Journal of APPLIED BIOMEDICINE
ISSN 1214-0287 (on-line)
ISSN 1214-021X (printed)
Volume 9 (2011), No 4, p 225-230
DOI 10.2478/v10136-011-0011-6
A comparison of the reactivating and therapeutic efficacy of the newly developed bispyridinium oxime K203 with currently available oximes, in sarin
poisoned rats and mice
Jiri Kassa, Jana Zdarova Karasova, Vendula Sepsova, Jiri Bajgar
Address: Jiri Kassa, Faculty of Military Health Sciences, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic
kassa@pmfhk.cz
Received 5th May 2011.
Revised 9th June 2011.
Published online 8th July 2011.
Full text article (pdf)
Summary
Key words
Introduction
Material and methods
Results
Discussion
Declaration of interest
Acknowledgements
References
SUMMARY
This study compares the abilities of the newly developed bispyridinium oxime K203 with currently available oximes (HI-6, obidoxime, and trimedoxime)
in the reactivation of sarin-inhibited acetylcholinesterase and the reduction of the acute toxicity of sarin. The percentage of reactivation of
sarin-inhibited rat blood and tissue acetylcholinesterase was determined in vivo and it was shown that the potency of bispyridinium oxime
K203 to reactivate sarin-inhibited acetylcholinesterase roughly corresponds to the relatively low reactivating efficacy of obidoxime and trimedoxime
except in the diaphragm where K203 was not effective. On the other hand, the oxime HI-6 was found to be a very efficient reactivator of
sarin-inhibited acetylcholinesterase in the peripheral as well as central compartment. The oxime HI-6 was able to reduce the acute toxicity of sarin
by more than four times, but the other oximes studied, including K203, decreased the acute toxicity of sarin by less than three times. Based on
these results, we can conclude that the reactivating and therapeutic efficacy of the oxime K203 is significantly lower compared to the oxime HI-6
and, therefore, it is not a suitable replacement for the oxime HI-6 in the antidotal treatment of acute sarin poisoning.
KEY WORDS
sarin; acetylcholinesterase; K203; HI-6; obidoxime; trimedoxime; rats; mice
INTRODUCTION
Highly toxic organophosphorus compounds, called
nerve agents, present a serious threat to both military
and civilian populations. They irreversibly inhibit the
enzyme acetylcholinesterase (AChE, EC 3.1.1.7)
leading to acetylcholine accumulation at synaptic
clefts and overstimulation of the muscarinic and
nicotinic cholinergic receptors located in the central
and peripheral cholinergic nervous system. Severe
poisoning can lead to death due to respiratory failure
(Marrs 1993, Lotti 2000).
The current antidotal treatment of nerve agent
poisoning usually consists of the combined
administration of an anticholinergic drug (preferably
atropine) and an oxime (preferably pralidoxime or
obidoxime). The anticholinergic drug blocks the
overstimulation of muscarinic receptor sites, while the
oxime repairs biochemical lesions by dephos-phonylating nerve agent-inhibited AChE and
restoring its activity (Taylor 2001, Kassa 2002).
Unfortunately, the oximes are not satisfactorily
effective against all nerve agents; in particular, the
antidotal treatment of acute poisonings with tabun,
cyclosarin and soman is very difficult (Dawson 1994,
Kassa 2002, Bajgar 2004) and therefore, the antidotal
treatment of nerve agent acute poisonings still
remains a serious problem. The development of a new
and more effective AChE reactivator is still a very
important issue.
The new bispyridinium asymmetric oxime K203
[1-(4-carbamoylpyridinium)-4-(4-hydroxyiminomethylpyridinium)-but-2-ene dibromide] (Fig. 1) has
been synthesized in our department (Musilek et al.
2008). The purpose was to increase the efficacy of
antidotal treatment of tabun poisoning which had
been shown to be resistant to conventional oxime
therapy due to the conformational changes in the
AChE-tabun complex prior ageing process at the
AChE active site (Ekstrom et al. 2006). As the oxime
K203 has been found to be a promising reactivator of
tabun-inhibited AChE (Kassa et al. 2008), we decided
to evaluate the reactivating and therapeutic efficacy
of K203 against other nerve agents including sarin
(isopropyl methylfluorophosphonate), because we are
still searching for a broad-spectrum oxime able to
sufficiently counteract the acute toxicity of all nerve
agents regardless of their chemical structure.
