Effect of a selective thromboxane A2 synthetase inhibitor on the systemic changes induced by circulating pancreatic phospholipase A2

Background In acute pancreatitis, pancreatic phospholipase A₂ (PLA2) in the circulating blood hydrolyzes phospholipids contained in plasma lipoproteins, liberating eicosanoid precursors that are subsequently converted to various eicosanoids. The pathophysiological significance of eicosanoid synthesis via this pathway is unknown. The aim of this study was to clarify the role of thromboxane A₂ (TXA₂) synthesis by circulating pancreatic PLA₂ in the pathogenesis of the systemic complications of acute pancreatitis. Methods Guinea pigs were divided into two groups: a control group and an ozagrel group, which received intravenous administration of ozagrel, a selective TXA₂ synthetase inhibitor. Pancreatic PLA₂ was infused intravenously in both groups for 30 min, and systemic changes during the infusion were examined. Results In the control group, there was an increase in plasma thromboxane B₂ (TXB₂) concentration, a decrease in mean arterial pressure and heart rate, a decrease in arterial base excess (BE), bicarbonate concentration (HCO₃ ⁻), and pH, a decrease in platelet count and plasma fibrinogen concentration, and a shortened prothrombin time during the infusion of pancreatic PLA₂. In the ozagrel-treated group, changes in plasma TXB₂ concentration, BE, HCO₃ ⁻, and platelet count were significantly inhibited. Conclusions TXA₂ synthesis by circulating pancreatic PLA₂ contributes to metabolic acidosis and thrombocytopenia during acute pancreatitis.     

Effects of a new thromboxane A2-antagonist (ONO-3708) and a new leukotriene-antagonist (ONO-1078) on thromboxane A2 analogue-, leukotriene C4-, and D4-induced regional myocardial blood flow reduction

Summary Effects of the administration of a thromboxane A₂ (TXA₂) analogue (STA₂), a leukotriene C₄ (LTC₄), and a leukotriene D₄ (LTD₄) on regional myocardial blood flow (RMBF) and hemodynamics were studied in anesthetized, open-chest dogs. The blocking ability of a recently synthesized TXA₂ selective antagonist, ONO-3708, and a peptidoleukotriene-selective antagonist, ONO-1078, was also investigated. RMBF was measured continuously in three areas: the left anterior descending coronary artery (LAD) area, the circumflex artery (Cx) area, and the area between LAD and Cx. STA₂, LTC₄, and LTD₄ caused a significant dose-dependent reduction of the RMBF in the LAD area. The peak percentage decrease in RMBF followed by a 10 µg dose of STA₂, 1 µg dose of LTC₄, and 1 µg dose of LTD₄ is 38.6%±3.0%, 39.0%±3.1%, and 36.2%±2.4%, respectively. ED₅₀ for the action of LTC₄, LTD₄, and STA₂ on RMBF is 3, 3, and 50 µg, respectively. Pretreatment with the newly developed TXA₂ antagonist, ONO-3708 (1 µg/kg/min for 10 min), completely inhibited the RMBF reduction induced by STA₂ (10 µg). Pretreatment with the peptidoleukotriene antagonist, ONO-1078 (1 mg), inhibited the RMBF reduction induced by LTC₄ or LTD₄ (0.3–3 µg). Following pretreatment with a 1 mg dose of ONO-1078, the peak percentage decrease of RMBF caused by a 1 µg dose of LTC₄ and LTD₄ was reduced to 21.1%±2.3% and 19.8%±3.1%, respectively. However, the LTC₄ (1 µg)-induced reduction of the RMBF was not affected by pretreatment with a TXA₂ antagonist, ONO-3708, or an inhibitor of the endogenous production of TXA₂, OKY-046. Likewise, the reduction of the RMBF induced by STA₂ (10 µg) was not affected by pretreatment with a leukotriene-antagonist, ONO-1078. These results suggest that the reduction of RMBF induced by TXA₂, LTC₄, and LTD₄ is mediated in vivo through different vascular receptors for each.     

