Novel anti-inflammatory peptides as cosmeceutical peptides

Peptides 32 (2011) 2134–2136

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Novel anti-inflammatory peptides as cosmeceutical peptides Youn-A Kang a,1 , Jung-Im Na a,1 , Hye-Ryung Choi a , Jee-Woong Choi a , Hee-Young Kang b , Kyoung-Chan Park a,∗ a b

Department of Dermatology, Seoul National University College of Medicine, Seoul National University Bundang Hospital, Seongnam-si, Republic of Korea Department of Dermatology, Ajou University School of Medicine, Suwon, Republic of Korea

a r t i c l e

i n f o

Article history: Received 25 July 2011 Received in revised form 19 August 2011 Accepted 20 August 2011 Available online 26 August 2011 Keywords: Cosmeceuticals Inflammation Peptides

a b s t r a c t Ultraviolet (UV) radiation induced inflammation plays an important role in the aging of human skin. Prostaglandin (PG) E2 is the primary mediator of UVB induced photoinflammation. We screened an internal library for dipeptides that inhibited UVB induced PGE2 synthesis but showed no cytotoxicity toward human keratinocytes. We identified three highly active inhibitory sequences, LE (Leu + Glu), MW (Met + Trp) and MY (Met + Tyr). To evaluate their efficacy in human skin, 24 sites of abdomen skin were irradiated with a 308 nm excimer laser (300 mJ/cm2 ), after which 2% LE, MW, MY or a control were applied to the irradiated sites for 24 h. The erythema index (EI) was measured before and 24 h after treatment. The results showed that LE and MW significantly decreased UVB induced erythema (p = 0.041 and p = 0.036, respectively), but ME did not. Overall, LE and MW are candidate cosmeceutical peptides that can protect skin from UVB induced photoinflammation. © 2011 Elsevier Inc. All rights reserved.

1. Introduction Bioactive peptides are short chains of amino acids that can trigger various cellular processes with protein interactions. When compared to monoclonal antibody drugs that also target protein interactions, bioactive peptides are easier to deliver and can access intracellular proteins. With the development of non-invasive transdermal delivery methods [10], topical peptides have become new options for the treatment of skin problems. In the literature, several types of peptides have been reported to have anti-inflammatory effects [1,2,4,6,11–13]. However, the effects of short chain peptides have not been thoroughly studied, even though better absorption is anticipated. Thus, the development of short chain peptides with active pharmacologic actions is needed. Ultraviolet radiation induced inflammation plays an important role in the aging of human skin [9]. The most prominent sign of inflammation resulting from ultraviolet light B (UVB) irradiation is erythema. It is well known that UVB leads to increased prostaglandin E2 (PGE2 ) synthesis, which is the primary mediator of erythema [8]. When skin is irradiated with UV, cycloxygenase is induced in keratinocytes, which in turn increases PGE2 [5].

∗ Corresponding author. Department of Dermatology, Seoul National University Bundang Hospital, 166 Gumi-ro, Bundang-gu, Seongnam-si, Gyeonggi-do 463-707, Republic of Korea. Tel.: +82 31 787 7311; fax: +82 2 3675 1187. E-mail address: [email protected] (K.-C. Park). 1 These authors contributed equally to this work. 0196-9781/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.08.017

In this study, we screened an internal library for dipeptides that inhibited UVB induced PGE2 synthesis but showed no cytotoxicity toward human keratinocytes. The photoinflammation inhibitory effects of these peptides were then tested on UVB irradiated human skin, in vivo.

2. Materials and methods Human keratinocytes were isolated from human foreskins obtained during circumcision in children. All samples were obtained with informed consent. Keratinocytes (105 cells) were cultured in 2 ml of keratinocyte growth medium (KGM, Clonetics, San Diego, CA, USA). After 24 h, the medium was replaced with KGM containing 0.1% BSA (Sigma, St. Louis, MO, USA). Twentyfour hours later, cells were pre-incubated with the same medium containing 10 ␮g/ml of peptide for additional 24 h. The cells were UVB-irradiated (100 mJ/cm2 , G8T5E, Sankyo Denki, Kanagawa, Japan) through the lid of the dish to remove the UVC. The applied energy was measured using a Waldmann UV meter (model No. 585100; Waldmann Co., VS-Schwenningen, Germany), after which the medium was replaced with 1 ml of phosphate-buffered saline (PBS) prior to UVB irradiation. The transferred medium was then returned to each well immediately after irradiation. After 24 h, 1 ml of medium was taken for PGE2 analysis and the cytotoxicity was measured by MTT assay. After the addition of 100 ␮l/well MTT solution (5 mg/ml), the plates were incubated for another 4 h. The supernatants were then removed and the formazan crystals were solubilized in 1 ml of dimethylsulfoxide, after which the

