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1.
JCI Insight ; 4(2)2019 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-30674715

RESUMO

The antiinflammatory effects of i.v. Ig (IVIG) in the treatment of autoimmune disease are due, in part, to the Fc fragments of Ig aggregates. In order to capitalize on the known antiinflammatory and tolerogenic properties of Ig Fc aggregates, we created a recombinant human IgG1 Fc multimer, GL-2045. In vitro, GL-2045 demonstrated high-avidity binding to Fc receptors, blocked the binding of circulating immune complexes from patients with rheumatoid arthritis to human Fcγ receptors (FcγRs), and inhibited antibody-mediated phagocytosis at log order-lower concentrations than IVIG. In vivo, administration of GL-2045 conferred partial protection against antibody-mediated platelet loss in a murine immune thrombocytopenic purpura (ITP) model. GL-2045 also suppressed disease activity in a therapeutic model of murine collagen-induced arthritis (CIA), which was associated with reduced circulating levels of IL-6. Furthermore, GL-2045 administration to nonhuman primates (NHPs) transiently increased systemic levels of the antiinflammatory cytokines IL-10 and IL-1RA, reduced the proinflammatory cytokine IL-8, and decreased surface expression of CD14 and HLA-DR on monocytes. These findings demonstrate the immunomodulatory properties of GL-2045 and suggest that it has potential as a treatment for autoimmune and inflammatory diseases, as a recombinant alternative to IVIG.

2.
PLoS One ; 12(9): e0185079, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28945765

RESUMO

C5-substituted 2,4-diaminoquinazoline inhibitors of the decapping scavenger enzyme DcpS (DAQ-DcpSi) have been developed for the treatment of spinal muscular atrophy (SMA), which is caused by genetic deficiency in the Survival Motor Neuron (SMN) protein. These compounds are claimed to act as SMN2 transcriptional activators but data underlying that claim are equivocal. In addition it is unclear whether the claimed effects on SMN2 are a direct consequence of DcpS inhibitor or might be a consequence of lysosomotropism, which is known to be neuroprotective. DAQ-DcpSi effects were characterized in cells in vitro utilizing DcpS knockdown and 7-methyl analogues as probes for DcpS vs non-DcpS-mediated effects. We also performed analysis of Smn transcript levels, RNA-Seq analysis of the transcriptome and SMN protein in order to identify affected pathways underlying the therapeutic effect, and studied lysosomotropic and non-lysosomotropic DAQ-DCpSi effects in 2B/- SMA mice. Treatment of cells caused modest and transient SMN2 mRNA increases with either no change or a decrease in SMNΔ7 and no change in SMN1 transcripts or SMN protein. RNA-Seq analysis of DAQ-DcpSi-treated N2a cells revealed significant changes in expression (both up and down) of approximately 2,000 genes across a broad range of pathways. Treatment of 2B/- SMA mice with both lysomotropic and non-lysosomotropic DAQ-DcpSi compounds had similar effects on disease phenotype indicating that the therapeutic mechanism of action is not a consequence of lysosomotropism. In striking contrast to the findings in vitro, Smn transcripts were robustly changed in tissues but there was no increase in SMN protein levels in spinal cord. We conclude that DAQ-DcpSi have reproducible benefit in SMA mice and a broad spectrum of biological effects in vitro and in vivo, but these are complex, context specific, and not the result of simple SMN2 transcriptional activation.


Assuntos
Endorribonucleases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Atrofia Muscular Espinal/tratamento farmacológico , Atrofia Muscular Espinal/enzimologia , Quinazolinas/farmacologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Inibidores Enzimáticos/química , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Atrofia Muscular Espinal/genética , Regiões Promotoras Genéticas , Quinazolinas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/deficiência , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Proteína 2 de Sobrevivência do Neurônio Motor/metabolismo
3.
J Med Chem ; 60(7): 3094-3108, 2017 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-28257199

