Editor's note: The treatment for nerve agent poisoning has been studied for many years. HI-6 has been studied for the last 50 or so years, yet it has not been selected by the various armies for a standard treatment. In this ASA Review article, some of the more senior researchers in nerve agent treatment highlight experiments that argue for the use of HI-6 as the replacement for the conventional oximes.


The Choice: HI-6, Pralidoxime or Obidoxime against Nerve Agents?

Kassa J., Cabal J., Bajgar J.
Purkyne Military Medical Academy, Hradec Kralove Czech Republic
Szinicz L
Akademie des Sanitaets und Gesundheitswesens der Bundeswehr, Muenchen, Deutschland

Introduction Although the basic mechanisms of action of the highly toxic nerve agents (sarin, soman, GF, VX) have been known for a long time, there are still major problems with the effective treatment of acute poisoning by them, especially by soman(1,2). The mechanisms are based mainly on the irreversible inhibition of the enzyme acetylcholinesterase (AChE, EC 3.1.1.7) thereby causing an accumulation of acetylcholine (ACh) in the synaptic clefts of the cholinergic nervous system. As a result, cholinergic receptors are excessively stimulated, leading to a cascade of postsynaptic events, which result in secondary toxic effects(3).
Conventional antidotal treatment of nerve agent intoxication consists of anticholinergic drugs (preferably atropine, to counteract the effects of accumulated ACh at the receptor) and oximes (preferably pralidoxime or obidoxime) to reactivate nerve agent-inhibited AChE(1,3). The increased international concern with both the military and terrorist threats from nerve agents has prompted us to critically consider the expected value of the antidotes currently available for treatment of nerve agent poisoning. The effectiveness of antidotal treatment is dependent on the reactivatability of AChE, i.e., on the effectiveness and speed of the reactivator used(4). Generally, the conventional oximes (pralidoxime or obidoxime) have been considered to be adequate against VX, sarin and GF(1,5,6) and ineffective against soman(1,2).
The differences in the oxime effectiveness are mainly due to the variation in aging rates; the process by which nerve agent-inhibited AChE is converted to a form that cannot be reactivated by oximes(7). The reactivation AChE inhibited by VX, sarin or GF is possible hours after the intoxication, while soman-inhibited AChE cannot be reactivated within minutes following intoxication and therefore the treatment of soman poisoning is much more difficult(4,8).
This fact led to the synthesis of a series of bisquaternary oximes, designated "H-oximes", that in combination with anticholinergic drugs have been relatively successful in antagonizing soman intoxication(9). Among the H-series oximes, HI-6 has been the most promising against soman poisoning and consequently has been the best studied(2,10). A model for the evaluation of the effectiveness should be chosen carefully and consider the type of nerve agent, reactivator and experimental animal. For our evaluation of effectiveness, we compared the efficacy of conventional oximes (pralidoxime and obidoxime) and the oxime HI-6 against three common nerve agents - sarin (O-isopropyl methylphosphonofluoridate), soman (O-pinacolyl methylphosphonofluoridate) and GF agents (O-cyclohexyl methylphosphonofluoridate both in vitro, using rat brain homogenates and in vivo, using rats to compare the therapeutic effects of the oximes and HI-6.

In Vitro Experiments
The effectiveness of the oxime in reactivating AChE inhibited by nerve agents in vitro was studied in rat brain homogenates 30 min following the onset of the rat brain AChE inhibition with 3xLD50 sarin or GF agent. In the case of soman, the oximes were added to the homogenate 5 min. before the addition of 2xLD50 soman, because of rapid aging of soman-inhibited AChE. A few minutes following soman inhibition of AChE, the reactivation of all oximes dramatically decreases and it is not possible to compare the reactivating efficacy among them(1). This would correspond to HI-6 given prophylactically, i.e., before any possible intoxication. In the work presented, the oxime concentrations maximally reactivating rat brain AChE were determined. The data are shown in Table 1. They clearly demonstrate large differences in the reactivating efficacies between HI-6 and pralidoxime and obidoxime, regardless of the nerve agents selected. In vitro, HI-6 is approximately 100 times more potent in reactivating rat brain AChE inhibited with sarin and GF. When soman is used, HI-6 is 2 to 5 times more effective a reactivator than pralidoxime or obidoxime, respectively.

In Vivo Experiments
To evaluate the maximal treatment efficacy of AChE reactivators in vivo, we have used their prophylactic administration (5 minutes before nerve agent). The prophylactic treatment should give much better results than treatment after poisoning and reduces the agent-specific influence of aging. Two mechanisms are considered:
1-shielding of the AChE active site from binding by nerve agent and
2-immediate reactivation by the oxime already present and subsequent indirect reduction of the rate of aging. We determined the i.m. LD50 values of oximes in rats and i.m. oxime doses that are sufficient for the 50% survival of rats (ED50) poisoned with 2xLD50 of soman and 3xLD50 of sarin or GF. The values were estimated by probit analysis based on 24 h mortality data in at least four groups of six animals each. For ED50 determinations, the oximes were administered i.m. in the same solution as atropine sulfate (21 mg/kg). Administration of atropine alone failed to prevent mortality following exposure soman, sarin or GF, although some clinical improvement was observed.
The results are shown in Table 2 and 3. They clearly demonstrate large differences in the efficacy between HI-6 and the conventional oximes. With pralidoxime used in therapeutic doses, it was not possible to save the rats intoxicated at supralethal doses regardless of the nerve agent. With obidoxime, sarin and GF poisoned rats could only be effectively treated with high doses (25% of LD50), but not with the doses supposed for humans (approximately 2% of LD50). Only HI-6 was effective at "human" doses in rats poisoned with supralethal doses, regardless of the nerve agent used. The much higher therapeutic potency of HI-6 in comparison with conventional oximes may be caused not only by the higher reactivating properties, but also by other antidotal mechanisms based on antimuscarinic, ganglion blocking, and post-junctional nondepolarizing actions, as well as effects on cardiovascular and respiratory systems(10). Various data suggest that pharmacological effects of HI-6 other than reactivation of AChE are most important for the survival of nerve agent intoxicated animals, regardless of the rate of aging of nerve agent-inhibited AChE(12).

