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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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Suggested Citation:"Appendix B. Digest Reports: Oximes." National Research Council. 1984. Possible Long-Term Health Effects of Short-Term Exposure To Chemical Agents, Volume 2: Cholinesterase Reactivators, Psychochemicals and Irritants and Vesicants. Washington, DC: The National Academies Press. doi: 10.17226/9136.
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APPENDIX B DIGEST REPORT OXIMES by J. Henry Wills INTRODUCTION The group of oximes that has been administered to human volun- teers by or under the auspices of the Biomedical Laboratory of the Edgewood Arsenal at Aberdeen Proving Ground includes several salts of N-methylpyridinium-2-formyl oxime, N,N'-trimethylene-bis-~4- formy~pyridinium oxime~biabromide and -bischloride, N,N'-methyI- eneoxymethylene-bis(4-formylpyridinium oxime~bischioride, and the ketoxime diacetylmonoxime. Two of the widely used salts of the monopyridini~ oxime are the chloride (pralido2cime chloride), referred to here as I, and the methane suffocate (contrathion), referred to as II ~ or P2S ~ . The bispyridinium bisoximes above are known, respectively, as trimedoxime bromide (III), trimedoxime chlo- ride, and obidoxime chloride (IV). The ketoxime is known as DAM (V). The designations by Roman numerals are used hereafter for these oximes. Other Batty of N-methy~pyridinium-2-formyl oxime are identified by the abbreviation 2-PAM followed by the common symbols for ele- mental anions (such as 2-PAM I for the iodide) or the names of orga- Other salts of N,N'-trimethyl- nic anions (such as 2-PAM tartrate). ene-bis-~4-formylpyridinium oxime) are identified by appending the designation for the anion to the abbreviation TMB-4. These and other oximes were developed to be Deactivators of cholinesterase that had been inhibited by organophosphorus anti- cholinesterase compounde;~4 they were considered initially and briefly to be complete antagonists of the toxic actions of these substances. This idea had to be abandoned when 2-PAM I was founders to be much more effective as an adjunct to atropine than as a sole therapeutic agent in antagonizing intoxication by organo- phosphorus anticho~ nesterase agents. Furthermore, 2-PAM I and I antagonized particularly the blockage of nicotinic chat inergic neuromuscular transmission at the motor endplate on skeletal muscle--an ef feet that can be reproduced to some extent with d-tubocurarine and other curare~imetic agents. The efficacy of crimes as adjuncts to atropine in treating intoxication by anticho~inesterase agents depends on both the agent -263-

and the oxime. For instance, I is potent as an adjunct to atropine in treating experimental animals intoxicated by sarin or VX, but is only mildly effective as an adjunct to atropine in treating intoxi- cation by tabun6~8 and almost completely ineffective in treating intoxication by soman,9 whereas III is moderately effective as an adjunct to atropine in treating experimental intoxication by tabun. None of the oximee considered here is outstandingly effective as an adjunct t' atropine in treating intoxication of laboratory animals by Roman. ~ The failure of these oximes to antagonize the alterations in normal function induced by Roman has been attributedl4 to hydroly- tic dealkylation of the phosphorus atom in the phosphony] residue attached to the active center of cholinesterase; that results in an alteration in the electronic field around the phosphorus atom that renders oximate ions unable to sever the bond between the phospho ws atom and the serine residue in the active center of the enzyme. The aging reaction, identified first with DFP,l5 has been found to pro- ceed particularly rapidly in the phosphonyl residue from Roman on inhibited cholinesterase.~3~6 A large dose of I was fouadi7 to stop the aging process in experimental animals, but reactivated only a part of the chow nesterase that had been inhibited. The amount of I required for this purpose was so large (104 mg/kg intravenously) as to carry a high hazard of toxic action. Furthermore, there is some uncertainty about the importance of aging in the response of an organism to Roman. For example, the repetitive depolarization of skeletal muscle fibers after a single indirect stimulus that follows a dose of Roman was stopped by TMB-4 Cl' without detectable reacti- vation of cholinesterase in the vicinity of the motor endplate. Similar results have been obtained with d-tubocurarine,l9 galla- mine,:9 and piperidyl methylandrostanediol.20 Crone23 reported that d-tubocurarine chloride in vitro at 10~4 M completely pre- vented for 6 h the aging of red-cell acety~cholinesterase that was inhibited by sarin and that gallamine triethiodide at the same con- centration markedly slowed the aging of similarly inhibited choli- nesterase. Because the response of skeletal muscle after a dose of Roman was affected by a dose of d-tubocurarine that would have yielded a concentration in the blood of no more than 0.45 ~ lo-6 M, it is difficult to believe that Crone's effect can explain fully the in viva action of d-tubocurarine in antagonizing the neuromuscular blocking action of soman. In 17 rats, twitch contraction of the anterior tibialis muscle in response to single indirect stimuli continued until a mean dose of soman had been given that was more than 2 100 times the minimal dose that altered the magnitude of the twitch. Eventual failure of the twitch response was seen to be always connected with marked alow- ing of the heart. This observation raises the possibility that the effect of Roman on the twitch response of skeletal muscle depends on -264—

a deficient supply of blood: to the muscle, rather than on an effect on neuromuscular transmission. However, a similar indirect action seems not to explain the effect of soman on the response of skeletal muscle to repetitive indirect stimulation. Skeletal muscles become unable to maintain a tetanic response to repetitive indirect stimu- lation at doses of Roman below those that affect significantly car- diovascular function. The indication that TMB-4 C1: can antagonize the effect without inducing reactivation of cholinesterase at the neuromuscular Junctionl~ illustrates the importance of knowing what actions other than reactivation of cholinesterase may reside in the molecules of not only III but also the other crimes with which this review is concerned. LETHALITY OF SINGLE DOSES IN EXPERIMENTAL ANIMALS Several compilations of the toxicities of oximes have been published.23~29 Additional information on the toxicities of the oxides under consideration is available from several sources, including Namba,30 Lindsey et al.,3' Wills,32 and Crook and Cresthul' .33 Tab' e 1 summarizes the avail ate' e information on the single-dose ~ ethalities of the oximes by giving typical values, with- out attribution to specific investigators. In comparing lethal doses of the various oximes, assuming that the oximate radical is the bio- logically active portion of the molecule, knowledge of the relative amounts of thi 8 radical in the various compounds is important. Tab] e 2 gives values for this measure; the last column of the table gives values for the relative lethal activities of the compounds derived from the data in Table 1. It is obvious that there 1e no measurement in Table 1 on which all eight oximes can be compared, so that the relative lethality values in Table 2 should not be taken too seriously; they may be approximately correct in order. Lethality after oral administration was omitted from the consideration of relative lethality because it obviously differed qualitatively from lethality by other routes of administration; e.g., all five of the oximes given to mice orally were less toxic than 2-PAM I, although all were more lethal than SPAN I by any other route of administra- Lion. When the two rankings in Table 2 are compared by means of Kendall's rank correlation coefficient, there is only a 90% chance that the ranks in the two measurements are significantly correlated Thus, the relative amounts of the oximate radical in the molecules of the different oximes may not explain completely their comparative lethalities . Seven of 14 dogs given sing' e intravenous doses at IB7 mg/kg of 2-PAM I died.33 Vomiting, weakness, tremor, salivation, loss of reflexes, and convulsion were the most common signs of intoxication in these animals. Seven of 16 dogs given single intravenous doses of III at 57.5 mg/kg by the same investigators died. Weakness, con- wlaion, tremor, salivation, loss of reflexes, and vomiting were the -2 65-

most common effects. In general, the 8igD8 of toxicity appeared in approximately the following order after each osime: staggering weak- ness, collapse, relaxation of muscles, urination, defecation, tremor, convulsion, gasping, salivation, loss of reaction to touch, sound, or pricking with a needle, loss of eye reflexes, apnea, cyanosis, and death. The principal difference in effects between 2-PAM I and III was that muscular weakness was a less immediate response after 2-PAM I than after III. Cholinolytic drugs have ~ en found34~35 to increase the letha- lities of II, III, and a 1:l mixture of II and III in mice given intramuscular injections. The changes in the Logos of II and III induced by a constant dose of atropine were 17.9% and 17.8X, respec- tively, despite the fact that the LDsos of the oximes alone dif- fered by a factor greater than 2. Parpanit and several mixtures of cholinoly tic drugs had effects qualitatively similar to those of atropine. Duke and deCandole36 reported that intramuscular indec- tion into rabbits of equal 408e8 (30 mg/kg) of I, II, and III resulted in peak plasma concentrations of the oximes about 9 min after the injections, the peak concentration of II] being consider- ably greater than those of I and II. These investigators reported also that, whereas the plasma concentrations of I and III after intravenous injections decreased more slowly than that of II, the pi asma concentration of all three after intramuscular injections decreased at about the same rate. Inasmuch as ~ and II had similar peak blood concentrations (lower than that of III) after intravenous injections, I and II seem to have somewhat larger volumes of distri- bution ire the body of the rabbit than Ill. In the rat, absorption of III from a single-loop intestinal prep- aration during ~ h was found to be only about 13% of that of 2-PAlI 1.37 The rate of absorption of I was somewhat lower than that of the iodide; II was absorbed at nearly the same rate as the iodide.38 Three hours after the oximes were put into intestinal loops, slightly more than one-third as much of III had been absorbed as of 2-PAM I. Brown39 found that lntracister~al injection of II, after injec- tion of sarin by the same route, was ineffective in overcoming respi- ratory paralysis and vasomotor stimulation resulting from satin, but that an intravenous dose of atropine was effective. Edery40 extended this sort of study with severe' organophosphorus compounds, atropine, I, III, and V. He found that intraventricular atropine and, to a minor extent, oximes were able to antagonize the effects of intraventricularly injected ethyl pyrophosphate. Intravenous injec- tion of ~ at 25 mg/kg I-2 min after intraventricular injection of ethyl pyrophosphate did not modify the effects induced by the organo- phosphorus compound. Intravenous injection of III at 20 mg/kg or, especially, of 2-PAM I at 50 mg/kg had definite antagonistic effects -266-

TABLE 1 Representatlve LD,o Values of Eight Osimes Administered to Seven Animal Species LD~. m~/kg YAK C1 P2S 2-PAM 1~-4 Br2 Obidoxime DAM Species Rout ea 2-PAM I (I) (I1) lactate (III) T~-4 C12 (IV) (V) Mouse IV 133 155 -- 122 44 -- 130 -- IP 210 140 -- 60 110 139 68 IM 230 180 231 - - 80 130 160 -- SC 257 222 16S -- 83 183 PO 1,650 2,S90 3,700 1,920 2,000 — 3,390 -- Rat IV 147 96 109 -- 89 104 140 IF 300 199 -- -- 165 -- 195 IM -- 150 218 -- 137 ~ 189 SC —— —— 332 —— — —— —— PO —- - - 7,000 - - - - - - 4,000 Guinea IM -- 168 305 pig 79 Rabbi t IV -- 94 133 -- -- 44 83 245 - - __ Cat IV -- -- -- -- -- -- 100 -- IM —— —— —— —— 117 —— 188 Don IV 190 - - - - -- 60 -- 70 IM -- -- 356 -- -- a IV, intravenous; IP, intraperitonea~l; IM, intramuscular; SC, subcutaneous; PO, by mouth. —2 67—

TABLE 2 Relative Oximate Content and Relative Lethality of Eight Oximes Relay ive Oximate Relat lve Oxime Content Lethality 2-PAM I 1.00 1.00 P2S (II) 1.13 1.27 2-PAM lactate 1.17 1.10 TMB-4 Br2 (III) 1.18 2.56 Obidoxime ( IV) 1. 47 1.39 TMB-4 C12. 1.48 1.67 2-PAM C1 (I) 1.53 1.33 DAM (V) 2.61 3.13 -268 -

on the responses induced by prior lntraventricular injection of ethyl pyrophosphate. In interpreting these findings, it is pertinent to point out that the doses of the various oximes used correspond with 0.044 mmol/kg of III, 0.~89 mmol/kg of 2-PAM I, and 0.248 mmol/kg of V. On the basis of these values, Edery' ~ conclusion that 2-PAM I was the most active antagonist of ethyl pyrophosphate may be quest ionable . These aM other findings have raised the question of whether qua- ternary pyridini~ osimes can enter the CNS from the general circula- tion. Kalser,43 in a study with rats and cats given intravenous injections of I tagged with i4C in the quateraizing me tiny] group, found that the cerebrum, the cerebellum, the medulla oblongata, and the spinal cord contained only traces of i4C at times at which the blood contained the label at 19-53 ~Ci/kg. However, Firemark et al.42 found that the brain of the rat had a concentration of i4--2-PAM I about one-tenth that in plasma 10 min after intravenous injection of labeled oxime at 20 mg/kg. In the brain, the cerebral and cerebellar cortices had the highest concentrations of the label; the caudate nucleus, the thalamus, and the hypothalamus contained concentrations a little less than half those in the cortices. Rats pretreated with the anthe~mintic organophosphate trichiorfon and killed 10 min after intravenous injection of labeled 2-PAM I had a concentration of the label in their brains about twice that found in normal rata. The brain appears, therefore, to be somewhat permeable by 2-PAM I, but distinctly less so than leg muscle, diaphragm, liver, and kidneys according to Ka1ser's data, and to have that permeability increased by an organophosphorus anticholinesterase compound. Freshly prepared 80iUtioU8 of II (150 mg/mI) in water or DMSO were applied to the skins of guinea pigs and rabbits, except that on the head awl legs.43 The II in DMSO entered ache blood of the rab- bit at a peak rate about 3 times that at which lI in water penetrated the skin. No uptake of II from the aqueous solution by the guinea pig was detected. The rate of uptake of II from the DMSO solution by the guinea pig was about two-thirds that by the rabbit. Guinea pigs that had been anointed with the DMSO solution of II were given sarin subcutaneously at 200 mI/kg and immediately thereafter atropine sul- fate intramuscularly at 15 mg/kg. Similarly anointed rabbits were given sarin intravenously at 100 ~g/kg and then atropine sulfate intramuscuairly at 15 mg/kg. For the guinea pigs, the shortest interval between skin application of II in DMSO and sarin administra- tion at which death occurred was 12 h. For the rabbits, the corre- sponding time was 5 h. It is apparent, therefore, that DMSO can facilitate the movement through guinea pig and rabbit skin of suffi- cient II to maintain protective blood concentrations of II for reasonab' e times. —269-

The LDso of I, injected intravenously, for rabbits was decreased by prior intravenous injections of d-tubocurarine at 0.1 mg/kg or atropine sulfate at 2 mg/kg by 63.4% and 9.3:, respec- ~ci~rely.44 Prior intravenous injection of neostigmine bromide at 0.1 mg/kg increased the LDso by 31.2X. These findings indicate that I exerts its principal action on the neuromuscular junction in skeletal muscles and has only a minor effect on muscarinlc choliner- gic junctions. This conclusion agrees generally with that from Kalser's work. It is reinforced by reports that I blocks transmission in the isolated rat phrenic nerve-diaphragm preparation;45 that it antagonizes the stimulant action of acety~choline on isolated frog muscle;46 that it decreases the response of the frog rectus abdo- minls muscle to decamethonium and to carbamylcholine, in addition to the response to acetylcholine, but in large concentrations increases partial blockade of transmission in the rat phrenic nerve-diaphragm preparation induced by prolonged exposure to decamethonium;47 and that it increases, and in high concentrations decreases, the endplate potential.48 Wagley48 found that V produced a dose-related decrease in the endplate potential of the curarized illofibularis muscle of the frog at 1-3 ~ 10-2 M. A similar effect of 2-PAM ~ was found at 1-3 ~ 10~3 M, whereas concentrations below 10-3 M produced dose-related increases in the endplate potential. Fleisher et al.47 found that III, unlike I, had no excitatory action on the isolated frog rectus abdominis, but had a more potent effect than I in inhibiting the response of that muscle to decamethonium, carbamylcholine, and acety~choline. Junket et al.49 found that intravenous administration of III at 5 and 10 mgTg had no effect on the response of the cat gastroc- nemius-soleus muscle group to supramaximal indirect stimulation at either slow (0.5/~) or tetanizing frequencies. An intravenous dose of 20 mg/kg produced a marked temporary decrease in the response to a tetanizing frequency without altering the twitch response. An intravenous dose of 40 mg/kg almost abolished for 25 min or more the response to delivery of a tetanizing frequency to the motor nerve and decreased by about BOX the tension developed in the twlLch response. That dose of III alto blocked partially the response of the heart to stinmiatioD of the vague nerves, but did not modify the changes in blood pressure induced by bilateral occlusion of the carotid arteries or by intravenous injection of acety~choline chloride or epinephrine chloride at 3 ~g/kg. The effect of III, like that of I, seems to be predominantly on nicotinic cholinergic junctions. The effects of TMB-4 Con on the response of the cat heart to stimulation of the vagi and on contraction of the nictitat- lng membrane after pregang~ionic stimulation of the superior cervical ganglion were more marked than those of the same intravenous dose (15 mg/kg) of I. 50 -27C-

