RESUMEN
Etelcalcetide, a novel peptide agonist of the calcium-sensing receptor, prevents vascular calcification in a rat model of renal insufficiency with secondary hyperparathyroidism. Vascular calcification occurs frequently in patients with chronic kidney disease (CKD) and is a consequence of impaired mineral homeostasis and secondary hyperparathyroidism (SHPT). Etelcalcetide substantially lowers parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) levels in SHPT patients on hemodialysis. This study compared the effects of etelcalcetide and paricalcitol on vascular calcification in rats with adenine-induced CKD and SHPT. Uremia and SHPT were induced in male Wistar rats fed a diet supplemented with 0.75% adenine for 4 weeks. Rats were injected with vehicle, etelcalcetide, or paricalcitol for 4 weeks from the beginning of adenine diet. Rats fed an adenine-free diet were included as nonuremic controls. Similar reductions in plasma PTH and parathyroid chief cell proliferation were observed in both etelcalcetide- and paricalcitol-treated rats. Serum calcium and phosphorus were significantly lower in etelcalcetide-treated uremic rats and was unchanged in paricalcitol-treated rats. Both serum FGF23 and aortic calcium content were significantly lower in etelcalcetide-treated uremic rats compared with either vehicle- or paricalcitol-treated uremic rats. The degree of aortic calcium content for etelcalcetide-treated rats was similar to that in nonuremic controls and corroborated findings of lack of histologic aortic mineralization in those groups. In conclusion, etelcalcetide and paricalcitol similarly attenuated progression of SHPT in an adenine rat model of CKD. However, etelcalcetide differentially prevented vascular calcification, at least in part, due to reductions in serum FGF23, calcium, and phosphorus levels.
Asunto(s)
Hiperparatiroidismo Secundario/complicaciones , Péptidos/farmacología , Insuficiencia Renal/complicaciones , Calcificación Vascular/etiología , Animales , Modelos Animales de Enfermedad , Ergocalciferoles/farmacología , Masculino , Ratas , Ratas WistarRESUMEN
AMG 416 is a novel D-amino acid-containing peptide agonist of the calcium-sensing receptor (CaSR) that is being evaluated for the treatment of secondary hyperparathyroidism in chronic kidney disease patients receiving hemodialysis. The principal amino acid residues and their location in the CaSR that accommodate AMG 416 binding and mode of action have not previously been reported. Herein we establish the importance of a pair of cysteine residues, one from AMG 416 and the other from the CaSR at position 482 (Cys482), and correlate the degree of disulfide bond formation between these residues with the pharmacological activity of AMG 416. KP-2067, a form of the CaSR agonist peptide, was included to establish the role of cysteine in vivo and in disulfide exchange. Studies conducted with AMG 416 in pigs showed a complete lack of pharmacodynamic effect and provided a foundation for determining the peptide agonist interaction site within the human CaSR. Inactivity of AMG 416 on the pig CaSR resulted from a naturally occurring mutation encoding tyrosine for cysteine (Cys) at position 482 in the pig CaSR. Replacing Cys482 in the human CaSR with serine or tyrosine ablated AMG 416 activity. Decidedly, a single substitution of cysteine for tyrosine at position 482 in the native pig CaSR provided a complete gain of activity by the peptide agonist. Direct evidence for this disulfide bond formation between the peptide and receptor was demonstrated using a mass spectrometry assay. The extent of disulfide bond formation was found to correlate with the extent of receptor activation. Notwithstanding the covalent basis of this disulfide bond, the observed in vivo pharmacology of AMG 416 showed readily reversible pharmacodynamics.
