In vitro pharmacological properties of a series of isoprenaline derivatives.

The p-trifluoromethylanilide congener of isoprenaline, tert-butyl N-[(S)[( 4-[(R)-6-[2-(3,4-dihydroxyphenyl-2- hydroxyethyl]amino]heptanamido]phenyl]methyl][(N-methylcarbamoy l) methyl]carbamoyl]methyl]carbamate (1S,4R)-4,7,7-trimethyl-3-oxo-2- oxabicyclo[2.2.1]heptane-1-carboxylate (1:1) (Ro 17-2218) was investigated for its effects in various pharmacological tests in vitro and compared to the parent compound. As Ro 17-2218 represented a mixture of four diastereomers, the pure isomers were synthesized. They had a purity of 97-98%. By pharmacological testing of the diastereomers the highest potency was found in the 6R,2'R-isomer Ro 17-8648, while the potency of the 6S,2'S-isomer, Ro 17-9651 was lower by three orders of magnitude. The amorphous hydrochloride Ro 17-8648/001 had 1/10 the potency of the respective crystalline camphanate Ro 17-8648/003. (R)-6-[(R)-[2-(3,4-Dihydroxyphenyl)-2-hydroxyethyl]amino]-N-[4- (trifluoromethyl) phenyl]heptan amide (Ro 17-8648/003) was found to have potent beta-agonist properties with clear beta 1-selectivity in radioligand binding studies. It exerted an extremely tight binding to membrane receptors. As a full beta-agonist it elicited positive inotropic effects in isolated cardiac tissues, with a potency 10-360 times that of isoprenaline and an extremely long duration of action. Electrophysiological studies in isolated guinea-pig papillary muscles confirmed the beta 1-receptor-mediated effects of the compound.

Cardiovascular effects of three calcium entry blockers in conscious dogs.

The cardiovascular effects of N-(3,4-dimethoxyphenethyl)-2-(3,4-dimethoxyphenyl-N-methyl-m-dithiane-2- propylamine-1,1,3,3-tetraoxide HCl (tiapamil, Ro 11-1781, Larocord), a new calcium entry blocker, were investigated in chronically-instrumented conscious dogs and compared with those of verapamil and nifedipine. Oral administration of tiapamil to normotensive dogs dilated preferentially the arterial vascular bed as evidenced by marked increases in the coronary and abdominal aortic blood flow in the absence of a depression of the myocardial contractile force. The vasodilator effects induced a reflex increase in heart rate and cardiac output, which prevented a decrease in blood pressure in normotensive, but not in renal hypertensive dogs. By contrast, verapamil and nifedipine decreased blood pressure in both normotensive and renal hypertensive dogs. At equieffective vasodilating doses, a negative inotropic effect was not seen with tiapamil. By contrast, nifedipine caused a marginal fall and verapamil a marked decrease in myocardial contractile force. This favourable pattern of hemodynamic properties makes tiapamil appear to be a useful agent for the treatment of hypertension and angina pectoris.

Cardiovascular profile of Ro 13-6438, a novel positive inotropic agent with vasodilating properties.

The cardiovascular properties of Ro 13-6438 (R-6-chloro-1,5-dihydro-3- methylimidazo -[2,1-b] quinazolin -2[ 3H]-one), a novel nonglycoside , noncatechol cardiotonic agent, were investigated in vitro and in vivo by both intravenous and oral administration. Ro 13-6438 increased tension development of isolated guinea pig left atria in a concentration-dependent manner with an EC50 of 30 microM, but had no stimulant effect on the spontaneous rate of right atria. The positive inotropic effect of Ro 13-6438 was additive to that of ouabain (0.1 microM); Ro 13-6438 suppressed the arrhythmogenic activity of high concentrations of ouabain (10 microM). IN anesthetized open-chest dogs 10-300 micrograms/kg Ro 13-6438 i.v. produced a significant and dose-dependent increase in myocardial force, with a duration of action exceeding 60 min following the highest dose. It also slightly increased heart rate, cardiac output, and blood flow. Ro 13-6438 decreased systolic and diastolic blood pressure, left ventricular end-diastolic pressure, and total peripheral resistance. Thus, the direct positive inotropic effects of Ro 13-6438 were supported by a decrease in preload and afterload. In chronically instrumented, conscious dogs Ro 13-6438 increased myocardial contractility after administration of 0.03-0.3 mg/kg i.v. or 3-10 mg/kg p.o. The effects persisted for greater than 8 h after oral administration of 10 mg/kg. The inotropic effects were accompanied by a modest increase in heart rate, which, however, had a clearly shorter duration of action than the former.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the mechanism of positive inotropic activity of Ro 13-6438, a structurally novel cardiotonic agent with vasodilating properties.

