Therapeutic efficacy of HI-6 in soman-poisoned marmoset monkeys

TOXICOLOGY

AND

APPLIED

Therapeutic

PHARMACOLOGY

115,50-56

( 1992)

Efficacy of HI-6 in Soman-Poisoned

HERMANP. M.

Marmoset

VAN HELDEN, HERMA J. VAN DER WIEL, JAN DE LANGE, BERT P. C. MELCHERS, AND OTTO L. WOLTHUIS

RUUD

Monkeys

W. BUSIER,

Medical Biological Laboratory TNO, P.O. Box 45, 2280 AA Rijswijk, The Netherlands Received September 27, 199 I ; accepted February 18, 1992

and the bispyridinium oxime HI-6. This result is important for two reasons: (i) oxime treatment may be effective after soman poisoning in primates and (ii) very low blood AChE levels in soman-poisoned animals, without any detectable recovery over a period of 4 hours, were encountered. Although this cannot be regarded as decisive evidence of a lack of AChE reactivation or protection by HI-6, it may suggest that the beneficial effects of HI-6 are not limited to AChE reactivation but are also due to some other action of HI-6. Such other oxime effects, henceforth called “direct effects,” have been reported before (Heilbronn and Tolagen, 1965; Oldiges and Schoene, 1970; Schoene et al., 1976; Smith and Muir, 1977; Clement, 1979, 1981; Kuhnen-Clausen et al., 1983; Van Helden et al., 1983) but were thought to be of little therapeutic value (Hobbiger, 1976) because of the fast elimination of HI-6 from the blood in several species including human (Kusic et al., 1985). Recently, organophosphate-poisoned rats were successfully treated with HI-6, without any AChE reactivation in blood, diaphragm or brain (Busker et al., 199 1). The present experiments have two goals: (i) We repeated the survival study of Hamilton and Lundy (1989) in another primate species, the marmoset, in order to give their results a more general bearing. (ii) We closely examined the levels of AChE-inhibition in marmoset monkeys after soman poisoning with or without HI-6 treatment, not only in the blood but also in muscle and brain, to investigate whether HI-6 reactivated AChE or protected the enzyme from inhibition by soman.

Therapeutic Efficacy of HI-6 in Soman-Poisoned Marmoset Monkeys. VAN HELDEN, H. P. M., VAN DER WIEL, H. J., DE LANGE, J., BUSKER, R. W., MELCHERS, B. P. C., AND WOLTHUIS, 0. L. (1992). Toxicol. Appl. Pharmacol. 115, 50-56. The therapeutic efficacy of the oxime HI-6 against intoxication with the irreversible cholinesterase (ChE) inhibitor soman was tested in marmoset monkeys. Five out of six marmosets, intoxicated with 5 x LDSo soman and treated immediately with diazepam (0.2 mg . kg-’ iv) and 15 set later with atropine (0.5 mg - kg-’ im) and HI-6 (50 mg - kg-’ im), survived for more than 24 hr. One of these animals died after 4 days. In the Hfd-treated marmosets blood ChE activity was inhibited at a rate slower than that in three animals treated similarly but with saline instead of HI-6. The latter marmosets died within 8 min after soman. HI-6 achieved its plasma peak 5 min after injection and was eliminated with a t1,2 of about 40 min. In a second experiment similarly treated marmosets were euthanized at 5 min (three saline-treated animals) or at 10 min (three Hid-treated animals) after the soman intoxication to enable the determination of acetylcholinesterase (AChE) activities in diaphragm and brain tissue. In addition, in these animals blood AChE and butyrylcholine esterase (BuChE) activities were determined. Low AChE activities were encountered in diaphragms and brains. These levels were not significantly different between saline- and HI-6-treated marmosets. In vitro treatment with HI-6 at 40 min after soman still led to an increase of the AChE activity, which was significant in diaphragm, suggesting that postmortem AChE inhibition had occurred. The ratio of AChE to BuChE in blood wassignificantly enhanced in Hid-treated animals, indicating that HI-6 preferentially reactivated AChE. It is concluded that (i) HI-6 is an effective treatment against soman poisoning in marmosets and (ii) AChE reactivation or protection by HI-6 contributed to the survival of the animals. 0 1992 Academic press, lnc.

