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Nerve agents pose a real threat to both the military and civil populations, but the current treatment of the poisoning is unsatisfactory. Thus, we studied the efficacy of prophylactic use of memantine alone or in combination with clinically used reversible acetylcholinesterase inhibitors (pyridostigmine, donepezil, rivastigmine) against soman. In addition, we tested their influence on post-exposure therapy consisting of atropine and asoxime. Pyridostigmine alone failed to decrease the acute toxicity of soman. But all clinically used acetylcholinesterase inhibitors administered alone reduced the acute toxicity, with donepezil showing the best efficacy. The combination of memantine with reversible acetylcholinesterase inhibitors attenuated soman acute toxicity significantly. The pretreatment administered alone or in combinations influenced the efficacy of post-exposure treatment in a similar fashion: (i) pyridostigmine or memantine alone did not affect the antidotal treatment, (ii) centrally acting reversible acetylcholinesterase inhibitors alone increased the antidotal treatment slightly, (iii) combination of memantine with reversible acetylcholinesterase inhibitors increased the antidotal treatment more markedly. In conclusion, memantine alone failed to decrease the acute toxicity of soman or increase post-exposure antidotal treatment efficacy. The combination of memantine with donepezil significantly increased post-exposure effectiveness (together 5.12, pretreatment alone 1.72). Both drugs, when applied together, mitigate soman toxicity and boost post-exposure treatment.

Oxime reactivators of acetylcholinesterase (AChE) represent an integral part of standard antidote treatment of organophosphate poisoning. Oxime K869 is a novel bisquaternary non-symmetric pyridinium aldoxime with two pyridinium rings connected by a tetramethylene bridge where two chlorines modify the pyridinium ring bearing the oxime moiety. Based on in vitro assays, K869 is a potent AChE and butyrylcholinesterase (BChE) reactivator. For the investigation of the basic pharmacokinetic properties of K869 after its intramuscular application, new HPLC-UV and LC-MS/MS methods were developed and validated for its determination in rat body fluids and tissues. In this study, the SPE procedure for sample pretreatment was optimized as an alternative to routine protein precipitation widely used in oxime pharmacokinetics studies. K869 oxime is quickly absorbed into the central compartment reaching its maximum in plasma (39 ± 4 μg/mL) between 15 and 20 min. The majority of K869 was eliminated by kidneys via urine when compared with biliary excretion. However, only a limited amount of K869 (65 ± 4 ng/g of brain tissue) was found in the brain 30 min after oxime administration. Regarding the brain/plasma ratio calculated (less than 1%), the penetration of K869 into the brain did not exceed conventionally used oximes.

Agent BZ (3-quinuclidinyl benzilate) is a centrally acting synthetic anticholinergic agent, considered as a potential military incapacitating chemical warfare agent. Despite its significance as a model compound in pharmacological research and its potential misuse in chemical attacks, few modern analytical methods for BZ determination in biological samples have been published. The goal of the present work is to develop and validate a sensitive and rapid LC-MS/MS method for the determination of agent BZ in rat plasma. The sample preparation was based on solid-phase extraction on C-18 cartridges. The reversed-phase HPLC coupled with the mass spectrometer with electrospray ionization in the positive ion-selective reaction monitoring mode was employed in the BZ analysis. Atropine was used as an internal standard. The presented method is selective, accurate, precise, and linear (r2 = 0.9947) in a concentration range from 0.5 ng/mL to 1 000 ng/mL and sensitive enough (limit of detection 0.2 ng/mL; limit of quantification 0.5 ng/mL) to determine the BZ plasma levels in rats exposed to 2 mg/kg and 10 mg/kg of BZ. The highest level of BZ in plasma was observed 5 minutes after intramuscular administration (154.6 ± 22.3 ng/mL in rats exposed to 2 mg/kg of BZ and 1024 ± 269 ng/mL in rats exposed to 10 mg/kg). After 48 h, no BZ was observed at detectable levels. This new method allows the detection and quantification of BZ in biological samples after exposure of an observed organism and it will be further optimized for other tissues to observe the distribution of BZ in organs.

Tacrine is a classic drug whose efficacy against neurodegenerative diseases is still shrouded in mystery. It seems that besides its inhibitory effect on cholinesterases, the clinical benefit is co-determined by NMDAR-antagonizing activity. Our previous data showed that the direct inhibitory effect of tacrine, as well as its 7-methoxy derivative (7-MEOTA), is ensured via a “foot-in-the-door” open-channel blockage, and that interestingly both tacrine and 7-MEOTA are slightly more potent at the GluN1/GluN2A receptors when compared with the GluN1/GluN2B receptors. Here, we report that in a series of 30 novel tacrine derivatives, designed for assessment of structure-activity relationship, blocking efficacy differs among different compounds and receptors using electrophysiology with HEK293 cells expressing the defined types of NMDARs. Selected compounds (4 and 5) potently inhibited both GluN1/GluN2A and GluN1/ GluN2B receptors; other compounds (7 and 23) more effectively inhibited the GluN1/GluN2B receptors; or the GluN1/GluN2A receptors (21 and 28). QSAR study revealed statistically significant model for the data obtained for inhibition of GluN1/Glu2B at 60 mV expressed as IC50 values, and for relative inhibition of GluN1/Glu2A at þ40 mV caused by a concentration of 100 mM. The models can be utilized for a ligand-based virtual screening to detect potential candidates for inhibition of GluN1/Glu2A and/or GluN1/Glu2B subtypes. Using in vivo experiments in rats we observed that unlike MK-801, the tested novel compounds did not induce hyperlocomotion in open field, and also did not impair prepulse inhibition of startle response, suggesting minimal induction of psychotomimetic side effects. We conclude that tacrine derivatives are promising compounds since they are centrally available subtype-specific inhibitors of the NMDARs without detrimental behavioral side-effects.

