New group of xylene linker-containing acetylcholinesterase reactivators as antidotes against the nerve agent cyclosarin

Abstract

Nerve agents such as sarin, cyclosarin and tabun are organophosphorus substances able to inhibit the enzyme acetylcholinesterase (AChE; EC 3.1.1.7). AChE reactivators and anticholinergics are generally used as antidotes in the case of intoxication with these agents. None of the known AChE reactivators is able to reactivate AChE inhibited by all nerve agents used. In this work, reactivation potency of nine newly developed AChE reactivators with an incorporated xylene ring in their structure was measured in vitro. Cyclosarin was chosen as an appropriate member of the nerve agent family. Reactivation potency of the tested AChE reactivators was compared with the gold standard of AChE reactivators – pralidoxime. Two oximes (K107 and K108) surpassed the reactivation potency of pralidoxime. Moreover, from the obtained results it could be deduced that AChE reactivators with a functional oxime group in position-2 are the most potent AChE reactivators in the case of cyclosarin intoxications.

Keywords::

• Nerve agent
• cyclosarin
• reactivation
• acetylcholinesterase
• oxime
• inhibition

Introduction

Organophosphorus compounds are needed in industry (softening agents, lubricants), veterinary and human medicine (drugs), agriculture (pesticides) etc. A special aspect of their utilization is military use as nerve agents [1]. History of nerve agents began before World War II in Germany. The first synthesized nerve agent – tabun (O-ethyl-N, N-dimethyl phosphoramidocyanidate) was synthesized in 1936 (Germany) and later, many other nerve agents such as sarin, cyclosarin, soman and VX were developed, stored and prepared for potential military use [2]. Fortunately, these substances were not used until recently in world conflicts. They were misused by Sadam Husain in Iraq in the Kurdish village of Birjinni (1988) and by the Japanese Aum Shinrikyo sect in Matsumoto (1994) and Tokyo (1995) [3,4].

Their toxic effect is based on the irreversible inhibition of an enzyme acetylcholinesterase (AChE; EC 3.1.1.7) by covalent binding of the nerve agent in its active site. Due to this inhibition, neuromediator acetylcholine accumulates at the synaptic clefts and overstimulates nerve receptors. Afterwards, cholinergic crisis occurs and in severe intoxications the affected organism could die due to respiratory failure [5].

Owing to the high toxicity of nerve agents, rapid use of antidotal treatment is necessary. Antidotal mean consists of anticholinergics (atropine mainly) to counteract muscarinic signs and AChE reactivators to liberate the free enzyme, which is again able to fullfill its physiological role in the organism [6].

Pralidoxime, obidoxime and HI-6 are currently the most used reactivators in the case of nerve agent intoxications. However, none from these is able to reactivate AChE inhibited regardless of nerve agent used [7]. For this reason, new AChE reactivators are still being developed 8-10.

In 2005, we prepared a new class of AChE reactivators with a xylene moiety introduced into the AChE reactivator structure. Due to the presence of the xylene linker, lipophilicity of this reactivator increased and, moreover, the rigidity in the reactivator structure increased, too. These substances were tested firstly as potential reactivators of AChE inhibited by the pesticide chlorpyrifos. The results showed that several of them were promising AChE reactivators [11].

In this work, we are interested in the in vitro evaluation of their potency in the case of reactivation of AChE inhibition using a real nerve agent. For this purpose, we have chosen cyclosarin as an appropriate member of the nerve agent family [12]. We have compared the potency of tested substances with pralidoxime, which is currently considered as gold standard for AChE reactivators [13].

Material and methods

Chemicals

All compounds were prepared as described earlier by Musilek et al [11]. Their structures are listed in Table I. Pralidoxime (2-hydroxyiminomethyl-1-methylpyridinium iodide) was used for comparison as the currently used AChE reactivator. Purities of the tested AChE reactivators were confirmed using TLC (DC-Alufolien Cellulose F; Merck, Germany; mobile phase BuOH-CH₃COOH-H₂O 5: 1: 2; detection by solution of Dragendorff reagent) and by NMR (Varian Gemini 300; ¹H 300 MHz, ¹³C 75 MHz, Palo Alto CA, USA) [11,14]. The nerve agent cyclosarin (GF; O-cyclohexylmethylflourophosphate) was obtained from the Military Facility Brno (97% purity). All other chemicals used were of reagent grade (Sigma-Aldrich, Czech Republic).

Table I.  Potency of newly developed acetylcholinesterase reactivators to reactivate cyclosarin-inhibited rat brain cholinesterases in vitro.

Name	R	Oxime position	Reactivation [%] (10^− 5 M)	Reactivation [%] (10^− 3 M)
Pralidoxime	–	2	0	0
K106		2,2	0	0
K109		3,3	3	9
K112		4,4	8	0
K107		2,2	29	0
K110		3,3	8	4
K113		4,4	7	0
K108		2,2	21	0
K111		3,3	2	0
K114		4,4	9	0

Source of the enzyme – rat brain homogenate; time of inhibition – 30 min; time of reactivation – 10 min, reactivation measured at two different reactivator concentrations – 0.00001 M and 0.001 M; pH 7.6; 25 °C

Enzyme

Rat brain cholinesterase was chosen as an appropriate source of cholinesterases. Its preparation was as follows. Lightly ether-narcotized animals were killed by bleeding from a carotid artery and then the brains were removed, washed with saline and homogenized using an Ultra-Turrax homogenizer in distilled water to make a 10% homogenate (w/v). The animals used in this study were handled under the supervision of the Ethics Committee of the Medical Faculty of Charles University and the Faculty of Military Medical Academy in Hradec Kralove, Czech Republic.

