RESUMEN
Veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) is a potentially life-threatening complication of hematopoietic cell transplantation (HCT), especially in severe cases. Patient outcomes are improved with prompt treatment; however, diagnosing this disease is challenging as many clinical symptoms of VOD/SOS overlap with other post-HCT complications. A biomarker-based prognostic test for the assessment of VOD/SOS risk, termed "VODCheck" was developed in a previous study but has not yet been validated. This study aimed to validate the accuracy of VODCheck as a prognostic test for VOD/SOS in an independent cohort of patients. VODCheck incorporates hyaluronan (HA), angiopoietin-2 (ANG-2), and thrombomodulin (TM) based on their association with VOD/SOS, their analytical characteristics, and their ability to complement each other in a multivariate prognostic model. To validate VODCheck we measured plasma biomarker levels from a subset of patients enrolled in the control arm of a phase 3 study that tested VOD/SOS prophylaxis. We used a hierarchical design with prespecified primary (day 7), secondary A (day 15), and secondary B (day 1) hypotheses to verify the prognostic accuracy of VODCheck post-HCT. The cases of VOD/SOS (n = 22) were age-matched â¼1:3 with controls (n = 65). VODCheck was prognostic of VOD/SOS risk at all 3 time points with an area under the curve (AUC) of .815 (P < .001) for day 7, .836 (P < .001) for day 15, and .706 (P = .002) for day 1 post-HCT. A multivariate analysis confirmed the prognostic accuracy of VODCheck after adjustment for confounders such as age, VOD/SOS risk status, primary disease, and ozogamicin treatment. VODCheck was validated as an independent predictor of risk for VOD/SOS within 15 days post-HCT and appears to provide clinicians with actionable information to improve patient care.
RESUMEN
Hepatic veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) is a potentially life-threatening complication of hematopoietic cell transplantation (HCT). This study aimed to determine a blood biomarker signature early post-HCT that identifies patients at high risk for VOD/SOS. A set of 23 plasma biomarkers, selected from the VOD/SOS literature, was measured on days 0, 7, and 14 after myeloablative HCT using blood samples from patients enrolled in the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) Protocol 1202. Eligible cases were diagnosed with VOD/SOS in BMT CTN 1202 using the Baltimore criteria. Controls (without VOD/SOS) were matched to cases for conditioning regimen and age. Significant biomarkers were identified using the Bonferroni-adjusted Wilcoxon rank-sum test (P ≤ .002). Thirty-three patients with mild or severe VOD/SOS were identified (cases) and matched to 107 controls. Two, 8, and 5 biomarkers measured from the plasma of these patients were significantly associated with the development of VOD/SOS at days 0, 7, and 14, respectively, with the strongest associations on days 7 and 14. Biomarker associations were stronger for severe VOD/SOS risk and were stronger prognostic markers for VOD/SOS cases occurring within 28 days of HCT. Hyaluronan was most strongly associated with VOD/SOS risk, with an area under the receiver operating characteristic curve (AUC) of .81 on day 7 and .79 on day 14. Multivariate models of up to 5 biomarkers generated AUCs ranging from .82 to .85. All associations with VOD/SOS risk were independent of clinical risk factors. This study confirms previously identified biomarkers of VOD/SOS risk and identified novel prognostic biomarker signatures that identify patients at risk for VOD/SOS shortly after HCT. Multivariate analysis suggests that a combination of up to 5 of these protein biomarkers may provide a prognostic tool for identifying patients at risk for VOD/SOS.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Enfermedad Veno-Oclusiva Hepática , Trasplantes , Humanos , Enfermedad Veno-Oclusiva Hepática/etiología , Médula Ósea , Pronóstico , Trasplante de Células Madre Hematopoyéticas/efectos adversosRESUMEN
INTRODUCTION: Increasing use of factor Xa (FXa) inhibitors necessitates effective reversal agents to manage bleeding. Andexanet alfa, a novel modified recombinant human FXa, rapidly reverses the anticoagulation effects of direct and indirect FXa inhibitors. OBJECTIVE: To evaluate the ability of andexanet to reverse anticoagulation in vitro and reduce bleeding in rabbits administered edoxaban. MATERIALS AND METHODS: In vitro studies characterized the interaction of andexanet with edoxaban and its ability to reverse edoxaban-mediated anti-FXa activity. In a rabbit model of surgically induced, acute hemorrhage, animals received edoxaban vehicle+andexanet vehicle (control), edoxaban (1 mg/kg)+andexanet vehicle, edoxaban+andexanet (75 mg, 5-minute infusion, 20 minutes after edoxaban), or edoxaban vehicle+andexanet prior to injury. RESULTS: Andexanet bound edoxaban with high affinity similar to FXa. Andexanet rapidly and dose-dependently reversed the effects of edoxaban on FXa activity and coagulation pharmacodynamic parameters in vitro. In edoxaban-anticoagulated rabbits, andexanet reduced anti-FXa activity by 82% (from 548±87 to 100±41 ng/ml; P<0.0001), mean unbound edoxaban plasma concentration by ~80% (from 100±10 to 21±6 ng/ml; P<0.0001), and blood loss by 80% vs. vehicle (adjusted for control, 2.6 vs. 12.9 g; P = 0.003). The reduction in blood loss correlated with the decrease in anti-FXa activity (r = 0.6993, P<0.0001) and unbound edoxaban (r = 0.5951, P = 0.0035). CONCLUSION: These data demonstrate that andexanet rapidly reversed the anticoagulant effects of edoxaban, suggesting it could be clinically valuable for the management of acute and surgery-related bleeding. Correlation of blood loss with anti-FXa activity supports the use of anti-FXa activity as a biomarker for assessing anticoagulation reversal in clinical trials.
