SBIR-STTR Award

There is significant interest in development of new antiplatelet agents as antithrombotic drugs that act directly and reversibly, avoiding documented drawbacks of the current drug of choice, clopidogrel. A synthetic ATP analog, which is a potent antagonist of one of the ADP- dependent platelet P2Y receptors, is in clinical testing as an antithrombotic drug. Diadenosine P1,P4-tetraphosphate (Ap4A) and its phosphonate and thiophosphonate analogs such as P Ap(S)pCHClp2(S)A ("Avathrom") inhibit platelet aggregation in vitro, and show antithrombotic activity in vivo, but at high doses. Existing data suggest P2Y1 targeting, but do not rule out P2Y12 inhibition as well. We have discovered new, highly efficient methods for synthesis of dinucleoside tetraphosphates and tetraphosphonates that will enable the efficient preparation of modified Ap4A derivatives for the first time. One method is based on a new reagent class - stable but reactive bis-imidazolides of pyrophosphate and methylenebisphosphonates - and inexpensive starting materials, resulting in symmetric products in high yield. The other method, suitable also for unsymmetric analogs, relies on an efficient reaction between nucleoside-5'-metatriphosphates or -5'-metatriphosphonates and nucleoside monophosphates. We propose to exploit the new synthetic methods to prepare symmetric and unsymmetric adenine-modified Ap4A analogs, with modifications based on existing SAR data of ATP and ADP analogs, with the aim to create potent antagonists of platelet P2Y1 and/or P2Y12 receptors, and, possibly, a unique class of antiplatelet agents which targets both receptors. The antiplatelet potency and platelet shape change activity will be determined, and antagonist/agonist properties of the new compounds toward P2Y1, P2Y12, and P2X1 receptors will be measured. We will also measure the stability of selected new analogs in rat, dog, and human plasma. Our immediate goals are to validate and further develop breakthrough methods for synthesis of bis-nucleoside polyphosphates, to determine if the class of Ap4A analogs targets P2Y1, P2Y12, or even better, both P2Y1 and P2Y12 platelet receptors, and to demonstrate the therapeutic potential and plasma stability of novel Ap4A analogs. Our long range goals are to discover novel compounds and methods for treatment of arterial thrombosis, and more particularly, a fast and reversibly acting antiplatelet agent targeting platelet P2Y1, or better, both P2Y1 and P2Y12 receptors, to complement existing antiplatelet therapeutics which mainly target the platelet P2Y12 receptor. This project will result in an effective antithrombotic drug that will be used to treat arterial thrombosis. The candidate drug will directly and reversibly inhibit one or both of important receptors involved in platelet aggregation, and will not have the drawbacks of slow and variable action of current drugs such as clopidogrel. The new drug will complement related drugs under development for arterial thrombosis.

Thesaurus Terms:
adenosine, analog, antithrombin, chemical synthesis, drug design /synthesis /production, method development, phosphate blood disorder chemotherapy, chemical structure function, chemoprevention, inhibitor /antagonist, phosphonate, platelet aggregation inhibitor, receptor, thrombosis human subject

