Závěrečná zpráva o řešení grantu Agentury pro zdravotnický výzkum MZ ČR
nestr.
Projekt je zaměřen na vývoj nových kombinovaných micelárních dekontaminačních systémů, založených na sloučeninách obsahující kvarterní dusík, které mají jak detergentní tak i aktivní dekontaminační vlastnosti, které napomohou rychlejší hydrolýze bojových chemických látek. V případě biologických agens tyto látky vykazují silné dezinfekční účinky, protože jsou schopny narušit membránové struktury patogenů. V rámci projektu budou připraveny dekontaminační směsi, u kterých budou metodami in vitro a in vivo hodnoceny jejich dekontaminační a dezinfekční vlastnosti vůči vybraným chemickým a biologickým agens. Předpokládaným výstupem projektu je účinné dekontaminační činidlo vhodné pro osobní dekontaminaci pokožky s dobrou snášenlivostí.; Project is aimed at the development of new combined micellar decontamination systems based on quaternary nitrogen compounds having detergent and active-decontamination properties, which will cause faster hydrolysis of chemical warfare agents. In the case of biological agents, these molecules are strong disinfectants, able to destabilize pathogen membrane structures. Several decontamination mixtures will be prepared and tested both in vitro and in vivo for their decontamination and disinfection properties against selected chemical and biological agents. The expected result of the project is efficient decontamination solution for personal skin decontamination with good tolerability.
- Klíčová slova
- dekontaminace, chemické agens, biologické agens, bojové chemické látky, viry, bakterie, decontamination, chemical agents, biological agents, chemical warfare agents, viruses, bacteria,
- NLK Publikační typ
- závěrečné zprávy o řešení grantu AZV MZ ČR
We have in vitro tested the ability of common, commercially available, cholinesterase reactivators (pralidoxime, obidoxime, methoxime, trimedoxime and HI-6) to reactivate human acetylcholinesterase (AChE), inhibited by five structurally different organophosphate pesticides and inhibitors (paraoxon, dichlorvos, DFP, leptophos-oxon and methamidophos). We also tested reactivation of human butyrylcholinesterase (BChE) with the aim of finding a potent oxime, suitable to serve as a "pseudocatalytic" bioscavenger in combination with this enzyme. Such a combination could allow an increase of prophylactic and therapeutic efficacy of the administered enzyme. According to our results, the best broad-spectrum AChE reactivators were trimedoxime and obidoxime in the case of paraoxon, leptophos-oxon, and methamidophos-inhibited AChE. Methamidophos and leptophos-oxon were quite easily reactivatable by all tested reactivators. In the case of methamidophos-inhibited AChE, the lower oxime concentration (10(-5) M) had higher reactivation ability than the 10(-4) M concentration. Therefore, we evaluated the reactivation ability of obidoxime in a concentration range of 10(-3)-10(-7) M. The reactivation of methamidophos-inhibited AChE with different obidoxime concentrations resulted in a bell shaped curve with maximum reactivation at 10(-5) M. In the case of BChE, no reactivator exceeded 15% reactivation ability and therefore none of the oximes can be recommended as a candidate for "pseudocatalytic" bioscavengers with BChE.
- Publikační typ
- časopisecké články MeSH
An high-performance liquid chromatography (HPLC) method for identification of quaternary and non-quaternary compounds (parent compounds, intermediates, by-products, and products) within the synthesis of the acetylcholinesterase reactivator HI-6, the most promising antidote of nerve agent poisonings, is described. This HPLC method could be of high interest as a quick purity control for those who are interested in development of new acetylcholinesterase reactivators as well as for those who are interested in the synthesis of HI-6 in laboratory or in large-scale production. An HPLC method for quaternary compounds without using common ion-pairing reagents was developed, too.
- Klíčová slova
- nervově paralytické látky, acetylcholinesteráza, cholinesterázy, reaktivátory, K027, K203, HI-6, autoinjektor,
- MeSH
- autoaplikace metody přístrojové vybavení MeSH
- chemické bojové látky škodlivé účinky MeSH
- cholinesterasové inhibitory farmakologie terapeutické užití MeSH
- financování organizované MeSH
- lékové formy MeSH
- lidé MeSH
- organofosfáty MeSH
- organofosforové sloučeniny farmakologie škodlivé účinky MeSH
- pesticidy MeSH
- reaktivátory cholinesterasy terapeutické užití MeSH
- Check Tag
- lidé MeSH
Bioscavengers are considered as promising antidotes against organophosphate poisoning. We focused on a bacterial phosphotriesterase (PTE) expressed in Escherichia coli. The main disadvantage of this non-human catalytic bioscavenger is its relatively short half-life in the organism and strong immunogenicity after repeated administration. Therefore, we prepared different methoxy polyethylene glycol (MPEG)-conjugated recombinant PTE as a potential catalytic bioscavenger with the aim to improve its biological properties. Enzyme was modified with two linear monofunctional MPEG derivatives with reactive aldehyde group of molecular weight 2 kDa and 5 kDa. We optimized reaction conditions (reagent ratios, temperature and duration of modification reaction) and we prepared homogeneous population of fully modified recombinant PTE with molecular weight around 52 kDa and 76 kDa, respectively. Modified PTE was characterized using SDS-PAGE and MALDI-TOF and by determining K(m) and V(max). We also investigated thermal stability of modified enzyme at 37 degrees C. Based on our results, for future in vivo evaluation of pharmacokinetics and pharmacodynamics properties, we selected recombinant PTE modified with 5 kDa MPEG aldehyde for its superior thermal stability.
