RESUMEN
Invariant natural killer T (iNKT) cells are innate-like T lymphocytes characterized by the expression of an invariant T cell receptor (iTCR) that recognizes glycolipid antigens presented by the MHC I-like CD1d molecule. Following antigenic stimulation, iNKT cells rapidly produce large amounts of cytokines that can trans-activate dendritic cells (DC) and promote the anti-tumor functions of cytotoxic lymphocytes, such as natural killer (NK) and CD8 T cells. Additionally, iNKT cells can mediate robust and direct cytotoxicity against CD1d+ tumor targets. However, many tumors down-regulate CD1d and evade iNKT cell attack. To circumvent this critical barrier to iNKT cell anti-tumor activity, a novel monoclonal antibody (mAb), NKT14 has been recently developed. This agonistic antibody binds directly and specifically to the iTCR of murine iNKT cells. In the current study, we demonstrate that NKT14m mediates robust activation, cytokine production and degranulation of murine iNKT cells, in vitro. Consistently, NKT14m also promoted iNKT cell activation and immunomodulatory functions, in vivo. Finally, administration of NKT14m with low dose interleukin (IL)-12 further augmented iNKT cell IFN-γ production in vivo, and this combination conferred superior suppression of tumor cell growth compared to NKT14m or IL-12 alone. Together, these data demonstrate that a combination treatment consisting of low dose IL-12 and iTCR-specific mAb may be an attractive alternative to activate iNKT cell anti-tumor functions.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Quimioterapia Combinada/métodos , Interferón gamma/metabolismo , Interleucina-12/farmacología , Linfoma/tratamiento farmacológico , Células T Asesinas Naturales/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Antígenos CD1d/inmunología , Línea Celular Tumoral , Citocinas/metabolismo , Citotoxicidad Inmunológica/efectos de los fármacos , Sinergismo Farmacológico , Inmunomodulación/efectos de los fármacos , Linfoma/inmunología , Ratones , Ratones Endogámicos C57BL , Células T Asesinas Naturales/efectos de los fármacos , Células T Asesinas Naturales/metabolismoRESUMEN
Invariant natural killer T cells (iNKTs) directly kill tumor cells and trans-activate the anti-tumor functions of dendritic cells (DC), natural killer (NK) cells, and T and B cells. As such, iNKTs serve as a powerful tool for use in cell-based cancer immunotherapy. iNKT cell activation commonly requires engagement of the invariant T cell receptor (iTCR) by CD1d presenting glycolipid antigens. However, transformed cells often down-regulate CD1d expression, which results in a reduction of iNKT cell anti-tumor functions. One approach to circumvent this critical barrier to iNKT cell activation is to develop an agonistic antibody that binds directly to the iTCR without the requirement for CD1d-mediated antigen presentation. To this end, we have characterized the iNKT cell stimulatory properties of NKTT320, a novel, recombinant, humanized, monoclonal antibody that binds selectively and with high affinity to human iTCRs. Strikingly, immobilized NKTT320 mediated robust iNKT cell activation (upregulation of CD25 and CD69) and proliferation (carboxyfluorescein succinimidyl ester (CFSE) dilution), as well as Th1 and Th2 cytokine production. Additionally, iNKTs stimulated by plate-bound NKTT320 exhibited increased intracellular levels of granzyme B and degranulation (exposure of CD107 on the cell surface). Furthermore, both soluble and immobilized NKTT320 induced iNKT cell-mediated activation of bystander immune cells, suggesting that this novel anti-iTCR antibody facilitates both direct and indirect iNKT cell cytotoxicity. These studies are significant, as they provide a framework by which iNKT cell anti-cancer functions could be enhanced for therapeutic purposes.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Células T Asesinas Naturales/efectos de los fármacos , Células T Asesinas Naturales/inmunología , Biomarcadores , Degranulación de la Célula/efectos de los fármacos , Degranulación de la Célula/inmunología , Línea Celular Tumoral , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inmunofenotipificación , Células T Asesinas Naturales/metabolismoRESUMEN
During toxic shock syndrome (TSS), bacterial superantigens trigger a polyclonal T -cell response leading to a potentially catastrophic "cytokine storm". Whether innate-like invariant natural killer T (iNKT) cells, with remarkable immunomodulatory properties, participate in TSS is unclear. Using genetic and cell depletion approaches, we generated iNKT cell-deficient, superantigen-sensitive HLA-DR4-transgenic (DR4tg) mice, which were compared with their iNKT-sufficient counterparts for responsiveness to staphylococcal enterotoxin B (SEB). Both approaches indicate that iNKT cells are pathogenic in TSS. Importantly, treating DR4tg mice with a TH2-polarizing glycolipid agonist of iNKT cells reduced SEB-inflicted morbidity/mortality. Therefore, iNKT cells may constitute an attractive therapeutic target in superantigen-mediated illnesses.