Since 2002, no clinical candidate against Alzheimer's disease has reached the market; hence, an effective therapy is urgently needed. We followed the so-called "multitarget directed ligand" approach and designed 36 novel tacrine-phenothiazine heterodimers which were in vitro evaluated for their anticholinesterase properties. The assessment of the structure-activity relationships of such derivatives highlighted compound 1dC as a potent and selective acetylcholinesterase inhibitor with IC50 = 8 nM and 1aA as a potent butyrylcholinesterase inhibitor with IC50 = 15 nM. Selected hybrids, namely, 1aC, 1bC, 1cC, 1dC, and 2dC, showed a significant inhibitory activity toward τ(306-336) peptide aggregation with percent inhibition ranging from 50.5 to 62.1%. Likewise, 1dC and 2dC exerted a remarkable ability to inhibit self-induced Aβ1-42 aggregation. Notwithstanding, in vitro studies displayed cytotoxicity toward HepG2 cells and cerebellar granule neurons; no pathophysiological abnormality was observed when 1dC was administered to mice at 14 mg/kg (i.p.). 1dC was also able to permeate to the CNS as shown by in vitro and in vivo models. The maximum brain concentration was close to the IC50 value for acetylcholinesterase inhibition with a relatively slow elimination half-time. 1dC showed an acceptable safety and good pharmacokinetic properties and a multifunctional biological profile.

3-Quinuclidinyl benzilate (BZ) ranks among incapacitating military warfare agents. It acts as a competitive inhibitor on muscarinic receptors leading to non-lethal mental impairment. The present study aimed to investigate toxicokinetics of BZ in rats. Moreover, BZ can be exploited to produce a pharmacological model of Alzheimer's disease; thus, this paper focuses mainly on the BZ distribution to the brain. Wistar rats were administered i.p. with BZ (2 and 10 mg/kg). The BZ concentration was determined using LC-MS/MS in plasma, urine, bile, brain, kidney and liver. The sample preparation was based on a solid phase extraction (liquids) or protein precipitation (organ homogenates). The plasma concentration peaked at 3 min (204.5 ± 55.4 and 2185.5 ± 465.4 ng/ml). The maximal concentration in the brain was reached several minutes later. Plasma elimination half-life was 67.9 ± 3.4 in the 2 mg/kg group and 96.6 ± 27.9 in the 10 mg/kg group. BZ concentrations remained steady in the brain, with slow elimination (t1/2 506.9 ± 359.5 min). Agent BZ is excreted mainly via the urine. Steady BZ concentration in the brain could explain the previously published duration of the significant impairment in passive avoidance tasks in rats after an injection of BZ.

To date, the only treatments developed for poisoning by organophosphorus compounds, the most toxic chemical weapons of mass destruction, have exhibited limited efficacy and versatility. The available causal antidotes are based on reactivation of the enzyme acetylcholinesterase (AChE), which is rapidly and pseudo-irreversibly inhibited by these agents. In this study, we developed a novel series of monoquaternary reactivators combining permanently charged moieties tethered to position 6- of 3-hydroxypyridine-2-aldoxime reactivating subunit. Highlighted representatives (21, 24, and 27; also coded as K1371, K1374, and K1375, respectively) that contained 1-phenylisoquinolinium, 7-amino-1-phenylisoquinolinium and 4-carbamoylpyridinium moieties as peripheral anionic site ligands, respectively, showed efficacy superior or comparable to that of the clinically used standards. More importantly, these reactivators exhibited wide-spectrum efficacy and were minutely investigated via determination of their reactivation kinetics in parallel with molecular dynamics simulations to study their mechanisms of reactivation of the tabun-inhibited AChE conjugate. To further confirm the potential applicability of these candidates, a mouse in vivo assay was conducted. While K1375 had the lowest acute toxicity and the most suitable pharmacokinetic profile, the oxime K1374 with delayed elimination half-life was the most effective in ameliorating the signs of tabun toxicity. Moreover, both in vitro and in vivo, the versatility of the agents was substantially superior to that of clinically used standards. Their high efficacy and broad-spectrum capability make K1374 and K1375 promising candidates that should be further investigated for their potential as nerve agents and insecticide antidotes.

Antidotes against organophosphates often possess physicochemical properties that mitigate their passage across the blood&ndash, brain barrier. Cucurbit[7]urils may be successfully used as a drug delivery system for bisquaternary oximes and improve central nervous system targeting. The main aim of these studies was to elucidate the relationship between cucurbit[7]uril, oxime K027, atropine, and paraoxon to define potential risks or advantages of this delivery system in a complex in vivo system. For this reason, in silico (molecular docking combined with umbrella sampling simulation) and in vivo (UHPLC&mdash, pharmacokinetics, toxicokinetics, acetylcholinesterase reactivation and functional observatory battery) methods were used. Based on our results, cucurbit[7]urils affect multiple factors in organophosphates poisoning and its therapy by (i) scavenging paraoxon and preventing free fraction of this toxin from entering the brain, (ii) enhancing the availability of atropine in the central nervous system and by (iii) increasing oxime passage into the brain. In conclusion, using cucurbit[7]urils with oximes might positively impact the overall treatment effectiveness and the benefits can outweigh the potential risks.

