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Neuroprotective efficacy of newly developed oximes in comparison with currently available oximes in tabun-poisoned rats
JAB-44; No. of Pages 8 journal of applied biomedicine xxx (2014) xxx–xxx
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Original Research Article
Neuroprotective efficacy of newly developed oximes in comparison with currently available oximes in tabun-poisoned rats Jiri Kassa *, Jan Misik, Jana Zdarova Karasova Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University of Defense, Hradec Kralove, Czech Republic
article info
abstract
Article history:
The ability of two newly developed oximes (K361, K378) to reduce tabun-induced acute
Received 10 August 2014
neurotoxic signs and symptoms was compared with the oxime K203 and trimedoxime using
Received in revised form
a functional observational battery. The neuroprotective effects of the oximes studied
9 October 2014
combined with atropine on rats poisoned with tabun at a sublethal dose (310 mg/kg i.m.;
Accepted 10 October 2014
90% of LD50 value) were evaluated. Tabun-induced neurotoxicity was monitored by func-
Available online xxx
tional 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
Keywords:
experiment. Both newly developed oximes (K361, K378) combined with atropine were able to
Tabun
decrease tabun-induced neurotoxicity in the case of sublethal poisonings although they did
Neurotoxicity
not eliminate all tabun-induced acute neurotoxic signs and symptoms. Their ability to
Functional observational battery
decrease tabun-induced acute neurotoxicity was slightly lower than that of trimedoxime
Oximes
and the oxime K203. Therefore, the newly developed oximes are not suitable for the
Rats
replacement of commonly used oximes (especially trimedoxime and obidoxime) in the treatment of acute tabun poisonings.
Abbreviations: A, atropine
# 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske Budejovice. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
AChE, acetylcholinesterase BBB, blood–brain barrier b.w., body weight CNS, central nervous system FOB, functional observational battery HPLC, high pressure liquid chromatography i.m., intramuscularly n, number of surviving animals PC, personal computer RRF, air righting reflex RRV, air righting reflex from vertical position x/M, average or modus value s, standard deviation * Corresponding author at: Faculty of Military Health Sciences, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic. Tel.: +420 973255164; fax: +420 495518094. E-mail address: [email protected] (J. Kassa). 1214-021X/$ – see front matter # 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske Budejovice. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.jab.2014.10.001 Please cite this article in press as: Kassa, J., et al., Neuroprotective efficacy of newly developed oximes in comparison with currently available oximes in tabun-poisoned rats. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.10.001
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N
Introduction Organophosphorus nerve agents are considered to be the most dangerous chemical warfare agents because of high toxicity, rapid onset of clinical signs and symptoms and rapid progression of acute poisoning to the death. Their acute toxic effects are based on the phosphonylation of acetylcholinesterase (AChE; EC 3.1.1.7), leading to the overstimulation of postsynaptic cholinergic receptors due to the accumulation of the neurotransmitter acetylcholine in the synapses of the central and peripheral nervous systems (Bajgar, 2004). A current standard treatment of poisoning with nerve agents usually consists of a combined administration of an anticholinergic drug (preferably atropine) and an oxime (preferably pralidoxime or obidoxime). Generally, anticholinergics are used for relieving muscarinic signs and symptoms whereas oximes are used for reactivation of nerve agent-inhibited AChE (Bajgar, 2004; Colovic et al., 2013). One of the most resistant nerve agents is tabun (O-ethyl-N, N-dimethyl phosphoramido-cyanidate) (Fig. 1). It differs from other highly toxic organophosphates in its chemical structure and by the fact that commonly used antidotes are not able to sufficiently prevent tabun-induced acute toxic effects. Deleterious effects of tabun are extraordinarily difficult to antagonize due to the changes in hydrogen bonding and conformational changes of AChE-tabun complex in the AChE active site that make the nucleophilic attack of oximes very difficult (Cabal and Bajgar, 1999; Ekström et al., 2006). In the case of severe intoxication, some nerve agents including tabun can cause centrally mediated seizure activity that can rapidly progress to status epilepticus and contribute to profound brain damage that is associated with long-lasting neurological and psychological injuries (Hoffman et al., 2007; Yamasue et al., 2007). Therefore, the ability of antidotes to counteract acute neurotoxic effects of nerve agents and prevent nerve agent-poisoned organisms from irreversible lesions in the central nervous system (CNS) is very important for the successful antidotal treatment of acute nerve agent poisonings. Generally, the oximes exert more potent effects in the peripheral nervous system compared to CNS due to their low penetration across the blood–brain barrier (BBB). However, the penetration of oximes into CNS and subsequent reactivation of
EtO
P
O N
tabun Fig. 1 – Chemical structure of tabun.
nerve agent-inhibited AChE in the brain was demonstrated in the literature (Lorke et al., 2008). Although the percentage of reactivation of nerve agent-inhibited AChE in the brain is lower compared to the peripheral system, the role of reactivation of nerve agent-inhibited AChE in the brain is important for survival from nerve agent exposure (Bajgar, 2004). Unfortunately, currently available antidotal treatment consisting of atropine and commonly used reactivator of inhibited AChE (pralidoxime, obidoxime, trimedoxime, HI-6) is not able to sufficiently counteract acute toxic effects of tabun because of very low ability of oximes to reactivate tabun-inhibited AChE (Jokanovic, 2012). Therefore, the replacement of commonly used oximes (pralidoxime, obidoxime, HI-6) with a more effective oxime has been a long-standing goal for the treatment of tabun poisoning. The oxime K203, developed at our Department of Toxicology and Military Pharmacy several years ago, was considered to be promising reactivator of tabun-inhibited AChE. However, the differences between the reactivating and therapeutic efficacy of the oxime K203 and commonly used bispyridinium oximes (obidoxime, trimedoxime) are relatively small (Kassa et al., 2008). Therefore, we are still searching for a more efficacious oxime able to sufficiently reactivate tabuninhibited AChE. For this purpose, two novel oximes, K361 [4-(1amino-1-hydroxyiminomethyl)-40 -hydroxyiminomethyl-1,10 (but-1,4-diyl)-bispyridinium dibromide] and K378 [1-(3-phenylpropyl-4-hydroxyiminomethyl)-pyridinium bromide] (Fig. 2) were synthesized at our Department of Toxicology and Military Pharmacy (Musilek et al., 2008) to improve the efficacy of antidotal treatment of tabun poisonings. The aim of this study was to evaluate the potential neuroprotective effects of two newly developed oximes (K361, K378) with the oxime K203 and trimedoxime in
O NH2 HON N
2 Br
NOH N
NOH HON
2 Br K361
N HON
2 Br K203
trimedoxime
N (CH 2 )4 N
N
Br N
NH 2
NOH K378
Fig. 2 – Chemical structure of oximes studied. Please cite this article in press as: Kassa, J., et al., Neuroprotective efficacy of newly developed oximes in comparison with currently available oximes in tabun-poisoned rats. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.10.001
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combination with an anticholinergic drug atropine in tabunpoisoned rats. The tabun-induced neurotoxic signs were determined using a functional observational battery, a noninvasive and relatively sensitive type of neurological examination for a wide range of neurobiological functions including measurements of sensory, motor and autonomic nervous functions (Frantik and Hornychova, 1995).
