Evaluation of oxime efficacy in nerve agent poisoning: development of a kinetic-based dynamic model

The widespread use of organophosphorus compounds (OP) as pesticides and the repeated misuse of highly toxic OP as chemical warfare agents (nerve agents) emphasize the necessity for the development of effective medical countermeasures. Standard treatment with atropine and the established acetylcholinesterase (AChE) reactivators, obidoxime and pralidoxime, is considered to be ineffective with certain nerve agents due to low oxime effectiveness. From obvious ethical reasons only animal experiments can be used to evaluate new oximes as nerve agent antidotes. However, the extrapolation of data from animal to humans is hampered by marked species differences. Since reactivation of OP-inhibited AChE is considered to be the main mechanism of action of oximes, human erythrocyte AChE can be exploited to test the efficacy of new oximes. By combining enzyme kinetics (inhibition, reactivation, aging) with OP toxicokinetics and oxime pharmacokinetics a dynamic in vitro model was developed which allows the calculation of AChE activities at different scenarios. This model was validated with data from pesticide-poisoned patients and simulations were performed for intravenous and percutaneous nerve agent exposure and intramuscular oxime treatment using published data. The model presented may serve as a tool for defining effective oxime concentrations and for optimizing oxime treatment. In addition, this model can be useful for the development of meaningful therapeutic animal models.     

An in vitro nerve agent brain poisoning transwell model for convenient and accurate antidote evaluation

Nerve agent (NA) can inhibit acetylcholinesterase (AChE) causing seriously injury at extremely low doses. However, the cruel reality is that the lack of effective cerebral antidotes for treatment of NA poisoning. There is an urgent requirement for the large-scale evaluation and screening of antidotes. An effective NA antidote should include two characteristics: a) to permeate the blood–brain barrier (BBB); 2) to reactivate the inhibited AChE in brain. Existing methods for evaluating reactivators in vitro can only examine the reactivation effect, while the current Transwell model can only evaluate the drug penetration performance for crossing the barrier. In this work, brain microvascular endothelial cells (RBMECs) were inoculated to establish a Transwell model. AChE, NAs and antidotes of reactivators were added into the different chambers to simulate central poisoning and peripheral drug administration. This method can evaluate the reactivation ability and brain penetration ability of compounds at same time, which is a rapidly and accurately way for drug preliminary screening. In addition to small-molecule drugs, a liposomal nanoantidote loaded with the reactivator Asoxime chloride (HI-6)was prepared. This nanoantidote show high reactivation rate against the NA (sarin), evaluated by both this modified model in vitro and animal test, gaining the consistence results.     

A structure-activity analysis of the variation in oxime efficacy against nerve agents

A structure-activity analysis was used to evaluate the variation in oxime efficacy of 2-PAM, obidoxime, HI-6 and ICD585 against nerve agents. In vivo oxime protection and in vitro oxime reactivation were used as indicators of oxime efficacy against VX, sarin, VR and cyclosarin. Analysis of in vivo oxime protection was conducted with oxime protective ratios (PR) from guinea pigs receiving oxime and atropine therapy after sc administration of nerve agent. Analysis of in vitro reactivation was conducted with second-order rate contants (k(r2)) for oxime reactivation of agent-inhibited acetylcholinesterase (AChE) from guinea pig erythrocytes. In vivo oxime PR and in vitro k(r2) decreased as the volume of the alkylmethylphosphonate moiety of nerve agents increased from VX to cyclosarin. This effect was greater with 2-PAM and obidoxime (>14-fold decrease in PR) than with HI-6 and ICD585 (<3.7-fold decrease in PR). The decrease in oxime PR and k(r2) as the volume of the agent moiety conjugated to AChE increased was consistent with a steric hindrance mechanism. Linear regression of log (PR-1) against log (k(r2)[oxime dose]) produced two offset parallel regression lines that delineated a significant difference between the coupling of oxime reactivation and oxime protection for HI-6 and ICD585 compared to 2-PAM and obidoxime. HI-6 and ICD585 appeared to be 6.8-fold more effective than 2-PAM and obidoxime at coupling oxime reactivation to oxime protection, which suggested that the isonicotinamide group that is common to both of these oximes, but absent from 2-PAM and obidoxime, is important for oxime efficacy.     

