Neuroactive steroids, WIN-compounds and cholesterol share a common binding site on muscarinic acetylcholine receptors.

Endogenous neurosteroids and their synthetic analogues-neuroactive steroids-have been found to bind to muscarinic acetylcholine receptors and allosterically modulate acetylcholine binding and function. Using radioligand binding experiments we investigated their binding mode. We show that neuroactive steroids bind to two binding sites on muscarinic receptors. Their affinity for the high-affinity binding site is about 100 nM. Their affinity for the low-affinity binding site is about 10 µM. The high-affinity binding occurs at the same site as binding of steroid-based WIN-compounds that is different from the common allosteric binding site for alcuronium or gallamine that is located between the second and third extracellular loop of the receptor. This binding site is also different from the allosteric binding site for the structurally related aminosteroid-based myorelaxants pancuronium and rapacuronium. Membrane cholesterol competes with neurosteroids/neuroactive steroids binding to both high- and low-affinity binding site, indicating that both sites are oriented towards the cell membrane..

Diabetes mellitus affects the duration of action of vecuronium in dogs.

OBJECTIVE: To compare the duration of action of vecuronium in diabetic dogs with a control group. STUDY DESIGN: Prospective clinical study. ANIMALS: Forty client-owned diabetic (n = 20) and non-diabetic dogs. METHODS: Dogs were considered free from other concurrent disease based on clinical examination and laboratory data. After pre-anaesthetic medication with acepromazine and methadone, anaesthesia was induced with intravenous (IV) propofol and maintained with isoflurane-nitrous oxide in oxygen. Neuromuscular blockade (NMB) was achieved with vecuronium, 0.1 mg kg(-1) IV and its effects recorded by palpation (pelvic limb digital extension) and electromyography (m. tibialis cranialis) of responses (twitches; T) to repeated train-of-four (TOF) nerve stimulation. Time to onset of NMB was the period between vecuronium injection and loss of fourth twitch (T4) in the TOF pattern recorded by EMG and palpation. Duration of NMB was defined as the time from drug administration to return of T1 by palpation (T1(tactile) ) and EMG (T1(EMG) ). Times to return of T2-4 were also recorded. Time from induction of anaesthesia to vecuronium injection was recorded. Heart rate, non-invasive mean arterial pressure, body temperature, end-tidal isoflurane and end-tidal CO(2) concentrations were recorded at onset of NMB and when T1(EMG) returned. Loss and return of palpable and EMG responses for diabetic and non-diabetic dogs were compared using t-tests and Mann Whitney U-tests. RESULTS: There were significant (p < 0.05) differences between diabetic and non-diabetic dogs for the return of all four palpable and EMG responses. Times (mean ± SD) for return of T1(tactile) were 13.2 ± 3.5 and 16.9 ± 4.2 minutes in diabetic and non-diabetic dogs respectively. There were no differences between diabetic and non-diabetic dogs in the time to onset of vecuronium with EMG or tactile monitoring. CONCLUSIONS AND CLINICAL RELEVANCE: The duration of action of vecuronium was shorter in diabetic dogs as indicated by both tactile and EMG monitoring.

Sugammadex: a novel approach to reversal of neuromuscular blockade.

Sugammadex is the first in a new class of medications termed selective relaxant binding agents. This medication acts to encapsulate free circulating steroidal nondepolarizing neuromuscular blocking agents. The encapsulation of neuromuscular agents effectively decreases the amount of neuromuscular blocker interacting at the neuromuscular receptor. This binding has a very high association rate, rendering the incidence of residual block extremely low, while avoiding the side effects associated with traditional reversal agents. Currently approved for clinical use in over 50 countries, sugammadex was not approved by the US FDA in 2008 due to concerns over potential hypersensitivity reactions. It is hoped that further study and clinical experience will help to better define the risk associated with sugammadex and eventually lead to the approval of this novel medication in the USA.

Aconitum and Delphinium alkaloids of curare-like activity. QSAR analysis and molecular docking of alkaloids into AChBP.

