Reduction by Pyridostigmine Pretreatment of the Efficacy of Atropine and 2-PAM Treatment of Sarin and VX Poisoning in Rodents

Reduction by Pyridostiginine Pretreatment of the Efficacy ofAtropine and 2-PAM Treatment of Sarin and VX Poisoning in Rodents.KOPLOVITZ, I., HARRIS, L. W., ANDERSON, D. R., LENNOX, W. J.,AND STEWART, J. R. (1992). Fundam. Appl. Toxicol. 18, 102–106. This study concerned the effect of pyridostigmine pretreatmenton (a) the antidotal efficacy of atropine and 2-PAM in sarin,tabun, and VX poisoning in mice and guinea pigs and on (b) theoxime-induced reactivation of VX-inhibited whole blood acetyicholinesterase(AChE) of guinea pigs. One hour prior to organophosphate (OP)challenge with sarin, tabun, or VX, animals were given oraldoses of pyridostiginine to induce approximately 30 and 60%inhibition of whole blood AChE; controls received vehicle. Micewere challenged im and guinea pigs sc with the OP compounds.Treatment with atropine (11.2 mg/kg to mice; 32 mg/kg to guineapigs) plus 2-PAM (25 mg/kg) was given im at 10 sec postchallengein mice and 1 min postchallenge in guinea pigs. In the reactivationexperiments, pyridostigmine or saline was given im to guineapigs 30 min prior to VX (8.24 µg/kg, sc), atropine (16mg/kg) was given im at 1 mm, and 2-PAM (25 mg/kg) at 16 minpostchallenge. Pyridostigmine significantly enhanced the efficacyof atropine and 2-PAM against tabun in both species. In contrast,pyridostigmine reduced or did not increase the efficacy of atropineand 2-PAM against sarin or VX in both species. Recovery of VX-inhibitedAChE by 2-PAM was decreased significantly in pyridostigminepretreated animals. The results suggest that pyridostigminepretreatment may adversely effect the efficacy of atropine and2-PAM as antidotes for VX and sarin intoxication.     

Effectiveness of Oximes 2-PAM and HI-6 in Recovery of Muscle Function Depressed by Organophosphate Agents in the Rat Hemidiaphragm: An in Vitro Study

Effectiveness of Oximes 2-PAM and HI-6 in Recovery of MuscleFunction Depressed by Organophosphate Agents in the Rat Hemidiaphragm:An in vitro Study. REDDY, V. K., DESHPANDE, S. S., CINTRA, W.M., SCOBLE, G. T., AND ALBUQUERQUE, E. X. (1991). Fundam. Appl.Toxicol. 17, 746–760. Phrenic nerve diaphragm musclesof young adult rats were used to study the ability of the oximes2-PAM and HI-6 to recover muscle function depressed by organophosphate(OP) agents. The single twitch of diaphragm muscles which wereexposed to soman (0.2 mm) recovered after washing with salinefor 3 hr, but the muscles pretreated with sarin (0.4 µM),VX (0.2 µM), or tabun (0.4 µM) showed only partialrecovery. In addition, after 3 hr washing, the muscles pretreatedwith soman as well as with tabun did not recover the tetanussustaining ability (TSA), yet complete recovery was observedwith muscles pretreated with sarin and VX. These results indicatethat the OPs have different effects on muscle contractile propertiesand that VX- and sarin-pretreated muscles recover equally wellafter wash with physiological solution. The recovery of twitchtension of diaphragm muscles by 2-PAM and HI-6 was similar tothat achieved by washing with saline for 3 hr for sarin- andsoman-exposed muscles. The most remarkable differences wereseen in the recovery of TSA. Both 2-PAM and HI-6 recovered theTSA of muscles that were pretreated with sarin and VX. Although2-PAM recovered the TSA after tabun pretreatment, HI-6 had nodiscernible effect. On the other hand, HI-6 recovered the TSAof soman-pretreated muscles but 2-PAM did not. The effectivenessof muscle function recovery was not related to the oximes' abilityto reactivate AChE, thus indicating that the recovery of musclecontractility may be attributed to a direct effect of thesecompounds on the muscle.     

