The Interleukin-1 -Converting Enzyme Inhibitor Pralnacasan Reduces Dextran Sulfate Sodium-Induced Murine Colitis and T Helper 1 T-Cell Activation

The proinflammatory cytokines interleukin (IL)-1 and IL-18 are supposed to play a crucial role in the pathogenesis of human inflammatory bowel disease. To exert biological activity, the precursors of both IL-1 and IL-18 need to be cleaved by the interleukin-1 -converting enzyme (ICE). IL-18 induces the synthesis of IFNin T cells and NK cells. In the present study, we investigated the effect of the specific ICE inhibitor pralnacasan in dextran sulfate sodium-induced murine colitis. Colitis was induced in BALB/c mice by 3.5% dextran sulfate sodium dissolved in drinking water for 10 days. Pralnacasan was administered either intraperitoneally or orally every day. To assess in vivo efficacy, a clinical disease activity score was evaluated daily. Colon length, expression of IL-18 in colonic tissue, expression of interferon(IFN) in paraaortal lymphocytes, and systemic production of IFNin splenocytes were analyzed post mortem. Intraperitoneally administered pralnacasan significantly reduced the clinical score compared with the dextran sulfate sodium control group from day 6 to day 10. Oral administration of pralnacasan also significantly reduced the clinical score at days 8 and 9. Administration of pralnacasan i.p. reduced the expression of intracolonic IL-18 significantly. Furthermore, pralnacasan reduced the number of IFN-positive lymphocytes in paraaortal lymph nodes. IFNsynthesis in stimulated splenocytes was significantly suppressed in all pralnacasan-treated groups. No side effects of pralnacasan were observed. In conclusion, pralnacasan is effective in the prevention of dextran sulfate sodium-induced colitis. This effect is probably mediated by suppression of the proinflammatory cytokines IL-18, IL-1 , and IFN. Cytokines orchestrate the pathogenesis of inflammatory bowel disease. Successful treatment of patients with steroidrefractory Crohn’s disease (CD) with anti-TNFantibody (van Dullemen et al., 1998; Rutgeerts et al., 1999; Sandborn and Hanauer, 1999) illustrates the potency in treating CD based on the specific blockade of this proinflammatory cytokine (for review, see Eigler et al., 1997). The specific blockade of type IV phosphodiesterase with mesopram decreases the synthesis of TNFand IFN, as well as ameliorates disease activity in the dextran sulfate sodium-induced murine colitis (Loher et al., 2003). Furthermore, these observations indicate the important role of a Th1-mediated immune response in the pathogenesis of CD, because TNFhas been shown to be produced as one of the final key mediators of a Th1 response. However, repeated administration of anti-TNFantibodies can lead to the production of autoantibodies (Sandborn and Hanauer, 1999) and anti-double-strand DNA antibodies (Elliott et al., 1994; Rutgeerts et al., 1999; Sandborn and Hanauer, 1999). Thus, in the treatment of human inflammatory bowel disease (IBD) further strategies to antagonize proinflammatory cytokines are warranted. IL-1 is produced by activated monocytes and macrophages. It stimulates B and T lymphocytes as well as bone marrow cells, induces the acute phase reaction, and acts as an endogenous pyrogen (Dinarello, 1996). Mononuclear cells isolated from colon specimens from patients with ulcerative colitis or CD produce more IL-1 compared with cells from This work was supported by Aventis Deutschland GmbH (Frankfurt, Germany) and Vertex Inc., Paramus, NJ. 1 These authors contributed equally to this work. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.103.057059. ABBREVIATIONS: CD, Crohn’s disease; TNF, tumor necrosis factor; IFN, interferon; Th1, T helper 1; IBD, inflammatory bowel disease; IL, interleukin; ICE, interleukin-1 -converting enzyme; PMA, phorbol 12-myristate 13-acetate; HEC, hydroxyethylcellulose; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting. 0022-3565/04/3082-583–590$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 308, No. 2 Copyright © 2004 by The American Society for Pharmacology and Experimental Therapeutics 57059/1125590 JPET 308:583–590, 2004 Printed in U.S.A. 583 at A PE T Jornals on D ecem er 5, 2017 jpet.asjournals.org D ow nladed from normal mucosa, providing circumstantial evidence for an active role of IL-1 in human IBD (Mahida et al., 1989). IL-18 is structurally related to IL-1 : both are allpleated sheet-folded proteins. In contrast with IL-1 , IL-18 mRNA and precursor protein are constitutively expressed in human peripheral blood mononuclear cells as well as in murine splenocytes and keratinocytes (Stoll et al., 1997). The production of