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Rationale: Idiopathic pulmonary fibrosis is a fatal and progressive disease with limited treatment options. Objectives: We sought to assess the efficacy and safety of CC-90001, an oral inhibitor of c-Jun N-terminal kinase 1, in patients with idiopathic pulmonary fibrosis. Methods: In a Phase 2, randomized (1:1:1), double-blind, placebo-controlled study (ClinicalTrials.gov ID: NCT03142191), patients received CC-90001 (200 or 400 mg) or placebo once daily for 24 weeks. Background antifibrotic treatment (pirfenidone) was allowed. The primary endpoint was change in the percentage of predicted FVC (ppFVC) from baseline to Week 24; secondary endpoints included safety. Measurements and Main Results: In total, 112 patients received at least one dose of study drug. The study was terminated early because of a strategic decision made by the sponsor. Ninety-one patients (81%) completed the study. The least-squares mean changes from baseline in ppFVC at Week 24 were -3.1% (placebo), -2.1% (200 mg), and -1.0% (400 mg); the differences compared with placebo were 1.1% (200 mg; 95% confidence interval: -2.1, 4.3; P = 0.50) and 2.2% (400 mg; 95% confidence interval: -1.1, 5.4; P = 0.19). Adverse event frequency was similar in patients in the combined CC-90001 arms versus placebo. The most common adverse events were nausea, diarrhea, and vomiting, which were more frequent in patients in CC-90001 arms versus placebo. Fewer patients in the CC-90001 arms than in the placebo arm experienced cough and dyspnea. Conclusions: Treatment with CC-90001 over 24 weeks led to numerical improvements in ppFVC in patients with idiopathic pulmonary fibrosis compared with placebo. CC-90001 was generally well tolerated, which was consistent with previous studies. Clinical trial registered with www.clinicaltrials.gov (NCT03142191).
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Fibrose Pulmonar Idiopática , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/fisiopatologia , Método Duplo-Cego , Masculino , Feminino , Idoso , Pessoa de Meia-Idade , Resultado do Tratamento , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , AdultoRESUMO
Approved treatments for idiopathic pulmonary fibrosis have tolerability concerns and limited efficacy. CC-90001, a c-Jun N-terminal kinase inhibitor, is under investigation as a therapy for fibrotic diseases. A Phase 1b safety, pharmacokinetics, and pharmacodynamics study of oral CC-90001 (100, 200, or 400 mg) administered once daily for 12 weeks was conducted in patients with pulmonary fibrosis (NCT02510937). Sixteen patients with a mean age of 68 years were studied. The most common treatment-emergent adverse events were nausea and headache; all events were of mild or moderate intensity. Pharmacokinetic profiles were similar between the patients in this trial and healthy adults in previous studies. Forced vital capacity increased in the 200- and 400-mg cohorts from baseline to Week 12, and dose-dependent reductions in fibrosis biomarkers were observed. Antifibrotic activity of CC-90001 was also evaluated in vitro in transforming growth factor beta 1 (TGF-ß1)-stimulated cells. CC-90001 reduced in vitro profibrotic gene expression in both lung epithelial cells and fibroblasts, supporting a potential direct antifibrotic action of c-Jun N-terminal kinase inhibition in either or both cell types. Overall, CC-90001 was generally safe and well tolerated, and treatment was associated with forced vital capacity improvement and reductions in profibrotic biomarkers.
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CC-90001 selectively inhibits c-Jun N-terminal kinase (JNK), a stress-activated protein implicated in fibrosis. In 3 phase 1 trials evaluating CC-90001 pharmacokinetics, pharmacodynamics, and safety, healthy adults (N = 184) received oral CC-90001 in a single dose (10-720 mg) or multiple doses (30-480 mg once daily for 7-18 days) or placebo. CC-90001 was rapidly absorbed (median time to maximum concentration, 1-4 hours) and eliminated with a mean terminal elimination half-life of 12-28 hours. Steady state was reached on day 5, with a mean accumulation ratio of 1.5- to 2-fold following daily dosing. Exposure was similar in fed versus fasted participants and in Japanese versus non-Japanese participants. CC-90001 demonstrated dose- and exposure-dependent inhibition of JNK as determined by histopathological analysis of c-Jun phosphorylation in ultraviolet-irradiated skin. The most common treatment-emergent adverse events were nausea and headache; all were mild or moderate in intensity. Based on exposure-response analysis using high-quality electrocardiogram data, no clinically relevant QT prolongation liability for CC-90001 was observed. Overall, single- and multiple-dose CC-90001 were generally safe and well tolerated at the tested doses and demonstrated JNK pathway engagement. These results support further clinical evaluation of CC-90001.
