Toxicology 244 (2008) 35–41

Available online at www.sciencedirect.com

Inhibition, reactivation and aging kinetics of highly toxicorganophosphorus compounds: Pig versus minipig

acetylcholinesterase

F. Worek a,∗, N. Aurbek a, J. Wetherell b, P. Pearce b, T. Mann b, H. Thiermann a

a Bundeswehr Institute of Pharmacology and Toxicology, Neuherbergstrasse 11, 80937 Munich, Germanyb Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire SP4 0JQ, UK

Received 5 September 2007; received in revised form 22 October 2007; accepted 23 October 2007Available online 30 October 2007

Dedicated to Professor Peter Eyer on the occasion of his 65th birthday.

Abstract

Organophosphorus compound-based (OP) chemical warfare agents (nerve agents) represent a continuing threat to military forcesand the civilian population. OPs act primarily by inhibiting acetylcholinesterase (AChE), the standard treatment for which includesAChE reactivators (oximes) in combination with antimuscarinic drugs. In the last decades, the efficacy of oximes has been investigatedmostly in small animal models. In order to increase the predictive value of animal studies it is desirable to measure numerousphysiological and biochemical parameters. This is difficult in small animals. Large animal models fulfil these requirements and swineare increasingly being used in toxicology studies. Swine breeds generally show considerable variability in different characteristicswhich may be minimised by the use of specially bred minipigs which have a known genetic background and health status. Acomparative study was, therefore, initiated to investigate the kinetic properties of the White Landrace pig and Gottingen minipigAChE in respect of inhibition by the pesticide paraoxon; the nerve agents cyclosarin, VX and VR; the reactivation of inhibited AChEby oximes (obidoxime, pralidoxime and HI 6); and the aging and spontaneous reactivation of inhibited AChE. The determination of

the respective kinetic constants found similarities between pig and minipig AChE which showed marked differences in comparisonwith human AChE values. This has to be considered in designing meaningful models for the investigation of oxime efficacy in pigor minipig experiments. The generated data indicate comparable kinetic properties of pig and minipig AChE and may provide akinetic basis for extrapolation of data from pig studies to humans.© 2007 Elsevier Ireland Ltd. All rights reserved.

pound

n

Keywords: Acetylcholinesterase; Pig; Human; Organophosphorus com

1. Introduction

The pertinent threat of using highly toxic organophos-phorus compound-based chemical warfare agents (OPs;

∗ Corresponding author. Tel.: +49 89 3168 2930;fax: +49 89 3168 2333.

E-mail address: FranzWorek@Bundeswehr.org (F. Worek).

toGiocta

0300-483X/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reservdoi:10.1016/j.tox.2007.10.021

s; Oximes; Inhibition; Reactivation; Aging

erve agents) during military operations and againsthe civilian population necessitates the developmentf effective medical countermeasures (Gosden andardener, 2005). OP poisoning primarily results in an

nhibition of acetylcholinesterase (AChE), accumulation

f the neurotransmitter acetylcholine and generalisedholinergic overstimulation, leading to death by respira-ory failure. Currently, the standard treatment for nervegent poisoning comprises a combination of atropine,

ed.

36 F. Worek et al. / Toxicology 244 (2008) 35–41

sphates

adHfcsCeo

ohtar1dt(2to

os

miaGactedssdo2s

Fig. 1. Structures of organopho

n oxime, e.g. obidoxime or pralidoxime, and a benzo-iazepine, e.g. avizafone (UK) (Fig. 1; Volans, 1996).owever, both oximes are considered to be rather inef-

ective against some nerve agents, e.g. soman andyclosarin (Marrs et al., 1996), which has led to theearch for more effective oximes in the last decades.urrently, the Hagedorn oxime HI 6 (Fig. 1) is consid-red to be the most promising compound for replacingbidoxime and pralidoxime (Wetherell et al., 2006).

