RESUMEN
Bone marrow (BM) metastasis remains one of the main causes of death associated with solid tumors as well as multiple myeloma (MM). Targeting the BM niche to prevent or modulate metastasis has not been successful to date. Here, we show that stromal cell-derived factor-1 (SDF-1/CXCL12) is highly expressed in active MM, as well as in BM sites of tumor metastasis and report on the discovery of the high-affinity anti-SDF-1 PEGylated mirror-image l-oligonucleotide (olaptesed-pegol). In vivo confocal imaging showed that SDF-1 levels are increased within MM cell-colonized BM areas. Using in vivo murine and xenograft mouse models, we document that in vivo SDF-1 neutralization within BM niches leads to a microenvironment that is less receptive for MM cells and reduces MM cell homing and growth, thereby inhibiting MM disease progression. Targeting of SDF-1 represents a valid strategy for preventing or disrupting colonization of the BM by MM cells.
Asunto(s)
Médula Ósea/patología , Quimiocina CXCL12/antagonistas & inhibidores , Mieloma Múltiple/terapia , Oligonucleótidos/farmacología , Animales , Médula Ósea/metabolismo , Neoplasias de la Médula Ósea/metabolismo , Neoplasias de la Médula Ósea/secundario , Ácidos Borónicos/farmacología , Bortezomib , Quimiocina CXCL12/biosíntesis , Quimiocina CXCL12/genética , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Masculino , Ratones , Ratones SCID , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Metástasis de la Neoplasia , Oligonucleótidos/química , Oligonucleótidos/genética , Polietilenglicoles/química , Pirazinas/farmacologíaRESUMEN
The sphingolipid S1P (sphingosine 1-phosphate) is known to be involved in a number of pathophysiological conditions such as cancer, autoimmune diseases and fibrosis. It acts extracellularly through a set of five G-protein-coupled receptors, but its intracellular actions are also well documented. Employing in vitro selection techniques, we identified an L-aptamer (Spiegelmer®) to S1P designated NOX-S93. The binding affinity of NOX-S93 to S1P had a Kd value of 4.3 nM. The Spiegelmer® shows equal binding to dihydro-S1P, but no cross-reactivity to the related lipids sphingosine, lysophosphatidic acid, ceramide, ceramide-1-phosphate or sphingosine phosphocholine. In stably transfected CHO (Chinese-hamster ovary) cell lines expressing the S1P receptors S1PR1 or S1PR3, NOX-S93 inhibits S1P-mediated ß-arrestin recruitment and intracellular calcium release respectively, with IC50 values in the low nanomolar range. The pro-angiogenic activity of S1P, and of the growth factors VEGF-A (vascular endothelial growth factor-A), FGF-2 (fibroblast growth factor-2) and IGF-1 (insulin-like growth factor-1), was effectively blocked by NOX-S93 in a cellular angiogenesis assay employing primary human endothelial cells. These data provide further evidence for the relevance of extracellular S1P as a central mediator of angiogenesis, suggesting pharmacological S1P neutralization as a promising treatment alternative to current anti-angiogenesis approaches.
Asunto(s)
Inhibidores de la Angiogénesis/aislamiento & purificación , Aptámeros de Nucleótidos/farmacología , Lisofosfolípidos/antagonistas & inhibidores , Esfingosina/análogos & derivados , Inhibidores de la Angiogénesis/farmacología , Animales , Arrestinas/metabolismo , Células CHO , Calcio/metabolismo , Cricetulus , Factor 2 de Crecimiento de Fibroblastos/antagonistas & inhibidores , Humanos , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Neovascularización Patológica , Receptores de Lisoesfingolípidos/metabolismo , Esfingosina/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , beta-ArrestinasRESUMEN
Complement factor C5a is a potent proinflammatory mediator that contributes to the pathogenesis of numerous inflammatory diseases. Here, we describe the discovery of NOX-D20, a PEGylated biostable mirror-image mixed (l-)RNA/DNA aptamer (Spiegelmer) that binds to mouse and human C5a with picomolar affinity. In vitro, NOX-D20 inhibited C5a-induced chemotaxis of a CD88-expressing cell line and efficiently antagonized the activation of primary human polymorphonuclear leukocytes (PMN) by C5a. Binding of NOX-D20 to the C5a moiety of human C5 did not interfere with the formation of the terminal membrane attack complex (MAC). In sepsis, for which a specific interventional therapy is currently lacking, complement activation and elevated levels of C5a are suggested to contribute to multiorgan failure and mortality. In the model of polymicrobial sepsis induced by cecal ligation and puncture (CLP), NOX-D20 attenuated inflammation and organ damage, prevented the breakdown of the vascular endothelial barrier, and improved survival. Our study suggests NOX-D20 as a new therapeutic candidate for the treatment of sepsis.