Development of the bisquaternary oxime HI-6 toward clinical use in the treatment of organophosphate nerve agent poisoning

The traditional therapeutic treatment of organophosphate cholinesterase inhibitor (nerve agents) poisoning consists of co-treatment with an antimuscarinic (atropine) and a reactivator of inhibited acetylcholinesterase (AChE), which contains a nucleophilic oxime function. Two oximes are presently widely available for clinical use, pralidoxime and obidoxime (toxogonin), but both offer little protection against important nerve agent threats. This has highlighted the real need for the development and availability of more effective oximes for human use, a search that has been going on for up to 30 years. However, despite the demonstration of more effective and safe oximes in animal experiments, no additional oximes have been licensed for human use. HI-6, (1-[[[4(aminocarbonyl)-pyridinio]methoxy]methyl]-2(hydroxyimino)pyridinium dichloride; CAS 34433-31-3) has been studied intensively and has been proved effective in a variety of species including non-human primates and appears from clinical experience to be safe in humans. These studies have led to the fielding of HI-6 for use against nerve agents by the militaries of the Czech republic, Sweden, Canada and under certain circumstances the Organisation for the Prohibition of Chemical Weapons. Nevertheless HI-6 has not been granted a license for clinical use, must be used only under restricted guidelines and is not available for civilian use as far as is known. This article will highlight those factors relating to HI-6 that pertain to the licensing of new compounds of this type, including the mechanism of action, the clinical and pre-clinical demonstration of safety and its efficacy against a variety of nerve agents particularly in non-human primates, since no relevant human population exists. This article also contains important data on the use of HI-6 in baboons, which has not been available previously. The article also discusses the possibility of successful therapy with HI-6 against poisoning in humans relative to doses used in non-human primates and relative to its ability to reactivate inhibited human AChE.     

Recent advances in oxazolidinone antibacterial agent research

The oxazolidinone class of antibacterial agents, as exemplified by linezolid (Zyvox^®, Pharmacia Corp.), represents the first completely new class of antibacterial agents to achieve regulatory approval in over 30 years. Due to their potent activity against multi-drug resistant Gram-positive bacteria, discovery research in this area has flourished, with many pharmaceutical companies worldwide beginning research programmes. With the expanded research efforts, new understanding of the oxazolidinone structure–activity relationship has led to an increased spectrum of activity to include the fastidious Gram-negative pathogens Haemophilus influenzae and Moraxella catarrhalis. In addition, a deeper understanding of the toxicology, mechanism of action, and mechanism of resistance has emerged.     

HI-6 in man: efficacy of the oxime in poisoning by organophosphorus insecticides.

The efficacy of the oxime HI-6 was studied as a treatment for organophosphorus poisoning. HI-6 was given four times daily as a single intramuscular injection of 500 mg accompanied by atropine and diazepam therapy. Oxime treatment was started on admission and continued for a minimum of 48 h and a maximum of 7 d. HI-6 rapidly reactivated human blood acetylcholinesterase inhibited by dimethoxy organophosphorus compounds, while the dimethoxy-inhibited enzyme was mainly resistant to the treatment by HI-6. Although both HI-6 and pralidoxime chloride reactivated the red blood cell cholinesterase in quinalphos-poisoned subjects, the return of enzyme activities was more rapid following the use of HI-6. The general improvement of poisoned patients, which was sometimes more rapid than the rise of acetylcholinesterase activity, pointed to direct pharmacological effects of HI-6. No undesirable side-effects were noted in patients when HI-6 plasma concentrations were maintained at levels far above the 'therapeutic' concentration for up to 7 d.     

