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Background C3H/HeJ mouse models progress gradually in hair loss from acute to chronic phase and reflect the symptoms of patients with alopecia areata (AA). However, the underlying pathological characteristics alteration associated with disease progression and autoantigens remain unclear. Objective We aimed at elucidating the pathological differences between acute and chronic-AA in the C3H/HeJ mouse model. Methods We analyzed populations of PBMCs, skin-draining lymph node (SDLN) cells, and cutaneous cells of AA mice using flow cytometry. The cytokine and chemokine expressions in the serum and skin were determined using multiplex assay and qPCR. The antibody serum levels were determined using ELISA and the antigen-specific T cells were detected using the MHC class I tetramer. Results The CD8+NKG2D+ T and CD8+ TEM cell percentage in the chronic-AA SDLNs or among the unaffected and acute-AA mice PBMCs increased. The Th1 and CD4+ TEM cell percentage in the SDLNs and among PBMCs increased in the unaffected and AA mice. The percentage of CD8+ TEM/TRM cells and MHC class I expression increased in the lesions of acute-AA or the non-lesions and lesions of chronic-AA. The Th1 cells, dendritic cell-related cytokines, CD11c+ cells and MHC class II expression increased in the skin of AA mice. The antibody levels and TYRP2 and tyrosinase-specific CD8+ T cell percentages were upregulated in AA mice. Conclusion These results suggest that the CD8+ and CD4+ T cell subpopulations, cytokine and chemokine expressions differ between the disease phases. Moreover, TYRP2 and tyrosinase are potential autoreactive targets in the AA mouse model.

Background Mucopolysaccharide polysulfate (MPS) is a heparinoid and MPS-containing formulations are widely used as moisturizers for dry skin and to treat peripheral vascular insufficiency. Although MPS has therapeutic effects in skin diseases with microvascular abnormalities, the effects of MPS on microvascular function remain incompletely understood. Objective The aim of this study was to evaluate the functional activities of MPS on human pericytes (HPC) and human dermal microvascular endothelial cells (HDMEC) in vitro, and on microvascular permeability of the skin. Methods The protein expression of angiopoietin (Ang)-1 in HPC, and platelet-derived growth factor-BB (PDGF-BB) and phosphorylated tyrosine-protein kinase receptor 2 (Tie2) in HDMEC were measured in the presence or absence of MPS. The vascular barrier was evaluated by the expressions of claudin-5 and vascular endothelial (VE)-cadherin, and transendothelial electrical resistance (TEER). Results In HPC, MPS dose-dependently enhanced Ang-1 secretion, which activated Tie2 in HDMEC. In HDMEC, MPS significantly increased the production of PDGF-BB, which is important for the recruitment of HPC to the vascular endothelium, and significantly increased the phosphorylation of Tie2, which results in the activation of the Ang-1/Tie2 signaling . MPS significantly increased the expression of tight junction protein claudin-5 and TEER in the HDMEC. Moreover, the intradermal injection of MPS prevented vascular endothelial growth factor-induced increase in vascular permeability in mouse skin. Conclusion We found that MPS promoted microvascular stabilization and barrier integrity in HDMEC via Ang-1/Tie2 activation. These results suggest that MPS might improve microvascular abnormalities in various diseases accompanied by disturbances in Ang-1/Tie2 signaling.

Background Alopecia areata (AA) is an autoimmune disease resulting in non-scarring hair loss. Animal models are useful means to identify candidates for therapeutic agents. The C3H/HeJ mouse AA model induced via transferring cultured lymphoid cells isolated from AA-affected mice is widely used for AA research. However, this conventional method requires the continuous breeding of AA mice. Objective We aimed to establish a new method to generate AA model using the transfer of cryopreserved cells, which allows the rapid induction of a large number of AA mice when needed. Methods We cryopreserved lymph node cells soon after isolation from AA-affected mice and injected thawed-cultured cells into recipient mice. H&E staining, immunohistochemical staining, quantitative real-time PCR and ELISA were conducted to identify pathological characteristics. Flow cytometry was performed to reveal the profile of transferred cells. Results More than 90 % of recipient mice developed AA-like hair loss and showed inflammatory cell infiltration around anagen hair follicles, markedly increased mRNA expressions of interferon-γ, CXCL11, and granzyme B, and elevated interferon-α protein levels in the skin compared with naive mice. Higher percentages of effector memory T cells and dendritic cells in transferred cells resulted in a higher incidence of AA. Conclusion This is the first report to establish a method for generating AA mice using cryopreserved lymphocytes. These AA mice have similar pathological characteristics to AA mice generated with the conventional method and AA patients. This convenient and reproducible method is expected to be valuable for AA study.

