WO2005023234A1

WO2005023234A1 - Molecules exhibiting anti-adipogenic activity on adipocyte human cells

Info

Publication number: WO2005023234A1
Application number: PCT/FR2004/002203
Authority: WO
Inventors: Anne-Marie Rodriguez; Christian Dani; Gérard Ailhaud; Joëlle Guesnet; Anne Guezennec
Original assignee: Yves Saint Laurent Parfums; Centre National De La Recherche Scientifique; Universite De Nice Sophia Antipolis
Priority date: 2003-08-29
Filing date: 2004-08-27
Publication date: 2005-03-17
Also published as: FR2859104A1

Abstract

The invention relates to the use of a nordihydroguaiaretic acid (C18H22O4) or the derivative thereof as an active principle for a cosmetic or food composition in the form of a human adipocytes formation inhibitor or as an active principle for a derma-pharmaceutical or pharmaceutical composition for preparing a drug used for treating or preventing pathologies which provoke the adipocytic differentiation dysfunction of a human being.

Description

USE OF NOR-DIHYDROGUAIARETIC ACID AS AN INHIBITOR OF HUMAN ADIPOCYTE FORMATION

The present invention relates to the use of nor-dihydroguaiaretic acid (“NDGA”) or a derivative thereof, as an anti-adipogenic agent in cosmetic, dermo-pharmaceutical, pharmaceutical and food preparations, in particular for human use.

The invention also relates to cosmetic, dermo-pharmaceutical, pharmaceutical and food compositions containing NDGA in an amount sufficient to inhibit the formation of human adipocytes. The invention also encompasses methods for the prevention or treatment of overweight or any other condition involving aberrant or undesirable adipocyte differentiation, in humans, using compositions based on

NDGA or its derivatives.

The adipocyte is the main cell of adipose tissue, and not only contains the equipment necessary for regulating the energy balance (under hormonal control), but also plays a complex and dynamic role interfering with immunological responses, vascular diseases and appetite regulation, thanks to its endocrine, autocrine and paracrine cell capacities. Adipocytes, highly specialized cells, store energy in the form of triglycerides and release it in the form of fatty acids. The weight development of adipose tissue is a direct reflection of the state of lipid energy reserves. The adipocyte precursor cells (the preadipocytes), can differentiate into adipocytes throughout life. This differentiation is stimulated by hormones or by nutrients such as fatty acids. The excessive development of adipose tissue leading to obesity involves on the one hand, adipocyte enlargement (increase in cell size) and on the other hand, cellular hyperplasia, ie an increase in the number of adipose cells .

The mature adipocyte, which is a differentiated cell, has lost all capacity for division. Consequently, the increase in the number of adipocytes, observed during the development of adipose tissue, results in fact from the overproliferation and the differentiation of cellular precursors into adipocytes, the precursors being present in the vascular fraction of the stroma of this tissue.

Insofar as the development of adipose tissue results in part from the differentiation of cellular precursors, it is interesting, in the search for molecules capable of being effective in the fight against overweight, to identify compounds capable of inhibiting adipocyte differentiation. To date, a number of inhibitors of adipocyte differentiation on 3T3-L1 cells in particular or other cell lines have been published, for example, [1, 25] -dihydroxy- vitamin D3 (Kelly, KA et al. , Ref. 1) or high concentration retinoic acid (Hilda et al., Ref. 2) while the latter is described as activator of adipocyte differentiation at low concentration (Safanova et al., Ref. 3 and 4) , octoanoate (Han et al., Ref. 5) and the vitamin

K2 (Palacios et al., Ref. 6). That said, these molecules are little or not adapted, or even prohibited, for use in cosmetics, pharmaceuticals or food.

There is therefore today a need to identify molecules having an inhibitory effect on the formation of human adipocytes, and which are suitable for cosmetic, therapeutic or food use.

The present inventors screened a large number of molecules for anti-adipogenic agents. They discovered, surprisingly, that nordihydroguaiaretic acid ("NDGA") is capable of significantly inhibiting human adipocyte differentiation, and this at a very early stage in the differentiation process.

The anti-adipogenic activity of NDGA on human cells is unexpected for several reasons. Although Thuillier P et al., (Reference 7) have recently shown that certain lipoxygenase inhibitors (LOX), including NDGA, quercetin and morine, inhibit the differentiation of murine keratinocytes, probably via an inhibitory action on PPA receptors α and PPARγ ("Peroxisome Proliferator-Activated Receptors α and γ" "), the present inventors have found that these results could not be extrapolated to human adipocyte differentiation. Indeed, tests carried out by the inventors in the context of the present invention demonstrate that quercetin and morine have no anti-adipogenic effects on human cells.

In addition, work carried out by Ye and Serrero (Reference 8), on a murine adipogenic cell line (line 1246) have shown that NDGA has no effect on the expression of ADRP ("adipose differentiation related protein") ) which is an early marker of adipocyte differentiation (Jang & Serrero, Reference 9).

It emerges from these various works that the effects of NDGA on human adipocyte differentiation could not be predicted on the basis of its effects on rodent cells.

NDGA has already been used in cosmetic and therapeutic preparations, in particular for its anti-hair regrowth effects (WO 00/28959), its anti-acne effects (FR 2830195), its antioxidant effects (Ref. 12), its anti-inflammatory effects (US 4,530,844 and US 4,568,696), and its anti-tumor effects (EP 297733). However, this compound has not been used to date as an adipocyte differentiation inhibitor.

