NOVEL C5A RECEPTOR ANTAGONISTS REGULATE NEUTROPHIL FUNCTIONS IN-VITROAND IN-VIVO

Novel recombinant human C5a receptor antagonists were discovered throu gh modification of the C terminus of C5a. The C5a(1-71)T(1)M,C27S,Q(71 )C monomer, (C5aRAM; CGS 27913), was a pure and potent functional anta gonist. The importance of a C-terminal cysteine at position 71 to anta gonist propel-ties of C5aRAM was confirmed by studying C5a(1-71) deriv atives with replacements of Q(71), C5a derivatives of various lengths (70-74) with C-terminal cysteines, and C5a derivatives of various leng ths (71-74) with Q(71)C replacements, The majority of C5a(1-71)Q(71) d erivatives were agonists (C5a-like) in the human neutrophil C5a-induce d intracellular calcium mobilization assay. The C5a(1-71)Q(71)C deriva tive was an antagonist. C5a derivatives of lengths 73 and 74 with C-te rminal cysteines were agonists, while lengths 70 to 72 were antagonist s. C5a derivatives of lengths 72, 73, and 74 with Q(71)C replacements were agonists, while, again, C5a(1-71)Q(71)C was an antagonist. C5aRAM and its adducts, including its dimer, C5aRAD (CGS 32359), were pure a ntagonists, Additionally, C5aRAM and C5aRAD inhibited binding of I-125 -labeled recombinant human C5a to neutrophil membranes (K-i = 79 and 2 pM, respectively), C5a-stimulated neutrophil intracellular calcium mo bilization (8 and 13 nM), CD11b integrin up-regulation (10 and 1 nM), superoxide generation (182 and 282 nM), lysozyme release (1 and 2 mu M ), and chemotaxis (11 and 7 mu M). In vivo, intradermal injection of C 5aRAM inhibited C5a-induced dermal edema in rabbits. Furthermore, a 5- mg/kg i.v. bolus of C5aRAD significantly inhibited C5a-induced neutrop enia in micropigs when challenged with C5a 30 min after C5aRAD adminis tration. C5aRAM and C5aRAD are novel, potent C5a receptor antagonists devoid of agonist or proinflammatory activity with demonstrated effica cy in vitro and in vivo.