RESUMEN
Glucose homeostasis is a vital and complex process, and its disruption can cause hyperglycaemia and type II diabetes mellitus. Glucokinase (GK), a key enzyme that regulates glucose homeostasis, converts glucose to glucose-6-phosphate in pancreatic ß-cells, liver hepatocytes, specific hypothalamic neurons, and gut enterocytes. In hepatocytes, GK regulates glucose uptake and glycogen synthesis, suppresses glucose production, and is subject to the endogenous inhibitor GK regulatory protein (GKRP). During fasting, GKRP binds, inactivates and sequesters GK in the nucleus, which removes GK from the gluconeogenic process and prevents a futile cycle of glucose phosphorylation. Compounds that directly hyperactivate GK (GK activators) lower blood glucose levels and are being evaluated clinically as potential therapeutics for the treatment of type II diabetes mellitus. However, initial reports indicate that an increased risk of hypoglycaemia is associated with some GK activators. To mitigate the risk of hypoglycaemia, we sought to increase GK activity by blocking GKRP. Here we describe the identification of two potent small-molecule GK-GKRP disruptors (AMG-1694 and AMG-3969) that normalized blood glucose levels in several rodent models of diabetes. These compounds potently reversed the inhibitory effect of GKRP on GK activity and promoted GK translocation both in vitro (isolated hepatocytes) and in vivo (liver). A co-crystal structure of full-length human GKRP in complex with AMG-1694 revealed a previously unknown binding pocket in GKRP distinct from that of the phosphofructose-binding site. Furthermore, with AMG-1694 and AMG-3969 (but not GK activators), blood glucose lowering was restricted to diabetic and not normoglycaemic animals. These findings exploit a new cellular mechanism for lowering blood glucose levels with reduced potential for hypoglycaemic risk in patients with type II diabetes mellitus.
Asunto(s)
Proteínas Portadoras/antagonistas & inhibidores , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/farmacología , Hipoglucemiantes/uso terapéutico , Proteínas Adaptadoras Transductoras de Señales , Animales , Glucemia/metabolismo , Proteínas Portadoras/metabolismo , Núcleo Celular/enzimología , Cristalografía por Rayos X , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/enzimología , Modelos Animales de Enfermedad , Hepatocitos , Humanos , Hiperglucemia/sangre , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/enzimología , Hipoglucemiantes/química , Hígado/citología , Hígado/enzimología , Hígado/metabolismo , Masculino , Modelos Moleculares , Especificidad de Órganos , Fosforilación/efectos de los fármacos , Piperazinas/química , Piperazinas/metabolismo , Piperazinas/farmacología , Piperazinas/uso terapéutico , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Ratas , Ratas Wistar , Sulfonamidas/química , Sulfonamidas/metabolismo , Sulfonamidas/farmacología , Sulfonamidas/uso terapéuticoRESUMEN
Sustained angiogenesis is essential for tumor growth as it provides the tumor with a network of blood vessels that supply both oxygen and essential nutrients. Limiting tumor-associated angiogenesis is a proven strategy for the treatment of human cancer. To date, the rapid detection and quantitation of tumor-associated endothelial cell (TAEC) proliferation has been challenging, largely due to the low frequency of endothelial cells (ECs) within the tumor microenvironment. In this report, we address this problem using a new multiparametric flow cytometry method capable of rapid and precise quantitation of proliferation by measuring bromodeoxyuridine (BrdUrd) uptake in mouse TAECs from established human tumor xenografts. We determined the basal proliferation labeling index of TAECs in two human tumor xenografts representing two distinct histologies, COLO 205 (colorectal cancer) and U-87 (glioblastoma). We then investigated the effects of two large-molecule antiangiogenic agents targeting different biochemical pathways. Blocking angiopoietin-Tie2 signaling with the peptide-Fc fusion protein, trebananib (AMG 386), inhibited proliferation of TAECs, whereas blocking Dll4-Notch signaling with an anti-Dll4-specific antibody induced hyperproliferation of TAECs. These pharmacodynamic studies highlight the sensitivity and utility of this flow cytometry-based method and demonstrate the value of this assay to rapidly assess the in vivo proliferative effects of angiogenesis-targeted agents on both the tumor and the associated vasculature.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Células Endoteliales/efectos de los fármacos , Citometría de Flujo/métodos , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Proteínas de la Membrana/antagonistas & inhibidores , Receptor TIE-2/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/farmacología , Animales , Anticuerpos Neutralizantes/uso terapéutico , Bromodesoxiuridina , Proliferación Celular/efectos de los fármacos , Neoplasias Colorrectales/patología , Femenino , Glioblastoma/patología , Humanos , Ratones Desnudos , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Proteínas Recombinantes de Fusión/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: The role of osteoprotegerin in vascular disease is unclear. Recent observational studies show that serum osteoprotegerin levels are associated with the severity and progression of coronary artery disease, atherosclerosis, and vascular calcification in patients. However, genetic and treatment studies in mice suggest that osteoprotegerin may protect against vascular calcification. METHODS AND RESULTS: To test whether osteoprotegerin induces or prevents vascular disease, we treated atherogenic diet-fed ldlr(-/-) mice with recombinant osteoprotegerin (Fc-OPG) or vehicle for 5 months. Vehicle-treated mice developed significant, progressive atherosclerosis with increased plasma osteoprotegerin levels, consistent with observational studies, and approximately 15% of these atherosclerotic lesions developed calcified cartilage-like metaplasia. Treatment with Fc-OPG significantly reduced the calcified lesion area without affecting atherosclerotic lesion size or number, vascular cytokines, or plasma cholesterol levels. Treatment also significantly reduced tissue levels of aortic osteocalcin, a marker of mineralization. CONCLUSIONS: These data support a role for osteoprotegerin in the vasculature as an inhibitor of calcification and a marker, rather than a mediator, of atherosclerosis.