Neuroparalysis and oxime efficacy in organophosphate poisoning: a study of butyrylcholinesterase

The temporal profile of butyrylcholinesterase (BuChE) and in vitro pralidoxime-reactivated BuChE was studied in a cohort of 25 organophosphate-poisoned patients to examine their relationship to the development of intermediate syndrome and to understand reasons for lack of efficacy of oxime treatment. The clinical severity of poisoning (assessed by the Namba Scale) correlated significantly with the severity of intermediate syndrome. BuChE activity increased significantly over time and showed significant relationship to muscle power. The temporal profile of the enzyme was correlated to the clinical severity of poisoning. Reactivation potentials of BuChE (the difference between oxime-reactivated and -unreactivated enzyme activity) declined significantly with time after organophosphate ingestion. The reactivation potential of the enzyme at admission decreased significantly with increasing severity of poisoning and was lower in patients who developed intermediate syndrome. Patients who received oxime prior to hospitalization had a higher rate of intermediate syndrome and lower levels of BuChE at admission than those who had not. The study suggests that (i) BuChE reflects the clinical course of poisoning, confirming earlier studies; (ii) intermediate syndrome may be associated with a persistent inhibition of BuChE; and (iii) the lack of oxime efficacy in our patients maybe due to their severity of poisoning and the timing of oxime treatment.     

Non-cholinergic intervention of sarin nerve agent poisoning

The protective effects of selected anesthetic regimens on sarin (GB) were investigated in domestic swine. At 30% oxygen, the toxicity of this agent in isoflurane anesthetized animals (LD(50)=10.1μg/kg) was similar to literature sited values in awake swine (LD(50)=11.8μg/kg) and slightly higher than that of both ketamine (LD(50)=15.6μg/kg) and propofol (LD(50)=15.3μg/kg) anesthetized swine. Use of 100% oxygen in ketamine anesthetized animals resulted in three-fold protective effects compared to 30% oxygen. Use of 100% oxygen in both isoflurane and propofol anesthetized animals, compared to 30% resulted in profound protection against GB poisoning (>33×). There were no differences in the severity of the poisoning or recovery time in animals treated over dose ranges of 10-350μg/kg (isoflurane) or 15-500μg/kg GB (propofol). Survivors of high GB challenges that were revived from propofol anesthetic exhibited no signs of cognitive impairment seven days later. Protective treatments did not attenuate cholinesterase (ChE) inhibition; survivors of otherwise supralethal GB concentrations exhibited very low blood ChE activities. This work indicates that propofol has protective effects against GB, and that oxygen tension may have an important role in treating nerve agent casualties. More importantly, it demonstrates that non-cholinergic protective mechanisms exist that may be exploited in the future development of medical countermeasures against organophosphorous nerve agents.     

靶向Ras蛋白类抗癌药物研究进展

大概1/3的人类癌症存在Ras原癌基因的突变,而Ras蛋白对于细胞的增殖分化等生理过程有着至关重要的作用,这使得Ras蛋白成为众人争相研究的对象。经过30多年的研究,发现Ras蛋白由于缺乏合适的药物分子结合口袋,难以成为有效的药物靶点蛋白,甚至认为"无药可成"。然而,近几年来结构生物学和生物信息学的不断发展,越来越多的证据表明Ras蛋白可以成为药物分子作用的靶点蛋白。本文综述了近几年来以Ras蛋白为靶点的抗癌药物研究进展,旨在为同类研究提供参考。     

Protection against nerve agent poisoning by a noncompetitive nicotinic antagonist

The acute toxicity of organophosphorus (OP) nerve agents arises from accumulation of acetylcholine (ACh) and overstimulation of ACh receptors. The mainstay of current pharmacotherapy is the competitive muscarinic antagonist, atropine. Nicotinic antagonists have not been used due to the difficulties of administering a dose of a competitive neuromuscular blocker sufficient to antagonise the effects of excessive ACh, but not so much that it paralyses the muscles. An alternative approach would be to use a noncompetitive antagonist whose effects would not be overcome by increasing ACh concentrations. This study demonstrates that the compound 1,1′-(propane-1,3-diyl)bis(4-tert-butylpyridinium), which blocks open nicotinic ion channels noncompetitively, is able to reverse the neuromuscular paralysis after nerve agent poisoning in vitro and to protect guinea pigs against poisoning by nerve agents when used as part of a therapeutic drug combination including a muscarinic antagonist. In contrast to the oxime HI-6, this compound was equally effective in protecting against poisoning by sarin or tabun. Further studies should identify more effective compounds with this action and optimise doses for protection against nerve agent poisoning in vivo.     