Antagonism of P2Y12 reduces physiological thromboxane levels

Antiplatelet therapy for the management of patients with cardiovascular risks often includes a combination therapy of aspirin and clopidogrel, acting through inhibition of thromboxane generation and blockade of G_i-coupled P2Y₁₂ receptor, respectively. We hypothesized that ADP acting through P2Y₁₂ regulates physiological thromboxane levels. The serum thromboxane levels in mice (n?=?3) dosed with clopidogrel and prasugrel were decreased by 83.1?±?5.3% and 94.26?±?1.75% respectively compared to untreated mice. Pre-treatment of human blood (n?=?3) ex vivo with active metabolites of clopidogrel or prasugrel led to a reduction in thromboxane levels to 16.3?±?3.2% and 4.9?±?0.8% respectively, compared to untreated human serum. We also evaluated serum thromboxane levels in P2Y receptor null mice (n?=?4). Whereas serum thromboxane levels in P2Y₁ null mice were similar to those in wild type littermates, those in the P2Y₁₂ null mice were inhibited by 83.15?±?3.8%. Finally, in a pilot study, serum thromboxane levels were reduced by 76.05?±?8.41% in healthy human volunteers (n?=?6) upon dosing with clopidogrel, compared to the levels before dosing. In conclusion, P2Y₁₂ antagonism alone can decrease physiological thromboxane levels. Thus, this study could pave way the for newer/modified treatment regimens for the management of patients with thrombotic complications who are allergic or non-responsive to aspirin.     

Effects of thromboxane A2 agonist STA2 on rapidly adapting pulmonary stretch receptors in vagotomized rabbits

We investigated the responses of rapidly adapting pulmonary stretch receptors (RARs) and tracheal pressure (P_T) to right atrial injections of the thromboxane A₂ (TXA₂) stable analogue STA₂ (0.3, 1.0, and 3.0 g/kg) before and after administration of atropine sulfate (1 mg/kg), isoprenaline (200 g/kg), indomethacin (1 mg/kg), or S-145 (0.5 mg/kg) in artificially ventilated, bilaterally vagotomized rabbits. The RARs increased their activity after STA₂ administration, and the increase was dose-dependent. However, intraatrial injections of STA₂ at all the doses examined had no significant effect on P_T. The excitatory responses of RAR activity to STA₂ (0.3–3.0 g/kg) were not significantly altered by administration of atropine sulfate (anticholinergic agent), isoprenaline (bronchodilator), or indomethacin (cyclooxygenase inhibitor). However, S-145 treatment (TXA₂ antagonist) blocked the STA₂-induced RAR stimulation. To determine whether or not administration of STA₂ causes release of acetylcholine (ACh), we also examined the effects of vagal efferent stimulation (10–15 V, 10 Hz, 1 ms), STA₂ administration (3.0 g/kg), and their combination on P_T in rabbits associated with both artificial ventilation and bilateral vagotomy. The vagally mediated bronchoconstriction that led to an increase in P_T was not enhanced by simultaneous administration of STA₂ at 3.0 g/kg in all of the tested animals. These results suggest that the stimulation of RARs by STA₂ is not mediated by the release of ACh from the nerve endings but is probably due to a local inflammatory bronchoconstriction that does not significantly alter the value of P_T.Offprint requests to: S. Matsumoto     

The contractile mechanism of beraprost sodium,a stable prostacyclin analog,in the isolated canine femoral vein

Summary The vascular contractile mechanism of prostacyclin (PGI₂) was investigated using beraprost sodium (BPS), a stable PGI₂ analog. Ring strips without endothelium isolated from canine femoral veins and arteries were used. BPS induced a dose-dependent contraction without precontraction and after precontraction with norepinephrine (NE) or 60 mM K⁺ in the veins. In contrast, BPS induced a dose-dependent relaxation after precontraction with U46619, a thromboxane A₂ (TXA₂) analog, or prostaglandin F₂ (PGF₂) in the veins. In the arteries, BPS induced contraction at higher concentrations after precontraction with NE. However, BPS relaxed arteries dose-dependently after precontraction with PGF₂. By pretreatment with 13-azaprostanoic acid (13-APA), a TXA₂/endoperoxide receptor antagonist, the dose-response curve of BPS in the veins was shifted to the right. Schild plot analysis resulted in a linear regression with a slope of 0.86 ± 0.13, which was not significantly different from unity, and the pA₂ value for 13-APA against BPS was 7.10 ± 0.06. By pretreatment with BPS, the dose-response curve of U46619 in the veins was shifted to the right. Kaumann plot analysis resulted in a linear regression with a slope of 0.89 ± 0.09, which was not significantly different from unity, and the pA₂ value for BPS against U46619 was 5.68 ± 0.04. These findings indicate that BPS is a partial agonist for the TXA₂/endoperoxide receptors.     

