Accepted Manuscript Title: The oximes HI-6 and MMB-4 fail to reactivate soman-inhibited human and guinea pig AChE: A kinetic in vitro study Authors: Franz Worek, Horst Thiermann, Timo Wille PII: DOI: Reference:

S0378-4274(17)31382-6 https://doi.org/10.1016/j.toxlet.2017.10.005 TOXLET 9971

To appear in:

Toxicology Letters

Received date: Revised date: Accepted date:

20-8-2017 26-9-2017 4-10-2017

Please cite this article as: Worek, Franz, Thiermann, Horst, Wille, Timo, The oximes HI-6 and MMB-4 fail to reactivate soman-inhibited human and guinea pig AChE: A kinetic in vitro study.Toxicology Letters https://doi.org/10.1016/j.toxlet.2017.10.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

The oximes HI-6 and MMB-4 fail to reactivate soman-inhibited human and guinea pig AChE: A kinetic in vitro study

Franz Worek*, Horst Thiermann, Timo Wille

Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany

*Corresponding author: Bundeswehr Institute of Pharmacology and Toxicology Neuherbergstrasse 11, 80937 Munich, Germany fax: +49-89-992692-2333 E-mail address: [email protected]

1

Highlights o We determined the in vitro kinetic interactions of human and guinea pig AChE, soman, HI-6 and MMB-4. o In a static assay, HI-6 retarded the inhibition of human AChE by soman. o Both oximes were not able to prevent complete inhibition of AChE by soman in a dynamic model. o HI-6 and MMB-4 could not reactivate soman-inhibited AChE in a simulated in vivo situation

Abstract

Acetylcholinesterase (AChE) inhibited by the organophosphorus nerve (OP) agent soman underlies a spontaneous and extremely rapid dealkylation (“aging”) reaction which prevents reactivation by oximes. However, in vivo studies in various, soman poisoned animal species showed a therapeutic effect of oximes, with the exact mechanism of this effect remaining still unclear. In order to get more insight and a basis for the extrapolation of animal data to humans, we applied a dynamic in vitro model with continuous online determination of AChE activity. This model allows to simulate the in vivo toxico- and pharmacokinetics between human and guinea pig AChE with soman and the oximes HI-6 and MMB-4 in order to unravel the species dependent kinetic interactions. It turned out that only HI-6 was able to slow down the ongoing inhibition of human AChE by soman without preventing final complete inhibition of the enzyme. Continuous perfusion of AChE with soman and simultaneous or delayed (8, 15 or 40 min) oxime perfusion did not result in a relevant reactivation of AChE (less than 2%). In conclusion, the results of the present study indicate a negligible reactivation of soman-inhibited AChE by oximes at conditions simulating the in vivo poisoning by soman. The observed therapeutic effect of oximes in soman poisoned animals in vivo must be attributed to alternative mechanisms which may not be relevant in humans.

2

Key words: acetylcholinesterase; organophosphorus compound; nerve agent; oxime; enzyme kinetics; human; guinea pig; reactivation

3

1. Introduction

Despite of more than fifty years of research poisoning by the organophosphorus (OP) nerve agent soman remains a major therapeutic challenge (Eyer and Worek, 2007). Soman is an extremely potent inhibitor of acetylcholinesterase (AChE) but more important soman-inhibited AChE underlies a deleterious post-inhibitory reaction, i.e. dealkylation of the pinacolyl residue, resulting in an “aged” phosphonylated AChE which cannot be reactivated by oximes (Worek et al., 2004). The half-time of aging of soman-inhibited human AChE is in the range of ~2 min (Berry and Davies, 1966) thus reducing the window of opportunity for a successful reactivation by oximes substantially (Luo et al., 2007). However, studies with guinea pigs and rats demonstrated a therapeutic effect of oximes, primarily HI-6, in soman poisoning ex vivo and in vivo (Smith et al., 1981; Wolthuis et al., 1981; Grubic and Tomazic, 1989; Hamilton and Lundy, 1989; Lundy et al., 1992). Such species differences may be attributed to the differential aging velocity between human and animal AChE. In fact, kinetic studies with rat and guinea pig erythrocyte AChE resulted in aging half-times of ~8 min thus being some four times longer compared to human AChE (Talbot et al., 1988). Hence, testing of oximes in soman poisoning with standard animal models, i.e. oxime administration either before or immediately after soman challenge may result in a sufficient reactivation of soman-inhibited AChE in rats and guinea pigs but does by no means reflect the situation in human soman poisoning. Unfortunately, standard protocols for therapeutic animal studies generally do not include the repeated measurement of AChE activity and thus do not provide a clear evidence for reactivation of somaninhibited AChE in vivo.

