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Colorectal cancer patients treated with irinotecan (IRI) often manifest acute cholinergic syndrome, which is the reason behind the growing use of legally prescribed cannabis preparations as well as illicit ones that can contain very high Δ9- tetrahydrocannabinol (THC) levels. IRI-induced cholinergic syndrome originates from the inhibition of acetylcholinesterase (AChE), while butyrylcholinesterase (BChE) plays an important role in the hydrolytic metabolism of this prodrug. On the other hand, existing reports of THC effects on cholinesterases are inconclusive. In this study, we aimed to investigate how the concomitant use of a high THC dose with IRI affects cholinesterase activities in blood of mice with experimental colon cancer induced by injection of CT26 cell line. Male BALB/c mice were assigned randomly to five study groups: (i) negative control, (ii) cancer control, (iii) irinotecan (i.p. 60 mg/kg ; 1st and 5th day of the experiment), (iv) THC (p.o. 7 mg/kg ; repeatedly for 7 days), (v) IRI + THC. ChE activity in blood was assayed by the spectrophotometric Ellman method, using acetylthiocholine as a substrate and inhibitors to distinguish between AChE and BChE activities. The results showed a time-dependent increase in total ChE and BChE activities in all experimental groups, including cancer control. In contrast, AChE activities decreased, with some minor fluctuations in the THC and IRI+THC group. Although there was a lack of statistically significant differences, the time course of changes for AChE and BChE activities suggested that these esterases may not only be indicators of metabolic status but also functionally important in neoplastic cell transformation.

Colorectal cancer patients treated with irinotecan (IRI) often manifest acute cholinergic syndrome, which is the reason behind the growing use of legally prescribed cannabis preparations as well as illicit ones that can contain very high Δ9- tetrahydrocannabinol (THC) levels. IRI-induced cholinergic syndrome originates from the inhibition of acetylcholinesterase (AChE), while butyrylcholinesterase (BChE) plays an important role in the hydrolytic metabolism of this prodrug. On the other hand, existing reports of THC effects on cholinesterases are inconclusive. In this study, we aimed to investigate how the concomitant use of a high THC dose with IRI affects cholinesterase activities in blood of mice with experimental colon cancer induced by injection of CT26 cell line. Male BALB/c mice were assigned randomly to five study groups: (i) negative control, (ii) cancer control, (iii) irinotecan (i.p. 60 mg/kg ; 1st and 5th day of the experiment), (iv) THC (p.o. 7 mg/kg ; repeatedly for 7 days), (v) IRI + THC. ChE activity in blood was assayed by the spectrophotometric Ellman method, using acetylthiocholine as a substrate and inhibitors to distinguish between AChE and BChE activities. The results showed a time-dependent increase in total ChE and BChE activities in all experimental groups, including cancer control. In contrast, AChE activities decreased, with some minor fluctuations in the THC and IRI+THC group. Although there was a lack of statistically significant differences, the time course of changes for AChE and BChE activities suggested that these esterases may not only be indicators of metabolic status but also functionally important in neoplastic cell transformation.

Currently we are faced with an ever-growing use of &Delta, 9-tetrahydrocannabinol (THC) preparations, often used as supportive therapies for various malignancies and neurological disorders. As some of illegally distributed forms of such preparations, like cannabis oils and butane hash oil, might contain over 80% of THC, their consumers can become intoxicated or experience various detrimental effects. This fact motivated us for the assessments of THC toxicity in vivo on a Wistar rat model, at a daily oral dose of 7 mg/kg which is comparable to those found in illicit preparations. The main objective of the present study was to establish the magnitude and dynamics of DNA breakage associated with THC exposure in white blood and brain cells of treated rats using the alkaline comet assay. The extent of oxidative stress after acute 24 h exposure to THC was also determined as well as changes in activities of plasma and brain cholinesterases (ChE) in THC-treated and control rats. The DNA of brain cells was more prone to breakage after THC treatment compared to DNA in white blood cells. Even though DNA damage quantified by the alkaline comet assay is subject to repair, its elevated level detected in the brain cells of THC-treated rats was reason for concern. Since neurons do not proliferate, increased levels of DNA damage present threats to these cells in terms of both viability and genome stability, while inefficient DNA repair might lead to their progressive loss. The present study contributes to existing knowledge with evidence that acute exposure to a high THC dose led to low-level DNA damage in white blood cells and brain cells of rats and induced oxidative stress in brain, but did not disturb ChE activities.

In this 28 day-study, we evaluated the effects of the insecticide chlorpyrifos orally administered to Wistar rats at doses 0.160, 0.015, and 0.010 mg/kg b. w./day. Following treatment, total cholinesterase activity and activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were measured. Oxidative stress responses were evaluated using a battery of endpoints to establish lipid peroxidation, changes in total antioxidant capacity, level of reactive oxygen species (ROS), glutathione (GSH) level and activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase. Using HPLC-UV DAD analysis, levels of the parent compound and its main metabolite 3,5,6-trichloro-2-pyridinol in plasma and brain tissue were measured. The genotoxic effect was estimated using alkaline comet assay in leukocytes and brain tissue. The exposure did not result in significant effects on total cholinesterase, AChE and BChE activity in plasma and brain tissue. Lipid peroxidation slightly increased both in plasma and brain tissue. Total antioxidant capacity, ROS and GSH levels were marginally influenced by the exposure. Treatment led to significant increases of GSH-Px activity in blood, SOD activity in erythrocytes and a slight increase of catalase activity in plasma. HPLC-UV DAD analysis revealed the presence of both the parent compound and its main metabolite in the plasma of all of the experimental animals and brain tissue of the animals treated at the two higher doses. All of the tested doses of chlorpyrifos were slightly genotoxic, both to leukocytes and brain tissue. Our results call for further research using other sensitive biomarkers of effect, along with different exposure scenarios.

