Understanding Cosmeceutical Peptides

COSMECEUTICAL PEPTIDES

JENNIFER LINDER, MD PCA SKIN®,6710 E. Camelback Rd, Suite 220, Scottsdale, AZ 85251, USA

Jennifer Linder Understanding cosmeceutical peptides

KEYWORDS: peptides; cosmeceuticals; palmitoyl tripeptide-38; palmitoyl tripeptide-5; wrinkling; laxity; photodamage; textural variances; hyperpigmentation

The development in recent years of novel synthetic cosmeceutical peptides has led to significant Abstractadvances in the clinical management of aging skin. Thanks to the outstanding collagen building capacity of some of these peptides, this category of compounds is rapidly becoming the cornerstone of the skin care industry, enabling the formulation of high-quality topical products that diminish lines and wrinkles, smooth texture, attenuate laxity, and reduce discoloration. Two agents, in particular, have revolutionized anti-aging skin care: palmitoyl tripeptide-38 and palmitoyl tripeptide-5. This article reviews the science behind peptides, the research supporting their use, and key factors to ensure the efficacy of peptide-based formulations and achieve optimal results.

INTRODUCTION ability to reduce free radical formation; and they can improve the functioning of the lipid barrier, reduce pore size, smooth texture, With age, the skin undergoes changes that affect the integrity of and brighten skin tone (7). For all these reasons, cosmeceuticals its support structure and the ability of its cells to communicate, have a wide range of applications in skin care, and are largely transfer nutrients, regenerate, and repair. These changes include used in formulations aimed at reducing the visible signs of aging – progressive thinning of the epidermis, a more compact stratum lines, wrinkles, laxity, coarsening, and discoloration (7). corneum, and reduced granular layer thickness (1, 2). There are six main categories of cosmeceuticals: antioxidants, The dermoepidermal junction fl attens, due to the retraction of growth factors, anti-infl ammatory agents, polysaccharides, the epidermal papillae as well as microprojections of basal cells pigment-lightening agents and peptides (6, 8). Antioxidants into the dermis (3, 2). protect against free radical-induced oxidative stress, which The production of collagen gradually diminishes (4). As a result is a major cause of photodamage, infl ammation and of these changes, the skin becomes thinner, lax, and less elastic, carcinogenesis (9, 10). Other benefi ts associated with their use which leads to the development of bags, lines, wrinkles, and include improved skin fi broblast synthesis, water retention and hyperpigmentation. cell renewal. Some of the most effective antioxidants are retinol

Facial wrinkles are typically the fi rst visible signs of aging – (vitamin A), niacinamide (vitamin B3), ascorbic acid (vitamin C), although factors other than senescence contribute to their tocopherol (vitamin E), ubiquinone and fl avonoids (e.g., grape appearance, including sun damage and muscle movement. seed and green tea extracts) (6). Growth factors have an active As the aging population is growing and set to rise in the coming role in dermal matrix wound healing. decades, the demand for products proven to attenuate and Additionally, fi ndings from clinical studies indicate that some prevent these and other visible manifestations of aging is steadily of these compounds may also have the ability to stimulate climbing. collagen production (6). Anti-infl ammatory agents inhibit the A testament to this phenomenon is the consistent increase in release of proinfl ammatory cytokines by blocking the activity revenue performed by premium anti-aging products. In 2011 this of the enzymes cyclo-oxygenase and lipo-oxygenase. Among market grew, despite the recession, by an impressive 14 percent, others, they include licochalcone A, derived from liquorice, reaching $3.3 billion (5). and silymarin, found in milk thistle. (6). Polysaccharides (e.g., Such increased demand and market growth have driven glycolic acid, lactic acid, gluconolactone) are known for their manufacturers in the skin care industry to develop new humectant, moisturizing and exfoliating properties (6). Pigment- advanced formulations and ingredients, within a group of lightening agents aid in fading hyperpigmentation and unifying topical products collectively known as cosmeceuticals. skin tone. Among the most frequently used is hydroquinone, which inhibits melanin synthesis by blocking the activity of the enzyme tyrosinase (6). Finally, we have peptides which consist COSMECEUTICALS of short amino acid sequences that are subunits of larger protein molecules. They are designed to interfere with some of The term “cosmeceutical” defi nes any bioactive agent that the most important processes responsible for skin aging and its exerts both cosmetic and pharmaceutical therapeutic effects visible manifestations. For example, they can increase collagen on the skin (6). Cosmeceuticals have been associated with many production, inhibit repeated muscle movements, and stimulate benefi ts: they help prevent ultraviolet (UV) damage, due their wound repair (6).