4

A proper analysis of such species differences is of utmost importance for the extrapolation of data from therapeutic animal experiments to humans and for the assessment of the potential therapeutic effect of oximes in human soman poisoning. In order to get more insight and a basis for the extrapolation of animal data to humans, we designed a kinetic in vitro study using a well-established dynamic model with continuous, online determination of AChE activity, which was applied previously for the detailed analysis of interactions of erythrocyte, brain and muscle AChE from different species with OP, oximes and carbamates (Eckert et al., 2007; Herkert et al., 2010; Herkert et al., 2011b; Herkert et al., 2012). This model allows to simulate to some extent the in vivo situation in order to unravel the species dependent kinetic interactions between human and guinea pig AChE, soman and the oximes HI-6 and MMB-4, which are the candidate compounds to supplement or even replace the established oximes obidoxime and 2-PAM (Worek and Thiermann, 2013).

5

2. Materials and Methods

2.1 Materials Acetylthiocholine iodide (ATCh) and 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) were obtained

from

Sigma

(Deisenhofen,

Germany).

HI-6

(1-[[[4-

(aminocarbonyl)pyridinio]methoxy]methyl]-2-[(hydroxyimino)methyl]pyridinium dichloride monohydrate) was made available by Dr. Clement (Defence Research Establishment Suffield, Ralston, Alberta, Canada) and MMB-4 dichloride (1,1’methylenebis(4-hydroxyiminomethyl)pyridinium dichloride) was kindly donated by Prof. Fusek (Purkyne Military Medical Academy, Hradec Kralove, Czech Republic). The organophosphorus compound (OP) soman (pinacolylmethylphosphonofluoridate; >98% by GC-MS, 1H-NMR and

31P-NMR)

was supplied by the Ministry of Defence

(Bonn, Germany). Millex®-GS, 0.22 µm (Millipore, Eschborn, Germany) were employed as particle filters. All other chemicals were purchased from Merck Eurolab GmbH (Darmstadt, Germany) at the purest grade available. Soman stock solutions (0.1% v/v) were prepared in acetonitrile, stored at ambient temperature and appropriately diluted in distilled water just before the experiment. HI6 and MMB-4 stock solutions (200 mM) were prepared in distilled water, stored at 80°C and diluted as required in phosphate buffer (0.1 M, pH 7.4) on the day of the experiment. All solutions were kept on ice until the experiment.

2.2 Preparation of packed erythrocytes and erythrocyte membranes Heparinized human and Dunkin-Hartley guinea pig blood (purchased from Charles River, Sulzfeld, Germany) was centrifuged at 3000 rpm and 4°C for 10 min, the plasma was removed and the erythrocytes were washed five times with an

6

approximately three-fold volume of phosphate buffer (0.1 M, pH 7.4) to obtain packed erythrocytes for the dynamic assay. Hemoglobin-free human and guinea pig erythrocyte ghosts served as AChE source for static cuvette assays and were prepared as described from heparinized human and guinea pig blood (Worek et al., 2010). Aliquots were stored at -80°C and were homogenized prior to use.

2.3 Static model – inhibition of human and guinea pig AChE by soman and oximes 2.3.1 Inhibition of human and guinea pig AChE by HI-6 and MMB-4 The inhibition of human and guinea pig AChE by HI-6 and MMB-4 was tested as described before (Horn et al., 2015). In brief, oximes (10 - 1000 µM final concentration) were transferred to tempered cuvettes (37°C) containing phosphate buffer (0.1 M, pH 7.4), DTNB (0.3 mM) and native human or guinea pig AChE. Then, ATCh (0.45 mM) was added and the AChE activity was measured. In addition, concentration-dependent oxime blanks were determined in the absence of AChE. All experiments were performed in duplicate. The IC50 values were calculated from semilogarithmic plots of the oxime concentration versus the AChE activity.

