OBJECTIVES: In this work, we hypothesized whether galantamine could interact with the cholinergic anti-inflammatory pathway and modulate immunity this way. BACKGROUND: Galantamine is a drug used for the therapy of Alzheimer disease. The drug inhibits enzyme acetylcholinesterase in the central nervous system, which causes better availability of neurotransmitter acetylcholine. METHODS: In the experiment, we immunized BALB/c laboratory mice by keyhole limpet hemocyanin (KLH) in combination with galantamine in a dose 0.02-0.5 mg/kg. The animals were sacrificed from 1 to 7 days after the substances applications and plasma was collected in order to examine immunochemical markers by enzyme-linked immunosorbent assay. RESULTS: We found significant drop in production of immunoglobulins and interleukin (IL) 4 level while IL2, IL4 and tumour necrosis factor alpha remained unaltered for the whole experiment. We infer that galantamine causes better availability of acetylcholine also in blood system, where the neurotransmitter interacts with nicotinic acetylcholine receptors on macrophages and initiates cholinergic anti-inflammatory pathway. CONCLUSIONS: In a conclusion, galantamine can cause lower efficacy of vaccination or immunity response to an infectious disease and the phenomenon should be taken into consideration in the current therapy (Tab. 1, Fig. 2, Ref. 24).
Title: Evaluation of possible inhibition of human liver drug metabolizing cytochromes P450 by two new acetylcholinesterase oxime-type reactivators Spicakova A, Anzenbacher P, Liskova B, Kuca K, Fusek J, Anzenbacherova E Ref: Food & Chemical Toxicology, 88:100, 2016 : PubMed
Two non-symmetric bispyridine oxime - based reactivators of acetylcholinesterase enzyme (AChE), labeled as K027 (1-(4-carbamoylpyridinium)-3-(4-hydroxyiminomethylpyridinium)-propane dibromide) and K203 ((E)-1-(4- carbamoylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide) were tested for their potential to inhibit activities of human liver microsomal cytochromes P450 (CYP). Both oximes are very potent reactivators of organophosphate-inhibited AChE. An interaction of both compounds with CYP in human liver microsomal preparation was detected using difference spectroscopy. The compounds were shown to bind to CYP enzymes with spectral binding constants of 5.04 +/- 1.79 nM (K027) and 5.2 +/- 2.6 nM (K203). Enzymology studies were subsequently performed aimed at determining which of the nine most important CYP involved in drug is affected by this interaction. The results have shown no prominent inhibition of individual CYP activities with either compounds except in the case of CYP2E1 and K203. Diagnostic Dixon plot revealed that K203 acted as an uncompetitive inhibitor of CYP2E1. Inhibition of this activity however is not as prominent as to make a potent drug interaction likely. Hence, the interaction of K027 and K203 oxime-type AChE reactivators with human liver microsomal CYP enzymes does not seem to be of prominent clinical importance and both compounds could be safely used in this respect as antidotes with low risk of drug interactions.
Reactivation effects of K203 and currently available oximes (obidoxime, HI-6) in combination with atropine on acetylcholinesterase activities in the brain parts of rats poisoned with tabun were studied. The activity was determined by quantitative histochemical and biochemical methods correlating between them very well. The tabun-induced changes in acetylcholinsterase activity as well as in reactivation potency of reactivators used were different in various parts of the brain. Pontomedullar area seems to be important for observed changes following tabun intoxication and its treatment. From the oximes studied, the reactivation effect of K203 was comparable with obidoxime; HI-6 was ineffective. Combination of bio- and histochemical methods allow fine differentiation among the action of different oximes following tabun poisoning.
The antidotal treatment of organophosphorus poisoning is still a problematic issue since no versatile antidote has been developed yet. In our study, we focused on an interesting property, which does not relate to the reactivation of inhibited acetylcholinesterase (AChE) of some oximes, but refers to their anti-muscarinic effects which may contribute considerably to their treatment efficacy. One standard reactivator (HI-6) and two new compounds (K027 and K203) have been investigated for their antimuscarinic properties. Anti-muscarinic effects were studies by means of an in vitro stimulated atrium preparation (functional test), the [(3)H]-QNB binding assay and G-protein coupled receptor assay (GPCR, beta-Arrestin Assay). Based on the functional data HI-6 demonstrates the highest anti-muscarinic effect. However, only when comparing [(3)H]-QNB binding results and GPCR data, K203 shows a very promising compound with regard to anti-muscarinic potency. The therapeutic impact of these findings has been discussed.
Current treatment of organophosphorus poisoning, resulting in overstimulation and desensitization of muscarinic and nicotinic receptors by acetylcholine (ACh), consists of the administration of atropine and oxime reactivators. However, no versatile oxime reactivator has been developed yet and some mortality still remains after application of standard atropine treatment, probably due to its lack of antinicotinic action. In our study, we focused on the interesting non-acetylcholinesterase property of oximes, i.e. antinicotinic effect of reactivators. Two standard reactivators (HI-6, obidoxime) and two new compounds (K027 and K203) were chosen for in vitro (patch clamp) and in vivo (nerve-evoked muscle contraction) testings. Both examinations showed antinicotinic effects of the reactivators. In vitro inhibition of acetylcholine-evoked currents by obidoxime, HI-6 and K203 was equivalent while K027 was less potent. Similar order of potency was observed by the in vivo examinations. We thus confirm previous in vitro results, which describe antinicotinic effects of oxime reactivators, and furthermore, we show in vivo antagonism of oxime reactivators exerted by the inhibition of ACh effect on the nicotinic receptor in the neuromuscular junction. Taking together, the effects of tested oxime reactivators indicate an antagonism on both embryonic and adult form of the muscle nicotinic receptors.
