A double-blind, randomized, vehicle-controlled efficacy assessment study of a skin care formulation for improvement of mild to moderately severe acne

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1 DOI: /jdv JEADV ORIGINAL ARTICLE A double-blind, randomized, vehicle-controlled efficacy assessment study of a skin care formulation for improvement of mild to moderately severe acne I. Angelova-Fischer, 1, * F. Rippke, 2 T.W. Fischer, 1 G. Neufang, 2 D. Zillikens 1 1 Department of Dermatology, University of L ubeck, Germany 2 Beiersdorf AG, Hamburg, Germany *Correspondence: I. Angelova-Fischer. irena.angelova-fischer@uk-sh.de Abstract Background Inflammation, increased sebum production and P. acnes colonization are key factors in acne pathogenesis. Cosmetic formulations based on a combination of active compounds with in vitro proven anti-inflammatory, sebum regulating and P. acnes reducing properties may therefore contribute to improve the clinical signs and associated burden of disease. Objective To provide in vivo proof-of-concept, we performed a 9-week, double-blind, randomized, vehicle-controlled study to assess the stand-alone efficacy of a skin care formulation containing licochalcone A, L-carnitine and 1,2-decanediol in volunteers with mild to moderately severe acne (10 25 inflammatory lesions) involving the face. Materials and methods After enrolment followed by a 1-week standardization of the cleansing procedure, 60 volunteers aged years (40 women and 20 men, mean age 22.4 years) were randomized into two groups of 30 volunteers each, to apply either the active formulation or the vehicle twice daily on the face for 8 weeks. Reduction in the lesion count, P. acnes and sebum levels, stratum corneum hydration, Dermatology Life Quality Index (DLQI) and skin tolerability, assessed after 4 and 8 weeks were defined as outcomes. Results Compared to baseline, the active formulation group showed at the end of the study a reduction in the mean total lesions count and papular lesions, significant reduction in the pustules (P < 0.05) and sebum levels (P < 0.01), marked reduction in P. acnes and improvement of DLQI. No significant changes in the respective parameters were found in the vehicle group. At the end of the study, greater reduction in the total lesion count, papules and pustules, P. acnes colonization, sebum production and more pronounced improvement of life quality in the active formulation group compared to the vehicle were found. Conclusions Our results provide evidence for improved outcomes in result of the application of the active formulation compared to the vehicle from both physician s and patient s perspective. Received: 2 April 2013; Accepted: 15 April 2013 Introduction Acne is a common skin disease with considerable psychological and social impact. 1 Several interrelated pathogenic factors such as abnormal follicular keratinization, sebum production, P. acnes follicular colonization and inflammation contribute to the morphology and clinical manifestations of acne that may vary from mild non-inflammatory forms to a widespread severe inflammatory condition with systemic symptoms. 2 6 Along with inflammation, local and systemic oxidative stress has been recently shown to play a role in disease initiation. 7 Combination treatment regimen directed towards two or more of the key pathogenic factors are first-line therapy to induce clinical improvement in most mild to moderately severe cases; the achievement of sustained remission, however, may require maintenance therapy supported by adjunctive and/or cosmetic treatments to maintain the level of improvement in the patient s skin condition. 8 In addition to the therapeutic strategies reviewed in the current guidelines, the use of adjuvant skin care is integral to the management of acne. 9 The treatment algorithm should consequently incorporate an appropriate daily skin care regimen to cleanse, soothe and moisturize the skin. 10 Considered a general measure to minimize the adverse effects of treatment, the evidence for the stand-alone efficacy of medical skin care formulations, developed to target relevant factors in acne pathogenesis has been limited and derived mostly from within-group comparisons while there have been no published, double-blind, randomized controlled clinical trials to assess the effects with respect to improvement of the clinical signs and life quality. Study aim The aim of this double-blind, randomized, vehicle-controlled study was to assess the efficacy of a skin care formulation

