Ingredients Technical File GREYVERSE - Redenhair

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ActiveingredientsTechnical FileGREYVERSE Zero shades of greyis also sexy!Patented a-MSH biomimeticpeptideStimulates melanogenesis andreduces oxidative stress in hairbulbReverses hair greying processto progressively recover naturalhair color for a younger andmore confident look!

Technical fileGreyverse SUMMARYINCI NAMEWater (1) (and) Glycerin (2) (and) Palmitoyl Tetrapeptide-20 (3)CAS(1) 56-81-5 (2) 7732-18-5 (3) -EINECS(1) 200-289-5 (2) 231-791-2 (3) -ORIGINBiomimetic peptide of the α-Melanocyte Stimulating Hormone(α-MSH)COSMETIC PROPERTIES Binds and stimulates the melanocortin 1 receptor (MC1-R) Stimulates the melanogenesis process Stimulates melanin synthesis ( MITF, TRP-1) Improves melanosome maturation ( Melan-A) and their transferto keratinocytes Decreases oxidative stress ( catalase, TRP-2) Decreases intracellular quantity of H2O2HAIR BENEFITS/POTENTIAL CLAIMS Prevents, delays or reverses the hair greying process Decreases grey hair density Provides a younger look Improves self-confidenceAPPLICATIONS Anti-aging hair care Premature grey hair coverage Post-coloration treatment Natural color fortifier Scalp-friendly alternative to chemical dye Beard & mustache careRECOMMENDEDDOSAGE0.5-1%: preventive hair care1-2%: intensive hair careUSAGE PH RANGE4.0 - 8.0INCORPORATIONAt the end of the formulation ( 40 C)Can be heated if needed (tested at 90 C for 2 hours)INCOMPATIBILITIESUnknown26.02.2018P a g e 1 48

Technical fileGreyverse INTRODUCTIONHair color contributes significantly to human social communications as it provides much informationregarding an individual’s race, ethnicity, age and health status, as well as physical and sexual attractiveness.It is not surprising, then, that many people desire full, shiny, lustrous hair that is still their natural color!The greying of hair is a natural biological process associated with aging. Men and women of every ethnicityare affected sooner or later. Recent studies show that up to a quarter of people worldwide have more than50% grey hair by the time they reach 50 years of age.While elderly people generally accept this new hair color, the same cannot be said for younger people,particularly those with dark hair who compose over 90% of the world’s population. Most people want toavoid, delay or hide this inevitable sign of aging as it makes them look older and affects their self-confidenceand self-esteem, and this is especially true in the case of young people suffering from premature grey hair.The anti-grey hair market is thus very promising. Most people who want to cover their grey hair choose tocolor it artificially, and there are several solutions available on the market:-Hair dye (chemical or vegetable), used by most people as an immediate 100% covering solution (63% of 45year-old women have dyed their hair within the last 6 months).-Compositions of reactive substances that reveal their color as a result of an oxidoreduction reaction oncethey are set on hair, instant root concealers to use in between two shampoos, and coloring vegetable extractsare some of the other types of products proposed.However, all these products color only the visible part of the hair shaft and the root inevitably grows grey orwhite, leading to an unsightly color contrast and obliging people to constantly renew the covering action.Until now, there has been no solution targeting the biological causes of the hair greying process in order tostop it or, even better, reverse it by natural repigmentation of the hair shaft.A breakthrough innovation from Lucas Meyer Cosmetics, Greyverse is the first anti-grey hair biomimeticpeptide. Clinically effective, it helps men and women suffering from greying hair look naturally younger byreversing the hair aging process and increase their self-esteem.26.02.2018P a g e 2 48

