USE OF BETA-L-ASPARTYL-L-ARGININE ON SENESCENT SKIN

Abstract

The present invention relates to β-L-aspartyl-L-arginine for use as an anti-inflammatory agent on the skin as well as the cosmetic use of β-L-aspartyl-L-arginine on mature skin. It particular, the β-L-aspartyl-L-arginine provides an antioxidative effect and/or improves mitochondrial function in senescent skin cells, in particular senescent fibroblasts, and/or restores firmness of the skin. Furthermore, the invention also provides a cosmetic method for the treatment of mature skin. The present invention also relates to β-L-aspartyl-L-arginine for use in a therapeutic method for the treatment of damaged mature skin. In particular, the β-L-aspartyl-L-arginine is provided for use in a therapeutic method to promote wound healing.

Claims

1-15. (canceled)

16. A method for treating inflammation of the skin comprising applying β-L-aspartyl-L-arginine to the skin of an individual.

17. The method of claim 16, wherein the method prevents or reduces secretion of one or more pro-inflammatory cytokines and/or and growth factors of skin cells.

18. The method of claim 17, wherein the pro-inflammatory cytokines and/or growth factors are chosen from interleukin 6 (IL-6), interleukin 8 (IL-8), vascular endothelial growth factor (VEGF), transforming growth factor (TGF), and combinations thereof

19. The method of claim 16, wherein the method treats and/or prevents chronic inflammation of the skin.

20. The method of claim 19, wherein the chronic inflammation is caused by the secretion of pro-inflammatory cytokines and/or growth factors by senescent skin cells.

21. The method of claim 16, wherein the method provides an antioxidative effect and/or improves mitochondrial function of mature skin in senescent skin cells.

22. The method of claim 21, wherein the method restores firmness to the skin.

23. The method of claim 21, wherein the individual is at least 40 years old.

24. The method of claim 21, wherein the β-L-aspartyl-L-arginine provides one or more effect(s) selected from: (a) protection of the senescent skin cells against mitochondrial reactive oxygen species (ROS)-induced damages; (b) protection against UV-A induced reactive oxygen species (ROS) in dermis; (c) protection against UV-induced oxidation; (d) reduction of mitochondrial reactive oxygen species (ROS) in the senescent skin cells; (e) reduction of mitochondrial DNA (mtDNA) in the senescent skin cells; reduction of cell size in the senescent skin cells; (g) reduction of a number of the senescent skin cells; (h) reduction of a nucleus to cell size ratio in the senescent skin cells; stimulation of collagen Type III production in the senescent skin cells; and stimulation of fibronectin production in the senescent skin cells.

25. The method of claim 21, wherein the β-L-aspartyl-L-arginine is applied topically to the skin.

26. The method of claim 21, wherein the method comprises applying to the skin of the individual a composition comprising: 0.001 to 2 wt.-% of the β-L-aspartyl-L-arginine, based on a total weight of the composition.

27. The method of claim 21, wherein the mature skin is damaged.

28. The method of claim 26, wherein the individual is at least 40 years old.

29. The method of claim 16, wherein the β-L-Aspartyl-L-Arginine promotes wound healing.

30. The method of claim 21, wherein the β-L-Aspartyl-L-Arginine promotes wound healing.

31. The method of claim 16, wherein the individual is at least 50 years old.

32. The method of claim 21, wherein the individual is at least 50 years old.

33. The method of claim 16, wherein the individual is an individual suffering from chronic inflammation.

34. The method of claim 21, wherein the individual is an individual suffering age-related photo-aging.

35. The method of claim 16, wherein the individual is in need of treatment for inflammation of the skin.

Description

[0069] Short description of the figures:

[0070] In the FIGS. β-L-aspartyl-L-arginine is designated by the batch numbers BIO 4618, BIO 4619 and BIO 4620.

[0071] FIG. 1 shows the reduction of mitochondrial ROS in senescent fibroblast by β-L-aspartyl-L-arginine as measured in the in vitro study described in example 1.

[0072] FIG. 2 shows the dosage dependence of the reduction of mitochondrial ROS in senescent fibroblast by β-L-aspartyl-L-arginine as measured in the in vitro study described in example 1.

[0073] FIG. 3 shows the reduction of cell size in senescent fibroblast by β-L-aspartyl-L-arginine as measured in the in vitro study described in example 2.

[0074] FIG. 4 shows the reduction of the nucleus to cell size (N/C) ratio in senescent fibroblasts by β-L-aspartyl-L-arginine as measured in the in vitro study described in example 3.

[0075] FIG. 5 shows the reduction of mitochondrial DNA (mtDNA) in senescent fibroblasts by β-L-aspartyl-L-arginine as measured in the in vitro study described in example 4.

