Clinical and Cytological Effects of Pimecrolimus

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Original Paper

Dermatology 2011;222:3648

DOI: 10.1159/000321711

Clinical and Cytological Effects of PimecrolimusCream 1% after Resolution of Active AtopicDermatitis Lesions by Topical Corticosteroids:A Randomized Controlled Trial

Christine Bangerta Bruce E. Stroberc Michael Corkd Jean-Paul Ortonnee

Thomas Lugerf Thomas Bieberg Adam Fergusond Rupert C. Eckerb

Tamara Koppa

Sophia Weise-Riccardih

Achim Guettnerh

Georg Stingla

aDepartment of Dermatology, Division of Immunology, Allergy and Infectious Diseases, Medical University

of Vienna, and bTissueGnostics GmbH, Vienna, Austria; cDepartment of Dermatology, New York University,

New York, N.Y., USA; dDivision of Genomic Medicine, University of Sheffield Medical School, Sheffield, UK;

eDepartment of Dermatology, Hpital de lArchet 2, Nice, France; fDepartment of Dermatology,

Universittsklinikum Mnster, Mnster, and gDepartment of Dermatology and Allergy, University of Bonn,

Bonn, Germany; hNovartis Pharma AG, Basel, Switzerland

with placebo cream (88.2 vs. 43.2 cells/mm2, respectively,

p = 0.047) and a slight but nonsignificant reduction in thenumber of dermal dendritic cells, Langerhans cells, T cells

and macrophages with pimecrolimus versus vehicle cream.

Limitations: This was an exploratory study. Conclusion:

Topical pimecrolimus was effective at maintaining beta-

methasone-17-valerate-induced AD remission by inhibit-

ing recurrences of the inflammatory infiltrate in the skin.

Copyright 2010 S. Karger AG, Basel

Introduction

Atopic dermatitis (AD) is a common inflammatorydisorder that runs a chronic or chronically relapsingcourse [13]. Clinical expression of the disease dependson the interactions between multiple susceptibility genes

Key Words

Atopic dermatitis Pimecrolimus cream 1% Topicalcorticosteroids Clinical and cytological response

Abstract

Background: Topical pimecrolimus may maintain remis-

sions of atopic dermatitis (AD) by inhibiting subclinical in-

flammation. Objective:To evaluate clinical and cytological

effects of pimecrolimus in topical corticosteroid-treated and

resolved AD lesions.Methods:Patients (n = 67) with resolved

AD lesions were randomized to 3-week double-blind treat-

ment with either pimecrolimus cream 1% or vehicle cream.

Outcome measures were reduction in Eczema Area and Se-verity Index (EASI) and number of leukocytes in skin biopsies

in all randomized patients who were evaluable at the end of

study. Results:The proportion of patients with a localized

EASI !2 at the end of study was higher with pimecrolimus

cream 1% than with vehicle cream (73.5 vs. 39.4%, respec-

tively). There was a significant decrease in the number of in-

filtrating CD45+ cells in pimecrolimus cream 1% compared

Received: February 23, 2010

Accepted after revision: September 27, 2010

Published online: December 8, 2010

Christine Bangert, MDDepartment of Dermatology, Division of Immunology, Allergy and InfectiousDiseases, Medical University of Vienna, Whr inger Grtel 1820

AT1090 Vienna (Austria), Tel. +43 1 404 007 704, Fax +43 1 404 007 574E-Mail christine.bangert @ meduniwien.ac.at

2010 S. Karger AG, Basel10188665/11/22210036$38.00/0

Accessible online at:www.karger.com/drm

Preliminary results were presented, in the form of a poster, at the

16th Congress of the European Academy of Dermatology and Vene-

reology, May 1620, 2007, Vienna, Austria.


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Dermatology 2011;222:3648 37

and the hosts environment [14]. The disorder is charac-terized by defective skin barrier function and cutaneoushyperreactivity to environmental triggers that are innoc-uous to normal individuals [1, 2, 4]. The immunopatho-logical features of the inflammatory infiltrate vary de-pending on the duration of the lesions [1, 2]. Allergen ex-

posure, scratching or the release of microbial toxinstrigger the extravasation of inflammatory cells into theskin through the release of proinflammatory cytokinesand chemokines from keratinocytes and resident mastcells, dendritic cells (DCs) and macrophages [2]. There isa biphasic response of helper T cells in the inf lammatoryinfiltrate [13], with a switching from cells that predomi-nantly produce a Th2 pattern of proinflammatory cyto-kines in the acute lesions to cells expressing the Th1 cy-tokine milieu in chronic lesions [58]. T-cell-derivedproducts such as interleukin- (IL) 4, IL-5 and granulo-cyte-macrophage colony-stimulating factor increase the

