10 SOFW-Journal | 136 | 6-2010

COSMETICSGHK-COPPER PEPT IDE

able to stimulate the synthesis of colla-gen in fibroblasts. The authors notedthat GHK sequence is present in the al-pha 2(I) chain of type I collagen and sug-gested that in the organism GHK mightbe liberated by proteases at the site of awound healing (3).

L. Pickart, A. Margolina*

GHK–Copper Peptide in Skin Remodeling and Anti-Aging

! GHK-Cu in Skin Remodeling

Although copper-binding tri-peptide gly-cyl-L-histidyl-L-lysine (GHK-Cu) was firstdiscovered as an activity that makes oldliver cells behave like young cells, thefollowing studies soon began to focus onits wound healing and skin remodelingactivity. These studies provided valuableinsight into GHK-Cu’s molecular actionsand revealed many mechanisms by whichGHK-Cu exhibits anti-aging and tissuerenewing properties. In particular it hasbeen established that GHK-Cu can in-crease production of key skin proteinssuch as collagen as well as other impor-tant components of dermal matrix. Inaddition it has been proven that GHK-Cumodulates the skin remodeling process,being able to stimulate both breakdownand synthesis of dermal matrix compo-nents. While GHK-Cu is used in moststudies, GHK without copper has been

used in a few instances. But all the avail-able evidence is that GHK exerts its bio-logical effects as its copper complex.In 1988, Maquart et al. from Universitéde Reims Champagne-Ardenne (France)found that GHK-Cu at concentrations of10-12 and 10-11 M, maximized at 10-9M was

Since the dawn of time, people have tried to discover the secret ofeternal youth and find a way to reverse aging. Among the most popu-lar ingredients of the elusive elixir of youth sought by ancient mages

and alchemists were herbs, powdered gems, gold, silver and mercury aswell as animal parts and extracts. The most common belief was that the»essence of youth« can be transferred from young to old people – often ina form of blood or other body fluids. Today it is known that plasma andother body fluids (such as saliva) indeed contain a multitude of solublegrowth factors that can enhance reparative and renewing activity of vari-ous cells and tissues (1). Of course neither blood nor saliva or other fluidsproduced by the human body can be used in cosmetics. Therefore the bio-logical regulators with proven scientific activity that can be purified or pro-duced by chemical synthesis are of particular interest for the cosmetic in-dustry. One of the anti-aging activities that is present in human plasma and salivais a human tri-peptide with an amino acid sequence glycyl-L-histidyl-L-ly-sine and high affinity for copper (II) (Fig. 1), isolated in 1973 by Dr. L. Pickart.First discovered as an activity in human plasma albumin that causes oldhuman liver tissue to synthesize proteins like younger tissue, today GHK-Cuis widely used as a cosmetic ingredient with confirmed anti-aging andreparative properties (2).

Introduction

Fig. 1 The molecular structure of thetri-peptide GHK-Copper

SOFW-Journal | 136 | 6-2010 3

COSMETICSGHK-COPPER PEPT IDE

In 1992 Maquart et al. used the methodof wound chambers to determine andquantify components of extracellularmatrix (ECM) produced during woundhealing in rats. GHK increased an accu-mulation of total ECM proteins, collagenand glycosaminoglycans as well as in-creased DNA synthesis. At the same time,another tri-peptide L-glutamyl-L-histidyl-L-proline had no significant effect, whichconfirmed that the observed stimulationof skin proteins production was a resultof a specific action of GHK-Cu (4). P. Ehrlich first observed that GHK simul-taneously increases both the synthesis ofnew type 1 collagen and the breakdownof existing collagens in wounds (5). In1999 Maquart and co-authors demon-strated for the first time that GHK-Curegulated skin remodeling process in ratsby modulating activity of different met-alloproteinases – enzymes that facilitatebreakdown of proteins of extracellularmatrix (6). In the next series of experiments, it wasshown that GHK-Cu not only modulatesactivity of different matrix metallopro-teinases, but also stimulates anti-pro-teases, maintaining balance betweenmatrix breakdown and synthesis. The ef-fect was reproduced by the addition ofcopper ions, but not by the GHK alone,which suggests that copper binding ac-tivity of GHK is more likely the key mech-anism in its tissue remodeling activity(7). The ability of GHK-Cu to stimulateboth proteases and anti-proteases indi-cates that it can balance the breakdownof ECM proteins, preventing excessiveskin damage.Maquart et al. also demonstrated thatinjection of GHK-Cu (2 mg per injection)into the rat experimental wounds result-ed in the increased production of colla-gen I and glycosaminoglycans (dermatansulfate and chondroitin sulfate). North-ern blot analysis also showed increasedproduction of two small proteoglycansof the dermis – biglycan and decorin. Ac-cording to the mRNA analysis, the levelof decorin continued to increase untilthe end of the GHK treatment (day 22nd).The ability of GHK-Cu to stimulate decorinproduction was also confirmed in fi-broblast culture (8). It was particularly interesting that GHK-Cu increased decorin, since this proteo-