Materials and methods Animals Male Wistar albino rats weighing 220–250 g were purchased from VELAZ (Prague, Czech Republic). They were kept in an airconditioned room (22 2 8C and 50 10% relative humidity, with lights from 7.00 to 19.00 h) and allowed access to standard food and tap water ad libitum. The rats were divided into groups of eight animals (N = 8). Handling of experimental animals was done under the supervision of the Ethics Committee of the Faculty of Military Health Sciences in Hradec Kralove (Czech Republic).
Chemicals Tabun (Fig. 1) was obtained from the Military Technical Institute in Brno (Czech Republic) and was 92% pure as assayed by acidimetric titration. The basic solution of tabun (1 mg/1 mL) was prepared in propyleneglycol 3 days before starting the experiments. Actual solution of tabun was prepared from its basic solution with the help of saline immediately before administration. All oximes studied (Fig. 2) were of 98.5% purity and were synthesized at the Department of Toxicology and Military Pharmacy of the Faculty of Military Health Sciences in Hradec Kralove (Czech Republic). Their purities were analyzed using HPLC (Jun et al., 2007). All other drugs and chemicals of analytical grade were obtained commercially and used without any further purification. The saline solution (0.9% NaCl) was used as a vehicle. All substances were administered intramuscularly (i.m.) at a volume of 1 mL/kg body weight (b.w.).
In vivo experiments Tabun was administered i.m. at a sublethal dose (310 mg/kg b. w. – 90% LD50). The relatively high dose of tabun was used to achieve maximum number of tabun-induced neurotoxic signs and symptoms at the time when the neurotoxicity of tabun was monitored by the functional observational battery. One minute following tabun poisoning, the rats were treated i.m. with atropine (10 mg/kg b.w.) in combination with the oxime K361, K378, K203 or trimedoxime at equitoxic doses corresponding to 5% of their LD50 values (Kassa et al., 2014). The neurotoxicity of tabun was monitored using the functional observational battery at 2 h following tabun poisoning. This time interval was chosen because we wanted to assess the ability of antidotal treatment to avoid or at least diminish all tabun-induced neurotoxic signs and symptoms during acute cholinergic crisis when the full clinical picture of acute poisoning with tabun is developed and visible. The evaluated
3
markers of tabun-induced neurotoxicity in experimental animals were compared with the parameters obtained from control rats given saline instead of tabun and antidotes at the same volume (1 mL/kg, b.w.). In addition, the markers of tabun-induced neurotoxicity in treated tabun-poisoned rats were compared with the parameters obtained from nontreated tabun-poisoned rats. The functional observational battery (FOB) consists of 43 measurements of sensory, motor and autonomic nervous functions. Some of them are scored (Table 1), the others are measured in absolute units (Frantik and Hornychova, 1995; Moser et al., 1997). The first evaluation was obtained when tabun-poisoned rats were in the home cage. The observer evaluated each animal's posture, palpebral closure and involuntary motor movements. Then, each rat was removed from the home cage and briefly hand-held. The exploratory activity, piloerection and other skin abnormalities were noted. Salivation and nose secretion were also registered and scored. Then, the rats were placed on a flat surface which served as an open field. A timer was started for 3 min during which the frequency of rearing responses was recorded. At the same time, gait characteristics were noted and ranked, and stereotypy and bizarre behaviours and abnormal posture were evaluated. At the end of the third minute, the number of faecal boluses and urine pools on the adsorbent pad was registered. Reflex testing comprising recording each rat's response to a frontal approach of the blunt end of a pen, a touch of the pen to the posterior flank and to an auditory click stimulus was also used. The response to a pinch on the tail and the ability of pupils to constrict in response to light were then assessed. These measures were followed by a test for the aerial righting reflex and by the measurements of forelimb and hindlimb grip strength, body weight, body temperature and finally hindlimb landing foot splay. The observer was blind to treatment condition.
Statistical evaluation Data collected with the FOB include categorial, ordinal and continuous values. Their statistical analyses were performed on a PC with a special interactive programme NTX (Frantik and Hornychova, 1995). The categorial and ordinal values were formulated as contingency tables and judged consecutively by chi-squared test of homogeneity, Concordance-Discordance test and Kruskal–Wallis test, respectively. The continual data were assessed by successive statistical tests: CI for Delta, Barlett test for Equality of Variance, Williams test and Test for Distribution Functions. The received data were evaluated at the significant level 2a = 0.05.
Results The results of the experiments related to the measurement of tabun-induced neurotoxicity at 2 h following tabun poisoning are divided into three parts (activity and neuromuscular measures, sensorimotor and excitability measures and autonomic measures) (Moser et al., 1997) and summarized in Tables 2a–2c. While three non-treated tabun-poisoned rats died before the evaluation of tabun-induced neurotoxicity by
Please cite this article in press as: Kassa, J., et al., Neuroprotective efficacy of newly developed oximes in comparison with currently available oximes in tabun-poisoned rats. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.10.001
Marker
Scored values only 2
1
0
Posture
1
2
3
4
Normal
Gait
Normal
Slightly impaired Slightly impaired Sporadic Stupor Stupor Body weaving Body
Somewhat impaired Somewhat impaired Reduced
Totally impaired Totally impaired Normal
None None None None
Normal Normal Very low Partial (ears) Partial (ears) Head weaving Head
Grooming Self-mutilation
Approach response
No reaction
Normal
Slow reaction
Circling Abnormal movements Energetic reaction
Touch response
No reaction
Normal
Slow reaction
Energetic reaction
Click response
No reaction
Normal
Slow reaction
Energetic reaction
Tail-pinch response
No reaction
Normal
Slow reaction
Energetic reaction
Mydriasis Normal reaction Slight mydriasis
Severe mydriasis
Normal
Slightly uncoordin.
