Acetylcholinesterase inhibition and anti-soman efficacy of homologs of physostigmine.

Inhibition of acetylcholinesterase (AChE) activity by physostigmine (PHY) is reversible due to spontaneous decarbamylation. Physostigmine has been shown to be effective as a pretreatment against potent anticholinesterase poisons (e.g., soman) in experimental animals, yet it is short acting and causes undesirable side effects in mammals. The two-fold purpose of this study was 1) to determine whether extension of the N-substituted alkyl chain (N-SAC) of PHY from N-methyl to N-ethyl (I), N-propyl (II), N-isopropyl (III), N-butyl (IV) or N-heptyl (V) affects anti-AChE potency and spontaneous decarbamylation of inhibited AChE of guinea pig blood in vitro and in vivo, and 2) to see whether chain extension affects efficacy as pretreatment in poisoning by soman. The in vitro AChE inhibition studies were done using whole blood incubated at 37 degrees C for 30 min. All 5 homologs possessed anti-AChE activity with I50s ranging from 1.1 to 27.6 x 10(-7)M; compound III was the least potent in vitro and in vivo. Lengthening of the N-SAC of PHY markedly extended the duration of anti-AChE activity when compared to PHY, but rendered the modified compounds ineffective as pretreatments against soman. These data support the premise that the decrease in decarbamylation rates observed upon extending the N-SAC of PHY is responsible for the loss of effectiveness of pretreatment regimens against soman. Perhaps, these homologs of PHY may have potential use in instances where sustained action of acetylcholine is required at cholinergic junctions because of disease conditions or drug overdosage.     

Scopolamine augments the efficacy of physostigmine against soman poisoning in guinea pigs

The efficacy of the subchronically administered cholinesterase-inhibitor physostigmine (PHY) (0.025 mg/kg/h) either with or without the muscarinergic receptor antagonist scopolamine (SCO) (0.018 mg/kg/h) in counteracting soman-induced lethality and incapacitation were determined in guinea pigs. This was tested in animals that either received atropine sulphate (AS, 17.4 mg/kg i.m.) or no postintoxication therapy. Behavioral and neurophysiological readout systems were used to measure postintoxication incapacitation. Only the pretreatment with PHY alone did not offer any protection against 2x LD50 soman intoxication. Animals that received the complete treatment (PHY + SCO + AS) did not show any abberations in the performance of learned behavior. The use of AS after soman intoxication resulted in an increase of the startle response, whereas the addition of SCO to the pretreatment led to a more persistent duration of the effect in time. In case one has to rely completely on the pretreatment, the addition of SCO to PHY is life-saving. However, some postintoxication incapacitation is still present. Therefore, the pretreatment regime may perhaps further be improved by the addition of a nicotinic antagonist.     

Protection of mice against soman by pretreatment with eptastigmine and physostigmine

Organophosphate (OP) compounds such as the nerve agents sarin, soman and VX are powerful inhibitors of acetylcholinesterases (AChEs), butyrylcholinesterases (BChEs), and carboxylesterases (CaEs) The acute toxicity of OPs is the result of their irreversible binding with AChEs in the nervous system, which elevates the acetylcholine (ACh) levels. In this study the protective actions of intravenously (i.v.), administered eptastigmine and physostigmine in acute soman intoxication were studied in mice. The mice received eptastigmine (0.9 mg/kg body weight) or physostigmine (0.1 mg/kg body weight) 10 min prior to the intraperitoneal (i.p.) administration of soman. To avoid possible signs of poisoning, the animals received atropine 37.5 mg/kg body weight subcutaneously (s.c.) in saline immediately after soman injection. Eptastigmine was the most effective carbamate against soman intoxication. The LD50 value of soman was 0.44 mg/kg, and the protective ratios of eptastigmine and physostigmine were 2.1- and 1.3-fold, respectively. Both eptastigmine and physostigmine had protected AChEs when measured 24 h after soman exposure. In this study, there was no inhibition of microsomal CaEs in soman treated mice. Nonetheless, the role of microsomal CaEs might be more important with prophylaxis at multiple LD50s of soman. In conclusion, these results indicate that eptastigmine treatment given i.v. protects better than physostigmine against soman exposure.     