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Anticoagulantes/farmacología , Antídotos/farmacología , Coagulación Sanguínea/efectos de los fármacos , Inhibidores del Factor Xa/farmacología , Factor Xa/farmacología , Hemorragia/tratamiento farmacológico , Piridinas/farmacología , Proteínas Recombinantes/farmacología , Tiazoles/farmacología , Animales , Modelos Animales de Enfermedad , Hemorragia/inducido químicamente , Masculino , ConejosRESUMEN
BACKGROUND: Janus kinases (JAKs) are regulators of signaling through cytokine receptors. The importance of JAK1/3 signaling on TH2 differentiation and development of lung allergic responses has not been investigated. OBJECTIVE: We sought to examine a selective JAK1/3 inhibitor (R256) on differentiation of TH subsets in vitro and on development of ovalbumin (OVA)-induced airway hyperresponsiveness (AHR) and inflammation in an experimental model of asthma. METHODS: A selective JAK1/3 inhibitor was used to assay the importance of this pathway on induction of TH1, TH2, and TH17 differentiation in vitro. In vivo, the effects of inhibiting JAK1/3 signaling were examined by administering the inhibitor during the sensitization or allergen challenge phases in the primary challenge model or just before provocative challenge in the secondary challenge model. Airway inflammation and AHR were examined after the last airway challenge. RESULTS: In vitro, R256 inhibited differentiation of TH2 but not TH1 or TH17 cells, which was associated with downregulation of signal transducer and activator of transcription (STAT) 6 and STAT5 phosphorylation. However, once polarized, TH2 cells were unaffected by the inhibitor. In vivo, R256 administered during the OVA sensitization phase prevented the development of AHR, airway eosinophilia, mucus hypersecretion, and TH2 cytokine production without changes in TH1 and TH17 cytokine levels, indicating that selective blockade of TH2 differentiation was critical. Inhibitor administration after OVA sensitization but during the challenge phases in the primary or secondary challenge models similarly suppressed AHR, airway eosinophilia, and mucus hypersecretion without any reduction in TH2 cytokine production, suggesting the inhibitory effects were downstream of TH2 cytokine receptor signaling pathways. CONCLUSIONS: Targeting the TH2-dependent JAK/STAT activation pathway represents a novel therapeutic approach for the treatment of asthma.
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Diferenciación Celular , Janus Quinasa 1/metabolismo , Janus Quinasa 3/metabolismo , Hipersensibilidad Respiratoria/inmunología , Hipersensibilidad Respiratoria/metabolismo , Transducción de Señal , Células Th2/citología , Células Th2/metabolismo , Alérgenos/administración & dosificación , Alérgenos/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 3/antagonistas & inhibidores , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Transgénicos , Inhibidores de Proteínas Quinasas/farmacología , Hipersensibilidad Respiratoria/patología , Transducción de Señal/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Células Th2/efectos de los fármacos , Células Th2/inmunologíaRESUMEN
Tissue injury following ischemia-reperfusion (I/R) occurs as a consequence of actions of soluble factors and immune cells. Growing evidence supports a role for platelets in the manifestation of tissue damage following I/R. Spleen tyrosine kinase has been well documented to be important in lymphocyte activation and more recently in platelet activation. We performed experiments to evaluate whether inhibition of platelet activation through inhibition of spleen tyrosine kinase prevents tissue damage after mesenteric I/R injury. Platelets isolated from C57BL/6J mice fed with R788 for 10 days were transfused into C57BL/6J mice depleted of platelets 2 days before mesenteric I/R injury. Platelet-depleted mice transfused with platelets from R788-treated mice before mesenteric I/R displayed a significant reduction in the degree of remote lung damage, but with little change in the degree of local intestinal damage compared with control I/R mice. Transfusion of R788-treated platelets also decreased platelet sequestration, C3 deposition, and immunoglobulin deposition in lung, but not in the intestine, compared with control groups. These findings demonstrate that platelet activation is a requisite for sequestration in the pulmonary vasculature to mediate remote tissue injury after mesenteric I/R. The use of small-molecule inhibitors may be valuable to prevent tissue damage in remote organs following I/R injury.
