RESUMEN
Prasugrel is an orally available thienopyridyl prodrug with more potent in vivo antiplatelet effects compared to clopidogrel. In the present study, we examined the effects of prasugrel in rat models of cerebral and peripheral arterial occlusive diseases. Cerebral arterial thrombosis was induced by photochemical irradiation of the middle cerebral artery. Prasugrel (3 and 10 mg/kg) dose-relatedly and significantly reduced thrombus-mediated cerebral infarction 24 h after the irradiation. The effect of prasugrel was further examined in an embolic infarction model. Four h after an oral administration of prasugrel, non-occlusive thrombus formation in the right common carotid artery was initiated. In this model, prasugrel (0.3-3 mg/kg) reduced incidence, total area, and total number of cerebral infarcts in a dose-related manner 24 h after the vascular injury. Clopidogrel (10 or 30 mg/kg) was less potent than prasugrel at the doses tested on these thrombotic and embolic infarctions. Finally, the effect of prasugrel on lauric acid-induced peripheral arterial occlusive diseases was evaluated. After injection of lauric acid into the femoral artery, the lesions were scored for the following 10 days as they gradually progressed from the toe throughout the leg. Prasugrel (0.03-3 mg/kg/day) administered from the day before the lauric acid injection for 11 successive days inhibited the progression of the disease in a dose-related manner. Clopidogrel (3-30 mg/kg/day) showed similar effect but its effect was less potent than prasugrel. These results suggest that prasugrel could be a useful drug for preventing thromboembolic diseases including cerebral infarction and peripheral arterial occlusive diseases.
Asunto(s)
Enfermedades Arteriales Cerebrales/tratamiento farmacológico , Enfermedades Vasculares Periféricas/tratamiento farmacológico , Piperazinas/farmacología , Antagonistas del Receptor Purinérgico P2 , Tiofenos/farmacología , Animales , Enfermedades Arteriales Cerebrales/etiología , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Masculino , Enfermedades Vasculares Periféricas/etiología , Clorhidrato de Prasugrel , Ratas , Ratas Sprague-DawleyRESUMEN
To develop a novel and effective anticoagulant with potent and selective factor Xa (FXa) inhibitory activity, a new series of cinnamyl derivatives with enhanced lipophilicity and prodrug forms were synthesized and their biological activities were evaluated. As a result, we found that cinnamyl derivative (N-[4-[1-(acetimidoyl)piperidin-4-yloxy]-3-carbamoylphenyl]-N-[(Z)-3-(3-amidinophenyl)-2-fluoro-2-propenyl]sulfamoyl)acetic acid dihydrochloride (26d, R-142086) with a fluorine atom on the double bond exhibited potent anticoagulant activity and no mutagenic potential. Moreover, orally administered R-142086 exhibited potent anti-FXa activity and anticoagulant activity in dogs.
Asunto(s)
Amidinas , Anticoagulantes , Antitrombina III , Cinamatos/química , Inhibidores del Factor Xa , Sulfonamidas , Administración Oral , Amidinas/síntesis química , Amidinas/química , Amidinas/farmacología , Animales , Anticoagulantes/síntesis química , Anticoagulantes/química , Anticoagulantes/farmacología , Antitrombina III/síntesis química , Antitrombina III/química , Antitrombina III/farmacología , Cricetinae , Perros , Humanos , Masculino , Estructura Molecular , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
To develop a potent and oral anticoagulant, a series of compounds with cinnamyl moiety was synthesized and their factor Xa (FXa) inhibitory activities were examined. As a result, some cinnamyl derivatives showed potent FXa inhibitory activities in vitro. Among them, compounds with substituent at the 3-position on the central benzene ring represented by (N-{4-[1-(acetimidoyl)piperidin-4-yloxy]-3-chlorophenyl}-N-[(E)-3-(3-amidinophenyl)-2-propenyl]sulfamoyl)acetic acid dihydrochloride (45b) and (N-{4-[1-(acetimidoyl)piperidin-4-yloxy]-3-carbamoylphenyl}-N-[(E)-3-(3-amidinophenyl)-2-propenyl]sulfamoyl)acetic acid dihydrochloride (45j) exhibited potent FXa inhibitory activities with IC(50) values of less than 10 nM in vitro. These compounds also showed potent anticoagulant activities both in vitro and ex vivo. Furthermore, these compounds exhibited no lethal toxicity (30 mg/kg, i.v.).
