Novel C5a Receptor Antagonists Regulate Neutrophil Functions In Vitro and In Vivo

Abstract

Novel recombinant human C5a receptor antagonists were discovered through modification of the C terminus of C5a. The C5a1–71T1M,C27S,Q71C monomer, (C5aRAM; CGS 27913), was a pure and potent functional antagonist. The importance of a C-terminal cysteine at position 71 to antagonist properties of C5aRAM was confirmed by studying C5a1–71 derivatives with replacements of Q71, C5a derivatives of various lengths (70–74) with C-terminal cysteines, and C5a derivatives of various lengths (71–74) with Q71C replacements. The majority of C5a1–71Q71 derivatives were agonists (C5a-like) in the human neutrophil C5a-induced intracellular calcium mobilization assay. The C5a1–71Q71C derivative was an antagonist. C5a derivatives of lengths 73 and 74 with C-terminal cysteines were agonists, while lengths 70 to 72 were antagonists. C5a derivatives of lengths 72, 73, and 74 with Q71C replacements were agonists, while, again, C5a1–71Q71C was an antagonist. C5aRAM and its adducts, including its dimer, C5aRAD (CGS 32359), were pure antagonists. Additionally, C5aRAM and C5aRAD inhibited binding of 125I-labeled recombinant human C5a to neutrophil membranes (Ki = 79 and 2 pM, respectively), C5a-stimulated neutrophil intracellular calcium mobilization (8 and 13 nM), CD11b integrin up-regulation (10 and 1 nM), superoxide generation (182 and 282 nM), lysozyme release (1 and 2 μM), and chemotaxis (11 and 7 μM). In vivo, intradermal injection of C5aRAM inhibited C5a-induced dermal edema in rabbits. Furthermore, a 5-mg/kg i.v. bolus of C5aRAD significantly inhibited C5a-induced neutropenia in micropigs when challenged with C5a 30 min after C5aRAD administration. C5aRAM and C5aRAD are novel, potent C5a receptor antagonists devoid of agonist or proinflammatory activity with demonstrated efficacy in vitro and in vivo.

Protection of innate immunity by C5aR antagonist in septic mice

“…These compounds have also decreased tissue injury in a model of ischemia and reperfusion (51). By modification of the carboxyl terminus of C5a, others found a monomer (C5aRam) and its dimer (C5aRad) to exhibit potent C5aR antagonistic activity (52). The latter antagonist reduced infarct size and attenuated the inflammatory response in a porcine model of cardiac ischemia/reperfusion injury (53).…”

Discovery and Characterization of a New Class of C5aR1 Antagonists Showing In Vivo Activity

“…These C5a-driven events ultimately result in a reactive increase in blood neutrophils (neutrophilia). 14,15 The increased neutrophil count, in the absence and presence of an hC5aR1 antagonist, was used as an index of neutrophil activation by exogenously administered hC5a. The vehicle or compound (1−10 mg/kg) was administered orally to transgenic KI rats 1.5, 7.5, or 15.5 h prior to being injected intravenously with hC5a.…”

“…Intravenous injection of hC5a to hC5aR1 KI rats activates neutrophils (and other myeloid cells), leading to a rapid and transient reduction in circulating blood neutrophils followed by release of neutrophils from the bone marrow. These C5a-driven events ultimately result in a reactive increase in blood neutrophils (neutrophilia). , The increased neutrophil count, in the absence and presence of an hC5aR1 antagonist, was used as an index of neutrophil activation by exogenously administered hC5a. The vehicle or compound (1–10 mg/kg) was administered orally to transgenic KI rats 1.5, 7.5, or 15.5 h prior to being injected intravenously with hC5a.…”

Disseminated Cryptococcosis Following Eculizumab Therapy: Insight Into Pathogenesis

“…The complement activation also results in the release of potent anaphylatoxins including C5a, which is a proinflammatory mediator [ 1 ]. Cytokine production and antimicrobial activity are influenced by C5a [ 2 ]. However, the harmful anaphylatoxin effect due to excess production of C5a, an uncontrolled host inflammatory response, facilitates infections caused by encapsulated bacteria [ 3 ].…”