cosmetic preparations for the skin is made using non-invasive methods using instruments based on electrical properties, such as conductance and capacitance, as well as spectroscopic techniques such as NIR (near-infrared spectroscopy ). However, there are problems associated with the reliability of measurements of electrical properties of the skin (capacitance is the most skin hydration) when it receives treatments with high concentrations of occlusive, supportive and non-hygroscopic ingredients. False negative measurements of the water status of the skin can be observed depending on the chemical composition of the formulation being studied. For this reason in some cases the combination of several instrumental techniques and even visual evaluations with the aid of photographic scales is recommended to demonstrate the benefits of cosmetic preparations for the skin.

Dryness is one of the most present and chronic conditions of the skin, resulting in "stiffness" of the skin and damage to the skin in the form of cracks and cracks. Thus, assessment of mechanical properties of the skin (using mechanical models) may be useful to understand how damage to dry skin occurs (eg, cracking).

Thus, the state of the art still requires new techniques to identify moisturizing products by more accurate and reliable means.

DESCRIPTION OF THE INVENTION

The present invention relates to a method for characterizing bioactive hydration by employing techniques for assessing gene and protein expression which allows the identification of novel ingredients or combination of efficient ingredients in stimulating the five endogenous skin mechanisms associated with the said hydration.

According to the present invention, "bioactive hydration", also referred to herein as biological hydration or biodynamic hydration or active hydration, is meant the ability of a balanced combination of ingredients to stimulate endogenous skin mechanisms to promote equilibrium between water and lipid content for efficient hydration and nutrition. The endogenous mechanisms that determine bioactive hydration according to the present invention are simultaneously:

(1) stimulating molecules that hold water in the deeper layers of the skin (dermis) such as hyaluronic acid and other components of the extracellular matrix;

(2) stimulating molecules that retain water on the surface of the skin as natural moisturizing factors, known as NMF, from the English "Natural Moisturizing Factor" (mineral salts);

[0021] (3) stimulating the production and metabolism of lipids in the skin surface;

[0022] (4) stimulation of proper and balanced removal of dead skin surface cells (desquamation);

(5) stimulating molecules carrying water and minerals to maintain the osmotic balance of the skin (such as, for example, aquaporins).

The effect on the five endogenous mechanisms above is measured by means of techniques for evaluation of gene and protein expression, which are highly accurate techniques and their results are independent of external factors as in commonly used techniques.

Thus, the method for characterizing bioactive hydration according to the present invention is to evaluate the expression of an ingredient or combination of ingredients (complexes) in a set of genes associated with the five endogenous mechanisms of the skin that promote the balance between the hydric and lipid content for efficient hydration and nutrition, which characterize bioactive hydration.

The inventor surprisingly identified that a set of specific genes is directly associated with the five endogenous mechanisms that characterize a bioactive hydration as identified in Table 1 below. Table 1. Gene pool associated with bioactive hydration

Gene symbol

18s RNA RNA, 18S ribosomal [reference gene]

ACACA Acetyl-CoA carboxylase 1

ACACB Acetyl-CoA carboxylase beta

AQP10 Aquaporin-10

AQP1 Aquaporin-1

AQP3 Aquaporin-3

AQP5 Aquaporin-5

ASAH1 Acid ceramidase

BGN Biglycan

CASP14 Caspase-14

CD44 CD44 molecule (Indian blood group)

CDH1 Cadherin-1

CDSN Corneodesmosin

CLDN1 Claudin-1

CLDN4 Claudin-4

CLDN7 Claudin-7

COL1A1 Collagen, type I, alpha 1

COL1A2 Collagen, type I, alpha 2

COL4A4 Collagen, type IV, alpha 4

COL7A1 Collagen, type VII, alpha 1

CRNN Cornulin

CST6 Cystatin 6

CTSB Cathepsin B

CTSD Cathepsin D

CTSE Cathepsin E

CTSL Cathepsin L

CTSV Cathepsin V

DCN Decorin

DSC1 Desmocollin 1

DSC2 Desmocollin 2

DSG1 Desmoglein 1 Gene symbol

DSG3 Desmoglein 3

DSG4 Desmoglein-4

DSP Desmoplakin

Eln Elastin

ELOVL3 ELOVL fatty acid elongase 3

EVPL Envoplakin

FASN Fatty acid synthase

FLG Filaggrin

FMOD Fibromodulin

GAPDH Glyceraldehyde-3-phosphate dehydrogenase [reference gene]

