TREATMENT OF AGING OR UV-DAMAGED SKIN

Abstract

The present invention provides a treatment of aging or UV-damaged skin comprising topically administrating a formulation comprising (i) cannabidiol and (ii) omega-3 fatty acid selected from eicosapentaenoic acid and docosahexaenoic acid and combinations thereof.

The invention further relates to a topical formulation comprising: cannabidiol; and, omega-3 fatty acid selected from eicosapentaenoic acid and docosahexaenoic acid and combinations thereof.

Claims

1. A topical formulation, comprising: (a) cannabidiol; and (b) omega-3 fatty acid selected from eicosapentaenoic acid and docosahexaenoic acid and combinations thereof.

2. The topical formulation according to claim 1, wherein the formulation comprises 0.05-3 wt. % cannabidiol.

3. The topical formulation according to claim 1, wherein the formulation comprises 0.0125-3 wt. % of the omega-3 fatty acid.

4. The topical formulation according to claim 3, wherein the formulation comprises at least 0.0125 wt. % eicosapentaenoic acid.

5. The topical formulation according to claim 1, wherein the formulation comprises cannabidiol and the omega-3 fatty acid in a weight ratio of 1:8 to 8:1, respectively.

6. The topical formulation according to claim 1, wherein the formulation further comprises one or more phenolic acids selected from danshensu, salvianolic acid A, salvianolic acid B and salvianolic acid C.

7. The topical formulation according to claim 6, wherein the formulation comprises 0.001-4 wt. % of the one or more phenolic acids.

8. The topical formulation according to claim 6, wherein the formulation comprises an extract of Salvia miltiorrhiza.

9. A method of treating ageing or UV-damaged skin, the treatment comprising topically administrating a formulation comprising cannabidiol and omega-3 fatty acid selected from eicosapentaenoic acid and docosahexaenoic acid and combinations thereof.

10. The method according to claim 9, wherein the treatment is for photo-aging skin.

11. The method according to claim 9, wherein the formulation comprises 0.05-3 wt. % cannabidiol.

12. The method according to claim 9, wherein the formulation comprises 0.0125-3 wt. % of the omega-3 fatty acid.

13. The method according to claim 12, wherein the formulation comprises at least 0.0125 wt. % eicosapentaenoic acid.

14. The method according to claim 9, wherein the formulation comprises cannabidiol and the omega-3 fatty acid in a weight ratio of 1:8. To 8:1, respectively.

15. The method according to claim 9, wherein the formulation additionally comprises one or more phenolic acids selected from danshensu, salvianolic acid A, salvianolic acid B and salvianolic acid C.

16. The method according to claim 15, wherein the formulation comprises 0.001-4 wt. % of the one or more phenolic acids.

17. The method according to claim 15, wherein the formulation comprises an extract of Salvia miltiorrhiza.

18. The method according to claim 9, wherein the method comprises topically administering the formulation to the skin of a human subject.

19. A method of preparing a topical formulation according to claim 1, the method comprising combining an extract of Cannabis and an omega-3 oil isolated from a source selected from fish, microalgae, lower fungi, marine bacteria and combinations thereof.

20. The method according to claim 19, wherein the method further comprises combining the extract of Cannabis and the omega-3 oil with an extract of Salvia miltiorrhiza.

Description

DETAILED DESCRIPTON OF THE INVENTION

[0027] A first aspect of the invention relates to the treatment of aging skin or UV-damaged skin, said treatment comprising topically administrating a formulation comprising (i) cannabidiol and (ii) omega-3 fatty acid selected from eicosapentaenoic acid and docosahexaenoic acid and combinations thereof.

[0028] The present treatment preferably comprises topical administration of the formulation to the skin of a human subject. According to a particularly preferred embodiment, the formulation the treatment comprises topical administration to the skin of a human subject. According to a particularly preferred embodiment, the formulation the treatment comprises topical administration to the face of a human subject.

[0029] The formulation that is employed in the treatment according to the present invention preferably contains 0.05-3 wt. % cannabidiol, more preferably 0.08-2 wt. % cannabidiol and most preferably 0.1-1 wt. % cannabidiol.

[0030] The omega-3 fatty acid selected from eicosapentaenoic acid and docosahexaenoic acid and combinations thereof is preferably present in the formulation in a concentration of 0.0125-3 wt. %, more preferably in a concentration of 0.025-2 wt. % and most preferably in a concentration of 0.05-1 wt. %.