MATERIALS Animals

Once approval was obtained from the Animal Experimentation Committee of TNO, 15 male marmosets (Cal(ilhrixjacchus; 365 + 14.4 g) were used for this study. The 9 animals used for the survival experiment (experiment 1) had been treated once with a single low dose of carbamate (physostigmine =Z0.04 mg. kg-’ im or pyridostigmine =S0.4 mg. kg-’ im) in the course of a behavioral experiment that ended 6 months before the present experiment. These doses of carbamates caused no overt symptoms. In view of the reversible nature of AChE inhibition by these carbamates it was felt that this time interval was sufficient to erase any possible effect of the previous carbamate treatment. The 6 marmosets used in experiment 2 (see below) were experimentally naive.

The treatment of poisoning with the irreversible acetylcholinesterase (AChE) inhibitor soman with oximes, although effective in rodents, is thought to be of little value in primates and man because of the rapid aging of the phosphorylated AChE in these species (Wolthuis et al., 1981). However, Hamilton and Lundy (1989) reported survival of three out of four rhesus monkeys after soman poisoning (5 X LD5,-,) and subsequent treatment with diazepam, atropine, 0041-008X/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

AND METHODS

50

HI-6 IN SOMAN-POISONED Pretreatment Animals received ketamine (8 mg - kg-‘, im) and were subsequently immobilized on a specially constructed cork “bed” at an angle of approximately 60” with the horizontal plane. After that they received another dose of ketamine (3 mg- kg-‘, im). Together, these doses of ketamine induced a light anesthesia of approximately half an hour, used to insert an indwelling cannula into the left femoral vein and to insert loose stitchings, to be used later for closing the small wound at the end of the acute phase of the experiment. Experimental Procedures The whole experimental protocol was identical to that used by Hamilton and Lundy (1989). Two hours after regaining consciousness all animals were im injected with 5 X LDSo soman (43.5 pg. kg’) and immediately treated with diazepam (0.2 mg . kg-’ iv) followed 15 set later by atropine (0.5 mg - kg-‘, im). All im injections were given into the right thigh. The estimated 48-hr im LDjo for soman is 8.7 pg. kg-’ (D’MelIo and Scott, 1986). Experiment 1. Three control animals received an additional im injection with saline and six experimental animals received an additional injection with HI-6 (50 mg. kg-i), in a11cases 15 set after soman. Blood samples (25 ~1) were drawn every 30 set to determine total blood cholinesterase (ChE) activity, starting just before soman was injected. In the animals treated with HI-6, samples of 150-200 ~1 blood were drawn at 5, 10, 15, 30,60,90, and 120 min after soman to determine ChE activities and HI-6 levels in plasma. After the last sample had been taken 1 ml saline was injected iv to compensate for the blood loss, the cannula was removed from the femoral vein, the small wound was closed with the stitches that had been inserted earlier, and the animals were moved to a plexiglass cage for close observation. On the night that followed the animals were monitored by a video camera, Experiment 2. Pretreatment, intoxication, drug treatment schedules, and doses were identical to those in experiment 1. Three control animals received saline and were euthanized at 5 min aher soman. Three experimental animals received HI-6 and were euthanized at 10 min after soman. This difference in time interval was chosen because the results of experiment 1 showed that in the HI&treated animals inhibition of total blood cholinesterase was slower than that in the controls. From the control animals blood samples (25 ~1) were obtained every 30 set, to determine acetylcholinesterase (AChE) and butyrylcholine esterase (BuChE) activities. Three larger blood samples (200 ~1) were taken to keep blood lossthe same as that in the experimental animals (see below). In the H&6-treated experimental animals blood samples (25 ~1) were also drawn at 30-set intervals during the first 5 min and from then on every minute. At I, 2.5, 5, and 10 min after soman the samples were 200 ~1, to enable determination of plasma HI-6 levels also. At the end of each experiment all animals were anesthetized with ether, the thorax was opened, a cannula was inserted through the left ventricle into the aorta, and the blood vesselswere flushed with Krebs-Ringer bicarbonate buffer at a pressure of approximately 100 mm Hg to remove remaining blood that still might contain soman or HI-6. The brain, the medulla oblongata, and a hemidiaphragm were used for the determination of AChE activity. Behavior The animals used in experiment 1 had 6 months previously been trained and subsequently tested on their performance of a two-choice visual discrimination task (N = 3) or a hand-eye coordination task (N = 1) (Wolthuis, 199 I). To get an impression of the condition (degree of incapacitation) of the surviving animals, their performance was tested from 9 days after intoxication. Briefly, to make a correct response in the discrimination test the animal had to choose and pull one of two handles indicated by a light; as a result a window opened and a reward was presented. In the hand-eye coordination test one window was used, it opened after a sound signal and the animal had to retrieve through the window a reward that moved with a speed of 8 cm * set-‘. For each test there was a control animal that had not been included in experiment 1.

