RESUMEN
PAR4 is a promising antithrombotic target with potential for separation of efficacy from bleeding risk relative to current antiplatelet therapies. In an effort to discover a novel PAR4 antagonist chemotype, a quinoxaline-based HTS hit 3 with low µM potency was identified. Optimization of the HTS hit through the use of positional SAR scanning and the design of conformationally constrained cores led to the discovery of a quinoxaline-benzothiazole series as potent and selective PAR4 antagonists. The lead compound 48, possessing a 2 nM IC50 against PAR4 activation by γ-thrombin in platelet-rich plasma (PRP) and greater than 2500-fold selectivity versus PAR1, demonstrated robust antithrombotic efficacy and minimal bleeding in the cynomolgus monkey models.
Asunto(s)
Fibrinolíticos , Trombosis , Animales , Fibrinolíticos/farmacología , Fibrinolíticos/uso terapéutico , Macaca fascicularis , Quinoxalinas/farmacología , Quinoxalinas/uso terapéutico , Receptores de Trombina , Trombina , Hemorragia , Trombosis/tratamiento farmacológico , Trombosis/prevención & control , Receptor PAR-1 , Plaquetas , Agregación PlaquetariaRESUMEN
This paper describes our continued efforts in the area of small-molecule apelin receptor agonists. Recently disclosed compound 2 showed an acceptable metabolic stability but demonstrated monodemethylation of the dimethoxyphenyl group to generate atropisomer metabolites in vitro. In this article, we extended the structure-activity relationship at the C2 position that led to the identification of potent pyrazole analogues with excellent metabolic stability. Due to the increased polarity at C2, the permeability for these compounds decreased. Further adjustment of the polarity by replacing the N1 2,6-dimethoxyphenyl group with a 2,6-diethylphenyl group and reoptimization for the potency of the C5 pyrroloamides resulted in potent compounds with improved permeability. Compound 21 displayed excellent pharmacokinetic profiles in rat, monkey, and dog models and robust pharmacodynamic efficacy in the rodent heart failure model. Compound 21 also showed an acceptable safety profile in preclinical toxicology studies and was selected as a backup development candidate for the program.
Asunto(s)
Receptores de Apelina/agonistas , Insuficiencia Cardíaca/tratamiento farmacológico , Pirimidinonas/farmacología , Animales , Perros , Descubrimiento de Drogas , Humanos , Pirimidinonas/química , Pirimidinonas/farmacocinética , Pirimidinonas/uso terapéutico , Ratas , Relación Estructura-ActividadRESUMEN
The apelin receptor (APJ) is a significant regulator of cardiovascular function and is involved in heart failure and other cardiovascular diseases. (Pyr1)apelin-13 is one of the endogenous agonists of the APJ receptor. Administration of (Pyr1)apelin-13 increases cardiac output in preclinical models and humans. Recently we disclosed clinical lead BMS-986224 (1), a C3 oxadiazole pyridinone APJ receptor agonist with robust pharmacodynamic effects similar to (Pyr1)apelin-13 in an acute rat pressure-volume loop model. Herein we describe the structure-activity relationship of the carboxamides as oxadiazole bioisosteres at C3 of the pyridinone core and C5 of the respective pyrimidinone core. This study led to the identification of structurally differentiated 6-hydroxypyrimidin-4(1H)-one-3-carboxamide 14a with pharmacodynamic effects comparable to those of compound 1.
RESUMEN
Heart failure (HF) treatment remains a critical unmet medical need. Studies in normal healthy volunteers and HF patients have shown that [Pyr1]apelin-13, the endogenous ligand for the APJ receptor, improves cardiac function. However, the short half-life of [Pyr1]apelin-13 and the need for intravenous administration have limited the therapeutic potential for chronic use. We sought to identify potent, small-molecule APJ agonists with improved pharmaceutical properties to enable oral dosing in clinical studies. In this manuscript, we describe the identification of a series of pyrimidinone sulfones as a structurally differentiated series to the clinical lead (compound 1). Optimization of the sulfone series for potency, metabolic stability and oral bioavailability led to the identification of compound 22, which showed comparable APJ potency to [Pyr1]apelin-13 and exhibited an acceptable pharmacokinetic profile to advance to the acute hemodynamic rat model.