RESUMO

The C-5 substituted 2,4-diaminoquinazoline RG3039 (compound 1), a member of a chemical series that was identified and optimized using an SMN2 promoter screen, prolongs survival and improves motor function in a mouse model of spinal muscular atrophy (SMA). It is a potent inhibitor of the mRNA decapping scavenger enzyme (DcpS), but the mechanism whereby DcpS inhibition leads to therapeutic benefit is unclear. Compound 1 is a dibasic lipophilic molecule that is predicted to accumulate in lysosomes. To understand if the in vivo efficacy is due to DcpS inhibition or other effects resulting from the physicochemical properties of the chemotype, we undertook structure based molecular design to identify DcpS inhibitors with improved physicochemical properties. Herein we describe the design, synthesis, and in vitro pharmacological characterization of these DcpS inhibitors along with the in vivo mouse CNS PK profile of PF-DcpSi (compound 24), one of the analogs found to be efficacious in SMA mouse model.


Assuntos
Desenho de Fármacos , Endorribonucleases/antagonistas & inibidores , Atrofia Muscular Espinal/tratamento farmacológico , Quinazolinas/química , Quinazolinas/uso terapêutico , RNA Mensageiro/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Endorribonucleases/genética , Endorribonucleases/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Quinazolinas/farmacocinética , Quinazolinas/farmacologia , RNA Mensageiro/genética , Proteína 2 de Sobrevivência do Neurônio Motor
4.
Blood Adv ; 1(8): 504-515, 2017 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-29296968

RESUMO

GL-2045 is a recombinant human immunoglobulin G1 (IgG1)-based Fc multimer designed to recapitulate the anti-inflammatory activities of intravenous immunoglobulin (IVIG) on the innate and adaptive immune responses. We used functional in vitro studies to determine if GL-2045 could mimic the modulatory activity of IVIG on complement activation. GL-2045, at log-order lower concentrations than heat-aggregated IgG (HAGG) and IVIG, protected antibody-opsonized cells from complement-dependent cytotoxicity. These protective effects were completely mediated by the higher order multimer fractions of GL-2045 and were partially dependent upon sequestration of C1q. Exposure of serum to GL-2045 and, to a lesser extent, IVIG, resulted in high levels of C4a, limited levels of C3a, and no C5a. In contrast, HAGG induced high levels of C4a, C3a, and C5a. The means by which GL-2045 governed complement activation was dependent on its ability to augment the function of factor H, alone and in combination with factor I, to indirectly limit the alternative form of C3 convertase, with resultant increases in the anti-inflammatory molecule, the "inactive" form of C3b, called iC3b. Although IVIG, like GL-2045, potentiated factor H function, it also directly inhibited the alternative form of C3 convertase. Our findings help elucidate how IVIG, GL-2045, and HAGG regulate complement function. Furthermore, the capacity of GL-2045 to sequester C1q and augment factor H activity, in combination with its ability to generate activation-induced immunomodulatory complement split products, such as iC3b, make it a viable drug candidate for the treatment of diverse complement-mediated diseases.

5.
PLoS One ; 10(1): e0116227, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25617631

RESUMO

INTRODUCTION: The cholinergic anti-inflammatory pathway can downregulate inflammation via the release of acetylcholine (ACh) by the vagus nerve. This neurotransmitter binds to the α7 subunit of nicotinic acetylcholine receptors (α7nAChR), expressed on macrophages and other immune cells. We tested the pharmacological and functional profile of two novel compounds, PMP-311 and PMP-072 and investigated their role in modulating collagen-induced arthritis (CIA) in mice. METHODS: Both compounds were characterized with binding, electrophysiological, and pharmacokinetic studies. For in vivo efficacy studies in the CIA model the compounds were administered daily by oral gavage from day 20 till sacrifice at day 34. Disease progression was monitored by visual clinical scoring and measurement of paw swelling. Inflammation and joint destruction were examined by histology and radiology. RESULTS: Treatment with PMP-311 was effective in preventing disease onset, reducing clinical signs of arthritis, and reducing synovial inflammation and bone destruction. PMP-072 also showed a trend in arthritis reduction at all concentrations tested. The data showed that while both compounds bind to α7nAChR with high affinity, PMP-311 acts like a classical agonist of ion channel activity, and PMP-072 can actually act as an ion channel antagonist. Moreover, PMP-072 was clearly distinct from typical competitive antagonists, since it was able to act as a silent agonist. It synergizes with the allosteric modulator PNU-120596, and subsequently activates desensitized α7nAChR. However, PMP-072 was less efficacious than PMP-311 at both channel activation and desensitization, suggesting that both conducting and non-conducting states maybe of importance in driving an anti-inflammatory response. Finally, we found that the anti-arthritic effect can be observed despite limited penetration of the central nervous system. CONCLUSIONS: These data provide direct evidence that the α7nAChR in immune cells does not require typical ion channel activation to exert its antiinflammatory effects.


