Anti‐inflammatory effect of lanoconazole on 12‐O‐tetradecanoylphorbol‐13‐acetate‐ and 2,4,6‐trinitrophenyl chloride–induced skin inflammation in mice

Summary Background Lanoconazole (LCZ) is a topical antifungal agent clinically used to treat fungal infections such as tinea pedis. LCZ has not only antifungal effects but also anti‐inflammatory effects, which have the potential to provide additional clinical benefits. However, the characteristic features of the inhibitory effects of LCZ on skin inflammation remain unclear. Objective We evaluated the inhibitory effects of topical application of LCZ, and compared the effects of LCZ with those of other antifungal agents including liranaftate, terbinafine and amorolfine. Methods Each antifungal agent was topically applied on 12‐O‐tetradecanoylphorbol‐13‐acetate‐induced irritant dermatitis and 2,4,6‐trinitrophenyl chloride‐induced contact dermatitis in mice (BALB/c). The ear thickness, myeloperoxidase activity and inflammatory mediator contents were evaluated. Results LCZ dose‐dependently suppressed 12‐O‐tetradecanoylphorbol‐13‐acetate‐induced irritant dermatitis, suppressed the production of neutrophil chemotactic factors such as keratinocyte‐derived chemokine and macrophage inflammatory protein‐2, and inhibited neutrophil infiltration to the inflammation site. Moreover, 1% LCZ reduced the ear swelling in mice with 2,4,6‐trinitrophenyl chloride‐induced contact dermatitis in accordance with the inhibition of interferon‐γ production. The inhibitory potency of LCZ on these types of dermatitis in mice was stronger than that of other types of antifungal agents. Conclusion The anti‐inflammatory effects of LCZ were exerted through the inhibition of inflammatory mediator production. These effects may contribute to the relief of dermatitis symptoms in patients with tinea pedis.


| INTRODUC TI ON
Tinea pedis, one of the most common skin diseases worldwide, is a superficial dermatomycosis that occurs secondary to infection by dermatophytes. [1][2][3] An inflammatory response induced by the dermatophyte is often observed at the infection site in patients with tinea pedis. This response is thought to be an irritant dermatitis caused by inflammatory mediators produced by epidermal keratinocytes and leucocytes that reacted to proteases and fungal components such as β-glucan. [4][5][6][7] Indeed, histological examination of the skin of patients with dermatophyte infection reveals infiltration of neutrophils and lymphocytes at the infection site. 8 12-O-Tetradecanoylphorbol-13-acetate (TPA) stimulates the activation of a wide variety of intracellular pathways through activation of protein kinase C, mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) followed by the generation of inflammatory mediators including tumour necrosis factor-α, interleukin (IL)-1β, keratinocyte-derived chemokine (KC), macrophage inflammatory protein (MIP)-2 and prostaglandins. 9 Moreover, TPA has been shown to induce a variety of histological changes in mouse (BALB/c) skin, including neutrophil infiltration in both the epidermis and dermis and epidermal hyperplasia. 10,11 In patients with tinea pedis, β-glucan (a component of the dermatophyte cell wall) reportedly binds to the host cell surface receptor dectin-1 and subsequently evokes the production of inflammatory mediators through activation of protein kinase C, MAPK and NF-κB inside the host cells. 12 Infiltration of inflammatory cells, mainly neutrophils, is also observed in the histological examination of skin affected by tinea pedis. These observations suggest that there are similarities in the intracellular mechanism between the inflammatory reaction in tinea pedis and TPA-induced inflammation.
Lanoconazole (LCZ) is an imidazole antifungal agent having potent in vitro antifungal activity against clinical dermatophyte isolates. 13,14 The clinical use of 1% LCZ cream, solution and ointment in Japan has demonstrated its therapeutic usefulness in the treatment of various dermatomycoses including tinea pedis, tinea corporis and cutaneous candidiasis. 15 Because tinea pedis is accompanied by inflammation at the infection site, an antifungal agent containing an anti-inflammatory effect would be therapeutically valuable. Indeed, some antifungal agents reportedly have anti-inflammatory effects. [16][17][18] Although our previous study demonstrated that LCZ suppressed TPA-induced irritant dermatitis in mice, 19 the characteristic features of these anti-inflammatory effects remain to be elucidated.
We also found that LCZ inhibited the production of IL-8, a neutrophil chemotactic factor, from epidermal keratinocytes. 19 Therefore, to characterise the anti-inflammatory effect of LCZ, we investigated whether LCZ affects the production of the neutrophil chemotactic factor and neutrophilic infiltration to the skin in TPA-induced irritant dermatitis in mice and compared its inhibitory potency with that of other antifungal agents. Furthermore, elevated levels of interferon (IFN)-γ mRNA expression and IFN-γ-positive cells in tinea pedis lesions suggest that a T helper cell type 1 (Th1)-driven allergic contact dermatitis is provoked in tinea pedis. 6,7 Therefore, we also compared the inhibitory potency of LCZ on Th1-type contact hypersensitivity induced by 2,4,6-trinitrophenyl chloride (PC) with that of other antifungal agents in mice.

