Use of a composition comprising avocado perseose in the protection of epidermal stem cells

Abstract

The invention concerns a use of C7 sugars and derivatives of formula (I), called avocado perseose, which have, inter alia, a protective activity to epidermal skin cells. These C7 sugars and derivatives are capable of maintaining the expression of markers of adult stem cells, in particular basal stem cells. Avocado perseose can also be used for preventing the deleterious effects of environmental damage. Avocado perseose has a beneficial effect on the conservation of the potential of epidermal stem cells and thus helps maintain skin homeostasis.

Claims

1. A method for treating skin cancer, consisting essentially of administering to a subject in need thereof an effective amount of a composition consisting essentially of at least one C7 sugar or derivative of the following formula (I) ##STR00004## wherein Ra is a hydrogen atom and Rb is an OR.sub.2 or CRaRb represents the CO radical; R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are, independently of one another a hydrogen atom or a (CO)R radical in which R is a saturated or unsaturated hydrocarbon chain containing from 11 to 24 carbon atoms, optionally substituted by one or more substituents selected from the group consisting of hydroxy radicals (OH), ethoxy radicals (OC.sub.2H.sub.5) and the SO.sub.3M group, wherein M is a hydrogen atom, an ammonium ion (NH.sub.4.sup.+) or a metal ion; or a (CO)R radical in which R is a saturated or unsaturated hydrocarbon chain containing from 2 to 10 carbon atoms, optionally substituted by one or more substituents selected from the group consisting of hydroxy radicals (OH), ethoxy radicals (OC.sub.2H.sub.5) and the SO.sub.3M group, wherein M is a hydrogen atom, an ammonium ion (NH.sub.4.sup.+) or a metal ion; and wherein the degree of esterification, for a sugar molecule, is between 0.2 and 1; and a pharmaceutically acceptable excipient, wherein said composition protects epidermal stem cells, wherein said stem cells are basal stem cells, and wherein said subject is a child.

2. The method of claim 1, wherein skin barrier function is maintained.

3. The method of claim 1, wherein aging of the skin is reduced.

4. The method of claim 1, wherein said composition promotes wound healing.

5. The method of claim 1, wherein expression of one or more stem cell markers is maintained.

6. The method of claim 5, wherein said marker is selected from the group consisting of Np63, FN1 (fibronectin 1), MCSP (melanoma-associated chondroitin sulfate proteoglycan), LRIG1 (leucine-rich repeats and immunoglobulin-like domains protein 1), GJA1 (connexin 43), NID1 (nidogen 1), NOTCH1 (Notch homolog 1, translocation-associated), KRT15 (keratin 15), KRT19 (keratin 19), EGFR (epidermal growth factor receptor), CD71 (transferrin receptor), DSG3 (desmoglein 3), ITGB1BP1 (integrin beta 1 binding protein), ITGA6 (integrin alpha 6) and ITGB4 (integrin beta 4).

7. The method of claim 5 wherein said marker is ITGA6 (integrin alpha 6).

8. The method of claim 1, wherein R is a fatty acid residue.

9. The method of claim 8, wherein the fatty acid residue is selected from the group consisting of a stearyl, linoleyl, oleyl, palmityl, lauryl, myristyl, arachidyl, behenyl, lauroleyl, myristoleyl, palmitoleyl, linolenyl in its and forms, and/or arachidonyl radical.

10. The method of claim 1, wherein the composition includes a C7 sugar selected from the group consisting of mannoheptulose, perseitol and mixtures thereof.

11. The method of claim 1, wherein said composition includes 0.001 to 30% by weight D-mannoheptulose or an acid derivative thereof, in relation to the total weight of said composition, and/or 0.001 to 30% by weight perseitol or an acid derivative thereof, in relation to the total weight of said composition.

12. The method of claim 10, wherein the source of mannoheptulose and/or perseitol is a water-soluble extract of avocado sugars.

13. The method of claim 12, wherein the avocado sugar water-soluble extract is obtained by a method comprising the following steps in the order shown: drying and extracting lipids to obtain an avocado oil cake; cryogrinding and total delipidation of said oil cake, then decanting and centrifugation in order to recover a soluble fraction rich in C7 sugars (elimination of the cake); demineralization on ionic resin of said soluble fraction obtained in the preceding step; ultrafiltration at 10,000 daltons; and concentration under vacuum and packaging.

