cosmeceuticals and peptides

Clinics in Dermatology (2009) 27, 485–494

Cosmeceuticals and peptidesLijuan Zhang, PhD, Timothy J. Falla, PhD⁎

Helix BioMedix Inc, 22118 20th Avenue SE, Bothell, WA 98021, USA

Abstract In nature, the majority of chemical reactions, biological responses, and regulatory processesare modulated in some part by specific amino acid sequences. The transfer of these interactive sequencesand the biological activities they induce to short, stable, and readily synthesized peptides has created adiverse new field of modulating molecules applicable to dermatology and skin care industries. Areassuch as inflammation, pigmentation, cell proliferation and migration, angiogenesis, innate immunity,and extracellular matrix synthesis have yielded peptide candidates for application to this area.© 2009 Published by Elsevier Inc.

Introduction

According to the United States Federal Food, Drug andCosmetic Act:

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0738doi:1

Cosmetics are defined as (1) articles intended to berubbed, poured, sprinkled, or sprayed on, introducedinto, or otherwise applied to the human body or any partthereof for cleansing, beautifying, promoting attractive-ness, or altering the appearance and (2) articles intendedfor use as a component of any such articles, except thatsuch term shall not include soap.

Cosmeceuticals are cosmetic products that deliver abiologic activity in support of cosmetic claims to providebeneficial topical actions. This market is expanding, partlydue to higher consumer demand for deliverables. As a result,bioactive peptides that are beneficial have been increasinglyused to supply such activities, and more than 25 differentpeptides are routinely found in a vast array of skin careproducts in the United States alone (Table 1). Many morepeptides are currently in development in company pipelines

Corresponding author. Tel.: +1 425 402 8400.-mail address: [email protected] (T.J. Falla).

-081X/$ – see front matter © 2009 Published by Elsevier Inc.0.1016/j.clindermatol.2009.05.013

worldwide that will expand not only the number of peptideingredients but also the diversity of their application.

There are a number of reasons for this surge in interest inbioactive peptides, the most important of which is activity.Peptides are important in many natural processes withrelevance to skin care, such as the modulation of cellproliferation, cell migration, inflammation, angiogenesis,melanogenesis, and protein synthesis and regulation. Themechanisms for such activities are primarily based on ligandbinding, of one type or another, and due to the immensediversity of sequence and structure provided by peptidesequences, vast arrays of activity are possible. In addition,the peptides used primarily consist of natural L-amino acids,which in general are not immunogenic and are readily brokendown over time to yield individual natural amino acids.Although the synthesis of peptides is straightforward andscalable, their price is highly dependant on the number ofamino acids required and the type. Certain amino acids aremore costly than others. This has significance, because theprice of an active ingredient effects how much of it can beincluded in a final product.

The issues that apply to any cosmeceutical ingredient alsoapply to bioactive peptides, including reproducible activity,stability, safety, formulation, and delivery. These issues are

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http://dx.doi.org/10.1016/j.clindermatol.2009.05.013

Table 1 Summary of bioactive peptides currently marketed for inclusion as active ingredients in skin care products

Company Name Activity Premix products Source

Atrium Tripeptide-2 ECM stimulation via MMP-1 inhibition ECM-protect UndisclosedAtrium Tripeptide-1 ECM stimulation via growth factor Kollaren HGFAtrium Acetyl tetrapeptide-2 Reduce loss of thymic factors Thymulen 4 ThymopoietenAtrium Acetylpeptide-1 Melanin increase via MSH regulation Melitane MSH agonistAtrium Nonapeptide-1 Tyrosinase activation inhibition Melanostatine MSH antagonistGrant Indust. Palmitoyl hexapeptide-6 Dermal repair Matrix Rebuilder Innate immunityGrant Indust. Oligopeptide-10 Dermal protection InvisaSkin-64 Innate immunityLipotec Tripeptide-1 Inhibits collagen glycation Aldenine, Trylagen Human serumLipotec Tripeptide-10 citrulline Collagen fibrillogenesis Decorinyl, Trylagen DecorinLipotec Acetyl tetrapeptide-5 Edema reduction by ACE inhibition Eyeseryl UndisclosedLipotec Pentapeptide-3 Botox-like via mimicing enkephalins Leuphasyl UndisclosedLipotec Acetyl hexapeptide-3 (or -8) Botox-like via SNARE inhibition Argireline SNAP-25Lipotec Acetyl octatapeptide-1 Botox-like via SNARE inhibition SNAP-8 SNAP-25Lipotec Hexapeptide-10 Increases cell proliferation and laminin V Serilesine LamininPentapharm Palmitoyl tripeptide-5 Collagen synthesis via TGF-β Syn-coll Thrombospondin IPentapharm Dipeptide diaminobutyroyl

