The benefit of combination of oximes for the neuroprotective efficacy of antidotal treatment of sarin-poisoned rats.

The potency of the oxime HI-6 and two combinations of oximes (HI-6 + trimedoxime, HI-6 + K203) to reduce sarin-induced acute neurotoxic signs and symptoms was evaluated in this study. Sarin-induced neurotoxicity and the neuroprotective effects of atropine alone or in combination with HI-6 alone and HI-6 combined with trimedoxime or K203 in rats poisoned with sarin at a sublethal dose (108 µg/kg i.m.; 90% of LD(50) value) were monitored by a functional observatory battery (FOB) 24 h following sarin administration. The results indicate that both mixtures of oximes combined with atropine were able to survive sarin-poisoned rats 24 h following sarin administration while two non-treated sarin-poisoned rats and one sarin-poisoned rat treated with atropine alone or with atropine in combination with the oxime HI-6 died within 24 h following sarin poisoning. All types of antidotal treatment were able to decrease sarin-induced neurotoxic signs and symptoms but not completely. While atropine alone and atropine in combination with the oxime HI-6 were able to eliminate some sarin-induced neurotoxic signs and symptoms, the neuroprotective efficacy of both combinations of oximes with atropine was slightly higher. Thus, both tested combinations of oximes in combination with atropine bring a small benefit for the neuroprotective efficacy of antidotal treatment of acute sarin poisonings.

In vitro binding and in vivo biodistribution studies of the neuroprotective peptide humanin using [125I]humanin derivatives.

Humanin (HN) and HN-derivatives are a family of peptides first reported in the last decade with potent in vitro and in vivo neuroprotective activity, which is mediated through a not completely elucidated mechanism. Recently, our group has evaluated the effect of various HN-derivatives on the 3-quinuclidinyl benzilate (QNB)-induced impairment of spatial orientation and memory in rats, by employing the T-maze test. In the present work four new, tyrosine containing HN-derivatives were synthesized (Y-PAGASRLLLTGEIDLP, peptide I; Y-PAGASRLLLLTGEIDLP, peptide II; Y-SALLRSIPAPAGASRLLLTGEIDLP, peptide III; Y-SALLRSIPAPAGASRLLLLTGEIDLP, peptide IV). The neuroprotective action of these peptides was evaluated in the T-maze test and the most active among them (peptides I and III) was radiolabeled with (125)I. The pure monoradioiodinated peptides were used in: (i) in vitro binding studies with various neuronal cell lines and with brain and stomach membranes from rats and mice and (ii) in vivo biodistribution studies in rats and mice. Moreover, the metabolic stability of the above radiolabeled peptides was studied. Under the experimental conditions used, our data do not confirm the existence of specific binding sites for HN on the neuronal tissue. Nevertheless, they are setting the basis for further relevant studies aiming at the clarification of the mode of the neuroprotective action of HN-peptides.

The multiple T-maze in vivo testing of the neuroprotective effect of humanin analogues.

Humanin (HN) and its analogues have been shown to protect cells against death induced by various Alzheimer's disease (AD) genes and amyloid-beta-peptides in vitro; the analogues [Gly(14)]-HN and colivelin have also been shown to be potent in reversing learning and memory impairment induced by scopolamine or quinuclidinyl benzilate (QNB) in mice or rats in vivo using the Y-maze or multiple T-maze tests. This paper describes the activity of new peptides of the HN family, after i.p. administration, on QNB-induced impairment of spatial memory in the multiple T-maze test in rats. The following peptides have been studied: HN analogues truncated either on the C- or N-terminus, or analogues having a tert-Leu in place of Leu in the central part of the molecule, the active HN core PAGASRLLLLTGEIDLP (RG-PAGA) and its analogues having three or five leucines instead of four, and finally the recently described hybrid peptide colivelin (i.e. a peptide having the activity-dependent neurotrophic factor SALLRSIPA attached to the N-terminus of the active RG-PAGA) and its des-Leu- and plus-Leu-analogues. While the truncated analogues and most of the tert-Leu containing analogues were devoid of activity, the analogues of the RG-PAGA were active, i.e. they reversed the impairment of spatial memory irrespective of the number of Leu present in their sequence. The highest activity was shown by colivelin and its des-Leu-analogue. These results demonstrate the potential of HN analogues in the modulation of the cholinergic system, which plays an important role in the cognitive deficits associated with AD and other neurodegenerative diseases.