Title: Tabun-inhibited rat tissue and blood cholinesterases and their reactivation with the combination of trimedoxime and HI-6 in vivo Bajgar J, Karasova JZ, Kassa J, Cabal J, Fusek J, Blaha V, Tesarova S Ref: Chemico-Biological Interactions, 187:287, 2010 : PubMed
Up to now, intensive attempts to synthesize a universal reactivator able to reactivate cholinesterases inhibited by all types of nerve agents/organophosphates were not successful. Therefore, another approach using a combination of two reactivators differently reactivating enzyme was used: in rats poisoned with tabun and treated with combination of atropine (fixed dose) and different doses of trimedoxime and HI-6, changes of acetylcholinesterase activities (blood, diaphragm and different parts of the brain) were studied. An increase of AChE activity was observed following trimedoxime treatment depending on its dose; HI-6 had very low effect. Combination of both oximes showed potentiation of their reactivation efficacy; this potentiation was expressed for peripheral AChE (blood, diaphragm) and some parts of the brain (pontomedullar area, frontal cortex); AChE in the basal ganglia was relatively resistant. These observations suggest that the action of combination of oximes in vivo is different from that observed in vitro.
Tabun belongs to the most toxic nerve agents. Its mechanism of action is based on acetylcholinesterase (AChE) inhibition at the peripheral and central nervous systems. Therapeutic countermeasures comprise administration of atropine with cholinesterase reactivators able to reactivate the inhibited enzyme. Reactivation of AChE is determined mostly biochemically without specification of different brain structures. Histochemical determination allows a fine search for different structures but is performed mostly without quantitative evaluation. In rats intoxicated with tabun and treated with a combination of atropine and HI-6, obidoxime, or new oxime K048, AChE activities in different brain structures were determined using biochemical and quantitative histochemical methods. Inhibition of AChE following untreated tabun intoxication was different in the various brain structures, having the highest degree in the frontal cortex and reticular formation and lowest in the basal ganglia and substantia nigra. Treatment resulted in an increase of AChE activity detected by both methods. The highest increase was observed in the frontal cortex. This reactivation was increased in the order HI-6 < K048 < obidoxime; however, this order was not uniform for all brain parts studied. A correlation between AChE activity detected by histochemical and biochemical methods was demonstrated. The results suggest that for the mechanism of action of the nerve agent tabun, reactivation in various parts of the brain is not of the same physiological importance. AChE activity in the pontomedullar area and frontal cortex seems to be the most important for the therapeutic effect of the reactivators. HI-6 was not a good reactivator for the treatment of tabun intoxication.
Title: New Bisquaternary Inhibitors of Acetylcholinesterase Used as a Prophylactics in Organophosphorus Poisoning Pavlikova R, Marek J, Musilek K, Fusek J, Kuca K, Karasova JZ Ref: Military Medical Science Letters, 79:46, 2010 : PubMed
The prophylactic antidotes are pharmaceutics which can prevent the intoxication of the human body or may improve its prognosis if administered before the exposure to a toxic substance. Pyridostigmine chloride, a reversible acetylcholinesterase(AChE) inhibitor, is used as a prophylactic antidote which may prevent the organism from organophosphorus poisoning (OP). This paper is focused on searching new AChE inhibitors that might be used in prophylactic treatment of OP intoxications. The aim of this study was to prepare 15 symmetric bisquaternary inhibitors and to evaluate their inhibition ability (IC50) via a standard in vitro method. The but-(2E)-en-1,4-diyl-1,1'-bis(4-(4-nitrobenzyl)-pyridinium) dibromide was selected, on the basis of IC50 value, the most potent AChE inhibitor.
The oxime reactivator K112 is a member of the new group of xylene linker-containing AChE reactivators. Its cholinergic properties could be of importance at OP poisoning and are not related to the AChE reactivation that has been studied. It has been found that, despite of reactivating potency, this compound has additional effects. These cholinergic effects include a weak inhibition of AChE (IC(50)=43.8 +/- 4.88 muM), inhibition of binding to the porcine muscarinic M2 receptor (IC(50)=4.36 muM) and finally, the inhibition of HACU (68.4 +/- 9.9%), a key regulatory step in the synthesis of ACh. The inhibition of the binding of (3H)-HC-3 (64.7 +/- 4.7%) and the influence on the membrane fluidity have also been observed. Blocking properties of K112 on the muscarinic receptors have been revealed in the in vitro experiment (rat urinary bladder) and in the in vivo experiment (rat heart BPM) as well. All these cholinergic properties could significantly contribute to the antidotal effect of K112 at the poisoning by the organophosphates.
The poisoning with organophosphorus compounds represents a life threatening danger especially in the time of terroristic menace. No universal antidote has been developed yet and other therapeutic approaches not related to reactivation of acetylcholinesterase are being investigated. This review describes the main features of the cholinergic system, cholinergic receptors, cholinesterases and their inhibitors. It also focuses on the organophosphorus nerve agents, their properties, effects and a large part describes various possibilities in treatments, mainly traditional oxime therapies based on reactivation of AChE. Furthermore, non-cholinesterase coupled antidotal effects of the oximes are thoroughly discussed. These antidotal effects principally include oxime interactions with muscarinic and nicotinic receptors.
