A novel JAK inhibitor JTE‐052 reduces skin inflammation and ameliorates chronic dermatitis in rodent models: Comparison with conventional therapeutic agents

Janus kinases (JAKs) are required for several inflammatory cytokine signalling pathways and are implicated in the pathogenesis of chronic dermatitis, including atopic dermatitis and psoriasis. JAK inhibitors are therefore promising therapeutic candidates for chronic dermatitis. In this study, we evaluated the effects of the novel JAK inhibitor JTE‐052 on inflammatory responses associated with chronic dermatitis, and compared its profile with those of conventional therapeutic agents in rodent models of chronic dermatitis. JTE‐052 inhibited the Th1‐, Th2‐ and Th17‐type inflammatory responses of human T cells and mast cells in vitro. Oral administration of JTE‐052 inhibited skin inflammation in hapten‐induced chronic dermatitis in mice, associated with reduced levels of inflammatory cytokines in the skin and immunoglobulin (Ig) E in serum. In contrast, although ciclosporin partly inhibited skin inflammation, it did not reduce interleukin (IL)‐4 production in skin, and enhanced IgE production in serum. Oral administration of JTE‐052 also inhibited skin inflammation in mouse models of atopic dermatitis and psoriasis induced by a mite extract, thymic stromal lymphopoietin or IL‐23. The maximal efficacy of JTE‐052 in these dermatitis models was superior to the conventional therapeutic agents, ciclosporin and methotrexate. Topical application of JTE‐052 ointment ameliorated hapten‐induced chronic dermatitis in rats more effectively than tacrolimus ointment. Furthermore, JTE‐052 ointment did not cause the thinning of normal skin associated with topical corticosteroids. These results indicate that JTE‐052 is a promising candidate as an anti‐inflammatory drug for various types of chronic dermatitis, with a distinctly different profile from conventional therapy following either oral or topical application.


| INTRODUCTION
Atopic dermatitis (AD) and psoriasis are the most common chronic inflammatory skin diseases. [1,2] They are characterised pathophysiologically by disrupted skin homeostasis and dysregulated immune response. [3,4] Several medications are used to treat these diseases with the aim of reducing skin inflammation. [5][6][7][8] Topical steroids and topical immunosuppressants are the main agents used for chronic dermatitis, and show definite therapeutic effects; however, their value is limited by local side effects and insufficient efficacy. [5,7] Oral steroids or immunosuppressants such as methotrexate and ciclosporin may also be used in patients with moderate-to-severe symptoms, but their usage is limited by systemic side effects. [6,8] Novel therapies with fewer side effects are therefore needed for the effective treatment of chronic dermatitis.
Biologics such as antitumor necrosis factor (TNF) monoclonal antibody, [9] ustekinumab [10] and secukinumab, [11] which have been approved for psoriasis, and dupilumab, [12] which is under development for AD, have recently shown impressive results with good efficacy in clinical trials. These trials suggested that inhibition of inflammatory cytokine signal transduction may offer a promising approach to the treatment of chronic dermatitis. Janus kinases (JAKs) comprise a non-receptortype tyrosine kinase family composed of four enzymes (JAK1, JAK2, JAK3 and Tyk2), which transduce signals from multiple type I and type II cytokine receptors and mediate various inflammatory responses. [13] Various JAK-related cytokines play roles in the pathophysiology of psoriasis (interleukin (IL)-12, IL-23, IL-22) and AD (IL-4, IL-13, IL-31, thymic stromal lymphopoietin (TSLP)). [14] Inhibition of JAKs may therefore represent a novel therapeutic approach for chronic dermatitis. Indeed, clinical trials of JAK inhibitors in patients with chronic dermatitis have already demonstrated definite effectiveness. [15,16] Furthermore, a preclinical study also reported efficacy of a JAK inhibitor in animal models of chronic dermatitis. [17][18][19][20] However, the difference between conventional therapies and JAK inhibitors has not been fully investigated.
We recently developed a novel JAK inhibitor, JTE-052, that was shown to be orally active and more potent than tofacitinib in mice. [21] In this study, we investigated the efficacy of oral JTE-052 in several rodent models of chronic dermatitis, and compared its profile with conventional therapeutic agents. We also investigated the efficacy of topical application of JTE-052 ointment in chronic dermatitis model, in the light of the likely reduced risk of systemic side effects of this mode of application.