Effect of galantamine on acetylcholinesterase and butyrylcholinesterase activities in the presence of L-carnitine in rat selected brain and peripheral tissues.

OBJECTIVES: The alkaloid galantamine (GAL), which exhibits a combined anticholinesterase and direct parasympathomimetic mechanism of action, is employed in conjunction with therapeutic interventions in the stimulation of central cholinergic transfer in cognitive diseases. We attempted to achieve pharmacologically-induced enhancement of the parasympathomimetic activity of GAL in the key areas of rat brain, using an interactive combination of the alkaloid with the transmembrane enhancer L-carnitine (CAR). METHODS: We investigated activities of acetylcholinesterase (AChE) in brain areas (frontal cortex, basal ganglia, septum and hippocampus) and the hypophysis, and that of butyrylcholinesterase (BuChE) in plasma and liver. RESULTS: Following administration of the highest of the GAL doses used (2.5; 5; 10 mg/kg i.m.), AChE activity decreased mainly in the frontal cortex, hippocampus and hypophysis. In the interaction of GAL and CAR, AChE inhibition was stronger but without any statistical significance. The peripheral inhibition of BuChE was found to be dose-dependent. Premedication by CAR led to a slight change in the values of the activities monitored. CONCLUSIONS: CAR in terms of positive modulation of GAL targeting to the central nervous system had no statistically significant effect.

Comparison of effects of different antidotes on tabun-induced cognitive impairment in rats using water maze.

In the past, scientists focused on the development of antidotes (mainly anticholinergics in combination with reactivators of inhibited acetylcholinesterase-oximes) to increase the number of surviving nerve agent-intoxicated individuals. Recently, they are interested in antidotes able not only to protect nerve agent-poisoned men from lethal toxic effects but also to improve their life quality by improvement of their central cognitive functions. In our study, the water maze was used to measure spatial working learning and memory in the case of tabun-induced cognitive impairment in albino Wistar rats. Antidotal treatment consisted of atropine alone or a combination of atropine with an oxime (obidoxime, trimedoxime or oxime HI-6). Our results suggest that atropine alone is not sufficient as a treatment for saving cognitive functions impaired by tabun. On the other hand, the addition of oxime to atropine contributes to improvement of cognitive performance in tabun-poisoned rats regardless of type of oxime.

The acute toxicity of acetylcholinesterase reactivators in mice in relation to their structure.

Oximes in combination with atropine, are an integral part of the treatment of acute intoxications with organophosphorus insecticides or with the nerve agents such as tabun, sarin, soman, cyclosarin or VX. Organophosphorus compounds are extremely potent inhibitors of the enzyme acetylcholinesterase (AChE, 3.1.1.7). The pharmacological action of oximes is multiple: they are able to reactivate the inhibited AChE, but they affect acetylcholine release in peripheral and central cholinergic synapses, allosterically modulate the muscarinic receptors in peripheral and central synapses, and influence the nicotinic receptor-associated ion-channels. In our study, we have determined the acute toxicity of different structures of oximes after intramuscular application in mice. The acute toxicity of oximes is crucial for the assesment of a dose applied as a treatment for organophosphorus intoxications. We have tested 7 oximes of different structures (HS-6, K033, BI-6, MMB-4, K048, HI-6 and obidoxime ) and during our experiments we have observed the intoxication process including typical signs of intoxication, and times of death. K033 was the most toxic oxime with an LD50 of only 48 mg/kg, while the least toxic oxime - HI-6 - has an LD50 value of 671 mg/kg. All the oximes tested were of the bispyridinium type, with different length or shape of the connecting chain and positions of oxime groups at the pyridinium rings. All these structural features play an important role in biological activity of these compounds performed by their acute toxicity as well as by their reactivation potency.

A comparison of the potency of newly developed oximes (K027, K048) and commonly used oximes (obidoxime, HI-6) to counteract tabun-induced neurotoxicity in rats.

