Human target validation of phosphoinositide 3-kinase (PI3K)ß: effects on platelets and insulin sensitivity, using AZD6482 a novel PI3Kß inhibitor.

BACKGROUND: Based on in vitro and animal data, PI3Kß is given an important role in platelet adhesion and aggregation but its role in insulin signaling is unclear. OBJECTIVE: To strengthen the PI3Kß target validation using the novel, short-acting inhibitor AZD6482. METHODS AND RESULTS: AZD6482 is a potent, selective and ATP competitive PI3Kß inhibitor (IC(50) 0.01 µm). A maximal anti-platelet effect was achieved at 1 µm in the in vitro and ex vivo tests both in dog and in man. In dog, in vivo AZD6482 produced a complete anti-thrombotic effect without an increased bleeding time or blood loss. AZD6482 was well tolerated in healthy volunteers during a 3-h infusion. The ex vivo anti-platelet effect and minimal bleeding time prolongation in the dog model translated well to data obtained in healthy volunteers. AZD6482 inhibited insulin-induced human adipocyte glucose uptake in vitro (IC(50) of 4.4 µm). In the euglycemic hyperinsulinemic clamp model, in rats, glucose infusion rate was not affected at 2.3 µm but reduced by about 60% at a plasma exposure of 27 µm. In man, the homeostasis model analysis (HOMA) index increased by about 10-20% at the highest plasma concentration of 5.3 µm. CONCLUSIONS: This is the first human target validation for PI3Kß inhibition as anti-platelet therapy showing a mild and generalized antiplatelet effect attenuating but not completely inhibiting multiple signaling pathways with an impressive separation towards primary hemostasis. AZD6482 at 'supratherapeutic' plasma concentrations may attenuate insulin signaling, most likely through PI3Kα inhibition.

Local proCPU (TAFI) activation during thrombolytic treatment in a dog model of coronary artery thrombosis can be inhibited with a direct, small molecule thrombin inhibitor (melagatran).

To test the hypothesis that the direct thrombin inhibitor, melagatran is able to inhibit local pro-carboxypeptidase U (proCPU) activation that occurs during thrombolytic treatment, t-PA alone, or in combination with melagatran, was given to dogs with a coronary artery thrombosis. Blood samples from the great cardiac vein and aorta were collected at baseline, during thrombus formation, throughout the t-PA+/-melagatran infusion and during the patency period, for analysis of CPU activity using a novel assay. A higher CPU activity in venous compared to arterial blood (V-A difference) indicates CPU activation in coronary vessels. Efficacy was assessed by determination of time to lysis, duration of patency and blood flow during patency. Dogs (n = 26) were randomized to receive either 1) t-PA, 1 mg/kg as an intravenous 20-min infusion; 2) t-PA as in group 1, +melagatran bolus, 0.3 mg/kg, followed by a 3-h infusion (0.15 mg/kg per h); 3) sham-operated but no coronary thrombus, and administered t-PA as for Group 1. All groups had similar baseline characteristics. Significant increases in CPU activity were observed in Groups 1 and 2 during thrombus formation, with V-A differences of 5.5 and 4.5 U/L, respectively. No significant V-A difference was observed in the sham-operated group. CPU activity increased in Group 1 during the t-PA infusion (V-A difference 15.9 U/L), whereas the V-A difference in Group 2 decreased to 2.6 U/L following melagatran treatment. These results demonstrate that melagatran attenuates generation of CPU in the coronary circulation. The mechanism is probably indirect, via inhibition of thrombin-mediated activation of proCPU.

Pharmacokinetics of new calcium channel antagonist clevidipine in the rat, rabbit, and dog and pharmacokinetic/pharmacodynamic relationship in anesthetized dogs.