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Plaquetas/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Pulmón/efectos de los fármacos , Oxazinas/farmacología , Proteínas Tirosina Quinasas/metabolismo , Piridinas/farmacología , Daño por Reperfusión/metabolismo , Aminopiridinas , Animales , Plaquetas/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/irrigación sanguínea , Intestinos/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Masculino , Ratones , Morfolinas , Recuento de Plaquetas , Transfusión de Plaquetas , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas , Quinasa SykRESUMEN
The activating mutations in JAK2 (including JAK2V617F) that have been described in patients with myeloproliferative neoplasms (MPNs) are linked directly to MPN pathogenesis. We developed R723, an orally bioavailable small molecule that inhibits JAK2 activity in vitro by 50% at a concentration of 2nM, while having minimal effects on JAK3, TYK2, and JAK1 activity. R723 inhibited cytokine-independent CFU-E growth and constitutive activation of STAT5 in primary hematopoietic cells expressing JAK2V617F. In an anemia mouse model induced by phenylhydrazine, R723 inhibited erythropoiesis. In a leukemia mouse model using Ba/F3 cells expressing JAK2V617F, R723 treatment prolonged survival and decreased tumor burden. In V617F-transgenic mice that closely mimic human primary myelofibrosis, R723 treatment improved survival, hepatosplenomegaly, leukocytosis, and thrombocytosis. R723 preferentially targeted the JAK2-dependent pathway rather than the JAK1- and JAK3-dependent pathways in vivo, and its effects on T and B lymphocytes were mild compared with its effects on myeloid cells. Our preclinical data indicate that R723 has a favorable safety profile and the potential to become an efficacious treatment for patients with JAK2V617F-positive MPNs.
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Antineoplásicos/uso terapéutico , Inhibidores Enzimáticos/uso terapéutico , Janus Quinasa 2/antagonistas & inhibidores , Trastornos Mieloproliferativos/tratamiento farmacológico , Trastornos Mieloproliferativos/genética , Anemia Hemolítica/inducido químicamente , Animales , Línea Celular , Células Cultivadas , Eritropoyesis/efectos de los fármacos , Femenino , Humanos , Janus Quinasa 2/genética , Leucemia/tratamiento farmacológico , Leucemia/genética , Leucocitosis/tratamiento farmacológico , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Mutación/efectos de los fármacosRESUMEN
Reperfusion injury to tissue following an ischemic event occurs as a consequence of an acute inflammatory response that can cause significant morbidity and mortality. Components of both the innate (complement, immunoglobulin, monocytes, and neutrophils) and adaptive (B and T lymphocytes) immune systems have been demonstrated to mediate tissue injury. Spleen tyrosine kinase (Syk) is responsible for membrane-mediated signaling in various cell types including B lymphocytes, macrophages, and T cells. We investigated the ability of a small drug Syk inhibitor, R788, to protect mice against mesenteric ischemia-reperfusion (I/R)-induced local (intestine) and remote lung injury. Mice were fed with chow containing a Syk inhibitor for 6 days before the performance of intestinal I/R, which resulted in silencing of the expression of the active phosphorylated Syk. Syk inhibition significantly suppressed both local and remote lung injury. The beneficial effect was associated with reduced IgM and complement 3 deposition in the tissues and significant reduction of polymorphonuclear cell infiltration. Our data place Syk upstream of events leading to the binding of natural antibodies to the ischemia-conditioned tissues and urge the consideration of the use of Syk inhibitors in the prevention or improvement of tissue injury of organs exposed to ischemia or hypoperfusion.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Daño por Reperfusión/enzimología , Daño por Reperfusión/patología , Circulación Esplácnica , Aminopiridinas , Animales , Complemento C3/antagonistas & inhibidores , Complemento C3/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Inmunoglobulina G/metabolismo , Inmunoglobulina M/metabolismo , Intestinos/irrigación sanguínea , Intestinos/efectos de los fármacos , Intestinos/patología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Pulmón/enzimología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Morfolinas , Infiltración Neutrófila , Oxazinas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Circulación Pulmonar , Piridinas/farmacología , Pirimidinas , Receptores de Superficie Celular/metabolismo , Daño por Reperfusión/metabolismo , Quinasa SykRESUMEN