Asunto(s)
Aterosclerosis/patología , Calcinosis/tratamiento farmacológico , Osteoprotegerina/farmacología , Enfermedades Vasculares/patología , Animales , Aorta , Aterosclerosis/etiología , Endotelio Vascular/química , Ratones , Ratones Noqueados , Osteoprotegerina/sangre , Ligando RANK/sangre , ARN Mensajero/sangre , Receptores de LDL/deficiencia , Proteínas Recombinantes , Enfermedades Vasculares/etiologíaRESUMEN
Sclerostin antibody (Scl-Ab) and parathyroid hormone (PTH) are bone-forming agents that have different modes of action on bone, although a study directly comparing their effects has not been conducted. The present study investigated the comparative quantitative effects of these two bone-forming agents over time on bone at the organ, tissue, and cellular level; specifically, at the level of the osteoblast (Ob) lineage in adolescent male and female rats. Briefly, eight-week old male and female Sprague-Dawley rats were administered either vehicle, Scl-Ab (3 or 50mg/kg/week subcutaneously), or human PTH (1-34) (75 µg/kg/day subcutaneously) for 4 or 26 weeks. The 50mg/kg Scl-Ab and the PTH dose were those used in the respective rat lifetime pharmacology studies. Using robust stereological methods, we compared the effects of these agents specifically at the level of the Ob lineage in vertebrae from female rats. Using RUNX2 or nestin immunostaining, location, and morphology, the total number of osteoprogenitor subpopulations, Ob, and lining cells were estimated using the fractionator or proportionator estimators. Density estimates were also calculated referent to total bone surface, total Ob surface, or total marrow volume. Scl-Ab generally effected greater increases in cancellous and cortical bone mass than PTH, correlating with higher bone formation rates (BFR) at 4 weeks in the spine and mid-femur without corresponding increases in bone resorption indices. The increases in vertebral BFR/BS at 4 weeks attenuated with continued treatment to a greater extent with Scl-Ab than with PTH. At 4 weeks, both Scl-Ab and PTH effected equivalent increases in total Ob number (Ob.N). Ob density on the formative surfaces (Ob.N/Ob.S) remained similar across groups while mineral apposition rate (MAR) was significantly higher with Scl-Ab at week 4, reflecting an increase in individual Ob vigor relative to vehicle and PTH. After 26 weeks, Scl-Ab maintained BFR/BS with fewer Ob and lower Ob.N/Ob.S by increasing the Ob footprint (bone surface area occupied by an Ob) and increasing MAR, compared with PTH. The lower Ob.N and Ob.N/Ob.S with Scl-Ab at 26 weeks were associated with decreased osteoprogenitor numbers compared with both vehicle and PTH, an effect not evident at week 4. Osteoprogenitor numbers were generally positively correlated with Ob.N across groups and timepoints, suggesting dynamic coordination between the progenitor and Ob populations. The time-dependent reductions in subpopulations of the Ob lineage with Scl-Ab may be integral to the greater attenuation or self-regulation of bone formation observed at the vertebra, as PTH required more Ob at the formative site with correlative increased numbers of progenitors compared with Scl-Ab indicating potentially greater stimulus for progenitor pool proliferation or differentiation.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Proteínas Morfogenéticas Óseas/inmunología , Huesos/efectos de los fármacos , Marcadores Genéticos/inmunología , Osteoblastos/efectos de los fármacos , Hormona Paratiroidea/administración & dosificación , Animales , Anticuerpos Monoclonales/química , Densidad Ósea/efectos de los fármacos , Resorción Ósea , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Femenino , Fémur/efectos de los fármacos , Humanos , Masculino , Osteogénesis/efectos de los fármacos , Hormona Paratiroidea/química , Ratas , Ratas Sprague-Dawley , Células Madre/citología , Tibia/efectos de los fármacos , Factores de TiempoRESUMEN
The glucokinase-glucokinase regulatory protein (GK-GKRP) complex plays an important role in controlling glucose homeostasis in the liver. We have recently disclosed a series of arylpiperazines as in vitro and in vivo disruptors of the GK-GKRP complex with efficacy in rodent models of type 2 diabetes mellitus (T2DM). Herein, we describe a new class of aryl sulfones as disruptors of the GK-GKRP complex, where the central piperazine scaffold has been replaced by an aromatic group. Conformational analysis and exploration of the structure-activity relationships of this new class of compounds led to the identification of potent GK-GKRP disruptors. Further optimization of this novel series delivered thiazole sulfone 93, which was able to disrupt the GK-GKRP interaction in vitro and in vivo and, by doing so, increases cytoplasmic levels of unbound GK.