In vitro measurement

Reactivation efficacy of the newly developed oximes was tested in vitro on the model of cholinesterases inhibited by cyclosarin using the standard reactivation test with electrometric instrumentation [15]. The rat brain homogenate (0.5 ml) was mixed with 0.5 ml of 0.01 μM cyclosarin solution in dry isopropanol and then incubated for 30 min at room temperature to reach 95% inhibition. Then the cyclosarin-inhibited AChE was incubated for 10 min with a solution of reactivator (1 ml) of given concentration (10^{− 3} M or 10^{− 5} M). Afterwards, 2.5 ml of 3M NaCl was added and the mixture diluted with distilled water to a volume of 23 ml. Then, 2 ml of 0.02 M acetylcholine bromide was added and enzyme activity was measured titrimetrically at pH 8.0 and 25 °C on an Autotitrator RTS 822 (Radiometer, Denmark).

The activities of intact (a_o), cyclosarin-inhibited (a_i) and reactivated AChE (a_r) were calculated from the slopes of the initial part of the titration curves. Each value represents the arithmetic mean from two independent measurements.

The percentage of reactivation (%) was calculated from measured data according to the formula:

Results

All results and the new AChE reactivators structures are summarized in Table I. Visualization of the obtained data is shown in Figure 1. As can be clearly seen, pralidoxime is not effective in the case of cyclosarin-inhibited AChE reactivation for both reactivator concentrations 10^{− 5} M and 10^{− 3} M. On the contrary, there are several new AChE reactivators with promising efficacy to reactivate cyclosarin-inhibited AChE; especially reactivators K107 and K108 which reached a very good reactivation potency (over 15% at concentration 10^{− 5} M), which is believed to save life in the case of severe intoxications[1,16]. Other reactivators tested were able to reactivate AChE at the human safe concentration of 10^{− 5} M just up to 10% (K112, K110, K113, and K114) or were ineffective (K106, K109 and K111) like pralidoxime.

Figure 1 Efficacy of tested oximes in reactivation of cyclosarin-inhibited AChE.

Discussion

Development of new AChE reactivators is still a very important aim, due to the fact that there is no single reactivator able to reactivate all nerve agents regardless of their structure, and many laboratories throughout the world are interested in this area of research8-1017–20. Pralidoxime, an AChE reactivator currently established in the USA, is generaly designated as the gold standard AChE reactivator [13], however, its potency to reactivate almost all nerve agent intoxications is poor 21-23. Our results confirm that pralidoxime is ineffective in the case of cyclosarin intoxications. For these purposes, new AChE reactivators are currently being synthesized 8-10.

All new AChE reactivators presented in this article were synthesized in 2005. Firstly, we have tested their in vitro potency to reactivate AChE inhibited by the pesticide chlorpyrifos and almost all these AChE reactivators (except K109, K110 and K111) had promising reactivation potency [11]. On the contrary, our new findings for the real nerve agent cyclosarin inhibition show that only two AChE reactivators (K107 and K108) seem to be promising. Moreover, their promising reactivation potency was reached at a concentration of 10^{− 5} M, which is believed to be attainable in human [1,16]. Higher reactivation potency at a lower concentration of AChE reactivators (10^{− 5} M) is caused, as was described earlier in this journal, by inhibition of reactivated AChE by the reactivator itself [11].

Generally, the presence of the xylene moiety in the reactivator's structure increases the lipophilicity in comparison with currently used AChE reactivators. Possibly due to this fact, these reactivators could theoretically penetrate the human blood brain barrier (BBB) in higher amount than pralidoxime, which goes through the BBB to the extent of 10% [24].

From the structural point of view, the only promising reactivators (K107 and K108) have a functional oxime group in position-2. This result is in very good agreement with the general rule that cyclosarin-inhibited AChE is preferably reactivated by reactivators with an oxime group in position-2 of the pyridinium ring [7,25].

The third compound (K106), having an oxime group in position-2 of the pyridinium ring, was not potent due to its steric properties; the ortho-xylene linking chain probably does not enable attack on the phosphorus atom of the cyclosarin connected to the enzyme.

In conclusion, synthesis of new AChE reactivators and subsequent evaluation of their in vitro potency to reactivate nerve agent-inhibited AChE are generally first steps in the development of new AChE reactivators [26]. The obtained results shows that two AChE reactivators (K107 and K108) seem to be promising AChE reactivators which should be tested in detail using other aspects of our development process such as in vivo and neurotoxicity studies 27-29.

Acknowledgement

This study was supported by the grant of Ministry of Defense No. ONVLAJEP 20031 and by a grant from the Grant Agency of Charles University No. 302/2005/B-CH/FaF.