Reduction in the appearance of facial hyperpigmentation after use of moisturizers with a combination of topical niacinamide and N-acetyl glucosamine: results of a randomized, double-blind, vehicle-controlled trial.

BACKGROUND: Topical niacinamide and N-acetyl glucosamine (NAG) each individually inhibit epidermal pigmentation in cell culture. In small clinical studies, niacinamide-containing and NAG-containing formulations reduced the appearance of hyperpigmentation. OBJECTIVES: To assess the effect of a combination of niacinamide and NAG in a topical moisturizing formulation on irregular facial pigmentation, including specific detection of changes in colour features associated with melanin. METHODS: This was a 10-week, double-blind, vehicle-controlled, full-face, parallel-group clinical study conducted in women aged 40-60 years. After a 2-week washout period, subjects used a daily regimen of either a morning sun protection factor (SPF) 15 sunscreen moisturizing lotion and evening moisturizing cream each containing 4% niacinamide + 2% NAG (test formulation; n = 101) or the SPF 15 lotion and cream vehicles (vehicle control; n = 101). Product-induced changes in apparent pigmentation were assessed by capturing digital photographic images of the women after 0, 4, 6 and 8 weeks of product use and evaluating the images by algorithm-based computer image analysis for coloured spot area fraction, by expert visual grading, and by chromophore-specific image analysis based on noncontact SIAscopy for melanin spot area fraction and melanin chromophore evenness. RESULTS: By all four measures, the niacinamide + NAG formulation regimen was significantly (P < 0.05) more effective than the vehicle control formulation regimen in reducing the detectable area of facial spots and the appearance of pigmentation. CONCLUSIONS: A formulation containing the combination of niacinamide + NAG reduced the appearance of irregular pigmentation including hypermelaninization, providing an effect beyond that achieved with SPF 15 sunscreen.

Topical palmitoyl pentapeptide provides improvement in photoaged human facial skin.

The palmitoyl pentapeptide palmitoyl-lysine-threonine-threonine-lysine-serine (pal-KTTKS) is a synthetic material that was designed as a topical agent to stimulate collagen production and thus provide a skin anti-wrinkle benefit. To determine if pal-KTTKS is effective, the clinical study reported here was conducted. Caucasian female subjects (n = 93, aged 35-55) participated in a 12-week, double-blind, placebo-controlled, split-face, left-right randomized clinical study assessing two topical products: moisturizer control product vs. the same moisturizer product containing 3 ppm pal-KTTKS. Pal-KTTKS was well tolerated by the skin and provided significant improvement vs. placebo control for reduction in wrinkles/fine lines by both quantitative technical and expert grader image analysis. In self-assessments, subjects also reported significant fine line/wrinkle improvements and noted directional effects for other facial improvement parameters.

Topical niacinamide reduces yellowing, wrinkling, red blotchiness, and hyperpigmented spots in aging facial skin.

Previous clinical testing of topical niacinamide (vitamin B3) has revealed a broad array of improvements in the appearance of aging facial skin. The study reported here was done to confirm some of those previous observations and to evaluate additional end points such as skin anti-yellowing. Caucasian female subjects (n = 50, aged 40-60 years) participated in a 12-week, double-blind, placebo-controlled, split-face, left-right randomized clinical study assessing two topical products: moisturizer control product versus the same moisturizer product containing 5% niacinamide. Niacinamide was well tolerated by the skin and provided significant improvements versus control in end points evaluated previously: fine lines/wrinkles, hyperpigmentation spots, texture, and red blotchiness. In addition, skin yellowing (sallowness) versus control was significantly improved. The mechanism by which this array of benefits is achieved with niacinamide is discussed.

The effect of niacinamide on reducing cutaneous pigmentation and suppression of melanosome transfer.