[0076] FIG. 6 shows the reduction of the ROS score by β-L-aspartyl-L-arginine after UVA irradiation of human skin explants as measured in the ex vivo study described in example 5.

[0077] FIG. 7 shows the stimulation of the production of fibronectin proteins by β-L-aspartyl-L-arginine in the dermis as measured in the ex vivo study described in example 6.

[0078] FIG. 8 shows the reduction of wrinkle intensity by β-L-aspartyl-L-arginine as measured in the clinical study described in example 7.

[0079] FIG. 9 shows the protection of human skin explants against carbonylation of proteins by β-L-aspartyl-L-arginine as measured in the ex vivo study described in example 8.

[0080] FIG. 10 shows the improvement of skin firmness by β-L-aspartyl-L-arginine as measured in the clinical study described in example 9.

[0081] FIG. 11 shows the improvement of skin elasticity by β-L-aspartyl-L-arginine as measured in the clinical study described in example 9.

[0082] FIG. 12 shows the stimulation of collagen Type III by β-L-aspartyl-L-arginine compared to untreated skin and a placebo.

EXAMPLE 1

Reduction of Mitochondrial ROS in Senescent Fibroblasts (In Vitro Study)

[0083] Using a fluorescent dye, the antioxidative potential of β-L-aspartyl-L-arginine with respect to mitochondrial reactive oxygen species (ROS) was quantified. Senescent cells were incubated with β-L-aspartyl-L-arginine for 24 hours. Subsequently, the treated and non-treated senescent cells as well as the treated and non-treated non-senescent cells were incubated with the fluorescent dye for 30 minutes at 37° C. After washing, a portion of the non-senescent cells was treated with the oxidative reference substance H.sub.2O.sub.2 (100 μM). The mitochondrial ROS production was measured immediately every 5 mins over a period of 60 minutes. A microtiter plate reader (Varioskan-Thermo) was used as measuring device. During the kinetic measurement, the cells were further incubated at 37° C. Using fluorimetry, the antioxidative activity was measured in parallel with the viability of the cell. The experiments comprised a blank as well as a negative and a positive control. The positive control consists of non-senescent cells, which were only treated with the vehicle (DMSO). These cells were compared to non-treated senescent cells (negative control). Senescent cells treated with resveratrol were used as reference for the comparison of the activity of β-L-aspartyl-L-arginine in the decrease of endogenous production of mitochondrial ROS in senescent cells. β-L-aspartyl-L-arginine was measured in a 4-fold measurement. The effects of β-L-aspartyl-L-arginine were simultaneously compared to the negative and the positive control.

[0084] As can be inferred from FIG. 1, with respect to non-treated senescent cells, the cells treated with 10 μM β-L-aspartyl-L-arginine show a significant reduction of mitochondrial ROS (mtROS), which is comparable to the effect achieved with resveratrol. Resveratrol is known to be able to reduce ROS and is used as a positive control. The values are shown with respect to non-treated replicative cells, which represent 100%.

[0085] In FIG. 2, it is shown that there is a dosage dependence of the mtROS reduction effect. Increasing amounts of β-L-aspartyl-L-arginine show a larger reduction. In this diagram, the values are shown with respect to the untreated senescent cells used as negative control representing 100%.

EXAMPLE 2

Reduction of Cell Size in Senescent Fibroblasts (In Vitro Study)

[0086] Senescent cells were incubated with β-L-aspartyl-L-arginine. Cell size and nuclear size were measured while cell density reflects the viability. The reference compound AZT (azidothymidine), a gamma polymerase inhibitor reduces mtDNA amount. See example 4 for the details of experimental procedure.

[0087] FIG. 3 shows that amounts of more than 1.0 mM β-L-aspartyl-L-arginine are able to reduce cell size (given in arbitrary units (A.U.)) of senescent fibroblasts compared to untreated senescent fibroblasts. By treatment with β-L-aspartyl-L-arginine, the cells size of senescent fibroblasts can be reduced so that it is comparable to that of young replicative fibroblasts.

EXAMPLE 3

Reduction of Nucleus to Cell Size (N/C) Ratio in Senescent Fibroblasts (In Vitro Study)

[0088] Senescent cells were incubated with β-L-aspartyl-L-arginine. Cell size and nuclear size were measured while cell density reflects the viability. The reference compound AZT (azidothymidine), a gamma polymerase inhibitor reduces mtDNA amount. See example 4 for the details of experimental procedure.

[0089] As can be inferred from FIG. 4, in the presence of β-L-aspartyl-L-arginine, the nucleus to cell size ratio in senescent fibroblasts is reduced compared to untreated senescent fibroblasts to reach the nucleus to cell size ratio of replicative young cells.