2.3.2 Inhibition of human and guinea pig AChE by soman The inhibition kinetics were determined in the presence of the substrate acetylthiocholine as described before (Aurbek et al., 2006). In brief, 10 µl human or guinea pig erythrocyte ghosts and 5 µl diluted soman (8 different concentrations) were added to a cuvette containing phosphate buffer, 300 µM DTNB and 450 µM ATCh (final volume 3.165 ml). The inhibition kinetics were determined in the absence or presence of 100 µM HI-6 or 100 µM MMB-4. ATCh hydrolysis was continuously monitored for up to 5 min. The recorded curves were analyzed by non-linear 7

regression analysis and used for the further determination of the bimolecular reaction constant ki = k2/Kd (Eq. 1). t Kd 1 1  *   ln v k 2 GD1    k 2

(1)

with Kd: dissociation constant; k2: unimolecular phosphylation rate constant; [GD]: soman concentration; α: [S] / (Km + [S]) where [S] is substrate concentration and Km is the species specific Michaelis constant. All experiments were performed in duplicate.

2.4 Dynamic model - general experimental procedure The well-established dynamic model (Eckert et al., 2006; Eckert et al., 2008) was used for the online determination of erythrocyte AChE activity. Packed erythrocytes were re-suspended in phosphate buffer and adjusted to a final hemoglobin concentration of 5 g/dL. This dilution was stored at -80°C until preparation of the enzyme reactor. For each experiment, 80 µL of diluted erythrocytes were further diluted to 5 mL with 0.1 M phosphate buffer. Subsequently, 3.2 mL were slowly layered onto the Millex syringe filter unit (Millex®-GS, 0.22 µm, Ø 33 mm) within 10 min with a peristaltic pump. To determine maximum AChE activity, the enzyme reactor was continuously perfused at 37°C with acetylthiocholine (ATCh, 0.45 mM), DTNB (Ellman’s reagent, 0.3 mM) and phosphate buffer (0.1 M, pH 7.4) containing 0.2 % gelatine from porcine skin (w/v). The total flow rate through the reactor was 0.500 mL/min with the effluent passing a photometer set at 470 nm. The digitized absorbance values were collected at intervals of 1.6 s. Two HPLC pumps with integrated quaternary low-pressure gradient formers set up the perfusion system that was programmed by a computer using commercial HPLC software.

8

2.4.1 Perfusion protocol for erythrocyte AChE inhibition and reactivation with oximes The detailed perfusion protocol is shown in Fig. 1. In brief, the enzyme reactor was inserted at t=0 min and perfused with phosphate-gelatine buffer. At t=15 min DTNB (300 µM) and the substrate acetylthiocholine (450 µM) were added to determine the control enzyme activity (t=30 min). AChE activity was inhibited with soman (human AChE: 22 nM; guinea pig AChE: 110 nM) for 30 min (t=30-60 min). Different perfusion profiles were applied for testing the reactivation of soman-inhibited AChE by HI-6 (100 µM) and MMB-4 (100 µM), respectively (Fig. 1). Oxime perfusion was started at t=30, t=38, t=45 or t=70 min, i.e. 0, 8, 15 or 40 min after start of soman perfusion, and was continued until t=160 min. All concentrations are final concentrations at the enzyme reactor.

2.4.2 Calculations Processing of experimental data from the dynamic model was performed as described before (Herkert et al., 2011a). In brief, absorbance values were collected at intervals of 1.6 sec and analysed by a curve-fitting program (Prism™ Vers. 4.0, GraphPad Software, San Diego, CA, USA). The time-dependent inhibition of AChE was calculated by applying Eq. (2)

At=A0 * e-kt

(2)

We assumed first order kinetics, when the goodness of fit exceeded r 2 > 0.995. In addition, the maximum reactivation of soman-inhibited AChE was calculated at t=150 min by using Eq. (3)

9

%react 

At  Ai * 100 A0  Ai

(3)

Data are presented as means ± standard deviation (SD; n=4). Differences in the soman inhibition kinetics and the maximum reactivation of soman-inhibited AChE were analyzed with one-way ANOVA with Bonferroni post hoc comparisons with GraphPad Prism Vers. 4.0. A p < 0.05 was considered to be significant.

