Cosmeceuticals and Peptides

Clinics in Dermatology (2009) 27, 485–494

Cosmeceuticals and peptides Lijuan 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 processes are modulated in some part by specific amino acid sequences. The transfer of these interactive sequences and the biological activities they induce to short, stable, and readily synthesized peptides has created a diverse new field of modulating molecules applicable to dermatology and skin care industries. Areas such 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 and Cosmetic Act: Cosmetics are defined as (1) articles intended to be rubbed, poured, sprinkled, or sprayed on, introduced into, or otherwise applied to the human body or any part thereof for cleansing, beautifying, promoting attractiveness, or altering the appearance and (2) articles intended for use as a component of any such articles, except that such term shall not include soap.

Cosmeceuticals are cosmetic products that deliver a biologic activity in support of cosmetic claims to provide beneficial topical actions. This market is expanding, partly due to higher consumer demand for deliverables. As a result, bioactive peptides that are beneficial have been increasingly used to supply such activities, and more than 25 different peptides are routinely found in a vast array of skin care products in the United States alone (Table 1). Many more peptides are currently in development in company pipelines ⁎ Corresponding author. Tel.: +1 425 402 8400. E-mail address: [email protected] (T.J. Falla). 0738-081X/$ – see front matter © 2009 Published by Elsevier Inc. doi:10.1016/j.clindermatol.2009.05.013

worldwide that will expand not only the number of peptide ingredients but also the diversity of their application. There are a number of reasons for this surge in interest in bioactive peptides, the most important of which is activity. Peptides are important in many natural processes with relevance to skin care, such as the modulation of cell proliferation, cell migration, inflammation, angiogenesis, melanogenesis, and protein synthesis and regulation. The mechanisms for such activities are primarily based on ligand binding, of one type or another, and due to the immense diversity of sequence and structure provided by peptide sequences, 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 broken down over time to yield individual natural amino acids. Although the synthesis of peptides is straightforward and scalable, their price is highly dependant on the number of amino acids required and the type. Certain amino acids are more costly than others. This has significance, because the price of an active ingredient effects how much of it can be included in a final product. The issues that apply to any cosmeceutical ingredient also apply to bioactive peptides, including reproducible activity, stability, safety, formulation, and delivery. These issues are

486 Table 1

L. Zhang, T.J. Falla Summary of bioactive peptides currently marketed for inclusion as active ingredients in skin care products

Company

Name

Activity

Premix products

Source

Atrium Atrium Atrium Atrium Atrium Grant Indust. Grant Indust. Lipotec Lipotec Lipotec Lipotec Lipotec Lipotec Lipotec Pentapharm Pentapharm

Tripeptide-2 Tripeptide-1 Acetyl tetrapeptide-2 Acetylpeptide-1 Nonapeptide-1 Palmitoyl hexapeptide-6 Oligopeptide-10 Tripeptide-1 Tripeptide-10 citrulline Acetyl tetrapeptide-5 Pentapeptide-3 Acetyl hexapeptide-3 (or -8) Acetyl octatapeptide-1 Hexapeptide-10 Palmitoyl tripeptide-5 Dipeptide diaminobutyroyl benzylamide diacetate Oligopeptide-20 Pentapeptide-3 Copper GHK/AHK Dipeptide-2 Palmitoyl oligopeptide Palmitoyl tetrapeptide-7 (formally -3) Palmitoyl pentapeptide-3 Palmitoyl oligopeptide Palmitoyl oligopeptide

ECM stimulation via MMP-1 inhibition ECM stimulation via growth factor Reduce loss of thymic factors Melanin increase via MSH regulation Tyrosinase activation inhibition Dermal repair Dermal protection Inhibits collagen glycation Collagen fibrillogenesis Edema reduction by ACE inhibition Botox-like via mimicing enkephalins Botox-like via SNARE inhibition Botox-like via SNARE inhibition Increases cell proliferation and laminin V Collagen synthesis via TGF-β Botox-like via acetycholine receptor

