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The potential for repositioning antithyroid agents as antiasthma drugs

2016; Elsevier BV; Volume: 138; Issue: 5 Linguagem: Inglês

10.1016/j.jaci.2016.04.047

1097-6825

Shoichi Suzuki, Masahiro Ogawa, Shoichiro Ohta, Kazuhiko Arima, Satoshi Nunomura, Yasuhiro Nanri, Yasutaka Mitamura, Tomohito Yoshihara, Yutaka Nakamura, Kohei Yamauchi, Kazuyuki Chibana, Yoshiki Ishii, James J. Lee, Yasuaki Aratani, Shigeru Kakuta, Sachiko Kubo, Yoichiro Iwakura, Hiroki Yoshida, Kenji Izuhara,

Allergic Rhinitis and Sensitization

Bronchial asthma is a common disease in which type 2 immunity is dominant.1Peters M.C. Mekonnen Z.K. Yuan S. Bhakta N.R. Woodruff P.G. Fahy J.V. Measures of gene expression in sputum cells can identify TH2-high and TH2-low subtypes of asthma.J Allergy Clin Immunol. 2014; 133: 388-394Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar Based on this immunological background, various antiasthma drugs targeting type 2 immune mediators, such as IL-4, IL-5, IL-13, and CRTH2, are now under development.2Izuhara K. Matsumoto H. Ohta S. Ono J. Arima K. Ogawa M. Recent developments regarding periostin in bronchial asthma.Allergol Int. 2015; 64: S3-S10Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar However, to develop novel drugs, particularly biologics, huge investments of time and money are required and safety risks are involved. Drug repositioning, which is the process of finding new therapeutic indications for existing drugs, is highly desirable as an alternative strategy.3Tobinick E.L. The value of drug repositioning in the current pharmaceutical market.Drug News Perspect. 2009; 22: 119-125Crossref PubMed Scopus (180) Google Scholar, 4Li Y.Y. Jones S.J. Drug repositioning for personalized medicine.Genome Med. 2012; 4: 27Crossref PubMed Scopus (5) Google Scholar However, to our knowledge, there has been no instance of drug repositioning in bronchial asthma. Airway peroxidase functions as a potent defense system against microbes by producing biocidal compounds, including hypothiocyanite (OSCN−), together with hydrogen peroxide generated by Duox1 and Duox2, which are members of the Nox/Duox family.5Hawkins C.L. The role of hypothiocyanous acid (HOSCN) in biological systems.Free Radic Res. 2009; 43: 1147-1158Crossref PubMed Scopus (71) Google Scholar, 6Barrett T.J. Hawkins C.L. Hypothiocyanous acid: benign or deadly?.Chem Res Toxicol. 2012; 25: 263-273Crossref PubMed Scopus (75) Google Scholar Three heme peroxidases—myeloperoxidase (MPO), eosinophil peroxidase (EPX), and lactoperoxidase (LPO) (expressed at neutrophils, eosinophils, and epithelial cells, respectively)—are involved in this system in the lung tissues. In contrast, the deleterious effects of the airway peroxidase system in bronchial asthma remain undetermined. To define the pathological roles of peroxidase in bronchial asthma, we applied heme peroxidase inhibitors widely used as antithyroid agents to a mouse model of allergic airway inflammation. We found that these agents efficiently inhibited allergic airway inflammation. These results suggest that antithyroid agents can be repositioned as antiasthma drugs. We first examined the expression of 3 airway peroxidase genes in a mouse model of allergic airway inflammation. The pulmonary expression of all 3 heme peroxidases (Mpo, Epx, and Lpo) was significantly upregulated in response to an allergen challenge (Fig 1, A). The peroxidase activities were also enhanced in the bronchoalveolar lavage fluids (BALFs) of allergen-challenged mice (Fig 1, B). We then collected bronchial biopsy samples from 10 patients with asthma (see Table E1 in this article's Online Repository at www.jacionline.org) to determine whether the expression of the heme peroxidases was also enhanced in the bronchial tissues of patients with