10

3. Results

The determination of the intrinsic inhibitory potential of the oximes HI-6 and MMB-4 with human and guinea pig AChE revealed marked differences between both oximes (Table 1). MMB-4 had an only marginal effect on both AChE species, calculated IC50 of >1000 µM, while HI-6 resulted in a relevant, concentration-dependent inhibition of human AChE and to less extent of guinea pig AChE. The impact of 100 µM HI-6 and 100 µM MMB-4 on the inhibition kinetics of soman was tested with human and guinea pig AChE (Fig. 2 and Table 2). It turned out, that MMB-4 had a negligible effect on soman inhibition kinetics with human AChE while HI-6 resulted in a substantial decrease of the calculated inhibition rate constant k i. With guinea pig AChE virtually no difference of ki values in the presence and absence of oximes was recorded. The determined inhibition rate constant were used to calculate the protective index of oximes (PI = ki of soman / ki of soman plus oxime), again demonstrating the substantial attenuation of HI-6 on soman inhibition kinetics of human AChE. It appears that HI-6 can partially shield the enzyme leading to a delayed phosphonylation. These data are in good agreement to the inhibition rate constants determined with the dynamic model (Table 3). Simultaneous perfusion of human AChE with soman and HI-6 resulted in a significant reduction of the inhibition rate constant k 1 in comparison to perfusion with soman alone. All other combinations did not result in a significant effect on AChE inhibition by soman. Fig 3 demonstrates the effect of soman and HI-6 perfusion on the inhibition of human AChE. Simultaneous soman and HI-6 perfusion resulted in a slower decrease of AChE activity but could not prevent total AChE inhibition.

11

Reactivation of soman-inhibited human and guinea pig AChE by 100 µM HI-6 or MMB-4 was negligible, irrespective of the start of oxime perfusion (Table 4 and Fig. 3). AChE activity at t=150 min was less than 1% of control activity with HI-6 and MMB-4 and human AChE and with HI-6 and guinea pig AChE. MMB-4 resulted in a guinea pig AChE activity of less than 2%.

12

4. Discussion

This kinetic in vitro study provided two major results. First, due to its intrinsic inhibitory activity HI-6 enables a transient attenuation of inhibition of human AChE by soman but does not prevent final complete inhibition. This effect was not observed with guinea pig AChE. Second, irrespective of the time of addition of HI-6 or MMB-4 no relevant reactivation of soman-inhibited human and guinea pig AChE could be recorded in a dynamic setup simulating to some extent the in vivo situation. Previous in vitro studies on the reactivation of soman-inhibited AChE by oximes gave rather variable results. In a number of studies the inhibition of AChE by soman was performed under conditions to prevent premature aging, i.e. pH 9-10 and cooling, with excess soman being removed before adding an oxime (de Jong and Wolring, 1980; Schoene et al., 1983; de Jong and Kossen, 1985; Worek et al., 1998; Luo et al., 2007). An in part remarkable reactivation was observed in these studies, HI-6 being in general the lead compound. Other studies used short (Puu et al., 1986) or undefined incubation times of soman with AChE (Wei et al., 2016). Hence, there is convincing evidence that oximes are able to reactivate soman-inhibited AChE under specific conditions, i.e. prevention of premature aging and absence of excess soman during the reactivation process. Due to the rather high volatility of soman, the most likely exposure route is by inhalation. Determination of the toxicokinetics of soman stereoisomers in guinea pigs after inhalation exposure demonstrated a rapid increase of agent concentrations in blood followed by a decrease with terminal half-times between 8 and 63 minutes, depending on the dose and exposure time (Langenberg et al., 1998). Hence, relevant soman concentrations are most likely present at the time of injection of oxime (and atropine) during emergency treatment or self- and buddy aid. In contrast to above 13

