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VOLUME 4 • NUMBER 1 • JANUARY 2005 Cosmetic Dermatology Edited by Zoe Diana Draelos Reprinted Article: Idebenone: a new antioxidant – Part I. Relative assessment of oxidativestress protection capacity compared to commonly known antioxidants D H McDaniel, B A Neudecker, J C DiNardo, J A Lewis, II & H I Maibach Blackwell Publishing, Ltd.
Idebenone: a new antioxidant – Part I. Relative assessment of oxidative
stress protection capacity compared to commonly known
antioxidants
D H McDaniel,1 B A Neudecker,2 J C DiNardo,3 J A Lewis, II3 & H I Maibach2
1Institute of Anti-Aging Research, Virginia Beach, VA, USA, Eastern Virginia Medical School, Norfolk, VA, USA
2Department of Dermatology, University of California San Francisco, San Francisco, CA, USA
3Pharma Cosmetix Research, LLC, Richmond, VA, USA
Topical applications of skin care products containing antioxidants have becomeincreasingly popular. Numerous studies have elucidated the biological effects of thesesubstances. General antiaging effects, anti-inflammatory properties, photoprotectiveproperties, and prevention of ultraviolet (UV) immunosuppression have been docu-mented. However, a standardized method to characterize and compare the propertiesand oxidative stress protection capacity of antioxidants was lacking. A multistep in vitroprocess utilizing a variety of biochemical and cell biological methods combined with invivo studies was designed to compare the oxidative stress protective capacity of com-monly used antioxidants. Data were presented for L-ascorbic acid, dl-alpha-tocopherol,kinetin, dl-alpha lipoic acid, ubiquinone, and idebenone. Methods included using UV-induced radical trapping/scavenging capacity measured by photochemiluminescence,pro-oxidative systems (LDL-CuSO , microsome-NADPH/ADP/Fe3+) with measurement of primary and secondary oxidation products, UVB irradiation of human keratinocytes,and in vivo evaluation, using the human sunburn cell (SBC) assay. Correlation and trendsbetween in vitro and in vivo results were established, and the standardized test protocolwas used to quantify oxidative stress protection capacity of antioxidants. Summarizingand totaling the data equally weighted for each oxidative stress study, the overall oxida-tive protection capacity scores of 95, 80, 68, 55, 52, and 41 were obtained for idebenone,dl-alpha tocopherol, kinetin, ubiquinone, L-ascorbic acid, and dl-alpha lipoic acid,respectively. The higher the score, the more effective the overall oxidative stress protec-tion capacity of the antioxidant became. This multistep protocol may serve as a standardin investigating and comparing new putative antioxidants for topical use as well as avaluable tool to assess the anti-inflammatory properties, photoprotective properties, andprevention of UV immunosuppression of topical antioxidants.
Keywords: idebenone, antioxidant, aging Ultraviolet radiation (UVR)-generated reactive oxygen Correspondence: David H. McDaniel, MD, Institute of Anti-Aging Research, species (ROS) and DNA photodamage play a critical LLC, 933 First Colonial Road, Suite 205, Virginia Beach, VA 23454. E-mail: role in the process of extrinsic environmentally ind- uced aging (photoaging) and photocarcinogenesis.1 In Accepted for publication January 26, 2005 addition to the well-known long-term effects such as 2005 Blackwell Publishing Ltd • Journal of Cosmetic Dermatology, 4, 1 0 – 17
Idebenone • D H McDaniel et al.
immunosuppression and skin cancer, photo-oxidative Q-10 that showed potent radical scavenging capacity and damage leads to alterations of cells and structural cell protection properties in previous studies.10 Five inde- macromolecules of the dermal connective tissue pendent biochemical, cell-biologic, and in vivo methods contributing significantly to photoaging with its clinical were combined to determine the antioxidant capacity of appearance of wrinkle formation, laxity, and pigment the single substances under different conditions in order dyschromias.2 The skin is constantly exposed to a pro- to demonstrate their overall performance. The studies oxidative environment such as UVR and air pollutants.