| Animals
Balb/c mice were obtained from Japan SLC, Inc. (Hamamatsu, Japan). C57BL/6 mice, Nc/Nga mice and BN rats were obtained from Charles River Japan, Inc. (Yokohama, Japan). Animals were maintained under specific pathogen-free conditions at a room temperature of 23±3°C and air humidity of 55±15% with a 12-h/12-h light/dark cycle. All procedures related to the use of animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee of Japan Tobacco Inc.

| Cellular assay
Human peripheral blood was obtained from healthy volunteers with informed consent, based on the Declaration of Helsinki. Human memory CD4 + T cells were isolated from peripheral blood using

| Hapten-induced chronic dermatitis in mice
Hapten-induced chronic dermatitis was induced in female BALB/c mice as described previously. [22] Briefly, mouse ears were treated with 25 μL of 0.15% 2, 4-dinitrofluorobenzene (DNFB) dissolved in acetone/olive oil (3:1) once a week for 5 weeks. Ear thickness as an index of ear swelling was measured with a digital thickness gauge (Digimatic Indicator; Mitutoyo Corporation, Kawasaki, Japan) before and 24 hours after DNFB application, and expressed as the increase in thickness from the baseline measurement. JTE-052 and ciclosporin were administered orally once a day from the day of first DNFB application. After the last measurement of ear thickness, mice were anesthetised, and blood samples were collected. The mice were then euthanised, and their ears were excised for analysis of cytokine expression and histological evaluation. Serum total IgE and anti-2,4-dinitrophenol (DNP)-specific IgE levels were assessed by ELISA (Shibayagi Co., Ltd. Shibukawa, Japan). [22] Ear sections were homogenised, and cytokine levels in the supernatant were assessed by ELISA (R&D Systems, Inc., Minneapolis, MN, USA).

| Mite extract-and cytokine-induced dermatitis model in mice
Mite extract-induced dermatitis was induced in female Nc/Nga mice as described previously. [23] Briefly, 5 μg of Dermatophagoides pteronyssinus extract (Cosmo Bio Co. Ltd., Tokyo, Japan) was injected intradermally in the ears of mice three times a week for 3 weeks, and ear thickness was measured 24 hours later. TSLP-induced dermatitis was induced in female C57BL/6 mice as described previously [18] with minor modifications. Briefly, 0.5 μg of recombinant mouse TSLP (R&D Systems, Inc.) was injected intradermally in the ears of mice on days 1, 3, 5, 8 and 10, and ear thickness was measured 24 hours after each injection. IL-23induced dermatitis was induced in female C57BL/6 mice as described previously [24] with minor modifications. Briefly, 0.25 μg of recombinant mouse IL-23 (R&D Systems, Inc.) was injected intradermally in the ears of mice on days 1, 3, 5, 8 and 10, and ear thickness was measured on days 1, 3, 5, 8, 10 and 12. In each model, JTE-052, ciclosporin and methotrexate, respectively, were administered orally from the day of the first injection.
Ear thickness was measured as an index of ear swelling 6 hours after DNCB application, and expressed as the increase in thickness from baseline. JTE-052 ointment, tacrolimus ointment or difluprednate ointment was administered topically once a day from the day of first DNCB application. After the last measurement of ear thickness, rats were euthanised, and their ears were excised for histological evaluation.

| Statistical analysis
Data are expressed as the mean+standard deviation of the indicated number of samples. The significance of differences between two groups was assessed by Student's t-tests (for homoscedastic data) or Aspin-Welch t-tests (for heteroscedastic data), after homoscedasticity analysis by F-tests. Differences among multiple groups were analysed by Dunnett's tests (for homoscedastic data) or Steel's tests (for heteroscedastic data) after homoscedasticity analysis by Bartlett's test.

| JTE-052 inhibits inflammatory responses including Th1-, Th2-and Th17-type responses
Th1, Th2 and Th17-type responses are thought to be involved in several chronic dermal diseases. [14] We therefore used a cell-based assay  Figure 1A). We also investigated the inhibitory effect of JTE-052 on Th-related cytokine production in non-T cells.
Mast cells have been reported to play an important role in the pathogenesis of allergic diseases by producing Th2-type cytokines. [26] We therefore determined the effect of JTE-052 on mast cells. JTE-052 dose-dependently inhibited IL-13 production from human cord bloodderived mast cells stimulated with IL-4 and IgE/anti-IgE ( Figure 1B).
These results indicated that JTE-052 inhibited Th1-, Th2-and Th17type cytokine production from both T cells and non-T cells.

