Ruxolitinib Cream Has Dual Efficacy on Pruritus and Inflammation in Experimental Dermatitis

Abstract

The goal of this study was to elucidate the anti-pruritic and anti-inflammatory efficacy of ruxolitinib cream in experimentally-induced dermatitis. Atopic dermatitis (AD), the most common chronic relapsing inflammatory skin disease, significantly impairs patients' quality of life, with pruritus being a common complaint. The sensation of itch results from the interplay between epidermal barrier dysfunction, upregulated immune signaling and the activation of the central nervous system. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway plays a central role in pro-inflammatory cytokine signaling in AD. Ruxolitinib cream is a potent and selective JAK1/2 inhibitor currently undergoing clinical evaluation in adults with mild-to-moderate AD (NCT03745638, NCT03920852 and NCT03745651). The efficacy of ruxolitinib cream was tested in murine models of acute and chronic dermatitis and was also characterized in an ex vivo human skin dermatitis model. Ruxolitinib cream was highly effective at ameliorating disease symptoms in multiple murine dermatitis models through downregulation of T helper (Th)2-driven inflammation, resulting in reduced skin thickening and decreased itch. Pathway analysis of mouse ear tissue and human skin explants underscored the role for ruxolitinib in ameliorating inflammation and reducing itch via modulation of the JAK-STAT pathway. Together, the data offer a strong rationale for the use of ruxolitinib cream as a potent therapeutic agent for the clinical management of atopic dermatitis.

Keywords: JAK/STAT signaling pathway; dermatitis; inflammation; pruritus; ruxolitinib.

Conflict of interest statement

All authors are employees and shareholders of Incyte Corporation.

Copyright © 2021 Scuron, Fay, Connell, Peel and Smith.

Figures

Figure 1

Ruxolitinib cream decreased inflammation and restored sleep duration in the acute thymic stromal lymphopoietin (TSLP)-induced dermatitis mouse model. (A) Repeated intradermal TSLP challenge induced acute ear swelling, which was significantly abrogated by ruxolitinib cream treatment. (B) Ruxolitinib cream efficacy was confirmed by a significant decrease in ear biopsy weight (% increase between right challenged ear and left control ear) at study termination. N = 16 mice per group. (C) Treatment with ruxolitinib cream restored sleep duration (sum of time points in a 24 h period where detectable motion ≤ 3) to baseline levels without causing sedation (D), as shown by unaltered wheel activity (sum of time spent on wheel during a 24 h period) across groups. Red arrows indicate TSLP immunizations. N = 10 mice per group (C, D). (E) Nanostring ear skin RNA analysis revealed differential gene expression between ruxolitinib cream and vehicle cream (baseline) treatment. Points above the gray and black dashed lines have adjusted p-values <0.05 and <0.01, respectively. (F) Inflammatory genes from multiple pathways were downregulated with ruxolitinib cream treatment. N = 8 mice per group. Data represents mean + SEM. (A) ***p < 0.001, ****p < 0.0001, vehicle vs. ruxolitinib. ####p<0.0001, vehicle vs. steroid. (B, C) *p < 0.05, ****p < 0.0001.

Figure 2

Ruxolitinib cream reduced chronic ear swelling and modulated inflammatory T-cell subsets in the fluorescein isothiocyanate (FITC)-induced dermatitis model. (A) Repeated FITC challenge did not induce inflammation-induced cachexia. Steroid (betamethasone) cream was poorly tolerated as indicated by the significant body weight loss. (B) Repeated FITC challenge resulted in chronic, non-resolving ear swelling in the vehicle group. (C) Ruxolitinib cream dose-dependently ameliorated ear swelling, which was confirmed by a significant decrease in ear biopsy weight (% increase between right challenged ear and left control ear) compared to the vehicle group at study termination. (D) Ruxolitinib cream treatment inhibited FITC-induced immune expansion in the auricular lymph node and resulted in lower proportions of Th1 (E) and Th2 (F) cells compared to vehicle. (G) Th17 cell proportions were not affected by ruxolitinib cream treatment in this model. Data represents mean + SEM. N = 6 mice per group. (A) ***p < 0.001, vehicle vs. steroid. (B) ****p < 0.0001, vehicle vs. ruxolitinib cream q.d. and b.i.d. (C–G) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 3

