Protective effects of M40403, a superoxide dismutase mimetic, in a rodent model of colitis

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Abstract

Inflammatory bowel disease is characterised by oxidative and nitrosative stress, leukocyte infiltration, and up-regulation of intercellular adhesion molecule 1 (ICAM-1) expression in the colon. The aim of the present study was to examine the effects of M40403, a superoxide dismutase mimetic, in rats subjected to experimental colitis. Colitis was induced in rats by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS). Rats experienced bloody diarrhoea and significant loss of body weight. At 4 days after TNBS administration, the colon damage was characterised by areas of mucosal necrosis. Neutrophil infiltration (indicated by myeloperoxidase activity in the mucosa) was associated with up-regulation of ICAM-1 and expression of P-selectin and high levels of malondialdehyde. Immunohistochemistry for nitrotyrosine and poly (ADP-ribose) synthetase showed an intense staining in the inflamed colon. Treatment with M40403 (5 mg/kg daily i.p.) significantly reduced the appearance of diarrhoea and the loss of body weight. This was associated with a remarkable amelioration of the disruption of the colonic architecture as well as a significant reduction of colonic myeloperoxidase activity and malondialdehyde levels. M40403 also reduced the appearance of nitrotyrosine and poly (ADP-ribose) synthetase immunoreactivity in the colon as well as reduced the up-regulation of ICAM-1 and the expression of P-selectin. The results of this study suggested that administration of a superoxide dismutase mimetic may be beneficial for treatment of inflammatory bowel disease.

Introduction

A growing body of data indicates that oxygen-derived free radicals such as superoxide (O2), nitric oxide (NO) and hydroxyl radicals (OH) have a role in mediating intestinal damage in inflammatory bowel disease. The intestine is well endowed with enzymes capable of producing such free radicals (Parks, 1989). Moreover, when inflammation is present the many phagocytic cells that are attracted and activated can produce large amounts of free radicals. Several studies suggest that peripheral blood monocytes (Kitahora et al., 1988) and isolated intestinal macrophages Verspaget and Beeken, 1985, Mahida et al., 1989 from patients with inflammatory bowel disease produce of free radicals. Also, high numbers of peripheral neutrophils, which are capable of producing large amounts of oxygen-derived free radicals (Verspaget et al., 1988), migrate into the intestinal wall of such patients (Crama-Bohbouth et al., 1988). Grisham and Granger (1988) hypothesised that in ulcerative colitis, transient ischemic episodes and subsequent reperfusion produce high levels of free radicals. This process initiates a cascade of events leading to the recruitment and activation of leucocytes, resulting ultimately in mucosal ulceration. Recently, Wakefield et al. (1989) presented evidence for multifocal infarctions in the intestine of patients with Crohn's disease, indicating that ischemic episodes may also occur in this disease.

Several studies indicate that sulphasalazine and its active metabolite 5-aminosalicylic acid are efficient scavengers of oxygen radicals in vitro Auroma et al., 1987, Miyachi et al., 1987. These scavenging potentials may be an important mode of action of these drugs in vivo Auroma et al., 1987, Miyachi et al., 1987, Dull et al., 1987. The pro-inflammatory roles of superoxide are well known: recruitment of neutrophils at sites of inflammation, formation of chemotactic factors (Fantone and Ward, 1982), DNA damage, depolymerization of hyaluronic acid and collagen (Schraufstatter et al., 1986), lipid peroxidation, release of cytokines such tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) and formation of peroxynitrite (ONOO−), which is a highly reactive oxidant produced by the combination of O2 and NO at rates approaching the diffusion limit Beckman et al., 1990, Ischiropoulos et al., 1992. Moreover, peroxynitrite can also cause DNA damage (Inoue and Kawanishi, 1995) resulting in the activation of the nuclear enzyme poly (ADP-ribose) synthetase and poly (ADP-ribose) synthase-driven cell death (Szabo and Dawson, 1998). Furthermore, ONOO− inhibits the activity of the endogenous superoxide dismutase enzymes, a factor that contributes to increased formation of O2 Yamakura et al., 1998, Macmillan-Crow and Thompson, 1999. Orgotein® (bovine CuZn superoxide dismutase) has been used in preliminary clinical trials in patients with various inflammatory disorders and improvement has been reported in four of four patients with Crohn's disease and three of four patients with ulcerative colitis (Niwa et al., 1985). Emerit et al., 1989 reported a beneficial effect in 82% of patients with Crohn's disease with long-term treatment with bovine superoxide dismutase. These results indicate that removal of O2 in humans by a superoxide dismutase enzyme has beneficial outcomes in these disorders. Despite encouraging clinical results in several other areas, Orgotein had to be removed from the market because of its origin (bovine), development of immunogenic responses in some individuals and other properties that made it an inadequate therapeutic agent (Flohe, 1988).

Based on the above, we have developed a series of superoxide dismutase mimetics that catalytically remove O2 as potential clinical candidates. The 1,4,7,10,13-pentaazacyclopentadecane containing the added bis(cyclohexylpyridine) functionalities (M40403) is a prototypic example of our low-molecular weight, manganese-containing, nonpeptidic molecule possessing the function and catalytic rate of native superoxide dismutase enzymes, but with the advantage of being a much smaller molecule (MW 483 vs. MW 30,000 for the mimetic and native enzyme, respectively) (Riley et al., 1996). M40403 is cytoprotective and stable in vivo, exerts anti-inflammatory properties in acute inflammation and is not deactivated by ONOO−, an added advantage over the native superoxide dismutase enzyme that is nitrated and deactivated by ONOO− Yamakura et al., 1998, Macmillan-Crow and Thompson, 1999. M40403 is not only a highly active catalyst for the dismutation of O2, but it is also highly selective for superoxide. M40403 does not react with hydrogen peroxide, nor does it directly react with other biologically relevant oxidants such as nitric oxide or peroxynitrite Riley et al., 1996, Salvemini et al., 1999a.

The objectives of the present study were to address whether M40403 exerted protection on the chronic inflammatory response (colitis) caused by injection of trinitrobenzene sulfonic acid (TNBS) in the rat, and if so, highlight possible mechanisms through which M40403 conferred protection.

Section snippets

Animals

Male Sprague–Dawley rats (300–350 g; Charles River, Milan, Italy) were housed in a controlled environment and provided with standard rodent chow and water. Animal care was in compliance with Italian regulations on protection of animals used for experimental and other scientific purpose (D.M. 116192) as well as with the EEC regulations (O.J. of E.C. L 358/1 12/18/1986).

Induction of experimental colitis

Colitis was induced using a technique of acid-induced colonic inflammation as described previously (Wallace et al., 1992). On

Effects of M40403 on colonic damage

Four days after intracolonic administration of TNBS, the colon appeared flaccid and filled with liquid stool. The cecum, colon and rectum all had evidence of mucosal congestion, erosion and hemorrhagic ulcerations (Fig. 1). Histological examination of lesion area of descending colon sections showed a complete alteration of the epithelial layer, muscularis mucosa and submucosal architecture. Diffuse inflammatory infiltration of neutrophils, lymphocytes and plasma cells was also observed in the

Discussion

Our data show that rats treated with M40403, a low molecular weight synthetic enzyme mimetic of superoxide dismutase, are significantly more resistant to death and pathological changes in the colon and rectum associated with TNBS induced colitis.

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