Technical fileGreyverse HAIR GREYING PROCESSRole of melaninHair color varies according to each individual’s genetic heritage. The wide range ofnatural hair colors (different shades of blond, red, brown or black) is the result of thevariable amounts and distribution of two types of melanin in the cortex, or middle layerof the hair shaft: eumelanin (black to brown pigments) and pheomelanin (red to yellowpigments). The composition of this blend is genetically determined based on the needfor hair protection one’s ancestors had as a result of their geographic location.Like in the skin, the role of melanin in hair is mainly to protect it from sun damage - e.g., dry and brittlestrands, broken or split ends, thinning and frizz - as the pigment is able to absorb UV rays.But melanin is also able to neutralize toxins, heavy metals, and other potentially toxic substances that canbecome highly accumulated in the protein structure of hair. Indeed, the selective and avid binding of thesecompounds to the melanin pigment limits their access (and thus potential damage) to the living tissue of thehighly vascularized scalp.Synthesis of melaninMelanin is only produced by melanocytes during the anagen ( growth phase) of the hair cycle. Melanocytesare located in the bulb of each hair follicle on the basement membrane surrounding the dermal papilla.Pigments are transferred through melanocyte dendrites into the surrounding keratinocytes in a continuousflow while they proliferate, building the new part of a pigmented hair shaft.Genetically programmed, the hair greying process, also called canities, usually starts in the 30s forCaucasians, later for Asians (late 30s) and even later for Africans (mid 40s). This unwanted event can alsoaffect some younger people suffering from premature grey hair, sometimes starting before they enter their20s. Greying usually first appears at the temples, then spreads to the vertex before spreading to theremainder of the scalp. Beard and body hair are usually affected later.Whatever the case, the appearance of grey color in a hair shaft is due to a gradual loss of melanin over timecaused by several factors. As the pigment level gets lower, the color of the hair becomes increasingly lightand the hair shaft appears grey. When the pigment level is even lower, it becomes undetectable to the eye(due to changes in light reflection caused by the low pigment level) and the transparent hair shaft appearswhite. This condition can be reversed as long as some melanocytes are still present in the bulb. Theirreversible final step of the hair greying process (a totally white hair shaft) occurs when hair contains nomore pigment due to a complete loss of all melanocytes.26.02.2018P a g e 3 48

Technical fileGreyverse Two factors have been identified as the main causes of the hair greying process: the decrease inmelanogenesis and the increase in oxidative stress in the bulb. Over time, the number of melanocytes in the hair bulb and their activity decrease, leading to a decline inthe melanogenesis process, which proceeds according to the “melanogenetic clock”.The synthesis of melanin in melanocytes is decreased due a lower stimulation of the process and a loweractivity of the enzymes involved (tyrosinase and TRP-1). Also, the transfer of melanosome (melanincontaining vesicle) into the surrounding keratinocytes is reduced due to some defect inmelanocyte/keratinocyte interaction. Therefore, the final quantity of pigments in hair shafts dims until hairappears grey, then white. Oxidative stress is increased by the decrease of two major enzyme activities:1) Catalase. Hair follicle cells produce small amounts of hydrogen peroxide (H202) as part of theoxygen cycle. This oxidative substance is naturally degraded by catalase into its harmless elementsof water and oxygen.catalaseH2022H20 02With aging, catalase enzyme activity decreases, leading to anaccumulation of H202 that bleaches the melanin pigments and thehair from the inside out, progressively leading to grey hair color.The buildup of H202 also inhibits the activity of tyrosinase, the enzyme involved in the synthesis of melanin,thus reducing the capacity of melanocytes to produce pigments.26.02.2018P a g e 4 48