Subchronic administration of various pretreatments of nerve agent poisoning. II. Compared efficacy against soman toxicity

OP nerve agents, such as soman, are potent irreversible inhibitors of central and peripheral acetylcholinesterases. Pretreatment of OP poisoning relies on the subchronic administration of a reversible acetylcholinesterase inhibitor. In the present study, the protective effects against soman toxicity of such compounds i.e. pyridostigmine, physostigmine (alone or associated with scopolamine) or huperzine are compared in guinea-pigs instrumented for EEG recording. Each medication is given via a subcutaneous mini-osmotic pump for 6 days at a delivery rate providing about 30% maximal inhibition of red cell acetylcholinesterase activity. The animals then receive iterative injections of soman (1/3 LD50) every 10 min. With pyridostigmine, reflecting a decreased overall tolerance to the poisoning, the cumulative doses of soman producing either tremors and convulsions or seizures are lower than those found in non-pretreated intoxicated controls. On the other hand, physostigmine does not afford satisfactory protection against the early mortality after intoxication. On this specific point, physostigmine + scopolamine and huperzine, although they do not prevent the appearance of seizures, give best results. The effects of each pretreatment on acetylcholinesterase, butyrylcholinesterase and carboxylesterase (these two latter enzymes may act as endogenous scavengers of OP compounds) are also examined in vitro and in the blood of each animal during subchronic administration. Huperzine appears as a selective inhibitor of red cell acetylcholinesterase activity while pyridostigmine or physostigmine additionally inhibit plasmatic butyrylcholinesterase. Considerations about huperzine or physostigmine + scopolamine as the most appropriate candidate for the pretreatment of OP poisoning are given.     

In vitro selection and efficacy of topical skin protectants against the nerve agent VX

Against highly toxic chemicals that are quickly absorbed in the skin, topical formulations could adequately complement specific protective suits and equipments. In this work, we evaluated in vitro and compared the skin protection efficacy against the nerve agent VX of four different topical formulations: oil-in-water and water-in-oil emulsions, a perfluorinated-based cream and a hydrogel. Semi-permeable silicone membrane, pig-ear and human abdominal split-thickness skin samples mounted in diffusion cells were compared as in vitro permeation tests. The results showed that silicone membrane could be used instead of skin samples to screen for potentially effective formulations. However, the results indicated that due to potentially significant interactions between formulations and skin, relevant ranking of formulations according to their protective efficacy could require tests with skin samples. The main phase of emulsions, water or oil, was not found to be critical for skin protective efficacy against VX. Instead, specific film-forming ingredients such as perfluorinated-based polymers and silicones could significantly affect the skin protective efficacy of formulations. We showed that a hydrogel containing specific hydrophilic polymers was by far the most effective of the formulations evaluated against VX skin permeation in vitro.     

Increasing oxime efficacy by blood-brain barrier modulation

One of the shortcomings of current treatment of nerve agent poisoning is that oximes hardly penetrate the blood-brain barrier (BBB), whereas nerve agents easily do. Increasing the concentration of oximes in the brain, would therefore provide an attractive approach to improve medical countermeasures. An explanation for limited penetration might be that oximes are substrates for the active P-glycoprotein (Pgp) efflux transporter located in the BBB.Using quantitative brain microdialysis in rats, the effect of i.v. injected tariquidar, a non-competitive, specific Pgp-inhibitor, on HI-6 levels in blood and brain was investigated. It appeared that tariquidar enhanced HI-6 levels in the brain approximately 2-fold during the first hour after HI-6 administration, whereas plasma levels did not differ between the treatment groups. A subsequent proof-of-concept study in rats showed that soman-induced seizures and convulsions were prevented almost completely when they were, in addition to HI-6 and atropine, pretreated with tariquidar. Moreover, twice as much AChE activity was present in their brains as compared to control rats.These results in rats indicate that modulation of the BBB by a drug like tariquidar, which is non-toxic by itself, is of great value in enhancing the efficacy of oximes.     