glycan functions by regulating assemblyof collagen fibrils, preventing scar for-mation and decreasing the level of TGF-beta, which is known to increase scarring(9-10). In addition GHK-Cu has beenshown to decrease TGF-beta productionin serum-free fibroblast culture (humancells obtained during facial surgery) (11).This may be explained through the in-creased decorin level, however it is alsopossible that GHK-Cu itself inhibits TGF-beta.Also, GHK-Cu was found to attract im-mune and endothelial cells to the site ofinjury, which is another explanation ofits wound healing promoting activity.The most recent study (2007) of woundhealing activity of GHK-Cu was conduct-ed in Taiwan. Huang et al. evaluated theeffect of GHK alone or in combinationwith LED irradiation (light emitting diodeirradiation, 625-635 nm) on human fi-broblasts. Combined GHK and LED treat-ment resulted in 12.5-fold increase incell viability, 230% increase in basic fi-broblast growth factor (bFGF) produc-tion, and 70% increase in collagen I mR-NA production compared with the LEDirradiation alone (13). Since fibroblastsare the key cells in skin reparative and re-newal processes, the ability of GHK-Cuto support these cells increasing theirfunctional activity may in part explain itswound healing and skin rejuvenating ac-tivity. GHK peptide belongs to a family of bio-logically active regulators that are natu-rally present in extracellular matrix (ECM)and released from it after injury. These»first emergency response« molecules arecalled matrikines (14). In addition to be-ing a part of collagen molecule, GHK se-quence is also found in a glycoproteinSPARC that is produced by endothelialcells in the site of injury. After an injuryGHK released from a SPARC molecule byproteolysis (15). Therefore GHK-Cu is not only naturallypresent in skin, but it is also liberatedfrom skin’s proteins after stress or injury.Considering its ability to stimulate thesynthesis of key skin proteins as well asto increase functionality of skin fibrob-lasts, it should be quite effective as awound healing agent. And indeed, ani-mal experiments fully support this state-ment.

! GHK Improves Wound Healingin Animals

A series of in vivo experiments confirmedwound healing activity of GHK-copper.In rabbits, GHK facilitated wound heal-ing, causing better wound contraction,faster development of granular tissueand improved vessel growth (16). AlsoGHK alone and in combination with highdose helium neon laser stimulated gran-ulation, increased the formation of newblood vessels and elevated the level ofantioxidant enzymes in the dermal woundsin rabbits (17). GHK-Cu was used to create a novel wounddressing – collagen membrane with in-corporated biotinylated GHK. This mate-rial has been shown to significantly im-prove wound healing processes in ratswhen compared to non-treated woundsand to wounds treated with collagenmatrix alone. Collagen dressing with in-corporated GHK stimulated wound con-traction and cell proliferation, as well asincreased expression of antioxidant en-zymes (18). The most important finding was GHK’sability to improve wound healing in dif-ficult to heal wounds, such as diabeticwounds in rats. GHK-Cu treatment re-sulted in faster wound contraction andepithelization, higher level of antioxi-dants such as glutathione and ascorbicacid, increased synthesis of collagen, andactivation of fibroblasts and mast cells(19). Also GHK-Cu improved healing ofischemic open wounds in rats. Woundsdisplayed faster healing, decreased con-centration of metalloproteinases 2 and9 as well as of TNF-alpha (a major in-flammatory cytokine) compared withvehicle alone or with untreated wounds(20). GHK-Cu triggered an acceleratedhealing of the injury after mild thermalburns to the backs of pigs. The rate of theburn wound healing gave a linear, dose-response up to 1% ionic copper in thecream. In pigs, punch biopsies were usedto create wounds. The defects were filledwith graded amounts of GHK-Cu in acream or control substances. In a dosedependent manner, GHK-Cu stimulatedwound healing and collagen synthesis.The effect was highly localized to theimmediate skin area that was treated(21).

4 SOFW-Journal | 136 | 6-2010

COSMETICSGHK-COPPER PEPT IDE

Animal experiments confirmed that GHK-copper has great potential to improveskin conditions. It speeds up skin repair,improves circulation, strengthens an-tioxidant defense, and increases produc-tion of skin proteins. All these qualitiesare highly desirable in cosmetics. Theability of GHK-Cu to improve difficult-to-heal wounds can be helpful in man-aging skin healing after plastic surgery,especially in advanced age patients andin patients with underlying health con-ditions.

