effect of reversible ligands on oxime-induced reactivation of sarin- and cyclosarin-inhibited human...

1

2 Q1

3 Q2

4

5

6

7

8

9

10

11

12

13

Q3

Toxicology Letters xxx (2014) xxx–xxx

G Model

TOXLET 8970 1–9

Effect of reversible ligands on oxime-induced reactivation of sarin- andcyclosarin-inhibited human acetylcholinesterase

Corinna Scheffel, Horst Thiermann, Franz Worek *Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany

H I G H L I G H T S

� We investigated the inhibition type and potency of human AChE by ligands in vitro.� We investigated the effect of AChE ligands on oxime reactivation of OP-inhibited human AChE.� AChE ligands did not improve reactivation of human AChE by obidoxime and HI-6.

A R T I C L E I N F O

Article history:Received 11 November 2014Received in revised form 11 December 2014Accepted 12 December 2014Available online xxx

Keywords:Organophosphorus compoundAcetylcholinesteraseOximesReversible acetylcholinesterase-ligandsReactivation

A B S T R A C T

Poisoning by organophosphorus compounds (OP) used as pesticides and nerve agents is due toirreversible inhibition of the enzyme acetylcholinesterase (AChE). Oximes have been widely recognizedfor their potency to reactivate the inhibited enzyme. The limited efficacy of currently available oximesagainst a broad spectrum of OP-compounds initiated novel research efforts to improve oxime-basedtreatment. Hereby, oxime-induced reactivation of OP-inhibited non-human AChE was reported to beaccelerated by different AChE-ligands. To investigate this concept with AChE from human source, theinhibitory potency, binding properties and the potential enhancement of oxime-induced reactivation ofOP-inhibited AChE by structurally different AChE-ligands was assessed. Several ligands competed withthe oxime for the AChE binding-site impairing reactivation of OP-inhibited AChE whereas a markedlyaccelerated reactivation of sarin-inhibited enzyme by obidoxime was recorded in the presence ofedrophonium, galanthamine and donepezil. Enhancement of oxime-induced reactivation with ligandswas presumably subject to prevention of re-inhibition by the reaction product phosphonyloxime (POX).In the end, the results of the present study did not confirm that AChE-ligands directly accelerate thereactivation of OP-inhibited AChE by oximes, but indirectly by prevention of re-inhibition by the reactionproduct POX. This may be due to species differences between human and non-human AChE of previousexperiments with non-human AChE.

ã 2014 Published by Elsevier Ireland Ltd.

Contents lists available at ScienceDirect

Toxicology Letters

journa l homepage: www.e lsev ier .com/ locate / toxlet

14

15

16

17

18

19

20

21

22

23

1. Introduction

Acetylcholinesterase (AChE) (EC 3.1.1.7), a serine hydrolasepresent at the synapses of the cholinergic nervous system,terminates cholinergic synaptic transmission by hydrolysis ofthe neurotransmitter acetylcholine (Quinn, 1987; Taylor et al.,1995). Irreversible inhibition of AChE is the primary mechanism ofaction of many organophosphorus (OP) esters, including pesticidesand highly toxic nerve agents. These compounds exert their acutetoxicity through phosphylation (denotes phosphorylation and

24

25

26

27* Corresponding author. Tel.: +49 89 31682930; fax: +49 89 992692 2933.E-mail address: [email protected] (F. Worek).

http://dx.doi.org/10.1016/j.toxlet.2014.12.0090378-4274/ã 2014 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Scheffel, C., et al., Effect of reversible ligahuman acetylcholinesterase. Toxicol. Lett. (2014), http://dx.doi.org/10.10

phosphonylation) of the g-oxygen of the AChE active site serine(Holmstedt, 1959; Taylor et al., 1995). Thus, impaired hydrolysis ofacetylcholine leads to accumulation of the neurotransmitter atmuscarinic and nicotinic receptors. The following overstimulationof peripheral and central cholinergic receptors causes disruption ofvital body functions, respiratory arrest and finally death (Grob andHarvey, 1953; Holmstedt, 1959; Wright, 1954).

Since the early 1950s, numerous nucleophilic oxime com-pounds, including monopyridinium and bis pyridinium com-pounds as pralidoxime, obidoxime and HI-6, were synthesized andtheir properties to reactivate the OP-inhibited enzyme shown(Eyer, 2003; Eyer and Worek, 2007; Worek et al., 2007). However,efficacy of oxime-induced reactivation of OP-inhibited AChE islimited in poisoning by different nerve agents, such as soman,

nds on oxime-induced reactivation of sarin- and cyclosarin-inhibited16/j.toxlet.2014.12.009

mailto:[email protected]

http://dx.doi.org/10.1016/j.toxlet.2014.12.009



http://www.sciencedirect.com/science/journal/03784274

www.elsevier.com/locate/toxlet

28 ta29 st30 W31 by32 ox33 sp34 1935 af36 de37 pa38 ox39 su40 ox41 du42 re43 et44 g.,45 co46 an47 Ow48 W49

50 m51 to52 (L53 st54 co55 ox56 us57 bo58 to59 us60 m61 in62 (F63 in

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

2 C. Scheffel et al. / Toxicology Letters xxx (2014) xxx–xxx

G Model

TOXLET 8970 1–9

bun and cyclosarin as reported by several in vivo and in vitroudies (Dawson, 1994; Eyer and Worek, 2007; Marrs et al., 2006;orek and Thiermann, 2013). One reason for impaired reactivation

oximes is the limited accessibility of the attacking nucleophilicime group to the phosphylated binding site as recorded by site-ecific mutagenesis studies (Ashani et al., 1995; Taylor et al.,95). In other cases, reactivation of the OP-enzyme-conjugate isfected by a post-inhibitory process known as aging comprising aalkylation reaction, which makes the phosphylated AChE,rticularly soman-inhibited AChE, resistant to reactivation byimes in living species (Segall et al., 1993). Another mechanismggested to explain the dramatically reduced reactivation rates ofimes is the inevitable formation of phosphylated oxime (POX)ring reactivation which is able to induce re-inhibition of thegenerated enzyme in vitro (de Jong and Ceulen, 1978; Harvey

