RESUMO
Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease driven by gain-of-function variants in activin receptor-like kinase 2 (ALK2), the most common variant being ALK2R206H. In FOP, ALK2 variants display increased and dysregulated signaling through the bone morphogenetic protein (BMP) pathway resulting in progressive and permanent replacement of skeletal muscle and connective tissues with heterotopic bone, ultimately leading to severe debilitation and premature death. Here, we describe the discovery of BLU-782 (IPN60130), a small-molecule ALK2R206H inhibitor developed for the treatment of FOP. A small-molecule library was screened in a biochemical ALK2 binding assay to identify potent ALK2 binding compounds. Iterative rounds of structure-guided drug design were used to optimize compounds for ALK2R206H binding, ALK2 selectivity, and other desirable pharmacokinetic properties. BLU-782 preferentially bound to ALK2R206H with high affinity, inhibiting signaling from ALK2R206H and other rare FOP variants in cells in vitro without affecting signaling of closely related homologs ALK1, ALK3, and ALK6. In vivo efficacy of BLU-782 was demonstrated using a conditional knock-in ALK2R206H mouse model, where prophylactic oral dosing reduced edema and prevented cartilage and heterotopic ossification (HO) in both muscle and bone injury models. BLU-782 treatment preserved the normal muscle-healing response in ALK2R206H mice. Delayed dosing revealed a short 2-day window after injury when BLU-782 treatment prevented HO in ALK2R206H mice, but dosing delays of 4 days or longer abrogated HO prevention. Together, these data suggest that BLU-782 may be a candidate for prevention of HO in FOP.
Assuntos
Modelos Animais de Doenças , Miosite Ossificante , Ossificação Heterotópica , Animais , Miosite Ossificante/tratamento farmacológico , Miosite Ossificante/metabolismo , Ossificação Heterotópica/tratamento farmacológico , Ossificação Heterotópica/metabolismo , Ossificação Heterotópica/prevenção & controle , Camundongos , Humanos , Receptores de Activinas Tipo II/metabolismo , Receptores de Ativinas Tipo I/metabolismo , Receptores de Ativinas Tipo I/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
Background and Aims: Fibrolamellar carcinoma (FLC) is a rare, difficult-to-treat liver cancer primarily affecting pediatric and adolescent patients, and for which precision medicine approaches have historically not been possible. The DNAJB1-PRKACA gene fusion was identified as a driver of FLC pathogenesis. We aimed to assess whether FLC tumors maintain dependency on this gene fusion and determine if PRKACA is a viable therapeutic target. Methods: FLC patient-derived xenograft (PDX) shRNA cell lines were implanted subcutaneously into female NOD-SCID mice and tumors were allowed to develop prior to randomization to doxycycline (to induce knockdown) or control groups. Tumor development was assessed every 2 days. To assess the effect of treatment with novel selective PRKACA small molecule kinase inhibitors, BLU0588 and BLU2864, FLC PDX tumor cells were implanted subcutaneously into NOD-SCID mice and tumors allowed to develop. Mice were randomized to treatment (BLU0588 and BLU2864, orally, once daily) or control groups and tumor size determined as previously. Results: Knockdown of DNAJB1-PRKACA reversed a FLC-specific gene signature and reduced PDX tumor growth in mice compared to the control group. Furthermore, FLC PDX tumor growth was significantly reduced with BLU0588 and BLU2864 treatment vs control (P = .003 and P = .0005, respectively). Conclusion: We demonstrated, using an inducible knockdown and small molecule approaches, that FLC PDX tumors were dependent upon DNAJB1-PRKACA fusion activity. In addition, this study serves as a proof-of-concept that PRKACA is a viable therapeutic target for FLC and warrants further investigation.