OBJECTIVE: Spleen tyrosine kinase (Syk) is involved in membrane-mediated signaling in various cells, including immune cells. It is overexpressed in T cells from patients with systemic lupus erythematosus (SLE), and its inhibition has been shown to improve T cell function as well as to improve disease manifestations in (NZB x NZW)F(1) lupus-prone mice and in patients with rheumatoid arthritis. While clinical trials examining Syk inhibition in patients with SLE are being considered, the aim of our experiments was to determine whether the therapeutic effects of Syk inhibition extend to other strains of lupus-prone mice and whether they result in improvement in skin disease and modification of established disease. METHODS: Female MRL/lpr or BAK/BAX mice were studied. Starting either at age 4 weeks (before disease) or at age 16 weeks (after established disease) and continuing for up to 16 weeks, mice were fed chow containing the Syk inhibitor R788 or control chow. RESULTS: We found that inhibition of Syk in MRL/lpr and BAK/BAX mice prevented the development of skin disease and significantly reduced established skin disease. Similarly, Syk inhibition reduced the size of the spleen and lymph nodes, suppressed the development of renal disease, and suppressed established renal disease. Discontinuation of treatment resulted in extended suppression of skin disease for at least 8 weeks and suppression of renal disease for 4 weeks. CONCLUSION: Syk inhibition suppresses the development of lupus skin and kidney disease in lupus-prone mice, suppresses established disease in lupus-prone mice, and may represent a valuable treatment for patients with SLE.
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Inhibidores Enzimáticos/farmacología , Lupus Eritematoso Sistémico/tratamiento farmacológico , Nefritis Lúpica/prevención & control , Oxazinas/farmacología , Profármacos/farmacología , Piridinas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Enfermedades de la Piel/prevención & control , Aminopiridinas , Animales , Modelos Animales de Enfermedad , Femenino , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/patología , Nefritis Lúpica/etiología , Nefritis Lúpica/patología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/patología , Ratones , Ratones Endogámicos MRL lpr , Ratones Noqueados , Morfolinas , Pirimidinas , Proteínas Tirosina Quinasas Receptoras/metabolismo , Enfermedades de la Piel/etiología , Enfermedades de la Piel/patologíaRESUMEN
Accumulating evidence suggests important roles for the receptor tyrosine kinase Axl in cancer progression, invasion, metastasis, drug resistance, and patient mortality, highlighting Axl as an attractive target for therapeutic development. We have generated and characterized a potent and selective small-molecule inhibitor, R428, that blocks the catalytic and procancerous activities of Axl. R428 inhibits Axl with low nanomolar activity and blocked Axl-dependent events, including Akt phosphorylation, breast cancer cell invasion, and proinflammatory cytokine production. Pharmacologic investigations revealed favorable exposure after oral administration such that R428-treated tumors displayed a dose-dependent reduction in expression of the cytokine granulocyte macrophage colony-stimulating factor and the epithelial-mesenchymal transition transcriptional regulator Snail. In support of an earlier study, R428 inhibited angiogenesis in corneal micropocket and tumor models. R428 administration reduced metastatic burden and extended survival in MDA-MB-231 intracardiac and 4T1 orthotopic (median survival, >80 days compared with 52 days; P < 0.05) mouse models of breast cancer metastasis. Additionally, R428 synergized with cisplatin to enhance suppression of liver micrometastasis. Our results show that Axl signaling regulates breast cancer metastasis at multiple levels in tumor cells and tumor stromal cells and that selective Axl blockade confers therapeutic value in prolonging survival of animals bearing metastatic tumors.