Asunto(s)
Antígeno HLA-DR4/genética , Células T Asesinas Naturales/inmunología , Choque Séptico/inmunología , Choque Séptico/prevención & control , Animales , Modelos Animales de Enfermedad , Enterotoxinas/inmunología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Superantígenos/sangre , Superantígenos/inmunologíaRESUMEN
iNKT cells and mast cells have both been implicated in the syndrome of allergic asthma through their activation-induced release of Th2 type cytokines and secretion of histamine and other mediators, respectively, which can promote airways hyperresponsiveness (AHR) to agents such as methacholine. However, a mechanistic link between iNKT cells and mast cell recruitment or activation has never been explored. Our objective was to determine whether iNKT cells are necessary for the recruitment of mast cells and if iNKT cells can influence the acute allergen induced bronchoconstriction (AIB) caused by mast cell mediator release. To do so, we pharmacologically eliminated iNKT cells using a specific antibody (NKT-14) and examined its impact on airway inflammation and physiological phenotype. In mice treated with NKT-14, the elimination of iNKT cells was sufficient to prevent AHR and pulmonary eosinophilic inflammation elicited by administration of the iNKT cell agonist αGalCer. In mice treated with NKT-14 and then sensitized and challenged with house dust mite extract (HDM), eliminating the iNKT cells significantly reduced both AHR and AIB but did not affect pulmonary inflammation, the mast cell population, nor the release of the mast cell mediators mast cell protease-1 and prostaglandin D2. We conclude that while iNKT cells contribute to the phenotype of allergic airways disease through the manifestation of AIB and AHR, their presence is not required for mast cell recruitment and activation, or to generate the characteristic inflammatory response subsequent to allergen challenge.
Asunto(s)
Broncoconstricción/inmunología , Mastocitos/metabolismo , Células T Asesinas Naturales/metabolismo , Hipersensibilidad Respiratoria/inmunología , Alérgenos/inmunología , Animales , Quimasas/metabolismo , Modelos Animales de Enfermedad , Eosinófilos/metabolismo , Femenino , Hipersensibilidad/inmunología , Inflamación/inmunología , Pulmón/inmunología , Pulmón/patología , Mastocitos/inmunología , Ratones , Ratones Endogámicos BALB C , Células T Asesinas Naturales/inmunología , Fenotipo , Prostaglandina D2/metabolismo , Pyroglyphidae/inmunologíaRESUMEN
OBJECTIVE: Aptamers are oligonucleotides targeting protein-protein interactions with pharmacokinetic profiles and activity reversal options. Although P-selectin and von Willebrand factor (vWF) have been implicated in the development of venous thrombosis (VT), no studies have directly compared aptamer efficacy with standard of care in VT. In this study, ARC5692, an anti-P-selectin aptamer, and ARC15105, an anti-vWF aptamer, were compared with low-molecular-weight heparin, enoxaparin, to test the efficacy of P-selectin or vWF inhibition in promoting thrombus resolution and preventing vein wall fibrosis, in a baboon model of VT. APPROACH AND RESULTS: Groups were as follows: treatment arm: animals received P-selectin or vWF aptamer inhibitors or enoxaparin (n=3 per group). Controls received no treatment (n=3). Prophylactic arm: animals received P-selectin inhibitor (n=4) or vWF inhibitor (n=3). Treatment arm: P-selectin-inhibitor demonstrated a significant improvement in vein recanalization by magnetic resonance venography (73% at day 21), and significantly decreased vein wall collagen, compared with all groups. Anti-P-selectin equaled enoxaparin in maintaining valve competency by ultrasound. All control animals had compromised valve competency post thrombosis. Prophylactic arm: animals receiving P-selectin and vWF inhibitors demonstrated improved vein recanalization by magnetic resonance venography versus controls (80% and 85%, respectively, at day 21). Anti-P-selectin protected iliac valve function better than anti-vWF, and both improved valve function versus controls. No adverse bleeding events were observed. CONCLUSIONS: The P-selectin inhibitor aptamer promoted iliac vein recanalization, preserved valve competency, and decreased vein wall fibrosis. The results of this work suggest that P-selectin inhibition maybe an ideal target in the treatment and prophylaxis of deep VT, warranting clinical trials.