RESUMO
BACKGROUND: Upregulation of cAMP-dependent and cAMP-independent PKA signaling is thought to promote cystogenesis in polycystic kidney disease (PKD). PKA-I regulatory subunit RIα is increased in kidneys of orthologous mouse models. Kidney-specific knockout of RIα upregulates PKA activity, induces cystic disease in wild-type mice, and aggravates it in Pkd1RC/RC mice. METHODS: PKA-I activation or inhibition was compared with EPAC activation or PKA-II inhibition using Pkd1RC/RC metanephric organ cultures. The effect of constitutive PKA (preferentially PKA-I) downregulation in vivo was ascertained by kidney-specific expression of a dominant negative RIαB allele in Pkd1RC/RC mice obtained by crossing Prkar1αR1αB/WT, Pkd1RC/RC , and Pkhd1-Cre mice (C57BL/6 background). The effect of pharmacologic PKA inhibition using a novel, selective PRKACA inhibitor (BLU2864) was tested in mIMCD3 3D cultures, metanephric organ cultures, and Pkd1RC/RC mice on a C57BL/6 × 129S6/Sv F1 background. Mice were sacrificed at 16 weeks of age. RESULTS: PKA-I activation promoted and inhibition prevented ex vivo P-Ser133 CREB expression and cystogenesis. EPAC activation or PKA-II inhibition had no or only minor effects. BLU2864 inhibited in vitro mIMCD3 cystogenesis and ex vivo P-Ser133 CREB expression and cystogenesis. Genetic downregulation of PKA activity and BLU2864 directly and/or indirectly inhibited many pro-proliferative pathways and were both protective in vivo. BLU2864 had no detectable on- or off-target adverse effects. CONCLUSIONS: PKA-I is the main PKA isozyme promoting cystogenesis. Direct PKA inhibition may be an effective strategy to treat PKD and other conditions where PKA signaling is upregulated. By acting directly on PKA, the inhibition may be more effective than or substantially increase the efficacy of treatments that only affect PKA activity by lowering cAMP.
Assuntos
Rim Policístico Autossômico Dominante , Rim Policístico Autossômico Recessivo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/farmacologia , Rim/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Doenças Renais Policísticas , Rim Policístico Autossômico Dominante/metabolismo , Receptores de Superfície Celular/genética , Canais de Cátion TRPP/genética , Canais de Cátion TRPP/metabolismoRESUMO
Fatty liver disease is a potential risk factor for drug-induced liver injury (DILI). Despite advances in nonclinical in vitro and in vivo models to assess liver injury during drug development, the pharmaceutical industry is still plagued by idiosyncratic DILI. Here, we tested the hypothesis that certain features of asymptomatic metabolic syndrome (namely hepatic steatosis) increase the risk for DILI in certain phenotypes of the human population. Comparison of the Zucker Lean (ZL) and Zucker Fatty rats fed a high fat diet (HFD) revealed that HFD-fed ZL rats developed mild hepatic steatosis with compensatory hyperinsulinemia without increases in liver enzymes. We then challenged steatotic HFD-fed ZL rats and Sprague-Dawley (SD) rats fed normal chow, a nonclinical model widely used in the pharmaceutical industry, with acetaminophen overdose to induce liver injury. Observations in HFD-fed ZL rats included increased liver injury enzymes and greater incidence and severity of hepatic necrosis compared with similarly treated SD rats. The HFD-fed ZL rats also had disproportionately higher hepatic drug accumulation, which was linked with abnormal hepatocellular efflux transporter distribution. Here, we identify ZL rats with HFD-induced hepatic steatosis as a more sensitive nonclinical in vivo test system for modeling DILI compared with SD rats fed normal chow.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Fígado Gorduroso , Síndrome Metabólica , Animais , Dieta Hiperlipídica/efeitos adversos , Fígado Gorduroso/induzido quimicamente , Humanos , Fígado , Síndrome Metabólica/induzido quimicamente , Ratos , Ratos Sprague-Dawley , Ratos ZuckerRESUMO
Glomerulopathy and body weight gain were noted after chronic oral administration of a novel nonsteroidal dissociated agonist of the glucocorticoid receptor compound, fosdagrocorat, to beagle dogs fed an ad libitum diet. To further investigate the role of diet and treatment with either fosdagrocorat or the glucocorticoid comparator, prednisone, on renal safety, a 13-week investigative study was conducted in beagle dogs. Renal histopathology, clinical chemistry, urinalysis, glomerular filtration rate (GFR), body weight, heart rate, blood pressure (BP), and hematology were investigated in restricted- and ad libitum-fed dogs administered prednisone (2.2 mg/kg/d), fosdagrocorat (5 mg/kg/d), or vehicle for 13 weeks. Glomerulopathy was primarily observed in fosdagrocorat- and prednisone-treated ad libitum but not in feed-restricted or ad libitum vehicle-treated dogs. Kidneys in dogs from the prednisone-treated ad libitum had the greatest incidence and severity of tubular degenerative changes. Increased urine volume and decreased urine-specific gravity were present in prednisone- and fosdagrocorat-treated dogs, regardless of diet. These changes were not associated with consistent changes in GFR. Fosdagrocorat or prednisone treatment ad libitum dogs had the greatest increase in body weight gain. Sporadic changes in systolic and diastolic BP were noted in fosdagrocorat- and prednisone-treated groups. Significant reductions in serum cortisol and absolute eosinophils were noted in both ad libitum- and restriction-fed prednisone- and fosdagrocorat-treated dogs. In conclusion, prednisone-treated dogs fed ad libitum had greater glucocorticoid-induced renal effects than those dosed with fosdagrocorat.