K048 is a member of K-oximes, a new oxime class that has recently been confirmed effective against poisoning by the nerve agent tabun and several pesticides. The toxicity profile of the K048 oxime has not been fully characterized and its optimal therapeutic dose has not yet been established. Earlier studies report excellent results with K048 in reactivating tabun-phosphorylated AChE and in the therapy of tabun-poisoned mice. It possesses a low acute toxicity and exerts an acceptable toxicity profile on isolated human peripheral blood lymphocytes in vitro. Intraperitoneal administration of K048 in rats resulted in an LD50 of 238.3 mg/kg. In this in vivo study, we investigated cholinesterase (ChE) activity and oxidative stress marker levels (lipid peroxidation and superoxide dismutase activity) in the plasma of exposed rats after administering the compound at 25% of its LD50. Lymphocyte viability was evaluated using an acridine orange/ethidium bromide in situ fluorescent assay. The levels of primary DNA damage in rat white blood cells were measured using the alkaline comet assay. The compound applied at 25% of its LD50 did not significantly affect ChE activity and lipid peroxidation and did not cause significant changes in the SOD activity in plasma. The cytotoxicity profile of K048 in the tested dose was also acceptable, and it did not possess significant DNA-damaging potential. The obtained results are promising for further evaluations of the K048 oxime, which should include tests on a broader concentration range and longer incubation times.

Some compounds, although not primarily designed as supportive drugs in chemotherapy, are promising candidates for clinical use. The ability of HI-6 oxime to relieve the side effects of irinotecan was recently determined in vitro. In this animal study, we investigated the efficacy of HI-6 in vivo, when given as a pre-treatment and concomitantly with irinotecan. We evaluated the cholinesterase (ChE)/acetylcholinesterase (AChE) activity, the levels of oxidative stress markers, DNA damage and the radical scavenging capacity of HI-6. Both HI-6 and irinotecan inhibited ChE/AChE activity but showed different levels of ChE inhibition in plasma and AChE inhibition in the liver and brain tissue. We also observed a weak antioxidant capacity of HI-6, undiscovered until now, and found an acceptable genotoxicity profile in three types of somatic cells in rats. The in vivo erythrocyte micronucleus assay showed that HI-6 did not significantly change either the frequency of micronuclei or the ratio of polychromatic and normorchromatic erythrocytes. Taken together, our results provide a good argument in favour of HI-6 as a promising molecule for further studies and eventual use in humans.

One of the therapeutic approaches to organophosphate poisoning is to reactivate AChE with site-directed nucleophiles such as oximes. However, pyridinium oximes 2-PAM, HI-6, TMB-4 and obidoxime, found as the most effective reactivators, have limiting reactivating potency in tabun poisoning. We tested oximes varying in the type of ring (pyridinium and/or imidazolium), the length and type of the linker between rings, and in the position of the oxime group on the ring to find more effective oximes to reactivate tabun-inhibited human erythrocyte AChE. Three of our tested pyridinium oximes K027, K048, K074, along with TMB-4, were the most promising for AChE reactivation. Promising oximes were further tested in vivo on tabun poisoned mice not only as antidotes in combination with atropine but also as pretreatment drug. Herein, we showed that a promising treatment in tabun poisoning by selected oximes and atropine could be improved if oximes are also used in pretreatment. Since the reactivating efficacy of the oximes in vitro corresponded to their therapeutic efficacy in vivo, it seems that pharmacological effect of these oximes is indeed primarily related to the reactivation of tabun-phosphorylated AChE.

Improving the efficacy of antidotal treatment of poisonings with nerve agents is still a challenge for the scientific community. This study investigated the interactions of four bispyridinium oximes with human erythrocyte acetylcholinesterase (AChE) and their effects on soman- and tabun-poisoned mice. Oximes HI-6 and TMB-4 were used for comparison. These oximes inhibited AchE with inhibitory potency (IC 50 ) ranging from 0.02 to 1.0 mM. The best reactivating potency (%R) was obtained with K074, when AChE was inhibited by tabun. The protective potency (P 50 ) of all oximes in human erythrocyte AChE inhibited by soman and tabun could not be determined. In tabun-poisoned mice very good antidotal efficacy was obtained with K027, K048, and K074, which makes them interesting for future investigation. The combination of HI-6 and atropine is the therapy of choice for soman poisoning.

Literature data on carbofuran genotoxicity in vitro and in vivo are very scarce. There are few papers indicating that occupational exposure to this AChE inhibiting insecticide might be connected to increased risk of developing non-Hodgkin’ s lymphoma and lung cancer. Other authors showed its genotoxicity in vitro. We used comet and CBMN micronucleus assay combined with centromere probes to evaluate genome damage in lymphocytes of workers employed in carbofuran production. Also, the level of AChE activity in blood and plasma was measured. Only few workers exhibited AChE activity below 85%. Comet assay parameters were slightly but significantly elevated compared to control subjects, especially the long-tailed nuclei ratio. We found poor correlation between AChE activity and comet assay parameters, but significant effect of smoking and alcohol intake on the latest. In binucleated lymphocytes of workers significantly increased number of micronuclei, nuclear buds, and nucleoplasmatic bridges was detected. Proportion of micronuclei with centromere, DAPI signal positive micronuclei was also elevated. Micronucleus assay parameters also appeared to be significantly influenced by duration of exposure to carbofuran. Together with published data on carbofuran’ s effect on health our results might indicate the need for further evaluations of its genotoxicity using a range of different cytogenetic techniques.