Early studies have shown that some of diterpenoid alkaloids, found in highly toxic plants of the genera Aconitum and Delphinium, act at neuronal nicotinic acetylcholine receptors (nAChRs) and exhibit potent N-cholinolytic activity. In the current study, GA-MLRA and GA-PLS approaches have been used to build QSAR models to predict N-cholinolytic activity measured in vivo (blockade of neuromuscular conductivity, BNMC and third eyelid relaxing activity, TYRA) and in vitro (suppression of frog's abdominal straight muscles on acetylcholine, SAM) for a series of diterpenoid alkaloids. Random splitting of a data set (five trials in total) produced QSAR models of a good level of correlation between experimental in vitro/in vivo and calculated N-cholinolytic activity expressed as log(1/ED(50)) with following average statistical parameters: log BNMC (r(2) = 0.87, s = 0.14, q(2) = 0.82), log TYRA (r(2) = 0.80, s = 0.29, q(2) = 0.67), log SAM (r(2) = 0.84, s = 29, q(2) = 0.64). QSAR results suggest descriptors accounting for H-bond capability of molecules influence all three type of N-cholinolytic activity with additional contribution of steric and reactivity features as identified for TYRA and SAM data, respectively. The alkaloid-receptor complexes were further analyzed by means of AutoDock Vina docking program using the binding site of MLA complexed with AChBP (homolog of the ligand binding domain of nAChRs) as template. All compounds were shown to be well fitted in the binding pocket of native MLA with good correlation exhibited between their ED(50) and AutoDock Vina binding free energy. An analysis of the possible factors significant for the ligand recognition has been enhanced by comparative docking studies performed for structurally related lycoctonine-type alkaloids (lappaconitine and aconitine) that are known to bind to voltage-gated Na(+) channel, but not to nAChRs.

cis-Atracurium in dogs with and without porto-systemic shunts.

OBJECTIVE: To evaluate the non-depolarizing neuromuscular blocking drug cis-atracurium in dogs with porto-systemic shunts, and to compare it in clinically normal animals. ANIMALS: Thirteen dogs of mixed breed and sex, aged between 3 and 31 months old, weighing 2.2-25.5 kg, with ASA physical status II-IV, and undergoing surgical attenuation of porto-systemic shunt. A control group of 11 bitches of mixed breed, between 8 and 60 months old, and weighing 4.5-41.0 kg, all ASA physical status I, undergoing routine ovarohysterectomy were also studied. MATERIALS AND METHODS: Pre-anaesthetic medication was an opioid analgesic, given either alone or in combination with acepromazine. Following induction of general anaesthesia with intravenous (IV) propofol and oro-tracheal intubation, anaesthesia was maintained using isoflurane in either oxygen or oxygen and nitrous oxide. Ventilation was controlled. The train of four (TOF) technique was used to monitor neuromuscular blockade. An initial dose of 0.1 mg kg(-1)cis-atracurium was given IV and additional doses of 0.03 mg kg(-1)cis-atracurium were administered when at least one twitch of the TOF was present. RESULTS: Except for one dog that was killed during surgery because its anomaly was inoperable, all animals recovered satisfactorily from anaesthesia and surgery. In dogs with porto-systemic shunt, onset of neuromuscular blockade was 3.1 +/- 1.1 minutes (mean +/- SD) and in control dogs was 3.4 +/- 0.7 minutes (not significantly different). Neuromuscular blockade lasted 34 +/- 13 minutes in dogs with porto-systemic shunt and 29 +/- 17 minutes in control dogs (not significantly different). CONCLUSIONS: The presence of porto-systemic shunt did not affect the rate of onset or duration of action of cis-atracurium. CLINICAL RELEVANCE: cis-Atracurium may have a use in veterinary anaesthesia for producing neuromuscular blockade in dogs with hepatic insufficiency, including those with porto-systemic shunt.

A survey of combined epidural-propofol anesthesia with noninvasive positive pressure ventilation as a minimally invasive anesthetic protocol.

