ORIGINAL RESEARCH

Novel Non-Steroidal Facial Cream DemonstratesAntifungal and Anti-Inflammatory Properties in ExVivo Model for Seborrheic Dermatitis

Corinne Granger . Anna Balato . Felipe Goni-de-Cerio .

Aurora Garre . Mridvika Narda

Received: May 21, 2019 / Published online: July 5, 2019� The Author(s) 2019

ABSTRACT

Introduction: Seborrheic dermatitis (SEBD) is achronic, recurrent skin disorder that typicallyoccurs as an inflammatory response to fungi ofthe genus Malassezia. The development of anex vivo model that mimics the fungal prolifer-ation and skin inflammation of SEBD wouldplay an important role in screening formula-tions for their efficacy in treating SEBD.Methods: An ex vivo model for SEBD usinghuman skin explants that had been mechani-cally manipulated to facilitate colonization ofMalassezia furfur was developed. This model wasused to evaluate the efficacy of a novel non-

steroidal facial cream (NSFC) in inhibitingM. furfur proliferation and reducing inflamma-tory cytokine levels.Results: This model reproduced some of thekey pathological features of SEBD, includingM. furfur proliferation and inflammatory cyto-kine production. Topical application of NSFCfacial cream reduced M. furfur counts by 92%(p\ 0.05) and levels of interleukin 8 (IL-8) andtumor necrosis factor alpha (TNF-a) by 82% and40%, respectively (p\0.05, both).Conclusion: The proposed ex vivo model forSEBD could be a useful tool to evaluate topicalantifungal treatments. The novel NSFC tested inthis study reduced M. furfur proliferation andinflammatory cytokine levels following topicalapplication and may be helpful in the man-agement of SEBD.Funding: ISDIN.

Keywords: Antifungal; Anti-inflammatory; Invitro model; Malassezia; Non-steroidal facialcream; Seborrheic dermatitis

INTRODUCTION

Seborrheic dermatitis (SEBD) is a commonlyoccurring, chronic papulosquamous dermatosischaracterized by red skin, dry white scales, andrash that affect sebaceous gland-rich areas of theface, scalp, and the upper chest, with a reportedprevalence between 1% and 3% in the adult

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Electronic supplementary material The onlineversion of this article (https://doi.org/10.1007/s13555-019-0311-4) contains supplementary material, which isavailable to authorized users.

C. Granger � A. Garre � M. Narda (&)ISDIN, Innovation and Development, Barcelona,Spaine-mail: mridvika.narda@isdin.com

A. BalatoDepartment of Advanced Biomedical Sciences,University of Naples Federico II, Naples, Italy

F. Goni-de-CerioBiotechnology Area, Gaiker Technology Centre,Bizkaia, Zamudio, Spain

Dermatol Ther (Heidelb) (2019) 9:571–578

https://doi.org/10.1007/s13555-019-0311-4

population [1, 2]. The etiology of SEBD is com-plex, involving interplay between genetic, hor-monal, immune, and environmental factors [3].A key pathogenic component of SEBD isbelieved to be proliferation of yeast of the genusMalassezia that concentrate in hair follicles andsebum-rich areas of the body [1, 3, 4]. Malasseziaare lipid-dependent fungi, requiring free fattyacids (FFAs) generated from the hydrolysis ofsebaceous triglycerides [5–9]. FFAs irritate theskin and damage the stratum corneum, therebydisrupting epithelial barrier function andinducing abnormal keratinization [10–13].Inflammatory cytokines have been reported inskin biopsies from SEBD lesions in patients,suggesting that an inflammatory response isalso involved [8].

