Ian A. Shiels

A Potent Human C5a Receptor Antagonist Protects against Disease Pathology in a Rat Model of Inflammatory Bowel Disease

The complement system is implicated in the pathogenesis of human inflammatory bowel disease, but the specific role of C5a has never been examined. We have compared the efficacy of an orally active human C5a receptor antagonist (AcPhe[Orn-Pro-d-cyclohexylalanine-Trp-Arg]), prednisolone, and infliximab against trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats. The drugs were administered either 2 days before or 24 h after TNBS instillation, and rats were then examined after 8 days. Drug-free colitis control rats showed severe disease pathology with significant mortality (39%). Rats pre or posttreated with the C5a antagonist (10 mg/kg/day peroral, 0.3 mg/kg/day s.c.) had reduced mortality and significantly improved macroscopic scores, colon edema, colon myeloperoxidase levels, reduced concentrations of TNF-α levels in the colon and serum, and had greater food intake resulting in greater weight gains than colitis-only rats. Rats pretreated with prednisolone (1 mg/kg/day s.c.) displayed significant improvement in parameters measured, but posttreatment was ineffective. Single dose pretreatment with the TNF-α inhibitor infliximab (3 mg/kg i.v.) also had significant improvements in the parameters measured. Rats pretreated with a combination of the C5a antagonist and prednisolone showed no greater improvements than either drug alone. These findings suggest a central role for complement, particularly C5a, in the pathology of TNBS-induced colitis in rats, indicating a possible therapeutic role for C5a antagonists in inflammatory bowel disease.

Therapeutic activity of C5a receptor antagonists in a rat model of neurodegeneration

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 Huntingtons 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 upregulation of C5a receptors on neuronal cells at the time of lesion formation. Inhibition of prostaglandins or TNF‐α 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.—Woodruff, T. M., Crane, J. W., Proctor, L. M., Buller, K. M., Shek, A. B., de Vos, K., Pollitt, S., Williams, H. M., Shiels, I. A., Monk, P. N., Taylor, S. M. Therapeutic activity of C5a receptor antagonists in a rat model of neurodegeneration. FASEB J. 20, 1407–1417 (2006)

Comparative anti-inflammatory activities of antagonists to C3a and C5a receptors in a rat model of intestinal ischaemia/reperfusion injury

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. C3aRA (IC50=0.15 μM) and C5aRA (IC50=0.32 μM) 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. 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−1); the C5aRA (1.0 mg kg−1); the C3aRA+C5aRA (each 1.0 mg kg−1); or vehicle, 45 min prior, or (B) the C3aRA (1.0 mg kg−1) or vehicle, 120 min prior to reperfusion. 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−1) caused transient neutropaenia, and the highest dose (10 mg kg−1) also caused a rapid and transient hypertension. The C3aRA (1.0 mg kg−1), 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. 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.

Effects of a new C5a receptor antagonist on C5a- and endotoxin-induced neutropenia in the rat.

A new C5a receptor antagonist, the cyclic peptide Phe‐[Orn‐Pro‐D‐cyclohexylalanine‐Trp‐Arg], (F‐[OPdChaWR]), was tested for its ability to antagonize the neutropenic effects of both C5a and endotoxin in rats. Human recombinant C5a (2 μg kg−1 i.v.) caused rapid neutropenia, characterized by an 83% decrease in circulating polymorphonuclear leukocytes (PMNs) at 5 min. Administration of F‐[OPdChaWR] (0.3–3 mg kg−1 i.v.), did not affect the levels of circulating PMNs but, when given 10 min prior to C5a, it inhibited the C5a‐induced neutropenia by up to 70%. Administration of E. Coli lipopolysaccharide (LPS, 1 mg kg−1 i.v.) also caused neutropenia with an 88% decrease in circulating PMNs after 30 min. When rats were pretreated with F‐[OPdChaWR] (0.3–10 mg kg−1 i.v.) 10 min prior to LPS, there was a dose‐dependent antagonism of the neutropenia caused by LPS, with up to 69% reversal of neutropenia observed 30 min after LPS administration. These findings suggest that C5a receptor antagonists may have therapeutic potential in the many diseases known to involve either endotoxin or C5a.

Complement C5a inhibition reduces atherosclerosis in ApoE−/− mice

The complement C5a receptor, CD88, is present on many of the cells found within human atherosclerotic plaques, but little is known about the role of C5a in atherogenesis. Using real‐time PCR, we determined that ApoE–/– mice fed a normal diet express more aortic CD88 mRNA compared with controls, and this increase coincides with atherosclerotic lesion development (P< 0.001 for 3‐ vs. 25‐wk‐old animals). Conversely, mRNA expression of the alternative C5a receptor, C5L2, in aortas of ApoE–/– mice, was lower than controls at all time points. Using immunohistochemistry, we confirmed the presence of CD88 on macrophages, smooth muscle cells, and activated endothelial cells in plaques from brachiocephalic arteries. Treatment of ApoE–/– mice with a CD88 antagonist (PMX53; 3 mg/kg s.c. 3×/wk plus 1 mg/kg/d p.o.) for 25 wk reduced lesion size and lipid content in the plaque by ~40% (P<0.05). Our study provides evidence for a proatherogenic role for C5a and identifies the CD88 antagonist PMX53 as a potential antiatherosclerotic drug.—Manthey, H. D., Thomas, A. C., Shiels, I. A., Zernecke, A., Woodruff, T. M., Rolfe, B., Taylor, S. M. Complement C5a inhibition reduces atherosclerosis in ApoE–/– mice. FASEB J. 25, 2447–2455 (2011). www.fasebj.org