MARMOSET

51

MONKEYS

Cholinesterase Determination Blood samples (5 ~1) from control and Hid-treated animals were immediately mixed with 1% saponin (BDH, Poole, England) and frozen in liquid Nz. After appropriate dilution, samples were assayedfor choline&erase activity using a modification of the Ellman method (Ellman et al., 1961). In all casesof low AChE activity a radiometric method was used (Johnson and Russell, 1975). The acetylcholine end concentration used was 12 NM; [3H]acetylcholine iodide (NEN, Dreieich, Germany) was diluted to a specific activity of 602 MBq . mmol-i. For determination of AChE and BuChE sep arately, ethopropazine (2.5 pM; Sigma, St. Louis, MO) and BW284C5 1 (10 pM; Sigma) wem used as specific inhibitors of BuChE and AChE, respectively. AChE from electric eel was used as a reference. To estimate the amount of reactivation and aging of soman-inhibited AChE (experiment 2), HI-6 (400 pM) was immediately added to parts of blood samples from control animals. This concentration was based on maximal plasma levels of HI-6 (150 pg.ml-‘) obtained (see Fig. 3). Brain or diaphragm were weighed subsequently and homogenized in 50 mM Tris-HCI (pH 7.4), 1 M NaCI, 5 mM EDTA and 1% Triton X-100 (l:lO, w/v) (Femandez and Stiles, 1984). In order to investigate possible postmortem cholinesterase inhibition by soman persisting in tissues, some of the organs were homogenized in the same buffer which also contained 0.1 U - ml-’ of electric eel AChE (Sigma), followed by a 30-min incubation (Van Helden et al., 1988). The homogenates were centrifuged for 10 min at 5OOOgand the supematant was collected. In some cases HI-6 (200 pM) was added to parts of the homogenates at 40 min after soman injection. All samples exposed to HI-6 were incubated for 15 min at 37°C. To study the course of inhibition of AChE and BuChE in vitro, 145 nM soman was added to heparinized blood from naive animals and 15 set later HI-6 (200 FM) or saline was added as well. This was incubated at 37°C under stirring, while at given times, 5-~1 ahquots were taken and frozen in liquid N2 for enzyme determination. HPLC Determination of HI-6 For the HI-6 determination the method described by Hamilton and Lundy (1989) was used. To plasma (100 ~1) or to 1:10 homogenates of diaphragm or brain (see under Cholinesterase Determination) 1 vol of 0.04 M trichloroacetic acid was added. After 15 min (O’C) this was centrifuged for 10 min and 20-~1 ahquots of the supematant were injected into a HPLC system. This comprised a Spectroflow 400 pump (ABI, Maarssen, The Netherlands), an ABI 757 absorbance meter, set at 304 nm, and a LDC Milton Roy (Hasselroth, Germany) CI 1OB integrator. The mobile phase contained 0.1 M KH2P0, (pH 4.8), 75 mg sodium octylsulfonate, and 5% (v/v) acetonitrile per liter Millipore-quality water and was pumped at 0.9 ml - min-‘. Compounds Soman (0pinacolyl methylphosphonylfluoridate) was synthesized by Dr. H. P. Benschop of the Prins Maurits Laboratorium TN0 (Rijswijk, The Netherlands). The chemical purity was 98.5%. HI-6 (2-hydroxyiminomethylpyridinium- 1-methyl-4’-carbamoylpyridinium- 1-methyl ether dichloride monohydrate) was made available by Dr. P. A. Lockwood and Dr. J. G. Clement (Defence Research Establishment, Suffield, Canada). HPLC analysis indicated that the purity of HI-6 was >98%. Atropine sulfate was purchased from Brocades Stheeman (Amsterdam, The Netherlands) and diazepam from Hoffman-La Roche (Basel, Switzerland). Statistics Data were analyzed by two-way repeated-measures ANOVA (Winer, 1971). For testing individual differences the Newman-Keuls method was used. Results were considered significant at the p < 0.05 level.