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Proteínas Quinases JNK Ativadas por Mitógeno , Adulto , Humanos , Voluntários Saudáveis , Meia-Vida , Relação Dose-Resposta a Droga , Método Duplo-CegoRESUMO
BACKGROUND: Mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MK2) is activated downstream of p38 MAPK and regulates stability of mRNAs encoding inflammatory cytokines. CC-99677 is a novel, irreversible, covalent MK2 inhibitor under development for the treatment of ankylosing spondylitis (AS) and other inflammatory diseases. As part of a phase I clinical trial to assess safety and tolerability, we evaluated target engagement, pharmacokinetics, and pharmacodynamics of CC-99677. METHODS: The MK2 inhibitor CC-99677 was evaluated for its effect on cytokine expression in vitro in peripheral blood mononuclear cells (PBMCs) from healthy donors and patients with a definitive AS diagnosis. A novel in vitro model was developed to compare the potential for tachyphylaxis of CC-99677 and p38 inhibitors in THP-1 cells. The effect of CC-99677 on tristetraprolin (TTP) and cytokine mRNA was assessed in stimulated human monocyte-derived macrophages. In a first-in-human study, thirty-seven healthy volunteers were randomly assigned to daily oral doses of CC-99677 or placebo, and blood was collected at pre-specified time points before and after dosing. CC-99677 concentrations were assessed in the plasma, and CC-99677 binding to MK2 was evaluated in PBMCs. Ex vivo stimulation of the whole blood was conducted from participants in the first-in-human study to assess the pharmacodynamic effects. RESULTS: In vitro, CC-99677 inhibited tumor necrosis factor (TNF), interleukin (IL)-6, and IL-17 protein production in samples of monocytes and macrophages from AS patients and healthy volunteers via an mRNA-destabilization mechanism. In the in vitro model of tachyphylaxis, CC-99677 showed a differentiated pattern of sustained TNF protein inhibition compared with p38 inhibitors. CC-99677 reduced TTP phosphorylation and accelerated the decay of inflammatory cytokine mRNA in lipopolysaccharide-stimulated macrophages. Administration of CC-99677 to healthy volunteers was safe and well-tolerated, with linear pharmacokinetics and sustained reduction of ex vivo whole blood TNF, IL-6, and chemokine synthesis. CONCLUSIONS: CC-99677 inhibition of MK2 is a promising approach for the treatment of inflammatory diseases and may overcome the limitations of p38 MAPK inhibition. TRIAL REGISTRATION: ClinicalTrials.gov NCT03554993 .
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Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Serina-Treonina Quinases , Citocinas/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , RNA Mensageiro , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
INTRODUCTION: Idiopathic pulmonary fibrosis (IPF) is a progressive and often fatal interstitial lung disease (ILD); other ILDs have a progressive, fibrotic phenotype (PF-ILD). Antifibrotic agents can slow but not stop disease progression in patients with IPF or PF-ILD. c-Jun N-terminal kinases (JNKs) are stress-activated protein kinases implicated in the underlying mechanisms of fibrosis, including epithelial cell death, inflammation and polarisation of profibrotic macrophages, fibroblast activation and collagen production. CC-90001, an orally administered (PO), one time per day, JNK inhibitor, is being evaluated in IPF and PF-ILD. METHODS AND ANALYSIS: This is a phase 2, randomised, double-blind, placebo-controlled study evaluating efficacy and safety of CC-90001 in patients with IPF (main study) and patients with PF-ILD (substudy). Both include an 8-week screening period, a 24-week treatment period, up to an 80-week active-treatment extension and a 4-week post-treatment follow-up. Patients with IPF (n=165) will be randomised 1:1:1 to receive 200 mg or 400 mg CC-90001 or placebo administered PO one time per day; up to 25 patients/arm will be permitted concomitant pirfenidone use. Forty-five patients in the PF-ILD substudy will be randomised 2:1 to receive 400 mg CC-90001 or placebo. The primary endpoint is change in per cent predicted forced vital capacity from baseline to Week 24 in patients with IPF. ETHICS AND DISSEMINATION: This study will be conducted in accordance with Good Clinical Practice guidelines, Declaration of Helsinki principles and local ethical and legal requirements. Results will be reported in a peer-reviewed publication. TRIAL REGISTRATION NUMBER: NCT03142191.
