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Faculty of Pharmacy, Lille II Pauline Fontaine Friday 12th, February 2010 Clotilde Bourdon Antoine Henninot The skin: an heterogeneous system 5 main functions: •Barrier : Langerhans cells + Sebaceous glands + Melanocytes + Hair follicles •Sens of touch, sensation : Nerve endings Merkel cells •Regulates body temperature •Excretes waste product and excess salt •Syntheses vit D Skin appendages: hair, nails, sweat glands, sebaceous glands CAUTION WITH THE TARGETS TOUGH TO PENETRATE Epidermis Skin's outer structure Stratified Stratum corneum = outermost layer, dead cells: not permeable Keratinocytes (++), melanocytes, Langerhans cells, Merkel cells Melanocytes (5% of the cells) Avascular Dermis Melanocytes underneath the basal layer i.e. within the dermis Contains: - blood vessels - lymph vessels - hair follicles - glands (produce sweat and sebum) => Rich capillary bed for systemic drug absorption 3 major routes to entry into the skin Sebum and surface material (1) Skin appendages (3) Epidermal route (2) -> Inter- or trans-cellular route Lipophilic substances Hydrophilic + Lipophilic substances Figure 1. Simplified representation of skin showing routes of penetration: (1) through the sweat ducts; (2) directly across the stratum corneum; (3) via the hair follicles.Reproduced with permission from Ref 10. Origin of cutaneous pigmentation Skin color determined by its content in pigments Melanins +++++, Oxygenated and reduced hemoglobin and Carotene Other factors epidermal thickness vascular supply Synthesis of Melanins In the melanocytes, within melanosomes Through fixation of α-MSH on MC1R with UVB rays Melanins absorb UV rays => protection α-Melanocyte-Stimulating Hormone Hypophysis Synthesis/Secretion POMC Hydrolysis ACTH α-MSH Peptide endogenous agonist for all Melanocortin Receptors regulator of skin and hair pigmentation Short t1/2 in circulation POMC ACTH Melanocyte ↗AMPc α-MSH Eumelanin + + - MC1R ASIP ↘AMPc Phemelanin Tyrosinase Tyrosine Dopaquinone DOPA Glutathion ou Cystein Leucodopaquinone TRP1 Cysteinyl dopa dopachrome DHICA DHI Indole-5,6quinone DHICA-melanin Eumelanin TRP2 1,4-benzothiazinyl Alanine Phaeomelanin Melanosome migration within melanocytes => Maturation (polymerisation) Migration of the melanosomes to the keratinocytes Melanins matured: Melanosomes -> keratinocytes Melanosomes accumulate above nucleus of keratinocytes Keratinocytes -> superficial layers of the epidermis Membrane of the melanosomes digested by enzymes Melanocytes Black skin White skin Similar number of melanocytes BUT melanin synthesis and melanocytes’ migration to the keratinocytes more significant for black skin Faculty of Pharmacy, Lille II Pauline Fontaine Friday 12th, February 2010 Clotilde Bourdon Antoine Henninot Overview of the diseases Melasma Lentigo Hyperpigmentation Iatrogenic hyperpigmentation Pregnancy mask Post-acneic or cicatricial hyperpigmentation Hypopigmentation Vitiligo Vitiligo Hyperpigmentation disorders Unknown etiologic origin High prevalence Lentigo: 90% of the population over 70 Melasma (chloasma): 8.8% of the latino american women Alter people’s quality of life hyperactivation of the melanocytes melanin Melasma: reemergence of the lesions as soon as the first exposition to the sunlight Melasma Lentigo Current treatments Old bleaching agents Hydroquinone (tyrosinase inhibitor) Azelaic acid Corticoïd Associated with « peeling » / dermabrasion products Vitamin A Glycolic acid Best treatment: Kligman trio Corticoïd (dexamethasone) Hydroquinone Retinoic acid Current treatments (continued) Satisfying BUT: - unconstant treatment - local treatment very long - numerous Adverse Events : irritation (hydroquinone (5%), retinoic acid) • Depigmenting duo: hydroquinone corticoid (hydrocortisone) less irritant BUT less effective • => B/R ratio