conducted were as follows: The skin is equipped with various antioxidant defense Study 1. Radical scavaging capacity measured by systems constituting a complex antioxidant network.1,3 photochemiluminescence: Utilizes Photochem®, a device Whereas UVB can damage DNA, proteins, and lipids that offers fast and reliable chemiluminometric assess- directly, UVA is believed to act largely via oxidative ment of the general antioxidative capacity of substances processes.4 Increased exposure to exogenous sources in their ability to scavenge free radicals via the measure- and/or endogenous production of ROS can provoke an ment of radicals generated (or lack thereof ) through their imbalance of the fragile pro-oxidant–antioxidant equilibrium, reaction with luminol and subsequent light emission.
resulting in oxidative damage of lipids, proteins, and Study 2. Low density lipoprotein (LDL) pro-oxidative DNA.5,6 For example, the superoxide (O ) radical can system measuring primary oxidation by-products: cause the "common deletion" mutation in mitochondrial assessment of antioxidant ability to protect LDL stressed DNA, which can be found in high numbers in photo- with copper sulphate (CuSO ) oxidative system. The damaged skin.7 According to the mitochondrial theory CuSO -LDL system was used to evaluate the protection of of aging, nonrepaired damage of mitochondrial DNA lipid bulks over time measuring the primary by-products and unstable electron transfer cause an important loss of of lipid peroxidation – the highly reactive and cytotoxic mitochondrial function in correlation with progression of age. Topical application of antioxidants is used to optimize Study 3. Microsome pro-oxidative system measuring the cutaneous antioxidative capacity and to limit ROS- secondary oxidation by-products: assessment of antioxi- induced skin damage. Numerous in vitro and in vivo dative ability to protect microsomal membrane stressed studies have demonstrated specific antioxidative capacity with NADPH/ADP/Fe3+-oxidative system measuring as well as their photoprotective properties.6,8 Antioxidants secondary oxidative by-products (malondialdehyde – applied topically before UV-irradiation on animal and MDA equivalents) utilizing the thiobarbituric acid-reactive human skin diminished UVA-induced polymorphous substances (TBARS) method.11 Antioxidants protecting light eruption, psoralen + ultraviolet A (PUVA)-induced bulky lipids, such as LDL, are not necessarily good protec- erythema, and sunburn cell formation.9 However, a stand- tors of cell membranes as a result of their hydrophilic/ ardized method to characterize and compare the complex lipophilic bilayer composition. Therefore, the pro-oxida- properties and effects of topical antioxidants is lacking. In tive NADPH/ADP/Fe3+-microsome system was used as this study, for the first time, a variety of biochemical and an in vitro model system more closely resembling natural cell biologic methods are combined with in vivo studies in cellular biological systems. Oxidation of cell membranes a protocol to compare protective capacity of commonly leads to serious consequences in altering cell membrane used antioxidant ingredients. The in vivo method was fluidity and cell function.
included to assess real biological effects in living tissue, Study 4. UVB irradiation of keratinocytes measuring as human skin itself contains lipophilic antioxidants such DNA damage: assessment of DNA damage in cell culture as vitamin E (tocopherols and tocotrienols), ubiquinones experiments under pro-oxidative conditions (UVB irradiation (coenzyme Q), carotenoids, and lipoic acid, as well as the of human keratinocytes) by measuring the positive cells hydrophilic antioxidants, vitamin C (ascorbate), uric acid for antithymine dimer antibodies. This experiment is thought (urate), superoxide dismutase (SOD), and glutathione.
to reflect a direct correlation to the in vivo-occurring DNA Reduction and oxidation (redox) cellular reactions cross linking damage following UVB exposure and the couple these antioxidants in a network together through protection of such nuclear damage by antioxidants.
a complex concerted action in which the antioxidants are Study 5. UVB irradiation of human skin measuring partly recycled by one another.5 damage by formation of sunburn cells (SBC): Exposure to For this multistep protocol, the following compounds UVR can cause damage of epidermal cells, resulting in were tested: L-ascorbic acid (vitamin C), kinetin (a plant the formation of sunburn cells.12 Because sunburn cells derivative), dl-alpha tocopherol (vitamin E), dl-alpha can be enumerated, their formation provides a relatively lipoic acid, ubiquinone (CoEnzyme Q-10), and ide- sensitive and quantitative measure of the extent of UVR benone, a lower molecular weight analog of CoEnzyme damage to the epidermis.