EXAMPLE 4

Reduction of Mitochondrial DNA (mtDNA) in Senescent Fibroblasts (In Vitro Study)

[0090] Disruptions of the nuclear and mitochondrial DNA were studied, analyzed by detecting nucleus fragments, the mitochondrial DNA content, the mitochondrial mass and the average cell density, the latter as representation of viability.

[0091] The cells were treated with β-L-aspartyl-L-arginine for 5 days. The cell images were taken after incubation. In order to label the nuclear and mitochondrial DNA, the cells were incubated with a fluorescent dye for 30 minutes. After removal of the dye, the images were taken with an epifluorescence microscope. Image analysis was performed in a 3-fold determination. Sampling 10 pictures per experimental condition or repetition, respectively.

[0092] Data collection: The images were taken with a Zeiss axioplan microscope. Image analysis was performed with a software developed by ICDD.

[0093] Data interpretation: The following data was collected: average cell size, mtDNA content, mitochondrial density, mitochondrial biogenesis, mitochondrial mass and cell nucelus size. The average cell number per field was used as control of cell viability for each experimental condition. The effect of active substances was also given as protective effect in comparison to non-treated cells. The effect of the positive control was included for comparison. FIG. 5 shows that the mtDNA in senescent cells is reduced by β-L-aspartyl-L-arginine to an amount comparable to young replicative cells.

[0094] FIG. 5 shows that the mtDNA in senescent cells is reduced by β-L-aspartyl-L-arginine 25 to an amount comparable to young replicative cells. The reference compound AZT (azidothymidine), a gamma polymerase inhibitor reduces mtDNA amount.

EXAMPLE 5

Reduction of ROS Score After UVA Irradiation on Human Skin Explants (Ex Vivo Study)

[0095] Cultivation of skin models: The skin was cut into about 8×3 mm large pieces (diameter×layer thickness of the models) and cultivated until the planned end of the experiment. Per treatment, six models each were used. The skin models were cultivated as air-liquid-interface cultures in a perforated stainless steel ring with contact to the culture medium (modified Williams 'E Medium) until the desired endpoint.

[0096] Treatment: Test substances and controls were applied topically. To this end, the skin models were carefully cleaned with a cotton pad. Subsequently, 4 μL of each test substance or the control, respectively, were applied to the models and covered with a membrane filter (diameter=6 mm).

[0097] The skin models were incubated for 18 h with test substance and positive control, respectively. Subsequently, the detection reagent (e.g. DCFH-DA) was added to the culture medium analog to the description of Marionnet et al. (Plos One, 9, 2014) After another 30 minutes, the DCFH-DA reagent was removed and the oxidative stimulation was performed (e.g UVA). Dichlorofluorescein diacetat reacts with ROS to form a fluorescent product.

[0098] UV-irradiation: The used sun simulator was a BIO-SUN-system from the company Vilber Lourmat. The system is based on a programmable microprocessor, which controls the UV-irradiation. The UV-light emission is constantly monitored and the system stops automatically when the pre-set energy quantity is reached. The irradiation cycles are therefore always reproducible—independent on the intensity loss of the UV lamp. In order to determine the UV-irradiation dose, the “biologically effective dose (BED)” was used, which is described in Del Bino et al. (Pigment Cell Research, 19, 2006).

[0099] Determination of ROS: At the end of the experiment, the skin models were harvested, cryoconserved and cut using a cryostat for the subsequent image capture and analysis. The fluorescent analysis was performed in the area of the dermis. The area selected for the analysis ran from the upper part of the dermis—following the basal layer—to the lower part of the dermis, while it was made sure that irregular structures such as blood vessels, sweat glands or hair follicles were avoided.

[0100] Of each skin model, two samples were used for image capture with the fluorescence analysis. Each picture was analyzed by determining the fluorescence using Image-J analysis software (NIH, USA). The measured values were normalized over the size of the selected surface.

[0101] Statistics: For each treatment of the skin models, the averages of the quantitative data was determined. For calculating the variations—standard deviation and standard error of the average (SEM)—the raw data was used. Differences between the treated skin models were calculated with one-way ANOVA with permutation test and subsequent Turkey and T-Test with permutation.

[0102] As can be inferred from FIG. 6, β-L-aspartyl-L-arginine is a potent UV-induced ROS scavenger in the dermis and significantly reduces the ROS score of UV irradiated skin in comparison to UV irradiated skin, which was not treated with β-L-aspartyl-L-arginine. Therefore, β-L-aspartyl-L-arginine provides a strong protection from UVA induced ROS damages in the dermis.

EXAMPLE 6

Stimulation of Fibronectin Proteins in the Dermis (Ex Vivo Study)

[0103] Collagen I, Collagen III, MMP1, Elastin, Fibrillin, Fibronectin

[0104] 4 μl of β-L-aspartyl-L-arginine were applied on the skin every day during 6 days of treatment at 37° C., 5% CO.sub.2 and 100% humidity. 12 skin cuts were immunohistochemically stained with selected antibodies. The papillary dermis was selected for the analysis because it is the part of the dermis, which changes the most in reaction to a previous treatment.

