RESUMO
Phosphoinositide 3-kinases (PI3K) have long been considered promising drug targets for the treatment of inflammatory and autoimmune disorders as well as cancer and cardiovascular diseases. But the lack of specificity, isoform selectivity and poor biopharmaceutical profile of PI3K inhibitors have so far hampered rigorous disease-relevant target validation. Here we describe the identification and development of specific, selective and orally active small-molecule inhibitors of PI3Kgamma (encoded by Pik3cg). We show that Pik3cg(-/-) mice are largely protected in mouse models of rheumatoid arthritis; this protection correlates with defective neutrophil migration, further validating PI3Kgamma as a therapeutic target. We also describe that oral treatment with a PI3Kgamma inhibitor suppresses the progression of joint inflammation and damage in two distinct mouse models of rheumatoid arthritis, reproducing the protective effects shown by Pik3cg(-/-) mice. Our results identify selective PI3Kgamma inhibitors as potential therapeutic molecules for the treatment of chronic inflammatory disorders such as rheumatoid arthritis.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Dioxóis/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase , Quinoxalinas/uso terapêutico , Tiazolidinedionas/uso terapêutico , Animais , Artrite Reumatoide/induzido quimicamente , Sítios de Ligação , Quimiotaxia de Leucócito/efeitos dos fármacos , Dioxóis/química , Modelos Animais de Doenças , Isoenzimas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos DBA , Camundongos Knockout , Dados de Sequência Molecular , Estrutura Molecular , Peritonite/induzido quimicamente , Peritonite/tratamento farmacológico , Fosfatidilinositol 3-Quinases/química , Quinoxalinas/química , Transdução de Sinais , Relação Estrutura-Atividade , Tiazolidinedionas/químicaRESUMO
We demonstrated recently that P8A-CCL2, a monomeric variant of the chemokine CCL2/MCP-1, is unable to induce cellular recruitment in vivo, despite full activity in vitro. Here, we show that this variant is able to inhibit CCL2 and thioglycollate-mediated recruitment of leukocytes into the peritoneal cavity and recruitment of cells into lungs of OVA-sensitized mice. This anti-inflammatory activity translated into a reduction of clinical score in the more complex inflammatory model of murine experimental autoimmune encephalomyelitis. Several hypotheses for the mechanism of action of P8A-CCL2 were tested. Plasma exposure following s.c. injection is similar for P8A-CCL2 and wild-type (WT) CCL2, ruling out the hypothesis that P8A-CCL2 disrupts the chemokine gradient through systemic exposure. P8A-CCL2 and WT induce CCR2 internalization in vitro and in vivo; CCR2 then recycles to the cell surface, but the cells remain refractory to chemotaxis in vitro for several hours. Although the response to P8A-CCL2 is similar to WT, this finding is novel and suggests that despite the presence of the receptor on the cell surface, coupling to the signaling machinery is retarded. In contrast to CCL2, P8A-CCL2 does not oligomerize on glycosaminoglycans (GAGs). However, it retains the ability to bind GAGs and displaces endogenous JE (murine MCP-1) from endothelial surfaces. Intravital microscopy studies indicate that P8A-CCL2 prevents leukocyte adhesion, while CCL2 has no effect, and this phenomenon may be related to the mechanism. These results suggest that oligomerization-deficient chemokines can exhibit anti-inflammatory properties in vivo and may represent new therapeutic modalities.
