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An exemplary claim substantiation for the firming / filling activity of a synthetic tripeptide

R. Campiche1, E. Jackson1, D. Imfeld1, E. Wandeler1, G. Laurent1, C. Wagner2, M. Roche3, P. Séroul3, M. Massironi4, S. Stroebel5, M. Gempeler1

1 DSM Nutritional Products Ltd., Personal Care & Aroma, Kaiseraugst, Switzerland, 2 proDERM GmbH, Schenefeld, Germany, 3 Newtone Technologies, Lyon, France, 4 CutechBiotech srl, Padova, Italy, 5 InSphero AG, Schlieren, Switzerland

* correspondance to: remo.campiche@dsm.com

Keywords: tripeptide, skin firming, filling, hyaluronic acid

Introduction

Material & Methods

Results in vivo

Figure 5: 2.5% active containing the tripeptidestimulated hyaluronic acid synthesis (bluestaining) in skin tissue microspheres. bFGFserved as a positive control. Shown arerepresentative tissue sections.

Figure 3: (A) A formulation containing 2.5% Activeled to improved firming as measured by DynaSKIN onthe cheeks of volunteers. It even outperformed aformulation containing a market relevant anti-ageingtechnology (Benchmark). *p<0.05 vs benchmark.*p<0.05 vs baseline. Error bars represent standarderror of the mean. (B) 3D false color images of acheek hole generated by the DynaSKIN airstream.Depth, but also circumference clearly improved after29 days of active formulation. A representativevolunteer is shown. Depth and circumference resultin negative volume.

Summary

References

We investigated the hyaluronic acid boosting, and firming / filling activity of the synthetic tripeptide TetradecylAminobutyroylvalylaminobutyric Urea Trifluoroacetate (trade name SYN®-HYCAN) in vitro, ex vivo and in vivo. Weprovide further evidence, that the tripeptide stimulates endogenous hyaluronic acid synthesis in facial skin tissue bothin vitro and ex vivo, also after UV-irradiation. In vivo we show that this leads to a filling and firming effect similar tohyaluronic acid injections at distinct facial sites. In addition, treatment with a formulation containing the tripeptideleads to increased cutaneous hydration, possibly due to the high water binding capacity of hyaluronic acid [4].

1) Heidl M, Gräub R: Nuovo tripeptide anti-età. In: Cosmetic Technology. vol. 13; 2010: 13-16.2) Dai G et al.: Chronic Ultraviolet B Irradiation Causes Loss of Hyaluronic Acid from Mouse Dermis Because of Down-Regulation of Hyaluronic Acid

Synthases. Am J Pathol 2007, 171(5):1451-14613) Kaya G et al.: Hyaluronate fragments reverse skin atrophy by a CD44-dependent mechanism. PLoS Med 2006, 3(12):e493.4) Oh JH, et al.: Intrinsic aging- and photoaging-dependent level changes of glycosaminoglycans and their correlation with water content in human skin.

J Dermatol Sci 2011, 62(3):192-201.5) Del Bino S, et al.: Relationship between skin response to ultraviolet exposure and skin color type. Pigment Cell Res 2006, 19(6):606-614.

Tetradecyl-Dab-Val-Dab (INCI: Tetradecyl Aminobutyroylvalylaminobutyric Urea Trifluoroacetate) is a tripeptide with anti-ageing effects in skin. In its product form it is combined with water, glycerine, and magnesium chloride (DSM product code503833304), and marketed under the trade name SYN®-HYCAN. It thus belongs to DSM’s SYN®-peptide family. Its sequence isderived from the activation domain of thrombospondin-1 and modeled by rational design to activate transforming growthfactor beta-1 (TGF 1). Its main activity in vitro is the stimulation of the skin’s endogenous synthesis of glycosaminoglycans,in particular hyaluronic acid (HA), and proteoglycans, in particular decorin and lumican [1]. There is a decrease inglycosaminoglycans with chronological as well as photo-ageing [2]. Dermal fillers, like collagen or hyaluronic acid fillers, arehigh on the agenda for many women to counteract cutaneous atrophy [3] leading to facial wrinkles, loss of skin firmness, andskin sagging. However, such fillers require beauty doctor’s visits and needle injections. This tripeptide, though, provides aneasy to use cosmetic solution. The data presented in this poster are from new clinical and pre-clinical studies. They providefurther evidence for the tripeptide’s outstanding activity for facial skin firming and moisturization [4] by acting as a needle-free hyaluronic acid booster. We propose that Tetradecyl Aminobutyroylvalylaminobutyric Urea Trifluoroacetate (hereaftercalled tripeptide) acts particularly in areas below the eyes, on the cheeks, and on the jawline (Fig.1).

