ArchToxicol(1992) 66:629-632
Archives ot
Toxicology 9 Springer-Vedag1992
Protection of guinea pigs against soman poisoning with ferrocene carbamate Nils Karlsson, Roland Larsson, and Gertrud Puu NationalDefenceResearchEstablishment, DepartmentofNBC Defence, S-901 82 Umeh, Sweden Received16 March 1992/Accepted22 July 1992
Abstract. The protective effect of ferrocene carbamate pretreatment against soman poisoning was studied in guinea pigs. At doses corresponding to 1 / 2 0 x a n d 1/10 x LDso of this carbamate a 20% and 45% decrease of the acetylcholinesterase in blood and brain, respectively, was obtained. In combination with additional pretreatment, diazepam, and therapy, HI-6 and atropine, the protective ratios (LD50 of soman in treated animals/LD50 of soman in untreated animals) were around 20 and 40, respectively. Animals pretreated with the high dose of the ferrocene carbamate that survived 10 x and 15 x LDsos of soman showed no remaining signs of poisoning after 24 h. Thus, the ferrocene carbamate afforded a better protection against soman than physostigmine. The explanation for this could be due to the properties of the ferrocene carbamate, not correlated to its cholinesterase inhibiting activity. This hypothesis is discussed. Key words: Ferrocene carbamate - Physostigmine Soman - Pretreatment - Guinea pigs
I~roducfion The idea to prevent intoxication caused by acetylcholinesterase inhibitors of organophosphorus type by administration of a "reversible inhibitor" of the enzyme in advance is almost as old as the organophosphates themselves. Koster, as early as 1946 (Koster 1946), showed that pretreatment of animals with physostigmine - at that time considered as a reversible cholinesterase inhibitor - gave protection against diisopropylfluorophosphate. Besides atropine, several other substances, including oximes were developed and tested for therapeutic countermeasures. As a
Correspondence to: N. Karlsson
result, atropine and an oxime was included in the antidotal arsenal of armed forces. Soman (1,2,2-trimethylpropyl methylphosphonofluoridate) is a nerve agent against which the oxime/atropine therapy proved less efficient. It was in the search for more effective protection against this nerve agent that carbamates such as pyridostigmine and physostigmine got a renewed interest. It has been shown very convincingly that the administration of a carbamate to give about 30% inhibition of blood cholinesterase results, especially when combined with oxime/atropine therapy, in good protection with no side-effects (Gall 1981). The positive and relatively long-lasting effect of these earbamates can be attributed to the fact that carbamates actually are irreversible inhibitors, and the half-life of the carbamylated enzyme is rather short. Pyridostigmine is nowadays a standard pretreatment regimen in several armed forces. The action of pyridostigmine is restricted to the peripheral nervous system. It could be advantageous to use a carbamate which penetrates the blood-brain barrier and thus prevents nerve agent-induced CNS injuries. On the other hand, a centrally active carbamate per se might give unacceptable mental side effects. One way to circumvent that problem could be to use a combination of physostigmine and aprophen (Leadbeater et al. 1985). Some years ago we investigated, as an alternative to physostigmine, a ferrocene-containing carbamate, which gave promising results as regards protection in mice against lethal doses of soman (Karlsson et al. 1984). The ferrocene carbamate has the advantage of being much less toxic than physostigmine and at least as good a blood-brain barrier penetrant. We have now studied protection in soman-intoxicated guinea-pigs, pretreated with the ferrocene carbamate and physostigmine, respectively, and which were also given supportive treatment with diazepam, HI-6 and atropine.
630
Materials and methods Animals Male guinea pigs (Duncan-Hartley, 290___40 g body weight, around 25 days of age) were purchased from Sahlins F6rs~Sksdjurfarm, Malm6, Sweden. The animals were acclimatized in the animal department for at least 5 days prior to the experiments and had free access to tap water and feed (Ewos AB, SOdertNje, Sweden).