benzylamide diacetateBotox-like via acetycholine receptor Syn-ake Waglerin 1

Pentapharm Oligopeptide-20 MMP inhibitor via TIMP Pepha-timp TIMP-2Pentapharm Pentapeptide-3 Botox-like via acetycholine receptor Vialox UndisclosedProcyte Copper GHK/AHK Wound healing Brand example Neova Human serumSederma Dipeptide-2 Lymph drainage via ACE inhibition Eyeliss RapeseedSederma Palmitoyl oligopeptide Collagen synthesis via signalling Eyeliss, Matrixyl 3000 Human serumSederma Palmitoyl tetrapeptide-7

(formally -3)Elasticity via IL6 reduction Matrixyl 3000, Rigin IgG/matrikine

Sederma Palmitoyl pentapeptide-3 Collagen stimulation via signalling Matrixyl ProcollagenSederma Palmitoyl oligopeptide Retinoic acid-like activity Biopeptide-CL CollagenSederma Palmitoyl oligopeptide Increases collagen and HA Biopeptide-EL Elastin

ACE, angiotensin I-converting enzyme; ECM, extracellular matrix; HA, hyaluronic acid; HGF, hepatocyte growth factor; IgG, immunoglobulin G; MMP,matrix metalloproteinases; MSH, melanocyte-stimulating hormone; SNARE, soluble N-ethylmaleimide sensitive factor attachment receptor; TGF-β,transforming growth factor-β; TIMP, tissue inhibitor of MMP.Companies: Atrium Biotechnologies (Quebec City, QC, Canada); Grant Industries (Elmwood, NJ, USA); Lipotec (Barcelona, Spain), Pentapharm (Basel,Switzerland); Procyte (Photomedix, Montgomeryville, PA, USA); Sederma (Le Perray en Yvelines, France).

486 L. Zhang, T.J. Falla

peptide-specific and marketers of such ingredients need tohave a strategy to create well validated and characterizedpeptide based active ingredients. This contribution outlinesthe role of peptides in nature in relation to skin care endpoints, the activities of such peptides, and their application tothe cosmeceutical industry.

Bioactive peptides

Generation and maintenance of dermal integrity

Skin is comprised of three broad layers: the epidermis, thedermis, and the hypodermis.1 The epidermis and dermis areseparated by a basement membrane that is rich inextracellular matrix (ECM) proteins, including collagens,epilugrin, laminin, fibronectin, elastins, nidogen, and heparinsulfate proteoglycans.2 In addition to anchoring cells andorgans together, the ECM serves as a mediator of receptor-induced interactions between cells. In cell proliferation and

morphogenesis, these interactions guide the growth anddifferentiation of cells.3

The dermis provides a supporting matrix, which includescollagen and elastin, for extensive vascular and nervenetworks. The largest class of fibrous ECM molecules isthe collagen family, which includes more than 16 differenttypes of collagen. Collagen in the dermal matrix is composedprimarily of type I (80% to 85%) and type III (8% to 11%),both of which are fibrillar or rod-shaped.4 The tensilestrength of skin is due predominately to these fibrillarcollagen molecules, which self-assemble into microfibrils ina head-to-tail and staggered side-to-side lateral arrangement.Collagen molecules become cross-linked to adjacent col-lagen molecules, creating additional strength and stability inthe fibers. Damage to the collagen network (eg, by enzymesor physical destruction) or its total collapse causes healing totake place by repair.

The ECM not only provides structural support but alsoinfluences cellular behavior such as differentiation andproliferation. Most of these functions are related tosignaling by matrix components to cells through cell-

1 Nomenclature for amino acids is by single- and triple-letter codesaccording to standard designation and written from N-terminus to C-terminus. Unless indicated by “d,” all amino acids are in the l-form.See Appendix.