[0031] In a preferred embodiment, the formulation contains the omega-3 fatty acid eicosapentaenoic acid. More preferably, the formulation contains at least 0.0125 wt. % eicosapentaenoic acid, even more preferably 0.025-3 wt. % eicosapentaenoic acid and most preferably 0.05-1 wt. % eicosapentaenoic acid.

[0032] In another preferred embodiment, the formulation contains the omega-3 fatty acid docosahexaenoic acid. More preferably, the formulation contains at least 0.0125 wt. % docosahexaenoic acid, even more preferably 0.025-2 wt. % docosahexaenoic acid and most preferably 0.05-1 wt. % docosahexaenoic acid.

[0033] Most preferably, the omega-3 fatty acid employed in accordance with the present invention is eicosapentaenoic acid.

[0034] Cannabidiol and the omega-3 fatty acid are preferably present in the formulation in a weight ratio of 1:10 to 10:1, more preferably in a weight ratio of 1:8 to 8:1.

[0035] The inventors have found that the effectiveness of the present formulation can be increased significantly by including one or more phenolic acids selected from danshensu, salvianolic acid A, salvianolic acid B and salvianolic acid C. Accordingly, in a particularly preferred embodiment, the formulation additionally contains one or more phenolic acids selected from danshensu, salvianolic acid A, salvianolic acid B and salvianolic acid C. More preferably, the formulation contains 0.0005-4 wt. % of the one or more phenolic acids, even more preferably 0.002-2 wt. % of the one or more phenolic acids, even more preferably 0.005-1.5 wt. % of the one or more phenolic acids and most preferably 0.01-1 wt. % of the one or more phenolic acids.

[0036] The chemical structures of these phenolic acids are shown below

##STR00001##

[0037] Salvia miltiorrhiza is a suitable source of the aforementioned phenolic acids. Since the phenolic acids are water soluble, they can be isolated from Salvia miltiorrhiza by aqueous extraction.

[0038] In a preferred embodiment, the formulation contains at least 0.0001 wt. % of danshensu, even more preferably 0.0003-0.3 wt. % danshensu and most preferably 0.001-0.1 wt. % danshensu.

[0039] The formulation preferably contains one or more salvianolic acids selected from salvianolic acid A, salvianolic acid B and salvianolic acid C in a concentration of 0.0001-3 wt. %, more preferably of 0.003-1 wt. and most preferably of 0.001-0.5 wt. %.

[0040] Cannabidiol and the one or more phenolic acids are preferably contained in the formulation in a weight ratio of 80:1 to 1:1, more preferably in a weight ratio of 40:1 to 2:1, most preferably in a weight ratio of 30:1 to 3:1.

[0041] The inventors have further found that the effectiveness of the present formulation can also be enhanced by including β-sitosterol. In a preferred embodiment, the composition contains 1-200 mg/kg, more preferably 2-150 mg/kg and most preferably 4-120 mg/kg of β-sitosterol.

[0042] Cannabidiol and β-sitosterol are typically contained in the formulation in a weight ratio of cannabidiol: β-sitosterol of 5:1 to 10,000:1, more preferably in a weight ratio of 20:1 to 5,000:1, most preferably in a weight ratio of 50:1 to 2,000:1.

[0043] β-Sitosterol is advantageously introduced in the present formulation in the form of hemp seed oil. Besides β-sitosterol, hemp seed oil also contains around 20 wt. % of the omega-3 fatty acid α-linolenic acid. This fatty acid is believed to also contribute to the anti-ageing effect of the present formulation. Typically, besides β-sitosterol, the formulation contains 0.001-0.5 wt. %, more preferably 0.005-0.4 wt. % and most preferably 0.01-0.2 wt. % of α-linolenic acid.

[0044] The present formulation preferably comprises a dermatologically acceptable carrier. Examples of dermatologically acceptable carriers include emulsion carriers, including, but not limited to, oil-in-water, water-in-oil, water-in-oil-in-water, and oil-in-water-in-silicone emulsions. Preferred carriers comprise an emulsion such as oil-in-water emulsions and water-in-oil emulsions.

[0045] The formulation of the present invention typically comprises 1 to 90 wt. % water. More preferably the formulation comprises 10 to 85 wt. % water, most preferably the formulation comprises 20 to 80 wt. % water.

[0046] The present formulation can be provided in different forms. Preferably, the formulation is a gel, a soap, a serum, a cream or a lotion.

