► Tabun exposure induced cholinergic and non-cholinergic effects in human myoblasts. ► Significant changes were observed on AChE activity but not on AChE mRNA level. ► Significant changes were ...observed in the HSP 27 level and secretion of IL-6. ► Oxime K048 as antidote was able to attenuate the tabun induced effects.
Organophosphorus compounds (OPs) and oximes may interfere with other molecules than AChE in the living systems, affecting in this way various cellular processes and underlying mechanisms. These non-cholinergic effects may contribute to the clinical status in OP poisoning and therefore deserve equal scientific attention. Here, we investigated the effects of tabun and oxime K048 on the processes known to be involved in muscle response to the environmental factors, like IL-6 release and the regulation of the heat shock proteins (HSPs). While IL-6 stimulates muscle regeneration, which follows well known OP-induced myopathy, HSPs have cytoprotective effect against various stress factors including xenobiotics. All our experiments were carried out on cultured human myoblasts, as the precursors of muscle regeneration. We found unchanged AChE mRNA level after tabun/K048 treatment meaning that tabun and K048 did not interfere with the transcription or stability of this mRNA in the time period tested, even if AChE catalytic activity was significantly affected. On the other hand, after myoblast exposure to tabun, we observed significant changes in the protein levels of HSP 27 and in the secretion of IL-6. Namely, secretion of IL-6 decreased to 53% and the level of HSP 27 increased by 34% compared to the control level. Both effects were attenuated if myoblasts were pretreated with oxime K048, but not if they were treated with K048 after exposure to tabun. The molecular mechanism underlying these effects remains to be elucidated. However, it seems that these effects could be associated with OPs and oximes as a specific group of compounds rather than as a specific compound itself. Overall, the effects of OPs and oximes demonstrated here might play an important role in muscle regeneration which importantly determines the final outcome of OP myotoxicity.
Even if organophosphorus (OP) nerve agents were banned entirely, their presence would remain a problem as weapons of terror (like in Syria). Oxime antidotes currently used in medical practice still ...fall short of their therapeutic purpose, as they fail to fully restore the activity of cholinesterases, the main target for OPs. As orphan drugs, these antidotes are tested too seldom for anybody's benefit. Over the last few decades, search for improved reactivators has reached new levels, but the translation of data obtained in vitro to in vivo application is still a problem that hinders efficient therapy. In this study, we tested the strengths and weaknesses of extrapolating pyridinium oxime antidotes reactivation efficiency from in vitro to in vivo application. Our results show that this extrapolation is possible with well-determined kinetic constants, but that it also largely depends on oxime circulation time and its tissue-specific distribution. This suggests that pharmacokinetic studies should be planned at the early stages of antidote development. Special attention should also be given to improving oxime distribution throughout the organism to overcome this major constraint in improving overall OP therapy.
A series of novel pyridinium oximes was prepared by reactions of quaternization of pyridoxal oxime with substituted phenacyl bromides in acetone at room temperature. The structures of compounds were ...determined according to the data obtained by IR spectroscopy, mass spectrometry,
1H and
13C nuclear magnetic resonance spectroscopy as well as by elemental analysis. We tested pyridoxal oxime (1) and five prepared oximes in 1
mM concentration as reactivators of human erythrocytes acetylcholinesterase (AChE) inhibited by organophosphorus compounds tabun and paraoxon: 1-phenacyl-3-hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methylpyridinium bromide (2), 1-(4′-chlorophenacyl)-3-hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methylpyridinium bromide (3), 1-(4′-fluorophenacyl)-3-hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methylpyridinium bromide (4), 3-hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methyl-1-(4′-methylphenacyl)pyridinium bromide (5), 3-hydroxy-4-hydroxyiminomethyl-5-hydroxymethyl-2-methyl-1-(4′-methoxyphenacyl)pyridinium bromide (6). However, tested oximes were not efficient in reactivation of either tabun or paraoxon inhibited AChE. The maximum restored enzyme activity in 24
h was below 25%. Therefore, this class of compounds cannot be considered as potential improvement in a search for new and more efficient antidotes against OP poisoning.