ECM-protect Kollaren Thymulen 4 Melitane Melanostatine Matrix Rebuilder InvisaSkin-64 Aldenine, Trylagen Decorinyl, Trylagen Eyeseryl Leuphasyl Argireline SNAP-8 Serilesine Syn-coll Syn-ake

Undisclosed HGF Thymopoieten MSH agonist MSH antagonist Innate immunity Innate immunity Human serum Decorin Undisclosed Undisclosed SNAP-25 SNAP-25 Laminin Thrombospondin I Waglerin 1

MMP inhibitor via TIMP Botox-like via acetycholine receptor Wound healing Lymph drainage via ACE inhibition Collagen synthesis via signalling Elasticity via IL6 reduction

Pepha-timp Vialox Brand example Neova Eyeliss Eyeliss, Matrixyl 3000 Matrixyl 3000, Rigin

TIMP-2 Undisclosed Human serum Rapeseed Human serum IgG/matrikine

Collagen stimulation via signalling Retinoic acid-like activity Increases collagen and HA

Matrixyl Biopeptide-CL Biopeptide-EL

Procollagen Collagen Elastin

Pentapharm Pentapharm Procyte Sederma Sederma Sederma Sederma Sederma Sederma

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).

peptide-specific and marketers of such ingredients need to have a strategy to create well validated and characterized peptide based active ingredients. This contribution outlines the role of peptides in nature in relation to skin care end points, the activities of such peptides, and their application to the cosmeceutical industry.

Bioactive peptides Generation and maintenance of dermal integrity Skin is comprised of three broad layers: the epidermis, the dermis, and the hypodermis.1 The epidermis and dermis are separated by a basement membrane that is rich in extracellular matrix (ECM) proteins, including collagens, epilugrin, laminin, fibronectin, elastins, nidogen, and heparin sulfate proteoglycans.2 In addition to anchoring cells and organs together, the ECM serves as a mediator of receptorinduced interactions between cells. In cell proliferation and

morphogenesis, these interactions guide the growth and differentiation of cells.3 The dermis provides a supporting matrix, which includes collagen and elastin, for extensive vascular and nerve networks. The largest class of fibrous ECM molecules is the collagen family, which includes more than 16 different types of collagen. Collagen in the dermal matrix is composed primarily of type I (80% to 85%) and type III (8% to 11%), both of which are fibrillar or rod-shaped.4 The tensile strength of skin is due predominately to these fibrillar collagen molecules, which self-assemble into microfibrils in a head-to-tail and staggered side-to-side lateral arrangement. Collagen molecules become cross-linked to adjacent collagen molecules, creating additional strength and stability in the fibers. Damage to the collagen network (eg, by enzymes or physical destruction) or its total collapse causes healing to take place by repair. The ECM not only provides structural support but also influences cellular behavior such as differentiation and proliferation. Most of these functions are related to signaling by matrix components to cells through cell-

Cosmeceuticals and peptides membrane receptors. Some of the signaling molecules are produced by proteolytic degradation of macromolecules of the ECM that release soluble peptides, termed “matrikines.”5