Chemicals Atropine sulphate was from Apoteksbolaget, Stockholm, Sweden and diazepam (DiazemulsR, batch no. 56916-52) was a gift from Kabi Vitrum, Stockholm, Sweden). HI-6 (1-(2-hydroxyiminomethylpyridinium)1-(4-carboxyimidopyridinium)dimethylether dichloride) was a gift from Dr. J Clement, DRES, Canada. Soman (1,2,2-trimethylpropyl methylphosphonofluoridate) and N-(ferrocenylmetyliden)-4-(N'-methyl-carbamoyloxy)-aniline (ferrocene carbamate no. 8, Karlsson et al. 1984) were synthesized at the Division of Chemistry at this Establishment. The latter compound was synthesized according to Hetnarski et al. (1980). Physostigmine sulphate, acetylthiocholine iodide and 4,4'-dithiodipyridine were all from Sigma Chemical Co., St Louis, Missouri.
25 min. The animals were decapitated at 30 rain and the cerebrums were dissected. Blood and brain were treated and analyzed for ChE activity as described.
Protection experiments. Animals were pretreated with ferrocene carbamate (I0 or 20 mg/kg body weight) or physostigmine (0.16 mg/kg body weight) alone (30 rain before soman), or a combination of ferrocene carbamate or physostigmine and diazepmn (2 mg/kg body weight, 60 min before soman). These pretreatments were always combined with therapeutic treatment with HI-6 (50 mg/kg body weight) and atropine (15 mg&g body weight), which was given 1 min after soman administration. The doses of physostigmine, HI-6 and atropine were selected from the literature for the sake of comparison of our results with the results of others in their studies of the protection against soman poisoning in guinea pigs (Berry and Davies 1970; Gordon et al. 1978; Inns and Leadbeater 1982). The LDs0 was calculated as above. The protective effect of the different treatments is expressed as the protective ratio, which is the ratio between the LDs0 in treated animals and the LDso in untreated animals. The combination of different treatments are shown in Table 1. Each animal was carefully observed for signs of anticholinesterase poisoning, i.e. motor incoordination, tremor, convulsion, fasciculation, salivation or lachrymation. The time for onset of any of these signs were noted. The time of death was assessed according to Larsson (1988).
Results Solutions
Acute toxicity of the ferrocene carbamate
Atropine, HI-6, physostigmine and soman were dissolved in distilled water, while ferrocene carbamate was dissolved in glycerol formal (Fluka AG, Buchs, Switzerland). Diazemuls was diluted in Diazemuls placebo (Kabi Vitrum, batch no. 1626-002B).
T h e 24 h LD50 o f the f e r r o c e n e c a r b a m a t e w a s f o u n d to be 201 m g / k g ( 1 6 8 - 2 4 2 m g / k g , 9 5 % c o n f i d e n c e interval). S i g n s o f p o i s o n i n g a p p e a r e d g e n e r a l l y w i t h i n 1 0 - 1 5 rain. T h e s e s i g n s c o n s i s t e d o f i n c o o r d i n a t i o n , t r e m o r , salivation and, at the h i g h e s t d o s e s , l a c h r y m a t i o n . A p a r t f r o m lachrym a t i o n t h e s e s i g n s a p p e a r e d s i m u l t a n e o u s l y . I n surviving a n i m a l s n o s i g n s o f p o i s o n i n g w e r e s e e n 24 h after administration.
Administration Diazepam (2 ml/kg body weight), physostigmine and ferrocene carbamate (1 ml/kg) were given intraperitoneally (i. p.). Soman (0.5 ml/kg) was given subcutaneously (s. c.) in the scruff of the neck and atropine and HI-6 intramuscularly (i. m.) in the same solution (1 ml/kg).
Cholinesterase assays in blood and brain tissue The enzyme activity was measured by the method described by Augustinsson et al. (1976), using 1 mM acetylthiocholine as substrate, at 22 ~C. Blood was obtained from the saphenous vein. A 50 lal aliquot was added to 950 lal of 0.1% (w/v) ice-cold Triton X-100. Enzyme activities were measured immediately in duplicate or triplicate. Cerebrum was rinsed in ice-cold water and homogenized with a Potter-Elvehjem homogenizer in 10 vol 0.1 M sodium phosphate buffer, pH 7.4, containing 0.1% (w/v) Triton X-100. Enzyme activities were measured as described for blood.
Experimental design Acute toxicity of the ferrocene carbamate. Ferrocene carbamate was given to four groups of guinea pigs with four animals per group. The animals were observed for 24 h and the LDso was calculated according to Weil (1952).