NOT acceptable for skin disorders • 1st TREATMENT= SOLAR PROTECTION!!! Meeting with Dr.Riboulet, Dermatologist Main ideas : Keratolytic peeling : caution with the concentration, otherwise -> inflammation, post –inflammatory hyperpigmentation Total depigmentation with MBEH (Mono Benzyl Ether Hydroquinone) prohibited in France to treat Melasma -> too hazardous Whitening agent : undesired hypopigmentation risk Hypopigmentation disorders Vitiligo High prevalence: 0.5 - 2% of the population Unknown etiology 3 major hypotheses, not exclusive of each other: immunologic factors oxidative stress sympathetic neurogenic disturbance VITILIGO Current treatments Puvatherapy (psoralen + UVA) no longer used (known carcinogenic risk) UVB narrow band For 10 years: unknown risk cancer Topical treatments Tacrolimus (off label) Corticoïd Surgery: too invasive technique If spot entirely healed, no relapsing Meeting with Dr.Riboulet, Dermatologist « patients with vitiligo have a bad observance with their treatments because they are restrictive and difficult. There is then definitely a market for new efficacious treatments » « So old treatments! » « Camouflage » Meeting with Dr.Riboulet, Dermatologist Conclusion : patients disappointed with their treatments the market exists, patients are waiting for efficacious treatments => new potential targets exploration Real diseases or just quality of life? Skin =body's envelope, reactions and emotions expressed via the skin Means of exercising power over others, captivating, influencing impressions and judgement, conquering Significant psychological impact Dermatology Life Quality Index (DLQI) Reasoning list of potential targets for each indication (hypo- + hyper- pigmentation) For each target Location Advantages / Inconvenients Drugs on the market Target’s mechanism of action Is the target interesting??? If YES => development Hypopigmentation TARGET LOCATION (skin or ubiquitous) Advantages Drawbacks Marketed medicines Mechanism of action POMC Ubiquitous, synthesis in the hypophysis, the hypothalamus and the melanocytes. Action through Alpha-MSH Production of alpha, betha gamma MSH, and some other different lipocortineq => Different levels of action, no specificity. ACTH Storage in the hypophysis Action through Alpha-MSH •Not specific •Production of mineralocorticoids, glucocorticoids, androgens by the surrenal glands Stimu-ACTH (diagnosis product) Binding to transmembrane MC2R of the surrenal glands’ cells Preo cAMP=> kinase activation Enzymatic activation and hormones production Alpha-MSH •Action in learning and memory, blood pressure, pigmentation, immune modulation,weight homeostasis, and others •Skin pigmentation with exposition to UV-B or sunlight •Potent anti-inflammatory effects •Repair of DNA photoproducts caused by UV • Reduce oxidative stress •Up-regulation of genes and their encoded proteins: Mitf • ErbB3 decreased by αMSH Peptide => parenteral administration (SC ) Topical administration: biodisponibility 0.05% Withdrawn of the market: •Afamelanotide •Menalotan •Natural ligand of MC1R, provoks increase in the intramelanocyte cAMP concentration and stimulates eumelanogenesis • UV induced oxidative DNA damage by inhibiting the generation of hydrogen peroxide, and enhances the repair of DNA photoproducts • Stimulates the differentiation of melanocytes MC1R Skin cells (melanocytes, keratinocytes,sebocytes and others), liver, testis, fat tissue, ovary (corpus luteum), intestin, red muscle, splin, kidney, heart, eyes, brain, immune cells (macrophages, fibroblasts, monocytes, mast cells, and neutrophils dendritic cells), the pituitary, placenta, endothelial cells, glioma cells, astrocytes, brain (low levels) • High constitutive activity • Involvement in endogenous control of some inflammatory processes •Mc1r is activated by all melanocortin mimetics •Several MCRs exist. The