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COSMETICSPEPT IDES

that PKEK is a powerful active ingredientwhich is able to balance the skin tone inmultiple ways. It is made available to themarket as a preservative- and glycol-free, water-soluble solution of 1600 ppmactive matter as TEGO® Pep 4-Even (INCI:Tetrapeptide-30, Glycerin).

n Tetrapeptide-21 –The ECM Boosting Peptide

The ECM is the structural backbone ofmany tissues, especially of the skin. Dur-ing regular turnover, ECM is degraded

M. Mentel*, J. Schild, U. Maczkiewitz, T. Koehler, M. Farwick

Innovative Peptide Technologies for Even, Young and Healthy Looking Skin

n Introduction

With age, skin performance is decreasingand as a consequence, overall skin ap-pearance is dramatically changed. Thisarticle describes two cosmetic peptideswhich were tailor-made to successfullycombat two of the most visible signs ofthe aging process, the appearance of wrin-kles and age spots.Tetrapeptide-21 (GEKG: Glycine – Glutam-ic Acid – Lysine – Glycine) is a tetrapep-tide-motif that is present in several hu-man extracellular matrix (ECM) proteins.Its topical application induces restora-

tion of the skin’s most important struc-tural dermal components. It is available asa preservative-free, clear, water-solublesolution of 2000 ppm active matter asTEGO® Pep 4-17 (INCI: Tetrapeptide-21,Glycerin, Butylene Glycol, Aqua).Besides the desire to counteract the de-pletion of different ECM components, asafe and skin-friendly way of inhibitingthe pigmentation process is of major cos-metic concern. Tetrapeptide-30 (PKEK:Proline – Lysine – Glutamic Acid – Lysine)is a skin-derived tetrapeptide which di-minishes post-inflammatory hyperpig-mentation. Several in vivo studies show

Abstract

Many peptides have been

developed and commer-

cialized for the dermato-

logical and skin care markets. The

present review summarizes the

development and bioactivity of

two such peptides, Tetrapeptide-

21 and Tetrapeptide-30. The effi-

cacy of the peptides was shown

in several in vitro, ex vivo and in

vivo studies.

The results show that Tetrapep-

tide-21 is highly efficacious in

boosting the major extracellular

matrix (ECM) components in a

well balanced way. As a conse-

quence, skin elasticity and volu-

me are improved and skin rough-

ness and scaliness are reduced.

The ingredient is effective in re-

ducing all kinds of wrinkles and

thus has a high anti-aging poten-

tial.

Tetraptide-30 has strong skin

tone modulating and skin brigh-

tening effects. It acts by a smart

mechanism via reducing kerati-

nocyte-induced activation of

melanocytes. It is active on all

kinds of skin types (Caucasian,

Asian and African) where it is

able to reduce acne lesions,

alleviate melasma and visibly

diminish hyperchromatic spots.

It acts in a safe and skin-friendly

way and as a result inhibits the

skin pigmentation process,

which is of major cosmetic con-

cern.

24 SOFW-Journal | 138 | 3-2012

COSMETICSPEPT IDES

and it is well-known that several of itsbreakdown products are able to stimu-late ECM resynthesis, in order to com-pensate for tissue destruction. An in sili-

co (computational modeling) approachwas used to develop peptides by identi-fying highly repetitive amino acid motifsin several ECM proteins (collagen I, II, III,IV, V, elastin, and proelastin) (1, 2). Ap-proximately 30 tetrapeptide motifs, par-ticularly abundant in different ECM pro-teins, have been identified. These pep-tides have been synthesized and testedfor induction of collagen protein pro-duction in human dermal fibroblasts ongenetic and protein level. The most ac-tive peptide had the sequence GEKG andwas subsequently assessed for its effectson the formation of ECM componentsin vitro and in vivo.

