In-Vitro Neutralization of the Neurotoxicity of Coastal Taipan Venom by Australian Polyvalent Antivenom: The Window of Opportunity.

Coastal taipan (Oxyuranus scutellatus) envenoming causes life-threatening neuromuscular paralysis in humans. We studied the time period during which antivenom remains effective in preventing and arresting in vitro neuromuscular block caused by taipan venom and taipoxin. Venom showed predominant pre-synaptic neurotoxicity at 3 µg/mL and post-synaptic neurotoxicity at 10 µg/mL. Pre-synaptic neurotoxicity was prevented by addition of Australian polyvalent antivenom before the venom and taipoxin and, reversed when antivenom was added 5 min after venom and taipoxin. Antivenom only partially reversed the neurotoxicity when added 15 min after venom and had no significant effect when added 30 min after venom. In contrast, post-synaptic activity was fully reversed when antivenom was added 30 min after venom. The effect of antivenom on pre-synaptic neuromuscular block was reproduced by washing the bath at similar time intervals for 3 µg/mL, but not for 10 µg/mL. We found an approximate 10-15 min time window in which antivenom can prevent pre-synaptic neuromuscular block. This time window is likely to be longer in envenomed patients due to the delay in venom absorption. Similar effectiveness of antivenom and washing with 3 µg/mL venom suggests that antivenom most likely acts by neutralizing pre-synaptic toxins before they interfere with neurotransmission inside the motor nerve terminals.

Preclinical Pharmacology in the Rhesus Monkey of CW 1759-50, a New Ultra-short Acting Nondepolarizing Neuromuscular Blocking Agent, Degraded and Antagonized by L-Cysteine.

WHAT WE ALREADY KNOW ABOUT THIS TOPIC: WHAT THIS ARTICLE TELLS US THAT IS NEW: BACKGROUND:: Structure-activity studies were performed to identify a new neuromuscular blocking agent retaining the ultra-short acting characteristics of gantacurium, including degradation and reversal by L-cysteine, but lacking its histaminoid properties in man. CW 1759-50 has emerged from this program. METHODS: Adduction of CW 1759-50 with L-cysteine was studied by high-performance liquid chromatography and mass spectrometry. Institutional Animal Care and Use Committee-approved comparisons of CW 1759-50 to gantacurium were performed in rhesus monkeys. ED95 for neuromuscular blockade was established. Spontaneous recovery was compared to reversal by L-cysteine in paired studies of boluses or infusions. In addition, changes in mean arterial pressure and heart rate after very large doses of 15 to 60 × ED95 were compared. RESULTS: The half-time of adduction of L-cysteine to CW 1759-50 in vitro was 2.3 min. The ED95 of CW 1759-50 was 0.069 ± 0.02 mg/kg; ED95 of gantacurium was 0.081 ± 0.05 mg/kg (P = 0.006). Duration of action (recovery to 95% twitch height after 98 to 99% blockade) was as follows: CW 1759-50, 8.2 ± 1.5 min; and gantacurium, 7.4 ± 1.9 min; (n = 8 and 9, P = 0.355). Administration of L-cysteine (30 mg/kg) shortened recovery (i.e., induced reversal) from CW 1759-50 after boluses or infusions (P always less than 0.0001). Recovery intervals (5 to 95% twitch) ranged from 6.1 to 6.7 min (and did not differ significantly) after boluses of 0.10 to 0.50 mg/kg, as well as control infusions (P = 0.426 by analysis of variance). Dose ratios comparing changes of 30% in mean arterial pressure or heart rate to ED95 for neuromuscular blockade (ED 30% Δ [mean arterial pressure or heart rate]/ED95) were higher for CW 1759-50 than for gantacurium. CONCLUSIONS: CW 1759-50, similar to gantacurium, is an ultra-short acting neuromuscular blocking agent, antagonized by L-cysteine, in the monkey. The circulatory effects, however, are much reduced in comparison with gantacurium, suggesting a trial in humans.

Toxic activity and protein identification from the parotoid gland secretion of the common toad Bufo bufo.

