RESUMEN
Inhibition of the Wnt antagonist sclerostin increases bone mass in patients with osteoporosis and in preclinical animal models. Here we show increased levels of the Wnt antagonist Dickkopf-1 (DKK-1) in animals treated with sclerostin antibody, suggesting a negative feedback mechanism that limits Wnt-driven bone formation. To test our hypothesis that co-inhibition of both factors further increases bone mass, we engineer a first-in-class bispecific antibody with single residue pair mutations in the Fab region to promote efficient and stable cognate light-heavy chain pairing. We demonstrate that dual inhibition of sclerostin and DKK-1 leads to synergistic bone formation in rodents and non-human primates. Furthermore, by targeting distinct facets of fracture healing, the bispecific antibody shows superior bone repair activity compared with monotherapies. This work supports the potential of this agent both for treatment and prevention of fractures and offers a promising therapeutic approach to reduce the burden of low bone mass disorders.
Asunto(s)
Anticuerpos Biespecíficos/administración & dosificación , Fracturas Óseas/tratamiento farmacológico , Fracturas Óseas/fisiopatología , Glicoproteínas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Densidad Ósea , Modelos Animales de Enfermedad , Femenino , Fracturas Óseas/genética , Fracturas Óseas/metabolismo , Glicoproteínas/genética , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Macaca fascicularis , Masculino , Ratones , Ratones Noqueados , Osteogénesis/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Vía de Señalización Wnt/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacosRESUMEN
OBJECTIVE: Sclerostin plays a major role in regulating skeletal bone mass, but its effects in articular cartilage are not known. The purpose of this study was to determine whether genetic loss or pharmacologic inhibition of sclerostin has an impact on knee joint articular cartilage. METHODS: Expression of sclerostin was determined in articular cartilage and bone tissue obtained from mice, rats, and human subjects, including patients with knee osteoarthritis (OA). Mice with genetic knockout (KO) of sclerostin and pharmacologic inhibition of sclerostin with a sclerostin-neutralizing monoclonal antibody (Scl-Ab) in aged male rats and ovariectomized (OVX) female rats were used to study the effects of sclerostin on pathologic processes in the knee joint. The rat medial meniscus tear (MMT) model of OA was used to investigate the pharmacologic efficacy of systemic Scl-Ab or intraarticular (IA) delivery of a sclerostin antibody-Fab (Scl-Fab) fragment. RESULTS: Sclerostin expression was detected in rodent and human articular chondrocytes. No difference was observed in the magnitude or distribution of sclerostin expression between normal and OA cartilage or bone. Sclerostin-KO mice showed no difference in histopathologic features of the knee joint compared to age-matched wild-type mice. Pharmacologic treatment of intact aged male rats or OVX female rats with Scl-Ab had no effect on morphologic characteristics of the articular cartilage. In the rat MMT model, pharmacologic treatment of animals with either systemic Scl-Ab or IA injection of Scl-Fab had no effect on lesion development or severity. CONCLUSION: Genetic absence of sclerostin does not alter the normal development of age-dependent OA in mice, and pharmacologic inhibition of sclerostin with Scl-Ab has no impact on articular cartilage remodeling in rats with posttraumatic OA.
Asunto(s)
Proteínas Morfogenéticas Óseas/genética , Cartílago Articular/lesiones , Cartílago Articular/fisiología , Marcadores Genéticos/genética , Glicoproteínas/genética , Osteoartritis de la Rodilla/fisiopatología , Proteínas Adaptadoras Transductoras de Señales , Adulto , Anciano , Envejecimiento/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Proteínas Morfogenéticas Óseas/inmunología , Proteínas Morfogenéticas Óseas/metabolismo , Condrocitos/fisiología , Femenino , Expresión Génica/fisiología , Marcadores Genéticos/inmunología , Glicoproteínas/inmunología , Glicoproteínas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intercelular , Traumatismos de la Rodilla/genética , Traumatismos de la Rodilla/metabolismo , Traumatismos de la Rodilla/fisiopatología , Articulación de la Rodilla/fisiopatología , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Osteoartritis de la Rodilla/genética , Osteoartritis de la Rodilla/metabolismo , Ovariectomía , Ratas , Ratas Sprague-Dawley , Bancos de TejidosRESUMEN
PURPOSE: To determine whether systemic treatment with AMG 386, a selective angiopoietin 1/2-neutralizing peptibody, inhibits neovascular processes in animal models of ocular disease. METHODS: AMG 386 was tested in a laser-induced choroidal neovascularization (CNV) model in monkeys using fluorescein angiography. The biodistribution of (125)I-AMG 386 was determined in cynomolgus monkeys by whole-body autoradiography and radioanalysis of ocular tissues. A murine retinopathy of prematurity (ROP) model was used to examine the effect of AMG 386 on established and newly formed retinal vessels, either as a single agent or when combined with VEGF inhibition.AMG 386 pharmacokinetics were evaluated in each model. RESULTS: In the CNV model, AMG 386 significantly decreased fluorescent angiographic leakage and reduced fibroplasia, indicating an impaired healing response consistent with angiogenesis blockade. Radiolabeled AMG 386 was widely distributed across ocular tissues, with highest concentrations in the choroid, cornea, retinal pigmented epithelium, iris/ciliary body, and sclera. In the ROP model, AMG 386 prevented pathologic retinal angiogenesis when administered from P8 to P16 but transiently impeded regression of these abnormal vessels when administered from P17 to P23. Combining AMG 386 with VEGF inhibition led to cooperative prevention of retinal angiogenesis in this model. No AMG 386-related ocular toxicities occurred, and no treatment-related clinical observations were made in any of the studies. CONCLUSIONS: In this study, AMG 386 inhibited angiogenesis in animal models of CNV and ROP, supporting investigation of AMG 386 for the treatment of ocular neovascular diseases in the clinical setting.
