RESUMEN
The epidemiology of tuberculin reactivity among physicians practicing in regions of moderate tuberculosis prevalence is unknown. We prospectively assessed the epidemiology of tuberculin skin test (TST) reactivity among physicians in training in St. Louis between 1992 and 1998. Of 1574 physicians who were tested, 267 (17%) had positive TST results. Older age, birth outside of the United States, prior bacille Calmette-Guérin (BCG) vaccination, and practice in the fields of medicine, anesthesiology, or psychiatry were associated with a positive TST result. Among physicians born in the United States, 63 (5.7%) had positive TST results. Among physicians with > or = 2 documented TSTs, 12 had conversion to a positive TST (1.6%; 1.03 conversions per 100 person-years). Physicians in this study had a high rate of tuberculin reactivity, despite a low conversion rate. The relationship between TST conversion and birth outside of the United States and BCG vaccination suggests a booster phenomenon rather than true new TST conversions.
Asunto(s)
Hospitales Universitarios , Médicos , Prueba de Tuberculina , Tuberculosis/epidemiología , Adulto , Vacuna BCG/inmunología , Femenino , Humanos , Masculino , Missouri/epidemiología , Valor Predictivo de las Pruebas , Estudios Prospectivos , Factores de TiempoRESUMEN
An efficient solid-phase synthesis of benzisothiazolone-1,1-dioxide-based serine protease inhibitors involving alkylation of carboxylic acids with N-(bromomethyl)benzisothiazolone-1,1-dioxide has been developed. An example using this procedure for preparation of a library of human mast cell tryptase inhibitors is described.
Asunto(s)
Inhibidores de Serina Proteinasa/síntesis química , Tiazoles/síntesis química , Quimasas , Humanos , Mastocitos/efectos de los fármacos , Mastocitos/enzimología , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Tiazoles/química , Tiazoles/farmacología , TriptasasRESUMEN
OBJECTIVE: To describe management and outcome of tuberculosis (TB) and current practices for isolation in two urban hospitals in the Midwest. DESIGN: Retrospective cohort study. SETTING: Barnes Hospital and Jewish Hospital, tertiary-care and community hospitals affiliated with Washington University School of Medicine in St Louis, Missouri. PATIENTS: All adult patients with a positive culture for Mycobacterium tuberculosis from 1988 to 1994. RESULTS: We identified 122 cases at Barnes and Jewish Hospitals (36.5/100,000 hospital discharges), median age was 59.0 years, 61.5% were non-Caucasian, and 54.9% resided within the city limits. Underlying risk conditions were common: substance abuse (25%), recent TB contact (24%), and foreign birth (13%). Coexistent human immunodeficiency virus infection (8%) was uncommon. Of skin-tested cases, 22% were anergic; of the rest, 22% tested negative. Almost 20% of cases had prior positive skin tests, and thus were preventable, but had not received adequate prophylaxis. Of hospitalized patients with pulmonary TB, 70% received respiratory isolation. Antibiotic resistance was recognized in 16%; only 19% of cases initially received four-drug therapy. TB-related death occurred in 16%. CONCLUSIONS: In this area, TB cases primarily involve traditional risk groups without HIV coinfection. Current infection control practices, diagnostic strategies, and initial treatment regimens are suboptimal. Education about local disease epidemiology is needed to prevent nosocomial TB transmission.
