Reactivator Report for: BI-6

Nerve agents are organophosphates acting as potent inhibitors of acetylcholinesterase (AChE), the enzyme responsible for the hydrolysis of acetylcholine and, consequently, the termination of the transmission of nerve impulses. The inhibition of AChE by an organophosphate can be reversed by a nucleophilic agent able to dephosphorylate a serine residue in the active site of AChE. In this sense, the oximes are compounds capable of removing the nerve agent and reactivate the enzyme. Here, we have applied a methodology involving theoretical docking and Quantum Mechanics/Molecular Mechanics, using the softwares Molegro((R)) and Spartan((R)), to evaluate the kinetic constants of reactivation and the interactions of the oxime BI-6 with AChE inhibited by different organophosphorus compounds in comparison to in vitro data. Results confirm that this method is suitable for the prediction of kinetic and thermodynamic parameters of oximes, which may be useful in the design and selection of new and more effective oximes.

1. The therapeutic efficacy of various oximes (pralidoxime, obidoxime, methoxime, HI-6, HL-7, BI-6) against supralethal nerve agent poisoning (soman, sarin, cyclosin) in mice was tested. 2. New oxime BI-6, synthesized in our laboratory, is significantly more efficacious than conventional oximes but a little less efficacious than other H-oximes (HI-6, HL-7). 3. H-oximes (HI-6, HL-7) seem to be the most efficacious reactivators of nerve agent-inhibited acetylcholinesterase for antidotal treatment of supralethal nerve agent poisoning in mice.

1. The influence of three oximes (obidoxime, HI-6 and the new asymmetric bispyridinium oxime BI-6) in combination with atropine on soman-induced cholinergic and stressogenic effects in rats was studied. 2. The oxime BI-6 produced significantly higher reactivation of soman-inhibited blood and diaphragm cholinesterases than obidoxime. On the other hand, its reactivating effect was not so high as the effect of the oxime HI-6. 3. There were not significant differences in the reactivation of soman-inhibited brain acetylcholinesterase among all three oximes tested. 4. The influence of the oxime BI-6 on soman-induced stressogenic effects was greater than the antistressogenic effects of HI-6 or obidoxime at 1 h or 3 h following soman poisoning. 5 These findings confirm that the oxime BI-6 has no definite advantages over HI-6 in the antidotal treatment of soman poisoning but BI-6 is significantly more effective in rats than obidoxime, one of the oximes presently in use.

Nerve agents are organophosphates acting as potent inhibitors of acetylcholinesterase (AChE), the enzyme responsible for the hydrolysis of acetylcholine and, consequently, the termination of the transmission of nerve impulses. The inhibition of AChE by an organophosphate can be reversed by a nucleophilic agent able to dephosphorylate a serine residue in the active site of AChE. In this sense, the oximes are compounds capable of removing the nerve agent and reactivate the enzyme. Here, we have applied a methodology involving theoretical docking and Quantum Mechanics/Molecular Mechanics, using the softwares Molegro((R)) and Spartan((R)), to evaluate the kinetic constants of reactivation and the interactions of the oxime BI-6 with AChE inhibited by different organophosphorus compounds in comparison to in vitro data. Results confirm that this method is suitable for the prediction of kinetic and thermodynamic parameters of oximes, which may be useful in the design and selection of new and more effective oximes.

The efficacy of H oximes (HI-6, HLo-7), the oxime BI-6, and currently used oximes (pralidoxime, obidoxime, trimedoxime) to reactivate acetylcholinesterase inhibited by two nerve agents (tabun, VX agent) was tested in vitro. Both H oximes (HI-6, HLo-7) and the oxime BI-6 were found to be more efficacious reactivators of VX-inhibited acetylcholinesterase than pralidoxime and obidoxime. On the other hand, their potency to reactivate tabun-inhibited acetylcholinesterase was low and did not reach the reactivating efficacy of trimedoxime and obidoxime. Thus, none of these compounds can be considered to be a broad-spectrum reactivator of nerve agent-inhibited acetylcholinesterase in spite of high potency to reactivate acetylcholinesterase inhibited by some nerve agents. More than one oxime may be necessary for the antidotal treatment of nerve agent-exposed individuals.