production of allergen-specific immunoglobulin E (IgE)and amplify the inflammatory reactions by promotingthe accumulation of eosinophils and monocytes-macro-phages at the tissue site [58]. Distinct subsets of DCs[913], which express the high-affinity receptor for IgE[1012], typically emerge in the lesional skin of AD pa-tients and inf luence both the extent of an initial allergen-specific inflammation and the persistence of the inflam-matory infiltrate by inducing the activation and furtherdifferentiation of specific subsets of helper T lympho-cytes and by modulating the levels of cytokines producedin the microenvironment [3, 11, 13]. The clinically unaf-

fected skin of patients with AD frequently shows a mildinf lammatory inf iltrate [14], mainly composed of helperT cells of the Th2 phenotype [5], that is absent in normalhealthy skin [5, 14]. IgE-bearing Langerhans cells (LCs)and inflammatory dendritic epidermal cells (IDECs)have also been detected in nonlesional skin [15].

The anti-inflammatory drugs currently available forthe intermittent treatment of active AD lesions includetopical corticosteroids of adequate potency and the topi-cal calcineurin inhibitors pimecrolimus cream 1% andtacrolimus ointment. Both topical corticosteroids and thetwo calcineurin inhibitors reduce the inflammatory in-

filtrate and the release of proinflammatory cytokines inthe lesional skin [1620], but the latter exhibit a more se-lective mode of action than topical corticosteroids [19, 21,22]. Because of the concerns about potential side effectsassociated with prolonged treatment [23], topical cortico-steroids are generally not used as long-term therapy espe-cially on nonlesional skin or during remission [2, 23].However, the nonlesional or postlesional skin of patients

with AD shows various degrees of subclinical inflamma-tion [2, 5, 14] that may need to be treated at an early stageto prevent relapses.

The use of pimecrolimus cream 1% as an early inter-vention, at the f irst signs and/or symptoms of a relapseduring the remission time, prevents the occurrence of

major flares and prolongs the f lare-free intervals [2429].With the present study, we began to explore the cellularevents underlying this phenomenon. We present for thefirst time data that correlate the clinical and cytologicaleffects of pimecrolimus cream 1%, when used to preventAD recurrence after resolution of active skin lesions by atopical corticosteroid.

Subjects and Methods

Study DesignThis multicenter study consisted of an open-label run-in pe-

riod of up to 2 weeks, during which patients with active AD le-sions were treated with a topical corticosteroid to induce diseaseremission, and a double-blind phase of up to 3 weeks, duringwhich patients applied either pimecrolimus cream 1% or thematched vehicle cream to the postlesional skin to prevent localdisease relapse. The study was conducted in accordance with theDeclaration of Helsinki and was approved by the appropriate localethics committee and institutional review board. All patients pro-

vided their informed consent. The trial was registered at http://www.clinicaltrials.gov on June 30, 2005, and the registration No.is NCT00117377.

Patients included in the study were 79 subjects aged 620years, with at least a 3-year history of mild or moderate AD. Toobtain control biopsies from normal skin, 12 healthy volunteers

aged 620 years were also included. Because it was initiallyplanned to perform both an analysis of the inflammatory infil-trate and an analysis of inflammatory cytokine gene expressionon replicate skin tissue samples, the study population (patientsand healthy volunteers) was limited to male patients to eliminatethe variation in gene and protein expression that may occur as aresult of including both sexes. However, this could not be per-formed as planned due to mRNA degradation of the skin biopsyspecimens. All subjects provided written informed consent toparticipate in the investigation.

At screening, the patients were required to have a diagnosis ofAD, according to the criteria of Hanifin and Rajka [30], and tosuffer from an acute episode of mild or moderate AD that hadstarted within the previous 5 days. AD severity was determined

using the Investigators Global Assessment (IGA) for the wholebody [31] and the Eczema Area and Severity Index (EASI) [32]. Awhole-body IGA score of 2 or 3 was necessary to confirm the di-agnosis of mild or moderate AD, respectively. In each patient, anAD lesional area of at least 10 cm2, present bilaterally on the armsor the legs, with a local EASI score of 18, was identified as thetarget site for assessment and was delineated with skin markers.

