RESUMO
BACKGROUND AND PURPOSE: DF 2156A is a new dual inhibitor of IL-8 receptors CXCR1 and CXCR2 with an optimal pharmacokinetic profile. We characterized its binding mode, molecular mechanism of action and selectivity, and evaluated its therapeutic potential. EXPERIMENTAL APPROACH: The binding mode, molecular mechanism of action and selectivity were investigated using chemotaxis of L1.2 transfectants and human leucocytes, in addition to radioligand and [(35) S]-GTPγS binding approaches. The therapeutic potential of DF 2156A was evaluated in acute (liver ischaemia and reperfusion) and chronic (sponge-induced angiogenesis) experimental models of inflammation. KEY RESULTS: A network of polar interactions stabilized by a direct ionic bond between DF 2156A and Lys(99) on CXCR1 and the non-conserved residue Asp(293) on CXCR2 are the key determinants of DF 2156A binding. DF 2156A acted as a non-competitive allosteric inhibitor blocking the signal transduction leading to chemotaxis without altering the binding affinity of natural ligands. DF 2156A effectively and selectively inhibited CXCR1/CXCR2-mediated chemotaxis of L1.2 transfectants and leucocytes. In a murine model of sponge-induced angiogenesis, DF 2156A reduced leucocyte influx, TNF-α production and neovessel formation. In vitro, DF 2156A prevented proliferation, migration and capillary-like organization of HUVECs in response to human IL-8. In a rat model of liver ischaemia and reperfusion (I/R) injury, DF 2156A decreased PMN and monocyte-macrophage infiltration and associated hepatocellular injury. CONCLUSION AND IMPLICATIONS: DF 2156A is a non-competitive allosteric inhibitor of both IL-8 receptors CXCR1 and CXCR2. It prevented experimental angiogenesis and hepatic I/R injury in vivo and, therefore, has therapeutic potential for acute and chronic inflammatory diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8B/antagonistas & inibidores , Sulfonamidas/farmacologia , Animais , Anti-Inflamatórios/farmacocinética , Anti-Inflamatórios/uso terapêutico , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Quimiotaxia de Leucócito/efeitos dos fármacos , Modelos Animais de Doenças , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucina-8/metabolismo , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Leucócitos/metabolismo , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Mutagênese Sítio-Dirigida , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Ratos , Ratos Sprague-Dawley , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologia , Pele/irrigação sanguínea , Sulfonamidas/farmacocinética , Sulfonamidas/uso terapêuticoRESUMO
BACKGROUND AND PURPOSE: Neutrophil migration into tissues is involved in the genesis of inflammatory pain. Here, we addressed the hypothesis that the effect of CXC chemokines on CXCR1/2 is important to induce neutrophil migration and inflammatory hypernociception. EXPERIMENTAL APPROACH: Mice were treated with a non-competitive allosteric inhibitor of CXCR1/2, DF 2162, and neutrophil influx and inflammatory hypernociception were assessed by myeloperoxidase assay and electronic pressure meter test, respectively, in various models of inflammation. KEY RESULTS: DF 2162 inhibited neutrophil chemotaxis induced by CXCR1/2 ligands but had no effect on CXCL8 binding to neutrophils. A single mutation of the allosteric site at CXCR1 abrogated the inhibitory effect of DF 2162 on CXCL-8-induced chemotaxis. Treatment with DF 2162 prevented influx of neutrophils and inflammatory hypernociception induced by CXCL1 in a dose-dependent manner. The compound inhibited neutrophil influx and inflammatory hypernociception induced by carrageenan, lipopolysaccharide and zymosan, but not hypernociception induced by dopamine and PGE(2). DF 2162 had a synergistic effect with indomethacin or the absence of TNFR1 to abrogate carrageenan-induced hypernociception. Treatment with DF 2162 diminished neutrophil influx, oedema formation, disease score and hypernociception in collagen-induced arthritis. CONCLUSIONS AND IMPLICATIONS: CXCR1/2 mediates neutrophil migration and is involved in the cascade of events leading to inflammatory hypernociception. In addition to modifying fundamental pathological processes, non-competitive allosteric inhibitors of CXCR1/2 may have the additional benefit of providing partial relief for pain and, hence, may be a valid therapeutic target for further studies aimed at the development of new drugs for the treatment of rheumatoid arthritis.
Assuntos
Analgésicos/farmacologia , Benzenoacetamidas/farmacologia , Hiperalgesia/prevenção & controle , Inflamação/complicações , Mesilatos/farmacologia , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Receptores de Interleucina-8A/antagonistas & inibidores , Receptores de Interleucina-8B/antagonistas & inibidores , Analgésicos/uso terapêutico , Animais , Artrite Experimental/complicações , Artrite Experimental/tratamento farmacológico , Benzenoacetamidas/uso terapêutico , Células Cultivadas , Quimiocina CXCL1/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/farmacologia , Relação Dose-Resposta a Droga , Humanos , Hiperalgesia/etiologia , Hiperalgesia/imunologia , Indometacina/farmacologia , Inflamação/tratamento farmacológico , Inflamação/imunologia , Interleucina-8/metabolismo , Masculino , Mesilatos/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Neutrófilos/imunologia , Medição da Dor , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , TransfecçãoRESUMO
BACKGROUND AND AIMS: Cholera toxin B subunit (CT-B) is a powerful modulator of immune responses. The authors have previously demonstrated that oral administration of recombinant CT-B (rCT-B) is able to prevent and cure the Crohn's disease (CD)-like trinitrobenzene sulfonic acid (TNBS) mediated colitis. In this study they extended their observations and examined if rCT-B interferes with the molecular signaling underlying the Th1 type response both in TNBS colitis and in ex vivo human CD explants. METHODS: TNBS treated mice were fed with rCT-B, and IFN-gamma and IL-12 production by colonic lamina propria mononuclear cells (LPMC) was examined by ELISA. In vitro culture of mucosal explants from CD patients and non-inflammatory bowel disease controls, pre-incubated with rCT-B, were examined for IFN-gamma and IL-12 production by ELISA and semiquantitative reverse transcription polymerase chain reactions. STAT-1, -4, -6 activation and T-bet expression were examined following rCT-B treatment by western blotting both in TNBS treated mice and in human mucosal explants. RESULTS: rCT-B significantly reduced IL-12 and IFN-gamma secretion by LPMC from TNBS treated mice. Consistent with this, rCT-B inhibited both STAT-4 and STAT-1 activation and downregulated T-bet expression. Inhibition of Th1 signaling by CT-B associated with no change in IL-4 synthesis and expression of active STAT-6 indicating that rCT-B does not enhance Th2 cell responses. Moreover, in vitro treatment of CD mucosal explants with rCT-B resulted in reduced secretion of IL-12/IFN-gamma and inhibition of STAT-4/STAT-1 activation and T-bet expression. CONCLUSIONS: These studies indicate that CT-B inhibits mucosal Th1 cell signaling and suggest that rCT-B may be a promising candidate for CD therapy.