The neuroprotective effects of newly developed oximes (K027, K048) and currently available oximes (obidoxime, HI-6) in combination with atropine in rats poisoned with tabun at a sublethal dose (170 microg kg(-1) i.m.; 80% of LD(50) value) were studied. The tabun-induced neurotoxicity was monitored using a functional observational battery and an automatic measurement of motor activity. The neurotoxicity of tabun was monitored at 24 h and 7 days following tabun challenge. The results indicate that the oxime HI-6 in combination with atropine was not able to protect the rats from the lethal effects of tabun. Two non-treated tabun-poisoned rats and one tabun-poisoned rat treated with atropine combined with HI-6 died within 2 h. On the other hand, all other tested oximes combined with atropine allowed all the tabun-poisoned rats to survive 7 days following tabun challenge. Both newly developed oximes combined with atropine seem to be sufficiently effective antidotes for a decrease in tabun-induced neurotoxicity in the case of sublethal poisoning although they are not able to eliminate tabun-induced neurotoxicity completely. The neuroprotective efficacy of obidoxime in combination with atropine approached the potency of newly developed oximes but the ability of the oxime HI-6 to counteract tabun-induced acute neurotoxicity was significantly lower, especially at 24 h after tabun poisoning. Due to their neuroprotective effects, both newly developed oximes appear to be suitable oximes for the antidotal treatment of acute tabun poisoning.

New quaternary pyridine aldoximes as casual antidotes against nerve agents intoxications.

In this work, the ability of four newly synthesized oximes--K005 (1,3-bis(2-hydroxyiminomethylpyridinium) propane dibromide), K027 (1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide), K033 (1,4-bis(2-hydroxyiminomethylpyridinium) butane dibromide) and K048 (1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide) to reactivate acetylcholinesterase (AChE, EC 3.1.1.7) inhibited by nerve agents is summarized. Reactivation potency of these compounds was tested using standard in vitro reactivation test. Tabun, sarin, cyclosarin and VX agent were used as appropriate testing nerve agents. Rat brain AChE was used as a source of the enzyme. Efficacies of new reactivators to reactivate tabun-, sarin-, cyclosarin- and VX-inhibited AChE were compared with the currently used AChE reactivators (pralidoxime, obidoxime and HI-6). Oxime K048 seems to be promising reactivator of tabun-inhibited AChE. Its reactivation potency is significantly higher than that of HI-6 and pralidoxime and comparable with the potency of obidoxime. The best reactivator of sarin-inhibited AChE seems to be oxime HI-6. None of the new AChE reactivators reached comparable reactivation potency. The same results were obtained for cyclosarin-inhibited AChE. However, oxime K033 is also potent reactivator of AChE inhibited by this nerve agent. In the case of VX inhibition, obidoxime and new oximes K027 and K048 seem to be the best AChE reactivators. None from the currently tested AChE reactivators is able to reactivate AChE inhibited by all nerve agents used and, therefore, the search for new potential broad spectrum AChE reactivators is needed.

Signs of cyclosarin-induced neurotoxicity and its pharmacological treatment with quaternary pyridinium-oximes reactivators.

Cyclosarin (GF-agent; O-cyclohexylmethylfluorophosphonate) belongs to highly toxic organophosphorus compounds. Potential for exposure to chemical warfare organophosphosphorus nerve agents, such as cyclosarin exists on the battlefield, or in the civilian sector as a threat by a terrorist group, as well as an accident as part of current demilitarization efforts. Cyclosarin was not in a front of scientific interest for long time. The research interest was increased after Operation Desert Shield and Desert Storm with the possibility (later confirmed by the UN special commission) that cyclosarin constituted the Iraqi chemical agent inventory. In this study, the neurotoxicity of cyclosarin and therapeutic efficacy of three oximes [HI-6(1-(2-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-2-oxa-propane dichloride), BI-6(2-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)-but-2-ene dibromide), HS-6(2-hydroxyiminomethylpyridinium)-3-(3-carbamoylpyridinium)-2-oxa-propane dichloride)] as acetylcholinesterase reactivators in combination with atropine was studied in rats. The therapy was administered intramusculary (i.m.) 1 min after i.m. GF-agent challenge (1 LD50). Testing of cyclosarin-induced neurotoxicity progress was carried out using the method of Functional observational battery (FOB). The experimental animals were observed at 24 h and 7 days following cyclosarin administration. The results were compared to the condition of control rats that received physiological solution instead of cyclosarin and treatment. All tested antidotal compounds induced neuroprotective efficacy, because decrease of neurotoxicity signs was recorded. There were no poisoned experimental group treated with atropine only, because our preliminary study showed no therapeutical effect of atropine alone. Cyclosarin caused a marked statistically significant change in most of the neurobehavioral parameters (FOB) at 24 h and 7 days after exposure, compared to the saline control group. Survival was 7/10 at 24 h and 5/10 at 7 days. Oxime (BI-6, HS-6 or HI-6) + atropine treatment caused a progressing recovery of the neurobehavioral disturbances caused by cyclosarin at 24 h and 7 days after exposure.

