RESUMEN
Intravascular clot formation is an important factor in a number of cardiovascular diseases. Therefore, the prevention of blood coagulation has become a major target for new therapeutic agents. One attractive approach is the inhibition of factor Xa (FXa), the enzyme directly responsible for thrombin activation. Herein we report a series of isoxazoline derivatives which are potent FXa inhibitors. Optimization of the side chain at the quaternary position of the isoxazoline ring led to SK549 which showed subnanomolar FXa potency (K(i) 0.52 nM). SK549 shows good selectivity for FXa compared to thrombin and trypsin, potent antithrombotic effect in the rabbit arterio-venous thrombosis model, and improved pharmacokinetics relative to other compounds evaluated from this series.
Asunto(s)
Inhibidores del Factor Xa , Fibrinolíticos/síntesis química , Isoxazoles/síntesis química , Tetrazoles/síntesis química , Animales , Derivación Arteriovenosa Quirúrgica , Sitios de Unión , Cristalografía por Rayos X , Perros , Fibrinolíticos/química , Fibrinolíticos/farmacología , Humanos , Isoxazoles/química , Isoxazoles/farmacología , Modelos Moleculares , Conejos , Relación Estructura-Actividad , Tetrazoles/química , Tetrazoles/farmacología , Trombina/antagonistas & inhibidores , Trombosis/tratamiento farmacológico , Tripsina/metabolismo , Inhibidores de Tripsina/síntesis química , Inhibidores de Tripsina/química , Inhibidores de Tripsina/farmacocinética , Inhibidores de Tripsina/farmacologíaRESUMEN
Thrombosis is a major cause of mortality in the industrialized world. Therefore, the prevention of blood coagulation has become a major target for new therapeutic agents. One attractive approach is the inhibition of factor Xa (FXa), the enzyme directly responsible for prothrombin activation. We report a series of novel biaryl-substituted isoxazoline derivatives in which the biaryl moiety was designed to interact with the S(4) aryl-binding domain of the FXa active site. Several of the compounds herein have low nanomolar affinity for FXa, have good in vitro selectivity for FXa, and show potent antithrombotic efficacy in vivo. The three most potent compounds (33, 35, and 37) have inhibition constants for human FXa of 3.9, 2.3, and 0.83 nM, respectively, and ID(50)'s ranging from 0.15 to 0.26 micromol/kg/h in the rabbit arterio-venous thrombosis model.
Asunto(s)
Acetatos/síntesis química , Inhibidores del Factor Xa , Fibrinolíticos/síntesis química , Isoxazoles/síntesis química , Acetatos/química , Acetatos/farmacología , Animales , Derivación Arteriovenosa Quirúrgica , Sitios de Unión , Compuestos de Bifenilo , Fibrinolíticos/química , Fibrinolíticos/farmacología , Humanos , Isoxazoles/química , Isoxazoles/farmacología , Modelos Moleculares , Conejos , Relación Estructura-Actividad , Trombosis/tratamiento farmacológicoRESUMEN
Substituted pyrazoles, 1,2,4-triazoles, and tetrazoles are good surrogates for cis-amide bonds in a series of boronate ester thrombin inhibitors.
Asunto(s)
Amidas/farmacología , Ácidos Borónicos/farmacología , Pirazoles/farmacología , Tetrazoles/farmacología , Trombina/antagonistas & inhibidores , Trombina/química , Triazoles/farmacología , Amidas/química , Sitios de Unión , Ácidos Borónicos/química , Cristalografía por Rayos X , Indicadores y Reactivos , Modelos Moleculares , Estructura Molecular , Pirazoles/síntesis química , Pirazoles/química , Estereoisomerismo , Relación Estructura-Actividad , Tetrazoles/síntesis química , Tetrazoles/química , Triazoles/síntesis química , Triazoles/químicaRESUMEN
First generation ELISA screening assays for antibodies to HTLV-III (HIV) generated between 0.1 and 1.0% false positive results. Western blot analysis in specialized reference centers is almost uniformly used as a method to confirm the specificity of the ELISA results. Yet, the high cost, time delay and lack of standardization in these systems cause a growing demand for tests that can be performed on site and that can at least reduce the number of sera that have to be sent to reference centers. Such tests thus should primarily be aimed at the detection of false positive results. Ancillary to the Vironostika anti-HTLV-III screening test, we developed a set of reagents (VERIFY) which can be used for the verification of initially or repeatedly positive screening results. The test employs a reagent specifically blocking true HTLV-III-anti HTLV-III reactions, a reagent blocking HLA-anti HLA reactions and a control reagent. Use of this test may reduce the number of sera found false positive by reference methods by more than 90%. The introduction of improved versions and second generation screening assays obviously will reduce the number of false positive results. Yet the significant results of this verification assay and the ease with which it can be integrated in the screening procedures will make it a valuable tool in the blood bank screening program.
