DRUG AND CHEMICAL TOXICOLOGY, 15(4), 285-294 (1992)
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The Effect of Pyridostigmine Pretreatment on Oxime Efficacy Against Intoxication by Soman or VX in Rats'.' Dana R. Anderson, Larrel W. Harris, Claude L. Woodard and Willard J. Lennox U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, MD 21010-5425 ABSTRACT This study was done to assess the effects of pyridostigmine (PYR) on a) the accumulation of labelled VX and soman within the brain, b) the therapeutic efficacy of atropine and oxime (2-PAM or HI-6) against intoxication by VX and soman and c) oxime-induced reactivation of inhibited acetylcholinesterase(AChE). In all experiments, rats were given PYR (131 pg/kg, im; Co dose for whole blood AChE) or vehicle 30 min prior to nerve agent. In estimating 3H-agentthe accumulation in the brain or estimating blood AChE activity, sufficient soman (47 pg/kg, iv) or VX (21.3 pg/kg, iv) was given to inhibit 50% of brain AChE activity. In assessing therapeutic efficacy and oxime-induced reactivation of blood AChE, rats were pretreated with PYR, challenged with agent and treated with atropine (16 mg/kg, im) and HI6 or 2-PAM (100 umoles/kg, im) 30 sec post agent. Whole blood was collected by tail bleeding to monitor peripheral AChE activity at various time points before and after PYR and challenge. Pyridostigminefailed to alter covalent binding of labelled VX or soman in the brain. The 24-hr suwival data showed that PYR reduced the therapeutic benefit of atropine and oxime against VX intoxication (but not soman). Protective ratios in VX-challenged rats given vehicle or PYR and treated with atropine + 2-PAM decreased slightly from 2.5 to 2.1 (p> .05), whereas with atropine + H I 6 they decreased significantlyfrom 3.8 to 2.4. Also, AChE reactivation by HI-6 in VX-challenged rats was greater (pc.05) in vehicle- than in PYR-pretreated rats. HI-6 significantly reactivatedAChE activity in both pretreatment groups (PYR or vehicle) given soman. The data suggest that PYR decreases the overall recovery of inhibited AChE in VX-challenged rats given HI-6; under the conditions used, this adverse effect decreases atropine + oxime efficacy against VX-induced lethality. ~~
' In conductingthe research described in this report, the investigatorsadhered to the "Guide for the Care and Use of Laboratory Animals," NIH Publication No. 85-23, revised 1985. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. This work was presented in part to the American Society for Pharmacology and Experimental Therapeutics, August 1990, Milwaukee, WI.
285 Copyright 0 1992 by Marcel Dekker, Inc.
ANDERSON ET AL.
286 INTRODUCTION
Pyridostigmine(PYR) pretreatmentfollowed by atropine t oxime therapy has been reported to be highly effective when compared to therapy alone against soman intoxication.’ Against oxime sensitive organophosphorus (OP) anticholinesteraseagents,
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Sydney on 08/19/13 For personal use only.
Gordon@
(1978f reported that the inclusion of the oxime P2S with atropine markedly
enhanced the efficacy of atropine against sarin and VX intoxication in guinea pigs pretreated with PYR. However, little information Is available concerning PYR pretreatment adversely affecting the therapeutic benefit of atropine + oxime therapy. It is known that oximes (e.g., 2-PAM) are good reactivators of Winhibitid acetylcholinesterase (AChEf and poor reactivators of carbamylated AChE.4-6 Furthermore, oximes do not readily penetrate the CNS to effect reactivation of inhibited AChE in the brain.7 Thus, it is postulated that, in animals not protected with PYR, peripheral AChE should be almost totally inhibited by VX, and virtually all will be susceptible to oxime-induced reactivation. Conversely, in PYR protected animals about 70% of the AChE will be carbamylated. and given that 2-PAM is a poor reactivator of PYR-inhibled AChE, a high percentage of the AChE will remain carbamylated initially, with only a limited amount of VX-inhibited AChE (30%) susceptible to reactivation. Also, with peripheralAChE carbamytated, more circulating agent should be
available for the
CNS. Thus, the aim of the work was to determine
1) whether PYR
pretreatment increasescovalent binding of3H-labelledVX or soman in the brain, 2) whether therapeutic administration of HI-6 and 2-PAM to PYR and vehicle pretreated animals results in similar reactivation of peripheral AChE activity and 3) whether PYR Pretreatment
adversely affects the therapeutic outcome of atropine and oxime therapy. METHODS
These experimentswere conducted using male Cri:CD(SD)BR rats (180-220 g) from Charles River Laboratories. The rats were housed singly in temperature and humidity controlled quarters and were maintained on a 12-hr light-dark cycle with lights on at 0600.