Lands on side
Lands on back
Catch difficulty Ease of handling Muscular tonus Lacrimation Palpebral closure Endo-exophthalmus Skin abnormalities Salivation Nose secretion Clonic movements
Atonia
Hypotonia
Endo
Gait score Mobility score Activity Tension Tension Stereotypy Bizarre behaviour
Pupil size Pupil response Pupil size Righting reflex
Miosis Severe miosis
Slight miosis
Normal None Normal Normal None None Normal
Normal No reaction Normal
6
7
Crouched over Aggrression
Head bobbing
Rearing
Asleep
Flattened
Lying on side
Normal Easy Rigidity Severe Slightly drooping
Defense Moderately difficult Fasciculations Crusta Half-way drooping
Flight Difficult
Escape
Coloured crusta Completely shut
Ptosis
Erythema Severe Severe Nonrhythmic quivers
Cyanosis
Pigmented
Cold
Injury
Coloured Mild tremors
Severe tremors
Myoclonic jerks
Clonic convulsions
Opisthotonus
Emprosthotonus
Explosive jumps
Overcompensation of hindlimbs movements
Feet point outwards from body
Forelimbs are extended
Tonic convulsions Walks on tiptoes
Hunched body
Enhanced
Permanent
Others Others Exaggerated reaction Exaggerated reaction Exaggerated reaction Exaggerated reaction
Body is flattened against surface
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Tonic movements
Sitting or standing Passive Very easy Hypertonia Slight Open Exo Pale Sllight Slight Repetitive movements of mouth and jaws Contraction of extensors Ataxia
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Please cite this article in press as: Kassa, J., et al., Neuroprotective efficacy of newly developed oximes in comparison with currently available oximes in tabun-poisoned rats. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.10.001
Table 1 – Functional observational battery.
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Table 2a – The values of tabun-induced activity and neuromuscular neurotoxic markers measured at 2 h following tabun challenge by the functional observational battery (Nos. 1–2, 4–14 – scored values, Nos. 3, 15–18 – values in absolute units). 2h No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Controls
Marker
x/M
Posture Muscular tonus Rearing Hyperkinesis Tremors Clonic movements Tonic movements Gait Ataxia Gait score Mobility score Activity RRF RRV Landing foot splay (mm) Forelimb grip strength (kg) Hindlimb grip strength (kg) Grip strength of all limbs (kg)
1.00 0.00 7.75 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 4.00 1.00 1.00 78.56 4.97 1.06 14.01
Tabun + A + trimedoxime
/+s
12.84 0.42 0.15 1.32
x
/+s
x/M x
1.00 2.00* 1.00* 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 4.00 1.00 1.00 77.63 5.05x 0.67* 13.34x
n=8 *
/+s
x/M x
4.23
Tabun + A + K203
1.77
11.95 0.83 0.17 1.99
1.00 2.00* 3.88* 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 4.00 1.00 1.00 80.13 5.00x 0.76* 14.46x
n=8
Tabun + A + K378 /+s
x/M *
2.03
15.08 0.75 0.17 2.73
3.00 2.00* 3.50* 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 4.00 1.00 1.00 71.25 3.54* 0.43* 12.23
n=8
Tabun + A + K361 /+s
x/M *
2.50
31.25 0.24 0.13 3.45
3.00 2.00* 1.25* 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 4.00 1.00 1.00 75.50 4.47* 0.60* 12.01*
n=8
Tabun /+s
x/M *
1.39
20.81 0.50 0.13 1.85
3.00 2.00* 2.50* 1.00* 2.00* 1.00* 0.00 7.00* 2.00* 2.00* 3.00* 3.00* 4.00* 4.00* 58.25 3.89* 0.65* 8.45*
n=8
4.57
27.42 0.35 0.25 3.74
n=5
Statistically significant as compared with the control. Statistically significant as compared with the tabun values.
FOB, all tabun-poisoned rats treated with atropine in combination with an oxime survived till the end of experiment. The evaluation of tabun-induced neurotoxic signs at 2 h following intoxication proved significant alteration of 28 observed parameters. Tabun produced passive behaviour of rats during handling and retention, miosis, slight nose secretion and a decrease in muscular tone. The posture of tabun-poisoned rats was altered and fur as well as skin abnormalities were observed. Exploratory and rearing activities were significantly reduced, righting reflex was altered, strong tremors and hyperkinesis were observed, gait and mobility were impaired
and ataxia was found. In addition, no reaction during a reflex testing consisting of recording each rat's response to the frontal approach of the blunt end of a pen, to the touch of the pen to the posterior flank, to an auditory click stimulus and to a pinch on the tail was found. The pupils of the tabun-poisoned rats did not constrict in response to light because of tabun-induced miosis. A significant decrease in forelimb and hindlimb grip strength and body temperature was also observed at 2 h following tabun challenge (Tables 2a–2c). Both newly developed oximes (K361, K378) in combination with atropine were able to prevent some tabun-induced signs
Table 2b – The values of tabun-induced sensorimotor and excitability neurotoxic markers measured at 2 h following tabun challenge by the functional observational battery (scored values). 2h No 1 2 3 4 5 6 7 8 9 10 11
Marker Catch difficulty Ease of handling Arousal (GSC) Tension Vocalization Stereotypy Bizarre behaviour Approach response Touch response Click response Tail-pinch response
Controls x/M
/+s
x
x/M x
2.00 2.00 1.00 0.00 0.00 0.00 0.00 2.00 2.00 2.00 2.00
/+s
Tabun + A + K203 x/M x
2.00 2.00x 1.00x 0.00 0.00 0.00 0.00 2.00x 2.00x 2.00x 2.00x n=8
*
Tabun + A + trimedoxime
2.00 2.00x 1.00x 0.00 0.00 0.00 0.00 2.00x 2.00x 2.00x 2.00x n=8
n=8
/+s
Tabun + A + K378 x/M *
1.00 2.00x 1.00x 0.00 0.00 0.00 0.00 2.00x 3.00* 2.00x 2.00x n=8
/+s
Tabun + A + K361 x/M *
1.00 2.00x 1.00x 0.00 0.00 0.00 0.00 2.00x 1.00* 2.00x 2.00x n=8
/+s
Tabun x/M
/+s
*
1.00 1.00* 3.00* 0.00 0.00 0.00 0.00 1.00* 1.00* 1.00* 1.00* n=5
Statistically significant as compared with the control. Statistically significant as compared with the tabun values.