Asunto(s)
Receptores de Apelina/agonistas , Fármacos Cardiovasculares/farmacología , Fármacos Cardiovasculares/farmacocinética , Péptidos y Proteínas de Señalización Intercelular/farmacología , Animales , Área Bajo la Curva , Fármacos Cardiovasculares/síntesis química , Diseño de Fármacos , Semivida , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Macaca fascicularis , Estructura Molecular , Pirimidinonas/química , Pirimidinonas/farmacología , Ratas , Relación Estructura-ActividadRESUMEN
Apelin-13 is an endogenous peptidic agonist of the apelin receptor (APJ) receptor with the potential for improving cardiac function in heart failure patients. However, the low plasma stability of apelin-13 necessitates continuous intravenous infusion for therapeutic use. There are several approaches to increase the stability of apelin-13 including attachment of pharmacokinetic enhancing groups, stabilized peptides, and Fc-fusion approaches. We sought a small-molecule APJ receptor agonist approach to target a compound with a pharmacokinetic profile amenable for chronic oral administration. This manuscript describes sequential optimization of the pyrimidinone series, leading to pyridinone 14, with in vitro potency equivalent to the endogenous ligand apelin-13 and with an excellent oral bioavailability and PK profile in multiple preclinical species. Compound 14 exhibited robust pharmacodynamic effects similar to apelin-13 in an acute rat pressure-volume loop model and was advanced as a clinical candidate.
Asunto(s)
Receptores de Apelina/agonistas , Piridonas/química , Piridonas/farmacología , Animales , Receptores de Apelina/metabolismo , Perros , Descubrimiento de Drogas , Haplorrinos , Humanos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Masculino , Modelos Moleculares , Piridonas/farmacocinética , Ratas , Ratas Sprague-DawleyRESUMEN
Antiplatelet agents are proven efficacious treatments for cardiovascular and cerebrovascular diseases. However, the existing drugs are compromised by unwanted and sometimes life-threatening bleeding that limits drug usage or dosage. There is a substantial unmet medical need for an antiplatelet drug with strong efficacy and low bleeding risk. Thrombin is a potent platelet agonist that directly induces platelet activation via the G protein (heterotrimeric guanine nucleotide-binding protein)-coupled protease-activated receptors PAR1 and PAR4. A PAR1 antagonist is approved for clinical use, but its use is limited by a substantial bleeding risk. Conversely, the potential of PAR4 as an antiplatelet target has not been well characterized. Using anti-PAR4 antibodies, we demonstrated a low bleeding risk and an effective antithrombotic profile with PAR4 inhibition in guinea pigs. Subsequently, high-throughput screening and an extensive medicinal chemistry effort resulted in the discovery of BMS-986120, an orally active, selective, and reversible PAR4 antagonist. In a cynomolgus monkey arterial thrombosis model, BMS-986120 demonstrated potent and highly efficacious antithrombotic activity. BMS-986120 also exhibited a low bleeding liability and a markedly wider therapeutic window compared to the standard antiplatelet agent clopidogrel tested in the same nonhuman primate model. These preclinical findings define the biological role of PAR4 in mediating platelet aggregation. In addition, they indicate that targeting PAR4 is an attractive antiplatelet strategy with the potential to treat patients at a high risk of atherothrombosis with superior safety compared with the current standard of care.
Asunto(s)
Anticuerpos/uso terapéutico , Fibrinolíticos/uso terapéutico , Hemorragia/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Receptores de Trombina/antagonistas & inhibidores , Administración Oral , Animales , Plaquetas/metabolismo , Cobayas , Células HEK293 , Humanos , Concentración 50 Inhibidora , Macaca fascicularis , Masculino , Dominios Proteicos , Receptor PAR-1/metabolismo , Accidente Cerebrovascular/tratamiento farmacológico , Trombina/química , Trombosis , Resultado del TratamientoRESUMEN
Current antithrombotic discovery efforts target compounds that are highly efficacious in thrombus reduction with less bleeding liability than the standard of care. Preclinical data suggest that P2Y1 antagonists may have lower bleeding liabilities than P2Y12 antagonists while providing similar antithrombotic efficacy. This article describes our continuous SAR efforts in a series of 7-hydroxyindolinyl diaryl ureas. When dosed orally, 4-trifluoromethyl-7-hydroxy-3,3-dimethylindolinyl analogue 4 was highly efficacious in a model of arterial thrombosis in rats with limited bleeding. The chemically labile CF3 group in 4 was then transformed to various groups via a novel one-step synthesis, yielding a series of potent P2Y1 antagonists. Among them, the 4-benzothiazole-substituted indolines had desirable PK properties in rats, specifically, low clearance and small volume of distribution. In addition, compound 40 had high i.v. exposure and modest bioavailability, giving it the best overall profile.
