- Email: [email protected]
Efficacy and safety of indigo naturalis ointment in Treating Atopic Dermatitis: A randomized clinical trial
Journal Pre-proof Efficacy and safety of indigo naturalis ointment in Treating Atopic Dermatitis: A randomized clinical trial Yin-Ku Lin, Shu-Han Chang, Chin-Yi Yang, Lai-Chu See, Be-Han Lee, I-Hsin Shih PII:
S0378-8741(19)32789-8
DOI:
https://doi.org/10.1016/j.jep.2019.112477
Reference:
JEP 112477
To appear in:
Journal of Ethnopharmacology
Received Date: 19 July 2019 Revised Date:
26 November 2019
Accepted Date: 10 December 2019
Please cite this article as: Lin, Y.-K., Chang, S.-H., Yang, C.-Y., See, L.-C., Lee, B.-H., Shih, I.-H., Efficacy and safety of indigo naturalis ointment in Treating Atopic Dermatitis: A randomized clinical trial, Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/j.jep.2019.112477. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Efficacy and Safety of Indigo naturalis Ointment in Treating Atopic Dermatitis: A Randomized Clinical Trial Short title: Indigo naturalis in Treating Atopic Dermatitis Yin-Ku Lina,b,g,*, Shu-Han Changa,g, Chin-Yi Yangc, Lai-Chu Seed,e, Be-Han Leea, I-Hsin Shihf Author Affiliations a
Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital,
Keelung, 222, Maijin Road, Keelung 20401, Taiwan b
School of Traditional Chinese Medicine, College of medicine, Chang Gung
University, 259, Wenhua 1st Rd., Guishan Dist., Taoyuan 33302, Taiwan c
Department of Dermatology, Chang Gung Memorial Hospital, Linkou, 5, Fuxing St.,
Guishan Dist., Taoyuan 33305, Taiwan d
Department of Public Health, and Biostatistics Core Laboratory, Molecular Medicine
Research Center, Chang Gung University, 259, Wenhua 1st Rd., Guishan Dist., Taoyuan 33302, Taiwan e
Department of Rheumatology and Allergy and Immunology, Chang Gung Memorial
Hospital, Linkou, 5, Fuxing St., Guishan Dist., Taoyuan 33305, Taiwan f
Department of Dermatology, Chang Gung Memorial Hospital, Taipei, 199, Dunhua N.
Rd., Taipei 10507, Taiwan
g
Co-first author
*Corresponding author Yin-Ku Lin, 222 Mai Chin Road, Keelung 20401, Taiwan; E-Mail: [email protected]; Phone: +886-2-24313131 ext.2777; Fax: +886-2-24332655 Other e-mail addresses: [email protected] (Shu-Han Chang), [email protected] (Chin-Yi Yang), [email protected] (Lai-Chu See), [email protected] (Be-Han Lee), [email protected] (I-Hsin Shih) Word count: 4879 words, 3 tables, 4 figures
Abstract Ethnopharmacological relevance: Indigo naturalis, a herbal medicine with a history of use dating back to ancient times, may be a good alternative topical treatment for atopic dermatitis (AD). Aim of the study: To provide empirical evidence of the efficacy and safety of Indigo naturalis ointment in treating AD. Materials and Methods: In this randomized double-blind clinical trial, participants aged 6 to 65 years with AD affecting less than 40% of their body surface area (BSA) and an Investigator’s Global Assessment (IGA) score of 2 to 4 were randomized (2:1) to receive either Lindioil ointment or a vehicle ointment twice daily for 6 weeks. The primary endpoint was the percentage change in the Eczema Area Severity Index (EASI) from baseline to week 6. Secondary endpoints were as follows: EASI improvement ≥ 50%, 75%, and 90%; IGA score; BSA affected by AD; pruritus severity; and Dermatology Life Quality Index. The safety assessment included adverse events (AEs), laboratory tests, and physical examinations. Results: The Lindioil group (32 participants) and vehicle group (16 participants) achieved mean percentage EASI reductions of 49.9% ± 36.5% (95% CI 36.8% to 63.1%) and 19.6% ± 52.2% (95% CI -8.2% to 47.4%), respectively (P = 0.0235). The Lindioil group also showed greater improvement in every secondary assessment category. No significant AEs
3
occurred. Conclusion: Indigo naturalis ointment is effective for treating mild to severe AD topically, and appears to be safe. This is the first clinical trial to provide evidence supporting topical indigo-based AD treatment. ClinicalTrials.gov identifier: NCT02669888. Keywords: Atopic dermatitis, Indigo naturalis, Indirubin, EASI Abbreviations: AD, atopic dermatitis; AE, adverse event; BSA, body surface area; CDLQI, Children’s Dermatology Quality of Life Index; CGMH, Chang Gung Memorial Hospital; CI, confidence interval; CRP, C-reactive protein; DLQI, Dermatology Quality of Life Index; EASI, Eczema Area Severity Index; FDA, Food and Drug Administration; IGA, Investigator’s Global Assessment; IgE, immunoglobin E; LOCF, last observation carried forward; TCSs, topical corticosteroids; TCIs, topical calcineurin inhibitors; TCM, traditional Chinese medicine; VAS, visual analogue scale.
4
1. Introduction Atopic dermatitis (AD) is a common skin disease. It begins most frequently in infancy but may flare up at any age. There is no cure. Currently, the standard treatments for moderate to severe AD involve topical corticosteroids (TCSs) and topical calcineurin inhibitors (TCIs) (Eichenfield et al., 2014). The risks of long-term TCS use are well known (Hengge et al., 2006). Safety concerns about TCIs have also been raised, and the US Food and Drug Administration (FDA) has issued a warning for the TCIs, tacrolimus and pimecrolimus (Ring et al., 2008). Many AD patients seek alternative treatments. Traditional Chinese medicine (TCM) is one such line of treatment with increasing trends of use in Taiwan (Lin et al., 2019); unfortunately, it is difficult to find empirical evidence regarding the effectiveness and safety of most herbal medicines used in TCM to treat AD (Tan et al., 2013). Indigo naturalis is a blue powder derived from indigo plants, such as Baphicacanthus cusia. Its centuries-long use in TCM to treat various infectious and inflammatory diseases is well documented (Tang and Eisenbrand, 1992). Because of its dark-blue colour, crude Indigo naturalis stains the skin and clothes when used topically. Consequently, despite the documentation of its use in treating skin disease, in modern times few patients have been willing to use it. In 2008, we successfully removed the dark-blue coloration by extracting it in olive oil, thus improving the formulation. The refined formulation, named Lindioil, does not cause staining (Lin et al., 2015; Lin et al., 2012).
5
In the past decade, our research has shown that Indigo naturalis repairs abnormal epidermal barriers by promoting differentiation, upregulating claudin-1 expression, restoring tight junction proteins, and inhibiting keratinocyte proliferation; additionally, we identified indirubin as its main active ingredient (Lin et al., 2013; Lin et al., 2009; Lin et al., 2007). Other research has shown that indirubin suppresses skin inflammation through the inhibition of cytokine production, nuclear factor kappa B activity, and mitogen-activated protein kinase activation (Kim et al., 2013). Thus, the anti-inflammatory, antimicrobial, antiviral and anticancer mechanisms of Indigo naturalis and its active ingredient have been explained by modern science (Chiang et al., 2013; Hoessel et al., 1999; Mak et al., 2004). Based on the modern understanding of these mechanisms, data from previous clinical trials demonstrating the efficacy and safety of both Indigo naturalis and Lindioil in treating psoriasis (Lin et al., 2008; Lin et al., 2018), and a historical document from TCM literature that mentions the use of Indigo naturalis to treat facial eczema in infants, we started using Lindioil ointment to treat AD (in addition to psoriasis) topically at our hospital several years ago. In this randomized, double-blind, vehicle-controlled clinical trial, we hoped to provide empirical evidence of our indigo-based ointment’s efficacy and safety in treating AD. 2. Materials and Methods 2.1 Study Design This was a randomized, double-blind, vehicle-controlled trial initiated by the principal
6
investigator and conducted at two branches (Taipei and Linkou) of Chang Gung Memorial Hospital (CGMH) in Taiwan. Participants were screened, enrolled, and randomized at a 2:1 ratio to receive either Lindioil ointment or placebo (vehicle ointment) for 6 weeks. All participants (and a parent for participants under 20 years old) signed an informed consent form. The protocol was approved by the institutional review boards of Chang Gung Medical Foundation (103-0472A) and the FDA in Taiwan and conducted in accordance with the ethical principles of the Declaration of Helsinki and Good Clinical Practice. The authors vouch for the study’s fidelity to the protocol. This study is registered with ClinicalTrials.gov (identifier NCT02669888). 2.2 Participants Patients who visited the TCM and dermatology outpatient clinics of CGMH and were diagnosed with AD based on the standard criteria of Williams et al (Williams, 2005) were invited to participate in this trial. They signed informed consent forms before being screened for eligibility. Patients ages 6 to 65 were approved for inclusion based on the following criteria: 40% or less body surface area (BSA) affected by the disease; and Investigator’s Global Assessment (IGA, ranging from 0 to 5, or completely clear to very severe) score of mild (2) to severe (4) (Eichenfield et al., 2002). Participants were ineligible for any of the following reasons: a history of topical or systemic hypersensitivity to Indigo naturalis or its excipient (e.g., beeswax) in ointment; a known malignancy (current or past);
7
aspartate aminotransferase or alanine aminotransferase levels greater than three times the upper normal limit; creatinine greater than 2.0 mg/dL; clinically significant haematological abnormalities; uncontrolled hypertension; uncontrolled metabolic diseases; psychiatric conditions; or human immunodeficiency virus infection. Women were not eligible if they were pregnant or lactating, and eligible female participants were asked to use birth control for the duration of the study. Participants were required to cease systemic treatments (e.g., immunosuppressive agents) or phototherapy at least 30 days before the first application of the study medication and were required to cease the use of TCSs and other topical medicines at least 7 days before beginning the study. For skin care needs other than AD treatment, participants were permitted to use a simple emollient, Vaseline lotion, which we provided. 2.3 Plant material and drug preparation The Indigo naturalis powder used in this study was purchased from Shufeng Shangrchia Indigo Farmer Cooperative, Xianyou county, Fujian province, China, and came from the leaves of Baphicacanthus cusia (Nees) Bremek. The quality of the Indigo naturalis powder was verified by Chuang Song Zong (CSZ) Pharmaceutical Co. Ltd. (Taiwan). Ms. Shu-Tuan Chiang (Research and Quality Control department manager of CSZ) identified and authenticated the material; sample vouchers were stored for reference under the code number M0813. Pure indigo blue was purchased from CHEM SERVICE (West Chester,
8
USA), and the indirubin was obtained from National Institutes for Food and Drug Control (NIFDC, Beijing, China). The Indigo naturalis powder used in this trial contained of 2.80% indigo blue and 0.15% indirubin determined by ultraviolet spectrophotometer, and high performance liquid chromatography (HPLC), respectively. The CMC report was made in accordance with the Guidance for Industry Botanical Drug Products. The preparation of Lindioil using an olive oil extraction process, and the process for HPLC analyses of Indigo naturalis and Lindioil (Fig. 1), have been described in detail previously (Lin et al., 2014). The vehicle ointment was prepared by mixing olive oil and beeswax with edible pigment to make the appearance identical to Lindioil ointment (Supplement, Table S1).
