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Antidotal action of pyridinium oximes in anticholinesterase poisoning; Comparative effects of soman, sarin, and neostigmine on neuromuscular function
TOXICOLOGY
ASD
APPLIED
Antidotal
Action
of
Anticholinesterase Effects
Pyridinium
Sarin,
Pharmacology,
LOOMIS
AND
November
in
Comparative Function’
B.
SALAFSKY
School of Medicine, Seattle 5, Washington
Received
Oximes
and Neostigmine
Neuromuscular
T. A. of
(1963)
Poisoning;
of Soman, on
Department
5, 685-701
PHARMACOLOGY
University
of
Washington,
16, 1962
Treatment of poisoning from organophosphate anticholinesterase agents with atropine plus an oxime is highly effective. Recently O’Leary et al. (1961) have shown that animals can be protected from 70 times the lethal dose of sarin (methyl isopropyl phosphonofluoridate) by treatment with a 1: 1 mixture of TMB-4 Cl [ 1,1’-trimethylenebis (Cformyl pyridinium) dioxime dichloride] and 2-PAMCl (l-methyl pyridinium-2-aldoxime chloride) plus atropine. In contrast to this effect of the oximes plus atropine in sarin poisoning, the oximes do not appear to be effective adjuvants to atropine in poisoning with the anticholinesterase agent soman (pinacolyl methyl phosphonofluoridate) . This report is concerned with the effects of soman on acetylcholinesterase and on neuromuscular function, and with the comparative effectiveness of some oximes on the effects of soman, sarin, and neostigmine. MATERIALS
AND
METHODS
The Warburg technique (Umbreit et al., 1957) was used to evaluate the inhibitor effect of sarin and soman on partially purified acetylcholinesterase (AChE, Nutritional Biochemicals Corporation) and the effects of several oximes on the sarin- and soman-inhibited enzyme. The reagents consisted of AChE, 0.0100 g per 100 ml in 0.0331 IM NaHC03; acetylcholine chloride (AChCl), 0.30 g in 25 ml 0.0298 IM NaHC03; oxime, 0.5 to 1O-4 M in propylene glycol: sarin or soman in propylene glycol. Tests were performed at 37°C. 1 Supported
by
ONR
Contract
Nonr-477(29), 685
NR-303-447.
656
T . A.
LOOMIS
AND
B.
SALAFSKY
Acute toxicity and therapeutic antidotal potency were determined on CSH strain mice weighing between 25 and 3j g. Toxicity was determined following either intraperitoneal injection or dermal absorption of sarin and soman. For the dermal absorption tests the anterior abdominal surface of mice was shaved over an area of about 2 cm0 and the mice were used on the following day. The organic phosphates in propylene glycol were applied to the shaved surface of the skin. The volume of solution applied was 0.01 ml. The animals were placed on a 12 X 30-cm surface of 6-mm mesh, galvanized screen which was supported in a nearly vertical position about 30 cm above the surface of a table. Time of onset of effect was recorded as the time interval between dermal application of the organic phosphate and the time when the animal dropped from the screen to the table. Control animals which were placed on the screen for 5 hours had no difficulty in holding themselves on its surface. Seuromuscular effects were determined on intact and isolated preparations. The isolated nerve-diaphragm preparation of the type described by Biilbring (1946) was prepared as follows: Stock mature rats were sacrificed by cervical vertebra crush, and a wedge-shaped section of the right diaphragm containing the entrance and about a 2-cm length of the phrenic nerve was removed from the animal and placed in a constant temperature water bath in Krebs-bicarbonate solution (ITmbreit c’f (11.. 1957) aerated with .5$ C&95:/( 0,. The phrenic nerve was supported above the level of the nutrient solution by a pair of platinum electrodes which served as stimulating electrodes. ‘4 second pair of platinum electrodes served to secure the costal margin of the diaphragm in the nutrient solution and served for direct stimulation of the muscle. Stimuli were supplied through an isolation transformer by a model Se-4 Grass Stimulator. A calibrated strain gauge was attached to the free membranous portion of the diaphragm? and isometric contractions of the muscle were recorded on a Sanborn recorder or on a Tektronix oscilloscope. The intact sciatic-anterior tibialis preparation in rats was prepared as follows: Stock rats were anesthetized with pentobarbital (40mg;kq, intraperitoneally). The internal jugular vein was cannulated, and all subsequent injections were made intravenously. Shielded bipolar platinum electrodes were placed on the right sciatic nerve and the nerve was transected proximal to the electrodes. The right leg was immobilized by means of a femur clamp. The distal tendon of the anterior tibialis muscle was transected and attached to a calibrated strain gauge. Stimuli were supplied and isometric contractions of the muscle were recorded
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with the same apparatus used for the isolated nerve muscle preparation. Surface action potentials of the muscle were recorded directly on an oscilloscope from a unipolar electrode made of stainless steel wire having a diameter of 1 mil. The electrode was shielded flush with its tip with “DUCO” cement (DuPont) and was placed in contact with the exposed anterior surface of the belly of the anterior tibialis muscle from which the fascia had been removed. -411 animals were atropinized (1 .O mg/kg) intravenously at the start of each experiment. Soman and sarin were kindly supplied by Dr. J. H. Wills, Army Chemical Center, Maryland, as solutions containing approximately 9970 pure organic phosphate. Stock solutions of these agents were made to contain 10 mg and 30 mg per milliliter in propylene glycol. Dilutions of the stock solutions were made in 0.97~ saline or in Krebs-bicarbonate nutrient solution at the time of use. The oximes were synthesized in this laboratory as described by Loomis et al. (1963) and were available in pure crystalline form. The oximes used in this study were 2-PAM (l-methyl pyridinium-2-aldoxime iodide), TClA (l-methyl pyridinium2-aldoxime trichloroacetate) , TMB-4 [ l,l’-trimethylenebis (4-formyl pyridinium) dioxime dibromide] , MTMB-4 [ l,l’-methyl isopropyl (4formyl pyridinium) dioxime dibromide] , and OX-2 [ 1,l’-O-dimethylbenzene (4-formyl pyridinium) dioxime dibromidej . Commercial preparations of the following drugs were used: neostigmine methanesulfate, d-tubocurarine chloride, U.S.P., and atropine sulfate, U.S.P. All solutions were made in 0.970 saline or in nutrient solution at the time of use. RESULTS
The effect of the oximes as “reactivators” of sarin- and soman-inhibited AChE, determined by the Warburg technique as shown in Table 1, was obtained by addition of 1 ml of sarin ( 10M5M) or soman (8 X 10F7 M) to 9 ml of the AChE; the mixture was allowed to come to temperature in the Warburg vesselsfor exactly 20 minutes. The reactivator was then added and the vesselswere gassedfor 8 minutes, the system was closed, and at 20 minutes the substrate (AChCl) was added. Carbon dioxide production was recorded at S-minute intervals for 50 minutes. The time intervals were kept constant between trials to avoid possible variations due to “aging” of the phosphorylated enzyme. The concentrations of inhibitors listed above were selectedso that they would produce 70-8070 inhibition of the enzyme. Per cent “reactivation” of the inhibited enzyme produced by each oxime was calculated on the basis of control activity
688
T. A. LOOMIS
AND
B. SALAFSKY
of uninhibited enzyme, and paired determinations were made at each trial. The table shows that sarin-phosphorylated-AChE is effectively reactivated by 1O-4 M concentrations of each of the oximes. However, soman-phosphorylated-AChE is only partially, but significantly (P = > O.OS), reactivated by the same concentrations of the oximes. TMB-4, MTMB-4, and OX-2 produced inhibition of the enzyme at a concentration of 10e2 M. The LDSO (intraperitoneal) of sarin in mice was found to be 0.059 4 0.023 mg/lOO g. The LDSO (intraperitoneal) of soman in mice was found to be 0.062 t- 0.020 mg/lOO g. Twenty mice were used for each deterTABLE EFFECT OF VARIOUS OXIMES ACETYLCHOLINESTERASE
AS “REACTIVATORS” AS DETERMINED
1 BY
OF SARINAND THE WARBURC
SOMAN-INHIBITED TECHNIQUES
% Reactivation Number of trials 6
6 6 6 6 6 6
Concentration of reactivator
Reactivator Z-PAM 2-PAM TClA TClA
IO-4 M
68 & 4
lo-“M
8?-+3
0.7
1.3 t
0.2
82 c
lo-4
M M
86 -+ 4 12 & 1
1.4 " 0.2 2.0 2 0.2
86 -c 2
1.3 '-t 0.2
89 2 4
5.3 2
lo-4 lo-4M 10-4 IO-5M.
