COMPOSITIONS FOR THE SKIN

Abstract

The present invention relates to a composition comprising hyaluronic acid, dermatan sulfate, at least one omega-3 fatty acid, and at least one nucleotide. It also relates to the new composition for use in the treatment or prevention of diseases, ailments, dysfunctions, or alterations of the skin, preferably atopic dermatitis. The composition may be in the form of a pharmaceutical composition, food supplement, functional food, or medical food.

Claims

1. A composition comprising hyaluronic acid, dermatan sulfate, at least one omega-3 fatty acid, and at least one nucleotide.

2. The composition according to claim 1, wherein a weight ratio of the omega-3 fatty acid to the hyaluronic acid is between 100:0.50 and 100:5.

3. The composition according to claim 1, wherein a weight ratio of the omega-3 fatty acid to the dermatan sulfate is between 100:0.10 and 100:2.

4. The composition according to claim 1, wherein a weight ratio of the omega-3 fatty acid to the nucleotide is between 100:20 and 100:200.

5. The composition according to claim 1, further comprising a zinc compound.

6. The composition according to claim 5, wherein the zinc compound is zinc oxide.

7. The composition according to claim 6, wherein a weight ratio of the omega-3 fatty acid to the zinc oxide is between 100:1 and 100:10.

8. The composition according to claim 1, further comprising at least one omega-6 fatty acid.

9. The composition according to claim 1, wherein the omega-3 fatty acid is selected from the group consisting of eicosapentaenoic acid, docosahexaenoic acid, alpha-linolenic acid, stearidonic acid, eicosatetraenoic acid, heneicosapentaenoic acid, docosapentaenoic acid and their mixtures.

10. The composition according to claim 8, wherein the omega-6 fatty acid is selected from the group consisting of gamma-linolenic acid, linoleic acid and their mixtures.

11. The composition according to claim 1, wherein the nucleotide is obtained from a yeast.

12. A food supplement, functional food, or medical food comprising the composition defined in claim 1, and at least one nutritional additive.

13. A pharmaceutical composition comprising the composition defined in claim 1, and at least one pharmaceutically acceptable excipient.

14. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

18. A method of treating or preventing a skin disease or lesion comprising administering the composition according to claim 1 to a subject in need thereof, wherein the skin disease or lesion is selected from the group consisting of atopic dermatitis, allergic dermatitis, demodicosis, psoriasis, wound, ulcer, and burn.

19. The method according to claim 18, wherein the treating or preventing a skin disease or lesion comprises at least one of restoring integrity of skin during or after dermatitis or demodicosis, preventing or reversing a psoriatic lesion, increasing hydration and flexibility of skin, preventing a formation of a wound or an ulcer, and improving quality of healing of a wound, an ulcer, or a burn.

20. The composition according to claim 6, wherein a weight ratio of the omega-3 fatty acid to the hyaluronic acid to the dermatan sulfate to the nucleotide to the zinc oxide is between 100:0.50:0.10:20:1 and 100:5:2:200:10.

21. The composition according to claim 6, further comprising at least one omega-6 fatty acid.

22. The method according to claim 18, wherein the composition further comprises zinc oxide.

23. The method according to claim 18, wherein the composition further comprises zinc oxide and a weight ratio of the omega-3 fatty acid to the hyaluronic acid to the dermatan sulfate to the nucleotide to the zinc oxide is between 100:0.50:0.10:20:1 and 100:5:2:200:10.

24. The method according to claim 18, wherein the composition further comprises zinc oxide and at least one omega-6 fatty acid.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0075] FIG. 1 shows, at the concentration of 0.156 mg/mL, the effect of a composition of the invention containing omega-3 fatty acids, hyaluronic acid, dermatan sulfate, and nucleotides (O-3+HA+DS+N), and the effect of a composition containing omega-3 fatty acids, hyaluronic acid, and dermatan sulfate (O-3+HA+DS) over the percentage of proliferation of human dermal fibroblasts after 48 hours. The Positive Control is also included (culture of human dermal fibroblasts in the presence of TGF-) and the Baseline Control (culture of human dermal fibroblasts in culture medium and in the absence of the compositions being studied).