2.4 Randomization and Masking After providing written consent, the participants were allocated by a permuted block randomization method with blocks of size 6 to receive Lindioil ointment containing 200 µg/g of indirubin (in a previous trial, 200 µg/g of indirubin was found to be the optimal concentration for treating psoriasis) (Lin et al., 2018) or a vehicle ointment. The two ointments and their containers were identical in appearance. A randomization code list generated by Contract Research Organization (CRO) of Formosa Biomedical Technology Corporation (FBC) was provided to CSZ for packaging and labelling the study medication. FBC CRO produced sealable, randomized envelopes for each randomization code (with the
9
codes shown on the envelopes), which were stored by sponsor personnel who were independent of the study. 2.5 Procedures Due to concerns expressed by Taiwan’s FDA, we excluded children from screening until the safety data for the first 5 adult participants (age range 20–40 years) showed no treatment-related adverse events. Thereafter, the age range was changed to 12–40 years and eventually broadened to 6–65 years. At baseline, the participants were instructed to apply 0.5 g of ointment per 100 cm2 of dermatitis lesion area twice daily for 6 weeks. The severity of AD and the safety of the treatment were assessed at the end of weeks 1, 2, 4, and 6. At the end of the 6th week, all medicine tubes containing Lindioil or vehicle ointment were collected from the participants, and the participants returned for a final follow-up assessment 2 weeks later. 2.6 Efficacy Outcomes and Adverse Events We chose the mean percentage change in Eczema Area Severity Index (EASI) scores at week 6 as our primary efficacy endpoint (EASI is a standardized, validated tool used by dermatologists to objectively combine surface area involvement, erythema, edema/papulation, excoriation, and lichenification into an overall assessment of dermatitis severity) (Hanifin et al., 2001). The secondary efficacy endpoints were (i) the proportions of patients with ≥ 50%, 75%, and 90% improvement in EASI scores (EASI-50, EASI-75, and EASI-90) at the end of
10
week 6; (ii) the proportion of participants who achieved ‘clear’ (IGA = 0) or ‘almost clear’ (IGA = 1) status or more than 2-point of improvement in IGA at week 6 (Thaci et al., 2016); (iii) the mean percentage reduction in the BSA affected by AD over the course of the study period; (iv) the mean change in the pruritus score (using a 10-cm visual analogue scale, VAS) (Reich et al., 2012); and (v) the mean change in the Dermatology Quality of Life Index (DLQI) or the Children’s DLQI (CDLQI) value from baseline to week 6 (Finlay, 1998). AEs and vital signs were assessed throughout the study at each participant’s visit. Haematology, biochemistry, urinalysis, immunoglobin E (IgE) and C-reactive protein (CRP) tests were performed at baseline and at the end of week 6. Any AEs occurring after the administration of the first dose of the study drug and at any time thereafter, up to the completion of the trial, were recorded. The investigating physician actively terminated study participation if a participant experienced significant worsening of dermatitis symptoms (50% increase in the EASI score from baseline); a significant treatment-related AE; failed to apply the medicine on more than 2 days per week; or used prohibited medications.
2.7 Statistical Analysis The descriptive statistics of continuous data are presented separately for the two groups (including the number of observations, mean, median, and standard deviation). The descriptive statistics of discrete variables (frequency, %) were tabulated separately for the two groups. An independent t-test was used to determine the difference in the percentage
11
change of the primary endpoint for the two groups. If the assumption of normality was violated, the Mann-Whitney U test was used instead. For the secondary endpoints, an independent t-test was used to compare continuous variables between the two groups. If the normality assumption was violated, the Mann-Whitney U test was used instead. A paired t-test was used to evaluate the mean change in continuous variables from baseline to the specified visit for each participant for comparison between the groups. If the normality assumption was violated, the Wilcoxon signed-rank test was used instead. The chi-square test or Fisher’s exact test was used to compare discrete variables between the two groups, and two-way ANOVA was used to evaluate the efficacy of continuous variables in relation to other categorical types (e.g., age) between the two groups. Intermittently missing efficacy data were imputed by last observation carried forward (LOCF), and no imputation method was used to estimate the missing values for safety data. All statistical analyses were conducted using SAS software (version 9.4).
3. Results From October 2015 to February 2017, 55 participants were screened, and 48 were determined to be eligible. Thirty-two of the eligible participants were randomly assigned to receive Lindioil ointment for treatment, and 16 were given a vehicle ointment (Fig. 2). The
12
demographic and baseline disease characteristics of the participants varied similarly within each of the groups (Table 1). Of the 48 participants whose data comprised the intention-to-treat analysis, 35 completed the full 6-week study period: 27/32 in the Lindioil group and 8/16 in the vehicle group. 3.1 Study Completion After randomization and the start of treatment, 16% (5/32) of the Lindioil participants and 50% (8/16) of the vehicle participants discontinued treatment. For 4 of the 8 vehicle group participants who discontinued, the reason for discontinuation was lack of efficacy (3/8) or significant worsening of disease characteristics (1/8). Only 1 participant in the Lindioil group discontinued due to lack of efficacy, and none experienced a significant worsening of symptoms. None of the adverse reactions experienced during the trial period were determined to be caused by Lindioil. Other reasons for discontinuation during the 6-week treatment period are listed in Fig. 2; further details of discontinued patients, including EASI and pruritus scores at baseline and last appointment before discontinuation, are listed in Supplement, Table S2. 3.2 Efficacy Our primary endpoint was the mean percentage change in the EASI score from baseline to the end of week 6. Fig. 3A and Table 2 show the results: reductions of 49.9% (95% CI 36.8–63.1) for the Lindioil group and 19.6% (95% CI -8.2–47.4) for the vehicle
13
group, a statistically significant difference (P = 0.0235), supporting the efficacy of Lindioil for treating AD. For the EASI-related secondary endpoints, Figs. 3B, 3C, and 3D show the proportions of participants who achieved EASI-50, EASI-75, and EASI-90, all supporting the efficacy of Lindioil. Figs. 4A, 4B, and 4C are before-and-after photos of participants in the Lindioil group who achieved EASI-50, EASI-75, and EASI-90 respectively. All other secondary endpoints also supported the efficacy of Lindioil. For the IGA score, 38% (12/32) of the Lindioil group achieved IGA 0 or 1, vs. 13% (2/16) of the vehicle group (P = 0.0983); 44% (14/32) of the Lindioil group achieved more than 2-point improvement in IGA score from baseline compared with 25% (4/16) of the vehicle group (P = 0.2059). The percent reduction in the mean IGA from baseline to the end of week 6 was 39.3% for Lindioil compared with 18.2% for vehicle (P = 0.0387). The mean percentage reductions in the BSA affected by AD from baseline to the end of week 6 were 50.4% (Lindioil) and 17.6% (vehicle) (P = 0.0241), and the mean changes in pruritus VAS score were -2.3 (Lindioil) and -1.3 (vehicle) (P = 0.0994). The mean changes in the DLQI/CDLQI from baseline to week 6 were -4.3 (Lindioil) and -2.5 (vehicle), with 53% (17/32) of the Lindioil participants and only 25% (4/16) of the vehicle participants achieving the minimal clinically important difference (a decrease of 4 points or more) (P = 0.0641) (Basra et al., 2015). Eosinophils, IgE, CRP, and other immune-related
14
markers showed no statistically significant differences in either group from baseline to week 6 (Supplement, Table S3). Thirty-three of the 35 participants who completed the treatment period (25/27 in the Lindioil group and 8/8 in the vehicle group) returned for the follow-up appointment at week 8. For the follow-up group, after the 2-week washout period, the IGA scores of 56% (14/25) of the Lindioil participants and 50% (4/8) of the vehicle participants had increased, as had the pruritus VAS scores of 60% (15/25) of the Lindioil participants and 25% (2/8) of the vehicle participants. 3.3 Safety During the study period, a total of 33 treatment-emergent AEs were reported by 24 participants (Table 3). The incidence of AEs was 53% (17/32) in the Lindioil group and 44% (7/16) in the vehicle group. All AEs were mild. The most commonly reported AEs, acne in 3/32 Lindioil participants (4 events) and nasopharyngitis in 2/16 vehicle participants, were clearly not treatment-related. 5 participants experienced a total of 6 mild skin AEs (acne, in 4 of these 6 cases). None of the skin AEs was determined to be treatment-related, and none resulted in discontinuation of treatment. Safety-related blood tests and urinalysis showed no statistically significant differences in either group from baseline to week 6 (data not shown). 4. Discussion
15
The results of this 6-week trial demonstrate that Lindioil ointment is effective in relieving all measurable AD symptoms, thereby improving the quality of life of AD patients, and is superior to a vehicle ointment. The primary endpoint data (a 49.9% mean EASI reduction for the participants who were given Lindioil ointment versus a 19.6% mean EASI reduction for those given a vehicle ointment) and data across the secondary endpoints provide strong evidence of Lindioil ointment’s efficacy. The 19.6% EASI improvement in the vehicle group may surprise some readers. In fact, it is consistent with other AD topical treatment studies, in which vehicle groups have shown a 20-25% EASI reduction at week 6 as well (Chapman et al., 2005; Schachner et al., 2005). This is because the moisturizers in the vehicle ointments, by themselves, increase skin hydration and reduce inflammation and severity of AD (Eichenfield et al., 2014). Olive oil, the main component in both our vehicle ointment and Lindioil ointment, is known to increase skin hydration and alleviate AD symptoms (Al-Waili, 2003; Verallo-Rowell et al., 2008). Our trial showed high standard deviation in the percentage reduction of EASI score. This is also consistent with other AD studies of similar sample size (Leung et al., 2009; Peppers et al., 2019). Just like many other chronic diseases, AD is influenced by environmental factors varying from patient to patient including mood and lifestyle, sometimes regardless of treatment. It is unfortunate that we ended up with such a small sample size. This limitation was due to budget and schedule constraints. A trial with larger sample size would likely show
16
a lower standard deviation. TCSs and TCIs comprise the vast majority of AD medicines worldwide, with TCSs yielding the best results. However, safety concerns regarding the risks of long-term corticosteroid use (such as skin atrophy and adrenal suppression) have led to many AD patients being unwilling to consider even short-term steroid-based treatment (Li et al., 2017). The most commonly used non-steroid topical medicines for AD treatment are TCIs. According to data from previous 6-week trials, the TCIs tacrolimus and pimecrolimus achieved 54.1% / 67.2% and 34.9% / 56.4% mean EASI reductions among adult/child patients, respectively (Paller et al., 2005). Lindioil ointment is the first topical herbal medicine to display efficacy results within this range. In addition, just as in the above-mentioned tacrolimus and pimecrolimus studies, the children in our trial displayed a better response than the adults. If indigo-based medicines continue to display these results, they may be an attractive alternative to TCIs for patients who prefer herbal medicines worldwide. The data from this 6-week trial alone should not be enough to assuage patients’ concerns about long-term safety, especially considering the limited sample size. However, Indigo naturalis has a documented history of use in TCM going back hundreds of years, with records indicating the topical use of Indigo naturalis to treat skin disease (specifically including infant eczema) and no mention of any side effects or safety concerns for long-term use, and modern
17
medical literature seems also to be lacking any reports of serious side effects from long-term topical use of Indigo naturalis. As other indigo-based medicines are developed and more trials conducted, more data will gradually become available over time. To the best of our knowledge, this is the first double-blind clinical trial to support the efficacy of Indigo naturalis for treating AD topically. Since this treatment also appears to be safe, we believe that Indigo naturalis has the potential to be developed into topical medicines for AD treatment worldwide. Acknowledgements We thank Daniel Teitler for proofreading and editing the manuscript. Funding Sources This principal investigator (Dr. Lin)-initiated phase IIa trial was supported by the Taiwan Ministry of Science and Technology (MOST105-2325-B-182A-003) and Chang Gung Medical Foundation (BMRP972A), Taiwan. The funding sponsors had no role in the design and conduct of the study; in the collection, analysis, and interpretation of data; or in the preparation, review, or approval of the manuscript. Conflict of Interest Disclosures Dr. Lin was supported by grants and personal fees from the Taiwan Ministry of Science and Technology and Chang Gung Foundation during the conduct of the study. In addition, Dr. Lin is the inventor of Lindioil.
