The impact of orthosteric radioligand depletion on the quantification of allosteric modulator interactions.

Radioligand binding assays remain a common method for quantifying the effects of allosteric modulators at G protein-coupled receptors. The allosteric ternary complex model (ATCM) is the simplest model applied to derive estimates of modulator affinity (K(B)) and cooperativity (alpha), which are necessary for understanding structure-activity relationships. However, the increasing drive toward assay miniaturization in modern drug discovery may lead to conditions where appreciable ligand depletion occurs in the assay. Theoretical simulations investigating the impact of orthosteric radioligand depletion on the estimation of ATCM parameters revealed the following. 1) For allosteric inhibitors, application of the standard ATCM to data obtained under depletion conditions leads to an underestimation of pK(B) and an overestimation of log alpha. 2) For allosteric enhancers, the opposite was noted, but not always; the nonlinear regression algorithm is more likely to struggle to converge to a satisfactory solution of (nondepletion) ATCM parameters in this situation. 3) Application of a novel ATCM that explicitly incorporates orthosteric ligand depletion will yield more reliable model estimates, provided the degree of depletion is not high (< approximately 50%). Subsequent experiments investigated the interaction between [3H]N-methylscopolamine and the allosteric enhancer, alcuronium, or inhibitor, gallamine, in the presence of increasing concentrations of M(2) muscarinic acetylcholine receptor and showed that application of an ATCM that explicitly incorporates radioligand depletion can indeed give more robust estimates of modulator affinity and cooperativity estimates than the standard model. These results have important implications for the quantification of allosteric modulator actions in binding-based discovery assays.

Structure/activity relationships of M2 muscarinic allosteric modulators.

Allosteric modulation of G protein-coupled receptors has been intensively studied at muscarinic acetylcholine receptors. Findings made with archetypal allosteric agents such as gallamine, alcuronium, and bis(ammonio)alkane-type agents revealed that binding of orthosteric ligands that attach to the acetylcholine site can be allosterically decreased or increased or left unaltered in a subtype-selective fashion. Analyses of structure/activity relationships (SARs) help to elucidate the molecular events underlying the allosteric action and they may pilot the development of new allosteric agents with improved properties and therapeutic perspectives. With a focus on SARs, this review illustrates the principles of muscarinic allosteric interactions, gives an overview of SARs in congeners of archetypal allosteric agents, and considers the topology of M(2) muscarinic allosteric interactions that are characterized by divergent binding modes.

Modes of allosteric interactions with free and [3H]N-methylscopolamine-occupied muscarinic M2 receptors as deduced from buffer-dependent potency shifts.

Muscarinic M2 acetylcholine receptors contain an allosteric site that is probably located at the entrance of the ligand binding pocket above the orthosteric binding site. With the orthosteric area not occupied, allosteric agents might gain access to this site. The interaction of allosteric agents with orthoster-occupied receptors is known to depend on the buffer conditions in an alloster-specific fashion. Utilizing the buffer-dependent potency shift as an indicator, we aimed to find out for two rod-like shaped and flexible allosteric agents whether or not there is evidence for a switch in the site of attachment in free compared with [3H]N-methylscopolamine ([3H]NMS)-occupied porcine heart M2 receptors. These agents are the bispyridinium compounds WDuo3 (1,3-bis[4-(phthalimidomethoxyimino-methyl)-pyridinium-1-yl] propane dibromide) and Duo3 (4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bis-pyridinium dibromide). The prototype allosteric agents gallamine and alcuronium were included. Inhibition of [3H]NMS association was taken to reflect alloster interaction with free receptors, inhibition of [3H]NMS dissociation indicated binding to [3H]NMS-occupied receptors. In Na,K,Pi buffer (4 mM Na2HPO4, 1 mM KH2PO4, pH 7.4 at 23 degrees C) compared with Mg,Tris,Cl,Pi buffer (45 mM Tris-HCl, 2.6 mM MgHPO4, pH 7.3 at 37 degrees C) WDuo3 underwent the same loss of potency for the interaction with either free or [3H]NMS-liganded receptors. The loss of potency was quantified by a potency ratio (PR), i.e. the ratio between the concentrations of the modulator leading to a half-maximal delay of [3H]NMS association or dissociation, respectively, in Mg,Tris,Cl,Pi compared with Na,K,Pi. For WDuo3 the ratios were PRass=27 and PRdiss=22, respectively. For Duo3, the interaction with free and [3H]NMS-occupied receptors only slightly depended on the composition of the incubation medium: PRass=1.3, PRdiss=2.8. In contrast to the other agents, the concentration-effect curves of which had slope factors nH not different from unity, the curves of Duo3 were steep (nH about -1.6). For alcuronium the shift factors amounted to PRass=29 and PRdiss=25, for gallamine to PRass=216 and PRdiss=159. In conclusion, there was a wide variation between the allosteric agents with regard to the respective buffer dependence of action. Yet, for a given allosteric agent, the interaction with either free or [3H]NMS-occupied receptors was always characterized by the same buffer-dependent shift. Thus, even the applied rod-shaped allosteric agents do not appear to switch to the orthosteric site in free compared with orthoster-occupied M2 receptors.

