Method for informing a purchaser of a topical skin care preparation of an oxidative stress protective capacity   

Abstract: A method for informing a purchaser of a topical skin care preparation of an oxidative stress protective capacity, relative to human skin, of a first antioxidative substance included in the topical skin care preparation, includes providing the topical skin care preparation, the topical skin care preparation including the first antioxidative substance. Information is provided including a total score indicative of an oxidative stress protective capacity, relative to human skin, of the first antioxidative substance. The total score is based on a combination of a first score representative of a performance of the first antioxidative substance in a first antioxidative efficacy test and a second score representative of a performance of the first antioxidative substance in a second antioxidative efficacy test.

This application is a divisional application of U.S. patent application Ser. No. 11/050,571, filed Feb. 3, 2005. The entire disclosure of this application is incorporated by reference herein.

The present invention relates in general to oxidative stress in human skin, and in particular to a method for characterizing the oxidative stress protective capacity of an antioxidative substance, such as an antioxidant, or of a topical skin preparation including an antioxidative substance.

Human skin is susceptible to oxidative stress due to various causes, such as external environmental stressors including exposure to ultraviolet radiation, air pollution, cigarette smoke, chemicals, cosmetics, drugs, ozone and even oxygen itself, as well as to internal stress due to natural biological processes.

For example, the damaging effect of the ultraviolet (UV) part of solar radiation on the skin is well recognized. UV radiation is known to be the predominant cause of premature aging of the skin. UV radiation may lead to photochemical reactions, wherein then the photochemical reaction products intervene in the skin mechanism. Predominantly such photochemical reaction products are free-radical compounds, for example hydroxy radicals. Also, undefined free-radical photoproducts, which are produced in the skin itself, may trigger uncontrolled side reactions due to their high reactivity. Furthermore, lipid peroxidation products, such as for example hydroperoxides and aldehydes, are produced. Free-radical chain reactions may be triggered, resulting in skin damage.

Moreover, UV radiation is ionizing radiation. Hence, there is the risk that ionic species are produced on UV exposure, which species in turn are able to intervene oxidatively in the biochemical processes.

In additional to externally generated oxidative stress, internally generated oxidative stress may occur as a natural by-product of cellular energy production. Both internal and external oxidative stress leads to inflammatory pathways mediated by the formation of free radicals (molecules with unpaired electrons that are highly reactive) that, left unchecked, can cause severe cellular damage to cell membranes, lipids, proteins and DNA. The superoxide radical, a natural by-product of metabolic energy production, causes serious deleterious effects to living cells if not quenched, neutralized or reduced almost immediately after production. It is known that lipid peroxidation is a major problem in biological systems. Protecting against cell membrane oxidation is of paramount importance in living biological systems since the cell membrane is the cell\'s first line of defense against oxidation

The lifetime summation of damage caused by run-away free radicals is one of the main theories of aging, “the damage accumulation theory of aging”. There is therefore a high interest in modern medicine regarding the use of antioxidants, substances that scavenge and eliminate free radicals, to counter the deleterious effects (i.e., aging) of the free radical mediated inflammatory pathways caused by oxidative stress.

Oxidative damage to the skin and its more detailed causes are discussed in J. Fuchs et al., “Skin Diseases Associated with Oxidative Injury” in “Oxidative Stress in Dermatology”, 1983, pp. 323 et seq.

Antioxidants are substances, to include free radical absorbers or scavengers, that prevent oxidation processes, including oxidation of a molecule such as a lipid, lipoprotein, protein or DNA, or autooxidation of fats containing unsaturated compounds. Various antioxidants are used in the field of cosmetics and pharmacy. These include, for example tocopherol, kinetin, ubiquinone, ascorbic acid, lipoic acid sesamol, colic acid derivatives, butylhydroxy anisole and butylhydroxy toluene. Antioxidants can thus help protect human cells, such as skin cells, from both externally and internally generated oxidative stress.

Topical application of antioxidants is used to optimize the cutaneous antioxidative capacity and to limit skin damage. In vitro and in vivo studies have demonstrated antioxidative and photoprotective properties of antioxidants. See K. Scharffetter-Kochanek et al., “Photoaging of the skin from phenotype to mechanisms”, Exp Gerontol 2000, 35:307-316; and J. Wenk et al., “UV-induced oxidative stress and photoaging”, Curr Probl Dermatol 2001, 29:83-94. Accordingly, antioxidants may be incorporated in the cosmetic or dermatological formulations.

U.S. Pat. No. 6,756,045 describes treating skin changes, for example involving oxidative processes, using idebenone.

