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Archives of Toxicology ISSN 0340-5761 Arch ToxicolDOI 10.1007/s00204-012-0977-1

The summary on non-reactivationcholinergic properties of oxime reactivators:the interaction with muscarinic andnicotinic receptors

O. Soukup, D. Jun, G. Tobin & K. Kuca

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MOLECULAR TOXICOLOGY

The summary on non-reactivation cholinergic properties of oximereactivators: the interaction with muscarinic and nicotinicreceptors

O. Soukup • D. Jun • G. Tobin • K. Kuca

Received: 31 March 2012 / Accepted: 7 November 2012

� Springer-Verlag Berlin Heidelberg 2012

Abstract Organophosphorus inhibitors (OP) of acetyl-

cholinesterase (AChE) represent a group of highly toxic

compounds. The treatment of OP intoxication is, however,

insufficiently ensured. Currently, two main categories of

drugs—anticholinergics and oxime reactivators— are

employed as antidotes. Oximes have been reported to act at

several levels of the cholinergic transmission, and among

the non-reactivation effects, the interaction with choliner-

gic receptors stands out. This review addresses issues

correlated with non-reactivating effects of oxime reacti-

vators with a special focus on the muscarinic and nicotinic

receptors, but involvement of other cholinergic structures

such as AChE and choline uptake carriers are discussed

too. It can be concluded that the oxime reactivators show a

variation in their antagonistic effect on the muscarinic and

nicotinic receptors, which is likely to be of significance in

the treatment of OP poisoning. In vitro data reported oxi-

mes to exert higher efficacy on the muscarinic M2 subtype

than on the AChE. However, this effect seemed to be

subtype specific since the antagonistic M3 effect was

lower. Also, and importantly, the antimuscarinic effect was

larger than that on nicotinic receptors. Even though atro-

pine showed a much higher muscarinic antagonism, it is

supposed that non-reactivation properties of oxime reacti-

vators play a significant role in the treatment of OP

poisoning.

Keywords Oxime reactivators � Organophosphate �Non-reactivation �Muscarinic receptor � Nicotinic receptor �Anticholinergics

Introduction

Organophosphorus inhibitors (organophosphates, OP) of

acetylcholinesterase (AChE) are widely used as pesticides,

and its potential use as chemical weapons is a matter of

high concern. These compounds bind to the AChE at the

serine hydroxyl group at its active site, and the binding

inhibits the enzyme’s physiological role in the organism—

to cleave the neuromediator acetylcholine (ACh) when

occurring in the synaptic clefts. Consequently, ACh accu-

mulates in the cholinergic synaptic junctions and the poi-

soning manifests as a cholinergic syndrome. In cases of

insufficient treatment, death is caused by paralysis of the

respiratory muscles and of the respiratory center (Marrs

1993; Bajgar 2004).

The treatment of the OP poisoning is ensured by two

main categories of drugs. (1) Anticholinergics, for exam-

ple, atropine, are able to antagonize the effects of excessive

ACh by a blockade of mAChRs. Other drugs, for example,

benactyzine or scopolamine might be used due to their

better penetration to CNS. (2) Compounds belonging to the

other group, reactivators of the AChE—oximes—are able

O. Soukup � D. Jun � K. Kuca (&)

Biomedical Research Center, University Hospital of Hradec

Kralove, Sokolska 581, 50005 Hradec Kralove, Czech Republic

e-mail: kucakam@pmfhk.cz

O. Soukup

Department of Public Health, University of Defence, Trebesska

1575, 50001 Hradec Kralove, Czech Republic

D. Jun � K. Kuca

Faculty of Military Health Sciences, Center of Advanced

Studies, University of Defence, Trebesska 1575, 50001 Hradec

Kralove, Czech Republic

G. Tobin

Department of Pharmacology, Sahlgrenska Academy, University

of Goteborg, Medicinaregatan 13, 405 30 Goteborg, Sweden

123

Arch Toxicol

DOI 10.1007/s00204-012-0977-1

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to restore the physiological function of inhibited AChE. In

addition to them, diazepam or other benzodiazepines can

be used as anticonvulsants. Anticholinergics and reactiva-

tors are usually co-administered because of their syner-

gistic effect (Kassa 2002; Bajgar 2004). Today, HI-6 and

obidoxime are the most commonly used oxime reactivators

in the treatment of OP poisoning; however, the reactivators

differ in their efficacy against individual nerve agents and

no universal antidote has been developed yet. Moreover,

the problem of phenomenon named ‘‘aging’’ (a time-

dependent loss of AChE ability to be reactivated) is an

important issue, since the therapeutic intervention must be

applied within a limited period time. Owing to this fact,

new, versatile AChE reactivators are thus required, or

alternative treatment approaches have to be developed.