Oximes are cholinesterase reactivators of use in poisoning with organophosphorus compounds. Pralidoxime (PRX) is used clinically as an adjunct to atropine in such exposure. Clinical experience with PRX (and other oximes) is, however, disappointing and routine use has been questioned. In addition it is known that oximes are not equally effective against all existing organophosphorus compounds. There is a clear demand for 'broad spectrum' cholinesterase reactivators with a higher efficacy than PRX. Over the years new reactivators of cholinesterase of potential clinical utility have been developed. Their chemical structures were derived from those of existing esterase reactivators, especially pralidoxime, obidoxime and HI-6. The purpose of the study was to quantify in vitro the extent of oxime (pralidoxime, K-27, K-33, K-48, methoxime and BI-6) conferred protection, using paraoxon as an inhibitor. Paraoxon (POX), the active metabolite of parathion (O,O-diethyl-O-p-nitro-phenyl phosphorothioate) is a non-neuropathic organophosphate. Red blood cell (RBC) acetylcholinesterase (AChE) activities in whole blood were measured photometrically in the presence of different POX concentrations and the IC50 was calculated. Determinations were repeated in the presence of increasing oxime concentrations. The IC50 of POX increases 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 alpha) was used to quantify the magnitude of the protective effect (nm IC50 increase per microm reactivator). Based on our determinations the new K series of reactivators is far superior to pralidoxime, methoxime and BI-6, K-27 being the outstanding compound with a tg alpha value of 3.7 (nm IC50 increase per microm reactivator) which is approximately 13 times the reactivator ability of PRX. In general there is an (expected) inverse relationship between the binding constant K and the slope of the IC50 shift curve (tg alpha) for all oximes examined. K-27 (the most protective substance judging by the tg alpha) has the lowest K value (highest affinity). In vivo testing of the new oximes as an organophosphate protective agent is necessary.

Oximes are cholinesterase reactivators used in organophosphorus poisoning. Clinical experience with pralidoxime (PRX) and other oximes is disappointing and their routine use has been questioned. In addition it is known that not all oximes are equally effective against all existing organophosphorus compounds. There is a demand for broad-spectrum reactivators with a higher efficacy than PRX. Based on our previous in vitro work the protection conferred by the various new oximes against inhibition by paraoxon as quantified by the IC(50) shift (nM increase in the IC(50) of the inhibitor per microM oxime present) is: 0.3 (PRX), 0.4 (methoxime; MMC-4), 1 (K-33), 1.2 (BI-6), 1.5 (K-48) and 3.7 (K-27). The purpose of the study was to quantify in vivo the extent of oxime-conferred protection, using paraoxon (POX) as a cholinesterase inhibitor and to test whether in vitro efficacy translates to protection from mortality. There were seven groups of six rats in each cycle of the experiment. Group 1 (G1) received 1 micromol POX (approximately LD(75)), the other groups (G2-G7) received 1 micromol POX + of one the six reactivators. The animals were monitored for 48 h and the time of mortality was recorded. The procedure was repeated five times (cycles). All substances were applied i.p. The experiments were repeated using 2, 3, 5 and 10 micromol POX. Mortality data were compared and hazards ratios (relative risks) ranked using the Cox proportional hazards model using POX dose and group (reactivator) as time-independent covariables. The relative risk of death estimated by Cox analysis (95% CI) in oxime treated animals when compared with untreated animals, adjusted for POX dose (high/low) was K-27: 0.26 (0.19-0.35); K-48: 0.34 (0.25-0.45); methoxime: 0.38 (0.29-0.50); BI-6: 0.53 (0.41-0.69); PRX: 0.70 (0.54-0.91); K-33: 0.82 (0.63-1.07). It is concluded that K-27 and K-48 are the most promising new oximes. The compounds with the best results in vitro also confer the best protection in vivo. Further testing using methyl- and propyl-organophosphates are needed.