- MeSH
- aktivace enzymů MeSH
- aldehydy chemie MeSH
- antidota chemie izolace a purifikace metabolismus farmakologie MeSH
- biokatalýza MeSH
- Caulobacteraceae enzymologie MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- hydrolasy triesterů kyseliny fosforečné chemie izolace a purifikace metabolismus farmakologie MeSH
- koncentrace vodíkových iontů MeSH
- organofosfáty metabolismus MeSH
- otrava organofosfáty MeSH
- polyethylenglykoly chemie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- stabilita enzymů MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Class of monoquaternary pyridinium oximes was in vitro tested as potential reactivators of acetylcholinesterase (AChE; EC 3.1.1.7) inhibited by nerve agent sarin. Human brain homogenate was used as an appropriate source of cholinesterases. Reactivation potency of novel oximes was compared with currently available reactivators - pralidoxime, obidoxime, and HI-6. According to the obtained results, only five reactivators were able to satisfactorily renew cholinesterase potency (pralidoxime, obidoxime, HI-6, 4-PAM, and K119). Unfortunately, none of the novel tested reactivators surpassed the reactivation potency of the currently most promising reactivator, HI-6. This study shows that monoquaternary reactivators are unable to reactivate nerve agent-inhibited AChE. Due to this, in future, only bisquaternary compounds derived from HI-6 or obidoxime should be designed as new potential cholinesterase reactivators.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- antidota farmakologie MeSH
- cholinesterasové inhibitory toxicita MeSH
- financování organizované MeSH
- lidé MeSH
- molekulární struktura MeSH
- mozek enzymologie účinky léků MeSH
- obidoxim chlorid farmakologie MeSH
- oximy farmakologie MeSH
- pralidoximové sloučeniny farmakologie MeSH
- pyridinové sloučeniny farmakologie MeSH
- reaktivátory cholinesterasy farmakologie MeSH
- sarin toxicita MeSH
- techniky in vitro MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
Organophosphorus pesticides (e.g. chlorpyrifos, malathion, and parathion) and nerve agents (sarin, tabun, and VX) are highly toxic organophosphorus compounds with strong inhibition potency against two key enzymes in the human body-acetylcholinesterase (AChE; EC 3.1.1.7) and butyrylcholinesterase (BuChE; EC 3.1.1.8). Subsequent accumulation of acetylcholine at synaptic clefts can result in cholinergic crisis and possible death of intoxicated organism. For the recovery of inhibited AChE, derivatives from the group of pyridinium or bispyridinium aldoximes (called oximes) are used. Their efficacy depends on their chemical structure and also type of organophosphorus inhibitor. In this study, we have tested potency of selected cholinesterase reactivators (pralidoxime, obidoxime, trimedoxime, methoxime and H-oxime HI-6) to reactivate human erythrocyte AChE and human plasma BuChE inhibited by pesticide paraoxon. For this purpose, modified Ellman's method was used and two different concentrations of oximes (10 and 100 microM), attainable in the plasma within antidotal treatment of pesticide intoxication were tested. Results demonstrated that obidoxime (96.8%) and trimedoxime (86%) only reached sufficient reactivation efficacy in case of paraoxon-inhibited AChE. Other oximes evaluated did not surpassed more than 25% of reactivation. In the case of BuChE reactivation, none of tested oximes surpassed 12.5% of reactivation. The highest reactivation efficacy was achieved for trimedoxime (12.4%) at the concentration 100 microM. From the data obtained, it is clear that only two from currently available oximes (obidoxime and trimedoxime) are good reactivators of paraoxon-inhibited AChE. In the case of BuChE, none of these reactivators could be used for its reactivation.
- MeSH
- acetylcholinesterasa MeSH
- butyrylcholinesterasa metabolismus MeSH
- cholinesterasové inhibitory toxicita MeSH
- enzymové reaktivátory farmakologie MeSH
- financování organizované MeSH
- lidé MeSH
- organofosforové sloučeniny toxicita MeSH
- oximy farmakologie MeSH
- paraoxon toxicita MeSH
- techniky in vitro MeSH
- Check Tag
- lidé MeSH
In this study, several thin-layer chromatography (TLC) methods are described for the identification of quaternary and non-quaternary compounds (parent compounds, intermediates, by-products, and products) arisen within the synthesis of the acetylcholinesterase reactivator HI-6, at present the most promising antidote in the case of nerve agent poisonings. Using the TLC technique, particular E and Z isomers of this compound on the oxime group are separated. These TLC methods could be of high interest as quick purity control for those who are interested in development of new acetylcholinesterase reactivators and the synthesis of HI-6 in laboratories or in large-scale production.