The major problem in the development of a newxime is the inability to test its antidotal efficacy inumans intentionally poisoned by nerve agents. Hence,he therapeutic effectiveness of oximes has to be evalu-ted in animal studies. In the last decades mostly mice,ats and guinea-pigs were used in such studies (Dawson,994). However, there is an increasing body of evi-ence that there are substantial species differences inhe kinetic properties of human and small animal AChE

Berry, 1971; Clement and Erhardt, 1994; Worek et al.,002). In addition, in small animals it is more difficulto carry out physiological and biochemical monitoringf different parameters, e.g. AChE activity, agent and

am

r

and oximes used in this study.

xime concentration, which limits the significance ofuch studies.

Swine are increasingly being used as a large animalodel in toxicology, particularly in studies concern-

ng percutaneous nerve agent poisoning (Dorandeu etl., 2007; Chilcott et al., 2005; Chilcott et al., 2003;oransson-Nyberg et al., 1995, 1998). The size of these

nimals allows for the design of experimental proto-ols with extensive monitoring of different parametershus enabling a more meaningful evaluation of the toxicffects of nerve agents and therapeutic effects of anti-otes. In previous studies mostly pure or hybrid juvenilewine breeds were used due to the large size of adultwine, i.e. up to 350 kg. A comparison of results fromifferent laboratories may be hampered by a variabilityf characteristics of different breeds (Dorandeu et al.,007). In order to overcome this shortcoming the use ofpecially bred minipigs may be an alternative. The small

dult size of minipigs, e.g. up to 35 kg with Gottingeninipigs, is advantageous for handling and housing.Recently, we determined the kinetics of inhibition,

eactivation and aging with human and White Landrace

icology

ioi

2

mEa1m3

biaphmas

pw1

2

wAwfloe

wp[td

s

2s

F. Worek et al. / Tox

pig AChE by using different highly toxic V-agents andoximes (Aurbek et al., 2006a). In order to compare thekinetic properties of White Landrace pig and Gottingenminipig AChE the present study was initiated to analysethe whole blood AChE and plasma BChE activities in pigand minipig blood samples, to determine the effects ofthe pesticide paraoxon and the nerve agents cyclosarin,VX and VR (Fig. 1) on pig and minipig AChE and toassess potential differences in aging, oxime-induced andspontaneous reactivation of inhibited AChE.

2. Materials and methods

2.1. Materials

Acetylthiocholine iodide (ATCh), S-butyrylthiocholineiodide (BTCh), 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB),paraoxon-ethyl (PXE), ethopropazine and pralidoxime chlo-ride (2-PAM) were obtained from Sigma and obidoximedichloride (obidoxime) were purchased from Duphar. HI 6dichloride was kindly provided by Dr. Clement (DefenceResearch Establishment Suffield, Ralston, Alberta, Canada)and HLo 7 dimethanesulfonate was a custom synthesis by Dr.Braxmeier. Soman, cyclosarin (GF), VX and VR (>98% byGC–MS, 1H NMR and 31P NMR) were made available by theGerman Ministry of Defence. All other chemicals were fromMerck Eurolab GmbH (Darmstadt, Germany).

Stock solutions of PXE, GF, VX, VR and soman (0.1%, v/v)were prepared weekly in 2-propanol, stored at 4 ◦C and wereappropriately diluted in distilled water just before the exper-iment. Oximes (200 mM) were prepared in distilled water,stored at −60 ◦C and diluted as required in distilled water onthe day of the experiment. All solutions were kept on ice untilthe experiment.

Heparinised Gottingen minipig whole blood was obtainedfrom Ellegaard (Dalmose, Denmark) and heparinised WhiteLandrace pig whole blood samples were obtained from thelocal slaughterhouse. Pig and minipig blood samples wereassayed immediately after receipt to determine whole bloodAChE and plasma BChE activities.

