MS-302三道测量分析系统的应用及HI-6在离体膈神经-膈肌上抗梭曼的作用机制

目的 呼吸肌麻痹是有机磷酸酯类化合物严重中毒致死的主要原因之一,由于膈肌是最主要的呼吸肌,因此,建立一种精确灵敏的实验方法评价药物对离体膈神经 膈肌的影响, 探讨相应的抗呼吸肌麻痹药物的实验显得尤为重要。本文在MS-302三道测量分析系统上探讨了抗神经毒有效化合物HI-6在离体膈神经-膈肌上抗梭曼的作用机制。方法 采用MS-302三道测量分析系统,观察大鼠离体膈神经-膈肌的收缩功能,此方法较既往测量方法实时、高效而灵敏。观察膈肌收缩功能的同时,待标本稳定30 min后取膈肌置-20℃冰箱,留待测定正常乙酰胆碱酯酶(AChE)活性,然后向麦氏槽中加入以台式液配置的药物,加入药液量一般不超过0.5 ml,使药物浓度为10 μmol·L^-1～1 mmol·L^-1, 对照组加入等量的生理盐水, 于加药后不同时间点留取膈肌样品测定AChE活性,观察药物对膈肌AChE活性的直接影响。结果 ① MS-302三道测量分析系统上,对膈神经-膈肌标本强直收缩曲线下的面积进行测量,能较为直接的反映膈神经-膈肌标本的生理功能,且结果精确。② HI-6 10和100 μmol·L^-1对正常膈神经-膈肌收缩功能影响不明显,当药物浓度为1 mmol·L^-1时, HI-6对正常膈神经-膈肌收缩功能有抑制作用;HI-6 10 μmol·L^-1对梭曼抑制的标本收缩功能既没有保护作用也没有拮抗作用; HI-6 100 μmol·L^-1有一定的保护和拮抗作用;当剂量增加到1 mmol·L^-1时,HI-6的保护作用和拮抗作用都明显增强,且保护作用在30 min内最佳。③ 预先给予HI-6不能减弱梭曼对大鼠离体膈肌AChE的抑制,提示HI-6对梭曼抑制的大鼠离体膈肌AChE无明显保护作用;梭曼中毒后给予HI-6对梭曼抑制的膈肌AChE活性无显著影响,提示HI-6对梭曼抑制的膈肌AchE无明显重活化作用。结论 HI-6对梭曼抑制的离体膈神经-膈肌收缩功能的恢复可能是直接生理对抗作用,在本实验条件下未见膈肌AChE活力有明显的恢复。     

HI-6对正常及梭曼抑制大鼠离体膈神经-膈肌标本收缩功能的影响

目的　探讨HI 6对抗梭曼中毒所致呼吸抑制重活化作用外的其他可能作用机制。方法　采用MS 30 2三道生理记录仪 ,建立大鼠离体膈神经 膈肌收缩标本 ,观察HI 6对梭曼中毒膈神经 膈肌标本收缩功能的影响。结果　①梭曼 (2mg·L- 1)中毒后 ,膈肌收缩功能明显下降 ,中毒 10min内强直收缩曲线下面积下降至最低点 ,且在 3h内无明显恢复。②梭曼中毒前给予不同浓度的HI 6 (0 .0 1,0 .1,1.0mmol·L- 1) ,发现HI 6 (0 .1,1.0mmol·L- 1)对梭曼中毒所致膈肌强直收缩具有明显保护作用 ,且保护作用随着浓度的增加逐渐增强。③梭曼中毒后再给予HI 6 (0 .0 1,0 .1,1.0mmol·L- 1) ,发现 0 .0 1mmol·L- 1HI 6对中毒大鼠离体膈肌收缩功能无任何拮抗作用 ;当浓度增至 0 .1mmol·L- 1时 ,对膈肌强直收缩有一定的拮抗作用 ,但无统计学意义 ;但 1.0mmol·L- 1HI 6可显著对抗梭曼中毒所致膈肌收缩功能下降。结论　HI 6对梭曼中毒所致大鼠离体膈神经 膈肌收缩功能抑制具有明显拮抗作用 ,提示HI 6对抗梭曼中毒所致呼吸抑制作用机制存在酶保护作用外的直接生理对抗作用     

