Toxicology and Applied Pharmacology 249 (2010) 231–237

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Toxicology and Applied Pharmacology

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Reactivation of organophosphate-inhibited human, Cynomolgus monkey, swine andguinea pig acetylcholinesterase by MMB-4: A modified kinetic approach

Franz Worek a,⁎, Timo Wille a, Nadine Aurbek a, Peter Eyer b, Horst Thiermann a

a Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germanyb Walther-Straub-Institute of Pharmacology and Toxicology, Ludwig-Maximilians-University, Goethestrasse 33, 80336 Munich, Germany

⁎ Corresponding author. Fax: +49 89 3168 2333.E-mail address: franzworek@bundeswehr.org (F. Wo

0041-008X/$ – see front matter © 2010 Elsevier Inc. Aldoi:10.1016/j.taap.2010.09.021

a b s t r a c t

a r t i c l e i n f o

Article history:Received 26 August 2010Revised 20 September 2010Accepted 23 September 2010Available online 1 October 2010

Keywords:MMB-4Nerve agentsAcetylcholinesteraseReactivationKineticsSpecies differences

Treatment of poisoning by highly toxic organophosphorus compounds (OP, nerve agents) is a continuouschallenge. Standard treatmentwith atropine and a clinically used oxime, obidoxime or pralidoxime is inadequateagainst various nerve agents. For ethical reasons testing of oxime efficacy has to be performed in animals. Now, itwas tempting to investigate the reactivation kinetics of MMB-4, a candidate oxime to replace pralidoxime, withnerve agent-inhibited acetylcholinesterase (AChE) from human and animal origin in order to provide a kineticbasis for the proper assessment of in vivo data. By applying a modified kinetic approach, allowing the use ofnecessary high MMB-4 concentrations, it was possible to determine the reactivation constants with sarin-,cyclosarin-, VX-, VR- and tabun-inhibited AChE. MMB-4 exhibited a high reactivity and low affinity towards OP-inhibited AChE, except of tabun-inhibited enzyme where MMB-4 had an extremely low reactivity. Speciesdifferences between human and animal AChE were low (Cynomolgus) to moderate (swine, guinea pig). Due tothe high reactivity of MMB-4 a rapid reactivation of inhibited AChE can be anticipated at adequate oximeconcentrations which are substantially higher compared to HI-6. Additional studies are necessary to determinethe in vivo toxicity, tolerability and pharmacokinetics of MMB-4 in humans in order to enable a properassessment of the value of this oxime as an antidote against nerve agent poisoning.

rek).

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© 2010 Elsevier Inc. All rights reserved.

Introduction

Treatment of poisoning by highly toxic organophosphorus com-pounds (OP, nerve agents) is still a challenging task (Worek et al.,2007b). Despite of research on treatment options since more than sixdecades the initial treatment of OP poisoning did not experiencemuch improvement. Atropine is used as basic, symptomatic antidotesupplemented by an oxime (Cannard, 2006). At present, pralidoxime(2-PAM) and obidoxime are the most important oximes used ascausal treatment of human OP poisoning.

Numerous in vitro and in vivo studies demonstrated that theseoximes have limited effectiveness in case of poisoning by variousnerve agents, e.g. soman, tabun and cyclosarin (Eyer and Worek,2007). This fact prompted an ongoing effort to develop more effectiveand broad-spectrum oximes and a huge number of compounds havebeen synthesized and tested in the past decades (Worek et al., 2007b).

Presently, Canada and several European countries are going tolicense the bispyridinium oxime HI-6 as OP antidote (Lundy et al.,2006) while the bispyridinium bis-oxime MMB-4 is under advanceddevelopment by the US Army to replace 2-PAM as nerve agentantidote (Luo et al., 2008).