Hemoglobin-free erythrocyte ghosts were prepared accord-ing to (Dodge et al., 1963) with minor modifications (Woreket al., 2002). Aliquots of the erythrocyte ghosts with an AChEactivity adjusted to that found in whole blood were stored at−60 ◦C until use. Prior to use, aliquots were homogenized onice with a Sonoplus HD 2070 ultrasonic homogenator (Ban-delin Electronic, Berlin, Germany), three times for 5 s with30 s intervals, to achieve a homogeneous matrix for the kineticstudies.

In order to prevent AChE denaturation during long-term◦

experiments at 37 C, AChE was stabilised by the addi-

tion of pig or minipig plasma with totally blocked BChE(Worek et al., 1999a). Plasma was obtained as describedabove and inhibited by soman (100 nM) for 30 min at 37 ◦Cto ensure complete inhibition and aging of BChE. The inhib-

tira

244 (2008) 35–41 37

ted plasma was dialyzed (phosphate buffer, 0.1 M, pH 7.4)vernight at 4 ◦C to adjust the pH and to remove any residualnhibitor.

.2. Enzyme assays

AChE and BChE activities were measured spectrophoto-etrically (Cary 3Bio, Varian, Darmstadt) with a modifiedllman assay (Worek et al., 1999b; Eyer et al., 2003). Thessay mixture (3.16 ml) contained 0.45 mM ATCh (AChE) or.0 mM BTCh (BChE) as substrate and 0.3 mM DTNB as chro-ogen in 0.1 M phosphate buffer (pH 7.4). Assays were run at

7 ◦C.The selective determination of AChE activity in whole

lood samples was achieved by adding the selective BChEnhibitor ethopropazine (20 �M) to the cuvette. The specificctivity of erythrocyte AChE activity in whole blood sam-les was calculated from the quotient of AChE activity andemoglobin content (mU/�mol HbFe). Hemoglobin was deter-ined by a cyanmethemoglobin method at 546 nm (Worek et

l., 1999b). BChE activity (mU/ml) was determined in plasmaamples.

For the determination of the Michaelis–Menten kinetics ofig and minipig AChE erythrocyte ghost samples were assayedith different ATCh concentrations ranging from 0.025 to.0 mM.

.3. Determination of inhibition rate constants (ki)

10 �l pig or minipig erythrocyte ghosts and 5 �l diluted OPas added to a cuvette containing phosphate buffer, DTNB andTCh (final volume 3.165 ml), the resultant OP concentrationsere 2–75 nM. ATCh hydrolysis was continuously monitored

or up to 30 min. The recorded curves were analysed by non-inear regression analysis and used for the further determinationf ki = k2/KD according to Forsberg and Puu (1984) and Aurbekt al. (2006a):

�t

� ln v= KD

k2× 1

[IX](1 − α)+ 1

k2(1)

here KD is the dissociation constant, k2 the unimolecularhosphylation rate constant and [IX] is OP-concentration. α:S]/(KM + [S]), where [S] is substrate concentration and KM ishe Michaelis constant. The experiments were performed inuplicate.

The analysis of the data was performed with PrismTM Ver-ion 4.00 (GraphPad Software, San Diego, CA, USA).

.4. Determination of rate constants for aging (ka) andpontaneous reactivation (ks)

OP-inhibited AChE was prepared by incubating ghosts withhe appropriate OP concentrations for 15 min at 37 ◦C result-ng in an inhibition of 95–98% of control activity. In order toemove excess OP after inhibition the samples were dialyzednd the absence of inhibitory activity was tested by incubation

38 F. Worek et al. / Toxicology 244 (2008) 35–41

omw

vdAmtP(VEtkbS

2

6(Aat

ssrS

ttpaprfsk

2a

ta

Table 1AChE and BChE activities in pig and minipig whole blood and plasma

AChE (mU/�mol Hb) BChE (mU/ml)

Pig 190 ± 6* 269 ± 10*

Minipig 297 ± 74* 271 ± 21*

Humana 651 ± 18 5675 ± 195

Data are given as mean ± S.D. from at least 10 determinations ofi

(

[

2

mt4

3

ah(takAw(Bp

Ain Table 2, human data were included for comparison.All OP had a comparable inhibitory activity with pigand minipig AChE. The potency decreased in the orderGF > VR > VX > PXE. Compared to human AChE, pig