Such an alternative approach presumes other mecha-

nisms of action of oximes, which are not related to the

reactivation. Oximes have been reported to act at several

levels of the cholinergic transmission, including the syn-

thesis, release, inactivation and reuptake of the transmitter,

but the interaction with muscarinic (mAChRs) and nico-

tinic (nAChRs) receptors seems to be the most plausible

alternative of mechanism of action (Vanhelden et al. 1996;

Soukup et al. 2010b).

This review deals mainly with the interaction of oxime

reactivators with muscarinic and nicotinic receptors, but

some other cholinergic properties were considered as well.

Particular focus is addressed to differences in efficacy,

anticholinergic potency of individual reactivators and the

correlation between in vitro and in vivo effects. To start

with, a review of literature concerning these effects of

interest is listed below.

Pharmacological effect of oximes

The major mechanism of action of the antidotes is the

reactivation of inhibited AChE. However, other mecha-

nisms are probably involved. The data derived from tabun

or soman lethally intoxicated primates suggests that, due to

a protocol ensuring low or no reactivation, other mecha-

nisms, which lead to survival via the recovery of neuronal

transmission, must be involved (Hamilton and Lundy 1989;

Vanhelden et al. 1992). Similar life-saving results were

obtained in a rat model, where rapidly ‘‘aging’’ crotylsarin

was used, and administration of HI-6 caused the recovery

of neuronal transmission (Busker et al. 1991).

The exact pharmacological effects of oximes are still an

open question. Oximes probably play a role at various

levels of the cholinergic transmission. Thus, the oximes

may exert effects by interactions with pre and postsynaptic

receptors or with the reuptake of transmitters, in addition to

possible effects by affecting the synthesis and release of

ACh. Clement reported that oxime HS-6 inhibits the syn-

thesis of ACh, its release and the reuptake of choline in

chicken (Clement 1979). The release of ACh from pre-

synaptic fibers can be modulated by oximes, suggesting an

interaction with presynaptic muscarinic (Kloog and Soko-

lovsky 1985) and/or nAChRs (Fossier et al. 1990) or via

the interaction with the high-affinity choline uptake carrier

(HACU), which is a key regulatory system in the synthesis

of ACh (Kristofikova et al. 2003). In the latter case, the

interaction with presynaptic mAChRs was also suggested

(Zahniser and Doolen 2001). Controversially, while a

decrease in the release have been reported in the rat brain

(Kloog et al. 1986), an increase has been reported in the rat

striated muscle (Melchers and Vanderlaaken 1991). Similar

contradictive data are reported by several authors, which

seem to depend on different experimental conditions

(Oydvin et al. 2005). Notably, some compounds are

effective against OP poisoning even when the compound

lacks the oxime moiety (Inns and Leadbeater 1983; Loke

et al. 2002; Seeger et al. 2012).

Antinicotinic action

Bis-pyridinium compounds in general were reported to

cause a recovery of neuromuscular transmission mediated

by nicotinic receptors in OP poisoning (Wolthuis et al.

1981). The effect of oximes at the neuromuscular junctions

on nAChRs has been examined in a phrenic nerve-dia-

phragm model (Busker et al. 1991). They observed that

HI-6 was able to counteract the failure of neuromuscular

transmission caused by crotylsarin. Since HI-6 had a sim-

ilar effect as pure cholinergic receptor antagonists (galla-

mine, mecamylamine), which are not involved in AChE

reactivation, a nicotinic antagonism of this compound was

proposed. Furthermore, HI-6 has been shown to be able to

counteract crotylsarin-induced excessive release of 3H-

ACh from an isolated endplate (Vanhelden et al. 1996). On

the contrary, under different experimental conditions,

enhancement of the release was observed as well (Aas

1996; Oydvin et al. 2005). Furthermore, the pharmaco-

logical action against soman poisoning was also due to the

channel blocking activity of HI-6, obidoxime and similar

compounds (Tattersall 1993). However, contrasting to

these studies, single channel studies have also revealed that

HI-6 and pralidoxime increases the opening probability of

nAChRs that are activated by ACh (Alkondon et al. 1988).

nAChRs (and muscarinic as well) in the rat brain were

blocked by some bis-pyridinium compounds as judged by

displacement examinations with tritiated QNB and a-bun-

garotoxin (Broomfield et al. 1987). Ganglion-blocking

properties have been reported by Lundy et al. (Lundy and

Tremblay 1979). Recently, interaction of bys-pyridinium

compounds derived from SAD-128 with orthosteric

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binding side of human a7 nAChR has been showed Nies-

sen et al. (2011).