HI-6对梭曼染毒大鼠膈肌胆碱酯酶的重活化作用

目的研究HI-6对膈肌局部梭曼染毒大鼠膈肌胆碱酯酶的重活化作用。方法将大鼠麻醉后固定,沿腹正中线切开腹腔,暴露膈肌。梭曼局部染毒膈肌1和10min后,局部或全身给予HI-6,于给药后24和72h处死大鼠,取出中毒部位膈肌。用硫胆碱酶法显示终板的AChE活性,观察HI-6对胆碱酯酶的重活化作用。结果膈肌局部梭曼中毒1和10min后．局部给予HI-624和72h对局部中毒膈肌胆碱酯酶均没有重活化作用．在光镜下观察不到棕褐色椭圆形的运动终板：而膈肌局部梭曼中毒1和10min后,全身给予HI-6对局部中毒膈肌胆碱酯酶有明显的重活化作用,在光镜下可见到肌纤维上又出现了棕褐色椭圆形的运动终板。结论HI-6全身给药对早期膈肌局部中毒的神经运动终板胆碱酯酶有重活化作用。     

HI-6对梭曼中毒活体膈肌胆碱酯酶的重活化作用

目的 研究HI-6对局部梭曼中毒活体膈肌胆碱酯酶(ChE)活性是否具有重活化作用。方法 大鼠戊巴比妥钠肌注40 mg·kg^-1麻醉后固定,沿腹正中线切开腹腔,暴露膈肌。以右膈神经穿过膈肌处为中心,在右侧膈肌上贴敷一片大小为0.5 cm×0.75 cm的棉纸。用微量进样器抽取10 μl 0.2%梭曼生理盐水溶液滴加其上。左侧膈肌为自体正常对照或全身系统给予HI-6的药物对照,中毒后暂闭腹腔,30 min后取出棉纸。实验分为两组：一组在大鼠膈肌局部梭曼中毒后1 min和10 min后肌注HI-6 144 mg·kg^-1;一组在大鼠膈肌局部梭曼中毒后1 min和10 min后局部给予180 ml（1 mmol·L^-1） HI-6, 分别于给药后24和72 h处死大鼠, 取出中毒部位膈肌,按Karnovsky等改良的硫胆碱法显示终板的AChE活性。将膈肌标本在4%甲醛2~4℃固定24 h后,冻切30 μm,37℃孵育30 min,在光镜下观察。结果 膈肌局部梭曼中毒后1 min和10 min后局部给予HI-6,24和72 h对局部中毒膈肌ChE均没有重活化作用,在光镜下均观察不到棕褐色椭圆形的运动终板;而膈肌局部梭曼中毒后1 min和10 min后,全身给予HI-6对局部中毒膈肌ChE有明显的重活化作用, 在光镜下可见到肌纤维上又出现了棕褐色椭圆形的运动终板。结论 HI-6对早期膈肌局部中毒的神经运动终板ChE有重活化作用。     

敌敌畏对大鼠膈肌的直接毒性作用

目的　观察敌敌畏是否对膈肌具有直接抑制作用。方法　用自制双功能电极剌激膈神经或直接剌激离体大鼠膈肌 ,引起膈肌收缩 ,观察农药敌敌畏对膈肌的直接抑制作用。用箭毒阻断神经肌肉接头后 ,剌激膈神经不再引起膈肌收缩 ,直接刺激膈肌引起的收缩与神经肌肉接头无关 ,对这种收缩的影响为对膈肌的直接作用。结果　 10V直接刺激膈肌出现类似于强直收缩的波形。敌敌畏浓度大于1.4 5mmol·L- 1时可使该收缩显著抑制 ,4 .34mmol·L- 1时抑制 80 % ,5 .79mmol·L- 1完全抑制 ,氯磷定不能减轻之。浓度大于 3.62mmol·L- 1的敌敌畏对单收缩也有明显抑制。结论　敌敌畏对膈肌具有与神经肌肉接头阻断无关的直接抑制作用     