Assuntos
Anilidas/farmacologia , Artrite Experimental/tratamento farmacológico , Oxazóis/farmacologia , Piperazinas/farmacologia , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Acetilcolina/metabolismo , Anilidas/química , Animais , Artrite Experimental/patologia , Artrite Experimental/prevenção & controle , Linhagem Celular , Inflamação/tratamento farmacológico , Articulação do Joelho/efeitos dos fármacos , Articulação do Joelho/patologia , Ligantes , Camundongos , Camundongos Endogâmicos , Oxazóis/química , Células PC12 , Piperazinas/química , Piridinas/química , Ratos , Xenopus
6.
Ann Rheum Dis ; 69(9): 1717-23, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20511609

RESUMO

BACKGROUND: The alpha7 subunit of nicotinic acetylcholine receptors (alpha7nAChR) can negatively regulate the synthesis and release of proinflammatory cytokines by macrophages and fibroblast-like synoviocytes in vitro. In addition, stimulation of the alpha7nAChR can reduce the severity of arthritis in murine collagen-induced arthritis (CIA). OBJECTIVE: To provide more insight into the role of the alpha7nAChR in the pathogenesis of arthritis by investigating the effect of the absence of alpha7nAChR in CIA in alpha7-deficient (alpha7nAChR(-/-)) compared with wild-type (WT) mice. METHODS: CIA was induced in alpha7nAChR(-/-) and WT littermate mice at day 0 by immunisation with chicken collagen type II (cCII) followed by a booster injection with cCII on day 20. Mice were killed on day 44 or day 63 and arthritis activity as well as radiological and histological damage were scored. The effects on the immune response were evaluated by measurement of antigen-specific antibodies and cytokines, and evaluation of the effects on antigen-specific stimulated spleen cells. RESULTS: In alpha7nAChR(-/-) mice a significant increase in the incidence and severity of arthritis as well as increased synovial inflammation and joint destruction were seen. Exacerbation of CIA was associated with elevated systemic proinflammatory cytokines and enhanced T-helper cell 1 (Th1)-cytokine and tumour necrosis factor alpha production by spleen cells. Moreover, a specific decrease in the collagen-specific 'Th1-associated' IgG2a response was seen, whereas IgG1 titres were unaffected. CONCLUSIONS: The results presented here indicate that immune cell function in a model of rheumatoid arthritis is regulated by the cholinergic system and, at least in part, mediated by the alpha7nAChR.


Assuntos
Artrite Experimental/imunologia , Artrite Reumatoide/imunologia , Receptores Nicotínicos/fisiologia , Doença Aguda , Animais , Artrite Experimental/complicações , Artrite Experimental/patologia , Artrite Reumatoide/complicações , Artrite Reumatoide/patologia , Doenças Ósseas Metabólicas/etiologia , Doenças Ósseas Metabólicas/imunologia , Doenças Ósseas Metabólicas/patologia , Doenças das Cartilagens/etiologia , Doenças das Cartilagens/imunologia , Doenças das Cartilagens/patologia , Quimiocina CCL2/sangue , Progressão da Doença , Feminino , Masculino , Camundongos , Camundongos Knockout , Receptores Nicotínicos/deficiência , Receptores Nicotínicos/genética , Células Th1/imunologia , Fator de Necrose Tumoral alfa/sangue , Receptor Nicotínico de Acetilcolina alfa7
7.
Arthritis Rheum ; 60(1): 114-22, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19116908