Please cite this article in press as: Kassa, J., et al., Neuroprotective efficacy of newly developed oximes in comparison with currently available oximes in tabun-poisoned rats. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.10.001
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Table 2c – The values of tabun-induced autonomic neurotoxic markers measured at 2 h following tabun challenge by the functional observational battery (Nos. 1–7, 10–11, 14 – scored values, Nos. 8–9, 12–13 – values in absolute units). 2h No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Controls
Marker
x/M
Lacrimation Palpebral closure Endo/exophtalmus Fur abnormalities Skin abnormalities Salivation Nose secretion Urination Defecation Pupil size Pupil response Body weight (g) Body temperature (8C) Respiration
0.00 1.00 0.00 0.00 0.00 0.00 0.00 1.13 0.00 0.00 1.00 205.38 37.49 0.00 n=8
* x
/+s
4.16
10.89 0.20
Tabun + A + trimedoxime x/M 0.00 1.00 0.00 0.00x 0.00x 0.00 0.00x 2.00 0.00 2.00* 0.00* 260.13 36.46* 0.00
/+s
3.16
15.03 0.41
n=8
Tabun + A + K203 x/M 0.00 1.00 0.00 0.00 0.00x 0.00 0.00x 4.88 0.00 2.00* 0.00* 243.63 36.49* 0.00 n=8
/+s
Tabun + A + K378 x/M
/+s
0.00 1.00 0.00
4.67
18.12 0.41
0.00x 0.00 0.00x 0.25 0.00 2.00* 0.00* 240.75 36.72* 0.00 n=8
Tabun + K361 x/M
/+s
0.00 1.00 0.00
0.71
11.21 0.80
0.00x 0.00 1.00* 3.00 0.00 2.00* 0.00* 240.38 36.10* 0.00 n=8
4.99
17.32 0.30
Tabun x/M
/+s
0.00 1.00 0.00 2.00* 1.00* 0.00 1.00* 3.63 0.00 2.00* 0.00* 212.13 35.65* 0.00
5.90
17.29 0.96
n=5
Statistically significant as compared with the control. Statistically significant as compared with the tabun values.
of neurotoxicity observed at 2 h following tabun challenge with the exception of alteration of posture, passive behaviour of rats during retention, miosis, a decrease in muscular tone and rearing activity, the absence of touch response and pupil response to light, a decrease in forelimb and hindlimb grip strength and body temperature (Tables 2a–2c). On the other hand, the ability of trimedoxime and the oxime K203 to eliminate tabun-induced signs of acute neurotoxicity was slightly higher compared to the novel oximes studied. They were not be able to eliminate or reduce miosis, the absence of pupil response to light and a decrease in muscular tone, body temperature, rearing activity as well as hindlimb grip strength (Tables 2a–2c).
Discussion The severe poisoning with nerve agents including tabun brings centrally mediated seizures that can rapidly progress to status epilepticus and cause irreversible seizure-related brain damage if left untreated (Chen, 2012). According to previously published data, atropine alone fails to prevent nerve agent-induced acute neurotoxic effects following an exposure to nerve agents at sublethal doses (Kassa and Kunesova, 2006). As the potential benefit of atropine alone to counteract the acute neurotoxicity of nerve agents is negligible, atropine should be combined with AChE reactivator for the antidotal treatment of nerve agent poisonings to improve its neuroprotective efficacy. Generally, the ability of currently available oximes to eliminate tabun-induced acute neurotoxic effects is relatively low (Kassa and Krejcova, 2003). Among them, trimedoxime seems to be the most effective to counteract tabun-induced acute neurotoxicity in rats although it is not able to completely eliminate or at least reduce tabun-induced signs of neurotoxicity in the case of sublethal tabun poisoning, either (Kassa and Kunesova, 2006). The evaluation of the neuroprotective
efficacy of the oxime K203 in tabun-poisoned rats brought relatively promising results but the differences between neuroprotective efficacy of the oxime K203 and commonly used oximes are not so high (Kassa et al., 2009). Therefore, new oximes with higher potency to counteract tabun-induced acute neurotoxicity are still searched to increase the efficacy of antidotal treatment of acute tabun poisonings. Monopyridinium oximes, such as pralidoxime, seem to be less effective to counteract tabun-induced acute toxicity than bispyridinium oximes (Kassa et al., 2007) despite of better penetration across BBB (Sakurada et al., 2003) Therefore, new pralidoxime analogues were developed to extend the properties of pralidoxime. The aromatic side chain (phenyl) was used for the synthesis of the oxime K378 in effort to improve its reactivation ability by the increase of its lipophilicity (Musilek et al., 2008). Moreover, the aromatic side chain was designed not only for the lipophilicity increase but also for the presence of p-electrons that are probably able to interact with the AChE active site via p–p or cation–p interactions (Musilek et al., 2007). Bispyridinium oximes are generally more effective to reactivate nerve agent-inhibited AChE than monopyridinium oximes, however, their ability to penetrate across BBB is lower, in maximum of 6% (Lorke et al., 2008). A design of another newly developed oxime (bispyridinium AChE reactivator K361) was based on the data obtained during the extensive work on oxime development and from structure-activity relationship studies realized at our Department of Toxicology and Military Pharmacy (Cabal et al., 2004; Kuca et al., 2006). Namely, the combination of two oxime groups in position 4 on both pyridinium rings and the tetracarbon chain linking two quaternary nitrogens was used to increase its ability to reactivate tabun-inhibited AChE (Kuca et al., 2006). To compare the neuroprotective efficacy of newly developed oximes (K361, K378) with trimedoxime and the oxime K203, the ability of both novel oximes to eliminate or reduce tabun-induced neurotoxic signs and symptoms was slightly
Please cite this article in press as: Kassa, J., et al., Neuroprotective efficacy of newly developed oximes in comparison with currently available oximes in tabun-poisoned rats. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.10.001
JAB-44; No. of Pages 8 journal of applied biomedicine xxx (2014) xxx–xxx
lower. As the neuroprotective efficacy of both novel oximes is not possible to explain by their central reactivating efficacy that was negligible (Kassa et al., 2014), their neuroprotective efficacy could be also caused by their direct pharmacological effects such as inhibition of acetylcholine release, interaction with presynaptic cholinergic nerve terminals and/or with postsynaptic receptors (Van Helden et al., 1996; Sürig et al., 2006; Niessen et al., 2011). However, the benefit of both novel oximes for neuroprotective efficacy of antidotal treatment of acute tabun poisonings is not so high to make the decision about the replacement of commonly used oximes (especially trimedoxime and obidoxime) in the antidotal treatment of acute tabun poisonings.
Conclusions The changes of the structure of commonly used oximes realized according to the postulated requirements (Kuca et al., 2006) are not able to markedly increase the potency of current antidotal treatment to eliminate tabun-induced acute neurotoxicity. Thus, it is necessary to find a new approach how to change the known structures of AChE reactivators to reach better entering into the active site of AChE-tabun complex and higher penetration through BBB. The higher brain concentration of AChE reactivator should bring higher reactivation of nerve agent-inhibited brain AChE and more effective elimination of acute neurotoxic signs and symptoms of nerve agents including tabun.
Conflict 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 to Mrs. E. Reslova and Mrs. J. Uhlirova for their skilful technical assistance. The study was supported by a grant of Ministry of Defense of the Czech Republic – ‘‘Long-term organization development plan 1011’’.