The ability of MMB-4 (Fig. 1) to reactivate OP-inhibited AChE andto protect animals from lethal OP effects was investigated first byHobbiger et al. (1960) and Hobbiger and Sadler (1959) some 50 yearsago and several reports on this oxime were published in the followingdecades (Bajgar et al., 1975; Harris et al., 1990; Luo et al., 2008, 2007;Bartling et al., 2007; Shih et al., 2009). Experimental data indicate thatMMB-4 is superior to 2-PAM in reactivating OP-inhibited AChE and inpreventing lethality in OP poisoned animals.

In view of the potential benefit of using MMB-4 it was tempting toperform a detailed kinetic study in order to evaluate the reactivatingefficiency of MMB-4 with nerve agent-inhibited AChE. In the presentstudy, human, Cynomolgus monkey, swine and guinea pig AChEinhibited by sarin, cyclosarin, tabun, VX and VRwas used to determinethe reactivation kinetics of MMB-4. With this effort a database on thekinetic properties of MMB-4 should be generated enabling theevaluation of potential species differences and the proper assessmentof experimental animal data for the extrapolation to humans.

Materials and methods

Materials. Sarin (GB), tabun (GA), cyclosarin (GF), VX and VR (N98%by GC-MS, 1H NMR and 31P NMR; Fig. 1) were made available by theGerman Ministry of Defence. MMB-4 dichloride (Fig. 1) was kindlyprovided by Prof. Fusek (Purkyne Military Medical Academy, HradecKralove, Czech Republic). Acetylthiocholine iodide (ATCh) and 5,5′-

P

O

CH3O

FCH3

CH3

P

O

CH3O

F

P

O

CH3 OS

N

CH3

CH3

CH3

CH3

CH3

P

O

CH3 OS

N

CH3

CH3

CH3

CH3

Sarin (GB) Cyclosarin (GF)

VX VR

P

O

C ON

CH3CH3

CH3

N

Tabun (GA)

2 Cl-

MMB-4

NOH

N+

CH2

N+

NOH

Fig. 1. Chemical structure of nerve agents and MMB-4.

232 F. Worek et al. / Toxicology and Applied Pharmacology 249 (2010) 231–237

dithiobis(2-nitrobenzoic acid) (DTNB) were supplied by Sigma-Aldrich (Deisenhofen, Germany). All other chemicals were fromMerck Eurolab GmbH (Darmstadt, Germany) at the purest gradeavailable.

Nerve agent stock solutions (0.1% v/v) were prepared in acetonitrile(ACN) and were stored at 20 °C. MMB-4 (200 mM) was prepared indistilled water and stored at −80 °C. Nerve agents and MMB-4 werediluted as required in distilled water immediately before use.

All solutions were kept on ice until the experiment. If not otherwisestated the buffer consisted of 0.1 M sodium phosphate, pH 7.4.

Blood samples. Heparinized human, Cynomolgus monkey (kindlydonated by Dr. Guy Lallement, CRSSA, La Tronche, France) andGerman landrace pig (obtained from the local slaughterhouse) wholeblood as well as heparinized blood from Dunkin–Hartley guinea pigs(supplied by Charles River, Sulzfeld, Germany) was centrifuged at3000 rpm and 4 °C for 10 min, the plasma was collected and theerythrocytes were washed five times with a three-fold volumephosphate buffer.

The washed erythrocytes were used for preparation of hemoglo-bin-free erythrocyte ghosts as AChE source (Worek et al., 2002).Aliquots of the erythrocyte ghosts with an AChE activity adjusted tothat found in whole blood of the respective species were stored at−80 °C. Prior to use, aliquots were homogenized on ice with aSonoplus HD 2070 ultrasonic homogenator (Bandelin electronic,Berlin, Germany), three-times for 5 s with 30-s intervals, to achieve ahomogeneous matrix for the kinetic studies.

Determination of AChE activity. AChE activities were measuredwith a modified Ellman assay (Worek et al., 1999; Ellman et al.,1961; Eyer et al., 2003) on a Cary 3Bio spectrophotometer (Varian,Darmstadt, Germany) at 436 nm using polystyrol cuvettes and0.45 mM ATCh as substrate and 0.3 mM DTNB as chromogen in0.1 M phosphate buffer (pH 7.4).