molecule has to be selective for MC1R in order to avoid AE •Perhaps Increased risk of skin cancers (regulation of melanocytes proliferation) •Several agonists have been synthetised by different laboratories. •MC1R activation via its ligands leads to the kinase A activation, increase in the intramelanocyte cAMP concentration and stimulates eumelanogenesis •stimulates the differentiation of melanocytes Production des differentes hormones selon la cellule ou l’on se trouve Hypophyse Melanotrope de l’hypophyse melanocytes Hypopigmentation α-MSH Synthesis POMC Central action Involved too early in the α-MSH pathway and involved in too many signalling pathways ACTH Central + Peripheral actions Involved in too many signalling pathways Production of mineralocorticoids, glucocorticoids, androgens by the surrenal glands Hypopigmentation Action on MC1R α-MSH Action on the different MCRs (MC1R) Activation of melanogenesis Anti inflammatory effect MC1R Present on inflammatory cells and melanocytes Role within melanocytes: melanogenesis alone POMC ACTH Melanocyte ↗AMPc α-MSH Eumelanin + + - MC1R ASIP ↘AMPc Phemelanin MC1R belongs to the MCRs family 5 G-protein-coupled transmembrane receptors (MC1R-5R), Positively coupled to adenylyl cyclase Pigmentation MC1R Adrenal function MC2R Sexual function MC4R Energy homeostasis MC3,4R MCR Immunity MC1R Sebaceous gland lipid production MC3,4R Weight control MC3,4R Melanocortin Receptor 1: MC1R Melanocytes Endothelial / Immune cells Testis- Ovaris Distribution Sebocytes Hypophysis α-MSH binds preferentially to MC1bR Binding Assay IC50a (nM) MC-1bR MC-3R MC-4R MC-5R 3.9 ±0.9 19 ± 2 19 ± 2 120 ± 19 Ideas to develop for Vitiligo MC1R : preferential target + Cutaneous pigmentation Antiinflammatory effect No carcinogenic effect MC1R agonist - Screening difficulty Type b receptors very difficult to screen EXCEPT for MC1bR!!! MC1R: Peptide Rc with an external loop (≠ Opiate Rcs) Action on MC1R Agonist MC1R discovered by BMS Peptidic agonist: Melanotan I Ac-Ser-Tyr-Ser-NorLeu-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 Some other peptidic agonists 1) Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Ava-Lys-Pro-Val-NH2 Ava9-10-NDP-αMSH 2 ) Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg–Gly-Lys-Pro-Val-NH2 desTrp9-NDP- α MSH MC1bR Agonists: Analogs of NDP- αMSH Binding Assay IC50a (nM) hMC-1bR hMC3R hMC4R 1 5.1 ± 0.5 >5000 2 16 ± 1 >2500 Assay EC50b hMC3R hMC5R hMC-1bR >5000 3500± 190 2.9 ± 0.21 11% at 2 µM >2500 >2500 9.6 ± 0.71 2% at 2 µM hMC4R hMC5R 0% at 2 µM >1500 1% at 2 µM 9% at 1 Clinical trials of Melanotan I Subcutaneous administration 2 significant findings: Melanotan I tolerable and effective at : melanin density in the skin (significant) UV-induced sunburn cells (significant) in subjects most at risk for UV induced skin damage 1% increase in melanin density => skin cancers roughly decreases by a factor of 2 MTI beneficial as a protection against UV-induced skin damage in populations most at risk for sunlightinduced skin damage and carcinogenesis Phase III, 1991 Melanotan I Melanotropins : the minimal message sequence (bioassays in frog and lizard skin) α-MSH : Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 minimal sequence required for agonistic activity, minimal chain length for observable biological activity Important potentiating AA, contibute significantly to the biopotency of alpha MSH A super potent melanotropin????? How??? Protect α-MSH from inactivation by oxidative mechanisms : Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 Norleucine : resulting analogue active or more active in most bioassays D-enantiomer instead of L-Phe : analoque proved to be 10-1000 times more active in one or more bio essays Melanotan II Lactam bridge between amino acids 5 and 10 Ac -Nle- Asp-His-D-Phe-Arg-Trp-Lys-NH2 Subcutaneous administration Much