The present invention therefore relates to the use of nor-dihydroguaiaretic acid (Cι ₈ H ₂₂ θ), or of its derivatives, as active ingredient in a cosmetic, dermopharmaceutical, pharmaceutical or food composition, as an inhibitor of the formation of human adipocytes.

In particular, the present invention relates to the use of nor-dihydroguaiaretic acid (C ₁₈ H ₂₂ θ ₄ ), or a derivative thereof, as active ingredient in a cosmetic composition, as an inhibitor of formation of human adipocytes or as active principle in a dermo-pharmaceutical composition for the preparation of a medicament for the treatment or prevention of pathologies involving a dysfunction of adipocyte differentiation in humans.

Nor-dihydroguaiaretic acid (C ₁₈ H ₂₂ θ ₄ ) has the following chemical formula

NDGA is also known under the names of: β'γ-dimethyl-α-δ-bis (3,4-dihydroxyphenyl) butane 1, 2-benzenediol, 4,4 '- (2,3-dimethyl-1, 4 -butane) 1,4-Bis (3,4-dihydroxyphenyl) -2,3-dimethylbutane 4.4 '- (2,3-dimethyI-1,4-butanediyl) bis (pyrocatechol) Butane, 1,4-bis (3,4-dihydroxyphenyl) -2,3-dimethyI- Masoprocol Dihydronorguaiaretic acid

In the context of the invention NDGA can be a natural molecule, obtained by plant extraction, for example from the plant Larrea divaricata or Larrea tridentata. NDGA can be purified from the plant extract, or the raw extract can be used. NDGA can also be produced by chemical synthesis.

In the context of the invention, an NDGA derivative means any molecule being structurally related to NDGA, and having an anti-adipogenic activity as defined below. Preferably, the derivatives have the formula I:

R ² 0

FORM I

in which R, 1, R are, independently of one another, either (-H) or a methyl radical

(-CH ₃ ) either an ethyl radical (-C ₂ H ₅ ) or a methoxy (-COCH ₃ ) or ethoxy radical (-

COC ₂ H ₅ ),

R ³ , R ⁴ , R ⁵ , R ⁶ , R ¹⁰ , R ¹¹ , R ¹² and R ¹³ are independently of each other (-H), or a methyl radical (-CH ₃ ) or an ethyl radical (-C ₂ H ₅ ),

R ⁷ , R ⁸ and R ⁹ are independently of each other either (-H), or (-OH), or a methyl radical (-CH ₃ ) or an ethyl radical (-C ₂ H ₅ ), or a radical methoxy (-COCH ₃ ) or ethoxy (-

COC ₂ H ₅ ).

Preferably, in formula I, R ¹ and R ² represent (-H), R ⁷ and R ⁸ represent (-OH), R ⁹ rreepprréésseente (H), and R ³ to R ⁶ and R ¹⁰ to R ¹³ represent independently (-H), or a methyl radical

(-CH ₃ ).

According to the invention, NDGA and its derivatives exert an inhibitory effect on the formation of human adipocytes. This effect, also designated as an “anti-adipogenic effect”, signifies an inhibition of the differentiation of human stem cells or of human preadipocyte precursor cells, into adipocytes. Adipocyte differentiation is mainly characterized by a morphological modification of the precursor cells (passage from the fibroblastic form to a round form with accumulation of triglyceride droplets), a phenotypic change, and the appearance of specific markers of the adipocyte, for example the hPPARγ2 (human γ2 receptor activated by proliferators of peroxisome or "human peroxisome proliferator activated receptor γ"), and haP2 (human fatty acid binding protein or "human adipocyte fatty acid binding protein"). Induction of the expression of certain enzymes, for example glycerophosphate dehydrogenase (GPDH), is also observed during adipocyte differentiation.

According to the invention, the inhibition by NDGA of the formation of adipocytes takes place at an early stage of differentiation, in particular before the appearance of specific markers of adipocytes, such as hPPARγ2 or haP2, before the expression of specific enzymatic activity, such as GPDH activity, and before the formation of adipocytes loaded with triglycerides.

The anti-adipogenic effect of NDGA and its derivatives can be demonstrated in vitro by measuring either the level of expression of adipocyte markers, or the level of specific enzymatic activity, presented by multipotent human cells in the presence of NDGA or its derivatives, after several days of culture in an adipocyte differentiation medium. These levels are compared with those presented by the same cells in the absence of NDGA or its derivatives. The cells having been exposed to the medium containing NDGA exhibit a level of expression of the adipocyte markers, or a level of enzymatic activity, much lower than the levels observed in the absence of NDGA or its derivatives. For measuring expression levels, hPPARγ2 or haP2 are suitable as specific adipocyte markers. Their expression levels can be determined by assaying mRNA or proteins. To measure the levels of enzyme activity, GPDH activity can be used.

Preferably, the anti-adipogenic effect of NDGA and its derivatives is demonstrated by measuring the glycerophosphate dehydrogenase (GPDH) activity, for example by implementing the tests described in the experimental protocols below.