Ozenoxacin is a topical quinolone showing potent antimicrobial activities against Gram-negative and Gram-positive bacteria and is widely used for the treatment of inflammatory acne. However, the anti-inflammatory activities of ozenoxacin have not been examined so far. In the present study, we investigated the in vitro and in vivo anti-inflammatory effects of ozenoxacin. The production of interleukin (IL)-6 and IL-8 by human epidermal keratinocytes stimulated by heat-killed Cutibacterium acnes was significantly inhibited by ozenoxacin at concentrations from 1 to 30 μg ml−1. Likewise, the production of IL-6, IL-8, and tumor necrosis factor alpha by stimulated THP-1 cells, a human monocyte cell line, was inhibited by ozenoxacin at concentrations from 1 to 30 μg ml−1. The production of IL-1β by THP-1 was also inhibited by ozenoxacin at the concentration of 30 μg ml−1. Phosphorylation of the mitogen-activated protein kinases and degradation of IκB-α, an inhibitory factor of NF-κB in keratinocytes and THP-1 cells, was increased by stimulation with heat-killed C. acnes. Of these activated intracellular pathways, the p38 phosphorylation pathway was remarkably reduced by ozenoxacin in both keratinocytes and THP-1 cells. In addition, the application of 2% ozenoxacin suppressed the increase in the ear thickness of rats induced by an intracutaneous injection of heat-killed C. acnes. These findings suggest that ozenoxacin possesses an anti-inflammatory activity, which may contribute to its therapeutic effects on inflammatory acne.

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.

Alopecia areata (AA) is an autoimmune non-scarring hair loss disease. Recently, several reports have suggested that innate immune systems such as interferon-α (IFN-α)-producing plasmacytoid dendritic cells and NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasomes play a role in the pathogenesis of AA. However, critical studies about their involvement in the initiation of AA have not yet been reported. Therefore, we investigated the expression of innate immune cytokines in serum and skin, and examined the effect of a selective NLRP3 inhibitor, MCC950, on AA in C3H/HeJ mice, induced by transferring cultured skin-draining lymph node cells. IFN-α production was upregulated in lesions of AA-affected mice, and interleukin-1β in serum and skin was highly expressed before onset as well as postonset. Furthermore, MCC950 treatment prevented AA development and promoted hair growth in AA mouse models by reducing NLRP3 signalling and Th1/Tc1 chemokines and cytokines in the skin. These results suggest that NLRP3 inflammasome contributes to AA onset and chronicity, and NLRP3 inhibitor may be a potential therapeutic agent for AA.

Background Amenamevir (AMNV) is a helicase-primase inhibitor with antiviral activity against herpesviruses [herpes simplex viruses (HSV)-1 and -2, and varicella-zoster virus], which are associated with the development of acute herpetic pain (AHP) and postherpetic neuralgia. However, the inhibitory effects of helicase-primase inhibitors on AHP and postherpetic neuralgia remain incompletely understood. Objective In this study, we investigated the effects of AMNV on AHP and postherpetic pain (PHP) in HSV-1-infected mice accompanied by zosteriform-like skin lesions. Methods HSV-1 was percutaneously infected on the femoral region of mice. AMNV was orally administered twice a day for 5 days. Pain-related response in the hind paw was evaluated using a paintbrush. The infiltration of inflammatory cells in dorsal root ganglion (DRG) and spinal cord (SC) was evaluated by hematoxylin and eosin staining. The viral load in DRG and the expression of pain-related genes in SC were measured by real-time PCR. Results Pain response was begun to be observed from day 3 post-infection (pi) in HSV-1-infected mice. AMNV administered repeatedly from day 3 pi or day 4 pi, but not day 5 pi, showed an inhibitory effect on the development of AHP and the transition to PHP. Repeated AMNV administration inhibited inflammatory cell infiltration and increases in the viral load and the expression of pain-related genes (ATF-3, TNF-α, COX-2). Conclusion These results demonstrate that AMNV potently suppresses the development of AHP and the transition to PHP as a consequence of decreased viral load in DRG and reduced expression of pain-related genes in SC.

