Reversal of Succinylcholine Induced Apnea with an Organophosphate Scavenging Recombinant Butyrylcholinesterase

Background

Concerns about the safety of paralytics such as succinylcholine to facilitate endotracheal intubation limit their use in prehospital and emergency department settings. The ability to rapidly reverse paralysis and restore respiratory drive would increase the safety margin of an agent, thus permitting the pursuit of alternative intubation strategies. In particular, patients who carry genetic or acquired deficiency of butyrylcholinesterase, the serum enzyme responsible for succinylcholine hydrolysis, are susceptible to succinylcholine-induced apnea, which manifests as paralysis, lasting hours beyond the normally brief half-life of succinylcholine. We hypothesized that intravenous administration of plant-derived recombinant BChE, which also prevents mortality in nerve agent poisoning, would rapidly reverse the effects of succinylcholine.

Methods

Recombinant butyrylcholinesterase was produced in transgenic plants and purified. Further analysis involved murine and guinea pig models of succinylcholine toxicity. Animals were treated with lethal and sublethal doses of succinylcholine followed by administration of butyrylcholinesterase or vehicle. In both animal models vital signs and overall survival at specified intervals post succinylcholine administration were assessed.

Results

Purified plant-derived recombinant human butyrylcholinesterase can hydrolyze succinylcholine in vitro. Challenge of mice with an LD₁₀₀ of succinylcholine followed by BChE administration resulted in complete prevention of respiratory inhibition and concomitant mortality. Furthermore, experiments in symptomatic guinea pigs demonstrated extremely rapid succinylcholine detoxification with complete amelioration of symptoms and no apparent complications.

Conclusions

Recombinant plant-derived butyrylcholinesterase was capable of counteracting and reversing apnea in two complementary models of lethal succinylcholine toxicity, completely preventing mortality. This study of a protein antidote validates the feasibility of protection and treatment of overdose from succinylcholine as well as other biologically active butyrylcholinesterase substrates.     

Effect of succinylcholine on responses of neurons in the motor cortex to natural activation of muscle spindle primary endings.

In locally anesthetized cats, the effects of intravenous administration of succinylcholine (SCh) in sub-paralytic dosages on the responses of single neurons in motor cortex to small dynamic muscle stretches were studied. A large transient enhancement of these cortical responses with a time course corresponding to the peripheral action of SCh on muscle spindles was observed. This finding is discussed in terms of the hypothesis that muscle spindle primary endings may activate projections to motor cortex.     

Effect of Dietary Magnesium on Post Mortem Phosphocreatine Utilization in Skeletal Muscle of Swine: A Non-Invasive Study Using 31P-NMR Spectroscopy

The effect of dietary magnesium on the post mortem PCr (phosphocreatine) decay in muscle of heterozygote malignant hyperthermia pigs was studied after in vivo exposure to a combination of halothane and succinylcholine. The pigs were anaesthetized with halothane and succinylcholine was injected in the ear vein. Immediately after initiation of the depolarizing neuromuscular blocking effect of succinylcholine the animals were captive-bolt stunned. The PCr decay, reflecting ATP turnover, was followed in situ by 31P-NMR spectroscopy in the biceps femoris muscle for the subsequent 40-70 min post mortem. In 3 of the 4 experiments, the Mg-fed pig had a significantly reduced rate of PCr hydrolysis compared to the control animal. The mechanism of this magnesium effect is unknown.     

Biochemical and genetic analysis of butyrylcholinesterase (BChE) in a family, due to prolonged neuromuscular blockade after the use of succinylcholine

Butyrylcholinesterase (BChE) is a plasma enzyme that catalyzes the hydrolysis of choline esters, including the muscle-relaxant succinylcholine and mivacurium. Patients who present sustained neuromuscular blockade after using succinylcholine usually carry BChE variants with reduced enzyme activity or an acquired BChE deficiency. We report here the molecular basis of the BCHE gene underlying the slow catabolism of succinylcholine in a patient who underwent endoscopic nasal surgery. We measured the enzyme activity of BChE and extracted genomic DNA in order to study the promoter region and all exons of the BCHE gene of the patient, her parents and siblings. PCR products were sequenced and compared with reference sequences from GenBank. We detected that the patient and one of her brothers have two homozygous mutations: nt1615 GCA > ACA (Ala539Thr), responsible for the K variant, and nt209 GAT > GGT (Asp70Gly), which produces the atypical variant A. Her parents and two of her brothers were found to be heterozygous for the AK allele, and another brother is homozygous for the normal allele. Sequence analysis of exon 1 including 5'UTR showed that the proband and her brother are homozygous for -116GG. The AK/AK genotype is considered the most frequent in hereditary hypocholinesterasemia (44%). This work demonstrates the importance of defining the phenotype and genotype of the BCHE gene in patients who are subjected to neuromuscular block by succinylcholine, because of the risk of prolonged neuromuscular paralysis.     

