Chemico-Biological Interactions 175 (2008) 173–179

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Chemico-Biological Interactions

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Oximes: Reactivators of phosphorylated acetylcholinesterase andantidotes in therapy against tabun poisoning

Zrinka Kovarik ∗, Maja Calic, Goran Sinko, Anita Bosak,Suzana Berend, Ana Lucic Vrdoljak, Bozica RadicInstitute for Medical Research and Occupational Health, Ksaverska c. 2,POB 291, HR-10001 Zagreb, Croatia

a r t i c l e i n f o

Article history:Available online 23 May 2008

Keywords:AntidoteBio-scavengerButyrylcholinesteraseNerve agentsPretreatment

a b s t r a c t

One of the therapeutic approaches to organophosphate poisoning is to reactivate AChEwith site-directed nucleophiles such as oximes. However, pyridinium oximes 2-PAM, HI-6,TMB-4 and obidoxime, found as the most effective reactivators, have limiting reactivatingpotency in tabun poisoning. We tested oximes varying in the type of ring (pyridinium and/orimidazolium), the length and type of the linker between rings, and in the position of theoxime group on the ring to find more effective oximes to reactivate tabun-inhibited humanerythrocyte AChE. Three of our tested pyridinium oximes K027, K048, K074, along withTMB-4, were the most promising for AChE reactivation. Promising oximes were furthertested in vivo on tabun poisoned mice not only as antidotes in combination with atropinebut also as pretreatment drug. Herein, we showed that a promising treatment in tabunpoisoning by selected oximes and atropine could be improved if oximes are also used in

pretreatment. Since the reactivating efficacy of the oximes in vitro corresponded to theirtherapeutic efficacy in vivo, it seems that pharmacological effect of these oximes is indeed

o the re

1

coiooteacai

0

primarily related t

. Introduction

Oximes have been investigated for many years asompounds with a great potential in the treatment ofrganophosphorus compounds (OPs) poisoning includingnsecticides and nerve agents [1]. Yet, to the present time,nly four pyridinium oximes, TMB-4, 2-PAM, HI-6 andbidoxime, have found clinical application [2,3]. However,reatment with these oximes still has disadvantages, inxample, they all have limited reactivation potency in nerve

gent tabun poisoning and some have pronounced toxi-ity [3,4]. Increased threat of the abuse of nerve agentsnd pesticides during military conflicts [5] and by terror-sts [6] creates an obligation for researches to search for

∗ Corresponding author. Tel.: +385 1 46 73 188; fax: +385 1 46 73 303.E-mail address: zrinka.kovarik@imi.hr (Z. Kovarik).

009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.cbi.2008.04.011

activation of tabun-phosphorylated AChE.© 2008 Elsevier Ireland Ltd. All rights reserved.

new, more effective oximes and treatment regimen againstthese poisonings.

Recently, we tested oximes varying in the type of ring(pyridinium and/or imidazolium), the length and type ofthe linker between rings, and in the position of the oximegroup on the ring to find more effective oximes to reactivatetabun-inhibited human erythrocyte acetylcholinesterase(AChE; EC 3.1.1.7) [7–10]. Promising oximes were furthertested in vivo on tabun poisoned mice not only as antidotesin combination with atropine but also as pretreatmentdrug [7,8]. We also investigated interactions of promisingoximes with human plasma butyrylcholinesterase (BChE;EC 3.1.1.8) because in vivo interactions with this endogenous

bio-scavenger could influence the result of applied ther-apy [8]. In this paper, we are compiling our previous resultsfrom in vitro and in vivo experiments with newly introducedoxime K074 [7–10]. Obtained results with K074 were com-pared with the most potent reactivators from our previous

gical In

concentration. The overall second-order rate constant ofreactivation (kr) is the ratio

kr = k+2

KOX(2)

174 Z. Kovarik et al. / Chemico-Biolo

studies, K027 and K048, and with TMB-4, currently usedoxime.