al., 1986; Schoene, 1973). However, only highly stable POX, as e. derived from obidoxime and other pyridinium-4-aldoximesnjugated with specific OP-residues, such as sarin, exertticholinesterase activity (Ashani et al., 2003; Hackley andens, 1959; Kiderlen et al., 2000, 2005; Worek et al., 2000;

orek and Thiermann, 2013).These drawbacks of oxime efficacy reinforced the search for

ore effective oximes and also initiated new therapeutic strategies provide an improved oxime-based treatment of OP poisoninguo et al., 1998; Musilek et al., 2011; Sit et al., 2011). Previousudies proposed that oxime-induced reactivation of AChE–OP-njugate may be accelerated in the presence of different non-ime AChE-ligands, such as edrophonium and decamethonium,ing AChE from diverse non-human sources, including fetalvine serum and mouse AChE (Luo et al., 1998, 1999a,b). In order

verify the potential acceleration by reversible AChE-compoundsing human erythrocyte AChE and to shed light into its underlyingechanism of action, the inhibitory potency and mode ofhibition by eight reversible, structurally different AChE-ligandsig. 1) as well as their effect in oxime-induced reactivation of OP-hibited AChE was investigated in the present study.

Fig. 1. Structures of AChE-l

Please cite this article in press as: Scheffel, C., et al., Effect of reversible lighuman acetylcholinesterase. Toxicol. Lett. (2014), http://dx.doi.org/10.1

2. Materials and methods

Acetylthiocholine iodide (ATCh) and 5,50-dithio-bis-2-nitro-benzoic acid (DTNB) were purchased from Sigma–Aldrich (Tauf-kirchen, Germany) and obidoxime (1,10-[oxybis(methylene)]bis[4-(hydroxyimino) methyl] pyridinium dichloride) from Merck(Darmstadt, Germany), HI-6 (1-[[[4-(aminocarbonyl) pyridinio]methoxy]methyl]-2-[(hydroxyimino)methyl]pyridinium dichlor-ide monohydrate) was provided by Dr. Clement (Defense ResearchEstablishment Suffield, Ralston, Alberta, Canada). Imipraminehydrochloride, donepezil hydrochloride, pancuronium bromideand propidium iodide were obtained from Sigma–Aldrich.Edrophonium chloride, itopride hydrochloride and desoxypega-nine hydrochloride were from Santa Cruz Biotechnology (SantaCruz, Dallas, Texas, USA) and galanthamine hydrobromide fromTocris Bioscience (Wiesbaden, Germany).

All other chemicals were purchased from Merck Eurolab GmbH(Darmstadt, Germany) at the purest grade available.

Sarin (isopropyl methylphosphonofluoridate;>98%byGC–MS,1HNMR and 31P NMR) and cyclosarin (cyclohexyl methylphosphono-fluoridate; >95% by GC–MS, 1H NMR and 31P NMR) were madeavailable by the German Ministry of Defense. Stock solutions of sarinand cyclosarin (0.1% v/v) were prepared in acetonitrile, stored at20 �C and appropriately diluted in distilled water just before use.

Oxime and ligand stock solutions were prepared in distilledwater and stored at �80 �C. Working solutions were diluted asrequired in 0.1 M phosphate buffer. All solutions were kept on iceuntil the experiment.

2.1. Blood samples

Hemoglobin-free human erythrocyte membranes (‘ghosts’)were prepared from human whole blood and served as sourceof human erythrocyte AChE (Worek et al., 2002). Aliquots oferythrocyte ghosts were adjusted to an AChE activity physiologi-cally found in whole blood (�9000 U/l) and stored at �80 �C. Prior

igands used in this study.

ands on oxime-induced reactivation of sarin- and cyclosarin-inhibited016/j.toxlet.2014.12.009


97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125 Q4126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

C. Scheffel et al. / Toxicology Letters xxx (2014) xxx–xxx 3

G Model

TOXLET 8970 1–9

to use, aliquots were thawed gently and homogenized on ice usinga Sonoplus HD 2070 ultrasonic homogenator (Bandelin electronic,Berlin, Germany), three times for 5 s with 30-s intervals to obtain ahomogeneous matrix for kinetic analysis. Subsequent determina-tion of AChE activity (see Section 2.2) corresponded to thephysiological activity obtained previous to freezing and sonicationconfirming that this had no effect on AChE activity of erythrocyteghosts.

2.2. AChE assay

A modified Ellman procedure was applied in all experiments(Ellman et al., 1961; Worek et al., 1999) to determine the catalyticAChE activity spectrophotometrically (Cary 50 Bio UV/VisibleSpectrophotometer) at 412 nm using polystyrol cuvettes prefilledwith the assay mixture (total volume 3.16 ml) containing 0.45 mM(ATCh) as substrate and 0.3 mM DTNB as chromogen in 0.1 Mphosphate buffer (pH 7.4). All measurements were performed atleast in duplicate at 37 �C and pH 7.4.

2.3. Determination of the inhibitory concentration (IC50) of reversibleAChE-ligands

IC50 was measured at a fixed substrate concentration usingdifferent ligand concentrations. In brief, 10 ml erythrocyte ghostsand 10 ml ligand were added simultaneously to a cuvette prefilledwith the assay mixture (see Section 2.2). Immediately after addingghosts and ligand, enzyme activity was continuously monitored forup to 3 min and IC50 values were calculated from semi-logarithmicdose-response curves of the ligand concentration versus AChEactivity.

The reversibility of AChE inhibition by the tested ligands wasinvestigated by incubating AChE with ligand concentrations of 2� IC50 for 5 min followed by extensive dilution (316 fold) of theincubate in phosphate buffer and subsequent determination ofAChE activity.

