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| Newsletter | Skinasensyl® - A new peptidic active ingredient to reduce discomfort and painful sensations in sensitive skin |
Gilles Pauly, Philippe Moussou, Jean-Luc Contet-Audonneau, Louis Danoux, Olga Freis, Mélanie Sabadotto, Isabelle Benoit and Andreas Rathjens |
Abstract
Approximately 40% of the population, of all categories and phototypes, complains of having sensitive skin. Sensitive skin is healthy but over-responsive, i.e. it will react faster and more intensely to several parameters (outside factors such as temperature changes and sun, use of cosmetic products, certain medicines): experiences of discomfort, tingling, burning and intolerance to certain types of products, a condition referred to as neurosensitivity, characterized by a lower threshold of tolerance. Currently all of the causes for this are not known yet, but an increase of the permeability of the stratum corneum as well as an exaggeration of the nerve response are considered to be involved in the phenomenon of sensitive skin. Lifestyle has also an effect: tobacco, alcohol, stress, fatigue, emotions.
Skinasensyl®, a new synthetic tetrapeptide mimicking a natural opioid peptide, has been developed with the aim to decrease the stimulation of skin's nerve endings. This tetrapeptide has demonstrated in vitro a reducing effect of cutaneous over-reactivity by decrease of the CGRP release from sensory neurons via an agonist effect on μ opioid receptors, and in vivo an improvement of sensitive skin comfort through a decrease of unpleasant sensations and pain induced by heat and capsaicin.
This tetrapeptide targeting at an exaggerated nerve response, will help to relieve sensitive skin by normalizing the tolerance threshold to environmental factors or to certain topically applied uncomfortable products or skin care treatments.
Introduction (Pr Laurent Misery, Laboratory of Skin Neurobiology and Department of Dermatology, University of Brest, France)
Sensitive skin is a condition of subjective cutaneous hyperreactivity to environmental factors or stimuli [1]. Approximately half of the population (40% of men and 60% of women) considers themselves to possess the characteristics of sensitive skin [2]. Consumers who perceive their skin as sensitive report exaggerated reactions to certain cosmetic products, soaps and sunscreens, and worsening after exposure to dry, cold, windy or warm climate, sun or UV irradiation, polluted environment, physical treatments as depilation, shaving, or stress. They react with subjective symptoms like itching, burning, stinging, prickling, tingling or pain sensations and sometimes erythema.
Sensitive skins have been classified into three different types based on physiological parameters [3] : type I defined as the low barrier function group with high trans epidermal water loss and abnormal desquamation; type II defined as the inflammation group with normal barrier function and inflammatory changes; and type III defined as the neurosensitive group in terms of normal barrier function, and no inflammatory changes.
Human skin involves many sensory nerve fibers, up to the epidermis, which allow the central nervous system to be aware of the skin state, and react to different chemical, thermal or physiological stimuli. An exaggeration of this nerve response and a low activation threshold are involved in the phenomenon of sensitive skin. This neurogenic hyperreactivity is usually associated with the action of TRPV1 and the release of neuropeptides, such as CGRP [4].
A synthetic tetrapeptide Skinasensyl®, derived from opioid peptides, has been developed with the aim to decrease the stimulation of the skin's nerve endings, and thus to help to relieve sensitive skin, and to enhance its tolerance.
Experiments
The efficacy of Skinasensyl® to increase the skin's tolerance threshold of neurosensitive skin and its mechanism of action were checked:
- in vitro, on the CGRP release from sensory neurons,
- in vitro, by a competition test with a μ opioid receptor antagonist on sensory neurons,
- in vivo, by measuring the influence on the threshold of cutaneous sensitivity to different stimuli:
capsaicin and temperature.
Active Ingredient
Skinasensyl® is a synthetic acetylated tetrapeptide: N-Acetyl-L-Tyrosyl-L-Prolyl-L-Phenylalanyl-L-Phenylalaninamide (Skinasensyl® ; INCI Name Acetyl Tetrapeptide-15) (Fig. 1).
Fig. 1: Synthetic acetylated tetrapeptide Skinasensyl® (INCI Name: Acetyl Tetrapeptide-15).
