Toxicology in Vitro 24 (2010) 1838–1844

Contents lists available at ScienceDirect

Toxicology in Vitro

journal homepage: www.elsevier .com/locate / toxinvi t

Passive diffusion of acetylcholinesterase oxime reactivators throughthe blood–brain barrier: Influence of molecular structure

Jana Zdarova Karasova a,*, Miroslav Pohanka b, Kamil Musilek a, Filip Zemek a, Kamil Kuca a,b

a Department of Toxicology, Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republicb Center of Advanced Studies, Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic

a r t i c l e i n f o

Article history:Received 1 February 2010Accepted 17 May 2010Available online 26 May 2010

Keywords:Immobilized artificial membraneBlood–brain barrierCNS penetrationReactivatorAcetylcholinesteraseHPLC

0887-2333/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.tiv.2010.05.009

* Corresponding author.E-mail address: karasova@pmfhk.cz (J.Z. Karasova)

a b s t r a c t

In this in vitro study, high-performance liquid chromatography (HPLC) was used to determinate the pen-etration of 30 acetylcholinesterase (AChE) reactivators through the blood–brain barrier (BBB). Accordingto our method, monoquaternary AChE reactivators were found to be able to penetrate the BBB. In addi-tion to molecular structure, molecular weight appears to be an important factor for passive transport ofoximes through the BBB. For bisquaternary reactivators, the connecting linker plays a key role in the abil-ity to penetrate into the central nervous system (CNS): simple, short linkers tend to facilitate permeation.The location of groups on the pyridine ring also influences passive transport into the brain; the optimumposition of the oxime group was found to be position four (para) and substitution of the oxime group onthe pyridine ring by carbamoyl or amidoxime group markedly decreased penetration of AChE reactivatorsinto the CNS.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

There is an increasing interest in predicting the passive transferof drugs from the systemic circulation into the central nervous sys-tem (CNS). In healthy individuals, the passive penetration of differ-ent compounds is restricted by the continuous layer of theendothelium lining the capillaries, commonly called the blood–brain barrier or ‘‘BBB” (Tsukita et al., 2001; Turksen and Troy,2004). The BBB presents both a physical and metabolic barrier.The latter being due to the presence of a range of degradation en-zymes (Lorke et al., 2008). Lipophilic drugs (including those whichtarget the CNS) may enter the brain by passive diffusion via thetranscellular route, although compounds may also penetrate theBBB by active processes involving influx and efflux transporters(Yoon et al., 2006).

Oxime reactivators play a key role in the treatment of poisoningcaused by organophosphate inhibitors (OPIs) (Marrs et al., 2006).The main target of OPIs is the enzyme acetylcholinesterase (AChE;E.C. 3.1.1.7). Active AChE is essential to the normal functioning ofcholinergic transmission in both the peripheral and central ner-vous systems (Reiner et al., 2007). However, the actual mechanismof action of OPIs within the central nervous system is likely due toa complex system of reactions, some of which are still not fullyunderstood (Balali-Mood and Balali-Mood, 2008).

ll rights reserved.

.

Strong inhibition of AChE in central nervous system (CNS) alsoleads to imbalances of other neurotransmitters. For example, cate-cholamine levels are also significantly changed following exposureto OPIs (Cassel et al., 1997). Interestingly, OPI intoxication influ-ences the permeability of the blood–brain barrier (BBB) which isclearly of toxicological and pharmacological relevance. Concomit-tant changes effected by exposure to OPIs within the central ner-vous system also include metabolic imbalances, changes in brainenergy metabolism, oxidative metabolism and ATP levels (Bajgar,2004).

