ORIGINAL RESEARCH

A Nonsteroidal Novel Formulation TargetingInflammatory and Pruritus-Related MediatorsModulates Experimental Allergic Contact Dermatitis

William C. Gordon . Virginia Garcı́a López . Surjyadipta Bhattacharjee . David Rodrı́guez Gil .

Javier Alcover Dı́az . Fernando Pineda de la Losa . Ricardo Palacios Peláez . Concha Tiana Ferrer .

Gabriela Silvina Bacchini . Bokkyoo Jun . Hélène Varoqui . Nicolas G. Bazan

Received: December 11, 2017 / Published online: February 16, 2018� The Author(s) 2018. This article is an open access publication

ABSTRACT

Introduction: A major clinical challenge intreating allergic contact dermatitis (ACD) is thatthe first line of treatment is based on the use of

corticosteroids. In this study, we aimed todevelop a formulation that is devoid of steroids.Methods: We used mouse ears treated withdinitrofluorobenzene (DNFB) to induce ACD.The efficacy of the test formulation to amelio-rate and to prevent induced ACD wasdetermined.Results: To treat this experimentally inducedACD, we developed a formulation containingBIPxine (a mixture of Rosa moschata and Crotonlechleri (antioxidants) and Aloe vera and D-pan-thenol (moisturizers), and hydroglycolic solu-tions of disodium cromoglycate. Our resultsshow that clear inhibition of ACD took place.The target of this formulation was PAR-2,TRPV4, and other mediators of the inflamma-tory and pain responses. However, this formu-lation must be evaluated in other modelsbesides the mouse to confirm its effectiveness.Conclusion: The formulation presented heremay provide new ACD therapies that do notinvolve the use of corticosteroids.

Keywords: Cromoglycate; Ear thickness;Experimental allergic contact dermatitis;Inflammation; Nonsteroidal therapy

INTRODUCTION

Dermatitis, a general term for skin inflamma-tion, is complex, with many subtypes, eachwith different etiologies, which may include dry

William C. Gordon, Virginia Garcı́a López, andSurjyadipta Bhattacharjee contributed equally.

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Electronic supplementary material The onlineversion of this article (https://doi.org/10.1007/s13555-018-0223-8) contains supplementary material, which isavailable to authorized users.

W. C. Gordon � S. Bhattacharjee � B. Jun �N. G. Bazan (&)Neuroscience Center of Excellence, School ofMedicine, Louisiana State University HealthSciences Center, New Orleans, LA, USAe-mail: nbazan@lsuhsc.edu

V. G. López � D. R. Gil � J. A. Dı́az �F. P. de la Losa � R. P. PeláezDiater, Av. Gregorio Peces Barba, Madrid, Spain

C. T. FerrerFarmalider, Calle La Granja, Madrid, Spain

G. S. BacchiniFerrer, Av. Diagonal 549, Barcelona, Spain

H. VaroquiOchsner North Shore Dermatology Practice, Slidell,LA, USA

Dermatol Ther (Heidelb) (2018) 8:111–126

https://doi.org/10.1007/s13555-018-0223-8

http://dx.doi.org/10.6084/m9.figshare.5798148http://dx.doi.org/10.6084/m9.figshare.5798148http://dx.doi.org/10.1007/s13555-018-0223-8http://dx.doi.org/10.1007/s13555-018-0223-8http://dx.doi.org/10.1007/s13555-018-0223-8http://dx.doi.org/10.1007/s13555-018-0223-8https://doi.org/10.1007/s13555-018-0223-8http://crossmark.crossref.org/dialog/?doi=10.1007/s13555-018-0223-8&domain=pdfhttp://crossmark.crossref.org/dialog/?doi=10.1007/s13555-018-0223-8&domain=pdf

skin, severe pruritus, pain, or lesions that, sub-sequently, may become infected [1, 2]. Atopicdermatitis (AD), resulting in inflamed, itchy,cracked skin, commences early in life, worsen-ing as it progresses into adulthood [3], requiringongoing, long-term, periodic treatments. Thecause of AD is associated with environmentalexposure, skin barrier dysfunction, immuno-logical responses, and a complex interplay ofsusceptibility genes [4], affecting up to 20% ofchildren and 3% of adults worldwide [5, 6].Allergic contact dermatitis (ACD), on the otherhand [7], represents 5–10% of consultations,including those related to irritation, and peri-odic treatment. Sensitizing allergens are diverse,can include metals such as nickel and gold,components of perfumes and soaps, or organiccompounds, and once sensitized, the presenceof the allergen will induce the characteristics ofACD [8–10]. Removal of the allergen results inloss of symptoms, but recovery can take weeks,during which treatment of symptoms is desir-able [10]. Treatment of severe reactions includescorticosteroids. However, their mechanism ofaction is not well defined; it is complex andaffects the immune system and skin barrier, andlong-term use may result in dermal atrophy,telangiectasia, striae, hypopigmentation, orcorticosteroid acne. Barrier disruption mayallow percutaneous absorption that could initi-ate adrenal suppression, altered growth, hyper-tension, hyperglycemia, insulin resistance, andcataracts [11–13].

Controversy remains as to whether long-term corticosteroid use poses increased risk forlymphoma or skin cancer, or whether it acti-vates herpes simplex. However, calcineurininhibitors have been developed as corticos-teroid alternatives [14, 15]. Also, immunosup-pressants tacrolimus and pimecrolimuscomplexing with FK-binding proteins (FKBP-12/macrophilin-12) reduce calcineurin andT lymphocyte activity [14–16]. Thus, cytokinesare inhibited and inflammatory signaling ishalted or reduced.