Phase I resulted in our discovery of low nanomolar level inhibitors of ADP-induced human platelet aggregation, with a unique mechanism of action, that warrant further development as first-in-class antithrombotic drugs. We developed a novel, efficient, high yield method to prepare a series of base and phosphate modified derivatives of diadenosine tetraphosphate (Ap4A). The proposed modifications revealed clear structure-activity trends, and led to the synthesis of highly potent inhibitors of human platelet aggregation that are also stable in plasma. We investigated for the first time the mechanism of action of this class at the level of the three platelet purinoreceptors (P2X1,P2Y1 and P2Y12), and showed that Ap4A and its analogs antagonize both P2Y1 and P2Y12. Moreover this unprecedented simultaneous inhibition of both platelet ADP dependent platelet receptors by a single ligand appears to be highly synergistic - IC50'sfor inhibition of ADP-induced platelet aggregation for the most active compounds is one to two order of magnitude lower than the corresponding IC50'sof inhibition of P2Y1 and P2Y12. Thus these compounds represent a new class of antiplatelet, and, ultimately, antithrombotic drugs with a novel mechanism of action. The specific aims are to 1, expand and tune the structure:activity relationship for inhibiting human platelet aggregation by synthesis and study of six additional Ap4A analogs, 2, evaluate the selectivity/specificity of the new class of Ap4A derivatives, by studying and quantifying the ability of 3-6 most active compounds to interact with non-platelet P2 receptors, utilizing tissue and recombinant receptor models, 3, optimize and scale up the chemical process of synthesis and purification of the class; 4, determine the basic pharmacokinetic parameters and identify metabolites of the 3 lead compounds candidates after IV administration in rats, and 5, study the aggregation properties of platelets after IV infusion of these candidates in catheterized rats, and 6, confirm the antithrombotic activity of the selected lead compound in the well-established Folts' canine thrombosis model. A single lead preclinical compound (plus backup) will be designated following these studies. Platelets play critical roles in hemostasis and its pathophysyology. Undesired platelet activation is a result of many common pathologies, e.g. hypertension and arteriosclerosis, and leads to excessive platelet aggregation and the generation of occlusive thrombi (thrombosis). The ischemic events that follow, such as myocardial infarction and stroke, are leading causes of death in the developed world, and antiplatelet drugs have been a major focus of drug development. Aspirin and clopidrogel (Plavix(R), $5.9B sales in 2005) are the most popular of the class today. Because of the vast interpatient variability in response to clopidogrel and newer single-receptor targeted drugs under clinical development, and because of the increased bleeding and morbidity in patients receiving clopidogrel, there is a need for the development of fast, direct acting, and rapidly cleared platelet ADP-receptor antagonists. The overall goal of this research is to identify a novel lead antithrombotic compound for human use, which can be licensed to a major pharmaceutical company for further pre-clinical and clinical development, or alternatively can be developed into a drug candidate by us after securing appropriate partnership or funding.

Public Health Relevance:
This project will result in an effective antithrombotic drug that will be used to treat arterial thrombosis in the acute setting. The candidate drug will directly and reversibly inhibit two important receptors involved in platelet aggregation, and will not have the drawbacks of slow and variable action of current drugs such as clopidogrel. The new drug will complement related drugs under development for arterial thrombosis.

