Cosmetology Are there any pharmalogical targets in cosmetology

Cosmetology Are there any « pharmalogical » targets in cosmetology for pigmentation disorders? Seminary of scientific communication 2009 -2010 Sophie Compagnon, Olivia Scaccia, Mélodie. Debacker, Catherine Lucas le 12/02/2010; Lille. 1

Outline 1. Definition 2. Hypopigmentation disorders a) b) c) d) Needs Currently solutions Possibilities in research Conclusion 3. Hyperpigmentation disorders a) b) c) d) Skin bleaching needs Currently solutions Possibilities in research Conclusion 2

Definition Cosmetics: Anysubstance or preparation intended to be placed in contact with the various external parts of the human body (epidermis, hair system, nails, lips and external genital organs) or with the teeth and the mucous membranes of the oral cavity with a view exclusively or mainly to cleaning them, perfuming them, changing their appearanceand/or correcting body odors and/or protecting them or keeping them in good condition. Medicines : Any substance or combination of substances presented as having properties for treatingor preventing disease in human beings; and any substance or combination of substances which may be used in or administered to human being either with a view to restoring, correcting or modifying physiological functions by exerting a pharmacological, immunological or metabolic action, or to making a 3 medical diagnosis.

Hypopigmentation disorders Self-tanners and esthetics solutions for vitiligo. 4

Hypopigmentation disorders 1. Needs 2. Currently solutions a) Covers up b) Self-tanners 3. Possibilities in research a) Increase precursor of melanin Induction of PAH b) Induction of tyrosinase 1. Lotus flower essential oil 2. Humanplacentalsphingolipid c) increaseeumelaninsynthesis Cysteinedeprivatio 4. Conclusion

Needs Vitiligo affects approximately 0, 5 -1% of the world population For about 50% of patients, the onset of skin disease is before the age of 20 years Most affected locations are the face, hands, and feet cause of severe psychological suffering like stress with social encounter slow self-esteem and poor body image Treatment of vitiligo is important May W. Linthorst. Homan et al, The burden of vitiligo: Patient characteristics associated with quality of life, J AM ACAD DERMATOL 2009 6

Cosmetic cover-up for vitiligo More simple possibility Hard and daily application, dificult for men Example : Avene: Couvrance® Vichy: Dermablend® 7

In association with cover-up • • Cavilon. TM 3 M No Sting Barrier Film : • siliconized material • a long-lasting and waterproof barrier • applied to cover cosmetic camouflage • quick and easy application, safe formulation Melasyn 100 : • • synthetic melanin • mimickes a natural tan soluble in water for cosmetic formulation daily application => with cover-up Reflection - Dermablend® Research possibilities for penetration in epidermis J. M. Pawelek, approaches to increasing skin melaninwith MSH analogs and syntheticsmelanins, pigment cellres 14, 2001, 155 -160. 8

Self-tanners Active ingredients = DHA = Di. Hydroxy. Acétone. Maillard reaction between DHA and AA of the stratum corneum → skin colouring Factors of variability : p. H, hydratation Permitted DHA content: 0, 5 – 10 % (usually: 3 - 5 %) 9

The Maillard reaction Bases de Schiff Réarrangements d’Amadori Aldosamines ou Cétosamines Dégradation Composés odorants (dont hétérocycles) Polymérisation Mélanoïdines 10

• doesn't cause irritation • doesn‘t cause allergy • only work son dead cells → no systemic penetration • declared « safe » by FDA and a study of « The EORTC Melanoma Cooperative group » • offensive odor • orange color • heterogeneous result → difficult to apply (thickness of the stratum corneum; scrubs, massage. . . ) • existence of an upper limit of the pigmentation • increases pigmentation of black heads • Hygroscopic → drying effect on skin • amadori products → free radicals • needs frequent applications Conclusion: a safe and effective substance, but which needs to be improved to stay longer and better color 11

DHA market First product in 1960: Man tan®

DHA market First product in 1960: Man tan® Now, a large offer of products:

Products associated with DHA : • Erythrulose. HOCH 2 -CO-CHOH-CH 2 OH • AHA R • Tyrosine � More homogeneous, natural and uniform tan 1. Erythrulose 1, 5% DHA 3, 5% 2. DHA 5% => After 14 days: more and longer lasting skin tan without undesired streaks, and 30% less dry 14 Source: Pentapharm