Technical fileGreyverse 2) TRP-2 (Tyrosine Related Protein-2) initially known as an enzyme involved in the synthesis melanin, is alsoan antioxidant enzyme that protects and defends melanocytes against reactive oxygen species generation.The decrease in TRP-2 intensifies the loss of melanocytes as they become more sensitive to aggressions fromfree radicals.PATENTED PEPTIDE FOR RECOVERING NATURAL HAIR COLORGreyverse is the result of a wide screening of several peptides tested on different biological targets involvedin the hair greying process.Greyverse is a patented Palmitoyl Tetrapeptide (4 amino acids grafted on palmitic acid). The activesequence is derived from the α-MSH structure, known to be the main molecule able to stimulatemelanogenesis.As an analogue of α-MSH, Greyverse is able to bind to MC1-R (Melanocortin-1 Receptor), a transmembranereceptor located at the surface of melanocytes. The activation of MC1-R stimulates the melanogenesisprocess, as well as the catalase expression and activity.Due to its complete mechanism of action, Greyverse is able to act simultaneously and efficiently on the twomain factors causing hair greying process:- It stimulates melanogenesis to increase melanin production in the bulb and favor a better transfer leadingto hair repigmentation.-It lowers oxidative stress by increasing the catalase expression and activity, thus reducing the accumulationof hydrogen peroxide to maintain the full pigment color.Contrary to skin melanocytes, which are able to produce a higher quantity of melanin than the constitutiveskin color if they are stimulated (facultative pigmentation), hair melanocytes cannot produce more than theyare genetically programmed (constitutive pigmentation only). Thus, Greyverse can only help hair recoverits natural color, it cannot darken it.26.02.2018P a g e 5 48

Technical fileGreyverse CLINICALLY PROVEN HAIR REPIGMENTATIONClinical evidence of the active ingredient’s effect is the most important argument, especially forapplications such as reversing grey hair.Tested on selected volunteers with early canities (35 years old, 20% grey hair), Greyverse has been provento decrease the overall whiteness of hair and to darken grey hair after 3 months of treatment, withoutstanding visible results: a 30% reduction in grey hair density.As a biological process, the hair greying process involves several genes. Evaluating the gene expression andsynthesis of related proteins is an innovative way to demonstrate product efficacy.Based on an analysis of hairs plucked from volunteers, Greyverse has demonstrated its ability to modulatesome of the main genes involved in pigmentation:- the increase in MC1-R gene expression increases the number of receptors responsible for inducingmelanogenesis- the decrease in ASIP gene expression (a competitor of α-MSH for the binding to MC1-R) favors the activationof MC1-R by α-MSH to better induce melanogenesis- the increase in MITF gene expression stimulates melanin synthesis26.02.2018P a g e 6 48

Technical fileGreyverse 26.02.2018P a g e 7 48

Technical fileGreyverse EFFICACY STUDIES26.02.2018P a g e 8 48

Technical fileGreyverse IN VITRO EFFECT OF GREYVERSE ON MELANIN SYNTHESIS INHUMAN MELANOCYTESBACKGROUNDHair pigmentation is due to the presence of a pigment, melanin, secreted by the melanocytes located in thehair bulb and then transferred into the surrounding keratinocytes [1]. The appearance of greying hair is aphenomenon directly linked to a lack of melanin in the hair bulb.OBJECTIVEThis study investigated Greyverse activity by measuring its effects on the production of melanin in a humanmelanocyte culture.PROTOCOLBiological materialsThe melanocytes used were a cell line of primary normal human epidermal melanocytes from adult tissue(HEMas). HEMas cells were grown in an adherent cell culture realized with CnT-40 CellnTec medium at 37 C,5% CO2 and 95% humidity.Tested products The Palmitoyl Tetrapeptide-20 (pure peptide contained in Greyverse ) was tested at the followingconcentration: 10-7 M (equivalent to 0.5% Greyverse ). The positive control, α-MSH, was tested at a concentration of 10-6 M.Evaluation of the activityHEMas cells were seeded in 6-well plates at the concentration of 3.105 cells/mL in a CnT-40 culture mediumand incubated at 37 C, 5% CO2. After 24 hours, the medium was removed and the cells were treated withPalmitoyl Tetrapeptide-20 (10-7 M) or α-MSH (10-6 M) for 72 hours.After 72 hours of treatment, cells were detached for counting, then incubated at 100 C for 10 minutes withNaOH at 1M to extract melanin.D0 - 0hCells seedingD4 - 72h of treatmentMelanin extraction and dosageD1 - 24hTreatment with Palmitoyl Tetrapeptide-20or α-MSHThen, melanin was detected by absorbance at 405 nm. Melanin content was expressed as the percentage ofmelanin production, compared to cell numbers.26.02.2018P a g e 9 48