Asunto(s)
Antiinflamatorios no Esteroideos/uso terapéutico , Aptámeros de Nucleótidos/uso terapéutico , Complemento C5a/antagonistas & inhibidores , Insuficiencia Multiorgánica/prevención & control , Peritonitis/tratamiento farmacológico , Sepsis/tratamiento farmacológico , Animales , Antiinflamatorios no Esteroideos/farmacocinética , Aptámeros de Nucleótidos/farmacocinética , Complemento C5a/inmunología , Complemento C5a/metabolismo , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Insuficiencia Multiorgánica/tratamiento farmacológico , Neutrófilos/inmunología , Neutrófilos/metabolismo , Peritonitis/complicaciones , Peritonitis/fisiopatología , Sepsis/complicacionesRESUMEN
Excessive secretion of glucagon, a functional insulin antagonist, significantly contributes to hyperglycemia in type 1 and type 2 diabetes. Accordingly, immunoneutralization of glucagon or genetic deletion of the glucagon receptor improved glucose homeostasis in animal models of diabetes. Despite this strong evidence, agents that selectively interfere with endogenous glucagon have not been implemented in clinical practice yet. We report the discovery of mirror-image DNA-aptamers (Spiegelmer®) that bind and inhibit glucagon. The affinity of the best binding DNA oligonucleotide was remarkably increased (>25-fold) by the introduction of oxygen atoms at selected 2'-positions through deoxyribo- to ribonucleotide exchanges resulting in a mixed DNA/RNA-Spiegelmer (NOX-G15) that binds glucagon with a Kd of 3 nm. NOX-G15 shows no cross-reactivity with related peptides such as glucagon-like peptide-1, glucagon-like peptide-2, gastric-inhibitory peptide, and prepro-vasoactive intestinal peptide. In vitro, NOX-G15 inhibits glucagon-stimulated cAMP production in CHO cells overexpressing the human glucagon receptor with an IC50 of 3.4 nm. A single injection of NOX-G15 ameliorated glucose excursions in intraperitoneal glucose tolerance tests in mice with streptozotocin-induced (type 1) diabetes and in a non-genetic mouse model of type 2 diabetes. In conclusion, the data suggest NOX-G15 as a therapeutic candidate with the potential to acutely attenuate hyperglycemia in type 1 and type 2 diabetes.
Asunto(s)
Aptámeros de Nucleótidos/farmacología , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 2/sangre , Glucagón/antagonistas & inhibidores , ARN/metabolismo , Animales , Aptámeros de Nucleótidos/sangre , Aptámeros de Nucleótidos/farmacocinética , Aptámeros de Nucleótidos/uso terapéutico , Glucemia/metabolismo , Peso Corporal/efectos de los fármacos , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/biosíntesis , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Modelos Animales de Enfermedad , Ayuno/sangre , Glucagón/metabolismo , Prueba de Tolerancia a la Glucosa , Humanos , Cinética , Masculino , Ratones , Ratones Endogámicos BALB CRESUMEN
Anemia of chronic inflammation is the most prevalent form of anemia in hospitalized patients. A hallmark of this disease is the intracellular sequestration of iron. This is a consequence of hepcidin-induced internalization and subsequent degradation of ferroportin, the hepcidin receptor and only known iron-export protein. This study describes the characterization of novel anti-hepcidin compound NOX-H94, a structured L-oligoribonucleotide that binds human hepcidin with high affinity (Kd = 0.65 ± 0.06 nmol/L). In J774A.1 macrophages, NOX-H94 blocked hepcidin-induced ferroportin degradation and ferritin expression (half maximal inhibitory concentration = 19.8 ± 4.6 nmol/L). In an acute cynomolgus monkey model of interleukin 6 (IL-6)-induced hypoferremia, NOX-H94 inhibited serum iron reduction completely. In a subchronic model of IL-6-induced anemia, NOX-H94 inhibited the decrease in hemoglobin concentration. We conclude that NOX-H94 protects ferroportin from hepcidin-induced degradation. Therefore, this pharmacologic approach may represent an interesting treatment option for patients suffering from anemia of chronic inflammation.