The aim of this study was therefore to determine
the reactivating and therapeutic efficacy of the newly
developed bispyridinium oxime K203 compared to
the currently available oximes (HI-6, obidoxime,
trimedoxime) against sarin in rats and mice.
MATERIAL AND METHODS
Animals
Male albino Wistar rats weighing 230-260 g (6 weeks
old) and NMRI male mice weighing between 20 and
24 g (4 weeks old) were purchased from Velaz
(Prague, Czech Republic). They were housed in
propylene cages (56 × 36 × 19 cm3, six rats or ten
mice per cage) in an air-conditioned room (22±2 °C
and 50±10% relative humidity, with lights from
7.00 a.m. to 7.00 p.m.) and were allowed access to
standard food and tap water ad libitum. The
laboratory animals were acclimatized in the
laboratory vivarium for 14 days before starting the
experiments and they were divided into groups of
8 animals. Handling of the experimental animals was
performed in compliance with relevant laws and
institutional guidelines and with the approval of the
Ethics Committee of the Faculty of Military Health
Sciences in Hradec Kralove (Czech Republic).
Chemicals
Sarin was obtained from the Military Technical
Institute in Brno (Czech Republic) and was 98% pure,
as assayed by an acidimetric titration. All oximes
(K203, HI-6, trimedoxime, obidoxime) were
synthesized at our Department of Toxicology of the
Faculty of Military Health Sciences (Czech Republic)
and they were more than 98% pure (Musilek et al.
2006, 2008). Their purity was analysed using the
HPLC technique (Jun et al. 2007). All other drugs and
chemicals of analytical grade were obtained
commercially and used without further purification.
All substances were administered intramuscularly
(i.m.) at a volume of 1 ml/kg body weight (b.w.) to
rats and 10 ml/kg b.w. to mice.
In vivo experiments
To investigate the reactivating efficacy of the oximes,
the rats were administered i.m. with either atropine
(21 mg/kg) alone or atropine (21 mg/kg) in
combination with one of the studied oximes at 5 min
before receiving sarin i.m. at a dose of 90 g/kg
(LD50). The oxime K203 (16.3 mg/kg), the oxime
HI-6 (39.3 mg/kg), obidoxime (10.5 mg/kg) and
trimedome (7.5 mg/kg) were administered in
equitoxic doses corresponding to 5% of their
previously published LD50 values (Kassa et al. 2008).
The rats were decapitated and exsanguinated to obtain
the blood 30 min after sarin poisoning. The blood was
haemolysed in Tris-HCl buffer (0.02 N; pH 7.6;
1:20). The tissues, diaphragm and brain were
removed and homogenized in Tris-HCl buffer
(0.02 N; pH 7.6; 1:10) to determine AChE activity by
the standard spectrophotometric method (Ellman et al.
1961). Acetylthiocholine was used as a substrate
(0.1 N Tris-HCl buffer; pH 7.6). A Helios Alpha
spectrophotometer (Thermo Electron Corporation,
UK), was used for the determination of absorbancy at
436 nm. The AChE activity was derived from
absorbance values with the help of the calibration
curve with cystein and expressed as kat/kg or l
(mol substrate hydrolyzed/kg wet tissue or l blood
within 1 second). The percentage of reactivation was
calculated using the AChE activity values: {1-
[(saline control) - (oxime + atropine))/((saline
control) - (atropine control))]} × 100 (equation
modified from Clement et al. 1992).
The ability of oximes in combination with
atropine to decrease the acute toxicity of sarin in mice
was determined as follows. The LD50 value of sarin
and its 95% confidence limit in mice were assessed using probit-logarithmical analysis of death occurring
within 24 h after i.m. administration of sarin at five
different doses with eight mice per dose (Tallarida
and Murray 1987). Sarin-poisoned mice were then
treated i.m. with one of the tested oximes in
combination with atropine (21 mg/kg) 1 min after i.
m. challenge of sarin. The oxime K203 (4.75 mg/kg),
the oxime HI-6 (33.6 mg/kg), obidoxime (9.4 mg/kg)
and trimedome (7.4 mg/kg) were administered in
equitoxic doses corresponding to 5% of their
previously published LD50 values (Kassa et al. 2008).
The efficacy of the tested antidotal treatment was
expressed as a protective ratio (LD50 value of sarin in
protected mice/ LD50 value of sarin in unprotected
mice).
Fig. 1. Chemical structure of oximes.