The principle of the GPDH assay is summarized in this diagram: GPDH

Dihydroxyacetone phosphate + NADH - Glycerophosphate + NAD

The initial rate of disappearance of NADH is determined at 340 nm (in the presence of NADH, DHAP and the cell lysate), thus making it possible to calculate the amount of degraded substrate, and therefore, the specific enzymatic activity (after assaying the proteins). In the context of the invention, a significant inhibition of adipocyte differentiation means a statistically significant decrease in the specific activity of GPDH, compared to cells, differentiated into adipocytes in the absence of treatment. This significant inhibition results, for example, in a reduction of at least 10%, or at least 15% or 30% of the specific activity of GPDH, compared with cells differentiated into adipocytes in the absence of treatment, preferably at least 50%, more particularly at least 60%, and preferably at least 80% of the specific activity of GPDH, relative to the control.

The process for demonstrating the anti-adipogenic effect preferably comprises the following steps (see Figure 1):

Multiplication of stem cells or precursors to confluence;

Initiation of differentiation (Day 0) by culturing the cells in an adipocyte differentiation medium, in the absence of fetal calf serum, and in the presence of NDGA or one of its derivatives, at the desired concentration, for 12 days ;

Maintaining the presence of NDGA or its derivatives until the determination of GPDH activity, this determination step being able to take place at any time between Day 0 and Day 12. A significant reduction in the specific activity of GPDH compared to the level of activity in the absence of treatment can be noted from Day 1, and generally reaches a reduction of at least 50% from Day 3. Preferably the determination of the GPDH activity is therefore performed between Day 3 and Day 12; The culture medium is changed every two days until Day 12.

The anti-adipogenic effects of NDGA, and its derivatives, are manifested on adult human stem cells, as well as on human preadipocytes. In the context of the invention, the term “stem cell” designates a multipotent cell having a high capacity for self-renewal, significant telomerase activity, and an ability to enter into quiescence. These multipotent cells are capable of differentiating into at least two cell types, and preferably into at least three or four different cell types. The term “preadipocyte” designates any precursor cell of the adipocyte already engaged in an adipocyte differentiation pathway. The preadipocytes are also called “adipocyte precursor cells”. As an example of human preadipocytes capable of being used for the demonstration in vitro of the antiadipogenic effect of NDGA or of its derivatives, there may be mentioned the cell line of human brown preadipocytes PAZ-6 (Ref. 10, and international patent application WO 9634100). This line was deposited with the National Collection of

Culture of Microorganisms (CNCM) of the Institut Pasteur, under the number 1-1531. Mention may also be made of cells called “SGBS Adipocytes” (Ref. 11). The anti-adipogenic effects of NDGA and its derivatives are particularly evident in adult stem cells derived from human adipose tissue. As an example of these preferred stem cells, mention may be made of multipotent cells described in international patent application No. PCT / FR 03/02439. More precisely, these multipotent cells, after reaching quiescence, stably exhibit the following phenotype in vitro:

HLA class I negative, HLA class II negative, CD3 negative,

CD13 positive, Oct-4 positive, Rex-1 positive, ABCG2 positive.

These phenotypic characteristics are associated with a normal karyotype, and with significant telomerase activity. Preferably the cells are also LIF-R negative. This phenotype is conserved in vitro in a stable manner, that is to say in the absence or in the presence of FGF-2, and at concentrations of fetal calf serum which can exceed 10%. The phenotype is also preserved at high seeding densities, and beyond

140 population doublings.

These multipotent human stem cells can be obtained by implementing a process comprising the following steps: a) enzymatic digestion of a sample of adipose tissue originating from a child aged less than ten years, for example aged less than five years ; b) recovery of a cell fraction devoid of adipocytes, containing all the cell types present in the preparation obtained according to (a), with the exception of adipocytes, c) in vitro culture for at least 12 hours, of the cell fraction obtained according to step (b), d) selection of two cellular subpopulations, called “CA” population and “CS” population, the CA population having an adhesion speed of less than 12 hours, and the “CS” population having an adhesion speed greater than 12 hours, e) enrichment of the “CA” population until a population of cells capable of entering a state of quiescence is obtained, f) possibly, induction of proliferation increased stem cells from the “CA” population, for example by adding a growth factor. In the context of the present invention, it has been demonstrated that NDGA significantly inhibits the differentiation of multipotent stem cells into adipocytes, and this at an early stage of differentiation, that is to say before the appearance of cellular or enzymatic markers, specific for adipocytes. As a result, NDGA, or a derivative thereof, is suitable for use as an active ingredient in a cosmetic, pharmaceutical or food composition, as an inhibitor of the formation of human adipocytes.

When the anti-adipogenic compositions of the invention are used in a cosmetic context, they are intended for the prevention or treatment of non-pathological overweight, the prevention or treatment of cellulite, the prevention or treatment of dimpling, or skin tightening.

The compositions based on NDGA, or on its derivatives, can also be used in dermo-pharmaceutical applications, in which case, they are intended for the prevention or treatment of pathological overweight implicating a dysfunction of adipocyte differentiation in humans. , such as overweight and obesity.

The concentrations of NDGA or of its derivatives used in the cosmetic or dermo-pharmaceutical compositions of the invention are of the order of 0.0001% to 10% by weight, relative to the total weight of the composition, for example at a concentration included between 0.001% and 5% by weight, preferably between 0.001% and 1% by weight.

The cosmetic and dermo-pharmaceutical anti-adipogenic compositions of the invention are generally formulated for administration by the cutaneous, percutaneous or transcutaneous route. Administration by the cutaneous route consists of administration to the skin, without breaking the skin, with a view to local action (topical effect) or not (systemic effect).