n Tetrapeptide-21 induces ECMGene Expression and Protein Poduction

Collagen protein expression was investi-gated in cell culture supernatant of hu-man dermal fibroblasts after stimulationfor 24 hours with 1 and 10 ppm Tetrapep-tide-21 or Palmitoyl Pentapeptide-4 (pal-KTTKS) as a reference standard. Tetrapep-tide-21 significantly (p<0.05) increasedthe amount of secreted collagen proteindose-dependently compared to control.When comparing to Palmitoyl Pentapep-tide-4, the collagen production was al-most doubled at either concentration(Fig. 1).On mRNA level, the tested ECM markergenes (COL1A1, Fibronectin and HAS1)were clearly and significantly (p<0.05)induced by treatment with Tetrapep-tide-21 at 1 ppm (data reported in 3).The well balanced induction of ECM con-stituents by Tetrapeptide-21 suggests astrong anti-aging effect of this mole-cule. This assumption was further testedin an in vivo biopsy study enrolling 10volunteers aged above 40 (average 48.2years). An O/W vehicle with and without50 ppm Tetrapeptide-21 was applied oncedaily on different areas of the uppergluteal region and on the inner forearmfor 8 weeks. Thereafter, punch biopsieswere taken from the treated sites and anuntreated site. Quantitative real-time PCR

(qRT-PCR) was used to quantify increas-es in COL1A1, the gene that encodes themajor component of type I collagen. Itwas demonstrated that topical applica-tion of Tetrapeptide-21 led to a statis-tically significant increase in expressionof COL1A1 (p<0.05). For immunohisto-chemical investigations, 4 representativesubjects were selected and their tissuesamples were analyzed for procollagen I,fibronectin and hyaluronic acid (Fig. 2).Compared to vehicle, a considerable in-duction of the ECM components colla-gen, fibronectin and hyaluronic acid wasobserved (3).

n Tetrapeptide-21 improves Skin Elasticity

Within the initial in vivo biopsy study,skin elasticity had been evaluated onthe inner forearm with a cutometer. Theimprovement of several important elas-ticity parameters was indicated by treat-ment with Tetrapeptide-21, but no sig-nificance was reached due to the smallpanel. Therefore, in a second in vivo

study 60 volunteers were recruited andeach 15 volunteers received the test for-mulation containing 10 ppm or 100 ppmTetrapeptide-21, 10 ppm Palmitoyl Pen-

Fig. 1 Collagen protein induction in fibroblast culture

Fig. 2 Photographical documentation of immunostaining of procollagen I (left),hyaluronic acid (centre) and fibronectin (right). Images obtained after 8 weeks ofapplication on buttock skin from a representative subject of the vehicle treatedgroup (top row) and Tetrapeptide-21 treated group (bottom row)

SOFW-Journal | 138 | 3-2012 25

COSMETICSPEPT IDES

tapeptide-4 or the O/W vehicle formula-tion. Test formulations were applied twicedaily over a period of 8 weeks on the leftinner forearm. A Visioscan VC 98 was usedto evaluate skin volume, skin roughnessand skin scaliness. Skin elasticity was as-sessed with a Cutometer.Treatment with 10 ppm Tetrapeptide-21significantly (p<0.10) reduced skin scali-ness and improved skin volume com-pared to vehicle. Further improvements(p<0.01) were detected when 100 ppmTetrapeptide-21 were employed. Increas-ing concentrations of Tetrapeptide-21also decreased skin roughness. Com-pared to Palmitoyl Pentapeptide-4, 10 ppmTetrapeptide-21 showed comparable ac-tivity whereas 100 ppm doubled the ef-fect (results are reported in (4)).R1, the remaining deformation after thefirst stretching cycle, was significantlyimproved after treatment. The improve-ment was maximal already at the lowestconcentration applied. The efficacy of10 ppm Tetrapeptide-21 was superior to10 ppm Palmitoyl Pentapeptide-4 (Fig. 3).