The main issues relating to AChE reactivator’s effect on the CNShave previously been discussed (van Helden et al., 1996; Yanagis-awa et al., 2006): according to basic pharmacokinetic principles,the BBB is relatively impermeable to higly hydrophilic drugs suchas AChE reactivators due to their quaternary structure and hydro-philic oxime group (Terasaki and Ohtsuki, 2005) and so these ther-apeutics are not generally considered to be candidates forefficacious reactivation of AChE within the CNS (Bajgar, 2004).Conversely, some authors have claimed that a central reactivationeffect does exist (Bajgar et al., 1972; Ligtenstein and Kosen, 1983),the evidences for this being based on recovery of AChE activityafter oxime administration (Berend et al., 2008; Karasova et al.,2008, 2009a). The ability of the monoquaternary oxime (2-PAM)to penetrate the BBB was directly confirmed by Sakurada et al.(2003). Therefore, it seems plausible that penetration of a rela-tively small quantity of reactivator to some parts of the CNS mightbe enough to elicit a pharmacological effect and subsequently reac-tivate a small percentage of the inhibited AChE. The ability of

J.Z. Karasova et al. / Toxicology in Vitro 24 (2010) 1838–1844 1839

bisquaternary oximes to penetrate the blood–brain barrier is hotlydiscussed nowadays (Lorke et al., 2008).

The aim of this study was to identify which specific aspects ofmolecular structure influence the penetration of AChE reactivatorsthrough the BBB. For this purpose, we choose an in vitro HPLCmethod which employs an immobilized artificial membrane(IAM). This particular technique has previously been used to pre-dict the passive transport of drugs across biological membranesand so has obvious application as a screening method for predict-ing BBB permeation. The method was initially validated on a set of21 therapeutics (Table 1) and consequently used for a set of 30AChE reactivators of varying structure including current AChEreactivators (HI-6, obidoxime, pralidoxime, etc.) and a range of no-vel reactivators with promising reactivation potency (K027, K075,etc.). Their structures are depicted in Table 2.

2. Experimental

2.1. Chemicals

Atenolol, b-estradiol, caffeine, cefuroxime, chlorpromazine,cimetidine, corticosterone, desipramine, enalapril, hydrocortisone,ibuprofen, imipramine, lomefloxacin, loperamide, nadolol, piroxi-cam, progesterone, promazine, propranolol, and testosterone werepurchased from Sigma Aldrich (Prague branch, Czech Republic).Their structures are shown in Table 1. Acetonitrile gradient gradeLiChrosolv was purchased from Merck (Darmstadt, Germany).KH2PO4, Na2HPO4, KCl, and NaCl were purchased from Lachema(Neratovice, Czech Republic). AChE reactivators were synthesizedpreviously in our laboratory. Their structures are shown in Table2. Water was obtained by reverse osmosis using a Milliporesystem.

2.2. Apparatus

The HPLC system consisted of a P200 gradient pump (Spectra-Physics Analytical, Fremont, USA), a 7125 injection valve – 10 ulloop (Rheodyne, Cotati, USA), an UV1000 detector (Spectra-PhysicsAnalytical, Fremont, USA) and a CSW Chromatography Station 1.5software (DataApex, Praha, Czech Republic).

Marvin was used for drawing, displaying and characterizingchemical structures, substructures and also for calculating of phys-ico-chemical properties (pKa, log P, PSA), Marvin 5.1.0, 2008,ChemAxon (http://www.chemaxon.com).

2.3. Chromatographic conditions

For analyses an IAM.PC.DD 2 (150 � 4.6 mm; 12 lm) column(Regis Technologies, Morton Grove, IL) was used. The mobile phasewas 80% PBS and 20% acetonitrile (v/v) at pH 7.4 (adjusted usingNa2HPO4). Phosphate-buffered saline (PBS) was prepared with2.7 mM KCl, 1.5 mM KH2PO4, 137 mM NaCl, and 8.1 mM Na2HPO4.It was delivered isocratically at a flow-rate of 1 ml/min. The absor-bance was measured at 210 nm. The temperature of column wasregulated and come up to 37 �C.

2.4. Method

Samples of each drug were maintained and measured at pH 7.4to ensure that the degree of chemical ionization was representativeof that expected in physiological fluids. In this study, we deter-mined kIAM (capacity factor of this special column) for the set of21 therapeutics (standards) and novel AChE reactivators. Thecapacity factor was calculated according to the equation, below.

kIAM ¼tr � t0

t0

tr is the retention time of the drug and t0 is the hold up time of thecolumn.

