Thesis 11 Prof

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Systemic Treatment in Atopic Dermatitis

Roekevisch, E.

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Download date:04 Oct 2021 Systemic Treatment in Atopic Dermatitis Uitnodiging

Voor het bijwonen van de openbare verdediging van het proefschrift: Systemic Treatment Systemic Treatment in in Atopic Dermatitis Atopic Dermatitis Door Evelien Roekevisch

in de aula der Universiteit, Singel 411, 1012 XM, te Amsterdam op woensdag 19 december om 13.00 uur

Evelien Roekevisch Maarten Harpertszoon Trompstraat 6 hs 1056 HZ Amsterdam [email protected] Evelien Roekevisch

Paranimfen Judith van Gemert Evelien Roekevisch [email protected]

Mina Noor [email protected]

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Systemic Treatment in Atopic Dermatitis

Evelien Roekevisch

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 1 Voor Mama en Papa

ISBN: 978-94-028-1286-2 Design/lay-out: Wendy Bour-van Telgen, Ipskamp Printing, Enschede Print: Ipskamp Printing, Enschede

© Evelien Roekevisch , 2018 All rights are reserved. No part of this book may be reproduced, distributed, stored in a retrieval system, or transmitted in any form or by any means, without prior written permission of the author.

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525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 3 *** Gooi een steen naar de dag, zo ver als je kunt. Spoel het zout van je huid, doof het vuur. Volg het spoor dat er ligt, zoek niet wat er nooit meer is. Was het zand uit je haar, geef een naam aan ieder jaar. Drink de tranen op je hand, zwijg ervan. Erf de ogen van je kind, kijk er door. Koester je geheime hart, tot het eind.

Reis ver, drink wijn, denk na, lach hard, duik diep, kom terug.

Droom een boot in de zon, geef hem zeilen en wind. Kus een droevige mond, heel zacht, voor de dag begint. Bewaar een steen in je tas, uit het land waar je sliep, waar je de wonden opliep, waar een koninkrijk verging. Haal de parels uit de zee, geef ze weg. Vecht met alles wat je hebt, verlies het goed. Wacht dan tot het lichter wordt, je hebt de tijd.

Reis ver, drink wijn, denk na, lach hard, duik diep, kom terug.

(Spinvis)

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 4 *** Gooi een steen naar de dag, zo ver als je kunt. Spoel het zout van je huid, doof het vuur. Volg het spoor dat er ligt, zoek niet wat er nooit meer is. Was het zand uit je haar, geef een naam aan ieder jaar. Drink de tranen op je hand, zwijg ervan. Erf de ogen van je kind, kijk er door. Koester je geheime hart, tot het eind.

Reis ver, drink wijn, denk na, lach hard, duik diep, kom terug.

(Spinvis)

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ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam op gezag van de Rector Magnificus prof. dr. ir. K.I.J. Maex ten overstaan van een door het College voor Promoties ingestelde commissie,

in het openbaar te verdedigen in de Aula der Universiteit op woensdag 19 december 2018, te 13.00 uur

door Evelien Roekevisch geboren te Lochem

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Promotor: Prof. dr. P. I. Spuls AMC-Universiteit van Amsterdam

Copromotores: Dr. M.A. Middelkamp Hup AMC-Universiteit van Amsterdam Dr. M.M.G. Leeflang AMC-Universiteit van Amsterdam

Overige leden: Dr. M.S. de Bruin Weller Universiteit van Utrecht Prof. dr. R.M. Luiten AMC-Universiteit van Amsterdam Prof. dr. A.H. Maitland-van der Zee AMC-Universiteit van Amsterdam Prof. dr. S.G.M.A. Pasmans Erasmus Universiteit Rotterdam Prof. dr. M.A. de Rie AMC-Universiteit van Amsterdam Prof. dr. T. Rustemeyer Vrije Universiteit van Amsterdam

Faculteit der Geneeskunde

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Promotor: Chapter 1 General introduction and outline of thesis 11 Prof. dr. P. I. Spuls AMC-Universiteit van Amsterdam 1.1 Atopic dermatitis 12 1.2 Systemic treatments in atopic dermatitis 13 Copromotores: 1.3 Efficacy and safety of methotrexate and azathioprine in 15 Dr. M.A. Middelkamp Hup AMC-Universiteit van Amsterdam atopic dermatitis Dr. M.M.G. Leeflang AMC-Universiteit van Amsterdam 1.4 Predictors and markers for severity and efficacy in atopic 15 dermatitis Overige leden: 1.5 Aims of the thesis 17 Dr. M.S. de Bruin Weller Universiteit van Utrecht Prof. dr. R.M. Luiten AMC-Universiteit van Amsterdam Part I Systemic treatments in atopic dermatitis 23 Prof. dr. A.H. Maitland-van der Zee AMC-Universiteit van Amsterdam Prof. dr. S.G.M.A. Pasmans Erasmus Universiteit Rotterdam Chapter 2 Ten years experience with oral immunosuppressive treatment in 25 Prof. dr. M.A. de Rie AMC-Universiteit van Amsterdam adult patients with atopic dermatitis in two academic centres Prof. dr. T. Rustemeyer Vrije Universiteit van Amsterdam Chapter 3 Efficacy and safety of systemic treatments for moderate-to-severe 41 atopic dermatitis: systematic review Faculteit der Geneeskunde Part II Efficacy and safety of methotrexate and azathioprine in atopic dermatitis 87

Chapter 4 A randomized trial of methotrexate and azathioprine for severe 89 atopic dermatitis

Chapter 5 Methotrexate versus azathioprine in patients with atopic dermatitis: 107 2-year follow-up data

Part III Predictors and markers for severity and efficacy in atopic dermatitis 127

Chapter 6 Biomarker analysis for disease severity and immunosuppressive 129 treatment responsiveness in adult atopic dermatitis patients

Chapter 7 Patients with atopic dermatitis with filaggrin loss-of-function 147 mutations show good but lower responses to immunosuppressive treatment

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Chapter 9 Summary and conclusions/ Samenvatting en conclusies 181

Addendum List of abbreviations 192 List of contributing authors 194 List of publications 197 PhD portfolio 199 Dankwoord 202 Curriculum vitae 208

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1.1 Atopic dermatitis

Atopic dermatitis (AD) is a chronic relapsing inflammatory disease characterized by exacerbations and remissions. Erythema and scaling of the skin with severe pruritus are characteristic, but the clinical pattern varies with age. Infants (<2 years of age) mostly present with acute lesions, like erythematous pruritic papules, plaques and vesicles, with preference localization on the cheeks, forehead or scalp, as well as the extensor sides of the extremities. From 2 years of age to puberty, exudative lesions are seen less frequently. Lichenified papules and plaques are more common, representing a more chronic disease. Preference locations are the hands, feet, wrists, ankles and antecubital and popliteal regions. Adults present with dry, scaly erythematous papules and plaques. Often large lichenified plaques are seen as well. Preference locations are the flexural folds, the face and neck, the upper arms and back, and the dorsal aspects of the hands, feet, fingers and toes.(1) More research is needed for defining the different phenotypes of AD. The prevalence of AD varies throughout the world, but is estimated to be 15-20% in children and 1-3% in adults, and the incidence has increased by 2- to 3-fold during the past decades in industrialized countries.(2) Several criteria have been developed to diagnose AD, however, the most widely used criteria for diagnosing AD, are the Hanifin and Rajka criteria (3) and the UK working party criteria.(4) The onset of AD occurs in 45% of cases between birth and 6 months of age. In 60% of the patients AD arises during the first year of life and in at least 85% before the age of 5 year.(5) Ten percent of the children with AD have persistent complaints of AD in adulthood. AD has great influence on quality of life of the patient and on the functioning of the family, mainly because of sleep deprivation due to pruritus, painful skin, employment loss, time to care and financial costs.(6) AD is associated with comorbidities like psychiatric (such as attention- deficit hyperactivity disorder, depression, anxiety and autism), cardiovascular (heart attack and stroke) and auto-immune diseases (alopecia areata, vitiligo, rheumatoid arthritis, and inflammatory bowel disease).(7)

A patient with chronic intermittent AD requires expert and long-term multidisciplinary guidance, and benefits from education including information and instructions about the clinical features, preventive measures, treatment possibilities and prognosis.(8)

The past years we have learned much more about the pathophysiology of AD. AD is characterized by an immune dysregulation favoring a Th2 cellular response (Interleukin (IL)- 4, IL-5, IL-13 and Thymic Stromal Lymphopoietin (TSLP)), which is dominant in the acute

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phase, while in the chronic phase there is a mixture of a Th1, Th2 and Th22 infiltrate .(9, 10) AD is characterized by an epidermal barrier dysfunction andfilaggrin FLG( ) gene mutations are a strong risk factor for developing AD. The skin microbiome plays an important role in our skin barrier and together with the immune system, it results in a complex and balanced ecosystem. Research is being done whether the microbiome influences AD, but the precise role is still unclear.(11, 12) Eczema is worsened an may be even initiated by factors like sweating, stress, infections (such as skin infections with Staphylococcus aureus), contact-allergens and aero-allergens.(13, 14)

1.2 Systemic treatments in atopic dermatitis 1

Standard treatment of AD includes the avoidance of irritants, regular use of emollients, and use of topical steroids and calcineurin inhibitors.(15) For more severe cases, treatment with ultraviolet light such as narrow-band ultraviolet B (NB-UVB) therapy, ultraviolet A1 (UVA1) therapy or phototherapy with oral psoralens and UVA (PUVA)) are used.(16) Still many patients do not seek treatment, while some patients are treated in primary and secondary care centres. Severe cases are often referred to tertiary care with intensive outpatients visits, day care treatment or even inpatient care if needed.

A subgroup of patients will not obtain total remission with the treatment options mentioned above. For those patients systemic treatment is needed to achieve adequate disease control. Because AD is a chronic disease, it is imperative that the proposed therapy for these patients has a positive risk benefit ratio with persistent efficacy and an acceptable adverse effect profile as it is used for a longer period of time.

Various systemic treatments are currently used in AD, however, these treatments have rarely been compared for efficacy and safety in adult patients with AD. In 2007, Schmitt et al. published a systematic review of the clinical trials on systemic treatment for severe AD to provide evidence-based treatment recommendations.(17) Forty-one percent (11/27) of the included studies showed efficacy for cyclosporine (CsA). CsA is a calcineurin inhibitor, which inhibits the proliferation of T lymphocytes. It inhibits IL-2, and an effect has been mentioned on IL-3, IL-4, GM-CSF, tumor necrosing factor (TNF-)alpha and interferon-gamma (INF-g). CsA is suggested to be used for short-term or intermittent long-term therapy. Although CsA has been proven to be effective, some patients have contra- indications, like renal impairment or poorly set arterial hypertension, or have to discontinue because of inefficacy or side- effects. In addition, long-term treatment with CsA is still doubtful because of concerns on

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nephrotoxicity. Some evidence was found for off-label systemic treatments with azathioprine (AZA), methotrexate (MTX) and mycophenolate mofetile (MMF).(17) AZA is a purine analog and inhibits the proliferation of B and T lymphocytes. It was primarily developed in the sixties to prevent organ rejection. It is a treatment option for multiple dermatologic diseases (off- label), as well as for AD.(18) AZA was investigated in one randomized controlled trial (RCT), where it showed significant improvements compared to placebo. It was concluded that AZA may be used for short-term treatment in patients with AD.(19) MTX is a folic acid antagonist and is widely used in chronic inflammatory diseases such rheumatoid arthritis. In 1951, the first result hinting towards effectiveness of MTX in dermatology was noticed when psoriatic skin lesions cleared in cancer patients undergoing treatment with the anti-metabolic drug aminopterin, a predecessor of MTX.(20) Over the years, only few data was published on the use of MTX in AD. A prospective open study with MTX showed a mean improvement in SASSAD score of 52% from baseline after 24 weeks of treatment.(21) A retrospective study showed a good response at 80% (16/20) of the patients after 8-12 weeks of treatment with MTX.(22). No RCTs were done comparing MTX with placebo or another agent when starting this thesis. MMF is a purine biosynthesis inhibitor. The direct result is a cytostatic effect on B and T lymphocytes, because B and T lymphocytes are highly dependent on de novo synthesis of purines for their proliferation. One head to head study of MMF and CsA showed equal efficacy. Besides this RCT some uncontrolled trials and case reports showed an effect in (23,AD. 24)

Other treatment options are intravenous immunoglobulins (IVIG), INF-y, oral pimecrolimus (17) and systemic glucocorticosteroids.(17) Systemic glucocorticosteroids are widely used for the treatment of AD, but evidence is sparse. One RCT showed that systemic glucocorticosteroids were less effective than CsA.(25) High rates of exacerbations and side effects were seen after discontinuation of treatment. Overall, short-term treatment is usually effective, but it is advised to give it as emergency intervention only, and to look for other treatment options because long term treatment is associated with severe side effects. INF-y and oral pimecrolimus are potential treatment options, but data is sparse and more research about efficacy and (long term safety) is needed. IVIG is not advised for the treatment of AD.(17) Other promising treatments are biologicals, but at the start of this thesis there was no biological available that had shown efficacy in AD. To date, Dupilumab showed its effectiveness and is licensed for AD.

To investigate what kind of systemic treatments are prescribed in patients with ADby dermatologists in a tertiary setting, we retrospectively reviewed medical records in two

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academic medical centers. The results will be discussed in chapter 2. In addition, as new RCTs on systemic treatments have been are published since the review of Schmitt et al., an updated systematic review was conducted to give a complete overview of the published data, which we will present in this thesis (chapter 3).

1.3 Efficacy and safety of methotrexate and azathioprine in atopic dermatitis

As literature on off-label treatment of AD with MTX and AZA is sparse and limited to some RCTs and several (retrospective) cohort studies, our department initiated a small RCT in 2009 1 (chapter 4) to compare MTX and AZA in patients with AD (Methotrexate versus Azathioprine for severe atopic dermatitis: MAcAD trial). After the initial 24 weeks patients were asked to participate in an open-label observational follow-up study to be evaluated 3 monthly for 2 (chapter 5) and 5 years.

1.4 Predictors and markers for severity and efficacy in atopic dermatitis

Biomarkers A method to objectively measure disease severity and to predict therapeutic response in AD is needed, and therefore a lot of research is being done to evaluate and develop biomarkers in AD. A systematic review and meta-analysis (26) showed that serum thymus and activation- regulated chemokine (TARC) is the superior biomarker for assessing disease severity. T-cell attracting chemokine (CTACK), sE-selectin, macrophage-derived chemokine (MDC), lactate dehydrogenase (LDH) and IL-18 could be useful as a severity biomarker as well, but need to be validated in additional studies. Three other promising biomarkers are B-cell activating factor of the TNF family (BAFF), a proliferation-inducing ligand (APRIL) and IL-31, although contradictory conclusions are published making the relevance unclear (27-36). IL-4 and IL-13 are believed to have a role in the pathogenesis of AD, because the frequency of IL-4 and IL-13- producing cells in AD patients is significantly higher than in healthy subjects (in both CD4+ and CD8+ T-cell subsets). In addition, Dupilumab, which blocks IL-4 and IL-13, has shown efficacy in patients with AD.(37)

Although promising results on potential biomarkers for AD have been published, more research is needed to find reliable biomarkers for disease severity and especially for predicting immunosuppressive treatment responsiveness in AD patients. To gather information about biomarker levels during MTX or AZA treatment we analyzed serum levels of chemokines and

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cytokines from patients that participated in the previously mentioned RCT on MTX and AZA (chapter 6).(18)

Filaggrine The discovery of the common loss-of-function variants in the FLG gene and their strong association with AD has led to an increased interest in the role of skin barrier impairment in the development of AD, allergic sensitization and also food and respiratory allergies. (38, 39) The stratum corneum, the outermost layer of the epidermis, forms the main barrier to protect against infection, dehydration, chemicals and mechanical stress. Filaggrin is a major structural protein of the stratum corneum, and plays a crucial role in proper barrier formation. TheFLG gene encodes for profilaggrin, which is dephosphorylated and processed into multiple copies of filaggrin.(39) During terminal differentiation of the keratinocytes in the granular layer to corneocytes, keratinocytes replace their plasma membrane by a solid layer called the “cornified envelope”, which is composed of various proteins, including filaggrin, involucrin and loricrin.(39) Filaggrin also leads to aggregation of the keratin filaments, thereby leading to compaction and flattening of the corneocyte. In the stratum corneum, filaggrin is further broken down by several enzymes including caspase-14 and bleomycin hydrolase into amino acids and their derivatives which are main constituents of the collectively known natural moisturizing factors (NMF).(40) Components of NMF contribute to the hydration of the skin and also play a role in regulating the pH-value, which is important for the local protease activity and defense against microorganisms such as Staphylococcus aureus, and therefore NMF contributes to the barrier function.(9, 38)

To date, loss-of-function mutations in FLG have been shown to be the most significant risk factor for developing AD.(38) The odds ratio of having AD in association with a FLG mutation were estimated to be 3.1 and 4.8 in two different studies published in 2009.(41, 42) However, FLG mutations are seen in less than a third of the total population withAD.(43, 44) Among patients with moderate-to-severe AD, up to 46% to 57% carry 1 or moreFLG mutations, while the population attributable risk fraction has been estimated to be between 4.2% and 15.1%. (45)

Specific phenotypic and biophysical characteristics are associated with AD in the context of carrying a FLG mutation. Palmar hyperlinearity, more severe and persistent eczema, a higher incidence of herpes simplex viral infections, as well as a greater risk of allergic sensitization and asthma are more frequently seen in patients with AD having a FLG mutation.(9) It has also been shown that atopic asthma patients carrying FLG mutations require more stringent

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asthma medication.(18) To date, around 47 mutations have been reported. The FLG loss-of- function mutations R501X, 2282del4, R2447X and S3247X appear frequently in European Caucasians (38, 46), while 3321delA mutation is more common in the Asian popula tion.(47, 48) By learning more about the pharmacogenomics of AD, we will be able to better focus on patient-centered care and personalized therapy. Although it is clear that FLG mutations play an important role in the development of AD, the relationship between FLG mutations and treatment outcome in patients with AD had not yet been studied. In the last chapter,chapter 7, the effect ofFLG loss or function mutations on immunosuppressive treatment outcome in AD patients is investigated. 1 1.5 Aims of the thesis

In part I “Systemic treatments in atopic dermatitis”, chapter 2 describes a retrospective review on prescribed systemic treatments by dermatologists over the past 10 years for AD patients in two Dutch academic hospitals. In chapter 3 we investigated the use of systemic immunosuppressive therapy in AD based on a systematic review.

In part II “Efficacy and safety of methotrexate and azathioprine in atopic dermatitischapter ”, 4 describes the results of a small RCT investigating the efficacy and safety of MTX and AZA after 24 weeks of treatment in patients with severe AD. In chapter 5 the long term use of MTX and AZA was analyzed to evaluate the efficacy and safety 2 years after start treatment.

In part III “Predictors and markers for severity and efficacy in atopic dermatitis”, chapter 6 describes a study investigating biomarkers for disease severity and immunosuppressive treatment responsiveness in adult AD patients. In the last chapter, chapter 7, the effect of FLG loss or function mutations on immunosuppressive treatment outcome in AD patients is investigated.

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Table 1: Outline of the thesis with corresponding chapters and aims.

Part I Systemic treatments in atopic dermatitis Ten years experience with oral immunosuppressive treatment in adult patients with Chapter 2 atopic dermatitis in two academic centres Aim: A 10-years overview of the use of oral immunosuppressive drugs in patients with severe AD Efficacy and safety of systemic treatments for moderate-to-severe atopic dermatitis: Chapter 3 systematic review Aim: An update of a systematic review to evaluate the efficacy and safety of systemic treatments for moderate-to-severe AD Part II Efficacy and safety of methotrexate and azathioprine in atopic dermatitis Chapter 4 A randomized trial of methotrexate and azathioprine for severe atopic dermatitis Aim: To compare the efficacy and safety of methotrexate versus azathioprine in adults with severe AD Methotrexate versus azathioprine in patients with atopic dermatitis: 2-year follow-up Chapter 5 data Aim: To assess the efficacy and safety of methotrexate versus azathioprine over a 2-year follow up period Part III Predictors and markers for severity and efficacy in atopic dermatitis Biomarkers for disease severity and immunosuppressive treatment responsiveness in Chapter 6 adult atopic dermatitis patients Aim: To identify biomarkers that reflect and/or predict therapeutic responsiveness in AD patients treated with systemic treatment, in the total study groups as well as in FLG mutation carriers versus non-FLG mutations carriers Patients with atopic dermatitis with filaggrin loss-of-function mutations show good Chapter 7 but lower responses to immunosuppressive treatment Aim: To assess the predictive value of FLG mutations on treatment outcome in patients with chronic severe AD on systemic immunosuppressive therapy

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M. Systemic treatment of severe AD: A, Oertel R, Augustin M, et al. Prednisolone a systematic review. Acta dermato- vs. ciclosporin for severe adult eczema. An venereologica. 2007;87(2):100-11. investigator-initiated double-blind placebo- 18. Schram ME, Roekevisch E, Leeflang MM, controlled multicentre trial. The British Bos JD, Schmitt J, Spuls PI. A randomized journal of dermatology. 2010;162(3):661-8. trial of methotrexate versus azathioprine for 26. Thijs J, Krastev T, Weidinger S, Buckens CF, severe AD. The Journal of allergy and clinical de Bruin-Weller M, Bruijnzeel-Koomen C, immunology. 2011;128(2):353-9. et al. Biomarkers for atopic dermatitis: a 19. Berth-Jones J, Takwale A, Tan E, Barclay G, systematic review and meta-analysis. Curr Agarwal S, Ahmed I, et al. Azathioprine in Opin Allergy Clin Immunol. 2015;15(5):453- severe adult atopic dermatitis: a double- 60. blind, placebo-controlled, crossover 27. Chen Y, Lind Enoksson S, Johansson C, trial. The British journal of dermatology. Karlsson MA, Lundeberg L, Nilsson G, et al. 2002;147(2):324-30. The expression of BAFF, APRIL and TWEAK 20. Shen S, O’Brien T, Yap LM, Prince HM, is altered in eczema skin but not in the McCormack CJ. The use of methotrexate circulation of atopic and seborrheic eczema in dermatology: a review. The Australasian patients. PloS one. 2011;6(7):e22202. journal of dermatology. 2012;53(1):1-18. 28. Dillon SR, Sprecher C, Hammond A, 21. Weatherhead SC, Wahie S, Reynolds NJ, Bilsborough J, Rosenfeld-Franklin M, Presnell Meggitt SJ. An open-label, dose-ranging SR, et al. Interleukin 31, a cytokine produced study of methotrexate for moderate-to- by activated T cells, induces dermatitis in severe adult AD. The British journal of mice. Nat Immunol. 2004;5(7):752-60. dermatology. 2007;156(2):346-51. 29. Matsushita T, Fujimoto M, Echigo T, 22. Lyakhovitsky A, Barzilai A, Heyman R, Baum Matsushita Y, Shimada Y, Hasegawa M, et S, Amichai B, Solomon M, et al. Low-dose al. Elevated serum levels of APRIL, but not methotrexate treatment for moderate-to- BAFF, in patients with atopic dermatitis. Exp severe atopic dermatitis in adults. Journal of Dermatol. 2008;17(3):197-202. the European Academy of Dermatology and 30. Niyonsaba F, Ushio H, Hara M, Yokoi H, Venereology : JEADV. 2010;24(1):43-9. Tominaga M, Takamori K, et al. Antimicrobial 23. Ballester I, Silvestre JF, Perez-Crespo M, peptides human beta-defensins and Lucas A. [Severe adult atopic dermatitis: cathelicidin LL-37 induce the secretion of a treatment with mycophenolate mofetil pruritogenic cytokine IL-31 by human mast in 8 patients]. Actas dermo-sifiliograficas. cells. J Immunol. 2010;184(7):3526-34. 2009;100(10):883-7. 31. Neis MM, Peters B, Dreuw A, Wenzel J, Bieber 24. Murray ML, Cohen JB. Mycophenolate T, Mauch C, et al. Enhanced expression mofetil therapy for moderate to severe levels of IL-31 correlate with IL-4 and IL-13 atopic dermatitis. Clinical and experimental in atopic and allergic contact dermatitis. The dermatology. 2007;32(1):23-7. Journal of allergy and clinical immunology. 25. Schmitt J, Schakel K, Folster-Holst R, Bauer 2006;118(4):930-7.

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32. Nobbe S, Dziunycz P, Muhleisen B, cell science. 2009;122(Pt 9):1285-94. Bilsborough J, Dillon SR, French LE, et al. 40. Harding CR, Aho S, Bosko CA. Filaggrin - IL-31 expression by inflammatory cells is revisited. International journal of cosmetic preferentially elevated in atopic dermatitis. science. 2013;35(5):412-23. Acta dermato-venereologica. 2012;92(1):24- 41. Palmer CN, Irvine AD, Terron-Kwiatkowski A, 8. Zhao Y, Liao H, Lee SP, et al. Common loss- 33. Raap U, Weissmantel S, Gehring M, of-function variants of the epidermal barrier Eisenberg AM, Kapp A, Folster-Holst R. IL-31 protein filaggrin are a major predisposing significantly correlates with disease activity factor for atopic dermatitis. Nature genetics. and Th2 cytokine levels in children with 2006;38(4):441-6. atopic dermatitis. Pediatr Allergy Immunol. 42. van den Oord RA, Sheikh A. Filaggrin gene 1 2012;23(3):285-8. defects and risk of developing allergic 34. Raap U, Wichmann K, Bruder M, Stander sensitisation and allergic disorders: S, Wedi B, Kapp A, et al. Correlation of IL- systematic review and meta-analysis. BMJ 31 serum levels with severity of atopic (Clinical research ed). 2009;339:b2433. dermatitis. The Journal of allergy and clinical 43. Brown SJ, McLean WH. Eczema genetics: immunology. 2008;122(2):421-3. current state of knowledge and future goals. 35. Siniewicz-Luzenczyk K, Stanczyk-Przyluska The Journal of investigative dermatology. A, Zeman K. Correlation between serum 2009;129(3):543-52. interleukin-31 level and the severity of 44. Weidinger S, Illig T, Baurecht H, Irvine AD, disease in children with atopic dermatitis. Rodriguez E, Diaz-Lacava A, et al. Loss-of- Postepy Dermatol Alergol. 2013;30(5):282-5. function variations within the filaggrin gene 36. Szegedi K, Kremer AE, Kezic S, Teunissen predispose for atopic dermatitis with allergic MB, Bos JD, Luiten RM, et al. Increased sensitizations. The Journal of allergy and frequencies of IL-31-producing T cells are clinical immunology. 2006;118(1):214-9. found in chronic atopic dermatitis skin. Exp 45. Basu K, Palmer CN, Lipworth BJ, McLean WH, Dermatol. 2012;21(6):431-6. Terron-Kwiatkowski A, Zhao Y, et al. Filaggrin 37. Beck LA, Thaci D, Hamilton JD, Graham null mutations are associated with increased NM, Bieber T, Rocklin R, et al. Dupilumab asthma exacerbations in children and young treatment in adults with moderate-to-severe adults. Allergy. 2008;63(9):1211-7. atopic dermatitis. The New England journal 46. Sandilands A, Terron-Kwiatkowski A, Hull of medicine. 2014;371(2):130-9. PR, O’Regan GM, Clayton TH, Watson RM, 38. Irvine AD, McLean WH, Leung DY. Filaggrin et al. Comprehensive analysis of the gene mutations associated with skin and allergic encoding filaggrin uncovers prevalent and diseases. The New England journal of rare mutations in ichthyosis vulgaris and AD. medicine. 2011;365(14):1315-27. Nature genetics. 2007;39(5):650-4. 39. Sandilands A, Sutherland C, Irvine AD, 47. Akiyama M. FLG mutations in ichthyosis McLean WH. Filaggrin in the frontline: role in vulgaris and AD: spectrum of mutations and skin barrier function and disease. Journal of population genetics. The British journal of

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dermatology. 2010;162(3):472-7. 48. Zhang H, Guo Y, Wang W, Shi M, Chen X, Yao Z. Mutations in the filaggrin gene in Han Chinese patients with atopic dermatitis. Allergy. 2011;66(3):420-7.

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F.M. Garritsen, E. Roekevisch, J. van der Schaft, J. Deinum, P.I. Spuls, M.S. de Bruin-Weller Journal of the European Academy of Dermatology and Venereology (JEADV). 2015; Oct;29(10):1905-12

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Abstract

Background There is a lack of information on the use oral immunosuppressive drugs in atopic dermatitis (AD) in daily practice.

Objective A 10-years overview of the use of oral immunosuppressive drugs in patients with severe AD.

Methods Medical charts of patients with AD, who received oral immunosuppressive drugs at the Academic Medical Center Amsterdam and in the University Medical Center Utrecht between January 2001 and January 2011, were analysed. Particular attention was paid to patient characteristics, prior treatment, prescribed oral immunosuppressive drugs, the order of use, doses and treatment durations and reasons for discontinuation of treatment.

Results 334 patients [53% male, mean age at start of an oral immunosuppressive drug 36.9 years (SD 13.6)] with AD received oral immunosuppressive treatment of which 102 (31%) participated in clinical trials. Cyclosporine A (CyA) was given in 80% of the patients, mycophenolate mofetil or enteric-coated mycophenolate (MMF/EC-MPS) in 31%, azathioprine (AZA) in 14%, methotrexate (MTX) in 11%, systemic glucocorticosteroids in 7% and systemic tacrolimus in 5%. In these academic centra, CyA was the first choice oral immunosuppressive in 252 patients. Reasons for discontinuation of oral immunosuppressive drugs were controlled AD disease, ineffectiveness and adverse events.

Conclusion Various types of oral immunosuppressive drugs have been used over the past 10 years for the treatment of severe AD with a prominent first choice for CyA. Adverse events and ineffectiveness were frequent reasons for discontinuation. A prospective database of patients using oral immunosuppressive treatments in daily practice will give more insight in the effectiveness and safety and may help to formulate future recommendations.

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Introduction

Atopic dermatitis (AD) is a chronic inflammatory skin disease. The prevalence varies between 2 and 10% in adults and 15–30% in children.1,2 A combination of emollients, topical glucocorticosteroids, calcineurin inhibitors, antihistamines and antibiotics, is often used in the management of AD.3 Different forms of phototherapy may also be effective.4–6 In severe AD patients, with insufficient response to topical treatment or needing continuous treatment with mild to high potent corticosteroids, oral immunosuppressive therapy is indicated.

Studies show that cyclosporine A (CyA), azathioprine (AZA), methotrexate (MTX), mycophenolate mofetil (MMF) and Enteric-Coated MycoPhenolate Sodium (EC- MPS) are effective in the treatment of severe AD patients. Long-term treatment 2 with oral glucocorticosteroids is not recommended.7–11 Guidelines for the use of oral immunosuppressive drugs have been published and include recommendations to support daily practice in the management of patients with7,11–18 AD. These guidelines are mostly based on results from clinical trials, preferably randomized clinical trials (RCT). However, patients participating in clinical trials often differ from daily practice patients due to the strict in- and exclusion criteria. In addition, patients participating in clinical trials are in general more compliant, probably resulting in better clinical efficacy of the drug. Daily practice data concerning oral immunosuppressive drugs used in AD patients may help to give additional insight in the effectiveness and safety. Therefore, a medical chart review was performed of all 334 AD patients treated with oral immunosuppressive drugs at the dermatology department of the Academic Medical Center Amsterdam (AMC) and University Medical Center Utrecht (UMCU) in the Netherlands over a period of 10 years (from January 2001 till January 2011). The aim of this study was to give an overview of the use of oral immunosuppressive drugs in patients with severe AD, with respect to patient characteristics, prior treatment, prescribed oral immunosuppressive drugs, the order of use, doses and treatment durations and reasons for discontinuation of treatment. Both daily practice data and trial (follow-up) datawere evaluated to provide a complete overview of systemic immunosuppressive drug use in AD patients.

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Materials and methods

Patient selection In both centres, AD patients were diagnosed by a dermatologist or validated diagnostic criteria.19 All patients using oral immunosuppressive drugs for AD between January 2001 and January 2011 were included. For all patients, follow-up ended on January 1st 2011, regardless whether treatment was continued after this date. Patients who started an oral immunosuppressive drug before January 1st 2001 and continued this treatment after this date were included. Only patients born before January 1st 1995, were investigated, excluding young children. In the AMC all patients diagnosed with AD in the digital registration system using oral immunosuppressive drugs were included. Additionally, patients treated in a RCT performed between July 2009 and December 2010, comparing AZA with MTX were included.8

In the UMCU, data of all adult patients diagnosed with AD and treated with oral immunosuppressive drugs from 1996 until now were retrospectively registered into a database. Patients who were treated with oral immunosuppressive drugs between January 2001 and January 2011 were selected. In addition, data from a RCT performed on EC-MPS vs. CyA from November 2005 to November 2007 were included.9 Excluded were patients who received oral immunosuppressive drugs as a therapy for a disease other than AD. When patients used the same oral immunosuppressive treatment more than once, only the first treatment episode was evaluated. When patients used different oral immunosuppressive drugs simultaneously, the combination of both treatments was evaluated. In case of monotherapy with systemic glucocorticosteroids, only patients who were treated for a continuous period of more than 3 months were included. Data-extraction Information about patient characteristics, prior oral immunosuppressive drug use and phototherapy were collected. Information about the order in which oral immunosuppressive drugs were prescribed, doses and durations of treatment, the reasons for discontinuation of treatment were collected. Also the manner of discontinuation (acute or stepwise) was investigated.

Study outcome The primary outcome parameter of this study was the reason for discontinuation of treatment and was subdivided in i) controlled disease, ii) adverse events iii) ineffectiveness, iv) end of study, v) miscellaneous. Controlled disease was defined as improvement of AD (in the opinion of the patient and/or physician) which resulted in discontinuation ofthe oral immunosuppressive drug. Adverse events leading to discontinuation of treatment included both subjective and objective adverse events, such as laboratory abnormalities.

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Ineffectiveness was registered as reason for discontinuation when patient and/or physician were not satisfied with the clinical response. Primary (not effective at all) and secondary (initially improvement but a loss of effect after a certain amount of time) ineffectiveness were not specified but the mean number on days on treatment were provided. End of the study was the reason for discontinuation in all patients participating in the RCT performed on EC- MPS vs. CyA as part of the study protocol. Patients participating in the RCT comparing AZA and MTX did not have to discontinue treatment after the trial period. These patients continued treatment with AZA or MTX as in daily practice. Any other reasons for discontinuation were summarized under miscellaneous. More than one reason for discontinuation per patient could be recorded. This study followed the STROBE guidelines as much as possible. 20

Statistical analysis 2 SPSS version 20 for windows (SPSS, Inc, Chicago, IL, USA) was used to calculate means, medians, standard deviations and interquartile ranges.

Results

Patient characteristics A total of 334 patients were included (85 from the AMC and 249 from the UMCU). Forty-two (13%) of them participated in the AMC RCT and 60 patients (18%) participated in the UMCU RCT.8,9 Patient characteristics are shown in Table 1.

Table 1 Patient characteristics N (%) (TOTAL=334) Male sex (%) 177 (53) Mean age 36.89 years (SD 13.64) Presence of asthma* (%) 180 (54) Presence of allergic rhinitis* (%) 211 (63) Presence of food allergy* (%) 146 (44) Serum IgE determined (%) 217 (65) Serum IgE increased (>100 kU/L) (%) 204 (61) Mean number of previous oral immunosuppressive treatment per 0.60 (SD 0.86) patient before referral to academic centers (SD) Patients who used more than one type of oral immunosuppresive 114 (34) treatment in the ten years period (%) n= number, *= anamnestic

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Prior treatment The mean number of oral immunosuppressive therapies per patient, used before referral to our academic centres, is 0.60 (SD 0.86) (Table 1). CyA was most frequently used (98 times, 37% of all patients) (Table 2), a requirement for starting in the MTX vs. AZA RCT.8

Table 2 An overview of prior immunosuppressive or UV interventions Therapy N (%) (TOTAL=262) Cyclosporin A(%) 98 (37) Systemic glucocorticosteroids (%)* 68 (26) Azathioprine (%) 7 (3) Mycophenolate mofetil / Enteric-Coated MycoPhenolate sodium(%) 4 (2) Efalizumab (%) 1 (0) Methotrexate (%) 2 (1) Neotigason (%) 1 (0) UVB (%) ** 50 (19) PUVA (%) ** 18 (7) UVA (%) ** 13 (5) * Data include short additional courses of a few days ** Data were only collected in the AMC UVB = ultraviolet B; UVA = ultraviolet A; PUVA= psoralen plus UVA

Oral immunosuppressive drugs More than one type of oral immunosuppressive drugs was used by 114 patients (34%) between January 2001 and January 2011. Table 3 shows an overview of the treatment characteristics, reasons for discontinuation and median duration (days) of treatment until discontinuation. In Table 4 the adverse events that caused discontinuation of the different drugs are described in more detail.

Cyclosporin A In total, 267 patients (80%) were treated with CyA. Thirty-seven patients (14%) received CyA in the UMCU RCT.9 Fifty-seven patients had more than one treatment episode with CyA, of which only the first episode was evaluated. The median duration of treatment till January 1st 2011 was 258 days (IQR 108–434). Of the 234 patients that discontinued treatment, 69 patients (29%) stopped because of controlled disease, 57 patients (24%) discontinued treatment because of adverse events. In 35 patients ineffectiveness (15%) was the reason for discontinuation of therapy. Thirty-seven patients (16%) discontinued because of the end of the trial in which they participated. One hundred and ninety-three other miscellaneous

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reasons caused discontinuation of treatment. Six patients used >3 months of prednisone simultaneously.

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 31 Chapter 2 294 (184-486) 217 (100-512) 244 (162-512) 50 (43-267) 377 (241-814) 177 (70-222) 114 (80-283) 217 (207-222) 270 (194-366) 27 (15-128) 146 (27-214) 261 96 (11-204) 175 (11-204) 138 (104-409) 2930 323 (118-1574) 223 77 (33-586) 91 (77-294) Reasons for discontinuation (%) and discontinuation for Reasons of treatment (days) median duration (IQR) discontinuation until disease: Controlled 69/234 (29) 57/234 events: Adverse (24) 35/234 (15) Not effective: End of trial: 37/234 (16) Miscellaneous: 193 disease: 11/82 Controlled (13) 18/82 events: Adverse (22) 36/82 (44) Not effective: End of trial: 23/82 (28) Miscellaneous: 35 disease: 5/26 Controlled (19) 10/26 events: Adverse (38) 4/26 (15) Not effective: Miscellaneous: 12 disease: Controlled 1/17(6) 7/17 events: Adverse (41) 11/17 (65) Not effective: Miscellaneous: 3 disease: 4/20 Controlled (20) 1/20 (5) events: Adverse 4/20 (20) Not effective: Miscellaneous: 31 disease: Controlled 1/16 (6) 9/16 (56) events: Adverse 7/16 (44) Not effective: Miscellaneous:9 Mode of discontinuation 108 Acute: 94 Stepwise: 32 Unknown: 64 Acute: 11 Stepwise: 7 Unknown: 14 Acute: 2 Stepwise: 10 Unknown: 11 Acute: 1 Stepwise: 5 Unknown: 0 Acute: 16 Stepwise: 4 Unknown: 15 Acute: 1 Stepwise: 0 Unknown:

st Dis- continuing before January 1 2011 (%) 234/267 (88) 82/104 (79) 26/46 (57) 17/37 (46) 20/24 (83) 16/18 (89) 2011 (%) st 33/267 (12) 22/104 (21) 20/46 (43) 20/37 (54) 4/24 (17) 2/18 (11) Continuing Continuing January after 1 258 (108-434) MMF: 84 (31.25- 151.75) and EC-MPS: 212 (113.75- 380.75) 208 (27.25- 340.25) 298 (147- 384.5) Prednisone: 266 (129.25- 492) and Celestone: 1363 (258.75- 2688.5) 106.5 (59- 556.5) Median of duration in treatment (IQR) days 4.27 mg/kg/ (0.91) day MMF: 1650 mg/day (474.34) EC-MPS: 1371.06 (275.72) 121.56 mg/day (64.09) 20.90 mg/week (18.50) Prednisone: 23.00 mg/day (9.51); Celestone: 1.50mg/day (0.5) 6.53 mg/day (SD 1.81) Mean maximum dose (SD) 4.23 mg/kg/ (0.95) day MMF: 1050 mg/day (437.80) EC-MPS: 1371.06 (275.72) 99.77 mg/day (43.91) 8.75 mg/week (2.57) Prednisone: 22.50 mg/day (9.93) Celestone: 1.83 mg/day (0.76) 6.53 mg/day (SD 1.81) Mean start Mean start dose (SD) 35.50 (12.90) 42.10 (12.17) 40.24 (14.03) 43.98 (14.42) Prednisone: 44.65 (18.78); Celestone: 36.40 (6.32) 38.67 (SD 11.97) Mean age at at Mean age in years start (SD) 2001 st 18/267 (7) 0 0 0 3/24 (13) 1 /18 (6) and continuing and continuing this date after (%) Started before before Started January 1 146 (55) 63(61) 22 (48) 19 (51) 14 (58) 13 (72) Male (%) 267 104 46 37 24 18 Total Total patients Cyclosporin A Cyclosporin Mycophenolate / mofetile Enteric-Coated MycoPhenolic Sodium Azathioprine Methotrexate gluco- Systemic corticosteroids Tacrolimus Table 3. An overview of the treatment characteristics, reasons for discontinuation and the median duration (days) of treatment until discontinuation until treatment (days) of duration and the median for discontinuation reasons characteristics, of the treatment 3. An overview Table Therapy

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Table 4 Adverse events that caused discontinuation of therapy Therapy Adverse events that caused discontinuation of therapy Cyclosporin A Serum creatinine increase 24/234 (10%) Hypertension 17/234 (7%) Neurological symptoms* 14/234 (6%) Fatigue 9/234 (4%) Gastrointestinal symptoms 9/234 (4%) Myalgia 7/234 (3%) Pitting oedema 5/234 (2%) Flu like symptoms 4/234 (2%) Hypertrichosis 2/234 (1%) Physical condition loss 2/234 (1%) Gum hyperplasia 2/234 (1%) Rushed feeling / mood changes 2/234 (1%) Recurrent viral infection with herpes simplex 1/234 (0%) 2 Flushing 1/234 (0%) Petechia 1/234 (0%) Joint problems 1/234 (0%) Anaemia 1/234 (0%) Mycophenolate Neurological symptoms* 8/82 (10%) mofetile/ Enteric- Gastro-intestinal symptoms 5/82 (6%) coated MycoPhenolic Flu like symptoms 3/82 (4%) Sodium Shortness of breath 3/82 (4%) Myalgia 2/82 (2%) Fatigue 1/82 (1%) Oedema 1/82 (1%) Hair loss 1/82 (1%) Azathioprine Nausea 7/26 (27%) Neurological symptoms* 2/26 (8%) Liver dysfunction 2/26 (8%) Fatigue 1/26(4%) Flu like symptoms 1/26 (4%) Anaemia 1/26 (4%) Lymphocytopenia 1/26 (4%) Pancytopenia 1/26 (4%) Methotrexate Neurological symptoms* 3/17 (18%) Nausea 2/17 (12%) Fatigue 2/17 (12%) Transaminase increase 1/17 (6%) Systemic corticosteroids Unknown 1/20 (5%) Tacrolimus Gastro-intestinal symptoms 3/16 (19%) Serum creatinine increase 3/16 (19%) Neurological problems* 2/16 (13%) Hypertension 1/16 (6%)

* Including trembling of the hands, visual impairments, tingling of the fingers, jactations, vertiginous, feeling weak and heaviness of the arms

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Mycophenolate mofetil and Enteric-Coated MycoPhenolate Sodium In total, 104 (31%) patients were treated with MMF and EC-MPS. Twenty-three patients (22%) received EC-MPS in the RCT that was performed in the UMCU.9 Four patients had more than one treatment episode with EC-MPS, of which only the first episode was evaluated. The median duration of treatment was 84 days (IQR 31.25–151.75) (MMF) and 212 days (IQR 113.75–380.75) (EC-MPS). Among the 82 patients (79%) who discontinued treatment before January 1st 2011, 11 patients (13%) stopped because of controlled disease, 18 patients (22%) because of adverse events. In 36 patients (44%) MMF or EC-MPS was ineffective. Twenty- three patients (28%) discontinued because of the end of the trial in which they participated. Thirty-five other miscellaneous reasons caused discontinuation of treatment. Twenty-eight patients used >3 months of prednisone simultaneously.

Azathioprine In total, 46 patients (14%) were treated with AZA. Twenty-three patients (50%) received AZA as part of the AMC RCT.8 One patient had more than one treatment episode with AZA of which only the first episode was evaluated. In our 10-year observation period, the median duration of treatment was 208 days (IQR 27.25–340.25). Twenty patients (43%) continued treatment with AZA after January 1st 2011. Of the 26 patients who stopped treatment, five patients (19%) stopped because of controlled disease, ten patients (38%) due to adverse events. In four patients (15%) AZA was ineffective. Twelve other miscellaneous reasons caused discontinuation of treatment. Twelve patients used >3 months of prednisone simultaneously.

Methotrexate Of the 37 patients (11%) who received MTX, 23 patients (62%) started with this therapy as part the AMC RCT.8 No patients had more than one treatment episode with MTX. In our 10-year observation period, the median duration of treatment was 298 days (IQR 147– 384.5). Twenty patients (54%) continued treatment with MTX after January 1st 2011. Of the seventeen patients who stopped treatment, one patient (6%) stopped because of controlled disease. Seven (41%) discontinued because of adverse events. Eleven patients discontinued treatment because of ineffectiveness(65%). Three other miscellaneous reasons caused discontinuation of treatment. Six patients used >3 months of prednisone simultaneously.

Systemic glucocorticosteroids monotherapy Twenty-four patients (7%) were treated with a monotherapy of systemic glucocorticosteroids, of which twenty patients (83%) used pred-nisone and four patients (17%) used celestone. Four patients had more than one treatment episode with systemic glucocorticosteroids, of

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which only the first episode was evaluated. All courses lasted longer than 3 months (inclusion criterium), with a median duration of treatment with prednisone of 266 days (IQR 129.25–492) and 1363 days (IQR 258.75–2688.5) for celestone. The mean starting dose was 22.50 mg/day (SD 9.93) for prednisone and 1.83 mg/day (SD 0.76) for Celestone. The mean maximum dose was 23.00 mg/day (SD 9.51) for prednisone and 1.50 mg/day (SD 0.5) for celestone. Twenty patients (83%) discontinued treatment, of which four patients (20%) discontinued treatment because of controlled disease. One patient discontinued treatment with celestone because of unknown adverseevents. In four patients (20%) systemic glucocorticosteroids were ineffective. Thirty- one other miscellaneous reasons caused discontinuation of treatment.

Tacrolimus Eighteen patients (5%) were treated with tacrolimus, with a median duration of treatment 2 of 107 days (IQR 59.0–556.5). One patient had more than one treatment episode with tacrolimus, of which only the first episode was evaluated. Of the sixteen patients (89%) who discontinued treatment, one patient (6%) discontinued because of controlled disease. Nine patients (56%) discontinued treatment because of adverse events. In seven patients (44%), the treatment was not effective (Table 3). Nine other miscellaneous reasons caused discontinuation of treatment.

Order of use The order of use of the different oral immunosuppressive drugs in the included patients is shown in Table 5. These data include only oral immunosuppressive drugs that were given to the patients in our centres and do not include previous oral immunosuppressive treatments, prescribed by dermatologists of peripheral hospitals or by general practitioners. First choice a treatment was CyA, given in 252 patients. The absolute number of patients receiving AZA, MTX or EC-MPS in first instance were comparable. Most of them participated in the earlier mentioned clinical trials.