Kamil Kuca,1,2 Jana Zdarova Karasova,2,3 Ondrej Soukup,2 Jiri Kassa,3 Eva Novotna,2 Vendula Sepsova,2,3 Anna Horova,2 Jaroslav Pejchal,3 Martina Hrabinova,2,3 Eva Vodakova,2 Daniel Jun,2,3 Eugenie Nepovimova,1,2 Martin Valis,4 Kamil Musilek1,2 1Department of Chemistry, Faculty of Science, University of Hradec Kralove, 2Biomedical Research Center, University Hospital Hradec Kralove, 3Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University of Defence, 4Department of Neurology, University Hospital Hradec Kralove, Hradec Kralove, Czech Republic Background: Intoxication by nerve agents could be prevented by using small acetylcholinesterase inhibitors (eg, pyridostigmine) for potentially exposed personnel. However, the serious side effects of currently used drugs led to research of novel potent molecules for prophylaxis of organophosphorus intoxication. Methods: The molecular design, molecular docking, chemical synthesis, in vitro methods (enzyme inhibition, cytotoxicity, and nicotinic receptors modulation), and in vivo methods (acute toxicity and prophylactic effect) were used to study bispyridinium, bisquinolinium, bisisoquinolinium, and pyridinium-quinolinium/isoquinolinium molecules presented in this study. Results: The studied molecules showed non-competitive inhibitory ability towards human acetylcholinesterase in vitro that was further confirmed by molecular modelling studies. Several compounds were selected for further studies. First, their cytotoxicity, nicotinic receptors modulation, and acute toxicity (lethal dose for 50% of laboratory animals [LD50]; mice and rats) were tested to evaluate their safety with promising results. Furthermore, their blood levels were measured to select the appropriate time for prophylactic administration. Finally, the protective ratio of selected compounds against soman-induced toxicity was determined when selected compounds were found similarly potent or only slightly better to standard pyridostigmine. Conclusion: The presented small bisquaternary molecules did not show overall benefit in prophylaxis of soman-induced in vivo toxicity. Keywords: AChE inhibitors, prophylaxis, pre-treatment, nerve agents, toxicity, soman

Background Based on in vitro and in vivo rat experiments, the newly developed acetylcholinesterase (AChE) reactivator, K203, appears to be much more effective in the treatment of tabun poisonings than currently fielded oximes. Methods To determine if this reactivating efficacy would extend to humans, studies were conducted in vitro using human brain homogenate as the source of AChE. The efficacy of K203 was compared with commercially available oximes; pralidoxime, obidoxime and asoxime (HI-6). Results Reactivation studies showed that K203 was the most effective reactivator with a second order kinetic constant (k r) of 2142 min− 1. M− 1, which was 51 times higher than that obtained for obidoxime (k r = 42 min− 1. M− 1). Both pralidoxime and asoxime (HI-6) failed to significantly reactivate tabun-inhibited human AChE. Discussion According to these results and previous studies, using K203, it appears that oxime K203 is the most effective reactivator of tabun-inhibited cholinesterase in several species including humans and should be considered as a possible medical countermeasure to tabun exposure.

The ability of two newly developed bispyridinium oximes (K920, K923) to reduce tabun-induced acute neurotoxic signs and symptoms was compared with the oxime K203 and trimedoxime using a functional observational battery (FOB). The neuroprotective effects of the oximes studied combined with atropine on rats poisoned with tabun at a sublethal dose (130 μg/kg i.m.; 80% of LD50 value) were evaluated. Tabun-induced neurotoxicity was monitored by FOB at 2 h after tabun administration. The results indicate that all tested oximes combined with atropine enable tabun-poisoned rats to survive till the end of experiment while one non-treated tabun-poisoned rat died within 2 h. Both newly developed oximes (K920, K923) combined with atropine were able to markedly decrease tabun-induced neurotoxicity in the case of sublethal poisoning although they did not eliminate all tabun-induced acute neurotoxic signs and symptoms. Their ability to decrease tabun-induced acute neurotoxicity did not prevail the neuroprotective efficacy of trimedoxime and the oxime K203. Therefore, the newly developed oximes are not suitable for the replacement of currently available oximes (especially trimedoxime) in the treatment of acute tabun poisonings.

Oxime-based acetylcholinesterase reactivators (briefly oximes) regenerate organophosphate-inactivated acetylcholinesterase and restore its function. Poor blood-brain-barrier passage and fast elimination from blood limit their actual use in treatment of patients exposed to organophosphates. Previous in vitro results implicated further testing of cucurbit[7]uril as a delivery vehicle for bisquaternary oximes. The present paper focuses on cell toxicity, in vivo safety and influence of cucurbit[7]uril on oxime pharmacokinetics and pharmacodynamics. Neither the K027 nor the complex caused any cell toxicity, changes in blood biochemistry or hepato- or nephrotoxicity in tested concentrations. The encapsulation of K027 increased and accelerated the blood-brain-barrier penetration. The peripheral oxime exposure also increased, supporting the suggestion that cucurbit[7]uril protects the circulating oxime from rapid renal clearance. Contrary to the comparable in vitro reactivation power of K027 and the encapsulated K027, we failed to confirm this in vivo. In theory, this might result from the non-specific binding of molecules to the cucurbit[7]uril or the interaction of K027 with cucurbit[7]uril being too strong for acetylcholinesterase reactivation. Precise explanation requires additional in silico, in vitro and also in vivo experiments.

The search for tacrine derivatives, as potential Alzheimer´s disease treatment, is still being at the forefront of scientific efforts. 7-MEOTA was found to be a potent, centrally active acetylcholinesterase inhibitor free of the serious side effects observed for tacrine. Unfortunately, a relevant argumentation about pharmacokinetics and potential toxicity is incomplete; information about tacrine derivatives absorption and especially CNS penetration are still rare as well as detailed toxicological profile

A combination of tacrine and tryptophan led to the development of a new family of heterodimers as multi-target agents with potential to treat Alzheimer's disease. Based on the in vitro biological profile, compound S-K1035 was found to be the most potent inhibitor of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE), demonstrating balanced IC50 values of 6.3 and 9.1 nM, respectively. For all the tacrine-tryptophan heterodimers, favorable inhibitory effect on hAChE as well as on hBChE was coined to the optimal spacer length ranging from five to eight carbon atoms between these two pharmacophores. S-K1035 also showed good ability to inhibit Aβ42 self-aggregation (58.6 ± 5.1% at 50 μM) as well as hAChE-induced Aβ40 aggregation (48.3 ± 6.3% at 100 μM). The X-ray crystallographic analysis of TcAChE in complex with S-K1035 pinpointed the utility of the hybridization strategy applied and the structures determined with the two K1035 enantiomers in complex with hBChE could explain the higher inhibition potency of S-K1035. Other in vitro evaluations predicted the ability of S-K1035 to cross blood-brain barrier and to exert a moderate inhibition potency against neuronal nitric oxide synthase. Based on the initial promising biochemical data and a safer in vivo toxicity compared to tacrine, S-K1035 was administered to scopolamine-treated rats being able to dose-dependently revert amnesia.

