Oximes-induced reactivation of rat brain acetylcholinesterase inhibited by VX agent

A comparison of one mono- and seven bisquaternary acetylcholinesterase (AChE) reactivators of acetylcholinesterase inhibited by VX agent was performed. As a source of the acetylcholinesterase, a rat brain homogenate was taken. There were significant differences in reactivation potency of all tested oximes. The oxime TO205 seems to be the most efficacious followed by TO046, HI-6, HS-6, K027, obidoxime, MMC and 2-PAM. In addition, the results of this study showed that the reactivation potency of the tested reactivators depends on many factors--such as the number of pyridinium rings, the number of oxime groups and their position, as well as the length and the shape of linkage bridge between two pyridinium rings.     

Analysis of inhibition, reactivation and aging kinetics of highly toxic organophosphorus compounds with human and pig acetylcholinesterase

Organophosphorus compounds (OP) are in wide spread use as pesticides and highly toxic OP may be used as chemical warfare agents (nerve agents). OP inhibit acetylcholinesterase (AChE), therefore, standard treatment includes AChE reactivators (oximes) in combination with antimuscarinic agents. In the last decades, the efficacy of oximes has been investigated in various animal models, mostly in rodents. However, extrapolating animal data to humans is problematical because of marked differences between rodents and humans concerning the toxicokinetics of nerve agents, the pharmacokinetics of antidotes and the AChE enzyme kinetics. In order to improve the understanding of species differences and to enable a more reliable extrapolation of animal data to humans a study was initiated to investigate the effect of highly toxic nerve agents, i.e. VX, Russian VX (VR) and Chinese VX (CVX), with human and pig erythrocyte AChE. Hereby, the rate constants for the inhibition of AChE by these OP (ki) and for the spontaneous dealkylation (ka) and reactivation (ks) of OP-inhibited AChE as well as for the oxime-induced reactivation of OP-inhibited AChE by the oximes obidoxime, 2-PAM, HI 6, HL? 7 and MMB-4 were determined. Compared to human AChE pig AChE showed a lower sensitivity towards the investigated OP. Furthermore, a slower spontaneous dealkylation and reactivation of pig AChE was recorded. The potency of the investigated oximes was remarkably lower with OP-inhibited pig AChE. These data may contribute to a better understanding of species differences and may provide a kinetic basis for extrapolation of data from pig experiments to humans.     

Rates of spontaneous reactivation and aging of acetylcholinesterase in human erythrocytes after inhibition by organophosphorus pesticides

The in vitro rates of spontaneous reactivation and aging in human erythrocyte acetylcholinesterase were studied after inhibition by a dimethoxy (R1R2) and diethoxy substituted (R1R2) organophosphate pesticide (OP) of general structure R1R2P(O)X. These have been compared with data for human plasma cholinesterase previously reported using a similar methodology. A significantly slower rate of aging for erythrocyte acetylcholinesterase was found compared to plasma cholinesterase, whether inhibited by dimethoxy or diethoxy substituted OPs. For diethoxy OPs the rate of spontaneous reactivation of the inhibited plasma enzyme was significantly slower than for the inhibited red cell enzyme. This acetylcholinesterase, and previously published plasma cholinesterase, data suggest that in practise a blood sample taken 30-40 h after significant acute OP exposure will still show inhibition in either plasma or erythrocyte cholinesterase when analysed, but that any inhibited plasma enzyme is more likely to be in the aged form. In contrast a substantial proportion of the erythrocyte acetylcholinesterase is found unaged and therefore sensitive to reactivation by oximes. Samples from an occupational exposure where depressions in plasma or erythrocyte cholinesterase activity from baseline measurements were reactivated ex vivo using the oxime 2-PAM support this hypothesis. These data also confirm that the plasma enzyme is a more sensitive than erythrocyte acetylcholinesterase as an indicator of OP exposure and thus the potential value of ex vivo oxime reactivation of erythrocyte acetylcholinesterase in a blood sample to indicate subclinical OP exposure may be limited. However, this study is too small to draw conclusions on the sensitivity of ex vivo oxime reactivation of acetylcholinesterase as a novel biomarker of excessive OP absorption. Given that there is a better relationship between anticholinergic symptoms and red cell acetylcholinesterase inhibition, and that the slower resynthesis rate of any aged or inhibited red cell enzyme may be interpretatively useful when venepuncture is delayed, it is suggested that red cell acetylcholinesterase activity does have a place in monitoring potential OP exposure.     