Asunto(s)
Antagonistas del Receptor Purinérgico P2Y/farmacología , Urea/análogos & derivados , Animales , Humanos , Espectroscopía de Resonancia Magnética , Antagonistas del Receptor Purinérgico P2Y/farmacocinética , Espectrometría de Masa por Ionización de Electrospray , Urea/farmacocinética , Urea/farmacologíaRESUMEN
Spiropiperidine indoline-substituted diaryl ureas had been identified as antagonists of the P2Y1 receptor. Enhancements in potency were realized through the introduction of a 7-hydroxyl substitution on the spiropiperidinylindoline chemotype. SAR studies were conducted to improve PK and potency, resulting in the identification of compound 3e, a potent, orally bioavailable P2Y1 antagonist with a suitable PK profile in preclinical species. Compound 3e demonstrated a robust antithrombotic effect in vivo and improved bleeding risk profile compared to the P2Y12 antagonist clopidogrel in rat efficacy/bleeding models.
Asunto(s)
Compuestos de Fenilurea/química , Inhibidores de Agregación Plaquetaria/química , Antagonistas del Receptor Purinérgico P2Y/química , Receptores Purinérgicos P2Y1/química , Tiazoles/química , Urea/análogos & derivados , Administración Oral , Animales , Perros , Semivida , Macaca fascicularis , Compuestos de Fenilurea/farmacocinética , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/uso terapéutico , Agregación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacocinética , Inhibidores de Agregación Plaquetaria/farmacología , Inhibidores de Agregación Plaquetaria/uso terapéutico , Antagonistas del Receptor Purinérgico P2Y/farmacocinética , Antagonistas del Receptor Purinérgico P2Y/farmacología , Antagonistas del Receptor Purinérgico P2Y/uso terapéutico , Ratas , Receptores Purinérgicos P2Y1/metabolismo , Relación Estructura-Actividad , Tiazoles/farmacocinética , Tiazoles/farmacología , Tiazoles/uso terapéutico , Trombosis/tratamiento farmacológico , Urea/farmacocinética , Urea/farmacología , Urea/uso terapéuticoRESUMEN
Preclinical antithrombotic efficacy and bleeding models have demonstrated that P2Y1 antagonists are efficacious as antiplatelet agents and may offer a safety advantage over P2Y12 antagonists in terms of reduced bleeding liabilities. In this article, we describe the structural modification of the tert-butyl phenoxy portion of lead compound 1 and the subsequent discovery of a novel series of conformationally constrained ortho-anilino diaryl ureas. In particular, spiropiperidine indoline-substituted diaryl ureas are described as potent, orally bioavailable small-molecule P2Y1 antagonists with improved activity in functional assays and improved oral bioavailability in rats. Homology modeling and rat PK/PD studies on benchmark compound 3l will also be presented. Compound 3l was our first P2Y1 antagonist to demonstrate a robust oral antithrombotic effect with mild bleeding liability in the rat thrombosis and hemostasis models.
Asunto(s)
Diseño de Fármacos , Conformación Molecular , Compuestos de Fenilurea/farmacología , Compuestos de Fenilurea/farmacocinética , Antagonistas del Receptor Purinérgico P2Y/farmacología , Antagonistas del Receptor Purinérgico P2Y/farmacocinética , Receptores Purinérgicos P2Y1/metabolismo , Compuestos de Espiro/farmacología , Compuestos de Espiro/farmacocinética , Urea/farmacología , Urea/farmacocinética , Animales , Disponibilidad Biológica , Humanos , Indoles/química , Modelos Moleculares , Compuestos de Fenilurea/química , Compuestos de Fenilurea/metabolismo , Antagonistas del Receptor Purinérgico P2Y/química , Antagonistas del Receptor Purinérgico P2Y/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2Y1/química , Homología de Secuencia de Aminoácido , Compuestos de Espiro/química , Compuestos de Espiro/metabolismo , Urea/química , Urea/metabolismoRESUMEN
Preclinical data suggests that P2Y1 antagonists, such as diarylurea compound 1, may provide antithrombotic efficacy similar to P2Y12 antagonists and may have the potential of providing reduced bleeding liabilities. This manuscript describes a series of diarylureas bearing solublizing amine side chains as potent P2Y1 antagonists. Among them, compounds 2l and 3h had improved aqueous solubility and maintained antiplatelet activity compared with compound 1. Compound 2l was moderately efficacious in both rat and rabbit thrombosis models and had a moderate prolongation of bleeding time in rats similar to that of compound 1.