Asunto(s)
Aptámeros de Nucleótidos/farmacología , Enoxaparina/farmacología , Fibrinolíticos/farmacología , Vena Ilíaca/efectos de los fármacos , Selectina-P/antagonistas & inhibidores , Trombosis de la Vena/prevención & control , Factor de von Willebrand/antagonistas & inhibidores , Animales , Coagulación Sanguínea/efectos de los fármacos , Colágeno/metabolismo , Modelos Animales de Enfermedad , Fibrina/metabolismo , Fibrosis , Vena Ilíaca/diagnóstico por imagen , Vena Ilíaca/metabolismo , Vena Ilíaca/patología , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Angiografía por Resonancia Magnética , Selectina-P/metabolismo , Papio , Flebografía/métodos , Agregación Plaquetaria/efectos de los fármacos , Factores de Tiempo , Ultrasonografía , Trombosis de la Vena/diagnóstico por imagen , Trombosis de la Vena/metabolismo , Trombosis de la Vena/patología , Válvulas Venosas/efectos de los fármacos , Válvulas Venosas/metabolismo , Válvulas Venosas/patología , Factor de von Willebrand/metabolismoRESUMEN
Hemophilia A and B are caused by deficiencies in coagulation factor VIII (FVIII) and factor IX, respectively, resulting in deficient blood coagulation via the intrinsic pathway. The extrinsic coagulation pathway, mediated by factor VIIa and tissue factor (TF), remains intact but is negatively regulated by tissue factor pathway inhibitor (TFPI), which inhibits both factor VIIa and its product, factor Xa. This inhibition limits clot initiation via the extrinsic pathway, whereas factor deficiency in hemophilia limits clot propagation via the intrinsic pathway. ARC19499 is an aptamer that inhibits TFPI, thereby enabling clot initiation and propagation via the extrinsic pathway. The core aptamer binds tightly and specifically to TFPI. ARC19499 blocks TFPI inhibition of both factor Xa and the TF/factor VIIa complex. ARC19499 corrects thrombin generation in hemophilia A and B plasma and restores clotting in FVIII-neutralized whole blood. In the present study, using a monkey model of hemophilia, FVIII neutralization resulted in prolonged clotting times as measured by thromboelastography and prolonged saphenous-vein bleeding times, which are consistent with FVIII deficiency. ARC19499 restored thromboelastography clotting times to baseline levels and corrected bleeding times. These results demonstrate that ARC19499 inhibition of TFPI may be an effective alternative to current treatments of bleeding associated with hemophilia.
Asunto(s)
Aptámeros de Nucleótidos/farmacología , Coagulación Sanguínea/efectos de los fármacos , Hemostasis/efectos de los fármacos , Lipoproteínas/antagonistas & inhibidores , Animales , Aptámeros de Nucleótidos/química , Tiempo de Sangría , Modelos Animales de Enfermedad , Factor VIII/metabolismo , Factor VIIa/metabolismo , Factor Xa/metabolismo , Hemofilia A/sangre , Hemofilia A/tratamiento farmacológico , Hemofilia B/sangre , Hemofilia B/tratamiento farmacológico , Humanos , Técnicas In Vitro , Macaca fascicularis , Proteínas Recombinantes/antagonistas & inhibidores , Trombina/biosíntesis , Tromboplastina/metabolismoRESUMEN
Adhesive interactions between circulating sickle red blood cells (RBCs), leukocytes, and endothelial cells are major pathophysiologic events in sickle cell disease (SCD). To develop new therapeutics that efficiently inhibit adhesive interactions, we generated an anti-P-selectin aptamer and examined its effects on cell adhesion using knockout-transgenic SCD model mice. Aptamers, single-stranded oligonucleotides that bind molecular targets with high affinity and specificity, are emerging as new therapeutics for cardiovascular and hematologic disorders. In vitro studies found that the anti-P-selectin aptamer exhibits high specificity to mouse P-selectin but not other selectins. SCD mice were injected with the anti-P-selectin aptamer, and cell adhesion was observed under hypoxia. The anti-P-selectin aptamer inhibited the adhesion of sickle RBCs and leukocytes to endothelial cells by 90% and 80%, respectively. The anti-P-selectin aptamer also increased microvascular flow velocities and reduced the leukocyte rolling flux. SCD mice treated with the anti-P-selectin aptamer demonstrated a reduced mortality rate associated with the experimental procedures compared with control mice. These results demonstrate that anti-P-selectin aptamer efficiently inhibits the adhesion of both sickle RBCs and leukocytes to endothelial cells in SCD model mice, suggesting a critical role for P-selectin in cell adhesion. Anti-P-selectin aptamer may be useful as a novel therapeutic agent for SCD.