Even if organophosphorus (OP) nerve agents were banned entirely, their presence would remain a problem as weapons of terror (like in Syria). Oxime antidotes currently used in medical practice still ...fall short of their therapeutic purpose, as they fail to fully restore the activity of cholinesterases, the main target for OPs. As orphan drugs, these antidotes are tested too seldom for anybody’s benefit. Over the last few decades, search for improved reactivators has reached new levels, but the translation of data obtained in vitro to in vivo application is still a problem that hinders efficient therapy. In this study, we tested the strengths and weaknesses of extrapolating pyridinium oxime antidotes reactivation efficiency from in vitro to in vivo application. Our results show that this extrapolation is possible with well-determined kinetic constants, but that it also largely depends on oxime circulation time and its tissue-specific distribution. This suggests that pharmacokinetic studies should be planned at the early stages of antidote development. Special attention should also be given to improving oxime distribution throughout the organism to overcome this major constraint in improving overall OP therapy.
Iako su organofosforni živčani bojni otrovi potpuno zabranjeni za upotrebu, njihova je prisutnost i dalje velik problem, posebice kao kemijsko oružje u terorističkim napadima (poput nedavnih u Siriji). Oksimi koji se danas koriste kao protuotrovi u tretmanu nemaju dostatno djelovanje na reaktivaciju aktivnosti kolinesteraza, glavnih meta djelovanja organofosfornih spojeva. Valja napomenuti kako se klinička testiranja ovih protuotrova rijetko provode zbog svoje iznimne specifičnosti. Tijekom zadnjih desetljeća učinjen je napredak u istraživanju novih učinkovitijih protuotrova, međutim još je uvijek veliki nedostatak u poboljšavanju terapije translacija in vitro dobivenih rezultata u in vivo primjenu. Ovom studijom ispitali smo mogućnosti ekstrapolacije reaktivacijske učinkovitosti određene za oksimske protuotrove iz in vitro u in vivo sustav. Naši rezultati pokazuju kako je ova translacija moguća uz detaljno određene kinetičke parametre in vitro i uz poznavanje distribucije oksima i vremena cirkulacije u organizmu. Takav rezultat ističe važnost planiranja i farmakokinetičkih istraživanja već u samom početku razvoja protuotrova. Također, poseban naglasak u istraživanju trebalo bi staviti i na poboljšanje tkivo-specifične distribucije oksima u organizmu čime bi se poboljšala cjelokupna terapijska učinkovitost.
Acetilkolinesteraza (AChE; E.C. 3.1.1.7) i butirilkolinesteraza (BChE; E.C. 3.1.1.8) enzimi su koji se zbog svoje uloge u organizmu intenzivno istražuju unutar područja biomedicine i toksikologije. ...Iako strukturno homologni, ovi enzimi razlikuju se prema katalitičkoj aktivnosti, odnosno specifičnosti prema supstratima koje mogu hidrolizirati te selektivnosti za vezanje mnogih liganada. U ovom radu dan je pregled dosadašnjih istraživanja kolinesteraza i njihovih interakcija s ligandima i inhibitorima te su izdvojene aminokiseline aktivnog mjesta koje sudjeluju u tim interakcijama.
Enzymes acetylcholinesterase (AChE; E.C. 3.1.1.7) and butyrylcholinesterase (BChE; E.C. 3.1.1.8) have intensively been investigated in biomedicine and toxicology due to important role in organisms. Even if structurally homologous, they differ in catalytic activity, specificity, for substrates, and selectivity in binding to many ligands. This paper compiles the results of research on cholinesterases and their interactions with ligands and inhibitors, and identifies amino acids of active sites involved in these interactions.
We studied bispyridinium oxime K203 (
)-1-(4-carbamoylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide with tabun-inhibited human acetylcholinesterase (AChE) and ...butyrylcholinesterase (BChE)
, and its antidotal effect on tabun-poisoned mice and rats
. 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
min
. This means that K203 is a very potent reactivator of tabun-inhibited AChE. In addition, K203 reversibly inhibited AChE (
= 0.090 mmol L
) and BChE (
= 0.91 mmol L
), and exhibited its protective effect against phosphorylation of AChE by tabun
, a quarter of the LD
K203 dose insured survival of all mice after the application of as many as 8 LD
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
- studirajući njegove interakcije s ljudskom acetilkolinesterazom (AChE) i butirilkolinesterazom (BChe) inhibiranim tabunom te u uvjetima
- 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
min
što ga svrstava u najučinkovitije reaktivatore AChE inhibirane tabunom. K203 je reverzibilno inhibirao AChE (
i = 0,090 mmol L
) i BChE (
i = 0,91 mmol L
) pokazujući svoja
zaštitna svojstva od inhibicije tabunom. Terapija dozom K203 od 1/4 njegove LD
omogućila je preživljavanje svih miševa nakon otrovanja dozom tabuna od 8,0 LD
. 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.
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