Vasodilator-stimulated phosphoprotein (VASP) is not a major mediator of platelet aggregation, thrombogenesis, haemostasis, and antiplatelet effect of prasugrel in rats.

Vasodilator-stimulated phosphoprotein (VASP) is a member of actin regulatory proteins implicated in platelet adhesion. In addition, phosphorylation of VASP is utilised for the assessment of platelet reactivity in patients treated with P2Y12 receptor antagonists, a class of antiplatelet agents. However, the role of VASP in platelet aggregation, thrombogenesis, haemostasis, and the antiplatelet effect of P2Y12 receptor antagonists remains unclear. We investigated these effects using heterozygous and homozygous VASP knockout rats generated with a CRISPR/Cas9 system. Baseline characteristics, such as haematology and other biochemical parameters, were comparable among the genotypes. In vitro platelet aggregation stimulated by adenosine diphosphate (ADP) or collagen, P-selectin expression of rat platelets treated with ADP, and in vivo thrombocytopenia induced by collagen were also comparable among the genotypes. In addition, in vivo thrombogenesis in a ferric chloride-induced arterial thrombosis model and bleeding time were also comparable among the genotypes. Furthermore, the in vitro antiplatelet effect of prasugrel, a third-generation P2Y12 receptor antagonist, was unaffected by VASP knockout. Although phosphorylated VASP is still an important surrogate marker specific for P2Y12 antagonists, our findings demonstrate that VASP is not a major mediator of platelet aggregation, thrombogenesis, haemostasis, and the antiplatelet effect of prasugrel in rats.

The active metabolite of prasugrel, R-138727, improves cerebral blood flow and reduces cerebral infarction and neurologic deficits in a non-human primate model of acute ischaemic stroke.

Previously, we showed preventive effects of prasugrel, a P2Y12 antagonist, in a non-human primate model of thrombotic middle cerebral artery occlusion (MCAO); however, it remains unclear if P2Y12 inhibition after MCAO reduces cerebral injury and dysfunction. Here we investigated the effects of R-138727, the major active metabolite of prasugrel, on ex vivo platelet aggregation at 5min, 15min, 60min, and 24h after administration to non-human primates (n=3). A single intravenous dose of R-138727 (0.03-0.3mg/kg) resulted in significant and sustained dose-related effects on platelets for up to 24h. R-138727 was administered 1h after MCAO induction, and its effects on thrombosis, cerebral infarction, and neurological deficits were determined (n=8-10). R-138727 (0.3mg/kg) significantly increased total patency rate of the MCA (P=0.0211). Although there was no effect on the patency rate before R-138727 dosing (P=0.3975), it increased 1h after dosing (P=0.0114). R-138727 significantly reduced total ischaemic infarction volumes (P=0.0147), including those of basal ganglia (P=0.0028), white matter (P=0.0393), and haemorrhagic infarction (P=0.0235). Additionally, treatment with R-138727 reduced overall neurological deficits (P=0.0019), including the subcategories of consciousness (P=0.0042), sensory system (P=0.0045), motor system (P=0.0079) and musculoskeletal coordination (P=0.0082). These findings support the possible utility of P2Y12 inhibition during early-onset MCAO to limit the progression and degree of cerebral ischaemia and infarction and also associated neurological deficits.

Prasugrel, a Platelet P2Y12 Receptor Antagonist, Improves Abnormal Gait in a Novel Murine Model of Thrombotic Hindlimb Ischemia.

BACKGROUND: The efficacy of P2Y12 inhibition for the prevention of cardiovascular events in patients with peripheral arterial disease (PAD) has been established. However, the therapeutic effects on ischemic limb complications are less clear. Accordingly, we aimed to develop a novel murine model of thrombotic hindlimb ischemia to reflect that found in patients with PAD exhibiting ischemic limb symptoms. We further investigated the effects of P2Y12 deficiency and P2Y12 inhibition by prasugrel in this model. METHODS AND RESULTS: Thrombus formation induced by application of ferric chloride to the femoral artery resulted in a significant reduction in blood flow in the injured limb. In gait analysis using the CatWalk system, moderate difficulties in grounding and weight bearing of the ischemic limb, including reduction of maximum contact area and stance phase duration and increasing in swing phase duration in the ischemic limb, were observed in this model. Blood flow reduction and gait abnormalities gradually recovered over 21 days to levels present before arterial injury. Compared to wild-type (WT) mice, significant increases in blood flow and improvement in gait were observed in P2Y12-deficient mice. In addition, daily oral administration of prasugrel (3 mg/kg per day) to WT mice resulted in significant inhibition of blood flow reduction and gait abnormalities to levels found in P2Y12 deficient mice. CONCLUSIONS: Acute femoral artery thrombosis resulted in hindlimb ischemia and moderate gait abnormalities in mice. In addition, the present study suggests a possible role of P2Y12 in the complications with thrombotic limb ischemia.