| JTE-052 inhibits skin inflammation in hapteninduced chronic dermatitis after oral administration
The repeated hapten-induced chronic dermatitis model is considered to involve Th2 cells and mast cells and is a useful model of human AD. [27,28] To determine if JTE-052 reduced skin inflammation in chronic dermatitis, we examined its effect on DNFB-induced chronic dermatitis and TNFα levels in ear skin but had no effect on IL-4. Moreover, ciclosporin potentiated serum anti-DNP IgE levels in this model, consistent with a previous report. [22]

| Comparison of efficacies of oral JTE-052 and conventional therapy in AD-like and psoriasis-like dermatitis in mice
We examined the effects of JTE-052 on other chronic dermatitis models related to AD and psoriasis, and compared its efficacy with conventional therapeutic agents used to treat these diseases. Mite extract-induced dermatitis in mice involves AD-like skin lesions and is reported to have an AD-like pathophysiology. [23] TSLP-induced dermatitis is also associated with the pathobiology of AD. [18] We therefore determined the effects of JTE-052 in these AD models.  Figure 3A,B). IL-23-induced dermatitis in mice has been reported to have a psoriasis-like pathophysiology, [24] and we therefore

| Topical JTE-052 ointment ameliorates hapteninduced chronic dermatitis in rats
We investigated the effect of topical administration of JTE-052 ointment on skin inflammation in a rat model of chronic dermatitis. The efficacies of JTE-052 ointment at 0.3% or 3% were superior to tacrolimus ointment 0.1% (Figure 4A,C). Administration of the topical corticosteroid difluprednate ointment 0.05% inhibited DNCB-induced ear swelling ( Figure 4C). We also investigated the local side effect of the ointments on normal rat skin. Treatment with JTE-052 ointment 3% for 22 days on the ear had no effect on normal ear thickness. In contrast, difluprednate ointment 0.05% decreased ear thickness in normal rats ( Figure 4D). These results indicate that JTE-052 ointment reduced ear swelling in a DNCB-induced dermatitis model at a concentration that did not affect normal skin thickness.