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Benzocicloheptenos/farmacología , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Carcinoma/mortalidad , Carcinoma/patología , Proteínas Oncogénicas/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Triazoles/farmacología , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Benzocicloheptenos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Carcinoma/tratamiento farmacológico , Femenino , Células HeLa , Humanos , Células K562 , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Metástasis de la Neoplasia , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas , Análisis de Supervivencia , Triazoles/uso terapéutico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Tirosina Quinasa del Receptor AxlRESUMEN
PURPOSE: Aurora kinases play a key role in mitotic progression. Over-expression of Aurora kinases is found in several human cancers and correlated with histological malignancy and clinical outcomes. Therefore, Aurora kinase inhibitors should be useful in the treatment of cancers. METHODS: Cell-based screening methods have an advantage over biochemical approaches because hits can be optimized to inhibit targets in the proper intracellular context. We developed a novel Aurora kinase inhibitor R763/AS703569 using an image-based phenotypic screen. The anti-proliferative effect was examined in a panel of tumor cell lines and primary cells. The efficacy was determined in a broad panel of xenograft models. RESULTS: R763/AS703569 inhibits Aurora kinases, along with a limited number of other kinases including FMS-related tyrosine kinase 3 (FLT3), and has potent anti-proliferative activity against many cell types accompanying unique phenotypic changes such as enlarged cell size, endoreduplication and apoptosis. The endoreduplication cycle induced by R763/AS703569 was irreversible even after the compound was withdrawn from the culture. Oral administration of R763/AS703569 demonstrated marked inhibition of tumor growth in xenograft models of pancreatic, breast, colon, ovarian, and lung tumors and leukemia. An acute myeloid leukemia cell line MV4-11, which carries a FLT3 internal tandem duplication mutation, is particularly sensitive to R763/AS703569 in vivo. CONCLUSIONS: R763/AS703569 is a potent inhibitor of Aurora kinases and exhibited significant anti-proliferative activity against a wide range of tumor cells both in vitro and in vivo. Inhibition of Aurora kinases has the potential to be a new addition to the treatment of cancers.
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Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Microscopía Fluorescente/métodos , Norbornanos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacología , Animales , Aurora Quinasas , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Células Cultivadas , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Citometría de Flujo , Células HL-60 , Células HeLa , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Endogámicos , Ratones Desnudos , Ratones SCID , Análisis de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
JAK3, a member of the Janus kinase family, is predominantly expressed in hemopoietic cells and binds specifically to the common gamma chain of a subfamily of cytokine receptors that includes IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Previous studies suggest that this tyrosine kinase plays key roles in mediating T cell functions, and inhibition of JAK3 has been shown to prevent graft rejection and decrease the severity of arthritis in rodent models. However, the functions of JAK3 in the development of skin immune responses and diseases such as psoriasis have not been determined. CD18 mutant PL/J mice develop spontaneous T cell-dependent psoriasiform skin disease with several similarities to human psoriasis. In this study, we treated mice with established skin disease with R348, a small molecule inhibitor of JAK3, and observed a marked attenuation of skin lesions following 6 wk of treatment. Histological analyses revealed major reductions of both epidermal and dermal lesion severity scores in R348-treated CD18-deficient PL/J mice compared with vehicle controls, which was associated with decreased CD4(+) T cell infiltration. In addition, systemic levels of IL-17, IL-22, IL-23, and TNF-alpha were significantly lower in mice receiving the compound, and T cells isolated from R348-treated mice also showed reduced phosphorylation of Stat5 after stimulation with IL-2. These findings suggest that small-molecule inhibitors of JAK3 may be useful in the treatment of inflammatory skin diseases such as psoriasis and strongly implicate JAK signaling events as important in the pathogenesis of this disease.
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Antígenos CD18/genética , Inflamación/tratamiento farmacológico , Janus Quinasa 3/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Psoriasis/tratamiento farmacológico , Animales , Linfocitos T CD4-Positivos/fisiología , Quimiotaxis , Citocinas/análisis , Janus Quinasa 3/inmunología , Janus Quinasa 3/fisiología , Ratones , Ratones Mutantes , Inhibidores de Proteínas Quinasas/farmacología , Psoriasis/patología , Factor de Transcripción STAT5/metabolismo , Resultado del TratamientoRESUMEN
We have generated mouse models of non-Hodgkin lymphoma (NHL) that rely on the cooperation between MYC overexpression and B-cell antigen receptor (BCR) signaling for the initiation and maintenance of B-cell lymphomas. Using these mouse models of NHL, we have focused on the identification of BCR-derived signal effectors that are important for the maintenance of NHL tumors. In the present study, we concentrate on Spleen tyrosine kinase (Syk), a nonreceptor tyrosine kinase required to transduce BCR-dependent signals. Using a genetic approach, we showed that Syk expression is required for the survival of murine NHL-like tumors in vitro and that tumor cells deficient in Syk fail to expand in vivo. In addition, a pharmacologic inhibitor of Syk was able to induce apoptosis of transformed B cells in vitro and led to tumor regression in vivo. Finally, we show that genetic or pharmacologic inhibition of Syk activity in human NHL cell lines are generally consistent with results found in the mouse models, suggesting that targeting Syk may be a viable therapeutic strategy.
