ABSTRACT. 1 P age LIKI VON OPPEN-BEZALEL, OLGA BEN-CHITRIT, FABIEN HAVAS, DANIT FISHBEIN IBR LTD.

Transcription

1 IBR CLEARLY BRIGHT Complex with Phyto(flu)ene the COLORLESS CAROTENOIDS, IBR-Dormin and IBR-Snowflake for better, outstanding Skin Lightening, Whitening & Evening of Skin Tone LIKI VON OPPEN-BEZALEL, OLGA BEN-CHITRIT, FABIEN HAVAS, DANIT FISHBEIN IBR LTD. ABSTRACT One of the strongest trends driving the global skin care market today is skin whitening, brightening and evening of the skin tone. This trend is dated as far back as ancient China and Japan, but even now whiter, flawless skin complexion is associated with younger perception of age, health, nobility and purity. It is commonly known among consumers and in the industry that whiter skin could hide some physically unattractive features of the face. Even and brighter skin tone is perceived as younger, more beautiful skin and overall appearance. All this time, women and men of all cultures across the globe seek effective lightening/whitening skin care products. Much effort has been invested by the cosmetic industry to make skin whitening easier and more effective. A number of actives are being used for skin whitening, some of which are extremely toxic and irritating. The need to find alternative materials that are not only safe but whose proven activity is scientifically based, is ever more challenging. This makes us proud to present IBR CLEARLY BRIGHT Complex with IBR-Phyto(flu)ene : the colorless carotenoids phytoene and phytofluene, IBR-Dormin (Narcissus Tazetta Bulb Extract) and IBR-Snowflake (Leucojum Aestivum Bulb Extract) as an advanced ingredients complex, whose recent clinical findings present it as outstanding and effective skin whitening and lightening ingredients complex. 1 P age

2 HYPER PIGMENTATION AND WAYS TO CONTROL IT Hyperpigmentation is the darkening of skin areas, caused by increased melanocyte proliferation, melanin synthesis and/or melanin deposition (Figure 1A). Although known to be associated with numerous conditions, hyperpigmentation is most closely linked to repeated irritation and inflammation of the skin. This inflammation is most commonly initiated by exposure to UV. Skin color is primarily determined by melanocytes distribution in the skin, the content level of melanin within these melanocytes and melanin transfer and distribution from the melanocytes to the keratinocytes surrounding them. Aging, acne, depilation, sun exposure, hormonal imbalance, various skin disorders form just a short list of repeated insults to the skin that result in hyperpigmentation. Such hyperpigmentation presents as darker skin tone, dark spots, freckles and uneven skin tone. The frequency of these undesired phenomena has driven science to seek for a solution. Preventive methods such as sun exposure avoidance and sunscreen use, attempt to reduce the UV contribution to the effect. Whitening or bleaching agents 2 P age

3 are implemented for the elimination of dark spots and lightening of skin tone. These agents act on the various steps involved in the pigmentation pathway (Figure 1A). 4"5.,&#6%"(!"#"$%&'( :2;*<#))* #(0&,(8%;* =5>)#$?&0)* &$'%* )*(!"#$%&'(%)* CDAE;*CDAF* +,-.//.0&,(!"#$%&')* +,-.(/)0*!"#$%&'(%)* &'$')*!"#"$%&'( 6!AE* 1&2,3( :"'.0,&#6%"( 657)8('&'%* 9)8('$%$7)%* 4"5.,&#6%"( 7'&589"'.0&,( FIGURE 1A: ILLUSTRATION OF PIGMENTATION PATHWAY 3 P age

4 IBR-Phyto(flu)ene, IBR-Dormin & IBR-Snowflake can inhibit melanin synthesis 4"5.,&#6%"(!"#"$%&'( :2;*<#))* #(0&,(8%;* =5>)#$?&0)* &$'%* )*(!"#$%&'(%)* CDAE;*CDAF* +,-.//.0&,(!"#$%&')* +,-.(/)0*!"#$%&'(%)* &'$')*!"#"$%&'( 6!AE* 1&2,3( :"'.0,&#6%"( 657)8('&'%* 9)8('$%$7)%* 4"5.,&#6%"( 7'&589"'.0&,( FIGURE 1B. IBR-PHYTO(FLU)ENE =, IBR-DORMIN = & IBR-SNOWFLAKE = CAN INHIBIT MELANIN SYNTHESIS THE ACTIVES CONCEPTS IN BRIEF IBR- PHYTO(FLU)ENE, THE COLORLESS CAROTENOIDS Phytoene and phytofluene, are the colorless carotenoids and the first carotenoid precursors in the biosynthetic pathway of other carotenoids. These molecules are devoid of color in the visible range, absorbing light in the UV range only and hence are invisible to the human eye. These colorless carotenoids are found in most carotenogenic organisms including microorganisms, algae, fungi and plants, where they act as a shield against excessive radiation and environmental stress. 4 P age