2. Materials and methods

2.1. Chemicals

Previously tested oximes were: HI-6 [1-(((4-carb-amoylpyridinium-1-yl)methoxy)methyl)-2-((hydroxyimi-no)methyl)pyridinium chloride], TMB-4 [N,N′-(propano)-bis(4-hydroxyiminomethyl)pyridinium bromide], DMB-4[N,N′-(ethano)bis(4-hydroxyiminomethyl)pyridiniummethano sulphonate], MMB-4 [N,N′-(methano)bis(4-hydroxyiminomethyl)pyridinium chloride], HLo-7 [1-(((4-carbamoylpyridinium-1-yl)methoxy)methyl)-2-((hy-droxyimino)methyl)-4-(hydroxyimino)methyl)pyridini-um) methane sulfonate], ICD-585 [1-(3-(4-carbamoylp-yridinium-1-yl)propyl)-2-((hydroxyimino)methyl)pyridi-nium chloride], ICD-692 [4-carbamoyl-1-(3-(2-((hyd-roxyimino)methyl)-1-methyl-1H-imidazol-3-ium-3-yl)-propyl)pyridinium chloride], ICD-467 [2-((hydroxyim-ino)methyl)-1-methyl-3-((3-methyl-3-nitrobutan-2-ylo-xy)methyl)-1H-imidazol-3-ium chloride], K033 [1,4-bis-(2-hydroxyiminomethylpyridinium) butane dibromide],K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carb-amoylpyridinium) propane dibromide], K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridi-nium) butane dibromide]. In this paper, we introducedoxime K074 [1,1′-(butane-1,4-diyl)bis(4-(hydroxyl-imin-omethyl) pyridinium) dibromide].

Oximes ICD-585, HLo-7, MMB-4, DMB-4, ICD-692 andICD-467 were obtained from US Army Medical ResearchInstitute of Chemical Defense, Aberdeen Proving Ground,MD, USA. Oximes K027, K033, K048 and K074 were pro-vided by Dr Kamil Kuca (Department of Toxicology, Facultyof Military Health Sciences, Hradec Kralove, Czech Repub-lic). HI-6 was synthesized in the Department for OrganicChemistry, Faculty of Science, University of Zagreb, Croatia,and TMB-4 was obtained from Bosnalijek, Sarajevo, Bosniaand Herzegovina. Oximes were dissolved in water justbefore use. Tabun [ethyl N,N-dimethylphosphoroamido-cyanidate] was purchased from NC Laboratory, Spiez,Switzerland. Tabun was diluted in isopropyl alcoholand further dilutions in water were made before use.Enzyme substrate acetylthiocholine iodide (ATCh) andassay reagent 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)were purchased from Sigma Chemical Co., St. Louis, MO,USA. All experiments were done in 0.1 M sodium phosphatebuffer, pH 7.4, at 25 ◦C. Atropine sulphate was purchasedfrom Kemika, Zagreb, Croatia. All chemicals used were ofanalytical grade.

2.2. Cholinesterase activity measurement andconformational analysis of oximes

Native non-heamolyzed human erythrocytes were the

source of AChE. Erythrocytes were obtained from hep-arinized blood after centrifugation (20 min, 2500 rpm) andwashed three times by 0.1 M sodium phosphate buffer (pH7.4) to remove residual plasma. The source of BChE (usualphenotype) was native human plasma obtained from blood

teractions 175 (2008) 173–179

as supernatant after centrifugation. Final dilution of ery-throcytes in experiments was 400-fold and plasma dilutionwas 100–300-fold.

The enzyme activity was measured spectrophotometri-cally according to the Ellman procedure [11] at 25 ◦C, withthe thiol reagent DTNB (0.3 mM final concentration) andATCh as substrate. The increase in absorbance was read at436 nm up to 2 min. All spectrophotometric measurementswere performed on a CARY 300 spectrophotometer (VarianInc., Australia) with temperature controller.

Conformational analysis was performed using MM2 andsemi-empirical calculations with MOPAC 2000 software(PM-3 method with COSMO solvation model) created byJ.J.P. Stewart (Fujitsu Ltd., Tokyo, Japan, 1999) according topublished procedures [12,13].

2.3. Reactivation of tabun-inhibited ChE

Undiluted erythrocytes, or plasma, were incubated with5 �M tabun up to 60 min achieving 90–100% inhibition.Incubation mixture was diluted 5–10 times with 0.1 Mphosphate buffer, pH 7.4, containing the oxime to start thereactivation. After a given time of reactivation aliquots werediluted 40 times, DTNB and ATCh (1.0 mM final concentra-tion) were added, and the enzyme activity was measured.An equivalent sample of uninhibited enzyme was diluted tothe same extent as the inhibited AChE, and control activitywas measured in the presence of oxime at concentrationsused for reactivation. Both activities of control and reactiva-tion mixture were corrected for oxime-induced hydrolysisof ATCh. No spontaneous reactivation of the phosphory-lated enzyme took place.