2.4. Determination of the inhibition type of reversible AChE-inhibitors

Determination of the inhibition type of AChE-ligands wasperformed according to Bisswanger (1979) using various ATChconcentrations from 100–1000 mM in the absence and presenceof different AChE-ligand concentrations. 10 ml native AChE and5 ml ligand were added to a polystyrol cuvette containingphosphate buffer and DTNB. Reaction was initiated by addingthe substrate ATCh into the cuvette and the determined enzymeactivity (v) was plotted versus substrate concentration [S] analyzedby non-linear regression analysis (Michaelis–Menten plot). Datawere transformed into a series of straight lines by plotting 1/vversus 1/[S] (Lineweaver–Burk plot). The mode of inhibition byAChE-ligands was obtained by the constants Vmax and Km

Table 1IC50 values, mode of inhibition and inhibitor constants Ki of reversible AChE-ligands. Al50 nM donepezil, 10 mM galanthamine, 10 mM itopride, 10 mM edrophonium, 10 mM pr

AChE-ligands IC50 (mM) Mode of inhibition

Donepezil 0.007 � 0.0004 Mixed

Galanthamine 1.0 � 0.01 Mixed

Itopride 1.1 � 0.03 Non-competitive

Edrophonium 4.6 � 0.03 Competitive

Propidium 4.7 � 0.03 Mixed

Pancuronium 9.3 � 0.05 Competitive

Desoxypeganine 12.4 � 0.01 Mixed

Imipramine 595.2 � 0.01 Mixed


calculated from Michaelis–Menten and double reciprocal Line-weaver–Burk plots and confirmed by the intersection point of thestraight lines of the Linewaever–Burk plot converging on theordinate, the abscissa or within the second quadrant of thecoordinate system.

Two secondary re-plots of the double reciprocal Lineweaver–Burk plot were generated by plotting the slope and the ordinateof these lines versus ligand concentration. Subsequently, thedissociation constants kic (slope versus ligand concentration) andkiu (ordinate versus ligand concentration) were determined fromthe x-intercept of the straight lines and the inhibitor constant Ki

was calculated from the ratio of the dissociation constants(kic/kiu) (Bisswanger, 1979). The Lineweaver–Burk- and thesecondary re-plots were analyzed by linear regressionanalysis.

In addition, two alpha factors (a and a0) were calculated usingequations Eqs. (1) and (2) to compare values of Vmax and Km ofcontrol AChE with those values of AChE in the presence of variousligand concentrations.

a ¼ 1 þ ½I�kic

(1)

a0 ¼ 1 þ ½I�kiu

(2)

2.5. Inhibition and reactivation of AChE in the absence and presence ofligands

Erythrocyte ghosts were incubated with appropriate concen-trations of sarin (25 nM) or cyclosarin (17 nM) for 15 min at 37 �C toobtain an AChE inhibition of >95%. Excess inhibitor was removedfrom the OP-treated ghosts by overnight dialysis against phosphatebuffer (0.1 M, pH 7.4) at 4 �C. Absence of residual inhibitor wastested by incubation of treated and control ghosts (30 min, 37 �C),subsequent measurement of AChE activity and aliquots werestored at �80 �C until the experiment. For reactivation, 150 ml OP-treated erythrocyte ghosts were incubated with 150 ml 0.2%gelatine in phosphate buffer in order to prevent denaturation ofAChE during prolonged incubation at 37 �C.

Formation of stable phosphonyloxime (POX) was suspected onthe base of a bi-phasic reactivation kinetic during obidoxime-induced reactivation of sarin-inhibited AChE. In this case, theenzyme sample was extensively pre-diluted (100 fold) prior to theexperiment to prevent AChE re-inhibition by the reaction productPOX. Then, reactivation was initiated by adding oxime (10 mMobidoxime or 10 mM HI-6) followed by ligand or vehicle (control)to the inhibited enzyme. An aliquot of the sample was transferredat different time intervals (1–45 min) to a pre-tempered cuvette

pha factors of Vmax and Km referred to control AChE activity and were obtained foropidium, 15 mM pancuronium, 20 desoxypeganine, 2000 mM imipramine.

Inhibitor constant Ki (mM) a0 � 1Vmax

a0

a� � 1

Km

0.72 � 0.05 3.1 0.70.02 � 0.002 1.5 0.061.06 � 0.08 5.3 1.00.001 � 0.0003 1.0 0.20.33 � 0.02 1.4 0.630.002 � 0.0002 1.0 0.050.63 � 0.04 2.52 0.730.14 � 0.009 1.6 0.2



183 co184 w

185 2.

186

187 tw188 th189 of190

191 ra192 ox193 th

ko

194195 In196 w197 de198 lig

ln

w199 ac200 (c201 (%202 AC

203 2.

204

205 an206 pr207 So208 ou209 m

210

211

212

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

TaFirco

A

I0151

E0111

G0111

D0000


G Model

TOXLET 8970 1–9

ntaining the assay mixture (see Section 2.2) and AChE activityas monitored over 3 min.

6. Reactivation kinetics of OP-inhibited AChE

Reactivation of OP-inhibited AChE by oximes proceeding via ao-step reaction can be determined by the affinity of the oxime toe OP-inhibited enzyme (1/KD) and the rate for the displacement

the phosphyl residue from AChE (kr).Thereby, the reactivation process follows a pseudo-first order

te equation (Eq. (3)) given that reactivation is complete and atime concentrations [OX] being higher than the concentration ofe phosphylated AChE (Su et al., 1986).

bs ¼kr � ½OX�KD þ ½OX� (3)

this study, the pseudo first-order reactivation rate constant kobsas calculated by linear-regression analysis (Table 2) for analyzingviations in reactivation kinetics in the absence and presence ofands, applying Eq. (4)

v0 � viv0 � vt

� �¼ �kobs � t (4)

ith vi = residual activity of AChE after inhibition, vt = AChEtivity at time t of reactivation and v0 = initial AChE activityontrol). In addition, plots of time (min) versus AChE reactivation

react) were drawn for experiments using pre-diluted inhibitedhE.

7. Data analysis

Processing of experimental data was performed by lineard non-linear regression analysis using line and curve fittingograms provided by GraphPad Prism 5.0 (GraphPadftware, San Diego, Calif. USA). All experiments were carriedt at a minimum of n = 2 and data were expressed aseans � SD.

ble 2st-order rate constant kobs calculated from semi-logarithmic plots of oxime-inducncentrations versus time.

Pseudo first-order reactivation rate cons

ChE-ligand concentration (mM) Reactivation of cyclosarin-inhibited ACh

HI-6 Obido

mipramine 125.3 � 6.9 17.4 �00 113.2 � 7.6 16.6 �00 98.4 � 8.6 15.6 �000 80.9 � 4.8 14.9 �

drophonium 123.4 � 4.0 19.4 � 99.0 � 1.9 20.2 �0 95.0 � 3.9 21.3 �00 46.6 � 3.5 18.6 �

alanthamine 90.9 � 4.3 12.5 � 96.1 � 3.2 12.4 �0 93.1 � 4.0 12.3 �00 64.3 � 2.7 10.7 �

onepezil 109.1 � 8.3 42.5 �.005 109.8 � 5.8 47.4 �.01 103.1 � 5.0 44.0 �.05 100.3 � 3.6 50.0 �


3. Results

3.1. Inhibitory potency of reversible AChE-ligands

The inhibitory potency of eight reversible AChE-ligands wasinvestigated by determining the IC50 values summarized in Table 1.

