Reduction in Serine Protease Activity Correlates with Improved Rosacea Severity in a Small, Randomized Pilot Study of a Topical Serine Protease Inhibitor Reduction in Serine Protease Activity Correlates with Improved Rosacea Severity in a Small, Randomized Pilot Study of a Topical Serine Protease Inhibitor Journal of Investigative Dermatology (2014) 134, 1143–1145; doi:10.1038/jid.2013.472; published online 12 December 2013 TO THE EDITOR Individuals with rosacea express high baseline levels of cathelicidin and kal- likrein 5 (KLK5; Yamasaki et al., 2007). The significance of elevated KLK5 is that this trypsin-like serine protease controls the cleavage of the cathelicidin precur- sor protein into LL-37 (Yamasaki et al., 2006, 2007). LL-37 is both proinflam- matory and angiogenic (Koczulla et al., 2003; Lande et al., 2007; Morizane et al., 2012). Based on these findings, we hypothesized that inhibition of KLK5 may improve the clinical signs of ro- sacea by decreasing LL-37 production. To test this hypothesis, we studied the effects of a known inhibitor of trypsin- like serine protease, e-aminocaproic acid (ACA). ACA is Food and Drug Administration approved for systemic administration to enhance hemostasis in cases of fibrinolytic bleeding, and is well-tolerated in humans when adminis- tered intravenously in doses up to 3 g kg�1. To determine whether ACA might be useful in rosacea, ACA was first tested in vitro using an Enzcheck Protease Assay Kit (Invitrogen) against the proteolytic activity of tissue kallikrein (100 mg ml�1, Sigma-Aldrich, St Louis, MO) or stratum corneum pro- tease activity (SPA) collected from tape- strips (D-100 CuDerm, Dallas, TX) of the facial skin of six healthy subjects. SPA was extracted from tape with 20 mM Tris-HCl (pH 8.0) containing 0.1% Triton X-100. Protease activity was measured at 37 1C for 2 hours after treatment with vehicle, ACA (1 M), or the known selective inhibitor of trypsin- related enzymes, aprotinin (50 mg ml�1, Sigma-Aldrich). Aprotinin strongly sup- pressed SPA, indicating predominance of trypsin-like proteases in the stratum corneum extract. The inhibitory effect of ACA was similar to that of aprotinin (Figure 1). These combined data sug- gested that ACA could be effective to inhibit stratum corneum trypsin-like pro- teases such as KLK5. Having confirmed that ACA was an effective inhibitor of SPA in vitro, we next sought to determine whether topi- cal application of a cream formulation containing equal weight of cream and ACA solution (2 M) decreased SPA in patients with rosacea. If active, this formulation, called SEI003, would per- mit us to test the hypothesis that inhibi- tion of SPA improves rosacea. We therefore conducted a proof-of-concept, randomized, double-blind, placebo- controlled study on 11 adult patients with papulopustular rosacea (clinical- trials.gov identifier NCT01398280). The study was conducted at the Uni- versity of California, San Diego (UCSD) dermatology clinic in accordance with the Declaration of Helsinki Principles and the regulations of the UCSD Institu- tional Review Board (reference number 100867). Written informed consent was obtained from all participants. The study was conducted between July 2011 and December 2012, and included 15 sub- jects with papulopustular rosacea ran- domized 2:1 to receive either SEI003 (the treatment group) or the base cream homogenized with an equal weight of the ACA vehicle (the placebo group). Subjects, study coordinators, and those performing clinical assessments were blinded. Of the 11 patients originally assigned to the treatment group, three subjects voluntarily withdrew from the study (one patient reported scheduling conflicts and two others were no longer interested in participating), and one was withdrawn from the study after starting doxycycline for ocular rosacea. The study numbers of participants who with- drew from the study were reassigned to new subjects after patient 11 was KLK inhibition Vehicle SEI003 Aprotinin Aprotinin 4,000,000 3,000,000 2,000,000 1,000,000 1,200,000 1,000,000 800,000 R F U /μ g p ro te in 600,000 400,000 200,000 0 R F U 0 0 30 60 90 120 Time (minutes) P = 0.0026 Stratum corneum protease inhibition P = 0.01 Vehicle SEI003 Figure 1. In vitro effects of SEI003 and aprotinin on KLK and human total stratum corneum protease activity. (a) Both SEI003 and aprotinin inhibit KLK activity compared to vehicle alone. SEI003 completely blocks KLK activity for the first 45 minutes of the assay. (b) Compared to vehicle alone, SEI003 and aprotinin both significantly decrease stratum corneum protease activity. KLK, kallikrein; RFU, relative fluorescent units. Accepted article preview online 8 November 2013; published online 12 December 2013 Abbreviations: ACA, e-aminocaproic acid; CEA, Clinician’s Erythema Assessment; IGA, Investigator’s Global Assessment; KLK, kallikrein; SPA, serine protease activity; UCSD, University of California, San Diego AM Two et al. Inhibition of Serine Protease in Rosacea www.jidonline.org 1143 http://www.jidonline.org enrolled. All four