Inhibition by suloctidil of [3H] nitrendipine binding to cerebral cortex membranes.

[3H] Nitrendipine binds specifically with high affinity and high capacity (KA congruent to 0.20 +/- 0.01 nM, Bmax = 4.4 +/- 0.3 pM/g tissue, means +/- S.E.M.; N = 4) to guinea-pig cerebral cortex membranes. Suloctidil fully inhibits [3H] nitrendipine binding with a Ki value of 0.45 microM. The interaction between suloctidil and the putative Ca2+ channels is allosteric as shown by competition experiments performed in the presence of D 600 or diltiazem. Comparison of the activity of some close analogs of suloctidil provides evidence for the importance of the amino group and of the hydrophobic amino substituent in the interaction of suloctidil and the putative Ca2+ channel. It is suggested that part of the previously reported blockade of Ca2+ entry induced by suloctidil is due to a blockade of the Ca2+ channels.

Effect of milacemide, a glycinamide derivative, on the rat brain gamma-aminobutyric acid system.

Milacemide (CP 1552 S, 2-n-pentylaminoacetamide), a drug with anti-epileptic potency, increases the gamma-aminobutyric acid (GABA) content specifically in the substantia nigra of rat brain. The effect is dose-related from 25 to 100 mg/kg p.o. The time course shows that at 100 mg/kg p.o. after 2, 3 and 4 hr the substantia nigra GABA content is significantly increased by 28, 33 and 38%, respectively. After 6 hr the GABA contents return to the control value. After repeated oral administration of milacemide a comparable effect to acute administration is obtained. After degeneration of the striato-nigral GABA-ergic pathway, milacemide no longer enhances the content of GABA in the substantia nigra. GABA-transaminase activity measured ex vivo in rat brain homogenate is not influenced by milacemide. On the other hand, the glutamate decarboxylase activity measured ex vivo 3 hr after 100 mg/kg of milacemide is significantly increased by 11% in homogenates of the whole rat brain. The results show that milacemide increases the GABA content in the GABA pool which is associated with the striato-nigral neurons. This increase is not due to GABA-transaminase inhibition but might be the result of an enhanced synthesis, possibly through glutamate decarboxylase activation.

Antiplatelet and antithrombogenic effects of suloctidil.

Platelet aggregation induced in mice or rats by i.v. ADP can be antagonized by oral administration of suloctidil. This effect on platelet behaviour appears to be sufficient to reduce the rate of thrombotic occlusion of the femoral artery in the dog and to protect the rat against occurrence of thrombophlebitis.

The adenylate cyclase activity in heart membranes from normotensive and spontaneously hypertensive rats, after chemical sympathectomy, suggests the presence of presynaptic secretin receptors.

Normotensive (WKY) and spontaneously hypertensive (SHR) male adult rats were sacrificed 2 and 3 weeks after 6-hydroxydopamine treatment. Untreated WKY and SHR rats served as controls. In rat heart membranes from WKY rats, 6-hydroxydopamine treatment increased guanosine 5'-O-(2-3-imido)-triphosphate (Gpp(NH)p)-, NaF-, D,L-isoproterenol- and glucagon-stimulated adenylate cyclase activities by 18-38% while secretin stimulation was unaffected. In heart membranes from SHR rats, Gpp(NH)p, NaF, D,L-isoproterenol, or glucagon stimulation of the enzyme was similarly increased by 14-38% whilst the low secretin responsiveness which is characteristic of these animals decreased even further (by 24-47%). These results are consistent with: (1) an up regulation of postsynaptic beta-adrenergic receptors coupled to adenylate cyclase after degeneration of adrenergic nerves, and (2) a differential response of secretin receptors coupled to adenylate cyclase in the two strains of rats: there was no change in WKY rats and a decreased response in SHR rats. The possible presence and contribution of presynaptic secretin cardiac receptors is considered.

Comparative effects of alpha-methyldopa, propranolol and hydralazine therapy on cardiac adenylate cyclase activity in normal and spontaneously hypertensive rats.

Normotensive (WKY) and spontaneously hypertensive (SHR) male rats were treated orally, one week after weaning and for 9 weeks, with alpha-methyldopa (100 mg/kg per day), propranolol (30 mg/kg per day) or hydralazine (10 mg/kg per day). Untreated WKY and SHR rats served as controls. The development of hypertension in SHR rats were attenuated by treatment but none of the drugs was able to restore the impairment in isoproterenol, secretin and glucagon responsiveness of cardiac adenylate cyclase activity which is characteristic of these animals. In heart membranes from both WKY and SHR rats, alpha-methyldopa treatment increased the number of beta-adrenoceptors by 20-32% and the maximal response of adenylate cyclase activity to isoproterenol and glucagon by 20-34%. By contrast, the beta-blocker propranolol was ineffective on these parameters. The results obtained are consistent with the hypothesis that the change in adenylate cyclase seen in SHR rats is genetic in origin and is not a consequence of hypertension.

In vitro inhibition of human liver drug metabolizing enzymes by second generation antihistamines.