Title: Characterization of the anticholinergic properties of obidoxime; functional examinations of the rat atria and the urinary bladder Soukup O, Tobin G, Kumar UK, Jun D, Fusek J, Kuca K Ref: Toxicol Mech Methods, 20:428, 2010 : PubMed
Obidoxime, a well-known bis-pyridinium reactivator, is often the preferred antidote of organophosphorus poisoning caused by pesticides and tabun. It is also considered to be an allosteric modulator of muscarinic receptors, preferably M2 sub-type. This study compared the effect of obidoxime and atropine in vivo and in vitro on the cholinergic stimulation of the rat heart (M2) and the urinary bladder (M3). The results showed that obidoxime exerts anti-muscarinic effects, that may play an important role in the treatment of organophosphourus poisoning, and that the muscarinic receptor inhibition profile shows M2 receptor selectivity. This anti-muscarinic effect is much smaller that the effect of atropine and might be due to the allosteric inhibition of the receptors. The results also indicate that the acetylcholinesterase inhibition and the muscarinic receptor antagonism occur at different concentrations and dose levels.
Title: Chemical aspects of pharmacological prophylaxis against nerve agent poisoning Bajgar J, Fusek J, Kassa J, Kuca K, Jun D Ref: Curr Med Chem, 16:2977, 2009 : PubMed
Prophylactic approaches against intoxication with organophosphates (OP)/nerve agents can be based on following principles: keeping acetylcholinesterase (AChE), the key enzyme for toxic action of OP/nerve agents, intact (protection of cholinesterases) is a basic requirement for effective prophylaxis. It can be reached using simple chemicals such as reversible inhibitors (preferably carbamates), which are able to inhibit AChE reversibly. AChE inhibited by carbamates is resistant to OP/nerve agent inhibition. After spontaneous recovery of the activity, normal AChE serves as a source of the active enzyme. Detoxification is realised by administration of the enzymes splitting the OP or exploitating specific enzymes (cholinesterases). OP/nerve agent is bound to the exogenously administered proteins (enzymes) and, thus, the agent level in the organism is decreased ("scavenger" effect). The antidotes currently used for the treatment of OP poisoning (also simple chemicals) can be tested as prophylactics. This principle can be considered as a treatment "in advance". The problem with their use is the timing, duration and achievement of sufficient levels of these antidotes after the administration. At present, PYRIDOSTIGMINE seems to be common prophylactic antidote; prophylactics PANPAL (tablets with pyridostigmine, trihexyphenidyle and benactyzine), TRANSANT (transdermal patch containing HI-6) are other means introduced into different armies as prophylactics. Future development will be focused on scavengers (cholinesterases and other enzymes) acting before the binding of nerve agent to the target sites, and on other drugs reversible cholinesterase inhibitors (e.g. huperzine A, physostigmine, acridine derivatives etc.) including non-traditional routes of administration.
Title: Different inhibition of acetylcholinesterase in selected parts of the rat brain following intoxication with VX and Russian VX Hajek P, Bajgar J, Slizova D, Krs O, Kuca K, Capek L, Fusek J Ref: Drug & Chemical Toxicology, 32:1, 2009 : PubMed
Differences between acetylcholinesterase (AChE) inhibition in the brain structures following VX and RVX exposure are not known as well as information on the possible correlation of biochemical and histochemical methods detecting AChE activity. Therefore, inhibition of AChE in different brain parts detected by histochemical and biochemical techniques was compared in rats intoxicated with VX and RVX. AChE activities in defined brain regions 30 min after treating rats with VX and Russian VX intramuscularly (1.0 x LD(50)) were determined by using biochemical and histochemical methods. AChE inhibition was less expressed for RVX, in comparison with VX. Frontal cortex and pontomedullar areas containing ncl. reticularis has been found as the most sensitive areas for the action of VX. For RVX, these structures were determined to be frontal cortex, dorsal septum, and hippocampus, respectively. Histochemical and biochemical results were in good correlation (R(xy) = 0.8337). Determination of AChE activity in defined brain structures was a more sensitive parameter for VX or RVX exposure than the determination of AChE activity in the whole-brain homogenate. This activity represents a "mean" of the activities in different structures. Thus, AChE activity is the main parameter investigated in studies searching for target sites following nerve-agent poisoning contributing to better understanding of toxicodynamics of nerve agents.
Substances K-48 and HI-6, oxime-type acetylcholinesterase (AChE) reactivators, were tested for their potential to inhibit the activities of human liver microsomal cytochromes P450 (CYP). The compounds were shown to bind to microsomal cytochromes P450 with spectral binding constants of 0.25+/-0.05 microM (K-48) and 0.54+/-0.15 microM (HI-6). To find which cytochrome P450 from the human liver microsomal fraction interacts with these compounds, an inhibition of enzyme activities specific for nine individual CYP enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) was studied. The results have shown no prominent inhibition of individual CYP activities with both compounds except the CYP2E1 activity and the HI-6 reactivator. However, the inhibition of this activity was less than 50% which makes the possible drug interactions highly unlikely. Hence, the interaction of K-48 and HI-6 oxime-type AChE reactivators with human liver microsomal CYP enzymes does not seem to be clinically significant and both compounds could be taken in this respect as antidotal drugs with low risk of drug interactions.
Title: An attempt to assess functionally minimal acetylcholinesterase activity necessary for survival of rats intoxicated with nerve agents Bajgar J, Fusek J, Kassa J, Jun D, Kuca K, Hajek P Ref: Chemico-Biological Interactions, 175:281, 2008 : PubMed
Acetylcholinesterase (AChE, EC 3.1.1.7) is an important enzyme for cholinergic nerve transmission. The action of toxic organophosphates such as nerve agents is based on AChE inhibition. The death following acute nerve agent poisoning is due to central or peripheral respiratory/cardiac failure. Therefore, the changes in AChE activity following nerve agents acting predominantly on the central (sarin, soman) or peripheral (VX) level were studied. It is known that AChE activity in different structures exists in relative excess. Female Wistar rats intoxicated with sarin, soman, and VX in different doses (0.5-2.0 x LD(50)) were divided into groups of survived and died animals. AChE activities in diaphragm, brain parts (pontomedullar area, frontal cortex, basal ganglia, in some cases other parts of the brain) were determined and the rest of activity (in %) was correlated with survival/death of animals. More precise elucidation of action of nerve agents and the assessment of minimal AChE activity in different organs compatible with the survival of organism poisoned with nerve agents were the aims of this study.