Oxime-induced reactivation of acetylcholinesterase inhibited by phosphoramidates

The reaction of human erythrocyte acetylcholinesterase (AChE) with a set of structurally related phosphoramidates was studied in order to investigate the properties of phosphorylated enzyme and the effects of 4 oximes PAM-2, TMB-4, HI-6 and BDB-106 on the reactivation of inhibited AChE. Second-order rate constant of the phosphorylation reaction of the compounds towards the active site of AChE ranged between 5.0 × 10² and 4.9 × 10⁶M⁻¹min⁻¹ and their inhibitory power (I₅₀) was from 7.3 × 10⁻⁵to 5.7 × 10⁻⁹M for 20 min incubation at 37 °C. The oximes used were weak reactivators of inhibited AChE except for (C₄H₉O)(NH₂)P(O)DCP (DCP, -O-2,5-dichlorphenyl group) and (C₆H₁₃O)(NH₂)P(O)SCH₃ where we have obtained good reactivation. Imidazole oxime BDB-106 proved to be a potent reactivator of tabun-inhibited AChE.     

Oxime-induced reactivation of acetylcholinesterase inhibited by organophosphinates

The comparative potency of oximes for reactivation of inhibited eel acetylcholinesterase (AChE) in vitro is dependent on the organophosphinate inhibitor. Some of the data, dealing with a reference organophosphonate, support the conclusion of other investigators that the oxime potency order is also dependent on the inhibiting phosphonate. This work was done to identify more clearly the nature of phosphinylated AChE with regard to oxime reactivation potency and the potential of phosphinates as pretreatment drugs to protect AChE against organophosphonate poisoning. We have determined the reactivation potency of four oximes--2-PAM, HI-6, TMB-4 and toxogonin--against four phosphinates--4-nitrophenyl methyl(phenyl)phosphinate (PMP), 4-nitrophenyl chloromethyl(phenyl)phosphinate (CPMP), 4-nitrophenyl trifluoromethyl(phenyl)phosphinate and 4-nitrophenyl bis(2-thienyl)phosphinate. For comparison, the phosphonate sarin (GB, isopropyl methylphosphonofluoridate) was included. Incubation of the inhibited enzyme (I-AChE) at 25 degrees C was with 0.30 microM oxime for PMP, 3.0 microM oxime for sarin and CPMP and 100 microM oxime for the two remaining phosphinates. AChE activity was assayed spectrophotometrically for 3.0 min at 272.5 nm at 25 degrees C in 0.10 M MOPS buffer (pH 7.60) using phenyl acetate as substrate. When sarin was the inhibitor (0% spontaneous recovery after a 2-h incubation), the order of oxime reactivation was 2-PAM (46%) greater than or equal to toxogonin (33%) = TMB-4 (31%) greater than HI-6 (9%) after 2-h incubations. For PMP (12% spontaneous recovery after a 2-h incubation) the oxime order was toxogonin (67%) greater than TMB-4 (53%) greater than 2-PAM (40%) after 2-h incubations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sarin poisoning in guinea pigs compared to reactivation of acetylcholinesterase in vitro as a basis for therapy

Contrary to the large number of publications dealing with treatment of organophosphate poisoning in a variety of animal species, there is no logic reason in the preference of one species, for this purpose. Guinea pigs were reported to respond better to treatment by oximes, than mice and rats. However, in the analysis of data on the effect of obidoxim and atropine or benactyzine on sarin poisoning it is demonstrated, that guinea pigs do not respond differently from mice and rats. Subcutaneous LD50's of sarin in mice ranged from 0.06 to 0.207 mg/kg, and those of guinea pigs from 0.04 to 0.112 mg/kg. The difference in the LD50's may be related to the different susceptibility of various animal species. The importance of "in vivo" dosage, mode of application, kinetics of antagonists, in correlation to the ability to reactivate "in vitro" is discussed.     

