pralidoxime and obidoxime
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Pralidoxime and ObidoximeTRANSCRIPT
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www.stmconnect.com/jeit Journal of Environmental Immunology and Toxicology 35
Journal of Environmental Immunology and Toxicology 1:1, 35-40; January/February/March 2014; 2014 STM Publishing
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AbstractThe acute toxicity of organophosphorus esters (organophosphates and organophosphonates) is due to inhibition of the enzyme acetylcholinesterase (achE), which metabolizes the neurotransmitter acetylcholine (ach). Esterase inhibition results from phosphylation (i.e. either phosphorylation or phosphonylation) of the serine hydroxyl group in the active center of the enzyme and translates into an endogenous acetylcholine poisoning.
The therapy of cholinesterase poisoning by organophosphorus inhibitors includes the use of oximes (such as pralidoxime in the US and obidoxime in Europe). Oximes reactivate the inhibited enzyme by dephosphylating it, thereby becoming themselves phosphylated. The phosphylated oxime thus generated can be itself a potent inhibitor of cholinesterases, which may reduce the efficacy of the reactivation attempt.
The present study estimates logP values of phosphylated pralidoxime and obidoxime after in-silico exposure to a number of organophosphorus esters [ethyl-paraoxon, methyl-paraoxon, diisopropyl-fluoro-phosphate, VX, soman, tabun, sarin, cyclosarin], compares them with the logP of native oximes and discusses possible therapeutic relevance. Our data indicate that phosphylation of oximes generally increases their lipophilicity, facilitating penetration into the brain where they can inhibit or re-inhibit enzymes. Estimated logP values of phosphylated pralidoxime increase on average by 0.6 compared to native pralidoxime, while for obidoxime the increase is on average 0.9. For both oximes, phosphylation by tabun shows the most significant effect on logP (increase by 0.9 and 1.9 respectively). Possible consequences with regard to blood-brain-barrier passage are discussed.
Journal of Environmental Immunology and Toxicology 2014; 1:35-40
Key words cholinesterase; obidoxime; organophosphorus esters; oxime; pralidoxime; phosphorylation; phosphonylation; logP
Pralidoxime and Obidoxime: Phosphylation-induced Changes in logP (partition coefficient)
Georg A Petroianu1, Dietrich E Lorke1, Gagani Athauda1, Ferenc Darvas1,2, Huba Kalasz3
1Department of cellular Biology and Pharmacology, herbert Wertheim college of Medicine, Florida International University, Miami, USa; 2Thales Nano, Budapest, hungary; 3Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, hungary
Correspondence to: Georg A Petroianu, Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida International University, Miami, USA; Email: [email protected]: 10/10/2013; Revised: 01/11/2013; Accepted: 29/11/2013DOI: 10.7178/jeit.8
Introduction
The acute toxicity of organophosphorus esters (organophosphates and organophosphonates) is due to inhibition of the enzyme acetylcholinesterase (AChE), which inactivates the neurotransmitter acetylcholine (ACh). The inhibition of esterases results from phosphylation. Phosphylation is the umbrella term used to describe phosphorylation (when the enzyme is inhibited by an organophosphate insecticide, such as paraoxon) or phosphonilation (when inhibited by an organophosphonate nerve gas).
Phosphylation of the serine hydroxyl group in the active center of the enzyme translates into an endogenous acetylcholine poisoning. The therapy of organophosphorus inhibitors of cholinesterase poisoning is known by the acronym A FLOP = Atropine, FLuids, Oxygen, Pralidoxime,1 reflecting the sometimes disappointing therapeutic results.
The history of the development of organophosphorus inhibitors
of cholinesterase has been described.2-4 Phosphylated AChE can be reactivated by oximes; all reactivators in clinical use are pyridinium oximes.5 Using 2-methyl-pyridine as a starting point, Wilson and Ginsburg working at Columbia University in the laboratory of David Nachmansohn synthesized a number of pyridine oximes. Among those was also the first aldoxime cholinesterase reactivator of clinical relevance, pralidoxime (2-pyridinium aldoxime or 2-PAM). Such research has also been conducted independently in Britain by Davies and Green [for a review see ref.5]. Obidoxime, developed by Luettringhaus and Hagedorn in Germany was initially known by the acronym LueH-6.6
The mode of action of pyridinium oximes has been recently reviewed. By interacting with the anionic site of the enzyme, the pyridinium moiety favors an optimal orientation of the reactivator at the catalytic site of the enzyme, thereby increasing efficacy.7,8 Whereas emergency treatment of nerve gas exposure with oximes is accepted doctrine, the therapeutic value of oximes in human organophosphate pesticide poisoning is doubtful.9,10
A possible reason for the questionable efficacy may be the generation of phosphylated oximes during AChE reactivation.10,11 Phosphylated oximes are themselves potent inhibitors of cholinesterase.12
The properties of the phosphylated oximes have therefore to be taken into account when evaluating kinetic studies on the
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reactivation and aging of organophosphate-AChE conjugates.13
One way to characterize biologically active compounds is the octanol-water partition coefficient [logP]. Partition of substances between oil and water, a concept introduced over a century ago by Berthelot [according to refs.14], is often correlated with their biological activities.15-18
Begley reviewed various mechanisms by which substances cross the blood-brain barrier. A strong correlation was identified for lipid solubility and BBB permeability; the importance of good solubility in the respective compartments (plasma and CSF) was also emphasized. He writes There is well-established relationship between lipid solubility, either calculated or determined as an oil-water partition coefficient, with brain penetration, which increases with increasing lipid solubility.19
Purpose of the study
The present work estimates logP values of phosphylated pralidoxime and obidoxime after in-silico exposure to a number of organophosphorus esters [ethyl-paraoxon, methyl-paraoxon, diisopropyl-fluoro-phosphate (DFP), VX, soman, tabun, sarin, cyclosarin], compares them with the logP of native oximes and discusses possible therapeutic relevance.