Y.-A. Kang et al. / Peptides 32 (2011) 2134–2136 Table 1 Cytotoxicity and ultraviolet (UV) B induced prostaglandin (PG)E2 synthesis inhibition by dipeptides. Samples were irradiated with100 mJ/cm2 of UVB and the effects of NS398 and dipeptides (LE, MW, and MY) were tested on cultured keratinocytes. None of the test products showed significant cytotoxicity in an MTT assay. PGE2 production was measured with a PGE2 assay kit. LE, MW, and MY effectively suppressed PGE2 synthesis, and this suppression was stronger than that of the positive control (NS398). Inhibition (%) was calculated as follows {(133.7 − concentration of PGE2 )/(133.7 − 75.8) × 100}. Results are the mean of duplicated samples and experiments were repeated two times. PGE2 assay MTT Control UVB (100 mJ/cm2 ) UVB (100 mJ/cm2 ) + NS398 (5 ␮M) LE (10 ␮g/ml) MW (10 ␮g/ml) MY (10 ␮g/ml)

100% 92.6% 89.1% 91.8% 96.3% 99.8%

Conc. (pg/ml) 75.8 133.7 81.5 55.42 39.0 69.9

% inhibition

Table 2 UVB induced erythema reduction by dipeptids. To evaluate the protective effect of topically applied dipeptides in vivo, 24 sites of abdomen skin were irradiated with a 308 nm excimer laser (300 mJ/cm2 ). Next, 2% LE, MW, and MY or a control were applied to the UVB irradiated sites (6 sites for each) for 24 h. The erythema index (EI) was measured before and 24 h after occlusion and EI differences (EI) were observed.

Control LE MW MY *

90.2 135.2 163.6 148.5

optical density was determined at 540 nm using an ELISA reader. The cycloxygenase-2 inhibitor, NS398 (5 ␮M, Sigma), was used as a positive control. The concentration of PGE2 was measured using a PGE2 Assay Kit (KGE004, R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s recommendations. All experiments were conducted twice and the mean results were used. Peptides that were more effective than the positive control were selected. LE (Leu + Glu, 95.6% purity), MW (Met + Trp, 99.2% purity) and MY (Met + Tyr, 98.1% purity) showed a highly active PGE2 inhibiting effect (Table 1). The anti-inflammatory actions of these small peptides were tested clinically. Twenty-four sites on the abdomen skin between breast and waist were irradiated with a single shot from a 308 nm excimer laser in one individual (corresponding author volunteered for the trial). In order to induce erythema in human skin, a higher dose of UV (300 mJ/cm2 ) was irradiated on abdomen skin instead of 100 mJ/cm2 , a dose for in vitro study. After UVB irradiation, 2% solution of each peptide and control (solvent, 2:1 mixture of 1,3butylene glycol and glycerin) were applied to the UVB irradiated sites (6 sites for each) for 24 h using Finn Chambers on Scanpor (Epitest, Ltd Tuusula, Finland). The erythema index (EI) was measured using a Mexameter MX18® (Courage-Khazaka Electronic GmbH, Cologne, Germany) before and 24 h after UVB irradiation and the EI differences (EI) were obtained. 3. Results In the MTT assay, 100 mJ/cm2 of UVB showed no significant cytotoxicity. NS-398, LE, MW and MY also showed no cytotoxicity towards keratinocytes (Table 1). Upon UVB irradiation, PGE2 synthesis from keratinocytes almost doubled. The positive control (NS-398) decreased UVB induced PGE2 synthesis to 39.0%, while LE, MW, and MY showed even better PGE2 synthesis inhibitory activity (Table 1). Clinically, topical application of LE and MW effectively reduced the EI increase after excimer laser irradiation (p = 0.041 and p = 0.036, respectively), while ME slightly increased the erythema (Table 2). 4. Discussion Cosmeceuticals are topical formulas with biologically active ingredients that are designed to improve the appearance of the skin [7]. Peptides are a rapidly expanding category of cosmeceuticals because they can modify many natural processes such as cell proliferation, inflammation, melanogenesis and protein synthesis. In addition, peptides composed of natural l-amino acids are generally not immunogenic and are readily broken down

2135

Before

24 h

EI

SD

P value vs control

167.44 172.72 174.72 166.22

363.50 353.22 350.72 382.11

196.06 180.50 176.00 215.89

20.12 22.03 20.79 36.56

0.041* 0.036* 0.097

p < 0.05 by the Mann–Whitney test.

into individual natural amino acids [14]. Peptides act through protein–protein interaction. In this study, the dipeptides LE, MW and MY inhibited UVB induced PGE2 synthesis effectively. The mechanism through which this occurred has not yet been investigated; however, they could act as signal peptides or precursors of other anti-inflammatory molecules. For clinical benefit, peptides must be delivered to the target. To be permeable, a relatively low molecular weight (<500 Da) is required [3]. All peptides tested in this study had a sufficiently low molecular weight (<400 Da). However, topically applied MY failed to prevent UVB induced erythema, in vivo. This may have been due to the hydrophilicity of tyrosine, which interferes with penetration through the corneal lipid layer. Topical application of LE and MW significantly reduced UV irradiation induced erythema, which means they can be transferred transdermally. In conclusion, our results demonstrate that short peptides such as LE and MW can have anti-inflammatory effects through anti-PGE2 effects. Thus, these findings suggest that small peptides have the potential for use in new drug development.

Acknowledgement This study was supported by a grant (A100179) from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea.

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