Organophosphorus pesticide‐induced butyrylcholinesterase inhibition and potentiation of succinylcholine toxicity in mice

Succinylcholine is the most important rapid‐acting depolarizing muscle relaxant during anesthesia. Its desirable short duration of action is controlled by butyrylcholinesterase, the detoxifying enzyme. There are two reported cases of prolonged paralysis from succinylcholine in patients poisoned with the organophosphorus insecticides parathion and chlorpyrifos. The present study examines the possibility that other organophosphorus and methylcarbamate pesticides might also prolong succinylcholine action by inhibiting butyrylcholinesterase using mice treated intraperitoneally as a model and relating inhibition of blood serum hydrolysis of butyrylthiocholine to potentiated toxicity (mouse mortality). The organophosphorus plant defoliant tribufos (4 h pretreatment, 160 mg/kg) and organophosphorus plant growth regulator ethephon (1 h pretreatment, 200 mg/kg) potentiate the toxicity of succinylcholine by seven‐ and fourfold, respectively. Some other pesticides or analogs are more potent sensitizers for succinylcholine toxicity with threshold levels of 0.5, 1.0, 1.7, 8, 10, and 67 mg/kg for phenyl saligenin cyclic phosphonate, profenofos, methamidophos, tribufos, chlorpyrifos, and ethephon, respectively. Enhanced mortality from succinylcholine is generally observed when serum butyrylcholinesterase is inhibited 55–94%. Mivacurium, a related nondepolarizing muscle relaxant also detoxified by butyrylcholinesterase, is likewise potentiated by at least threefold on 4 hour pretreatment with tribufos (25 mg/kg) or profenofos (10 mg/kg). © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 113–118, 1999     

In Vivo Studies on Fast and Slow Muscle Fibers in Cat Extraocular Muscles

In anesthetized in vivo preparations, responses of two types of extraocular muscle fibers have been studied. The small, multiply innervated slow fibers have been shown to be capable of producing propagated impulses, and thus have been labeled slow multi-innervated twitch fibers. Fast and slow multi-innervated twitch fibers are distinguished by impulse conduction velocities, by ranges of membrane potentials, by amplitudes and frequencies of the miniature end plate potentials, by responses to the intravenous administration of succinylcholine, by the frequency of stimulation required for fused tetanus, and by the velocities of conduction of the nerve fibers innervating each of the muscle fiber types.     

Anaesthetic-induced Malignant Hyperpyrexia: A Suggested Method of Treatment

Experiments in susceptible Landrace pigs have shown that procaine blocks the initiation of anaesthetic-induced malignant hyperpyrexia by both halothane and succinylcholine. Pretreatment with curare prevents only the trigger action of succinylcholine. In a preliminary study procaine was used to treat the established syndrome in five pigs, two of which survived. On the basis of these findings a treatment regimen can be suggested for patients who develop malignant hyperpyrexia.     

Ethnic differences in the frequency of distribution of serum cholinesterase activity in the Iranian population

One thousand Iranians belonging to 5 different Iranian ethnic groups were tested for butyrylcholinesterase (BChE) activity and phenotype. The phenotype was measured as percent inhibition in the presence of dibucaine. It was found that the Iranian population had an extraordinarily high frequency of the atypical variant of butyrylcholinesterase. 70% to 80% of Iranians carried the atypical mutation (Asp70Gly) on one allele. This contrasts with European and American populations where only 4% carry the atypical allele. The atypical variant of butyrylcholinesterase is known to be associated with prolonged apnea after administration of the muscle relaxants succinylcholine and mivacurium, and is also thought to be associated with abnormal sensitivity to cocaine toxicity. This study demonstrates that the ethnic background of a person has an important role in a person's response to drugs.     

Rapid determination of succinylcholine in human plasma by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method with fluorometric detection has been developed for the determination of succinylcholine in human plasma. Succinylcholine shows fluorescence at 282 nm with an excitation at 257 nm. The assay is sensitive, reproducible and linear for concentrations ranging from 100 ng/ml to 100 μg/ml of succinylcholine. In a pilot study the plasma concentration—time curve showed a triphasic elimination, with half-lives of 0.4, 1.2 and 8 min, respectively. In a clinical setting, drugs commonly administered during anaesthesia did not interfere with the assay. This method provides a simple and time-saving alternative to existing methods.     