Asunto(s)
Anemia de Células Falciformes/fisiopatología , Aptámeros de Nucleótidos/farmacología , Adhesión Celular/efectos de los fármacos , Rodamiento de Leucocito/efectos de los fármacos , Selectina-P/antagonistas & inhibidores , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Técnica SELEX de Producción de Aptámeros , Resonancia por Plasmón de SuperficieRESUMEN
OBJECTIVE: We investigated the stability, pharmacokinetic, and pharmacodynamic profile of the 2(nd) generation anti-von Willeband factor aptamer ARC15105. METHODS AND RESULTS: Platelet plug formation was measured by collagen/adenosine diphosphate-induced closure time with the platelet function analyzer-100 and platelet aggregation by multiple electrode aggregometry. Platelet adhesion was measured on denuded porcine aortas and in a flow chamber. Aptamer stability was assessed by incubation in nuclease rich human, monkey, and rat serum for up to 72 hours. Pharmacokinetic and pharmacodynamic profiles were tested in cynomolgus monkeys after IV and SC administration. The median IC(100) and IC(50) to prolong collagen/adenosine diphosphate-induced closure timewere 27 nmol/L and 12 nmol/L, respectively. ARC15105 (1.3 µmol/L) completely inhibited ristocetin-induced platelet aggregation in whole blood (P<0.001), but also diminished collagen, ADP, arachidonic acid, and thrombin receptor activating peptide-induced platelet aggregation to some extent (P<0.05). ARC15105 (40 nmol/L) decreased platelet adhesion by >90% on denuded porcine aortas (P<0.001), which was comparable to the degree of inhibition obtained with abciximab. ARC15105 (100 nmol/L) also inhibited platelet adhesion to collagen under arterial shear in a flow chamber by >90% (P<0.001). The IV and SC terminal half-lives in cynomolgus monkeys were 67 and 65 hours, respectively, and the SC bioavailability was ≈98%. Allometric scaling estimates the human T(1/2) would be ≈217 hours. Pharmacodynamic analysis confirms that ARC15105 inhibits von Willeband factor activity >90% in blood samples taken 300 hours after a 20 mg/kg IV or SC dose in monkeys. CONCLUSIONS: The potency, pharmacokinetic profile, and SC bioavailability of ARC15105 support its clinical investigation for chronic inhibition of von Willeband factor -mediated platelet activation.
Asunto(s)
Aptámeros de Nucleótidos/uso terapéutico , Aptámeros de Péptidos/farmacología , Plaquetas/efectos de los fármacos , Infarto del Miocardio/sangre , Activación Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Factor de von Willebrand/antagonistas & inhibidores , Anciano , Animales , Aptámeros de Nucleótidos/administración & dosificación , Aptámeros de Nucleótidos/farmacocinética , Aptámeros de Péptidos/administración & dosificación , Aptámeros de Péptidos/farmacocinética , Austria , Disponibilidad Biológica , Plaquetas/metabolismo , Boston , Estudios de Casos y Controles , Colágeno/metabolismo , Estudios Transversales , Relación Dosis-Respuesta a Droga , Estabilidad de Medicamentos , Femenino , Semivida , Humanos , Inyecciones Intravenosas , Inyecciones Subcutáneas , Macaca fascicularis , Masculino , Persona de Mediana Edad , Inhibidores de Agregación Plaquetaria/administración & dosificación , Inhibidores de Agregación Plaquetaria/farmacocinética , Pruebas de Función Plaquetaria , Unión Proteica , Quebec , Ratas , Porcinos , Factor de von Willebrand/metabolismoRESUMEN
Invariant natural killer T-lymphocytes (iNKT) are unique immunomodulatory innate T cells with an invariant TCRα recognizing glycolipids presented on MHC class-I-like CD1d molecules. Activated iNKT rapidly secrete pro-and anti-inflammatory cytokines, potentiate immunity, and modulate inflammation. Here, we report the effects of in vivo iNKT activation in Mauritian-origin cynomolgus macaques by a humanized monoclonal antibody, NKTT320, that binds to the invariant region of the iNKT TCR. NKTT320 led to rapid iNKT activation, increased polyfunctionality, and elevation of multiple plasma analytes within 24 hours. Flow cytometry and RNA-Seq confirmed downstream activation of multiple immune subsets, enrichment of JAK/STAT and PI3K/AKT pathway genes, and upregulation of inflammation-modulating genes. NKTT320 also increased iNKT frequency in adipose tissue and did not cause iNKT anergy. Our data indicate that NKTT320 has a sustained effect on in vivo iNKT activation, potentiation of innate and adaptive immunity, and resolution of inflammation, which supports its future use as an immunotherapeutic.
RESUMEN
High plasma levels of soluble P-selectin are associated with thrombotic disorders and may predict future cardiovascular events. Mice with high levels of soluble P-selectin have more microparticles in their plasma than do normal mice. Here we show that chimeras of P-selectin and immunoglobulin (P-sel-Ig) induced formation of procoagulant microparticles in human blood through P-selectin glycoprotein ligand-1 (PSGL-1; encoded by the Psgl1 gene, officially known as Selpl). In addition, Psgl1-/- mice produced fewer microparticles after P-sel-Ig infusion and did not spontaneously increase their microparticle count in old age as do wild-type mice. Injected microparticles specifically bound to thrombi and thus could be involved in thrombin generation at sites of injury. Infusion of P-sel-Ig into hemophilia A mice produced a 20-fold increase over control immunoglobulin in microparticles containing tissue factor. This significantly improved the kinetics of fibrin formation in the hemophilia A mice and normalized their tail-bleeding time. P-sel-Ig treatment could become a new approach to sustained control of bleeding in hemophilia.