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Fibrose Pulmonar Idiopática , Doenças Pulmonares Intersticiais , Inibidores de Proteínas Quinases , Ensaios Clínicos Fase II como Assunto , Fibrose , Humanos , Fibrose Pulmonar Idiopática/tratamento farmacológico , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Doenças Pulmonares Intersticiais/tratamento farmacológico , Doenças Pulmonares Intersticiais/etiologia , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Ensaios Clínicos Controlados Aleatórios como Assunto , Capacidade VitalRESUMO
BACKGROUND: Apremilast, an oral phosphodiesterase (PDE) 4 inhibitor, has demonstrated efficacy in psoriasis, while its efficacy in atopic dermatitis (AD) was found to be modest. AD is a chronic inflammatory skin disease associated with activation of T helper (Th) 2 and Th17 immunity and a compromised epidermal barrier. OBJECTIVE: The objectives of this study were to examine the expression of PDE4 isoforms in skin from healthy subjects and AD patients, and to determine the effects of apremilast on AD-related inflammatory markers in vitro and in murine models of AD. METHODS: The expression of PDE4 isoforms (A, B, C, and D) in skin biopsies from healthy subjects and AD patients was evaluated using immunohistochemistry and digital image analysis. Using quantitative real-time reverse-transcriptase polymerase chain reaction, we evaluated the effects of apremilast on gene expression in adult human epidermal keratinocytes (HEKa) stimulated by Th2 and Th17 cytokines, and in two mouse models of antigen-induced AD. RESULTS: Expression of PDE4 isoforms increased up to three-fold in the epidermis of AD patients versus healthy skin. In interleukin (IL)-4 and IL-17-stimulated HEKa cells, apremilast significantly changed the expression of ILs, including IL-12/IL-23p40 and IL-31, and alarmins S100A7, S100A8, and S100A12. In mouse models of AD, apremilast significantly reduced ear swelling and monocyte chemoattractant protein-1 expression. CONCLUSION: PDE4 is overexpressed in AD skin compared with normal skin, and inflammatory gene expression by human keratinocytes and mouse dermatitis can be modulated by apremilast.
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Citocinas/genética , Dermatite Atópica/tratamento farmacológico , Expressão Gênica/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , Inibidores da Fosfodiesterase 4/uso terapêutico , Talidomida/análogos & derivados , Adulto , Animais , Células Cultivadas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Dermatite Atópica/imunologia , Modelos Animais de Doenças , Voluntários Saudáveis , Humanos , Interleucina-17/genética , Interleucina-4/genética , Queratinócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Cultura Primária de Células , Isoformas de Proteínas , Talidomida/uso terapêuticoRESUMO
OBJECTIVE: To evaluate the safety and efficacy of pomalidomide (POM) on forced vital capacity (FVC), modified Rodnan skin score (mRSS), and gastrointestinal (GI) symptomatology over 52 weeks of treatment in patients with interstitial lung disease due to systemic sclerosis (SSc). METHODS: Twenty-three adult patients diagnosed with SSc were randomized 1:1 POM:placebo (PBO). RESULTS: Mean change at Week 52 from baseline in predicted FVC% -5.2 and -2.8; mRSS -2.7 and -3.7; and UCLA Scleroderma Clinical Trial Consortium Gastrointestinal Tract (SCTC GIT 2.0) score 0.1 and 0.0, with POM and PBO, respectively. Statistical significance was not achieved for any of these 3 primary endpoints at 52 weeks. CONCLUSION: Because of recruitment challenges, subject enrollment was discontinued early. In an interim analysis, the study did not meet its Week 52 primary endpoints. Therefore, a decision was made to terminate all study phases. POM was generally well tolerated, with an adverse event profile consistent with the known safety and tolerability profile of POM in other diseases. Study results were neither positive nor negative because too few subjects were enrolled to make meaningful conclusions. Clinical Trials number: NCT01559129.