Antimuscarinic action

Concerning the antimuscarinic effects, binding studies of

some H-oximes have been performed on mice smooth

muscle tissue in vitro (Kuhnen-Clausen et al. 1983). The

inhibitory constants were determined to occur at the

micromolar range. However, observed nicotinic inhibitory

potency did not correlate with the efficacy against soman

in vivo (Kuhnen-Clausen et al. 1983). Functional musca-

rinic antagonism of some reactivators has been observed at

guinea pig (Kuhnen-Clausen 1970; Fusek and Patocka

1976; Kuhnen-Clausen et al. 1983). The mechanism of

binding to the mAChRs is still not fully understood, but an

allosteric manner has been suggested (Lee and Elfakahany

1991; Ellis and Seidenberg 1992; Tucek and Proska 1995;

Kostenis et al. 1996; Christopoulos et al. 1998). Moreover,

in vitro functional examination showed the oximes HGG-

12 and HGG-42 to be allosteric inhibitors (Kloog and

Sokolovsky 1985).

AntiAChE action

The stimulatory effect on cholinergic system by oximes,

which has been described by several authors, should be

more likely ascribed to a partial inhibition of AChE by the

oxime rather than to a direct agonism at muscarinic ACh

receptors (Reithmann et al. 1991). The antiAChE activity

of bis-pyridinium compounds has also been observed when

measured physiologically in the guinea pig ileum (Amitai

et al. 1980) or spectrophotometrically in vitro (Calic et al.

2006). Thus, it can be concluded that pharmacological

effects of oximes are complex and not yet well defined.

There is a hypothesis that oxime reactivators used in the

treatment of OP poisoning exert both antimuscarinic and

antinicotinic properties. However, many questions still

need to be answered. Are the non-reactivation properties of

oximes significant for the treatment of OP poisoning? Do

differences among reactivators in anticholinergic efficacy

exist? Why is there a difference between in vitro and

in vivo effects of the oximes? This review tries to sum-

maries findings that may put some light on these questions.

Our observations

Antimuscarinic and antiAChE findings

According to the literature and according to our observa-

tions, oxime reactivators have very complex mechanisms

of action and influence the cholinergic nervous systems at

various levels. An antagonistic effect on both muscarinic

and nicotinic receptors has been observed. However, cho-

linomimetic action of these drugs cannot be definitely

excluded. As mentioned above, an inhibitory effect on

AChE results in this type of action (Busker et al. 1991).

Ellman’s method, characterizing the potency of oxime re-

activators to inhibit AChE (Table 1) revealed donepezil (a

classical AChE inhibitor used in the treatment of Alzhei-

mer’s disease) to possess approximately 10,000 times

higher inhibition potency than HLo-7 and HI-6—the most

potent classical reactivators. However, in case of OP poi-

soning, the (repeated) dosage of HI-6 may result in

approximately 10 lM concentrations in the human body

(Kassa 2002), so the IC50 of 70 lM describing the potency

of HI-6 to inhibit AChE may cause a significant cholino-

mimetic effect at this compound.

However, cholinolytic effects are the most frequently

reported (sometimes both cholinolytic and cholinomimetic

effects were observed). Displacing experiments using (3H)-

QNB revealed an affinity of oximes to the mAChRs. The

affinity increases in following order HI-6 \ K203 &K027 \ Obidoxime (Table 2). However, the correlation

with a muscarinic standard antagonist—atropine—showed

an approximately threefold lower affinity of obidoxime, the

most potent compound tested (Soukup et al. 2011b). This

finding is in agreement with most other similar studies

(Kuhnen-Clausen et al. 1983). Binding to the mAChRs is a

prerequisite for the transduction of the signal through the

receptor, which, in the case of G protein coupled receptors

(GPCR), leads to the activation of the G protein and the

subsequent intracellular cascade. In the GPCR activation–

based experiment, the oximes antagonize the activation

induced by oxotremorine (muscarinic agonist; control). No

intrinsic activity of the oximes was observed, so the direct

cholinomimetic type of action was excluded (Soukup et al.