Assuntos
Rim/efeitos dos fármacos , Organofosfatos/farmacologia , Fenantrenos/farmacologia , Prednisona/farmacologia , Receptores de Glucocorticoides/agonistas , Animais , Pressão Sanguínea/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Cães , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Privação de Alimentos , Taxa de Filtração Glomerular/efeitos dos fármacos , Frequência Cardíaca/efeitos dos fármacos , Rim/patologia , Organofosfatos/administração & dosagem , Organofosfatos/efeitos adversos , Fenantrenos/administração & dosagem , Fenantrenos/efeitos adversos , Prednisona/efeitos adversos , Receptores de Glucocorticoides/efeitos dos fármacosRESUMO
Targeting oncogenic kinase drivers with small-molecule inhibitors can have marked therapeutic benefit, especially when administered to an appropriate genomically defined patient population. Cancer genomics and mechanistic studies have revealed that heterogeneous mutations within a single kinase can result in various mechanisms of kinase activation. Therapeutic benefit to patients can best be optimized through an in-depth understanding of the disease-driving mutations combined with the ability to match these insights to tailored highly selective drugs. This rationale is presented for BLU-285, a clinical stage inhibitor of oncogenic KIT and PDGFRA alterations, including activation loop mutants that are ineffectively treated by current therapies. BLU-285, designed to preferentially interact with the active conformation of KIT and PDGFRA, potently inhibits activation loop mutants KIT D816V and PDGFRA D842V with subnanomolar potency and also inhibits other well-characterized disease-driving KIT mutants both in vitro and in vivo in preclinical models. Early clinical evaluation of BLU-285 in a phase 1 study has demonstrated marked activity in patients with diseases associated with KIT (aggressive systemic mastocytosis and gastrointestinal stromal tumor) and PDGFRA (gastrointestinal stromal tumor) activation loop mutations.
Assuntos
Mutação/genética , Medicina de Precisão , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/química , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/químicaRESUMO
OBJECTIVE: To investigate whether the effects of nerve growth factor (NGF) inhibition with tanezumab on rats with medial meniscal tear (MMT) effectively model rapidly progressive osteoarthritis (RPOA) observed in clinical trials. METHODS: Male Lewis rats underwent MMT surgery and were treated weekly with tanezumab (0.1, 1 or 10â mg/kg), isotype control or vehicle for 7, 14 or 28â days. Gait deficiency was measured to assess weight-bearing on the operated limb. Joint damage was assessed via histopathology. A second arm, delayed onset of treatment (starting 3-8â weeks after MMT surgery) was used to control for analgesia early in the disease process. A third arm, mid-tibial amputation, evaluated the dependency of the model on weight-bearing. RESULTS: Gait deficiency in untreated rats was present 3-7â days after MMT surgery, with a return to normal weight-bearing by days 14-28. Prophylactic treatment with tanezumab prevented gait deficiency and resulted in more severe cartilage damage. When onset of treatment with tanezumab was delayed to 3-8â weeks after MMT surgery, there was no increase in cartilage damage. Mid-tibial amputation completely prevented cartilage damage in untreated MMT rats. CONCLUSIONS: These data suggest that analgesia due to NGF inhibition during the acute injury phase is responsible for increased voluntary weight-bearing and subsequent cartilage damage in the rat MMT model. This model failed to replicate the hypotrophic bone response observed in tanezumab-treated patients with RPOA.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Cartilagem Articular/lesões , Fator de Crescimento Neural/antagonistas & inibidores , Lesões do Menisco Tibial/tratamento farmacológico , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Monoclonais Humanizados/toxicidade , Artrite Experimental/induzido quimicamente , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Cartilagem Articular/fisiopatologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Avaliação Pré-Clínica de Medicamentos/métodos , Marcha , Masculino , Meniscos Tibiais/diagnóstico por imagem , Meniscos Tibiais/patologia , Radiografia , Ratos Endogâmicos Lew , Lesões do Menisco Tibial/diagnóstico por imagem , Lesões do Menisco Tibial/patologia , Lesões do Menisco Tibial/fisiopatologia , Suporte de Carga , Microtomografia por Raio-XRESUMO
In Friedreich's ataxia (FRDA) patients, diminished frataxin (FXN) in sensory neurons is thought to yield the predominant pathology associated with disease. In this study, we demonstrate successful usage of RNA transcript therapy (RTT) as an exogenous human FXN supplementation strategy in vitro and in vivo, specifically to dorsal root ganglia (DRG). Initially, 293 T cells were transfected with codon optimized human FXN mRNA, which was translated to yield FXN protein. Importantly, FXN was rapidly processed into the mature functional form of FXN (mFXN). Next, FXN mRNA, in the form of lipid nanoparticles (LNPs), was administered intravenously in adult mice. Examination of liver homogenates demonstrated efficient FXN LNP uptake in hepatocytes and revealed that the mitochondrial maturation machinery had efficiently processed all FXN protein to mFXN in ~24 h in vivo. Remarkably, greater than 50% mFXN protein derived from LNPs was detected seven days after intravenous administration of FXN LNPs, suggesting that the half-life of mFXN in vivo exceeds one week. Moreover, when FXN LNPs were delivered by intrathecal administration, we detected recombinant human FXN protein in DRG. These observations provide the first demonstration that RTT can be used for the delivery of therapeutic mRNA to DRG.