487Cosmeceuticals and peptides

membrane receptors. Some of the signaling molecules areproduced by proteolytic degradation of macromoleculesof the ECM that release soluble peptides, termed“matrikines.”5

Matrikines

Two classes of matrikines have been characterized:natural matrikines signal directly from the extracellularmilieu, and cryptic matrikines (or matricryptins) requireproteolytic processing to reveal the ligand or to release theligand from its ECM protein.6,7 Matrikines and matricryptinsare classes of ligand that have been characterized assubdomains of various ECM proteins capable of signalingto the cell through receptors, such as growth factor receptors.

Members of the noncollagenous ECM proteins includelaminins, fibronectins, and tenascins. Both tenascin-C andlaminin-5 have epidermal growth factor (EGF) like repeats.Select EGF-like repeats of tenascin-C elicit mitogenesis andepidermal growth factor receptor (EGFR) autophosphoryla-tion in an EGFR-dependent manner.8 The EGF-like repeatsof laminin-5 also act as cryptic ligands revealed by matrixmetalloproteinase-2 (MMP-2) degradation of the surround-ing extracellular matrix.9

Other matrikines in collagen, elastin, decorin, andlaminin-1 can promote chemotaxis, mitogenesis, and metas-tasis in conditions such as melanoma.10 Unlike traditionalgrowth factors, these individual matrikine domains seem topossess relatively low binding affinity (high nanomolar ormicromolar) and multiple valences.11 A study in 2004showed ECM components contained domains that caninteract with and activate receptors with intrinsic tyrosinekinase activity.11 These receptor tyrosine kinases are strongmediators of the cell response, such as proliferation,migration, differentiation, and dedifferentiation.

Fibrillar collagens are synthesized as precursor moleculescontaining N-terminal and C-terminal propeptides that arecleaved off extracellularly by specific peptidases. Work by agroup at the University of Tennessee identifying the activitiesof small peptide fragments within procollagen I resulted inthe identification of KTTKS, a subfragment within thecarboxy-terminal propeptide (residues 197-241) capable ofstimulating the production of both collagen and fibronec-tin.12-14 This peptide fragment dramatically augmented ECMproduction in subconfluent fibroblasts. It also stimulated typeI and type III collagens and fibronectin production in a dose-and time-dependent manner with no effect on total proteinsynthesis or on the ratio of secreted proteins to cell-associatedproteins.13,14

Sederma brought this peptide to market in a lipidated formunder the International Nomenclature of Cosmetic Ingredi-ents name palmitoyl pentapeptide-3 as part of the premixproduct Matrixyl. The application of this peptide to skin carewas made by Dr Karl Lintner of Sederma, and the associatedintellectual property was filed in 1999. Within this nowpublished patent is the demonstration that at 50 parts per

million (ppm), palmitoyl pentapeptide-3 produces a sig-nificant benefit to lines and wrinkles around the eyescompared to vehicle alone control in a randomized study(Table 1).

Elastic fibers are an important component of the extra-cellular matrix and consist of two elements, the microfibrilsand matrix elastin, providing elasticity and resilience totissues that require the ability to deform repetitively andreversibly. Elastin has a unique repeating sequence in thehydrophobic region. VGVAPG1 is a hexapeptide repeatedmultiple times in human, bovine, and porcine elastinmolecules.15-17 Elastase-mediated elastolysis liberates elastinfragments called elastokines, which display a wide range ofbiologic activities in a number of normal and transformedcells18 and are best known for their chemokine-like activitiesthat are chemotactic for fibroblasts, macrophages, mono-cytes, and polymorphonuclear neutrophils.19-22 They alsopromote cell cycle progression and induce release ofproteolytic enzymes by stromal and cancer cells.23

Chemotactic sites on the elastin molecule have beenidentified containing the XGXXPG motif.24-26 SuchXGXXPG sites are also present in multiple copies amongfibrillin-1, -2, and -3, fibronectin, laminin, and severaltenascins and collagens.7

The best studied elastin-peptide, VGVAPG, is known forits chemotactic activity and MMP up-regulation properties.Elastin-derived peptides stimulated the growth of humanskin fibroblasts and accelerated angiogenesis in the chickchorioallantoic membrane in an in vivo model.27 Thishexapeptide stimulates pseudotube formation from humanvascular and microvascular, endothelial cells in the matrigeland collagen models, as well as cell migration in an in vitrowound healing assay.28 Elastin-derived peptide effects wereattributed to up-regulation of pro-MT1-MMP and pro-MMP-2 expression and activation at both the messenger RNA(mRNA) and protein levels.27 The Sederma premix product,Biopeptide-EL, contains such a fragment of elastin and isincorporated into products to provide “reconstruction of thedermis” and “to produce chemotaxis for restructuring andrepair” (Table 1).

