in vitro oxime reactivation of red blood cell acetylcholinesterase inhibited by methyl-paraoxon

168 G. A. PETROIANU ET AL.

Copyright © 2007 John Wiley & Sons, Ltd. J. Appl. Toxicol. 2007; 27: 168–175

DOI: 10.1002/jat

JOURNAL OF APPLIED TOXICOLOGYJ. Appl. Toxicol. 2007; 27: 168–175Published online 30 January 2007 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/jat.1189

In vitro oxime reactivation of red blood cellacetylcholinesterase inhibited by methyl-paraoxon

G. A. Petroianu,1,* K. Arafat,1 S. M. Nurulain,1 K. Kuca2 and J. Kassa2

1 UAE University, Al Ain-UAE2 University of Defense, Faculty of Military Health Sciences, Hradec Kralove, Czech Republic

Received 27 June 2006; Revised 9 October 2006; Accepted 9 October 2006

ABSTRACT: Oximes are cholinesterase reactivators of use in poisoning with organophosphorus ester enzyme inhibitors.

Pralidoxime (PRX) is the oxime used in the United States. Clinical experience with pralidoxime (and other oximes) is

disappointing and the routine use has been questioned. Furthermore oximes are not equally effective against all existent

enzyme inhibitors. There is a clear demand for ‘broad spectrum’ cholinesterase reactivators with a higher efficacy than

those clinically available. To meet this need over the years new reactivators of cholinesterase of potential clinical utility

have been developed.

The purpose of the study was to quantify ‘in vitro’ the extent of protection conferred by available (pralidoxime and

methoxime) and experimental (K-27, K-33 and K-48) oximes, using methyl-paraoxon (methyl-POX) as an esterase inhibitor

and to compare the results with those previously obtained using paraoxon (POX) as an inhibitor.

Red blood cell (RBC) acetylcholinesterase (AChE) activities in whole blood were measured photometrically in the pres-

ence of different methyl-POX concentrations and IC50 values calculated. Determinations were repeated in the presence of

increasing oxime concentrations. The IC50 of methyl-POX (59 nM) increased with the oxime concentration in a linear

manner. The calculated IC50 values were plotted against the oxime concentrations to obtain an IC50 shift curve. The slope

of the shift curve (tg ααααα) was used to quantify the magnitude of the protective effect (nM IC50 increase per µµµµµM reactivator).

Based on our determinations the new K-series of reactivators is superior to pralidoxime (tg ααααα ===== 1.9) and methoxime

(tg ααααα ===== 0.7), K-27 and K-48 being the outstanding compounds with a tg ααααα value of 10 (nM IC50 increase per µµµµµM

reactivator), which is ≈≈≈≈≈ five times the reactivator ability of PRX. The tg ααααα value determined for K-33 was 6.3.

The ranking of reactivator potencies of the examined oximes determined with methyl-POX as an inhibitor (K-27 ===== K-

48 >>>>> K-33 >>>>> pralidoxime >>>>> methoxime) is similar to the ranking previously reported by us using POX as an inhibitor (K-

27 ≥≥≥≥≥ K-48 >>>>> K-33 >>>>> methoxime ===== pralidoxime). There is an (expected) inverse relationship between the binding constant

K and the slope of the IC50 shift curve (tg ααααα ) for all oximes examined. K-27 and K-48 (the most protective substances

judging by the tg ααααα ) having the lowest K value (highest affinity).

In vivo testing of the new oximes as methyl-paraoxon protective agents is necessary. Copyright © 2007 John Wiley &

Sons, Ltd.

KEY WORDS: cholinesterase; organophosphate; methyl-paraoxon; oxime; pralidoxime; methoxime; K-oxime

* Correspondence to: Georg A. Petroianu, Faculty of Medicine and Health

Sciences, United Arab Emirates University, Department of Pharmacology

and Therapeutics, P.O. Box 17 666, Al Ain-United Arab Emirates.