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Fatores Imunológicos/efeitos adversos , Fatores Imunológicos/uso terapêutico , Doenças Pulmonares Intersticiais/tratamento farmacológico , Doenças Pulmonares Intersticiais/etiologia , Escleroderma Sistêmico/complicações , Talidomida/análogos & derivados , Adulto , Método Duplo-Cego , Término Precoce de Ensaios Clínicos , Feminino , Fibrose/tratamento farmacológico , Seguimentos , Volume Expiratório Forçado/efeitos dos fármacos , Humanos , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Pele/patologia , Talidomida/efeitos adversos , Talidomida/uso terapêutico , Resultado do Tratamento , Capacidade Vital/efeitos dos fármacosRESUMO
BACKGROUND: Lung remodeling and pulmonary fibrosis are serious, life-threatening conditions resulting from diseases such as chronic severe asthma and idiopathic pulmonary fibrosis (IPF). Preclinical evidence suggests that JNK enzyme function is required for key steps in the pulmonary fibrotic process. However, a selective JNK inhibitor has not been investigated in translational models of lung fibrosis with clinically relevant biomarkers, or in IPF patients. METHODS: The JNK inhibitor CC-930 was evaluated in the house dust mite-induced fibrotic airway mouse model, in a phase I healthy volunteer pharmacodynamic study, and subsequently in a phase II multicenter study of mild/moderate IPF (n = 28), with a 4-week, placebo-controlled, double-blind, sequential ascending-dose period (50 mg QD, 100 mg QD, 100 mg BID) and a 52-week open-label treatment-extension period. RESULTS: In the preclinical model, CC-930 attenuated collagen 1A1 gene expression, peribronchiolar collagen deposition, airway mucin MUC5B expression in club cells, and MMP-7 expression in lung, bronchoalveolar lavage fluid, and serum. In the phase I study, CC-930 reduced c-Jun phosphorylation induced by UV radiation in skin. In the phase II IPF study, there was a CC-930 dose-dependent trend in reduction of MMP-7 and SP-D plasma protein levels. The most commonly reported adverse events were increased ALT, increased AST, and upper respiratory tract infection (six subjects each, 21.4 %). A total of 13 subjects (46.4 %) experienced adverse events that led to discontinuation of study drug. Nine out of 28 subjects experienced progressive disease in this study. The mean FVC (% predicted) declined after 26-32 weeks at doses of 100 mg QD and 100 mg BID. Changes in MMP-7, SP-D, and tenascin-C significantly correlated with change in FVC (% predicted). CONCLUSIONS: These results illustrate JNK enzymatic activity involvement during pulmonary fibrosis, and support systemic biomarker use for tracking disease progression and the potential clinical benefit of this novel intervention in IPF. Trial registration ClinicalTrials.gov NCT01203943.
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Phosphodiesterases 4 (PDE4) act as proinflammatory enzymes via degradation of cAMP, whereas PDE4 inhibitors play an anti-inflammatory role in vitro and in vivo. In particular, apremilast has been recently approved for the treatment of psoriasis and psoriatic arthritis. However, little is known on the expression pattern of PDE4 in psoriasis. We report that PDE4B and PDE4D mRNA are overexpressed in peripheral blood mononuclear cells (PBMC) from psoriasis, as compared with normal controls, while apremilast reduces PBMC production of a number of pro-inflammatory cytokines and increases the levels of anti-inflammatory mediators. PDE4 expression is up-regulated in psoriatic dermis as compared with normal skin, with particular regard to fibroblasts. This is confirmed in vitro, where both dermal fibroblasts (DF) and, to a greater extent, myofibroblasts (DM) express all PDE4 isoforms at the mRNA and protein level. Because PDE4 interacts with the nerve growth factor (NGF) receptor CD271 in lung fibroblasts, we evaluated the relationship and function of PDE4 and CD271 in normal human skin fibroblasts. All PDE4 isoforms co-immunoprecipitate with CD271 in DM, while apremilast inhibits apoptosis induced by ß-amyloid, a CD271 ligand, in DM. Furthermore, apremilast significantly reduces NGF- and transforming growth factor-ß1 (TGF-ß1)-induced fibroblast migration, and inhibits DF differentiation into DM mediated by NGF or TGF-ß1. Finally, in DM, apremilast significantly reduces cAMP degradation induced by treatment with ß-amyloid. Taken together, these results indicate that PDE4 play an important role in psoriasis. In addition, the study reveals that the PDE4/CD271 complex could be important in modulating fibroblast functions.