Asunto(s)
Aminopiridinas/farmacología , Proteínas Portadoras/antagonistas & inhibidores , Glucoquinasa/antagonistas & inhibidores , Hipoglucemiantes/farmacología , Hígado/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Sulfonas/química , Aminopiridinas/química , Animales , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Glucoquinasa/metabolismo , Glucosa/metabolismo , Hipoglucemiantes/química , Hígado/citología , Hígado/metabolismo , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Ratas , Ratas Sprague-Dawley , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Sulfonas/farmacologíaRESUMEN
The HTS-based discovery and structure-guided optimization of a novel series of GKRP-selective GK-GKRP disrupters are revealed. Diarylmethanesulfonamide hit 6 (hGK-hGKRP IC50 = 1.2 µM) was optimized to lead compound 32 (AMG-0696; hGK-hGKRP IC50 = 0.0038 µM). A stabilizing interaction between a nitrogen atom lone pair and an aromatic sulfur system (nN â σ*S-X) in 32 was exploited to conformationally constrain a biaryl linkage and allow contact with key residues in GKRP. Lead compound 32 was shown to induce GK translocation from the nucleus to the cytoplasm in rats (IHC score = 0; 10 mg/kg po, 6 h) and blood glucose reduction in mice (POC = -45%; 100 mg/kg po, 3 h). X-ray analyses of 32 and several precursors bound to GKRP were also obtained. This novel disrupter of GK-GKRP binding enables further exploration of GKRP as a potential therapeutic target for type II diabetes and highlights the value of exploiting unconventional nonbonded interactions in drug design.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Glucoquinasa/metabolismo , Hipoglucemiantes/química , Sulfonamidas/química , Tiofenos/química , Transporte Activo de Núcleo Celular , Animales , Glucemia/metabolismo , Núcleo Celular/metabolismo , Cristalografía por Rayos X , Citoplasma/metabolismo , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Masculino , Ratones , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Conformación Molecular , Unión Proteica , Ratas Sprague-Dawley , Estereoisomerismo , Relación Estructura-Actividad , Sulfonamidas/farmacocinética , Sulfonamidas/farmacología , Tiofenos/farmacocinética , Tiofenos/farmacologíaRESUMEN
Small molecule activators of glucokinase have shown robust efficacy in both preclinical models and humans. However, overactivation of glucokinase (GK) can cause excessive glucose turnover, leading to hypoglycemia. To circumvent this adverse side effect, we chose to modulate GK activity by targeting the endogenous inhibitor of GK, glucokinase regulatory protein (GKRP). Disrupting the GK-GKRP complex results in an increase in the amount of unbound cytosolic GK without altering the inherent kinetics of the enzyme. Herein we report the identification of compounds that efficiently disrupt the GK-GKRP interaction via a previously unknown binding pocket. Using a structure-based approach, the potency of the initial hit was improved to provide 25 (AMG-1694). When dosed in ZDF rats, 25 showed both a robust pharmacodynamic effect as well as a statistically significant reduction in glucose. Additionally, hypoglycemia was not observed in either the hyperglycemic or normal rats.
Asunto(s)
Proteínas Portadoras/metabolismo , Glucoquinasa/metabolismo , Hipoglucemiantes/química , Piperazinas/química , Animales , Sitios de Unión , Proteínas Portadoras/química , Cristalografía por Rayos X , Glucoquinasa/química , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Hipoglucemia/inducido químicamente , Hipoglucemiantes/efectos adversos , Hipoglucemiantes/farmacología , Piperazinas/efectos adversos , Piperazinas/farmacología , Conformación Proteica , Transporte de Proteínas , Ratas , Ratas Zucker , Estereoisomerismo , Relación Estructura-Actividad , Sulfonamidas/efectos adversos , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
In the previous report , we described the discovery and optimization of novel small molecule disruptors of the GK-GKRP interaction culminating in the identification of 1 (AMG-1694). Although this analogue possessed excellent in vitro potency and was a useful tool compound in initial proof-of-concept experiments, high metabolic turnover limited its advancement. Guided by a combination of metabolite identification and structure-based design, we have successfully discovered a potent and metabolically stable GK-GKRP disruptor (27, AMG-3969). When administered to db/db mice, this compound demonstrated a robust pharmacodynamic response (GK translocation) as well as statistically significant dose-dependent reductions in fed blood glucose levels.