2 Efficacy of a skin care formulation in acne 7 containing licochalcone A, L-carnitine and 1,2-decanediol in volunteers with mild to moderately severe inflammatory acne. The outcomes were measured by physician assessment of the clinical signs, non-invasive bioengineering measurements and dermatology-specific life quality measures. Materials and methods Study population Sixty volunteers aged years (40 women and 20 men, mean age 22.4 years) with mild to moderately severe papulopustular acne (10 25 papular and/or pustular lesions) affecting the face were enrolled in the study. The exclusion criteria were defined as follows: (i) Use of topical or systemic acne medication in the last 4 weeks preceding the study; (ii) special forms of acne; (iii) intensive UV-light exposure within the preceding 4 weeks, as well as for the entire duration of the study; (iv) another skin disorder in the test area; (v) known or suspected contact sensitization to the test formulation ingredients; (vi) severe concomitant diseases; (vii) pregnancy or lactation and (viii) participation in another study within the preceding month. The study was performed in agreement with the Helsinki Declaration principles. The protocol and group size were approved by the local ethics committee (no ) and all participants gave written informed consent/assent beforehand. Study design The study was a 9-week, prospective, double-blind, randomized, active formulation to vehicle comparison test. After recruitment, the participants entered a pre-conditioning phase to standardize the daily cleansing routines, limited to the use of the same cleansing gel and facial tonic for the entire duration of the study. After 1 week (D0), the volunteers were randomized into two groups of 30 volunteers each to receive either the active formulation, a light o/w emulsion containing licochalcone A, L-carnitine and 1,2-decanediol or the vehicle, applied on the face twice daily for 8 weeks. The skin condition in the test area was assessed by the same investigator at baseline (D0), at 4 weeks (D28) and 8 weeks (D56). The test formulations were supplied in identical neutral package. The use of other skin care products or acne medication throughout the study was not allowed. The study was performed between November 2009 and April 2011; there was no recruitment in the months May October Outcomes and methods of assessment The outcomes included reduction in the inflammatory lesions, P. acnes and skin surface sebum content, skin hydration (capacitance), life quality and assessment of the skin tolerability. The lesion count was based on assessment of the papules, pustules and total number of inflammatory lesions in the test area by visual inspection and palpation. In addition, standardized cross-polarized facial photographs (FotoFinder Mediscope; FotoFinder Systems GmbH, Bad Birnbach, Germany) were taken. The skin surface sebum content was measured with the Sebumeter SM815 and skin hydration was assessed by measuring capacitance with the Corneometer CM825, both devices from Courage and Khazaka Electronics (Cologne, Germany). For each parameter, three consecutive measurements on defined fields on the forehead and cheek were performed by the same observer under controlled environmental conditions (room temperature 20 2 C; average relative humidity 40% 45%) and according to the published guidelines. 11,12 The measurement of sebum production was performed by means of adhesive tapes (Sebutape â, CuDerm Corp., Dallas, TX, USA) applied on the forehead for 30 minutes and assessed by a 7-point scale as previously described. 13 The P. acnes counts were assessed following a previously established protocol. Briefly, ml phosphate buffer solution with Triton X-100 was pipetted on the skin with the help of a teflon ring and the skin surface was carefully rubbed with a spatula for two times 1 minute. The samples were transferred into sterile Sarstedt tubes (Sarstedt, N umbrecht, Germany); 100 ll of the original suspension as well as 1 : 100 and 1 : 1000 dilutions of the same were plated in duplicate on columbia agar plates, supplemented with defibrinated sheep blood and furazolidone and the colonies were counted after 96-hour cultivation at 37 C under anaerobe conditions. Life quality was measured by Dermatology Life Quality Index (DLQI). 