Cetirizine, terfenadine, loratadine, astemizole and mizolastine were compared for their ability to inhibit marker activities for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and for some glucuronidation isoenzymes in human liver microsomes. The most pronounced effects were observed with terfenadine, astemizole and loratadine which inhibited CYP3A4-mediated testosterone 6beta-hydroxylation (IC50 of 23, 21 and 32 microM, respectively) and CYP2D6-mediated dextromethorphan O-demethylation (IC50 of 18, 36 and 15 microM, respectively). In addition, loratadine markedly inhibited the CYP2C19 marker activity, (S)-mephenytoin 4-hydroxylation (Ki of 0.17 microM). Furthermore, loratadine activated the CYP2C9-catalyzed tolbutamide hydroxylation (ca. 3-fold increase at 30 microM) and inhibited some glucuronidation enzymes. Mizolastine appeared to be a relatively weak and unspecific inhibitor of CYP2E1, CYP2C9, CYP2D6 and CYP3A4 (IC50Ss in the 100 micromolar range). Cetirizine demonstrated no effect on the investigated activities. A comparison of the inhibitory potencies of cetirizine, terfenadine, loratidine, astemizole and mizolastine with their corresponding plasma concentrations in humans suggests that these antihistamines are not likely to interfere with the metabolic clearance of coadministered drugs, with the exception of loratidine, which appears to inhibit CYP2C19 with sufficient potency to warrant additional investigation.

Deoxyuridine suppression test on isolated rat bone marrow cells and the in vitro effect of bidisomide.

The deoxyuridine suppression test was performed on isolated rat bone marrow cells in order to study the effect of bidisomide, a new Class I antiarrhythmic agent, on folate-dependent DNA synthesis. Methotrexate and 5-fluorouracil, two known inhibitors of DNA synthesis, were included in the study to validate the test system. Methotrexate and 5-fluorouracil, at a concentration of 5.5 mum, decreased thymidine incorporation into DNA by way of the de novo pathway (thymidylate synthase activity). The salvage pathway of DNA synthesis (thymidine kinase activity), however, was not affected by these anticancer drugs. Bidisomide up to 1 mm did not affect the folate-dependent thymidylate synthase activity, nor the thymidine kinase activity of isolated rat bone marrow cells.

The use of spontaneously hypertensive rats for the study of anti-hypertensive agents.

Hypertension studies using laboratory animals have been conducted since 1930. These were not completely satisfactory because either surgery or pharmacologic induction were required to produce hypertensive animals. Many attempts have been made to breed spontaneously hypertensive rats, mainly from the Okamoto strain. The cause of hypertension in the rat, with specific reference to genetic aspects and pathogenicity, were reviewed. The hypertensive rat is an acceptable model for hypertension studies because of the stability of the hypertensive state and the reproducibility of experimental effects. It is a particularly useful model for screening antihypertensive agents. Development of mutant Okamato stran rats which have brain softening, cerebral hemorrhages, and myocardial infarctions would permit the screening of specific therapeutic agents with fewer side-effects. Mutants which develop obesity, hyperlipidism, and early atherosclerosis have been reported in Okamoto strain X Sprague-Dawley rat crosses.

Comparison of [3H]nitrendipine binding to heart membranes of normotensive and spontaneously hypertensive rats.

We compared the binding properties of [3H]nitrendipine in heart membranes from 9- and 24-week-old spontaneously hypertensive rats (SHR) and their normotensive controls (WKY). In native membranes of 9-week-old SHR and WKY and of 24-week-old WKY, [3H]nitrendipine binds to a single class of binding sites with high affinity (Kd, approximately equal to 0.20 nM) and high capacity (Bmax, approximately equal to 96 fmol X mg protein-1). By contrast, in membranes of 24-week-old, SHR, Kd and Bmax increased significantly to 0.30 nM and 137 fmol X mg protein-1, respectively. Addition of 1 mM Ca2+ had no effect on [3H]nitrendipine binding in WKY but decreased the Kd value to 0.18 nM in 24-week-old SHR. Bmax remained 40% above the value in WKY. Inhibition and activation of specific binding by nifedipine and dilitiazem are identical in both strains. We conclude that the differences in [3H]nitrendipine binding in hearts of aged SHR are due to a decreased Ca2+ content and might reflect a functional adaptation.

In vivo antispasmodic activity of Sulcotidil.

Suloctidil was tested in vivo for its antispasmodic activity on peripheral and cerebral circulations. In the perfused dog hind limb preperation, suloctidil was found to inhibit after 2 and 5 min, the vasospasm induced by norepinephrine or angiotensin; at equal dose, and at the game time intervals, it was more potent than cinnarizine and papaverine. BaCl(2) induced spasms of the pial arterial of the rabbit were also rapidly alleviated by i.a. suloctidil (3.5 mug/kg or more), in conditions where papaverine and vincamine were inactive.

Effect of folate supplementation on clinical chemistry and hematologic changes related to bidisomide administration in the rat.