Matrikines Two classes of matrikines have been characterized: natural matrikines signal directly from the extracellular milieu, and cryptic matrikines (or matricryptins) require proteolytic processing to reveal the ligand or to release the ligand from its ECM protein.6,7 Matrikines and matricryptins are classes of ligand that have been characterized as subdomains of various ECM proteins capable of signaling to the cell through receptors, such as growth factor receptors. Members of the noncollagenous ECM proteins include laminins, fibronectins, and tenascins. Both tenascin-C and laminin-5 have epidermal growth factor (EGF) like repeats. Select EGF-like repeats of tenascin-C elicit mitogenesis and epidermal growth factor receptor (EGFR) autophosphorylation in an EGFR-dependent manner.8 The EGF-like repeats of laminin-5 also act as cryptic ligands revealed by matrix metalloproteinase-2 (MMP-2) degradation of the surrounding extracellular matrix.9 Other matrikines in collagen, elastin, decorin, and laminin-1 can promote chemotaxis, mitogenesis, and metastasis in conditions such as melanoma.10 Unlike traditional growth factors, these individual matrikine domains seem to possess relatively low binding affinity (high nanomolar or micromolar) and multiple valences.11 A study in 2004 showed ECM components contained domains that can interact with and activate receptors with intrinsic tyrosine kinase activity.11 These receptor tyrosine kinases are strong mediators of the cell response, such as proliferation, migration, differentiation, and dedifferentiation. Fibrillar collagens are synthesized as precursor molecules containing N-terminal and C-terminal propeptides that are cleaved off extracellularly by specific peptidases. Work by a group at the University of Tennessee identifying the activities of small peptide fragments within procollagen I resulted in the identification of KTTKS, a subfragment within the carboxy-terminal propeptide (residues 197-241) capable of stimulating the production of both collagen and fibronectin.12-14 This peptide fragment dramatically augmented ECM production in subconfluent fibroblasts. It also stimulated type I and type III collagens and fibronectin production in a doseand time-dependent manner with no effect on total protein synthesis or on the ratio of secreted proteins to cell-associated proteins.13,14 Sederma brought this peptide to market in a lipidated form under the International Nomenclature of Cosmetic Ingredients name palmitoyl pentapeptide-3 as part of the premix product Matrixyl. The application of this peptide to skin care was made by Dr Karl Lintner of Sederma, and the associated intellectual property was filed in 1999. Within this now published patent is the demonstration that at 50 parts per

487 million (ppm), palmitoyl pentapeptide-3 produces a significant benefit to lines and wrinkles around the eyes compared to vehicle alone control in a randomized study (Table 1). Elastic fibers are an important component of the extracellular matrix and consist of two elements, the microfibrils and matrix elastin, providing elasticity and resilience to tissues that require the ability to deform repetitively and reversibly. Elastin has a unique repeating sequence in the hydrophobic region. VGVAPG1 is a hexapeptide repeated multiple times in human, bovine, and porcine elastin molecules.15-17 Elastase-mediated elastolysis liberates elastin fragments called elastokines, which display a wide range of biologic activities in a number of normal and transformed cells18 and are best known for their chemokine-like activities that are chemotactic for fibroblasts, macrophages, monocytes, and polymorphonuclear neutrophils.19-22 They also promote cell cycle progression and induce release of proteolytic enzymes by stromal and cancer cells.23 Chemotactic sites on the elastin molecule have been identified containing the XGXXPG motif. 24-26 Such XGXXPG sites are also present in multiple copies among fibrillin-1, -2, and -3, fibronectin, laminin, and several tenascins and collagens.7 The best studied elastin-peptide, VGVAPG, is known for its chemotactic activity and MMP up-regulation properties. Elastin-derived peptides stimulated the growth of human skin fibroblasts and accelerated angiogenesis in the chick chorioallantoic membrane in an in vivo model.27 This hexapeptide stimulates pseudotube formation from human vascular and microvascular, endothelial cells in the matrigel and collagen models, as well as cell migration in an in vitro wound healing assay.28 Elastin-derived peptide effects were attributed to up-regulation of pro-MT1-MMP and pro-MMP2 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 is incorporated into products to provide “reconstruction of the dermis” and “to produce chemotaxis for restructuring and repair” (Table 1). The era of the short bioactive peptide being applied to dermatology started with discoveries made in the field of wound healing. The work of Dr Loren Pickart in the 1970s on the discovery and subsequent characterization of the GHK copper-binding peptide isolated from human plasma29-31 led to studies demonstrating clinical benefit in acute and chronic wound healing.32-34 The GHK peptide exhibits a high affinity for copper (II) ions, with which it forms spontaneously a tripeptide-copper complex (GHK-Cu). GHK-Cu was initially described as a growth factor for a variety of differentiated cells.35,36 A 1 Nomenclature for amino acids is by single- and triple-letter codes according to standard designation and written from N-terminus to Cterminus. Unless indicated by “d,” all amino acids are in the l-form. See Appendix.