1.4 k 0.2 10.7 c
M
ox-2
=
Somaninhibited enzyme (mean -C SD)
10-z
MTMB-4 TMB-4
a Sarin concentration
Sarininhibited enzyme (mean -+ SD)
A4
Soman concentration
3
= 8X
0.6
IO-7M.
mination, and the LDjO was calculated by the method of Litchfield and Wilcoxon (1949). Survival was considered complete if the animals survived for 24 hours. Those animals which died following injection of sarin or soman showed generalized peripheral muscle tremors within 3-5 minutes after injection of the organic phosphate. The tremors were followed by generalized clonic convulsions and gasping respiratory motions. Respiration usually ceased within 8-10 minutes after injection of the lethal dose. Prophylactic effects of TMB-4, atropine, TCIA, and the combination of oxime and atropine were determined in mice poisoned with 2 LDco of sarin or soman; the results are shown in Table 2. All injections were made intraperitoneally and all volumes of injected material were from 0.2 to 1.0 ml. Groups of 20 mice were pretreated with either atropine,
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POISONING
TMB-4, TClA, or combinations of atropine plus the oximes as listed in the table. Ten minutes later, 2 animals from each group were given 0.9% saline (1.0 ml), 9 animals were given soman, and the remaining 9 animals were given sarin (2 LDjo intraperitoneally). Mortality figures are based on survival for 24 hours. The table shows that mortality following injection of soman was not altered by any of the 6 treatments TABLE EFFECT ox
OF ATROPINE, THE
MORTALITY
OXIME,
AND
OF 2 LD,,
ATROPINE
2 PLUS
OXIME
OF SARIN
AND
SOMAN
MIXED
SEXY
AS PROPYLACTIC IN
CsH
STRAIN
TREATMENT MICE
OF
$i& Mortality Treatment 1. 2. 3. 4.
5.
6.
Dose (m&100
.itropine TMB-4 TCIA TMB-4 plus Itropine TClA plus Atropine TMB-4 PIUS TCIA plus .\tropine
a Sine animals were tested were injected intraperitoneally organophosphorous compound 24 hours.
0.4 3 .o 3.0 3.0
g)
Saline (1.0 ml)
Soman LD,,)
(2
(2
Sarin LD,,)
0 0 0
100 100
100 100
100
100
0
100
I1
0
100
22
0
100
0
0.4 3 .o 0.4 1.5 1.5 0.4 for
each treament listed. The various treatment 10 minutes prior to intraperitoneal injection or 0.99 saline. Mortality figures based on death
agents of the within
whereas the combinations of atropine plus oxime were effective antidotes in the sarin-injected animals. The animals pretreated with atropine plus oxime and then injected with soman showed less severe convulsions than those pretreated with atropine or oxime alone, and 3 animals survived for about 4 hours. Soman and sarin are absorbed from the intact skin so that systemic effects similar to those seen after intraperitoneal injection of these organic phosphates are manifested within 3-6 minutes after their application to the skin. The LDBO’s (dermal) for sarin and som.an in CsH strain
690
T. A. LOOMIS
AND
B. SALAFSKY
mice weighing between 25 and 30 g were found to be 0.92 f 0.08 mg and 0.78 +- 0.10 mg per 100 g, respectively, when the solvent is propylene glycol and the volume is 0.01 ml applied to an area of approximately 2 cm2 on the anterior abdominal skin. It was observed in these tests that when animals died following dermal application of the organophosphorous compounds, they died within 4 hours or they survived for at least 24 hours; they were then sacrificed. Therefore subsequent mortality tests were based on survival for at least 4 hours. Onset of toxic effects following dermal application of 1.3 LD 50 (calculated LDge) of the organic phosphates was determined in mice and prophylactic antidotal effect of atropine, two oximes, and ,atropine plus the oximes was determined in the organophosphate-poisonedanimal. The results are shown in Table 3. Groups of 6 or 10 mice were pretreated by intraperitoneal injection of atropine, TMB-4 plus TCIA, or atropine plus TMB-4 plus TCIA as a 1: 1: 1 mixture. Ten minutes later the animals were administered sarin or soman by dermal application. Table 3 shows that onset of systemic effect of the sarin and soman was not significantly altered by prior treatment with each of the three treatment schedules.However, mortality of both sarin and soman was altered by prior administration of each of the three treatment schedules.Atropine plus oxime produced significant protection from the soman, and those animals that died within 4 hours after application of soman survived significantly longer than untreated animals. Soman or sarin produces progressive impairment of twitch and tetanic responseas measured in the isolated nerve-diaphragm preparation of the rat when the muscle is stimulated either indirectly through the nerve or by direct stimulation of the musclefibers. The preparation can be washed repeatedly with fresh nutrient solution without recovery of function. Sarin-inhibited twitch and tetanic response is reverted to normal by addition of the oximes. CompIete recovery of function occurs in 20-30 minutes after addition of either MTMB-4 or TMB-4. This effect of MTMB-4 on the sarin responseis shown in Fig. 1. In contrast to this, these oximes are not completely effective in reversing the soman-induced blockade of twitch and tetanic response.After the addition of the oxime there is either no improvement or gradual minor improvement in twitch response.The blocked tetanic responseis converted to a single potentiated twitch followed by muscular relaxation. An example of these results as obtained in a single experiment in which TMB-4 was used are shown in Fig. 2. Results of this type were obtained in 20 experiments using oxime concentrations of 0.05 t0 0.5 mM.
OXIMES
IN
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0
0
t
4 +I h?
\o
POISONING
$1 t r-2
w
692
T. A. LOOMIS
AND
B. SALAFSKY
FIR 1. Irt vitro effect of MTMB-4 on sarin-inhibited twitch and tetanic response on the isolated nerve-diaphragm of the rat. Stimuli (supramasimal voltage) applied continuously to phrenic nerve at a rate of J per crcond except where indicated, when the rate vvaa 10 per second
-“+.: .__ ;,I t-; ._,“__. I. 1, ;-. ‘ __ : : ,&_ /--& _. .* .i --i-i”-.t
.._
,,,:,.
’ ‘“-9.
_d._
_,
.“T-’
FIG. 2. Effect of soman on the twitch and tetanic response of the isolated phrenic nerve-diaphragm preparation of the rat and the failure of TMB-4 to reverse somaninduced effects. Supramaximal stimuli applied tcl ncrvc in the upper row r~i rct~~rrds and to the muscle in lower row of records. Stimuli continuous and at a rate S-Ii.! per second except \vherc indicated, when the rate \vas 100 per scc~nd.