Table 2Rate constant (ki) for the inhibition of pig and minipig AChE byparaoxon-ethyl (PXE), cyclosarin (GF), VX and VR

PXE GF VX VR

Pig 1.07 × 106 4.84 × 108 4.43 × 107a 1.88 × 108a

Minipig 1.23 × 106 4.84 × 108 5.61 × 107 1.95 × 108

Scheme 1.

f treated and control enzyme (15 min at 37 ◦C) followed by theeasurement of residual AChE activity. OP-treated samplesere stored in aliquots at −60 ◦C until use.

OP-treated pig and minipig ghosts were mixed with equalolumes of soman-treated pig or minipig plasma to preventenaturation of AChE during long-term experiments at 37 ◦C.liquots were taken after various time intervals for deter-ination of AChE activity (“spontaneous reactivation”) and

he decrease of oxime-induced reactivation (“aging”). Hereby,XE-treated samples were incubated with 500 �M obidoxime30 min), GF-treated samples with 500 �M HI 6 (20 min) andX- and VR-treated samples with 1 mM HLo 7 (15 min).xperiments were performed in duplicate and data were related

o control activities and the pseudo-first-order rate constantss (spontaneous reactivation) and ka (aging) were calculatedy a non-linear regression model (Worek et al., 1999a, 2004;krinjaric-Spoljar et al., 1973).

.5. Oxime reactivation of OP-inhibited AChE

The reactivation kinetics of obidoxime, pralidoxime and HIwere determined by a standard procedure as described before

Worek et al., 2004). In brief, OP-inhibited pig and minipigChE was incubated with 8–10 different oxime concentrationsnd the increase in AChE activity was determined at differentimes.

Reactivation data were used for the calculation of the dis-ociation constant (KD), the reactivity constant (kr) and theecond-order reactivation rate constant (kr2). Hereby, oximeeactivation of OP-inhibited AChE proceeds according tocheme 1.

In this scheme [EP] is the phosphonylated AChE, [EPOX]he Michaelis-type phosphonyl-AChE–oxime complex, [OX]he reactivator, [E] the reactivated enzyme and [POX] the phos-honylated oxime. KD is equal to the ratio [EP]×[OX]/[EPOX]nd approximates the dissociation constant which is inverselyroportional to the affinity of the oxime to [EP], and kr theate constant for the displacement of the phosphonyl residuerom [EPOX] by the oxime, indicating the reactivity. Theecond-order reactivation rate constant kr2 can be derived fromr2 = kr/KD (Worek et al., 2004).

.6. Calculation of oxime concentration and AChEctivity

The oxime concentration necessary to obtain a cer-ain fraction of reactivated enzyme at a given time in thebsence of excess inhibitor was calculated by using Eq. (2)

H

D

ndividual samples from five animals.a From Worek et al. (1999b).* Significantly different to human ChE (p < 0.05).

Worek et al., 2002).

OX] = − KD

1 + ((t × kr)/ ln((V0 − Vt)/(V0 − Vi)))(2)

.7. Statistical analysis

Analysis of significant differences between human, pig andinipig AChE and BChE activities was performed with the

wo-tailed Mann–Whitney test provided by PrismTM Version.00.

. Results

The determination of the specific erythrocyte AChEctivity in heparinised whole blood resulted in slightlyigher values in minipig compared to pig samplesTable 1). Pig and minipig AChE activities were substan-ially lower compared to human whole blood AChE. Thenalysis of the kinetic properties, i.e. Michaelis–Menteninetics, gave comparable KM values for pig and minipigChE (67.2 ± 2.3 �M and 65.1 ± 2.4 �M, respectively)hich were somewhat lower than those for human AChE

95.4 ± 1.6 �M; Mast, 1997). Pig and minipig plasmaChE activities were much lower than those for humanlasma (Table 1).