[0105] The area to be analyzed was carefully selected while it was made sure that irregular structures such as blood vessels, sweat glands or hair follicles were avoided.

[0106] For the assessment of the results, both, the color intensity and the color distribution were measured using Image-J analysis software (NIH, USA)—thus a semi-quantitative assessment was possible.

[0107] FIG. 7 shows that β-L-aspartyl-L-arginine is able to stimulate the fibronectin production in the dermis compared to untreated skin. It is therefore able to support skin renewal and wound healing.

[0108] FIG. 12 shows that β-L-aspartyl-L-arginine is able to stimulate the collagen Typ III production compared to untreated skin. It is therefore able to support skin renewal and wound healing.

EXAMPLE 7

Reduction of Wrinkle Intensity (Clinical Study)

[0109] The study was conducted with 26 female volunteers of age 53 +/−3 years. The application was done twice daily for two months. On one side of the face, the product is applied, on the other side the placebo.

[0110] Imaging: The evaluation is based on high-resolution digital photography under controlled conditions. Pictures were taken at the start of the study, after 28 days and after 56 days of product application.

[0111] The subjects were positioned in front of the optical arrangement and the face was photographed as portrait or close-up. The persons were photographed seated at controlled lighting. The conditions were kept constant during all photographs to ensure comparability.

[0112] Reduction of wrinkles and lines: The assessment of wrinkles and lines was done in a limited area around the eye of the subject. The intensity of the wrinkles and lines was measured as parameter R. The larger the value for R, the more pronounced are the wrinkles and lines. The relative reduction of wrinkles is calculated according to the following formula:

R%=100(R.sub.i−RF)/R.sub.i(R=wrinkle intensity, i=initial; f=final)

[0113] Data analysis and interpretation: For data analysis the following software packages were used:

[0114] Microsoft® Office Excel 2010 (Mircrosoft Corp., EUA, 2010)

[0115] GraphPad™ Prism® 6.00 (GraphPad Software, San Diego Calif. USA)

[0116] It can be inferred from FIG. 8, that β-L-aspartyl-L-arginine visibly smoothes wrinkles and fine lines on crow feet after only four weeks of twice daily use of 0.1%. After eight weeks, 85% of volunteers have observed a significant 6.8% reduction of wrinkle intensity compared to a placebo.

EXAMPLE 8

Protection Against Carbonylation of Proteins (Ex Vivo Study)

[0117] Detection of carbonylation: After removing the fatty and connective tissue, the frozen skin samples are weighed and cut. The skin pieces of the tested samples are combined and homogenized in a cold extraction buffer. Cell lysates are collected by centrifugation.

[0118] The measurement of the carbonylated protein is performed with the “OxyBlot Protein_Oxidation Kit by Millipore”, (#S7150) according to the protocol of the producer. Only the protein bands of a size between 50 kDa and 150 kDa were taken into account for the calculation. The obtained amount of carbonylated protein is normalized with the actin band (which is constant) and given with respect to the non-treated sample.

[0119] As can be inferred from FIG. 9, β-L-aspartyl-L-arginine is a potent antioxidant at protein level and is able to prevent the formation of carbonylated proteins in an ex vivo model comparing untreated UV stimulated skin and UV stimulated skin treated with 0.2% and 0.5% β-L-aspartyl-L-arginine. The positive control was treated with vitamin C and vitamin B, which are known to provide an antioxidative effect.

EXAMPLE 9

Improvement of Skin Firmness and Elasticity (Clinical Study)

[0120] The study was conducted with 26 female volunteers of age 53 +/−3 years. The application was done twice daily for two months. On one side of the face, the product is applied, on the other side the placebo.

[0121] The method of measurement relies on suction. A vacuum is created in the gauge head of the cutometer, which sucks the skin into the measuring device. An optical arrangement, consisting of a light source and a light detector, measures the light intensity, which enters dependent on how much skin is sucked in. The resulting parameters are elasticity and skin firmness.

[0122] Skin firmness represents the resistance, which the skin creates against the suction by the vacuum. Elasticity, is the time, which the skin needs to return to its original state.

[0123] FIG. 10 shows how β-L-aspartyl-L-arginine visibly improves skin firmness after only four weeks of twice daily use of 0.1%. After eight weeks, 92% of volunteers observed a significant enhancement of skin firmness (+10.1%) compared to the placebo.

[0124] FIG. 11 shows how β-L-aspartyl-L-arginine visibly improves skin elasticity after only four weeks of twice daily use of 0.1%. After eight weeks, 100% of volunteers observed a significant enhancement of skin elasticity (+8.5%) compared to the placebo.