Asunto(s)
Cinamatos/síntesis química , Cinamatos/farmacología , Inhibidores del Factor Xa , Animales , Coagulación Sanguínea/efectos de los fármacos , Cricetinae , Humanos , Técnicas In Vitro , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Masculino , Espectrometría de Masas , Inhibidores de Tripsina/farmacologíaRESUMEN
In serine proteases, Gly193 (chymotrypsin numbering) is conserved with rare exception. Mutants of blood coagulation proteases have been reported with Glu, Ala, Arg or Val substitutions for Gly193. To further understand the role of Gly193 in protease activity, we replaced it with Ala or Val in coagulation factor XIa (FXIa). For comparison to the reported FXIa Glu193 mutant, we prepared FXIa with Asp (short side chain) or Lys (opposite charge) substitutions. Binding of p-aminobenzamidine (pAB) and diisopropylfluorphosphate (DFP) were impaired 1.6-36-fold and 35-478-fold, respectively, indicating distortion of, or altered accessibility to, the S1 and oxyanion-binding sites. Val or Asp substitutions caused the most impairment. Salt bridge formation between the amino terminus of the mature protease moiety at Ile16 and Asp194, essential for catalysis, was impaired 1.4-4-fold. Mutations reduced catalytic efficiency of tripeptide substrate hydrolysis 6-280-fold, with Val or Asp causing the most impairment. Further studies were directed toward macromolecular interactions with the FXIa mutants. kcat for factor IX activation was reduced 8-fold for Ala and 400-1100-fold for other mutants, while binding of the inhibitors antithrombin and amyloid beta-precursor protein Kunitz domain (APPI) was impaired 13-2300-fold and 22-27000-fold, respectively. The data indicate that beta-branching of the side chain of residue 193 is deleterious for interactions with pAB, DFP and amidolytic substrates, situations where no S2'-P2' interactions are involved. When an S2'-P2' interaction is involved (factor IX, antithrombin, APPI), beta-branching and increased side chain length are detrimental. Molecular models indicate that the mutants have impaired S2' binding sites and that beta-branching causes steric conflicts with the FXIa 140-loop, which could perturb the local tertiary structure of the protease domain. In conclusion, enzyme activity is impaired in FXIa when Gly193 is replaced by a non-Gly residue, and residues with side chains that branch at the beta-carbon have the greatest effect on catalysis and binding of substrates.
Asunto(s)
Factor XIa/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Precursor de Proteína beta-Amiloide/farmacología , Antitrombinas/farmacología , Benzamidinas/química , Carbamatos/química , Factor IX/metabolismo , Factor XIa/antagonistas & inhibidores , Factor XIa/genética , Factor XIa/metabolismo , Humanos , Isoflurofato/farmacología , Cinética , Modelos MolecularesRESUMEN
Antiplatelet and antithrombotic activity of multiple oral dosing of prasugrel were evaluated in several animal species. Prasugrel's active metabolite concentration-relatedly inhibited in vitro ADP-induced aggregation of rat, rabbit, dog, monkey and human platelets. Oral administration of prasugrel to dogs (0.03-0.3 mg/kg/day) and monkeys (0.1 and 0.3 mg/kg/day) once a day for 14 days resulted in potent, dose-related and cumulative inhibition of ADP-induced platelet aggregation. The inhibitory effects reached a plateau on days 3 to 5 and thereafter were maintained during dosing. Inhibition decreased gradually after cessation of dosing with near full recovery by 7 days after last dose. Antiplatelet and antithrombotic activity of prasugrel and clopidogrel were further examined in rats. Multiple oral dosing of prasugrel (0.3-3 mg/kg/day) to rats resulted in more potent inhibition of platelet aggregation compared to clopidogrel (3-30 mg/kg/day) and ticlopidine (30-300 mg/kg/day). Separate experiments confirmed that platelet inhibition was associated with inhibition of [(3)H]-2-methylthio-ADP binding to rat platelets. In a rat model of electrically-induced arterial thrombosis, prasugrel (0.1-1 mg/kg/day, p.o.) significantly prolonged the time to arterial occlusion and increased the duration of arterial patency. The inhibition of platelet aggregation of prasugrel was about 10 and 300 times more potent than clopidogrel and ticlopidine, respectively. Overall these results show that in several species multiple oral administration of prasugrel results in more potent inhibition of platelet aggregation and thrombus formation than clopidogrel and ticlopidine, and that these effects are mediated by inhibition of platelet ADP receptors.