All experiments were performed at 37 °C and pH 7.4. Allconcentrations refer to final concentrations.

Preparation of OP-inhibited AChE. Erythrocyte ghosts were incubat-ed with a small volume (≤1%, v/v) of appropriate OP concentrationsin distilled water (20–50 nM) for 15 min at 37 °C to achieve an AChEinhibition by N95%. Then, the treated ghosts were dialyzed against0.1 M phosphate buffer, pH 7.4, overnight at 4 °C to remove residualinhibitor. Finally, the absence of inhibitory activity was tested byincubation of treated and control ghosts (30 min, 37 °C) and aliquotswere stored at −80 °C until use.

Reactivation of OP-inhibited AChE. The reactivation rate constantsof MMB-4 were determined by a discontinuous procedure (Worek etal., 2004). Sixty parts OP-inhibited AChE were incubated with twoparts MMB-4 solution (10–5000 μM final concentration) and one partATCh (0.45 mM final concentration). Ten microliters (human andCynomolgus AChE) or 20-μl aliquots (swine and guinea pig AChE)were transferred after specified time intervals (1–30 min) to tem-pered cuvettes containing 3000 μl phosphate buffer and 100 μl DTNB.

Different oxime concentrations (8–15) were used for the deter-mination of the reactivation rate constants in duplicate experiments.AChE activities of OP-inhibited AChE and after time-dependentreactivation were referred to maximum reactivation, i.e. the maxi-mum AChE activity at the end of the observation period, which wasclose to control AChE activity in all cases.

Kinetics of oxime reactivation.

EP½ � + OX½ �⇌KD EPOX½ �→kr E½ � + POX½ � ðScheme 1Þ

The ability of oximes to reactivate OP-inhibited AChE can bequantified by the determination of reactivation rate constants.

Fig. 2. Time- and concentration-dependent reactivation of sarin-inhibited human AChEby MMB-4. Sarin-inhibited human AChE was incubated with 10–2000 μM MMB-4 (10,30, 50, 100, 150, 200, 300, 500, 1000, 1500 and 2000 μM) and AChE activity wasdetermined after 1–9 min. Data were analyzed by linear (A; method A) and non-linearregression (B; method B) to determine kobs. Plot of kobs vs. [MMB-4] enabled thecalculation of KD and kr (C).

233F. Worek et al. / Toxicology and Applied Pharmacology 249 (2010) 231–237

According to Scheme 1 oxime-induced reactivation of OP-inhibitedAChE may be described by a two-step reaction (Aldridge and Reiner,1972). In this scheme [EP] is the OP-inhibited AChE, [EPOX] theMichaelis-type OP-AChE-oxime complex, [OX] the oxime, [E] thereactivated enzyme and [POX] the phosphylated oxime. KD approx-imates the dissociation constant which is inversely proportional to theaffinity of the oxime to [EP], and kr the rate constant for thedisplacement of the OP residue from [EPOX] by the oxime, indicatingthe reactivity. The hybrid reactivation rate constant kr2 was calculatedfrom the ratio of kr and KD; the dimension resembles a second-orderrate constant.

In case of complete reactivation and with [Ox]N [EP]0 a pseudo-first-order rate equation can be derived for the reactivation processEq. (1):

kobs =kr⁎ OX½ �

KD + OX½ � ð1Þ

Initially, kobs values were calculated for each oxime concentrationby linear regression analysis (method A), applying Eq. (2)

lnv0−vtv0−vi

� �= −kobs⁎t ð2Þ

with vi=velocity of inhibited AChE, vt=velocity at time t andv0=maximum velocity (control).

Alternatively, kobs was calculated by non-linear regression analysis(method B) using Eq. (3)

vt = v0* 1−e−kobs⁎t� �

ð3Þ

Finally, kr and KD were obtained by the nonlinear fit of therelationship between kobs vs. [OX].

Data analysis. Processing of experimental data for the determina-tion of the kinetic constants was performed by non-linear regressionanalysis using curve fitting programs provided by Prism™ Vers. 4.03(GraphPad Software, San Diego, CA).