Asunto(s)
Proteínas Portadoras/metabolismo , Glucoquinasa/metabolismo , Hipoglucemiantes/química , Piperazinas/síntesis química , Sulfonamidas/síntesis química , Alquinos/síntesis química , Alquinos/farmacocinética , Alquinos/farmacología , Animales , Glucemia/metabolismo , Proteínas Portadoras/química , Glucoquinasa/química , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/farmacología , Ratones , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Morfolinas/síntesis química , Morfolinas/farmacocinética , Morfolinas/farmacología , Piperazinas/farmacocinética , Piperazinas/farmacología , Unión Proteica , Transporte de Proteínas , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologíaRESUMEN
Erythropoietin (Epo) is a cytokine that binds and activates an Epo receptor (EpoR) expressed on the surface of erythroid progenitor cells to promote erythropoiesis. While early studies suggested EpoR transcripts were expressed exclusively in the erythroid compartment, low-level EpoR transcripts were detected in nonhematopoietic tissues and tumor cell lines using sensitive RT-PCR methods. However due to the widespread use of nonspecific anti-EpoR antibodies there are conflicting data on EpoR protein expression. In tumor cell lines and normal human tissues examined with a specific and sensitive monoclonal antibody to human EpoR (A82), little/no EpoR protein was detected and it was not functional. In contrast, EpoR protein was reportedly detectable in a breast tumor cell line (MCF-7) and breast cancer tissues with an anti-EpoR polyclonal antibody (M-20), and functional responses to rHuEpo were reported with MCF-7 cells. In another study, a functional response was reported with the lung tumor cell line (NCI-H838) at physiological levels of rHuEpo. However, the specificity of M-20 is in question and the absence of appropriate negative controls raise questions about possible false-positive effects. Here we show that with A82, no EpoR protein was detectable in normal human and matching cancer tissues from breast, lung, colon, ovary and skin with little/no EpoR in MCF-7 and most other breast and lung tumor cell lines. We show further that M-20 provides false positive staining with tissues and it binds to a non-EpoR protein that migrates at the same size as EpoR with MCF-7 lysates. EpoR protein was detectable with NCI-H838 cells, but no rHuEpo-induced phosphorylation of AKT, STAT3, pS6RP or STAT5 was observed suggesting the EpoR was not functional. Taken together these results raise questions about the hypothesis that most tumors express high levels of functional EpoR protein.
Asunto(s)
Proteínas de Neoplasias/genética , Neoplasias/genética , Receptores de Eritropoyetina/análisis , Factores de Transcripción/genética , Animales , Biopsia , Línea Celular Tumoral , Reacciones Falso Positivas , Femenino , Expresión Génica , Humanos , Inmunoensayo , Masculino , Ratones , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Fosforilación , Receptores de Eritropoyetina/deficiencia , Receptores de Eritropoyetina/genética , Sensibilidad y Especificidad , Factores de Transcripción/metabolismoRESUMEN
OBJECTIVE: Antibodies to human granulocyte colony-stimulating factor receptor (HuG-CSFR) are widely available and have been used in numerous studies to evaluate the expression of this protein on normal and malignant cells of hematopoietic and nonhematopoietic origin. Spurred by recent studies that demonstrated that two commonly used antibodies against the erythropoietin and thrombopoietin receptors can in fact bind to completely unrelated and more broadly expressed proteins, we screened 27 commercially available monoclonal and polyclonal antibodies with claimed specificity to HuG-CSFR to determine if they are specific to this receptor. MATERIALS AND METHODS: Antibodies were evaluated by Western blotting, flow cytometry, and immunohistochemistry using 293T cells engineered to overexpress HuG-CSFR protein, immortalized human hematopoietic cell lines expressing endogenous G-CSFR, and purified human neutrophils. RESULTS: Only two monoclonal antibodies and one polyclonal antibody could be employed using defined Western blotting or flow cytometry protocols to detect G-CSFR protein in cell lysates or on the surface of cells that express G-CSFR messenger RNA with no binding to cells that did not express the gene. None of the antibodies were suitable for immunohistochemistry. Competitive inhibition with soluble G-CSFR extracellular domain and small inhibitory RNA-mediated knock-down of G-CSFR messenger RNA further demonstrated the limited specificity of these antibodies for HuG-CSFR expressed on the cell surface. CONCLUSIONS: Most commercially available anti-HuG-CSFR antibodies do not bind specifically to this protein. These studies highlight the need for investigators to validate antibodies in their own systems to avoid the inadvertent use of nonspecifically binding antibodies that could lead, as exemplified in this case with a hematopoietic growth factor receptor, to erroneous conclusions about protein expression.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Receptores de Factor Estimulante de Colonias de Granulocito/inmunología , Anticuerpos Monoclonales/metabolismo , Unión Competitiva/inmunología , Western Blotting , Línea Celular Tumoral , Células Cultivadas , Citometría de Flujo , Regulación Leucémica de la Expresión Génica , Células HEK293 , Humanos , Inmunohistoquímica , Leucemia/genética , Leucemia/metabolismo , Leucemia/patología , Neutrófilos/citología , Neutrófilos/inmunología , Neutrófilos/metabolismo , Unión Proteica/inmunología , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Células U937RESUMEN