Spontaneous and oxime-induced reactivation of acetylcholinesterase inhibited by phosphoramidates

Methamidophos (CH₃O(NH₂)P(O)SCH₃) and phosphoramidates, with the general structure RO(NH₂)P(O)OC₆H₄-p-NO₂, in which R = C₂H₅, ClCH₂CH₂, FCH₂CH₂ and F₃CCH₂, as well as (NH₂)₂P(O)OC₂H₄-p-NO₂ were synthesized to investigate the relationship between the rates of inhibition and of spontaneous reactivation of AChE inhibited by these organophosphates and their potential as prophylactics against nerve agent poisoning. The phosphoramidates inhibit electric eel acetylcholinesterase (EEAChE), the bimolecular inhibition rate constants ranging from 5×l0⁴ to 3×l0⁶ M^–1·min^–1 at pH 7.5, 25° C. The inhibited enzymes reactivate spontaneously, with half-lives ranging from 1.3 to 15 h at pH 7.5, 25° C. These half-lives increase 2–4 fold when the temperature is raised to 37° C. Reactivation is accelerated by micromolar concentrations of oximes such as obidoxime and HI-6. Aging of the inhibited enzymes was not observed. Nevertheless, reactivation appears to be incomplete for some of the inhibited enzymes. The title compounds seem promising as prophylactic agents against nerve agent intoxication.     

Profenofos insecticide bioactivation in relation to antidote action and the stereospecificity of acetylcholinesterase inhibition,reactivation, and aging

Poisoning signs in chicks administered the organophosphorus insecticide profenofos correlated with in vivo inhibition of brain acetylcholinesterase (AChE) activity. Mixtures of atropine with eserine, pyridinium oximes, or the bispyridinium compound SAD-128 increased the LD50 of coadministered profenofos by up to sevenfold in chicks and fourfold in mice. Atropine and the oximes were less effective as profenofos antidotes, indicating that profenofos-inhibited AChE may undergo rapid aging. Brain AChE from chicks poisoned with profenofos was not reactivated by pralidoxime methanesulfonate, although it was from chicks poisoned with the phosphoramidothiolate, methamidophos. Similarly, eel AChE, inhibited in vitro by bioactivated (?)-profenofos, the most toxic isomer, did not reactivate in contrast to that inhibited by methamidophos, nonbioactivated (?)-profenofos, and (+)-profenofos, with or without bioactivation. It appears that the action of eserine and possibly SAD-128 was due to protecting AChE or cholinergic receptors from profenofos or bioactivated profenofos and that oximes may work in the same way rather than as reactivators due to rapid aging of the inhibited AChE.     

In vitro reactivation potency of novel symmetrical bis-pyridinium oximes for electric eel acetylcholinesterase inhibited by nerve agent sarin

This communication describes synthesis and in vitro evaluation of a series of novel bis-pyridinium oximes connected by bis-methoxymethyl benzene, 1,4-bis-methoxymethyl (cis)-but-2-ene and 1,4-bis-methoxymethyl but-2-yne linkers as reactivators of sarin inhibited acetylcholinesterase (AChE). The reactivation data of synthesized oximes were compared with those of 2-PAM and obidoxime. The efficacy of oximes such as 1,4-dimethoxy cis-but-2-ene bis-[4,4′-(hydroxyiminomethyl)-pyridinium] dichloride (3g), 1,4-dimethoxy benzene bis-[3,3′-(hydroxyimino-methyl) pyridinium] dichloride (3b) and 1,3-dimethoxy benzene bis-[3,3′-(hydroxy-iminomethyl) pyridinium] dichloride (3e) were found to be more than that of obidoxime in reactivating sarin inhibited AChE. The oxime 3g was able to reactivate 25% of AChE activity in comparison to 20% and 5% reactivation exhibited by 2-PAM and obidoxime respectively at a concentration of 10⁻⁴ M. The pKa of the oximes were determined and correlated with the reactivation potential.     