RESULTS

Survival, Animals Condition, and Behavior The control animals died respectively at 6, 6, and 8 min postsoman. One of the HI-6-treated monkeys died 5 min

52

VAN HELDEN

after soman; however, the other five survived. During the first hours after treatment surviving animals were semicomatose and showed tremors and fasciculations, but reacted to sound and touch. The next morning the animals were all conscious and alert. One animal did not regain righting reflexes. It had to be fed by hand, and drinking appeared difficult; fluid was administered by injecting saline subcutaneously. This condition was stationary until the animal died after four nights. The remaining four animals were in an upright position the next morning, but still had difficulty using their hind legs. They greedily ate and drank. Twentyfour hours after intoxication these animals were returned to their home cage in fairly good condition, although climbing and especially jumping were still a bit clumsy during the first 2 days. One animal still occasionally showed some rapid muscle fibrillations, but after a week all four animals were indistinguishable from “normal.” Table 1 shows the performance of the animals in the behavioral tasks, both 6 months before and 9 days after the soman intoxication. Nine days after intoxication their performance was compared with that of nonintoxicated control animals in tasks that they had acquired 6 months before. The few results obtained show that upon repeated testing of the soman-intoxicated animals (B, C, and D), the initially lowered performance in the discrimination test improves again. Performance in the hand-eye coordination test (animal E) seemed little affected by the intoxication. Blood ChE, AChE, and BuChE Blood ChE was rapidly inhibited in control and oximetreated monkeys (Fig. 1). In control animals it decreased to

ET AL.

60

40

20

0 0

1

2

time

3

(mid

4

after

5

soman

6

1015306090120

injection

FIG. 1. Total blood cholinesterase (ChE) activity (means f SE) in marmoset monkeys after poisoning with 5 X LDSo soman. Closed circles show values from three control animals that received therapy consisting of atropine and diazepam; open circles represent values from five animals that received HI-6 in addition. In all blood samples the ChE activity in Hid-treated animals was higher than that in control animals (p < 0.05).

1, 3, and 4% of pretreatment values, respectively, at 6 min after soman. In each blood sample from the HI-6-treated animals this enzyme activity was significantly higher (p < 0.05) than that in samples from control animals. In the oxime-treated group a steady-state inhibition was reached at 10 min postsoman, at which about 5% of ChE activity remained. The AChE activity (experiment 2) in blood from control animals dropped to less than 1% within 2 min after soman injection (Fig. 2A). The AChE activity of the three Hid-treated animals varied widely and was not significantly different from that of controls; in two monkeys AChE-in-

TABLE 1 Performance (Percentage Correct/40 Responses) of a Discrimination or a Hand-Eye Coordination Task by Trained Marmosets Measured on 4 Subsequent Days, 6 Months before Experiment 1 and from Day 9 on after this Experiment % correct/40 responses

Discrimination Day Animal: A

Hand-eye coordination task

task

B

C

D

(B + C + D)/3

E

F

Performance on 4 subsequent days, 6 months before experiment 1

1 2 3 4

53 65 58 58

53 83 63 80

85 65 75 75

50 38 63 65

63 k 62? 67+ 73F

16 19 6 6

93 83 90 90

80 63 68 80

Performance on 4 subsequent days, from Day 9 after experiment I

1 2 3 4

63 35 63 65

40 73 43 63

43 40 58 58

20 28 35 63

34+47+ 45+ 61+

10 19 10 2

75 88 90 78

78 83 88 88

Note. Animals B. C, D, and F were in experiment I; A and E had also acquired these tasks 6 months before but did not participate in the present experiment (controls). The average value (&SD) for animals B, C, and D is also presented.

