Evaluation of possible inhibition of human liver drug metabolizing cytochromes P450 by two new acetylcholinesterase oxime-type reactivators.

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.

Melatonin Regulates Oxidative Stress Initiated by Freund's Complete Adjuvant.

Melatonin is a hormone with strong antioxidant properties. In this experiment, Freund's complete adjuvant was used as a stressogenic substance given to laboratory outbred mice, whereas melatonin was investigated as a protectant against the stressogenic effect. Levels of low molecular weight antioxidants, thiobarbituric acid reactive substances, and tumor necrosis factor α and activity of glutathione reductase were determined in blood from the animals. Surprisingly, melatonin was not involved in direct regulation of antioxidants, thiobarbituric acid reactive substances and tumor necrosis factor α. On the other hand, melatonin regulated glutathione reductase activity. We can conclude on regulation of metabolism caused by melatonin in the model. The effect was more important than the expected regulation of immunity and basal oxidative homeostasis.

Combined approach to demonstrate acetylcholinesterase activity changes in the rat brain following tabun intoxication and its treatment.

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.

A comparison of tabun-inhibited rat brain acetylcholinesterase reactivation by three oximes (HI-6, obidoxime, and K048) in vivo detected by biochemical and histochemical techniques.

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.

Characterization of the anticholinergic properties of obidoxime; functional examinations of the rat atria and the urinary bladder.

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.

Tabun-inhibited rat tissue and blood cholinesterases and their reactivation with the combination of trimedoxime and HI-6 in vivo.

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.

Chemical aspects of pharmacological prophylaxis against nerve agent poisoning.

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.

Methylacridinium and its cholinergic properties.

10-Methylacridinium iodide (methylacridinium; MA) is an inhibitor of cholinesterases. Inhibitors of acetylcholinesterase (AChE) are used in the treatment of myasthenia gravis, Alzheimer's disease, and in the prophylaxis of poisoning with organophosphates. Using spectrophotometric Ellman's method at 436 nm and commercial enzymes we found that MA inhibits AChE by binding with relatively high potency to the peripheral anionic site (IC(50) = 1.68 +/- 0.14 1M; human recombinant AChE) and equally to its inhibition of butyrylcholinesterase (BuChE; IC(50) = 3.54 +/- 0.27 1M; BuChE from human serum). MA also inhibits the binding of [(3)H]N-methylscopolamine to the muscarinic M2 receptor subtype, possibly in an allosteric manner (IC(50) = 1.90 1M). Functional effects on both the enzyme and the receptor could be observed in contractile studies on the isolated rat bladder. The ability of MA to cross the blood-brain barrier (log P = -0.32; polar surface area 3.88) provides prerequisites for a potential use of the drug in the treatment of neural disorders.

Different inhibition of acetylcholinesterase in selected parts of the rat brain following intoxication with VX and Russian VX.

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.

Effect of acetylcholinesterase oxime-type reactivators K-48 and HI-6 on human liver microsomal cytochromes P450 in vitro.

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.

The effect of HI-6 on cholinesterases and on the cholinergic system of the rat bladder.

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.

An attempt to assess functionally minimal acetylcholinesterase activity necessary for survival of rats intoxicated with nerve agents.

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.

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

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

Treatment of organophosphate intoxication using cholinesterase reactivators: facts and fiction.

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.

Changes of acetylcholinesterase activity in different rat brain areas following intoxication with nerve agents: biochemical and histochemical study.

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.

Changes of cholinesterase activities in the plasma and some tissues following administration of L-carnitine and galanthamine to rats.

Changes of acetylcholinesterase (AChE) activities in the hypophysis and brain (frontal cortex, hippocampus, medial septum and basal ganglia), and butyrylcholinesterase in plasma and liver following galanthamine (GAL) administration were studied in rats pretreated with L-carnitine (CAR). Following only GAL administration (10 mg/kg, i.m.), both cholinesterases (without clinical symptoms of GAL overdosage) were significantly inhibited. Pretreatment with CAR (3 consecutive days, 250 mg/kg, p.o.) followed by GAL administration showed higher AChE inhibition in comparison with single GAL administration. However, a statistically significant difference was observed for AChE in the hippocampus only. The activity of peripheral cholinesterases was not influenced by CAR pretreatment. Thus, pretreatment with CAR enhanced AChE inhibition in some brain parts of the rat following GAL administration.

Equine butyrylcholinesterase protects rats against inhalation exposure to sublethal sarin concentrations.

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.

Biochemical and behavioral effects of soman vapors in low concentrations.

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.

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

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.