Table 5 Order of use in oral immunosuppressive treatments in steps Therapy First step Second step Third step Fourth step Total CyA 252 13 2 - 267 Syst cort 16 4 3 1 24 MMF - 6 3 1 10 AZA 22 8 13 3 46 MTX 21 9 6 1 37 EC-MPS 19 66 9 0 94 Tacrolimus 4 9 3 2 18

Syst cort = systemic corticosteroids

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Discussion

The present study provides an overview of oral immunosuppressive drugs used in the treatment of adult AD patients in two academic centres in a time period of 10 years. These data reflect the situation in the Netherlands and may be different from other European countries. CyA was the most commonly used oral immunosuppressive drug, followed by MMF/EC-MPS, AZA, MTX, systemic glucocorticosteroids and tacrolimus. Although long-term treatment with oral glucocorticosteroids is not recommended in guidelines, 27 patients were treated with oral glucocorticosteroids for more than 3 months. These patients were monitored well and only one patient had to discontinue due to unknown side-effects. The mean amount of 0.60 previous oral immunosuppressive treatments per patient indicates that the use of oral immunosuppressive drugs is not limited to our academic centres. Although the major part of the patients used oral immunosuppressive drugs in a daily practice setting, about 30% of the patients participated in one of the earlier mentioned trials. These patients may have a different profile due to the inclusion and exclusion criteria of the trials. The aim of the present study was to provide a complete overview of the oral immunosuppressive drugs that were prescribed in AD patients over a period of 10 years. It was not the intention to compare the efficacy of the different oral immunosuppressive drugs, as reliable efficacy measures, such as clinical scores were not available in all patients (only in trial patients) and bias could influence the data. Therefore, we chose the reason for discontinuation ofthe oral immunosuppressive drug as primary outcome measure. In our opinion this is the most reliable measure in retrospective studies. Still, this outcome has its shortcomings, because no predefined definitions for controlled disease and inefficacy were used. Discontinuation of treatment may have been influenced by different factors like local differences in treatment strategies and physician’s experience. Guidelines and insights have changed during the 10- year evaluation period. For instance in the first part of the evaluation period only CyA and oral steroids were described for severe AD, while the prescription of second-line drugs like MTX, AZA and MMF/MPA started in the last part of the evaluation period. This may partly explain the high number of finished CyA treatment episodes, while relatively more patients on second-line drugs are still under treatment at the moment of datalock. Adverse events were reported often as a reason for discontinuation of treatment. The lowest percentages were seen in patients treated with systemic glucocorticosteroids (5%), MMF/EC-MPA (22%) and CyA (24%). In CyA-treated patients objective adverse events (serum creatinine rise and hypertension) were the most common reason for discontinuation, while subjective side-effects as a reason for discontinuation were more prominent in the other treatment modalities. A relatively high percentage of patients discontinued treatment with AZA (38%),

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MTX (41%) and tacrolimus (56%) due to subjective adverse events, especially gastro-intestinal (AZA and MTX) and neurological (tacrolimus) side-effects. Although these side-effects are reported in clinical trials, the percentage of patients that discontinued treatment in this daily practice population is higher compared to published clinical trials.8–10 The most logical explanation is the difference in patients selection and probably compliance between trials and daily practice. Although AD is a chronic disease, it is in general not the intention to use oral immunosuppressive drugs for many years, despite good clinical efficacy. This in contrast to the treatment with biologicals in for instance psoriasis patients where continuous treatment for many years in stable disease is more common. Therefore, we also focussed on adequate disease control (controlled disease) as a reason for discontinuation of treatment. Controlled disease was the reason for discontinuation in relatively few patients. The highest number was seen in patients treated with CyA (29%), followed by systemic glucocorticosteroids (20%) 2 and AZA (19%). For MMF/EC-MPS, MTX and tacrolimus the percentages of discontinuation due to controlled disease were 13%, 6% and 6% respectively. It is important to realize that discontinuation due to controlled disease is not the same as clinical effectiveness, as patients who were still under treatment at the moment of data analysis, probably have agood response to treatment. In the future, these patients may also discontinue their treatment because of controlled AD disease. In the present study, patients with multiple comorbidities and difficult treatable AD are included. This in contrast to RCTs where guidelines are based on. Due to the strict inclusion criteria, patients participating in RCTs have less comorbidity and are often more motivated and compliant. A recent study of anti-TNF-a in psoriasis patients, showed that patients who received prior anti-TNF-a in a clinical trial setting, had a better drug survival rate afterwards when treated in a daily practice setting compared to patients who had not participated in clinical trials before. Possible explanations couldbe that former trial patients may be healthier and have fewer comorbidities, or show a better adherence to treatment due to positive experiences in a previous trial.21 Another important difference between clinical trials and daily practice is the duration of treatment: in clinical trials treatments are often limited to 3–6 months, while treatment periods in daily practice are much longer. There is a need for more practical guidelines in the treatment of patients with AD with oral immunosuppressive drugs. A recent published systematic review provided an evidence-based systemic treatment algorithm for patients with moderate-to-severe AD and the recent AAD guideline gives an overview of the existing literature.7,11 In children, the TREAT survey gives an indication on which systemic immunomodulating drugs are used.22 Data from the present study provide practical information on dosing, order of use and reasons for discontinuation of oral immunosuppressive drugs in adult AD patients, which is valuable for future recommendations in guidelines.

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Limitations of this study In this retrospective study, the quality of the included data depended on the completeness of medical records. Some patients used prednisolone >3 months simultaneously with other oral immunosuppressive drugs. It was not possible to evaluate these treatments separately. The use of oral steroids was supportive. Therefore, the data of these combinations were described in the evaluation of the main compound. In the result section of the amount, long-term treatment with prednisone is mentioned for each oral immunosuppressive drug. To provide an extensive complete overview of oral immunosuppressive drugs that were prescribed in the last 10 years, both daily practice and RCT data were included. We were encouraged to do so because the trials were set up as much as possible according to daily standard care. Nevertheless, it should be taken into account that the data of trial patients are influenced by the trial process. Table 5 for instance gives an overview of the order of use of oral immunosuppressive drugs in our centres, but it should be taken in consideration that the order has been influenced by the randomization process that was applied in the AMC and UMCU RCTs. Finally, we are aware of the fact that these data may not be generalizable for the Netherlands and may show differences with other European academic AD centres.

Recommendations Besides clinical trial data, observational daily practice data should be taken into account in updating existing guidelines and formulating recommendations. A prospective database of adults and children with AD using oral immunosuppressive drugs in daily practice, including validated and standardized diagnostic criteria, outcome domains and measurements, could be of great value for further recommendations and is currently being developed.

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References 9. Haeck IM, KnolMJ, ten BergeO, van Velsen SG, de Bruin-WellerMS, Bruijnzeel-Koomen CA.

1. Asher MI, Montefort S, Bjorksten et al. Enteric-coatedmycophenolate sodiumversus en B et al. Worldwide time trends in cyclosporine A as long-term treatment in the prevalence of symptoms of asthma, adult patients with severe atopic dermatitis: allergic rhinoconjunctivitis, and eczema in a randomized controlled trial. J AmAcad childhood: ISAAC Phases One and Three Dermatol 2011; 64: 1074–1084. repeat multicountry cross-sectional surveys. 10. Schmitt J, Sch€akel K, F€olster-Holst R et al. Lancet 2006; 368: 733–743. Prednisolone vs. ciclosporin for severe adult 2. Bieber T. Atopic dermatitis. Ann Dermatol eczema. An investigator-initiated double- 2010; 22: 125–137. blind placebocontrolled multicenter trial. Br 3. Ring J, Alomar A, Bieber T et al. Guidelines J Dermatol 2010; 162: 661–668. for treatment of AD (atopic dermatitis) 11. Roekevisch E, Spuls PI, Kuester D, Limpens 2 part I. J Eur Acad Dermatol Venereol 2012; J, Schmitt J. Efficacy and safety of systemic 26:1045–1060. treatments for moderate-to-severe atopic 4. Gambichler T. Management of atopic dermatitis: a systematic review. J Allergy Clin dermatitis using photo(chemo)therapy. Arch Immunol 2014; 133: 429–438. Dermatol Res 2009; 301: 197–203. 12. Hanifin JM, Cooper KD, Ho VC et al. Guidelines 5. Meduri NB, Vandergriff T, Rasmussen H, of care for atopic dermatitis, developed in Jacobe H. Phototherapy in the management accordance with the American Academy of of atopic dermatitis: a systematic review. Dermatology (AAD)/American Academy of Photodermatol Photoimmunol Photomed Dermatology Association “Administrative 2007; 23: 106–112. Regulations for Evidence- Based Clinical 6. Garritsen FM, Brouwer MW, Limpens J, Spuls Practice Guidelines”. J Am Acad Dermatol PI. Photo(chemo)therapy in the management 2004; 50: 391–404. of atopic dermatitis: an updated systematic 13. Hoare C, Li Wan Po A, Williams H. Systematic review with implications for practice and review of treatments for AD. Health Technol research. Br J Dermatol 2014; 170:501–513. Assess 2000; 4: 1–191. 7. Sidbury R, Davis DM, Cohen DE et al. 14. 14 Schmitt J, Sch€akel K, Schmitt N, Meurer Guidelines of care for the management of M. Systemic treatment of severe AD: a atopic dermatitis: section 3. Management systemic review. Acta Derm Venereol 2007; and treatment with phototherapy and 87: 100–111. systemic agents. J Am Acad Dermatol 2014; 15. Saeki H, Furue M, Furukawa F et al. Guidelines 71: 327–349. for management of atopic dermatitis. J 8. Schram ME, Roekevisch E, Leeflang MM, Bos Dermatol 2009; 36: 563–577. JS, Schmitt J, Spuls PI. A randomized trial of 16. Williams HC. Clinical practice. Atopic methotrexate versus azathioprine for severe dermatitis. N Engl J Med 2005; 352: 2314– AD. J Allergy Clin Immunol 2011; 128: 353– 2324. 359. 17. Dutch Society of Dermatology and

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Venereology, Quality Institute for Healthcare CBO. Dutch Guidelines for the Treatment of Atopic Dermatitis, Utrecht, the Netherlands, 2014. 18. Ring J, Alomar A, Bieber T et al. Guidelines for treatment of AD (atopic dermatitis) Part II. J Eur Acad Dermatol Venereol 2012; 26:1176–1193. 19. Brenninkmeijer EE, Schram ME, Leeflang MM, Bos JD, Spuls PI. Diagnostic criteria for atopic dermatitis: a systematic review. Br J Dermatol 2008; 158: 754–765. 20. von Elm E, Altman DG, Egger M et al. The strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies. J Clin Epidemiol 2008; 61: 344–349. 21. van den Reek JM, van L€umig PP, Driessen RJ et al. Determinants of drug survival for etanercept in a long-term daily practice cohort of patients with psoriasis. Br J Dermatol 2014; 170: 415–424. 22. Proudfoot LE, Powell AM, Ayis S et al. The European TREatment of severe AD in children Taskforce (TREAT) survey. Br J Dermatol 2013; 169: 901–909.

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A systematic review

E. Roekevisch, P.I. Spuls, D. Kuester, J. Limpens, J. Schmitt Journal of Allergy and Clinical Immunology (JACI). 2014 Feb;133(2):429-38

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Abstract

Background Many patients with moderate-to-severe atopic dermatitis (AD) require systemic immunomodulating treatment to achieve adequate disease control.

Objective We sought to systematically evaluate the efficacy and safety of systemic treatments for moderate-to-severe AD.

Methods A systematic literature search was performed in MEDLINE, EMBASE, and CENTRAL (until June 2012). Randomized controlled trials (RCTs) evaluating systemic immunomodulating treatments for moderate-to-severe AD were included. Selection, data extraction, quality assessment, and generation of treatment recommendations using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach were performed independently by 2 reviewers. Efficacy outcomes were clinical signs, symptoms, quality of life, and the course of AD. Safety data were compared by calculating the weekly incidence rates (as percentages) for adverse events.

Results Thirty-four RCTs with 12 different systemic treatments and totaling 1653 patients were included. Fourteen trials consistently indicate that cyclosporin A efficaciously improves clinical signs of AD. Cyclosporin A is recommended as first-line treatment for short-term use. A second-line treatment option is azathioprine, but efficacy is lower, and evidence is weaker. Methotrexate can be considered a third-line treatment option. Recommendations are impossible for mycophenolate, montelukast, intravenous immunoglobulins, and systemic glucocorticosteroids because of limited evidence. A meta-analysis was not performed because of a lack of standardization in outcome measures.

Conclusion Although 12 different interventions for moderate-to-severe AD have been studied in34 RCTs, strong recommendations are only possible for the short-term use of cyclosporin A. Methodological limitations in the majority of trials prevent evidence-based conclusions. Large head-to-head trials evaluating long-term treatments are required.

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Introduction

In many patients with moderate-to-severe atopic dermatitis (AD), disease activity requires systemic treatment to achieve adequate disease control. Data from routine clinical care suggest that more than 10% of all patients with AD receive systemic anti-inflammatory treatment.1 Various immunomodulating therapies are currently used, including glucocorticosteroids, cyclosporin A (CsA), methotrexate (MTX), azathioprine (AZA), IFN-g, intravenous immunoglobulin (IVIG), montelukast, Enteric-coated mycophenolate sodium (EC- MPS), and traditional Chinese herbal medicine (TCHM). We conducted a systematic review to critically appraise the evidence on the benefits and harms of systemic treatments for AD, to provide clinically relevant recommendations, and to inform future guideline development.

Methods 3

We conducted a systematic review on the efficacy and safety of immunomodulating systemic treatments (further noted as ‘‘systemics’’) for moderate-to severe AD.

Eligibility criteria All fully published randomized controlled trials (RCTs) or open-label extensions of RCTs reporting on systemics in patients with moderate-to severe AD were included. Because of the absence of an established definition of moderate-to-severe AD, RCTs were eligible when including subjects defined as ‘‘patients with moderate-to-severe AD,’’ ‘‘patients with non -adequately controlled AD despite the use of topical anti-inflammatory therapy,’’ or patients with moderate-to-severe AD according to severity criteria (Rajka and Langeland score2 >4.52, SCORAD score3 >20%, or BSA4 >10%). RCTs without original data (ie. reviews) or a full report available (eg. letters and abstracts) in which a mixed population of different dermatologic conditions was studied without AD being analyzed separately, including subgroups of AD, and RCTs not reporting a clinical outcome (clinical signs) or patient- reported outcome (eg, quality of life and symptoms) were excluded. There were no language restrictions.

Literature search A medical librarian (J.L.) searched MEDLINE (OVID, from 1948), EMBASE (OVID, from 1980), and the Cochrane Central Register of Controlled Trials (CENTRAL, from inception) to June 2012. The search strategy consisted of (1) free-text words and subject headings related to AD,

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(2) specific immunomodulatory drugs or systemic therapy, and (3) a filter to find RCTs.5 The search strategy for MEDLINE is shown in Table E1 in this article’s Online Repository at www. jacionline.org. The search included an iterative process to refine the search strategy through adding search terms as new relevant citations were identified. Reference Manager software (version 12.0) was used to manage and deduplicate all identified references.

Study selection and data extraction At least 2 reviewers (E.R., D.K., or J.S.) independently conducted the selection and data extraction. Any discrepancies were resolved with a fourth reviewer (P.I.S.). Author, country, year of publication, number of patients, age range, sex, severity of AD, study design, dosage and duration of treatment, concurrent treatment, clinical outcome measures, and results were extracted. For chronic diseases, such as AD, it is necessary to distinguish between short- term (ie, to induce remission) and long-term treatment. For this review, short-term treatment is defined as active treatment of less than 16 weeks’ duration.

Quality assessment The Risk of Bias tool was used for quality assessment with review Manager 5.1 (Cochrane Collaboration) software to conduct the analyses.6 Extra clarifying rules were defined as follows. If the observer was blinded but the patient and RCT personnel were not blinded, we judged the risk of bias as ‘‘unclear’’ because of insufficient blinding. If an RCT had dropouts and an intention-to-treat analysis was used but no information on the handling of the missing data was given, the risk of bias was scored ‘‘unclear’’ because of incomplete outcomes. We made a separate column for authors’ and sponsors’ conflict of interest as a potential source for bias.7 When no information on funding or conflict of interest was provided, we assumed that there was none.

Data synthesis In accordance with the Harmonising Outcome Measures for Eczema core outcome domains for AD trials,8 outcomes concerning clinical signs, symptoms, health-related quality of life, and course of AD were abstracted as efficacy outcomes. Additionally, the primary outcome measures of included RCTs and changes in IgE levels and eosinophil counts were extracted. If the primary outcome measure was not explicitly defined, the first outcome mentioned in the results section was assumed to be the primary outcome. The mean change in efficacy outcome was defined as investigator-rated measurement9 from baseline to the end of active treatment. If not mentioned in the article, the mean change in clinical severity was calculated by using absolute scores at baseline and the end of active treatment extracted from the text,

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a presented figure, or graph. In crossover RCTs only the period before crossover was used to avoid information bias because of carryover effects. To compare safety data, the incidence rates (as percentages) per patient per week for adverse events (AEs), serious adverse events (SAEs), and withdrawals because of AEs or SAEs were calculated as follows:

Number of events/(Number of patients x Duration of RCT in weeks) x 100.

We intended to pool the mean change in clinical severity of AD from baseline until the end of active treatment of qualitatively homogeneous RCTs in a random-effects meta-analysis.

Strength of evidence and recommendations Two reviewers (E.R. and J.S.) graded the quality of evidence and the strength of the recommendation using the Grading of Recommendations Assessment, Development and Evaluation (GRADE)10 methodology. However, in this review GRADE was used in an adjusted 3 way. The evidence per treatment was graded instead of the evidence per outcome. GRADE could only be applied for the effect of treatments on clinical signs because of poor reporting of results in other outcome domains. Discrepancies were resolved with a third reviewer (P.I.S.). The definitions of ‘‘high,’’ ‘‘moderate,’’ ‘‘low,’’ and ‘‘very low’’ were used in grading the quality of evidence, according to the GRADE methodology (Table I and see Appendix E1 in this article’s Online Repository at www.jacionline.org).10

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 45 Chapter 3 CsA is effective in patients with moderate-to-severe AD. Further with moderate-to-severe patients in CsA is effective in impact on our confidence an important have to is likely research recommendation. our may change and effect of the estimate Higher dosages of CsA are more effective than lower dosages of CsA dosages lower than effective more of CsA are Higher dosages to change very unlikely is research Further AD. with severe in patients effect. of in the estimate our confidence formulation older of CsA efficacy comparative the on evidence Current equal weak and suggests very for AD is formulation CsA newer vs. effect in of estimate Any formulations. of both CsA effectiveness uncertain. is very research future CsA is more effective than prednisolone to induce stable remission stable to induce prednisolone than effective CsA is more an to have very likely is research Further AD. with severe in patients effect and is of in the estimate impact on our confidence important our recommendation. change to likely CsA and MMF may be equally effective as a maintenance treatment in treatment as a maintenance be equally effective CsA and MMF may very effect is in the confident but our AD, with severe some patients very uncertain. is research in future effect of estimate Any low. for the use of IVIG recommend to is insufficient evidence Current of CsA effectiveness on the comparative evidence Current AD. severe in effect of estimate CsA. Any and favours weak AD is very IVIG for vs. uncertain. is very research future CsA and topical tacrolimus 0.1% may be equally effective in patients patients in be equally effective 0.1% may tacrolimus CsA and topical low. is effect in the but our confident AD, with moderate-to-severe impact on our an important have to likely is very Further research our to change likely effect and is of in the estimate confidence recommendation. AD, severe with patients UVAB in than effective be more CsA may effect in future of estimate Any low. is effect in the but our confident uncertain. is very research AZA is effective in many patients with moderate-to-severe AD. Any AD. with moderate-to-severe patients in many is effective AZA very uncertain. is research in future effect of estimate MTX and AZA are both equally effective in patients with severe AD and severe with patients in effective equally both are AZA and MTX to is likely research Further option. as a treatment be considered may effect of in the estimate impact on our confidence an important have our recommendation. change and may Conclusion

Moderate quality Moderate High quality quality low Very Low quality Low Very low quality low Very quality low Very Low quality Low quality low Very Very low quality low Very Moderate quality Moderate GRADE Dose response # # response Dose

0 1 0 0 0 0 0 0 0 0 Publication bias* Publication

0 0 0 0 -1 -1 -1 -1 -1 -1 Imprecision*

0 0 0 -2 -1 -2 -1 -1 -1 -1 Indirectness*

0 0 0 0 0 0 0 0 0 0 Inconsistency*

0 0 0 0 0 0 0 0 0 -1 Risk of bias* bias* of Risk 0 0 0 0 0 -1 -2 -1 -1 -1 Comparison CsA v Placebo CsA higher dose v CsA lower dose CsA higher dose v lower formulation CsA older v newer CsA v prednisolone CsA v MMF CsA v IVIG CsA v topical tacrolimus CsA v topical CsA vUVAB AZA v Placebo AZA AZA v MTX AZA

(35;40;45;47;49) (24;51) (36) (25) (41) (20;34) (52) (26) (19) (42) 2 (182) 1(14) 1 (38) 1(50) 1(14) 1 (30 ) 1 ( 72) 2 (98) 1(42) 5( 146) No. of (total RCTs participants) Table 1: GRADE Table

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 46 Efficacy and safety of systemic treatments for moderate-to-severe atopic dermatitis downgrade -2 downgrade à -1/ Two or -1/ more items “high” Two à Systemic pimecrolimus is effective in some patients with moderate- patients in some is effective pimecrolimus Systemic Further option. as a treatment be considered AD and may to-severe in impact on our confidence an important have to is likely research recommendation. our may change and effect of the estimate INF is effective in some patients with severe AD and may be AD and severe with patients in some INF is effective an to have is likely research Further option. as a treatment considered may effect and of in the estimate impact on our confidence important our recommendation. change Systemic glucocorticosterodis (beclomethasone diproprionate and diproprionate (beclomethasone glucocorticosterodis Systemic with patients remission in to induce effective be flunosolide) may moderate-to- very uncertain. is research in future effect of estimate Any AD. severe in a is effective or not montelukast whether do not know We of estimate Any AD. with moderate-to-severe of patients subgroup very uncertain. is research in future effect We do not know whether or not montelukast is more, equally, or less equally, is more, or not montelukast whether do not know We steroid topical antihistamine, with treatment combined as effective of estimate Any AD. with moderate-to-severe patients in +- antibiotic very uncertain. is research in future effect AD and is not severe with patients for is not effective M. Vaccae to is likely Further research evidence. based on current recommended effect of in the estimate impact on our confidence an important have our recommendation. change and may Current evidence does not recommend the use of TP-5 for patients patients for the use of TP-5 does not recommend evidence Current an important have to likely is very Further research AD. with severe to likely effect and is of in the estimate impact on our confidence our recommendation. change Current evidence is insufficient to recommend the use of XFS or the use of recommend to is insufficient evidence Current effect of estimate Any AD. with moderate-to-severe patients for TCHM uncertain. is very research in future Current evidence is insufficient to recommend the recommend to is insufficient evidence Current estimate Any AD. with severe patients use of IVIG for very uncertain. is research in future of effect 3 downgrade -1/ If sum of patients for one comparison in all trials < 20 < trials all in comparison one for patients of sum If -1/ downgrade à no downgrading/ One item “high” in majority of studies à Moderate quality Moderate Moderate quality Moderate Very low quality low Very quality low Very Very low quality low Very quality Moderate Low Quality Very low quality low Very quality low Very 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 -1 -1 -1 0 0 0 -1 -1 -1 -1 -1 -1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 -2 -2 -2 No item “high” in majority of studies 0 0 -1 -1 -1 -1 -1 -1 -2 Maximal downgrading: -1/ patient population highly selected (e.g. only subgroup of AE-patients): -1 AE-patients): of (e.g. only subgroup highly selected population -1/ patient downgrading: Maximal If dose-response relationship is present: +1 is present: relationship If dose-response Maximal downgrading: -2 / Downgrading, if substantial variation in effect between studies, only quantitative: -1, also qualitative: -2 -1, also qualitative: studies, only quantitative: between effect in variation if substantial -2 / Downgrading, downgrading: Maximal Maximal downgrading: -2/ If sum of patients for one comparison in all trials is < 100: < is trials all in comparison one for patients of sum If -2/ downgrading: Maximal -2 à Pimecrolimus v placebo Pimecrolimus INF v placebo Systemic glucocortico-sterodis vs placebo glucocortico-sterodis Systemic v Placebo Montelukast Montelukast v antihistamine, topical topical v antihistamine, Montelukast +- antibiotic steroid v placebo M. Vaccae TP-5 v Placebo TP-5 TCHM v Placebo TCHM control IVIG v placebo /waiting Does not apply to RCTs Does not apply to Maximal downgrading: -2 downgrading: Maximal Does not apply to RCTs Does not apply to (33)

(27;31) (21) (32;37) (50) (29) (38;48) (22;39) (23;30;43;44) (46) 2 (134) 2 (67) 2 (74) 2 (63) 1 (166) 1(39) 4 (79) 1(103) 2 (49) Downgrading rules: Downgrading Risk of ofBias items(consideration 1-6 (withoutconflict ofinterest): in majority of studies of the results: Inconsistency of the evidence: Indirectness results: the of Imprecision bias: Publication rules: Upgrading effect: overall Large : relationship Dose-response Confounder:

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Results

Results of the literature search Of the 925 references retrieved by using the systematic search, 34 RCTs11-44 were selected for review, including a total of 1653 patients (see Fig E1 in this article’s Online Repository at www.jacionline.org).

Study characteristics Twelve different types of systemic treatments for moderate-to-severe AD were investigated. Fourteen RCTs evaluated CsA.11, 16-18, 20, 27, 28, 32, 33, 37, 39, 41, 43, 44. Five RCTs were placebo controlled27,32,37,39,41; six were head-to-head comparisons with IVIG,11 UV, 17 EC-MPS,18 tacrolimus,28 and prednisolone33; and 1 compared a new (Sandimmune) versus an old CsA formulation (Neoral).44 Three RCTs were dose finding.16,20,43 Three RCTs12,26,34 evaluated AZA, including 82 patients treated with AZA. Two were placebo- controlled trials,12,26 and 1 RCT34 was a head-to-head RCT versus MTX (n 5 20). IFN-g was evaluated in 2 RCTs.19,23 Three RCTs21,25,33 evaluated systemic glucocorticosteroids, including 20 children treated with flunisolide25 and an unreported number of children with beclomethasone diproprionate.21 Twentyone adults were treated with prednisolone.33 Three RCTs11,24,29 evaluated IVIG, including 45 patients treated with IVIG. Montelukast was investigated in 4 RCTs.14,30,31,40 Two RCTs30,40 were placebo-controlled trials, and 2 RCTs14,31 were head-to-head trials against antihistamine, a topical steroid/antibiotic. Four RCTs15,22,35,36 evaluated TCHM. Mycobacterium vaccae,13 pimecrolimus,42 and thymopentin (TP-5)38 were investigated in 1 RCT each, respectively. Twenty-two trials were conducted in Europe, 2 in the United States,19,38 and 7 in Asia,11,15,22-24,30,31 and 3 RCTs were international.13,17,24 The number of patients ranged from 10 to 166. Most RCTs (94%) were small and recruited less than 100 patients. Long-term treatment of 16 weeks or greater was assessed in 5 (15%) studies. Topical corticosteroids as concomitant therapy were allowed in 24 (71%) RCTs. Ninety-seven percent of the RCTs appeared to have the domain ‘‘clinical signs’’ as the primary outcome domain, and 1 (3%) RCT32 used ‘‘quality of life’’ as the primary outcome domain. The 34 selected RCTs applied a total of 12 different clinical sign scales as the primary outcome measure. The (objective) SCORAD score was most frequently used (11 [32%] RCTs),11, 14, 17, 18, 22, 24, 28, 29, 31, 33, 34 the Eczema Area and Severity Index (EASI) was used in 2 RCTs.40,42 In 17 (50%) RCTs 15, 16, 19, 21-23, 25, 27, 30, 35-39, 41, 43, 44 no validated clinical sign scores were used. For further details on study characteristics, see Table E2in this article’s Online Repository atwww.jacionline.org . Twelve RCTs14, 15, 18, 19, 21-24, 28, 29, 36, 37 mentioned changes in IgE levels, eosinophil counts, or both after treatment. One RCT19 showed a significant reduction in eosinophil counts and a possible

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trend in IgE levels after treatment with IFN-g. A decrease in total serum IgE levels was seen in patients treated with EC-MPS,18 and the difference in IgE levels between those receiving EC-MPS and those receiving CsA treatment was significant. Most RCTs 12, 15, 19, 22-24, 26, 28, 31, 32, 35, 36, 38-40 (44%) included both children and adults, 5 11,13,20,25,30 (15%) included exclusively children, and 14, 16-18, 21, 27, 29, 33, 34, 37, 41-44 (41%) RCTs included only adults (age > 18 years). Details on study characteristics, study eligibility criteria, and baseline characteristics are summarized in Tables E2 and E3 in this article’s Online Repository at www.jacionline.org.

Study quality/risk of bias Authors often reported that the trial was randomized without stating how the randomization sequence was generated. An unclear or high risk of bias was found in 60%11, 14, 17, 19, 20, 23-25, 27, 28, 31, 32, 35-43 (21/34 RCTs) for sequence generation, 79% 11, 14-21, 23-25, 27-29, 31, 32, 34-41, 43, 44 (27/34 RCTs) for allocation concealment, 63% 11, 13, 14, 17-20, 23-25, 29, 31-34, 36,39-44 (22/34 RCTs) for blinding, 71% 11-14, 17, 19-21, 23-25, 27, 29, 30, 32, 35-41, 43, 44 (24/34 RCTs) for incomplete outcome data, 24%11, 12, 14, 18, 23, 26, 3 29, 32 (8/34 RCTs) for selective outcome reporting, and 32% (11/34 RCTs)11, 12, 14-16, 18, 21, 23, 33, 35, 36 for other biases (see Figs E2 and E3 in this article’s Online Repository atwww.jacionline.org).

Efficacy and safely of systemic treatments for moderate-to-severe AD An overview of the (modified) efficacy results can be found in Table II. The rest of the efficacy and safety results can be found in Tables E4 and E5 in this article’s Online Repository atwww. jacionline.org. Recommendations according to the GRADE approach are provided in Table I.

CsA. Benefit. CsA, which inhibits the proliferation of T lymphocytes, was superior to placebo in 5 RCTs, with a mean clinical improvement in severity between 53% and 95% in different clinical severity scores after short-term treatment (10 days to 8 weeks).27,37,39,41 One RCT used quality of life as the primary outcome and found CsA to be superior to placebo.32 In head- to-head trials CsA was superior to prednisolone,33 superior to IVIG,11 superior to UVAB,17 and similarly efficacious as EC-MPS.18 Higher CsA dosages (5 mg/kg per day) lead to a more rapid response and are more efficacious than lower dosages (2.5-3 mg/kg per day) in the short- term treatment of AD.43 Long-term use of CsA up to 1 year can be recommended based on 4 trials,17,18,20,43 but evidence is limited because of the open-label design 17,20,43 and high dropout rates.17,43 Harm. The weekly rate of any AEs ranged between 1.0%41 and 28.2%.33 The weekly rate of withdrawals because of AEs ranged between 0% 16, 20, 27, 28, 32, 37, 41, 44 and 2.0%.33 The weekly rate of any SAE ranged from 0.0% 16-18, 27, 28, 33, 41, 43, 44 to 2.2%.39 In 9 RCTs no severe SAEs were

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reported. 16-18, 27, 28, 33, 41, 43, 44 Unclear information on the occurrence of SAEs per group was provided in 2 RCTs.11,20 Recommendation. CsA is currently recommended as the first-line short-term treatment option for moderate-to-severe AD because of moderate- and high-quality studies based on the GRADE approach and the efficacy and safety shown for short-term use, including large patient numbers.

AZA. Benefit. AZA, a purine analog, was superior to placebo, with a mean improvement in Six Area, Six Sign Atopic Dermatitis scores of 26%12 and 37%26 at week 12. AZA and MTX were found to be equally efficacious, with a mean SCORAD score improvement of approximately 39%34 for AZA and a mean improvement of 20% in quality of life (SKINDEX) after 12 weeks. Sixteen (84%) patients in the AZA group continued. At week 24, the relative reduction in the mean SCORAD score was 43%. Harm. The weekly rate of any AE ranged between 5.6%26 and 22.9%.34 Abnormalities in blood counts, such as lymphocytopenia, were most frequently seen. The weekly rate of withdrawals because of AEs ranged between 0.2%8 and 0.4%.26 SAEs were not observed12,34 or not reported.26 After 24 weeks, no SAEs were seen, in 9% AEs led to withdrawal, and in 9% dose adjustments were related to AEs. On the basis of the RCT by Meggitt,26 the AZA dosage should be determined on the basis of thiopurine S-methyltransferase activity to limit myelosuppression. Recommendation. AZA is currently recommended as a second-line treatment option for moderate-to-severe AD because of a moderate-quality study based on the GRADE approach and the efficacy and safety shown for short- and long-term use (24 weeks), including large patient numbers.

MTX. Benefit. MTX, a folic acid antagonist, was equally efficacious as AZA, with a mean improvement in SCORAD score of 42% and a mean improvement of 26% in quality of life (SKINDEX) after 12 weeks of treatment in a head-to-head trial.34 Eighteen (95%) patients in the MTX group continued. At week 24, the relative reduction in the mean SCORAD score was 48%. Harm. The weekly rate of any AE was 23.5%. The weekly rate of withdrawals because of AEs was 0.4%. SAEs were not observed. After 24 weeks, no SAEs were seen, in 5% AEs led to withdrawal, and in 10% dose adjustments were related to AEs. Recommendation. MTX is recommended as a third-line treatment for adults with severe AD because of a moderate quality study based on the GRADE approach and the efficacy and

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safety shown for short- and long-term use (24 weeks), including large patient numbers.

IFN-g. Benefit. Both RCTs19,23 investigating IFN-g, which downregulates TH2 cell function, versus placebo found IFN-g to be efficacious after 12 weeks of treatment based on unvalidated outcome measures. One trial23 observed a positive dose-response relationship. Harm. The weekly rate of any AE was 16.3% in one trial19 and not reported in the other trial.23 Recommendation. The quality of evidence of efficacy of IFN-g versus placebo is moderate for short-term use. IFN-g can be considered a third-line treatment option for patients with severe AD, but safety and tolerability need to be monitored closely.

EC-MPS. Benefit. One small trial18 (n550) investigated the efficacy of EC-MPS, a purine biosynthesis inhibitor, versus CsA as a maintenance treatment after induction treatment with CsA. Within 3 30 weeks of maintenance treatment, EC-MPS and CsA were equally effective. Harm. The weekly rate of any AE was 4.5%. The weekly rate of withdrawals because of AEs was 0.3%. SAEs were not observed. Recommendation. Only a very weak recommendation is possible for EC-MPS as a maintenance treatment for severe AD after induction of remission by CsA for long-term use up to 30 weeks.

Systemic glucocorticosteroids. Benefit. Two small trials investigated the short-term efficacy of the systemic glucocorticosteroids beclomethasone diproprionate21 and flunisolide25 in children with severe AD based on unvalidated outcome measures. Systemic prednisolone was less efficacious than CsA in adults with severe AD and induced stable remission in only 1 of 21 patients.33 Harm. The weekly rate of any AE ranged between 0%25 and 20.4%.33 The study that compared prednisolone versus CsA was stopped prematurely because of high rates of exacerbations and adverse effects after discontinuation of prednisolone treatment.33 Recommendation. Systemic steroids are currently not recommended for moderate-to-severe AD.

IVIG. Benefit. IVIG was less efficacious than placebo and CsA.11 Harm. The weekly rate of any AE ranged between 0.6%24 and2.8%.11 Recommendation. IVIG is currently not recommended for the treatment of severe AD.

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Monteluklast. Benefit. Four RCTs14,30,31,40 investigated the efficacy of montelukast for moderate-to-severe AD. The results of these trials are inconsistent, indicating beneficial effects compared with placebo based on an unvalidated score30 but no difference in efficacy compared with placebo in mean EASI change.40 Montelukast was equally effective as a combination of cetirizine, clarithromycin, and a topical glucocorticosteroid,14 with a mean SCORAD score improvement of 51% at week 6, and it was superior to a combination of an antihistamine and topical glucocorticoid after 4 weeks.31 Harm. None of the trials reported AEs of montelukast. Recommendation. Because of the contradictory results from the published trials, montelukast is currently not recommended for the treatment of moderate-to-severe AD.

Pimecrolimus. Benefit. Systemic pimecrolimus, a calcineurin inhibitor, was superior to placebo in a dose- dependent manner with a mean clinical improvement in EASI of 35% and 47% after 12 weeks of treatment with 20 and 30 mg of pimecrolimus, respectively.42 Harm. The weekly rate of any AE was 7.7% in the 30-mg group. Recommendation. Systemic pimecrolimus could be a possible treatment option for moderate- to-severe AD in short-term use.

TCHM. Benefit. TCHM was shown to be superior to placebo in several trials, with a mean improvement in an unvalidated score of 56%,22 60%,36 and 82%35 after 8 weeks of treatment. In 1 RCT placebo tended to be more efficacious than TCHM, with a mean improvement in SCORAD score of 15% (TCHM) versus 19% (placebo) after 12 weeks of treatment. For further information on the formulations used in the different trials, see Fig E1. Harm. The weekly rate of any AE ranged between 0.5%35 and 10.8%.22 The weekly rate of withdrawals because of AEs ranged between 0.0%35 and 0.2%.15 Recommendation. Because of the inconsistent results from the published trials, TCHM is currently not recommended for the treatment of moderate-to-severe AD.

M vaccae. Benefit. M vaccae was found not to be more efficacious than placebo in children with severe AD.13 Harm. AEs per group were not reported. SAEs occurred with a weekly rate of 0.2%. Recommendation. M vaccae is not recommended for the treatment of severe AD.

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TP-5. Benefit. One RCT38 based on 39 children and adults with severe AD reported a mean improvement in an unvalidated clinical score of 21% in the TP-5 group versus 12% in the placebo group (P 5.055). Harm. AEs were not reported.Withdrawals because of AEs or SAEs were not observed. Recommendation. TP-5 is currently not recommended for the treatment of severe AD.

Discussion

This systematic review provides an evidence-based treatment algorithm for patients with moderate-to-severe AD. By ranking the quality of evidence based on the GRADE approach45 and by taking into account the efficacy and safety shown for short-term (and long-term) use and the number of participants, this review extends previous research and guidelines 3 in which less specific treatment recommendations were made based on expert opinion46 or less systematic approaches, such as the Goodman method47 or the Category of Evidence and Strength of Recommendation– based approach.48-50

Recommendations for systemic treatment of moderate-to-severe AD Fourteen trials 11, 16-18, 20, 27, 28, 32, 33, 37, 39, 41, 43, 44 consistently indicate that CsA efficaciously improves clinical signs of AD in children and adults. Although higher doses (5 mg/kg body weight) of CsA are more effective, lower starting doses (3 mg/kg body weight) with stepwise adjustment to the individual minimum effective dose are preferable because most side effects are dose related.16,43 Overall, CsA is recommended as first-line treatment for short-term use. Long-term use of CsA up to 1 year can be recommended based on the results of 4 trials.17,18,20,43 However, the long-term safety of CsA cannot be concluded from the trial evidence. As a second-line treatment option, AZA is recommended for short-term induction treatment and long-term treatment up to 24 weeks. The dosage should be determined on the basis of thiopurine S-methyltransferase activity to limit myelosuppression.26 Indirect comparisons suggest that the efficacy of AZA is lower than that of CsA. MTX can be considered a third-line treatment option for shortterm induction treatment and long-term treatment up to 24 weeks, but the evidence is limited to a rather small single trial that found MTX to be similarly efficacious as AZA.34 According to placebo-controlled trials, IFN-g is also efficacious for severe AD. IFN-g can be considered a third-line treatment option, but safety and tolerability need to be monitored closely. EC-MPS might be a treatment option for maintenance treatment of AD after induction

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treatment with CsA, but this recommendation can currently be based only on a single study.18 Systemic glucocorticosteroids are the most frequently used systemic treatment for severe AD in routine care.1 In contrast, trial evidence concerning efficacy is very limited. One small trial33 indicated that systemic prednisolone is not suitable to induce long-term remission and also less efficacious than CsA. However, it is unclear whether this RCT investigated the ‘‘right’’ systemic steroid treatment regimen. The suggestions on how to apply systemic steroids differ substantially, with some authors suggesting treatment only for a few days and others over a period of months. Montelukast and TCHM are currently not recommended for the treatment of moderate-to-severe AD in routine care because the results from the trials published are inconsistent. One difficulty in assessing TCHM is its heterogeneity. Standard regimens include a variety of herbs, which can be called ‘‘polypharmaceuticals,’’ and are used in the form of tea. Potential concerns are the side effects of TCHM, such as liver failure, cardiomyopathy, and hepatotoxity,51-54 although none of these effects were reported in the included RCTs. IVIG, M vaccae, and TP-5 are currently not recommended because trials do not suggest that these treatments are efficacious in the treatment of moderate-to-severe AD.

Limitations of this review and published trials Although 12 different interventions have been studied in 34 RCTs, strong recommendations are only possible for the short-term use of CsA. Methodological limitations, such as small sample size, short duration of most of the trials conducted, a lack of head-to-head trials on key comparators, and unclear or high risk of bias in many important domains in a substantial proportion of trials, prevent strong evidence-based recommendations in accordance with the GRADE approach.45 Meta-analysis was not indicated because of the clinical and methodological heterogeneity, such as the lack of standardization in outcome measures, the substantial differences in trial designs, and the wide use of unvalidated outcome measures. Because of the lack of reporting of quality-of-life outcomes and symptoms, GRADE could only be applied for clinical efficacy based on measures of clinical signs (measures). Most systemics included in our review are known to be associated with potential adverse drug reactions.47,49,55-57 However, mainly short-term RCTs were included in this review; 85% (29/34) of the RCTs had a total RCT period of less than 16 weeks. Important AEs, SAEs, and withdrawals that occur after long-term treatment or follow-up could have been missed. Many RCTs provided inadequate information on AEs. Better arrangements need to be made on how to document short- and long-term safety data. Prospective registries with these drugs could detect rare and long-term side effects. Research recommendations More head-to-head trials or prospective registries are required to draw relevant conclusions for routine care and clarify the comparative effectiveness, safety,

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and tolerability of CsA, AZA, MTX, IFN-g, systemic steroids, TP-5, and EC-MPS. Such trials should consider the 4 core outcome domains for clinical trials, as defined by the Harmonising Outcome Measures for Eczema initiative.8 Although the prevalence of AD is highest among children, RCTs on children are missing for many relevant interventions, and more research in this age group is very important

Clinical implications: CsA is recommended as first-line treatment for short-term use in patients with moderate-to-severe AD. AZA is the second-line and MTX the third-line recommendation.

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Online Repository

Online Repository 1 Literature search: The search terms and strategy for MEDLINE are shown in the Table E1: Search strategy for MEDLINE.

Online Repository 2 Strength of evidence and recommendations Proposed rules for applying GRADE to systemic treatments for eczema RCTs

There are 4 categories of quality evidence: Grade Definition Further research is very unlikely to change our confidence in the estimate High of effect. Further research is likely to have an important impact on our confidence Moderate in the estimate of effect and may change the estimate. Further research is very likely to have an important impact on our Low confidence in the estimate of effect and is likely to change the estimate. Very low Any estimate of effect is very uncertain. RCT starts always with the level > High quality

Downgrading: Risk of Bias (consideration of items 1-6 (without conflict of interest): - No item “high” in majority of studiesà no downgrading - One item “high” in majority of studies à -1 - Two or more items “high” in majority of studies à -2

Inconsistency of the results - Maximal downgrading: -2 - Downgrading, if substantial variation in effect between studies, o only quantitative: -1 o also qualitative: -2

indirectness of the evidence - Maximal downgrading: -1

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- patient population highly selected (e.g. only subgroup of AE-patients): -1 imprecision of the results - Maximal downgrading: -2 - If sum of patients for one comparison in all trials is < 100: à downgrade -1 - If sum of patients for one comparison in all trials < 20 à downgrade -2

publication bias - Maximal downgrading: -2 - Strong evidence for publication bias is present (e.g. funnel plot regression): -2 - ALL studies on the comparison are n<100 AND funded by industry AND studies are in favor of investigated drug: -1

Upgrading: Large overall effect 3 - Does not apply to RCTs Dose-response relationship - If dose-response relationship is present: +1 Confounder Does not apply to RCTs

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Online repository 1

Table E1 Search strategy for MEDLINE Search Results 1 exp Eczema/ 8438 2 Dermatitis, Atopic/ 13126 3 eczem*.tw. 12724 4 (atopic adj5 (dermatit* or neurodermati*)).tw. 11772 5 (disseminated adj2 neurodermatit*).tw.0 35 6 or/1-5 29254 7 animals/ not humans/ 3642688 8 6 not 7 27818 9 exp cyclosporins/ 34887 10 (c?closporin* or CyA or Cy-A or CsA or Cs-A or csaneoral or neoral or sandimmun*).tw. 49720 11 exp Aminopterin/ 30688 12 (aminopterin or MTX or methotrexate).tw,ot. 30364 13 Azathioprine/ 12640 14 (az?thioprine or im?uran).tw. 11668 15 Leukotriene Antagonists/ or Quinolines/ 17923 (antileukotriene* or anti-leukotriene* or (leukotriene adj3 (antagonist* or block* or inhibitor*))). 16 3256 tw,ot. 17 leukast.tw,ot. or (zafirlukast or zileuton or montelukast or quinoline*).tw,ot,rn. 20873 18 Peptide Fragments/tu 2695 19 exp thymus hormones/ 4122 (thymopentin* or thymopoietin* or thymoptin* or thymosin* or thymostimulin* or 20 3749 thymomodulin*).rn,tw,ot. or (TP-5 or TP5).tw,ot. 21 exp Recombinant Proteins/ 264780 22 Interferon-gamma/ad, tu, pd, ae or interferon-gamma, recombinant/ 15733 (rIFN* or bioferon or biogen or immuneron or imukin or kw-2202 or polyferon or ru-42369 or 23 1779 ru42369 or s-6810 or sch-36850 or sun-4800).tw. 24 ((r or recombinant) adj3 (interferon* or IFN or IFNg or IFNgamma)).tw. 7677 ((Interferon* or IFN or IFNg or IFNgamma) adj3 (therap* or treat* or administ* or given or deliver* 25 27969 or systemic* or oral*)).tw. 26 (etanercept or enbrel).tw,ot. or tnfr fc fusion protein.rn. 4531 27 alefacept.tw,ot,rn. 391 28 ((((tnf or tumor necrosis factor) adj2 receptor) or tnfr) adj2 (fusion adj protein*)).ti,ab. 204 29 Tumor Necrosis Factor-alpha/ai, ag 9182 30 (anti-TNF* or anti tumo?r necrosis factor).tw. 7550 31 antibodies, monoclonal/tu, ad, ae 42418 ((humanized adj8 (monoclonal* or antibod* or MoAb* or mAb or mAbs or Fab or Fab’)) or 32 3748 rhuMAb*).tw. 33 (chim?eric adj3 (monoclonal* or antibod* or MoAb* or mAb or mAbs)).tw. 2734

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34 ((against or anti) adj IgE adj2 (monoclonal* or antibod* or MoAb* or mAb or mAbs)).tw. 837 ((anti or against or block* or MoAb* or mAb or mAbs or antibod* or monoclonal*) adj3 (IL5 or IL-5 35 2276 or interleukin-5 or LFA1 or LFA-1 or CD11a)).tw. 36 omalizumab.tw,ot,rn. or (olizumab or hu-901 or hu901 or tnx-901 or tnx901 or xolair).tw,ot. 805 37 infliximab.tw,ot,rn. or (avakine or remicade).tw,ot. 7398 38 efalizumab.tw,or,rn. or (raptiva or xanelim or hu1124 or hu-1124).tw,ot. 602 39 adalimumab.tw,ot,rn. or (humira or D2E7 or trudexa).tw,ot. 2585 40 rituximab.tw,ot,rn. or (idec c2b8 or mabthera or rituxan or rituxin).tw,ot. 9274 41 keliximab.tw,ot,rn. or (sb 210396 or sb210396).tw,ot. 19 42 mepolizumab.tw,ot,rn. or (bosatria or sb 240563 or sb240563).tw,ot. 122 43 mycophenolic acid/ 5550 (mycophen?lat* or mycophenol* or mycofen?lat* or mycofenol* or mofetil* or MMF or erl-080* 44 8647 or erl080* or melbex or myfortic or nsc-129185 or nsc129185).tw. 45 Immunoglobulins, Intravenous/ 8688 (((intravenous or IV) adj (immune globulin* or IG or immun?globulin* or antibod*)) or IVIG or 46 9354 HdIVIg).tw. 3 exp Prednisolone/ or Beclomethasone/ or exp Fluocinolone Acetonide/ or Adrenal Cortex 47 93594 Hormones/ (prednison* or methylprednis* or dehydrocortisone or dexamethason* or beclomethasone or 48 115327 flunisolide).tw,ot,rn. ((systemic or oral* or sublingual* or “per os” or inhal* or nasal* or parenteral*) adj (steroid* or 49 16955 glucosteroid* or corticosteroid* or glucocorticosteroid*)).tw. (biologicals or biologics or ((biological or biologic) adj (treatment or therap* or medicine* or drug* 50 13825 or agent* or product*))).tw. 51 (biologic* response modifier* or BRM*).tw. 2978 52 targeted therap*.tw. 12434 53 administration, inhalation/ or administration, intranasal/ or exp administration, oral/ 137999 ((systemic* or oral* or sublingual* or “per os” or inhal* or nasal* or parenteral*) adj3 (treat* or 54 agent* or drug*1 or monotherap* or medication* or medicine* or administ* or given or deliver* 193200 or immunosuppres* or antihistamin* or anti-histamin*)).tw. 55 or/9-54 939405 56 8 and 55 3079 (randomized controlled trial or controlled clinical trial).pt. or (randomi?ed or placebo or randomly). 57 704713 tw. or trial.ti. 58 56 and 57 455

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 59 Chapter 3 Random sequenceRandom (selection generation bias) Allocation concealment (selection bias) bias)Blinding participants of and personnel (performance bias) (attrition data outcome Incomplete bias) Selective (reporting reporting Other bias Other Conflict author interest of Conflict sponsor interest of . Bemanian 2005 ? ? – ? ? ? + ?