The ability of two newly developed oximes (K361, K378) to reduce tabun-induced acute neurotoxic signs and symptoms was compared with the oxime K203 and trimedoxime using a functional observational battery. The neuroprotective effects of the oximes studied combined with atropine on rats poisoned with tabun at a sublethal dose (310 μg/kg i.m.; 90% of LD50 value) were evaluated. Tabun-induced neurotoxicity was monitored by functional observational battery at 2 h following tabun challenge. The results indicate that all tested oximes combined with atropine enable tabun-poisoned rats to survive till the end of experiment. Both newly developed oximes (K361, K378) combined with atropine were able to decrease tabun-induced neurotoxicity in the case of sublethal poisonings although they did not eliminate all tabun-induced acute neurotoxic signs and symptoms. Their ability to decrease tabun-induced acute neurotoxicity was slightly lower than that of trimedoxime and the oxime K203. Therefore, the newly developed oximes are not suitable for the replacement of commonly used oximes (especially trimedoxime and obidoxime) in the treatment of acute tabun poisonings.

The ability of two newly developed oximes (K305, K307) to protect tabun-poisoned rats from tabun-induced inhibition of brain acetylcholinesterase, acute neurotoxic signs and symptoms and brain damage was compared with that of the oxime K203 and trimedoxime. The reactivating and neuroprotective effects of the oximes studied combined with atropine on rats poisoned with tabun at a sublethal dose were evaluated. The reactivating efficacy of a newly developed oxime K305 is lower compared to the reactivating efficacy of the oxime K203 and trimedoxime while the ability of the oxime K307 to reactivate tabun-inhibited acetylcholinesterase (AChE) in the brain roughly corresponds to the reactivating efficacy of the oxime K203 and it is slightly lower compared to trimedoxime. In addition, only one newly developed oxime (K307) combined with atropine was able to markedly decrease tabun-induced neurotoxicity although it did not eliminate all tabun-induced acute neurotoxic signs and symptoms. These results correspond to the histopathological evaluation of tabun-induced brain damage. Therefore, the newly developed oximes are not suitable for the replacement of commonly used oximes (especially trimedoxime) in the treatment of acute tabun poisonings.

Standard treatment of organophosphorus compounds (OPs) poisoning includes administration of an anti-muscarinic (atropine), anticonvulsive (diazepam) and acetylcholinesterase reactivator (oxime). From a wide group of newly synthesized oximes, oxime K027 and oxime K203 seem to be perspective compounds in some specific OPs intoxication. The available in vitro and in vivo preclinical data indicate that both oximes may be considered for potential human use. The main aim of this study was to establish plasmatic concentration curves of both oximes after intramuscular (i.m.) and intragastric (i.g.) application with subsequent pharmacokinetic analysis and study distribution after (i.m.) application on a non-rodent animal model (experimental pigs; 1500mg/animal). According to the results, both oximes had similar Cmax (K027: 106±19μg/mL and K203: 111±8μg/mL) in Tmax 19±5min, respectively, in 22±3min. Bioavailability of oxime K027 calculated as AUCtotal (8389±1024minμg/mL) was halved compared to oxime K203 (16938±795minμg/mL). The highest concentration from peripheral tissues was found in the kidney and lung, but the brain concentrations stay very low, the plasma/brain ratio being approximately 1%. The applied doses were derived from the recommendation where it is possible to use three autoinjectors to save human life. The results provide us with knowledge about the pharmacokinetics and distribution of these new oximes and may help us to better estimate the human pharmacokinetic profile.

The activity of human cholinesterases, erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7) and plasma butyrylcholinesterase (BChE; EC 3.1.1.8) represents an important marker when monitoring exposure to pesticides/nerve agents, and may also be used in occupational medicine in diagnosis and prognosis of some diseases. In this study "normal/baseline" AChE and BChE activity has been investigated in a young and healthy population, with subsequent evaluation of several intra-population factors including sex, age (categories 18-25, 26-35 and 36-45 years old) and smoker status. The modified Ellman's method was used for enzyme activity assessment in 387 young and healthy individuals (201 males and 186 females aged 18-45). A significant inter-sexual difference in AChE and BChE activity was found (AChE: 351±67 for males and 377±65 for females, (μmol/min)/(μmol of hemoglobin), p0.001; BChE: 140±33 for males and 109±29 for females, μkat/l, p0.001; mean±SD). Despite the finding that mean AChE activity somewhat decreased whereas BChE activity grew within the age categories of the tested subjects, no significant effect of age on cholinesterase activity was found (p0.05). Smoking influenced cholinesterase activity - AChE activity in smokers was elevated (approx. 3% in males; 8% in females) relative to that in non-smokers (p0.05). Smoking was found not to have any effect on BChE activity. Reference values based on confidence intervals for AChE and BChE activity were established. The presented results might be useful in routine clinical practice where the monitoring of blood AChE and plasma BChE activity is crucial for prognosis and diagnosis of organophosphate poisoning, in occupational medicine and in relevant mass casualty scenarios.

Summary The objective of the experiment was to verify that HI-6 dimethanesulphonate (HI-6 DMS) is able to penetrate the skin in amounts sufficient to protect against organophosphate poisoning using a rat model. HI-6 2Cl is a major component of Transant, a transdermal patch, used as a protective agent against organophosphate intoxication in the Czech and Slovak armies, although there is little evidence that HI-6 would penetrate the skin in sufficient amounts. HI-6 DMS at a total amount of 127 mg or 635 mg was applied as a buffer solution on the Transant patch which was fixed on the back of the rat. Two, seven or twenty-four hours later, rats were sacrificed and blood samples were collected to determine the levels of HI -6 in plasma by HPLC on reversed phase with isocratic elution and UV/VIS detection. HI-6 was not detectable in plasma samples of animals exposed to 127 mg of HI-6 DMS. The highest levels of HI-6 (20.6 ± 18.8 ng/ml) were found in plasma of animals exposed to 635 mg of HI-6 DMS 2 hours after patch application, whereas after 7 or 24 hours the levels were very low. Based on these results, the ability of HI-6 DMS to penetrate the skin is discussed and some possibilities of improving the transdermal penetration are suggested.

