Antieosinophilic Activity of Orazipone □ S Hannu Kankaanranta, Pinja Ilmarinen, Xianzhi Zhang, Erkki Nissinen, and Eeva Moilanen The Immunopharmacology Research Group, Medical School, University of Tampere, Tampere, Finland (H.K., P.I., X.Z., E.M.); the Center for Infection and Immunity, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China (X.Z.); Department of Respiratory Medicine (H.K.) and Research Unit (E.M.), Tampere University Hospital, Tampere, Finland; and OrionPharma Ltd., Espoo, Finland (E.N.) Received November 25, 2005; accepted March 15, 2006 ABSTRACT Orazipone is a novel sulfhydryl-reactive compound that has been previously shown to reduce lung eosinophilia in guinea pigs and rats and to inhibit degranulation in mast cells and cytokine production in monocytes and T-cells. However, the effects of orazipone on granulocyte longevity are unknown. Orazipone and its derivative 3-(4-chloro-3-nitro-benzylidene)- pentane-2,4-dione (OR-2370) reversed interleukin-5-afforded survival of human eosinophils by inducing apoptosis. In con- trast, orazipone did not affect granulocyte macrophage-colony- stimulating factor–induced survival of human neutrophils. The effect of orazipone on eosinophil apoptosis is different from that of glucocorticoids in that even high con-centrations of interleukin-5 were not able to overcome the effect of orazipone. Orazipone further enhanced spontaneous apoptosis as well as that induced by CD95 ligation without inducing primary necro- sis. OR-2370-induced DNA fragmentation was shown to be dependent on caspases 3 and 6 and c-jun-N-terminal kinase, whereas extracellular regulated kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase as well as caspases 4, 8, and 9 seem not to mediate its actions. Our results suggest that orazipone and its derivative OR-2370 pos- sess strong antieosinophilic activity and thus may have anti- inflammatory efficacy in the treatment of asthma and/or allergic conditions. Eosinophils are thought to play a critical role in allergic diseases, such as allergic rhinitis, asthma, and atopic derma- titis (Giembycz and Lindsay, 1999; Gleich, 2000). In asth- matic patients, activation of eosinophils in the airways is believed to cause epithelial tissue injury, contraction of air- way smooth muscle and increased bronchial responsiveness. Apoptosis or programmed cell death is regarded as an impor- tant feature in the resolution of asthmatic inflammation (Kankaanranta et al., 2005). Apoptosis is characterized by specific biochemical and morphological changes, including cell shrinkage, surface blebbing, DNA fragmentation and loss of nucleoli (Kankaanranta et al., 2005), so that the apo- ptotic cell is phagocytosed intact without release of its con- tents. In vitro, eosinophil apoptosis is inhibited by cytokines, such as interleukin-3, interleukin-5, and granulocyte macro- phage– colony-stimulating factor (GM-CSF) (Giembycz and Lindsay, 1999; Kankaanranta et al., 2005). Eosinophil apo- ptosis is up-regulated by Fas (CD95/APO-1), a 45-kDa trans- membrane protein belonging to the tumor necrosis factor receptor family (Kankaanranta et al., 2005). Eosinophil apoptosis is delayed in patients with asthma or inhalant allergy (Wedi et al., 1997; Kankaanranta et al., 2000a). Fur- thermore, the number of eosinophils in asthmatic lung is elevated and is inversely correlated with the number of apoptotic eosinophils (Vignola et al., 1999). Thus, pharmaco- logical induction of eosinophil apoptosis is considered an in-teresting possibility to treat eosinophilic inflammatory conditions such as asthma and/or allergic diseases. Orazipone and its derivatives OR-1958 and OR-2370 (Fig. This study was supported by Tampere Tuberculosis Foundation (Finland), the Finnish Anti-Tuberculosis Association Foundation (Finland), the Academy of Finland, the Medical Research Fund of Tampere University Hospital (Fin- land), OrionPharma Ltd. (Finland) and the National Technology Agency (TEKES, Finland). □ S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.021170. ABBREVIATIONS: GM-CSF, granulocyte macrophage– colony-stimulating factor; JNK, c-jun-N-terminal kinase; DMSO, dimethyl sulfoxide; Z-, N-benzyloxycarbonyl-; FMK, fluoromethyl ketone; Ac-, N-acetyl-; CHO, aldehyde; MAPK, mitogen-activated protein kinase; LY294002, 2-(4- morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PD098059, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; PD169316, 4-(4-fluorophe- nyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole; PI3K, phosphatidylinositol 3-kinase; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)- 5-(4 pyridyl)-1H-imidazole; DCB, [(2,6-dichlorobenzoyl)oxy]methane; L-JNKI1, GRKKRRQRRR-PP-RPKRPTTLNLFPQVPRSQD-amide; L-TAT, RKKRRQRRR-amide, negative control for L-JNKI1; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; IL, interleukin; OR-2370, 3-(4-chloro-3-nitro-benzylidene)-pentane-2,4-dione; OR-1958, 3-(3-chloro-4-methanesul- fonyl-benzylidene)-pentane-2,4-dione; OR-2149, 3-(4-methanesulfonyl-benzyl)-pentane-2,4-dione. 0026-895X/06/6906-1861–1870$20.00 MOLECULAR PHARMACOLOGY Vol. 69, No. 6 Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics 21170/3116533 Mol Pharmacol 69:1861–1870, 2006 Printed in U.S.A. 1861 http://molpharm.aspetjournals.org/content/suppl/2006/03/16/mol.105.021170.DC1.html Supplemental material to this article can be found at: at ASPET Journals on October 25, 2016 molpharm.aspetjournals.org Downloaded from