2005 Blackwell Publishing Ltd • Journal of Cosmetic Dermatology, 4, 1 0 – 17
Idebenone • D H McDaniel et al.
trifugation from pooled plasma of healthy donors using Methods, materials, and results
an established protocol. After isolation, LDLs wereextensively dialyzed against a degassed and nitrogen- Study 1. Radical scavenging capacity measured by saturated tris-hydrochloric (HCl) buffer (5 pH 7.4) containing 1 mmol/L ethylenediaminetetraacetic The individual antioxidant capacity of the putative acid (EDTA). Before oxidation by CuSO , EDTA was antioxidant substances was estimated by the Photo- removed from LDL by dialysis against a tris-HCl buffer chem® system (Analytik Jena AG, Jena, Germany, and (5 mmol/L, pH 7.4) without added EDTA.
Analytik Jena USA, Inc., TX). The system combines thegeneration of radicals through photochemical excitation Incubation LDL with the pro-oxidant system Ham's F-10/ with highly sensitive luminometric detection via the radical reaction with luminol to produce measurable The LDL oxidation was achieved by incubating (37 °C, light emission. Samples are diluted with premade buffers 95% O , 5% CO ) 1 g of LDL protein/L with and without (standardized kits) and applied to the device. The relative the putative antioxidant substances (at equivalent antioxidative capacity is determined by comparison to 100 µmol concentrations) in 2 mL of serum-free Ham's a standardized blank (without antioxidants) and a F-10 medium (BioSource International, Camarillo, CA) standard provided with the kit. The ACL-kit (integral in the presence of 20 µmol/L CuSO for the times antioxidative capacity of lipid-soluble substances) and indicated in the figure legends.
the ACW-kit (integral antioxidative capacity of water-soluble substances) were used. Antioxidant concen- Measurement of lipid hydroperoxides trations effective to eliminate radical formulation were Lipid hydroperoxides were determined with the Cayman established for each antioxidant as indicated in Table 1.
Lipid Peroxidation (LPO) Assay Kit (Cayman Chemical,Ann Arbor, MI) which measures the hydroperoxides directly utilizing the redox reactions with ferrous ions to Idebenone, dl-alpha-tocopherol, and L-ascorbic acid all produce ferric ions which can be detected using thiocy- demonstrated effective neutralization of radicals as mea- anate ion as the chromogen. The antioxidative effect of sured by photochemiluminescence at a relatively low con- the substances is shown as percentage compared to the centration of 10 nmol/L. Ubiquinone was somewhat less Blank (incubation without addition of antioxidants).
effective, requiring 100 nmol/L for effective concentration,kinetin required 1,000 nmol/L for effective concentration, and alpha lipoic acid was ineffective in this system (see Kinetin and idebenone demonstrated a consistent protection against lipid peroxidation over 24 h. Other substances likeubiquinone, lipoic acid, and ascorbic acid showed only a com-paratively short-lasting protective efficiency (see Fig. 1).
Study 2. LDL pro-oxidative system measuring primary oxidation by-products Study 3. Microsome pro-oxidative system measuring Isolation of LDL secondary oxidation by-products Low density lipoproteins (d = 1.019–1.063 kg/L) wereisolated in clean Beckman one-way Quick-Seal Tubes Preparation of microsomes (Beckman Coulter Inc., Palo Alto, CA) by ultracen- Livers were obtained from male Wistar rats weighingbetween 250 and 400 g. Tissue was homogenized in50 mmol/L N-2-hydroxyethylpiperazine-N-2-ethanesulphonic Table 1 Radical scavenging capacity measured by
acid (HEPES), 250 mmol/L sucrose buffer, pH 7.4, containing 150 mmol/L potassium chloride (KCl) and Antioxidant substance Effective concentration (nmol/L) 500 µmol/L EDTA using a Kinematica Polytron PT3000(Brinkmann Instruments, Westbury, NY) 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 sedi- > 1000 (not detectable) mented at 105,000 g for 60 min at 4 °C. The pellet was 2005 Blackwell Publishing Ltd • Journal of Cosmetic Dermatology, 4, 1 0 – 17

Idebenone • D H McDaniel et al.
Figure 1 Low density lipoprotein (LDL)
pro-oxidative system measuring primary
oxidation by-products (lipid
hydroperoxides).
