Pharmacology of a potent, new antithrombotic agent, 1,3-dihydro-7,8-dimethyl-2H-imidazo[4,5-b]quinolin-2-one (BMY-20844).

The effects of 1,3-dihydro-7,8-dimethyl-2H-imidazo[4,5-b]quinolin-2-one (BMY-20844) on platelet function and experimental thrombosis were evaluated in a series of in vitro, ex vivo and in vivo experiments. The compound inhibited platelet aggregation in vitro in platelet rich plasma obtained from humans, rats and rabbits with EC50s of less than 1 microgram/ml when aggregation was induced by ADP, collagen or thrombin. Supra-additive interaction against ADP aggregation was also observed when BMY-20844 was combined with prostacyclin. BMY-20844 was orally active with an ex vivo ED50 in the rat of 3.2 mg/kg vs ADP. Significant antithrombotic activity was observed in two animal models (laser induced thrombosis in the microcirculation of the rabbit ear and coronary artery thrombosis in the dog). Inhibitions of 52% at 3 mg/kg p.o. in the laser model and 100% at 1 mg/kg i.d. in the coronary artery thrombosis model were obtained. Modest inotropic and hemodynamic effects were observed in ferrets and dogs. BMY-20844 was found to be a potent, specific inhibitor of platelet low Km cyclic AMP phosphodiesterase.

The anesthetized ferret, an in vivo model for evaluating inotropic activity: effects of milrinone and anagrelide.

The anesthetized ferret model is introduced as an in vivo acute model for evaluating inotropic effects of new cardiovascular compounds. The effects of anagrelide and milrinone were compared to vehicle. Validation was accomplished using intravenous isoproterenol, which elicited a dose-dependent inotropic response. This suggests that the anesthetized ferret model is both reproducible and dependable. Additionally, in vivo inotropic activity can be assessed on small amounts of compound in a relatively inexpensive species.

Pharmacology of BMY 20064, a potent Ca2+ entry blocker and selective alpha 1-adrenoceptor antagonist.

BMY 20064 is a dihydropyridine Ca2+ entry blocker with potent and selective alpha 1-adrenoceptor antagonist properties. The drug was equal in potency to nifedipine as a Ca2+ entry blocker in depolarized smooth muscle preparations. It was less active than nifedipine in antagonizing Ca2+-induced contractions of isolated guinea pig papillary muscles paced at 0.2, 1.0, or 2 Hz. BMY 20064 was a potent (0.1-0.2 X prazosin) and selective alpha 1-adrenoceptor antagonist in radioligand binding assays and in ganglion-blocked anesthetized rats challenged with phenylephrine. BMY 20064 blocked both the K+ and alpha 1-adrenergic agonist-induced increases in 45Ca uptake into rabbit aortic rings. The drug was more effective than nifedipine, prazosin, or combinations of the drugs in preventing ATP depletion of the rat heart during global ischemia. BMY 20064 was a potent antihypertensive agent in normotensive rats and in SHR. BMY 20064 administered intraarterially (i.a.) dilated both femoral and coronary arterial beds of the dog. Hemodynamic changes elicited by BMY 20064 in anesthetized dogs were similar to those induced by nifedipine. BMY 20064 appears to be a more effective myocardial antiischemic agent than nifedipine, prazosin, or combinations of nifedipine and prazosin. A drug of this type may be more efficacious than dihydropyridines in the management of ischemic episodes.

Effects of anagrelide on platelet cAMP levels, cAMP-dependent protein kinase and thrombin-induced Ca++ fluxes.

Anagrelide (BL-4162A, 6,7-dichloro-1,5-dihydroimidazo[2, 1-6] quinazolin-2[3H]one monohydrochloride hydrate) is a potent and broad spectrum inhibitor of platelet aggregation. Prior studies showed that anagrelide inhibited platelet cyclic AMP (cAMP) phosphodiesterase activity but did not appreciably elevate platelet cAMP levels. We examined the effects of anagrelide on washed human platelets and found that anagrelide caused significant elevation of cAMP levels. Anagrelide treatment also resulted in activation of the platelet cAMP-dependent protein kinase at anagrelide concentrations of 0.1 to 1 microgram/ml, which inhibited platelet aggregation but caused only small increases in platelet cAMP content. When whole platelets were incubated with radiolabeled phosphate, anagrelide increased phosphorylation of platelet proteins with relative molecular weights of 22, 26, 50 and 80 kilodaltons. The pattern of protein phosphorylation stimulated by anagrelide treatment was similar to that observed when the platelets were treated with forskolin. Anagrelide also inhibited the rise in intracellular Ca++ caused by thrombin, as measured using Fura-2-loaded platelets. The inhibition of increased intracellular Ca++ resulted from block of thrombin-induced mobilization of intracellular Ca++, as well as prevention of Ca++ influx through the plasma membrane. Anagrelide itself had no influence on inositol 1,4,5-trisphosphate-induced Caz5++ release from isolated platelet membrane vesicles. These studies suggest that anagrelide inhibits platelet phosphodiesterase activity in intact platelets resulting in an elevation in cAMP levels sufficient to activate the cAMP-dependent protein kinase and inhibit agonist-activated Ca++ fluxes.