When selecting external medicines for the treatment of skin diseases, it is thought to be very important to consider differences in characteristics of their bases, because the bases may influence the clinical efficacy of the medicines. In this study, we investigated whether the differences in characteristics of three kinds of bases, white petrolatum, macrogol ointment, and aqueous gel affect wound healing. In vitro moisture permeability tests demonstrated that these bases have different characteristics in coatability and water retentivity, with the rank order of the intensity of coatability as white petrolatum>macrogol ointment>aqueous gel, and that of water retentivity as macrogol ointment>white petrolatum>aqueous gel. Similar rank order of these bases was observed for transepidermal water loss and stratum corneum water content in the dry skin on the abdomen of guinea pigs induced by topical application of acetone/ether mixture, followed by water. In addition, we found that treatment with macrogol ointment, but not white petrolatum or aqueous gel, significantly accelerated wound healing in rat skin, and that the contents of basic fibroblast growth factor and epidermal growth factor in the skin treated with macrogol ointment were significantly higher compared with non-treated skin. In conclusion, these results imply an important role of the bases of external medicines in the treatment of skin diseases.

Atopic dermatitis (AD) is a chronic inflammatory skin disease accompanied by severe itching and eczematous lesion. In this study, we applied an ointment containing Dermatophagoides farinae body (Dfb) extract repeatedly on the dorsal skin of NC/Nga mice with barrier disruption to investigate the characteristics of this murine model of human AD. Following repeated topical application of Dfb ointment twice weekly for 2 weeks, the dermatitis score increased gradually, accompanied by an elevation of total immunoglobulin E level in plasma. Topical application of Dfb ointment also caused epidermal hyperplasia and accumulation of inflammatory cells in the lesional skin and increased expression of T-helper (Th) 1/Th2/Th17 cytokines in axillary lymph node cells. Furthermore, increased sprouting of intraepidermal nerve fibres was observed with an increase in the content of nerve growth factor and decrease in that of semaphorin 3A in the lesional skin. These findings suggest that the characteristics in this model were similar to those observed in patients with AD. Interestingly, it was observed for the first time that scratching behaviour increased in a biphasic fashion by topical application of Dfb ointment in addition to an increase in spontaneous scratching behaviour in this model. It is also suggested that further clarifying the underlying mechanisms of scratching behaviour in this model leads not only to elucidating the pathogenesis of AD but also to discovering novel therapeutic drugs for AD.

Atopic dermatitis (AD)-like dermatitis can be induced by repeated topical application of an ointment containing Dermatophagoides farinae body (Dfb) extract in NC/Nga mice. This AD-like murine model also exhibits a biphasic increase in the number of scratching behaviour after topical application of Dfb ointment. In this study, we investigated the possible mechanisms underlying the scratching behaviour in each phase. An increase in the content of mast cell-derived mediators such as histamine and 5-hydroxytryptamine in the lesional skin and increased vascular permeability were observed in the early phase after the Dfb ointment application. Chlorpheniramine (H1 receptor antagonist) and cromoglycate (mast cell stabilizer) reduced the scratching behaviour in the early phase but not that in the later phase. Furthermore, the content of various endogenous pruritogens such as interleukin-31 and thymic stromal lymphopoietin in the lesional skin was increased 1 or 24 hours after the Dfb ointment application. Elevated expression of proteinase-activated receptor-2 (PAR-2) was also observed in the epidermis. Finally, gabexate (serine protease inhibitor) reduced the scratching behaviour in both phases, and anti-PAR2 antibody also showed a tendency to reduce both scratching behaviours. These findings suggest that immediate-type allergic reactions caused by mast cell degranulation and PAR-2 activation by proteases are involved in the scratching behaviour in this AD-like model.