Material and method
Chemical structures of all compounds were drawn using ChemDraw Ultra 12.0 (CambridgeSoft Software, PerkinElmer Inc. Waltham, Massachusetts). LogP values of organophosphorus esters, oximes, phosphylated pralidoxime and phosphylated obidoxime were estimated using the PrologP module of the Pallas 3413 software (CompuDrug Inc., Sedona, AZ, USA). Details of the algorithm used for calculations are given by.20 The program takes into account all lipophilic and hydrophilic fragments of a specific compound and makes minor corrections based on octanol-water partition data, as available from the literature. The authors emphasize that their neural network-based method (pseudo-linear algorithms) combines the precision of non-linear approaches with the transparency of the early linear methods. The logP value of a substance is most relevant for neutral substances and is also useful as a general reference point to help compare overall hydrophobicity trends of compounds.
Results
Structure and logP values of organophosphorus pesticides (ethyl-paraoxon, methyl-paraoxon), diisopropyl-fluoro-phosphate and nerve gases (sarin, cyclosarin, soman, tabun, VX) are listed in table 1. Their logP values range from -0.02 (tabun) to =2.18 (ethyl-paraoxon). All compounds except tabun are therefore lipophilic (positive logP values).
Table 2 shows the chemical formulas and logP values of pralidoxime [2-PAM] and of pralidoxime phosphylated by various organophosphates (ethyl-paraoxon, methyl-paraoxon, sarin, cyclosarin, soman, tabun, VX). The logP value of pralidoxime is -1.960.19 reflecting its hydrophilicity. Phosphylation of pralidoxime significantly reduces hydrophilicity to values between
-1.700.01 (phosphylation by ethyl-paraoxon) and -1.020.01 (phosphylation by tabun), the most significant increase in logP. Phosphylated pralidoxime derivatives are thus less hydrophilic than unphosphylated pralidoxime.
Chemical formulas and logP values of obidoxime and of obidoxime phosphylated by various organophospates are listed in table 3. Having a logP value of -3.400.26, obidoxime is markedly more hydrophilic than pralidoxime. As observed for pralidoxime, phosphylation of obidoxime significantly reduces its hydrophilicity, except for obidoxime phosphylation by soman. LogP values of phosphylated obidoxime range between =-3.630.01 (phosphylation by soman) and =-1.470.01 (phosphylation by tabun).
Discussion
Oximes in general have disappointed clinically.9 Although, theoretically, their mode of action is reasonably well understood, their practical value remains uncertain.21 Oximes are polar molecules with a negative logP, indicating hydrophilicity. Since the blood brain barrier generally only allows free passage of small lipophilic compounds, oximes have only a limited brain penetration.22 For the monopyridinium aldoxime pralidoxime, the brain concentration is only 10% of the blood concentration, while penetration of bis-pyridinium aldoximes, such as obidoxime, is one order of magnitude lower.23,24
Among the fundamental questions still unanswered is the relationship between oxime efficacy and brain penetration: is the limited efficacy of oximes due to their limited brain penetration? Would an increase in brain penetration translate into superior efficacy? It appears that initially the answer must have been yes, as evidenced by Nicholas Bodors attempt to develop pro-2-PAM, a pro-drug dihydropyridine derivative of pralidoxime (2-PAM) penetrating the brain.25 Although pralidoxime brain levels were considerably higher when using the brain penetrating pro-drug, overall results were disappointing.26 In a recent review, it was concluded that Increasing the BBB penetration by oximes does not actually lead to significant benefits of survival rate, but certainly amplifies the neurotoxic risks.27
Our own animal work suggests that a good predictor for low oxime toxicity (as assessed by survival) is a high negative logP (strongly hydrophilic), which would imply that limited brain penetration is actually desirable.17,18 A putative explanation for these surprising findings is the intrinsic toxicity of phosphylated oximes generated by the reaction of oximes with organophosphorus-inhibited enzymes.11
Phosphylated oximes are themselves potent inhibitors of AChE, sometimes much more potent than the initial offending organophosphate or organophosphonate (organophosphates do not contain a direct phosphorus-carbon link, while organophosphonates contain one), which may translate into high toxicity.7,11,13,28,29