We investigated the possible mechanisms involved in the positive inotropic activity of Ro 13-6438 (R-6-chloro-1,5-dihydro-3- methylimidazo -[2,1-b] quinazolin -2[ 3H]-one), a structurally novel cardiotonic agent with vasodilating properties. The positive inotropic response to Ro 13-6438 of the isolated guinea pig papillary muscle was accompanied by inhibition of myocardial phosphodiesterase (PDE) activity and elevation of intracellular cyclic AMP (cAMP) levels Ro 13-6438 had no effect on Na+,K+-stimulated or Ca2+-stimulated ATPase activity and did not influence the rate of 45Ca uptake in cardiac membrane vesicles. Ro 13-6438 caused a concentration-dependent increase in the upstroke velocity, overshoot, and duration of slow action potentials evoked in partially depolarized papillary muscles. Pretreatment of guinea pigs with reserpine did not prevent the effects of Ro 13-6438 on slow action potentials, but slightly decreased its positive inotropic activity. The muscarinic agonist carbachol reversed the Ro 13-6438-induced enhancement of contractility and changes in the slow action potential in an atropine-sensitive manner. These results indicate that the positive inotropic effects of Ro 13-6438 are correlated with PDE inhibition, increased cAMP levels, and an increase of the slow action potential in ventricular myocardium. It is suggested that the elevated cAMP levels resulting from the Ro 13-6438-induced inhibition of PDE enhance the slow inward Ca2+ current.

The effects of bufuralol, a beta-adrenoceptor antagonist with predominant beta 2-adrenoceptor agonistic activity, in the cat and the dog.

The effects of bufuralol and its carbinol metabolite have been compared with those of propranolol in the anaesthetised and conscious cat and dog. Bufuralol and its carbinol metabolite are nonselective beta-adrenoceptor antagonists; the former has equivalent potency to propranolol, whereas the latter is six times more potent. In anaesthetised animals both bufuralol and its metabolite exhibited partial agonistic activity, resulting in tachycardia and vasodilation. In conscious cats there was no change in heart rate or slight bradycardia, whereas in dogs both compounds again produced tachycardia. In anaesthetised and conscious cats and conscious dogs, both bufuralol and the metabolite increased abdominal aortic blood flow. There was a reduction in blood pressure in the conscious dog. It is concluded that the partial agonistic activity of bufuralol and its carbinol metabolite is exerted mainly at the beta 2-adrenoceptor, producing vasodilation and reducing peripheral resistance, resulting in a reduction in blood pressure with a long duration of action.

Progesterone-controlled growth hormone overproduction and naturally occurring canine diabetes and acromegaly.

Female pet dogs exhibiting either glucose intolerance alone or glucose intolerance and acromegaly were investigated. Some dogs developed the disorder(s) during dioestrus and some animals developed the disorder(s) after they were given medroxyprogesterone acetate (MPA). Elevated fasting plasma glucose levels (12.3 +/- 1.9 mM, mean +/- SEM) were accompanied by fasting hyperinsulinaemia (144 +/- 21 microU/ml, mean +/- SEM) and drastic elevation of plasma growth hormone (GH) levels (112.6 +/- 45 ng/ml, mean +/- SEM). An iv glucose tolerance test (IVGTT) performed on all dogs revealed non-suppressibility of GH levels and glucose intolerance. Plasma concentrations of glucose, insulin and GH during IVGTT in affected dogs differed significantly from the concentrations measured in normal dogs during the same test. MPA withdrawal and/or ovariohysterectomy (OVx-HYx) in affected animals was followed by reversal of GH levels to normal and improved glucose tolerance. Acromegaly associated soft tissue changes were also reversible after MPA withdrawal and/or OVx-HYx when GH levels had dropped. In 5 dogs which had developed diabetes during dioestrus and in which a spontaneous decrease in plasma progesterone occurred during the investigation a concomittant decrease in GH levels was observed. Plasma GH measured at different stages of pregnancy in 45 dogs was found to be elevated in one animal only. The results show that the development of spontaneous diabetes/acromegaly occurring in some female dogs is related to progestagen (progesterone/MPA) exposure and that reversal of the signs is achieved by progesterone/MPA withdrawal. The results suggest that diabetes/acromegaly in the dogs studied was caused by progesterone/MPA-evoked GH elevation. Finally, the findings also suggest that the GH axis normally not appreciably responsive to progestagen exposure in some dogs becomes and/or is paradoxically controlled by physiologic levels of endogenous progesterone or low doses of MPA.

