Archlvu of

TOXICOLOGY

Arch. Toxicol. 36, 71-81 (1976)

9 by Springer-Verlag 1976

Therapeutic Effects of HS-3, HS-6, Benaetyzine, and Atropine in Soman Poisoning of Dogs* J. Schenk, W. L6fller, and N. Weger** Federal Armed Forces Medical College, Department of Toxicology and Pharmacology, Infanteriestr. 17, D-8000 Miinchen 40, Federal Republic of Germany

Abstract. Investigations into the therapeutic properties of various combinations of the bispyridinium salts HS-3 and HS-6 and the cholinolytics atropine and benactyzine against soman poisoning in unanesthetized male beagles were performed. In our investigations we observed that: 1. The most effective protection against soman poisoning was attained if both oximes were applied early i.m. 6 min after intoxication together with the cholinolytics. 2. On the basis of clinical symptoms HS-6 proved to have a more intensive therapeutic effect than HS-3 upon early application. 3. If HS-3 was applied early after s.c. intoxication with low concentrations of soman (up to 3 LDs0), a significant protection or reactivation effect on serum cholinesterase was measured. 4. When HS-3 was applied at the beginning of convulsions - generally 28 min after s.c. intoxication - it also raised the rate of surviving animals. 5. The maximal blood levels for HS-3 and HS-6 were measured 2 0 - 3 0 min after i.m. injection; the half-life values of HS-3 and HS-6 in plasma were 45--60 min. Key words: Antidotes - Dogs - Oximes -

Soman.

Zusammenfassung. In Versuchen an nicht narkotisierten, m/innlichen BeagleHunden wurden die therapeutischen Wirkungen verschiedener Kombinationen der Bispyridiniumsalze HS-3 und HS-6 mit den Cholinolytica Atropin und Benactyzin gegen/iber der Somanvergiftung untersucht. Die Untersuchungen erbrachten folgende Ergebnisse: 1. Die beste therapeutische Wirkung war zu erzielen, wenn beide Oxime zusammen mit den Cholinolytica fr/ihzeitig - 6 min nach Vergiftung - verabfolgt wurden. * The results were briefly reported at the 16th Meeting of the Deutsche Pharmakologische Gesellschaft, Mainz, 1975 (Naunyn-Schmieberg's Arch. Pharmacol. 287, R 98) ** To whom offprint requests should be sent

72

J. Schenk et al. 2. HS-6 zeigte gegeniiber HS-3 bei friiher Gabe unter Beriicksichtigung des klinischen Bildes und der Uberlebensrate eine bessere therapeutische Wirkung. 3. HS-3 wies dagegen bei frfiher Gabe bei Vergiftungen mit niedrigen Somankonzentrationen (bis 3 DLs0) einen mel3baren Schutz- oder Reaktivierungseffekt auf die Serumcholinesterase auf. 4. HS-3 erh/Shte auch noch bei sp/iter Gabe zu Krampfbeginn - durchschnittlich 28 min nach Vergiftung - die 1Jberlebensrate. 5. Nach i.m. Applikation wurde fiir HS-3 der maximale Blutspiegel nach 20 rain und eine Halbwertszeit von 45 min, ffir HS-6 der maximale Blutspiegel nach 30 min und eine Halbwertszeit yon 60 min gemessen. Schliisselwiirter: Antidote - Hunde - Oxime - Soman.

Introduction

Nucleophilic agents such as obidoxim (Toxogonin | and TMB-4 have no therapeutic effect on soman poisoning in vivo (Heilbronn and Tolagen, 1965; Boskovi6 and Jovi6, 1969; Boscovi6 and Stern, 1970), whereas 2-PAM is known to have certain antidotal properties in dogs and cats poisoned by soman when administered shortly after intoxication (Fleisher et al., 1967; Jovi6 and Vojvodi6, 1971). In statements of success or failure of antidotal therapy against soman poisoning several authors pointed to species' differences in response to anticholinesterase agents as well as antidotes (Davies et al., 1959; Berry and Davies, 1970; Jovi6 and Vojvodi6, 1971). Studying the antidotal effect of 35 pyridinium and imidazolium salts on atropinized mice poisoned by soman, Oldiges and Schoene (1970) described a protective effect of HS-3 and HS-6 against soman. The purpose of the present study was to establish the therapeutic efficiency of bispyridinium salts HS-3 and HS-6 (Fig. 1) together with the cholinolytics atropine and benactyzine in soman-poisoned dogs. ~ i

OH

CH=NOH

2cte H2"- 0 - CH~-~N~) Toxogonin(~ (Obidoxim)

CH=NOH I

QN'--- CHz - 0--r

N~

HS3

HS6

Fig. 1. Strucural formulas of pyridinium oximes HS-3 and HS-6 comparedto obidoxim

Antidotal Effects in Soman Poisoning

73

T h e effect o f a n t i d o t e c o m b i n a t i o n s w a s e v a l u a t e d o n t h e b a s i s o f clinical s y m p t o m s ( r a t e o f s u r v i v a l , s e r u m C P K , G O T , G P T a c t i v i t y ) a n d t h e effect o n t h e activity of serum cholinesterase taking into account the time interval between the onset of intoxication and the beginning of therapy.