H&PC Today - Household and Personal Care Today, Vol. 9 nr. 1 January/February 2014 23 PEPTIDE CHARACTERISTICS AND TYPES therine-lysine-serine (KTTPS) increases the production of collagen and fibronectin (22). Further, in combination with palmitic acid, Similarl to proteins, peptides are organic compounds made of it has recently been reported to attenuate facial wrinkles, chains of amino acids connected by peptide bonds. However, in both in vitro skin models and clinical patient cohorts (23). they differ from proteins in that they are markedly smaller. They Available signal peptides also include palmitoyl tetrapeptide-7, consist of two, three, a few (3 to 10) or many amino acids, and dipeptide-2, and the complex palmitoyl oligopeptide–palmitoyl are referred to as dipeptides, tripeptides, oligopeptides and tetrapeptide-7 (Matrixyl 3000), which have been associated polypeptides, respectively (11). Proteins are typically made of with improved skin firmness and elasticity, enhanced toxin one or more polypeptides. elimination, and increased ECM synthesis, respectively (24). There is a characteristic resemblance between the amino Some signal peptides have demonstrated anti-collagenase acid sequences of peptides and those contained in larger activity and, consequently, have the potential to reduce proteins like collagen. This has led to the idea that peptides collagen breakdown (25). Finally, palmitoyl tripeptide-38 and could increase the production of this particular component of palmitoyl tripeptide-5 have been shown to be highly effective at the extracellular matrix (ECM) and, consequently, mitigate the stimulating the production of collagen and hyaluronic acid, and visible signs of aging associated with collagen depletion (12). promoting dermal thickening (24). Indeed, studies have confirmed that peptides have excellent collagen building properties (13), which places them among the most groundbreaking anti-aging ingredients available. Although NEXT-GENERATION AGENTS they are not tested in the same way as most mainstream FDA- regulated compounds (14), the evidence supporting their While cosmeceutical peptides in general are considered a benefits is increasing, making their use in skin care extremely major advance in the clinical management of aging skin, desirable. Three main types of cosmeceutical peptides are arguably the most revolutionary are the newest synthetic signal available: carrier peptides, neurotransmitter-affecting peptides, sequences palmitoyl tripeptide-38 and palmitoyl tripeptide-5. and signal peptides (14). The research to date suggests that these compounds are among the most scientifically advanced ingredients for use in Carrier Peptides high-performance skin care formulations that aim at promoting Carrier peptides play a key role in delivering essential metals the key processes that lead to healthier, younger-looking skin. to the skin, particularly copper, an important cofactor in the biosynthesis of collagen and elastin via activation of Palmitoyl Tripeptide-38 lysyl oxidase, downregulation of matrix metalloproteinases Palmitoyl tripeptide-38 (Matrixyl synthe’6) is a matrikine-mimetic (MMPs) and anti-collagenase activity (6, 14). The tripeptide compound with significant ECM building and strengthening glycyl-L-histidyl-L-lysine (GHK) spontaneously binds copper properties, that regulates cell activity, wound repair, and to form a copper-tripeptide complex, which facilitates the collagen tissue remodeling (26). As a matrikine mimetic, uptake of the metal at the cellular level. This, in turn, has been palmitoyl tripeptide-38 activates signaling pathways that initiate shown to stimulate collagen synthesis, resulting in reduced the cascade of biochemical processes involved in the synthesis wrinkles, increased skin firmness, and improved texture (14, of the following key constituents of the ECM (26, 13): 15). Notably, copper tripeptide also increases levels of MMP- -- Together with type