Technical fileGreyverse RESULTSAfter 72h of treatment, Greyverse significantly increased the melanin synthesis by 19% at the concentrationof 10-7 M.The results indicate that Greyverse has an activity stronger than the positive control α-MSH .CONCLUSIONA biomimetic peptide of α-MSH, Greyverse stimulates the melanin synthesisto improve grey hair repigmentation.26.02.2018P a g e 10 48

Technical fileGreyverse EX VIVO EFFECT OF GREYVERSE ON PIGMENTATIONIN HUMAN HAIR FOLLICLESBACKGROUNDMelanosomes are typically divided into four maturation stages (I–IV) determined by their structure and thequantity, quality, and arrangement of the melanin produced.- Stage I melanosomes have an early matrix organization, are spherical, do not contain tyrosinaseactivity, and are localized at the periphery of the nucleus.- Stage II melanosomes are tyrosinase-containing elongated organelles with an organized filamentousmatrix. In addition, Mart-1 protein, also known as Melan-A, localized in stage I and/or II, contributesto the melanosome formation.- Stage III melanosomes, where melanin is uniformly deposited.- Stage IV melanosomes are melanosomes that are electron-opaque, fully melanized and have a lowtyrosinase activity; they are the melanosomes supplied from the dendrites to the neighbouringkeratinocytes.Within melanosomes, at least three key enzymes, tyrosinase, Tyrosinase-related protein 1 (TRP-1), andTyrosinase-related protein 2 (TRP-2), are absolutely required for the synthesis of different types of melanin.Moreover, the quality of the pigment depends of the balance of the Agouti signaling protein (ASIP, alsoknown as PARD3) and alpha-MSH expression. The binding of ASIP to MC1R precludes alpha-MSH initiatedsignaling and thus blocks production of cAMP, leading to a down regulation of eumelanogenesis(brown/black pigment) and increasing synthesis of pheomelanin (yellow/red pigment).OBJECTIVEThe aim of this study was to determine the effects of Greyverse on the pigmentation of isolated humangrey hair follicles.To this end, we checked the hair melanosome biogenesis by melanosomal protein immunohistochemistry(Melan-A and TRP-1) and by the nature of the pigment expressed by the immunostaining of ASIP/PARD3.PROTOCOLTested productsPalmitoyl Tetrapeptide-20 (pure peptide containing in Greyverse ) was tested at the concentrations of10-9 M and 10-7 M, equivalent to 0.005% and 0.5% Greyverse , respectively.26.02.2018P a g e 11 48