BACKGROUND: Combined epidural-propofol anesthesia with use of noninvasive positive pressure ventilation (NPPV) via the nose has been used routinely in our operating theaters. The purpose of this report was to present a survey of this anesthesia. MATERIAL/METHODS: 265 adult patients undergoing lower extremity or lower abdominal gynecological surgery during 1999 were examined. After epidural anesthesia, patients were given propofol infusion. NPPV was applied with an inspiratory/expiratory positive airway pressure of 14/8 cm H2O, a respiratory rate of 10 breaths/min, and oxygen delivery into the nasal mask resulting in a concentration of 40% or an inspiratory oxygen fraction of 0.35. Epidural anesthesia was continuously applied after surgery for postoperative pain relief. Various data related to the surgery or anesthesia were evaluated both on the day of surgery and on postoperative day 1. RESULTS: Of 265 patients, 3 patients could not receive our anesthetic protocol. Of the residual 262 patients, no patients showed serious clinical problems during anesthesia, excluding for hypotension, which was observed in 31-56% patients and was treated with ephedrine injection. Patients informed us of good analgesia (98%), feelings (78%) and dreams (47%). On postoperative day 1, postoperative analgesia and mood conditions were satisfactory. There were no patients complaining of intraoperative awareness. CONCLUSIONS: The principle of our anesthesia consists of epidural anesthesia, sole propofol infusion and noninvasive airway management, so as to provide an anesthetic technique with minimal invasiveness. Although airway maintenance by NPPV is not always suitable, our anesthesia is practicable for certain kinds of operations.

Competition between acetylcholine and a nondepolarizing muscle relaxant for binding to the postsynaptic receptors at the motor end plate: simulation of twitch strength and neuromuscular block.

UNLABELLED: The goal of the study was to simulate twitch strength and neuromuscular block produced by nondepolarizing muscle relaxants. METHODS: In the proposed model, affinities of the two binding sites at a single postsynaptic receptor for acetylcholine (A) and the muscle relaxant (D) define the formation of three complexes with A only, three complexes with D only, and two complexes with both A and D. Twitch strength was postulated to be a function of the receptors with both binding sites occupied by A, and two constants. Neuromuscular block (NMB) was calculated from NMB = 1-twitch. RESULTS: Stimulus-induced release of A results in rapid, but transient, changes in the concentrations of free A, the eight complexes, and the unoccupied receptors. Muscle relaxants that display either a congruous or an inverse pattern of affinities for the binding sites relative to those of A produce NMB vs. [D] curves with slightly different slopes but markedly different estimates for IC50. Depending on the number of activated receptors at the end plates of muscle fibers, the simulations represent the distributions of contracting fibers in a whole muscle. CONCLUSION: Simulations of competition between A and D for binding to two sites at a receptor reveal that the potencies of muscle relaxants, defined by IC50, and the slopes of the NMB vs. [D] curves depend on (1) the affinities of D for the two binding sites, (2) the orientation of the affinities relative to those of A, and (3) the affinities of A for the same two sites.

Curariform antagonists bind in different orientations to the nicotinic receptor ligand binding domain.

Curariform alkaloids competitively inhibit muscle acetylcholine receptors (AChR) by bridging the alpha and non-alpha subunits that form the ligand-binding site. Here we delineate bound orientations of d-tubocurarine (d-TC) and its methylated derivative metocurine using mutagenesis, ligand binding measurements, and computational methods. When tested against a series of lysine mutations in the epsilon subunit, the two antagonists show marked differences in the consequences of the mutations on binding affinity. The mutations epsilon L117K, epsilon Y111K, and epsilon L109K decrease affinity of metocurine by up to 3 orders of magnitude but only slightly alter affinity of d-TC. At the alpha subunit face of the binding site, the mutation alpha Y198T decreases affinity of both antagonists, but alpha Y198F preferentially enhances affinity of d-TC. Computation of antagonist docking orientations, based on our structural model of the alpha-epsilon site of the human AChR, indicates distinct orientations of each antagonist; the flatter metocurine fits into a pocket formed principally by the epsilon subunit, whereas the more compact d-TC spans the narrower crevasse between alpha and epsilon subunits. The side chains of epsilon Tyr-111 and epsilon Thr-117 juxtapose one of two quaternary nitrogens in metocurine but are remote from the equivalent quaternary nitrogen in d-TC, which instead closely approaches alpha Tyr-198. The different docked orientations arise through tilt of the curariform scaffold by approximately 60 degrees normal to the nitrogen-nitrogen axis, together with a 20 degrees rotation about the axis. The overall mutagenesis and computational results show that despite their similar structures, d-TC and metocurine bind in distinctly different orientations to the adult human AChR.

Laudanosine, an atracurium and cisatracurium metabolite.