Asunto(s)
Hemofilia A/terapia , Hemostasis/fisiología , Glicoproteínas de Membrana/metabolismo , Selectina-P/metabolismo , Adulto , Animales , Coagulación Sanguínea/fisiología , Modelos Animales de Enfermedad , Factor VIII/metabolismo , Hemofilia A/metabolismo , Humanos , Inmunoglobulinas/genética , Inmunoglobulinas/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Persona de Mediana Edad , Selectina-P/genética , Fenotipo , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
BACKGROUND: In thrombotic thrombocytopenic purpura (TTP), ultralarge von Willebrand factor (VWF) multimers bind platelet (PLT) glycoprotein Ib and lead to the formation of disseminated fibrin-poor, VWF-rich PLT thrombi. The aptamer ARC1779 blocks binding of the VWF A1 domain to PLT glycoprotein Ib. We evaluated whether ARC1779 inhibits the excessive VWF activity and VWF-mediated PLT function in patients with TTP. STUDY DESIGN AND METHODS: We studied the ex vivo concentration response curves for ARC1779 on PLT function analyzer (PFA-100, Dade Behring) and cone-and-plate analyzer (CPA, Impact-R) PLT function tests, agonist-induced PLT aggregation, and VWF activity of TTP patients (n = 11, three in acute phase and eight in remission) and healthy controls (n = 44). RESULTS: VWF activity and VWF-dependent PLT plug formation were increased in TTP patients relative to healthy controls, but agonist-induced PLT aggregation was not. ARC1779 blocked collagen/adenosine 5'-diphosphate (ADP)-induced PLT plug formation as measured by PFA-100 with an inhibitory concentration (IC)(100) of approximately 1 microg/mL in citrate-anticoagulated samples and approximately 3 to 4 microg/mL in hirudin-anticoagulated samples. A similar concentration of ARC1779 was necessary to block shear-dependent PLT adhesion in both TTP patients and healthy controls using the CPA assay (IC(100) of approx. 1 microg/mL for both). ARC1779 blocked VWF activity with an IC(90) of approximately 3 to 4 microg/mL in all subjects, but did not inhibit PLT aggregation by ADP, collagen, or arachidonic acid even at concentrations much greater than those that fully inhibited VWF-dependent PLT function. CONCLUSIONS: ARC1779 potently and specifically inhibits VWF activity and VWF-dependent PLT function. ARC1779 may be a promising novel therapeutic for the treatment of TTP.
Asunto(s)
Plaquetas/efectos de los fármacos , Púrpura Trombocitopénica Trombótica/tratamiento farmacológico , Factor de von Willebrand/antagonistas & inhibidores , Adulto , Aptámeros de Nucleótidos , Plaquetas/fisiología , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Agregación Plaquetaria/efectos de los fármacos , Púrpura Trombocitopénica Trombótica/sangreRESUMEN
The first therapeutic aptamer was approved for human use in 2004, and a range of chemical substitutions that improve the drug-like properties of aptamers has recently been shown to increase the utility of this modality. Currently there are both anticoagulant and antithrombotic aptamers in the clinic, and additionally there are a number of earlier stage projects in which a variety of cardiovascular targets are inhibited by specific aptamers and for which a wide range of therapeutic applications has been suggested.
Asunto(s)
Aptámeros de Nucleótidos/farmacología , Fármacos Cardiovasculares/farmacología , Enfermedades Cardiovasculares/tratamiento farmacológico , Animales , Anticoagulantes/farmacología , Aptámeros de Nucleótidos/uso terapéutico , Fármacos Cardiovasculares/uso terapéutico , Enfermedades Cardiovasculares/fisiopatología , Adhesión Celular/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Fibrinolíticos/farmacología , HumanosRESUMEN
ARC1779 is an aptamer, which blocks binding of the von Willebrand Factor (VWF) A1 domain to platelet GPIb receptors. VWF is increased in the elderly an in the setting of acute myocardial infarction (AMI), as reflected by increased shear-dependent platelet function. We hypothesized that ARC1779 concentration-dependently inhibits ex vivo platelet function, and that this concentration effect relationship may be shifted in patients with AMI. We studied ex vivo dose response curves for ARC1779 on VWF activity, shear-dependent platelet function, and agonist-induced platelet aggregation. We included patients with AMI on standard treatment (n = 40), young (n = 20) and elderly controls (n = 20) in this ex vivo dosing study. AMI patients displayed approximately 2-fold increased plasma levels of VWF activity as compared to controls. ARC1779 inhibited VWF activity (IC90: approximately 3-4 microg/mL) and shear dependent platelet function (Platelet Function Analyzer (PFA-100), IC50: approximately 0.5-0.9 microg/mL and Cone and Plate Analyzer (CPA), IC50: approximately 0.1-0.4 microg/mL in citrated blood) at comparable concentrations in all groups. In contrast to GPIIb/IIIa antagonists, ARC1779 did not inhibit platelet aggregation induced by ADP, collagen or arachidonic acid at concentrations (10 microg/mL) that fully inhibited VWF dependent platelet function. ARC1779 potently and specifically inhibits VWF activity and VWF dependent platelet function, even in the setting of AMI where VWF activity is increased.
