RESUMEN
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
BACKGROUND: In recent years, a number of tissue factor pathway inhibitor (TFPI) antagonists have been developed to serve as bypassing agents to improve hemostasis in hemophilia A. Since TFPI antagonists and FVIII concentrates are procoagulants, their combined effect on spatial clot formation could be potentially pro-thrombotic. OBJECTIVE: To investigate the cooperative effect of TFPI inhibition and supplementation of FVIII in hemophilia A in a spatial, reaction-diffusion experiment in vitro. METHODS: Plasma was collected at different time points from hemophilia A patients undergoing prophylaxis and was supplemented in vitro with TFPI inhibitor BAX499 (formerly ARC19499) at concentrations from 0 up to 600nM. Clotting propagation in recalcified plasma activated by a surface with immobilized tissue factor (TF) was monitored by videomicroscopy. RESULTS: Increasing concentration of BAX499 improved coagulation for all hemophilia A plasma samples activated with TF at 1.6pmole/m(2) by shortening lag time and increasing initial clot growth velocity and clot size. In contrast, plasma concentration of FVIII had little effect on lag time, but increased spatial clot growth velocity. There was a decrease in the BAX499 efficiency as FVIII concentration increased (lag time shortened by 50% if FVIII:C<5%, but the effect was only 25% if FVIII:C>30%). CONCLUSIONS: The results indicate that BAX499 has an effect on clotting in hemophilia A plasma at low FVIII concentrations, however has little effect at high FVIII concentrations.
Asunto(s)
Aptámeros de Nucleótidos/farmacología , Factor VIII/farmacología , Fibrina/metabolismo , Hemofilia A/sangre , Hemofilia A/tratamiento farmacológico , Aptámeros de Nucleótidos/farmacocinética , Interacciones Farmacológicas , Factor VIII/farmacocinética , Humanos , Lipoproteínas/antagonistas & inhibidoresRESUMEN
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
Hemophilia is a family of rare bleeding disorders. The two primary types, hemophilia A and hemophilia B, are caused by recessive X-chromosome linked mutations that result in deficiency of coagulation factor VIII (FVIII) or factor IX (FIX), respectively. Clinically, hemophilia is manifested by spontaneous bleeding, particularly into the joints (haemarthrosis) and soft tissue, and excessive bleeding following trauma or surgery. The total overall number of hemophilia patients worldwide is approximately 400,000, however only about 100,000 of these individuals are treated. The first treatment of hemophilia was initiated when it was determined that the clotting deficiency could be corrected by a plasma fraction taken from normal blood. The discovery of factor VIII enrichment by cryoprecipitation of plasma opened a new era of therapy which eventually led to the production of factor concentrates and the subsequent development of highly purified forms of plasma factors. The most significant improvements have been the availability of recombinant forms of factors VIII and IX. Unfortunately, recombinant factors still retain some of the limitations of plasma concentrates. These limitations include development of antibody responses in patients and the relatively short half-life of the molecules requiring frequent injection to maintain effective concentration. Treatment beyond replacement of native factors has been tried. They include the development of modified factor VIII and IX molecules with improved potency, stability and circulating half-life and enhancement of a prothrombotic responses and/or stabilization of coagulation factors via inhibition of key negative regulatory pathways. These approaches will be reviewed in this commentary.
Asunto(s)
Factores de Coagulación Sanguínea/orina , Hemofilia A/tratamiento farmacológico , Animales , Factor IX/uso terapéutico , Factor VIII/uso terapéutico , Factor VIIa/uso terapéutico , Factor Xa/genética , Humanos , Lipoproteínas/antagonistas & inhibidores , Inhibidor de Proteína C/uso terapéutico , Proteínas Recombinantes/uso terapéuticoRESUMEN
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
The von Willebrand factor (vWF) aptamer, ARC1779 that blocks the binding of vWF A1-domain to platelet glycoprotein 1b (GPIb) at high shear, may deliver a site-specific antithrombotic effect. We investigated the efficiency of ARC1779 on platelet function in patients with coronary artery disease (CAD) on double antiplatelet therapy. Blood from patients taking aspirin and clopidogrel and from normal volunteers was treated ex vivo with ARC1779 or abciximab, either prior to perfusion (pretherapy) or 10 minutes following the initiation of perfusion (posttherapy) on damaged arteries. Under pre- but not posttherapy, platelet adhesion was significantly reduced by ARC1779 at 83 and 250 nmol/L and by abciximab (100 nmol/L) versus placebo (4.8, 3.8, and 2.9 vs 7.3 platelets × 10(6)/cm(2), P < .05). In contrast to abciximab, ARC1779 did not significantly affect platelet aggregation, P-selectin expression, and platelet-leukocyte binding. These proof-of-concept data may constitute the framework for randomized clinical investigations of this novel antiplatelet therapy among patients with CAD.