Oxime-assisted reactivation of phosphorylated ChEproceeds according to Scheme 1: where EP is the phos-phorylated enzyme, [EP][OX] is the reversible Michaelistype complex between EP and the oxime (OX), E is theactive enzyme and P-OX the phosphorylated oxime, k+2 isthe maximum first-order rate constant and kr is the over-all second-order rate constant of reactivation. Scheme 1 isdefined by the following equation:

ln[EP]0

[EP]t= k+2[OX]

KOX + [OX]t = kobst (1)

where [EP]0 and [EP]t are concentrations of phosphorylatedenzyme at time zero and at time t, respectively. KOX is equalto the ratio (k−1 + k+2)/k+1, and it approximates the dissocia-tion constant of the [EP][OX] complex. kobs is the observedfirst-order rate constant of reactivation at a given oxime

Scheme 1.

gical In

Ev

R

wtb1(ttElw

2

isctt

oa

K

Kaeieaac

2

wstcptf

(

l

wtbi

P

Z. Kovarik et al. / Chemico-Biolo

xperimental data were presented as percentage of reacti-ation

eactivation (%) = v(EP+OX)t

v(E+OX)× 100 (3)

here v(EP+OX)tis activity of reactivated enzyme at

ime t and v(E+OX) stands for activity of enzyme incu-ated with oxime. Since (100 − Reactivation) is equal to00·[EP]t/[EP]0, one can relate the experimental data to Eq.1). At each oxime concentration, kobs was calculated fromhe slope of the initial portion of log (100 − Reactivation) vs.ime of reactivation. Reactivation with each oxime followedq. (1). Therefore, k+2 and KOX were obtained by the non-inear fit of the relationship between kobs vs. [OX], while kr

as calculated using Eq. (2).

.4. Reversible inhibition of ChE

Reversible inhibition of ChE by oxime was measuredn a medium which contained erythrocytes or plasmauspended in buffer, DTNB, the oxime and ATCh. The con-entrations of the oxime and substrate were chosen so thathe oxime-induced ATCh hydrolysis does not interfere withhe studied interactions [14,15].

The inhibition constants were evaluated from the effectf substrate concentration (s) on the degree of inhibitionccording to the equation:

app = vi[OX]v0 − vi

= Ki + Ki

Ks[S] (4)

app is the apparent enzyme–oxime dissociation constantt a given substrate concentration (S), calculated from thenzyme activities v0 and v measured in the absence andn the presence of the oxime (OX), respectively. Ki is thenzyme–oxime dissociation constant of a complex formedt the catalytic site. Ks is the enzyme–substrate dissoci-tion constant which should correspond to the Michaelisonstant, if oxime binds only to the catalytic site [7].

.5. Protection of AChE against phosphorylation

Protection of AChE against phosphorylation by tabunas measured in a medium which contained erythrocytes

uspended in 0.1 M phosphate buffer, DTNB, the oxime, andabun (100 nM). The concentration of oxime for protectionorresponded to its determined Ki. After a given time ofhosphorylation (up to 5 min) ATCh (1.0 mM final concen-ration) was added and the increase in absorbance was reador 1 min. Control samples contained no oxime.

The second-order rate constant of inhibition by tabunki) was calculated from the equation:

nv0

vOP= ki[OP]t (5)

here vOP and v0 are enzyme activities with and withoutabun, OP is the concentration of tabun and t is time of inhi-

ition. Protection was expressed in terms of the protective

ndex (PI) which corresponds to the ratio:

I = ki

k′i

(6)

teractions 175 (2008) 173–179 175

The second-order rate constant of inhibition in the presenceof the oxime (k

′i) was calculated using the Eq. (5) where v0

and vOP denote the enzyme activities in absence of both theoxime and tabun, and in their presence, respectively.

Protective index was also determined theoreticallyfrom:

PItheor = 1 + [OX]Ki

(7)

where OX stands for oxime concentration, and Ki is theexperimentally determined enzyme–oxime dissociationconstant if oxime binds to the enzyme catalytic site [16].

2.6. Animals

Male BALB-C mice (purchased from Agency for Medic-inal Products and Medical Devices Zagreb, Croatia) wereselected by body weight (18–25 g), fed on a standard diet(Pliva, Zagreb, Croatia) and had free access to water. Micewere kept in Macrolone cages at 21 ◦C maintained by a ther-mostat, with exchanging light and dark cycles every 12 h,and were randomly distributed in groups of four animals.

