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[Combined therapy with glycosides and Ca++-antagonistic agents (author's transl)].

Ca-antagonistic drugs increase cardiac oxygen supply by a coronary vasodilator action whilst they simultaneously lower myocardial oxygen demand. The latter drug effect is due to a decrease in cardiac contractile energy expenditure both directly (restriction of myocardial contractility) and indirectly (reduction of arterial blood pressure resulting from systemic vasodilation). In coronary disease all these factors can contribute to an improvement of the disturbed myocardial energy balance. As to the coronary vasodilator action of Ca-antagonistic drugs in particular, it is important to note that they readily relax the smooth musculature of the major extramural stem arteries where most of the occlusive atherosclerotic intima processes (about 95%) are located. In contrast, other coronary vasodilators such as adenosine, dipyridamole, chromonar, lidoflazine, theophylline, which lack Ca-antagonistic properties, are primarily acting on the small intramural resistance vessels of the heart whereas vascular smooth musculature originating from the major extramural arteries is less affected. Therefore, in elderly atherosclerotic patients the possible risk of glycoside-induced vasoconstriction in extramural coronary stem arteries (and also in arteries of systemic circulation) can be sufficiently eliminated only with the help of Ca-antagonists.

Animals↗

Effect of carbocromen induced coronary vasodilatation on myocardial metabolism in coronary artery disease.

Myocardial metabolism was studied during coronary vasodilatation by carbocromen during atrial pacing in patients with coronary arteriosclerotic disease. Coronary sinus flow was measured by the continuous infusion thermodilution technique. Carbocromen increased myocardial flow by the same amount at rest and during pacing-induced tachycardia. The administration of carbocromen did not interfere with myocardial oxygen consumption, free fatty acid (FFA) uptake, pulmonary or systemic hemodynamics. Atrial pacing above the anginal threshold increased lactate and hypoxanthine excretion to the coronary sinus, but to a greater extent during carbocromen treatment. Thus, carbocromen is a specific coronary vasodilator acting independently of pacing induced vasodilatation. Metabolic data indicate a small but unfavourable effect on ischemic metabolism which might be due to a "coronary steal".

Cardiac Pacing, Artificial↗

Changes in vessel ultrastructure during ischemia and reperfusion of rabbit hindlimb: implications for therapeutic intervention.

Peripheral ischemia was induced in the rabbit by occlusion of the left iliac artery for 6 hr, followed by 24 hr of reperfusion. Biochemical and morphological investigations were performed to evaluate the extent of vascular and tissue injury. Blood samples for plasma enzyme determinations (creatine kinase (CK) and lactate dehydrogenase (LDH) activities) were obtained at times t = 0, t = 6, t = 30 hr. Plasma CK and LDH activities in ischemic animals were approximately twice as high as those in sham-operated animals at the end of reperfusion, although no difference was observed at the end of the period of ischemia. Morphological and morphometric analysis of extensor digitorum longus muscle from ischemic animals showed a reduction in the number of patent capillary vessels per muscle fiber (1.54 +/- 0.1 and 1.04 +/- 0.09, P < 0.05, in sham and ischemic groups, respectively; mean +/- SEM). In addition, the number of microvilli on endothelial surfaces were considerably increased in the ischemic group (0.14 +/- 0.02 and 0.41 +/- 0.01 microns -2, P < 0.001, in sham and ischemic groups, respectively). A great number of adhered leucocytes were found on the vessel surface with some leucocytes having migrated through the vessel wall. Microcirculatory damage was accompanied by the formation of microthrombi which sometimes occluded the entire vessel lumen. The infusion of 1 mg/kg/hr of cloricromene for 6 hr prevented ischemic injury in microvessels and also prevented swelling of muscle mitochondria. In the treated group the number of patent capillaries per muscle fiber was very similar to that found in sham-operated animals (1.49 +/- 0.08; P < 0.01 vs. ischemic control). In conclusion, several different cell types are involved in the pathophysiological changes which occur in microvessels during ischemia/reperfusion injury. Pharmacological interventions, which inhibit the interactions of blood cells with endothelium, may be of value in the treatment of peripheral ischemia/reperfusion injury.

Animals↗

Differential inhibition of polymorphonuclear leucocyte functions by cloricromene.