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Original Research Article
Neuroprotective efficacy of newly developed oximes in comparison with currently available oximes in tabun-poisoned rats Jiri Kassa *, Jan Misik, Jana Zdarova Karasova Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, University of Defense, Hradec Kralove, Czech Republic
article info
abstract
Article history:
The ability of two newly developed oximes (K361, K378) to reduce tabun-induced acute
Received 10 August 2014
neurotoxic signs and symptoms was compared with the oxime K203 and trimedoxime using
Received in revised form
a functional observational battery. The neuroprotective effects of the oximes studied
9 October 2014
combined with atropine on rats poisoned with tabun at a sublethal dose (310 mg/kg i.m.;
Accepted 10 October 2014
90% of LD50 value) were evaluated. Tabun-induced neurotoxicity was monitored by func-
Available online xxx
tional 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
Keywords:
experiment. Both newly developed oximes (K361, K378) combined with atropine were able to
Tabun
decrease tabun-induced neurotoxicity in the case of sublethal poisonings although they did
Neurotoxicity
not eliminate all tabun-induced acute neurotoxic signs and symptoms. Their ability to
Functional observational battery
decrease tabun-induced acute neurotoxicity was slightly lower than that of trimedoxime
Oximes
and the oxime K203. Therefore, the newly developed oximes are not suitable for the
Rats
replacement of commonly used oximes (especially trimedoxime and obidoxime) in the treatment of acute tabun poisonings.
Abbreviations: A, atropine
# 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske Budejovice. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.
AChE, acetylcholinesterase BBB, blood–brain barrier b.w., body weight CNS, central nervous system FOB, functional observational battery HPLC, high pressure liquid chromatography i.m., intramuscularly n, number of surviving animals PC, personal computer RRF, air righting reflex RRV, air righting reflex from vertical position x/M, average or modus value s, standard deviation * Corresponding author at: Faculty of Military Health Sciences, Trebesska 1575, 500 01 Hradec Kralove, Czech Republic. Tel.: +420 973255164; fax: +420 495518094. E-mail address: [email protected] (J. Kassa). 1214-021X/$ – see front matter # 2014 Faculty of Health and Social Studies, University of South Bohemia in Ceske Budejovice. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved. http://dx.doi.org/10.1016/j.jab.2014.10.001 Please cite this article in press as: Kassa, J., et al., Neuroprotective efficacy of newly developed oximes in comparison with currently available oximes in tabun-poisoned rats. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.10.001
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N
Introduction Organophosphorus nerve agents are considered to be the most dangerous chemical warfare agents because of high toxicity, rapid onset of clinical signs and symptoms and rapid progression of acute poisoning to the death. Their acute toxic effects are based on the phosphonylation of acetylcholinesterase (AChE; EC 3.1.1.7), leading to the overstimulation of postsynaptic cholinergic receptors due to the accumulation of the neurotransmitter acetylcholine in the synapses of the central and peripheral nervous systems (Bajgar, 2004). A current standard treatment of poisoning with nerve agents usually consists of a combined administration of an anticholinergic drug (preferably atropine) and an oxime (preferably pralidoxime or obidoxime). Generally, anticholinergics are used for relieving muscarinic signs and symptoms whereas oximes are used for reactivation of nerve agent-inhibited AChE (Bajgar, 2004; Colovic et al., 2013). One of the most resistant nerve agents is tabun (O-ethyl-N, N-dimethyl phosphoramido-cyanidate) (Fig. 1). It differs from other highly toxic organophosphates in its chemical structure and by the fact that commonly used antidotes are not able to sufficiently prevent tabun-induced acute toxic effects. Deleterious effects of tabun are extraordinarily difficult to antagonize due to the changes in hydrogen bonding and conformational changes of AChE-tabun complex in the AChE active site that make the nucleophilic attack of oximes very difficult (Cabal and Bajgar, 1999; Ekström et al., 2006). In the case of severe intoxication, some nerve agents including tabun can cause centrally mediated seizure activity that can rapidly progress to status epilepticus and contribute to profound brain damage that is associated with long-lasting neurological and psychological injuries (Hoffman et al., 2007; Yamasue et al., 2007). Therefore, the ability of antidotes to counteract acute neurotoxic effects of nerve agents and prevent nerve agent-poisoned organisms from irreversible lesions in the central nervous system (CNS) is very important for the successful antidotal treatment of acute nerve agent poisonings. Generally, the oximes exert more potent effects in the peripheral nervous system compared to CNS due to their low penetration across the blood–brain barrier (BBB). However, the penetration of oximes into CNS and subsequent reactivation of
EtO
P
O N
tabun Fig. 1 – Chemical structure of tabun.
nerve agent-inhibited AChE in the brain was demonstrated in the literature (Lorke et al., 2008). Although the percentage of reactivation of nerve agent-inhibited AChE in the brain is lower compared to the peripheral system, the role of reactivation of nerve agent-inhibited AChE in the brain is important for survival from nerve agent exposure (Bajgar, 2004). Unfortunately, currently available antidotal treatment consisting of atropine and commonly used reactivator of inhibited AChE (pralidoxime, obidoxime, trimedoxime, HI-6) is not able to sufficiently counteract acute toxic effects of tabun because of very low ability of oximes to reactivate tabun-inhibited AChE (Jokanovic, 2012). Therefore, the replacement of commonly used oximes (pralidoxime, obidoxime, HI-6) with a more effective oxime has been a long-standing goal for the treatment of tabun poisoning. The oxime K203, developed at our Department of Toxicology and Military Pharmacy several years ago, was considered to be promising reactivator of tabun-inhibited AChE. However, the differences between the reactivating and therapeutic efficacy of the oxime K203 and commonly used bispyridinium oximes (obidoxime, trimedoxime) are relatively small (Kassa et al., 2008). Therefore, we are still searching for a more efficacious oxime able to sufficiently reactivate tabuninhibited AChE. For this purpose, two novel oximes, K361 [4-(1amino-1-hydroxyiminomethyl)-40 -hydroxyiminomethyl-1,10 (but-1,4-diyl)-bispyridinium dibromide] and K378 [1-(3-phenylpropyl-4-hydroxyiminomethyl)-pyridinium bromide] (Fig. 2) were synthesized at our Department of Toxicology and Military Pharmacy (Musilek et al., 2008) to improve the efficacy of antidotal treatment of tabun poisonings. The aim of this study was to evaluate the potential neuroprotective effects of two newly developed oximes (K361, K378) with the oxime K203 and trimedoxime in
O NH2 HON N
2 Br
NOH N
NOH HON
2 Br K361
N HON
2 Br K203
trimedoxime
N (CH 2 )4 N
N
Br N
NH 2
NOH K378
Fig. 2 – Chemical structure of oximes studied. Please cite this article in press as: Kassa, J., et al., Neuroprotective efficacy of newly developed oximes in comparison with currently available oximes in tabun-poisoned rats. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.10.001
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combination with an anticholinergic drug atropine in tabunpoisoned rats. The tabun-induced neurotoxic signs were determined using a functional observational battery, a noninvasive and relatively sensitive type of neurological examination for a wide range of neurobiological functions including measurements of sensory, motor and autonomic nervous functions (Frantik and Hornychova, 1995).