GBA Glucosylceramidase beta

GJA1 Gap junction protein, alpha 1

GJB2 Gap junction protein, beta 2

GPC3 Glypican 3

HAL Histidine ammonia-lyase

GUSB Glucuronidase, beta [reference gene]

HAS1 Hyaluronan synthase 1

HAS2 Hyaluronan synthase 2

HAS3 Hyaluronan synthase 3

HMGCR 3-hydroxy-3-methylglutaryl-CoA reductase

HMGCS1 3-hydroxy-3-methylglutaryl-CoA synthase 1

HMMR Hyaluronan mediated motility receptor

HYAL1 Hyaluronidase 1

HYAL2 Hyaluronidase 2

HPRT Hypoxanthine phosphoribosyltransferase [reference gene]

ITGA6 Integrin alpha-6

ITGB4 Integrin beta-4

IVL Involucrin

KLF4 Krueppel-like factor 4

KLK5 Kallikrein 5 Gene symbol

KLK7 Kallikrein 7

KRT1 Keratin 1

KRT10 Keratin 10

LAMA5 Laminin subunit alpha-5

LCE2B Late cornified envelope protein 2B

LOR Loricrin

LOX Lysyl oxidase

LUM Lumican

MATN2 Matrilin 2

MMP1 Matrix metallopeptidase 1

MMP12 Matrix metallopeptidase 12

MMP9 Matrix metallopeptidase 9

OCLN Occludin

PADI1 Peptidyl arginine deiminase, type I

PADI3 Protein-arginine deiminase type 3

PCNA Proliferating cell nuclear antigen

PKP1 Plakophilin 1

PLEC Plectin-1

PPL Periplakin

SDC1 Syndecan 1

SMPD1 Sphingomyelin phosphodiesterase / Acid sphingomyelinase

SOD2 Superoxide dismutase 2, mitochondrial

SPINK5 Serine peptidase inhibitor, Kazal type 5

SPTLC2 Serine palmitoyltransferase, long chain base subunit 2

SREBF2 Sterol regulatory element-binding protein 2

ST14 Suppression of tumorigenicity protein 14

TGM1 Transglutaminase-1

TGM3 Transglutaminase-3

TGM5 Transglutaminase-5

TIMP1 TIMP metallopeptidase inhibitor 1

TIMP2 TIMP metallopeptidase inhibitor 2 Gene symbol

TJP1 Tight junction protein 1

UGCG Ceramide glucosyltransferase

VCAN Versican

VCL Vinculin

In particular embodiment, the set of genes associated with each endogenous mechanism according to the present invention is:

1. Stimulating molecules that retain water in the deeper layers of the skin (hyaluronic acid and other extracellular matrix components): BGN, CD44, COL1A1, COL1A2, COL4A4, DCN, ELN, FMOD, GPC3, HAS1, HAS2, HAS3, HMMR, HYAL1, HYAL2, LOX, LUM, MATN2, MMP12, MMP1, MMP9, SDC1, TIMP1, TIMP2, VCAN;

2. Incubation to surface water retaining molecules (filaggrin processing and NMF synthesis): CASP14, FLG, HAL, PA-DM, PADI3, ST14, TGM1, TGM3, TGM5;

3. Stimulating the production of lipid molecules on the skin surface: ACACA, ACACB, ASAH1, ELOVL3, FASN, GBA, HMGCS, HMGCS1, SMPD1, SPTLC2, SREBF2, UGCG;

4. Stimulation of appropriate and balanced removal of dead skin surface cells (desquamation): CRNN, CST6, CTSB, CTSD, CTSE, CTSL, CTSV, DSC1, DSG1, DSG3, KLF4, KLK5, KLK7, SPINK5; and

5. Stimulating molecules carrying water and minerals to maintain the osmotic balance of the skin: AQP10, AQP1, AQP3, AQP5, CLDN1, CLDN4, CLDN7, GJA1, GJB2, TJP1.

In another embodiment, the present invention also comprehensively contemplates cosmetic compositions for the modulation of genes responsible for bioactive hydration of the skin, either the body or the face, which comprises at least one ingredient or combination of ingredients that act simultaneously in the five endogenous mechanisms of the invention and less a cosmetically acceptable vehicle.

The cosmetically acceptable excipients according to the present invention are those known to those skilled in the art for the composition of cosmetic bases in various forms, for example, emulsions, creams, gels, sils, and others known to those skilled in the art. For example, without limitation, cosmetically acceptable excipients may be selected from those quoted in the International Cosmetic Ingredient Dictionary & Handbook, 16th Edition.