Asunto(s)
Fibrinolíticos/química , Compuestos de Fenilurea/química , Antagonistas del Receptor Purinérgico P2Y/química , Piridinas/química , Receptores Purinérgicos P2Y1/química , Urea/química , Animales , Células CACO-2 , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Fibrinolíticos/síntesis química , Fibrinolíticos/farmacocinética , Semivida , Humanos , Microsomas Hepáticos/metabolismo , Tiempo de Tromboplastina Parcial , Compuestos de Fenilurea/farmacocinética , Compuestos de Fenilurea/uso terapéutico , Agregación Plaquetaria/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2Y/farmacocinética , Antagonistas del Receptor Purinérgico P2Y/uso terapéutico , Piridinas/farmacocinética , Piridinas/uso terapéutico , Conejos , Ratas , Receptores Purinérgicos P2Y1/metabolismo , Solubilidad , Relación Estructura-Actividad , Trombosis/tratamiento farmacológico , Urea/farmacocinética , Urea/uso terapéutico , Agua/químicaRESUMEN
Two distinct G protein-coupled purinergic receptors, P2Y1 and P2Y12, mediate ADP-driven platelet activation. The clinical effectiveness of P2Y12 blockade is well established. Recent preclinical data suggest that P2Y1 and P2Y12 inhibition provide equivalent antithrombotic efficacy, while targeting P2Y1 has the potential for reduced bleeding liability. In this account, the discovery of a 2-(phenoxypyridine)-3-phenylurea chemotype that inhibited ADP-mediated platelet aggregation in human blood samples is described. Optimization of this series led to the identification of compound 16, 1-(2-(2-tert-butylphenoxy)pyridin-3-yl)-3-4-(trifluoromethoxy)phenylurea, which demonstrated a 68 ± 7% thrombus weight reduction in an established rat arterial thrombosis model (10 mg/kg plus 10 mg/kg/h) while only prolonging cuticle and mesenteric bleeding times by 3.3- and 3.1-fold, respectively, in provoked rat bleeding time models. These results suggest that a P2Y1 antagonist could potentially provide a safe and efficacious antithrombotic profile.
Asunto(s)
Fibrinolíticos/síntesis química , Compuestos de Fenilurea/síntesis química , Antagonistas del Receptor Purinérgico P2Y/síntesis química , Piridinas/síntesis química , Urea/análogos & derivados , Animales , Arteriopatías Oclusivas/sangre , Arteriopatías Oclusivas/tratamiento farmacológico , Tiempo de Sangría , Fibrinolíticos/química , Fibrinolíticos/farmacología , Células HEK293 , Humanos , Masculino , Compuestos de Fenilurea/química , Compuestos de Fenilurea/farmacología , Agregación Plaquetaria/efectos de los fármacos , Antagonistas del Receptor Purinérgico P2Y/química , Antagonistas del Receptor Purinérgico P2Y/farmacología , Piridinas/química , Piridinas/farmacología , Ratas , Relación Estructura-Actividad , Trombosis/sangre , Trombosis/tratamiento farmacológico , Urea/síntesis química , Urea/química , Urea/farmacologíaRESUMEN