Administration by percutaneous or transcutaneous route leads to diffusion through the skin and subcutaneous structures, and possibly to a systemic effect, after blood absorption. When the anti-adipogenic compositions of the invention are intended for pharmaceutical use other than dermo-pharmaceutical, they are formulated for administration by the intradermal, subcutaneous or oral route.

According to the cosmetic and dermo-pharmaceutical aspects of the invention, the composition based on NDGA, or its derivatives, is applied directly to the skin of the parts of the body to be treated, for example on the hips, thighs, lower legs. belly, belly, arms, chest.

Alternatively, the cosmetic or dermo-pharmaceutical composition can be incorporated or adsorbed in textiles capable of being placed directly in contact with the skin in order to allow continuous topical delivery.

The compositions of the invention are presented in the form of an emulsion, milk, lotion, gel, ointment, body oil, soap, spray, stick, capsules, tablets, patch, syrup, solutes, or solutions. The active principle (s) is (or are) incorporated (s) as is (s) in the composition in the form of liposomes, macro-, micro-, nanoparticles, or are absorbed (s) on an organic or inorganic support .

Apart from NDGA, or its derivatives, the compositions of the invention generally contain other ingredients, for example an excipient which is acceptable from the cosmetic or physiological point of view.

For cosmetic use, the compositions of the invention may contain any ingredient usually used in cosmetics, such as gelling polymers, surfactants, emulsifying agents, plant extracts, marine extracts, water or liposoluble active principles, biotechnological extracts, sun filters , and essential oils.

In addition, the cosmetic and dermo-pharmaceutical compositions of the invention may contain a second active principle. In the case of a cosmetic composition, the second active principle can be any active agent commonly used in cosmetics. It is preferably chosen from the following active principles: a slimming active, a draining active, an activator of the microcirculation, a protector of the blood capillaries, or else a mixture of at least two of these active principles.

When the compositions based on NDGA, or its derivatives, contain, as an additional active principle, a slimming active, the latter is preferably an activator of lipolysis, such as caffeine, or a derivative of the latter such as siloxanetriol. caffeine alginate, or xanthic base promoting lipolysis. The slimming active can also be an inhibitor of lipoprotein lipase.

As an example of a second active principle stimulating cutaneous microcirculation, mention may be made of extracts of plant origin, for example containing flavonoids, ruscogenins and derivatives, and aescin contained in the extracts of Ruscus aculeatus.

According to another variant of the invention, nor-dihydroguaiaretic acid, or a derivative thereof, is used as active ingredient in a slimming food composition, for use human. According to this aspect of the invention, the composition based on NDGA, or its derivatives, is formulated for oral ingestion, for example in the form of tablets, granules, rusks, capsules, drinks, cakes, etc. The content of NDGA, or its derivatives, is preferably of the order of 0.0001% to 10% by weight, relative to the total weight of the composition, for example at a concentration of between 0.001% and 5% by weight, of preferably between 0.001% and 1% by weight. In the context of the invention, the term “food composition” includes food supplements for nutritional or physiological use.

According to the present invention, a composition is said to be slimming when it allows, in the more or less long term, to make or make an individual appear thinner. Such an effect is for example obtained by reducing the volume or density of adipose tissue, or by modifying its appearance. It is for example measured by reducing the diameter of the thighs, belly, hips, arms, etc.

Different aspects of the invention are illustrated in the figures:

Figure 1: schematic representation of the GPDH activity test at 340 nm (measurement of the NAD consumed in the presence of dihydroxyacetone phosphate). Abbreviations: D = day; GPDH = Glycerophosphate dehydrogenase.

Figure 2: schematic representation of the enzymatic reaction used in the GPDH activity tests. Abbreviations: DHAP = dihydroxyacetone phosphate.

Figure 3: Standard curve: NDGA and Morine.

Figure 4: Standard curve: Quercetin.

Figure 5: GPDH specific activity in nmoles / min / mg of protein in the presence of 2.5 μM NDGA and 10 μM NDGA.

Figure 6: Specific GPDH activity in nmoles / min / mg of protein in the presence of 2.5 μM

Morine and 10 μM Morine.

Figure 7: GPDH specific activity in nmoles / min / mg of protein in the presence of 2.5 μM Quercetin and 10 μM Quercetin

Figure 8: NDGA dose-response curve, illustrating the specific GPDH activity in nmoles / min / mg of protein in the presence of different concentrations of NDGA (0 to 10 μM NDGA). The inhibition of GPDH activity by NDGA is concentration-dependent. EXAMPLES

MATERIAL AND METHODS

A.1 CULTURE OF HUMAN MULTIPOTENT CELLS:

Multipotent stem cells (called “Primo 2CA cells”) are obtained from human adipose tissue, by implementing the method described in international patent application PCT / FR 03/02439. More particularly, the stem cells are obtained by the following process: a) enzymatic digestion of a sample of adipose tissue coming from a healthy child, aged less than ten years, for example from a newborn or a child aged 2 or 3 months to 8 years. They may be male or female children; b) recovery of a cell fraction devoid of adipocytes, containing all the cell types present in the preparation obtained according to (a), (for example preadipocytes, stem cells, endothelial cells, pericytes, mast cells ...) with the exception adipocytes. Fat cells can be removed, for example, by centrifugation; c) in vitro culture for at least 12 hours, of the cell fraction obtained according to step (b), and preferably for 12 to 80 hours, for example 12 to 72 hours. The seeding of the cells is preferably carried out at a density of between