Dultz et al.23 infused V intravenously into dogs at 50 mg/kg per minute and I at 30 mg/kg per minute. The mean times to death after infusions of the two oximes were 10 and about 33 min. respec- tive~y. These times correspond with relative lethal doses of 100 and 196.5, respectively. These values are not far from those for intraperitoneal injections of the two opines into mice.~00~206 After V, the heart rate rose initially and then, after about 3 min of infusion, began a slow decrease. The diastolic pressure decreased progressively from the start of infusion, whereas the systolic pressure remained fairly steady during the first 4 min of infusion and then began to decrease sharply. The rate of breathing and the tidal volume were reasonably constant during the first 5 min of infusion; the rate of breathing then increased, after an initial brief decrease, to nearly 3 times its original value by 9.5 min after the start of infusion. During the same period, the tidal volume decreased to about one-tenth its original value. At 10 min after the start of infusion, the animal became apneic, the pulse pressure fell to zero, there was a brief period of arrhythmia associated ~ th an elevation of the J segment of the EGG, and then the dog died. The most striking changes during the early minute e of infusion of I were increases in systolic and pulse pressures. These were accompanied by an increase in breathing rate without much change in tidal volume or heart rate. After about 28 min of infusion, systolic pressure and heart rate began to decrease. About 3 min later, both systolic and diastolic blood pressures fell precipitously with heart rate and tide' volume. Breathing rate had begun to decrease sharply after about 28.5 min of infusion. Terminally, the T wave of the ECG was increased and prolonged, and the voltage of the QRS complex was markedly reduced. Death followed apnea by only a few minutes. The changes reported by these investigators suggest that V kills by CNS depression, whereas I kills by interfering with Depolarization and contraction of cardiac muscle. Ba11 ant yore et al. 5' gave rabbits intramuscul ar or intravenous injections of II at the LDso. They found that plasma II maintained at less than 90 ~g/~1 was not lethal. Concentrations of 90-160 l~g/ml were not lethal if they persisted for only a few minutes. If concen- tratioDe in that range were maintained for 40-SO min. there might be a sudden increase to above 200 ug/mi. A plasma II concentration above that loci t generally led to death. The II concentrat ion in the aqueous humor of the eye increased slowly, but an hour after injection was nearly the same as that in the plasma. Thereafter, the II concentration in the plasma and-in the aqueous humor decreased at similar rates. TOXICITY OF REPEATED DOSES IN EXPERIMENTAL ANIMALS . . Rats and rabbits received intramuscular injections of solutions of II in saline 5 d/wk for ll and 9 wk. respectively.S2 Dogs were -271—

given gelatin capsules of II by mouth 5 d/wk for 17 wk. Control groups of rats and rabbits were given physiologic saline intramuscu- larly on the same schedules as those which received II. No control dogs were included in the experiment; comparisons were made between the dogs given II and "normal" animals. The daily doses of II injected into rats were 50 and 150 mg/kg; those injected into the rabbits were 50 and 100 mg/kg. Dogs weighing 13-17 kg received daily doses of ~ g, or about 59-77 mg/kg. The rats given II seemed to be normal both grossly and by micro- acopic esaminatlon of sections of tissues at the end of the study. The rabbits had no abnormality clearly attributable to lI other than puNIent, indurative myositis at the site of injection in nine of 10 animals. Inasmuch as the solutions injected were not stated to have been sterilized, the myositis is not astonishing. The stomachs of the three dogs all had fibrosed mucous membranes at the cardiac and/or pylorlc regions. The plasmas of these dogs had subnormal concentra- tions of albumin and total protein and low albumin: globulin ratios. lathe rats given either of the doses of II grew normally, and indeed somewhat more than the controls. The rabbits, as is not unusual, had coccidial infestations of their livers and intestines, but had no lesions attributable specifically to the oxime. The plasma II concentrations of the dogs shortly before they were killed at the end of the exposure period were around 60-90 ~g/mI. One of the three dogs had roundworms (Tosascaris leonine) in its intestines and granulomatous nodules in its kidneys and pancreas due to this infestation. The same investigators made a comparative study of the toxicities of I and TMB-4 CI2 in rabbits and dogs, published as an interim report by FOA ~ (Cl024-FlOO) in April 1963 and as a paper in April 1964.53 Groups of eight rabbits received intramuscular injections 5 d/wk for 12 wk of I at 65 mg/kg, 11IB-4 C12 at 30 mg/kg, or physi- ologic saline at 0.2 mI;/kg. The solutions were sterilized by filtra- tion through a Seitz filter. Groups of four young beagles (9-~1 kg) were given capsules containing ~ g of IMB-4 Cl2 or of I 5 d/wk. For TMB-4 CI2, this dose was continued throughout the 15 wk of the study; for I, the daily dose was reduced to 0.75 g after the first 2 wk and kept there until the end of the 15-wk study. Except for local changes at the site of injection, the rabbits given I intravenously suffered no detectable toxic effect other than a minor loss of weight. Those given TMB-4 Cl2 began to die during the third week of the study, six of eight rabbits having died by the end of the 15-wk period. The blood of the rabbits given either oxime was seen to clot rapidly, the effect being more marked after TMB-4 C12 at 30 mg/kg than after I at 65 mg/kg and lasting for 4-5 h after an intramuscular injection. The dogs given capsules of TMB-4 Cl2 had no signs of intoxication by the oxime, whereas those given cannules of I had dime nished activity, ataxia, and head drop starting —272—

2-3 h after they were given their capsules. After the daily dose of I was reduced, these Aims of intoxication disappeared; they became evident again in three of the four dogs during weeks 10-12 of the study. The signs of renewed intoxication appeared on only a few days in each dog and then disappeared again, although administration of I continued. The curves of gain of body weight by the dogs were unaf- fected by the oximes, and all were judged to be in a normal state of nutrition at the end of the 15-wk period. At the sites of injection of oxime in the rabbits, various extents of hemorrhage and of purulent, indurative myositis and muscle necroals were seen. The rabbits that received physiologic saline and five that received intramuscular injections of 2.15 M sodium chloride on ~ ~ in a satellite experiment had waxy degeneration of muscle at the site of injection. Five rabbits that received in~cramuscular injections of 2.15 M I on ~ ~ also had wary degeneration of muscle at the site of injection. This was stated to be more extensive than that seen in the rabbits given equimolar sodium chloride. No other lesions in the rabbits that seem to be attributable to the oximes were described. The dogs given capsules of the o~cimes were found to have hypere- mia in their stomachs and intestines. Four of the eight dogs had erosions of gastric mucosa, evident particularly in the apices of the rugae in the fundus and found in animals given I an well as in those given TMB-4 CI:. ~ ~ the dogs had epithet al defects and proliferation of the connective tissue of the lamina propria. Atro- phy of stomach glands was seen sometimes. The brains of both the dogs and the rabbits were described as having peculiar, slightly granular, basophilic structures in the white matter of the brain stem. These could appear as spheres or clouds. The figure purport- ing to demonstrate these structures does not do so clearly enough to permit a guess as to their nature; they may be nothing more than fix- ation artifacts. Inasmuch as myositls was reported in the second paper53 as well as in the first,52 this response must be induced by the oxides and not by bacteria. The second paper showed that oral doses of I and TMB-4 Cl;: induced the same sort of scar formation as II in the gastric mucosa, 80 that this response may be induced by either the oximino group or the quater~ary nitrogen atom. It would be informa- tive in this regard to have the results of an experiment in which capsu] es of pyridine-2-a~ doxime and of N-methy~pyridinium chloride were administered in a similar fashion. The toxicities of repeated intravenous doses of I and II in rab- bits and dogs have been estimated.54 Six rabbits were given I at 50 mg/kg 5 d/wk for 6 wk; four rabbits were given II at the same dose on the same schedule. Three 60g8 were given I at 25 mg/kg b.i.~. 5 -27 3—

d/wk for 6 wk; three other dogs were treated similarly with II. Dur- ing the 6 wk of the experiment, 91 observations of signs of toxicity were recorded for the dogs given II and Il5 for those given I. In both groups of dogs, the most common observation was hyperventila- tion. This sign accounted for 60% of the observations recorded for the dogs given I and 7BX of those for the dogs given IT. The next most common sign for I was ataxia; for II it was vomiting. Collapse was the third most common sign with both oximes, but accounted for less than 6% of all the observed signs of toxicity. Tremor and jerk- ing of the head were equally frequent Signs of intoxication in fourth place with I, whereas tremor and ataxia had equal incidences in fourth place among the dogs given II. The only other sign reported was salivation, which accounted for less than 2% of the total of recorded signs. Hyperventilation, mentioned above as the most fre- quent sign of intoxication, was stated to occur only during injec- tions of the oximes and to stop immediately after the end of the injection. All the dogs given intravenous injections of an osime had decreases in their blood concentrations of hemoglobin (mean, -23.2X). Two of the dogs given I had increases in their hem~tocrits, whereas only one ot the dogs given 11 nao t=s sort or c=nge. "1 three dogs given I had decreases in their leukocyte counts, whereas two of the dogs given II had increases in their leukocyte counts. Of the four dogs that had decreases in their total leukocyte counts, two had increased percentages of polymorphonuclear leukocytes and two decreased percentages. The two dogs with increased percentages of PAN leuko- cytes had subnormal percentage of lymphocytes. lathe two dogs with decreased percentages of PMN leucocytes had increased percentages of lymphocytes. ~ . _ _ ~ ~ ~ ~ ~ · ~ ~ Two rabbits died after the third dose of I, and two other rab- bits from this group died during a weekend after having developed diarrhea that fasted 2-7 d. One rabbit given II died after the fourth injection. Another rabbit in this group developed diarrhea during the fifth week and died during the following weekend. Three rabbits and three dogs given ~ and two rabbits and three dogs given II were subjected to necropay at the end of the experi- ment. No lesions attributable to the oximes were found. Other rab- bits housed in the same areas as those used in this study were reported to have developed diarrhea and in some instances to have died, so that the deaths of rabbits during the experiment may have been unrelated to the experimental procedures. The decreased hemo- g~obin concentration and the diminished white cell counts in dogs may have been induced by the oxides, but the larger dose of II given to rabbits be Albanus et al.52 during a longer period did not result in decreases in hemoglobin concentration or tn the reo-ce~1 count, although it may have induced a decrease in the white-cell count. Crook _ al.54 concluded that I and II have relatively low —274—

toxicities for dogs and rabbits when they are given intravenously in daily doses of-50 mg/kg on 5 d/wk for 6 wk and that I is more likely to induce ataxia than II, but is less likely to induce vomiting. A comparative study of the toxicities of I, III, and IV a~mini- stered to rats by gavage and to dogs in capsules has been reported.55 Groups of five rats were given daily doses of I, III, or IV at 20 mg/kg through intragastric catheters 5 d/wk for 4 wk. Wheezing was the most frequent observation, even occurring in the control group. Wheezing was observed more frequently among the rats given oximes than among the control animals; it was less frequent in the group given IV than in those given the other two oximes. Chronic inflam- mation of the lower respiratory tract was found in almost all the rate. Irritation of the eyes occurred in a few rats of each group, including the control group. Hematologic measurements, organ weights, body weights, food consumption, and gross and microscopic surveillance of organs and tissues at necropsy revealed no oxime-related changes. The control dogs (given empty capsules on the Awe schedule on which other dogs received oxime-containing capau~es)-and most of those given oximes seemed to be in good health throughout the study. The exceptions were one dog in the group given I that exhibited fasciculations and tremors 10 min after receiving its first capsule, one dog in the group given III that retched 10 min after receiving its capsule on the twelfth day of the study, and one dog in the group given IV that retched about 4 h after being given its capsule on the ninth day of the experiment e No dogs died, arid all gained weight at about the same rate as during a preliminary observation period. No alterations that seemed to be related to ingestion of the oxides were found in hematologic values, blood chemistry, urinalysis, organ weights, organ: body weight ratios, or gross and microscopic appearances of tissues and organs removed at necropsy. AIL additional study of daily intravenous injection into rats and dogs of IV at 35 mg/kg 5 d/wk for 4 wk used groups of 10 rats and four doga--two males arid two females.56 The only visible signs of toxicity observed in the rats were wheezing, hype Apnea, and "awell- ing in the throat." Wheezing, recorded I] times during the total of 200 rat-days, was the most common sign among the an male given IV; it was recorded once in the control group and was the only abnormal observation for that group. "Swelling in the throat" was recorded four times among the rats given IV, and hyperpnea three times. The mean consumption of food and the mean rate of gain of body weight were significantly lower in rats given TV than in the control group, which was not given sham injections. Hemato~ogic measurements disclosed no significant differences between the two groups of rats. The mean liver and kidney weights were reduced in the rats given IV; the mean adrenal weight in these animals was identical with that in -275-

controls. When organ:body weight ratios were calculated, the ratios for liver aM kidney in rats given IV did not differ signifi- cantly from those in the control rats. The ratio for the adrenals in rats given IV was above that for the ccatrol group. One concludes that the anorexic a resulting from injection of IV into the rats affected the weights of the liver and kidneys in proportion to its effect on the overall body weight, but did not alter the size of the adrenals. The only sign of toxicity recorded for the dogs given IV was retching, which was recorded six times during the 80 dog~ays of obeer~ration within the second, third, and fourth weeks of the experi- ment, but not during the first week. The dogs given IV lost 2.34% of their original mean body weight during the experiment, whereas the control dogs lost only I.6BX. One bitch among the dogs given IV had increased alkaline phosphatase and transaminase activities in its serum after the fourth week of the experiment, but not after the second week; microscopic study of sections of this animal's liver recreated degenerative changes in the periphery of the lobules. The livers of the other three dogs in this group were free of significant pathologic findings, so that the changes in the liver of one of the bitches may have been unrelated to administration of the oxime. No other significant pathologic findings were reported, except for the finding of intestinal parasites (hook worms, round worms, tape worms, or whipworma) in most of the dogs Hematologic, blood chemical, and urinanalytic studies revealed no significant differences between the control group and that given IV other than those mentioned above for one bitch. The toxic effects of 2-PAM I and III on rabbits and dogs, liven intravenous injections 5 d/wk for 6-8 wk. have been evaluated. 7 Both rabbits and dog e received daily doses of 2-PAM T at 30 mg/kg and of III at 10 mg/kg. Two dogs and two rabbits received intra- venous injections of physiologic saline on the same schedule fo1- lowed for injecting oximes. Groups of three dogs and three rabbits were given the offices. All animals were observed for signs of toxic effect after the injections and were weighed weekly. Hematologic add blood chemical studies were performed on the dogs. Rectal temperatures of the dogs were measured once each week. All animals were subjected to necropay at the end of the Study. Samples of abdominal and leg muscles and of blood from the dogs were analyzed for o~cimes. All rabbits gained weight during the 6~ periods of injection of 2-PAM I (0.26-0.84 mg/kg) and of III (0.18-1.14 mg/kg). The dogs ate well and seemed to be in good physical condition during the 8-wk period of injection of 2-PAM I. Two dogs maintained their original body weights, and the third gained about 0~5 kg. The dogs neither lost nor gained weight during the 6~k period of injection of III. -276-

Neither the rabbits - nor the dogs gave visible evidence of toxic ef fects during the experiment. The only indicator that suggested an effect on the composition of the blood of the dogs was the white-cel' count after ~ wk of in] ec tion of 2-PAM I . This increased by a mean of 27 . 6%, whereas it decreased by 5. 5% in the control group during the same period. If the plasma concentration of an oxime 5 min after intravenous injection was taken as the initial value, at 30 minutes it had fallen to 21.~% (2-PAM I) or 28.3: (III) and at 60 min it had fallen further to 10.7X (2-PAM I) or 14.4% (III). It is apparent, therefore, that III is removed initially from the plasma somewhat more slowly than 2-PAM I, but that the difference is not great and that there may be an increase in the rate of removal of III somewhere between 30 and 60 min after the initial value. The percentages of the initial concen- trations of 2-PAM I and III revaluing in the plasma at 155 min after estimation of the initial values differed by only about 0.1% Abdominal and thigh muscles examined 20 h after the last intra- venous injection of oxime contained no detectab] e amount of either oxime. Samples of muscles collected 30 and 90 min after the last dose of oxime contained 2-PAM I at higher concentration than III. At both these times, thigh muscle contained a higher concentration of 2-PAM I than abdominal muscle, but a lower concentration of III. Dogs and rabbits appear, therefore, to tolerate repeated daily intra- venous doses of 2-PAM I at 30 mg/kg or of Ill at 10 mg/kg during a period of 6-8 wk when the daily doses are suspended during each week- end. Because in this and the other studies reviewed the animals were killer at or soon after the end of the period of administration of an oxime, there has been no opportunity to judge whether repeated administration of an oxime may initiate some alteration in normal structure or function that will result eventually in a definite lesion. No truly chronic study of the toxicity of an oxime has been found. Thus, possible cryptic toxic effects of this type of compound have never been assessed. SIDE OF OXIMES IN MAN Hop ff and Waser58 have listed mechanisms whereby reacti~rators of inhibited cholinesterase could be harmful to persons to whom they are admi nistered in treatment of intoxication by anticho 11 nesterase compounds. The following is a slightly modified version of their list: · The Deactivator may affect enzymes other than those involved directly in the actions of the inhibitor of cholinesterase. · The Deactivator may itself affect some part of the active center of cholinesterase. -277—