Spectroscopic determination of succinylcholine in dosage forms using eosin Y

Two simple and sensitive analytical assay methods using spectrophotometry and spectrofluorimetry techniques were developed for the estimation of succinylcholine chloride (SUC) in pharmaceutical preparations. The suggested methods are based on the formation of an ion pair complex formed between the drug and eosin Y spectrophotometrically (Method I), or the suppressive effect of succinylcholine on the native fluorescence property of eosin Y (Method II). The spectrophotometric method (Method I) involves measuring the absorbance of the complex between succinylcholine and eosin Y at 550 nm in Britton Robinson buffer of pH 3. However, the spectrofluorimetric method (Method II) involves measuring the quenching effect of the studied drug on the native fluorescence property of eosin Y at the same pH at 550 nm after excitation at 480 nm. The absorbance versus concentration of the drug is rectilinear over the range of 0.5 to 15 μg/ml. The formation constant was 3.5 × 10⁴ and the Gibb's free energy change was ?2.5 × 10⁴ J/mol. In Method II, the relative fluorescence intensity was directly proportional to SUC concentration over the range of 0.05 to 1 μg/ml. The proposed methods allowed a successful application to the estimation of succinylcholine ampoules. An explanation of the reaction pathway was postulated.     

Decreased Serum Choline Concentrations in Humans after Surgery,Childbirth, and Traumatic Head Injury

The serum levels of choline decreased by approximately 50% in patients having a surgery under general as well as epidural anesthesia. The decrease is lasts for two days after surgery. Intravenous administration of succinylcholine, either by a single bolus injection or by a slow continuous infusion, increased the serum choline levels several folds during surgery. In these patients, a significant decrease in the serum choline levels was observed one and two days after surgery. In 16 pregnant women at the term, serum choline levels were higher than the value observed in 19 non-pregnant women. The serum choline levels decreased by about 40% or 60% after having a child-birth either by vaginal delivery or caesarean section, respectively. Serum choline levels in blood obtained from 9 patients with traumatic head injury were significantly lower than the observed levels in blood samples obtained from healthy volunteers. These observations show that serum choline levels increase during pregnancy and decrease during stressful situations in humans.     

Chemical Restraint of Exotic Animals in a Disease Regulatory Program

The African red tick (Rhipicephalus evertsi Neuman) is widely distributed in Africa but had never been identified in North America until September 1960 when it was found in a wild animal compound in Florida. The red tick is a vector of cattle fever (piroplasmosis) and several other exotic diseases including the highly fatal East Coast fever. The 130 acre compound contained more than 300 African and Asian animals, including giraffes, camels, zebras, elands, nilghais, blackbuck, aoudads, ostriches and Abyssinian asses. To determine the extent of the infestation, representative numbers of animals were immobilized with succinylcholine chloride for visual and manual examination. The grounds were treated with 2 lbs. of DDT per acre at three week intervals for six weeks and another group of animals were immobilized to determine the efficacy of the program. In all more than 50 wild animals were successfully immobilized with succinylcholine.     

A note on suxamethonium sensitivity and serum cholinesterase variants

Summary Sera from 21 cases of prolonged apnoea which showed normal phenotype (UU) on the basis of dibucaine and fluoride inhibition were re-examined by replacing the substrate benzoylcholine with succinylcholine (suxamethonium). 9 samples had normal enzyme activity but low dibucaine number (DN=<20) indicating the atypical variant; 6 sera showed no detectable enzyme activity. The remaining 6 samples had enzyme activity and DN comparable with healthy controls. The occurence of new variants of serum cholinesterase sensitive only to succinylcholine is suggested.Dedicated to Prof. Dr. med. J. Kühnau on his 75th birthday.     

Cholinesterase Newfoundland: a new succinylcholine-sensitive variant of cholinesterase at locus 1.

A family from Newfoundland was found to have a new rare variant for plasma cholinesterase (E.C.3.1.1.8) recognized by a high-percentage inhibition by dibucaine (DN), particularly when succinyldithiocholine was used as substrate (DNSDTC) but also somewhat high when benzoylcholine was substrate (DNBZCH). The family data demonstrated that the variant is determined by an allele of the usual and atypical alleles at locus 1, and the new allele is designated CHE1*NFLD. The proband who was heterozygous for the Newfoundland and atypical alleles had shown sensitivity to succinylcholine. It is postulated that cholinesterase Newfoundland (NFLD) has a reduced affinity for succinylcholine. Samples selected for high DNs with a benzoylcholine from 200 Canadian Caucasians and 70 Newfoundlanders did not have the variant, and, therefore, it is assumed that the remainder of the samples did not have the variant.     

Biochemical Basis of Malignant Hyperpyrexia

Pharmacologically-induced muscle contracture in vitro has been used as a model to study the biochemical basis of malignant hyperpyrexia. In 15 susceptible subjects halothane, succinylcholine, and potassium chloride all produced an abnormal muscle contracture, and the caffeine-induced contracture was greater than normal. The contractures were reproducible only in the presence of extracellular calcium ions. The fact that such dissimilar pharmacological stimuli all induced contracture in the affected muscle suggests that the essential abnormality in the muscle cell in malignant hyperpyrexia is an impaired binding of calcium ions to the membranes of the sarcoplasmic reticulum and the sarcolemma. Exposure of these membranes to halothane, succinylcholine, and other anaesthetic agents then leads to a rapid and abnormally large release of calcium into the myoplasm, which in turn gives rise to all the clinical features of the syndrome.     