asthma. The peroxidase activities and the expression levels of LPO were not statistically enhanced. No expression of EPX or MPO was detected or was invariant between the patients and normal donors in contrast to the mouse analyses (Fig 1, C, data not shown). However, some patients showed distinctly high peroxidase activities and LPO expression. This may be due to the background (steps 2-4 according to the GINA2014 criteria), effects of the treatment for the patients (under good control of inhaled corticosteroids [ICSs]), and/or heterogeneity among patients with asthma regarding airway peroxidase expression. These results suggest the involvement of the airway peroxidase system at least in the mouse model of allergic airway inflammation and possibly in some patients with asthma. We applied the heme peroxidase inhibitors to examine the role of the airway peroxidase system in allergic airway inflammation. The approach we took to understand the role of this production system during allergic inflammatory responses involved inhibiting all peroxidase-mediated events in the lung because no inhibitors specific for LPO, MPO, or EPX have been developed. We examined the effects of 2-mercapto-1-methylimidazole (methimazole) and 6-propyl-2-thiouracil (PTU), which are agents that inhibit all peroxidases and are widely used as antithyroid agents targeting thyroid peroxidase. For the experiments of methimazole or PTU ingestion, 0.2 mg/mL of methimazole (Wako Pure Chemical Industries, Osaka, Japan) or 0.5 mg/mL of PTU (Sigma-Aldrich, St Louis, Mo) in drinking water was administered orally every day for the indicated times. We adopted 2 protocols of methimazole administration, a long and a short administration strategy (Met-L and Met-S, respectively) (Fig 2, A). Methimazole was administered orally every day from the start of sensitization (day 0) in the long administration and from 2 days before the start of the allergen airway challenge (day 20) in the short administration. The parameters of ovalbumin (OVA)-induced airway inflammation (ie, airway hyperresponsiveness [AHR], infiltration of inflammatory cells in BALF, and histological changes) were completely inhibited in the long administration and less so (yet significantly) in the short administration (Fig 2, B-D). The use of another peroxidase-inhibiting antithyroid agent, PTU, showed effects similar to but smaller than those of methimazole. Nonetheless, these effects showed a statistically significant improvement in AHR at the dosages used in this experiment (0.2 mg/mL of methimazole vs 0.5 mg/mL of PTU) (see Fig E1 in this article's Online Repository at www.jacionline.org). These results strongly suggest that heme peroxidase activities are critical for the setting of allergic airway inflammation in the model mice. The Met-L decreased free T4 in serum by inhibiting thyroid peroxidase (1.32 ± 0.14 ng/dL vs 2.0 ± 0.27 ng/dL; P < .001) (see Fig E2 in this article's Online Repository at www.jacionline.org). We complemented the thyroid function by administering thyroxine to exclude the possibility that the inhibition of airway inflammation by methimazole was due to impaired thyroid function; however, administering thyroxine had no effect on airway responsiveness (see Fig E3 in this article's Online Repository at www.jacionline.org). Next, we used mice deficient in each of the 3 peroxidases (Mpo, Epx, and Lpo) to determine which peroxidase dominantly contributes to the setting of allergic airway inflammation. Because Lpo-deficient mice were not available, we generated Lpo-deficient animals on a BALB/c background in preparation for these studies (see Fig E4 in this article's Online Repository at www.jacionline.org). Epx- and Lpo-deficient mice showed a nominal but not statistically significant decrease in AHR compared with their control littermates (see