DHA and vitiligo • Cosmetically camouflage • Low cost • Improves quality of life in vitiligo • Minimum sides effects • No homogenous • Hard to do a perfect application => develop new formulation Example: (a) Before DHA (b) After DHA Satisfying result Not acceptable result 15 Natta Rajatanavin et al, dihydroxyacetone: a safe camoufling option in vitiligo, inter journal of derm 2008, 47, 402 -406

SWOT of cover up and self-tanners Strenghts : Weaknesses : • no side effects • daily application • performing • heterogenity of color • strong odor • comedogenic • increase UV bad effects • don’t prevent lesions extention Opportunities : Threats : • possibilities of new formula: patch • difficult to be accepted by men • wide impact of vitiligo : 0, 5 to 1% of population world wilde = 60 M potential patients. • saturated market 16

Conclusion To limit weaknesses and threats we should leverage the Natural synthesis of melanin Induce melanogenesis • wide market • reach male market • homogenous and natural color • spaced out applications • no comedogenic 17

Possibilities in cosmetic research 18

Different possibilities L-phe PAH 19

Different possibilities L-phe PAH 20

Differentpossibilities 1) Increase local concentration of Melanin precursor → Inducephenylalaninehydroxylase 2) Induction of tyrosinase → Lotus flower essential oil → Humanplacentalsphingolipid PSL 3) Expose skin to free radicals → Dangerous 4) Bring melanogenesis around to EUMELANIN synthesis → Cysteinedeprivation → MC 1 R agonists : α-MSH analog ACTH analog

1) Increase local concentration of melanin precursors Disruption of PAH mechanism in vitiligo 22

Augmentation of H 2 O 2 and ROS in vitiligo The loss of existing skin color from the epidermis suggested the involvement of reactive oxygen species. Epidermal H 2 O 2 concentrations in the 10 -3 range were demonstrated. Indeed, these patients present a decreased catalase and gluthationperoxidase level. H 2 O 2 interferes with synthesis and recycling of 6 -biopterin. 23 Karin U. Schallreuter et al. Decreasedphenylalalineuptake and turnover in patients with vitiligo. Moleculargenetics and metabolism 86 S 27, S 33, 2005

Disruptation with H 2 O 2 10 -3 M melanin K. U. Shallreuter et al, perturbed 6 -tetrahydrobiopterinrecycling via decreaseddihydropteridinereductase in vitiligo : more evidence for H 2 O 2 stress. The journal of investigativedermatology, 2004, 122, 307 -313. 24

With H 2 O 2 Dimininution of PCD decreasedrecycling of 6 BH 4 accumlation of 4αOHBH 4 formation of 7 BH 4 Favors formation of 7 BH 4 potentcompetitor of 6 BH 4 inhibition of PAH decreased epidermal enzyme activities in patients with vitiligo (n=8) compared to healthy controls (n=10) skin phototype III (Fitz- patrick classi. Wcation). K. U. Shallreuter et al, Epidermal H 2 O 2 Accumulation Alters Tetrahydrobiopterin (6 BH 4) Recycling in Vitiligo: Identification of a General Mechanism in Regulation of All 6 BH 4 -Dependent Processes? The journal of investigative dermatologt, 2001, vol 116; 167 -174

With H 2 O 2 30μM Diminution of PCD Favors formation of 7 BH 4 Diminution of DHPR decreased recycling of 6 BH 4 miss cofactor of PAH and TH The influence of H 2 O 2 on the Vmax of DHPR activity. Enzyme activities were determined in the presence of H 2 O 2. Decrease of Vmax for DHPR with H 2 O 2 concentrations of greater than 30 mm. The decrease of Vmax suggests deactivation of the enzyme. N. B. , Vmax without the addition of H 2 O 2 is 4. 76 m. U. K. U. Shallreuter et al, perturbed 6 -tetrahydrobiopterin recycling via decreased dihydropteridine reductase in vitiligo : more evidence for H 2 O 2 stress. The journal of investigative dermatology, 2004, 122, 307 -313.

