C5a, but not C3a, increases VEGF secretion in ARPE-19 human retinal pigment epithelial cells.

PURPOSE: We examined the potential for the pro-inflammatory complement proteins C5a and C3a to increase VEGF expression in ARPE-19 cells. MATERIALS AND METHODS: Expression of complement receptors in ARPE-19 cells was evaluated by RT-PCR. VEGF secretion from ARPE-19 cells treated with C5a or C3a was determined by ELISA. RESULTS: C5a and C3a receptor, but not C5L2, were detected in human eye tissue and ARPE-19 cells. C5a, but not C3a, treatment increased VEGF secretion from ARPE-19 cells, an effect inhibited by the C5aR antagonist, NDT 9513727. CONCLUSIONS: C5a receptor mediates increased VEGF secretion from ARPE-19 cells, suggesting a role for the C5a receptor in the pathogenesis of macular degeneration.

Characterization of N-(adamantan-1-ylmethyl)-5-[(3R-amino-pyrrolidin-1-yl)methyl]-2-chloro-benzamide, a P2X7 antagonist in animal models of pain and inflammation.

Recent evidence suggests that the P2X(7) receptor may play a role in the pathophysiology of preclinical models of pain and inflammation. Therefore, pharmacological agents that target this receptor may potentially have clinical utility as anti-inflammatory and analgesic therapy. We investigated and characterized the previously reported P2X(7) antagonist N-(adamantan-1-ylmethyl)-5-[(3R-amino-pyrrolidin-1-yl)methyl]-2-chloro-benzamide, hydrochloride salt (AACBA; GSK314181A). In vitro, AACBA was a relatively potent inhibitor of both human P2X(7)-mediated calcium flux and quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triemethylammonio)propyl]-diiodide (YO-PRO-1) uptake assays, with IC(50) values of approximately 18 and 85 nM, respectively. Compared with the human receptor, AACBA was less potent at the rat P2X(7) receptor, with IC(50) values of 29 and 980 nM in the calcium flux and YO-PRO-1 assays, respectively. In acute in vivo models of pain and inflammation, AACBA dose-dependently reduced lipopolysaccharide-induced plasma interleukin-6 release and prevented or reversed carrageenan-induced paw edema and mechanical hypersensitivity. In chronic in vivo models of pain and inflammation, AACBA produced a prophylactic, but not therapeutic-like, prevention of the clinical signs and histopathological damage of collagen-induced arthritis. Finally, AACBA could not reverse L(5) spinal nerve ligation-induced tactile allodynia when given therapeutically. Consistent with previous literature, these results suggest that P2X(7) receptors do play a role in animal models of pain and inflammation. Further study of P2X(7) antagonists both in preclinical and clinical studies will help elucidate the role of the P2X(7) receptor in pain and inflammatory mechanisms and may help identify potential clinical benefits of such molecules.

Identification and characterization of NDT 9513727 [N,N-bis(1,3-benzodioxol-5-ylmethyl)-1-butyl-2,4-diphenyl-1H-imidazole-5-methanamine], a novel, orally bioavailable C5a receptor inverse agonist.

The complement system represents an innate immune mechanism of host defense that has three effector arms, the C3a receptor, the C5a receptor (C5aR), and the membrane attack complex. Because of its inflammatory and immune-enhancing properties, the biological activity of C5a and its classical receptor have been widely studied. Because specific antagonism of the C5aR could have therapeutic benefit without affecting the protective immune response, the C5aR continues to be a promising target for pharmaceutical research. The lack of specific, potent and orally bioavailable small-molecule antagonists has limited the clinical investigation of the C5aR. We report the discovery of NDT 9513727 [N,N-bis(1,3-benzodioxol-5-ylmethyl)-1-butyl-2,4-diphenyl-1H-imidazole-5-methanamine], a small-molecule, orally bioavailable, selective, and potent inverse agonist of the human C5aR. NDT 9513727 was discovered based on the integrated use of in vitro affinity and functional assays in conjunction with medicinal chemistry. NDT 9513727 inhibited C5a-stimulated responses, including guanosine 5'-3-O-(thio)triphosphate binding, Ca(2+) mobilization, oxidative burst, degranulation, cell surface CD11b expression and chemotaxis in various cell types with IC(50)s from 1.1 to 9.2 nM, respectively. In C5a competition radioligand binding experiments, NDT 9513727 exhibited an IC(50) of 11.6 nM. NDT 9513727 effectively inhibited C5a-induced neutropenia in gerbil and cynomolgus macaque in vivo. The findings suggest that NDT 9513727 may be a promising new entity for the treatment of human inflammatory diseases.