Anuran toxins released from the skin glands are involved in defence against predators and microorganisms. Secretion from parotoid macroglands of bufonid toads is a rich source of bioactive compounds with the cytotoxic, cardiotoxic and hemolytic activity. Bufadienolides are considered the most toxic components of the toad poison, whereas the protein properties are largely unknown. In the present work, we analysed the cardio-, myo-, and neurotropic activity of extract and the selected proteins from Bufo bufo parotoids in in vitro physiological bioassays carried out on two standard model organisms: beetles and frogs. Our results demonstrate a strong cardioactivity of B. bufo gland extract. The toad poison stimulates (by 16%) the contractility of the insect heart and displays the cardioinhibitory effect on the frog heartbeat frequency (a 27% decrease), coupled with an irreversible cardiac arrest. The gland extract also exhibits significant myotropic properties (a 10% decrease in the muscle contraction force), whereas its neuroactivity remains low (a 4% decrease in the nerve conduction velocity). Among identified peptides present in the B. bufo parotoid extract are serine proteases, muscle creatine kinase, phospholipid hydroperoxide glutathione peroxidase, cytotoxic T-lymphocyte protein, etc. Some proteins contribute to the cardioinhibitory effect. Certain compounds display the paralytic (myo- and neurotropic) properties. As the toad gland extract exhibits a strong cardiotoxic activity, we conclude that the poison is a potent agent capable of slaying a predator. Our results also provide the guides for the use of toad poison-peptides in therapeutics and new drug development.

Rattling the border wall: Pathophysiological implications of functional and proteomic venom variation between Mexican and US subspecies of the desert rattlesnake Crotalus scutulatus.

While some US populations of the Mohave rattlesnake (Crotalus scutulatus scutulatus) are infamous for being potently neurotoxic, the Mexican subspecies C. s. salvini (Huamantlan rattlesnake) has been largely unstudied beyond crude lethality testing upon mice. In this study we show that at least some populations of this snake are as potently neurotoxic as its northern cousin. Testing of the Mexican antivenom Antivipmyn showed a complete lack of neutralisation for the neurotoxic effects of C. s. salvini venom, while the neurotoxic effects of the US subspecies C. s. scutulatus were time-delayed but ultimately not eliminated. These results document unrecognised potent neurological effects of a Mexican snake and highlight the medical importance of this subspecies, a finding augmented by the ineffectiveness of the Antivipmyn antivenom. These results also influence our understanding of the venom evolution of Crotalus scutulatus, suggesting that neurotoxicity is the ancestral feature of this species, with the US populations which lack neurotoxicity being derived states.

Pharmacogenomics in Anesthesia.

A significant number of commonly administered medications in anesthesia show wide clinical interpatient variability. Some of these include neuromuscular blockers, opioids, local anesthetics, and inhalation anesthetics. Individual genetic makeup may account for and predict cardiovascular outcomes after cardiac surgery. These interactions can manifest at any point in the perioperative period and may also only affect a specific system. A better understanding of pharmacogenomics will allow for more individually tailored anesthetics and may ultimately lead to better outcomes, decreased hospital stays, and improved patient satisfaction.

Thiadiazolodiazepine analogues as a new class of neuromuscular blocking agents: Synthesis, biological evaluation and molecular modeling study.

The synthesis, biological evaluation and molecular modeling study of 6,7-dihydro-[1,3,4] thiadiazolo[3,2-a][1,3]diazepine analogues as new class of neuromuscular blocking agents are described. The new compounds act via competitive mechanism with ACh which could be reversed by the anticholinesterase - Physostigmine. Compounds GS-53 (30) and AAH1 (33) induced dose-dependent neuromuscular blockade with onset time of 3 and 10 min, ED50 0.15 and 0.36 mmol/kg i.p., respectively, in rats. Compound 30 proved to be as twice as potent as 33 with rapid onset and shorter duration (P < 0.05). Docking profile of 30 and 33 closely resembles HIE-124 (3), in α7ß2 nAChR receptor. Molecular modeling analysis indicated that hydrogen bonding to Thr120 and Thr124 beside hydrophobic interactions play effective role incorporating the active ligands to nAChR. The obtained model could be useful for further development of new skeletal muscle relaxants.

Sugammadex: cyclodextrins, development of selective binding agents, pharmacology, clinical development, and future directions.

Neuromuscular blocking agents are widely used in perioperative medicine to aid in endotracheal intubation, facilitate surgery, and in critical care/emergency medicine settings. Muscle relaxants have profound clinical uses in current surgical and intensive care and emergency medical therapy. This article reviews cyclodextrins, development of selective binding agents, clinical development, and future directions of sugammadex.

Neuromuscular action of venom from the South American colubrid snake Philodryas patagoniensis.

