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Background and purpose Multiple members of the thiol isomerase (TI) family of enzymes are present in, and released by platelets. Inhibition of these enzymes results in diminished platelet responses including aggregation, adhesion and thrombus formation. In recent years, the therapeutic potential of TI inhibition has been recognised and drug-development technologies used to identify selective small molecule inhibitors. To date, few pan-TI inhibitors have been characterised and the most studied, bacitracin is known to be nephrotoxic which prohibits its systemic therapeutic usage. Experimental approach We therefore sought to identify novel broad-spectrum inhibitors of these enzymes and test their effects in vivo. 3641 compounds were screened for inhibitory effects on the redox activity of ERp5, PDI, ERp57, ERp72 and thioredoxin (TRX) in an insulin turbidity assay. Of the lead compounds identified, zafirlukast (ZFL) was selected for further investigation. Key results When applied to platelets, ZFL diminished platelet responses in vitro. ZFL was antithrombotic in murine models of thrombosis but did not impair responses in a model of haemostasis. Since thiol isomerases are known to modulate adhesion receptor function, we explored the effects of ZFL on cell migration. This was inhibited independently of cysteinyl leukotriene receptor expression and was associated with modulation of cell-surface free thiol levels consistent with alterations in redox activity on the cell surface. Conclusion and implications We identify zafirlukast to be a novel, potent, broad-spectrum TI inhibitor, with wide ranging effects on platelet function, thrombosis and integrin-mediated cell migration. ZFL is antithrombotic but does not cause bleeding.

Background and purpose: Cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory and cardiovascular disorders. Their actions are mediated by CysLT1 and CysLT2 receptors. Here we report the discovery of 3-({[(1S,3S)-3-carboxycyclohexyl]amino}carbonyl)-4-(3-{4-[4-(cyclo-hexyloxy)butoxy]phenyl}propoxy) benzoic acid (HAMI3379), the first potent and selective CysLT2 receptor antagonist. Experimental approach: Pharmacological characterization of HAMI3379 was performed using stably transfected CysLT1 and CysLT2 receptor cell lines, and isolated, Langendorff-perfused, guinea pig hearts. Key results: In a CysLT2 receptor reporter cell line, HAMI3379 antagonized leukotriene D4- (LTD4-) and leukotriene C4- (LTC4-) induced intracellular calcium mobilization with IC50 values of 3.8 nM and 4.4 nM respectively. In contrast, HAMI3379 exhibited very low potency on a recombinant CysLT1 receptor cell line (IC50 > 10 000 nM). In addition, HAMI3379 did not exhibit any agonistic activity on both CysLT receptor cell lines. In binding studies using membranes from the CysLT2 and CysLT1 receptor cell lines, HAMI3379 inhibited [3H]-LTD4 binding with IC50 values of 38 nM and >10 000 nM respectively. In isolated Langendorff-perfused guinea pig hearts HAMI3379 concentration-dependently inhibited and reversed the LTC4-induced perfusion pressure increase and contractility decrease. The selective CysLT1 receptor antagonist zafirlukast was found to be inactive in this experimental setting. Conclusions and implications: HAMI3379 was identified as a potent and selective CysLT2 receptor antagonist, which was devoid of CysLT receptor agonism. Using this compound, we showed that the cardiac effects of CysLTs are predominantly mediated by the CysLT2 receptor.