488 number of other biologic effects have since been reported, and GHK-Cu appears to be a potent activator of wound healing. It is a potent chemotactic agent for monocytes/ macrophages and mast cells.37,38 It stimulates nerve tissue regeneration,39 angiogenesis in vivo,40,41 and stimulates the expression of different components of extracellular matrix both in vitro and in vivo. 31,34,42 When injected into superficial wounds, GHK-Cu accelerates wound closure.43 Clinical trials showed that treatment with GHK-Cu might significantly improve the healing of skin ulcers in diabetic patients. 44 The copper peptide also enhances wound remodeling by modulating expression of MMPs.45 It has been speculated that at least a proportion of circulating GHK may be derived from the ECM-binding protein SPARC (secreted protein, acidic, rich in cysteine).41 This protein is expressed by endothelial cells during development and tissue remodelling and yields the GHK sequence specifically, upon degradation by proteases such as elastase, stromelysin, trypsin, and subtilisin. These are generally present in situations of matrix turnover and may endow GHK with a matrikine-like role.

Innate immunity Matrikines are created and released as a result of damage or injury that may have been caused by a wide range of factors. The body, however, also has a system targeted as an immediate response to such damage or threat where the trigger for such a response is the threat itself. This system is innate immunity, and peptides contribute significantly to its operation in protection against pathogens and restoration of barrier function.46 Although the skin was formerly considered an inactive physical protective barrier that participates in host immune defense merely by blocking entry of microbial pathogens, it is now apparent that a major function of the skin is to defend the body by rapidly mounting an innate immune response to injury and microbial insult. Resident and infiltrating cells in the skin synthesize and secrete small peptides that exhibit a wide range of bioactivities aimed at restoring barrier function and protecting the body until that function has been restored. Human skin is comprised of two major classes of innate immunity peptide, β-defensins and cathelicidins (LL-37).47 The β-defensins are cysteine-rich peptides of 36 to 42 amino acids and are stabilized by three disulfide bonds. The three best-characterized human β-defensins—human β-defensin (hBD-1), hBD-2, and hBD-3—have been detected in human skin and cultured keratinocytes.48 The only endogenous cathelicidin in humans, hCAP-18/LL-37, is found at high concentrations in its unprocessed form (hCAP-18) in the granules of neutrophils and is processed upon degranulation and release.49 Innate immunity peptides are multieffectors that are capable of recruiting and activating antigen-presenting cells and serve as early warning signals to activate innate and adaptive immune systems. Defensins and LL-37 are both