Clevidipine is a new vascular selective calcium channel antagonist of the dihydropyridine type, structurally related to felodipine. Clinical trials have shown that the drug can be used to effectively control the blood pressure in connection with cardiac surgical procedures. The compound is tailored to be a short-acting drug and, due to incorporation of an ester linkage into the drug molecule, clevidipine is rapidly metabolized by ester hydrolysis. The pharmacokinetics of clevidipine and its primary metabolite, H 152/81, were studied in rats, rabbits, and dogs. In addition, the influence of the pharmacokinetics on the effect on mean arterial blood pressure was evaluated in anesthetized dogs. Compartmental nonlinear mixed effect regression analysis was used to calculate the population mean and individual pharmacokinetics of clevidipine, whereas nonlinear regression analysis of individual data was used to determine the pharmacokinetics of the primary metabolite. A linked Emax model was fitted to the individual pharmacodynamic/pharmacokinetic data in dogs. According to the results, clevidipine is a high-clearance drug with a relatively small volume of distribution, resulting in an extremely short half-life in all species studied. The median initial half-life of the individual value (Bayesian estimates) is 12, 20, and 22 s in the rabbit, rat, and dog, respectively. The primary metabolite is a high-clearance compound in the dog, whereas it is a low-clearance compound in the rat. A significant gender difference in the clearance of the metabolite was observed in the rat. The mean maximum reduction in arterial blood pressure is 38 +/- 12% (Emax) and is achieved at 85 +/- 46 nM (EC50). The half-life for reaching equilibrium between the central and the effect compartment (T1/2ke0) is 47 +/- 49 s.

Characterization and molecular cloning of vascular neuropeptide Y receptor subtypes in pig and dog.

Cloning with subsequent in vitro and in vivo characterization of vascular neuropeptide Y (NPY) receptor subtypes in porcine and canine peripheral tissues was performed. RT-PCR with Y1 and Y2 receptor-specific primers, indicated expression of Y1 receptors in both kidney and spleen of dog and pig, and expression of Y2 receptors in pig spleen. In pig kidney, expression of Y1 receptor mRNA was located to intrarenal arteries, as demonstrated with in situ hybridization using human probes. The cloned and sequenced canine Y1, porcine Y1 and Y2 receptors revealed high homologies to previously characterized mammalian NPY receptors. Membrane and autoradiographic receptor binding studies showed specific high-affinity binding sites for the purported Y1-selective radioligands 125I-[Leu31Pro34]peptide YY (PYY) and 3H-BIBP 3226 in dog spleen, and for the putative Y2-selective 125I-PYY(3-36) in dog and pig spleen. In the pig in vivo, [Leu31Pro34]PYY, administered i.v., evoked vasoconstriction in spleen and kidney, actions that were potently inhibited by the non-peptide Y receptor antagonist SR 120107A. In contrast, PYY(3-36) evoked vasoconstriction only in spleen and this effect was not influenced by SR 120107A. NPY evoked renal and splenic vasoconstriction in the dog in vivo, vascular responses that were inhibited by both BIBP 3226 and SR 120107A. Furthermore, the Y1 receptor agonist [Leu31Pro34]NPY also caused vasoconstriction in dog kidney and spleen, whereas the putative Y2 agonist N-acetyl[Leu28Leu31]NPY(24-36) evoked no such vascular responses. It is concluded that the pig spleen is likely to contain Y1 and Y2 receptors, both involved in splenic vasoconstriction. In contrast, the Y1 receptor seems to be the sole vascular NPY receptor subtype in pig kidney. Moreover, Y1 receptors predominate in dog spleen and kidney. Furthermore, the cloned canine Y1 receptor and the porcine Y1 and Y2 receptors show great homologies to, and possess ligand requirement profiles in accordance with, the human forms.

Ischemic and nonischemic tissue concentrations of felodipine after coronary venous retroinfusion during myocardial ischemia and reperfusion: an experimental study in pigs.