寡核苷酸药物定量分析方法的研究进展

寡核苷酸作为治疗药物在抗肿瘤、病毒治疗、免疫调节等方面有着良好的应用前景。寡核苷酸药物的药代动力学的研究和质量控制均要求具备相应的定量分析方法。本文综述了近年来在寡核苷酸药物药代动力学评价及生产质量控制中常用的几种定量分析方法,主要包括质谱法、毛细管电泳法、定量PCR法以及杂交技术相关的方法,阐述了各方法的特点、应用前景及其最新研究进展。     

Development of next generation medical countermeasures to nerve agent poisoning

Medical countermeasures provide a key role in the UK integrated approach to chemical defence and are aimed at preventing or mitigating the effects of exposure to nerve agents. It is UK policy that medical countermeasures will be licensed products. Demonstration of efficacy relies on extrapolation of animal-derived data to man which means that species selection is extremely important. For the foreseeable future it is likely that a combination of pretreatment and therapy will be required to provide protection against nerve agent poisoning. There is a longer-term aspiration to develop a post poisoning-therapy which would reduce the reliance on pretreatment, prevent or mitigate the effects of exposure to all nerve agents and decrease the requirement for three autoinjectors. Immediate therapy comprising physostigmine (0.2mg/kg), hyoscine hydrobromide (4mg/kg) and HI-6 (93.6mg/kg) protected all animals against the lethal effects of a supralethal dose of GD, when given 1min after nerve agent poisoning in the absence of any pretreatment. In contrast when hyoscine hydrobromide was replaced with hyoscine methyl nitrate most of the animals died within 24h, whereas when an equal mixture of hyoscine hydrobromide and hyoscine methyl nitrate was used all the animals survived. None of these animals had an intussusception. It would not be possible to deliver these doses of HI-6 to a human from a single autoinjector device. Recent studies have shown that a lower dose of HI-6 (7mg/kg) which can be delivered via an autoinjector, in combination with physostigmine and hyoscine hydrobromide provides good protection against the lethal effects of a supralethal dose of GD. A number of animals died between 6 and 24h and had an intussusception. The surviving animals did not begin to regain weight until 48h after poisoning. In contrast when a mixture of hyoscine hydrobromide and hyoscine methyl nitrate was used, one animal died within 15min, the other animals all survived, regained weight from 24h and did not have an intussusception. These studies will now be extended to include other agents and will be taken forward to studies in non-human primates where the incidence of intussusception will be closely monitored.     

In vitro evaluation of acetylcholinesterase reactivators as potential antidotes against tabun nerve agent poisonings

Searching for new potent acetylcholinesterase (AChE; E.C. 3.1.1.7) reactivators (oximes) is a very time-consuming process. At our department, we are able to synthesize more than 50 new AChE reactivators per year. Owing to this fact, we have to select promising reactivators using our in vitro method (potentiometric titration, pH 8 and temperature 25 degrees C; source of cholinesterases, rat brain homogenate; time of inhibition by nerve agents, 30 min; time of reactivation, 10 min) prior to in vivo experiments. For this purpose, we are using two-phase in vitro evaluation of reactivator potency. In the first phase, reactivation potency of all newly synthesized AChE reactivators is tested at two concentrations: 10(-3) M and 10(-5) M. Afterwards, all reactivators achieving reactivation potency over 15% (especially at the concentration 10(-5) M) are tested. The second phase consists of the measurement of the relationship between concentration of the oxime and its reactivation ability. In most cases, the reactivation bell-shaped curve is obtained. The most potent AChE reactivators are selected and provided for further experiments during our development process.