Berth Jones 2002 + + + ? ? ? – +

Berth Jones 2006 + + ? – + + + ?

Capella 2001 ? ? – ? ? ? + +

Cheng 2010 + – + + + – + ?

Czech 2002 + – + + + – – –

Granlund 2001 ? ? – ? + + + –

Haeck 2011 + ? ? + – – + –

Hanifin 1993 ? ? ? ? + + + –

Harper 2000 ? ? – ? + + + –

Heddle 1984 + ? + ? + – + –

Hon 2007 + + + + + + + ?

Jang 2000 ? ? ? – ? ? + –

Jee 2011 ? ? – ? + + + –

Meggitt 2006 + + + + ? + + –

Munro 1994 ? ? + – + + + –

Pacor 2004 ? ? + + + + + ?

Paul 2002 + ? ? – – + + –

Pei 2001 + + + ? + + + ?

Rahman 2006 ? ? – + + + + +

Rosa, la 1995 ? ? ? ? + + + –

Salek 1993 ? ? ? ? – + + –

Schmitt 2010 + + ? + + ? – –

Schram 2011 + ? ? + + + + –

Sheehan 1992 ? ? + – + ? + ?

Sheehan 1992b ? ? ? – + ? + ?

Sowden 1991 ? ? + ? + + + –

Stiller 1994 ? ? + ? + + + –

Van Joost 1994 ? ? ? – + + + ?

Veien 2005 ? ? ? ? + + + –

Wahlgren 1990 ? ? ? ? + + + –

Wolff 2005 ? + ? + + + + –

Zonneveld ? ? – ? + + + –

Zurbriggen 1999 + ? ? ? + + – – Online Repository 3 Online Repository each included RCT for about each risk of bias item authors’ judgments Figure E1: Risk of bias summary: review

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 60 Efficacy and safety of systemic treatments for moderate-to-severe atopic dermatitis primary outcome primary outcome measure** Mean change in Mean change from SASSAD 12 baseline to weeks Mean change in Mean change from SASSAD 12 baseline to weeks Mean change in Mean change from SCORAD 12 baseline to weeks Mean change Mean change from in TBSA 8 baseline to weeks Mean change in Mean change from SCORAD 8 baseline to * weeks Trial duration \ duration Trial treatment; (active total) 12 weeks; 24 weeks 12 weeks; 12 weeks; 36 weeks 36 weeks 12 weeks; 12 weeks; 12 weeks 12 weeks; 8 weeks ; 12 weeks ; 12 weeks 8 weeks 8 weeks (intermittent); (intermittent); 8 weeks 52 weeks Completed RCT RCT Completed 7 14 35 19 8 6 53 50 32 16 3 Study size (number of patients) Study size Randomized Randomized and started treatment 19 18 41 20 8 6 53 53 36 36 Concurrent Concurrent medication steroids, Topical histamines Anti Topical steroids Topical Topical steroids, steroids, Topical histamines Anti Systemic Systemic and topical antibiotics histamines, Anti biotics Systemic Topical steroids Topical

2,5mg; OD A: Azathioprine B: placebo; OD 0,5 mg/kgA: Azathioprine or 1,0 mg/kg 1,0 mg/kg dose.Then start week 4 first daily or 2,5 mg/kg;OD B: Placebo;OD 4 mg/kg/day;A: Cyclosporin OD B: Iv immunoglobulines 2 gr/kg; Single dose 2 After 150 mg/A: Cyclosporin OD. day; minimum to adjustment stepwise weeks dose effective B: Cyclosporin 300 mg/ day; OD, After 2 After 300 mg/B: Cyclosporin OD, day; minimum to adjustment stepwise weeks dose effective 4 mg/kg/day;A: Cyclosporin OD B: UVAB Phototherapy up to maximal maximal up to Phototherapy B: UVAB and 0.26 J/ of UVA doses of 15 J/cm2 or decreaments increments cm2 of UVB, week ; 2 ± 3 times a

cross-over Parallel / Parallel Intervention Dose; Frequency/Control Dose; Dose; Frequency/Control Intervention Frequency C P P P P Ref. Country year Berth-Jones UK 2002 Meggit UK 2006 Bemanian Iran 2005 Czech Germany 2000 Granlund Finland/ Norway 2001 Online Repository 4 Online Repository characteristics E2 Summary of study Table

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 61 Chapter 3 Mean change in Mean change Under the Area Curve SCORAD baseline to from 6 weeks Mean change in Mean change SCORAD objective baseline to from 30 weeks Mean change Mean change of active in area from eczema 8 baseline to weeks Mean change from in UKSIP baseline 8 weeks to UNSKP (QoL measurement) Mean change Mean change from in SASSAD 52 baseline to weeks Mean change in Mean change SCORAD objective baseline to from 6 weeks Mean change in Mean change Disease severity( six defined from body sites) 8 baseline to weeks 3 5 ; 48 weeks 2 ; 9 weeks ; 9 weeks 4 6 weeks 8 weeks; 16 weeks 8 weeks; 30 weeks 30 weeks 52 weeks; 52 weeks 52 weeks; (A)6 weeks (B) 2 weeks (B) 2 weeks (A)6 weeks 18 weeks / 4 weeks; 8 weeks; 26 weeks 8 weeks; 8 weeks; 20 weeks 8 weeks; 10 15 15 17 20 20 15 10 11 14 9 10 17 14 12 15 15 17 26 24 21 16 17 19 12 21 17 16 Anti Anti histamines Topical steroids Topical Topical setroids Topical Prednison 0.5 Prednison mg/kg, of max 2 courses steroids Topical Topical steroids, steroids, Topical histamines Anti Topical steroids Topical Topical steroids Topical B: Placebo; OD 3 mg/kg;A: Cyclosporin OD / Placebo no dose; BID of Tacrolimus B: Tacrolimus 0,1%; BID/ Placebo of B: Tacrolimus no dose; OD Cyclosporin 5 mg/kg/day;A: Cyclosporin OD A: Cyclosporin (5mg for 6 weeks run-in); weeks 6 (5mg for Cyclosporin A: 3 mg/kg; OD B: (Cyclosporin 5mg for 6 weeks run-in) 6 weeks 5mg for B: (Cyclosporin sodium mycophenolate Enteric-coated 720 mg; BD 5 mg/kg;A: Cyclosporin OD B: Placebo; OD 2,7–4,0 mg/kg;A: Cyclosporin OD B: Cyclosporin 5 mg/kg, B: Cyclosporin weeks 4 after minimum to adjustment stepwise dose; OD effective 5 mg/kg/day;A: Cyclosporin OD B: Prednisolon 0,5–0,8 mg kg/1, B: Prednisolon tapered nil; OD/ Placebo no dose; OD to 5 mg/kg;A: Cyclosporin OD B: Placebo; OD P C P P P C C Pacor Italy 2004 Salek UK 1993 Haeck Holland 2011 Harper UK 2000 Schmitt Germany 2010 Munro UK 2004 Sowden UK 1991

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 62 Efficacy and safety of systemic treatments for moderate-to-severe atopic dermatitis Mean change in Mean change baseline from TCS 2 weeks to Mean change in Mean change disease severity( six defined from body sites) 8 baseline to weeks Mean change in Mean change disease 20 area from severity 10 baseline to days Mean change Mean change from in TBSA 6 baseline to weeks Mean change in Mean change baseline from TCS 12 weeks to Mean change in Mean change disease severity( six defined from body sites) 52 baseline to weeks Mean change in Mean change baseline from TCS 12 weeks to 7 ; 16 weeks 8 ; 5 weeks 9 ; 41 weeks 6 6 weeks; 6 weeks 6 weeks; 12 weeks; 12 weeks 12 weeks; 2 weeks 2 weeks 52 weeks; 52 weeks 52 weeks; 10 days 12 weeks; 12 weeks 12 weeks; 2x8 weeks 2x8 weeks 14 20 38 7 20 5 14 9 5 40 20 16 7 18 10 3 23 20 40 7 20 5 38 23 5 43 21 40 7 20 10 Anti histamines Anti Topical steroid, steroid, Topical Systemic antibiotics, Acetaminophen Anti histamines Anti Topical steroids, steroids, Topical histamines Anti Topical sterois Topical Prednison 10 Prednison mg/day( 2 with patients asthma) Acetaminophen Systemic Systemic antibiotics steroids Topical A: Cyclosporin 5 mg/kg;A: Cyclosporin OD B: Placebo, BID B: Placebo, 50 ug/m2;A: INF-y OD B: Neoral (new formulation of formulation (new B: Neoral 4-4,5 mg/kg;Cyclosporin) BID A: Flunosolide 0.64 mg/day 2 years) (age BID 1.2 mg/day 3–6 years); (age B: Placebo; OD 3 mg/kg/perA: Cyclosporin day, 5 mg/kg to increasing per day B: Placebo; OD 5 mg/kg;A: Cyclosporin OD B: Placebo, OD B: Placebo, 1.5 × 106 IU/m3; 3 /week A: INF-y B: Cyclosporin 5 mg/kg/per day, 5 mg/kg/perB: Cyclosporin day, 3 mg/kg to decreasing per day of A: Sandimmun (old formulation 4-4,5 mg/kg;Cyclosporin) BID B: INF-Y 0.5 × 106 IU/m3; 3 /week B: INF-Y C: Placebo ; 3 × /week P P C P C P C van Joost van Holland 1994 Hanifin USA 1993 Rosa, la Rosa, Italy 1995 Zonneveld Holland 1996 Wahlgren Sweden 1990 Jang Korea 2000 Zurbriggen Switzerland 1999

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 63 Chapter 3 Mean change in Mean change from SCORAD 12 baseline to weeks Mean change in Mean change from SCORAD 4 baseline to weeks Mean change in Mean change from SASSAD 12 baseline to weeks Mean change in Mean change from SCORAD 12 baseline to weeks Mean change in Mean change from SCORAD 6 baseline to weeks Mean change in Mean change from SCORAD baseline to 4weeks 3x in 12 weeks; 36 3x in 12 weeks; weeks 1 cycle for 2 for 1 cycle consecutive at days(evaluation 12 30); (90 days) day weeks 1 day; 24 weeks 1 day; 12 weeks; 24 weeks 12 weeks; 6 weeks; 6 weeks 6 weeks; 4 weeks; 4 weeks 4 weeks; 25 8 4 5 52 54 49 19 19 16 16 16 15 30 10 4 5 58 56 52 20 22 16 16 16 15 Acetaminophen Topical steroids Topical Topical steroids Topical Topical steroids, steroids, Topical histamines Anti Prednisolone Prednisolone of (in case exacerbation) drugs Intranasal or eyedrops (including corticosteroids) None A: IVIG 2.0 g/kg 3 body weight/month; × /week B: Placebo (topical moisturizing lotion, moisturizing B: Placebo (topical × antihistamines);3 1% hydrocortisone, /week 1 g/kgA: IVIG 8-h infusion -1 daily ( days consecutive two group) immediate B: IVIG 8-h infusion 1 g/kg-1B: IVIG 8-h infusion daily ( 30/ day at within 2 days group) delayed 31 (SRP299) 0.1mg, one A: M.vaccae injection intradermal A: M.vaccae (SRP299) 1 mg, one A: M.vaccae injection intradermal B: placebo, one intradermal injection one intradermal B: placebo, 10-22.5 mg/wk, 2.5 to A: Methotrexate per 5 mg per scheduled visit; one day per week 5 mg; one day /Folate week B: Azathioprine 1.5-2.5 mg/kg,B: Azathioprine 0.5 mg/ kg/d 2.5 mg/kg/d until reached; OD was 10 mg/day OD/ Placebo A: Monteluklast BID gel TID/ Non greasy B: Cetrizine 10 mg BID /Claritromycin 10 mg BID /Claritromycin B: Cetrizine or 250 mg BID/ momentasone OD/Hydrating methylprednisolone OD preparations topical 10mg/day 14), (age:>= A: Monteluklast 5mg/day 6-14); OD (age: B: Anti histamines 0,10%; NR/ histamines B: Anti ;NR Hydrocortison P P P P P P Jee Korea 2011 Paul France 2002 Berth-Jones UK/Croatia 2006 Schram Holland 2011 Capella Italy 2001 Rahman Bangladesh 2006

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 64 Efficacy and safety of systemic treatments for moderate-to-severe atopic dermatitis Mean change in Mean change disease 20 area score severity baseline to from 4 weeks Mean change from in EASI 12 baseline to weeks Mean change in Mean change disease severity( defined eight from body sites) 4 baseline to weeks Mean change in geometric for mean score erythema from 8 baseline to weeks Mean change in Mean change from SCORAD 12 baseline to weeks Mean change in Mean change EASI Validated baseline to from 4 weeks Mean change Severity in Total from Score 12 baseline to weeks 14 12 ; 18 weeks 13 4 weeks; 8 weeks 4 weeks; 24 weeks 12 weeks; 4 weeks; 4weeks 4 weeks; 16 weeks 12 weeks; 8 weeks; 20 weeks 20 weeks 8 weeks; 12 weeks 4 weeks; 10 weeks 4 weeks; 17 17 17 17 18 18 NR NR 21 18 17 (16) 21 (19) 16 (14) 6 17 43 14 26 20 (16) 17 42 5 3 18 18 29 20 NR 24 26 27 9 NR 20 43 20 30 26 19 42 6 None steroids, Topical histamines Anti Anti histamines Anti Topical steroids, steroids, Topical Topical steroids Topical Topical steroids Topical steroids, Topical histamines Anti Topical steroids Topical A: Monteluklast 10 mg/day; OD A: Monteluklast weekly 50 mg; 3 times A: Thymopentin B: Rotacaps + nasal aerosos; 4 times daily + nasal aerosos; B: Rotacaps 20 mg; BID A: Primecrolimus A: Primecrolimus 30 mg; BID A: Primecrolimus B: Placebo; BID B: Placebo; OD + nasal beclomethasone A: oral 0,8 mg v 0.4 mg/day; 4 diproprionate times daily no of inert plant B: Placebo mixture to AD; NR benefit known B: Placebo; TD 3 capsules of 10 formulation Standardized A: TCHM NR (Zemaphyte) herbs B: Placebo; OD 10 mg; BID A: Primecrolimus weekly B: Placebo; 3 times of 5 formulation Standardized A: TCHM TD 3 capsules herbs; A: Monteluklast 5 mg/day; OD A: Monteluklast P P C C P P C

Veien Denmark 2005 Wolff Austria/ Belgium/ Canada/ Finland/ Poland/UK/ USA 2004 Stiller USA 1994 Heddle UK 1984 Sheehan UK 1992 Pei China 2001 Hon China 2007

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 65 Chapter 3 Mean change in Mean change Lesion Clinical from Score 8 baseline to weeks Mean change Mean change in geometric for mean score erythema from 8 baseline to weeks 15 8 weeks; 20 weeks 20 weeks 8 weeks; 12 weeks 8 weeks; Nr NR 35 16 16 47 NR NR Topical steroids Topical None A: Xiao-Feng-San standardized standardized A: Xiao-Feng-San dose materials, of plant formulation TID depending on age; A: TCHM Standardized formulation of 10 formulation Standardized A: TCHM ; NR (Zemaphyte) herbs B: Placebo mixture of inert plant no of inert plant B: Placebo mixture to AD; NR benefit known P C Cheng Taiwan 2010 Sheehan UK 1992 * All studies were RCTs as defined in inclusion criteria for this systematic review systematic for this in inclusion criteria as defined RCTs were * All studies is the 2; based on which outcome as primairy outcome, by the authors is mentioned 1. what criteria: following to the according ** Selection of primary outcome calculation sample size patients, of run-in phase with 5mg CsA 55 weeks stopped, 2; plus 6 was treatment or was given dose effective last 2 weeks 1; last washout weeks by 2-3 wash-out period, 6;followed weeks 2 of run-in, 5; plus a preceding regimens (phase II), 4; plus 7 days of dose reduction 3; including 10 weeks of the administration period separated washout 9; One week treatments, between 8; no wash-out wash-out, 2-3 weeks by seperated 7; treatments cross-over, before wash-out, weeks run-in, 14; including 4 weeks wash-out, 13; plus 2 weeks 12; including 2 or continued, switched stopped al, 11; pt et 10; see Stevens each treatment, patients more breckets, in treatmentphase of completion RCT), 17; completed (47 of which 37 all participants for 16; number only given wash-out, 15; including 4 weeks RCT) completed (27 of which 26 all participants for 18; number only given treatment, completetd than have follow-up entered

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 66 Efficacy and safety of systemic treatments for moderate-to-severe atopic dermatitis ** Baseline characteristics Baseline characteristics placebo a) Placebo b) N randomized b) N randomized c) Mean age; female (%) female c) Mean age; d) Mean severity at at d) Mean severity baseline a) Placebo b) NR c) NR; NR 33.6 d) SASSAD a) Placebo b) 21 c) 36:20% 32.7 d) SASSAD

** Baseline characteristics intervention intervention characteristics Baseline II (III) a) Intervention b) N randomized b) N randomized c) Mean age; female (%) female c) Mean age; d) Mean severity at baseline at d) Mean severity a) IVIG b) 6 c) 6.44; 50% 79* d) SCORAD a) CsA 300mg b) 53 c) NR; NR 60.7 d) TBSA a) UVAB b) 36 c) 33.2;58% 46.8 d) SCORAD 3 ** Baseline characteristics intervention intervention characteristics Baseline I a) Intervention b) N randomized b) N randomized c) Mean age; female (%) female c) Mean age; d) Mean severity at baseline at d) Mean severity a) AZA a) AZA a) CsA b) NR c) NR; NR 39.7 d) SASSAD b) 42 c) 30; 54% 32.3 d) SASSAD b) 8 c) 11.91; 50% 79* d) SCORAD a) CsA 150mg b) 53 c) NR; NR 59.0 d) TBSA a) CsA b) 36 c)33.3; 42% 48.5 d) SCORAD Severity of eczema of eczema Severity (definition) Severe (Inadequately (Inadequately Severe topical by controlled by not defined treatment, means of score) (Active Moderate-to-severe optimum disease despite SASSAD therapy, topical =>10) respond to (Failed Severe line and second first to > 70) SCORAD therapy, Severe (Refractory to to (Refractory Severe therapies, conventional >=30) TBSA NR (7-9 Rajka & Langeland) NR (7-9 Rajka Inclusion criteria Age ≥ 16 years 16-65 years NR (Children) ≥ 18 years 18 -70 years 18 -70 years Reference Berth-Jones Meggit Bemanian Czech Granlund Online Repository 5 Online Repository inclusion and baseline characteristics E3: Summary of study Table

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a) Placebo b) 12 c) NR; NR 19.2 eczema of active d) Area a) Placebo b) 16 c) 29; 37.5% d) NR a) Placebo b) 16 c) 29; 37.5% ( six d) Disease severity 46* body sites) defined a) Placebo b) 23 c) 31; 52.2 54 d) TBSA a) MMF b) 24 c)36.2; 45.8% 24.0 d) obj. SCORAD a) CsA, continuous b) 19 c) 10; NR 50 d) SASSAD a) Tacrolimus b) 15 c) 27.1; 66.7% 69.0 d)SCORAD a) Prednisolon/placebo b) 17 c)28.8; 47.6% 57.6 d) SCORAD a) CsA b) 26 c) 36.9;34.6% 20.5 d) obj. SCORAD a) CsA, short course b) 21 c) 10.1; NR 52 d) SASSAD a) CsA a) CsA b) 12 c) NR; NR 19.2 eczema of active d)Area b) 15 c) 26.6; 73.3% 73.7 d)SCORAD a) CsA a) CsA b) 17 c) 30; 35% d) NR b) 21 c) 30.1; 41.2% 54.7 d) SCORAD a) CsA a) CsA b) 17 c) 30; 35.3% body ( six defined d) Disease severity 40 * sites) b) 23 c) 31.6; 47.8% 57 d) TBSA Severe (Inadequately (Inadequately Severe topical by controlled by not defined treatment, means of score) Severe (Refractory to topical topical to (Refractory Severe by not defined steroids, means of score) Severe (Not defined by (Not defined Severe means of score) to-severe Moderate- controlled (Inadequately defined steroids, topical by and Raijka criteria by Langeland) Severe (Resistant to to (Resistant Severe not therapy, conventional by means of score) defined NR (Not be adequately topical by controlled and corticosteroids calcineurin topical by objective inhibitors, > > 40 and DLQI SCORAD 10) Severe (Inadequately (Inadequately Severe conventional by controlled by not defined therapies, means of score) other to (Resistant Severe by defined therapiesnot means of score) ≥ 18 years 2-16 years 2-16 years NR (Adults) NR (Adults + children) NR (Adults + children) 18-55 years NR (Adults) 17-68 years Haeck Harper Munro Pacor Salek Schmitt Sowden Joost van

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 68 Efficacy and safety of systemic treatments for moderate-to-severe atopic dermatitis a) Placebo b) NR c) NR; NR chart 23* severity d) 20 area

a) Placebo b) 10 c) NR; NR 75* d) TCS a) Placebo b) 43 c) 28.2; 47% 12.2 d) TCS a) Placebo b) 10 c) 21; 65% d) TCS 18.4 d) TCS a) Placebo b) 10 c) 6.5; 60% 42.1 d) SCORAD

a) Placebo b) 12 c) NR; NR 19.2 eczema of active d) Area a) Placebo b) 16 c) 29; 37.5% d) NR a) Placebo b) 16 c) 29; 37.5% ( six d) Disease severity 46* body sites) defined a) Placebo b) 23 c) 31; 52.2 54 d) TBSA dose a) CsA, decreasing b) 40 c) NR; NR 49.7 score severity d) Mean eczema of CsA) formulation (new a) Neoral b) 7 c) 32; 42.9% body ( six defined d) Disease severity 43 sites) a) INF 0.5 × 106 IU/m4 b) 20 c) 23; 71.4% d) TCS 20.1 d) TCS a) IVIG delayed b) 5 c) 27; 60% 76 d) SCORAD 3 a) MMF b) 24 c)36.2; 45.8% 24.0 d) obj. SCORAD a) CsA, continuous b) 19 c) 10; NR 50 d) SASSAD a) Tacrolimus b) 15 c) 27.1; 66.7% 69.0 d)SCORAD a) Prednisolon/placebo b) 17 c)28.8; 47.6% 57.6 d) SCORAD a) CsA dose a) CsA, increasing b) NR c) NR; NR chart 24* severity d) 20 area b) 38 c) NR; NR 48.5 score severity d) Mean eczema of a) Sandimmun (old formulation CsA) b) 7 c) 38; 28.6% body ( six defined d) Disease severity 55 sites) a) Flunosolide a) INF a) INF 1.5 × 106 IU/m3 b) 10 c) NR; NR 76* d) TCS b) 40 c) 37.2; 35% 12.7 d) TCS b) 21 c) 21; 70% d) TCS 19.4 d) TCS a) IVIG a) IVIG b) 30 c)6.3 ; 40% 61.5 d) SCORAD b) 4 c) 29; 20% 75 d) SCORAD a) CsA b) 26 c) 36.9;34.6% 20.5 d) obj. SCORAD a) CsA, short course b) 21 c) 10.1; NR 52 d) SASSAD a) CsA a) CsA b) 12 c) NR; NR 19.2 eczema of active d)Area b) 15 c) 26.6; 73.3% 73.7 d)SCORAD a) CsA a) CsA b) 17 c) 30; 35% d) NR b) 21 c) 30.1; 41.2% 54.7 d) SCORAD a) CsA a) CsA b) 17 c) 30; 35.3% body ( six defined d) Disease severity 40 * sites) b) 23 c) 31.6; 47.8% 57 d) TBSA Moderate-to-severe (Not Moderate-to-severe by means of score) defined to (Resistant Severe and/ therapy conventional disabling or significantly by means AE, not defined of score) Severe (Not defined by (Not defined Severe means of score) Severe (Inadequately (Inadequately Severe topical by controlled by not defined steroids, means of score) Severe(Inadequately conventional by controlled by not defined therapies, means of score) (Inadequately Severe conventional by controlled a least involved therapies, 20% of the body) Moderate-to-severe Moderate-to-severe controlled (Inadequately , therapies conventional by >30% of the body surface) (Inadequately Severe conventional by controlled > 50) SCORAD therapies, Severe (Inadequately (Inadequately Severe topical by controlled by not defined treatment, means of score) Severe (Refractory to topical topical to (Refractory Severe by not defined steroids, means of score) Severe (Not defined by (Not defined Severe means of score) to-severe Moderate- controlled (Inadequately defined steroids, topical by and Raijka criteria by Langeland) Severe (Resistant to to (Resistant Severe not therapy, conventional by means of score) defined NR (Not be adequately topical by controlled and corticosteroids calcineurin topical by objective inhibitors, > > 40 and DLQI SCORAD 10) Severe (Inadequately (Inadequately Severe conventional by controlled by not defined therapies, means of score) other to (Resistant Severe by defined therapiesnot means of score) NR (Adults) NR (Adults) NR (Adults) NR (Children) 2-65 years > 15 years > 2 years 18-50 years ≥ 18 years 2-16 years 2-16 years NR (Adults) NR (Adults + children) NR (Adults + children) 18-55 years NR (Adults) 17-68 years Wahlgren Zonneveld Zurbriggen Rosa, la Rosa, Hanifin Jang Jee Paul Haeck Harper Munro Pacor Salek Schmitt Sowden Joost van

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a) Placebo b) 30 c) 30; 70% 9.5 score EASI d) validated a) Placebo a) Rotacaps + nasal aerosos a) Rotacaps a) M vaccae high dose a) M vaccae b) NR c) NR; NR d) NR b) 56 c) 9.6; (54%) d) SASSAD 33 d) SASSAD a) AZA b) 22 c) 37; 45% 58.4 d) SCORAD a)Cetrizine/Claritromycin/ or momentasone methylprednisolone/Hydrating preparations topical b) 16 c) 30.1; 31% 48.2 d) SCORAD Hydrocortison histamines/ a) Anti b) 15 c) 18; 33% 53.31 d) SCORAD 2 a) oral + nasal beclomethasone + nasal beclomethasone a) oral diproprionate a) M vaccae low dose low a) M vaccae c) 9.67; 60% (six signs score d)Disease severity of AD) 78 b) NR c) 6.5; 48.1% d) NR b) 58 c) 9.3; ( 47%) d) SASSAD 30 d) SASSAD a) MTX/ Folate b) 20 c) 43; 50% 57.2 d) SCORAD Placebo/ Non a) Monteluklast/ gel greasy b) 16 c) 28.9; 56% 49.1 d) SCORAD a) Monteluklast b) 16 c) 19.81; 56% 52.70 d) SCORAD a) Monteluklast a) Monteluklast b) 29 c) 28; 75.9 % 8.9 score EASI d) validated b) 6 Moderate-to- severe(Inadequately conventional by controlled by , not defined therapies means of score) Severe (SASSAD >= 20) (SASSAD Severe Severe (Unresponsive, (Unresponsive, Severe or contraindicated, cyclosporin to intolerant and Raijka treatment, Langeland) Moderate to severe severe to Moderate controlled (Inadequately , treatments previous by >= 30) SCORAD >= 30) NR (SCORAD Moderate-to-severe Moderate-to-severe 4,5, Rajka (Minimal score ) and Langeland Moderate-to-severe by controlled (Inadequately , six therapies conventional signs of AD >40) NR (Adults) 5-16 years ≥ 18 years ≥ 18 years ≥ 6 years 16-70 years NR (Children) Heddle Berth-Jones Schram Capella Rahmen Veien Pei

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 70 Efficacy and safety of systemic treatments for moderate-to-severe atopic dermatitis a) Placebo b) 27 c) 37; 54% d) EASI 23.7 d) EASI a) Placebo b) 19 c) 38; 22.9% 6.32 score severity d) Total a) Placebo b) 43 c) 11.7; 46.5% 56.9 d) SCORAD a) Placebo b) 20 c) NR; NR d) NR a) Placebo b) NR c) NR; NR for mean score d) Geometric damage erythema/surface 110/108 a) Placebo b) 24 c) 13.6; 50% lesion score/ d) Clinical Erythema score/Surface 120/44/84 score damage

3 a) Pimecrolimus 20mg a) Pimecrolimus 30mg) (Pimecrolimus b) 24(26) c) 35 (37); 58% (69%) d) EASI 20.7 (23.6) d) EASI a) Pimecrolimus 10mg a) Pimecrolimus b) 26 c) 35; 35 % d) EASI 24.7 d) EASI a) TP-5 a) TCHM a) TCHM a) TCHM a) XFS b) 20 c) 42; 25.7% 6.06 score severity d) Total b) 42 c) 11.7; 45.2% 58.3 d) SCORAD b) 20 c) NR; NR d) NR b) NR c) NR; NR for mean score d) Geometric 120/ 121 damage erythema/surface b) 47 c) 12.2; 46.8% lesion score/Erythema d) Clinical score damage score/Surface 142/42/90.5 Moderate-to-severe (Not Moderate-to-severe by means of score) defined Severe (>20% cutaneous (>20% cutaneous Severe surface) to-severe Moderate- >15) SCORAD (objective and NR (Extensive disease, > 20% refractory BSA) to NR (Resistant and therapies, conventional a warrant enough to severe treatment) trial of a novel (Inadequately Severe conventional by controlled , > 20% of the therapies area) body surface NR (Adults) > 2 years 5-21 years 16-65 years NR (Adults + children) NR (Adults + children) Wolff Stiller Hon Sheehan Sheehan Cheng * Taken out of Figure ** Defined by named atopic-specific score considering the severity of clinical signs of clinical severity considering the score atopic-specific by named ** Defined out of Figure * Taken RCT completed who patients for 11 given only 2 Baseline characteristics RCT; completed who for patients only given 1 Baseline characteristics

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Online Repository 6 Figure E2 –Flow chart: Identification of relevant RCTs for inclusion in the systematic review (*) number of times discussed in selected RCTs

Online Repository 7

Figure E3: Risk of bias graph: review authors’ judgments about each risk of bias item presented as percentages across all included RCTs

Random sequence generation (selection bias)

Allocation concealment (selection bias)

Blinding of participants and personnel (performance bias)

Incomplete outcome data (attrition bias)

Selective reporting (reporting bias)

Other bias

Conflict of interest author

Conflict of interest sponsor

0% 25% 50% 75% 100%

Low risk of bias Unclear risk of bias High risk of bias

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 72 Efficacy and safety of systemic treatments for moderate-to-severe atopic dermatitis a) Placebo Efficacy placebo Efficacy signs in clinical b) Change in quality of life c) Change a) Placebo 8 weeks at of 3% in SASSAD b) Mean improvement work daytime VAS of 20%* in c) Mean deterioration activity a) Placebo 12 weeks at in of 20% SASSAD b) Mean improvement 8 weeks at of 26% in DLQI c) Mean improvement

3 Efficacy drug B Efficacy a) Intervention signs in clinical b) Change in quality of life c) Change

a) IVIG of 30% in SCORAD b) Mean improvement 12 weeks at c) NR a) CsA 300mg at of 59% in TBSA b) Mean improvement 8 weeks at of 59% in DLQI c) Mean improvement 8 weeks a) UVAB of 34% in SCORAD b) Mean improvement 8 weeks at 8 of 39% in EDI at c) Mean improvement weeks Efficacy drug A Efficacy a) Intervention signs in clinical b) Change in quality of life c) Change a) AZA at of 26% in SASSAD b) Mean improvement 8 weeks work of 55%* in VAS c) Mean improvement weeks at 8 activity daytime a) AZA in of 37% SASSAD b) Mean improvement 12 weeks at 8 at of 61% in DLQI c) Mean improvement weeks a) CsA at of 68% in SCORAD b) Mean improvement 12 weeks c) NR a) CsA 150mg 8 at of 48% in TBSA b) Mean improvement weeks 8 at of 41% in DLQI c) Mean improvement weeks a) CsA at of 54% in SCORAD b) Mean improvement 8 weeks 8 of 56% in EDI at c) Mean improvement weeks Summary of efficacy of systemic treatments for moderate-to-severe AD in the RCTs included RCTs AD in the for moderate-to-severe treatments systemic of Summary of efficacy Reference Berth-Jones Meggit Bemanian Czech Granlund Online Repository 8 Online Repository E4 Table

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a) Placebo 8 weeks at of 19% in score** b) Mean improvement c) NR

a) Placebo of disease) extent b) NR (Disease activity and 8 weeks of 21% in EDI at c) Mean improvement

at 1 c) Mean improvement of 71% in DLQI c) Mean improvement 6 weeks

a) MMF of 0% in SCORAD b) Mean improvement and phase 0-30 weeks the maintenance at including in SCORAD 39% improvement 30 to (-6 weeks run in phase of 6 weeks weeks) c) NR a) CsA, continuous of 56% in SASSAD b) Mean improvement 52 weeks at c) NR (DLQI)

a) Tacrolimus of 89% in Area b) Mean improvement 6 weeks at Curve Under the SCORAD c) NR

a) Prednisolon/placebo of 43% in objective b) Mean improvement 6 weeks at SCORAD50 at 6 at 1 weeks a) CsA at of 53% in score** b) Mean improvement 8 weeks c) NR a) CsA of 17% in SCORAD ib) Mean Improvement and phase 0-30 weeks the maintenance at including run in in SCORAD 42% improvement 30 weeks) to (-6 weeks phase of 6 weeks c) NR a) CsA, short course at of 42% in SASSAD b) Mean improvement 52 weeks c)NR(DLQI) a) CsA at of 95% in score** b) Mean improvement 8 weeks c) NR a) CsA Under of 88% in Area b) Mean improvement 6 weeks at Curve the SCORAD c) NR a) CsA of disease) extent b) NR (Disease activity and 8 of 52% in EDI at c) Mean improvement weeks a) CsA of 55% in objective b) Mean improvement 6 weeks at SCORAD50 of 76% in DLQI c) Mean improvement Sowden Haeck Harper Munro Pacor Salek Schmitt

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 74 Efficacy and safety of systemic treatments for moderate-to-severe atopic dermatitis a) Placebo 6 weeks at of 11% in TBSA b) Mean improvement c) NR a) Placebo at 10 days of 4% in score** b) Mean deterioration c) NR

a)Placebo 2 weeks at of 16*% in TCS b) Mean improvement c) NR a) Placebo b) NR c) NR a)Placebo 12 weeks at 2*% in TCS b) Mean improvement c) NR a)Placebo 12 weeks at of 40% in SCORAD b) Mean improvement c) NR

a) CsA, decreasing dose a) CsA, decreasing of 29% in score** b) Mean improvement 52 weeks at c) NR a) Neoral (new formulation of CsA) formulation (new a) Neoral of 72% in score** b) Mean improvement 8 weeks at c) NR

a) INF 0.5 × 106 IU/m4 12 at 36*% in TCS b) Mean improvement weeks c) NR

a) IVIG delayed of 15% in SCORAD b) Mean improvement 4 weeks at c) NR a) CsA 6 at of 56% in TBSA b) Mean improvement weeks c) NR a) CsA at of 58% in score** b) Mean improvement 10 days c) NR a) CsA increasing dose a) CsA increasing at of 46% in score** b) Mean improvement 52 weeks c) NR a) Sandimmun (old formulation of CsA) a) Sandimmun (old formulation at of 73% in score** b) Mean improvement 8 weeks c) NR a) Flunosolide 2 at of 54*% in TCS b) Mean improvement weeks c) NR a) INF than in is greater in TCS b) Improvement weeks at 12 but not significant placebo group c) NR a) INF 1.5 × 106 IU/m3 12 at 48*% in TCS b) Mean improvement weeks c) NR a) IVIG at of 24% in SCORAD b) Mean improvement 12 weeks c) NR a) IVIG at of 15% in SCORAD b) Mean improvement 4 weeks c) NR van Joost van Wahlgren Zonneveld Zurbriggen Rosa, la Rosa, Hanifin Jang Jee Paul

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a)Placebo 4 at EASI of 20% in validated b) Mean improvement weeks c) NR a)Placebo 4 weeks at of 33% in score** b) Mean improvement c)NR(CDLQI) a) M vaccae high dose a) M vaccae of 26% in SASSAD b) Mean improvement 12 weeks at c) NR + nasal aerosos a) Rotacaps b) NR c) NR a) AZA of 39% in SCORAD b) Mean improvement 12 weeks at of 20% in SKINDEX c) Mean improvement 12 weeks at a) Cetrizine/Claritromycin/ momentasone momentasone a) Cetrizine/Claritromycin/ topical or methylprednisolone/Hydrating preparations of 50% in SCORAD b) Mean improvement 6 weeks at c) NR a) Anti histamines/ Hydrocortison histamines/ a) Anti of 9% in SCORAD b) Mean improvement 4 weeks at c) NR

a) M vaccae low dose low a) M vaccae at of 25% in SASSAD b) Mean improvement 12 weeks c) NR diproprionate + nasal beclomethasone a) oral on improvements greater b) significantly beclomethasone 4 weeks than on placebo at diproprionate c)NR a) MTX/ Folate at of 42% in SCORAD b) Mean improvement 12 weeks of 26% in SKINDEX at c) Mean improvement 12 weeks a) Monteluklast/ Placebo/ Non greasy gel Placebo/ Non greasy a) Monteluklast/ at of 51% in SCORAD b) Mean improvement 6 weeks c) NR a) Monteluklast at of 29% in SCORAD b) Mean improvement 4 weeks c) NR a) Monteluklast of 24% in validated b) Mean improvement 4 weeks at EASI c)NR a) Monteluklast at of 42% in score** b) Mean improvement 4 weeks c)NR(CDLQI) Berth-Jones Heddle Schram Capella Rahmen Veien Pei

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 76 Efficacy and safety of systemic treatments for moderate-to-severe atopic dermatitis a)Placebo 12 weeks at of 20% in EASI b) Mean improvement c)NR a)Placebo at Score Severity of 14% in Total b) Mean improvement 12 weeks c)NR a)Placebo 12 weeks at of 19% in SCORAD b) Mean improvement 12 weeks at of 2% in DLQI C) Mean improvement a)Placebo 8 weeks at of 20% in score** b) Mean improvement c) NR a)Placebo 8 weeks at of 11% in score** b) Mean improvement c)NR a)Placebo 8 weeks at of 11% in score** b) Mean improvement c)NR

3 a) Pimecrolimus 20mg (Pimecrolimus 20mg (Pimecrolimus a) Pimecrolimus 30mg) at of 35% in EASI b) Mean improvement of 47% in (Mean improvement 12 weeks 12 weeks) at EASI c)NR

a) Pimecrolimus 10mg a) Pimecrolimus 12 at of 21% in EASI b) Mean improvement weeks c)NR a) TP-5 of 21% in Total b) Mean improvement 12 weeks at Score Severity c)NR a) TCHM at of 15% in SCORAD b) Mean improvement 12 weeks 12 at of 32% in DLQI C) Mean improvement weeks a) TCHM at of 82% in score** b) Mean improvement 8 weeks c) NR a) TCM at of 60% in score** b) Mean improvement 8 weeks c)NR a) XFS at of 56% in score** b) Mean improvement 8 weeks c)NR Wolff Stiller Hon Sheehan Sheehan Cheng * Values taken out of Figure/graph ** Non validated score. ** Non validated out of Figure/graph taken * Values in DLQI improvement) relevant (i.e. minimum improvement of people with ≥ 5 point 1. proportion Area, Six Sign Six SASSAD Dermatitis; Atopic SCOring SCORAD Index; and Severity Area Eczema EASI disability index; EDI Eczema Index; Quality of Life DQLI Dermatological scale VAS Visual analog Profile; Sickness Impact Kingdom UKSIP United Severity; Clinical Total TCS Area; Body Surface Total TBSA Dermatitis; Atopic

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 77 Chapter 3 2 2 0% 0% 0.% NR NR 0.3% 0.3% 0.1% 0.0% 0.0% 0.0% 0.4% 0.1% 0.0% 0.0% 0.0% 0.7% 0.2% 0.0% 0.0% 2.0% 3.6% Withdrawals Withdrawals adverse due to trial (total events (n; duration)any per incidence rate week*) patient 3 3 NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR 2.1% 2.8% 4.7% 5.9% Related Adverse Adverse Related trial (total events (n; any dration); per incidence rate week*) patient 1 1 NR NR NR NR 7.4% 4.5% 7.9% 5.0% 8.1% 2.2% 3.0% 3.0% 7.4% 5.6% 4.0% 2.1% 2.8% 3.1% 17.7% 21.4% 28.2% 20.4% Total Adverse Adverse Total events trial (total duration); (n; any incidence rate per patient week*) 6 6 NR NR NR NR NR NR NR NR 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% NR NR 0.7% Related Serious Related events adverse trial (total (n; any duration); per incidence rate week*) patient 5 5 NR NR NR NR 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% NR NR 0.4% 0.8% 0.0% 0.0% 0.0% 0.5% Total Serious adverse Serious adverse Total trial (total events (n; any duration); per incidence rate week*) patient CsA Short course CsA MMF CsA CsA UVAB CsA Placebo CsA Tacrolimus Placebo CsA Treatment 1 Treatment 2 Treatment AZA AZA Placebo CsA IVIG CsA150 mg CsA 300 mg Placebo CsA Continiously Prednison

NR NR 0% 0.6% 0.5% 0.6% 0.0% 0.2% 1.4% 1.1% 0.0% 2.4% 6.1% 1.5% 0.0% 0.7% 0.6% 3.9%

Overall withdrawals withdrawals Overall reasons any due to trial duration) (total (n; incidence rate week*) per patient 4 Harper Haeck Salek Bemanian Chech Granlund Munro Pacor Schmitt Author Berth-Johnes Meggit Online Repository 9 Online Repository appraised AD in the RCTs moderate-to-severe for of systemics E5 Summary of safety/tolerability Table

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0 0 NR NR NR NR NR NR 0% 0% 0% 0% 0% 0% 0% 0% 0.4% 0.2% 0.2% 0.1% 0.2% 0.5% 0.8% 0.4% 1.0% 0.0% 0.3% NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR 0% 0% 0% 2.9% 2.3% 0.0% 0.0% 2.7% 2.0% 10.9% Unclear NR NR NR NR NR NR NR NR NR 0% 0% 0.0% 0.0% 2.5% 9.3% 4.5% 9.8% 0.6% 6.5% 1.0% 0.5% 8.2% 9.5% 23.5% 22.9% 11.5% 20.3% 16.3% NR NR NR NR NR NR NR NR NR 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.1% 3 NR NR NR NR NR 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0.0% 0.0% 0.3% 0.0% 0.2% 0.2% 0.6% 2.2% 2.2% 0.1% Monteluklast Cetrizine/ Claritromycin/ or momentasone methylprednisolone/ topical Hydrating preparations MTX AZA CsA dose low M.vaccae high dose M.vaccae Placebo Placebo IVIG CsA Placebo dose CsA increasing dose CsA decreasing Sandimmun (old of CsA) formulation (new Neoral of CsA) formulation INF INF Placebo IVIG Placebo IVIG CsA Placebo Flunosolide Placebo INF Placebo

NR 0% 0.4% 1.2% 0.3% 8.3% 1.2% 0.4% 0.5% 0.5% 0.8% 0.0% 0.5%

2006 Capella Schram Sowden Berth-Johnes Van Joost Van Zonneveld Zurbriggen la Rosa, Jang Jee Paul Wahlgren Hanifin

525295-L-bw-Roekevisch Processed on: 16-11-2018 PDF page: 79 Chapter 3 NR NR NR NR 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0.2% 0.3% 0.3% 0.2% 0.2% 0.2% NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR NR 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 7 NR NR NR NR NR NR NR NR NR NR 0.0% 0.0% 0.0% 0.0% 5.6% 6.1% 6.9% 0.5% 7.7% 0.5% 7.7% 10.8% NR NR NR 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.0% 0.3% 0.0% 0.0% 0.2% 0.0% 0.0% 0.2% 0.0% 0.0% 0.3% 0.0% 0.0% 0.0% Authors reported only the total related SAE, unclear amount per group unclear amount SAE, related only the total reported Authors 6 AE only reported up to week 8 week up to AE only reported 4 One extra withdrawal appeared, unclear which group; unclear which group; appeared, withdrawal One extra 2 Monteluklast histamines/ Anti Hydrocortison Monteluklast Placebo Monteluklast Placebo beclomethasone Oral diproprionate + nasal Rotacaps aerosos 10 mg Pimecrolimus Placebo Pimecrolimus 20 mg Pimecrolimus TCHM Pimecrolimus 30 mg Pimecrolimus Placebo Placebo Placebo TP-5 Placebo TCHM TCHM Placebo XFS 0.0% 5.1% 2.7% 0.5% 2.1% 1.1% 0.9% 0.3% 1.1% 0.4%

Authors reported only about the most frequent AE; frequent only about the most reported Authors AE; related frequent only about the most reported Authors per group; unclear amount SAE, only the total reported Authors Assuming equally distributed loss-to-follow up and constant risk for adverse event / withdrawal over time over / withdrawal event adverse risk for up and constant loss-to-follow Assuming equally distributed AE reported by more than one patient more by AE reported Rahman Veien Pei Heddle Wolff Sheehan

Stiller Hon Sheehan Cheng * 1 3 5 7

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of atopic dermatitis. Mymensingh Med J thymopentin as an adjunctive treatment 2006;15:85-8. in atopic dermatitis. J Am Acad Dermatol 32. Salek MS, Finlay AY, Luscombe DK, 1994;30:597-602. Allen BR, Berth-Jones J, Camp RD, et 39. van Joost T, Heule F, Korstanje M, van al. Cyclosporin greatly improves the den Broek MJ, Stenveld HJ, van Vloten quality of life of adults with severe atopic WA. Cyclosporin in atopic dermatitis: a dermatitis. A randomized, double-blind, multicentre placebo-controlled study. Br placebo-controlled trial. Br J Dermatol J Dermatol 1994;130:634-40. 1993;129:422-30. 40. Veien NK, Busch-Sorensen M, Stausbol- 33. Schmitt J, Schakel K, Folster-Holst R, Bauer Gron B. Montelukast treatment of A, Oertel R, Augustin M, et al. Prednisolone moderate to severe atopic dermatitis vs. ciclosporin for severe adult eczema. in adults: a randomized, double-blind, An investigator-initiated double-blind placebocontrolled trial. J Am Acad placebo-controlled multicentre trial. Br J Dermatol 2005;53:147-9. Dermatol 2010;162:661-8. 41. Wahlgren CF, Scheynius A, Hagermark O. 3 34. Schram ME, Roekevisch E, Leeflang MM, Antipruritic effect of oral cyclosporine A Bos JD, Schmitt J, Spuls PI. A randomized in atopic dermatitis. Acta Derm Venereol trial of methotrexate versus azathioprine 1990;70:323-9. for severe AD. J Allergy Clin Immunol 42. Wolff K, Fleming C, Hanifin J, Papp K, 2011;128:353-9. Reitamo S, Rustin M, et al. Efficacy and 35. Sheehan MP, Rustin MH, Atherton DJ, tolerability of three different doses of Buckley C, Harris DW, Brostoff J, et al. oral pimecrolimus in the treatment of Efficacy of traditional Chinese herbal moderate to severe atopic dermatitis: a therapy in adult atopic dermatitis [erratum randomized controlled trial. Br J Dermatol appears in Lancet 1992;340:188]. Lancet 2005;152:1296-303. 1992;340:13-7. 43. Zonneveld IM, De Rie MA, Beljaards RC, 36. Sheehan MP, Atherton DJ.Acontrolled Van Der Rhee HJ, Wuite J, Zeegelaar J, et trial of traditional Chinese medicinal al. The long-term safety and efficacy of plants in widespread non-exudative AD. cyclosporin in severe refractory atopic Br J Dermatol 1992;126:179-84. dermatitis: a comparison of two dosage 37. Sowden JM, Berth-Jones J, Ross JS, Motley regimens. Br J Dermatol 1996; 135(Suppl. RJ, Marks R, Finlay AY, et al. Double-blind, 48):15-20. controlled, crossover study of cyclosporin 44. Zurbriggen B, Wuthrich B, Cachelin AB, in adults with severe refractory atopic Wili PB, Kagi MK. Comparison of two dermatitis. Lancet 1991;338:137-40. formulations of cyclosporin A in the 38. Stiller MJ, Shupack JL, Kenny C, Jondreau treatment of severe atopic dermatitis. A L, Cohen DE, Soter NA. A doubleblind, double-blind, single-centre, cross-over placebo-controlled clinical trial to pilot study. Dermatology 1999;198:56-60. evaluate the safety and efficacy of 45. GRADE working group. Available at:

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http://www.gradeworkinggroup.org/ Cobden I, Hayes PC. Hepatotoxicity of herbal publications. Accessed October 17, 2013. remedies. BMJ 1989;299:1156-7. 46. Akdis CA, Akdis M, Bieber T, Bindslev-Jensen 54. Mostefa-Kara N, Pauwels A, Pines E, Biour C, Boguniewicz M, Eigenmann P, et al. M, Levy VG. Fatal hepatitis after herbal tea. Diagnosis and treatment of atopic dermatitis Lancet 1992;340:674. in children and adults: European Academy 55. Ring J, Alomar A, Bieber T, Deleuran M, Fink- of Allergology and Clinical Immunology/ Wagner A, Gelmetti C, et al. Guidelines for American Academy of Allergy, Asthma and treatment of AD (atopic dermatitis) part I. J Immunology/PRACTALL Consensus Report. J Eur Acad Dermatol Venereol 2012;26:1045- Allergy Clin Immunol 2006;118:152-69. 60. 47. Hanifin JM, Cooper KD, Ho VC, Kang S, 56. Saeki H, Furue M, Furukawa F, Hide M, Krafchik BR, Margolis DJ, et al. Guidelines Ohtsuki M, Katayama I, et al. Guidelines of care for atopic dermatitis, developed in for management of atopic dermatitis. J accordance with the American Academy of Dermatol 2009;36:563-77. Dermatology (AAD)/American Academy of 57. Williams HC. Clinical practice. Atopic Dermatology Association ‘‘Administrative dermatitis. N Engl J Med 2005;352: 2314-24. Regulations for Evidence-Based Clinical Practice Guidelines.’’ J Am Acad Dermatol 2004;50:391-404. 48. Leung TF, Wong KY, Wong CK, Fung KP, Lam CW, Fok TF, et al. In vitro and clinical immunomodulatory effects of a novel Pentaherbs concoction for atopic dermatitis. Br J Dermatol 2008;158:1216-23. 49. Ring J, Alomar A, Bieber T, Deleuran M, Fink- Wagner A, Gelmetti C, et al. Guidelines for treatment of AD (atopic dermatitis) Part II. J Eur Acad Dermatol Venereol 2012;26:1176- 93. 50. Schneider L, Tilles S, Lio P, Boguniewicz M, Beck L, LeBovidge J, et al. Atopic dermatitis: a practice parameter update 2012 .JAllergyClinImmunol2013;131:295-9. 51. Graham-Brown R. Toxicity of Chinese herbal remedies. Lancet 1992;340:673-4. 52. Hon KL, Chan BC, Leung PC. Chinese herbal medicine research in eczema treatment. Chin Med 2011;6:17. 53. MacGregor FB, Abernethy VE, Dahabra S,

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E1. Schram ME, Roekevisch E, Leeflang MM, Bos JD, Schmitt J, Spuls PI. A randomized trialof methotrexate versus azathioprine for severe AD. J Allergy Clin Immunol 2011;128:353-9. E2. Rajka G, Langeland T. Grading of the severity of atopic dermatitis. Acta Derm Venereol Suppl (Stockh) 1989;144:13-4. Brenninkmeijer EE, Schram ME, Leeflang MM, Bos JD, Spuls PI. Diagnostic criteria for atopic dermatitis: a systematic review. Br J Dermatol 2008;158:754-65. E4. Severity scoring of atopic dermatitis: the SCORAD index. Consensus Report of the European Task Force on Atopic Dermatitis. Dermatology (Basel, Switzerland) 1993;186:23-31. E5. Hanifin JM, Thurston M, Omoto M, Cherill R, Tofte SJ, Graeber M. The eczema area and severity index (EASI): assessment of reliability in atopic dermatitis. EASI Evaluator Group. Exp Dermatol 2001;10:11-8. E6. Charman CR, Venn AJ, Williams HC. The patient-oriented eczema measure: development and initial validation of a new tool for measuring AD severity from the patients’ perspective. Arch Dermatol 2004;140:1513-9. E7. Nijsten TE, Sampogna F, Chren MM, Abeni DD. Testing and reducing skindex-29 using Rasch analysis: Skindex-17. J Invest Dermatol 2006;126:1244-50. 3 E8. National Cancer Institute (U.S.). Common terminology criteria for adverse events (CTCAE), Rev. U.S. Dept. of Health and Human Services, National Institutes of Health, National Cancer Institute; 2009.