5 Interestingly, phytoene and phytofluene have been demonstrated to reach our skin, preferentially, as a result of carotenoids rich diet perhaps to protect the skin in a similar manner it does to the plants. Phytoene and phytofluene present a beneficial efficacy enhancing skin anti inflammatory capacity, protecting against UV and oxidative damage stabilizing other actives like sunscreens and boosting their effect. Recently, their observed effect on inhibition of melanin synthesis resulting in pigmentation reduction was demonstrated and proven in a clinical study as well. The effect of phytoene and phytofluene to reduce pigmentation is most probably related to the ability to inhibit melanin synthesis in melanocytes and the dual protection against UV radiation and free radicals subsequent damage and inflammation (figure 1B). IBR-Phyto(flu)ene technology comprises natural extracts from microalgae (IBR-CLC ) or tomato (IBR-TCLC ) sources for topical application and PhytoflORAL, a nutriceutical for Beauty from Within. All three platforms, IBR-CLC, IBR-TCLC and PhytoflORAL, are rich in the colorless carotenoids Phytoene and Phytofluene able to provide protection to skin. Activity of these IBR- Phyto(flu)ene members in relation to control of pigmentation has been established as (a) inhibition of melanin synthesis (figure 2, table 1), (b) absorbance of UV light both in the UVB and UVA (figure 4), (c) reduction in transmittance of the UVB and A, (d) anti inflammatory activity (figure 1B), (e) excellent anti oxidation mostly against hydroxyl radical and (figure 3, table 2) (f) prevention of damage to DNA (table 3). IBR- DORMIN NARCISSUS TAZETTA BULB EXTRACT Dormins are natural extracts from plants and plant organs in their dormant stage which are able to slow down cell proliferation to maintain younger healthier skin. IBR-Dormin (INCI: Narcissus Tazetta Bulb Extract) specifically capture and transfer flower bulbs dormancy and rejuvenation to skin. Flower bulbs go dormant through the winter like beauty sleep to allow beautiful, rejuvenated blossom in the spring. The IBR Ltd. anti-aging technology via implementation of DORMINs features slowdown of skin aging as a result of slowing down cell proliferation thereby preserving youth cell capital and improving skin appearance and maturation. IBR-Dormin ability to slowdown melanocytes proliferation was shown and both inhibition of melanin synthesis and melanocytes proliferation is affected via Endothelin 1 (ET-1) expression inhibition (figure 1B). IBR- SNOWFLAKE LEUCOJUM AESTIVUM BULB EXTRACT Natural, aqueous extract from dormant Summer Snowflake bulbs. Slowdown skin aging by reduction of cell proliferation through dormancy and increasing the natural defence against external aggression by enhancing SOD production. Clinically proven of smoothing the skin and reducing wrinkles - affecting muscle cell contraction. Contributing to brightening and whitening of the skin through dormancy of the melanocytes. As IBR-Snowflake boosts Super Oxide Dismutase (SOD) expression in the skin, it is strengthening the skin ability to defend itself from oxidative damage that in many cases, also leads to pigmentation. This along with its ability to slowdown melanocytes proliferation and 5 P age

