A Nonsteroidal Novel Formulation Targeting Inflammatory and Pruritus-Related Mediators Modulates Experimental Allergic Contact Dermatitis

Abstract

Introduction: A major clinical challenge in treating allergic contact dermatitis (ACD) is that the first line of treatment is based on the use of corticosteroids. In this study, we aimed to develop a formulation that is devoid of steroids.

Methods: We used mouse ears treated with dinitrofluorobenzene (DNFB) to induce ACD. The efficacy of the test formulation to ameliorate and to prevent induced ACD was determined.

Results: To treat this experimentally induced ACD, we developed a formulation containing BIPxine (a mixture of Rosa moschata and Croton lechleri (antioxidants) and Aloe vera and D-panthenol (moisturizers), and hydroglycolic solutions of disodium cromoglycate. Our results show that clear inhibition of ACD took place. The target of this formulation was PAR-2, TRPV4, and other mediators of the inflammatory and pain responses. However, this formulation must be evaluated in other models besides the mouse to confirm its effectiveness.

Conclusion: The formulation presented here may provide new ACD therapies that do not involve the use of corticosteroids.

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

Fig. 1

Outlines of experimental protocols. Protocol for the a curative, b preventative, and c experimental corticosteroid treatments. These protocols generally cover a 2-week period. Ear tissue was harvested at the times indicated by “killed”

Fig. 2

Ear thickness profiles for curative, preventative, and corticosteroid experimental treatments: novel formulations promote resolution of inflammation-induced ear thickening. a Ear thickness averages of individual responses (Suppl S1) for each curative experimental treatment showed increases induced by ACD. Following onset of secondary treatments, thickness rapidly declined with MAM, formulation A, and formulation B. Formulation B produced the best effect. No changes were observed in controls, while ear thickness in ACD-alone animals continued to increase. b Ear thickness averages for each curative experimental treatment based on the 14-day protocol (Fig. 1a; see Suppl S2 for additional graphs). Ear thickness averages for each experimental treatment showed increases of about 0.1 mm induced by ACD during the first week. Following onset of secondary treatments during week 2, thicknesses generally stabilized or were reduced. Treatment with a combination of MAM and cromoglycate 5% induced the most dramatic reduction, returning thicknesses to near starting levels. Onset of the secondary treatment occurred after 1 week. Measurements were collected at time zero and at 1 and 2 weeks from both ears of seven mice for each treatment. Graphs represent two independent experiments (total n = 28). The efficacy of formulation B in reducing the effects of ACD was similar to that shown for experiments in a. c Parallel curative and preventative experiments induced ACD for 1 week, with simultaneous treatment, or was followed by 1 week of treatment with two steroids, hydrocortisone (H) and desoximetasone (D) (Fig. 1c). Both compounds maintained or reduced ear thickness to naïve levels. Measurements were collected at time zero and at 1 and 2 weeks from both ears of five mice for each treatment (n = 10). d A preventative experiment was performed by simultaneously treating ears with the DNFB ACD inducer and the test compounds (Fig. 1b). ACD alone increased ear thickness as in all other experiments while co-treatment with MAM plus cromoglycate 2% or 5% maintained ear thicknesses very close to naïve control and vehicle-alone ears, demonstrating a preventative effect on the development of ACD. Arrows indicate onset of treatment. 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

Fig. 3

Novel formulation treatment attenuated inflammatory markers. a Sensitization of skin promoted upregulation of PAR-2, while novel formulation attenuated PAR-2 production. Ears treated with vehicle alone did not develop ACD, while DNFB sensitization (D) triggered upregulation of the itch marker PAR-2. Test substances attenuated the PAR-2 response by 22% and 69%. Typical Western blots are shown. GAPDH, house-keeping protein. D, ACD; MAM + CGDS 2% (formulation A); MAM + CGDS 5% (formulation B). PAR-2 means + SEM, n = 7. *p = 0.002, ^#p = 0.016. b Skin sensitization promoted upregulation of TRPV4, while novel formulation prevented TRPV4 production. Similarly, ears treated with vehicle alone did not develop ACD; however, DNFB sensitization (D) triggered upregulation of the itch marker TRPV4. Test substance formulation B reduced TRPV4 formation to naïve control levels. Two typical Western blots are shown. GAPDH, house-keeping protein. D, ACD; MAM + CGDS 2% (formulation A); MAM + CGDS 5% (formulation B). TRPV4 means + SEM, n = 7. *p = 0.007, ^#p = 0.026. c Skin sensitization induced an inflammatory response, while novel formulation blocked inflammation onset. DNFB sensitization triggered upregulation of the inflammatory marker TNFα, but formulation B prevented inflammation onset. Two typical Western blots are shown. GAPDH, house-keeping protein. D, ACD; MAM + CGDS 2% (formulation A); MAM + CGDS 5% (formulation B). TNFα means + SEM, n = 7. *p = 0.249, ^#p = 0.210

Fig. 4

Naïve control ears exhibit dense sensory fibers and a distinctive PAR-2 band. a Merged immunohistochemistry image showing sensory fibers (green) within the epidermis and distinctive PAR-2 labeling (red). b Green fluorescence channel revealing PGP 9.5 plus beta III tubulin-labeled complex sensory fibers within the epidermis, arising from underlying fiber tracts within the dermis. c Red fluorescence channel demonstrating a narrow bright dense PAR-2-labeled band near the epidermis/dermis interface. Light background label also appears within the stratum corneum of all immunopreparations. d Blue fluorescence channel showing the DAPI-labeled nuclei within this section. Scale bar, 50 μm. e Dermatitic ears generally exhibit increases in PAR-2 deeper within the tissue, when compared to naïve control and vehicle-alone ears (red label), indicating that inflammation has been triggered. The green channel reveals the extent of the sensory fibers within the epidermis, also seen within the merged images at the right. Scale bar, 25 μm

Fig. 5

Mass spectrometric lipidomic analysis of ear tissue revealed synthesis of neuroprotective mediators. Mass spectrometric analysis of ear tissues permitted quantification of the inflammatory markers a PGE2, LTB4, and LTC4, 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 compounds NPD1 and its isomers. Following 1 week of treatment after ACD induction, tissues responded to test compounds in unexpected manners. Inflammatory markers increased, AA, DHA, and their associated metabolites remained unchanged. However, NPD1 and its isomers tended to increase as expected of protectant molecules. Means + SEM, n = 8