In this study, the kIAM was determined for standards mentionedbefore as the first parameter. The knowledge about penetration(Table 1) of these drugs through the BBB was compared with mea-sured kIAM. The results were corrected by power function transfor-mation of molecular weight according to the equation, below.

X ¼ kIAM

MW4

The results for the standard drugs were subsequently correlatedwith physico-chemical constants. Specifically, logarithm of octanolwater partition coefficient (log P), molecular polar surface area(PSA) and molecular weight (MW) (Table 1). The same processwas performed for the novel AChE reactivators (Table 2).

The log P values indicate that the majority of test substanceswere more soluble in the water phase. The PSA parameter is thesum of surface areas of polar atoms (oxygen, nitrogen and hydro-gen) within a molecule. These two parameters are useful for theprediction of drug transport properties and are important for eval-uating the ability of different drugs to penetrate through the BBB(Chen et al., 2009; Lanevskij et al., 2009). The calculated parame-ters, when plotted as a function of X, demonstrated good correla-tion and hence the validity of this approach (Figs. 1 and 2).

The PSA has been previously shown to correlate with humanintestinal absorption (Palm et al., 1998; Zhu et al., 2002). WhenPSA is applied to a larger and more diverse compound set, misin-terpretation of the achieved data become more frequent (Clark,1999). However, good correlation was observed between PSA andkIAM/MW4 for drugs used in this study (linear correlation coeffi-cient = 0.720; Fig. 2).

The CNS� drugs (which do not readily penetrate the BBB) lackedability to bind the phosphatidylcholine column correlated by theirMW (low permeability values <0.64). In contrast, CNS+ drugs(drugs with higher passive penetration through the BBB) have ahigher column affinity and so their permeability values (correlatedby their MW) were consistently >1.61. Thus, based on this study of21 therapeutic agents, a permeability value >1.61 represents thethreshold at which drugs can readily penetrate the BBB whereasa value is below 0.64 is indicative that the drug will not crossthe BBB.

Following method validation, the set of 30 structurally differentnovel AChE reactivators were measured in triplicate under similarconditions. kIAM values were calculated from the retention times ofthe AChE reactivators and correlated with molecular weights.Using this approach, it was possible to predict the BBB permeabil-ity of the novel reactivators.

3. Results

Several molecular characteristics correlate with the ability ofoximes to penetrate the BBB primarily molecular structure andmolecular weight (Table 2). According to our methodology, themonoquaternary oximes are able to penetrate through the BBB10–15-fold more effectively than bisquaternary oximes. The reasonmay be explained by presence of only one quaternary nitrogen ineach molecule of novel AChE reactivator and lower molecularweight when compared with bisquaternary or triquaternaryreactivators.

The position of the oxime group on the pyridinium ring alsoinfluenced the ability of AChE reactivators to penetrate into CNS.For monoquaternary structures (e.g. compounds 5, 29 and 30; Ta-ble 2), the best results were obtained with the para position on the

Table 1Formulas and physico-chemical parameters of standards.

No. MW pKa log P PSA (Å2) tr (min) Result (X)

1. CefuroximeCNS�

424 3.15 �1.44 201.89 1.82 0.13

2. EnalaprilCNS�

376 3.18/5.19 2.10 98.77 1.71 0.16

3. LomefloxacineCNS�

352 5.65/8.70 2.06 80.29 2.90 0.80

4. PiroxicameCNS�

331 4.27 1.02 110.81 3.45 1.38

5. NadololCNS�

309 9.76 0.37 86.53 3.01 1.44

6. AtenololCNS�

266 9.67 0.26 84.58 2.24 1.44

7. HydrocortisoneCNS�

362 12.74 1.47 94.83 5.51 1.90

8. CimetidineCNS�

252 6.92 0.24 114.19 2.39 2.08

9. CorticosteroneCNS�

346 – 2.48 74.60 8.90 4.08

10. LoperamideCNS�/CNS+

477 9.41 4.71 44.98 57.33 8.32

11. CaffeineCNS+

194 1.50 �0.79 58.44 1.99 3.75

12. IbuprofeneCNS+

206 4.86 3.83 40.13 5.67 18.67

13. PropranololCNS+

259 9.67 2.50 46.07 21.30 34.19

14. Progesterone 314 – 4.63 34.14 56.07 43.34

1840 J.Z. Karasova et al. / Toxicology in Vitro 24 (2010) 1838–1844

Table 1 (continued)