II and III, this protein accounts for 80 to 2 and MMP-2 messenger RNA, as well as the secretion of the 90 percent of the total amount of collagen in the human metalloproteinase inhibitors TIMP-1 and TIMP-2, which suggests a body. It is the major component of the dermis and is role in collagen remodeling (14, 16). Another carrier peptide with typically found in the skin, tendons, and bone ligaments, MMP synthesis suppressing activity used in skin care formulations among other tissues (27). is dipalmitoyl hydroxyproline (17). -- Often referred to as “collagen of youth” because of its ability to enhance skin smoothness and elasticity, this type Neurotransmitter-Affecting Peptides of collagen is produced by immature mesenchymal cells in The hexapeptide sequence acetyl-glutamyl-glutamyl- the earliest phases of wound healing, before type I collagen methyoxyl-glutamynyl-arginyl-arginyl-arginylamide (acetyl- synthesis initiation (28). It is mostly found in elastic tissues, hexapeptide-3 or Argireline) is a well-known member of the including the skin, muscles, and blood vessels (27). The neurotransmitter-affecting group. Its main clinical effect is to amount of type III collagen in the skin diminishes with age in reduce the appearance of lines and wrinkles caused by the favor of collagen I (29). repeated movement of facial muscles over time (14). There -- Collagen IV. This is the primary component of the is evidence that the mechanism of action is related to the extracellular basement membrane that separates epithelial inhibition of calcium-dependent release of acetylcholine at and endothelial cells, and of the dermoepidermal the neuromuscular junction, via interference with the soluble junction (30). It has important wound healing stimulation N-ethylmaleimide-sensitive factor attachment protein receptor properties (30). These result from its capacity to interact with (SNARE) complex (18, 19). keratinocytes, which induce the secretion of granulocyte- macrophage colony stimulating factor (GM-CSF) and, Signal Peptides consequently, epidermal cell proliferation immediately after Signal peptides are involved in the repair mechanisms of human a skin injury (31). dermal fibroblasts following skin injury. One such compound -- Fibronectin. A non-collagenous glycoprotein relatively is the elastin-derived palmitoyl oligopeptide. A combination abundant in the ECM (32), fibronectin has been shown of palmitic acid and the hexapeptide valine-glycine-valine- to increase in vivo activity levels of bone morphogenetic alanine-proline-glycine (VGVAPG), palmitoyl oligopeptide has protein-1-like proteinases, which play a critical role demonstrated the ability to stimulate fibroblast synthesis and in ECM production (32). The proline-histidine-serine- proliferation (21). The pentapeptide sequence lysine-therine- arginine-asparagine (PHSRN) sequence contained in this

24 H&PC Today - Household and Personal Care Today, Vol. 9 nr. 1 January/February 2014 glycoprotein regulates fibroblast adhesion, migration, Palmitoyl Tripeptide-5 growth, and differentiation (33). It also appears to have Palmitoyl tripeptide-5 mimics the action of thrombospondin 1 a role in the stimulation and acceleration of in vivo tissue (TSP-1), a multifunctional adhesive glycoprotein of the ECM (43, repair (34). 4) that stimulates the production of type I and II collagens, and -- Hyaluronic acid. Also referred to as hyaluronan or HA, fibronectin via the activation of tissue growth factor β (TGF-β) hyaluronic acid is a highly versatile component of the ECM (44, 4). For this reason, palmitoyl tripeptide-5 is considered a with excellent bio-compatibility, non-immunogenicity, key collagen building ingredient for anti-aging skin formulations biodegradability, and viscoelasticity (35). Known to facilitate aiming at attenuating lines and wrinkles, and correcting laxity. wound repair, hyaluronic acid is thought to improve skin The collagen building properties of