BACKGROUND: Cutaneous hyperpigmentation occurs in multiple conditions. In addition, many Asian women desire a lighter skin colour. Thus, there is a need for the development of skin lightening agents. Niacinamide is a possible candidate. OBJECTIVES: To investigate the effects of niacinamide on melanogenesis in vitro and on facial hyperpigmentation and skin colour in vivo in Japanese women. METHODS: Melanin production was measured in a purified mushroom tyrosinase assay, cultured melanocytes, a keratinocyte/melanocyte coculture model, and a pigmented reconstructed epidermis (PREP) model. The clinical trials included 18 subjects with hyperpigmentation who used 5% niacinamide moisturizer and vehicle moisturizer in a paired design, and 120 subjects with facial tanning who were assigned to two of three treatments: vehicle, sunscreen and 2% niacinamide + sunscreen. Changes in facial hyperpigmentation and skin colour were objectively quantified by computer analysis and visual grading of high-resolution digital images of the face. RESULTS: Niacinamide had no effect on the catalytic activity of mushroom tyrosinase or on melanogenesis in cultured melanocytes. However, niacinamide gave 35-68% inhibition of melanosome transfer in the coculture model and reduced cutaneous pigmentation in the PREP model. In the clinical studies, niacinamide significantly decreased hyperpigmentation and increased skin lightness compared with vehicle alone after 4 weeks of use. CONCLUSIONS: The data suggest niacinamide is an effective skin lightening compound that works by inhibiting melanosome transfer from melanocytes to keratinocytes.

Synergistic topical photoprotection by a combination of the iron chelator 2-furildioxime and sunscreen.

BACKGROUND: Iron is a factor in skin photodamage, apparently by way of its participation in oxygen radical production. Certain topical iron chelators are photoprotective. OBJECTIVE: Our purpose was to determine the level of topical photoprotection provided by the iron chelator 2-furildioxime (FDO) in combination with sunscreen in short- and long-term photoprotection models. METHODS: Guinea pigs were treated topically with FDO, sunscreen, and a combination of the two and were then exposed to varying doses of UV radiation to determine the sun protection factor (SPF). Hairless mice were treated topically with FDO, sunscreen, and a combination of the two and then subjected to long-term exposure to a suberythemal dose of UV radiation. The mice were evaluated for skin wrinkling and skin tumors. RESULTS: In guinea pigs, topical FDO combined with sunscreen provided more than additive protection; 5% FDO alone provides approximately SPF 4, whereas 5% FDO combined with an SPF 4 sunscreen product yielded an SPF of more than 30. In hairless mice exposed long term to UV radiation, 5% FDO and sunscreen delayed tumor onset by a mean of 8 and 12 weeks, respectively. The combination of FDO and sunscreen delayed tumor onset by a mean of 58 weeks. A similar more than additive level of protection was observed for skin wrinkling. CONCLUSION: Topical FDO combined with sunscreen is a potent photoprotection system against both short- and long-term UV radiation exposure.

Lysozyme binds to elastin and protects elastin from elastase-mediated degradation.

Lysozyme has been shown to be associated with damaged elastic fibers in many tissues and organs. To better characterize this interaction, binding of lysozyme to elastin was studied using solution-based binding assays. Under physiologic conditions, radio-labeled lysozyme bound specifically to elastin in a time- and concentration-dependent manner. Binding was reversible and was inhibited by unlabeled human and hen lysozyme but not by other proteins. Lysozyme had no elastolytic activity as assessed by a standard tritium-release assay, but, importantly, prevented the proteolytic degradation of elastin by human leukocyte elastase, pancreatic elastase, thermolysin, and Pseudomonas elastase. A striking feature of lysozyme's anti-elastase activity was that it did not function in the classical sense of inhibiting directly the enzymatic activity of the protease. Instead, by binding to elastin, lysozyme prevented the protease from interacting with the elastin substrate in ways that normally favor proteolysis. These results show that lysozyme binds to the elastin component of elastic fibers and that this interaction has important biological consequences for elastic fiber degradation. By preventing degradation of elastin, lysozyme can function as an important natural inhibitor that exerts a protective effect on elastic fibers at sites of tissue injury.

Effect of topically applied tocopherol on ultraviolet radiation-mediated free radical damage in skin.