Cholinesterase activities. A blood sample was collected from each guinea pig (four animals per group) immediately before the administration of the carbamate and served as control (100% activity). The guinea pigs were treated with ferrocene carbamate (10 or 20 mg/kg i.p.) or physostigmine (0.16 mg/kg). A second blood sample was taken at
Table 1. Protective effect of pretreatment with ferrocene carbamate or physostigmine and diazepam in combination with HI-6 and atropine therapy against soman poisoning in guinea pig Treatment None
LD50 of soman (lag&g) 22 (18-
Protective ratio
27)
1
Ther
101 (74- 138)
5
Diaz + Ther
115 (75- 175)
5
FClow + Ther Diaz + FClow+ Ther
249 (203- 305) 413 (310- 551)
11 19
FChigh + Ther Diaz + FChigh + Ther
413 (310- 551) 716 (173-3343)" 930 (668 - 1294) a
19 35 42
Physo + Ther Diaz + Physo + Ther
197 (129- 300) 374 (274- 509)
9 17
Ther = HI-6, 50 mg/kg and atropine, 15 mg/kg, i. m. 1 min after soman Diaz = diazepam, 2 mg/kg, i.p., 60 min before soman FClow = ferrocene carbarnate, 10 mg/kg, i.p., 30 min before soman FChigh = ferrocene carbamate, 20 mg/kg, i.p., 30 rain before soman Physo = physostigmine sulphate, 0.16 mg/kg, i.p., 30 min before soman Soman was given s. c. a Two separate experiments
631 Table2. Inhibition of cholinesterasein blood and brain after administra-
tionof ferrocene-carbamateand physostigmine Blood (25 min)a
Brain (30 rain)a
Ferrocene-carbamate 10mg/kg,i.p. Ferrocene-carbamate 20mg/kg, i.p.
25.1___ 8.1
17.2+ 4.0
48.9 -+- 7.9
43.9-4- 6.0
Physostigminesulphate 0.16mg/kg, i.p.
64.5 -+-10.0
36.0 + 12.3
Thedataare given as mean values of percentageinhibition, with standard deviations,4 animals in each group. The blood values were calculatedin relationto blood sample taken from the same animal before carbamate administration,while the brain values are in relation to the ones obtained fromthree control animals a 25 and 30 min is the time elapsed between administration and withdrawalof blood and decapitation,respectively
Cholinesterase activities in blood and brain tissue In preliminary experiments it was found that, independent of the dose of ferrocene carbamate, maximum inhibition of ChE activity in the blood occurred around 30 min after administration (data not shown). From these experiments two doses of the ferrocene carbamate were chosen for later experiments, namely 10 and 20 mg/kg body weight, corresponding to 1/20 and 1/10 of the 24 h LD50 of the compound. We also compared the inhibition of cholinesterase caused by the ferrocene carbamate with that caused by physostigmine. As shown in Table 2, the degree of inhibition of the ChE activity with the ferrocene carbamate is roughly the same in blood and brain tissue, while with physostigmine this inhibition is significantly higher in the blood.
Clinical signs in soman treated animals All animals receiving doses of soman higher than 1 x LDs0 showed signs of cholinergic overstimulation, i.e. at least one of the following symptoms: chewing, tremor, incoordination, excess salivation, urination, defecation, convulsions, prostration, etc. The time till appearance of signs of poisoning was inversely proportional to the dose of soman. At doses of 1.5 x and 2 x LDs0, symptoms occurred within 1 0 - 2 0 min, when no treatment was given (i. e. in controls). At doses above 4.5 x LD50, symptoms generally appeared within 2 - 3 min, irrespective of supportive treatment. At doses above 10xLD50, signs of poisoning were seen within 1 min or less. In animals treated with diazepam in combination with therapy, clinical signs of poisoning occurred earlier than in animals given therapy only, at comparable doses of soman. This finding was unexpected, and to the best of our knowledge similar observations have not been reported in the literature. In animals pretreated with ferrocene carbamate the severity of the symptoms observed was noticeably lower than in other animals. In accordance with the findings of Gordon et al. (1978) the signs of poisoning were much less pronounced by 2 h. In six out of the nine experimental groups there was a tendency to an inverse relation between the dose of soman and the time until the animal died. It was also observed that the addition of diazepam resulted in a prolonged survival time after poisoning with soman. A similar effect of the carbamates could not be seen in this study, because the doses of soman were much higher than in comparable control groups. Surviving animals in the control group and animals given therapy showed no signs of poisoning 24 h after the administration of soman (doses up to 3 x LD50). All animals pretreated with the high dose of the ferrocene carbamate, diazepam and given therapy, that survived I0 x and 15 x LD50 of soman, did not show evident signs of poisoning at the end of the observation period. With the low dose of ferrocene carbamate or with physostigmine, most animals showed some remaining sign of poisoning.