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

A conjugate of pyridine-4-aldoxime and atropine (ATR-4-OX) was synthesized and its antidotal efficiency was tested in vitro on tabun- or paraoxon-inhibited acetylcholinesterase (AChE) of human erythrocytes as well as in vivo using soman-, tabun- or paraoxon-poisoned mice. Its genotoxic profile was assessed on human lymphocytes in vitro and was found acceptable for further research. ATR-4-OX showed very weak antidotal activity, inadequate for soman or tabun poisoning. Conversely, it was effective against paraoxon poisoning both in vitro and in vivo. All animals treated with 5 % or 25 % LD(50) doses of the new oxime survived after administration of 10.0 or 16.0 LD(50) doses of paraoxon, respectively. Based on the persistence of toxicity symptoms in mice, the atropine moiety had questionable effects in attenuating such symptoms. It appears that ATR-4-OX has a therapeutic effect related to the reactivation of phosphylated AChE, but not to receptor antagonization.

Organophosphate pesticides and nerve agents are characterized as compounds influencing cholinergic nerve transmission via inhibition of acetylcholinesterase (AChE). The brain is primary target, since the induced toxic effects on brain are life threatening. Identifying the brain regions that are most vulnerable to organophosphates can facilitate the development of effective antidotes that will reduce damage after exposure. The aim of this study was to determine AChE activity in selected brain regions and whole brain at four different time points (0.5, 1, 6 and 24 h) in paraoxon poisoned rats as well as reactivatability of paraoxon-inhibited AChE after treatment with oxime K048 and atropine. Paraoxon administration markedly inhibited cortical AChE activity (around 50%). The rank order of inhibition in other regions from high to low was septum>pontomedullar area (PM)≥cerebellum>thalamus and hypothalamus (TH). Such inhibition was maintained till 24 h after poisoning which correlates well with the inhibition of enzyme activity in the whole brain. Paraoxon inhibited AChE was generally not affected by the oxime treatment, with three exceptions. In the cortex, PM and TH treatment with K048 and atropine elevated AChE activity during the first hour but the reactivation potency did not exceed 20%. Observed different levels of inhibition underline the fact that protection of AChE have different importance for various brain structures. No marked differentiation of AChE activity between poisoned and treated rats could be due to oxime's limiting penetration through blood-brain barrier.

Qualitative GC-MS Assessment of TCP and Tamorf Elimination in RatsNerve agents are highly toxic organophosphorus (OP) compounds. They inhibit acetylcholinesterase (AChE), an enzyme that hydrolyses acetycholine (ACh) in the nervous system. Pathophysiological changes caused by OP poisonings are primarily the consequence of surplus ACh on cholinergic receptors and in the central nervous system. Standard treatment of OP poisoning includes combined administration of carbamates, atropine, oximes and anticonvulsants. In order to improve therapy, new compounds have been synthesised and tested. Tenocyclidine (TCP) and its adamantane derivative 1-[2-(2-thienyl)-2-adamantyl] morpholine (TAMORF) have shown interesting properties against soman poisoning. In this study, we developed a qualitative GC-MS method to measure elimination of TCP and TAMORF through rat urine in order to learn more about the mechanisms through which TCP protects an organism from OP poisoning and to determine the duration of this protective effect. GC-MS showed that six hours after treatment with TCP, rat urine contained only its metabolite 1-thienylcyclohexene, while urine of rats treated with TAMORF contained both TAMORF and its metabolites.

Rats have been used in study of temporal distribution of the cholinesterase activity after exposure to tabun and treatment with oxime 1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide, K048. In the event of poisoning by organophosphorus compounds (OP), immediate therapeutic treatment usually consists of combined administration of an anticholinergic drug, such as atropine, and an oxime-reactivator of acetylcholinesterase. However, the treatment is very limiting in case of nerve agent tabun poisoning. Since our recent in vitro and in vivo studies on mice showed that oxime 1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide (K048) is very promising as reactivator of tabun-inhibited human erythrocyte acetylcholinesterase and as antidote of tabun poisoned mice, we tested antidotal potency of K048 in tabun-poisoned rats. We measured acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity in plasma after 0.5, 1, 6 and 24 h long oxime therapy. In view of the possible oxidative stress involved in OP poisoning, it has been decided to estimate the levels of oxidative stress (lipid peroxidation), and to see whether any difference exists in this parameter upon tabun poisoning and administrated therapy in rats. Male rats received a subcutaneous (s.c.) dose of 3/4 of the tabun LD50. Oxime was given intraperitoneally (i.p.) at doses of 1/4 of its LD50 after 1 min of tabun poisoning. Animals were divided into 5 groups of four animals receiving: tabun (I), oxime (II), atropine (III) tabun+oxime+atropine (IV) and saline as control group. Atropine was administered i.p. (10.0 mg/kg body weight) together with oxime. All animals were sacrificed after 0.5, 1, 6 and 24 h. Plasma was diluted 50 times for AChE and BChE activity measurement by Ellman assay at 25 °C using acetylthiocholine as substrate. AChE and BChE activity was determined using selective inhibitors BW284C51 and ethopropazine, respectively. As a measure of lipid peroxidation the concentration of thiobarbituric reactive substances (TBARS) was determined using modification of the method by Drury et al. (1997). Activity of AChE and BChE (control group and group II) ranged from 1.5-2.9 and 0.6-1.8  mol min-1 per g of protein, respectively, with no matter of sacrifice time. Activity of group I and III of both enzymes was below 0.3  mol min-1 per g of protein up to 6 h, while in 24 h both activities were completely restored. AChE activity of group IV in 30 min and 1 h was in same range as control confirming the K048 reactivation potency, while in 6 h the therapy by K048 was depleted - AChE activity was low as in group I. Treatment with K048 did not preserve BChE activity that is also in accordance with previous K048 in vitro study, due to limiting BChE reactivation by K048. The levels of plasma TBARS progressively increased up to 6 h for all treatment groups. The major increase was detected after tabun administration while only minor time-dependent differences were observed after treatment with K048. Although the levels of TBARS decreased up to 24 h, they remained above the range of the control for most of the treatments. The significant improvement was noticed in the AChE activity of tabun-poisoned rats with applied K048 therapy. Poisoning did not significantly induce the oxidative stress.