All ligands inhibited AChE in a concentration-dependentmanner ranging from IC50 values of 0.007 to 595.2 mM. Amongall tested ligands, donepezil was the most potent enzymeinhibitor (Fig. 2A). Comparable IC50 values were recorded forthe following compounds increasing in the given order: galanth-amine (Fig. 2B) < itopride (Fig. 2C) < edrophonium (Fig. 2D)< propidium < pancuronium (Fig. 2E) < desoxypeganine. Imipra-mine was shown to be the weakest AChE-inhibitor of all ligands(Fig. 2F).

Incubation of AChE with ligand concentrations of 2 � IC50

followed by extensive dilution (316 fold) resulted in enzymeactivities comparable to control AChE activity indicating areversibility of all tested ligands (data not shown).

3.2. Determination of the inhibition type of reversible AChE-ligands

The nature of reversible inhibition can be divided into four maintypes: competitive, non-competitive, uncompetitive and mixedcompetitive–non-competitive inhibition, an example for themixed AChE-inhibition by imipramine is shown in Fig. 3A–D(Bisswanger, 1979).

Vmax and Km of this compound decreased with increasingconcentration indicated by the alpha factors a and a0, which wassimilar to galanthamine, propidium, desoxypeganine, and done-pezil. These compounds yielded a mixed competitive–non-competitive type of inhibition as demonstrated by an intersectionpoint within the second quadrant of the Lineweaver–Burk plot.However, a different tendency of inhibition of a competitive ornon-competitive manner was observed as indicated by theinhibition constant Ki calculated from the ratio of the dissociationconstants kic to kiu obtained from the x-intercept of the secondary

ed reactivation of inhibited AChE in the absence and presence of different ligand

tant (kobs)

E (�10�3) Reactivation of sarin-inhibited AChE (�10�3)

xime HI-6 Obidoxime

0.3 100.3 � 2.2 9.8 � 0.7 0.4 76.9 � 1.4 10.8 � 0.4 0.3 55.5 � 1.9 15.4 � 1.1 0.3 46.7 � 1.9 14.0 � 1.1

0.4 76.1 � 4.4 13.4 � 0.7 0.2 60.7 � 5.2 14.6 � 0.9 0.4 54.7 � 3.7 19.5 � 1.9 0.6 24.2 � 2.7 21.3 � 0.9

0.3 87.6 � 2.2 7.7 � 0.5 0.5 76.3 � 2.5 9.0 � 0.5 0.5 74.5 � 3.2 10.8 � 0.9 0.4 66.1 � 3.5 14.2 � 1.0

2.0 100.7 � 6.9 5.4 � 0.8 1.3 106.5 � 7.7 8.6 � 1.3 1.5 103.7 � 6.6 11.5 � 1.0 1.7 96.8 � 7.6 13.7 � 1.3



243

244

245

246

247

248

249

250

Fig. 2. Concentration-dependent inhibition by reversible AChE-ligands. Six–eleven different ligand concentrations ranging from 0.0001 to 10,000 mM were tested. Data werepresented as % AChE activity and expressed as mean values � SD of two independent experiments.


G Model

TOXLET 8970 1–9

re-plots (Fig. 3C and D). Thereby, a decreasing value of Ki

demonstrating an increasing competitive inhibition type wasrecorded in the order: donepezil > desoxypeganine > propidium >imipramine > galanthamine (Table 1).

Fig. 3. Type of AChE inhibition by imipramine. Top graphs: The direct (A) and double reci^ 1500 mM; ^ 2000 mM) versus substrate concentration (100–1000 mM) Bottom graphs:Data were expressed as mean values � SD of two independent experiments.


Direct Michaelis–Menten and double reciprocal Lineweaver–Burk plots revealed a competitive type of inhibition for pancuro-nium and edrophonium (data not shown). These compounds didnot influence the maximum hydrolysis rate Vmax of AChE but

procal (B) plots of AChE inhibition by imipramine (& 0 mM;4 500 mM; 1000 mM; Re-plots of slope (C) and y-intercepts (D) from (B) versus imipramine concentration.



251 en252 Li253

254 co255 th256 an257 Th258 fu259 Ta260 a

261 ed

262

263

264

265

266

267

268

269

270

271

Fig. 4. Reactivation kinetics of concentrated cyclosarin- (A and B) and sarin- (C and D) inhibited AChE by 10 mM obidoxime (B and D) or 10 mM HI-6 (A and C) in the absenceand presence of edrophonium. AChE activity was determined by the Ellman assay (412 nm) at different time intervals (1–45 min) after incubation with different edrophoniumconcentrations (& 0 mM, ! 1 mM, 4 10 mM, 100 mM). Data were analyzed by linear regression and expressed as mean values � SD of two independent experiments.

Figwa10


G Model

TOXLET 8970 1–9

hanced Km concentration-dependently, i.e., straight lines of theneweaver–Burk plot converged on the y-intercept.In contrast, itopride was found to be an inhibitor of a non-mpetitive type demonstrated by a common intersection point one abscissa of the double-reciprocal plot revealing a reduced Vmax

d an unchanged Km in the presence of ligand (data not shown).e determination of the mode of inhibition by these ligands wasrther confirmed with the inhibition constants Ki summarized inble 1. Hereby, non-competitive inhibition by itopride resembledKi > 1, whereas competitive inhibition by pancuronium androphonium was indicated by a Ki< 0.005.