E-mail: [email protected]

signs and symptoms, PRX is supposed to shorten the

duration of the respiratory muscle paralysis by reactiva-

tion of cholinesterases (Johnson et al., 2000; Alston,

2005; Petroianu, 2005). Clinical experience with PRX is,

however, disappointing and its routine use has been ques-

tioned (van Helden et al., 1996; Peter and Cherian, 2000;

Eddleston et al., 2002; Buckley et al., 2005; Peter et al.,

2006). There is a clear demand for ‘broad spectrum’

cholinesterase reactivators with a higher efficacy than

PRX.

Over the years new potential reactivators of cholines-

terase inhibited by organophosphorus compounds were

developed by different groups. Methoxime (MMC-4) was

synthesized and tested by Hobbiger and Sadler in the UK.

Currently, this reactivator is used by the Czech Army

after nerve agent exposure (Hobbiger and Sadler, 1959).

Introduction

Poisoning with organophosphorus cholinesterase inhibi-

tors is common and the effects have been described ex-

tensively (Namba, 1971; Namba et al., 1971; Zoch, 1971;

Petroianu et al., 1998). Oximes are the only enzyme

reactivators clinically available (Wilson and Ginsburg,

1955; Kassa, 2002). Pralidoxime (PRX) is commonly

used as an adjunct to atropine in the treatment of such

poisonings. Clinically, while atropine relieves muscarinic

OXIME REACTIVATION OF ACETYLCHOLINESTERASE 169


DOI: 10.1002/jat

The K-series of reactivators was developed by Kuca

and later Musilek in the Department of Toxicology at

the Faculty of Military Health Sciences (University of

Defense), Hradec Kralove, Czech Republic (Kuca et al.,

2003 a, b, 2004; Bajgar, 2004).

From a chemical point of view, the newly developed

oximes are bisquaternary symmetric (K-33 and

methoxime) or asymmetric (K-27 and K-48) pyridinium

aldoximes with the functional aldoxime group at position

two (K-33) or four (K-27, K-48 and methoxime) of the

pyridine (Fig. 1).

The newly synthesized AChE reactivators were pre-

viously examined with respect to their respective

abilities to reactivate paraoxon (POX) inhibited enzymes

Figure 1. Chemical structure of established and experimental oxime reactivators of organophosphorus inhibitedcholinesterase. From a chemical point of view, the newly developed oximes are bisquaternary symmetric (K-33 andmethoxime) or asymmetric (K-27 and K-48) pyridinium aldoximes with the functional aldoxime group at positiontwo (K-33) or four (K-27, K-48 and methoxime) at the pyridine rings



DOI: 10.1002/jat

(Petroianu et al., 2006). In this paper, in an attempt to

ellucidate the effect of the substituent (methyl vs ethyl)

on the ability of the oximes to reactivate an inhibited

enzyme, the efficacy of the compounds in reactivating

methyl-paraoxon (methyl-POX) inhibited RBC AChE

was examined.

Purpose of the Study

1. To determine in vitro in human blood the IC50 values

of methyl-POX

2. To quantify in vitro the protective effect of increasing

oxime concentrations on RBC-AChE activity when

exposed to methyl-POX, as assessed by the IC50 shift

3. To quantify in vitro the binding constant K of the new

oximes for RBC-AChE using methyl-POX inhibition

data (Schild plot)

Material and Methods

RBC-AChE Activity

The RBC-AChE activity was measured in diluted whole

blood samples in the presence of the selective butyryl-

cholinesterase inhibitor, ethopropazine, as previously

described (Worek et al., 1999). The assay which is

based on Ellman’s method, measures the reduction of

dithiobis-nitrobenzoic acid (DTNB) to nitrobenzoate

(TNB−) by thiocholine, the product of acetylthiocholine

hydrolysis (Ellman et al., 1961). Freshly drawn venous

blood samples were diluted in 0.1 M phosphate buffer

(pH 7.4) and incubated with DTNB (10 mM) and

ethopropazine (6 mM) for 20 min at 37 °C prior to addi-

tion of acetylthiocholine. The change in the absorbance

of DTNB was measured at 436 nm. The AChE activity

was calculated using an absorption coefficient of TNB−

at 436 nm (ε = 10.6 mM−1 cm1). The values were normal-

ized to the hemoglobin (Hb) content (determined as

cyanmethemoglobin) and expressed as mU µmol−1 Hb

(van Kampen and Zijlstra, 1961).