Asunto(s)
Aptámeros de Nucleótidos/uso terapéutico , Infarto del Miocardio/sangre , Agregación Plaquetaria/efectos de los fármacos , Factor de von Willebrand/antagonistas & inhibidores , Adulto , Factores de Edad , Anciano , Aptámeros de Nucleótidos/farmacología , Células Sanguíneas , Plaquetas/fisiología , Estudios de Casos y Controles , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Infarto del Miocardio/tratamiento farmacológico , Adulto JovenRESUMEN
Invariant natural killer T (iNKT) cells are a small population of T lymphocytes that expresses an invariant T cell receptor with a unique specificity for glycolipid antigens. Their activation using the glycolipid α-galactosylceramide (α-GalCer) triggers innate and adaptive immune responses. The use of α-GalCer in preclinical models as a single antitumor treatment showed moderate effect, but its efficacy in cancer patients was less effective. In addition, this glycolipid induces long-term iNKT-cell anergy precluding the possibility of retreatment. Recently, the first murine iNKT-cell agonistic antibody, NKT14m, has been developed. Here, we analyzed, for the first time, the antitumor efficacy of NKT14m in a B-cell lymphoma model. In a therapeutic setting, a single dose of NKT14m had a moderate antitumor efficacy that was associated with an increase of IFN-γ producing iNKT cells even after a second dose of the NKT14m antibody. Importantly, the combination of a single dose of NKT14m with cyclophosphamide had a potent antitumor efficacy and long-lasting immunity in vivo. Our findings provide the first evidence of the in vivo antitumor efficacy of NKT14m antibody, showing that, either alone or in combination with chemotherapy, induces an effective antitumor response. These results open new opportunities for iNKT-cell mediated immunotherapy to treat B-cell lymphoma.
RESUMEN
BACKGROUND: ARC1779 is a therapeutic aptamer antagonist of the A1 domain of von Willebrand Factor (vWF), the ligand for receptor glycoprotein 1b on platelets. ARC1779 is being developed as a novel antithrombotic agent for use in patients with acute coronary syndromes. METHODS AND RESULTS: This was a randomized, double-blind, placebo-controlled study in 47 healthy volunteers of doses of ARC1779 from 0.05 to 1.0 mg/kg. Pharmacodynamic effects were measured by an ELISA for free vWF A1 binding sites and by a platelet function analyzer. In terms of pharmacokinetics, the concentration-time profile of ARC1779 appeared monophasic. The observed concentration and area under the curve were dose proportional. The mean apparent elimination half-life was approximately 2 hours, and mean residence time was approximately 3 hours. The mean apparent volumes of distribution (at steady state and during terminal phase) were approximately one half the blood volume, suggesting that ARC1779 distribution is in the central compartment. The mean clearance ranged from approximately 10% to approximately 21% of the glomerular filtration rate, suggesting that renal filtration may not be a major mechanism of clearance of ARC1779. Inhibition of vWF A1 binding activity was achieved with an EC(90) value of 2.0 mug/mL (151 nmol/L) and of platelet function with an EC(90) value of 2.6 mug/mL (196 nmol/L). ARC1779 was generally well tolerated, and no bleeding was observed. Adverse events tended to be minor and not dose related. CONCLUSIONS: This is the first-in-human evaluation of a novel aptamer antagonist of vWF. ARC1779 produced dose- and concentration-dependent inhibition of vWF activity and platelet function with duration of effect suitable for the intended clinical use in acute coronary syndromes.
Asunto(s)
Síndrome Coronario Agudo/tratamiento farmacológico , Aptámeros de Nucleótidos/farmacocinética , Fibrinolíticos/farmacocinética , Factor de von Willebrand/antagonistas & inhibidores , Adolescente , Adulto , Anciano , Aptámeros de Nucleótidos/efectos adversos , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Femenino , Fibrinolíticos/efectos adversos , Humanos , Masculino , Persona de Mediana Edad , Pruebas de Función Plaquetaria , Complejo GPIb-IX de Glicoproteína Plaquetaria/agonistas , Estructura Terciaria de Proteína , Factores de TiempoRESUMEN
P-selectin plays a significant and well documented role in vascular disease by mediating leukocyte and platelet rolling and adhesion. This study characterizes the in vitro activity, pharmacokinetic properties, and the anti-inflammatory and antithrombotic efficacy of the orally active P-selectin small-molecule antagonist PSI-697 [2-(4-chlorobenzyl)-3-hydroxy-7,8,9,10-tetrahydrobenzo[h] quinoline-4-carboxylic acid; molecular mass, 367.83]. Biacore and cell-based assays were used to demonstrate the ability of PSI-697 to dose dependently inhibit the binding of human P-selectin to human P-selectin glycoprotein ligand-1, inhibiting 50% of binding at 50 to 125 microM. The pharmacokinetics of PSI-697 in rats were characterized by low clearance, short half-life, low volume of distribution, and moderate apparent oral bioavailability. A surgical inflammation model, using exteriorized rat cremaster venules, demonstrated that PSI-697 (50 mg/kg p.o.) significantly reduced the number of rolling leukocytes by 39% (P < 0.05) versus vehicle control. In a rat venous thrombosis model, PSI-697 (100 mg/kg p.o.) reduced thrombus weight by 18% (P < 0.05) relative to vehicle, without prolonging bleeding time. Finally, in a rat carotid injury model, PSI-697 (30 or 15 mg/kg p.o.) administered 1 h before arterial injury and once daily thereafter for 13 days resulted in dose-dependent decreases in intima/media ratios of 40.2% (P = 0.025) and 25.7% (P = 0.002) compared with vehicle controls. These data demonstrate the activity of PSI-697 in vitro and after oral administration in animal models of both arterial and venous injury and support the clinical evaluation of this novel antagonist of P-selectin in atherothrombotic and venous thrombotic indications.