Asunto(s)
Anemia/tratamiento farmacológico , Anemia/etiología , Inflamación/complicaciones , Inflamación/tratamiento farmacológico , Oligorribonucleótidos/uso terapéutico , Anemia/patología , Animales , Antiinflamatorios/administración & dosificación , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Péptidos Catiónicos Antimicrobianos/antagonistas & inhibidores , Células Cultivadas , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Hemoglobinas/análisis , Hemoglobinas/efectos de los fármacos , Hepcidinas , Interleucina-6/administración & dosificación , Interleucina-6/efectos adversos , Hierro/sangre , Hierro/metabolismo , Trastornos del Metabolismo del Hierro/inducido químicamente , Macaca fascicularis , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Oligorribonucleótidos/administración & dosificación , Oligorribonucleótidos/farmacologíaRESUMEN
High mobility group A1 (HMGA1) proteins belong to a group of architectural transcription factors that are overexpressed in a range of human malignancies, including pancreatic adenocarcinoma. They promote anchorage-independent growth and epithelial-mesenchymal transition and are therefore suggested as potential therapeutic targets. Employing in vitro selection techniques against a chosen fragment of HMGA1, we have generated biostable l-RNA oligonucleotides, so-called Spiegelmers, that specifically bind HMGA1b with low nanomolar affinity. We demonstrate that the best binding Spiegelmers, NOX-A50 and NOX-f33, compete HMGA1b from binding to its natural binding partner, AT-rich double-stranded DNA. We describe a formulation method based on polyplex formation with branched polyethylenimine for efficient delivery of polyethylene glycol-modified Spiegelmers and show improved tissue distribution and persistence in mice. In a xenograft mouse study using the pancreatic cancer cell line PSN-1, subcutaneous administration of 2 mg/kg per day NOX-A50 formulated in polyplexes showed an enhanced delivery of NOX-A50 to the tumor and a significant reduction of tumor volume. Our results demonstrate that intracellular targets can be successfully addressed with a Spiegelmer using polyethylenimine-based delivery and underline the importance of HMGA1 as a therapeutic target in pancreatic cancer.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Aptámeros de Nucleótidos/farmacología , Sistemas de Liberación de Medicamentos , Proteínas HMGA/antagonistas & inhibidores , Neoplasias Pancreáticas/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Línea Celular Tumoral , ADN de Neoplasias/metabolismo , Relación Dosis-Respuesta a Droga , Proteínas HMGA/metabolismo , Humanos , Ratones , Ratones Mutantes , Unión ProteicaRESUMEN
The monocyte chemoattractant protein CCL2 is crucial for monocyte and T cell recruitment from the vascular to the extravascular compartment at sites of inflammation. CCL2 is expressed in human lupus nephritis and was shown to mediate experimental lupus; therefore, CCL2 antagonists may be beneficial for therapy. This study describes the l-enantiomeric RNA oligonucleotide mNOX-E36, a so-called Spiegelmer that binds murine CCL2 with high affinity and neutralizes its action in vitro and in vivo. The mirror image configuration of the Spiegelmer confers nuclease resistance and thus excellent biostability. mNOX-E36 does not induce type I IFN via Toll-like receptor-7 or cytosolic RNA receptors, as recently shown for certain synthetic D-RNA. Autoimmune-prone MRL(lpr/lpr) mice that were treated with a polyethylene glycol form of mNOX-E36 from weeks 14 to 24 of age showed prolonged survival associated with a robust improvement of lupus nephritis, peribronchial inflammation, and lupus-like inflammatory skin lesions. Thus, mNOX-E36-based inhibition of CCL2 represents a novel strategy for the treatment of autoimmune tissue injury, such as lupus nephritis.