The protective effect of ferrocene carbamate and supportive treatment against soman The protection against soman poisoning afforded by the different treatments is shown in Table 1. The pretreatment was always combined with therapy, consisting of HI-6 and atropine. The protective ratio for the HI-6/atropine treatment was around five. The addition o f diazepam as a pretreatment 60 min before soman did not improve the protective effect of HI-6/atropine. However, the combination ofpretreatment with a carbamate 30 rain before soman and therapy with HI-6/atropine significantly increased the protective effect and the protective ratio obtained was 9 for physostigmine and 11 and 19 for the two doses of ferrocene carbamate. When diazepam was added as a pretreatment to the carbamates and HI-6/atropine a further significant increase of the protective ratio was obtained, namely 17 for physostigmine and 19 for the low dose (10 mg/kg) of the ferrocene carbamate. With the high dose of the ferrocene carbamate the protective ratio was found to be 35 and 42, respectively, in two separate experiments.
Discussion
The main purpose of the present investigation was to assess the efficacy of a ferrocene carbamate as pretreatment against organophosphates. This type of compound has been shown to pass the blood brain barrier in mice (Karlsson et al. 1984). As shown in Table 2, this holds true also in guinea pigs. A dose-related inhibition of the acetylcholinesterase activity in blood and brain tissue was found with roughly the same degree of inhibition in blood and brain. This was, however, not the case with the tertiary carbamate physostigmine. As shown in the table, the degree of inhibition with this carbamate was two times lower in the brain than in the blood. The two doses of the ferrocene carbamate (1/20 and 1/10 of the LD50 dose of the compound) did not induce any clinical sign of poisoning in the animals and they were therefore considered as "sign free" doses.
632 In a study of the protective effect of physostigmine against soman poisoning in guinea pigs, Berry and Davies (1970) and Lennox et al. (1985) found that the protection correlated with the dose of the carbamate. This was also found to be the case in the present study with the ferrocene carbamate. Thus the lower dose of the compound gave a protective ratio of 19, while this value was 35 and 42 with the higher dose (Table 1). It is interesting to note that in spite of a significantly lower degree of inhibition of acetylcholinesterase, both in blood and brain tissue, the low dose of the ferrocene carbamate gave as good protection as that obtained with physostigmine. When the degree of enzyme inhibition was in the same order of magnitude the ferroeene carbamate afforded a better protection than physostigmine. In contrast to our data and the data cited above, Gordon et al. (1978) found that the dose of carbamate is not critical for the protection of guinea pigs against soman lethality. Carbamates differ in potency in their protective action against organophosphates (see, e.g. Berry and Davies 1970). Thus, both pyridostigmine and physostigmine provide good protection of peripheral cholinesterase against inhibition by soman. In addition to that, physostigmine gives protection of brain cholinesterase, since this tertiary carbamate penetrates the blood-brain barrier. Harris et al. (1984) showed that rats pretreated with physostigmine recovered more rapidly from the incapacitating effect of soman than did animals pretreated with pyridostigmine, although both carbamates provided equivalent protection against the lethal effects of soman. Similar findings were reported by Leadbeater et al. (1985). Our finding that animals surviving doses of 10 x and 15 x LDs0 of soman did not show signs of poisoning is probably due to a central action of the ferrocene carbamate. The same most probably also goes for the finding of a prolonged survival time for rats pretreated with physostigmine before the administration of DFP (Husain et al. 1990). Green (1983) has, in an excellent theoretical analysis, been able to correlate m a n y of the observations made in animal experiments to some simple kinetic parameters. He identifies two important factors for the protection. One is the "minimal essential cholinesterase" necessary for survival, which is low ( 0 . 