PARTICULAR PYRIDINEDICARBOXYLIC ACID DERIVATIVE/ANTIOXIDANT COMBINATION

Abstract

The invention relates to a cosmetic composition comprising one or more pyridinedicarboxylic acid derivatives of general formula (I) or a salt thereof and one or more antioxidants chosen from flavone heterosides, polyhydroxylated stilbenes, pyrimidinecarboxylic acid derivatives and ascorbic acid esters.

##STR00001##

Claims

1. Cosmetic composition comprising: one or more pyridinedicarboxylic acid derivatives of general formula (I) or a salt thereof: ##STR00004## in which R.sub.1 and R.sub.2 represent, independently of each other, OH, OR′, —NH.sub.2, —NHR′ or —NR′R and R′ and R″ represent, independently of each other, a linear or branched, saturated or unsaturated C.sub.1-C.sub.18 alkyl group, or an aryl group, this alkyl or aryl group being optionally substituted with an OH, alkoxy, acyloxy, amino or alkylamino group, or R′ and R″ together represent a heterocycle, and one or more antioxidants chosen from flavone heterosides, polyhydroxylated stilbenes, pyrimidinecarboxylic acid derivatives and ascorbic acid esters.

2. Cosmetic composition according to claim 1, characterized in that R.sub.1 and R.sub.2 represent, independently of each other, —OH, —OCH.sub.3, —O—CH.sub.2—CH.sub.3, —O—CH(CH.sub.3).sub.2, —O—CH.sub.2—O—COCH.sub.3, —NH—CH.sub.2—CH.sub.2—CH.sub.3, —NH—CH.sub.2—CH.sub.2OH, —NH—CH.sub.2—CH.sub.2—CH.sub.2OH and —NH—CH.sub.2—CH.sub.2OCH.sub.3.

3. Cosmetic composition according to claim 1 or 2, characterized in that —COR.sub.1 and —COR.sub.2 are, respectively, in positions 2 and 3, or 2 and 4, of the pyridine nucleus.

4. Cosmetic composition according to any one of the preceding claims, characterized in that the salt of the derivative of formula (I) is a salt chosen from the sodium or potassium double salt, the zinc (Zn.sup.2+), calcium (Ca.sup.2+), copper (Cu.sup.2+), iron (Fe.sup.2+), strontium (Sr.sup.2+), magnesium (Mg.sup.2+) or manganese (Mn.sup.2+) salts, the triethanolamine, monoethanolamine, diethanolamine, hexadecylamine, N,N,N′,N′-tetrakis(2-hydroxypropyl)ethylenediamine and tris(hydroxylmethyl)aminomethane salts, hydroxides, carbonates and chlorides.

5. Composition according to one of the preceding claims, characterized in that the pyridinedicarboxylic acid derivative is chosen from: 2,4-pyridinedicarboxylic acid or the zinc or sodium salt thereof, 2,3-pyridinedicarboxylic acid or the zinc or sodium salt thereof, dimethyl 2,4-pyridinedicarboxylate, dimethyl 2,3-pyridinedicarboxylate, diethyl 2,4-pyridinedicarboxylate, diethyl 2,3-pyridinedicarboxylate, diisopropyl 2,4-pyridinedicarboxylate, 2,4-bis(n-propylamido)pyridine, di(acetyloxymethyl) 2,4-pyridinedicarboxylate, diethyl 2,5-pyridinedicarboxylate, dimethyl 2,5-pyridinedicarboxylate, 2,4-bis(2-hydroxyethylamido)pyridine, and 2,4-bis(3-hydroxypropylamido)pyridine.

6. Composition according to any one of the preceding claims, characterized in that the derivative is in ester form.

7. Composition according to one of the preceding claims, characterized in that the derivative is diethyl 2,4-pyridinedicarboxylate.

8. Composition according to one of the preceding claims, characterized in that the derivative(s) or a salt thereof are present in an amount ranging from 10.sup.−3% to 10% by weight, preferably from 0.1% to 8% by weight and better still from 0.5% to 5% by weight, relative to the total weight; of the composition.

9. Composition according to any one of the preceding claims, characterized in that the antioxidant is chosen from baicalin, resveratrol, ectoin and ascorbyl palmitate, and mixtures thereof.

10. Composition according to any one of the preceding claims, characterized in that the antioxidant(s) are present in an amount ranging from 0.1% to 15% by weight, preferably from 0.5% to 10% by weight and even better still from 0.7% to 6% by weight, relative to the total weight of the composition.

11. Cosmetic process for treating human keratin fibres and/or the scalp, comprising the application of a composition according to any one of the preceding claims to said fibres and/or the scalp.

12. Use of the composition according to any one of claims 1 to 10, for inducing and/or stimulating the growth of human keratin fibres such as the hair and the eyelashes, and/or for curbing their loss.