四逆汤对家兔膈肌疲劳的保护作用

目的 观察四逆汤对家兔膈肌疲劳的保护作用。方法 家兔乌拉坦麻醉,耳静脉注射油酸０．１ｍｌ／ｋｇ复制膈肌疲劳模型,剑突下分离膈肌条。分别记录膈肌收缩力及电活动变化。结果 腹腔注射四逆汤（２ｍｌ／ｋｇ）后可使正常家兔膈肌收缩力增（Ｐ〈０．０１）,膈肌放电积分曲线峰值（ｐｅａｋ）、积分曲线斜率（ｓｌｏｐ）增加（Ｐ〈０．０１）;复制膈肌疲劳后,腹腔注射四逆汤（２ｍｌ／ｋｇ）,可使疲劳膈肌收缩力增加（Ｐ〈０     

碘化二甲基木防己碱对神经肌肉接头传递的作用

碘化二甲基木防己碱(dimethyltrilobine iodide简称DTI)有降压作用,抗心律失常作用和骨骼肌松驰作用。本文采用离体大白鼠膈神经膈肌和小鸡颈二腹肌标本,进一步观察了DTI对神经肌肉系统的作用,并对其作用机制作了初步分析。选体重250～350 g大白鼠,按文献方法制备离体膈神经膈肌标本,固定在含循环Tyrode液的肌槽中,用JSD-731-C型刺激器,超强方波脉冲(波宽0.2ms,频率6/min)刺激膈神经,膈肌收缩用XWT-204型平衡记录仪描记。     

在不均匀牵拉的离体大鼠膈肌标本上梭曼的突触前和突触后作用

在离体大鼠膈肌上制备了一种不均匀牵拉肌肉以制动,便于用微电极记录终板电位(EPP)的标本(INSMP),避免了常规制动方法带来的制动药物、台氏液Ca~(2+),Mg~(2+)浓度改变或钳压肌肉导致膜电位(MP)下降的种种干扰。INSMP的MP和小终板电位(MEPP)正常,EPP幅度高达30mV以上,是研究药物对接头作用的较好标本。在INSMP上,梭曼(5.5μM)使MEPP频率加快,串刺激(50Hz)诱发的平均串EPPs幅度及其平均ACh 量子含量减少80％和77％。小剂量三碘季铵酚和六烃季铵可部分对抗梭曼的作用。梭曼引起强直收缩抑制主要原因为终板区蓄积的ACh作用于突触前N受体、负反馈地抑制ACh 量子释放;次要原因为作用突触后N受体、使其对ACh敏感性降低。     

MS-302三道生理信号测量分析系统应用及神经刺激装置的改进

目的 呼吸肌麻痹是有机磷酸酯类化合物严重中毒致死的主要原因之一，由于膈肌是最主要的呼吸肌，因此，建立在离体或在体膈神经-膈肌标本上寻找相应的抗呼吸肌麻痹药物的实验显得尤为重要。以往观察离体膈神经-膈肌标本的实验，多是在记纹鼓或平衡记录仪上进行的。它们均有一定的缺点。为此，我们率先引进了MS-302三道生理信号测量分析系统，该系统具有精确性高、可控性强及灵敏度好等优点，很好的满足了实验的要求。此外，实验中我们发现MS-302三道记录仪系统中的神经刺激器存在一定的局限性。如该刺激器只存在刺激膈神经的电极，只能刺激膈神经而不能刺激膈肌；只能用于研究整个膈神经-膈肌肌肉系统的功能状态，不能对神经-肌肉接头及膈肌本身的功能状态进行分别观察。针对上述缺点，我们对原有的单功能刺激电极进行了改进，改进后的刺激电极，可对肌肉和神经同时，分别或交替进行连续或不连续的刺激，并能将两者明确分开，可广泛应用于医学和生物学领域的各种实验，检测、生产和临床医疗。上述技术革新成果已成功申请专利(专利号为02247761．6)。     