Asunto(s)
Péptidos/farmacología , Receptores Sensibles al Calcio/agonistas , Regulación Alostérica , Animales , Cisteína , Disulfuros/química , Perros , Células HEK293 , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Receptores Sensibles al Calcio/química , Relación Estructura-Actividad , Porcinos , Porcinos EnanosRESUMEN
BACKGROUND: AMG 416 is a novel peptide agonist of the calcium-sensing receptor (CaSR). This report describes the activity of AMG 416 in two different rodent models of uremia, compared in each case to cinacalcet, an approved therapeutic for secondary hyperparathyroidism (SHPT) in patients with chronic kidney disease on dialysis. METHODS: AMG 416 was administered as a single intravenous (IV) bolus in a severe, acute model of renal insufficiency (the "1K1C" model) and plasma parathyroid hormone (PTH) and serum calcium levels were monitored for 24 hours. In a chronic, less severe model of renal dysfunction, the 5/6 nephrectomy (5/6 Nx) model, AMG 416 was administered as a once-daily IV bolus for 28 days. Both studies included a control (vehicle) group and a comparison cinacalcet group (po dosing at 30 mg/kg and 10 mg/kg for the 1K1C and 5/6 Nx studies, respectively). RESULTS: Administration of AMG 416 by IV bolus injection into rats with acute renal dysfunction (1K1C model) resulted in a sustained reduction in plasma PTH from the initial elevated values. Following a single IV bolus (0.5 mg/kg), AMG 416 caused a substantial drop in PTH levels which remained approximately 50% below their initial level at 24 hrs. In the same model, oral treatment with cinacalcet (30 mg/kg) resulted in an acute drop in PTH which almost returned to the starting level by 24 hours after dosing. In the 5/6 Nx chronic uremia model, daily IV dosing of AMG 416 over 4 weeks (1 mg/kg) resulted in a sustained reduction in PTH, with approximately 50% of the initial level observed 48 hours post treatment throughout the study. Cinacalcet treatment (10 mg/kg) in the same model resulted in acutely lowered plasma PTH levels which returned to placebo levels by 24 hours post-dose. Consistent with the reductions in plasma PTH, reductions in serum calcium were observed in both AMG 416- and cinacalcet-treated animals. CONCLUSIONS: As a long-acting CaSR agonist suitable for administration by the IV route, AMG 416 is a potential new therapy for the treatment of CKD patients with SHPT receiving hemodialysis.
Asunto(s)
Calcimiméticos/administración & dosificación , Modelos Animales de Enfermedad , Naftalenos/administración & dosificación , Péptidos/administración & dosificación , Receptores Sensibles al Calcio/agonistas , Uremia/tratamiento farmacológico , Animales , Cinacalcet , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/efectos de los fármacos , Resultado del Tratamiento , Uremia/fisiopatologíaRESUMEN
A novel peptide, AMG 416 (formerly KAI-4169, and with a United States Adopted Name: velcalcetide), has been identified that acts as an agonist of the calcium-sensing receptor (CaSR). This article summarizes the in vitro and in vivo characterization of AMG 416 activity and the potential clinical utility of this novel compound. AMG 416 activates the human CaSR in vitro, acting by a mechanism distinct from that of cinacalcet, the only approved calcimimetic, since it can activate the CaSR both in the presence or the absence of physiologic levels of extracellular calcium. Administration of AMG 416 in vivo into either normal or renally compromised rats results in dose-dependent reductions in parathyroid hormone (PTH) levels and corresponding decreases in serum calcium, regardless of the baseline level of PTH. Treatment of 5/6 nephrectomized rats with AMG 416 resulted in dramatic improvements in their metabolic profile, including lower PTH and serum creatinine levels, reduced amounts of vascular calcification, attenuated parathyroid hyperplasia, and greater expression of the parathyroid gland regulators CaSR, vitamin D receptor, and FGF23 receptor compared with vehicle-treated animals. No drug accumulation was observed under this dosing regimen, and the terminal half-life of AMG 416 was estimated to be 2-4.5 hours. As a long-acting CaSR agonist, AMG 416 is an innovative new therapy for the treatment of hemodialysis patients with secondary hyperparathyroidism.
Asunto(s)
Hiperparatiroidismo Secundario/tratamiento farmacológico , Péptidos/uso terapéutico , Receptores Sensibles al Calcio/agonistas , Animales , Femenino , Factor-23 de Crecimiento de Fibroblastos , Células HEK293 , Humanos , Hiperparatiroidismo Secundario/etiología , Hiperparatiroidismo Secundario/metabolismo , Riñón/fisiopatología , Masculino , Nefrectomía , Glándulas Paratiroides/efectos de los fármacos , Glándulas Paratiroides/metabolismo , Hormona Paratiroidea/sangre , Péptidos/farmacocinética , Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Diálisis Renal/efectos adversos , Distribución Tisular , Uremia/tratamiento farmacológico , Uremia/etiologíaRESUMEN
Heart failure (HF) and cardiac arrhythmias share overlapping pathological mechanisms that act cooperatively to accelerate disease pathogenesis. Cardiac fibrosis is associated with both pathological conditions. Our previous work identified a link between phytosterol accumulation and cardiac injury in a mouse model of phytosterolemia, a rare disorder characterized by elevated circulating phytosterols and increased cardiovascular disease risk. Here, we uncover a previously unknown pathological link between phytosterols and cardiac arrhythmias in the same animal model. Phytosterolemia resulted in inflammatory pathway induction, premature ventricular contractions (PVC) and ventricular tachycardia (VT). Blockade of phytosterol absorption either by therapeutic inhibition or by genetic inactivation of NPC1L1 prevented the induction of inflammation and arrhythmogenesis. Inhibition of phytosterol absorption reduced inflammation and cardiac fibrosis, improved cardiac function, reduced the incidence of arrhythmias and increased survival in a mouse model of phytosterolemia. Collectively, this work identified a pathological mechanism whereby elevated phytosterols result in inflammation and cardiac fibrosis leading to impaired cardiac function, arrhythmias and sudden death. These comorbidities provide insight into the underlying pathophysiological mechanism for phytosterolemia-associated risk of sudden cardiac death.