palmitoyl tripeptide-5 were moisture and elasticity. Additionally, it has been associated originally observed in in vitro studies of human fibroblasts. These with long-lasting smoothing effects and facial wrinkle found that fibroblast cultures treated with the novel tripeptide reduction due to its capacity to attract and retain water showed an increase in collagen synthesis of 119 percent, and fill space (17, 35). Hyaluronic acid has a demonstrated compared with cultures treated with standard TGF-β (4). In a ability to facilitate the delivery of active ingredients to clinical study, 60 volunteers received twice daily applications of the inner layers of the skin, enabling them to exert their a product containing 2.5 percent palmitoyl tripeptide-5. After 84 beneficial effects from the inside (35). Its free radical days, the study participants showed significant visible reductions scavenging properties indicate that it may also have a role in average skin roughness and wrinkle depth (45). The skin in UV protection (35). smoothing effects of the tripeptide were confirmed in another -- Laminin-5. Alongside collagens and proteoglycans, laminins clinical study that included 37 volunteers with mild to moderate are important components of the basement membrane. fine and coarse periocular and perioral wrinkles (17). Twice daily Laminin-5 is an adhesion glycoprotein specific to epithelial applications of a palmitoyl tripeptide-5-containing formulation tissues where it complexes with hemidesmosomes, thus on the affected areas significantly reduced (p<0003) mean contributing to the mechanical processes that anchor wrinkle severity by more than 50 percent. Visible improvements the epidermis to the dermis (36, 37). It also regulates were observed within as little as 15 minutes from the first keratinocyte migration, facilitating the healing of injured application and peaked at three months – the duration of the epidermis (38). treatment. In both clinical studies, palmitoyl tripeptide-5 was -- Heat shock protein 47 (HSP47). One of the glycoproteins found to be safe and well tolerated, with no major side effects produced in the endoplasmic reticulum and Golgi (17, 45). apparatus of collagen-producing cells (39), the expression of HSP47 is upregulated in stress conditions such as exposure to extreme temperatures, heavy metals and oxidative ACHIEVING OPTIMAL PERFORMANCE stress (39). HSP47 is an essential player in the biosynthesis of collagen (40). It has a well-documented role of “molecular While strong collagen building properties make newer peptides chaperone” in the dissociation of newly-produced essential ingredients of modern anti-aging skin care formulations, procollagen molecules (41) and their subsequent folding a number of key factors need to be considered to ensure and assembling after injury (42). maximum efficacy and safety. Using the correct amounts of Quantitative measures of the stimulatory activity of palmitoyl peptide is one such factor. For example, the recommended use tripeptide-38 on the above ECM components – type I, III, and level is 10 percent for Argireline (24), two percent for palmitoyl IV collagens; fibronectin; hyaluronic acid; laminin-5; and HSP47 tripeptide-38, and one to three percent for palmitoyl tripeptide-5 – have been performed in in vitro tests (13) that examined the (13, 45). Practicing dermatologists should proactively seek effects of twice daily applications of a formulation containing this information from manufacturers, or refer to the product two percent Matrixyl synthe’6 on skin explants for five days. They monograph of the specific peptide they are interested in using. found that type I, III, and IV collagens significantly increased A further important factor to consider is that, although peptides (p<0.01) by 105, 104, and 42 percent, respectively (13). Hyaluronic have intrinsic efficacy, this can be enhanced to achieve optimal acid, HSP47, laminin-5, and fibronectin also