patients originally assigned to the placebo group com- pleted the study in its entirety. Patients were instructed to apply their assigned medication to their entire face twice daily for the 12-week duration of the study. Clinical assessments including the Investigator’s Global Assessment (IGA) and the summed total of the 5- point Clinician’s Erythema Assessment (CEA) score of five different target sites (left cheek, right cheek, nose, chin, and glabella), as well as digital photography were completed at baseline. These procedures were repeated 2, 6, 9, and 12 weeks after the baseline visit along with safety monitoring and tape strip sampling for SPA. The average age and baseline IGA and CEA scores for subjects in the two groups were similar (Figure 2a). The IGA scores at weeks 0 and 12 Subject demographic information Treatment group Placebo group 56–6830–80 57 2 (29%) 5 (71%) 6 (86%) 1 (14%) 1.8 9 63.5 1 (33%) 2 (67%) 4 (100%) 0 (0%) 2.0 7 Baseline IGA score Baseline CEA score CEA scores at weeks 0 and 12 Week 0 Week 12 Week 0 Week 12 P = 0.05 P = 0.15 P = 0.98P = 0.05 2.5 2.0 1.5 IG A s co re C E A s co re 1.0 0.5 0.0 15 10 5 0 0 12 0 12 0 12 0 12 Placebo 2,500 Serine protease activity in all patients by week P = 0.005 P = 0.53 P = 0.04 P = 0.001 SEI003 (N =7) Placebo (N =4) 2,000 S P A ( R F U ) S P A ( R F U ) S P A ( R F U ) 1,500 1,000 500 0 1,500 1,500 4,000 6,000 8,000 1,000 1,000 500 500 0 0 Placebo Treatment group Treatment group SEI003 2 2 6 6 9 9 12 12 2 6 9 12 Weeks of application Weeks of SEI003 application Weeks of placebo application SEI003 SPA of patients in the SEI003 treatment group over time SPA of patients in the placebo group over time Age Gender Ethnicity Caucasian Hispanic Male Range Mean Female Figure 2. Response of rosacea patients to SEI003 treatment. (a) Demographic data for participants completing the trial. (b) Stratum corneum protease activity (SPA) levels were not significantly different in the treatment groups at week 2, however differences at weeks 6, 9, and 12 were statistically significant. (c) SPA levels of individual subjects in the treatment group tended to decrease over time. (d) SPA levels of individual subjects in the placebo group remained relatively unchanged over time. (e) IGA scores decreased throughout the study for participants in both groups, however scores were only significant for time and the interaction between time and treatment group. (f) CEA scores also trended down as participants progressed through the study, however a two-way analysis of variance of CEA scores for participants in both groups was significant only for time. (g) Decreased erythema is seen in patients in the treatment group at week 12. (h) Decreased erythema is also appreciated in this control subject over time, however this finding is less pronounced in these patients. Error bars represent mean±SE of the mean. Treatment group n¼7, control group n¼4. CEA, Clinician’s Erythema Assessment; IGA, Investigator’s Global Assessment; KLK, kallikrein; RFU, relative fluorescent units. AM Two et al. Inhibition of Serine Protease in Rosacea 1144 Journal of Investigative Dermatology (2014), Volume 134 average of the SEI003 treatment group had significantly lower SPA compared with the placebo group at weeks 6, 9, and 12 after the start of therapy. A two- way analysis of variance comparing the SPA of subjects in the two groups at all visits showed that treatment group (P¼0.002), time (P¼0.002), and the interaction between time and treatment group (P¼0.001) all had a significant effect on subjects’ SPA (Figure 2b). As this was a small pilot study, and to better illustrate the individual response of SPA over the course of the study, SPA levels were also plotted for each subject over time. SPA levels of the two subjects with the highest baseline SPA had the most pronounced decrease (Figure 2c). In contrast, SPA levels of subjects in the placebo group remained relatively stable during the course of the study (Figure 2d). Overall improvements in IGA and CEA scores were seen in subjects in both the placebo and SEI003 groups over the 12-week course of the study. Although there were no significant dif- ferences in IGA and CEA scores between the two groups at any time point, a correlation between activity to inhibit SPA and clinical improvement was seen as IGA and CEA scores of subjects in the SEI003 group differed significantly at week 12 compared to baseline (P¼0.05, Figure 2e and f). This significant change was not seen in the placebo group (P¼0.15, Figure 2e; P¼0.98, Figure 2f). These findings are likely related to the small sample size and short duration of the study, as well as a placebo effect from the base cream. Digital photographs taken of partici- pant’s faces at the beginning of each visit showed the pronounced improve- ment in rosacea symptoms in some patients receiving SEI003 (Figure 2g and h). Interestingly, the two subjects with the highest baseline SPA values were among the subjects with the most noticeable changes on digital photogra- phy, and both are pictured in Figure 2g. No