Very-low-dose twice-daily aspirin maintains platelet inhibition and improves haemostasis during dual-antiplatelet therapy for acute coronary syndrome

Higher aspirin doses may be inferior in ticagrelor-treated acute coronary syndrome (ACS) patients and reducing bleeding risk whilst maintaining antithrombotic benefits could improve outcomes. We characterized the pharmacodynamics of a novel dual-antiplatelet-therapy regimen consisting of very-low-dose twice-daily (BD) aspirin with standard-dose ticagrelor. A total of 20 ticagrelor-treated ACS patients entered a randomized crossover to take aspirin 20 mg BD (12-hourly) during one 14-day period and 75 mg once-daily (OD) in the other. After 14 days of treatment, serum thromboxane (TX)B₂ and light-transmittance aggregometry were assessed pre- and 2 h post-morning-dose, bleeding time was measured post-dose, and TXA₂ and prostacyclin stable metabolites were measured in urine collected 2 h post-morning-dose. Data are expressed as mean ± SD. After 14 days treatment, serum TXB₂ levels were significantly greater 2 h post-dosing with aspirin 20 mg BD vs. 75 mg OD (3.0 ± 3.6 ng/mL vs. 0.8 ± 1.9 ng/mL; p = 0.018) whereas pre-dosing levels were not significantly different (3.5 ± 4.1 ng/mL vs. 2.5 ± 3.1 ng/mL, p = 0.23). 1-mmol/L arachidonic acid-induced platelet aggregation was similarly inhibited by both regimens pre-dose (8.5 ± 14.3% vs. 5.1 ± 3.6%, p = 0.24) and post-dose (8.7 ± 14.2% vs. 6.6 ± 5.3%; p = 0.41). Post-dose bleeding time was shorter with 20 mg BD (680 ± 306 s vs. 834 ± 386 s, p = 0.02). Urinary prostacyclin and TX metabolite excretion were not significantly different. In conclusion, compared to aspirin 75 mg OD, aspirin 20 mg BD provided consistent inhibition of platelet TXA₂ release and aggregation, and improved post-dose hemostasis, in ticagrelor-treated ACS patients. Further studies are warranted to assess whether this regimen improves the balance of clinical efficacy and safety.     

Lack of thromboxane A2 involvement in the arrhythmias occurring during acute myocardial ischemia in dogs

Summary Coronary artery occlusion (CAO) followed by reperfusion of the ischemic myocardium has been associated with the onset of ventricular arrhythmias. It has been suggested that platelet aggregates in the ischemic area may release thromboxane A₂ (TxA₂) which may then be responsible for the arrhythmias that occur during reperfusion. To study this possibility, the effect of TxA₂ synthetase inhibition on arrhythmias was examined in anesthetized dogs during occlusion and for 60 minutes following release. Imidazole (30 mg/kg) was infused intravenously for 10 minutes, followed by continuous infusion of 100 mg/kg/hr for 125 minutes. The left anterior descending coronary artery was occluded, 5 minutes after the initial dose, for 60 minutes. Three minutes after release of CAO, TxB₂ concentrations were significantly higher in the arterial blood of vehicle-treated animals (2.06±0.53 pmoles/ml) than in either CAO + imidazole (0.66±0.16 pmoles/ml) or sham-CAO animals receiving imidazole (0.66±0.09 pmoles/ml). However, CAO dogs whether receiving imidazole or 0.9% NaCl generated a significantly greater number of ectopic beats during and after occlusion than sham-CAO animals. Therefore, release of TxA₂ does not appear to be a major causative factor in the generation of reperfusion arrhythmias in dogs following coronary artery occlusion.Predoctoral Fellow of the Ischemia-Shock Research Center of Jefferson Medical College.     