1000 and 5000 cells / cm ² , for example 1000 to 3500 cells / cm ² . The culture medium used for this stage of the process is normally a DMEM type culture medium, supplemented with fetal serum without addition of other growth factors. For example, a particularly suitable medium is the following: DMEM + 10% of decomplemented fetal calf serum + antibiotics (100 U / ml of penicillin, 100 μg / ml of streptomycin) .; d) selection of two cellular subpopulations, called “CA” population and “CS” population, the CA population having an adhesion speed less than 12 hours, and the “CS” population having an adhesion speed greater than 12 hours (for example from 48 to 72 hours). The CS population is found after 12 hours of culture, in suspension in the culture medium, while the CA population adheres to the dishes. Stem cells are found only in the CA subpopulation; e) enrichment of the “CA” population until a population of cells capable of entering a state of quiescence is obtained. This enrichment is obtained by culturing the “CA” population, in a medium without added growth factors, for example the DMEM culture medium + 10% of decomplemented fetal calf serum + antibiotics (100 U / ml of penicillin, 100 μg / ml streptomycin). Cell transfer is performed when the cells reach 80% confluence and the seeding is carried out at high density, that is to say at a density between

1000 to 5000 cells / cm ² , preferably between 2000 and 2500 cells / cm ² . At each cell transfer, the cells are diluted by 2 or 3 at most, for approximately

50-80 population doublings (stage at which the CA population is highly enriched in stem cells.) The CA population can be considered to have reached quiescence when there is spontaneous arrest of proliferation at approximately 70% confluence; f) induction of an increased proliferation of stem cells from the “CA” population, for example by addition of a growth factor, such as human FGF-2. After reaching quiescence, the proliferation of cells of the CA population is induced by trypsinization and dilution of the cells in new dishes.

The multipotent cells (“stem”) are always maintained at 37 ° C in an incubator ensuring a humid atmosphere (95% air; 5% C0 ₂ ).

Culture medium for proliferating stem cells:

Multipotent cells are cultured in DMEM medium enriched with 10% fetal calf serum (FCS) decomplemented (heating 20 minutes at 56 ° C), supplemented with antibiotics (62 μg / ml of penicillin and 50 μg / ml of streptomycin) and in human FGF-2 at a final concentration between 2.5 ng / ml and 10ng / ml (Sigma F0291).

Interview:

The maintenance cultures are sown in boxes 100 mm in diameter at the rate of 2.5 × 10 ⁵ cells per box. The environment is changed every two days. The cells are trypsinized (Gibco Trypsin-EDTA 1X Invitrogen Corporation) when they reach approximately 70% confluence. They thus retain their cellular plasticity.

Adipocyte differentiation protocol:

When the cells reach confluence, they are maintained for an additional 24 to 48 hours in the culture medium in the absence of FGF-2. At this stage, adipocyte differentiation can then be induced.

At D0 (start of differentiation), the culture medium is changed and the cells are now cultured in DMEM / F12 medium (1: 1), in the absence of fetal calf serum (FCS) but then supplemented with antibiotics (62 μg / ml of penicillin and 50 μg / ml of streptomycin) and in various products detailed in the following table:

th -

The medium is then changed every two days until day 12.

A.2 Screening of products - MTT test for toxicity

Preparation of stock solutions of the tested products:

The preadipocyte cells (called “Primo 2CA” stem cells) are seeded in 96-well plates at the rate of 5.10 ³ cells per well in the proliferation medium (DMEM + 10% FCS) in the absence of human FGF-2. 24 hours later, the differentiation is induced according to the differentiation protocol described above by adding or not adding the products to be tested in the culture medium.

The MTT test ((3-4,5-dimethylthiazol-2,5-diphenyl tetrazolium bromide) makes it possible to eliminate all the products which prove to be toxic for the cells. MTT is a soluble yellow dye which is said to be supravital with a metabolization step, i.e. it is absorbed by the cells alive then metabolized by mitochondrial enzymes (succinate dehydrogenase) into a dark blue compound: formazan, which is in the form of purple crystals insoluble in aqueous medium. The formation of formazan depends directly on the activity of succinate dehydrogenase and therefore on the number of viable cells.

In order to obtain final concentrations of the order of μM, 1 μl of the mother solutions previously prepared is taken in 1 ml of adipocyte differentiation medium. 100 μl of these dilutions are then deposited per well in duplicate.

For the positive controls, the medium is devoid of product but contains DMSO.

The wells located around the 96-well plate are not sown. Otherwise, they could generate errors when reading with a spectrophotometer. The wells representing the blank remain empty. They measure the background noise. The wells representing the positive controls (basic mortality taken as 0% mortality) do not receive any product. The wells receiving the products to be tested (1/1000 of the mother solutions prepared) are seeded at 5.10 ³ cells per well.

The differentiation medium with or without the drugs to be tested is changed every other day until ^day 12.

On D12, add 10 μl of MTT per well (ROCHE Cell Proliferation kit I MTT). The plates are placed in an incubator ensuring a humid atmosphere (95% air; 5% C0 ₂ ) for 4 hours. Then add 100 μl per well of the solubilization solution (ROCHE Cell Proliferation kit I MTT). They are then placed in the incubator overnight.