n Tetrapeptide-21 – Facial Anti-Wrinkle Study

To further manifest the beneficial effectsof the ECM boosting tetrapeptide in acosmetically relevant context, an in vivo

facial anti-wrinkle study was conducted

on 60 volunteers. One half of the panelreceived an O/W formulation containing80 ppm Tetrapeptide-21, the other halfreceived vehicle only. Test formulationswere applied twice daily in the face for8 weeks. Cutaneous roughness was as-sessed with a Primos Pico, and digital im-ages of the periorbital region were record-ed before treatment and after 4 and 8weeks of application, respectively.The parameters Sa (arithmetic average ofsurface roughness) and Sz (average ofthe 5 highest peaks and 5 deepest val-leys form the entire measuring field)

were significantly reduced after 4 and 8weeks of application, while the vehiclewas not effective in reducing wrinkles(Fig. 4).The following images indicate the effi-cacy of the tetrapeptide in reducing wrin-kles (Fig. 5). It is clearly visible that theproduct is able to reduce fine lines aswell as medium or deep wrinkles notably.First effects were visible after 4 weeksand were further manifested with in-creasing application time.Taken the results of the various in vitro

and in vivo studies together, Tetrapep-

Fig. 3 Improvement of skin elasticity parameter R1

Fig. 4 Skin roughness parameters Sa and Sz

tide-21 shows superior collagen, hyaluron-ic acid and fibronectin boosting activityand thus improves skin elasticity and re-duces skin roughness. The skin is strong-ly smoothed and firmed, and the appear-ance of all kinds of wrinkles is minimized.The high anti-aging potential of Tetrapep-tide-21 turned out to be particularly suit-able for anti-wrinkle and anti-aging eyecare applications.

n Tetrapeptide-30 –Balancing Skin Tone

The skin pigment melanin is synthesizedin melanocytes in the basal layer of theepidermis, by a process which is con-trolled by a plethora of mechanisms andreactions (melanogenesis). One of theseprocesses depends on the interaction be-tween keratinocytes and melanocytes,which can be regulated by the pigmen-tation-inducing soluble factor melano-cyte-stimulating hormone (α-MSH). It isgenerated by cleavage of the precursorpeptide proopiomelanocortin (POMC) andsecreted by the keratinocytes. Addition-ally, pro-inflammatory cytokines secret-ed from keratinocytes modulate melano-cyte activation and proliferation.Skin brightening and the pigmentationprocess can be influenced in many dif-ferent ways. A commonly used approachis inhibition of the main responsible en-zyme for melanin synthesis (tyrosinase).Inhibition of melanocyte activation andblocking of melanin transfer into ker-atinocytes are alternative and equallyeffective approaches.Tetrapeptide-30 acts by a smart mecha-nism via reducing keratinocyte-inducedactivation of melanocytes. The tetrapep-tide contains the KEK motif, which ispresent in the antimicrobial polypeptidecathelicidin LL-37 (1, 2). In vitro and invivo studies indicate a strong efficacy ofTetrapeptide-30 in balancing skin tone.

n Reduction of Keratinocyte-Secreted Factors of Melanogenesis

The effect of Tetrapeptide-30 on ker-atinocyte-secreted factors of melano-genesis was investigated in vitro on UVB-irradiated primary normal human epi-

dermal keratinocytes (NHEKs) from neona-tal foreskin. NHEKs were cultivated inmedium containing 0.7 ppm or 7 ppmTetrapeptide-30 or vehicle for 24 hoursprior to exposure to a dose of 160 J/m2

UVB radiation. Afterwards, cells were cul-tivated for another 6 hours and 24 hours,respectively. Gene expression was as-sessed by qRT-PCR applying total RNA.It was found that irradiation of NHEKswith UVB led to up-regulation of inter-leukin-6 (IL-6) and interleukin-8 (IL-8)gene expression 6 hours post irradiation,an effect that was significantly (p<0.05)reduced by the action of Tetrapeptide-30. Interleukin increase was followed byan induction of the α-MSH precursorPOMC gene expression after 24 hours, aneffect that was completely and signifi-cantly (p<0.05) blocked with either 0.7or 7 ppm Tetrapeptide-30 (Fig. 6). Thus,

the induction of POMC gene expressionremained at basal level (5).The results of this study show thatTetrapeptide-30 efficiently reduces UVB-induced melanogenesis in cultured ker-atinocytes by reducing POMC/α-MSHas well as interleukin gene expression.The data suggests that the activation ofmelanocytes and thus production of theskin pigment melanin is efficiently re-duced by a mechanism involving ker-atinocyte to melanocyte communica-tion.