Asunto(s)
Hospitales Urbanos/estadística & datos numéricos , Aislamiento de Pacientes , Tuberculosis Pulmonar/diagnóstico , Tuberculosis Pulmonar/epidemiología , Adulto , Farmacorresistencia Microbiana , Femenino , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Missouri/epidemiología , Estudios Retrospectivos , Factores de Riesgo , Tuberculosis Pulmonar/terapiaRESUMEN
A library of compounds were prepared by reacting 2-(bromomethyl)-1, 2-benzisothiazol-3(2H)-one 1,1-dioxide (5) with commercially available carboxylic acids in the presence of potassium carbonate or a tertiary amine base. From this library, (1,1-dioxido-3-oxo-1, 2-benzisothiazol-2(3H)-yl)methyl N-[(phenylmethoxy)carbonyl]-beta-alanate (7b) emerged as a potent inhibitor of human mast cell tryptase (IC50 = 0.85 microM). Extension of the side chain of 7b by two carbons gave (1, 1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methyl 5-[[(phenylmethoxy)carbonyl]amino]pentanoate (7d) which was an 8-fold more potent inhibitor (IC50 = 0.1 microM). Further modification of this series produced benzoic acid derivative (1, 1-dioxido-3-oxo-1,2-benzisothiazol-2(3H)-yl)methyl 4-[[(phenylmethoxy)carbonyl]amino]benzoate (7n) which is the most potent inhibitor identified in this series (IC50 = 0.064 microM). These compounds exhibit time-dependent inhibition consistent with mechanism-based inhibition. For 7b, the initial enzyme velocity is not a saturable function of the inhibitor concentration and the initial Ki could not be determined (Ki > 10 microM). The steady-state rate constant, Ki, was determined to be 396 nM. On the other hand, compounds 7d and 7n are time-dependent inhibitors with a saturable initial complex. From these studies, an initial rate constant, Ki, for 7d and 7n was found to be 345 and 465 nM, respectively. The steady-state inhibition constants, Ki, for 7d and 7n were calculated to be 60 and 52 nM, respectively. Compound 7n is a 13-fold more potent inhibitor than 7b, and these kinetic studies indicate that the increase in inhibitory activity is due to an increase in initial affinity toward the enzyme and not an increase in chemical reactivity. These inhibitors generally show high selectivity for tryptase, being 40-fold weaker inhibitors of elastase, being 100-fold weaker against trypsin, and showing no inhibition against thrombin. These compounds are not inhibitors of thrombin, plasmin t-PA, urokinase, and factor Xa (IC50 > 33 microM). In the delayed-type hypersensitivity (DTH) mouse model, a model of skin inflammation, a 5% solution of 7d reduced edema by 69% compared to control animals.
Asunto(s)
Antiinflamatorios no Esteroideos/síntesis química , Óxidos S-Cíclicos/síntesis química , Mastocitos/enzimología , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/síntesis química , Tiazoles/síntesis química , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacología , Quimasas , Óxidos S-Cíclicos/química , Óxidos S-Cíclicos/farmacología , Dermatitis por Contacto/patología , Femenino , Humanos , Hipersensibilidad Tardía/patología , Cinética , Ratones , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Relación Estructura-Actividad , Tiazoles/química , Tiazoles/farmacología , TriptasasRESUMEN
[3-[4-(4,5-diphenyl-2-oxazolyl)-5-oxazolyl]phenoxy]acetic acid (BMY 45778) inhibits human (IC50 = 35 nM), rabbit (136 nM) and rat (1.3 microM) platelet aggregation. This compound activates adenylyl cyclase (ED50 = 6-10 nM) and stimulates GTPase in human platelet membrane preparations. The potency (EC50) of BMY 45778 stimulating adenylyl cyclase is comparable to iloprost. However, maximal stimulation of GTPase by BMY 45778 is approximately half the iloprost-stimulated activity, and BMY 45778 limits the GTPase stimulation by iloprost suggesting that BMY 45778 is a partial agonist at the IP receptor. BMY 45778 completely prevents [3H]]Iloprost binding to platelet membranes (IC50 = 7 nM). In whole platelets, BMY 45778 causes elevation of platelet cAMP levels (cAMP content doubles at 13 nM) and activation of the cAMP-dependent protein kinase (cAMP-protein kinase ratio is twice basal at 2 nM). BMY 45778 treatment of whole platelets also desensitizes the adenylyl cyclase activation by iloprost. These results indicate that BMY 45778, which is structurally different from prostacyclin and most prostacyclin agonists, acts by stimulating prostacyclin (IP) receptors.