A Novel Model of Intravital Platelet Imaging Using CD41-ZsGreen1 Transgenic Rats.

Platelets play pivotal roles in both hemostasis and thrombosis. Although models of intravital platelet imaging are available for thrombosis studies in mice, few are available for rat studies. The present effort aimed to generate fluorescent platelets in rats and assess their dynamics in a rat model of arterial injury. We generated CD41-ZsGreen1 transgenic rats, in which green fluorescence protein ZsGreen1 was expressed specifically in megakaryocytes and thus platelets. The transgenic rats exhibited normal hematological and biochemical values with the exception of body weight and erythroid parameters, which were slightly lower than those of wild-type rats. Platelet aggregation, induced by 20 µM ADP and 10 µg/ml collagen, and blood clotting times were not significantly different between transgenic and wild-type rats. Saphenous arteries of transgenic rats were injured with 10% FeCl3, and the formation of fluorescent thrombi was evaluated using confocal microscopy. FeCl3 caused time-dependent increases in the mean fluorescence intensity of injured arteries of vehicle-treated rats. Prasugrel (3 mg/kg, p.o.), administered 2 h before FeCl3, significantly inhibited fluorescence compared with vehicle-treated rats (4.5 ± 0.4 vs. 14.9 ± 2.4 arbitrary fluorescence units at 30 min, respectively, n = 8, P = 0.0037). These data indicate that CD41-ZsGreen1 transgenic rats represent a useful model for intravital imaging of platelet-mediated thrombus formation and the evaluation of antithrombotic agents.

Prevention of occlusive arterial thrombus formation by a single loading dose of prasugrel suppresses neointimal hyperplasia in mice.

The present study examined the effects of prasugrel in a mouse model of thrombosis-induced neointimal hyperplasia. Following carotid artery injury by application of ferric chloride solution, thrombus formation was assessed on Day 1 and neointimal thickening was assessed on Day 21. Single administrations of prasugrel at 0.3-3mg/kg (p.o.) resulted in a dose-related and sustained inhibition of ADP-induced platelet aggregation through 24h. Single and multiple (1 and 3 weeks) administration of prasugrel (3mg/kg loading and 1mg/kg/day maintenance doses) resulted in a marked inhibition of neointimal thickening in the injured artery. In the dose-response study, a single administration of prasugrel at 0.3-3mg/kg (p.o.) dose-relatedly inhibited thrombus formation and neointimal thickening on Days 1 and 21, respectively. The degree of neointimal hyperplasia in the injured artery correlated significantly with the thrombus indices, time to occlusion and patency rate. To explore possible mechanisms of inhibition of neointimal hyperplasia by prasugrel, mRNA expression levels of inflammatory and fibrosis markers were determined in injured arteries. Prasugrel treatment resulted in reduced MCP-1, ICAM-1 and TGF-ß mRNA levels on Day 2 (24h after the injury) and Day 8 (1 week after the injury) in the target arteries. In conclusion, we found that a single oral loading dose of prasugrel markedly prevented neointimal hyperplasia by inhibiting platelet activation and thrombus formation and was associated with inhibition of the expression of inflammatory and fibrosis markers, including MCP-1, ICAM-1 and TGF-ß, in the injured arteries.

Prasugrel reduces ischaemic infarct volume and ameliorates neurological deficits in a non-human primate model of middle cerebral artery thrombosis.