Previously, we demonstrated by electron paramagnetic resonance (EPR) spectroscopy that ultraviolet radiation induces free-radical formation in Skh-1 hairless mouse skin. Because free-radical oxidative stress is thought to play a principal role in skin photoaging and cancer, oxidative stress and subsequent photodamage should be decreased by supplementation of skin with antioxidants. Using both the ascorbate free radical and an EPR spin-trapping system to detect short-lived radicals, we evaluated the effect of the topically applied antioxidants tocopherol sorbate, alpha-tocopherol, and tocopherol acetate on ultraviolet radiation-induced free-radical formation. We show that tocopherol sorbate significantly decreases the ultraviolet radiation-induced radical flux in skin. With our chronically exposed mouse model, tocopherol sorbate was also found to be significantly more protective against skin photoaging than alpha-tocopherol and tocopherol acetate. These results extend our previous observations of ultraviolet radiation-induced free-radical generation in skin and indicate the utility of tocopherol sorbate as an antioxidant in providing significant protection against ultraviolet radiation-induced oxidative damage.

Evaluation of a topical iron chelator in animals and in human beings: short-term photoprotection by 2-furildioxime.

BACKGROUND: In previous work we found that iron is a factor in skin photodamage, apparently by way of its participation in oxygen radical production. Here we report topical photoprotection with the iron chelator 2-furildioxime (FDO). OBJECTIVE: Our purpose was to determine the level of photoprotection provided by FDO in both animal and human testing. METHODS: Mice, guinea pigs, and human beings were treated topically with 5% simple vehicle solutions of FDO versus vehicle. The skin was then exposed to doses of simulated solar UV radiation greater than the minimal erythema dose. Mouse skin was harvested for analysis of ornithine decarboxylase (ODC); guinea pig skin was graded for erythema; and human skin was graded for erythema and biopsy specimens taken for analysis of ODC and for histologic evaluation. RESULTS: In animal testing with simulated solar UV radiation, topical 5% FDO provided 90% protection against induction of ODC in the hairless mouse and sun protection factor 3.5 against erythema in the guinea pig. In a double-blind, paired-comparison, vehicle-controlled clinical test, 5% FDO applied topically before a single dose of simulated solar radiation at three times minimal erythema dose prevented UV-induced erythema, sunburn cell formation, epidermal thickening, infiltration of inflammatory cells, and induction of epidermal ODC. CONCLUSION: The high level of protection provided by FDO indicates that metal chelation is a significant approach to providing photoprotection.

Chronic ultraviolet radiation-induced increase in skin iron and the photoprotective effect of topically applied iron chelators.

In the skin of albino hairless mice (Skh:HR-1) there is a basal level of non-heme iron. Chronic exposure of mice to sub-erythemal doses of ultraviolet (UV) B radiation results in an increased skin level of non-heme iron. The iron increase may be the result of a UVB radiation-induced increase in vascular permeability, which we measured in vivo with the dye marker Evans Blue. We also observed greater non-heme iron in sun-exposed vs non-exposed body sites of human skin, suggesting that similar events occur in man. Iron may have a role in skin photodamage by participating in formation of reactive oxygen species. These species have been implicated in skin photodamage. It is known that iron can contribute to oxygen radical production by acting catalytically in the formation of species such as hydroxyl radical. While the basal level of skin iron may be available for catalysis, the elevated iron content of UV-exposed skin increases the potential for iron-catalyzed radical production. Topical application of certain iron chelators to Skh albino hairless mice dramatically delayed the onset of UVB radiation-induced skin photodamage. Non-chelating analogs provided no significant protection.

Time-dependent decrease in sunscreen protection against chronic photodamage in UVB-irradiated hairless mouse skin.

To determine the time dependence of sunscreen protection against chronic photodamage in hairless mice, the time was varied (0-8 h) between topical sunscreen treatment and UVB radiation exposure. Sunscreen products with labeled sun protection factor (SPF) values of 2, 4 and 8 were evaluated; these values were verified in a guinea pig model for SPF determinations. When applied immediately prior to UVB radiation exposure, these sunscreen products were very effective in prevention of skin wrinkling and tumor formation. Onset of photodamage was delayed, the delay being greater with higher SPF values. However, the sunscreen actives were rapidly lost from the skin surface, and their protective effect diminished strikingly as the time between treatment and irradiation increased. For daily protection against chronic photodamage, this suggests a need for photoprotectants with greater substantivity to achieve a high level of protection throughout the day.