OXIM.ES
IN
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The anticholinesterase agents sarin, soman, and neostigmine result in two consistently reproducible effects on the intact nerve muscle preparations as observed in a total of 37 preparations. These effects are dose dependent and occur prior to complete neuromuscular paralysis as the dose is progressively increased. The earliest effect is that of potentiation of the indirectly induced twitch response. This is usually accompanied by
FIG. 3. Effect of sarin and soman on the twitch and tetanic response of anterior tibialis muscle to indirect stimulation via the sciatic nerve in the atropinized rat. Effect of Z-PAM on the sarin- and soman-induced response. Stimuli supramaximal and continuous at a rate of 1 per second except where indicated by T, when the rate was 100 per second. Record A obtained from 1 animal; records B, C, and D obtained from a different animal. Ten-minute interval between records B and C, and 2-hour interval between records C and D. All doses in milligrams per kilogram, and all injections made intravenously.
some blockade of indirectly induced tetanic response. As the dose of anticholinesterase agent is increased, the twitch response is depressed and tetanic response is abolished. This later effect is persistent, but both these effects of sarin and neostigmine can be reversed by the oximes. Figure 3, part A, shows these effects of sarin on the intact nerve muscle preparation of the r.at. The figure also shows the prompt recovery produced by administration of 2-PAM. Parts B, C, and D of Fig. 3
694
T. A. LOOMIS
AND
B. SALAFSKY
show the potentiating effect on twitch tension and the nearly complete blockade of tetanic contraction produced by soman on a separate preparation. The effect of 2-PAM on the soman-treated animal was that of a prompt transient inhibition of the potentiated twitch and a partial recovery of the inhibited tetanic response. Figure 4 shows that the poten-
FIG. 4. Potentiating effect of neostigmine on the twitch response with partial blockade of the tetanic response in the intact anterior tibialis muscle preparation of the atropinized rat. Effect of TMB-4 and d-tubocurarine on the neostigmineinduced response. All records from the same preparation. Upper and middle rows continuous except for time intervals indicated. One-hour interval between middle and lower record. Supramaximal stimuli applied continuously to the nerve at a rate of 1 every 2 seconds except when indicated by T, when the rate was 100 per second. AI1 does in milligrams per kilogram, and all injections made intravenously.
tiated twitch response and partial blockade of tetanic response produced by neostigmine is promptly reverted to normal by TMB-4. The figure also shows that the neostigmine-potentiated twitch response is promptly reverted to control by d-tubocurarine. Figure 5 shows the sarin-induced potentiated twitch and blockade of tetanus. The figure also shows the inhibition of the potentiated twitch produced by d-tubocurarine without an influence on the blocked tetanic response. Subsequent injection of the
OXIMES
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oxime, TMB-4, produced prompt transient impairment of twitch response and subsequent recovery of both twitch and tetanic response. The anticholinesterase-induced potentiation of indirectly induced twitch response in the intact nerve muscle preparation is accompanied by an action potential afterdischarge which is oscillatory in nature as recorded
FIG. 5. Potentiating effect of sarin on twitch response with blockade of tetanic response in the intact anterior tibialis muscle preparation in the atropinized rat. Effect of d-tubocurarine and TMB-4 on the sarin-induced responses. All records from a single preparation and continuous except where indicated. Supramaximal stimuli continuously applied to the nerve at a rate of 1 every 2 seconds except where indicated by T, when the rate was 100 per second. All doses in milligrams per kilogram, and all injections made intravenously.
from the surface electrodes. Figure 6 consists of oscilloscope recordings of the surface action potentials from the intact anterior tibialis muscle, which was indirectly stimulated via the motor nerve. A potenti,ated twitch response which was produced by injection of neostigmine was accompanied by an oscillatory afterpotential, and both the potentiated twitch response and afterpotential were promptly abolished by injection of TMB-4 (upper row of recordings, Fig. 6). Impaired tetanic response in-
696
T. A. LOOMIS
AND
B. SALAFSKY
duced by the neostigmine is accompanied by low voltage action potentials (lower row of recordings, Fig. 6) which are promptly reversed to near control values by administration of TMB-4. Figure 7 consists of a series of copies of oscilloscope recordings of action potentials resulting from twitch stimuli in a similar neuromuscular preparation. The twitch response (grams tension) following each action potential is recorded in parentheses beneath each action potential. The
FIG. 6. Recordingsof surfaceaction potentialsobtained from intact anterior tibialismuscle of the atropinizedrat in response to singletwitch stimuli (upperrow) and in response to tetanic stimuli (lower row). Stimulusrecordedon lower beam in each oscilloscope picture.The first picture in eachrow is control, the secondduring potentiatedtwitch response followingneostigmine, and the third 1 minuteafter TMB-4. All dosesin milligramsper kilogram,and all injections made intravenously.
recordings in column’ 1 are control potentials, those in column 2 are during potentiated twitch response, and those in column 3 are after administration of the oxime or d-tubocurarine. The figure indic,ates that the potentiated twitch which had been induced by sarin or soman is accompanied by an action potential which is followed by the oscillatory afterpotential, and that both the potentiated twitch response and the oscillatory after-potential are abolished by 2-PAM or d-tubocurarine. Figure 8 is a record obtained from an intact nerve muscle preparation in which sarin was initially injected in sufficient dose to produce partial blockade of the twitch and tetanic responseto indirectly induced stimuli.
OXIMES
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A/--- 170) FIG. 7. Copies of a series of surface action potentials and the accompanying twitch tension obtained from intact anterior tibialis muscle of the atropinized rat in response to single twitch stimuli applied to the sciatic nerve. The records were obtained from 3 animals (A, B, and C). Column 1 is control, column 2 is 5-8 minutes after administration of the indicated agent, and cohunn 3 is 30 seconds to 1 minute after the indicated agent. All drugs injected intravenously; doses are in milligrams per kilogram of animal weight.
Fro 8. Protective effect of prior administration of sarin on toxicity of soman in the intact anterior tibialis muscle preparation of the atropinized rat. Records are from a single preparation and are continuous except for a 30-minute time interval between B and C. Supramaximal stimuli applied continuously to the nerve at a rate of 3 per second except where indicated by T, when the rate was 100 per second. AI1 doses in milligrams per kilogram, and all injections made intravenously.