Materials and methods Animals Male Wistar albino rats weighing 220–250 g were purchased from VELAZ (Prague, Czech Republic). They were kept in an airconditioned room (22 2 8C and 50 10% relative humidity, with lights from 7.00 to 19.00 h) and allowed access to standard food and tap water ad libitum. The rats were divided into groups of eight animals (N = 8). Handling of experimental animals was done under the supervision of the Ethics Committee of the Faculty of Military Health Sciences in Hradec Kralove (Czech Republic).
Chemicals Tabun (Fig. 1) was obtained from the Military Technical Institute in Brno (Czech Republic) and was 92% pure as assayed by acidimetric titration. The basic solution of tabun (1 mg/1 mL) was prepared in propyleneglycol 3 days before starting the experiments. Actual solution of tabun was prepared from its basic solution with the help of saline immediately before administration. All oximes studied (Fig. 2) were of 98.5% purity and were synthesized at the Department of Toxicology and Military Pharmacy of the Faculty of Military Health Sciences in Hradec Kralove (Czech Republic). Their purities were analyzed using HPLC (Jun et al., 2007). All other drugs and chemicals of analytical grade were obtained commercially and used without any further purification. The saline solution (0.9% NaCl) was used as a vehicle. All substances were administered intramuscularly (i.m.) at a volume of 1 mL/kg body weight (b.w.).
In vivo experiments Tabun was administered i.m. at a sublethal dose (310 mg/kg b. w. – 90% LD50). The relatively high dose of tabun was used to achieve maximum number of tabun-induced neurotoxic signs and symptoms at the time when the neurotoxicity of tabun was monitored by the functional observational battery. One minute following tabun poisoning, the rats were treated i.m. with atropine (10 mg/kg b.w.) in combination with the oxime K361, K378, K203 or trimedoxime at equitoxic doses corresponding to 5% of their LD50 values (Kassa et al., 2014). The neurotoxicity of tabun was monitored using the functional observational battery at 2 h following tabun poisoning. This time interval was chosen because we wanted to assess the ability of antidotal treatment to avoid or at least diminish all tabun-induced neurotoxic signs and symptoms during acute cholinergic crisis when the full clinical picture of acute poisoning with tabun is developed and visible. The evaluated
3
markers of tabun-induced neurotoxicity in experimental animals were compared with the parameters obtained from control rats given saline instead of tabun and antidotes at the same volume (1 mL/kg, b.w.). In addition, the markers of tabun-induced neurotoxicity in treated tabun-poisoned rats were compared with the parameters obtained from nontreated tabun-poisoned rats. The functional observational battery (FOB) consists of 43 measurements of sensory, motor and autonomic nervous functions. Some of them are scored (Table 1), the others are measured in absolute units (Frantik and Hornychova, 1995; Moser et al., 1997). The first evaluation was obtained when tabun-poisoned rats were in the home cage. The observer evaluated each animal's posture, palpebral closure and involuntary motor movements. Then, each rat was removed from the home cage and briefly hand-held. The exploratory activity, piloerection and other skin abnormalities were noted. Salivation and nose secretion were also registered and scored. Then, the rats were placed on a flat surface which served as an open field. A timer was started for 3 min during which the frequency of rearing responses was recorded. At the same time, gait characteristics were noted and ranked, and stereotypy and bizarre behaviours and abnormal posture were evaluated. At the end of the third minute, the number of faecal boluses and urine pools on the adsorbent pad was registered. Reflex testing comprising recording each rat's response to a frontal approach of the blunt end of a pen, a touch of the pen to the posterior flank and to an auditory click stimulus was also used. The response to a pinch on the tail and the ability of pupils to constrict in response to light were then assessed. These measures were followed by a test for the aerial righting reflex and by the measurements of forelimb and hindlimb grip strength, body weight, body temperature and finally hindlimb landing foot splay. The observer was blind to treatment condition.
Statistical evaluation Data collected with the FOB include categorial, ordinal and continuous values. Their statistical analyses were performed on a PC with a special interactive programme NTX (Frantik and Hornychova, 1995). The categorial and ordinal values were formulated as contingency tables and judged consecutively by chi-squared test of homogeneity, Concordance-Discordance test and Kruskal–Wallis test, respectively. The continual data were assessed by successive statistical tests: CI for Delta, Barlett test for Equality of Variance, Williams test and Test for Distribution Functions. The received data were evaluated at the significant level 2a = 0.05.
Results The results of the experiments related to the measurement of tabun-induced neurotoxicity at 2 h following tabun poisoning are divided into three parts (activity and neuromuscular measures, sensorimotor and excitability measures and autonomic measures) (Moser et al., 1997) and summarized in Tables 2a–2c. While three non-treated tabun-poisoned rats died before the evaluation of tabun-induced neurotoxicity by
Please cite this article in press as: Kassa, J., et al., Neuroprotective efficacy of newly developed oximes in comparison with currently available oximes in tabun-poisoned rats. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.10.001
Marker
Scored values only 2
1
0
Posture
1
2
3
4
Normal
Gait
Normal
Slightly impaired Slightly impaired Sporadic Stupor Stupor Body weaving Body
Somewhat impaired Somewhat impaired Reduced
Totally impaired Totally impaired Normal
None None None None
Normal Normal Very low Partial (ears) Partial (ears) Head weaving Head
Grooming Self-mutilation
Approach response
No reaction
Normal
Slow reaction
Circling Abnormal movements Energetic reaction
Touch response
No reaction
Normal
Slow reaction
Energetic reaction
Click response
No reaction
Normal
Slow reaction
Energetic reaction
Tail-pinch response
No reaction
Normal
Slow reaction
Energetic reaction
Mydriasis Normal reaction Slight mydriasis
Severe mydriasis
Normal
Slightly uncoordin.