New developments in nondepolarizing muscle relaxants.

Anesthesiologists perceive that the ideal muscle relaxant is not yet available, particularly the nondepolarizing one with a rapid onset and a short duration of action. There is also a need for relaxants with different durations of action but which would be free from side effects. During the process of this development several new compounds have been tested and four have reached an advanced state of study; three of these, doxacurium, pipecuronium, and mivacurium are already licensed and rocuronium is likely to be licensed in the near future. Doxacurium and pipecuronium are slow onset and long duration of action compounds but singularly free from cardiovascular side effects. Mivacurium has an onset comparable to that of atracurium and vecuronium but with a duration of action which is intermediate in duration between these drugs and succinylcholine. Rocuronium is a drug with a fast onset of action capable of being used in place of succinylcholine but with a duration of action which is similar to that of vecuronium.     

FIELD IMMOBILIZATION OF YOUNG WILDEBEEST WITH SUCCINYLCHOLINE CHLORIDE

The results of 112 cases of field immobilization of wildebeest, Gorgon taurinus hecki (Neumann), * with succinylcholine chloride are given, and compared with the results given by Talbot and Lamprey (1961), who reported field dose rates of 0.023 mg/lb. body weight as ineffective, 0.028–0.029 mg/lb. as effective, and 0.036 mg/lb. as lethal, when given to adult wildebeest. Doses in the present series were determined empirically in the field and it was found that the youngest age group of wildebeest tried with the drug (49 cases, 6–12 months old) survived 'lethal' doses of 0.036–0.037 mg/lb. Field experience suggested that not only were young wildebeest capable of surviving greater doses of succinylcholine than adults, but there was an absence of secondary complications. Adult animals suffered from regurgitation of ruminal contents and difficulty with breathing.
In the field it proved easier to produce consistent results with young wildebeest, and it is suggested that in testing the possible use of succinylcholine for immobilizing other game animals the effect of the drug on both young and old animals should be compared.
Use of an effective marking method, by branding, followed by field recording of marked animals has shown that six animals out of a total of 87 disappeared shortly after release. The casualty rate associated with the field marking is given.     

Effects of hydrocortisone on electrical activity of the cat spinal cord

The effect of the corticosteroid hormone hydrocortisone on electrical activity in the lumbosacral portion of the spinal cord was studied in acute experiments on cats anesthetized with urethane and chloralose and immobilized with succinylcholine. The amplitude of mono- and polysynaptic discharges arising in the ventral roots in response to stimulation of various afferents of the animal's hind limb was increased by a statistically significant degree after intravenous injection of the hormone. The potentiating action of the hormone was strongest and most stable with respect to early and late postsynaptic potentials of the spinal cord. The dorsal cord potentials were not significantly changed by hydrocortisone. Spontaneous unit activity in the intermediate nucleus of the spinal cord rose sharply after administration of hydrocortisone. Before the action of the hormone the mean frequency of spontaneous discharges of 46 neurons was 7.91/sec, rising to 20/sec after the injection. The number of neurons with a high spontaneous firing rate also was increased. Prolonged extracellular recording of the spontaneous activity of the same neuron before and after administration of hydrocortisone also revealed a marked increase in the frequency of its discharges. The results are evidence of the activating effect of hydrocortisone on spinal interneuronal activity.     

Interaction of Diazepam with the Muscle-relaxant Drugs

Diazepam has been found to increase the duration of the neuromuscular block produced by gallamine and to reduce that produced by succinylcholine. Preliminary studies suggest that the action is at the presynaptic site.     

The interaction of cyproheptadine with agents affecting neuromuscular transmission.

Using the rat phrenic nerve diaphragm, cyproheptadine at concentrations of 1 to 8 mug/ml did not affect or slightly augmented indirect muscle twitches, but potentiated blockade by tubocurarine, decamethonium and succinylcholine, and antagonized the augmentation of twitches by neostigmine. Ketamine, choline and tetraethylammonium at concentrations causing no blockade produced, when given after cyproheptadine (6 mug/ml), a high degree of blockade. At concentrations of 9 to 20 mug/ml, cyproheptadine induced neuromuscular blockade which was slow in onset, more apparent at higher rate of stimulation and was not reversed by neostigmine, choline or tetraethylammonium. In the cat tibialis anterior muscle, it potentiated blockade by tubocurarine, decamethonium and succinylcholine, and blocked acetylcholine twitches. In the chick biventer cervicis muscle, the durg was more effective in blocking indirect twitches than responses to carbachol.