Asunto(s)
Anticuerpos Antivirales/análisis , Donantes de Sangre , Ensayo de Inmunoadsorción Enzimática , Seropositividad para VIH , VIH/inmunología , Reacciones Falso Positivas , Anticuerpos Anti-VIH , Humanos , Inmunoensayo , Valor Predictivo de las PruebasRESUMEN
Inhibition of the phosphorylation of the synaptic plasma membrane (SPM) protein B50 by [D-Trp8]-somatostatin in vitro is time-dependent. Increasing the time of incubation of hippocampal synaptic plasma membranes with the peptide from 15 sec to 30 min prior to addition of 7.5 microM [gamma-32P]ATP results in a complete reduction of B50 phosphorylation. Incubation of synaptic plasma membranes for 30 min in the absence of peptide does not alter basal B50 phosphorylation. Neither ACTH nor beta-endorphin produces similar effects--inhibition of B50 phosphorylation by ACTH is maximal at 15 sec and beta-endorphin produces only a small inhibition, even after 30 min. [D-Trp8]-somatostatin is not activating a membrane-bound protease, since maximal inhibition of B50 phosphorylation by the peptide is seen in the presence of leupeptin or bacitracin. Hippocampal synaptic plasma membranes contain protein phosphatase activity. Assays of B50 phosphorylation in synaptic plasma membranes done under conditions that favor either net phosphorylation or dephosphorylation are consistent with inhibition of protein phosphatase activity by [D-Trp8]-somatostatin. This evidence suggests that [D-Trp8]-somatostatin interacts with SPM binding sites in the hippocampus, which may alter the activity of an endogenous protein phosphatase to determine the degree of B50 phosphorylation.
Asunto(s)
Hipocampo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Somatostatina/análogos & derivados , Membranas Sinápticas/metabolismo , Animales , Membrana Celular/metabolismo , Proteína GAP-43 , Hipocampo/efectos de los fármacos , Cinética , Leupeptinas/farmacología , Fosforilación , Ratas , Somatostatina/farmacología , Membranas Sinápticas/efectos de los fármacosRESUMEN
Rat hippocampal slices were incubated with [3H]uridine in vitro to analyze the metabolism of nuclear RNA and the RNA precursor fractions. Labeling of total nuclear RNA was linear for 4 h of incubation and proportional to the concentration of labeled uridine in the incubation medium. Addition of 3.5 X 10(-8) M corticosterone to the incubation medium produced an enhancement of nuclear RNA labeling with no significant effect on the labeling of the RNA precursor fraction. Progesterone and dexamethasone, at the same concentration, had no effect on either variable. Labeling of RNA by cerebellar slices under the same conditions was approximately one-half the value obtained using hippocampal slices and the cerebellar RNA precursor fraction accumulated only 65% of the radioactivity from [3H]uridine found in the hippocampal pool. Corticosterone had no effect on the labeling of total nuclear RNA in cerebellar slices. Nuclear poly(A)-containing RNA constituted 19% of the total labeled nuclear RNA in these incubations, as estimated by oligo (dT)-cellulose chromatography. Cordycepin (3'-deoxyadenosine) at a concentration of 25 micrograms/ml inhibited to some extent the labeling of total nuclear RNA and the RNA precursor fraction, but preferentially diminished the amount of labeled RNA bound to oligo (dT)-cellulose. Corticosterone increased the amount of [3H]RNA which bound to oligo (dT)-cellulose, while progesterone had no effect. These results show that hippocampal slices maintained in vitro, can be used to analyze nuclear RNA metabolism, one positive regulator of which in the rat hippocampus is the adrenal steroid, corticosterone.