Asunto(s)
Arritmias Cardíacas/patología , Muerte Súbita Cardíaca/patología , Fibrosis/patología , Insuficiencia Cardíaca/patología , Hipercolesterolemia/complicaciones , Inflamación/patología , Enfermedades Intestinales/complicaciones , Errores Innatos del Metabolismo Lipídico/complicaciones , Fitosteroles/efectos adversos , Fitosteroles/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/fisiología , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/fisiología , Animales , Arritmias Cardíacas/etiología , Arritmias Cardíacas/metabolismo , Citocinas/sangre , Muerte Súbita Cardíaca/etiología , Fibrosis/etiología , Fibrosis/metabolismo , Insuficiencia Cardíaca/etiología , Insuficiencia Cardíaca/metabolismo , Inflamación/etiología , Inflamación/metabolismo , Lipoproteínas/fisiología , Proteínas de Transporte de Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Cardiovascular disease is expected to remain the leading cause of death worldwide despite the introduction of proprotein convertase subtilisin/kexin type 9 inhibitors that effectively control cholesterol. Identifying residual risk factors for cardiovascular disease remains an important step for preventing and clinically managing the disease. Here we report cardiac injury and increased mortality occurring despite a 50% reduction in plasma cholesterol in a mouse model of phytosterolemia, a disease characterized by elevated levels of dietary plant sterols in the blood. Our studies show accumulation of stigmasterol, one of phytosterol species, leads to left ventricle dysfunction, cardiac interstitial fibrosis and macrophage infiltration without atherosclerosis, and increased mortality. A pharmacological inhibitor of sterol absorption prevents cardiac fibrogenesis. We propose that the pathological mechanism linking clinical sitosterolemia to the cardiovascular outcomes primarily involves phytosterols-induced cardiac fibrosis rather than cholesterol-driven atherosclerosis. Our studies suggest stigmasterol is a potent and independent risk factor for cardiovascular disease.
Asunto(s)
Hipercolesterolemia/complicaciones , Enfermedades Intestinales/complicaciones , Errores Innatos del Metabolismo Lipídico/complicaciones , Miocardio/patología , Fitosteroles/efectos adversos , Estigmasterol/farmacología , Disfunción Ventricular Izquierda/inducido químicamente , Disfunción Ventricular Izquierda/mortalidad , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 5/genética , Transportador de Casete de Unión a ATP, Subfamilia G, Miembro 8/genética , Animales , Aterosclerosis , Supervivencia Celular/efectos de los fármacos , Suplementos Dietéticos , Fibrosis , Células Endoteliales de la Vena Umbilical Humana , Humanos , Lipoproteínas/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
B lymphocyte stimulator (BLyS), also known as B cell-activating factor, is a key positive regulator of B cell homeostasis, and elevated levels of BLyS have been observed in systemic lupus erythematosus (SLE) patients. Given that anti-chromatin auto-antibodies are one of the hallmarks of SLE, we examined the role of BLyS and its receptors in the regulation of anti-chromatin B cells. We demonstrate that exogenous BLyS treatment leads to an increase in B cell numbers, particularly anti-chromatin B cells; yet, their localization in the spleen and auto-antibody production remain unaffected. We also examined transmembrane activator and CAML interactor (TACI), BLyS receptor 3 (BR3) and B cell maturation antigen expression on anti-chromatin B cells before and after receiving T cell help. Interestingly, in the absence of T cell help, TACI expression is greater on immature anti-chromatin B cells compared with immature Tg(-) B cells, whereas BR3 levels are comparable. After receiving T cell help, the anti-chromatin B cells that have differentiated into short-lived plasma cells no longer express BR3 but retain TACI. These data suggest a novel role for TACI in anti-chromatin B cell homeostasis and differentiation.