14. The method of claim 13, wherein the avocado oil cake is obtained from avocado fruit.

15. The method of claim 13, wherein the water-soluble extract of avocado sugars includes by weight, in relation to the total weight of dry matter of the extract (relative composition determined by HPLC): TABLE-US-00013 D-mannoheptulose 5 to 80% Perseitol 5 to 80% Sucrose less than 10% Glucose less than 10% Fructose less than 10%.

16. The method of claim 1, wherein said composition is administered topically or orally.

17. A method for treating skin cancer, consisting essentially of administering to a subject in need thereof an effective amount of a composition consisting essentially of at least one C7 sugar or derivative of the following formula (I) ##STR00005## and one additional active agent; wherein Ra is a hydrogen atom and Rb is an OR.sub.2 or CRaRb represents the CO radical; R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 and R.sub.7 are, independently of one another a hydrogen atom or a (CO)R radical in which R is a saturated or unsaturated hydrocarbon chain containing from 11 to 24 carbon atoms, optionally substituted by one or more substituents selected from the group consisting of hydroxy radicals (OH), ethoxy radicals (OC.sub.2H.sub.5) and the SO.sub.3M group, wherein M is a hydrogen atom, an ammonium ion (NH.sub.4+) or a metal ion; or a (CO)R radical in which R is a saturated or unsaturated hydrocarbon chain containing from 2 to 10 carbon atoms, optionally substituted by one or more substituents selected from the group consisting of hydroxy radicals (OH), ethoxy radicals (OC.sub.2H.sub.5) and the SO.sub.3M group, wherein M is a hydrogen atom, an ammonium ion (NH.sub.4+) or a metal ion; wherein at least one of the hydroxyl groups is esterified; and a pharmaceutically acceptable excipient, wherein said composition protects epidermal stem cells, wherein said stem cells are basal stem cells, wherein said subject is a child, and wherein said additional active agent is: a dermatological active agent selected from the group consisting of emollients, moisturizing active agents, keratin synthesis activators, keratoregulators, keratolytics, agents that repair the cutaneous barrier, peroxisome proliferator-activated receptor (PPAR) agonists, RXR or LXR agonists, scarring agents, sebum-regulating agents, anti-irritation agents, soothing agents, anti-inflammatory agents, antioxidant agents and anti-aging agents, depigmenting or hypodepigmenting agents, pigmenting agents, lipolytic agents or lipogenesis inhibitors or anti-cellulitis or slimming agents, organic or inorganic sun screens or filters, antifungal compounds, preservatives, antibacterial agents, prebiotics and probiotics, antibiotics, immunomodulators and mixtures thereof; selected from the group consisting of prebiotics and probiotics, antibacterial agents, antifungal compounds, preservatives, immunomodulators, growth factors, and mineral or organic sun filters or screens (pigmentary or ultrafine); selected from the group consisting of wound-healing agents and/or agents that repair the cutaneous barrier, anti-inflammatory and/or anti-irritations and/or soothing agents, sebum-regulating agents, anti-inflammatory and/or anti-irritations and/or soothing agents, hypopigmenting or depigmenting agents, inorganic or organic sun filters and screens, and preservatives; selected from the group consisting of plant oils, oleodistillates or concentrates of plant or animal oil, unsaponifiables of plants or plant oil, peptides or complexes of plant amino acids, butyl avocadate, extracts rich in polyphenols, lupeol, Cupuacu butter, oxazolines, and mixtures thereof; or selected from the group consisting of diethyl pyridine-2,4-dicarboxylate, sodium cocoyl alaninate, pentapeptide-31, ctyldodecanol, Echium Planta-gineum Seed Oil, Cardiospermum Halicacabum Flower/Leaf/Vine Extract, Helianthus Annuus Sunflower Seed Oil Unsaponifiables, Helianthus Annuus Sunflower Seed Oil, Ethyl Ferulate, Polyglyceryl-5 Trioleate, Rosmarinus Officinalis Leaf Extract, Aqua, Disodium Uridine Phosphate, hydrolyzed alginic acid, extracts of Laminaria digitata, extracts of plant stem cells of Apline rose, extracts of plant stem cells of Gamay Freaux teinturier grape, extracts of plant stem cells of Uttwiler spatlauber apple (Malus domestica), extracts of plant stem cells of argan, extracts of plant stem cells of Vitis vinifera vines, and plant stem cells of Christe Marine shoots.

