Gene Therapy (2004) 11, 317–324 & 2004 Nature Publishing Group All rights reserved 0969-7128/04 $25.00 www.nature.com/gt RESEARCH ARTICLE Improvement of dermatitis by iontophoretically delivered antisense oligonucleotides for interleukin-10 in NC/Nga mice T Sakamoto1, E Miyazaki1, Y Aramaki1, H Arima1,3, M Takahashi1, Y Kato2, M Koga2 and S Tsuchiya1 1School of Pharmacy, Tokyo University of Pharmacy and Life Science, Horinouchi, Hachioji, Tokyo, Japan; and 2Department of Dermatology, Tokyo Medical University, Shinjuku, Shinjuku-ku, Tokyo, Japan IL-10 is overexpressed in skin lesions of atopic dermatitis distributed in the epidermis and upper dermis. Topically (AD) patients and believed to be an important factor in the delivered AS6 decreased the levels of mRNA and protein of pathogenesis of the disease. Thus the regulation of IL-10 IL-10 in the lesions of NC/Nga mice, with no effect on IL-4 production is a potential solution for immunotherapeutic levels. The dorsal lesions of NC/Nga mice disappeared with intervention in AD. We examined the topical delivery of an repeated topical application of AS6. Topically delivered AS6 antisense oligonucleotide for mouse IL-10 (AS6) and the showed an inhibitory effect on the production of IL-10 in the therapeutic effect on the skin lesions of NC/Nga mice, a skin lesions of NC/Nga mice and had a therapeutic effect on human AD model. Using an iontophoresis system, about the established dermatitis. 30% of the applied dose of AS6 penetrated the skin and was Gene Therapy (2004) 11, 317–324. doi:10.1038/sj.gt.3302171 Keywords: interleukin-10; antisense oligonucleotides; atopic dermatitis; NC/Nga mice; topical Introduction factor in the pathogenesis of AD.12,13 Thus, IL-10 is an important factor in the pathogenesis of AD, and the Atopic dermatitis (AD) is a chronically relapsing regulation of its production is a potential solution for inflammatory disorder with pruritic and eczematous immunotherapeutic intervention. skin lesions usually associated with elevated serum IgE NC/Nga mice are an inbred strain established from levels.1,2 The skin lesions of AD patients are character- Japanese fancy mice in 1957 by Kondo (Nagoya ized by the presence of an inflammatory infiltrate University, Nagoya, Japan). Matsuda et al14 proposed consisting of T lymphocytes, monocytes/macrophages, that the NC/Nga mouse would make an excellent eosinophils, and mast cells.3,4 These cells are involved in animal model for human AD. When kept in specific the pathogenesis and development of AD through the pathogen-free (SPF) conditions,14–16 the mice remain release of various cytokines and chemokines including healthy. But when kept under conventional conditions, IL-4, IL-5, IL-10, IL-13,5 GM-CSF,6 and RANTES (regu- they started to scratch themselves at about 8 weeks, at lated upon activation, normal T expressed and secreted).7 which time their skin became dry and scaly. Within the Of these cytokines/chemokines, IL-10 is overexpressed next several weeks, the mice developed lesions on the by monocytes/macrophages and the Th2 lymphocytes in ears, back, neck, and face. The lesions on the back spread the skin lesions of AD patients.8 In addition, IL-10 from the ears down to the middle of the back and augments the production of thymus and activation reached the posterior axillary line. Immunohistochemical regulated chemokine (TARC), which is a Th2-specific examination of the lesions in conventional NC/Nga mice chemokine and is known for overexpression in AD reveal hyperkeratosis, acanthosis, and parakeratosis, all lesions9 and in human keratinocytes treated by TNF-a of which resemble the typical features of the skin and IFN-g.10 Furthermore, as IL-10 inhibits the apoptosis observed in patients with AD.9,15 The lesions show of T cells activated by ligation of T-cell receptors and IL-2 lymphocyte infiltration with a high CD4/CD8 ratio, treatment,11 IL-10 may be involved in the dysregulated macrophage infiltration, and mast cell and eosinophil apoptosis of T cells in the skin lesions of AD. This degranulation. In addition, the level of IgE in the blood dysregulated apoptosis is considered to be an important gradually increases to high levels.2 Although these immunological, histological, and biochemical changes of NC/Nga mice kept in conventional conditions Correspondence: S Tsuchiya, School of Pharmacy, Tokyo University of resemble human AD,14 the mechanisms are not fully Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, clear yet. Japan 3Present address: Graduate School of Pharmaceutical Sciences, Kumamoto Antisense oligonucleotides (AS-ODNs) designed to University, 5-1, Oe-honmachi, Kumamoto, Kumamoto 862-0973, Japan hybridize specifically to heterogenous nuclear RNA or Received 07 February 2003; accepted 