! Cosmetic Use of GHK-Cu

Today peptide based cosmetic productscontinue gaining popularity. However,not all peptides that are used in today’scosmetic products have enough scientif-ic data confirming their efficacy. Alsothe main problem with peptide-basedcosmetics is that most peptides cannotpass through stratum corneum andtherefore cannot reach viable layers ofepidermis. Since peptides are used fortheir cell regulatory activity, inability topermeate stratum corneum is a serioushandicap. Mazurowska et al. demonstrated thatGHK-Cu is able to pass through the lipidbarrier of the stratum corneum and toreach epidermal cells (22). Moreover,GHK-Cu complex appears to be the onlypeptide-copper complex that can pene-trate stratum corneum membranes (23). At present, GHK-Cu undoubtedly hasmore scientific support data than anypeptide used in today’s cosmetic practice(24). Its efficacy was confirmed in sever-al placebo-controlled independent trials. A study of 20 women compared theskin's production of collagen after ap-plying creams containing GHK-Cu, vita-min C, or retinoic acid to thighs daily forone month. New collagen productionwas determined by skin biopsy samplesusing immunohistological techniques.After one month, GHK-Cu increased col-lagen in 70% of those treated against50% treated with vitamin C and 40%treated with retinoic acid (25). A GHK-Cu facial cream reduced visiblesigns of aging after 12 weeks of applica-tion to the facial skin of 71 women withmild to advanced signs of photoaging.The cream improved skin laxity, clarity,

and appearance, reduced fine lines andthe depth of wrinkles, and increased skindensity and thickness comparing withplacebo (26). A GHK-Cu eye cream, tested on 41 womenfor twelve weeks with mild to advancedphotodamage, was compared to a place-bo control and an eye cream containingvitamin K. The GHK-Cu cream performedbetter than both controls in terms of re-ducing lines and wrinkles, improvingoverall appearance, and increasing skindensity and thickness (27). In another 12 week facial study of 67women between 50-59 years with mildto advanced photodamage, a GHK-Cucream was applied twice daily and im-proved skin laxity, clarity, firmness andappearance, reduced fine lines, coarsewrinkles and mottled hyperpigmenta-tion, and increased skin density andthickness. The result was assessed visual-ly by a trained technician (wrinkles, pig-mentation, laxity, roughness, overall ap-pearance) as well as using ballistometer(firmness of the skin) and ultrasound(skin density). The GHK-Cu cream alsostrongly stimulated dermal keratinocyteproliferation as determined by histologi -cal analysis of biopsies. At the same time,GHK-containing cream has proved to bevery safe. GHK-Cu complex at 20x uselevel was proved to be non-allergenic. Italso did not produce eye irritation (28).

! Stimulation of Hair Growth

From the very first experiments withGHK-Cu, we observed exceptionally largehair follicles developing at the peripheryof wounds treated with GHK-Cu. Todayit is known that hair follicles are the im-portant source of stem cells that active-ly participate in skin repair and are ca-pable of differentiating into many skincell lineages (29). The hair growth stimulating effect ofGHK-Cu was confirmed in the followingexperiment. A 25 days old C3H mousewas shaved and injected in three spotswith GHK-Cu. According to the radioiso-tope analysis, GHK-Cu was present at theinjection site for about 30 sec, beforeclearing from the area. However, thisbrief exposure was enough to stimulatehair growth. In 12 days there was stronghair growth stimulation at the injectionsite (Fig. 2) (30). In fuzzy rats a copper binding peptidePC1031, a structural analog of GHK(ghkvfv-copper complex) affected hairgrowth with its effect comparable tothat of minoxidil. Both 5% minoxidil and5% PC1031 almost doubled follicle sizeafter 3-4 months of treatment, andcaused 80% increase in the number ofanagen hair follicles. An increased DNAsynthesis and cell proliferation was con-firmed in the enlarged hair follicles (31).

Fig. 2 A 25 day-old mouse was shaved and injected intradermally in three spotswith GHK-Cu. Twelve days later, there was a very strong stimulation of hair growthat the injection sites

SOFW-Journal | 136 | 6-2010 5

COSMETICSGHK-COPPER PEPT IDE

The mechanism of GHK-Cu induced hairgrowth was investigated in experimentswith GHK-Cu analog – AHK (L-alanyl-L-histidyl-L-lysine-Cu2+). It was found thatAHK-Cu stimulated dermal papilla cells(DPCs) – specialized fibroblasts impor-tant in the morphogenesis and growth ofhair follicles. AHK caused elongation ofhair follicles, stimulated proliferation ofDPCs and prevented their apoptosis. Today, analogs of GHK-Cu with hy-drophobic amino-acids are used to en-hance hair growth in the commercialproduct Tricomin (Procyte Corp). Also aproduct GraftCyte (Procyte Corp) is usedto increase success of hair transplantswith clinically confirmed efficiency (33).