Exogenous proteases or endogenous tryp-tases from mast cells activate protease-activatedreceptor-2 (PAR-2), localized in skin C-fiberterminals, mediating local inflammation[17–19]. Activated PAR-2 subsequently activates

transient receptor potential vanilloids 1 and 4(TRPV1, TRPV4) [20]. Ca2? then enters thesenon-selective ion channels to promote inflam-mation and pain through calcitonin gene-re-lated peptide (CGRP) and substance P [21]. PAR-2 sensitizes TRPV ion channels [22, 23], whichare associated with itch [24].

Sodium cromoglycate is a potent free radicalscavenger [25]; some naturally occurring chro-mones exhibit free radical inhibition [26]. Sig-nificantly, adverse effects from cromoglycatetreatment have not been reported. We haveevaluated treatments based on compounds withantioxidant properties, a physical barrier effect,and a protective capability of inhibitinginflammatory mediator release, as a possiblealternative to steroid and immunosuppressant-related treatments in our mouse model byinduction of ACD with repeat application ofdinitrofluorobenzene (DNFB).

METHODS

Animals

Male Balb/C mice (23–25 g) were obtained fromCharles River Laboratories (Wilmington, MA)and acclimated to the Louisiana State Univer-sity Health Sciences Center (LSUHSC) animalcolony for 1 week. This facility is maintained ona 12:12 h light/dark cycle (06:00 h ON; 18:00 hOFF), with an average light intensity at cagelevel of 20 lx. Mice were supplied with normalmouse chow and water ad libitum. All animalprocedures were approved by the InstitutionalAnimal Care and Use Committee and Institu-tional Review Board, LSUHSC, New Orleans, allinstitutional and national guidelines for thecare and use of laboratory animals were fol-lowed, and this study has conformed with theHelsinki Declaration of 1964, as revised in 2013,concerning human and animal rights.

General Reagents and Antibodies

Acetone was purchased from Fisher Scientific(Waltham, MA), 2,4-dinitrofluorobenzene(DNFB) was purchased form Sigma-Aldrich (St.

112 Dermatol Ther (Heidelb) (2018) 8:111–126

Louis, MO), and olive oil was purchased locally.The following lists detail where the primary andsecondary antibodies were obtained. Primaryantibodies: PAR-2 (sc-8207) 1:1000, Santa Cruz;TRPV4 (GTX54764) 1:500, GeneTex; TNFa (LS-C104816) 1:1000, LS Biosciences; GAPDH(MAB374) 1:500, EMD Millipore. Secondaryantibodies: Donkey anti-goat IgG-HRP (sc-2020)1:5000; Donkey anti-rabbit IgG-HRP (sc-2313)1:5000; Donkey anti-mouse IgG-HRP (sc-2314)1:5000, Santa Cruz.

Ear Skin Sensitization and Inductionof ACD

An established method for sensitization andACD induction was utilized [27, 28]. Threeprotocols were utilized (Fig. 1). Mice were sen-sitized by application of 0.15% 2,4-dinitrofluo-robenzene (DNFB) (Sigma-Aldrich, St Louis,MO) in acetone and olive oil (3:1) (vehicle) withcotton applicators to the outer surface of bothears every other day.

Four test compounds were supplied ascreams (Dermogen Farma, Madrid, Spain): (1)BIPxine (a mixture of Rosa moschata and Crotonlechleri (antioxidants) and Aloe vera and D-pan-thenol (moisturizers), referred to hereafter as‘‘mixture of antioxidants and moisturizers’’(MAM); (2) 2% hydroglycolic solution of dis-odium cromoglycate (CGDS); (3) BIPxine andCGDS 2% (MAM ? CGDS 2%, formulation A);(4) BIPxine and CGDS 5% (MAM ? CGDS 5%,formulation B).

Two approaches were tested: a curative and apreventative approach. In the curative model,ACD was induced first, followed by applicationof the test compounds to determine their effi-cacy at restoring homeostasis (Fig. 1a).

In the curative model, as ACD developed andthen as the test compounds were applied, earthickness (millimeters) of 36 mice was measureddaily with a digital caliper; ears graduallythickened as the ACD response developed. Inthis first experiment, DNFB treatment was hal-ted at 19 days, when ear thicknesses surpassed0.50 mm, and application of test formulationscommenced.

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Fig. 1 Outlines of experimental protocols. Protocol forthe a curative, b preventative, and c experimental corti-costeroid treatments. These protocols generally cover a2-week period. Ear tissue was harvested at the timesindicated by ‘‘killed’’

Dermatol Ther (Heidelb) (2018) 8:111–126 113

In addition to the mice undergoing treat-ment with the test compounds, two additionalsets of mice served as controls. One received notreatment throughout the experiment (control),while the second set was sensitized with DNFBonly (ACD control), but received no test creamapplication. Test substances were applied bycotton applicators for 7 days and the ears col-lected for analysis. Six mice (12 ears) wereemployed for each set of conditions.

In a second repeat experiment, mice weresimilarly sensitized by application of DNFB tothe outer surface of both ears. However, thesensitization period was shortened to 7 days.Test compounds were again applied for 7 daysand the ears collected for analysis. During thisexperiment, ear thicknesses were collected onlyon days 0, 7, and 14 (Fig. 1a). Here, seven mice(14 ears) were employed for each set ofconditions.