SUMMARY

Antioxidants have become increasingly popular in anti-aging cosmeceuticals. Consumers may be generally familiar with some or many of the topical antioxidants marketed today, including vitamin C, vitamin E, co-enzyme Q10, and alpha lipoic acid. However, consumers do not understand which antioxidants are the most effective, as little scientific research has been conducted on the wide variety of skincare products that contain these ingredients.

Additionally, consumer products may contain various substances that function to protect the skin from external environmental stressors, including oxygen itself, by blocking a stressor from contacting or penetrating the skin. For example, sun screen preparations may contain substances that help protect the skin by blocking all or some ultraviolet radiation from reaching and/or penetrating the skin. Such environmental stressor blocking agents may therefore have a protective, including preventive, effect against oxidative stress in the skin related to external environmental stressors.

What is needed is a standard way to compare the efficacy of antioxidative substances or antioxidative substance-containing preparations in a way that consumers can understand. Such antioxidative substances may include an antioxidant, photoprotective, or another substance that has a protective effect against externally generated and/or internally-generated oxidative stress.

The present invention provides a method for characterizing an oxidative stress protective capacity of an antioxidative substance. The method includes assigning a first score indicative of an oxidative stress protective capacity of a first antioxidative substance. The first score is based on a performance of the first antioxidative substance in a first antioxidative efficacy test relative to a performance of a second antioxidative substance in the first antioxidative efficacy test.

The present invention also provides another method for characterizing an oxidative stress protective capacity of an antioxidative substance. The method includes:

assigning a first score indicative of an oxidative stress protective capacity of a first antioxidative substance, the first score being based on a performance of the first antioxidative substance in a first antioxidative efficacy test;

assigning a second score indicative of an oxidative stress protective capacity of the first antioxidative substance, the second score being based on a performance of the first antioxidative substance in a second antioxidative efficacy test; and

combining the first and second scores so as to provide a total score characterizing the oxidative stress protective capacity of the first antioxidative substance.

The present invention also provides another method for characterizing an oxidative stress protective capacity of an antioxidative substance. The method includes assigning a first score indicative of an oxidative stress protective capacity of a first antioxidative substance. The first score is based on a performance of the first antioxidative substance in a first antioxidative efficacy test relative to a first standard indicative of unprotected human skin.

The present invention also a method for informing a purchaser of a topical skin care preparation of an oxidative stress protective capacity, relative to human skin, of a first antioxidative substance included in the topical skin care preparation, includes providing the topical skin care preparation, the topical skin care preparation including the first antioxidative substance. Information is provided including a total score indicative of an oxidative stress protective capacity, relative to human skin, of the first antioxidative substance. The total score is based on a combination of a first score representative of a performance of the first antioxidative substance in a first antioxidative efficacy test and a second score representative of a performance of the first antioxidative substance in a second antioxidative efficacy test.

Also provided by the present invention is a method for marketing a topical skin preparation. The method includes providing the preparation, the preparation including a first antioxidative substance, and providing information including a score indicative of an oxidative stress protective capacity of the first antioxidative substance.

The method according to the present invention addresses the need for standardized information regarding the topical efficacy of antioxidative substances, including antioxidants and other oxidative stress protective substances, as well as preparations containing them. The present invention provides a standardized comparison of different antioxidative substances regarding their topical oxidative stress protective capacity, i.e., their oxidative stress protective capacity in topical applications. The present invention thus provides a simple way for consumers to recognize the level of oxidative/environmental stress protection to be expected from an antioxidative skincare product, and to compare efficacy of topical antioxidative cosmeceutical products.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graph depicting percent reduction in sunburn cell (SBC) formation with application of test substances under ultraviolet B (UVB) irradiation of human skin.

FIG. 2 shows a graph depicting the relative amount of primary oxidation by-products (lipid hydroperoxides) in the presence of test substances as percentage compared to the BLANK (control, i.e., incubation without addition of antioxidants) in a low density lipoprotein (LDL) pro-oxidative system.

FIG. 3 shows a graph depicting the relative amount of secondary oxidative by-products (MDA equivalents) in the presence of test substances as percentage compared to the BLANK (control, i.e., incubation without addition of antioxidants) in a microsome pro-oxidation system.

DETAILED DESCRIPTION

In an embodiment of the present invention six antioxidants were each subjected to five independent antioxidative efficacy tests, or studies. The following antioxidants were tested: idebenone (6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone) dl-alpha tocopherol (vitamin E); kinetin (a plant derivative); ubiquinone (coenzyme Q-10); 1-ascorbic acid (vitamin C) and dl-alpha lipoic acid. The five tests were used to assess the oxidative stress protective capacity of each antioxidant. The results of the five tests for each antioxidant were combined so as to provide a total score indicative of overall performance of each antioxidant.