. 5. Reactivation kinetics of concentrated sarin-inhibited AChE by obidoxime in the as determined by the Ellman assay (412 nm) at different time intervals (1–45 min) a0 mM). Data were analyzed by non-linear (A and B) and linear regression (C and D


3.3. Oxime-induced reactivation of OP-inhibited AChE in the absenceand presence of ligands

The time- and concentration-dependent effect of ligands onreactivation of sarin- and cyclosarin-inhibited AChE was testedwith all eight ligands and was analyzed by linear regressionanalysis calculating kobs, the enzyme reactivation velocity, which issummarized in Table 2 for oxime-induced reactivation in theabsence and presence of imipramine, edrophonium, galanthamineand donepezil. Itopride, propidium, pancuronium and desoxype-ganine did not accelerate reactivation of sarin-inihibited AChE by

bsence and presence of galanthamine (A and C) or donepezil (B and D). AChE activityfter incubation with different ligand concentrations (& 0 mM, ! 1 mM, 4 10 mM, ) and expressed as mean values � SD of two independent experiments.



272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

Fig. 6. Reactivation kinetics of pre-diluted sarin-inhibited AChE by obidoxime in the absence and presence of galanthamine or donepezil. AChE activity was determined by theEllman assay (412 nm) at different time intervals (1–45 min) after incubation with different ligand concentrations (& 0 mM, ! 1 mM, 4 10 mM, 100 mM)). Data wereanalyzed by non-linear (A and B) and linear regression (C and D) and expressed as mean values � SD of two independent experiments.


G Model

TOXLET 8970 1–9

obidoxime. Deviations of oxime-induced reactivation in thepresence of ligands all proceeded in a concentration-dependentmanner as shown for edrophonium in Fig. 4A–D.

Comparison of reactivation velocity with controls (i.e., in theabsence of ligands) revealed that none of the ligands couldpromote restoration of cyclosarin-inhibited AChE by either oxime,HI-6 or obidoxime, or of sarin-inhibited AChE by HI-6. Hereby,addition of ligands resulted in an impaired reactivation velocity(Fig. 4A–C) although to a different degree.

As illustrated in Table 2, imipramine and edrophonium reducedvelocity of HI-6-induced reactivation of cyclosarin-inhibited AChEby approximately 60% and up to 30% in case of galanthamine.Restoration of sarin-inhibited enzyme by HI-6 in the presence ofedrophonium markedly decreased kobs by approximately 70%,whereas a less pronounced impairment was recorded withimipramine (54%) and galanthamine (24%). Only a minorimpairment (<15%) in reactivation velocity of cyclosarin-inhibitedAChE induced by obidoxime together with imipramine, galanth-amine and edrophonium was observed. In contrast, donepezil didnot markedly evoke deviations in reactivation at all.

Acceleration of reactivation was recorded with the testedligands (Figs. 4D and 5), only in case of obidoxime-inducedreactivation of sarin-inhibited enzyme. Imipramine and edropho-nium yielded an enhancement by approximately 50% compared toreactivation without ligands, followed by galanthamine with anincrease of kobs by a factor of 2 (Table 2). The most pronouncedacceleration in reactivation was recorded with donepezil compris-ing a 2.5 fold increased constant (Table 2).

The reactivation of sarin-inhibited AChE by obidoxime, in thepresence and absence of AChE-ligands, followed a biphasic patternwith a rapid initial increase followed by a substantially slowerphase (Fig. 5A and B). In consequence, the linear regressionanalysis of the data (Fig. 5C and D) did not intersect the origin andresulted in a marked y-intercept.

The bi-phasic reactivation indicates a re-inhibition of thereactivated enzyme by the reaction product POX formed during thereactivation and was only observed in obidoxime-inducedreactivation of sarin-inhibited AChE. AChE-ligands did not prevent


the POX effect but resulted in a higher maximum reactivation ofsarin-inhibited AChE (Fig. 5).

In order to investigate the hypothesis of re-inhibition ofreactivated AChE by POX, additional experiments with highlydiluted (100 fold) sarin-inhibited AChE were performed (Fig. 6).

It turned out, that the reactivation of sarin-inhibited AChE byobidoxime, in the presence and absence of AChE-ligands, wasobviously mono-phasic as can be derived from the linear and semi-logarithmic presentation (Fig. 6).

4. Discussion

The inhibitory potency, type of inhibition by structurallydiverse AChE-ligands and the potential enhancement of oxime-induced reactivation of OP-inhibited human AChE in the presenceof AChE-ligands was assessed. It was shown that reactivation couldnot be accelerated by the tested ligands directly, but enhancedindirectly by a protective effect of the re-inhibition by the formedPOX at high enzyme concentration.

The reversible AChE-ligands, most of them having effectivemedical applications in various neurological disorders, were foundto inhibit human AChE at different concentrations. This result is inagreement with previous results generated with AChE fromdifferent species (Delini-Stula et al., 1995; Iwanaga et al., 1994;Lockhart et al., 2000; Loewenick et al., 2001; Woltjer and Milatovic,2006; Zeldowicz and Buckler, 1965). Donepezil was the mostpotent reversible AChE-inhibitor in the series of compounds testedas summarized in Table 1. This observation was consistent withIC50 values reported by Galli et al. (1994) for electric eel AChE.Comparative data for IC50 values of galanthamine, itopride,edrophonium, propidium, pancuronium and desoxypeganine wereobtained and were found ranging from 1.0 to 12.4 mM and therebybeing more than 100 times higher than that of donepezil.Comparable IC50 values of these compounds with the exceptionof propidium were reported by several authors using enzymederived from different sources including human erythrocyte andelectric eel AChE (Andrisano et al., 2001; Iwanaga et al., 1994;Lockhart et al., 2001; Schuh,1977; Taylor and Lappi,1975; Thomsen



346 an347 To348 pr349 be350 La351 lig352 ag353

354 w355 sim356 He357 of358 Vm359 19360 ed361 su362 ac363 th364 no365 Iw366 de367 co368 co369 Bi370 th371 ca372 bi373 w374 19375 de376 lim377 in378 et379 of380 as381

382 pr383 in384 et385 m386 in387 ob388 pr389 ac390 ga391 re392 to393 re394 hu395 co396 le397 4