| Animals
Female BALB/c mice aged 7 weeks were purchased from Charles River Laboratories Japan, Inc. All of the animal experimental procedures were approved by the Ethics Committee for Animal Experiments of Maruho Co., Ltd. and conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals at Maruho Co., Ltd.

| TPA-induced irritant dermatitis
TPA-induced irritant dermatitis was induced in mice according to a previously described method. 19 Briefly, 10 µL of 0.01% TPA (20 µL for ear) was applied to the inner and outer surfaces of the right ear of each mouse. Negative control mice received only acetone instead of TPA. Immediately after the TPA application, 20 µL of 0.3%, 1% or 3% LCZ; 1% LNF; 1% TBF; 1% AMO; or vehicle (acetone) was applied to the same ear. The ear thickness was measured 6 hours after the application of TPA using a dial thickness gauge (Series 547-401 customised; Mitutoyo Corporation). Thereafter, 5-mm-diameter punch biopsies were obtained from the skin of the right ear to measure the myeloperoxidase (MPO) activity and inflammatory mediator contents, and 3-mm-diameter punch biopsies were obtained from the skin of the right ear, fixed with 10% neutral formalin and embedded in paraffin. Tissue sections were stained with haematoxylin and eosin for microscopic examination.

| PC-induced contact hypersensitivity
The mice were sensitised and challenged with PC according to a previously described method. 19 Sensitisation was performed by application of 100 µL of 3% PC to their shaved abdomen. Negative control mice received acetone instead of PC. Six days later, the skin of the mice was challenged to elicit a contact hypersensitivity response by the application of 10 µL of 1% PC to the inner and outer surfaces of the right ear. Next, 20 µL of 1% LCZ, 1% LNF, 1% TBF, 1% AMO or vehicle (acetone) was applied to the same ear. The ear thickness was measured 24 hours after the PC application using a dial thickness gauge (Mitutoyo Corporation). Punch biopsies of 5-mm diameter were then obtained from the skin of the right ear to determine the IFN-γ content.

| Measurement of MPO activity and chemokine content in the skin
The ear biopsy was homogenised in lysis buffer (phosphate-buffered saline containing 0.1% Tween-20 and protease inhibitor cocktail) and centrifuged for 10 minutes (10 000 g, 4ºC). The MPO activity and the KC, MIP-2 and IFN-γ contents in the supernatants were measured according to the manufacturer's instructions.

| Statistical analysis
Data are expressed as mean ± standard error of the mean. A significant difference between two groups was evaluated by Student's t test or the Aspin-Welch t test following the F-test. Dunnett's multiple-comparison test was used to analyse differences among three or more groups. A P value of <.05 was considered statistically significant.