Standard treatment options for SEBD includetopical therapy with antifungal and anti-in-flammatory agents such as ketoconazole orcorticosteroids [14]. Non-steroidal treatmentoptions that help maintain skin free of Malas-sezia may help prevent flare-ups and can behelpful in the management of the disease.However, as a result of the complex nature ofSEBD, no in vitro models currently exist toscreen products for possible efficacy in themanagement of SEBD. Here we describe thedevelopment of an ex vivo model for SEBDusing human skin explants infected withMalassezia spp. fungi. This model reproducesthe fungal proliferation and inflammatoryresponses of SEBD. Following its validation, wetested the efficacy of a novel non-steroidal facialcream (NSFC), containing stearyl glycyrrheti-nate, piroctone olamine, and biosaccharidegum-2, that is designed to reinforce the skinbarrier and reduce fungal proliferation andinflammation. Our data suggest that this SEBDmodel may prove useful for the future devel-opment of SEBD treatments.

METHODS

Human Skin Explants

Human skin explants were obtained, withinformed consent from healthy female donorsundergoing abdominoplasty, under

authorization granted by the French govern-ment ethical committee according to Frenchlaw L.1245 CSP. The explants were sourced fromBiopredic, France. Within 2 h of surgery, skinwas cut into 0.8 cm2 pieces and placed epider-mis side up in 6-well Transwell plates contain-ing 1.5 ml of Dulbecco’s Modified Eagle’sMedium (DMEM; GIBCO, Grand Island, NY,USA), supplemented with 2 mM L-glutamine(GIBCO) and antibiotics (100 IU/ml penicillin Gand 100 lU/ml streptomycin; GIBCO). Explantswere incubated at 37 �C in a humidified atmo-sphere containing 5% CO2 for 48 h prior tostudy initiation.

Malassezia furfur

M. furfur (DSM 6170) was obtained from LeibnizInstitute DSMZ-German Collection of Microor-ganisms and Cell Cultures (Brunswick, Ger-many) and cultivated in specific modifiedDixon medium (m-Dixon; Sigma Aldrich, St.Louis, USA). Fungal cultures were incubated at30 �C for 42–48 h.

Topical Products

The investigational product tested was an NSFCcontaining stearyl glycyrrhetinate, piroctoneolamine, and biosaccharide gum-2 (full com-position in Table 1). NSFC without anti-in-flammatory ingredients (NSFC-AI) had a similarcomposition to NSFC, with the exception ofingredients with known or suspected anti-in-flammatory effects (Table 1). Petrolatum (Vase-lina pura filante Acofarderm), ketoconazole 2%cream (Fungarest 20 mg/g�), and hydrocorti-sone (HC) 1% cream (Cortaid HydrocortisoneMaximum Strength Anti-Itch Cream) werepurchased at a local pharmacy.

SEBD Model Development and ProductTesting

Skin explants were stripped using D-squametapes (Clinical and Derm LLC, Dallas, USA) toremove approximately 40% of the stratum cor-neum (confirmed by tape absorbance measure-ment). Control skin samples were not stripped.

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At 48 h after stripping, skin explants wereinoculated with 1 9 106 colony forming units(CFU) of M. furfur and incubated at 37 �C, 5%CO2, and 95% humidity. At 24 h after inocula-tion, test products (dose 2 mg/cm2) wereapplied topically to explants using a micropip-ette and spread with a microspatula. Controlexplants were not treated. Two independentstudies and at least three replicates for eachproduct were performed.

Determination of Skin Viability

Skin viability was determined using a commer-cially available lactate dehydrogenase (LDH)

assay kit (CytoTox 96� Non-Radioactive Cyto-toxicity Assay, Promega, WI, USA) according tothe manufacturer’s specifications. Skin viabilitywas additionally measured using a resazurinassay. Briefly, skin explants were treated with6 lM of resazurin NaCl solution (Sigma Aldrich,St. Louis, USA) for 1 h, and the concentration ofresorufin (RES) formed was quantified in a flu-orometer plate reader (530 nm excitationwavelength and 590 nm emission wavelength).

Malassezia furfur Quantificationand Visual Analysis

Viable fungal cells were recovered from skinexplants by tape stripping. Tape strips wereimmersed in a solution of physiological buf-fered saline and 0.1% Triton X-100 and thenumber of CFUs determined by serial dilutionin m-Dixon followed by direct plating. Plateswere incubated at 30 �C for 42–48 h. Presence ofM. furfur on skin explants was visualized bystaining with Crystal Violet (0.5%; SigmaAldrich, St. Louis, USA).