HI-6 IN SOMAN-POISONED

hibition tended to be less, and in the third marmoset (marmoset 2; not specifically shown) the course of AChE inhibition was similar to that of the controls. The BuChE activities of Hid-treated marmosets and controls were similar (Fig. 2B). The ratio between AChE and BuChE activity of the HI-6-treated animals, however, was significantly higher than that of the control animals (0.5’ < t < 5’; Fig. 2C). The inhibition of AChE and BuChE by soman in blood from naive marmosets was studied in vitro (Fig. 2D). A significant decrease of the AChE-BuChE ratio was observed in absence of HI-6. HI-6 (200 yM), added 15 set after soman, significantly increased this ratio. From Table 2 it can be seen that shortly after soman injection, inhibited blood AChE can still be reactivated to a considerable degree by addition of HI-6. From the data in this table the half-life of aging of soman-inhibited AChE was estimated (using probit transformation) to be approximately 1.5 min. AChE Activity in Diaphragm

and Brain

The mean activities of AChE present in diaphragms and brains from control and HI-6-treated marmosets were low and did not differ significantly (Table 3). Addition of HI-6 (200 PM) to homogenates of diaphragm at 40 min after soman injection resulted for both HI-6- and saline-treated marmosets in a small but significant increase of the AChE activity. In brain homogenates AChE activity increased after

MARMOSET

In

vitro

TABLE 2 Reactivation with HI-6 (200 PM) of Blood AChE from Marmosets Intoxicated with 5 X LD,, Soman

Time after soman (min) 1 3 5

0

(mid

2

time

after

4

(mid

after

6

soman

% “Aged“ ’

83 +4 91 F 2 99 t 2

44 f 6 8+1 3+_1

39 + 8 89 iY I 96 + 1

2

time

10

% Reactivated with HI-6

HI-6 treatment, but not significantly @ = 0.053). As in vitro addition of HI-6 took place approximately 28 half-lives of aging after euthanizing the marmosets, at least part of the in vitro reactivated enzyme must have been inhibited postmortem by still persisting soman. As this phenomenon was perceived only when experiment 2 was almost completed, it could only be investigated in more detail with marmosets 5 and 6 (see Table 3). Indeed, purified electric eel AChE, added to parts of the diaphragms and the brains before homogenization, was substantially inhibited (58-95%).

soman

6

% Inhibited

Note. AChE is expressed as a percentage of the value obtained before soman injection (mean f SE of three marmosets). Immediately after bloodsampling, HI-6 or saline was added to the blood, and the mixture was incubated at 37°C for 15 min. ’ Note that the percentage of “aged” AChE has been calculated as the difference between inhibited and reactivated AChE. The half-life of aging was estimated from these data using probit analysis.

0

time

53

MONKEYS

0

4

(mid

2

time

6

after

4

(mid

10

soman

6

after

6

6

10

soman

FIG. 2. Blood acetylcholinesterase (AChE) (A) and butyrylcholine esterase (BuChE) activity (B) in marmosets after poisoning with 5 X LD,o soman. Closed circles show values from three control animals that received therapy consisting of atropine and diazepam: open circles represent values from three animals that in addition received HI-6 (150 prnol. kg-‘). The ratio between these enzyme activities is expressed in (C). (*. p i 0.05: **, p < 0.0 1: F test on simple main effects). D shows this ratio in blood from naive marmosets following administration of soman (145 nM) and 15 set later HI-6 (200 j.cM) (open circles) or saline (closed circles) (*, p < IO-“: F test on simple main effects).(Means * SE).

54

VAN HELDEN

ET AL.

TABLE 3 AChE Activities (mUa ml-‘) in Diaphragms and Brains Isolated from Marmoset Monkeys Intoxicated with 5

X LDsO Soman (im) and Treated, in Addition to Atropine and Diazepam, with Saline (Control) or HI-6 (50 mg. kg-‘, im)

Saline in vitro Marmoset Saline treated

Diaphragm

1

Electric eel activity

Brain

29

25 17 3

19 17 6

3 5

Mean + SE

14-t4

HI-6 treated

2 4 6

Electric eel activity

Diaphragm

Brain

5

31 22 8

45 22 8

15?6

7 16 8

Mean _+ SE

HI-6 in vitro

20 f 7

4 II 12

42

lo? 3

25

7 23 14

17

9k3

f 11 6 15

18

15 f 5

13*4

Note. Control animals were euthanized at 5 min and Hid-treated animals at 10 min postintoxication. Homogenates of these target organs were treated 40 min after the soman injection with either saline or HI-6 (200 PM). Part of the diaphragms and the brains of marmosets 5 and 6 were homogenized in the presence of 100 mU * ml-’ AChE from electric eel, followed by a 30-min incubation.