Snakes of the opisthoglyphous genus Philodryas are widespread in South America and cause most bites by colubrids in this region. In this study, we examined the neurotoxic and myotoxic effects of venom from Philodryas patagoniensis in biventer cervicis and phrenic nerve-diaphragm preparations and we compared the biochemical activities of venoms from P. patagoniensis and Philodryas olfersii. Philodryas patagoniensis venom (40 microg/mL) had no effect on mouse phrenic nerve-diaphragm preparations but caused time-dependent neuromuscular blockade of chick biventer cervicis preparations. This blockade was not reversed by washing. The highest concentration of venom tested (40 microg/mL) significantly reduced (p<0.05) the contractures to exogenous acetylcholine (55 microM and 110 microM) and K(+) (13.4 mM) after 120 min; lower concentrations of venom had no consistent or significant effect on these responses. Venom caused a concentration- and time-dependent release of creatine kinase (CK) from biventer cervicis preparations. Histological analysis showed contracted muscle fibers at low venom concentrations and myonecrosis at high concentrations. Philodryas venoms had low esterase and phospholipase A(2) but high proteolytic activities compared to the pitviper Bothrops jararaca. SDS-PAGE showed that the Philodryas venoms had similar electrophoretic profiles, with most proteins having a molecular mass of 25-80 kDa. Both of the Philodryas venoms cross-reacted with bothropic antivenom in ELISA, indicating the presence of proteins immunologically related to Bothrops venoms. RP-HPLC of P. patagoniensis venom yielded four major peaks, each of which contained several proteins, as shown by SDS-PAGE. These results indicate that P. patagoniensis venom has neurotoxic and myotoxic components that may contribute to the effects of envenoming by this species.

Effects of hypothermia on drug disposition, metabolism, and response: A focus of hypothermia-mediated alterations on the cytochrome P450 enzyme system.

OBJECTIVES: Therapeutic hypothermia has been shown to decrease neurologic damage in patients experiencing out-of-hospital cardiac arrest. In addition to being treated with hypothermia, critically ill patients are treated with an extensive pharmacotherapeutic regimen. The effects of hypothermia on drug disposition increase the probability for unanticipated toxicity, which could limit its putative benefit. This review examines the effects of therapeutic hypothermia on the disposition, metabolism, and response of drugs commonly used in the intensive care unit, with a focus on the cytochrome P450 enzyme system. DATA SOURCES AND STUDY SELECTION: A MEDLINE/PubMed search from 1965 to June 2006 was conducted using the search terms hypothermia, drug metabolism, P450, critical care, cardiac arrest, traumatic brain injury, and pharmacokinetics. DATA EXTRACTION AND SYNTHESIS: Twenty-one studies were included in this review. The effects of therapeutic hypothermia on drug disposition include both the effects during cooling and the effects after rewarming on drug metabolism and response. The studies cited in this review demonstrate that the addition of mild to moderate hypothermia decreases the systemic clearance of cytochrome P450 metabolized drugs between approximately 7% and 22% per degree Celsius below 37degreesC during cooling. The addition of hypothermia decreases the potency and efficacy of certain drugs. CONCLUSIONS: This review provides evidence that the therapeutic index of drugs is narrowed during hypothermia. The magnitude of these alterations indicates that intensivists must be aware of these alterations in order to maximize the therapeutic efficacy of this modality. In addition to increased clinical attention, future research efforts are essential to delineate precise dosing guidelines and mechanisms of the effect of hypothermia on drug disposition and response.

Glycogen synthase kinase 3 activation is essential for the snake phospholipase A2 neurotoxin-induced secretion in chromaffin cells.

Neuroendocrine chromaffin cells were used to study the mechanism of the snake phospholipase A2 (PLA2) neurotoxin enhancement of exocytosis. Notexin, beta-bungarotoxin, taipoxin or textilotoxin enhanced the fast release of catecholamines elicited by flash photolysis of cytosolic caged calcium. Such an increase correlates with the capacity of these neurotoxins to cause fragmentation of the F-actin cortical barrier with subsequent accumulation of vesicles in the proximity of the plasma membrane. These PLA2 neurotoxins do not act via protein kinase C activation, which is known to promote F-actin fragmentation. Lithium, RO31-8220 and SB216763, three inhibitors of the glycogen synthase kinase 3, prevent both the alteration of the F-actin peripheral cortex and the enhancement of fast release elicited by these neurotoxins. In addition, glycogen synthase kinase 3 has been detected by immunolocalization in a membranous compartment of the chromaffin cell endoplasmic reticulum (ER). These results suggest that the activation of this enzyme plays a major role in the enhancement of exocytosis of the readily releasable granules caused by PLA2 neurotoxins in neuroendocrine chromaffin cells.