2010c). The inhibitory properties of the signal transduc-

tion increased in the following order: HI-6 \ K027

Table 1 Inhibitory potency of standard and newly in house-synthesized oxime reactivators

Compound Hlo-7 HI-6 Pralidoxime Trimedoxime Obidoxime Methoxime K113 K114 K112 K203 K027 Done pezil

IC50 (lM) 67 70 198 287 434 538 10 27 44 311 391 0.007

Error (± lM) 13 6 43 10 71 89 5 12 5 16 23 N/A

Values are expressed as IC50 ± error. IC50 for donepezil, classical AChE inhibitor was obtained from a literature (Ogura et al. 2000)

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\ K203 & Obidoxime. This roughly corresponds to the

binding affinities at least in that HI-6 has the smallest and

obidoxime has the largest pronounced effect. Similarly to

AChE inhibition, IC50 values in units or tens of micro-

moles, with regard to achievable concentrations in a human

body, may testify of an important antimuscarinic compo-

nent in the reactivator’s complex mechanism of action. The

correlation of anticholinesterase activity with the affinity to

the mAChRs was shown to be of inversely related (studied

for 4 reactivators (Soukup et al. 2010c). However, in

another report 7 of 8 OP inhibitors showed a direct cor-

relation between affinity to AChE and to the muscarinic

receptors (Ward et al. 1993). Therefore, it is hard to con-

clude whether a general relationship between AChE

inhibitors and their effect on mAChRs exists.

Organ bath and in vivo experiments

In in vitro contraction studies on the isolated rat urinary

bladder strips, HI-6 and obidoxime showed an antagonistic

effect on the M3 mAChRs (Soukup et al. 2008, 2010c).

However, this effect was only present at the higher con-

centrations of the oximes (from 10 to 100 lM and higher).

At lower concentrations, an opposite effect was observed

(increase in contraction) (Fig. 1). This is probably due to

an inhibition of AChE caused by the reactivators (Reith-

mann et al. 1991). In the atrial preparations, an antagonistic

effect on M2 mAChRs, revealed as increase in the heart

frequency, was observed for three different reactivators

(Soukup et al. 2010b). An explanation to this fact seems

not far to seek. For stimulation of M2 mAChRs metha-

choline (agonist), whose effect resulted in a drop of heart

frequency, was used. However, methacholine is not broken

down by AChE, so the potential inhibition of AChE did not

resulted in the accumulation of agonist in the synapse.

Notably, methacholine was used to stimulate the bladders

as well, and here, the effect on AChE was observed. This

could be due to the fact that in the bladder a pronounced

non-neuronal production and a release of endogenous ACh

from the urothelium occur. The mechanisms that induce

the release are largely unknown, but are most likely to be

induced by shear stress, that is, stretch of the urothelium

may be caused by the methacholine-evoked contraction

(Andersson and Hedlund 2002). Also, the activation of

urothelial mAChRs may contribute to the release. It means

that the observed effect is due to the AChE inhibition and

its influence on ACh’s break down, not that one of meth-

acholine. Interestingly, the efficacy comparison of indi-

vidual agents showed a high potency of HI-6, which does

not correlate with the affinity studies, where HI-6 showed

the lowest affinity to mAChRs. An explanation could be

found in the interaction with presynaptic receptors, the

reuptake system or in another mechanism that influence the

release of endogenous ACh. The same type of experiments

using atropine instead of a reactivator show, as could be

expected, only the antimuscarinic type of action. A com-

parison of cholinergic effects of atropine and obidoxime on

bladder strips can be seen in Fig. 1. Furthermore, a notably

higher potency of atropine compared to that of reactivators

has been reported. Comparing the in vitro antimuscarinic

efficacy of the reactivators and atropine, atropine showed

approximately 7-order and 2-order higher potency for

atropine in the bladder and heart atria, respectively (So-

ukup et al. 2010c).