2.7. Pretreatment/therapy by atropine and oxime againsttabun poisoning

Mice received a subcutaneous (s.c.) dose of 1.0–50.0multiples of the tabun LD50 (356.6 �g/kg body weight; [7]).Oxime was dissolved in water just before use and givenintraperitoneally (i.p.) at doses of 25% or 5% of its LD50[7]. Atropine was dissolved in water (5.0 mg/ml) and alsoadministered i.p. (10.0 mg/kg body weight).

Acute toxicity of K074 was determined herein and itwas based upon 24 h mortality rates determined accord-ing to Thompson [17] and Weil [18]. LD50 was evaluatedfrom the results obtained with four doses of K074 (dis-solved in water); four animals were injected per dose. The95% confidence limits were evaluated from tables describedelsewhere [17,18].

The therapeutic efficacy of antidote (oxime) was testedas pretreatment given 15 min before tabun poisoningand/or as therapy (oxime with atropine) given 1 minafter tabun poisoning. We chose 15 min before poisoningbecause a recent study has shown that it takes 15 minfor K048 to reach its peak plasma concentration followingi.p. administration [19]. Since K074, used for pretreat-ment, is similar to K048, we related these findings to ourexperiments. The efficacy of antidote was expressed astherapeutic factor (TF) with 95% confidence limits, andtherapeutic dose (TD). TF was the ratio between the LD50of tabun with and without treatment. TD was the highestmultiple of the LD50 of tabun, which was fully counteracted(survival of all animals) by the administered treatment.

This study was performed with the approval of the Eth-ical Committee of the Institute for Medical Research andOccupational Health in Zagreb, Croatia.

3. Results and discussion

Oximes differing in a ring type (imidazolium vs. pyri-dinium), length of the linker between rings and the

176 Z. Kovarik et al. / Chemico-Biological Interactions 175 (2008) 173–179

re the m

should not be obstructed if K027, K048, K074 are applied asantidotes, however possible interaction of phosphorylatedBChE with TMB-4 could influence its concentration in theblood circulation.

Fig. 1. Minimised structure of tested oximes that we

position of the oxime group/s on the ring/s were evalu-ated as reactivators of tabun-inhibited human erythrocyteAChE [7,10]. High percentage of reactivation achieved ina short time was the main criteria for selection amongthe tested oximes. From all tested oximes, only pyri-dinium oximes TMB-4 (bis-oxime, propane-like linker),K027 (mono-oxime, propane-like linker), K048 (mono-oxime, butane-like linker), along with, in this paperintroduced, K074 (bis-oxime, butane-like linker), had theability to efficiently reactivate AChE (Fig. 1) [7]. Their over-all reactivation rate constants (kr) were 306, 376, 673 and2375 M−1 min−1, respectively [7]. The difference betweenthese oximes was observed in both, the affinity of the phos-phorylated AChE for the oxime (1/KOX) and the rate of thenuchleophilic displacement of the phosphoryl-moiety fromthe active site serine (kmax) (Fig. 2a). Maximal AChE reacti-vation rate constant, kmax, of K074 was two and four timeslower than that of K048 and TMB-4, respectively [7]. But,as it can be seen, K074 was the most efficient oxime atthe lowest concentration applied (Fig. 2b). Apparently, inthe case of K074, a high binding affinity of phosphorylatedAChE for K074 (the lowest KOX of 0.016 mM) dictates a highefficiency in the reactivation of tabun-inhibited AChE. How-ever, K074-assisted reactivation did not completely restoreenzyme activity as did TMB-4, K048 or K027. With K074,reactivation maximum of 90% was obtained in 80 min, andthe kinetics at longer time intervals deviated from the first-order process shown in one representative experiment inFig. 3. This deviation could be a sign of post-inhibitorydealkylation of phosphorylated enzyme known as aging.The other reason could be the re-inhibition of reactivatedenzyme by the phosphorylated K074, if the conjugate is sta-ble. In the literature, there are no data on the stability orinhibitory potency of K074-tabun conjugate. However, it isknown that conjugates of tabun and monopyridinium para-oxime 4-PAM or ortho-oxime 2-PAM are poor inhibitors ofAChE, which is attributed to a combination of steric factorsand a reduction in the electro-positivity of the phosphorusatom [20].

Since in vivo interactions of oxime with BChE, endoge-nous bio-scavenger, could influence the result of applied

therapy, we evaluated reactivation of tabun-inhibited BChEwith K074. Results were compared with the oximes fromour previous studies, K027 and K048, and with TMB-4 [8].Overall reactivation rate constants (kr) were from around5 M−1min−1 for K027, K048, K074 and slightly higher,

ost efficient in reactivation of tabun-inhibited AChE.