Assuntos
Anti-Inflamatórios/farmacologia , Quimiocina CCL2/farmacologia , Quimiocinas/fisiologia , Leucócitos/fisiologia , Animais , Encéfalo/citologia , Circulação Cerebrovascular/fisiologia , Quimiocina CCL2/genética , Quimiocinas/química , Quimiotaxia de Leucócito , Feminino , Engenharia Genética , Heparina/farmacologia , Imunização , Leucócitos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Tioglicolatos/farmacologiaRESUMO
OBJECTIVE: The purpose of this study was to assess the tocolytic effect of AS604872, an orally active, potent, and selective prostanoid prostaglandin F2alpha receptor (FP) antagonist. STUDY DESIGN: Compound AS604872 was characterized and tested for its ability to block uterine contraction and delay preterm parturition in rodent models. RESULTS: AS604872 inhibited spontaneous uterine contractions in pregnant rat near term. In pregnant mouse, AS604872 delayed parturition induced by either the antiprogesterone RU-486 or the endotoxin lipopolysaccharide. Pups from treated mothers were delivered alive. The efficacy of AS604872 was superior to the beta-mimetic drug ritodrine. Combination of AS604872 and ritodrine showed an additive inhibitory effect on spontaneous uterine contractions in rat. CONCLUSION: A selective antagonist of the FP receptor suppresses uterine contractility and delays labor. Our findings identify a new potential modality for the pharmacological management of preterm labor.
Assuntos
Compostos de Bifenilo/farmacologia , Trabalho de Parto Prematuro/prevenção & controle , Receptores de Prostaglandina/antagonistas & inibidores , Sulfonamidas/farmacologia , Tocolíticos/farmacologia , Contração Uterina/efeitos dos fármacos , Animais , Quimioterapia Combinada , Feminino , Humanos , Camundongos , Mifepristona/farmacologia , Gravidez , Ratos , Ratos Sprague-Dawley , Ritodrina/farmacologia , Resultado do TratamentoRESUMO
BACKGROUND: Management of preterm labor by tocolysis remains an unmet medical need. Prostaglandins play a major role in regulation of uterine activity and in molecular mechanisms of human labor and parturition. There is some circumstantial evidence that prostaglandin F2alpha by action through the prostaglandin receptor subtype FP is effective in key events during labor uterine contraction, rupture of membranes and cervical dilation. This role of FP is briefly reviewed. In this study, we tested the hypothesis that an orally active and selective FP antagonist may arrest labor and delay parturition in animal models. METHODS: We examined the effects of a small molecule selective antagonist of the FP receptor (AS604872) in inhibition of spontaneous uterine contraction in pregnant rat near term. We tested AS604872 for its ability to delay preterm birth in a mouse model in which the anti-progestin agent RU486 triggered parturition. RESULTS: By oral or intravenous dosing AS604872 reduced markedly and dose-dependently the spontaneous uterine contractions in late-term pregnant rats at gestational days 19-21. In pregnant mice, AS604872 delayed the preterm birth caused by RU486 administration. The effect was dose-dependent with a significant increase in the mean delivery time of 16 and 33 hours at oral doses of 30 mg/kg and 100 mg/kg, respectively, in the case of labor triggered at gestational day 14. In both models AS604872 appeared more effective than the beta-agonist ritodrine. CONCLUSION: The tocolytic activity displayed by a selective FP receptor antagonist supports a key role for the FP receptor in the pathophysiology of premature birth and demonstrates the therapeutic potential of an FP antagonist for the treatment of preterm labor cases in which uterine hyperactivity plays a dominant role.
Assuntos
Compostos de Bifenilo/administração & dosagem , Dinoprosta/antagonistas & inibidores , Prenhez/efeitos dos fármacos , Nascimento Prematuro/prevenção & controle , Sulfonamidas/administração & dosagem , Tocolíticos/administração & dosagem , Contração Uterina/efeitos dos fármacos , Administração Oral , Animais , Preparações de Ação Retardada , Relação Dose-Resposta a Droga , Feminino , Injeções Intravenosas , Gravidez , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento , Contração Uterina/fisiologiaRESUMO
Several lines of evidence support the hypothesis that c-Jun N-terminal kinase (JNKs) plays a critical role in a wide range of diseases including cell death (apoptosis)-related disorders (neurodegenerative diseases, brain, heart, and renal ischemia, epilepsy) and inflammatory disorders (multiple sclerosis, rheumatoid arthritis, inflammatory bowel diseases). Screening of our internal compound collection for inhibitors of JNK3 led to the identification of (benzothiazol-2-yl)acetonitrile derivatives as potent and selective JNK1, -2, -3 inhibitors. Starting from initial hit 1 (AS007149), the chemistry and initial structure-activity relationship (SAR) of this novel and unique kinase inhibitor template were explored. Investigation of the SAR rapidly revealed that the benzothiazol-2-ylacetonitrile pyrimidine core was crucial to retain a good level of potency on rat JNK3. Therefore, compound 6 was further optimized by exploring a number of distal combinations in place of the chlorine atom. This led to the observation that the presence of an aromatic group, two carbons away from the aminopyrimidine moiety and bearing substituents conferring hydrogen bond acceptor (HBA) properties, could improve the potency. Further improvements to the biological and biopharmaceutical profile of the most promising compounds were performed, resulting in the discovery of compound 59 (AS601245). The in vitro and in vivo anti-inflammatory potential of this new JNK inhibitor was investigated and found to demonstrate efficacy per oral route in an experimental model of rheumatoid arthritis (RA).
