ABSTRACT
Inhibition of the hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) represents a promising strategy for discovering next-generation treatments for renal anemia. We discovered DS44470011 in our previous study, which showed potent in vitro activity and in vivo efficacy based on HIF-PHD inhibition. However, DS44470011 was also found to exert genotoxic effects. By converting the biphenyl structure, which is suspected to be the cause of this genotoxicity, to a 1-phenylpiperidine structure, we were able to avoid genotoxicity and further improve the in vitro activity and in vivo efficacy. Furthermore, through the optimization of pyrimidine derivatives, we discovered DS-1093a, which has a wide safety margin with potent in vitro activity and an optimal pharmacokinetic profile. DS-1093a achieved an increase in hemoglobin levels in an adenine-induced rat model of chronic kidney disease after its continuous administration for 4 days.
Subject(s)
Anemia , Hypoxia-Inducible Factor-Proline Dioxygenases , Prolyl-Hydroxylase Inhibitors , Animals , Rats , Hypoxia-Inducible Factor-Proline Dioxygenases/antagonists & inhibitors , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Anemia/drug therapy , Prolyl-Hydroxylase Inhibitors/pharmacology , Prolyl-Hydroxylase Inhibitors/chemistry , Humans , Administration, Oral , Structure-Activity Relationship , Renal Insufficiency, Chronic/drug therapy , Drug Discovery , Molecular Structure , Pyrimidines/chemistry , Pyrimidines/pharmacology , Pyrimidines/chemical synthesis , Dose-Response Relationship, DrugABSTRACT
Inhibition of the hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) represents a promising strategy for discovering next-generation treatments for renal anemia. We identified a pyrimidine core with HIF-PHD inhibitory activity based on scaffold hopping of FG-2216 using crystal structures of HIF-PHD2 in complex with compound. By optimizing the substituents at the 2- and 6- positions of the pyrimidine core, we discovered DS44470011, which improves the effectiveness of erythropoietin (EPO) release in cells. Oral administration of DS44470011 to cynomolgus monkeys increased plasma EPO levels.
Subject(s)
Anemia , Hypoxia-Inducible Factor-Proline Dioxygenases , Macaca fascicularis , Prolyl-Hydroxylase Inhibitors , Animals , Anemia/drug therapy , Hypoxia-Inducible Factor-Proline Dioxygenases/antagonists & inhibitors , Hypoxia-Inducible Factor-Proline Dioxygenases/metabolism , Administration, Oral , Humans , Prolyl-Hydroxylase Inhibitors/pharmacology , Prolyl-Hydroxylase Inhibitors/chemistry , Prolyl-Hydroxylase Inhibitors/chemical synthesis , Pyrimidines/chemistry , Pyrimidines/pharmacology , Pyrimidines/chemical synthesis , Structure-Activity Relationship , Molecular Structure , Erythropoietin , Drug Discovery , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/chemical synthesisABSTRACT
We evaluated the effects of ministrokes targeted to individual pial arterioles on motor function in Thy-1 line 18 channelrhodopsin-2 (ChR2) transgenic mice within the first hours after ischemia. Using optogenetics, we directly assessed both the excitability and motor output of cortical neurons in a manner independent of behavioral state or training. Occlusion of individual arterioles within the motor cortex led to a ministroke that was verified using laser speckle contrast imaging. Surprisingly, ministrokes targeted to a relatively small region of the forelimb motor map, with an ischemic core of 0.07 ± 0.03 mm(2), impaired motor responses evoked from points across widespread areas of motor cortex even 1.5 mm away. Contrasting averaged ChR2-evoked electroencephalographic, spinal (ChR2 evoked potential), and electromyographic responses revealed a mismatch between measures of cortical excitability and motor output within 60 min after stroke. This mismatch suggests that apparently excitable cortical neurons (even >1 mm into peri-infarct areas, away from the infarct core) were impaired in their capacity to generate spinal potentials leading to even more severe deficits in motor output at muscles. We suggest that ischemia, targeted to a subset of motor cortex, leads to relatively small reductions in excitability within motor cortex, and cumulative depression of both descending spinal circuits and motor output in response to the activation of widespread cortical territories even outside of the area directly affected by the ischemia.