The tests included one in vivo test (Test 1) and four in vitro tests (Tests 2-5).

Test 1, UVB irradiation of human skin measuring damage by formation of sunburn cells (“sunburn cell assay”) tested in vivo the ability of each antioxidant to protect against UV induced oxidative stress. In Test 2, radical scavenging capacity measured by photochemilluminescence (“photochemilluminescence”), the ability of the antioxidant to suppress free radical formation was assessed. Test 3, LDL pro-oxidative systems measuring primary oxidation by-products (“primary oxidative products”), assessed the antioxidant\'s ability to suppress lipid oxidation. Test 4, microsome pro-oxidative system measuring secondary oxidation by-products (“secondary oxidative products”), assessed the antioxidant\'s ability to protect against cell membrane oxidation. In in vitro Test 5, UVB irradiation of keratinocytes measuring DNA damage (“UVB irradiated keratinocytes”), the antioxidant\'s ability to protect against UV induced oxidative stress was assessed.

Each oxidant was scored in each test, the results were then totaled for each antioxidant for each of the tests on an equal weighted basis. The total score of a particular antioxidant is reflective of the overall oxidative stress protective capacity of the antioxidant.

The five tests, which were equally weighted at 20 points each, allowed for the maximum highest possible score of 100 points. Using this scale, the higher score indicates higher oxidative stress protective capacity of the antioxidant. Equal weight was given to an antioxidant\'s performance in each study because each study tested the antioxidant\'s ability to protect against a unique different set of oxidative stress parameters. To assign scores, the performance of the antioxidant that demonstrated the greatest benefit for a specific test conducted was used as the standard for that test and received a score of 20 points. The results of the remaining antioxidants in that test were normalized to the standard by assigning each antioxidant a score equal to a percentage of the 20 points based on their performance relative to the standard in that test.

The following provides a brief summary of the protocol for each test conducted:

Exposure to UV radiation can cause damage of epidermal cells, resulting in the formation of sunburn cells. Since sunburn cells can be counted, their formation provides a relatively sensitive and quantitative measure of the extent of UV radiation damage to the epidermis.

All applications were made to a 5×10 cm area site over the mid-back region once a day for two weeks. Each putative antioxidant was applied to five (n=5) different healthy adult volunteers between the ages of 18 and 60. All Antioxidants were dissolved in ethanol/water at 0.5% w/w concentrations. Additionally, one test site was left untreated and served as a control. Approximately ten minutes after the last application, test sites were irradiated to1.5 MED (Minimal Erythema Dose) of UVB light, a shave biopsy taken and prepared histologically, and the number of sun burn cells (SBC) evaluated microscopically per High Power Field.

The light source used was a 150 watt xenon arc solar simulator equipped with a UV reflecting dichromic mirror and a 1 mm thick Schott WG-320 (BES Optics Inc., W. Warwick, R.I.) filter to produce simulation of the solar spectrum. A 1 mm thick UG5 filter was added to remove reflected heat and remaining visible radiation.

The MED for each subject was determined by exposing a 1 cm diameter circle to untreated areas to a series of exposures in 25% dose increments from the solar simulator. The MED was defined as the time of exposure required to produce a minimally perceptible erythema 20±4 hours after exposure. Biopsies: Approximately ten minutes after the last topical application of the putative antioxidant, a circular area measuring 1 cm in diameter was exposed to a single dose of 1.5 MED using the solar simulator. Approximately 20 hours later, a shave biopsy (˜4×4 mm) was obtained from each irradiated and untreated control sites. Following injections of a local anesthetic (lidocaine). The skin specimens were immediately fixed in 10% buffered formalin.

The fixed specimens were processed routinely, embedded in paraffin and then sectioned and stained with hematoxylin-eosin. The numbers of sun burn cells (SBC) were determined in at least 12 sections at 50 u intervals. A minimum of 70 high power fields (HPF) was counted from each biopsy and the average number of SBCs per HPF determined. All specimens were counted in a blinded manner.

FIG. 3 expresses the photoprotective benefits of the antioxidants tested based on the percent change over baseline (delta percent) for the number of SBC per HPF.

This test assessed the general antioxidative capacity of substances in their ability to scavenge free radicals via the measurement of radicals generated (or lack thereof) through their reaction with luminol and subsequent light emission.