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Adapaleno/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Derme/patologia , Inflamação/enzimologia , Miofibroblastos/metabolismo , Psoríase/sangue , Psoríase/enzimologia , Talidomida/análogos & derivados , Adulto , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Citocinas/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Humanos , Imuno-Histoquímica , Imunoprecipitação , Inflamação/sangue , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Psoríase/patologia , Talidomida/farmacologiaRESUMO
The arginine-glycine-aspartate (RGD)-binding integrins alphavbeta6 and alphavbeta8 activate latent TGFbeta1 and TGFbeta3 in vivo, but it is uncertain whether other RGD-binding integrins such as integrins alphavbeta5 and alphavbeta3 activate these TGFbeta isoforms. To define the combined role of alphavbeta6- and alphavbeta8-integrin in TGFbeta activation, we analyzed mice lacking function of both integrins by means of gene deletion and/or pharmacologic inhibition. Most Itgb6-/-;Itgb8-/- embryos die at mid-gestation; those that survive develop cleft palate-as observed in Tgfb3-/- mice. Itgb8-/- mice treated with an anti-alphavbeta6-integrin antibody develop severe autoimmunity and lack Langerhans cells-similar to Tgfb1-null mice. These results support a model in which TGFbeta3-mediated palate fusion and TGFbeta1-mediated suppression of autoimmunity and generation of Langerhans cells require integrins alphavbeta6 and alphavbeta8 but not other RGD-binding integrins as TGFbeta activators.
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Antígenos de Neoplasias/metabolismo , Fissura Palatina/metabolismo , Integrinas/metabolismo , Palato/anormalidades , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta3/metabolismo , Animais , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Autoimunidade/genética , Autoimunidade/imunologia , Fissura Palatina/genética , Fissura Palatina/imunologia , Integrinas/genética , Integrinas/imunologia , Estimativa de Kaplan-Meier , Células de Langerhans/imunologia , Camundongos , Camundongos Knockout , Oligopeptídeos/metabolismo , Palato/imunologia , Palato/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/imunologia , Fator de Crescimento Transformador beta3/genética , Fator de Crescimento Transformador beta3/imunologiaRESUMO
Deletion of integrin alphav beta6 has been associated with significant protection in experiments where lung injury was induced by bleomycin, lipophilic polysaccharides, and high tidal volume ventilation. This has led to the suggestion that antibody blockade of this integrin is a novel therapy for acute lung injury. We questioned whether beta6 gene deletion would also protect against Pseudomonas aeruginosa-induced acute lung injury. Wild-type and littermate beta6-null mice, as well as recombinant soluble TGF-beta receptor type II-Fc (rsTGF-betaRII-Fc) and anti-alphav beta6 treated wild-type mice, were instilled with live P. aeruginosa. Four or 8 h after bacterial instillation, the mice were euthanized, and either bronchoalveolar lavage fluid or lung homogenates were obtained. Deletion of the beta6 gene resulted in an overall increase in inflammatory cells in the lungs. Bacterial numbers from the lung homogenates of infected beta6-null mice were significantly decreased compared to the numbers in the wild-type mice (1.6 x 10(6) CFU versus 4.2 x 10(6) CFU [P < 0.01]). There were no significant differences in P. aeruginosa-mediated increases in lung endothelial permeability between wild-type and beta6-null mice. Similarly, pretreatment with the alphav beta6 antibody or with rsTGF-betaRII-Fc did not significantly affect the P. aeruginosa-induced lung injury or rate of survival compared to pretreatment with control immunoglobulin G. We conclude that deletion or inhibition of the integrin alphav beta6 did not protect animals from P. aeruginosa-induced lung injury. However, these therapies also did not increase the lung injury, suggesting that host defense was not compromised by this promising new therapy.
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Antígenos de Neoplasias/metabolismo , Integrinas/metabolismo , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/microbiologia , Animais , Anticorpos Bloqueadores/imunologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/imunologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/análise , Citocinas/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Integrinas/genética , Integrinas/imunologia , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Knockout , Fagocitose/genética , Infecções por Pseudomonas/microbiologia , Receptores de Fatores de Crescimento Transformadores beta/uso terapêutico , Síndrome do Desconforto Respiratório/prevenção & controle , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Interleukin (IL)-1beta has previously been shown to be among the most biologically active cytokines in the lungs of patients with acute lung injury (ALI). Furthermore, there is experimental evidence that lung vascular permeability increases after short-term exposure to IL-1 protein, although the exact mechanism is unknown. Therefore, the objective of this study was to determine the mechanisms of IL-1beta-mediated increase in lung vascular permeability and pulmonary edema following transient overexpression of this cytokine in the lungs by adenoviral gene transfer. Lung vascular permeability increased with intrapulmonary IL-1beta production with a maximal effect 7 days after instillation of the adenovirus. Furthermore, inhibition of the alphavbeta6 integrin and/or transforming growth factor-beta attenuated the IL-1beta-induced ALI. The results of in vitro studies indicated that IL-1beta caused the activation of transforming growth factor-beta via RhoA/alphavbeta6 integrin-dependent mechanisms and the inhibition of the alphavbeta6 integrin and/or transforming growth factor-beta signaling completely blocked the IL-1beta-mediated protein permeability across alveolar epithelial cell monolayers. In addition, IL-1beta increased protein permeability across lung endothelial cell monolayers via RhoA- and alphavbeta5 integrin-dependent mechanisms. The final series of in vivo experiments demonstrated that pretreatment with blocking antibodies to both the alphavbeta5 and alphavbeta6 integrins had an additive protective effect against IL-1beta-induced ALI. In summary, these results demonstrate a critical role for the alphavbeta5/beta6 integrins in mediating the IL-1beta-induced ALI and indicate that these integrins could be a potentially attractive therapeutic target in ALI.
