Non-depolarizing neuromuscular blocking activity of bisquaternary amino di- and tripeptide derivatives.

Herein we describe the synthesis of novel di- and tripeptide derivatives with two quaternary nitrogen groups attached and the biological testing of these compounds for neuromuscular blocking (NMB) activity in vitro and in vivo. The short peptide scaffold was selected because it offers potential for desired distance between the two pharmacophoric quaternary nitrogen groups, short duration of action, straightforward synthesis, and compatibility with an injectable formulation. From a small series of compounds 20c,e are identified as effective non-depolarizing NMB agents in vitro and in vivo in anesthetized cats and Rhesus monkeys with potencies similar to those of the clinical reference compounds rocuronium (4) and suxamethonium (2) (monkey ED(90) = 0.68, 0.23, 0.16, 5.04 micromol/kg, respectively). These new peptide derivatives 20c,e have similar potency and onset time but longer duration and slower recovery than the clinically used reference compounds. The structure-activity relationships described for this chemical series lead to the conclusion that the di- or tripeptide fragment can be regarded as an alternative template to the steroid or aliphatic ester of previously reported NMBs and within this tripeptide-derived series clog P correlates well with in vitro NMB activity.

Neuromuscular blocking and cardiovascular effects of Org 9487, a new short-acting aminosteroidal blocking agent, in anaesthetized animals and in isolated muscle preparations.

This study was undertaken to investigate the neuromuscular blocking profile and cardiovascular effects of Org 9487, a new aminosteroidal, non-depolarizing, neuromuscular blocking agent structurally related to vecuronium, in anaesthetized animals and in isolated muscle preparations. In in vitro functional assays of neuromuscular blocking activity, Org 9487 was between eight and 15 times less potent than vecuronium. In cats and monkeys the potency of Org 9487 was approximately one-seventh and one-twentieth, respectively, that of vecuronium. In both species, Org 9487 induced rapidly developing (onset times between 1.5 min and 1.9 min) neuromuscular paralysis, which was shorter-lasting than that of vecuronium and similar in time course to suxamethonium. The vagal: neuromuscular blocking dose ratio for Org 9487 was 3 and ganglion block was seen only at approximately 20 times the neuromuscular blocking dose. There was no evidence in the rat that Org 9487, administered at doses up to 3 mg kg-1, inhibited noradrenaline re-uptake. In anaesthetized dogs, Org 9487 (3 x 90% blocking dose) induced only relatively small and transient haemodynamic effects. The administration of clinically relevant doses of neostigmine or pyridostigmine shortened the time-course profile of Org 9487, even when administered during profound neuromuscular block. In animals, Org 9487 is a low potency, nondepolarizing neuromuscular blocking agent with a time course profile similar to that of suxamethonium. Although Org 9487 is less selective than vecuronium for the neuromuscular junction, it is unlikely to produce prohibitive cardiovascular side effects in man.

Research and development of aminosteroid neuromuscular blocking agents: past and future.

This article attempts to summarize more than 25 years of chemistry and pharmacology research aimed at the search for clinically useful muscle relaxants based on an amino-androstane skeleton. This research strategy has produced three clinically useful drugs (pancuronium, vecuronium and rocuronium), and in addition has demonstrated that it is indeed possible to design a non-depolarizing neuromuscular blocker with the onset/offset characteristics of suxamethonium. Lastly, much has been learned about the variable clinical predictiveness of data obtained in various animal species with these compounds. This factor is probably mainly responsible for the difficulties and delay in producing the non-depolarizing suxamethonium equivalent.

An overview of the pharmacology of rocuronium bromide in experimental animals.

In various animal species anaesthetized with a-chloralose (cats and pigs) or pentobarbitone (Beagle dogs and Rhesus monkeys), rocuronium has been shown to be a readily reversible, non-depolarizing neuromuscular blocking agent with a similar duration of action as vecuronium but a 6-10 fold lower potency. The outstanding features of its action is rapidity of onset. It is not expected to have any marked cardiovascular or autonomic side-effects when used in the neuromuscular blocking dose range. There is no evidence of any selective pre-junctional effect and there is no clinically relevant inhibition of acetycholinesterase. Screening in rats has not demonstrated any oestrogenic, androgenic, anabolic, glucocorticoid-like or gonad-inhibiting properties, although there was a slight increase in pituitary weight in male rats.