No. MW pKa log P PSA (Å2) tr (min) Result (X)

CNS+

15. TestosteroneCNS+

288 – 3.54 37.30 64.12 70.24

16. ImipramineCNS+

280 9.19 4.01 7.68 89.71 110.00

17. DesipramineCNS+

266 10.02 3.64 19.85 95.09 143.29

18. p-ToluidineCNS+

107 5.46 2.78 3.24 4.08 163.14

19. PromazineCNS+

284 9.20 4.04 32.98 153.76 180.28

20. ChlorpromazineCNS+

319 9.19 4.56 32.98 274.48 203.29

21. b-EstradiolCNS+

272 10.33 3.71 40.46 267.90 372.58

J.Z. Karasova et al. / Toxicology in Vitro 24 (2010) 1838–1844 1841

pyridinium ring. The less effective position was ortho (Karasovaet al., 2010, in press). In the bisquaternary group, the same rulewas confirmed (compounds 19, 21, 3 or 25, 26, 4 or 7, 13; Table2 respectively).

Passive penetration into the CNS may also be strongly influ-enced by the linker connecting two pyridinium rings. Extensionof the carbon linker is accompanied with a reduced ability to pen-etrate the BBB (Table 2; compare compounds 23, 4, 27 or 17, 3).The inclusion of other moieties into the linker also leads to a reduc-tion in predicted permeability. Furthermore, substituents such asxylene (compounds 13 and 17), double bond (compounds 20 and23) or oxygen (compounds 3 and 4) all significantly reduced pre-dicted permeation through the BBB. If cis/trans butane configura-tion is discussed, cis isomer prefers to penetrate into the CNSmuch more than trans isomer.

Increasing the number of oxime groups also had a detrimentaleffect on predicted diffusivity (compounds 1, 2, 3 and 28). The typeof substituents (as well as position on the pyridinium ring) wasalso a factor influencing predicted BBB diffusivity. For example,substitution of the oxime group by carbamoyl group or amidoxime

group markedly decreased the predicted penetration of AChE reac-tivators into the CNS (compounds 10, 12 and 20). This phenome-non is clearly observed if the oxime group is replaced bycarbamoyl group (compounds 9, 17 or 11, 24 or 3, 18).

4. Discussion

The ability of AChE oxime reactivators to penetrate into thebrain plays the key role in therapy of organophosphate intoxica-tions (Karasova et al., 2009b). The results of this study indicatethat bisquaternary AChE reactivators have a relatively low pre-dicted permeability value (range 0.538–1.780) which indicatesthat these compounds may be poorly absorbed across the BBB.However, the monoquaternary AChE reactivators are predictedto more effectively penetrate into the CNS which is in agreementwith previous studies which report oxime penetration of �10%for monoquaternary and 1–2% for bisquaternary compounds(Gyenge et al., 2007; Kalász et al., 2009; Lorke et al., 2007; Petro-ianu et al., 2007).

Table 2Formulas and physico-chemical parameters of tested AChE reactivators.