EFFECT OF PYRTDOSTIGMINE PRETREATMENT
287
Food and water were providedad kb. Pyridostigminebromide (PYR), atropine sulfate and oximes (pralidoxime chloride (2-PAM) and 1-[(2-hydroxyimino)methylpyridinio]-3-(4carbamoyLpyridinio)-2-oxapropanedichloride(HI-6)) were obtainedthrough the Walter Reed
Army Instituteof Research: the purities of the drugs were all in excess of 99%. The source
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Sydney on 08/19/13 For personal use only.
for tritiated soman and VX was MRI, Kansas City, MO. Non-labelledsoman and VX were obtained from the Chemical Research, Development and Engineering Center, Aberdeen Proving Ground, MD, and their purities were >96%. Appropriate solutions of soman and
VX were prepared in normal saline just before use; the agent solutions were stored on cracked ice during use. Atropine, PYR and oximes were prepared in twice distilled water just before use.
The dosage of PYR (131 pg/kg, im) used in all experiments was expected to produce 70% inhibtion (bo) of whole blood AChE activity.’ In examining the effects of PYR pretreatment on the distribution of 3H-VX and 3H-sornan in rats treated with atropine, animals were pretreated with PYR 30 min before challenge with labelled soman (47pg/kg, iv) or W (21.3 yg/kg, iv) followed by atropine (8 rng/kg, im) alone, 30 sec later. Fifteen minutes after agent administration, animals were injected with 0.2 ml of heparin solution,
im (1000 USP units/ml), and at 30 min post agent, they were euthanized with CQ. The thoracic cavity was opened and the vascular system was rapidly perfused with heparinized saline; all resulting fluids were contained and disposed of as liquid radioactivewaste. The brain (a hemisphere) and diaphragm were processed for total covalently bound labelled agent moiety. These samples were processed in a Packard 306 Sample Oxidizer and counted in a scintillation counter. Data were expressed as DPM/g tissue and were analyzed using analysis of variance (ANOVA) methods. A p value of less than 0.05 was used to denote statistical significance. To assess the effect of PYR pretreatment on oxime reactivation of peripheralAChE activity, whole blood (50~1)was collected by tail bleeding before (this served as control for
288
ANDERSON ET AL.
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Sydney on 08/19/13 For personal use only.
Table 1. Tritium Labelled VX (21.3 pg/kg, iv) or Soman (47 pg/kg, 9)in Brain or Diaohraam of Rats Sacrificed 30 min Post-Aaent. Brain DiaDhraam VEH K R &t VEH’ PYR -
vx
1726g 16640
GD
38926 34777
‘
8334
8132
55606 63071
Vehicle (saline) or pyridostigmine (PYR; 131 pg/kg, im) 30 min before agent. All rats received atropine (8 mg/kg, im) 30 sec after agent.
*
Average DPM/g tissue (N = 4 per group). No vehicle group is different from its corresponding PYR group (p>0.05).
each animal) and at 28 min after PYR or vehicle administration and again at 2, 8, 15, 25, 40. 60,120 and 180 min after agent. The doses of non-labelled nerve agent in this study
were 37pg/kg. iv for soman and 8.5pg/kg, iv for VX. Atropine (8 mg/kg) and 2-PAM or HI-6 (100 umdes/kg) or vehicle (0.5 ml/kg) were administered (im) 1 min after agent.
AChE activity was measured by a radiometric method using acetylg-methyl choline as substrate. Each animal served as its own control; a repeated measures ANOVA was used
to analyze the data and statistical significance was indicated by p
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Sydney on 08/19/13 For personal use only.