Laudanosine is a metabolite of the neuromuscular-blocking drugs atracurium and cisatracurium with potentially toxic systemic effects. It crosses the blood-brain barrier and may cause excitement and seizure activity. Its interest in recent years has increased because of the recognized interaction with gamma-aminobutyric acid, opioid and nicotinic acetylcholine receptors. It has been shown to produce analgesia in animals. In the cardiovascular system, high plasma concentrations produce hypotension and bradycardia. In hepatic failure, its elimination half-life is prolonged but only moderate accumulation occurs in adults, whereas in infants and children plasma concentration are greater. In patients undergoing liver transplantation, laudanosine concentrations are increased during preanhepatic, anhepatic and postanhepatic stages. Patients with renal failure have higher plasma concentrations and a longer mean elimination half-life. In pregnancy, laudanosine crosses the placental barrier. The mean transplacental transfer is 14% of maternal blood concentrations. Except for prolonged administration of atracurium in intensive care units, laudanosine accumulation and related toxicity seem unlikely to be achieved in clinical practice. When cisatracurium is used, plasma concentrations of laudanosine are lower. Further studies are needed, especially around the interactions with gamma-aminobutyric acid, opioid and nicotinic acetylcholine receptors.

The effect of fluoride and hypothermia on the in vitro metabolism of mivacurium.

IMPLICATIONS: The fluoride inhibition of mivacurium hydrolysis by pseudocholinesterase increases in hypothermia, but it will very rarely occur in clinical practice because it requires rather large fluoride concentrations (>50 micromol/L) and very low temperatures (<28 degrees C).

Plasma cholinesterase activity and duration of action of mivacurium in phenotypically normal patients.

BACKGROUND: The short duration of action of mivacurium is due to its rapid hydrolysis by plasma cholinesterase (pChe). In patients with normal phenotype, low pChe activity because of, for instance, disease or intake of drugs may prolong the duration of action of mivacurium. The purpose of this study was to evaluate the relationship between pChe activity and the duration of action of mivacurium 0.2 mg/kg in phenotypically normal patients. MATERIAL: Forty-three adult patients with normal pChe phenotype and low or normal pChe activity, undergoing a variety of surgical procedures were included in the study with their informed consent and Ethics Committee approval. The neuromuscular block was monitored using TOF stimulation every 12 s and mechanomyography. The time to reappearance of the first response to TOF stimulation was measured. RESULTS: The patients pChe activities ranged from 45 to 1272 U/l (normal range 660-1620 U/l) and the time to first response to TOF from 8.1 to 62.7 min. An inverse relationship between enzyme activity and duration of action of mivacurium was found. The relationship was described by the equation: log10 (time) =alpha- beta log10 (pChe), where alpha (SD) is 2.547 (0.186) and beta (SD) 0.454 (0.069). CONCLUSION: In patients with phenotypically normal pChe, prediction of the duration of action of mivacurium is possible from the patients actual pChe activity. In patients with pChe activities below the normal range, the time to reappearance of the first response to TOF stimulation may vary from 10 to 180 min Only patients with pChe activities <220 U/l had a significantly prolonged duration of action of mivacurium.

[Carboxyperitoneum and clinical efficacy of nondepolarizing relaxants with different types of metabolism]. / Karboxyperitoneum i klinicheskaia éffektivnost' nedepoliarizuiushchikh relaksantov s razlichnymi tipami metabolisma.

A total of 108 patients operated on the abdominal cavity were examined with laparoscopic equipment or via laparotomic assess. The recovery time of neuromuscular conduction was defined for a myorelaxant with organ-depended metabolism (pipecuronium) and, predominantly, nonorgan-depended metabolism (athracurium and cisathracurium) in relation to the type of surgical technique (laparoscopy and laparotomy). It is concluded that carboxyperitoneum prolongs the recovery of neuromuscular conduction for pipecuronium without affecting this parameter in athracurium and cisathracurium. It is more expedient to use these myorelaxants during laparoscopic operations than pipecuronium, a myorelaxant having organ-dependent metabolism.

New approaches to reversal of neuromuscular block.

Although numerous reversal agents for neuromuscular block (NMB) have been known for some time, investigations on new approaches were initiated only recently. The different approaches used in an attempt to avoid the muscarinic side effects associated with the antagonists of NMB that are currently available are reviewed.