Background: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by skin barrier dysfunction and abnormal immune response. House dust mites (HDM) are a major source of allergens, some of which have cysteine and serine protease activities. Keratinocytes stimulated by HDM-derived proteases have been suggested to contribute to the pathogenesis of AD by producing various cytokines. However, whether keratinocytes contribute to the induction of pruritus in AD, especially by producing pruritus-related mediators upon stimulation with HDM-derived proteases, has not been fully elucidated. Methods: We examined whether the production of endothelin-1 (ET-1), matrix metalloproteinase (MMP)-2, and MMP-9 in keratinocytes can be induced by stimulation with Dermatophagoides farinae extracts, and if so, whether pretreatment with a protease inhibitor or proteinase-activated receptor-2 (PAR-2) antagonist affects the production of these mediators in keratinocytes. Results: Although MMP-2 levels were undetectable in the culture supernatants, the production of ET-1 and MMP-9 was increased upon stimulation with HDM extracts in a concentration- and time-dependent manner and suppressed by pretreatment of HDM extracts with serine protease inhibitor, but not with cysteine protease inhibitor. Mite-derived serine proteases also induced ET-1 and MMP-9 production in a concentration- and time-dependent manner. Moreover, pretreatment with a PAR-2 antagonist inhibited the production of ET-1 and MMP-9 in keratinocytes. Conclusion: These results suggest that the activation of PAR-2 on keratinocytes by HDM-derived serine proteases induces the production of ET-1 and MMP-9, and may contribute to the induction of pruritus in AD.

1. TAK-242 is a novel compound that suppresses nitric oxide and cytokine production by selectively inhibiting intracellular signals from toll-like receptor (TLR)-4. In the present study, we investigated the effectiveness of TAK-242 against sepsis using an endotoxaemia model in conscious and unrestricted guinea-pigs. Measures examined included muscle tension paralysis of the intestine, blood pressure, high morbidity group box (HMGB)-1 levels and survival rate. 2. Tension of the longitudinal muscle of the colon was monitored continuously by telemetry. Arterial blood pressure was monitored via a carotid artery catheter. TAK-242 was administered intravenously through a jugular vein catheter. Guinea-pigs were divided into a control group, given vehicle (placebo emulsion), and the experimental group, administered 3 or 10 mg/kg TAK-242, 1 h before administration of 10 mg/kg lipopolysaccharide (LPS). 3. In the control group, the tension of the longitudinal muscle of the colon decreased in a time-dependent manner and blood pressure was reduced, with maximal effects observed 1-3 h after administration of LPS. In the TAK-242-treated group, LPS-induced relaxation of the intestine and hypotension were significantly inhibited. In the control group, HMGB-1 levels were increased after LPS administration and this reaction was significantly blocked in the TAK-242-treated group. Importantly, survival rate was increased after TAK-242 treatment. 4. In conlusion, the results of the present study show that TAK-242 inhibited the symptoms associated with endotoxaemia in a guinea-pig model of sepsis and that it may, therefore, be an effective treatment for sepsis.

Topical antifungal agents which have anti-inflammatory effects have the potential to provide additional clinical benefits. Therefore, an anti-inflammatory activity of lanoconazole (LCZ), a topical antifungal agent, was investigated against in vitro and in vivo models of inflammation. The release of interleukin-8 (IL-8) from human epidermal keratinocytes stimulated by the addition of 100 μg ml(-1) β-glucan of Saccharomyces cerevisiae was significantly inhibited by LCZ at the concentration of 10(-5) mol l(-1). The release of interferon-γ and IL-2 from human peripheral blood mononuclear cells stimulated by the addition of 30 and 100 μg ml(-1) phytohemagglutinin was significantly inhibited by LCZ at the concentrations of 10(-7) and 10(-6) mol l(-1), respectively. The increase in the ear thickness induced by topical application of 0.01% 12-O-tetradecanoyl phorbol-13-acetate and 1% 2,4,6-trinitrochlorobenzene (TNCB) after sensitisation with 3% TNCB were established as the mouse models of irritant and contact dermatitis, respectively. Application of 1% and 3% LCZ showed a significant anti-inflammatory activity against both the irritant and contact dermatitis models. These findings suggest that LCZ possesses an anti-inflammatory activity, which may be partially helpful in the treatment of dermatomycoses.