Inhibition of immune‐complex mediated dermal inflammation in rats following either oral or topical administration of a small molecule C5a receptor antagonist

Initiation of a peritoneal Arthus reaction by deposition of immune‐complexes results in vascular leakage, polymorphonuclear leukocyte (PMN) infiltration, and tumour necrosis factor α (TNFα) and interleukin‐6 (IL‐6) production. We now demonstrate in rats that oral administration of the C5a receptor antagonist AcPhe[Orn‐Pro‐D‐Cyclohexylalanine‐Trp‐Arg] (AcF‐[OPdChaWR]; 1 – 10 mg kg−1 30 min prior to immune‐complex deposition) inhibits these inflammatory markers in the peritoneal Arthus reaction. Initiation of a dermal Arthus reaction resulted in a significant increase in vascular leakage, PMN infiltration, systemic production of TNFα and pathological changes in the dermis. Pretreatment of rats with AcF‐[OPdChaWR] either intravenously (1 mg kg−1 10 min prior to immune‐complex deposition) or orally (1 – 10 mg kg−1 30 min prior to immune‐complex deposition) significantly inhibited immune‐complex mediated dermal vascular leakage and systemic cytokine production. Topical pretreatment with AcF‐[OPdChaWR] (400 μg site−1 in 10% dimethyl sulphoxide 10 min prior to immune‐complex deposition) also inhibited vascular leakage, as well as histopathological changes associated with a dermal Arthus reaction. Oral administration of 3 mg kg−1 AcF‐[OPdChaWR] resulted in the appearance of the drug in plasma within 5 min, with peak blood levels ∼0.3 μM reached within 20 min. The plasma elimination half‐life was ∼70 min. The oral activity and bioavailability of AcF‐[OPdChaWR], its activity when applied topically to the skin, suggest that small molecule C5a receptor antagonists may have therapeutic utility in dermal inflammatory disorders involving complement activation. This is the first demonstration for either an orally or topically active C5a receptor antagonist, and suggests that small molecule C5a antagonists may have therapeutic utility when given by multiple routes of application.

Treatment of osteoarthritis

This invention relates to methods of treatment of osteoarthritis, and especially to treatment of this condition with cyclic peptidic and peptidomimetic compounds which have the ability to modulate the activity of G protein-coupled receptors. The compounds preferably act as antagonists of the C5a receptor, and are active against C5a receptors on polymorphonuclear leukocytes and macrophages. Particularly preferred compounds for use in the invention are disclosed.

Comparative protection against rat intestinal reperfusion injury by a new inhibitor of sPLA2, COX‐1 and COX‐2 selective inhibitors, and an LTC4 receptor antagonist

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.

The Effects of Salbutamol, Beclomethasone, and Dexamethasone on Fibronectin Expression by Cultured Airway Smooth Muscle Cells

Possible mechanisms of adverse drug effects in asthma include worsening of cellular hyperplasia and stimulation of extracellular matrix deposition. In this study, salbutamol, dexamethasone and beclomethasone were investigated to ascertain their ability to induce mitogenesis and stimulate fibronectin expression in cultured canine airway smooth muscle cells. In cells maintained in serum-free media for 72 h, salbutamol (1 nM–10 μM) caused mitogenesis. The control cells had 2.57 ± 0.34 × 105 cells per ml (mean ± SEM, N = 13), while salbutamol (1 μM) caused a maximal increase in cell number to 3.57 ± 0.23 × 105 cells/ml (P < 0.01). In cells stimulated to replicate by addition of either fetal bovine serum or canine serum, no additional mitogenic effect of salbutamol was seen. Salbutamol did not have a detectable quantitative effect on fibronectin matrix expression. The glucocorticoids, beclomethasone and dexamethasone, significantly altered fibronectin expression by cultured airway smooth muscle cells. Beclomethasone increased fibronectin expression, while dexamethasone decreased expression.

Airway smooth muscle proliferation in asthma: The potential of vascular leakage to contribute to pathogenesis

Proliferation of the non-vascular smooth muscle in the walls of the bronchi and bronchioles is a prominent histopathological feature of asthma and is thought to contribute to airway hyperreactivity and narrowing. Increased vascular permeability with plasma leakage is also a feature of asthma pathology and causes submucosal oedema. We hypothesize that, in asthmatics, the accumulation of enriched plasma in the environment surrounding airway smooth muscle promotes respiratory smooth muscle mitogenesis and hyperplasia. This situation represents the in vivo correlate of the increase in airway smooth muscle cell growth seen in vitro with increasing concentrations of serum in the culture medium. Thus, we hypothesize that vascular leakage in the airways in asthma is a primary pathogenic event leading to airway smooth muscle hyperplasia and hypertrophy, and consequently airway narrowing, promoting the characteristic bronchial hyperreactivity associated with narrowing of the airway lumen.