ABSTRACT
Antiplatelet drugs are used to prevent aberrant platelet activation in pathophysiologic conditions such as myocardial infarction and ischemic stroke. The key role that ADP plays in this process has led to the development of antiplatelet drugs that target the P2Y12 receptor. The aim of this study was to characterize the pharmacodynamic (PD) and pharmacokinetic (PK) properties of the novel P2Y12 receptor antagonists, BX 667 and BX 048. BX 667 blocks ADP-induced platelet aggregation in human, dog and rat blood (IC50=97, 317 and 3000 nM respectively). BX 667 had nominal effects on collagen-induced aggregation and weakly inhibited arachidonic acid-induced aggregation. BX 667 has an active metabolite, BX 048, that also potently inhibits ADP-induced aggregation (IC50=290 nM) in human blood. BX 667 was shown to have high oral bioavailability in both dog and rat unlike BX 048. Administration of BX 667 resulted in a rapid and sustained inhibition of platelet aggregation where the extent and duration of platelet inhibition was directly proportional to circulating plasma levels. This report describes the PK/PD properties of BX 667 showing that it has the properties required for a potential antiplatelet therapeutic agent.
Subject(s)
Blood Platelets/drug effects , Blood Platelets/metabolism , Keto Acids/pharmacokinetics , Platelet Aggregation Inhibitors/pharmacokinetics , Purinergic P2 Receptor Antagonists , Quinolines/pharmacokinetics , Receptors, Purinergic P2/metabolism , Animals , Dogs , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Humans , Ligands , Male , Models, Biological , Platelet Aggregation/drug effects , Protein Binding , Rats , Receptors, Purinergic P2Y12 , Species SpecificityABSTRACT
ADP plays a key role in platelet aggregation which has led to the development of antiplatelet drugs that target the P2Y12 receptor. The aim of this study was to characterize the effects of two novel P2Y12 receptor antagonists, BX 667 and its active metabolite BX 048, on platelets. BX 667 and BX 048 block the binding of 2MeSADP to platelets and antagonize ADP-induced platelet aggregation in human, dog and rat washed platelets. Both compounds were shown to be reversible inhibitors of platelet aggregation. BX 048 prevents the decrease in cAMP induced by treatment of platelets with ADP. The specificity of BX 667 and BX 048 was demonstrated against cell lines expressing P2Y1 and P2Y6 as well as against a panel of receptors and enzymes. Taken all together these data show that both BX 048 and BX 667 are potent P2Y12 antagonists with high specificity which, in the accompanying paper are demonstrated to behave predictably in vivo.
Subject(s)
Blood Platelets/drug effects , Blood Platelets/metabolism , Keto Acids/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Purinergic P2 Receptor Antagonists , Quinolines/pharmacology , Receptors, Purinergic P2/metabolism , Adenosine Diphosphate/chemistry , Animals , Calcium/metabolism , Dogs , Drug Evaluation, Preclinical , Humans , In Vitro Techniques , Ligands , Models, Biological , Platelet Aggregation/drug effects , Protein Binding , Rats , Receptors, Purinergic P2Y12 , Species SpecificityABSTRACT
A novel series of cyclic potent, selective, small molecule, thiol-based inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) and the crystal structures of TAFIa inhibitors bound to porcine pancreatic carboxypeptidase B are described. Three series of cyclic arginine and lysine mimetic inhibitors vary significantly in their selectivity against other human basic carboxypeptidases, carboxypeptidase N and carboxypeptidase B. (-)2a displays TAFIa IC50 = 3 nM and 600-fold selectivity against CPN. Inhibition of TAFIa with (rac)2a resulted in dose dependent acceleration of human plasma clot lysis in vitro and was efficacious as an adjunct to tPA in an in vivo rabbit jugular vein thrombolysis model.
Subject(s)
3-Mercaptopropionic Acid/pharmacology , Carboxypeptidase B2/antagonists & inhibitors , Fibrinolytic Agents/chemical synthesis , Animals , Arginine , Carboxypeptidase B/antagonists & inhibitors , Crystallography, X-Ray , Fibrinolytic Agents/pharmacokinetics , Fibrinolytic Agents/pharmacology , Humans , Lysine , Lysine Carboxypeptidase/antagonists & inhibitors , Molecular Mimicry , Peptides, Cyclic , Rabbits , Structure-Activity Relationship , Substrate Specificity , SwineABSTRACT
This paper presents the crystal structure of porcine pancreatic carboxypeptidase B (pp-CpB) in complex with a variety of thiol-based inhibitors that were developed as antagonists of activated thrombin-activatable fibrinolysis inhibitor (TAFIa). Recent studies have indicated that a selective inhibitor of TAFIa could enhance the efficacy of existing thrombolytic agents for the treatment of acute myocardial infarction, one of the most prevalent forms of heart attacks. Unfortunately, activated TAFIa rapidly degrades in solution and cannot be used for crystallographic studies. In contrast, porcine pancreatic CpB is stable at room temperature and is available from commercial sources. Both pancreatic CpB and TAFIa are zinc-based exopeptidases, and the proteins share a 47% sequence identity. The homology improves considerably in the active site where nearly all of the residues are conserved. The inhibitors used in this study were designed to mimic a C-terminal arginine residue, one of the natural substrates of TAFIa. The X-ray structures show that the thiol group chelates the active site zinc, the carboxylic acid forms a salt bridge to Arg145, and the guanidine group forms two hydrogen bonds to Asp255. A meta-substituted phenyl was introduced into our inhibitors to reduce conformational freedom. This modification vastly improved the selectivity of compounds against other exopeptidases that cleave basic residues. Comparisons between structures indicate that selectivity derives from the interaction between the guanidine group in the inhibitors and an acidic active site residue. The location of this acidic residue is not conserved in the various carboxypeptidases.