Lands on side
Lands on back
Catch difficulty Ease of handling Muscular tonus Lacrimation Palpebral closure Endo-exophthalmus Skin abnormalities Salivation Nose secretion Clonic movements
Atonia
Hypotonia
Endo
Gait score Mobility score Activity Tension Tension Stereotypy Bizarre behaviour
Pupil size Pupil response Pupil size Righting reflex
Miosis Severe miosis
Slight miosis
Normal None Normal Normal None None Normal
Normal No reaction Normal
6
7
Crouched over Aggrression
Head bobbing
Rearing
Asleep
Flattened
Lying on side
Normal Easy Rigidity Severe Slightly drooping
Defense Moderately difficult Fasciculations Crusta Half-way drooping
Flight Difficult
Escape
Coloured crusta Completely shut
Ptosis
Erythema Severe Severe Nonrhythmic quivers
Cyanosis
Pigmented
Cold
Injury
Coloured Mild tremors
Severe tremors
Myoclonic jerks
Clonic convulsions
Opisthotonus
Emprosthotonus
Explosive jumps
Overcompensation of hindlimbs movements
Feet point outwards from body
Forelimbs are extended
Tonic convulsions Walks on tiptoes
Hunched body
Enhanced
Permanent
Others Others Exaggerated reaction Exaggerated reaction Exaggerated reaction Exaggerated reaction
Body is flattened against surface
journal of applied biomedicine xxx (2014) xxx–xxx
Tonic movements
Sitting or standing Passive Very easy Hypertonia Slight Open Exo Pale Sllight Slight Repetitive movements of mouth and jaws Contraction of extensors Ataxia
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Table 1 – Functional observational battery.
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Table 2a – The values of tabun-induced activity and neuromuscular neurotoxic markers measured at 2 h following tabun challenge by the functional observational battery (Nos. 1–2, 4–14 – scored values, Nos. 3, 15–18 – values in absolute units). 2h No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Controls
Marker
x/M
Posture Muscular tonus Rearing Hyperkinesis Tremors Clonic movements Tonic movements Gait Ataxia Gait score Mobility score Activity RRF RRV Landing foot splay (mm) Forelimb grip strength (kg) Hindlimb grip strength (kg) Grip strength of all limbs (kg)
1.00 0.00 7.75 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 4.00 1.00 1.00 78.56 4.97 1.06 14.01
Tabun + A + trimedoxime
/+s
12.84 0.42 0.15 1.32
x
/+s
x/M x
1.00 2.00* 1.00* 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 4.00 1.00 1.00 77.63 5.05x 0.67* 13.34x
n=8 *
/+s
x/M x
4.23
Tabun + A + K203
1.77
11.95 0.83 0.17 1.99
1.00 2.00* 3.88* 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 4.00 1.00 1.00 80.13 5.00x 0.76* 14.46x
n=8
Tabun + A + K378 /+s
x/M *
2.03
15.08 0.75 0.17 2.73
3.00 2.00* 3.50* 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 4.00 1.00 1.00 71.25 3.54* 0.43* 12.23
n=8
Tabun + A + K361 /+s
x/M *
2.50
31.25 0.24 0.13 3.45
3.00 2.00* 1.25* 0.00 0.00 0.00 0.00 0.00 0.00 1.00 1.00 4.00 1.00 1.00 75.50 4.47* 0.60* 12.01*
n=8
Tabun /+s
x/M *
1.39
20.81 0.50 0.13 1.85
3.00 2.00* 2.50* 1.00* 2.00* 1.00* 0.00 7.00* 2.00* 2.00* 3.00* 3.00* 4.00* 4.00* 58.25 3.89* 0.65* 8.45*
n=8
4.57
27.42 0.35 0.25 3.74
n=5
Statistically significant as compared with the control. Statistically significant as compared with the tabun values.
FOB, all tabun-poisoned rats treated with atropine in combination with an oxime survived till the end of experiment. The evaluation of tabun-induced neurotoxic signs at 2 h following intoxication proved significant alteration of 28 observed parameters. Tabun produced passive behaviour of rats during handling and retention, miosis, slight nose secretion and a decrease in muscular tone. The posture of tabun-poisoned rats was altered and fur as well as skin abnormalities were observed. Exploratory and rearing activities were significantly reduced, righting reflex was altered, strong tremors and hyperkinesis were observed, gait and mobility were impaired
and ataxia was found. In addition, no reaction during a reflex testing consisting of recording each rat's response to the frontal approach of the blunt end of a pen, to the touch of the pen to the posterior flank, to an auditory click stimulus and to a pinch on the tail was found. The pupils of the tabun-poisoned rats did not constrict in response to light because of tabun-induced miosis. A significant decrease in forelimb and hindlimb grip strength and body temperature was also observed at 2 h following tabun challenge (Tables 2a–2c). Both newly developed oximes (K361, K378) in combination with atropine were able to prevent some tabun-induced signs
Table 2b – The values of tabun-induced sensorimotor and excitability neurotoxic markers measured at 2 h following tabun challenge by the functional observational battery (scored values). 2h No 1 2 3 4 5 6 7 8 9 10 11
Marker Catch difficulty Ease of handling Arousal (GSC) Tension Vocalization Stereotypy Bizarre behaviour Approach response Touch response Click response Tail-pinch response
Controls x/M
/+s
x
x/M x
2.00 2.00 1.00 0.00 0.00 0.00 0.00 2.00 2.00 2.00 2.00
/+s
Tabun + A + K203 x/M x
2.00 2.00x 1.00x 0.00 0.00 0.00 0.00 2.00x 2.00x 2.00x 2.00x n=8
*
Tabun + A + trimedoxime
2.00 2.00x 1.00x 0.00 0.00 0.00 0.00 2.00x 2.00x 2.00x 2.00x n=8
n=8
/+s
Tabun + A + K378 x/M *
1.00 2.00x 1.00x 0.00 0.00 0.00 0.00 2.00x 3.00* 2.00x 2.00x n=8
/+s
Tabun + A + K361 x/M *
1.00 2.00x 1.00x 0.00 0.00 0.00 0.00 2.00x 1.00* 2.00x 2.00x n=8
/+s
Tabun x/M
/+s
*
1.00 1.00* 3.00* 0.00 0.00 0.00 0.00 1.00* 1.00* 1.00* 1.00* n=5
Statistically significant as compared with the control. Statistically significant as compared with the tabun values.