| DISCUSSION
In this study, we investigated the effects of the novel JAK inhibitor JTE-052 on chronic dermatitis in rodent models, and compared it with existing therapeutic agents. Oral administration of JTE-052 inhibited skin inflammation in various types of dermatitis, along with suppression of inflammatory cell activation. The maximal efficacy of oral JTE-052 in skin inflammation was superior to that of ciclosporin and methotrexate in dermatitis models related to AD and psoriasis.
Topical administration of JTE-052 ointment was also effective in chronic dermatitis, and its efficacy was superior to tacrolimus ointment. Furthermore, it did not cause the thinning of normal skin associated with topical corticosteroids such as difluprednate ointment.
In this study, oral administration of JTE-052 improved skin inflammation in all the tested models of chronic dermatitis. In contrast, the T cell activation inhibitor ciclosporin only reduced skin inflammation in hapten-induced chronic dermatitis in mice and had little effect on mite extract-induced or TSLP-induced AD-like dermatitis. Cutaneous T cells have been reported to contribute to skin inflammation in a hapteninduced chronic allergic dermatitis model. [22] However, although T cells contribute to mite extract-induced dermatitis, [29] mite components can also induce local inflammation directly through the activation of non-T cells. [30,31] TSLP was also reported to induce the activation of non-T cells such as dendritic cells, mast cells and innate lymphoid cells type 2 directly, but not T cells in the absence of antigens. [32] The rela- Several pharmacological mechanisms of action have been proposed for methotrexate, and its anti-inflammatory effect has largely been attributed to the reduction of conventional T cell proliferation. [33] The partial efficacy of methotrexate in IL-23-induced dermatitis might thus F I G U R E 4 Topical application of JTE-052 ointment ameliorates hapten-induced chronic dermatitis in rats. Rats received topical application of 0.5% DNCB in acetone/olive oil or vehicle on the ear three times a week for 3 wk. JTE-052 ointment (placebo, 0.03%, 0.3% or 3%) or tacrolimus ointment (0.12%) was administered topically once daily for 20 d from the day of first DNCB application. (A) Ear thickness was assessed 6 h after DNCB application and expressed as the increase in thickness from baseline. (B) Histological analysis on day 21. (C) Effect of difluprednate ointment 0.05% on hapten-induced chronic dermatitis was determined in a separate experiment. (D) Normal rats were administered topical JTE-052 ointment (placebo, 3%), tacrolimus ointment 0.1% or difluprednate ointment 0.05%, and ear thickness was assessed on day 21. The results are expressed as mean+standard deviation (n=6-9). NA: nonadministration *P<.05, **P<.01 vs placebo by Dunnett's test. # P<.05, ## P<.01 vs placebo by Steel's test. $$ P<.01 vs non-administration by Student's t test. ++P<.01 vs non-administration by Aspin-Welch t test be explained by its limited effect on non-T cells. Overall, these findings indicate that JTE-052 exerts anti-inflammatory effects in different types of dermatitis by inhibiting various types of inflammatory cells.
In this study, serum IgE levels were increased by ciclosporin in a hapten-induced dermatitis model, as described previously. [22] In a previous report, selective inhibition of Th1 but not Th2 by ciclosporin in vivo enhanced Th2-induced IgE production. Similarly, ciclosporin failed to inhibit IL-4 production in the ear in the current study, suggesting that partial inhibition of the Th response enhanced IgE production in this hapten-induced dermatitis model. In contrast, JTE-052 reduced IL-4 production in the ear in this model, consistent with inhibition of IL-4 in vitro, indicating that JTE-052 inhibited Th2 cells in vivo. In addition, we previously revealed that JTE-052 inhibited B-cell proliferation induced by IL-21, a cytokine indispensable for the germinal centre reaction, [21] suggesting that its direct inhibitory effect on B cells might contribute to the decrease in serum IgE production induced by JTE-052. These findings might explain the difference between ciclosporin and JTE-052 in terms of their effects on serum IgE production. IgE is considered to play an important role in the pathogenesis of allergic diseases such as AD. [34] Indeed, in contrast to ciclosporin that showed reduced efficacy in the late phase of the experiment in association with serum IgE elevation, the effect of JTE-052 on ear swelling lasted throughout the experiment in hapten-induced dermatitis in mice.
Taken together, these findings indicate that JTE-052 may have distinct therapeutic effects from ciclosporin in these diseases via inhibition of IgE production.
Systemic immunomodulating agents such as ciclosporin and methotrexate are prevalent treatment options for the management of severe symptoms in patients with AD and psoriasis, but their usage is limited by organ toxicities including nephrotoxicity, hepatotoxicity and pulmonary fibrosis. [6,35] In contrast, there have been no noticeable reports of organ toxicity associated with systemic JAK inhibitors in several clinical trials. [36] JAK inhibitors may therefore offer a beneficial systemic medication option for chronic dermatitis, with a lower risk of side effects than conventional systemic agents. However, systemic administration of JAK inhibitors is associated with potential risks of infection and malignancies, as for other systemic agents, [37] and topical application of JAK inhibitors may thus offer a preferable option for chronic dermatitis. Although topical corticosteroids and calcineurin inhibitors are widely used for treating chronic dermatitis without systemic toxicity, they are associated with a risk of local cutaneous side effects such as skin atrophy. [38] Indeed, topical administration of the corticosteroid difluprednate ointment 0.05% in the current study reduced skin thickness in normal rats, while topical treatment of JTE-052 ointment 3% had no effect on normal skin, but showed comparable efficacy to difluprednate ointment in terms of skin inflammation in a DNCB-induced rat dermatitis model. In addition, it was recently recognised that skin barrier disruption contributes to the pathophysiology of chronic dermatitis, such as AD. [39,40] As JAK inhibition by JTE-052 was revealed to improve skin barrier disruption by directly affecting epidermal keratinocytes in our previous study, [20] topical application may be an effective dosage form for this JAK inhibitor to exert its effects on the skin barrier function. Topical application of JTE-052 may therefore be an effective novel therapy for chronic dermatitis, with a better risk profile than conventional therapies.
In conclusion, the results of this study demonstrate that JTE-052, by either oral or topical application, is a good candidate as an anti-inflammatory drug for the treatment of various types of chronic dermatitis, including AD and psoriasis, with a distinct profile from conventional therapies.