The second approach was designed to deter-mine the ability of the test compounds to pre-vent ACD in the presence of the sensitizingagent. In this preventative experiment, micewere sensitized, as before, for 1 week. However,the test compounds were applied throughoutthe 2-week period. Ear thickness was measureddaily and the results compared to untreatedcontrols (Fig. 1b). Seven mice (14 ears) wereemployed for each set of conditions here aswell.

A final experiment was designed to test theACD model with intermediate- and low-strength steroids. Two groups of mice weresensitized with DNFB for 7 days, but one groupreceived simultaneous treatment with des-oximetasone or hydrocortisone. The othergroup was sensitized for 7 days and then treatedwith desoximetasone or hydrocortisone for anadditional 7 days (Fig. 1c). Ear thicknesses wereobtained at days 0, 7, and 14. Each group con-sisted of 5 mice each.

Immunoblot Analysis of Ear Tissue

Two proteins that are associated with theinflammatory and pain responses, PAR-2 andthe transient receptor potential vanilloidreceptor 4 (TRPV4), were quantitated by

comparison to an unchanging house-keepingprotein GAPDH. Because ACD is associated withthe inflammatory response, the inflammatorymarker TNFa was also quantitated. Ear tissuewas minced and homogenized (glass-on-glass)at 4 �C using RIPA buffer (Thermo-Fisher Sci-entific) consisting of Complete Protease andPhosphatase Inhibitor Cocktail (Roche Diag-nostics, Indianapolis, IN). Samples were soni-cated and centrifuged for 10 min at14,000g. The protein concentration was deter-mined with a Bradford assay (Bio-Rad, Hercules,CA). The supernatant was used for SDS-PAGEand the pellet stored at - 20 �C. Equal amountsof boiled protein (20 lg per lane) were dilutedwith the SDS sample buffer, loaded onto BioRadCriterion Pre-cast Gels (4–12%) for SDS-PAGE(BioRad, Hercules, CA), and electrophoresed at125 V for 1.5 h on ice. Proteins were electro-blotted to polyvinylidene difluoride (PVDF)membranes using the BioRad Criterion Trans-Blot system. Transference took place in a TurboTransfer system (BioRad) following the manu-facturer’s instructions. Gel retention was asses-sed by staining with Coomassie blue (Pierce,Rockford, IL). Nonspecific binding was blockedfor 1 h at room temperature with SuperBlockblocking solution (Thermo-Fisher Scientific).Membranes were incubated overnight at 4 �Cwith primary antibodies to PAR-2, TRPV4, andTNFa in TBS. Bound primary antibody wasdetected by HRP-linked secondary antibody at55, 98, and 17 kDa, respectively. Protein bandswere visualized with a Fujifilm LAS-3000 digitalscanner, following the manufacturer’s instruc-tions, and quantitated using ImageQuant TLsoftware (GE Healthcare). Membranes were thenstripped for 30 min, re-probed with GAPDH(38 kDa, mouse monoclonal IgG) as a loadingcontrol (EMD Millipore), and detected with HRPanti-mouse IgG (Santa Cruz).

Labeling and Quantificationof the Primary Afferent Arborizations

We found that neither PGP 9.5 nor beta IIItubulin alone (both good markers for neuronalfibers) would label the fine neuronal processeswithin the ear. However, by combining both

114 Dermatol Ther (Heidelb) (2018) 8:111–126

markers and incubating for prolonged periodsin briefly fixed tissue (1 h), labeling wasdetectable with a 639 objective. While beta IIItubulin will also label epidermal Langerhanscell dendrites, suggesting a mixed population oflabeled fibers here, this method likely labeled allneuronal fibers. It is important to mention herethat we were interested in learning whether anyfibers near the surface were altered, not whethersensitization affected specific fiber subgroups.To determine whether inflammation had beeninduced following sensitization with DNFB, theprotein PAR-2 was also probed. Briefly, verticalcryosections of mouse pinna (30 lm thickness)were prepared for immunolocalization of pri-mary afferent fiber arborizations in fixed controland treated ears. Following Triton X-100 per-meablization and blocking, samples were incu-bated with PGP 9.5 (1:500, Abcam, ab108986),beta III tubulin (1:200, Abcam, ab52901), andPAR-2 (1:200, Santa Cruz) in blocking buffer for3 days, washed, and then incubated for 1 h indonkey anti-rabbit IgG Alexa Fluor 488 (Invit-rogen) for simultaneous labeling of both PGP9.5 and beta III tubulin, and donkey anti-goatIgG Alexa Fluor 633 (Invitrogen) for PAR-2.After labeling with DAPI for 10 min, slides werecoverslipped and viewed with an Olympus Flu-oview 1200 confocal microscope.

To determine the area of nerve fibers withinsections of the sensitized ear, the green channelwas isolated to reveal only the labeled fibers,and the contrast adjusted until the backgroundwas black. ImageJ was used to outline thearborized region within the image, and thenumber of green pixels, determined by theColor Pixel Counter plugin, was presented asthe percentage of total specified area. Percent-ages were averaged, standard errors were calcu-lated, and p values were determined bycomparing control untreated ears with ears thatunderwent sensitization and subsequenttreatment.