Statistical evaluation
The differences between groups were calculated using
means ± SD and differences were tested by one-way
ANOVA test with Scheffe's post hoc test at the
significance level 2alpha=0.05.
RESULTS
The potency of oximes to reactivate sarin-inhibited
AChE in rat blood, diaphragm and brain in vivo is
shown in Table 1. The oxime K203 is a relatively
weak reactivator of sarin-inhibited AChE. It was able
to increase the activity of sarin-inhibited AChE in
blood by 6.3% and in brain by 3.1% while its ability
to reactivate sarin-inhibited AChE in the diaphragm
was negligible, Its potency to reactivate
sarin-inhibited AChE was comparable to the
reactivating efficacy of obidoxime and trimedoxime
with the exception of the diaphragm where the
potency of obidoxime and trimedoxime to reactivate
sarin-inhibited AChE was significantly higher. All the
above mentioned oximes are markedly weaker
reactivators of sarin-inhibited AChE than the oxime
HI-6 which seems to be the most effective reactivator
of sarin-inhibited AChE in the peripheral as well as
central compartment; it was able to increase the
activity of sarin-inhibited AChE by more than 70% in
blood, by almost 50% in the diaphragm and by 19%
in the brain. The differences between HI-6 and the
other oximes studied (K203, obidoxime, and
trimedoxime) in the blood, the diaphragm as well as
the brain were significant (Table 1).
The potency of oximes studied in reducing the
acute toxicity of sarin is shown in Table 2. The oxime
K203 was able to decrease the acute toxicity of sarin
by almost 2.5 times. Its therapeutic efficacy roughly
corresponds to the potency of obidoxime and
trimedoxime in decreasing the acute toxicity of sarin.
On the other hand, the oxime HI-6 showed
significantly higher potency in the reduction of the
acute lethal toxic effects of sarin in mice than all
other oximes studied. It decreased the acute toxicity
of sarin by more than four times (Table 2).
Table 1. Percentage of reactivation of sarin-inhibited AChE by oximes in rat blood, diaphragm and brain in vivo.
| Treatment |
AChE activity (kat/l or kat/kg) | | Blood |
Diaphragm |
Brain | | Atropine |
6.83±0.62a |
5.69±1.42a |
27.29±3.51a | | Atropine + K203
(% reactivationb) |
7.43±0.49
(6.3) |
5.37±1.12
(0) |
31.39±3.69
(3.1) | | Atropine + HI-6
(% reactivation) |
13.69±1.41
(71.6*X) |
10.88±1.39
(48.7*X) |
52.49±4.38
(19.3*X) | | Atropine + obidoxime
(% reactivationb) |
7.35±0.96
(5.4) |
6.16±0.95
(4.4) |
33.42±9.29
(4.6) | | Atropine + trimedoxime
(% reactivation) |
7.66±0.54
(8.6) |
6.92±1.16
(11.6) |
36.41±7.19
(6.9) |
a means ± SD, N = 8. The untreated control value (saline control) for rat blood AChE activity was 16.41 kat/l, for
diaphragm AChE activity 16.35 kat/kg and for brain AChE activity 157.60 kat/kg
b percent reactivation was determined using the AChE activity values: {1- [((saline control) - (oxime + atropine))/((saline control)
- (atropine control))]} × 100
* significantly different from the atropine group
X significantly different from the atropine + obidoxime (trimedoxime, K203) group
Table 2. The influence of the type of oxime on the potency of antidotal treatment to reduce acute toxicity of sarin in mice.
| Treatment |
LD50 (g/kg)±95% confidence limit |
Protective ratio | | - |
116.1 (96.8-139.3) |
- | | K203 + atropine |
271.0 (217.3-311.2)* |
2.33 | | HI-6 + atropine |
523.3 (423.0-617.1)*X |
4.56 | | Obidoxime + atropine |
334.0 (272.6-378.5)* |
2.88 | | Trimedoxime + atropine |
308.4 (265.6-338.1)* |
2.66 |
* significantly different from the untreated group
X significantly different from the group treated by atropine in combination with obidoxime (trimedoxime, K203)
DISCUSSION
Due to the threat of misuse of various nerve agents
for military as well as terrorist purposes, a
broad-spectrum oxime, sufficiently effective against
nerve agents regardless of their chemical structure, is
urgently required as a satisfactorily effective antidotal
treatment of nerve agent exposure. Generally, it is
known that no currently available oxime is able to
satisfactorily reactivate the AChE inhibited by all nerve agents (Marrs et al. 2006, Kassa et al. 2007,
Szinicz et al. 2007). The oxime HI-6 seems to be the
most effective and broadest oxime among the
commonly used oximes, but it is a weak reactivator of
tabun-inhibited AChE (Lundy et al. 2006, Kuca et al.