Exclusion criteria for AD patients were concurrent skin dis-ease (other than AD) in the treatment area, active skin infections,unsatisfactory response to previous treatment with topical tacro-


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limus or pimecrolimus, current topical therapy known or sus-pected to have an effect on AD, any systemic therapy known orsuspected to have an effect on AD within 4 weeks prior to thescreening visit. Healthy volunteers were excluded for personal orfamily history of atopy, erythroderma or topical therapy at thetarget sites within 5 weeks prior to the screening visit. Furtherexclusion criteria for both groups consisted of any history or cur-

rent diagnosis of an immunocompromised status, a history ofrheumatic fever, a known hypersensitivity to the study medica-tions, a history of serious reactions to anesthetics and any otherclinical condition that could interfere with study evaluations,such as treatment with phototherapy, systemic corticosteroids orantihistamines within 7 days prior to the screening visit.

During the open-label run-in period, AD patients applied be-tamethasone 17-valerate (BMV) cream or ointment 0.1% to thetarget sites, as well as to all the other affected areas of the body,twice daily until the lesions became clear/almost clear, as demon-strated by a localized EASI of 1 for each individual sign of AD,or for a maximum of 2 weeks. Patients who had a localized EASIof 1 after treatment with the topical corticosteroid were thenrandomized to double-blind treatment of the postlesional targetsites, as well as of all the other affected areas of the body, with ei-ther pimecrolimus cream 1% or vehicle cream twice daily for 3weeks. The methods used for randomization and to ensure theblinding required for the double-blind phase of the study weresimilar to those adopted in previous randomized, vehicle-con-trolled trials with pimecrolimus cream 1% [2426]. Briefly, therandomization list was produced using a validated system thatautomates the random assignment of treatment groups to ran-domization number in the specified ratio; randomization datawere kept strictly confidential until the time of unblinding andwere not accessible to anyone involved in the study; the identityof the treatments was concealed by the use of drugs that werematched in packaging, labeling, schedule of administration, ap-pearance and odor. Investigators involved in immunohistochem-ical processing were unblinded until the end of analysis.

After clinical examination and scoring of the severity of ADon the whole body and at the target sites, using the IGA scale andthe EASI scoring system, respectively, 2 bilateral punch biopsies(4 mm) of the target sites were performed at the screening visit, atthe baseline visit (end of the run-in period or within 2 days of le-sion clearance if this condition was fulfilled earlier) and at theend-of-study visit (end of the double-blind phase) or at the timeof discontinuation (for patients who discontinued treatment pre-maturely). The second and third bilateral skin biopsies were to beperformed at a distance of at least 1.5 cm from the previous biop-tic areas. Control skin biopsies were also performed in the 12healthy volunteers at the screening visit.

Prohibited therapies for the duration of the study were all top-ical and systemic agents known or suspected to be effective in

treating AD (tacrolimus, corticosteroids other than the topicalcorticosteroid used in the open-label run-in period or sedatingantihistamines) and phototherapy. Bland emollients could be ap-plied if used throughout the entire study period. Treatment withoral and topical antibiotics was allowed, if necessary.

Clinical Efficacy AssessmentThe change in the overall severity of AD over time was as-

sessed by using the whole-body IGA, a 6-point ordinal scale rang-ing from 0 (clear) to 5 (very severe disease) [31]. The change in AD

severity at the target site was determined by using the localizedEASI, a composite variable incorporating the severity (rated 03)of ery thema, inf iltration/papulation, excoriations and lichenifi-cation at the target sites as assessed by the investigator [32] .

Skin BiopsyBilateral 4-mm punch biopsies were performed to the level of

the subcutaneous tissue at all 3 visits for AD patients and at thescreening visit for healthy volunteers. The biopsy samples wereimmediately snap-frozen in liquid nitrogen, stored at 70 C andshipped on dry ice to the appropriate laboratory (Division of Im-munology, Allergy and Infectious Diseases, Department of Der-matology, Medical University of Vienna) for further processing.

Immunofluorescence AnalysisThe frozen biopsies were embedded in OCT Tissue-Tek (Saku-

ra Finetek Europe BV, Zoeterwounde, the Netherlands) and snap-frozen in liquid nitrogen again. Frozen tissue was cut into 5-m-thick sections and mounted on capillary gap microscope slides(Dako, Glostrup, Denmark). The cryostat sections were air-driedfor 20 min, fixed in ice-cold acetone for 10 min and either stainedimmediately or stored at 20 C. To determine the phenotype andnumber of different leukocyte subsets, multicolor immunofluo-rescence stainings were performed as follows. Primary antibodiesagainst specif ic cell markers were either biotinylated or conjugatedwith the following fluorochromes: phycoerythrin (PE), allophyco-cyanin (APC) or PE-cyanin 5. Total leukocytes and monocytes-macrophages were identified by using a PE-conjugated anti-CD45(clone J33, Immunotech, Coulter, Marseille, France) and an APC-conjugated anti-CD14 antibody (clone RMO52, Immunotech),singly or in combination. Total T cells, helper T cells and cyto-toxic/suppressor T cells were characterized by using APC-conju-gated anti-CD3 (clone UCHT1, Immunotech), PE-conjugated an-ti-CD4 (clone 13B8.2, Immunotech) and PE-cyanin-5-conjugatedanti-CD8 antibodies (clone B9.11, Immunotech). LCs were identi-fied by double-staining with APC-conjugated anti-CD1a (clone