398

399 un400 do401 in402 py403 su404 pr405 20406 ni407 th408 PO409 en410 co411 be

412

413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

435

436

437

438

439

440

441

442

443

444

445

446

447

448

449

450

451

452

453

454

455

456

457

458

459

460

461

462Q5463

464

465466467468469470


G Model

TOXLET 8970 1–9

d Kewitz, 1990). Previous studies using AChE isolated fromrpedo californica obtained a lower inhibitory concentration foropidium compared to results of the current study, which could

attributed to species differences in AChE kinetics (Taylor andppi, 1975). The lowest inhibitory potency among all AChE-ands tested was found for imipramine, which was in goodreement with a previous report by Spinedi et al. (1989).The present study showed that pancuronium and edrophonium

ere inhibitors of a competitive type and thus, preventingultaneous substrate binding, indicated by an elevated Km.nce, increasing substrate concentration leads to a displacement

the AChE-ligand from the enzyme resulting in a comparableax. According to previous studies (Robaire and Kato,1975; Schuh,77) the competitive nature of inhibition by pancuronium androphonium was suggested to be due to binding to the anionicbsite of the active center involved in binding and hydrolysis ofetylcholine. In contrast, similar affinities for the free enzyme ande substrate-enzyme complex were found for itopride revealing an-competitive type of inhibition consistent with studies byanaga et al. (1994) and indicated by an unaffected Vmax and acreased Km in the presence of ligand. A mixed type of inhibition,mprising different affinities toward the free and the substrate-njugated enzyme, was observed with the residual AChE-ligands.nding studies proposed that imipramine and propidium bind toe peripheral anionic subsite inducing a conformational modifi-tion that leads to inhibition of substrate hydrolysis, whereasnding of donepezil is thought to involve hydrophobic bindingith aromatic residues within the active site (Shafferman et al.,92; Snape et al., 1999; Szegletes et al., 1998). In case ofsoxypeganine, the mode of binding is not resolved and onlyited published data is available with respect to the type of

hibition suggesting a competitive type of inhibition (Lockhart al., 2001). Galanthamine was identified as mixed type inhibitor human AChE while Pilger et al. (2001) classified this compound

a competitive inhibitor of Torpedo californica AChE.Oxime-induced reactivation of OP-inhibited AChE in theesence of ligands was investigated in order to gain more insightto the previously proposed acceleration phenomenon (Galli al., 1994; Harris et al., 1978; Luo et al., 1998) and the underlyingechanism of action by these ligands. On one hand an impairment

reactivation of cyclosarin-inhibited AChE by either oxime,idoxime or HI-6, and of sarin-inhibited AChE by HI-6 in theesence of ligands was found. On the other hand, a pronouncedceleration of reactivation was observed with edrophonium,lanthamine and donepezil in case of obidoxime-inducedactivation of sarin-inhibited enzyme. Hereby, one mechanism explain the enhancement of reactivation by ligands wasported by Harris et al. (1978). When using sarin-inhibitedman erythrocyte AChE it was suggested that the bisquaternarympound SAD-128 produced an allosteric modification of AChEading to a better accessibility for oximes, thereby allowing TMB-and obidoxime restoration of enzyme activity.In the current study, this mode of action was assessed to belikely because acceleration by edrophonium, galanthamine andnepezil could only be achieved in case of reactivation of sarin-hibited AChE by obidoxime. Since obidoxime and otherridinium-4-aldoximes conjugated with specific OP-residues,ch as sarin, lead to the inevitable formation of a stable reactionoduct, POX, with high anticholinesterase activity (Ashani et al.,03; Hackley and Owens, 1959; Kiderlen et al., 2000), edropho-um, galanthamine and donepezil could prevent re-inhibition ofe reactivated enzyme by POX. Thereby, ligands competed withX for the binding site of AChE, formed a complex with thezyme and arrested within the catalytic machinery. Then, thempounds were removed by a nucleophilic attack of the oximefore POX reacted with the enzyme. Accordingly, a study by Luo


et al. (1999a) proposed that enhancement of reactivation byobidoxime of MEPQ (7-(methylethoxyphosphinyl-oxy)-1-methyl-quinolinium iodide)-inhibited FBS AChE in the presence ofedrophonium which was attributed to the formation of a stablereversible complex between AChE and the ligand preventing re-inhibition of the active site serine by POX. As the extent of re-inhibition by POX decreases with increasing dilutions of inhibitedenzyme (Kiderlen et al., 2000; Worek et al., 2000), reactivation ofextensively (100 fold) diluted sarin-inhibited enzyme samples byobidoxime were investigated, and compared to correspondingreactivation kinetics of concentrated samples to verify thehypothesis of prevention of POX re-inhibition by AChE-ligands.As expected, the reactivation of highly diluted sarin-inhibited AChEby obidoxime followed a mono-phasic pattern supporting thehypothesis of POX re-inhibition in concentrated samples. Inaddition, the experiment with diluted AChE showed a negligibleeffect of AChE-ligands on obidoxime reactivation further support-ing the assumption of a protective effect of the ligands in case ofPOX formation.

Different experimental conditions of previous studies (Luoet al., 1998; Harris et al., 1978) have to be considered for thedetermination of reproducible and comparable AChE activitieswith the Ellman assay and may contribute to differences in thereactivation kinetics. Thus, another buffer composition and pH wasapplied during the assay by Luo et al. (1998) which couldcontribute to altering effects on AChE activity.

In previous experiments (Luo et al., 1998; Harris et al., 1978)residual OP inhibitors were not completely removed before theaddition of oxime and AChE-ligand so that the OP could re-inhibitthe enzyme or ligands could reduce re-inhibition by OPs. Althoughcomparable ligand concentrations were applied for reactivationkinetics in the previous studies, the experimental conditions andthe fact that a different structure of OP moiety as well as non-human AChE was used in these experiments, may have led todifferent results of the present and the previous studies.

In conclusion, the addition of AChE-ligands to OP-inhibitedhuman AChE resulted in a reduced reactivation indicating acompetition between oximes and AChE-ligands for the bindingsite. The exception was the reactivation of sarin-inhibited humanAChE by obidoxime. Here, AChE-ligands prevented the re-inhibition of the reactivated AChE by POX. In the end, the resultsof the present study did not confirm that AChE-ligands acceleratethe reactivation of OP-inhibited AChE by oximes, but indirecty byprevention of re-inhibition by the reaction product POX.

Conflict of interest

The authors declare that there are no conflicts of interest.

Transparency document

The Transparency document associated with this article can befound in the online version.

Acknowledgements

The study was funded by the German Ministry of Defense. Theauthors are grateful to T. Hannig for expert technical assistance.