2009). Therefore, new structural analogues of
currently available oximes have been developed to
find a broad-spectrum reactivator of nerve
agent-inhibited AChE (Kuca et al. 2004, 2006). Their
reactivating efficacy depends on the chemical
structure of a linker connecting both pyridinium rings
(in the case of bispyridinium oximes), the position of
an oxime group, the chemical structure and the
position of a functional group situated on the
non-oximated pyridinium ring (Kuca et al. 2006,
2007, Jokanović and Prostran 2009). On the other
hand, a higher number of aldoxime groups is not
necessary. The oxime HI-6 has only one oxime group
and it is significantly more efficacious to reactivate
soman and cyclosarin-inhibited AChE than
bispyridinium oximes with two oxime groups such as
obidoxime, methoxime or trimedoxime (Kassa and
Cabal 1999a, b, Lundy et al. 2006).
The oxime K203 was primarily synthesized to
increase the effectiveness of the antidotal treatment of
tabun poisonings. In vivo evaluation of its reactivating
efficacy against tabun showed that K203 is a really
promising oxime for the antidotal treatment of acute
tabun poisonings (Kassa et al. 2008, Kovarik et al.
2009). Unfortunately, based on the results obtained,
its potency to reactivate sarin-inhibited AChE is
relatively low and similar to the reactivating efficacy
of obidoxime and trimedoxime, probably due to its
chemical structure that is advantageous for its potency
in reactivating tabun-inhibited AChE, but is
disadvantageous for their potency in reactivating
AChE inhibited by fluorophosphonates such as
soman, sarin and cyclosarin (Kuca et al. 2006, 2007).
While the potency of bispyridinium oximes with the
oxime groups in position 4 (obidoxime, trimedoxime,
K203) in reactivating sarin-inhibited AChE is
relatively low, the oxime HI-6 with the oxime group
in position 2 showed the high potency in reactivating
sarin-inhibited AChE in the peripheral as well as the
central compartment and in reducing its acute
toxicity.
The findings clearly showed that the oxime K203
as well as all other currently available oximes are not
suitable as broad-spectrum reactivators of nerve
agent-inhibited AChE. Therefore, the combination of
two oximes with a different spectrum of their
reactivating and therapeutic efficacy seems to be a
possible approach to increasing the efficacy of
antidotal treatment of acute poisonings with nerve
agents regardless of their chemical structure (Clement
et al. 1987, Maksimovic and Kovacevic 1989,
Kovacevic et al. 1991, Worek et al. 2007). To
combine two oximes with a different spectrum of high
reactivating efficacy seems to be an appropriate way
to cover the whole spectrum of nerve agents.
Although the potency of K203, obidoxime and
trimedoxime in reactivating sarin-inhibited AChE in
rats is relatively low, they are able to reduce the acute
toxicity of sarin in mice by more than twice, probably
due to other beneficial effects than reactivation of
sarin-inhibited AChE. It has previously been
demonstrated that oximes are able to directly
influence the activity of muscarinic as well as
nicotinic receptors (Van Helden et al. 1996, Soukup
et al. 2010).
The results obtained confirm that none of the
oximes studied can be considered as a broad-spectrum
reactivator able to sufficiently counteract acute
toxicity of all nerve agents regardless of their
chemical structure. The newly developed
bispyridinium oxime K203, satisfactorily effective
against tabun (Kassa et al. 2008), was not found to be
sufficiently effective against sarin. In contrast, the
oxime HI-6 is a much more effective oxime for the
reactivation of sarin-inhibited AChE in rats and for
the reduction of acute toxicity of sarin in mice than all
the other oximes studied. Therefore, it is still the most
promising oxime for the antidotal treatment of acute
sarin poisoning.
DECLARATION OF INTEREST
The authors report no conflict of interest. The authors
alone are responsible for the content and writing of
the paper.
ACKNOWLEDGEMENTS
The authors would like to thank Mrs. J. Uhlirova for
her skilful technical assistance. The study was
supported by the grant of Ministry of Defence,
MO0FVZ0000501.
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