Obidoxime was investigated in the in vivo muscarinic

experiment where the heart frequency in anesthetized rat

was examined (Soukup et al. 2010c). Once again, effects

on both AChE and M2 mAChRs were observed. Although

only obidoxime was examined, qualitatively similar effects

can be expected from other reactivators too. Interestingly,

it occured the other way around than in the bladder, that is,

at lower doses the effect on M2 mAChRs could be seen and

at higher doses AChE is inhibited. It tentatively indicates

the following in vivo selectivity of the obidoxime in rat:

M2 [ AChE [ M3. The relative M2 selectivity of allo-

steric modulators (including obidoxime) has been observed

too (Ellis and Seidenberg 2000). Recently, the interaction

of bys-piridinium compounds with the human mAChR

subtype 5 has been reported. The effect on AChE can be

observed due to the fact that the stimulation of the vagal

nerve leads to the release of endogenous ACh, which is not

broken down by reactivator-inhibited AChE.

Table 2 Inhibitory potency in the binding and functional GPCR activation–based experiments of selected oxime reactivators on the mAChRs

(Soukup, Kumar et al. 2011b)

Reactivator IC50 (lM) 3H-QNB

Replacement

95 % Confidence intervals

(lM)

IC50 (lM) Inhibition of

activation

95 % confidence intervals

(lM)

HI-6 656 156–2760 167 45–290

K203 131 65–262 4.7 1.4–15

K027 117 49–275 18 11–28

Obidoxime 31 16–63 3.5 2–6.3

Values are expressed as IC50 (lM) with respective 95 % confidence intervals. Inhibition of activation refer to an effect caused by the agonist

(Oxotremorine-M; 15 lM)

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Antinicotinic findings: patch clamp and in vivo

The interaction of oximes with nAChRs has been investi-

gated too (Soukup et al. 2011a). In vivo observations

showed a clear-cut dose-dependent inhibition on nerve-

evoked twitches, which indicate a blockade of nicotinic

receptors at the neuromuscular endplate (Fig. 2). At 5 Hz

stimulation of the nerve, smaller inhibitory effects of oxi-

mes appeared, probably due to a larger availability of ACh

at this frequency. Even though no statistical significance

appeared at 5 Hz, still clear dose-dependent effects

occurred. Concerning the individual compounds, we

observed only small differences in potency. If any differ-

ence in potency occurred between the various oximes, it

was somewhat less for K027. It should be stressed that

spontaneous fading of twitches may occur during a repet-

itive stimulation, probably due to a changed sensitivity of

the nicotinic receptor on the motor end-plate (Vanderkloot

et al. 1994). However, a decrease by 23 % was reported

after delivery of 3,000 stimuli. In our study, only about

1,000 stimuli were delivered and the reduction observed

was up to 50 %. Nevertheless, the current results indicate a

direct antagonistic effect of oxime reactivators on the

nAChRs similarly to what has been reported earlier (Tat-

tersall 1993). To the best of our knowledge, this is the first

in vivo report showing that reactivators inhibit the muscle

type of nAChR. The current antinicotinic therapy in the OP

treatment is problematic, and antinicotinic properties of

oximes, especially that of non-competitive and open-

channel blocking origin, are welcome (Sheridan et al.

2005; Turner et al. 2011). However, we did not deal with

the binding mechanism in our study and further investi-

gations are needed in this area.

Interestingly, in the case of neuromuscular transmission,

no effect on AChE has been noticed, as in the case of the

in vivo muscarinic examination (Soukup et al. 2010c). It

must be considered that the physiological processes at

central and neuromuscular synapse differ. The AChE

occurs in different molecular forms due to a posttransla-

tional process, which has the same activity, but differ in the

affinity to AChE inhibitors (Massoulie 2000). Moreover,

the approximate maximum response to autonomic nerve

stimulation occurs in the range 15–40 Hz. The corre-

sponding interval to maximum responses to motoric stim-

ulation occurs above 40 Hz (40–70 Hz or even higher).

The quantal theory of ACh release approximates the

release of 1,000 molecules of ACh per quantum in the CNS

and 6,000–10,000 per quantum in the neuromuscular

junction (Karczmar 2007). In the current experiments,

10 Hz was applied for the vagal stimulation and only 1 and

5 Hz for neuromuscular stimulation. One explanation

could be that oximes have a more pronounced antagonistic

effect on the nAChRs than on the mAChRs in relation to

the AChEi effect. However, it has been shown that

H-oxime has lower affinity to the nAChRs than to the

mAChRs (Kuhnen-Clausen et al. 1983). More likely, these

two situations are rather incomparable and various theories

are at hand concerning the effect on AChE. Nevertheless,

the antagonistic effect on nAChRs and on the mAChRs is

doubtless.