Material and Methods 1. Animals. Unanesthetized, fasted, male beagles, weight 8 - 1 8 kg, were used. 2. Organophosphorus Compound and Antidotes. Soman (pinacolyl-methylphosphonofluoridate, Institute for Aerobiology of the Fraunhofer Gesellschaft, Grafschaft). HS-3 (N,N'-oxydimethylene-bis(pyridinium-2-4-aldoxime)-dichloride) and HS-6 (N,N'-oxydimethylene-bis(pyridinium-2-aldoxime-3carboxamido)-dichloride) (synthesized by Professor I. Hagedorn, Freiburg) (Fig. 1); atropine sulfate (Merck, Darmstadt); benactyzine hydrochloride (Boehringer, Ingelheim). Soman was dissolved in isopropanol weekly to give final concentrations of 0.02--0.2 g/100 ml. The dogs were poisoned subcutaneously with doses of 2.3 x 10-7 mol/kg (about 3 LDs0), 3.85 x 10-7 mol/kg (about 5 LDs0), 6.16 • 10 7 mol/kg (about 8 LD~0), and 7.7 x 10-7 mol/kg (about 10 LD~0). The antidotes were stored in the form of powder and were dissolved into doses of 50 mg HS-3/kg, 100 mg HS-6/kg, 2 mg atropine sulfate/kg, and 2 mg benactyzine hydrochloride/kg in 2 ml of 0.9% of saline solution immediately before use. 3. Determination of Enzymatic Activities. Venous blood was drawn from a femoral vein. Serumcholinesterase, CPK, GOT, and GPT were measured photometrically using the test combinations of Boehringer, Mannheim, the cholinesterase in erythrocytes with the Titrigraph (Radiometer, Copenhagen). 4. Determination of Oxime Concentration in Blood. According to Creasy and Green (1959), the concentration of oximes in blood was determined by measuring the increase in absorbance at 356 nm: 1 ml of blood was mixed with 1 ml 10% trichloracetic acid and centrifuged for 10 min at 30 000 g at 0 ~ C, and 0.1-0.5 ml of the clarified serum was added to 2.4-2.0 ml of 0.01 n NaOH (HS-3 determination) or 0.01 n Na2HPO 4 at pH 8.8 (HS-6 determination). The concentrations of HS-3 and HS-6 in plasma were read from standard curves established for both oximes. 5. Experiments. After s.c. intoxication with soman the antidotes were injected i.m. in the following combinations and at the following times. a) 2 mg atropine/kg and 2 mg benactyzine/kg at the beginning of convulsions, 1 mg atropine/kg 60 rain thereafter. b) 50 mg HS 3/kg, 2 mg atropine/kg, and 2 mg benactyzine/kg at the beginning of convulsions, 25 mg HS-3/kg and 1 mg atropine/kg 60 min after applying the cholinolytics. c) 50 mg HS 3/kg 6 min after intoxication, 2 mg atropine/kg and 2 mg benactyzine/kg at the beginning of convulsions, and 25 mg HS-3/kg and 1 mg atropine/kg 60 min after applying the cholinolytics. d) 100 mg HS-6/kg, 6 min after intoxication, 2 mg atropine/kg and 2 mg benactyzine/kg at the beginning of convulsions, 1 mg atropine/kg 60 min after applying the cholinolytics. e) 100 mg HS-6/kg, 50 mg HS-3/kg, 2 mg atropine/kg, and 2 mg benactyzine/kg 6 min after intoxication, 25 mg HS-3/kg, and 1 mg atropine/kg 60 min thereafter. 6. Statistical comparisons were made during analysis of variance.

Results 1. C l i n i c a l C o u r s e T h e clinical s y m p t o m s a n d t h e p h y s i c a l signs o f t h e d o g s p o i s o n e d b y s o m a n depended on the dose of the poison, the selection of the antidotes, and the time of administration of the antidotes.