Asunto(s)
Acetatos/farmacología , Adenilil Ciclasas/efectos de los fármacos , AMP Cíclico/metabolismo , Iloprost/metabolismo , Oxazoles/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Adenilil Ciclasas/metabolismo , Animales , Unión Competitiva , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Epoprostenol/agonistas , GTP Fosfohidrolasas/efectos de los fármacos , GTP Fosfohidrolasas/metabolismo , Humanos , Iloprost/farmacología , Imidazoles/farmacología , Fenoxiacetatos/farmacología , Proteínas Quinasas/efectos de los fármacos , Proteínas Quinasas/metabolismo , Conejos , RatasRESUMEN
A peptide-based structure-activity study is reported leading to the discovery of novel potent thrombin receptor antagonists. Systematic substitution of nonproteogenic amino acids for the second and third residues of the human thrombin receptor "tethered ligand" sequence (SFLLR) led to a series of agonists with enhanced potency. The most potent pentapeptide agonist identified was Ser-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-NH2, 9 (EC50 approximately 0.04 microM for stimulation of human platelet aggregation, approximately 10-fold more potent than the natural pentapeptide). Systematic substitution of the NH2-terminal Ser in 9 with neutral hydrophobic NH2-acyl groups led to partial agonists and eventually antagonists with unprecedented potency (greater than 1000-fold increase over the previously reported antagonist 3-mercaptopropionyl-Phe-Cha-Cha-Arg-Lys-Pro-Asn-Asp-Lys-NH2). In the series of NH2-acyl tetrapeptide antagonists, N-transcinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-NH 2, 41 (BMS-197525), was identified as the tightest binding (IC50 approximately 8 nM) and most potent with an IC50 approximately 0.20 microM for inhibition of SFLLRNP-NH2-stimulated platelet aggregation. Systematic single substitutions in 41 indicated that, in addition to the NH2-terminal acyl group, the side chains at the second and third positions were also responsible for important and specific receptor interactions. The p-fluoroPhe and p-guanidinoPhe residues in the second and third positions of 41 were observed to be optimal in both the agonist and antagonist series. In the case of antagonists, however, an appropriately positioned positively charged group (i.e., protonated base) at the third residue was required. In contrast, such a substitution was not required for potent agonist activity. An even more potent antagonist resulted when 41 was extended at the C-terminus by a single Arg residue giving rise to analog 90 (BMS-200261) which had an IC50 approximately 20 nM for inhibition of SFLLRNP-NH2-stimulated platelet aggregation. When the C-terminal Arg of 90 was replaced by an Orn-(Ndelta-propionyl) residue, the resulting antagonist 91 (BMS-200661) was suitable for use in radioligand binding assays (Kd = 10-30 nM). Antagonist activity observed for selected compounds was verified through secondary assays in that these analogs prevented SFLLRNP-NH2-stimulated GTPase activity in platelet membranes and Ca2+ mobilization in cultured human smooth muscle cells and mouse fibroblasts. Furthermore, this inhibition occurred at concentrations that had no effect on thrombin catalytic activity indicating a specific activity attributable to receptor binding and not enzyme inhibition.
Asunto(s)
Péptidos/química , Péptidos/farmacología , Receptores de Trombina/antagonistas & inhibidores , Animales , Diseño de Fármacos , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Relación Estructura-ActividadRESUMEN
Thrombin's proteolytically activated "tethered-ligand" receptor is widely expressed and mediates many of thrombin's actions on cells. Its central role in thrombin-stimulated human platelet activation and vascular smooth muscle proliferation as well as location in atherosclerotic plaques suggests receptor involvement in arterial thrombosis and atherosclerosis. Thrombin receptor antagonists, should they be effective, could be more selective than thrombin active site inhibitors in antithrombotic therapy as well as other indications. Blocking antibodies to peptides derived from the thrombin receptor have been used as prototypical thrombin receptor antagonists in vitro and have been useful in implicating this receptor in thrombin's actions on a variety of cell types. These antibodies have also shown the involvement of the receptor in arterial thrombosis models in nonhuman primates. Amino acid substitution studies have shown the structural requirements for receptor activation of peptides homologous to the new NH2-terminus. Peptide-based partial agonists and antagonists have been synthesized by NH2-terminal replacements of the serine in the receptor activating peptides. Current thrombin receptor antagonists lack potency and some are partial agonists; however, it is expected that more potent compounds will result from further investigation. The potency limitations are important to overcome before serious evaluation of their efficacy can be determined.