Results

Initially, the time- and concentration-dependent reactivation ofOP-inhibited AChE was analyzed by method A, i.e. linear regressionusing Eq. (2). However, the goodness of fit decreased with increasingMMB-4 concentrations. Fig. 2A exemplifies this for the reactivation ofsarin-inhibited human AChE with MMB-4 concentrations from 10 μM(R2 0.99) to 1500 μM(R2 0.73) due to progressive deviation of the datafrom a straight line (Fig. 2A). In addition, with rising MMB-4concentrations the linear regression did not intersect the originleading to a substantial increase of the Y-intercept.

Subsequent analysis of the data by non-linear regression analysis(method B) provided a markedly improved goodness of fit, especiallyat high MMB-4 concentrations, e.g. R2 0.99 at 2000 μM, again sarin-inhibited human AChE serving as an example (Fig. 2B).

The analysis of the data with methods A and B resulted in markeddifferences of the reactivation constants (Fig. 2C). The secondary plot,kobs vs. [MMB-4], provided kr and KD values of 0.44 min−1 and 306 μM(method A) and 1.87 min−1 and 1541 μM (method B). However, thecalculation of the hybrid reactivation rate constant kr2 from the ratioof kr and KD gave comparable values for methods A and B with1.43 mM−1 min−1 and 1.21 mM−1 min−1, respectively.

The reactivation rate constants determined with method A and Bwere used to calculate the theoretical reactivation of sarin-inhibitedAChE at different MMB-4 concentrations. Fig. 3 shows that despite ofremarkable differences of reactivation constants between both

methods the impact on reactivation was small at potentiallytherapeutic oxime concentrations.

The determination of the reactivation constants of sarin-, cyclo-sarin-, VX- and VR-inhibited AChE resulted in a low affinity, i.e. highKD, with AChE from all species (Table 1). The reactivity of MMB-4 wasvery high, krN1 min−1, in most cases. Only with cyclosarin-inhibitedswine AChE and cyclosarin- and VX-inhibited guinea pig AChE lowervalues were recorded. The low affinity of MMB-4 to OP-inhibitedAChE outweighed the high reactivity and resulted in rather low hybridreactivation rate constants kr2 (Table 1).

MMB-4 had a low reactivity and affinity towards tabun-inhibitedAChE with all species resulting in exceptionally low hybrid reactiva-tion rate constants kr2 (Table 1). With guinea pig AChE no reactivationconstants could be determined at the experimental conditions used inthis study due to negligible reactivation.

Species differences in the reactivation of OP-inhibited AChE byMMB-4 are exemplified by graphs showing the time- and concentra-tion-dependent reactivation of cyclosarin-inhibited AChE (Fig. 4). By

Fig. 3. Illustration of the concentration-dependent reactivation of sarin-inhibitedhuman AChE by MMB-4. The reactivation constants KD and kr as determined bymethods A and B were used to calculate kobs according to Eq. (1). Applying these data,the expected reactivation of sarin-inhibited human AChE by MMB-4 (1–1000 μM)within 10 min was calculated (Eq. (3)) and depicted as Hill-plot (n variable) withbottom set to 0% and top to 100%. The Hill coefficient was 1.18 (method A) and 1.46(method B) and the EC50 was calculated was 56.4 and 56.5 μM for methods A and B,respectively.

234 F. Worek et al. / Toxicology and Applied Pharmacology 249 (2010) 231–237

forming the ratio between human and animal reactivation constantsthe species differences could be quantified (Table 2). Only slightdifferences were found between human and Cynomolgus AChE whiledifferences between human and swine or guinea pig AChE were morepronounced.

The availability of reactivation constants allowed further calcula-tions to evaluate the reactivation efficiency of MMB-4. Fig. 5 showsthe calculated MMB-4 concentration necessary to obtain 40%reactivation of inhibited AChE in 10 min in the absence of excessOP. This model calculation indicates that MMB-4 concentrations ofless than 50 μM could be sufficient to meet this goal with human andCynomolgus AChE while higher but potentially therapeutic concen-trations (except of VX-inhibited guinea pig AChE) could be effectualwith swine and guinea pig AChE.