Thesaurus Terms:
2-(Acetyloxy)Benzoic Acid; Adp Receptors; Acetylsalicylic Acid; Active Follow-Up; Acute; Adverse Effects; Affinity; Agents, Antiplatelet; Animals; Antiplatelet Drugs; Aorta; Ap4a; Apoplexy; Appppa; Arteries; Arteriosclerosis; Aspergum; Aspirin; Assay; Binding; Binding (Molecular Function); Bioassay; Biologic Assays; Biological Assay; Bizzozero's Corpuscle/Cell; Bladder; Bleeding; Bleeding Time; Bleeding Time Procedure; Blood; Blood Plasma; Blood Platelet Aggregation Inhibitors; Blood Platelet Antiaggregants; Blood Platelets; Blood Pressure, High; Body Tissues; Brain Region; Cangrelor; Canine Species; Canis Familiaris; Cardiac Infarction; Cause Of Death; Cavia; Cell Line; Cell Lines, Strains; Cell Model; Cellline; Cellular Model; Cerebral Stroke; Cerebrovascular Apoplexy; Cerebrovascular Stroke; Cerebrovascular Accident; Chemicals; Chemistry; Clinical; Common Rat Strains; Complement; Complement Proteins; Contracting Opportunities; Contracts; Coronary Thrombosis; Deetjeen's Body; Dependence; Development; Dogs; Dose; Drip Infusions; Drip, Intravenous; Drug Delivery; Drug Delivery Systems; Drug Kinetics; Drug Targeting; Drug Targetings; Drugs; Dysfunction; Ear; Ear Structure; Ecotrin; Empirin; Endothelium; Entericin; Evaluation; Event; Extren; Functional Disorder; Funding; Generations; Goals; Guinea Pigs; Hayem's Elementary Corpuscle; Hemorrhage; Hemostasis; Hemostatic Function; Human; Human, General; Hypertension; Iv Infusion; Infusion; Infusion Procedures; Infusions, Intravenous; Intermediary Metabolism; Intravenous Infusion Procedures; Lead; Licensing; Ligands; Metbl; Mammals, Dogs; Mammals, Guinea Pigs; Mammals, Rabbits; Mammals, Rats; Man (Taxonomy); Man, Modern; Marrow Platelet; Measurin; Medication; Membrane; Metabolic Processes; Metabolism; Methods; Modeling; Modification; Molecular Interaction; Monitor; Morbidity; Morbidity - Disease Rate; Myocardial Infarct; Myocardial Infarction; Nucleotides; Operation; Operative Procedures; Operative Surgical Procedures; Oryctolagus Cuniculus; P(1), P(4)-Diadenosine-5'tetraphosphate; P2x; P2x-Receptor; Pathology; Patients; Pb Element; Pharmaceutic Preparations; Pharmaceutical Agent; Pharmaceutical Preparations; Pharmaceuticals; Pharmacokinetics; Pharmacologic Substance; Pharmacological Substance; Phase; Phosphates; Physiopathology; Plasma; Platelet Activation; Platelet Aggregation Inhibitors; Platelet Antiaggregants; Platelet Aggregation; Platelets; Plavix; Play; Preparation; Process; Property; Property, Loinc Axis 2; Rabbit, Domestic; Rabbits; Rat; Rattus; Receptor Protein; Recombinants; Relaxation; Reproducibility; Research; Reticuloendothelial System, Blood; Reticuloendothelial System, Platelets; Reticuloendothelial System, Serum, Plasma; Role; Safety; Sales; Science Of Chemistry; Secure; Series; Serum, Plasma; Specificity; Stroke; Structure; Structure-Activity Relationship; Surgical; Surgical Interventions; Surgical Procedure; Therapeutic; Thieno(3,2-C)Pyridine, 5-((2-Chlorophenyl)Methyl)-4,5,6,7-Tetrahydro-; Thrombocytes; Thrombosis; Thrombus; Ticlopidine; Time; Tissue Model; Tissues; Treatment Side Effects; Urinary System, Bladder; Vascular Accident, Brain; Vascular Hypertensive Disease; Vascular Hypertensive Disorder; Work; Adenosine 5'-(Pentahydrogen Tetraphosphate), 5'-5'-Ester With Adenosine; Adenosine(5')Tetraphospho(5')Adenosine; Analog; Analytical Method; Base; Bis(5'-Adenosyl)Tetraphosphate; Blood Loss; Brain Attack; Canine; Cardiac Infarct; Cerebral Vascular Accident; Chemical Structure Function; Clopidogrel; Coronary Attack; Coronary Infarct; Coronary Infarction; Cultured Cell Line; Diadenosine 5',5'''-P(1),P(4)--Tetraphosphate; Diadenosine Tetraphosphate; Domestic Dog; Drug Candidate; Drug Development; Drug/Agent; Follow-Up; Heart Attack; Heart Infarct; Heart Infarction; Heavy Metal Pb; Heavy Metal Lead; Hyperpiesia; Hyperpiesis; Hypertensive Disease; In Vivo; Inhibitor; Inhibitor/Antagonist; Inorganic Phosphate; Intervention Development; Membrane Structure; Novel; Pathophysiology; Pre-Clinical; Preclinical; Public Health Relevance; Purinoceptor P2y1; Purinoceptor P2y6; Radioligand; Receptor; Receptor, P2y(6); Receptor, P2y1; Receptor, P2y6; Response; Scale Up; Side Effect; Social Role; Stroke; Structure Function Relationship; Success; Surgery; Therapy Adverse Effect; Therapy Development; Thienopyridine; Thrombocyte/Platelet; Treatment Adverse Effect; Treatment Development; Trend; Urinary Bladder