Asunto(s)
Piperazinas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2 , Tiofenos/farmacología , Trombosis/tratamiento farmacológico , Administración Oral , Adulto , Animales , Clopidogrel , Perros , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Humanos , Técnicas In Vitro , Macaca fascicularis , Masculino , Persona de Mediana Edad , Piperazinas/administración & dosificación , Inhibidores de Agregación Plaquetaria/administración & dosificación , Clorhidrato de Prasugrel , Conejos , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Tiofenos/administración & dosificación , Ticlopidina/análogos & derivados , Ticlopidina/farmacologíaRESUMEN
A series of cinnamylindoline derivatives were synthesized, and their factor Xa (FXa) inhibitory activities and selectivity over trypsin were evaluated. Among them, some novel derivatives showed potent FXa inhibitory activities and good selectivity over trypsin. Especially, (E)-2-{5-[1-(acetimidoyl)piperidin-4-yloxy]-2-[2-(5-amidino-2-hydroxyphenyl)ethen-1-yl]indolin-1-ylsulfonyl}acetic acid (22f) having 2-hydroxycinnamyl moiety exhibited the most potent FXa inhibitory activity in vitro. Furthermore, 22f also exhibited potent anticoagulant activities in vitro.
Asunto(s)
Anticoagulantes/farmacología , Cinamatos/farmacología , Inhibidores del Factor Xa , Indoles/farmacología , Anticoagulantes/síntesis química , Cinamatos/síntesis química , Cinamatos/química , Humanos , Indoles/síntesis química , Indoles/química , Concentración 50 Inhibidora , Espectroscopía de Resonancia Magnética , Modelos Químicos , Relación Estructura-Actividad , Tripsina/metabolismoRESUMEN
Prasugrel is a novel orally active thienopyridine with faster, higher and more reliable inhibition of platelet aggregation than clopidogrel reflecting its metabolism in vivo to an active metabolite with selective P2Y(12) antagonistic activity. Several lines of evidence support the contention that prasugrel provides selective P2Y(12) receptor antagonistic activity. To date, however, direct evidence of P2Y(12) specific action by prasugrel in vivo is limited. In the present study, effects of prasugrel on ex vivo platelet aggregation were examined in wild type (WT) and P2Y(12)(-/-) mice. In WT mice, prasugrel showed platelet inhibition that was 8.2 times more potent than clopidogrel. In P2Y(12)(-/-) mice, ADP induced platelet aggregation was minimal, and its extent was similar to that in prasugrel-treated WT mice. In addition, no further inhibition of platelet aggregation was observed after administration of prasugrel to P2Y(12)(-/-) mice. Furthermore, prasugrel-treated WT mice showed similar aggregation patterns using collagen- and murine PAR-4 agonist peptide to those of P2Y(12)(-/-) mice treated with vehicle or prasugrel. Overall, these results clearly provide additional in vivo evidence that prasugrel has selective P2Y(12) antagonistic activity.