RESUMO

OBJECTIVE: The parasympathetic nervous system, through the vagus nerve, can down-regulate inflammation in vivo by decreasing the release of cytokines, including tumor necrosis factor alpha (TNFalpha), by activated macrophages. The vagus nerve may exert antiinflammatory actions via a specific effect of its principal neurotransmitter, acetylcholine, on the alpha7 subunit of nicotinic acetylcholine receptors (alpha7nAChR) on macrophages. The present study was undertaken to obtain insight into the role of the cholinergic antiinflammatory pathway in arthritis. METHODS: To inhibit the cholinergic antiinflammatory pathway, mice were subjected to unilateral cervical vagotomy or sham surgery, after which arthritis was induced with type II collagen. In a separate study, nicotine was added to the drinking water of mice with collagen-induced arthritis (CIA). In addition, we investigated the effects of intraperitoneally (IP)-injected nicotine and the specific alpha7nAChR agonist AR-R17779. RESULTS: Clinical arthritis was exacerbated by vagotomy and ameliorated by oral nicotine administration. Moreover, oral nicotine inhibited bone degradation and reduced TNFalpha expression in synovial tissue. Both IP-injected nicotine and AR-R17779 ameliorated clinical arthritis and reduced synovial inflammation. This was accompanied by a reduction of TNFalpha levels in both plasma and synovial tissue. The effect of AR-R17779 was more potent compared with that of nicotine and was associated with delayed onset of the disease as well as with protection against joint destruction. CONCLUSION: These data provide the first evidence of a role of the cholinergic antiinflammatory pathway in the murine CIA model of rheumatoid arthritis.


Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Macrófagos/metabolismo , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/metabolismo , Administração Oral , Animais , Artrite Experimental/patologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Cartilagem/patologia , Modelos Animais de Doenças , Injeções Intraperitoneais , Masculino , Camundongos , Camundongos Endogâmicos DBA , Sistema Nervoso Parassimpático/fisiologia , Compostos de Espiro/farmacologia , Membrana Sinovial/metabolismo , Sinovite/tratamento farmacológico , Sinovite/metabolismo , Sinovite/patologia , Fator de Necrose Tumoral alfa/metabolismo , Nervo Vago/fisiologia , Receptor Nicotínico de Acetilcolina alfa7
8.
Gastroenterology ; 133(4): 1219-28, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17919496

RESUMO

BACKGROUND & AIMS: We previously showed that intestinal inflammation is reduced by electrical stimulation of the efferent vagus nerve, which prevents postoperative ileus in mice. We propose that this cholinergic anti-inflammatory pathway is mediated via alpha7 nicotinic acetylcholine receptors expressed on macrophages. The aim of this study was to evaluate pharmacologic activation of the cholinergic anti-inflammatory pathway in a mouse model for postoperative ileus using the alpha7 nicotinic acetylcholine receptor-agonist AR-R17779. METHODS: Mice were pretreated with vehicle, nicotine, or AR-R17779 20 minutes before a laparotomy (L) or intestinal manipulation (IM). Twenty-four hours thereafter gastric emptying was determined using scintigraphy and intestinal muscle inflammation was quantified. Nuclear factor-kappaB transcriptional activity and cytokine production was assayed in peritoneal macrophages. RESULTS: Twenty-four hours after surgery IM led to a delayed gastric emptying compared with L (gastric retention: L(saline) 14% +/- 4% vs IM(saline) 38% +/- 10%, P = .04). Pretreatment with AR-R17779 prevented delayed gastric emptying (IM(AR-R17779) 15% +/- 4%, P = .03). IM elicited inflammatory cell recruitment (L(saline) 50 +/- 8 vs IM(saline) 434 +/- 71 cells/mm(2), P = .001) which was reduced by AR-R17779 pretreatment (IM(AR-R17779) 231 +/- 32 cells/mm(2), P = .04). An equimolar dose of nicotine was not tolerated. Subdiaphragmal vagotomy did not affect the anti-inflammatory properties of AR-R17779. In peritoneal macrophages, both nicotinic agonists reduced nuclear factor kappaB transcriptional activity and proinflammatory cytokine production, with nicotine being more effective than AR-R17779. CONCLUSIONS: AR-R17779 treatment potently prevents postoperative ileus, whereas toxicity limits nicotine administration to ineffective doses. Our data further imply that nicotinic inhibition of macrophage activation may involve other receptors in addition to alpha7 nicotinic acetylcholine receptor.