· The Deactivator may form, either with the inhibitor or with its residue in inhibited cholinesterase, a stable secondary inhibitor of cholinesterase. · The Deactivator or a stable complex between the Deactivator and the inhibitor may affect important functional systems of the body other than those related to cholinesterase. · The Deactivator may be metabolized to harmful products. The last three mechanisms of action, and possibly the other two as well, are involved in the causation of the side effects that have been reported to occur either in normal subjects to whom reactiva- tors had been administered during research projects or in patients who had been given reactivators as therapy for intoxication by pesti- cidal anticholinesterases or other inhibitors of cholinesterases. Some side effects seem to be common to all six deactivators with which this report is concerned. For example, complaint of a bitter, metallic, salty or musty taste has been made by people who have been given any of the six oximes, whether by mouth or by injection. Intravenous injections of any of the oximes, if the solutions were too concentrated or were given too rapidly, have resulted in pain along the vein. Dizziness, nausea possibly progressing to vomiting, blurred vision with impaired accommodation, and muscular weakness have been complained about often. Convulsions have been reported after V.59~63 Moderately marked increases in systolic and diastolic blood pressures with increased pulse pressures and tachycardia have been reported to follow intravenous administration of 2-PAM I, I, and II.59~63~69~72~73 The increases after II were not as great as those after I. The two bisquater~ary bis-oximes (III and IV) may produce initial increases in blood pressure, but these are followed by marked and prolonged hypotension, the pulse pressure being reduced progressively after administration of the oxime.72~77 Obi- doxime given by mouth did not alter blood pressure.~] Another symptom of some practical importance is gastrointestinal distress, evident especially when oximes were given repetitively dur- ing several days. This symptom has been particularly bothersome with II an`1 III,72,80 but has been reported after large doses of I also.76 III and IV have induced symptoms that suggested local anesthetic ef fecta: sensations of heat or coolness in the nose and throat, circ~oral numbness, and facial paresthesia.72~73~77~80 III has given- rise to ic~cerus, petechi~ bleeding with increases in pro~rombin time and sedimentation rate, increases in serum alkaline phosphatase, go utamic-oxaloacetic traneaminase, and glutamic-pyru~ric transaminase activities, an increase in serum bilirubin concentra- tion, and a fine macular rash on the face and arma.72 IV may have resulted In cholestatic hepatosis,73 but ll persons (six men and -278-

five women) given two intramuscular injections of 250 mg of IV 2 h apart on one day had no significant increases in their serum alka- 1 ine phosphatase, glutamic-oxaloacetic transaminase, g'utamic-pyru- vic transaminase, glutamate dehydrogenase, or sorbito} dehydrogenase activities thereafter.78 Also, four oral doses maklug a total of 7.36-g of IV on one day induced no significant alterations in serum glutamic-o2raloacetic ard glutamic-pyruvic transaminase activities in 10 people who were given that dose, although four of them complained of some side effect. Facial paresthesia and headache were the principal complaints of the 13 subjects, in a group of 24, who men- tioned symptoms after oral doses of 1.84-7.36 g of IV. Sidel1 _ al.~° reported that intramuscular injection of I or sodium chloride into normal human subjects resulted in increases in serum creatine phosphokinase activity. When the concentrations of the solutes in the solutions were expressed as mi]]losmols per liters sodium chloride and I yielded nearly parallel relationships between the increase in serum creatine phosphokin~se activity and the amount of solute administered, I being 2.4-3.5 times as active as sodium chloride in inducing an increase in activity. The data furnished by these investigators indicate that, when the volume of solution injected into muscle was constant, the release of creatine phosphoki- nase from the muscle was related directly to the o~molarity of the solution; when the osmolarity was kept constant, the amount of crea- tine phosphoklaase released was a function of the amount of material injected into the muscle. A graph of the increase in serum creatine phosphokinase activity as a function of the amount of I injected extrapolates to a point close to the origin, whereas the line of the increase in serum creatine phosphokinase activity as a function of the concentration of sodium chloride injected extrapolates to zero at a concentration of 4%, indicating that I had an effect on the integrity of cellular membranes beyond that due simply to osmotic relationships. The report by Wedd and Burgesses that intramuscular injection of a 25% solution of II produced no more damage than intra- muscular injection of a 7.5% solution of sodium chloride suggests that II may not have the extra damaging effect on cellular membranes that I seems to have. In this regard, consideration should be given to ache possibility that the biochemical indicator of effect used by Sidell et al. may be more sensitive than the histologic indicator used by Wedd and Burges ~ . Obi~oXime ~8 been stated8] not to have any local irritant action when injected intramuscularly; it did produce facial pares- thesia, headache, a sensation of coo' nese in the mouth, genera- lized weakness, nausea and vomiting, pallor, pyrosis, and sore throat in 13 of 24 subjects given tablets of IY in doses of 1. 84-7 .36 g. Administration of IV in tab' eta with enteric coatings probably did not alter the incidence of side reactions--only four subjects and two doses of the oxime were used in this part of the study. -279-

MODIFICATIONS OF NOT UNIONS BY OXIDES The oximes were developed originally as reactivators of inhi- bited cholinesterase by evolution from hydroxyIamine through hydrox- amic acids to offices. Kinetic studies with hydroxamic acids indi- cated that the hydroxamate ions were the reactivating agenta.82- 0n the assumption that the anionic site in inhibited cholinesterase is still operational, so that a positively charged site in a reac- tivator could be used as a directing group to guide the active group in a molecule of a Deactivator to the inhibited esteratic site of the enzyme, compounds containing quatemized amino groups were made. This chemical change in, for example, pyridine-2-aldoxime also lowered the pK of the resulting pyrld~ium oxime and increases its ability to move into tissues. Me thy] nicotir~ium hydrodynamic acid iodide ah later 2-PAM I~both having a quaternized nitrogen atom removed by about two carbon atoms from a hydroxylated nitrogen atom-- were found to be effective Deactivators of chollnesterase inactivated by organophosphorus inhibitors.2~4~82 Although the truth of the basic assumption was not demonstrated until 1959,84 the general success of quateraization in increasing the reactivating potencies of hydroxamic acids and oxides with pyridine skeletons made it almost certain before then. Oximes were found to be much more active than the corresponding hydroxamic acids.83 At about the same time, hydroxamic acids and oximes were found to react directly with organophosphorus compounda.84~85 2-PAM I was found to react in vitro with sarin with marked deviation from firat-order kinetics; that suggested that the reaction actually consists of (at least) two sequential reactions. Green86 showed that quateratzed pyridine aldoximes react with an orsanoDhosohorus (OP) compound in three steps: ~ ~ ~ _ 0 Formation of a phosphorylated or phosphonylated oxime by reaction of the OP compound with the oximate ion. O Hydroxylation of the phosphorylated or phosphonylated oxime to produce an N-alky~cyanopyridiDium salt. ~ Hydrolysis of the N-alky~cyanopyridIalum compound to an N-alky~pyridone and hydrogen cyanide. The second-order rate constant for the reaction between sarin and either 2-PAM I or II was found to be 170 L/mol per minute. If a phosphorylated or phosphonylated oxime that does not enter rapidly into the second step above is formed, that product may be an inhibitor of cholinesterase.87~8 Hydrolysis of sarin in the presence of 20 - for concentrations of V and I] took place more rapidly in plasma from rats with the former osime than with the -280—

latter.89 In human plasma, sarin Was hydrolyzed more rapidly in the presence of II than in that of V. The protective activity of IV against lethal intoxication by OP compounds has been thought to be exerted principally by promoting reactivation of inhibited cholinesterase. O IV has been found to produce either activation or inhibition of acetylcholinesterase, depending on the simultaneous concentrations of that osime and of acety~cho~ine in the vicinity of the enzyme.9i With acety~choline at 5 ~ 10~4 M, concentrations of I1J below about 3 ~ 10~4 M produced activation of cholinesterase; greater concentrations of the oxime inhibited cholinesterase. Increasing the concentration of acety~cho~ine to lo-2 M decreased progressively both the activat- ing and inhibitory actions of IV. With the substrate at lo-2 M, the activity of the enzyme was not altered appreciably by IV at 5 10-6 M to 7 s 10~3 M. These findings suggest that acety~cho1 ~ ne and IV react with different, but interdependent, receptors on the molecule of acety~cholinesterase; the data contain no evidence of competition between IV and acety~choline for a common receptor. Ho lees and Robins45 reported that 2-PAM I overcame neuromus- cular blockade induced by DFP, TEPP, or satin, but this could be demonstrated with the isolated phrenic nerve-diaphragm preparation from the rat only after washing away excess OP compound. Intravenous injection of 2-PAM I overcame slowly neuromuscular blockade induced by OP compounds. The oxime, they found, also had a direct toxic action on muscle, reducing the ability of muscle to shorten and decreasing the ability of muscle fibers to conduct impulses. lathe same investigators reported92 that ~ (intraperitoneally at 150 mg/kg) had little, if any, effect on a sarin-induced blockade of neuromuscular transmission. Wills aul Kunke193 studied a group of 46 compounde-~-tubocura- rine chloride, decamethonium bromide, gallamine triethiodide, a series of quaternized bisoxamide salts, a group of tertiary and qua- ternary anticho~inergic compounds, and a group of hydroxamic acids and o~cimes--for their ability to antagonize a sarin-~duced blockade of neuromuscular transmission to the cat's gastrocnemius-soleus mus- cle group during indirect stimulation at a frequency of ~ shock every 2 a. They identified d-tubocurarine chloride. two of the oxamide derivatives, and a quaternary anticholinergic and antihistaminic com- pound as particularly active antagonists of sarin's effect. 2-PAM I and 2-PAM benzy] bromide had comparatively low activity. V had only slight, if any, activity in this test . When the frequency of indi- rec~c stimulation was raised to 40/s, 2-PAM I became much more effec- tive in antagonizing blockade of neuromuscular tranamission than an atropinium salt that had had about the same activity as 2-PAM I against blockade at the lower frequency of stimulation. -281-

Pleisher94 reported that both sarin and VX increased the sensi- tivity of the isolated frog's rectus abdominls to external applica- tion of acety~cho Une and at the same time decreased the activity of cholinesterase in the external surfaces of the muscle cells. Sarin at 5 ~ 10~7 M reduced the threshold concentration of acety~choline for inducing contraction of the muscle to OX of that required before application of sarin. The same concentration of VX reduced the threshold concentration of acety~choline to 6.7% of that needed pre- viously. Contemporaneously, the activity of cholinesterase in the external surfaces of the muscle cell was reduced to 8.~% and OF of that before application of sarin and VX, respectively. Addition of 2-PAM I at 5 s 10~4 M to the baths in which the muscles were sus- pended had little effect on the activity of the enzyme in homogenates of the muscles, but restored 75% and 91%, respectively, of the acti- vity of cholinesterase in the external surfaces of the muscles exposed to sarin and VX. At the same time, the concentration of ace- tylcholine required to induce contraction of the muscles was raised to 53.3Z and 58.3% of the original threshold concentrations, respec- tively, for the muscles exposed to sarin and to VX. Rats poisoned by subcutaneous injection of VX at twice the LDso were kept alive for 20 min with artificial ventilation-of the lunge when necessary.95 At 20 min after the dose of VX, some rats were given intraperitoneal injections of either atropine sulfate (7 mg/kg) or atropine sulfate plus 2-PAM I (17.5 mg/kg). Untreated rats were killed 20 min after injection of VX; samples of parotid gland, gas- trocnemius muscle, add brain were collected for examl~ation for cho- linesterase activity. The treated rats were killed 3 h after treat- meet. Organ samples were collected and analyzed for chow nesterase activity. Reactivation of cholinesterase was calculated as 100 times the ratio of the difference between cholinesterase activities 3 h after therapy and 20 min after VX to the difference between cholines- terase activities in unpoisoned rats and in poisoned rats 20 men after VX; it is shown in Table 3 for the two modes of therapy. Because atropine has never been found to have reactivating activity in vitro, the reactivation that occurred in the rats treated with atropine Fate is assumed to be spontaneous. It Is apparent from Table 3 that addition of 2-PAM I to atropine increased cholinesterase reactivation by 40.5% in the parotid gland, by 127.~% in the gastroc- nemius muscle, and by 8.2% in the brain. The especially large change in cholinesterase activity in skeletal muscle suggests tat this may be the principal site at which 2-PAM ~ antagonizes inhibition of cho linest era se . The finding by Fleisher et a].47 that 2-PAM I facilitated response by skeletal muscle to acetylcholine and depressed responses to de came Phonic and carbamylcholine, whereas IIT had only the dePres- sant actions, suggests that 2-PAM - cated in Ill. The nature of this ., ~ ~ has an activity that is not dupli- difference is not entirely clear. -282-

TABLE 3 Recovery of Cholinesterase Activity in Parotid Glands, Gastrocnemius Muscles, and Brains of Rats Poisoned with Subcutaneous Vat at 40 llg/kg and Treated 20 Min Later with Intraperitoneal Atropine Sulfate at 7 mg/kg or with Intraperitoneal Atropine Sulfate and 2-PAM I at 17.5 mg/kga Reactivation, X of inhibition by VX Parotid Gastrocnemius Go and Muscle Therapy Atropine sulfate 39.0 Atropine sulfate + 2-PAM I 54.8 25.2 57.4 Brain 8.5 9.2 a Samples of tissue were removed 3 h after therapy, or 20 min after VX if no therapy was administered. Groups of rats contained a mean of ~ (6-15) animals each. —283-

Because 2-PAM adds to the depressant effect of d-tubocurarine on the response of skeletal muscle to indirect stimulation, as weU as to those of decame~chonim and carbamy~choline, the facilitating action canon be based on depolarizing activity. __ that the facilitating component of the action of salts of 2-PAM depends on the ability of this oxime to inhibit cho~nesterase, rather than on a direct depolarizing action. The finding that salts of 2-PAM depress the response of eserinized muscle to acety~choline and to the inhibiting action of d-tubocurarine polats to the posses- sion by 2-PAM of not only a chollnestera~e-inhibiting action but also a competitive interference with acety~choline uptake by the motor endplate analogous to that of d-tubocurarine itself. The accentua- tion by salts of 2-PAM of blockade of neuromuscular transmission by such depolarizing compounds as decamethonium and carbamy~cho~ ne would be analogous to that of d-tubocurarine in increasing Phase II blockade by depolarizing compounds. Although III is a more potent inhibitor of acety~cholinesterase in vitro than the salts of 2-PAM, its effects on muscle seem to be those of a competitor with acetyI- choline for access to the motor endplate. Edery96 studied the effects of V on neuromuscular blockade induced by ethyl pyrophosphate or neostigmine methylaulfate in the cat, finding only an insignificant effect. The skeletal muscle responses to both direct and indirect excitation were altered by V in the absence of any other active chemical. The effect of V on the response of indirectly stimulated muscle to d-tubocurarine was an increase in the blockade of neuromuscular tranamisaion similar to that of the salts of 2-PAM and of III. ~ , _ ~ ~ Fleisher et al. suggested Table 3 shows that cholinesterase inhibition in brat" is affected only slightly by a dose of 2-PAM I that produces nigDifi- cant reactivation of the enyzme in muscle and parotid glares. Indeed, there has been considerable controversy about the ability of oximes to enter the CNS and reactivate inhibited choUnesterase there. Several clinical observations suggest that comparatively small doses of olives have significant effects on cerebral function. For e~cam- ple, Schuchter et al.97 reported a case of poisoning by parathion treated initially with atropine alone. After administration of 30.5 me of atropine sulfate during the 14 h after admission to hospital, the apnea, convulsions, and arrhythmia tachycardia that had characte- rized the patient' condition on admission had stopped. The patient was still unconscious, however, with markedly constricted pupils. At that time, intravenous injection of 0.5 g of 2-PAM I in a 1% solution relieved both residual effects immediately, and no further treatment was required. Several similar reports are available in the medical literature; they suggest that, even though the quatern~zed oximes may not be able to cross the blood-brain barrier in large amounts, either the small quantities that penetrate that barrier are capable of affecting the function of crucial parts of the brain or the oximes -~.84—

affect the functions of peripheral structures (perhaps receptors of sensory input) that modify the functions of crucial parts of the brain. The latter possibility is related to the proposal of Erdmann _ al.98 that restoration by 2-PAM I of the righting reflex after its abolition by parathion is effected by the oxime's modifying inhi- bition of chow nesterase in some peripheral site important for acti- vity of the refiles. Rajapurkar and Koelle99 reported that intravenous V at 40 mg/kg, but not at 4 mg/kg, induced reactivation of cholinesterase in the sur- faces of cells of the casts superior cervical ganglion after the ani- mal had been given DFP at 3.7 mg/kg 20 min earlier; there was no sig- nificant reactivation of the cholinesterase in the ganglion as a whole (after homogenization). These findings suggest that, even when an oxide is able to make contact with the surfaces of nerve cells, it is not able to penetrate into the neurons; this is similar to the situation for muscle cells described by Pleisher.94. Scha~a~iOO found that pretreatment of mice ~ th 2-PAM I reduced inhibition of acety~cholinesterase in brain by parao~on much more effectively than those by DFP and 217-AO. The finding of some protection against all three OP compounds could depend on direct reac- tion between the last two inhibitors and the oxime, with a reduction in inhibition of the enzyme. A similar considera~cion applies to the report by Bisa et al.~°l that IV protected serum and brain choli- nesterase from inhibition by paraozon administered later at twice the LD,o. Although the same intraperitonea] dose of IV (7 ma) was found to protect the cholinesterase of rat serum and brain only incom- pletely from inhibition by DEP at 5 times the LD,o, that of serum recovered its normal activity by 20 h after the dose of DEP, whereas that of brain required 26 h for recovery. On comparison of cholinesterase activities in the brain on various days after savage with parathion at 35 mg/kg and after exposure of a homogenate of brain in vitro to 10 3 M 2-PAM I, the reactivation _ , accomplished by the oxime was found . ~ to decrease progressively as spontaneous reactivation increased: .= Brain Cho~nesterase, % of noroal Before 2-PAM I After 2-PAM I . 0 6 100 1 20 59 4 44 79 8 63 76 -?~85- 9