Asunto(s)
Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Piperazinas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Receptores Purinérgicos P2/deficiencia , Receptores Purinérgicos P2/genética , Tiofenos/farmacología , Animales , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Femenino , Eliminación de Gen , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Agregación Plaquetaria/efectos de los fármacos , Clorhidrato de Prasugrel , Antagonistas del Receptor Purinérgico P2 , Receptores Purinérgicos P2Y12 , Caracteres SexualesRESUMEN
Factor Xa (FXa) is well known to play a pivotal role in blood coagulation, so FXa inhibitor is a promising drug candidate for prophylaxis and treatment of thromboembolic diseases. In the course of our research, we have found that (R)-5-[1-(acetimidoyl)piperidin-4-yloxy]-2-(7-amidinonaphthalen-2-yl)-1-(ethanesulfonyl)indoline ((R)-1) showed potent FXa inhibitory activity in vitro. However, single oral administation (RS)-1 showed high toxicity in mice. Among newly synthesized compounds, ({(RS)-5-[1-(acetimidoyl)piperidin-4-yloxy]-2-(7-amidinonaphthalen-2-yl)indolin-1-yl}sulfonyl)acetic acid ((RS)-11d) showed more potent FXa inhibitory activity and higher safety than (RS)-1. The R-isoform of compound 11d ((R)-11d) exhibited potent in vitro anticoagulant activity in human and hamster plasma. Orally administered (R)-11d also showed dose-dependent potent anticoagulant activity in hamsters, marmosets and cynomolgus monkeys. Compound (R)-11d with potent anticoagulant activity and high safety is therefore favorable as a novel oral FXa inhibitor.
Asunto(s)
Anticoagulantes/síntesis química , Anticoagulantes/farmacología , Inhibidores del Factor Xa , Indoles/síntesis química , Indoles/farmacología , Animales , Callithrix , Cricetinae , Relación Dosis-Respuesta a Droga , Humanos , Macaca fascicularis , Ratones , Estructura MolecularRESUMEN
A series of bisamidine derivatives each having a ring structure in the center of the molecule was synthesized and their Factor Xa (FXa) inhibitory activities were evaluated. Among them, some indoline derivatives showed potent inhibitory activities in vitro. In particular, (R)-18a having an (R)-configuration at the 2-position of the indoline ring exhibited the most potent FXa inhibitory activity in vitro, more potent than DX-9065a. Furthermore, (R)-18a exhibited more potent anticoagulant activity than DX-9065a. We also succeeded in obtaining an X-ray crystal structure of FXa bound with (R)-18a.
Asunto(s)
Inhibidores del Factor Xa , Indoles/síntesis química , Indoles/farmacología , Adulto , Animales , Coagulación Sanguínea/efectos de los fármacos , Fenómenos Químicos , Química Física , Cricetinae , Cristalografía por Rayos X , Humanos , Técnicas In Vitro , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Masculino , Modelos Moleculares , Conformación Molecular , Relación Estructura-ActividadRESUMEN
CS-747 (Prasugrel, LY640315) is a thienopyridine antiplatelet prodrug that is metabolized to the thiol-containing active metabolite R-138727,which binds to and irreversibly inhibits the platelet P2Y12ADP receptor. R-138727 is composed of 4 stereo-isomers, (R, S)-, (R, R)-, (S, S)-, and (S, R)-isomers (the first letter for the configuration of a chiral center at the sulfur-bearing position and the second for that at the benzylic position). In the present study, we determined the stereoselectivity of P2Y12 antagonist effects by assessing the antagonism of the [3H]-2-MeS-ADP that binds to human P2Y12 receptors expressed in Chinese hamster ovary cells as an affinity assay, and by the inhibition of ADP-induced aggregation of washed human platelets as a functional assay. R-138727 and its 2 components, R-99224, a mixture of (R, S)- and (S, R)-isomers and R-100364, a mixture of (R, R)- and (S, S)-isomers, inhibited [3H]-2-MeS-ADP binding and platelet aggregation. The rank order of potency of these compounds were identical in both assays: R-99224>R-138727>> R-100364. Inhibition of ADP-induced platelet aggregation by R-138727 and R-99224 was concentration- and time-related. In experiments using the 4 single stereo-isomers, all isomers inhibited ADP-induced platelet aggregation, but the (R, S)-isomer was found to be the most potent, followed by the (R, R)-isomer. These in vitro studies indicate that R- 138727 is an effective antagonist of P2Y12 and potent inhibitor of ADP-induced platelet aggregation, and that these antiplatelet activities of R-138727 are largely dependent on its (R, S)-isomer. This suggests that the (R)-configuration of the reactive thiol group of the active metabolite of CS-747 is critical for P2Y12 and platelet inhibitory activities.