Photoprotective effect of topical anti-inflammatory agents against ultraviolet radiation-induced chronic skin damage in the hairless mouse.

Albino hairless mice (Skh:HR-1) exposed chronically to suberythemal doses of ultraviolet (UV) radiation display visible and histological alterations in the skin. One alteration is an increase in dermal cellularity, including inflammatory cells. This suggested a role for inflammation in chronic photodamage. We evaluated the photoprotective effect of topical hydrocortisone, ibuprofen, and naproxen against photodamage. All 3 agents protected against UVB radiation-induced visible wrinkling, tumor formation, and histological alterations. Hydrocortisone and naproxen were also evaluated for protection against UVA radiation-induced visible skin sagging and histological alterations. Both were very effective. These data indicate that chronic topical application of anti-inflammatory agents provides broad solar UV spectrum photoprotection.

Protective effect of topically applied conjugated hexadienes against ultraviolet radiation-induced chronic skin damage in the hairless mouse.

Albino hairless mice (SkH:HR-1) exposed chronically to suberythemal doses of ultraviolet B (UVB) radiation display visible skin wrinkling and tumors. Topical treatment of mice with solutions of conjugated dienes (2,4-hexadien-1-ol and derivatives of it) prior to each UVB radiation exposure reduces significantly the severity of these visible alterations. Chronic suberythemal doses of ultraviolet A radiation induce skin sagging, a distinctly different visible skin alteration. The severity of skin sagging is not reduced by topical application of the conjugated dienes tested here.

Photoprotective effect of superoxide-scavenging antioxidants against ultraviolet radiation-induced chronic skin damage in the hairless mouse.

Albino hairless mice (Skh:HR-1) exposed chronically to suberythemal doses of ultraviolet radiation develop visible skin changes, histological alterations, and tumors. Topical treatment of mice with solutions of superoxide-scavenging antioxidants (such as alpha-tocopherol, ascorbic acid, propyl gallate and Trolox) prior to each UVB radiation exposure reduced significantly the severity of these events. Tocopherol esters and ascorbyl palmitate were not as effective as the parent compounds in providing protection. The data suggest a role for superoxide in UVB radiation-induced skin photoaging and the protective potential of superoxide scavengers. In contrast, the severity of UVA radiation-induced mouse skin damage was not reduced by topical application of the antioxidants tested here.

Acute and chronic ultraviolet radiation induction of epidermal ornithine decarboxylase activity in hairless mice.

The effects of acute, multiple, and chronic exposure of hairless mice to ultraviolet radiation (UVR) on induction of epidermal ornithine decarboxylase (ODC) (EC 4.1.1.17) activity were investigated. Acute UVR exposure results in a biphasic time course of induction of epidermal ODC activity. Enzyme activity maxima occur at 3 and 24 h postirradiation. The biphasic time course is observed in two different strains of hairless mice (Skh:HR-1 and Jackson HRS/J) when the UVR source is either UBV fluorescent tubes or a solar simulator. The ratio of 24-h/3-h postirradiation ODC activity increases with increasing UVR dose. UVR induction of ODC activity was not significant below the mouse minimum erythemal dose (MED). The 3- and 24-h ODC activities have similar apparent Kms for ornithine (34 and 50 microM, respectively), and thermal stabilities at 52 degrees C (t1/2 = 23 and 18 min, respectively), and exhibit similar half-lives in vivo (t1/2 = 15 and 18 min, respectively). Multiple UVR exposure experiments showed 24-h ODC activity is sensitive to the preexposure history of the mouse, while 3-h ODC is not. Preexposure of hairless mice to several sub-MED levels of simulated solar radiation (SSR) specifically suppresses induction of 24-h ODC by a follow-up 2 x MED of SSR. Preexposure to a single 2 x MED of SSR specifically enhances induction of 24-h ODC induced by a second 2 x MED of SSR administered 48 h after the first. The 3-h ODC was not significantly affected by either preexposure regimen. Preexposure to a single high or low dose of UVA radiation did not affect epidermal ODC activity nor had an effect on ODC induction by UVB radiation. Several weeks of chronic exposure to UVB radiation elevated basal levels of epidermal ODC substantially (up to 350-fold). In these chronically irradiated mice, exposure to 2 x MED SSR resulted in a further 3.5-fold increase in ODC activity over the elevated basal level. These data reveal novel properties of epidermal cell expression of ODC activity in response to acute and chronic UVR insult. The results provide additional insight into the use of ODC as a marker for skin photodamage.