离体膈肌疲劳模型及其沙美特罗和咖啡因的干预作用

目的 :建立低氧高碳酸性大鼠离体膈肌疲劳模型 ,探讨沙美特罗、咖啡因对此种大鼠膈肌疲劳的作用。方法 :用低氧高二氧化碳混合气造成膈肌疲劳模型 ,观察沙美特罗 (10 -9～ 10 -6mol·L-1)及咖啡因 (10 -5～ 10 -3 mol·L-1)对膈肌收缩力的影响。结果 :混合气降低了 5～ 12 0Hz所有频率下的张力 ,且使峰颤搐张力、维持张力、肌刺张力均下降 ;沙美特罗 10 -9～ 10 -6mol·L-1提高了力 -频率关系曲线中4 0～ 12 0Hz下的张力 ;咖啡因 10 -4和 10 -3 mol·L-1明显增强了 5～ 12 0Hz所有频率下的张力 (P <0 .0 1) ;同时增强了峰颤搐张力、维持张力及肌刺张力。结论 :通低氧高二氧化碳混合气 4min能够造成低氧高碳酸性大鼠离体膈肌疲劳模型 ,沙美特罗和咖啡因对此种大鼠膈肌疲劳有一定的改善作用     

Effect of HI-6, applied into the cerebral ventricles, on the inhibition of brain acetylcholinesterase by soman in rats

When applied to rats (intraperitoneally) immediately after subcutaneous injection of soman (120 micrograms/kg) HI-6 (100 mg/kg) protected about 40% of the activity of acetylcholinesterase (AChE) in the motor end plate region of the diaphragm but did not protect AChE in the brain. However, a partial protection of AChE in brain against inhibition by soman was obtained in anaesthetized, atropinized rats by the oxime injected into the cerebral ventricle 5 min before parenteral exposure to soman. The AChE activity in brain of rats pretreated with HI-6, analyzed 60 min after the injection of soman was between 10 and 19%, while that in non-protected animals did not exceed 1% of the control. The degree of protection of AChE in brain was dose-dependent. Large doses of HI-6 (greater than or equal to 100 micrograms) were tolerated by animals because of the pentobarbital anaesthesia which counteracted the lethal action of HI-6. The rate of "aging" of AChE in brain inhibited by soman was analyzed by intracerebroventricular injection of 200 micrograms of HI-6 at different time intervals after the subcutaneous injection of soman. A statistically-significant reactivation of inhibited AChE activity in brain was demonstrated when HI-6 was applied up to 20 min after soman. The protection and reactivation by HI-6 of both AChE in brain and AChE in muscle end plates in poisoning with soman appear to be quite similar.     

Mechanism of action of HI-6 on soman inhibition of acetylcholinesterase in preparations of rat and human skeletal muscle; comparison to SAD-128 and PAM-2

HI-6 is presently considered the most potent oxime antidote against soman poisoning in mice, rats, dogs and monkeys. However, it is still an open question whether efficiency of HI-6, observed in experimental animals, can be extrapolated to soman intoxicated humans. In this paper efficiency of HI-6 and possible mechanisms of action were compared in rat and human fresh muscle preparations. In rat muscle, about 50% of control AChE activity could be recovered by both therapeutic (5 min after soman) and prophylactic (5 min before soman) application of HI-6. On the other hand, in human muscle therapeutic treatment restored only 5%, while prophylactic application of HI-6 again resulted in about 50% recovery of control AChE activity. As revealed by comparison of the prophylactic effects of HI-6 and the non-oxime bispyridinium compound SAD-128, competitive inhibition of AChE plays a minor role as a protective mechanism. Immediate reactivation of rapidly aging human AChE must therefore be instituted for successful protective treatment by HI-6. Retardation of aging, a direct effect of SAD-128, was roughly estimated to improve reactivation by HI-6 for about 10% of control AChE activity of the human muscle. PAM-2 proved completely inefficient as a therapeutic and as a prophylactic agent on both rat and human muscle preparations.     