Prophylactic ruxolitinib dosing regimen ameliorated pruritus-induced behaviors and skin histopathology in IL-33tg mice. (A) In the IL-33tg dermatitis model, prophylactic treatment with ruxolitinib cream significantly ameliorated body weight loss compared to the vehicle cream group. Steroid (clobetasol) cream was poorly tolerated, as evident in significant body weight loss that required euthanasia at study midpoint. All mice in the steroid group were therefore euthanized at study midpoint, and their tissues were processed for histology. IL-33tg mice treated with vehicle cream exhibited abnormal scratching (B) and grooming behavior (C), while prophylactic treatment with topical ruxolitinib significantly ameliorated these behaviors at both study midpoint and endpoint. Eyelid skin samples were stained with hematoxylin and eosin and visualized under (D) 20x (scale bar = 200 µm) and (E) 50x (scale bar = 100 µm) magnification. The black arrow in vehicle panel (D) indicates lymphocyte infiltration. The black arrow in vehicle panel (E) indicates epidermal acanthosis. (F) Toluidine blue-stained mast cells were assessed under 200x magnification (scale bar = 50 µm). The black arrow in vehicle panel (F) indicates mast cell granule staining. Prophylactic application of ruxolitinib cream significantly reduced mast cell frequency (G) and cumulative histology score (H). Data represents mean + SEM. N = 10 in vehicle and ruxolitinib groups. N = 5 in WT and steroid groups. *p < 0.05, ***p < 0.001, ****p < 0.0001, vehicle vs. ruxolitinib.

Figure 4

Therapeutic ruxolitinib dosing regimen resolved dermatitis symptoms, pruritus-induced behaviors and skin histopathology in IL-33tg mice. (A) Therapeutic ruxolitinib cream treatment significantly resolved dermatitis symptoms in IL-33tg mice, while steroid (betamethasone) cream only prevented further disease progression over time. The vehicle group exhibited abnormal scratching (B) and grooming (C) behavior. Topical ruxolitinib significantly reduced both scratching and grooming by week 24. Eyelid skin samples were stained with hematoxylin and eosin and visualized under (D) 20x (scale bar = 200 µm) and (E) 50x (scale bar = 100 µm) magnification. The black arrow in vehicle panel (E) indicates epidermal acanthosis. The number of mast cells was scored from toluidine blue stain at 200x magnification (scale bar = 50 µm) (F). The black arrow in vehicle panel (F) indicates mast cell granule staining. Therapeutic ruxolitinib cream treatment significantly reduced mast cell frequency (G) and cumulative histology score (H). There was no significant difference between the vehicle and steroid group. Data represents mean + SEM. N = 10 in vehicle and ruxolitinib groups. N = 5 in WT and steroid groups. (A) ****p < 0.0001, vehicle vs. ruxolitinib. (B) **p < 0.01, ****p < 0.0001, vehicle vs. ruxolitinib; ##p < 0.01, vehicle vs. steroid. (C) *p < 0.05, **p < 0.01, ***p < 0.001, vehicle vs. WT; #p < 0.05, vehicle vs. ruxolitinib. (D, E) *p < 0.05, **p < 0.01.

Figure 5

Therapeutic ruxolitinib cream treatment downregulated inflammatory gene expression in IL-33tg mice. (A) In the IL-33tg mouse model, Nanostring eyelid skin RNA pathway analysis showed differential gene expression between therapeutic ruxolitinib cream and vehicle cream (baseline) treatment. Points above the gray and black dashed lines have adjusted p-values <0.05 and <0.01, respectively. (B) Inflammatory genes from multiple pathways were downregulated with ruxolitinib cream treatment. N = 7 mice per group.

Figure 6

Ruxolitinib cream downregulated expression of inflammatory genes in Th2-stimulated human skin. (A) Nanostring RNA pathway analysis showed differential gene expression between ruxolitinib cream and vehicle cream (baseline) treatment in human skin explants stimulated in Th2 conditions. Points above the gray and black dashed lines have adjusted p-values <0.05 and <0.01, respectively. (B) Inflammatory genes from multiple pathways were downregulated with ruxolitinib cream treatment. N = 6 donors per treatment.