698
T. A. LOOMIS
AND
B. SALAFSKY
Soman was then injected in amounts calculated to produce complete blockade of tetanic response.Injection of TMB-4 produced initial partial recovery and subsequent complete recovery of tetanic response. Similar results were obtained in six different preparations. After the oximeinduced recovery of neuromuscular function, subsequent administration of soman produced neuromuscular blockade as shown in Fig. 8. These results indicate that pretreatment of the neuromuscular preparation with sarin, the effects of which can be reversed by the oxime, protects the neuromuscular mechanism from the effects of soman, which can be only partially reversed by the oxime. DISCUSSION
Phosphorylated AChE which has been allowed to “age” either in vitro or in viva undergoes conversion to a form which is essentially nonreactivatable (Hobbiger, 1956; Davies and Green, 1956). The “aging” process which occurs when the enzyme is phosphorylated with DFP (diisopropyl fluorophosphate) is believed to involve loss of an isopropyl group (Behrends et al., 1959). Sarin-inhibited AChE is half converted to the nonreactivatable form in 2-3 hours, but this rate of conversion is dependent on the media in which the test is performed (Hobbiger, 1956). No information is available on the “aging” process for soman-inhibited AChE, but if such a processoccurs and is sufficiently rapid it could account for the failure of the oximes to effectively reactivate the phosphorylated enzyme. Reactivation of inhibited AChE is believed to be the major mechanism by which the oximes act as effective antidotes in poisoning from the organic phosphates. Hobbiger and Sadler (1959) have shown that 2-PAM (0.095 mmole/kg i.p.) will raise the LDsO of neostigmine (given subcutaneously) in mice approximately threefold, and there is an accompanying transient increase in AChE activity. Grob and Johns (1958) have shown in humans that 2-PAM reverses the effect of neostigmine on neuromuscular transmission. The data obtained in this study indicate that the oximes are effective in reversing some of the actions of neostigmine, which is a nonphosphorylating anti-AChE agent. In the current studies, soman produced an inhibited AChE which w,as only slightly reactivated by concentrations of the oximes, which were highly effective reactivators of the sarin-inhibited enzyme. However, in the intact animal studies soman-, sarin-, or neostigmine-induced potentiated twitch response as well as the accompanying post action
OXIMES
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699
potential discharge in the muscle were promptly reversed by the oxime, Z-PAM. Sarin-induced blockade of tetanic response in the neuromuscular preparation was promptly reversed by the oxime, but soman-induced blockade of the tetanic response was only partially reversed by the oxime. Although the oximes were inefficient reactivators of the soman-inhibited enzyme, as determined in the in vitro tests, reactivation of only a small percentage of the total amount of AChE present at the neuromuscular mechanism may be sufficient to account for the observed functional effects of the oxime in the soman-treated neuromuscular preparation. The effectiveness of atropine plus oximes as antidotes to dermal administration of soman and the lack of effectiveness when soman was injected intraperitoneally suggest that this therapy may be effective following minimal exposure to soman by routes of administration which would not result in overwhelmingly rapid absorption. However, the oximes may have effects in addition to the “reactivator” action on the phosphorylated enzyme. The potentiated twitch response produced by 3-hydroxyphenyl triethylammonium compounds and by physostigmine has been described by Riker et al. (1959) as an effect of these compounds on the motor nerve terminal. This effect is that of repetitive discharge of the motor nerve terminal in response to a single twitch stimulus applied to the motor nerve, and the result is conversion of the twitch to a brief asynchronous tetanus. The current experiments demonstrate that an oscillatory post action potential discharge at the muscle accompanied by a potentiated-twitch response is produced by soman, sarin, or neostigmine and that these effects are reversed by the oximes. A partial blockade-type of effect of the oximes at the motor nerve terminal would best describe the observed effect on function. Lindren and Sundwall (1960) and Loomis et ~2. (1963) have shown that the oximes in high doses produce an atropine-like effect on autonomic mechanisms. However, Koelle (1962) has suggested that endogenous acetylcholine (from nerve stimulation) may act to produce liberation of additional amounts of acetylcholine at the nerve terminal. In organophosphate poisoning, endogenous acetylcholine would be expected to accumulate owing to inhibition of AChE. Reactivation of the inhibited (phosphorylated) enzyme by the oximes would result in lowering the accumulated free ACh concentration at the nerve terminal, thereby removing the stimulus for liberation of additional ACh. The nonphosphorylating anti-AChE agent physostigmine is known to protect AChE from phosphorylation by certain organophosphates (Koelle.
700
T. A. LOOMIS
AND
B. SALAFSKY
1946). The current experiments indicate that injection of sarin which leads to the formation of a reactivatable phosphorylated AChE will protect the animal from the effects of soman, which produces a relatively nonreactivatable phosphorylated AChE. Such a protected enzyme can then be “reactivated” by the oximes. SUMMARY
AND
CONCLUSIONS
Soman (pinacolyl
methyl phosphonofluoridate) is an effective inhibitor of acetylcholinesterase in vitro with potency greater than that of sarin (methyl isopropyl phosphonofluoridate). Soman and sarin are equally toxic in the intact rat. Soman, like sarin, is rapidly absorbed after topical application or after intraperitoneal injection. The mono and bis pyridinium oximes, which are effective antidotes when given with atropine in sarin-poisoned mice, are not effective antidotes in mice poisoned by intraperitoneal injection of 2 LD,, of soman. However, mortality of mice poisoned by dermal application of minimal doses of soman is decreased by prior intraperitoneal administration of atropine, the oximes, or a mixture of these agents. Soman produces neuromuscular effects in intact and isolated preparations from rats which are similar to those produced by sarin and neostigmine. Potentiation of twitch response to indirectly induced stimuli produced by these anticholinesterase agents is accompanied by an oscillatory post-action potential discharge in the muscle ceIIs. Both the potentiated twitch and the afterpotential are abolished by administration of the pyridinium oximes. The sarin-inhibited tetanic response of the intact and isolated nerve muscle preparations of the rat is promptly reversed by the pyridinium oximes. The soman-inhibited tetanic response in the same preparations is only partially reversed by the pyridinium oximes. ACKNOWLEDGMENT The authors wish to acknowledge the technical assistance of Miss Jeanne I. Nelson. REFERENCES POSTHUMUS, C. H., SLUYS, V. D. I., and DJIERKAUF, F. A. (1959). Biochim. Biophys. Acta 34, $76-580. B~~LBRING,E. (1946). Observations on the isolated phrenic nerve diaphragm of the rat. Brit. J. Pharmacol. 1, 38-61. DAVIES, D. R., and GREEN, A. L. (1956). The kinetics of reactivation by oximes of cholinesterase inhibited by organophosphorous compounds. Biochem. J. 63, 529535. GROB, D., and JOHNS, R. J. (1958). Use of oximes in the treatment of intoxication by anticholinesterase compounds in normal subjects. Am. J. Med. 24, 497-511. HOBBIGER, F. (1956). Chemical reactivation of phosphorylated human and bovine true choiinesterases. Brit. J. Pharmacol. 11, 295-303. HOBBIGER, F., and SADLER, P. W. (1959). Protection against lethal organophosphate poisoning by quaternary pyridine aldoximes. Brit. J. Pharmacol. 14, 192-201. KOELLE, G. B. (1962). A new general concept of the neurohumoral functions of acetylcholine and acetylcholinesterase. J. Pharm. Pharmacol. 14, 65-90. BEHRENDS,
F.,
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P., and SUNDWALL, A. (1960). Parasympatholytic effects of TMB-4 [l,ltrimethylene-bis (4-formyl pyridinium bromide) dioximel and some related oximes in the cat. Acta Pharmacol. Toxicol. 17, 69-83. LITCHFIELD, J. T., and WILCOXON, F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exptl. Therap. 95, 99-113. LOOMIS, T. A., WELSH, M. J., JR., and MILLER, G. T. (1963). A comparative study of some pyridinium oximes as reactivators of phosphorylated acetylcholinesterase and as antidotes in sarin poisoning. Toxicol. Appl. Pharmacol. 5, 588-598. O’LEARY, J. F., KUNKEL, A. M., and JONES, A. H. (1961). Efficiency and limitations of oxime-atropine treatment of organophosphorous anticholinesterase poisoning. J. Pharmacot. Exptl. Therap. 132, 50-56. RIKER, W. F., JR., WERNER, G., ROBERTS, J., and KUPPERMAN, A. (1959). Pharmacologic evidence for the existence of a presynaptic event in neuromuscular transmission. J. Phavmacol. Exptl. Therap. 125, 150-158. UMBREIT, W. W., BURRIS, R. H., and STAUFFER, J. F. (1957). Manometric Techniques. Burgess, Minneapolis, Minnesota. LINDGREN,
ASD
APPLIED
Antidotal
Action
of
Anticholinesterase Effects
Pyridinium
Sarin,
Pharmacology,
LOOMIS
AND
November
in
Comparative Function’
B.