Evaluation of several oximes as reactivators of unaged soman-inhibited whole blood acetylcholinesterase in rabbits

The antidotal benefit of oximes against organophosphorus (OP) anticholinesterase intoxication is thought to be due to reactivation of the OP-inhibited acetylcholinesterase (AChE). This study was conducted to determine whether the antidotal efficacy against soman by the oximes 2-hydroxyiminomethyl-3-methyl-1-[2-(3-methyl-3-nitrobutyl o Cl (ICD 467) and 1,1′-methylenebis[4-(hydroxyiminomethyl) pyridinium] di-Cl (MMB-4) resulted, in part, from reactivation of the inhibited AChE. These oximes were tested in parallel with pralidoxime Cl (2-PAM) and 1-(2-hydroxyiminomethyl-1-pyridinio-3-(4-carbamoyl-1-pyridinio)-2-oxapropane di-Cl (HI-6). Rabbits were atropinized (8 mg/kg, i.m.) and intoxicated with soman (13 μg/kg, i.V.; 1.2 × ₅₀) 5 min later. Three minutes after soman, animals were treated with oxime (50, 100 or 150 μmol/kg, i.m.). Whole blood was collected from a catheter in the central artery of the ear just before soman, at 2 min after soman and at 2, 5, 10, 15, 30, and 60 min after oxime or vehicle for determination of AChE activity. Shortly thereafter, animals were anesthetized and exsanguinated with immediate flushing using heparinized saline. AChE activity was also determined on the cortex, medulla-pons and diaphragm to assess central and peripheral reactivation. Treatment with HI-6 or MMB-4 (50 μmol/kg, i.m.) resulted in significant (P < 0.05) reactivation of soman-inhibited whole blood AChE and diaphragm cholinesterase (ChE), but not brain AChE. In contrast, 2-PAM was completely ineffective in reactivating somaninhibited AChE. HI-6 was significantly better than MMB-4 in reactivating blood AChE; they were essentially equal against soman-inhibited diaphragm ChE. Three animals exposed to soman and treated with ICD 467 died within 15 min. When animals not exposed to soman were treated with ICD 467 (25 μmol/kg, i.m.), whole blood AChE activity was depressed by 60% within 5–10 min after treatment. Furthermore, ICD 467 failed to reactivate significantly unaged soman-inhibited erythrocyte AChE, in vitro. These observations indicate that ICD 467 would be contraindicated as a therapy for anti-ChE intoxication and that the efficacy of HI-6 or MMB-4 can be explained, in part, by reactivation of somaninhibited AChE.     

The efficacy of some bis-pyridinium oximes as antidotes to soman in isolated muscles of several species including man

Previous results had shown that bis-pyridinium oximes, particularly HI-6 are quite effective therapeutically in soman-poisoned rats and mice in vivo and in the rat diaphragm preparation in vitro. The aim of the present study was to investigate the efficacy of bis-pyridinium oximes on soman-inhibited neuromuscular transmission in muscle preparations from several species including man. The muscles tested were preparations of rat diaphragm and intercostal muscle, guinea-pig diaphragm, dog external intercostal muscle and human external interscotal muscle. These muscles were stimulated indirectly with field stimulation. With a few exceptions the preparations were exposed to soman for 2.5 or 15 min. In some cases different exposure times were employed or the organophosphate sarin was administered instead of its analogue soman. After the degree of inhibition of neuromuscular transmission had been established, oximes were added to the bath fluid. After washout 15 min later, recovery of neuromuscular transmission was tested. Subsequently, a second dose of soman was administered to investigate whether the recovery observed had been caused by cholinesterase reactivation. The results of these experiments indicate that the oximes tested, mostly HI-6, were quite effective as soman antidotes in muscle preparations of rats, guinea-pigs and dogs. In the human preparation while these oximes were quite effective after sarin intoxication they were essentially without effect against soman.     