| Anti-inflammatory effect of LCZ on TPAinduced irritant dermatitis in mice
We first assessed the anti-inflammatory effect of LCZ on the TPAinduced irritant dermatitis in mice. As shown in Figure 1, topical application of TPA to the mouse ear resulted in an increase in the ear thickness. LCZ dose-dependently reduced the TPA-induced F I G U R E 1 Inhibitory effect of LCZ on ear swelling induced by topical application of TPA in mice. TPA (0.01%, 20 µL) was topically applied to both sides of the right ear. LCZ (0.3%, 1% or 3%) or vehicle (acetone) was topically applied (20 µL/ear) immediately after the treatment with TPA. Negative control mice received acetone instead of TPA. (A) The ear thickness was measured 6 h after the treatment with TPA. Each column represents the mean ± standard error of the mean (n = 6). **P < .01 compared with TPA alone (Dunnett's multiple-comparison test, two-sided); † † P < .01 compared with TPA (−) (Aspin-Welch t test, two-sided). increase in ear thickness, and the inhibitory effect of LCZ at concentrations of 1% and 3% was significantly stronger than that of vehicle ( Figure 1A,B). Additionally, histological examination showed TPAinduced oedema and infiltration of inflammatory cells such as neutrophils and lymphocytes in the dermis with suppression of these phenomena in the LCZ treatment group ( Figure 1C).
Next, we assessed MPO activity in the TPA-applied ear as the index of neutrophil infiltration. We found that topical application of TPA induced an increase in MPO activity in the ear skin and that LCZ dose-dependently suppressed the increase in MPO activity ( Figure 2A). Furthermore, we investigated the changes in the KC and MIP-2 contents in the TPA-induced irritant dermatitis because mouse KC and MIP-2 are reportedly the functional analogues of human IL-8 (a neutrophil chemotactic factor). [20][21][22] As shown in Figure 2B and 2C, the TPA application resulted in an increase in the KC and MIP-2 protein levels in the ear skin and LCZ significantly reduced both the KC and MIP-2 levels in a dose-dependent manner.
We then compared the anti-inflammatory effect of LCZ with that of various antifungal agents in the TPA-induced irritant dermatitis model. As shown in Figure 3A, treatment with LCZ or TBF significantly inhibited the increase in ear thickness induced by TPA.
However, treatment with LNF or AMO showed no inhibition of the increase in ear thickness. The increase in MPO activity induced by TPA was also suppressed by treatment with LCZ or TBF, and treatment with LNF or AMO showed no inhibition of them ( Figure 3B).
Furthermore, the increase in the KC and MIP-2 contents in TPAtreated ear tissue was significantly inhibited by all antifungal agents tested, and the inhibitory effects of LCZ and TBF were stronger than those of the other agents ( Figure 3C,D). We then analysed the relationship between ear thickness and each anti-inflammatory index (MPO activity, KC content and MIP-2 content). A significantly positive correlation with each of these anti-inflammatory indexes was found (r = .74, .63 and .56, respectively).

| Anti-inflammatory effect of LCZ on PCinduced contact dermatitis in mice
We  Ketoconazole, another imidazole antifungal agent, also reportedly inhibits the increase in NF-κB activity in human epidermal keratinocytes stimulated by tumour necrosis factor-α, and its inhibitory effect is dependent on PGE 2 production by keratinocytes. 31 Therefore, based on these reports of the anti-inflammatory effects of sertaconazole and ketoconazole, LCZ may also inhibit the production of KC Furthermore, this study demonstrated the inhibitory effect of LCZ on PC-induced contact dermatitis in mice as previously reported. 19 We also found that LCZ reduced the increase in IFN-γ content in the ear tissue of this dermatitis model. PC-induced contact dermatitis is thought to be a Th1-driven immune response, and IFN-γ produced from T cells is considered to play an important role in the development of dermatitis. 32 In addition, we previously reported that LCZ inhibited IFN-γ production by human peripheral blood mononuclear cells in vitro. 19 These observations suggest that LCZ suppresses PC-induced contact dermatitis in mice by inhibiting IFN-γ production F I G U R E 4 Inhibitory effects of topical antifungal agents on ear swelling of PC-induced contact dermatitis in mice. Mice were sensitised by topical application of PC (3%, 100 µL) to the shaved abdominal skin. Negative control mice received acetone instead of PC. Six days later, PC (1%, 20 µL) was topically applied to both sides of the right ear. An antifungal agent solution (1%, 20 µL) or vehicle (acetone) was applied immediately after the challenge. (A) The ear thickness was measured using a thickness gauge 24 h after the challenge. (B) Punch biopsies (5-mm diameter) were obtained after measuring the ear thickness, and the IFN-γ content in the ear biopsy was measured by enzyme-linked immunosorbent assay. Each column represents the mean ± standard error of the mean (n = 5). **P < .01 compared with PC alone (Student's t test, two-sided); # P < .05, ## P < .01 compared with non-sensitisation (Student's t test, two-sided). AMO, amorolfine; IFN, interferon; LCZ, lanoconazole; LNF, liranaftate; ND, not detected; PC, 2,4,6-trinitrophenyl chloride; TBF, terbinafine from T cells that have infiltrated the skin. Furthermore, whether LCZ can inhibit skin inflammation induced by dermatophytes as well as PC should be examined to further clarify contribution of anti-inflammatory effect of LCZ to relief of the dermatitis symptoms in patients with tinea pedis, and then in vivo examination using dermatophyte-induced skin inflammation in mice will need to be done in the next study.

| D ISCUSS I ON
We also compared the effects of LCZ versus other antifungal agents on PC-induced allergic contact dermatitis in mice. Only LCZ showed inhibitory effects in this dermatitis model, which differs from the results in the TPA-induced dermatitis model. Although the reason for the discrepancy in the effects of other antifungal agents between the two dermatitis models is not clear, the differences in factors involved in the dermatitis model may affect the results.
In conclusion, the present study demonstrated that LCZ sup-

CO N FLI C T O F I NTE R E S T
All authors are employees of Maruho Co., Ltd.