INTRODUCTION: Published evidence suggests that phosphoinositide 3 kinase-ß (PI3K-ß) plays an important role in platelet aggregation and shear activation. TGX-221 is a selective PI3K-ß inhibitor with a good separation of anti-thrombotic efficacy and bleeding (therapeutic index) in rats. Our goal was to further evaluate potential of a PI3K-ß inhibitor as an anti-thrombotic agent by determining the therapeutic index in another species and efficacy model. Reported effects of TGX-221 in rats were also confirmed. MATERIALS AND METHODS: TGX-221 (0.3 + 0.3, 1 + 1, 3 + 3 mg/kg + mg/kg/hr, i.v.) or vehicle was given to mice starting 15 min prior to FeCl(3) arterial thrombosis (AT), tail or kidney bleeding time (BT) procedures. RESULTS: Integrated blood flow over 30 min (%baseline mean ± SEM) improved (p < 0.05) with TGX-221 doses 1 + 1 (49 ± 13.9%) and 3+3 (88 ± 10.6%) versus 0.3 + 0.3 (10 ± 0.8%) and vehicle (10 ± 0.6%). Vascular patency (non-occluded/total arteries) improved (p < 0.01) with TGX-221 doses of 3 + 3 (7/8), but not 0.3 + 0.3 (0/8) or 1 + 1 (4/8) versus vehicle (0/8). Tail BT (sec) increased (p < 0.05) with TGX-221 doses of 3 + 3 (median 1560) and 1 + 1 (1305) versus vehicle (225). Mean renal BT (sec) increased (p < 0.05) in all TGX-221 groups (3 + 3: 510 + 26; 1 + 1: 478 + 41; 0.3 + 0.3: 246 + 37) versus vehicle (123 + 9). For comparison, a reference agent, aspirin (30 mpk, i.p.) increased tail BT 1.9X and renal BT 2.6X. CONCLUSIONS: The novel finding of a clear impact on hemostasis by TGX-221 was demonstrated by increased bleeding in two models in mice at anti-thrombotic doses. The results suggest a narrower therapeutic index for this PI3K-ß inhibitor than previously recognized, at least for this species.
Asunto(s)
Plaquetas/efectos de los fármacos , Fibrinolíticos/farmacología , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Pirimidinonas/farmacología , Animales , Tiempo de Sangría , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Trombosis de las Arterias Carótidas/enzimología , Modelos Animales de Enfermedad , Fibrinolíticos/sangre , Fibrinolíticos/toxicidad , Hemorragia/inducido químicamente , Humanos , Riñón/irrigación sanguínea , Masculino , Ratones , Ratones Endogámicos C57BL , Morfolinas/sangre , Morfolinas/toxicidad , Fosfatidilinositol 3-Quinasas/sangre , Pirimidinonas/sangre , Pirimidinonas/toxicidad , Ratas , Ratas Sprague-Dawley , Cola (estructura animal)/irrigación sanguíneaRESUMEN
Apixaban is an oral, direct, and highly selective factor Xa inhibitor in late-stage clinical development for the prevention and treatment of thromboembolic diseases. Apixaban was evaluated in rat thrombosis and hemostasis models. Thrombosis was produced in the carotid artery by FeCl2 application, in the vena cava by either FeCl2 application or tissue factor injection, and in an arterial-venous shunt. Hemostasis was assessed using cuticle, renal cortex, and mesenteric artery bleeding times. Intravenous apixaban infusions of 0.1, 0.3, 1, and 3 mg/kg per hour increased the ex vivo prothrombin time to 1.24, 1.93, 2.75, and 3.98 times control, respectively. The 0.3, 1, and 3-mg/kg per hour doses inhibited thrombosis in all models. Concentrations for 50% thrombus reduction ranged from 1.84 to 7.57 microM. The 3-mg/kg per hour dose increased cuticle, renal, and mesenteric bleeding times to 1.92, 2.13, and 2.98 times control, respectively. Lower doses had variable (1 mg/kg per hour) or no effect (0.1, 0.3 mg/kg per hour) on hemostasis. Heparin's prolongation of renal and cuticle bleeding time was twice that of apixaban when administered at a dose that approximated apixaban (3 mg/kg per hour) efficacy in arterial thrombosis. In summary, apixaban was effective in a broad range of thrombosis models at doses producing modest increases in multiple bleeding time models.