Lipidomic Analysis of Ear Tissueby LC–MS/MS

Mouse samples were homogenized in MeOH(3 ml) followed by the addition of CHCl3 (6 ml)

containing an internal standard mixture of AA-d8, PGD2-d4, EPA-d5, 15HETE-d8, LTB4-d4, andPC(28:0), sonicated in an ice water bath, andthen stored at - 80 �C overnight. After vortex-ing and centrifugation, the supernatant wasremoved and the pellet washed with CHCl3/MeOH (1 ml, 2:1), centrifuged, and the super-natants combined. Distilled water (2 ml, pH 3.5)was then added to the supernatant, centrifuged,and the pH of the upper phase adjusted to3.5–4.0 with HCl. The lower phase was thendried down under N2, resuspended in methyl-formate (200 ll), transferred to a vial, driedunder N2, resuspended in MeOH (10 ll), andthen water (5 ll) was added.

A Xevo TQ-S, equipped with Acquity I ClassUPLC (Waters), was used for liquid chromatog-raphy–mass spectrometry (LC–MS/MS) analysis.An Acquity UPLC HSS T3 1.8 lm 2.1 9 50 mmcolumn was used for fatty acids and theirderivatives. Seventy-five percent of solvent A(H2O ? 0.1% acetic acid) and 25% of solvent B(90% acetonitrile, 10% isopropanol) with a0.6 ml/min flow rate were used for the firstminute, then graduated to 100% of solvent B for7.5 min, followed by 100% solvent B for2.5 min. The column was then re-equilibratedto 75% A and 25% B for 2 min. The capillaryvoltage was - 2.5 kV, the desolvation tempera-ture was set at 600 �C, the desolvation gas flowat 1100 l/h, the cone gas at 150 l/h, and thenebulizer pressure at 7.0 bar with the sourcetemperature at 150 �C.

Eicosapentaenoic acid (EPA), arachidonicacid (AA), docosahexaenoic acid (DHA),14HDHA, 17HDHA, neuroprotectin D1 (NPD1)and its isomers, 12HETE, 15HETE, PGE2, LTB4,and LTC4 were quantified for each of thetreatments.

Statistics

Ear Measurements and Western BlottingAll results were expressed as mean ± SEM. Datafrom all experiments were evaluated using one-way ANOVA (analysis of variance) (n = 28;n = 6, respectively) followed by Sidak’s multiplecomparisons post hoc test. Statistical analyseswere performed using Graphpad Prism software

Dermatol Ther (Heidelb) (2018) 8:111–126 115

Version 7.04. A value of p\0.05 was consideredto be statistically significant.

Mass spectrometric analysisResults were expressed as means ? SEM. p valueswere obtained with Student’s t test (two-tailed,two-sample equal variance conditions). Calcu-lations were done with Microsoft Excelprogram.

RESULTS

Ear Thickness

In our initial curative experimental design, asDNFB treatment proceeded, ear thicknessesincreased from 0.20 mm to about 0.48 mm (a0.28 mm increase, or 100%) by 22 days. WhenDNFB treatment was stopped and test sub-stances applied, the increase in ear thicknessceased. Thicknesses remained relatively con-stant for 3 days, then decreased. However, theears of the ACD control mice (DNFB alone; notest substances applied) continued to thickenfor 5 days (up to 0.68 mm) and began to thinafterwards. Ear thickness in the MAM, CGDS2%, and formulation A (MAM CGDS 2%) treat-ment mice declined similarly, reaching0.30 mm (a decrease of 0.18 mm, or 64%) by6 days. However, treatment with formulation B(MAM CGDS 5%) declined more rapidly to0.27 mm (a decrease of 0.21 mm, or 75%). Atthis point, ear tissue was collected for furtheranalysis (Fig. 2a, Suppl 1a–f). To corroboratethese results, this experiment was repeatedusing four mice (eight ears). The same treat-ments produced similar results (Suppl 1g).

A second, modified curative experiment wasconducted, but measurements of ear thicknesswere obtained only at the start of the experi-ment at day 0, 7 days after the DNFB sensitiza-tion at the start of the test treatments, and after1 week of treatments at the conclusion (day 14).Seven mice were included in each treatment. Asbefore, one naı̈ve control group received notreatments. A second group received onlyvehicle for the first week, then no treatment forthe second week. All other groups were sensi-tized with DNFB for 1 week and then treated

with test compounds for the second week.Treatments were DNFB for 1 week only; DNFBfor 1 week with no treatment for the second

Fig. 2 Ear thickness profiles for curative, preventative, andcorticosteroid experimental treatments: novel formulationspromote resolution of inflammation-induced ear thicken-ing. a Ear thickness averages of individual responses (SupplS1) for each curative experimental treatment showedincreases induced by ACD. Following onset of secondarytreatments, thickness rapidly declined with MAM, formu-lation A, and formulation B. Formulation B produced thebest effect. No changes were observed in controls, while earthickness in ACD-alone animals continued to increase.b Ear thickness averages for each curative experimentaltreatment based on the 14-day protocol (Fig. 1a; see SupplS2 for additional graphs). Ear thickness averages for eachexperimental treatment showed increases of about 0.1 mminduced by ACD during the first week. Following onset ofsecondary treatments during week 2, thicknesses generallystabilized or were reduced. Treatment with a combinationof MAM and cromoglycate 5% induced the most dramaticreduction, returning thicknesses to near starting levels.Onset of the secondary treatment occurred after 1 week.Measurements were collected at time zero and at 1 and2 weeks from both ears of seven mice for each treatment.Graphs represent two independent experiments (totaln = 28). The efficacy of formulation B in reducing theeffects of ACD was similar to that shown for experimentsin a. c Parallel curative and preventative experimentsinduced ACD for 1 week, with simultaneous treatment, orwas followed by 1 week of treatment with two steroids,hydrocortisone (H) and desoximetasone (D) (Fig. 1c).Both compounds maintained or reduced ear thickness tonaı̈ve levels. Measurements were collected at time zero andat 1 and 2 weeks from both ears of five mice for eachtreatment (n = 10). d A preventative experiment wasperformed by simultaneously treating ears with the DNFBACD inducer and the test compounds (Fig. 1b). ACDalone increased ear thickness as in all other experimentswhile co-treatment with MAM plus cromoglycate 2% or5% maintained ear thicknesses very close to naı̈ve controland vehicle-alone ears, demonstrating a preventative effecton the development of ACD. Arrows indicate onset oftreatment. Control (no treatment), D (dermatitis), V(vehicle), MAM (mixture of antioxidants and moisturizer),CGDS 2% (cromoglycate 2%), formulation A(MAM ? CGDS 2%), and formulation B(MAM ? CGDS 5%). ± SEMs are indicated,#p\0.0001, §p 0.0016–0.0021, ns not significant