The individual antioxidant capacity of the putative antioxidant substances was estimated by the Photochem® system (Analytik Jena AG, Jena, Germany, and Analytik Jena USA, Inc., Texas). The system combines the generation of radicals through photochemical excitation with highly sensitive luminometric detection via the radical reaction with luminol to produce measurable light emission. Samples are diluted with pre-made buffers (standardized kits) and applied to the device. The relative antioxidative capacity is determined by comparison to a standardized blank (without antioxidants) and a standard provided with the kit. The ACL-kit (Integral Antioxidative Capacity of lipid-soluble substances) and the ACW-kit ((Integral Antioxidative Capacity of water-soluble substances) were used.

Antioxidant concentrations effective to eliminate radical formulation were established for each antioxidant. Table 1 summarizes the results.

TABLE 1 Radical Scavenging Capacity Measured by Photochemilluminescence Antioxidant Substance Effective Concentration (nmol/L) Idebenone 10 Ascorbic acid 10 Tocopherol 10 Ubiquinone 100 Kinetin 1000 lipoic acid >1000 (not detectable)

This test assessed the ability of the antioxidant to protect LDL stressed with Copper Sulfate (Cu—SO4) oxidative system. The CuSO4-LDL system was used to evaluate the protection of lipid bulks over time measuring the primary by-products of lipid peroxidation—the highly reactive and cytotoxic lipid hydroperoxides.

Low Density Lipoproteins (d=1.019-1.063 kg/L) were isolated in clean Beckman one-way QuickSeal© Tubes (Beckman Coulter Inc., Palo Alto, Calif.) by ultracentrifugation from pooled plasma of healthy donors using an established protocol. After isolation, LDLs were extensively dialyzed against a degassed and nitrogen-saturated Tris-Hydrochloric (HCl) buffer (5 mmol/L, pH7.4) containing 1 mmol/L EDTA. Before oxidation by CuSO4, EDTA was removed from LDL by dialysis against a Tris-HCl buffer (5 mmol/L, pH7.4) without added EDTA.

Incubation LDL with the pro-oxidant system Ham\'sF-10/CuSO4

LDL oxidation was achieved by incubating (37° C., 95% O2, 5% CO2) 1 g of LDL protein/L with and without the putative antioxidant substances (at equivalent 100 μmol concentrations) in 2 ml of serum-free Ham\'s F-10 medium (BioSource International, Camarillo, Calif.) in the presence of 20 μmol/L CuSO4 for the times indicated in the figure legends.

Lipid hydroperoxides were determined with the Cayman Lipid Peroxidation (LPO) Assay Kit (Cayman Chemical, Ann Arbor, Mich.) which measures the hydroperoxides directly utilizing the redox reactions with ferrous ions to produce ferric ions which can be detected using thiocyanate ion as the chromogen.

FIG. 2 shows the antioxidative effect of the substances as percentage compared to the BLANK control (incubation without addition of antioxidants).

Test 4: Microsome Pro-Oxidative System measuring Secondary Oxidation By-Products

In this test, an assessment of antioxidative ability to protect microsomal membrane stressed with NADPH/ADP/Fe3+-oxidative system measuring secondary oxidative by-products (Malondialdehyde—MDA equivalents) was performed utilizing the Thiobarbituric Acid Reactive Substances (TBARS) method. See E. Wieland et al., “Idebenone protects hepatic microsomes against oxygen radical-mediated damage in organ preservation solutions”, Transplantation, September 1995, 15, 60(5):444-51, the subject matter of which is hereby incorporated by reference herein. Antioxidants protecting bulky lipids, such as low density lipoproteins, are not necessarily good protectors of cell membranes due to their hydrophilic/lipophilic bilayer composition. Therefore the pro-oxidative NADPH/ADP/Fe3+-microsome system was used as an in vitro model system more closely resembling natural cellular biological systems. Oxidation of cell membranes leads to serious consequences in altering cell membrane fluidity and cell function.

Livers were obtained from male Wistar rats weighing between 250 and 400 g. Tissue was homogenized in 50 mmol/L N-2-Hydroxyethylpiperazine-N-2-Ethanesulfonic Acid (HEPES), 250 mmol/L sucrose buffer, pH 7.4, containing 150 mmol/L Potassium Chloride (KCl) and 500 μmol/L EDTA using a kinematica polytron PT3000 (Brinkmann Instruments, Westbury, N.Y.) homogenizer. Microsomal vesicles were isolated by removal of the nuclear fraction at 8,000×g for 10 min at 4° C. and removal of the mitochondrial fraction at 18,000×g for 10 min at 4° C. using a Beckman L8-55 ultracentrifuge and a 50Ti-13 rotor. The microsomal fraction was sedimented at 105,000×g for 60 min at 4° C. The pellet was washed once in 50 mmol/L HEPES and 150 mmol/L KCl, pH7.4, and collected again at 105,000×g for 30 min. The resulting microsomal pellet was resuspended in HEPES/KCl, pH7.4, by careful sonication in ice and stored in portions (10 mg protein/ml) at −80° C. until use.