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Inhibidores de la Angiogénesis/farmacología , Neovascularización Coroidal/prevención & control , Modelos Animales de Enfermedad , Proteínas Recombinantes de Fusión/farmacología , Neovascularización Retiniana/prevención & control , Retinopatía de la Prematuridad/prevención & control , Inhibidores de la Angiogénesis/farmacocinética , Angiopoyetina 1/antagonistas & inhibidores , Angiopoyetina 2/antagonistas & inhibidores , Animales , Animales Recién Nacidos , Autorradiografía , Permeabilidad Capilar/efectos de los fármacos , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Ojo/metabolismo , Femenino , Angiografía con Fluoresceína , Humanos , Hibridación in Situ , Recién Nacido , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/farmacocinética , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Vasos Retinianos/efectos de los fármacos , Retinopatía de la Prematuridad/metabolismo , Retinopatía de la Prematuridad/patología , Distribución TisularRESUMEN
Calcimimetics are positive allosteric modulators to the calcium-sensing receptor (CaSR). Activation of the CaSR inhibits the secretion of parathyroid hormone (PTH), stimulates the secretion of calcitonin, and decreases serum calcium (Ca(2+)). Cinacalcet, a second-generation calcimimetic, is used therapeutically to control PTH in patients with chronic kidney disease who are on dialysis with secondary hyperparathyroidism. A calcimimetic that displays increased separation of PTH versus Ca(2+) lowering in patients would potentially allow the use of calcimimetics to treat patients in earlier stages of renal disease because hypocalcemia can develop in this population. Toward this end, we developed a third-generation calcimimetic, determined the molecular pharmacological properties of it using an operation model of allosteric modulation/agonism, and measured the compound effects on PTH, serum ionized Ca(2+), and calcitonin levels in 5/6 nephrectomized rats. We found the new molecule effectively reduced PTH levels without promoting calcitonin secretion or hypocalcemia. Furthermore, our third-generation molecule was less efficacious at promoting calcitonin secretion from human thyroid carcinoma cells compared with 3-(2-chlorophenyl)-N-((1R)-1-(3-methoxyphenyl)ethyl)-1-propanamine (R-568), a first-generation calcimimetic. These data provide evidence that calcimimetics with increased potency can be used to lower PTH without production of significant hypocalcemia because the threshold for inhibition of PTH secretion is much lower than the threshold for calcitonin secretion.