Interaction between rocuronium bromide and some drugs used during anaesthesia.

In cats anaesthetized with i.p. chloralose and pentobarbitone, neuromuscular blockade produced by various doses of rocuronium was measured and dose response curves constructed in the presence of halothane, enflurane, nitrous oxide, propofol, alfentanil, thiopentone, ketamine, diazepam, chlorpromazine, morphine or streptomycin. In general, when a shift in the dose response curve was produced, it was a parallel shift to the left, indicating potentiation. Both halothane and enflurane produced a left shift and a small increase in the time from maximum block to 90% recovery. Nitrous oxide had no effect on the depth of block but delayed recovery. Thiopentone and ketamine potentiated the blocking effect of rocuronium but propofol and alfentanil had no effect. Chlorpromazine and morphine caused potentiation which took 1-1.5 h to develop. Streptomycin had a potentiating effect in four cats but not in two others. Diazepam displaced the dose-response curve to the right in four cats. Prior treatment with suxamethonium had no effect.

Antiarrhythmic, electrophysiological and haemodynamic effects of prolonged oral dosing with Org 7797 in the anaesthetized rat.

The antiarrhythmic, electrophysiological and haemodynamic effects of chronic oral administration of Org 7797 ((16 alpha,17 beta)-17-methylamino-oestra-1,3,5(10)-triene-3, 16-diol-(Z)-2-butonedioate) were studied in rats. During dosing (10 mg kg-1 twice a day for 10 days) no effects on the electrocardiogram, monitored in conscious animals, were observed despite modest reductions (15-18%) in the maximum rate of depolarization of papillary muscle excised 1 or 6 h after completion of the dosing regime. Following anaesthesia, Org 7797 reduced the severity of arrhythmias induced by coronary artery occlusion and prevented the accompanying decrease in the ventricular fibrillation threshold (VFT) at 1 h after completion of dosing. By 6 h the effect on VFT had waned but protection against ischaemia-induced arrhythmias was retained despite a substantial decrease in Org 7797 plasma levels. Drug treatment did not modify arterial blood pressure, heart rate or stroke volume. We conclude that Org 7797 given chronically via the oral route exerts antiarrhythmic actions which may, at least in part, be due to sodium-channel block. In addition, our results suggest the presence of an active metabolite. The protective effects of Org 7797 were seen in the absence of electrocardiographic or haemodynamic changes suggesting that multiple oral doses of Org 7797 do not compromise normal cardiac function.

Effects of Org 7797 on early, late and inducible arrhythmias following coronary artery occlusion in rats and dogs.

1. The class Ic steroidal antiarrhythmic agent, Org 7797, was compared with two other Ic agents, flecainide and propafenone for intravenous activity against ischaemia-related cardiac arrhythmias and for electrophysiological actions in vivo. In addition the haemodynamic effects of Org 7797 were assessed in greyhounds. 2. Org 7797 (0.5 mg kg-1) significantly reduced the expected incidence of early ischaemia-induced ventricular fibrillation (VF) in rats and greyhound dogs and at doses of 0.5-1.0 mg kg-1 antagonized reperfusion-induced arrhythmias. Comparative studies in rats showed Org 7797 to be 2-4 times more potent than flecainide or propafenone. 3. Org 7797 (0.5 mg kg-1) slowed intracardiac conduction in anaesthetized beagles and again was at least 2-4 times more potent than flecainide or propafenone. 4. Org 7797 (0.5 and 2.0 mg kg-1), flecainide (1.0 and 2.0 mg kg-1) or propafenone (0.5 and 2.0 mg kg-1), did not significantly prevent induction of tachyarrhythmias (VT) in dogs with 5-6 day old myocardial infarcts although all 3 drugs appeared to prevent induced VF. All 3 drugs (notably flecainide) did however reduce the VT rate. 5. All 3 drugs (1-2 mg kg-1) suppressed spontaneous tachyarrhythmias in conscious beagle dogs with 1-2 day old infarcts. Propafenone was the least effective. 6. In an antifibrillatory dose (0.5 mg kg-1), the major haemodynamic effect of Org 7797 was a 10% increase in peripheral vascular resistance. Stroke volume, cardiac output and coronary blood flow were unchanged. In therapeutic doses, Org 7797 was also less negatively chronotropic than flecainide.7. It was concluded that Org 7797 is a potent antifibrillatory agent which is haemodynamically well tolerated. Higher doses are required to suppress late ischaemia-induced tachyarrhythmias which suggest that its antifibrillatory effects are the consequence of an action other than, or in addition to, sodium channel block.