With H 2 O 2 Diminution of PCD Favors formation of 7 BH 4 Diminution of DHPR Diminution of PAH activity accumulation de L-phe augmentation of 6 BH 4 synthesis by GTPCH 1/GFRP Consequently the short circuit to q-BH 2 causes the formation of H 2 O 2 from O 2 and 6 BH 4.

What can we do? Reduction of ROS : superoxide dismutase SOD + O 2 - + M(n+1)+ SOD + O 2 + Mn+ Reduction of H 2 O 2 : catalase 2 H 2 O 2 2 H 2 O + O 2 Reduces local concentration of H 2 O 2 and restores synthesis/recycling cycle of 6 BH 4 and thus restore production of tyrosine and consequently melanin too in vitiligo. H 2 O 2 passes cell membrane, maybe we can use catalase for indirect action. Reduction of extracellular H 2 O 2 concentration is going to reduce intracellular H 2 O 2 concentration. Already used in Vitise® (mediderma laboratory) with melon extract. (no study of efficiency found) Or use a pseudo catalase like PC-KUS which passes cell membrane. Already used in Vitix® (ACM laboratoire) which works in several studies 28

2) Induction of tyrosinase Lotus flower essential oil Human placental shingolipid 29

Lotus flower essential oil This oil increased melanogenesis in normal human melanocytes Stimulates melanin synthesis and tyrosinase activity in a dose dependant manner. More specifically with augmentation of expression of tyrosinase, MITF and TRP 2. Augmentation of melanin Augmentation of tyrosinase, MITF, TRP 1 and TRP 2 expression (with 10μg/ml of oil) 30

Main ingredients of lotus flower essential oil (petals, stamens) : Palmitic acid methyl ester 22, 66% Linoleic acid methyl ester 11, 16% … Effect of ingredients on tyrosinase expression Effect of PA and m. PA on tyrosinase activity, melanin content and cell viability Songhee. Jeon et al, Lotus (Nelumbonuficera) flower essential oilincreasedmelanogenesis in normal humanmelanocytes, Exp. Mol. Med vol 41, 2009, 517 -524 31

Maybe, the lotus oil can be exploited for a cosmetic product like tan agent. BUT miss information about : • side effects, • skin color after use, • frequency of applications. Informations important to know if oil is better than DHA 32

Human Placental Sphingolipid SPHINGOLIPIDES céramides, sphingomyélines, glycosphingolipides… Ex : SPHINGOMYELINE Sphingosine Phosphorylcholine AG (acide lignocérique) 33

Sphingolipid : functions RE O F BE Only: Constituent of the plasmic membrane Substrates for β-oxidation and Glycolisis NOW But also: Major biological effects in numerous biological processes. Ceramides, Sphingosine-1 -P Cellular proliferation Apoptosis/survival Cellular migration Inflammatory phenomenon Melanogenesis Martin Claire, Rôle de la signalisation Sphingosine kinase-1 / Sphingosine 1 -Phosphate dans les interactions stroma-cellules épithéliales au sein des métastases osseuses du cancer de la prostate, 2009. http: //thesesups. ups-tlse. fr/513/1/Martin_Claire. pdf 34

Human Placental Sphingolipid A sphingolipid isolated from hydroalcoholic extract of fresh term human placenta = Placental Sphingo. Lipid = PSL In vitro culture of mouse melanoma B 16 F 10 cells • Increase in melanin cellular content • Induction of tyrosinase activity • 10 μg/ml = optimum dose with 24 h of treatment as the period of stimulation. • 10 μg/ml = optimum dose with 24 h 35 of treatment as the period of stimulation.