Incubation of Microsomes with the Pro-Oxidant System NADPH/ADP/Fe3+

The microsomal preparations were incubated in the presence of the pro-oxidant system NADPH/ADP/Fe3+, consisting of 0.20 mmol/L NADPH, 50 mmol/L ADP, and 0.25 mmol/L FeCl3 in HEPES/KCl buffer (150 mmol/L KCl, 50 mmol/L HEPES) with and without the putative antioxidant substances. Oxidation of 1-ml aliquots containing 1 mg of protein was started at 37° C. by the addition of NADPH and was stopped with EDTA (10 mmol/L) after the times indicated in the figure legends. Control incubations without the pro-oxidant system were performed at 37° C. in the presence of EDTA. All Antioxidants were dissolved in water or ethanol and added to the incubations at equivalent 100 μmol concentrations.

Microsomal preparations (500 μl) were mixed with 1 ml of thiobarbituric acid (0.67 g/100 ml, 0.05 mol/L Sodium Hydroxide—NaOH). After the addition of trichloroacetic acid (50% w/v), the samples were heated to 90° C. for 30 min. After cooling and extraction of the samples with 1 ml of butanol, the absorbance of the butanol phase was determined spectrophotometrically at 532 nm. For quantification, an external standard curve was prepared using 1,1,3,3-tetraethoxypropane, which yields MDA.

FIG. 3 shows the antioxidative effect of the substances as percentage compared to the BLANK control (incubation without addition of antioxidants).

This test assessed DNA damage in cell culture experiments under pro-oxidative conditions (UVB irradiation of human keratinocytes) by measuring the positive cells for anti-thymine dimer antibodies. This experiment is thought to reflect a direct correlation to the in vivo occurring DNA cross linking damage following UVB exposure and the protection of such nuclear damage by antioxidants.

Human primary foreskin keratinocytes (second passage) were grown in 6-well plates containing cover slips to 60% confluence in serum free medium (KGM, Clonetics) containing 0.07 mM CaCl2. Six hours before UVB-radiation medium was removed and replaced by fresh growth medium with or without the antioxidative substances. The concentrations of the antioxidants are indicated in the figures.

Keratinocyte cultures were irradiated with a single dose of 200 mJ/cm2 UVB, using FS-20/T-12 bulbs (emission range: 280-340 nm; 305 nm max.). Immediately prior to irradiation, the medium was replaced with 1 ml sterile PBS (pH 7.4, 37° C.), and after irradiation, PBS was replaced with fresh growth medium. UVB exposure was quantitated using a Goldilux Ultraviolet radiometer. Cells were maintained at 37° C. (5% CO2) for 1 hour until fixation with paraformaldehyde (PFA).

Cells were fixed with 4% PFA in PBS for 30 min at RT, washed with PBS and permeabilized by incubation with EtOH/PBS (90/10; v %/v %) for 30 min at ˜10° C. After fixation and permeabilization cells were washed twice with PBS containing 1% of bovine serum albumin (BSA). They were then incubated for 30 min with 10 μg/ml anti-thymine Dimer Ab (clone KTM53; Kamiya) at RT. After the incubation period cells were washed twice with PBS-BSA and incubated for 30 min with 20 μg/ml secondary FITC-conjugated anti-mouse IgG at RT. After the incubation with the secondary antibody, cells were washed twice with PBS-BSA and fixed again with 4% PFA for 15 min at RT. Slides were analyzed by confocal microscopy.

This experiment is thought to reflect the in-vivo occurring DNA damage following UVB exposure and the protection of such nuclear damage by antioxidants. Table 2 shows the results, which may be seen as approximate estimations of the occurrence of nuclear thymine dimer photo products.

TABLE 2 UVB Irradiation of Keratinocytes Measuring DNA Damage Inhibition of Positive cells after photoproduct generation Antioxidant Substance UVB radiation* (protective effect) No-radiation control  0% 100%  Idebenone 29% 45% Ascorbic acid 34% 36% Kinetin 34% 36% Tocopherol 35% 34% Ubiquinone 51%  4% lipoic acid 53%  0%

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