Acetylcholinesterase (AChE) reactivators were developed for the treatment of organophosphate intoxication. Standard care involves the use of anticonvulsants (e.g., diazepam), parasympatolytics (e.g., atropine) and oximes that restore AChE activity. However, oximes also bind to the active site of AChE, simultaneously acting as reversible inhibitors. The goal of the present study is to determine how oxime structure influences the inhibition of human recombinant AChE (hrAChE). Therefore, 24 structurally different oximes were tested and the results compared to the previous eel AChE (EeAChE) experiments. Structural factors that were tested included the number of pyridinium rings, the length and structural features of the linker, and the number and position of the oxime group on the pyridinium ring.

Although the information about Alzheimer’s disease (AD) etiology is still unclear; acetylcholinesterase inhibitors still play a major role in symptomatic treatment of AD. Unfortunately, a relevant argumentation is complicated since information about real drug concentration in the brain or time-dependent blood-brain barrier (BBB) distribution studies are still quite rare. In this in vitro study, high-performance liquid chromatography (HPLC) method with special (IAM – immobilized artificial membrane) column was used to determine the ability of cholinesterase inhibitors to penetrate through BBB. Set of 8 structurally different cholinesterase inhibitors applicable to AD treatment was evaluated throughout this study. According to our method, all molecules are able to penetrate BBB by passive transport. However, some molecules such as huperzine A and galanthamine have lower ability to penetrate the BBB directly. These molecules may be delivered into the brain via active transport. Other molecules probably use passive transport to permeate into the central nervous system; tacrine and 7-methoxytacrine exert the highest passive permeation from this set of compounds. Key words: Blood-brain barrier, central nervous system, acetylcholinesterase, Alzheimer disease.

Summary In this study, 21 structurally diverse drugs with examined central nervous system penetration were used for prediction of blood-brain barrier penetration using HPLC-UV instrument. Drugs' chromatographic capacity factors were measured by the immobilized artificial membrane presented by phosphatidylcholine column. The correlation between chromatographic capacity factor, octanol-water partition coefficient (log P) and molecular polar surface area (PSA) were determined at the physiological pH. The correlation factor 0.6677 with respect to log P and 0.7199 in reference to PSA was assigned. The developed in vitro prediction method may be used as a screening tool for blood-brain barrier penetration of drugs with passive transport mechanism.

The ability of two newly developed bispyridinium oximes (K456, K458) to reduce tabun-induced acute neurotoxic signs and symptoms was compared with oxime K203 and trimedoxime using the functional observational battery. The neuroprotective effects of the oximes studied combined with atropine on rats poisoned with tabun at a sublethal dose (200 μg/kg i.m.; 85% of LD50 value) were evaluated. Tabun-induced neurotoxicity was monitored by the functional observational battery and automatic measurement of motor activity at 2 hr after tabun challenge. The results indicate that all tested oximes combined with atropine enable tabun-poisoned rats to survive till the end of experiment. Both newly developed oximes (K456, K458) combined with atropine were able to decrease tabun-induced neurotoxicity in the case of sublethal poisonings although they did not eliminate all tabun-induced acute neurotoxic signs and symptoms. Their ability to decrease tabun-induced acute neurotoxicity was slightly higher than that of trimedoxime and oxime K203, but the difference in neuroprotective efficacy among all oximes studied is not large enough to make a decision about replacement of commonly used oximes (especially trimedoxime and obidoxime) in the treatment of acute tabun poisonings.

The reactivating and therapeutic efficacy of two combinations of oximes (HI‑6 + trimedoxime and HI‑6 + K203) was compared with the effectiveness of antidotal treatment involving single oxime (HI‑6, trimedoxime, K203) using in vivo methods. In vivo determined percentage of reactivation of cyclosarin‑inhibited blood and tissue acetylcholinesterase in poisoned rats showed that the reactivating efficacy of both combinations of oximes is slightly higher than the reactivating efficacy of the most effective individual oxime in blood, diaphragm as well as in brain. Moreover, both combinations of oximes were found to be slightly more efficacious in the reduction of acute lethal toxic effects in cyclosarin‑poisoned mice than the antidotal treatment involving single oxime. Based on the obtained data, we can conclude that the antidotal treatment involving chosen combinations of oximes brings a beneficial effect for its ability to counteract the acute poisoning with cyclosarin.

A simple, rapid and sensitive method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been developed and validated for the quantitative determination in rat plasma of a new candidate for AD treatment, namely PC 48 (a 7-MEOTA-donepezil like compound) in rat plasma. Sample preparation involved pH adjustment with sodium hydroxide followed by solvent extraction with ethyl acetate:dichloromethane (80:20, v/v). The chromatographic separation was achieved on an Ascentis Express RP-Amide column (75 mm × 2.1mm, 2.7 μm) with a gradient mobile phase consisting of 0.05 M aqueous formic acid and acetonitrile. Detection was carried out using positive-ion electrospray tandem mass spectrometry on an LTQ XL system using the MS/MS CID (collision-induced dissociation) mode. The method was linear in the range 0.1-1000 ng/ml (r(2)=0.999) with a lower limit of quantitation of 0.1 ng/mL. Extraction recovery was in the range 63.5-72.1% for PC 48 and 70.5% for reserpine (internal standard, IS). Intra- and inter-day precisions measured as relative standard deviation were below 10.8% and accuracy was from -7.2% to 7.4%. The method was successfully applied to a pharmacokinetic study involving intramuscular application of 3.86 mg/kg PC 48 to rats for the first time. Pharmacokinetic parameters for PC 48 include Cmax 39.09 ± 4.45 ng/mL,Tmax 5.00 ± 3.08 min, AUC0-t 23374 ± 4045 min ng/mL and t1/2 1065 ± 246 min.