The rate constants for the inhibition of pig and minipigChE (ki) by PXE, GF, VX and VR are summarized

uman 3.30 × 106 4.21 × 108 9.91 × 107a 4.60 × 108a

ata are given as ki in M−1 min−1 (mean of two to three experiments).a From Aurbek et al. (2006a).

F. Worek et al. / Toxicology 244 (2008) 35–41 39

Table 3Rate constants for the spontaneous dealkylation (ka) and reactivationof AChE (ks) inhibited by paraoxon-ethyl (PXE), cyclosarin (GF), VXor VR

Inhibitor Pig Minipig Humanb

ka (h−1)PXE 0.021 0.016 0.022GF 0.049 0.043 0.099VX 0.0081a 0.0072 0.019VR 0.0026a 0.0025 0.005

ks (h−1)PXE 0.020 0.016 0.022GF – – –VX 0.0086a 0.0084 0.021VR 0.0073a 0.0076 0.039

Table 5Reactivation constants of cyclosarin-inhibited pig and minipig AChE

Obidoxime 2-PAM HI 6

Pigkr (min−1) 0.11 0.04 0.38KD (�M) 2670 2045 228.3kr2 (mM−1 min−1) 0.04 0.02 1.67

Minipigkr (min−1) 0.09 0.04 0.30KD (�M) 3657 1928 176.8kr2 (mM−1 min−1) 0.03 0.02 1.70

Humana

kr (min−1) 0.39 0.18 1.3KD (�M) 946 3159 47.2

peAAcwht

toasa

Data are mean of two to three experiments.a From Aurbek et al. (2006a).b From Worek et al. (2004).

and minipig AChE was somewhat less sensitive towardsthe tested OP (with the exception of GF).

The aging and spontaneous reactivation kineticsof OP-inhibited pig and minipig (as well as human)AChE followed first-order kinetics. The investigationdemonstrated marked differences concerning aging andspontaneous reactivation half-time, depending on thespecies and the inhibitor used (Table 3). Hereby, compa-rable ka and ks values were obtained for pig and minipigAChE. When compared to human AChE, the half-time ofaging and spontaneous reactivation of GF-, VX- and VR-inhibited AChE was substantially longer with pig andminipig AChE. Apart from GF-inhibited enzyme, spon-

taneous reactivation was observed, its velocity beingeven higher than aging in case of VR-inhibited AChE.

The determination of reactivation constants ofobidoxime, pralidoxime and HI 6 with OP-inhibited

Table 4Reactivation constants of paraoxon-inhibited pig and minipig AChE

Obidoxime 2-PAM HI 6

Pigkr (min−1) 0.55 0.49 0.08KD (�M) 39.7 262.9 324.3kr2 (mM−1 min−1) 13.9 1.9 0.26

Minipigkr (min−1) 0.49 0.39 0.1KD (�M) 52.1 152.3 411.8kr2 (mM−1 min−1) 9.32 2.59 0.24

Humana

kr (min−1) 0.81 0.17 0.20KD (�M) 32.2 187.3 548.4kr2 (mM−1 min−1) 25.1 0.9 0.37

a From Worek et al. (2004).

cah

TR

P

M

H

kr2 (mM−1 min−1) 0.42 0.06 27.5

a From Worek et al. (2004).

ig, minipig and human AChE revealed marked differ-nces depending on the inhibitor and oxime (Tables 4–7).

close similarity was observed with pig and minipigChE. In contrast, the second-order reactivation rateonstants (kr2) determined with pig and minipig AChEere, in part substantially, lower when compared touman AChE. The difference was especially evident inhe case of HI 6 and GF-, VX- and VR-inhibited AChE.