Asunto(s)
Antitrombina III/uso terapéutico , Trombosis/tratamiento farmacológico , Animales , Anticoagulantes/uso terapéutico , Tiempo de Sangría , Coagulación Sanguínea/efectos de los fármacos , Trastornos de la Coagulación Sanguínea/tratamiento farmacológico , Hemostasis/efectos de los fármacos , Heparina/uso terapéutico , Masculino , Tiempo de Protrombina , Pirazoles , Piridonas , Ratas , Ratas Sprague-Dawley , Trombosis/prevención & controlRESUMEN
The effect of inhibiting activated blood coagulation factor XIa was determined in rat models of thrombosis and hemostasis. BMS-262084 is an irreversible and selective small molecule inhibitor of factor XIa with an IC(50) of 2.8 nM against human factor XIa. BMS-262084 doubled the activated thromboplastin time in human and rat plasma at 0.14 and 2.2 microM, respectively. Consistent with factor XIa inhibition, the prothrombin time was unaffected at up to 100 microM. BMS-262084 administered as an intravenous loading plus sustaining infusion was effective against FeCl(2)-induced thrombosis in both the vena cava and carotid artery. Maximum thrombus weight reductions of 97 and 73%, respectively (P<0.05), were achieved at a pretreatment dose of 12 mg/kg+12 mg/kg/h which increased the ex vivo activated thromboplastin time to 3.0 times control. This dose level also arrested growth of venous and arterial thrombi when administered after partial thrombus formation. BMS-262084 was most potent in FeCl(2)-induced venous thrombosis, decreasing thrombus weight 38% (P<0.05) at a threshold dose of 0.2 mg/kg+0.2 mg/kg/h. In contrast, doses of up to 24 mg/kg+24 mg/kg/h had no effect on either tissue factor-induced venous thrombosis or the ex vivo prothrombin time. Doses of up to 24 mg/kg+24 mg/kg/h also did not significantly prolong bleeding time provoked by either puncture of small mesenteric blood vessels, template incision of the renal cortex, or cuticle incision. These results demonstrate that pharmacologic inhibition of factor XIa achieves antithrombotic efficacy with minimal effects on provoked bleeding.
Asunto(s)
Azetidinas/uso terapéutico , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Factor XIa/antagonistas & inhibidores , Fibrinolíticos/uso terapéutico , Hemostáticos/uso terapéutico , Piperazinas/uso terapéutico , Trombosis de la Vena/tratamiento farmacológico , Animales , Trombosis de las Arterias Carótidas/fisiopatología , Humanos , Masculino , Tiempo de Tromboplastina Parcial , Tiempo de Protrombina , Ratas , Ratas Sprague-Dawley , Triptasas/antagonistas & inhibidores , Trombosis de la Vena/fisiopatologíaRESUMEN
Target levels of ex vivo inhibition of platelet aggregation (IPA) induced by adenosine diphosphate (ADP) that produce clinically relevant effects of clopidogrel, a P2Y12 antagonist, are unclear. We examined standard and modified IPA and P2Y12 receptor occupancy as predictors of antithrombotic (% thrombus weight reduction) and bleeding time (BT, fold-increase over control) effects of clopidogrel in rabbit models of carotid artery thrombosis and cuticle bleeding, respectively. Standard and modified IPA with 20 microM ADP were measured in the absence and presence of partial P2Y1 blockade, respectively. Clopidogrel maximally produced standard IPA of 57% +/- 5%, antithrombotic effect of 85% +/- 1%, BT increase of 6.0 +/- 0.4-fold and P2Y12 receptor occupancy of 87% +/- 5%. Surprisingly, a clopidogrel dose that produced a low standard IPA of 17% +/- 4% and P2Y12 receptor occupancy of 39% +/- 5% achieved a significant antithrombotic activity of 55% +/- 2% with a moderate increase in BT of 2.0 +/- 0.1-fold. This underestimation of clopidogrel efficacy by standard IPA was improved by measuring either modified IPA or P2Y12 receptor occupancy. These results suggest that in clopidogrel-treated rabbits, low standard IPA is associated with significant antithrombotic effects. Moreover, modified IPA and P2Y12 receptor occupancy appear to better predict the magnitude of clopidogrel's efficacy compared with standard IPA, which may be a better predictor of BT.