14 The assessment of the skin tolerability was based on a standardized questionnaire including global assessment and record of the reported symptoms. Statistical analysis Statistical analysis was performed using GraphPrism Version 4 (GraphPad Software Inc, San Diego, CA, USA). A P-value < 0.05 was considered statistically significant. The changes in the lesion counts, sebum content, capacitance, P. acnes and DLQI within each group over time were analysed by repeated measures ANOVA or Friedmann test for the respective parameter; for P-values less than 0.05 a post hoc test was performed. The differences between the groups at baseline, at 4 weeks and at 8 weeks were analysed by unpaired t-test or Mann Whitney test. All values are expressed as mean and standard error of the mean (SEM) except for the lesion counts and DLQI expressed as median. Results Fifty-one volunteers completed the study according to the protocol. Therefore, the analysis and comparison of the outcomes was based on data from 25 volunteers for the active formulation group and 26 volunteers for the vehicle (Fig. 1). The reasons for drop out were as follows: vehicle group lack of compliance (n = 2), worsening of the skin condition in the pre-conditioning phase (n = 2); active formulation group lack of compliance

3 8 Angelova-Fischer et al. (n = 3), consent withdrawal (n = 1), erythema in the test area at the end of the pre-conditioning phase (n = 1). At baseline, there were no significant differences in the total inflammatory lesions count, skin surface sebum content, stratum corneum hydration and DLQI between the groups. The clinical assessment of the skin condition at the end of the study found no significant differences in the total number of inflammatory lesions in result of the application of the vehicle (median total lesion count on D0 and D56, respectively, 19.2 and 19.4). In the active formulation group, significant reduction in the total inflammatory lesions count on D56 compared to D28 was found (median counts on D28 and D56: 20.9 and 18.9, P < 0.05). The percentage reduction in the total lesions counts at the end of the study (D56) compared to baseline (D0) was greater in the active formulation ( 6.9%) than the vehicle group (+1.4%), the difference between the groups (8.3%), however, was not significant. In the vehicle group, the clinical assessment with regard to the type of inflammatory lesions showed no significant reduction in the number of papules and pustules on D28 and D56 compared to baseline (D0). The application of the active formulation resulted in significant decrease in the number of papules on D56 compared to D28 Enrolled volunteers /n = 60/ Baseline evaluation /D0/ Active formulation (n = 29) Vehicle (n = 28) Evaluation D28 (n = 26) Evaluation D56 (n = 25) Figure 1 +4 weeks +8 weeks 1 week pre-conditioning phase Evaluation D28 (n = 26) Evaluation D56 (n = 26) Analysed n = 25 Analysed n = 26 Study flowchart. (median counts on D28 and D56, respectively, 20.4 and 18.3, P < 0.05) as well as significant reduction in the pustular lesions on D56 compared to D0 (P < 0.05; Figs 2 and 3). The changes in the skin surface sebum content in the vehicle and active formulation group throughout the study are presented in Fig. 4. At the end of the study, the sebum levels in the vehicle group were comparable to baseline ( and AU, respectively, on D0 and D56). In contrast, the measured sebum levels in the active formulation group were significantly reduced ( and AU; P < 0.01); the difference in percentage reduction between the groups, however, was not significant. The Sebutape â analysis at the end of the study showed, in addition, a more pronounced decrease in the sebum production in result of application of the active formulation than the vehicle (mean score difference compared to D0, respectively, 0.42 and 0.21). The capacitance values at the end of the study increased compared to baseline in both groups (vehicle: D AU, D AU, P < 0.01; active formulation, respectively, AU and AU, P > 0.05). No significant differences between the groups throughout the study were found. The effects of the active formulation and vehicle on P. acnes counts are shown in Fig. 5. Although the differences were not significant, compared