[0076] FIG. 2 shows, at the concentration of 0.078 mg/mL, the effect of a composition of the invention containing omega-3 fatty acids, hyaluronic acid, dermatan sulfate, nucleotides, and zinc oxide (O-3+HA+DS+N+ZnO), and the effect of a composition containing omega-3 fatty acids, hyaluronic acid, and dermatan sulfate (O-3+HA+DS) over the percentage of proliferation of human dermal fibroblasts after 24 hours. The Positive Control is also included (culture of human dermal fibroblasts in the presence of TGF-) and the Baseline Control (culture of human dermal fibroblasts in culture medium and in the absence of the compositions being studied).

[0077] FIG. 3 shows the % of the area covered with cells, 48 hours after applying 0.0818 mg/mL of nucleotides (N), 0.0027 mg/mL of ZnO, O-3+HA+DS (0.156 mg/mL O-3+0.0017 mg/mL HA+0.0004 mg/mL DS) or O-3+HA+DS+N+ZnO (0.156 mg/mL O-3+0.0017 mg/mL HA+0.0004 mg/mL DS+0.0818 mg/mL N+0.0027 mg/mL ZnO) to the wound. The negative control refers to untreated cells.

[0078] FIG. 4 shows the % of the area covered with cells, 48 hours after applying 0.156 mg/mL of O-3, 0-3+HA+DS+N (0.156 mg/mL O-3+0.0017 mg/mL HA+0.0004 mg/mL DS+0.0818 mg/mL N) or O-3+HA+DS+N+ZnO (0.156 mg/mL O-3+0.0017 mg/mL HA+0.0004 mg/mL SD+0.0818 mg/mL N+0.0027 mg/mL ZnO) to the wound. The two compositions of the invention with the omega-3 fatty acids (O-3) are compared. The negative control refers to untreated cells.

[0079] FIG. 5 shows the % of the area covered with cells, 48 hours after applying 0.0027 mg/mL of ZnO, O-3+HA+DS+N (0.156 mg/mL O-3+0.0017 mg/mL HA+0.0004 mg/mL DS+0.0818 mg/mL N) or O-3+HA+DS+N+ZnO (0.156 mg/mL O-3+0.0017 mg/mL HA+0.0004 mg/mL DS+0.0818 mg/mL N+0.0027 mg/mL ZnO) to the wound. The two compositions of the invention with ZnO are compared. The negative control refers to untreated cells.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0080] The following examples are provided for the purposes of illustration and do not represent any limitation of the scope of the present invention.

Example 1: Soft Gelatin Capsules

[0081] The capsules were prepared following conventional procedures.

[0082] Each capsule contained:

TABLE-US-00001 omega-3 fatty acids 572.00 mg hyaluronic acid* 6.50 mg dermatan sulfate* 1.34 mg nucleotides 300.00 mg zinc oxide 13.88 mg omega-6 fatty acids 75.00 mg *The hyaluronic acid and dermatan sulfate may be substituted by 10 mg of Dermial, which contains hyaluronic acid and dermatan sulfate.

Example 2: Soft Gelatin Capsules with Vitamin E

[0083] The capsules were prepared following conventional procedures.

[0084] Each capsule contained:

TABLE-US-00002 omega-3 fatty acids 572.00 mg hyaluronic acid* 6.50 mg dermatan sulfate* 1.34 mg nucleotides 300.00 mg zinc oxide 13.88 mg omega-6 fatty acids 75.00 mg vitamin E 18.00 mg *The hyaluronic acid and dermatan sulfate may be substituted by 10 mg of Dermial, which contains hyaluronic acid and dermatan sulfate.

Example 3: Stimulation Activity of the Proliferation of Fibroblasts of a Composition of the Invention Containing Omega-3 Fatty Acids, Hyaluronic Acid, Dermatan Sulfate, and Nucleotides. Comparison with a Composition that does not Contain Nucleotides

[0085] Fibroblasts, along with keratinocytes, are the principal cells in the dermis and epidermis. Their migration and proliferation are involved in the process of re-epithelialization, which is very necessary in skin pathologies in which the dermal barrier is altered and patients present lesions caused by the pathology itself or by scratching due to itching. The aim of this study was to determine the stimulation activity of the proliferation of human dermal fibroblasts of a composition of the invention that contained omega-3 fatty acids, hyaluronic acid, dermatan sulfate, and nucleotides, comparing it with a composition that contained omega-3 fatty acids, hyaluronic acid, and dermatan sulfate. The two compositions were compared at the same final concentration of 0.156 mg/mL.