Assuntos
Acetonitrilas/síntese química , Anti-Inflamatórios não Esteroides/síntese química , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Tiazóis/síntese química , Acetonitrilas/química , Acetonitrilas/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Antirreumáticos/síntese química , Antirreumáticos/química , Antirreumáticos/farmacologia , Artrite Experimental/tratamento farmacológico , Benzotiazóis , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/química , Células Jurkat , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos DBA , Ratos , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossínteseRESUMO
We report a novel chemical class of potent oxytocin receptor antagonists showing a high degree of selectivity against the closely related vasopressin receptors (V1a, V1b, V2). An initial compound, 7, was shown to be active in an animal model of preterm labor when administered by the intravenous but not by the oral route. Stepwise SAR investigations around the different structural elements revealed one position, the arenesulfonyl moiety, to be amenable to structural changes. Consequently, this position was used to introduce a variety of substituents to improve the physicochemical properties. Some of the resulting analogues were found to be superior to 7 both in terms of potency in vitro and aqueous solubility, which translated into significantly improved efficacy in the animal model after intravenous and oral administration. The best compound, 73, potently inhibited oxytocin-induced uterine contractions in nonpregnant rats and reduced spontaneous uterine contractions in late-term pregnant rats.
Assuntos
Hidrazinas/síntese química , Receptores de Ocitocina/antagonistas & inibidores , Sulfonamidas/síntese química , Administração Oral , Animais , Antagonistas dos Receptores de Hormônios Antidiuréticos , Ligação Competitiva , Linhagem Celular , Cricetinae , Cricetulus , Feminino , Humanos , Hidrazinas/química , Hidrazinas/farmacologia , Técnicas In Vitro , Trabalho de Parto Prematuro/fisiopatologia , Trabalho de Parto Prematuro/prevenção & controle , Gravidez , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Contração Uterina/efeitos dos fármacosRESUMO
1 Myocardial ischemia/reperfusion is associated with inflammation, apoptosis and necrosis. During this process, c-jun N-terminal kinase is activated in cardiac myocytes resulting in apoptosis. 2 This study investigates the effects of AS601245, a nonpeptide ATP competitive JNK inhibitor, on infarct size caused by myocardial ischemia/reperfusion in anaesthetized rats. The left descending coronary artery of anaesthetized rats was occluded for 30 min and then reperfused for 3 h. AS601245 was administered 5 min before the end of the ischemia period as an i.v. bolus (1.5, 4.5 or 15 mg kg(-1) i.v.) followed by continuous i.v. infusion (18, 55 and 183 microg kg(-1) min(-1), respectively) during reperfusion. Controls received saline only. 3-Aminobenzamide, a poly(ADP-ribose) polymerase inhibitor, was used as reference compound at 10 mg kg(-1) i.v. bolus plus 0.17 mg kg(-1) min(-1) continuous infusion. 3 AS601245 significantly reduced infarct size at 4.5 mg kg(-1) (-44%; P<0.001) and 15 mg kg(-1) i.v. (-40.3%; P<0.001) similarly to 3-aminobenzamide (-44.2%; P<0.001). This protective effect was obtained without affecting hemodynamics or reducing ST-segment displacement. 4 The beneficial effects on infarct size correlated well with the reduction of c-jun phosphorylation (-85%; P<0.001 versus control) and of TUNEL-positive cells (-82.1%; P<0.001) in post-ischemic cardiomyocytes. No change in the phosphorylation state of p38 MAPK and ERK in post-ischemic heart was observed in the presence of AS601245 in comparison to the vehicle-treated group. 5 These results demonstrate that blocking the JNK pathway may represent a novel therapeutic approach for treating myocardial ischemia/reperfusion-induced cardiomyocyte death.