The era of the short bioactive peptide being applied todermatology started with discoveries made in the field ofwound healing. The work of Dr Loren Pickart in the 1970son the discovery and subsequent characterization of the GHKcopper-binding peptide isolated from human plasma29-31 ledto studies demonstrating clinical benefit in acute and chronicwound healing.32-34

The GHK peptide exhibits a high affinity for copper (II)ions, with which it forms spontaneously a tripeptide-coppercomplex (GHK-Cu). GHK-Cu was initially described as agrowth factor for a variety of differentiated cells.35,36 A


number of other biologic effects have since been reported,and GHK-Cu appears to be a potent activator of woundhealing. It is a potent chemotactic agent for monocytes/macrophages and mast cells.37,38 It stimulates nerve tissueregeneration,39 angiogenesis in vivo,40,41 and stimulates theexpression of different components of extracellular matrixboth in vitro and in vivo.31,34,42 When injected intosuperficial wounds, GHK-Cu accelerates wound closure.43

Clinical trials showed that treatment with GHK-Cu mightsignificantly improve the healing of skin ulcers in diabeticpatients.44 The copper peptide also enhances woundremodeling by modulating expression of MMPs.45

It has been speculated that at least a proportion ofcirculating GHK may be derived from the ECM-bindingprotein SPARC (secreted protein, acidic, rich in cysteine).41

This protein is expressed by endothelial cells duringdevelopment and tissue remodelling and yields the GHKsequence specifically, upon degradation by proteases such aselastase, stromelysin, trypsin, and subtilisin. These aregenerally present in situations of matrix turnover and mayendow GHK with a matrikine-like role.

Innate immunity

Matrikines are created and released as a result of damageor injury that may have been caused by a wide range offactors. The body, however, also has a system targeted as animmediate response to such damage or threat where thetrigger for such a response is the threat itself. This system isinnate immunity, and peptides contribute significantly to itsoperation in protection against pathogens and restoration ofbarrier function.46 Although the skin was formerly con-sidered an inactive physical protective barrier that partici-pates in host immune defense merely by blocking entry ofmicrobial pathogens, it is now apparent that a major functionof the skin is to defend the body by rapidly mounting aninnate immune response to injury and microbial insult.Resident and infiltrating cells in the skin synthesize andsecrete small peptides that exhibit a wide range ofbioactivities aimed at restoring barrier function and protect-ing the body until that function has been restored.

Human skin is comprised of two major classes of innateimmunity peptide, β-defensins and cathelicidins (LL-37).47

The β-defensins are cysteine-rich peptides of 36 to 42 aminoacids and are stabilized by three disulfide bonds. The threebest-characterized human β-defensins—human β-defensin(hBD-1), hBD-2, and hBD-3—have been detected in humanskin and cultured keratinocytes.48 The only endogenouscathelicidin in humans, hCAP-18/LL-37, is found at highconcentrations in its unprocessed form (hCAP-18) in thegranules of neutrophils and is processed upon degranulationand release.49

Innate immunity peptides are multieffectors that arecapable of recruiting and activating antigen-presenting cellsand serve as early warning signals to activate innate andadaptive immune systems. Defensins and LL-37 are both

known for their chemotactic role on various cell types andstimulation of cytokines.50 Endogenous LL-37 stimulateswound vascularization and reepithelialization of healing skinand angiogenesis in an animal model.51 The ability of thispeptide to close wounds has partly been attributed to aninduction of keratinocyte migration through transactivationof the EGFR.52 The immumodulatory activity of LL-37 hasbeen dissected from its antimicrobial activity at the sequencelevel, and the resulting peptide subunits induced proliferationand migration mediated through EGFR.49,51