Asunto(s)
Plaquetas/efectos de los fármacos , Proteínas de la Membrana/antagonistas & inhibidores , Piperazinas/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Profármacos/metabolismo , Antagonistas del Receptor Purinérgico P2 , Tiofenos/farmacología , Animales , Unión Competitiva , Células CHO , Cricetinae , Cricetulus , Ciclopropanos/química , Ciclopropanos/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Piperazinas/química , Piperazinas/metabolismo , Piperidinas/química , Piperidinas/farmacología , Inhibidores de Agregación Plaquetaria/química , Inhibidores de Agregación Plaquetaria/metabolismo , Clorhidrato de Prasugrel , Profármacos/química , Profármacos/farmacología , Receptores Purinérgicos P2Y12 , Estereoisomerismo , Relación Estructura-Actividad , Tiofenos/química , Tiofenos/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
The effects of R-102444 ((2R, 4R)-4-lauroyloxy-2-[2-[2-[2-(3-methoxy)phenyl]ethyl]phenoxy]ethyl-1-methylpyrrolidine hydrochloride) and its active metabolite R-96544 ((2R, 4R)-2-[2-[2-[2-(3-methoxy)phenyl]ethyl]phenoxy]ethyl-4-hydroxy-1-methylpyrrolidine hydrochloride), potent and selective 5-hydroxytryptamine 2A (5-HT2A) receptor antagonists, on development of pancreatitis were investigated in experimental models of acute and chronic pancreatitis. Rat acute pancreatitis was induced by caerulein (20 microg/kg) intraperitoneal injection and by pancreatic duct ligation. In both the models, serum amylase and lipase activities were markedly increased. R-102444 dose-dependently reduced these enzyme activities at a dose range of 10 to 100 mg/kg (p.o.) for the caerulein model and 0.3 to 10 mg/kg (p.o.) for the ligation model. In a mouse model of acute pancreatitis induced by a choline-deficient, ethionine (0.5%)-supplemented diet, subcutaneous administration of R-96544 (10-100 mg/kg, bid) reduced serum amylase activity. Histological analysis showed that R-96544 dose-dependently attenuated pancreatic necrosis, inflammation and vacuolization. The effect of R-102444 was further examined in male Wistar Bonn/Kobori rats (4-9 months of age) which spontaneously show pancreatic fibrosis and parenchymal destruction compatible with human chronic pancreatitis. In Wistar Bonn/Kobori rats (from 3 to 9 months of age) fed a diet containing 0.017% and 0.17% of R-102444, pancreatic weight, pancreatic protein and amylase content were higher compared to those in non-treated pancreatitis control rats. Histological analysis showed that R-102444 suppressed parenchymal destruction and replacement with adipose tissue, indicating inhibition of pancreatic atrophy. These results clearly indicate that R-102444 and R-96544 inhibit the progression of acute and chronic pancreatitis and support the contention of possible involvement of 5-HT2A receptors in the progression of experimental pancreatitis.