The effects of a 16-N-homopiperidino analogue of vecuronium on neuromuscular transmission in anaesthetized cats, pigs, dogs and monkeys, and in isolated preparations.

Org 9991, a 16-N-homopiperidinium substituted vecuronium analogue, has been tested for neuromuscular blocking activity in anaesthetized cats, pigs, dogs and monkeys, and in isolated nerve-muscle preparations. Org 9991 exhibited non-depolarizing neuromuscular blocking activity of the competitive type, being reversible by neostigmine and showing no endplate channel blocking action in isolated preparations. In cats, 50% vagal block was observed at doses of Org 9991 approximately 10 times those producing 50% neuromuscular block; no ganglion block was seen at these doses. Effects on blood pressure or heart rate at 90% twitch blocking doses were either minor or absent. The potency and time course of action of Org 9991 remained similar in all four species: i.e. 90% block at ca 200-300 micrograms kg-1; onset time ca 1.2-1.9 min; duration 90% ca 4.5-8.9 min. This study suggests that 16-N-homopiperidinium analogues of vecuronium may provide leads in the quest for a potent non-depolarizing replacement for suxamethonium.

Effects of a new neuromuscular blocking agent (Org 9426) in anaesthetized cats and pigs and in isolated nerve-muscle preparations.

The effects of Org 9426 (the 2-morpholino, 3-hydroxy, 16N-allyl pyrrolidino analogue of vecuronium) were studied in anaesthetized cats and pigs and in isolated nerve--muscle preparations using tension and intracellular recording techniques. In isolated preparations, the effects of Org 9426 were antagonized by neostigmine. No contracture of the chick muscle preparation occurred. Org 9426 reduced the amplitude of endplate currents (EPC) in rat and snake muscle, but had no major effects on EPC decay characteristics, indicating a lack of endplate channel blocking action. In anaesthetized animals, no fasciculations were observed and the neuromuscular block was associated with tetanic and train-of-four fade and was antagonized by neostigmine. In anaesthetized cats and pigs, Org 9426 was approximately 20% as potent as vecuronium, its onset of action was twice as rapid as that of vecuronium in the cat and its duration of action was similar to that of vecuronium in both cats and pigs. It blocked the bradycardia produced by vagal stimulation only in doses greater than those necessary to produce neuromuscular block (ratios 7.2 in the cat and 4.4 in the pig--10-14% of the corresponding ratios for vecuronium). Ganglion block was seen only at doses several times those producing vagal block. In general the effects of Org 9426 on the cardiovascular system were slight, a small depressor effect occurring at high doses in the cat. The 17-hydroxy analogue, the potential metabolite of Org 9426, was approximately 20 times less potent than Org 9426 and is thus unlikely to contribute to the neuromuscular block produced by the parent compound.

A comparison of the neuromuscular blocking and autonomic effects of two new short-acting muscle relaxants with those of succinylcholine in the anesthetized cat and pig.

The actions of two new steroidal nondepolarizing neuromuscular blocking agents, structurally related to vecuronium, have been compared with those of succinylcholine in anesthetized cats and pigs. Both new compounds (Org 7617 and Org 9616) exhibited properties typical of nondepolarizing relaxants in each species. Org 9616 was one-fifth (ED50 cat tibialis 154 micrograms.kg-1) and Org 7617 was one-tenth (ED50 cat tibialis 287 micrograms.kg-1) as potent as vecuronium as a neuromuscular blocking drug. Both compounds produced rapidly developing muscle relaxation in cats that, like that of succinylcholine, was transient in time course (onset/duration of action--tibialis anterior muscle: Org 7617 1.6/3.9 min; Org 9616 1.5/4.3 min; succinylcholine 1.7/5.7 min). In pigs that were used as a predictor of duration of action in humans, both Org 7617 and Org 9616 also produced short-lived neuromuscular blockade. The neuromuscular blocks produced by Org 7617 and Org 9616 were readily reversed by neostigmine. Both compounds blocked the heart rate responses to vagal stimulation at doses higher than those required to produce neuromuscular block. The vagal:neuromuscular blocking ratio for Org 7617 was 10, and for Org 9616 was 17. These compare to approximate published values for vecuronium, atracurium, and pancuronium of 60, 25, and 3, respectively. Ganglion block was only seen at 30-60 times the neuromuscular blocking doses. Both compounds produced a decrease in arterial blood pressure. This was more pronounced with Org 7617. In addition, Org 9616 produced a slight increase in heart rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative neuromuscular blocking effects of vecuronium, pancuronium, Org 6368 and suxamethonium in the anaesthetized domestic pig.