The MMB-4 reactivation constants determined with human AChEwere compared with the corresponding constants of HI-6 (Woreket al., 2004). Hereby, it became obvious that with sarin-, cyclosarin-,VX- and VR-inhibited human AChE MMB-4 had a 2.8–6.5 fold higherreactivity (Fig. 6A) but a 30–103 fold lower affinity (Fig. 6B).Consequently, HI-6 had amore than 10 fold higher hybrid reactivationrate constant kr2 (Fig. 6C).

The differences between MMB-4 and HI-6 are also reflected by thecalculated oxime concentrations necessary to obtain 40% reactivationof inhibited AChE in 10 min in the absence of excess OP (Fig. 7). Here,

Table 1Reactivation constants for MMB-4-induced reactivation of OP-inhibited AChE.

OP Constant Human AChE

Sarin kr (min−1) 1.87KD (μM) 1544kr2 (mM−1 min−1) 1.21

Cyclosarin kr (min−1) 4.47KD (μM) 2467kr2 (mM−1 min−1) 1.81

VX kr (min−1) 1.56KD (μM) 1196kr2 (mM−1 min−1) 1.31

VR kr (min−1) 4.01KD (μM) 574kr2 (mM−1 min−1) 6.98

Tabun kr (min−1) 0.02KD (μM) 2418kr2 (mM−1 min−1) 0.01

n.d. not determinable due to low reactivation.

a 10–15 fold higher MMB-4 concentration would be necessarycompared to HI-6 to achieve the benchmark.

Discussion

Modified kinetic approach

In previous studies, the reactivation kinetics of oximes wasdetermined by two different methods, depending on the reactivityand affinity of the oximes (Worek et al., 2002, 2004). Thediscontinuous procedure, i.e. determination of enzyme activity afterdifferent reactivation time and subsequent calculation of kobs valuesby linear regression analysis (method A), was applicable for oximeswith low affinity and low to moderate reactivity, e.g. 2-PAM andcyclosarin-inhibited human AChE. By incubating concentrated AChEwith oxime followed by excessive dilution for activity determinationit was possible to use high oxime concentrations (up to 5 mM) duringincubation.

With the continuous procedure, presented first by Kitz et al.(1965), kobs was determined from the recorded reaction curve as theamount of hydrolyzed substrate after a given time interval. Thisapproach proved to be adequate for oximes with moderate to highreactivity and high affinity, e.g. HI-6 and cyclosarin-inhibited humanAChE. Since the reactivation was followed during measurement ofAChE activity the maximum oxime concentration was limited to 50–100 μM.

The continuous procedure was not applicable with MMB-4 due toits low affinity, requiring testing of high oxime concentrations. Thehigh reactivity of MMB-4 was a challenge for the discontinuousprocedure especially at high oxime concentrations. Hence, theanalysis of the time-dependent reactivation by non-linear regressionanalysis (method B) proved to be the most appropriate method todetermine the reactivation constants of MMB-4.

MMB-4 reactivation kinetics

The reactivation constants, kr and KD, determined in the presentstudy are in part markedly different to data published by our groupand others before (Eyer and Worek, 2007; Luo et al., 2007). A closerlook on the experimental details may provide an explanation for thediscrepancies. Luo et al. reported that they failed to calculate kr and KD

values due to a linear relationship of kobs versusMMB-4 concentrationin case of sarin-, cyclosarin- and VR-inhibited human AChE. This maybe due to the use of inadequateMMB-4 concentrations, e.g. 5 to 40 μMMMB-4 were used for the determination of reactivation kinetics ofcyclosarin-inhibited human AChE (Luo et al., 2007). A similar

Cynomolgus AChE Swine AChE Guinea pig AChE

1.99 1.19 1.171299 1923 11091.53 0.62 1.052.64 0.77 0.761085 1406 16822.43 0.55 0.452.76 1.19 0.272480 1552 10531.11 0.77 0.263.83 3.49 1.45555 2575 15986.91 1.36 0.910.04 0.01 n.d.3129 23350.01 0.002

Fig. 4. Time- and concentration-dependent reactivation of cyclosarin-inhibited AChE by MMB-4. Human (A), Cynomolgus monkey (B), swine (C) and guinea pig AChE (D) wasincubated with different MMB-4 concentrations and AChE activity was determined after 1–9 min. Data were analyzed by non-linear regression (method B) to determine kobs.