Asunto(s)
Pancreatitis/prevención & control , Pirrolidinas/farmacología , Antagonistas del Receptor de Serotonina 5-HT2 , Antagonistas de la Serotonina/farmacología , Enfermedad Aguda , Amilasas/sangre , Amilasas/metabolismo , Animales , Ceruletida/administración & dosificación , Ceruletida/toxicidad , Colina/administración & dosificación , Enfermedad Crónica , Suplementos Dietéticos , Etionina/administración & dosificación , Inyecciones Intraperitoneales , Ligadura/efectos adversos , Lipasa/sangre , Lipasa/metabolismo , Masculino , Tamaño de los Órganos/efectos de los fármacos , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Páncreas/patología , Conductos Pancreáticos/cirugía , Pancreatitis/etiología , Ratas , Ratas Endogámicas , Ratas Sprague-Dawley , Ratas Wistar , Receptor de Serotonina 5-HT2A/fisiología , Factores de TiempoRESUMEN
Studies of the mechanisms of blood coagulation zymogen activation demonstrate that exosites (sites on the activating complex distinct from the protease active site) play key roles in macromolecular substrate recognition. We investigated the importance of exosite interactions in recognition of factor IX by the protease factor XIa. Factor XIa cleavage of the tripeptide substrate S2366 was inhibited by the active site inhibitors p-aminobenzamidine (Ki 28 +/- 2 microM) and aprotinin (Ki 1.13 +/- 0.07 microM) in a classical competitive manner, indicating that substrate and inhibitor binding to the active site was mutually exclusive. In contrast, inhibition of factor XIa cleavage of S2366 by factor IX (Ki 224 +/- 32 nM) was characterized by hyperbolic mixed-type inhibition, indicating that factor IX binds to free and S2366-bound factor XIa at exosites. Consistent with this premise, inhibition of factor XIa activation of factor IX by aprotinin (Ki 0.89 +/- 0.52 microM) was non-competitive, whereas inhibition by active site-inhibited factor IXa beta was competitive (Ki 0.33 +/- 0.05 microM). S2366 cleavage by isolated factor XIa catalytic domain was competitively inhibited by p-aminobenzamidine (Ki 38 +/- 14 microM) but was not inhibited by factor IX, consistent with loss of factor IX-binding exosites on the non-catalytic factor XI heavy chain. The results support a model in which factor IX binds initially to exosites on the factor XIa heavy chain, followed by interaction at the active site with subsequent bond cleavage, and support a growing body of evidence that exosite interactions are critical determinants of substrate affinity and specificity in blood coagulation reactions.
Asunto(s)
Factor IX/metabolismo , Factor XIa/metabolismo , Aprotinina/metabolismo , Benzamidinas/metabolismo , Catálisis , Dominio Catalítico , Factor XIa/química , Humanos , Hidrólisis , Especificidad por SustratoRESUMEN
R-102444 is a prodrug that is metabolized into R-96544, a potent and selective 5-hydroxytryptamine2A (5-HT2A) receptor antagonist. The effects of R-102444 on peripheral vascular disease were examined using two different rat models: one induced by lauric acid and the other by ergotamine plus epinephrine. R-96544 (0.3-30 nM) relaxed the 5-HT (3 microM)-precontracted rat caudal artery in a concentration-dependent manner. The intravenous administration of R-96544 (0.3-3 microg/kg) to anesthetized rats inhibited the pressor response to 5-HT (50 microg/kg i.v.) dose dependently. The oral administration of R-102444 (1 mg/kg) to rats resulted in a marked inhibition of platelet aggregation induced by 5-HT plus ADP, and statistically significant inhibition was still evident 8 h after the dosing. In contrast, sarpogrelate, at a dose of 100 mg/kg p.o., produced only a moderate antiplatelet effect. Oral administration of R-102444 (1 mg/kg/day, o.d.) significantly prevented the progression of peripheral vascular lesion induced by the injection of lauric acid into a rat femoral artery, whereas sarpogrelate (100 mg/kg/day) showed only a minimal effect. Both 5-day treatments with R-102444 (1-30 mg/kg/day p.o., o.d.), one commenced 1 h before the injection of epinephrine plus ergotamine and one just after injection, resulted in the prevention of rat tail gangrene in a dose-dependent manner, whereas sarpogrelate (100 mg/kg) produced a minimal protection in this model. Based on these results, we conclude that 5-HT2A receptor activation is involved in peripheral vascular disease in the rat and that R-102444 is a useful oral agent for the investigation of diseases involving 5-HT2A receptor activation.