Subject(s)
Motor Cortex/physiopathology , Neurons/physiology , Recovery of Function/physiology , Stroke/physiopathology , Animals , Channelrhodopsins , Disease Models, Animal , Electrophysiology , Female , Male , Mice , Mice, Transgenic , Neurons/pathology , Optogenetics/methodsABSTRACT
We evaluated the effects of prasugrel, a third-generation thienopyridyl prodrug, on P2Y12 receptors, adenosine 5'-diphosphate (ADP)-induced platelet aggregation, and myocardial infarction (MI) in rats. Oral administration of prasugrel (0.3-3 mg/kg) resulted in the dose-related inhibition of washed platelet aggregation induced by ADP (1-10 µM). Ex vivo [H]-2-MeS-ADP binding to platelet P2Y12 receptors was also inhibited by prasugrel in a similar dose range. The antiaggregatory effects of prasugrel correlated strongly with P2Y12 blockade with correlation coefficients of 0.85-0.92, suggesting that the antiaggregatory activity of prasugrel largely reflected P2Y12 blockade achieved in vivo. We further examined the effects of the in vivo P2Y12 inhibition by prasugrel (1-10 mg/kg, po) on MI induced by thrombotic coronary artery occlusion in rats. In surviving rats, infarct size at 24 hours after photoirradiation was evaluated. In the vehicle group, necrosis area/total left ventricular area was 37.9% ± 6.8% (mean ± SE, n = 7). At all prasugrel doses tested (n = 7 for each dose), necrosis area/total left ventricular area was significantly smaller than that in the vehicle group: 14.4% ± 4.0% for 1 mg/kg (P < 0.01), 19.8% ± 4.5% for 3 mg/kg (P < 0.05), and 14.8% ± 3.6% for 10 mg/kg (P < 0.01). At the highest administered dose of prasugrel (10 mg/kg), blood pressure and heart rate were unchanged. Arrhythmia was observed in 5 of 7 animals in the vehicle group at 24 hours after irradiation; in contrast, no arrhythmia was found in the group treated with prasugrel (10 mg/kg). Taken together, these results demonstrate that prasugrel is a selective P2Y12 inhibitor in vivo, providing effective inhibition of platelet aggregation and MI in rats.
Subject(s)
Drug Evaluation, Preclinical , Myocardial Infarction/prevention & control , Piperazines/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Purinergic P2 Receptor Antagonists/pharmacology , Receptors, Purinergic P2Y12/metabolism , Thiophenes/pharmacology , Animals , Blood Platelets , Blood Pressure , Coronary Occlusion/drug therapy , Coronary Occlusion/pathology , Coronary Thrombosis/drug therapy , Coronary Thrombosis/pathology , Disease Models, Animal , Electrocardiography , Heart Rate , Male , Myocardial Infarction/drug therapy , Myocardial Infarction/metabolism , Myocardial Infarction/pathology , Platelet Aggregation , Prasugrel Hydrochloride , Protein Binding , Rats , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
Cerebral Arterial Diseases/drug therapy , Peripheral Vascular Diseases/drug therapy , Piperazines/pharmacology , Purinergic P2 Receptor Antagonists , Thiophenes/pharmacology , Animals , Cerebral Arterial Diseases/etiology , Disease Models, Animal , Dose-Response Relationship, Drug , Male , Peripheral Vascular Diseases/etiology , Prasugrel Hydrochloride , Rats , Rats, Sprague-DawleyABSTRACT
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.
Subject(s)
Amidines , Anticoagulants , Antithrombin III , Cinnamates/chemistry , Factor Xa Inhibitors , Sulfonamides , Administration, Oral , Amidines/chemical synthesis , Amidines/chemistry , Amidines/pharmacology , Animals , Anticoagulants/chemical synthesis , Anticoagulants/chemistry , Anticoagulants/pharmacology , Antithrombin III/chemical synthesis , Antithrombin III/chemistry , Antithrombin III/pharmacology , Cricetinae , Dogs , Humans , Male , Molecular Structure , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/chemistry , Sulfonamides/pharmacologyABSTRACT
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.
Subject(s)
Piperazines/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation/drug effects , Purinergic P2 Receptor Antagonists , Thiophenes/pharmacology , Thrombosis/drug therapy , Administration, Oral , Adult , Animals , Clopidogrel , Dogs , Dose-Response Relationship, Drug , Drug Administration Schedule , Humans , In Vitro Techniques , Macaca fascicularis , Male , Middle Aged , Piperazines/administration & dosage , Platelet Aggregation Inhibitors/administration & dosage , Prasugrel Hydrochloride , Rabbits , Rats , Rats, Sprague-Dawley , Species Specificity , Thiophenes/administration & dosage , Ticlopidine/analogs & derivatives , Ticlopidine/pharmacologyABSTRACT
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.