HI-6 Pharmacokinetics

DISCUSSION

The results of experiment 1 show that the absorption of HI-6 from the im site was relatively rapid; the highest measured plasma concentration of HI-6 ( 160 pg. ml-‘) was at 5 min (Fig. 3). The small number of observations prevents detailed pharmacokinetic analysis. However, some parameters were calculated, assuming a two-compartment open model (Stemler et al., 199 1): rate constant of the elimination phase, 0.016 * 0.003 min-’ (mean f SE; N = 4); half-time of elimination, 43 + 7 min; area under the curve, 11 f 2.4 tnM . min; total body clearance, 14 + 3 ml . min-’ . kg-‘. Table 4 shows that l-20 pg HI-6 is found per gram diaphragm and 2-3.6 pugper gram brain. The mean plasma levels of HI6 at 2.5, 5, and 10 min postintoxication (experiment 2, Table 4) are not statistically different from those in Fig. 3.

The major aim of the present study was to determine whether the therapeutic efficacy of HI-6 against soman poisoning in rhesus monkeys, as reported by Hamilton and Lundy (1989), could be reproduced in another primate species. The secondary aim was to examine AChE reactivation by HI-6 in these animals. The results indicate that HI-6, in combination with atropine and diazepam, was effective against 5 X LDSo soman in marmoset monkeys and confirm the findings by Hamilton and Lundy (1989). The results further indicate that atropine and diazepam alone (control experiment) are ineffective. This was expected, since Lipp (1972) and Lipp and Dola ( 1980) had shown that this combination protected only up to about 1.5 X LD5,, soman in rhesus monkeys. The data from the

TABLE 4 HI-6 Concentrations in Plasma(/lg. ml-‘) and Homogenates

(pg. g-’ Wet Weight) of Target Organs from Hid-Treated

Mar-

mosets(Experiment 2) Marmoset

T Location

Time (min) postintoxication

Plasma 0) 0

20

40

60 time

80

100

10.0

120

(min)

FIG. 3. Plasma concentrations of HI-6 following im administration of 50 mg. kg-’ in the five marmosets that survived 5 X LD5,, soman following treatment with diazepam, atropine, and HI-6. (Points represent means f SE).

5.0 5.0

Diaphragm Brain Medulla ’ Not determined.