Cytokine Quantification

Cytokine levels (TNF-a, IL-8, and IL-6) inexplant culture media were determined using acommercially available ELISA kit according tothe manufacturer’s specifications (ThermoFisher Scientific, MA, USA). Results wereexpressed as average interleukin concentration(picograms per milliliter).

Statistical Analyses

Results are reported as mean ± standard devia-tion (SD). The homogeneity of variance wasconfirmed by the Levene’s test and the nor-mality confirmed by the Anderson–Darling test.Unpaired t tests and one-factor analysis ofvariance (ANOVA) with Bonferroni–Dunn’scorrection were performed to assess differencesbetween groups. A p value less than 0.05 wasconsidered significant.

Table 1 Ingredient list for topical treatments NSFC andNSFC-AI

NSFC NSFC-Al

Aqua (water), glycerin,

acetamide MEA, isodecyl

neopentanoate,

cyclopentasiloxane,

pentylene glycol, cetyl

alcohol, cyclohexasiloxane,

sclerotium gum, zinc

PCA, piroctone olamine,

polyacrylamide,

polymethyl methacrylate,

butylene glycol, stearyl

glycyrrhetinate, C13–14

isoparaffin, glyceryl

stearate, PEG-100 stearate,

acrylates/C10–30 alkyl

acrylate crosspolymer,

dimethicone/vinyl

dimethicone crosspolymer,

sodium hydroxide,

laureth-7, biosaccharide

gum-2, disodium EDTA,

hydroxyphenyl

propamidobenzoic acid,

ascorbyl palmitate

Aqua (water), glycerin,

acetamide MEA, isodecyl

neopentanoate,

cyclopentasiloxane,

pentylene glycol, cetyl

alcohol,

cyclohexasiloxane,

sclerotium gum, zinc

PCA, piroctone olamine,

polyacrylamide,

polymethyl methacrylate,

C13–14 isoparaffin,

glyceryl stearate, PEG-

100 stearate, acrylates/

C10-30 alkyl acrylate

crosspolymer,

dimethicone/vinyl

dimethicone

crosspolymer, sodium

hydroxide, laureth-7,

disodium EDTA

NSFC non-steroidal facial cream, NSFC-Al non-steroidalfacial cream without anti-inflammatory ingredients

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RESULTS

Model Development

Although Malassezia spp. are part of normalskin microbiota, they manifest themselvespathogenically only in some individuals, sug-gesting that compromised skin integrity may beresponsible for facilitating colonization byMalassezia spp. in SEBD. Considering this, wedecided to mechanically disrupt the integrity ofthe skin by tape stripping to facilitate fungalinvasion. To confirm that stripping the skin didnot negatively impact skin viability, we quan-tified LDH levels in the culture media and RESlevels in skin tissue. LDH and RES levels in tape-stripped skin were higher and lower, respec-tively, than those of unstripped control skin at24 h but had returned to normal by 48 h (Sup-plementary Table S1). Levels of IL-6, IL-8, andTNF-a were higher at 24 h but had returned tonormal by 48 h (Supplementary Table S2).Together these data suggest that by 48 h theskin had recovered sufficiently from the processof tape stripping to examine its impact uponM. furfur colonization.

At 48 h after tape stripping, explants wereinoculated with M. furfur and colonies counted24 h later. Fungal colony counts in tape-strip-ped skin were increased by 204.35% comparedto non-inoculated skin. This was significantlymore than non-tape-stripped skin (?61.24%,p\0.05), confirming that skin surface damagewas necessary to achieve the high fungal countsrepresentative of SEBD. Levels of inflammatorycytokines were also increased in response tofungal inoculation (Supplementary Table S3),confirming the inflammatory effect of fungalinfection in SEBD. TNF-a, IL-8, and IL-6 levels,however, did not differ between stripped andunstripped skin (Supplementary Table S3), sug-gesting that inflammation is a direct result ofM. furfur colonization and not tape stripping.