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Marcación de Gen , Péptidos y Proteínas de Señalización Intracelular/fisiología , Linfoma no Hodgkin/genética , Linfoma no Hodgkin/terapia , Proteínas Tirosina Quinasas/fisiología , Animales , Apoptosis/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Linfoma no Hodgkin/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Interferencia de ARN/fisiología , ARN Interferente Pequeño/farmacología , Quinasa Syk , Células Tumorales CultivadasRESUMEN
Diminished expression of TCR zeta and reciprocal up-regulation and association of FcRgamma with the TCR/CD3 complex is a hallmark of systemic lupus erythematosus (SLE) T cells. In this study we explored whether differential molecular associations of the spleen tyrosine kinase Syk that preferentially binds to FcRgamma contribute to pathological amplification of signals downstream of this "rewired TCR" in SLE. We detected higher amounts of Syk expression and activity in SLE compared with normal T cells. Selective inhibition of the activity of Syk reduced the strength of TCR-induced calcium responses and slowed the rapid kinetics of actin polymerization exclusively in SLE T cells. Syk and ZAP-70 also associated differently with key molecules involved in cytoskeletal and calcium signaling in SLE T cells. Thus, while Vav-1 and LAT preferentially bound to Syk, phospholipase C-gamma1 bound to both Syk and ZAP-70. Our results show that differential associations of Syk family kinases contribute to the enhanced TCR-induced signaling responses in SLE T cells. Thus, we propose molecular targeting of Syk as a measure to control abnormal T cell responses in SLE.
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Señalización del Calcio/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Lupus Eritematoso Sistémico/inmunología , Proteínas Tirosina Quinasas/inmunología , Linfocitos T/inmunología , Regulación hacia Arriba/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adulto , Anciano , Complejo CD3/inmunología , Complejo CD3/metabolismo , Femenino , Humanos , Lupus Eritematoso Sistémico/enzimología , Masculino , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Fosfolipasa C gamma/inmunología , Fosfolipasa C gamma/metabolismo , Unión Proteica/inmunología , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Proto-Oncogénicas c-vav/inmunología , Proteínas Proto-Oncogénicas c-vav/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Quinasa Syk , Linfocitos T/enzimología , Proteína Tirosina Quinasa ZAP-70/inmunología , Proteína Tirosina Quinasa ZAP-70/metabolismoRESUMEN
OBJECTIVE: To assess whether R788, an orally bioavailable small molecule inhibitor of spleen tyrosine kinase (Syk)-dependent signaling, could modulate disease in lupus-prone (NZB x NZW)F1 (NZB/NZW) mice via inhibition of Fc receptor (FcR) and B cell receptor signaling. METHODS: R788 was administered to NZB/NZW mice before and after disease onset. Proteinuria, blood urea nitrogen levels, and autoantibody titers were examined periodically, and overall survival and renal pathologic features were assessed following long-term treatment (24-34 weeks). The distribution and immunophenotype of various splenic T cell and B cell subpopulations were evaluated at the time of study termination. Arthus responses in NZB/NZW mice pretreated with R788 or Fc-blocking antibody (anti-CD16/32) were also examined. RESULTS: When R788 was administered prior to or after disease onset, it delayed the onset of proteinuria and azotemia, reduced renal pathology and kidney infiltrates, and significantly prolonged survival of lupus-prone NZB/NZW mice; autoantibody titers were minimally affected throughout the study. Dose-dependent reductions in the numbers of CD4+ activated T cells expressing high levels of CD44 or CD69 were apparent in spleens from R788-treated mice. Minimal effects on the numbers of naive T cells expressing CD62 ligand and total CD8+ T cells per spleen were observed following long-term drug treatment. R788 pretreatment resulted in reduced Arthus responses in NZB/NZW mice, similar to results obtained in mice pretreated with FcR-blocking antibody. CONCLUSION: We demonstrate that a novel Syk-selective inhibitor prevents the development of renal disease and treats established murine lupus nephritis. These data suggest that Syk inhibitors may be of therapeutic benefit in human lupus and related disorders.
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Lupus Eritematoso Sistémico/prevención & control , Oxazinas/uso terapéutico , Piridinas/uso terapéutico , Administración Oral , Aminopiridinas , Animales , Progresión de la Enfermedad , Lupus Eritematoso Sistémico/mortalidad , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NZB , Morfolinas , Pirimidinas , Tasa de Supervivencia , Factores de TiempoRESUMEN
Spleen tyrosine kinase (Syk), a key mediator of immunoreceptor signaling in inflammatory cells, is essential for immune complex-mediated signal transduction initiated by activated receptors for immunoglobulin G. In collagen-induced arthritis, R788/R406, a novel and potent small molecule Syk inhibitor suppressed clinical arthritis, bone erosions, pannus formation, and synovitis. Serum anti-collagen type II antibody levels were unaltered, while the half-life of exogenous antibody was extended when co-administered with R406. Expression of the targeted kinase (Syk) in synovial tissue correlated with the joint level of inflammatory cell infiltrates and was virtually undetectable in treated rats. Syk inhibition suppressed synovial cytokines and cartilage oligomeric matrix protein (COMP) in serum, suggesting a sensitive and reliable biomarker for R406 activity. These results highlight the role of activating Fcgamma receptors in inflammatory synovitis and suggest that interruption of the signaling cascade with a novel Syk inhibitor may be a useful addition to immunosuppressive disease-modifying anti-rheumatic drugs currently used in the treatment of human autoimmune diseases such as rheumatoid arthritis.