Antagonism of physostigmine induced lethality by a combination of scopolamine and methscopolamine

The antagonistic effects of scopolamine, methscopolamine, and a combination of methscopolamine and scopolamine were evaluated in preventing physostigmine induced lethality in Swiss Webster mice. Low dose scopolamine was found to be highly effective in reversing high dose (3 X the LD100) physostigmine induced lethality, in contrast to methscopolamine, which was ineffective except in very high doses. A combination of scopolamine and methscopolamine was more effective than either drug alone.     

梭曼和毒扁豆碱引起大鼠脑电癫痫波的M和N胆碱能成分

在加拉碘铵麻痹, 人工呼吸维持和甲基阿托品预防的大鼠上, 梭曼0.1 mg·kg^-1 im (n=24)或毒扁豆碱30.0 mg·kg^-1 iv (n=18)使全部大鼠脑电图(EEG)上出现早期持续很短的紧张性癫痫波和随后持续很长的阵挛性癫痫波. 中枢N受体激动剂烟碱1.0 mg·kg^-1 iv (n=38)和中枢M受体激动剂槟榔碱150 mg·kg^-1 iv (n=46)或匹鲁卡品380 mg·kg^-1 iv (n=24)能分别在EEG癫痫波的特征, 出现时间和持续时间上模拟出上述早期紧张性癫痫波和随后的阵挛性癫痫波. 预先小剂量iv烟碱使中枢N受体脱敏或给小剂量N受体拮抗剂美加明保护中枢N受体后, 梭曼只能引起潜伏 期较长且持续很久的阵挛性癫痫波. 胆碱酯酶抑制剂梭曼和毒扁豆碱较大剂量引起大鼠EEG癫痫波可能的机理是过量的乙酰胆碱作用于潜伏期短, 容易脱敏的N受体出现早期持续很短的EEG紧张性癫痫波, 又作用于潜伏期较长, 不容易脱敏的M受体, 出现稍后的EEG阵挛性癫痫波.     

Acetylcholinesterase Inhibition by (+)Physostigmine and Efficacy against Lethality Induced by Soman

ABSTRACT

The optical isomer (+)Physostigmine [(+) Phy] is a very weak anticholinesterase. In a recent report, pretreatment with (+)Phy, at a dose which failed to inhibit acetylcholinesterase (AChE), and atropine provided efficacy against a lethal dose of sarin (SYNAPSE:2,139,1988). It was of interest to see whether (+)Phy could protect against soman at a dose which caused only marginal inhibition of the whole blood (WB) AChE in guinea pigs (GPs). (-)Phy (0.15 rog/kg, im) and (+)Phy (10.0 mg/kg, im) produced nearly 70% inhibition of WB AChE at 30 min whereas (+)Phy (0.15 rog/kg, im) caused only marginal inhibition. Groups of guinea pigs (20/group) were dosed, im, with (-)Phy (0.15 mg/kg), (+)Phy (0.15 rog/kg), (+)Phy (10.0 mg/kg) or vehicle (0.5 ml/kg) respectively in one thigh while the mild anticholinergic trihexyphenidyl (THP), 2.0 mg/kg, was injected into the other thigh of 10 animals from each of the respective groups. Thirty min after pretreatment, all animals were challenged with soman (60 ug/kg, sc; 2 LD₅₀S); this dose of soman is lethal in unprotected animals. (-)Phy or (+)Phy (10 rog/kg) alone protected nearly 50% from soman lethality, and in combination with THP, all animals survived. In contrast, (+)Phy (0.15 mq/kq; alone or together with THP) was completely ineffective against a 2 LD₅₀ challenge of soman. These data support the hypothesis that protection against soman-induced lethality is related to the degree of carbamylation of the AChE just prior to challenge.     