No. MW pKa log P PSA (Å2) tr (min) Result (X)

1. HLö-7 331.33 3.68 �6.83 105.31 2.17 0.555

2. HI-6 Cl 288.30 3.87 �5.97 85.0 2.24 1.047

3. Obidoxime 288.30 8.28 �6.09 82.17 2.54 1.381

4. Trimedoxime 286.33 9.4 �6.61 72.94 2.77 1.682

5. Pralidoxime 137.16 5.58 �3.12 39.3 2.03 15.866

6. K 388 464.54 9.16 �10.05 112.65 3.19 0.312

7. K 107 348.40 5.58 �4.73 78.6 2.33 0.538

8. K 108 348.40 5.15 �4.73 78.6 2.68 0.720

9. K 127 316.36 8.72 �7.45 92.67 2.41 0.852

10. K 269 313.35 8.85 �6.98 98.96 2.46 0.926

11. K 048 300.36 9.86 �7.15 83.44 2.35 0.992

12. K 203 298.34 8.9 �7.12 83.44 2.33 1.000

13. K 113 348.40 9.22 �4.98 72.94 3.25 1.018

14. K 206 298.34 8.9 �7.12 83.44 2.39 1.058

15. BI-6 296.34 4.77 �1.52 68.4 2.38 1.077

16. K 237 288.30 3.83 �3.88 95.5 2.31 1.125

1842 J.Z. Karasova et al. / Toxicology in Vitro 24 (2010) 1838–1844

Table 2 (continued)

No. MW pKa log P PSA (Å2) tr (min) Result (X)

17. K 117 316.36 8.62 �6.96 82.17 2.79 1.144

18. K 027 286.33 8.00 �7.6 83.44 2.34 1.190

19. K 316 288.30 3.53 �5.85 87.83 2.49 1.325

20. K 075 298.34 8.51 �6.13 72.94 2.69 1.350

21. K 317 288.30 6.86 �6.09 87.83 2.53 1.370

22. K 282 298.34 8.51 �6.13 72.94 2.76 1.418

23. K 074 300.36 9.48 �6.16 72.94 2.81 1.427

24. K 033 300.36 5.03 �5.91 78.6 2.85 1.465

25. K 099 286.33 7.65 �6.61 72.94 2.61 1.499

26. K 005 286.33 4.91 �6.37 78.6 2.64 1.534

27. Metoxime 258.28 8.23 �6.3 72.94 2.33 1.780

28. K 156 243.30 9.79 �6.25 40.35 3.35 4.500

29. 3-PAM 137.16 8.28 �3.24 36.47 2.04 16.083

30. 4-PAM 137.16 9.66 �3.24 36.47 2.09 17.170

Fig. 1. Correlation between log P and kIAM/MW4 for tested 21 drugs. Goodcorrelation was found with the correlations coefficient (r2) being 0.668 at pH 7.4.

Fig. 2. Correlation between polar surface area (PSA) and kIAM/MW4 determined atthe mobile phase pH of 7.4 for the tested drugs.

J.Z. Karasova et al. / Toxicology in Vitro 24 (2010) 1838–1844 1843

Fig. 3. Structure of proposed oxime.

1844 J.Z. Karasova et al. / Toxicology in Vitro 24 (2010) 1838–1844

Considering the hydrophilicity of oxime reactivators, penetra-tion through the BBB cannot be explained by simple diffusion. In-deed, monoquaternary oximes (such as pralidoxime) are known toundergo active transport by specific Na+-dependent pumps (Saku-rada et al., 2003). In contrast, there are no known transport sys-tems for bisquaternary oximes. The results of this study indicatethat there is a dependency between passive CNS penetration andthe structure of reactivators. The least penetrating compoundwas K388 which has three quaternary nitrogens, a branched linkerand three hydrophilic oxime groups. In contrast, K156 seems to bethe most promising bisquaternary reactivator. It bears a short car-bon linker with only one oxime group (in the para ring position).Unfortunately, this oxime is relatively toxic. Although a carbamoylgroup could be introduced to its structure to reduce this adverseeffect (Bajgar et al., 2007). However, one would predict that sucha modification would undergo reduced transport across the BBB.

According to our understanding of molecular factors whichinfluence BBB transport, enhanced flux across the BBB (and thusimproved efficacy) could be attained by designing a reactivatorwith a short carbon linker (C1–C2) in the absence of additionalfunctional groups and preferably with one or two oxime groupsin the para position (Fig. 3).

In summary, structural factors affecting BBB penetration werepredicted using HPLC methodology. The method described in ourstudy will be incorporated into future work to develop AChE reac-tivators to focus discovery on centrally active oximes. However, itshould be mentioned that our method can be used only for passivetransport prediction. Clearly, more sophisticated methods shouldbe used to assess the contribution of active transport mechanismsacross the BBB.

