Reactivation of acetylcholinesterase inhibited by 1,2,2'-trimethylpropyl methylphosphonofluoridate (soman) with HI-6 and related oximes

doi:10.1016/0006-2952(80)90273-7

Biochemical Pharmacology

Volume 29, Issue 17, 1 September 1980, Pages 2379-2387

https://doi.org/10.1016/0006-2952(80)90273-7 Get rights and content

Abstract

Bovine erythrocyte acetylcholinesterase inhibited by 1,2,2'-trimethylpropyl methylphosphonofluoridate (soman) is reactivated rapidly but incompletely by 1 mM of the bispyridinium mono-oximes HI-6, HS-6, HGG-12 and HGG-42 (pH 7.5, 25°). These oximes, especially HI-6 and HS-6, show a higher reactivating potency than conventional reactivators, like P2S, obidoxime or TMB4. The incomplete reactivation is studied in more detail with HI-6, which is found to be the most potent reactivator. Rapid reactivation and aging take place in the presence of the oxime (10–70 μM). The reactions are not consistent with a minimum scheme involving simultaneous aging of the inhibited enzyme and reactivation and aging of an inhibited enzyme-oxime complex. The formation of aged enzyme can be described as a first-order process. The formation of reactivated enzyme proceeds in a more complicated manner. HI-6 appears to have no effect on aging of the soman-inhibited enzyme. It is concluded that HI-6, being able to compete with the rapid aging of soman-inhibited acetylcholinesterase, has an extremely high reactivating potency, on which the therapeutic effect of the oxime is based.

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Cited by (66)