The determined reactivation rate constants enabledhe calculation of oxime concentrations necessary tobtain a certain portion of the inhibited AChE withingiven time (Table 8). These data demonstrate, in part

ubstantial, differences between human and pig AChEnd indicate that in most cases markedly higher oxime

oncentrations would be necessary in pigs/minipigs tochieve a comparable level of reactivation compared toumans.

able 6eactivation constants of VX-inhibited pig and minipig AChE

Obidoxime 2-PAM HI 6

iga

kr (min−1) 0.449 0.229 0.249KD (�M) 31.19 239.1 379.0kr2 (mM−1 min−1) 14.39 0.96 0.66

inipigkr (min−1) 0.229 0.194 0.152KD (�M) 20.38 186.2 184.4kr2 (mM−1 min−1) 11.26 1.04 0.82

umanb

kr (min−1) 0.892 0.215 0.242KD (�M) 27.35 28.07 11.53kr2 (mM−1 min−1) 32.63 7.67 20.98

a From Aurbek et al. (2006a).b From Worek et al. (2004).

40 F. Worek et al. / Toxicology

Table 7Reactivation constants of VR-inhibited pig and minipig AChE

Obidoxime 2-PAM HI 6

Piga

kr (min−1) 0.143 0.157 0.430KD (�M) 374.0 993.8 225.6kr2 (mM−1 min−1) 0.38 0.16 1.91

Minipigkr (min−1) 0.140 0.151 0.472KD (�M) 364.0 759.8 168.0kr2 (mM−1 min−1) 0.38 0.19 2.81

Humanb

kr (min−1) 0.630 0.058 0.712KD (�M) 105.9 30.65 9.15kr2 (mM−1 min−1) 5.95 1.90 77.79

4

tLtcttdreB

smco

sraTeaBTpm

AapaHeipakn

TC

I

P

G

V

V

Tcc

a From Aurbek et al. (2006a).b From Worek et al. (2004).

. Discussion

The main purpose of the present study was to inves-igate and compare the kinetic properties of Whiteandrace pig and Gottingen minipig AChE. It was shown

hat both enzyme species exhibit similar properties con-erning the inhibition by the pesticide paraoxon andhe nerve agents cyclosarin, VX and VR. In addition,he reactivation of inhibited AChE by obidoxime, prali-

oxime and HI 6 as well as the aging and spontaneouseactivation was comparable between pig and minipignzyme. Moreover, whole blood AChE and plasmaChE activities were comparable in pig and minipig

obao

able 8alculated oxime concentration for the reactivation of OP-inhibited AChE to

nhibitor Species Oxime (�M)

Obidoxime 2-PAM

XEHuman 1.9 68.0Pig 3.5 25.9Minipig 5.3 19.4

FHuman 120.4 1,028Pig 1892 >10,000Minipig 3137 >10,000

XHuman 1.4 7.3Pig 3.4 57.9Minipig 4.9 55.5

RHuman 8.1 100.2Pig 169.8 394.3Minipig 169.4 319.4

he data represent oxime concentrations necessary to reactivate 20% inhibitedalculated by applying Eq. (2), assuming AChE being fully inhibited and beoncentration (pig/minipig vs. human) is given. n.a.: not applicable.

244 (2008) 35–41

amples and the Michaelis–Menten kinetics of pig andinipig AChE were almost identical. These data indi-

ate that the interactions between AChE, tested OP andximes are congruent with pig and minipig enzyme.

Recently, it has been shown that there are in partubstantial differences between human and pig AChEelated to the kinetics of inhibition, reactivation andging with different V-agents (Aurbek et al., 2006a).he data presented here demonstrate that these differ-nces are also present with other OP and that there arelso differences in the whole blood AChE and plasmaChE activities between human and pig/minipig blood.he markedly lower BChE activity in pig and minipiglasma indicates that compared to humans, pig blooday have a lower capacity to scavenge circulating OP.Different kinetic properties of animal and human

