615 Pentapeptide to Control Acne
biotin-conjugated anti-collagen-IV antibody for 1 hour, followed by the substrate solution
(containing streptavidin-phycoerythrin conjugate) for 15 minutes at room temperature. The
reaction was then stopped, and absorbance was measured at 450 nm using a microplate
reader.
EQUIVALENT SKIN MODEL
Human Living Skin Equivalents (LSE) were produced in-house (adapted of method described
by Asselineau et al.25). Briefly, for equivalent-dermis, NHF were seeded into collagen-I
matrix (Jacques Boy, France) in 6-well plate inserts (Falcon™, New York, USA) and kept
emerged for 48 hours to allow dermal contraction. Human keratinocytes (NHK, CellnTec)
were seeded on top of the equivalent dermis. Models were cultivated for 7 days in immersion
media, followed by 7 days of air-liquid interphase to obtain a stratified epidermis. 9 ppm of
pKTSKS or its solvent (0.1% DMSO) were applied on top of the epidermis. After 48 hours
of treatment, LSE were embedded in freezing media (Leica, UK) and snap-frozen in liquid
nitrogen. 5–7 µm sections were prepared using a cryostat (CM15105, Leica, Germany). LSE
histology was evaluated by H&E (Sigma Aldrich, Germany) staining. Thickness of stratum
corneum was quantified on pictures captured under the microscope (Eclipse TI) using NIS
Element Software (both Nikon, Japan).
CLINICAL TRIALS
Two single-blind studies were performed on volunteers with acne-prone skin and acne scars.
The first study conducted on a panel of 30 volunteers made of 7 men and 23 women aged
between 18–39 years old with a mean age of 24 years old and a phototype III, was aimed
to measure pKTSKS effects on C. acnes and S. epidermidis populations. The second study
dedicated to topographic measurements was included a panel of 18 volunteers comprising
one man and 17 women aged from 20–58 years old with a mean age of 32, phototype II–IV
to evaluate the peptide’s effects on skin texture and scar improvement. Creams containing
12 ppm pKTSKS or placebo were formulated (see the formula in Figure 1). Volunteers
applied placebo on one cheek and pKTSKS-cream on the other, twice a day for up to 2
months. To study effects on C. acnes and S. epidermidis populations, RT-qPCR (real time
quantitative polymer chain reaction) analysis was performed on skin microbiota sampled
from 30 volunteers, before and after 1 month treatment. In the topographic variation
study, acquisitions were taken on 18 volunteers before and after 2 months treatment (this
relatively low number of volunteers was due to many dropouts linked to the Covid-19
pandemic). An Antera3D camera was used for analysis of various topographic parameters
such as inflammatory marks, pockmark, and skin roughness.
STATISTICS
ANOVA was used to determine whether there was any significant difference between
the means of two or more independent groups. Difference between two means with
similar variances was performed with Student’s t-test. A p-value p 0.05 or p 0.01 was
considered statistically significant.

616 JOURNAL OF COSMETIC SCIENCE
RESULTS
From a preliminary screening of peptides performed on both NHK, NHF, and C. acnes,
we selected the pKTSKS peptide which modulated pre-inflammatory mediators release,
reduced the growth of C. acnes cells and improved extracellular matrix protein synthesis
without any observed cytotoxicity.
EFFECT OF PKTSKS AND ITS COMPOUNDS ON C. ACNES AND S. EPIDERMIDIS GROWTH
Growth of C. acnes with pKTSKS. C. acnes RT-1 cells grew regularly from 21–53 hours in
their cell culture medium with 0.1% DMSO (control). The presence of 0.1% DMSO had
no impact on C. acnes growth. On the other hand, pKTSKS (6–12 ppm) strongly reduced
C. acnes growth (Figure 2) with a dose-dependent effect. When treated with 6 ppm,
9 ppm and 12 ppm of peptide, C. acnes reached an OD
600nm
of 0.5 at respectively 52 hours,
77 hours, and 96 hours. All these values were significant compared to solvent control
(p 0.01). The peptide slows down the growth of this bacterium, limits its ability to reach
its quorum and to potentially acquire a more virulent phenotype.
In a second set of assays, RT-4, and RT-5 strains of C. acnes were tested in the same way
as RT-1. RT-5 and RT-4 strains required 3X and 2X more time respectively than RT-1 to
reach an OD
600nm
of 0.5 (data not shown). Therefore, it appears that, in vitro, the effects of
pKTSKS differ according to the strain used.
The in vitro activity was validated via a clinical trial. Analysis demonstrated that C. acnes
population of the pKTSKS treated zones of the face was reduced by 18.5% versus the zones
treated with the placebo cream (p =0.067), hence confirming the in vitro results.
Growth of C. acnes with KTSKS and/or palmitic acid. To further validate the interest of the
peptide pKTSKS, C. acnes RT-1 cells were put in contact with either the palmitic acid
alone (4 ppm), the “naked” peptide (8 ppm KTSKS), the association of 4 ppm palmitic acid
+8 ppm KTSKS (note that in the latter, palmitic acid and KTSKS do not form a single
INCI Placebo cream Active cream
%%
Water Qsp 100 Qsp 100
Squalane 3.00 3.00
C12-15 Alkyl Benzoate 2.00 2.00
Glycerin 2.00 2.00
Polysorbate 20 1.00 1.00
Phenoxyethanol 0.80 0.80
Caprylyl Glycol 0.50 0.50
Sodium Hydroxide 0.50 0.50
carbomer 0.45 0.45
Acrylates/C10-30
Alkyl Acrylate
Crosspolymer
0.20 0.20
Potassium Sorbate 0.15 0.15
Fragrance 0.10 0.10
pKTSKS 0.00 0.0012
Figure 1. Formulation composition of the placebo and active creams used for the two clinical studies.