Involvement of thromboxane and leukotriene in arachidonate induced coronary constriction in diabetic rats

Summary Vascular responsiveness to sodium arachidonate was examined in isolated perfused hearts from rats with streptozotocin-induced diabetes. In diabetic rats arachidonate induced a biphasic coronary vascular response characterized by initial vasoconstriction followed by prolonged vasodilation. Non-diabetic rats showed only a vasodilator response. The vasoconstrictor phase found in diabetic rats was abolished by ONO-3708, a selective thromboxane A₂ antagonist. Indomethacin partly inhibited the vasoconstrictor response, the residual response being abolished by a leukotriene antagonist, ONO-1078. The vasodilator response, however, was completely abolished by indomethacin in both diabetic and non-diabetic rats. Furthermore, the coronary constrictor response to leukotriene D₄ was enhanced in diabetic compared to non-diabetic rats. These results suggest an involvement of leukotriene in the vasoconstrictor response to arachidonate in diabetic rats, especially when cyclooxygenase is inhibited.     

Tannin is the major agent present in cotton mill dust responsible for human platelet 5-hydroxytryptamine secretion and thromboxane formation

Aqueous extracts of cotton mill dust were examined for their ability to activate washed human platelets as indicated both by the secretion of 5-hydroxytryptamine and the generation of thromboxane A₂. The cotton dust extract promoted 5-hydroxytryptamine secretion in a dose-dependent manner. When expressed in terms of its tannin concentration, the dose curve for cotton dust extract was identical to that obtained with purified tannin. Cotton dust extract also promoted the generation of low levels of thromboxane A₂. Fractionation of the extract by Amicon ultrafiltration into a > 10,000 MW fraction and a < 10,000 MW fraction revealed that the < 10,000 MW fraction contained an inhibitor of the thromboxane assay. The dose-response curve for thromboxane A₂ generation mediated by the > 10,000 MW cotton dust extract fraction was indistinguishable from that of purified tannin. Thus, 2 criteria show that platelet activation by cotton mill dust appears due to the tannin present in the dust.     

Involvement of Nitric Oxide and Potassium Channels in the Reduction of Basal Tone Produced by Blockade of Thromboxane A2/Prostaglandin H2/ Receptors in Aortic Rings Of Hypertensive Rats

This study was designed to investigate involvement of potassium channels in the action of nitric oxide facilitating reduction of basal tone by thromboxane A₂/prostaglandin H₂receptor blockade with ifetroban in rings of thoracic aorta taken from rats with aortic coarctation-induced hypertension. Ifetroban-induced reduction of basal tone in aortic rings without drug pretreatment was attenuated (P<0.05) in rings pretreated with the nitric oxide synthesis inhibitor Nω-nitro-L-arginine methyl ester (L-NAME; 3 × 10<^-4mol/L; 0.55±0.09 g versus 0.23±0.07 g). The vasorelaxing effect of ifetroban also was decreased (P<0.05) in preparations pretreated with a potassium channel blocker, either tetraethylammonium (TEA; 10^-2mol/L) or 4-aminopyridine (4-AP; 3 × 10^-3mol/L). Ifetroban-induced reduction of basal tone was not attenuated in preparations pretreated first with L-NAME and then with sodium nitroprusside (SNP; 6±1 nmol/L) to compensate for the loss of endogenous nitric oxide. However, the facilitatory effect of SNP on ifetroban-induced relaxation of aortic rings pretreated with L-NAME alone was not demonstrable in rings pretreated with L-NAME plus TEA or 4-AP. These observations suggest that a mechanism involving nitric oxide and potassium channels facilitates the reduction in basal tone produced by ifetroban in aortic rings of rats with aortic coarctation-induced hypertension     

