RESUMO
The list of potential cardiovascular biomarkers has expanded dramatically in recent years; however, the number of regulatory agency-approved diagnostic tests that guide treatment has been relatively unchanged compared with this growth in the discovery of putative biomarkers. Surrogate biochemical endpoints such as LDL and HDL are included in the current guidelines of various regulatory agencies for the management of cardiovascular diseases, as a result of many years of research. Inclusion of tests for these markers, as well as any future tests, in treatment guidelines requires data obtained from large-scale clinical trials comparing these endpoints with 'hard' clinical endpoints, such as morbidity and mortality. Consequently, current guidelines are limited to conventional in vitro tests and incorporate few novel tests for guiding or modifying treatment. Despite the failure to include newer in vitro tests in cardiovascular treatment and prevention paradigms, ongoing biomarker discovery and assay optimization has provided many improvements in drug discovery and development, and has afforded opportunities for the optimized medical treatment of patients with cardiovascular disease.
Assuntos
Biomarcadores/análise , Fármacos Cardiovasculares/uso terapêutico , Doenças Cardiovasculares/diagnóstico , Doenças Cardiovasculares/tratamento farmacológico , Proteína C-Reativa/análise , Descoberta de Drogas , Humanos , Peptídeo Natriurético Encefálico/análise , Troponina I/análiseRESUMO
Aiming to improve upon previously disclosed Factor Xa inhibitors, a series of 4,4-disubstituted pyrrolidine-1,2-dicarboxamides were explored with the intent of increasing the projected human half-life versus 5 (projected human t(1/2)=6 h). A stereospecific route to compounds containing a 4-aryl-4-hydroxypyrrolidine scaffold was developed, resulting in several compounds that demonstrated an increase in the half-life as well as an increase in the in vitro potency compared to 5. Reported herein is the discovery of 26, containing a (2R,4S)-4-hydroxy-4-(2,4-difluorophenyl)-pyrrolidine scaffold, which is a selective, orally bioavailable, efficacious Factor Xa inhibitor that appears suitable for a once-daily dosing (projected human t(1/2)=23 h).
Assuntos
Pirrolidinas/farmacologia , Administração Oral , Cristalografia por Raios X , Meia-Vida , Humanos , Pirrolidinas/administração & dosagem , Pirrolidinas/farmacocinéticaRESUMO
The objective of this study was to determine if orally-administered PD0348292, a direct specific factor Xa inhibitor, inhibits thrombosis following porcine carotid arterial injury comparably to aspirin or clopidogrel alone or in combination. We further sought to determine whether the antithrombotic efficacy in vivo could be predicted using an ex-vivo perfusion chamber. Oral treatments included: PD0348292 (0.4, 0.9, or 4.3 mg/kg); PD0348292 (0.4 mg/kg) plus aspirin (325 mg); aspirin; clopidogrel (75 mg); aspirin plus clopidogrel; or vehicle (n = 6-10/group). Aspirin and clopidogrel were administered 27 and four hours pre-injury and PD0348292 or vehicle was administered four hours pre-injury. Both carotid arteries were crush-injured, and thrombus was measured by detection of (111)In-platelets over 30 minutes. Prior to injury, the antithrombotic efficacy was assessed by ex-vivo perfusion chamber platelet deposition. PD0348292 produced dose-dependent prothrombin time (0.9- to 2.9-fold) and aPTT (1.4- to 2.5-fold) prolongations. Bleeding times were significantly prolonged in each active drug group compared to vehicle, but were not significantly different between drug groups. PD0348292 significantly inhibited arterial platelet deposition (x10(6)/cm(2)) at 4.3(549 +/- 1,066), 0.9 (399 +/- 162) and 0.4 mg/kg (531 +/- 470) compared to vehicle (2,242 +/- 1,443). Aspirin (992 +/- 973), clopidogrel (537 +/- 483), clopidogrel plus aspirin (228 +/- 66) or PD0348292 plus aspirin (558 +/- 317) also significantly inhibited platelet deposition, although these values were not significantly different than with any dose of PD348292. Perfusion chamber platelet deposition correlated significantly with in-vivo anti-thrombotic response. In conclusion, PD0348292 inhibited arterial thrombosis comparable to aspirin plus clopidogrel. Perfusion chamber methodology may be useful in predicting in-vivo antithrombotic efficacy.