adverse events were reported during the study. Previous studies have correlated high KLK5 activity with rosacea disease state (Yamasaki et al., 2007). Recent studies of known rosacea treatments suggest that clinical improvements correlate with decreases in this protease cas- cade, suggesting that KLK5 has a key role in mediating the inflammatory response seen in rosacea by its ability to activate cathelicidin. A recent open- label study of 15% azelaic acid gel showed that rosacea patients with high baseline SPA had a significant reduction in their SPA by week 16 of treatment, and this correlated with clinical impro- vement (Coda et al., 2013). Our data show a similar trend, with subjects with the highest baseline SPA correlating with maximal improvement. In another recent study, the antibiotic doxycycline was found to indirectly inhibit SPA and block LL-37 generation in vitro (Kanada et al., 2012). Subsequently, a report of a multi-center clinical trial showed that the therapeutic benefits of doxycycline when taken as a formulation consisting of both immediate release and delayed release beads also correlated with decreased protease activity and LL-37 (Huang et al., 2013). Our study agrees with the associations observed in these other studies, and further suggests that direct targeted inhibition of SPA can result in a reduction of erythema and inflammatory papules in some patients with rosacea. In conclusion, this pilot study shows early clinical evidence that directly sup- ports a causative role for high protease activity in the pathogenesis of rosacea. Although we failed to show clear evi- dence of clinical benefit over placebo for this formulation of serine protease inhibitor, this study was designed only as a proof-of-concept trial with a small number of patients. This work supports the KLK-cathelicidin inflammation hypothesis and the need for adequately powered clinical trial of the therapeutic benefit of direct targeting of SPA in the treatment and control of rosacea. CONFLICT OF INTEREST RLG serves as an unpaid member of the advisory board and consultant to Skin Epibiotics. EH is acting chief executive officer and chief medical officer of Skin Epibiotics. Skin Epibiotics is a virtual corporate entity designated to assess and document potential intellectual property associated with active pharmaceutical ingredients the company identifies for use in new dermatologic-related indications. Although SEI003 was developed by RLG and EH while both were employed by UCSD, the product was eventually named SEI003 after Skin Epibiotics. ACKNOWLEDGMENTS In vitro analysis described in this work was supported in part by the United States National Institutes of Health (NIH) grant R01-AR052728 to RLG. Neither Therapeutics nor Skin Epibiotics provided any financial compensation for the study or to any members of the study team with the exception of EH, who left his position at UCSD for employment opportunities at these companies part-way through the study. Aimee M. Two1, Tissa R. Hata1, Teruaki Nakatsuji1, Alvin B. Coda1, Paul F. Kotol1, Wiggin Wu1, Faiza Shafiq1, Eugene Y. Huang1,2,3 and Richard L. Gallo1,3 1Division of Dermatology, University of California, San Diego, San Diego, California, USA; 2Therapeutics Clinical Research, San Diego, California, USA and 3Skin EpiBiotics Inc., San Diego, California, USA E-mail: rgallo@ucsd.edu REFERENCES Coda AB, Hata T, Miller J et al. (2013) Cathelici- din, KLK5 and serine protease activity is inhibited during treatment of rosacea with azelaic acid 15% gel. J Am Acad Dermatol 69:570–7 Huang E, DiNardo A, Gallo R et al. (2013) Surface cathelicidin expression is a predictor of treat- ment success in papulopustular rosacea. J Invest Dermatol 133:S23 Kanada KN, Nakatsuji T, Gallo RL (2012) Doxycy- cline indirectly inhibits proteolytic activation of tryptic kallikrein-related peptidases and activation of cathelicidin. J Invest Dermatol 132:1435–42 Koczulla R, von Degenfeld G, Kupatt C et al. (2003) An angiogenic role for the human peptide antiobitic LL-37/hCAP-18. J Clin Invest 111: 1165–72 Lande R, Gregorio J, Facchinetti V et al. (2007) Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide. Nature 449:564–9 Morizane S, Yamasaki K, Muhleisen B et al. (2012) Cathelicidin antimicrobial peptide LL-37 in psoriasis enables keratinocyte reactivity against TLR9 ligands. J Invest Dermatol 132: 135–43 Yamasaki K, Di Nardo A, Bardan A et al. (2007) Increased serine protease activity and cathe- licidin promotes skin inflammation in rosa- cea. Nat Med 13:975–80 Yamasaki K, Schauber J, Coda A et al. (2006) Kallikrein mediated proteolosis regulates the antimicrobial effects of cathelicidins in skin. FASEB J 20:2068–80 AM Two et al. Inhibition of Serine Protease in Rosacea www.jidonline.org 1145 http://www.jidonline.org Reduction In Serine Protease Activity Correlates Withimproved Rosacea Severity In A Small, Randomized Pilotstudy Of A Topical Serine Protease Inhibitor������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� References����������������������������������������������