A toxic effect of highly toxic nervous agents is irreversible inhibition of vitally important enzyme acethylcholinesterase (AChE). Inhibition of AChE results in accumulation of acetylcholine (ACh) at the synaptic cleft of the cholinergic neurons, leading to overstimulation of cholinergic receptors. The highly toxic nature of tabun has been known for many years, but there are still serious limitations to the antidotal therapy. In this paper a bispyridinium compound K027 [1-(4-hydroxyiminomethylpyridinium)-3-(-4-carbamoylpyridinium) propane dibromide] was tested as potential antidote in tabun poisoned mice. Oxime TMB-4 was included for comparison. The therapeutic efficacy of applied antidotal regimens was tested as pretreatment given 15 min before tabun poisoning and/or as therapy given 1 min after tabun poisoning. Using oxime K027 (25% of its LD 50 ) plus atropine as both, pretreatment and therapy, we showed that this combination can protect mice 8 times better than the therapy alone. Under these experimental conditions we confirmed good antidotal efficacy of K027. Moreover, its low acute toxicity is as much as beneficial effect in contrast to high toxicity of currently used TMB-4.

Alzheimer’s disease (AD) is the most common cause of age-associated memory deficit. It is a degenerative neurological disorder characterized by senile plaques containing amyloid β protein and loss of cholinergic neuromediators in the brain. The most remarkable biochemical change in the brain of AD patients is a reduction of acetylcholine (ACh) levels. Inhibition of acetylcholinesterase (AChE), the enzyme responsible for hydrolysis of ACh, is one of the most established therapeutic approaches in AD treatment. In recent years the free radical hypothesis of aging has brought a better understanding of biochemical events that occur with Alzheimer’s disease. The brain is considered very sensitive to damage caused by oxidative stress for a variety of reasons including, high uptake of oxygen, low glutathione content and high membrane content of polyunsaturated fatty acids. The fact that age is a key risk factor in AD provides considerable support for the free radical hypothesis because effects of the attacks by free radicals, particularly those produced by reactive oxygen species (ROS), can accumulate over the years. Numerous plants and their constituents are reputed in traditional practices of medicine to enhance cognitive function and to alleviate other symptoms of AD, including depression. As a part of our continuing study into discovery of new cholinesterase inhibitors from Croatian plants, the aim of this work was to determine inhibitory potential on acetylcholinesterase as well as antioxidant activity of 39 extracts from 13 selected Croatian plants. AChE inhibitory activities were tested by Ellman method, while antioxidant activities were evaluated using DPPH and FRAP assays. Salix alba L. methanol extract showed the best inhibition of AChE (EC50 =1, 2 g/L), as well as good antioxidant activity correlated with high phenol content determined by Folin - Ciocalteu method.

Evaluation of Oxime K203 as Antidote in Tabun Poisoning We studied bispyridinium oxime K203 [(E)-1-(4-carbamoylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide] with tabun-inhibited human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in vitro, and its antidotal effect on tabun-poisoned mice and rats in vivo. We compared it with oximes K048 and TMB-4, which have proven the most efficient oxime antidotes in tabun poisoning by now. Tabun-inhibited AChE was completely reactivated by K203, with the overall reactivation rate constant of 1806 L mol-1 min-1. This means that K203 is a very potent reactivator of tabun-inhibited AChE. In addition, K203 reversibly inhibited AChE (K i = 0.090 mmol L-1) and BChE (K i = 0.91 mmol L-1), and exhibited its protective effect against phosphorylation of AChE by tabun in vitro. In vivo, a quarter of the LD50 K203 dose insured survival of all mice after the application of as many as 8 LD50 doses of tabun, which is the highest dosage obtained compared to K048 and TMB-4. Moreover, K203 showed high therapeutic potency in tabun-poisoned rats, preserving cholinesterase activity in rat plasma up to 60 min after poisoning. This therapeutic improvement obtained by K203 in tabun-poisoning places this oxime in the spotlight for further development.

We studied bispyridinium oxime K203 [(E)-1-(4-carbamoylpyridinium)-4-(4-hydroxyimino methylpyridinium)- but-2-ene dibromide] with tabun-inhibited human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in vitro, and its antidotal effect on tabun-poisoned mice and rats in vivo. We compared it with oximes K048 and TMB-4, which have proven the most effi cient oxime antidotes in tabun poisoning by now. Tabun-inhibited AChE was completely reactivated by K203, with the overall reactivation rate constant of 1806 L mol-1 min-1. This means that K203 is a very potent reactivator of tabun-inhibited AChE. In addition, K203 reversibly inhibited AChE (Ki = 0.090 mmol L-1) and BChE (Ki = 0.91 mmol L-1), and exhibited its protective effect against phosphorylation of AChE by tabun in vitro. In vivo, a quarter of the LD50 K203 dose insured survival of all mice after the application of as many as 8 LD50 doses of tabun, which is the highest dosage obtained compared to K048 and TMB-4. Moreover, K203 showed high therapeutic potency in tabun-poisoned rats, preserving cholinesterase activity in rat plasma up to 60 min after poisoning. This therapeutic improvement obtained by K203 in tabun-poisoning places this oxime in the spotlight for further development., Proučavali smo bispiridinijski oksim K203 [(E)-1-(4-karbamilpiridinij)-4-(4-hidroksiiminometilpiridinij)- but-2-ene dibromid] u uvjetima in vitro - studirajući njegove interakcije s ljudskom acetilkolinesterazom (AChE) i butirilkolinesterazom (BChe) inhibiranim tabunom te u uvjetima in vivo - određivanjem njegova antidotskog učinka na miševe i štakore otrovane tabunom. Radi usporedbe uključili smo rezultate dobivene s oksimima K048 i TMB-4 kao najučinkovitijim oksimima kod otrovanja tabunom. K203 je potpuno reaktivirao AChE inhibiranu tabunom sa sveukupnom brzinom reaktivacije od 1806 L mol-1 min-1 što ga svrstava u najučinkovitije reaktivatore AChE inhibirane tabunom. K203 je reverzibilno inhibirao AChE (Ki = 0,090 mmol L-1) i BChE (Ki = 0,91 mmol L-1) pokazujući svoja in vitro zaštitna svojstva od inhibicije tabunom. Terapija dozom K203 od ¼ njegove LD50 omogućila je preživljavanje svih miševa nakon otrovanja dozom tabuna od 8,0 LD50. Time je K203 pokazao bolju učinkovitost u usporedbi s K048 ili TMB-4. K tome, K203 je značajno zaštitio štakore od otrovanja tabunom kompenzirajući toksični učinak tabuna na aktivnost kolinesteraze i do 60 min nakon trovanja. Pokazano poboljšanje terapeutske učinkovitosti K203 ističe ovaj oksim pretečom za daljnji razvoj antidota u otrovanju tabunom.