1 - 1 0 % ) . The other is the ratio between the rate constant for decarbamylation (k2) and the rate of inhibition by the organophosphorous compound (i. e. the product of the inhibition constant and the concentration of the agent), a ratio called "enzyme conservation index". The best protection is obtained when the first factor is low and the second one is high. W e find that the "enzyme conservation index" should be the same for the two carbamates used in the present study, as both are monomethylcarbamates, thus giving the same k2. There was a difference in cholinesterase inhibition, caused by the carbamates, before soman challenge, but this difference is of minor importance for protection, as judged from Green's calculations. W e would rather attribute the more efficient protection from ferrocene carbamate pretreatment, as compared to physostigmine pretreatment, to the first-mentioned critical factor, i.e. " m i n i m u m essential cholinester-
ase". As Green points out, this can be lowered by the administration of anticholinergic drugs. It is well known that carbamates have direct effects on cholinergic receptors. These effects might be of much greater importance with the ferrocene carbamate than with physostigmine. This hypothesis finds some support in our earlier experiments (Karlsson et al. 1984), in which we showed that mice could be protected against 5 - 6 • doses of soman, when pretreated with only ferrocene carbamate and atropine. In summary, the ferrocene carbamate used in the present study was inferior to physostigmine as a cholinesterase inhibitor. In spite of this, the former compound gave better protection against soman. W e suggest that this might be explained by properties not related to the cholinesterase inhibiting activity, e. g. direct receptor effects.
References Augustinsson K-B, Eriksson H, Faijersson Y (1978) A new approachto determining cholinesterase activities in samples of whole blood.Clin Chim Acta 89" 239-252 Berry WK, Davies DR (1970) The use of carbamates and atropinein the protection of animals against poisoning by 1,2,2-trimethylpropyl methylphosphonofluoridate.Biochem Pharmacol 19:927 - 934 Gall D (1981) The use of therapeutic mixtures in the treatment of cholinesterase inhibition. Fundam Appl Toxicol 1: 214- 216 Gordon JJ, Leadbeater L, Maidment MP (1978) The protection of animals against organophosphatepoisoning by pretreatment with a carbamate. Toxicol Appl Pharmaco143: 207-216 Green AL (1983) A theoretical kinetic analysis of the protective action exerted by eserine and other carbamate anticholinesterases against poisoning by organophosphorus compounds. Biochem Pharmac01 32: 1717-1722 Harris LW, McDonough Jr JH, Stitcher DL (1984) Protection against both lethal and behavioural effects of soman. Drug Chem Toxicol7: 605 - 624 Hetnarski B, Lajhta A, Wisniewski HM (1980) On some derivativesof ferrocene, novel acetylcholinesterase inhibitors. J Neurosci Res 5: 1-5 Husain R, VijayaraghavanR, Marjit DN (1990) The effect of pyridostigmine and physostigmine on acute toxicity of diisopropyl fluor0phosphate in rats. Arh Hig Rada Toksiko141:19-24 Inns RH, Leadbeater L (1983) The efficacy of bispyridinium derivatives in the treatment of organophosphonate poisoning in the guinea-pig. J Pharm Pharmcol 35:427-433 Karlsson N, Larsson R, Puu G (1984) Ferrocene-carbamate as prophylaxis against soman poisoning. Fundam Appl Toxicol 4: S 184-S 189 Koster R (1946) Synergism and antagonism between physostigmineand di-isopropyl fluorophosphate in cats. J Pharmacol Exp Ther 88: 39-46 Larsson R (1988) Andningsregistrering - en metod frr 6vervakningav sm~tdjur rid toxictetstester (Registration of respiration - of method for the surveillanceof small animals in toxicity studies). Scand-LAS, Nordic Symposiumon Laboratory Animal Science, Stockholm,Sweden Leadbeater L, Inns RH, Rylands JM (1985) Treatment of poisoningby soman. Fundam Appl Toxicol 5:$225 - $231 Lennox WJ, Harris LW, Talbot BG, Anderson DR (1985) Relationship between reversible acetylcholinesterase inhibition and efficacy against soman lethality. Life Sci 37:793-798 Weil CS (1950) Tables for convenient calculation of median-effective dose (LDso or EDso) and instruction in their use. Biometrics 8: 249-263
Archives ot
Toxicology 9 Springer-Vedag1992
Protection of guinea pigs against soman poisoning with ferrocene carbamate Nils Karlsson, Roland Larsson, and Gertrud Puu NationalDefenceResearchEstablishment, DepartmentofNBC Defence, S-901 82 Umeh, Sweden Received16 March 1992/Accepted22 July 1992