Leucocytes play an essential role in the pathogenesis of ischaemia and reperfusion injury and inhibition of their adhesion and of mediator release can reduce vascular and tissue damage. Previous studies have shown that cloricromene modifies several granulocyte as well as monocyte/macrophage functions and it has been shown that cloricromene administration exerts a clear protective action in several experimental models of ischaemia. The present work describes new data on polymorphonuclear leukocyte (PMN) inhibition exerted by cloricromene and compares these observations with earlier ones. Human washed PMN and human whole blood (HWB) were studied in vitro upon stimulation with f-MLP in the presence of cytochalasin B, with opsonized zymosan and with a phorbol ester (PMA). Amongst free radicals, superoxide anions were chosen as index of oxidative burst. Phagocytosis and beta-glucuronidase, as lysosomal release indicators, were measured to characterize PMN function: cloricromene inhibited concentration-dependently all the parameters upon stimulation by each activator tested. Experiments performed in rabbit whole blood (RWB) showed that cloricromene inhibited free radical generation with IC50 values similar to those obtained in human whole blood. Comparing the action of cloricromene on human cells in different tests, we found that some parameters were more sensitive than others, even when the same stimulus was used. In particular, free radical generation was inhibited by cloricromene with IC50 values below 36 microM, while other functions, like lysosomal release and phagocytosis were inhibited with IC50 values over 100 microM. These data confirm that cloricromene exerts a notable inhibitory effect on PMN and may explain the activity of the compound, observed in vivo in several experimental models of ischaemia-reperfusion and shock.

Animals↗

Inhibition of vitamin D3-induced vascular calcification by carbocromen.

High doses of vitamin D3 induce myocardial lesions, necrotic alterations, and calcification of the vascular smooth muscle, (Mönckeberg arteriosclerosis) (6). These effects are accompanied by an increased calcium influx into the wall of the aorta and mesenterial vessels (1). Prophylactic application of MgCl2 nearly prevents this calcium overload, whereas KCl or calcium antagonists are less active. In our studies, the uptake of calcium-45 4 days after a single dose of vitamin D3 (90,000 IU/200 g) was increased in rat heart and aorta by a factor of 3 to 7, and up to 30-fold in mesenterial vessels. Application of carbocromen (10 mg/kg b.i.d.) for 4 days decreases the vitamin D3-induced radiocalcium uptake by 44 (aorta) to 67% (mesenterial vein), MgCl2 (15 mmol/kg b.i.d.) by 46 (mesenterial vein) to 92% (mesenterial artery), and verapamil (5 mg/kg b.i.d.) by 15 (aorta) to 44% (mesenterial vein).

Animals↗

In vitro inhibition of human polymorphonuclear cell function by cloricromene.

Cloricromene, a coumarin derivative with antiaggregating and vasodilating properties, was tested in vitro on polymorphonuclear cell (PMN) adhesion to the endothelium, superoxide anion generation and chemotaxis. PMN adhesion was measured using cultured human umbilical vein endothelial cells (EC) either untreated or previously activated with interleukin-1 (IL-1). Cloricromene (5-50 microM) induced dose-related inhibition of PMN adhesion to untreated and IL-1 treated EC. Cloricromene also inhibited PMN superoxide generation induced by the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or by N-formyl-methionyl-leucyl-phenylalanine (FMLP). PMN and monocyte chemotaxis was evaluated by a modification of the Boyden chamber technique. Cloricromene inhibited both types of cell motility induced by FMLP in a concentration-dependent fashion. The major cloricromene metabolite (cloricromene acid) had no effect on any of the biological parameters studied up to a concentration of 500 microM. HPLC measurement showed that cloricromene accumulated in PMN within a few minutes and levels of the drug were still high after 60 min. In contrast its acid metabolite was not taken up in a significant amount during incubation periods up to 60 min. We conclude that cloricromene inhibits a series of PMN activities in vitro. This effect might be of pharmacological interest in view of the role of PMN activation in different pathophysiological conditions.

Cell Adhesion↗

Multiple actions of the coumarine derivative cloricromene and its protective effects on ischemic brain injury.

The effects of different doses (0.25, 0.5, 1 and 2 mg/kg i.p.) of cloricromene, a coumarine derivative, have been investigated on brain malondialdehyde levels, brain edema, myeloperoxidase activity, survival, locomotor hyperactivity and hippocampal neuronal loss following transient cerebral ischemia induced by temporary bilateral carotid occlusion in the Mongolian gerbil. Cloricromene reduced brain lipid peroxidation, measured through the evaluation of malondialdehyde (-82.9% with the highest dose), and the formation of post-ischemic brain edema, evaluated by water content. The increase in myeloperoxidase activity observed in the hippocampus of postischemic animals was also reduced: 0.7 +/- 0.3 U x 10(-3) vs. 3.3 +/- 0.3 U x 10(-3)/g tissue. The same treatment increased survival and reduced hyperactivity linked to neurodegeneration induced by cerebral ischemia and reperfusion. Histological observations of the pyramidal layer of CA1 showed a reduction of neuronal loss in animals that received the drug before occlusion but not in those that were treated after the occlusion. These results show that cloricromene, a drug with multiple actions, improves brain injury induced by transient cerebral ischemia.