c

116 Dermatol Ther (Heidelb) (2018) 8:111–126

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week; and DNFB for 1 week then MAM ? CGDS2% (formulation A), or MAM ? CGDS 5% (for-mulation B) (Figs. 1a, 2b, Suppl 2). This wasrepeated a second time. Figure 2b illustrates theaverage results of this experiment (two repli-cates of 7 mice per group, n = 28 ears). Formu-lation B was remarkable in reducing earthickness to almost naı̈ve control levels.

As an additional experiment, ACD wasinduced and hydrocortisone (H) or des-oximetasone (D) was applied every other dayeither simultaneously with DNFB (D) sensitiza-tion (1 week only, DH, DD) or for 1 week fol-lowing the sensitization period (D–H, D–D)(Fig. 1c). Both compounds reduced ear thick-ness to control levels by the end of the secondweek or prevented the onset of ear thickness by1 week (Fig. 2c).

In a modification of the experimental pro-tocol, DNFB sensitization was begun and treat-ment with the test compounds was appliedsimultaneously to determine whether these

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Fig. 3 Novel formulation treatment attenuated inflam-matory markers. a Sensitization of skin promoted upreg-ulation of PAR-2, while novel formulation attenuatedPAR-2 production. Ears treated with vehicle alone did notdevelop ACD, while DNFB sensitization (D) triggeredupregulation of the itch marker PAR-2. Test substancesattenuated the PAR-2 response by 22% and 69%. TypicalWestern blots are shown. GAPDH, house-keeping protein.D, ACD; MAM ? CGDS 2% (formulation A);MAM ? CGDS 5% (formulation B). PAR-2means ? SEM, n = 7. *p = 0.002, #p = 0.016. b Skinsensitization promoted upregulation of TRPV4, whilenovel formulation prevented TRPV4 production. Simi-larly, ears treated with vehicle alone did not develop ACD;however, DNFB sensitization (D) triggered upregulationof the itch marker TRPV4. Test substance formulation Breduced TRPV4 formation to naı̈ve control levels. Twotypical Western blots are shown. GAPDH, house-keepingprotein. D, ACD; MAM ? CGDS 2% (formulation A);MAM ? CGDS 5% (formulation B). TRPV4means ? SEM, n = 7. *p = 0.007, #p = 0.026. c Skinsensitization induced an inflammatory response, whilenovel formulation blocked inflammation onset. DNFBsensitization triggered upregulation of the inflammatorymarker TNFa, but formulation B prevented inflammationonset. Two typical Western blots are shown. GAPDH,house-keeping protein. D, ACD; MAM ? CGDS 2%(formulation A); MAM ? CGDS 5% (formulation B).TNFa means ? SEM, n = 7. *p = 0.249, #p = 0.210

c

118 Dermatol Ther (Heidelb) (2018) 8:111–126

compounds would prevent the onset of ACD(Fig. 1b). Except with DNFB sensitization alone,ear thickening generally failed to take place.Only minimal thickening occurred in theDNFB-sensitized mice treated with either for-mulation A or formulation B. The best effectwas obtained again with formulation B. Nochanges were observed in controls (Fig. 2d).

Protein Abundance by Immunoblotting

Mice were treated as in Fig. 1a. The inflamma-tory response indicators PAR-2 and TRPV4,while low in the naı̈ve control animals, wereupregulated in all ACD animals (Fig. 3). In thepresence of formulation B (MAM ? CGDS 5%),PAR-2 levels were about 50% below the ACDmouse values, whereas TRPV4 remained atcontrol levels. TNFa, also an inflammatorymarker, was upregulated in the ACD mice butwas maintained at naı̈ve mouse levels by for-mulation B.

Immunohistochemistry

Mice were treated as in Fig. 2a, and ears werecollected and cryosectioned. Immunolabelingwith antibodies containing a mixture of PGP 9.5and beta III tubulin delimited fibers in the epi-dermis which exhibited extensive branching(Fig. 4a–d). The percentage of these arboriza-tions within the dermatitic ears, including allears that had undergone 1 week of treatmentwith the test compounds, was reduced by about30% (data not shown). However, values fromthe naı̈ve (untreated) ears matched those fromthe vehicle-alone treatment (p = 0.03), indicat-ing that the acetone/olive oil mixture did notinduce the dermatitic response.