18
Authors’ Contributions Dr. Lin had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Lin. Acquisition of data: Lin, Yang, and Shih. Analysis and interpretation of data: Lin and See. Drafting of the manuscript: Lin, Chang, and Lee. Critical revision of the manuscript for important intellectual content: Lin, See, Chang, Yang, and Shih. Statistical analysis: See and Lee. Obtained funding: Lin. Administrative support: Lin. Study supervision: Lin.
19
References Al-Waili, N.S., 2003. Topical application of natural honey, beeswax and olive oil mixture for atopic dermatitis or psoriasis: partially controlled, single-blinded study. Complement Ther Med 11(4), 226-234, 10.1016/s0965-2299(03)00120-1 Basra, M.K., Salek, M.S., Camilleri, L., Sturkey, R., Finlay, A.Y., 2015. Determining the minimal clinically important difference and responsiveness of the Dermatology Life Quality Index (DLQI): further data. Dermatology 230(1), 27-33, 10.1159/000365390 Chapman, M.S., Schachner, L.A., Breneman, D., Boguniewicz, M., Gold, M.H., Shull, T., Linowski, G.J., Jaracz, E., Group, U.S.T.O.S., 2005. Tacrolimus ointment 0.03% shows efficacy and safety in pediatric and adult patients with mild to moderate atopic dermatitis. J Am Acad Dermatol 53(2 Suppl 2), S177-185, 10.1016/j.jaad.2005.04.061 Chiang, Y.R., Li, A., Leu, Y.L., Fang, J.Y., Lin, Y.K., 2013. An in vitro study of the antimicrobial effects of Indigo naturalis prepared from Strobilanthes formosanus Moore. Molecules 18(11), 14381-14396, 10.3390/molecules181114381 Eichenfield, L.F., Lucky, A.W., Boguniewicz, M., Langley, R.G., Cherill, R., Marshall, K., Bush, C., Graeber, M., 2002. Safety and efficacy of pimecrolimus (ASM 981) cream 1% in the treatment of mild and moderate atopic dermatitis in children and adolescents. J Am Acad Dermatol 46(4), 495-504, 10.1067/mjd.2002.122187 Eichenfield, L.F., Tom, W.L., Berger, T.G., Krol, A., Paller, A.S., Schwarzenberger, K.,
20
Bergman, J.N., Chamlin, S.L., Cohen, D.E., Cooper, K.D., Cordoro, K.M., Davis, D.M., Feldman, S.R., Hanifin, J.M., Margolis, D.J., Silverman, R.A., Simpson, E.L., Williams, H.C., Elmets, C.A., Block, J., Harrod, C.G., Smith Begolka, W., Sidbury, R., 2014. Guidelines of care for the management of atopic dermatitis: section 2. Management and treatment of atopic dermatitis with topical therapies. J Am Acad Dermatol 71(1), 116-132, 10.1016/j.jaad.2014.03.023 Finlay, A.Y., 1998. Quality of life assessments in dermatology. Semin Cutan Med Surg 17(4), 291-296, 10.1016/s1085-5629(98)80026-6 Hanifin, J.M., Thurston, M., Omoto, M., Cherill, R., Tofte, S.J., Graeber, M., 2001. The eczema area and severity index (EASI): assessment of reliability in atopic dermatitis. EASI Evaluator Group. Exp Dermatol 10(1), 11-18, 10.1034/j.1600-0625.2001.100102.x Hengge, U.R., Ruzicka, T., Schwartz, R.A., Cork, M.J., 2006. Adverse effects of topical glucocorticosteroids. J Am Acad Dermatol 54(1), 1-15; quiz 16-18, 10.1016/j.jaad.2005.01.010 Hoessel, R., Leclerc, S., Endicott, J.A., Nobel, M.E., Lawrie, A., Tunnah, P., Leost, M., Damiens, E., Marie, D., Marko, D., Niederberger, E., Tang, W., Eisenbrand, G., Meijer, L., 1999. Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases. Nat Cell Biol 1(1), 60-67, 10.1038/9035 Kim, M.H., Choi, Y.Y., Yang, G., Cho, I.H., Nam, D., Yang, W.M., 2013. Indirubin, a purple
21
3,2- bisindole, inhibited allergic contact dermatitis via regulating T helper (Th)-mediated immune system in DNCB-induced model. J Ethnopharmacol 145(1), 214-219, 10.1016/j.jep.2012.10.055 Leung, D.Y., Hanifin, J.M., Pariser, D.M., Barber, K.A., Langley, R.G., Schlievert, P.M., Abrams, B., Hultsch, T., 2009. Effects of pimecrolimus cream 1% in the treatment of patients with atopic dermatitis who demonstrate a clinical insensitivity to topical corticosteroids: a randomized, multicentre vehicle-controlled trial. Br J Dermatol 161(2), 435-443, 10.1111/j.1365-2133.2009.09145.x Li, A.W., Yin, E.S., Antaya, R.J., 2017. Topical Corticosteroid Phobia in Atopic Dermatitis: A Systematic Review. JAMA Dermatol 153(10), 1036-1042, 10.1001/jamadermatol.2017.2437 Lin, P.Y., Chu, C.H., Chang, F.Y., Huang, Y.W., Tsai, H.J., Yao, T.C., 2019. Trends and prescription patterns of traditional Chinese medicine use among subjects with allergic diseases: A nationwide population-based study. World Allergy Organ J 12(2), 100001, 10.1016/j.waojou.2018.11.001 Lin, Y.K., Chang, C.J., Chang, Y.C., Wong, W.R., Chang, S.C., Pang, J.H., 2008. Clinical assessment of patients with recalcitrant psoriasis in a randomized, observer-blind, vehicle-controlled trial using Indigo naturalis. Arch Dermatol 144(11), 1457-1464, 10.1001/archderm.144.11.1457
22
Lin, Y.K., Chang, Y.C., Hui, R.C., See, L.C., Chang, C.J., Yang, C.H., Huang, Y.H., 2015. A Chinese Herb, Indigo naturalis, Extracted in Oil (Lindioil) Used Topically to Treat Psoriatic Nails: A Randomized Clinical Trial. JAMA Dermatol 151(6), 672-674, 10.1001/jamadermatol.2014.5460 Lin, Y.K., Chen, H.W., Leu, Y.L., Yang, Y.L., Fang, Y., Su Pang, J.H., Hwang, T.L., 2013. Indigo naturalis upregulates claudin-1 expression in human keratinocytes and psoriatic lesions. J Ethnopharmacol 145(2), 614-620, 10.1016/j.jep.2012.11.044 Lin, Y.K., Leu, Y.L., Yang, S.H., Chen, H.W., Wang, C.T., Pang, J.H., 2009. Anti-psoriatic effects of iIndigo naturalis on the proliferation and differentiation of keratinocytes with indirubin as the active component. J Dermatol Sci 54(3), 168-174, 10.1016/j.jdermsci.2009.02.007 Lin, Y.K., See, L.C., Huang, Y.H., Chang, Y.C., Tsou, T.C., Leu, Y.L., Shen, Y.M., 2012. Comparison of refined and crude Indigo naturalis ointment in treating psoriasis: randomized, observer-blind, controlled, intrapatient trial. Arch Dermatol 148(3), 397-400, 10.1001/archdermatol.2011.1091 Lin, Y.K., See, L.C., Huang, Y.H., Chang, Y.C., Tsou, T.C., Lin, T.Y., Lin, N.L., 2014. Efficacy and safety of Indigo naturalis extract in oil (Lindioil) in treating nail psoriasis: a randomized, observer-blind, vehicle-controlled trial. Phytomedicine 21(7), 1015-1020, 10.1016/j.phymed.2014.02.013
23
Lin, Y.K., See, L.C., Huang, Y.H., Chi, C.C., Hui, R.C., 2018. Comparison of indirubin concentrations in Indigo naturalis ointment for psoriasis treatment: a randomized, double-blind, dosage-controlled trial. Br J Dermatol 178(1), 124-131, 10.1111/bjd.15894 Lin, Y.K., Wong, W.R., Chang, Y.C., Chang, C.J., Tsay, P.K., Chang, S.C., Pang, J.H., 2007. The efficacy and safety of topically applied Indigo naturalis ointment in patients with plaque-type psoriasis. Dermatology 214(2), 155-161, 10.1159/000098576 Mak, N.K., Leung, C.Y., Wei, X.Y., Shen, X.L., Wong, R.N., Leung, K.N., Fung, M.C., 2004. Inhibition of RANTES expression by indirubin in influenza virus-infected human bronchial epithelial cells. Biochem Pharmacol 67(1), 167-174, 10.1016/j.bcp.2003.08.020 Paller, A.S., Lebwohl, M., Fleischer, A.B., Jr., Antaya, R., Langley, R.G., Kirsner, R.S., Blum, R.R., Rico, M.J., Jaracz, E., Crowe, A., Linowski, G.J., Group, U.S.C.T.O.S., 2005. Tacrolimus ointment is more effective than pimecrolimus cream with a similar safety profile in the treatment of atopic dermatitis: results from 3 randomized, comparative studies. J Am Acad Dermatol 52(5), 810-822, 10.1016/j.jaad.2004.12.038 Peppers, J., Paller, A.S., Maeda-Chubachi, T., Wu, S., Robbins, K., Gallagher, K., Kraus, J.E., 2019. A phase 2, randomized dose-finding study of tapinarof (GSK2894512 cream) for the treatment of atopic dermatitis. J Am Acad Dermatol 80(1), 89-98 e83, 10.1016/j.jaad.2018.06.047 Reich, A., Heisig, M., Phan, N.Q., Taneda, K., Takamori, K., Takeuchi, S., Furue, M.,