In vitro, addition of IV and DFP simultaneously to red-cell cholinenterase90 protected the enzyme against inhibition by DFP to a greater extent than the reactivation that the same concentration of the oxime (10-3 M) was capable of effecting after echo sure of the enzyme to the same concentrations of DFP (~-3 ~ 10~ g/mI). In guinea pigs, simultaneous injections of IV (intramuscularly at 100 mg/kg) and DEP (subcutaneously at 1.5 mg/kg) limited inhibition of red-cell chow nesterase to 20%, whereas the same dose of DFP alone inhibited it by about 65X. Half that dose of IV injected intramuscularly 90 man after the same dose of DFP stopped inhibition of red-cell cholinesterase by DFP and returned the activity of that enzyme to 93% of its normal value by 24 h after the dose of DFP. The red-cell cholinesterase of guinea pigs that were not given oxide after the dose of DEP had risen to only 87% of its normal value by the twenty-first day after injection of DFP. In dogs poisoned with soman (intravenously at 30 vg/kg) and treated with I at 104 mg/kg (la/ravenously 3 1/2 min after soman), the large dose of I stopped aging of inhibited cholineaterase and reactivated 24.0Z and 35.6Z of the red-cell and diaphragm cholineste- rase activities, respectively. It failed to reactivate brain choli- neaterase. Indeed, the brain acety~cholinesterase activity after the treatment with I was lower than that Just before the injection of I. The last finding indicates the inability of I to cross the blood- brain barrier in significant quantities. Apparently on the other aide of the picture iB a report by Meeter 04 that intraperitoneal injection of either III or IV at 40 mg/kg, given to rats when their body temperatures had fallen 2-2.5°C after intravenous injections of DFP at I.2 mg/kg, blunted the fan , which might proceed in untreated rats to a decrease of nearly 6 °C. These doses also shortened the return of body temperature to its nor- mal value. Larger doses of the same oilmen blunted the fall less than the smaller dose, but had more effect on the rate of recovery of body temperature to its normal value. From the fact that atropine also is a good antagonist of the hypothermic action of OP compounds, whereas an equivalent dose of me tiny} atropinium nitrate has essentially no antagonistic activity, the hypothermia seems to result from some effect of an OP compound in the CNS. On this basis, one supposes that the moderate doses of II! and IV antagonize some effect of OP com- pounds on the CNS that leads to hypothermia and that larger doses of the same oximes exert initially a toxic action that may actually increase hypothermia. As the concentration of the oxime in the body falls back toward that established by the modest dose, the antago- nistic activity becomes evident. It would be useful to know whether oximes are able to increase the partial antagosdem of loception of hypothermia that atropine has been found to exert. -~86-

A dose of IV given intramuscularly to atropinized guinea pigs 0.5 h after a sublethal dose of sarin produced less reactivation of retinal and brain cholinesterase than an equimolar dose of I.~05 reactivation of 23.9% of the inhibited cholinesterase in the retina by I was accompanied by one of only 3.7: in the brain. If IV pro- duces less reactivation of inhibited cho~nesterase in the brain than I, any central mechanism related to the induction of hypothermia by OF compounds must be either very sensitive to the concentration of acety~choline within the brain or more permeable to the quaternized oilmen ached the brain in general. Atropine has been foundl06 to reduce markedly the increase in the concentration of total brain acetylcholine in rats later given parao~on at 0.4 mg/kg. Rats given a dose of paraozon and than treated with IV intraperitonea1ly had brain concentrations of free and tote' acety~choline that were essentially the same as those in rats given parao~on alone, but no tremors or convulsions were observed. These animals survived; those given paragon alone al, died in convulsions within 3-8 min. Badgar et al.~07 found that acety'cholinesterase activity in the pontomedul~ary area of the mouse brain was possibly a direct linear function of the dose of IV (2.5-35.0 mg/kg) injected intra- muscu~arly with a constant dose (21 mg/kg) of atroplne sulfate 30 after an intramuscular dose (400 ug/kg) of sarin. They found no significant relation between the dose of IV and acety~cholinesterase activity in mesencephalon, diencephalon, and basal ganglia in the same experiments. Mortalities in the groups of mice given various doses of IV seemed to be inversely related to the dose of the oxime and, therefore, to cholinesterase activity in the pontomedullary region. Vasic et al. ,108 using armies as the OP compound, found that IV (intraperitoneally at 25 mg/kg 5 min after subcutaneous injection of armin at 0.4 mg/kg) resulted in a cholinesterase acti- vity in the pontomedul~ary region of the rat's brain 45.3% of that in control anir - In, whereas this region in rats given armin alone contained only 18.7Z of the cho~inesterase activity of controls. The decrease in cholinestera~e activity was accompanied by increases in the total acety~cho~ine concentration in the pontomedullary region of 52: in the rats given armin alone and of 25% in those given both armin and IV. 2-PAM I seems to be less effective than IV in reactivating brain cholinesterase after its inactivation by parao~on or other OP compounda.l09 There was early evidence that 2-PAM I produced more reactivation of cholinesterase in the pontomedul~ary region and the area postrema that had been inhibited by paragon than in the cere- bellum and the cerebral cortexli° and that it could prevent the appearance of grand mar-like discharges in EEGs of rabbits after Closes of sarin that evoked such discharges in rabbits not protected —287—

with 2-PAM I.~} This report by Longo et al. is not completely satisfying in indicating penetration of a quaternary oxime into the brain inasmuch as the protective effect could arise from direct reac- tion between the once and sarin in the blood; in that case, only a part of the satin administered would be available to affect cholines- terase in the brain and other tissues. The protective effect of the dose of 2-PAM I (30-50 mg intravenously in rabbits weighing 2-3 kg) was found to be overcome by doubling or tripling the dose of sarin. This finding suggests that the protective action of the 2-PAM I was due primarily to direct reaction with satin. - In the final study to be mentioned in regard to the possible penetration of pyridini~ oximes into brain, anesthetized, atropi- nized cats were given intravenous injections of sarin at 27,~Lg/kg.l'2 Thirty minutes later, to allow clearance of unreacted satin from the tissues, some cats received saline injections into one common carotid artery an others received similar injections of I at 15 mg/kg. The cholinesterase activity of cerebral cortex was measured. In animals given I, nearly 20% of the cholinesterase in their cerebral cortices that had been inhibited by sarin was calculated to have been reactivated by the oxime. The only functional changes noticed in cats and dogs (anesthetized with sodium pentobarbital) after intravenous 2-PAM I at 5-60 mg/kg were stimulation of respiration (principally increased depth) and an increase in pulse prensure.~3 The mean blood pressure, after a small dip immediately after the injection, returned approximately to the control value. With repeated doses of 5 and 10 mg/kg, the heart rate decreased after each dose and then returned gradually to its normal value e After a cumulative dose of 100 mg/kg, there was marked lowering of the heart rate and a decrease in the voltage of the T-wave of the EGG. Isolated hearts from rabbits perfused with 2-PAM I at 1.3-2.7 g/L in the Ringer-Locke solution underwent no alteration of normal activity other than a alight decrease in the rate of beating, even when the perfusion was continued for more than an hour. In cats, intravenous injection of 2-PAM I at 40 mg/kg or more markedly increased the peristaltic activity of the intestines. Atropine stopped this ef feet. Supramaxim~1 indirect stimulation at 30/min of the cat's gastroc- nemiun-soleus muscle group was blocked by close intra-arterial injec- tion of 2-PAN I at 80 mg/kg. By the intravenous route, the oxime at 80-100 mg/kg induced a persistent increase in the tension developed during a twitch. An intravenous dose of 300 mg/kg resulted in a brief increase in the tension developed during a twitch followed by a decrease in the tension iaBt~g for about 2 min. ~i8 dose resulted also in a sequence of changes in blood pressure: brief hypertension, hypotension with increasing pulse pressure, and slight hypertension with greatly increased pulse pressure. Respiration, recorded from the —288—

pressure in the side arm of a T-shaped tracheal cannula, went through a sequence of changes similar to those in blood pressure: a brief increase in depth of respiration, a brief decrease in depth of respi- ration, increasing depth of respiration with an increased rate, and greatly increased depth of respiration at a rate somewhat below the original rate. Mydriasis and muscular fasciculation were observed also after this large dose of 2-PAM I. Blockade of neuromuscular transmlasion produced by an intrave- nous dose of d-tubocurarine chloride at 0.3 mg/kg, but not that due to 0.5 mg/kg, was antagonized by intravenous 2-PAM I at 5 mg/kg. Edrophordum bromide (intravenously at 0.2 mg/kg) was a more potent antagonist of d-tubocurarine than this dose of 2-PAM I. Partial blockades of neuromuscular transmission induced by intravenous injec- tions of decamethonium bromide at 20 ~g/kg, neostigmine bromide at 1 mg/kg, or succiny~choline chloride at 50 ~g/kg were intensified by intravenous injection of 2-PAM I at 5 mg/kg, in about the same way in which they were enhanced by intravenous doses of edrophonium bro- mide at 0.2 mg/kg. Intravenous doses of 2-PAM I at up to 150 mg/kg had no effect on the muscle response to direct stimulation in anes- thetized cats. Doses of 2-PAM I larger than 40 mg/kg produced brief blockade of the cardiac response to peripheral vaga' stimulation and of the nic- titating membrane's response to preganglionic, but not to postgang- lionic, stimulation of the cervical sympathetic trunk. Reactions in which postganglionic adrenergic effecters participate--such as mydriasis, presser response to injected acetylcholine by atropinized animals, presser response to injected epinephrine, and presser response to bilateral occlusion of the common carotid arteries--were augmented definitely by intravenous injection of 2-PAM I at 20 mg/kg or more. No significant effect of 2-PAM I on the EEGe of curarized cats was recorded. In sugary, 2-PAM I was found by Kunkel et al.il3 to act as a depolarizing compound at the neuromyal junction and to have acetyI- cholinomimetic properties in large doses. It had some ganglionic blocking activity, but no direct effect on the ENS was detected. A later paper presented the results of a study of the mechanism of the sympathomimetic cardiovascular actions of I. ii4 Cats and dogs anesthetized with allobarbital-urethane were used for measure- ments of pressure near the bifurcation of the descending aorta and of blood flow with electromagnetic probes. Intravenous injection of I at 20 mg/kg was found to produce an immediate, sharp increase in blood pressure lasting for about 25 ~ and followed after a lag of about 6 ~ by an increase in blood flog. A slow drift downward of the peak systolic pressure followed. Repetition of the dose of I after an hour yielded responses similar to those after the first dose, but -289-

th a somewhat larger and more persistent immediate effect on blood flow. Calculation of the peripheral resistance and graphing of simultaneous plots of peripheral resistance, peak systolic blood pressure, blood flow, and contractile force of the heart (from Walton strain-gauge arches attached to the outer surface of the ventricles) revealed that the peak systolic blood pressure paralleled more faith- fully the peripheral resistance than it did any of the other vari- ables and that the peripheral resistance increased more abruptly after a third dose of I at 30 mg/kg than after the second such dose, which in turn induced a larger increase than the first dose. The blood flow decreased to 55% of the original value during the 76 min between the peak flow after the second dose of I and the third dose. It increased only slightly after the third dose. A dose of I at 30 mg/kg increased the effects of intravenous doses of epinephrine at 5 g/kg and of dI-aorepinephrine at 10 ~g/kg on both blood flow and blood pressure. Intravenous phenoxybenzamine at 15 mg/kg plus tolazoline at 2 mg/kg prevented almost completely the actions of I or blood pressure add blood flop. Intravenous reserpine at 2 mg/kg increased markedly the effects of I at 30 mg/kg on blood pressure add peripheral resistance, but converted the usual immediate, small, temporary increase in blood flow into an imme- diate, small, temporary decrease. These various responses would be expected from either a mild sympathomimetic amine or an inhibitor of the breakdown of endogenous catecholamines. Indeed, I at 10~4 M, was found to inhibit the monoamineoxidase of the rat's liver. If the done of I used in these experiments were distributed into the same fraction of the body water as that estimated for the human body,li5 the concentration in the plasma would be about 9 times that stated above as the effective concentration for inhibiting the monoamineoxidase. It is possible that inhibition of monoamineoxidase by I plays a part in inducing the ef fects of the oxime on blood ves- sels ad blood pressure. It is possible also that I interferes with reuptake of catecholamines by nerve endings; this possibility seems not to have been explored. Another study of the effects of I on the cardiovascular systemii6 concluded that, in dogs anesthetized with sodium pento- barbital, the response of blood pressure to intravenous admini- stration of I is a resultant of two separate effects: a direct myocardial stimulation that was stopped with dichloroisoproterenol and a stimulation of vascular smooth muscle that results in a slight increase in renal arterial pressure and a slight decrease in renal arterial flow. Neither atropine nor dichloroisoproterenol affected theme vascular effects. Injections of I into a Jugular vein or a renal artery had no consistent effect on catecholamine concentra- tions in plasma taken from a femoral artery or a renal vein. In seven experiments in which I at 21-35 mg/kg was injected into a jugular vein, the mean blood pressure increased from 176/125 + 22/11 -290-

to 186/124 ~ 53/39 mm Hg, and the mean concentration of catecholamines in the plasma of femoral arterial blood went from 1.7 + 0.7 to l.9 ~ I.3 ~g/L. In three experiments in which 100 mg of I was injected into the renal arteries of ca~ne kidneys that weighed a mean of 62.3 g (body weights not stated), renal blood flow changed from a mean of 233 + 2.9 mI/min deco 227 + 8.2 ml/mint Mean _ _ catecholamine concentrations in renal arterial be Cod changed from 1.5 + 0.3 to 2.l ~ 0.4 g/L of plasma, and those in renal vein blood from l.5 + 0.5 to 2.0 + 0.3 vg/~. Another study of the hypertensive action of I was made by DiPa~oa and associates.il7 Dogs anesthetized with a11obarbi~cal- urethane and cats anesthetized with urethane or by section of the cervical spinal cord during anesthesia with ether were used. In 11 dogs, intravenous injection of I at 20 mg/kg induced an increase in mean blood pressure from 160/103 + 28/22 to 201/130 + 28/23 mm Hg at 2 min after injections by 20 min after injection, the mean blood pressure was I85/~19 + 35/27 mm Hg. Phenoxybenzamine (intravenously at ~ mg/kg) and phentolamine (intravenously at ~ mg/kg) consistently blocked the hypertensive action of I when they were injected a few minutes before I. Hexamethonium (intravenously at I-2 mg/kg 10 min before I) did not prevent the increase in blood pressure, but did eliminate the initial period of bradycardia and the initial spike of hypertension; slow development of an increase in blood pressure still occurred. Pentolinium tartrate (at 2 mg/kg), succinylcho~ine chloride (at ~ mg/kg), guanethidine (at I-3 mg/kg), and d-tubocura- rine chloride (at I.2 mg/kg) were ineffective in preventing the increase in blood pressure. Dogs given norepinephrine by infusion at 0.~85-2.04 ~g/kg per minute for long periods ~ 9-35 h) until their blood pressures became approximately the same as they had been before the infusions began were then given intravenous injections of I at 20 or 40 mg/kg, tyra- mine at 0.l or 0.5 mg/kg, or nicotine at 0.5 mg/kg.il~ The concen- tration of norepinephrine in carotid arterial blood was estimated before and at intervals after the injection of one of the test com- pouada. Except after the lower dose of tyramine, when the maximal locrease in the concentration of norepinephrine in the blood appeared at 5 min after the injection, the peak concentration of norepine- phrine in carotid arterial blood occurred at 3 min after injection. The release of norepinephrine induced by I at 40 mg/kg ~ s about 1.35 times that produced by the lower dose. The release of norepinephrine induced by tyramine at 0.5 mg/kg was 1.47 times that by I at 40 mg/kg. The release by nicotine at 0.5 mg/kg was 1.59 times that by the larger dose of I. The action of I in inducing release of norepinephrine from tissues in which it has been stored, although it is slightly more pro- longed (20 min vat 15 min) than those of the classical releasers of norepinephrine, is much weaker than those of tyramine and nicotine. —291—