Inhibitors of inositol trisphosphate-induced Ca2+ release from isolated platelet membrane vesicles.

Platelet membrane vesicles accumulated Ca2+ in an ATP-dependent fashion, and 25-50% of the accumulated Ca2+ was released by the addition of 10 microM inositol 1,4,5-trisphosphate (IP3). The concentration of IP3 required for half-maximal Ca2+ release was approximately 0.5 microM. The inhibition of IP3-induced Ca2+ release from these membrane vesicles by various agents was examined. Of the plasma membrane Ca2+ channel blockers, cinnarizine and flunarizine were found to be potent inhibitors of IP3-induced Ca2+ release while having no effect on ATP-dependent Ca2+ uptake. The IC50 value for both cinnarizine and flunarizine as inhibitors of IP3-induced Ca2+ release was below 10(-6) M. Nifedipine, verapamil, bepridil, and diltiazem did not significantly inhibit IP3-induced Ca2+ release at the highest concentration tested (50 microM). The "intracellular Ca2+ antagonists" ryanodine, TMB-8 (8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate), dantroline, trifluoperazine and chlorpromazine were not inhibitors of IP3-induced Ca2+ release at 50 microM. The local anesthetics benzocaine and lidocaine weakly inhibited the IP3-induced Ca2+ release with IC50 values of approximately 5 and 50 microM, respectively, whereas other local anesthetics tested were less potent inhibitors. The potent inhibitors described may prove useful as probes of the IP3-induced Ca2+ release channels.

In vitro effects of beta adrenoceptor agonists and antagonists on the rat ovarian suspensory ligament.

The mesovarian suspensory ligament of the rat was used to compare the activities of beta adrenoceptor agonists and antagonists. The following beta adrenoceptor agonists, in descending order of potency, inhibited spontaneous activity in a dose-related manner: zinterol greater than isoproterenol much greater than dobutamine. Several noncardioselective, beta-2 adrenoceptor antagonists with intrinsic sympathomimetic activity (ISA) also inhibited the activity of the ligament: pindolol greater than alprenolol = bucindolol = oxprenolol greater than labetalol. Maximal relaxation induced by the antagonists was equivalent to that caused by the beta receptor agonists. Two cardioselective, beta adrenoceptor antagonists with ISA, acebutolol and practolol, did not inhibit the activity of the suspensory ligament but did increase the rate of the isolated right atrium of the rat. The maximal increases in atrial rate evoked by the antagonists were significantly less than those induced by the beta adrenoceptor agonists. Studies with ICI 118,551 or atenolol as beta-2 or beta-1 selective adrenoceptor blockers, respectively, suggest that the beta adrenoceptors of the suspensory ligament are predominantly of the beta-2 subtype. The possible relevance of these results to the induction of mesovarian leiomyomas in rats by noncardioselective beta adrenoceptor agonists and antagonists with ISA is discussed.

Evaluation of cardiac anoxia and ischemia models in the rat using calcium antagonists.

The effects of severe global ischemia on cardiac high energy phosphate (HEP) stores were investigated in an in vitro rat model. The heart was removed from the rat in this model, sealed in a plastic bag and incubated for varying times and temperatures (20-45 degrees C). The rat, in the in vivo anoxic model, was subjected to cervical dislocation which resulted in respiratory arrest. In both models the hearts were removed and analyzed for HEP at appropriate times following the onset of anoxia or ischemia. Verapamil and nifedipine, administered intravenously 10 minutes before the start of the experiments, preserved HEP stores in both models. The degree of protection provided by the Ca+2 blockers was related to both the dose of drug and the duration of the ischemia/anoxia. Verapamil was more active than nifedipine in both models.