Cardiovascular effects of tiapamil (Ro 11-1781), a new calcium-entry blocker.

We studied the hemodynamic effects of the new calcium antagonist tiapamil (Ro 11-1781) in anesthetized open-chest dogs and compared them with those of verapamil. Increasing doses of tiapamil injected intravenously caused the following hemodynamic effects: increase in coronary flow and decrease in coronary vascular resistance, followed by decreases in heart rate, blood pressure, and total peripheral resistance. Tiapamil did not depress myocardial contractility over a rather wide dose range, while verapamil did. Tiapamil was more effective in increasing coronary flow than verapamil. Cardiac autonomic denervation did not modify the tiapamil-induced decrease in coronary vascular resistance and did not cause tiapamil to display negative inotropism. Both tiapamil and verapamil reduced the extent of S-T segment elevation in the epicardial electrocardiogram produced by transient occlusion of the left anterior descending coronary artery. Neither drug had this effect when heart rate was kept constant by atrial pacing. In contrast to nitroglycerin, tiapamil dilated small but not large coronary arteries. Tiapamil raised PO2 relatively more in the subendocardial than in the subepicardial layers of the myocardium. The hemodynamic and electrocardiographic effects of tiapamil were not changed by diphenylhydantoin and disopyramide or by pindolol, a beta-adrenoceptor blocking agent with intrinsic sympathomimetic activity. However, the decreases in heart rate and blood pressure in response to propranolol were enhanced by tiapamil.

Radioimmunoassay of canine growth hormone.

A sensitive radioimmunoassay (RIA) for canine growth hormone (GH) was developed. Antibodies were elicited in rhesus monkeys. One antiserum exhibited a working titer at a dilution of 1: 500 000. Radioiodination was performed enzymatically employing lactoperoxidase. Logit-log transformation and least squares fitting resulted in straight line fitting of the standard curve between 0.39 and 50 ng/ml. Formation of large-molecular [125I]GH during storage caused diminished assay sensitivity. Therefore [125I]GH was re-purified by gel chromatography. Using this procedure, high and reproducible assay sensitivity was obtained. Tracer preparations were used for as long as 3 months after iodination. Diluted plasma from normal and acromegalic dogs resulted in a dose-response curve parallel to the standard curve. Canine prolactin exhibited a cross-reactivity of 2%. The within-assay coefficient of variation (CV) was 3.8 and the between-assay CV was 7.2%. Mean plasma GH concentration in normal dogs was 1.92 +/- 0.14 ng/ml (mean +/- SEM). GH levels in acromegalic dogs were appreciably higher. Insulin-induced hypoglycaemia, arginine and ornithine administration resulted in inconsistent and sluggish GH increment. A better response was obtained by injecting a low dose of clonidine. Clonidine administration to hypopituitary dogs resulted in absent or poor GH increment.

Influence of medroxyprogesterone acetate (Provera) on plasma growth hormone levels and on carbohydrate metabolism. II. Studies in the ovariohysterectomized, oestradiol-primed bitch.

The combined effects of oestradiol and medroxyprogesterone acetate on growth hormone (GH) levels and carbohydrate metabolism were studied in 6 ovariohysterectomized dogs, which previously had shown moderate increments in GH after medroxyprogesterone acetate (MPA) administration. Oestradiol (Oe2) implants were administered 5 months after the last MPA injection, when MPA and GH levels tended to decrease. Following Oe2 administration GH levels rose significantly. Single MPA injections (100 mg) given 20 days after Oe2-priming were followed by still further increased GH levels. These GH levels were several-fold higher than GH levels achieved by previous MPA administration alone. GH levels decreased in 3 dogs after 35 days and remained elevated in the other 3 dogs as long as 70 days after MPA administration with Oe2 priming. Glucose assimilation became impaired and insulin response to a glucose load increased in relation to elevated GH levels. Oe2-primed control dogs, which received no MPA, failed to develop elevated GH levels. These findings indicate (1) that Oe2 and MPA induce overproduction in ovariohysterectomized dogs synergistically (2) that GH levels of the magnitude evoked are associated with glucose intolerance and insulin resistance.