Subject(s)
Membrane Proteins/deficiency , Membrane Proteins/genetics , Piperazines/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Receptors, Purinergic P2/deficiency , Receptors, Purinergic P2/genetics , Thiophenes/pharmacology , Animals , Blood Platelets/drug effects , Blood Platelets/metabolism , Female , Gene Deletion , Male , Membrane Proteins/antagonists & inhibitors , Mice , Mice, Inbred C57BL , Platelet Aggregation/drug effects , Prasugrel Hydrochloride , Purinergic P2 Receptor Antagonists , Receptors, Purinergic P2Y12 , Sex CharacteristicsABSTRACT
CS-747 (prasugrel, LY640315) is a member of the thienopyridine class of oral platelet aggregation inhibitors that includes ticlopidine and clopidogrel. A single oral administration of CS-747 produced a dose-related inhibition of platelet aggregation in rats that was approximately 10- and 100-fold more potent than that of clopidogrel and ticlopidine, respectively. The antiaggregatory effect of CS-747 was evident at 30 minutes and lasted until 72 hours after dosing, indicating fast onset and long duration of action. CS-747 showed more potent antithrombotic activity compared with clopidogrel and ticlopidine with the same rank order as the antiaggregatory potencies. Combined administration of CS-747 with aspirin to rats produced substantially greater inhibition of both platelet aggregation and thrombus formation compared with each agent alone. The antiplatelet action of CS-747 is due to irreversible and selective blockade of platelet P2Y (12) adenosine diphosphate (ADP) receptors by its active metabolite R-138727. In phase I studies, a single oral dose of CS-747 (30 and 75 mg) produced > 50% inhibition of ADP-induced platelet aggregation, with rapid onset (1 hour) and long duration (> 48 hours) of action. In healthy volunteers, once-daily administration of 10 mg CS-747 for 10 days showed significant cumulative inhibition of platelet aggregation from 2 days after the first dose until at least 2 days after the final dose. Studies conducted to date indicate that CS-747 is a highly effective antiplatelet and antithrombotic agent and is anticipated to be effective in the treatment of atherothrombotic and other ischemic vascular diseases.
Subject(s)
Membrane Proteins/antagonists & inhibitors , Piperazines/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Purinergic P2 Receptor Antagonists , Thiophenes/pharmacology , Adenosine Diphosphate/pharmacology , Adult , Animals , Aspirin/pharmacology , Biotransformation , Clinical Trials, Phase I as Topic , Clopidogrel , Collagen/pharmacology , Drug Evaluation, Preclinical , Drug Synergism , Fibrinolytic Agents/pharmacology , Fibrinolytic Agents/therapeutic use , Hemostasis/drug effects , Humans , Male , Membrane Proteins/physiology , Molecular Structure , Piperazines/therapeutic use , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/therapeutic use , Prasugrel Hydrochloride , Prodrugs/pharmacokinetics , Pyridines/pharmacology , Rats , Receptors, Purinergic P2/physiology , Receptors, Purinergic P2Y12 , Thiophenes/therapeutic use , Thrombin/pharmacology , Thrombosis/drug therapy , Ticlopidine/analogs & derivatives , Ticlopidine/pharmacology , Ticlopidine/therapeutic useABSTRACT
The temporary three-vessel occlusion (3VO) technique with a surgical approach for middle cerebral artery (MCA) produces consistent cerebral infarction in the neocortex in normotensive rats. The intraluminal thread-occlusion technique with an endovascular approach targeting the MCA occlusion (MCAO) is more widely used since it does not require complicated intracranial procedures. The aim of this study was to review the methods/models for MCAO stroke in normotensive rats and to evaluate a 3VO stroke model that provides consistent degrees and variance of cortical stroke injury for additional discussion. First, we analyzed a model with modified temporary 3VO technique requiring less complicated procedures than the temporary 3VO model, i.e., temporary occlusion of the bilateral common carotid arteries (CCAs) superimposed on a permanent occlusion of the MCA, in Sprague-Dawley rats or C57BL/6J mice. In the microvascular tissue (cerebral) perfusion study, significant reductions in regional cerebral perfusion during the 3VO accompanied a rapid return to baseline after release of the CCAs, showing that the technique induces temporary focal ischemia. The average sizes and variances of the neocortical infarction in this model, together with those in the other normotensive rat models caused by the 3VO technique in the literature, indicated a standard size and variance of infarcted lesion in the control groups relative to the specific ischemic period. However, stroke injuries in the neocortex induced by the thread occlusion technique showed greater variability with less consistent lesion sizes. Inclusion/exclusion criteria to avoid inappropriate cases with too mild (no/faint infarction) or too great (huge/fatal infarction) severity in the ischemic injury may differ between laboratories in the thread occlusion model.