to baseline, at the end of the study there was a marked reduction in P. acnes in the active formulation group ( 49.4%) and minor changes in the vehicle group ( 3.2%). The median DLQI in the active formulation and vehicle group on D0 was 2.5 and 2.0 respectively. The improvement of life quality, assessed by the DLQI decrease at the end of the study compared to baseline, was greater in the active formulation group than the vehicle though the difference between the groups was not significant (in both groups, median DLQI on D56 1.5; reduction compared to baseline, respectively, 1.0 and 0.5). The tested formulations were well tolerated. The most common symptoms were mild skin dryness and tightness reported in each case by 12% of the volunteers in the active formulation group as well as, respectively, by 7.7% and 3.8% of the volunteers in the vehicle group. Discussion The management of acne requires the correct choice of a treatment regimen with respect to the type and severity of the clinical manifestations, supported by general measures and patient education to enhance compliance and reduce the psychological burden of disease. 15 Skin irritation and dryness are common side-effects of acne medications and the use of skin care, adjuvant to treatment, is an established approach to optimize tolerability and improve adherence to therapy in the induction as well as the maintenance phase. 10 In addition to enhancement of compliance, well-designed skin care formulations may possibly contribute to improvement of the skin condition and

4 Efficacy of a skin care formulation in acne 9 D * % change of baseline D0 D28 D56 D0 D28 D56 Vehicle Active formulation Figure 3 Significant reduction in the pustules in the active formulation group at the end of the study (D56) compared to baseline (D0). The data are presented as percentage change from baseline, level of significance set at 0.05; *P < D ** Skin surface sebum /AU/ D0 D28 D56 D0 D28 D56 Vehicle Active formulation Figure 4 Sebum levels in the active formulation and vehicle groups at baseline (D0), at 4 weeks (D28) and 8 weeks (D56) measured by Sebumeter. Mean standard error of the mean (SEM), level of significance <0.05; **P < 0.01; AU-arbitrary units. Figure 2 Cross-polarized photographs of the test area at the beginning (D0) and end of the study (D56): active formulation group. patient s well-being. 16 Therefore, even though not a regulatory requirement for skin care, double-blind, randomized, controlled clinical trials based on validated measures may be important evidence-based sources for the exerted effects. Inflammation, sebum production and P. acnes follicular colonization are relevant factors in the pathogenesis of acne. Consequently, cosmetic formulations based on a combination of active compounds with in vitro proven anti-inflammatory, sebum

5 10 Angelova-Fischer et al. cfu D0 D28 D56 D0 D28 D56 Vehicle Active formulation Figure 5 Propionibacterium acnes counts in the active formulation and vehicle groups at baseline (D0), at 4 weeks (D28) and at 8 weeks (D56). Mean standard error of the mean (SEM), level of significance <0.05. regulating and P. acnes reducing properties may contribute to the improvement of symptoms in patients with inflammatory disease forms. The improved outcomes as a result of the 8-week application of the active formulation containing licochalcone A, L-carnitine and 1,2-decanediol compared to the vehicle, as shown in our study, provide in vivo evidence in support of this concept. The anti-inflammatory and anti-oxidative properties of licochalcone A, a reversely constructed chalcone specific for the Xynjiang licorice root Glycyrrhiza inflata have been previously described The pharmacological activity of licochalcone A-rich extracts from G. inflata and synthetic licochalcone A was shown to be directed towards suppression of PGE 2, LTB 4 as well as IL-6 and TNF-a production in in vitro systems relevant to the skin such as dermal fibroblasts, granulocytes, dendritic cells and human skin equivalents The reduction in the pro-inflammatory mediators in vitro has been shown to translate into in vivo efficacy by reducing experimentally induced erythema and improving the inflammatory signs in patients with rosacea and atopic dermatitis. 