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Compuestos de Anilina/farmacología , Compuestos de Bifenilo/farmacología , Calcitonina/metabolismo , Calcio/agonistas , Calcio/metabolismo , Dietilaminas/farmacología , Hiperparatiroidismo Secundario/tratamiento farmacológico , Hormona Paratiroidea/metabolismo , Receptores Sensibles al Calcio/metabolismo , Animales , Compuestos de Bifenilo/administración & dosificación , Células CHO , Calcitonina/sangre , Calcio/sangre , Cricetinae , Cricetulus , Dietilaminas/administración & dosificación , Células HEK293 , Humanos , Hiperparatiroidismo Secundario/etiología , Hipocalcemia/complicaciones , Fosfatos de Inositol/metabolismo , Fallo Renal Crónico/complicaciones , Masculino , Glándulas Paratiroides/efectos de los fármacos , Hormona Paratiroidea/sangre , Fenetilaminas , Fosforilación/efectos de los fármacos , Propilaminas , Ratas , Ratas Sprague-Dawley , Diálisis Renal/efectos adversosRESUMEN
INTRODUCTION: Rats with adjuvant-induced arthritis (AIA) were necropsied on 14 occasions during preclinical, acute clinical and chronic clinical stages of AIA progression to characterize local (joint protein extracts) and systemic (serum) levels of mediators regulating inflammation and bone erosion in conjunction with lymphoid tissue-specific leukocyte kinetics. RESULTS: Systemic increases in alpha1 acid glycoprotein, tumor necrosis factor-alpha (TNFalpha), interleukin (IL)-17, transforming growth factor beta (TGFbeta), and chemokine (C-C motif) ligand 2 (CCL2) together with local IL-1alpha/beta and TGFbeta enrichment and local lymphoid hyperplasia preceded the onset of clinical disease and joint damage. Systemic upregulation of TNFalpha, IL-6, IL-17, TGFbeta, IL-18, CCL2, receptor activator of nuclear factor-kappabeta ligand (RANKL), and prostaglandin E(2) during acute and/or chronic AIA coincided with systemic leukocytosis and CD4+ T cell increase in blood and spleen. In contrast, progression of joint erosions during clinical AIA was associated with intra-articular increases in IL-1alpha/beta, IL-6, RANKL, IL-17, TGFbeta, CCL2, and KC/GRO and also a dramatic decline in osteoprotegerin. CONCLUSION: These data indicate that systemic and local events in inflammatory arthritis are discrete processes, driven by multiple cellular and humoral mediators with distinct kinetic profiles.
Asunto(s)
Artritis Experimental/inmunología , Artritis Experimental/patología , Citocinas/inmunología , Inmunoglobulinas/sangre , Leucocitos/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Artritis Experimental/inducido químicamente , Biomarcadores/sangre , Biomarcadores/metabolismo , Citocinas/biosíntesis , Progresión de la Enfermedad , Leucocitos/metabolismo , Masculino , Osteoprotegerina/sangre , Ligando RANK/sangre , Ratas , Ratas Endogámicas LewRESUMEN
The interleukin (IL)-1 family members IL-1alpha, -1beta, and -18 are potent inflammatory cytokines whose activities are dependent on heterodimeric receptors of the IL-1R superfamily, and which are regulated by soluble antagonists. Recently, several new IL-1 family members have been identified. To determine the role of one of these family members in the skin, transgenic mice expressing IL1F6 in basal keratinocytes were generated. IL1F6 transgenic mice exhibit skin abnormalities that are dependent on IL-1Rrp2 and IL-1RAcP, which are two members of the IL-1R family. The skin phenotype is characterized by acanthosis, hyperkeratosis, the presence of a mixed inflammatory cell infiltrate, and increased cytokine and chemokine expression. Strikingly, the combination of the IL-1F6 transgene with an IL1F5 deficiency results in exacerbation of the skin phenotype, demonstrating that IL-1F5 has antagonistic activity in vivo. Skin from IL1F6 transgenic, IL1F5(-/-) pups contains intracorneal and intraepithelial pustules, nucleated corneocytes, and dilated superficial dermal blood vessels. Additionally, expression of IL1RL2, -1F5, and -1F6 is increased in human psoriatic skin. In summary, dysregulated expression of novel agonistic and antagonistic IL-1 family member ligands can promote cutaneous inflammation, revealing potential novel targets for the treatment of inflammatory skin disorders.
Asunto(s)
Inflamación/fisiopatología , Interleucina-1/fisiología , Enfermedades de la Piel/fisiopatología , Animales , Cápsulas Bacterianas , Humanos , Interleucina-1/genética , Interleucina-1/inmunología , Ligandos , Ratones , Ratones Transgénicos , Polisacáridos Bacterianos/genética , Regiones Promotoras Genéticas , Piel/patologíaRESUMEN
PURPOSE: To evaluate the activity of palifermin (rHuKGF) in a murine model of mucosal damage induced by a radiotherapy/chemotherapy (RT/CT) regimen mimicking treatment protocols used in head-and-neck cancer patients. METHODS AND MATERIALS: A model of mucosal damage induced by RT/CT was established by injecting female BDF1 mice with cisplatin (10 mg/kg) on Day 1; 5-fluorouracil (40 mg/kg/day) on Days 1-4, and irradiation (5 Gy/day) to the head and neck on Days 1-5. Palifermin was administered subcutaneously on Days -2 to 0 (5 mg/kg/day) and on Day 5 (5 mg/kg). Evaluations included body weight, organ weight, keratinocyte growth factor receptor expression, epithelial thickness, and cellular proliferation. RESULTS: Initiation of the radiochemotherapeutic regimen resulted in a reduction in body weight in control animals. Palifermin administration suppressed weight loss and resulted in increased organ weight (salivary glands and small intestine), epithelial thickness (esophagus and tongue), and cellular proliferation (tongue and salivary glands). CONCLUSIONS: Administration of palifermin before RT/CT promotes cell proliferation and increases in epithelial thickness in the oral mucosa, salivary glands, and digestive tract. Palifermin administration before and after RT/CT mitigates weight loss and a trophic effect on the intestinal mucosa and salivary glands, suggesting that palifermin use should be investigated further in the RT/CT settings, in which intestinal mucositis and salivary gland dysfunction are predominant side effects of cytotoxic therapy.