Changes of Cholinesterase Activities in the Rat Blood and Brain After Sarin Intoxication Pretreated with Butyrylcholinesterase

After sarin inhalation exposure of rats pretreated with equine serum butyrylcholinesterase (EqBuChE), cholinesterase activities of the whole blood, acetylcholinesterase (AChE) in erythrocytes, pontomedullar area, frontal cortex, and striatum of the brain, and plasma butyrylcholinesterase (BuChE) were determined. Using different doses of EqBuChE as a pretreatment (intraperitoneal injection), dose-dependent increases in plasma BuChE activity and no changes in the erythrocyte and brain AChE activities were demonstrated. Decreases in plasma BuChE activity and red blood cells (RBC) and brain AChE activities were observed in control rats after sarin inhalation exposure without EqBuChE pretreatment. In rats pretreated with EqBuChE, this inhibition was lower compared with control animals not only in the blood but also in the brain structures studied. These results demonstrate protective effects of EqBuChE pretreatment in rats intoxicated with sublethal concentrations of sarin by inhalation.     

Changes of cholinesterase activities in the rat blood and brain after sarin intoxication pretreated with butyrylcholinesterase

After sarin inhalation exposure of rats pretreated with equine serum butyrylcholinesterase (EqBuChE), cholinesterase activities of the whole blood, acetylcholinesterase (AChE) in erythrocytes, pontomedullar area, frontal cortex, and striatum of the brain, and plasma butyrylcholinesterase (BuChE) were determined. Using different doses of EqBuChE as a pretreatment (intraperitoneal injection), dose-dependent increases in plasma BuChE activity and no changes in the erythrocyte and brain AChE activities were demonstrated. Decreases in plasma BuChE activity and red blood cells (RBC) and brain AChE activities were observed in control rats after sarin inhalation exposure without EqBuChE pretreatment. In rats pretreated with EqBuChE, this inhibition was lower compared with control animals not only in the blood but also in the brain structures studied. These results demonstrate protective effects of EqBuChE pretreatment in rats intoxicated with sublethal concentrations of sarin by inhalation.     

Comparison of Cholinergic and Neuromuscular Toxicity following Acute Exposure to Sarin and VX in Rat

Comparison of Cholinergic and Neuromuscular Toxicity followingAcute Exposure to Sarin and VX in Rat. GUPTA, R. C, PATTERSON,G. T., AND DETTBARN, W-D. (1991). Fundam. Appl. Toxicol. 16,449–458. Male Sprague-Dawley rats injected with a sublethalsc dosage of 110 µg/ kg of sarin (isopropyl methylphosphonofluoridate),or 12 µg/kg of VX (S-{2-diisopropylaminoethyl) O-ethylmethylphosphonothioate), developed severe toxic signs within5–15 min after sarin and 20–50 min after VX lastingfor 5 to 7 hr. Myonecrotic lesions were seen in soleus and diaphragmmuscles within 1 hr. A maximum number of lesions had developedafter 24 hr, and lesions were also present in extensor digitorumlongus (EDL) at this time. Regeneration of muscle fibers wasslow since lesions were still evident past 7 days of treatment.Within 1 hr following VX, AChE activity was reduced to 8, 12,and 17% of control activity in soleus, diaphragm, and EDL, respectively,whereas with sarin the enzyme activity was reduced to 23, 48,and 82% of control. A still greater inhibition was seen 24 hrafter sarin when AChE activity was reduced to 19, 13, and 43%in these muscles. In skeletal muscles the different molecularforms of AChE, such as 16 S, 12 S, 10 S, and 4 S vary in locationand functional importance with the 16 S form highly concentratedat the neuromuscular junction. All forms in a given muscle wereequally sensitive to the inhibitors. In EDL, sarin was the leasteffective in reducing AChE or its molecular forms. In the brainstructures (cortex, brain stem, striatum, and hippocampus),AChE activity was reduced to 1–6% of control by sarinand VX with the exception that following VX striatal AChE wasreduced to only 41% of control activity. AChE activity in thebrain cortex following either of the agents was maximally affected(1%). A slow but significant recovery of brain AChE was evidentafter 24 hr and more so after Day 7. Butyrylcholinesterase (BuChE)activity was less sensitive to inhibition by both inhibitorscompared to AChE activity and showed a rapid recovery. Basedon the equitoxic doses (toxic signs of similar magnitude), VXwas found to be 10 times more toxic than sarin. The mechanismsof this disparity may be due to differences in rate of uptake,circulation, susceptibility to hydrolysis, and reactivity withnonspecific binding sites.     