Figure 1: (A) Face of a young woman. (B,C) She has an augmented cheekbone area(fields 1 and 2), firm cheeks (field 3), anda well defined jawline (field 4). Fields 1to 4 denote areas of filling mainly to liftor augment the cheek area. Hyaluronicacid filling in these four facial areas isoften done in plastic surgery to achieve awell defined young looking contour.

In vivo study: This was a full-face, base formula controlled, randomized single center study. It took place from October 18th toDecember 15th, 2017, in Schenefeld/Hamburg, Germany. We recruited female Caucasian volunteers age 41 to 60 (mean 52.9 ± 4.8years) with self-perceived sagging of the skin. We had three cohorts (Base, Active, Benchmark) consisting of 30 volunteers each.Volunteers were instructed to apply their respective formulation twice daily to the entire face for 29 days. Instrumentmeasurements to assess various skin parameters were done at baseline, day 15, and day 29. Composition of the three formulationsis listed here:

Skin firmness was measured by DynaSkin (Eotech, France). An air stream is blown perpendicularly to a defined spot on the cheek.Depth, circumference, and negative volume of the as such generated whole in the cheek are measured.

Skin filling was measured by acquisition of 3D-facial scans using the AEVA-HE device (Eotech, France). Images were processed andusing a defined algorithm positive volume was assessed.

Skin hydration was measured by Corneometer (Courage & Kazaka, Germany). Five distinct spots each on right and left cheekbonewere measured and mean values calculated.

In vitro study: We grew 3D-microtissue (microspheres) resembling human skin. They consisted of a fibroblast core (dermis) and anouter keratinocyte layer (epidermis). The skin microspheres were treated for ten days with the active (consisting of the peptideproduct) and a positive control (basicFGF). Culture medium was replaced every other day. After ten days skin microspheres wereharvested, formalin fixed and paraffin embedded. They were cut into 6micron tissue sections for staining of hyaluronic acid (byalcian blue kit, Chroma 8GS, AppliChem GmbH, Germany).

Ex vivo study: Abdominal skin samples from a Caucasian female donor age 46 were harvested after obtaining informed consentand following Helsinki declaration. The solar simulator adopted for daily UV-irradiation was a BIO-SUN system produced by VilberLourmat (Eberhardzell, Germany). The selected UV irradiation intensity was 3 J/cm2 (= 0.1 J/cm2 UVB + 2.9 J/cm2 UVA)corresponding to 40% biologically effective dose (BED) for daylight UV [5]. Hyaluronic acid was stained by alcian blue (Alcian Blue8GX staining kit, Sigma- Aldrich cat #A3157). Semiquantitative evaluation of staining intensity was done by Image J software(NIH, Bethesda, US).

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Figure 4: A formulationcontaining 2.5% Active led toimproved skin hydration asmeasured by Corneometer onthe cheekbone of volunteers.*p<0.05 vs placebo. **p<0.01 vsbaseline. Error bars representstandard error of the mean.

Figure 6: 10 ppm tripeptide stimulated hyaluronic acid content in skin tissue ex vivo(blue staining) after UV-irradiation. Retinoic acid served a positive control and thetripeptide palmGHK as anti-ageing reference. *p<0.05 vs vehicle. Error barsrepresent standard error of the mean.

Figure 2: (A) A formulation containing 2.5% Active led toincreased volume as measured by AEVA-HE and distance perpixel in the area below the eyes of volunteers. Aformulation containing 2.5% Active led to a significantincrease in volume compared to base formulation. *p<0.05(B) 3D false color images of the area below the eyes (field1). Displayed are images of volunteers representingapproximately the mean values shown in A. (C) Volumemeasured by AEVA-HE 3D-image scans. A formulationcontaining 2.5% SYN-HYCAN led to a significantly increasedvolume compared to baseline after 29 days. *p<0.05 vsbaseline. Error bars represent standard error of the mean.

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% w/w % w/w % w/wBase Formulation (emulsion) 97.5 100 97Glycerin, Aqua, Butylene Glycol, Carbomer, Polysorbate 20, Palmitoyl Tripeptide-1, Palmitoyl Tetrapeptide-7 0 0 3

Glycerin, Aqua, Magnesium chloride, Tetradecyl Aminobutyroylvalylaminobutyric urea trifluoroacetate (tripeptide) 2.5 0 0

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