BL6, Immunotech) and PE-conjugated antilangerin antibodies(DCGM4, Immunotech). Total dermal DCs were characterized byusing biotinylated anti-CD1c antibody (clone M241, Ancell, Bay-port, Minn., USA), and IDECs were identified by double-stain-ing with PE-conjugated anti-CD11b (clone ICRF44, BD, San Jose,Calif., USA) and biotinylated anti-CD1c antibodies. The slideswere initially incubated for 20 min with 2% bovine serum albumin(Sigma-Aldrich, St. Louis, Mo., USA) in phosphate-buffered salineto block nonspecific staining. Each primary antibody was mixedwith 2% bovine serum albumin in phosphate-buffered saline (Gib-co, Invitrogen GmbH, Vienna, Austria) and applied to frozen sec-tions in a humid chamber overnight at 4 C. After washing withphosphate-buffered saline, the binding of biotinylated antibodieswas revealed by incubating the sections with streptavidin-Alexa

660 (Molecular Probes, Eugene, Oreg., USA) for 45 min at roomtemperature. Cell nuclei were counterstained with the nuclear dyeSytox Green (Molecular Probes). Slides were dehydrated in 96%ethanol, mounted in Vectashield Mounting Medium (Vector Lab-oratories, Burlingame, Calif., USA) and cover slipped. In all stain-ing experiments, substitution of the primary antibodies with iso-type-matched IgG (either biotinylated or PE-, APC- or PE-cyanin-5-conjugated IgG) served as negative controls.

Data acquisition and image cytometry were performed by us-ing the TissueFAXS technology (TissueGnostics, Vienna, Aus-


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Dermatology 2011;222:3648 39

tria), based on a motorized confocal laser scanning microscope(CLSM 510, Zeiss, Jena, Germany) and the image cytometry soft-ware Tissue Quest (TissueGnostics), as previously described [19].TissueFAXS is a validated technology, also referred to as micros-copy-based multicolor tissue cytometry [33, 34], which providesin situ, observer-independent analyses on a single-cell basis of cellsubpopulations labeled with fluorescent antibodies in tissue sec-

tions. Image acquisition, including stage movement and autofo-cus, object recognition and discrimination of individual cell pop-ulations were done automatically. Each tissue section was scannedunder a!63 objective lens. Depending on the tissue size, 100400individual fields/section (1 field, 0.079153 mm2) were recordedusing the confocal laser scanning microscope. The various cellsubpopulations were identified on the basis of their staining pat-tern. The mean relative fluorescence intensity per cell for eachfluorescence channel was measured and displayed in scatter-grams. The areas of positivity were automatically defined on thebasis of the cutoff values determined by analysis of sectionsstained with the isotype-matched control for each antibody (neg-ative controls). Results were expressed as the number of specifi-cally stained cells per mm2of tissue. Positive cells in the epider-mal and dermal compartments were not counted separately.

Safety and Tolerability AssessmentThe assessment of treatment safety and tolerability consisted

of monitoring and recording all serious and nonserious adverseevents, with their severity and relationship to treatments. Eventsoccurring after the onset of the biopsy procedure (healthy volun-teers) or after starting treatments (AD patients) were consideredadverse events. Medical conditions or diseases present prior totreatment with BMV were only considered adverse events if theyworsened during the open-label or double-blind period. Abnor-mal laboratory data that resulted in clinical signs or symptoms orrequired therapy were considered adverse events.

Statistical Analysis

The data were analyzed by an independent clinical researchorganization (PPD Development, Melbourne, Australia). De-scriptive analysis of the changes in IGA score and EASI during thestudy was performed in all randomized patients who received atleast 1 dose of the study drug in the double-blind phase and whohad at least 1 safety assessment after the baseline visit (safety pop-ulation). The analysis of cell profile in skin biopsies was separate-ly performed in the safety population and in the population of ADpatients who did not suffer from a clinically significant diseaserelapse during the double-blind phase of the study. The latter pop-ulation included all randomized patients who had a localizedEASI of 1 for each individual sign of AD at baseline, were com-pliant with the treatment regimen, and completed the double-blind phase of the study without showing moderate or severe signs

of AD (localized EASI of!