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Schram ME, Roekevisch E, Leeflang MM, Bos JD, Schmitt J, Spuls PI. Journal of Allergy and Clinical Immunology (JACI). 2011 Aug;128(2):353-9

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Abstract

Background Patients with severe atopic eczema/dermatitis (AD) frequently require systemic treatment to control their disease. Methotrexate and azathioprine are proposed as off-label treatment options, but direct comparisons are lacking.

Objectives We sought to compare the efficacy and safety of methotrexate versus azathioprine in adults with severe AD.

Methods Patients with severe AD were randomly assigned in a 1:1 ratio to receive either methotrexate (dosage, 10-22.5 mg/wk) or azathioprine (dosage, 1.5-2.5 mg/kg/d) for 12 weeks, followed by a 12-week follow-up period. Primary outcome was the mean change in the severity scoring of atopic dermatitis (SCORAD) index after 12 weeks. Efficacy assessors blinded for allocation of treatment were used to perform clinical outcome assessment. Analyses were done on an intention-to-treat basis.

Result Of the 45 patients screened, 42 were included. At week 12, patients in the methotrexate group had a mean relative reduction in the SCORAD index of 42% (SD, 18%) compared with 39% (SD, 25%) in the azathioprine group (P .52). Proportions of patients achieving at least mild disease and reductions on impact of quality of life, symptoms, and levels of thymus and activation-regulated chemokine (TARC) were similar in both groups at weeks 12 and 24. No statistically significant differences were found in the number and severity of adverse events. Abnormalities in blood count were more common in the azathioprine group. No serious adverse events occurred.

Conclusion Both treatments achieved clinically relevant improvement and were safe in the short term. Methotrexate and azathioprine are appropriate options for the treatment of severe AD.

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Introduction

Atopic eczema/dermatitis (AD) is a chronic inflammatory skin disorder that affects approximately 3% to 5% of the adult population in the western world.1 AD can result in impairment of skin function, poor sleep, and social stigma over a long period of time. Patients often have to bear the burden of considerable psychological comorbidity.2 Patients with severe AD require prolonged treatment with large amounts of highly potent topical corticosteroids, systemic treatment, or both. Frequently used options for systemic treatment of AD include cyclosporine and systemic corticosteroids. Although proved effective,3 a proportion of patients have a contraindication for cyclosporine or discontinue treatment because of ineffectiveness or side effects. Moreover, long-term use of cyclosporine raises concerns over (nephro)toxicity.4 Systemic corticosteroids are used frequently to suppress exacerbations, although high-level evidence is lacking.5Arecent randomized controlled trial comparing short-term cyclosporine versus prednisolone was interrupted because of an unsuspected high proportion of severe rebound in the prednisolone group.6 Medium- to long-term treatment with prednisolone is relatively contraindicated because of the cumulative effect of the side effects.5 This illustrates 4 the need for novel medium- to long-term treatment options for patients with severe AD. However, commercial interest for research in eczema is low, and thus investigator-initiated studies are needed. As health care costs are increasing, dermatologists are looking for cheaper alternatives. Long-existing and relatively cheap disease-modifying antirheumatic drugs seem to be beneficial for AD. Two of those drugs are methotrexate and azathioprine. Azathioprine, a purine synthesis inhibitor that inhibits the proliferation of leukocytes, and methotrexate, a folic acid antagonist that targets several key T-cell activities, are currently used off-label in some (referral) centers. Despite several case series and open-label studies for methotrexate,7-10 there have been no randomized controlled trials supporting a role for methotrexate in the management of AD. The role of azathioprine in AD was established by 2 randomized controlled trials in which azathioprine was significantly superior to placebo, with mean improvements of 26% and 37% on clinical outcome scales after 3 months.11,12 Numerous uncontrolled studies on azathioprine in adult and juvenile patients showed similar results.13 To our knowledge, no comparison of methotrexate with azathioprine in a randomized controlled fashion has been performed. With the present study, we conducted a randomized comparison of methotrexate with azathioprine for the treatment of severe AD evaluating efficacy, safety, and effect on quality of life.

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Methods

Design This study was an investigator-initiated, single-blind, parallel-group (ratio 1:1), randomized controlled trial evaluating efficacy, safety, and quality of life with methotrexate versus azathioprine over a 12-week period. The trial was conducted between July 2009 and December 2010 at the Department of Dermatology of the AcademicMedical Center in Amsterdam, The Netherlands. Patients were evaluated every 2 weeks in the firstmonth and monthly thereafter. The follow-up phase consisted of another 12 weeks in which study drugs could be continued, stopped, or switched, reflecting normal clinical practice. The study protocol was reviewed and approved by the local medical ethics committee (institutional review board) and was performed in accordance with the Good Clinical Practice Guidelines of the International Conference of Harmonisation, Declaration of Helsinki. The trialwas registered in the Dutch Trial Register (NTR1916). Written informed consent was obtained from all patients before study-related procedures were commenced.

Patients Patients were recruited from the inpatient and outpatient clinics of the Academic Medical Center of Amsterdam (referral center for severe AD) or were referred by regional dermatologists. Patients with AD (with and without the presence of allergen-specific IgE) defined according to the Millennium Criteria and the UK Working Party criteria14 were eligible if they were 18 years or older; the severity grading by the Rajka and Langeland criteria was severe15; the patients were unresponsive, contraindicated, or intolerant to cyclosporine treatment; and the patients had not previously been treated with azathioprine or methotrexate. Excluded were patients who were pregnant, breast-feeding, or planning pregnancy (men and women) until 3 months after discontinuation; those with a history of cancer, alcohol abuse, organ transplantation, chronic or recurrent infectious diseases, or any severe and uncontrolled disease; those with a history of herpes zoster infection within 2 months of baseline or current bacterial skin infection; and those who had received phototherapy, any systemic medication, or a potent topical medication within the last 2 weeks. Because thiopurine methyltransferase (TPMT) is a key enzyme in the purine metabolism and genetic variation in the gene that transcribes TPMT is linked to interpersonal differences in toxicity of azathioprine, patients randomized to the azathioprine group were tested for TPMT activity. When TPMT activity was low or absent (<21 nmol/g/hour), indicating homozygous TPMT mutations and a subsequent risk for life-threatening myelotoxicity, patients were excluded. Patients randomized to receive methotrexate were excluded if abnormal laboratory results were discovered after

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they had taken a test dose of 5 mg of methotrexate. At every study visit (weeks 0, 2, 4, 8, 12, and 24), laboratory tests were done, including a full blood count and kidney and liver function measurement. Women of childbearing potential underwent a serum pregnancy test at every visit. Treatment regimens Treatment with methotrexate was initiated at 10 mg/ wk and administered as a single oral dose. Dose escalation with 2.5 to 5 mg per scheduled visit was allowed until 22.5 mg/wk was reached. Because folate supplementation reduces the risk for hepatotoxicity in patients with rheumatoid arthritis, each patient randomized to methotrexate received 5 mg of folate 1 day after methotrexate intake.16 Azathioprine was initiated at 1.5 mg/kg/d in a single dose, and the dosage could be escalated at each visit with 0.5 mg/kg/d until a maximum of 2.5 mg/kg/d was reached. Dosage was escalated if patients did not achieve at least a 25% reduction in disease activity at a study visit. The dosage could be decreased according to protocol in case of abnormal findings on physical examination, laboratory markers, and/or adverse events. After the first 12 weeks, dosages in responders were reduced to find the optimum dosage. Patients were allowed to continue or start with concomitant topical triamcinolone acetonide ointment (body), hydrocortisone ointment (face), and oral antihistamines. In case of an exacerbation 4 or postponed treatment effect in the first 8 weeks of treatment, patients were allowed to receive a maximum of 2 courses of rescue medication: 30 mg/d oral prednisolone for 1 week and a 1-week reduction schedule (20-20-15-15-10-10-5 mg).

Outcomes Efficacy. The primary efficacy outcome parameter was the mean relative andabsolute change in the severity of AD at week 12 assessed by means of the severity scoring for atopic dermatitis (SCORAD) score.17 The SCORAD score combines both objective items as affected area and intensity of the lesions (erythema, edema/induration, excoriation, oozing/ crusting, lichenification, and dryness) and subjective items as extent of pruritus and sleep loss on a visual analog scale (VAS). Scores range from 0 to 108 points.18 Secondary outcome parameters included the number of patients with a SCORAD score reduction of 50% or more (SCORAD50) and the number of patients achieving mild disease (defined as mild, minimal, or no disease activity on investigator global assessment [IGA]), IGA and patient global assessment (PGA), mean change in the eczema area and intensity index (EASI), patient-oriented eczema measurement (POEM), itch and sleeplessness on a VAS, Skindex-17, levels of thymus and activation-regulated chemokine (TARC), amount of concomitant topical corticosteroids, and number of courses of rescue medication used. The IGA and PGA were assessed by using a 6-point Likert scale: 0, clear; 1, almost clear; 2, mild disease; 3, moderate disease; 4, severe disease; and 5, very severe disease. The EASI is based on the extent of the eczematous

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involvement of the body surface area, as well as the intensity of the lesions (range, 0-72).19 The POEM includes 7 questions regarding skin symptoms (range, 0-28).20 Change in quality of life was assessed by the use of the Dutch version of the Skindex-17.21 Scores range from 0 to 85 points, with higher scores indicating more significantly impaired quality of life. Clinical outcome parameters and quality of life were assessed at each visit. Furthermore, at baseline and week 12, serum TARC levels were measured.22,23

Safety The number and severity of adverse events were assessed at each visit by the safety assessor to address safety. Adverse events that were transient and easily tolerated by the patient were considered mild. Moderate adverse events were defined as causing discomfort and interrupting the subject’s usual activities. Adverse events were severe if the event caused considerable interference with the subject’s usual activities and could be incapacitating or life-threatening. Serious adverse events were defined as life-threatening events, death, prolonged or initial hospitalization, disability, or permanent damage. The safety assessor defined adverse events as not, possible, probably, or definitely related to treatment.

Blinding Concealment of allocation was achieved by using a computerized program (see the Statistical analysis section). Clinical outcome measurements were assessed by trained efficacy assessors, who were blinded for allocation. Statistical analysis was performed by the third author, who was also blinded for allocation. Patients and safety assessors were not blinded.

Statistical analysis In the primary analysis the difference in mean SCORAD scores between the treatment groups at week 12 was analyzed by using intention-to-treat analysis. The criterion for including patients in the intention-to-treat analysis was receiving at least 1 dose of study medication. Randomization was performed in a 1:1 ratio by using a computerized program (TENALEA Clinical Trial Data Management System) with the (nondeterministic) minimization method described by Pocock and Simon.24 Patient factors (strata) did not influence the allocation scheme. If a patient missed a visit, we used the score fromthe previous visit for the intention- to-treat analysis. Without the availability of a formally calculated minimally clinically important difference, we deemed it appropriate to use an 8-point difference in SCORAD scores between groups as the nonequivalence limit.25,26 Assuming an SD of 10 points in both groups, the power analysis showed that 42 participants were needed for a study with 80% power and 5% significance. No interim analysis was performed. Two-sided P-values of less than .05 were considered to indicate statistical significance. Specific statistical tests used are

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indicated in the legends of the tables. SPSS 18.0 for Windows (SPSS, Inc, Chicago, Ill) was used to perform data analysis.

Results

Patients’ characteristics Between September 2009 and May 2010, 45 patients were screened for enrollment, 43 of whom were randomized (Fig 1). Twenty patients were assigned to the methotrexate group, and 23 patients were assigned to the azathioprine group. One patient randomized to the azathioprine group withdrew informed consent before the initiation of study medication and was not included in the analyses. Baseline characteristics of included patients are shown in Table 1. Fifty-two percent of the patients were male, the mean age was 40 years, and the mean duration of eczema was 36 years over the 2 groups. The mean SCORAD score was 58 points at baseline.18 The mean Skindex-17 score was 51 points. Mean dosage of methotrexate was 20 mg/wk at week 12 and 17.5 mg/wk at week 24. Mean dosage of azathioprine was 2.2 mg/kg/d at week 12 and 2.1 mg/kg/d at week 24. During the study, 1 patient in the 4 methotrexate group dropped out after 4 weeks because of nausea and fatigue. Three patients were withdrawn in the azathioprine group: 1 patient because of lymphocytopenia, increased liver enzyme values, and worsening of preexisting mild anemia at week 4; 1 patient because of nausea and vomiting at week 5; and 1 patient because of failure to adhere to the study protocol at week 2. No patients were lost to follow-up.

Table 1. Demographics and baseline characteristics of the included participants

Treatment group Methotrexate (n=20) Azathioprine (n=22) Age in years (mean (SD)) 43.0 (14.7) 37.0 (14.1) Male sex 10 (50%) 12 (55%) Presence of asthma or allergic rhinitis 19 (95%) 18 (82%) Presence of allergen specific IgE 20 (100%) 21 (95%) Duration of AD (mean years ± SD) 39.8 (16.2) 33.1 (16.8) Disease activity SCORAD (mean (SD)) 57.2 (11.8) 58.4 (10.4) IGA (mean (SD)) 3.8 (0.6) 3.6 (0.6) PGA (mean (SD)) 3.9 (0.6) 3.8 (0.8) EASI (mean (SD)) 27.9 (12.3) 30.4 (14.2) POEM (mean (SD)) 19.8 (5.3) 19.5 (4.0) Quality of life (mean Skindex-17 (SD)) 50.2 (11.7) 51.7 (8.6)

EASI; eczema area-and-severity index, IGA; investigator global assessment, IQR; interquartile range, ITT; intention to treat, PGA; patient global assessment, POEM; patient oriented eczema measurement, SCORAD; scoring of atopic dermatitis index, SD; standard deviation, TARC; thymus and activation-regulated chemokine, VAS; visual analogue scale.

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Figure 1. Screening, randomization, treatment and follow-up of study participants

*Data per-protocol analysis not shown. AE; adverse event

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Efficacy At week 12, mean SCORAD scores in the patients in the methotrexate group changed from 57.2 (SD, 11.8) to 34.4 (SD, 13.0), representing a relative reduction of 42% (P < .001, Table 2). SCORAD scores in patients randomized to the azathioprine group changed from 58 (SD, 10.4) to 36.3 (SD, 16.9), representing a relative reduction of 39% (P < .001). The P-value for the absolute difference between the groups is .89. Fig 2 shows the mean SCORAD scores during the study period. Eight (40%) patients in the methotrexate group versus 10 (45%) patients in the azathioprine group achieved a SCORAD50 response (P 5 .76). Fifteen patients in each group (75% in the methotrexate group vs 68% in the azathioprine group) achieved at least mild disease on IGA (P 5 .74). On global assessment, the mean IGA score was reduced to 1.8 (SD, 0.7) in the methotrexate group versus 1.4 (SD, 0.9) in the azathioprine group (P 0.20), and the mean PGA score was reduced to 1.3 (SD, 0.9) in the methotrexate group versus 1.2 (SD, 1.3) in the azathioprine group (P0 .95). The EASI score was reduced to 17.4 (SD, 6.6) points in the methotrexate group compared with 17.2 (SD, 14.1) points in the azathioprine group (P5.95). Reduction in the mean POEM score was 6.9 (SD, 5.7) in the methotrexate group versus 7.9 (SD, 7.7) in the azathioprine group (P 5 .65). Clinical improvement was 4 paralleled by a decrease in symptoms. Mean VAS itch scores decreased to 2.5 (SD, 2.2) in the methotrexate group versus 2.6 (SD, 2.2) in the azathioprine group (P 5.78). Mean VAS scores for sleeplessness decreased to 2.8 (SD, 2.6) in the methotrexate group versus 3.8 (SD, 2.8) in the azathioprine group (P 0.24). When comparing quality of life, the mean Skindex-17 score was reduced from 50.2 (SD, 11.7) at baseline to 37.8 (SD, 9.8) at week 12 in the methotrexate group (P <.001). In the azathioprine group the mean Skindex-17 score was reduced from 51.7 (SD, 8.6) to 41.5 (SD, 13.1; P < .001). This equals a reduction of 26% versus 20% (P 5.65) Median TARC levels decreased to 1215 ng/mL (interquartile range, 302- 2496 ng/mL) in the methotrexate group and 885 ng/mL (interquartile range, 122-3107 ng/ mL) in the azathioprine group (P 5.61). Patients in the methotrexate group used a median of 115.2 g of concomitant topical corticosteroids versus 79.1 g in the azathioprine group. Two (10%) patients in the methotrexate group required a course of rescue medication at weeks 1 and 2, respectively, and 4 (18%) patients in the azathioprine group required a course at weeks 1, 2, 4, and 5, respectively (P 5.67).

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Table 2. Clinical response at week 12 (intention to treat analysis)

Treatment group Methotrexate Azathioprine Variable P-value (n=20) (n=22) Improvement in SCORAD Absolute reduction, mean (SD) 22.7 (7.9) 22.2 ± 16.5 0.89* Relative reduction, mean (SD) 42 (18%) 39 (25%) 0.70* At least 50% (SCORAD50), no (%) 8 (40%) 10 (45%) 0.76¥ Improvement IGA, no (%) Reduction, mean (SD) 1.8 (0.7) 1.4 (0.9) 0.20* Cleared, minimal or mild disease (IGA <2), no (%) 15 (75%) 15 (68.2%) 0.74¥ Improvement other outcomes Reduction in PGA, mean (SD) 1.3 (0.9) 1.2 (1.3) 0.95* Reduction in EASI, mean (SD) 17.4 (6.6) 17.2 (14.1) 0.95* Reduction in POEM, mean (SD) 6.9 (5.7) 7.9 (7.7) 0.65* Reduction in VAS itch score, mean (SD) 2.5 (2.2) 2.6 (2.2) 0.78* Reduction in VAS sleeplessness score, mean (SD) 2.8 (2.6) 3.8 (2.8) 0.24* Reduction in Skindex-17, mean (SD) 12.9 (8.8) 10.3 (12.9) 0.46* Reduction in TARC levels (median (IQR) 1215 (302 to 2496) 885 (122 to 3107) 0.61** Use of concomitant topical steroids (g), median (IQR) 115.2 (45 to173) 79.1 (22 to 121) 0.16§ No. of rescue medication, no (%) 2 (10%) 4 (18%) 0.67¥

EASI; eczema area-and-severity index, IGA; investigator global assessment, IQR; interquartile range, PGA; patient global assessment, POEM; patient oriented eczema measurement, SCORAD; scoring of atopic dermatitis index, SD; standard deviation, TARC; thymus and activation-regulated chemokine, VAS; visual analogue scale. * T-test for independent groups § Mann-Whitney U test for independent groups ¥ Fisher’s exact test ** based on ln_TARC

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Figure 2. Mean (SD) scores for the SCORAD at baseline, during treatment and follow -up Figure 2. Mean (SD) scores for the SCORAD at baseline, during treatment and follow-up

MTX; methotrexate, AZA; azathioprine

Safety Table 3 shows an overview of the safety results. Abnormalities in blood count (mostly lymphocytopenia) were statistically significantly more frequent in the azathioprine group (P 5 .002). Infections, gastrointestinal adverse events, and increased liver enzyme levels occurred in equal proportion in both groups. Fourteen patients in each group experienced infections, mainly upper airway infections, common colds, and mild skin infections. Skin infections occurred in 5 (25%) patients in the methotrexate group and 7 (32%) patients in the azathioprine group; all were mild. Five infections in the methotrexate group and 8 in the azathioprine group were considered moderate of intensity. Three (15%) patients in the methotrexate group had an exacerbation of their AD during the study compared with 2 (9%) patients in the azathioprine group. No severe and serious adverse events occurred.

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Table 3. Adverse events and other key safety data through week 24

Treatment group Variable Methotrexate Azathioprine P-value* Total number of patients 20 22 Total number of patients with adverse events, no. of 20 (100%) 22 (100%) 1.00 patients (%) Serious and severe adverse events¥ 0 0 - Adverse events, total (events)** 113 121 Adverse events leading to withdrawal 1 (5%) 2 (9%) 1.00 Adverse event requiring dose adjustment, no. of 2 (10%)*** 2 (9%) 1.00 patients (%) Treatment-related adverse events§, no. of patients 14 (70%) 12 (64%) 0.66 (%) Categorized adverse events, no. of patients (%) Infections 14 (70%) 14 (64%) 0.19 Gastro-intestinal adverse events 11 (55%) 13 (59%) 0.79 Exacerbation of AD 3 (15%) 2 (9%) 0.66 Abnormalities in blood count 6 (30%) 17 (77%) 0.002 Increased liver enzymes 7 (35%) 8 (36%) 0.93

* P-values were calculated using Chi-square test or Fisher’s exact test. § Treatment-related adverse events are those classified as possibly, probably or definitely related to the study drug by the safety assessor. ¥ Adverse events were classified as serious and severe according to pre-defined definition (see methods section). ** Total number of adverse events: all patients displayed adverse events, the number of events per patient ranging from 1 to 14 (median 5, interquartile range 3.25 to 8). If a patient presented twice with the same adverse effect (e.g. twice migraine), then this was counted as one adverse event. This was the case in 17 out of the 42 patients; 2 patients presented a certain symptom three times (which was also counted as one time). ***One patient had twice dose-lowering

Follow-up After 12 weeks, 18 (95%) patients in the methotrexate group continued their treatment, and 1 patient discontinued after induction of remission. In the azathioprine group 16 (84%) patients continued, 1 switched to methotrexate because of lack of efficacy, and 2 discontinued because of induction of remission. At week 24, the mean SCORAD score on intention-to-treat analysis was 30.4 (SD, 14.3) in the methotrexate group and 33.7 (SD, 16.9) in the azathioprine group (P 5 .58), representing a relative reduction from baseline of 48% versus 43%. After 24 weeks of treatment, there was no statistically significant difference between the 2 groups in all outcome measurements

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Discussion

General conclusion Disease-modifying anti-rheumatic drugs, such as methotrexate, azathioprine, and mycophenolate, offer a range of off-label therapeutic options for patients with severe AD. In the present randomized controlled trial methotrexate and azathioprine were compared. Patients in the methotrexate group experienced a statistically significant overall improvement of 42% in1 mean SCORAD score at week 12. Patients in the azathioprine group showed a statistically significant overall improvement of 39% in mean SCORAD score. In addition, intensity of symptoms and TARC levels were reduced in a similar fashion in both groups. Effect on quality of life reduced by 26% in the methotrexate group versus 20% in the azathioprine group. Overall, there was no statistical difference between both therapies in any primary or secondary efficacy outcome measures assessed at weeks 12 and 24. In this study the number and severity of adverse events, including laboratory abnormalities, appeared to be generally similar in short-term treatment, with the exception of mild myelosuppression in the azathioprine group. No severe or serious adverse events occurred. 4 Because of the relatively slow onset of action of both treatments, it is common in some clinical practices to give patients concomitant oral corticosteroids to support them in the first weeks of treatment. In our study both treatments were given as single systemic therapy with the possibility of adjuvant rescue medication. This was needed in 6 of the 42 patients treated. This might indicate that routine administration of concomitant oral corticosteroids is not necessary. Our results are in concordance with the previous results of 2 placebo-controlled studies regarding azathioprine, in which improvements of 26% and 37% on the 6-area, 6-sign atopic dermatitis score at 12 weeks were found.11,12 Similar results were found for the effect of methotrexate in cohort studies, although in some the effect was more pronounced: 52% at week 24 in one study and a greater than 70% reduction in outcome parameters in 65% of the patients at week 12 in another.7,9 Studies on cyclosporine, which is the first-choice systemic treatment, showed a more marked response. A systematic review showed that relative improvements were consistently greater than 50% at 6 to 8 weeks.3 In conclusion, methotrexate and azathioprine can be considered equally effective for the treatment of severe AD in adults. Overall, this study is limited to conclusions about the safety for medium- to long-term use. Nevertheless, because both drugs have been available for more than 50 years, they have a well-known toxicity profile, and dermatologists are familiar with the use of these drugs in the treatment of psoriasis or bullous diseases. Patients treated with azathioprine should be monitored for myelosuppression, and preferably, TPMT levels should be measured before the initiation. Patients receiving methotrexate should be monitored for

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hepatic and pulmonary toxicity andmyelosuppression. Individual treatment decisions should be based on patients’ preferences and comorbidity.

Strengths and limitations Important strengths of our trial include that our study is the first head-to-head comparison of methotrexate versus azathioprine in patients with severe atopic dermatitis. Thereby, the study was investigator initiated, with the invest igators having no conflict ofinterest; the study included patients naive for methotrexate or azathioprine; and validated outcome parameters were used. Our trial had certain limitations. Power analysis was based on the SCORAD score. Because the minimally clinically important difference of the SCORAD score is unknown, we decided that a difference of at least 8 points would represent a clinical meaningful difference. This was based on other studies and on personal experience with the scale. Nevertheless, it could have been possible that a smaller SCORAD score difference was clinically meaningful. In that case our sample size would not have been sufficient to detect a meaningful difference between the therapies. In our study the washout period for systemic treatment was relatively short. This might have resulted in a lower baseline SCORAD score and in a smaller difference between baseline and week 12 efficacy outcomes. At baseline, patients were allowed to start or increase the amount of topical steroids. This could have influenced the treatment effect. However, considering the low total amount and low potency of the topical steroids used during the trial, the effect on efficacy outcomes will be minimal. Continuing with a stable dose or implementation of a stabilization period would have been more methodologically sound. Ideally, patients should be blinded for allocation of treatment to avoid a performance bias. Because each patient received active treatment in this study, this effect will be less pronounced than in a placebo-controlled design. Concerning the nonblinded safety assessors, patient management could have been biased. However, it should be noted that safety assessors did not have personal preferences or conflicts of interest Apatient-blinded design could not be performed because of a lack of funding and other financial resources. In the absence of international guidelines for these treatments, our dosing schedule was based on earlier performed studies and experience with the drugs in other indications. Therefore it could be argued that the interventions were not similar. Currently, there is an international initiative to harmonize outcome assessment in AD: the Harmonizing Outcome Measurement in Eczema initiative. During a Delphi round on core outcome domains, it was shown that symptoms, physician assessed clinical signs, and measurement for long-term control were of particular interest.27 Symptoms were addressed by means of VASs on itch and sleeplessness and by means of the POEM. Physician-assessed clinical signs were addressed by using the SCORAD and EASI. Both were sufficiently validated

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according to a recently published systematic review on the validity of clinical outcome measures.28 Although we have performed a 3-month follow-up period to indicate long-term control, we could not totally comply with the measurement for long-term control because of the design of the study. An observational follow-up study, which is currently being conducted, is directed at that particular outcome domain.

Generalizability This study was performed in adults (18-75 years of age) with severe AD who were unresponsive or contraindicated for cyclosporine treatment. Sex was equally distributed. Because the included patients were 40 years old and had a mean duration of disease of 36 years, we can conclude it concerned a very chronic population. In some cases included patients were very severely affected and had frequent hospitalizations for disease management in the past. All except 1 of the patients showed the presence of allergen-specific IgE on a Phadiatop test (Pharmacia, Uppsala, Sweden). We believe that our data are applicable to all adults with difficult-to-treat chronic AD, depending on their comorbidity and preferences. Studies in children should be performed to establish the role of these treatments in children. 4

Clinical research implications We have demonstrated that both methotrexate and azathioprine are effective and short-term safe treatment options for adults with severe AD. We believe that the results from this study justify treatment with these drugs when regular treatment is insufficient. Furthermore, the results from this study can be used to update or formulate clinical practice guidelines for the treatment of severe AD. In light of the rules and regulations concerning off-label treatment, it is important to generate evidence-based guidelines for its use. Future studies should be performed to confirm the long-term safety profile of both methotrexate and azathioprine, to confirm their role in children, and to compare both treatments with other therapies, such as cyclosporine and oral corticosteroids.

Clinical implications: Methotrexate and azathioprine can be considered alternative treatment options for patients with severe atopic dermatitis when regular treatment is insufficient.

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References for the treatment of adult atopic dermatitis. Eur J Dermatol 2006;16: 155-8.

1. Williams H, Robertson C, Stewart A, it-Khaled 10. Shaffrali FC, Colver GB, Messenger AG, N, Anabwani G, Anderson R, et al. Worldwide Gawkrodger DJ. Experience with lowdose varia;tions in the prevalence of symptoms of methotrexate for the treatment of AD in the International Study of Asthma and eczema in the elderly. J Am Acad Dermatol Allergies in Childhood. J Allergy Clin Immunol 2003;48:417-9. 1999;103:125-38. 11. Berth-Jones J, Takwale A, Tan E, Barclay G, 2. Schmitt J, Romanos M, Pfennig A, Leopold K, Agarwal S, Ahmed I, et al. Azathioprine in Meurer M. Psychiatric comorbidity in adult severe adult atopic dermatitis: a double- eczema. Br J Dermatol 2009;161:878-83. blind, placebo-controlled, crossover trial. Br 3. Schmitt J, Schmitt N, Meurer M. Cyclosporin J Dermatol 2002;147:324-30. in the treatment of patients with AD—a 12. Meggitt SJ, Gray JC, Reynolds NJ. Azathioprine systematic review and meta-analysis. J Eur dosed by thiopurine methyltransferase Acad Dermatol Venereol 2007;21:606-19. activity for moderate-to-severe AD: a 4. Behnam SM, Behnam SE, Koo JY. Review of double-blind, randomized controlled trial. cyclosporine immunosuppressive safety data Lancet 006;367:839-46. in dermatology patients after two decades of 13. Schram ME, Borgonjen RJ, Bik CJMJ, van der use. J Drugs Dermatol 2005;4:189-94. Schroeff JG, van Everdingen JJE, Spuls PhI. 5. Schmitt J, Schakel K, Schmitt N, Meurer Off-label use of azathioprine in dermatology: M. Systemic treatment of severe AD: a a systematic review. Arch Dermatol systematic review. Acta Derm Venereol 2011;147:474-88. 2007;87:100-11. 14. Brenninkmeijer EE, Schram ME, Leeflang 6. Schmitt J, Schakel K, Folster-Holst R, Bauer MM, Bos JD, Spuls PhI. Diagnostic criteria for A, Oertel R, Augustin M, et al. Prednisolone atopic dermatitis: a systematic review. Br J vs. ciclosporin for severe adult eczema. An Dermatol 2008;158:754-65. investigator-initiated double-blind placebo- 15. Rajka G, Langeland T. Grading of the severity controlled multicentre trial. Br J Dermatol of atopic dermatitis. Acta Derm Venereol 2010;162:661-8. Suppl (Stockh) 1989;144:13-4. 7. Weatherhead SC, Wahie S, Reynolds NJ, 16. Hoekstra M, van Ede AE, Haagsma CJ, van de Meggitt SJ. An open-label, dose-ranging study Laar MA, Huizinga TW, Kruijsen MW, et al. of methotrexate for moderate-to-severe Factors associated with toxicity, final dose, adult AD. Br J Dermatol 2007;156:346-51. and efficacy of methotrexate in patients 8. Zoller L, Ramon M, Bergman R. Low dose with rheumatoid arthritis. Ann Rheum Dis methotrexate therapy is effective in late- 2003;62:423-6. onset atopic dermatitis and idiopathic 17. Severity scoring of atopic dermatitis: the eczema. Isr Med Assoc J 2008;10: 413-4. SCORAD index. Consensus Report of the 9. Goujon C, Berard F, Dahel K, Guillot I, European Task Force on Atopic Dermatitis. Hennino A, Nosbaum A, et al. Methotrexate Dermatology 1993;186:23-31.

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18. Oranje AP, Glazenburg EJ, wolkerstorfer A, de Biometrics 1975;31:103-15. Waard-van der Spek FB. Practical issues on 25. Paul C, Lahfa M, Bachelez H, Chevret S, interpretation of scoring atopic dermatitis: Dubertret L. A randomized controlled the SCORAD index, objective SCORAD and evaluator-blinded trial of intravenous the three-item severity score. Br J Dermatol immunoglobulin in adults with severe atopic 2007;157:645-8. dermatitis. Br J Dermatol 2002;147:518-22. 19. Tofte SJ, Graeber M, Cherill R, Omoto M, 26. Schmitt J, Meurer M, Schwanebeck U, Thurston M, Hanifin JM. Eczema area and Grahlert X, Schakel K. Treatment following severity index (EASI): A new tool to evaluate an evidence-based algorithm versus atopic dermatitis. J Eur Acad Dermatol individualised symptom-oriented treatment Venereol 1998;11(suppl 2):S197. for AD. A randomised controlled trial. 20. Charman CR, Venn AJ, Williams HC. Dermatology 2008;217:299-308. The patient-oriented eczema measure: 27. Schmitt J, Williams H. Harmonising Outcome development and initial validation of a Measures for Eczema (HOME). Report from new tool for measuring AD severity from the First International Consensus Meeting the patients’ perspective. Arch Dermatol (HOME 1), 24 July 2010, Munich, Germany. 2004;140:1513-9. Br J Dermatol 010;163:1166-8. 4 21. Nijsten TE, Sampogna F, Chren MM, Abeni 28. Schmitt J, Langan S, Williams HC. What are DD. Testing and reducing skindex-29 using the best outcome measurements for AD? Rasch analysis: Skindex-17. J Invest Dermatol A systematic review. J Allergy Clin Immunol 2006;126:1244-50. 2007;120:1389-98. 22. Hijnen D, De Bruin-Weller M, Oosting B, Lebre C, De Jong E, Bruijnzeel-Koomen C, et al. Serum thymus and activation-regulated chemokine (TARC) and cutaneous T cell- attracting chemokine (CTACK) levels in allergic diseases: TARC and CTACK are disease- specific markers for atopic dermatitis. J Allergy Clin Immunol 2004;113:334-40. 23. Kakinuma T, Nakamura K, Wakugawa M, Mitsui H, Tada Y, Saeki H, et al. Thymus and activation-regulated chemokine in atopic dermatitis: Serum thymus and activation- regulated chemokine level is closely related with disease activity. J Allergy Clin Immunol 2001;107:535-41. 24. Pocock SJ, Simon R. Sequential treatment assignment with balancing for prognostic factors in the controlled clinical trial.

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Evelien Roekevisch, Mandy Elvira Schram, Mariska Maria Geertruida Leeflang, Marijke Willemijn Dorothée Brouwer, Louise Anna Andrea Gerbens, Jan Dositheus Bos, Phyllis Ira Spuls

Journal of Allery and Clinical Immunology (JACI). 2011 Aug;128(2):353-9

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Methotrexate versus azathioprine in patients with atopic dermatitis: 2-year follow-up data

To the Editor:

Long-term data of methotrexate (MTX) and azathioprine (AZA) in atopic dermatitis (AD) are lacking. Previously, we published the results of a randomized controlled trial (RCT) of 42 adults with severe AD treated with MTX or AZA for 12 to 24 weeks. Both MTX and AZA induced significant reductions in SCORing Atopic Dermatitis (SCORAD) index at 12 and 24 weeks after baseline, without a significant difference between treatments.1 To assess long- term efficacy and safety of MTX versus AZA, all 42 patients were asked to participate in an observational follow-up study to be evaluated 3 monthly for 2 years. After 12 weeks of treatment with MTX or AZA, treatments were continued, stopped, or switched, reflecting normal clinical practice. The same outcomes as in the original RCT were investigated, which correspond to the 4 core outcome domains for AD suggested by the Harmonising Outcome Measures for Eczema initiative. Patients were scored by trained blinded investigators. One of the primary outcomes was the difference in mean absolute and relative change in SCORAD index after 2 years compared with baseline. An intention-to-treat (ITT) analysis and a per- protocol (PP) analysis were performed. Adverse events (AEs) were coded according to the Medical Dictionary for Regulatory Activities/Common Terminology Criteria for Adverse Events.2 In addition, the effect of filaggrin gene(FLG) mutation status on treatment outcome (R501X, 2282del4, R2447X, S3247X, -3321delA) was evaluated by collecting buccal mucosa swabs. Patients were divided into a FLG mutation group (FMG) and a non-FMG group. For detailed methods on dosing and monitoring, we refer to this article’s Online Repository at www.jacionline.org and a previous publication.1 For the MTX and AZA groups, an overview of primary and secondary outcomes after 2 years compared with baseline is presented in Table I (ITT, n 5 35) and Table II (PP, n 5 18). In Table E1 in this article’s Online Repository at www. jacionline.org we present the baseline characteristics. Treatment and dosage overview are presented in Tables E2 and E3 and Fig E1 in this article’s Online Repository atwww.jacionline . org. Fig E2, A (ITT) and B (PP), in this article’s Online Repository at www.jacionline.org shows the mean 6 SD total SCORAD index. Table E4 (ITT) and Table E5 (PP) in this article’s Online Repository at www.jacionline.org show AEs and other key safety data. Fig E3, A (ITT) and B (PP), in this article’s Online Repository at www.jacionline.org shows the mean SCORAD index after 2 years compared with baseline for the FMG and non-FMG groups. Thirty-five patients, 17 primary randomized to MTX and 18 to AZA, participated in this follow-up study. Two years after baseline, 10 patients still used the allocated MTX and 11 the allocated AZA, of which

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8 continuously since randomization. Reasons for patients in the ITT analysis to discontinue treatment (17 of 35) were controlled AD (7 of 35), inefficacy (7 of 35), or AEs (3 of 35). These AEs concerned fatigue, nausea, recurrent herpes infections in the MTX group (2 of 35), and increased liver enzyme values and worsening of preexisting mild anemia in the AZA group (1 of 35), both reversible after stopping AZA. Two years after baseline, improvement was found for all primary and secondary outcomes. ITT analysis showed a mean absolute reduction in total SCORAD index of 37.8 6 13.8 and 32.6 6 SD 17.4 in the MTX group and the AZA group, respectively, representing a relative reduction of 63% (P < .01) and 53% (P < .01), respectively. There was no significant difference in SCORAD index between groups (P 5 .33). PP analysis showed a mean absolute reduction in total SCORAD index of 41.7 6 10.6 and 43.6 6 18.5 in the MTX group and the AZA group, respectively, representing a relative reduction of 67% (P < .01) and 65% (P < .01), respectively. There was no significant difference in SCORAD index between groups (P 5 .79). Also, for other outcome measures, there was no significant difference between treatments, except for the Patient Global Assessment in the PP analysis, which showed a favorable effect for the AZA group. Because the Patient Global Assessment gives insight into the patient’s own health experience, this may be an important finding. Interestingly, although not significant, the Investigator Global Assessment in the PP analysis also showed a trend of more efficacy (P 5 .07) in the AZA group. Amounts of highly 5 potent (classes III-IV, scales I-IV) topical immunosuppressive treatment (total gram, P 5 .17) and oral corticosteroids needed as rescue medication (P 5 .70) were comparable between groups during the course of 2 years (P 5 .17). Oral antibiotics used for bacterial skin infections were more often used in the MTX group than in the AZA group (P 5.03). Concerning safety, 3 serious AEs occurred, 2 exacerbations of AD and 1 hospitalization because of psychiatric comorbidity. No life-threatening serious AEs occurred. No malignancies were observed. Most AEs were found in the group of infections and infestations disorders. Common cold and liver enzyme disorders were most frequently seen in the MTX group. Influenza was significantly more present in the AZA group. Concerning FLG mutation status, 1 patient declined to undergo DNA testing; therefore, information was gathered from 34 patients. Previously, we showed a better response of systemic treatment in the non-FMG group over the course of 24 weeks.3 However, in this study, we observe that this difference in effect fades away over time, and after 2 years the response in both groups is comparable. In the non-FMG group, there is a steep curve in reduction in SCORAD index in the first 3 months; in other words, not much further improvement is to be expected after 3 months of therapy (absolute SCORAD index reduction of only 4 points), whereas in the FMG group significant effect can still be expected (absolute SCORAD index reduction of 25 points).