Acknowledgements

Authors would like to thank the Ministry of Health (CzechRepublic) – ‘‘The Evaluation of Penetration of Different Acetylcho-linesterase Reactivators through Blood–Brain Barrier”. Thanks aredue to Mrs. M. Hrabinova and Mr. P. Stodulka for skilled technicalassistance and R.P. Chilcott (Chemical Hazards and Poisons Divi-sion, Health Protection Agency, Oxfordshire, United Kingdom) forlanguage correction.

References

Bajgar, J., 2004. Organophosphates/nerve agent poisoning: mechanism of action,diagnosis, prophylaxis, and treatment. Advances in Clinical Chemistry 38, 151–216.

Bajgar, J., Jakl, A., Hrdina, V., 1972. The influence of obidoxime onacetylcholinesterase activity in different parts of the mouse brain followingisopropylmethyl phosphonofluoridate intoxication. European Journal ofPharmacology 19, 199–202.

Bajgar, J., Kuca, K., Jun, D., Bartosova, L., Fusek, J., 2007. Cholinesterase reactivators:the fate and effects in the organism poisoned with organophosphates/nerveagents. Current Drug Metabolism 8, 803–809.

Balali-Mood, M., Balali-Mood, K., 2008. Neurotoxic disorders of organophosphoruscompounds and their managements. Archives of Iranian Medicine 11, 65–89.

Berend, S., Vrdoljak, A.L., Radic, B., Kuca, K., 2008. New bispyridinium oximes:in vitro and in vivo evaluation of their biological efficiency in soman and tabunpoisoning. Chemico–Biological Interactions 175 (2008), 413–416.

Cassel, G., Karlsson, L., Waara, L., Ang, K.W., Göransson-Nyberg, A., 1997.Pharmacokinetics and effects of HI 6 in blood and brain of soman-intoxicatedrats: a microdialysis study. European Journal of Pharmacology 332, 43–52.

Chen, Y., Zhu, Q.J., Pan, J., Yang, Y., Wu, X.P., 2009. A prediction model for blood–brain barrier permeation and analysis on its parameter biologically. ComputerMethods and Programs in Biomedicine 95, 280–287.

Clark, D.E., 1999. Rapid calculation of polar molecular surface area and itsapplication to the prediction of transport phenomena. 2. Prediction of blood–brain barrier penetration. Journal of Pharmaceutical Sciences 88, 815–821.

Gyenge, M., Kalasz, H., Petroianu, G.A., Laufer, R., Kuca, K., Tekes, K., 2007.Measurement of K-27, an oxime-type cholinesterase reactivator by high-performance liquid chromatography with electrochemical detection fromdifferent biological samples. Journal of Chromatography A 1161, 146–151.

Kalász, H., Szöko, E., Tábi, T., Petroianu, G.A., Lorke, D.E., Omar, A., Alafifi, S., Jasem,A., Tekesz, K., 2009. Analysis of pralidoxime in serum, brain and CSF of rats.Medicinal Chemistry 5, 237–241.

Karasova, J.Z., Kassa, J., Jung, Y.S., Musilek, K., Pohanka, M., Kuca, K., 2008. Effect ofseveral new and currently available oxime cholinesterase reactivators ontabun-intoxicated rats. International Journal of Molecular Sciences 9, 2243–2252.

Karasova, J.Z., Kassa, J., Musilek, K., Pohanka, M., Novotny, L., Kuca, K., 2009a. Effectof seven newly synthesized and currently available oxime cholinesterasereactivators on cyclosarin-intoxicated rats. International Journal of MolecularSciences 10, 3065–3075.

Karasova, J.Z., Bajgar, J., Jun, D., Pavlikova, R., Kuca, K., 2009b. Time-course changesof acetylcholinesterase activity in blood and some tissues in rats afterintoxication by Russian VX. Neurotoxicity Research 16, 356–360.