more active than Melanotan I in stimulating melanogenesis Very long t1/2 Melanotan I and II Advantages of those peptides : More active Resitant to enzimatic inactivation Capacity of increasing melanin production => Potent stimulators of melanogenesis of skin cancer incidence Photoprotective strategy: «tanning without the sun» Melanotan I and II side effects: Racoon effect «I got some completely new spots. Got a few facial freckles and some black spots elsewhere. So I suppose neither I or II are completely free of hyperpigmentation side effects » Epitan trial in 2003 for the melanotan implant Side effects (continued) Spots and hyperpigmentation Nausea => Flushing TOPICAL ADMINISTRATION Action on sexual arousal Despite all those AE, Melanotan (normally used for Congenital Protoporphyria) is misued to get a tan Inconvenients of peptides Peptides-> unstable orally Subcutaneous administration too complicated if repeated Topical administration => skin permeation poor Epidermal delivery: stratum corneum = skin’s barrier to peptide penetration How to increase skin permeability ? Formulation Liposome Avoid polypeptide : try to find a small molecule ???? - Graft palmitic acid : lipidic tail =>increase penetration Cutaneous penetration methods Peeling first : decrease cornea thickness Skin electroporation (electropermeabiliz ation /Iontophoretic route) Microneedles ac glycolique,vit A - complicated if daily application Microneedles (1) 2 categories Solid microneedles - pierce the skin then penetration of the drug from an extendedrelease patch - drug coated on microneedles -> rapid release - drug encapsulated within microneedles -> controlled release Hollow microneedles delivery by infusion Microneedles (2) A variety of needle sizes, shapes and materials Made out of silicon, metals and polymers • risk of breakage •expensive more robust •sufficient strength to insert into skin •safe alternative •water-soluble <-> dissolve in the skin over a timescale of minutes Low-cost manufacturing methods Microneedles: mechanism Microneedle array applied to the skin surface Microneedles penetrate the epidermis to bypass the stratum corneum Microneedles deliver drug directly to the viable epidermis Microneedles (3) Advantages: - Minimally invasive technique - Painlessness (epidermis: absence of nociceptors) - Well tolerated - Minimal irritation, short lasting - Insertion by hand + not damaged during skin insertion Roles - skin permeability => delivery M.A.P: topical molecule Safety profile Sun protector Short t1/2, controlled released Acceptable cost Compatibility with textile Administration rhythm: max once daily Satisfactory organoleptic characteristics (odor, taste, color, water resistant…) for acceptability NO systemic absorption, cannot go over the dermis Proposed drug Based on α-MSH and melanotan structure Short t1/2 : • NorLeu • Melanotan II Melanotan I Method increasing skin permeability : Microneedles Controled liberation : encapsulated microneedles made of polymers bandage of microneedles Sun protector : add chemical filter (benzylidene) SWOT Strenghts -First-in-class in this indication - Action on several different etiologies of Vitiligo -“tanning without the sun” => photoprotective strategy against skin cancer -A lot of work done on MC1R but no marketed product - Numerous adverse effects Opportunities Threats - No effective treatment already on the market Weaknesses -Currently significant research on pigmentation disorders Hyperpigmentation TARGET LOCATION (skin or ubiquitous) Advantages Drawbacks Agouti signaling protein (ASIP) •A secreted factor •Skin, heart, reproductive tract, fat tissue, liver, and kidney •Down-regulation of genes and their encoded proteins involved in biosynthesis (e.g., pigmentation, translation, transporters, alcohol metabolism, DNA replication, GSH anabolism, and mitochondrial redox