Fig E5, A, in this article's Online Repository at www.jacionline.org). The Mpo-deficient mice showed no change in AHR. Furthermore, infiltration of eosinophils and T cells was decreased in the BALF of the Lpo-deficient mice, whereas there was no change in infiltration in the Mpo- or Epx-deficient mice (Fig E5, B). These results suggest that the contributions of the 3 peroxidases are redundant in the setting of allergic airway inflammation. However, Lpo appears to be dominant among the peroxidases. Our results may help explain the findings of several reports showing the beneficial effects of the accidental administration of antithyroid agents to patients with asthma,7Settipane G.A. Schoenfeld E. Hamolsky M.W. Asthma and hyperthyroidism.J Allergy Clin Immunol. 1972; 49: 348-355Abstract Full Text PDF PubMed Scopus (35) Google Scholar, 8Nakazawa T. Kobayashi S. Influence of antithyroidal therapy on asthma symptoms in the patients with both bronchial asthma and hyperthyroidism.J Asthma. 1991; 28: 109-116Crossref PubMed Scopus (5) Google Scholar although there is a conflicting report.9Grembiale R.D. Naty S. Iorio C. Crispino N. Pelaia G. Tranfa C.M. Bronchial asthma induced by an antithyroid drug.Chest. 2001; 119: 1598-1599Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar It is of note that in most patients, bronchial asthma was exacerbated by discontinuing or tapering off of antithyroid agents.7Settipane G.A. Schoenfeld E. Hamolsky M.W. Asthma and hyperthyroidism.J Allergy Clin Immunol. 1972; 49: 348-355Abstract Full Text PDF PubMed Scopus (35) Google Scholar, 8Nakazawa T. Kobayashi S. Influence of antithyroidal therapy on asthma symptoms in the patients with both bronchial asthma and hyperthyroidism.J Asthma. 1991; 28: 109-116Crossref PubMed Scopus (5) Google Scholar Currently, it remains unknown how heme peroxidase plays a role in the pathogenesis of allergic airway inflammation. We found that OSCN−, a product of airway peroxidase, activates nuclear factor kappa B in airway epithelial cells (unpublished data), which partly explains the role of peroxidase in bronchial asthma. Drug repositioning is now expected as an alternative to drug discovery and development. The use of antithyroid agents could be the first example of drug repositioning in bronchial asthma. We thank Dr Dovie R. Wylie for the critical review of this manuscript and Prof. Hidenobu Soejima and Drs Kenichi Nishioka and Hidetaka Watanabe for the instruction of Droplet Digital PCR. We also thank Maki Futamata, Tameko Takahashi, Chizuko Kondo, Kazuyo Yoshida, and Seiji Kawasaki for technical assistance. Seven- to 10-week-old female mice were used. Animal studies were undertaken following the guidelines for care and use of experimental animals of the Japanese Association for Laboratory Animals Science (1987) and were approved by the Saga University Animal Care and Use Committee (Saga, Japan). The mouse model of airway allergic inflammation was generated as previously describedE1Matsushita H. Ohta S. Shiraishi H. Suzuki S. Arima K. Toda S. et al.Endotoxin tolerance attenuates airway allergic inflammation in model mice by suppression of the T-cell stimulatory effect of dendritic cells.Int Immunol. 2010; 22: 739-747Crossref PubMed Scopus (30) Google Scholar with minor modifications. Mice were sensitized by intraperitoneal injections of 50 μg OVA (Sigma-Aldrich) and 1 mg alum (LSL, Tokyo, Japan) in 500 μL saline on days 0 and 12. Mice inhaled 1% OVA in saline on days 22, 26, and 30 or on days 22, 23, and 24, followed by assessment of airway responsiveness on the following day and of BALF, lung histology, and gene expression 2 days later. In some experiments, 0.05 μg/mouse thyroxine (Sigma-Aldrich) was administered intraperitoneally every day for the indicated times. Airway responsiveness to methacholine (Sigma-Aldrich) was