Animals↗

Treatment of haemodynamic and electrocardiographic side-effects resulting from imipramine toxicity in rats and dogs.

This study was designed to analyze the effects of carbocromene and dipyridamole on the haemodynamic and electrocardiographic side-effects resulting from imipramine infusion in anaesthetised rats and dogs. Imipramine was infused at 1 mg/kg/min until cardiac failure and vascular collapse terminated the experiment at 21 +/- 2.3 min in rats and at 29.5 +/- 2.1 min in dogs. This was characterized by hypotension, bradycardia, intraventricular conduction delay, cardiac tachyarrhythmia and A-V block. Carbocromene (4 mg/kg i.v., followed by 80 micrograms/kg/min) protected the animals against heart failure. This was associated with delayed hypotension and negative inotropy, and lower incidence of heart block. Survival time increased to 37 +/- 1.5 min (P less than 0.05), and 54.2 +/- 2.6 min (P less than 0.02) in rats and dogs, respectively. Dipyridamole (0.5 mg/kg i.v., followed by 80 micrograms/kg/min) failed to decrease imipramine toxicity as judged by the haemodynamic and electrocardiographic parameters and did not alter survival time of imipramine controls. These results suggest that carbocromene is an effective treatment for imipramine-induced cardiovascular collapse and cardiac arrhythmias, the beneficial effects being largely due to metabolic and membrane stabilizing effects. Carbocromene has both therapeutic and prophylactic value and appears to be superior to dipyridamole therapy.

Animals↗

The cardiodynamic and metabolic effects of carbochromen and propranolol on the isolated dog heart.

In the isolated canine heart-lung preparation modified to measure coronary outflow and myocardial oxygen consumption, an infusion of carbochromen of 1 mg/min produced an increase in coronary outflow of 120.0% and a decrease in myocardial oxygen consumption of 11.0% at a cumulative dose of 28.0 +/- 3.8 mg, resulting in an increase in efficiency of 22.4%. At a total dose of 40 mg, the corresponding figures were +181.0%, -15.0% and +38.0% respectively. No change in heart rate was observed. An infusion of propranolol of 1 mg/min, on the other hand, was characterized by decreases in myocardial contractility and heart rate starting with cumulative doses of 20 mg and 40 mg respectively. At a cumulative dose of 62.9 +/- 3.0 mg a marked degree of myocardial failure occurred (peak failure) characterized by a decrease in cardiac output (-53.3%), dp/dt (-50.8%), heart rate (-18.5%) and myocardial oxygen consumption (-25.5%). In a separate series exposed to the same infusion of propranolol but in which the decrease in heart rate was prevented by pacing, identical changes were observed except that myocardial oxygen consumption decreased only by 7.7%. It may be concluded from this study that carbochromen, in confirmation of reports by other investigators, increases coronary outflow markedly. Carbochromen decreases myocardial oxygen consumption by a direct effect on myocardial metabolism as is evident from the fact that this decrease is not accompanied by changes in any of the parameters which are known to influence myocardial oxygen consumption such as heart rate. Propranolol has no effect on myocardial oxygen consumption unless it is given in a dose which produces myocardial failure and a decrease in heart rate. The decrease in heart rate following propranolol bears a close relationship to the decrease in myocardial oxygen consumption.

Animals↗

Action of AD6 (8-monochloro-3-beta-diethylaminoethyl-4-methyl-7-ethoxycarbonylmet hox y coumarin) on human platelets in vitro.