! GHK’s Effect on Skin Stem Cells

The ability of GHK-Cu to rejuvenate ag-ing skin and to stimulate hair growth in-dicates its important role in skin and hairrenewal process. Recent studies revealedone possible mechanism of this rejuve-nating action. It has been discovered that

GHK is able to restore proliferative po-tential of adult stem cells residing in skin.In 2009, a group of researchers from theSeoul National University (Republic ofKorea) used multilayered skin equivalent(SE) model to demonstrate that GHK-copper is able to increase proliferativepotential of basal human keratinocytesas well as expression of epidermal stemcell markers.Epidermal stem cells are slow cyclingcells in a basal layer that maintain non-differentiated state through the entirelife time of an organism. They give riseto transient stem cells that in turn dif-ferentiate into keratinocytes. In case ofan injury, epidermal stem cells are ableto differentiate into all types of cellsfound in the skin, including fibroblasts,melanocytes, endothelial cells etc. Although not all characteristics of epi-dermal stem cells are known, their mainfeatures are believed to be a cuboidalshape, high integrin expression and anexpression of p63 protein – a member ofp53 family – that is often used as a stemcell marker. Loss of proliferative poten-

tial of skin stem cells is associated withflattened shape and decreased expres-sion both integrins and p63. Integrins aresurface proteins that play an importantrole in maintaining an attachment ofstem cells to the basement membrane. Itis believed that such attachment is re-quired for maintaining immature, non-committed state of stem cells – their»stemness«. In cell cultures GHK copper (0.1-10 mi-croM) stimulated proliferation of ker-atinocytes in a dose dependent mannerand was not toxic in these concentra-tions. An addition of GHK-copper result-ed in noticeable changes in epidermalbasal cells. Their integrins and p63 ex-pression markedly increased and theirshape became more cuboidal, as is char-acteristic for stem cells. The authors con-cluded that GHK-copper is able to main-tain »stemness« of basal keratinocytes aswell as to revive their proliferative po-tential (34). This discovery is especially important inthe light of the recent findings that sug-gest that age-related decline of the pro-

Fig. 3 GHK-Cu effects on aged skin

6 SOFW-Journal | 136 | 6-2010

liferative capacity of stem cells may bedue to disrupted regulation from theaged environment rather than to stemcells senescence. In contrast to the preva-lent theory, according to which all hu-man cells have limited proliferative ca-pacity of approximately 50 divisions (socalled Hayflick limit), in some renewingtissues stem cells have been shown toundergo more than 1000 divisions with-out showing signs of senescence (35).New findings suggest that epidermalstem cells may be intrinsically aging re-sistant and that local microenvironment,rather than cellular senescence, is respon-sible for their functional decline (36). GHK’s ability to revive proliferative po-tential of epidermal stem cells makes ita powerful player in skin repair and re-juvenation process (Fig 3).

! GHK-Cu Restores Function in Damaged Cells

A recent study showed GHK-Cu's abilityto restore function of irradiated fibrob-lasts to that of intact cells. The re-searchers used cultured human fibrob-lasts obtained from cervical skin that waseither intact or exposed to radioactivetreatment (5000 rad). GHK copper (10-

9M) was added in a serum free mediumdirectly to the cell culture. An equivalentamount of plain serum-free medium wasadded to control cells (37). Although irradiated fibroblasts survivedand replicated in the cell culture, theirgrowth dynamics were markedly differ-ent from that of intact cells. The growthof the irradiated cells was especially de-layed at 24 and 48 hour measurements.However, the irradiated fibroblasts treat-ed with GHK showed much faster growth,similar to the normal (non-irradiatedcontrol cells). In addition GHK-treatedirradiated fibroblasts showed increasedproduction of growth factors bFGF andVEGF, which was significantly higherthan that of irradiated and intact con-trol cells (37).Fibroblasts are central cells in both woundhealing and tissue renewal processes. Theynot only synthesize different compo-nents of ECM, but also produce a num-ber of growth factors that regulate cellmigration and proliferation, angiogene-

sis, epithelialization etc. However, fi-broblasts are sensitive cells that can bedamaged by many factors. The fact thatGHK-Cu restored damaged fibroblasts’activity up to a level to that of normal,non-irradiated fibroblasts, opens newpossibilities in enhancing fibroblasts’function in aged skin.