6 putting them to sleep, helps reducing the amount of total melanin in the skin, hence resulting in lighter and more even skin tone (figure 1B). IBR CLEARLY BRIGHT COMPLEX The IBR CLEARLY BRIGHT Complex is bringing a multiple approach to lightening and evening of the skin tone addressing most aspects causing pigmentation along with multiple protection, prevention and skin beautification activities derived from this unique complex of patented active ingredients. Any agent attempting to battle pigmentation would have at least one of these activities an excellent agent would have them all. ACTIVITY ASPECTS IBR-PHYTO(FLU)ENE, SKIN LIGHTENING EFFECTS Skin color is primarily determined by the amount of the pigment melanin present in melanocytes. Melanin production in response to the stimulus of UVA light leads to the well-known tanning effect of the skin. Aging, exposure to sun, hormonal abnormalities, and various skin disorders increase the deposition of the melanin pigment in the skin, resulting in dark spots and freckles. Such dark spot are not only considered to be unattractive in many societies, they are also known to be unhealthy and, therefore, undesired. For those seeking elimination of dark spots or lightening of skin tone, whitening or bleaching compositions are useful. Typical whitening agents in cosmetic formulations are kojic acid, arbutin, licorice extract, and vitamin C. These agents act as either effective tyrosinase inhibitors or anti-oxidants, but commonly unstable, easily oxidized and degraded. Moreover, the high risk of skin irritation and subsequent inflammation, so closely associated with the use of these compounds, may result in an opposite effect than desired. IBR-Phyto(flu)ene (phytoene and phytofluene) q Inhibition of melanin synthesis, a direct activity (table 1) q Control of pigmentation by anti inflammatory activity q Reduction of melanin production by reduction of oxidative stress (figure 1, table 2) q Reduction of skin darkening due to sun exposure by UVA/B absorption (figure 2) q Pigmentation control by reduction of UVB induced inflammation by UVB absorption Their evident activity, natural source and lack of known side effects make them an ideal candidate for skin whitening agent. 6 P age

7 IBR-DORMIN SKIN LIGHTENING EFFECTS q Inhibition of melanocytes proliferation (figure 3) q Control of melanin synthesis and pigmentation by inhibition of Endothelin 1 (ET-1), whose expression in keratinocytes is induced by UV q Reduction of melanin production by reduction of oxidative stress affecting nitric oxide Their evident activity, natural source and lack of known side effects make them an ideal candidate for skin whitening agent. IBR-SNOWFLAKE SKIN LIGHTENING EFFECTS q Inhibition of melanocytes proliferation q Natural control of melanin synthesis and pigmentation - reducing oxidative damage by boosting Super Oxide Dismutase (SOD) expression leading to elimination of SO ions Their evident activity, natural source and lack of known side effects make them an ideal candidate for skin whitening agent. We are proud to propose IBR CLEARLY BRIGHT TM Control of hyper pigmentation by: Complex for skin whitening and lightening. IN VITRO AND IN VIVO: þ Inhibition of melanin synthesis directly þ Inhibition of melanocytes proliferation þ þ Anti Inflammatory - Inflammation which leads to pigmentation. Anti inflammatory activity controls pigmentation Anti Oxidant - Free radicals enhance melanin synthesis. Anti oxidants help controlling pigmentation þ Boosting the skin natural anti oxidant system by expression of Super Oxide Dismutase (SOD) increased protection against oxidative damage þ Protection Against UVA/B - UVA exposure leads to tanning, UVB increase inflammation protection against UVB and UVA prevents darkening of skin à IBR CLEARLY BRIGHT TM Complex able to reduce multiple causes of pigmentation 7 P age

8 IBR CLEARLY BRIGHT Complex - A multi functional complex of actives (IBR-Dormin all natural 0305, IBR-Snowflake all natural 1003, IBR-TCLC in jojoba oil 0705) effectively and significantly lightens the skin tone protects the skin and keeps skin younger and younger looking These suggestions were substantiated both in vitro and in vivo proving to lighten the skin color after continuous use. IN VITRO IBR-PHYTO(FLU)ENE SKIN LIGHTENING RELATED ACTIVITIES EFFECT ON MELANIN SYNTHESIS IN CULTURES OF NORMAL HUMAN AND MURINE B16 MELANOCYTES The effect of IBR-TCLC (tomato phytoene & phytofluene) on Melanin Synthesis in Cultures of Normal human and Murine B16 Melanocyte could be seen in figure 2. In one study, the compound IBR-TCLC tested at 0.5% decreased to 71% the melanin content of normal human Melanocytes compared to control (International Patent Application/Publication No. WO2008/004206) In an additional study the whitening effect of IBR-TCLC in Squalane on B16 mouse melanoma cells was measured resulting in a dose dependent response for increased inhibition of melanin synthesis with increased COLORLESS CAROTENOIDS concentration described below (table 1). Moreover, for evaluation of IBR-TCLC in Squalane whitening effect of on melanoma cells, cells were treated with IBR-TCLC in Squalane (0.25, 0.50, 1.25, 3.75, 5.00%) for 96h and cells were harvested. Harvested cells were centrifuged and evaluated the color of the cell pellets. The cells treated with more than 0.5% TCLC in Squalane showed whitening effect (International Patent Application/Publication No. WO2008/004206). WHITENING EFFECT OF TOMATO PHYTOENE AND PHYTOFLUENE ON MELANOMA CELLS Concentration Whitening Effect Cell Viability TABLE 1: WHITENING EFFECT OF TOMATO PHYTOENE AND PHYTOFLUENE ON MELANOMA CELLS - No effect + Obviously white compared to the non treated + Extremely white compared to the non treated 8 P age