Assuntos
Acetonitrilas/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Infarto do Miocárdio/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Tiazóis/farmacologia , Anestesia , Animais , Benzotiazóis , Pressão Sanguínea/efeitos dos fármacos , Western Blotting , Doença das Coronárias/fisiopatologia , Fragmentação do DNA/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/complicações , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PURPOSE: This study was conducted to investigate the potential neuroprotective effect of IL-6 on chemotherapy induced neuropathy (CIN). IL-6 was compared to four-methylcatechol (4-MC)-a known inducer of NGF secretion previously shown to exhibit neuroprotective effects in CIN models. METHODS: Three CIN models were used; two in rats (cisplatin and vincristine) and one in mice (paclitaxel). IL-6 was delivered in four different doses in rats (0.3, 1, 3, 10 microg/kg, sc) every day from the first day of chemotherapeutic agent intoxication until the end of the study (day 37 for cisplatin protocol and day 30 for vincristine procedure). In mice, IL-6 was delivered at 10 microg/kg, sc either daily or three times a week from the first day of intoxication until the end of the study (day 19). Behavioral testings (hot plate and rotarod), nerve conduction studies (CMAP, SNCV, H-wave) and histo-morphometric analysis were done for all models. In addition, we tested whether IL-6 interfered with the tumor-reducing effects of the chemotherapeutic agents. RESULTS: IL-6 treatment prevented the behavioral and electrophysiological abnormalities produced by vincristine, cisplatin and Taxol intoxication, and similarly prevented the pathological changes in peripheral nerves. The neuroprotective action of chronic IL-6 treatment was at least equal to that of 4-MC. In addition, IL-6 neither inhibited the antitumour activity of cisplatin, nor stimulated tumour growth. CONCLUSION: IL-6 at low doses (10 microg/kg) provided protection against the development of CIN without demonstrating interference with the anti tumoural activity of these anti-mitotic drugs.
Assuntos
Antineoplásicos/toxicidade , Cisplatino/toxicidade , Interleucina-6/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Paclitaxel/toxicidade , Doenças do Sistema Nervoso Periférico/prevenção & controle , Vincristina/toxicidade , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Idoso , Animais , Antineoplásicos/uso terapêutico , Ataxia/induzido quimicamente , Ataxia/prevenção & controle , Catecóis/farmacologia , Catecóis/uso terapêutico , Cisplatino/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Avaliação Pré-Clínica de Medicamentos , Feminino , Humanos , Interleucina-6/administração & dosagem , Interleucina-6/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Condução Nervosa/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/farmacologia , Paclitaxel/uso terapêutico , Limiar da Dor/efeitos dos fármacos , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Equilíbrio Postural/efeitos dos fármacos , Ratos , Ratos Endogâmicos , Transplante Heterólogo , Vincristina/uso terapêuticoRESUMO
A novel high-affinity inhibitor of tumor necrosis factor (TNF) is described, which is created by the fusion of the extracellular domains of TNF-binding protein 1 (TBP-1) to both the alpha and beta chains of an inactive version of the heterodimeric protein hormone, human chorionic gonadotropin. The resulting molecule, termed TNF-soluble high-affinity receptor complex (SHARC), self-assembles into a heterodimeric protein containing two functional TBP-1 moieties. The TNF-SHARC is a potent inhibitor of TNF-alpha bioactivity in vitro and has a prolonged pharmacokinetic profile compared with monomeric TBP-1 in vivo. Consistent with the long half-life, the duration of action in an lipopolysaccharide-mediated proinflammatory mouse model is prolonged similarly. In a collagen-induced arthritis mouse model, this molecule demonstrates improved efficacy over monomeric TBP-1. Based on these results, we demonstrated that inactivated heterodimeric protein hormones are flexible and efficient scaffolds for the creation of soluble high-affinity receptor complexes.