The reading is made the next morning with a spectrophotometer at 550 nm.

The ODs are then compared with the OD of the control taken as 0% mortality.

% mortality = [(D.O (0% mortality)) - (D.O (product)) * 100] / (D.O (0% mortality)

A.3 GLYCEROPHOSPHATE DEHYDROGENASE (GPDH) ACTIVITY SCREENING

Once the MTT cytotoxicity test was completed, certain products were selected for their absence of cytotoxicity. In order to monitor the possible effect of these products on adipocyte differentiation, they are then screened by GPDH activity tests at 340 nm (measurement of the reduced NAD consumed in the presence of dihydroxyacetone phosphate). (See Figure 1) Principle and protocol for assaying Glycerophosphate dehydrogenase GPDH: Adipocyte differentiation is carried out on 24-well plates. The starting seeding is 10 ⁵ cells per well, 24 hours before the start of differentiation.

At D12, the plates are put on ice. The culture medium is aspirated. The wells are gently rinsed with PBS (Gibco BRL, Life Technologies) so as not to peel off the cell mat. 200 μl of cold homogenization buffer (Tris 242g (20 mM); EDTA 37.2 mg (1mM); β-mercaptoethanol 7 μl (1mM); H ₂ 0 100 ml qs; adjust to pH 7.3) are added per well. The cells are then recovered with a Policeman or a pipette tip and then placed in 1.5 ml tubes. The homogenate obtained is subjected to a sonic treatment between 20 and 30 Hz for 10 to 15 seconds while keeping the tubes in ice. The initial rate of disappearance of NADH at 340 nm is determined, which makes it possible to calculate the amount of substrate consumed per well, then the specific enzymatic activity after assaying the proteins (see FIG. 2).

The dosage :

In a spectrophotometer tank are added: 830 μl of water, 100 μl of assay buffer (Triethanolamine 14.9g (1 M); β-mercaptoethanol 7 μl (1 mM); EDTA 930 mg (25mM); H ₂ 0 100 ml qs; adjust the pH to 7.7) at room temperature, 10 μl of NADH at 12.5 mM in initial concentration (Roche).

It is checked that the basic O.D is around 0.6 to 0.8. Then 50 μl of homogenate and 10 μl of DHAP at 20 mM initial are added (Roche).

The variation of D.O at 340 nm is then read.

Calculations :

The extinction coefficient of NADH at 340 nm is 6220 (the OD at 340 nm of a 1 M NADH solution = 6220). Hence for a 1 nmol / ml solution → 0.00622

D.O / min (oxidation rate of NADH) * Volume of the homogenization buffer (200 μl)

0.00622 * Volume of homogenate dosed (generally 50 μl)

expressed in nmol / min / well Example of calculation:

Activity = 12 nmol / min for 50 μl of homogenate In total for 200 μl of homogenate: (12 * 200) 150 = 48 nmol / min / well

Protein determination: for 5 μl of homogenate we obtain a D.O = 0.1 So for 200 μl of homogenate (1 well) = (200 * 0.1) / 5 = 4 BSA standard curve: 1 μg of protein = 0.05 D.O

So for 1 well: (4 * 1) / 0.05 = 80 μg of protein

AS = (48 * 1000) / 80 = 600 nmol / min / mg protein

A.5 DOSE-RESPONSE CURVE

The cells are seeded in a 24-well plate at the rate of 10 ⁵ cells per well in “conventional” culture medium used for maintenance but without human FGF-2. 24 hours later, the cells are placed in differentiation medium containing or not containing the retained product (NDGA) at increasing concentrations ranging from 2 to 10 μM.

The differentiation medium, whether or not containing NDGA, is changed every two days. The ddiifffféérreenncciiaattiioonn eesstt aarrrrêêttééee tthhee 1I2 ^th day and activity assays dehydrogenase glycerophosphatase are made.

B. RESULTS

B.1 MTT CYTOTOXICITY TEST

All the compounds are tested in duplicate at different final concentrations (10; 5; 1; 0.5; 0.1 μM).

The results obtained with the MTT cytotoxicity test are reported in Table 1.

It is noted that the three compounds tested (quercetin, nor-dihydroguaiaretic acid, morine), do not exhibit any toxicity for the cells.

All the compounds are therefore tested at the end of adipocyte differentiation for their anti-adipogenic effect by GPDH activity tests. B.2 SCREENING BY GLYCEROPHOSPHATE DEHYDROGENASE (GPDH) ACTIVITY ASSAYS

The selected compounds are tested in triplicate at two different concentrations

The results obtained are reported in Tables 2 and 3 and in Figures 3 and 4. Figures 5, 6 and 7 present the histograms revealing the specific activity in nmoles / min / mg of proteins of all the non-cytotoxic compounds tested.

NDGA appears to be non-cytotoxic and anti-adipogenic (Table 4) at the concentrations tested.

B.4 DOSE-RESPONSE CURVE

The dose-response curve provides information on the specific activity (in nmoles / min / mg of protein) as a function of the different concentrations of NDGA (0; 2; 4; 6; 8 and 10 μM). The results are reported in Table 4 and Figure 8. The IC50 (concentration required for 50% inhibition) is 4.5 μM.

In conclusion, nor-dihydroguaiaretic acid appears to be non-toxic and has an anti-adipogenic effect on human cells during their differentiation into adipocytes. It is therefore suitable for use in therapy and in cosmetics, in pharmaceuticals (or in therapy) and in food.