梭曼和毒扁豆碱引起大鼠脑电癫痫波的M和N胆碱能成分

在加拉碘铵麻痹，人工呼吸维持和甲基阿托品预防的大鼠上，梭曼ＦＳ０．１ｍｇ·ｋｇ－１ｉｍ（ｎ＝２４）或毒扁豆碱３０．０ｍｇ·ｋｇ－１ｉｖ（ｎ＝１８）使全部大鼠脑电图（ＥＥＧ）上出现早期持续很短的紧张性癫痫波和随后持续很长的阵挛性癫痫波．中枢＃ＦＳＮ＃ＦＫ受体激动剂烟碱１．０ｍｇ·ｋｇ－１ｉｖ（ｎ＝３８）和中枢Ｍ受体激动剂槟榔碱１５０ｍｇ·ｋｇ－１ｉｖ（ｎ＝４６）或匹鲁卡品３８０ｍｇ·ｋｇ－１ｉｖ（ｎ＝２４）能分别在ＥＥＧ癫痫波的特征，出现时间和持续时间上模拟出上述早期紧张性癫痫波和随后的阵挛性癫痫波．预先小剂量ｉｖ烟碱使中枢Ｎ受体脱敏或给小剂量Ｎ受体拮抗剂美加明保护中枢Ｎ受体后，梭曼只能引起潜伏期较长且持续很久的阵挛性癫痫波．胆碱酯酶抑制剂梭曼和毒扁豆碱较大剂量引起大鼠ＥＥＧ癫痫波可能的机理是过量的乙酰胆碱作用于潜伏期短，容易脱敏的Ｎ受体出现早期持续很短的ＥＥＧ紧张性癫痫波，又作用于潜伏期较长，不容易脱敏的Ｍ受体，出现稍后的ＥＥＧ阵挛性癫痫波     

Antidotal efficacy of quinuclidinium oximes against soman poisoning

The efficiency of newly synthesized oxime derivatives of quinuclidinium were tested in vitro on soman inhibited acetylcholinesterase (AChE) of human erythrocytes and in vivo using soman poisoned mice. For this purpose, the inhibitory power of oximes (IC₅₀), acute toxicity (LD₅₀) as well as reactivating and protective capacities with respect to soman-inhibited AChE were determined for each of the oximes. All oximes tested were ineffective in vitro but protected mice very efficiently (BM-1 protects against 4LD₅₀ of soman). The results indicate that the in vivo effectiveness of quinuclidinium oximes against soman poisoning may not be related to reactivation or protection of AChE but rather to some other mechanism of the cholinergic system. Received: 7 August 1996 / Accepted: 21 January 1997     

Antidotal efficacy of pyridinium chloride derivatives against soman poisoning

Acetylcholinesterase (AChE; EC 3.1.1.7.) is an extremely active enzyme necessary for terminating the action of acetylcholine in cholinergic synapses. The aim of this study was to evaluate the efficacy of four mono-pyridinium compounds 1-phenacylpyridinium chloride (I), 1-phenacyl-2-methylpyiridinium chloride (II), 1-benzoylethylpyridinium chloride (III), and 1-benzoylethylpyridinium-4-aldoxime chloride (IV) in the therapy of soman poisoning. Their effect was compared with HI-6 and TMB-4 oximes. The inhibitory potency (IC50) of compounds as well as reactivating (%R) and protective potency (P50) with respect to soman-inhibited AChE were determined for each of the compounds. Their acute intraperitoneal toxicity (LD50 with 95% confidence limits) was tested in mice and observed for 24 hr. The therapeutic effect was expressed as the protective index and as the therapeutic dose. The tested compounds were found to be reversible inhibitors of AChE. In vivo results show that the tested compounds are relatively toxic (their LD50 was from 74.9 to 210.0 mg/kg body weight). The best antidotal efficacy was obtained with compound II, which had the highest affinity for AChE (IC50 was 1.9 x 10(-5) mol l(-1)) and seems to be an adequate antidote in soman poisoning (its protective index and therapeutic dose were 2.8 and 2, respectively). Our results indicate that its antidotal effect is related to the reactivation or protection of AChE. The type of the substituent in the pyridinium ring generally has a significant influence on toxicity in vitro and in vivo, and on the antidotal efficacy of all new tested compounds.     