19 September 2003 mature mRNA sequences have been shown to inhibit Antisense oligonucleotides for IL-10 alleviate dermatitis T Sakamoto et al 318 the expression of numerous genes both in vitro and Distribution and stability of AS6 in vivo.17,18 We have reported that AS6, the 18-mer We evaluated the ability of an iontophoresis system to phosphorothioate AS-ODN directed against the se- deliver AS6 into conventional NC/Nga mouse skin at 12 quence in the 30-untranslated region of mouse IL-10 weeks of age in vivo. On topical application of 1 mCi of mRNA, powerfully inhibited the production of IL-10 in [32P]-AS6 with iontophoresis, approximately 30% of the RAW264.7, a mouse macrophage-like cell line, by an dose was observed in the skin under the cathodal gel, antisense mechanism.19 In addition, Ohmen et al8 while about 35% remained in the cathodal gel (Figure revealed that the IL-10-producing cells were CD14 þ 2a). Only a negligible level of radioactivity was observed monocytes localized at the dermal perivascular infiltrate in the anodal gel, the skin under it, and other tissues. On in human AD lesions. Therefore, AS6 may have potential the other hand, no radioactivity of [32P]-AS6 was in the treatment of AD if it can be delivered to the site of observed in the skin under the cathodal gel when current the skin where IL-10-producing cells are located. How- was not applied (data not shown). ever, AS-ODNs have difficultly permeating the skin To evaluate the localization of AS6 in the skin, skin through the stratum corneum due to a lower lipophilicity was cross-sectioned after iontophoresis of AS6 labeled and a relatively high molecular weight. To date, with rhodamine (Rho) at the 50 end (Rho-AS6). Fluores- iontophoresis is known as a method of positively cence of Rho was observed at the epidermis and upper delivering ionized drugs, which enables the transdermal dermis, and iontophoresis allowed Rho-AS6 to penetrate administration of compounds with large molecular the stratum corneum although Rho-AS6 remained in the weights.20,21 Accordingly, the method is now attracting stratum corneum (Figure 2b, IP ( þ )). On the other hand, attention as a noninvasive method, and allows AS-ODNs no fluorescence was observed in the stratum corneum, to penetrate the stratum corneum and be distributed in when no current was applied (Figure 2b, IP (À)). the skin.22 Thus, we expect that iontophoresis can deliver To check the stability of AS6 in the skin of NC/Nga AS6 to regions where target cells are producing IL-10 in mice, AS6 was extracted from the skin following the lesions of AD. In the present study, we evaluated the iontophoresis and subjected to gel electrophoresis. In therapeutic effects of topically delivered AS6 on derma- Figure 2c, radioactivity gave a single band at the same titis on the dorsal skin of NC/Nga mice, as a human AD mobility as a marker, [32P]-AS6 (18 mer) (lane 1). The model. Results Cytokine and IgE levels in the skin lesion of NC/Nga mice NC/Nga mice have been reported to develop eczema- tous skin lesions when kept in conventional surround- ings14–16 but cytokine levels in the lesions of NC/Nga mice are not reported. IL-10, IL-4, and IgE levels in the lesions were determined in mice kept under conven- tional and SPF conditions for different feeding periods. IL-10 and IL-4 productions increased after 8 weeks of age (Figure 1). In addition, the IgE level in the skin of conventional mice at 12 weeks of age was much higher than that of mice kept under SPF conditions (Figure 1). Figure 2 Distribution and stability of topically delivered AS6 in NC/Nga mice. [32P]-AS6 was applied at 20 nmol including 1 mCi in the cathodal gel. Iontophoresis was carried out under conventional conditions (10 V, 0.3 mA, 2.5 h) in NC/Nga mice. (a) Radioactivity recovered after topical application of [32P]-AS6 with iontophoresis. Others included blood, liver, spleen, and kidney. Each value represents the mean7s.e. for six mice in each group. (b) Cross-sections showing the distribution of Rho-AS6 in NC/ Nga mouse skin. Rho-AS6 was applied at 20 nmol in the cathodal gel. Fluorescent images were obtained immediately after application using confocal laser scanning microscopy. IP ( þ ), iontophoresis was carried out; IP (À), iontophoresis was not carried out. Similar results were obtained in three separate experiments. (c) The stability of [32P]-AS6 in the skin of NC/ Figure 1 Determination of IL-10, IL-4, and IgE productions in the skin Nga mice. Lane 1, mixture of intact 18 mer [32P]-AS6 and [g-32P]-ATP; lesions of NC/Nga mice. IL-10, IL-4, and IgE productions were determined lane 2, immediately; lane 3, 24 h; lane 4, 48 h after topical application of in the skin of NC/Nga mice of various ages under SPF (&) and [32P]-AS6.