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Antígenos de Neoplasias/metabolismo , Integrinas/metabolismo , Interleucina-1beta/farmacologia , Receptores de Vitronectina/metabolismo , Síndrome do Desconforto Respiratório/etiologia , Adenoviridae/genética , Albuminas/metabolismo , Animais , Antígenos de Neoplasias/genética , Permeabilidade Capilar/efeitos dos fármacos , Bovinos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Técnicas de Transferência de Genes , Humanos , Integrinas/genética , Interleucina-1beta/metabolismo , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vison , Edema Pulmonar/etiologia , Edema Pulmonar/metabolismo , Edema Pulmonar/patologia , Ratos , Receptores de Vitronectina/genética , Síndrome do Desconforto Respiratório/metabolismo , Síndrome do Desconforto Respiratório/patologia , Fator de Crescimento Transformador alfa/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The alpha(v)beta(6) integrin is up-regulated on epithelial malignancies and has been implicated in various aspects of cancer progression. Immunohistochemical analysis of alpha(v)beta(6) expression in 10 human tumor types showed increased expression relative to normal tissues. Squamous carcinomas of the cervix, skin, esophagus, and head and neck exhibited the highest frequency of expression, with positive immunostaining in 92% (n = 46), 84% (n = 49), 68% (n = 56), and 64% (n = 100) of cases, respectively. We studied the role of alpha(v)beta(6) in Detroit 562 human pharyngeal carcinoma cells in vitro and in vivo. Prominent alpha(v)beta(6) expression was detected on tumor xenografts at the tumor-stroma interface resembling the expression on human head and neck carcinomas. Nonetheless, coculturing cells in vitro with matrix proteins did not up-regulate alpha(v)beta(6) expression. Detroit 562 cells showed alpha(v)beta(6)-dependent adhesion and activation of transforming growth factor-beta (TGF-beta) that was inhibited >90% with an alpha(v)beta(6) blocking antibody, 6.3G9. Although both recombinant soluble TGF-beta receptor type-II (rsTGF-beta RII-Fc) and 6.3G9 inhibited TGF-beta-mediated Smad2/3 phosphorylation in vitro, there was no effect on proliferation. Conversely, in vivo, 6.3G9 and rsTGF-beta RII-Fc inhibited xenograft tumor growth by 50% (n = 10, P < 0.05) and >90% (n = 10, P < 0.001), respectively, suggesting a role for the microenvironment in this response. However, stromal collagen and smooth muscle actin content in xenograft sections were unchanged with treatments. Although further studies are required to consolidate in vitro and in vivo results and define the mechanisms of tumor inhibition by alpha(v)beta(6) antibodies, our findings support a role for alpha(v)beta(6) in human cancer and underscore the therapeutic potential of function blocking alpha(v)beta(6) antibodies.