24
Blome, C., Augustin, M., Stander, S., Szepietowski, J.C., 2012. Visual analogue scale: evaluation of the instrument for the assessment of pruritus. Acta Derm Venereol 92(5), 497-501, 10.2340/00015555-1265 Ring, J., Mohrenschlager, M., Henkel, V., 2008. The US FDA 'black box' warning for topical calcineurin inhibitors: an ongoing controversy. Drug Saf 31(3), 185-198, 10.2165/00002018-200831030-00001 Schachner, L.A., Lamerson, C., Sheehan, M.P., Boguniewicz, M., Mosser, J., Raimer, S., Shull, T., Jaracz, E., Group, U.S.T.O.S., 2005. Tacrolimus ointment 0.03% is safe and effective for the treatment of mild to moderate atopic dermatitis in pediatric patients: results from a randomized, double-blind, vehicle-controlled study. Pediatrics 116(3), e334-342, 10.1542/peds.2004-2638 Tan, H.Y., Zhang, A.L., Chen, D., Xue, C.C., Lenon, G.B., 2013. Chinese herbal medicine for atopic dermatitis: a systematic review. J Am Acad Dermatol 69(2), 295-304, 10.1016/j.jaad.2013.01.019 Tang, W., Eisenbrand, G., 1992. Chinese drugs of plant origin: chemistry, pharmacology, and use in traditional and modern medicine. Springer-Verlag Berlin Heidelberg, 10.1007/978-3-642-73739-8 Thaci, D., Simpson, E.L., Beck, L.A., Bieber, T., Blauvelt, A., Papp, K., Soong, W., Worm, M., Szepietowski, J.C., Sofen, H., Kawashima, M., Wu, R., Weinstein, S.P., Graham, N.M.,
25
Pirozzi, G., Teper, A., Sutherland, E.R., Mastey, V., Stahl, N., Yancopoulos, G.D., Ardeleanu, M., 2016. Efficacy and safety of dupilumab in adults with moderate-to-severe atopic dermatitis inadequately controlled by topical treatments: a randomised, placebo-controlled, dose-ranging phase 2b trial. Lancet 387(10013), 40-52, 10.1016/S0140-6736(15)00388-8 Verallo-Rowell, V.M., Dillague, K.M., Syah-Tjundawan, B.S., 2008. Novel antibacterial and emollient effects of coconut and virgin olive oils in adult atopic dermatitis. Dermatitis 19(6), 308-315, 10.2310/6620.2008.08052 Williams, H.C., 2005. Clinical practice. Atopic dermatitis. N Engl J Med 352(22), 2314-2324, 10.1056/NEJMcp042803
26
TABLE LEGENDS Table 1: Demographic and clinical characteristics at baseline (n=48). Patients
Lindioil (n=32)
Vehicle (n=16)
20 (63)
9 (56)
21.9 (8.3-37.8)
23.0 (9.2-35.1)
Adult (≥ 18 years old), n (%)
18 (56)
12 (75)
Children (< 18 years old), n (%)
14 (44)
4 (25)
Body mass index, mean (range), kg/m2
21.1 (13.9-28.7)
21.7 (16.2-28.7)
Age at onset of AD, mean (range), y
4.4 (0.0-32.0)
4.8 (0.0-16.0)
Duration of AD, mean (range), y
17.5 (1.3-36.8)
18.2 (3.5-30.6)
History of IgE reactivity, n (%)
26 (81)
15 (94)
History of allergic rhinitis, n (%)
26 (81)
10 (63)
History of asthma, n (%)
16 (50)
6 (38)
Topical therapy
32 (100)
16 (100)
Systemic therapies
27 (84)
16 (100)
Other therapies
30 (94)
14 (88)
IGA 3
13 (41)
6 (38)
IGA 4
19 (59)
10 (63)
12.3 (4.7-28.9)
12.3 (2.9-29.3)
Mild (EASI ≤ 7.0), n (%)
8 (25)
7 (44)
Moderate (7.1 ≤ EASI ≤ 21.0), n (%)
19 (59)
5 (31)
Severe (EASI ≥ 21.1), n (%)
5 (16)
4 (25)
15.5 (3.0-39.5)
13.0 (3.5-36.0)
Pruritus VAS, mean (range)
7.5 (6.0-9.0)
6.8 (4.0-9.0)
DLQI/CDLQI, mean (range)
11.5 (1.0-26.0)
10.3 (2.0-23.0)
Males, n (%) Age, mean (range), y
Prior treatments, n (%)
IGA, n (%)
EASI, mean (range)
BSA, mean (range)
Abbreviations: AD, atopic dermatitis; EASI, Eczema Area Severity Index; BSA, body surface area; VAS, visual analogue scale; DLQI/CDLQI, Dermatology Quality of Life Index/children’s DLQI; IGA, Investigator’s Global Assessment.
27
Table 2: Efficacy outcomes, baseline to week 6 (n=48). Patients
P valuea
Lindioil (n=32)
Vehicle (n=16)
Baseline, mean ± SD (median)
12.3 ± 7.1 (8.7)
12.3 ± 9.1 (7.5)
0.4183
Week 6, mean ± SD (median)
6.4 ± 6.5 (5.1)
9.8 ± 7.9 (5.8)
0.1857
Change from baseline, mean ± SD (median)
-5.8 ± 5.2 (-5.2)†
-2.6 ± 6.9 (-2.4)
0.0279*
% reduction from baseline, mean ± SD (median)
49.9 ± 36.5 (58.2)
19.6 ± 52.2 (20.2)
0.0235*
Baseline, mean ± SD (median)
3.6 ± 0.5 (4.0)
3.6 ± 0.5 (4.0)
0.8465
Week 6, mean ± SD (median)
2.2 ± 1.2 (2.0)
2.9 ± 1.1 (3.0)
0.0468*
39.3 ± 34.0 (33.3)
18.2 ± 29.7 (0.0)
0.0387*
No. (%) of IGA 0 or 1 at Week 6
12 (38)
2 (13)
0.0983
No. (%) of ≥ 2-point improvement at Week 6
14 (44)
4 (25)
0.2059
EASI score
IGA
% reduction from baseline, mean ± SD (median)
Percent body surface area with atopic dermatitis involvement Baseline, mean ± SD (median)
15.5 ± 13.0 (8.0)
13.0 ± 11.2 (6.3)
0.5045
Week 6, mean ± SD (median)
8.9 ± 11.6 (3.8)
11.5 ± 11.2 (6.4)
0.2886
50.4 ± 37.7 (63.8)
17.6 ± 48.6 (15.3)
0.0241*
Baseline, mean ± SD (median)
7.5 ± 0.9 (7.0)
6.8 ± 1.3 (7.0)
0.1028
Week 6, mean ± SD (median)
5.2 ± 2.2 (4.5)
5.5 ± 2.6 (6.5)
0.5210
-2.3 ± 2.0 (-2.0)†
-1.3 ± 2.5 (0.0)
0.0994
Baseline, mean ± SD (median)
11.5 ± 5.9 (10.5)
10.3 ± 5.7(9.5)
0.4770
Week 6, mean ± SD (median)
7.2 ± 5.8 (5.5)
7.8 ± 5.8 (6.5)
0.6446
-4.3 ± 5.3 (-4.0)†
-2.5 ± 4.9 (0.0)†
0.1557
17 (53)
4 (25)
0.0641
% reduction from baseline, mean ± SD (median) Pruritus VAS
Change from baseline, mean ± SD (median) DLQI/CDLQI score
Change from baseline, mean ± SD (median) No. (% ) that achieved MCID
Abbreviations: AD, atopic dermatitis; SD, standard deviation; EASI, Eczema Area Severity Index; VAS, visual analogue scale; DLQI/CDLQI, Dermatology Quality of Life Index/Children’s DLQI; IGA, Investigator’s Global Assessment; MCID, minimal clinically important difference (a decrease of 4 points or more). a Comparison between the Lindioil group and the vehicle group. *Statistical significance, P <0.05. †Significant change from baseline at Week 6, P <0.05.
28
Table 3: Adverse events during the overall study period (n=48). Event
Lindioil (n=32)
Vehicle (n=16)
17 (53)
7 (44)
Total AEs
22
11
Participant discontinued due to AE
0
0
Treatment-related AE
0
0
Carbuncle
1 (5)
0 (0)
Hordeolum
1 (5)
0 (0)
Nasopharyngitis
1 (5)
2 (18)
Oral herpes
0 (0)
1 (9)
Otitis externa candida
1 (5)
0 (0)
Upper respiratory tract infection
2 (9)
1 (9)
Constipation
1 (5)
0 (0)
Diarrhoea
1 (5)
0 (0)
Gastric disorder
0 (0)
1 (9)
Gastrointestinal disorder
2 (9)
0 (0)
Irritable bowel syndrome
1 (5)
0 (0)
Peptic ulcer
1 (5)
0 (0)
Cough
0 (0)
1 (9)
Nasal turbinate hypertrophy
0 (0)
1 (9)
Allergic rhinitis
1 (5)
0 (0)
Rhinorrhoea
1 (5)
1 (9)
Laceration
1 (5)
0 (0)
Headache
1 (5)
0 (0)
Insomnia
1 (5)
1 (9)
Irregular menstruation
0 (0)
1 (9)
4 (18)1
0 (0)
Dermatitis
1 (5)
0 (0)
Dyshidrotic eczema
0 (0)
1 (9)
Participant experienced at least one AE, n (%)
Type of AE, n (%)
Acne
1
One participant reported two separate acne occurrences.
29
FIGURE LEGENDS Fig. 1: (A) HPLC fingerprint of Lindioil, (B) HPLC fingerprint of standard reference for indirubin.
Fig. 2: Flow chart of the trial.
Fig. 3: (A) Mean percentage change from baseline to week 6 for EASI, (B) Proportion of patients achieving EASI-50, (C) EASI-75, or (D) EASI-90.
Fig. 4: Before-and-after photos of participants treated with Lindioil ointment achieving (A) EASI-50, (B) EASI-75, and (C) EASI-90. (A) score 6.2 at baseline to 3 at week 6. (B) score 13.8 at baseline to 1.7 at week 6. (C) score 18.7 at baseline to 1.6 at week 6.