OBJECTIVES: The current standard treatment of organophosphate poisoning consists of an administration of anticholinergic drugs and cholinesterase reactivators (oximes). Oximes can react - except their reactivating effect on cholinesterases - directly with cholinoreceptors. HI-6 is an oxime that may have an inhibitory effect on the muscarinic receptors, too. METHODS: In our work, we have investigated an influence of HI-6 on the acetylcholinesterase (AChE), butyrylcholinesterase (BChE) and on the muscarinic receptors in vitro. The study was conducted using biosensor technique and on the rat bladder using in vitro test (tissue bath; methacholine as muscarinic agonist). IC50 for BChE from human serum was determined to be 1.01x10-6 M and for human erythrocytes AChE 3.31x10-6 M, respectively. CONCLUSION: We assume that the demonstrated contractile response can be attributed to the inhibition of the AChE at the lower concentration and to a predominant inhibition of muscarinic receptor at higher concentration of compound tested.
Title: Changes of acetylcholinesterase activity in different rat brain areas following intoxication with nerve agents: biochemical and histochemical study Bajgar J, Hajek P, Slizova D, Krs O, Fusek J, Kuca K, Jun D, Bartosova L, Blaha V Ref: Chemico-Biological Interactions, 165:14, 2007 : PubMed
Acetylcholinesterase activity in defined brain regions was determined using biochemical and histochemical methods 30 min after treating rats with sarin, soman or VX (0.5 x LD(50)). Enzyme inhibition was high in the pontomedullar area and frontal cortex, but was low in the basal ganglia. Histochemical and biochemical results correlated well. Determination of the activity in defined brain structures was a more sensitive parameter than determination in whole brain homogenate where the activity was a "mean" of the activities in different structures. The pontomedullar area controls respiration, so that the special sensitivity of acetylcholinesterase to inhibition by nerve agents in this area is important for understanding the mechanism of death caused by nerve agents. Thus, acetylcholinesterase activity is the main parameter investigated in studies searching for target sites following nerve agent poisoning.
Title: Changes of cholinesterase activities in the rat blood and brain after sarin intoxication pretreated with butyrylcholinesterase Bajgar J, Bartosova L, Kuca K, Jun D, Fusek J Ref: Drug & Chemical Toxicology, 30:351, 2007 : PubMed
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.
Title: Cholinesterase reactivators: the fate and effects in the organism poisoned with organophosphates/nerve agents Bajgar J, Kuca K, Jun D, Bartosova L, Fusek J Ref: Curr Drug Metab, 8:803, 2007 : PubMed
Understanding the mechanism of action of organophosphates (OP)/nerve agents -- irreversible acetylcholinesterase (AChE, EC 3.1.1.7) inhibition at the cholinergic synapses followed by metabolic dysbalance of the organism -- two therapeutic principles for antidotal treatment are derived. The main drugs are anticholinergics that antagonize the effects of accumulated acetylcholine at the cholinergic synapses and cholinesterase reactivators (oximes) reactivating inhibited AChE. Anticonvulsants such as diazepam are also used to treat convulsions. Though there are experimental data on a good therapeutic effects of reactivators, some attempts to underestimate the role of reactivators as effective antidotes against OP poisoning have been made. Some arguments on the necessity of their administration following OP poisoning are discussed. Their distribution patterns and some metabolic and pharmacological effects are described with the aim to resolve the question on their effective use, possible repeated administration in the treatment of OP poisoning, their peripheral and central effects including questions on their penetration through the blood brain barrier as well as a possibility to achieve their effective concentration for AChE reactivation in the brain. Reactivation of cholinesterases in the peripheral and central nervous system is described and it is underlined its importance for the survival or death of the organism poisoned with OP. Metabolization and some other effects of oximes (not connected with AChE reactivation) are discussed (e.g. forming of the phosphonylated oxime, parasympatholytic action, hepatotoxicity, behavioral changes etc.). An universality of oximes able to reactivate AChE inhibited by all OP is questioned and therefore, needs of development of new oximes is underlined.
Title: Treatment of organophosphate intoxication using cholinesterase reactivators: facts and fiction Bajgar J, Fusek J, Kuca K, Bartosova L, Jun D Ref: Mini Rev Med Chem, 7:461, 2007 : PubMed
Basic part of the current standard treatment of organophosphate (OP) agent poisoning is administration of cholinesterase reactivators. It includes different types of oximes with a similar basic structure differing by the number of pyridinium rings and by the position of the oxime group in the pyridinium ring. Oximes hydrolytically cleave the organophosphates from acetylcholinesterase (AChE), restoring enzymatic function. This reactivation of AChE is dependent on the type of the agent and, on the reactivator used. From the common oximes, mono- and bisquaternary pyridinium oximes are more or less frequently used in clinical practice such as pralidoxime, obidoxime, trimedoxime, and HI-6. Though there are data on a good therapeutic effects of reactivators, some attempts to undermine the role of reactivators as effective antidotes against OP poisoning have been made. Some arguments on the necessity of their administration following OP poisoning are discussed with the aim to resolve the question on their effective use, possible repeated administration in the treatment of OP poisoning, their peripheral and central effects including questions on their penetration through the blood brain barrier as well as a possibility to achieve their effective concentration for AChE reactivation in the brain. Reactivation of cholinesterases in the peripheral and central nervous system is described and it is underlined its importance for the survival or death of the organism poisoned with OP. An universality of oximes able to reactivate AChE inhibited by all OP is questioned and trends (molecular modelling using neural network, structure-activity relationship, combination of reactivation and anticholinergic properties in one molecule) for future research are characterized.