Abstract. The protective effect of ferrocene carbamate pretreatment against soman poisoning was studied in guinea pigs. At doses corresponding to 1 / 2 0 x a n d 1/10 x LDso of this carbamate a 20% and 45% decrease of the acetylcholinesterase in blood and brain, respectively, was obtained. In combination with additional pretreatment, diazepam, and therapy, HI-6 and atropine, the protective ratios (LD50 of soman in treated animals/LD50 of soman in untreated animals) were around 20 and 40, respectively. Animals pretreated with the high dose of the ferrocene carbamate that survived 10 x and 15 x LDsos of soman showed no remaining signs of poisoning after 24 h. Thus, the ferrocene carbamate afforded a better protection against soman than physostigmine. The explanation for this could be due to the properties of the ferrocene carbamate, not correlated to its cholinesterase inhibiting activity. This hypothesis is discussed. Key words: Ferrocene carbamate - Physostigmine Soman - Pretreatment - Guinea pigs
I~roducfion The idea to prevent intoxication caused by acetylcholinesterase inhibitors of organophosphorus type by administration of a "reversible inhibitor" of the enzyme in advance is almost as old as the organophosphates themselves. Koster, as early as 1946 (Koster 1946), showed that pretreatment of animals with physostigmine - at that time considered as a reversible cholinesterase inhibitor - gave protection against diisopropylfluorophosphate. Besides atropine, several other substances, including oximes were developed and tested for therapeutic countermeasures. As a
Correspondence to: N. Karlsson
result, atropine and an oxime was included in the antidotal arsenal of armed forces. Soman (1,2,2-trimethylpropyl methylphosphonofluoridate) is a nerve agent against which the oxime/atropine therapy proved less efficient. It was in the search for more effective protection against this nerve agent that carbamates such as pyridostigmine and physostigmine got a renewed interest. It has been shown very convincingly that the administration of a carbamate to give about 30% inhibition of blood cholinesterase results, especially when combined with oxime/atropine therapy, in good protection with no side-effects (Gall 1981). The positive and relatively long-lasting effect of these earbamates can be attributed to the fact that carbamates actually are irreversible inhibitors, and the half-life of the carbamylated enzyme is rather short. Pyridostigmine is nowadays a standard pretreatment regimen in several armed forces. The action of pyridostigmine is restricted to the peripheral nervous system. It could be advantageous to use a carbamate which penetrates the blood-brain barrier and thus prevents nerve agent-induced CNS injuries. On the other hand, a centrally active carbamate per se might give unacceptable mental side effects. One way to circumvent that problem could be to use a combination of physostigmine and aprophen (Leadbeater et al. 1985). Some years ago we investigated, as an alternative to physostigmine, a ferrocene-containing carbamate, which gave promising results as regards protection in mice against lethal doses of soman (Karlsson et al. 1984). The ferrocene carbamate has the advantage of being much less toxic than physostigmine and at least as good a blood-brain barrier penetrant. We have now studied protection in soman-intoxicated guinea-pigs, pretreated with the ferrocene carbamate and physostigmine, respectively, and which were also given supportive treatment with diazepam, HI-6 and atropine.
630
Materials and methods Animals Male guinea pigs (Duncan-Hartley, 290___40 g body weight, around 25 days of age) were purchased from Sahlins F6rs~Sksdjurfarm, Malm6, Sweden. The animals were acclimatized in the animal department for at least 5 days prior to the experiments and had free access to tap water and feed (Ewos AB, SOdertNje, Sweden).
Chemicals Atropine sulphate was from Apoteksbolaget, Stockholm, Sweden and diazepam (DiazemulsR, batch no. 56916-52) was a gift from Kabi Vitrum, Stockholm, Sweden). HI-6 (1-(2-hydroxyiminomethylpyridinium)1-(4-carboxyimidopyridinium)dimethylether dichloride) was a gift from Dr. J Clement, DRES, Canada. Soman (1,2,2-trimethylpropyl methylphosphonofluoridate) and N-(ferrocenylmetyliden)-4-(N'-methyl-carbamoyloxy)-aniline (ferrocene carbamate no. 8, Karlsson et al. 1984) were synthesized at the Division of Chemistry at this Establishment. The latter compound was synthesized according to Hetnarski et al. (1980). Physostigmine sulphate, acetylthiocholine iodide and 4,4'-dithiodipyridine were all from Sigma Chemical Co., St Louis, Missouri.
