An anti-inflammatory ditriazine inhibiting leukocyte functions and expression of inducible nitric oxide synthase and cyclo-oxygenase-2

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Abstract

A ditriazine derivative (4,10-dichloropyrido[5,6:4,5]thieno[3,2-d′:3,2-d]-1,2,3-ditriazine (DTD)) inhibited neutrophil functions, including degranulation, superoxide generation, and leukotriene B4 production, without any effect on 5-lipoxygenase activity. This compound reduced nitric oxide (NO) and prostaglandin E2 production in mouse peritoneal macrophages stimulated with lipopolysaccharide, whereas no influence on the activity of inducible NO synthase, cyclo-oxygenase-2 or cyclo-oxygenase-1 was observed. DTD significantly reduced mouse paw oedema induced by carrageenan and also markedly reduced NO and prostaglandin E2 levels in exudates from 24-h zymosan-stimulated mouse air pouch. Western blot analysis showed that DTD reduced the expression of inducible NO synthase and cyclo-oxygenase-2. Our results indicate that DTD exerts anti-inflammatory effects related to the inhibition of neutrophil functions and of NO and prostaglandin E2 production, which could be due to a decreased expression of inducible NO synthase and cyclo-oxygenase-2.

Introduction

Prostaglandins and nitric oxide (NO) are ubiquitous mediator systems exerting numerous vascular and inflammatory effects. Production of prostaglandins or NO by the constitutive isoenzymes, cyclo-oxygenase-1, or endothelial NO synthase, is implicated in the physiological regulation of vascular tone and homeostatic functions. In contrast, cyclo-oxygenase-2 and inducible NO synthase are not generally expressed in resting cells, but are induced following appropriate stimulation with pro-inflammatory agents such as cytokines and lipopolysaccharide in many cell types including macrophages Salvemini et al., 1993, MacMicking et al., 1997. The activity of these inducible enzymes results in overproduction of prostaglandins and NO, which play a key role in the pathophysiology of arthritis and other inflammatory conditions Kaur and Halliwell, 1994, Vane et al., 1994, Kang et al., 1996.

It is known that simultaneous production of NO and superoxide by phagocytic cells leads to formation of peroxynitrite, a very reactive species mediating tissue injury Demiryurek et al., 1998, Kröncke et al., 1997. In addition, NO is also able to enhance the production of a variety of mediators, such as tumour necrosis factor-α and interleukin-1α, which participate in the macrophage-dependent inflammatory response (Marcinkiewicz et al., 1995).

On the other hand, neutrophils are essential for host defense and their contribution to the propagation and maintenance of acute and chronic inflammation includes several mechanisms. Activated neutrophils also release granule constituents Smith, 1994, Salvemini et al., 1996 and produce leukotrienes, which participate in the inflammatory response through stimulation of leukocyte functions and regulation of smooth muscle tone and vascular permeability Lewis et al., 1990, Henderson, 1994. Thus, the suppression of neutrophil functions could control the inflammatory response and has been implicated in the mechanism of action of some non-steroidal anti-inflammatory agents (Kankaanranta et al., 1994).

Little is known of the biological activity of 1,2,3-triazine derivatives and previous studies have focused mainly on their antifungal and antianaphylactic properties Guerrera et al., 1993, Wagner et al., 1993. Nevertheless, there is increasing interest in the pharmacological potential of these types of heterocyclic compounds due to the recent finding by our group that a series of 8-cyanopyridothienotriazines were able to inhibit NO and prostaglandin E2 synthesis in murine peritoneal macrophages stimulated with bacterial endotoxin (Quintela et al., 1999).

The present study was undertaken to examine the effects of a new ditriazine derivative, 4,10-dichloropyrido[5,6:4,5]thieno[3,2-d′:3,2-d]-1,2,3-ditriazine (DTD) (Fig. 1), on murine macrophage and human neutrophil functions as well as on several enzymes relevant to the inflammatory process. The results demonstrated the in vitro inhibitory effects on cell functions exerted by this compound, which also exhibited anti-inflammatory activity in vivo. This is the first report concerning the pharmacological properties of the compound.

Section snippets

Preparation of human neutrophils

Venous blood was obtained, with informed consent, from healthy volunteers. Leukocytes were obtained and purified as previously was described (Bustos et al., 1995). Viability was greater than 95% according to the trypan blue exclusion test. The mitochondrial dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan (Gross and Levi, 1992) was used to assess the possible cytotoxic effect of DTD on human neutrophils.

Isolation and culture of mouse peritoneal macrophages

Female Swiss mice weighing 25–30 g were

Chemiluminescence by human neutrophils and murine peritoneal macrophages

DTD inhibited in a concentration-dependent manner the chemiluminescence response induced by stimulation of neutrophils with TPA, with an IC50 value and confidence limits of 3.9 (1.4–5.5) and 4.4 (1.5–6.7) μM when luminol or lucigenin was used as substrate, respectively (Fig. 2). In contrast, DTD showed a weak inhibition of the chemiluminescence generated by the hypoxanthine/xanthine oxidase system (22.9±3.5**% inhibition at 10 μM), indicating a poor scavenging effect on reactive oxygen species

Discussion

It is generally accepted that recruitment and activation of leukocytes contribute to tissue damage in inflammation. Neutrophils migrate to the site of inflammation and upon activation by different stimuli, generate large amounts of reactive oxygen species, and release granular enzymes such as elastase and myeloperoxidase, which mediate tissue injury (Smith, 1994). Accordingly, inhibition of neutrophil functions can participate in the mechanism of action of a number of drugs, including some

Acknowledgments

This work was supported by grant SAF97-0249 from CICYT and by grant XUGA 10308B95 and 20908B97.

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