Nerve agents can be divided into G-agents (sarin, soman, tabun, cyclosarin etc.) and V-agents. The studies dealing with V-agents (O-alkyl S-2-dialkylaminoethyl methyl phosphonothiolates) are limited to one or two representatives only (VX, Russian VX). Anticholinesterase properties of 11 V-agents were studied in rats in vivo. Following intoxication with these agents in doses of 1 x LD(50) (intramuscular administration), activities of cholinesterases in the blood were continuously monitored and half-lives (t(0.5)) of inhibition were determined. These values varied from 3 min (VX and some other agents) to 10-14 min (derivatives substituted on the phosphorus head by O-ethyl- or O-isopropyl-, and by dimethyl-, diethyl- and dibutyl- on the nitrogen). Acetylcholinesterase activities in selected parts of the brain and diaphragm (30 min after the intoxication) were also detected. A correlation between toxicities and rates of inhibition of the blood enzymes was demonstrated. A similar relationship between acetylcholinesterase inhibition in vitro (from literature data) and half-lives of the blood cholinesterases was also observed. Though the chemical similarity of V compounds is evident, marked differences were observed among different derivatives; however, all agents examined had high inhibition potency corresponding to their toxicities.
Title: Evaluation of reactivation test in anaesthetized dogs with experimental intoxication with nerve agents Bajgar J, Fusek J, Bartosova L, Jun D, Kuca K Ref: J Appl Toxicol, 26:439, 2006 : PubMed
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.
Title: Equine butyrylcholinesterase protects rats against inhalation exposure to sublethal sarin concentrations Bajgar J, Bartosova L, Fusek J, Jun D, Kuca K Ref: Arh Hig Rada Toksikol, 57:391, 2006 : PubMed
Protection experiments were conducted using different doses of equine serum butyrylcholinesterase (Eq BuChE) as pretreatment in rats. Cholinesterase activities were determined in blood [whole blood, red blood cells (RBC) acetylcholinesterase (AChE), and plasma BuChE] before and after sarin inhalation exposure in untreated rats and those pretreated with Eq BuChE. Brain AChE activity was also determined in the frontal cortex, basal ganglia and pontomedullar areas following exposure. Dose-dependent increases in plasma BuChE activity and no changes in the RBC and brain AChE activities were demonstrated following i.p. injection of different amounts of Eq BuChE. Decreases in plasma BuChE activity and RBC and brain AChE activities were observed in control rats following sarin inhalation exposure. In rats pretreated with Eq BuChE this inhibition was lower than in control animals. These results demonstrate protective effects of Eq BuChE pretreatment in rats intoxicated with sublethal concentrations of sarin by inhalation.
The aim of this study was to demonstrate changes in acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities, tyrosine aminotransferase activity (TAT) and plasma corticosterone level, neuroexcitability and behavior following 24 hours and 4 weeks of soman sublethal inhalation exposure at low level. AChE activity in erythrocytes and BuChE activity in plasma was decreased (dependent on the concentration of soman) 24 h and 4 weeks after the exposure. Similar decrease in AChE activity in different brain parts was observed. One of stressogenic parameters (TAT) was changed after 24 h exposure only. 4 weeks after the exposure, these parameters (corticosterone and TAT) were in the range of normal values. Behaviour of experimental animals was changed 24 h after the exposure persisting 4 weeks after the exposure as well as neuroexcitability.
Soman belongs to the most dangerous nerve agents because of the low effectiveness of the presently available antidotes. Soman acts by inhibiting acetylcholinesterase (AChE) both peripherally and centrally, with a subsequent accumulation of neuromediator acetylcholine and other metabolic changes. From the data published in literature it can be concluded that exposure to nerve agents leading to acute effects or chronic exposure to nerve agents may lead to delayed and persistent adverse effects. The aim of this study was to demonstrate changes in AChE and butyrylcholinesterase (BuChE) activities, stressogenic markers (i.e., tyrosine aminotransferase [TAT] activity, and plasma corticosterone level), and neuroexcitability and behavior 24 h and 4 wk following a single soman inhalation exposure at low level. AChE activity in erythrocytes and BuChE activity in plasma was decreased (dependent on the dose of soman) 24 h and 4 wk after the exposure. A similar decrease in AChE activity in different brain parts was observed. One of the stressogenic parameters, TAT, was changed 24 h after exposure only. Behavior of experimental animals was changed 24 h after the exposure, and 4 behavioral parameters persisted 4 wk after the exposure. Neuroexcitability was increased at 24 h after the exposure and had become about normal 4 wk after the exposure. Summarizing, long-term effects (4 wk) were observed after inhalation exposure of guinea pigs to sublethal concentrations of soman.