Acceleration of would healing has been demonstratedwith other non-antimicrobial innate immunity peptides suchas HB-107, a derivative of cecropin B.53 This peptideproduced a 64% improvement in wound repair comparedwith scrambled peptide and vehicle controls, an effectcomparable to treatment with recombinant human platelet-derived growth factor.53 Subunits and subunit analogs ofsuch peptides have been identified that exhibit specificactivities that contribute to this wound healing activity.Palmitoyl hexapeptide-6, a peptide designed using an innateimmunity peptide template (Grant Industries, Elmwood, NJ)stimulates fibroblast proliferation and scaffolding, collagensynthesis, and cell migration, and is currently marketed forinclusion in antiwrinkle skin care products. The multi-functional nature of innate immunity peptides and theirfragments hold potential for a wide range of applications.

The sweat protects the epidermal surface by secreteddermcidin and its natural proteolytic derivatives,54 and theupper epidermis of normal human skin expresses hBDs andLL-37 in addition to human neutrophil protein released byrecruited neutrophils and mast cells.55 After exposure tomicrobe-derived molecules, monocytes and lymphocytesboth stimulate the epidermal expression of hBD-1, hBD-2,and hBD-3 through distinct mechanisms.56 These peptidesand their derivatives are broad spectrum and synergisticagainst Staphylococcus aureus and Escherichia coli in theextracellular milieu.57 Some may perform more effectivelyin vivo, because different cleavage forms of LL-37 have beenfound in physiologic conditions and are more active againstCandida albicans than the full length LL-37.58

The cathelicidin (cathelicidin-related antimicrobial pep-tide [CRAMP]) knockout mouse becomes more susceptibleto necrotic skin infection caused by group A Streptococ-cus,59 but transgenic mice expressing porcine PR-39 aremore resistant to the same pathogen.60 Since the firstobservation that patients with atopic dermatitis are relativelydeficient in cathelicidin and hBD-2 and demonstrateincreased susceptibility to bacterial and viral superinfectionof the involved skin, the sweat of the patients was found alsoto have reduced level of, in addition to LL-37, dermcidinswhich, as mentioned above, are predominantly secreted intothe sweat of healthy skin.61,62 Atopic dermatitis affects 15million people in the United States and 90% suffer long-termStaphylococcal skin infection. Restoration of innate peptidesto normal levels via external application could providesignificant benefit.


Inflammation is often associated with skin conditions thathave bacterial involvement, which is partly caused bylipopolysaccahride released from the outer membrane ofgram-negative bacteria, and lipotechoic acid released fromgram-positive bacteria. Innate immunity peptides such asdefensins and LL-37 are well known for binding andneutralizing bacterial debris, including lipopolysaccahrideand lipotechoic acid responsible for inflammation, resultingin down-regulation of proinflammatory cytokines.63 Con-sistent with this, LL-37 has also been shown to modulate theinflammatory response mediated by toll-like receptor.63

Another example comes from granulysin-derived peptidesthat suppress cytokine release stimulated by Propionibac-terium acnes.64 A synthetic peptide designed to bindlipotechoic acid, oligopeptide-10, has been developed forinclusion in anti-acne products (Grant Industries, NJ).

Up-regulation of LL-37 has been reported to be a naturalprotective mechanism associated with ultraviolet (UV) sun-light exposure. Excessive sunlight exposure is an importantetiologic factor in the development of acute inflammationcharacterized by erythema, edema, immunosuppression, skinaging, and cancer development. UVB is a well known riskfactor for the development of acute inflammation as well asnonmelanoma skin cancer in the epidermis. The UVB bandstimulates expression of hCAP-18 in human skin. Individualswho were exposed to UVA (300 to 400 nm) and UVB (280 to315 nm) exhibited an increase in the amount of both hCAP-18mRNA and vitamin D receptor in their skin.65 Topicaladministration of a vitamin D analog (calcipotriol) onto theskin of volunteers increased the expression of fully processedLL-37 peptide within 24 hours of administration.65 Lowdoses of UVB appear to coordinately suppress keratinocyteproliferation and stimulate cellular maturation as well ascertain arms of the adaptive immune system.66 When sunlightslows the adaptive immune system within the skin, the innatedefense system kicks in. Functional deficits in the defense ofthe skin against microbes caused by suppression of localadaptive immunity could be compensated for by stimulationof antimicrobial peptide expression. This provided a rationalefor potential application of peptides such as LL-37 amongcertain ethnic groups that are prone to sunburn to preventphotochemical alterations in both the DNA and membranelipids of epidermal cells caused by UV radiation.