Figure 2 Microsome pro-oxidation system
measurement of secondary oxidative by-
products (MDA equivalents).
washed once in 50 mmol/L HEPES and 150 mmol/L KCl, Measurement of secondary oxidation products, MDA pH 7.4, and collected again at 105 000 g for 30 min. The equivalents or TBARS method resulting microsomal pellet was resuspended in HEPES/ Microsomal preparations (500 µL) were mixed with KCl, pH 7.4, by careful sonication in ice and stored in 1 mL of thiobarbituric acid (0.67 g/100 mL, 0.05 mol/L portions (10 mg protein/mL) at −80 °C until use.
sodium hydroxide – NaOH). After the addition oftrichloroacetic acid (50% w/v), the samples were heated Incubation of microsomes with the pro-oxidant system to 90 °C for 30 min. After cooling and extraction of the samples with 1 mL of butanol, the absorbance of the The microsomal preparations were incubated in the butanol phase was determined spectrophotometrically at presence of the pro-oxidant system NADPH/ADP/Fe3+, 532 nm. For quantification, an external standard curve consisting of 0.20 mmol/L NADPH, 50 mmol/L ADP, and was prepared using 1,1,3,3-tetraethoxypropane, which 0.25 mmol/L FeCl in HEPES/KCl buffer (150 mmol/L yields MDA. The antioxidative effect of the substances is KCl, 50 mmol/L HEPES) with and without the putative shown as percentage compared to the control (incu- antioxidant substances. Oxidation of 1-mL aliquots con- bation without addition of antioxidants).
taining 1 mg of protein was started at 37 °C by the addi-tion of NADPH and was stopped with EDTA (10 mmol/L) after the times indicated in the figure legends. Control Lipoic acid and idebenone showed the most effective incubations without the pro-oxidant system were per- protection against oxidation of the cell membrane lipids.
formed at 37 °C in the presence of EDTA. All antioxidants Kinetin, which showed favorable results in protecting bulky were dissolved in water or ethanol and added to the lipids (LDL), showed only a weak protective effect in the incubations at equivalent 100 µmol concentrations.
ability to protect microsomal membranes (see Fig. 2).
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Idebenone • D H McDaniel et al.
Table 2 UVB irradiation of keratinocytes measuring DNA damage.
Study 4. UVB irradiation of keratinocytes measuring DNA Human primary foreskin keratinocytes (second passage) Antioxidant substance (protective effect) were grown in 6-well plates containing cover slips to 60%confluence in serum free medium (KGM, Clonetics; No radiation control Cambrex Cooperation, E. Rutherford, NJ) containing 0.07 mm calcium chloride (CaCl ). Six hours before UVB- radiation, the medium was removed and replaced by fresh growth medium with or without the antioxidative substances. Each antioxidant concentration was 10 µmol 200 mJ/cm2 UVB radiation Ultraviolet light (UVB) irradiation of keratinocytes *Percentage of positive cells (above threshold) in three fields Keratinocyte cultures were irradiated with a single dose (counted cell number ∼120–150).
of 200 mJ/cm2 UVB, using FS-20/T-12 bulbs (emissionrange: 280 –340 nm; 305 nm max.). Immediately prior Study 5. UVB irradiation of human skin measuring to irradiation, the medium was replaced with 1 mL sterile damage by formation of sunburn cells (SBC) phosphate buffered saline (PBS) (pH 7.4, 37 °C), andafter irradiation, PBS was replaced with fresh growth medium without antioxidants. The UVB exposure was All applications were made to a 5 × 10 cm area site over quantified using a Goldilux™ Ultraviolet Radiometer (Oriel the mid-back region once a day for 2 weeks. Each putative Instruments, Stratford, CT). Cells were maintained at antioxidant was applied to five (n = 5) healthy adult 37 °C (5% CO ) for 1 h until fixation with paraform- volunteers between the ages of 18 and 60. All antioxidants aldehyde (PFA).
were dissolved in ethanol/water at 0.5% w/w concen-trations. Additionally, one test site was left untreated and Fixation and nuclear thymine–dimer staining of keratinocytes served as a control. Approximately 10 min after the last Cells were fixed with 4% PFA in PBS for 30 min at application, test sites were irradiated to 1.5 minimal room temperature (RT), washed with PBS and per- erythema dose (MED) of UVB light, a shave biopsy taken meabilized by incubation with EtOH/PBS (90/10; v%/v%) and prepared histologically, and the number of sun burn for 30 min at ∼10 °C. After fixation and permeabilization, cells (SBC) evaluated microscopically per high power field.