18. The method of claim 17, wherein said composition is administered topically or orally.

19. The method of claim 17, wherein skin barrier function is maintained.

20. The method of claim 17, wherein aging of the skin is reduced.

21. The method of claim 17, wherein said composition promotes wound healing.

22. The method of claim 17, wherein expression of one or more stem cell markers is maintained.

23. The method of claim 22, wherein said marker is selected from the group consisting of Np63, FN1 (fibronectin 1), MCSP (melanoma-associated chondroitin sulfate proteoglycan), LRIG1 (leucine-rich repeats and immunoglobulin-like domains protein 1), GJA1 (connexin 43), NID1 (nidogen 1), NOTCH1 (Notch homolog 1, translocation-associated), KRT15 (keratin 15), KRT19 (keratin 19), EGFR (epidermal growth factor receptor), CD71 (transferrin receptor), DSG3 (desmoglein 3), ITGB1BP1 (integrin beta 1 binding protein), ITGA6 (integrin alpha 6) and ITGB4 (integrin beta 4).

24. The method of claim 22 wherein said marker is ITGA6 (integrin alpha 6).

25. The method of claim 17, wherein the degree of esterification, for a sugar molecule, is between 0.2 and 1.

26. The method of claim 17, wherein R is a fatty acid residue.

27. The method of claim 26, wherein the fatty acid residue is selected from the group consisting of a stearyl, linoleyl, oleyl, palmityl, lauryl, myristyl, arachidyl, behenyl, lauroleyl, myristoleyl, palmitoleyl, linolenyl in its and forms, and/or arachidonyl radical.

28. The method of claim 17, wherein the composition includes a C7 sugar selected from the group consisting of mannoheptulose, perseitol and mixtures thereof.

29. The method of claim 17, wherein said composition includes 0.001 to 30% by weight D-mannoheptulose or an acid derivative thereof, in relation to the total weight of said composition, and/or 0.001 to 30% by weight perseitol or an acid derivative thereof, in relation to the total weight of said composition.

30. The method of claim 28, wherein the source of mannoheptulose and/or perseitol is a water-soluble extract of avocado sugars.

31. The method of claim 30, wherein the avocado sugar water-soluble extract is obtained by a method comprising the following steps in the order shown: drying and extracting lipids to obtain an avocado oil cake; cryogrinding and total delipidation of said oil cake, then decanting and centrifugation in order to recover a soluble fraction rich in C7 sugars (elimination of the cake); demineralization on ionic resin of said soluble fraction obtained in the preceding step; ultrafiltration at 10,000 daltons; and concentration under vacuum and packaging.

32. The method of claim 31, wherein the avocado oil cake is obtained from avocado fruit.

33. The method of claim 31, wherein the water-soluble extract of avocado sugars includes by weight, in relation to the total weight of dry matter of the extract (relative composition determined by HPLC): TABLE-US-00014 D-mannoheptulose 5 to 80% Perseitol 5 to 80% Sucrose less than 10% Glucose less than 10% Fructose less than 10%.

Description

FIGURE LEGENDS

(1) FIG. 1: Evolution of genes of stem cells in the presence of avocado perseose.

(2) FIG. 2: Immunostaining of integrin alpha 6, marker for basal layer stem cells; A: control not irradiated or treated; B: UVA+UVB irradiation; C: UVA+UVB irradiation in the presence of avocado perseose.

(3) FIG. 3: Immunostaining of integrin beta 1, marker for basal layer stem cells; A: control not irradiated or treated; B: UVA+UVB irradiation; C: UVA+UVB irradiation in the presence of avocado perseose.

(4) The examples which follow illustrate the invention but are not restrictive.