This study aimed to evaluate the antidotal potency of tenocyclidine (TCP) that probably might protect acetylcholinesterase (AChE) in the case of organophosphate poisoning. TCP was tested alone as a pretreatment or in combination with atropine as a therapy in rats poisoned with (1/4) and (1/2) of LD(50) of soman. Possible genotoxic effects of TCP in white blood cells and brain tissue were also studied. Results were compared with previous findings on the adamantyl tenocyclidine derivative TAMORF. TCP given alone as pretreatment, 5 min before soman, seems to be superior in the protection of cholinesterase (ChE) catalytic activity in the plasma than in brain, especially after administration of the lower dose of soman. Plasma activities of the enzyme after a joint treatment with TCP and soman were significantly increased at 30 min (P0.001) and 24 h (P=0.0043), as compared to soman alone. TCP and atropine, given as therapy, were more effective than TCP administered alone as a pretreatment. The above therapy significantly increased activities of the enzyme at 30 min (P=0.046) and 24 h (P0.001), as compared to controls treated with (1/4) LD(50) of soman alone. Using the alkaline comet assay, acceptable genotoxicity of TCP was observed. However, the controversial role of TCP in brain protection of soman-poisoned rats should be studied further.

The function of acetylcholinesterase (AChE) is the rapid hydrolysis of the neurotransmitter acetylcholine (ACh), which is involved in the numerous cholinergic pathways in both the central and the peripheral nervous system. Therefore, AChE measurement is of high value for therapy management, especially during the course of intoxication with different chemicals or drugs that inhibit the enzyme. Pyridinium or bispyridinium aldoximes (oximes) are able to recover the activity of the inhibited enzyme. Since their adverse effects are not well elucidated, in this study the efficiency of HI-6 oxime in protection and/or reactivation of human erythrocyte AChE inhibited by the antineoplastic drug irinotecan as well as its cyto/genotoxicity in vitro were investigated. HI-6 was effective in protection of AChE and increased its activity up to 30%; the residual activity after irinotecan inhibition was 7%. Also, it reactivated the enzyme previously inhibited by 50% irinotecan (4.6 microg/ml) applied at 1/4 of the IC50 value. The tested concentrations of HI-6 exhibited acceptable genotoxicity towards white blood cells, as estimated by the alkaline comet assay, DNA diffusion assay and cytogenetic endpoints (structural chromosome aberrations and cytokinesis-block micronucleus assay). The results obtained warrant the further investigation of HI-6 in vivo, as well as its development for possible application in chemotherapy.

Improving the efficacy of antidotal treatment of poisonings with nerve agents is still very important task. Organophosphates-nerve agents are characterized as compounds influencing cholinergic nerve transmission via inhibition of acetylcholinesterase (AChE), an extremely active enzyme that hydrolyses acetylcholin. The clinical signs of AChE inhibition manifest as hypersalivation, lacrimation, diarrhoea, tremor, respiratory distress, convulsions and seizures. Signs are dose-dependent, leading to severe incapacitation and rapid death. Pyridinium oximes are nowadays used as successful treatment of poisoning with many organophosphates. Reason for that lies in the mechanism of oxime activity, which is based on protection of unphosphorylated AChE and/or reactivation of phosphorylated AChE. The aim of this investigation was to define the biochemical parameters of three bispyridinium oximes 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]. For each of the compounds it was determined the toxic effect on human erythrocytes AChE in vitro (IC50) and their reactivating (%R) and protective potency (P50) with respect to soman- and tabun- inhibited AChE. Earlier we described in vivo antidotal activity of tested oximes against soman and tabun in male mice. The new tested oximes were toxic to human erythrocytes AChE ; in decreasing order from K033, K048 to K027. IC50 ranged from 0.02 to 1.0 mM. The best reactivating potency was obtained with K048 when AChE was inhibited by tabun. The protective potency of all oximes on human erythrocytes AChE inhibited by soman and tabun could not be determined. Our results indicate a good affinity of the tested compounds for AChE. The oximes are reversible ihibitors of AChE in vitro and show pharmacological properties which are related to reactivation of AChE.

The function of acetylcholinesterase (AChE) is the rapid hydrolysis of the neurotransmitter acetylcholine (ACh), which is involved in the numerous cholinergic pathways in both, the central and the peripheral nervous system. Therefore, AChE measurement is of high value for therapy management, especially during the course of the intoxication with different chemicals or drugs that inhibits activity of enzyme. For the recovery of inhibited enzyme, derivatives from the group of pyridinium or bispyridinium aldoximes (oximes) are used. Adverse effects of these substances are not well elucidated, because of their narrow and one-shot usage. In this study we evaluated the in vitro efficacy of oxime HI-6 to protect and/or reactivate human erythrocyte AChE inhibited by anticancer drug irinotecan, which cause acute cholinergic syndrome after its administration. Moreover, using alkaline comet assay possible genotoxic effects of HI-6 were evaluated on human peripheral blood leukocytes. HI-6 increases the activity of AChE to 30% ; residual activity after irinotecan inhibition was 7%. Also, it reactivated the enzyme by 40% when applied in a concentration of ¼ of the IC50 value. None of the tested concentrations of HI-6 was genotoxic as estimated by evaluation of three main comet parameters: tail length, tail intensity and tail moment. The results obtained sustain the further investigation of HI-6 in vivo, as well as its development for possible application in chemotherapy.