Mode of action of ergonovine on isolated porcine coronary artery: the role of Ca2+.

Coronary vasoconstrictor responses to ergonovine were examined in helical coronary arterial strips of young swine. Both ergonovine and serotonin (5-hydroxytryptamine) produced dose-dependent contractions of the strips. The distal region (less than 1.00 mm outer diameter) of the circumflex coronary artery was most sensitive to the responses of serotonin and ergonovine. Methysergide and nifedipine significantly depressed the contractions induced by ergonovine and serotonin. Atropine, propranolol, and the alpha 1 blocker, prazosin, did not antagonize ergonovine-induced contractions. The ergonovine response may depend entirely upon extracellular Ca2+ while the effect of serotonin may be mediated in part through the mobilization of intracellular Ca2+ stores. Increases in 45Ca2+ cellular contents occurred after ergonovine or serotonin and these increases were blocked by methysergide or nifedipine at concentrations which blocked mechanical responses to the agonist. It is concluded that the contractions of the porcine coronary artery produced by ergonovine and serotonin are as follows: (i) regionally sensitive; (ii) blocked by Ca2+ antagonists and therefore may utilize Ca2+ channels similar to those described in other excitable tissues; (iii) blocked by methysergide. These studies indicate that the major mechanism of ergonovine's action in the porcine coronary artery is through the activation of serotonin receptors on coronary arteries which are, in turn, linked to Ca2+ channels. However, this mechanism of action may be different in an intact animal.

Pharmacology and neurochemistry of buspirone.

Biochemically, buspirone is unlike the benzodiazepines in that it neither stimulates nor inhibits 3H-benzodiazepine binding, does not affect the influence of GABA or halide anion on 3H-benzodiazepine binding, and does not interfere with GABA binding or uptake. Buspirone lacks anticonvulsant activity, interacts minimally with CNS depressants, and does not cause muscle relaxation. Its tranquilizing activity is characterized by the ability to (1) tame aggressive rhesus monkeys, (2) block conditioned avoidance responding in the rat, (3) inhibit shock-induced fighting in the mouse, and (4) attenuate shock-induced suppression of drinking in the rat. In vitro binding experiments indicate that buspirone lacks significant activity at several binding sites. It appears to interact only with the dopaminergic system and possesses properties that are similar to both dopamine agonists and dopamine antagonists.

Dopamine and antianxiety activity.

Clinical trials have indicated that buspirone (Buspar) is effective in the treatment of anxiety with efficacy and dosage comparable to diazepam. Until recently it has been thought that antianxiety drugs must alter benzodiazepine receptor binding in vitro. However, buspirone lacks any structural similarity to te benzodiazepines and does not interact with the benzodiazepine/gamma-aminobutyric acid (GABA) axis. Specifically, buspirone neither stimulates nor inhibits [3H]benzodiazepine binding, does not affect the influence o GABA or halide anions on benzodiazepine binding, and does not interfere with GABA binding or uptake. Behavioral testing has revealed that buspirone does not produce muscle weakness, does not control seizures, does not potentiate the impairment of psychophysiological function or lethality produced by administration of CNS depressants, does not produce sedation/hypnosis and does not appear to possess any abuse potential or liability for physical dependence. Thus, buspirone has been termed an anxioselective agent. Buspirone appears to only interact with the dopaminergic system with reasonable potency and exhibits properties of both a dopamine agonist and a dopamine antagonist. This suggests that dopamine is implicated in the etiology and expression of anxiety. A discussion of this implication is presented with a review of the clinical efficacy of nonbenzodiazepine drugs, especially dopamine agonists and dopamine antagonists, in the management of anxiety. In addition, neuropharmacological studies which have investigated the role of dopamine in animal models of anxiety are considered. Finally, the multiplicity of dopamine receptors and their regional localization in the brain are considered in the formulation of an hypothesis which features a role for the dopaminergic agents in the pharmacotherapy of anxiety.

Factors to consider when selecting animal models for postnatal teratology studies.