Assuntos
Inibidores do Fator Xa , Inibidores da Agregação Plaquetária/farmacologia , Piridonas/farmacologia , Pirrolidinas/farmacologia , Trombose/prevenção & controle , Administração Oral , Animais , Aspirina/administração & dosagem , Aspirina/farmacologia , Tempo de Sangramento , Coagulação Sanguínea/efeitos dos fármacos , Lesões das Artérias Carótidas/complicações , Lesões das Artérias Carótidas/tratamento farmacológico , Trombose das Artérias Carótidas/etiologia , Trombose das Artérias Carótidas/prevenção & controle , Clopidogrel , Modelos Animais de Doenças , Feminino , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/administração & dosagem , Piridonas/administração & dosagem , Pirrolidinas/administração & dosagem , Sus scrofa , Trombose/etiologia , Ticlopidina/administração & dosagem , Ticlopidina/análogos & derivados , Ticlopidina/farmacologiaRESUMO
OBJECTIVE: The clinical use of venous stents is increasing dramatically. Although antiplatelet agents are required for arterial stent patency, optimal thrombo-prophylaxis after venous stenting remains undefined. To address this issue, PD0348292, a direct Factor Xa inhibitor, was compared with antiplatelet therapy in a porcine venous stent model. METHODS AND RESULTS: Four hours before stent deployment, pigs (n=5 to 6 per group) received oral PD0348292 at 0.4, 0.9, 4.3 mg/kg, or 0.4 mg/kg plus aspirin (325 mg). Aspirin, clopidogrel (75 mg), aspirin plus clopidogrel, or vehicle (n=10) were administered daily for 2 days before the procedure. Two hours after stent placement, thrombi were quantified by autologous (111)In-platelet content and weights. Thrombus weight and platelet deposition were significantly reduced by PD0348292 at 0.4 (49+/-79 mg and 110+/-145x10(6)/cm2), 0.9 (5+/-6 mg and 107+/-128x10(6)/cm2), 4.3 mg/kg (0+/-0 mg and 87+/-125x10(6)/cm2), and PD348292 plus aspirin (20+/-40 mg and 157+/-70x10(6)/cm2) compared with vehicle (402+/-226 mg; 584+/-454x10(6)/cm2). Despite prolonging bleeding times and inhibiting platelet aggregation, neither aspirin (567+/-683 mg and 533+/-622x10(6)/cm2), clopidogrel (404+/-349 mg and 178+/-101x10(6)/cm2), nor aspirin plus clopidogrel (247+/-261 mg and 231+/-266x10(6)/cm2) significantly decreased stent thrombosis. CONCLUSIONS: PD0348292 completely inhibited thrombosis after venous stenting. Platelet accretion in these venous thrombi appear to involve pathways distinct from arachidonate metabolism or ADP P2Y12 receptor activation.
Assuntos
Antitrombina III/farmacologia , Aspirina/farmacologia , Piridonas/administração & dosagem , Pirrolidinas/administração & dosagem , Stents , Ticlopidina/análogos & derivados , Trombose Venosa/tratamento farmacológico , Administração Oral , Angioplastia/métodos , Animais , Clopidogrel , Constrição Patológica/prevenção & controle , Modelos Animais de Doenças , Quimioterapia Combinada , Feminino , Veia Ilíaca/cirurgia , Cuidados Pré-Operatórios/métodos , Probabilidade , Distribuição Aleatória , Valores de Referência , Ticlopidina/farmacologia , Resultado do Tratamento , Grau de Desobstrução Vascular/efeitos dos fármacos , Trombose Venosa/cirurgiaRESUMO
Herein, we report the discovery of novel, proline-based factor Xa inhibitors containing a neutral P1 chlorophenyl pharmacophore. Through the additional incorporation of 1-(4-amino-3-fluoro-phenyl)-1H-pyridin-2-one 22, as a P4 pharmacophore, we discovered compound 7 (PD 0348292). This compound is a selective, orally bioavailable, efficacious FXa inhibitor that is currently in phase II clinical trials for the treatment and prevention of thrombotic disorders.