Still, release of catecholamines may play a part in the genesis of hypertension after injection of a pyridinium oxime. Dogs anesthetized with sodium pentobarbital were used in experi- ments in which the lungs were mechanically ventilated after the dogs' chests had been opened to place electromagnetic flowmeters around the ascending aorta and catheters into the left atrial appendage.~9 Dose-response curves for doses of ~ at 5-40 mg/kg in the absence and presence of blockade of 6-adrenergic receptors (phentolamine), of blockade of ~adrenergic receptors (propranolol), or of depletion of catecholamines (reserpine) were constructed. The heart rate was decreased progressively by intravenous I at 5 and 10 mg/kg; larger doses of 20 and 40 mg/kg yielded serially smaller effects on heart rate. Phentolamine (l mg/kg) increased the effects of the two lower doses of I on heart rate, but antagonized those of the two larger doses, converting the usual slight bradycardiac effect of 40 mg/kg into a mild tachycardiac one. Both propranolol (0.5 mg/kg) and reSerpine (0.5 mg/kg during 3 d) tended to increase the bradycardiac ef feet of all doses of I. The increase in peripheral resistance induced by I reached significance only after the largest two doses; this effect was antagonized by phentolamine and was increased by both propranolo} and reserpine. The stroke volume of the heart was increased progressively by the four doses of I used; this effect was antagonized to some extent by all three drugs and especially by the two blockers of adrenergic receptors. Cardiac output was increased progressively by increasing doses of I and also was antagonized to some extent by all three drugs. Propranolol and reserplne had espe- cially marked antagonistic actions. Arterial pressure, which is a resultant of cardiac output and peripheral resistance, was increased progressively by the four doses of I. The hypertensive action was antagonized especially by reserpine and phentolamine, propranolol having only a minor antagonistic action, according to Barnes et _·~Ig _ Taken together with the results obtained by DiPa~ma's group,li7~8 the finding by Barges et a1.~9 of the parti- cularly large effect of prior treatment with reserpine on the -response of the arterial pressure to I seems to confirm the involve- ment of catecholamine release in this hypertensive response. It is clear, however, that I ai80 has a direct inotropic effect on the heart, in that the increase in stroke volume wan blocked only partially by any of the three possible antagonists used by Barnes et al. In view of the fact that none of the possible antagonists was able to prevent more than about 68.5% of the increase in peri- pheral resistance induced by I, this oxime may well have direct - stimulant effects on vascular smooth muscle. - III, unlike I, was fouad49 to lower blood pressure and produce bradycardia in cats anesthetized with sodium pentobarbital. Partial -292-

heart block and inversion of the T wave of the EGG followed the admi nis~crat ion of large doses of this oxime . Moderate doses ~ 5 and 10 mg/kg) produced no noticeable changes in peristaltic or respira- tory activities; ITI at 20 mg/kg induced temporary decreases in both these activities. Doses of 40-80 mg/kg produced respiratory failure and spastic contraction of lntestina' muscles. Neuromuscular trans- mission of either single or repetitive stimuli was not affected by doses of 5 or 10 mg/kg. A dose of 20 mg/kg resulted in a marked, temporary decrease in the tension developed during indirect tetanlc stimulation, but was without effect on the twitch response of the gastrocnemius-so~eus muscle group. Doses to and including 40 mg/kg did root alter responses to bilateral occlusion of the carotid arte- ries and to intravenous injections of acety~choline or eplnephrine at 3,ug/kg; 40 mg/kg partially blocked the cardiovascular response deco stimulation of the peripheral vagi. This onetime was found to be an effective adjunct to atropine in both prophylaxis and treatment of intoxication by sarin and VX in anesthetized cats and dogs. Bayl20 nude a more extensive study of the erects of III on blood pressure, respiration, and ganglionic and neuromuscular trans- mission. Intravenous doses of 25 mg/kg or more decreased systolic and diastolic pressure in anesthetized cats and dogs. In the cat, a 408e of 30 mg/kg slightly increased the presser effect of epinephrine at 5 ~g/kg and slightly decreased the depressor effect of acety~cho- ~ine at 3 ~g/kg. III doses of 2.5-40 mg/kg were found to yield linear log dose-response curves for inhibition of effects induced by preganglionic stimulation of sympathetic and parasympathetic ganglia; the effect on the response to stimulation of preganglionic neurons by sympathetic ganglia was I.4-2.2 times that on the response to similar stimulation by parasympathetic ganglia (the larger factor applies to the lower doses of the oxime). The transient hypotension was attributed largely to the action of III in blocking ganglionic transmission and partially to a direct relaxant action on vascular smooth muscle. Moderate doses of III temporarily increased the breathing rate and transiently decreased the tension developed dur- ing an indirectly stimulated twitch response by the gastrocnemius muscle. Doses of III greater than 50 mg/kg abolished the indirectly stimulated twitch response of the muscle and reduced its ability to maintain a tetanic response to repetitive indirect stimulation. The response of skeletal muscle to direct stimulation was not altered. Neostigmine, edrophosium, and R+ ions antagonized, whereas d-tubocurarine increased, the effects of III on the neuromya1 Junc- tion, which seemed to arise from competition with acety~choline for access to the receptor. In cats anesthetized with sodium pentobarbital, an intravenous dose of III at 25 mg/kg was found to be able to block almost comietely the cardiac response to stimulation of the peripheral right vague nerve.l2' Small doses (2-8 mg/kg) did not inhibit perista~tlc activity; in -2 93-

fact, an increase in the rate of contractions was seen. II and V had effects on response by the heart to stimulation of the vague that were similar quantitatively to that of III, but were weaker. Intestine contraction induced by Bra gel stimulation was blocked by III, but not by twice as large a dose of V. Lindgren and Sundwalii2\ found that the response of the nictitating mem- brane to stimulation of the cervical sympathetic chain was almost unaffected by III; thus, they differed with Bay on the relative sympatholytic and parsaympatholytic activities of Ill. These inves- tigators concluded that III probably exerts its vagolytic action by competition with acety~choline. Welihor~er et a1.~22 reported that intravenous injection of III at 25 mg/kg led to marked hypotension in rats, guinea pigs, rabbits, and cats. The h~rpotensive effect was particularly marked in old cats that had high blood pressures initially; it was not altered by bila- teral vagotomy, evisceration, or removal of the carotid bodies, but was reduced or abolished by decapitation. These investigators sug- gested that the hypotension may result from some effect of the oxime on the CNS. They found also that prior doses of III prevented the hypertensive action of norepinephrine. Kunkel et al. 50 found that both ~ and III at 15 mg/kg blocked the response of the heart to stimulation of the vague nerve. Although I increased the depressor action of acety~choline, III did not alter it . ~ increased the ef fects of injected epinephrine on blood pressure aM contraction of the nictitating membrane, but decreased the response of the nictitating membrane to stimulation of the cervical sympathetic trunk. These findings fairly well loca- ~ized the blocking action of this oxime to the ganglion itself. In contrast, III had no effect on the pressor action of injected epine- phrine, partially blocked stimulation of the nictitating membrane by injected epinephrine, and blocked the response of the nictitating membrane to preganglioDic stimulation of the superior cervical gang- lion. On the basis of these observations, one would think that III affected the receptors in both the ganglion and the nictitating mem- brane. The pressor effect of acety~choline in atropi"ized dogs was increased by I, but not by II]. III decreased the presser effect of bilateral occlusion of the carotid arteries in cats, whereas I did not. It is possible, therefore, that III affects the inferior petro- sal ganglion, which includes the somas of the pressoreceptors in the carotid sinus, or the reticular formation of the pons and medulla, which contains the vasomotor area. Previously, the pontomedullary area has been mentioned as the part of the brain that is the most readily accessible to pyridinium oxi~es.~07~~0 Erdma~ aM Engelhardi23~24 reported that in vitro IV was a more potent Deactivator of acety~cho~inesterase inhibited by DFP or -294—

paraoxon than I or III. IV also reacted directly with these OP com- pounds, particularly parao~on, at high rates. IV was found to enter the CSF more rapidly and to a greater extent than I and to be excreted in the urine comparatively slowly (~% during 6 h). The maximal con- centration of IV in the blood of the rabbit occurred about 20 mln after an intramuscular injection. Removal of the oxime from the blood bet- ween ~ and 4 h after an intravenous injection amounted to about BOX of the oxime present ~ h after the injection. After a lethal dose of IV, there was somnolence, paralysis of the extremities, and terminal para- lysis of respiration. A single intraperitoneal dose of IV at 150 mg/kg resulted in diffuse fatty infiltration of the liver, which disappeared within 1-2 d. No other important pathologic effects were reported. Dilute solutions of IV (36 mg/IOO mid were not irritating to the in, times that concentra- eyes.~4 An isotonic solution containing 1O tion of IV was not hemolytic. A solution containing 3.6: of IV was not irritating to the hairless skin inside the ear of the rabbit. IV at 10-4 M increased the ability of acety~choline to stimulate the rectus abdominis muscle of the frog; a concentration of 3 ~ 10~3 M paralyzed neuromuscular transmission in the isolated rat phrenic nerve-diaphragm preparation. The isolated jeJunum of the rabbit was stimulated fleetingly by IV, but this oxime relaxed segments of jejunum that had been caused to contract by acety~choline or carbamoy~choline. Intravenous injection of IV into a cat at 15 mg/kg decreased the response of the heart to stimulation of the vague nerve and completely blocked contractions of the nictitating membrane in response to stimu- lation of the cervical sympathetic chain. An intravenous dose of 50 mg/kg decreased blood pressure am1 respiratory activity. When mechard- cal ventilation of the lungs was provided, cats survived doses of 200 mg/kg . Repetitive administrat ion on 3 successive days of 50-70% of the LDso resulted in muscular weakness, Swayback, and exophthalmos. Daily injection during 30 ~ of 30% of the LDso had no evident deleterious effect on the health of the animals. IV has been fo''ndi25 to antagonize stimulation of the isolated ileum by carbamoy~choline and ethyl arecaidinate (a homologue of arecoline ~ and to cause parallel shif ts of the dose-response curves of each of these agonists. This oxime was considered to be a functional . . . . . ~ . ~ . antagonist of cho n nergic agonists, ratner cnan a compete one, because various mixtures of IV with the competitive anticho~inergic drug, N-methylatropinium nitrate, shifted the dose-response curves of the agonists by amounts that were not proportional to the sum of the individual displacements by the two antagonists. The parasympatholy- tic action of IV is suggested, therefore, to arise from contact bet- ween IV and a specific receptor for that molecule different from the molecule(~) with which the agonists and the N~methylatropinium salt interact, but functionally interdependent with it. This interdepen- dence may involve an allosteric mechanism, but that has not been demonstrated. -~.95-

METABOLISM OF OXIMES 2-PAM I in dilute hydrochloric acid (0.1-0. 4 N) is hydrolyzed in a firat~order reaction.l26 The rate of acid hydrolysis at a constant H+ ion concentration had a Clo of about 2 when the temperature was increased from 37.~°C to 47.6°C. The hydrolysis of 2-PAM I was not catalyzed by hydroxyl ions, but was rapid at high pH. The syD form of the opine was converted to the anti form more rapidly at a pH of 13 than at one of 7 or l. Within the range of pH from ~ to 13, 2-P~ I was found to have its maximal stability with respect to hydrolyele in aqueous solvents at a pH of 1, whereas III was most stable at a pH Of 3.327 Even at the indicated pHs, 30: of 2-PAM I and 20% of III were hydrolyzed before equilibria was reached. After the oximee apparently reached an equilibria with their hydrolytic products, degradation of the oximes continued at a low rate. Ellini28 suggested the following sequence of reactions during degradation of 2-PAM I in aqueous solution: the oxime is dehydrated to N-methy~pyridinium~2~itrile (A), which is converted to N~ethy~pyridinium-2-hydroxylate (B) and then to N~ethyl- pyridinium-2-one, hydrogen cyanide being released during the conversion of A to B. Creasey and Greeni29 reported that II is soluble in water to the extent of ~ g in 2 mI; like I, it is more stable in aqueous Solution at low pH, the pH of maximal stability being 4-5. When II in a solu- tion at a pH of 4.5 had been freeze-dried and stored in a sealed glass container for 6 ma, no cyanide wee detectable in the container when it was opened. Similar treatment of a solution at a pH of 6.0 resulted in detection of cyanide by odor when the container was opened. Heat sterilization of an aqueous solution of II was found to result in the formation of small amounts of cyanide and in a change in the color of the solution from pale yellow to orangebrown. A homogenate of liver from the rat was found to decompose II slowly; other biologic sam- ples (whole blood-, kidney, skeletal muscle, urine, and feces) did not. Kondritzer et al.~30 in searching for a salt of 2-PAM that would be more soluble than 2-PAM I in water, made a number of other malts. Most of these were considerably more soluble in water than 2-PAM I. One of the most soluble was the lactate, with a volubility of 1 g/mO. Unfortunately, this salt was found to be quite unstable in aqueous solution with respect to heat. ~ was less soluble-in water than the lactate, but more than 13 times as soluble as the iodide. Furthermore, solutions of I at a pH of 3.5-4.3 could be autoclaved at 120°C for 15 min with only a 4% loss. Solutions of this salt stored at 50-70°C for 1-3 ma contained at least 7BX of the original oxime and no more than 0.1% of hydrogen cyanide. When the aged solu- tions were examined for lethality to experimental animals, their lethal activities were exactly those expected on the basis of their oxime concentrations. —296—

Barkmanl31 compared the stabilities of I and II at various pHs and storage temperatures. Storage of a solution of I at a pH of 3 and 50°C for a year resulted in some increase in pH and in loss of 37-60% of the oxime in the original solution. Similar storage of a solution of II at the same pH and at 45°C for 2 ye resulted in 108s of 80% of the oxime. Storage of solutions of these two salts at the same origi- nal pH, but at a temperature of only 25°C, resulted in loss of only 5% of I during 1 yr and of 4.8Z of II during 2 ye. The pH increased by ~ 16 during storage of the solution of II at 45°C for 2 yr, but by 0 19 during storage of that of I at 50°C for ~ yr. The principal products of degradation of these oximes were found to be N-methylpyri- dinium-2-Aminocarbonyl aIld N~methy~pyridinium-2~itrile. These end products have low toxicities, so the deteriorated solutions of I and II were less toxic than freshly made ones (see also Barkman, et al.l32) If the temperature of storage of solutions of either of these 2-PAM salts can be kept below 25°C, the solutions should have shelf-lives of several years. When stored at 10-20 °C, solutions of III had their greatest stabilities at an initial pH of 6.~33 When the temperature of storage was 30°C or more, the initial pH that provided the greatest stability of the oxime in an aqueous solution was 5. With that ini- tial pH, the estimated half-life of III in solutions stored at 40°C was 9.6 ye. The half-life of I in solutions at the pH of greatest stability, initially 4, for storage at 40°C was estimated to be 9 yr. In a solution containing also atropine and benactyzine at a pH of 2.S, I.4% of the protective potency due to Ill was lost after 1 yr at 250C.~34 There seems to be little basis for choice among I, II, and III on the grounds of relative stability in solutions, so long as the storage temperature can be held below 40°C and the initial pH of the solution is adjusted to the value that yields the greatest stabi- lity for the particular osime. For the salts of 2-PAM in solutions with pHe above those of maximal stability, the sequence of hydrolytic products may be N-methy~pyridindum-2-aminocarbonyI, N-methy~pyridinium- 2-nitrile, N-methy~pyridinium-2-hydroxide, and N-methy~pyridinium-2-one, hydrogen cyanide being a side product. At pHs at or below those of maximal stability, the end products of hydrolysis of N-methylpyridinium- 2-formyl oxime are N-methy~pgridinium-2-aldehyde and hydroxylamine. Information on the atorage stabilities of IV and V has not been found, other than a note that, when stored at 37°C at an unspecified pH, both 2-PAM I and V had half-lives greater than 7 mot After oral administration of I to dogs at nearly Too mg/kg in an aqueous solution, the -concentration of oxime in the plasma ~ h later was as great as was measured at any later time.~35 By 5 h after the dose, the plasma concentration had decreased to 51.3: of that at ~ h; by 13 h, it was only I8X of that at ~ h. When approximately the same -297~

dose Was given in tablets, the peak plasma concentration did not occur until 2 h later; it was more than 2.3 times the plasma concentration found ~ h after administration of the solution. This last finding suggests that the oxime is absorbed rapidly from a solution introduced into the gastrointestinal tract, so that the peak plasma concentration of office after oral administration of the solution may have passed before the first blood sample was taken ~ h after administration of the solution. By 5 h after administration of the tablets, the plasma concentration was slightly more than 39% of the peak concentration, but still nearly Price that measured at the same time after administration of I in solution. By 13 h after the tablets had been given, the plasma concentration was only 7.6X of the peak concentration and was the same as that measured at the same time after the solution had been administered. Inclusion of atropine in the preparation of the oxime was found not to influence absorption of the oxime. Administration of a nearly lethal dose of sarin to rabbits before the intramuscular injection of II or III nearly doubled the mean time (9 min vat 5 min) for attainment of the peak plasma concentration. i36 The concentra- tion of III in the blood was nearly twice that of II when equal doses of the two oxides were administered. However, Erdmann and Okoneki37 found that introduction of parathion into the lumen of a dog's intes- tine increased the absorption of Ilt from the Amen. In the rat, ore' administration of disod:ium EDTA with IV nearly doubled the rate of absorption of the osime. The peak plasma concentration of oxime at ~ h after administration of that mixture ranged from 6.9 to 17.4 ~g/m1. Sundwalli38 regarded a concentration of oximate ion in the blood of 4 ug/ml as minimal to overcome the bradyeardia and apnea induced by lethal doses of isopropoxymethy~phosphory~thiocho1ine in an anesthe- tized cat. Kunkel et al.l39 found that three of six rabbits Riven intramuscular injections of atropine sulfate at 6 mg/kg 55 min before intravenous injection of nomad at l.5 times the LD,o lived for 7 ~ thereafter. When the atropine was accompanied by I at 15 mg/kg, yield- ing a plasma concentration of office of about 4.8 ~g/mI, two of six rab- bits lived for 7 d. When the dose of atropine sulfate was increased to 10 mg/kg and the time between prophylaxis and poi8ODiDg was shortened to 5 min. only one of six rabbits challenged with soman at 2 times the LDso lived for 7 de When I at 15 mg/kg accompanied the same dose of atropine, the plasma concentration of the osimate ion Just before Roman was administered was about 25.4 Gym, and three of six rabbits given intravenous injections of soman at 2 times the LDso lived. Larger doses of I, up to 45 mgtkg, yielded a plasma concentration of oxime of about 50 ~g/ml and increased survival after soman at 2 times the IDso to four of sis. Accepting that lethal intoxication with Roman is unusually difficult to prevent or treat, one nonethelese sees in these results an indication that the plasma concentration of office is important in determining the severity of intoxication by OF compounds that can be withstood. —298-