The antagonistic effect on the nAChRs has also been

confirmed by using in vitro patch clamp technique (Soukup

et al. 2011b. The results obtained on the TE671 cell line

expressing the human embryonic muscle-like acetylcholine

receptor (Schoepfer et al. 1988) showed antagonistic

activity of all tested compounds on the muscle type of

nAChRs (Table 3). Once again, an apparent lower potency

Fig. 1 Effect of increasing concentrations of obidoxime and atropine

on the methacholine-evoked contractions at the isolated rat bladder

strip: Effect of increasing concentrations of obidoxime. Values are

presented as a mean ± SEM (n = 6). Figure adapted from data

presented in Soukup et al. (2010c)

Fig. 2 The effect of different reactivators (at the dose 10 mg/kg,

n = 5) on the amplitude of the muscular twitches produced by

electrical stimulation (1 and 5 Hz). Data are presented as a

mean ± SEM. Figure adapted from data presented in Soukup et al.

(2011b)

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of K027 was observed. Comparing this efficacy with

in vitro muscarinic examinations, the potency to affect the

mAChRs is approximately one order higher. This agrees

well with previous observations (Kuhnen-Clausen et al.

1983). In these experiments, antagonism on both the

embryonic and the adult form of the nAChRs was shown.

Interaction with HACU

As has been mentioned above, reactivators have a complex

mechanism of action. The presynaptic activity differs among

individual reactivators, and this effect influences the release

of ACh (Aas 1996; Oydvin et al. 2005). In another experi-

ment, the interaction of oximes with the HACU transport

system and their effect on membrane fluidity were examined

(Kristofikova et al. 2003; Soukup et al. 2010a, 2012). This

system is a key regulatory step (rate limiting) in the syn-

thesis of new ACh. We investigated standard reactivators

(obidoxime, HI-6, trimedoxime, methoxime and pralidox-

ime) and a new oxime—K112. In general, the results

showed that oxime reactivators are able to interact with this

carrier at low (10 lM; K112 and trimedoxime) or at high

concentrations (50 lM; HI-6, obidoxime and pralidoxime).

While methoxime did not significantly affect the carrier, an

influence on the fluidity of membrane was observed

(Table 4). It should be noticed that even though we observed

significant effects at micromolar concentrations, much lower

concentrations (picomolar or nanomolar) estimated in the

experiments in vitro will be relevant for the in vivo use

(Kristofikova et al. 1992, 2001; Kristofikova and Klaschka

1997). Due to this fact, prolonged administration would

need to be considered. However, a correlation between

HC-3 sensitive carriers and an impairment of cholinergic

neurotransmitter system was observed in various diseases

(e.g., Alzheimer’s), where the prolonged HACU inhibition is

connected with toxic detrimental effects. Therefore, a sig-

nificant inhibition of the carriers mediated by trimedoxime

and K112 could be a risk factor considering the prolonged

administration. On contrary, methoxime did not influence

the carriers, and its easy penetration into the membrane

could be an advantage since positively charged reactivators

hardly penetrate across the blood–brain barrier (Sakurada

et al. 2003). These results also show that reactivators are

able to modulate the release of ACh by the presynaptic

activity. Whether it is a result of direct interaction with

HACU, presynaptic cholinergic receptors or membrane

fluidity impairment is a matter of discussion; however, a

cooperation of M2 muscarinic autoreceptors and presynap-

tically localized HC-3 sensitive carriers through G proteins

has also been suggested (Zahniser and Doolen 2001).

Concerning the antidotal efficacy of individual reactiva-

tors, it is well known that results from in vitro examinations

do not always correlate with in vivo findings (Kuhnen-

Clausen et al. 1983; Tattersall 1993).For example, HI-6 can

handle in vivo tabun poisoning (Hamilton and Lundy 2007),

whereas in vitro it exerts only a minute reactivation potency

(Kuca et al. 2007). In our study, we observed a similar

situation. We performed experiments employing commonly

used reactivators and some of our new, promising com-

pounds, and the efficacy differed for individual compounds

when various methods are applied. Trimedoxime and oxime

K112 exerted high potency to inhibit choline reuptake,

Table 3 The effect of selected reactivators on the nicotinic receptors

expressed by the TE671 cell line in the whole-cell mode patch clamp

(Soukup et al. 2011b)