9 ANTI INFLAMMATORY ACTIVITY Reduction of inflammation in an area susceptible to constant environmental stress from repeated irritation of skin from shaving, friction or other sources is suggested to reduce long time damage effect such as darkening. Anti inflammatory activity of the IBR-CLC (Colorless Carotenoids from Alga Dunaliella sp.) - Significant reductions in expression of IL-6 and IL-12 in activated lymphocytes by the IBR-CLC were demonstrated. No LDH leakage was seen with IBR-CLC indicating no loss of viability at the doses tested (Oppen-Bezalel L. et al., Fragrance Journal, 34, part 3, pp (2006)) A significant reduction in PGE-2 expression in IL-1 induced normal human fibroblasts of 46 percent by IBR- TCLC was observed and published (Fuller B. B., et al J. Cos. Derm., 5(1), pp (2006)). ANTIOXIDANT ACTIVITY Colorless carotenoids quench hydroxyl radicals - of the most destructive free radicals in nature. We propose use of abovenebtioned extracts for reduction of oxidative stress, linked to inflammatory conditions, damage to DNA that induces melanin syntheis leading to hyperpigmentation and skin damage. FIGURE 1. HYDROXYL RADICALS TRAPPING BY IBR-CLC PROTECT DNA FROM FREE RADICALS DAMAGE Compo und Phytoe ne and Phytofl uene Positive Control Conc. mcg/ml % of inhibition hydroxyl radicals % of non specific inhibition % of specific inhibition Conc. giving 50% of activity 11.2 mcg/ml 80 mcg/ml TABLE: 2 COLORLESS CAROTENOIDS PROTECT DNA FROM FREE RADICALS DAMAGE 9 P age

10 In the study results (table 2) above Phytoene and Phytofluene provide an effective DNA protection activity against free radical damage. UV LIGHT ABSORBTION FIGURE 2: PHYTOENE AND PHYTOFLUENE ABSORPTION SPECTRA IBR-DORMIN SKIN LIGHTENING RELATED ACTIVITIES FIGURE 3: IBR-DORMIN REDUCES PROLIFERATION OF MELANOMA CELL IN CULTURE 10 P age

11 Narcissus Tazetta bulb extract (IBR-Dormin ) potently inhibits Endothelin-1 production by normal human keratinocytes in vitro. C. Lasserre, M. Mildner, C. Wallaert, E. Tschachler Skin Research and Technology 2004; 10: Abstracts, 2004 US Symposium of the International Society for Bioengineering and the Skin Among several samples tested Narcissus Tazetta bulb extract was found to significantly inhibit ET-1 production/release in a dose dependent manner. In addition this extract also completely inhibited the IL1ainduced up-regulation of ET1 production by normal human keratinocytes in culture. Therefore Narcissus Tazetta bulb extract represents an interesting active ingredient for products used to attenuate and prevent pigment spots of the skin. IBR-Dormin inhibition of Nitric Oxide Sample IBR-Dormin Quercetin Control IC50 (µg/ml) 4.41µg/ml (0.05%) & 6.21µg/ml (0.07%) <10 mg/ml IBR-SNOWFLAKE SKIN LIGHTENING RELATED ACTIVITIES In nature, skin eliminates oxidative stress through enzymes like SOD; IBR Snowflake significantly increases MnSOD in keratinocytes (*20% increase at 0.15% IBR-Snowflake ) improving the natural skin protection and reducing oxidative damages % 0,15% Mn SOD FIGURE 4: IBR-SNOWFLAKE INDUCE SUPER OXIDE DISMUTASE (SOD) EXPRESSION IN KERATINOCYTES 11 P age