L. Zhang, T.J. Falla known for their chemotactic role on various cell types and stimulation of cytokines.50 Endogenous LL-37 stimulates wound vascularization and reepithelialization of healing skin and angiogenesis in an animal model.51 The ability of this peptide to close wounds has partly been attributed to an induction of keratinocyte migration through transactivation of the EGFR.52 The immumodulatory activity of LL-37 has been dissected from its antimicrobial activity at the sequence level, and the resulting peptide subunits induced proliferation and migration mediated through EGFR.49,51 Acceleration of would healing has been demonstrated with other non-antimicrobial innate immunity peptides such as HB-107, a derivative of cecropin B.53 This peptide produced a 64% improvement in wound repair compared with scrambled peptide and vehicle controls, an effect comparable to treatment with recombinant human plateletderived growth factor.53 Subunits and subunit analogs of such peptides have been identified that exhibit specific activities that contribute to this wound healing activity. Palmitoyl hexapeptide-6, a peptide designed using an innate immunity peptide template (Grant Industries, Elmwood, NJ) stimulates fibroblast proliferation and scaffolding, collagen synthesis, and cell migration, and is currently marketed for inclusion in antiwrinkle skin care products. The multifunctional nature of innate immunity peptides and their fragments hold potential for a wide range of applications. The sweat protects the epidermal surface by secreted dermcidin and its natural proteolytic derivatives,54 and the upper epidermis of normal human skin expresses hBDs and LL-37 in addition to human neutrophil protein released by recruited neutrophils and mast cells.55 After exposure to microbe-derived molecules, monocytes and lymphocytes both stimulate the epidermal expression of hBD-1, hBD-2, and hBD-3 through distinct mechanisms.56 These peptides and their derivatives are broad spectrum and synergistic against Staphylococcus aureus and Escherichia coli in the extracellular milieu.57 Some may perform more effectively in vivo, because different cleavage forms of LL-37 have been found in physiologic conditions and are more active against Candida albicans than the full length LL-37.58 The cathelicidin (cathelicidin-related antimicrobial peptide [CRAMP]) knockout mouse becomes more susceptible to necrotic skin infection caused by group A Streptococcus,59 but transgenic mice expressing porcine PR-39 are more resistant to the same pathogen.60 Since the first observation that patients with atopic dermatitis are relatively deficient in cathelicidin and hBD-2 and demonstrate increased susceptibility to bacterial and viral superinfection of the involved skin, the sweat of the patients was found also to have reduced level of, in addition to LL-37, dermcidins which, as mentioned above, are predominantly secreted into the sweat of healthy skin.61,62 Atopic dermatitis affects 15 million people in the United States and 90% suffer long-term Staphylococcal skin infection. Restoration of innate peptides to normal levels via external application could provide significant benefit.

Cosmeceuticals and peptides Inflammation is often associated with skin conditions that have bacterial involvement, which is partly caused by lipopolysaccahride released from the outer membrane of gram-negative bacteria, and lipotechoic acid released from gram-positive bacteria. Innate immunity peptides such as defensins and LL-37 are well known for binding and neutralizing bacterial debris, including lipopolysaccahride and lipotechoic acid responsible for inflammation, resulting in down-regulation of proinflammatory cytokines.63 Consistent with this, LL-37 has also been shown to modulate the inflammatory response mediated by toll-like receptor.63 Another example comes from granulysin-derived peptides that suppress cytokine release stimulated by Propionibacterium acnes.64 A synthetic peptide designed to bind lipotechoic acid, oligopeptide-10, has been developed for inclusion in anti-acne products (Grant Industries, NJ). Up-regulation of LL-37 has been reported to be a natural protective mechanism associated with ultraviolet (UV) sunlight exposure. Excessive sunlight exposure is an important etiologic factor in the development of acute inflammation characterized by erythema, edema, immunosuppression, skin aging, and cancer development. UVB is a well known risk factor for the development of acute inflammation as well as nonmelanoma skin cancer in the epidermis. The UVB band stimulates expression of hCAP-18 in human skin. Individuals who were exposed to UVA (300 to 400 nm) and UVB (280 to 315 nm) exhibited an increase in the amount of both hCAP-18 mRNA and vitamin D receptor in their skin.65 Topical administration of a vitamin D analog (calcipotriol) onto the skin of volunteers increased the expression of fully processed LL-37 peptide within 24 hours of administration.65 Low doses of UVB appear to coordinately suppress keratinocyte proliferation and stimulate cellular maturation as well as certain arms of the adaptive immune system.66 When sunlight slows the adaptive immune system within the skin, the innate defense system kicks in. Functional deficits in the defense of the skin against microbes caused by suppression of local adaptive immunity could be compensated for by stimulation of antimicrobial peptide expression. This provided a rationale for potential application of peptides such as LL-37 among certain ethnic groups that are prone to sunburn to prevent photochemical alterations in both the DNA and membrane lipids of epidermal cells caused by UV radiation.