! GHK-Cu Suppresses Cancer

This area of GHK-copper’s activity maybelong to medicine rather than cosme-tology, however it sheds an importantlight on the protective and reparativerole of GHK-Cu in the organism. In the study published in 2010 by Hong Y.et al. (Department of Colorectal Surgery,Singapore General Hospital, Singapore)GHK-Cu was selected out of 1309 bio-logically active substances as an activitythat was able to reverse expression ofcertain genes involved in metastaticspreading of colon cancer. Another sub-stance that produced such effect wasplant alkaloid securinine. GHK-Cu wasnot toxic and produced effect at a verylow concentration - 1mkM (securininewas active at 18 microM) (38). This research is the first that reports di-rect anti-cancer activity of GHK-copper.However, there is also indirect evidencesuggesting an important role of GHK-copper in preventing cancer. First of all, GHK-Cu can exhibit anti-tu-mor activity by increasing decorin (10).It has been shown that stimulation ofdecorin expression leads to the regres-sion of certain tumors, such as gliomas.Decorin also was found to inhibit growthof various tumors and to prevent metas-tasis of breast cancer (39). One of the im-portant qualities of decorin is its abilityto inactivate TGF-beta. It is known thatTGF-beta helps the tumor to escape theaction of the host’s immune system bysuppressing immune response, causingapoptosis of immune cells and modulat-ing the activity of growth factors (40).GHK-Cu is also proved to decrease TGF-beta, although this effect may be sec-ondary – due to the increased decorinlevel. Another contribution of GHK-copper in-to anti-tumor defense of an organism aswell as into prevention of other age re-

lated degenerative conditions is its an-tioxidant and anti-inflammatory action.

! Anti-inflammatory and Antioxidant Action

It is known that inflammation is alwaysaccompanied by increased oxidative dam-age. One reason is that immune cells usereactive oxygen species (ROS) and otherpowerful oxidants (such as hypochloriteand peroxinitrite) to combat the bacte-ria. Since free radicals play an importantrole in the aging process, antioxidantand anti-inflammatory ingredients areoften included in anti-aging cosmeticformulations (41). It was found that GHK-Cu suppresses in-flammation by lowering the level of acutephase inflammatory cytokines such asTGF-beta and TNF-alpha (42). GHK alsoreduces oxidative damage by modulat-ing iron levels (43) and by quenchingtoxic products of fatty acids lipid peroxi-dation (44-45). Since lipid peroxidationplays an important role in skin aging andUV-induced skin damage, the ability ofGHK to form complexes and sequestertoxic by-products of lipid peroxidationhas a protective effect against skin ag-ing and photo-damage. One of the pos-sible application of GHK-Cu is sun-pro-tective cosmetics. It is known that manysynthetic UV-filters can generate freeradicals and increase oxidative damageto the skin. An addition of GHK-Cu tosun-protective cosmetics may decreasethe level of harmful by-products of lipidperoxidation, reduce inflammation andskin damage.

! How Does GHK-Cu Work?

GHK tri-peptide has a strong affinity forcopper and more often exists in a formof GHK-Cu. On the basis of available da-ta it was proposed that GHK-Cu mightfunction by modulating copper intakeinto cells (46). It is now well established that the bio-logical significance of GHK-Cu primarilyresults from its unique relationship withcopper. Since copper ions are used bymore than a dozen enzymes involved indifferent biological processes in the cell,

GHK-COPPER PEPT IDECOSMETICS

SOFW-Journal | 136 | 6-2010 7

supplying the tissues with copper canimprove many aspects of tissue metabo-lism including antioxidant defense, tis-sue repair, oxygenation etc. Among en-zymes that use copper are an anti-oxidant enzyme superoxide dismutase(SOD), lysyl oxidase is required for con-nective tissue formation, tyrosinaze re-sponsible for melanin synthesis andmany others (47). Another important function of copper issignaling. For example, low tissue copperprompts proliferation of stem cells, whilesufficient tissue copper is required forstem cell differentiation into cells need-ed for tissue repair (48). An inadequatelevel of copper can prevent stem cellsfrom differentiation and hamper tissuerepair (49). By modulating copper levelGHK-Cu may act as a »switch« that curbsinflammation and prompts skin healingand remodeling process (Fig. 4). Smallsize and mobility allow GHK-copper easyaccess to molecular receptors and speedytravel in the extracellular space. Fig. 5 GHK-Cu and »The Copper switch«.

Fig. 6 Proposed mechanism of action of GHK-Cu

GHK-COPPER PEPT IDECOSMETICS

8 SOFW-Journal | 136 | 6-2010

However, it would be a mistake to viewGHK-Cu simply as a copper transportingprotein. As it has been discovered re-cently, its biological activity goes wellbeyond copper binding.A number of studies show that tri-pep-tide is able to bind not only with copper,but also with a variety of biological mol-ecules as well as promote cell attach-ment to different substrates. Rabenstein et al. demonstrated that GHKis able to form complexes with heparin(50). It is known that heparin shares com-mon characteristics with heparin sulfate– one of the main GAGs of the epider-mal basement membrane (51). The abili-ty of GHK to bind with heparin indicatesits ability to bind also with heparin sul-fate, and therefore – with basement mem-brane and ECM. Since GHK also can bindwith cellular receptor, its ability to bindwith ECM allows it to facilitate cell ad-hesion to ECM structures, stimulatingtheir migrating and differentiating (52)(Fig. 5). By modulating copper level and facili-tating cellular interaction with extracel-lular matrix, GHK-Cu is able to exhibitbroad reparative, protective and rejuve-nating activity acting as a natural skinwellness and age reversal agent.