The following examples, without limiting the particular embodiments of the present invention, as well as demonstrating the efficacy of the complex according to the present invention in bioactive hydration.

EXAMPLES

Example 1. Example 2. Analysis of the efficacy in bioactive hydration of a potential ingredient

Gene and protein expression analysis was used to verify the modulation of the genes involved in the stimulation of the endogenous mechanisms of the skin, which promote the balance between water and lipid content for efficient hydration and nutrition, that is, bioactive hydration.

A large-scale gene expression study was performed, followed by confirmation of the protein expression to prove the benefit.

The sample was tested on human skin explants obtained after plastic surgery (blepharoplasty) and evaluated efficacy in modulating a total of 92 genes related to biological mechanisms determined to be relevant to skin hydration, as well as for some additional benefits such as integrity of the skin barrier, antioxidant, adhesion and cohesion between cells, cell renewal and anti-aging (elasticity, firmness, filling and dermo-epidermal cohesion).

In this experiment, skin fragments from 3 different donors were divided into two portions and inserted, in triplicate from each donor, immediately into culture medium. The explants were treated with 2mg / cm ² of the topically applied sample without any dilution and maintained for 24 hours (portion 1) and 72 hours (portion 2) in a humid atmosphere at 37 ° C in the presence of 5% CO2 . In addition, a control condition was performed by maintaining the explants in triplicate of each donor in culture medium alone. A previous viability analysis was performed to ensure tissue integrity throughout the study protocol.

Portion (1) was collected and subjected to total RNA extraction. From the RNA, the cDNA was prepared and this (50ng) was submitted to a real-time RT-PCR (Polymerase-Chain Reaction) step to evaluate the expression of 96 genes. The gene expression profile and selection of the differentially expressed genes were performed with the aid of Expression Suite Software v.1.0.3 (Life Technologies). The ACt values for the reference genes and the target genes were calculated by subtracting the values from the experimental groups. Subsequently, the ACT of the experimental group was subtracted from the control group (untreated skin explant) to obtain the AACt. Finally, the relative quantification of the target genes was determined by the equation: RQ = 2 -AACt. Only genes with a threshold of 1, 3, that is, 30% increase or decrease in relation to the control were selected. Statistical significance was assessed by the t-test followed by the FDR (false-rate) method, with p-value <0.05 being considered significant. For detection by immunofluorescence, the samples were fixed in 4% paraformaldehyde (pH 7.4) for 24 hours and cryoprotected in 30% sucrose solution for 48 hours. Then, serial cuts of 10 m were collected directly on silanized slides with the aid of Cryostat (Leica - CN1850). At the end of the Collection of the cuts, they were subjected to washings with 0.1 M PB and incubated overnight with antibodies referring to the selected markers of interest. Afterwards, the slides were analyzed in a Fluorescence Microscope (Leica - DM 1000) with the aid of LAS Software (Leica Application Suite). The parameter evaluated was fluorescence intensity emitted by specific antibody labeling. For statistical analysis, analysis of variance (ANOVA) was used. In all groups studied, those whose P values were less than 0.05 were considered statistically significant.

For the detection by Elisa, commercially available kits (R & D Systems, USA) were used. The capture monoclonal antibody against the marker of interest was added to a 96-well plate which was then incubated for 12 hours at room temperature. Samples were added and the plate was incubated for 2 hours at room temperature. The detection antibody against the marker of interest was then incubated for an additional 2 hours (RT). Streptavidin-peroxidase solution was added and incubated for 1 hour (RT). Finally, the substrate solution (H2O2 and TMB-tetramethylbenzidine) was added to the plate and a blue color developed over a period of 20 minutes. The staining reaction was stopped by adding 2N H2 SO4 and the reading was performed on a microplate reader at 450 nm. Protein levels were expressed in pg / ml or g / ml and calculated from reference values obtained with a standard curve constructed at known concentrations of the protein. For statistical analysis, analysis of variance (ANOVA) was used. In all groups studied, those whose P values were less than 0.05 were considered statistically significant.