C COSMETIC FORMULATIONS

NDGA-based compositions can be included in cosmetic formulations of the following types: SLIMMING CREAM

INCI name Quantity

WATER QSP 100

GLYCERINE 5.00

CARBOPOL 0.20

SODIUM SALT OF ETHYLENE DIAMINE TETRACETIC ACID 0.10

LEAF OIL from CAMELLIA SINENSIS 2.00

POLYISOBUTENE HYDROGENE 8.00

STEARATE DE GLYCERYLE SE 2.00

DIMETHICONE 1.00

GLYCERYLE STEARATE AND PEG-100 STEARATE 1.50

TRIETHYLHEXANOINE 5.00

STEARIC ACID 2.00

CETYL ALCOHOL 1.00

SILICA 1.00

DICAPRYLE MALEATE 6.00

GLYCERYLE STEARATE 1.00

DIMETHICONE 3.50

WATER 2.30

TRIETHANOLAMINE 0.50

PHENOXYETHANOL, METHYL p-HYDROXYBENZOATE, PROPYL p-HYDROXYBENZOATE, BUTYL p-HYDROXYBENZOATE, 0.70 p-HYDROXYBENZOATE

FRAGRANCE 0.30

TOCOPHEROL ACETATE 0.50

CAFEINE 2.00

SODIUM HYALURONATE 0.04

EXTRACT FROM CENTELLA ASIATICA 1.00

EXCERPT FROM RUSCUS ACULEATUS 1.00

NDGA or derivatives 0.001 to 10 SLIMMING GEL

INCI name Quantity

WATER QSP 100

SODIUM SALT OF ETHYLENE DIAMINE TETRACETIC ACID 0.20

CARBOPOL 0.50

GLYCERINE 3.00

POLYGLYCERYLMETHACRYLATE AND PROPYLENEGLYCOL 5.00

DIPROPYLENEGLYCOL 3.00

BUTYLENEGLYCOL 5.00

METHYL p-HYDROXYBENZOATE, PROPYL p-HYDROXYBENZOATE, BUTYL p-HYDROXYBENZOATE, 0.65 p-HYDROXYBENZOATE

TRIETHANOLAMINE 0.50

CAFEINE 2.00

EXCERPT FROM RUSCUS ACULEATUS 1.00

NDGA or derivatives 0.001 to 10

SLIMMING SERUM

INCI name Quantity

WATER QSP 100

TRISODIC SALT OF ETHYLENE DIAMINE TETRACETIC ACID 0.10

HYDROXYETHYLCELLULOSE 0.10

XANTHANE GUM 0.30

BUTYLENEGLYCOL 5.00

SORBITANE POLYOXYETHYLENIC MONOSTEARATE (20) 1.00

FRAGRANCE 0.05

TOCOPHEROL ACETATE 0.10

SODIUM CITRATE 0.65

CAFEINE 2.00

NDGA or derivatives 0.001 to 10 SLIMMING TINTED CREAM

INCI name Quantity

WATER QSP 100

DOUBLE SILICATE OF ALUMINUM AND MAGNESIUM 0.50

CELLULOSE GUM 0.35

GLYCERINE 3.00

POLYVINYLPYRROLIDONE (PVP) 5.00

DIPROPYLENEGLYCOL 0.05

PROPYLENEGLYCOL 2.00

METHYL p-HYDROXYBENZOATE, PROPYL p-HYDROXYBENZOATE, BUTYL p-HYDROXYBENZOATE, 1.00 p-HYDROXYBENZOATE

XANTHAN GUM 0.20

TRIETHANOLAMINE 0.70

ISOPROPYLE PALMITATE 4.00

MINERAL OIL 2.00

HEXYLDECANOL 3.00

STEARATE DE GLYCERYLE 2.00

STEARIC ACID (STEARIC ACID) 2.40

OLEIC ACID (OLEIC ACID) 0.50

SORBITANE POLYOXYETHYLENIC MONOSTEARATE (60) 0.70

TOCOPHEROL 0.50

TITANIUM DIOXIDE (TITANIUM DIOXIDE) 7.00

IRON OXIDE (IRON OXIDE) 4.00

NYLON-2 6.00

SODIUM HYALURONATE 0.01

FRAGRANCE 0.50

PROPYLENEGLYCOL DICAPRYLATE / DICAPRATE 7.20

NDGA or derivatives 0.001 to 10

Table 1

r

Table 2

Table 4

BIBLIOGRAPHICAL REFERENCES

1 Kelly KA, Gimble JM (Endocrinology, 1998 May; 139 (5): 2622-8)

2 Hida Y, Kawada T, Kayahashi S, Ishihara T, Fushili T (Life Sci. 1998; 62 (14); PL205-11)

„Safonova I, Reichert U, Shroot B, (Biochem Biophys Res Commun. 1994 Oct 28; 204 (2): 498- 504)

. Safonova I, Darimont C, Amri EZ, Grimaidi P, Ailhaud G, Reichert U, Shroot B, (Mol Cell Endocrinol, 1994 Sep; 104 (2): 201-11)

,. Han J, Farmer SR, Kirkiand JL, Corkey BE, Yoon R, Pirtskhalava T, Ido Y, Guo W, (J Nutr. 2002 May; 132 (5): 904-10)