30
A
Lindioil
B
Standard
55 screened
7 screen failure 2 not eligible 3 withdrew consent 2 other
48 randomized (2:1) 48 safety population
Treatment period (6 weeks)
32 Lindioil 5 discontinued 1 poor compliance 1 withdrew consent 1 lack of efficacy 2 used prohibited medications
Follow-up period (2 weeks)
27 completed
16 Vehicle 8 discontinued 2 poor compliance 1 withdrew consent 3 lack of efficacy 1 deterioration of AD 1 lost contact without reason 8 completed
2 discontinued 1 lost contact without reason 1 poor compliance 25 completed
8 completed
A
C
Proportion of patients achieving EASI-75 response 30
25
20
15
10
5
0
Lindioil Vehicle
0 1 2
Week
4
*
6
B
Proportion of patients achieving EASI-50 response
D
Proportion of patients achieving EASI-90 response
60
50
40
30
20
10
0
20 18 16 14 12 10 8 6 4 2 0
Lindioil Vehicle
0
Lindioil Vehicle
0
1
2
2 Week
4
4
6
6
*
1 Week
A
B
C
S0378-8741(19)32789-8
DOI:
https://doi.org/10.1016/j.jep.2019.112477
Reference:
JEP 112477
To appear in:
Journal of Ethnopharmacology
Received Date: 19 July 2019 Revised Date:
26 November 2019
Accepted Date: 10 December 2019
Please cite this article as: Lin, Y.-K., Chang, S.-H., Yang, C.-Y., See, L.-C., Lee, B.-H., Shih, I.-H., Efficacy and safety of indigo naturalis ointment in Treating Atopic Dermatitis: A randomized clinical trial, Journal of Ethnopharmacology (2020), doi: https://doi.org/10.1016/j.jep.2019.112477. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
Efficacy and Safety of Indigo naturalis Ointment in Treating Atopic Dermatitis: A Randomized Clinical Trial Short title: Indigo naturalis in Treating Atopic Dermatitis Yin-Ku Lina,b,g,*, Shu-Han Changa,g, Chin-Yi Yangc, Lai-Chu Seed,e, Be-Han Leea, I-Hsin Shihf Author Affiliations a
Department of Traditional Chinese Medicine, Chang Gung Memorial Hospital,
Keelung, 222, Maijin Road, Keelung 20401, Taiwan b
School of Traditional Chinese Medicine, College of medicine, Chang Gung
University, 259, Wenhua 1st Rd., Guishan Dist., Taoyuan 33302, Taiwan c
Department of Dermatology, Chang Gung Memorial Hospital, Linkou, 5, Fuxing St.,
Guishan Dist., Taoyuan 33305, Taiwan d
Department of Public Health, and Biostatistics Core Laboratory, Molecular Medicine
Research Center, Chang Gung University, 259, Wenhua 1st Rd., Guishan Dist., Taoyuan 33302, Taiwan e
Department of Rheumatology and Allergy and Immunology, Chang Gung Memorial
Hospital, Linkou, 5, Fuxing St., Guishan Dist., Taoyuan 33305, Taiwan f
Department of Dermatology, Chang Gung Memorial Hospital, Taipei, 199, Dunhua N.
Rd., Taipei 10507, Taiwan
g
Co-first author
*Corresponding author Yin-Ku Lin, 222 Mai Chin Road, Keelung 20401, Taiwan; E-Mail: [email protected]; Phone: +886-2-24313131 ext.2777; Fax: +886-2-24332655 Other e-mail addresses: [email protected] (Shu-Han Chang), [email protected] (Chin-Yi Yang), [email protected] (Lai-Chu See), [email protected] (Be-Han Lee), [email protected] (I-Hsin Shih) Word count: 4879 words, 3 tables, 4 figures
Abstract Ethnopharmacological relevance: Indigo naturalis, a herbal medicine with a history of use dating back to ancient times, may be a good alternative topical treatment for atopic dermatitis (AD). Aim of the study: To provide empirical evidence of the efficacy and safety of Indigo naturalis ointment in treating AD. Materials and Methods: In this randomized double-blind clinical trial, participants aged 6 to 65 years with AD affecting less than 40% of their body surface area (BSA) and an Investigator’s Global Assessment (IGA) score of 2 to 4 were randomized (2:1) to receive either Lindioil ointment or a vehicle ointment twice daily for 6 weeks. The primary endpoint was the percentage change in the Eczema Area Severity Index (EASI) from baseline to week 6. Secondary endpoints were as follows: EASI improvement ≥ 50%, 75%, and 90%; IGA score; BSA affected by AD; pruritus severity; and Dermatology Life Quality Index. The safety assessment included adverse events (AEs), laboratory tests, and physical examinations. Results: The Lindioil group (32 participants) and vehicle group (16 participants) achieved mean percentage EASI reductions of 49.9% ± 36.5% (95% CI 36.8% to 63.1%) and 19.6% ± 52.2% (95% CI -8.2% to 47.4%), respectively (P = 0.0235). The Lindioil group also showed greater improvement in every secondary assessment category. No significant AEs
3
occurred. Conclusion: Indigo naturalis ointment is effective for treating mild to severe AD topically, and appears to be safe. This is the first clinical trial to provide evidence supporting topical indigo-based AD treatment. ClinicalTrials.gov identifier: NCT02669888. Keywords: Atopic dermatitis, Indigo naturalis, Indirubin, EASI Abbreviations: AD, atopic dermatitis; AE, adverse event; BSA, body surface area; CDLQI, Children’s Dermatology Quality of Life Index; CGMH, Chang Gung Memorial Hospital; CI, confidence interval; CRP, C-reactive protein; DLQI, Dermatology Quality of Life Index; EASI, Eczema Area Severity Index; FDA, Food and Drug Administration; IGA, Investigator’s Global Assessment; IgE, immunoglobin E; LOCF, last observation carried forward; TCSs, topical corticosteroids; TCIs, topical calcineurin inhibitors; TCM, traditional Chinese medicine; VAS, visual analogue scale.
4
1. Introduction Atopic dermatitis (AD) is a common skin disease. It begins most frequently in infancy but may flare up at any age. There is no cure. Currently, the standard treatments for moderate to severe AD involve topical corticosteroids (TCSs) and topical calcineurin inhibitors (TCIs) (Eichenfield et al., 2014). The risks of long-term TCS use are well known (Hengge et al., 2006). Safety concerns about TCIs have also been raised, and the US Food and Drug Administration (FDA) has issued a warning for the TCIs, tacrolimus and pimecrolimus (Ring et al., 2008). Many AD patients seek alternative treatments. Traditional Chinese medicine (TCM) is one such line of treatment with increasing trends of use in Taiwan (Lin et al., 2019); unfortunately, it is difficult to find empirical evidence regarding the effectiveness and safety of most herbal medicines used in TCM to treat AD (Tan et al., 2013). Indigo naturalis is a blue powder derived from indigo plants, such as Baphicacanthus cusia. Its centuries-long use in TCM to treat various infectious and inflammatory diseases is well documented (Tang and Eisenbrand, 1992). Because of its dark-blue colour, crude Indigo naturalis stains the skin and clothes when used topically. Consequently, despite the documentation of its use in treating skin disease, in modern times few patients have been willing to use it. In 2008, we successfully removed the dark-blue coloration by extracting it in olive oil, thus improving the formulation. The refined formulation, named Lindioil, does not cause staining (Lin et al., 2015; Lin et al., 2012).
5
In the past decade, our research has shown that Indigo naturalis repairs abnormal epidermal barriers by promoting differentiation, upregulating claudin-1 expression, restoring tight junction proteins, and inhibiting keratinocyte proliferation; additionally, we identified indirubin as its main active ingredient (Lin et al., 2013; Lin et al., 2009; Lin et al., 2007). Other research has shown that indirubin suppresses skin inflammation through the inhibition of cytokine production, nuclear factor kappa B activity, and mitogen-activated protein kinase activation (Kim et al., 2013). Thus, the anti-inflammatory, antimicrobial, antiviral and anticancer mechanisms of Indigo naturalis and its active ingredient have been explained by modern science (Chiang et al., 2013; Hoessel et al., 1999; Mak et al., 2004). Based on the modern understanding of these mechanisms, data from previous clinical trials demonstrating the efficacy and safety of both Indigo naturalis and Lindioil in treating psoriasis (Lin et al., 2008; Lin et al., 2018), and a historical document from TCM literature that mentions the use of Indigo naturalis to treat facial eczema in infants, we started using Lindioil ointment to treat AD (in addition to psoriasis) topically at our hospital several years ago. In this randomized, double-blind, vehicle-controlled clinical trial, we hoped to provide empirical evidence of our indigo-based ointment’s efficacy and safety in treating AD. 2. Materials and Methods 2.1 Study Design This was a randomized, double-blind, vehicle-controlled trial initiated by the principal
6
investigator and conducted at two branches (Taipei and Linkou) of Chang Gung Memorial Hospital (CGMH) in Taiwan. Participants were screened, enrolled, and randomized at a 2:1 ratio to receive either Lindioil ointment or placebo (vehicle ointment) for 6 weeks. All participants (and a parent for participants under 20 years old) signed an informed consent form. The protocol was approved by the institutional review boards of Chang Gung Medical Foundation (103-0472A) and the FDA in Taiwan and conducted in accordance with the ethical principles of the Declaration of Helsinki and Good Clinical Practice. The authors vouch for the study’s fidelity to the protocol. This study is registered with ClinicalTrials.gov (identifier NCT02669888). 2.2 Participants Patients who visited the TCM and dermatology outpatient clinics of CGMH and were diagnosed with AD based on the standard criteria of Williams et al (Williams, 2005) were invited to participate in this trial. They signed informed consent forms before being screened for eligibility. Patients ages 6 to 65 were approved for inclusion based on the following criteria: 40% or less body surface area (BSA) affected by the disease; and Investigator’s Global Assessment (IGA, ranging from 0 to 5, or completely clear to very severe) score of mild (2) to severe (4) (Eichenfield et al., 2002). Participants were ineligible for any of the following reasons: a history of topical or systemic hypersensitivity to Indigo naturalis or its excipient (e.g., beeswax) in ointment; a known malignancy (current or past);
7
aspartate aminotransferase or alanine aminotransferase levels greater than three times the upper normal limit; creatinine greater than 2.0 mg/dL; clinically significant haematological abnormalities; uncontrolled hypertension; uncontrolled metabolic diseases; psychiatric conditions; or human immunodeficiency virus infection. Women were not eligible if they were pregnant or lactating, and eligible female participants were asked to use birth control for the duration of the study. Participants were required to cease systemic treatments (e.g., immunosuppressive agents) or phototherapy at least 30 days before the first application of the study medication and were required to cease the use of TCSs and other topical medicines at least 7 days before beginning the study. For skin care needs other than AD treatment, participants were permitted to use a simple emollient, Vaseline lotion, which we provided. 2.3 Plant material and drug preparation The Indigo naturalis powder used in this study was purchased from Shufeng Shangrchia Indigo Farmer Cooperative, Xianyou county, Fujian province, China, and came from the leaves of Baphicacanthus cusia (Nees) Bremek. The quality of the Indigo naturalis powder was verified by Chuang Song Zong (CSZ) Pharmaceutical Co. Ltd. (Taiwan). Ms. Shu-Tuan Chiang (Research and Quality Control department manager of CSZ) identified and authenticated the material; sample vouchers were stored for reference under the code number M0813. Pure indigo blue was purchased from CHEM SERVICE (West Chester,
8
USA), and the indirubin was obtained from National Institutes for Food and Drug Control (NIFDC, Beijing, China). The Indigo naturalis powder used in this trial contained of 2.80% indigo blue and 0.15% indirubin determined by ultraviolet spectrophotometer, and high performance liquid chromatography (HPLC), respectively. The CMC report was made in accordance with the Guidance for Industry Botanical Drug Products. The preparation of Lindioil using an olive oil extraction process, and the process for HPLC analyses of Indigo naturalis and Lindioil (Fig. 1), have been described in detail previously (Lin et al., 2014). The vehicle ointment was prepared by mixing olive oil and beeswax with edible pigment to make the appearance identical to Lindioil ointment (Supplement, Table S1).