A new group IIa sPLA2 inhibitor was compared with selective inhibitors of COX-1, COX-2 and an LTC4 antagonist for effects on local and remote tissue injuries following ischaemia and reperfusion (I/R) of the small intestine in rats. In an acute model of ischaemia (30 min) and reperfusion (150 min) injury in the absence of inhibitors, there was significant intestinal haemorrhage, oedema and mucosal damage, neutropenia, elevated serum levels of aspartate aminotransferase (AST) and hypotension. Preischaemic treatment with the inhibitor of sPLA2 (Group IIa), at 5 mg kg−1 i.v. or 10 mg kg−1 p.o. significantly inhibited I/R-induced neutropenia, the elevation of serum levels of AST, intestinal oedema and hypotension. Pretreatment with the COX-2 inhibitor celebrex (10 mg kg−1 i.v.) and the LTC4 antagonist zafirlukast (1 mg kg−1 i.v.) also showed marked improvement with I/R-induced AST, oedema and neutropenia. Hypotension was only reduced by the LTC4 antagonist. The COX-1 inhibitor flunixin (1 mg kg−1 i.v.) did not effect improvement in the markers of tissue injury. Histological examination of rat I/R injury showed that all of the drugs offered some protection to the mucosal layer damage compared to no drug treatment. Given i.v., the sPLA2 inhibitor was more effective than either the COX-1 or COX-2 inhibitors in preventing rat I/R injury. These results indicate that a potent new inhibitor of sPLA2 (group IIa) protects the rat small intestine from I/R injury after oral or intravenous administration. COX-2 and LTC4 inhibitors also showed some beneficial effects against intestinal I/R injury. Our study suggests that sPLA2 (Group IIa) may have a pathogenic role in intestinal I/R in rats. Keywords: Gut ischaemia–reperfusion, sPLA2, COX-1, COX-2, LTC4, neutropenia, inflammation, histopathology, pharmacokinetics Introduction Intestinal ischaemia occurs as a result of inadequate systemic blood flow or local vascular abnormalities, and the metabolic demand of the tissue exceeds the delivery of oxygen. Bowel obstruction, abdominal aortic aneurism, haemorrhagic shock, sepsis and traumatic injury can all induce intestinal ischaemia (Fink, 1991; Christenson et al., 1996; Tadros et al., 2000; Wattanasirichaigoon et al., 2000). Diseases such as necrotising enterocolitis, mesenteric insufficiency in the elderly and intestinal dysfunction following bowel transplantation are thought to have a component of ischaemia–reperfusion (I/R) in their pathogenesis (Haglund et al., 1987; Schoenberg & Beger, 1993). Reperfusion of blood to an ischaemic tissue further increases acute ischaemic injury (Granger et al., 1981). In addition to damaging the bowel, intestinal I/R injury can induce pathology at sites remote from the initial injury (Chiu et al., 1970; Koike et al., 1992b; Poggetti et al., 1992; Sun et al., 1999). Intestinal I/R can lead to adult respiratory distress syndrome and multiple organ dysfunction syndrome (MODS) (Sheng et al., 1991). Reperfusion injury is caused by the release of a variety of endogenous agents including oxygen radicals (Granger et al., 1986; Arumugam et al., 2002a), polymorphonuclear leucocytes (PMNs) (Grisham et al., 1986), tumour necrosis factor-alpha (TNF-α) (Caty et al., 1990), leukotrienes (Karasawa et al., 1991), platelet activating factor (PAF) (Kim et al., 1995) and complement products (Wada et al., 2001; Arumugam et al., 2002b). Phospholipases A2 (PLA2) are also important components of the inflammatory response in intestinal I/R injury, although it is not known precisely which specific subtype(s) of this enzyme family are involved. PLA2-mediated tissue injury results through either a direct action of the enzyme(s) or through subsequent actions of its products, which include PAF, leukotrienes, lipoxins, prostaglandins and thromboxanes (Chang et al., 1987). Evidence in support of the role of PLA2 in intestinal I/R has been shown in several studies using the nonspecific PLA2 inhibitor quinacrine (Otamiri et al., 1987, 1988; Otamiri & Tagesson, 1989; Koike et al., 1992a), which reduced manifestations of gut I/R injury. The PLA2 inhibitor used in the present study is an orally active, potent inhibitor of group IIa secretory PLA2 (sPLA2) (Hansford et al., 2003). Group IIa sPLA2 is a human enzyme reported to induce lung injury after intestinal I/R (Koike et al., 2000). Although numerous agents are reported to inhibit ‘PLA2' activity via different mechanisms, there are actually only a handful of bona fide inhibitors (Balsinde et al., 1999) of this specific isoform found in human platelets and synoviocytes. We have shown that this particular sPLA2 (group IIa) inhibitor is highly selective for the group IIa enzyme and has potent anti-inflammatory activity in rats (Hansford et al., 2003; unpublished observations). PLA2 hydrolyses membrane phosphoglycerides to liberate free fatty acids (arachidonic acid) and lysophospholipids (Scheuer, 1989). Cyclooxygenases are involved in the biosynthesis of prostaglandins from arachidonic