The neuromuscular blocking and cardiovascular effects of three competitive neuromuscular blocking drugs, vecuronium, pancuronium and Org 6368, and the depolarizing drug suxamethonium, have been evaluated in the anaesthetized domestic pig. Both the relative time-course and the order of potency of the drugs agreed well with published data in man. In addition, the cardiovascular side-effects of pancuronium and Org 6368 were identified readily. These preliminary results suggest that the pig may provide more reliable neuromuscular blocking time-course data than does the more commonly utilized cat model.

Effects of diet-induced hypokalaemia on the efficacy of antiarrhythmic drugs against ventricular arrhythmias evoked by coronary artery ligation in the anaesthetised rat.

The antiarrhythmic efficacies of intravenous quinidine (Q), disopyramide (D), prenylamine (P), bepridil (B), and practolol were compared in normokalaemic and dietary-induced hypokalaemic anaesthetised rats. Hypokalaemia markedly increased the severity of arrhythmias induced by coronary artery ligation and reduced the antiarrhythmic efficacy of all five drugs tested. After anaesthesia, hypokalaemia was associated with hypotension and bradycardia but not QTc prolongation. However, bradycardia was not seen in conscious hypokalaemic animals. Papillary muscles taken from hypokalaemic rats displayed a longer action potential duration but no increase in Vmax compared with tissue taken from normokalaemic animals. It is concluded that hypokalaemia in the rat causes electrophysiological disturbances which may contribute both to the observed exacerbation of ischaemia-induced arrhythmias and to the reduced efficacy of Q, D, P, and B. The reduced efficacy of practolol might be explained by other consequences of hypokalaemia.

Effects of antiarrhythmic drugs on ventricular fibrillation thresholds of normal and ischaemic myocardium in the anaesthetized rat.

1 The effects of agents which produce membrane stabilization (class I), beta 1-adrenoceptor blockade (class II), prolongation of the cardiac action potential (class III) or inhibition of the slow inward current (class IV) were investigated for their ability to increase the ventricular fibrillation threshold (VFT) or to modify the fall in VFT consequent upon coronary artery ligation in the anaesthetized rat. 2 The class I agent, Org6001, increased VFT of normal myocardium and in lower doses reduced the postligation fall in VFT. 3 The class II agent, metoprolol, failed to increase VFT of normal myocardium but reduced the postligation fall. 4 The class III agent, melperone, increased VFT of both normal and ischaemic myocardium whereas the class IV agent, nifedipine failed to influence VFT in either region. 5 Bepridil (class I and IV) was similar to Org6001 and sotalol (class II and III) in that it increased VFT of normal myocardium and in lower doses reduced the postligation fall in VFT. 6 Measurement of VFT before and after coronary artery ligation in the rat constitutes a rapid and reproducible screen to detect antifibrillatory activity. 7 The results also suggest that in the rat, the low currents used (approximately 400 microA) do not release substantial quantities of catecholamines whereas these may be released by coronary artery ligation.

The effects of postligation administration of ORG 6001 and disopyramide on early ischaemia-induced arrhythmias in the anaesthetized rat.

The effects of intravenous doses of Org 6001 and disopyramide (10 mg/kg) known to confer protection against early postligation-induced arrhythmias in the anaesthetized rat when given prophylactically, were assessed following postligation administration. When given 1 min after ligation both drugs greatly reduced the incidence of ventricular fibrilloflutter and completely prevented electrical deaths (45% in the controls). Protection was also seen when the drugs were given just before the expected onset of arrhythmias (4.5 min postligation).

Influences of hypothermia, cold, and isolation stress on the severity of coronary artery ligation-induced arrhythmias in rats.