ChE are a critical factor for the extrapolation of in vivonimal data to humans. Such differences were shownreviously for rat, guinea-pig and rabbit AChE (Worek etl., 2002) and are also evident for pig and minipig AChE.owever, in contrast to small animals, swine models

nable an extensive monitoring of different parametersn a single animal which is a prerequisite for the inter-retation of data on the therapeutic efficacy of oximesnd other antidotes. By knowing the in vitro enzymeinetics, the OP toxicokinetics and oxime pharmacoki-etics it may be possible to calculate equi-effective

xime concentrations and doses by means of kinetic-ased computer models (Aurbek et al., 2006b, 2007). Inddition, the availability of enzyme kinetic data, OP andxime concentrations enables the calculation of AChE

20% within 5 min

Ratio of oxime concentration(pig/minipig vs. human AChE)

HI 6 Obidoxime 2-PAM HI 6

154.9367.1 1.8 0.4 2.4335.5 2.8 0.3 2.2

1.730.2 15.7 n.a. 18.030.7 26.0 n.a. 18.3

2.682.9 2.4 7.9 31.876.7 3.4 7.6 29.4

0.626.1 21.0 3.9 42.717.6 21.0 3.2 28.7

AChE within 5 min in the absence of excess inhibitor. The data wereing 100% reactivatable. In addition, the ratio of the necessary oxime

icology

D

E

E

F

G

G

G

M

M

S

V

W

W

W

W

W

toxic organophosphates. Arch. Toxicol. 76, 523–529.Worek, F., Thiermann, H., Szinicz, L., Eyer, P., 2004. Kinetic analysis

F. Worek et al. / Tox

activities and the comparison to in vivo AChE activi-ties. This approach may be used for the validation of invitro kinetic data which can serve as a basis for definingappropriate oxime doses in animals (Worek et al., 2007;Eyer and Worek, 2007).

In conclusion, the results of this study demonstratethat the kinetic properties of White Landrace pig andGottingen minipig AChE are comparable in view ofinteractions with different OP and oximes. These datamay serve as a basis for the refinement of swine modelsfor the investigation of oxime efficacy in OP poisoning.

Acknowledgements

The authors are grateful to T. Hannig, J. Inwich andL. Windisch for their expert technical assistance.

References

Aurbek, N., Thiermann, H., Szinicz, L., Eyer, P., Worek, F., 2006a.Analysis of inhibition, reactivation and aging kinetics of highlytoxic organophosphorus compounds with human and pig acetyl-cholinesterase. Toxicology 224, 91–99.

Aurbek, N., Thiermann, H., Szinicz, L., Eyer, P., Worek, F., 2006b.Application of kinetic-based computer modelling to evaluate theefficacy of HI 6 in percutaneous VX poisoning. Toxicology 224,74–80.

Aurbek, N., Thiermann, H., Szinicz, L., Worek, F., 2007. Evaluationof HI 6 treatment after percutaneous VR exposure by use of akinetic-based dynamic computer model. Toxicology 233, 173–179.

Berry, W.K., 1971. Some species differences in the rates ofreaction of diaphragm particulate acetylcholinesterase withtetraethyl pyrophosphate and pralidoxime. Biochem. Pharmacol.20, 1333–1334.

Chilcott, R.P., Dalton, C.H., Hill, I., Davidson, C.M., Blohm, K.L.,Hamilton, M.G., 2003. Clinical manifestations of VX poisoningfollowing percutaneous exposure in the domestic white pig. Hum.Exp. Toxicol. 22, 255–261.

Chilcott, R.P., Dalton, C.H., Hill, I., Davison, C.M., Blohm, K.L.,Clarkson, E.D., Hamilton, M.G., 2005. In vivo skin absorp-tion and distribution of the nerve agent VX (O-ethyl-S-[2(diiso-propylamino)ethyl]methylphosphonothioate) in the domesticwhite pig. Hum. Exp. Toxicol. 24, 347–352.

Clement, J.G., Erhardt, N., 1994. In vitro oxime-induced reactivation ofvarious molecular forms of soman-inhibited acetylcholinesterasein striated muscle from rat, monkey and human. Arch. Toxicol. 68,648–655.

Dawson, R.M., 1994. Review of oximes available for treatment ofnerve agent poisoning. J. Appl. Toxicol. 14, 317–331.