Asunto(s)
Modelos Animales de Enfermedad , Enfermedades Vasculares Periféricas/tratamiento farmacológico , Pirrolidinas/administración & dosificación , Antagonistas del Receptor de Serotonina 5-HT2 , Antagonistas de la Serotonina/administración & dosificación , Administración Oral , Animales , Relación Dosis-Respuesta a Droga , Masculino , Enfermedades Vasculares Periféricas/fisiopatología , Ratas , Ratas Sprague-Dawley , Ratas Wistar , Receptor de Serotonina 5-HT2A/fisiología , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
In serine proteases, Gly(193) is highly conserved with few exceptions. A patient with inherited deficiency of the coagulation serine protease factor XI (FXI) was reported to be homozygous for a Gly(555) --> Glu substitution. Gly(555) in FXI corresponds to Gly(193) in chymotrypsin, which is the numbering system used subsequently. To investigate the abnormality in FXI(G193E), we expressed and purified recombinant FXIa(G193E), activated it to FXIa(G193E), and compared its activity to wild type-activated FXI (FXIa(WT)). FXIa(G193E) activated FIX with approximately 300-fold reduced k(cat) and similar K(m), and hydrolyzed synthetic substrate with approximately 10-fold reduced K(m) and modestly reduced k(cat). Binding of antithrombin and the amyloid beta-precursor protein Kunitz domain inhibitor (APPI) to FXIa(G193E) was impaired approximately 8000- and approximately 100000-fold, respectively. FXIa(G193E) inhibition by diisopropyl fluoro-phosphate was approximately 30-fold slower and affinity for p-aminobenzamidine (S1 site probe) was 6-fold weaker than for FXIa(WT). The rate of carbamylation of NH(2)-Ile(16), which forms a salt bridge with Asp(194) in active serine proteases, was 4-fold faster for FXIa(G193E). These data indicate that the unoccupied active site of FXIa(G193E) is incompletely formed, and the amide N of Glu(193) may not point toward the oxyanion hole. Inclusion of saturating amounts of p-aminobenzamidine resulted in comparable rates of carbamylation for FXIa(WT) and FXIa(G193E), suggesting that the occupied active site has near normal conformation. Thus, binding of small synthetic substrates or inhibitors provides sufficient energy to allow the amide N of Glu(193) to point correctly toward the oxyanion hole. Homology modeling also indicates that the inability of FXIa(G193E) to bind antithrombin/APPI or activate FIX is caused, in part, by impaired accessibility of the S2' site because of a steric clash with Glu(193). Such arguments will apply to other serine proteases with substitutions of Gly(193) with a non-glycine residue.
Asunto(s)
Factor XIa/genética , Factor XIa/metabolismo , Glicina/metabolismo , Mutación Puntual , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Animales , Quimotripsina/química , Quimotripsina/genética , Quimotripsina/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/metabolismo , Factor XIa/química , Humanos , Modelos Moleculares , Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Serina Endopeptidasas/químicaRESUMEN
We examined the pharmacology of (2R,4R)-4-hydroxy-2-[2-[2-[2-(3-methoxy)phenyl]ethyl]phenoxy]ethyl-1-methylpyrrolidine hydrochloride (R-96544), the active form of a novel 5-HT(2A) receptor antagonist, (2R,4R)-4-lauroyloxy-2-[2-[2-[2-(3-methoxy)phenyl]ethyl]phenoxy]ethyl-1-methylpyrrolidine hydrochloride (R-102444). R-96544 produced a concentration-dependent inhibition of platelet aggregation induced by serotonin (5-hydroxytryptamine, 5-HT) alone or in combination with ADP in platelets from humans, monkeys, cats, rabbits, rats and mice. An intravenous administration of R-96544 to rabbits significantly inhibited ex vivo platelet aggregation induced by 5-hydroxytryptamine (5-HT) combined with epinephrine. An oral administration of R-102444 to rats also resulted in significant inhibition of ex