BACKGROUND Acute poisoning with the highly toxic organophosphorus agent, soman, is not treated satisfactorily even by the most modern antidotes. METHODS AND RESULTS: In experiments on rats, the reactivating effect of a new asymmetric bispyridinium oxime BI-6 was compared with widely used oximes HI-6 and obidoxime by investigating the changes of soman-inhibited acetylcholinesterase activity in the diaphragm and various parts of the brain in rats up to three hours following soman challenge. Our findings confirm that the new oxime BI-6 is a more effective reactivator of soman-inhibited acetylcholinesterase than obidoxime but not as effective as the oxime HI-6 especially in the peripheral compartment.
CONCLUSIONS: The new oxime BI-6 is not as effective as HI-6 which seems to have definite advantages over other oximes in the treatment of soman poisoning.

The therapeutic efficacy of the new asymmetric bispyridinium oxime BI-6 against acute toxicity of the highly toxic organophosphate soman and the organophosphorus insecticide fosdrin by means of affecting the LD50 values of these noxiores substances was compared with the effect of the hitherto most perspective oxime HI-6 and the classic obidoxime always in combination with the identical dose of atropine. At the equimolar level the effect of oxime BI-6 against fosdrin completely equals the effects of both oximes HI-6 and obidoxime. The effect of oxime BI-6 against soman is even more marked than the effect of HI-6 but this difference is not statistically significant. On the other hand, at the equi-effective level, the effect of oxime BI-6 against soman is statistically significantly lower than the effect of HI-6, and against fosdrin it is even lower than the effect of both remaining oximes. The effects of the new oxime BI-6 equal, or slightly exceed the therapeutic effect of HI-6 but at the equimolar level only. At the equi-effective level which respects the toxicity of the oxime and is therefore more important for practical use, it is a therapeutically weaker reactivator of acetylcholinesterase than HI-6.

1. The reactivating and therapeutic efficacy of a new acetylcholinesterase reactivator, designated BI-6 (1-/2-hydroxyiminomethylpyridinium/-4-/carbamoylpyridinium/- 2-butene dibromide), against organophosphate sarin was compared with presently used oximes (pralidoxime, obidoxime, methoxime) and H oximes (HI-6, HL-7) by in vitro and in vivo methods. 2. Our results confirm that the new oxime BI-6 is a significantly more efficacious acetylcholinesterase reactivator than currently available pralidoxime and obidoxime but not as effective as H oximes (HI-6, HL-7) in vitro as well as in vivo. In addition, the oxime BI-6 is able to protect supralethal sarin poisoned rats at human-relevant doses. 3. Our data also suggest that the potency of oximes tested to reactivate sarin-inhibited acetylcholinesterase in vitro closely corresponds to their reactivating efficacy in vivo and their ability to protect rats poisoned with supralethal doses of sarin.

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, HL-7) using in vitro and in vivo methods. H oximes HI-6 and HL-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.

1. The therapeutic efficacy of various oximes (pralidoxime, obidoxime, methoxime, HI-6, HL-7, BI-6) against supralethal nerve agent poisoning (soman, sarin, cyclosin) in mice was tested. 2. New oxime BI-6, synthesized in our laboratory, is significantly more efficacious than conventional oximes but a little less efficacious than other H-oximes (HI-6, HL-7). 3. H-oximes (HI-6, HL-7) seem to be the most efficacious reactivators of nerve agent-inhibited acetylcholinesterase for antidotal treatment of supralethal nerve agent poisoning in mice.

1. The influence of three oximes (obidoxime, HI-6 and the new asymmetric bispyridinium oxime BI-6) in combination with atropine on soman-induced cholinergic and stressogenic effects in rats was studied. 2. The oxime BI-6 produced significantly higher reactivation of soman-inhibited blood and diaphragm cholinesterases than obidoxime. On the other hand, its reactivating effect was not so high as the effect of the oxime HI-6. 3. There were not significant differences in the reactivation of soman-inhibited brain acetylcholinesterase among all three oximes tested. 4. The influence of the oxime BI-6 on soman-induced stressogenic effects was greater than the antistressogenic effects of HI-6 or obidoxime at 1 h or 3 h following soman poisoning. 5 These findings confirm that the oxime BI-6 has no definite advantages over HI-6 in the antidotal treatment of soman poisoning but BI-6 is significantly more effective in rats than obidoxime, one of the oximes presently in use.