[0047] A preferred embodiment of the present invention relates to the treatment of UV-damaged skin.

[0048] Another preferred embodiment of the present invention relates to the treatment of photo-aging skin. The term “photo-aging” refers to extrinsic aging of skin caused by exposure to UV irradiation.

[0049] Another aspect of the invention relates to a topical formulation comprising: [0050] cannabidiol; and, [0051] omega-3 fatty acid selected from eicosapentaenoic acid and docosahexaenoic acid and combinations thereof.

[0052] Advantageous embodiments of this formulation have already been described above.

[0053] Yet another aspect of the invention relates to a method of preparing a topical formulation as described herein before, said method comprising combining an extract of Cannabis and an omega-3 oil isolated from a source selected from fish, microalgae, lower fungi, marine bacteria, plant and combinations thereof.

[0054] The extract of Cannabis that is employed in this method preferably contains at least 20%, more preferably at least 30% and most preferably at least 40% cannabidiol by weight of dry matter.

[0055] The omega-3 oil used in the method typically contains at least 30 wt. %, more preferably at least 50 wt. %, more preferably at least 65 wt. % and most preferably at least 80 wt. % of glycerides selected from triglycerides, diglycerides, monoglycerides, phosphoglycerides and combinations thereof.

[0056] Omega-3 fatty acids selected from eicospentaenoic acid (EPA), docosahexaenoic acid (DHA) and combinations thereof preferable constitute at least 20 wt. %, even more preferably at least 30 wt. % and most preferably at least 40 wt. % of the fatty acids contained in the omega-3 oil.

[0057] In one advantageous embodiment, EPA constitutes at least 20 wt. %, more preferably at least 30 wt. % and most preferably at least 40 wt. % of the fatty acids contained in the omega-3 oil.

[0058] In another advantageous embodiment, DHA constitutes at least 20 wt. %, more preferably at least 30 wt. % and most preferably at least 40 wt. % of the fatty acids contained in the omega-3 oil.

[0059] The effectiveness of the present formulation for treating aging or UV-damaged skin may be further enhanced by the additional inclusion of an extract of S. miltiorrhiza. Preferably, the formulation contains 0.01-8 wt. %, more preferably 0.02-4 wt. % and most preferably 0.1-2 wt. % of an extract of S. miltiorrhiza.

[0060] In a preferred embodiment, the extract of S. miltiorrhiza contains at least 0.5%, more preferably 1-50% and most preferably 5-30% by weight of dry matter, of one or more phenolic acids selected from danshensu, salvianolic acid A, salvianolic acid B and salvianolic acid C.

[0061] Preferably, the extract of S. miltiorrhiza is an extract that has been obtained by extraction with a polar liquid selected from water, methanol, ethanol, iso-propanol and combinations thereof. Most preferably the extract of S. miltiorrhiza is an aqueous extract.

[0062] The effectiveness of the present formulation can also be enhanced by including hemp seed oil. Preferably, the formulation contains 0.001-3 wt., more preferably 0.005-2 wt. % and most preferably 0.01-1 wt. % of hemp seed oil.

[0063] The hemp seed oil used in the present method typically contains at least 60 wt. %, more preferably at least 70 wt. % and most preferably at least 75 wt. % of glycerides selected from triglycerides, diglycerides, monoglycerides, phophoglycerides and combinations of

[0064] The hemp seed oil typically contains 0.5-5 wt. %, more preferably 1-4 wt. % and most preferably 1.2-3 wt. % β-sitosterol.

[0065] Besides β-sitosterol, the hemp seed oil preferably contains 8-40 wt. %, more preferably 10-35 wt. % and most preferably 12-28 wt. % of α-linolenic acid.

[0066] The invention is further illustrated by the following non-limiting examples.

EXAMPLES

Example 1

[0067] The impact of cannabidiol, eicosapentaenoic acid (EPA) and combinations of these components on the UV-B induced secretion of prostaglandin E2 (PGE.sub.2) was investigated in keratinocytes HaCaT cells.

[0068] The specifications of the aforementioned components are provided in Table 1.

TABLE-US-00001 TABLE 1 Supplier Characterisitcs Cannabidiol Echo Pharmaceuticals >95% pure extracted CBD BV, the Netherlands EPA KD Pharma, Germany Acid value 3 mg KOH/g, 67.2 wt. % EPA (as FFA), 0.2% mixed natural tocopherol

[0069] UV-8 Induced Secretion of PGE2

[0070] This assay was conducted on Human Epidermal Keratinocyte cells (HaCaT) and was carried out in triplicates. After reaching the required confluency, the cells were treated with pre-prepared growth medium containing the test component(s) at a final volume of 200 μL/well. Growth medium without test component was used to prepare the blank samples.