increased significantly performance by adding other cosmeceuticals to formulations; (p<0.01), by 174, 123, 75, and 59 percent, respectively. Other in antioxidants are especially important in this regard. As described vitro analyses found significant increases (p<0.01) of 20 and 37.5 earlier, antioxidants can attenuate and prevent the negative percent in type I collagen synthesis in non-aged skin explants and effects of oxidative stress (9, 10), which is not only a cause of experimentally aged ones (29). premature skin aging and disease, it can also damage the Importantly, the above effects translate into visible improvements active ingredients that may be present in a formulation. Hence, on the skin’s surface. Of particular significance in this regard are the addition of antioxidants helps ensure that the peptide will the results of a placebo-controlled clinical study of 25 women actually work, while minimizing the risk of photodamage to aged 42 to 70 years. In this study, a formulation containing two the skin. Examples of compounds with these properties are percent palmitoyl tripeptide-38 was applied twice daily for two grape seed extract and epigallocatechin gallate (EGCG), months on two facial skin areas especially prone to wrinkling: a key polyphenol contained in green tea leaves. Both have the forehead and the crow’s feet by the eyes. In the former, demonstrated potent protective effects against oxidative stress wrinkle volume and depth decreased by 31 and 16.3 percent, (46, 47). In addition, there is some evidence that EGCG can respectively, whereas lifting improved by 28 percent. In the latter, increase dermal thickness by inducing keratinocyte proliferation, wrinkle surface, volume, and maximum depth diminished by whereas grape seed extract can promote dermal wound 28.5, 21.1, and 15 percent, respectively; lifting improved by 12.6 repair via the stimulation of vascular endothelial growth factor percent. It is worth noting that the reductions in wrinkle depth expression (6). observed in both facial skin areas are especially important, given Several other antioxidants are considered important co- that this parameter is the most significant factor influencing the ingredients of peptide-containing formulations. For example, visual perception of wrinkles (13). retinol has been associated with improved water retention,

H&PC Today - Household and Personal Care Today, Vol. 9 nr. 1 January/February 2014 25 cell renewal, and fibroblast synthesis, as well as reduced 9. Masaki H., Journal of Dermatological Science, 58, 85-90 (2010). collagen breakdown, resulting in fewer lines and wrinkles (6). 10. Rahman K., Clinical Interventions in Aging, 2, 219-236 (2007). The antioxidant niacinamide reduces transepidermal water loss 11. Voet D., and Voet J., Biochemistry, Wiley (1995). (TEWL), increases collagen production, and inhibits oxidative 12. Alam M., Gladstone H.B., et al., Cosmetic Dermatology, Saunders (2009). glycation of proteins. Overall, these effects contribute to 13. Matrixyl synthe’6 Sederma, Product Monograph (2010). improved skin tone and texture, reduced lines and wrinkles, and 14. Lupo M.P., and Cole A.L., Dermatologic Therapy, 20, 343-349 diminished yellowing of the skin (Maillard reaction) (6). Ascorbic (2007). acid promotes wound repair and fibroblast regeneration, 15. Lintner K., in Draelos Z.D., Cosmetic Dermatology, Blackwell prevents inflammation, and is thought to have skin lightening Publishing (2010). properties (6). Tocopherol provides photoprotective effects and 16. Siméon A., Emonard H., et al., Life Sciences, 67, 2257-2265 (2000). acts as a humectant (6). 