! Conclusion

Human tri-peptide GHK-Cu is a naturalskin wellness molecule that exhibits abroad range of reparative, anti-aging,and protective actions. By regulatingprocesses of skin renewal and repair, itensures not only fast healing of dermalwounds, but also exhibits rejuvenatingaction by increasing water-holding mol-ecules in dermal matrix, stimulating col-lagen synthesis, balancing the action ofskin proteases and reducing oxidativedamage. Particularly interesting is itsability to maintain epidermal stem cellsas well as to restore viability of damagedskin cells such as irradiated fibroblasts.Since fibroblasts and adult stem cellsplay a crucial role in skin renewal andwound healing process, GHK-Cu presentsitself as a natural skin renewing, repair-ing and rejuvenating ingredient. Possible applications of cosmetics con-taining GHK-copper include:

1. Anti-aging topical formulations – slow-ing down aging, reducing wrinkles,improving skin complexion, reducingunwanted pigmentation, improvingskin structure.

2. Cosmetic formulations intended to beused before and after aggressive cos-metic procedures and plastic surgery –speeding up skin healing, reducingthe risk of side effect, improving out-come. Such formulations would beespecially valuable for patients of ad-vanced age or with underlying healthconditions.

3. Topical formulations for reducing in-flammation and redness associatedwith various cosmetic manipulations.

4. Sun-protective formulations.

References

(1) Zelles T, Purushotham KR, Macauley SP, Ox-ford GE, Humphreys-Beher MG. Saliva andgrowth factors: the fountain of youth residesin us all. J Dent Res. 1995;74(12):1826-32.Review

(2) Pickart L. The human tri-peptide GHK and tis-sue remodeling. J. Biomater. Sci. Polymer Edn.2008; 19(8):969-988

(3) Maquart FX, Pickart L, Laurent M, Gillery P,Monboisse JC, Borel JP. Stimulation of colla-gen synthesis in fibroblast cultures by thetripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+.FEBS Lett. 1988; 10;238(2):343-6

(4) Wegrowski Y, Maquart FX, Borel JP. Stimula-tion of sulfated glycosaminoglycan synthesisby the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+. Life Sci. 1992;51(13):1049-56

(5) Ehrlich P. Symposium on collagen and skin re-pair. Reims. Sept 1991 (12)

(6) Siméon A, Monier F, Emonard H, Gillery P,Birembaut P, Hornebeck W, Maquart FX. Ex-pression and activation of matrix metallo-proteinases in wounds: modulation by thetripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+.J Invest Dermatol. 1999;112(6):957-64

(7) Siméon A, Emonard H, Hornebeck W, MaquartFX. The tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu2+ stimulates matrix met-alloproteinase-2 expression by fibroblast cul-tures. Life Sci. 2000; 22;67(18):2257-65

(8) Siméon A, Wegrowski Y, Bontemps Y, Ma-quart FX. Expression of glycosaminoglycansand small proteoglycans in wounds: modula-tion by the tripeptide-copper complex glycyl-L-histidyl-L-lysine-Cu(2+).J Invest Dermatol.2000;115(6):962-8

(9) Sayani K, Dodd CM, Nedelec B, Shen YJ, Gha-hary A, Tredget EE, Scott PG. Delayed ap-pearance of decorin in healing burn scars.Histopathology 200; 36(3): 262-272

(10) Zhang Z, Li XJ, Zhang X, Li YY, Xu WS. Re-combinant human decorin inhibits cell pro-liferation and downregulates TGF-beta pro-duction in hypertrophic scar fibroblasts. Burns2007; 33(5):634-641

(11) McCormack MC, Nowak KC, Koch RJ. The ef-fect of copper peptide and tretinoin ongrowth factor production in a serum-free fi-broblast model. Arch facial Plast. Surg. 2001;3(1):28-32

(12) Buffoni F, Pino R, Dal Pozzo. A Effect of tri-peptide-copper complexes on the process ofskin wound healing and on cultured fibrob-lasts. Arch Int Pharmacodyn Ther. 1995 Nov-Dec; 330(3):345-60

(13) Huang PJ, Huang YC, Su MF, Yang TY, HuangJR, Jiang CP. In vitro observations on the in-fluence of copper peptide aids for the LEDphotoirradiation of fibroblast collagen syn-thesis. Photomed Laser Surg. 2007;25(3):183-90