The genes that had their expression evaluated by the study were: ACACA, ACACB, AQP1, AQP3, AQP5, ASAM, BGN, CASP14, CD44, CDH1, CDSN, CLDN1, CLDN4, CLDN7, COL1A1, COL1A2, COL4A4, COL7A1, CRNN, CST6, CTSB, CTSD, CTSE, CTSL, CTSV, DCN, DSC1, DSC2, DSG1, DSG3, DSG4, DSP, ELN, ELOVL3, EVPL, FASN, FLG, FMOD, GBA, GJA1, GJB2, HAL, HAS1, HAS2, HAS3, HMGCR, HMGCS1, HYAL1, HYAL2, ITGA6, ITGB4, IVL, KLF4, KLK5, KLK7, KRT1, KRT10, LAMA5, LCE2B, LOR, LOX, LUM, MATN2, MMP1, MMP12, MMP9, OCLN, PADI1, PADI1, PADI3, PCM1, PKP1, PLEC, PPL, SDC1, SMPD1, SOD2, SPINK5, SPTLC2, SREBF2, ST14, TGM1, TGM5, TIMP1, TIMP2, TJP1, UGCG, VCAN, VCL.

Figure 1 shows the gene expression results obtained from a potential active ingredient compared to other market ingredients.

As can be seen, only one ingredient was able to act simultaneously on the five endogenous mechanisms that determine the occurrence of bioactive hydration.

The man skilled in the art will readily appreciate, by means of the teachings contained in the text and in the examples presented, advantages of the invention and propose variations and equivalent alternatives of attainment without departing from the scope of the invention as defined in the appended claims

Claims

A method for evaluating bioactive hydration of an ingredient or mixture of ingredients, characterized by checking the expression of the genes ACACA, ACACB, AQP10, AQP1, AQP3, AQP5, ASAH1, BGN, CASP14, CD44, CDH1, CDSN, CLDN1, CLDN4, CLDN7, COL1A1, COL1A2, COL4A4, COL7A1, CRNN, CST6, CTSB, CTSD, CTSE, CTSL, CTSV, DCN, GBA, GJA1, GJB2, GPC3, HAL, HAS1, HAS2, HAS3, HMGCR, HMGCS1, HMMR, HYAL1, HYAL2, ITGA6, ITGB4, IVL, KLF4, KLK5, KLK7, KRT1, KRT10, LA- MA5, LCE2B, LOR, LOX, LUM, MATN2, MMP1, MMP12, MMP9, OCLN, PADI1, PADI3, PCNA, PKP1, PLEC, PPL, SDC1, SMPD1, SOD2, SPINK5, SPRELC2, SREBF2, ST14, TGM1, TGM3, TGM5, TIMP1, TIMP2 , TJP1, UGCG, VCAN, VCL.

A method according to claim 1, characterized by the correlation of gene expression with at least one of the following five endogenous mechanisms:

- stimulating molecules that hold water in the deeper layers of the skin: BGN, CD44, COL1A1, COL1A2, COL4A4, DCN, ELN, FMOD, GPC3, HAS1, HAS2, HAS3, HMMR, HYAL1, HYAL2, LOX, LUM, MATN2, MMP12, MMP1, MMP9, SDC1, TIMP1, TIMP2, VCAN;

- Stimulating molecules that retain water on the surface: CASP14, FLG, HAL, PADI1, PADI3, ST14, TGM1, TGM3, TGM5;

- Stimulating the production of lipid molecules on the skin surface: ACACA, ACACB, ASAH1, ELOVL3, FASN, GBA, HMGCS, HMGCS1, SMPD1, SPTLC2, SREBF2, UGCG;

- Stimulation of adequate and balanced removal of dead skin surface cells: CRNN, CST6, CTSB, CTSD, CTSE, CTSL, CTSV, DSC1, DSG1, DSG3, KLF4, KLK5, KLK7, SPINK5; and Stimulating molecules carrying water and minerals to maintain osmotic skin balance: AQP10, AQP1, AQP3, AQP5, CLDN1, CLDN4, CLDN7, GJA1, GJB2, TJP1.

Cosmetic compositions for the modulation of genes responsible for bioactive hydration of the skin, characterized in that they comprise at least one ingredient or combination of ingredients which act simultaneously on the following five endogenous mechanisms:

- stimulation of molecules that hold water in the deeper layers of the skin;

- stimulation of molecules that retain water on the surface of the skin;

- stimulation of the production of lipids on the surface of the skin;

- stimulation of proper and balanced removal of dead skin surface cells;

- stimulating molecules that carry water and minerals to maintain the osmotic balance of the skin (such as aquaporins).

A method for modulating the expression of genes responsible for bioactive hydration of the skin, characterized in that it comprises a step of administering a cosmetic composition to the skin of an individual.