PAR-2 is expressed in mast cells and increasesin chronic skin inflammation; therefore, welooked for indications of PAR-2 in ear tissue thathad undergone treatment with the test com-pounds. PAR-2 localized to two regions withinthe ear sections of all treated animals. Thestratum corneum consistently demonstratednon-specific labeling, as did the upper-mostlayer of the epidermal cells. Moreover, intenselabeling was often roughly correlated with the

inner-most layer of epidermal cells near thebasement membrane (Fig. 4c, e). This innerlabel appeared as a thin band in the naı̈ve (un-treated) and vehicle-alone-treated ears, butappeared to thicken and somewhat diffuse inthe 2-week ACD ears.

Lipid Mediators

Neuroprotectin D1 (NPD1), a docosanoid, isderived from docosahexaenoic acid (DHA)under conditions of oxidative stress andinflammation, and it is highly neuroprotectivein the brain and retina [29–31]. Analysis showsthat treatment with formulation A(MAM ? CGDS 2%) and formulation B(MAM ? CGDS 5%) resulted in some produc-tion of NPD1, as well as its isomers (Fig. 5).

Following 1 week of treatment after ACDinduction, tissues responded to test compoundsin unexpected ways. Inflammatory markersincreased (PGE2, LTB4, and LTC4) whilearachidonic acid (AA), DHA, and their associ-ated metabolites remained unchanged. NPD1and its isomers tended to increase. When theNPD1 isomers were totaled, treatment withformulation A and formulation B resulted inborderline production of these docosanoidsafter only 1 week.

DISCUSSION

DNFB sensitization of ears induces thickening,which will continually progress, eventuallycausing ear tissue loss. Ear thickness measure-ments reveal the extent of sensitization, and theefficacy of treatment. We show that ear thick-ness changes from 0.2 to 0.9 mm. In the firstsets of experiments in which sensitization pre-ceded treatment, ear thickness of mice treatedwith test substances gradually decreased.Moreover, in the second group of experiments,with simultaneous sensitization and treatment,test substances prevented ear thickening. Bothmethods demonstrated an improving or pre-ventive effect of the test compounds. Theseresults were corroborated by Western blotanalysis and mass spectrometry.

Dermatol Ther (Heidelb) (2018) 8:111–126 119

Naive

Vehicle

D+1w

D+1w+1w

D+MAM

D+CGDS 2%

D+MAM+CGDS 2%

D+MAM+CGDS 5%

Merge

a

PGP 9.5+beta III tubulin-PGP 9.5+beta III tubulin-neuronal processesneuronal processesPGP 9.5+beta III tubulin-neuronal processes

fiber tracts

b

PAR-2

epidermis

dermis

stratum corneumc

DAPI-nuclei

d

e

120 Dermatol Ther (Heidelb) (2018) 8:111–126

Overall, immunoblotting revealed thatupregulation of inflammation-related mole-cules was greatly reduced by the MAM andCGDS compounds (formulation B,MAM ? CGDS 5%, greatest; formulation A,MAM ? CGDS 2%, little effect).

PAR-2, a mediator of neurogenic inflamma-tion and pain, is activated by injury andinflammation. This activates TRPV4, resultingin mediation of the PAR-2 proinflammatoryresponse. Consequently, both PAR-2 and TRPV4are good indicators of inflammation and pain[21, 32]. These indicators were upregulated withACD, but PAR-2 far exceeded TRPV4. TheTRPV4 ion channel can enable Ca2? entry,permitting inflammation- and pain-associatedCGRP and substance P release [21]. Whileinflammation was indicated, we observed min-imal upregulation of the inflammatory markerTNFa, perhaps due to reduced TRPV4 activa-tion. These findings suggest that pain andinflammation may be separately triggered andprogress independently in this ACD model.

PAR-2 is associated with inflammation andichthyosis, but these are completely negatedwith a PAR-2 knockout genotype [33]. Trypsinand tryptase activate PAR-2, but, while topicalapplication of trypsin or agonists of PAR-2peptides trigger itch, the response is histamine-

independent [34, 35]. The Mas-related GPCRitch receptor MrgprC11 [36] triggers scratching,but PAR-2 elicits thermal pain hyperalgesia.Combinations of several PARs can exist; PAR-2can be activated by thrombin-cleaved PAR-1[37], suggesting a link between MrgprC11 andPAR-2 [38]. PAR-2 is greatly increased in DNFB-sensitized skin and decreased by formulation B(MAM ? CGDS 5%).

PAR-2 is expressed by mast cells [39] but isincreased in chronic skin inflammation [40] andpsoriatic lesions [41]. In humans, cell typeslocalized throughout the dermis and epidermisalso produce PAR-2 [39], and PAR-2 is alsohighly expressed in epidermal keratinocytes[41] within this stratum. PAR-2 is localized tothe stratum corneum and basal cells within themouse ear epidermis; its expression is increasedby DNFB, and formulation B (MAM ? CGDS5%) reduces PAR-2 to control levels. Since PAR-2is intimately associated with inflammation,reduced expression indicates amelioration ofACD.

Neuronal processes from the dermis extendinto the epidermis, ramify, and terminate justbelow the stratum corneum. Noxious com-pounds on the skin activate PAR-2 on mast cellsand initiate inflammation. These sensoryC-fibers respond to PAR-2 signaling compromise[17–19]. The branching pattern of these fibers iscomplex, but changes in their density suggestthat DNFB-induced ACD decreases their massbelow naı̈ve levels within all treatments. How-ever, though treatment with formulation B(MAM ? CGDS 5%) reduced PAR-2, TRPV4, andTNFa to near naı̈ve values (indicating reductionof pain and resolution of inflammation), 1 weekof treatment may not be enough for full tissuerecovery. Importantly, it should be noted thatproteins can be quantified by Western blot, butthe same proteins can only be localized byimmunohistochemistry without extreme carein quantifying fluorescence, so while Westernblots indicated a reduction in signal (e.g., PAR-2), our immunohistochemistry only revealedthat PAR-2 was present.