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TABLE I. Clinical response after 2 years compared to baseline (intention-to-treat analysis)

Mean Difference (95% MTX AZA Variable Confidence Interval of P-value (n=17) (n=18) the Difference) Baseline values of SCORAD ¥ SCORAD - Total SCORAD, mean (SD) 59.4 (11.4) 59.7 (11.0) -0.3 (-8.0 to 7.4) 0.93* - Objective SCORAD, mean (SD) 46.6 (10.3) 46.3 (10.1) 0.3 (-6.7 to 7.3) 0.93* - Subjective SCORAD, mean (SD) 12.8 (4.0) 13.4 (3.4) -6.2 (-2.9 to 1.7) 0.59* Primary outcomes, after 2 years compared to baseline SCORAD Absolute reduction - Total SCORAD, mean (SD) 37.8 (13.8) 32.6 (17.4) 5.3 (-5.6 to 16.1) 0.33* - Objective SCORAD, mean (SD) 29.0 (12.7) 25.0 (13.8) 3.9 (-5.2 to 13.0) 0.39* - Subjective SCORAD, mean (SD) 8.8 (4.1) 7.5 (5.5) 1.3 (-2.1 to 4.7) 0.43* Relative reduction - Total SCORAD, mean (SD) 63% (17%) 53% (24%) 9.9 (-4.3 to 24.2) 0.16* - Objective SCORAD, mean (SD) 61% (19%) 52% (23%) 8.3 (-6.2 to 22.7) 0.25* - Subjective SCORAD, mean (SD) 49% (33%) 37% (41%) 12.6 (-13.2 to 38.4) 0.33* SCORAD50, no (%) 15 (88%) 11 (61%) - 0.12^ Reduction IGA, median (range) 2 (0-3) 2 (-1-4) - 0.86# Cleared, minimal or mild disease (IGA <3), 15 (88%) 14 (78%) - 0.66^ no (%) Secondary outcomes, after 2 years compared to baseline Reduction in PGA, median (range) 2(0-4) 1(-2-4) - 0.27# Reduction in EASI, mean (SD) 24.9 (13.0) 26.4 (14.7) -1.5 (-11.1 to 8.1) 0.75* EASI50, no (%) 17 (100%) 17 (94%) - 1.0 EASI75, no (%) 14 (82%) 14 (78%) - 1.0 Reduction in POEM, mean (SD) 10.6 (5.4) 10.2 (9.1) 0.4 (-4.8 to 5.5) 0.89* Reduction in VAS pruritus, mean (SD) 3.1 (2.1) 2.4 (3.3) 0.7 (-1.3 to 2.6) 0.49* Reduction in VAS sleeplessness, mean (SD) 4.2 (2.1) 4.1 (2.8) 0.1 (-1.6 to 1.8) 0.88* Reduction in Skindex-17 - Total Skindex, mean (SD) 8.8 (6.9) 7.4 (7.4) 1.4 (-3.5 to 6.3) 0.56* - Symptoms, mean (SD) 2.4 (2.1) 1.7 (2.5) 0.6 (-0.9 to 2.2) 0.43* - Psychosocial, mean (SD) 6.5 (5.6) 5.7 (5.5) 0.8 (-3.0 to 4.6) 0.67*

Table includes all patients who participated in the observational follow-up study (n=35). Last available observation value was carried forward (ITT analysis). EASI, Eczema Area and Severity Index; EASI50, Improvement in EASI with at least 50%; EASI75, Improvement in EASI with at least 75; IGA, Investigator Global Assessment; LOCF, last observation carried forward; PGA, Patient Global Assessment; POEM, Patient-Oriented Eczema Measure; SCORAD, SCORing Atopic Dermatitis; SCORAD50, Improvement in SCORAD with at least 50%; SD, Standard Deviation; VAS, Visual Analogue Scale. ¥ Baseline SCORAD values are values at the first visit of the original study before starting MTX or AZA; * T-test for independent groups; # Fisher’s exact test; ^ Pearson Chi-Square.

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TABLE II. Clinical response after 2 years compared to baseline (per-protocol analysis)

Mean Difference (95% P MTX AZA Variable Confidence Interval of (n=10) (n=8) the Difference) Value Baseline values of SCORAD ¥ SCORAD - Total SCORAD, mean (SD) 61.9 (9.4) 67.0 (8.3) -5.0 (-14.0 to 3.9 ) 0.25* - Objective SCORAD, mean (SD) 48.5 (8.2) 52.7 (9.0) -4.1 (-12.7 to 4.5) 0.83* - Subjective SCORAD, mean (SD) 13.4 (3.5) 14.4 (3.4) -1.0 (-4.4 to2.5) 0.56* Primary outcomes, after 2 years compared to baseline SCORAD Absolute reduction - Total SCORAD, mean (SD) 41.7 (10.6) 43.6 (18.5) -1.9 (-16.6 to 12.8) 0.79* - Objective SCORAD, mean (SD) 32.4 (9.5) 33.8 (14.9) -1.3 (-13.6 to 10.9) 0.82* - Subjective SCORAD, mean (SD) 9.3 (4.7) 9.9 (5.9) -0.6 ( -5.8 to 4.7) 0.82* Relative reduction - Total SCORAD, mean (SD) 67% (8%) 65% (25%) 1.9 (-16.1 to 19.3) 0.83* - Objective SCORAD, mean (SD) 66% (10%) 63% (24%) 2.5 (-15.5 to 20.5) 0.77* - Subjective SCORAD, mean (SD) 45% (30%) 39% (36%) 5.6 (-27.5 to 38.8) 0.72* SCORAD50, no(%) 10 (100%) 6 (75%) - 0.18^ Reduction IGA, median (range) 2 (1-3) 3 (-1-4) - 0.07# 5 Cleared, almost clear or mild disease (IGA 9 (90%) 7 (88%) - 1.00^ <3), no (%) Secondary outcomes, after 2 years compared to baseline Reduction in PGA, median (range) 1.5 (0-4) 3 (-2-3) - 0.01# Reduction in EASI, mean (SD) 28.1 (12.6) 34.9 (13.8) -6.8 (-20.0 to 6.5) 0.29* EASI50, no (%) 10 (100%) 8 (100%) - - EASI75, no (%) 10 (100%) 6 (94%) - 0.18 Reduction in POEM, mean (SD) 12.9 (4.4) 13.0 (10.1) -0.1 (-7.6 to7.4) 0.98* Reduction in VAS pruritus, mean (SD) 3.2 (2.5) 2.6 (3.2) 0.6 (-2.2 to 3.4) 0.68* Reduction in VAS sleeplessness, mean (SD) 4.6 (2.0) 5.0 (3.3) -0.4 (-3.0 to 2.2) 0.75* Reduction in Skindex-17 - Total, mean (SD) 11.1 (7.8) 10.0 (8.6) 1.1 (-7.1 to 9.3) 0.78* - Symptoms, mean (SD) 2.8 (2.3) 2.6 (2.1) 0.2 (-2.0 to 2.8) 0.77* - Psychosocial, mean (SD) 8.3 (6.5) 7.4 (6.7) 0.9 (-5.7 to 7.6) 0.87*

Table includes all patients who were continuously on the randomized treatment till 2 years after baseline (n=18). EASI, Eczema Area and Severity Index; EASI50, Improvement in EASI with at least 50%; EASI75, Improvement in EASI with at least 75; IGA, Investigator Global Assessment; IGA <2 = Cleared or minimal disease; LOCF, last observation carried forward; PGA, Patient Global Assessment; POEM, Patient-Oriented Eczema Measure; SCORAD, SCORing Atopic Dermatitis; SCORAD50, Improvement in SCORAD with at least 50%; SD, Standard Deviation; VAS, Visual Analogue Scale. ¥ Baseline SCORAD values are values at the first visit of the original study before starting MTX or AZA; * T-test for independent groups; # Fisher’s exact test; ^ Pearson Chi-Square.

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FLG mutation status may therefore be important in relation to the duration of systemic treatment. In conclusion, because AD is a chronic disease, it is imperative that any proposed therapy has persistent efficacy and acceptable side effects during years of use. This study demonstrates that MTX and AZA seem to be appropriate and safe options for the treatment of adult patients with severe AD. As with most observational prospective studies, a limitation is the interpretation of the ITT analysis as patients stop treatment for various reasons over the course of 24 months. Patients with aFLG mutation seemed to have slower but prolonged effects of therapy compared with patients without a mutation.

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REFERENCES

1. Schram ME, Roekevisch E, Leeflang MM, Bos JD, Schmitt J, Spuls PI. A randomized trial of methotrexate versus azathioprine for severe AD. J Allergy Clin Immunol 2011;128:353-9. 2. National Cancer Institute (U.S.). Common terminology criteria for adverse events (CTCAE), Rev. U.S. Dept. of Health and Human Services, National Institutes of Health, National Cancer Institute; 2009. 3. Roekevisch E, Leeflang MM, Schram ME, Campbell LE, Irwin McLean WH, Kezic S, et al. Atopic dermatitis patients with filaggrin loss- of-function mutations show good but lower responses to immunosuppressive treatment. Br J Dermatol 2016 [Epub ahead of print].

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Online repository

METHODS

The methods of the original randomized controlled trial (RCT) have been published previously. E1

Study design

This was an open-label observational follow-up study of patients who participated in a single- blind RCT evaluating the efficacy and safety of MTX versus AZA over a 12-week period. This RCT was conducted between September 2009 and May 2010 at the Academic Medical Center of Amsterdam, the Netherlands.E1 Eligible patients had severe AD, graded at baseline by the Rajka and LangelandE2 criteria and defined by the millennium and UK Working Party Criteria.E3 They were unresponsive, contraindicated, or intolerant to Cyclosporin A and were not treated with MTX or AZA before. Concealment of MTX or AZA was done by a computerized program. Trained investigators and the epidemiologist who did the statistics were blinded for the allocated treatments. Patients and treating physician were not blinded for allocation. All 42 randomized patients who used MTX or AZA were offered the opportunity to enroll in the observational follow-up study in which they would be evaluated 3 monthly over a total period of 2 years (and eventually 5 years) after baseline. Patients had the option to remain under treatment at the Academic Medical Center or to be referred back to their non-academic dermatologist. The last patient had data point 2-year check in April 2012. The local medical ethics committee (institutional review board) approved the observational follow-up study protocol, which was performed in accordance with the Good Clinical Practice Guidelines of the International Conference of Harmonization, Declaration of Helsinki. The trial was registered in the Dutch Trial Register (NTR1916). Written informed consent was obtained from all patients for this observational follow-up study before study-related procedures were commenced. This study report followed the Consolidated Standards of Reporting Trials guidelines.

Treatment regimens Twelve weeks after randomization, patients could continue, switch, or stop the treatment they were randomized to, reflecting normal practice. MTX dose increments were restricted to a maximum of 22.5 mg/wk in combination with 5 mg of folic acid the following day, once a week. AZA dosage was restricted to a maximum of 2.5 mg/kg/d. There was no limitation in

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the use of concomitant topical medication or oral treatment with corticosteroids (if needed as rescue medication). Oral antibiotics were giving according to standard clinical practice usually when the eczema seemed to be infected.

Figure EI. Flowchart

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Figure E2 A. Mean (SD) total SCORAD scores at baseline, 3 months and 24 months (2 years) (intention- to-treat analysis)

AZA, Azathioprine; MTX, Methotrexate.

Figure E2 B. Mean (SD) total SCORAD scores at baseline, 3 months and 24 months (2 years) (per- protocol analysis)

AZA, Azathioprine; MTX, Methotrexate

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Figure E3 A. Mean (SD) SCORAD scores up to 2 years after baseline in patients with and without a filaggrin mutation (intention-to-treat analysis)

FMG, filaggrin mutation group; non-FMG, non filaggrin mutation group. 5

Figure E3 B. Mean (SD) SCORAD scores up to 2 years after baseline in patients with and without a filaggrin mutation (per-protocol analysis)

FMG, filaggrin mutation group; non-FMG, non filaggrin mutation group.

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Treatment outcomes Primary outcomes were differences in mean absolute and relative change in SCORAD indexE4 and Investigator Global Assessment (IGA; 6-point scale, range, 0-5, relative change in IGA, data no shown) between groups after 2 years compared with baseline. Also, the number of patients with a SCORADindex reduction of 50% or more and the difference in number of patients with an IGA score of less than 3 (clear/almost clear/mild disease) were compared between groups after 2 years compared with baseline. Deviating from the study protocol, the SCORAD index was subdivided into objective SCORAD index and subjective SCORAD index. Secondary outcomes were the absolute and relative change in Eczema Area and Severity Index,E5 Patient-Oriented Eczema Measure,E6 Patient Global Assessment (PGA, 5 grades), pruritus and sleeplessness scores on a Visual Analogue Scale, quality of life assessed by the Skindex-17,E7 and the frequency of remissions (PGA <2, clear/almost clear) between groups after 2 years compared with baseline. Deviating from the study protocol, the Skindex-17 was subdivided into symptomatic and psychosocial subscales.E7 Reasons of discontinuation of treatment were subdivided into (1) controlled AD, (2) inefficacy, (3) AE, (4) relapse, and (5) miscellaneous. Controlled AD was defined as improvement in AD (in the opinion of the patient and/or physician) leading to discontinuation of the oral immunosuppressive drug. Inefficacy was registered as a reason for discontinuation when the patient and/or the physician were not satisfied with the clinical response and the treatment was stopped. AEs leading to discontinuation of treatment included both subjective and objective (such as laboratory abnormalities) AEs. A relapse was defined as aworsening of AD (in the opinion of the patient and/or the physician) that resulted in (re)starting an oral immunosuppressive drug. Any other reasons for discontinuation were registered as miscellaneous.

Safety AEs (type, frequency, duration, severity, and relation to MTX or AZA) were determined. AEs that were transient and easily tolerated were considered mild. Moderate AEs were defined as causing discomfort and interrupting the subject’s usual activities. AEs were severe if the event caused considerable interference with the subject’s usual activities and could be incapacitating or life-threatening. A serious AE was defined as a life-threatening event, death, prolonged or initial hospitalization, disability, or permanent damage. AEs were retrospectively coded according to the standardized and recognized Medical Dictionary for Regulatory Activities/Common Terminology Criteria for Adverse Events.E8

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Data collection Treating physicians checked 3 monthly all prescribed concomitant medication (ie, topical calcineurin inhibitors, topical and/or oral antibiotics, corticosteroids [rescue medication]) and AEs. When indicated, routine laboratory evaluations were done (ie, hematology, biochemistry, and urinalysis [including serum pregnancy test]). Blood chemistry included procollagen-3 N-terminal peptide while on MTX and thiopurine methyltransferase before starting AZA. An yearly FibroScan to quantify liver fibrosis was performed when on MTX (FibroScan and P3NP data not shown). Treatment regimens were adjusted according to daily practice. Trained investigators evaluated 3 monthly the objective SCORAD index, IGA, and the Eczema Area and Severity Index. Patients evaluated the subjective SCORAD index, PGA, Patient-Oriented Eczema Measure, pruritus and sleeplessness Visual Analogue Scale, and Skindex-17. Patients and treating physician were not blinded for allocation, but the trained investigators were blinded for the allocated treatments.

TABLE EI. Baseline characteristics of the included and non-included participants

P- Value (of total P-Value MTX AZA Total Total included (of MTX (n=17) (n=18) (n=35) (n=7) vs vs AZA) 5 total not included) Age (y), mean (SD) 43.3 (15.8) 39.5 (14.4) 41.3 (15) 0.46 32.4 (9.6) 0.14* Male sex (%) 10 (59%) 11 (61%) 21 (60%) 1.00# 1 (14%) 0.04# Presence of asthma or allergic 16 (94%) 17 (94%) 33 (94%) 1.00# 4 (57%) 0.03# rhinitis (%) Duration of AD (y), mean (SD) 39.6 (17.5) 34.8 (18.1) 37.1 (17.7) 0.43* 31.9 (9.7) 0.45* Outcomes SCORAD, mean (SD) -Total SCORAD, mean (SD) 59.4 (11.4) 59.7 (11.0) 59.5 (11.0) 0.93* 49.2 (5.8) 0.02* -Objective SCORAD, mean (SD) 46.6 (10.3) 46.3 (10.1) 46.4 (10.1) 0.93* 38.5 (5.2) 0.052* -Subjective SCORAD, mean (SD) 12.8 (4.0) 13.4 (3.4) 13.1 (3.6) 0.59* 10.7 (4.2) 0.10* IGA, median (range) 4.0 (3.0-5.0) 4.0 (3.0-5.0) 4.0 (3.0-5.0) 0.60# 3.0 (3.0-4.0) 0.82# PGA, median (range) 4.0 (3.0-5.0) 4.0 (2.0-5.0) 4.0 (2.0-5.0) 0.84# 4.0 (3.0-4.0) 0.56# EASI, mean (SD) 29.2 (12.8) 31.1 (14.6) 30.2 (13.6) 0.67* 24.4 (10.8) 0.30* POEM, mean (SD) 20.6 (5.2) 20.3 (3.5) 20.4 (4.36) 0.84* 15.7 (4.4) 0.01* Skindex-17, mean (SD) 20.4 (6.2) 20.9 (5.1) 19.9 (5.5) 0.78* 20.7 (5.6) 0.72*

These baseline values apply to patients included in the observational follow-up study (n=35) and are values at the first visit of the original study before starting MTX or AZA. Significant values are shown in bold. * T-test for independent groups; # Fisher’s exact test.

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TABLE EIIA. Two years treatment and dosage overview: Methotrexate group

Weight Patient 2w 4w 8w 3m 6m 9m 1y 15m 18m 21m 2y at BL 1 73 kg M10 M15 M20 M22.5 M20 M22.5 M22.5 M20 M20 M20 M17.5 2 87 kg M10 M15 M20 M22.5 M22.5 M20 M20 M20 M20 M20 M17.5 3 59 kg M103 T T T T T T T T T T 4 143 kg M10 M15 M20 M22.5 M22.5 M20 M20 M20 M20 M20 M20 5 100 kg M10 M15 M20 M22.5 M22.5 M10 M12.5 M12.5 M12.5 M12.5 M10 6 71 kg M10 M15 M20 M22.5 M22.5 M20 M12.5 M12.5 M12.5 M12.5 M10 7 85 kg M10 M15 M20 M22.5 M10 M12.5 M12.5 M15 M15 M15 M15 8 67 kg M10 M15 M20 M22.5 M22.5 M12.5 M12.5 M152 A50 A50 A50 9 55 kg M10 M15 M15 M20 M20 M15 M15 M15 M12.5 M12.5 M10 10 94 kg M10 M15 M20 M22.5 M20 M17.5 M10 M10 M10 M102 T 11 76 kg M10 M15 M15 M20 M15 M153 T T T T T 12 64 kg M10 M15 M15 M15 M15 M15 M15 M15 M12.5 M12.5 M12.5 13 65 kg M10 M15 M20 M22.5 M22.5 M22.5 M151 T T T T 14 85 kg M10 M15 M20 M20 M202 A150 A1504 T T T T 15 85 kg M10 M15 M20 M22.5 M22.5 M17.5 M15 M15 M15 M15 M12.5 16 52 kg M10 M15 M20 M22.5 M17.5 M20 M20 M7.51 T T T 17 65 kg M10 M15 M20 M22.5 M22.5 M22.5 M22.5 M17.5 M20 M20 M20 Mean dosis MTX 14.5 mg

A150, Azathioprine 150 mg; m, months; MTX, Methotrexate; M10, Methotrexate 10 mg; T, topical; w, week;; y, year. Reasons for discontinuation of systemic treatment: 1) controlled AD, 2) inefficacy, 3) adverse event (AE), 4) miscellaneous. Reasons to start systemic treatment: 5) relapse. SAE Framed-months stands for the months in which the patient experienced a SAE.

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TABLE EIIB. Two years treatment and dosage overview: Azathioprine group

Weight Patient 2w 4w 8w 3m 6m 9m 12m 15m 18m 21m 24m at BL 1 75 kg A125 A150 A175 A175 A175 A150 A1501 T T T 5 A125 2 78 kg A125 A150 A200 A200 A2001 T T T 5 A100 A100 A100 3 55 kg A50 A753 M10 M15 M17.5 M17.5 M12.5 M12.5 M12.5 M10 M10 4 70 kg A100 A150 A175 A200 A2001 T T T T T T 5 74 kg A125 A150 A175 A175 A175 A125 A150 A175 A175 A175 A200 6 63 kg A100 A125 A125 A125 A1252 MTX?2 P 1 T P P P 7 87 kg A125 A175 A175 A175 A200 A175 A175 A150 A150 A150 A175 8 73 kg A100 A150 A175 A1752 M152 C2 T T T T T 9 89 kg A125 A175 A225 A225 A200 A200 A200 A200 A200 A175 A100 10 68 kg A100 A125 A150 A150 A150 A125 A125 A125 A150 A150 A150 11 81 kg A125 A150 A200 A125 A125 A1251 T T T T 5 A100 12 79 kg A125 A150 A200 A200 A200 A175 A200 A200 A200 A200 A200 13 105 kg A150 A200 A275 A275 A250 A225 A2502 T T T T 14 87 kg A125 A150 A200 A200 A200 A200 A150 A175 A150 A125 A125 15 95 kg A150 A125 A175 A175 A150 A150 A150 A125 A125 A150 A100 5 16 65 kg A100 A125 A125 A1252 T T T T T T T 17 68 kg A100 A100 A100 A100 A75 A50 A251 T T T T 18 90 kg A125 A175 A175 A175 A175 A175 A175 A150 A150 A125 A125 Mean dosis AZA 136.4 mg

AZA, Azathioprine; A150, Azathioprine 150 mg; C, Cellcept; m, months; MTX?, Methotrexate unknown dose; M10, Methotrexate 10 mg; P, Prednisolone; T, topical; w, week . Reasons for discontinuation of systemic treatment: 1) controlled AD, 2) inefficacy, 3) adverse event (AE), 4) miscellaneous. Reasons to start systemic treatment: 5) relapse. SAE Framed-months stands for the months in which the patient experienced a SAE.

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TABLE EIII A Adverse events and other key safety data up to 2 years after baseline in patients (intention-to-treat analysis)

Total AEs Total Total AEs Total Total Total AZA AEs MTX MTX AEs AZA Variable AEs AEs/pt group/ P-value group group/pt group (n=35)a (n=35) pt (n=17) (n=17) (n=18) (n=18) AE, total (events)*, no. of pt 422 1.06 220 12,94 202 11.22 0.14 - Mild, no. of pt 372 10.63 198 11,65 174 9.67 0.07 - Moderate, no. of pt 45 1.29 21 1,24 24 1.33 0.80 - Severe, no. of pt 5 0.14 1 0,06 4 0.22 0.20 SAE, total (events)*, no. of pt 3 0.09 2 0,12 1 0.06 0.53 Treatment-related AE§, no. 252 7.20 133 7,82 119 6.61 0.18 of pt AE requiring dose adjustment, 6 0.17 4 0,24 2 0.11 0.38 no. of pt Type of AE coded according to MedDRA, no. of pt Blood and lymphatic system 34 0.97 11 0.65 23 1.28 0.058 disorders Cardiac disorders 9 0.26 4 0.24 5 0.28 0.80 Eye disorders 7 0.20 5 0.29 2 0.11 0.23 Gastrointestinal disorders 44 1.26 26 1.53 18 1.00 0.16 General disorders and 46 1.31 22 1.29 24 1.33 0.92 administration site conditions Hepatobiliary disorders 20 0.57 11 0.65 9 0.50 0.57 Immune system disorders 1 0.03 0 0.00 1 0.06 0.33 Infections and infestations 98 2.80 52 3.06 46 2.56 0.37 Ear and Labyrinth • 2 0.06 1 0.06 1 0.06 0.97 disorders • Eye disorders 2 0.06 2 0.12 0 0.00 0.15 • General disorders and administration site 11 0.31 2 0.12 9 0.50 0.04 conditions • Musculoskeletal and connective tissue 9 0.26 3 0.18 6 0.33 0.36 disorders • Neoplasms benign, malignant and 4 0.11 4 0.24 0 0.00 0.04 unspecified ** Renal and urinary • 5 0.14 4 0.24 1 0.06 0.16 disorders Reproductive system and • 1 0.03 0 0.00 1 0.06 0.33 breast disorders Respiratory, thoracic and • 27 0.77 15 0.88 12 0.67 0.46 mediastinal disorders

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Skin and subcutaneous • 37 1.06 21 1.24 16 0.89 0.31 tissue disorders - Bacterial 19 0.54 11 0.65 8 0.44 0.41 - Viral 10 0.29 5 0.29 5 0.28 0.93 - Fungal 6 0.17 3 0.18 3 0.17 0.94 Injury, poisoning and 8 0.23 4 0.24 4 0.22 0.94 procedural complications Metabolism and nutrition 1 0.03 0 0.00 1 0,06 0.33 disorders Musculoskeletal and 28 0.80 16 0.94 12 0.67 0.36 connective tissue disorders Neoplasms benign, malignant 9 0.26 3 0.18 6 0.33 0.36 and unspecified ** Nervous system disorders 34 0.97 20 1.18 14 0.78 0.23 Psychiatric disorders 2 0.06 0 0.00 2 0.11 0.17 Renal and urinary disorders 11 0.31 6 0.35 5 0.28 0.69 Reproductive system and 4 0.11 2 0.12 2 0.11 0.95 breast disorders Respiratory, thoracic and 16 0.46 10 0.59 6 0.33 0.27 mediastinal disorders Skin and subcutaneous tissue 47 1.34 26 1.53 21 1.17 0.36 disorders Surgical and medical 3 0.09 2 0.12 1 0.06 0.53 5 procedures Type of SAE coded according to MedDRA, no. of pt Injury, poisoning and 1 0,03 1 0.06 0 0.00 0.30 procedural complications Skin and subcutaneous tissue 2 0,06 1 0.06 1 0.06 0.97 disorders

*Adverse events were classified as mild, moderate, severe or serious according to predefined definitions (see the Methods section) . **No malignant neoplasm were found in both groups.. § Treatment-related adverse events are those classified as possibly, probably, or definitely related to the study drug by the safety assessor. Significant values are shown in bold. AE, Adverse Event; AZA, Azathioprine; MedDRA, Medical Dictionary for Regulatory Activities; MTX, Methotrexate; SAE, Serious adverse events; pt, patient.

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TABLE EIIIB. Adverse events and other key safety data up to 2 years after baseline in patients who were continuously on allocated treatment (per-protocol analysis)

Total Total AEs Total Total AEs Total Total AEs AZA P-value AEs MTX MTX Variable AEs AEs/pt AZA group/ MTX vs group group/pt (n=18) (n=18) group pt AZA (n=10) (n=10) (n=8) (n=8) Total AE*, no. of pt 203 11.3 122 12.2 81 10.1 0.19 • Mild, no. of pt 183 10.2 107 10.7 76 9,5 0,43 • Moderate, no. of pt 18 1 15 1.5 3 0.4 0,02 • Severe, no. of pt 2 0.1 0 0 2 0.3 0.11 Total serious AE*, no. of pt 2 0.1 1 0.1 1 0.1 0.87 Treatment-related AE §, no. 131 7.3 76 7.6 55 6.9 0.57 of pt AE requiring dose adjustment, 4 0.2 3 0.3 1 0.1 0.43 no. of pt Type of AE coded according to MedDRA, no. of pt Blood and lymphatic system 13 0.7 5 0.5 8 1 0.22 disorders Cardiac disorders 4 0.2 1 0.1 3 0.4 0.22 Eye disorders 4 0.2 3 0.3 1 0.1 0.43 Gastrointestinal disorders 23 1.3 16 1.6 7 0.9 0.18 General disorders and 24 1.3 9 0.9 15 1.9 0.08 administration site conditions Hepatobiliary disorders 13 0.7 11 1.1 2 0.3 0.04 Immune system disorders 1 0.1 0 0 1 0.1 0.26 Infections and infestations 44 2.4 29 2.9 15 1.9 0.17 • Eye disorders 1 0.1 1 0.1 0 0 0.37 • General disorders and administration site 7 0.4 1 0.1 6 0.8 0.03 conditions • Musculoskeletal and connective tissue 2 0.1 0 0 2 0.3 0.11 disorders Neoplasm benign, malig- • 1 0.1 1 0.1 0 0 0.37 nant and unspecified ** Renal and urinary • 3 0.2 3 0.3 0 0 0.12 disorders Reproductive system and • 1 0.1 0 0 1 0.1 0.26 breast disorders Respiratory, thoracic and • 14 0.8 12 1.2 2 0.3 0.02 mediastinal disorders Skin and subcutaneous • 15 0.8 11 1.1 4 0.5 0.17 tissue disorders - Bacterial 11 0.6 7 0.7 4 0.5 0.59 - Viral 3 0.2 3 0.3 0 0 0.12

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Injury, poisoning and 5 0.3 4 0.4 1 0.1 0.27 procedural complications Metabolism and nutrition 1 0.1 0 0 1 0.1 0.26 disorders Musculoskeletal and 12 0.7 7 0.7 5 0.6 0.85 connective tissue disorders Neoplasms benign, malignant 8 0.4 3 0.3 5 0.6 0.30 and unspecified ** Nervous system disorders 18 1 11 1.1 7 0.9 0.64 Renal and urinary disorders 7 0.4 4 0,4 3 0.4 0.93 Respiratory, thoracic and 10 0.6 7 0,7 3 0.4 0.36 mediastinal disorders Skin and subcutaneous tissue 14 0.8 10 1 4 0.5 0.23 disorders Surgical and medical 2 0.1 2 0.2 0 0 0.21 procedures Type of SAE coded according to MedDRA, no. of pt Injury, poisoning and 1 0.06 1 0.1 0 0 0.37 procedural complications Skin and subcutaneous tissue 1 0.06 0 0 1 0.1 0.26 disorders

*Adverse events were classified as mild, moderate, severe or serious according to predefined definitions (see the Methods section). **No malignant neoplasm were found in both groups.. § Treatment-related adverse events are 5 those classified as possibly, probably, or definitely related to the study drug by the safety assessor. Significant values are shown in bold. AE, Adverse event; AZA, Azathioprine; MedDRA, Medical Dictionary for Regulatory Activities; MTX, Methotrexate; SAE, Serious adverse event; pt, patient.

Filaggrin During this observational follow-up study, a buccal swab was taken to investigate the FLG mutation status (R501X, 2282del4, R2447X, S3247X, and 3321delA mutations). Patients were divided into an FMG group and a non-FMG group to assess the effect of FLG mutations on primary and secondary outcomes.

Statistical analysis In the ITT analysis we included all patients who participated in this observational follow- up study (35 patients), with the last available observation carried forward method between baseline and 2 years. In the PP analysis, we included patients who were continuously on the allocated systemic treatment for 2 years. We used the t test for normally distributed continuous outcome measures and Fisher exact test for dichotomous outcome measures. AEs were analyzed by using the chi-square test. Two-sided P-values of less than.05 were considered to indicate statistical significance. Analyses were done in SPSS 22.0 for Windows (SPSS, Inc, Chicago, Ill).

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Roekevisch E*, Szegedi K*, Hack DP, Schram ME, Res PCJM, Bos JD, Leeflang MMG, Luiten RM, Kezic S, Spuls PI, Middelkamp-Hup MA. * Contributed equally

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ABSTRACT

Background Biomarkers to objectively measure disease severity and predict therapeutic responses are needed in atopic dermatitis (AD).

Objective Primary aim: to identify biomarkers reflecting therapeutic response in patients withAD treated with systemic treatment. Secondary aims: i) to identify a biomarker predicting responsiveness to systemic treatment. ii) to identify differences in biomarkers in filaggrin-gene (FLG) mutation carriers versus non- FLG mutations carriers.

Methods Thirty-eight severe AD patients treated with methotrexate or azathioprine participated in this study. Blood samples were collected to analyze serum levels of APRIL, BAFF, TARC (CCl- 17), IL-1RA, IL-1β, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-18, IL-31, IFN-γ, TNF-α, VEGF, MIG (CXCL-9), IP-10 (CXCL-10), MCP-1 (CCL-2), MIP-1β (CCL-4), RANTES (CCL-5), CTACK (CCL- 27), TSLP, IL-5, IL-1α and G-CSF by ELISA and Luminex. The primary outcomes were differences in mean absolute change of SCORAD between groups after 12 weeks compared to baseline. Responders to treatment were defined by a SCORAD reduction of ≥50%. Buccal mucosa swabs were collected to determine the FLG genotype status.

Results Serum levels of TARC, CTACK, IL-13 and VEGF showed a significant decrease after treatment with methotrexate or azathioprine. However, decreased cytokine levels were not significantly correlated with change in outcome parameters. Baseline biomarker levels were not significantly different between responders and non-responders, and FLG and non-FLG mutants showed similar biomarker profiles.

Conclusion TARC and CTACK were confirmed as potential biomarkers. VEGF and IL-13 have a potential value as well. Biomarkers could not be used to discriminate at baseline between responders and non-responders, or FLG genotype status.

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Introduction

Atopic dermatitis (AD) is characterized by an epidermal barrier dysfunction and an immune dysregulation favoring a Th2 cellular response. (1) A method to objectively measure disease severity and to predict therapeutic responses in AD is wanted, and much research is being done to evaluate biomarkers in AD. A systematic review and meta-analysis (2) showed that serum thymus and activation-regulated chemokine (TARC) is the superior biomarker for assessing disease severity. T-cell attracting chemokine (CTACK), sE-selectin, macrophage- derived chemokine (MDC), lactate dehydrogenase (LDH) and interleukin (IL)-18 could be useful as a severity biomarker as well, but need to be validated in additional studies. Three other promising biomarkers are BAFF (B-cell activating factor of the TNF family), APRIL (a proliferation-inducing ligand) and IL-31, although contradictory conclusions are published which makes the importance unclear (3-12). IL-4 and IL-13 are believed to have a role in the pathogenesis of AD, because the frequency of IL-4 and IL-13-producing cells in AD patients is significantly higher than in healthy subjects (in both CD4+ and CD8+ T-cell subsets).(13) In addition, Dupilumab, which blocks IL-4 and IL-13, has shown efficacy in patients with (14)AD. Although some promising results on potential biomarkers for AD have been published, more research is needed to find a reliable biomarker for disease severity and especially for predicting immunosuppressive treatment responsiveness in AD patients, that can be used in clinical practice. 6 In the present study, the primary aim was to identify biomarkers that reflect the therapeutic response in patients with AD that were treated with systemic immunosuppressive treatment. Secondary aims were i) to identify a biomarker that predicts responsiveness to systemic immunosuppressive treatment, and ii) to identify differences in biomarker profiles in filaggrine-gene (FLG) mutation carriers versus non-carriers. For this purpose, we analysed blood samples of patients with severe AD who participated in a single blinded randomized controlled trial (RCT) with methotrexate (MTX) or azathioprine (AZA), after 12 weeks of treatment.(15)

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Materials and methods

The methods of the original RCT, containing inclusion and exclusion criteria, concealment of treatment and treatment regimens have been published previously.(15)

Study population and treatment protocol This is a sub-study of patients who participated in a single blinded RCT evaluating efficacy and safety of MTX versus AZA over a 12-week period.(15) All 42 randomized patients were asked to donate blood samples for biomarker determinations. Healthy volunteers were also recruited. Inclusion criteria for the healthy volunteers were no personal or family history of AD, asthma, allergic rhinitis or allergic conjunctivitis. Primary outcomes of treatment were differences in mean absolute change of SCORing Atopic Dermatitis (SCORAD) between groups after 12 weeks compared to baseline. Deviating from the study protocol, the SCORAD was also subdivided in objective SCORAD. Secondary outcomes were the absolute change of Eczema Area And Severity Index (EASI), pruritus and sleeplessness scores on a Visual Analogue Scale (VAS, range 0-10). EASI and SCORAD assessments were provided by a blinded assessor. VAS itch and VAS sleeplessness were provided by the patient. As both MTX and AZA induced a significant reduction in SCORAD at 12 weeks without a significant difference in effect between treatments(15), all patients were pooled together and divided in responders and non-responders. Responders to treatment were defined as patients with a SCORAD reduction of 50% or more 12 weeks after baseline (SCORAD50), and biomarkers of responders and non-responders were compared at baseline. This study was conducted with institutional research ethics committee approval (Academic Medical Center Amsterdam) and all patients and healthy volunteers signed informed consent which complied with all the Declaration of Helsinki Principles.

Blood sample collection Peripheral serum blood samples were collected by venipuncture at enrollment before starting treatment (week 0, baseline) and 12 weeks after using MTX or AZA. Peripheral serum blood samples of healthy volunteers were collected by venipuncture at one not further specified time point.

Filaggrin genotyping A buccal swab was taken to investigate the FLG mutation status (R501X, 2282del4, R2447X, S3247X and 3321delA mutations). Patients were pooled together and divided into FLG

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mutation carriers and non-FLG mutation carriers. During the RCT, no one was aware of the mutation status. More information about the methods have been published previously.(16)

Cytokine and chemokine analyses IL-31, APRIL and BAFF in serum samples were measured using specific ELISA kits. IL-31 measurement (R&D Systems Europe Ltd., Abingdon, UK) was performed according to manufacturer’s instructions with the following modifications: diluent was composed of PBS (Fresenius Kabi, Zeist, The Netherlands) supplemented with 50% fetal bovine serum (Lonza Verviers, Sprl., Verviers, Belgium) and used for dilution of standard and detection antibody. Blocking step was performed with 3% Milk (Nutricia Nederland BV, Zoetermeer, The Netherlands) in PBS. Capture antibody was diluted in a working concentration of 1.6 µg/ mL instead of 0.8 µg/mL. Streptavidin-HRP was diluted to a working concentration of 1:300 instead of 1:200. The limit of detection was 63-4000 pg ⁄ ml. APRIL (eBioscience, Hatfield, UK) measurement was performed according to the manufacturer’s instructions. The limit of detection was 780-25000 pg/mL. BAFF (Antigenix America Inc., Huntington Station, NY, USA) assay was conducted according tothe manufacturer’s instructions with the following modification: the blocking step was performed with 3% Milk in PBS. The limit of detection was 156-10000 pg/mL. For all ELISAs the stop solutions were added after monitoring the color development at 620 nm. The substrate reaction was stopped when the highest standard had reached an OD of 9.0-9.5. 6 Levels of TARC were assessed using a Luminex-based multiplex system (Millipore BV, Amsterdam, The Netherlands), according to the manufacturer’s instructions. The limit of detection was 9.8-10.000 pg/mL. A Luminex-based multiplex system (Bio-Rad Laboratories BV, Veenendaal, The Netherlands) was used to determine the levels of IL-1α, IL-1RA, IL-1β, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-18, IFN-γ, TNF-α, VEGF, G-CSF, MIG (CXCL-9), IP-10 (CXCL-10), MCP-1 (CCL-2), MIP-1β (CCL-4), RANTES (CCL-5) and CTACK (CCL-27). Levels of TSLP were assessed using a Luminex-based multiplex system (Millipore BV, Amsterdam, The Netherlands), according to the manufacturer’s instructions. The limit of detection was 9.8-10.000 pg/mL. If the Luminex panel measured a value below the detection limit than half of that value was assigned to the cytokine/chemokine. A patient was excluded for analysis if that patient had a missing value of a cytokine/chemokine. Besides that, only cytokines/chemokines with serum levels that were significantly different (P < 0.05) from the healthy volunteer group were analysed, unless stated otherwise. If a cytokine/chemokine had more than 50% values below the lower limit of detection (LLD) evaluated in the total AD group, than that specific cytokine/chemokine would not participate

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in the analyses. This criterion is based on the article by Domthong et al. (17) More than 50% of the AD patients had values below the lower limit of detection (LLD) at baseline for TSLP (60%), IL-5 (62%), IL-1α (>95%) and G-CSF (>95%) and these cytokines were excluded from analysis.

Statistical analysis Data on patients characteristics were analysed using SPSS 23.0 for Windows (SPSS, Inc., Chicago, Ill).) The distribution of dependent data was assessed by D’Agostino & Pearson omnibus normality test. Medians with 1st and 3rd inter-quartiles (IQR: Q1, Q3) were used for non-normal distributed data and means with standard deviation were used for normal distributed data. Biomarker data obtained by ELISA and Luminex data were analysed using Graphpad Prism 6.07 (GraphPad Software, Inc, San Diego, Cal). All investigated biomarkers showed a non- normal distribution of data. If the natural logarithms (Ln) of the biomarker values showed a normal distribution, then these values were used in the analyses. Correlations were performed with the Pearson correlation coefficient on normal-distributed data and Spearman rank test on non-normal distributed data. Comparison analysis of non- normal distributed data was performed with Wilcoxon matched-pairs signed rank tests (paired data) and Mann-Whitney U tests (unpaired data). Comparison analysis of normal distributed data was performed with two-sided paired t-tests (paired data) and unpaired t-test (unpaired data). Due to multiple testing the P-value was adjusted using the Bonferroni correction (alfa/ m) were alpha was 0.05 and m is the number of tested hypotheses.

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RESULTS

Patients’ characteristics.

MTX vs AZA Of the 43 randomized patients, 38 patients were included in this sub-study (Figure 1). In the AZA group, 1 patient did not start on study medication, 1 did not adhere to the study protocol and 2 discontinued because of adverse events. In the MTX group 1 patient did not complete the study due to an AE. As no blood samples were collected from these 5 patients at week 12, these patients were excluded. Patients’ characteristics and baseline values of these 38 patients are shown in Table 1.

Figure 1. Flow chart of the randomization, treatment and blood sample collection of study participants

AE Adverse Event

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Table 1. Patients’ baseline characteristics FLG non-FLG Non- Total MTX AZA Responders* mutation mutation Responders (n=38) (n=19) (n=19) (n=15) carriers carriers (n=23) (n=12) (n=22) Age (y), mean 39.8 (14.2) 41.5 (13.7) 38.0 (14.9) 39.3 (13.1) 40.0 (15.2) 50.6 (12.9) 34.8 (12.4) (SD)

Male sex (%) 21 (55%) 10 (53%) 11 (58%) 8 (53%) 13 (57%) 7 (58%) 13(59%)

Presence of asthma or 35 (92%) 18 (95%) 17 (89%) 14 (93%) 21 (91%) 12 (100%) 19 (86%) allergic rhinitis (%) Received MTX 19 (50%) 19 (100%) - 7 (47%) 12 (52%) 6 (50%) 11 (50%) (%) Received AZA 19 (50%) - 19 (100%) 8 (53%) 11 (48%) 6 (50%) 11 (50%) (%) Presence of allergen-specific 38 (100%) 19 (100%) 19 (100%) 15 (100%) 23 (100%) 12 (100%) 22 (100%) IgE (%) Duration of AD (y), 35.9 (16.6) 38.3 (15.1) 33.5 (18.1) 34.9 (14.7) 36.6 (18.1) 50.3 (13.1) 28.8 (14.0) mean (SD) OUTCOME Total SCORAD, 57.8 (11.4) 57.1 (12.2) 58.4 (10.8) 59.8 (11.1) 56.4 (11.6) 59.7 (13.2) 57.6 (10.7) mean (SD) Objective SCORAD, 45.4 (10.1) 45.0 (10.4) 45,6 (10.3) 47.9 (9.9) 43.8 (10.2) 49.6 (11.3) 43.9 (9.5) mean (SD)

EASI, mean (SD) 28.2 (13.4) 27.6 (12.6) 30.1 (14.6) 30.7 (13.9) 27.6(13.4) 33.6 (14.1) 26.6 (13.7)

VAS-itch, mean 7.4 (1.7) 7.2 (1.8) 7,6 (1,6) 7,8 (1,0) 7.1 (2.0) 6.7 (2.4) 7.7 (1.2) (SD) VAS-sleep loss, 5.5 (2.6) 4.9 (2.7) 6.0 (2,5) 5.3 (2.3) 5.5 (2.8) 5.0 (2.8) 5.9 (2.3) mean (SD)

AZA, Azathioprine; MTX, Methotrexate; FLG, Filaggrin-gene; Y, Year; SD, Standard Deviation. * Responders to treatment were defined by a SCORAD reduction of ≥50% 12 weeks after baseline. Non-responders: SCORAD <50% 12 weeks after baseline.

Response to treatment In the original study, after 12 weeks of treatment, both the MTX group and AZA group showed significant reductions in SCORAD, without a significant difference between treatments.(15) In this sub-study, of the total group of 38 AD patients, 15 (39%) were responders and 23 (61%) were non-responders. Of the responders 7 (47%) received MTX and 8 (53%) received AZA.

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FLG mutation carriers versus non-carriers Of the 38 AD patients, 12 (38%) had a FLG-mutation, 22 had no FLG mutation (58%) and in 4 (11%) patients the mutation could not be determined because they were lost to follow up, could not be reached by phone or email or were unwilling to participate. Of theFLG mutation carriers 6 (47%) received MTX and 6 (53%) received AZA. Of the non-FLG mutation carriers 11 (50%) received MTX and 11 (50%) received AZA. Age was significantly higher in patients with a FLG mutation (P=0.001, unpaired t-test) compared to the non-FLG mutation carriers with 50.7 and 34.8 years (given as means), respectively.

TARC, VEGF, IL-13 and CTACK reflect responsiveness to systemic immunosuppressive treatment, as levels decrease significantly during treatment. At baseline, 16 cytokines/chemokines were significantly different in AD patients compared to healthy volunteers, namely APRIL, BAFF ,TARC (CCL-17), IL-1RA, IL-9, IP-10 (CXCL-10), VEGF, IL-1β , IL-6, IL-7, IL-13, MCP-1 (CCL-2), MIP-1β (CCL-4), IL-18, CTACK (CCL-27), MIG (CXCL-9). These biomarkers were included in subsequent data analysis. Differences between baseline and week 12 were considered significant if P-value <0.0031 (Bonferroni correction, 0.05/16 (number of selected cytokines)) An overview of the cytokines/chemokines levels at baseline and after 12 weeks of systemic treatment with MTX and AZA are shown in Table 2. 6 In the total AD group, after 12 weeks of treatment , TARC (CCL-17) (Mean Difference (MD) -403.8 (-734.5 - -53.72, P < 0.0001), VEGF (MD -9.54 (-28.64-0.0), P <0.0001), IL-13 (MD -0.47 (-1.20-0.0), P= 0.003) and CTACK (MD -434.7 (-1381 - -196.7), P <0.0001) showed a significant (P<0.0031) decrease. Without Bonferroni correction, IL-6, MCP-1 (CCL-2), MIP-1β (CCL-4), IL-18 were found significant as well in this group. In the MTX group this reduction in levels was significant for IL-18 (MD -9.23 (-19.70 - -0.97), P= 0.001) and CTACK (MD -562.5 (-1340 - -191.1), P=0.0002). In the AZA group this was significant for VEGF (MD -7.19 (-28.71-0.0), P=0.001). The P-value (P <0.05) for the absolute difference between the MTX and AZA groups was only significant for TARC (P=0.02), as TARC levels decreased more under treatment with MTX than with AZA.