Protection by sustained release of physostigmine and procyclidine of soman poisoning in rats

The efficacy of a combinational prophylactic regimen on the lethality, convulsions, and loss of morphological and functional integrities of the brain induced by an organophosphate soman was investigated in rats. The rats were implanted subcutaneously with osmotic minipumps containing the combinational prophylactic regimen composed of physostigmine, a reversible cholinesterase inhibitor, and procyclidine, an N-methyl-D-aspartate antagonist possessing anticholinergic action, for 3 days, and intoxicated subcutaneously with soman (160 microg/kg, 1.3 LD50). The doses of combinational regimen in minipumps were optimized to achieve 30-35% inhibition of blood cholinesterase activity by physostigmine and 50-100 ng/ml of blood concentrations of procyclidine as clinically available doses, respectively. In comparison, 1-[([4-(aminocarbonyl)pyridinio]methoxy)methyl]-2-[(hydroxyimino)methyl]pyridinium (HI-6, 125 mg/kg) was administered intraperitoneally 30 min prior to the soman challenge in control groups to reduce mortality of rats without affecting convulsions. Soman induced profound limbic convulsions and 30% mortality, leading to increased blood-brain barrier permeability, neural injuries, learning and memory impairments, and physical incapacitation of survived rats pretreated with HI-6. The combinational regimen, at optimal doses without adverse effects on passive avoidance performances (72 microg/kg/h of physostigmine plus 432 microg/kg/h of procyclidine), exerted full protective effects against lethality, convulsions, blood-brain barrier opening, brain injuries, learning and memory impairments, and physical incapacitation induced by soman. Taken together, it is suggested that the combination of physostigmine and procyclidine, at adequate doses, could be a choice to provide the victims of organophosphate poisoning with chance of intensive care for survival and neuroprotection.     

Neuroprotective efficacy of caramiphen against soman and mechanisms of its action

BACKGROUND AND PURPOSE

Caramiphen is a muscarinic antagonist with potent anticonvulsant properties. Here, we investigated the efficacy of caramiphen against behavioural seizures and neuropathology induced by the nerve agent soman, and revealed two mechanisms that may underlie the anticonvulsant efficacy of caramiphen.

EXPERIMENTAL APPROACH

Rats were given caramiphen at 30 or 60 min after treatment with soman. Neuronal loss in the basolateral amygdala (BLA) and neuronal degeneration in the amygdala, hippocampus, piriform cortex, entorhinal cortex and neocortex, were investigated 24 h after soman, using design-based stereology and FluoroJade-C staining. The effects of caramiphen on NMDA-, AMPA- and GABA-evoked currents were studied in the BLA region of in vitro brain slices from un-treated rats, using whole-cell recordings.

KEY RESULTS

Caramiphen given either 30 min or 60 min after soman, suppressed behavioural seizures within 10 min, but required 1∼4.5 h for complete cessation of seizures. Neuronal loss and degeneration were significantly reduced in the caramiphen-treated, soman-exposed rats. Postsynaptic currents evoked by puff-application of NMDA on BLA principal cells were reduced by caramiphen in a dose-dependent manner (100 µM, 300 µM and 1 mM), while GABA-evoked currents were facilitated by 100 µM and 300 µM, but depressed by 1 mM caramiphen. AMPA-evoked currents were not affected by caramiphen.