Conclusions
The data show the oxime HI-6 is effective against supralethal intoxication of rats, when given in very low doses corresponding to those proposed for humans. On the other hand, pralidoxime, used in the US Army, and obidoxime, used in European Armies, are not effective for treatment of supra lethal intoxication with nerve agents. Similar findings have been presented by many other authors on the basis of the experiments using better animal models than rats for predicting effects in man, such as guinea pigs and primates (5, 13, 14). Their data also suggest that HI-6 had definite advantages over conventional oximes in the treatment of nerve agent intoxication. Based on the presented data, the question of whether it is reasonable to use pralidoxime or obidoxime against nerve agents for us is a simple answer: They are not effective; it makes no sense to use them. HI-6 appears to be the nerve agent therapy of choice. This oxime is being considered to replace pralidoxime and obidoxime in military injectors, although a final decision has not been made(12). In our opinion, the time has come to withdraw the conventional oximes and to replace them with HI-6 for military provision.

Acknowledgement.
The support of "Scientific and Environmental Affairs Division" of the NATO is gratefully acknowledged.

References:
1. Dawson, R.M. (1994), J. Appl. Toxicol., 14, 317 - 331.
2. Koplovitz, I. and Stewart, J.R. (1994) Toxicol. Lett., 70, 269 - 279.
3. Marrs, T.C. (1993) Pharmac. Ther., 51 - 66.
4. Bajgar, J., et al (1994) ASA Newsletter, 94-4,10 - 11.
5. Koplovitz, et al (1992) Arch. Toxicol., 66, 622 - 628.
6. Clement, J.G. (1994), Arch. Toxicol., 68, 64 - 66.
7. Berends F. (1987), In: De Matteis, F. and Lock, E.A.(eds.) Structure molecular mechanisms toxicity, McMillan Press, UK, pp. 125 - 152.
8. Coult, D.B., Marsh, D.J. and Read, G. (1966), Biochem. J., 98, 869 - 873.
9. Shih, T.-M., Whalley, Ch. E. and Valdes, J.J. (1991), Toxicol. Lett., 55, 131 - 147.
10. Rousseaux, C.G. and Dua, A.K. (1989), Can. J. Physiol. Pharmacol., 67, 1183 - 1189.
11. Grubi, Z. and Tomazi, A. (1989), Arch. Toxicol. 63, 68 - 71.
12. van Helden, H.P.M., et al (1996), Arch. Toxicol., 70, 779 - 786
13. Inns,.R.H. and Leadbeater, L. (1983), J.Pharm. Pharmacol., 35, 427-433.
14. Lallement, G. (1997), Pharmocol.Biochem.Behav. 56, 326-332.

Table 1: Reactivating efficacy of oximes of nerve agent - inhibited rat brain acetyl-cholinesterase in vitro.
  Rmax (%) / Cmax (mM)
OXIME SARIN GF AGENT SOMAN
PRALIDOXIME 65/5 30/50 53/1
OBIDOXIME 90/5 10/50 10/1
HI-6 75/0.02 96/0.1 90/1
Rmax = oxime concentration that maximally reactivated nerve agent - inhibited enzyme


Table 2: Toxicity and therapeutic doses of various oximes
  LD50 Therapeutic doseb
Oxime mg/kg µmol/kg mg/kg
pralidoxime 150a 584 >100
  (136-174)    
obidoxime 158.4 441 37.4 - 100
  (138-178)    
HI-6 781.3 2071 0.55 - 20
  (738-827)    
a These values are from Dawson (1994)  
b For detailed dosage, see Table 3  


Table 3: Prophylactic antidotal potency (ED50) and safety ratio (SR=LD50/ED50) in poisoned rat.
ED50 PRALIDOXIME OBIDOXIME HI-6
  SOMAN
mg/kg >100 >100 20.0 (17.2-29.2)
µmol/kg >389.1 >278.4 53.0 (45.6-77.4)
SR <1.5 <1.6 39.1
  SARIN
mg/kg >100 37.4 (21.9-64.1) 0.67 (0.49-0.91)
µmol/kg >389.1 104.1 (61.0- 178.4) 1.8 (1.3-2.4)
SR <1.5 4.2 1200
  GF
mg/kg >100 37.4 (34.3-55.2) 0.67 (0.26-1.16)
µmol/kg >389.1 121.1(95.5-153.7) 0.55(0.7-3.1)
SR <1.5 3.6 1400
NOTES
The doses of the various nerve agents were :
soman 160 µg/kg i.m. (2x LD50)
sarin 345 µg/kg i.m. (3x LD50)
GF 240µg/kg i.m. (3xLD50)

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