13. Use according to claim 12, for treating androgenic alopecia.

Description

EXAMPLES

[0074] The following protocol was followed: the tests were performed on human keratinocytes in culture, seeded on Greiner brand 48-well plates, coated with bovine collagen I.

[0075] The plates were prepared according to the following procedure: the solution of bovine collagen I at 0.1 mg/ml was prepared by dilution in phosphate-buffered saline (PBS) of the bovine collagen I solution sold by the company Life Technologies. Each well was immersed with 1 ml of this dilution, which was left at the bottom of the wells for 1 hour at 37° C. At the end of the incubation, the collagen solution was removed and the wells were rinsed twice with 1 ml of PBS. The plates were then stored at 4° C. until the time of use.

[0076] The tests are performed using primary human keratinocytes at a rate of 23 800 cells/cm′ of wells coated with bovine collagen I as explained previously, followed by culturing for 72 hours in the presence of 500 μl of KGM medium sold by the company Lonza, supplemented with: [0077] 0.1% by weight of gentamicin sulfate/amphotericin mixture sold under the commercial brand GA-1000 by the company Lonza (CC-3101/CC-4131), [0078] 0.4% by weight of bovine pituitary gland extract sold under the commercial brand BPE by the company Lonza, [0079] 0.1% by weight of insulin sold under the commercial brand insulin by the company Lonza, [0080] 0.1% by weight of hydrocortisone and [0081] 0.1% by weight of epidermal growth factor (or recombinant human EGF sold under the commercial brand Epidermal growth factor by the company Lonza),
at 37° C. under an atmosphere saturated with water and containing 5% CO.sub.2.

[0082] The cells were then treated with the various compounds (stemoxydine, vitamin C, resveratrol, baicalin, ectoin, ascorbyl palmitate and mixtures thereof with stemoxydine) in concentrations ranging up to the highest non-cytotoxic concentration, for 72 hours under normoxia (21% oxygen).

[0083] Following this culturing and treatment, the cell lawns were washed with phosphate-buffered saline (or PBS) and then lysed using a lysis buffer proposed in the kit from the supplier Qiagen. The RNAs were then extracted using the RNeasy isolation kit and the Qiacube robotic workstation, both sold by the company Qiagen, according to the manufacturer's instructions.

[0084] The quantity and quality of RNAs were controlled using the LabChip® GX bioanalyser from Perkin-Elmer before performing reverse transcription (RT) using the Qiagen kit and according to the supplier's recommendations (QuantiTect reverse transcription kit). The cDNA obtained following the RT was then amplified by real-time quantitative PCR using a specific kit sold under the commercial brand LightCycler® 480 SYBR Green Master Mix by the company Roche (Cat. No. 14123920) and an LC480 thermocycler (Roche). The PCRs were performed in triplicate (n=3). The information relating to the primers used is presented below.

[0085] Priming was performed using specific standard primers sold by the company Qiagen, the references for which are BNIP3/QT00024178/Hs BNIP3-1-SG Quantitect Assay Primer; CA9/QT00011697/Hs_CA9-1-SG Quantitect Assay Primer; EGNL3/QT00025900/Hs_EGNL3-1-SG Quantitect Assay Primer; RPL13A/QT00089915/Hs_RPL13A-1-SG Quantitect Assay Primer, and the fluorescent probe of brand name SYBR Green sold by the company specified previously.

[0086] The PCR was performed in three phases: [0087] denaturing phase for 10 minutes at 95° C., [0088] amplification phase which consists of 45 cycles comprising: [0089] a step of denaturing for 30 seconds at 95° C., [0090] a step of hybridization for 30 seconds at 60° C., and [0091] a step of elongation at 72° C. for 30 seconds, [0092] melting phase for ensuring the quality of the hybridizations.

[0093] The incorporation of SYBR Green into the amplified DNA was measured continuously during the amplification cycles. These measurements make it possible to obtain curves of fluorescence intensity as a function of the PCR cycles and thus to evaluate the relative expression of each marker from the cycle thresholds (Ct), corresponding to the number of cycles required to appropriately detect a fluorescence level. For each marker and for each condition, the relative expression (RE) value was normalized relative to the expression of the reference gene RPL13.

[0094] The expression of each gene is normalized by that of the “stable referent gene” (or “housekeeping gene” RPL13A, ribosomal gene). The results (“fold change” (Fc)) are expressed relative to the control.

[0095] The genes used are hypoxia-sensitive (HIF-1 alpha signalling pathway) and are collated in Table 1 below.