Several clinical trials have demonstrated the benefits of thienopyridine monotherapy in ischaemic stroke patients. Non-human primate models of ischaemic stroke have been used for various antithrombotic agents; however, to the best of our knowledge, there is no data on the effects of P2Y12 antagonists in models, such as the thrombotic middle cerebral artery occlusion (MCAO) monkey model. Accordingly, it remains unclear what level of inhibition of platelet aggregation (IPA) is required for optimal treatment of ischaemic stroke. In the present study, we investigated the effects of prasugrel, a third-generation thienopyridine antiplatelet drug, on platelet aggregation, thrombus formation and cerebral infarct volume in a non-human primate model. Daily oral administration of prasugrel resulted in significant and stable platelet inhibitory effects on Day 3, with IPA values ranging from 31% to 36% at 0.3mg/kg/day and from 44% to 50% at 1mg/kg/day. These IPA levels encompassed values observed in clinical trials of clopidogrel, and were thus selected for further study. In the thrombotic MCAO model, prasugrel increased MCA patency in a dose-dependent manner and significantly reduced ischaemic infarct volume by approximately 70% at 0.3mg/kg/day and 90% at 1mg/kg/day without increasing haemorrhagic infarction. Prasugrel also significantly reduced neurological deficit scores by 60% at 0.3mg/kg/day and 80% at 1mg/kg/day. In conclusion, prasugrel treatment resulted in effective reduction of ischaemic infarction and an associated improvement in neurological function without increasing haemorrhagic infarction. These data suggest that prasugrel monotherapy would be effective for the prevention of thrombotic stroke.

Characterization of platelet aggregation responses in microminipigs: Comparison with miniature pigs and the influence of dual antiplatelet administration of aspirin plus prasugrel.

We aimed to characterize platelet aggregation responses and the impact of dual antiplatelet therapy in microminipigs. In this in vitro study, both adenosine-5'-diphosphate (ADP, 5-50µM) and collagen (2-20µg/ml) induced concentration-related platelet aggregation in the microminipigs; 20µM ADP and 5 and 12.5µg/ml collagen were selected for further ex vivo studies. Aspirin plus prasugrel were administered orally for 7days (n=4/each group). Ex vivo platelet aggregation was analyzed on Day 1 (1 and 4h after administration), Day 4 (4h), and Day 7 (4h) under three different prasugrel dosing regimens: LD0/MD1 (1mg/kg/day), LD0/MD3 (3mg/kg/day), and LD10/MD1 (10mg/kg loading dose and 1mg/kg/day maintenance dose). Aspirin (10mg/kg/day) was administered to all groups. In the presence of aspirin, prasugrel at 3 and 10mg/kg significantly inhibited ADP-induced platelet aggregation on Day 1. On Days 4 and 7, significant inhibition of platelet aggregation (IPA) was also observed in each group. With 5µg/ml collagen-induced platelet aggregation, all three groups showed significant IPA at 4h on Day 1 or later. In 12.5µg/ml collagen-induced platelet aggregation, all groups showed significant effects on Days 4 and 7; however, the 30%-35% IPA was considerably lower than that (50%-60%) found with 5µg/ml collagen. In Clawn miniature pigs, similar inhibitory patterns were observed for both ADP- and collagen-induced ex vivo platelet aggregation. In conclusion, these results indicated that microminipigs as well as miniature pigs may represent useful experimental animals for thrombosis research.

Platelet activation biomarkers in Berkeley sickle cell mice and the response to prasugrel.

Vaso-occlusive crisis (VOC) is a common complication that occurs in sickle cell disease (SCD) patients. Although underlying mechanisms of VOC remain unclear, platelet activation has been associated with VOC. In the present study, plasma adenine nucleotide measurements using LC-ESI-MS/MS showed that plasma ADP in the Berkeley murine model of SCD was significantly higher (applox. 2.7-fold increase) compared with control mice. Assessment of platelet activation markers using flow cytometry indicated that in SCD mice at steady state (8 weeks old), circulating platelets were partially activated and this tended to increase with age (15 weeks old). The administration of prasugrel, a thienopiridyl P2Y12 antagonist, did not affect the activation state of circulating platelets suggesting P2Y12 independent mechanism of activation. In this murine SCD model, ex vivo addition of ADP or PAR4 TRAP resulted in further platelet activation as assessed by expression of activated GPIIb/IIIa and P-selectin both at 8 and 15 weeks. In 15 weeks old SCD mice, agonist-induced increases in activation markers were enhanced compared to control mice. Oral administration of prasugrel effectively inhibited ex vivo platelet activation consistent with clinical data in patients with SCD. In conclusion, in the Berkeley murine model of SCD, we found evidence of basal and agonist-stimulated platelet activation which could in part be attenuated by prasugrel. These data are consistent with observations made in patients with SCD and suggest possible utility of this murine model and prasugrel therapy in exploring treatment options for patients with SCD.