CONCLUSIONS AND IMPLICATIONS

Caramiphen offered partial protection against soman-induced seizures and neuropathology, even when given 60 min after soman. NMDA receptor antagonism and facilitation of GABAergic inhibition in the BLA may play a key role in the anticonvulsive and neuroprotective properties of caramiphen.     

Protection by a transdermal patch containing physostigmine and procyclidine of soman poisoning in dogs

The prophylactic efficacy of a combinational patch system containing physostigmine and procyclidine against soman intoxication was evaluated using dogs. Female beagle dogs (body weights 9-10 kg) were shaved on the abdominal side, attached with a matrix-type patch (7x7 cm) containing 1.5% of physostigmine plus 6% procyclidine for 2 days, and challenged with subcutaneous injection of serial doses (2-10 LD50) of soman. Separately, in combination with the patch attachment, atropine (2 mg/dog) plus 2-pralidoxime (600 mg/dog) or atropine plus 1-[([4-(aminocarbonyl)pyridinio]methoxy)methyl]-2-[(hydroxyimino)methyl]pyridinium (HI-6, 500 mg/dog) were injected intramuscularly 1 min after soman poisoning. The LD50 value of soman was determined to be 9.1 microg/kg, and high doses (> or = 1.4 LD50) of soman induced salivation, emesis, defecation and diarrhea, tremors and seizures, and recumbency of dogs, leading to 100% mortality in 24 h. The prophylactic patch, which led to mean 18.5-18.8% inhibition of blood cholinesterase activity by physostigmine and mean 7.9-8.3 ng/ml of blood concentration of procyclidine, exerted a high protection ratio (4.7 LD50), in comparison with relatively-low effects of traditional antidotes, atropine plus 2-pralidoxime (2.5 LD50) and atropine plus HI-6 (2.7 LD50). Noteworthy, a synergistic increase in the protection ratio was achieved by the combination of the patch with atropine plus HI-6 (9 LD50), but not with atropine plus 2-pralidoxime (5 LD50). In addition, the patch system markedly attenuated the cholinergic signs and seizures induced by soman, especially when combined with atropine plus HI-6, leading to elimination of brain injuries and physical incapacitation up to 6 LD50 of soman poisoning. Taken together, it is suggested that the patch system containing physostigmine and procyclidine, especially in combination with atropine and HI-6, could be a choice for the quality survival from nerve-agent poisoning.     

Prophylactic and therapeutic efficacy of memantine against seizures produced by soman in the rat.

Male Sprague-Dawley rats injected sc with a single sublethal dose of the organophosphate nerve agent, soman (100 micrograms/kg), had motor limbic seizures within 5-15 min. Pretreatment with a single dose of memantine HCl (MEM, 18 mg/kg, sc), alone or in combination with atropine sulfate (ATS, 16 mg/kg, sc), before soman prevented seizures without sedation or ataxia. Rats appeared normal or demonstrated increased exploratory activity. Excessive salivation, a peripheral manifestation of soman intoxication, was decreased by ATS, but pretreatment with ATS alone did not prevent seizures. After seizure onset, MEM +/- ATS, but not ATS, abolished seizures. Acetylcholinesterase (AChE) activity in several brain regions (cortex, stem, striatum, and hippocampus) was markedly reduced by soman, but not by MEM, ATS, or MEM + ATS. Preadministration of MEM + ATS in vivo significantly protected AChE from inhibition by soman. Memantine reduced inhibition of AChE activity in crude brain homogenates by soman, but not by edrophonium (anionic site inhibitor) or decamethonium (peripheral site inhibitor). Thus, MEM may bind to a different modulatory site, not yet characterized, to protect AChE. When given after onset of soman-induced seizures, treatment with MEM +/- ATS did not reactivate AChE although seizures were controlled, suggesting additional anticonvulsant mechanisms of action. At concentrations (10(-4) to 5 x 10(-4) M) which did not significantly alter the spontaneous firing of action potentials (APs), MEM limited sustained high frequency repetitive firing (SRF) induced by depolarization of spinal cord (mouse and rat) and neocortical (mouse) neurons in monolayer-dissociated cell culture. In the same range of concentrations, ATS both limited SRF and suppressed spontaneous activity, suggesting toxicity. In addition, MEM and ATS reversibly produced use-dependent block of depolarizing responses to acetylcholine (ACh) applied by pressure ejection to spinal cord neurons. Thus, the anticonvulsant efficacy of MEM, with or without ATS, may have resulted from a combination of actions, including protection of AChE from inhibition by soman, limitation of high frequency firing of APs, and blockade of excitatory postsynaptic responses to ACh.     