Jager et al.59~1l5 reported that infusion of 2-PAM I into dogs at 0.5 mg/kg per minute resulted after about 2 1/2 h in a relatively stable serum concentra~cion of oxime of about 40 t~g/mI. Ligation of the renal pedicles resulted in a rapid increase, during the 30 min thereafter, in the serum concentration of oxime to nearly 120 ~g/mi. In one dog in which the infusion was continued for another 90 min. the serum concentration rose to nearly 140 ~g/mI. In nephrectomized rats given intravenous 2-PAM I at 50 mg/kg during 2 min. the serum concentration of oxide decreased during an hour at about two-thirds the rate at which it decreased in control rata. Furthermore, the extrapolated serum concentration of oxime in the nephrectomized rats at zero time was one-third greater than that in the control rata. Evidently, therefore, the kidneys are important organs for the remove' of 2-PAM salts from the circulating blood. These investigators found also59~5 that homogenates of rat liver were able to degrade both 2-PAM I and ~ if oxygen was present during the incubation; anaerobic incubation left the concentrations of the oxides in mixtures with the homogenate the same as they had been before incubation. Dultz et al.23 found that dog kidneys took up from the blood more than 5.4 times as much 2-PAM I as ~ and that V disappeared from the serum of nephrectomized rats at essen- tia1ly the same rate as from that of normal rata. Excretion into urine seems to be a much more important route for removal of 2-PAM salts than of V from the body. On comparing various organs of the dog other than the kidneys for ability to take up btoodborae 2-PAM I and V, Dultz et a' .23 found that V was abstracted from the blood by the brain slightly more than I' times as readily as 2-PAM I, whereas liver, spleen, skeletal muscle, add cardiac muscle removed the two oximes in ratios between 1.0 and l.7. Fat took up 4.5 times as much 2-PAM I from the blood as V. Because of its comparatively great ability to enter the brain, V has been considered by some people to be an especially good antagonist of inhibition of brain choLinesterase by OP compounds. ~~ser4} found that excretion of the label in intravenously injected 2-PAM I having ~4C in the methyl group attached to the N atom in the pyridine ring was largely in the urine of mice; the feces accounted for less tin 5% of the total excretion. The label was excreted rapidly (38.7: in 145 min) in the urine of one cat. lrhe urine of mice was believed to contain at least six chromatographical~y separable metabolites of 2-PAM I, in addition to the unchanged oxide, whereas the urine of the one cat seemed deco contain only unchanged oximee Only O.~8X of the label was found in the expired air of mice during 6 h after intravenous injection of labeled onetime. The label disappeared from the blood of the cat rapidly: by 2.5 h after injection of the oxime, only about 17.5: of the peak concentration of the osime —299—

in the plasma remained there. Correspondingly, about 38. 8: of the label had been excreted in the urine during the same time. In the cat, comparatively large concentrations of the label were found in the gall bladder and bile, the urinary bladder, the small intestine, the feces, and the liver. Crook et al.57 reported that the plasma concentration of III after its injection into a dog at 10 mg/kg followed essentially the same course as that of 2-PAM I injected intravenously at 30 mg/kg. Inasmuch as the plasma concentrations at approximately the same times after injection of the two oximes were almost identical, despite the difference between the doses, one must assume that III has a much smaller volume of distribution than 2-PAM I and that either the kine- tics of removal of 2-PAM I from its larger volume of distribution were correspondingly greater than those of III or the 2-PAM I in extravas- cular components of its volume of distribution was held there tena- ciously. The fairly rapid changes in the 2-PAM I concentrations found by Kalser4t in various organs and tissues of the mouse argue against the validity of the latter possibility. Enander, Sundwall, and Sorboi40~~42 found that either oral or intramuscular administration of I] to rats at 500 or 100 mg/kg, respectively, increased by many times the urinary excretion of thiocyanate. From the amount of thiocy~anate excreted above the base line, the quantity of hydrogen cyanide produced in metabolism of the oxime was calculated to be 0.l mg/kg--a little more than one-third the LD,o for rats by intraperitoneal injection. Urine from rats given 120 Amok of II intramuscularly or 400 Amok by mouth contained 3.9-7.8X N-methylpyridiDium-2-nitrile methanesulfonate. When this compound was injected intramuscularly into rats at 90 mg/kg, thiocyanate was excreted in the urine in increased amounts. Also present in the urine was a metabolic product that yielded cyanide on acidification of the urine, similar to a cyanide-yielding metabolize of II found earlier. Studies with N-~4C]methy~pyridinium-2-aldosime iodide administered to rats by intramuscular injection at 40 or 100 mg/kg or by mouth at 100 mg/kg revealed that about 87% of the label was excreted in the urine during the 24 h after injection, but that only 52% was excreted during the same time after oral administration. The label excreted in the urine during this period was largely (8O-9OX) in the form of unaltered oxime. Other metabolic products identified in the urine were N-methylpyridinium-2-nitrile and N-methylpridinium-2- carbosylic acid. Two other peaks in the radio-chromatograms of the urine were thought to represent N-methyl-a-picolinium amine and the cyanide-yielding metabolize mentioned earlier, but the identities of the compounds were not established, because there was too little of the substances represented by these radiochromatographic leaks. No N-methylpyridinium-2-one was found. Enander et al.l4 presented a acheme for the metabolic transformations undergone by 2-PAM whereby the — 300 —

oxime is converted to either the rtitrile, the aminocarbony' derivative, or the a1dehyde. The aminocarbonyl compound and the aldehyde were considered to be converted to the carboxylic acid as the end product. End products thought to be derived from the nitrite are the pyridone and the unidentified cyanide-yielding metabolitee Way criticized this work as being superficial and inept and developed a chromatographic method for removing from a reaction mixture or solution all compounds containing a pyridi~um group. The pyridini~-containing comports could then be resolved at pHs permitting the molecules to exist without degradation. Using this method, Way et al.~43~~46 identified N-methy~pyridini ~-2-Ditri~e, _ _ _ N-methy~pyridinium-2-one, and N-methylpyridinium-2-aminocarbonyI-4- one as metabolic products of 2-PAM in the perfusate from isolated rat livers. They also found, but did not identify, an N-methylpyridini~'m- 2-0-conjugate, which was not hydrolyzed by either ~glucuroni- case or phenol sulfatase. When exposed to 0.3 N NaOH, the conjugate released N-methy~pyridiniumr2-one. N-methy~pyridintum-2-ethoxy iodide - Wa8 synthesized and found to yield the pyridone on exposure to 0.3 N NaOH. However, the metabolic product and the synthetic compound were found to have different nobilities on paper chromatograms with various solvent systems. The synthetic compound may be a model of the natural conjugate, but is not identical with it. Way presented a scheme for the metabolism of 2-PAM wherein the oxime is converted to either a 2-nitrile-4-one or a 2-acetiminomethyl or a 2-phosphoryliminomethyl compound. The acetimino or phosphorylimino compound is converted to the 2-nitrile, which is changed into either the 2-cyanohydrin or the 2-nitrile-4-one derivative. The 2-aitrile-4- one is metabolized to ache 2-aminocarbony1-4-one. The 2-cyanohydrin is proposed as the precursor of the 2-one derivative and also of the 2-0-conjugate through a 2-cyano-0-condugate. Both reactions of the cyanohydrin would release ON- ion. Way et al.~45 found N-me thy' ridinium-2-Ditrile in human urine _ Pa as a metabolite of 2-PAM. Kramer reported that five men given 2-PAM I at 2 g/day by mouth on 2 consecutive days while they were on a closely supervised regimen of dietary and other intakes excreted in their urine not only unaltered 2-PAM, but also a material that had a bright blue fluorescence when exposed to W light (366 nary). This Valerie was shown not to result from simple exposure to urine by incu- bation of 2-PAM I in normal hen urine under several different condi- tioDs for up to 7 d. It was thought possibly to be a derivative of N-methy~piqolinic acid. Bergland et al.~48 studied the handling of II by the dogma kid- neys and found that the amount of the oxime excreted always was greater than the amount filtered through the glomeruli. At a plasma concentra- tion of TI in one dog of about 6.2 ng/ml, the difference between the —301—

i amount excreted in the urine and the amount filtered was about 1.2 mg/min in a total excretion of about 1.75 mg/min. This plasma concen- tration of the oxime gave the Tm for tubular secretion in that animal (the Tm was reached in other dogs at plasma concentrations of II of 6-8 ~/ml). Loading dogs with ammonium chloride (6 Aged by mouth for 3 d) before the experiment increased excretion of the oxime without altering appreciably its filtration. Conversely, induction of alkalosis by intravenous infusion of 0.6 M sodium bicarbonate decreased excretion of the oxime without changing significantly its filtration. Alkalinization of the urine by administration of ace~cazolamide had no effect on the urinary excretion of the osime. Probenecid (priming with 25 mg~kg followed by infusion at 40 mg/kg per hour) lowered the urinary- excretion of the oxime from 1.66 mg/min to 1.61 mg/min and tubular secretion from 0.96 mg/min to 0.~8 mg/min. These differences are not statistically significant. Apart from a conclusion that the tubular epithelium has a more effective trans- fer system for the oximate form of II than for the N-oxide form, the meaning of these findings is obscure. The only other oxime on which any metabolic information was found is III. Way_ al.~49 perfused III labeled with i4C in the ~ and 3 positions of the 3-carbon chain between the pyridinium moieties through isolated rat livers. The only metabolite that they reported finding in the perfusate was l-~4-aldoximinopyrldinium)-3-~4-cyano-pyridinium) propane ion, which was identified by comparison with the authentic synthetic compound. Later, DeMiranda et al. i50 found the same com- pound as the principal metabolize in the urine of rats that had been given doses of IlI. III seems to be treated metabolically as though it were one molecule of 2-PAM attached to a large inert group. In an attempt to find a way of extending the protective action of oxides, Stera and Boskovic]5t explored the possibility that an inhibitor of drug processing enzymes, diethylaminoethyl dipheny~propyl- acetate (SKF 525A), can increase the ability of oximes to antagonize the lethal actions of tabun. In the Scheme used, SKE 525A and atropine were injected into experimental animals 40 min before ta bun was injected; oximes were injected 10 min before tabun. SKF 525A approximately doubled the protective activity of Clotures of atropine with 2-PAM I III, and a mixture of 2-PAM I and V. Later, Milose~lc and Tersici5} compared other inhibitors of microsomal enzymes with SKF 525A for potency in increasing the ability of III to prevent the lethality of parao~on. The principal difference between III alone and with SKF 525A was that SKF 525A significantly prolonged the time bet- ween injection of III and of parao~on at which the oxime would remain protective. For example, injection of ITI at 25 mg/kg 30 min before paraozon yielded 30X survivors in the group. When the same dose of III was injected with SKF 525A at 50 mg/kg 60 min before the same dose of parao~con, al' the animals lived. These investigators forms also that —3 02—

one of a group of related inhibitors of microsomal enzymes, diethyI- a~inoethyl phenyldiallylacetate (CFT 1201), was at least as effective as SKF 525A in a much Smaller dose. The same people carried the study of CFT 1201 further.~53 When rats were given CFT 1201 ~ h before intravenous injection of III, the blood concentration of III was only 21.6Z greater than when III was administered alone. When mice were given osimes subcutane- ously and CFT 1201 intraperitoneally simultaneousI, 15 min before a challenge with parao~on at 3 times the LDso, the protective dose of 2-PAM I was 24.3X of that when 2-PAM I was given alone, and that of III was 20% of that when III was injected alone. CFT 1201 and SKF 525A alone had almost no protective actions against tabun or parao~on. STUDIES WITH EIU~N SUBJECTS _ Jager _ a1.59~115 injected 2-PAM I intravenously into five human subjects during 2-4 min at 15 mg/kg and I into five other subjects at the same dose. One patient with a plasma concentration of urea nitro- gen of 165 mg/lOO ml was given the same dose of 2-PAM I. During 4 h after these doses, essentially all the 2-PAM I left the serum of the five normal subjects, whereas the serum concentration of this oxime in the azotemic patient decreased by only about B.6%. The mean concentra- tion of V in the serum of normal subjects decreased by about 13.5% dur- ing 4 h. Correspondingly, three subjects given 2-PAM I excreted in their urine a mean of 80.9% of their doses of the oxime during the 6 h that followed the injections, whereas two subjects given V excreted a mean of only about 6.8X of their doses of that oxime during the 6 h after administration of the oxime. Urinary excretion of 2-PAM I by the azotemic subject seems not to have been measured. During a continuous intravenous infusion of V into a subject 0.27 mg/kg per minute~sampies of spinal fluid taken at 60 and 150 after the start of the infusion contained a mean of 68.3% + 2.4X serum concentration of the oxime at the same time.l15 A subject given an infusion of 2-PAM I for 60 min at 0.73 mg/kg per minute had a serum concentration of the oxime of 2.8 mg/100 ml at the end of the infusion, but had no detectable concentration of that oxime in the spinal fluid. These findings corroborate the idea that V may have readier access than the pyridinium oximes to the brain. at min of the In 1964, Calesnickl54 and Calesnick et al.155 reported that, when I was injected intravenously into normal male and female volun- teer~ at 15 mg/kg (three subjects), both systolic and diastolic blood pressures were increased immediately after the end of the infusion (usual duration, 15 min.). The hypertension lasted for 1.5-4 h and was -accompanied by a alight increase in heart rate. The plasma concentra- -303-

Lion of oxime at the end of the infusion ranged from 12.3 to 17.3 ~g/mI. Two subjects given similar infusions of I at 30 mg/kg had no Immediate increases in blood pressure, but the systolic aM diastolic pressures rose siowly--e.g., to about 32 and ~4 mm He above the origi- nal pressures, respectively, at about 35 min after the end of the infu- sion in one subject; subnormal blood pressures developed about 3 h later. The subjects given the smaller dose of I also developed subnor- mal pressure after the hypertensive phase of the response. The mean plasma concentration of I at the end of the infusions at 30 mg/kg was 17.6 + 0.3 ug/mI. Infusions of I at 45 mg/kg into two subjects resulted in marked increases in systolic and diastolic blood pressures immediately after completion of the infusions. The hypertensive response lasted for about 1.5 h and was followed by the development of subnormal blood pressure about 2.5 h later. Increases in the voltage of the T waves and in the length of the PR interval of the EGG were reported after this 608e. Administration of a second dose of I after the blood pressures had returned to the control values yielded a second hypertensive response. Ephedrine intensified the hypertensive response to I, whereas phento' amine and reserpine reduced it. Injection of I intramuscularly at 15 mg/kg into two subjects gave a plasma concentration of oxime of 4 ~g/mi, but had no effect on blood pressure.l54 Two other subjects given intramuscular doses of 30 mg/kg developed hypertension about 1.5-2 h after the end of the infusions. A subject who inhaled aerosols of lob and 50% aqueous solutions of I did not develop hypertension or detectable oximemia. Oral administration of 1 or 2 g every 6 h for 5 ~ to eight subjects resulted 1n no significant changes in blood pressure or heart rate. Infusion of II at 45 mg/kg into one subject resulted in a h~per- tensive response immediately after the infusion was completed.~5 Oral doses of 2 and 4 g of II had no detectable effect on blood pressure in another subject. When 4 g of III were given by mouth to one subject, there were Blight decreases in systolic and diastolic blood pressure 6 h later. Infusions of III at 15 or 30 mg/kg into one subject produced immediate, brief hypertensive episodes at the end of the infusions, followed by a period of subnormal pressures lasting from about 22 min to more than 363 min after the end of the infusion for the systolic pressure; the diastolic pressure did not fall below normal until about Il7 min after the end of the infusion, but wee still below normal at 363 min after completion of the infusion. More detailed data from these experiments and from others in which I and 2-PAM maleate were administered orally and by intraduo- denal tubes, deco compare rates of absorption of the two salts by the two methods of administration, were given by DiPalma and Caleenick.l56 As a result of these studies, Caleanick et al. ]57 concluded that I - 304 -

was more satisfactory than II or III, in that the last two oximes caused marked gastrointestinal disturbances that limited both the amounts of these oxides that could be administered and the durations of their administration that could be tolerated. This limitation was especially marked with III, which also caused nervousness, malaise, dizziness, paresthesia of the face and arms, rash, and icterus in some subjects. Details of studies in which propranolol and phentolamine were found to antagonize the hypertensive effect of infusions of I at 2S-45 mg/k~ were given in the same report by DiPa~ma add Caleanick. 57 Doses of 5 me of phentolamine, probably as the methane sulfonate although they did not identify the salt used, and of 4 or 5 me of propranolol were used in four subjects (one woman and three men) aged 23-32 yr. In three subjects given I by intra- venous infusion at 25 and 30 mg/kg during 15 kin, phentolamine was the more potent antagonist of the hypertensive effect of I; in one subject given ~ at 45 mg/kg, propranolol was a better antagonist than phentolamine. It is of some interest that the adrenergic blocking agent, phentolamine, usually had a greater antagonistic action against the hypertensive effect of I than the nonselective ~adrenergic blocking agent, propranolo1. This suggests that the principal action of I is directly on the vascular smooth musc~es--a posesibility that is supported by the finding that the hypertensive action of I usually does not involve a marked change in heart rate. Kondritzer et al.iS~ administered I. 2-PAM lactate. 2-PAM dihY- drogen phosphate, II, 2-PAM I, and III to human subjects as aqueous solutions. The subjects were primed with 400 ml of water during the hour before they drank the solution of oxime and drank 100 m] of wincer during each of 5 consecutive hours after taking the oxime. The peak plasma concentrations of oxime occurred at about 2 h after drinking of a solution of 2-PAN I that contained a mean dose of 71.4 mg/kg, at about 2.5 h when the dose was increased to Il4.3 mg/kg, and at about 3 h when the dose was increased further to 142.9 mg/kg. The lactate gave a greater peak concentration of oxime in the plasma than the same dose of the dthydrogen phosphate, but required a longer time to induce it (3 h vat 2 h); the peak concentrations differed by I8.9X of that established by the dihydrogen phosphate, although the molecular weight of that salt was greater than that of the lactate by only 3.4% of its own molecular weight. When I and II were administered in equimolar doses, the peak plasma concentrations of oxime were reached ater 2 h. With doses of 0.31 mmol/kg, IT gave a peak concentration greater than that given by I by about 4.6 M. When the dose was 0.12 m~ol/kg, the peak concentration established by I was about I.5 UM above that established by II. These two salts are probably absorbed and eliminated from the plasma at closely similar rates. -305-

The biologic half-lives of the five salts of 2-PAM calculated from both the data on plasma concentrations at various times after ingestion and those on urinary excretion are given in Table 4. The table demonatrates that the iodide was cleared from the plasma more slowly than the other salts and that the dihydrogen phosphate was cleared from the plasma a little more rapidly than the others. The only undesirable effects observed during these studies were signs and complaints of iodinism by the subjects given 2-PAM I and decreases in red-cell and plasma cholinesterase activities of about 20% in the subjects given III. Less of III was excreted in the urine (3% during 24 h) than of the monoquaternary compounds (27% in 14 h). Sidell et al.76 gave ore] doses of 3-9 g of I to a total of 28 men. Although these doses were not adjusted to the body weights of the subjects, there was a general tendency for the mean peak plasma concentration of oxime to increase as the dose increased. The mean peak concentration varied from 4.20 ug/ml after 3 g of I was ingested by four subjects to 9.15 ~g/m1 after 9 g was taken by two subjects. The time for attainment of the perk concentration varied from 30 min to 3 h, without any discernible relation with dose or any other vari- able in the experiment. The mean half-life of the oxime, calculated from concentrations measured in the plasma at various times, was 2.66 h in 25 subjects; the mean half-life calculated from the amounts of oxime excreted in urine at various times was 2.44 h in 21 subjects. In five experiments in which the mean peak plasma concentration of oxime of 6.] ug/ml was attained 2 h after ingestion, the mean 6 h after ingestion was 2.0 ~g/mI--~1ightly lese than 32.~% of the mean peak concentration. When these investigatora76 administered oral doses of I every 6 h for 48 h, the plasma concentration of oxime proceeded as a series of peaks at 3, 9, 15, 2l, 27, 33, 39, 45, and 51 h. There was a tendency for each peak to be silghtly greater than the pre- ceding one. When the interval between doses was shortened to 4 h, the peak concentrations were about i.3 times those when the same dose was given every 6 h. -306-

TABLE 4 Biologic Half-Li~res of 2-PAM Salts Given Orally to Man Half-Life, h Concentration Based on Urinary 2-PAM Salt in Plasma Excretion Iodide 2. O (9) 2.2 (5) Chloride (I) 1.7 (7) 1.7 (7) Metha su one e ( ) ~ 7 (10) ~ 6 (10) Lactate 1 7 ( 9) 7 ( 9) Phosphate 1.3 (5) 1.5 (5) 1 a Figures in parentheses are numbers of subjects —307- .