Melanogenesis UV radiation stimulates melanogenesis by human epidermal melanocytes, both in the skin and in cultured cells.67 The synthesis and distribution of melanin contributes to skin and hair color; however, increased levels of epidermal melanin synthesis can darken the skin and cause cosmetic problems. Melanins in mammalian melanocytes are synthesized within melanosomes and mainly controlled by the expression and activation of tyrosinase. There is evidence that the melanocortin-1 receptor (MC1-R) is a key control point for both constitutive and facultative skin pigmentation. On binding to

489 the MC1-R, melanocortin peptide, α-melanocyte-stimulating hormone (α-MSH) activates adenylate cyclase (cyclic adenosine monophosphate [cAMP]), which in turn causes an increase in intracellular cAMP. Increases in cAMP result, via protein kinase C, in the activation of tyrosinase, which was often reviewed as the classic pathway.68,69 The use of analogs of α-MSH that function as MC1-R agonists has been attempted for potential topical agents to prevent skin photocarcinogenesis. Tetrapeptide α-MSH analogs, Ac-His-D-Phe-Arg-Trp-NH2, N-pentadecanoyland 4-phenylbutyryl-His-d-Phe-Arg-Trp-NH2, have been shown on cultured human melanocytes to be more potent than α-MSH in stimulating the activity of melanogenesis, reducing apoptosis and release of hydrogen peroxide, and enhancing repair of DNA photoproducts in melanocytes exposed to UV radiation.70 Skin keratinocytes also express factors that are involved in melanogenesis. Keratinocyte protease-activated receptor 2 (PAR-2) has been shown to affect melanosome transfer from melanocyte to keratinocyte. The PAR-2 activating peptide SLIGRL enhanced melanosome ingestion by keratinocytes, thus increasing pigmentation.71 In contrast, human homolog of agouti-signaling protein blocked the binding of α-MSH to the MC1-R and inhibited the effects of α-MSH on human melanocytes.72 Treatment of human melanocytes with recombinant mouse or human agouti-signaling protein blocked the stimulatory effects of α-MSH on cAMP accumulation, tyrosinase activity, and cell proliferation.72 Melanogenesis is regulated by many factors. In melanocytes, protein kinase C-β (PKC-β) was shown to be associated with melanosomes and regulate human melanogenesis through activation of tyrosinase.73 Depletion of PKC by chronic treatment of cells with phorbol esters markedly reduces the basal melanin level and tyrosinase activity in human melanocytes and murine S91 melanoma cells.73,74 Sequence analysis of a complimentary DNA clone for human tyrosinase revealed the phosphorylation site for PKC-β within an 11-amino-acid sequence located to the C-terminus of tyrosinase.75 This 11mer tyrosinase mimetic peptide has been shown to compete for PKC-β binding, resulting in inhibition of phosphorylation of tyrosinase by PKC-β.76 These findings support the notion that peptide compounds that inhibit PKC-β might lead to the development of cosmeceuticals for potential skin and hair lightening. The search for tyrosinase inhibitors is another path that has lead to numerous chemical compounds with potential inhibitory activities. Very few peptides, however, meet such a demand due to the nature of tyrosinase. The early finding of cyclic peptide, cyclo(Pro-Val-Pro-Tyr), although an effective tyrosinase inhibitor, has not been proceeded to any clinical study due to the difficulty in large scale synthesis.77 Among many chemicals, kojic acid is the most effective inhibitor of human tyrosinase and has been already incorporated into many cosmetic products. Attachment of kojic acid to peptides improved the effectiveness of tyrosinase inhibitory activity of kojic acid. The best

490 compound, kojic acid-FWY, exhibited 100-fold tyrosinase inhibitory activity compared with kojic acid alone.78 In addition, storage stability was approximately 15 times higher and toxicity was lower than that of kojic acid.78,79