[0086] A composition of the invention that contained 65.38% of omega-3 fatty acids (O-3), 0.64% hyaluronic acid (HA), 0.15% dermatan sulfate (DS), and 34.13% nucleotides (N) (weight ratio O-3:HA:DS:N 100:0.98:0.23:52.20) was used.

[0087] The three-component composition contained 98.65% of omega-3 fatty acids (O-3), 1.12% hyaluronic acid (HA) and 0.23% dermatan sulfate (DS) (weight ratio O-3:HA:DS 100:1.13:0.23).

[0088] Materials and Methods

[0089] The degree of proliferation was quantified by measuring the incorporation of bromodeoxyuridine (BrdU) during DNA synthesis in fibroblasts in proliferation.

[0090] The human dermal fibroblasts were seeded at 5,500 cells/well in dishes with 96 wells, and after 6-7 hours, they were left with a growth-factor deprived culture medium overnight. The next day, the cells were treated with a composition of the present invention, at a concentration of 0.156 mg/mL, that contained omega-3 fatty acids, hyaluronic acid, dermatan sulfate, and nucleotides (O-3+HA+DS+N), or with a composition at a concentration of 0.156 mg/mL that contained omega-3 fatty acids, hyaluronic acid, and dermatan sulfate (O-3+HA+DS). The quantity of absorbance was determined by spectrophotometry after 48 hours of exposure of the culture to the compositions being studied.

[0091] As Baseline Control, the cells were not treated; and as Positive Control, the fibroblasts were exposed to TGF-1 (25 ng/mL).

[0092] Results

[0093] As shown in FIG. 1, the composition of the present invention (0-3+HA+DS+N) at the dosage of 0.156 mg/mL and after 48 hours, showed a statistically significant stimulating effect (p=0.0297) of the proliferation of fibroblasts, if compared with the Baseline Control. Specifically, proliferation increased by 40.70%. However, at the same dosage of 0.156 mg/mL, the composition that contained omega-3 fatty acids, hyaluronic acid, and dermatan sulfate (O-3+HA+DS), but did not contain nucleotides did not show efficacy (a tendency to stimulate fibroblast proliferation was observed, but it was not statistically significant when compared with the Baseline Control). FIG. 1 also shows the statistically significant difference (p=0.0263) between the two compositions.

Example 4: Stimulation Activity of the Proliferation of Fibroblasts of a Composition of the Invention Containing Omega-3 Fatty Acids, Hyaluronic Acid, Dermatan Sulfate, Nucleotides, and ZnO. Comparison with a Composition that does not Contain Nucleotides or ZnO

[0094] The aim of this study was to determine the stimulation activity of the proliferation of human dermal fibroblasts of a composition of the present invention that contained omega-3 fatty acids, hyaluronic acid, dermatan sulfate, nucleotides, and ZnO, comparing it with a composition that contained omega-3 fatty acids, hyaluronic acid, and dermatan sulfate. The two compositions were compared at the same final concentration of 0.078 mg/mL.

[0095] A composition of the invention that contained 64.10% of omega-3 fatty acids (O-3), 0.76% hyaluronic acid (HA), 0.12% dermatan sulfate (DS), 33.33% nucleotides (N) and 1.28% of ZnO (weight ratio O-3:HA:DS:N:ZnO 100:1.20:0.20:52:2) was used.

[0096] The composition of three components contained 98.65% of omega-3 fatty acids (O-3), 1.12% hyaluronic acid (HA) and 0.23% dermatan sulfate (DS) (weight ratio O-3:HA:DS 100:1.13:0.23).

[0097] Materials and Methods

[0098] The same methodology used in Example 3 was applied, but in this case, the culture was exposed for 24 hours to the compositions being studied, which were prepared with a final concentration of 0.078 mg/mL.

[0099] Results

[0100] As shown in FIG. 2, at the dose of 0.078 mg/mL and after 24 hours, there was a statistically significant difference (p=0.0001) between the composition of the present invention ((O-3+HA+DS+N+ZnO) and the composition that contained omega-3 fatty acids, hyaluronic acid, and dermatan sulfate (0-3+HA+DS), but that did not contain nucleotides or zinc oxide.