cells were washed twice with PBS containing 1% ofbovine serum albumin (BSA). They were then incubated Light source for 30 min with 10 µg/mL antithymine dimer Ab (clone The light source used was a 150-W xenon arc solar KTM53; Kamiya Biomedical Company, Seattle, WA) at simulator equipped with a UV reflecting dichromic RT. After the incubation period, the cells were washed mirror and a 1-mm thick Schott WG-320 (BES Optics twice with PBS-BSA and incubated for 30 min with Inc., W. Warwick, RI) filter to produce simulation of the 20 µg/mL secondary fluorescein-isothiocyanate (FITC)- solar spectrum. A 1-mm thick UG5 filter was added to conjugated antimouse immunoglobulin G (IgG) at RT.
remove reflected heat and remaining visible radiation.
After the incubation with the secondary antibody, cellswere washed twice with PBS-BSA and fixed again with Minimal Erythema Dose (MED) determination 4% PFA for 15 min at RT. Slides were analyzed by The MED for each subject was determined by exposing a circle 1 cm in diameter to untreated areas to a seriesof exposures in 25% dose increments from the solar simulator. The MED was defined as the time of exposure This experiment is thought to reflect the in vivo occurring required to produce a minimally perceptible erythema DNA damage following UVB exposure and the protection 20 ± 4 h after exposure.
of such nuclear damage by antioxidants. The results (seeTable 2) have to be seen as approximate estimations of the occurrence of nuclear thymine dimer photo products.
Approximately 10 min after the last topical application of Idebenone provided the highest level of inhibition.
the putative antioxidant, a circular area measuring 1 cm 2005 Blackwell Publishing Ltd • Journal of Cosmetic Dermatology, 4, 1 0 – 17

Idebenone • D H McDaniel et al.
Figure 3 UVB irradiation of human skin
measuring DNA damage by formation of
SBC.
in diameter was exposed to a single dose of 1.5 MED using antioxidative capacities. In this study, a multistep pro- the solar simulator. Approximately 20 h later, a shave tocol is presented to allow the comparison of different biopsy (∼4 × 4 mm) was obtained from each irradiated antioxidants regarding their usefulness in topical appli- and untreated control site following injections of a local cations. This combination of biochemical, cell-biologic, anesthetic (lidocaine). The skin specimens were imm- and in vivo methods allows the determination of various ediately fixed in 10% buffered formalin.
independent aspects of antioxidant substances, such asanti-inflammatory properties, photoprotective properties, or protection of cell membranes. The results demonstrate The fixed specimens were processed routinely, embedded the diversity of actions and the value of utilizing a com- in paraffin, and then sectioned and stained with bination of entirely different methods when comparing hematoxylin-eosin. The numbers of SBC were deter- the relative efficacy of antioxidant activity. Kinetin for mined in at least 12 sections at 50-µ intervals. A example showed a very weak antioxidant effect when minimum of 70 high power fields (HPF) was counted evaluated by chemiluminometric detection of antioxidative from each biopsy, and the average number of SBCs per capacity yet showed the strongest effect in the LDL/ HPF determined. All specimens were counted in a blinded CuSO -oxidation system. Another example is lipoic acid which showed a strong effect in the microsome-NADPH/ADP/Fe3+-system while showing minimal response in every other method employed. Even tocopherol, which Figure 3 expresses the photoprotective benefits of the showed good results in most experiments, revealed a antioxidants tested based on the percent change over weakness regarding lipid protection over time (LDL/ baseline (delta percent) for the number of SBC per high CuSO -oxidation system). This is most likely because its power field. Idebenone was the most effective antioxidant pro-oxidative metabolites appear through reduction of in the study in its ability to protect human skin from radicals by hydrogen donation. The implementation of an sunburn cell formation post-UVR exposure.
in vivo approach employing the human SBC assay gavecrucial additional clinical information, as the capacity ofeach compound to penetrate the upper skin layers may vary. These results confirm that favorable in vitro results Previous studies comparing different antioxidants for use do not necessarily reflect the in vivo situation. An example in topical applications primarily focused on certain thereof is ascorbic acid which surprisingly had no biochemical or cell-biologic aspects of those substances.
protective effect (at the concentration tested) on in vivo Because the currently popular topical antioxidants are SBC formation. Idebenone, while to date had demonstrated of very heterogeneous structure and origin (vitamins, only the ability to protect against ROS-mediated damage flavonoids, coenzymes, etc.), a protocol to compare their in organ preservation solutions and to treat Alzheimer's properties should consist of a variety of methods aiming disease,13 showed a strong overall performance throughout to elucidate the overall picture regarding their specific all experiments conducted.