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Modelos Animales de Enfermedad , Hidroxiquinolinas/uso terapéutico , Selectina-P , Vasculitis/tratamiento farmacológico , Trombosis de la Vena/tratamiento farmacológico , Animales , Células HL-60 , Humanos , Hidroxiquinolinas/química , Hidroxiquinolinas/farmacología , Masculino , Selectina-P/metabolismo , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Ratas , Ratas Sprague-Dawley , Vasculitis/metabolismo , Trombosis de la Vena/metabolismoRESUMEN
This study aimed to evaluate a small-molecule PAI-1 inhibitor (PAI-039; tiplaxtinin) in a rodent stenosis model of venous thrombosis in a two-phase experiment. Phase 1 determined the efficacy of tiplaxtinin against Lovenox (LOV), while phase 2 determined the dose-dependent efficacy. For both phases, drug treatment began 24 hours after surgically induced venous thrombosis and continued for four days. Phase 1 animals (n = 24) receiving low-dose (LD; 1 mg/kg oral gavage) PAI-1 inhibitor demonstrated a 52% decrease in thrombus weight (TW) versus controls (p < 0.05) with significant reductions in active plasma PAI-1, while the high-dose (HD; 10 mg/kg oral gavage) group demonstrated a 23% reduction in TW versus controls. Animals treated subcutaneously with LOV (3 mg/kg) showed a 39% decrease in TW versus controls (p < 0.05). Coagulation tests (aPTT and TCT) were significantly different in LOV compared to PAI-1 inhibitor groups. PAI-039 treatment was also associated with significantly increased return of inferior vena cava blood flow four days post-thrombosis versus controls (p < 0.05). In phase 2 (n = 30), TW was reduced from the 0.5 mg/kg to 5 mg/kg experimental groups, with the 10 mg/kg group demonstrating a paradoxical increase. The 5 mg/kg group showed statistically significant decreases in TW versus controls after four treatment days (p < 0.05). This is the first study to demonstrate dose related effects of PAI-039 on increasing thrombus resolution and inferior vena cava blood flow without adverse effects on anti-coagulation in a rat stenosis model of venous thrombosis.
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Ácidos Indolacéticos/administración & dosificación , Inhibidor 1 de Activador Plasminogénico/sangre , Trombosis de la Vena/sangre , Trombosis de la Vena/tratamiento farmacológico , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Enoxaparina/antagonistas & inhibidores , Fibrosis , Inflamación/tratamiento farmacológico , Inflamación/patología , Masculino , Ratas , Ratas Sprague-Dawley , Túnica Íntima/patología , Vena Cava Inferior/patología , Trombosis de la Vena/patologíaRESUMEN
P-selectin inhibition has been evaluated as a therapeutic for prevention and treatment of venous thrombosis. In this study, a novel oral small-molecule inhibitor of P-selectin, PSI-421, was evaluated in a baboon model of stasis induced deep vein thrombosis (DVT). Experimental groups included i) primates receiving a single oral dose of 1 mg/kg PSI-421 two days prior and continued six days after thrombosis (n = 3); ii) primates receiving a single daily subcutaneous dose of 0.57 mg/kg enoxaparin sodium two days prior and continued six days post thrombosis (n = 3); and iii) primates receiving no treatment (n = 3). PSI-421 treated primates had greater percent vein reopening and less vein wall inflammation than the enoxaparin and controls at day 6. Microparticle tissue factor activity (MPTFA) was significantly lower in the animals receiving PSI-421 immediately after thrombosis (T+6 hours day 0) suggesting lower potential for thrombogenesis in these animals. PSI-421 also reduced soluble P-selectin levels versus controls at T+6 hours day 0, day 2 and 6. Experimental animals in any group showed no adverse effects on coagulation. This study is the first to demonstrate a reduction in MPTFA associated with vein reopening and reduced vein inflammation due to oral P-selectin inhibition in a baboon model of DVT.