Melanogenesis

UV radiation stimulates melanogenesis by human epider-mal melanocytes, both in the skin and in cultured cells.67 Thesynthesis and distribution of melanin contributes to skin andhair color; however, increased levels of epidermal melaninsynthesis can darken the skin and cause cosmetic problems.Melanins in mammalian melanocytes are synthesized withinmelanosomes and mainly controlled by the expression andactivation of tyrosinase. There is evidence that the melano-cortin-1 receptor (MC1-R) is a key control point for bothconstitutive and facultative skin pigmentation. On binding to

the MC1-R, melanocortin peptide, α-melanocyte-stimulatinghormone (α-MSH) activates adenylate cyclase (cyclicadenosine monophosphate [cAMP]), which in turn causesan increase in intracellular cAMP. Increases in cAMP result,via protein kinase C, in the activation of tyrosinase, whichwas often reviewed as the classic pathway.68,69

The use of analogs of α-MSH that function as MC1-Ragonists has been attempted for potential topical agents toprevent skin photocarcinogenesis. Tetrapeptide α-MSHanalogs, Ac-His-D-Phe-Arg-Trp-NH2, N-pentadecanoyl-and 4-phenylbutyryl-His-d-Phe-Arg-Trp-NH2, have beenshown on cultured human melanocytes to be more potentthan α-MSH in stimulating the activity of melanogenesis,reducing apoptosis and release of hydrogen peroxide, andenhancing repair of DNA photoproducts in melanocytesexposed to UV radiation.70

Skin keratinocytes also express factors that are involvedin melanogenesis. Keratinocyte protease-activated receptor 2(PAR-2) has been shown to affect melanosome transfer frommelanocyte to keratinocyte. The PAR-2 activating peptideSLIGRL enhanced melanosome ingestion by keratinocytes,thus increasing pigmentation.71 In contrast, human homologof agouti-signaling protein blocked the binding of α-MSH tothe MC1-R and inhibited the effects of α-MSH on humanmelanocytes.72 Treatment of human melanocytes withrecombinant mouse or human agouti-signaling proteinblocked the stimulatory effects of α-MSH on cAMPaccumulation, tyrosinase activity, and cell proliferation.72

Melanogenesis is regulated by many factors. In melano-cytes, protein kinase C-β (PKC-β) was shown to beassociated with melanosomes and regulate human melano-genesis through activation of tyrosinase.73 Depletion of PKCby chronic treatment of cells with phorbol esters markedlyreduces the basal melanin level and tyrosinase activity inhuman melanocytes and murine S91 melanoma cells.73,74

Sequence analysis of a complimentary DNA clone for humantyrosinase revealed the phosphorylation site for PKC-βwithin an 11-amino-acid sequence located to the C-terminusof tyrosinase.75 This 11mer tyrosinase mimetic peptide hasbeen shown to compete for PKC-β binding, resulting ininhibition of phosphorylation of tyrosinase by PKC-β.76

These findings support the notion that peptide compoundsthat inhibit PKC-β might lead to the development ofcosmeceuticals for potential skin and hair lightening.

The search for tyrosinase inhibitors is another path thathas lead to numerous chemical compounds with potentialinhibitory activities. Very few peptides, however, meet sucha demand due to the nature of tyrosinase. The early findingof cyclic peptide, cyclo(Pro-Val-Pro-Tyr), although aneffective tyrosinase inhibitor, has not been proceeded toany clinical study due to the difficulty in large scalesynthesis.77 Among many chemicals, kojic acid is the mosteffective inhibitor of human tyrosinase and has been alreadyincorporated into many cosmetic products. Attachment ofkojic acid to peptides improved the effectiveness oftyrosinase inhibitory activity of kojic acid. The best


compound, kojic acid-FWY, exhibited 100-fold tyrosinaseinhibitory activity compared with kojic acid alone.78 Inaddition, storage stability was approximately 15 timeshigher and toxicity was lower than that of kojic acid.78,79