Comparison of antiplatelet effects of prasugrel and ticagrelor in cynomolgus monkeys by an ELISA-based VASP phosphorylation assay and platelet aggregation.

Prasugrel is the third generation thienopyridine prodrug, and ticagrelor is a non-competitive direct acting P2Y12 antagonist. In phase 3 studies, both agents reduced ischaemic event rates compared to clopidogrel. The present in vitro human and monkey studies showed that ticagrelor's active metabolite (AM) was more potent than ticagrelor and prasugrel's AM on inhibition of ADP-induced platelet aggregation by light transmission aggregometry and ELISA-based vasodilator-stimulated phosphoprotein (VASP) phosphorylation assay. In contrast, on an oral dosage basis (mg/kg), prasugrel showed more potent platelet inhibition compared to ticagrelor on ex vivo aggregation and VASP phosphorylation assays in monkeys. Single oral doses of prasugrel (0.3 and 1 mg/kg) resulted in robust antiplatelet effects, which were sustained up to 24 hours after administration. Ticagrelor (3 and 10 mg/kg, p.o.) also showed significant antiplatelet effects but its effects were diminished at 24 hours after the dosing. Repeat administration of prasugrel (1.8 mg/kg loading dose [LD], 0.3 mg/kg once daily maintenance dose [MD]) showed more rapid antiplatelet effects and longer duration of action throughout the entire day. Twice a day repeat administration of ticagrelor (10 mg/kg bid MD following a single 20 mg/kg LD) also showed significant antiplatelet effects but with more intra-day variability compared to prasugrel. The in vitro and ex vivo studies showed strong correlations between platelet aggregation and VASP phosphorylation for prasugrel, ticagrelor and their AMs. These strong correlations between platelet aggregation and VASP phosphorylation in non-human primates also suggest that ELISA-based human VASP assay can be utilised for non-human primate platelet studies.

Nitroxide derivatives for imaging of hypercholesterolemia-induced kidney dysfunction and assessing the effectiveness of antilipidemic drugs.

The present study was designed to clarify the possibility for application of nitroxide derivatives in magnetic resonance imaging (MRI) of hypercholesterolemia-mediated renal dysfunction in mice, as well as to assess the effectiveness of antilipidemic drugs (cholestyramine and ezetimibe). The mice were separated in four groups: (i) on a normal diet (ND) without medication (control); (ii) on a high cholesterol diet (CD) without medication; (iii) CD mice receiving cholestyramine; and (iv) CD mice receiving ezetimibe. In CD mice without medication, a hypercholesterolemia was developed, detected by the increasing of total plasma cholesterol and non-HDL cholesterol, and decreasing of HDL cholesterol. The hypercholesterolemia compromised renal function: blood urea nitrogen, creatine and uric acid increased significantly, accompanied with development of glomerulosclerosis, enhancement of the amount of neutrophils and overexpression of metalloproteinase-9. The mice were subjected to anesthesia and MR imaging was performed on 7 T magnet (T1-weighted incoherent gradient-echo sequence; fast low-angle shot). The region-of-interest was selected within the kidney. The images were obtained before and after injection of contrast probe [carbamoyl-PROXYL (CMP) or Gd-DTPA]. In the kidney of ND mice, the MRI signal intensity increased after injection of CMP, reached a maximum (very well-defined renal filtration peak) and decreased to the baseline level within 14 min. In kidney of CD mice, the CMP-mediated enhancement of MRI signal was not detected. Antilipidemic drugs patially abolished the effect of hypercholesterolemia on CMP-enhanced MRI in the kidney. The kinetic curves of Gd-enhanced MRI signal had also different profiles in the kidney of ND and CD mice. They were similar to the profiles of the kinetic curves, obtained from MR urography of healthy human and human with renal pathology, respectively. The present study suggests that CMP is a suitable MRI contrast probe for visualization of hypercholesterolemia-induced renal dysfunction in intact animals and the assessment of the efficacy of antilipidemic drugs. The probe was applied at a concentration that was 3 times lower than the LD50 for intravenous administration in mice. Since the probe is excreted by the kidney, it could be considered harmless for mammalians in the selected dose and appropriate candidate for translational research.