The Effect of Pyridostigmine Pretreatment on Oxime Efficacy Against Intoxication by Soman or VX in Rats'.' Dana R. Anderson, Larrel W. Harris, Claude L. Woodard and Willard J. Lennox U.S. Army Medical Research Institute of Chemical Defense Aberdeen Proving Ground, MD 21010-5425 ABSTRACT This study was done to assess the effects of pyridostigmine (PYR) on a) the accumulation of labelled VX and soman within the brain, b) the therapeutic efficacy of atropine and oxime (2-PAM or HI-6) against intoxication by VX and soman and c) oxime-induced reactivation of inhibited acetylcholinesterase(AChE). In all experiments, rats were given PYR (131 pg/kg, im; Co dose for whole blood AChE) or vehicle 30 min prior to nerve agent. In estimating 3H-agentthe accumulation in the brain or estimating blood AChE activity, sufficient soman (47 pg/kg, iv) or VX (21.3 pg/kg, iv) was given to inhibit 50% of brain AChE activity. In assessing therapeutic efficacy and oxime-induced reactivation of blood AChE, rats were pretreated with PYR, challenged with agent and treated with atropine (16 mg/kg, im) and HI6 or 2-PAM (100 umoles/kg, im) 30 sec post agent. Whole blood was collected by tail bleeding to monitor peripheral AChE activity at various time points before and after PYR and challenge. Pyridostigminefailed to alter covalent binding of labelled VX or soman in the brain. The 24-hr suwival data showed that PYR reduced the therapeutic benefit of atropine and oxime against VX intoxication (but not soman). Protective ratios in VX-challenged rats given vehicle or PYR and treated with atropine + 2-PAM decreased slightly from 2.5 to 2.1 (p> .05), whereas with atropine + H I 6 they decreased significantlyfrom 3.8 to 2.4. Also, AChE reactivation by HI-6 in VX-challenged rats was greater (pc.05) in vehicle- than in PYR-pretreated rats. HI-6 significantly reactivatedAChE activity in both pretreatment groups (PYR or vehicle) given soman. The data suggest that PYR decreases the overall recovery of inhibited AChE in VX-challenged rats given HI-6; under the conditions used, this adverse effect decreases atropine + oxime efficacy against VX-induced lethality. ~~
' In conductingthe research described in this report, the investigatorsadhered to the "Guide for the Care and Use of Laboratory Animals," NIH Publication No. 85-23, revised 1985. The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Army or the Department of Defense. This work was presented in part to the American Society for Pharmacology and Experimental Therapeutics, August 1990, Milwaukee, WI.
285 Copyright 0 1992 by Marcel Dekker, Inc.
ANDERSON ET AL.
286 INTRODUCTION
Pyridostigmine(PYR) pretreatmentfollowed by atropine t oxime therapy has been reported to be highly effective when compared to therapy alone against soman intoxication.’ Against oxime sensitive organophosphorus (OP) anticholinesteraseagents,
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Sydney on 08/19/13 For personal use only.
Gordon@
(1978f reported that the inclusion of the oxime P2S with atropine markedly
enhanced the efficacy of atropine against sarin and VX intoxication in guinea pigs pretreated with PYR. However, little information Is available concerning PYR pretreatment adversely affecting the therapeutic benefit of atropine + oxime therapy. It is known that oximes (e.g., 2-PAM) are good reactivators of Winhibitid acetylcholinesterase (AChEf and poor reactivators of carbamylated AChE.4-6 Furthermore, oximes do not readily penetrate the CNS to effect reactivation of inhibited AChE in the brain.7 Thus, it is postulated that, in animals not protected with PYR, peripheral AChE should be almost totally inhibited by VX, and virtually all will be susceptible to oxime-induced reactivation. Conversely, in PYR protected animals about 70% of the AChE will be carbamylated. and given that 2-PAM is a poor reactivator of PYR-inhibled AChE, a high percentage of the AChE will remain carbamylated initially, with only a limited amount of VX-inhibited AChE (30%) susceptible to reactivation. Also, with peripheralAChE carbamytated, more circulating agent should be
available for the
CNS. Thus, the aim of the work was to determine
1) whether PYR
pretreatment increasescovalent binding of3H-labelledVX or soman in the brain, 2) whether therapeutic administration of HI-6 and 2-PAM to PYR and vehicle pretreated animals results in similar reactivation of peripheral AChE activity and 3) whether PYR Pretreatment
adversely affects the therapeutic outcome of atropine and oxime therapy. METHODS
These experimentswere conducted using male Cri:CD(SD)BR rats (180-220 g) from Charles River Laboratories. The rats were housed singly in temperature and humidity controlled quarters and were maintained on a 12-hr light-dark cycle with lights on at 0600.