Determination in vitro in Human Blood of theIC50 value of Methyl-POX for RBC-AChE

Blood from human volunteers was used (n = 5, 2 males

and 3 females). None of the volunteers was on any drugs.

Enzyme activities were determined in the absence of and

then after the addition of methyl-POX. The inhibitor was

added before the incubation period. For the graphical

representation and IC50 calculation the SlideWrite™

(Advanced Graphics Software Inc, Encinitas, CA-USA)

software was used (user defined equation y = a0/[1 +(x/a1)exp a2]), where a1 corresponds to the IC50 value.

Enzyme activities were corrected for oxime induced

thiocholine-esteratic activity (Petroianu et al., 2004).

Quantification of the Protective Effect ofIncreasing Oxime Concentrations on RBC-AChEagainst Methyl-POX Inhibition, as assessed bythe IC50 Shift in vitro in Human Blood

IC50 determinations (POX for RBC-AChE) as described

above were repeated in the absence of and then in the

presence of increasing oxime concentrations. Enzyme

activities were corrected for oxime induced thiocholine-

esterase activity (Petroianu et al., 2004). The calculated

IC50 values were plotted against the oxime concentrations

to obtain an IC50 shift curve. For the graphical represen-

tation and calculations the SlideWrite™ (Advanced

Graphics Software Inc, Encinitas, CA-USA) software

was used (equation y = a0 + a1x) where a1 represents

the slope (tangent; tg α) of the IC50 shift graph. The slope

of the shift curve (tg α) was used to quantify the magnitude

of the protective effect (nM IC50 increase per µM reacti-

vator). The IC50 shift [tg α (nM/µM)] has no units.

Calculation of the Binding Constant K of Oximefor RBC-AChE

The performed measurements (IC50 shift) allow the calcu-

lation of the binding constant K of oxime for RBC-AChE.

K (the estimated amount of free substance required to

half saturate the maximal binding capacity of RBC-AChE)

was calculated using the Schild plot. The graphical method

requires plotting of log (dose ratio − 1) vs − log concen-

tration, where the dose ratio is defined as IC50 of methyl-

POX determined in the presence of oxime divided by the

IC50 of the methyl-POX determined in the absence of

oxime (Cheng, 2001; Arunlakshana and Schild, 1959).

For the graphical representation and calculations the

SlideWrite™ (Advanced Graphics Software Inc, Encinitas,

CA-USA) software was used (equation y = a0 + a1x).

Results

IC50 Value of Methyl-POX for RBC-AChE

IC50 value of Methyl-POX for RBC-AChE = 59 ± 1 nm

[95% confidence interval (CI) 55–62] (Fig. 2).

IC50 Shift [tg ααααα (nM/µµµµµM)]

Results are summarized in Table 1 and presented in

Figs 3–7. The differences between the oximes are signifi-

cant, reaching one order of magnitude.



DOI: 10.1002/jat

Figure 3. IC50 shift of methyl-POX for RBC-AChE in thepresence of increasing concentrations of PRX: increas-ing the PRX concentration from 0 to 50 µM increasesthe IC50 of methyl-POX for RBC-AChE from 59 to165 nM (∆ = 107 nM); tg α = 1.9 ± 0.1 (95% CI = 1.6–2.2). When using POX as an inhibitor the protectiveeffect of PRX was shown to be tg α = 0.3 ± 0.01 (95%CI = 0.29–0.33) (Petroianu et al., 2006). This figure isavailable in colour online at www.interscience.wiley.com/journal/jat

with PRX (and other available oximes) is mixed at best,

giving the impression of a therapeutic equivalent to ‘the

emperor has no clothes’ story.