Acetylcholine transmission There is increasing evidence that the cutaneous nervous system modulates physiologic and pathophysiologic effects, including cell growth and differentiation, immunity and inflammation, and tissue repair. Cutaneous nerve fibers and inflammatory cells are both able to release neuromediators and thereby activate specific receptors on target cells in the skin or transient immunocompetent cells. Endocytosis and exocytosis, the major transport system of these neurotransmitters, involve tightly orchestrated mechanisms ensuring trafficking of numerous molecules and particles through the formation of membrane carriers, often vesicles.80 Membrane fusion between vesicles and target membranes, a critical step in these processes, is mediated by a large number of so-called soluble N-ethylmaleimide sensitive factor attachment protein (SNAP) receptor (SNARE) proteins.81 The SNARE protein complex is involved in synaptic vesicle exocytosis from many types of neurons. Cutaneous neuromediators include classic neurotransmitters such as catecholamines and acetylcholine being released from the automatic nervous system or cutaneous cells.82 Acetylcholine can be stored in both small and large synaptic vesicles and contribute to vasodilator responses over a wide range of stimulation frequencies. Botulinum neurotoxins (Botox) cause muscle paralysis by blocking acetylcholine release at nerve-muscle junctions through a very specific and exclusive endopeptidase activity against SNAP-25 of the presynaptic exocytosis machinery. The toxins bind to acceptor sites on the neuronal membrane, are taken up into the cell by a calcium- and pH-dependent translocation process, and cleave specific SNARE proteins in a temperature- and zincdependent manner.83 Treatment with botulinum neurotoxin has to be under strict medical control because it is the most potent toxin known to mankind. To circumvent this limitation, small peptide molecules that mimic the action of Botox are attracting an increasing amount of attention. In this regard, synthetic peptides that emulate the amino acid sequence of the synaptic protein SNAP-25 were shown to be specific inhibitors of neurosecretion at micromolar concentrations. To facilitate membrane permeability, new sequences that are shorter, while preserving a biologic activity, have been pursued. A 6-mer peptide (Ac-EEMQRR-NH2), derived from the N-terminal domain of SNAP-25 (aa 12-17), interferes with the assembly of the SNARE ternary complex and inhibits Ca2+-dependent catecholamine release from chromaffin cells (Table 1). This hexapeptide (acetyl hexapeptide-3) was marketed under the name of Argireline (Lipotec, Barcelona, Spain). Although the evidence to support the benefits of Argireline is slim, a clinical study84

L. Zhang, T.J. Falla suggested a 10% concentration of acetyl hexapeptide-3 reduces the depth of wrinkles up to 30% after 30 days of use.84 These findings suggest that the hexapeptide is a biosafe cosmetic alternative to attenuate facial wrinkles and it is feasible to follow a rational strategy to identify peptidebased mimetics of toxin effects. Another source of such bioactive peptides is the toxins from venomous organisms that disrupt neuromuscular communication to paralyze their prey. Waglerin-1 from the venom of Wagler's pit viper (Tropidolaemus wagleri) is a 22amino acid peptide selective for the adult form of nicotinic acetylcholine receptors (nAChRs) and causes paralysis by competitively antagonizing muscle nAChRs.85 A synthetic tripeptide that mimics the effect of Waglerin-1 has recently been marketed as SYN-AKE (Pentapharm, Basel, Switzerland) for reducing wrinkles by inhibiting muscle contractions. Acting at the postsynaptic membrane, SYN-AKE is a reversible antagonist of the muscular nAChR. It was assumed that this peptide binds to the epsilon subunit of the muscular nAChR, which prevents binding of acetylcholine to the receptor. More products based on peptides with such neurotransmitter blocking activity are expected to affect the cosmeceutical industry in the next few years.