SALAFSKY
School of Medicine, Seattle 5, Washington
Received
Oximes
and Neostigmine
Neuromuscular
T. A. of
(1963)
Poisoning;
of Soman, on
Department
5, 685-701
PHARMACOLOGY
University
of
Washington,
16, 1962
Treatment of poisoning from organophosphate anticholinesterase agents with atropine plus an oxime is highly effective. Recently O’Leary et al. (1961) have shown that animals can be protected from 70 times the lethal dose of sarin (methyl isopropyl phosphonofluoridate) by treatment with a 1: 1 mixture of TMB-4 Cl [ 1,1’-trimethylenebis (Cformyl pyridinium) dioxime dichloride] and 2-PAMCl (l-methyl pyridinium-2-aldoxime chloride) plus atropine. In contrast to this effect of the oximes plus atropine in sarin poisoning, the oximes do not appear to be effective adjuvants to atropine in poisoning with the anticholinesterase agent soman (pinacolyl methyl phosphonofluoridate) . This report is concerned with the effects of soman on acetylcholinesterase and on neuromuscular function, and with the comparative effectiveness of some oximes on the effects of soman, sarin, and neostigmine. MATERIALS
AND
METHODS
The Warburg technique (Umbreit et al., 1957) was used to evaluate the inhibitor effect of sarin and soman on partially purified acetylcholinesterase (AChE, Nutritional Biochemicals Corporation) and the effects of several oximes on the sarin- and soman-inhibited enzyme. The reagents consisted of AChE, 0.0100 g per 100 ml in 0.0331 IM NaHC03; acetylcholine chloride (AChCl), 0.30 g in 25 ml 0.0298 IM NaHC03; oxime, 0.5 to 1O-4 M in propylene glycol: sarin or soman in propylene glycol. Tests were performed at 37°C. 1 Supported
by
ONR
Contract
Nonr-477(29), 685
NR-303-447.
656
T . A.
LOOMIS
AND
B.
SALAFSKY
Acute toxicity and therapeutic antidotal potency were determined on CSH strain mice weighing between 25 and 3j g. Toxicity was determined following either intraperitoneal injection or dermal absorption of sarin and soman. For the dermal absorption tests the anterior abdominal surface of mice was shaved over an area of about 2 cm0 and the mice were used on the following day. The organic phosphates in propylene glycol were applied to the shaved surface of the skin. The volume of solution applied was 0.01 ml. The animals were placed on a 12 X 30-cm surface of 6-mm mesh, galvanized screen which was supported in a nearly vertical position about 30 cm above the surface of a table. Time of onset of effect was recorded as the time interval between dermal application of the organic phosphate and the time when the animal dropped from the screen to the table. Control animals which were placed on the screen for 5 hours had no difficulty in holding themselves on its surface. Seuromuscular effects were determined on intact and isolated preparations. The isolated nerve-diaphragm preparation of the type described by Biilbring (1946) was prepared as follows: Stock mature rats were sacrificed by cervical vertebra crush, and a wedge-shaped section of the right diaphragm containing the entrance and about a 2-cm length of the phrenic nerve was removed from the animal and placed in a constant temperature water bath in Krebs-bicarbonate solution (ITmbreit c’f (11.. 1957) aerated with .5$ C&95:/( 0,. The phrenic nerve was supported above the level of the nutrient solution by a pair of platinum electrodes which served as stimulating electrodes. ‘4 second pair of platinum electrodes served to secure the costal margin of the diaphragm in the nutrient solution and served for direct stimulation of the muscle. Stimuli were supplied through an isolation transformer by a model Se-4 Grass Stimulator. A calibrated strain gauge was attached to the free membranous portion of the diaphragm? and isometric contractions of the muscle were recorded on a Sanborn recorder or on a Tektronix oscilloscope. The intact sciatic-anterior tibialis preparation in rats was prepared as follows: Stock rats were anesthetized with pentobarbital (40mg;kq, intraperitoneally). The internal jugular vein was cannulated, and all subsequent injections were made intravenously. Shielded bipolar platinum electrodes were placed on the right sciatic nerve and the nerve was transected proximal to the electrodes. The right leg was immobilized by means of a femur clamp. The distal tendon of the anterior tibialis muscle was transected and attached to a calibrated strain gauge. Stimuli were supplied and isometric contractions of the muscle were recorded
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687
with the same apparatus used for the isolated nerve muscle preparation. Surface action potentials of the muscle were recorded directly on an oscilloscope from a unipolar electrode made of stainless steel wire having a diameter of 1 mil. The electrode was shielded flush with its tip with “DUCO” cement (DuPont) and was placed in contact with the exposed anterior surface of the belly of the anterior tibialis muscle from which the fascia had been removed. -411 animals were atropinized (1 .O mg/kg) intravenously at the start of each experiment. Soman and sarin were kindly supplied by Dr. J. H. Wills, Army Chemical Center, Maryland, as solutions containing approximately 9970 pure organic phosphate. Stock solutions of these agents were made to contain 10 mg and 30 mg per milliliter in propylene glycol. Dilutions of the stock solutions were made in 0.97~ saline or in Krebs-bicarbonate nutrient solution at the time of use. The oximes were synthesized in this laboratory as described by Loomis et al. (1963) and were available in pure crystalline form. The oximes used in this study were 2-PAM (l-methyl pyridinium-2-aldoxime iodide), TClA (l-methyl pyridinium2-aldoxime trichloroacetate) , TMB-4 [ l,l’-trimethylenebis (4-formyl pyridinium) dioxime dibromide] , MTMB-4 [ l,l’-methyl isopropyl (4formyl pyridinium) dioxime dibromide] , and OX-2 [ 1,l’-O-dimethylbenzene (4-formyl pyridinium) dioxime dibromidej . Commercial preparations of the following drugs were used: neostigmine methanesulfate, d-tubocurarine chloride, U.S.P., and atropine sulfate, U.S.P. All solutions were made in 0.970 saline or in nutrient solution at the time of use. RESULTS
The effect of the oximes as “reactivators” of sarin- and soman-inhibited AChE, determined by the Warburg technique as shown in Table 1, was obtained by addition of 1 ml of sarin ( 10M5M) or soman (8 X 10F7 M) to 9 ml of the AChE; the mixture was allowed to come to temperature in the Warburg vesselsfor exactly 20 minutes. The reactivator was then added and the vesselswere gassedfor 8 minutes, the system was closed, and at 20 minutes the substrate (AChCl) was added. Carbon dioxide production was recorded at S-minute intervals for 50 minutes. The time intervals were kept constant between trials to avoid possible variations due to “aging” of the phosphorylated enzyme. The concentrations of inhibitors listed above were selectedso that they would produce 70-8070 inhibition of the enzyme. Per cent “reactivation” of the inhibited enzyme produced by each oxime was calculated on the basis of control activity
688
T. A. LOOMIS
AND
B. SALAFSKY
of uninhibited enzyme, and paired determinations were made at each trial. The table shows that sarin-phosphorylated-AChE is effectively reactivated by 1O-4 M concentrations of each of the oximes. However, soman-phosphorylated-AChE is only partially, but significantly (P = > O.OS), reactivated by the same concentrations of the oximes. TMB-4, MTMB-4, and OX-2 produced inhibition of the enzyme at a concentration of 10e2 M. The LDSO (intraperitoneal) of sarin in mice was found to be 0.059 4 0.023 mg/lOO g. The LDSO (intraperitoneal) of soman in mice was found to be 0.062 t- 0.020 mg/lOO g. Twenty mice were used for each deterTABLE EFFECT OF VARIOUS OXIMES ACETYLCHOLINESTERASE
AS “REACTIVATORS” AS DETERMINED
1 BY
OF SARINAND THE WARBURC
SOMAN-INHIBITED TECHNIQUES
% Reactivation Number of trials 6
6 6 6 6 6 6
Concentration of reactivator
Reactivator Z-PAM 2-PAM TClA TClA
IO-4 M
68 & 4
lo-“M
8?-+3
0.7
1.3 t
0.2
82 c
lo-4
M M
86 -+ 4 12 & 1
1.4 " 0.2 2.0 2 0.2
86 -c 2
1.3 '-t 0.2
89 2 4
5.3 2
lo-4 lo-4M 10-4 IO-5M.