14 M−1min−1, for TMB-4 [8]. None of the oximes displayeda significant reactivation of tabun-inhibited BChE. It tookthem up to 20 h to reach the maximum of 60–70%. Since themaximal BChE reactivation rate constants were similar forall oximes (kmax = 0.003 min−1) the reason for higher rateobtained in the case of TMB-4 lies in the higher affinity ofphosphorylated BChE for this oxime [8]. Interestingly, theaffinity of phosphorylated BChE was only for TMB-4 higherthan of phosphorylated AChE [8]. From this we can con-clude that in vivo fast reactivation of phosphorylated AChE

Fig. 2. (a) Maximal obtained tabun-inhibited AChE reactivation rate con-stant kmax, for the most efficient oximes and comparison of affinities ofnative (1/KOX) and phosphorylated AChE (1/Ki) for these oximes. (b) Reacti-vation (%) obtained after 30 min with different concentrations of the mostefficient oximes.

Z. Kovarik et al. / Chemico-Biological In

Fig. 3. Oxime K074-assisted reactivation of tabun-inhibited human ery-tdto

assosoiiptRpb

K074, was 21.4 mg/kg body weight. With LD this low K074

hrocyte AChE. (a) A single data point indicates reactivation (%) afteresignated time of reactivation with K074. (b) Slopes of the initial part ofhe reactivation curve yield kobs constants. (c) kobs is plotted as a functionf K074 wherefrom reactivation constants were determined.

The important oxime characteristic that we evaluateds well was the overall flexibility of the oxime molecule,ince it determines how well the oxime will span the activeite of the enzyme and will it gain a position for the nucle-philic attack on the phosphorylated serine. Minimisedtructure of para-oximes and the spatial arrangement of thexime groups bonded to the pyridinium ring is presentedn Fig. 1. Conformational analysis showed that oxime K074s the most flexible oxime out of all oximes that we testedreviously [10] due to a low rotation barrier of bonds in

he linker connecting two pyridinium rings (12.9 kcal/mol).otation barrier of the bond between oxime group andyridinium ring of K074 was 6.0 kcal/mol, while of theond between linker and nitrogen in the pyridinium ring

teractions 175 (2008) 173–179 177

was 0.86 kcal/mol that corresponded to energies calculatedfor the other para-pyridinium oximes [9,14]. Generally,aldoximes with the oxime group in the ortho-position ICD-585, HI-6, K033, ICD-692 and ICD-467 were less flexiblethan those with the oxime group in the para-position K027,K048, TMB-4, DMB-4 and MMB-4 due to a higher rotationbarrier of the pyridinium/imidazolium ring [9,14].

Since oximes are also reversible inhibitors ofcholinesterases we determined dissociation constantsof enzyme–oxime complex (Ki) from the degree of enzymeinhibition in the presence of different substrate concentra-tions [7,10]. The Ki constants for binding of K027, K048 andTMB-4 to AChE ranged from 0.07 to 0.18 mM [7], however,compared to these oximes, the affinity of AChE for K074was up to seven times higher (Ki = 0.024 mM) (Fig. 2a). Atstudied substrate acetylthiocholine concentration range,reversible inhibition by K074 was competitive as it waswith similar oximes tested previously [7,10]. Possiblebinding of oxime K074 to the peripheral site could not bestudied, because at higher acetylthiocholine concentra-tions oxime-induced the hydrolysis of acetylthiocholine ata rate which interfered with the studied inhibition [14,15].The binding affinity of K074 to the phosphorylated AChEwas slightly higher than that to the free enzyme (Fig. 2a).Usually, phosphorylated AChE has a lower affinity foroxime binding than native AChE [9,21–23]. In the caseof BChE reversible inhibition, enzyme-K074 dissociationconstant, Ki, was 0.13 mM which indicates its preferencefor binding to the free AChE (for AChE was 0.024 mM). Thesame preference for native AChE was also observed forsimilar, previously tested, pyridinium oximes, K048 andK027 [8]. The exception was TMB-4 which had a similaraffinity for both enzymes (Ki ≈ 0.20mM) [7].