[0071] The cells were incubated for 24 hr. Then, the medium was replaced with PBS (containing the test component) and the cells were subjected to 25 mJ/cm.sup.2 (Table 2) or 12.5 mJ/cm.sup.2 (Table 3) UV-B irradiation. The flux was determined by an external AccuMAX™ Meter.

[0072] Immediately after irradiation, the PBS was aspirated, and the cells were further incubated with freshly prepared media incubated for 24 hr. at 37° C. with 5% CO.sub.2. After incubation, the conditioned medium of the different treatment groups was collected under standardized conditions and centrifuged at 250×g for 5 min to remove particulates. Clear supernatants were frozen at −70° C. until analyses.

[0073] The UVB-induced cytokines secretion of PGE.sub.2 was measured by commercial ELISA, according to the manufacturer's instructions. The percentage inhibition was determined by comparing PGE.sub.2 levels in the test samples with PGE.sub.2 level in the stimulated controls. The results are shown in Table 2.

TABLE-US-00002 TABLE 2 μg/ml Inhibition (%) Sample Cannabidiol EPA PGE.sub.2 1 1.25 0 0 2 10 0 17 3 0 1.25 −11 4 0 10 1 5 1.25 1.25 21 6 10 1.25 22 7 10 10 34

Example 2

[0074] Example 1 was repeated in HaCaT cells, using the lower UV-B dosage (12.5 mJ/cm.sup.2 flux). In addition, an extra test component was used (docosahexaenoic acid). The specification of the latter component is provided in Table 3.

TABLE-US-00003 TABLE 3 Supplier Characterisitcs DHA KD Pharma, Acid value 1.5 mg KOH/g, 72.7 Germany wt. % DHA (as FFA), 0.2% mixed natural tocopherol

[0075] The results of the measurements are summarized in Table 4.

TABLE-US-00004 TABLE 4 μg/ml Inhibition (%) Sample Cannabidiol EPA DHA PGE.sub.2 1 1.25 0 0 −11 2 10 0 0 −8 3 0 1.25 0 −3 4 0 10 0 5 5 0 0 1.25 15 6 1.25 1.25 0 45 7 1.25 10 0 43 8 10 1.25 0 45 9 10 0 1.25 48

Example 3

[0076] The impact of cannabidiol, EPA oil and combinations of these components on the UV-B induced secretion of interleukin 8 (IL-8) was investigated in HaCaT cells using the same ingredients and assay as in Example 1, except that this time secretion of IL-8 was determined.

[0077] UV-8 Induced Secretion of IL-8

[0078] The percentage inhibition was determined by comparing IL-8 levels in the test samples with the IL-8 level in the stimulated controls. The results are summarized in Table 5.

TABLE-US-00005 TABLE 5 μg/ml Inhibition (%) Sample Cannabidiol EPA IL-8 1 1.25 0 −1 2 10 0 3 3 0 1.25 −6 4 0 10 −16 5 10 1.25 24 6 10 10 22

Example 4

[0079] Inhibition of PGE2 was also tested on murine macrophage cell line (RAW 264.7). Beside cannabidiol and EPA, an additional test component was used (extract of S. miltiorrhiza). The specification of the latter component is provided in Table 6.

TABLE-US-00006 TABLE 6 Supplier Characteristics S. miltiorrhiza Draco Natural Salvia Powdered extract 1% Products, USA Danshensu by HPLC. Root extract. Spray dried, fine powdered botanical extract of Salvia miltiorrhiza which has been extracted in a lower-temperature process. Extraction ratio around 8:1.

[0080] The results of the measurements are summarized in Table 7.

TABLE-US-00007 TABLE 7 μg/ml Inhibition (%) Sample Cannabidiol EPA S. miltiorrhiza PGE.sub.2 1 2.5 2.5 0 32 2 2.5 2.5 2.5 49

Example 5

[0081] The effect of a combination of cannabidiol and eicosapentaenoic acid (EPA) on the secretion of nitric oxide was determined in macrophage RAW 264.7 cells. The cannabidiol and EPA used were the same as in Example 1. The effect on nitric oxide reduction was also determined for a combination of cannabidiol, EPA and S. miltiorrhiza extract, and for a combination of cannabidiol, EPA, S. miltiorrhiza extract and hemp oil. The S. miltiorrhiza extract used was the same as in Example 4. The specification of the hemp oil is provided in Table 8.