Reference Substances
Capsazepine: vanilloid receptor 1 (TRPV1) antagonist
Verapamil: calcium channel blocker
Naloxone: opioid receptor antagonist
Abbreviations
ADC: adenylate cyclase
cAMP: cyclic adenosine monophosphate
CGRP: calcitonin gene-related peptide
DRG: dorsal root ganglion
ELISA: enzyme-linked immunosorbent assay
PKA: protein kinase A
SP: Substance P
TRPV1: transient receptor potential vanilloid 1
MTT: 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
CGRP Release From Cultured Sensory Neurons [5]
The neuropeptide release is proportional to the electrical activity of sensory neurons and was used as a parameter in order to evaluate the appeasing effect of Skinasensyl®.
The inhibiting effect of Skinasensyl® on CGRP release was evaluated by ELISA method on cultured sensory neurons (Fig. 2) (DRG differentiated by incubation for 10 days culture) after incubation for 6 hours at 37° C and stinging during 25 minutes by capsaicin (TRPV1 agonist, pungent constituent of chili peppers), or membrane depolarization by KCl. As positive controls, capsazepine and verapamil have been used as standard inhibitors of CGRP release, respectively for capsaicin and KCl stimulated neurons (Fig. 3). Lack of cytotoxicity of the acetylated tetrapeptide on the cell viability was determined by measurement of MTT.
Fig. 2: Culture of sensory neurons (DRG) (neuron cells labeled with anti-ß-tubulin antibody, nuclei stained with Hoechst solution).
Fig. 3: Protocol
Enlarged version
Inhibition Test of CGRP Release Via µ Opioid Receptor
To validate the action of Skinasensyl® via µ opioid receptor, a competition test was conducted between the peptide and naloxone (opioid receptor antagonist) on sensory neuron culture after capsaicin stimulation by measurement of CGRP release (ELISA method) (Fig. 4).
Fig. 4: Protocol.
Enlarged version
Clinical Test [6]
Cutaneous Capsaicin Sensitivity
Cutaneous application of capsaicin stimulates nerve endings via the vanilloid receptor 1 and causes the appearance of uncomfortable sensations such as itching, burning or stinging. The normalization of these sensations on the skin has been evaluated on the face of 20 human volunteers presenting exaggerated sensitivity to capsaicin, by determination of the detection threshold to increasing concentrations of capsaicin [7] before and after 4 days bi-daily application of 0.0015% Skinasensyl® (Fig. 5). This threshold was determined by successive hemi-face application on the nasolabial fold of solutions of capsaicin with increasing concentration: 0.316 µg/ml (noted 1), 1 µg/ml (noted 2), 3.16 µg/ml (noted 3), 10 µg/ml (noted 4) and 31.6 µg/ml (noted 5), scheduled each 3 minutes. The test was stopped as soon as the subject had reported a sensation (stinging, burning, itching...) on the side of capsaicin application. The corresponding applied concentration, from 1 to 5, is the capsaicin sensitivity threshold.
Fig 5: Protocol.
Enlarged version
Cutaneous Thermal Sensitivity
The soothing effect on the skin can be evaluated by measurement of the Cutaneous Thermal Sensitivity (CTS) using a thermal probe applied on the skin. The heat perception involves sensitive anatomical elements such as corpuscles and vanilloid receptors on terminal nerve fibers. The CTS method allows to study physical sensations, such as discomfort and pain.
An in vivo study was conducted to investigate the soothing effect of Skinasensyl® on the cutaneous sensitivity via the CTS method. The experiment was performed on the back of 16 female human volunteers and the soothing effect was determined as an increase of tolerance threshold to temperature for parameters discomfort and pain, determined before and up to 120 minutes after a single application on the same area of an emulsion containing 0.001% of Skinasensyl® in comparison to the placebo emulsion (Fig. 6).
Fig. 6: Protocol.
Enlarged version
Results and discussion
CGRP Release from Cultured Sensory Neurons
Fig. 7: Decrease of CGRP release from cultured sensory neurons.
Enlarged version
Capsaicin at 1 µM and KCl at 40 mM have strongly and significantly stimulated the release of CGRP, validating the in vitro model. Skinasensyl® has decreased the rate of released CGRP in resting, capsaicin-stimulated and KCl-depolarized neurons (Fig. 7). The acetylated tetrapeptide activity is significant and dose-dependent in the µM range. At the dose of 0.001% (16.3 µM), it is as effective as the TRPV1 antagonist capsazepine (10 µM) on the capsaicin-stimulated neurons, and as the calcium channel blocker verapamil (100 µM) on the KCl-depolarized neurons.
Inhibition Test of CGRP Release Via µ Opioid Receptor
Fig. 8: Inhibiting effect of CGRP release via µ opioid receptor.