Asunto(s)
Factor 7 de Crecimiento de Fibroblastos/uso terapéutico , Estomatitis/prevención & control , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/efectos de la radiación , División Celular/efectos de los fármacos , Cisplatino , Esófago/efectos de los fármacos , Esófago/metabolismo , Esófago/efectos de la radiación , Femenino , Humanos , Yeyuno/efectos de los fármacos , Yeyuno/metabolismo , Yeyuno/efectos de la radiación , Antígeno Ki-67/análisis , Ratones , Boca/efectos de los fármacos , Boca/metabolismo , Boca/efectos de la radiación , Fármacos Sensibilizantes a Radiaciones , Radioterapia/efectos adversos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Estomatitis/etiología , Estomatitis/metabolismoRESUMEN
Mutations affecting the activity of the Wnt co-receptors LRP5 and LRP6 that cause alterations in skeletal biology confirmed the involvement of Wnt signaling in bone formation. We evaluated the potential role of Dkk1, an inhibitor of LRP5/6 activity, in bone formation by examining the normal expression pattern of Dkk1 in normal young mice and by assessing the consequences of osteoblast overexpression of Dkk1 in transgenic mice. Endogenous Dkk1 expression was detected primarily in osteoblasts and osteocytes. Transgenic over-expression of Dkk1 using two different rat collagen 1A1 promoters resulted in distinct bone phenotypes. More widespread Dkk1 expression (driven by the Col1A1 3.6 kb promoter) yielded osteopenia with forelimb deformities and hairlessness, while expression restricted to osteoblasts (driven by the Col1A1 2.3 kb promoter) induced severe osteopenia without limb defects or alopecia. The decrease in bone mass in vivo resulted from a significant 49% reduction in osteoblast numbers and was reflected in a 45% reduction in serum osteocalcin concentration; an in vitro study revealed that Dkk1 caused a dose-dependent suppression of osteoblast matrix mineralization. These data indicate that Dkk1 may directly influence bone formation and suggest that osteopenia develops in mice over-expressing Dkk1 at least in part due to diminished bone formation resulting from reduced osteoblast numbers.
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Enfermedades Óseas Metabólicas/fisiopatología , Huesos/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Proteínas Wnt/fisiología , Células 3T3 , Animales , Densidad Ósea , Enfermedades Óseas Metabólicas/genética , Enfermedades Óseas Metabólicas/metabolismo , Huesos/patología , Huesos/fisiopatología , Células Cultivadas , Embrión de Mamíferos/metabolismo , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/fisiología , Proteínas Relacionadas con Receptor de LDL/metabolismo , Masculino , Ratones , Ratones Transgénicos , Osteoblastos/citología , Osteoblastos/metabolismo , Osteocalcina/sangre , Osteogénesis/genética , Osteogénesis/fisiología , Embarazo , Ratas , Proteínas Recombinantes/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/metabolismoRESUMEN
Angiopoietin-2 (Ang2) exhibits broad expression in the remodeling vasculature of human tumors but very limited expression in normal tissues, making it an attractive candidate target for antiangiogenic cancer therapy. To investigate the functional consequences of blocking Ang2 activity, we generated antibodies and peptide-Fc fusion proteins that potently and selectively neutralize the interaction between Ang2 and its receptor, Tie2. Systemic treatment of tumor-bearing mice with these Ang2-blocking agents resulted in tumor stasis, followed by elimination of all measurable tumor in a subset of animals. These effects were accompanied by reduced endothelial cell proliferation, consistent with an antiangiogenic therapeutic mechanism. Anti-Ang2 therapy also prevented VEGF-stimulated neovascularization in a rat corneal model of angiogenesis. These results imply that specific Ang2 inhibition may represent an effective antiangiogenic strategy for treating patients with solid tumors.