Competition between positive and negative allosteric effectors on muscarinic receptors.

Alcuronium allosterically increases the affinity of cardiac muscarinic receptors for methyl-N-scopolamine (NMS), whereas gallamine has the opposite effect. We discovered that strychnine also increases the affinity of muscarinic receptors in rat heart atria for NMS. It is not known whether the positive and the negative allosteric effectors bind to the same binding site. To investigate this question, we elaborated on a theoretical model predicting changes in the binding of a classic radiolabeled ligand occurring in the presence of a positive and a negative allosteric effector that compete for the allosteric binding site. The model is based on data obtained at equilibrium and avoids uncertainties associated with the use of nonequilibrium methods for the evaluation of interactions between allosteric ligands. We examined changes in the binding of [3H]NMS to membranes of rat heart atria exposed to various concentrations of a positive allosteric effector (alcuronium or strychnine) and of a negative allosteric effector (gallamine) simultaneously. The binding data obtained were in perfect agreement with the model assuming competition between gallamine and alcuronium and gallamine and strychnine, strongly suggesting that these positive and negative allosteric effectors bind to identical or overlapping sites.

Protection by alcuronium of muscarinic receptors against chemical inactivation and location of the allosteric binding site for alcuronium.

We have found earlier that the neuromuscular blocker alcuronium binds to cardiac muscarinic receptors simultaneously with their specific antagonist [3H]methyl-N-scopolamine ([3H]NMS) and allosterically increases their affinity to this ligand. Nothing is known about the allosteric site with which alcuronium interacts. To gain an insight, we have now investigated how the binding of [3H]NMS is affected by agents known to modify specific residues in proteins and how their effects are altered by alcuronium. Reagents that covalently modify the tyrosyl residues (p-nitrobenzenesulfonyl fluoride and 4-chloro-7-nitrobenzofurazan) and the carboxyl groups of aspartate and glutamate [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-dicyclohexylcarbodiimide, and N-ethyl-5-phenylisoxazolium-3'-sulfonate] blocked the binding of [3H]NMS to receptors in rat heart atria. Their action was probably due to the modification of tyrosyl and aspartyl residues directly in the muscarinic binding sites because it was antagonized by atropine and carbamoylcholine. Alcuronium and gallamine, another allosteric ligand, also protected the [3H]NMS binding sites against the inactivation by tyrosine- and carboxyl-directed chemical modifiers just as well as by benzilylcholine mustard, known to attach covalently to the muscarinic binding sites. Protection by alcuronium has also been observed on cerebrocortical muscarinic receptors. The effect of alcuronium indicates that the drug interferes with the access of chemical modifiers to the muscarinic sites. In view of the unspecific nature of most of the modifiers used (with regard to muscarinic mechanisms), the protection by alcuronium appears to be best explained on the assumption that the drug binds in close vicinity of the "classical" muscarinic site and sterically blocks the access to this site.

Alcuronium pharmacodynamics in dogs: effect-concentration relationships in the diaphragmatic and limb muscles.