Technical fileGreyverse Biological materialsOn a scalp plasty coming from a 71-year-old Caucasian woman who originally had dark hair,grey hair follicles (from the infundibulum to the bulb) were isolated by micro dissection onday 0 (D0). These hair follicles were placed separately in a 48-well plate and kept in survivalfor seven days in William’s medium (Sigma-Aldrich, W1878) supplemented with Lglutamine, insulin, hydrocortisone, serum and antibiotics, at 37 C in a 5% CO2 atmosphere.Eleven hair follicles were used per condition (untreated or treated). The PalmitoylTetrapeptide-20 was diluted in the culture medium on day 0, day 1, day 4 and day 7whereas the untreated medium was renewed.MethodAt the end of survival (day 7), the hair shafts were fixed in buffered formalin solution, dehydrated, andparaffin impregnated. 7-µm-thick paraffin sections were mounted on glass slides for immunostainings andstainings. For fluorescent immunostainings, the primary antibodies used were anti-PARD3/ASIP (Abcam, ref.ab64646). The sections were then incubated for 30 minutes at room temperature with the Alexa Fluor488-labeled antibody (Lifetechnologies, ref. A11078). The nuclei were counter-stained using propidiumiodide at 0.2µg/mL. The slides were finally mounted using Vectashield mounting medium (Vector, ref.H-1400). For Immunohistochemistry, the hair sections were pre-incubated with hydrogen peroxide (VWR, ref.23619.264) to inactivate endogenous peroxidase activity. The primary antibodies used were anti-TRP1(Eurogentec, ref. SIG-38150-1000) and anti-Melan-A (Santa Cruz biotechnologies, ref. sc-20032). Then,the sections were pre-incubated with a biotinylated secondary antibody and incubated with astreptavidin-labeled peroxidase (Vector, Vectastain Universal ABC kit, ref. PK7200). The staining wasrevealed by a substrate of peroxidase, VIP (Vector, ref. SK-4600). The nuclei were counter-stained usingMayer hemalun (RAL diagnostics, ref. 320550). The slides were finally mounted using Eukitt mountingmedium (VWR, ref. KIND01250).The microscopic observations were performed using a Leica DMLB or Olympus BX43 microscope. Pictureswere digitized with a numeric DP72 Olympus camera with CellD storing software. Different hair parts wereanalyzed: the infundibulum, upper shaft, bulge, lower shaft and bulb.Hair follicle areas26.02.2018P a g e 12 48

Technical fileGreyverse RESULTSAfter 7 days of treatments with 10-9 M and 10-7 M Palmitoyl Tetrapeptide-20, the expression of themelanosome protein, Melan-A, was increased especially in the upper sheath and in the bulb area, a site ofmelanocyte stem cells, attesting an improvement in the melanosome maturation, necessary for an optimaltransfer.Moreover, the expression of the Agouti signaling protein, ASIP/PARD3, was decreased in the lower part ofthe hair follicle (lower root sheath and bulb). This result indicates that Greyverse directs the nature ofpigment synthesis: it preferentially increases eumelanin synthesis over pheomelanin synthesis, allowing thenatural dark hair color of the donor to be recovered.26.02.2018P a g e 13 48

Technical fileGreyverse The key enzyme of melanin synthesis, tyrosinase-related proteins 1 (TRP1), was induced in the upper rootsheaths, attesting the greater capacity to produce pigments.ConclusionGreyverse promotes hair pigmentation by increasing melanosome transfer andmelanin synthesis, preferably eumelanin.26.02.2018P a g e 14 48

Technical fileGreyverse IN VITRO EFFECT OF GREYVERSE ON MELANOSOME TRANSFERIN A CO-CULTURE OF KERATINOCYTES AND MELANOCYTESBACKGROUNDHair pigmentation can be influenced by the concentration of melanin as well as by the number, size anddistribution of melanosomes around the nucleus of keratinocytes. Melanin is synthesized, stored andtransported within a specialized organelle called a melanosome in melanocyte. The transfer of melanin frommelanocytes to keratinocytes (melanosome transfer phagocytosis) is a critical step in skin pigmentation,one that depends on various parameters such as melanocyte dendricity.OBJECTIVEIn this study, the potential pro-pigmenting effect of Greyverse was evaluated in a co-culture of normalhuman epidermal keratinocytes (NHEK) and normal epidermal melanocytes (NHEM).In brief, the effect of Greyverse on melanosome transfer was evaluated using flow cytometry to quantify amelanosome specific marker (Pmel17) inside keratinocytes identified by the expression of the cell surfacemarker CD49f.PROTOCOLTested products Palmitoyl tetrapeptide-20 (pure peptide contained in

Technical file Greyverse 2 6 . 0 2 . 2 018 P a g e 4 48 Two factors have been identified as the main causes of the hair greying process: the decrease in melanogenesis and the increase in oxidative stress in the bulb. Over time, the number of melanocytes in the hair

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