20,23,24 To the best of our knowledge, the effects of licochalcone A in mild to moderately severe inflammatory acne forms have not been previously investigated. The more pronounced clinical improvement in the active formulation group found in our study thus extends the published knowledge and the spectrum of conditions that may be positively influenced by the application of licochalcone A-containing skin care formulations due to their anti-inflammatory and anti-oxidative properties. Increased sebum is a common associated clinical feature and one of the key pathogenic factors that contribute to the development of acne lesions. 25 The reduced skin surface sebum levels in the active formulation group therefore, attributed to L-carnitine, are important findings of the study. L-carnitine has been recently shown to reduce sebum production through concentrationdependent stimulation of ß-oxidation and consequent decrease in the amount of the intracellular lipid content in the immortalized human sebaceous gland SZ95 line. 26,27 The sebum regulating properties of L-carnitine have been additionally investigated in a vehicle-controlled split-face study in human volunteers with oily facial skin that showed significant reduction in the sebum amount after 3-week application of a cosmetic formulation containing 2% L-carnitine. 26 The results of our study are in agreement with these findings and provide further evidence for the sebum regulating efficacy of L-carnitine in vivo. Similarly, the pronounced reduction in P. acnes in result of the application of the active formulation found in the study correlated with the results of in vitro suspension tests showing a rapid decrease in P. acnes in the presence of the 1,2-decanediol containing formulation compared to the vehicle (own unpublished data). Although important for improved quality of care, the use of life quality measures in randomized controlled studies assessing the efficacy of skin care formulations in patients with acne has been rare so far. Scherdin et al. 16 investigated the changes in life quality measured by the acne disability index 28 in a control study comparing the effects of different skin care regimen including the application of a cleansing gel, cleansing gel and tonic or cleansing gel, cream gel and tonic in volunteers with acne grades I and II. The use of the full combination (cleansing gel, cream gel and tonic) resulted in greater improvement of life quality, in particular with respect to the social and psychological aspects, compared to only the cleansing gel or the combination of cleansing gel and tonic. In another open study, the same group showed an improvement by 45% in the Cardiff acne disability index due to regular skin cleansing and care. 29 Whereas the reported outcomes may have been possibly influenced by the open test design, the double-blind, vehicle-controlled design of our study excludes assessment bias and provides evidence for greater improvement of life quality in the active formulation compared to the vehicle group. As might be expected, the effects exerted by the active formulation were not comparable to the ones observed in clinical trials for topical drug formulations developed to treat acne. Although no references regarding the minimal differences to assess the efficacy of medical skin care formulations have been published so far, the improvement of DLQI parallel to the greater reduction in the lesion count, sebum levels and P. acnes in the active formulation group are relevant endpoints from both physician s and patient s perspective. As the influence of confounding factors throughout the study, such as differences in the individual skin cleansing routines or the use of acne medication were excluded by the protocol, the comparison to the vehicle provides a direct measure for the effects of the active formulation that would be consistent inde-