2 for any individual sign of AD at theend-of-study visit). The reason for performing a separate cyto-logical analysis in the patients who did not show a clinically sig-nificant exacerbation of skin lesions was to detect early recur-rences of the inflammatory infiltrate in postlesional skin that pre-ceded the clinical manifestation of a ful l disease relapse.

The differences in cell counts between treatment groups dur-ing the double-blind phase of the study were compared by analy-sis of covariance with localized EASI score at screening, treat-ment and cell count at baseline as covariates, and no interaction

term. Between-treatment comparisons of the cell counts weremade using Wilcoxons rank sum test. Statistical comparison be-tween AD patients before treatment with BMV and healthy vol-unteers was made by using the unpaired two-tailed t test. Thedata are presented as the means and standard errors of the means(SEMs).

The sample size was calculated on the basis of previous data

on the inf lammatory infiltrate and patterns of gene expression inAD that allowed a meaningful pharmacogenomic data interpre-tation as per the objectives of the study [3537]. From these previ-ous studies, baseline variation in immunofluorescence was as-sumed to be 0100% with a mean of 50%, and the average percent-age was estimated to be reduced to 12.5% at the end of theopen-label period. Further, it was anticipated that patients treatedwith pimecrolimus cream 1% would maintain their level of posi-tively stained cells while placebo (vehicle)-treated patients wouldincrease their level twofold to 25%. Thus, assuming a standarddeviation of 0.152, it was estimated that 25 patients per treatmentgroup during the double-blind phase of the study would give 80%power at a level of significance of 0.05 (two-sided t test).

Results

PatientsA diagram showing the flow of AD patients through

each stage of the study is shown in figure 1. Of the 79 pa-tients screened, 74 entered the open-label run-in period,during which they were treated with BMV for up to 2weeks. Subsequently, 67 patients were randomized andexposed to double-blind study medications (eitherpimecrolimus cream 1% or vehicle cream) for up to 3weeks. The most common reasons for discontinuation

during the open-label run-in period were failure to meetthe criteria for randomization (i.e. BMV-induced ADclearing with a localized EASI 1) and no improvementof AD (unsatisfactory effect of BMV), which were eachreported in 3 patients (fig. 1). During the double-blindtreatment period, the discontinuation rate was higher inthe vehicle group (42.4%) than in the pimecrolimus group(11.8%), and all premature discontinuations were due toan unsatisfactory therapeutic effect. Therefore more pa-tients in the pimecrolimus group (88.2%) than in the ve-hicle group (57.6%) completed the study.

The mean age of AD patients was 32.7 years (range:

1967 years) and 68.9% were Caucasian. The mean age ofhealthy volunteers was 32.3 years (range: 2047 years)and 91.7% were Caucasians. The demographic character-istics of patients randomized to treatment with eitherpimecrolimus cream 1% or vehicle cream during the dou-ble-blind phase of the study were comparable. All pa-tients met the eligibility criteria for disease severity(whole-body IGA of 2 or 3) at screening, most patients


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having moderate AD (table 1). The mean total localizedEASI at the target sites were comparable between the twotreatment groups (table 2).

During the open-label run-in period of the study,compliance with BMV treatment was adequate. Approx-imately 7075% patients subsequently randomized totreatment with either pimecrolimus cream 1% or vehiclecream received more than 12 days of treatment. The me-dian exposure to the topical corticosteroid was 15.0 daysin both groups, and ranged from 5 to 43 days in thepimecrolimus group and from 8 to 42 days in the vehicle

group. Five patients had more than 20 days of exposureto BMV. Treatment was extended beyond the allowedtime because lesions had not been cleared (2 patients) orbecause of scheduling difficulties (3 patients).

Despite the differences in the discontinuation rates,drug exposure was similar in the pimecrolimus and ve-hicle groups during the double-blind phase of the study.In the pimecrolimus group, the mean and median treat-ment days were 20.9 days and 22.0 days, respectively

(range: 928 days). The corresponding values for the ve-hicle group were 18.8 days and 21.0 days (range: 624days). During the double-blind phase of the study, 29.4%patients in the pimecrolimus group and 33.3% patients inthe vehicle group used emollients. There were no majordifferences between treatment groups in terms of use ofantibiotics (29.4% patients in the pimecrolimus arm and33.3% patients in the vehicle arm).

79 screened

74 entered open-labelrun-in (BMVtreatment)

IGA = 2 or 3Localized EASI = 18

67 randomizedLocalized EASI 1

Clinical and Cytological Effects of Pimecrolimus

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