One of the therapeutic approaches to organophosphate poisoning is to reactivate AChE with site-directed nucleophiles such as oximes. However, pyridinium oximes 2-PAM, HI-6, TMB-4 and obidoxime found as the most effective reactivators have limiting reactivating potency in tabun poisoning. We tested oximes varying in the type of ring (pyridinium and/or imidazolium), the length and type of the linker between rings, and in the position of the oxime group on the ring to find more effective oximes to reactivate tabun-inhibited human erythrocyte AChE and studied their effects on tabun-poisoned mice. Using molecular mechanics we performed conformational analysis to determine the flexibility of the oxime molecules. Tabun-inhibited human erythrocyte AChE was completely reactivated by three flexible bispyridinium oximes with the oxime group in para-position and with the linker of three or four methylene groups. Although oximes with imidazolium ring or with the oxime group in the ortho-position on the pyridinium ring had similar electron density on the oxygen of the oxime group as para-bispyridinium oximes, they did not show significant reactivation potency. However, as inhibitors they had higher affinity for the native human erythrocyte AChE. Promising oximes were further tested in vivo on tabun poisoned mice not only as antidotes in combination with atropine but also as pretreatment drug. Without pretreatment the best antidotal efficacy was obtained with para-oximes that protected mice from 5-fold LD50 of tabun. Pretreatment by the oxime insured survival of all tested animals after the application of 10 LD50 of tabun. Herein we show that already promising treatment in tabun poisoning by oximes and atropine could be improved if oximes are also used in pretreatment. Furthermore, low toxicity of these oximes makes these compounds leading in the therapy of tabun poisoning. Since the reactivating efficacy of the oximes in vitro corresponded to their therapeutic efficacy in vivo, it seems that pharmacological effect of these oximes is indeed primarily related to the reactivation of tabun-phosphorylated AChE.

The function of acetylcholinesterase (AChE) is the rapid hydrolysis of the neurotransmitter acetylcholine (ACh), which is involved in the numerous cholinergic pathways in both, the central and the peripheral nervous system. Therefore, AChE measurement is of high value for therapy management, especially during the course of the intoxication with different chemicals or drugs that inhibits activity of enzyme. For the recovery of inhibited enzyme, derivatives from the group of pyridinium or bispyridinium aldoximes (oximes) are used. Adverse effects of these substances are not well elucidated, because of their narrow and one-shot usage. In this study we evaluated the in vitro efficacy of oxime HI-6 to protect and/or reactivate human erythrocyte AChE inhibited by anticancer drug irinotecan, which cause acute cholinergic syndrome after its administration. Moreover, using alkaline comet assay possible genotoxic effects of HI-6 were evaluated on human peripheral blood leukocytes. HI-6 increases the activity of AChE to 30% ; residual activity after irinotecan inhibition was 7%. Also, it reactivated the enzyme by 40% when applied in a concentration of ¼ of the IC50 value. None of the tested concentrations of HI-6 was genotoxic as estimated by evaluation of three main comet parameters: tail length, tail intensity and tail moment. The results obtained sustain the further investigation of HI-6 in vivo, as well as its development for possible application in chemotherapy.

The function of acetylcholinesterase (AChE) is the rapid hydrolysis of the neurotransmitter acetylcholine (ACh), which is involved in the numerous cholinergic pathways in both, the central and the peripheral nervous system. Therefore, AChE measurement is of high value for therapy management, especially during the course of the intoxication with different chemicals or drugs that inhibits activity of enzyme. Different substances, pyridinium or bispyridinium aldoximes (oximes) among them, are able for the recovery of inhibited enzyme. Since their adverse effects are not well elucidated, in this study the efficiency of HI-6 oxime in protection and/or reactivation of human erythrocyte (AChE) inhibited by antineoplastic drug irinotecan as well as its cyto / genotoxicity in vitro were investigated. HI-6 increases the activity of AChE to 30% ; residual activity after irinotecan inhibition was 7%. Also, it reactivated the enzyme previously inhibited by irinotecan (4.6 mg/ml) by 50% when applied in a concentration of ¼ of the IC50 value. Tested concentrations of HI-6 exhibited acceptable genotoxicity on white blood cells, as estimated by the alkaline comet assay, DNA diffusion assay and cytogenetic endpoints (structural chromosome aberrations and cytokinesis-block micrunucleus assay). The results obtained sustain the further investigation of HI-6 in vivo, as well as its development for possible application in chemotherapy.

Oximes K033 [1, 4-bis(2-hydroxyiminomethylpyridinium) butane dibromide] and K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide] were tested as pretreatment drugs in tabun poisoned mice followed by treatment with atropine plus K033, K048, K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], TMB-4 [1, 3-bis(4-hydroxyiminomethylpyridinium) propane dibromide] and HI-6 [(1-(2-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-2-oxapropane dichloride)]. Oxime doses of 25% or 5% of its LD50 were used for pretreatment 15 minutes before tabun-poisoning and for treatment one minute after tabun administration to mice. The best therapeutic effect was obtained when oxime K048 (25% of its LD50) was used in both pretreatment and treatment with atropine. This regiment insured survival of all tested animals after the application of 10 LD50 of tabun. In addition, since BChE is considered an endogenous bio-scavenger of anticholinesterase compounds and its interactions with oximes could be masked by AChE interactions, we evaluated kinetic parameters for interactions of tested oximes with native and tabun-inhibited human plasma butyrylcholinesterase (BChE ; EC 3.1.1.8) and compared them with results obtained previously for human erythrocyte acetylcholinesterase (AChE ; EC 3.1.1.7). Progressive inhibition of BChE by tabun was slightly faster than that of AChE. The reactivation of tabun-inhibited BChE by oximes was very slow, and BChE binding affinity for oximes was lower than AChE's. Therefore, BChE could scavenge tabun prior to AChE inhibition, but fast oxime-assisted reactivation of tabun-inhibited AChE or protection of AChE by oxime against inhibition with tabun would not be obstructed by interaction between BChE and oximes.