Acetylcholine transmission

There is increasing evidence that the cutaneous nervoussystem modulates physiologic and pathophysiologic effects,including cell growth and differentiation, immunity andinflammation, and tissue repair. Cutaneous nerve fibers andinflammatory cells are both able to release neuromediatorsand thereby activate specific receptors on target cells in theskin or transient immunocompetent cells. Endocytosis andexocytosis, the major transport system of these neurotrans-mitters, involve tightly orchestrated mechanisms ensuringtrafficking of numerous molecules and particles through theformation of membrane carriers, often vesicles.80 Membranefusion between vesicles and target membranes, a critical stepin these processes, is mediated by a large number of so-calledsoluble N-ethylmaleimide sensitive factor attachment protein(SNAP) receptor (SNARE) proteins.81

The SNARE protein complex is involved in synapticvesicle exocytosis from many types of neurons. Cutaneousneuromediators include classic neurotransmitters such ascatecholamines and acetylcholine being released from theautomatic nervous system or cutaneous cells.82 Acetylcho-line can be stored in both small and large synaptic vesiclesand contribute to vasodilator responses over a wide range ofstimulation frequencies. Botulinum neurotoxins (Botox)cause muscle paralysis by blocking acetylcholine release atnerve-muscle junctions through a very specific and exclusiveendopeptidase activity against SNAP-25 of the presynapticexocytosis machinery. The toxins bind to acceptor sites onthe neuronal membrane, are taken up into the cell by acalcium- and pH-dependent translocation process, and cleavespecific SNARE proteins in a temperature- and zinc-dependent manner.83

Treatment with botulinum neurotoxin has to be understrict medical control because it is the most potent toxinknown to mankind. To circumvent this limitation, smallpeptide molecules that mimic the action of Botox areattracting an increasing amount of attention. In this regard,synthetic peptides that emulate the amino acid sequence ofthe synaptic protein SNAP-25 were shown to be specificinhibitors of neurosecretion at micromolar concentrations.To facilitate membrane permeability, new sequences that areshorter, while preserving a biologic activity, have beenpursued. A 6-mer peptide (Ac-EEMQRR-NH2), derivedfrom the N-terminal domain of SNAP-25 (aa 12-17),interferes with the assembly of the SNARE ternary complexand inhibits Ca2+-dependent catecholamine release fromchromaffin cells (Table 1). This hexapeptide (acetylhexapeptide-3) was marketed under the name of Argireline(Lipotec, Barcelona, Spain). Although the evidence tosupport the benefits of Argireline is slim, a clinical study84

suggested a 10% concentration of acetyl hexapeptide-3reduces the depth of wrinkles up to 30% after 30 days ofuse.84 These findings suggest that the hexapeptide is abiosafe cosmetic alternative to attenuate facial wrinkles andit is feasible to follow a rational strategy to identify peptide-based mimetics of toxin effects.

Another source of such bioactive peptides is the toxinsfrom venomous organisms that disrupt neuromuscularcommunication to paralyze their prey. Waglerin-1 from thevenom ofWagler's pit viper (Tropidolaemus wagleri) is a 22-amino acid peptide selective for the adult form of nicotinicacetylcholine receptors (nAChRs) and causes paralysis bycompetitively antagonizing muscle nAChRs.85 A synthetictripeptide that mimics the effect of Waglerin-1 has recentlybeen marketed as SYN-AKE (Pentapharm, Basel, Switzer-land) for reducing wrinkles by inhibiting muscle contrac-tions. Acting at the postsynaptic membrane, SYN-AKE is areversible antagonist of the muscular nAChR. It wasassumed that this peptide binds to the epsilon subunit ofthe muscular nAChR, which prevents binding of acetylcho-line to the receptor. More products based on peptides withsuch neurotransmitter blocking activity are expected to affectthe cosmeceutical industry in the next few years.