Assuntos
Anti-Inflamatórios/farmacologia , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Gastroenterite/prevenção & controle , Íleus/prevenção & controle , Intestinos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Complicações Pós-Operatórias/prevenção & controle , Compostos de Espiro/farmacologia , Animais , Anti-Inflamatórios/uso terapêutico , Anti-Inflamatórios/toxicidade , Hidrocarbonetos Aromáticos com Pontes/uso terapêutico , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Terapia por Estimulação Elétrica , Feminino , Esvaziamento Gástrico/efeitos dos fármacos , Gastroenterite/metabolismo , Gastroenterite/fisiopatologia , Íleus/metabolismo , Íleus/fisiopatologia , Intestinos/inervação , Intestinos/fisiopatologia , Intestinos/cirurgia , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Nicotina/farmacologia , Nicotina/toxicidade , Agonistas Nicotínicos/uso terapêutico , Agonistas Nicotínicos/toxicidade , Complicações Pós-Operatórias/metabolismo , Complicações Pós-Operatórias/fisiopatologia , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/metabolismo , Compostos de Espiro/uso terapêutico , Transcrição Gênica/efeitos dos fármacos , Vagotomia , Nervo Vago/cirurgia , Receptor Nicotínico de Acetilcolina alfa7
9.
Shock ; 28(6): 700-3, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17621262

RESUMO

The cholinergic nervous system can inhibit the release of proinflammatory cytokines such as TNF-alpha from LPS-stimulated macrophages. Acetylcholine, the principal neurotransmitter of the vagus nerve, is the key mediator of this so-called cholinergic anti-inflammatory pathway, specifically interacting with alpha7 cholinergic receptors expressed by macrophages and other cell types to inhibit TNF-alpha production. The aim of the current study was to determine the capacity of the selective alpha7 cholinergic receptor agonist 3-(2,4-dimethoxybenzylidene) anabaseine (GTS-21), administered locally into the airways, to inhibit LPS-induced inflammatory responses in the mouse lung in vivo. GTS-21 dose-dependently inhibited LPS-induced TNF-alpha release by MH-S mouse alveolar macrophages in vitro. Intranasal inoculation with GTS-21 also dose-dependently inhibited TNF-alpha release into the lung compartment after intrapulmonary delivery of LPS in mice in vivo, whereas IL-6 concentrations were not affected. However, GTS-21 did not influence the influx of neutrophils into bronchoalveolar lavage fluid elicited by LPS and increased the concentrations of the neutrophil-attracting chemokines cytokine-induced neutrophil chemoattractant and macrophage inflammatory protein 2. These data indicate that local administration of GTS-21 inhibits TNF-alpha release in the lung during LPS-induced inflammation.


Assuntos
Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Agonistas Nicotínicos/farmacologia , Receptores Nicotínicos/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Compostos de Benzilideno/farmacologia , Linhagem Celular , Relação Dose-Resposta a Droga , Feminino , Pulmão/citologia , Pulmão/metabolismo , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos Alveolares/citologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Piridinas/farmacologia , Receptor Nicotínico de Acetilcolina alfa7
10.
Shock ; 28(5): 570-5, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17577142

RESUMO

Ethyl pyruvate (EP) is a stable pyruvate derivative that has been shown to exert anti-inflammatory effects in various models of systemic inflammation including endotoxemia. We here sought to determine the local effects of EP, after intrapulmonary delivery, in models of lung inflammation induced by instillation via the airways of either lipopolysaccharide (LPS, a constituent of the gram-negative bacterial cell wall) or lipoteichoic acid (LTA, a component of the gram-positive bacterial cell wall). For this, we first established that EP dose dependently reduced the responsiveness of mouse MH-S alveolar macrophages and mouse MLE-15 and MLE-12 respiratory epithelial cells to stimulation with LPS or LTA in vitro. We then showed that intranasal administration of EP dose dependently inhibited tumor necrosis factor alpha release in bronchoalveolar lavage fluid of mice challenged with either LPS or LTA via the airways. Moreover, EP reduced the recruitment of neutrophils into the bronchoalveolar space after either LPS or LTA administration. These data suggest that intrapulmonary delivery of EP diminishes lung inflammation induced by LPS or LTA, at least in part by targeting alveolar macrophages and respiratory epithelial cells.