TABLE-US-00008 TABLE 8 Supplier Characteristics Hemp oil Vitaprom Hemp seed oil from (senior vita/ Cannabis Sativa L Boca Raton) (Hemp). Contains 18.4% alpha linoleic acid, 3% stearidonic fatty acid and 52.9% linoleic acid

[0082] Secretion of Nitric Oxide

[0083] RAW 264.7 cells (approx. 2.5×10.sup.5 ¢/ml, by counting) were seeded in 96 well plates containing 170 μl/well of complete growth medium. The cells were incubated at 37° C. with 5% CO.sub.2 for 24 hr. Then, the medium was aspirated and replaced by LPS-containing medium (12.5 ng/ml) without or with the test components. In addition, naïve cells, vehicle-treated cells, Stimulated Control and Stimulated Vehicle Control served as negative controls. Dexamethasone served as a positive control for anti-inflammatory assays. A Blank control group was included in the assay.

[0084] The cells were incubated at 37° C. with 5% CO.sub.2 for 24 hr. At the end of incubation, the viability of the cells was measured using the MTT assay, according to SOP. In addition, the spent media from all test groups were collected under standardized conditions and centrifuged at 250 g for 5 min to remove particulates. Clear supernatants were frozen at −70° C. until nitric oxide analysis.

[0085] The results of the measurements are summarized in Table 9.

TABLE-US-00009 TABLE 9 μg/ml Hemp Inhibition (%) Sample Cannabidiol EPA S. miltiorrhiza oil nitric oxide 1 1.25 1.25 0 0 13 2 1.25 1.25 1.25 0 26 3 1.25 1.25 1.25 0.625 39

Example 6

[0086] The effect on epidermal viability and the effect on UV-B induced secretion of PGE.sub.2 and IL-8 secretion was determined for a combination of cannabidiol, EPA, S. miltiorrhiza extract and hemp oil. The specifications of these ingredients were the same as in Examples 1, 4 and 5.

[0087] Fixed size skin explant pieces (0.64 cm.sup.2) were cut from the skin tissue, using a designated press apparatus. The skin pieces were prepared and maintained in air liquid interphase. The explants were laid in 6-well culture plates containing skin culture medium (DMEM supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin), dermal side down in the medium and epidermis phasing up. The pieces were left to recover overnight at 37° C. with 5% CO.sub.2.

[0088] Anti-Aging Evaluation

[0089] The assay was carried out in triplicate.

[0090] Skin pieces were treated with skin creams having the compositions shown Table 10.

TABLE-US-00010 TABLE 10 Skin cream 3 Skin cream 1 Skin cream 2 (Placebo) Cannabidiol 0.2 0.5 0 EPA 0.2 0.5 0 S. miltiorrhiza 0.2 0.5 0 Hemp oil 0.1 0.25 0 Cream base remainder 100.0

[0091] 15 min post application, skin pieces were transferred to PBS and subjected to 400 mJ/cm.sup.2 UVB irradiation, and then returned to the growth medium. After 24 hr, the spent medium was collected under standardized conditions and centrifuged at 250×g for 5 min to remove particulates. The supernatants were frozen at −70° C. until cytokine analysis.

[0092] UVB-induced cytokines secretion (PGE2 and IL-8) was quantified by ELISA, according to the manufacturer's instructions. The ability to attenuate UVB-induced-reduction in viability was measured after 48 hr, by the MTT assay, according to standard operating procedure. Lastly, histological evaluation of collagen and elastin deposits were performed.

[0093] The results of these analysis are summarized in Table 11 (viability and inhibition of secretion PGE.sub.2 and IL-8 were determined in comparison to the placebo skin cream). The results demonstrate that the ingredient mixture of the present invention is beneficial and effective in reducing UV induced inflammation when applied as a cream and on human skin transplant, the closest model to human application/clinical trial.

TABLE-US-00011 TABLE 11 Epidermal Inhibition Inhibition Skin cream viability (%) PGE.sub.2 (%) IL-8 (%) 1 131 25 26 2 138 51 51 3 (Placebo) 100 0 0

[0094] Histological evaluation showed that the skin creams according to the invention protected the skin from elastin and collagen reduction following UV exposure and reduced photo-aging effects.