The group of Worek from Muenchen and others had some success in the attempt at synthesizing phosphylated oximes. For example they isolated isolated mono(diethylphosphoryl) obidoxime (DEP-obidoxime) showing that the compound displayed remarkable stability under physiological conditions.30
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Table 1. LogP values of organophosphorus esters [phosphylating agents]. All compounds with the exception of tabun are lipophilic (positive logP values).Ethyl-paraoxon
N+
O
O
O
P
O
O O
logP=2.18
Methyl-paraoxon
N+
O
O
O
P
O
O O
logP=1.51
Di-isopropyl-fluoro-phosphate
P
O
O
F O
logP=1.08
Sarin
O
P
O
F
logP=0.84
Cyclosarin
P
O
O
F
logP=1.78
Soman
P
O
O
F
logP=1.89
Tabun
P
O
N
OC
N
logP=-0.02
VX
P
O
O
SN
logP=2.10
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Table 2. LogP values of pralidoxime [2-PAM] and of the phosphylated-pralidoxime. Pralidoxime is a hydrophilic compound; phosphylation significantly reduces hydrophilicity [on average by 0.6]. Phosphylation by tabun shows the most significant effect on logP2-PAM
N+
NOH logP=-1.960.19
Phospylated Oxime Structure logP phosphylated Oxime
2-PAM phosphylated by ethyl-paraoxon
N+
NO
O
P
O
O
logP=-1.700.01logP=0.26
2-PAM phosphylated by methyl-paraoxon
N+
NO P O
O
O
logP=-1.070.01logP=0.89
2-PAM phosphylated by di-isopropyl-fluoro-phosphate
N+
NO P O
O
O
logP=-1.350.01logP=0.61
2-PAM phosphylated by sarin
N+
NO P
O
O
logP=-1.570.01logP=0.39
2-PAM phosphylated by cyclosarin
N+
NO P
O
O
logP=-1.220.01logP=0.74
2-PAM phosphylated by soman
N+
NO P
O
O
logP=-1.50.01logP=0.46
2-PAM phosphylated by tabun
N+
NO P
O
N
O
logP=-1.020.01logP=0.94
2-PAM phosphylated by VX
N
N+
O P
O
O
logP=-1.360.01logP=0.6
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Table 3. LogP values of obidoxime and of phosphylated-obidoxime. Obidoxime is a highly hydrophilic compound; phosphylation significantly reduces hydrophilicity [on average by 0.9]. Oxime phosphylation by tabun shows the most significant effect on logP.Obidoxime
HON
N+ O N+
NOH
logP=-3.400.26
Phospylated Oxime Structure logP phosphylated Oxime
Obidoxime phosphylated by ethyl-paraoxon
HON
N+ O N+
NO P
O
O
O
logP=-2.510.01logP=0.89
Obidoxime phosphylated by methyl-paraoxon
HON
N+ O N+
NO P
O
O
O
logP=-1.820.01logP=1.58
Obidoxime phosphylated by di-isopropyl-fluoro-phosphate
N+ O N+
NO
NHO P
O
O
O
logP=-2.810.01logP=0.59
Obidoxime phosphylated by sarin
HON
N+ O N+
NO P
O
O
logP=-2.600.01logP=0.8
Obidoxime phosphylated by cyclosarin
HON
N+ O N+
NO P
O
O
logP=-3.000.01logP=0.4
Obidoxime phosphylated by soman
HON
N+ O N+
NO P
O
O
logP=-3.630.01logP=0.23
Obidoxime phosphylated by tabun N
+N+ O
NHO
NO P
O
N
O
logP=-1.470.01logP=1.93
Obidoxime phosphylated by VX
HON
N+ O N+
NO P
O
O
logP=-2.200.01logP=1.2
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4-pyridinium aldoximes (MMB-4 [methoxime], TMB-4 [trimedoxime] and LuH-6 [obidoxime]), have phosphylated forms that decompose slower than those of 2-pyridinium aldoximes (2-PAM [pralidoxime] and HI-6 [asoxime]).7,31 While some of the compounds have indeed short t
1/2 some others dont; considering
the vast number of possible phosphylated oximes any attempt at generalization is bound to have serious limitations.
LogP: Pyridinium oximes are hydrophilic compounds (large negative value of LogP) with very limited CNS penetration.22,23 Phosphylation results in a significant reduction in the absolute value of LogP, corresponding to a reduction in hydrophilicity, i.e. increase in lipophylicity. This decrease in hydrophilicity favors penetration into the brain, where phosphylated oximes might phosphylate AChE, thereby becoming trapped. For pralidoxime, phosphylation on average results in a change towards lipophilicity of 0.6 (which indicates a six-fold increase), with the maximum noticed for phosphylation induced by tabun. For obidoxime an average change towards lipophilicity of 0.9 was estimated (which indicates a nine-fold increase), with the maximum noticed for phosphylation induced by tabun.
Numerous other parameters such as logD have been correlated with BBB penetration. LogD does not appear to have advantages over log P in the context analyzed. A thorough analysis is offered by Vilar.32
Conclusion
Based on preliminary data derived in silico we conclude that the ideal oxime must not only be non-toxic itself, but should also yield non- toxic products after phosphylation, since phosphylation generally increases brain penetration. Any such conclusions is in need of experimental confirmation.
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