Effects of hypophysectomy on acetylcholinesterase and butyrylcholinesterase in the rat

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities were examined in several tissues of normal and hypophysectomized male and female rats. Significant sex differences in the mean AChE activities of normal rats were observed in the superior cervical ganglion (three times more activity in males) and in serum (50% more activity in females). Sex differences in the BuChE activity of serum and liver were even larger (ten times more activity in females), but the activity of other tissues was similar in both sexes. Hypophysectomy had little effect on the mean activity of AChE but did alter BuChE activity in certain tissues. Most of the effects of hypophysectomy on mean BuChE activity were opposite in direction in the two sexes. For example, in males hypophysectomy caused increases in the BuChE activity of serum (300%) and liver (43%), while in females it caused decreases in both tissues (25 and 30% respectively). In rats of a given group, the AChE activity of each tissue appeared to be regulated independently of the activity in other tissues. By contrast, BuChE activity showed statistically significant correlations in more than half of the tissue-pairs examined in control rats of either sex. These correlations can be considered to reflect a tendency toward body-wide regulation. In female rats, the cross-tissue correlations were largely eliminated by hypophysectomy. This finding indicates that the regulation of BuChE mav be strongly affected by hormones under the control of the pituitary gland. However, in male rats, only the correlations involving atria were altered by hypophysectomy. Therefore, the effects of hormones on BuChE are probably both sex and tissue dependent.     

Differential sensitivity of blood, peripheral, and central cholinesterases in beagle dogs following dietary exposure to chlorpyrifos

Two studies were performed to find out whether exposure limits that protect brain acetylcholinesterase (AChE) will protect peripheral tissue AChE after exposure to chlorpyrifos (CPF), an organophosphate insecticide. In a methods-development study, male dogs (3/dose) were exposed to 0.0, 0.3, 0.6, or 1.2mg/kg/day CPF in their diets for 4 weeks. Mixed cholinesterase (mChE), AChE, and butyrylcholinesterase (BuChE) activities were measured in plasma, RBC, brain, left atrium and ventricle, diaphragm, quadriceps, and nodose ganglia. Plasma, brain and peripheral tissue BuChE was inhibited at all dose levels. While RBC AChE was inhibited at all doses, brain and peripheral AChE activities were unaffected. In the main study, dogs (4/sex/dose) were exposed to 0.0, 0.5, 1.0, or 2.0mg/kg/day CPF in their diets for six weeks and RBC AChE was significantly inhibited at all doses in both sexes. Diaphragm, quadriceps, and nodose ganglia AChE was unaffected by treatment. Brain AChE was decreased by approximately 6% compared to controls in high-dose groups, and this was considered a threshold effect. Left atrium AChE in high-dose dogs was 25.5% less (males) and 32.1% greater (females) than controls; these differences were attributed to chance. While peripheral tissue and brain AChE were not affected following exposure to 1.0mg/kg/day, RBC AChE was inhibited at all doses. These results show that RBC AChE is more sensitive than brain or peripheral tissue AChE to inhibition by CPF, and that protection of brain AChE would protect peripheral tissue AChE.     

Studies of the amplification of carbaryl toxicity by various oximes.

The administration of 2-pyridine aldoxime methyl chloride (2-PAM Cl) is a standard part of the regimen for treatment of human overexposure to many organophosphorus pesticides and nerve agents. However, some literature references indicate that poisoning by carbaryl (1-naphthyl N-methyl carbamate), an insecticide in everyday use, is aggravated by the administration of 2-PAM Cl. This effect has been reported in the mouse, rat, dog and man. We have found that the inhibition of both eel acetylcholinesterase (eel AChE, EC 3.1.1.7) and human serum cholinesterase (human BuChE, EC 3.1.1.8) by carbaryl was enhanced by several oximes. Based on 95% confidence limits the rank order of potentiation with eel AChE was TMB-4 = Toxogonin > HS-6 = HI-6 > 2-PAM Cl. By the same criterion, the rank order of potentiation with human BuChE was TMB-4 > Toxogonin > HS-6 = 2-PAM Cl. Carbaryl-challenged mice also reflected a potentiation since TMB-4 exacerbated the toxicity more than 2-PAM Cl. Our hypothesis is that certain oximes act as allosteric effectors of cholinesterases in carbaryl poisoning, resulting in enhanced inhibition rates and potentiation of carbaryl toxicity.     