The role of the amino acid residue at alpha1:189 in the binding of neuromuscular blocking agents to mouse and human muscle nicotinic acetylcholine receptors.

BACKGROUND AND PURPOSE: Nicotinic acetylcholine receptors (AChRs) are valuable therapeutic targets. To exploit them fully requires rapid assays for the evaluation of potentially therapeutic ligands and improved understanding of the interaction of such ligands with their receptor binding sites. EXPERIMENTAL APPROACH: A variety of neuromuscular blocking agents (NMBAs) were tested for their ability to inhibit the binding of [(125)I]alpha-bungarotoxin to TE671 cells expressing human muscle AChRs. Association and dissociation rate constants for vecuronium inhibition of functional agonist responses were then estimated by electrophysiological studies on mouse muscle AChRs expressed in Xenopus oocytes containing either wild type or mutant alpha1 subunits. KEY RESULTS: The TE671 inhibition binding assay allowed for the rapid detection of competitive nicotinic AChR ligands and the relative IC(50) results obtained for NMBAs agreed well with clinical data. Electrophysiological studies revealed that acetylcholine EC(50) values of muscle AChRs were not substantially altered by non-conservative mutagenesis of phenylalanine at alpha1:189 and proline at alpha1:194 to serine. However the alpha1:Phe189Ser mutation did result in a 3-4 fold increase in the rate of dissociation of vecuronium from mouse muscle AChRs. CONCLUSIONS AND IMPLICATIONS: The TE671 binding assay is a useful tool for the evaluation of potential therapeutic agents. The alpha1:Phe189Ser substitution, but not alpha1:Pro194Ser, significantly increases the rate of dissociation of vecuronium from mouse muscle AChRs. In contrast, these non-conservative mutations had little effect on EC(50) values. This suggests that the AChR agonist binding site has a robust functional architecture, possibly as a result of evolutionary 'reinforcement'.

Pharmacogenetics of anesthetic and analgesic agents.

Predicting a patient's response to a particular drug has long been a goal of clinicians. Rapid advances in molecular biology have enabled researchers to identify associations between an individual's genetic profile and drug response. Pharmacogenetics is the study of the molecular mechanisms that underlie individual differences in drug metabolism, efficacy, and side effects. The pharmacogenetics of commonly used anesthetic and analgesic agents are reviewed.

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.

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.

The role of zinc binding in the biological activity of botulinum toxin.

Botulinum toxin is a zinc-dependent endoprotease that acts on vulnerable cells to cleave polypeptides that are essential for exocytosis. To exert this poisoning effect, the toxin must proceed through a complex sequence of events that involves binding, productive internalization, and intracellular expression of catalytic activity. Results presented in this study show that soluble chelators rapidly strip Zn(2+) from its binding site in botulinum toxin, and this stripping of cation results in the loss of catalytic activity in cell-free or broken cell preparations. Stripped toxin is still active against intact neuromuscular junctions, presumably because internalized toxin binds cytosolic Zn(2+). In contrast to soluble chelators, immobilized chelators have no effect on bound Zn(2+), nor do they alter toxin activity. The latter finding is because of the fact that the spontaneous loss of Zn(2+) from its coordination site in botulinum toxin is relatively slow. When exogenous Zn(2+) is added to toxin that has been stripped by soluble chelators, the molecule rebinds cation and regains catalytic and neuromuscular blocking activity. Exogenous Zn(2+) can restore toxin activity either when the toxin is free in solution on the cell exterior or when it has been internalized and is in the cytosol. The fact that stripped toxin can reach the cytosol means that the loss of bound Zn(2+) does not produce conformational changes that block internalization. Similarly, the fact that stripped toxin in the cytosol can be reactivated by ambient Zn(2+) or exogenous Zn(2+) means that productive internalization does not produce conformational changes that block rebinding of cation.

Neuromuscular blockers in surgery and intensive care, Part 2.