Therapeutic efficacy of HI-6 in soman-poisoned marmoset monkeys.

The therapeutic efficacy of the oxime HI-6 against intoxication with the irreversible cholinesterase (ChE) inhibitor soman was tested in marmoset monkeys. Five out of six marmosets, intoxicated with 5 x LD50 soman and treated immediately with diazepam (0.2 mg.kg-1 iv) and 15 sec later with atropine (0.5 mg.kg-1 im) and HI-6 (50 mg.kg-1 im), survived for more than 24 hr. One of these animals died after 4 days. In the HI-6-treated marmosets blood ChE activity was inhibited at a rate slower than that in three animals treated similarly but with saline instead of HI-6. The latter marmosets died within 8 min after soman. HI-6 achieved its plasma peak 5 min after injection and was eliminated with a t1/2 of about 40 min. In a second experiment similarly treated marmosets were euthanized at 5 min (three saline-treated animals) or at 10 min (three HI-6-treated animals) after the soman intoxication to enable the determination of acetylcholinesterase (AChE) activities in diaphragm and brain tissue. In addition, in these animals blood AChE and butyrylcholine esterase (BuChE) activities were determined. Low AChE activities were encountered in diaphragms and brains. These levels were not significantly different between saline- and HI-6-treated marmosets. In vitro treatment with HI-6 at 40 min after soman still led to an increase of the AChE activity, which was significant in diaphragm, suggesting that postmortem AChE inhibition had occurred. The ratio of AChE to BuChE in blood was significantly enhanced in HI-6-treated animals, indicating that HI-6 preferentially reactivated AChE. It is concluded that (i) HI-6 is an effective treatment against soman poisoning in marmosets and (ii) AChE reactivation or protection by HI-6 contributed to the survival of the animals.     

Soman and sarin inhibition of molecular forms of acetylcholinesterase in mice. Time course of recovery and reactivation by the oxime HI-6.

The in vivo sensitivity of the molecular forms of the enzyme acetylcholinesterase to inhibition by either soman or sarin, reactivation by HI-6 and the time course of recovery following inhibition by soman were investigated in mice. Administration of HI-6 (50 mg/kg, i.p.) immediately after soman (100 micrograms/kg, s.c.) or sarin (150 micrograms/kg, s.c.) resulted in an apparent selective reactivation of the 10S and 16S molecular forms of acetylcholinesterase and no reactivation of the 4S form of diaphragm acetylcholinesterase. The apparent selectivity of the reactivation of the molecular forms of the acetylcholinesterase was probably due to the fact that the 10S and 16S forms of acetylcholinesterase are located primarily extracellularly and the 4S form intracellularly. The HI-6 was restricted primarily to the extracellular compartment due to its quaternary, hydrophilic nature. If the administration of HI-6 was delayed until 60 min following soman (100 micrograms/kg, s.c.) injection, no reactivation of any of the molecular forms of acetylcholinesterase could be found in the diaphragm. The soman-inhibited acetylcholinesterase had probably aged and, thus, was not susceptible to reactivation by HI-6. The time course of recovery of the molecular forms in the diaphragm occurred rather quickly with the smaller 4S and 10S forms recovering to control levels faster than the larger 16S form. It took between 8 and 16 days for the 16S form to recover to normal. In the brain, hypothalamic acetylcholinesterase molecular forms such as the 4S recovered faster than the 10S form which had not recovered to control 16 days after soman administration; the 16S form of acetylcholinesterase was not detected in the brain.     