6 Efficacy of a skin care formulation in acne 11 pendently of whether it is applied as stand-alone or adjuvant to treatment skin care. Funding sources The study was funded by Beiersdorf AG. Conflict of interest I. Angelova-Fischer has been investigator for and received honoraria as speaker from Beiersdorf AG. References 1 Bhate K, Williams HC. Epidemiology of acne vulgaris. Br J Dermatol 2013; 168: Zaenglein A, Graber E, Thiboutot D. Acne vulgaris and acneiform eruptions. In: Fitzpatrick s Dermatology in General Medicine (Goldsmith L, Katz S, Gilchrest B, Paller A, Leffell D, Wolff K, eds.), McGraw Hill, New York, 2012: Kurokawa I, Danby FW, Qiang Ju et al. New developments in our understanding of acne pathogenesis and treatment. Exp Dermatol 2009; 18: Thiboutot D, Gollnick H, Bettoli V et al. New insights into the management of acne: an update from a Global Alliance to Improve Outcomes in acne. J Am Acad Dermatol 2009; 60:S1 S50. 5 Jeremy A, Holland D, Roberts S et al. Inflammatory events are involved in acne lesion initiation. J Invest Dermatol 2003; 121: Makrantonaki E, Ganceviciene R, Zouboulis C. An update on the role of the sebaceous gland in the pathogenesis of acne. Dermatoendocrinology 2011; 3: Bowe W, Patel N, Logan A. Acne vulgaris: the role of oxidative stress and the potential therapeutic value of local and systemic antioxidants. J Drugs Dermatol 2012; 11: Nast A, Dreno B, Bettoli V et al. European evidence-based (S3) guidelines for the treatment of acne. J Eur Acad Dermatol Venereol 2012; 26 (Suppl.1): Nast A, Bayerl C, Borelli C et al. S2-guideline for therapy of acne. J Dtsch Dermatol Ges 2010; 8:S1 S Goodmann G. Cleansing and moisturizing in acne patients. Am J Clin Dermatol 2009; 10(Suppl. 1): Berardesca E, EEMCO Group. EEMCO guidance for the assessment of stratum corneum hydration: electrical methods. Skin Res Tech 1997; 3: Pierard G, Pierard-Franchimont C, Marks R et al. EEMCO guidance for the in vivo assessment of skin greasiness. Skin Pharmacol Appl Skin Physiol 2000; 13: Kligman A, Miller D, McGinley K. Sebutape: a device for visualizing and measuring human sebaceous secretion. J Soc Cosmet Chem 1986; 37: Finlay AY, Khan GK. Dermatology Life Quality Index (DLQI) a simple practical measure for routine clinical use. Clin Exp Dermatol 1994; 19: Gollnick H, Cunliffe WJ, Berson D et al. Management of acne: a report from a global alliance to improve outcomes in acne. J Am Acad Dermatol 2003; 49: S1 S Scherdin U, Presto S, Rippke F et al. In vivo assessment of the efficacy of an innovative face care system in subjects with mild acne vulgaris. Int J Cosmet Sci 2004; 26: Shibata S, Inoue H, Iwata S et al. Inhibitory effects of licochalcone A isolated from Glycyrrhiza inflata root on inflammatory ear edema and tumour promotion in mice. Planta Med 1991; 57: Furusawa J, Funakoshi-Tago M, Mashino T et al. Glycyrrhiza inflataderived chalcones, licochalcone A, licochalcone B and licochalcone D, inhibit phosphorylation of NF-kappa B p65 in LPS signalling pathway. Int Immunopharmacol 2009; 9: Kolbe L, Immeyer J, Eggers K, Neufang G. Anti-oxidative properties of licochalcone A from Glycyrrhiza inflata on human skin in vitro and in vivo. J Am Acad Dermatol 2006; 54: AB1 AB Kolbe L, Immeyer J, Batzer J et al. Anti-inflammatory efficacy of licochalcone A: correlation of clinical potency and in vitro effects. Arch Dermatol Res 2006; 298: Funakoshi-Tago M, Nakamura K, Tsuruya R et al. The fixed structure of licochalcone A by alpha, beta-unsaturated ketone is necessary for antiinflammatory activity through the inhibition of NF-kappa B activation. Int Immunopharmacol 2010; 10: Furuhashi I, Iwata S, Shibata S et al. Inhibition by licochalcone A, a novel flavonoid isolated from liquorice root, of IL-1 beta-induced PGE2 production in human skin fibroblasts. J Pharm Pharmacol 2005; 57: Weber T, Ceilley R, Buerger A et al. Skin tolerance, efficacy and quality of life of patients with red facial skin using a skin care regimen containing licochalcone A. J Cosmet Dermatol 2006; 5: Udompataikul M, Srisatwaja W. Comparative trial of moisturizer containing licochalcone A vs. hydrocortisone lotion in the treatment of childhood atopic dermatitis: a pilot study. J Eur Acad Dermatol Venereol 2011; 25: Zouboulis CC. Acne and sebaceous gland function. Clin Dermatol 2004; 22: Peirano R, Hamann T, D using HJ et al. Topically applied L-carnitine effectively reduces sebum secretion in human skin. J Cosmet Dermatol 2012; 11: Zouboulis CC, Seltman H, Neitzel H et al. Establishment and characterisation of an immortalized human sebaceous gland cell line (SZ95). J Invest Dermatol 1999; 113: Motley RJ, Finlay AY. Practical use of a disability index in the routine management of acne. Clin Exp Dermatol 1992; 17: Scherdin U, Treder-Conrad C, Berger B et al. Medical skin care significantly improves quality of life in subjects with mild acne vulgaris. J Eur Acad Dermatol Venereol 2004; 18 (Suppl.2): 202.