235F. Worek et al. / Toxicology and Applied Pharmacology 249 (2010) 231–237

situation may have led to the low kr (0.024 min−1) and KD values(137 μM) with VX-inhibited AChE in this study. In our previous study,kobs was determined by linear regression analysis (method A)preventing the use of sufficiently high MMB-4 concentrations due toprogressive curving and deviation from the origin. Despite of the hugedifferences of the reactivation constants kr and KD between theprevious and the present study the hybrid reactivation rate constantskr2 are in good agreement, cf. Table 1 and Eyer and Worek (2007).

The deviation of the reactivation kinetics of sarin-inhibited humanAChE by higher concentrations of MMB-4 from a mono-exponentialcurve as observed with both methods (cf. Fig. 2) points to a hithertounrevealed phenomenon. Conceivably, formation of a meta-stablephosphonyloxime may re-inhibit the reactivated enzyme, if thereactivation is much faster than the decomposition of the suspectedphosphonyloxime. Since the amount of phosphonyloxime is equiva-lent to the amount of reactivated enzyme, the re-inhibition willincrease with the square of the reactivated enzyme concentration.From this it can be expected that this phenomenon may not beobserved at low MMB-4 concentrations. Alternatively, high MMB-4concentrations may exert additional, e.g. allosteric effects thatinfluence the reactivation process. The deviation of the Hill-slope

Table 2Ratio of MMB-4 reactivation constants between human and animal AChE.

Sarin Cyclosarin VX VR

Ratio of human vs. Cynomolgus kr 0.94 1.69 0.57 1.05KD 1.19 2.27 0.48 1.04kr2 0.79 0.74 1.18 1.01

Swine kr 1.56 5.82 1.31 1.15KD 0.8 1.75 0.77 0.22kr2 1.94 3.32 1.70 5.14

Guinea pig kr 1.60 5.89 5.72 2.77KD 1.39 1.47 1.14 0.36kr2 1.15 4.02 5.03 7.71

Ratio of MMB-4 reactivation constants between human and animal AChE wascalculated by using the data shown in Table 1.

from unity may be suggestive of such an interaction (cf. Fig. 3).Interestingly, such effects were not observed with cyclosarin-inhibited AChE of all the species tested (cf. Fig. 4).

MMB-4 exhibited a high reactivitywith sarin-, cyclosarin-, VX- andVR-inhibited AChE. In this respect, MMB-4 may be considered as anoxime with a broader spectrum than 2-PAM or obidoxime, similar toHI-6. However, the high reactivity of MMB-4 could not outweigh itslow affinity resulting in markedly lower hybrid reactivation rateconstants kr2 compared to HI-6 (Fig. 6). Consequently, substantiallyhigherMMB-4 concentrations would be necessary to achieve a similarlevel of reactivation as with HI-6 (Fig. 7). Up to now, data ontherapeutically relevant MMB-4 concentrations in vivo are scarce.Administration of 16.5 mg/kgMMB-4 i.m. resulted in a Cmax of 150 μMin rabbits and 32 mg/kg MMB-4 i.m. in a Cmax of 255 μM in swine(Woodard and Lukey, 1991; Stemler et al., 1991). The administeredMMB-4 dose was well tolerated by the pigs. From these limited data it

Fig. 5. MMB-4 concentrations necessary to obtain 40% reactivation of nerve agent-inhibited AChE within 10 min. Oxime concentrations were calculated according toWorek et al. (2002) using determined reactivation constants (Table 1). The hatchedarea resembles the range of clinically used oxime concentrations.