Pyridinium compounds also exhibit their protectiveeffect against phosphorylation by organophosphorus com-pounds by reversibly binding to the AChE active siteinterfering with the binding of the phosphorylating agents[16]. The ratio of the rate constant of phosphorylation of theenzyme by organophosphorus compound in the absenceand in the presence of oxime (protective index) is importantparameter for oxime treatment especially when reactiva-tion is slow or aging is very fast. Therefore, we measuredprogressive inhibition of AChE by tabun in the absenceand in the presence of K074 in concentration correspond-ing to its Ki (0.02 mM). Similar to other tested oximes,K074 decreased the rate of the inhibition by tabun abouttwo times and in that way protected AChE from phos-phorylation. Since the experimentally obtained protectiveindex corresponded to the theoretically calculated protec-tive index (by Eq. (7)) we concluded that K074, as well asother oximes, protected the enzyme primarily by bindingto the catalytic site [10].

Promising in vitro results led us to test a treatment reg-imen in vivo, in mice, against tabun poisoning where K074was applied not only as a therapy but also as a pretreatmentin a dose of 25% and 5% of its LD50. Acute toxicity of oxime,

50ranks as a very toxic oxime to mice (3.5 times more thanTMB-4 and 11 times more than K048; Ref. [7]).

Therapeutic effect against tabun poisoning was firsttested by giving the studied oxime, K074, together with

178 Z. Kovarik et al. / Chemico-Biological Interactions 175 (2008) 173–179

Table 1Therapeutic effect of the oxime K074a given as treatment with atropine and as pretreatment upon tabun toxicityb in male mice

Pretreatment: 15 min before tabun Therapy: 1 min after tabun LD50 (�g/kg) 95% confidence limits (�g/kg) TF TD

None 25% K074 + atropine 2853 1227–6636 8.0 5.0None 5% K074 + atropine 2016 1764–2304 6.4 5.025% K074 None 566.1 474–676 1.6 1.35% K074 None 423.8 355–505 1.3 1.025% K074 Atropine 1425 565–3592 4.0 2.55% K074 Atropine 565.7 294–1088 1.6 1.325% K074 25% K074 + atropine 4493 3760–5368 12.6 10.05% K074 5% K074 + atropine 1604 1210–2124 5.0 3.2

a i.p. LD50 of K074 was 21.4 mg/kg.b s.c. LD50 of tabun was 356.6 �g/kg [7].

atropine only as treatment immediately after tabun poison-ing. When K074 was applied, animals survived fivefold LD50of tabun (Table 1). The same was observed in the case of themost efficient oxime from our previous studies, K048 [7].The combined antidotal therapy (pretreatment/treatment)of tabun-poisoned mice with 25% of LD50 of K074, resultedwith a high therapeutic dose, TD = 10 (Table 1). How-ever, the therapeutic factor, TF, of this regimen was 12.6,which is only the half of TF obtained with K048 [8].Pretreatment/treatment by K048 resulted with a consid-erable increase in protection of mice compared to oximetreatment alone (TF and TD 22.5 and 10.0, respectively)[8]. On the other hand, pretreatment with 5% of K074or K048 LD50 and therapy following poisoning failed todecrease tabun-induced acute toxicity in mice [8]. It seemsthat a substantial concentration of a potent reactivator-oxime needs to be applied in the treatment to overcomesuch, probably, synergistic effect of tabun, oxime andatropine.

Since the therapeutic efficacy of the oximes in vivocorresponded to their reactivating efficacy in vitro, itseems that pharmacological effect of these oximes isindeed primarily related to the reactivation of tabun-phosphorylated AChE. Whatever are the shortcomings ofusing the reversible inhibitors as protectors, we have shownthat already promising treatment in tabun poisoning byoximes and atropine could be significantly improved ifoximes are also used in pretreatment. Promising oxime mayprovide a platform for further modifications and devel-opment of more potent protectors and reactivators inorganophosphorus poisoning.

Acknowledgement

We wish to thank Dr. Irwin Koplovitz (US Army MedicalResearch Institute of Chemical Defense, Aberdeen ProvingGround, MD, USA) and Professor Palmer Taylor (Universityof California at San Diego, La Jolla, CA, USA) for supplying uswith oximes ICD-585, HLo-7, MMB-4, DMB-4, ICD-692 andICD-467. We wish to thank Dr. Kamil Kuca (Faculty of Mil-itary Health Sciences, Hradec Kralove, Czech Republic) for

preparing and supplying the oximes K027, K033, K048 andK074. This work was supported by the NATO ReintegrationGrant (EAP.RIG.981791) and by the Croatian Ministry of Sci-ence, Education and Sports Grants (No. 022-0222148-2889to Z.K. and No. 022-0222148-2139 to B.R.).

[

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