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Table 2. Serum levels of cytokines/chemokines under systemic treatment with MTX and AZA P-value MTX vs AZA of Mean Difference the absolute P- Week 0 Week 12 between week 12 difference value and week 0 between week 12 and week 0 APRIL (pg/mL), median (IQR) Total AD group (n=38) 1391 (1109-1920) 1397 (953-1986) 0.68 28.0 (-2993-124.8) 0.72 MTX (n=19) 1430 (863-1921) 1397 (911-1970) 0.98 36.0 (-198.0–123.0) AZA (n=19) 1360 (1120-1920) 1397 (962-2002) 0.65 20.0 (-335.0–143.0) BAFF (pg/mL), median (IQR) Total AD group (n=35) 96.0 (26.0-364.0) 222.0 (42.0-410.0) 0.47 0.0 (-44.0–106.0) 0.06 MTX (n=17) 70.0 (29.0-212.0) 192.0 (33.0-282.0) 0.07 84.0 (-26.0–194.0) AZA (n=18) 160.0 (26.0-670.0) 248.0 (46.50-557.0) 0.46 0.0 (-151.5–38.50) TARC (pg/mL), median (IQR) Total AD group (n=38) 1254 (895.4-2455) 827.2 (479.9-1733) 0.0001 -403.8 (-734.5 - -53.72) 0.02 MTX (n=19) 2166 (1142-2809) 828.3 (492.5-1717) 0.004 -700.0 (-1595 - -125.0) AZA (n=19) 1038 (604.6-2113) 826.1 (381.0-2098) 0.03 -231.9 (-471.0–30.36) IL-1RA (pg/mL), median (IQR) Total AD group (n=38) 7.83 (0.07-29.37) 4.77 (0.07-16.79) 0.15 0.0 (-7.91–0.0) 0.80 MTX (n=19) 7.05 (0.07-16.79) 5.51 (0.07-13.46) 0.21 -1.13 (-9.74–2.50) AZA (n=19) 10.21 (0.07-49.03) 1.20 (0.07-30.70) 0.37 0.0 (-2.50-0.0) IL-9 (pg/mL), median (IQR) Total AD group (n=38) 23.91 (3.05-43.13) 18.42 (3.52-30.61) 0.24 0.0 (-12.73-3.55) 0.89 MTX (n=19) 23.91 (15.17-40.84) 16.25 (4.75-30.61) 0.50 0.0 (-13.35-8.91) AZA (n=19) 21.70 (0.92-66.52) 21.70 (0.92-30.61) 0.39 0.0 (-12.52-1.10) IP-10 (pg/mL), median (IQR) Total AD group (n=38) 685.6 (523.7-1042) 636.3 (501.5-898.5) 0.27 -40.21 (-214.0-96.59) 0.89 MTX (n=19) 686.3 (613.3-1183) 858.4 (531.3-922.4) 0.62 -33.82 (-266.7-187.3) AZA (n=19) 680.8 (452.3-863.4) 596.2 (470.7-791.0) 0.23 -42.95 (-134.3-71.23) VEGF (pg/mL), median (IQR) Total AD group (n=38) 32.38 (8.14-60.10) 14.40 (0.95-37.68) <0.0001 -9.54 (-28.64-0.0) 0.72 MTX (n=19) 39.11 (15.57-94.06) 29.70 (0.95-66.50) 0.005 -12.84 (-24.07-0.0 AZA (n=19) 29.56 (8.14-48.61) 4.06 (0.95-22.76) 0.001 -7.19 (-28.71-0.0) IL-1β (pg/mL), median (IQR) Total AD group (n=38) 0.14 (0.03-0.43) 0.11 (0.03-0.43) 0.44 0.0 (-0.12 -0.05) 0.67 MTX (n=19) 0.14 (0.08-0.31) 0.14 (0.03-0.43) 0.45 0.0 (-0.17-0.06) AZA (n=19) 0.08 (0.03-0.66) 0.08 (0.03-0.54) 0.81 0.0 (-0.12-0.05) IL-6 (pg/mL), median (IQR) Total AD group (n=38) 2.01 (0.38-3.45) 1.41 (0.17-2.09) 0.02 -0.26 (-1.71-0.09) 0.42 MTX (n=19) 2.09 (0.72-3.45) 1.41 (0.72-2.09) 0.45 -0.34 (-1.71-0.69) AZA (n=19) 1.92 (0.03-3.79) 1.07 (0.03-1.92) 0.007 0.0 (-1.71-0.0)

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IL-7 (pg/mL), median (IQR) Total AD group (n=38) 8.40 (7.20-10.40) 8.27 (6.70-10.46) 0.67 -0.13 (-2.14-1.59) 0.79 MTX (n=19) 8.27 (7.01-11.27) 8.27 (6.50-9.27) 0.64 0.0 (-1.75-0.75) AZA (n=19) 8.52 (7.26-10.27) 8.77 (6.76-12.02) 0.95 -0.51 (-2.53-2.99) IL-13 (pg/mL), median (IQR) Total AD group (n=38) 1.81 (0.85-3.17) 1.18 (0.04-2.76) 0.003 -0.47 (-1.20-0.0) 0.95 MTX (n=19) 1.81 (0.33-3.02) 1.65 (0.15-2.57) 0.10 -0.34 (-1.47-0.11) AZA (n=19) 1.50 (1.02-3.61) 0.85 (0.04-3.61) 0.008 -0.60 (-0.98-0.0) MCP1 (pg/mL), median (IQR) Total AD group (n=38) 50.67 (38.18-85.12) 53.40 (33.04-68.95) 0.01 -9.46 (-26.26-5.94) 0.95 MTX (n=19) 45.65 (39.40-78.55) 50.97 (41.70-69.33) 0.07 -8.92 (25.09-5.35) AZA (n=19) 54.21 (31.06-96.61) 55.64 (30.44-67.29) 0.07 -10.24 (-27.92-9.57) MIP-1b (pg/mL), median (IQR) Total AD group (n=38) 114.3 (91.98-152.1) 111.8 (80.28-134.7) 0.004 -12.01 (-25.89 - -1.82) 0.91 MTX (n=19) 111.1 (91.14-180.3) 109.1 (86.38-142.0) 0.02 -11.31 (-27.36 - -2.65) AZA (n=19) 117.5 (92.26-151.8) 112.1 (64.52-131.8) 0.07 -13.29 (-25.73-0.87) IL-18 (pg/mL), median (IQR) Total AD group (n=38) 57.75 (45.64-80.94) 53.61 (36.51-68.93) 0.01 -7.62 (-20.0-2.57) 0.37 MTX (n=19) 57.50 (44.92-77.15) 48.76 (36.86-62.42) 0.001 -9.23 (-19.70 - -0.97) AZA (n=19) 58.98 (45.88-82.46) 54.58 (34.99-99.68) 0.35 -2.89 (-25.85-11.14) CTACK (pg/mL), median (IQR) Total AD group (n=38) 2125 (1420-3108) 1423 (1046-2047) <0.0001 -434.7 (-1381 - -196.7) >0.99 MTX (n=19) 2195 (1396-3059) 1435 (1049-2004) 0.0002 -562.5 (-1340 - -191.1) AZA (n=19) 2035 (1428-3258) 1411 (1037-2175) 0.004 -425.8 (-1545 - -245.7) 6 MIG (pg/mL), median (IQR) Total AD group (n=38) 1091 (765.3-2030) 1077 (643.1-1695) 0.31 -30.99 (-365.4-161.5) 0.08 MTX (n=19) 1932 (1004-2175) 1111 (703.1-1915) 0.06 -263.0 (-1112-106.5) AZA (n=19) 921.6 (672.1-1641) 839.4 (579.4-1621) 0.60 -7.59 (-108.8-268.7)

AD, Atopic Dermatitis; AZA, Azathioprine; IQR, InterQuartile Range; MTX, Methotrexate Differences between baseline and week 12 were considered significant with a P-value <0.0031 (Bonferroni correction, 0.05/16). The P-value of week 0 vs week 12 was analysed by the Wilcoxon matched-pairs signed rank test. The P-value of Δ absolute value (MTX vs AZA) was analysed with the Man-Whitney U test.

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Decreased cytokine levels were not significantly correlated with the change in outcome parameters. Cytokines TARC (CCL-17), VEGF, CTACK (CCL-27) and IL-13 that in the total group showed a significant decrease on systemic treatment were analysed to evaluate if the decrease correlated with the change in outcome parameters. The total SCORAD consists of the oSCORAD, VAS-itch and VAS- sleep loss. For the Bonferroni correction they were clustered so 2 outcome parameters (SCORAD and EASI) and 4 cytokines/ chemokines resulted in 8 correlations leading to a Bonferroni correction with a P-value of 0.05/8=0.006. Based on a Bonferroni corrected P-value of 0.006, we found that decreased cytokine levels were not significantly correlated with change in primary and secondary outcome parameters (Figure 2).

Figure 2 Correlations between ΔTARC (CCL-17), ΔVEGF, ΔCTACK (CCL-27) and ΔIL-13 and change in SCORAD over 12 weeks of treatment with MTX or AZA in patients with AD

AD, Atopic Dermatitis; AZA, Azathioprine; MTX, Methotrexate; R, Rho; P, P-value

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None of the biomarkers could identify or discriminate between responders and non- responders. We investigated if cytokine/chemokine profiles differed between responders and non- responders at baseline, to find a predictive biomarker that could differentiate these two groups. Based on a Bonferroni corrected P-value of 0.0031, none of the baseline biomarker levels were significantly different between responders and non-responders, indicating that none of the cytokines/chemokines were able to distinguish between responders and non-responders (Table 3).

FLG and non-FLG mutation carriers show similar biomarker profiles. After 12 weeks of treatment, all the outcome parameters showed a significant decrease for both the FLG mutation carriers and non-FLG carriers (data not shown). No significant differences could be observed in cytokine/chemokine levels at baseline, after 12 weeks of treatment and between the change in cytokine levels after treatment between carriers and non-carriers of FLG mutations.

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Table 3. Baseline serum levels of cytokines/chemokines in responders and non-responders.

Responders Non-responders P-value Cytokines/chemokines (n=15) (n=23) R vs NR week 0 APRIL (pg/mL), median (IQR) 1298 (953.0-1609) 1587 (1146-2100) 0.055 BAFF (pg/mL), median (IQR) 74.0 (26.0-240.0)* 109.0 (29.50-508.0)** 0.57 TARC (pg/mL), median (IQR) 1160 (920.1-2690) 1723 (647.5-2445) 0.75 IL-1RA (pg/mL), median (IQR) 8.61 (0.07-21.91) 7.05 (0.07-41.60) 0.74 IL-9 (pg/mL), median (IQR) 29.48 (17.33-66.52) 21.70 (0.29-40.84) 0.58 IP-10 (pg/mL), median (IQR) 714.1 (670.1-866.3) 618.1 (431.5-1154) 0.31 VEGF (pg/mL), median (IQR) 23.90 (0.95-57.69) 35.19 (8.75-67.34) 0.59 IL-1β (pg/mL), median (IQR) 0.26 (0.08-0.37) 0.08 (0.03-0.60) 0.41 IL-6 (pg/mL), median (IQR) 1.75 (0.72-3.45) 2.09 (0.38-3.45) 0.62 IL-7 (pg/mL), median (IQR) 8.77 (5.74-9.77) 8.27 (7.26-11.77) 0.38 IL-13 (pg/mL), median (IQR) 2.12 (1.02-3.61) 1.50 (0.33-2.72) 0.35 MCP-1 (pg/mL), median (IQR) 44.71 (31.06-76.06) 71.20 (39.40-91.95) 0.41 MIP-1β (pg/mL), median (IQR) 107.6 (90.25-181.2) 117.5 (95.52-145.4) 0.88 IL-18 (pg/mL), median (IQR) 57.50 (45.88-77.15) 57.99 (44.92-90.12) 0.79 CTACK (pg/mL), median (IQR) 2572 (1778-3271) 1812 (1387-3059) 0.13 MIG (pg/mL), median (IQR) 1024 (718.6-2248) 1111 (780.8-2025) 0.74

AD, Atopic Dermatitis; AZA, Azathioprine; IQR, InterQuartile Range; MTX, Methotrexate; R: Responder; NR: non-responder. *n=35 ** n=20 Responders to treatment were defined as patients with a SCORAD reduction of 50% or more (SCORAD50) 12 weeks after baseline. Differences at baseline was considered significant with a P-value <0.0031 (Bonferroni correction, 0.05/16).

DISCUSSION

Biomarkers may play an important role in the personalized treatment of AD, since it is assumed that treatments will be more effective when targeting the patient’s specific biological signature as determined by biomarkers. Moreover, biomarkers may provide tools to predict and monitor therapeutic response.(18)

In this study, TARC (CCL-17), VEGF, IL-13, and CTACK showed a significant decrease in the 38 evaluated AD patients who completed 12 weeks of systemic treatment with MTX or AZA, and

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therefore these markers showed to be potential severity biomarkers. The decreased cytokine levels over 12 weeks of time were not significantly correlated with the change in outcome parameters over 12 weeks of time. None of the biomarkers could be used to discriminate between responders and non-responders, or FLG genotype status, and therefore no potential predictive biomarkers were found. Maybe a combination of biomarkers is needed to find a predictive value.

TARC (CCL-17) and IL-13 are mediated by Th2 cells which play an important role in the pathogenesis of AD in the acute phase of AD. TARC (CCL-17) is expressed by keratinocytes in the epidermis, vascular endothelial cells, T cells and dendritic cells. TARC is described at this moment to be the best single biomarker for assessing disease severity. (2) IL-13 is secreted by Th2 cells and eosinophils and it’s a central regulator in the IgE synthesis. Dupilumab blocks the receptor for IL-4 and IL-13 which results in inhibition of the inflammation. It has shown good efficacy in patients with(14) AD.

Like TARC, VEGF is also derived from endothelial cells, but as well as from fibroblasts, smooth muscle cells and macrophages and it takes part in different stages of the angiogenesis, as well as vasodilatation. It promotes survival, migration and proliferation of endothelial cells and keratinocytes and increases the vascular permeability of the skin. Vascular remodeling is (19) known in chronic inflammatory skin diseases. VEGF was also found to play a role in inducing 6 pruritus via epidermal hyper innervation.(20) An active eczema with high inflammation levels could therefore be associated with a higher VEGF and possibly more itch, which detracts when it is suppressed by systemic immunosuppressive treatment.

Serum CTACK (CCL27) has previously been put forward as a severity biomarker in adults, and showed to have a strong correlation with disease severity.(2) It belongs, like TARC, to the CC family, and is thought to play an important role in skin inflammation as it attracts cutaneous lymphocyte antigen-positive memory T cells into the inflammatory sites. CTACK (CCL27) is only expressed in the skin, mostly in the epidermal keratinocytes.(21) It is expected that by suppressing inflammation with systemic treatment, CTACK is reduced.

Besides TARC and CTACK, which we found in this study, also sE-selectin, MDC, LDH and IL-18 were shown to be promising biomarkers.(2) From these biomarkers, only IL-18 was analysed in this study and showed a significant decrease in the 38 evaluated AD patients whom completed 12 weeks of systemic treatment with MTX or AZA, but only if it was not corrected for Bonferroni. IL-18 is a member of IL-1 cytokine family and its levels have been shown to

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correlate with disease severity in AD children and adult AD patients.(22, 23)

Interestingly, TARC decreased significantly more under MTX than AZA despite of their similar treatment efficacy as assessed by decrease in disease severity.(15) Studies with MTX and AZA have been done in patients with rheumatoid arthritis (considered a Th-1 dominant condition), but the mechanism of action is still not fully understood.(24, 25) It has been shown that serum IL-1β, IL-6 and IL-8 decrease upon MTX treatment.(25) Concerning AZA, one study found that serum levels of IL-6 in rheumatoid arthritis patients did not change upon treatment(26) . The mechanism of actions of these treatments on serum level of cytokines/chemokines is hardly described in the literature and is therewith inconclusive. Treatment response (SCORAD50) could not be predicted based on the cytokine/chemokine profile of a patient at baseline. Also, the changes in the cytokine/chemokine profiles of responders compared to non-responders were similar for MTX and AZA.

Our study has several limitations. It is a relative small study and more than 50% of the AD patients had values below the lower limit of detection (LLD) at baseline for TSLP, IL-5 IL-1α and G-CSF. These cytokines were excluded from analysis and therewith we could have missed some potential biomarkers. A combination of biomarkers shows a better correlation with disease severity(27) , however in this explorative study multivariable analysis was not sensible as the number of subjects was low. Furthermore, six patients had used rescue medication in the form of oral corticosteroids and patients were allowed to start or continue concomitant topical steroids during systemic treatment with MTX and AZA. This may have influenced clinical outcome parameters , and their relations to cytokines/chemokines profiles.

In conclusion, serum levels of TARC, CTACK, IL-13 and VEGF significantly decreased upon treatment. None of the investigated biomarkers could discriminate between responders and non-responders and biomarker profiles betweenFLG mutation carriers were similar to those in non-FLG mutation carriers.

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1. McAleer MA, Irvine AD. The multifunctional preferentially elevated in atopic dermatitis. role of filaggrin in allergic skin disease. J Acta Derm Venereol. 2012;92(1):24-8. Allergy Clin Immunol. 2013;131(2):280-91. 9. Raap U, Weissmantel S, Gehring M, 2. Thijs J, Krastev T, Weidinger S, Buckens CF, Eisenberg AM, Kapp A, Folster-Holst R. IL-31 de Bruin-Weller M, Bruijnzeel-Koomen C, significantly correlates with disease activity et al. Biomarkers for atopic dermatitis: a and Th2 cytokine levels in children with systematic review and meta-analysis. Curr atopic dermatitis. Pediatr Allergy Immunol. Opin Allergy Clin Immunol. 2015;15(5):453- 2012;23(3):285-8. 60. 10. Raap U, Wichmann K, Bruder M, Stander 3. Chen Y, Lind Enoksson S, Johansson C, S, Wedi B, Kapp A, et al. Correlation Karlsson MA, Lundeberg L, Nilsson G, et al. of IL-31 serum levels with severity of The expression of BAFF, APRIL and TWEAK atopic dermatitis. J Allergy Clin Immunol. is altered in eczema skin but not in the 2008;122(2):421-3. circulation of atopic and seborrheic eczema 11. Siniewicz-Luzenczyk K, Stanczyk-Przyluska patients. PLoS One. 2011;6(7):e22202. A, Zeman K. Correlation between serum 4. Dillon SR, Sprecher C, Hammond A, interleukin-31 level and the severity of Bilsborough J, Rosenfeld-Franklin M, Presnell disease in children with atopic dermatitis. SR, et al. Interleukin 31, a cytokine produced Postepy Dermatol Alergol. 2013;30(5):282-5. by activated T cells, induces dermatitis in 12. Szegedi K, Kremer AE, Kezic S, Teunissen mice. Nat Immunol. 2004;5(7):752-60. MB, Bos JD, Luiten RM, et al. Increased 6 5. Matsushita T, Fujimoto M, Echigo T, frequencies of IL-31-producing T cells are Matsushita Y, Shimada Y, Hasegawa M, et found in chronic atopic dermatitis skin. Exp al. Elevated serum levels of APRIL, but not Dermatol. 2012;21(6):431-6. BAFF, in patients with atopic dermatitis. Exp 13. Teraki Y, Hotta T, Shiohara T. Increased Dermatol. 2008;17(3):197-202. circulating skin-homing cutaneous 6. Niyonsaba F, Ushio H, Hara M, Yokoi H, lymphocyte-associated antigen (CLA)+ Tominaga M, Takamori K, et al. Antimicrobial type 2 cytokine-producing cells, and peptides human beta-defensins and decreased CLA+ type 1 cytokine-producing cathelicidin LL-37 induce the secretion of a cells in atopic dermatitis. Br J Dermatol. pruritogenic cytokine IL-31 by human mast 2000;143(2):373-8. cells. J Immunol. 2010;184(7):3526-34. 14. Beck LA, Thaci D, Hamilton JD, Graham 7. Neis MM, Peters B, Dreuw A, Wenzel J, Bieber NM, Bieber T, Rocklin R, et al. Dupilumab T, Mauch C, et al. Enhanced expression levels treatment in adults with moderate-to- of IL-31 correlate with IL-4 and IL-13 in atopic severe atopic dermatitis. N Engl J Med. and allergic contact dermatitis. J Allergy Clin 2014;371(2):130-9. Immunol. 2006;118(4):930-7. 15. Schram ME, Roekevisch E, Leeflang MM, Bos 8. Nobbe S, Dziunycz P, Muhleisen B, JD, Schmitt J, Spuls PI. A randomized trial of methotrexate versus azathioprine for severe

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AD. J Allergy Clin Immunol. 2011;128(2):353- dermatitis in children. Allergy and asthma 9. proceedings. 2004;25(3):181-4. 16. Roekevisch E, Leeflang MMG, Schram ME, 23. Zedan K, Rasheed Z, Farouk Y, Alzolibani AA, Campbell LE, Irwin McLean WH, Kezic S, et al. Bin Saif G, Ismail HA, et al. Immunoglobulin Patients with atopic dermatitis with filaggrin e, interleukin-18 and interleukin-12 in loss-of-function mutations show good but patients with atopic dermatitis: correlation lower responses to immunosuppressive with disease activity. Journal of clinical and treatment. Br J Dermatol. 2017;177(6):1745- diagnostic research : JCDR. 2015;9(4):Wc01- 6. 5. 17. Domthong U, Parikh CR, Kimmel PL, Chinchilli 24. Hildner K, Marker-Hermann E, Schlaak VM. Assessing the agreement of biomarker JF, Becker C, Germann T, Schmitt E, et al. data in the presence of left-censoring. BMC Azathioprine, mycophenolate mofetil, and Nephrol. 2014;15:144. methotrexate specifically modulate cytokine 18. Thijs JL, van Seggelen W, Bruijnzeel-Koomen production by T cells. Ann N Y Acad Sci. C, de Bruin-Weller M, Hijnen D. New 1998;859:204-7. Developments in Biomarkers for Atopic 25. Kremer JM, Lawrence DA, Hamilton R, Dermatitis. J Clin Med. 2015;4(3):479-87. McInnes IB. Long-term study of the impact 19. Samochocki Z, Bogaczewicz J, Sysa- of methotrexate on serum cytokines and Jedrzejowska A, McCauliffe DP, Kontny lymphocyte subsets in patients with active E, Wozniacka A. Expression of vascular rheumatoid arthritis: correlation with endothelial growth factor and other pharmacokinetic measures. RMD Open. cytokines in atopic dermatitis, and 2016;2(1):e000287. correlation with clinical features. Int J 26. Crilly A, McInnes IB, Capell HA, Madhok R. Dermatol. 2016;55(3):e141-6. The effect of azathioprine on serum levels 20. Wong LS, Otsuka A, Yamamoto Y, Nonomura of interleukin 6 and soluble interleukin 2 Y, Nakashima C, Honda T, et al. Vascular receptor. Scand J Rheumatol. 1994;23(2):87- endothelial growth factor partially induces 91. pruritus via epidermal hyperinnervation in 27. Thijs JL, Nierkens S, Herath A, Bruijnzeel- imiquimod-induced psoriasiform dermatitis Koomen CA, Knol EF, Giovannone B, et in mice. Journal of dermatological science. al. A panel of biomarkers for disease 2016;83(2):148-51. severity in atopic dermatitis. Clinical and 21. Machura E, Rusek-Zychma M, Jachimowicz experimental allergy : journal of the British M, Wrzask M, Mazur B, Kasperska-Zajac Society for Allergy and Clinical Immunology. A. Serum TARC and CTACK concentrations 2015;45(3):698-701. in children with atopic dermatitis, allergic asthma, and urticaria. Pediatr Allergy Immunol. 2012;23(3):278-84. 22. Sohn MH, Lee KE, Kim KE. Interleukin-18 is associated with increased severity of atopic

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Evelien Roekevisch, Mariska M.G. Leeflang, Mandy E. Schram, Linda E. Campbell, W. H. Irwin McLean, Sanja Kezic, Jan D. Bos, Phyllis I. Spuls, Maritza A. Middelkamp-Hup

British Journal of Dermatology (BJD). 2017 Dec;177(6):1745-1746

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Research letter

Patients with atopic dermatitis with filaggrin loss-of-function mutations show good but lower responses to immunosuppressive treatment

DEAR EDITOR, Filament-aggregating protein (filaggrin) mutations are a strong risk factor for developing atopic dermatitis but the relationship has not been thoroughly studied. To investigate whether filaggrin mutations influence the outcome of immunosuppressive treatment we studied patients with severe atopic dermatitis in a single-blinded randomized controlled trial with methotrexate or azathioprine during a 24-week regimen.1 Buccal mucosa swabs were collected 2 years after randomization from 36 of the 42 participants in the randomized controlled trial (86%) to determine their filaggrin genotype status (R501X, 2282del4, R2447X, S3247X and 3321delA mutations). In the original trial both methotrexate and azathioprine induced significant reductions in the Scoring of Atopic Dermatitis index (SCORAD) at 12 and 24 weeks, with no significant difference found between the treatments.1 We therefore pooled all patients together for this substudy and divided them into a filaggrin mutation group (FMG) and a non-FMG. The primary outcome parameter was the SCORAD, which was obtained at baseline and weeks 2,4,8,12 and 24, and analysed as repeated measurements (lme4 package in R, version 3.2.5, https://www.r-project.org). All SCORAD values were obtained from patients who had been receiving systemic immunosuppressive treatment with or without topical immunosuppressive treatment, except for three values in three patients. Two patients stopped systemic treatment after experiencing adverse events (FMG, week 2; non-FMG, week 4) and their last scores, obtained within 2 weeks of stopping systemic therapy, were for topical immunosuppressive treatment alone. One (FMG) patient stopped systemic treatment at week 12, and the values at week 24 were for patients undergoing only topical immunosuppressive treatment. As the patients’ mean age was significantly higher in the FMG than in the non-FMG (P < 0,01, Table E1; see Supporting Information), all analyses were corrected for age as a possible confounder, and age was included in the models as a potential confounder variable. As the treatment regimen (azathioprine or methotrexate) did not influence treatment success in the FMG compared with the non-FMG, we included in the mixed models as covariates visit, mutation, age and an interaction term for visit * mutation. The main limitation of this substudy was the small sample size and our subsequent inability to randomize on filaggrin mutation status.1 Filaggrin mutations were found in 13 of the 36 patients (36%, Table E2; see Supporting Information). Every patient in the FMG and the non- FMG showed an improvement of SCORAD at week 24 compared with baseline (Fig. E1; see Supporting Information). The mean SCORAD in the FMG decreased by 20,7 points (95%

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confidence interval 14,7–26,6, P < 0,01) and in the non-FMG it decreased by 31,3 points (95% confidence interval 26,3–36,2, P < 0,01). However, over the course of 24 weeks the FMG showed less improvement in SCORAD than the non-FMG (P= 0,02) and the improvement in the SCORAD of patients in the FMG stagnated during the course of treatment (Fig. 1). When the three SCORAD values obtained under topical treatment alone were excluded from data analysis, the FMG showed a trend towards less improvement in the course of 24 weeks than the non- FMG, as a significant difference was not reached (P= 0,08). Median dosages of topical immunosuppressive treatment (FMG 132 g, non-FMG 120 g, P= 0,51) methotrexate (FMG 83,8 g, non-FMG 90 g, P= 0,26) and azathioprine (FMG 837,5 g, non-FMG 825 g, P= 0,75) were comparable between groups over the course of 24 weeks (Mann–Whitney U-test). It is possible that T-helper-cell-2 cytokines can downregulate filaggrin protein expression2 and, in patients with atopic dermatitis and severe skin inflammation, skin barrier impairment seems similar in patients with and without filaggrin mutations and is correlated with the SCORAD.3 At the initiation of the study, levels of skin barrier impairment and inflammation were probably comparable in the two groups, which is supported by their similar mean SCORAD. When decreasing inflammation by immunosuppressive treatment, downregulation of filaggrin expression by the inflammatory infiltrate will be abrogated, allowing the skin barrier to be restored and, probably, the establishment of normal filaggrin protein levels in the non-FMG. In contrast, patients with filaggrin mutations will also experience an improvement of their atopic dermatitis due to the immunosuppression but the impairment of the barrier function and reduced filaggrin protein levels will still be present in the end because of their genetically caused filaggrin protein deficiency. This may explain both the lower therapeutic responses in the FMG over the course of 24 weeks and why the initial improvement stagnates during the 7 treatment period. Although both groups showed a significant improvement in SCORAD compared with baseline, filaggrin mutations may have a negative impact on the success of immunosuppressive treatment in severe atopic dermatitis. Additional research investigating the relationship between filaggrin mutation status and treatment success is necessary to confirm these findings.

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Fig 1. Mean (SD) reduction in Scoring of Atopic Dermatitis index (SCORAD) from baseline to 24 weeks, adjusted for age. Differences in outcomes between the filaggrin mutation group (FMG, n=13) and non-FMG (n=23) when on combined systemic and topicalimmunosuppressive treatment or topical immunosuppressive treatment alone. All data points are included. Ggplot package in R was used to draw the figures. The grey area represents the 95% confidence interval for the mean reduction in SCORAD in the FMG and non-FMG. Dark grey represents the overlap between the scores of the groups. These results show that both groups showed an improvement in SCORAD compared with baseline. However, patients in the FMG showed less improvement in SCORAD in the course of24 weeks than those in the non-FMG (P= 0,02), and their improvement stagnated during the course of the treatment.

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Online Supporting Information

Materials and methods

Study population Between September 2009 and May 2010, 42 patients with severe AD were included in an investigator-initiated, single-blind, parallel-group (ratio 1:1) RCT evaluating efficacy, safety and quality of life of MTX versus AZA over a 12-week period.(1) The follow-up phase consisted of another 12 weeks in which study drugs could be continued, stopped, or switched. Study visits took place at baseline, 2, 4, 8, 12 and 24 weeks. Subjects that ended study participation prior to week 12 had an early termination visit. AD was defined according to the Millennium Criteria and the UK Working Party criteria.(5) The severity of AD, graded at baseline by the Rajka and Langeland criteria, was severe. Two years after the randomization all 42 patients were asked to participate in this sub-study on FLG mutations. Thirty six patient accepted to participate. This study was conducted with institutional research ethics committee approval (Academic Medical Center Amsterdam) and all patients signed informed consent which complied with the Declaration of Helsinki Principles. For in- and exclusion criteria we refer to the original RCT.(1)

Treatment regimes In the original RCT, patients were randomised (concealed allocation) to treatment with MTX or AZA. MTX was initiated at 10 mg/week, together with folate 5 mg the next day. AZA was initiated at 1.5 mg/kg/day. During the total period of 24 weeks, dose increments with 7 2.5 to 5 mg MTX were allowed till a maximum of 22.5 mg/week. For AZA, dosage could be increased with 0.5 mg/kg/day till a maximum of 2.5 mg/kg/day. In the first 12 weeks, topical triamcinolone acetonide 0.1% ointment (body), hydrocortisone acetate 1% ointment (face) and oral antihistamines were allowed and in case of an exacerbation or postponed treatment effect in the first 8 weeks of treatment, patients were allowed to receive a maximum of 2 courses of prednisolone 30 mg/day for 1 week, followed by 1-week reduction schedule 20- 20-15-15-10-10-5 mg. From week 12 to 24, there was no limitation in the use of concomitant topical or systemic treatment with corticosteroids. All concomitant medication was recorded.

Filaggrin genotyping Patients of West-European ethnicity were genotyped for the 4 most common mutations in European Caucasians: R501X, 2282del4, R2447X and S3247X. Because three patients were of Asian ethnicity, 3321delA (6) was genotyped additionally. One patient was of

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Afro-Caribbean ethnicity, but no additionally genotyping, besides the 4 most common in European Caucasians, was performed as no specific mutations have been reported for this population. DNA material was obtained from buccal mucosa cells with buccal swabs (Geneticlab Diagnostic & Research, Pordenone, Italy). For each subject, two swabs were obtained and 2 mL lysis buffer (Puregene® Cell Lysis Solution; Gentra Systems, Minneapolis, MN, U.S.A.) was added to each swab to disrupt the cells and stabilize the DNA. Extraction and genotyping for R501X, R2447X, S3247X and 3321delA were performed by KBioscience (http://www.kbioscience.co.uk). Genotyping was performed using the KASP single nucleotide polymorphism genotyping system (KBioscience), a homogeneous fluorescent resonance energy transfer-based system, coupled with competitive allele-specific polymerase chain reaction (PCR) as described previously.(7;8)

Treatment outcomes The primary outcome parameter of our study was the SCOring Atopic Dermatitis (SCORAD) in the course of 24 weeks. Secondary outcome parameters were changes in eczema area and intensity index (EASI), patient-oriented eczema measurement (POEM), itch and sleeplessness scores on a visual analog scale (VAS), Skindex-17, the number of patients achieving no or minimal disease (investigator global assessment (IGA)<2, patient global assessment (PGA)<2). Efficacy assessors were blinded for allocation. During the RCT, no one was aware ofthe mutation status.

Data analysis As in the original RCT both MTX and AZA induced a significant reduction in SCORAD at 12 and 24 weeks without a significant difference in effect between treatments (1), all patients were pooled together and were divided into a FLG mutation group (FMG) and non-FMG. Primary and secondary outcomes were analysed as repeated measures by using the lme4 package in R (version 3.2.5), as we were interested in the course over 24 weeks. The lme4 package was used to test whether response to immunosuppressive treatment differed between FMG and non-FMG. Lme4 provides functions for fitting and analyzing linear mixed models. A significant higher mean age and longer mean duration of AD was found in the FMG compared to the non-FMG (P<0.01, Table E1). As it is known that FLG mutation patients tend to have a more severe and persistent AD phenotype(10), this may have resulted in an overrepresentation of older patients in the FMG and influenced the results. Age was therefore included as a variable in the models, to prevent age as a confounder on all outcomes. AD duration was not included separately as a variable, as there was a high correlation between AD duration and age (Pearson’s R=0.8; P<0.001). Treatment regimen (randomization to AZA or MTX)

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did not influence treatment success in FMG vs. non-FMG (results not shown). Therefore, covariates in the model were visit, mutation, age and an interaction term for visit * mutation. We assumed a random slope and intercept for each patient. Because the actual curves were not exactly linear, we introduced natural cubic splines with two degrees of freedom. Spline functions are used to represent smoothly varying relationships between a predictor (visit) and the response (e.g. SCORAD), which can take on virtually any shape. As lme4 does not provide P-value, we tested whether adding the interaction term significantly improved the model by comparing the fit of the model without the interaction term to the fit of the model with the interaction term. This means we report the P-value for model improvement, meaning that a significant P-value indicates that responses to immunosuppressive treatment are statistically significant different between FMG and non-FMG over the entire period of 24 weeks. All patients were included in the model, as mixed models allow missing values. All SCORAD values were obtained from patients receiving systemic immunosuppressive treatment with or without topical immunosuppressive treatment, except for three values in three patients. Two patients stopped systemic treatment because of adverse events (FMG, week 2; non-FMG, week 4), and their last scores, obtained within 2 weeks of stopping systemic therapy, were under topical immunosuppressive treatment alone. One patient (FMG) stopped systemic treatment at week 12, and values at week 24 were under topical immunosuppressive treatment alone. We therefore analysed results in two ways, i.e. effect of FLG mutations on combined systemic and topical immunosuppressive treatment plus on topical treatment alone (including all SCORAD values), as well as effect of FLG mutation on combined systemic and topical immunosuppressive treatment only (excluding the 3 SCORAD values obtained under topical treatment alone). Baseline characteristics were analysed using 7 SPSS 22.0 for Windows (SPSS, Inc., Chicago, Ill). We used the T-test for normally distributed continuous outcome measures and Fisher’s exact test for dichotomous outcome measures. Dosages of MTX and/or AZA were analysed. In the FMG, 7 patients used MTX. One patient stopped MTX because of an AE and was excluded for analyses. Eleven patients were using MTX in the non-FMG, none of them were excluded. In the FMG, 6 patients used AZA. One patient stopped AZA because of an AE and one patient switched AZA at week 12 to MTX, both were excluded. Twelve patients used AZA in the non FMG. One patient stopped AZA at week 12 and was excluded from analyses. For this analysis and the amount (total grams) of topical immunosuppressive treatment, the Mann–Whitney U test was used, as these were not normally distributed. P-values below 0.05 were considered to be statistically significant. All other analyses were done in R version 3.2.5 (R Foundation for Statistical Computing, Vienna, Austria). (9) Ggplot package in R was used to draw the figures.

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Table E1. Demographics and baseline characteristics of severe atopic dermatitis patients with and without filaggrin gene mutations

FMG (N=13) non-FMG (N= 23) P-value Age, mean (95% CI for mean) 52.2 (44.0-63.3) 34.8 (29.6-40.1) <0.01* Male (%) 7 (54%) 13 (57%) 1.00¥ Presence of asthma or allergic rhinitis (%) 13 (100%) 20 (87%) 0.29¥ Duration of AD (y), mean (95% CI for mean) 51.8 (43.5-60.0) 29.0 (23.1-35.0) <0.01* Non-European ethnicity (%) 0 4 (17%) 0.27¥ Outcomes (week 0) SCORAD, mean (95% CI for mean) 59.6 (52.0-67.2) 58.1 (53.5-62-7) 0.70* IGA, median (range) 4 (3-5) 4 (3-5) 0.35¥ PGA, median (range) 4 (3-5) 4 (2-5) 0.80¥ EASI, mean (95% CI for mean) 33.6 (25.4-41.7) 26.9 (21.1-32.7) 0.16* POEM, mean (95% CI for mean) 18.3 (15.1-21.6) 21.4 (19.9-22.8) 0.04* Skindex-17, mean (95% CI for mean) 19.8 (15.4-24.1) 20.5 (18.5-22.6) 0.72*

*T-test for two independent groups; ¥ Fisher’s exact test. AD; Atopic Dermatitis, EASI; Eczema Area and Severity Index, FMG; filaggrin mutation group, IGA; Investigator Global Assessment, non- FMG; non filaggrin mutation group, PGA; Patient Global Assessment, POEM; Patient-oriented eczema measurement, SCORAD; SCOring Atopic Dermatitis, SD; Standard Deviation, VAS; Visual Analogue Scale. Patients in the FMG showed less improvement in the secondary outcome parameters IGA, PGA, EASI, POEM, and Skindex-17 in the course of 24 weeks than the non-FMG (data not shown), but differences were not statistically significant. Our model failed to converge IGA <2 and PGA <2 in the course of 24 weeks when we included all covariates in the model. IGA and PGA therefore could not be corrected for age.

Table E2. Type of filaggrin gene mutations among the 13 filaggrin gene mutation carriers

FLG null FLG null FLG null FLG Mutations compound heterozygous homozygous heterozygous R501X - 4 (31%) 1 (8%) 2282del4 4 (31%) 2 (15%) 1* (8%) R2447X 1 (8%) - S3247X - - - 3321DelA - - -

Percentages given in the table correspond with the amounts of that specific mutation divided by the total amount of patients (13). Fourteen mutations found in 13 patients. One patient (*) was compound heterozygous for 2282del4 and R2447X. FLG= filaggrin gene.

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Figure E1. Overview of SCORAD values measured at baseline (week 0), week 2, 4, 8, 12 and 24 in severe atopic dermatitis patients with (n=13) and without filaggrin (n=23) mutations

Differences in SCOring Atopic Dermatitis (SCORAD) between filaggrin mutation group (FMG) and non- FMG (n=36) when on of combined systemic and topical immunosuppressive treatment, or topical 7 immunosuppressive treatment alone. Number surrounded by a square means “Randomised to Azathioprine”, without a square “Randomised to Methotrexate”. -Two patients (patient 24 in FMG at week 2 and patient 32 in non-FMG at week 4) stopped systemic treatment because of adverse events. End of study visits were completed within 2 weeks, so the last observations were under topical immunosuppressive treatment alone. After that these two patients were lost to follow up. -One patient (patient 2 in FMG) stopped systemic treatment at week 12, values at week 24were obtained under topical immunosuppressive treatment alone. -All other patients had data points from baseline till week 24 under systemic and topical treatment. This figure shows that every single patient, both in FMG and non-FMG, showed improvement of SCORAD at week 24 compared to baseline.

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REFERENCES

1. Schram ME, Roekevisch E, Leeflang MM et al. A randomized trial of methotrexate versus azathioprine for severe atopic eczema. J Allergy Clin Immunol 2011; 128:353–9. 2. Howell MD, Kim BE, Gao P et al. Cytokine modulation of atopic dermatitis filaggrin skin expression. J Allergy Clin Immunol 2007; 120:150–5. 3. Mocsai G, Gaspar K, Nagy G et al. Severe skin inflammation and filaggrin mutation similarly alter the skin barrier in patients with atopic dermatitis. Br J Dermatol 2014; 170:617–24.

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General discussion

In this thesis we evaluated several aspects of the use, efficacy and safety of systemic therapy in atopic dermatitis (AD), both in literature, daily practice and in randomized controlled trials (RCTs). We focused particularly on the short and long-term efficacy and safety of methotrexate (MTX) and azathioprine (AZA).

Systemic treatment in AD: prescription behavior in 2 academic medical centers

AD is a chronic relapsing inflammatory skin disease characterized by exacerbations and remissions. Topical treatment and phototherapy are treatment options, but when these fails, systemic immunosuppressive treatment may be required. There is a lack of information about which oral immunosuppressive treatments are prescribed in AD in daily practice. To get more insights in this field we retrospectively analyzed medical charts in 2 academic medical centers over a period of 10 years (chapter 2).(1) Both daily practice data and trial (follow- up) data were evaluated to provide a complete overview of systemic immunosuppressive drugs used in AD patients. 334 AD patients received oral immunosuppressive treatment of which 102 patients (31%) participated in clinical trials. Cyclosporine A (CyA) was the most commonly used oral immunosuppressive drug, followed by mycophenolate mofetil (MMF) and Enteric-Coated MycoPhenolate Sodium (EC-MPS), AZA, MTX, systemic glucocorticosteroids and tacrolimus. CyA was the most prescribed oral immunosuppressive treatment in 267 (80%) patients, MMF or EC-MPS in 104 (31%), AZA in 46(14%), MTX in 37(11%), systemic glucocorticosteroids in 24 (7%) and systemic tacrolimus in (18)5%. Reasons for discontinuation of oral immunosuppressive drugs were controlled AD, ineffectiveness and adverse events (AEs). Although long-term treatment with oral glucocorticosteroids is not recommended in guidelines (2-4), it was remarkable that 24 (7%) patients were treated with long-term oral glucocorticosteroids. Some of these patients used prednisolone more than 3 months simultaneously with other oral immunosuppressive drugs. Oral glucocorticosteroids, like prednisolon, can be effective as an emergency intervention when patients havean exacerbation of their AD and a rapid improvement is needed. It can also be used to bridge a period when starting a new immunosuppressive treatment like MTX, AZA or MMF, as these treatments have their mode of action between 12 and 16 weeks. However, long-term use of oral corticosteroids is associated with multiple side effects and AD may exacerbate after discontinuation.(5) Participation in 1 of the RCTs may have affected the prescribing behavior. Included were patients were randomized to either CsA versus EC-MPS or MTX versus AZA.

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To investigate what kind of oral immunosuppressive treatment is being prescribed in children and adults with AD, also online surveys have been done among dermatologists and pediatricians. These TREatment of severe AD Taskforce (TREAT) surveys, were done so far in Europe and the United States (US) and Canada for children.(6, 7) For adults they were done in the UK.(8) In two of these surveys CsA was found to be the first choice of systemic therapy in AD.(6, 7) In the UK AZA was the first choice for AD.(8) This choice may be influenced by the growing evidence that AZA is a safe and effective option for dermatological disorders, including AD. It is also possible that the choice for AZA is made because it is more suitable as a long-term treatment than CsA for refractory moderate to severe AD. Retrospective data and data from online surveys are of great value to obtain information on the actual choices of therapies of treating physicians. However, high levels of safety and efficacy data are scarce. Therefore we systematically reviewed all available RCTs which were published since the systematic review by Schmitt et al in 2007.(4)

Systemic treatment in AD: evidence of efficacy and safety in literature

Over the past years many RCTs on systemic immunosuppressive treatments in AD were published. Schmitt et al. published a systematic review in 2007 which included in total 27 studies, of which 15 were RCTs and 12 were open uncontrolled studies, totaling 979 patients and 8 different systemic treatments, namely CsA, systemic corticosteroids, interferon-g (IFN-g), intravenous immunoglobulines (IVIG), MMF, AZA, infliximab and traditional Chinese herbal therapy.(4) In 2013 we updated this review. The difference with the review of Schmitt et al. was that only RCTs were included. 34 RCTs were included, containing 12 different systemic treatments (chapter 3), including 1653 patients. Besides the treatments included by Schmitt et al, we included monteluklast, pimecrolimus, mycobacterium vaccae (m. vaccae) and 8 thymopentin (TP-5) as systemic treatments. Efficacy outcomes were clinical signs, symptoms, quality of life, and the course of AD. Safety data were compared by calculating the weekly incidence rates (as percentages) for AEs. We graded the quality of evidence and the strength of the recommendation using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) methodology to provide an evidence-based treatment algorithm(9) However, in our review, GRADE was used in an adjusted way whereby the evidence per treatment was graded instead of the evidence per outcome. GRADE could only be applied for the effect of treatments on clinical signs because of poor reporting of results in other outcome domains. After our systematic review, several other reviews and guidelines were published containing information about systemic treatments in AD. The systemic immunosuppressive treatments prescribed frequently, will be discussed.

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Cyclosporin A (CsA) CsA is a calcineurin inhibitor that inhibits the proliferation of T lymphocytes. CsA was found to be an effective immunosuppressant of T cells in the ‘70s and treatment for AD was first reported in 1991.(10) CsA has been licensed for the short-term treatment of severe refractory AD in many European countries since the ‘90s. It is not licensed in children and pregnant women, but may be used. (11) We found in our systematic review that 14 trials consistently indicated that CsA efficaciously improves clinical signs of AD in children and adults. 5 RCTs, in which CsA was compared to placebo, showed a mean clinical improvement in severity for CsA between 53% and 95% in different clinical severity scores after short-term treatment (10 days to 8 weeks).(2) Concerning safety, the weekly rate of withdrawals because of AEs ranged between 0% and 2.0%. The weekly rate of any SAE ranged from 0.0% to 2.2%. In 9 RCTs no severe SAEs were reported. Unclear information on the occurrence of SAEs per group was provided in 2 RCTs. Although higher doses (5 mg/kg body weight) of CsA are more effective, lower starting doses (3 mg/kg body weight) with stepwise adjustment to the individual minimum effective dose are preferable because most side effects are dose(2) related. In general, most common AEs in CsA are increased serum creatinine levels and hypertension. AEs such as infections, leukopenia, hyperglycemia, hyperuricemia, hyperkaliemia, gingival hyperplasia hypertrichosis, gastrointestinal disorders, fatigue and nausea are often described as well. Regular measurements of blood pressure, renal and liver function tests, and blood cell counts are advised.(12) Overall, CsA is recommended as first-line treatment for short-term use. The use of CsA for up to 1 year can be recommended based on the results of 4 RCTs. Since the publication of our systematic review, several new RCTs were published. One head to head study showed that CsA was similarly effective as MTX, with a mean SCORAD reduction of 49% and 45% respectively.(13) Two studies were published where CsA in combination with glucosamine was tested.(14, 15) CsA with glucosamine was superior in both trials. An RCT, where CsA was compared with extracorporeal photopheresis (ECP) showed that the reduction in SCORAD for CsA was 34% compared to 46% for ECP after 30 weeks of treatment. With the publication of these new data, new treatment options for AD have possibly been found, but long term data on the use of CsA beyond 1 year are still lacking. Maintenance treatment for AD is often needed to prevent exacerbations of the disease. A drug survival study of a retrospective cohort showed the long-term use of CsA in 356 adults with AD. Overall drug survival rates were 34% after 1 year to 4% after 6 years. The reason for discontinuation was in ¼ of the patients controlled AD and approximately the same amount stopped because of AEs and ineffectiveness, 16% because of ineffectiveness alone. Of the patients who stopped because of controlled disease 68% stopped after 6 years. The most common AEs were hypertension and abnormal serum creatinine levels. (16) These abnormal serum creatinine

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levels have been the reason that treatment with CsA was restricted to the use of a one year because no long-term safety data was available. A retrospective study evaluated serum levels of creatinine during and after long-term treatment with CsA. After 3 weeks of treatment with CsA, a significant increase in creatinine was seen, which stabilized during the maintenance phase. Patients who did show a serum creatinine increase of more than 30% were also significantly older. In the follow-up (mean duration 357 days) all patients showed less than 30% increase compared to baseline. The duration of the treatment was not related to the levels of serum creatinine.(17) Guidelines do advise now to switch CsA after 2 years. Longer duration of treatment with CsA may be possible and tolerated but more research is needed.(18)

Pediatric considerations CsA was effective and well tolerated in children aged from 2-16 who were continuously or intermittently treated for a year.(19) A study comparing CsA (2.5 mg/kg/day) with MTX (7.5 mg/week) showed that CsA is effective, relatively safe and well tolerated when using low dosages. After 12 weeks of treatment no significant differences were seen between groups.(20)

Azathioprine (AZA) AZA is a purine analog that affects the proliferation of B and T lymphocytes. It was primarily developed in the ‘60s to prevent organ rejection. It is a treatment option for multiple dermatologic diseases, including AD. Two RCTs were included in the systematic review and showed that AZA was superior to placebo, with a mean improvement in Six Area, Six Sign Atopic Dermatitis (SASSAD) scores of 26% and 37% at week 12.(21, 22) AZA and MTX (chapter 4) were found to be equally efficacious, with a mean SCORAD score improvement of approximately 39% for AZA.(23) Concerning safety, the weekly rate of withdrawals because of AEs ranged between 0.2% and 0.4%. SAEs were not observed or not reported. In general, 8 most frequently seen AEs in AZA are abnormalities in blood counts, such as lymphocytopenia, and mild side effects like headache and gastro intestinal disorders. Patients with homozygous thiopurine methyltransferase (TPMT) (low or absent TPMT activity) are unusually sensitive to the myelosuppressive effect of AZA. Patients with a supra normal activity are more sensitive to hepatotoxicity.(10) Patients who are heterozygous do not have an abnormal response to AZA. It is therefore highly recommended to determine TPMT levels before the start of AZA(22) During treatment, regular checks of blood cell counts and liver function tests are advised(10) Overall, AZA is recommended as a second-line treatment option.(9) Since the publication of our systematic review, no new RCTs were published concerning AZA. Two and 5 years prospective follow-up data concerning AZA was published by our group end will be discussed under the heading “Short and long-term follow-up of MTX and AZA in AD“.