PAR-2, TRPV4, and TNFa upregulation indi-cates pain and inflammation induction byDNFB sensitization. Mass spectrometric analysispermitted quantification of inflammatory

bFig. 4 Naı̈ve control ears exhibit dense sensory fibers anda distinctive PAR-2 band. a Merged immunohistochem-istry image showing sensory fibers (green) within theepidermis and distinctive PAR-2 labeling (red). b Greenfluorescence channel revealing PGP 9.5 plus beta IIItubulin-labeled complex sensory fibers within the epider-mis, arising from underlying fiber tracts within the dermis.c Red fluorescence channel demonstrating a narrow brightdense PAR-2-labeled band near the epidermis/dermisinterface. Light background label also appears within thestratum corneum of all immunopreparations. d Bluefluorescence channel showing the DAPI-labeled nucleiwithin this section. Scale bar, 50 lm. e Dermatitic earsgenerally exhibit increases in PAR-2 deeper within thetissue, when compared to naı̈ve control and vehicle-aloneears (red label), indicating that inflammation has beentriggered. The green channel reveals the extent of thesensory fibers within the epidermis, also seen within themerged images at the right. Scale bar, 25 lm

Dermatol Ther (Heidelb) (2018) 8:111–126 121

Allergiccontact determitis (D)D+MAM CGDS 2%D+MAM CGDS 5%

50

40

30

20

10

0

Rat

io to

inte

rnal

sta

ndar

d

EPA AA DHA

n.s.

n.s.n.s.

n.s.

n.s.n.s.

4

3

2

1

0

Rat

io to

inte

rnal

sta

ndar

d

12HETE 15HETE

n.s.

n.s.

n.s.

n.s.

0.20

0.15

0.10

0.05

0.00

Rat

io to

inte

rnal

sta

ndar

d

14HDHA 17HDHA

n.s.

n.s.

e

c

b

0.4

0.3

0.2

0.1

0.0

Rat

io to

inte

rnal

sta

ndar

d

NPD1 NPD1 and isomers

n.s.

n.s.

n.s. (p=0.06)

n.s. (p=0.07)

2.0

1.5

1.0

0.5

0.0

Rat

io to

inte

rnal

sta

ndar

d

PGE2 LTB4 LTC4

p = 0.02

p < 0.01

p = 0.05

n.s.

n.s.

n.s.

d

a

122 Dermatol Ther (Heidelb) (2018) 8:111–126

markers (PGE2, LTB4, and LTC4) and precursormolecules, which initiate stress-induced fattyacid signaling cascades from EPA, AA, and DHA(AA products: 12HETE and 15HETE; DHAproducts: 14HDHA, 17HDHA; and the DHA-derived neuroprotective compound NPD1 andits isomers). Following 1 week of treatment afterACD induction, tissues responded to test com-pounds in unexpected manners. Inflammatorymarkers increased while AA, DHA, and theirmetabolites remained unchanged. However,NPD1 and its isomers tended to increase, asexpected of protectant molecules. NPD1, ahighly protective docosanoid, is produced fromDHA when cells undergo oxidative stress andhomeostatic changes. When the NPD1 isomerswere totaled, treatment with formulation A andformulation B resulted in borderline productionof these docosanoids after only 1 week, sug-gesting that these experimental compounds addto the normal tissue protective response elicitedby DNFB alone, stimulating additional upregu-lation of neuroprotective molecules. Impor-tantly, increases in inflammatory markers PGE2,LTB4, and LTC4 indicate continual inflamma-tion, suggesting that 1 week may be too shortan interval to affect full ameliorative benefitsfrom the test compounds, and that analysis ofadditional time points would resolve this issue.These lipid mediators reflect inflammatory cellinflux plus activation of resident cells in thismodel. These results represent a snapshot of the

ongoing changes from a steady state, andshould progress as treatment continues; withadditional time, indicators of inflammationshould decrease and neuroprotective elementsincrease. These are issues that we are currentlystudying.

In summary, following DNFB treatment withno additional therapy, eventual decline inthickness occurred from normal tissue repair.However, subsequent treatment with MAM,disodium cromoglycate 2%, and formulation A(MAM ? CGDS 2%) reduced ear thickness morerapidly by as much as 60%, whereas treatmentwith formulation B (MAM ? CGDS 5%) causeda thickness reduction of up to 70%. Impor-tantly, simultaneous sensitization and testcompound treatment, especially with formula-tion B, prevented onset of the inflammationand pain response. Mass spectrometry suggeststhat both formulations are linked to the syn-thesis of NPD1 isomers, all of which are potentneuroprotective agents. Immunoblottingdemonstrated that development of ACD isaccompanied by upregulation of inflammatoryand pain-related PAR-2 and TRPV4 and theinflammatory marker TNFa. Moreover, thepresence of PAR-2, TRPV4, and TNFa wassomewhat reduced in the presence of formula-tion A, but significantly prevented by formula-tion B. Finally, this supports studies which haveshown that mediation of PAR-2 can regulate itsproinflammatory actions [21]. Overall, thisstudy suggests that the experimental com-pounds formulation A (MAM ? CGDS 2%) and,particularly, formulation B (MAM ? CGDS 5%)effectively decrease pain, reduce inflammation,ameliorate tissue swelling, and increase neuro-protective docosanoid levels.