Enlarged version
The increase of the released CGRP after stimulation of neurons by capsaicin comparatively to control conditions confirmed the functionality of testing model. Skinasensyl® has decreased the rate of released CGRP in capsaicin-stimulated neurons, but this effect was abrogated in the presence of the opioid receptor antagonist, naloxone. These results show that the inhibitory effect of Skinasensyl® on the capsaicin-induced CGRP release is mediated via the μ opioid receptor (Fig. 8).
Clinical Test
Cutaneous Capsaicin Sensitivity
The acetylated tetrapeptide Skinasensyl® has significantly increased the tolerance threshold to topically applied capsaicin concentration which provokes uncomfortable sensations as itching, burning or stinging (Fig. 9). The cutaneous reactivity to capsaicin was decreased of one concentration in mean, i.e. from 1µg/ml% to 3.16µg/ml in mean.
Fig. 9: Increase of perception threshold after treatment with Skinasensyl® at 0.0015% in hydroalcoholic solution.
Enlarged version
Cutaneous Thermal Sensitivity
Fig. 10: Clinical evaluation of the soothing effect of Skinasensyl® .
A: Increase of perception threshold with time after treatment with the emulsion with Skinasensyl® at 0.001%.
B: Increase of perception threshold for discomfort sensation and pain sensation by Skinasensyl® at 0.001% versus placebo
Enlarged version
A perceptible increase in the tolerance threshold was observed for sensorial discomfort and pain (significant 60 minutes after application of the emulsion with active ingredient (Fig. 10A)). The tendency of the soothing effect of the emulsion with Skinasensyl® versus placebo was observed 60 and 90 minutes after application. 120 minutes after application, an increase of sensorial discomfort threshold of 2.5°C (p=0.025 vs placebo) and of pain threshold of 1.7°C (p=0.073 vs placebo) was registered (Fig. 10B).
Discussion
A stimulation of the skin nerve response is a determining step in the phenomenon of sensitive skin. The skin includes many nerve structures like nervous corpuscles and nerve fibers, in particular the C fibers present in the epidermis up to the stratum granulosum. They allow a control by the central nervous system, of all the skin functions.
The stimulation of the nerve fibers by several stimuli (environmental or chemical) generates a nervous influx to the central nervous system inducing subjective symptoms like itching, burning, stinging, prickling, tingling or discomfort and even pain sensations (Fig. 11).
Simultaneously the nerve endings activation locally provokes the release of neuromediators like CGRP which then activates the release of cytokines, inducing a vasodilatation and an inflammatory process.
Fig. 11: Schematic representation of the mechanism and consequences of sensitive neuron activation by stress on skin.
Enlarged version
To fight against skin neurosensitivity, we propose a new peptidic active ingredient, Skinasensyl® which is derived from endomorphin 2, a highly selective natural agonist for the µ opioid receptor [8]. This endogenous peptide has been reported for its anti-nociceptive effect on inflammatory, chronic and neuropathic pain [9].
We have shown on cultured sensory neurons that Skinasensyl® attenuates the nervous response by inhibiting the release of CGRP.
Fig. 12: Inhibitory action of Skinasensyl® on capsaicin-evoked TRPV1 response via the μ opioid receptor.
Enlarged version
An important receptor for the stimulation of nervous response is the vanilloid receptor 1 (TRPV1), which is an ionic channel (Ca2+) activated by capsaicin, as well as protons and heat [10]. We have demonstrated that Skinasensyl® reduces the potentiation of capsaicin-evoked TRPV1 response via the µ opioid receptor which are co-expressed with TRPV1 receptors on DRG [11].
The signal pathway involved goes through the inhibition of adenylate cyclase to inhibit cAMP-dependent PKA which potentiates the TRPV1 response (Fig. 12) [11, 12]. This mechanism of action of Skinasensyl® is in correlation with the benefit obtained in vivo: an increase of sensitive tolerance threshold of skin, in analogy to a decrease of cutaneous hyperreactivity and a normalization of its resistance to external stimuli.
Conclusion
A new tetrapeptide, agonist of the µ opioid receptor, has demonstrated in vitro a reducing effect of cutaneous overreactivity by decrease of CGRP release from sensory neurons, and in vivo an increase of skin’s tolerance threshold to different nerve ending’s stimuli, capsaicin and heat.
The main complaints of sensitive skin such as sensation of discomfort and pain are perceivably reduced.