References

Andrisano, V., Bartolini, M., Gotti, R., Cavrini, V., Felix, G., 2001. Determination ofinhibitors’ potency (IC50) by a direct high-performance liquid chromatographicmethod on an immobilised acetylcholinesterase column. J. Chromatogr. BBiomed. Sci. Appl. 753, 375–383.

Ashani, Y., Bhattacharjee, A.K., Leader, H., Saxena, A., Doctor, B.P., 2003. Inhibition ofcholinesterases with cationic phosphonyl oximes highlights distinctive


doi:10.1016/j.toxlet.2014.12.009


471472473

474475476477478479480481482483484485486487488489490491492493494495496497498499500501502503504505506507508509510

511512513514515516517518519520521522523524525526527528529530531532533534535536537538539540541

542543544545546547548549550551552553554555556557558559560561562563

564565566

567568569570571572573574575576577578579580581582583584585586587588589590591592593594595596597598599600601602603604605606607608609


G Model

TOXLET 8970 1–9

properties of the charged pyridine groups of quaternary oxime reactivators.Biochem. Pharmacol. 66, 191–202.

Ashani, Y., Radic, Z., Tsigelny, I., Vellom, D.C., Pickering, N.A., Quinn, D.M., Doctor, B.P., Taylor, P., 1995. Amino acid residues controlling reactivation oforganophosphonyl conjugates of acetylcholinesterase by mono- andbisquaternary oximes. J. Biol. Chem. 270, 6370–6380.

Bisswanger, H., 1979. Enzymkinetik. Theorie und Methoden der Enzymkinetik.Verlag Chemie, Weinheim, Deerfield Beach (Florida), pp. S. 55–131.

Dawson, R.M., 1994. Review of oximes available for treatment of nerve agentpoisoning. J. Appl. Toxicol. 14, 317–331.

de Jong, L.P., Ceulen, D.I.,1978. Anticholinesterase activity and rate of decompositionof some phosphylated oximes. Biochem. Pharmacol. 27, 857–863.

Delini-Stula, A., Mikkelsen, H., Angst, J., 1995. Therapeutic efficacy ofantidepressants in agitated anxious depression – a meta-analysis ofmoclobemide studies. J. Affect. Disord. 35, 21–30.

Ellman, G.L., Courtney, K.D., Andres Jr., V., Featherstone, R.M., 1961. A new and rapidcolorimetric determination of acetylcholinesterase activity. Biochem.Pharmacol. 7, 88–95.

Eyer, P., 2003. The role of oximes in the management of organophosphorus pesticidepoisoning. Toxicol. Rev. 22, 165–190.

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

Galli, A., Mori, F., Benini, L., Cacciarelli, N., 1994. Acetylcholinesterase protection andthe anti-diisopropylfluorophosphate efficacy of E2020. Eur. J. Pharmacol. 270,189–193.

Grob, D., Harvey, A.M., 1953. The effects and treatment of nerve gas poisoning. Am. J.Med. 14, 52–63.

Hackley, B.E., Owens, O.O., 1959. Preparation of O-(isopropylmethylphosphono)-4-formyl-1-methylpyridinium iodide oxime. J. Org. Chem. 24, 1120.

Harris, L.W., Heyl, W.C., Stitcher, D.L., Broomfield, C.A., 1978. Effects of 1,10-oxydimethylene bis-(4-tert-butylpyridinium chloride) (SAD-128) anddecamethonium on reactivation of soman- and sarin-inhibited cholinesteraseby oximes. Biochem. Pharmacol. 27, 757–761.

Harvey, B., Scott, R.P., Sellers, D.J., Watts, P., 1986. In vitro studies on the reactivationby oximes of phosphylated acetylcholinesterase–I: on the reactions of P2S withvarious organophosphates and the properties of the resultant phosphylatedoximes. Biochem. Pharmacol. 35, 737–744.

Holmstedt, B., 1959. Pharmacology of organophosphorus cholinesterase inhibitors.Pharmacol. Rev. 11, 567–688.

Iwanaga, Y., Kimura, T., Miyashita, N., Morikawa, K., Nagata, O., Itoh, Z., Kondo, Y.,1994. Characterization of acetylcholinesterase-inhibition by itopride. Jpn. J.Pharmacol. 66, 317–322.

Kiderlen, D., Eyer, P., Worek, F., 2005. Formation and disposition ofdiethylphosphoryl-obidoxime: a potent anticholinesterase that is hydrolyzedby human paraoxonase (PON1). Biochem. Pharmacol. 69, 1853–1867.

Kiderlen, D., Worek, F., Klimmek, R., Eyer, P., 2000. The phosphoryl oxime-destroying activity of human plasma. Arch. Toxicol. 74, 27–32.

Lockhart, B., Closier, M., Howard, K., Steward, C., Lestage, P., 2001. Differentialinhibition of [3H]-oxotremorine-M and [3H]-quinuclinidyl benzilate binding tomuscarinic receptors in rat brain membranes with acetylcholinesteraseinhibitors. Naunyn Schmiedebergs Arch. Pharmacol. 363, 429–438.

Lockhart, B., Iop, F., Closier, M., Lestage, P., 2000. (S)-2,3-dihydro-[3,4]cyclopentano-1,2,4-benzothiadiazine-1,1-dioxide: (S18986-1) a positive modulator of AMPAreceptors enhances (S)-AMPA-mediated [3H]noradrenaline release from rathippocampal and frontal cortex slices. Eur. J. Pharmacol. 401, 145–153.

Loewenick, C.V., Krampfl, K., Schneck, H., Kochs, E., Bufler, J., 2001. Open channel andcompetitive block of nicotinic receptors by pancuronium and atracurium. Eur. J.Pharmacol. 413, 31–35.

Luo, C., Ashani, Y., Doctor, B.P., 1998. Acceleration of oxime-induced reactivation oforganophosphate-inhibited fetal bovine serum acetylcholinesterase bymonoquaternary and bisquaternary ligands. Mol. Pharmacol. 53, 718–726.

Luo, C., Saxena, A., Ashani, Y., Leader, H., Radic, Z., Taylor, P., Doctor, B.P., 1999a. Roleof edrophonium in prevention of the re-inhibition of acetylcholinesterase byphosphorylated oxime. Chem. Biol. Interact. 119–120, 129–135.