Inflammation

The cosmetics industry generally assumes that inflamma-tion has a negative effect on the condition and appearance ofskin. Dehydroepiandrosterone (DHEA), a secretary productof the human adrenal gland, has been characterized asexhibiting a wide array of therapeutic benefits, includingslowing of the aging process.86 Although DHEA levelsdecline in the elderly, the supplementation of DHEAresulting in benefit in this population remains to be provenin humans. Among the properties exhibited by DHEA areacceleration of wound healing and the reduction ofinterleukin-6 (IL-6) levels in inflamed cells.87,88

The tetin class of peptides, first described in the 1990s, arefragments of immunoglobulins, interferons, ILs, or growthfactors that modulate cytokine levels.89 A subgroup of thisfamily, the rigins, derived from immunoglobulin G, also hasbeen shown to down-regulate IL-6.90 One such peptide,palmitoyl tetrapeptide-7 (Pal-GQPR) has been developed asan active ingredient by Sederma and marketed as RIGIN.The ability of RIGIN to down-regulate IL-6 in resting andinflamed cells was compared with DHEA in vitro, and thetwo actives were comparable. Marketing materials related toRIGIN indicate that this reduction in IL-6 can produceincreased skin firmness, smoothness, and elasticity.

Angiotensin-converting enzyme inhibition

A number of dipeptides and tripeptides have beenidentified as effective angiotensin-converting enzyme(ACE) inhibitors, and these have been isolated from a varietyof natural sources.91,92 One of the most potent of such


inhibitors is valine-tryptophan (VW), which has been shownto reduce blood pressure when delivered orally.93 ACEinhibitors act by preventingACE from converting angiotensinI to angiotensin II, a potent vasoconstrictor, and by preventingthe enzyme from inactivating the vasodilator bradykinin. Thepeptide VW has been marketed as dipeptide-2 (or DipeptideVW) by Sederma. Under the intellectual property protectingthis and related peptides for skin care use, it can only be usedin combination with a second peptide and hesperidin, acompound that protects vasculature, is anti-inflammatory, andis an antioxidant.94,95 The premix is marketed in such acombination as Eyeliss, in which the second peptide ispalmitoyl tetrapeptide-7 (see Inflammation).

Application of activities tocosmeceutical products

An aging population is seeking an ever increasing breadthof bioactivity and increased potency from the ingredients inskin care products. Consumers also have an ever increasingneed to understand the theory of the technology and activity,if not the details. This has led to a more science-based focuson active ingredients. Activities that result in diminished linesand wrinkles, smoother skin texture, and a reduction inredness and skin discoloration are the key target end points.Providing this functionality are antioxidants, growth factors,peptides, anti-inflammatories, polysaccharides, and agents tolighten pigments. As has been described, the role of peptidesin a wide range of processes is becoming more fullyunderstood and the potential application of such activitiesmore fully appreciated. To maintain that activity in a productand subsequently translate that activity into a beneficial effectfor the consumer creates issues that also need to be addressed.

The compatibility and stability of a bioactive peptidewithin a cosmetic formulation can provide significant cluesabout whether it can be delivered in an active form.Binding, particularly of charged peptides, by other ingre-dients may prevent release from the formulation or preventthe peptide being released in an active form. If the desiredbioactivity can be demonstrated by the formulated peptideor, by the use of a Franz Cell, demonstrated to be releasedfrom the formulation in an active form, a level of comfortcan be achieved.

Human skin has unique properties, of which functioningas a physicochemical barrier is one of the most important.The traditional assumption has been that molecules exceed-ing 500 mw are unable to traverse the barriers of the skin and,in particular, the stratum corneum.96 More recent studieshave demonstrated that this paradigm does not hold true,particularly in the case of dry or aged skin.97,98 In addition,with the advent of newer and more potent penetrationenhancers, either peptide or chemical in origin, larger andlarger molecules are being transported.99,100

The application of bioactive peptides to the cosmeticsindustry holds great promise due to the wide range of

activities, chemistries, and indications that can be developed.The cost-benefit ratio is key in this area, and the balance ofeconomics, peptide concentration and, reproducible, stable,deliverable bioactivity, will be the key driver for the successof each peptide on an individual basis.

Appendix A. Amino acid symbols

A
mino acid Symbol
3 Letter
1 Letter
Alanine
Ala A Arginine Arg R Asparagine Asn N Asparagine or aspartic acid Asx B Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glutamine or glutamic acid Glx Z Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Leucine or Isoleucine Xle J Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Unspecified or unknown Xaa X Valine Val V
References

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