Model Validation

Next, we sought to determine the repro-ducibility of the model by performing multipleinfection replicates in different explant

samples. On the basis of four separate studies,fungal colony counts did not significantly differ(92–105% of mean) up to 96 h post-inoculation(Supplementary Table S4), indicating goodrepeatability and reproducibility of the model,and suggesting that M. furfur proliferates in acontrolled manner in human skin explants.

Having developed and validated our model,we sought to test its utility to screen topicalagents for their antifungal activity. Topicaltreatment with ketoconazole 2% (a productwith known antifungal properties [15]) reducedfungal CFUs by 76.3%. Petroleum jelly, how-ever, did not decrease CFUs.

NSFC Antifungal Effect

We then used our model to test the antifungalactivity of a novel NSFC for treating SEBD. NSFCcontains stearyl glycyrrhetinate, piroctone ola-mine, and biosaccharide gum-2, and is designedto reinforce the skin barrier and reduce fungalproliferation and inflammation. At 24 h afterapplication, NSFC reduced the number ofM. furfur colonies by 92% compared to non-treated control skin (p\0.05; Fig. 1). Todemonstrate that this was a direct result of theantifungal activity of NSFC rather than its anti-inflammatory properties, we also treatedM. furfur-infected skin explants with NSFC-AI(NSFC without its anti-inflammatory compo-nents) and HC 1% (a known anti-inflammatoryagent). NSFC-AI reduced M. furfur numbers by91.5% compared to non-treated control skin.The HC 1% cream did not reduce colonycounts, however. Together these data suggestthat the ability of NSFC to reduce M. furfurnumbers in skin is directly attributable to itsantifungal properties.

NSFC Anti-Inflammatory Effect

To examine the anti-inflammatory propertiesNSFC, we measured TNF-a and IL-8 levels in theculture media 24 h after product application.Both NSFC and NSFC-AI reduced TNF-a and IL-8to levels equivalent to those of HC (Fig. 2). SinceNSFC-AI does not contain any anti-inflamma-tory agents, this observation suggests that the

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reduction in inflammation by NSFC is a directresult of its antifungal effect rather than its anti-inflammatory properties.

DISCUSSION

Here we report the development of a novelex vivo model that reproduces a number of thesalient pathological features of SEBD. Thismodel, based on human skin explants inocu-lated with M. furfur, can be established in a rel-atively short time span (3 days) and issuitable for testing the efficacy of agents intreating SEBD. To our knowledge an ex vivomodel of SEBD that mimics the fungal prolif-eration and cutaneous inflammation of SEBDhas not been described previously.

In order to establish fungal colonies inpathologically relevant numbers in this model,the integrity of the skin first had to be com-promised. Although stripping of the skinexplants caused an inflammatory response thatcould potentially interfere with the testing oftopical products, skin viability and cytokinelevels had returned to their basal levels within48 h, suggesting that skin homeostasis had beenrestored by this time. Although several species

Fig. 1 Antifungal effect. a M. furfur CFUs in SEBDmodel control vs. treated with NSFC, NSFC-AI, and HC1% cream (*p\ 0.05). b Macroscopic pictures of SEBDmodel taken after staining with Crystal Violet stain beforeM. furfur inoculation (i) No M. furfur inoculation; (ii)M. furfur control; (iii) M. furfur ? NSFC; (iv) M. furfur? NSFC-AI; (v)M. furfur ? hydrocortisone 1% cream;(vi) M. furfur ? Petrolatum group

Fig. 2 Anti-inflammatory effect. IL-8 (a) and TNF-a(b) levels in SEBD model control vs. treated with NSFC,NSFC-AI, and HC 1% cream (*p\ 0.05)

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of the Malassezia genus have been associatedwith SEBD, M. furfur has been shown to be oneof the predominant species in SEBD [16, 17] andthus was chosen to generate this model. Futuremodels using different Malassezia species oreven clinical isolates would further extend itssuitability.