Summary Acetylcholinesterase (AChE) reactivators (oximes) are generally used as antidotes in case of nerve agent poisoning. Because of their affinity to AChE, they may also act as weak inhibitors of AChE. Their inhibition potency against AChE was determined by an in vitro method based on the interaction between AChE and oxime reactivator in the concentration range 10 -1 to 10 -8 M. We used eel AChE for these assays. We found that AChE inhibition strongly depends on the oxime structure. The aim of the present study is to describe the structure-activity relationship (SAR) between oxime structure and inhibition of AChE. AChE reactivators tested include both monoquaternary and bisquaternary structures with the oxime group in different positions on the pyridine ring and with changes in the connecting linker in the case of the bisquaternary compounds. We found AChE inhibition to be highest in bisquaternary oximes that have a longer linker length and have the oxime group in the ortho position. Increased AChE inhibition in monoquaternary oximes was highest when the meta position was occupied by the oxime nucleophile. In addition, different substituents in the connecting chain (in case of bisquaternary oximes) modulated their inhibition potency.

Asoxime (HI-6) is a well known oxime reactivator used for counteracting intoxication by nerve agents. It is able to reactivate acetylcholinesterase (AChE) inhibited even by sarin or soman. The present experiment was aimed to determine markers of oxidative stress represented by thiobarbituric acid reactive substances and antioxidants represented by ferric reducing antioxidant power, reduced and oxidized glutathione in a Beagle dog model. Two groups of dogs were intramuscularly exposed to single (11.4 mg/kg.b.wt.) or tenfold (114 mg/kg.b.wt.) human therapeutically doses of HI-6. HI-6 affinity for AChE in vitro was evaluated in a separate experiment. Complete serum biochemistry and pharmacokinetics were also performed with significant alteration in blood urea nitrogen, creatine phosphokinase, glucose and triglycerides. Blood samples were collected before HI-6 application and after 30, 60, and 120 min. The overall HI-6 impact on organism is discussed.

K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)–propane dibromide] is a promising new reactivator of organophosphate- or organophosphonate-inhibited acetylcholinesterase (AChE) with low acute toxicity and broad spectrum efficacy. The aim of the present study was to compare the pharmacokinetics of both compounds. Male Wistar rats (body weight = 320 ± 10 g) were administered a single intramuscular dose of K027 (22.07 mg kg−1) and an equimolar dose of trimedoxime. Blood was collected at various time intervals until 180 min. Plasma samples were analyzed by reversed-phase HPLC with ultraviolet (UV) detection. The recovery of both oximes from the plasma was approximately 90% and a linear relationship (R2 > 0.998) was observed between the peak areas and concentrations of calibrated standards in the range 1–100 µg ml−1. Near-identical plasma profiles were obtained for both compounds. No differences were found in the mean ± SD values of Cmax (18.6 ± 2.5 vs 20.0 ± 6.3 µg ml−1, P = 0.72) and AUC0–180min (2290 ± 304 vs 2269 ± 197 min µg ml−1, P = 0.84). However, the percentage coefficient of variation of the first-order rate constant of absorption (ka) was 3-fold higher (P

Summary Plasma proteins such as human serum albumin (HSA) and alpha 1-acid glycoprotein (AGP) play an important role in the pharmacokinetic and pharmacodynamic properties of all endogenous or exogenous substances presented in the blood vessels. Their abundance in human blood not only helps to maintain hemostasis, but also makes them ideal candidates as transporters or possible depot reservoirs. Numerous methods e.g. equilibrium dialysis, ultrafiltration. ultracentrifugation, high-performance affinity chromatography were introduced to determine potential interactions with plasma proteins and possible quantification. In this work method was developed for the determination of drug-human serum albumin interactions and applied to obtain pharmacokinetic profile of AChE modulators prepared at the Faculty of Military Health Sciences. For method validation commercially used compounds HI-6 and obidoxime were chosen. HI-6 did not bind at all while obidoxime showed 7 % binding potency.

The present study is aimed at construction of biosensors consisting of a screen printed sensor with a platinum working electrode and immobilized acetylcholinesterase (AChE) as a biorecognition element. Several immobilization protocols such as precipitation with glutaraldehyde, immobilization onto spherical graphite microparticles, sorption, interception into gelatin, and sol-gel were used in order to construct biosensors suitable for practical performance. The activity of AChE was evaluated by means of voltammetric oxidation of thiocholine originating from acetylthiocholine. Square wave and cyclic voltammetry were used throughout the experiments. Suitability of the constructed biosensors was evaluated using the toxic organophosphate diisopropylfluorophosphate.

The penetration of acetylcholinesterase reactivators (oximes) into the central nervous system is typically restricted by the blood–brain barrier. Although oximes are highly hydrophilic compounds, some contradictory results confirming permeation into the brain exist. The aim of this study is to verify the penetration of oximes through the blood–brain barrier and to detect their levels achieved in different brain regions 60 min after the administration. It was confirmed that oximes are able to penetrate into the brain after injection of therapeutic doses corresponding with 5% of LD50. The level in whole brain was 0.58% for trimedoxime and 0.85% for the experimental drug oxime K074 as the percentage of their plasma concentration. The highest concentration was found in frontal cortex (trimedoxime 2.27%; oxime K074 0.95%) and lowest in basal ganglia (trimedoxime 0.86%; oxime K074 0.42%). Entry of oximes into the brain is minimal, but some low reactivation effect should be expected. The reactivation potency of oximes might be higher or lower, depending on the real oxime concentration in a given area.