Therapy of organophosphate poisoning in the rat by direct effects of oximes unrelated to ChE reactivation

Isolated rat diaphragm preparations treated with soman or with the irreversible and oxime resistant cholinesterase (ChE) inhibitor S₂₇ (see Compounds) showed a considerable recovery of neuromuscular transmission (NMT) during incubation with the (bis)pyridinium oximes HI-6, HGG-12, P₂S and obidoxime. In the soman-treated preparations this NMT recovery was predominantly caused by reactivation of acetylcholinesterase (AChE) but in the S₂₇-treated preparations it was caused by a direct (pharmacological) effect unrelated to enzyme reactivation. Atropinized rats were artificially ventilated after injection with 3×LD₅₀ soman for 3 h and then treated with HI-6, i.e. at a time when oxime reactivation of soman inhibited ChE is no longer possible. Nevertheless, these rats started to breathe spontaneously and 50–60% survived more than 24 h, whereas all control animals (saline instead of HI-6) died within 10 min after artificial ventilation was terminated. In such animals no significant reactivation of ChE activity at various time intervals following HI-6 treatment was found, either in the diaphragms or in the brains. There was a significant amount of NMT (50%) in vitro in diaphragms obtained from these animals. This NMT did not improve in vitro in the presence of HI-6 and was not inhibited by soman administered to the medium. It is concluded that in this case the NMT found was based on synaptic adaptation to the continued inhibition of ChE and that the survival of the animals might be due to a combination of this synaptic adaptation and central direct effects of HI-6.     

Organophosphate poisoning: a method to test therapeutic effects of oximes other than acetylcholinesterase reactivation in the rat.

A method was developed to study exclusively those therapeutic effects of oximes that are not related to reactivation of organophosphate-inhibited acetylcholinesterase (AChE). The model uses the organophosphorus compound crotylsarin (CRS), which proved to be a potent, irreversible, peripherally and centrally active AChE inhibitor with a very short biological half-life. CRS-inhibited AChE appeared to age very rapidly, because in vitro addition of oximes immediately following inhibition, did not result in any AChE reactivation. Anaesthetized, atropinized and artificially ventilated rats were intoxicated with 3 x LD50 CRS and treated 5 min later with the bispyridinium oxime HI-6. Fifty percent of these animals survived more than 24 h following termination of artificial ventilation at 10 min after oxime treatment. The mean survival time of the remaining animals was 66 min, whereas all untreated animals died within 4 min. HI-6, when added in vitro to isolated intact hemidiaphragms, or to diaphragm or brain homogenates from rats which had been killed 1 min following 3 x LD50 CRS, failed to reactivate the inhibited AChE. If blood was sampled (before and) after HI-6 administration to CRS-intoxicated rats, no HI-6-induced AChE reactivation was observed. Yet, a clear improvement of the neuromuscular transmission in the hindleg muscles of these animals was found following HI-6 injection. With this model, decisive evidence is obtained that non-reactivating effects of HI-6 by themselves are therapeutically relevant.     

HI-6 therapy of soman and tabun poisoning in primates and rodents

The bis-pyridinium oxime HI-6, in conjunction with atropine, was found to offer significant protection against multiple LD₅₀ challenges with the organophosphorus compounds soman and tabun. In adult rhesus macaques, the therapeutic administration of HI-6 with atropine and diazepam protected three of four animals from the lethal effects of 5 × LD₅₀ of soman and three of three animals from 5 × LD₅₀ of tabun. However, when toxogonin was substituted for HI-6 in the therapeutic mixture, all three animals poisoned with 5 × LD₅₀ of soman died. In rats, the 24 h protective ratios against tabun and soman with HI-6 were 2 and 3.5, respectively, whereas in guinea pigs these values were between 4 and 6 for both agents. No evidence was obtained for acetylcholinesterase (AChE) reactivation by HI-6 in tissue from tabun-poisoned rodents or following soman or tabun in primate plasma. The results underscore the significant therapeutic benefit of HI-6 in primates, a species specific efficacy against tabun, and argue for some mechanism of action of HI-6 at least partly unrelated to AChE reactivation.