Tissue and plasma concentrations of felodipine, a dihydropyridine (DHP) calcium antagonist, retroinfused through the coronary venous system were studied in 27 pigs. The animals underwent 45-min myocardial ischemia followed by 4-h reperfusion. Felodipine (7 nmol/kg body weight) was administered in the coronary vein for 30 min, 5 min before reperfusion. Concentrations of felodipine in the ischemic and nonischemic myocardium and in plasma were determined by gas chromatography. In the ischemic area, felodipine concentration at start of reperfusion was 304 +/- 285, 171 +/- 160, and 52 +/- 47 nmol/kg (mean +/- SD) in the subepicardial, midmyocardial, and subendocardial layer, respectively. Corresponding concentrations in the nonischemic area were 15 +/- 13, 17 +/- 14, and 16 +/- 15 nmol/kg (p < 0.05 vs. ischemic area). Subepicardial concentration was highest at start of reperfusion, whereas concentrations in other layers peaked at the end of retroinfusion. The transmural concentration gradient of felodipine in the ischemic area decreased progressively during the reperfusion period. The nonischemic tissue concentration increased slightly during the reperfusion period. The plasma concentration was very low throughout the study (peak = 3.2 +/- 1.4 nM at 30 min). Coronary venous retroinfusion of felodipine resulted in profound accumulation of the drug, specifically in ischemic myocardium. The plasma concentration was low and did not affect systemic hemodynamics. Coronary venous retroinfusion is considered an advantageous technique for selective drug delivery.

Myocardial uptake and release of lactate after high dose neurolept endotracheal intubation in coronary surgery.

To evaluate the relationship between the hemodynamic and ECG variables used in routine surveillance of coronary surgery and myocardial lactate metabolism, 23 middle-aged, male, beta 1-blocked patients about to undergo coronary surgery were monitored before and after endotracheal intubation with high dose (30 micrograms/kg) fentanyl-midazolam anesthesia. The induction of anesthesia was followed by a mean arterial pressure decrease (from 98 +/- 4 to 76 +/- 3 mm Hg) and heart rate increase (from 53 +/- 3 to 66 +/- 2 beats/min). After intubation the hemodynamic variables were stable except for a further, transient increase in heart rate (to 69 +/- 2 beats/min). The myocardial uptake of lactate decreased after intubation, from 48 +/- 5 mumol/min to a lowest level of 24 +/- 3 mumol/min. A lactate release was exhibited in 7/23 patients (30%). No ST-segment changes were observed. The correlation between the myocardial lactate uptake/release and hemodynamic or ECG variables was unimpressive or non-existent (r < or = 0.20). Thus, a reduced uptake and even a release of lactate occurred irrespective of the ST-segment, heart rate, or systemic or pulmonary artery pressures. In conclusion, endotracheal intubation in patients with coronary disease was consistently (17/23 patients) followed by a reduced myocardial uptake of lactate, in spite of high dose neurolept anesthesia and beta 1-blockade. This metabolic event was not consistently related to hemodynamic changes.

Prostacyclin synthesis in relation to sympathoadrenal activation. Effects of beta-blockade.

Results from several studies suggest that beta-adrenoceptor blockade causes increased biosynthesis of the vasodilator prostanoid prostacyclin in the arterial wall. This effect may contribute to the clinical effects of beta-blockers in hypertension and coronary heart disease. Studies in hypertensive patients and in animals indicate that the arterial pressure reduction after beta-blockade is related to the associated increase of prostacyclin biosynthesis, regarding both magnitude and time of onset of effect. Some observations suggest that the effects of beta-blockers in myocardial ischemia may in part be due to an improvement of coronary blood flow caused by increased prostacyclin biosynthesis.

Superoxide dismutase and catalase do not improve recovery of regional myocardial contractile function when given at the time of reperfusion after reversible regional ischemia in anesthetized dogs.

Earlier studies have demonstrated an improvement in the recovery of the regional myocardial function after reversible myocardial ischemia when dogs were treated with superoxide dismutase (SOD) + catalase (CAT). In all these studies, drug administration was started prior to the ischemic period. The aim of this study was to investigate the effects of SOD and CAT on the recovery of the regional contractile function in anesthetized beagle dogs when the drugs were administered at the time of reperfusion. The animals were subjected to 20 min of left coronary artery occlusion followed by 3 h reperfusion. The regional myocardial contractile function, measured as subendocardial segment shortening (SS, sonomicrometry) decreased to below zero and the regional blood flow in the ischemic subendocardium was reduced to about 5% of pre-ischemic values during the coronary artery occlusion period. The size of the occluded bed was similar in the two groups. Saline (n = 8) or SOD (10 mg/kg) + CAT (3.4 mg/kg) (n = 8) were infused into the left atrium from 2.5 min prior to until 20 min after the start of reperfusion. The peak plasma level of SOD was 102 +/- 15 mg/l at 20 min reperfusion. There were no significant differences in the arterial blood pressure, cardiac contractile function and regional blood flow between the two groups at any time during the experiment. During reperfusion in the dogs given vehicle, SS recovered to 48 +/- 7% (mean +/- SEM) after the first hour of reperfusion, and to 51 +/- 6% of pre-ischemic values after 3 h of reperfusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute haemodynamic effects of felodipine, verapamil and hydralazine in the anaesthetized dog.

