Galantamine has impact on immunity in mice exposed to keyhole limpet hemocyanin.

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 α 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).

Freund´s complete adjuvant effect on BALB/c mice: an insight into inflammation and oxidative stress after immunity challenge.

Freund´s complete adjuvant (FCA) is a mean used for improving immunization efficacy in experiments and veterinary medicine. Despite high efficacy, it is not used in human vaccination due to number of adverse effects. Arthritis can be an example of a typical adverse consequence. In this work, we decided to investigate the link between oxidative stress and inflammation in animals exposed to FCA. BALB/c mice received either saline or FCA in doses 10-50 µl and were euthanized from one to three days after infection. Interleukins (IL) IL-2, IL-4 and tumor necrosis factor α (TNF-α) were determined in plasma. Ferric reducing antioxidant power (FRAP), malondialdehyde by thiobarbituric acid reactive substances assay (TBARS), reduced glutathione (GSH) and caspase-3 were measured in livers, muscles and other organs. We determined significant effect of FCA on TBARS and GSH in the livers. Beside this, TBARS were altered in muscles as well. Inflammation was confirmed by increased level of TNF-α. Taking the results into account, we infer that the harmful effect of FCA is mediated not only by inflammation but also by oxidative. This should be considered when pathological consequences of FCA are scored.

The effect of oxime reactivators on muscarinic receptors: functional and binding examinations.

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.

Oxime reactivators and their in vivo and in vitro effects on nicotinic receptors.

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.

Macrophage-assisted inflammation and pharmacological regulation of the cholinergic anti-inflammatory pathway.

Macrophages play an important role in the immune system. They also participate in multiple processes including angiogenesis and triggering of inflammation. The present study summarizes pieces of knowledge on the importance of macrophages in disease, especially the inflammation. Special attention is paid to the cholinergic anti-inflammatory pathway (CAP) associated with the nicotinic acetylcholine receptor (nAChR) and the parasympathetic nervous system. The current pharmacological effectiveness in suppressing the inflammation in general and the septic shock in particular, is limited. CAP was discovered recently and it seems to be a suitable target for the development of new drugs. Moreover, available drugs binding to either nAChR or acetylcholinesterase (AChE) are candidates for either an inhibition or enhancement of CAP. Though the current scientific databases do not include all necessary data on the association of CAP with body functions and the research is quite intensive, the objective of the present review is to introduce the current trends and to critically evaluate CAP and macrophage-associated pathways.

Novel acetylcholinesterase reactivator K112 and its cholinergic properties.

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 µM), inhibition of binding to the porcine muscarinic M2 receptor (IC(50)=4.36 µM) 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.

Interaction of nerve agent antidotes with cholinergic systems.

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.

Inhibition of blood cholinesterases following intoxication with VX and its derivatives.

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.

Cholinesterase reactivators: the fate and effects in the organism poisoned with organophosphates/nerve agents.

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.

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

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

Biochemical effects of low level exposure to soman vapour.

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.

Chemical agents and chemical terrorism.

Chemical terrorism is a new threat to the security of mankind, which scale essentially exceeds the impact of use of the most modem firearms. At present time all over the world threats from different radical elements to use radioactive materials, potent poisonous substances and pathogenic microorganisms for terrorist purposes became more frequent. High-toxic chemical substances can fall in terrorist hands through wide range of sources. Potentially misused types of chemical compounds are discussed in this article.

The influence of anticholinergic drug selection on the efficacy of antidotal treatment of soman-poisoned rats.

The influence of some anticholinergic drugs (atropine, benactyzine, biperiden, scopolamine) on the efficacy of antidotal treatment to eliminate soman (O-pinacolyl methylphosphonofluoridate)-induced disturbance of respiration and circulation and to protect experimental animals poisoned with supralethal dose of soman (1.5 x LD(50)) was investigated in a rat model with on-line monitoring of respiratory and circulatory parameters. While the oxime HI-6 in combination with atropine prevented soman-induced changes in monitored physiological parameters insufficiently and very shortly, the combination of HI-6 with benactyzine or biperiden is able to prevent soman-induced alteration of respiration and circulation much more longer. Nevertheless, only rats treated with HI-6 in combination with scopolamine were fully protected against the lethal toxic effects of soman within 2 h following soman challenge. Our findings confirm that anticholinergic drugs with the strong central antimuscarinic activity, such as benactyzine, biperiden and especially scopolamine, seem to be more effective adjuncts to HI-6 treatment of severe acute soman-induced poisoning than atropine.

3-Nitropropionic acid and similar nitrotoxins.

3-Nitropropionic acid as well as 3-nitro-1-propanol and its beta-D-glucopyranoside (miserotoxin) are the plant and fungal toxins reported to interrupt mitochondrial electron transport resulting in cellular energy deficit. These nitrotoxins induce neurological degeneration in ruminants and humans. 3-Nitropropionic acid-intoxicated rats serve as the animal model for Huntington's disease.

The positive influence of a cholinergic-anticholinergic pretreatment and antidotal treatment on rats poisoned with supralethal doses of soman.

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.

Effect of Panpal pretreatment and antidotal treatment (HI-6 plus benactyzine) on respiratory and circulatory function in soman-poisoned rats.

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.

Changes of cholinesterase activity in the erythrocytes, plasma, diaphragm, liver and various parts of the brain in the rabbit following transfusion of erythrocytes with soman inhibited acetylcholinesterase.

1. The changes of cholinesterase activity in rabbit blood, peripheral tissues and the central nervous system following transfusion of erythrocytes with soman inhibited acetylcholinesterase we were demonstrated. 2. After incubation with soman for 0.5 or 24 h, erythrocytes without acetylcholinesterase activity were injected to intact rabbits and cholinesterase activity in the erythrocytes, plasma, diaphragm, liver and various parts of the brain were evaluated 24 h following blood-transfusion. 3. When erythrocytes were incubated with soman for 24 h, no changes of cholinesterase activity in the rabbit following blood-transfusion were observed with an exception of erythrocyte acetylcholinesterase. 4. When erythrocytes were incubated with soman for 0.5 h, a significant decrease in cholinesterase activity in the erythrocytes, plasma, diaphragm and liver following blood-transfusion was found. These data show that soman is able to release from erythrocytes and inhibit cholinesterase activities not only in vitro but also in vivo although the significant inhibition of cholinesterase activities by soman was only observed in the peripheral compartment.

Toxicities of O-alkyl S-(2-dialkylaminoethyl) methyl phosphonothiolates (V-compounds).

Toxicities expressed as LD50 values of O-alkyl S-(2-dialkylaminoethyl) methyl phosphonothiolates in different species and different routes of administration were determined. Rats were more sensitive to these compounds than mice, rabbits and guinea-pigs were more sensitive than mice and rats. The most sensitive to these compounds were dogs. LD50 values varied in a wide range, however, a decrease of toxicity was observed in the following rank: i.v., i.m., s.c., i.p., p.o. and p.c., respectively. Depending on the route of administration, LD50 values varied from 5 g/kg (i.m., dogs) through tens of microgram/kg for the most part of compounds and routes of administration to hundreds, rarely thousands of g/kg (some chemicals, p.c. administration).

Use of hemoperfusion in experimental intoxication with nerve agents.

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.