The action of AD6 was tested in vitro on human platelets by measuring beta-thromboglobulin (BTG), platelet factor 4 (PF4) and thromboxane B2 (TXB2) release as well as aggregation. BTG and PF4 release from blood anticoagulated with sodium citrate was inhibited by AD6 during a 3 h incubation. Platelet rich plasma (PRP) was stimulated with ADP, collagen, sodium arachidonate, PAF, A23187 and epinephrine, while resuspended washed platelets (WP) were stimulated by thrombin. AD6 (5-100 microM) inhibited dose dependently aggregation, BTG, PF4 and TXB2 release induced by threshold concentration of all the tested aggregating agents; however AD6 action could be overcome by increasing the concentration of the stimulating agents. After cyclo-oxygenase blockade by acetylsalicylic acid (ASA), PRP was stimulated by a supramaximal concentration of PAF. Under these circumstances we could observe a reversible aggregation and a partial release of BTG and PF4, AD6 was able to further reduce aggregation and release. Cyclic AMP accumulation induced in WP by prostacyclin was not modified by AD6 (100 microM), while theophylline greatly potentiated prostacyclin action. We conclude that AD6 is an inhibitor of platelet activation in vitro. Its mode of action is different from cyclo-oxygenase blockade and provides inhibition of platelet activation by a number of different stimuli.

Anticoagulants↗

Further evidence for the involvement of cAMP in central blood pressure regulation.

In order to test the hypothesis that the activity of cardiovascular centres is determined by their content of cAMP a number of drugs which influence the activity of either phosphodiestase or adenylcyclase were injected in doses of 100-1000 mug/kg into the lateral cerebral ventricle of cats. The effects on blood pressure and heart rate were studied. The phosphodiesterase inhibitors papaverine, carbocromene, theophylline and caffeine caused hypertension and tachycardia which increased with the dose while the phosphodiesterase activator imidazole exerted opposite effects. Sodium fluoride which activates adenylcyclase increased blood pressure and heart rate substantially. The results confirm the above-mentioned hypothesis.

3',5'-Cyclic-AMP Phosphodiesterases↗

Pharmacological alterations of coronary collateral circulation; implication to the steal-phenomenon.

Measurements of resistances were performed on different parts of coronary vessels, including spontaneous collaterals in anesthetized dogs, following ligation and embolization of the descending branch of the left coronary artery. The normal situation was compared with the state of maximal pharmacological dilatation. The pharmacological dilatation decreased the collateral resistance by 24% in spite of the fact that the collaterals supplied an infarcted area in this case; the precollateral resistance, which chiefly consists of large vessels was also diminished. The collateral perfusion pressure is not only dependent on the aortic pressure, but also on the flow rate in the corresponding large coronary vessel and thereby on the ratio of precollateral to postcollateal resistance. A decrease of the collateral perfusion pressure causes an insufficient perfusion of the area supplied by the collaterals only when the pressure fall is not compensated for by an adequate decrease in the collateral resistance and when the ratio of the collateral to the nutritive resistance increases. Thus, both, the extent of the pressure fall across the precollateral resistance and the dilating capacity of the collaterals determine whether is not a pharmacological dilatation of the coronary vascular bed results in an insufficient supply of an infarcted area, i.e. in a so called "Steal-phenomenon". The results show further, that the given physical model of the microcirculation satisfactorily approximates the observed behaviour of the vascular system.

Animals↗

Density of plasma-perfused capillaries in the rat heart during carbocromene-induced vasodilation.

Urethane-anesthetized thoracotomized rats were used to ascertain whether the density of plasma-perfused capillaries increases in the heart during pharmacologically induced vasodilation. Carbocromene, in a dose of 3.0 mg/(kg x min) i.v., infused for 5 min, raised coronary blood flow from 6.2 +/- 0.6 to 15.6 +/- 0.1 ml/(min x g); heart rate and blood pressure were only slightly changed. Capillary density was determined by timed infusions of a plasma label (FITC or RB 200 coupled with gamma-globulin), infused for different periods of time in the same animal. No significant difference could be observed in the number of capillaries marked for 10 min in the carbocromene-treated rats as compared to the controls (carbocromene-treated rats: 3,630 +/- 90 cap/mm2 subepicardium, 3,360 +/- 70 cap/mm2 subendocardium; controls: 3,750 +/- 140 cap/mm2 subepicardium, 3,210 +/- 90 cap/mm2 subendocardium). In those rats treated with the vasodilator the filling of the microcirculatory system was nearly complete within a labelling period of 1 sec (3,500 +/- 170 cap/mm2 subepicardium, 3,070 +/- 110 cap/mm2 subendocardium), whereas significantly lower values were found when the dye was infused for 1 sec in the controls (2,560 +/- 460 cap/mm2 subepicardium, 1,960 +/- 400 cap/mm2 subendocardium). The results indicate that the filling of the cardiac microcirculatory system is accelerated by a pharmacologically induced vasodilation, the maximal density of plasma-perfused capillaries is not raised, however.

Animals↗

Alpha-receptor constriction induced by atrial fibrillation during maximal coronary dilatation.