Assuntos
Antitrombina III/síntese química , Antitrombina III/farmacologia , Piridonas/síntese química , Piridonas/farmacologia , Pirrolidinas/síntese química , Pirrolidinas/farmacologia , Animais , Anticoagulantes/síntese química , Anticoagulantes/farmacocinética , Anticoagulantes/farmacologia , Antitrombina III/farmacocinética , Cristalografia por Raios X , Cães , Humanos , Masculino , Piridonas/farmacocinética , Pirrolidinas/farmacocinética , Coelhos , Ratos , Relação Estrutura-AtividadeRESUMO
A novel series of pyrrolidine-1,2-dicarboxamides was discovered as factor Xa inhibitors using structure-based drug design. This series consisted of a neutral 4-chlorophenylurea P1, a biphenylsulfonamide P4 and a D-proline scaffold (1, IC(50) = 18 nM). Optimization of the initial hit resulted in an orally bioavailable, subnanomolar inhibitor of factor Xa (13, IC(50) = 0.38 nM), which was shown to be efficacious in a canine electrolytic model of thrombosis with minimal bleeding.
Assuntos
Antitrombina III/química , Química Farmacêutica/métodos , Ácido Pirrolidonocarboxílico/farmacologia , Administração Oral , Animais , Antitrombina III/farmacologia , Cristalização , Cães , Desenho de Fármacos , Humanos , Concentração Inibidora 50 , Modelos Químicos , Modelos Moleculares , Ligação Proteica , Ácido Pirrolidonocarboxílico/química , Relação Estrutura-Atividade , Fatores de TempoRESUMO
The activated factor VII/tissue factor complex (FVIIa/TF) is known to play a key role in the formation of blood clots. Inhibition of this complex may lead to new antithrombotic drugs. A fluoropyridine-based series of FVIIa/TF inhibitors was discovered which utilized a diisopropylamino group for binding in the S2 and S3 binding pockets of the active site of the enzyme complex. In this series, an enhancement in binding affinity was observed by substitution at the 5-position of the hydroxybenzoic acid sidechain. An X-ray crystal structure indicates that amides at this position may increase inhibitor binding affinity through interactions with the S1'/S2' pocket.
Assuntos
Inibidores Enzimáticos/farmacologia , Fator VIIa/antagonistas & inibidores , Piridinas/farmacologia , Tromboplastina/antagonistas & inibidores , Cristalografia por Raios X , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Modelos Moleculares , Estrutura Molecular , Piridinas/síntese química , Piridinas/química , Relação Estrutura-AtividadeRESUMO
The activated Factor VII/tissue factor complex (FVIIa/TF) plays a key role in the formation of blood clots. Inhibition of this complex may lead to new antithrombotic drugs. An X-ray crystal structure of a fluoropyridine-based FVIIa/TF inhibitor bound in the active site of the enzyme complex suggested that incorporation of substitution at the 5-position of the hydroxybenzoic acid side chain could lead to the formation of more potent inhibitors through interactions with the S1'/S2' pocket.