Assuntos
Células Epiteliais/metabolismo , Aromatizantes/farmacologia , Lipopolissacarídeos/toxicidade , Macrófagos Alveolares/metabolismo , Pneumonia/tratamento farmacológico , Piruvatos/farmacologia , Ácidos Teicoicos/toxicidade , Animais , Líquido da Lavagem Broncoalveolar , Linhagem Celular , Relação Dose-Resposta a Droga , Células Epiteliais/patologia , Feminino , Macrófagos Alveolares/patologia , Camundongos , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Pneumonia/patologia , Fator de Necrose Tumoral alfa/metabolismo
11.
Shock ; 27(4): 443-7, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17414429

RESUMO

The cholinergic nervous system controls inflammation by inhibiting the release of proinflammatory cytokines such as tumor necrosis factor (TNF) alpha from lipopolysaccharide (LPS)-stimulated macrophages. The key endogenous mediator of this so-called cholinergic anti-inflammatory pathway is acetylcholine, the principal neurotransmitter of the vagus nerve, which specifically interacts with alpha7 cholinergic receptors expressed by macrophages and other cell types to inhibit TNF-alpha production. We here investigated the capacity of the selective alpha7 cholinergic receptor agonist 3-(2,4-dimethoxybenzylidene) anabaseine (GTS-21) to inhibit LPS-induced inflammatory responses in mice in vivo. To this end, mice received an intraperitoneal injection of LPS (from Escherichia coli, 200 microg) preceded by GTS-21 (4 mg/kg) or vehicle. GTS-21 strongly inhibited LPS-induced TNF-alpha release into the peritoneal cavity and the circulation. In addition, GTS-21 attenuated the influx of neutrophils into peritoneal fluid upon administration of LPS. This inhibitory effect on neutrophil recruitment by GTS-21 was independent of its effect on TNF-alpha release, considering that etanercept, a potent TNF-alpha-blocking protein containing the extracellular domain of the p75 TNF-alpha receptor, did not influence LPS-induced neutrophil influx either in the presence or in the absence of GTS-21 treatment. GTS-21 did not reduce the local secretion of macrophage inflammatory protein 2 and keratinocyte-derived cytokine, suggesting that altered concentrations of these neutrophil-attracting chemokines did not contribute to GTS-21-induced inhibition of neutrophil migration. These data identify a novel anti-inflammatory effect of chemical alpha7 cholinergic receptor stimulation that is independent from its capacity to inhibit TNF-alpha production.


Assuntos
Inibição de Migração Celular , Lipopolissacarídeos/antagonistas & inibidores , Infiltração de Neutrófilos/imunologia , Receptores Nicotínicos/metabolismo , Fator de Necrose Tumoral alfa/fisiologia , Animais , Compostos de Benzilideno/farmacologia , Citocinas/metabolismo , Feminino , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Agonistas Nicotínicos/farmacologia , Piridinas/farmacologia , Receptores Nicotínicos/fisiologia , Receptor Nicotínico de Acetilcolina alfa7
12.
Crit Care Med ; 35(4): 1139-44, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17334244