Felodipine, a potent dihydropyridine calcium antagonist with a pronounced vascular selectivity, was given intravenously (0.006-0.025 mumol kg-1) to anaesthetized, open-chest dogs with denervated hearts. The result was a dose-dependent decrease in mean arterial pressure (MAP) and total peripheral resistance (TPR), while heart rate (HR), stroke volume (SV) and left ventricular end-diastolic pressure remained relatively unchanged. Cardiac tension work (TTI) and oxygen consumption (MVO2) were reduced, probably due to the decrease in afterload. The relative reduction of the coronary vascular resistance (CVR) was greater than that of TPR. The hypotensive effect of verapamil (0.05-0.20 mumol kg-1) was small and MAP decreased mainly via a decrease in HR and SV. Higher doses of verapamil which induced vasodilatation could not be given without the development of complete atrio-ventricular dissociation. Hydralazine (11-45 mumol kg-1) decreased TPR and CVR in parallel but the decrease in MAP was partly counteracted by a powerful increase in HR, SV and cardiac inotropy which was associated with elevated catecholamine levels in plasma. When MAP and HR were maintained constant by means of aortic balloon inflation and atrial pacing, felodipine markedly increased coronary blood flow and coronary sinus oxygen saturation while SV, TTI, inotropy and MVO2 remained relatively unchanged. It is concluded that felodipine markedly dilates peripheral resistance vessels, and in particular those in the coronary vascular bed, without any cardiodepressant effects.

Derivation of haemodynamic information from ultrasonic recordings of aortic diameter changes.

An ultrasound phase-locked echo-tracking system was used for noninvasive measurements of diameter changes in the upper abdominal aorta of the anaesthetised cat. Comparisons were made between the noninvasively recorded diameter changes and central haemodynamic variables measured with inserted catheters and transducers. It was found that noninvasive observations of aortic diameter changes give reliable information on the direction and relative magnitude of the blood pressure change both in systole and in diastole. Indications of the direction of change of stroke volume, cardiac output, and aortic flow acceleration (a measure of cardiac inotropy) could also be gained. The information, taken together, comprises a pattern of response reflecting cardiovascular adjustments likely to have occurred. It is suggested that the technique is suitable for interpretation of (patho-) physiological changes in the foetus, as well as for determinations of great vessel compliance in man, i.e., in atherosclerosis research.

The role of sympathetic activity in atherogenesis: effects of beta-blockade.

Clinical and experimental evidence points to potential antiatherosclerotic effects of certain beta-adrenoreceptor antagonists. Long-term treatment with metoprolol resulted in significant reductions of total and cardiovascular mortality or morbidity due to decreased incidence of coronary and cerebrovascular complications both in a primary prevention trial in hypertensive patients and in a secondary prevention trial in patients surviving myocardial infarction. The observations suggest that a retardation of atherosclerosis development might have contributed to the reduced incidence of cardiovascular complications. An antiatherosclerotic effect of beta-blockers has been directly demonstrated in animal studies. In cholesterol-fed rabbits, metoprolol significantly reduced the development of atherosclerotic plaques in the aortic intima in the absence of any changes in blood lipids. Similar findings were reported for propranolol, which prevented psychosocial stress-induced atherosclerosis of the coronary artery in monkeys. Furthermore, beta-blockers have been shown to prevent stress-induced endothelial injury and platelet accumulation to intima at atherosclerotic predilection sites in animal models. These antiatherogenic effects may be due to biochemical and hemodynamic factors. Two biochemical effects of beta-blockade may lead to reduced cholesterol accumulation in arterial intima at unchanged serum cholesterol levels. One is a beta-blocker-induced increase of prostacyclin biosynthesis, and the other a metabolic change of low-density lipoprotein, reducing its potential for deposition in the arterial wall. The antiatherogenic effect of these factors may be reinforced by beta-blocker-induced hemodynamic changes leading to reductions of arterial flow aberrations and pressure-related wall stress.