EXPERIMENTAL EXAMPLES

1. Example 1: Preparation of a Water-Soluble Extract of Avocado Sugars

(5) Fresh avocados, of the Hass variety, are cut into thin slices 2-5 mm thick, seed included, using a circular-blade slicer. The drying apparatus is a temperature controlled hot air drying oven. The sliced avocados are distributed in a thickness of 4-5 cm on stacked racks. Drying is for 48 hours at a temperature of 80 C. Once dried, the fruits are subjected to cold pressing. This operation is carried out on a small Komet laboratory press. Thus, oil and oil cake are obtained.

(6) The oil cake is then crushed and then extracted, in the presence of 70% ethanol or water.

(7) The liquid and solid portions are separated by centrifugation, for example. The soluble fraction (liquid) is taken up to be purified and concentrated according to the following procedure: Demineralization using ion-exchange resins: demineralization of heptuloses by passing over OH.sup. resins and then over H.sup.+ resin. Ultrafiltration at 10,000 Da: ultrafiltration is carried out with a system equipped with four 10 kDa cut-off membranes. Vacuum concentration: the purified extract is concentrated using a vacuum evaporator until a dry matter content of near 4% is obtained. Packaging: the concentration of the extract is adjusted to 5% dry matter and preservative is added, and then it is sterile filtered on a 0.2 m cut-off membrane and packaged.

(8) Table 3 gives the composition of the extract of avocado C7 sugars, with 5% dry matter, prepared according to the method described above:

(9) TABLE-US-00005 TABLE 3 Appearance Pale yellow solution Analytical criteria Dry matter 5% pH ( dilution) 7.0 Composition (%/dry matter) Sucrose 3.0 Glucose 7.5 D-mannoheptulose 40.0 Fructose 8.6 Perseitol 40.0

(10) According to this same method, two other extracts were prepared, whose pH, absorbance and C7 sugar content values in are given in table 4. The C7 sugar content corresponds to the sum of perseitol and D-mannoheptulose analyzed by HPLC.

(11) TABLE-US-00006 TABLE 4 Batch 1 2 Dry matter 5% 5% pH ( dilution) 5.9 5.4 C7 sugars/dry matter 80.5 83.4

(12) The avocado sugars prepared by the method of example 1 and called avocado perseose hereafter were used to study the expression of stem cell markers under various conditions.

2. Effect of Avocado Perseose on the Expression Profile of Stem Cell Genes and Markers

(13) The effect of avocado perseose on the expression of stem cell markers was first tested in a keratinocyte culture model.

(14) 2.1. Materials and Methods

(15) 2.1.1. Biological Model and Treatment

(16) Skin samples were taken (from circumcision or mammaplasty, according to the sex of the donor) from donors aged from 1 month, 3 months, 3 years, 6 years and 11 years, as well as from adults. From these skin samples, epidermal keratinocytes were extracted and grown in supplemented SFM, at 37 C. and 5% CO.sub.2.

(17) The various keratinocytes were grown for 24 hours. After incubation, the medium was replaced by medium containing 0.005% avocado perseose or not (control). The cells were then incubated for 24 hours. All the experimental conditions were carried out in triplicate. At the end of incubation, the culture supernatants were removed and the cell layers were immediately freeze dried at 80 C.

(18) 2.1.2. Analysis of the Differential Expression of Genes of Interest

(19) The expression of markers was evaluated by RT-qPCR on the messenger RNA extracted from the cell layers of each donor. Total RNA was extracted from each sample using TriPure Isolation Reagent (Roche) according to the protocol recommended by the manufacturer. The extracted RNA was quantified using the NanoVue spectrophotometer (GE Healthcare). For the analysis by quantitative RT-PCR (RT-qPCR), the prepared RNA was first reverse-transcribed into cDNA in the presence of oligo(dT) using the Superscript II enzyme. The cDNA obtained was quantified using the NanoVue spectrophotometer (GE Healthcare) and their concentration adjusted to 5 ng/l.

(20) The PCR reactions were carried out by quantitative PCR with the Light Cycler system (Roche Molecular Systems Inc.) according to the procedures recommended by the supplier.

(21) The reaction mixture (10 l final) for each sample contains 2.5 l of 5 ng/l cDNA, the primers for the various markers used, the reaction mixture (Roche) containing Taq DNA polymerase enzyme, SYBR Green I marker and MgCl.sub.2.