Acyl derivatives of coenzyme A inhibit platelet function via antagonism at P2Y1 and P2Y12 receptors: A new finding that may influence the design of anti-thrombotic agents

We have performed a detailed investigation of the effects on platelet function of coenzyme A (CoA) and several acyl-CoAs. Platelet aggregation was measured by turbidimetry and by platelet counting; platelet shape change was measured using light scattering; P-selectin, Ca²⁺ mobilization and vasodilator-stimulated phosphoprotein (VASP) phosphorylation were measured by flow cytometry. The compounds investigated inhibited ADP-induced platelet aggregation; those with saturated acyl groups containing 16-18 carbons were most effective. The effects of palmitoyl-CoA (16:0) were studied in depth. It inhibited platelet shape change and Ca²⁺ mobilization brought about by ADP (but not other agonists) indicating antagonism at P2Y₁ receptors, and also inhibited ADP-induced P-selectin expression. Effects of palmitoyl-CoA on the platelet aggregation and Ca²⁺ mobilization induced by several different agonists and agonist combinations were compared with those of MRS 2179 (a P2Y₁ antagonist) and AR-C69931 (a P2Y₁₂ antagonist), and were consistent with palmitoyl-CoA acting mainly at P2Y₁ but also with partial antagonism at P2Y₁₂ receptors. Antagonism at P2Y₁₂ receptors was confirmed in studies of VASP-phosphorylation. Palmitoyl-CoA did not act as an antagonist at P2X₁ receptors. The results are discussed in relation to the possibility that acyl-CoAs may contribute as endogenous modulators of platelet function and might serve as lead compounds for the design of novel antithrombotics.     

A randomized open-label trial of on-demand rabeprazole vs ranitidine for patients with non-erosive reflux disease

AIM: To compare the efficacy of the proton-pump inhibitor, rabeprazole, with that of the H₂-receptor antagonist, ranitidine, as on-demand therapy for relieving symptoms associated with non-erosive reflux disease (NERD).METHODS: This is a single center, prospective, randomized, open-label trial of on-demand therapy with rabeprazole (group A) vs ranitidine (group B) for 4 wk. Eighty-three patients who presented to the American University of Beirut Medical Center with persistent gastroesophageal reflux disease (GERD) symptoms and a normal upper gastrointestinal endoscopy were eligible for the study. Patients in group A (n = 44) were allowed a maximum rabeprazole dose of 20 mg twice daily, while those in group B (n = 39) were allowed a maximum ranitidine dose of 300 mg twice daily. Efficacy was assessed by patient evaluation of global symptom relief, scores of the SF-36 quality of life (QoL) questionnaires, total number of pills used, and number of medication-free days.RESULTS: Among the 83 patients who were enrolled in the study, 76 patients (40 in the rabeprazole group and 36 in the ranitidine group) completed the 4-wk trial. Baseline characteristics were comparable between both groups. After 4 wk, there was no significant difference in the subjective global symptom relief between the rabeprazole and the ranitidine groups (71.4% vs 65.4%, respectively; P = 0.9). There were no statistically significant differences between mean cumulative scores of the SF-36 QoL questionnaire for the two study groups (rabeprazole 22.40 ± 27.53 vs ranitidine 17.28 ± 37.06; P = 0.582). There was no significant difference in the mean number of pills used (rabeprazole 35.70 ± 29.75 vs ranitidine 32.86 ± 26.98; P = 0.66). There was also no statistically significant difference in the mean number of medication-free days between both groups.CONCLUSION: Rabeprazole has a comparable efficacy compared to ranitidine when given on-demand for the treatment of NERD. Both medications were associated with improved quality of life.     