1.4 k 0.2 10.7 c
M
ox-2
=
Somaninhibited enzyme (mean -C SD)
10-z
MTMB-4 TMB-4
a Sarin concentration
Sarininhibited enzyme (mean -+ SD)
A4
Soman concentration
3
= 8X
0.6
IO-7M.
mination, and the LDjO was calculated by the method of Litchfield and Wilcoxon (1949). Survival was considered complete if the animals survived for 24 hours. Those animals which died following injection of sarin or soman showed generalized peripheral muscle tremors within 3-5 minutes after injection of the organic phosphate. The tremors were followed by generalized clonic convulsions and gasping respiratory motions. Respiration usually ceased within 8-10 minutes after injection of the lethal dose. Prophylactic effects of TMB-4, atropine, TCIA, and the combination of oxime and atropine were determined in mice poisoned with 2 LDco of sarin or soman; the results are shown in Table 2. All injections were made intraperitoneally and all volumes of injected material were from 0.2 to 1.0 ml. Groups of 20 mice were pretreated with either atropine,
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POISONING
TMB-4, TClA, or combinations of atropine plus the oximes as listed in the table. Ten minutes later, 2 animals from each group were given 0.9% saline (1.0 ml), 9 animals were given soman, and the remaining 9 animals were given sarin (2 LDjo intraperitoneally). Mortality figures are based on survival for 24 hours. The table shows that mortality following injection of soman was not altered by any of the 6 treatments TABLE EFFECT ox
OF ATROPINE, THE
MORTALITY
OXIME,
AND
OF 2 LD,,
ATROPINE
2 PLUS
OXIME
OF SARIN
AND
SOMAN
MIXED
SEXY
AS PROPYLACTIC IN
CsH
STRAIN
TREATMENT MICE
OF
$i& Mortality Treatment 1. 2. 3. 4.
5.
6.
Dose (m&100
.itropine TMB-4 TCIA TMB-4 plus Itropine TClA plus Atropine TMB-4 PIUS TCIA plus .\tropine
a Sine animals were tested were injected intraperitoneally organophosphorous compound 24 hours.
0.4 3 .o 3.0 3.0
g)
Saline (1.0 ml)
Soman LD,,)
(2
(2
Sarin LD,,)
0 0 0
100 100
100 100
100
100
0
100
I1
0
100
22
0
100
0
0.4 3 .o 0.4 1.5 1.5 0.4 for
each treament listed. The various treatment 10 minutes prior to intraperitoneal injection or 0.99 saline. Mortality figures based on death
agents of the within
whereas the combinations of atropine plus oxime were effective antidotes in the sarin-injected animals. The animals pretreated with atropine plus oxime and then injected with soman showed less severe convulsions than those pretreated with atropine or oxime alone, and 3 animals survived for about 4 hours. Soman and sarin are absorbed from the intact skin so that systemic effects similar to those seen after intraperitoneal injection of these organic phosphates are manifested within 3-6 minutes after their application to the skin. The LDBO’s (dermal) for sarin and som.an in CsH strain
690
T. A. LOOMIS
AND
B. SALAFSKY
mice weighing between 25 and 30 g were found to be 0.92 f 0.08 mg and 0.78 +- 0.10 mg per 100 g, respectively, when the solvent is propylene glycol and the volume is 0.01 ml applied to an area of approximately 2 cm2 on the anterior abdominal skin. It was observed in these tests that when animals died following dermal application of the organophosphorous compounds, they died within 4 hours or they survived for at least 24 hours; they were then sacrificed. Therefore subsequent mortality tests were based on survival for at least 4 hours. Onset of toxic effects following dermal application of 1.3 LD 50 (calculated LDge) of the organic phosphates was determined in mice and prophylactic antidotal effect of atropine, two oximes, and ,atropine plus the oximes was determined in the organophosphate-poisonedanimal. The results are shown in Table 3. Groups of 6 or 10 mice were pretreated by intraperitoneal injection of atropine, TMB-4 plus TCIA, or atropine plus TMB-4 plus TCIA as a 1: 1: 1 mixture. Ten minutes later the animals were administered sarin or soman by dermal application. Table 3 shows that onset of systemic effect of the sarin and soman was not significantly altered by prior treatment with each of the three treatment schedules.However, mortality of both sarin and soman was altered by prior administration of each of the three treatment schedules.Atropine plus oxime produced significant protection from the soman, and those animals that died within 4 hours after application of soman survived significantly longer than untreated animals. Soman or sarin produces progressive impairment of twitch and tetanic responseas measured in the isolated nerve-diaphragm preparation of the rat when the muscle is stimulated either indirectly through the nerve or by direct stimulation of the musclefibers. The preparation can be washed repeatedly with fresh nutrient solution without recovery of function. Sarin-inhibited twitch and tetanic response is reverted to normal by addition of the oximes. CompIete recovery of function occurs in 20-30 minutes after addition of either MTMB-4 or TMB-4. This effect of MTMB-4 on the sarin responseis shown in Fig. 1. In contrast to this, these oximes are not completely effective in reversing the soman-induced blockade of twitch and tetanic response.After the addition of the oxime there is either no improvement or gradual minor improvement in twitch response.The blocked tetanic responseis converted to a single potentiated twitch followed by muscular relaxation. An example of these results as obtained in a single experiment in which TMB-4 was used are shown in Fig. 2. Results of this type were obtained in 20 experiments using oxime concentrations of 0.05 t0 0.5 mM.
OXIMES
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0
0
t
4 +I h?
\o
POISONING
$1 t r-2
w
692
T. A. LOOMIS
AND
B. SALAFSKY
FIR 1. Irt vitro effect of MTMB-4 on sarin-inhibited twitch and tetanic response on the isolated nerve-diaphragm of the rat. Stimuli (supramasimal voltage) applied continuously to phrenic nerve at a rate of J per crcond except where indicated, when the rate vvaa 10 per second
-“+.: .__ ;,I t-; ._,“__. I. 1, ;-. ‘ __ : : ,&_ /--& _. .* .i --i-i”-.t
.._
,,,:,.
’ ‘“-9.
_d._
_,
.“T-’
FIG. 2. Effect of soman on the twitch and tetanic response of the isolated phrenic nerve-diaphragm preparation of the rat and the failure of TMB-4 to reverse somaninduced effects. Supramaximal stimuli applied tcl ncrvc in the upper row r~i rct~~rrds and to the muscle in lower row of records. Stimuli continuous and at a rate S-Ii.! per second except \vherc indicated, when the rate \vas 100 per scc~nd.