TABLE-US-00001 TABLE 1 Accession Name Abbreviation number Function Carbonic CA9 NM_001216 Regulation of the anhydrase IX intracellular pH BCL2/adenovirus BNIP3 NM_004052 Control of the E1B 19 kDa- cell apoptosis interacting protein Prolyl EGLN3 (or NM_022073 Hydroxylation of hydroxylase Egg-laying collagen and HIF- nine homolog 1alpha 3, or PHD3) Ribosomal RPL13 NM_000977 Referent gene protein L13

[0096] Three hypoxia-sensitive genes (BNIP3, EGLN3 and CA9) were used in the following examples. Three tests were performed according to the protocol described above for each gene and for each compound or combination. The results are expressed as a function of the expression of the referent gene RPL13 (control) (1.00) and indicated in the tables below, the mean and the standard deviation being indicated in parentheses.

Example 1 (Comparative): Effect of Stemoxydine Alone or Combined with Vitamin C on the Expression of a Selection of Genes in Relation with the Described Effects of Hypoxia

[0097]

TABLE-US-00002 TABLE 2 Vitamin C Vitamin C Stemoxydine Gene 100 μM 10 μM 300 μM Control BNIP3 0.98/1.32/1.07 1.02/1.13/0.89 1.51/1.65/2.13 1 ± 0 (1.12 ± 0.18) (1.01 ± 0.12) (1.76 ± 0.32) EGLN3 1.05/1.51/1.16 0.92/1.41/0.89 2.15/2.09/3.56 1 ± 0 (1.24 ± 0.24) (1.07 ± 0.29) (2.60 ± 0.83) CA9 1.43/1.51/0.74 1.19/1.43/0.43 3.87/3.86/5.86 1 ± 0 (1.22 ± 0.42) (1.02 ± 0.52) (4.53 ± 1.15) Vitamin C 100 μM + Vitamin C 10 μM + Gene Stemoxydine 300 μM Stemoxydine 300 μM BNIP3 1.72/1.98/2.3 1.63/1.9/2   (2 ± 0.23) (1.84 ± 0.19) EGLN3 3.01/3.35/4.33 2.15/2.64/2.43 (3.56 ± 0.68) (2.40 ± 0.24) CA9 6.39/6.74/8.13 5.33/4.8/4.93 (7.09 ± 0.92) (5.02 ± 0.28)

Example 2 (Invention): Effect of Stemoxydine Alone or Combined with Resveratrol on the Expression of a Selection of Genes in Relation with the Described Effects of Hypoxia

[0098]

TABLE-US-00003 TABLE 3 Resveratrol Resveratrol Stemoxydine Genes 1 μM 0.3 μM 300 μM Control BNIP3 1.10/2.35/2.57 1.05/1.53/1.21 1.51/1.65/2.13 1 ± 0 (2.01 ± 0.79) (1.26 ± 0.24) (1.76 ± 0.32) EGLN3 1.62/4.65/4.69 1.31/2.36/2.10 2.15/2.09/3.56 1 ± 0 (3.65 ± 1.76) (1.92 ± 0.55) (2.60 ± 0.83) CA9 2.34/1.97/1.75 1.82/1.75/1.31 3.87/3.86/5.86 1 ± 0 (2.02 ± 0.33) (1.63 ± 0.28) (4.53 ± 1.15) Resveratrol 1 μM + Resveratrol 0.3 μM + Genes Stemoxydine 300 μM Stemoxydine 300 μM BNIP3 2.28/4.68/7.23 1.9/2.21/3.39 (4.73 ± 2.48) (2.5 ± 0.79) EGLN3 5.04/13.13/13.18 4.68/5.46/11.53 (10.45 ± 4.69) (7.22 ± 3.75) CA9 8.99/10.87/10.38 9.98/7.96/11 (10.08 ± 0.98) (9.65 ± 1.55)

[0099] The results show a synergistic effect on the expression of the genes BNIP3, EGLN3 and CA9 when stemoxydine is combined with resveratrol. In addition, these effects are higher than those for the stemoxydine+vitamin C combination.

Example 3 (Invention): Effect of Stemoxydine Alone or Combined with Baicalin on the Expression of a Selection of Genes in Relation with the Described Effects of Hypoxia

[0100]

TABLE-US-00004 TABLE 4 Baicalin Baicalin Stemoxydine Gene 30 μM 3 μM 300 μM Control BNIP3 1.14/1.41/1.15 0.99/1.27/1.06 1.51/1.65/2.13 1 ± 0 (1.23 ± 0.15) (1.11 ± 0.15) (1.76 ± 0.32) EGLN3 1.70/2.47/2.03 1.00/1.33/1.25 2.15/2.09/3.56 1 ± 0 (2.07 ± 0.39) (1.19 ± 0.17) (2.60 ± 0.83) CA9 1.06/1.07/0.6 1.27/1.18/0.74 3.87/3.86/5.86 1 ± 0 (0.91 ± 0.27) (1.06 ± 0.28) (4.53 ± 1.15) Baicalin 30 μM + Baicalin 3 μM + Gene Stemoxydine 300 μM Stemoxydine 300 μM BNIP3 2.10/2.19/2.07 2.10/1.99/2.77 (2.12 ± 0.06) (2.29 ± 0.42) EGLN3 5.94/8.65/5.87 4.61/3.88/6.21 (6.82 ± 1.59) (4.90 ± 1.19) CA9 8.25/7.95/7.71 8.89/6.75/9.23 (7.97 ± 0.27) (8.29 ± 1.34)

[0101] The results show a synergistic effect on the expression of the genes BNIP3, EGLN3 and CA9 when stemoxydine is combined with baicalin. In addition, these effects are higher than those for the stemoxydine+vitamin C combination.