Cardiovascular effects of a novel vasoactive antihypertensive agent, Ro 12-4713.

Ro 12-4713, an oxadiazolopyrimidine derivative, lowered blood pressure of conscious spontaneously hypertensive rats and renal hypertensive dogs in a dose-dependent manner from 10 to 100 mg/kg p.o. The antihypertensive effect was slow in onset, had a long duration of action and was not subject to the development of tachyphylaxis. Ro 12-4713 did not influence blood pressure in conscious normotensive rats, cats and dogs. Several observations suggest that metabolites formed from the inactive parent compound are responsible for the cardiovascular effects. A hemodynamic analysis in anesthetized dogs showed that Ro 12-4713 lowered blood pressure and peripheral vascular resistance. These effects were associated with an increase in cardiac output and flow in various vascular beds as well as with an increase in myocardial contractility. This pattern of hemodynamic activity together with the absence of effects on the autonomic neuroeffector system allow the classification of Ro 12-4713 as a vasoactive antihypertensive agent like e.g. hydralazine or minoxidil. However, in contrast to other vasoactive antihypertensives, Ro 12-4713 virtually did not produce tachycardia or water and sodium retention. Ro 12-4713 may represent a considerable improvement in current vasodilator antihypertensive therapy.

Telemetry of cardiac left ventricular pressure in conscious dogs.

A fully implantable telemetric system for the measurement of cardiac left ventricular pressure and derived parameters in conscious dogs is presented. It provides the following advantages: ethical acceptability, scientific relevance, economical experimentation, avoidance of stress-induced situations for the animal, experiments without anaesthesia or sedation. The system has an operation time which allows the sequential pharmacological evaluation of a great number of drugs in individual dogs as has been shown by testing the effect of the positive inotropic agent, dobutamine, and the calcium antagonists, verapamil and nifedipine.

Tiapamil, a new calcium antagonist. 1. Demonstration of calcium antagonistic activity and related studies.

N-(3,4-Dimethoxyphenethyl)-2-(3,4-dimethoxyphenyl)-N-methyl-m-dithiane-2-propylamine-1,1,3,3-tetraoxide hydrochloride (tiapamil, Ro 11-1781), a new homoveratrylamine derivative, was studied under various experimental conditions which allow the recognition of calcium antagonistic activity, and compared with the reference calcium antagonist verapamil. Similar to verapamil, tiapamil inhibited in a concentration-dependent manner calcium-induced contractions in isolated, potassium-depolarized preparations of rat renal artery, dog coronary artery and rabbit main pulmonary artery. The inhibitory effects of tiapamil and verapamil were overcome by raising the calcium concentration of the bath fluid and a competitive antagonism between calcium ions and both compounds was found. In the rabbit main pulmonary artery both tiapamil and verapamil reduced 45Ca influx into the potassium-depolarized vascular smooth muscle cells. Calcium-mediated slowly rising potentials were evoked in partially depolarized guinea-pig papillary muscles by electrical stimulation in the presence of isoprenaline. Tiapamil, like verapamil, inhibited the slow potentials, an effect which was antagonized by elevation of the concentration of calcium or isoprenaline in the bath fluid. Both tiapamil and verapamil decreased contractile force in isolated guinea-pig atria and papillary muscles as well as in isolated cat hearts. In the last preparation the two compounds also reduced heart rate and increased coronary flow. These results characterize tiapamil as a calcium antagonist in both vascular smooth muscle and cardiac muscle. A comparison between tiapamil and verapamil indicated that both drugs are of approximately equal calcium antagonistic potency in coronary vascular smooth muscle, but verapamil is 5--10 times more potent in smooth muscle of other arterial beds and in cardiac muscle. Thus tiapamil appears to be a calcium antagonist with some preference for coronary vascular smooth muscle.