2.4 Randomization and Masking After providing written consent, the participants were allocated by a permuted block randomization method with blocks of size 6 to receive Lindioil ointment containing 200 µg/g of indirubin (in a previous trial, 200 µg/g of indirubin was found to be the optimal concentration for treating psoriasis) (Lin et al., 2018) or a vehicle ointment. The two ointments and their containers were identical in appearance. A randomization code list generated by Contract Research Organization (CRO) of Formosa Biomedical Technology Corporation (FBC) was provided to CSZ for packaging and labelling the study medication. FBC CRO produced sealable, randomized envelopes for each randomization code (with the
9
codes shown on the envelopes), which were stored by sponsor personnel who were independent of the study. 2.5 Procedures Due to concerns expressed by Taiwan’s FDA, we excluded children from screening until the safety data for the first 5 adult participants (age range 20–40 years) showed no treatment-related adverse events. Thereafter, the age range was changed to 12–40 years and eventually broadened to 6–65 years. At baseline, the participants were instructed to apply 0.5 g of ointment per 100 cm2 of dermatitis lesion area twice daily for 6 weeks. The severity of AD and the safety of the treatment were assessed at the end of weeks 1, 2, 4, and 6. At the end of the 6th week, all medicine tubes containing Lindioil or vehicle ointment were collected from the participants, and the participants returned for a final follow-up assessment 2 weeks later. 2.6 Efficacy Outcomes and Adverse Events We chose the mean percentage change in Eczema Area Severity Index (EASI) scores at week 6 as our primary efficacy endpoint (EASI is a standardized, validated tool used by dermatologists to objectively combine surface area involvement, erythema, edema/papulation, excoriation, and lichenification into an overall assessment of dermatitis severity) (Hanifin et al., 2001). The secondary efficacy endpoints were (i) the proportions of patients with ≥ 50%, 75%, and 90% improvement in EASI scores (EASI-50, EASI-75, and EASI-90) at the end of
10
week 6; (ii) the proportion of participants who achieved ‘clear’ (IGA = 0) or ‘almost clear’ (IGA = 1) status or more than 2-point of improvement in IGA at week 6 (Thaci et al., 2016); (iii) the mean percentage reduction in the BSA affected by AD over the course of the study period; (iv) the mean change in the pruritus score (using a 10-cm visual analogue scale, VAS) (Reich et al., 2012); and (v) the mean change in the Dermatology Quality of Life Index (DLQI) or the Children’s DLQI (CDLQI) value from baseline to week 6 (Finlay, 1998). AEs and vital signs were assessed throughout the study at each participant’s visit. Haematology, biochemistry, urinalysis, immunoglobin E (IgE) and C-reactive protein (CRP) tests were performed at baseline and at the end of week 6. Any AEs occurring after the administration of the first dose of the study drug and at any time thereafter, up to the completion of the trial, were recorded. The investigating physician actively terminated study participation if a participant experienced significant worsening of dermatitis symptoms (50% increase in the EASI score from baseline); a significant treatment-related AE; failed to apply the medicine on more than 2 days per week; or used prohibited medications.
2.7 Statistical Analysis The descriptive statistics of continuous data are presented separately for the two groups (including the number of observations, mean, median, and standard deviation). The descriptive statistics of discrete variables (frequency, %) were tabulated separately for the two groups. An independent t-test was used to determine the difference in the percentage
11
change of the primary endpoint for the two groups. If the assumption of normality was violated, the Mann-Whitney U test was used instead. For the secondary endpoints, an independent t-test was used to compare continuous variables between the two groups. If the normality assumption was violated, the Mann-Whitney U test was used instead. A paired t-test was used to evaluate the mean change in continuous variables from baseline to the specified visit for each participant for comparison between the groups. If the normality assumption was violated, the Wilcoxon signed-rank test was used instead. The chi-square test or Fisher’s exact test was used to compare discrete variables between the two groups, and two-way ANOVA was used to evaluate the efficacy of continuous variables in relation to other categorical types (e.g., age) between the two groups. Intermittently missing efficacy data were imputed by last observation carried forward (LOCF), and no imputation method was used to estimate the missing values for safety data. All statistical analyses were conducted using SAS software (version 9.4).
3. Results From October 2015 to February 2017, 55 participants were screened, and 48 were determined to be eligible. Thirty-two of the eligible participants were randomly assigned to receive Lindioil ointment for treatment, and 16 were given a vehicle ointment (Fig. 2). The
12
demographic and baseline disease characteristics of the participants varied similarly within each of the groups (Table 1). Of the 48 participants whose data comprised the intention-to-treat analysis, 35 completed the full 6-week study period: 27/32 in the Lindioil group and 8/16 in the vehicle group. 3.1 Study Completion After randomization and the start of treatment, 16% (5/32) of the Lindioil participants and 50% (8/16) of the vehicle participants discontinued treatment. For 4 of the 8 vehicle group participants who discontinued, the reason for discontinuation was lack of efficacy (3/8) or significant worsening of disease characteristics (1/8). Only 1 participant in the Lindioil group discontinued due to lack of efficacy, and none experienced a significant worsening of symptoms. None of the adverse reactions experienced during the trial period were determined to be caused by Lindioil. Other reasons for discontinuation during the 6-week treatment period are listed in Fig. 2; further details of discontinued patients, including EASI and pruritus scores at baseline and last appointment before discontinuation, are listed in Supplement, Table S2. 3.2 Efficacy Our primary endpoint was the mean percentage change in the EASI score from baseline to the end of week 6. Fig. 3A and Table 2 show the results: reductions of 49.9% (95% CI 36.8–63.1) for the Lindioil group and 19.6% (95% CI -8.2–47.4) for the vehicle
13
group, a statistically significant difference (P = 0.0235), supporting the efficacy of Lindioil for treating AD. For the EASI-related secondary endpoints, Figs. 3B, 3C, and 3D show the proportions of participants who achieved EASI-50, EASI-75, and EASI-90, all supporting the efficacy of Lindioil. Figs. 4A, 4B, and 4C are before-and-after photos of participants in the Lindioil group who achieved EASI-50, EASI-75, and EASI-90 respectively. All other secondary endpoints also supported the efficacy of Lindioil. For the IGA score, 38% (12/32) of the Lindioil group achieved IGA 0 or 1, vs. 13% (2/16) of the vehicle group (P = 0.0983); 44% (14/32) of the Lindioil group achieved more than 2-point improvement in IGA score from baseline compared with 25% (4/16) of the vehicle group (P = 0.2059). The percent reduction in the mean IGA from baseline to the end of week 6 was 39.3% for Lindioil compared with 18.2% for vehicle (P = 0.0387). The mean percentage reductions in the BSA affected by AD from baseline to the end of week 6 were 50.4% (Lindioil) and 17.6% (vehicle) (P = 0.0241), and the mean changes in pruritus VAS score were -2.3 (Lindioil) and -1.3 (vehicle) (P = 0.0994). The mean changes in the DLQI/CDLQI from baseline to week 6 were -4.3 (Lindioil) and -2.5 (vehicle), with 53% (17/32) of the Lindioil participants and only 25% (4/16) of the vehicle participants achieving the minimal clinically important difference (a decrease of 4 points or more) (P = 0.0641) (Basra et al., 2015). Eosinophils, IgE, CRP, and other immune-related
14
markers showed no statistically significant differences in either group from baseline to week 6 (Supplement, Table S3). Thirty-three of the 35 participants who completed the treatment period (25/27 in the Lindioil group and 8/8 in the vehicle group) returned for the follow-up appointment at week 8. For the follow-up group, after the 2-week washout period, the IGA scores of 56% (14/25) of the Lindioil participants and 50% (4/8) of the vehicle participants had increased, as had the pruritus VAS scores of 60% (15/25) of the Lindioil participants and 25% (2/8) of the vehicle participants. 3.3 Safety During the study period, a total of 33 treatment-emergent AEs were reported by 24 participants (Table 3). The incidence of AEs was 53% (17/32) in the Lindioil group and 44% (7/16) in the vehicle group. All AEs were mild. The most commonly reported AEs, acne in 3/32 Lindioil participants (4 events) and nasopharyngitis in 2/16 vehicle participants, were clearly not treatment-related. 5 participants experienced a total of 6 mild skin AEs (acne, in 4 of these 6 cases). None of the skin AEs was determined to be treatment-related, and none resulted in discontinuation of treatment. Safety-related blood tests and urinalysis showed no statistically significant differences in either group from baseline to week 6 (data not shown). 4. Discussion
15
The results of this 6-week trial demonstrate that Lindioil ointment is effective in relieving all measurable AD symptoms, thereby improving the quality of life of AD patients, and is superior to a vehicle ointment. The primary endpoint data (a 49.9% mean EASI reduction for the participants who were given Lindioil ointment versus a 19.6% mean EASI reduction for those given a vehicle ointment) and data across the secondary endpoints provide strong evidence of Lindioil ointment’s efficacy. The 19.6% EASI improvement in the vehicle group may surprise some readers. In fact, it is consistent with other AD topical treatment studies, in which vehicle groups have shown a 20-25% EASI reduction at week 6 as well (Chapman et al., 2005; Schachner et al., 2005). This is because the moisturizers in the vehicle ointments, by themselves, increase skin hydration and reduce inflammation and severity of AD (Eichenfield et al., 2014). Olive oil, the main component in both our vehicle ointment and Lindioil ointment, is known to increase skin hydration and alleviate AD symptoms (Al-Waili, 2003; Verallo-Rowell et al., 2008). Our trial showed high standard deviation in the percentage reduction of EASI score. This is also consistent with other AD studies of similar sample size (Leung et al., 2009; Peppers et al., 2019). Just like many other chronic diseases, AD is influenced by environmental factors varying from patient to patient including mood and lifestyle, sometimes regardless of treatment. It is unfortunate that we ended up with such a small sample size. This limitation was due to budget and schedule constraints. A trial with larger sample size would likely show
16
a lower standard deviation. TCSs and TCIs comprise the vast majority of AD medicines worldwide, with TCSs yielding the best results. However, safety concerns regarding the risks of long-term corticosteroid use (such as skin atrophy and adrenal suppression) have led to many AD patients being unwilling to consider even short-term steroid-based treatment (Li et al., 2017). The most commonly used non-steroid topical medicines for AD treatment are TCIs. According to data from previous 6-week trials, the TCIs tacrolimus and pimecrolimus achieved 54.1% / 67.2% and 34.9% / 56.4% mean EASI reductions among adult/child patients, respectively (Paller et al., 2005). Lindioil ointment is the first topical herbal medicine to display efficacy results within this range. In addition, just as in the above-mentioned tacrolimus and pimecrolimus studies, the children in our trial displayed a better response than the adults. If indigo-based medicines continue to display these results, they may be an attractive alternative to TCIs for patients who prefer herbal medicines worldwide. The data from this 6-week trial alone should not be enough to assuage patients’ concerns about long-term safety, especially considering the limited sample size. However, Indigo naturalis has a documented history of use in TCM going back hundreds of years, with records indicating the topical use of Indigo naturalis to treat skin disease (specifically including infant eczema) and no mention of any side effects or safety concerns for long-term use, and modern
17
medical literature seems also to be lacking any reports of serious side effects from long-term topical use of Indigo naturalis. As other indigo-based medicines are developed and more trials conducted, more data will gradually become available over time. To the best of our knowledge, this is the first double-blind clinical trial to support the efficacy of Indigo naturalis for treating AD topically. Since this treatment also appears to be safe, we believe that Indigo naturalis has the potential to be developed into topical medicines for AD treatment worldwide. Acknowledgements We thank Daniel Teitler for proofreading and editing the manuscript. Funding Sources This principal investigator (Dr. Lin)-initiated phase IIa trial was supported by the Taiwan Ministry of Science and Technology (MOST105-2325-B-182A-003) and Chang Gung Medical Foundation (BMRP972A), Taiwan. The funding sponsors had no role in the design and conduct of the study; in the collection, analysis, and interpretation of data; or in the preparation, review, or approval of the manuscript. Conflict of Interest Disclosures Dr. Lin was supported by grants and personal fees from the Taiwan Ministry of Science and Technology and Chang Gung Foundation during the conduct of the study. In addition, Dr. Lin is the inventor of Lindioil.