Karasova, J.Z., Stodulka, P., Pohanka, M., Kuca, K., in press. In vitro screening ofblood–brain barrier penetration of monoquaternary acetylcholinesterasereactivators. Analytical Letters. doi: 10.1080/00032710903502082.

Karasova, J.Z., Stodulka, P., Kuca, K., 2010. In vitro screening of blood–brain barrierpenetration of clinically used acetylcholinesterase reactivators. Journal ofApplied Biomedicine 8, 35–40.

Lanevskij, K., Japertas, P., Didziapetris, R., Petrauskas, A., 2009. Ionization-specificprediction of blood–brain permeability. Journal of Pharmaceutical Sciences 98,122–134.

Ligtenstein, D.A., Kosen, S.P., 1983. Kinetic profile in blood and brain of thecholinesterase reactivating oxime HI-6 after intravenous administration to therat. Toxicology and Applied Pharmacology 71, 177–183.

Lorke, D.E., Hasan, M.Y., Nurulain, S.M., Sheen, R., Kuca, K., Petroianu, G.A., 2007.Entry of two new asymmetric bispyridinium oximes (K-27 and K-48) into therat brain: comparison with obidoxime. Journal of Applied Toxicology 27, 482–490.

Lorke, D.E., Kalasz, H., Petroianu, G.A., Tekes, K., 2008. Entry of oximes into thebrain: a review. Current Medicinal Chemistry 15, 743–753.

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

Palm, K., Luthman, K., Ungell, A.L., Strandlung, G., Beigi, F., Lundahl, P., 1998.Evaluation of dynamic polar molecular surface area as predictor of drugabsorption: comparison with other computational and experimental predictors.Journal of Medicinal Chemistry 41, 5382–5392.

Petroianu, G.A., Lorke, D.E., Hasan, M.Y., Adem, A., Sheen, R., Nurulain, S.M., Kalasz,H., 2007. Paraoxon has only a minimal effect on pralidoxime brainconcentration in rats. Journal of Applied Toxicology 27, 350–357.

Reiner, E., Radic, Z., Simeon-Rudolf, V., 2007. Mechanisms of organophosphatetoxicity and detoxication with emphasis on studies in Croatia. Arhiv za HigijenuRada i Toksikologiju 58, 329–338.

Sakurada, K., Matsubara, K., Shimizu, K., Shiono, H., Seto, Y., Tsuge, K., Yoshino, M.,Sakai, I., Mukoyama, H., Takatori, T., 2003. Pralidoxime iodide (2-pAM)penetrates across the blood–brain barrier. Neurochemical Research 28, 1401–1407.

Terasaki, T., Ohtsuki, S., 2005. Brain-to-blood transporters for endogenoussubstrates and xenobiotics at the blood–brain barrier: an overview of biologyand methodology. Neurotherapeutics 2, 63–72.

Tsukita, S., Furuse, M., Itoh, M., 2001. Multifunctional strance in tight junctions.Nature Reviews Molecular Cell Biology 2, 285–293.

Turksen, K., Troy, T.C., 2004. Barriers built on claudins. Journal of Cell Science 117,2435–2447.

van Helden, H.P., Busker, R.W., Melchers, B.P., Bruijnzeel, P.L., 1996.Pharmacological effects of oximes: how relevant are they? Archives ofToxicology 70, 779–786.

Yanagisawa, N., Morita, H., Nakajima, T., 2006. Sarin experiences in Japan: acutetoxicity and long-term effects. Journal of the Neurological Sciences 249, 76–85.

Yoon, H.C., Kim, S.J., Shin, B.S., Lee, K.C., Yoo, S.D., 2006. Rapid screening of blood-brain barrier penetration of drugs using the immobilized artificial membranephosphatidylcholine column chromatography. Journal of BiomolecularScreening 11, 13–20.

Zhu, C., Juany, L., Chen, T.M., Hwang, K.K., 2002. A comparative study of artificialmembrane permeability assay for high throughput profiling of drug absorptionpotential. European Journal of Medicinal Chemistry 37, 399–407.