A novel mechanism of action for hypertension control: Moxonidine as a selective I1-imidazoline agonist

Sympathoadrenal inhibition by a direct action within the central nervous system is an advantageous route to blood pressure control. Stimulation of brain ₂-adrenergic receptors is one mechanism for sympathoadrenal suppression, but comes at the cost of nonspecific depression of CNS function, including sedation and decreased salivary flow. Evidence is accumulating for a second pathway for pharmacological control of sympathoadrenal outflow, mediated by a novel receptor specific for imidazolines. First-generation central antihypertensive agents, which are imidazolines such as clonidine, act primarily to stimulate these I₁-imidazoline receptors in the rostral ventrolateral medulla oblongata (RVLM) to lower blood pressure, but have sufficient agonism at ₂-adrenergic receptors to produce side effects. Second-generation centrally acting antihypertensive agents, such as moxonidine and rilmenidine, are selective for I₁ relative to ₂ receptors. The reduced ₂ potency of these agents correlates with reduced severity of side effects. In this study we further established the selectivity of moxonidine for I₁-imidazoline sites by characterizing the direct interaction of [³H]moxonidine with these receptors in the RVLM and in adrenomedullary chromaffin cells. [³H]Moxonidine preferentially labeled I₁-imidazoline sites relative to ₂-adrenergic sites, only a small portion of which were labeled in the RVLM. [³H]Moxonidine binding to I₁-imidazoline sites was modulated by guanine nucleotides, implying that I₁-imidazoline sites may be membrane receptors coupled to guanine nucleotide binding regulatory proteins (G proteins). Receptor autoradiography with [¹²⁵I]p-iodoclonidine confirmed the presence of I₁-imidazoline sites in the RVLM and other areas of the brainstem reticular formation. In contrast, ₂-adrenergic sites were mainly localized to the nucleus of the solitary tract. Moxonidine selectively displaced [¹²⁵I]p-iodoclonidine binding from reticular areas, including the RVLM. In vivo studies in SHR rats confirmed the ability of moxonidine to normalize hypertension by an action within the RVLM and confirmed the correspondence of I₁ binding affinity and antihypertensive efficacy. We also discuss prior literature on the cardiovascular pharmacology of imidazolines, reinterpreting previous studies that only considered alpha-adrenergic mechanisms.     

Changes of systemic prostacyclin and thromboxane A2 in sodium taurocholate-and cerulein-induced acute pancreatitis in rats

Systemic prostacyclin and thromboxane A₂ production in rat experimental acute pancreatitis has been evaluated by measuring the urinary excretion of the 2,3-dinor 6-keto prostaglandin F₁ and 2,3-dinor thromboxane B₂, respectively. Acute pancreatitis was induced by intraductal administration of 4.5% sodium taurocholate (0.1 ml/100 mg body weight) and intravenous cerulein perfusion (5 g/kg/hr) for 6 hr, respectively. Urinary excretion of 2,3-dinor 6-keto prostaglandin F₁ and 2,3-dinor thromboxane B₂ were much more important in sodium taurocholate- than in cerulein-induced acute pancreatitis. These data confirm an altered prostacyclin and thromboxane metabolism occurring in experimental acute pancreatitis. Phospholipase A₂ activity and the effect of gabexate mesilate on the arachidonate metabolism were also evaluated.This work was supported by the Fondo de Investigación Sanitaria (89/386).     

In Vivo Effect of Pancreatic Phospholipase A2 on the Arachidonic Acid Cascade

Summary Background. In acute pancreatitis, pancreatic phospholipase A₂ increases in systemic circulation. Yet the pathophysiological significance is controversial, because previous in vitro studies have shown that the enzyme has little cytotoxicity or ability to activate the arachidonic acid cascade by itself in contrast to other isozymes. Aim of the Study. The aims of this study are to examine the effect of pancreatic phospholipase A₂ on the arachidonic acid cascade in vivo; to explain the discrepancy, if present, between in vitro and in vivo findings; and to reassess the pathophysiological significance of circulating pancreatic phospholipase A₂. Methods. Pancreatic phospholipase A₂ was infused intravenously in guinea pigs, and changes in the arachidonic acid cascade, plasma lipoprotein, and cardiopulmonary function were investigated. Results. Plasma concentrations of 6-keto-prostaglandin F_1α, prostaglandin E₂, and thromboxane B₂ increased after intravenous (iv) infusion of pancreatic phospholipase A₂. Some of the plasma phospholipids such as phosphatidylcholine and phosphatidylethanolamine decreased, and free dihomo-γ-linolenic acid, arachidonic acid, and eicosapentaenoic acid were detected in plasma. These changes were accompanied with decreases in blood pressure, heart rate, and base excess. Conclusion. Circulating pancreatic phospholipase A₂ activates the arachidonic acid cascade, probably by supplying free eicosanoid precursors from plasma lipoprotein to eicosanoid-producing cells. It is supposed to be a cause of systemic complications in acute pancreatitis.     