Carbamoyl-PROXYL-enhanced MRI detects very small disruptions in brain vascular permeability induced by dietary cholesterol.

BACKGROUND: Gd-DTPA-enhanced magnetic resonance imaging (MRI) is a conventional method for non-invasive investigation of blood-brain-barrier (BBB) permeability in animal models. It allows the visualization of serious injury to the BBB. We developed a novel approach for detecting very small disruptions in BBB permeability induced by dietary cholesterol by using carbamoyl-PROXYL (CMP) as an MRI contrast probe. METHODS: Mice were separated into two groups: normal diet (ND-mice) and high cholesterol diet (CD-mice). MRI-signal dynamics, plasma cholesterol, matrix metalloproteinase (MMP-9, MMP-2), and the white blood cell profile were analyzed. For the MRI analysis, two regions-of-interest (ROI) were selected: brain (ROI-1) and surrounding area (ROI-2). RESULTS: In the ROI-2 of ND-mice, CMP- or Gd-enhanced MRI-signal followed typical kinetics with a half-life of signal decay (τ(1/2)) approximately 8 or approximately 15 min, respectively. In CD-mice, the MRI-signal increased continuously without decay. In the ROI-1 of ND- and CD-mice, MRI-signal enhancement was not detected by Gd-DTPA. In the ROI-1 of ND-mice, CMP-induced MRI-signal enhancement was negligible, while in CD-mice, it was significant (τ(1/2)>15 min). Hypercholesterolemia increased the plasma levels of MMP-9 and neutrophils. CONCLUSIONS: Hypercholesterolemia increases vascular permeability, which is mediated by MMP-9 and neutrophils. GENERAL SIGNIFICANCE: Even very small disruptions in brain vascular permeability could be detected by CMP-enhanced MRI but not by Gd-DTPA-enhanced MRI.

Selective blockade of P2Y12 receptors by prasugrel inhibits myocardial infarction induced by thrombotic coronary artery occlusion in rats.

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.

Pharmacodynamic and pharmacokinetic evaluation of CS-526 in cynomolgus monkeys.

In the present study, we evaluated the effect of the novel acid pump antagonist 7-(4-fluorobenzyloxy)-2,3-dimethyl-1-{[(1S,2S)-2-methylcyclopropyl]methyl}-1H-pyrrolo[2,3-d]pyridazine (CS-526) on the intragastric acidity of cynomolgus monkeys. The study was performed in a crossover manner with five male animals. CS-526 was administered orally or intravenously at doses of 3.0, 10 and 30 mg/kg, or 0.3, 1.0 and 3.0 mg/kg, respectively. The time period in which the intragastric pH was 4.0 or more (Time(pH > or = 4.0)) and the median pH were calculated for 24 h after the administration. The intragastric pH was elevated after CS-526 treatment. The Time(pH > or = 4.0) was increased in a dose-dependent manner (p = 0.0292) in the oral administration, and the median pH was also increased in a dose-dependent fashion (p = 0.0491) in the intravenous administration. The plasma concentration of CS-526 and its metabolite R-130185 was increased after oral and intravenous administration of CS-526, except for one animal which did not show any detectable amount of R-130185 after intravenous administration at the lowest dose. The area under the time-concentration curve of the active component was increased in the dose proportional manner after oral and intravenous administration. The absolute bioavailability of the active component was estimated to be approximately 1%. Correlation between the pharmacodynamic parameters and the pharmacokinetic parameters was observed in oral (p = 0.0029-0.0745), but not in intravenous administration (p = 0.0558-0.2789). In conclusion, oral and intravenous administration of CS-526 showed inhibition on gastric acidity in cynomolgus monkeys using intragastric pH-metry and some pharmacokinetic and pharmacodynamic parameters were well correlated.