In vitro reactivation potency of some acetylcholinesterase reactivators against sarin- and cyclosarin-induced inhibitions.

In our study, we have tested six acetylcholinesterase (AChE) reactivators (pralidoxime, obidoxime, HI-6, trimedoxime, BI-6 and Hlö-7) for reactivation of sarin- and cyclosarin-inhibited AChE using an in vitro reactivation test. We have used rat brain homogenate as the suitable source of enzyme. All oximes are able to reactivate sarin-inhibited AChE. On the other hand, only HI-6 is able to reactivate satisfactorily cyclosarin-inhibited AChE.

Effectivity of new acetylcholinesterase reactivators in treatment of cyclosarin poisoning in mice and rats.

The present study was performed to assess and compare a therapeutic efficacy of obidoxime, HI-6, BI-6 and HS-6 administered in equimolar doses and combined with atropine in cyclosarin-poisoned mice and rats. It was demonstrated that all the therapeutic regimens tested, were able to decrease the cyclosarin-induced toxicity significantly - at least 1.5 times. Higher therapeutic ratios, almost three times, were achieved in rats in comparison with mice. The highest therapeutic ratio was achieved for therapeutic regimen consisting of HI-6 and atropine in both mice and rats. Obidoxime was the least effective oxime in the treatment of cyclosarin intoxication. The BI-6 oxime was significantly more efficacious than obidoxime (in both mice and rats) and HS-6 (in rats) but its effectiveness did not reach the efficacy of HI-6.

Strategy for the development of new acetylcholinesterase reactivators - antidotes used for treatment of nerve agent poisonings.

The mechanism of intoxication with organophosphorus compounds, including highly toxic nerve agents, is based on the formation of irreversibly inhibited acetylcholinesterase (AChE; EC 3.1.1.7) that could be followed by a generalized cholinergic crisis. Nerve agent poisoning is conventionally treated using a combination of a cholinolytic drug (atropine mostly) to counteract the accumulation of acetylcholine at muscarinic receptors and AChE reactivators (pralidoxime or obidoxime) to reactivate inhibited AChE. At the Department of Toxicology, the strategy of the development of new more potent AChE reactivators consists of several steps: description of the nerve agent intoxication mechanism on the molecular basis (molecular design), prediction of the biological active structure of AChE reactivators (artificial neural networks), their synthesis, in vitro evaluation of their potencies (potentiometric titration and Ellman's method), in vivo studies (therapeutic index, LD(50) of newly synthesized reactivators, reactivation in different tissues, neuroprotective efficacy).

Comparison of the potency of newly developed and currently available oximes to reactivate nerve agent-inhibited acetylcholinesterase in vitro and in vivo.

Reactivation potency of three newly developed oximes K027, K033 and K048 was tested using standard in vitro and in vivo reactivation tests. K027 and K048 seem to be efficacious reactivators of tabun-inhibited acetylcholinesterase. K033 is sufficient reactivator of cyclosarin-inhibited AChE. However, its potency is poor compared with current "gold standard" oxime HI-6.

Comparison of ability of some oximes to reactivate sarin-inhibited brain acetylcholinesterase from different species.

The aim of this work was the comparison of reactivation potency of four oxime acetylcholinesterase (AChE) reactivators (pralidoxime, HI-6, K027 and K033) on three resources of the enzyme (human, pig and rat brain homogenate) inhibited by nerve agent sarin. The results demonstrate remarkable differences in the reactivation of inhibited brain AChE, depending on the oxime and species

Bispyridinium oximes as antidotal treatment of cyclosarin poisoning-in vitro and in vivo testing.