Toxicity of anticholinesterases: interactions of pyridostigmine and physostigmine with soman

This investigation was conducted to assess the potential of carbamate pretreatment to exacerbate the ill effects of low doses of soman. Ambulatory Activity in a photocell cage (AA) and performance time on an accelerating rotarod (ARR) were used to test for interactions between pyridostigmine or physostigmine and soman. ED50s (i.e., dosages sufficient to reduce ARR time and AA to 50% of control level) of each carbamate (IM) and soman (SC) were determined. The ED50 values (mg/kg) in the ARR test were 3.2, 0.21, and 0.072 for pyridostigmine, physostigmine and soman, respectively, while in the AA test the corresponding values were 1.8, 0.072 and 0.060. The matrix of 16 combinations of 0, 1, 2/3, and 1/3 ED50 each of carbamate and soman was studied in each test system, as well as the effect of behavioral deficit free (BDF) dosages of each carbamate on the ED50s of soman. In both the AA and ARR tests the matrix of combinations of pyridostigmine and soman indicated an additive effect. In contrast, physostigmine produced one instance of potentiation in each test system and anatagonism in two combinations in the AA procedure. A BDF dosage of each carbamate (0.056 mg/kg of pyridostigmine and 0.026 mg/kg of physostigmine) gave no evidence of adding to the deficit in AA induced by soman. In the ARR test, the ED50 of soman was lower by 11% with pyridostigmine pretreatment and by 14% with physostigmine; the latter just reached statistical significance (p less than 0.05). Although additivity was most often found at higher dosages of pyridostigmine and physostigmine, at the BDF dosages little or no adverse interaction was found between Pyridostigmine or Physostigmine and low levels of soman.     

Stress adversely affects efficacy of physostigmine-scopolamine pretreatment against soman in guinea pigs

During military operations, soldiers may be exposed to mixtures of chemicals and to physical, emotional and psychological stress factors, which all may influence efficacy of any treatment, including the nerve agent pretreatment regimen. The purpose of this study was therefore to investigate the influence of chronic intermittent, variable, unpredictable and uncontrollable stress conditions on the side effects and therapeutic efficacy of the combination of physostigmine (0.025 mg/kg/h) and scopolamine (0.018 mg/kg/h) as a pretreatment against 2 x LD50 soman intoxication in guinea pigs. Stress during pretreatment led to an increase of motor activity and an increase of power in the EEG delta2 frequency band. After chronic stress, exposure of pretreated animals to 2 x LD50 soman resulted in more severe intoxication symptoms, a more persistent effect on the startle response, and considerable more severe and persistent effects on the EEG power-spectrum, indicating irreversible brain damage.     

Effects of physostigmine, paraoxon and soman on brain GABA level and metabolism

The effects of sublethal doses of physostigmine, paraoxon and soman on GABA levels and metabolism were studied in small rat brain areas (hypothalamus, striatum, cerebellum, rest of the brain). Physostigmine induced a significant decrease in striatal GABA level and a reduction of synthesis index in the rest of the brain while organophosphates have little or no effect on GABA level and metabolism. This work provides new data about the physostigmine effect on brain GABA which could be related to the action of the anticholinesterase agents on other non-cholinergic neurotransmitters.     