Not only is the efficacy of the available reactivators

low, certainly none of them can be viewed as ‘broad

spectrum’, i.e. efficacious against organophosphates and

organophosphonates (Bajgar, 2004; Kassa, 2002). In view

of the real or perceived threat of organophosphorus

agent exposure the need for a highly efficacious ‘broad

Figure 4. IC50 shift of methyl-POX for RBC-AChE in thepresence of increasing concentrations of K-27: increas-ing the K-27 concentration from 0 to 10 µM increasesthe IC50 of methyl-POX for RBC-AChE from 59 to 161 nM

(∆ ≈ 102 nM); tg α = 10 ± 0.4 (95% CI = 9–12). Whenusing POX as an inhibitor the protective effect of K-27was shown to be tg α = 3.9 ± 0.2 (95% CI = 3.4–4.4)(Petroianu et al., 2006). This figure is available in colouronline at www.interscience.wiley.com/journal/jat

Table 1. IC50 shift induced by oximes

tg α [(nM/µM)]

Methoxime (MMC-4) 0.7 ± 0.03 (CI 0.6–08)

Pralidoxime (PRX) 1.9 ± 0.1 (CI 1.6–2.2)

K-33 6.3 ± 0.5 (CI 5–8)

K-48 9.9 ± 0.7 (CI 812)

K-27 10 ± 0.4 (CI 9–12)

Binding Constant K [µµµµµM] of Oximes for RBC-AChE

The results are summarized in Table 2 and presented

in Figs 8–12. The differences between the oximes are

significant, reaching one order of magnitude.

Discussion

Present Treatment Options

Pralidoxime (PRX) and related oxime class reactivators

are used in the treatment of poisoning by certain cholin-

esterase inhibitors. Clinically, while atropine relieves

muscarinic signs and symptoms, PRX is supposed to

shorten the duration of the respiratory muscle paralysis

by reactivation of cholinesterases. The clinical experience

Figure 2. Determination in vitro in human blood ofthe IC50 value of methyl-POX for RBC-AChE. The IC50

value of methyl-POX for RBC-AChE is about four timeshigher than that of POX determined under identicalconditions (59 nM vs 15 nM). This figure is available incolour online at www.interscience.wiley.com/journal/jat

Table 2. Binding constant K of oximes for RBC AChE

Binding constant K [µM]

Methoxime (MMC-4) 63

Pralidoxime (PRX) 35

K-33 9

K-27 7

K-48 6



DOI: 10.1002/jat


(∆ ≈ 67 nM); tg α = 6.3 ± 0.5 (95% CI = 5–8). When usingPOX as an inhibitor the protective effect of K-33 wasshown to be tg α = 1 ± 0.04 (95% CI = 0.89–1.04)(Petroianu et al., 2006). This figure is available incolour online at www.interscience.wiley.com/journal/jat

Figure 7. IC50 shift of methyl-POX for RBC-AChE inthe presence of increasing concentrations of MMC-4:increasing the MMC-4 concentration from 0 to 100 µM

increases the IC50 of methyl-POX for RBC-AChE from 59to 121 nM (∆ ≈ 62 nM); tg α = 0.7 ± 0.03 (95% CI = 0.6–0.8). When using POX as an inhibitor the protectiveeffect of MMC-4 was shown to be tg α = 0.4 ± 0.02(95% CI = 0.37–0.48) (Petroianu et al., 2006). This figureis available in colour online at www.interscience.wiley.com/journal/jat

IC50 Shift Determinations

Oximes are capable of increasing the IC50 values of an

organophosphorus enzyme inhibitor thus causing a dose

dependent shift. In most of the cases the shift is linear

and allows the use of the slope of the line (tg α) to quan-

tify the magnitude of the protective effect (nM IC50

increase per µM reactivator). Based on our determina-

tions the new K series of reactivators is superior to

pralidoxime and methoxime, K-27 and K-48 being the

outstanding compounds with a value of ≈ 10 (nM IC50

increase per µM reactivator) which is ≈ 5 times the

reactivator ability of PRX.

Binding Constant K

The slope of the Schild plot (a1) for all new oximes is

close to 1, indicative of a competitive mechanism of

interaction (methyl-POX and oxime). For competitive

mechanism situations (slope of the Schild plot tg α ≈ 1)

the dissociation equilibrium constant (binding constant)

K is equal to the inhibitory constant (Arunlakshana and

Schild, 1959; Cheng, 2001). Methoxime has possibly a

different mode of action.