Title: The influence of oxime selection on the efficacy of antidotal treatment of soman-poisoned rats Kassa J, Fusek J Ref: Acta Medica (Hradec Kralove), 45:19, 2002 : PubMed
1. The influence of some acetylcholinesterase reactivators (HI-6, obidoxime, pralidoxime) on the efficacy of antidotal treatment to eliminate soman-induced disturbance of respiration and circulation and to protect experimental animals poisoned with supralethal dose of soman (1.5 x LD50) was investigated in a rat model with on-line monitoring of respiratory and circulatory parameters. 2. Obidoxime or pralidoxime in combination with atropine were insufficient to enable soman-poisoned rats to survive for 2 hours when given 1 minute after the administration of soman. 3. On the other hand, the ability of the oxime HI-6 in combination with atropine to prevent soman-induced alteration of respiration and circulation was significantly higher. Some rats treated with HI-6 in combination with atropine were fully protected against the lethal toxic effects of soman within 2 hours following soman administration. 4. Our findings confirm that the oxime HI-6 seems to be a much more suitable and efficacious acetylcholinesterase reactivator for the antidotal treatment of severe acute soman-induced poisoning than currently used obidoxime or pralidoxime.
Title: The positive influence of a cholinergic-anticholinergic pretreatment and antidotal treatment on rats poisoned with supralethal doses of soman Kassa J, Fusek J Ref: Toxicology, 128:1, 1998 : PubMed
The influence of pretreatment with the drug mixture (pyridostigmine, benactyzine and trihexyphenidyle), and antidotal treatment (the oxime HI-6 in combination with benactyzine) on respiration, circulation and survival of experimental animals poisoned with supralethal doses of soman (2 x LD50) was investigated in a rat model with on-line monitoring of respiratory and circulatory parameters. Untreated soman poisoning caused rapid respiratory depression, progressive bradycardia and a short-term increase in the mean arterial pressure, followed immediately by hypotension. The poisoned rats died on average within 10 min from respiratory and circulatory insufficiency. A cholinergic-anticholinergic pretreatment alone partially prevented changes in monitored physiological variables, caused by soman, but only for a few minutes; post-poisoning treatment with antidotes, also alone, had similar effects. The rats died on average within 30 min from respiratory and circulatory failure. When rats were pretreated by the drug mixture and treated by antidotes, respiration as well as circulation were completely restored and the rats survived at least 120 min following soman challenge. The results of the investigation suggest that cholinergic-anticholinergic pretreatment seems able to enhance the efficacy of antidotal treatment in restoring respiratory and circulatory changes induced by soman.
Title: Effect of Panpal pretreatment and antidotal treatment (HI-6 plus benactyzine) on respiratory and circulatory function in soman-poisoned rats Kassa J, Fusek J Ref: Hum Exp Toxicol, 16:563, 1997 : PubMed
1 The effect of pharmacological pretreatment (pyridostigmine, benactyzine and trihexyphenidyle), designated Panpal, and antidotal treatment (the oxime HI-6 plus benactyzine) in soman poisoning was investigated in a rat model with on-line monitoring of respiratory and circulatory parameters. 2 Soman poisoning caused a high decrease in respiratory rate as well as minute respiratory volume and an increase in mean arterial pressure from 30-120 min following soman challenge. Soman at sublethal dose also significantly inhibited acetylcholinesterase activity in diaphragm and various brain parts. 3 Panpal pretreatment as well as antidotal treatment were effective in improving the respiratory and circulatory function disturbed by soman without the ability to increase significantly soman-inhibited acetylcholinesterase activity in all brain parts studied. 4 The efficacy of combined Panpal pretreatment and antidotal treatment against sublethal soman poisoning was not different from the efficacy of Panpal pretreatment or antidotal treatment alone. 5 The results of this investigation suggest that Panpal pretreatment as well as antidotal treatment are able to restore respiratory and circulatory function in soman-poisoned rats without significant reactivation of brain acetylcholinesterase.
Title: Changes of cholinesterases in the blood and some tissues following administration of tacrin and its two derivatives to rats Bajgar J, Fusek J, Skopec F Ref: Neurochem Int, 24:555, 1994 : PubMed
Tacrin, its 7-methoxy-(MEOTA) and 7-hydroxy-(HYOTA) derivatives were i.m. administered to rats in a dose of 1.2 x LD50 and acetylcholinesterase (blood, hippocampus, frontal cortex, basal ganglia, septum and diaphragm) or butyrylcholinesterase (liver) activities were detected. The inhibitory effect of the examined substances in vivo decreased in the following order: tacrin HYOTA > MEOTA. The marked inhibition of the enzymes studied following administration of all three compounds in the frontal cortex could suggest importance of this structure for action of these drugs.
Title: Treatment of intoxication with GV compound in laboratory rats Fusek J, Bajgar J Ref: Sb Ved Pr Lek Fak Karlovy Univerzity Hradci Kralove, 37:57, 1994 : PubMed
Changes of some physiological functions in rats were studied following intoxication with new type of highly toxic organophosphate GV [2-dimethylaminoethyl-(dimethylamido)-fluorophosphate] in doses of 2x and 4x LD50 i.m. The intoxication has begun with increasing motoric activity followed by increased salivation, rumination and bristling. Tachypnoe and fasciculations followed by convulsions and death were observed later. Therapy of intoxication with GV compound (in dose of 2x LD50) in rats demonstrated the best antidotal effect of combination of benactyzine, atropine and HI-6. In GV intoxication in higher doses (4x LD50), therapeutic efficacy (survival of experimental animals) was limited for 24 hours. HI-6 can be therefore considered as potentially universal reactivator for the treatment of nerve agents intoxication.
The aim of this study was to evaluate the efficiency of hemoperfusion (HP) through coated resin adsorbent Synachrom E-5 in animal intoxications with organophosphate inhibitors of cholinesterases type of nerve agents. Five anesthetized dogs were intoxicated with 2 to 6 LD50 of VX substance and another 4 with 2 to 3 LD50 sarine. Both nerve agents were given i.m. after starting 5 h HP. The clinical and laboratory tests were monitored during each HP. HP therapy prevented the development of serious signs of intoxication provided that the administered quantity of both sarine and the VX substance was only 2 doses of LD50. Specific antidote therapy was necessary to prevent cardiorespiratory failure in animals intoxicated with a higher dose of poison. The results obtained show that HP through Synachrom E-5 in intoxication with nerve agents sarine and the VX type is only partially successful.