(14) Schultz GS, Wysocki A. Interactions betweenextracellular matrix and growth factors inwound healing. Wound Repair Regen. 2009Mar-Apr;17(2):153-62

(15) Lane TF, Iruela-Arispe ML, Johnson RS, SageEH. SPARC is a source of copper-binding pep-tides that stimulate angiogenesis. J Cell Biol.1994 May;125(4):929-43

(16) Cangul IT, Gul NY, Topal A, Yilmaz R. Evalua-tion of the effects of topical tripeptide-cop-per complex and zinc oxide on open-woundhealing in rabbits. Vet Dermatol. 2006;17(6):417-23

(17) Gul NY, Topal A, Cangul IT, Yanik K. The ef-fects of topical tripeptide copper complexand helium-neon laser on wound healing inrabbits. Vet Dermatol. 2008;19(1):7-14

(18) Arul V, Gopinath D, Gomathi K, Jayakumar R.Biotinylated GHK peptide incorporated col-lagenous matrix: A novel biomaterial for der-mal wound healing in rats. J Biomed MaterRes B Appl Biomater. 2005;73(2):383-91

(19) Arul V, Kartha R, Jayakumar R. A therapeuticapproach for diabetic wound healing usingbiotinylated GHK incorporated collagen ma-trices. Life Sci. 2007;2;80(4):275-84

GHK-COPPER PEPT IDECOSMETICS

SOFW-Journal | 136 | 6-2010 9

COSMETICSGHK-COPPER PEPT IDE

(20) Canapp SO Jr, Farese JP, Schultz GS, GowdaS, Ishak AM, Swaim SF, Vangilder J, Lee-Am-brose L, Martin FG. The effect of topicaltripeptide-copper complex on healing of is-chemic open wounds. Vet Surg. 2003;32(6):515-23

(21) Counts, D, Hill E, Turner-Beatty M, GrotewielM, Fosha-Thomas S, Pickart L. Effect of laminon full thickness wound healing. Fed Am SocExp Biol. 1992; A1636

(22) Mazurowska L, Mojskin M. Biological activi-ties of selected peptides: skin penetrationability of copper complexes with peptides. JCosmet Sci. 2008:59-69

(23) Mazurowska L, Mojski M. ESI-MS study of themechanism of glycyl-l-histidyl-l-lysine-Cu(II)complex transport through model membraneof stratum corneum. Talanta. 2007;30;72(2):650-4

(24) Gorouhi F, Maibach HI. Role of topical pep-tides in preventing and treating aged skin. Int.J. Cosm. Sci., 2009;31:327-345

(25) Abdulghani, AA, Sherr S, Shirin S, SolodkinaG, Tapia EM,Gottlieb AB. Effects of topicalcreams containing vitamin C, a copper-bind-ing peptide cream and melatonin comparedwith tretinoin on the ultrastructure of nor-mal skin - A pilot clinical, histologic, and ul-trastructural study. Disease Manag Clin Out-comes. 1998;1:136-141

(26) Leyden J, Stephens T, Finkey MB, Appa, Y,Barkovic S. Skin Care Benefits of Copper Pep-tide Containing Facial Cream. Amer AcademyDermat Meeting, February 2002, Abstract P68

(27) Leyden J, Stephens T, Finkey MB, Barkovic S.Skin Care Benefits of Copper Peptide Con-taining Eye Creams. Amer Academy DermatMeeting, February 2002, Abstract P69

(28) Finkley MB, Appa Y, Bhandarkar S. CopperPeptide and Skin. Cosmeceuticals and ActiveCosmetic, 2nd Edition, P. Eisner and HI.Maibach (Eds.) Marcel Dekker, New York.2005:549-563

(29) Ohyama M. Hair follicle bulge: a fascinatingreservoir of epithelial stem cells. J DermatolSci. 2007 May;46(2):81-9

(30) Trachey R, Fors TD, Pickart L, Uno H. The hairfollicle stimulating properties of peptide cop-per complexes. Results in C3H mice. Ann N YAcad Sci. 1991;642:468-4

(31) Uno H, Kurata S. Chemical agents and pep-tides affect hair growth. J Invest Dermatol.1993;101(1 Suppl):143S-147S

(32) Pyo HK, Yoo HG, Won CH, Lee SH, Kang YJ, EunHC, Cho KH, Kim KH. The effect of tripeptide-copper complex on human hair growth in vit-ro. Arch Pharm Res. 2007;30(7):834-9

(33) Perez-Meza D, Leavitt M, Trachy R. Clinicalevaluation of GraftCyte moist dressings onhair graft viability and quality of healing. In-ter. J. Cos. Surg. 1988;6:80-84

(34) Kang YA, Choi HR, Na JI, Huh CH, Kim MJ,Youn SW, Kim KH, Park KC. Copper-GHK in-creases integrin expression and p63 positivi-ty by keratinocytes. Arch Dermatol Res. 2009Apr;301(4):301-6

(35) Rubin H. The disparity between human cellsenescence in vitro and lifelong replication invivo. Nat Biotechnol. 2002;20(7):675-81

(36) Giangreco A, Qin M, Pintar JE, Watt FM. Epi-dermal stem cells are retained in vivo through-out skin aging. Aging Cell. 2008;7(2):250-9.