The historical development, pharmacology, pharmacodynamics, pharmacokinetics, clinical applications, pharmacologic basis for selection, adverse effects, and cost of neuromuscular blockers (NMBs) are discussed. The first NMB to be used was tubocurarine. During neurotransmission, acetylcholine is synthesized, stored in vesicles at the neuromuscular junction, released into the synapse, and bound to nicotinic receptors in the muscle end plate. For muscle contraction to occur, the impulse generated in a neuron's cell body must create an action potential that is chemically transmitted across the synapse. The postsynaptic nicotinic receptor at the neuromuscular junction is the major site of action of depolarizing and nondepolarizing NMBs. All NMBs have the potential for cross-reactivity at other nicotinic and muscarinic sites. Drug interactions most commonly occur between NMBs and inhalation anesthetics, certain antimicrobials, calcium-channel blockers, and anticholinesterases. When selecting an NMB, an agent's onset and duration of action must be considered. NMBs can be used on a short-term or long-term basis. Apart from cost, the choice of an NMB is made on the basis of its adverse-reaction profile, pharmacokinetics, and indications for use. Monitoring tools, their use, the rationale for their use, and the interpretation of the results they provide are unique. The patterns of peripheral nerve stimulation vary and elicit different characteristics of nondepolarizing neuromuscular blockade. The effectiveness of reversal agents is proportional to the degree of blockade. The mechanism of action of anticholinesterases involves inhibition of acetylcholinesterase. The expensive NMBs should be conserved for use in surgery, while the cheaper, long-acting [corrected] agents should be used in the intensive care unit. An understanding of the pharmacology, pharmacodynamics, and pharmacokinetics of NMBs will help health care providers gain expertise in the selection and use of these agents.

C-terminal half of tetanus toxin fragment C is sufficient for neuronal binding and interaction with a putative protein receptor.

Tetanus neurotoxin (TeNT) is a powerful bacterial protein toxin that cleaves VAMP/synaptobrevin, an essential protein of the synaptic vesicle fusion machinery, and consequently blocks neurotransmission. The extreme neurospecificity of TeNT is determined by the binding of its C-terminal domain (fragment C or H(C)) to neuronal receptors. Whereas polysialogangliosides are known acceptors for the toxin, the existence of additional protein receptors has also been suggested. We have reported previously on a 15 kDa cell-surface glycoprotein that interacts with TeNT in neuronal cell lines and motoneurons [Herreros, Lalli, Montecucco and Schiavo (2000) J. Neurochem., in the press]. Here, on the basis of the structural information provided by the crystallization of fragment C of TeNT, we have expressed its C-and N-terminal halves as recombinant proteins and analysed their binding abilities to rat phaeochromocytoma (PC12) cells differentiated with nerve growth factor. We found that the C-terminal subdomain of the fragment C of TeNT is necessary and sufficient for cell binding and for the interaction with the 15 kDa putative receptor. In contrast, the N-terminal half showed a very poor interaction with the cell surface. These results restrict the binding domain of TeNT to the C-terminal half of the fragment C and highlight the importance of this domain for the neurospecific interaction of the toxin with the synapse. Furthermore, these findings support the use of this portion of TeNT as a neurospecific targeting device, pointing to an involvement of the N-terminal subdomain in later steps of the intoxication pathway.

Recent advances in neuromuscular blocking agents.

Factors driving the development of neuromuscular blocking agents are discussed. The goal of recent development of neuromuscular blocking agents is to develop agents with fewer adverse effects than succinylcholine and greater control. Greater control can be achieved through a short duration of action and a fast onset, similar to that found with succinylcholine. Duration control can be achieved through rapid, reliable metabolism that is organ independent, as with cisatracurium, or that occurs in the liver, because this will fail only in patients with severe hepatic disease. Rapid onset can be achieved by giving higher doses of drugs that have a fast, reliable metabolism or a rapid distribution to tissue. This has been done with succinylcholine, cisatracurium, and mivacurium. It has been shown that a low-potency drug has a faster onset than a higher-potency drug, even at the same relative dose. Rocuronium was the first neuromuscular blocking agent marketed that was developed specifically as a low-potency drug for achieving a rapid onset. In a comparison with two other intermediate-duration neuromuscular blocking agents at equipotent doses, rocuronium's onset was about one minute, which was two minutes faster than that of either vecuronium or atracurium. Rapacuronium is an investigational intermediate-duration agent and is even less potent than rocuronium. Clinical studies have shown it to have a very rapid onset (about 50 seconds) and a short duration of action (15-25 minutes, depending on the dose). Its short duration of action will make it potentially useful for short surgical procedures and procedures in which nerve integrity must be monitored. At the same time, its onset time approaches that of succinylcholine. Recent advances have made the administration of neuromuscular blocking agents much easier because the newer nondepolarizing relaxants offer faster onset and greater control.