17. Trookman N.S., Rizer R.L., et al., The Journal of Clinical and Aesthetic Another key ingredient is calcium hydroxymethionine Dermatology, 2, 38-43 (2009). (hydroxymethionine-Ca). A natural analogue of methionine, 18. Blanes-Mira C., Clemente J., et al., International Journal of this compound can chelate calcium, acquiring the ability to Cosmetic Science, 24, 303-310 (2002). stimulate keratinocyte proliferation, which plays a key role in 19. Blanes-Mira C., Merino J.M., et al., Journal of Neurochemistry, 88, 124-135 (2004). maintaining epidermal cell cohesion (48). Since the proliferation 20. Morrissette G., Germain L., et al., British Journal of Dermatology, and turnover of keratinocytes naturally diminishes with age 156, 433-439 (2007). (49), hydroxymethionine-Ca represents a desirable addition to 21. Kamoun A., Landeau J.M., et al., Cell Adhesion and anti-aging products, increasing firmness and consequently the Communication, 3, 273-81 (1995). appearance of loose skin. 22. Katayama K., Armendariz-Borunda J., et al., The Journal of Biological Chemistry, 268, 9941-9944 (1993). 23. Mondon P., Doridot E., et al., Reduced Appearance of Facial CONCLUSION Wrinkles with PAL-KTTKS peptide containing moisturizer, http://bit. ly/1gcavHl (Accessed 12/02/2013). Cosmeceutical peptides have become increasingly popular 24. Linder J., Plastic Surgical Nursing, 32, 71-72 (2012). 25. Njieha F.K., Morikawa T., et al., Biochemistry, 21, 757-764 (1982). in dermatology in recent years. They combine strong collagen 26. Matrixyl synthe’6 Sederma, Customer Information, http://bit. building properties with the ability to reduce repeated ly/1f9a7gl (Accessed 12/02/2013). muscle movements and promote wound repair, conferring 27. Lodish H., Berk A., et al., Molecular Cell Biology, 4th Ed., New York: excellent performance to skin formulations aimed at mitigating W.H. Freeman (2000). the visible signs of aging. Two newer peptides in particular 28. Gay S., Vijanto J., et al., Acta Chirurgica Scandinavica, 144, 205- have been associated with significant benefits in studies: 2011 (1978). palmitoyl tripeptide-38 and palmitoyl tripeptide-5. Both are 29. No author listed. Personal Care (2011) http://bit.ly/198FrZN considered essential agents for the production of collagen, (Accessed 12/03/2013). a key component of the ECM. As such, they can assist in 30. Abreu-Velez A.M., and Howard M.S. North America Journal of maintaining and improving the natural elasticity, thickness, and medical Sciences, 4, 1-8 (2012). smoothness of the skin; reduce the appearance of lines and 31. Mann A., Breuhahn K., et al., The Journal of Investigative Dermatology, 117, 1382-1390 (2001). wrinkles; and promote wound repair and regeneration after 32. Huang G., Zhang Y growth., et al., The Journal of Biological injury. These effects are enhanced by the remarkable ability Chemistry, 284, 25879-25888 (2009). of palmitoyl tripeptide-38 to stimulate the production of other 33. Yamada K.M., Journal of Cell Science, 115, 3861-3863 (2002). major constituents of the ECM, including fibronectin, hyaluronic 34. Yamada K.M., The Journal of Clinical Investigation, 105, 1507-1509 acid, laminin-5, and HSP47. To ensure efficacy and safety, it is (2000). important to verify that formulations contain the amount of a 35. Brown M.B., and Jones S.A., Journal of The European Academy of particular peptide recommended by the manufacturer. Optimal Dermatology and Venereology, 19, 308-318 (2005). product performance can be achieved by using peptides 36. Baker S.E., Hopkinson S.B., et al., Journal of Cell Science, 109, 2509- in combination with other cosmeceuticals, such as certain 2520 (1996). antioxidants (e.g., grape seed extract, epigallocatechin gallate, 37. Matsui C., Wang C.K., et al., The Journal of Biological Chemistry, 270, 23496-23503 (1995). retinol, and niacinamide) and hydroxymethionine-Ca, among 38. Frank D.E., and Carter W.G. Journal of Cell Science, 117, 1351-1363 others. (2004). 39. Hart D.A., Reno C., et al., Biochemistry and Cell Biology, 78, 511-518 (2000). REFERENCES AND NOTES 40. Nagai N., Hosokawa M., et al., The Journal of Cell Biology, 150, 1499-1506 (2000). 1. Bhawan J., Andersen W., et al., Journal of Cutaneous Pathology, 41. 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