n Reduction of MelanogenesisActivation in Human Skin

The in vivo relevance of these findingswas explored in the context of a biopsystudy enrolling 10 volunteers aged 25-

26 SOFW-Journal | 138 | 3-2012

COSMETICSPEPT IDES

Fig. 5 Wrinkles in the periorbital region from 3 representative subjects before (left)and after 8 weeks of application of 50 ppm Tetrapeptide-21 (right)

69. The volunteers were treated oncedaily over a period of 4 weeks with ve-hicle or formulation containing 40 ppmTetrapeptide-30 on the upper medial

quarter of the buttock. Thirty minutesafter the final application, skin color wasmeasured with a colorimeter and volun-teers were irradiated with a dose of 1.5

MED (minimal erythema dose) of broad-band UVB. One day after irradiation, thechange in skin color was recorded andpunch biopsies were taken from a UVB-

COSMETICSPEPT IDES

Fig. 6 Effect of Tetrapeptide-30 treatment on gene expression 6 hours (IL-6, IL-8 ) and 24 hours (POMC) post irradiation withUVB

SOFW-Journal | 138 | 3-2012 27

treated and -untreated area from each avehicle and Tetrapeptide-30 treated re-gion.It was found that expression of key in-flammation marker genes IL-6 and IL-8,TNF-α and COX-2, as well as expressionof genes for tyrosinase and POMC wasinduced in UVB-irradiated, vehicle-treat-ed skin. This effect was significantly re-duced by treatment with Tetrapeptide-30 (Fig. 7) (5).

UVB-irradiation led to a significant de-crease in ITA° values, which represents anincrease in skin melanin content (tan-ning). Treatment with Tetrapeptide-30led to a moderate change of ITA°, thuspreventing UV-induced melanin forma-tion by trend (data reported in (5)).

n Lightening the Skin Tone on the Hand

The previous results confirmed the rele-vance of the in vitro findings in an in vi-

vo context. In a next step, a broader in-vestigation of in vivo effects was startedand 38 panelists aged 35-59 were enrolledin a skin lightening study. A hand creamcontaining 1.5% sodium ascorbyl phos-phate (SAP; A commonly accepted light-ening active ingredient and known in-hibitor of tyrosinase activity) or a combi-

nation of 1.5% SAP plus 40 ppm Tetrapep-tide-30 was applied twice daily for 8weeks. Before and after 4 and 8 weeks ofapplication, skin color was determinedon the back of the hand with a colori-meter, and skin redness and erythemawere assessed with a Mexameter.The combination of Tetrapeptide-30 andSAP led to a strong and significant(p<0.01) reduction of skin redness (a*),yellow skin color (b*) and erythema, while

SAP alone did not show significant ef-fects. Also a significant (p<0.10) increasein ITA° (skin lightening) was detected forthe combination of active ingredientsbut not for SAP alone (5) (Fig. 8).In summary, it could be clearly demon-strated that a combination of 1.5% SAPplus 40 ppm Tetrapeptide-30 is effectivein visibly reducing hyperchromatic agespots on the hands and in lightening thehand skin tone.