Acetylcholinesterase (AChE: EC 3.1.1.7.) is an extremely active enzyme necessary for terminating the action of acetylcholine in cholinergic synapses. Inhibition of AChE enzyme by organophosphorus compounds (pesticides, nerve agents in chemical warfare) leads to an accumulation of neurotransmitter acetylcholine (ACh) resulting in an over-stimulation of the whole cholinergic system. The clinical signs of AChE inhibition manifest as hypersalivation, lacrimation, diarrhoea, tremor, respiratory distress, convulsion and seizures. Together with atropine, pyridinium oximes are known to be successfully used to treat poisoning with many organophosphorus compounds. In this paper new bis-pyridinium oxime K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide] was tested as potential antidote in vitro using human erythrocyte AChE inhibited by tabun and in vivo using tabun poisoned mice. Its antidotal effect was compared with TMB-4 [1, 3-bis (4-hydrxyiminomethylpyridinium) propane dibromide], which is the best-known antidote in tabun poisoning. K048 was poor inhibitor of human erythrocyte AChE in vitro (its IC50 value could not be determined). Its protective potency expressed as P50 was 3.3 x 10-3 M, and the reactivating potency (applied in final concentration of 1.4 x 10-3 M) was 98%. In all experiments on mice, oxime doses of ¼ or 5% of its LD50 were used for pre-treatment 15 minutes before tabun-intoxication and for treatment together with atropine one minute after tabun administration. The antidotal efficacy of tested compound was expressed as therapeutic factor (TF) and therapeutic dose (TD). The highest TF and TD (22.5 and 10.0, respectively) were obtained when K048 was used in dose of ¼ of its LD50 in both, pre-treatment and treatment with atropine. Herein we showed that already promising treatment in tabun poisoning by oximes and atropine could be improved if oximes are also used in pre-treatment. Low acute toxicity of K048 ; about three times lower than of TMB-4, makes it interesting for further investigation of tabun poisoning treatment. K048 may even provide a platform for further modifications and development of more potent protectors and reactivators in organophosphorus poisoning.

The increased concern about terrorist use of nerve agents prompted us to search for new more effective oximes against tabun and soman poisoning. We investigated the interactions of five bispyridinium oximes: K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide], K033 [1, 4-bis(2-hydroxyiminomethylpyridinium) butane dibromide], TMB-4 [1, 3-bis(4-hydroxyiminomethylpyridinium) propane dibromide] and HI-6 [(1-(2-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-2-oxapropane dichloride)] with human erythrocyte acetylcholinesterase (AChE ; E.C. 3.1.1.7) and their effects on tabun- and soman-poisoned mice. All the oximes reversibly inhibited AChE, and the enzyme-oxime dissociation constants were between 17 and 180  M. Tabun-inhibited AChE was completely reactivated by TMB-4, K027 and K048, with the overall reactivation rate constants of 306, 376 and 673 min-1M-1, respectively. The reactivation of tabun-inhibited AChE by K033 reached 50 % after 24 hours, while HI-6 failed to reactivate any AChE at all. Soman-inhibited AChE was resistant to reactivation by 1 mM oximes. All studied oximes protected AChE from phosphorylation with both soman and tabun. In vivo experiments showed that the studied oximes were relatively toxic to mice ; K033 was the most toxic (LD50=33.4 mg/kg), while K027 was the least toxic (LD50=672.8 mg/kg). The best antidotal efficacy was obtained with K048, K027 and TMB-4 for tabun poisoning, and HI-6 for soman poisoning. Moreover, all tested oximes showed no cytotoxic effect on several cell lines in concentrations up to 0.8 mM. The potency of the oximes K048 and K027 to protect mice from 5-fold LD50 of tabun and their low toxicity make these compounds leading in the therapy of tabun poisoning. The combination of HI-6 and atropine is the therapy of choice for soman poisoning.

The nerve agent soman, a powerful inhibitor of acetylcholinesterase (AChE; EC 3.1.1.7), causes an array of toxic effects in the central nervous system. Adamantyl tenocyclidine derivative Tamorf (1-[2-(2-thienyl)-2-adamantyl] morpholine), a compound with potential activity at the N-methyl-D-aspartate (NMDA) receptors and with neuroprotective properties, is effective against convulsions and brain lesions related to soman poisoning. The objective of this study was to evaluate the antidotal potency of Tamorf (2.5 mg kg(-1)), which was tested alone as a pretreatment or in combination with atropine (10.0 mg kg(-1)) as a therapy in rats poisoned with two different sub-lethal doses of soman (1/4 and 1/2 of LD50). The effect of Tamorf was compared with carbamate physostigmine (0.1 mg kg(-1)). The study also determined the possible genotoxic effects of Tamorf and physostigmine, especially primary DNA damage in white blood cells, liver and brain tissue. Tamorf administered 5 min before poisoning stopped soman-induced seizures, was successful against sub-lethal doses of soman and protected AChE activity in the brain (P = 0.0014, P = 0.0019), and in plasma (P = 0.0464, P = 0.0405). Compared with Tamorf, physostigmine was slightly effective in the elimination of soman-induced poisoning in rats. The pharmacological effect of Tamorf and atropine was less effective as therapy, but did not increase soman toxicity (P0.05 for all interactions). The results obtained indicate that Tamorf and physostigmine are not genotoxic to rats in the concentrations tested. Treatment with Tamorf seems to be a good alternative for current pretreatment in soman poisoning. Its antidotal mechanism is complex and is based on combined biochemical and receptor properties.