Previous studies suggest that the muscles of the diaphragm are less sensitive to neuromuscular blocking agents than the limb muscles. However, this difference has not been characterized directly in terms of relaxant drug plasma concentrations. The pharmacodynamics of the non-depolarizing muscle relaxant alcuronium were therefore investigated in nine dogs using a constant-rate infusion regimen with simultaneous measurement of muscle paralysis in the limb and diaphragm. Maximum paralysis between 95 and 100% was achieved in both muscle groups, within approximately the same time interval. However, during onset of and offset of effect, the pharmacodynamic parameters ECp50 and ECp95 for the limb muscle were lower than in the diaphragm. From a pharmacodynamic effect model it was also predicted that Css(50) and Css(95) for the limb muscles are half those values for the diaphragm. Thus, the diaphragm is less sensitive to the action of alcuronium than are limb muscles. The half-time for equilibration of alcuronium between plasma and the effect site was two-fold lower for the diaphragm, and the rate of recovery from paralysis in diaphragmatic muscles was twice that observed in limb muscles. Collectively, these data suggest that there is a greater margin of safety in the diaphragmatic muscles and that the response of the peripheral limb muscles to nerve stimulation provides only a conservative index of recovery from competitive neuromuscular block in the diaphragmatic muscles.

[Course of the neuromuscular block under atracurium. Comparison with alcuronium]. / Evolution du bloc neuromusculaire sous atracurium. Comparaison avec l'alcuronium.

Using a standardized anaesthetic protocol, the continuous monitoring of the twitch height after a 0.1 Hz stimulus was used to follow the evolution of curarization following injection of either atracurium (0.6 mg . kg-1) or alcuronium (0.2 mg . kg-1). The maximum twitch height inhibition was always greater than 99% of the control value and occurred after 107 +/- 50 s with atracurium (n = 30) and 172 +/- 120 s for alcuronium (n = 30) (p less than 0.02). Although surgical stage of muscular relaxation (twitch height less than 25% of reference value) was the same for both drugs (55 +/- 15 min for alcuronium versus 52 +/- 10 min for atracurium; n = 30 for both groups), the clinical duration (spontaneous restoration of twitch height to 90% of the reference value) was significantly longer (p less than 0.005) for alcuronium: 89 +/- 20 min (n = 10) versus 62 +/- 9 min for atracurium (n = 10). The spontaneous return to normal of the train of four was also significantly longer (p less than 0.001) for alcuronium: 118 +/- 23 min (n = 10) versus 69 +/- 7 min for atracurium (n = 10). The recovery index (the time required for twitch height to rise from 25% to 75%) was three times quicker (p less than 0.01) for atracurium (10 +/- 3 min; n = 10) than for alcuronium (30 +/- 13 min; n = 10).(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacokinetics of alcuronium in elderly patients undergoing total hip replacement or aortic reconstructive surgery.

The pharmacokinetic behaviour of alcuronium was studied in three patients undergoing resection of an aortic aneurysm, and in another two patients undergoing total hip replacement (group I). A control group of five elderly patients undergoing relatively minor surgery was included (group II). In group I patients, the values of the pharmacokinetic parameters such as plasma clearance, elimination half-life and the apparent volume of distribution of the drug were found to be comparable to those obtained in normal young patients in previous studies. The group II patients, however, were found to have a prolonged elimination half-life as a result of reduced plasma clearance, possibly an age-related effect. The differences between these two groups of patients may be explained by the differences in the extent of haemorrhage and fluid replacement or changes in blood circulation, or both. However, alcuronium must still be used cautiously in both groups of patients, especially in the light of a recent finding that patients undergoing aortic reconstructive surgery have a high frequency of functional renal failure after operation.

Alcuronium kinetics and plasma concentration-effect relationship.

The kinetics and dynamics of the neuromuscular blocker alcuronium were investigated in 12 surgical patients who received bolus and infusion regimens. In six patients the duration of the infusion was sufficiently long so that a steady-state alcuronium plasma concentration was reached (mean SD, 0.80 +/- 0.23 micrograms/ml). In the remaining six patients a steady state was not reached but the alcuronium concentration at the end of the infusion was 0.91 +/- 0.39 micrograms/ml. Alcuronium kinetic parameters did not differ between the two groups or from those obtained previously after bolus doses. In six patients for whom sufficient alcuronium concentration-time response data were available over the 0 to 100% response range, various mathematic models were used to characterize the concentration-effect relationship. A dynamic model incorporating a separate effect compartment connected to the central compartment was found to be the most appropriate. The (mean +/- SD) rate constant for equilibration of alcuronium concentration and effect was found to be 0.24 +/- 0.11 min-1, whereas the steady-state concentration required to induce 95% paralysis was 0.91 +/- 0.35 micrograms/ml (mean +/- SD).