Assuntos
Gonadotropina Coriônica/genética , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Proteínas Recombinantes de Fusão/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacocinética , Anti-Inflamatórios não Esteroides/farmacologia , Artrite Experimental/tratamento farmacológico , Artrite Experimental/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Gonadotropina Coriônica Humana Subunidade beta/genética , Eletroforese em Gel de Poliacrilamida , Feminino , Subunidade alfa de Hormônios Glicoproteicos/genética , Humanos , Interleucina-6/sangue , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C3H , Peso Molecular , Fragmentos de Peptídeos/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacocinética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Leukocyte trafficking to inflammatory sites is a gradual process, which is dominated in its early phases by chemokine- and cytokine-mediated neutrophil recruitment. The chemokine regulated on activation normal T cell expressed and secreted (RANTES) has been shown to be highly expressed in the joints of patient with rheumatoid arthritis and to promote leukocyte trafficking into the synovial tissue. In this study, we investigated the effect of RANTES in a murine model of peritoneal chemotaxis, and we found that RANTES dose-dependently induces neutrophil recruitment. Then, through morphological and histological analyses, we observed that activated neutrophils represent the major infiltrating population in response to RANTES chemotactic stimulus. Furthermore, we demonstrated that oral administration of either nonisoform-specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (morpholin-4-yl-8-phenylchromen-4-one) or selective PI3Kgamma inhibitor AS041164 (5-benzo[1,3]dioxol-5-ylmethylene-thiazolidine-2,4-dione) blocks RANTES-induced chemotaxis and reduces the level of AKT phosphorylation. Because the two compounds showed a similar pharmacokinetic profile in terms of bioavailability and half-life after oral route administration, the selective inhibition of the PI3Kgamma-isoform pathway through AS041164 was three times more potent in reducing neutrophil recruitment. Finally, to confirm the blockade of neutrophil infiltration that occurs in the early phase of the inflammatory response, AS041164 was also tested in a model of carrageenan-induced paw edema in rats. Therefore, the PI3Kgamma pathway plays an important role in controlling neutrophil chemotaxis during early steps of inflammation.
Assuntos
Quimiotaxia/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inflamação/tratamento farmacológico , Neutrófilos/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Administração Oral , Animais , Disponibilidade Biológica , Quimiocina CCL5/farmacologia , Classe Ib de Fosfatidilinositol 3-Quinase , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacocinética , Isoenzimas/antagonistas & inibidores , Camundongos , PeritônioRESUMO
We have used in vitro oligodendrocyte differentiation and the in vivo remyelination model, the cuprizone model, to identify genes regulating oligodendrocyte function and remyelination. One of the genes we identified, osteopontin (opn), is a secreted glycoprotein with cytokine-like, chemotactic, and anti-apoptotic properties that contains an Arg-Gly-Asp (RGD) cell adhesion motif-mediating interactions with several integrins. Both microglia and astrocytes in demyelinating brain regions of cuprizone-fed mice expressed OPN protein. Recombinant OPN protein produced in a baculovirus expression system induced proliferation of both the rat CG-4 and the mouse Oli-neu oligodendrocyte precursor (OLP)-like cell lines in a dose-dependent manner. In addition, recombinant OPN treatment stimulated both myelin basic protein (MBP) synthesis and myelin sheath formation in mixed cortical cultures from embryonic mouse brain, an in vitro primary culture model of myelination. Interestingly, myelinating mixed cultures prepared from OPN(-/-) mice contained significantly less MBP compared to wild-type cultures after 17 days in culture. We propose that in the central nervous system, OPN may act as a novel regulator of myelination and remyelination.