The effect of subchronic administration of 7-(4-fluorobenzyloxy)-2,3-dimethyl-1-{[(1S,2S)-2-methylcyclopropyl]methyl}-1H-pyrrolo[2,3-d]pyridazine (CS-526), a novel acid pump antagonist, on gastric acid secretion and gastrin levels in rats.

In the present report, we evaluated the effect of the novel acid pump antagonist 7-(4-fluorobenzyloxy)-2,3-dimethyl-1-{[(1S,2S)-2-methylcyclopropyl]methyl}-1H-pyrrolo[2,3-d]pyridazine (CS-526) and 2-[3-methyl-4-(2,2,2-trifluoro-ethoxy)-pyridin-2-ylmethanesulfinyl]-1H-benzimidazole (lansoprazole) on rebound gastric acid secretion, using an intragastric dialysis membrane perfusion model and on the serum and antral gastrin level after a 14-day treatment in rats. The effect of CS-526 on gastric acid secretion was almost constant during the 14 days of treatment. After the 14-day treatment, gastric acid secretion had returned to pretreatment levels. However, CS-526 slightly increased and lansoprazole potently increased gastric acid secretion thereafter. In the posttreatment period, the influence on rebound gastric acid secretion by lansoprazole treatment was significant, but that by CS-526 was not. The serum gastrin concentration after the 14-day treatment with CS-526 did not increase significantly, even at 100 mg/kg/day. On the other hand, lansoprazole at 100 mg/kg/day significantly elevated the serum gastrin concentration. After the 14-day treatment with CS-526 at 100 mg/kg/day, the antral gastrin content significantly increased. Lansoprazole at the doses of 30 and 100 mg/kg/day also significantly increased the antral gastrin content after the 14-day treatment. The elevation of the serum gastrin level after the lansoprazole treatment was suppressed by the concomitant administration of CS-526. In conclusion, CS-526 has a potent antisecretory effect on gastric acid secretion without rebound gastric hypersecretion. Moreover, CS-526 had minimal effects on the serum and antral gastrin elevation. It is suggested that these effects on gastric acid secretion and serum gastrin after subchronic treatment with CS-526 would be beneficial in clinical use.

Pharmacological profile of novel acid pump antagonist 7-(4-fluorobenzyloxy)-2,3-dimethyl-1-{[(1S,2S)-2-methyl cyclopropyl]methyl}-1H-pyrrolo[2,3-d]pyridazine (CS-526).

The pharmacological profiles of the novel acid pump antagonist 7-(4-fluorobenzyloxy)-2,3-dimethyl-1-{[(1S,2S)-2-methylcyclopropyl]methyl}-1H-pyrrolo[2,3-d]pyridazine (CS-526) were investigated in terms of hog gastric H+,K+-ATPase activity, gastric acid secretion, and acute gastroesophageal lesions in comparison with other proton pump inhibitors (PPIs). CS-526 inhibited H+,K+-ATPase activity in a concentration-dependent manner, with an IC50 value of 61 nM. The inhibitory effect of CS-526 on H+,K+-ATPase activity was more potent than that of any of the other PPIs examined. The inhibitory mechanism of CS-526 on H+,K+-ATPase was a competitive antagonism to the K+ binding site of H+,K+-ATPase, and it was also a reversible inhibition. In pylorus-ligated rats, intraduodenal or oral administration of CS-526 inhibited gastric acid secretion in a dose-dependent manner, and the ID50 values were 2.8 or 0.7 mg/kg, respectively. In Heidenhain pouch dogs, intrapouch administration of CS-526 inhibited histamine-stimulated gastric acid secretion in a dose- and retention time-dependent manner. In a reflux esophagitis model, intraduodenal and oral administration of CS-526 prevented esophageal lesions with ID50 values of 5.4 and 1.9 mg/kg, respectively. Lansoprazole prevented esophagitis only by intraduodenal administration (ID50 = 2.2 mg/kg). Furthermore, CS-526 inhibited acute gastric mucosal lesions. These data demonstrate that the novel acid pump antagonist CS-526 has potent antisecretory and antiulcer effects. These findings indicate that CS-526 would have a curative effect on gastroesophageal reflux disease via its potent antisecretory and antiulcer actions.