There were some limitations in this study. Itwas initially determined that induction of der-matitis increased ear thickness from about0.2 mm to more than 0.6 mm, with eventualtissue destruction and loss of ear tips. To avoidtissue loss, we chose an intermediate thicknessof about 0.50 mm, at which to apply the testcompounds, and then applied treatment for7 days, during which ear thicknesses weredecreased. However, there were indications thatsome inflammatory markers (e.g., PGE2 andarachidonic acid) remained elevated. This

bFig. 5 Mass spectrometric lipidomic analysis of ear tissuerevealed synthesis of neuroprotective mediators. Massspectrometric analysis of ear tissues permitted quantifica-tion of the inflammatory markers a PGE2, LTB4, andLTC4, b the precursor molecules initiating the stress-induced fatty acid signaling cascades, EPA, AA, and DHA,c products of the AA cascade 12HETE and 15HETE,d products of the DHA cascade 14HDHA, 17HDHA,and e the DHA-derived neuroprotective compoundsNPD1 and its isomers. Following 1 week of treatmentafter ACD induction, tissues responded to test compoundsin unexpected manners. Inflammatory markers increased,AA, DHA, and their associated metabolites remainedunchanged. However, NPD1 and its isomers tended toincrease as expected of protectant molecules.Means ? SEM, n = 8

Dermatol Ther (Heidelb) (2018) 8:111–126 123

suggests that treatment had not completelyameliorated the dermatitis; by prolonging thetreatment phase in some mice, more informa-tion could have been obtained about the actionof these compounds and their effective timecourse. Also, there was variability between earsof the same animal and among the mice withineach treatment, as evidenced by the size of errorbars in some data groups. Obviously, more than12 mice (24 ears) per group would likely haveproduced less variability within groups. Finally,to achieve visible neurite labeling we had tocombined two markers, PGP 9.5 and beta IIItubulin. However, tubulin also labels the den-dritic components of Langerhans cells, whichlikely contribute to the green fluorescentlylabeled processes of Fig. 4. Also, with all neu-ronal processes labeled, subpopulations couldnot be distinguished.

CONCLUSIONS

The significance of this study is twofold: BIPx-ine (a mixture of Rosa moschata and Crotonlechleri (antioxidants) and Aloe vera and D-pan-thenol (moisturizers), i.e., the mixture ofantioxidants and moisturizers (MAM)), whencombined with the hydroglycolic solution ofdisodium cromoglycate (CGDS) to produce thetreatment formulations applied to the ACD,ameliorated inflammation, reduced swelling,and initiated docosanoid protection. Moreover,this treatment offers an alternative to steroidtherapy and the possible side effects. However,additional studies are needed to determine theideal duration of treatment and the most effi-cacious concentrations of the active compo-nents in the test formulations which will bestalleviate ACD.

ACKNOWLEDGEMENTS

Funding. This work was supported by theNational Institute of General Medical SciencesP30GM103340 (NGB). Article processing char-ges were funded by the LSU Neuroscience Cen-ter of Excellence.

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. Ricardo PalaciosPeláez, Nicolas G. Bazan, Hélène Varoqui, andWilliam C. Gordon designed the experiments.Concha Tiana Ferrer, Fernando Pineda de laLosa, and Gabriela Silvina Bacchini supplied thetest compounds. William C. Gordon and Sur-jyadipta Bhattacharjee performed the experi-ments. Bokkyoo Jun performed the massspectrometry analyses. William C. Gordon,Ricardo Palacios Peláez, Nicolas G. Bazan, Vir-ginia Garcı́a López, David Rodrı́guez Gil, JavierAlcover Dı́az, and Concha Tiana Ferrer wrotethe paper.

Disclosures. William C. Gordon, VirginiaGarcı́a López, Surjyadipta Bhattacharjee, DavidRodrı́guez Gil, Javier Alcover Dı́az, FernandoPineda de la Losa, Ricardo Palacios Peláez,Concha Tiana Ferrer, Gabriela Silvina Bacchini,Bokkyoo Jun, Hélène Varoqui, and Nicolas G.Bazan have nothing to disclose.

Compliance with Ethics Guideline. All ani-mal procedures were approved by the Institu-tional Animal Care and Use Committee andInstitutional Review Board, LSUHSC, NewOrleans, all institutional and national guideli-nes for the care and use of laboratory animalswere followed, and this study has conformedwith the Helsinki Declaration of 1964, as revisedin 2013, concerning human and animal rights.

Data Availability. The data sets generatedduring and/or analyzed during the currentstudy are available from the correspondingauthor on reasonable request.

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 any

124 Dermatol Ther (Heidelb) (2018) 8:111–126

http://creativecommons.org/licenses/by-nc/4.0/http://creativecommons.org/licenses/by-nc/4.0/

medium, 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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A Nonsteroidal Novel Formulation Targeting Inflammatory and Pruritus-Related Mediators Modulates Experimental Allergic Contact DermatitisAbstractIntroductionMethodsResultsConclusion

IntroductionMethodsAnimalsGeneral Reagents and AntibodiesEar Skin Sensitization and Induction of ACDImmunoblot Analysis of Ear TissueLabeling and Quantification of the Primary Afferent ArborizationsLipidomic Analysis of Ear Tissue by LC--MS/MSStatisticsEar Measurements and Western BlottingMass spectrometric analysis

ResultsEar ThicknessProtein Abundance by ImmunoblottingImmunohistochemistryLipid Mediators

DiscussionConclusionsAcknowledgementsReferences