Sldell _ a1.159 found that, when doses of 600 me of I and 2 mg of atropine sulfate were injected intramuscularly simultaneously but separately into the two arms of a man, his heart rate rose more sharply and to a higher value than when the two compounds were injected in one solution. The mised solution seemed to produce less pain than intramuscular injection of I alone. On the basis of this study, there is no evident disadvantage in filling atropine and I in a single solution for lutramuscular injection. There is a question, however, whether the doses of atropine and I should be linked in a fixed ratio, as they would be if these two compounds were provided only as a mixed solution. Swartz and Sidelli60 administered I to subjec~ce under various conditions of ambient temperature and exercise (walking on a tread- mill). Resting at 21°C was the baseline condition. The renal clearances of creatinine, E:-aminohippurate, and I were measured dur- ing rest at 40.6°C, during walking at 3 mph during 20 of each 30 min in a period of 3 h at 21°C, and during walking at 3 mph during 20 of each 30 min in a period of 3 h at 40.6°C. Whereas the renal clearance of creatinine was decreased progressively by exercise, by exposure to an increased ambient temperature, and by exercise at the higher ambient temperature, the clearances of both p-aminohippurate and ~ were increased during rest at the higher ambient temperature. Walking on the treadmill in the heated room, however, decreased the clearances of ~-aminohippurate and I even more than walking on the treadmill at the lower temperature. That the effects on these two renal clearances were similar is not astonishing when one considers that tubular secretion is the most important part of the mechanism of clearance of both substances from the plasma. It is likely that exposure to the higher temperature at rest resulted in increased blood flow to the kidneys by vasodilatation, that exercise at the lower ambient temperature decreased blood flow to the kidneys by shunting of blood to the active muscles and possibly to the skin to permit dissipation of the extra heat produced during the exercise, that the greater heat load imposed by the combination of higher ambient temperature aM exercise resulted in an eared larger shunting of blood away from tile kidneys to the skin, and that the resultant of vasodilatatioD in the active skeletal muscles and in the skin was a sharply decreased blood flow to the kidneys and, consequently, lower tubular secretion of p-~minohippurate and I. After being found to be healthy on a thorough physical examina- tion accompanied by a broad range of laboratory examinations, 22 men were used in studies of the renal clearance of I, after intravenous injection at 5 mg/kg under a variety of conditions.~6' Alkaliniza- tion of the urine to a pH above 7.5 by administration of bicarbonate and acidification of the urine to a pH below 5.0 by administration of ammonium chloride both reduced urinary excretion of I. When 200 mg of thiamine was injected intramuscularly 20-30 min before intra- venous injection of I, urinary excretion of I during the 5 h after -308-

its injection was decreased to the greatest extent (by almost 24%~. Thiamine resulted in an increase in the total volume of distribution of I of about 47.4%, the expansion of the central volume being about 13.3% greater than that of the peripheral volume. The mechanism of the changes in the volume of distribution of I is not apparent. During the first 3 h after intravenous injection of I that fol- lowed administration of thiamine, urinary excretion of the oxime was about 12.7% below that during the corresponding period of the control experiment; during the remainder of the run, it was 62.2% above that during the same period of the control experiment. Inasmuch as intra- venous lndection of 900 me of sodium p-aminohippurate with I decreased by only 6.3% the urinary excretion of I during the first 3 h after its administration, the tubular transport mechanisms for I and for ~-amino- hippurate probably are dif ferent. Josselson and Sidel1~62~364 extended study of the effect of thiamine on the pharmacody~amics of I in the human body. They found initiallyi62 that an infusion of thiamine at 100 mg/h during 2.5 h led to a greater plasma concentration of oxime after intravenous injection of I at 5 mg/kg than when the injection of I was not accom- panied by an infusion of thiamine. The renal clearance of I and the urinary excretion of I were greater during the first 1.5 h of infu- sion of thiamine than during the corresponding period after injection of I without infusion of thiamine. The investigators concluded that thiamine decreased the peripheral volume of distribution of I in this study. However, their published value for the standard deviation of the mean for this variable during the infusion of thiamine was 0.01 times the red value; that may have contributed to this unjustified conclusion. The sole barely significant change in the volume of dis- tribution of I was an increase in the central volume of distribu- tion. The most significant changes in the men given thiamine infu- sions were a decrease in the removal of I from the central compart- ment of the volume of distribution, a decrease in the movement of I from the central to the peripheral compartment, and a decrease in the value of the exponential constant for the rapid phase of the decrease in the plasma concentration of I. Infusions of thiamine at two different rates during intravenous injections of a constant dose of I produced sequentially Slower remo- val of I from plasma.~63 The half-time for the rapid phase of removal of I from plasma was 4.2 min in the control runs; it was increased to B.4 min during the infusion of thiamine at both rates. The ha~f-time for the Slow phase of removal of I from plasma was 59.8 min in the control runs; it was increased to 87.6 min and 91.2 min. respectively, by the lower and higher rates of infusion of thiamine. The rate constant for removal of I from the central compartment was 2.94 in the control nms and was decreased to 1.32 by infusion of thiamine at the lower rate; the higher rate of infusion of thiamine -309-

(twice the lower one) induced no significant further reduction of this rate constant. The rate constant for movement of I from the peripheral compartment to the central one was 4.95 in the control rune; it was decreaed to 2.49 by the lower rate of infusion of thiamine, but rose to 3.21 with the higher rate of infusion. The last value had a very large standard deviation and consequently was not significantly different from either ache rate during the control runs or that during infusion of thiamine at the lower rate. The higher rate of infusion of thiamine, but not the lower one, decreased significantly the peripheral and total Holmes of distribution of I. The higher rate of infusion of thiamine markedly prolonged side effects of the osime, including lethargy, but was tolerated well by the subjects. Infusions of ~ g of I, with and without 200 mg of thiamine hydrochloride, into five men during 30 min produced hypertension in two subjects during the infusions that included thiamine.~64 In the trials without thiamine, the only effects detected by the investigators or reported by the subjects were transient blurring of vision, diplopia, and a sensation of expansion of the eyeballs, never tatting for more than about 3-5 min. in four of the fire subjects. During the infusions that included thiamine, in addition to the hypertension in two subjects, one other subject had an increased heart rate, and all complained of the same visual effects that were reported during the control infusion. These were more marked and lasted for up to 2 h, instead of for a few minutes as during the control runs. Four of the men also complained of fatigue and drowainess; two complained of "vitamin gustatory sensation." The two men who had hypertension after the infusion of I with thiamine had mean increases in systolic blood pressure of 34 mm Hg and in diastolic pressure of 24 mm Hg. lathe mean maximal increase in heart rate of these two men and of the one other man who had only an increase in heart rate was 25 beats/mint In all five men, the mean plasma concentration of oxime was always greater in the experiments in which the infusion included thiamine than in those In which ache infusion contained I alone, by about l-~1 ug/mI. During the first I.5 h after the beginning of the infusions, those with I alone excreted in their urine a mean of 73% of the total 24-h excretion of the oxime, whereas when the infusions included thiamine only 34% of the 24-h excretion of the oxime took place during the fires l.5 h after the beginning of the infusions. During ache first 3 h after the beginning of the infusions, 89% of the total 24-h urinary excre- tion of I given- alone occurred, compared with only 55% of that of I accompanied by thiamine. During the remainder of the 24 h after the start of the infusions, I1X of the causative urinary excretion of I not accompanied by thiamine appeared in the urine, compared with 45X of that of I mixed with thiamine. The total urinary excretion of I —310—

during the 24-h collection period was 83.6% of that infused alone and 72.3X of that given with thiamine. It is unfortunate that these investigators never measured the excretion of thiamine alone and accompanied by I, because such information would allow one to judge whether I and thiamine use the same transport system across the tubu- 1ar epithelium. This is a distinct possibility suggested, but not proved, by these studies. WaIkeri35 reported the results of giving 12 subjects I orally in tablets at a mean of 58.6 mg/kg-(experiment performed by G. Marier, P. Bussault, and J.M. Orr). Blood samples taken at inter- vals thereafter contained the following mean concentrations of I: 1 h, 6.48 ~g/ml; 1.5 h, 6.33 ~g/ml; 2 h, 6.75 ~g/ml; 3 h, 5.77 ~g/ml; 5 h, 3.03 ug/ml; and 7 h, 1.60 vg/ml. The mean time to the maximal plasma concentration of I was 1.67 h, and the mean value of that con- centration was 7.28 ~g/ml. The mean half-life of I in the plasma after reaching the maximal concentration was 125 min. Sundwalll38 found that plasma concentrations of II established by intravenous and latramuscular injections of that oxime became approximately identical about 1 h after the injections, although the courses during that hour were quite different--one descended, at first sharply and then progressively more slowly, and the other ascended to a maxims and thereafter descended slowly. An intramus- cular injection of a 25% solution of II caused some pain at the site of injection; similar administration of a 12.5% solution did not eli- cit pain. When 10 subjects got intramuscular injections of II at 30 mg/kg, three gave evidence of rapid absorption of the oxime, its plasma~concentration rising to about 20 ug/ml within 5 min. The others men seemed to have slower absorption of the oxime, its plasma concentration rising to 12-27 ~g/m] after 20-30 min. In one subject, ache peak concentration in the plasma was not achieved until 90 min after the injection. On oral administration of II at 45 mg/kg in gelatin capsules, the peak plasma concentration of about 5 i~g/m was reached after a mean (six subjects) of 130 min. Sidell et al.75 used two types of tablets containing II one designed to disintegrate rapidly in the gastrointestinal tract and the other intended to provide along, sustained release of oxime. The time courses of the plasma concentrations of the oxime with the two types of tablets were fairly similar, the peak concentrations being attained at the name time after ingestion. lrhe peak concentra- tions after the rapid-release tablets were somewhat above those pro- duced by the same tote' amounts of II in the sustatned-release tab- lets and the descent from the peak concentration was less precipitous after the sustained-release tablets than after the rapid-release ones. Oral doses of I (form not specified) produced peak plasma concentra- tions of oxime sooner after ingestion than after ingestion of the tab- lets of II; the peak plasma concentration from a given dose of I usu- ally was larger than that from a similar dose of II in the form of -311-

either of the tablets. The mean half-time for elimination of II from the body, starting at the peak plasma concentration, was 144 man for the rapid-release tablets and 135 min for the sustained-release tab- ~ets. The mean biologic half-life, calculated from analyses of urine, were 132 min for the rapid-release tablets and 138 min for the alow- release ones. Holland et al.~65 performed a similar study, using a single dose of 4 g of II, whereas Sidel1 et al.75 had used doses of 3, 5, 7, and 9 g. The two types of tablets used in these two studies were made by Glaxo Laboratories Ltd. Ho1 land et al. used a total of 51 subjects and found that the rapid-release tablets yielded more rapidly (by about ~ h) a slightly larger peak plasma concentration of oxime (means for 42 subjects: 6.63 and 6.32 ~g/m] for the rapid-release and siow- release tablets, respectively) and that the decrease from the peak concentration after the siow-release tablets was Blower than that after the rapid-release ones. They concluded that use of a mixture of the two types of tablets would be preferable to use of oily one type in facilitating both a rapid increase in plasma concentration of oxime to an effective point and a relatively prolonged maintenance of such a concentration. In a later paper, Holland and Parkesi66 reported that an intramuscular injection of 500 mg of II is needed to establish an effective plasma concentration. They found that three such doses given 20 mire apart could be tolerated by normal people without complaints of visual disturbances. When intramuscular injection of II was added deco an oral prophylactic regimen with II (4 g every 6 h), complaints of a feeling of enlargement of the eyeballs, blurred vision, and difficulty in accommodation after sudden head movement were expressed. Such complaints were more common from the subjects who received the larger doses of II. Sidell et al.~67 used 20 subjects in a study of tablets of II prepared by Philipa-Duphar in the Netheriands. Fourteen subjects took the tablets on empty stomachs; the other six ate a breakfast of eggs, bacon, and toast about 30-45 min before they took the tablets, which were coated with a methacrylate ester. Doses of 2, 4, 6, and g of II were taken by the fasted subjects, and doses of 4 and 6 g by those who had broken their fasts. Peak plasma concentrations of the onetime were reached within 2-3 h after the tablets were ingested. The ingested doses resulted in the following order of decreasing peak plasma concentrations of onetime: fed, 6 g; fasted, ~ g; fasted, 6 g; fed, 4 g; fasted, 4 g; and fasted, 2 g. The only surprise in this list is the high positions held by the two groups of fed subjects. Urinary excretion of II by the fed subjects was greater than that by the fasted subjects. The plasma concentration of II was a linear function of the logarithm of the dose of oxime for both groups of sub- dects, but the slope of the line for the fed subjects was greater than that for the fasted subjects. Although the difference was never -312-

large, the mean volume of distribution of II in the fasted men was greater than that in the fed ones. The mean first-order rate con- stant for absorption of II by the fasted subjects was greater than that for the fed subjects, and the mean first-order rate constant for elimination of II was greater for the fasted subjects than for the fed ones. Comparison of the pi asma concentrations of II at various times after ingestion of the Netherlands tablets with those after ingestion of the English tablets reveals that comparable doses of II in the English tablets produced the lower plasma concentrations according to the data provided by Sidell et al.,75 but the higher concentrations according to the data of Holland et al.~65 Values measured after the rapid-release Eng1ish tablets were used for these comparisons. Because more ci08e~y similar methods were probably used in the two studies by Sidell et al., comparison of the Two sets of tablets based on the data of the two reports by Sidell et al.75~67 may be more valid than that based on the data of Holland et a1.l65 and of Sidell_ al.~67 Simon _ al.~3 presented data that indicate, by a Small extra- polation, that an oral dose of 4 g of IV in tablets would induce a peak plasma concentration of oxime in a human subject of about 6.2 Ug/ml. This is about the same figure found by Sidel1 et al.~67 af ter 4 g of II in the Netheriands tablets, somewhat less than that reported by Holland et al.~65 after 4 g of lI in the English tab- Jets, and almost I.5 times that found by Sidell et al.75 with the English tablets. Sidel:t add Groff77 gave 10 men intramuscular injections of IV at 2.5, 5.0, 7.5, or 10 mg/kg. These doses produced mean peak plasma concentrations of the oxime of 12.1, 19.8, 34.9, and 39.3 Gym. These concentrations were achieved after 20-30 min. The logarithm of the peak concentration was a linear function of the logarithm of the dose injected. The half-time of removal from the plasma was about 81.3 + 4.3 min. The half-eime of urinary excretion was about 84.5 + 10 min. Dose-re~ated increases in the heart rate and systolic and diastolic blood pressures were alto produced by IV. A comparison of the dose-plasma concentration relationships for IV and I reveals that the plan concentration achieved by a given dose of oxime is 4.7-~.0 times as great for IV as for I. An apparently all-or~nothing symptom complex induced in some subjects by IV consisted of a feeling of warmth ire the upper body that gradually became localized circumorally, a feeling of warmth in the throat that according to three of nine complainants was associated with a taste similar to that of menthol, numbness in and around the mouth and a definite hypoalgesia of that area in response to pricking with a pin, and a feeling that the eye- balle had enlarged and become heavy. Most of these symptoms subsided within 1-2 h. In a later report, 79~168 Sidell and Groff told of giving a 25: aqueous 801UtiOll of IV to 13 men by mouth, using doses of 1, 3, 5, '' -313- 7