Enzyme-kinetic investigation of different sarin analogues reacting with human acetylcholinesterase and butyrylcholinesterase

The pertinent threat of using organophosphorus compound (OP)-type chemical warfare agents (nerve agents) during military conflicts and by non-state actors requires the continuous search for more effective medical countermeasures. OP inhibit acetylcholinesterase (AChE) and therefore standard treatment of respective poisoning includes AChE reactivators (oximes) in combination with antimuscarinic agents. Hereby, standard oximes, 2-PAM and obidoxime, are considered to be rather insufficient against various nerve agents. Numerous experimental oximes have been investigated in the last decades by in vitro and in vivo models. Recently, we studied the reactivating potency of several oximes with human AChE inhibited by structurally different OP and observed remarkable differences depending on the OP and oxime. In order to investigate structure-activity relationships we determined the various kinetic constants (inhibition, reactivation, aging) for a series of sarin analogues bearing a methyl, ethyl, n-propyl, n-butyl, i-propyl, i-butyl, cyclohexyl or pinacolyl group with human AChE and BChE. The rate constants for the inhibition of human erythrocyte AChE and plasma BChE by these OP (k(i)), for the spontaneous dealkylation (k(a)) and reactivation (k(s)) of OP-inhibited AChE and BChE as well as for the oxime-induced reactivation of OP-inhibited AChE and BChE by the oximes obidoxime, 2-PAM, HI 6, HL? 7 and MMB-4 were determined. With compounds bearing a n-alkyl group the inhibition rate constant increased with chain length. A relation between chain length and spontaneous reactivation velocity was also observed. In contrast, no structure-activity dependence could be observed for the oxime-induced reactivation of AChE and BChE inhibited by the compounds tested. In general, OP-inhibited AChE and BChE were susceptible towards reactivation by oximes. HL? 7 was the most potent reactivator followed by HI 6 and obidoxime while 2-PAM and MMB-4 were rather weak reactivators. These data indicate a potential structure-activity relationship concerning inhibition and spontaneous reactivation but not for oxime-induced reactivation.     

Evaluation of cognitive and biochemical effects of low-level exposure to sarin in rhesus and African green monkeys

We investigated the potential of low-level exposures to the chemical warfare nerve agent, sarin, to produce adverse effects. Rhesus (Macaca mulatta) and African green monkeys (Chlorocebus acthiops) were trained on a serial probe recognition (SPR) task before IM administration of a low-level concentration (5.87 microg/kg or 2.93 microg/kg) of sarin. Blood was sampled before agent administration and at various times following administration. Sarin administration did not disrupt performance on the SPR task in either species. Major dependent measures characterizing performance (accuracy, number of completed trials per session, average choice response time) were largely unaffected on the day sarin was administered as well as on subsequent testing sessions occurring over several weeks following administration. Analyses of red blood cell (RBC) and plasma samples revealed that sarin administration produced a substantial degree of inhibition of circulating acetylcholinesterase (AChE) in RBC fractions and butyrylcholinesterase (BChE) in plasma fractions, which only slowly recovered. In this regard, AChE activity was inhibited to a greater extent than BChE activity. Blood samples were also evaluated for regenerated sarin, which was found in RBC and plasma fractions in both species and showed orderly elimination functions. More sarin was regenerated from RBC fractions than from plasma fractions. Elimination of regenerated sarin was much slower in RBC than plasma and exceeded the expected time of AChE aging, suggesting the presence of additional sarin binding sites. In general, effects were similar in both species. Taken together, our results show that while the concentrations of sarin administered were clearly biochemically active, they were below those that are required to produce a disruption of behavioral performance.     