Assuntos
Encéfalo/metabolismo , Doenças Desmielinizantes/metabolismo , Bainha de Mielina/metabolismo , Regeneração Nervosa/genética , Sialoglicoproteínas/metabolismo , Regulação para Cima/fisiologia , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/patologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/genética , Células Cultivadas , Técnicas de Cocultura , Cuprizona , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/genética , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/citologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Osteopontina , Ratos , Proteínas Recombinantes de Fusão/farmacologia , Sialoglicoproteínas/deficiência , Sialoglicoproteínas/genética , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
A hallmark of autoimmunity and other chronic diseases is the overexpression of chemokines resulting in a detrimental local accumulation of proinflammatory immune cells. Chemokines play a pivotal role in cellular recruitment through interactions with both cell surface receptors and glycosaminoglycans (GAGs). Anti-inflammatory strategies aimed at neutralizing the chemokine system have to-date targeted inhibition of the receptor-ligand interaction with receptor antagonists. In this study, we describe a novel strategy to modulate the inflammatory process in vivo through mutation of the essential heparin-binding site of a proinflammatory chemokine, which abrogates the ability of the protein to form higher-order oligomers, but retains receptor activation. Using well-established protocols to induce inflammatory cell recruitment into the peritoneal cavity, bronchoalveolar air spaces, and CNS in mice, this non-GAG binding variant of RANTES/CCL5 designated [44AANA47]-RANTES demonstrated potent inhibitory capacity. Through a combination of techniques in vitro and in vivo, [44AANA47]-RANTES appears to act as a dominant-negative inhibitor for endogenous RANTES, thereby impairing cellular recruitment, not through a mechanism of desensitization. [44AANA47]-RANTES is unable to form higher-order oligomers (necessary for the biological activity of RANTES in vivo) and importantly forms nonfunctional heterodimers with the parent chemokine, RANTES. Therefore, although retaining receptor-binding capacity, altering the GAG-associated interactive site of a proinflammatory chemokine renders it a dominant-negative inhibitor, suggesting a powerful novel approach to generate disease-modifying anti-inflammatory reagents.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Líquido da Lavagem Broncoalveolar/imunologia , Quimiocina CCL5/antagonistas & inibidores , Quimiocina CCL5/metabolismo , Heparina/metabolismo , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Peritônio/imunologia , Animais , Anti-Inflamatórios não Esteroides/metabolismo , Sítios de Ligação/genética , Sítios de Ligação/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Inibição de Migração Celular , Quimiocina CCL5/administração & dosagem , Quimiocina CCL5/genética , Dessensibilização Imunológica , Feminino , Humanos , Mediadores da Inflamação/administração & dosagem , Leucócitos/citologia , Leucócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia de Vídeo , Mutação , Peritônio/citologia , Peritônio/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
We have discovered a new, potent, selective, and orally active oxytocin receptor antagonist, (2S,4Z)-N-[(2S)-2-hydroxy-2-phenylethyl]-4-(methoxyimino)-1-[(2'-methyl[1,1'-biphenyl]-4-yl)carbonyl]-2-pyrrolidinecarboxamide (compound 1). We report the biochemical, pharmacological, and pharmacokinetic characterization in vitro and in vivo of this compound. Compound 1 competitively inhibits binding of [3H]oxytocin and the peptide antagonist 125I-ornithine vasotocin analog to human and rat oxytocin receptor expressed in human embryonic kidney 293-EBNA or Chinese hamster ovary cells with nanomolar potency. Selectivity against vasopressin receptor subtypes is >6-fold for V1a and >350-fold for V2 and V1b. Compound 1 inhibits oxytocin-evoked intracellular Ca2+ mobilization (IC50 = 8 nM). Compound 1 has no intrinsic agonist activity at the oxytocin receptor. Oxytocininduced contraction of isolated rat uterine strips is blocked by compound 1 (pA2 = 7.82). In anesthetized nonpregnant rats, single administration of compound 1 by i.v. or oral routes causes dose-dependent inhibition of contractions elicited by repeated injections of oxytocin with ED50 = 3.5 mg/kg i.v. and 89 mg/kg p.o., respectively. Compound 1 significantly inhibits spontaneous uterine contractions in pregnant rats near term when administered intravenously or orally. We conclude that compound 1 is a potent, selective, and orally active nonpeptide oxytocin receptor antagonist, which is a suitable candidate for evaluation as a potential tocolytic agent for the management of preterm labor.