RESUMO

OBJECTIVE: Tumor necrosis factor and high mobility group box 1 are critical cytokine mediators of inflammation. The efferent vagus nerve inhibits cytokine release through alpha7-nicotinic acetylcholine receptor-mediated cholinergic signaling. Here we studied whether GTS-21, a selective alpha7-nicotinic acetylcholine receptor agonist, inhibits proinflammatory cytokines in vitro and in vivo and improves survival in murine endotoxemia and severe sepsis. DESIGN: Randomized and controlled in vitro and in vivo study. SETTINGS: Research laboratory and animal facility rooms. SUBJECTS: RAW 264.7 cells and BALB/c mice treated with endotoxin or subjected to cecal ligation and puncture (CLP). INTERVENTIONS: RAW 264.7 cells were exposed to endotoxin (4 ng/mL or 10 ng/mL) in the presence or absence of GTS-21 (1-100 muM), and tumor necrosis factor and high mobility group box 1 release and nuclear factor-kappaB activation were analyzed. Mice were treated with GTS-21 (0.4 mg/kg or 4 mg/kg, intraperitoneally) or saline 30 mins before endotoxin (6 mg/kg, intraperitoneally), and serum tumor necrosis factor was analyzed 1.5 hrs after the onset of endotoxemia. In survival experiments, mice were treated with GTS-21 (0.4 or 4.0 mg/kg, intraperitoneally) or saline 30 mins before and 6 hrs after endotoxin and then twice daily for 3 days. Severe sepsis was induced by CLP. Mice were treated with GTS-21 (4 mg/kg) or saline immediately and 6 hrs and 24 hrs after CLP, and serum high mobility group box 1 was analyzed 30 hrs after CLP. In survival experiments, GTS-21 (0.4 or 4 mg/kg) treatment was initiated 24 hrs after CLP and continued twice daily for 3 days. MEASUREMENTS AND MAIN RESULTS: GTS-21 dose-dependently inhibited tumor necrosis factor and high mobility group box 1 release and nuclear factor-kappaB activation in vitro. GTS-21 (4 mg/kg) significantly inhibited serum tumor necrosis factor during endotoxemia and improved survival (p < .0001). GTS-21 (4 mg/kg) significantly inhibited serum high mobility group box 1 levels in CLP mice and improved survival (p < .0006). CONCLUSION: These findings are of interest for the development of alpha7-nicotinic acetylcholine receptor agonists as a new class of anti-inflammatory therapeutics.


Assuntos
Compostos de Benzilideno/farmacologia , Endotoxemia/tratamento farmacológico , Agonistas Nicotínicos/farmacologia , Piridinas/farmacologia , Receptores Nicotínicos/metabolismo , Sepse/tratamento farmacológico , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Endotoxemia/mortalidade , Proteína HMGB1/antagonistas & inibidores , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/antagonistas & inibidores , Sepse/mortalidade , Índice de Gravidade de Doença , Taxa de Sobrevida , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Receptor Nicotínico de Acetilcolina alfa7
13.
Bioorg Med Chem Lett ; 17(11): 3141-5, 2007 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-17395464

RESUMO

The identification, optimization, and structure-activity relationship (SAR) of small-molecule CCR4 antagonists is described. An initial screening hit with micromolar potency was identified that was optimized to sub-micromolar binding potency by enantiomer resolution, halogenation of the naphthalene ring, and extension of the alkyl chain linker between the central piperidine ring and the terminal aryl group. An antagonist was identified that showed good cross-reactivity against the mouse receptor and inhibited CCR4-based cell recruitment in dose-dependent fashion.


Assuntos
Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Naftalenos/química , Naftalenos/farmacologia , Receptores de Quimiocinas/antagonistas & inibidores , Sulfonamidas/química , Sulfonamidas/farmacologia , Animais , Anti-Inflamatórios não Esteroides/síntese química , Camundongos , Camundongos Endogâmicos BALB C , Naftalenos/síntese química , Receptores CCR4 , Relação Estrutura-Atividade , Sulfonamidas/síntese química
14.
J Leukoc Biol ; 81(1): 129-36, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16980509

RESUMO

High mobility group box protein 1 (HMGB1) exerts different biological functions dependent on its cellular localization. Nuclear HMGB1 maintains chromatin architecture and is required for undisturbed transcription activity, and extracellularly released HMGB1 mediates inflammation and tissue regeneration. A present paucity of readily accessible methods to quantify released HMGB1 represents a problem concerning the exploration of HMGB1 biology. We have now developed a HMGB1-specific ELISPOT assay enabling enumeration of individual HMGB1-releasing cells. The method also allows automated, semiquantitative assessment of released HMGB1 by evaluating areas of single HMGB1 spots. Actively secreted HMGB1 as well as cells passively releasing the protein following necrotic cell death are visualized distinctly using this ELISPOT assay. Kinetics of HMGB1 secretion after different stimuli was studied using cell lines of various lineages. IFN-gamma already induced substantial HMGB1 secretion from the monocytic cell line RAW 264.7 within 24 h and even more so after 48 h. LPS only stimulated a modest HMGB1 release within 24 h, but this increased considerably by 48 h. TNF-induced HMGB1 release was unexpectedly low. Mast cells, which share the secretory, lysosomal pathway with macrophages/monocytes, did not secrete HMGB1 in response to any studied mode of activation. Most transformed cells overexpress HMGB1, but the ELISPOT assay revealed that all transformed cell lines will not actively secrete the protein. We believe the ELISPOT method provides a novel tool to study pathways promoting or inhibiting HMGB1 secretion.