_R Garcia Palacios V, Morita I, Murota S, (Prostagiandins Leukos Essent Fatty Acids, 2001 Oct; 65 (4): 215-21)

7 Thuillier P., et al (Biochem. J. 2002, 366, 901-910)

8 Ye H. & Serrero G., (Biochem. J. 1998, 330, 803-809)

g Jiang HP & Serrero G, (Proc. Nat. Acad. Sci. USA 1992, 89, 7856-7860). ₀ Zilberfarb V. et al. (J. Cell Se, 1997, 110, 801-807) 1 Wabitsch M et al., (Int. J. Obes. Relat. Metab. Disord. 2001, Jan., 25 (1): 8-15)

Harry's Cosmeticology, JB Wilkinson and RJ Moore (Eds), 7th Edition 1982, Chemical ² Publishing, New York, pp. 718, 722-724.

Claims

1. Use of nor-dihydroguaiaretic acid (C ₁₈ H ₂₂ O ₄ ), or a derivative thereof, as active ingredient as an inhibitor of the formation of human adipocytes, in a composition cosmetic, or in a dermo-pharmaceutical composition for the preparation of a medicament for the treatment or prevention of pathologies involving a dysfunction of adipocyte differentiation in humans.

2. Use according to claim 1, characterized in that the active principle is a natural molecule, obtained by plant extraction.

3. Use according to claim 1 or 2, characterized in that the active principle is contained in a plant extract.

4. Use according to claim 1, characterized in that the active principle is a synthetic molecule.

5. Use according to any one of claims 1 to 4, characterized in that the cosmetic composition is intended for the prevention or treatment of non-pathological overweight, the prevention or treatment of cellulite, the prevention or to the treatment of dimples, or to skin tightening.

6. Use according to any one of claims 1 to 5, characterized in that the dermo-pharmaceutical composition is intended for the prevention or treatment of pathological overweight, such as overweight and obesity.

7. Use according to any one of claims 1 to 6, characterized in that the cosmetic or dermo-pharmaceutical composition contains nor-dihydroguaiaretic acid (C ₁₈ H ₂₂ O ₄ ), or the derivative thereof, at a concentration of between 0.0001% and 10% by weight, preferably between 0.001% and 5% by weight, and more preferably between 0.001% and 1.0% by weight, relative to the total weight of the composition

8. Use according to any one of claims 1 to 9, characterized in that the cosmetic or dermo-pharmaceutical composition is applied by the cutaneous, percutaneous or transcutaneous route.

9. Use according to any one of claims 1 to 8, characterized in that the cosmetic or dermo-pharmaceutical composition is in the form of an emulsion, milk, lotion, gel, ointment, body oil, soap, spray, stick, capsules, tablets, patch, syrup, solutes, solutions.

10. Use according to any one of claims 1 to 9, characterized in that the active ingredient (s) are incorporated (s) in the composition in the form of liposomes, macro-, micro-, nanoparticles, or are absorbed (s) on an organic or mineral support.

11. Use according to any one of claims 1 to 10, characterized in that the cosmetic composition also contains a cosmetically acceptable excipient, and optionally any other ingredient usually used in cosmetics, such as gelling polymers, surfactants, emulsifying agents, plant extracts, marine extracts, water- or fat-soluble active ingredients, biotechnological extracts, sun filters, essential oils.

12. Use according to any one of claims 1 to 11, characterized in that the dermo-pharmaceutical composition additionally contains an excipient which is acceptable from the physiological point of view.

13. Use according to claim 1, characterized in that the cosmetic or dermo-pharmaceutical composition contains a second active principle.

14. Use according to claim 13, characterized in that the second active principle is chosen from the following active principles: a slimming active, a draining active, an activator of the microcirculation, a protector of the blood capillaries.

15. Use according to claim 14, characterized in that the slimming active is an activator of lipolysis, such as caffeine, or an inhibitor of Lipoprotein Lipase.

16. Use according to any one of claims 1 to 15, characterized in that the cosmetic or dermo-pharmaceutical composition is incorporated or bound or adsorbed in textiles capable of being brought directly into contact with the skin in order to allow delivery topical continues.

17. Slimming cosmetic composition comprising nor-dihydroguaiaretic acid (C ₁₈ H ₂₂ O ₄ ), or a derivative thereof, in combination with a lipolytic agent such as caffeine or a derivative thereof such as caffeine siloxanetriol alginate, or a xanthic base promoting lipolysis.

18. Slimming cosmetic composition comprising nor-dihydroguaiaretic acid (C ₁₈ H ₂₂ θ ₄ ), or a derivative thereof, in combination with a Lipoprotein Lipase inhibitor.

19. Slimming cosmetic composition comprising nor-dihydroguaiaretic acid (C ₁₈ H ₂₂ O ₄ ), or a derivative thereof, in combination with a plant extract stimulating the skin microcirculation such as flavonoids, ruscogenins and derivatives, l aescin contained in the extracts of Ruscus aculeatus.

20. Use of nor-dihydroguaiaretic acid, or a derivative thereof, as active ingredient in a slimming food composition for human use.

21. Use of nor-dihydroguaiaretic acid, or a derivative thereof, as active ingredient in a pharmaceutical composition for the preparation of a medicament for the treatment or prevention of pathologies involving a dysfunction of adipocyte differentiation in humans, said drug being formulated for intradermal, subcutaneous or oral administration.