Asunto(s)
Arteriosclerosis/sangre , Activación Plaquetaria , Receptores de Trombina/antagonistas & inhibidores , Trombosis/sangre , Animales , Arteriosclerosis/patología , Arteriosclerosis/fisiopatología , Humanos , Trombosis/patología , Trombosis/fisiopatologíaRESUMEN
Thrombin receptor activation, by thrombin or SFLLR-containing peptides, stimulates GTPase activity in platelet and CHRF-288 membranes. Polyclonal antibodies to peptides derived from the thrombin receptor (anti-TR52-69 and anti-TR36-49), which block many of thrombin's actions on platelets and endothelial cells, also block thrombin activation of membrane GTPase (as does thrombin active site and anion-binding exosite inhibitors). Most of the receptor-activated GTPase, stimulated by both thrombin and SFLLRNP in platelet membranes, was inhibited by prior treatment with pertussis toxin or N-ethylmaleimide, suggesting that under these conditions much of the thrombin receptor-stimulated GTPase in platelet membranes is a member of the pertussis toxin-sensitive G alpha i family. In platelet membrane preparations, the peptide agonists stimulated approximately twice as much GTPase activity as stimulated by alpha-thrombin. In contrast, the membranes prepared from CHRF-288 cells showed similar maximal SFLLRNP- and alpha-thrombin-stimulated GTPase activity. Stimulation of the platelet membrane GTPase by a variety of different peptide agonists correlated with their ability to stimulate platelet aggregation. Several peptide-based agonists were more potent than the wild-type sequence. The most potent was Ser-(p-fluoro-Phe)-(2-Napthyl-Ala)-Leu-Arg-NH2, which stimulated platelet aggregation (EC50 = 80 nM) and GTPase activity (EC50 = 110 nM). The peptide YFLLRN stimulated GTPase activity but only to approximately 40% of the activity observed with optimal concentrations of other receptor agonists. YFLLRN also limited the stimulation observed with SFLLRNP in a competitive fashion, indicating that YFLLRN is a competitive partial agonist at the thrombin receptor. These studies show that the tethered-ligand receptor mediates the GTPase activation by thrombin in platelet and CHRF-288 cell membranes, and this provides a specific, reliable, and convenient cell-free assay system with which one can evaluate agonists and partial agonists.
Asunto(s)
Plaquetas/efectos de los fármacos , GTP Fosfohidrolasas/metabolismo , Péptidos/farmacología , Receptores de Trombina/efectos de los fármacos , Trombina/farmacología , Secuencia de Aminoácidos , Anticuerpos/farmacología , Plaquetas/metabolismo , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Activación Enzimática , Etilmaleimida/farmacología , GTP Fosfohidrolasas/antagonistas & inhibidores , Humanos , Datos de Secuencia Molecular , Péptidos/inmunología , Toxina del Pertussis , Receptores de Trombina/inmunología , Receptores de Trombina/metabolismo , Trombina/antagonistas & inhibidores , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
A series of 3-(3-guanidinopropyl)-azetidin-2-one derivatives was prepared and evaluated as inhibitors of cleavage of synthetic substrates in vitro by the serine proteases thrombin, trypsin and plasmin. The N-unsubstituted, 4-phenethyl derivative 9a demonstrated weak inhibition of these enzymes but acetylation of the beta-lactam N atom afforded 9b, an effective, time-dependent inhibitor of thrombin and a potent inhibitor of plasmin. Variation of the 4-position of the beta-lactam ring was examined in conjunction with different N-substituents to provide a series of potent, time-dependent inhibitors of thrombin. A C-4 substituent was essential for good inhibitory properties and, in general, polar C-4 substituents enhanced the selectivity of inhibition for thrombin compared to plasmin. A trans relationship between the C-4 and C-3 substituents was found to be superior to a cis disposition whilst homologation of the guanidinopropyl side chain to that of a guanidinobutyl moiety reduced activity. Several compounds were effective inhibitors of thrombin-induced clot formation in human plasma in vitro but activity in this assay did not correlate well with inhibition of thrombin-induced cleavage of a synthetic substrate, presumably a consequence of inherent chemical instability and degradation in plasma.
Asunto(s)
Antitrombinas/síntesis química , Azetidinas/síntesis química , Inhibidores de Serina Proteinasa/síntesis química , Trombina/antagonistas & inhibidores , Antitrombinas/química , Antitrombinas/farmacología , Azetidinas/química , Azetidinas/farmacología , Diseño de Fármacos , Fibrinolisina/antagonistas & inhibidores , Guanidinas/síntesis química , Guanidinas/química , Guanidinas/farmacología , Humanos , Indicadores y Reactivos , Cinética , Espectroscopía de Resonancia Magnética , Estructura Molecular , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Espectrometría de Masa Bombardeada por Átomos Veloces , Espectrofotometría Infrarroja , Relación Estructura-Actividad , Inhibidores de Tripsina/síntesis químicaRESUMEN
A series of N-arylsulfonylarginine amides was synthesized wherein the guanidine or arginine moiety was isosterically replaced by a number of heterocyclic functionalities. These compounds were evaluated as potential active-site inhibitors of thrombin. Bisamidines 11a-n showed a similar SAR to that of simple arginine compounds. The ex vivo clotting time measurement of 11d after ip dosing showed prolongation of clotting time in rats.