We were interested in RCS (rabbit aorta contracting substance) and SRS-A (slow reacting substance of anaphylaxis) and their involvement in human bronchial asthma. When we started our anti-asthmatic drug research in the 1970's. We synthesized a lot of chemical compounds and eventually discovered that AA-861 inhibited the generation of SRS-A from the lung tissue of actively sensitized guinea pigs. AA-861 was found to be a potent 5-lipoxygenase inhibitor. This compound reduced experimental allergic asthma in guinea pigs, but it is easily metabolized in the body. More recently, we found a novel compound, AA-2414 (seratrodast), which is not metabolized in the body. AA-2414 proved to be not a 5-lipoxygenase inhibitor, but a thromboxane A2 (TXA2) receptor antagonist. Seratrodast is the first receptor antagonist that is being developed as an anti-asthmatic drug. Seratrodast inhibits both immediate-, late asthmatic responses in guinea pigs, and also reduces airway hyperresponsiveness in dogs. The anti-asthmatic action of seratrodast in animal models indicates that the drug should be of use in the treatment of human asthmatics. In clinical studies, seratrodast showed a marked effect to improve clinical parameters in bronchial asthma. It is also reported that seratrodast is free from harmful aftereffects. Clinical trials are under way in the US.

To investigate the potency of an adenosine A3 receptor (A3AR) antagonist as an anti-asthmatic drug, a novel series of 4-phenyl-5-pyridyl-1,3-thiazole derivatives was synthesized and evaluated in human adenosine A1, A2A and A3 receptor and rat adenosine A3 receptor binding assays. From investigation of the SAR study, compound 7af was identified as a highly potent human and rat A3AR antagonist. This compound inhibited IB-MECA-induced plasma protein extravasation in the skin of rats and showed good oral absorption. Also, compound 7af significantly inhibited antigen-induced hyper-responsiveness to acetylcholine in actively sensitized Brown Norway rats. These results show that 4-phenyl-5-pyridyl-1,3-thiazole derivatives are potential candidates to enable the evaluation of A3AR antagonists. Further evaluation of this class of compounds may afford a novel anti-inflammatory agent such as an anti-asthmatic drug.

The human thromboxane A2 receptor (TXA2-R)-coding gene was introduced into Chinese hamster ovary cells and a cell line (TCHO-25) stably expressing TXA2-R, at a level of 3 x 10(5)/cell, was obtained. An anti-asthmatic agent AA-2414 [(+-)-7-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-7- phenylheptanoic acid] competitively inhibited the specific binding of a TXA2 mimic ([3H]U-46619) to the TCHO-25 cells, with an IC50 of 6.0 x 10(-8) M, indicating that the drug is an antagonist of human TXA2-R. The TCHO-25 cells offer a tool for the screening and characterization of human TXA2-R antagonists.

We studied changes in airway responsiveness to acetylcholine (ACh) after antigen inhalation in Ascaris suum (A. suum)-allergic dogs. Airway responsiveness was determined by obtaining a dose-response curve of lung resistance plotted against increasing concentrations of ACh aerosol before and after inhalation of A. suum antigen. To determine the role of thromboxane A2 (TXA2) in the airway response, we tested the effect of a TXA2 receptor antagonist, AA-2414, in A. suum-allergic dogs. The procedure was repeated in each dog at an interval of 2 weeks to evaluate the effect of AA-2414 in a crossover manner. The dogs showing an airway response to antigen showed an increase in airway responsiveness to ACh 2, 4 and 6 h after antigen inhalation. The increase in airway responsiveness was significantly inhibited by administration of AA-2414 (5 mg/kg, i.v.) before antigen inhalation. These results suggest that TXA2 may be involved in antigen-induced airway hyperresponsiveness (AHR) in dogs.

