Title: Oral Administration of Chymase Inhibitor Improves Dermatitis in NC/Nga Mice
Abstract: stem cell factor TO THE EDITOR Chymase has been thought to play an important role in the pathogenesis of atopic dermatitis; for example, (i) an intradermal injection of chymase to mouse skin elicits edema as well as inflammatory cell accumulation (Tomimori et al., 2002aTomimori Y. Muto T. Fukami H. Saito K. Horikawa C. Tsuruoka N. et al.Chymase participates in chronic dermatitis by inducing eosinophil infiltration.Lab Invest. 2002; 82: 789-794Crossref PubMed Scopus (51) Google Scholar, Tomimori et al., 2002cTomimori Y. Tsuruoka N. Fukami H. Saito K. Horikawa C. Saito M. et al.Role of mast cell chymase in allergen-induced biphasic skin reaction.Biochem Pharmacol. 2002; 64: 1187Crossref PubMed Scopus (27) Google Scholar), (ii) there are several reports suggesting a correlation between a certain single-nucleotide polymorphism of the chymase gene and incidence of atopic dermatitis (Mao et al., 1996Mao X.Q. Shirakawa T. Yoshikawa T. Yoshikawa K. Kawai M. Sasaki S. et al.Association between genetic variants of mast-cell chymase and eczema.Lancet. 1996; 348: 581-583Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar; Tanaka et al., 1999Tanaka K. Sugiura H. Uehara M. Sato H. Hashimoto-Tamaoki T. Furuyama J. Association between mast cell chymase genotype and atopic eczema: comparison between patients with atopic eczema alone and those with atopic eczema and atopic respiratory disease.Clin Exp Allergy. 1999; 29: 800-803Crossref PubMed Scopus (57) Google Scholar; Weidinger et al., 2005Weidinger S. Rummler L. Klopp N. Wagenpfeil S. Baurecht H.J. Fischer G. et al.Association study of mast cell chymase polymorphisms with atopy.Allergy. 2005; 60: 1256-1261Crossref PubMed Scopus (45) Google Scholar). In fact, a chymase inhibitor SUN C8257 has been shown to improve dermatitis in several animal models (Tomimori et al., 2002aTomimori Y. Muto T. Fukami H. Saito K. Horikawa C. Tsuruoka N. et al.Chymase participates in chronic dermatitis by inducing eosinophil infiltration.Lab Invest. 2002; 82: 789-794Crossref PubMed Scopus (51) Google Scholar, Tomimori et al., 2002cTomimori Y. Tsuruoka N. Fukami H. Saito K. Horikawa C. Saito M. et al.Role of mast cell chymase in allergen-induced biphasic skin reaction.Biochem Pharmacol. 2002; 64: 1187Crossref PubMed Scopus (27) Google Scholar; Watanabe et al., 2002Watanabe N. Tomimori Y. Saito K. Miura K. Wada A. Tsudzuki M. Fukuda Y. Chymase inhibitor improves dermatitis in NC/Nga mice.Int Arch Allergy Immunol. 2002; 128: 229-234Crossref PubMed Scopus (54) Google Scholar). However, the effect of the compound was shown exclusively when administered intraperitoneally (Tomimori et al., 2002aTomimori Y. Muto T. Fukami H. Saito K. Horikawa C. Tsuruoka N. et al.Chymase participates in chronic dermatitis by inducing eosinophil infiltration.Lab Invest. 2002; 82: 789-794Crossref PubMed Scopus (51) Google Scholar, Tomimori et al., 2002cTomimori Y. Tsuruoka N. Fukami H. Saito K. Horikawa C. Saito M. et al.Role of mast cell chymase in allergen-induced biphasic skin reaction.Biochem Pharmacol. 2002; 64: 1187Crossref PubMed Scopus (27) Google Scholar) or given as drinking water (Watanabe et al., 2002Watanabe N. Tomimori Y. Saito K. Miura K. Wada A. Tsudzuki M. Fukuda Y. Chymase inhibitor improves dermatitis in NC/Nga mice.Int Arch Allergy Immunol. 2002; 128: 229-234Crossref PubMed Scopus (54) Google Scholar), as this compound has a very low bioavailability and short half-life in vivo. Recently, we have synthesized a novel chymase inhibitor with an improved profile, SUN13350 (free form of SUN13834). (Hiroshi Maruoka, Tsuyoshi Muto, Taisaku Tanaka, Seiichi Imajo, Yoshiaki Tomimori, Yoshiaki Fukuda, and Takashi Nakatsuka: development of 6-benzyl substituted 4-aminocarbonyl-1,4-diazepane-2,5-diones as orally active human chymase inhibitors, manuscript in preparation). In this letter, we report the effect of SUN13350 on NC/Nga mice which spontaneously develop dermatitis in conventional (nonspecific pathogen-free) conditions (Matsuda et al., 1997Matsuda H. Watanabe N. Geba G.P. Sperl J. Tsudzuki M. Hiroi J. et al.Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC/Nga mice.Int Immunol. 1997; 9: 461-466Crossref PubMed Scopus (652) Google Scholar). To examine the effect of SUN13350 on NC/Nga mice, the compound was administered orally once a day for 3 weeks, starting at 8 weeks old (the medical ethical committee of The Jikei University approved all described studies). As shown in Figure 1a, the clinical scores increased in the control mice with aging and were reduced significantly and completely by the treatment with SUN13350 at 30 mg/kg. The effect of SUN13350 was obvious by macroscopic observation (the photo of the representative mice is shown in Figure 1b). Histological analysis of the skin revealed that administration of SUN13350 significantly inhibited the number of mast cells and eosinophils (Figure 1c and d) compared to control mice. In addition, administration of SUN13350 at 30 mg/kg significantly reduced the serum level of IgE compared to the control mice (Figure 1e). Chymase is known to participate in mast cell accumulation by releasing soluble stem cell factor (SCF) from membrane-bound SCF (Longley et al., 1997Longley B.J. Tyrrell L. Ma Y. Williams D.A. Halaban R. Langley K. et al.Chymase cleavage of stem cell factor yields a bioactive, soluble product.Proc Natl Acad Sci USA. 1997; 94: 9017-9021Crossref PubMed Scopus (161) Google Scholar; Tomimori et al., 2002bTomimori Y. Muto T. Fukami H. Saito K. Horikawa C. Tsuruoka N. et al.Mast cell chymase regulates dermal mast cell number in mice.Biochem Biophys Res Commun. 