Asunto(s)
Antitrombinas/síntesis química , Arginina , Guanidinas , Inhibidores de Serina Proteinasa/síntesis química , Trombina/antagonistas & inhibidores , Animales , Antitrombinas/química , Antitrombinas/farmacología , Sitios de Unión , Coagulación Sanguínea/efectos de los fármacos , Diseño de Fármacos , Humanos , Indicadores y Reactivos , Cinética , Espectroscopía de Resonancia Magnética , Estructura Molecular , Ratas , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Relación Estructura-ActividadRESUMEN
Proteolytic action of alpha-thrombin on human thrombin receptor results in cleavage of a portion of the N-terminus, thereby generating a 'tethered ligand' at the newly exposed N-terminus, which then activates the receptor in an intramolecular fashion. Agonist peptides incorporating the amino acid sequence of the newly exposed N-terminal portion of the cleaved receptor cause receptor activation without requiring prior cleavage of the receptor by thrombin. The pentapeptide amide Ser-Phe-Leu-Leu-Arg-NH2, which retains the N-terminal sequence of the 'tethered ligand' of the receptor, has been shown to be the minimum sequence to cause receptor activation. To understand the importance of the side chains of various residues within the pentapeptide amide, we carried out an extensive structure-activity study of the ability of peptides to stimulate gel-filtered platelet aggregation. In this study 106 pentapeptide amides were synthesized, utilizing naturally occurring L-amino acids, unnatural amino acids, D-amino acids and N-methyl amino acids for replacements. At position-1, charged residues (acidic or basic) were not tolerated, and the size and shape of the residue were important. Position-2 tolerated only aromatic residues. Position-3 accommodated various residues. A significant finding of this study was that two very different residues, [3-(2-naphthyl)]-L-alanine and L-arginine, when substituted for leucine residue at position-3, resulted in more active agonists. At position-4 aromatic and aliphatic residues were well tolerated, whereas basic and acidic residues were less tolerated. Position-5 mimicked position-3 in its ability to tolerate a wide range of residues.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Plaquetas/efectos de los fármacos , GTP Fosfohidrolasas/metabolismo , Oligopéptidos/química , Oligopéptidos/farmacología , Agregación Plaquetaria/efectos de los fármacos , Receptores de Trombina/agonistas , Secuencia de Aminoácidos , Sitios de Unión , Plaquetas/enzimología , Plaquetas/fisiología , Humanos , Ligandos , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Relación Estructura-Actividad , Trombina/metabolismoRESUMEN
A thrombin receptor has been described that is activated by thrombin cleavage generating a new N-terminus. The newly exposed SFLLR-containing "tethered-ligand" then activates the receptor. In these studies, we used 3-mercapto-propionyl-Phe-Cha-Cha-Arg-Lys-Pro-Asn- Asp-Lys-amide (Mpapeptide) as a thrombin receptor antagonist. This compound was capable of preventing both thrombin- and SFLLR-peptide-induced platelet aggregation with little effect on collagen-induced platelet aggregation. It also prevented thrombin- and SFLLRNP-induced calcium mobilization with little effect on thromboxane receptor-activated platelet Ca2+ mobilization. Platelet membrane GTPase could be activated by peptides that activated the thrombin receptor, and the thrombin receptor antagonist also prevented receptor-stimulated GTPase activity. Platelet phospholipase A2 (PLA2) activity (measured as the release of radiolabeled arachidonic acid) and Na+/H+ exchange activation were stimulated by alpha-thrombin and by SFLLR-containing peptides. Activation of both processes with low concentrations of thrombin required thrombin's anion-binding exosite, as they were not activated by similar concentrations of gamma-thrombin, and the alpha- and zeta-thrombin activation was blocked by peptides mimicking the C-terminal region of hirudin. Stimulation of PLA2 and Na+/H+ exchange by both thrombin and SFLLR-containing peptides was inhibited by the thrombin receptor antagonist Mpa-peptide. These results support the hypothesis that thrombin stimulation of PLA2 activity and Na+/H+ exchange occurs via activation of the thrombin tethered-ligand receptor. Moreover, these data are consistent with the tethered-ligand receptor mediating most actions elicited by low concentrations of alpha-thrombin involved in human platelet activation.