18
Authors’ Contributions Dr. Lin had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Lin. Acquisition of data: Lin, Yang, and Shih. Analysis and interpretation of data: Lin and See. Drafting of the manuscript: Lin, Chang, and Lee. Critical revision of the manuscript for important intellectual content: Lin, See, Chang, Yang, and Shih. Statistical analysis: See and Lee. Obtained funding: Lin. Administrative support: Lin. Study supervision: Lin.
19
References Al-Waili, N.S., 2003. Topical application of natural honey, beeswax and olive oil mixture for atopic dermatitis or psoriasis: partially controlled, single-blinded study. Complement Ther Med 11(4), 226-234, 10.1016/s0965-2299(03)00120-1 Basra, M.K., Salek, M.S., Camilleri, L., Sturkey, R., Finlay, A.Y., 2015. Determining the minimal clinically important difference and responsiveness of the Dermatology Life Quality Index (DLQI): further data. Dermatology 230(1), 27-33, 10.1159/000365390 Chapman, M.S., Schachner, L.A., Breneman, D., Boguniewicz, M., Gold, M.H., Shull, T., Linowski, G.J., Jaracz, E., Group, U.S.T.O.S., 2005. Tacrolimus ointment 0.03% shows efficacy and safety in pediatric and adult patients with mild to moderate atopic dermatitis. J Am Acad Dermatol 53(2 Suppl 2), S177-185, 10.1016/j.jaad.2005.04.061 Chiang, Y.R., Li, A., Leu, Y.L., Fang, J.Y., Lin, Y.K., 2013. An in vitro study of the antimicrobial effects of Indigo naturalis prepared from Strobilanthes formosanus Moore. Molecules 18(11), 14381-14396, 10.3390/molecules181114381 Eichenfield, L.F., Lucky, A.W., Boguniewicz, M., Langley, R.G., Cherill, R., Marshall, K., Bush, C., Graeber, M., 2002. Safety and efficacy of pimecrolimus (ASM 981) cream 1% in the treatment of mild and moderate atopic dermatitis in children and adolescents. J Am Acad Dermatol 46(4), 495-504, 10.1067/mjd.2002.122187 Eichenfield, L.F., Tom, W.L., Berger, T.G., Krol, A., Paller, A.S., Schwarzenberger, K.,
20
Bergman, J.N., Chamlin, S.L., Cohen, D.E., Cooper, K.D., Cordoro, K.M., Davis, D.M., Feldman, S.R., Hanifin, J.M., Margolis, D.J., Silverman, R.A., Simpson, E.L., Williams, H.C., Elmets, C.A., Block, J., Harrod, C.G., Smith Begolka, W., Sidbury, R., 2014. Guidelines of care for the management of atopic dermatitis: section 2. Management and treatment of atopic dermatitis with topical therapies. J Am Acad Dermatol 71(1), 116-132, 10.1016/j.jaad.2014.03.023 Finlay, A.Y., 1998. Quality of life assessments in dermatology. Semin Cutan Med Surg 17(4), 291-296, 10.1016/s1085-5629(98)80026-6 Hanifin, J.M., Thurston, M., Omoto, M., Cherill, R., Tofte, S.J., Graeber, M., 2001. The eczema area and severity index (EASI): assessment of reliability in atopic dermatitis. EASI Evaluator Group. Exp Dermatol 10(1), 11-18, 10.1034/j.1600-0625.2001.100102.x Hengge, U.R., Ruzicka, T., Schwartz, R.A., Cork, M.J., 2006. Adverse effects of topical glucocorticosteroids. J Am Acad Dermatol 54(1), 1-15; quiz 16-18, 10.1016/j.jaad.2005.01.010 Hoessel, R., Leclerc, S., Endicott, J.A., Nobel, M.E., Lawrie, A., Tunnah, P., Leost, M., Damiens, E., Marie, D., Marko, D., Niederberger, E., Tang, W., Eisenbrand, G., Meijer, L., 1999. Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases. Nat Cell Biol 1(1), 60-67, 10.1038/9035 Kim, M.H., Choi, Y.Y., Yang, G., Cho, I.H., Nam, D., Yang, W.M., 2013. Indirubin, a purple
21
3,2- bisindole, inhibited allergic contact dermatitis via regulating T helper (Th)-mediated immune system in DNCB-induced model. J Ethnopharmacol 145(1), 214-219, 10.1016/j.jep.2012.10.055 Leung, D.Y., Hanifin, J.M., Pariser, D.M., Barber, K.A., Langley, R.G., Schlievert, P.M., Abrams, B., Hultsch, T., 2009. Effects of pimecrolimus cream 1% in the treatment of patients with atopic dermatitis who demonstrate a clinical insensitivity to topical corticosteroids: a randomized, multicentre vehicle-controlled trial. Br J Dermatol 161(2), 435-443, 10.1111/j.1365-2133.2009.09145.x Li, A.W., Yin, E.S., Antaya, R.J., 2017. Topical Corticosteroid Phobia in Atopic Dermatitis: A Systematic Review. JAMA Dermatol 153(10), 1036-1042, 10.1001/jamadermatol.2017.2437 Lin, P.Y., Chu, C.H., Chang, F.Y., Huang, Y.W., Tsai, H.J., Yao, T.C., 2019. Trends and prescription patterns of traditional Chinese medicine use among subjects with allergic diseases: A nationwide population-based study. World Allergy Organ J 12(2), 100001, 10.1016/j.waojou.2018.11.001 Lin, Y.K., Chang, C.J., Chang, Y.C., Wong, W.R., Chang, S.C., Pang, J.H., 2008. Clinical assessment of patients with recalcitrant psoriasis in a randomized, observer-blind, vehicle-controlled trial using Indigo naturalis. Arch Dermatol 144(11), 1457-1464, 10.1001/archderm.144.11.1457
22
Lin, Y.K., Chang, Y.C., Hui, R.C., See, L.C., Chang, C.J., Yang, C.H., Huang, Y.H., 2015. A Chinese Herb, Indigo naturalis, Extracted in Oil (Lindioil) Used Topically to Treat Psoriatic Nails: A Randomized Clinical Trial. JAMA Dermatol 151(6), 672-674, 10.1001/jamadermatol.2014.5460 Lin, Y.K., Chen, H.W., Leu, Y.L., Yang, Y.L., Fang, Y., Su Pang, J.H., Hwang, T.L., 2013. Indigo naturalis upregulates claudin-1 expression in human keratinocytes and psoriatic lesions. J Ethnopharmacol 145(2), 614-620, 10.1016/j.jep.2012.11.044 Lin, Y.K., Leu, Y.L., Yang, S.H., Chen, H.W., Wang, C.T., Pang, J.H., 2009. Anti-psoriatic effects of iIndigo naturalis on the proliferation and differentiation of keratinocytes with indirubin as the active component. J Dermatol Sci 54(3), 168-174, 10.1016/j.jdermsci.2009.02.007 Lin, Y.K., See, L.C., Huang, Y.H., Chang, Y.C., Tsou, T.C., Leu, Y.L., Shen, Y.M., 2012. Comparison of refined and crude Indigo naturalis ointment in treating psoriasis: randomized, observer-blind, controlled, intrapatient trial. Arch Dermatol 148(3), 397-400, 10.1001/archdermatol.2011.1091 Lin, Y.K., See, L.C., Huang, Y.H., Chang, Y.C., Tsou, T.C., Lin, T.Y., Lin, N.L., 2014. Efficacy and safety of Indigo naturalis extract in oil (Lindioil) in treating nail psoriasis: a randomized, observer-blind, vehicle-controlled trial. Phytomedicine 21(7), 1015-1020, 10.1016/j.phymed.2014.02.013
23
Lin, Y.K., See, L.C., Huang, Y.H., Chi, C.C., Hui, R.C., 2018. Comparison of indirubin concentrations in Indigo naturalis ointment for psoriasis treatment: a randomized, double-blind, dosage-controlled trial. Br J Dermatol 178(1), 124-131, 10.1111/bjd.15894 Lin, Y.K., Wong, W.R., Chang, Y.C., Chang, C.J., Tsay, P.K., Chang, S.C., Pang, J.H., 2007. The efficacy and safety of topically applied Indigo naturalis ointment in patients with plaque-type psoriasis. Dermatology 214(2), 155-161, 10.1159/000098576 Mak, N.K., Leung, C.Y., Wei, X.Y., Shen, X.L., Wong, R.N., Leung, K.N., Fung, M.C., 2004. Inhibition of RANTES expression by indirubin in influenza virus-infected human bronchial epithelial cells. Biochem Pharmacol 67(1), 167-174, 10.1016/j.bcp.2003.08.020 Paller, A.S., Lebwohl, M., Fleischer, A.B., Jr., Antaya, R., Langley, R.G., Kirsner, R.S., Blum, R.R., Rico, M.J., Jaracz, E., Crowe, A., Linowski, G.J., Group, U.S.C.T.O.S., 2005. Tacrolimus ointment is more effective than pimecrolimus cream with a similar safety profile in the treatment of atopic dermatitis: results from 3 randomized, comparative studies. J Am Acad Dermatol 52(5), 810-822, 10.1016/j.jaad.2004.12.038 Peppers, J., Paller, A.S., Maeda-Chubachi, T., Wu, S., Robbins, K., Gallagher, K., Kraus, J.E., 2019. A phase 2, randomized dose-finding study of tapinarof (GSK2894512 cream) for the treatment of atopic dermatitis. J Am Acad Dermatol 80(1), 89-98 e83, 10.1016/j.jaad.2018.06.047 Reich, A., Heisig, M., Phan, N.Q., Taneda, K., Takamori, K., Takeuchi, S., Furue, M.,
24