assessed with a 4-chamber whole-body plethysmograph from Buxco Electronics (Wilmington, NC) or flexiVent from SCIREQ (Montreal, Canada). Droplet Digital PCR was performed on QX200 Droplet Digital PCR system (Bio-Rad, Hercules, Calif) using ddPCR supermix for probes or ddPCR EVAGreen supermix. The sequences of primers are as follows: Mpo, 5′-CCCTTCCTAAACTGAACCTGAC-3′ and 5′-ATGGCCTCCGTCCTTCTC-3′; Epx, 5′-CTCCTGACTAACCGCTCTGC-3′ and 5′-TCACTTGACCGAGTGTCACC-3′. Predesigned TaqMan Gene Expression Assays (Life Technologies, Carlsbad, Calif) for Lpo (Mm00475466_m1) and LPO (Hs00976400_m1) were used. After mice were anesthetized, lungs were lavaged with 500 μL of BAL liquid (0.1% BSA/50 μmol/L EDTA/PBS), and BALF was collected. This procedure was repeated 3 times. BALF cell counts were determined with a particle counter (CDA500, Sysmex, Japan). Eosinophils, neutrophils, T cells, and macrophages were defined as siglec-F+ cells (BD Bioscience, San Diego, Calif), Gr-1high cells (eBioscience, San Diego, Calif), CD3+ cells (eBioscience), and strongly autofluorescent/bigger-size cells,E2van Rijt L.S. Kuipers H. Vos N. Hijdra D. Hoogsteden H.C. Lambrecht B.N. A rapid flow cytometric method for determining the cellular composition of bronchoalveolar lavage fluid cells in mouse models of asthma.J Immunol Methods. 2004; 288: 111-121Crossref PubMed Scopus (150) Google Scholar respectively. Paraffin-embedded lung sections were prepared and stained with hematoxylin and eosin or periodic acid-Schiff. For immunostaining, sections were incubated with anti-Lpo antibodies (Thermo Fisher Scientific, Waltham, Mass) followed by visualization according to a standard procedure using 3,3′-diaminobenzidine. One hundred microliter of TMB solution (1-step Ultra TMB-ELISA, Thermo Fisher Scientific) was added to 100 μL of BALF in the presence or absence of 1 mmol/L sodium azide (Wako Pure Chemical Industries). The reaction was stopped by adding 50 μL of 2 mol/L sulfuric acid and the enzyme activity was evaluated by the absorbance at 450 nm. The peroxidase activity was converted to peroxidase concentrations using a standard curve generated with commercial human EPX (Lee BioSolutions, Maryland Heights, Mo). Heme peroxidase activity was calculated by subtracting azide-insensitive peroxidase activity from total peroxidase activity. Free T4 was measured by Elecsys FT4II (Roche Diagnostics, Basel, Switzerland). Ten patients with asthma and 10 healthy donors were recruited from the Iwate Medical University Hospital. The backgrounds of the patients with asthma are described in Table E1. The patients with asthma had no other medical disorders and were not current smokers; the 10 control subjects were nonallergic and nonasthmatic patients. All the patients with asthma were at step 2 to 4 according to the GINA2014 criteria and were well controlled with inhaled corticosteroids. Biopsy samples were taken from the bifurcations of the subsegmental bronchi under bronchoscopy. This study was approved by the Iwate Medical University Hospital Ethics Committee. Healthy donors had both normal spirometry and required a lung resection for treatment of lung carcinoma. They were not current smokers and had no history of asthma and allergy or systemic diseases. Mpo- or Epx-deficient mice were prepared as previously described (Fig E6).E3Aratani Y. Koyama H. Nyui S. Suzuki K. Kura F. Maeda N. Severe impairment in early host defense against Candida albicans in mice deficient in myeloperoxidase.Infect Immun. 1999; 67: 1828-1836Crossref PubMed Google Scholar, E4Denzler K.L. Borchers M.T. Crosby J.R. Cieslewicz G. Hines E.M. Justice J.P. et al.Extensive eosinophil degranulation and peroxidase-mediated oxidation of airway proteins do not occur in a mouse ovalbumin-challenge model of pulmonary inflammation.J Immunol. 