There is an (expected) inverse relationship between

the binding constant K and the slope of the IC50 shift

curve (tg α) for all oximes examined. K-27 and K-48

(the most protective substances judging by the tg α) have

the lowest K value (highest affinity).

spectrum’ agent is urgent. Development of such sub-

stances beyond the experimental setting is hampered by

the impossibility of conducting clinical trials. While it is

understandably difficult to argue in favour of new sub-

stances without any human data, the in vitro and rodent

data strongly suggest that the new K-oximes are superior

to the clinically available ones. The results presented

here, while not definitive, are in our view a further piece

in the mosaic establishing this superiority.


(∆ ≈ 100 nM); tg α = 9.9 ± 0.7 (95% CI = 8–12). Whenusing POX as an inhibitor the protective effect of K-48was shown to be tg α = 1.4 ± 0.2 (95% CI = 0.8–2.0)(Petroianu et al., 2006). This figure is available incolour online at www.interscience.wiley.com/journal/jat



DOI: 10.1002/jat

Figure 8. Estimation of the dissociation equilibriumconstant (binding constant) K of PRX for RBC-AChEusing the Schild equation and IC50 methyl-POX shiftdata. According to the Schild equation K = [PRXconcentration]/(DR-1)]. DR, the dose ratio, is the IC50 ofthe inhibitor (methyl-POX) determined in the presenceof a protective agent (PRX), divided by the IC50 ofmethyl-POX. When using the Schild plot for graphicalestimation of the K value, the y data are log (DR-1)whereas the x data are −log [PRX]. The curve fit (r2 =0.97) is a line y = a0 + a1x where a1 represents the slopeof the line [a1 = −1.2; 95% CI = −(0.1–1.4)]. The slopeof the Schild plot (a1) is essentially 1, indicative of acompetitive mechanism of interaction (methyl-POXand PRX). The intercept of the graph with the x-axis(−log K) is ≈ 4.45. Therefore the calculated dissociationequilibrium constant (binding constant) K of PRX forRBC-AChE is ≈ 35 µM. For competitive mechanism situ-ations (slope of the Schild plot a1 ≈ 1) the dissociationequilibrium constant (binding constant) K is equal tothe inhibitory constant. The calculated binding con-stant K of PRX for RBC-AChE using POX data was ≈50 µM (Petroianu et al., 2006). This figure is available incolour online at www.interscience.wiley.com/journal/jat

Figure 9. Estimation of the dissociation equilibriumconstant (binding constant) K of K-27 for RBC-AChEusing the Schild equation and IC50 methyl-POX shiftdata. The curve fit (r2 = 0.99) is a line y = a0 + a1xwhere a1 represents the slope of the line [a1 = −1.1;95% CI = −(1–1.2)]. The intercept of the graph withthe x-axis (−log K) is ≈ 5.15. Therefore the calculateddissociation equilibrium constant (binding constant)K of K-27 for RBC-AChE is ≈ 7 µM. The calculated bind-ing constant K of K-27 for RBC-AChE using POX datawas ≈ 6 µM (Petroianu et al., 2006). This figure is avail-able in colour online at www.interscience.wiley.com/journal/jat

Comparison with Data obtained using POX as anInhibitor

The comparison is based on data previously published by

our group (Petroianu et al., 2006).

The ranking of reactivator potencies of the examined

oximes determined with methyl-POX as an inhibitor (K-27

= K-48 > K-33 > pralidoxime > methoxime) is essentially

the same as the ranking obtained using POX as an inhibi-

tor (K-27 ≥ K-48 > K-33 > methoxime = pralidoxime).

The ratios of tg α determined using methyl-POX and

POX are K-27 (2.6), K-48 (7.1), K-33 (6.3), methoxime

(1.8) and pralidoxime (5.8), which are of the same order

of magnitude as the ratio of the IC50 values of methyl-

POX and POX ≈ 4. In the in vitro model used, the pres-

ence of ethyl substituents (POX) vs methyl substituents

(methyl-POX) does not seem radically to alter the ability

of the examined oximes to protect/reactivate the esterase.