(22) The incorporation of fluorescence into the amplified DNA is measured continuously during PCR cycles. These measurements make it possible to obtain fluorescence intensity curves as a function of PCR cycles and to thus evaluate a value of relative expression for each marker.

(23) The number of cycles is determined from the exit points of the fluorescence curves. For a given marker used, the later a sample leaves (high number of cycles), the lower the initial number of copies of mRNA.

(24) The relative expression (RE) value is expressed in arbitrary units according to the following formula:
RE=(.sup.number of cycles)10.sup.6

(25) The reference gene for stable expression regardless of the experimental conditions, which makes it possible to normalize the results is GAPDH.

(26) 2.2. Results

(27) Genomic analysis showed that the expression of genes of stem cells, KRT15, NOTCH1, KRT19, ITGBP1 and ITGA6, is high after birth and decreases with age. This expression profile was maintained in the presence of avocado perseose (FIG. 1), demonstrating that avocado perseose is well tolerated by cells.

3. Evaluation of the Protective Effect of Epidermal Stem Cells from Avocado Perseose with Respect to UVA+UVB Irradiation

(28) The protective effect of avocado perseose on stem cell markers was tested with a model of skin irradiated with UV or not irradiated.

(29) 3.1. Materials and Methods

(30) 3.1.1. Biological Model and Treatment

(31) Skin explants are allowed to stabilize for 4 hours at 37 C. in DMEM with 5% serum.

(32) The explants are placed in a 24-well plate containing DMEM with 5% serum. The skin samples are pretreated with 0.01% avocado perseose applied topically to the center of the explant. PBS is deposited on the untreated explants considered as controls.

(33) The skin samples are also pretreated with 0.005% avocado perseose in the culture medium. Each condition is evaluated in duplicate. The skin samples are left in the oven for 24 hours. The explants are rinsed with PBS and then transferred to 6-well lates to be irradiated. The irradiation dose is 10 J/cm.sup.2 in UVA and 200 mJ/cm.sup.2 in UVB.

(34) The explants are treated again as before and then returned to the incubator for 24 hours.

(35) 3.1.2. Protein Immunostaining within Tissue

(36) The inclusion technique preserves the biopsy in a frozen state without chemical fixing (which could alter the epitopes of the antigens of interest). The inclusion of skin is carried out in two steps: a step of molding the skin in a histocassette filled with OCT (Tissue-Tek), a substance that makes it possible to preserve skin biopsies in frozen form in order to prepare sections with a cryostat. And a second step in liquid nitrogen makes it possible to freeze and harden the preparation. Next, 10 m sections are prepared with a cryostat (a refrigerated enclosure for preparing frozen sections on a microtome). The sections of skin mounted on microscope slides are fixed in acetone for 10 minutes. The nonspecific sites are saturated. The primary antibodies (anti-p63, anti-6 and anti-1) are left to incubate overnight at 4 C. Then the signal is developed with the secondary antibodies coupled to a fluorochrome for 1 hour at room temperature in the dark. At the same time as this incubation, the cell nuclei are stained with DAPI.

(37) The skin samples are observed by fluorescence optical microscopy using the Olympus CK 40 microscope. Visualization of the fluorescence employs the filters Rhodamine (TRITC, red), FITC (green) and Dapi (blue). The skin samples are observed under 40 magnification. The image acquisition software is called Archimed (Microvision).

(38) The images were acquired with an automated high-resolution imaging system (INCell Analyzer1000, GE Healthcare). For each condition, two skin explants were analyzed and five digital images were captured per explant, for a total of 10 analyzed images.

(39) 3.2. Results

(40) Staining was quantified by measuring the intensity of fluorescence of integrin alpha 6 in relation to the number of nuclei identified by DAPI.

(41) As indicated in table 5, the cells that were irradiated lost the expression of integrin alpha 6. On the other hand, the expression of this marker is maintained when the cells are treated with avocado perseose, indicating that avocado perseose makes it possible to preserve the potential of epidermal stem cells.