Asunto(s)
Oligopéptidos/antagonistas & inhibidores , Fosfolipasas A/antagonistas & inhibidores , Activación Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Receptores de Trombina/antagonistas & inhibidores , Intercambiadores de Sodio-Hidrógeno/antagonistas & inhibidores , Trombina/antagonistas & inhibidores , Inhibidores de Adenilato Ciclasa , Secuencia de Aminoácidos , Plaquetas/metabolismo , Humanos , Datos de Secuencia Molecular , Oligopéptidos/farmacología , Fosfolipasas A2 , Inhibidores de Agregación Plaquetaria/farmacologíaRESUMEN
The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.
Asunto(s)
Antitrombinas/metabolismo , Oligopéptidos/metabolismo , Conformación Proteica , Trombina/antagonistas & inhibidores , Trombina/metabolismo , Secuencia de Aminoácidos , Antitrombinas/química , Sitios de Unión , Cristalografía por Rayos X , Hirudinas/análogos & derivados , Hirudinas/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oligopéptidos/química , Fragmentos de Péptidos/metabolismo , Trombina/químicaRESUMEN
The effects of a thrombin active-site inhibitor on arterial and venous thrombosis, and thrombin-induced thrombocytopenia were determined in anesthetized rats. Desamino D-Phe-Pro-Arg-aldehyde (BMY 44621) was administered before experimental intervention as a loading i.v. dose plus continuous i.v. infusion. Carotid artery thrombosis was produced by transmural vessel injury and vena cava thrombosis was produced by partial stasis of blood flow combined with endothelial injury. Thrombocytopenia was induced by an i.v. injection of human alpha-thrombin. BMY 44621 inhibited arterial and venous thrombosis in a dose-dependent manner. Its threshold antithrombotic dose for venous thrombosis was half of that for arterial thrombosis. Maximum reductions in thrombus weight were greater for venous (> 90%) compared to arterial (57%) thrombosis and correlated with 2-and 9-fold prolongation of ex vivo thrombin clotting time, respectively. A 40% reduction in platelet counts induced by thrombin injection was abolished by the threshold dose of BMY 44621 for inhibiting venous thrombosis. These experiments demonstrate that thrombin's active-site is an effective target for inhibiting venous and arterial thrombosis, although venous thrombosis is more sensitive to this therapeutic strategy than arterial thrombosis.
Asunto(s)
Aldehídos/uso terapéutico , Antitrombinas/uso terapéutico , Trombosis de las Arterias Carótidas/prevención & control , Dipéptidos/uso terapéutico , Trombina , Tromboflebitis/prevención & control , Aldehídos/administración & dosificación , Secuencia de Aminoácidos , Animales , Antitrombinas/administración & dosificación , Trombosis de las Arterias Carótidas/inducido químicamente , Dipéptidos/administración & dosificación , Relación Dosis-Respuesta a Droga , Hemodinámica/efectos de los fármacos , Humanos , Infusiones Intravenosas , Masculino , Datos de Secuencia Molecular , Tiempo de Protrombina , Ratas , Ratas Sprague-Dawley , Trombina/administración & dosificación , Trombina/fisiología , Trombocitopenia/inducido químicamente , Tromboflebitis/inducido químicamenteRESUMEN
BMY 42393, (2-[3-[2-(4,5-diphenyl-2-oxazolyl)ethyl]phenoxy]acetic acid), is a new prostacyclin partial agonist that inhibited ADP, collagen and thrombin-induced platelet aggregation (IC50 range 0.3 - 2.0 microM). BMY 42393 stimulated platelet adenylate cyclase activity (EC50 = 25 nM), however, the maximal activation was 75-80% of that observed with maximal iloprost or PGE1. Platelets treated with BMY 42393 showed an elevation of cAMP levels and activation of cAMP-dependent protein kinase. BMY 42393 also inhibited thrombin-induced elevation of intracellular free calcium. BMY 42393 competed for radiolabeled iloprost and PGE1 binding to platelet membranes (IC50; 170 nM and 130 nM, respectively); however, it had little effect on radiolabeled PGE2, PGD2, or SQ 29548 binding. These studies indicate that BMY 42393 is a novel platelet aggregation inhibitor which acts by stimulation of platelet prostacyclin receptors to elevate platelet cAMP levels.