vivo platelet aggregation, whereas R-102444 was ineffective in an in vitro platelet aggregation assay. These antiplatelet effects of R-96544 and R-102444 were more potent than those of two other 5-HT(2A) receptor antagonists, sarpogrelate and its active metabolite (+/-)-1-[2-[2-(3-methoxyphenyl)ethyl]phenoxy]-3-(dimethylamino)-2-propanol hydrochloride (M-1). A binding study using cat platelet membranes showed that R-96544 has high affinity for 5-HT(2A) receptors but no effect on non-serotonergic [3H]ketanserin-binding sites. R-96544 caused a parallel shift to the right of concentration-response curves for 5-HT in rat caudal artery contraction mediated by 5-HT(2A) receptors. Schild plot analysis gave a pA(2) value of 10.4 with a slope near unity (1.04). R-96544 also inhibited 5-HT(2A) receptor-mediated contraction of guinea pig trachea but not 5-HT(3) receptor-mediated contraction of guinea pig ileum and 5-HT(2B) receptor-mediated contraction of rat fundus preparation. R-96544 (i.v.) attenuated the pressor responses evoked by 5-HT (15 microg/kg, i.v.) but not by phenylephrine (5 microg/kg, i.v.) and angiotensin II (0.1 microg/kg, i.v.), after ganglionic blockade in anesthetized spontaneously hypertensive rats. These results show that R-96544, the active form of R-102444, is a novel 5-HT receptor antagonist with potent, competitive, and 5-HT(2A)-selective activity.
Asunto(s)
Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Pirrolidinas/farmacología , Receptores de Serotonina/metabolismo , Antagonistas de la Serotonina/farmacología , Adulto , Animales , Sitios de Unión , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Plaquetas/fisiología , Presión Sanguínea/efectos de los fármacos , Gatos , Relación Dosis-Respuesta a Droga , Humanos , Hipertensión/metabolismo , Hipertensión/fisiopatología , Técnicas In Vitro , Inyecciones Intravenosas , Ketanserina/metabolismo , Ketanserina/farmacología , Macaca fascicularis , Masculino , Ratones , Ratones Endogámicos , Inhibidores de Agregación Plaquetaria/administración & dosificación , Pirrolidinas/administración & dosificación , Conejos , Ratas , Ratas Endogámicas SHR , Ratas Sprague-Dawley , Receptor de Serotonina 5-HT2A , Antagonistas de la Serotonina/administración & dosificación , Especificidad de la EspecieRESUMEN
Platelet-agonist interaction results in aggregatory and secretory responses. While the activation of glycoprotein (GP) IIb/IIIa plays an essential role in platelet aggregation, its role in granule secretion is not clear. The present study was performed to examine the effect of 3-[[[[1-[4-(aminoiminomethyl) phenyl]-2-oxo-3S-pyrrolidinyl]amino]carbonyl]amino]-propanoate monohydrochloride salt (SC-57101A), a GPIIb/IIIa antagonist, on platelet alpha-granule secretion responses to collagen, ADP, and thrombin receptor activating peptide (TRAP). Both SC-57101A and prostaglandin E(1) (PGE(1)) inhibited collagen-, ADP-, and TRAP-induced platelet aggregation in a concentration-dependent manner. SC-57101A inhibited the collagen- and ADP-induced release of platelet-derived growth factor (PDGF) and beta-thromboglobulin (beta-TG) from platelets, but not TRAP-induced secretion of these granule contents. On the other hand, PGE(1) inhibited the release of PDGF and beta-TG from platelets activated with all the agonists used. ADP and TRAP elicited P-selectin expression in the absence of platelet aggregation, while collagen produced no such reaction. SC-57101A only moderately inhibited P-selectin expression induced by ADP and had no inhibitory effect on that induced by TRAP. The inhibition of ADP-induced secretion of alpha-granule contents by SC-57101A was abolished when platelets were pretreated with aspirin. These results suggest that GPIIb/IIIa activation plays a minor role, if any, in alpha-granule secretion in human platelets.