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COSMETICSPEPT IDES

Fig. 8 Visible fading of hyperchromatic spots and yellow skin color after applicationof 1.5% SAP plus 40 ppm Tetrapeptide-30 (from left to right: before, after 4 and 8weeks)

Fig. 7 Effect of Tetrapeptide-30 on gene expression of POMC, Tyrosinase and inflammation markers COX-2 and TNF-α 24 hourspost irradiation with UVB

n Reduction of Facial Age Spots

Subsequently, we investigated if the ac-tivity profile of Tetrapeptide-30 was al-so extendable to facial applications. Ahalf-face study was performed on fortywomen aged 30-70 with hyperchro-matic spots. They applied either vehicle,40 ppm Tetrapeptide-30, 1.5% SAP or acombination of both twice daily for 6weeks. Skin color of hyperchromatic spotsand their surrounding area was deter-mined and the severeness of spots is rep-resented by the contrast (difference inITA°). The fading of age spots was de-scribed as the change in contrast be-tween the spot and the surrounding areabefore and after treatment (Fig. 9).A visible fading of hyperchromatic spotswas detected, with similar efficacy, whenTetrapeptide-30 or SAP were applied.The fading was further increased when acombination of Tetrapeptide-30 and SAPwas used (Fig. 10).

SOFW-Journal | 138 | 3-2012 29

COSMETICSPEPT IDES

Fig. 9 Fading of hyperchromatic age spots

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n Improvement of Evenness ofSkin Tone on Asian Skin

The proven skin lightening efficacy ofTetrapeptide-30 on caucasian skin indi-cated its potential also on asian skin,where skin brightening applications andapplications targeting hyperchromaticspots and an uneven skin tone are a hugemarket need.Thus, a mixed panel consisting of 27 vol-unteers aged 28-60 with Japanese back-ground and an uneven skin tone wererecruited. A test formulation contain-ing 1.5% SAP as the vehicle compared tothe same formulation containing 40 ppmTetrapeptide-30 was applied twice dailyfor 8 weeks on one side of the face, whilethe other side remained untreated. Aclinical assessment of skin conditions wasperformed, and skin color was recordedbefore and after 4 and 8 weeks of appli-cation.Changes in ITA° values among the SAP(vehicle) group almost remained un-changed and skin tone was not affected,while Tetrapeptide-30 treatment signif-icantly (p< 0.01) lightened the skin com-pared to the vehicle (Fig. 11).No change in hyperpigmentation wasobserved among the SAP group by ex-pert grading after 8 weeks, while ratingvalues were reduced by 15% in theTetrapeptide-30 group (5) (Fig. 12).The study demonstrated that Tetrapep-tide-30 is able to reduce some of the ma-

jor concerns of asian skin. Skin tone islightened, skin tone evenness is im-proved and hyperchromatic age spotsare reduced. Therefore, it can be con-cluded that Tetrapeptide-30 is also high-ly recommended for cosmetic treatmentof skin tone among asian population.

n Improvement of Evenness ofSkin Tone on African Skin

Finally, the high activity profile of Tetrapep-tide-30 raised the question, if the active

ingredient was also able to reduce melas-ma and/or post-inflammatory hyperpig-mentation lesions among African eth-nicities, who often suffer from an unevenskin tone. Melasma is a tan or dark skindiscoloration that mainly affects women,and is especially pronounced in regionswhere the skin is exposed to intense sunirradiation. Post-inflammatory hyperpig-mentation is the main cause of spot de-velopment after acne lesions.Fifty women aged 18-50 with mild acneand/or melasma (Fitzpatrick type IV-V)

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COSMETICSPEPT IDES

Fig. 11 Changes in skin tone by increase of ITA° (skin lightening)

Fig. 10 Pictures of each two representative panelists who applied Tetrapeptide-30 alone (top row) or a combination of Tetrapep-tide-30 + SAP (bottom row)

were recruited and panelists were askedto avoid excessive sunlight. Test formu-lations with either 40 ppm Tetrapeptide-30 or vehicle were applied twice dailyover a period of 12 weeks on the fullface. A clinical assessment of overall skincondition, evenness of skin tone, area ofmelasma (percentage values) and num-ber of lesions (divided into three groups:<5, 5-10, >10 lesions) was performedon a five-level scale by experts beforeand after 2, 4, 8 and 12 weeks of appli-cation. Skin overall appearance was significant-ly (p<0.05) improved after 12 weeks oftreatment with Tetrapeptide-30 (Fig. 13),