2001; 167: 1672-1682Crossref PubMed Scopus (109) Google Scholar Lpoflox/+ chimeric mice were obtained by injecting Lpotm1a(EUCOMM)Wtsi embryonic stem cells (JM8.N4, C57BL/6N background, European Mouse Mutant Cell Repository, Neuherberg, Germany) into blastocysts from ICR mice (Japan SLC, Hamamatsu, Japan). Lpoflox/+ mice were selected by mating Lpoflox/+ chimeric mice and C57BL/6 mice (Kyudo, Tosu, Japan). Then, Lpo-deficient mice were generated by mating Lpoflox/+ mice and Cre transgenic mice (BALB/c-Tg(CMV-cre)1Cgn/J #3465, The Jackson Laboratory, Bar Harbor, Maine) and were backcrossed onto BALB/c mice (Japan SLC) for 8 generations.Fig E2Effects of methimazole on free T4 in mice. Serum-free T4 in methimazole-free (Met(−)), Met-L, and Met-S mice. Serum was taken on day 31. *P < .05, #P < .001 (vs Met(−) mice).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Effects of thyroxine on the improvement in allergic airway inflammation by methimazole in mice. A, The experimental protocol. Oral administration of 0.2 mg/mL methimazole (Met) in drinking water alone or with intraperitoneal (ip) administration of 0.05 μg thyroxine per mouse every day from day 0 to day 31. B, Airway reactivity assessed by flexiVent (SCIREQ, Montreal, Canada). Open circle, without OVA inhalation; closed circle, with OVA inhalation; closed triangle, methimazole alone; and closed square, methimazole and thyroxine. *P < .05, **P < .01, (vs with OVA inhalation). C, Serum-free T4 in Met(−), Met-L, and Met-L supplemented with thyroxine mice. Serum was taken on day 31. **P < .01, #P < .001 (vs Met(−) mice).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Genotyping of Lpo-deficient mice. A, Genomic structures of the Lpo-floxed allele and the Lpo-deficient allele generated by cleavage with Cre recombinase. The positions and the directions of 3 primers (Lpo-5'arm, Lpo-3'arm, and LAR3) are depicted. B, Genotyping of Lpo+/+, Lpo+/−, and Lpo−/− mice. The Lpo-5'arm/Lpo-3'arm band (543 bp) and the Lpo-5'arm/LAR3 (278 bp) correspond to the wild-type and knockout alleles, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5Redundancy of contribution to airway allergic inflammation in 3 peroxidases. A, Airway reactivity as assessed by the flexiVent. Open circle, WT littermates without OVA inhalation; closed circle, WT littermates with OVA inhalation; and closed square, knocked-out mice with OVA inhalation. B, Analyses of BALF. OVA, WT littermates without OVA inhalation; WT, wild-type littermates with OVA inhalation; KO, knocked-out mice with OVA inhalation. *P < .05, **P < .01, and NS: not significant (vs WT littermates with OVA inhalation).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E6Genotyping of Epx- and Mpo-deficient mice. Genotyping of Epx+/+, Epx+/−, and Epx−/− mice (left panel) and Mpo+/+, Mpo+/−, and Mpo−/− mice (right panel). The 687 bp and the 991 bp bands for Epx and the 608 bp and the 361 bp bands for Mpo correspond to the wild-type (WT) and knockout (KO) alleles, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Clinical backgrounds, LPO expression, and heme peroxidase activity in patients with asthmaS. no.Age (y)SexFeno (ppb)TreatmentIgE (IU/mL)FEV1 (%)Eosinophil (/μL)LPO mRNA (copy number/μg)Heme peroxidase activity (ng/mL)161M44FP200 μg/d52898.03311,600809235M134FP500 μg/d8253.32864,1002,792336M52FP500 μg/d265796.8659130367438M34FP400 μg/d128663.02966408,317540M34FP200 μg/d29396.328016,400567636F30BUD200 μg/d57282.834176,500483765M39FP200 μg/d90948.125212,300392848M25FP400 μg/d8865.9391ND550939M62FP100 μg/d134104.8293ND6,0831040F46BUD400 μg/d19059.1179960967BUD, Budesonide; F, female; Feno, fractional exhaled nitric oxide; FP, fluticasone propionate; M, male; ND, not determined. Open table in a new tab BUD, Budesonide; F, female; Feno, fractional exhaled nitric oxide; FP, fluticasone propionate; M, male; ND, not determined.