(37) Pollard JD, Quan S, Kang T, Koch RJ. Effects ofcopper tripeptide on the growth and expres-sion of growth factors by normal and irradi-ated fibroblasts. Arch Facial Plast Surg.2005;7(1):27-31

(38) Hong Y, Downey T, Eu KW, Koh PK, Cheah PY.A 'metastasis-prone' signature for early-stagemismatch-repair proficient sporadic colorec-tal cancer patients and its implications forpossible therapeutics. Clin Exp Metastasis.2010 Feb 9. [Epub ahead of print]

(39) Goldoni S, Seidler DG, Heath J, Fassan M, Baf-fa R, Thakur ML, Owens RT, McQuillan DJ, Ioz-zo RV. An anti-metastatic role for decorin inbreast cancer. Am J Pathol. 2008 Sep;173(3):844-55. Epub 2008 Aug 7

(40) Ständer M, Naumann U, Wick W, Weller M.Transforming growth factor-beta and p-21:multiple molecular targets of decorin-medi-ated suppression of neoplastic growth. CellTissue Res. 1999;296(2):221-7

(41) Cuzzocrea S, Riley DP, Caputi AP, Salvemini D.Antioxidant therapy: a new pharmacologicalapproach in shock, inflammation, and is-chemia/reperfusion injury. Pharmacol Rev.2001;53(1):135-59

(42) Canapp SO Jr, Farese JP, Schultz GS, GowdaS, Ishak AM, Swaim SF, Vangilder J, Lee-Am-brose L, Martin FG. The effect of topicaltripeptide-copper complex on healing of is-chemic open wounds. Vet Surg. 2003;32(6):515-23

(43) Miller DM, DeSilva D, Pickart L, Aust SD. Ef-fects of glycyl-histidyl-lysyl chelated Cu(II)on ferritin dependent lipid peroxidation. AdvExp Med Biol. 1990;264:79-84

(44) Beretta G, Artali R, Regazzoni L, Panigati M,Facino RM. Glycyl-histidyl-lysine (GHK) is aquencher of alpha,beta-4-hydroxy-trans-2-nonenal: a comparison with carnosine. in-sights into the mechanism of reaction byelectrospray ionization mass spectrometry,1H NMR, and computational techniques.Chem Res Toxicol. 2007 Sep;20(9):1309-14

(45) Beretta G, Arlandini E, Artali R, Anton JM,Maffei Facino R. Acrolein sequestering abili-ty of the endogenous tripeptide glycyl-his-tidyl-lysine (GHK): characterization of conju-gation products by ESI-MSn and theoreticalcalculations. J Pharm Biomed Anal. 2008 Jul15;47(3):596-602

(46) Pickart L, Freedman JH, Loker WJ, et al.Growth-modulating plasma tripeptide mayfunction by facilitating copper uptake intocells. Nature 1980;288:715-7

(47) Lalioti V, Muruais G, Tsuchiya Y, Pulido D.Sandoval IV Molecular mechanisms of copperhomeostasis. . Front Biosci. 2009;1;14:4878-903

(48) Rodríguez JP, Ríos S, González M. Modulationof the proliferation and differentiation of hu-man mesenchymal stem cells by copper. J CellBiochem. 2002;85(1):92-100

(49) Peled T, Fibach E, Treves A. Methods of con-trolling proliferation and differentiation ofstem and progenitor cells. U.S. Patent 7,429,489, September 30, 2008

(50) Rabenstein DL, Robert JM, Hari S. Binding ofthe growth factor glycyl-L-histidyl-L-lysineby heparin. FEBS Lett. 1995;4;376(3):216-20

(51) Mulloy B, Forster MJ. Conformation and dy-namics of heparin and heparan sulfate. Gly-cobiology. 2000 Nov;10(11):1147-56

(52) Godet D, Marie PJ. Effects of the tripeptideglycyl-L-histidyl-L-lysine copper complex onosteoblastic cell spreading, attachment andphenotype. Cell Mol Biol (Noisy-le-grand).1995 Dec;41(8):1081-91

Address of the authors’:Loren Pickart PhD

Anna Margolina PhDSkin Biology, Inc

4122 Factoria Boulevard - Suite 200Bellevue, WA 98006

Email:lorenpickart@skinbiology.com

anna@amargolina.com

!