The influence of the combinations of oximes on the reactivating and therapeutic efficacy of antidotal treament of acute sarin poisoning was evaluated in this study. The ability of two combinations of oximes (HI-6 + trimedoxime and HI-6 + K203) to reactivate sarin-inhibited acetylcholinesterase and reduce acute toxicity of sarin was compared with the reactivating and therapeutic efficacy of antidotal treatment involving single oxime (HI-6, trimedoxime, K203) using in vivo methods. Studies determining percentage of reactivation of sarin-inhibited blood and tissue acetylcholinesterase in poisoned rats showed that the reactivating efficacy of the combination of oximes involving HI-6 and K203 is slightly higher than the reactivating efficacy of the most effective individual oxime in diaphragm and brain but the difference between them is not significant. The ability of combination of oximes involving HI-6 and trimedoxime to reactivate sarin-inhibited acetylcholinesterase roughly corresponds to the reactivating effects of the most effective individual oxime in blood as well as tissues. Moreover, both combinations of oximes were found to be as efficacious in the reduction of acute lethal toxic effects in sarin-poisoned mice as the most effective individual oxime. A comparison of reactivating and therapeutic efficacy of individual oximes showed that the oxime HI-6 is markedly more effective than the oxime K203 and trimedoxime. Based on the obtained data, we conclude that the antidotal treatment involving chosen combinations of oximes does not significantly influence the ability of the most effective individual oxime (HI-6) to reactivate sarin-inhibited rat acetylcholinesterase and to reduce acute toxicity of sarin in mice.

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.

The HPLC method for prediction of drug penetration into the CNS was examined in our study. In this in vitro study, we determined the ability of monoquaternary AChE reactivators to penetrate through blood-brain barrier (BBB). Three reactivators varying in the position of oxime group on the pyridinium ring (2-PAM, 3-PAM and 4-PAM) were evaluated. As a result, the position of the oxime group on the pyridine ring plays a role for penetration of oximes into brain. The best position of oxime group seems to be position four. The location on the position two and three is not favorable for penetrating through BBB.

The influence of the combination of oximes on the reactivating and therapeutic efficacy of antidotal treatment of acute soman poisoning was evaluated. The ability of two combinations of oximes (HI-6 + trimedoxime and HI-6 + K203) to reactivate soman-inhibited acetylcholinesterase and reduce acute toxicity of soman was compared with the reactivating and therapeutic efficacy of antidotal treatment involving single oxime (HI-6, trimedoxime, K203) using in vivo model. Studies determining percent of reactivation of soman-inhibited blood and diaphragm acetylcholinesterase in poisoned rats showed that the reactivating efficacy of both combinations of oximes is slightly greater than the reactivating efficacy of the most effective individual oxime, but the difference among them is not significant. Both combinations of oximes were found to be as effective in the reduction of acute lethal toxic effects in soman-poisoned mice as the antidotal treatment involving the most efficacious individual oxime. Thus, the efficacy of oximes is comparative in rats vs mice. A comparison of reactivating and therapeutic efficacy of individual oximes showed that the newly developed oxime K203 is approximately as effective as commonly used trimedoxime; nevertheless, their reactivating and therapeutic efficacy is markedly lower compared to the oxime HI-6. Based on the obtained data, one can conclude that the antidotal treatment involving chosen combinations of oximes does not significantly influence the potency of the most effective individual oxime (HI-6) to reactivate soman-inhibited rat acetylcholinesterase and to reduce acute toxicity of soman.

Newly developed acetylcholinesterase reactivators K117 [1,5-bis(4-hydroxyiminomethylpyridinium)-3-oxapentane dichloride] and K127 [(1-(4-hydroxyiminomethylpyridinium)-5-(4-carbamoylpyridinium)-3-oxapentane dibromide)] were tested for their potency to reactivate tabun-inhibited human brain cholinesterases. Pralidoxime and trimedoxime were chosen as standard reference reactivators. Human tissue was used, as that was closer on the real treatment of human beings. As a result, oxime K127 was found as the best tested reactivator according to the constant kr, characterizing the overall reactivation process. On the contrary, the maximal reactivation ability expressed as percentage of reactivation was the best for trimedoxime. This differences were caused as a result of using the enzyme from different species. Due to this, experiments on human tissue should be conducted after in vitro and in vivo tests on animals to eliminate such important failures of promising oximes.

Oxime reactivators HI-6, obidoxime, trimedoxime, K347 and K628 were investigated as drugs designed for treatment of tabun intoxication. The experiments were performed on rats in order to simulate real conditions. Rats were intoxicated with one LD50 of tabun and treated with atropine and mentioned reactivators. Activities of erythrocyte acetylcholinesterase (AChE), plasma butyrylcholinesterase (BChE) and brain AChE were measured as markers of reactivation efficacy. An estimation of low molecular weight antioxidant levels using cyclic voltammetry was the second examination parameter. The evaluation of cholinesterases activity showed good reactivation potency of blood AChE and plasma BChE by commercially available obidoxime and newly synthesized K347. The potency of oximes to reactivate brain AChE was lower due to the poor blood–brain barrier penetration of used compounds. Commercially available reactivator HI-6 and newly synthesized K628 caused oxidative stress measured by cyclic voltammetry as antioxidant level. The oxidative stress provoked by HI-6 and K628 was found to be significant on probability level P = 0.05. The others reactivators did not affect antioxidant levels. Copyright © 2009 John Wiley & Sons, Ltd.

Seven new oxime-based acetylcholinesterase reactivators were compared with three currently available ones (obidoxime, trimedoxime, HI-6) for their ability to lessen cholinesterase inhibition in blood and brain of cyclosarin-treated rats. Oximes were given at doses of 5% their LD(50) along with 21 mg/kg atropine five min before the LD(50) of cyclosarin (120 ug/kg) was administered. Blood and brain samples were collected 30 minutes later. The greatest difference between acetylcholinesterase inhibition in blood of cyclosarin-treated rats was found after administration of HI-6 (40%), compared to 22% for trimedoxime and 6% for obidoxime. Only two of the seven newly synthesized oximes had any effect (K203 at 7%, K156 at 5%). Effective oximes against cyclosarin-inhibited plasma butyrylcholinesterase were HI-6 (42%), trimedoxime (11%), and K156 (4%). The oximes were less effective in brain than in blood, with reactivation values for HI-6 30% against acetylcholinesterase and 10% against butyrylcholinesterase. Values for newly synthesized oximes were less than 10% for K206, K269 and K203.

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.