Figure 10. Estimation of the dissociation equilibriumconstant (binding constant) K of K-33 for RBC-AChEusing the Schild equation and IC50 methyl-POX shiftdata. The curve fit (r2 = 0.97) is a line y = a0 + a1 xwhere a1 represents the slope of the line [a1 = −1.16;95% CI = −(0.8–1.5)]. The intercept of the graph withthe x-axis (−log K) is ≈ 5.05. Therefore the calculateddissociation equilibrium constant (binding constant) Kof K-33 for RBC-AChE is ≈ 9 µM. The binding constant Kof K-033 for RBC-AChE (Schild plot) using POX inhibi-tion data is ≈ 16 µM (Petroianu et al., 2006). This figureis available in colour online at www.interscience.wiley.com/journal/jat



DOI: 10.1002/jat

Interestingly enough the mathematical product of bind-

ing constant K and tg α is equal to the IC50 value of the

organophosphorus enzyme inhibitor used to generate the

data, in this case methyl-POX. This observation holds

true also for the previously published data using POX as

an inhibitor.

Conclusion

Newer K-oximes have, according to our in vitro results,

superior enzyme protective properties than the presently

used substances pralidoxime and methoxime when inhi-

bition is due to the organophosphate methyl-paraoxon.

This was previously shown to be true also for inhibition

due to paraoxon.

Further in vitro work using structurally different

organophosphorus enzyme inhibitors is needed in order to

consolidate the conclusions. In vivo testing of the new

oximes as organophosphate protective agents is also

necessary and unavoidable in order to validate the

in vitro results.

References

Alston TA. 2005. Pralidoxime rescues both muscarinic and nicotinicsystems. Anesth. Analg. 101: 926–927.

Arunlakshana O, Schild HO. 1959. Some quantitative uses of drugantagonists. Br. J. Pharmacol. 14: 48–58.

Bajgar J. 2004. Organophosphates/nerve agent poisoning: mechanism ofaction, diagnosis, prophylaxis, and treatment. Adv. Clin. Chem. 38:151–216.

Buckley N, Eddleston M, Szinicz L. 2005. Oximes for acute organo-phosphate pesticide poisoning. Cochrane Database Syst Rev. 1:CD005085.

Cheng HC. 2001. The power issue: determination of KB or Ki fromIC50. A closer look at the Cheng-Prusoff equation, the Schild plot andrelated power equations. J. Pharmacol. Toxicol. Methods 46: 61–71.

Eddleston M, Szinicz L, Eyer P, Buckley N. 2002. Oximes inacute organophosphorus pesticide poisoning: a systematic review ofclinical trials. Q J Med. 95: 275–283.

Ellman GL, Courtney KD, Feather-Stone RM, Andres V Jr. 1961. Anew and rapid colorimetric determination of acetyl-cholinesteraseactivity. Biochem. Pharmacol. 7: 88–95.

Hobbiger F, Sadler PW. 1959. Protection against lethal organophosphatepoisoning by quaternary pyridine aldoximes. Br. J. Pharmacol. 14:192–201.

Johnson MK, Jacobsen D, Meredith TJ, Eyer P, Heath AJ, LigtensteinDA, Marrs TC, Szinicz L, Vale JA, Haines JA. 2000. Evaluation ofantidotes for poisoning by organophosphorus pesticides. Emerg. Med.

12: 22–37.Kassa J. 2002. Review of oximes in the antidotal treatment of poison-

ing by organophosphorus nerve agents. J. Toxicol. Clin. Toxicol. 6:803–816.

Kuca K, Bielavsky J, Cabal J, Bielavska M. 2003a. Synthesis of apotential reactivator of acetylcholinesterase — 1-(4-hydroxyimin-omethylpyridinium)-3-(carbamoylpyridinium)-propane dibromide.Tetrahedron Lett. 44: 3123–3125.

Kuca K, Bielavsky J, Cabal J, Kassa J. 2003b. Synthesis of a newreactivator of tabun-inhibited acetylcholinesterase. Bioorg. Med.