Epidermal growth factor receptor (EGFR) kinase domain mutations cause hyperresponsiveness to ligand and hypersensitivity to small-molecule tyrosine kinase inhibitors. However, little is known about how these mutations respond to antibodies against EGFR. We investigated the activity of panitumumab, a fully human anti-EGFR monoclonal antibody, in vitro in mutant EGFR-expressing non-small cell lung carcinoma (NSCLC) cells and in vivo with chemotherapy in xenograft models. Mutant EGFR-expressing NSCLC cells (NCI-H1975 [L858R+T790M] and NCI-H1650 [Delta746-750]) and CHO cells were treated with panitumumab before EGF stimulation to assess the inhibition of EGFR autophosphorylation. Established tumors were treated with panitumumab (25, 100, or 500 mug/mouse twice a week) alone or with docetaxel (10 or 20 mg/kg once a week) or cisplatin (7.5 mg/kg once a week). Antitumor activity and levels of proliferation markers were analyzed. Treatment of mutant EGFR-expressing CHO and NSCLC cells with panitumumab inhibited ligand-dependent autophosphorylation. In NCI-H1975 and NCI-H1650 xenografts, treatment with panitumumab alone or with cisplatin inhibited tumor growth compared with control (P < 0.0003). With panitumumab plus docetaxel, enhanced antitumor activity was seen in both xenografts versus panitumumab alone. Panitumumab treatment alone decreased Ki-67 and phospho- mitogen-activated protein kinase (pMAPK) staining in both xenografts compared with control. Docetaxel enhanced panitumumab activity in NCI-H1650 xenografts (decreased Ki-67 and pMAPK staining by >60%) when compared with either agent alone. Panitumumab inhibits ligand-induced EGFR phosphorylation, tumor growth, and markers of proliferation alone or with docetaxel in NSCLC cell lines that express clinically observed EGFR kinase domain mutations, including the small-molecule tyrosine kinase inhibitor-resistant T790M mutation.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Western Blotting , Células CHO , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/administración & dosificación , Cricetinae , Cricetulus , Docetaxel , Receptores ErbB/genética , Receptores ErbB/inmunología , Humanos , Inmunohistoquímica , Antígeno Ki-67/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Panitumumab , Fosforilación/efectos de los fármacos , Taxoides/administración & dosificación , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
INTRODUCTION: Rat adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) feature bone loss and systemic increases in TNFalpha, IL-1beta, and receptor activator of NF-kappaB ligand (RANKL). Anti-IL-1 or anti-TNFalpha therapies consistently reduce inflammation in these models, but systemic bone loss often persists. RANKL inhibition consistently prevents bone loss in both models without reducing joint inflammation. Effects of these therapies on systemic markers of bone turnover and inflammation have not been directly compared. METHODS: Lewis rats with established AIA or CIA were treated for 10 days (from day 4 post onset) with either PBS (Veh), TNFalpha inhibitor (pegsunercept), IL-1 inhibitor (anakinra), or RANKL inhibitor (osteoprotegerin (OPG)-Fc). Local inflammation was evaluated by monitoring hind paw swelling. Bone mineral density (BMD) of paws and lumbar vertebrae was assessed by dual X-ray absorptiometry. Markers and mediators of bone resorption (RANKL, tartrate-resistant acid phosphatase 5b (TRACP 5B)) and inflammation (prostaglandin E2 (PGE2), acute-phase protein alpha-1-acid glycoprotein (alpha1AGP), multiple cytokines) were measured in serum (day 14 post onset). RESULTS: Arthritis progression significantly increased paw swelling and ankle and vertebral BMD loss. Anti-TNFalpha reduced paw swelling in both models, and reduced ankle BMD loss in AIA rats. Anti-IL-1 decreased paw swelling in CIA rats, and reduced ankle BMD loss in both models. Anti-TNFalpha and anti-IL-1 failed to prevent vertebral BMD loss in either model. OPG-Fc reduced BMD loss in ankles and vertebrae in both models, but had no effect on paw swelling. Serum RANKL was elevated in AIA-Veh and CIA-Veh rats. While antiTNFalpha and anti-IL-1 partially normalized serum RANKL without any changes in serum TRACP 5B, OPG-Fc treatment reduced serum TRACP 5B by over 90% in both CIA and AIA rats. CIA-Veh and AIA-Veh rats had increased serum alpha1AGP, IL-1beta, IL-8 and chemokine (C-C motif) ligand 2 (CCL2), and AIA-Veh rats also had significantly greater serum PGE2, TNFalpha and IL-17. Anti-TNFalpha reduced systemic alpha1AGP, CCL2 and PGE2 in AIA rats, while anti-IL-1 decreased systemic alpha1AGP, IL-8 and PGE2. In contrast, RANKL inhibition by OPG-Fc did not lessen systemic cytokine levels in either model. CONCLUSIONS: Anti-TNFalpha or anti-IL-1 therapy inhibited parameters of local and systemic inflammation, and partially reduced local but not systemic bone loss in AIA and CIA rats. RANKL inhibition prevented local and systemic bone loss without significantly inhibiting local or systemic inflammatory parameters.