The mechanism of coronary vasoconstriction induced by atrial fibrillation during maximal coronary dilatation was studied in 19 chloralose-urethane anesthetized dogs. Maximal coronary dilatation was achieved by carbochromene (5 mg/kg i.v.) or dipyridamole (0.2 mg/kg i.v.). Left circumflex coronary blood flow was measured with an electromagnetic flowmeter. Atrial fibrillation was compared with rhythmic atrial pacing at similar heart rates (207 +/- 12 vs. 204 +/- 12 beats/min). During maximal coronary dilatation, coronary resistance was 0.38 +/- 0.05 mm Hg X min X 100 g/ml (RU) at sinus rhythm, 0.41 +/- 0.06 RU at atrial pacing, and 0.52 +/- 0.07 RU at atrial fibrillation, that was significantly (p less than 0.005) higher than during sinus rhythm and atrial pacing. Accordingly, coronary oxygen extraction was 14 +/- 1% at sinus rhythm, 17 +/- 1% at atrial pacing (p less than 0.005 vs. sinus rhythm) and 27 +/- 2% at atrial fibrillation (p less than 0.001 vs sinus rhythm and atrial pacing). Beta-adrenoceptor blockade with propranolol (1 mg/kg i.v.) did not prevent this coronary vasoconstrictive effect. Following alpha-blockade with phenoxybenzamine (10 mg/kg i.v.), however, coronary resistance was 0.52 +/- 0.08 RU during sinus rhythm, 0.54 +/- 0.10 RU during atrial pacing and 0.57 +/- 0.09 RU during atrial fibrillation. The data suggest coronary vasoconstriction induced by atrial fibrillation mediated by an alpha-adrenoceptor mechanism.

Animals↗

The effect of carbocromene on strophanthin-induced ventricular tachycardia in dogs.

The antiarrhythmic effect of carbocromene (Intensain) was studied in 17 anaesthetized dogs. Ventricular tachycardia was induced by infusion of K-strophanthin (mean dose: 154 mug/kg i.v.). In 25 out of 26 experiments carbocromene (4 mg/kg i.v.) was effective. Ventricular tachycardia was converted to sinus rhythm in 16 experiments, to atrial tachycardia in 6 experiments and to junctional tachycardia in 3 experiments. The favourable effect started 8 to 50 seconds after the injection with lower doses of carbocromene (3 and 2 mg/kg i.v.) in time intervals of 5 minutes resulted in similar positive effects leading after 2 or 3 injections finally to a second phase of drug effect where ventricular tachycardia was strongly modified for about 30 minutes. The results favour the therapeutic use of this drug in patients with arrhythmia especially with arrhythmias caused by digitalis.

Animals↗

Effect of AD6 (8-monochloro-3-beta-diethylamino-ethyl-4-methyl-7-ethoxycarbonylme tho xy coumarin) on cyclic nucleotide phosphodiesterases in human platelets.

The effect of AD6 (8-monochloro-3-beta-diethylamino-ethyl-4-methyl-7-ethoxycarbonylmeth oxy coumarin), an inhibitor of platelet aggregation, on cyclic nucleotide metabolism was investigated. AD6 inhibited selectively human platelet cyclic GMP phosphodiesterase, which was separated from cyclic AMP phosphodiesterase by DEAE-cellulose chromatography. Addition of AD6 to washed platelets increased cyclic GMP levels significantly in two minutes. These results could be useful in elucidating the action of AD6 on intact platelets.

3',5'-Cyclic-GMP Phosphodiesterases↗

The coumarin derivative AD6 inhibits the release of arachidonic acid by interfering with phospholipase A2 activity in human platelets stimulated with thrombin.

AD6 is a coumarin derivative which is able to inhibit platelet aggregation and release due to various agonists as adrenaline, PAF, Ca++ ionophore and others. It has been demonstrated that this compound reduces the production of free arachidonate and diglyceride from human platelets pulse-labeled with radioactive arachidonic acid thus suggesting a possible interference with the activity of phospholipase A2 and/or phospholipase C. The present report indicates that the drug has no effect on the increase of the labeling of phosphatidic acid which takes place when platelets pulse-labeled with arachidonic acid are stimulated with thrombin. Furthermore, AD6 is not able to cause changes on the metabolism of phosphoinositides monitored using platelets pre-labeled with [3H] inositol. These observations exclude the possibility that AD6 interferes with phospholipase C activity. Experiments with platelets pulse-labeled with arachidonate suggest that AD6 inhibits phospholipase(s) A2 activity or modulate negatively one or more processes involved in its activation.

Arachidonic Acid↗