12 UV LIGHT ABSORBTION CLINICAL EVALUATION OF THE LIGHTENING EFFECT OF A COSMETIC PRODUCT CONTAINING CLEARLY BRIGHT TM COMPLEX A randomized, double blinded, placebo-controlled study evaluating the lightening effect of a cream containing IBR-TCLC 5% and cream containing IBR-TCLC NEW! CLINICAL 3%, EVALUATION IBR-Dormin OF THE LIGHTENING 2% and EFFECT IBR-Snowflake OF A COSMETIC 2% PRODUCT (CLEARLY CONTAINING BRIGHT TM PHYTOENE AND PHYTOFLUENE Complex) was performed. A twice a day application during a 3 month course of treatment resulted in a significant skin whitening effect in both DAY-42 and DAY-84 (n=21). * Full study report is available upon request. RESULTS FIGURE 4: PHYTOENE AND PHYTOFLUENE ABSORPTION SPECTRA A randomized, double blinded, placebo-controlled study evaluating the lightening effect of a cream containi IBR-TCLC 5% was performed. A twice a day application during a 3 month course of treatment resulted in significant skin whitening effect in both DAY-42 and DAY-84 (n=21). * Full study report is available upon request. RESULTS after 42 and 84 days of twice daily use Effect of CLEARLY BRIGHT Complex on lightening the skin tone in vivo after 42 and 84 days of twice daily use SIGNIFICANT LIGHTENING EFFECT: ITA parameter variations +14% +13% Variations in % +12% +10% +8% +6% +4% +7% +3% +10% +2% +0% D42 D (active product) (placebo) IBR-Dormin all natural 0305 / IBR-Snowflake all natural 1003 / IBR-TCLC in jojoba oil 0705 (IBR-PHYTO(FLU)ENE ) CLEARLY BRIGHT Complex has SIGNIFICANT LIGHTENING EFFECT, higher than IBR-Phyto(flu)ene alone. 12 P age

13 VISUAL IMPROVEMENT IN THE CUTANEOUS STATE: SATISFIED THE MAJORITY OF THE SUBJECTS: 13 P age

14 14 P age

15 PHOTOGRAPHY Illustrations of a Subject #3, who presented the best visual results with active product "#367960" are presented below Illustrations of Subjects #24, who presented the best visual results with placebo "#514345" are presented below SUBJECT #24 ON D0 15 P a g e SUBJECT #24 ON D84

16 CLINICAL STUDY CONCLUSIONS The active product "#367960" (CLEARLY BRIGHT Complex) induced a significant lightening effect associated to a better improvement in the visual cutaneous state than placebo "#514345" OVERALL CONCLUSIONS The demonstrated report and studies substantiate CLEARLY BRIGHT Complex, a complex comprising of: IBR-DORMIN all natural 0305, IBR-SNOWFLAKE all natural 1003 & IBR-TCLC in jojoba oil 0705 (IBR- PHYTO(FLU)ENE ) as most effective and significantly able to lighten and whiten the skin tone. The complex acts on whitening and lightening the skin tone through multiple mechanisms and pathways, making it an ideal active complex addressing whitening in a complete aproach that is safe and natural. SAFETY ASPECTS Irritation Potential Non Irritating (Neutral Red Release method) Skin sensitization Cutaneous tolerance Does not induce sensitization (HRIPT, Marzulli and Maibach method) Very well tolerated (Patch test) Very well tolerated (HRIPT Marzulli and Maibach method) Skin Compatibility Photo-irritation Potential Phototoxic Potential Allergens Good skin compatibility, Patch Test Non Photo-irritating, Clinical evaluation Non Phototoxic, Clinical evaluation 26 allergens described in Directive 2003/15/CE - None detected REGULATORY ASPECTS The IBR COLORLESS CAROTENOIDS products, including the IBR-TCLC (Colorless Carotenoids from Tomato) and IBR-CLC (colorless carotenoids from algae) are listed in PCPC (formerly CTFA) International Cosmetic Ingredient Dictionary and Handbook. Both Ingredients are labeled by the JCIA. The products are natural, GMO and BSE free. No harmful solvents are used for production of these ingredients. IBR substances are preregistered under REACH and so are REACh compliant until P age

17 CONTACT Business Development and Marketing: Liki von Oppen-Bezalel, Ph.D. Tel: Fax: Office: 4 Pekeris street Rehovot 76702, Israel Tel: Fax: ibroffice@ibrweb.com 17 P age