Human Placental Sphingolipid On the one hand: PSL increases tyrosinase expression: • [tyrosinase] • [tyrosinase. ARNm] On the other hand, activity of the promotor of tyrosinase, after treatment with PSL. Conclusion : PSL up regulates tyrosinase gene expression, at transcription level, through promoter activation. Bidisha. Saha et al, Transcriptional activation of tyrosinase gene by human placental sphingolipid, DOI 10. 1007/s 10719 -006 -7931 -5 36

3) Expose skin to ROS To forget: dangerous!! Several risks: burn marks, … 37

4) Bring melanogenesis around to EUMELANIN synthesis A) MC 1 R agonists: α-MSH analogs ACTH analog Pharmacological issue 38

4) Bring melanogenesis around to EUMELANIN synthesis B) Cysteine deprivation 39

4) Bring melanogenesis around to EUMELANIN synthesis B) Cysteine deprivation - cysteine EUMELANIN Brown pigmentation + cysteine PHEOMELANIN Red-orange pigmentation 40

4) Bring melanogenesis around to EUMELANIN synthesis B) Cysteine deprivation - cysteine EUMELANIN Brown pigmentation +++ + cysteine PHEOMELANIN Red-orange pigmentation 41

Decrease cysteine and GSH cellular contents HBL = eumelanotic cells LND 1 = pheomelanotic cells Intracellular [Cys] decreased while extracellular [Cys] was voluntary decreased. Intracellular [GSH] is also sensitive to cysteine deprivation. Veronique del Marmol et al, Cysteine Deprivation promotes eumelanogenesis in Human Melanoma Cells, Society for investigative dermatology 42

Cystein deprivation promotes eumelanogenesis HBL = eumelanotic cells decrease in pheomelanin synthesis increase in eumelanin synthesis BUT LND 1 = pheomelanotic cells increase in both melanins synthesis… Conclusion: the results are those expected, but only in the cell lines which preferentially product eumelanin. However, increase in melanin (pheo+eu) synthesisis an acceptable result. 43

Problem: In each scientific article about this, experiences were conducted IN VITRO. No cysteine in the culture medium Expulsion of intracellular cysteine No cysteine available for melanogenesis Increase in eumelanin synthesis 44

Problem: In each scientific article about this, experiences were conducted IN VITRO. No cysteine in the culture medium Expulsion of intracellular cysteine No cysteine available for melanogenesis Increase in eumelanin synthesis How is it possible to decrease intracellular cysteine concentration IN VIVO? 45

How is it possible to decrease intracellular cysteine concentration IN VIVO? • Alkylating agents, to make cysteine turn to R-S. • Oxydizing agents, to make cysteine turn to S-S. Major problem : risk to affect cysteine protease… 46

How is it possible to decrease intracellular cysteine concentration IN VIVO? • Alkylating agents, to make cysteine turn to R-S. • Oxydizing agents, to make cysteine turn to S-S. Major problem : risk to affect cysteine protease… Decrease cysteine extracellular level, to make cells expulse cysteine = INDIRECT ACTION Alkylating/Oxydizing agents, but which are totally unable to enter the epidermal/dermal cells 47

Conclusion Several potential active substances Only focused on four ones In our point of view : • lotus flower essential oil, would have a better marketing impact than placental extract. • pharmacological indirect mecanism concept is very hopeful • catalase and SOD • cysteine deprivation It would deserve to be improved 48

Last solution More 50% body depigmentationvitiligo induced Total bleaching Before After 49

Hyperpigmentation disorders Improve of skin whitening agents 50

Hyperpigmentation disorders 1. Skin bleachingneeds 2. Differencesbetween. Asian, European and Africanmarket 3. Currently solution a) Hydroquinone b) Kojicacid c) Dermocorticoid 4. Possibilities in research a) b) c) d) Local change in p. H of the skin Inhibition of tyrosinase by SH function Improving the stability of kojic acid Strutural modification improved properties of Hydroquinone 5. Conclusion

Needs in whitening products Skin lightening products have a bright future a head of them. Use very wide spread in Asia, especially Japan and South Korea, and in Africa. Growing preoccupation in Europe ( age spots) 52

Differences between asian, european and african market Different issues : ancestral traditions (in Asia) + differences of skin East/West. In Asia : clear skin = sophistication, innocence, woman hood and high social level. asian skin : thick, becoming dull, yellowish, brown spots from 20 years. 53

Differences between asian, european and african market Precocious hyperpigmentation in comparison with wrinkles and loss of firmness Research of a more "transparent “ skin, with products more "Brightening" than "Whitening". The lightening products represent 35% of the total market care 54

Differences between asian, european and african market European skin : thin skin, wrinkles and sagging skin appear before the age spots main preoccupation is the fight against wrinkles, but concern about pigment spots and radiance is growing recently, increased market bleaching of 15 to 20%! 55