In a chronic toxicity study in the rat, bidisomide administered as a dietary admixture produced a dose-related lowering of reticulocytes and leucocytes. Plasma alanine aminotransferase activity was increased at 300 mg/kg and decreased at 900 mg/kg. The potential mechanisms of these effects were investigated by comparing the responses in groups of male Sprague-Dawley rats receiving a control diet, or 300 or 1200 mg/kg/day bidisomide. Subsets of these groups were co-treated subcutaneously with folinic acid or with a vitamin B1, B6, B12 complex. Subsets of control and 300 mg/kg groups were maintained on a 20-25% feed restriction regimen for 3 months, to mimic the depression in body weight gain observed in animals receiving 1200 mg/kg. Body weight gains were significantly reduced at 1200 mg/kg and in all feed-restricted animals. Plasma and liver alanine aminotransferase (ALT) and plasma aspartate aminotransferase (AST) levels were also reduced at this dose level. At 300 mg/kg, plasma transaminases, glutamate dehydrogenase (GLDH) and sorbitol dehydrogenase (SDH) activities were increased. These changes were prevented in animals receiving folinic acid supplementation. Plasma glucose, triglycerides, and unsaturated and total iron binding capacities were decreased, while plasma iron levels tended to increase, mainly at the high dose. Vitamin supplementation prevented a decrease in reticulocyte counts at 300 mg/kg. Bidisomide increased urinary formimino-glutamic acid (FIGLU) excretion but did not affect methylmalonic acid (MMA) or taurine excretion. The effect on FIGLU at 1200 mg/kg was prevented by folinic acid co-treatment. Absolute liver weight was lowered at both dose levels and in feed-restricted animals. However, the relative liver weights were unaffected. Thymidine kinase and thymidylate synthase activity of the bone marrow cells were not altered by the bidisomide treatment. Except for the increase in plasma transaminase, GLDH and SDH levels at 300 mg/kg, changes in clinical chemistry parameters are considered to result mainly from nutritional restrictions. Changes in hematologic parameters appear to be related to the combination of decreased feed consumption (leukocytes) and decreased availability or utilization of folates (reticulocytes). This alteration, however, did not affect DNA synthesis in bone marrow. The prevention by folinic acid, but not by feed restriction, of the elevation of liver enzymes at 300 mg/kg is an intriguing, yet unexplained finding. There was no evidence that bidisomide affected B6 and B12 availability.

A simple method for performing routine histopathological examination of the cardiac conduction tissue in the dog.

In standard toxicity studies, the cardiac conduction tissue is not systematically sampled and examined for histopathological changes. Most methods described use serial sectioning perpendicular to the long axis of the sinoauricular node (SAN) and atrioventricular node (AVN). Dozens of slides are needed to allow examination of a significant portion of the SAN and AVN. A simple method was developed to be used in routine histopathologic examination of the dog heart. With a plane parallel to the external wall and the upper edge of the right auricle, the SAN and its arterial supply were easily sectioned and examined. The frontal plane parallel to the interventricular crest was the most appropriate plane for observing a large portion of the AVN and the bundle of His. Based on these results, the heart of 240 dogs from toxicology studies were successfully sampled and processed utilizing this technique. An average of 5 slides per node was needed to perform a satisfactory examination of each of the SANs and AVNs.

Anticonvulsant activity of milacemide.

The anticonvulsant activity of a new drug, milacemide (2-(pentylamino)-acetamide), has been studied in animal models of convulsions like those induced by bicuculline, pentylenetetrazol, picrotoxin, strychnine, inhibitors of GABA synthesis as 3-mercaptopropionic acid, allylglycine, isoniazid and thiosemicarbazide and electroshock. Milacemide is particularly effective in inhibiting the convulsions induced by bicuculline. The ED50 is 5.7 mg/kg by oral route and the activity lasts for more than 48 hr. It is less active against pentylenetetrazol and only marginally active against electroshock. It has not be found active against the other types of convulsions. Milacemide has a low toxicity (LD50: 2585 mg/kg in the mouse) and alters the behaviour of mouse, rat and monkey, only at high doses (greater than or equal to 1000 mg/kg). Milacemide seems to be specially free of sedative potential.

Antihypertensive activity of tibalosine (CP 804 S) in the rat. Possible involvement of a central alpha 1-adrenergic receptor blockade.

The effect of tibalosine (CP 804 S) on systolic blood pressure and heart rate of unanaesthetized normotensive, spontaneously hypertensive (SHR) and DOCA-salt and Goldblatt hypertensive rats has been examined. After a single oral dose, tibalosine (1.9 to 15 mg/kg) elicited dose-dependent reductions in blood pressure in the four models tested. These reductions are accompanied by a tachycardia except in the SHR where no variation in heart rate is observed. The same result is obtained in SHR after oral, i.v. or i.c.v. administration. After repeated treatment (9 to 14 weeks), the blood pressure lowering effect of tibalosine (10 mg/kg, p.o.) is observed only in hypertensive rats. No variation in heart rate is observed in normotensive, DOCA-salt and Goldblatt rats. A significant bradycardia is observed in the SHR. The antihypertensive effect of tibalosine in SHR is suppressed by naloxone, like that of clonidine and unlike that of prazosin. The present study suggests that the antihypertensive activity of tibalosine is at least partly centrally mediated.