2002; 290: 1478-1482Crossref PubMed Scopus (26) Google Scholar). To understand the mechanism of inhibition of mast cell accumulation in NC/Nga mice, the effect of SUN13350 was examined on the chymase-induced processing of SCF in vitro. Recombinant human SCF with 165 amino-acid residues (SCF165) was detected as a single and sharp band with an apparent molecular weight of approximately 21 kDa in SDS-PAGE (Figure 2a). Incubation of human SCF165 with human chymase resulted in the production of a shorter species (∼20 kDa) of the protein, consistent with the data of the previous report of Longley et al., 1997Longley B.J. Tyrrell L. Ma Y. Williams D.A. Halaban R. Langley K. et al.Chymase cleavage of stem cell factor yields a bioactive, soluble product.Proc Natl Acad Sci USA. 1997; 94: 9017-9021Crossref PubMed Scopus (161) Google Scholar, who showed that chymase cleaves membrane-bound SCF at a site close to the membrane to yield a soluble bioactive form. SUN13350 inhibited the production of the shorter species of SCF (Figure 2a), with a complete inhibition at more than 0.5 μM. This result demonstrates that SUN13350 inhibits the chymase-induced processing of membrane-bound SCF. Similar results were obtained using mouse recombinant SCF164 and mouse chymase mMCP-4 (mouse mast cell protease-4), whereas SUN13350 at 0.5 μM showed partial effects (Figure 2a). These data suggest that the reduction of mast cell accumulation in NC/Nga mice by SUN13350 may be mediated by inhibition of the release of soluble SCF. Chymase is also known as a chemoattractant for various inflammatory cells including eosinophils (He and Walls, 1998He S. Walls A.F. Human mast cell chymase induces the accumulation of neutrophils, eosinophils and other inflammatory cells in vivo.Br J Pharmacol. 1998; 125: 1491-1500Crossref PubMed Scopus (130) Google Scholar; Tani et al., 2000Tani K. Ogushi F. Kido H. Kawano T. Kunori Y. Kamimura T. et al.Chymase is a potent chemoattractant for human monocytes and neutrophils.J Leukoc Biol. 2000; 67: 585-589PubMed Google Scholar). Therefore, the effect of SUN13350 on chymase-induced eosinophil migration was examined by chemotaxis analysis in vitro. As shown in Figure 2b, human chymase induced migration of human eosinophils, in agreement with the previous report (Tani et al., 2000Tani K. Ogushi F. Kido H. Kawano T. Kunori Y. Kamimura T. et al.Chymase is a potent chemoattractant for human monocytes and neutrophils.J Leukoc Biol. 2000; 67: 585-589PubMed Google Scholar). SUN13350 significantly inhibited the chymase-induced cell migration at more than 1.0 μM, and complete inhibition was observed at 100 μM. Similarly, mouse chymase mMCP-4 induced migration of mouse eosinophils isolated from peritonitis mice, and SUN13350 inhibited mMCP-4-induced chemotaxis (Figure 2c). These results suggest that eosinophil infiltration to the skin observed in NC/Nga mice may be mediated at least in part by chymase, and that the inhibition of eosinophil accumulation by SUN13350 may be due to the inhibition of chymase-induced eosinophil migration. Administration of SUN13350 reduced the serum level of IgE in NC/Nga mice (Figure 1e), which may be the result of the improvement of the dermatitis. However, chymase is known to stimulate IgE production from mouse B cells in vitro (Yoshikawa et al., 2001Yoshikawa T. Imada T. Nakakubo H. Nakamura N. Naito K. Rat mast cell protease-I enhances immunoglobulin E production by mouse B cells stimulated with interleukin-4.Immunology. 2001; 104: 333-340Crossref PubMed Scopus (25) Google Scholar). Thus it is also possible that SUN13350 inhibits IgE production in NC/Nga mice as well and in turn ameliorates dermatitis. In conclusion, it was shown that a novel chymase inhibitor SUN13350 ameliorates dermatitis in NC/Nga mice when administered orally, and that inhibition of SCF-processing and eosinophil infiltration might be involved in the effect of the compound. Most of the existing therapies for atopic dermatitis (such as corticosteroids and immunosuppressants) are ointments or creams, as they exert adverse effects when administered systemically. SUN13350 was shown to inhibit the processing of human SCF as well as chemotaxis of human eosinophils, and therefore is thought to be promising as a novel oral agent for human atopic dermatitis. The authors state no conflict of interest. Materials and Methods References