The oximes HI-6 and MMB-4 fail to reactivate soman-inhibited human and guinea pig AChE: A kinetic in vitro study
2018, Toxicology Letters
Citation Excerpt :
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.
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.
Y124 at the peripheral anionic site is important for the reactivation of nerve agent-inhibited acetylcholinesterase by H oximes
2010, Biochemical Pharmacology
Citation Excerpt :
The difficulty in reactivating GD-inhibited AChE was once believed to be solely due to the ‘aging’ of the inhibited enzyme. However, HI-6 and HLo-7, were shown to be able to rapidly reactivate GD-inhibited AChE prior to ‘aging’ as compared to other bis-pyridinium oximes [6,8]. Therefore, steric hindrance from the bulky pinacolyl group of GD is also thought to be a significant factor for the slow reactivation of GD-inhibited AChE.
The toxicity of organophosphorus (OP) nerve agents is manifested through irreversible inhibition of acetylcholinesterase (AChE) at the cholinergic synapses, which stops nerve signal transmission, resulting in a cholinergic crisis and eventually death of the poisoned person. Oxime compounds used in nerve agent antidote regimen reactivate nerve agent-inhibited AChE and halt the development of this cholinergic crisis. Due to diversity in structures of OP nerve agents, none of the currently available oximes is able to reactivate AChE inhibited by different nerve agents. To understand the mechanism for the differential activities of oximes toward AChE inhibited by diverse nerve agents in order to aid the design of new broad-spectrum AChE reactivators, we undertook site-directed mutagenesis and molecular modeling studies. Recombinant wild-type and mutant bovine (Bo) AChEs were inhibited by two bulky side-chain nerve agents, GF and VR, and used for conducting reactivation kinetics with five oximes. A homology model for wild-type Bo AChE was built using the recently published crystal structure of human AChE and used to generate models of 2-PAM and HI-6 bound to the active-sites of GF- and VR-inhibited Bo AChEs before nucleophilic attack. Results revealed that the peripheral anionic site (PAS) of AChE as a whole plays a critical role in the reactivation of nerve agent-inhibited AChE by all 4 bis-pyridinium oximes examined, but not by the mono-pyridinium oxime 2-PAM. Of all the residues at the PAS, Y124 appears to be critical for the enhanced reactivation potency of H oximes.
Comparison of oxime reactivation and aging of nerve agent-inhibited monkey and human acetylcholinesterases
2008, Chemico-Biological Interactions
Citation Excerpt :
For example, the bis-pyridinium oxime obidoxime is used in European countries, while the mono-pyridinium oxime pralidoxime (2-PAM) is used in the United States. The dry powder form of HI-6 is used in the auto-injectors in Canada, a potent reactivator of AChE inhibited by nerve agents GD and GF [4–6]. However, HI-6 does not reactivate AChE inhibited by tabun [7–9] and OP pesticides such as paraoxon [10,11].
Non-human primates are valuable animal models that are used for the evaluation of nerve agent toxicity as well as antidotes and results from animal experiments are extrapolated to humans. It has been demonstrated that the efficacy of an oxime primarily depends on its ability to reactivate nerve agent-inhibited acetylcholinesterase (AChE). If the in vitro oxime reactivation of nerve agent-inhibited animal AChE is similar to that of human AChE, it is likely that the results of an in vivo animal study will reliably extrapolate to humans. Therefore, the goal of this study was to compare the aging and reactivation of human and different monkey (Rhesus, Cynomolgus, and African Green) AChEs inhibited by GF, GD, and VR. The oximes examined include the traditional oxime 2-PAM, two H-oximes HI-6 and HLo-7, and the new candidate oxime MMB4. Results indicate that oxime reactivation of all three monkey AChEs was very similar to human AChE. The maximum difference in the second-order reactivation rate constant between human and three monkey AChEs or between AChEs from different monkey species was 5-fold. Aging rate constants of GF-, GD-, and VR-inhibited monkey AChEs were very similar to human AChE except for GF-inhibited monkey AChEs, which aged 2–3 times faster than the human enzyme. The results of this study suggest that all three monkey species are suitable animal models for nerve agent antidote evaluation since monkey AChEs possess similar biochemical/pharmacological properties to human AChE.
Kinetic analysis of the protection afforded by reversible inhibitors against irreversible inhibition of acetylcholinesterase by highly toxic organophosphorus compounds
2006, Biochemical Pharmacology
Citation Excerpt :
Reactivating oximes are probably of limited value since the time frame for reactivation is utmost narrow. Looking at Fig. 14 shows that under these conditions reactivating oximes such as HI 6 or HLö 7 [42–46] should reach effective systemic concentrations within 10 min after massive exposure. Otherwise aging prevents any significant reactivation in case of soman poisoning.