Asunto(s)
Aptámeros de Nucleótidos/uso terapéutico , Aspirina/uso terapéutico , Enfermedad de la Arteria Coronaria/tratamiento farmacológico , Adhesividad Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/uso terapéutico , Trombosis/tratamiento farmacológico , Ticlopidina/análogos & derivados , Abciximab , Adulto , Anticuerpos Monoclonales/uso terapéutico , Clopidogrel , Enfermedad de la Arteria Coronaria/sangre , Femenino , Humanos , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Masculino , Persona de Mediana Edad , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Trombosis/sangre , Ticlopidina/uso terapéutico , Factor de von Willebrand/antagonistas & inhibidores , Factor de von Willebrand/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
Previously, we reported the discovery of PSI-697 (1a), a C-2 benzyl substituted quinoline salicylic acid-based P-selectin inhibitor. It is active in a variety of animal models of cardiovascular disease. Compound 1a has also been shown to be well tolerated and safe in healthy volunteers at doses of up to 1200 mg in a phase 1 single ascending dose study. However, its oral bioavailability was low. Our goal was to identify a back up compound with equal potency, increased solubility, and increased exposure. We expanded our structure-activity studies in this series by branching at the alpha position of the C-2 benzyl side chain and through modification of substituents on the carboxylic A-ring of the quinoline. This resulted in discovery of PSI-421 with marked improvement in aqueous solubility and pharmacokinetic properties. This compound has shown oral efficacy in animal models of arterial and venous injury and was selected as a preclinical development compound for potential treatment of such diseases as atherosclerosis and deep vein thrombosis.
Asunto(s)
Traumatismos de las Arterias Carótidas/tratamiento farmacológico , Hidroxiquinolinas/síntesis química , Selectina-P/antagonistas & inhibidores , Salicilatos/síntesis química , Trombosis de la Vena/tratamiento farmacológico , Administración Oral , Animales , Células CACO-2 , Permeabilidad de la Membrana Celular , Perros , Estabilidad de Medicamentos , Humanos , Hidroxiquinolinas/farmacocinética , Hidroxiquinolinas/farmacología , Rodamiento de Leucocito/efectos de los fármacos , Macaca fascicularis , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Papio , Ratas , Ratas Sprague-Dawley , Salicilatos/química , Salicilatos/farmacología , Solubilidad , Relación Estructura-ActividadRESUMEN
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
BACKGROUND: P-selectin antagonism has been shown to decrease thrombogenesis and inflammation in animal models of deep venous thrombosis (DVT). OBJECTIVE: To determine the effectiveness of P-selectin inhibitors versus saline and enoxaparin in venous thrombus resolution in nonhuman primate models of venous thrombosis. METHODS: Studies reporting vein re-opening, inflammation expressed as Gadolinium enhancement and coagulation parameters were searched in the literature and pooled into a meta-analysis using an inverse variance with random effects. RESULTS: Five studies were identified comparing P-selectin/ PSGL-1 inhibitors versus saline or enoxaparin regarding venous thrombosis resolution. Vein re-opening was significantly higher on P-selectin/ PSGL-1 compounds, when compared to saline (Inverse Variance [IV] 95% CI; 44.37 [17.77-70.96], p=0.001, I(2)=97%) and similar to enoxaparin (IV 95% CI; 5.03 [-8.88-18.95], p=0.48, I(2)=41%). Inflammation, reflected as Gadolinium enhancement at magnetic resonance venography (MRV), was significantly decreased in the P-selectin treated group when compared to saline (IV 95% CI; -17.84 [-14.98-(-8.30)], p<0.00001, I(2)=80%). No significant differences on vein wall inflammation were observed between P-selectin/ PSGL-1 inhibitors and enoxaparin treated animals (IV95% CI; -3.59 [-10.67-3.48], p=0.32, I(2)=66%). In addition, there was no differences in the coagulation parameters (aPTT, TCT, BT, D-Dimer, fibrinogen, platelets) between P-selectin/ PSGL-1 inhibitors and enoxaparin (IV 95% CI; -1.12[-2.36-0.11], p=0.07, I(2)=92%), although there was a trend showing less of a prolongation in TCT with P-selectin/PSGL-1 inhibitors compared to enoxaparin (p<0.0001). CONCLUSION: P-selectin antagonism successfully paralleled the low-molecular-weight-heparin enoxaparin, for the treatment of DVT in nonhuman primate models, by decreasing both thrombus burden and inflammation without causing any bleeding complications and without increasing coagulation times.