Assuntos
Inibidores Enzimáticos/síntese química , Fator VIIa/química , Fibrinolíticos/síntese química , Piridinas/síntese química , Tromboplastina/química , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/química , Fator VIIa/antagonistas & inibidores , Inibidores do Fator Xa , Fibrinolíticos/química , Fibrinolíticos/farmacologia , Humanos , Concentração Inibidora 50 , Ligação Proteica , Tempo de Protrombina , Piridinas/química , Relação Estrutura-Atividade , Tromboplastina/antagonistas & inibidoresRESUMO
The enzyme complex prothrombinase plays a pivotal role in fibrin clot development through the production of thrombin, making this enzyme complex an attractive target for therapeutic regulation. This study both functionally and structurally characterizes a potent, highly selective, active site directed inhibitor of human factor Xa and prothrombinase, PD0313052, and identifies structurally conserved residues in factor Xa and prothrombinase. Analyses of the association and dissociation of PD0313052 with human factor Xa identified a reversible, slow-onset mechanism of inhibition and a simple, single-step bimolecular association between factor Xa and PD0313052. This interaction was governed by association (k(on)) and dissociation (k(off)) rate constants of (1.0 +/- 0.1) x 10(7) M(-1) s(-1) and (1.9 +/- 0.5) x 10(-3) s(-1), respectively. The inhibition of human factor Xa by PD0313052 displayed significant tight-binding character described by a Ki* = 0.29 +/- 0.08 nM. Similar analyses of the inhibition of human prothrombinase by PD0313052 also identified a slow-onset mechanism with a Ki* = 0.17 +/- 0.03 nM and a k(on) and k(off) of (0.7 +/- 0.1) x 10(7) M(-1) s(-1) and (1.7 +/- 0.8) x 10(-3) s(-1), respectively. Crystals of factor Xa and PD0313052 demonstrated hydrogen bonding contacts within the S1-S4 pocket at residues Ser195, Asp189, Gly219, and Gly216, as well as interactions with aromatic residues within the S4 pocket. Overall, these data demonstrate that the inhibition of human factor Xa by PD0313052 occurs via a slow, tight-binding mechanism and indicate that active site residues of human factor Xa, including the catalytic Ser195, are effectively unaltered following assembly into prothrombinase.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores do Fator Xa , Fator Xa/química , Piperidinas/farmacologia , Tromboplastina/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Modelos Moleculares , Conformação ProteicaRESUMO
A systematic modification of the C(3) side-chain of the beta-aminoester class of factor Xa inhibitors and a survey of P(4) variations is described. These changes have resulted in the identification of sub-nanomolar inhibitors with improved selectivity versus related proteases. Coagulation parameters (i.e., APTT doubling concentrations) are also improved.
Assuntos
Inibidores do Fator Xa , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , ÉsteresRESUMO
Further optimization of the beta-aminoester class of factor Xa (fXa) inhibitors is described culminating in the identification of 9c (FXV673), a potent and selective factor Xa inhibitor with excellent in vivo anticoagulant activity. An X-ray structure of FXV673 bound to human fXa is also presented. Based on its selectivity, potent in vivo activity and favorable pre-clinical safety profile, FXV673 was selected for further development and is currently undergoing clinical trials.
Assuntos
Anticoagulantes/química , Anticoagulantes/farmacologia , Óxidos N-Cíclicos/química , Óxidos N-Cíclicos/farmacologia , Inibidores do Fator Xa , Piridinas/química , Piridinas/farmacologia , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/farmacologia , Cristalografia por Raios X , Ésteres , Humanos , Modelos Moleculares , Estrutura MolecularRESUMO
In an effort to understand the modulation of G protein-coupled receptor (GPCR)-mediated signalling in platelets, we sought to identify which regulators of G protein signalling proteins (RGSs) are present in human platelets. Using degenerate oligonucleotides, we performed RT-PCR with human platelet and megakaryocytic cell line RNA. In addition to confirming the presence of several known RGS transcripts, we found a novel RGS domain-containing transcript in platelet RNA. Northern blot analysis of multiple human tissues indicates that this transcript is most abundantly expressed in platelets compared to other tissues examined. Full-length cloning of this novel RGS, which we now term RGS18, demonstrates that this transcript is predicted to encode a 235-amino acid protein that is most closely related to RGS5 (46% identity) and that has approximately 30-40% identity to other RGS proteins. RGS18 is expressed in platelet, leukocyte, and megakaryocyte cell lines and binds to endogenous Galphai1, Galphai2, Galphai3, and Galphaq but not Galphaz, Galphas or Galpha12 in vitro.