In organophosphate poisoning, the underlying mechanism of the therapeutic efficacy of carbamate prophylaxis, which was successfully tested in animal experiments, still awaits complete understanding. In particular, it is unclear whether survival is improved by increased acetylcholinesterase activity during the acute phase, when both carbamate and organophosphate are present. This question should be solved experimentally by means of a dynamically working in vitro model. Immobilized human erythrocytes were continuously perfused while acetylcholinesterase activity was monitored in real-time by a modified Ellman method. The concentrations of reversible inhibitors and of paraoxon were varied to assess the influence of both components on the enzyme activity under steady-state conditions. Physostigmine, pyridostigmine and huperzine A were tested for their prophylactic potential. Upon pretreatment with these reversible inhibitors the enzyme was inhibited by 20–90%. Additional perfusion with 1 μM paraoxon for 30 min resulted in a residual activity of 1–4%, at low and high pre-inhibition, respectively. The residual activity was significantly higher than in the absence of reversibly blocking agents (0.3%). After discontinuing paraoxon, the activity increased even in the presence of the reversible blockers. Stopping the reversibly blocking agents resulted in 10–35% recovery of the enzyme activity, depending on the degree of pre-inhibition. The experimental results agreed with computer simulations upon feeding with the essential reaction rate constants, showing that physostigmine was somewhat superior to pyridostigmine in enhancing residual activity in the presence of 1 μM paraoxon for 30 min. The model predicts that inhibitors with a faster dissociation rate, e.g. huperzine A, may be superior in case of a ‘hit-and-run’ poison such as soman.
A comparison of the efficacy of a new asymmetric bispyridinium oxime BI-6 with currently available oximes and H oximes against soman by in vitro and in vivo methods
1999, Toxicology
The reactivating and therapeutic efficacy of a new acetylcholinesterase reactivator, designated BI-6(1-/2-hydroxyiminomethylpyridinium/-4-/carbamoylpyridinium/-2-butene dibromide), against the organophosphate soman was compared with oximes at present used (pralidoxime, obidoxime, methoxime) and H oximes (HI-6, HLö-7) using in vitro and in vivo methods. H oximes HI-6 and HLö-7 seem to be the most efficacious acetylcholinesterase reactivators against soman according to the evaluation of their reactivating and therapeutic efficacy in vitro as well as in vivo. The new oxime BI-6 is not as effective as the H oximes against soman, nevertheless it is significantly more effective against soman than the currently available oximes, pralidoxime, obidoxime and methoxime, which failed to protect rats poisoned with supralethal doses of soman. Our results confirm that the reactivating efficacy of oximes evaluated by the methods in vitro closely correlates not only with the potency of oximes in vivo in reactivating soman-inhibited acetylcholinesterase but also with the ability to protect rats poisoned with supralethal doses of soman.
In vitro effects of toxogonin, HI-6 and HLö-7 on the release of [<sup>3</sup>H]acetylcholine from peripheral cholinergic nerves in rat airway smooth muscle
1996, European Journal of Pharmacology
The purpose of this work was to evaluate the possible non-reactivating effects of toxogonin (1,1'[oxybis(methylene)]bis[4-[(hydroxyimino)methyl]pyridinium]-dichloride), HI-6 (1-[[[(4-aminocarbonyl)pyridinio]methoxy]methyl]-2-[(hydroxyimino)methyl]pyridinium-dichloride) and HLö-7 (pyridinium, 1-[[[4-(aminocarbonyl)pyridino]methoxy]methyl]-2,4-bis-[(hydroxyimino)methyl]diiodide) on the release of acetylcholine from cholinergic nerves. The oximes have been tested in our rat bronchial smooth muscle model, with respect to the effects of oximes on the K⁺ (51 mM)-evoked release of [³H]acetylcholine in the presence and absence of soman (1.0 μM). Toxogonin (100 μM) had no effect on the K⁺-evoked release of [³H]acetylcholine in the presence or absence of soman (1.0 μM). Similar results were found for HI-6 (100 μM). In contrast, HLö-7 (100 μM) enhanced the K⁺-evoked release of [³H]acetylcholine in the absence of soman. In the presence of soman HLö-7 did not alter the release of [³H]acetylcholine induced by K⁺ stimulation. The potentiating effect of HLö-7 on the release of [³H]acetylcholine could be blocked by the L-, N- and P-Ca²⁺ channel blockers verapamil (0.1 and 1.0 μM), ω-conotoxin GVIA (1.0 μM) and ω-agatoxin IV-A (0.2 μM), respectively. Muscarinic receptor antagonists (atropine (10 μM), pirenzepine (M₁) (1.0 μM) and methoctramine (M₂) (1.0 μM)) had no effects on the HLö-7 (100 μM)-enhanced release of [³H]acetylcholine. Protein kinase inhibitors (H-7 (20 μM), calphostin C (1.0 μM) and KN-62 (10 μM)) inhibited the HLö-7 (100 μM)-enhanced K⁺-evoked release of [³H]acetylcholine. The results showed that only HLö-7 had a direct enhancing effect on the release of acetylcholine through activation or opening of Ca²⁺ channels and a subsequent protein phosphorylation in the nerve terminal.

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Reactivation of acetylcholinesterase inhibited by 1,2,2'-trimethylpropyl methylphosphonofluoridate (soman) with HI-6 and related oximes

Abstract

Biochem. Pharmac.

J. Pharmac. exp. Ther.

Archs Biochem. Biophys.

Biochem. Pharmac.

Toxic. appl. Pharmac.

Naunyn-Schmiedeberg's Arch. exp. Path. Pharmak.

Arch. Tox.

Arch. Tox.

Eur. J. Pharmac.

Eur. J. Pharmac.

Eur. J. Pharmac.

Arch. Tox.

Arch. Tox. Suppl.