Kinetics of human acetylcholinesterase inhibition by the novel experimental Alzheimer therapeutic agent, tolserine

Characterization of the kinetic parameters of tolserine, a novel acetylcholinesterase (AChE) inhibitor of potential in the therapy of Alzheimer's disease, to inhibit purified human erythrocyte AChE was undertaken for the first time. An IC(50) value was estimated by three methods. Its mean value was found to be 8.13 nM, whereas the IC(100) was observed to be 25.5 nM as calculated by single graphical method. The Michaelis-Menten constant (K(m)) for the hydrolysis of the substrate acetylthiocholine iodide was found to be 0.08 mM. Dixon as well as Lineweaver-Burk plots and their secondary replots indicated that the nature of the inhibition was of the partial non-competitive type. The value of K(i) was estimated as 4.69 nM by the primary and secondary replots of the Dixon as well as secondary replots of the Lineweaver-Burk plot. Four new kinetic constants were also investigated by polynomial regression analysis of the relationship between the apparent K(i) (K(Iapp)) and substrate concentration, which may open new avenues for the kinetic study of the inhibition of several enzymes by a wide variety of inhibitors in vitro. Tolserine proved to be a highly potent inhibitor of human AChE compared to its structural analogues physostigmine and phenserine.     

Carbamates with differential mechanism of inhibition toward acetylcholinesterase and butyrylcholinesterase

Most carbamates are pseudoirreversible inhibitors of cholinesterases. Phenothiazine carbamates exhibit this inhibition of acetylcholinesterase but produce reversible inhibition of butyrylcholinesterase, suggesting that they do not form a covalent bond with the catalytic serine. This atypical inhibition is attributable to pi-pi interaction of the phenothiazine moiety with F329 and Y332 in butyrylcholinesterase. These residues are in a helical segment, referred to here as the E-helix because it contains E325 of the catalytic triad. The involvement of the E-helix in phenothiazine carbamate reversible inhibition of butyrylcholinesterase is confirmed using mutants of this enzyme at A328, F329, or Y332 that show typical pseudoirreversible inhibition. Thus, in addition to various domains of the butyrylcholinesterase active site gorge, such as the peripheral anionic site and the pi-cationic site of the Omega-loop, the E-helix represents a domain that could be exploited for development of specific inhibitors to treat dementias.     

Spontaneous reactivation and aging kinetics of acetylcholinesterase inhibited by dichlorvos and diazinon

Organophosphorus (OP) are among the most toxic of all substances that cause poisoning in food animals and are the most frequently encountered insecticides, commonly detected in agricultural products, animal-derived foodstuffs, environmental samples, and home use and represent a significant potential health risk. The first-order rate constants obtained for spontaneous reactivation (k(s)) was found to be higher in sheep compared to cattle, pig, and ranged between 0.133 to 0.323 hr?1 and between 0.021 to 0.088 hr?1 for dichlorvos (DDVP) and diazinon (DZN) respectively. Aging of phosphorylated acetylcholinesterase (AChE) follows the kinetics of a first-order reaction with rate constants of aging (k(a)) higher in cattle compared to sheep and pig, and ranged between 0.013 to 0.021 hr?1 and between 0.009 to 0.01 hr?1 for DDVP and DZN respectively. Half-time (t?) for spontaneous reactivation and aging are higher in DZN compared to DDVP and ranged from 2.3 to 85.3 hr (sheep), 3.2 to 76.3 hr (cattle), and 2.9 to 58.3 hr (pig), respectively.     