Asunto(s)
Anticuerpos Antinucleares/inmunología , Factor Activador de Células B/farmacología , Linfocitos B/efectos de los fármacos , Receptores del Factor de Necrosis Tumoral/metabolismo , Traslado Adoptivo , Animales , Anticuerpos Antinucleares/sangre , Formación de Anticuerpos/efectos de los fármacos , Formación de Anticuerpos/inmunología , Receptor del Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/metabolismo , Antígeno de Maduración de Linfocitos B/genética , Antígeno de Maduración de Linfocitos B/metabolismo , Subgrupos de Linfocitos B/efectos de los fármacos , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Femenino , Expresión Génica , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Receptores CXCR5 , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/trasplante , Proteína Activadora Transmembrana y Interactiva del CAML/genética , Proteína Activadora Transmembrana y Interactiva del CAML/metabolismoRESUMEN
Autoreactive B cells may become activated in a T-independent manner via synergistic engagement of the BCR and TLRs. Using the VH3H9 Ig H chain transgene to track anti-chromatin B cells, we demonstrate that VH3H9/Vlambda1 anti-chromatin B cells proliferate in response to stimulatory oligodeoxynucleotides containing CpG motifs, suggesting that these autoreactive B cells are responsive to TLR9 signaling. Strikingly, some VH3H9 B cells, but not the well-characterized VH3H9/Vlambda1 B cells, proliferate spontaneously in culture medium. This proliferation is blocked by inhibitory CpG oligodeoxynucleotides, implicating the TLR9 (or possibly TLR7) pathway. Most hybridomas generated from the proliferating cells are polyreactive, and one exhibits binding to nuclear Ags but not to the other Ags tested. Thus, B cells carrying autoreactive and/or polyreactive specificities may be susceptible to T cell-independent activation via dual engagement of the BCR and TLRs.
Asunto(s)
Autoanticuerpos/biosíntesis , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Cromatina/inmunología , Activación de Linfocitos/inmunología , Receptor Toll-Like 9/metabolismo , Adyuvantes Inmunológicos/metabolismo , Adyuvantes Inmunológicos/fisiología , Animales , Reacciones Antígeno-Anticuerpo , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/efectos de los fármacos , Proliferación Celular , Células Cultivadas , Islas de CpG/inmunología , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Ligandos , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Modelos Biológicos , Oligodesoxirribonucleótidos/metabolismo , Oligodesoxirribonucleótidos/farmacología , Receptor Toll-Like 9/fisiologíaRESUMEN
Anti-double-stranded DNA (anti-dsDNA) B cells are regulated in non-autoimmune mice. While some are deleted or undergo receptor editing, a population of anti-dsDNA (VH3H9/V lambda 1) B cells that emigrate into the periphery has also been identified. These cells have an altered phenotype relative to normal B cells in that they have a reduced lifespan, appear developmentally arrested, and localize primarily to the T/B-cell interface in the spleen. This phenotype may be the consequence of immature B cells encountering antigen in the absence of T-cell help. When provided with T-cell help, the anti-dsDNA B cells differentiate into antibody-forming cells. In the context of the autoimmune-prone lpr/lpr or gld/gld mutations, the VH3H9/V lambda 1 anti-dsDNA B cells populate the B-cell follicle and by 12 weeks of age produce serum autoantibodies. The early event of anti-dsDNA B-cell follicular entry, in the absence of autoantibody production, is dependent upon CD4(+) T cells. We hypothesize that control of autoantibody production in young autoimmune-prone mice may be regulated by the counterbalancing effect of T-regulatory (T(reg)) cells. Consistent with this model, we have demonstrated that T(reg) cells are able to prevent autoantibody production induced by T-cell help. Additional studies are aimed at investigating the mechanisms of this suppression as well as probing the impact of distinct forms of T-cell-dependent and -independent activation on anti-dsDNA B cells.
Asunto(s)
Linfocitos B/inmunología , Lupus Vulgar/inmunología , Activación de Linfocitos , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Linfocitos B/metabolismo , Linfocitos B/patología , ADN/inmunología , Humanos , Lupus Vulgar/genética , Lupus Vulgar/patología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/patologíaRESUMEN
Autoreactive thymocytes can be eliminated by clonal deletion during their development in the thymus. The precise developmental stage(s) at which clonal deletion occurs in a normal thymus has been difficult to assess, in large part because of the absence of a specific marker for TCR-mediated apoptosis. In this report, we reveal that Nur77 expression can be used as a specific marker of clonal deletion in an unmanipulated thymus and directly identify TCRintCD4+CD8+ and semimature CD4+CD8- thymocytes as the principal targets of deletion. These data indicate that clonal deletion normally occurs at a relatively late stage of development, as cells mature from CD4+CD8+ thymocytes to single-positive T cells.