The positive inotropic effect of felodipine in isovolumically beating dog heart.

Felodipine is a potent arteriolar vasodilator. Its possible myocardial effects were studied by placing dogs anaesthetized with chloralose on cardiopulmonary bypass. A balloon was then inserted into the left ventricular cavity with a vent alongside it. Isovolumic contractions (developed pressure or maximal rate of rise of pressure) were studied as the intraventricular balloon was inflated to various levels. Total coronary venous return was collected to measure coronary blood flow and felodipine concentrations. In seven vagotomized and beta-blocked preparations, a small positive inotropic effect was found [32 +/- 11 (SEM)%, p less than 0.05]; the maximum effect was reached at arterial plasma felodipine concentrations of between 7 and 20 nM and was accompanied by appreciable coronary vasodilatation. In five dogs, infusion of the solvent vehicle (5% polyethylene glycol) alone had no effect. In three more dogs, an infusion of nitroprusside caused coronary vasodilatation, but no positive inotropic effect. The results show that at low concentrations, felodipine has a positive inotropic effect in vivo. These findings suggest that the properties of myocardial calcium agonist and vascular smooth muscle antagonist properties may coexist in the same dihydropyridine molecule. The favorable effects of this drug in the treatment of heart failure can be explained not only by reduction of peripheral resistance, but also by a moderate positive inotropic effect.

Cardiac anti-ischemic effect of metoprolol: role of beta-blockade within the ischemic region.

The distribution of metoprolol and atenolol into ischemic and nonischemic myocardium was studied in anesthetized dogs, pigs, and cats. The beta-blockers were administered intravenously after coronary artery occlusion. Metoprolol was found to be significantly more efficiently distributed to the ischemic myocardium than atenolol in all three species. To investigate the functional implications of this difference in tissue distribution, the anti-ischemic effects of the two beta-blockers were studied in the 2-h period following coronary artery occlusion in anesthetized cats, in which heart rate was kept at a constant level. In this model, metoprolol (0.3 mg.kg-1 + 0.15 mg.kg-1.h-1) was found to attenuate or delay the developing ischemic process. This is shown by its significant reduction of (a) the decline of CK activity in ischemic myocardium, (b) the ST elevation in a precordial ECG lead, and (c) the decrease of arterial pressure and cardiac output. In contrast to metoprolol, atenolol (0.3 mg.kg-1 + 0.15 mg.kg-1.h-1) caused no significant anti-ischemic effect in this cat model. The difference in the effectiveness of the two drugs can most probably be explained by their differential distribution in the ischemic heart. Furthermore, the anti-ischemic effect of metoprolol shows that the presence of a beta-blocker in ischemic left ventricular myocardium can favorably affect the early phase of developing infarction.

Studies on cardiac distribution and function of neuropeptide Y.

High concentrations of a novel peptide, neuropeptide Y, have been demonstrated in the guinea-pig and canine heart and in the latter, a particularly high concentration was found in the region of the coronary vasculature (126 +/- 31 pmol g-1). Intra-arterial infusion of neuropeptide Y for 30 s into the coronary artery of the intact, innervated dog heart resulted in a rapid and short-lasting reduction of blood flow from 38 +/- 4 to 31 +/- 3 ml min-1 (P less than 0.05) to resume control level, 39 +/- 5 ml min-1, within 5 min. These injections were unaccompanied by changes in heart rate and aortic pressure, while there was an associated small reduction in dP/dt, used as a measure for changes in contractility. In vitro studies using the isolated, paced papillary muscle from cat, guinea-pig and rat, and spontaneously beating right atria from the guinea-pig, demonstrated no effect of NPY on active tension or beating frequency. The results indicate that NPY has vasoconstrictor properties, but under the test circumstances to lack both positive and negative inotropic and chronotropic effects.