acid. Two isoforms of the enzyme have been described: cyclooxygenase-1 (COX-1), which is constitutively expressed in most cells and required for physiological functions, and cyclooxygenase-2 (COX-2), which is an inducible form arising in response to inflammatory stimuli (Feng et al., 1993). sPLA2 regulates the release of arachidonic acid (AA) from membrane phospholipids, while COX converts AA to prostaglandins. Accumulating evidence suggests that sPLA2-IIa and sPLA2-V are functionally coupled with COX-1 and COX-2 pathways for prostaglandin biosynthesis (Murakami et al., 1999). To determine the roles of COX-1 and COX-2 in intestinal I/R injury, the present study used flunixin meglumine (Flunixin, Mavlab P/L, Brisbane, Australia) and celecoxib (Celebrex, Pfizer, Australia). Flunixin is a relatively selective COX-1 inhibitor (Brideau et al., 2001) commonly used for the management of intestinal ischaemia, colic and endotoxemia in equids (Jochle et al., 1989; Semrad et al., 1993), while celebrex is a relatively selective inhibitor of COX-2 approved for the treatment of rheumatism and osteoarthritis (Davies et al., 2000). The other major metabolites of the arachidonate pathway are the leukotrienes, which are generated by the action of lipoxygenases (Bingham & Austen, 1999). Lipoxygenase inhibitors and leukotriene B4 receptor antagonists have frequently been investigated in animal models of intestinal I/R (Karasawa, et al., 1991; Goldman et al., 1992; Mangino et al., 1994; Kirschner et al., 1995). The cysteinyl leukotrienes are also elevated in the bronchoalveolar lavage fluid of patients with adult respiratory distress syndrome (Stephenson et al., 1988), a common consequence of intestinal I/R. Zafirlukast (Accolate, Zeneca) is a potent and selective cysteinyl leukotriene receptor antagonist (Calhoun, 1998) and was also used in the present study as a comparator drug. This study sought to test the effectiveness of a new sPLA2 (group IIa) inhibitor in alleviating intestinal I/R-induced injury. As a comparison to this sPLA2 blockade, this study also investigated the relative contribution of a number of inflammatory mediators in intestinal I/R by selectively blocking different stages of the eicosanoid inflammatory cascade. This was achieved with a relatively selective COX-2 inhibitor, a predominantly COX-1 inhibitor and a cysteinyl leukotriene receptor LTC4 antagonist.

1. We investigated the inhibitory effects of the cysteinyl leukotriene (CysLT1) receptor antagonists, pranlukast and zafirlukast, on 35SO4 labelled mucus output, in vitro, in guinea-pig trachea, induced by leukotriene D4 (LTD4) or by antigen challenge of sensitized animals. Agonists and antagonists were administered mucosally, except in selected comparative experiments where drugs were administered both mucosally and serosally to assess the influence of the epithelium on evoked-secretion. 2. LTD4 increased 35SO4 output in a concentration-related manner with a maximal increase of 23 fold above controls at 100 microM and an approximate EC50 of 2 microM. Combined mucosal and serosal addition of LTD4 did not significantly affect the secretory response compared with mucosal addition alone. Neither LTC4 nor LTE4 (10 microM each) affected 35SO4 output. Pranlukast or zafirlukast significantly inhibited 10 microM LTD4-evoked 35SO4 output in a concentration-dependent fashion, with maximal inhibitions of 83% at 10 microM pranlukast and 78% at 10 microM zafirlukast, and IC50 values of 0.3 microM for pranlukast and 0.6 microM for zafirlukast. Combined mucosal and serosal administration of the antagonists (5 microM each) gave degrees of inhibition of mucosal-serosal 10 microM LTD4-evoked 35SO4 output similar to those of the drugs given mucosally. Pranlukast (0.5 microM) caused a parallel rightward shift of the LTD4 concentration-response curve with a pKB of 7. Pranlukast did not inhibit ATP-induced 35SO4 output. 3. Ovalbumin (10-500 microg ml(-1) challenge of tracheae from guinea-pigs actively sensitized with ovalbumin caused a concentration-related increase in 35SO4 output with a maximal increase of 20 fold above vehicle controls at 200 microg ml(-1). The combination of the antihistamines pyrilamine and cimetidine (0.1 mM each) did not inhibit ovalbumin-induced 35SO4 output in sensitized guinea-pigs. Neither mucosal (10 microM or 100 microM) nor mucosal-serosal (100 microM) histamine had any significant effect on 35SO4 output. 4. Pranlukast or zafirlukast (5 microM each) significantly suppressed ovalbumin-induced secretion in tracheae from sensitized guinea-pigs by 70% and 65%, respectively. 5 We conclude that LTD4 or ovalbumin challenge of sensitized animals provokes mucus secretion from guinea-pig trachea in vitro and this effect is inhibited by the CysLT1 receptor antagonists pranlukast and zafirlukast. These antagonists may be beneficial in the treatment of allergic airway diseases in which mucus hypersecretion is a clinical symptom, for example asthma and allergic rhinitis.