Asunto(s)
Epoprostenol , Oxazoles/farmacología , Fenoxiacetatos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Receptores de Prostaglandina/efectos de los fármacos , Adenilil Ciclasas/sangre , Plaquetas/enzimología , Calcio/sangre , AMP Cíclico/sangre , Proteínas Quinasas Dependientes de AMP Cíclico/sangre , Humanos , Estructura Molecular , Oxazoles/química , Fenoxiacetatos/química , Inhibidores de Agregación Plaquetaria/química , Ensayo de Unión Radioligante , Receptores de Epoprostenol , Relación Estructura-ActividadRESUMEN
The oral activity and antithrombotic efficacy of BMY 42393 was examined in ex vivo platelet aggregation studies and arterial thrombosis animal models. In a heterologous ex vivo platelet aggregation assay, ADP-induced human platelet aggregation was inhibited when washed human platelets were combined with rat platelet-poor plasma, taken from rats previously orally-dosed with BMY 42393. The IC50 for platelet aggregation inhibition was approximately 10 mg/kg. In a laser-induced thrombosis model, thrombus formation in a revascularized rabbit ear chamber was prevented in a dose-dependent fashion with an ED50 of about 2 mg/kg. A relatively long duration of anti-thrombotic activity was observed in the rabbit ear laser-induced thrombus study and the ex vivo platelet studies. Inhibition of thrombus formation was also demonstrated in a canine model of electrically-induced coronary artery thrombosis. BMY 42393 also prevented cyclic flow reductions in a monkey stenotic renal artery model. These studies indicate that BMY 42393 is orally active and capable of preventing laser and electric current-induced thrombus formation in animal models of arterial thrombosis.
Asunto(s)
Trombosis Coronaria/tratamiento farmacológico , Oxazoles/uso terapéutico , Fenoxiacetatos/uso terapéutico , Administración Oral , Animales , Chlorocebus aethiops , Modelos Animales de Enfermedad , Perros , Humanos , Macaca fascicularis , Masculino , Conejos , Ratas , Ratas Sprague-Dawley , Obstrucción de la Arteria Renal/tratamiento farmacológicoRESUMEN
Different pharmacological approaches to thrombin inhibition were compared for their effects on thrombosis and bleeding time in anesthetized rats. Thrombosis was induced in the carotid artery by transmural vessel injury and in the vena cava by partial blood flow stasis combined with mild endothelial disruption. Small mesenteric arteries were punctured with a hypodermic needle to measure the bleeding time. Dose-response relationships were determined with a thrombin active site inhibitor, N-methyl (GYKI 14,766); a thrombin exosite inhibitor, succinyl-Phe-Glu-Pro-Ile-Pro-Glu-Glu-Tyr-cyclohexylalanine-Gln (BMS 180,742); and heparin. BMS 180,742 interferes with fibrinogen binding to the thrombin exosite but, unlike GYKI 14,766, it does not block thrombin's catalytic site. The effects on thrombosis and bleeding time were correlated with ex vivo clotting times using the activated partial thromboplastin time for heparin and the thrombin time for GYKI 14,766 and BMS 180,742. Venous thrombosis was inhibited more than 90% by all three inhibitors at doses that either produced threshold increases or had no effect on bleeding and clotting times. Arterial thrombosis was inhibited 82% by GYKI 14,766 and 63% by heparin but it was not inhibited by BMS 180,742. These antithrombotic activities were accompanied by a maximal activated partial thromboplastin time increase and doubling of the bleeding time with heparin and a maximal thrombin time prolongation and 35% increase in bleeding time with GYKI 14,766. These results suggest that thrombin inhibitors, which act at the active site or exosite or through antithrombin III, are equally efficacious against venous thrombosis but active site inhibitors are the most effective against arterial thrombosis.