Example 4 (Invention): Effect of Stemoxydine Alone or Combined with Ascorbyl Palmitate on the Expression of a Selection of Genes in Relation with the Described Effects of Hypoxia

[0102]

TABLE-US-00005 TABLE 5 Ascorbyl Ascorbyl palmitate palmitate Stemoxydine Gene 30 μM 10 μM 300 μM Control BNIP3 1.04/1.6/0.81 1.01/1.35/0.95 1.51/1.65/2.13 1 ± 0 (1.15 ± 0.41) (1.10 ± 0.22) (1.76 ± 0.32) EGLN3 1.02/2.04/0.43 1.4/1.78/0.87 2.15/2.09/3.56 1 ± 0 (1.16 ± 0.81) (1.35 ± 0.46) (2.60 ± 0.83) CA9 1.09/1.73/0.14 1.65/2.00/1.25 3.87/3.86/5.86 1 ± 0 (0.99 ± 0.80) (1.63 ± 0.38) (4.53 ± 1.15) Ascorbyl palmitate Ascorbyl palmitate 30 μM + 10 μM + Gene Stemoxydine 300 μM Stemoxydine 300 μM BNIP3 2.21/2.51/2.34 2.42/2.25/2.50 (2.35 ± 0.15) (2.39 ± 0.13) EGLN3 4.05/3.93/3.50 5.42/3.77/4.82 (3.83 ± 0.29) (4.67 ± 0.84) CA9 8.23/8.21/6.56 9.42/8.36/11.14 (7.67 ± 0.96) (9.64 ± 1.40)

[0103] The results show a synergistic effect on the expression of the genes BNIP3, EGLN3 and CA9 when stemoxydine is combined with vitamin C palmitate. These effects are higher in all respects than those for the stemoxydine+vitamin C combination.

Example 5 (Invention): Effect of Stemoxydine Alone or Combined with Ectoin on the Expression of a Selection of Genes in Relation with the Described Effects of Hypoxia

[0104]

TABLE-US-00006 TABLE 6 Ectoin Ectoin Stemoxydine Gene 300 μM 100 μM 300 μM Control BNIP3 1.40/1.79/1.66 1.31/1.77/1.77 1.51/1.65/2.13 1 ± 0 (1.62 ± 0.2)  (1.62 ± 0.27) (1.76 ± 0.32) EGLN3 2.07/2.82/2.97 2.48/2.76/3.46 2.15/2.09/3.56 1 ± 0 (2.62 ± 0.48) (2.90 ± 0.50) (2.60 ± 0.83) CA9 2.23/2.54/2.93 2.73/2.55/3.74 3.87/3.86/5.86 1 ± 0 (2.57 ± 0.35) (3.01 ± 0.64) (4.53 ± 1.15) Ectoin 300 μM + Ectoin 100 μM + Gene Stemoxydine 300 μM Stemoxydine 300 μM BNIP3 2.30/2.56/2.98 2.20/2.37/2.65 (2.61 ± 0.34) (2.41 ± 0.23) EGLN3 3.88/3.51/4.01 5.03/3.56/4.18 (3.80 ± 0.26) (4.25 ± 0.74) CA9 6.86/5.86/7.27 8.38/5.87/7.56 (6.66 ± 0.73) (7.27 ± 1.28)

[0105] The results show a synergistic effect on the expression of the genes BNIP3, EGLN3 and CA9 when stemoxydine is combined with ectoin.

[0106] The compositions of the invention may be used, for example, as a lotion applied at a rate of 1 ml of lotion to the scalp, at a frequency of one to two times per day. The compositions described below may also be thickened using a thickening polymer.

Example 6: Example of Lotion

[0107]

TABLE-US-00007 Diethyl 2,4-pyridinedicarboxylate 0.5 g to 5 g Resveratrol  0.2 g Propylene glycol 10.0 g Isopropyl alcohol qs 100.0 g

Example 7: Example of Lotion

[0108]

TABLE-US-00008 Diethyl 2,4-pyridinedicarboxylate 0.5 g to 5 g Ascorbyl palmitate   2 g Propylene glycol 10.0 g Isopropyl alcohol qs 100.0 g