Fig. 6. Ratio of reactivation constants of MMB-4 and HI-6. The ratio of kr (A), KD (B) andkr2 (C) was formed between MMB-4 (Table 1) and HI-6 (Worek et al., 2004).

236 F. Worek et al. / Toxicology and Applied Pharmacology 249 (2010) 231–237

may be assumed that MMB-4 concentrations comparable to that ofestablished oximes, obidoxime and pralidoxime, i.e. up to 100 μM(Eddleston et al., 2009; Thiermann et al., 2009), may be consideredclinically relevant. In view of this assumption, MMB-4 should be ableto reactivate sarin-, cyclosarin-, VX- and VR-inhibited human AChEsufficiently if no residual OP is present (cf. Fig. 5).

Species differences

TheMMB-4 reactivation kinetics of Cynomolgus, swine and guineapig AChE showed only low to moderate differences compared to

Fig. 7. MMB-4 and HI-6 concentrations necessary to obtain 40% reactivation of nerveagent-inhibited AChE within 10 min. Oxime concentrations were calculated accordingtoWorek et al. (2002) using determined reactivation constants of MMB-4 (Table 1) andHI-6 (Worek et al., 2004).

human AChE (Table 2). Regarding guinea pig AChE this is in contrastto results obtained with HI-6 (Worek et al., 2002) giving a ratio of kr2between human and guinea pig AChE of 41, 145 and 150 with sarin-,cyclosarin- and VX-inhibited AChE, respectively. Compared to HI-6,MMB-4 had a 3.2 fold higher kr2 with sarin- and cyclosarin-inhibitedguinea pig AChE and a 1.4 fold higher value in case of VX.

Such oxime-related species differences have to be taken intoaccount for the evaluation of oxime efficiency in animal experimentsin vivo. Recently, Shih et al. (2009) investigated the reactivation ofnerve agent-inhibited guinea pig AChE by different oximes in vivo.The animals were challenged by 1xLD50 sarin, cyclosarin, VX and VR,were treated by equimolar MMB-4 and HI-6 (58 μmol/kg) and bloodand tissue samples were taken 60 min later and both oximes provideda comparable level of AChE reactivation. However, these results do notnecessarily reflect the human in vivo situation due to the differentialspecies differences in reactivation kinetics of MMB-4 and HI-6.

MMB-4 and tabun-inhibited AChE

MMB-4 was an extremely weak reactivator of tabun-inhibitedAChE in all species, which was primarily due to an outstanding lowreactivity (Table 1). Previous work showed already that MMB-4 is aweak oxime against tabun in vitro and in vivo (Luo et al., 2007;Sevelova and Vachek, 2003). Experimental data and model calcula-tions indicate that millimolar MMB-4 concentrations would benecessary to achieve a partial reactivation of tabun-inhibited AChE.In this respect, MMB-4 is comparable to HI-6 which was shown to beunable to reactivate tabun-inhibited AChE (Luo et al., 2007; Woreket al., 2004; Worek et al., 2007a).

Conclusions

The bispyridinium bis-oxime MMB-4 is a reactivator of nerveagent-inhibited AChE exhibiting some specific properties. It combinesa high reactivity and low affinity towards sarin-, cyclosarin-, VX- andVR-inhibited AChE resulting in a rather low overall reactivatingefficiency. Due to the high reactivity of MMB-4 a rapid reactivation ofinhibited AChE can be anticipated at adequate oxime concentrations,which are substantially higher compared to HI-6. Additional studiesare necessary to determine the in vivo toxicity, tolerability andpharmacokinetics of MMB-4 in humans in order to enable a properassessment of the value of this oxime as an antidote against nerveagent poisoning.

Conflict of interest statementThe authors declare that there are no conflicts of interest.

Acknowledgments

The study was funded by the German Ministry of Defence. Theauthors are grateful to T. Hannig and L. Windisch for expert technicalassistance.

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