This presentation identifies some of the factors which should be considered when choosing a particular animal as a model to predict postnatal teratogenic hazards to man. The developmental pharmacologist-toxicologist must consider in concert the potential interactions of a xenobiotic on the mother, her placenta, her fetus, and her nursing young. Since the physiology of each of these components is in a state of dynamic flux during gestation and/or postnatal development, the critical time at which to expose the mother or her progeny to a chemical will vary widely from species to species and organ to organ. The identification of critical periods during the perinatal period may be quite difficult because the physiological or behavioral changes in the adult organism which signal that perinatal teratogenic effects have occurred are frequently subtle. Much research is still needed on the comparative aspects of maternal, placental, fetal, and neonatal pharmacokinetics and pharmacodynamics. We are not ready to intelligently evaluate large numbers of chemicals as potential postnatal teratogens for man.

Management program for improving sow and gilt performance by feeding dichlorvos.

Treated sows were on a special management program which consisted of feeding 1,000 mg of dichlorvos to the sow each day for the last 30 days of gestation. The dichlorvos was delivered as a slow-release formulation. The late gestation treatment signigicantly (P less than 0.01) improved the productivity of the sows by improving the performance of their litters. The litters from treated sows were superior in 4 ways: (1) increased number of pigs born alive per litter (2.3%); (2) increased pig birth weights (9.6%); (3) improved survival of pigs born alive; and (4) improved growth rate to market weight. The litters from treated sows were 11.6%, 16.5%, and 12.4% heavier than control litters at birth, weaning (35 days), and market (160 days), respectively.

Performance of swine chilled during artificial rearing.

There were more deaths among neonatal swine artificially reared for 21 days in individual cages at 27.9 C than among pigs reared under similar conditions at thermoneutrality (34.6 C). Furthermore, these deaths occurred at a younger age in the chilled animals. Chilled swine gained less body weight than did warm pigs for the first 15 days of life, although the survivors of the 27.9 C environment weighed the same as warm survivors at 22 days of age. Plasma glucose, liver, and skeletal muscle glycogen concentrations were significantly lower in neonatal swine exposed to 27.9 C from 1 to 4 days of age. Plasma nonesterified fatty acids, glycerol, cholesterol, and triglyceride concentrations were not altered by chilling. However, these lipid variables were significantly higher in 4-day-old nursig pigs than in animals reared artificially for the same period on artificial food. Adrenal gland weights and adrenal medullary catecholamine-synthetic enzyme activities were not altered by exposure to 27.9 C in pigs 1 to 4 days of age.

The need for comparative pharmacologists in industrial and academic research.

Comparative pharmacologists solidly trained in the principles of modern pharmacology, comparative biology, and comparative medicine are needed in various facets of drug research. Although this type of training would ideally be obtained in the professional medical environment of a veterinary college, the discipline of pharmacology has been neglected in these schools and must be upgraded by staffing and funding before quality training programs can be established. An interim solution would be to offer postgraduate training in comparative pharmacology and medicine at selected veterinary schools. Trainees would be recruited from pharmacology centers of excellence in medical or pharmacy schools. We assume that some of these trainees wound join veterinary school faculties and eventually establish quality Ph.DTRAINING PROGRAMS IN COMPARATIVE PHARMACOLOGY AT THEIR INSTITUTIONS. Comparative pharmacologists trained in these pre- or postdoctoral programs would be valuable additions to any team responsible for evaluating the safety and efficacy of drugs or chemicals in man or animals.

Sympathoadrenal Neurochemistry and early weaning of swine.

Three litters of pigs were weaned at 21 days of age, and 3 others were left with the sow. Pigs were killed at 21, 23, 28, or 39 days of age. Weaned pigs exhibited anxiety, gastrointestinal dysfunction, and decreased rate of body weight gain. Plasma glucose or liver glycogen concentrations were not decreased by weaning. Adrenal gland weights and tyrosine hydroxylase (EC 1.14.3a), dopamine beta-hydroxylase (EC 1.14.2.1), phenethanolamine-N-methyl transferase (EC 2.1.1), and monoamine oxidase (EC 1.4.3.4) activities were increased after weaning. Adrenal catecholamine and cortisol levels and dopa decarboxylase (EC 4.1.1.26) and catechol-o-methyl transferase (EC 2.1.1.6) activities were not significantly altered, although some increases were indicated. Cranial cervical ganglionic choline acetyltransferase (EC 2.3.1.6) and tyrosine hydroxylase activities were increased after weaning. Weaning of swine at 21 days of age is a stressful experience, and many effects persist for at least 18 days; however, growth was no longer impaired 18 days after weaning.