Chronic ultraviolet B radiation-induced biochemical changes in the skin of hairless mice.

Skh:HR-1 hairless mice were irradiated chronically with sub-erythemal doses of UVB radiation, and a number of biochemical parameters in the skin were determined after 6, 12, 18, and 24 wk of exposure. The parameters measured were water, collagen, elastin, and glycosaminoglycan content; collagenase and elastase levels; and Bz-Tyr-OEt (N-benzoyl-L-tyrosine ethyl ester) and BAPNA (alpha-N-benzoyl-DL-arginine-p-nitroanilide) hydrolyzing activities. Data for UVB radiation-exposed and chronological age-matched control mice were compared with respect to unit area and to unit mass of skin. On a unit area of skin basis, UVB radiation exposure increased the level of most parameters. The particular exceptions were collagen and collagenase which remained constant. On a mass of skin basis, though, there is an apparent decrease in collagen content because of the increase in the other skin components. This suggests that there is insufficient collagen in UVB radiation-exposed skin to support the increasing mass of the tissue.

Wavelength dependence of histological, physical, and visible changes in chronically UV-irradiated hairless mouse skin.

Albino hairless mice (Skh: HR-1) exposed chronically to sub-erythemal doses of UV radiation display physical, visible and histological alterations. Using narrow bandwidth radiation covering the UV radiation spectrum from 280-380 nm, the wavelength dependence of these alterations was determined. The wavelength dependence spectra indicate that for all but one parameter measured (skin sagging), UV-B radiation is considerably more efficient than UV-A radiation in producing changes in the skin. However, in natural sunlight there is considerably more UV-A than UV-B radiation, providing the potential for UV-A to have a larger contribution to skin damage than UV-B. This argues in favor of using broad spectrum photoprotective agents to shield the skin adequately from UV-induced aging. The spectra were also used to develop potential associations among events by determining which events occur at similar wavelengths. There seems to be a correspondence between mouse visible skin wrinking (UV-B event) and two histological events: increase in glycosaminoglycans and alteration in collagen. There was no obvious correspondence among UV-A-induced events.

Preparation and characterization of a reconstituted stratum corneum film as a model membrane for skin transport.

A zwitterionic surfactant, 6-eicosyldimethyl ammoniohexanoate (C20AH), completely disaggregates stratum corneum into individual cells. The cells can be cast into a film of reconstituted stratum corneum (RSC). Such films prepared from pig and human skin mimic intact human stratum corneum in microscopic, mechanical, and barrier evaluations. The films are useful as model membranes for skin transport experiments.

Role of protein and calcium in stratum corneum cell cohesion.

The work reported here indicates that protein and calcium have roles in stratum corneum cell cohesion. A zwitterionic surfactant, 6-octadecyldimethyl ammoniohexanoate (C18AH), was found to completely disaggregate pig and human stratum corneum into intact, individual cells. This method of disaggregation provided a tool to determine the role of tissue components in cell cohesion. The C18AH disaggregation of pig and human stratum corneum was accelerated by proteolytic enzyme and ethylenediaminetetraacetate (EDTA). The C18AH disaggregation could be blocked by pretreatment of the stratum corneum with the serine-type proteolytic enzyme inhibitor phenylmethylsulfonyl fluoride (PMSF). The blockage could be overcome by addition of proteolytic enzyme or divalent metal ion chelator. These and other data indicate the importance of protein and calcium in stratum corneum cell cohesion.