With a view to increasing the severity of the arrhythmias that arise following coronary artery ligation in anesthetized rats, the influences of exposure to lowered environmental temperature (LET) (21 to 13 degrees C) and isolation stress were examined. Housing rats singly for 48 hr, prior to the induction of arrhythmias, caused the most marked changes in the severity of the model; the incidence of ventricular fibrillation and mortality were increase from 43 to 90% and 9 to 60%, respectively. Exposure of multiply housed rats to LET for 48 hr also significantly increased mortality from fibrillation. A combination of LET plus isolation stress, however, did not further increase the severity of the arrhythmias following ligation. Acute hypothermia (body temperature reduced to 30 or 25 degrees C) did not significantly enhance the severity of the model.

Comparative antidysrhythmic and haemodynamic effects of orally or intravenously administered mexiletine and ORG 6001 in the anaesthetized rat.

1 The antidysrhythmic and haemodynamic effects of the aminosteroid, Org 6001, were studied in the rat anaesthetized with pentobarbitone. Mexiletine was used for comparison. 2 Both Org 6001 (2--10 mg/kg) and mexiletine (1 mg/kg) given intravenously antagonized the development of dysrhythmias evoked by acute coronary artery ligation in rats. 3 In antidysrhythmic doses, Org 6001 and mexiletine exerted only moderate and transient hypotension and depression of cardiac contractility (assessed from LV dP/dtmax). Org 6001 did, however, induce a more sustained bradycardia. 4 Effective oral doses of Org 6001 (20--100 mg/kg) were similar to those of mexiletine, disopyramide and propafenone. 5 Oral Org 6001 (100 mg/kg) was effective for 18 h whereas mexiletine (100 mg/kg) failed to protect against evoked dysrhythmias 3 h after dosing. 6 Org 6001 and mexiletine differed in their actions on ventricular fibrillation threshold (VFT). Org 6001 (100 mg/kg orally 12 h before ligation) prevented the decrease in VFT produced by coronary ligation whereas mexiletine (100 mg/kg orally) had no effect. When administered intravenously, mexiletine (but not ORg 6001) increased VFT in normal ventricular muscle.

Development of a severe model of early coronary artery ligation-induced dysrhythmias in the anaesthetized rat.

1. The potential use of catecholamines to increase the severity of dysrhythmias evoked by coronary artery ligation in the anaesthetized rat was investigated. Drugs were given intravenously prior to ligation. 2 Pressor doses of adrenaline (5 microgram/kg) noradrenaline (1 microgram/kg) phenylephrine (5-10 microgram/kg), and angiotensin (0.25 microgram/kg) conferred protection against the development of dysrhythmias. 3 Atropine (1 mg/kg) increased mortality from ventricular fibrilloflutter (VF) and abolished the protective effects of phenylephrine (10 micrograms/kg). 4 Administration of isoprenaline (10 microgram/kg) significantly increased the incidence of and the mortality from VF. 5 The order of antidysrhythmic drug potency of Org 6001 (1-10 mg/kg), disopyramide (2-10 mg/kg) and practolol (2-10 mg/kg) was similar in both the standard (without isoprenaline) and modified (with isoprenaline) models. 6 Use of the modified method for antidysrhythmic screening purpose allows demonstration of statistically meaningful results with the use of relatively few animals. 7 Comparison of the pattern of VF in the rat heart induced by various means suggests that the diagnosis of ventricular fibrillation can be made with more confidence in the modified method compared to the standard method.

The beneficial actions of bepridil in acute myocardial infarction in anaesthetized dogs.

1 When administered intravenously shortly before acute coronary ligation in dogs anaesthetized with chloralose, bepridil (5 mg/kg) produced immediate and transient falls in coronary and systemic vascular resistance which were accompanied by marked decreases in myocardial oxygen extraction. These effects were followed by sustained decreases in heart rate and myocardial oxygen consumption. 2 This dose of bepridil reduced the number of premature ventricular beats and abolished fibrillation induced by coronary artery ligation without modifying the haemodynamic or metabolic consequences (lactate production) of myocardial ischaemia. 3 When administered 1.5-2 h after ligation, bepridil did not compromise the critical perfusion of the acutely ischaemic zone but reduced the lactate production and ST-segment elevation in the ischaemic zone. 4 These results suggest that bepridil may be a useful drug in the chronic treatment of angina pectoris and in this respect may possess advantages over beta-adrenoceptor antagonists.