Differences between asian, european and african market African skin: compact, heavy, sensitive to dehydratation, large melanosomes, more dispersed, and in greater quantity, irritation -> discoloration. the reasons for use included : (a) a desire to even out the skin tone, (b) a desire to lighten the complexion, (c) a desire to improve the appearance of the skin Widespread use of lightening agents. J-J. Morand, E. Lightburne, Dermatologie des peauxgénétiquementhyperpigmentées(dites «peauxnoires» ), 98 -850 -A-10. 56

Products currently on the market • Hydroquinone : long-considered as the reference Oxidative damage of membrane lipids and proteins (including tyrosinase) + depletion of gluthation Exogenous ochronosis, post inflammatory hyperpigmentation. . . 57

Products currently on the market • Kojic Acid : present in traditional Japanese fermented food + effective for inhibiting melanin production Post inflammatory hyperpigmentation. . . 58

Products currently on the market Dermocorticoid : depigmenting power = secondary effect striae, peri-oral dermatitis, rosacea-like eruption, cutaneous infections, ophtalmic problems. . . 59

Swot of skin whitening agents Strengths : Weaknesses : - numerous targets for action - treat a large number of skin’s disorders - positive impact on quality of life - many side effects - products sometimes instable - dangerous for health - risk of systemic transfer Opportunities : Threats : - important market in development - trend effect - demand not served - emergence of new competing products 60

DP Products not irritating, not comedogenic, not allergenic, not toxic Rapidly metabolized by cytochromeno systemic transfer Long-term use and for everyone Without risk when exposed to sunlight Skin application Reasonable price 61

Possibilities in cosmetic research 62

Possibilities in research Local change in p. H of the skin Kojic acid-amino acid conjugates To promote the formation of phaeomelanin Glucoside derivative of hydroquinone : Deoxyarbutin 63

Local change in p. H of the skin Tyrosinase activity is affected by melanosomal p. H In melanosoms, p. H is modulated by protons’ pumps and Na+/H+ exchangers In black skin: Tyrosinase activity high because p. H of melanosoms is neutral whereas In white skin : p. H acid in melanosoms -> low tyrosinase activity Dustin R. Smith, Deborah T. Spaulding, The relationship between Na+/H+ exchanger expression and tyrosinase activity in human melanocytes, Experimental Cell Research 298 (2004) 521– 534 64

Local change in p. H of the skin Objective = Increase of the acidity to decrease tyrosinase activity ➡ activation of protons’ pump or NHEs inhibitors ex : NHEs inhibitors : amiloride, ethyl-isopropyl amiloride (EIPA). 65

Local change in p. H of the skin • To confirm this hypothesis : • EIPA doesn’t decrease tyrosinase abundance compared to untreated cells • Tyrosinase activity in EIPA-treated cells is markedly reduced 66

SH functions inhibit tyrosinase? 67

SH functions inhibit tyrosinase? Ideas : bring glutathione or cystein, bring molecules with SH functions, use N-acetylcystein. 68

SH functions inhibit tyrosinase? Mechanisms of action of GSH and cysteinon melanogenesis GSH Cystein 69

SH functions inhibit tyrosinase? Mechanisms of action of GSH and cystein on melanogenesis: Ø reaction of the thiol groups with dopaquinone : formation of sulfhydryl-dopa conjugates and finally sulfur-containing pigments. Ø direct interaction between the sulfhydryl compounds and the tyrosinase active site. Cystein : inhibition of the tyrosinasehydroxylase and dopaoxidase activities of melanoma tyrosinase. this inhibition becoming greater as the cystein concentration increases GSH : - <3 m. M : activation of the tyrosine hydroxylase activity. - >3 m. M : inhibition of the tyrosine hydroxylase activity. - powerful antioxidant. 70

SH functions inhibit tyrosinase? Bring molecules with SH functions : SH-containing compounds are effective inhibitors of polyphenoloxidase Ø weak inhibition of o-quinone formation Ø significantly inhibition of ROS generation Ø positively modulation of the ratio of GSH/GSSG = GSH. Ex: 1 -Propylmercaptan H 3 C - CH 2– CH 2 – SH ªPotent candidate for cosmetic application sickening odor : unusable! 71