Blome, C., Augustin, M., Stander, S., Szepietowski, J.C., 2012. Visual analogue scale: evaluation of the instrument for the assessment of pruritus. Acta Derm Venereol 92(5), 497-501, 10.2340/00015555-1265 Ring, J., Mohrenschlager, M., Henkel, V., 2008. The US FDA 'black box' warning for topical calcineurin inhibitors: an ongoing controversy. Drug Saf 31(3), 185-198, 10.2165/00002018-200831030-00001 Schachner, L.A., Lamerson, C., Sheehan, M.P., Boguniewicz, M., Mosser, J., Raimer, S., Shull, T., Jaracz, E., Group, U.S.T.O.S., 2005. Tacrolimus ointment 0.03% is safe and effective for the treatment of mild to moderate atopic dermatitis in pediatric patients: results from a randomized, double-blind, vehicle-controlled study. Pediatrics 116(3), e334-342, 10.1542/peds.2004-2638 Tan, H.Y., Zhang, A.L., Chen, D., Xue, C.C., Lenon, G.B., 2013. Chinese herbal medicine for atopic dermatitis: a systematic review. J Am Acad Dermatol 69(2), 295-304, 10.1016/j.jaad.2013.01.019 Tang, W., Eisenbrand, G., 1992. Chinese drugs of plant origin: chemistry, pharmacology, and use in traditional and modern medicine. Springer-Verlag Berlin Heidelberg, 10.1007/978-3-642-73739-8 Thaci, D., Simpson, E.L., Beck, L.A., Bieber, T., Blauvelt, A., Papp, K., Soong, W., Worm, M., Szepietowski, J.C., Sofen, H., Kawashima, M., Wu, R., Weinstein, S.P., Graham, N.M.,
25
Pirozzi, G., Teper, A., Sutherland, E.R., Mastey, V., Stahl, N., Yancopoulos, G.D., Ardeleanu, M., 2016. Efficacy and safety of dupilumab in adults with moderate-to-severe atopic dermatitis inadequately controlled by topical treatments: a randomised, placebo-controlled, dose-ranging phase 2b trial. Lancet 387(10013), 40-52, 10.1016/S0140-6736(15)00388-8 Verallo-Rowell, V.M., Dillague, K.M., Syah-Tjundawan, B.S., 2008. Novel antibacterial and emollient effects of coconut and virgin olive oils in adult atopic dermatitis. Dermatitis 19(6), 308-315, 10.2310/6620.2008.08052 Williams, H.C., 2005. Clinical practice. Atopic dermatitis. N Engl J Med 352(22), 2314-2324, 10.1056/NEJMcp042803
26
TABLE LEGENDS Table 1: Demographic and clinical characteristics at baseline (n=48). Patients
Lindioil (n=32)
Vehicle (n=16)
20 (63)
9 (56)
21.9 (8.3-37.8)
23.0 (9.2-35.1)
Adult (≥ 18 years old), n (%)
18 (56)
12 (75)
Children (< 18 years old), n (%)
14 (44)
4 (25)
Body mass index, mean (range), kg/m2
21.1 (13.9-28.7)
21.7 (16.2-28.7)
Age at onset of AD, mean (range), y
4.4 (0.0-32.0)
4.8 (0.0-16.0)
Duration of AD, mean (range), y
17.5 (1.3-36.8)
18.2 (3.5-30.6)
History of IgE reactivity, n (%)
26 (81)
15 (94)
History of allergic rhinitis, n (%)
26 (81)
10 (63)
History of asthma, n (%)
16 (50)
6 (38)
Topical therapy
32 (100)
16 (100)
Systemic therapies
27 (84)
16 (100)
Other therapies
30 (94)
14 (88)
IGA 3
13 (41)
6 (38)
IGA 4
19 (59)
10 (63)
12.3 (4.7-28.9)
12.3 (2.9-29.3)
Mild (EASI ≤ 7.0), n (%)
8 (25)
7 (44)
Moderate (7.1 ≤ EASI ≤ 21.0), n (%)
19 (59)
5 (31)
Severe (EASI ≥ 21.1), n (%)
5 (16)
4 (25)
15.5 (3.0-39.5)
13.0 (3.5-36.0)
Pruritus VAS, mean (range)
7.5 (6.0-9.0)
6.8 (4.0-9.0)
DLQI/CDLQI, mean (range)
11.5 (1.0-26.0)
10.3 (2.0-23.0)
Males, n (%) Age, mean (range), y
Prior treatments, n (%)
IGA, n (%)
EASI, mean (range)
BSA, mean (range)
Abbreviations: AD, atopic dermatitis; EASI, Eczema Area Severity Index; BSA, body surface area; VAS, visual analogue scale; DLQI/CDLQI, Dermatology Quality of Life Index/children’s DLQI; IGA, Investigator’s Global Assessment.
27
Table 2: Efficacy outcomes, baseline to week 6 (n=48). Patients
P valuea
Lindioil (n=32)
Vehicle (n=16)
Baseline, mean ± SD (median)
12.3 ± 7.1 (8.7)
12.3 ± 9.1 (7.5)
0.4183
Week 6, mean ± SD (median)
6.4 ± 6.5 (5.1)
9.8 ± 7.9 (5.8)
0.1857
Change from baseline, mean ± SD (median)
-5.8 ± 5.2 (-5.2)†
-2.6 ± 6.9 (-2.4)
0.0279*
% reduction from baseline, mean ± SD (median)
49.9 ± 36.5 (58.2)
19.6 ± 52.2 (20.2)
0.0235*
Baseline, mean ± SD (median)
3.6 ± 0.5 (4.0)
3.6 ± 0.5 (4.0)
0.8465
Week 6, mean ± SD (median)
2.2 ± 1.2 (2.0)
2.9 ± 1.1 (3.0)
0.0468*
39.3 ± 34.0 (33.3)
18.2 ± 29.7 (0.0)
0.0387*
No. (%) of IGA 0 or 1 at Week 6
12 (38)
2 (13)
0.0983
No. (%) of ≥ 2-point improvement at Week 6
14 (44)
4 (25)
0.2059
EASI score
IGA
% reduction from baseline, mean ± SD (median)
Percent body surface area with atopic dermatitis involvement Baseline, mean ± SD (median)
15.5 ± 13.0 (8.0)
13.0 ± 11.2 (6.3)
0.5045
Week 6, mean ± SD (median)
8.9 ± 11.6 (3.8)
11.5 ± 11.2 (6.4)
0.2886
50.4 ± 37.7 (63.8)
17.6 ± 48.6 (15.3)
0.0241*
Baseline, mean ± SD (median)
7.5 ± 0.9 (7.0)
6.8 ± 1.3 (7.0)
0.1028
Week 6, mean ± SD (median)
5.2 ± 2.2 (4.5)
5.5 ± 2.6 (6.5)
0.5210
-2.3 ± 2.0 (-2.0)†
-1.3 ± 2.5 (0.0)
0.0994
Baseline, mean ± SD (median)
11.5 ± 5.9 (10.5)
10.3 ± 5.7(9.5)
0.4770
Week 6, mean ± SD (median)
7.2 ± 5.8 (5.5)
7.8 ± 5.8 (6.5)
0.6446
-4.3 ± 5.3 (-4.0)†
-2.5 ± 4.9 (0.0)†
0.1557
17 (53)
4 (25)
0.0641
% reduction from baseline, mean ± SD (median) Pruritus VAS
Change from baseline, mean ± SD (median) DLQI/CDLQI score
Change from baseline, mean ± SD (median) No. (% ) that achieved MCID
Abbreviations: AD, atopic dermatitis; SD, standard deviation; EASI, Eczema Area Severity Index; VAS, visual analogue scale; DLQI/CDLQI, Dermatology Quality of Life Index/Children’s DLQI; IGA, Investigator’s Global Assessment; MCID, minimal clinically important difference (a decrease of 4 points or more). a Comparison between the Lindioil group and the vehicle group. *Statistical significance, P <0.05. †Significant change from baseline at Week 6, P <0.05.
28
Table 3: Adverse events during the overall study period (n=48). Event
Lindioil (n=32)
Vehicle (n=16)
17 (53)
7 (44)
Total AEs
22
11
Participant discontinued due to AE
0
0
Treatment-related AE
0
0
Carbuncle
1 (5)
0 (0)
Hordeolum
1 (5)
0 (0)
Nasopharyngitis
1 (5)
2 (18)
Oral herpes
0 (0)
1 (9)
Otitis externa candida
1 (5)
0 (0)
Upper respiratory tract infection
2 (9)
1 (9)
Constipation
1 (5)
0 (0)
Diarrhoea
1 (5)
0 (0)
Gastric disorder
0 (0)
1 (9)
Gastrointestinal disorder
2 (9)
0 (0)
Irritable bowel syndrome
1 (5)
0 (0)
Peptic ulcer
1 (5)
0 (0)
Cough
0 (0)
1 (9)
Nasal turbinate hypertrophy
0 (0)
1 (9)
Allergic rhinitis
1 (5)
0 (0)
Rhinorrhoea
1 (5)
1 (9)
Laceration
1 (5)
0 (0)
Headache
1 (5)
0 (0)
Insomnia
1 (5)
1 (9)
Irregular menstruation
0 (0)
1 (9)
4 (18)1
0 (0)
Dermatitis
1 (5)
0 (0)
Dyshidrotic eczema
0 (0)
1 (9)
Participant experienced at least one AE, n (%)
Type of AE, n (%)
Acne
1
One participant reported two separate acne occurrences.
29
FIGURE LEGENDS Fig. 1: (A) HPLC fingerprint of Lindioil, (B) HPLC fingerprint of standard reference for indirubin.
Fig. 2: Flow chart of the trial.
Fig. 3: (A) Mean percentage change from baseline to week 6 for EASI, (B) Proportion of patients achieving EASI-50, (C) EASI-75, or (D) EASI-90.
Fig. 4: Before-and-after photos of participants treated with Lindioil ointment achieving (A) EASI-50, (B) EASI-75, and (C) EASI-90. (A) score 6.2 at baseline to 3 at week 6. (B) score 13.8 at baseline to 1.7 at week 6. (C) score 18.7 at baseline to 1.6 at week 6.
30
A
Lindioil
B
Standard
55 screened
7 screen failure 2 not eligible 3 withdrew consent 2 other
48 randomized (2:1) 48 safety population
Treatment period (6 weeks)
32 Lindioil 5 discontinued 1 poor compliance 1 withdrew consent 1 lack of efficacy 2 used prohibited medications
Follow-up period (2 weeks)
27 completed
16 Vehicle 8 discontinued 2 poor compliance 1 withdrew consent 3 lack of efficacy 1 deterioration of AD 1 lost contact without reason 8 completed
2 discontinued 1 lost contact without reason 1 poor compliance 25 completed
8 completed
A
C
Proportion of patients achieving EASI-75 response 30
25
20
15
10
5
0
Lindioil Vehicle
0 1 2
Week
4
*
6
B
Proportion of patients achieving EASI-50 response
D
Proportion of patients achieving EASI-90 response
60
50
40
30
20
10
0
20 18 16 14 12 10 8 6 4 2 0
Lindioil Vehicle
0
Lindioil Vehicle
0
1
2
2 Week
4
4
6
6
*
1 Week
A
B
C