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Artritis Reumatoide/tratamiento farmacológico , Resorción Ósea/prevención & control , Inflamación/prevención & control , Péptidos y Proteínas de Señalización Intracelular/fisiología , Oxazinas/farmacología , Profármacos/farmacología , Proteínas Tirosina Quinasas/fisiología , Piridinas/farmacología , Aminopiridinas , Animales , Artritis Reumatoide/inmunología , Reacción de Arthus/tratamiento farmacológico , Colágeno Tipo II/inmunología , Modelos Animales de Enfermedad , Femenino , Inmunoglobulina G/sangre , Inflamación/inmunología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Morfolinas , Oxazinas/administración & dosificación , Oxazinas/metabolismo , Profármacos/administración & dosificación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Piridinas/administración & dosificación , Piridinas/metabolismo , Pirimidinas , Ratas , Ratas Sprague-Dawley , Receptores de IgG/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Quinasa Syk , Líquido Sinovial/inmunología , Sinovitis/tratamiento farmacológicoRESUMEN
Recent compelling evidence has lead to renewed interest in the role of antibodies and immune complexes in the pathogenesis of several autoimmune disorders, such as rheumatoid arthritis. These immune complexes, consisting of autoantibodies to self-antigens, can mediate inflammatory responses largely through binding and activating the immunoglobulin Fc receptors (FcRs). Using cell-based structure activity relationships with cultured human mast cells, we have identified the small molecule R406 [N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine] as a potent inhibitor of immunoglobulin E (IgE)- and IgG-mediated activation of Fc receptor signaling (EC(50) for degranulation = 56-64 nM). Here we show that the primary target for R406 is the spleen tyrosine kinase (Syk), which plays a key role in the signaling of activating Fc receptors and the B-cell receptor (BCR). R406 inhibited phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 bound to the ATP binding pocket of Syk and inhibited its kinase activity as an ATP-competitive inhibitor (K(i) = 30 nM). Furthermore, R406 blocked Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and BCR-mediated activation of B lymphocytes. R406 was selective as assessed using a large panel of Syk-independent cell-based assays representing both specific and general signaling pathways. Consistent with Syk inhibition, oral administration of R406 to mice reduced immune complex-mediated inflammation in a reverse-passive Arthus reaction and two antibody-induced arthritis models. Finally, we report a first-inhuman study showing that R406 is orally bioavailable, achieving exposures capable of inhibiting Syk-dependent IgE-mediated basophil activation. Collectively, the results show R406 potential for modulating Syk activity in human disease.
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Complejo Antígeno-Anticuerpo/fisiología , Inhibidores Enzimáticos/farmacología , Inflamación/tratamiento farmacológico , Oxazinas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Piridinas/farmacología , Receptores Fc/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Bazo/enzimología , Animales , Artritis Experimental/patología , Reacción de Arthus/fisiopatología , Linfocitos B/efectos de los fármacos , Linfocitos B/fisiología , Basófilos/efectos de los fármacos , Western Blotting , Células Cultivadas , Cristalografía , Método Doble Ciego , Inhibidores Enzimáticos/farmacocinética , Inmunoensayo de Polarización Fluorescente , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/inmunología , Inflamación/patología , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Mastocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Oxazinas/farmacocinética , Agregación Plaquetaria/efectos de los fármacos , Piridinas/farmacocinética , Estimulación Química , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Spleen tyrosine kinase (Syk) is a key regulator of cell signaling induced by cytokines or Fc receptor engagement. However, the role of Syk in rheumatoid arthritis (RA) is not known yet. We investigated the pathways activated by Syk in tumor necrosis factor-alpha (TNFalpha)-stimulated fibroblast-like synoviocytes (FLS) using the novel Syk inhibitor N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine (R406). Using immunohistochemistry, Syk was detected in RA synovial tissue (ST), primarily in the synovial intimal lining. Western blot analysis demonstrated significantly greater amounts of phospho-Syk expression in RA ST compared with osteoarthritis ST. The kinase was expressed and functionally activated by TNFalpha in FLS and was blocked by R406. Western blot analysis demonstrated that Syk inhibition by R406 markedly suppressed TNFalpha-induced c-Jun N-terminal kinase (JNK) phosphorylation in FLS, with a modest decrease in extracellular signal-regulated kinase phosphorylation. Surprisingly, p38 activation was not affected by R406. The Syk inhibitor also decreased TNFalpha-induced mitogen-activated protein kinase kinase (MKK) 4 phosphorylation but not MKK3 and MKK6 phosphorylation, which is consistent with its selective sparing of p38. The connection between Syk and JNK was confirmed by demonstrating decreased phospho-c-Jun protein expression and complete inhibition of JNK function in R406-treated cells. R406 also suppressed downstream actions of JNK, as determined by activator protein 1 binding, as well as matrix metalloproteinase 3 gene expression. These data demonstrate that Syk activation plays an essential role in TNFalpha-induced cytokine and matrix metalloproteinase production in RA FLS, especially by suppressing activation of the JNK pathway.