Dodge, J.T., Mitchell, C., Hanahan, D.J., 1963. The preparation andchemical characteristics of hemoglobin-free ghosts of human ery-throcytes. Arch. Biochem. Biophys. 100, 119–130.

244 (2008) 35–41 41

orandeu, F., Mikler, J.R., Thiermann, H., Tenn, C., Davidson,C., Sawyer, T.W., Lallement, G., Worek, F., 2007. Swine mod-els in the design of more effective medical countermeasuresagainst organophosphorus compounds poisoning. Toxicology 233,128–144.

yer, P., Worek, F., 2007. Oximes. In: Marrs, T.C., Maynard, R.L.,Sidell, F.R. (Eds.), Chemical Warfare Agents: Toxicology andTreatment. John Wiley & Sons Ltd., Chichester, pp. 305–329.

yer, P., Worek, F., Kiderlen, D., Sinko, G., Stuglin, A., Simeon-Rudolf, V., Reiner, E., 2003. Molar absorption coefficients forthe reduced Ellman reagent: reassessment. Anal. Biochem. 312,224–227.

orsberg, A., Puu, G., 1984. Kinetics for the inhibition of acetyl-cholinesterase from the electric eel by some organophosphates andcarbamates. Eur. J. Biochem. 140, 153–156.

oransson-Nyberg, A., Cassel, G., Jeneskog, T., Karlsson, L., Larsson,R., Lundstrom, M., Persson, S.A., 1995. Treatment of organophos-phate poisoning in pigs: antidote administration by a new binaryautoinjector. Arch. Toxicol. 70, 20–27.

oransson-Nyberg, A., Fredriksson, S.A., Karlsson, B., Lundstrom,M., Cassel, G., 1998. Toxicokinetics of soman in cerebrospinalfluid and blood of anaesthetized pigs. Arch. Toxicol. 72, 459–467.

osden, C., Gardener, D., 2005. Weapons of mass destruction-threatsand responses. Br. Med. J. 331, 397–400.

arrs, T.C., Maynard, R.L., Sidell, F.R., 1996. Chemical WarfareAgents—Toxicology and Treatment. John Wiley & Sons, Chich-ester.

ast, U., 1997. Reaktivierung der Erythrozyten-Acetylcholinesterasedurch Oxime. Thesis. University Munich.

krinjaric-Spoljar, M., Simeon, V., Reiner, E., 1973. Spontaneous reac-tivation and aging of dimethylphosphorylated acetylcholinesteraseand cholinesterase. Biochim. Biophys. Acta 315, 363–369.

olans, A.P., 1996. Sarin: guidelines on the Management of Victimsof a Nerve Gas Attack. J. Accid. Emerg. Med. 13, 202–206.

etherell, J., Price, M., Mumford, H., 2006. A novel approach formedical countermeasures to nerve agent poisoning in the guinea-pig. Neurotoxicology 27, 485–491.

orek, F., Diepold, C., Eyer, P., 1999a. Dimethylphosphoryl-inhibitedhuman cholinesterases: inhibition, reactivation, and aging kinetics.Arch. Toxicol. 73, 7–14.

orek, F., Eyer, P., Aurbek, N., Szinicz, L., Thiermann, H., 2007.Recent advances in evaluation of oxime efficacy in nerve agentpoisoning by in vitro analysis. Toxicol. Appl. Pharmacol. 219,226–234.

orek, F., Mast, U., Kiderlen, D., Diepold, C., Eyer, P., 1999b.Improved determination of acetylcholinesterase activity in humanwhole blood. Clin. Chim. Acta 288, 73–90.

orek, F., Reiter, G., Eyer, P., Szinicz, L., 2002. Reactivation kineticsof acetylcholinesterase from different species inhibited by highly

of interactions between human acetylcholinesterase, structurallydifferent organophosphorus compounds and oximes. Biochem.Pharmacol. 68, 2237–2248.