74

J. Schenk et al.

Characteristic for poisoning by organophosphates are tonic-clonic cramps of the striated muscle. Following s.c. intoxication by a 3-, 5-, 8-fold LDso of soman without antidote administration (therapy schedule A/B) a mean interval of 28 min elapsed before convulsions were detectable; after poisoning with a 10-fold LDso of soman, convulsions started after a latency period of 16 min. The convulsions were accompanied by restlessness, ensuing exhaustion, and dyspnoea. The early application of HS-3, only 6 min after administration of the poison (therapy schedule C), had a distinct influence on the time of onset of the tonic-clonic cramps. As a consequence of poisoning with a 3-fold LDs0 of soman, convulsions ensued after a mean time of 52 min and the animals recovered quickly. Higher doses (5- and 8-fold LDs0) of soman caused convulsions after 18 and 15 min respectively and were followed by respiratory insufficiency and general physical exhaustion. The administration of HS-6, 6 min after experimental poisoning (therapy schedule D), however, was in no case followed by a prolongation of the latent period between the poisoning and onset of convulsions. In this group poisoning with a 3and 5-fold LDso of soman resulted in convulsions after a mean time of 18 min. Despite an initial phase of exhaustion and marked respiratory insufficiency the animals recovered quickly. After poisoning with an 8-fold LDso of soman, convulsions ensued after 11.5 min (mean time) and were soon followed by increasing exhaustion and respiratory insufficiency. Experimental poisoning with a 10-fold LDs0 of soman followed by early administration of both oximes and cholinolytics 6 min after poisoning (therapy schedule E) caused no (two animals) or barely detectable convulsions (four animals). After treatment, signs of moderate physical exhaustion and respiratory distress subsisted.

2. Survival Rate If the survival rate is considered as an efficiency criterion the result of treatment with antidotes of dogs poisoned by soman depends on the selection and time of administration of the antidotes. The LD50 of soman could be increased about 3-fold by the sole application of the cholinolytics atropine and benactyzine during the onset of muscular cramps (therapy schedule A). The concomitant administration of HS-3 (therapy schedule B) or the early administration of HS-6 before cholinolytic treatment (therapy schedule D) further expanded LDs0 of soman 5 - 8 times. The early administration of HS-3 6 min after intoxication (therapy schedule C) lowered the lethality of dogs poisoned with a 5-fold LD~0 of soman only during the first 24 h. Nevertheless, if both oximes and cholinolytics are administered as early as 6 min after poisoning (therapy schedule E) the LDs0 of soman can be increased at least 10fold (Table 1).

3. Activity of Soman-Inhibited Cholinesterase in Serum and in Erythroeytes in the Presence of Antidotes Only if administered 6 min after poisoning with 2.3 x 10-7 mol of soman/kg s.c. (3 LDso) did HS-3 alter the activity of serum cholinesterase (therapy schedule C); the decrease in enzyme activity 16-66 min after intoxication was significantly differ-

Antidotal Effects in Soman Poisoning

75

Table 1. Protection of dogs against s.c. poisoning with soman by treatment with various combinations of cholinolytics and oximes LD50 Soman

3x

5x

lOx

8x

val Rate Id

7d

Id

7d

ld

7d

5/6

5/6

416

316

216

0/5

0/5

6/6

6/6

5/6

216

316

116

5/5

515

6/6

516

114

0/4 5/6

4/6

Id

7d

a/6

216

5/6

Antidotes: HS-3 50 mg/kg; HS-6 100 mg/kg; Atropine 2 mg/kg; Benactyzine 2 mg/kg Antidote combinations: A Administration of atropine and benactyzine at beginning of convulsions;B Administration of HS-3, atropine and benactyzine at beginningof convulsions; C Administration of HS-3, 6 rain after intoxication, atropine and benactyzine at beginning of convulsions;D Administration of HS-3, 6 min after intoxication, atropine and benactyzine at beginning of convulsions; E Administration of HS-3, HS-6, atropine and benactyzine 6 min after intoxication

ent (P < 0.05) from that in all other groups. The cholinesterase activity decreased about 20% from the original value in about 2 h. This positive influence of HS-3 on cholinesterase activity was not found at higher doses of soman. In this and in all the other groups of soman-poisoned dogs serum cholinesterase activity decreased to 6 - 1 9 % in the first 36 min and to 4 - 1 4 % after 66 min. After 24 h the activity of serum cholinesterase in the surviving dogs increased to 34 + 9%, after 48 h to 49 + 13%, and after 152 h to 71 + 13%. Recovery was independent of the particular scheme of antidotal therapy (Table 2, Fig. 2). Cholinesterase in erythrocytes decreased independent of the dose of soman or of the therapy schedule during 5 - 1 1 min until zero, and remained 2 - 3 weeks in nonmeasurable concentrations. After 2 - 3 weeks a small increase in enzyme concentration was measured (not more than 20%) until 6 - 8 weeks after poisoning.

4. Serum Enzymes: CPK, GOT, and G P T The activity of serum enzymes C PK, GOT, and GPT were determined in order to see the reactions on liver or muscle. Figure 3 shows the concentrations of serum enzymes after poisoning with soman and the antidote therapy 4 - 2 4 h after soman intoxication. GOT increased 4 times the original values. 2 h after intoxication, C PK activity increased, about 4 times the original values, but 4 and 24 h after intoxication the CPK activity was 14 times that of the original values, decreasing again in 48 h to about twice the original values.

76

J. Schenk

e t al.

. ,0- ,

~,~

~

~

~

to

to

to

I ~

-.

J

_

I

,~

J [:

~

~

~

to

to

. . . . . . . t~

to

~

I ~x

to

i

~

I

+

+

i

i

i

i

i

,

j 1 0

I

] 8

c

co

eo

r

I

I

to ~

j

l

'