Reactivator Inhibition (200 lM) (%) S.E.M (%)

Obidoxime 51 2

HI-6 45 1

K027 31 1

K203 50 1

Inhibition reflects the drop (in %) in a response caused by reactivators

at the concentration of 200 lM on the ACh 10-lM-evoked response

(0 % = no effect, 100 % = complete inhibition). n = 4 for obidox-

ime and HI-6, n = 3 for K027 and K203

Table 4 Effect of reactivators on the high-affinity choline uptake (HACU) system and membrane anisotropy

Reactivator (3H)-choline (% of control) 10 lM (3H)-HC-3 (% of control) 50 lM Scatchard analysis TMA-DPH (%) DPH (%)

HI-6 91.0 ± 28.1 71.4 ± 12.9* : Kd 101.4 ± 2.6 97.1 ± 2.4

Trimedoxime 26.7 ± 6.2*** 48.4 ± 13.8*** : Kd 102.8 ± 2.6* 99.5 ± 1.7

Methoxime 85.9 ± 46.7 97.3 ± 27.2 – 103.1 ± 2.7* 94.4 ± 2.1**

Obidoxime 69.4 ± 26.1 40.2 ± 23.3*** ; Bmax 102.5 ± 2.0* 97.1 ± 2.4

Pralidoxime 71.8 ± 11.7 49.5 ± 25.7*** ; Bmax 106.1 ± 0.3*** 103.2 ± 1.1

K112 68.4 ± 9.9* 64.7 ± 4.7** : Kd 96.5 ± 1.0** 97.5 ± 1.1

All values with asterisk(s) represent a statistical difference from the control. Scatchard analysis refers to (3H)-hemicholinium-3 experiments.

Anisotropy was estimated in the region of the hydrophilic heads of phospholipid bilayer (TMA-DPH) or in the hydrocarbon core (DPH). Kd

characterizes the affinity between the carrier and its competitive inhibitor, and Bmax represents maximal number of binding sites. Table adapted

from Soukup et al. (2012) and extended

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obidoxime showed high affinity to muscarinic receptors, all

tested oximes exerted similar antinicotinic effect, whereas

oxime K027 showed considerably lower potency etc.

Therefore, it is very hard to choose the best reactivator

according to our examinations, but this has not been the aim

for our work at the present stage of understanding. However,

the generally accepted broad spectrum usage of HI-6 should

be pointed out (Kassa 2002), despite of the fact that this

versatility is not fully understood. Similarly, we observed

that even though HI-6 was less effective in some in vitro

examinations, it showed considerably higher potency when

examined in the whole (isolated) organ or in vivo. Unfor-

tunately, we have no exact explanation of this phenomenon,

but tentatively, the interaction with presynaptic receptors

(Vanhelden et al. 1996), or with other, so far, unrevealed

cholinergic mechanisms, may be the cause.

Concluding comments

In conclusion, one part of the mechanism of action of the

oxime reactivators that shows a pronounced variation is

their antagonistic effect on muscarinic and nicotinic

receptors. This effect is likely to be of significance in the

treatment of OP poisoning, where the relative ACh excess

can be counteracted by the blocking properties of oximes

on cholinergic receptors. The comparison of the in vitro

data reported oximes to exert higher efficacy on the mus-

carinic M2 subtype than on the AChE. However, this effect

seemed to be subtype specific since the antagonistic M3

effect was lower (i.e., M2 [ AChE [ M3). Also, and

importantly, the antimuscarinic effect was larger than that

on nicotinic receptors. Albeit oxime reactivators show

much lower muscarinic antagonism than another drug used

in the treatment, the classical antidote atropine, it is sup-

posed that non-reactivation properties of oxime reactiva-

tors play a significant role in the treatment of OP

poisoning. Another important phenomenon may occur in

the treatment with reactivators—a dual effect on cholin-

ergic receptors and on AChE simultaneously. This property

is of course not in favor in the antidotal treatment, and it

should be stressed that this effect on AChE has not been

observed at the nicotinic transmission. Since overstimula-

tion of nicotinic receptors is considered to be the cause of

death in the OP poisoning, clear antinicotinic action is of

advantage in this matter.

Acknowledgments This work was supported by the Grant No.

NT12062 from the Ministry of Health, Czech Republic and the

Postdoctoral project No. CZ.1.07/2.3.00/30.0012.

Conflict of interest The authors declare that they have no conflict

of interest.

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