OXIM.ES
IN
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693
The anticholinesterase agents sarin, soman, and neostigmine result in two consistently reproducible effects on the intact nerve muscle preparations as observed in a total of 37 preparations. These effects are dose dependent and occur prior to complete neuromuscular paralysis as the dose is progressively increased. The earliest effect is that of potentiation of the indirectly induced twitch response. This is usually accompanied by
FIG. 3. Effect of sarin and soman on the twitch and tetanic response of anterior tibialis muscle to indirect stimulation via the sciatic nerve in the atropinized rat. Effect of Z-PAM on the sarin- and soman-induced response. Stimuli supramaximal and continuous at a rate of 1 per second except where indicated by T, when the rate was 100 per second. Record A obtained from 1 animal; records B, C, and D obtained from a different animal. Ten-minute interval between records B and C, and 2-hour interval between records C and D. All doses in milligrams per kilogram, and all injections made intravenously.
some blockade of indirectly induced tetanic response. As the dose of anticholinesterase agent is increased, the twitch response is depressed and tetanic response is abolished. This later effect is persistent, but both these effects of sarin and neostigmine can be reversed by the oximes. Figure 3, part A, shows these effects of sarin on the intact nerve muscle preparation of the r.at. The figure also shows the prompt recovery produced by administration of 2-PAM. Parts B, C, and D of Fig. 3
694
T. A. LOOMIS
AND
B. SALAFSKY
show the potentiating effect on twitch tension and the nearly complete blockade of tetanic contraction produced by soman on a separate preparation. The effect of 2-PAM on the soman-treated animal was that of a prompt transient inhibition of the potentiated twitch and a partial recovery of the inhibited tetanic response. Figure 4 shows that the poten-
FIG. 4. Potentiating effect of neostigmine on the twitch response with partial blockade of the tetanic response in the intact anterior tibialis muscle preparation of the atropinized rat. Effect of TMB-4 and d-tubocurarine on the neostigmineinduced response. All records from the same preparation. Upper and middle rows continuous except for time intervals indicated. One-hour interval between middle and lower record. Supramaximal stimuli applied continuously to the nerve at a rate of 1 every 2 seconds except when indicated by T, when the rate was 100 per second. AI1 does in milligrams per kilogram, and all injections made intravenously.
tiated twitch response and partial blockade of tetanic response produced by neostigmine is promptly reverted to normal by TMB-4. The figure also shows that the neostigmine-potentiated twitch response is promptly reverted to control by d-tubocurarine. Figure 5 shows the sarin-induced potentiated twitch and blockade of tetanus. The figure also shows the inhibition of the potentiated twitch produced by d-tubocurarine without an influence on the blocked tetanic response. Subsequent injection of the
OXIMES
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oxime, TMB-4, produced prompt transient impairment of twitch response and subsequent recovery of both twitch and tetanic response. The anticholinesterase-induced potentiation of indirectly induced twitch response in the intact nerve muscle preparation is accompanied by an action potential afterdischarge which is oscillatory in nature as recorded
FIG. 5. Potentiating effect of sarin on twitch response with blockade of tetanic response in the intact anterior tibialis muscle preparation in the atropinized rat. Effect of d-tubocurarine and TMB-4 on the sarin-induced responses. All records from a single preparation and continuous except where indicated. Supramaximal stimuli continuously applied to the nerve at a rate of 1 every 2 seconds except where indicated by T, when the rate was 100 per second. All doses in milligrams per kilogram, and all injections made intravenously.
from the surface electrodes. Figure 6 consists of oscilloscope recordings of the surface action potentials from the intact anterior tibialis muscle, which was indirectly stimulated via the motor nerve. A potenti,ated twitch response which was produced by injection of neostigmine was accompanied by an oscillatory afterpotential, and both the potentiated twitch response and afterpotential were promptly abolished by injection of TMB-4 (upper row of recordings, Fig. 6). Impaired tetanic response in-
696
T. A. LOOMIS
AND
B. SALAFSKY
duced by the neostigmine is accompanied by low voltage action potentials (lower row of recordings, Fig. 6) which are promptly reversed to near control values by administration of TMB-4. Figure 7 consists of a series of copies of oscilloscope recordings of action potentials resulting from twitch stimuli in a similar neuromuscular preparation. The twitch response (grams tension) following each action potential is recorded in parentheses beneath each action potential. The
FIG. 6. Recordingsof surfaceaction potentialsobtained from intact anterior tibialismuscle of the atropinizedrat in response to singletwitch stimuli (upperrow) and in response to tetanic stimuli (lower row). Stimulusrecordedon lower beam in each oscilloscope picture.The first picture in eachrow is control, the secondduring potentiatedtwitch response followingneostigmine, and the third 1 minuteafter TMB-4. All dosesin milligramsper kilogram,and all injections made intravenously.
recordings in column’ 1 are control potentials, those in column 2 are during potentiated twitch response, and those in column 3 are after administration of the oxime or d-tubocurarine. The figure indic,ates that the potentiated twitch which had been induced by sarin or soman is accompanied by an action potential which is followed by the oscillatory afterpotential, and that both the potentiated twitch response and the oscillatory after-potential are abolished by 2-PAM or d-tubocurarine. Figure 8 is a record obtained from an intact nerve muscle preparation in which sarin was initially injected in sufficient dose to produce partial blockade of the twitch and tetanic responseto indirectly induced stimuli.
OXIMES
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A/--- 170) FIG. 7. Copies of a series of surface action potentials and the accompanying twitch tension obtained from intact anterior tibialis muscle of the atropinized rat in response to single twitch stimuli applied to the sciatic nerve. The records were obtained from 3 animals (A, B, and C). Column 1 is control, column 2 is 5-8 minutes after administration of the indicated agent, and cohunn 3 is 30 seconds to 1 minute after the indicated agent. All drugs injected intravenously; doses are in milligrams per kilogram of animal weight.
Fro 8. Protective effect of prior administration of sarin on toxicity of soman in the intact anterior tibialis muscle preparation of the atropinized rat. Records are from a single preparation and are continuous except for a 30-minute time interval between B and C. Supramaximal stimuli applied continuously to the nerve at a rate of 3 per second except where indicated by T, when the rate was 100 per second. AI1 doses in milligrams per kilogram, and all injections made intravenously.