Asunto(s)
Plaquetas/metabolismo , Trombosis de las Arterias Carótidas/prevención & control , Trombosis de las Arterias Carótidas/fisiopatología , Arteria Carótida Común/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/efectos de los fármacos , Receptores Purinérgicos P2/metabolismo , Ticlopidina/análogos & derivados , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Análisis de Varianza , Animales , Aspirina/farmacología , Biomarcadores/sangre , Tiempo de Sangría , Plaquetas/efectos de los fármacos , Trombosis de las Arterias Carótidas/sangre , Arteria Carótida Común/fisiopatología , Clopidogrel , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Hemostasis/efectos de los fármacos , Masculino , Valor Predictivo de las Pruebas , Unión Proteica/efectos de los fármacos , Conejos , Receptores Purinérgicos P2/efectos de los fármacos , Flujo Sanguíneo Regional/efectos de los fármacos , Tromboxano B2/sangre , Ticlopidina/farmacologíaRESUMEN
We determined the dose response of the ADP antagonist clopidogrel (0.3-50 mg/kg p.o.) in rat models of thrombosis and provoked bleeding and correlated these activities to ex vivo platelet activation. Carotid artery thrombosis was induced by FeCl(2). Bleeding time was measured by mesenteric vessel puncture and renal cortex or cuticle incision. Platelet biomarkers included standard ADP-induced aggregation, P2Y(12) receptor occupancy, and phosphorylation of vasodilator-stimulated phosphoprotein. Clopidogrel decreased thrombus weight up to 78%, caused maximal prolongation of cuticle and mesenteric bleeding, but had little effect on renal bleeds. Due to the steep mesenteric dose response, further comparisons concentrated on cuticle bleeding. The half-maximal inhibitory dose (ED(50)) for thrombus reduction was 2.4 +/- 0.4 mg/kg, with 10 mg/kg providing optimal blood flow preservation and thrombus reduction. The ED(50) for bleeding was 10.5 +/- 3.4 mg/kg. Increased bleeding was intermediate (3-fold) at 10 mg/kg and maximal (6-fold) at 30 mg/kg. All biomarkers were affected, but with differing sensitivity. ED(50)s for peak platelet aggregation to 10 microM ADP (11.9 +/- 0.4 mg/kg) and the vasodilator-stimulated phosphoprotein index (16.4 +/- 1.3 mg/kg) approximated the higher ED(50) for bleeding. ED(50)s for ligand binding (3.0 +/- 0.3 mg/kg) and late aggregation (5.1 +/- 0.4 mg/kg) better matched the lower ED(50) for antithrombotic activity. Aspirin exerted lesser effects on bleeding (42-70% increase in all models) and thrombosis (24% inhibition). In summary, antithrombotic doses of clopidogrel have limited effects on bleeding and standard measures of platelet aggregation. Other biomarkers may be more sensitive for tracking antithrombotic efficacy.
Asunto(s)
Fibrinolíticos/farmacología , Hemostáticos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Ticlopidina/análogos & derivados , Adenosina Difosfato/farmacología , Animales , Aspirina/farmacología , Biomarcadores , Moléculas de Adhesión Celular/metabolismo , Clopidogrel , Relación Dosis-Respuesta a Droga , Masculino , Proteínas de la Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Agregación Plaquetaria/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y12 , Ticlopidina/farmacologíaRESUMEN
INTRODUCTION: TAFI indirectly reduces the action of tPA on plasminogen. Whether exogenous tPA is necessary for TAFI inhibitor efficacy is unclear. Potato carboxypeptidase inhibitor (PCI), a TAFI inhibitor, has shown variable tPA dependence in rat models of arteriovenous shunt thrombosis (required) and microthrombosis (not required). This study was designed to further explore the importance of exogenous tPA in revealing PCI activity in rat models of venous and arterial thrombosis and provoked bleeding. METHODS: PCI was given as a bolus (5, 10 mg/kg) +/- infusion (5, 10 mg/kg/h) and with or without low dose tPA (5, 10, 25 microg/kg/min). In each instance tPA was adjusted to produce subthreshold thrombus reduction. Arterial thrombosis was induced by FeCl2; venous thrombosis by tissue factor or FeCl2. Bleeding was induced by kidney incision with PCI given (5 mg + 5 mg/kg/h) in the presence or absence of tPA (10, 150, 200 microg/kg/min). RESULTS: PCI was ineffective without exogenous tPA in all tested thrombosis models. With exogenous tPA, PCI decreased thrombus weight 85% in tissue factor thrombosis, 59% in FeCl2 thrombosis, and 46% in arterial thrombosis. PCI prolonged bleeding only when combined with a relatively high tPA dose (200 microg/kg/min) that increased bleeding alone. CONCLUSIONS: If the current results predict clinical efficacy, the need for exogenous tPA in combination with TAFI inhibition is a potential problem. However, in acute settings where intravenous fibrinolytics are administered, or indications in which tPA production increases, TAFI inhibitors may prove to be safe and moderately effective profibrinolytic agents.