The complement system is thought to be involved in the pathogenesis of numerous neurological diseases, although its precise role remains controversial. In this study we used orally active C5a receptor antagonists (PMX53 and PMX205) developed in our laboratories in a rat model of 3-nitropropionic acid (3-NP) -induced Huntington's disease. Administration of the C5a antagonists (10 mg/kg/day, oral) either 48 h pre- or 48 h post-toxin significantly reduced body weight loss, anorexia, and behavioral and motor deficits associated with 3-NP intoxication. Striatal lesion size, apoptosis, neutrophil infiltration, and hemorrhage were also significantly reduced in C5a antagonist-treated rats. Immunohistochemical analysis demonstrated marked deposition of C3 and C9, and up-regulation of C5a receptors on neuronal cells at the time of lesion formation. Inhibition of prostaglandins or TNF-alpha with ibuprofen or infliximab had no effect in this model. The C5a antagonists did not affect 3-NP-induced cell death when added directly to rat striatal neuronal cultures, indicating a secondary mechanism of action in vivo. Our findings demonstrate for the first time that complement activation in the brain, particularly C5a, is a key event in the pathogenesis of this disease model, and suggest a future role for inhibitors of C5a in the treatment of neurodegenerative diseases.

Human C5a is a plasma protein with potent chemoattractant and pro-inflammatory properties, and its overexpression correlates with severity of inflammatory diseases. C5a binds to its G protein-coupled receptor (C5aR) on polymorphonuclear leukocytes (PMNLs) through a high-affinity helical bundle and a low-affinity C terminus, the latter being solely responsible for receptor activation. Potent and selective C5a antagonists are predicted to be effective anti-inflammatory drugs, but no pharmacophore for small molecule antagonists has yet been developed, and it would significantly aid drug design. We have hypothesized that a turn conformation is important for activity of the C terminus of C5a and herein report small cyclic peptides that are stable turn mimics with potent antagonism at C5aR on human PMNLs. A comparison of solution structures for the C terminus of C5a, small acyclic peptide ligands, and cyclic antagonists supports the importance of a turn for receptor binding. Competition between a cyclic antagonist and either C5a or an acyclic agonist for C5aR on PMNLs supports a common or overlapping binding site on the C5aR. Structure-activity relationships for 60 cyclic analogs were evaluated by competitive radioligand binding with C5a (affinity) and myeloperoxidase release (antagonist potency) from human PMNLs, with 20 compounds having high antagonist potencies (IC 50 , 20 nM-1 M). Computer modeling comparisons reveal that potent antagonists share a common cyclic backbone shape, with affinitydetermining side chains of defined volume projecting from the cyclic scaffold. These results define a new pharmacophore for C5a antagonist development and advance our understanding of ligand recognition and receptor activation of this G proteincoupled receptor.

1 Complement activation is implicated in the pathogenesis of intestinal ischaemia-reperfusion injury (I/R), although the relative importance of individual complement components is unclear. A C3a receptor antagonist N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (C3aRA) has been compared with a C5a receptor antagonist (C5aRA), AcF-[OPdChaWR], in a rat model of intestinal I/R. 2 C3aRA (IC 50 ¼ 0.15 mM) and C5aRA (IC 50 ¼ 0.32 mM) bound selectively to human polymorphonuclear leukocyte (PMN) C3a and C5a receptors, respectively. Effects on circulating neutrophils and blood pressure in the rat were also assessed. 3 Anaesthetised rats, subjected to intestinal ischaemia (30 min) and reperfusion (120 min), were administered intravenously with either (A) the C3aRA (0.1-1.0 mg kg À1 ); the C5aRA (1.0 mg kg À1 ); the C3aRA þ C5aRA (each 1.0 mg kg À1 ); or vehicle, 45 min prior, or (B) the C3aRA (1.0 mg kg À1 ) or vehicle, 120 min prior to reperfusion. 4 The C3aRA and C5aRA, administered 45 min prior to reperfusion, displayed similar efficacies at ameliorating several disease markers (increased oedema, elevated ALT levels and mucosal damage) of rat intestinal I/R. The combination drug treatment did not result in greater injury reduction than either antagonist alone. However, doses of the C3aRA (0.01-10 mg kg À1 ) caused transient neutropaenia, and the highest dose (10 mg kg À1 ) also caused a rapid and transient hypertension. 5 The C3aRA (1.0 mg kg À1 ), delivered 120 min prior to reperfusion to remove the global effect of C3aRA-induced neutrophil sequestration, did not attenuate the markers of intestinal I/R, despite persistent C3aR antagonism at this time. 6 C3aR antagonism does not appear to be responsible for the anti-inflammatory actions of this C3aRA in intestinal I/R in the rat. Instead, C3aRA-mediated global neutrophil tissue sequestration during ischaemia and early reperfusion may account for the protective effects observed.

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We have previously shown that complement factor 5a (C5a) plays a role in the pathogenesis of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats by using the selective,

1 A new group IIa sPLA 2 inhibitor was compared with selective inhibitors of COX-1, COX-2 and an LTC 4 antagonist for effects on local and remote tissue injuries following ischaemia and reperfusion (I/R) of the small intestine in rats.2 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. 3 Preischaemic treatment with the inhibitor of sPLA 2 (Group IIa), at 5 mg kg À1 i.v. or 10 mg kg À1 p.o. significantly inhibited I/R-induced neutropenia, the elevation of serum levels of AST, intestinal oedema and hypotension.4 Pretreatment with the COX-2 inhibitor celebrex (10 mg kg À1 i.v.) and the LTC 4 antagonist zafirlukast (1 mg kg À1 i.v.) also showed marked improvement with I/R-induced AST, oedema and neutropenia. Hypotension was only reduced by the LTC 4 antagonist. The COX-1 inhibitor flunixin (1 mg kg À1 i.v.) did not effect improvement in the markers of tissue injury. 5 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 sPLA 2 inhibitor was more effective than either the COX-1 or COX-2 inhibitors in preventing rat I/R injury. 6 These results indicate that a potent new inhibitor of sPLA 2 (group IIa) protects the rat small intestine from I/R injury after oral or intravenous administration. COX-2 and LTC 4 inhibitors also showed some beneficial effects against intestinal I/R injury. Our study suggests that sPLA 2 (Group IIa) may have a pathogenic role in intestinal I/R in rats.

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