Effects on platelet function of an EP3 receptor antagonist used alone and in combination with a P2Y12 antagonist both in-vitro and ex-vivo in human volunteers

EP3 receptor antagonists may provide a new approach to the treatment of atherothrombotic disease by blocking the ability of prostaglandin E₂ (PGE₂) to promote platelet function acting via EP3 receptors. DG-041 is an EP3 antagonist in the early stage of clinical development. Here, we quantitated effects on platelet function of DG-041 in-vitro and ex-vivo after administration to man when given alone and concomitantly with clopidogrel or clopidogrel and aspirin. With its unique mechanism of action, it was anticipated that DG-041 would potentiate inhibition of platelet function when given in combination with clopidogrel without materially increasing bleeding time. Initially, in-vitro studies were performed to determine inhibitory effects of DG-041 (3?µM) used alone or in combination with the P2Y₁₂ antagonist cangrelor (1?µM), both without and with aspirin (100?µM). Platelet aggregation and P-selectin expression were measured in whole blood (n?=?10) following stimulation with the thromboxane A₂ (TXA₂) mimetic U46619 (0.3 or 1?µM) in combination with either the EP3 agonist sulprostone (0.1?µM), or PGE₂ (1?µM). DG-041 alone partially inhibited platelet function in-vitro, as did cangrelor. Addition of both DG-041 and cangrelor in combination provided significantly greater inhibition. An ex-vivo study was then performed using the same experimental approaches. This clinical study was a prospective, randomised, blinded (for DG-041/matching placebo), blocked, crossover study designed to compare the effects of DG-041, clopidogrel, or the combination of DG-041 with either clopidogrel or clopidogrel and aspirin. Healthy volunteers (n?=?42) were randomly assigned to receive no background treatment, clopidogrel (300?mg loading dose plus 75?mg daily) or clopidogrel and aspirin (75?mg daily) for 10 days alongside DG-041 (200?mg twice daily) or placebo for 5 days, crossed over to placebo or DG-041 for the next 5 days. Platelet effects and bleeding time were measured at baseline, days 5 and 10. DG-041 partially inhibited platelet function ex-vivo, as did clopidogrel, while administration of both DG-041 and clopidogrel provided significantly greater inhibition. Administration of DG-041 alone did not increase bleeding time, and did not significantly affect the increased bleeding time seen with clopidogrel or clopidogrel with aspirin. Using these experimental approaches, the antiplatelet effects of DG-041 and a P2Y₁₂ antagonist used alone and in combination can be determined both in-vitro and ex-vivo. Results show inhibitory effects of DG-041 on platelet function acting via EP3 receptor blockade, confirmed to be additional to those brought about by P2Y₁₂ blockade. In both in-vitro and ex-vivo studies, aspirin neither promoted nor negated the effects of the other drugs.     

Extent of beta1- and beta2-receptor occupancy in plasma assesses the antagonist activity of metoprolol,pindolol, and propranolol in the elderly