but not in case of vehicle-treated skin.Similar improvements were observed forevenness of skin tone after 8 and 12weeks (p<0.10 and 0.01, respectively),and the number and appearance of postinflammatory hyperpigmentation/acnelesions already showed a significant de-crease after 4 weeks of Tetrapeptide-30treatment (p<0.05) (Fig. 14). Furtherimprovements were registered after 8and 12 weeks (p< 0.01).The study demonstrated that Tetrapep-tide-30 is also able to reduce some of themajor cosmetic concerns of African skin.There is much potential of the compoundto significantly and visibly improve the

skin’s evenness and overall appearance.In addition, the number of spots causedby acne lesions are reduced (6).Therefore, it can be concluded thatTetrapeptide-30 is highly recommendedfor treating skin tone problems on eth-nic skin.

AcknowledgementsStudies were conducted at the Institutefor Molecular Preventive Medicine (IUF)at the Heinrich Heine University in Dues-seldorf, Germany, the Institute of Skinand Product Evaluation (ISPE) in Milan,Italy and at the Photobiology Laborato-

32 SOFW-Journal | 138 | 3-2012

COSMETICSPEPT IDES

Fig. 14 Panelist with reduced number of acne lesions (left:before, right: after 12 weeks of Tetrapeptide-30 treatment)

Fig. 13 Panelist with reduced melasma intensity (left: be-fore, right: after 12 weeks of Tetrapeptide-30 treatment)

Fig. 12 Three representative panelists with reduced hyperpigmentation by 1.5% SAP plus 40 ppm Tetrapeptide 30 (Top row:Before and bottom row: After 8 weeks of application)

ry, Medunsa Campus in South Africa. Theresults of these studies were a con-tributing factor for TEGO® Pep 4-17 forreceiving the Innovation Award at thePCHi 2011 in Shenzen, China.

References

(1) Harris, S.M., T.J. Falla and L. Zhang (inventors).

Peptide fragments for inducing synthesis of ex-

tracellular matrix proteins. US 20070299105,

PCT⁄WO2007⁄146269

(2) Farwick, M. and P. Lersch (inventors). Personal

care and cosmetic composition containing

tetrapeptides with motifs GX1X2G, PX1X2P, or

PX1X2K. PCT⁄WO2009⁄068351

(3) Farwick, M., S. Grether-Beck, A. Marini, U.

Maczkiewitz, J. Lange, T. Koehler, P. Lersch, T.

Falla, I. Felsner, H. Brenden, T. Jaenicke, S. Fran-

ke and J. Krutmann. 2011. Exp Dermatol 20:

600-613

(4) Farwick, M., U. Maczkiewitz, P. Lersch, T. Falla,

S. Grether-Beck and J. Krutmann. 2009. An

ECM-derived Tetrapeptide to Counterbalance

ECM Degeneration. Cosmetics & Toiletries

124:51-54

(5) Marini, A., M. Farwick, S. Grether-Beck, H. Bren-

den, I. Felsner, T. Jaenicke, M. Weber, J. Schild,

U. Maczkiewitz, T. Koehler, A. Bonfigli, V. Pagani

and J. Krutmann. 2011. Modulation of skin pig-

mentation by the tetrapeptide PKEK: in vitro

and in vivo evidence for skin whitening effects.

Exp Dermatol 21:140-146

(6) Farwick, M., U. Maczkiewitz, P. Lersch, B. Sum-

mers and A.V. Rawlings. 2011. Facial skin-light-

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* Authors’ address:

Matthias Mentel, PhDJennifer Schild

Ursula MaczkiewitzTim Koeler, PhD

Mike Farwick, PhDConsumer Specialties

Innovation Management ActiveIngredients

Evonik Industries AGGoldschmidtstrasse 100

45127 EssenGermany

Email: matthias.mentel@evonik.com

n

SOFW-Journal | 138 | 3-2012 33

COSMETICSPEPT IDES

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