698
T. A. LOOMIS
AND
B. SALAFSKY
Soman was then injected in amounts calculated to produce complete blockade of tetanic response.Injection of TMB-4 produced initial partial recovery and subsequent complete recovery of tetanic response. Similar results were obtained in six different preparations. After the oximeinduced recovery of neuromuscular function, subsequent administration of soman produced neuromuscular blockade as shown in Fig. 8. These results indicate that pretreatment of the neuromuscular preparation with sarin, the effects of which can be reversed by the oxime, protects the neuromuscular mechanism from the effects of soman, which can be only partially reversed by the oxime. DISCUSSION
Phosphorylated AChE which has been allowed to “age” either in vitro or in viva undergoes conversion to a form which is essentially nonreactivatable (Hobbiger, 1956; Davies and Green, 1956). The “aging” process which occurs when the enzyme is phosphorylated with DFP (diisopropyl fluorophosphate) is believed to involve loss of an isopropyl group (Behrends et al., 1959). Sarin-inhibited AChE is half converted to the nonreactivatable form in 2-3 hours, but this rate of conversion is dependent on the media in which the test is performed (Hobbiger, 1956). No information is available on the “aging” process for soman-inhibited AChE, but if such a processoccurs and is sufficiently rapid it could account for the failure of the oximes to effectively reactivate the phosphorylated enzyme. Reactivation of inhibited AChE is believed to be the major mechanism by which the oximes act as effective antidotes in poisoning from the organic phosphates. Hobbiger and Sadler (1959) have shown that 2-PAM (0.095 mmole/kg i.p.) will raise the LDsO of neostigmine (given subcutaneously) in mice approximately threefold, and there is an accompanying transient increase in AChE activity. Grob and Johns (1958) have shown in humans that 2-PAM reverses the effect of neostigmine on neuromuscular transmission. The data obtained in this study indicate that the oximes are effective in reversing some of the actions of neostigmine, which is a nonphosphorylating anti-AChE agent. In the current studies, soman produced an inhibited AChE which w,as only slightly reactivated by concentrations of the oximes, which were highly effective reactivators of the sarin-inhibited enzyme. However, in the intact animal studies soman-, sarin-, or neostigmine-induced potentiated twitch response as well as the accompanying post action
OXIMES
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699
potential discharge in the muscle were promptly reversed by the oxime, Z-PAM. Sarin-induced blockade of tetanic response in the neuromuscular preparation was promptly reversed by the oxime, but soman-induced blockade of the tetanic response was only partially reversed by the oxime. Although the oximes were inefficient reactivators of the soman-inhibited enzyme, as determined in the in vitro tests, reactivation of only a small percentage of the total amount of AChE present at the neuromuscular mechanism may be sufficient to account for the observed functional effects of the oxime in the soman-treated neuromuscular preparation. The effectiveness of atropine plus oximes as antidotes to dermal administration of soman and the lack of effectiveness when soman was injected intraperitoneally suggest that this therapy may be effective following minimal exposure to soman by routes of administration which would not result in overwhelmingly rapid absorption. However, the oximes may have effects in addition to the “reactivator” action on the phosphorylated enzyme. The potentiated twitch response produced by 3-hydroxyphenyl triethylammonium compounds and by physostigmine has been described by Riker et al. (1959) as an effect of these compounds on the motor nerve terminal. This effect is that of repetitive discharge of the motor nerve terminal in response to a single twitch stimulus applied to the motor nerve, and the result is conversion of the twitch to a brief asynchronous tetanus. The current experiments demonstrate that an oscillatory post action potential discharge at the muscle accompanied by a potentiated-twitch response is produced by soman, sarin, or neostigmine and that these effects are reversed by the oximes. A partial blockade-type of effect of the oximes at the motor nerve terminal would best describe the observed effect on function. Lindren and Sundwall (1960) and Loomis et ~2. (1963) have shown that the oximes in high doses produce an atropine-like effect on autonomic mechanisms. However, Koelle (1962) has suggested that endogenous acetylcholine (from nerve stimulation) may act to produce liberation of additional amounts of acetylcholine at the nerve terminal. In organophosphate poisoning, endogenous acetylcholine would be expected to accumulate owing to inhibition of AChE. Reactivation of the inhibited (phosphorylated) enzyme by the oximes would result in lowering the accumulated free ACh concentration at the nerve terminal, thereby removing the stimulus for liberation of additional ACh. The nonphosphorylating anti-AChE agent physostigmine is known to protect AChE from phosphorylation by certain organophosphates (Koelle.
700
T. A. LOOMIS
AND
B. SALAFSKY
1946). The current experiments indicate that injection of sarin which leads to the formation of a reactivatable phosphorylated AChE will protect the animal from the effects of soman, which produces a relatively nonreactivatable phosphorylated AChE. Such a protected enzyme can then be “reactivated” by the oximes. SUMMARY
AND
CONCLUSIONS
Soman (pinacolyl
methyl phosphonofluoridate) is an effective inhibitor of acetylcholinesterase in vitro with potency greater than that of sarin (methyl isopropyl phosphonofluoridate). Soman and sarin are equally toxic in the intact rat. Soman, like sarin, is rapidly absorbed after topical application or after intraperitoneal injection. The mono and bis pyridinium oximes, which are effective antidotes when given with atropine in sarin-poisoned mice, are not effective antidotes in mice poisoned by intraperitoneal injection of 2 LD,, of soman. However, mortality of mice poisoned by dermal application of minimal doses of soman is decreased by prior intraperitoneal administration of atropine, the oximes, or a mixture of these agents. Soman produces neuromuscular effects in intact and isolated preparations from rats which are similar to those produced by sarin and neostigmine. Potentiation of twitch response to indirectly induced stimuli produced by these anticholinesterase agents is accompanied by an oscillatory post-action potential discharge in the muscle ceIIs. Both the potentiated twitch and the afterpotential are abolished by administration of the pyridinium oximes. The sarin-inhibited tetanic response of the intact and isolated nerve muscle preparations of the rat is promptly reversed by the pyridinium oximes. The soman-inhibited tetanic response in the same preparations is only partially reversed by the pyridinium oximes. ACKNOWLEDGMENT The authors wish to acknowledge the technical assistance of Miss Jeanne I. Nelson. REFERENCES POSTHUMUS, C. H., SLUYS, V. D. I., and DJIERKAUF, F. A. (1959). Biochim. Biophys. Acta 34, $76-580. B~~LBRING,E. (1946). Observations on the isolated phrenic nerve diaphragm of the rat. Brit. J. Pharmacol. 1, 38-61. DAVIES, D. R., and GREEN, A. L. (1956). The kinetics of reactivation by oximes of cholinesterase inhibited by organophosphorous compounds. Biochem. J. 63, 529535. GROB, D., and JOHNS, R. J. (1958). Use of oximes in the treatment of intoxication by anticholinesterase compounds in normal subjects. Am. J. Med. 24, 497-511. HOBBIGER, F. (1956). Chemical reactivation of phosphorylated human and bovine true choiinesterases. Brit. J. Pharmacol. 11, 295-303. HOBBIGER, F., and SADLER, P. W. (1959). Protection against lethal organophosphate poisoning by quaternary pyridine aldoximes. Brit. J. Pharmacol. 14, 192-201. KOELLE, G. B. (1962). A new general concept of the neurohumoral functions of acetylcholine and acetylcholinesterase. J. Pharm. Pharmacol. 14, 65-90. BEHRENDS,
F.,
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P., and SUNDWALL, A. (1960). Parasympatholytic effects of TMB-4 [l,ltrimethylene-bis (4-formyl pyridinium bromide) dioximel and some related oximes in the cat. Acta Pharmacol. Toxicol. 17, 69-83. LITCHFIELD, J. T., and WILCOXON, F. (1949). A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exptl. Therap. 95, 99-113. LOOMIS, T. A., WELSH, M. J., JR., and MILLER, G. T. (1963). A comparative study of some pyridinium oximes as reactivators of phosphorylated acetylcholinesterase and as antidotes in sarin poisoning. Toxicol. Appl. Pharmacol. 5, 588-598. O’LEARY, J. F., KUNKEL, A. M., and JONES, A. H. (1961). Efficiency and limitations of oxime-atropine treatment of organophosphorous anticholinesterase poisoning. J. Pharmacot. Exptl. Therap. 132, 50-56. RIKER, W. F., JR., WERNER, G., ROBERTS, J., and KUPPERMAN, A. (1959). Pharmacologic evidence for the existence of a presynaptic event in neuromuscular transmission. J. Phavmacol. Exptl. Therap. 125, 150-158. UMBREIT, W. W., BURRIS, R. H., and STAUFFER, J. F. (1957). Manometric Techniques. Burgess, Minneapolis, Minnesota. LINDGREN,