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Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Oxazinas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Piridinas/farmacología , Membrana Sinovial/efectos de los fármacos , Células Cultivadas , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Metaloproteinasa 3 de la Matriz/genética , Fosforilación , Unión Proteica , Proteínas Tirosina Quinasas/genética , Quinasa Syk , Membrana Sinovial/citología , Membrana Sinovial/enzimología , Factor de Transcripción AP-1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
RATIONALE: Spleen tyrosine kinase (Syk) is important for Fc and B-cell receptor-mediated signaling. OBJECTIVE: To determine the activity of a specific Syk inhibitor (R406) on mast cell activation in vitro and on the development of allergen-induced airway hyperresponsiveness (AHR) and inflammation in vivo. METHODS: AHR and inflammation were induced after 10 d of allergen (ovalbumin [OVA]) exposure exclusively via the airways and in the absence of adjuvant. This approach was previously established to be IgE, FcepsilonRI, and mast cell dependent. Alternatively, mice were passively sensitized with OVA-specific IgE, followed by limited airway challenge. In vitro, the inhibitor was added to cultures of IgE-sensitized bone marrow-derived mast cells (BMMCs) before cross-linking with allergen. RESULTS: The inhibitor prevented OVA-induced degranulation of passively IgE-sensitized murine BMMCs and inhibited the production of interleukin (IL)-13, tumor necrosis factor alpha, IL-2, and IL-6 in these sensitized BMMCs. When administered in vivo, R406 inhibited AHR, which developed in BALB/c mice exposed to aerosolized 1% OVA for 10 consecutive d (20 min/d), as well as pulmonary eosinophilia and goblet cell metaplasia. A similar inhibition of AHR was demonstrated in mice passively sensitized with OVA-specific IgE and exposed to limited airway challenge. CONCLUSION: This study delineates a functional role for Syk in the development of mast cell- and IgE-mediated AHR and airway inflammation, and these results indicate that inhibition of Syk may be a target in the treatment of allergic asthma.
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Hiperreactividad Bronquial/inmunología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Mastocitos/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Animales , Linfocitos B/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Péptidos y Proteínas de Señalización Intracelular/inmunología , Mastocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Proteínas Tirosina Quinasas/inmunología , Receptores Fc/inmunología , Transducción de Señal , Quinasa SykRESUMEN
We evaluated the role of Syk, using an inhibitor, on allergen-induced airway hyperresponsiveness (AHR) and airway inflammation in a system shown to be B cell- and mast cell-independent. Sensitization of BALB/c mice with ovalbumin (OVA) and alum after three consecutive OVA challenges resulted in AHR to inhaled methacholine and airway inflammation. The Syk inhibitor R406 (30 mg/kg, administered orally, twice daily) prevented the development of AHR, increases in eosinophils and lymphocytes and IL-13 levels in bronchoalveolar lavage (BAL) fluid, and goblet cell metaplasia when administered after sensitization and before challenge with OVA. Levels of IL-4, IL-5, and IFN-gamma in BAL fluid and allergen-specific antibody levels in serum were not affected by treatment. Because many of these responses may be influenced by dendritic cell function, we investigated the effect of R406 on bone marrow-derived dendritic cell (BMDC) function. Co-culture of BMDC with immune complexes of OVA and IgG anti-OVA together with OVA-sensitized spleen mononuclear cells resulted in increases in IL-13 production. IL-13 production was inhibited if the BMDCs were pretreated with the Syk inhibitor. Intratracheal transfer of immune complex-pulsed BMDCs (but not nonpulsed BMDCs) to naive mice before airway allergen challenge induced the development of AHR and increases in BAL eosinophils and lymphocytes. All of these responses were inhibited if the transferred BMDCs were pretreated with R406. These results demonstrate that Syk inhibition prevents allergen-induced AHR and airway inflammation after systemic sensitization and challenge, at least in part through alteration of DC function.