Asunto(s)
Antirreumáticos/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Osteoprotegerina/uso terapéutico , Ligando RANK/antagonistas & inhibidores , Animales , Artritis Experimental/patología , Artritis Reumatoide/patología , Densidad Ósea/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Inflamación/patología , Proteína Antagonista del Receptor de Interleucina 1 , Interleucina-1/antagonistas & inhibidores , Ratas , Ratas Endogámicas Lew , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
RANKL is a TNF family member that mediates osteoclast formation, activation, and survival by activating RANK. The proresorptive effects of RANKL are prevented by binding to its soluble inhibitor osteoprotegerin (OPG). Recombinant human OPG-Fc recognizes RANKL from multiple species and reduced bone resorption and increased bone volume, density, and strength in a number of rodent models of bone disease. The clinical development of OPG-Fc was discontinued in favor of denosumab, a fully human monoclonal antibody that specifically inhibits primate RANKL. Direct binding assays showed that denosumab bound to human RANKL but not to murine RANKL, human TRAIL, or other human TNF family members. Denosumab did not suppress bone resorption in normal mice or rats but did prevent the resorptive response in mice challenged with a human RANKL fragment encoded primarily by the fifth exon of the RANKL gene. To create mice that were responsive to denosumab, knock-in technology was used to replace exon 5 from murine RANKL with its human ortholog. The resulting "huRANKL" mice exclusively express chimeric (human/murine) RANKL that was measurable with a human RANKL assay and that maintained bone resorption at slightly reduced levels versus wildtype controls. In young huRANKL mice, denosumab and OPG-Fc each reduced trabecular osteoclast surfaces by 95% and increased bone density and volume. In adult huRANKL mice, denosumab reduced bone resorption, increased cortical and cancellous bone mass, and improved trabecular microarchitecture. These huRANKL mice have potential utility for characterizing the activity of denosumab in a variety of murine bone disease models.
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Anticuerpos Monoclonales/uso terapéutico , Densidad Ósea/efectos de los fármacos , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/fisiopatología , Técnicas de Sustitución del Gen , Ligando RANK/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Afinidad de Anticuerpos/efectos de los fármacos , Especificidad de Anticuerpos/efectos de los fármacos , Huesos/efectos de los fármacos , Huesos/patología , Denosumab , Humanos , Hipercalcemia/tratamiento farmacológico , Ratones , Datos de Secuencia Molecular , Osteoclastos/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Osteoprotegerina/metabolismo , Fenotipo , Unión Proteica/efectos de los fármacos , Ligando RANK/química , Ligando RANK/genética , Ligando RANK/farmacocinética , Ligando RANK/farmacología , Ligando RANK/uso terapéutico , Microtomografía por Rayos XRESUMEN
Receptor activator of NF-kappaB ligand (RANKL) is an essential mediator of osteoclast formation, function, and survival. The effects of RANKL are inhibited by a soluble decoy receptor called osteoprotegerin (OPG). Total ablation of RANKL in knockout mice leads to high bone mass, lymph node agenesis, and altered lymphocyte differentiation. In contrast, RANKL inhibition via OPG suppresses bone resorption but not inflammation in animal models of inflammatory bone loss. This suggests that the immune phenotype of RANKL knockout mice is related to total RANKL ablation. We hypothesized that prenatal RANKL inhibition via OPG overexpression would suppress bone resorption without influencing lymph node formation or subsequent immune responses. Transgenic rats were created, wherein soluble OPG was overexpressed by 100-fold vs wild type (WT) controls, by gestational day 11 (i.e., before lymph node formation). The structure of lymph nodes, spleen, and thymus of OPG-transgenic (OPG-Tg) animals were comparable to those of age-matched WT rats at gestational day 19 and in adulthood. The OPG-Tg neonates had elevated bone mass, confirming the prenatal inhibition of RANKL. Adult OPG-Tg rats and OPG-Tg mice exhibited no significant functional alterations relative to WT controls when subjected to immune challenges to test for altered innate and humoral responses (e.g., contact hypersensitivity to oxazolone, IgM response to Pneumovax, IgG response to keyhole limpet hemocyanin, or cytokine response to LPS). In summary, prenatal RANKL inhibition did not impair lymph node development, nor did continuous life-long RANKL inhibition cause obvious changes in innate or humoral immune responses in mice or rats.
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Osteoprotegerina/fisiología , Ligando RANK/antagonistas & inhibidores , Animales , Resorción Ósea/inmunología , Modelos Animales de Enfermedad , Inmunidad/genética , Lipopolisacáridos/farmacología , Ganglios Linfáticos/crecimiento & desarrollo , Ganglios Linfáticos/inmunología , Linfocitos/inmunología , Ratones , Ratones Endogámicos , Ratones Noqueados , Ratones Transgénicos , Osteoprotegerina/biosíntesis , Osteoprotegerina/genética , Fenotipo , Ratas , Ratas Sprague-DawleyRESUMEN
Investigators using anti-EpoR antibodies for immunoblotting and immunostaining have reported erythropoietin receptor (EpoR) expression in nonhematopoietic tissues including human tumors. However, these antibodies detected proteins of 66 to 78 kDa, significantly larger than the predicted molecular weight of EpoR (56-57 kDa). We investigated the specificity of these antibodies and showed that they all detected non-EpoR proteins. C-20 detected 3 proteins in tumor cell lines (35, 66, and 100 kDa). Sequences obtained from preparative gels had similarity to the C-20-immunizing peptide. The 66-kDa protein was a heat shock protein (HSP70) to which antibody binding was abrogated in peptide competition experiments. Antibody M-20 readily identified a 59-kDa EpoR protein. However, neither M-20 nor C-20 was suitable for detection of EpoR using immunohistochemical methods. We concluded that these antibodies have limited utility for detecting EpoR. Thus, reports of EpoR expression in tumor cells using these antibodies should be viewed with caution.