abilities of melanocytes): Mitf, Tyr, Tyrp1, Dct, Si , Mart-1, Gpr143, Rab27a, Met and Cdk2 •Inhibition of other pigment genes: Rab38, P and Gpnmb •Modulated expresion of genes involved in other cellular pathways: gluthathione synthesis and redox metabolism (up-regulation of genes involved in nervous sytem, skeletal, bone, cartilage, kidney, muscle development; vasculogenesis; differentiation; cell adhesion; motility; and extracellular matrix-Rc interactions) •Tcf4, Lef1, Tcfap2a/Ap2a are upregulated • ErbB3 •AGRP (agouti related protein) •Responsible for lighter phenotypes in humans => ↗ risk of skin cancer • Down- regulation of genes encoding proteins involved in repair of DNA damage. => genes increased by ASIP implicated in various cancers TRP1 (implicated in the eumelanogenesis) Dopachrome tautomerase (DCT ou TRP2) enzymes that influence the metabolism of these sulfhydryls Removal of the enzyme leads to hypopigmentation. • Melanocyte specificity Marketed medicines Mechanism of action •Acts as a competitive antagonist of α-MSH: when binding to MC1R, it prevents α-MSH from increasing intramelanocyte cAMP levels and then stimulates pheomelanogenesis •ASIP also suppresses the basal, ligandindependent activity of hMC1R •Inhibition of melanocyte differentiation (↘ in expression of numerous melanogenic proteins) TRP-1: catalyses oxydation: DHICA -> acide indole 5,6-quinone2-carboxylique TRP-2: activity of dopachrome tautomérase, catalyses transformation: dopachrome -> acide 5,6dihydroxyindole-2carboxylique (DHICA) (not DHI) Glutamine or cystéine bind to DOPAquinone => availability of sulfhydryls=primary Hyperpigmentation TARGET LOCATION (skin or ubiquitous) Advantages Drawbacks Marketed medicines Mechanism of action Tyrosinase (most essential enzyme in the melanin biosynthetic pathway) • Ubiquitous amino acid tyrosine= substrate • melanocye specificity •Inhibitor leading to hypopigmentation •Reversible effect •DOPA: proceed spontaneously at physiological pH => AE • activated by ferrous ions to hydroxylate tyrosine •Metabolism of melanin: catalyses 3 different chemical reactions • Lack of direct correlation between tyrosinase synthesis and expression and pigment production. •↘ transcription: Vitamin A and derivates, glucosamine •Inhibition: Hydroquinone, Azelaic acid, Kojic acid •↗degradation: Elagic acid • indirect inhibition: Nacetyl glucosamine (a sugar itself) Catalyses the 3 following transformations: - tyrosine -> 3,4dihydroxyphénylalanine (DOPA) - DOPA -> dopaquinone - DHI -> indole-quinone •melanosomal association melanosome Motility By binding to diverse effector molecules, Rabs regulate vesicule budding, vesicule delivery, vesicule tethering, and vesicule membrane fusion with the membrane of the target compartment Down-regulated by ASIP Exocytic transport of lysosome-related organelles such as lytic granules in cytotoxic T lymphocytes Blockage of Rab 27a leads to IMMUNODEFICIENCY Rab 27 subfamily (Rab27a and b) Multiple molecules have a Nterminal homologous Rabbinding region and then function as Rab 27 effectors (exophilin, Slp/Slac2) Rab 27a/b effectors: role in a broad range of regulated exocytic pathways Granuphilin, another Rab27a effector: regulates the exocytosis of of classical secretory granules Gene encoding proteins involved in melanosome transport Hyperpigmentation TARGET LOCATION (skin or ubiquitous) Advantages Drawbacks Melanosomes’ transfer NCK5 (a member of the potassiumdependent sodium calcium exchanger family) Mechanism of action Inhibition: Linoleic acid Niacinamide (=vitamin B3) Epidermal turnover Slc24a5 (solute carrier family gene) Marketed medicines Chromosome 15 intracellular compartments in skin and the pigmented epithelium of the eye (+++) also a broad tissue distribution ↗ cutaneous permeability ↘ melanin concentration => ↘ pigmentation Risk of inflammatory