Summary We estimated antagonist activity of metoprolol, pindolol, and propranolol in elderly cardiovascular patients by determining the extent to which the drugs occupied rabbit lung beta₁- and rat reticulocyte beta₂-adrenoceptors in plasma samples during drug treatment. The randomized, double-blind, crossover study was carried out by administering twice daily 100 mg metoprolol, 5 mg pindolol, and 80 mg propranolol for 7 days to 20 hypertensive subjects with a mean age of about 70 years. A 2-week interval was kept between administration of the different regimens. Receptor occupancy was measured at 1 hour before and 2 hours after administration of the last dose of each regimen by adding rabbit lung beta₁- and rat reticulocyte beta₂-receptors to plasma samples and by labeling the receptors with a radiolabeled beta-antagonist, (–)-[³H]CGP-12177. The results and conclusions were the following: (a) The extent to which metoprolol, pindolol, and propranolol occupied rabbit lung beta₁-and rat reticulocyte beta₂-adrenoceptors in plasma samples estimated accurately the intensity of beta-receptor antagonism in the patients who did not tolerate physiological and pharmacological tests measuring the degree of beta₁- and beta₂-adrenoceptor blockade. (b) The mean beta₁- and beta₂-receptor occupancy of pindolol and propranolol varied between 76% and 99% during the treatments. The mean beta₁-receptor occupancy of the metoprolol regimen varied between 54% and 92%, and its beta₂-receptor occupancy varied between 6% and 38%. Thus the antagonist activity of the metoprolol regimen differed significantly from that of the other regimens (ANOVA for repeated measures, p<0.05 and 0.001, for the beta₁- and beta₂-occupancy, respectively). (c) The extent of beta₁- and beta₂-receptor occupancy in plasma samples was in conformity with the literature on the intensity, selectivity, and duration of beta-blockade after similar drug doses. (d) The data on the receptor occupancy of beta-blocking drugs in plasma samples appear to be valuable in analyzing their effects, and it may be a method for optimizing drug therapy for aged cardiovascular patients.     

A proposed potential role for increasing atmospheric CO2 as a promoter of weight gain and obesity

Human obesity has evolved into a global epidemic. Interestingly, a similar trend has been observed in many animal species, although diet composition, food availability and physical activity have essentially remained unchanged. This suggests a common factor—potentially an environmental factor affecting all species. Coinciding with the increase in obesity, atmospheric CO₂ concentration has increased more than 40%. Furthermore, in modern societies, we spend more time indoors, where CO₂ often reaches even higher concentrations. Increased CO₂ concentration in inhaled air decreases the pH of blood, which in turn spills over to cerebrospinal fluids. Nerve cells in the hypothalamus that regulate appetite and wakefulness have been shown to be extremely sensitive to pH, doubling their activity if pH decreases by 0.1 units. We hypothesize that an increased acidic load from atmospheric CO₂ may potentially lead to increased appetite and energy intake, and decreased energy expenditure, and thereby contribute to the current obesity epidemic.     

P2Y12 and EP3 antagonists promote the inhibitory effects of natural modulators of platelet aggregation that act via cAMP

Several antiplatelet drugs that are used or in development as antithrombotic agents, such as antagonists of P2Y₁₂ and EP3 receptors, act as antagonists at G_i-coupled receptors, thus preventing a reduction in intracellular cyclic adenosine monophosphate (cAMP) in platelets. Other antiplatelet agents, including vascular prostaglandins, inhibit platelet function by raising intracellular cAMP. Agents that act as antagonists at G_i-coupled receptors might be expected to promote the inhibitory effects of agents that raise cAMP. Here, we investigate the ability of the P2Y₁₂ antagonists cangrelor, ticagrelor and prasugrel active metabolite (PAM), and the EP3 antagonist DG-041 to promote the inhibitory effects of modulators of platelet aggregation that act via cAMP. Platelet aggregation was measured by platelet counting in whole blood in response to the TXA₂ mimetic U46619, thrombin receptor activating peptide and the combination of these. Vasodilator-stimulated phosphoprotein phosphorylation (VASP-P) was measured using a cytometric bead assay. Cangrelor always increased the potency of inhibitory agents that act by raising cAMP (PGI₂, iloprost, PGD₂, adenosine and forskolin). Ticagrelor and PAM acted similarly to cangrelor. DG-041 increased the potency of PGE₁ and PGE₂ as inhibitors of aggregation, and cangrelor and DG-041 together had more effect than either agent alone. Cangrelor and DG-041 were able to increase the ability of agents to raise cAMP in platelets as measured by increases in VASP-P. Thus, P2Y₁₂ antagonists and the EP3 antagonist DG-041 are able to promote inhibition of platelet aggregation brought about by natural and other agents that raise intracellular cAMP. This action is likely to contribute to the overall clinical effects of such antagonists after administration to man.