Asunto(s)
Enoxaparina/farmacología , Selectina-P/efectos de los fármacos , Selectina-P/inmunología , Trombosis/tratamiento farmacológico , Trombosis de la Vena/patología , Animales , Coagulación Sanguínea/efectos de los fármacos , Coagulación Sanguínea/inmunología , Pruebas de Coagulación Sanguínea , Plaquetas/efectos de los fármacos , Plaquetas/inmunología , Plaquetas/patología , Enoxaparina/inmunología , Productos de Degradación de Fibrina-Fibrinógeno/metabolismo , Gadolinio/farmacología , Hemorragia/complicaciones , Hemorragia/tratamiento farmacológico , Hemorragia/inmunología , Heparina de Bajo-Peso-Molecular/inmunología , Heparina de Bajo-Peso-Molecular/farmacología , Inflamación/complicaciones , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Angiografía por Resonancia Magnética/efectos adversos , Selectina-P/farmacología , Tiempo de Tromboplastina Parcial , Flebografía/efectos adversos , Ratas , Selectinas/inmunología , Selectinas/farmacología , Tiempo de Trombina , Trombosis/complicaciones , Trombosis/inmunología , Venas/efectos de los fármacos , Venas/inmunología , Venas/patología , Trombosis de la Vena/sangre , Trombosis de la Vena/etiologíaRESUMEN
ARC1172 is a 41-mer DNA aptamer selected to bind the A1 domain of von Willebrand factor (VWF). A derivative of ARC1172 with modifications to increase intravascular survival inhibits carotid artery thrombosis in a Cynomolgus macaque model and inhibits VWF-dependent platelet aggregation in humans, suggesting that such aptamers may be useful to prevent or treat thrombosis. In the crystal structure of a VWF A1-ARC1172 complex, the aptamer adopts a three-stem structure of mainly B-form DNA with three noncanonical base pairs and 9 unpaired residues, 6 of which are stabilized by base-base or base-deoxyribose stacking interactions. The aptamer-protein interface is characterized by cation-pi interactions involving Arg, Lys, and Gln residues, often stabilized by H-bonds with adjacent bases. The ARC1172 binding site on the A1 domain overlaps with that of botrocetin and clashes with glycoprotein Ibalpha binding at an adjacent site, which accounts for the antithrombotic activity of ARC1172 and related aptamers.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Fibrinolíticos/metabolismo , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Factor de von Willebrand/metabolismo , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Sitios de Unión/genética , Cristalografía , Fibrinolíticos/química , Fibrinolíticos/farmacología , Humanos , Complejos Multiproteicos/química , Unión Proteica , Factor de von Willebrand/químicaRESUMEN
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
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.
Asunto(s)
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
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.
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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
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
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.
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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
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.