SH functions inhibit tyrosinase? Use N-acetylcystein : • NAC is an amino acid produced by the body that stimulates production of glutathione, a powerful antioxidant • inhibitory effect on tyrosinase • ➞ bleaching effect • ex: Westerhof’s Formula: 4. 7% N-acetylcystein (NAC), 2% HQ, and 0. 1% triamcinoloneacetonide improved melasma after 4 to 8 weeks of application in a left–right, placebo-controlled clinical study of 12 female patients 72

Kojic acid Trio D Melanex trio Use of Kojic acid in cosmetics has been limited because of the skin irritation unstability during storage. 73

Derivatives of Kojic acid with improved Properties Acid-amino acid conjugates have increased stability during storage When the C-terminus was converted to the amide form stability was further improved. Jin-Mi Noh a, Seon-Yeong. Kwak a, Hyo-Suk. Seo, Kojic acid–amino acid conjugates as tyrosinase inhibitors, Bioorganic & Medicinal Chemistry Letters 19 (2009) 5586– 5589. 74

Storage stabilities of KA and KA-F-NH 2. KA-AA-NH 2 was almost stable for 3 months 75

Derivatives of Kojic acid with improved Properties tyrosinase inhibitory activity was also increased 76

Spectrum of mushroom tyrosinase and L-tyrosine reaction of L-tyrosine and L-DOPA dopachrome 77

Interactions near the tyrosinase binding site • hydrophobic interactions between the aromatic rings of KA-AA-NH 2 and the hydrophobic side chains in the tyrosinase active site. • these interactions block the accessibility of the substrate to the active site. 78

Hydroquinone molecular mechanism of cytotoxicity to melanocytes : rapid oxidation in the extracellular milieu to generate: o-quinone and ROS, Rapid oxidation by tyrosinase in the intracellular milieu to generate : an intermediate substrate to tyrosine, The substitute in the para-position of phenol to confer : high cellular permeability, high susceptibility to autooxidation. 79

Derivative synthesis of hydroquinone Deoxyarbutin = 4 -[(tetrahydro-2 H-pyran-2 -yl) oxyl] phenol • Has synthesized by removal hydroxyls from the glucose side-chain of arbutin : • Increase hydrophobic property • increase inhibition of tyrosinase activity 80

Effects of D-arb on melanogenesis of murine melanocytes Inhibition of tyrosinase is dose-dependent. D-arbutinis a potent melanogenic inhibitor like this HQ or arbutin. Zhi-Ming Hua, et al. Effects of hydroquinone and its glucoside derivatives on melanogenesis and antioxidation: Biosafety as skin whitening agents. Journalof Dermatological Science 55 (2009) 179– 184 81

Derivative synthesis of hydroquinone Deoxyarbutin = 4 -[(tetrahydro-2 H-pyran-2 -yl) oxyl] phenol • Has synthesized by removal hydroxyls from the glucose side-chain of arbutin : • increase hydrophobic property • increase inhibition of tyrosinase activity, • is safer and less cytotoxic compared with hydroquinone. 82

Effects of D-arb on the viabilities of murinemelanocytes with or without UVA irradiation D-arbutin is more photo-stable against UVA-mediated oxidative toxicity compared with arbutin. 83

Effects of D-arb on intracellular ROS level of murinemelanocytes with or without UVA irradiation D-arbutin exerts an anti-oxidative potential due to its resistance to oxidation. 84

Structural modification improved properties of Hydroquinone D-arb is a novel candidate to serve as a skin whitening ingredient: tyrosinase inhibition, effective skin penetration, less cytotoxicity antioxidation. 85

Conclusion Strong demand, several products but dangerous. Our propositions are interesting but need to be improved Nevertheless, the most probably: N-acetylcystein Product decreasing p. H Deoxyarbutin 86

Are there any « pharmalogical » targets in cosmetology for pigmentation disorders? ANY QUESTIONS ? 87 Sophie Compagnon, Olivia Scaccia, Mélodie. Debacker, Catherine Lucas le 12/02/2010, Lille.

Thank you for your attention