lesions following by a hyperpigmentation if too powerful •up-regulated by MSH •SLC24A5 expression is required for melanin synthesis •SLC24A5 knockdown with two different siRNA duplexes in greater than a 20% reduction in the total normalized melanin content of these cells with no effect on cell viability decreased by ASP •NCKX5directly regulates human epidermal melanogenesis and natural skin color •Removal of the NCKX5 protein through small interfering RNAmediated knockdown disrupts melanogenesis causing a significant presence in multiple tissues -> contribute to other Ca2+ signaling events in addition to those involved in skin pigmentation Increased: Vitamins C, E, A, Glycolic acid melanogenesis (encode melanosomal proteins) gene that encodes NCKX5 •intracellular potassiumdependent exchanger activity •extrude cytosolic Ca2+ and K+ in exchange for Na+ ions Hyperpigmentation MC1R Antagonism ASIP Decrease in skin pigmentation Involvement in multiple pathways Inflammatory risk Related to numerous cancers POMC ACTH Melanocyte ↗AMPc α-MSH Eumelanin + + - MC1R ASIP ↘AMPc Phemelanin Hyperpigmentation Epidermal Turnover Increase in the epidermal turn-over Found in certain « peeling » products Risk of lesions => unpleasant Risk of post inflammatory hyperpigmentation Melanosome transfer Rab27 subfamily - Too many effectors involved in exocytic pathways such as exocytic transport of lytic granules in cytotoxic T Lc => risk of IMMUNODEFICIENCY Hyperpigmentation Melanin Synthesis Tyrosinase Enzyme involved in the 2 melanins synthesis Not specific of the eumelanogenesis Activity variation not always well porportional with the pigmentation variation TRP1 and TRP2 Enzymes specific of the melanocytes Enzymes involved in the eumelanogenesis Tyrosinase Tyrosine Dopaquinone DOPA Glutathion ou Cystein Leucodopaquinone TRP1 Cysteinyl dopa dopachrome DHICA DHI Indole-5,6quinone DHICA-melanin Eumelanin TRP2 1,4-benzothiazinyl Alanine Phaeomelanin Ideas to develop for hyperpigmentation MC1R = target? Proinflammatory effect of a MC1R antagonist => MC1R Seems safer to act later on the melanin synthesis Potential targets: INHIBITION of TRP1 TRP2 Screening of TRP1 TRP2 inhibitors Cells with recombinant TRP1, TRP2 TRP substrates modification TRP inhibitors Evolution of the skin coloration (easy to mesure) Other way of TRP inhibition: siRNA Small interfering RNA (siRNA), double-stranded RNA: inhibition of mRNA translation about 20-nucleotides siRNA for the mRNA of TRP1 and/or TRP2 by topical administration Specific action Main issue: siRNA very fragile => Might be an asset when topical action siRNA (continued) siRNA structure requirements: - low G+C content (30-50%) - lack of internal repeats - an A/U-rich 5' end - minimum distance of the siRNA from the start codon and the stop codon (set at 100 nt by default) +++ Target site accessibility siRNA: mechanism Nucleus Cytoplasm Transfection agent Entry into the cells -> transfection agent Different types of transfection reagents: nanoparticule polymer cationic lipid M.A.P: inhibition of TRP1 and 2 less exigent talking about absorption Sun filter Safety profile Acceptable cost Absolute specificity on TRP1-2 Compatibility with textile if topical Administration rhythm: max once daily Satisfactory organoleptic characteristics (odor, taste) for acceptability SWOT Strenghts - Melanogenesis specific enzyme Very specific of eumelanin No risk if eventual systemic absorption Opportunities • No existing effective treatment for hyperpigmentati on disorders Weaknesses • No work on these targets Threats Currently significant research on pigmentation disorders Overall conclusion High prevalence, existing market Altered quality of life Of the patients with vitiligo, 32.9% would pay more than 5000 € in order to achieve complete disease remission Not only a cosmetical disease but also a REAL disease