J. Cosmet. Sci., 63, 259–265 ( July/August 2012) 259 Inhibition of sebum production and Propionibacterium acnes lipase activity by fullerenol, a novel polyhydroxylated fullerene: Potential as a therapeutic reagent for acne SHIGEKI INUI, HISAE AOSHIMA, MASAYUKI ITO, KEN KOKUBO, and SATOSHI ITAMI, Department of Regenerative Dermatology, Graduate School of Medicine, Osaka University, 2-2, G2 Yamadaoka, Suita, Osaka, 565-0871 Japan (S. In., S. It.) Vitamin C60 BioResearch Corporation, 1-3-19 9F Yaesu Chuo-ku, Tokyo, 103-0028 Japan (H.A., M.I.) and Division of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871 Japan (K.K.). Accepted for publication December 27, 2011. Synopsis Oxidative stress plays a major role in acne formation this suggests that oxygen-radical scavengers could be potential therapeutic agents. Fullerenol C60(OH)44, a recently developed polyhydroxylated fullerene, is a spherical carbon molecule that has many hydroxyl groups capable of potent radical-scavenging activity. We have investigated its inhibitory effects in vitro on sebum production in hamster sebocytes and in Propionibacterium acnes lipase activity. Sebum production was signifi cantly reduced by 1.5 μM of fullerenol in cells that had been irradiated with 10 mJ/cm2 UVB, although it was not altered in the non-irradiated cells, indicating that fullerene is a sebum suppressor for sebocytes under oxidative stress, such as that induced by UVB. It was also found that fullerenol has inhibitory activity against P. acnes lipase. These results suggest that fullerenol could be a beneficial skin care reagent for control- ling acne vulgaris by suppressing sebum in the infl ammatory response and by reducing P. acnes lipase activity. INTRODUCTION Acne vulgaris is a common infl ammatory skin disease of the pilosebaceous follicles it is characterized by a multifactorial pathogenesis that includes sebaceous gland hyperplasia, increased sebum production, hyperkeratosis of hair follicle pores, and Propionibacterium acnes colonization. However, previous studies have shown the major roles of oxidative stress in the pathophysiology of acne (1–6). Superoxide dismutase (SOD) activity is lower, and hydrogen peroxide generation increased (4) from the Address all correspondence to Shigeki Inui. Address all e-mail to inui@r-derma.med.osaka-u.ac.jpo

JOURNAL OF COSMETIC SCIENCE 260 leukocytes of acne patients. Further, alteration of oxidative stress parameters such as catalase, glucose-6-phosphate dehydrogenase, SOD, and malondialdehyde (MDA) in the venous blood of acne patients has been reported, favoring a role of ROS in acne (5), and patients with severe acne reportedly show higher MDA levels in the skin tissues and plasma (6). From therapeutic aspects, tetracycline, erythromycin, and minocycline are preferable, compared with other antibiotics, because of their ability to suppress the generation of ROS from neutrophils (7,8). Moreover, although ben- zoyl peroxide (BPO), one of well-established acne treatments, exerts its anti-acne effect through antibacterial activity due to the production of ROS (9), it possess di- rect cytotoxic effects on leukocytes, conversely resulting in the inhibition of ROS generation by neutrophils in a dose-dependent manner (10). This evidence to support the major pathogenic roles of oxidative stress in acne suggests that oxygen radical scavengers, including fullerene and its derivatives, could be potential therapeutic agents. Fullerene is a spherical carbon molecule that exhibits powerful radical-scavenging activ- ity because of its unique “cage” structure. Consequently, the antioxidant activity of fuller- ene is several-hundred-fold higher than that of other antioxidants (11). Thus, fullerene has been reported to be benefi cial in the treatment of neurodegenerative disorders (12) and arthritis (13–15) furthermore, it is expected to be applicable to various oxidative diseases (16). It has been reported that fullerene exhibits protective activity against kera- tinocyte apoptosis caused by the reactive oxygen species (ROS) that arise from UV expo- sure (17). Fullerene penetrates well into the epidermis (18) without skin irritation or toxicity (19,20), and its potential as a topical drug for acne vulgaris has been proposed (21). Indeed, our open clinical trial showed its effi cacy on acne vulgaris (22). However, fullerene is problematic in biological applications because it has poor solubility in polar solvents such as H2O. In recent studies, a polyhydroxylated fullerene derivative, fuller- enol, which is produced by a simple synthesis method, has been shown to have excellent water-soluble properties (23,24). In addition, our previous studies have demonstrated that fullerenol exhibits antimicrobial activity against P. acnes in addition to its potent antioxidant activity (25–28). These discoveries have prompted us to investigate whether fullerenol could be useful for the treatment of acne vulgaris. In this study, we have exam- ined the ability of fullerenol to suppress sebum production, and to inhibit the lipase ac- tivity of P. acnes. EXPERIMENTAL MATERIALS Fullerenol was synthesized according to the method reported in a previous study (24). Hamster sebocytes, derived from the sebaceous glands of the auricles of fi ve-week-old male golden hamsters, HuMedia-BB, and an assay kit for lipid production (SE-3001) were purchased from Kurabo Co. (Osaka, Japan). Clinically isolated P. acnes was ob- tained from Nicoderm Research Inc. (Osaka, Japan). GAM medium and adapalene were purchased from Nissui Pharmaceutical Co. Ltd. (Tokyo, Japan) and Funakoshi Co. Ltd. (Tokyo, Japan), respectively. We purchased 4-methylumbelliferone (4-MU) and 4-MU-oleate (4-MUO) from Sigma (St. Louis, MO).