In vitro reactivation of acetylcholinesterase using the oxime K027

The ability of a new bisquaternary oxime, K027 (1-[4-hydroxyiminomethylpyridinium]-3-[carbamoylpyridinium] propane dibromide), to reactivate the enzyme acetylcholinesterase (AChE) inhibited by the nerve agents Tabun, sarin and VX was evaluated. Its reactivation potency was compared to the AChE reactivators pralidoxime (2-PAM), obidoxime and HI-6; K027 seems a good reactivator of organophosphates-inhibited AChE. Its reactivation potency is lower compared to the other oximes for reactivation of sarin-inhibited AChE, but it is sufficient to significantly increase the activity of sarin-inhibited AChE. Its reactivation ability is comparable to obidoxime for reactivation of VX- and tabun-inhibited AChE and is higher than the reactivation potency of HI-6, for tabun-inhibited AChE. HI-6 is currently regarded the most promising reactivator of organophosphates-inhibited AChE.     

Comparison of the oxime-induced reactivation of erythrocyte and muscle acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity

The purpose of these experiments was to compare oxime-induced reactivation rate constants of acetylcholinesterase from different human tissue sources inhibited by organophosphorus compounds. To this end, preliminary testing was necessary to generate a stable system both for working with erythrocytes and musculature. We established a dynamically working in vitro model with a fixed enzyme source in a bioreactor that was perfused with acetylthiocholine, Ellman's reagent and any agent of interest (e.g. nerve agents, oximes) and analyzed in a common HPLC flow-through detector. The enzyme reactor was composed of a particle filter (Millex-GS, 0.22 microm) containing a thin layer of membrane-bound acetylcholinesterase and was kept at constant temperature in a water bath. At constant flow the height of absorbance was directly proportional to the enzyme activity. To start with, we applied this system to human red cell membranes and then adapted the system to acetylcholinesterase of muscle tissue. Homogenate (Ultra-Turrax and Potter-Elvehjem homogenizer) of human muscle tissue (intercostal musculature) was applied to the same particle filter and perfused in a slightly modified way, as done with human red cell membranes. We detected no decrease of acetylcholinesterase activity within 2.5h and we reproducibly determined reactivation rate constants for reactivation with obidoxime (10 microM) or HI 6 (30 microM) of sarin-inhibited human muscle acetylcholinesterase (0.142+/-0.004 min(-1) and 0.166+/-0.008 min(-1), respectively). The reactivation rate constants of erythrocyte and muscular acetylcholinesterase differed only slightly, highlighting erythrocyte acetylcholinesterase as a proper surrogate marker.     

VX染毒小鼠乙酰胆碱酯酶活性和含量与作用时间的相互关系

目的评价不同剂量VX对小鼠脑和外周血乙酰胆碱酯酶(AChE)的活性和含量随时间变化的影响。方法小鼠sc给予VX 5,10和16μg·kg^-1,分别于10 min,1 h,4 h,24 h,48 h和72 h记录体重和脑重,计算脑系数;Ellman法测定AChE活性;ELISA测定AChE含量;并计算被VX抑制的AChE含量和VX消耗量。结果在染毒后72 h内,各组小鼠体重、脑重和脑系数均未见明显变化。与同剂量下染毒10 min相比,VX 5,10和16μg·kg^-1组外周血AChE活性抑制率在各时间点均明显下降(P<0.05,P<0.01)。与同剂量染毒10 min比较,VX 16μg·kg^-1组脑AChE活性抑制率在4～72 h显著下降(P<0.01);10μg·kg^-1组在48和72 h显著下降(P<0.01);5μg·kg^-1组在24 h显著上升(P<0.01),而在其他时间点无明显变化。各剂量组脑和外周血中VX的消耗量均在48 h达到最低,...