Subchronic physostigmine pretreatment in marmosets: absence of side effects and effectiveness against soman poisoning with negligible postintoxication incapacitation.

Subchronic pretreatment with physostigmine (PHY) (0.0125 mg/kg/h) leading to a blood acetylcholinesterase inhibition of about 30% caused no side effects when applied to marmoset monkeys. This was evident on behavioral parameters and on EEG and cortical visual evoked response. Furthermore, this treatment regime, followed by atropine as postintoxication therapy, protected the marmosets against lethality after a 2 x LD50 dose of soman with negligible postintoxication incapacitation. These findings suggest that a symptom-free pretreatment with subchronic PHY could protect man sufficiently against severe soman intoxication.     

Minimum effective drug concentrations of a transdermal patch system containing procyclidine and physostigmine for prophylaxis against soman poisoning in rhesus monkeys

A transdermal patch system containing procyclidine, an N-methyl-d-aspartate receptor antagonist possessing anticholinergic action, and physostigmine, a reversible cholinesterase inhibitor, was developed, and its prophylactic efficacy against soman intoxication was investigated. Male rhesus monkeys were shaved on the dorsal area, attached with a matrix-type patch with various sizes (2 × 2 to 7 × 7 cm) for 24 or 72 h, and challenged with 2 × LD₅₀ doses (13 μg/kg) of soman. The smallest patch size for the protection against lethality induced by soman intoxication was 3 × 3 cm, resulting in blood procyclidine concentration of 10.8 ng/ml, blood physostigmine concentration of 0.54 ng/ml, which are much lower concentrations than maximum sign-free doses, and blood cholinesterase inhibition of 42%. The drug concentrations and enzyme inhibition rate corresponding to a diverging point of survivability were presumably estimated to be around 7 ng/ml for procyclidine, 0.35 ng/ml for physostigmine, and 37% of enzyme inhibition. Separately, in combination with the patch treatment, the post treatment consisting of atropine (0.5 mg/kg) plus 1-[([4-(aminocarbonyl)pyridinio]methoxy)methyl]-2-[(hydroxyimino)methyl]pyridinium (HI-6, 50 mg/kg) exerted protection against 5 × LD₅₀ challenge of soman, which means the posttreatment remarkably augmented the efficacy of the patch. Additionally, it was found that brain injuries induced by soman toxicity were effectively prevented by the patch treatment according to histopathological examinations. These results suggest that the patch system could be an effective alternative for diazepam, an anticonvulsant, and the current pyridostigmine pretreatment, and especially in combination with atropine plus HI-6, could be a choice for quality survival from nerve-agent poisoning.     

Quantification of tremor in rats induced by physostigmine

A simple and accurate device for recording tremor intensity in unanaesthetized and unrestrained rats is described. The physical measures of tremor are shown to have several advantages over previous devices. First, the new apparatus, unlike some earlier ones, does not restrict the animal's movements to an unusually small cage, with weighty mechanical devices or with electrical leads. Second, most earlier methods for measuring tremor use a subjective rating scale. However, the present method uses objective and reliable measures. In a double-blind, illustrative experiment involving complete crossover, Wistar rats were randomly injected IP with physostigmine in doses of 0.2, 0.4, 0.6 and 0.8 mg/kg or the corresponding amount of 0.9% NaCl solution as control. The recorded tremor intensity showed a clear dose-response relationship for physostigmine. Moreover, linear regression of the dose-response relationship showed that tremor intensity increased linearly with increasing doses of physostigmine. The cholinergic antagonist atropine (0.3 mg/kg SC) antagonized physostigmine-induced tremor, whereas methylatropine (0.3 mg/kg SC) tended to potentiate it. These results show that the technique described is suitable for quantification of tremor intensity in rats and for testing drug interactions on physostigmine-induced tremor.