and 9 g. The smallest dose produced a plasma concentration of the oxime so low that measurements of it were of questionable signifi- cance; the results from that dose were not analyzed further. The peak plan concentrations produced by the doses of 3, 5, 7, and 9 g were i.9, 3.19, 4.41, and 5.56 regal, respectively. Both I and II produced larger plasma concentrations with a given dose than IV; the ratio between the plasma concentrations produced by a given dose of II and the same dose of IV varied between 1.44 and 2.0, and the same ratio for I and IV varied between 1.44 and 2.6. The half-time for removal of IV from plasma was about 159 min. and that for urinary excretion of IV was about 288 min. The latter time is not only considerably longer than the corresponding times for I and II, but also much longer than that for removal from the plasma. The last discrepancy may indicate metabolic breakdown of the compound in the kidney. Karyotypic study of three of the subjects found no unusual number of gaps and breaks or increased mitotic index. Sidell et a1.~69 compared I, IT, and IV with respect to absorp- tion, distribution, and excretion in human subjects. The oximes were injected intravenously, I and II at 5 mg/kg and IV at 0.5 and laO mg/kg. The lines for the plasma concentration of osime at various times after the injections of I, II, and the larger dose of IV were nearly identical. The smatter dose of IV yielded plasma concentra- tions 0.5-5~0 ~g/ml below those produced by the larger dose. The renal clearances of I and II were 606 and 644 mI/min, respectively, whereas that of IV was 95 mI/min. Although no independent measure of giomerular filtration was reported, the last value for renal clearance is well below the generally accepted mean for g~omerular filtration in the hope kidney of 126 mI/min and could be smaller than glomerular filtrat ion in the particular subJ ects used is the Study. If the clearance of IV really is smaller than the glomerular filtration, that would mean that the kidneys have a tubular reabsorption mechanism for IV, whereas the tubular epithelium secretes I and II into the tubular Humid. The role of distribution of I and II were 815 and 775 ml/kg, respectively, whereas that of IV was only 173 ml/kg. The cen- tral compartment accounted for 33.2% and 25.3% of the total volumes of distribution of ~ and II, respectively, and for 58.0% of that for IV. The half-times for removal of oxide from the plasma were 79 min for I, 85 min for II, and 72 min for IV. Half-times for urinary excretion of the oximes were not given, but values for I and II can be estimated from curves in the report of the c~ulative percentages of the doses excreted in the urine during various times. lrhe half-times for uri- nary excretion estimated from these curves were 39 min for I and 48 min for II. After intramuscular injection of IV, the half-time for its urinary excretion was about 85 minutes;77 the half-tiD~e for excretion after ln~cravenous injection might be smaller by about 20 min. ache minimal time required for reaching the peak plasma concer~t- ration of the oxime after intramuscular injection. The half-time for urinary excretion of IV may be about ~ h after intravenous injection. - -314—

GENERAL COGENT ~ . _ None of the oxides used in experiments with human subjects by the personnel of the Biomedical Laboratory at Edgewood Arsenal or of its contractors has given evidence of being dangerously toxic in the single doses used. The studies detailed in the body of this review have shown that the letbalities of the various oximes are not due solely to the content of the animate radical in these compounds. The bispyridinium biso~lmes (III and IV) seem to be more curaremimetic and more dlaturbing of normal gastrointestinal function than the monopyridinium monoxides (2-PAM I, I, and Il) and the ketoxime (V). The most marked effect of both the monopyridinium monoxides and the bispyridinium bisoximes on functions in subjects poisoned by OP compounds is on neuromuscular tranamission. The bispyridinium bisoximes are absorbed from the gastrointestinal tract lese rapidly than the monopyridinium monoximes, but are more efficient in yielding a comparatively high plasma concentration of oxime than the monopyri- dictum monoximes, by virtue of having smaller volumes of distribution within the body and being excreted less rapidly by the kidneys. The oximes, although they are metabolized to some extent, are removed from the body largely by excretory processes. Particularly for the monopyridinium monoximes, the kidneys are important excretory organs. Renal insufficiency has been found to increase the toxicities of these oximes significantly, but to have no definite effect on that of the ketoxime. Most of these compounds are removed from the human body so rapidly that there is comparatively little hazard of cumulative toxicity from administration of reasonable doses of the oximes. The principal excep- tion is it, which has been found to act synergistically with depressant barbituratesi70 and to induce significant changes in CHS function when repetitive doses were administered. The discovery that IV also appears in the CSF comparatively rapidly after its administration sug- gests that its potential for exerting toxic effects on CNS function during a course of repetitive administrations should be evaluated carefully. III also has been found to produce bothersome, but apparently not particularly dangerous, effects after repetitive doses. Whereas the most striking cardiovascular response to the monopyri- dinium monoximes is hypertension, that to the bispyridinium bisoximes is prolonged hypotension after an initial, short hypertensive response. The hypertension and tachycardia that have been induced by initial —315—

doses of oximes have been found not to persist during a series of repe- titlve administrations. Furthermore, sympatholytic compounds have been found to be effective antagonists of theme actions that could be used to control bothersome cardlova~cular symptoms after the use of oximes. The principal lack in the available information perceived in this review is the absence of any significant attempt to determine whether a delayed toxic effect becomes evident after administration of these compounds. Although hydrogen cyanide is a metabolic product of the monopyridinium monoximes and a nitrite has been found in the urine of rats given III, the production of these compounds has not been great enough to cause obvious toxic effects near the times of administration of the oximes. There is a slight possibility that repetitive admini- strations of oximes during sufficient periods could induce damage to crucial organs by continued production of low concentrations of hydro- gen cyanide and nitrites in the body. Studies of the toxicities of these compounds during long-term administrations should be designed to afford information not only on chronic tosiclty itself, but also on the possibility that oximes may have some degree of carcinogenic or mutagenic potenital. Their teratogenic potential should also be evaluated, although the existing informatloni7] suggests that there will be no effect of that sort. REFERENCES Davies, D.R., Green, A.L. 1955. General discussion. Disc. Faraday Soc. 20:269. 2. Wilson, I.B 1955. Promotion of acety~cholinesterase activity by the anionic site. Disc. Faraday Sac. 20:~19-125. 3. Wilson, I.B., Ginsburg, S. 1955. A powerful deactivator of alky~phosphate-inhibited acety~cholinesterase. Biochem. Biophys. Acta l8 :168-170. 4. Childs, A.F., Davies, D.R., Green, A.L., Rutiand, J.P. 1955. The reactivation by oxides and hydrosamlc acids of cholinesterase inhibited by organophosphorus compound. Brit. J. Pharmacol. Chemotherap . 10: 462-465. 5. Loomis, T.A. 1956. The effect of an aldoxime on acute sarin poi- soning. J. Pharmacol. Exp. Therap. ll8 :123-128. 6. Wills, J.H., Kunkel, A.M., Brown, R.V., GroblewaXi, G.E. 1957. Pyridine-2-aldosime methiodide and poisoning by anticholineste- rases. Science 125: 743-744. - 316

7. Fleisher, J.H., Michel, H.O., Yates, L., Harrison, C.S. 1960. 1,1'-trimethylene bis (4-formylpyridinium bromide) dio~cime (TMB- 4) and 2-pyridine aldoxime me~chiodide (2-PAM) as adjuvants to atropine in the treatment of anticholinesterase poisoning. J. Pharmacol. Exp. Therap. 129: 31-35. B. O'Leary, J.F., Kunkel, A.M., Jones, A.H. 1961. Efficacy and limitations of oxime-atropine treatment of organophosphorus poi- soDlag. J. Pharmacol. Exp. Therap. 132:50-56. 9. Jones, A.H., Kunkel, A.~. 1960. unpublished, quoted by Ellin, R.I., Wills, J.H. 1964: Oximes antagonistic to inhibitors of cholinesterase. J. Pharmaceut Sci. 53:995-1007, I]43-1150. 10. Deyi, X., Linxiu, W., Shuqui, P. 1963. Unpublished, quoted by Deyi, X., Linxiu, W., Shuqui, P. 1981: The inhibition and protection of cho~nesterase by physostigmine and pyridostigmine againat soman poisoning in vivo. Fundam. Appl. Toxicol. 1: 217-221. I' . Loomis, T.A., Salafaky, B. 1963. Antidotal action of pyridi- nium o~cimes in anticholinesterase poisoning; comparative effects of soman, sarin, and neostigmine on neuromuscular function. Toxicol. Appl. Pharmacol. 5: 685-701 . 12. Heilbronn, E ., Tolagen, B. 1965. To~ogonin in sarin , soman, and tabun poisoning. Biochem. Pharmacol. 14: 73-77. 13. Berry, W.K., Da~ries, D.R. 1966. Factors inf~ uencing ~che rate of "aging" of a series of alky! methy~phosphonylacety~cholinesterases. Biochem. J. 100:572-576. 14. FIeisher, J.H., Harria, L.W. 1965. Dealkylation a~ a mechaDism for aging of cholinesterase after poisoning with pinacolyl methylphosphonofluoridate. Biochem. Pharmacol. 14:641-650. 15. Berends, F., Posthumus, C.H., van der Sluys, I., Deierkauf, F.A. 1959. The chemical basis of the "ageing" process of DFP-inhlbited cholinesterase. Biochim. Biophys. Acta 34:576-578. 16. Coult, D.B., Marsh, D.J., Read, G. 1966. Dealkylation studies on inhibited acety~cholinesterase. Biochem. J. 98:869-873. 17. F1eisher, J.H., Harris, L.W., Murtha, E.F. 1967. Reactivation by pyridinium aldoxime methochloride (PAM) of inhibited cho~i- nesterase activity in dogs after poisoning with pinacolyl methyl- phosphonofluoridate (soman). J. Pharmacol. Exp. Therap. 156: 345-351. -317—

l8. Loomis, T.A. 1966. Reirereal of a soman-induced ef feet on neuromuscular function by oximes. Life Scl. 5 :1255-1261. 9. Wills, J.H. 1965. Unpublished. 20. Loomis, T.A., Johnson, D.~. 1966. Reversal of a soman-induced effect on neuromuscular function without reactivation of choli- nesterase. Toxicol. Appl. Pharmacol . B: 528-532. 21. Crone, H.D. 1974. Can allosteric effecters of acety~choli- nesterase control the rate of ageing of the phosphony' ated enzyme? Biochem. Pharmacol. 23: 460-463. 22. I`oomis, T.A., Salafaky, B. 1964. Some effects of soman on neuro- scular function and on acety~cholinesterase in the rat. J. Pharmacol. Exp. The rap . 144: 301-309 . 23. Dultz , L., Epstein, M.A., Freeman, G., Gray, E.H., Weil, W.B. 1957. Studies on a group of osimes as therapeutic compounds in sarin poisoning. J. Pharmcol. Exp. Therap. Il9: 522-531. 24. Hobbiger, F. 1963. Reactivation of phosphonyla~ced acety~cho' i- nesterase. In, Koelle, G. B. (ed) 1963: Cholinesterases and Ar~ticholinesterase Agents, Ber'in~Gottingen-Heide' berg, Springer- Veriag, pp. 921-988. 25. Elmira, R.I., WiUs, J.H. 1964. Osimes antagonistic to inhibitors of cholinesterase. J. Pharmaceut. Sei. 53:995-1007, 1143-~150. 26. Wills, J.H. 1970. Treatment of poisoning by anticholinesterases . In, Karczmar, A.G. (ed) 1970: Anticholinestearee Agents, Oxford, Pergamon Press Ltd., pp. 400-436. 27. Bay, E., Steinberg, G.M. 1973. Evaluation of chemotherapeutic compounds in nerve agent poisoning. EATR 4716. 28. McNamara, B.P. 1976. Osimes as antidotes in poisoning by anticho~nesterase compounds. EB-SP-76004. 29. Benschop, H.P., DeJong, L.R.A., Vink, J.A.J., Kienhuis, H., Berends, F., Elakamp, D.M.W., Repner, L.A., Heeter, E., Visser, R.P.L.S. 1976. The prophylactic value of oximes against organo phosphorus poisoning. In, Stares, J. (ed) 1976: Medical Protection against Chemical-Warfare Agents, Stockholm, Almquist & Wiksell International, pp. 120-133. 30. Namba, T. 1958. Toxicity of PAM (pyridine-2-aldoxime methio- dide) . Naika no Ryoiki 6: 437-441. —318-

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44. O'Leary, J.F. 1964. Unpublished work. 45 . Holmes , R., Robins , E. L. 1955. The reversal by osimea of neuromuscular block produced by anticholinesterases. Brit. J. Pharmacol. 10: 490-495. 46. Fleisher, J.H., Howard, J.~., Corrlgan, J. P. 1958. Effect of pyridine aldoximes on response of frog rectus muscle to acetyI- choline . Brit. J. Pharmacol. 13: 288-290. 47. Fleisher, J.H., Moen, T.H., Ellingaon, N.R. 1965. Effects of 2-PAM and IMB-4 on neuromuscular transmission. J. Pharmaeol. Exp . Therap. 149: 311-319. ~ 48. Wagley, P. F. 1957. A study of end-plate potentials. Observa- tione on the actions of certain oximes. CWLR 2145. 49. Kunkel, A.M., Oikemus, A.H., Brown , R.V. 1957. Preliminary pharmacology of EA IS14. CWL1tM 23-5. 50. KuDlcel, A.M., O'Leary, J.F., Jones, A.H. 196Q. Observations on autonomic responses of 2-PAM, EA 1814, and a mixture (1:1) of these two oxides. COLTS 23-1R. 51. Ballantyne, B., Gazzard, M.F., Robson, D.C., Swanston, D.W. 1975. . Concentrations of 2-hydrosyimHnomethyl-N-methylpyridinium ion in plasma and aqueous humor as indices of the toxicity of pralidosime mesylate (P2S) for the rabbit. Toxicol. Appl. Phar- macol. 33:559-567. 52. Albanus, L., Jarplid, B., Sundwall, A. 1961. The chronic toxi- city of N-methylpyridinium-2-aldosime methane sulphonate (P2S), a Deactivator of phosphorylated cholinestera~e. Acta Pharmacol. Toslcol. I8: 321-328. - 53. Albanus, L., Jarplid, B., Sundwall, A. 1964: The toxicity of some cholinesterase reactivating offices. Belt. J. Esp. Path. 45:120-127. - 54. Crook, J.W. , Cresthull, P., O'Neil, H.W. , Oberat, F.W. 1962. Chronic intravenous toxicity of offices 2-PAM chloride and 2-PAM methanesulfonate to dogs and rabbits. CRDLR 3153. 55. Hasleton Laboratories, Inc. 1965. Preclinical pharmacology study-rats and dogs: EA 3475 (tosogonin), TMB-4, and 2-PAM C1. Report Group No. 15, Contract No. DA-18-035-AMC-120(A). 56. Hazleton Laboratories, Inc. 1965. Preclinical pharmacology study-rats and dogs: EA 3475 (tozogonin). Report Group No. 15, Contract No. DA-~-035-AMC-120(A). — 320 —

57. Crook , J .~., Colbourn , J. L., Sicks , M. E., Grof f ~ W.A., Yevich , P.P., Z^rblis , P., Hackley, E., Sternberger, L., Oberat , F.W. 1960. Chronic toxicity studies on 2-PA}1 and EA 1814 in doge and rabbi ts . CRDLR 3026 . 58. Hopff, W.H., Waser, P.G. 1970. Warn~m konnen Reaktivatoren schadlich sein? Abgehandelt am BeispieJ der Reaktivierung der b' ockierten Acety~cholinesterase . Pharmaceut . Acta Hel~r. 45: 414-423. 59. Jager, B.V., Stagg, G. 1957. Toxicity of DAM aIld PAM in man. Progress Report, Jan. -Sept. 1957, Contract No ~ DA-13-cml-5421. 60. Namba, T. ~ 953. The effectivenese of pyridine-2-aldoxime methiodide ~ PAM) against parathion poisoning in thirty~ine human cases. Naika no Ryoiki 6:84-95. 61. Wedd, G.D., Burgess, F. 1958. Local effects of i.m. injection of hype rtortic solutions of P2S and sodium chloride. PTP 630. 62. Holmes , J.H., Gaon, M., Coulson, E. 1958. Treatment of expo- sures to antichol inest era se agents with 2-PAM. Semi-Annual Pro- gress Report, Sept. 1957-March 195B, Contract No. DA-18-108-cml- 5586. 63. Jager, B.V., Stagg, G.N. ~ 958. To~cicity of diacetyl monoxime and of pyridine-2-aldoxime methiodide in man. Bu~l. Johns Hopkins Hosp. 102: 203-211. 64. Holmes ~ J.H. 1958. Treatment with 2-PAM. Report of two cases . Progress Report, urldated, Contract No. DA-18-108-cml -5586. 65. Holmes, J.H., Gaon, M. 1959. Case report of treatment with 2- PAM. Semi-Annual Progress Report, Oct. 1958-llarch 1959, Con- trac t No. DA-1 B-1 08-405-cml-264. 66. Grob , D.-, Johns , R. J. 1958. Use of oximes in the treatment of intoxication by anticholinesterase compounds in normal subjecte. Am. J. Med. 24: 497-510. 67. Grob, D., Johns , R. J. 1958. Treatment of anticho~ nesterase intoxication with oximes. Use in normal subjects and in patients with myasthenia gravis. J. Am. Med. Assu. 166:~855- iB58. 68. Ladell, W. S.S . 1958. Treatment of antichottnesterase poison- ing. Brit. Med. J. 2 :141-142. -321-

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