Title: [Treatment of tardive dyskinesias with 7-methoxycrine. II] Hanus H, Tuma I, Fusek J, Patocka J Ref: Sb Ved Pr Lek Fak Karlovy Univerzity Hradci Kralove Suppl, 36:47, 1993 : PubMed
In an open clinical study the authors have verified the effectivity and tolerance of 7-metoxytacrine cholinergic agent (7-MEOTA) in a total consisting of 14 patients with schizophrenic or schizoaffective psychoses suffering from tardive dyskinesias in long-termed treatment with neuroleptics. In the whole group consisting of 14 patients the seven days' administration of the experimental preparation in a dose of 100-150 mg pro die was evaluated. The 7-MEOTA preparation was administered to 7 patients for 2 weeks. In the seven and fourteen days' administration a favourable effect of 7-MEOTA on tardive dyskinesias was observed. A fast onset of the efficiency as early as in the first days of the treatment was recorded. In final evaluation a minimal reduction of 50% of the intensity of the dyskinesias (using the AIMS scale) in 29% of the patients treated for a fortnight was found. The 7-MEOTA preparation was well tolerated, no undesirable marked side effects being observed. A transient increase of ALT was found in 1 patient only in the 2nd week of the treatment. In experimental treatment the maintenance neuroleptical therapy was not discontinued. An improvement of the dyskinesias overlasted in some patients as long as 2 month g after the discontinuation of the administration of 7-MEOTA preparation.
Title: [Treatment of tardive dyskinesias with 7-methoxytacrine. I] Hanus H, Tuma I, Fusek J, Patocka J Ref: Sb Ved Pr Lek Fak Karlovy Univerzity Hradci Kralove Suppl, 36:37, 1993 : PubMed
The 7-metoxytacrine (7-MEOTA) is an original Czech cholinergic agent synthetized in the labs of the Military Medical Academy in Hradec Kralove. The treatment of tardive dyskinesias is one of the possible indications of the use of 7-MEOTA in clinical practice. The authors have summed up their experience from the first phase of clinical tests with 7-MEOTA in psychiatrical patients suffering from tardive dyskinesias in long-termed administration of cholinergic agents. The clinical efficiency and tolerance of 7-MEOTA have been evaluated after a single administration of 100 mg of 7-MEOTA per os in 19 patients. A reduction of dyskinesias was observed as early as 4 h following the testing dose. In 5 patients, viz. in 26% a reduction of dyskinesias was observed of more than 50% of the original value of the total score of the testing scale AIMS. Except for a slight decrease of the blood pressure and a mild somnolence in 3 patients no other undesirable effects have been observed. In some patients a slight euphorization effect of 7-MEOTA has been found.
Title: Pharmacokinetics and tolerance of 7-methoxytacrine following the single dose administration in healthy volunteers Filip V, Vachek J, Albrecht V, Dvorak I, Dvorakova J, Fusek J, Havluj J Ref: Int J Clinical Pharmacology & Therapeutics Toxicol, 29:431, 1991 : PubMed
7-methoxy-tetrahydroaminoacridine (7-MEOTA) is a new reversible cholinesterase inhibitor. Forty-eight young male volunteers divided into six dosage groups were included into a single-dose pharmacokinetic study with either oral (p.o.) or intramuscular (i.m.) administration. The dose of 7-MEOTA was 2, 4 or 8 mg/kg body weight p.o. or 0.5, 1 or 2 mg/kg body weight i.m. in the respective six dosage groups. The plasma levels data were fitted to an open one-compartmental model. The compound showed cholinomimetic adverse effects in 2 subjects with the blood levels exceeding 1,500 micrograms/l. The red blood cells levels paralleled those in plasma and were 2.5 times higher. The tmax was 4 hours and 1 h, t1/2 8.7 +/- 3.9 hours and 6.5 +/- 5.8 hours in case of p.o. and i.m. administration, respectively. The apparent clearance (D/AUC) was 5 times higher following p.o. administration, reflecting the differences in bioavailability.
Title: Tacrine in Alzheimer's disease Patocka J, Fusek J Ref: Homeost Health Dis, 33:161, 1991 : PubMed
Title: First experience with the application of 7-methoxytacrine to psychiatric patients Zapletalek M, Hanus H, Fusek J, Hrdina V Ref: Activitas Nervosa Superior (Praha), 31:305, 1989 : PubMed
Title: [Initial experience with the administration of 7-methoxyacrine to patients with psychological disorders] Zapletalek M, Hanus H, Fusek J, Hrdina V Ref: Cesk Psychiatr, 85:155, 1989 : PubMed
The cholinomimemtic substance 7-methoxyacrine was administered to eight patients, incl. 5 patients with tardive dyskinesias after neuroleptic drugs. In all patients an europhorizing effect was recorded, all five patients with tardive dyskinesias improved markedly after a single dose, in one female patient after repeated administration symptoms of tardive dyskinesia were eliminated. The incidence of side effects is minimal. Based on our modest experience we consider 7-methoxyacrine an important extension of therapeutic possibilities, in particular in tardive dyskinesias. The authors plan to test 7-methoxyacrine also in other indications, in particular in Alzheimer's disease and in side-effects caused by psychopharmaceutical preparations.