Assuntos
Ataxia de Friedreich/genética , Gânglios Espinais/metabolismo , Proteínas de Ligação ao Ferro/genética , Lipídeos , Nanopartículas , RNA Mensageiro , Animais , Modelos Animais de Doenças , Feminino , Ataxia de Friedreich/diagnóstico , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/terapia , Expressão Gênica , Genes Reporter , Humanos , Injeções Espinhais , Proteínas de Ligação ao Ferro/metabolismo , Lipídeos/química , Fígado/metabolismo , Medições Luminescentes , Camundongos , Imagem Molecular , Nanopartículas/administração & dosagem , Nanopartículas/química , Biossíntese de Proteínas , RNA Mensageiro/administração & dosagem , RNA Mensageiro/química , Transdução de Sinais , Transfecção , FrataxinaRESUMO
PURPOSE: Study the impact of CXCL13 neutralization on germinal center (GC) response in vivo, and build quantitative relationship between target coverage and pharmacological effects at the target tissue. METHODS: An anti-CXCL13 neutralizing monoclonal antibody was dosed in vivo in a T-dependent mouse immunization (TDI) model. A quantitative site-of-action (SoA) model was developed to integrate antibody PK and total CXCL13 levels in serum and spleen towards estimating target coverage as a function of dose. To aid in the SoA model development, a radio-labeled study using [I(125)] CXCL13 was conducted in mice. Model estimated target coverage was linked to germinal center response using a sigmoidal inhibitory effect model. RESULTS: In vivo studies demonstrated that CXCL13 inhibition led to an architectural change in B-cell follicles, dislocation of GCs and a significant reduction in the GC absolute numbers per square area (GC/mm(2)). The SoA modeling analysis indicated that ~79% coverage in spleen was required to achieve 50% suppression of GC/mm(2). The 3 mg/kg dose with 52% spleen coverage resulted in no PD suppression, whereas 30 mg/kg with 93% coverage achieved close to maximum PD suppression, highlighting the steepness of PD response. CONCLUSIONS: This study showcases an application of SoA modeling towards a quantitative understanding of CXCL13 pharmacology.
Assuntos
Anticorpos Neutralizantes/farmacologia , Quimiocina CXCL13/imunologia , Centro Germinativo/efeitos dos fármacos , Linfócitos T/imunologia , Animais , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Feminino , Centro Germinativo/imunologia , Centro Germinativo/ultraestrutura , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Ratos , Ratos Sprague-Dawley , Baço/efeitos dos fármacos , Baço/imunologia , Linfócitos T/efeitos dos fármacosRESUMO
The infiltration of neutrophils and monocytes is a prominent feature of inflammatory diseases including human rheumatoid arthritis. Understanding how neutrophil recruitment is regulated during pathogenesis is crucial for developing anti-inflammatory therapies. We optimized the K/B×N serum-induced mouse arthritis model to study neutrophil trafficking dynamics in vivo using two-photon microscopy. Arthritogenic serum was injected subcutaneously into one hind footpad to induce a local arthritis with robust neutrophil recruitment. Using this approach, we showed that the depletion of monocytes with clodronate liposomes impaired neutrophil recruitment specifically at the transendothelial migration step. The depletion of CCR2(+) monocytes with the monoclonal antibody MC-21 reproduced these effects, implicating CCR2(+) monocytes as key regulators of neutrophil extravasation during arthritis initiation. However, monocyte depletion did not prevent neutrophil extravasation in response to bacterial challenge. These findings suggest that anti-inflammatory therapies targeting monocytes may act in part through antagonizing neutrophil extravasation at sites of aseptic inflammation.