This tetrapeptide, targeting an exaggerated nerve response, will help to relieve sensitive skin by a reduction of the cutaneous hyperreactivity to external stimuli.
Sensitive skin is significantly soothed, and the tetrapeptide can be offered in the latest skin care treatments without concern.
Acknowledgments
This work was supported by the contribution of K. Tiveddu and P. Wolff for their technical assistance, and also A. Courtois for her editorial assistance.
Notes
Skinasensyl®, Synthetic acetylated tetrapeptide Ac-YPFF-NH2 (INCI Name: Acetyl Tetrapeptide-15) is a registered trademark of Laboratoires Sérobiologiques - Division de Cognis France.
This article was presented as a poster at the IFSCC 2008, Barcelona, 6-9/10/08 and published in IFSCC Magazine Vo l. 12, No. 1 /2009, pp.25-30.
References
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[2] Saint Martory, C., Roguedas Contios, A.M., Sibaud, V., Degouy, A., Schmitt, A.M., Misery, L., Sensitive skin is not limited to the face, Br J Dermatol, 158 (2008) 130-133.
[3] Yokota, T., Matsumoto, M., Sakamaki, T., Hikima, R., Hayashi, S., Yanagisawa, M., Kuwahara, H., Yamazaki, S., Ogawa, T., Hayase, M., Classification of sensitive skin and development of a treatment system appropriate for each group, IFSCC Mag., 6 (2003) 303-307.
[4] Boulais, N., Misery, L., The epidermis: a sensory tissue, Eur J Dermatol, 18 (2008) 119-127.
[5] Steinschneider, R., Quelven, E., Grousson, J., Laurent, J.C., Screening of molecules with anti-inflammatory properties and for sensitive skin applications using rat sensory neurons cultured or not with human keratinocytes, COSM'ING, (2001) 211-219.
[6] Girard, P., Etude de la sensibilité thermique cutanée, Journal de la société française de cosmétologie, 13 (1992) 7-12.
[7] Jourdain, R., Bastien, P., De Lacharriere, O., Rubinstein, G., Detection thresholds of capsaicin: a new test to assess facial skin neurosensitivity. J. Cosmet. Sci., 56 (2005) 153-166.
[8] Zadina, J.E., Hakler, L., Ge, L.J., Kastin, A.J., A potent and selective endogenous agonist for the mµ-opiate receptor, Nature, 386 (1997) 499-502.
[9] Zadina, J.E., Isolation and distribution of endomorphins in the central nervous system, J. Pharmacol., 89 (2002) 203-208.
[10] Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D., Julius, D., The capsaicin receptor: a heat-activated ion channel in the pain pathway, Nature, 389 (1997) 816-824.
[11] Vetter, I., Wyse, B.D., Monteith, G.R., Roberts Thomson, S.J., Cabot, P.J., The mµ opioid agonist morphine modulates potentiation of capsaicin-evoked TRPV1 responses through a cyclic AMP-dependent protein kinase A pathway, Molecular Pain, 22 (2006) 16p.
[12] Endres-Becker, J., Heppenstall, P.A., Mousa, S.A., Labuz, D., Oksche, A., Schäfer, M., Stein, C., Zöllner, C., Mµ-opioid receptor activation modulates transient receptor potential vanilloid 1 (TRPV1) currents in sensory neurons in a model of inflammatory pain, Mol. Pharmacol., 71 (1) (2007) 12-18.
Author
Dr. Gilles Pauly
Dr Gilles Pauly, medical doctor, dermatologist and immuno-allergist, was co-founder in 1982 and Research Director of the Research Center of Laboratoires Sérobiologiques. He is co-author of more than 88 patents and 98 publications and scientific presentations in the cosmetic field. After the acquisition of Laboratoires Sérobiologiques by Cognis in 1999, Gilles Pauly was successively R&D Manager, and has been Scientific Director since 2005. The R&D Department has developed a strong know-how in in-vitro technologies of cell cultures and molecular biology, ex-vivo/ in vivo efficacy tests, histology, immunohistochemistry with 2D and 3D image analysis and in phytochemistry.
Cognis France, Division Laboratoires Sérobiologiques, 3 rue de Seichamps, CS71040 Pulnoy 54272 ESSEY LES NANCY CEDEX, France.
Tel: +33(0)3 83 29 08 02,
Fax: +33(0)3 83 21 12 15
E-mail: Gilles.pauly@cognis.com
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