Assuntos
Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática/métodos , Proteína HMGB1/metabolismo , Animais , Apoptose , Linhagem Celular , Linhagem da Célula , Sinergismo Farmacológico , Humanos , Interferon gama/farmacologia , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Mastócitos/metabolismo , Camundongos , Transporte Proteico , Fator de Necrose Tumoral alfa/metabolismo
15.
Gastroenterology ; 130(6): 1822-30, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16697744

RESUMO

BACKGROUND & AIMS: The nervous system, through the vagus nerve, controls inflammation by decreasing the release of tumor necrosis factor-alpha from endotoxin stimulated macrophages. This anti-inflammatory effect is mediated by an interaction of acetylcholine, the principal neurotransmitter of the vagus nerve, with macrophage cholinergic nicotinic receptors expressing the alpha7 subunit. METHODS: To determine the role of this "nicotinic anti-inflammatory pathway" in experimental pancreatitis, we induced pancreatitis in mice by 12 hourly intraperitoneal injections of cerulein. Pancreatitis was preceded by unilateral left cervical vagotomy or pretreatment with the nicotinic receptor antagonist mecamylamine or by pretreatment with the selective alpha7 nicotinic receptor agonist 3-(2,4-dimethoxybenzylidene) anabaseine (GTS-21). RESULTS: Vagotomy or pretreatment with mecamylamine resulted in an enhanced severity of pancreatitis, as reflected by histology, edema, plasma hydrolases, and interleukin-6 levels. Furthermore, the number of neutrophils migrated to the pancreas was increased in these mice, as shown by myeloperoxidase content and intrapancreatic staining of neutrophils. Conversely, GTS-21 pretreatment strongly decreased the severity of pancreatitis. Pancreatitis-associated pulmonary inflammation was independent of the integrity of the vagus nerve and nicotinic receptors. CONCLUSIONS: This study provides the first evidence for a therapeutic potential of the vagus nerve and the "nicotinic anti-inflammatory pathway" in attenuating inflammation and injury during experimental pancreatitis.


Assuntos
Antagonistas Nicotínicos/farmacologia , Pancreatite/tratamento farmacológico , Pancreatite/patologia , Receptores Nicotínicos/metabolismo , Nervo Vago/fisiologia , Animais , Biópsia por Agulha , Ceruletídeo , Modelos Animais de Doenças , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Pancreatite/fisiopatologia , Probabilidade , Fatores de Risco , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Vagotomia
16.
J Med Chem ; 49(9): 2669-72, 2006 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-16640325

RESUMO

Activation of CCR8 by its ligand CCL1 may play an important role in diseases such as asthma, multiple sclerosis, and cancer. The study of small molecule CCR8 antagonists will help establish the validation of these hypotheses. We report the design, synthesis, and progress toward optimization of potent small molecule CCR8 antagonists identified from a high-throughput screen. These analogues exhibit good potency in binding and chemotaxis assays, show good selectivity versus the hERG channel, and have good eADME (early absorption, distribution, metabolism, and excretion) profiles.


Assuntos
Desenho de Fármacos , Receptores de Quimiocinas/antagonistas & inibidores , Aminação , Linhagem Celular , Quimiotaxia/efeitos dos fármacos , Éter/química , Humanos , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Piperidinas/farmacologia , Pirrolidinas/síntese química , Pirrolidinas/química , Pirrolidinas/farmacologia , Receptores CCR8 , Relação Estrutura-Atividade
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