Inflammation The cosmetics industry generally assumes that inflammation has a negative effect on the condition and appearance of skin. Dehydroepiandrosterone (DHEA), a secretary product of the human adrenal gland, has been characterized as exhibiting a wide array of therapeutic benefits, including slowing of the aging process.86 Although DHEA levels decline in the elderly, the supplementation of DHEA resulting in benefit in this population remains to be proven in humans. Among the properties exhibited by DHEA are acceleration of wound healing and the reduction of interleukin-6 (IL-6) levels in inflamed cells.87,88 The tetin class of peptides, first described in the 1990s, are fragments of immunoglobulins, interferons, ILs, or growth factors that modulate cytokine levels.89 A subgroup of this family, the rigins, derived from immunoglobulin G, also has been shown to down-regulate IL-6.90 One such peptide, palmitoyl tetrapeptide-7 (Pal-GQPR) has been developed as an active ingredient by Sederma and marketed as RIGIN. The ability of RIGIN to down-regulate IL-6 in resting and inflamed cells was compared with DHEA in vitro, and the two actives were comparable. Marketing materials related to RIGIN indicate that this reduction in IL-6 can produce increased skin firmness, smoothness, and elasticity.

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

Cosmeceuticals and peptides inhibitors is valine-tryptophan (VW), which has been shown to reduce blood pressure when delivered orally.93 ACE inhibitors act by preventing ACE from converting angiotensin I to angiotensin II, a potent vasoconstrictor, and by preventing the enzyme from inactivating the vasodilator bradykinin. The peptide VW has been marketed as dipeptide-2 (or Dipeptide VW) by Sederma. Under the intellectual property protecting this and related peptides for skin care use, it can only be used in combination with a second peptide and hesperidin, a compound that protects vasculature, is anti-inflammatory, and is an antioxidant.94,95 The premix is marketed in such a combination as Eyeliss, in which the second peptide is palmitoyl tetrapeptide-7 (see Inflammation).

Application of activities to cosmeceutical products An aging population is seeking an ever increasing breadth of bioactivity and increased potency from the ingredients in skin care products. Consumers also have an ever increasing need to understand the theory of the technology and activity, if not the details. This has led to a more science-based focus on active ingredients. Activities that result in diminished lines and wrinkles, smoother skin texture, and a reduction in redness and skin discoloration are the key target end points. Providing this functionality are antioxidants, growth factors, peptides, anti-inflammatories, polysaccharides, and agents to lighten pigments. As has been described, the role of peptides in a wide range of processes is becoming more fully understood and the potential application of such activities more fully appreciated. To maintain that activity in a product and subsequently translate that activity into a beneficial effect for the consumer creates issues that also need to be addressed. The compatibility and stability of a bioactive peptide within a cosmetic formulation can provide significant clues about whether it can be delivered in an active form. Binding, particularly of charged peptides, by other ingredients may prevent release from the formulation or prevent the peptide being released in an active form. If the desired bioactivity can be demonstrated by the formulated peptide or, by the use of a Franz Cell, demonstrated to be released from the formulation in an active form, a level of comfort can be achieved. Human skin has unique properties, of which functioning as a physicochemical barrier is one of the most important. The traditional assumption has been that molecules exceeding 500 mw are unable to traverse the barriers of the skin and, in particular, the stratum corneum.96 More recent studies have 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 penetration enhancers, either peptide or chemical in origin, larger and larger molecules are being transported.99,100 The application of bioactive peptides to the cosmetics industry holds great promise due to the wide range of

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

Appendix A. Amino acid symbols Amino acid

Alanine Arginine Asparagine Asparagine or aspartic acid Aspartic acid Cysteine Glutamic acid Glutamine Glutamine or glutamic acid Glycine Histidine Isoleucine Leucine Leucine or Isoleucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Unspecified or unknown Valine

Symbol 3 Letter

1 Letter

Ala Arg Asn Asx Asp Cys Glu Gln Glx Gly His Ile Leu Xle Lys Met Phe Pro Ser Thr Trp Tyr Xaa Val

A R N B D C E Q Z G H I L J K M F P S T W Y X V

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