Title: Anticholinergic action of cholinesterase reactivator methoxime (bis-/4- hydroxyaminomethylpyridinium/-methane dichloride) Jakl A, Fusek J, Bajgar J Ref: Sb Ved Pr Lek Fak Karlovy Univerzity Hradci Kralove, 29:457, 1986 : PubMed
Title: Some possibilities of protection against acetylcholinesterase inhibition by organophosphates in vivo Bajgar J, Patocka J, Fusek J, Hrdina V Ref: Sb Ved Pr Lek Fak Karlovy Univerzity Hradci Kralove, 27:425, 1984 : PubMed
Title: Protective effect of 9-amino-7-methoxy-1,2,3,4-tetra-hydroacridine against inhibition of acetylcholinesterase by O-ethyl s-(2- dimethylaminoethyl) methylphosphonotioate in vivo Bajgar J, Fusek J, Patocka J, Hrdina V Ref: Archives of Toxicology, 54:163, 1983 : PubMed
Title: The influence of some drugs on the rat blood phosphodiesterase activity Bajgar J, Fusek J, Patocka J, Hrdina V Ref: Sb Ved Pr Lek Fak Karlovy Univerzity Hradci Kralove, 26:423, 1983 : PubMed
Title: Exploitation of the antagonistic properties of some drugs against poisoning with psychochemicals Koupilova M, Hrdina V, Fusek J Ref: Activitas Nervosa Superior (Praha), 23:292, 1981 : PubMed
Title: In vivo kinetics of blood cholinesterase inhibition by 9-amino-1, 2, 3, 4-tetrahydroacridine, its 7-methoxy derivative and physostigmine in rats Bajgar J, Fusek J, Patocka J, Hrdina V Ref: Physiol Bohemoslov, 28:31, 1979 : PubMed
In vivo inhibition of blood acetylcholinesterase activity by 9-amino-1, 2, 3, 4-tetrahydroacridine, its 7-methoxy derivative and physostigmine was studied in rats. Changes of enzyme activity in the blood were continually registered using an automatic colorimeter Auto Analyzer system. The dependence of % enzyme inhibition upon time in semilogarithmic transformation was characterized by a two phase curve. The first phase reflected the increase in the concentration of inhibitors in the blood during their resorption; this was followed by a slow second phase of inhibition. The inhibitory effect decreased in the order: physostigmine greater than tacrine greater than 7-methoxytacrine. The relationship between the anticholinesterase and antipsychotomimetic action of the examined substances is discussed.
Title: The interaction of anticholinesterases and diazepam in the treatment of anticholinergic syndrome in dogs Fusek J, Herink J, Koupilova M, Patocka J, Bajgar J, Hrdina V Ref: Activitas Nervosa Superior (Praha), 21:183, 1979 : PubMed
Title: Diazepam in combination with tacrine derivatives antagonizes behavioural effects of anticholinergic psychotomimetics in rats Koupilova M, Fusek J, Hrdina V Ref: Activitas Nervosa Superior (Praha), 21:182, 1979 : PubMed
1. The effects of acetylcholine and methylfurmetide on isolated guinea-pigs heart atria in the presence or absence of O-ethyl-S-(2-dimethylaminoethyl)-methylphosphonothioate (EDMM) were investigated. The influence of three cholinesterase reactivators on the heart atria pretreated with EDMM has been studied. 2. In the presence of EDMM, the negative inotropic effect of acetylcholine on the isolated heart atria was not significantly increased. This effect of methylfurmetide on heart atria was significantly decreased in the presence of EDMM. 3. EDMM alone caused negative inotropic and negative chronotropic effects depending on its concentration. These pharmacological effects were accompanied with inhibition of acetylcholinesterase activity in the heart muscle. 4. Addition of cholinesterase reactivators normalized the inotropic response of the isolated guinea-pig heart atria altered by EDMM and also increased the acetylcholinesterase activity in the heart.
Title: Interaction of imipramine and 3-quinuclidyl benzilate with 9-amino-7-methoxy-1,2,3,4-tetrahydroacridine on the after-discharges in the limbic system Herink J, Fusek J, Bajgar J, Patocka J, Hrdina V Ref: Activitas Nervosa Superior (Praha), 20:79, 1978 : PubMed
Title: Tacrine and its derivatives antagonize cholinergic psychotomimetics. Behavioural study in rats Koupilova M, Fusek J, Hrdina V Ref: Activitas Nervosa Superior (Praha), 20:76, 1978 : PubMed
Title: Continual determination of acetylcholinesterase inhibition following organophosphate poisoning using an auto analyzer Bajgar J, Fusek J, Patocka J, Hrdina V Ref: Acta Biologica et Medica Germanica, 36:231, 1977 : PubMed
A modification for continual monitoring of the blood acetylcholinesterase activity in the rat on an auto analyzer system was described. With using this technique, inhibition of the enzyme in vitro with O-ethyl-S-(2-dimethylaminoethyl)-methylphosphonothioate was determined. Bimolecular rate constant and I50 values were 1.66-10(6) M-1-min-1 and 1.4-10(-8)M, respectively. Rats treated i.m. with 0.043 mg/kg of this compound showed fast and rapid inhibition of the blood acetylcholinesterase after intoxication. The bimolecular rate constant of inhibition in vivo, calculated analogously as that in vitro was 1.37-10(6) M-1-min-1. On the presumption that both rates of inhibition (in vitro and in vivo) are identical, the concentration of organophosphate producing inhibition effect in vivo was 82% of the dose administered.
Title: The effects of some cholinesterase reactivators on contractility of the isolated guinea-pig heart atria Fusek J, Patocka J Ref: Acta Biologica et Medica Germanica, 35:657, 1976 : PubMed