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Anticuerpos Monoclonales/química , Especificidad de Anticuerpos , Neoplasias/metabolismo , Receptores de Eritropoyetina/biosíntesis , Animales , Anticuerpos Monoclonales/inmunología , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/inmunología , Humanos , Inmunohistoquímica/métodos , Ratones , Péptidos/química , Péptidos/inmunología , Valor Predictivo de las Pruebas , Receptores de Eritropoyetina/inmunologíaRESUMEN
We describe regulatory effects that a novel neurotrophin-1/B cell-stimulating factor-3 (NNT-1/BSF-3; also reported as cardiotrophin-like cytokine) has on B cell function. NNT-1/BSF-3 stimulates B cell proliferation and Ig production in vitro. NNT-1/BSF-3-transgenic mice, engineered to express NNT-1/BSF-3 in the liver under control of the apolipoprotein E promoter, show B cell hyperplasia with particular expansion of the mature follicular B cell subset in the spleen and the prominent presence of plasma cells. NNT-1/BSF-3-transgenic mice show high serum levels of IgM, IgE, IgG2b, IgG3, anti-dsDNA Abs, and serum amyloid A. NNT-1/BSF-3-transgenic mice also show non-amyloid mesangial deposits that contain IgM, IgG, and C3 and are characterized by a distinctive ultrastructure similar to that of immunotactoid glomerulopathy. NNT-1/BSF-3-transgenic mice produce high amounts of Ag-specific IgM, IgA, and IgE and low amounts of IgG2a and IgG3. Normal mice treated with NNT-1/BSF-3 also produce high amounts of Ag-specific IgE. NNT-1/BSF-3 regulates immunity by stimulating B cell function and Ab production, with preference for Th2 over Th1 Ig types.
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Linfocitos B/fisiología , Citocinas/fisiología , Animales , Formación de Anticuerpos , Femenino , Riñón/patología , Activación de Linfocitos , Ratones , Ratones Transgénicos , Bazo/patología , Células TH1/inmunología , Células Th2/inmunologíaRESUMEN
OBJECTIVE: To assess the capacities of the cytokine inhibitors interleukin-1 receptor antagonist (IL-1Ra; anakinra) and PEGylated soluble tumor necrosis factor receptor I (PEG sTNFRI; pegsunercept) to suppress neovascularization. METHODS: A corneal angiogenesis assay was performed by implanting nylon discs impregnated with an angiogenic stimulator (basic fibroblast growth factor or vascular endothelial growth factor) into one cornea of female Sprague-Dawley rats. Animals were treated with IL-1Ra or PEG sTNFRI for 7 days, after which new vessels were quantified. In a parallel study, male Lewis rats with mycobacteria-induced adjuvant-induced arthritis were treated with IL-1Ra or PEG sTNFRI for 7 days beginning at disease onset, after which scores for inflammation and bone erosion as well as capillary counts were acquired from sections of arthritic hind paws. RESULTS: Treatment with IL-1Ra yielded a dose-dependent reduction in growth factor-induced corneal angiogenesis, while PEG sTNFRI did not. IL-1Ra, but not PEG sTNFRI, significantly reduced the number of capillaries in arthritic paws, even though both anticytokines reduced inflammation and bone erosion to a similar degree. CONCLUSION: These data support a major role for IL-1, but not TNFalpha, in angiogenesis and suggest that an additional antiarthritic mechanism afforded by IL-1 inhibitors, but not anti-TNF agents, is the suppression of the angiogenic component of pannus.
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Artritis Experimental/tratamiento farmacológico , Neovascularización de la Córnea/tratamiento farmacológico , Interleucina-1/antagonistas & inhibidores , Polietilenglicoles/farmacología , Receptores Tipo I de Factores de Necrosis Tumoral/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Artritis Experimental/inmunología , Neovascularización de la Córnea/inmunología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Expresión Génica/inmunología , Proteína Antagonista del Receptor de Interleucina 1 , Masculino , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Ratas , Ratas Endogámicas Lew , Receptores de Interleucina-1/genética , Receptores Tipo I de Interleucina-1 , Receptores Tipo II de Interleucina-1 , Receptores del Factor de Necrosis Tumoral , Sialoglicoproteínas/farmacología , Receptores Señuelo del Factor de Necrosis TumoralRESUMEN
We have identified a novel chordin-like protein, CHL2, which is structurally most homologous to CHL/neuralin/ventroptin. When injected into Xenopus embryos, CHL2 RNA induced a secondary axis. Recombinant CHL2 protein interacted directly with BMPs in a competitive manner to prevent binding to the type I BMP receptor ectodomain, and inhibited BMP-dependent induction of alkaline phosphatase in C2C12 cells. Thus, CHL2 behaves as a secreted BMP-binding inhibitor. In situ hybridization revealed that CHL2 expression is restricted to chondrocytes of various developing joint cartilage surfaces and connective tissues in reproductive organs. Adult mesenchymal progenitor cells expressed CHL2, and its levels decreased during chondrogenic differentiation. Addition of CHL2 protein to a chondrogenic culture system reduced cartilage matrix deposition. Consistently, CHL2 transcripts were weakly detected in normal adult joint cartilage. However, CHL2 expression was upregulated in middle zone chondrocytes in osteoarthritic joint cartilage (where hypertrophic markers are induced). CHL2 depressed chondrocyte mineralization when added during the hypertrophic differentiation of cultured hyaline cartilage particles. Thus, CHL2 may play negative roles in the (re)generation and maturation of articular chondrocytes in the hyaline cartilage of both developing and degenerated joints.
