Etrasimod, Once-Daily, Oral, Selective Sphingosine 1-Phosphate Receptor Modulator Improves Skin Inflammation in a Contact Hypersensitivity Model of DermatitisCatherine M Crosby, H Kiyomi Komori, John W Adams

Arena Pharmaceuticals, San Diego, CA

Presented at Revolutionizing Atopic Dermatitis (RAD) virtual symposium, April 5, 2020

Conclusions• Etrasimod effectively reduced ear skin inflammation and dermatitis in the FITC-induced

hypersensitivity dermatitis mouse model. — Etrasimod reduced the trafficking of antigen-presenting dendritic cells from the skin

to the lymph nodes, which correlated with a reduction of T cell activation in the lymph node.

— Etrasimod treatment led to a statistically significant reduction in multiple immune cell types in the skin, including both CD4+ and CD8+ T cells.

— Cytokine production was significantly decreased in the tissue.

• These in vivo proof of concept studies support the unique mechanism of action of etrasimod, which acts upstream of treatments that specifically target cytokines within the tissue. Etrasimod reduced the trafficking of dendritic cells into and T cells out of lymph nodes into circulation, which produced a downstream reduction in immune cells, cytokine production, and dermatitis in the skin. These data encourage further study of etrasimod as a novel therapy for atopic dermatitis.

References1. Buzard, D. J. et al. Discovery of APD334: Design of a Clinical Stage Functional Antagonist of the Sphingosine-1-phosphate-1 Receptor. ACS Med Chem

Lett 5, 1313-1317, doi:10.1021/ml500389m (2014).

2. Brinkmann, V. et al. Fingolimod (FTY720): discovery and development of an oral drug to treat multiple sclerosis. Nat Rev Drug Discov 9, 883-897, doi:10.1038/nrd3248 (2010).

3. Rathinasamy, A., Czeloth, N., Pabst, O., Forster, R. & Bernhardt, G. The origin and maturity of dendritic cells determine the pattern of sphingosine 1-phosphate receptors expressed and required for efficient migration. J Immunol 185, 4072-4081, doi:10.4049/jimmunol.1000568 (2010).

ResultsFigure 5. Etrasimod Reduced Cytokine Protein in Ear Skin

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Etrasimod significantly reduced ear protein content of (A) IL-4, (B) IFNγ, (C) IL-6, and (D) TNFα in a dose-dependent manner. Data mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 for comparison vs vehicle. ns = not statistically significant vs vehicle. IL-4 = interleukin-4, IL-6 = interleukin-6, IFNγ = interferon gamma, TNFα = tumor necrosis factor alpha.

Figure 1. Etrasimod Dose-Dependently Reduced Ear Histopathological Score

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In Study 1 and Study 2, etrasimod treatment resulted in a similar dose-dependent reduction in ear thickness (data not shown). Etrasimod treatment also resulted in a significant, dose-dependent reduction in histopathological score (A) on Day 13. Results are shown for Study 1. There was no significant difference between etrasimod 3 mg/kg and dexamethasone. Histopathological score was based on the main changes in dermatitis, pustules, and epidermal hyperplasia (0 = no inflammation, 1 = mild, 2 = moderate, 3 = severe). Representative H&E stained ear sections from animals treated with vehicle (B), dexamethasone 1 mg/kg (C), or etrasimod 3 mg/kg (D). Data are mean ± SEM. **P < 0.01, ****P < 0.0001 for comparison vs vehicle.

Figure 2. Etrasimod Reduced Dendritic Cell Influx and T cell Activation in the Cervical Lymph Node

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Etrasimod 1 and 3 mg/kg treatments similarly reduced the trafficking of both Langerin+ (Langerhan’s cells) (A) and Langerin– (non-Langerhan’s DCs) (B) dendritic cells. (C) Etrasimod 1 and 3 mg/kg reduced the number of T cells in the cervical lymph node, indicating reduced T cell expansion. (D) Etrasimod 1 and 3 mg/kg reduced the number of tissue resident CD69+ T cells, indicating reduced T cell activation. Data mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001 for comparison vs vehicle. DC = dendritic cells.

Figure 3. Etrasimod Reduced T Cells, B Cells, and Eosinophils in Ear Skin

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Etrasimod 1 and 3 mg/kg significantly the number of T cells (A), including skin-homing CLA+ T cells (B) and tissue resident CD69+ (activated) T-cells in ear skin (C). However, only the 3 mg/kg treatment substantially reduced the expansion of B cells (D) and eosinophils (E) in the skin. Data mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 for comparison vs vehicle. ns = not statistically significant vs vehicle.

Figure 4. Etrasimod Reduced Activation and Expansion of CD4 and CD8 T Cells in Ear Skin

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Etrasimod 1 and 3 mg/kg doses significantly reduced the number of T cells (A), as well as activated CD4+ T cells (B) and CD8+ T cells (C). Additionally, etrasimod 1 and 3 mg/kg doses significantly reduced tissue-resident CD69+ CD4+ T cells (D) and tissue-resident CD69+ CD8+ T cells (E), indicating reduced T cell expansion into ear tissue. Data mean ± SEM. ****P < 0.0001 for comparison vs vehicle.

Introduction• Etrasimod (APD334) is a once-daily, orally administered, selective sphingosine 1-phosphate

receptor 1,4,5 (S1P1,4,5) modulator1 in development for multiple immune-mediated inflammatory disorders, including an ongoing Ph 2b ADVISE trial in atopic dermatitis.

• S1P1 is a cell surface G protein-coupled receptor (GPCR) that has been shown to regulate lymphocyte egress from lymph nodes2 and dendritic cell trafficking3.

• Upon binding to S1P1, synthetic modulators such as etrasimod act as functional antagonists by inducing and sustaining receptor internalization. This prevents cell migration along S1P gradients, resulting in lymphocyte retention within lymphoid tissue, and a reduction in peripheral blood lymphocytes available to be recruited to sites of inflammation2.

Objective• The goal of these preclinical studies was to establish a proof of concept for the etrasimod mechanism

of action in a dermatology model. We used a fluorescein isothiocyanate (FITC)-induced dermatitis mouse model to evaluate how the reduction in circulating lymphocytes produced by etrasimod affects immune cell trafficking and the ultimate impact on skin inflammation.

MethodsExperimental Design and Treatment Groups

• Beginning on Day -1, female BALB/c mice were orally dosed daily with the indicated treatments in phosphate buffered saline (PBS). Etrasimod doses were based on potency determined by previous in vivo studies.

• On Days 0 and 5, mice were sensitized with 1% fluorescein isothiocyanate (FITC) in Acetone: Dibutyl phalate (ADBP) on the hind flank skin, and subsequently challenged on the ear skin on Days 10, 11, and 12.

SENSITIZATION

Prophylactic Daily Oral Dosing: Vehicle/Dexamethasone/Etrasimod

CHALLENGE

Day: –1 0 5 10 11 12 13

Treatment Group

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Sham 5 10 • Complete blood count (CBC) with differential

• Cervical lymph node cellularity analyzed by flow cytometry

• One ear histologically scored (standard H&E)

• Ear tissue cellularity analyzed by flow cytometry

• Complete blood count (CBC) with differential

• Ear tissue analyzed for cytokine protein

• Ear tissue cellularity analyzed by flow cytometry

Vehicle (PBS) 10 15

Etrasimod 1 mg/kg 10 15

Etrasimod 3 mg/kg 10 15

Dexamethasone 1 mg/kg 10 X