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Anticorpos Monoclonais/farmacologia , Carcinoma de Células Escamosas/patologia , Proliferação de Células/efeitos dos fármacos , Integrina alfa5/imunologia , Neoplasias Faríngeas/patologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Carcinoma de Células Escamosas/metabolismo , Células Cultivadas , Progressão da Doença , Feminino , Humanos , Fragmentos Fc das Imunoglobulinas/farmacologia , Integrina alfa5/metabolismo , Integrina alfa5/fisiologia , Camundongos , Camundongos Nus , Vison , Neoplasias Faríngeas/metabolismo , Isoformas de Proteínas/imunologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/farmacologia , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/química , Proteínas Recombinantes de Fusão/farmacologia , Transdução de Sinais/genética , Proteínas Smad/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RATIONALE: In experimental models, lung fibrosis is dependent on transforming growth factor (TGF)-beta signaling. TGF-beta is secreted in a latent complex with its propeptide, and TGF-beta activators release TGF-beta from this complex. Because the integrin alpha(v)beta6 is a major TGF-beta activator in the lung, inhibition of alpha(v)beta6-mediated TGF-beta activation is a logical strategy to treat lung fibrosis. OBJECTIVES: To determine, by genetic and pharmacologic approaches, whether murine radiation-induced lung fibrosis is dependent on alpha(v)beta6. METHODS: Wild-type mice, alpha(v)beta6-deficient (Itgb6-/-) mice, and mice heterozygous for a Tgfb1 mutation that eliminates integrin-mediated activation (Tgfb1(+/RGE)) were exposed to 14 Gy thoracic radiation. Some mice were treated with an anti-alpha(v)beta6 monoclonal antibody or a soluble TGF-beta receptor fusion protein. Alpha(v)beta6 expression was determined by immunohistochemistry. Fibrosis, inflammation, and gene expression patterns were assessed 20-32 weeks postirradiation. MEASUREMENTS AND MAIN RESULTS: Beta6 integrin expression increased within the alveolar epithelium 18 weeks postirradiation, just before onset of fibrosis. Itgb6-/- mice were completely protected from fibrosis, but not from late radiation-induced mortality. Anti-alpha(v)beta6 therapy (1-10 mg/kg/wk) prevented fibrosis, but only higher doses (6-10 mg/kg/wk) caused lung inflammation similar to that in Itgb6-/- mice. Tgfb1-haploinsufficient mice were also protected from fibrosis. CONCLUSIONS: Alpha(v)beta6-mediated TGF-beta activation is required for radiation-induced lung fibrosis. Together with previous data, our results demonstrate a robust requirement for alpha(v)beta6 in distinct fibrosis models. Inhibition of alphavbeta6-mediated TGF-beta activation is a promising new approach for antifibrosis therapy.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias/farmacologia , Modelos Animais de Doenças , Fibrose Pulmonar/prevenção & controle , Pneumonite por Radiação/prevenção & controle , Fator de Crescimento Transformador beta/metabolismo , Animais , Antígenos de Neoplasias/genética , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Haplótipos , Heterozigoto , Integrinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/patologia , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Pneumonite por Radiação/genética , Pneumonite por Radiação/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
RATIONALE: Transforming growth factor (TGF)-beta has a central role in driving many of the pathological processes that characterize pulmonary fibrosis. Inhibition of the integrin alpha(v)beta6, a key activator of TGF-beta in lung, is an attractive therapeutic strategy, as it may be possible to inhibit TGF-beta at sites of alpha(v)beta6 up-regulation without affecting other homeostatic roles of TGF-beta. OBJECTIVES: To analyze the expression of alpha(v)beta6 in human pulmonary fibrosis, and to functionally test the efficacy of therapeutic inhibition of alpha(v)beta6-mediated TGF-beta activation in murine bleomycin-induced pulmonary fibrosis. METHODS: Lung biopsies from patients with a diagnosis of systemic sclerosis or idiopathic pulmonary fibrosis were stained for alpha(v)beta6 expression. A range of concentrations of a monoclonal antibody that blocks alpha(v)beta6-mediated TGF-beta activation was evaluated in murine bleomycin-induced lung fibrosis. MEASUREMENTS AND MAIN RESULTS: Alpha(v)beta6 is overexpressed in human lung fibrosis within pneumocytes lining the alveolar ducts and alveoli. In the bleomycin model, alpha(v)beta6 antibody was effective in blocking pulmonary fibrosis. At high doses, there was increased expression of markers of inflammation and macrophage activation, consistent with the effects of TGF-beta inhibition in the lung. Low doses of antibody attenuated collagen expression without increasing alveolar inflammatory cell populations or macrophage activation markers. CONCLUSIONS: Partial inhibition of TGF-beta using alpha(v)beta6 integrin antibodies is effective in blocking murine pulmonary fibrosis without exacerbating inflammation. In addition, the elevated expression of alpha(v)beta6, an activator of the fibrogenic cytokine, TGF-beta, in human pulmonary fibrosis suggests that alpha(v)beta6 monoclonal antibodies could represent a promising new therapeutic strategy for treating pulmonary fibrosis.