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Proteínas Portadoras/genética , Cartílago/embriología , Condrocitos/fisiología , Osteoartritis/genética , Xenopus/embriología , Fosfatasa Alcalina/biosíntesis , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Proteínas Morfogenéticas Óseas/farmacología , Proteínas Portadoras/metabolismo , Secuencia Conservada , Cartilla de ADN , Embrión no Mamífero/fisiología , Proteínas de la Matriz Extracelular , Femenino , Humanos , Ratones , Datos de Secuencia Molecular , Placenta/fisiología , Embarazo , Ratas , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
Artemin (ART) signals through the GFR alpha-3/RET receptor complex to support sympathetic neuron development. Here we show that ART also influences autonomic elements in adrenal medulla and enteric and pelvic ganglia. Transgenic mice over-expressing Art throughout development exhibited systemic autonomic neural lesions including fusion of adrenal medullae with adjacent paraganglia, adrenal medullary dysplasia, and marked enlargement of sympathetic (superior cervical and sympathetic chain ganglia) and parasympathetic (enteric, pelvic) ganglia. Changes began by gestational day 12.5 and formed progressively larger masses during adulthood. Art supplementation in wild type adult mice by administering recombinant protein or an Art-bearing retroviral vector resulted in hyperplasia or neuronal metaplasia at the adrenal corticomedullary junction. Expression data revealed that Gfr alpha-3 is expressed during development in the adrenal medulla, sensory and autonomic ganglia and their projections, while Art is found in contiguous mesenchymal domains (especially skeleton) and in certain nerves. Intrathecal Art therapy did not reduce hypalgesia in rats following nerve ligation. These data (1) confirm that ART acts as a differentiation factor for autonomic (chiefly sympathoadrenal but also parasympathetic) neurons, (2) suggest a role for ART overexpression in the genesis of pheochromocytomas and paragangliomas, and (3) indicate that ART is not a suitable therapy for peripheral neuropathy.
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Sistema Nervioso Autónomo/embriología , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Fármacos Neuroprotectores/metabolismo , Nervios Periféricos/embriología , Adulto , Animales , Sistema Nervioso Autónomo/efectos de los fármacos , Sistema Nervioso Autónomo/fisiología , Enfermedades del Sistema Nervioso Autónomo/patología , Southern Blotting , Células Cultivadas , Femenino , Receptores del Factor Neurotrófico Derivado de la Línea Celular Glial , Humanos , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Nervios Periféricos/efectos de los fármacos , Nervios Periféricos/fisiología , Enfermedades del Sistema Nervioso Periférico/patología , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas , Proteínas Proto-Oncogénicas c-ret , Ratas , Proteínas Tirosina Quinasas Receptoras , Transducción de Señal/fisiologíaRESUMEN
We have identified and cloned a novel human cytokine with homology to cytokines of the interleukin-17 (IL-17) family, which we have termed human IL-17E (hIL-17E). With the identification of several IL-17 family members, it is critical to understand the in vivo function of these molecules. We have generated transgenic mice overexpressing hIL-17E using an apolipoprotein E (ApoE) hepatic promoter. These mice displayed changes in the peripheral blood, particularly, a 3-fold increase in total leukocytes consisting of increases in eosinophils, lymphocytes, and neutrophils. Splenomegaly and lymphoadenopathy were predominant and included marked eosinophil infiltrates and lymphoid hyperplasia. CCR3(+) eosinophils increased in the blood and lymph nodes of the transgenic mice by 50- and 300-fold, respectively. Eosinophils also increased 8- to 18-fold in the bone marrow and spleen, respectively. In the bone marrow, most of the eosinophils had an immature appearance. CD19(+) B cells increased 2- to 5-fold in the peripheral blood, 2-fold in the spleen, and 10-fold in the lymph nodes of transgenic mice, whereas CD4(+) T lymphocytes increased 2-fold in both blood and spleen. High serum levels of the cytokines IL-2, IL-4, IL-5, granulocyte colony-stimulating factor, eotaxin, and interferon gamma were observed. Consistent with B-lymphocyte increases, serum immunoglobulin (Ig) M, IgG, and IgE were significantly elevated. Antigenic challenge of the transgenic mice with keyhole limpet hemocyanin (KLH) resulted in a decrease in anti-KLH IgG accompanied by increases of anti-KLH IgA and IgE. In situ hybridization of transgenic tissues revealed that IL-17Rh1 (IL-17BR/Evi27), a receptor that binds IL-17E, is up-regulated. Taken together, these data indicate that IL-17E regulates hematopoietic and immune functions, stimulating the development of eosinophils and B lymphocytes. The fact that hIL-17E overexpression results in high levels of circulating eosinophils, IL-4, IL-5, eotaxin, and IgE suggests that IL-17E may be a proinflammatory cytokine favoring Th2-type immune responses.