Comparative evaluation of antidotal efficacy of 2-PAM and HNK-102 oximes during inhalation of sarin vapor in Swiss albino mice

Abstract

Efficacy of two oximes treatments evaluated during inhalation of sarin vapor (LCt₅₀, 755.9?mg/min/m³) in simulated real scenario in vivo. Majority of mice either became moribund or died within 1–2?min during exposure to multifold-lethal concentrations of sarin vapor. Protection indices were determined by exposing to sarin vapor in two sessions, 1?min exposure followed by treatments with or without HNK-102 (56.56?mg/kg, im) or 2-PAM (30?mg/kg, im) and atropine (10?mg/kg, ip), and again exposed for remaining 14?min. Protection offered by HNK-102 was found to be four folds higher compared to 2-PAM in the same toxic environment. Secondly, sub-lethal concentration of sarin vapor (0.8?×?LCt₅₀ or 605?mg/min/m³), 24?h post investigations revealed that the oximes could not reactivate brain and serum acetylcholinesterase (AChE) activity. The treatments prevented increase in protein concentration (p?<?.05) and macrophages infiltration compared to sarin alone group in broncho-alveolar lavage fluid. Lung histopathology showed intense peribronchial infiltration and edema with desquamating epithelial lining and mild to moderate alveolar septal infiltration in sarin and atropine groups, respectively. Noticeable peeling-off observed in epithelial lining and sporadic mild infiltration of epithelial cells at bronchiolar region in 2-PAM and HNK-102 groups, respectively. The oximes failed to reactivate AChE activity; however, the mice survived up to 6.0?×?LCt₅₀, proved involvement of non-AChE targets in sarin toxicity. Atropine alone treatment was found to be either ineffective or increased the toxicity. HNK-102, exhibited better survivability with lung protection, can be considered as a better replacement for 2-PAM to treat sarin inhalation induced poisoning.     

Synthesis and in vitro and in vivo inhibition potencies of highly relevant nerve agent surrogates

Four nonvolatile nerve agent surrogates, 4-nitrophenyl ethyl dimethylphosphoramidate (NEDPA, a tabun surrogate), 4-nitrophenyl ethyl methylphosphonate (NEMP, a VX surrogate), and two sarin surrogates, phthalimidyl isopropyl methylphosphonate (PIMP) and 4-nitrophenyl isopropyl methylphosphonate (NIMP), were synthesized and tested as acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors. These surrogates were designed to phosphorylate cholinesterases with the same moiety as their respective nerve agents, making them highly relevant for the study of cholinesterase reactivators. Surrogates were characterized by liquid chromatography-mass spectrometry and nuclear magnetic resonance. NEMP, PIMP, and NIMP were potent inhibitors of rat brain, skeletal muscle, diaphragm, and serum AChE as well as human erythrocyte AChE and serum BuChE in vitro. PIMP was determined to degrade quickly in aqueous solution, making it useful for in vitro assays only, and NEDPA was not a potent inhibitor of AChE or BuChE in vitro; therefore, these two surrogates were not tested in subsequent in vivo studies. Sublethal dosages (yielding about 80% brain AChE inhibition) were determined for both the stable sarin surrogate, NIMP (0.325 mg/kg ip), and the VX surrogate, NEMP (0.4 mg/kg ip), in adult male rats. Time course studies indicated the time to peak brain AChE inhibition for both NIMP and NEMP to be 1 h postexposure. Both surrogates yielded severe cholinergic signs. These dosages did not require the addition of atropine to prevent lethality, and the rate of AChE aging was slow, making these surrogates useful for reactivation studies both in vitro and in vivo. The surrogates synthesized in this study are potent yet safer to test than nerve agents and are useful tools for initial screening of nerve agent oxime therapeutics.     

A comparison of in vivo and in vitro rates of aging of soman-inhibited erythrocyte acetylcholinesterase in different animal species