Assuntos
Anticorpos Monoclonais/farmacologia , Modelos Animais de Doenças , Integrinas/antagonistas & inibidores , Fibrose Pulmonar/imunologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Antígenos de Neoplasias/fisiologia , Colágeno/metabolismo , Relação Dose-Resposta a Droga , Integrinas/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/imunologia , Fibrose Pulmonar/patologia , Fibrose Pulmonar/terapia , Escleroderma Sistêmico/imunologia , Escleroderma Sistêmico/patologia , Escleroderma Sistêmico/terapia , Fator de Crescimento Transformador beta/fisiologiaRESUMO
The transforming growth factor (TGF)-beta-inducible integrin alpha v beta6 is preferentially expressed at sites of epithelial remodeling and has been shown to bind and activate latent precursor TGF-beta. Herein, we show that alpha v beta6 is overexpressed in human kidney epithelium in membranous glomerulonephritis, diabetes mellitus, IgA nephropathy, Goodpasture's syndrome, and Alport syndrome renal epithelium. To assess the potential regulatory role of alpha v beta6 in renal disease, we studied the effects of function-blocking alpha v beta6 monoclonal antibodies (mAbs) and genetic ablation of the beta6 subunit on kidney fibrosis in Col4A3-/- mice, a mouse model of Alport syndrome. Expression of alpha v beta6 in Alport mouse kidneys was observed primarily in cortical tubular epithelial cells and in correlation with the progression of fibrosis. Treatment with alpha v beta6-blocking mAbs inhibited accumulation of activated fibroblasts and deposition of interstitial collagen matrix. Similar inhibition of renal fibrosis was observed in beta6-deficient Alport mice. Transcript profiling of kidney tissues showed that alpha v beta6-blocking mAbs significantly inhibited disease-associated changes in expression of fibrotic and inflammatory mediators. Similar patterns of transcript modulation were produced with recombinant soluble TGF-beta RII treatment, suggesting shared regulatory functions of alpha v beta6 and TGF-beta. These findings demonstrate that alpha v beta6 can contribute to the regulation of renal fibrosis and suggest this integrin as a potential therapeutic target.
Assuntos
Antígenos de Neoplasias/biossíntese , Integrinas/biossíntese , Nefrite Hereditária/metabolismo , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Antígenos de Neoplasias/imunologia , Modelos Animais de Doenças , Matriz Extracelular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Humanos , Imuno-Histoquímica , Integrinas/antagonistas & inibidores , Integrinas/imunologia , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Knockout , Células NIH 3T3 , Nefrite Hereditária/tratamento farmacológico , Nefrite Hereditária/etiologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/metabolismo , Regulação para CimaRESUMO
The therapeutic effects of the Sonic hedgehog (Shh) have been difficult to evaluate because of its relatively short serum half-life. To address this issue polyethylene glycol modification (PEGylation) was investigated as an approach to improve systemic exposure. Shh was PEGylated by a targeted approach using cysteines that were engineered into the protein by site-directed mutagenesis as the sites of attachment. Sixteen different versions of the protein containing one, two, three, or four sites of attachment were characterized. Two forms were selected for extensive testing in animals, Shh A192C, which provided a single site for PEGylation, and Shh A192C/N91C, which provided two sites. The PEGylated proteins were evaluated for reaction specificity by SDS-PAGE and peptide mapping, in vitro potency, pharmacokinetic and pharmacodynamic properties, and efficacy in a sciatic nerve injury model. Targeted PEGylation was highly selective for the engineered cysteines and had no deleterious effect on Shh function in vitro. Systemic clearance values in rats decreased from 117.4 mL/h/kg for unmodified Shh to 29.4 mL/h/kg for mono-PEGylated Shh A192C that was modified with 20 kDa PEG-maleimide and to 2.5 mL/h/kg for di-PEGylated Shh A192C/N91C modified with 2, 20 kDa PEG vinylsulfone adducts. Serum half-life increased from 1 h for unmodified Shh to 7.0 and 12.6 h for the mono- and di-PEGylated products. These changes in clearance and half-life resulted in higher serum levels of Shh in the PEG-Shh-treated animals. In Ptc-LacZ knock-in mice expressing lacZ under regulation of the Shh receptor Patched, about a 10-fold lower dose of PEG-Shh was needed to induce beta-galactosidase than for the unmodified protein. Therapeutic treatment of mice with PEG-Shh enhanced the regeneration of injured sciatic nerves. These studies demonstrate that targeted PEGylation greatly alters the pharmacokinetic and pharmacodynamic properties of Shh, resulting in a form with improved pharmaceutical properties.