Assuntos
Artrite Experimental/imunologia , Monócitos/imunologia , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Animais , Anticorpos Monoclonais/farmacologia , Artrite Experimental/induzido quimicamente , Artrite Experimental/patologia , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Ácido Clodrônico/farmacologia , Feminino , Humanos , Injeções Subcutâneas , Lipossomos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica , Monócitos/efeitos dos fármacos , Monócitos/patologia , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/patologia , Receptores CCR2/biossínteseRESUMO
OBJECTIVE: The mechanistic link between Janus kinase (JAK) signaling and structural damage to arthritic joints in rheumatoid arthritis (RA) is poorly understood. This study was undertaken to investigate how selective inhibition of JAK with tofacitinib (CP-690,550) affects osteoclast-mediated bone resorption in a rat adjuvant-induced arthritis (AIA) model, as well as human T lymphocyte RANKL production and human osteoclast differentiation and function. METHODS: Hind paw edema, inflammatory cell infiltration, and osteoclast-mediated bone resorption in rat AIA were assessed using plethysmography, histopathologic analysis, and immunohistochemistry; plasma and hind paw tissue levels of cytokines and chemokines (including RANKL) were also assessed. In vitro RANKL production by activated human T lymphocytes was evaluated by immunoassay, while human osteoclast differentiation and function were assessed via quantitative tartrate-resistant acid phosphatase staining and degradation of human bone collagen, respectively. RESULTS: Edema, inflammation, and osteoclast-mediated bone resorption in rats with AIA were dramatically reduced after 7 days of treatment with the JAK inhibitor, which correlated with reduced numbers of CD68/ED-1+, CD3+, and RANKL+ cells in the paws; interleukin-6 (transcript and protein) levels were rapidly reduced in paw tissue within 4 hours of the first dose, whereas it took 4-7 days of therapy for RANKL levels to decrease. Tofacitinib did not impact human osteoclast differentiation or function, but did decrease human T lymphocyte RANKL production in a concentration-dependent manner. CONCLUSION: These results suggest that the JAK inhibitor tofacitinib suppresses osteoclast-mediated structural damage to arthritic joints, and this effect is secondary to decreased RANKL production.
Assuntos
Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Janus Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Ligante RANK/metabolismo , Animais , Artrite Experimental/imunologia , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/imunologia , Reabsorção Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Janus Quinases/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Osteoclastos/citologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/enzimologia , Piperidinas , Ratos , Ratos Endogâmicos Lew , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/enzimologiaRESUMO
The glucocorticoid-induced TNFR-related (GITR) protein is a coactivating receptor that is constitutively expressed on Treg cells and induced on activated T cells. To better under-stand the role of long-term GITR signaling, we generated a mouse that constitutively expresses GITR ligand (GITRL) on APCs that mimics the physiological distribution of GITRL in vivo. Despite a five-fold expansion of the Treg-cell pool, there is increased activation and depletion of naive T cells in the transgenic (Tg) mice, suggesting that the increased number of Treg cells cannot fully suppress T-cell activation. Interestingly, GITRL Tg mice have multiorgan lymphocytic infiltrates yet display no overt autoimmunity, indicating the existence of a compensatory immunoregulatory mechanism(s). In the spleens and tissue infiltrates ofGITRL Tg mice, we found increased numbers of Foxp3(-) IL-10-producing type 1 regulatory T (Tr-1)-like cells that suppress naïve T-cell proliferation in an IL-10-dependent fashion. Increased IL-27 production from Tg APCs and activation of c-Maf in the Tr1-like cells suggest a possible mechanism for their induction. Our results demonstrate that enhanced GITR/GITRL interactions have a pleiotropic role on the regulation of T-cell responses, which includes promoting the differentiation of Tr-1-like cells, which contribute to the maintenance of peripheral T-cell tolerance.