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Pediatric considerations No RCTs of AZA in children are published so far. Prospective and retrospective studies show that AZA might be effective in children.(24-26) Data about dosage and duration of treatment and side-effects in children are scarce.(10, 18, 27)

Methotrexate (MTX) MTX is a folic acid antagonist that is widely used in chronic inflammatory diseases such rheumatoid arthritis. In 1951, the first effective results of MTX in dermatology were noticed when psoriatic skin lesions cleared in cancer patients undergoing treatment with the anti- metabolic drug aminopterin, a predecessor of MTX.(28) We found in our head-to-head trial that MTX was equally efficacious as AZA (chapter 4), with a mean improvement in SCORAD score of 42% after 12 weeks of treatment. After 24 weeks of treatment, the relative reduction in the mean SCORAD score was 48%. Concerning safety, the weekly rate of withdrawals because of AEs was 0.4%. SAEs were not observed. MTX was always followed by folic acid to prevent AEs. In general, most frequently seen AEs in MTX are bone marrow suppression (leukopenia, anemia, and thrombocytopenia) and hepatotoxicity. Other common side effects include stomatitis, abdominal pain, nausea, vomiting, diarrhea, fatigue, pneumonia and oral ulcers.(22) During treatment regular checks of blood cell counts and liver function test are advised. Overall, we considered MTX as a third-line treatment.(2) A combination treatment of MTX and AZA was applied to 7 patients who were non-responder or poor-responder with either monotherapy. In 4 out of 7 (57%) patients this combination of treatments proved to be effective. If a monotherapy with MTX or AZA fails, a combination therapy may be considered but first more data are needed.(29) This combination treatment has been described in rheumatoid arthritis.(29) Since the publication of our systematic review, 2 RCTs were published concerning MTX in comparison to CsA in children with AD(20) and in adults with AD.(13) Results of these RCTs are discussed in the section about CsA. Two and 5 years prospective follow-up data concerning MTX was published by our group end will be discussed under the heading “Short and long-term follow-up of MTX and AZA in AD“.

Pediatric considerations The study comparing CsA (2.5 mg/kg/day) with MTX (7.5 mg/week) showed that MTX is effective, relatively safe and well tolerated when using low dosages. After 12 weeks of treatment no significant differences were seen between groups.(20) In psoriasis, much more experience with MTX in children is available which shows that it is safe, effective and well tolerated.(10)

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Mycophenolate mofitil (MMF) MMF, or EC-MPS, blocks the purine biosynthesis pathway and affects B cells and T cells. It might be a treatment option for maintenance treatment of AD after induction treatment with CsA, but this recommendation is based only on a single RCT.(30) Advised dosages are 1.0-1.5 g orally twice daily.(10) Concerning safety, the weekly rate of withdrawals because of AEs was 0.3%. SAEs were not observed.(2) The most common AEs are gastrointestinal side effects (nausea, vomiting, diarrhea, abdominal pain), fatigue, headache and infections (candidiasis, herpes infections of the skin). When taking EC-MPS, significantly fewer gastrointestinal side effects are seen as compared to MMF. Hematological side effects (leucopenia, thrombocytopenia, anemia) are rare in MMF. During treatment, regular checks of blood cell counts and liver function tests are advised.(10) A systematic review about the safety and efficacy of MMF in systemic sclerosis, suggested MMF to be safe. Mostly gastrointestinal AEs were seen. The duration of treatment with MMF ranged from 3–60 months.(31) Since the publication of our review, no RCTs have been published concerning MMF.

Pediatrics consideration No RCTs were published. Two retrospective trials showed that MMF might be effective and safe in children with AD.(32, 33) Continuous treatment up to 1 year has been reported. More research is needed.

Other systemic treatments According to placebo-controlled trials, IFN-y (2 RCTs) and systemic pimecrolimus (1 RCT) may also efficacious for severe AD in short-term use and can be considered as a third-line treatment option, but safety and tolerability need to be monitored closely. Montelukast and traditional Chinese herbal therapy are currently not recommended for the treatment of moderate-to- 8 severe AD in routine care because the results from the trials published are inconsistent. IVIG, m. vaccae, and thymopentin (TP-5) are currently not recommended because trials do not suggest that these treatments are efficacious in the treatment of moderate-to-severe AD.

Biologics Although used extensively in psoriasis for a longer period of time, there were no RCTs available on biologics for the treatment of AD by the time we published our review. However, in the meantime a systematic review summarizing the evidence of efficacy and safety of biologic agents was published, including 13 RCTs. The most studied biologics were dupilumab, omalizumab and ustekinumab.(34) Dupilumab (Dupixent®) was approved for AD in 2017, and is a fully human monoclonal antibody that blocks interleukin (IL)-4 and 13. Dupilumab was

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first shown to be effective in asthma and now several studies have shown its efficacy in AD. Dupilumab is approved in Europe and the USA for the treatment of moderate to severe AD.(35) In this recently published systematic review, 5 RCTs were included investigating dupilumab compared to to placebo in adult patients with AD.(34) Dupilumab was dosed ranging from 100 mg every month to 300 mg every week for 3 months to 1 year. Dupilumab was superior compared to placebo in all studies except when dupilumab was dosed at 100 mg monthly. Effects remained until 52 weeks after baseline. Dupilumab outcomes were compared with other studies. Dupilumab showed an improvement in SCORAD of 54 –62% compared to baseline and CsA an improvement of 42–88%, which was comparable. Dupilumab was superior compared to AZA (39%) and MTX (42%).(34) Concerning safety, dupilumab was well tolerated over a short-term period of 16 weeks and a long-term period of 52 weeks. The most frequently observed AEs were injection-site reactions, allergic conjunctivitis and oral herpes. No clinically significant laboratory abnormalities were seen.(36-38) Besides dupilumab, other biologics and small molecules have entered the world of AD or will arrive soon. Nemolizumab (anti-IL31), lebrikizumab (anti-IL13), and tralokinumab (anti- IL13) showed promizing results but further data are needed.(34) Janus Kinase (JAK) inhibitor tofazitinib showed promising results in 6 adults with moderate to severe AD after 14 weeks of treatment. Besides the SCORAD scores, pruritis and sleep loss scores improved significantly as well.(39) Much more research is done in the field of biologics, treatments such as anti- IL-5, anti-IgE, anti-IL-31, anti-IL-6, anti-IL-17, anti-IL-12/23, anti-IL-22 antibodies are currently being investigated in patients with AD, and these are just some examples of many others.(39)

Pediatric consideration Data regarding dupilumab in children is not published yet, but several trials have been conducted.(40)

The need for standardization of future cohort studies and outcome measures in ad trials

While performing our systematic review on systemic treatments for moderate-to-severe AD, we faced the problem that multiple outcome instruments were used in trials. We were therefore not able to perform a meta-analyzes in our systematic review because of the clinical and methodological heterogeneity, such as the substantial differences in trial designs, the lack of standardization in outcome measures, and the wide use of unvalidated outcome measures. Evidence-based decision making is therefore difficult. This emphasizes the need for standardization of outcome measures. In recent years, a lot of

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effort is put in international harmonization of trials. The Harmonising Outcome Measures in Eczema (HOME) initiative started in 2010 to develop a COS (core outcome set )for eczema. Clinical signs, symptoms, quality of life and long-term control of eczema were agreed on as the core outcome domains which should be applied in all future eczema trials.(41) There may also be a need of large real-life cohort studies as not all our research questions will be answered by RCTs. New RCTs are published, but long-term data is lacking and often small groups of patients are included. RCTs with large patients numbers are available but most studies are initiated or sponsored by pharmaceutical industries. In addition, study subjects are often difficult to compare with patients in daily clinical practice, as young patients with minimal co-morbidities are frequently included in these trials. Prospective registries are currently developed, to compare effectiveness, safety, and tolerability of photo and systemic immunosuppressive therapies and to enable the evaluation of health economics. A platform that pursues prospective registration of data on systemic therapies and phototherapy in AD is the TREatment of AD (TREAT) registry Taskforce.(42) Their goal was to harmonize data collection among national registries in order to be able to pool and compare data.By implementing standardized laboratory testing and obtaining specific genetic or biomarker information, an important biobank can be created.

Short and long-term follow-up of methotrexate and azathioprine in AD

By the time we reviewed the evidence on systemic treatments for AD, long-term data was scarce for MTX and AZA. Schmitt et al. found 1 RCT in their systematic review in which AZA was compared to placebo.(21) No RCT was found in which MTX was investigated, and no-head-to head trials were done. In daily clinical practice we needed more treatment options than CsA and systemic steroids for our severe AD patients and found the need to augment and improve 8 the evidence for the off-label drugs MTX and AZA in AD. We initiated a small mono-center RCT comparing MTX and AZA (Methotrexate versus Azathioprine for severe atopic dermatitis: MAcAD trial) (chapter 4). Only patients with severe AD were eligible, graded at baseline by the Rajka and Langeland (43) criteria and defined by the millennium and UK Working Party Criteria.(44) They were not treated with MTX or AZA before and unresponsive, contraindicated or intolerant to CsA. Because of these last inclusion criteria, it was a difficult (severe AD patient unresponsive, contraindicated or intolerant to CsA) population to treat. Patients were randomly assigned to receive either MTX or AZA for 12 weeks, followed by a 12-week follow- up period in which they could stop, switch or continue their treatment. Concealment of MTX or AZA was done by a computerized program. Trained investigators and the statistical annalist were blinded for the allocated treatments. Patients and treating physician were not blinded

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for allocation. Both treatments achieved a clinically relevant improvement and were safe over a period of 24 weeks. We concluded that MTX and AZA could be appropriate options for the treatment of severe AD at least for the duration of 6 months. However, AD is a chronic disease and it is imperative that the proposed therapy for these patients has a persistent effectiveness and an acceptable adverse effect profile when used for longer periodsof time. That is why all 42 randomized patients that participated in this RCT were offered the opportunity to enroll in the observational follow-up study in which they would be evaluated 3 monthly over a total period of 2 years (and eventually 5 years) after baseline. 35 of 42 patients were included in this follow-up study. After 2 years of treatment with MTX and AZA both therapies still seemed to be effective and safe for adult patients with severe AD (chapter 5).(45) This was the first head to head RCT including MTX versus AZA, and long-term follow-up study and therewith unique data. After 2 years of study 59% patients were continuously using allocated MTX. 41% patients discontinued MTX, 12% because of controlled AD, 18% because of inefficacy and 12% because of AEs. A drug survival study for MTX, in which 89 patients were included, showed a drug survival of 34% after 2 years.(46) 6% discontinued because of controlled AD, 15% because of ineffectiveness, 25 % of AEs, and 7% because of other reasons. Concerning AZA, in our study, 62% patients were using allocated AZA, of which 44% had been using AZA continuously since baseline. 56% discontinued AZA, 28% because of controlled AD, 22% because of inefficacy and 6% because of AEs. A drug survival study for AZA, in which 94 patients were included, showed a drug survival of 26% after 2 years (16); 21% discontinued because of controlled AD, 36 % because of ineffectiveness, 44% of AEs in that study. Although there were generally no major differences between the drug survival data and our study, our data was slightly more favorable in some fields. In the meantime, the 5 year follow-up data of the RCT has also been published.(47) After 5 years of study there were only 5 people who had continuously been treated on MTX and only 1 patient who was continuously treated with AZA. The reason for stopping the medication was mainly controlled AD, however, there were also several subjects who stopped because of side effects or inefficacy of the drug. This low drug survival rate requires optimization of these 2 treatments, for example by optimizing life standards and additive topical therapies, to carefully search for an optimal treatment dose, or possibly by a combination of systemic treatments. Analyzation of AEs needs to be optimized as well so one can prevent them in the future. Garritsen et al. investigated the use of allopurinol in 15 patients treated with AZA who experienced inefficacy, AEs or a skewed metabolism. The amount of responders increased from 27% to 57%, which was a significant finding.(48) It is also important to evaluate the patients who stopped their treatment because of controlled disease in order to know how they are doing after discontinuation. Patients who stopped because of ineffectiveness need analyzes as well, for example, it is important

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to know if they were treated with an optimal dose. In our RCT, MTX was initiated at 10 mg/ week, dose increments were restricted to a maximum of 22.5 mg/week in combination to 5 mg of folic acid the following day, once a week. AZA was initiated at 1.5 mg/kg/day, and restricted to a maximum of 2.5 mg/kg/day. After 2 years of study 10 of 17 (59%) patients were continuously using allocated MTX with a mean dosage of 14.5 mg/week. 11 of 18 (62%) patients were using allocated AZA with a mean dosage of 136.4 mg/day, of which 8 were using AZA continuously since baseline. We performed dose elevation and reduction based on clinical practice. In general, when a patient achieves controlled disease, continuity in dose is possible but the aim is to ultimately reduce to the lowest possible dose while maintaining controlled disease. When there is a flare up of disease you can temporarily increase the dose, and reduce again when the disease is controlled again. When a patient is experienced in handling his/her medication, self-regulation of medication might be possible with supervision by a doctor. Interesting is the study that was recently published by Goujon et al.(13) Patients were randomized to receive oral MTX (15 mg/wk) or CsA (2.5 mg /kg/d) for 8 weeks. If no SCORAD reduction of 50% had occurred after 8 weeks, MTX was increased to 25 mg/ week and CsA to 5 mg/kg/day during the following 8 weeks. MTX 15 mg/week was primary inferior to CsA 2.5 mg/ kg/day in the first 8 weeks. Increasing the dose to 25 mg MTX/ week gave a significant improvement versus CsA at week 20. Safety aspects over a short time (24 weeks) and a long time period (2 and 5 years) were analyzed in our studies regarding MTX and AZA. After 24 weeks of treatment no statistically significant differences were found in the number and severity of AEs. Abnormalities in blood count were more common in the AZA group. After 2 years, in the intention-to-treat-analyzes (ITT) and per-protocol (PP) analyzes, no life threatening SAEs and AEs occurred in both MTX and AZA group. In the PP analyzes, most AEs were found in the infections and infestations disorders. Common cold and liver enzyme disorders were most frequently seen in the 8 MTX group. Influenza (the flu) was significantly more often seen in the AZA group. After 5 years, for MTX, AEs concerned mainly ‘hepatobiliary’ (i.e. abnormality in liver enzymes) and ‘gastrointestinal disorders’ (i.e. nausea), and their severity was mild to moderate. For AZA it mainly concerned ‘blood and lymphatic system disorders’ (i.e. lymphocytopenia and anemia) and were mild to moderate in severity. After 24 weeks besides AEs, no SAEs were seen. The 2 year data showed 3 SAEs, 2 exacerbations of AD (possibly related) and 1 hospitalization (not related) because of psychiatric comorbidity. The 5 year follow-up data showed 12 SAE; 4 had a possible causal relationship and concerned 2 exacerbation of AD, 1 pneumonia and 1 bladder carcinoma. This last patient had a treatment history at the time the carcinoma was detected with CsA for 3 months, MTX for 25 months and AZA for 21 months. Therefore a causal relationship with 1 of these drugs could be possible and not be excluded. Long-

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term safety data of MTX and AZA may be extrapolated from other diseases A systematic review of the long-term safety of MTX as monotherapy in patients with rheumatoid arthritis showed a good long-term safety. Side effects found were described as mild. Possible risk of liver cirrhosis and malignancies, including lymphoma, remained inconclusive.(49) A Cochrane review evaluating AZA for multiple sclerosis showed that gastrointestinal disturbances, bone marrow suppression and hepatic toxicity was seen more frequently comparing to placebo. However, it was indicated that these AEs were easy to anticipate. Withdrawals as a result of AEs were not frequently seen and mainly concerned gastrointestinal intolerance.(50) Among AZA used for inflammatory bowel disease, there is an increased risk found for lymphoma and non-melanoma skin cancer, data on other malignancies are less clear, although more recent studies are reassuring.(51)

Towards personalized treatment in AD

Biomarkers may play an important role in personalized treatment of AD in the near future, since it is assumed that treatments will be more effective when targeting the patient’s specific biological signature as determined by biomarkers. A biomarker (or biological markers) is an objective outcome measure. They are biological markers such as genes, molecules and proteins, that are characteristic and can be detected and measured in for example blood, urine or in tissue. Reliable biomarkers can contribute to a better understanding of the pathogenesis of AD, accelerate the diagnostic process and contribute to predict the effect of a treatment. Nowadays, the reason of discontinuation of treatment is often ineffectiveness. Systemic treatments are not targeted on the individual, but are prescribed based on a general approach. The more we know about the functioning of the human body on a molecular, gene and protein level, the clearer it becomes that each individual is biologically different. This implies that treatments can also differ per individual and the effect may differ. As a doctor you want to give the right therapy to the right patient at the right moment in time. With new therapies such as biologicals and small molecules this targeted/personalized therapy may be possible as they may very specifically target molecules and proteins involved in the pathophysiology of AD. An example of such therapy is dupilumab, which targets IL-4 and IL-13. But as it concerns expensive medication, it is of increasing importance to know which treatment is suitable for which patient, in order to avoid unnecessary costs.

Some promising results on potential biomarkers for AD have been published. Thymus and activation-regulated chemokine (TARC) is the superior biomarker for assessing disease severity. T-cell attracting chemokine (CTACK), sE-selectin, macrophage-derived chemokine

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(MDC), lactate dehydrogenase (LDH) and IL-18 could be useful as a severity biomarker, but need to be validated in additional studies.(52) More research is needed to find a reliable biomarker for disease severity and especially for predicting immunosuppressive treatment responsiveness in AD patients, that can be used in clinical practice. To gather information about biomarker levels while treated with MTX or AZA we analyzed serum levels of chemokines (chapter 6) and cytokines by ELISA and Luminex in 38 patients that participated in the previously mentioned RCT on MTX and (23) AZA. We aimed to identify biomarkers that reflect the therapeutic response in patients with AD that were treated with systemic immunosuppressive treatments MTX or AZA. Our secondary aim was to identify a biomarker that predicted responsiveness to systemic immunosuppressive treatment, and to identify differences in biomarker profiles inFLG mutation carriers versus non-carriers. TARC (CCL-17), Vascular Endothelial Growth Factor (VEGF), IL-13, and CTACK showed a significant decrease after 12 weeks of systemic treatment and were therefore considered as potential severity biomarkers. Although the cytokine levels of these biomarkers decreased over 12 weeks, the decrease was not significantly correlated with the decrease in outcome parameters. None of the biomarkers could be used to discriminate between responders and non-responders, which was defined as a reduction of SCORAD of 50%, or discriminate betweenFLG genotype status, and therefore no potential predictive biomarkers were found. TARC (CCL-17) is mediated by Th-2 cells that play an important role in the pathogenesis of AD in the acute phase of AD cells. TARC (CCL-17) is expressed by keratinocytes in the epidermis, vascular endothelial cells, T cells and dendritic cells. Although TARC has been shown to correlate with AD activity, a variation in TARC levels has also been found between patients in cross-sectional cohorts of patients with similar disease activity.(53) It could be that the variation in TARC between patients might be due to the diversity of biologic pathways involved in AD pathogenesis. AD is a disease with a heterogeneous character and different entities. A panel of various biomarkers shows a better 8 correlation with disease severity compared to a single biomarker in AD patients.(54) IL-13 also showed a significant decrease after 12 weeks of systemic treatment. IL-13 is secreted by Th2 cells and eosinophils and it is a central regulator in IgE synthesis. IL-13 is believed to have a role in the pathogenesis of AD, because the frequency of IL-13-producing cells in AD patients is significantly higher than in healthy subjects (in both CD4+ and CD8+ T-cell subsets).(55) The first licensed biological for AD, dupilumab, blocks the receptor for IL-13 (and IL-4) which results in inhibition of the inflammation. Like TARC, VEGF is also derived from endothelial cells, as well as from fibroblasts, smooth muscle cells and macrophages. It takes part in different stages of the angiogenesis, as well as in vasodilatation. It promotes survival, migration and proliferation of endothelial cells and keratinocytes and increases the vascular permeability of the skin. Vascular remodeling is

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known in chronic inflammatory skin diseases.(56) VEGF was also found to play a role in inducing pruritus via epidermal hyper innervation.(57) An active eczema with high inflammation levels could therefore be associated with a higher VEGF and possibly more pruritus, which detracts when it is suppressed by systemic immunosuppressive treatment. There are some studies investigating the role of VEGF in AD. A recent study investigated systemic and stratum corneum biomarkers in AD and showed that there was a significant difference between VEGF in healthy skin and AD skin.(58) The role between AD and VEGF was also confirmed by other studies.(59, 60) However, one study also finding increased VEGF levels in AD skin discredited its usefulness as a severity biomarker, because they assumed that other cells might also release the cytokine, which makes VEGF non-specific.(61) On the other hand, serum CTACK (CCL27) has been put forward as a severity biomarker in adults, and showed to have a strong correlation with disease severity.(52) It belongs, like TARC, to the CC family, and is thought to play an important role in skin inflammation as it attracts cutaneous lymphocyte antigen- positive memory T cells into the inflammatory sites. CTACK (CCL27) is only expressed in the skin, mostly in the epidermal keratinocytes.(62) It is expected that by suppressing inflammation with systemic treatment CTACK is reduced. Besides TARC and CTACK, which we found in this study, also sE-selectin, MDC, LDH and IL-18 were shown to be promising biomarkers.(52) From these biomarkers, only IL-18 was analyzed in this study. It showed a significant decrease in the 38 evaluated AD patients whom completed 12 weeks of systemic treatment with MTX or AZA, but only if the analyzes was not corrected for multiple testing with the Bonferroni correction. IL-18 is a member of IL-1 cytokine family and its levels have been shownto correlate with disease severity in children and adult AD patients (63, 64) Interestingly, TARC decreased significantly more under MTX than AZA despite of their similar treatment efficacy as assessed by decrease in disease severity.(23) It could be that the mode of action concerning MTX plays part in this finding, compared to the mode of action of AZA. But the mechanism of actions of these treatments on serum level of cytokines/chemokines is hardly described in the literature and is therefore inconclusive. Studies with MTX and AZA have been done in patients with rheumatoid arthritis (considered a Th-1 dominant condition), but the mechanism of action is still not fully understood.(65, 66) It has been shown that serum IL-1β, IL-6 and IL-8 decrease upon MTX treatment.(66) Concerning AZA, one study found that serum levels of IL-6 in rheumatoid arthritis patients did not change upon treatment.(67) We were not able to predict treatment outcome based on the cytokines that we analyzed. Also, none of the biomarkers could be used to discriminate between FLG genotype status, as levels did not show significant differences. To date, loss-of-function mutations inFLG have been shown to be the most significant risk factor for AD.(68) FLG mutations are seen in less than a third of the total population with AD.(69, 70) Among patients with moderate-to-severe

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AD, up to 46% to 56% carry 1 or more FLG mutations, while the population attributable risk has been estimated to be between 4.2% and 15.1%.(70) Although it is clear that FLG mutations play an important role in the development of AD, the relationship between FLG mutations and treatment outcome in patients with AD has not been thoroughly studied. Preliminary results of a small prospective open-label trial showed that none of the AD patients with a FLG mutation responded to Omalizumab (P=0.05), which seems to indicate that FLG mutations may influence treatment outcome. We evaluated whether FLG genotype status had any influence on treatment outcome in severe ADchapter ( 7). For this purpose, we analyzed the patients with severe AD who participated in the previous described RCT with MTX or AZA during a 24 weeks treatment regimen.(ref) DNA material was obtained from buccal mucosa cells with buccal swabs. At least one FLG mutation was found in 13 patients (36%). Ten patients were heterozygous, two patients were homozygous and one patient was compound heterozygous. In the course of 24 weeks, patients with a FLG mutation showed less improvement in SCORAD than patients without a FLG mutation (P=0.02) However, we also evaluated treatment response after 2 year of treatment in the ITT group (n=35) and the PP group (n=18) (chapter 5). When analyzing treatment response after two years of treatment, patients with and without FLG mutations both showed a significant reduction in SCORAD compared to baseline (P <0.01), and no difference in response was seen between the two groups. So although the patients with a FLG mutation initially showed significantly less reduction in SCORAD compared to patients without FLGa mutation at 24 weeks of treatment, after two years we observed that this difference in effect had vanished and the response in both groups was completely equal. FLG mutation status may therefore be of major clinical importance in the determination of systemic treatment duration, and therefore needs to be taken into account when evaluating treatment outcome in the first 24 weeks of treatment, as patients withFLG mutations may need to be treated for a longer duration of time in order to 8 obtain the same therapeutic effect as patients withoutFLG mutations. Our results may have implications for the management of AD patients in clinical practice. In patients having a FLG mutation, the initial effectiveness of treatment may be less, and in view of the above-mentioned hypothesis, putting more emphasis on adding topical barrier restoring therapies to the treatment regimen, may lead to better therapeutic responses in this patient group when on immunosuppressive treatment.(71) On the other hand, immunosuppressive treatment may be needed to be given at higher starting dosages to obtain the same therapeutic effect as in patients without FLG mutations in the early stages of the treatment. These observations may have implications for clinical research as well. If patients with FLG mutations show lower initial clinical responses to treatment, it may be important to

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determine the FLG mutation status of patients participating in clinical trials, as this may impact the generalizability of the results. In that case, determination of the FLG mutation status before randomization should be performed and studies should consider to stratificate. Further studies are needed to elucidate these questions and considerations.

CONCLUSIONS

Part I Systemic treatments in atopic dermatitis

- CyA was the most commonly used oral immunosuppressive drug in 2 academic medical centres in the Netherlands. - CsA is recommended as first-line treatment, it is a well-studied, tolerated and effective drug in adults and children for the use up to 1 year. Retrospective data shows that low dose CsA for longer use is well tolerated in some adults. When CsA is not effective or contra-indicated: ** AZA and MTX may be used in patients with AD. RCT data shows that MTX in children seems to be safe for the use up to 12 weeks. No AZA RCTs are published in children with AD, prospective and retrospective data show that AZA might be effective. ** MMF may be used for the treatment of AD in children and adults, but efficacy and safety data is scarce. - Well known side effects of CsA, MTX, AZA and MMF must be carefully monitored.

Part II Efficacy and safety of MTX and AZA in atopic dermatitis - Both off-label treatments seem to be well tolerated and effective in adults up to 5 years of use. - Low drug survival rate requires optimization of treatment with MTX and AZA. - Folic acid supplementation is recommended while taking MTX. - Before starting AZA, TPMT levels should be measured. - Well known side effects of MTX and AZA must be carefully monitored.

Part III Predictors and markers for severity and efficacy in atopic dermatitis - TARC and CTACK were confirmed as potential biomarkers. VEGF and IL-13 have a potential value as well. Biomarkers could not be used to discriminate at baseline

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between responders and non-responders, or FLG genotype status. - FLG mutations seem to have a negative impact on the initial treatment outcome measured at 24 weeks of immunosuppressive treatment, but after 2 years of treatment both patients with and without FLG mutations show similar treatment effects. - FLG genotype status determination may be useful in daily practice to tailor treatment regimens, as well as in research settings, as it may influence outcome parameters.

FUTURE PERSPECTIVES

An update of the systematic review summarizing the evidence for systemic immunosuppressive in AD is needed using the right methodology such as advised by the GRADE working group. As new systemic treatments are investigated, it will be essential to compare these treatments with existing treatments. But head-to-head comparison in trials is often not done, as drugs are mostly compared against placebos. A network meta-analyses (NMA) may provide more insight. By using NMA, available evidence from trials can by compared, directly and indirectly, allowing a convincing conclusion about safety and efficacy to be made.(72) Currently, both CsA and dupilimab are approved for the treatment of AD when topical treatment or phototherapy is not sufficient or contraindicated. But there is still a lack of high quality evidence concerning the treatments of AD, which highlights the need for large head to head studies or long-term prospective follow-up studies incorporating patient reported outcomes. As new treatment modalities for AD are emerging, it is important to get evidence for the effectiveness, cost-effectiveness and safety of currently used systemic treatments for AD or to compare them with these new generation systemic treatment options like dupilumab. 8 As prospective databases not have stringent inclusion criteria, also data can be gathered of sub-groups, like children, pregnant women, patients with comorbidities or eldery.(73) The use of core outcome domains and instruments according to the COS developed by HOME in future research is important in these prospective databases, to create homogeneous data and to make it possible to compare studies. When utilizing these aspects mentioned above, possibly a systemic immunosuppressive treatment algorithm can be made which makes treatment of a patient with moderate to severe AD more clear for the treating physician. Biomarkers and therewith targeted therapy should help us towards personalized medicine. While gathering information regarding biomarkers, standardization of methods is essential. When the same technics are used, biomarker data can be compared with each other. By collecting body material specimens in a prospective databases, a biobank can be created.

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This data can be used for further stratification of phenotypes in (74,AD. 75) When collected with standardized methods, data will be suitable to compare. In that way AD can be classified better, and treatment can be optimized.

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REFERENCES practices for systemic agents in the treatment of severe pediatric atopic dermatitis in the

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of Dermatologists’ guidelines for the safe of clinical dermatology. 2018;19(2):145-65. and effective prescribing of azathioprine 35. Seegraber M, Srour J, Walter A, Knop M, 2011. The British journal of dermatology. Wollenberg A. Dupilumab for treatment of 2011;165(4):711-34. atopic dermatitis. Expert review of clinical 28. Shen S, O’Brien T, Yap LM, Prince HM, pharmacology. 2018;11(5):467-74. McCormack CJ. The use of methotrexate 36. Blauvelt A, de Bruin-Weller M, Gooderham in dermatology: a review. The Australasian M, Cather JC, Weisman J, Pariser D, et al. journal of dermatology. 2012;53(1):1-18. Long-term management of moderate-to- 29. Vedie AL, Ezzedine K, Amazan E, Boralevi severe atopic dermatitis with dupilumab and F, Milpied B, Taieb A, et al. Long-term Use concomitant topical corticosteroids (LIBERTY of Systemic Treatments for Moderate- AD CHRONOS): a 1-year, randomised, to-Severe Atopic Dermatitis in Adults: A double-blinded, placebo-controlled, phase Monocentric Retrospective Study. Acta 3 trial. Lancet (London, England). dermato-venereologica. 2016;96(6):802-6. 2017;389(10086):2287-303. 30. Haeck IM, Knol MJ, Ten Berge O, van 37. de Bruin-Weller M, Thaci D, Smith CH, Velsen SG, de Bruin-Weller MS, Bruijnzeel- Reich K, Cork MJ, Radin A, et al. Dupilumab Koomen CA. Enteric-coated mycophenolate with concomitant topical corticosteroid sodium versus cyclosporin A as long-term treatment in adults with atopic dermatitis treatment in adult patients with severe with an inadequate response or intolerance atopic dermatitis: a randomized controlled to ciclosporin A or when this treatment is trial. Journal of the American Academy of medically inadvisable: a placebo-controlled, Dermatology. 2011;64(6):1074-84. randomized phase III clinical trial (LIBERTY 31. Omair MA, Alahmadi A, Johnson SR. Safety AD CAFE). The British journal of dermatology. and effectiveness of mycophenolate in 2018;178(5):1083-101. systemic sclerosis. A systematic review. PloS 38. Simpson EL, Bieber T, Guttman-Yassky E, Beck one. 2015;10(5):e0124205. LA, Blauvelt A, Cork MJ, et al. Two Phase 3 8 32. Heller M, Shin HT, Orlow SJ, Schaffer JV. Trials of Dupilumab versus Placebo in Atopic Mycophenolate mofetil for severe childhood Dermatitis. The New England journal of atopic dermatitis: experience in 14 patients. medicine. 2016;375(24):2335-48. The British journal of dermatology. 39. Levy LL, Urban J, King BA. Treatment of 2007;157(1):127-32. recalcitrant atopic dermatitis with the oral 33. Waxweiler WT, Agans R, Morrell DS. Systemic Janus kinase inhibitor tofacitinib citrate. treatment of pediatric atopic dermatitis with Journal of the American Academy of azathioprine and mycophenolate mofetil. Dermatology. 2015;73(3):395-9. Pediatric dermatology. 2011;28(6):689-94. 40. https://clinicaltrials.gov. 34. Snast I, Reiter O, Hodak E, Friedland R, 41. Schmitt J, Williams H. Harmonising Outcome Mimouni D, Leshem YA. Are Biologics Measures for Eczema (HOME). Report Efficacious in Atopic Dermatitis? A Systematic from the First International Consensus Review and Meta-Analysis. American journal Meeting (HOME 1), 24 July 2010, Munich,

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Germany. The British journal of dermatology. Hijnen DJ, Bruijnzeel-Koomen CAF, van den 2010;163(6):1166-8. Broek MPH, et al. Allopurinol Co-prescription 42. Gerbens LAA, Apfelbacher CJ, Irvine AD, Improves the Outcome of Azathioprine Barbarot S, de Booij RJ, Boyce AE, et al. Treatment in Chronic Eczema. Acta dermato- TREatment of AD (TREAT) Registry Taskforce: venereologica. 2018;98(3):373-5. an international Delphi exercise to identify 49. Salliot C, van der Heijde D. Long-term safety a core set of domains and domain items for of methotrexate monotherapy in patients national AD photo- and systemic therapy with rheumatoid arthritis: a systematic registries. The British journal of dermatology. literature research. Annals of the rheumatic 2018. diseases. 2009;68(7):1100-4. 43. Rajka G, Langeland T. Grading of the 50. Casetta I, Iuliano G, Filippini G. Azathioprine severity of atopic dermatitis. Acta dermato- for multiple sclerosis. The Cochrane database venereologica Supplementum. 1989;144:13- of systematic reviews. 2007(4):Cd003982. 4. 51. Camus M, Seksik P, Bourrier A, Nion- 44. Brenninkmeijer EE, Schram ME, Leeflang Larmurier I, Sokol H, Baumer P, et al. Long- MM, Bos JD, Spuls PI. Diagnostic criteria term outcome of patients with Crohn’s for atopic dermatitis: a systematic review. disease who respond to azathioprine. The British journal of dermatology. Clinical gastroenterology and hepatology 2008;158(4):754-65. : the official clinical practice journal of the 45. Roekevisch E, Schram ME, Leeflang MMG, American Gastroenterological Association. Brouwer MWD, Gerbens LAA, Bos JD, et al. 2013;11(4):389-94. Methotrexate versus azathioprine in patients 52. Thijs J, Krastev T, Weidinger S, Buckens CF, with atopic dermatitis: 2-year follow-up de Bruin-Weller M, Bruijnzeel-Koomen C, data. The Journal of allergy and clinical et al. Biomarkers for atopic dermatitis: a immunology. 2018;141(2):825-7.e10. systematic review and meta-analysis. Curr 46. Politiek K, van der Schaft J, Coenraads Opin Allergy Clin Immunol. 2015;15(5):453- PJ, de Bruin-Weller MS, Schuttelaar ML. 60. Drug survival for methotrexate in a daily 53. Landheer J, de Bruin-Weller M, Boonacker C, practice cohort of adult patients with severe Hijnen D, Bruijnzeel-Koomen C, Rockmann atopic dermatitis. The British journal of H. Utility of serum thymus and activation- dermatology. 2016;174(1):201-3. regulated chemokine as a biomarker for 47. Gerbens LAA, Hamann SAS, Brouwer MWD, monitoring of atopic dermatitis severity. Roekevisch E, Leeflang MMG, Spuls PI. Journal of the American Academy of Methotrexate and azathioprine for severe Dermatology. 2014;71(6):1160-6. atopic dermatitis: a 5-year follow-up study 54. 54. Thijs JL, Nierkens S, Herath A, of a randomized controlled trial. The British Bruijnzeel-Koomen CA, Knol EF, Giovannone journal of dermatology. 2018;178(6):1288- B, et al. A panel of biomarkers for disease 96. severity in atopic dermatitis. Clinical and 48. Garritsen FM, van der Schaft J, de Graaf M, experimental allergy : journal of the British

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Society for Allergy and Clinical Immunology. factor in the lesions of atopic dermatitis. 2015;45(3):698-701. Archives of dermatological research. 55. Teraki Y, Hotta T, Shiohara T. Increased 2006;297(9):425-9. circulating skin-homing cutaneous 61. Koczy-Baron E, Jochem J, Kasperska-Zajac A. lymphocyte-associated antigen (CLA)+ type Increased plasma concentration of vascular 2 cytokine-producing cells, and decreased endothelial growth factor in patients CLA+ type 1 cytokine-producing cells in with atopic dermatitis and its relation to atopic dermatitis. The British journal of disease severity and platelet activation. dermatology. 2000;143(2):373-8. Inflammation research : official journal of 56. Samochocki Z, Bogaczewicz J, Sysa- the European Histamine Research Society Jedrzejowska A, McCauliffe DP, Kontny [et al]. 2012;61(12):1405-9. E, Wozniacka A. Expression of vascular 62. Machura E, Rusek-Zychma M, Jachimowicz endothelial growth factor and other M, Wrzask M, Mazur B, Kasperska-Zajac cytokines in atopic dermatitis, and A. Serum TARC and CTACK concentrations correlation with clinical features. Int J in children with atopic dermatitis, allergic Dermatol. 2016;55(3):e141-6. asthma, and urticaria. Pediatr Allergy 57. Wong LS, Otsuka A, Yamamoto Y, Nonomura Immunol. 2012;23(3):278-84. Y, Nakashima C, Honda T, et al. Vascular 63. Sohn MH, Lee KE, Kim KE. Interleukin-18 is endothelial growth factor partially induces associated with increased severity of atopic pruritus via epidermal hyperinnervation in dermatitis in children. Allergy and asthma imiquimod-induced psoriasiform dermatitis proceedings. 2004;25(3):181-4. in mice. Journal of dermatological science. 64. Zedan K, Rasheed Z, Farouk Y, Alzolibani AA, 2016;83(2):148-51. Bin Saif G, Ismail HA, et al. Immunoglobulin 58. McAleer MA, Jakasa I, Hurault G, Sarvari P, e, interleukin-18 and interleukin-12 in McLean WHI, Tanaka RJ, et al. Systemic and patients with atopic dermatitis: correlation stratum corneum biomarkers of severity with disease activity. Journal of clinical and 8 in infant AD include markers of innate and diagnostic research : JCDR. 2015;9(4):Wc01- Th-related immunity and angiogenesis. The 5. British journal of dermatology. 2018. 65. Hildner K, Marker-Hermann E, Schlaak 59. Zablotna M, Sobjanek M, Glen J, JF, Becker C, Germann T, Schmitt E, et al. Niedoszytko M, Wilkowska A, Roszkiewicz Azathioprine, mycophenolate mofetil, and J, et al. Association between the -1154 G/A methotrexate specifically modulate cytokine promoter polymorphism of the vascular production by T cells. Ann N Y Acad Sci. endothelial growth factor gene and atopic 1998;859:204-7. dermatitis. Journal of the European Academy 66. Kremer JM, Lawrence DA, Hamilton R, of Dermatology and Venereology : JEADV. McInnes IB. Long-term study of the impact 2010;24(1):91-2. of methotrexate on serum cytokines and 60. Zhang Y, Matsuo H, Morita E. Increased lymphocyte subsets in patients with active production of vascular endothelial growth rheumatoid arthritis: correlation with

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pharmacokinetic measures. RMD Open. C, Nierkens S, Giovannone B, Csomor E, 2016;2(1):e000287. et al. Moving toward endotypes in atopic 67. Crilly A, McInnes IB, Capell HA, Madhok R. dermatitis: Identification of patient clusters The effect of azathioprine on serum levels based on serum biomarker analysis. The of interleukin 6 and soluble interleukin 2 Journal of allergy and clinical immunology. receptor. Scand J Rheumatol. 1994;23(2):87- 2017;140(3):730-7. 91. 68. Irvine AD, McLean WH, Leung DY. Filaggrin mutations associated with skin and allergic diseases. The New England journal of medicine. 2011;365(14):1315-27. 69. Palmer CN, Irvine AD, Terron-Kwiatkowski A, Zhao Y, Liao H, Lee SP, et al. Common loss- of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis. Nature genetics. 2006;38(4):441-6. 70. Weidinger S, Illig T, Baurecht H, Irvine AD, Rodriguez E, Diaz-Lacava A, et al. Loss-of- function variations within the filaggrin gene predispose for atopic dermatitis with allergic sensitizations. The Journal of allergy and clinical immunology. 2006;118(1):214-9. 71. Kezic S, Jakasa I. Filaggrin and Skin Barrier Function. Current problems in dermatology. 2016;49:1-7. 72. Lv J, Zhou D, Wang Y, Zhao J, Chen Z, Zhang J, et al. Quantitative evaluation to efficacy and safety of therapies for psoriasis: A network meta-analysis. Molecular pain. 2018;14:1744806918762205. 73. Tanei R. ATOPIC DERMATITIS IN THE ELDERLY. Arerugi = [Allergy]. 2015;64(7):918-25. 74. Brunner PM, Guttman-Yassky E, Leung DY. The immunology of atopic dermatitis and its reversibility with broad-spectrum and targeted therapies. The Journal of allergy and clinical immunology. 2017;139(4s):S65-s76. 75. Thijs JL, Strickland I, Bruijnzeel-Koomen

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Summary and conclusions

In this thesis we evaluated systemic therapy in atopic dermatitis (AD). In the first part we give an outline of prescription behavior of systemic treatments in two academic centers in the Netherlands and provide a systematic overview of systemic immunosuppressive treatments for AD. In part II we concentrated particularly on the treatment of chronic severe AD with methotrexate (MTX) and azathioprine (AZA), looking at the short and long-term efficacy and safety. In part III we identify predictive values on treatment outcome in patients with chronic severe AD when on systemic immunosuppressive therapy.

The main aims of this thesis were: 1. to provide a systematic overview of systemic treatments for AD in a clinical trial setting and to obtain an overview of prescription behavior in daily academic clinical practice. 2. to gather more information concerning short and long-term treatment with MTX and AZA in AD. 3. to identify predictive values on treatment outcome in patients with chronic severe AD when on systemic immunosuppressive therapy.

In the general introduction in chapter 1, an overview is given regarding AD. We discuss the clinical features and patterns, diagnostic criteria, epidemiology and pathogenesis.

Part I Systemic treatments in atopic dermatitis Many patients with moderate-to-severe AD require systemic immunosuppressive treatment to achieve adequate disease control. Because there is a lack of information on the use oral immunosuppressive drugs in AD in daily practice, in chapter 2 we evaluated prescription behavior in two academic medical centers, namely the Amsterdam Academic Medical Center and the University Medical Center Utrecht. Medical charts of patients with AD, who received oral immunosuppressive were analyzed. Over a total of 10 years, 334 patients with AD received oral immunosuppressive treatment of which 102 also participated in clinical trials. The mean age at the start of an oral immunosuppressive drug is about 37 years. CsA was given in 80% of the patients, mycophenolate mofetil or enteric-coated mycophenolate in 31%, AZA in 14%, MTX in 11%, systemic glucocorticosteroids in 7% and systemic tacrolimus in 5%. Frequent reasons for discontinuation were adverse events and inefficacy. In chapter 3 we systematically evaluated literature for the efficacy and safety of systemic treatments for moderate-to-severe AD. Thirty-four randomized controlled trials (RCTs)

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evaluating systemic treatments for moderate-to-severe AD were included. Although 12 different interventions for moderate-to-severe AD have been studied in 34 RCTs, strong recommendations are only possible for the short-term use of cyclosporin A (CsA), which efficaciously improves clinical signs of AD. CsA is recommended as first-line treatment for short-term use (one year). AZA is mentioned as second-line treatment option, but its efficacy is lower, and the evidence is weaker. MTX can be considered a third-line treatment option. Recommendations were impossible to give for mycophenolate, montelukast, intravenous immunoglobulins, and systemic glucocorticosteroids because of limited evidence. Unfortunately we have detected many methodological limitations in the majority of trials. Well-designed, large head-to-head trials evaluating short and long-term treatments are required.

Part II Efficacy and safety of MTX and AZA in atopic dermatitis MTX and AZA are proposed as off-label treatment options, but direct comparisons are lacking. In chapter 4, adult patients with severe AD were randomly assigned in a 1:1 ratio to receive either MTX or AZA for 12 weeks, followed by a 12-week follow-up period. Of the 45 patients screened, 42 were included. At week 12, patients in the MTX grouphad a mean relative reduction in the SCORing Atopic Dermatitis(SCORAD) of 42% (SD,18%) compared with 39% (SD, 25%) in the AZA group (P 0.52). Proportions of patients achieving at least mild disease and reductions on impact of quality of life, symptoms were similar in both groups at weeks 12 and 24. No statistically significant differences were found in the number and severity of adverse events. Abnormalities in blood count were more common in the AZA group. No serious adverse events occurred. With this study we concluded that both treatments achieved clinically relevant improvement and were safe on the short term. But because AD is a chronic disease, it is imperative that the proposed therapy for these patients has a positive risk benefit ratio with persistent efficacy and an acceptable adverse effect profile when used for longer periods of time. That is why we investigated the long-term 9 outcomes of MTX and AZA over 2 and 5 years. In chapter 5 we present the 2-years follow-up data of these patients. All 42 patients were asked to participate in an open-label observational follow-up study to be evaluated 3 monthly for 2 years. After 12 weeks of treatment with MTX or AZA, treatments were continued, stopped, or switched, reflecting normal clinical practice. The primary outcomes were difference in mean absolute and relative change of SCORAD and Investigator Global Assessment (IGA) between groups, after 2 years compared to baseline. Thirty-five out of 42 patients were included. Two years after baseline, 10 patients had used MTX and 8 patients AZA, continuously. Both groups maintained a significant reduction in SCORAD and there was no significant difference in effect between groups (both intention-

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to-treat and per-protocol population). No important differences were found in type, number and severity of adverse events. Three serious adverse events occurred, 2 exacerbations of AD and 1 hospitalization because of psychiatric comorbidity. With this study we concluded that MTX and AZA seem to be effective and safe long-term therapies for adult patients with severe AD.

Part III Predictors and markers for severity and efficacy in atopic dermatitis Another way to look for predictive values on treatment outcome in patients is to search for biomarkers to objectively measure disease severity and predict therapeutic responses in AD. This was done in chapter 6. Thirty-eight severe AD patients treated with MTX or AZA were included in this study. Blood samples were collected to analyze serum levels of APRIL, BAFF, TARC (CCl-17), IL-1RA, IL-1β, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-18, IL-31, IFN-γ, TNF-α, VEGF, MIG (CXCL-9), IP-10 (CXCL-10), MCP-1 (CCL-2), MIP-1β (CCL-4), RANTES (CCL- 5), CTACK (CCL-27), TSLP, IL-5, IL-1α and G-CSF by ELISA and Luminex. Responders to treatment were defined by a SCORAD reduction of ≥50%. Buccal mucosa swabs were collected to determine the FLG genotype status. Serum levels of TARC, CTACK, IL-13 and VEGF showed a significant decrease after treatment with MTX or AZA. However, decreased cytokine levels were not significantly correlated with change in outcome parameters. Baseline biomarker levels were not significantly different between responders and non-responders, andFLG and non-FLG mutants showed similar biomarker profiles. With this study we confirmed TARC and CTACK as potential biomarkers. VEGF and IL-13 have a potential value as well. Biomarkers could not be used to discriminate at baseline between responders and non-responders, or FLG genotype status. Identification of predictive values on treatment outcome in patients with chronic severe AD when on systemic immunosuppressive therapy are needed. In chapter 7 we aimed to assess the effect of the filaggrin gene (FLG) mutations on treatment outcome in patients with chronic severe AD treated with combined systemic and topical immunosuppressive treatment, or topical immunosuppressive treatment alone. Thirty-six severe AD patients treated with systemic (either MTX or AZA) and topical immunosuppressive treatment or topical immunosuppressive treatment alone participated in this study. Buccal mucosa swabs were collected to determine the FLG genotype status (R501X, 2282del4, R2447X, S3247X and 3321delA mutations). The primary outcome was the SCORAD in the course of 24 weeks. We used mixed models and splines to investigate the difference in SCORAD between the patients with and without mutations. As age was significantly different at baseline, all analyses were corrected for age. At least one FLG mutation was found in 13 patients (36%). Ten patients were heterozygous, two patients were homozygous and one patient was compound heterozygous.