Please cite this article in press as: Kassa, J., et al., Neuroprotective efficacy of newly developed oximes in comparison with currently available oximes in tabun-poisoned rats. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.10.001
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Table 2c – The values of tabun-induced autonomic neurotoxic markers measured at 2 h following tabun challenge by the functional observational battery (Nos. 1–7, 10–11, 14 – scored values, Nos. 8–9, 12–13 – values in absolute units). 2h No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Controls
Marker
x/M
Lacrimation Palpebral closure Endo/exophtalmus Fur abnormalities Skin abnormalities Salivation Nose secretion Urination Defecation Pupil size Pupil response Body weight (g) Body temperature (8C) Respiration
0.00 1.00 0.00 0.00 0.00 0.00 0.00 1.13 0.00 0.00 1.00 205.38 37.49 0.00 n=8
* x
/+s
4.16
10.89 0.20
Tabun + A + trimedoxime x/M 0.00 1.00 0.00 0.00x 0.00x 0.00 0.00x 2.00 0.00 2.00* 0.00* 260.13 36.46* 0.00
/+s
3.16
15.03 0.41
n=8
Tabun + A + K203 x/M 0.00 1.00 0.00 0.00 0.00x 0.00 0.00x 4.88 0.00 2.00* 0.00* 243.63 36.49* 0.00 n=8
/+s
Tabun + A + K378 x/M
/+s
0.00 1.00 0.00
4.67
18.12 0.41
0.00x 0.00 0.00x 0.25 0.00 2.00* 0.00* 240.75 36.72* 0.00 n=8
Tabun + K361 x/M
/+s
0.00 1.00 0.00
0.71
11.21 0.80
0.00x 0.00 1.00* 3.00 0.00 2.00* 0.00* 240.38 36.10* 0.00 n=8
4.99
17.32 0.30
Tabun x/M
/+s
0.00 1.00 0.00 2.00* 1.00* 0.00 1.00* 3.63 0.00 2.00* 0.00* 212.13 35.65* 0.00
5.90
17.29 0.96
n=5
Statistically significant as compared with the control. Statistically significant as compared with the tabun values.
of neurotoxicity observed at 2 h following tabun challenge with the exception of alteration of posture, passive behaviour of rats during retention, miosis, a decrease in muscular tone and rearing activity, the absence of touch response and pupil response to light, a decrease in forelimb and hindlimb grip strength and body temperature (Tables 2a–2c). On the other hand, the ability of trimedoxime and the oxime K203 to eliminate tabun-induced signs of acute neurotoxicity was slightly higher compared to the novel oximes studied. They were not be able to eliminate or reduce miosis, the absence of pupil response to light and a decrease in muscular tone, body temperature, rearing activity as well as hindlimb grip strength (Tables 2a–2c).
Discussion The severe poisoning with nerve agents including tabun brings centrally mediated seizures that can rapidly progress to status epilepticus and cause irreversible seizure-related brain damage if left untreated (Chen, 2012). According to previously published data, atropine alone fails to prevent nerve agent-induced acute neurotoxic effects following an exposure to nerve agents at sublethal doses (Kassa and Kunesova, 2006). As the potential benefit of atropine alone to counteract the acute neurotoxicity of nerve agents is negligible, atropine should be combined with AChE reactivator for the antidotal treatment of nerve agent poisonings to improve its neuroprotective efficacy. Generally, the ability of currently available oximes to eliminate tabun-induced acute neurotoxic effects is relatively low (Kassa and Krejcova, 2003). Among them, trimedoxime seems to be the most effective to counteract tabun-induced acute neurotoxicity in rats although it is not able to completely eliminate or at least reduce tabun-induced signs of neurotoxicity in the case of sublethal tabun poisoning, either (Kassa and Kunesova, 2006). The evaluation of the neuroprotective
efficacy of the oxime K203 in tabun-poisoned rats brought relatively promising results but the differences between neuroprotective efficacy of the oxime K203 and commonly used oximes are not so high (Kassa et al., 2009). Therefore, new oximes with higher potency to counteract tabun-induced acute neurotoxicity are still searched to increase the efficacy of antidotal treatment of acute tabun poisonings. Monopyridinium oximes, such as pralidoxime, seem to be less effective to counteract tabun-induced acute toxicity than bispyridinium oximes (Kassa et al., 2007) despite of better penetration across BBB (Sakurada et al., 2003) Therefore, new pralidoxime analogues were developed to extend the properties of pralidoxime. The aromatic side chain (phenyl) was used for the synthesis of the oxime K378 in effort to improve its reactivation ability by the increase of its lipophilicity (Musilek et al., 2008). Moreover, the aromatic side chain was designed not only for the lipophilicity increase but also for the presence of p-electrons that are probably able to interact with the AChE active site via p–p or cation–p interactions (Musilek et al., 2007). Bispyridinium oximes are generally more effective to reactivate nerve agent-inhibited AChE than monopyridinium oximes, however, their ability to penetrate across BBB is lower, in maximum of 6% (Lorke et al., 2008). A design of another newly developed oxime (bispyridinium AChE reactivator K361) was based on the data obtained during the extensive work on oxime development and from structure-activity relationship studies realized at our Department of Toxicology and Military Pharmacy (Cabal et al., 2004; Kuca et al., 2006). Namely, the combination of two oxime groups in position 4 on both pyridinium rings and the tetracarbon chain linking two quaternary nitrogens was used to increase its ability to reactivate tabun-inhibited AChE (Kuca et al., 2006). To compare the neuroprotective efficacy of newly developed oximes (K361, K378) with trimedoxime and the oxime K203, the ability of both novel oximes to eliminate or reduce tabun-induced neurotoxic signs and symptoms was slightly
Please cite this article in press as: Kassa, J., et al., Neuroprotective efficacy of newly developed oximes in comparison with currently available oximes in tabun-poisoned rats. J. Appl. Biomed. (2014), http://dx.doi.org/10.1016/j.jab.2014.10.001
JAB-44; No. of Pages 8 journal of applied biomedicine xxx (2014) xxx–xxx
lower. As the neuroprotective efficacy of both novel oximes is not possible to explain by their central reactivating efficacy that was negligible (Kassa et al., 2014), their neuroprotective efficacy could be also caused by their direct pharmacological effects such as inhibition of acetylcholine release, interaction with presynaptic cholinergic nerve terminals and/or with postsynaptic receptors (Van Helden et al., 1996; Sürig et al., 2006; Niessen et al., 2011). However, the benefit of both novel oximes for neuroprotective efficacy of antidotal treatment of acute tabun poisonings is not so high to make the decision about the replacement of commonly used oximes (especially trimedoxime and obidoxime) in the antidotal treatment of acute tabun poisonings.
Conclusions The changes of the structure of commonly used oximes realized according to the postulated requirements (Kuca et al., 2006) are not able to markedly increase the potency of current antidotal treatment to eliminate tabun-induced acute neurotoxicity. Thus, it is necessary to find a new approach how to change the known structures of AChE reactivators to reach better entering into the active site of AChE-tabun complex and higher penetration through BBB. The higher brain concentration of AChE reactivator should bring higher reactivation of nerve agent-inhibited brain AChE and more effective elimination of acute neurotoxic signs and symptoms of nerve agents including tabun.
Conflict 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 to Mrs. E. Reslova and Mrs. J. Uhlirova for their skilful technical assistance. The study was supported by a grant of Ministry of Defense of the Czech Republic – ‘‘Long-term organization development plan 1011’’.
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