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Trombosis Coronaria/prevención & control , Proteínas de la Membrana/uso terapéutico , Activación Plaquetaria/efectos de los fármacos , Proteínas Recombinantes de Fusión/farmacología , Factor de von Willebrand/antagonistas & inhibidores , Animales , Tiempo de Sangría , Trombosis Coronaria/tratamiento farmacológico , Desamino Arginina Vasopresina/farmacología , Perros , Relación Dosis-Respuesta a Droga , Humanos , Glicoproteínas de Membrana , Pruebas de Función Plaquetaria , Complejo GPIb-IX de Glicoproteína Plaquetaria , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/uso terapéutico , Factor de von Willebrand/análisisRESUMEN
OBJECTIVES: We evaluated whether serum myeloperoxidase (MPO) levels are associated with the risk of future development of coronary artery disease (CAD) in apparently healthy individuals. BACKGROUND: An enzyme of the innate immune system, MPO exhibits a wide array of proatherogenic effects. These include induction of oxidative damage to low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol and promotion of plaque vulnerability. Recent studies revealed that MPO independently predicts adverse outcomes in patients with chest pain or suspected acute coronary syndrome. METHODS: Myeloperoxidase was measured in baseline samples of a case-control study nested in the prospective EPIC (European Prospective Investigation into Cancer and Nutrition)-Norfolk population study. Case subjects (n = 1,138) were apparently healthy men and women who developed CAD during 8-year follow-up. Control subjects (n = 2,237), matched for age, gender, and enrollment time, remained free of CAD. RESULTS: The MPO levels were significantly higher in case subjects than in control subjects and correlated with C-reactive protein (CRP) (rho = 0.25; p < 0.001) and white blood cell count (rho = 0.33; p < 0.001). Risk of future CAD increased in consecutive quartiles of MPO concentration, with an odds ratio (OR) of 1.49 in the top versus bottom quartile (95% confidence interval [CI] 1.20 to 1.84; p < 0.001). After adjustment for traditional risk factors, the OR in the top quartile remained significant at 1.36 (95% CI 1.07 to 1.73). Elevated MPO levels (>728 pmol/l) similarly predicted increased risk of future CAD among participants with either LDL-cholesterol <130 mg/dl, HDL-cholesterol >50 mg/dl, or CRP <2.0 mg/l (OR 1.52 [95% CI 1.21 to 1.91], 1.59 [95% CI 1.24 to 2.05], and 1.42 [95% CI 1.14 to 1.77)], respectively). CONCLUSION: Elevated MPO levels predict future risk of CAD in apparently healthy individuals. This study suggests that inflammatory activation precedes the onset of overt CAD by many years.
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Enfermedad de la Arteria Coronaria/sangre , Peroxidasa/sangre , Anciano , Biomarcadores/sangre , Presión Sanguínea , Estudios de Casos y Controles , Femenino , Humanos , Lípidos/sangre , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Factores de RiesgoRESUMEN
P-selectin inhibition has been shown to decrease thrombogenesis in multiple animal species. In this study, we show that a novel oral small-molecule inhibitor of P-selectin, PSI-697, promotes thrombus resolution and decreases inflammation in a baboon model of venous thrombosis. Experimental groups consisted of the following: 1) primates receiving a single oral dose of PSI-697 (30 mg/kg) daily starting three days pre-iliac vein balloon occlusion, and continued for six days; 2) primates receiving a single treatment dose of a low-molecular-weight-heparin (LMWH) (1.5 mg/kg) daily starting one day pre-iliac balloon occlusion, and continued for six days; and 3) primates receiving a single oral dose of a vehicle control daily starting three days pre-iliac vein balloon occlusion, and continued for six days. Animals receiving PSI-697, although thrombosed after balloon deflation, demonstrated greater than 80% vein lumen opening over time, with no opening (0%) for vehicle control (p < 0.01). LMWH opening evident after balloon deflation slightly deteriorated over time compared to PSI-697. PSI-697 therapy also significantly decreased vein wall inflammation determined by magnetic resonance venography (MRV). Importantly, this beneficial opening occurred without measured anticoagulation. Animals receiving PSI-697 demonstrated significantly increased plasma D-dimer levels versus LMWH and control animals six hours post thrombus induction (p < 0.01). This study is the first to demonstrate the effectiveness of oral P-selectin inhibition to modify venous thrombogenesis, increase vein lumen opening, and decrease inflammation in a large animal model.