This study was conducted to assess differences in the rate of aging of soman-inhibited erythrocyte (RBC) acetylcholinesterase (AChE) from different species and to determine whether the rate of aging in vitro approximates that in vivo following a single exposure to soman. The logarithm of the percentage RBC AChE reactivated by 2-PAM after soman exposure was plotted as a function of time. Linear regression analysis was used to determine the half-time (t1/2) for aging. In the in vivo experiments, the rapidity of aging increased from rat to guinea pig to marmoset; the corresponding t1/2 values (mean +/- standard error) were 8.6 (+/- 0.94) min, 7.5 (+/- 1.7) min and 0.99 (+/- 0.10) min. The in vitro values of t1/2 in marmoset and guinea pig were 1.1 (+/- 0.08) min and 8.0 (+/- 0.82) min, respectively. Other t1/2 values computed for in vitro experiments were 1.4 (+/- 0.11) min for cynomolgus monkeys and 0.88 (+/- 0.03) min for squirrel monkeys. The results indicate that, for marmoset and guinea pig, the in vitro values closely approximate the in vivo. Since aging of soman-inhibited human and monkey RBC AChE occurs rapidly (1 min) in vitro, it is reasonable to assume that in man rapid aging will be crucial in delimiting successful treatment of soman intoxication.     

In vitro evaluation of bis-pyridinium oximes bearing methoxy alkane linker as reactivators of sarin inhibited human acetylcholinesterase

A series of bis-pyridinium oximes connected by methoxy alkane linkers were synthesized and their in vitro reactivation efficacy was evaluated against sarin-inhibited human AChE, and data were compared with 2-PAM and obidoxime. Among the synthesized compounds, 1,2-dimethoxy ethylene bis-[4,4′-(hydroxyiminomethyl) pyridinium] dichloride (4P-2) and 1,2-dimethoxy ethylene bis-[3,3′-(hydroxyiminomethyl) pyridinium] dichloride (3P-2) were found to be the most potent reactivators of human AChE inhibited by nerve agent sarin. The oximes 4P-2 and 3P-2 exhibited 41% and 36% regeneration of sarin-inhibited AChE, respectively, whereas 2-PAM showed 32% regeneration. The higher reactivation efficacy of the oximes was attributed to their acid dissociation constants (pK_a). The pK_a values of all the oximes were determined by UV–vis spectrophotometric method and correlated with their observed reactivation potential. Overall, the study reveals that the oxime 4P-2 may have therapeutic potential in the reactivation of human AChE inhibited by sarin.     

Determination of miosis threshold from whole-body vapor exposure to sarin in African green monkeys

We determined the threshold concentration of sarin vapor exposure producing miosis in African green monkeys (Chlorocebus aethiops). Monkeys (n=8) were exposed to a single concentration of sarin (0.069-0.701mg/m3) for 10min. Changes in pupil size were measured from photographs taken before and after the exposure. Sarin EC50 values for miosis were determined to be 0.166mg/m3 when miosis was defined as a 50% reduction in pupil area and 0.469mg/m3 when miosis was defined as a 50% reduction in pupil diameter. Monkeys were also evaluated for behavioral changes from sarin exposure using a serial probe recognition test and performance remained essentially unchanged for all monkeys. None of the concentrations of sarin produced specific clinical signs of toxicity other than miosis. Sarin was regenerated from blood sampled following exposure in a concentration-dependent fashion. Consistent with a predominant inhibition of acetylcholinesterase (AChE), more sarin was consistently found in RBC fractions than in plasma fractions. Further, elimination of regenerated sarin from RBC fractions was slower than from plasma fractions. Blood samples following exposure also showed concentration-dependent inhibition of AChE activity and, to a lesser extent, butyrylcholinesterase activity. At the largest exposure concentration, AChE inhibition was substantial, reducing activity to approximately 40% of baseline. The results characterize sarin exposure concentrations that produce miosis in a large primate species in the absence of other overt signs of toxicity. Further, these results extend previous studies indicating that miosis is a valid early indicator for the detection of sarin vapor exposure.     

In vitro reactivation of sarin inhibited electric eel acetylcholinesterase by bis-pyridinium oximes bearing methoxy ether linkages.

Bis-pyridinium oximes connected by methoxy alkane ether linker were synthesized and their in vitro reactivation efficacy was evaluated for sarin inhibited AChE. Reactivation efficacy of synthesized compounds was compared with 2-PAM and obidoxime. Among the synthesized compounds, 1,2-dimethoxy ethylene bis-[3,3'-(hydroxyiminomethyl) pyridinium] dichloride (3P-2) and 1,3-dimethoxy propylene bis-[3,3'-(hydroxyiminomethyl) pyridinium] dichloride (3P-3) were found to be most potent reactivators for AChE inhibited by nerve agent sarin. 3P-2 and 3P-3, respectively exhibited 80% and 69% regeneration of inhibited AChE, whereas 2-PAM (well known antidote for nerve agent poisoning) showed 42% regeneration.     