mentioned studies using static test tube protocols the dynamic model allows the continuous perfusion of AChE with soman and oxime thus enabling the application of different perfusion protocols to determine the dynamic changes of AChE activity online. Perfusion of AChE with 100 µM HI-6 or MMB-4 had no significant effect on the inhibition of AChE by soman, except with HI-6 and human AChE. This may be attributed to the intrinsic inhibitory potency of HI-6 (IC50 of 218 µM) resulting in a transient shielding of the enzyme. Due to the reversible nature of HI-6 inhibition and the higher affinity of soman to the active site of AChE HI-6 appears not able to prevent complete inhibition of the enzyme within short time. In vitro studies with soman-inhibited, non-aged AChE revealed a species dependent reactivating potency of HI-6 and MMB-4. With guinea pig AChE second order reactivation rate constants of 0.051 and 0.038 mM-1min-1 were determined for HI-6 and MMB-4, respectively (Luo et al., 2007). Corresponding values for human AChE were 21.51 and 0.785 mM-1min-1, demonstrating a rather low reactivating potency of both oximes with guinea pig AChE, which may be attributed to distinct structural differences between human and guinea pig AChE (Cadieux et al., 2010), and an almost 30fold higher potency of HI-6 compared to MMB-4 with human AChE. Continuous perfusion of AChE with soman for 30 min and start of oxime perfusion at 0, 8, 15 and 40 min did not result in a relevant AChE reactivation (Table 4). This result was not unexpected with human AChE due to the rapid aging of somaninhibited human AChE. Nominally, a perfusion of human AChE with soman for 30 min resembles 15 aging half-times. Even simultaneous perfusion with soman and HI-6 did not prevent complete AChE inhibition within 20 min (Fig. 3) and perfusion with soman for another 10 min obviously resulted in completely aged AChE.

14

To some extent surprising were the results with guinea pig AChE. Due to a slower aging velocity, t½ ~8 min, a small portion of soman-inhibited AChE should be in a reactivatable state at the end of soman perfusion. The failure of HI-6 and MMB-4 to reactivate inhibited AChE may be attributed to the low reactivating potency of both oximes (Luo et al., 2007). On the base of the published reactivation rate constants and the applied oxime concentration (100 µM) a reactivation half-time of ~50 min and 260 min can be calculated for HI-6 and MMB-4, respectively. Hence, the reactivation velocity was obviously too slow to prevent ongoing and complete aging of somaninhibited guinea pig AChE. Numerous in vivo studies in soman poisoned mice, rats and guinea pigs investigated the therapeutic effect of atropine and oxime combinations and protective ratios (PR = LD50 with treatment / LD50 without treatment) up to 9 were reported, while single atropine treatment resulted in a PR of less than 3 in these species (Dawson, 1994). These data imply a therapeutic effect of oximes in vivo, but this effect cannot be attributed to reactivation of AChE (Busker et al., 1996). The difficulty of correlating in vitro reactivation with in vivo efficacy data prompted the search for additional mechanisms of oximes and it was postulated that oximes may have a direct effect not related to AChE reactivation (van Helden et al., 1996). In fact, with isolated rat and guinea pig respiratory muscles a therapeutic effect of high doses of oximes, primarily HI-6, was demonstrated despite complete aging of inhibited AChE (Melchers et al., 1991; van Helden et al., 1991; Tattersall, 1993). Unfortunately, this effect was not reproducible in soman treated human intercostal muscle preparations despite of using up to 1000 µM HI-6 (Seeger et al., 2011). In conclusion, the results of the present study indicate a negligible reactivation of soman-inhibited human and guinea pig AChE by oximes at conditions simulating the in vivo poisoning by soman. The observed therapeutic effect of oximes in soman 15

poisoned animals in vivo must be attributed to alternative mechanisms which may not be relevant in humans. Hence, further attempts to improve treatment of human soman poisoning must be directed to a prevention or retardation of aging and to the development of generic therapeutics, e.g. antinicotinics or stoichiometric or catalytic scavengers of incorporated soman.

16

Acknowledgements

The study was funded by the German Ministry of Defence. The authors are grateful to M. Baumann, T. Hannig and J. Letzelter for expert technical assistance.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

17

Legends

Fig. 1

Perfusion protocol of pumps A and B for the treatment of erythrocyte-loaded enzyme reactors with constant soman and oxime concentrations. 1st row: Simultaneous start of soman and oxime perfusion; 2 nd row: Start of oxime perfusion 8 min after soman; 3rd row: Start of oxime perfusion 15 min after soman; 4th row: Start of oxime perfusion 40 min after soman.

Fig. 2

Soman (GD) inhibition kinetics of human (A) and guinea pig AChE (B) in the absence and presence of 100 µM HI-6 or MMB-4. Secondary plot of 1/k1 versus 1/([GD](1- α)). [GD] is the inhibitor concentration, whereas α stands for [S] / (Km + [S]) with [S] substrate concentration and Km Michaelis constant.