Asunto(s)
Anticoagulantes/uso terapéutico , Trombosis de las Arterias Carótidas/prevención & control , Heparina/uso terapéutico , Oligopéptidos/uso terapéutico , Péptidos/uso terapéutico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Trombina/antagonistas & inhibidores , Tromboflebitis/prevención & control , Secuencia de Aminoácidos , Animales , Sitios de Unión , Tiempo de Sangría , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Masculino , Arterias Mesentéricas/efectos de los fármacos , Arterias Mesentéricas/fisiología , Datos de Secuencia Molecular , Ratas , Ratas Sprague-DawleyRESUMEN
1-(Cyclohexylmethyl)-4-[4-[(2,3-dihydro-2-oxo-1H-imidazo[4,5-b] quinolin-7-yl)oxy]-1-oxobutyl]piperazine (2) was previously identified as a potent, water-soluble inhibitor of human blood platelet cAMP phosphodiesterase and of induced aggregation in vitro that demonstrated effective antithrombotic activity in animal models of thrombosis. Although 2 exhibited 25% oral bioavailability in rats, pharmacokinetic studies conducted in monkeys revealed that the parent compound was less than 5% bioavailable, the result of extensive first-pass biotransformation in the liver. In an effort to identify potent platelet aggregation inhibitors with enhanced metabolic stability, the side-chain amide moiety of 2 was replaced with chemically more stable urea (6a-s), sulfonamide (13a-m), sulfone (19a-r), and tetrazole (23a-s) moieties. Many representatives from each of these structural types effectively combined potent inhibition of ADP-induced human platelet aggregation in vitro with excellent aqueous solubility, and several are superior to 2. Within each series, the N-(cyclohexylmethyl)-, N-(2-ethylbutyl)-, N-benzyl-, and N-(4-fluorobenzyl)-substituted derivatives were evaluated for in vitro metabolic stability by incubating with the S-9 fraction of monkey liver for 2 h, and the extent of biotransformation was compared with that of the prototype 2. The sulfone 19e and the tetrazoles 23e, 23g, 23j, and 23q were significantly more stable than 2 under these conditions, and 19e and 23e were selected for evaluation in vivo. Tetrazole 23e exhibited 72% bioavailability following ip administration to rats compared with 35% bioavailability for 2 and 19e under the same conditions. However, the oral bioavailability of 19e and 23e in the rat was estimated to be only 3%, suggesting that 19e and 23e are less readily absorbed from the gastrointestinal tract than 2.
Asunto(s)
3',5'-AMP Cíclico Fosfodiesterasas/antagonistas & inhibidores , Plaquetas/enzimología , Imidazoles/síntesis química , Imidazoles/farmacología , Inhibidores de Agregación Plaquetaria/síntesis química , Quinolonas/síntesis química , Quinolonas/farmacología , 3',5'-AMP Cíclico Fosfodiesterasas/sangre , Adenosina Difosfato/farmacología , Animales , Plaquetas/efectos de los fármacos , Chlorocebus aethiops , Ratas , Solubilidad , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/farmacología , Sulfonas/síntesis química , Sulfonas/farmacología , Tetrazoles/síntesis química , Tetrazoles/farmacología , Urea/análogos & derivados , Urea/síntesis química , Urea/farmacología , AguaRESUMEN
We determined the effects of two prostacyclin agonists (octimibate and BMY 42393) on the progression of the fatty streak in vivo and on macrophage function in vitro. Hamsters were fed chow plus 0.05% cholesterol and 10% coconut oil. Control hamsters were compared with animals receiving either octimibate (10 or 30 mg/kg per day) or BMY 42393 (30 mg/kg per day). After 10 weeks of treatment, octimibate decreased plasma total cholesterol and triglycerides by 43% and 32%, respectively. Neither agonist affected blood pressure or heart rate. Lesion-prone aortic arches were stained with hematoxylin and oil red O and examined en face. Compared with controls, octimibate and BMY 42393 on average decreased mononuclear cells attached to the luminal surface by 44% and reduced subendothelial macrophage-foam cell number by 56%, foam cell size by 38%, and fatty streak area by 63%. Since octimibate is a putative inhibitor of acyl coenzyme A cholesterol acyltransferase, we studied the effect of both agents on cholesteryl ester metabolism in murine macrophages. At 10 microM, octimibate and BMY 42393 decreased cholesteryl ester accumulation in macrophages by 90% and 41%, respectively. Octimibate inhibited cholesteryl ester synthesis by 96% and increased the rate of cholesteryl ester degradation by 52%. Both prostacyclin agonists reduced macrophage scavenger receptor-mediated uptake of acetylated low density lipoprotein by 24-66% and increased cyclic adenosine monophosphate levels. Octimibate and BMY 42393 inhibited the secretion of tumor necrosis factor by 80-88% when macrophages were activated with lipopolysaccharide. At 10 microM, both agents decreased human monocyte chemotaxis to N-formyl-methionyl-leucyl-phenylalanine by 64-79%. The in vitro results with octimibate and BMY 42393 are consistent with the low number of small foam cells quantified in vivo. We suggest that octimibate and BMY 42393 suppress monocyte-macrophage atherogenic activity and cytokine production and thus inhibit the development of early atherosclerosis.