New Safe Method for Preparation of Sarin-Exposed Human Erythrocytes Acetylcholinesterase Using Non-Toxic and Stable Sarin Analogue Isopropyl p-Nitrophenyl Methylphosphonate and its Application to Evaluation of Nerve Agent Antidotes

Introduction A non-toxic and stable sarin analogue, isopropyl p-nitrophenyl methylphosphonate (INMP), was synthesized for safe preparation of sarin-exposed acetylcholinesterase (AChE). Results and Discussion This agent was stable for years, able to be handled in an ordinary laboratory without special care, and its 50% inhibitory concentration (IC₅₀) on 0.04 U/ml human erythrocytes AChE was 15 nM. This reagent was thought to be especially useful since it enables experiments that require sarin-inhibited AChE, such as the development of antidotes for sarin, in a usual laboratory. To demonstrate the usefulness of this method, 40 known and novel pyridinealdoxime methiodide (PAM)-type oxime antidotes were synthesized, and their reactivation activities to INMP-exposed AChE and structure–activities correlation were studied. Conclusion Among the antidotes tested in this experiment except for 2-PAM, the compound found to have the highest reactivation activity, was the novel hydrophobic 2-PAM-type compound, 2-[(hydroxyimino)methyl]-1-[4-(tert-butyl)benzyl] pyridinium bromide.     

Reactivating and protective effects of Pro-2-PAM in mice poisoned with paraoxon

Acute toxicity of N-methyl-1,6-dihydropyridine-2-carbaldoxime hydrochloride (Pro-2-PAM) in mice, its concentrations in blood and brain and its protective and reactivating effects in diethyl p-nitrophenyl phosphate (paraoxon) poisoning were determined. Comparative studies with N-methylpyridinium-2-aldoxime chloride (PAM-2 Cl) and a mixture of two oximes were also performed. Pro-2-PAM was much less toxic than was PAM-2 Cl. In contrast to this, the Pro-compound penetrates well into central nervous system (CNS), and effectively reactivates brain acetylcholinesterase (AChE) in vivo inhibited by paraoxon. The two oximes were equally effective in reactivating blood AChE. However, protective effects of Pro-2-PAM were unexpectedly lower than those of PAM-2 Cl. The observation suggests that there is no correlation between brain AChE reactivating power and protective effects of Pro-2-PAM. Contrary to the present belief, it seems that Pro-2-PAM undergoes slow conversion in vivo into its active form PAM-2 Cl. The possible use of Pro-2-PAM as a model oxime for penetration into CNS requires further evaluation with regard to therapy of organophosphorus poisoning.     

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.     

Evaluation of reactivation test in anaesthetized dogs with experimental intoxication with nerve agents

Following repeated antidotal treatment of anaesthetized dogs (1 min with atropine, 10 min with atropine and obidoxime, 60 min with atropine and obidoxime) after the intoxication with soman, sarin and VX (1 x LD50, i.m.), the blood cholinesterases (erythrocyte, whole blood, plasma) were monitored and their reactivatability (whole blood) was determined. During this treatment, the activities of erythrocyte acetylcholinesterase (AChE), plasma butyrylcholinesterase (BuChE) and whole blood cholinesterases were monitored. Atropine and obidoxime did not affect cholinesterase activities in control animals, whereas administration of obidoxime to dogs intoxicated with nerve agent caused an increase in the cholinesterase activities. The sensitivity of cholinesterases decreased in the order erythrocyte AChE > whole blood cholinesterases > plasma BuChE, respectively. Following sarin intoxication, blood cholinesterases were increased after the obidoxime administration. Intoxication with VX showed a similar picture but reactivation after the obidoxime administration was greater. In soman intoxication, the picture of cholinesterase changes was similar during the first 30 min of treatment. Then the increase in AChE activity following obidoxime administration was not as high as in the case of sarin and VX intoxication. Thus, the reactivation efficacy of obidoxime during nerve agent intoxication indicates that its repeated administration could be easily monitored using the reactivation test.