Assuntos
Proteína Relacionada a TNFR Induzida por Glucocorticoide/fisiologia , Interleucina-10/biossíntese , Interleucinas/biossíntese , Linfócitos T Reguladores/fisiologia , Fatores de Necrose Tumoral/fisiologia , Animais , Autoimunidade , Fatores de Transcrição Forkhead/análise , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Inhibitors of the JAK family of nonreceptor tyrosine kinases have demonstrated clinical efficacy in rheumatoid arthritis and other inflammatory disorders; however, the precise mechanisms by which JAK inhibition improves inflammatory immune responses remain unclear. In this study, we examined the mode of action of tofacitinib (CP-690,550) on JAK/STAT signaling pathways involved in adaptive and innate immune responses. To determine the extent of inhibition of specific JAK/STAT-dependent pathways, we analyzed cytokine stimulation of mouse and human T cells in vitro. We also investigated the consequences of CP-690,550 treatment on Th cell differentiation of naive murine CD4(+) T cells. CP-690,550 inhibited IL-4-dependent Th2 cell differentiation and interestingly also interfered with Th17 cell differentiation. Expression of IL-23 receptor and the Th17 cytokines IL-17A, IL-17F, and IL-22 were blocked when naive Th cells were stimulated with IL-6 and IL-23. In contrast, IL-17A production was enhanced when Th17 cells were differentiated in the presence of TGF-ß. Moreover, CP-690,550 also prevented the activation of STAT1, induction of T-bet, and subsequent generation of Th1 cells. In a model of established arthritis, CP-690,550 rapidly improved disease by inhibiting the production of inflammatory mediators and suppressing STAT1-dependent genes in joint tissue. Furthermore, efficacy in this disease model correlated with the inhibition of both JAK1 and JAK3 signaling pathways. CP-690,550 also modulated innate responses to LPS in vivo through a mechanism likely involving the inhibition of STAT1 signaling. Thus, CP-690,550 may improve autoimmune diseases and prevent transplant rejection by suppressing the differentiation of pathogenic Th1 and Th17 cells as well as innate immune cell signaling.
Assuntos
Imunidade Adaptativa , Artrite Experimental/imunologia , Proteínas Aviárias/toxicidade , Colágeno Tipo II/toxicidade , Imunidade Inata , Pirimidinas/administração & dosagem , Pirróis/administração & dosagem , Imunidade Adaptativa/genética , Animais , Artrite Experimental/tratamento farmacológico , Artrite Experimental/enzimologia , Células Cultivadas , Galinhas , Humanos , Imunidade Inata/genética , Janus Quinase 3/antagonistas & inibidores , Janus Quinase 3/deficiência , Janus Quinase 3/genética , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Piperidinas , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/uso terapêutico , Pirimidinas/uso terapêutico , Pirróis/uso terapêuticoRESUMO
K/BxN mice develop a spontaneous destructive arthritis driven by T cell dependent anti-glucose-6-phosphate isomerase (GPI) antibody production. In this study, a modified version of the K/BxN model, the KRN-cell transfer model (KRN-CTM), was established to determine the contribution of Th17 cells in the development of chronic arthritis. The transfer of naive KRN T cells into B6.TCR.Cα(-/-)H-2(b/g7) T cell deficient mice induced arthritis by day 10 of transfer. Arthritis progressively developed for a period of up to 14 days following T cell transfer, thereafter the disease severity declined, but did not resolve. Both IL-17A and IFNγ were detected in the recovered T cells from the popliteal lymph nodes and ankles. The transfer of KRN Th17 polarized KRN CD4(+) T cells expressing IL-17A and IFNγ induced arthritis in all B6.TCR.Cα(-/-)H-2(b/g7) mice however the transfer of Th1 polarized KRN CD4(+) T cells expressing IFNγ alone induced disease in only 2/3 of the mice and disease induction was delayed compared to Th17 transfers. Th17 polarized KRN/T-bet(-/-) cells induced arthritis in all mice and surprisingly, IFNγ was produced demonstrating that T-bet expression is not critical for arthritis induction, regardless of the cytokine expression. Neutralization of IFNγ in KRN Th17 transfers resulted in earlier onset of disease while the neutralization of IL-17A delayed disease development. Consistent with K/BxN mice, naive KRN T cell transfers and Th17 polarized KRN/T-bet(-/-) transfers induced anti-GPI IgG(1) dominant responses while KRN Th17 cells induced high levels of IgG(2b). These data demonstrate that Th17 cells can participate in the production of autoantibodies that can induce arthritis.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/imunologia , Linfócitos B/imunologia , Células Th17/imunologia , Transferência Adotiva , Animais , Tornozelo/patologia , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide/patologia , Separação Celular , Citometria de Fluxo , Interleucina-17/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Th1/imunologia , Células Th1/transplante , Células Th17/transplanteRESUMO