The potency of newly developed bispyridinium compounds (K250, K251) in reactivating tabun-inhibited acetylcholinesterase and reducing tabun-induced lethal toxic effects was compared with currently available oximes (obidoxime, trimedoxime, the oxime HI-6) using in vivo methods. Studies determined percentage of reactivation of tabun-inhibited blood and tissue AChE in poisoned rats and showed that the reactivating efficacy of both newly developed oximes is comparable with the oxime HI-6 but it is significantly lower than the reactivating effects of obidoxime and trimedoxime, especially in diaphragm and brain. Both newly developed oximes were also found to be able to slightly reduce lethal toxic effects in tabun-poisoned mice. Their therapeutic efficacy is higher than the potency of the oxime HI-6 but it is lower than the therapeutic effects of trimedoxime and obidoxime. Thus, the reactivating and therapeutic potency of both newly developed oximes (K250, K251) does not prevail over the effectiveness of currently available oximes and, therefore, they are not suitable for their replacement for the treatment of acute tabun poisoning.

Cholinesterase activity in blood of laboratory rats was monitored. Rats were intoxicated with paraoxon at dosis of 0 - 65 - 125 - 170 - 250 - 500 nmol. The 250 nmol dose was found to be the LD(50). An electrochemical sensor was found useful to provide information about cholinesterase activity. The decrease of cholinesterase activity was correlated to intoxication symptoms and mortality level. It was found that the symptoms of intoxication are not observed while at least 50% of cholinesterase activity in blood remains. The minimal cholinesterase activity essential to survival is around 10%, when compared with the initial state. No changes in levels of low moleculary weight antioxidants were observed.

The neuroprotective effects of newly developed oximes (K203, K206) and commonly used oximes (obidoxime, HI-6) in combination with atropine in rats poisoned with tabun at a sublethal dose (180 microg/kg i.m.; 80% LD(50)) were studied. The tabun-induced neurotoxicity was monitored by using a functional observational battery and an automatic measurement of motor activity. The neurotoxicity of tabun was monitored at 24 hours and 7 days following tabun challenge. The results indicate that K203 and obidoxime in combination with atropine allow all tabun-poisoned rats to survive within 7 days following tabun challenge, while 2 nontreated tabun-poisoned rats and 1 tabun-poisoned rat treated with K206 or HI-6 in combination with atropine died within 7 days. Only one of the newly developed oximes (K203) combined with atropine seems to be effective for a decrease in tabun-induced neurotoxicity within 24 hours after tabun sublethal poisoning, although it is not able to eliminate tabun-induced neurotoxicity completely. On the other hand, the neuroprotective efficacy of commonly used oximes (obidoxime and HI-6), as well as one of the new synthesized oximes (K206), is significantly lower in comparison with K203, according to the number of eliminated tabun-induced neurotoxic signs at 24 hours after tabun challenge. Due to its neuroprotective effects, K203 appears to be a suitable oxime for the antidotal treatment of acute tabun poisonings.

The neuroprotective effects of newly developed oximes (K206, K269) and currently available oximes (obidoxime, HI-6) in combination with atropine in rats poisoned with cyclosarin were studied. The cyclosarin-induced neurotoxicity was monitored using a functional observational battery at 24 hr following cyclosarin challenge. The results indicate that a newly developed oxime K206 is able to counteract cyclosarin-induced neurotoxicity while the neuroprotective potency of another newly developed oxime (K269) is negligible. The neuroprotective efficacy of K206 is markedly higher than commonly used obidoxime; nevertheless, its potency to eliminate cyclosarin-induced neurotoxicity is slightly lower compared to the oxime HI-6. Thus, a newly developed oxime K206 seems to be a better oxime for the antidotal treatment of cyclosarin poisonings than obidoxime due to higher neuroprotective potency although the oxime HI-6 is still the most suitable oxime for the antidotal treatment of acute poisonings with cyclosarin.

The ability of currently available reactivators to reactivate cyclosarin is low. The aim of this study was to determine the reactivating and therapeutic efficacy of newly developed oximes (K206, K269) compared with currently available oximes against cyclosarin.Rats and mice received atropine or atropine + oxime intramuscularly (i.m.) before or after an i.m. dose of cyclosarin. Acetylcholine activity levels in blood and tissues were measured to calculate the reactivation efficacy and potency.In vivo determined percentage of reactivation of cyclosarin-inhibited blood and tissue acetylcholinesterase (AChE) in poisoned rats showed that the potency of both newly developed oximes (K206, K269) to reactivate cyclosarin-inhibited AChE is comparable with that of obidoxime in blood and diaphragm, but slightly higher than that of obidoxime in brain. Their reactivating efficacy is significantly lower compared with that of the oxime HI-6. K206 and K269 are relatively effective in reducing cyclosarin-induced lethal toxic effects in mice. Their therapeutic efficacies exceed the therapeutic potency of obidoxime but not that of HI-6.K206 and K269 are as effective in the reactivation of cyclosarin-inhibited AChE in rats and in the reduction of lethal toxic effects of cyclosarin in mice as obidoxime, but because their reactivating and therapeutic potency is significantly lower than that of HI-6, they are not suitable replacements for the currently available oximes for the treatment of cyclosarin poisoning.

The therapeutical efficacies of eleven oxime-based acetylcholinesterase reactivators were compared in an in vivo (rat model) study of treatment of intoxication caused by tabun. In this group there were some currently available oximes (obidoxime, trimedoxime and HI-6) and the rest were newly synthesized compounds. The best reactivation efficacy for acetylcholinesterase in blood (expressed as percent of reactivation) among the currently available oximes was observed after administration of trimedoxime (16%) and of the newly synthesized K127 (22432) (25%). The reactivation of butyrylcholinesterase in plasma was also studied, the best reactivators were trimedoxime, K117 (22435), and K127 (22432), with overall reactivation efficacies of approximately 30%. Partial protection of brain ChE against tabun inhibition was observed after administration of trimedoxime (acetylcholinesterase 20%, butyrylcholinesterase 30%) and obidoxime (acetylcholinesterase 12%, butyrylcholinesterase 16%).