Asunto(s)
Quimiocina CCL2/antagonistas & inhibidores , Terapia Genética/métodos , Nefritis Lúpica/terapia , Oligorribonucleótidos/farmacocinética , Animales , Autoinmunidad , Médula Ósea/inmunología , Quimiocina CCL2/metabolismo , ADN/inmunología , Femenino , Nefritis Lúpica/inmunología , Nefritis Lúpica/mortalidad , Trastornos Linfoproliferativos/inmunología , Trastornos Linfoproliferativos/mortalidad , Trastornos Linfoproliferativos/terapia , Ratones , Ratones Endogámicos MRL lpr , Monocitos/inmunología , Oligorribonucleótidos/sangre , Tasa de SupervivenciaRESUMEN
A class of diuretic/aquaretic agents based on mirror-image oligonucleotides (so-called Spiegelmers) has been identified. These molecules directly bind and inhibit the neuropeptide vasopressin (AVP). AVP is the major regulatory component of body fluid homeostasis mediated through binding to the renal V(2) receptor. Elevated plasma levels of AVP are implicated in several pathological conditions, mainly cardiovascular diseases. In congestive heart failure, AVP is part of a neuroendocrine imbalance that is responsible for progressive worsening of the disease. Employing in vitro selection techniques, RNA aptamers that bind to the unnatural d-configuration of AVP were isolated. The best aptamer displayed an affinity to d-AVP of approximately 560 pM at 37 degrees C. The corresponding Spiegelmer, a 38-mer mirror-image oligonucleotide (l-RNA) termed NOX-F37, inhibits vasopressin-dependent activation of V(1a) as well as V(2) receptors with IC(50) values of 6.1 nM and 1 nM, respectively. NOX-F37 administered to healthy rats effectively neutralized AVP and increased diuresis dose-dependently for 24 h. The mode of action was strictly aquaretic, i.e., the increase in urine volume was not accompanied by an increase in electrolytes. These results clearly prove the in vivo efficacy of NOX-F37 and points out its potential as a drug in the treatment of diseases that are associated with body fluid overload.
Asunto(s)
Oligorribonucleótidos/farmacología , ARN/farmacología , Vasopresinas/antagonistas & inhibidores , Animales , Antagonistas de los Receptores de Hormonas Antidiuréticas , Secuencia de Bases , Sitios de Unión , Conformación de Carbohidratos , Línea Celular , Riñón/efectos de los fármacos , Riñón/metabolismo , Masculino , Datos de Secuencia Molecular , Mutación/genética , Oligorribonucleótidos/química , ARN/química , ARN/genética , Ratas , Ratas Sprague-Dawley , Receptores de Vasopresinas/metabolismo , Porcinos , Vasopresinas/farmacologíaRESUMEN
Bacterial staphylococcal enterotoxin B is involved in several severe disease patterns and it was therefore used as a target for the generation of biologically stable mirror-image oligonucleotide ligands, so called Spiegelmers. The toxin is a 28 kDa protein consisting of 239 amino acids. Since the full-length protein is not accessible to chemical peptide synthesis, a stable domain of 25 amino acids was identified as a suitable selection target. DNA in vitro selection experiments were carried out against the equivalent mirror-image D-peptide domain resulting in high affinity D-DNA aptamers. As expected, the corresponding enantiomeric L-DNA Spiegelmer showed comparable binding characteristics to the L-peptide domain. Moreover, the Spiegelmer bound the whole protein target with only slightly reduced affinity. Dissociation constants of both peptide-oligonucleotide complexes were measured in the range of 200 nM, whereas the Spiegelmer binding to the full-length protein was determined at approximately 420 nM. These data demonstrate the possibility to identify Spiegelmers against large protein targets by a domain approach.