In this study, a chronic yet synchronized version of the K/BxN mouse, the KRN-cell transfer model (KRN-CTM), was developed and extensively characterized. The transfer of purified splenic KRN T cells into T cell-deficient B6.TCR.Calpha(-/-)H-2(b/g7) mice induced anti-glucose 6-phosphate isomerase antibody-dependent chronic arthritis in 100% of the mice with uniform onset of disease 7 days after T cell transfer. Cellular infiltrations were assessed by whole-ankle transcript microarray, cytokine and chemokine levels, and microscopic and immunohistochemical analyses 7 through 42 days after T cell transfer. Transcripts identified an influx of monocytes/macrophages and neutrophils into the ankles and identified temporal progression of cartilage damage and bone resorption. In both serum and ankle tissue there was a significant elevation in interleukin-6, whereas macrophage inflammatory protein-1 alpha and monocyte chemotactic protein-1 were only elevated in tissue. Microscopic and immunohistochemical analyses revealed a time course for edema, synovial hypertrophy and hyperplasia, infiltration of F4/80-positive monocytes/macrophages and myeloperoxidase-positive neutrophils, destruction of articular cartilage, pannus invasion, bone resorption, extra-articular fibroplasia, and joint ankylosis. The KRN cell transfer model replicates many features of chronic rheumatoid arthritis in humans in a synchronized manner and lends itself to manipulation of adoptively transferred T cells and characterizing specific genes and T cell subsets responsible for rheumatoid arthritis pathogenesis and progression.
Assuntos
Artrite Reumatoide/patologia , Modelos Animais de Doenças , Articulações/patologia , Linfócitos T/patologia , Linfócitos T/transplante , Animais , Artrite Reumatoide/etiologia , Artrite Reumatoide/metabolismo , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imuno-Histoquímica , Inflamação , Articulações/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Camundongos Transgênicos , Monócitos/metabolismo , Monócitos/patologia , Linfócitos T/metabolismoRESUMO
INTRODUCTION: Standard measurements used to assess murine models of rheumatoid arthritis, notably paw thickness and clinical score, do not align well with certain aspects of disease severity as assessed by histopathology. We tested the hypothesis that non-invasive optical tomographic imaging of molecular biomarkers of inflammation and bone turnover would provide a superior quantitative readout and would discriminate between a disease-modifying anti-rheumatic drug (DMARD) and a non-DMARD treatment. METHODS: Using two protease-activated near-infrared fluorescence imaging agents to detect inflammation-associated cathepsin and matrix metalloprotease activity, and a third agent to detect bone turnover, we quantified fluorescence in paws of mice with collagen antibody-induced arthritis. Fluorescence molecular tomographic (FMT) imaging results, which provided deep tissue detection and quantitative readouts in absolute picomoles of agent fluorescence per paw, were compared with paw swelling, clinical scores, a panel of plasma biomarkers, and histopathology to discriminate between steroid (prednisolone), DMARD (p38 mitogen-activated protein kinase (MAPK) inhibitor) and non-DMARD (celecoxib, cyclooxygenase-2 (COX-2) inhibitor) treatments. RESULTS: Paw thickness, clinical score, and plasma biomarkers failed to discriminate well between a p38 MAPK inhibitor and a COX-2 inhibitor. In contrast, FMT quantification using near-infrared agents to detect protease activity or bone resorption yielded a clear discrimination between the different classes of therapeutics. FMT results agreed well with inflammation scores, and both imaging and histopathology provided clearer discrimination between treatments as compared with paw swelling, clinical score, and serum biomarker readouts. CONCLUSIONS: Non-invasive optical tomographic imaging offers a unique approach to monitoring disease pathogenesis and correlates with histopathology assessment of joint inflammation and bone resorption. The specific use of optical tomography allowed accurate three-dimensional imaging, quantitation in picomoles rather than intensity or relative fluorescence, and, for the first time, showed that non-invasive imaging assessment can predict the pathologist's histology inflammation scoring and discriminate DMARD from non-DMARD activity.