biologically active IL-18 is induced by different stimuli such as bacterial lipopolysaccharides or cytokines such as TNFor IL-1 . IL-18 induces the production of IFNin T cells and NK cells (Dinarello, 1999). It acts synergistically with IL-12, which up-regulates IL-18 receptors in target cells. In BALB/c mice, induction of the expression of IFNby IL-18 leads to a shift to a Th1 response of the immune system (Szabo et al., 1997). This shift is thought to be relevant in the pathogenesis of human CD and might also contribute to murine colitis induced by dextran sulfate sodium as an enhancing factor. Both IL1and IL-18 are produced as biologically inactive precursor proteins that require cleavage by the interleukin-1 converting enzyme (ICE), which is a member of the family of aspartate-specific cysteine proteases (caspases). ICE is produced as a 45-kDa proenzyme that lacks proteolytic activity and is present in monocytic cells (Ayala et al., 1994). Upon cell stimulation proenzymes aggregate and subsequently undergo autocatalytic activation that presumably leads to a tetramer consisting of two 10-kDa and two 20-kDa polypeptide fragments of ICE with the active site Cys-285 localized near to the C terminus of the 20-kDa peptide chain (Wilson et al., 1994; Gu et al., 1995; Schonbeck et al., 1997). Active ICE prevails in the plasma membrane where it cleaves pro-IL-1 and pro-IL-18. It is complexed with other proteins that are involved in the secretion of the activated cytokines (Singer et al., 1995). Several classes of ICE inhibitors exist, which can be divided into irreversible and reversible inactivators of this protease (Livingstone et al., 1997). Irreversible inactivators react with the active site sulfhydryl group in the manner of a nucleophile substitution and therefore must provide leaving groups in their chemical structure. Such inhibitors belong chemically to the halomethylketones, diazomethylketones, or acyloxymethylketones. Reversible inactivators of ICE are phenyalkylketones and peptide aldehydes, which have a high affinity for binding to the enzyme and thus are also highly effective protease inhibitors (Thornberry et al., 1994; Livingstone et al., 1997). Dextran sulfate sodium-induced colitis is characterized histopathologically by mucosal infiltration of inflammatory cells, focal crypt damage, epithelial injury, and ulceration (Cooper et al., 1993; Dieleman et al., 1997; Okayasu et al., 2002). The pathological mechanism of dextran sulfate sodium-induced colitis includes both toxic effects on the epithelium and production of proinflammatory cytokines by macrophages that are activated after phagocytosis of dextran sulfate sodium. The purpose of this study was to investigate the disease-modifying activity of the ICE inhibitor pralnacasan (previously known as HMR 3480/VX-740) in a murine model of dextran sulfate sodium-induced colitis (Siegmund and Zeitz, 2003). Pralnacasan is an ethyl-hemiacetal orally bioavailable prodrug. Once absorbed, it is rapidly hydrolyzed to the active but reversible aldehyde protease inhibitor (M. D. Mullican and D. J. Lauffer, manuscript submitted for publication). The active compound AcYVAD-CHO was structure based designed using crystallographic structural data to model the interaction between ICE and the acetylated tetrapeptide ICE inhibitor AcYVAD-CHO (Siegmund and Zeitz, 2003). Materials and Methods Mice. Female, 8-week-old BALB/c mice (Harlan Winkelmann, Borchen, Germany) weighing 20 to 22 g were housed in temperaturecontrolled rooms with a 12-h light/dark cycle. Mice were fed standard mice chow pellets, had access to bottled tap water ad libitum, and were acclimatized to the environmental conditions 10 days before they were studied in the experiment. Mice were killed by cervical dislocation under isoflurane anesthesia (Forene; Abbott GmbH, Wiesbaden, Germany). All experiments were approved by the regional animal study committee and are in agreement with the guidelines for the proper use of animals in biomedical research. Both animal handling and clinical and histological scoring of colitis were performed in a blinded experimental design. Reagents. Brefeldin A, phorbol 12-myristate 13-acetate (PMA), and ionomycin were purchased from Sigma Chemie (Munich, Germany). RPMI 1640 medium was from Biochrom KG (Berlin, Germany) and fetal calf serum (FCS) was from Invitrogen (Karlsruhe, Germany). For i.p. use, pralnacasan was dissolved in 25% Cremophor EL solution and filtered through syringe filters (0.2 m) purchased from Gelman Sciences (Ann Arbor, MI). For p.o. use, pralnacasan was homogenously suspended in 0.5% hydroxyethylcellulose solution (HEC, lot S29089015; HEC, Schuchard, Hohenbrunn, Ger-