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Table 3 Global relative antioxidant activity: total oxidative stress protection capacity scores (environmental protection factor; EPF of
common antioxidants).
Sun burn cell assay Primary oxidative products Secondary oxidative products UVB irradiated keratinocytes Total points (EPF score) damage) and in vivo (study 5; UVB irradiation of human Establishing a standardized way to summarize results skin measuring damage by formation of SBC) test The scoring system introduced in this study is designed to methods. Therefore, overall, equal weighting of study provide, for the first time, a standardized comparison results seems to be most appropriate at this time. To of the protective capacity of different antioxidant sub- assign values, the active antioxidant that demonstrates stances used in topical applications against oxidative the greatest benefit for the test conducted became stress. Five tests, which were equally weighted at 20 the standard for the study, and received 20 points. The points each, allowed for the maximum highest possible remaining antioxidants were assigned a percentage of score of 100 points. Using this scale, the higher score the 20 points based on their efficacy relationship to the indicates higher oxidative stress protection capacity highest scoring antioxidant in each independent study.
of the antioxidant. Equal weight was given to the Example: idebenone produced the greatest benefit in antioxidant's performance in each study because each the SBC assay, a 38% reduction in SBCs. Therefore, it study tested the antioxidant's ability to protect against a became the standard and received 20 points. Tocopherol unique different set of oxidative stress parameters.
was second, producing a 31% reduction. To determine the Without knowing the direct correlation of each study to relative activity (efficacy) of tocopherol to the standard, actual living biological systems (with one exception, the idebenone, one calculates 31/38 × 100 = 82% relative SBC study was conducted in vivo), the equal weight activity. Therefore, tocopherol would receive 82% of approach was selected for the initial introduction of this the 20 points or 16 points (rounded to the nearest whole concept. Future experiments and clinical correlation should allow refinement of this concept and the Because the photochemiluminescence assay results weighting may be adjusted, if data warrant, for optimal are expressed as the lowest effective concentration, and a clinical correlation. For example, it is known that the base 10 serial dilution was used, the antioxidant scores superoxide radical, a natural by-product of metabolic were assigned as follows: 10 nmol/L = 20; 100 nmol/ energy production, causes serious deleterious effects L = 15; 1000 nmol/L = 10; > 1000 nmol/L = 5.
to living cells if not quenched, neutralized, or reduced The "relative value" of each of the five tests was arbi- almost immediately after production. In study 1, we trarily assigned equal weight. That is, each test con- tested the ability of the antioxidant to suppress super- tributed equally to the antioxidant scale. Further clinical oxide radical formation. It is also known that lipid testing in the future may allow additional refinement of peroxidation is a major problem in biological systems. In this scoring system and weight. The overall scores, and study 2 we assessed the antioxidants' ability to suppress thus relative oxidative stress protection capacity of the lipid oxidation. Protecting cell membrane oxidation is respective antioxidants tested, are summarized in Table 3.
of paramount importance to living biological systemsbecause the cell membrane is the cell's first line of defense against oxidation. In study 3, we assessed this protec-tive parameter. Because UV light is known to be the These studies compared the protective capacity of five predominant cause of premature aging of the skin, commonly used antioxidant ingredients and one novel the antioxidants' ability to protect against UV-induced new antioxidant for skin care, idebenone, in both in vitro oxidative stress was tested under both in vitro (study and in vivo methods. A standardized testing protocol that 4; UVB irradiation of keratinocytes measuring DNA quantifies the oxidative stress protection capacity of the 2005 Blackwell Publishing Ltd • Journal of Cosmetic Dermatology, 4, 1 0 – 17
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substances studied was developed, and a scoring system 5 Berneburg MH, Plettenberg J, Krutmann J. Photoaging of to compare relative activity was presented.
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