The mechanism of intoxication with organophosphorus compounds, including highly toxic nerve agents and less toxic pesticides, is based on the formation of irreversibly inhibited acetylcholinesterase, which causes cumulation of neuromediator acetylcholine in synaptic clefts and subsequent overstimulation of cholinergic receptors, that is followed by a generalized cholinergic crisis. Nerve agent poisoning is conventionally treated using a combination of a cholinolytic (atropine mostly) to counteract the accumulation of acetylcholine and acetylcholinesterase reactivators (pralidoxime or obidoxime) to reactivate inhibited acetylcholinesterase. In this study of cyclosarin poisoning treatment, oximes of different chemical structures (obidoxime, HI-6, BI-6, and HS-6) were tested in vitro on rat brain acetylcholinesterase (enzyme source: rat brain homogenate), and afterwards, they were tested in vivo in equimolar doses, in mice and rats. The HI-6 oxime appeared to be the most effective oxime in vitro and in vivo.

Antidotal treatment of GF-agent intoxication in mice with bispyridinium oximes.

It was shown that intoxications with GF-agent are rather resistant to convential oxime therapy; therefore, the development of new oximes in an effort to improve this unsatisfactory situation continues. Upon screening in vitro reactivation test for oximes, that were either newly synthesized at our department, or those that have never been tested for reactivation of GF-inhibited acetylcholinesterase (AChE), three oximes {(1,4-bis(4-hydroxyiminomethylpyridinium)butane dibromide) (K033); (1-(2-hydroxyiminomethylpyridinium)-3-(3-carbamoylpyridinium)-2-oxa-propane dichloride) (HS-6); and (1-(2-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)-but-2-ene dibromide) (BI-6)} with the highest reactivation potency were chosen for in vivo testing in our study. 1,3-Bis(4-hydroxyiminomethylpyridinium)-2-oxa-propane dibromide) (obidoxime); (1-(2-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-2-oxa-propane dichloride) (HI-6); and (1,1-bis(4-hydroxyiminomethylpyridinium)-methane dibromide) (methoxime) were chosen for comparison as a standard antidotal treatment. All the oximes were applied at the same proportion of their LD50 value (5%), and because of the different acute toxicity of the oximes, the molar concentrations of their solutions for intramuscular (i.m.) administration were considerably different. The highest therapeutic ratio was achieved for therapeutic regimen consisting of HI-6 and atropine. The significantly (P < 0.05) lowest effectivity in treatment of supralethal GF-agent poisoning in comparison with all the other therapeutic regimens, was surprisingly observed for methoxime. HS-6, K033 and BI-6 as well as obidoxime were comparably effective antidotes against GF-agent intoxication and their therapeutic ratios were similar.

In vitro reactivation of acetylcholinesterase inhibited by cyclosarin using bisquaternary pyridinium aldoximes K005, K033, K027 AND K048.

We have tested four new bisquaternary pyridinium acetylcholinesterase (AChE; EC 3.1.1.7) reactivators - K005 (1,3-bis(2-hydroxyiminomethylpyridinium) propane dibromide), K033 (1,4-bis(2-hydroxyiminomethylpyridinium) butane dibromide), K027 (1 -(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide) and K048 (1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide) as the potential reactivators of AChE inhibited by cyclosarin. Their reactivation potencies were studied using standard in vitro reactivation test. Rat brain homogenate was used as the source of the enzyme. Oxime K033 seems to be the most potent reactivator of cyclosarin-inhibited AChE. Its reactivation potency is significantly higher than the efficacy of all other tested AChE reactivators.

Therapeutic efficacy of different antidotal mixtures against poisoning with GF-agent in mice.

The toxicity of cyclohexyl methylphosphonofluoridate (GF-agent; cyclosarin) and therapeutic efficacy of four oximes (trimedoxime, methoxime, obidoxime and HI-6) in combination with atropine or benactyzine (BNZ) was studied in mice. The oxime therapy combined with anticholinergic drug was administered intramusculary (i.m.) 1 or 2 min after i.m. GF-agent challenge. All the drugs were applied in dose of 20% of LD50. Obidoxime and trimedoxime that were combined with atropine were less effective than methoxime and HI-6 in combination with BNZ when applied 2 minutes after GF-agent poisoning. When the drugs were administered 1 min after GF-agent challenge already, in case of methoxime, the faster application of therapy resulted in significantly higher protective ratio, while for obidoxime the therapeutic effectivity did not depend significantly on the seasonableness of therapeutic intervention. The present findings show that all four combinations of oxime with anticholinergic drug decrease the GF-agent toxicity more than twofold regardless of the time of treatment administration.