The increased concern about terrorist use of nerve agents prompted us to search for new, more effective oximes against tabun and soman poisoning. We investigated the interactions of five bispyridinium oximes: K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide], K033 [1, 4-bis(2-hydroxyiminomethylpyridinium) butane dibromide], TMB4 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) propane dibromide] and HI-6 [(1-(2’ -hydroxyiminomethyl-1'-pyridinium)-3-(4''-carbamoyl-1''-pyridinium)-2-oxapropane dichloride)] with human erythrocyte acetylcholinesterase (AChE ; E.C. 3.1.1.7) and their effects on tabun and soman poisoned mice. All the oximes reversibly inhibited AChE, and the enzyme-oxime dissociation constants were between 17 and 180  M. Tabun-inhibited AChE was completely reactivated by TMB4, K027 and K048, with the overall reactivation rate constants of 311, 381 and 659 min-1M-1, respectively. The reactivation of tabun-inhibited AChE by K033 reached 50 % after 24 hours, while HI-6 failed to reactivate any AChE at all. Soman-inhibited AChE was resistant to reactivation by 1 mM oximes. All studied oximes protected AChE from phosphorylation by both soman and tabun. Our results obtained from in vivo experiments showed that the studied oximes were relatively toxic to mice ; K033 was the most toxic (LD50=33.4 mg/kg), while K027 was the least toxic (LD50=672.8 mg/kg). The best antidotal efficacy was obtained with K048, K027 and TMB4 for tabun poisoning, and HI-6 for soman poisoning. Therefore, the potency of the oximes to protect mice from 5-fold LD50 of tabun makes these compounds important in the therapy of tabun poisoning. Whatever its shortcomings, the combination of HI-6 and atropine has been the therapy of choice for soman poisoning.

Organophosphorus compounds are widely used as pesticides and unfortunately as nerve agents in chemical warfare. They are known inhibitors of acetylcholinesterase (AChE, EC 3.1.1.7) an enzyme that hydrolizes the neurotransmitter acetylcholine in the nervous system. The clinical signs of AChE inhibition manifest as hypersalivation, lacrimation, diarrhoea, tremor, respiratory distress, convulsion and seizures. Signs are dose-dependent, leading to severe incapacitation and rapid death. Together with atropine, pyridinium oximes are known to be successfully used to treat poisoning with many organophosphorus compounds. In this paper three new bis-pyridinium compounds: K033 [1, 4-bis(2-hydroxyiminomethylpyridinim)butane dibromide], K027 [1, 4-hydroksyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide] were tested as potential antidotes in tabun poisoned mice. Their antidotal effect was compared with TMB-4 [1, 3-bis(4-hydrxyiminomethylpyridinium) propane dibromide], which is the best-known antidote in tabun poisoning. In all experiments, oxime K033 in doses of 1/4 or 5% of its LD50 was used for the pre-treatment 15 minutes before tabun-intoxication. Also, one or 5 minutes after tabun application experimental animals received oxime K027, K033 or K048 (5% or 1/4 of its LD50) plus atropine sulphate as therapy. The antidotal efficacy of tested compounds was expressed as therapeutic factor (TF) and therapeutic dose (TD). Under same experimental conditions, our experiment selected compound K048 as the most reactivator of tabun inhibited AChE. Namely, this study has shown that the therapeutic regimen consisting of K033 in dose 5% of its LD50 as preatretment and f of LD50 of K048 plus atropine as treatment had the highest TF and TD. The TF was 13.3 LD50 of tabun, TD was 10 LD50 of tabun and insurance survival of all tested animals. In conclusion, treatment with these new bis-pyridinium oximes seems to be a very good alternative for current treatment in tabun poisoning. For this reason, these and other similar compounds require further investigation.

The progressive inhibition of acetylcholinesterase (AChE [EC 3.1.1.7]) by organophosphates (OPs), such as the nerve agents tabun and soman, is due to phosphorylation of the active center serine characterized by the formation of conjugates and inactivation of this essential enzyme involved in neurotransmission. Presently, a combination of an antimuscarinic agent, e.g., atropine, and an AChE reactivator, oxime, is used for the treatment of organophosphorus compound poisoning. The increased concern about terrorist use of nerve agents prompted us to search for new, more effective oximes against tabun and soman poisoning. We investigated the interactions of five bispyridinium oximes with human erythrocyte AChE and their effects on tabun- and soman-poisoned mice.

Organophosphorous (OP) pesticides are in wide use around the world and a significant number of poisonings and deaths are reported each year as a result of exposure to them. OP insecticide parathion, whose active metabolite is paraoxon, has a major contribution to occupational and accidental intoxications. The acute toxicity of OP pesticides is primarily attributed to irreversible inhibition of acetylcholinesterase (AChE), an enzyme vital to normal nerve function. The inability of the conventional therapy to provide adequate protection against these intoxications calls for a synthesis of new compounds with the characteristic oxime group. In the present study, we investigated efficacy of oxime K048 by using a rat model. ChE/AChE activity was measured in rat plasma, brain and diaphragm 0.5, 1, 6 and 24 h upon paraoxon poisoning and applied oxime plus atropine therapy. Degree of inhibition and temporal distribution of the cholinesterase activity after exposure to paraoxon were similar in all observed organs but it is important to stress that enzyme activity in both plasma and tissues did not return to normal even after 24 h (max. 37%). A combination of K048 and atropine afforded high potency in restoring the activity of ChE in plasma by achieving 50-60% of reactivation efficacy up to 6 h and by completely reactivating plasma ChE after 24 h. The applied therapy also increased AChE activity in tissues although the reactivation potency did not surpassed 20%. Our results reveal marked improvement in counteracting paraoxon poisoning probably due to good reactivation potential of oxime K048.