Luo, C., Saxena, A., Smith, M., Garcia, G., Radic, Z., Taylor, P., Doctor, B.P., 1999b.Phosphoryl oxime inhibition of acetylcholinesterase during oxime reactivationis prevented by edrophonium. Biochemistry 38, 9937–9947.

Marrs, T.C., Rice, P., Vale, J.A., 2006. The role of oximes in the treatment of nerveagent poisoning in civilian casualties. Toxicol. Rev. 25, 297–323.

Musilek, K., Dolezal, M., Gunn-Moore, F., Kuca, K., 2011. Design: evaluation andstructure-activity relationship studies of the AChE reactivators againstorganophosphorus pesticides. Med. Res. Rev. 31, 548–575.


Pilger, C., Bartolucci, C., Lamba, D., Tropsha, A., Fels, G., 2001. Accurate prediction ofthe bound conformation of galanthamine in the active site of Torpedocalifornica acetylcholinesterase using molecular docking. J. Mol. Graph. Model.19, 288.

Quinn, D.M., 1987. Acetylcholinesterase: enzyme structure reaction dynamics, andvirtual transition states. Chem. Rev. 87, 955–979.

Robaire, B., Kato, G., 1975. Effects of edrophonium eserine, decamethonium, d-tubocurarine, and gallamine on the kinetics of membrane-bound andsolubilized eel acetylcholinesterase. Mol. Pharmacol. 11, 722–734.

Schoene, K., 1973. Phosphonyloxime of soman; formation and reaction withacetylcholinesterase in vitro. Biochem. Pharmacol. 22, 2997–3003.

Schuh, F.T., 1977. On the inhibition of cholinesterases by pancuronium. Anaesthesist26, 125–129.

Segall, Y., Waysbort, D., Barak, D., Ariel, N., Doctor, B.P., Grunwald, J., Ashani, Y., 1993.Direct observation and elucidation of the structures of aged and nonagedphosphorylated cholinesterases by 31P NMR spectroscopy. Biochemistry 32,13441–13450.

Shafferman, A., Velan, B., Ordentlich, A., Kronman, C., Grosfeld, H., Leitner, M.,Flashner, Y., Cohen, S., Barak, D., Ariel, N., 1992. Substrate inhibition ofacetylcholinesterase: residues affecting signal transduction from the surface tothe catalytic center. EMBO J. 11, 3561–3568.

Sit, R.K., Radic, Z., Gerardi, V., Zhang, L., Garcia, E., Katalinic, M., Amitai, G., Kovarik,Z., Fokin, V.V., Sharpless, K.B., Taylor, P., 2011. New structural scaffolds forcentrally acting oxime reactivators of phosphylated cholinesterases. J. Biol.Chem. 286, 19422–19430.

Snape, M.F., Misra, A., Murray, T.K., De Souza, R.J., Williams, J.L., Cross, A.J., Green, A.R., 1999. A comparative study in rats of the in vitro and in vivo pharmacology ofthe acetylcholinesterase inhibitors tacrine, donepezil and NXX-066.Neuropharmacology 38, 181–193.

Spinedi, A., Pacini, L., Luly, P., 1989. A study of the mechanism by which someamphiphilic drugs affect human erythrocyte acetylcholinesterase activity.Biochem. J. 261, 569–573.

Su, C.T., Wang, P.H., Liu, R.F., Shih, J.H., Ma, C., Lin, C.H., Liu, C.Y., Wu, M.T., 1986.Kinetic studies and structure-activity relationships of bispyridinium oximes asreactivators of acetylcholinesterase inhibited by organophosphoruscompounds. Fundam. Appl. Toxicol. 6, 506–514.

Szegletes, T., Mallender, W.D., Rosenberry, T.L., 1998. Nonequilibrium analysis altersthe mechanistic interpretation of inhibition of acetylcholinesterase byperipheral site ligands. Biochemistry 37, 4206–4216.

Taylor, P., Lappi, S., 1975. Interaction of fluorescence probes withacetylcholinesterase: the site and specificity of propidium binding.Biochemistry 14, 1989–1997.

Taylor, P., Radic, Z., Hosea, N.A., Camp, S., Marchot, P., Berman, H.A., 1995. Structuralbases for the specificity of cholinesterase catalysis and inhibition. Toxicol. Lett.82–83, 453–458.

Thomsen, T., Kewitz, H., 1990. Selective inhibition of human acetylcholinesterase bygalanthamine in vitro and in vivo. Life Sci. 46, 1553–1558.

Woltjer, R.L., Milatovic, D., 2006. Therapeutic Uses of Cholinesterase Inhibitors inNeurodegenerative Diseases. In: Gupta, R.C. (Ed.), Toxicology ofOrganophosphate and Carbamate Compounds. Elsevier Academic Press,Amsterdam, Boston, pp. 25–34.

Worek, F., Aurbek, N., Thiermann, H., 2007. Reactivation of organophosphate-inhibited human AChE by combinations of obidoxime and HI 6 in vitro. J. Appl.Toxicol. 27, 582–588.

Worek, F., Diepold, C., Eyer, P., 1999. Dimethylphosphoryl-inhibited humancholinesterases: inhibition reactivation, and aging kinetics. Arch. Toxicol. 73, 7–14.

Worek, F., Eyer, P., Kiderlen, D., Thiermann, H., Szinicz, L., 2000. Effect of humanplasma on the reactivation of sarin-inhibited human erythrocyteacetylcholinesterase. Arch. Toxicol. 74, 21–26.

Worek, F., Reiter, G., Eyer, P., Szinicz, L., 2002. Reactivation kinetics ofacetylcholinesterase from different species inhibited by highly toxicorganophosphates. Arch. Toxicol. 76, 523–529.

Worek, F., Thiermann, H., 2013. The value of novel oximes for treatment of poisoningby organophosphorus compounds. Pharmacol. Ther. 139, 249–259.

Wright, P.G., 1954. An analysis of the central and peripheral components ofrespiratory failure produced by anticholinesterase poisoning in the rabbit. J.Physiol. 126, 52–70.

Zeldowicz, L.R., Buckler, W.S., 1965. Myasthenia gravis: medical aspects. Can. Med.Assoc. J. 93, 189–197.



effect of reversible ligands on oxime-induced reactivation of sarin- and cyclosarin-inhibited human...

Documents