Assuntos
Antirreumáticos/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Experimental/patologia , Inibidores de Ciclo-Oxigenase/uso terapêutico , Pirazóis/uso terapêutico , Sulfonamidas/uso terapêutico , Tomografia Óptica , Animais , Artrite Experimental/metabolismo , Catepsinas/metabolismo , Celecoxib , Modelos Animais de Doenças , Progressão da Doença , Feminino , Glucocorticoides/uso terapêutico , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Prednisolona/uso terapêutico , Resultado do TratamentoRESUMO
14-Deoxyandrographolide (14-DAP) is a labdane diterpene isolated from Andrographis paniculata with previously reported calcium channel blocking activity. Its potential platelet activating factor (PAF) antagonistic activity in bovine neutrophils was assessed. 14-DAP, in concentrations between 10-100 microM, reduced the extracellular acidification rate and the intracellular alkalinization in a dose-dependent manner. In addition, 14-DAP reduced PAF-induced calcium flux in the presence of extracellular calcium, and tyrosine phosphorylation of a 44 kDa protein corresponding to the MAPK(ERK1). However, 14-DAP reduced the 3H-PAF binding with a Ki of 7.8 x 10 (- 9)M, and a Hill slope of 0.63, suggesting that there is more than one binding site for 14-DAP. We concluded that 14-DAP is an effective antagonist of PAF-mediated processes in bovine neutrophils, probably by virtue of its calcium channel blocking property.
Assuntos
Andrographis , Diterpenos/farmacologia , Neutrófilos/efeitos dos fármacos , Fitoterapia , Fator de Ativação de Plaquetas/antagonistas & inibidores , Animais , Bloqueadores dos Canais de Cálcio/administração & dosagem , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Bovinos , Diterpenos/administração & dosagem , Diterpenos/uso terapêutico , Relação Dose-Resposta a Droga , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacosRESUMO
1. Platelet-activating factor (PAF) is known to stimulate a variety of neutrophil activities, including chemotaxis, phagocytosis, degranulation, reactive oxygen species production and intracellular pH increase. The purpose of this study was to investigate the effect of PAF on pH((i)), specifically if these changes in pH are the result of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathway activation in bovine neutrophils. 2. PAF caused intracellular alkalinization in 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester-loaded bovine neutrophils. This phenomenon seems to be mediated by amiloride-sensitive Na(+)/H(+) exchange, and is inhibited by WEB2086 (a selective PAF receptor antagonist), genistein (a tyrosine kinase inhibitor), wortmannin and LY294002 (PI3K inhibitors), and PD98059 and UO126 (MEK inhibitors). 3. PAF 100 nm induced an increase in tyrosine phosphorylation of proteins 62, 44 and 21 kDa with a maximum response at 2 min of incubation. 4. Unlike human neutrophils, bovine neutrophils are strongly stimulated by PAF via phosphorylation of ERK1/2 (extracellular-signal-regulated protein kinase) with an EC(50) of 30 and 13 nm, respectively. 5. PAF MAPK activation was also inhibited by WEB2086, pertussis toxin (PTX), genistein, wortmannin, LY294002, PD98059 and UO126 in bovine neutrophils. The ERK1/2 activation is dependent on PI3K pathway, because protein kinase B was phosphorylated by PAF and inhibited by wortmannin and LY294002, but not by U0126. 6. Our results suggest that PAF induces intracellular alkalinization via PI3K-MAPK activation. This effect is upstream regulated by PAF receptor, PTX-sensitive G protein, tyrosine kinase, PI3K and MEK1/2 in bovine neutrophils.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neutrófilos/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Fator de Ativação de Plaquetas/fisiologia , Animais , Bovinos , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Neutrófilos/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 QuinaseRESUMO
In this study, the effect of 14-deoxyandrographolide (14-DAP) on calcium channel-dependent rat uterine smooth muscle contraction was evaluated. Using a tissue bath preparation, 14-DAP was able to reduce the contractile response to 0.3 and 3.0 mm of CaCl(2), with an IC(50) of 1.24 +/- 0.23 x 10(-5) m and 5.94 +/- 0.29 x 10(-5) m, respectively. 14-DAP shifted the CaCl(2) cumulative dose response curve to the right, increasing the EC(50) from 2.08 +/- 0.20 x 10(-4) m to 4.22 +/- 0.22 x 10(-4) m (5 micrometer 14-DAP) and 2.5 +/- 1.0 x 10(-3) m (50 micrometer 14-DAP). In order to determine if 14-DAP had any effect on intracellular calcium, the relaxant response to 14-DAP following contraction by oxytocin, PGF(2alpha) and vanadate in Ca(+2)-free solution was compared with that of isoproterenol and phenylbutazone. While isoproterenol and phenylbutazone relaxed the smooth muscle in a dose-dependent manner, 14-DAP did not have any effect on either the oxytocin, PGF(2alpha) or vanadate-induced smooth muscle contraction. Based on these data, it appears that 14-DAP is an uterine smooth muscle relaxant which produces a selective blockade of voltage operated calcium channels.