EFFECT OF PYRTDOSTIGMINE PRETREATMENT
287
Food and water were providedad kb. Pyridostigminebromide (PYR), atropine sulfate and oximes (pralidoxime chloride (2-PAM) and 1-[(2-hydroxyimino)methylpyridinio]-3-(4carbamoyLpyridinio)-2-oxapropanedichloride(HI-6)) were obtainedthrough the Walter Reed
Army Instituteof Research: the purities of the drugs were all in excess of 99%. The source
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Sydney on 08/19/13 For personal use only.
for tritiated soman and VX was MRI, Kansas City, MO. Non-labelledsoman and VX were obtained from the Chemical Research, Development and Engineering Center, Aberdeen Proving Ground, MD, and their purities were >96%. Appropriate solutions of soman and
VX were prepared in normal saline just before use; the agent solutions were stored on cracked ice during use. Atropine, PYR and oximes were prepared in twice distilled water just before use.
The dosage of PYR (131 pg/kg, im) used in all experiments was expected to produce 70% inhibtion (bo) of whole blood AChE activity.’ In examining the effects of PYR pretreatment on the distribution of 3H-VX and 3H-sornan in rats treated with atropine, animals were pretreated with PYR 30 min before challenge with labelled soman (47pg/kg, iv) or W (21.3 yg/kg, iv) followed by atropine (8 rng/kg, im) alone, 30 sec later. Fifteen minutes after agent administration, animals were injected with 0.2 ml of heparin solution,
im (1000 USP units/ml), and at 30 min post agent, they were euthanized with CQ. The thoracic cavity was opened and the vascular system was rapidly perfused with heparinized saline; all resulting fluids were contained and disposed of as liquid radioactivewaste. The brain (a hemisphere) and diaphragm were processed for total covalently bound labelled agent moiety. These samples were processed in a Packard 306 Sample Oxidizer and counted in a scintillation counter. Data were expressed as DPM/g tissue and were analyzed using analysis of variance (ANOVA) methods. A p value of less than 0.05 was used to denote statistical significance. To assess the effect of PYR pretreatment on oxime reactivation of peripheralAChE activity, whole blood (50~1)was collected by tail bleeding before (this served as control for
288
ANDERSON ET AL.
Drug and Chemical Toxicology Downloaded from informahealthcare.com by University of Sydney on 08/19/13 For personal use only.
Table 1. Tritium Labelled VX (21.3 pg/kg, iv) or Soman (47 pg/kg, 9)in Brain or Diaohraam of Rats Sacrificed 30 min Post-Aaent. Brain DiaDhraam VEH K R &t VEH’ PYR -
vx
1726g 16640
GD
38926 34777
‘
8334
8132
55606 63071
Vehicle (saline) or pyridostigmine (PYR; 131 pg/kg, im) 30 min before agent. All rats received atropine (8 mg/kg, im) 30 sec after agent.
*
Average DPM/g tissue (N = 4 per group). No vehicle group is different from its corresponding PYR group (p>0.05).
each animal) and at 28 min after PYR or vehicle administration and again at 2, 8, 15, 25, 40. 60,120 and 180 min after agent. The doses of non-labelled nerve agent in this study
were 37pg/kg. iv for soman and 8.5pg/kg, iv for VX. Atropine (8 mg/kg) and 2-PAM or HI-6 (100 umdes/kg) or vehicle (0.5 ml/kg) were administered (im) 1 min after agent.
AChE activity was measured by a radiometric method using acetylg-methyl choline as substrate. Each animal served as its own control; a repeated measures ANOVA was used
to analyze the data and statistical significance was indicated by p