Central hemodynamic effects of adrenaline with special reference to beta 2-adrenergic influence on heart rate and cardiac afterload in anesthetized cats.

Central hemodynamic responses evoked by i.v. infusions of adrenaline and noradrenaline were studied in normovolemic anesthetized cats with intact adrenoceptors, after selective beta 2-blockade (ICI 118,551), and after nonselective beta-blockade propranolol). The results demonstrated the presence of an important beta 2-adrenergic component in the integrated response to 'physiological' doses of adrenaline contributing to increased cardiac output, decreased total peripheral resistance and virtually unchanged mean arterial blood pressure. Corresponding beta 2-adrenergic effects of noradrenaline were small. The beta 2-adrenergic effects of adrenaline on the heart seemed to be both direct and indirect. A moderate direct chronotropic response mediated by beta 2-adrenoceptors apparently was present but there was no evidence of a direct beta 2-adrenergic inotropic effect. An indirect, quite marked effect on the heart was accomplished by a beta 2-adrenergic vasodilator interaction with the alpha-adrenergic vasoconstrictor influence on the systemic resistance vessels. This caused a net decrease in total peripheral resistance, thereby preventing an undue increase in cardiac afterload (arterial pressure) which seemed to be essential for evoking 'optimal' increases in cardiac output. It is suggested that such adrenaline evoked indirect, beta 2-adrenergic improvement of cardiac performance is of functional importance in reflex sympatho-adrenal circulatory control.

The ulcerogenic effect on the oesophagus of three beta-adrenoceptor antagonists, investigated in a new porcine oesophagus test model.

Drug-induced oesophageal lesions have attracted increasing attention during the past few years. A test model is presented for assessing irritative or ulcerogenic effects of potential drugs on the oesophagus. In this model the pig oesophagus is used, as it is more similar to the human oesophagus than models used in other techniques that have been suggested. The beta-blockers, alprenolol and propranolol, displayed the same ulcerogenic properties with this test model as reported with other test models in the literature. The beta 1-selective blocker metoprolol, did not have such ulcerogenic effects. The pig model is suggested as the method of choice in evaluating the potential irritative or ulcerogenic effects of pharmaceutical formulations intended for human use.

The haemodynamic effects of intravenous prenalterol and ouabain in conscious dogs.

Experiments were performed on 5 resting conscious dogs supplied with an electromagnetic flow probe on the ascending aorta and a chronic aortic catheter for pressure recording. The animals were used repeatedly in four different types of experiment involving i.v. administration of 1. saline (controls), 2. prenalterol 45 nmol/kg (approximately 10 micrograms/kg) followed by an additional dose of 135 nmol/kg 20 min later, 3. ouabain 50 nmol/kg (approximately 30 micrograms/kg) and 4. a combination of protocols 2. and 3. Ouabain and the low dose of prenalterol exerted clear-cut positive inotropic effects as reflected in increased stroke volume and max dF/dt without significant changes in heart rate or arterial pressure. The PQ interval increased with ouabain but decreased with prenalterol. The higher dose of prenalterol caused a further rise in max dF/dt, a further shortening of the PQ time, increased heart rate and reduction in systemic vascular resistance. Higher doses of ouabain could not be given due to side-effects (vomiting). The combined treatment with ouabain and prenalterol showed their inotropic responses to be additive. Arrhythmias did not occur in any of the animals at the applied dose levels of the drugs. The experiments show that prenalterol through its beta 1-adrenoceptor stimulating action exerts a positive inotropic effect which surpasses that of emetic doses of ouabain. The inotropic response at moderate doses occurs without a change in heart rate. This fact and the apparent lack of influence of prenalterol on vascular alpha- and beta 2-adrenoceptors make the substance potentially useful clinically as an inotropic agent in cardiac failure, particularly in view of its relatively long duration of action.