Fig. 3

Inhibition and reactivation of immobilized human erythrocyte AChE. Perfusion of erythrocyte-loaded enzyme reactors with soman (t=30-60 min) followed by HI-6 (100 µM; t=30, t=38, t=45 or t=70 min until t=160 min). The inset shows the inhibition of AChE by soman (t=35-55 min) in the presence of different HI6

perfusion

protocols.

Data

are

representative single experiments.

18

shown

as original recordings of

Table 1

Static inhibition of human and guinea pig AChE by HI-6 and MMB-4

Oxime

IC50 (µM)

HI-6

218 ± 7#

MMB-4

>>1000#

HI-6

635 ± 47

MMB-4

>>1000

Human AChE

Guinea pig AChE

The inhibition of native human and guinea pig AChE by HI-6 and MMB-4 was tested with 10 concentrations (1–1000 µM) and was referred to control AChE activity. The IC50 values were calculated from semi-logarithmic plots of the oxime concentration versus the AChE activity (n = 2). # from Winter et al. (2016).

19

Table 2

Static inhibition kinetics of soman with human and guinea pig AChE

ki (M-1min-1)

PI

Soman

1.7 ± 0.05*108

1

Soman + HI-6

3.4 ± 0.11*107

5.1

Soman + MMB-4

1.3 ± 0.09*108

1.3

Soman

4.6 ± 0.21*107

1

Soman + HI-6

4.2 ± 0.15*107

1.1

Soman + MMB-4

5.6 ± 0.30*107

0.8

Inhibitor / oxime

Human AChE

Guinea pig AChE

Second order inhibition rate constants ki are given as means ± SD (n=2) in the absence and presence of 100 µM HI-6 or MMB-4. The protective index (PI) was calculated from the ratio of ki in the absence of oxime and ki in the presence of oxime.

20

Table 3

Dynamic inhibition kinetics of soman with human and guinea pig AChE

k1 (min-1) No oxime

0.53 ± 0.01

Start of oxime perfusion

HI-6

MMB-4

0 min

0.17 ± 0.03$

0.45 ± 0.09

8 min

0.42 ± 0.06

0.51 ± 0.01

15 min

0.49 ± 0.03

0.55 ± 0.01

40 min

0.56 ± 0.01

0.58 ± 0.01

Human AChE

No oxime

0.58 ± 0.01

Start of oxime perfusion Guinea pig AChE

HI-6

MMB-4

0 min

0.43 ± 0.05

0.57 ± 0.03

8 min

0.46 ± 0.07

0.50 ± 0.03

15 min

0.58 ± 0.04

0.54 ± 0.03

40 min

0.57 ± 0.02

0.53 ± 0.09

The first-order inhibition rate constants (k1) were calculated by non-linear regression analysis from original recordings of the dynamic model by using Eq. (2). Oxime treatment (100 µM) 0, 8, 15 or 40 min after start of soman perfusion. Data are given as means ± SD (n=4). $ p < 0.05 to soman only.

21

Table 4

Maximum reactivation of soman-inhibited human and guinea pig AChE

%reactivation (t=150 min) Start of oxime perfusion

HI-6

MMB-4

0 min

0.11 ± 0.11

0.38 ± 0.23

8 min

0.10 ± 0.04

0.38 ± 0.20

15 min

0.27 ± 0.34

0.28 ± 0.09

40 min

0.52 ± 0.22

0.08 ± 0.18

0 min

0.48 ± 0.07

1.80 ±0.23

8 min

0.50 ± 0.05

1.73 ± 0.14

15 min

0.49 ± 0.10

1.93 ± 0.39

40 min

0.34 ± 0.16

1.53 ± 0.30

Human AChE

Guinea pig AChE

Perfusion of erythrocyte-loaded enzyme reactors with soman followed by oxime treatment (100 µM) 0, 8, 15 or 40 min after start of soman perfusion. Maximum reactivation at t=150 min is given as % reactivation (means ± SD; n=4).

22

Fig. 1

23

Fig. 2

24

Fig. 3

25

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Horn, G., Wille, T., Musilek, K., Kuca, K., Thiermann, H., Worek, F., 2015. Reactivation kinetics of 31 structurally different bispyridinium oximes with organophosphate-inhibited human butyrylcholinesterase. Arch.Toxicol. 89, 405– 414. De Jong, L., Kossen, S.P., 1985. Stereospecific reactivation of human brain and erythrocyte

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30

A

modified

kinetic

approach.