Alcuronium kinetics in patients undergoing cardiopulmonary bypass surgery.

1 The disposition of alcuronium was investigated in 10 patients undergoing surgery involving cardiopulmonary bypass (CPB) and compared with results from a group of non-cardiac patients studied previously. 2 After intravenous administration of a combined bolus and infusion dosage regimen, plasma concentrations fell in a bi-exponential fashion to a mean value of 0.55 micrograms/ml immediately before the start of extracorporeal circulation. 3 During CPB an apparent steady-state of alcuronium was reached immediately after commencement of CPB, however plasma concentrations were some 50% higher than those noted prior to commencement of CPB and those predicted using previous pharmacokinetic data from normal surgical patients. 4 Once CPB was completed and the alcuronium infusion terminated, post-infusion alcuronium plasma concentrations again appeared to decline bi-exponentially with time. 5 Of the pharmacokinetic parameters which were calculated model-independently, the apparent volume of distribution (Vss) was unchanged (329 vs 313 ml/kg) and the elimination half-life (t1/2,z) (532 vs 199 min) was prolonged and the plasma clearance (CL) (0.8 vs 1.34 ml min-1kg-1) markedly reduced in these patients compared to non-cardiac surgical patients. 6 As a result of these changes in alcuronium concentration during CPB and the diminished elimination of alcuronium following CPB, a closer monitoring of neuromuscular function may be necessary in cardiac patients undergoing CPB.

Caesarean section and placental transfer of alcuronium.

The placental transfer of alcuronium was studied in twelve patients undergoing elective or emergency caesarean section. Umbilical cord vein and maternal plasma was analysed for alcuronium at dose-delivery time intervals ranging from 5 to 10.5 minutes. The mean umbilical vein concentration of the relaxant was 0.41 +/- 0.18 (SD) microgram/ml, and the mean foetal/maternal concentration ratio was 0.26 +/- 0.11. A positive correlation between foetal and maternal concentrations of alcuronium was demonstrated. Although alcuronium appears to cross the placenta rapidly and in reasonably high concentration, no apparent adverse effects on the neonates was evident as judged by measurement of Apgar scores.

Clinical pharmacokinetics of alcuronium chloride in man.

The pharmacokinetic behaviour of alcuronium is described for nineteen patients undergoing anaesthesia for elective surgery. Eleven patients received a single bolus intravenous dose of 0.25 mg/kg, while 8 patients required additional doses of 0.125 mg/kg. A two-compartment open model was found to describe adequately both the single dose and multiple dose data for the majority of patients. No significant differences were found in the model-independent pharmacokinetic parameters between the single and multiple dose studies. Mean values for the pooled data for the half-life (t 1/2 beta), apparent volume of distribution (Vd beta), volume of distribution at steady-state (Vdss), volume of the central compartment (Vc) and plasma clearance (Clp) were 198.75 min, 24.261, 20.891, 8.181 and 90.22 ml/min respectively. Evoked muscle twitch response was monitored in 17 of the patients to assess the degree of relaxant blockade. The bolus dose of alcuronium produced complete block in 9 patients and between 95 and 99% block in the remainder. The time of onset to maximum block ranged from 3 to 30 min with the concurrently measured plasma levels of alcuronium being 0.79 to 2.25 microgram/ml. The time taken following bolus administration to 5% recovery (95% paralysis) was a mean of 42 min and the corresponding mean alcuronium plasma concentration was 0.78 microgram/ml.

Studies on muscle relaxants during haemodialysis.

Signs of neuromuscular block were evident more than 20 hours after the administration of alcuronium to an anuric patient. Complete recovery occurred during haemodialysis. We therefore decided to study the dialysance of three radioactive non-depolarising relaxants during haemodialysis of four patients with chronic renal failure. Although dimethyl tubocurarine and alcuronium were equal as regards dialysance, the concentration of the former, in plasma, falls faster than does alcuronium. It is believed that a larger volume of distribution occurs with dimethyl tubocurarine. In spite of the fact that the dialysance of muscle relaxants is small, haemodialysis might lower the concentration of these substances in the plasma to a level below the critical point which produces paralysis.