Our model demonstrated good repro-ducibility, with consistent M. furfur numbersbetween experimental replicates, making ithighly suitable for preclinical testing. A poten-tial limitation of this model, however, is that ituses skin derived from anatomical areas notnormally affected by SEBD. Since skin-residentimmune cell populations and skin-associatedmicrobiota are influenced by skin region[18, 19], it is likely that differences exist in skintaken from regions normally affected by SEBD.Another limitation of the model is that it doesnot represent all aspects of pathogenesis ofseborrheic dermatitis which is clearly verycomplex. However it represents part of it as MFcolonization is a well-established component ofseborrheic dermatitis pathogenesis, althoughaspects of why Malassezia is pathogenic in someindividuals and commensal in others and therole played by the rest of the skin microbiota inpathogenesis are not clear today. Nevertheless,we believe that the simplicity of this model andthe ready availability of abdominal skin make itsuitable for screening. Indeed, the efficacy ofthe novel NSFC tested in this study is supportedby results from clinical trials. Here, NSFCdemonstrated significant effects on desquama-tion, erythema, and pruritus in subjects withmild-to-moderately inflamed facial SEBD [20].

Topical application of NSFC in this modelled to a significant reduction in M. furfur countsand a subsequent reduction in inflammatorycytokine levels. Interestingly, anti-inflamma-tory effects were also observed with NSFC-AI.NSFC-AI comprises the same formulation asNSFC but without its anti-inflammatory ingre-dients, suggesting that skin inflammation inSEBD is predominantly secondary to fungalproliferation.

CONCLUSIONS

We have generated a novel ex vivo model forSEBD, based on human skin explants inoculatedwith M. furfur. The SEBD model was employedto test the antifungal efficacy of an NSFC thatuses skin barrier enhancing properties to protectagainst fungal proliferation. The NSFC demon-strated remarkable efficacy in reducing fungalload and decreasing levels of cutaneousinflammation. This non-steroidal cream withskin barrier enhancing properties can be helpfulin the management of SEBD and complementthe current topical armamentarium for SEBD.

ACKNOWLEDGEMENTS

Funding. This study and the Rapid ServiceFee were funded by ISDIN, Barcelona, Spain.The authors would like to acknowledge thecontribution of Ms. G. Martinez-Masana inmanagement of the experimental work per-formed in this study. All authors had full accessto all of the data in this study and take completeresponsibility for the integrity of the data andaccuracy of the data analysis.

Authorship. All named authors meet theInternational Committee of Medical JournalEditors (ICMJE) criteria for authorship for thisarticle, take responsibility for the integrity ofthe work as a whole, and have given theirapproval for this version to be published.

Author Contributions. F. Goni-de-Cerioperformed the research. C. Granger and A. Garredesigned the research study. M. Narda andA.Garre analyzed the data. M. Narda wrote thepaper.

Disclosures. Dr. C. Granger is an employeeof ISDIN. Dr. A. Garre is an employee of ISDIN.Dr. M. Narda is an employee of ISDIN. Dr.A. Balato has given conferences in symposiasponsored by Abbvie, Janssen, Lilly, Sanofi, andhas been a consultant for Abbvie, ISDIN,Novartis, Lilly. F. Goni-de-Cerio has nothing todisclose.

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Compliance with Ethics Guidelines. Humanskin explants were obtained, with informedconsent, from healthy female donors undergo-ing abdominoplasty, under authorization gran-ted by French government ethical committeeaccording to French law L.1245 CSP. Theexplants were sourced from Biopredic, France.The anonymity of patients is maintained andpatients sign an informed consent form beforedonating their samples.

Data Availability. All data generated oranalyzed during this study are included in thispublished article/as supplementary informationfiles.

Open Access. This article is distributedunder the terms of the Creative CommonsAttribution-NonCommercial 4.0 InternationalLicense (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommer-cial use, distribution, and reproduction in anymedium, provided you give appropriate creditto the original author(s) and the source, providea link to the Creative Commons license, andindicate if changes were made.

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