Chem. Lett. 13: 3545–3547.Kuca K, Cabal J, Patocka J, Kassa J. 2004. Synthesis of bisquaternary

symmetric bis (2-hydroxyiminomethylpyridinium) alkane dibromides

Figure 11. Estimation of the dissociation equilibriumconstant (binding constant) K of K-48 for RBC-AChEusing the Schild equation and IC50 methyl-POX shiftdata. The curve fit (r2 = 0.96) is a line y = a0 + a1xwhere a1 represents the slope of the line [a1 = −1.2;95% CI = −(0.8–1.7)]. The intercept of the graph withthe x-axis (−log K) is ≈ 5.2. Therefore the calculateddissociation equilibrium constant (binding constant) Kof K-48 for RBC-AChE is ≈ 6 µM. The calculated bindingconstant K of K-48 for RBC-AChE using POX data was≈ 10 µM (Petroianu et al., 2006). This figure is availablein colour online at www.interscience.wiley.com/journal/jat

Figure 12. Estimation of the dissociation equilibriumconstant (binding constant) K of MMC-4 for RBC-AChEusing the Schild equation and IC50 methyl-POX shiftdata. The curve fit (r2 = 0.96) is a line y = a0 + a1xwhere a1 represents the slope of the line [a1 = −1.6;95% CI = −(1–2.2)]. The slope of the Schild plot (a1) is≠ 1, indicative of a complex mechanism of interaction(methyl-POX and MMC-4). The intercept of the graphwith the x-axis (−log K) is ≈ 4.2. Therefore the calcu-lated dissociation equilibrium constant (binding con-stant) K of MMC-4 for RBC-AChE is ≈ 63 µM. Thecalculated binding constant K of MMC-4 for RBC-AChEusing POX data is ≈ 29 µM (Petroianu et al., 2006).This figure is available in colour online atwww.interscience.wiley.com/journal/jat



DOI: 10.1002/jat

and their reactivation of cyclosarin-inhibited acetylcholinesterase.Lett. Org. Chem. 1: 84–86.

Namba T. 1971. Cholinesterase inhibition by organophosphoruscompounds, and its clinical effects. Bull WHO 44: 289–307.

Namba T, Nolte CT, Jackrel J, Grob D. 1971. Poisoning due toorganophosphate insecticides: acute and chronic manifestations. Am.

J. Med. 50: 457–492.Peter JV, Cherian AM. 2000. Organic insecticides. Anaesth. Intensive

Care 28: 11–21.Peter JV, Moran JL, Graham P. 2006. Oxime therapy and outcomes in

human organophosphate poisoning: an evaluation using meta-analytictechniques. Crit. Care Med. 34: 502–510.

Petroianu GA. 2005. Pralidoxime rescues both muscarinic and nicotinicsystems: In response. (letter) Anesth. Analg. 101: 926–927.

Petroianu GA, Arafat K, Kuca K, Kassa J. 2006. Five oximes(K-27, K-33, K-48, BI-6 and methoxime) in comparison withpralidoxime: in vitro reactivation of red blood cell acetylcholines-terase inhibited by paraoxon. J. Appl. Toxicol. 26: 64–71.

Petroianu GA, Missler A, Zuleger K, Thyes C, Ewald V, Maleck WH.2004. Enzyme reactivator treatment in organophosphate exposure:

the clinical relevance of thiocholine-esteratic activity of pralidoxime.J. Appl. Toxicol. 24: 429–435.

Petroianu G, Toomes LM, Petroianu A, Bergeler W, Rüfer R. 1998.Control of blood pressure, heart rate and haematocrit duringhigh-dose intravenous paraoxon exposure in mini pigs. J. Appl.

Toxicol. 18: 293–298.van Helden HP, Busker RW, Melchers BPC, Bruijnzeel PLB. 1996.

Pharmacological effects of oximes: how relevant are they? Arch.

Toxicol. 70: 779–786.van Kampen EJ, Zijlstra WG. 1961. Standardization of hemo-

globinometry II. The hemoglobincyanide method. Clin. Chim. Acta 6:538–544.

Wilson IB, Ginsburg S. 1955. A powerful reactivator of alkylphosphate.Biochim. Biophys. Acta 18: 168–170.

Worek F, Mast U, Kiderlen D, Diepold C, Eyer P. 1999. Improveddetermination of acetylcholinesterase activity in human whole blood.Clin. Chim. Acta 288: 73–90.

Zoch E. 1971. Die Wirkung organischer Phosphorsäureester bzw.Phosphorsäureester auf versch. Enzyme. Arzneimittelforschung. 21:181–187.

in vitro oxime reactivation of red blood cell acetylcholinesterase inhibited by methyl-paraoxon

Documents