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In the course of 24 weeks, patients with aFLG mutation showed less improvement in SCORAD than patients without a FLG mutation (P=0.02). With this study we concluded that patients with a FLG mutation treated with combined systemic and topical immunosuppressive treatment, or topical immunosuppressive treatment alone, tend to have lower treatment responses compared to patients withoutFLG mutations at 24 weeks of treatment.

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Samenvatting en conclusies

Dit proefschrift beschrijft het gebruik van systemische therapie bij atopisch eczeem (AD). Het eerste deel bevat een overzicht van het voorschrijfgedrag van dermatologen betreffende systemische behandelingen in twee academische centra in Nederland en een systematische review van systemische immunosuppressieve behandelingen voor AD. In deel II bespreken we een gerandomiseerde gecontroleerde studie (RCT) naar de werkzaamheid en veiligheid op korte en lange termijn van methotrexaat (MTX) versus azathioprine (AZA) bij patiënten met chronische ernstige AD. In deel III identificeren we voorspellende waarden (zogenaamde biomarkers) voor het behandelresultaat bij patiënten met chronische ernstige AD bij systemische immunosuppressieve therapie.

De doelstellingen van dit proefschrift waren: 1. Meer inzicht krijgen in het gebruik en van systemische behandelingen voor AD en het voorschrijfgedrag aangaande deze middelen in de dagelijkse academische klinische praktijk; 2. Het beoordelen van de veiligheid en effectiviteit van MTX en AZA op zowel de korte als lange termijn; 3. De identificatie van biomarkers die bij patiënten met chronische ernstige AD de ernst van AD en eventueel behandelsucces van systemische therapie kunnen voorspellen.

In de algemene inleiding in hoofdstuk 1 wordt een overzicht gegeven van AD. We bespreken de klinische kenmerken en patronen, diagnostische criteria, epidemiologie en pathogenese.

Deel I Systemische behandelingen bij atopische dermatitis Veel patiënten met matig tot ernstige AD hebben een systemische immunosuppressieve behandeling nodig om de ziekte onder controle te krijgen. Omdat er een gebrek aan informatie is over het gebruik van orale immunosuppressiva bij AD in de dagelijkse praktijk, evalueerden we in hoofdstuk 2 het voorschrijfgedrag in twee academische medische centra, namelijk het Academisch Medisch Centrum Amsterdam en het Universitair Medisch Centrum Utrecht. De medische dossiers van patiënten met AD die orale immunosuppressiva kregen werden geanalyseerd. In totaal kregen 334 patiënten met AD gedurende een periode van 10 jaar een orale immunosuppressieve behandeling waarvan 102 patiënten ook deelnamen aan klinisch onderzoek. De gemiddelde leeftijd bij starten van een oraal immunosuppressivum was ongeveer 37 jaar. Ciclosporine A (CsA) werd aan 80% van de patiënten gegeven, mycofenolaatmofetil (MMF) of mycofenolzuur aan 31%, AZA aan 14%, MTX aan 11%,

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systemische glucocorticosteroïden aan 7% en tacrolimus aan 5%. Meest voorkomende redenen voor het staken van de medicatie waren bijwerkingen en ineffectiviteit. hoofdstukIn 3 hebben we de bestaande medische literatuur over de werkzaamheid en veiligheid van systemische behandelingen voor matig tot ernstige AD op systematische wijze samengevat. Vierendertig gerandomiseerde gecontroleerde studies (RCTs) waarin systemische behandelingen onderzocht werden voor matig tot ernstig AD waren geïncludeerd. Omdat er 12 verschillende systemische behandelingen voor matige tot ernstige AD werden onderzocht in 34 RCTs, konden we alleen betrouwbare aanbevelingen doen voor het korte termijngebruik van CsA. CsA geeft een duidelijke verbetering van de klinische verschijnselen van AD. CsA wordt daarom aanbevolen als eerste behandeling voor kortdurend gebruik (1 jaar) voor AD. AZA is een tweede behandeloptie, de werkzaamheid lijkt minder te zijn en het bewijs is zwakker dan voor CsA. MTX kan worden beschouwd als een derde behandeloptie. Er waren geen goede aanbevelingen te doen voor MMF, montelukast, intraveneuze immunoglobulinen en systemische glucocorticosteroïden vanwege beperkt bewijs. Helaas hebben we in de meeste onderzoeken veel methodologische beperkingen ontdekt. Goed opgezette, grote RCTs die korte- en lange termijn behandelingen evalueren zijn daarom hard nodig.

Deel II Werkzaamheid en veiligheid van methotrexaat en azathioprine voor atopische dermatitis MTX en AZA worden regelmatig off-label gebruikt als behandeling voor AD, maar goede studies ontbreken. In hoofdstuk 4 beschrijven we een kleine RCT waarin volwassen patiënten met ernstige AD 1:1 gerandomiseerd worden voor MTX of AZA. Zij krijgen deze behandeling gedurende 12 weken, gevolgd door een follow-up periode van 12 weken. Van de 45 patiënten die werden gescreend, werden er 42 geïncludeerd in de studie. Twaalf weken na behandeling hadden patiënten in de MTX-groep een gemiddelde relatieve afname van de SCORing atopische dermatitis (SCORAD) index van 42% (SD 18%) vergeleken met 39% (SD 25%) in de AZA-groep (P 0,52). Het aantal patiënten waarbij de ziekte afnam tot ten minste 9 “milde AD” en waarbij er een vermindering van de impact op de kwaliteit van leven bereikt werd, was vergelijkbaar in beide groepen, zowel 12 als 24 weken na start van de behandeling. Er werden geen statistisch significante verschillen gevonden in het aantal en de ernst van de bijwerkingen. Afwijkingen van het bloedbeeld kwamen vaker voor in de AZA-groep. Er zijn geen ernstige bijwerkingen opgetreden. Uit dit onderzoek concludeerden we dat beide behandelingen resulteerden in een klinisch relevante verbetering en op korte termijn veilig waren. Omdat AD een chronische ziekte is, is het noodzakelijk dat de voorgestelde therapie voor deze patiënten een positieve risico-batenverhouding heeft; namelijk aanhoudende werkzaamheid en een aanvaardbaar bijwerkingenprofiel bij gebruik voor de langere tijd.

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Daarom hebben we de lange termijn resultaten van MTX en AZA na 2 en 5 jaar onderzocht. In hoofdstuk 5 presenteren we de 2-jaar follow-up gegevens van deze patiënten. Alle 42 patiënten werden gevraagd deel te nemen aan een open-label observationele follow- up studie die gedurende 2 jaar plaatsvond met 3-maandelijkse evaluaties. Na 12 weken behandeling met MTX of AZA werd er in overleg met patiënt besloten de behandeling voort te zetten, te stoppen of om te zetten naar het andere middel, hetgeen de normale klinische praktijk weerspiegelt. De primaire uitkomstmaat was het verschil in gemiddelde absolute en relatieve verandering van de SCORAD en Investigator Global Assessment (IGA) tussen de 2 groepen 2 jaar na start van behandeling in vergelijking met deze maten bij de start van de studie. Vijfendertig van de 42 patiënten werden geïncludeerd. Twee jaar na baseline hadden 10 patiënten continu MTX en 8 patiënten continu AZA gebruikt. Beide groepen hadden ook na twee jaar een significante reductie in de SCORAD en er was geen significant verschil in effect tussen de twee groepen (zowel in de intention-to-treat als per-protocol analyse). Er werden geen belangrijke verschillen gevonden in type, aantal en ernst van de bijwerkingen. Drie ernstige bijwerkingen traden op; 2 exacerbaties van AD en 1 hospitalisatie vanwege psychiatrische comorbiditeit. Uit dit onderzoek concludeerden we daarom dat MTX en AZA effectieve en veilige lange termijn therapieën lijken te zijn voor volwassen patiënten met ernstige AD.

Deel III Voorspellers en markers voor de ernst en werkzaamheid bij atopische dermatitis Om in de toekomst beter en vooral eerder te kunnen voorspellen of een behandeling aanslaat of niet, om eventueel bijwerkingen te kunnen voorspellen en om de ernst van de aandoening objectief te meten zouden geschikte biomarkers van grote toegevoegde waarde zijn. In hoofdstuk 6 hebben wij diverse biomarkers geanalyseerd bij patiënten met AD die behandeld worden met systemische therapie. Achtendertig ernstige AD patiënten die behandeld werden met MTX of AZA konden geïncludeerd worden in deze studie. Er werden bloedmonsters verzameld om serumspiegels van APRIL, BAFF, TARC (CCI-17), IL-1RA, IL-1ß, IL-4, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-12, IL-13, IL-18, IL-31, IFN-y, TNF-α, VEGF, MIG (CXCL-9), IP-10 (CXCL-10), MCP-1 (CCL- 2), MIP-lp (CCL-4), RANTES (CCL-5), CTACK (CCL-27), TSLP, IL- 5, IL-1α en G-CSF te meten met behulp van ELISA en Luminex. “Responders”, patiënten die goed reageerden op behandeling, werden gedefinieerd als een SCORAD-reductie van ≥50%. Buccale mucosa-swabs werden verzameld om de filaggrine-gen (FLG) genotypestatus te bepalen. Serumspiegels van TARC, CTACK, IL-13 en VEGF namen significant af na behandeling met MTX of AZA. Verlaagde cytokineniveaus waren echter niet significant gecorreleerd met een verandering in de uitkomstparameters. Baseline biomarker spiegels waren niet significant verschillend tussen responders en niet-responders, en FLG en niet-FLG mutanten vertoonden

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vergelijkbare biomarkerprofielen. Mogelijk zijn TARC, CTACK, IL-13 en VEGF dus potentiële biomarkers omdat zij ziekte-ernst kunnen weerspiegelen, maar om dit te bevestigen is meer onderzoek nodig. Wij konden geen biomarkers identificeren die voorafgaand aan start van de behandeling voorspellend waren voor respons op systemische therapie. In hoofdstuk 7 hebben we onderzocht of een (FLG) -mutatie effect heeft op de behandelingsresultaten bij patiënten met chronische ernstige AD. Zesendertig ernstige AD patiënten behandeld met systemische (MTX of AZA) en/of lokale immunosuppressieve behandeling namen deel aan deze studie. Buccale mucosa-swabs werden verzameld om de FLG-genotypestatus (R501X, 2282del4, R2447X, S3247X en 3321delA-mutaties) te bepalen. De primaire uitkomstmaat was het verloop van de SCORAD na 24 weken behandeling. We gebruikten gemengde statistische modellen en splines om het verschil in SCORAD tussen de patiënten met en zonder mutaties te onderzoeken. Omdat de leeftijd bij aanvang van de studie significant verschilde tussen de twee onderzochte groepen, werden alle analyses gecorrigeerd voor leeftijd. Er werd ten minste één FLG-mutatie gevonden bij 13 patiënten (36%). Tien patiënten waren heterozygoot, twee patiënten waren homozygoot en één patiënt was samengesteld heterozygoot. Na 24 weken vertoonden patiënten met eenFLG -mutatie een significant lagere verbetering in SCORAD dan patiënten zonder een FLG-mutatie (P= 0,02). We concludeerden dan ook dat patiënten met een FLG-mutatie die behandeld worden met systemische en/of lokale immunosuppressieve behandeling na 24 weken dus minder goed op therapie lijken te reageren dan AD patiënten zonder FLG-mutatie(s).

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List of abbreviations List of contributing authors List of publications PhD portfolio Dankwoord Curriculum vitae

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List of abbreviations

AD Atopic dermatitis AE Adverse event AMC Academic Medical Centre, Amsterdam APRIL A PRoliferation-Inducing Ligand AZA Azathioprine BAFF B-cell Activating Factor of the TNF Family COS Core outcome set CsA Cyclosporin CTACK(CCL-27) Cutaneous T cell-Attracting Chemokine (Chemokine (C-C motif) ligand 27) EASI D.K. Denise Kuester DLQI Dermatological Quality of Life Index EASI Eczema Area and Severity Index EDI Eczema disability index E.R. Evelien Roekevisch FLG Filaggrin-gene FMG Filaggrin Mutation Group G-CSF Granulocyte-Colony Stimulating Factor GRADE Grading of Recommendations Assessment, Development and Evaluations HOME Harmonising Outcome Measures for Eczema IFN-g Interferon-gamma IGA Investigator Global Assessment IL Interleukin IL-1RA Interleukin-1 Receptor Antagonist IL-1α Interleukin-1 alfa IL-1β Interleukin-1 bèta IL-x (number) Interleukin-x (number) IP-10(CXCL-10) Interferon gamma-induced Protein 10 (C-X-C motif chemokine Ligand 10) ITT Intention-to-treat IVIG Intravenous immunoglobulin JAK Janus kinase J.S. Jochen Schmitt LDH Lactate DeHydrogenase LOCF Last observation carried forward LRM Linear Regression Model m Months MAcAD Methotrexate versus Azathioprine for severe Atopic Dermatitis MCP-1(CCL-2) Monocyte Chemoattractant Protein-1 (Chemokine (C-C Motif) Ligand 2) MDC (CCL-22) Macrophage-Derived Chemokine ( C-C Motif Chemokine Ligand 22) MedDRA Medical Dictionary for Regulatory Activities MIG(CXCL-9) Monokine Induced by interferon Gamma (Chemokine (C-X-C motif) Ligand 9)

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MIP-1β(CCL-4) Macrophage Inflammatory Protein- 1 beta (Chemokine (C-C motif) Ligand 4) MMF Mycophenolate mofitil MTX Methotrexate NB-UVB Narrow band Ultraviolet B NMF Natural Moisturizing Factors OLE Open Label Extension P3NP Procollagen-3 N-terminal peptide PARC (CCL18) Pulmonary and Activation-Regulated Chemokine (Chemokine (C-C motif) ligand 18) PGA Patient Global Assessment POEM Patient-Oriented Eczema Measure PP Per-protocol P.I.S Phyllis Spuls PUVA Psoralen and ultraviolet A Regulated on Activation Normal T cell Expressed and Secreted (Chemokine (C-C motif) RANTES(CCL-5) ligand 5) RCT Randomized controlled trial RoB Risk of Bias SAE Serious adverse event SASSAD Six Area, Six Sign Atopic Dermatitis SCORAD SCORing Atopic Dermatitis index SCORAD50 Improvement of SCORAD with at least 50% SD Standard deviation SR Systematic review TARC (CCL-17) Thymus and activation-regulated chemokine (Chemokine (C-C motif) ligand 17 TBSA Total Body Surface Area TCHM Traditional Chinese Herbal Medicine TCS Total Clinical Severity Th2 T helper 2 TNF-α Tumor Necrosis Factor- alpha TP-5 Thymopentin TPMT Thiopurine S-methyltransferase TREAT TREatment of AD Treg cells Regulatory T cells TSLP Thymic Stromal Lymphopoietin UKSIP United Kingdom Sickness Impact Profile UV Ultra Violet UVA1 Ultraviolet A-1 UVB Ultraviolet B VAS Visual Analogue Scale VEGF Vascular Endothelial Growth Factor w weeks XFS Xiao-Feng-San y years

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List of contributing authors

J.D. Bos Department of Dermatology, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands

M.S. de Bruin-Weller Department of Dermatology and Allergology, University Medical Center Utrecht, the Netherlands

M.W.D. Brouwer Department of Dermatology, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands

L.E. Campbell Centre for Dermatology and Genetic Medicine, University of Dundee, Dundee, U.K.

J. Deinum Department of Dermatology, University Medical Center, Utrecht, the Netherlands

F.M. Garritsen Department of Dermatology, Academic Medical Center, University of Amsterdam, Amsterdam, the Netherlands Department of Dermatology, University Medical Centre, Utrecht, the Netherlands

L.A.A. Gerbens Department of Dermatology, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands

D.P. Hack Department of Dermatology, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands

S. Kezic Academic Medical Center, Coronel Institute of Occupational Health, Amsterdam, the Netherlands

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D. Kuester Centre for Evidence-based Healthcare, Medizinische Fakultät Carl Gustav Carus, Technical University, Dresden, Germany, University Allergy Center, University Hospital Carl Gustav Carus Dresden, Germany.

M.M.G. Leeflang Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands

J. Limpens Medical Library, Academic Medical Centre, Amsterdam, the Netherlands

R.W. Luiten Department of Dermatology and Netherlands Institute for Pigment Disorders (NIPD), Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands

W.H.I. McLean Centre for Dermatology and Genetic Medicine, University of Dundee, Dundee, U.K.

M.A. Middelkamp-Hup Department of Dermatology, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands

P.C.J.M. Res Department of Dermatology, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands

J. van der Schaft Department of Dermatology, University Medical Center, Utrecht, the Netherlands

J. Schmitt Centre for Evidence-based Healthcare, Medizinische Fakultät Carl Gustav Carus, Technical University, Dresden, Germany, University Allergy Center, University Hospital Carl Gustav Carus Dresden, Germany.

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M.E. Schram Department of Dermatology, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands

P.I. Spuls Department of Dermatology, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands

K. Szegedi Department of Dermatology, Academic Medical Centre, University of Amsterdam, Amsterdam, the Netherlands

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List of Publications

Hurault G, Schram ME, Roekevisch E, Spuls PI, Tanaka RJ. Relationship and probabilistic stratification of Eczema Area and Severity Index and objective Scoring Atopic Dermatitis severity scores for atopic dermatitis. Br J Dermatol. 2018 Oct;179(4):1003-1005.

Gerbens LAA, Hamann SAS, Brouwer MWD, Roekevisch E, Leeflang MMG, Spuls PI. Methotrexate and azathioprine for severe atopic dermatitis: a 5-year follow-up study of a randomized controlled trial. Br J Dermatol. 2018 Jun;178(6):1288-1296.

Roekevisch E, Schram ME, Leeflang MMG, Brouwer MWD, Gerbens LAA, Bos JD, Spuls PI. Methotrexate versus azathioprine in patients with atopic dermatitis: 2-year follow-up data. J Allergy Clin Immunol. 2018 Feb;141(2):825-827.

Roekevisch E, Leeflang MMG, Schram ME, Campbell LE, Irwin McLean WH, Kezic S, Bos JD, Spuls PI, Middelkamp-Hup MA. Patients with atopic dermatitis with filaggrin loss-of-function mutations show good but lower responses to immunosuppressive treatment. Br J Dermatol. 2017 Dec;177(6):1745-1746.

Garritsen FM, Roekevisch E, van der Schaft J, Deinum J, Spuls PI, de Bruin-Weller MS. Ten years experience with oral immunosuppressive treatment in adult patients with atopic dermatitis in two academic centres. J Eur Acad Dermatol Venereol. 2015 Oct;29(10):1905- 12.

Schmitt J, Spuls P, Boers M, Thomas K, Chalmers J, Roekevisch E, Schram M, Allsopp R, Aoki V, Apfelbacher C, Bruijnzeel-Koomen C, Bruin-Weller M, Charman C, Cohen A, Dohil M, Flohr C, Furue M, Gieler U, Hooft L, Humphreys R, Ishii HA, Katayama I, Kouwenhoven W, Langan S, Lewis-Jones S, Merhand S, Murota H, Murrell DF, Nankervis H, Ohya Y, Oranje A, Otsuka H, Paul C, Rosenbluth Y, Saeki H, Schuttelaar ML, Stalder JF, Svensson A, Takaoka R, Wahlgren CF, Weidinger S, Wollenberg A, Williams H. Towards global consensus on outcome measures for AD research: results of the HOME II meeting. Allergy. 2012 Sep;67(9). Schram ME, Roekevisch E, Leeflang MM, Bos JD, Schmitt J, Spuls PI.Response to a randomized trial of methotrexate vs. azathioprine for severe AD: a critical appraisal. Br J Dermatol. 2012. Apr;166(4):704.

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Schram ME, Roekevisch E, Leeflang MM, Bos JD, Schmitt J, Spuls PI. A randomized trial of methotrexate versus azathioprine for severe AD. J Allergy Clin Immunol. 2011 Aug;128(2):353-9.

Roekevisch E, de Vries HJC. Proctitis. Soaaidsmagazine. 2010 April; 1:7-9.

Roekevisch E, de Vries HJC. Consistent use of condoms offers modest protection against genital herpes. Ned Tijdschr Geneeskd. 2009; 153: A10

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PhD Portfolio

PhD student: E. Roekevisch PhD period: 2010-2018 Promotor: prof. dr. P.I. Spuls Copromotores: Dr. M.A. Middelkamp Hup and Dr. M.M.G. Leeflang

Year Workload (ECTS)

1. PhD training General courses Clinical Data Management 2010 0.3 Project management 2010 0.6 BROK/Good Clinical Practice 2010 0.9 PubMed 2010 0.2 Reference Manager 2010 0.2 Evidence-Based Searching 2010 0.3 AMC World of Science 2010 0.7

Oral presentations at (inter)national conferences Filaggrin mutations as predictors for treatment outcome in Atopic 2012 0.9 Dermatitis. Georg Rajka Symposium, International Symposium on Atopic Dermatitis (ISAD) Moshi, Tanzania Filaggrin mutations as predictors for treatment outcome in Atopic 2012 0.7 Dermatitis. International Dermato-Epidemiology Association (IDEA) Congress, Malmö, Sweden Efficacy and safety of systemic treatments for moderate-to-severe atopic 2014 0.9 dermatitis: a systematic review. European Academy of Dermatology and Venereology (EADV) Congress, Amsterdam, the Netherlands. Atopic dermatitis patients with filaggrin loss-of-function mutations show 2016 0.7 good but lower responses to immunosuppressive treatment. Nederlandse Vereniging voor Experimentele Dermatologie (NVED), Lunteren, the Netherlands Methotrexate versus azathioprine in patients with atopic dermatitis: two 2018 0.8 years follow up data. – “Breaking news” session. Dermatologendagen, Amsterdam, the Netherlands

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Methotrexate and azatioprine in severe atopic dermatitis; A 2018 0.9 randomized controlled trial and open label extension study. E u ro p e a n A ca d e my o f D e r m ato l o g y a n d Ve n e re o l o g y ( EA DV ) Congress, Paris, France.

Poster presentations at (inter)national conferences Filaggrin mutations as predictors for treatment outcome in Atopic 2012 0.3 Dermatitis. Georg Rajka Symposium, International Symposium on Atopic Dermatitis (ISAD) Moshi, Tanzania Filaggrin mutations as predictors for treatment outcome in Atopic 2014 0.3 Dermatitis. Academy of Dermatology and Venereology (EADV) Congress, Amsterdam, the Netherlands Methotrexate versus azathioprine in patients with atopic dermatitis. 2017 0.2 European Dermato-Eidemiology Netwerk Forum (EDEN , Madrid, Spain. Methotrexate versus azathioprine in patients with atopic dermatitis. 2018 0.3 International Symposium on Atopic Dermatitis (ISAD), Utrecht, the Netherlands.

International Conferences 41th European Society for Dermatological Research (ESDR)meeting, 2011 0.1 Barcelona, Spain 7th Georg Rajka Symposium, International Symposium on Atopic Dermatitis 2012 1.2 (ISAD) Moshi, Tanzania 6th International Dermato-Epidemiology Association (IDEA) Congress, 2012 0.7 Malmö, Sweden

National Conferences 23rd and 26th Academy of Dermatology and Venereology (EADV) Congress, 2014/2016 2.1 Amsterdam, the Netherlands Dermatologen in Opleiding (DIO) dagen, Amersfoort 2012-2015 0.9 Nederlandse Vereniging voor Experimentele Dermatologie (NVED), 2013/2016 0.9 Lunteren, the Netherlands Refereeravond AMC-VUMC, Amsterdam 2015 0.1 Wetenschappelijke vergadering Nederlandse Vereniging voor Dermatologie 2012-2018 1.4 en Venereologie (NVDV), several places, the Netherlands Nederlandse Vereniging voor Dermatologie en Venereologie (NVDV) 2014/2017 1.0 Dermatologendagen, Arnhem en Amsterdam International Symposium on Atopic Dermatitis (ISAD), Utrecht, the 2018 0.6 Netherlands.

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2. Teaching Selma Ataly, extracurricular internship Medical Student, AMC/UVA 2010 1.0 Amsterdam, the Netherlands Floor Garritsen, scientific internship Medical Student, UMC Utrecht, 2011 2.0 the Netherlands Maryam Soltanipoor, extracurricular internship Medical Student, AMC/UVA 2014 1.0 Amsterdam, the Netherlands Dennis Hack, scientific internship Medical Student, AMC/UVA Amsterdam, 2016 2.0 the Netherlands

3. Parameters of Esteem Awards Methotrexate versus azathioprine in patients with atopic dermatitis: two 2018 years follow up data. – “Breaking news” session. Dermatologendagen, Amsterdam, the Netherlands.

4. Others Sub investigator clinical trials, pace II, III and IV, Department of 2010-2012 Dermatology, AMC, Amsterdam, the Netherlands Member steering group “Allergie en Eczeem”, Department of Dermatology 2010-2013 0.3 AMC, Amsterdam, the Netherlands Organising committee Second International Consensus Meeting 2011 2.0 Harmonising Outcome Measures for Eczema (HOME II), Amsterdam, the Netherlands. Clinical scientific meeting (weekly), Department of Dermatology, AMC, 2012-2016 2.1 Amsterdam, the Netherlands Contribution “ Richtlijn Constitutioneel Eczeem” 2014 1.8 DARC (Dermatologie Assistenten Refereer Club) 2015-present 0.2 AMC Dermatology (ex)residents Journal Club AMC Dermatology residents 2017- present 0.2

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Dankwoord

En met dit hoofdstuk kom ik bijna aan het einde van dit proefschrift en tevens aan het einde van een tijdperk. Een leerzame enerverende periode die me veel positieve energie heeft gegeven maar me tevens af en toe de keerzijde van de medaille heeft laten zien. Ik kan niet anders dan zeggen dat zonder de begeleiding en hulp van veel mensen dit proefschrift niet tot stand was gekomen en daarom wil ik hierbij graag gebruik maken van de mogelijkheid om een aantal van hen in het bijzonder te bedanken.

De allereerste alinea wil ik wijden aan alle patiënten, die mee hebben gedaan aan de onderzoeken in dit proefschrift. Een groot gedeelte van jullie is 5 jaar lang, 3 maandelijks, ter controle op de poli gekomen of zelfs vaker. Jullie deden mee aan diverse onderzoeken resulterend in data waar dit proefschrift op gebaseerd is. Ik kan niet anders dan jullie enorm dankbaar zijn voor jullie medewerking.

Prof Ph. I. Spuls, lieve Phyllis. Wat ben ik dankbaar dat jij in mijn leven bent gekomen. Ik heb bewondering voor alles wat je doet. Een duizendpoot, hardwerkend en evenwichtig op vele fronten. Vanaf het eerste uur heb je me opgenomen in je team. Je bent nooit veroordelend en altijd probleemoplossend. Je bent enorm lief, betrokken, begripvol en ook nog eens heel leuk en gezellig. Ik kon wanneer dan ook bij je terecht. In het AMC, Amstelveen, Malmö, Madrid, een hotelkamer samen in Moshi of Parijs, ik voel me altijd op mijn gemak bij jou. Dank je wel voor wat je me allemaal hebt geleerd en hebt gegeven.

Dr. M.M.A Middelkamp-Hup, lieve Pina, jij werd mijn copromotor en ik jouw eerste PhD student. In een van de eerste maanden stelde je voor onze relatie “uit” te maken (mijn woorden) omdat je het idee had mij niet te kunnen geven wat ik nodig had als PhD student. Maar niets bleek minder waar. Wat een bijdrage heb jij geleverd en wat heb jij in mij geïnvesteerd. Ik heb enorm veel van je geleerd en ben enorm dankbaar dat jij aan mijn zijde hebt gestaan. Je zat er bovenop en dat is precies wat ik nodig had. Door de hele reis van mijn proefschrift heb ik je meer en meer leren kennen en je bent enorm lief, slim, doordenkend en enorm behulpzaam. Ik heb je in mijn hart gesloten.

Dr. M.M. Leeflang, lieve Mariska, gedurende mijn promotietraject was het duidelijk. Jij moest mijn copromotor zijn. Zoveel tijd je in mij en mijn onderzoeken hebt geinvesteerd. Meestal last minute moesten er weer opnieuw analyses worden gedaan, of laat ik het anders zeggen, moesten analyses weer voor de zoveelste keer herhaald worden, en jij stond werkelijk altijd voor me klaar. Lief, rustig en geduldig boetseerde jij uit mijn relaas van woorden en vragen

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weer een duidelijk beeld waardoor ik verder kon. Dank je wel hiervoor.

Prof. Bos, hartelijk dank voor uw begeleiding van mijn promotietraject. Ik heb dit altijd als heel prettig en bijdragend ervaren. Als een pater waakte u over mij en beschermde en corrigeerde, uw grote kennis over eczeem was daarbij bijdragend. Dankzij u werd mijn academische weg in de dermatologie mogelijk doordat u mij aannam voor de functie in het trialteam, daar zal ik u eeuwig dankbaar voor zijn.

Leden van de promotiecommissie, u wil ik hartelijk danken voor het zitting nemen in mijn promotiecommissie en voor de genomen tijd om mijn proefschrift te beoordelen.

Co-auteurs, dank voor jullie inzet en medewerking. Lieve Sanja, bron van rust en kennis, dank je wel voor al je input tijdens mijn promotie.

Dear dr Jochen Schmitt en Denise Kuester, thank you for al the hard work and cooperation, this was very valuable.

Lief trailteam, lieve Marleen, Gabrielle, Anna Christa, Dominique, Anne en Sanna, dank je wel lieve collegas voor de enorme fijne tijden van samenwerking in het trialteam. Gabrielle zo rustig, zacht, lief en betrokken, Marleen onuitputtelijk doorzettend en geïnteresseerd. Ook als ik straks weg ben uit het AMC, hoop ik jullie te blijven zien.

Dr M.E. Schram, lieve Mandy, waar was ik zonder jou geweest? Jij hebt een groot gedeelte van de onderzoeken in dit proefschrift opgezet en ik ben in jouw voetsporen verder gegaan. Eerst zaten we samen in het trialtream, en daarna ook in opleiding tot dermatoloog. Tranen van het lachen als ik samen met je was en ben. Je bent een hardwerkende dame met een doel voor ogen. Ik hoop op nog veel gezelligheid in de toekomst. Dank Mandy!

Lieve Marijke, dank je wel voor het opvolgen van de trial-patiënten nadat ik in opleiding ging. We go way back. Wat ben jij een fijne collega. Jij bent en blijft voor mij “de jonge leeuw“ en wie weet in de toekomt wel de “gouden“.

Lieve Louise, jij hebt de MAcAD afgerond en je bent het afgelopen jaar ook gepromoveerd, waarvoor mijn complimenten. Je bent geweldig in hoe je dingen ziet, aanpakt en doet.

Lieve Floor, knapperd van me. Wat ben je een kanjer en wat ben ik toch stiekem altijd trots op jou. Als student kwam je bij me en jij deed het gewoon. Jij bijt je op een bewonderingswaardige manier in iets vast en dan zorg je ook gewoon dat het er komt. Wat was het gezellig en fijn samenwerken met jou.

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Lieve Krisztina, dank je wel voor alle overleg momenten en onze samenwerking betreffende de biomarkers. En lieve Dennis, evenveel dank aan jou. Door jou stage werd de grootste basis van het biomarker hoofdstuk gelegd waarvoor veel dank!

Lieve Bernice, Carmen en Mariska, DE dames van het secretariaat. Goud waard en altijd meedenkend. Dank jullie wel voor al jullie hulp en voor de altijd aanwezige gezelligheid.

Lieve ondersteuning, doktersassistenten en verpleegkundigen. Dank voor jullie hulp en samenwerking!

Alle stafleden van de afdeling Dermatologie uit het AMC, OLVG, Flevoziekenhuis en VUMC, dank voor de samenwerking en alle leermomenten.

Dr. Mekkes, op zo’n 2000 meter hoogte werd de vraag gesteld: ”Wanneer wordt Dr .Mekkes “Jan”, en wordt U jij?” In Oostenrijk werd door jou besloten dat voor Hansje Eva en mij het moment wel daar was. Wij doorgewinterde AIOS. Ik ben je enorm dankbaar voor de opleiding die je mij gegeven heb. Door jouw betrokkenheid, buigzaamheid, rekbaarheid en meedenkendheid heb ik dit promotietraject tot een goed einde weten te brengen. Naast de beste opleider die ik me kan wensen ben je ook nog eens de meest slimme dermatoloog die ik ken, en ben ik dankbaar dat ik van je kennis heb mogen leren. Je humor is daarnaast iets wat ik, naast dit alles, enorm ga missen als ik het AMC verlaat.

Dr. Kemperman, lieve Patrick. We werkten samen in het Waterlandziekenhuis, later en tot op heden ook in het AMC. Hoe fijn en bijdragend! Jij straalt altijd “als een zonnetje”. Heerlijk is dat!

Dr. van de Linden, lieve Mireille. Heel graag wil ik jou noemen. Autoritten vol gesprekken of gewoon tussen de bedrijven door. Waardevol en fijn. Je ben een enorm lief en betrokken mens en ik dank je voor al het geloof dat je altijd in mij hebt gehad en hebt getoond. Je altijd aanwezige energie is aanstekelijk. De etentjes en gebreide kindersokjes in de kleur van het babykaartje zijn onvergetelijk.

Prof. Rustemeyer, lieve Thomas, jou ken ik al vanaf het eerste uur. Wat waren die 4 maanden op de allergologie fijn. Ik ben heel blij dat je deel uitmaakt van mijn commissie.

Prof de Vries, lieve Henry, dermatologisch onderzoek begon bij jou. Dank je wel dat je mij deze mogelijkheid hebt gegeven en veel dank voor jouw aanbeveling van mij bij de dermatologie in het AMC.

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Dear Dr. Carsten Flohr and prof. Catherine Smith, you guided me when I was there with you in London for a five-month during clinical Dermatology fellowship. Thank you for that!

Lieve (oud) collega’s uit het AMC, OLVG, Flevoziekenhuis en VUMC. Jullie creëer(d)en een warm bad met veel collegialiteit en gezelligheid. Extra dank voor jullie inzet, vooral aan het einde van mijn promotie, en dan met name een specifiek aantal van jullie die mijn poli’s op hebben gevangen zodat ik mijn deadlines kon halen. Jullie zijn top!

Lieve Hansje, mijn buddy. Samen op een hotelkamer tijdens COCOM. Elkaar bijpratend. Of elkaar opzoekend in de gang of assistentenkamer om even ons hart te luchten. Jouw rustige stem brengt mij altijd tot rust.

Lieve Favery-tje, lieve Mark. Jij kwam eigenlijk pas heel laat op m’n pad. In het einde van jouw opleiding begonnen we met praten en dat is nooit opgehouden. Uren vliegen voorbij als ik met jou ben. Naast een fijne collega en goede dokter, ben je een vriend die ik graag nog lang zie en spreek.

Lief Zonnetje , liefste vrienden, hoe had mijn leven er uitgezien zonder jullie… maar saai en leeg. Hoeveel moois geven jullie mij en hebben jullie mij gegeven. De boottochten tijdens Koninginnedag/ Koningsdag en LOS, Ardennenweekenden waarwe dineren en dansen tot diep in de nacht, verjaardagen, etentjes, rond een vuur of leunend tegen het aanrechtblad in de keuken, gesprekken vol betrokkenheid en interesse. Lieve Inge, Eva, Martina, Vera en Karin, lieve meisjes van me. Voordat zonnetje ontstond waren wij al samen. Rond de tafel gesprekken of gewoon heel hard mee zingen met “Turn around”. Dank voor alle herinneringen die jullie me geven. Lieve Erika, dank je wel voor de Engelse check van mijn proefschrift en Michelle dank je wel voor het maken van mijn toto PDF J. En een speciaal woord aan lieve drs. Grasman, lieve Thijs, veeg dat s-je snel weg en sluit je promoverende weg af, you can do it!

Lieve cultuursnuivers, lieve Thijs, Mo, Michiel, Marlies, Chip, Tammie en Pim, wat een fijne momenten in Zoutelande, “De Tuin” en daarbuiten. Dierbaar, dat zijn jullie.

Lieve Rus, je bent mijn altijd trouwe liefste en beste vriend. Hoewel ik altijd druk aan het rennen ben, zijn alle momenten die ik wel met je heb altijd fijn. Je bent goud waard.

Lieve Katy, mijn altijd lieve Katy. Onze vriendschap begon op de middelbare school en is alle jaren doorgegaan. Je bent waardevol voor me in alle opzichten. Jouw trouwe aard is onbeschrijfelijk. Dank voor de Engelse check van mijn proefschrift!

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Lieve Jacqueline, Jet, Karin, Janneke en Suus, geneeskundevriendinnetjes van het eerste uur. De uren tijdens onze etentjes vliegen voorbij en zijn eigenlijk altijd te kort. Lieve Jacq, wij woonden samen aan de Zeilstraat en wat hadden we het goed daar. Lieve Jet, bestuur 2002, onvergetelijk. Like a prayer en de zeemannen, wat kwamen ze vaak voorbij. Momenten van alleen maar geluk.

Lieve Simoon, mijn chica bonita, wat wij hebben kan niemand ons afnemen. Mexico, Honduras, Guatamala, Niguragua, El Salvador, Costa Rica, New York, Marokko (meine gute die kameel in de Sahara), Spanje en ik vergeet er vast nog wel wat. Nog nooit een woord van onenigheid. Hoe kostbaar is dat.

Lieve Anouk, op de SEH in het IJsselmeerziekenhuis ontstond onze vriendschap. Elk moment met jou is een feest.

Lieve Stef, mijn (peri)paranimf, graag herhaal ik de woorden die ik je eerder toesprak tijdens je promotiediner: “In je leven kom je veel mensen tegen. De meeste van die mensen vind je aardig, er kunnen er wat bij zijn die wat minder zijn, maar een enkele keer raken mensen je hart, en daar hou je steeds een beetje meer van elke keer als je ze ziet. Die mensen koester je in je hart. Stef jij bent een van die mensen voor mij. “

En dan mijn lieve paranimfjes. Lieve Jud en Mien, jullie zijn beide ongelooflijk sterke vrouwen, waardevolle vriendinnen, als jullie er maar zijn, dan is het goed. Lieve Judith, bij jou thuis staat de deur altijd voor me open. Je biedt me een luisterend oor, houdt me in de gaten en bent er voor me. Met drie kindjes, een baan in de Haag, en een drukke man, kijk ik met veel respect naar je. Lieve Mina, bezig bijtje van me. Ook jij luistert altijd naar me en waakt over me. Je bent loyaal en warm en denkt altijd aan alles en iedereen, hoe druk je ook bent. Met drie prachtige kindjes en Matthijs woon je nu voor 4 jaar in Sarajevo, en oh wat mis ik je. Wat fijn lieve vriendinnetjes dat jullie op deze belangrijke dag in mijn leven naast mestaan. Lieve Sas en Matthijs, met zijn zessen op vakantie, samen met de kindjes, is onmisbaar en heerlijk.

Lieve Lanne, je bent een enorm slim meisje en je hebt een hartje van goud. Je bent me heel dierbaar. Nu de drukte hopelijk wat minder wordt hoop ik op veel meer tijd samen, lief zusje van me. Lieve Louren en Florin, wat een lieverds zijn jullie. Het is altijd fijn om jullie te zien. Lieve Harry, Mike en Lisa, dank voor jullie liefde aan mijn zusje.

Lieve familie en schoonfamilie, dank voor jullie betrokkenheid en interesse!

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Lieve Henri, Joran, Thijn en Maus, m’n lieve zus. Ik mis jullie met heel mijn hart, ik hoop op een hereniging in de toekomst.

Lieve Addi en Rob, kerst 2001 kwamen jullie voor het eerst in mijn leven en ik kan daar alleen maar zo dankbaar voor zijn. Ik werd opgenomen in jullie warme familie. Jullie zijn evenwichtig, behulpzaam en bieden altijd een luisterend oor. En wat lief zijn jullie voor Jonas en Pepijn. Dank voor alles wat jullie mij geven en hebben gegeven. Lieve Koen, wat ben jij toch een kanjer. Alles goed op een rij, met 2 leuke banen, leuke vrienden en een en al passie voor sport en muziek. Dank je voor je altijd goede humeur en daarbij je interesse in alles wat ik doe.

Lieve papa en mama, jullie onvoorwaardelijke liefde en trouw is onbeschrijfbaar. Wat ik in mijn leven heb bereikt heb ik aan jullie te danken. Jullie hebben me altijd op alle vlakken gestimuleerd en achter mij gestaan, mij alles gegeven wat ik nodig had, en altijd gelooft in mijn kunnen. Jullie waken over mij, maar laten me ook los en vrij in de beslissingen die ik neem. Dank je voor het zijn van een enorm lieve opa en oma. Ik hou van met jullie met heel mijn hart en had geen betere papa en mama kunnen wensen dan jullie.

Het is ongelooflijk te beseffen hoeveel je van 2 mensjes kan houden. Lieve Pepijn en Jonas, m’n lieve mannetjes. Als ik bij jullie ben voel ik alleen maar geluk, jullie vullen mijn leven. Ik ben zo trots op jullie. En jij, , kleintje die nog in mijn buik zit. Ik kijk uit om je in mijn armen te sluiten en je alle liefde van de wereld te geven.

En dan ten slotte,

mijn liefste Marc. Ik weet werkelijk niet waar ik zou zijn zonder jou. Je bent mijn rots in de branding, je stabiliseert, relativeert en stimuleert op de momenten wanneer ikdat nodig hebt. Je humor is onmiskenbaar. Je enthousiasme en doorzettingsvermogen bewonderingswaardig. Je bent de liefste papa. Met niemand ben ik zoveel verbonden als met jou. Samen met onze mannen hebben we het fijnste leven. Ik hou van je met heel mijn hart.

Daarom wil ik dit dankwoord afsluiten met deze laatste zinnen aan jou:

Ik heb je lief….. Ik heb je liever. Liever dan mijn leven. Dan om het even wat. Ik heb je lief…. Ik heb je liever. Liever liefste elke dag. (Stef Bos)

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Curriculum vitae

Evelien Roekevisch was born in Lochem, the Netherlands, on October 19, 1979. She, graduated from secondary school (VWO) in Deventer in 1999. That same year she moved to Amsterdam where she attended medical school at the VU University of Amsterdam (VUmc). In the summer of 2000 she did her nursing internship in St. Luke’s Hospital in Malta. She joined the committee of the MFVU (Medical Faculty of the Free University) of Amsterdam as a sponsor coordinator in 2002. Between 2004 and 2008 she worked as a biometrist at the gynecology department of VUmc and also conducted research on the topic of uterine ruptures. In addition she performed a study on chronic abdominal pain in the Sint Lucas Andreas Hospital, at the department of gynecology. In 2005 she obtained her doctoral degree. Her scientific internship was done in Mexico City at the University of Anahuac on child psychiatry. Together with Simone Pulskens, she studied the social and health status of the youth in an American school in Mexico City which led to a research report written in collaboration with the University of Yale and Harvard. After her medical internships between 2005 and 2007, she worked for four months in the Trinity Hospital in Muona, Malawi, together with Marc Jansen. In September 2007 she graduated from medical school and started working at the emergency department of the Ijsselmeer hospital and after one year she moved to the emergency department of the VUmc. She started in 2008 at the STD department of the GGD (Medical and Health Service) in Amsterdam as seasonal doctor, and combined it with a research project on lymfogranuloma venereum under supervision of Jannie van der Helm and prof H.J. de Vries. In 2009, she worked as ANIOS in the Dermatology department of the Waterland Hospital in Purmerend. In 2010 she joined the trial team of the Dermatology department of the Amsterdam Medical Center (AMC) Amsterdam, where she worked as a sub-investigator of clinical trials and started her thesis under supervision of Prof. J.D. Bos, Prof. Ph.I. Spuls, Dr. M.M.G. Leeflang and Dr. M.A. Middelkamp-Hup. From April till October 2012 she worked as a doctor at the dermatology department of St. John’s Institute of Dermatology, Guy’s & St. Thomas, in London. In 2012 she started her residency Dermatology at the AMC in Amsterdam under supervision of Dr. J.R. Mekkes. In December 2018 she will complete her training as a dermatologist, together with her dissertation. Evelien lives in Amsterdam, together with her partner Marc Jansen and her sons Pepijn (5 years) and Jonas (3 years). She expects her third son in March 2019.

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Evelien Roekevisch werd geboren op 19 oktober 1979 in Lochem, Nederland. Ze studeerde af aan het VWO in Deventer in 1999. Datzelfde jaar verhuisde ze naar Amsterdam waar ze geneeskunde aan de Vrije Universiteit van Amsterdam (VUmc) ging studeren. In de zomer van 2000 deed ze haar verpleeghulpstage in het St. Luke’s Hospital te Malta. Zij trad in 2002 toe tot de commissie van de Medische Faculteit van de Vrije Universiteit (MFVU) als sponsorcoördinator. Tussen 2004 en 2008 werkte ze als biometriste op de afdeling gynaecologie van het VUMC en deed ze tevens onderzoek naar baarmoederrupturen. In het Sint Lucas Andreas Ziekenhuis, op de afdeling gynaecologie, deed ze onderzoek naar chronische buikpijn. In 2005 behaalde zij haar doctoraat Geneeskunde. Ze deed haar wetenschappelijke stage in de kinderpsychiatrie in Mexico aan de Universiteit van Anahuac. Daar onderzocht ze, samen met Simone Pulskens, de sociale- en gezondheidsstatus van de jeugd op een Amerikaanse school in Mexico-stad. Ze schreven een onderzoeksrapport in samenwerking met de Universiteit van Yale en Harvard. Na haar coschappen tussen 2005 en 2007 werkte ze 4 maanden samen met haar partner Marc Jansen, in het Trinity Hospital in Muona, Malawi. In september 2007 behaalde zij haar geneeskundebul en ging ze werken op de afdeling spoedeisende hulp van het IJsselmeerziekenhuis en na een jaar op de spoedeisende hulpafdeling van het VUmc. Ze startte in 2008 op de SOA-afdeling van de GGD (Geneeskundige en Gezondheidsdienst Amsterdam) in Amsterdam als seizoens- arts en combineerde dit met onderzoek naar lymfogranuloma venereum onder begeleiding van Jannie van der Helm en prof. H.J. de Vries. In 2009 ging ze als ANIOS werken op de afdeling Dermatologie van het Waterlandziekenhuis. In 2010 startte ze als onderzoeker in het trialteam van de afdeling dermatologie in het AMC in Amsterdam en begon ze tevens haar promotieonderzoek onder begeleiding van prof. J.D. Bos, prof. Ph.I. Spuls dr. M.M.G. Leeflang en dr. M.A. Middelkamp-Hup. Van april tot oktober 2012 was ze werkzaam als arts op de afdeling dermatologie van het St. John’s Institute of Dermatology, Guy’s & St. Thomas, in Londen. In 2012 begon zij met de opleiding Dermatologie in het AMC in Amsterdam onder toezicht van dr. J.R. Mekkes. In december 2018 zal ze haar opleiding tot dermatoloog afronden, naast haar promotietraject. Evelien woont in Amsterdam, samen met haar partner Marc Jansen en haar zoons Pepijn (5 jaar) en Jonas (3 jaar). Ze verwacht haar derde zoon in maart 2019.

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Systemic Treatment in Atopic Dermatitis Uitnodiging

Voor het bijwonen van de openbare verdediging van het proefschrift: Systemic Treatment Systemic Treatment in in Atopic Dermatitis Atopic Dermatitis Door Evelien Roekevisch

in de aula der Universiteit, Singel 411, 1012 XM, te Amsterdam op woensdag 19 december om 13.00 uur

Evelien Roekevisch Maarten Harpertszoon Trompstraat 6 hs 1056 HZ Amsterdam [email protected] Evelien Roekevisch

Paranimfen Judith van Gemert Evelien Roekevisch [email protected]

Mina Noor [email protected]

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