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Anticoagulantes/farmacología , Enoxaparina/farmacología , Fibrinolíticos/farmacología , Hidroxiquinolinas/farmacología , Vena Ilíaca/efectos de los fármacos , Selectina-P/efectos de los fármacos , Trombosis de la Vena/prevención & control , Administración Oral , Animales , Anticoagulantes/administración & dosificación , Coagulación Sanguínea/efectos de los fármacos , Plaquetas/efectos de los fármacos , Modelos Animales de Enfermedad , Enoxaparina/administración & dosificación , Inhibidores del Factor Xa , Fibrinolíticos/administración & dosificación , Fibrinolíticos/sangre , Hidroxiquinolinas/administración & dosificación , Hidroxiquinolinas/sangre , Vena Ilíaca/metabolismo , Vena Ilíaca/patología , Vena Ilíaca/fisiopatología , Inflamación/metabolismo , Inflamación/prevención & control , Inyecciones Subcutáneas , Masculino , Selectina-P/metabolismo , Papio anubis , Flebografía , Tromboplastina/metabolismo , Factores de Tiempo , Ultrasonografía Doppler en Color , Grado de Desobstrucción Vascular , Trombosis de la Vena/sangre , Trombosis de la Vena/metabolismo , Trombosis de la Vena/patología , Trombosis de la Vena/fisiopatologíaRESUMEN
P-selectin-PSGL-1 interaction causes rolling of leukocytes on the endothelial cell surface, which subsequently leads to firm adherence and leukocyte transmigration through the vessel wall into the surrounding tissues. P-selectin is upregulated on the surface of both platelets and endothelium in a variety of atherosclerosis-associated conditions. Consequently, inhibition of this interaction by means of a small molecule P-selectin antagonist is an attractive strategy for the treatment of atherosclerosis. High-throughput screening and subsequent analoging had led to the identification of compound 1 as the lead candidate. Herein, we report the continuation of this work and the discovery of a second-generation series, the tetrahydrobenzoquinoline salicylic acids. These compounds have improved pharmacokinetic properties, and a number of them have shown oral efficacy in mouse and rat models of atherogenesis and vascular injury. The lead 31 (PSI-697), is currently in clinical development for the treatment of atherothrombotic vascular events.
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Aterosclerosis/prevención & control , Fibrinolíticos/síntesis química , Hidroxiquinolinas/síntesis química , Selectina-P/metabolismo , Quinolinas/síntesis química , Salicilatos/síntesis química , Administración Oral , Animales , Apolipoproteínas E/genética , Estenosis Carotídea/prevención & control , Perros , Fibrinolíticos/química , Fibrinolíticos/farmacología , Hidroxiquinolinas/farmacocinética , Hidroxiquinolinas/farmacología , Indoles/química , Indoles/farmacocinética , Indoles/farmacología , Rodamiento de Leucocito/efectos de los fármacos , Masculino , Ratones , Ratones Noqueados , Quinolinas/farmacocinética , Quinolinas/farmacología , Ratas , Ratas Sprague-Dawley , Salicilatos/farmacocinética , Salicilatos/farmacología , Relación Estructura-ActividadRESUMEN
The interaction between von Willebrand factor (VWF) and platelet glycoprotein Ibalpha (GPIbalpha) is a critical step that allows platelet adhesion, activation and subsequent thrombus formation to the injured vessel wall under high-shear conditions. In this study, we sought to investigate 1) whether GPG-290, a recombinant human GPIbalpha chimeric protein, would prevent thrombosis in a canine model of coronary thrombosis by blocking VWF-GPIbalpha interaction; and 2) whether desmopressin (DDAVP), a VWF release stimulant, could reduce the prolonged bleeding time caused by a 10x efficacious dose of GPG-290. The antithrombotic efficacy of GPG-290 was evaluated by the in-vivo ability to prevent cyclic flow reductions (CFRs) and ex-vivo inhibition of platelet adhesion/aggregation reflected by prolongation of Platelet Function Analyzer (PFA-100) collagen/ADP closure time. The anti-hemostatic effect was assessed by template bleeding time. GPG-290 at doses of 25, 50 and 100 microg/kg abolished CFRs in 67%, 100% and 100% of the treated dogs without bleeding time prolongation, respectively; GPG-290 dose-dependently prolonged the ex-vivo collagen/ADP-closure time, while it had no effects on plasma VWF antigen level (VWF:Ag) and VWF-collagen binding activity (VWF:CB); the prolonged template bleeding time caused by 500 microg/kg of GPG-290 was prevented by intravenous infusion of DDAVP (0.3 microg/kg). In conclusion, GPG-290 appears to be an effective agent for treating arterial thrombosis without bleeding time prolongation.