2,3,5-Trimethyl-6-(3-pyridylmethyl)-1,4-benzoquinone (CV-6504) has inhibitory activities on both thromboxane A2 synthase and 5-lipoxygenase as well as scavenging activity against active oxygen species. The latter two activities are closely related to the quinone moiety, which is reduced to a hydroquinone in the living body. Comparison of these two activities for both the quinone and hydroquinone showed that the hydroquinone form had superior activities. Concerning the reduction mechanism by PB-3c cells we can see that superoxide dismutase (SOD) has no influence on the rate of reduction, but dicumarol almost completely inhibits the reduction at a concentration greater than 1 x 10(-6) M. Therefore, it can be concluded that CV-6504 is reduced mainly by two electron donating enzymes without the intermediary of a semiquinone radical and that the resulting hydroquinone inhibits lipid peroxidation as well as 5-lipoxygenase activity.

The anti-asthmatic activity of AA-2414 [(±)-7-(3,5,6-trimethyl-1,4-benzoquinon-2-yl-7-phenylheptanoic acid)] has been studied in vivo and in vitro . Experimental allergic asthma was inhibited by orally administered AA-2414 in a dose-dependent manner. AA-2414, 0.08∼1.25 mg/kg (p.o.), inhibited the bronchoconstriction in guinea pigs induced by a prostaglandin endoperoxide analogue (U-46619), leukotriene D 4 (LTD 4 ), and platelet activitating factor (PAF) with a long duration of action. The compound did not inhibit histamine-induced bronchoconstriction. AA-2414 reduced the induction of pulmonary inflation caused by LTD 4 aerosol inhalation. AA-2414 competitively inhibited the contractile response to U-46619 in guinea pig tracheal and parenchymal strips and dog saphenous vein strips with pA2 values of 7.69, 8.29 and 6.79, respectively. Furthermore, the contractile responses of guinea pig tracheal strip to PGD 2 , 9 a , 11s-PGF 2 and PGF 2 a were inhibited with pA2 values of 7.20, 7.79 and 5.71, respectively. These results suggest that AA-2414, a quinone derivative, is a novel, potent and orally active antagonist of a variety of spasmogenic prostanoids.

Background Refractory chronic pain dramatically reduces the quality of life of patients. Existing drugs cannot fully achieve effective chronic pain control because of their lower efficacy and/or accompanying side effects. Voltage-gated potassium channels (KCNQ) openers have demonstrated their analgesic effect in preclinical and clinical studies, and are thus considered to be a potential therapeutic target as analgesics. However, these drugs exhibit a narrow therapeutic window due to their imposed central nerve system (CNS) side effects. To clarify the analgesic effect by peripheral KCNQ channel activation, we investigated whether the analgesic effect of the KCNQ channel opener, retigabine, is inhibited by intracerebroventricular (i.c.v.) administration of the KCNQ channel blocker, 10, 10-bis (4-Pyridinylmethyl)-9(10H) -anthracenone dihydrochloride (XE-991) in rats. Results Oral administration (p.o.) of retigabine showed an anticonvulsant effect on maximal electronic seizures and an analgesic effect on complete Freund’s adjuvant-induced thermal hyperalgesia. However, impaired motor coordination and reduced exploratory behavior were also observed at the analgesic doses of retigabine. Administration (i.c.v.) of XE-991 reversed the retigabine-induced anticonvulsant effect, impaired motor coordination and reduced exploratory behavior but not the analgesic effect. Moreover, intraplantar administration of retigabine or an additional KCNQ channel opener, N-(6-Chloro-pyridin-3-yl)-3,4-difluoro-benzamide (ICA-27243), inhibited formalin-induced nociceptive behavior. Conclusions Our findings suggest that the peripheral sensory neuron is the main target for KCNQ channel openers to induce analgesia. Therefore, peripheral KCNQ channel openers that do not penetrate the CNS may be suitable analgesic drugs as they would prevent CNS side effects.