Example 5: Study of the Healing Activity and Synergy of a Composition of the Invention Containing Omega-3 Fatty Acids, Hyaluronic Acid, Dermatan Sulfate, Nucleotides, and ZnO

[0101] One aim of this study was to determine the healing activity of a composition of the invention with five components, comparing it with a composition that contained only three components (omega-3 fatty acids, hyaluronic acid, and dermatan sulfate) through an in vitro trial on wound closure. Another aim was to determine whether the composition of the invention with five components (omega-3 fatty acids, hyaluronic acid, dermatan sulfate, nucleotides, and zinc oxide) was synergistic.

[0102] Materials and Methods

[0103] Human dermal fibroblasts (HDF) were used, which were cultivated in dishes with 24 wells with a surface area of 1.99 cm.sup.2, with a density of 710.sup.4 cells/well. A mark was made in a straight line approximately 2 mm wide on the surface of the single layer of cells using a sterile plastic tip, creating an area free of cells. Immediately afterward, the compounds or compositions were applied to the cultures in the following concentrations: [0104] Omega-3 fatty acids (O-3): 0.156 mg/mL [0105] Hyaluronic acid (HA): 0.0017 mg/mL [0106] Dermatan sulfate (DS): 0.0004 mg/mL [0107] Nucleotides (N): 0.0818 mg/mL [0108] Zinc oxide (ZnO): 0.0027 mg/mL [0109] Three-component composition: 0.156 mg/mL O-3+0.0017 mg/mL HA+0.0004 mg/mL DS (weight ratio O-3:HA:DS 100:1.09:0.25) [0110] Four-component composition of the invention: 0.156 mg/mL O-3+0.0017 mg/mL HA+0.0004 mg/mL DS+0.0818 mg/mL N (weight ratio 0-3:HA:DS:N 100:1.09:0.25:52.43) [0111] Five-component composition of the invention: 0.156 mg/mL O-3+0.0017 mg/mL HA+0.0004 mg/mL DS+0.0818 mg/mL N+0.0027 mg/mL ZnO (weight ratio O-3:HA:DS:N:ZnO 100:1.09:0.25:52.43:1.73)

[0112] The untreated cells were used as the negative control. The positive control was the Dulbeco's medium (DMEM) culture supplemented with fetal bovine serum (FBS) at 0.1%.

[0113] The cells were then allowed to migrate to the center of the dish, to cover the area free of cells, in the presence of the compounds being studied, and the area covered with cells was quantified after 48 hours using the TIRF Automated Inverted MicroscopeScanR Olympus (Leica) and a 4 objective lens (total amplification 40). Each experimental condition was carried out in triplicate.

[0114] To determine the healing activity of each treatment, the area covered by cells was quantified, in m.sup.2, before (T0 h) and after (48 h) the treatment, using image analysis using the software Image J. The results were compared with those obtained for the negative and positive controls.

[0115] Results

[0116] FIG. 3 shows that the composition of the invention O-3+HA+DS+N+ZnO is synergistic. The combination of nucleotides, ZnO and the three-component composition (O-3+HA+DS) presented a synergistic healing effect, exceeding the sum of the individual components separately.

[0117] FIG. 4 shows that the omega-3 fatty acids did not cause a significant increase in healing, but a significant increase in healing (p<0.05) was observed when HA+DS+N was added to obtain the four-component composition of the invention (O-3+HA+DS+N) and when HA+DS+N+ZnO was added to the omega-3 fatty acids to obtain the five-component composition of the invention (O-3+HA+DS+N+ZnO). Also, when the increase in healing was compared between groups, a significant difference was found between the four-component invention and the O-3 (p=0.023459), and between the five-component invention (O-3+HA+DS+N+ZnO) and the O-3 (p=0.017714).

[0118] Also, FIG. 5 shows that the ZnO did not cause a significant increase in healing and that the four-component compositions of the invention (O-3+HA+DS+N) and five-component compositions of the invention (O-3+HA+DS+N+ZnO) presented a significant difference with respect to the ZnO (p=0.024106 and p=0.018395, respectively).