40 40 40

2

4

6

82 97 123

107 140 145

87 116 133

1.0 3.0

a

19.5 3.0 3.6

12.6 2.2 2.1

HI-6 IN SOMAN-POISONED

MARMOSET

MONKEYS

55

behavioral experiment have evidently only a limited value. tabun in primate plasma. Persisting tabun or soman in the Nevertheless they suggest that the four surviving marmosets body could also have masked any in vivo enzyme reactivation were not severely mentally or physically incapacitated. In in their case. our control marmosets the ChE activity (experiment 1) deIn conclusion, HI-6 appears to be an effective therapeutic creased rapidly (within 3 min) following soman injection, against soman poisoning in marmoset monkeys. On the basis but in the Hid-treated animals the course of enzyme inhiof our data we cannot decide whether direct effects of HI-6 bition was delayed. This significant difference in total ChE are involved in the beneficial effects of this agent. However, activity was clearly not caused by a difference in the extent since the data strongly suggest that HI-6 reactivates AChE, of BuChE inhibition (Fig. 2B). Although the individual values it is likely that this AChE increase contributes to the survival of AChE activity suggested reactivation of this enzyme in of the marmosets. two of the three HI-6-treated animals, the large interindividual variation hampered interpretation (Fig. 2A). The ratio REFERENCES between AChE and BuChE activity appeared to be a more sensitive parameter and was significantly different between Busker, R. W., Zijlstra, J. J., Van der Wiel, H. J., Melchers, B. P. C., and Van Helden, H. P. M. (199 1). Otganophosphate poisoning: A method to control and oxime-treated animals, indicating that HI-6 test therapeutic effects of oximes other than acetylcholinesterase reactipreferably reactivated AChE (Fig. 2C). Such an effect was vation in the rat. Toxicology 69, 33 l-344. also found in vitro with blood obtained from naive mar- Clement, J. G. (1979). Pharmacological action of HS-6, an oxime, on the mosets, by adding soman and subsequently HI-6 or saline neuromuscular junction. Eur. J. Pharmacol. 53, 135- 14 I. (Fig. 2D). Apparently, in marmoset blood AChE is more Clement, J. G. (198 1). Toxicology and pharmacology of bispyridinium oxsusceptible to soman than BuChE, because the ratio of these imes-Insight into the mechanisms of action versus soman poisoning in viva. Fundam. Appl. Toxicol. 1, 193-202. enzymes decreased after administration of soman (Fig. 2D). D’Mello, G. D., and Scott, E. A. M. (1986). Toxicity of GB and GD in This was also observed in human blood (Karczmar, 1970). marmosets (Callithrixjacchus). Technical Paper 458. Chemical Defence The increase in the ratio between AChE and BuChE upon Establishment, Porton Down, United Kingdom. addition of HI-6 suggests that AChE is better reactivated G. I., Courtney, K. D., Andres, V., Jr., and Featherstone, R. M. than BuChE. The subsequent decrease (at t > 3 min) in the Ellman, ( 196 1). A new and rapid colorimetric determination of acetylcholinesterase ratio between AChE and BuChE in soman-poisoned and activity. Biochem. Pharmacol. 7, 88-95. Hid-treated marmosets (Fig. 2C) is probably due to rein- Femandez, H. L., and Stiles, J. R. (1984). Intra- versusextracellular recovery hibition of AChE by soman delivered from the body (see of 16s acetylcholinesterase following organophosphate inactivation in the rat. Neurosci. Lett. 49, I 17-22. below; Van Helden et al., 1988). Hamilton, M. G., and Lundy, P. M. (1989). HI-6 therapy of soman and It is not surprising that HI-6 treatment 15 set after soman tabun poisoning in primates and rodents. Arch. Toxicol. 63, 144-149. injection results in reactivation of AChE since we estimated Heilbronn, E., and Tolagen, B. (1965). Toxogonin in sarin, soman and tabun. the half-life of aging of soman-inhibited AChE in marmosets Biochem. Pharmacol 14, 73-78. to be 1.5 min, which is in accordance with the literature Hobbiger, F. (1976). In Neuromuscular Junction. (E. Zaimis, Ed.), p. 553. (Talbot et al., 1988). Springer-Verlag, Berlin. The activity of AChE in target organs such as diaphragm Johnson, C. D., and Russell, R. L. (1975). A rapid, simple radiometric assay and brain is functionally of more importance than that in for cholinesterase, suitable for multiple determinations. Anal. Biochem. blood. In this respect, no significant differences were found 64,229-238. between both treatment groups. However, from earlier ex- Karczmar, A. G. (1970). In International Encyclopedia of Pharmacology and Therapeutics. Sect. 13. Anticholinesterase Agents (A. G. Karczmar, periments with marmosets (Van Helden et al., 1988) it is Ed.), pp. 157- 158. Pergamon Press, Oxford. known that free soman can persist in tissues for several hours Kuhnen-Clausen, P.. Hagedorn, I., Gross, G., Bayer, H., and Huchs, F. postintoxication so that the remaining AChE activity (non(1983). Interactions of bisquatemary pyridine salts (H-oximes) with choinhibited or HI-6 reactivated) may become inhibited upon linergic receptors. Arch. Toxicol. 54, 17 l- 177. homogenization. This probably interfered with our AChE Kusic, R., Boskovic, B., Vojvodic, V., and Jovanovic, D. (1985). HI-6 in determination, because even 40 min after termination of the man: Blood levels, urinary excretion, and tolerance after intramuscular marmosets (i.e., 28 half-lives of aging), we still observed sigadministration of the oxime to healthy volunteers. Fundam. Appl. Toxicol. nificant AChE reactivation, suggesting postmortem inhibi5, S89-S97. tion. Clear evidence for the presence of unbound intact so- Lipp, J. A. (1972). Effect of diazepam upon soman-induced seizure activity and convulsions. Electroencephalogr. Clin. 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