(42) TABLE-US-00007 TABLE 5 Quantification of the staining of integrin alpha 6 in the basal layer of skin explants INTEGRIN ALPHA 6 % in relation to (fluorescence intensity the control Control not irradiated or 49854 100% treated UVA + B irradiation 10103 20% Avocado perseose - UVA + B 40021 80% irradiation

4. Examples of Formulations

(43) 4.1. Moisturizing Cream

(44) TABLE-US-00008 TABLE 6 Raw material/Trade name % CAPRYLO CAPRATE GLYC 1 to 15% SUNFLOWER OIL SR 1 to 15% PURE CETYL ALCOHOL 1 to 5% GLYCERYL STEARATE CITRATE 1 to 10% BEESWAX 1 to 5% EUMULGIN SG 0 to 2% VITAMIN E ACETATE 0 to 1% PURIFIED WATER QSP 100% CARBOPOL ULTREZ 20 0 to 1% GLYCEROL 1 to 10% XANTHAN GUM 0 to 1% SOUDEXI DETERGENT 0 to 1% PRESERVATIVE 0 to 2% AVOCADO PERSEOSE 0 to 1%

(45) 4.2. Restructuring Milk

(46) TABLE-US-00009 TABLE 7 Raw material/Trade name % PURIFIED WATER QSP 100% SUNFLOWER OIL SR 1 to 10% HYDRO COPRA OIL 1 to 10% SWEET ALMOND OIL 1 to 10% CORN OIL 1 to 10% MONOSTEARATE GLYCEROL 1 to 10% STEARIC ACID 1 to 10% PRESERVATIVE 0 to 2% C16-C18 CETYL ALCOHOL 0 to 2% VITAMIN E ACETATE 0 to 1% SOUDEXI DETERGENT 0 to 1% AVOCADO PERSEOSE 0 to 1%

(47) 4.3. Cleansing Water

(48) TABLE-US-00010 TABLE 8 Raw material/Trade name % PURIFIED WATER QSP 100% GLYCEROL 1 to 10% SODIUM COCOYL 1 to 10% GLUTAMATE PRESERVATIVE 0 to 2% RICINUS HYDROBUTETH 26 0 to 2% ALLANTOIN 0 to 2% TARTARIC ACID 0 to 2% ALOE VERA POWDER SR 0 to 2% SOAPWORT EXTRACT SR 0 to 2% SOUDEXI DETERGENT 0 to 2% AVOCADO PERSEOSE 0 to 1%

(49) 4.4. Shampoo

(50) TABLE-US-00011 TABLE 9 Raw material/Trade name From PURIFIED WATER QSP 100% COCAMIDOPROPYL BETAINE 1 to 10% GLYCEROL 1 to 10% COCOGLUCOSIDEXI 1 to 10% SODIUM MYRETH SULFATE 1 to 10% PRESERVATIVE 0 to 2% PEG6000 DISTEARATE 0 to 2% POLYQUARTERNIUM JR400XI 0 to 2% DEXTROROTATORY PANTHENOL 0 to 2% HYDXI A CITRIC CID 0 to 2% AVOCADO PERSEOSE 0 to 1%

(51) 4.5. SPF 50+ Sun Cream

(52) TABLE-US-00012 TABLE 10 Raw material/Trade name From COPRAH CAPRYLATE/CAPRATE 5 to 20% DICAPRYLYL CARBONATE 5 to 20% CAPRYLOCAPRATE GLYC 5 to 20% MIGLYOL GELB 5 to 20% TITANDIOXID JOJOBA ESTERS 1 to 15% TINOSORBS 1 to 5% DIETAMIN HYDBENZO YHEXBENZ 1 to 10% ETHYL HEXYL TRIAZONE 1 to 10% HAREF AVOCADO OIL 1 to 5% ALPHA TOCOPHEROL 0.05 to 1% LAURYL GLUCOSE-GLYSTEARATXI 2 to 12% PURIFIED WATER QSP 100% GLYCEROL 1 to 10% XANTHAN GUM 0 to 1% PRESERVATIVE 0 to 2% POTASSIUM CETYL PHOSPHATE 0 to 2% SOUDEXI DETERGENT 0 to 2% PHENYL BENZIMIDAZO SULFONAC 1 to 5% AVOCADO PERSEOSE 0 to 1%