25 min. The animals were decapitated at 30 rain and the cerebrums were dissected. Blood and brain were treated and analyzed for ChE activity as described.
Protection experiments. Animals were pretreated with ferrocene carbamate (I0 or 20 mg/kg body weight) or physostigmine (0.16 mg/kg body weight) alone (30 rain before soman), or a combination of ferrocene carbamate or physostigmine and diazepmn (2 mg/kg body weight, 60 min before soman). These pretreatments were always combined with therapeutic treatment with HI-6 (50 mg/kg body weight) and atropine (15 mg&g body weight), which was given 1 min after soman administration. The doses of physostigmine, HI-6 and atropine were selected from the literature for the sake of comparison of our results with the results of others in their studies of the protection against soman poisoning in guinea pigs (Berry and Davies 1970; Gordon et al. 1978; Inns and Leadbeater 1982). The LDs0 was calculated as above. The protective effect of the different treatments is expressed as the protective ratio, which is the ratio between the LDs0 in treated animals and the LDso in untreated animals. The combination of different treatments are shown in Table 1. Each animal was carefully observed for signs of anticholinesterase poisoning, i.e. motor incoordination, tremor, convulsion, fasciculation, salivation or lachrymation. The time for onset of any of these signs were noted. The time of death was assessed according to Larsson (1988).
Results Solutions
Acute toxicity of the ferrocene carbamate
Atropine, HI-6, physostigmine and soman were dissolved in distilled water, while ferrocene carbamate was dissolved in glycerol formal (Fluka AG, Buchs, Switzerland). Diazemuls was diluted in Diazemuls placebo (Kabi Vitrum, batch no. 1626-002B).
T h e 24 h LD50 o f the f e r r o c e n e c a r b a m a t e w a s f o u n d to be 201 m g / k g ( 1 6 8 - 2 4 2 m g / k g , 9 5 % c o n f i d e n c e interval). S i g n s o f p o i s o n i n g a p p e a r e d g e n e r a l l y w i t h i n 1 0 - 1 5 rain. T h e s e s i g n s c o n s i s t e d o f i n c o o r d i n a t i o n , t r e m o r , salivation and, at the h i g h e s t d o s e s , l a c h r y m a t i o n . A p a r t f r o m lachrym a t i o n t h e s e s i g n s a p p e a r e d s i m u l t a n e o u s l y . I n surviving a n i m a l s n o s i g n s o f p o i s o n i n g w e r e s e e n 24 h after administration.
Administration Diazepam (2 ml/kg body weight), physostigmine and ferrocene carbamate (1 ml/kg) were given intraperitoneally (i. p.). Soman (0.5 ml/kg) was given subcutaneously (s. c.) in the scruff of the neck and atropine and HI-6 intramuscularly (i. m.) in the same solution (1 ml/kg).
Cholinesterase assays in blood and brain tissue The enzyme activity was measured by the method described by Augustinsson et al. (1976), using 1 mM acetylthiocholine as substrate, at 22 ~C. Blood was obtained from the saphenous vein. A 50 lal aliquot was added to 950 lal of 0.1% (w/v) ice-cold Triton X-100. Enzyme activities were measured immediately in duplicate or triplicate. Cerebrum was rinsed in ice-cold water and homogenized with a Potter-Elvehjem homogenizer in 10 vol 0.1 M sodium phosphate buffer, pH 7.4, containing 0.1% (w/v) Triton X-100. Enzyme activities were measured as described for blood.
Experimental design Acute toxicity of the ferrocene carbamate. Ferrocene carbamate was given to four groups of guinea pigs with four animals per group. The animals were observed for 24 h and the LDso was calculated according to Weil (1952).
Cholinesterase activities. A blood sample was collected from each guinea pig (four animals per group) immediately before the administration of the carbamate and served as control (100% activity). The guinea pigs were treated with ferrocene carbamate (10 or 20 mg/kg i.p.) or physostigmine (0.16 mg/kg). A second blood sample was taken at
Table 1. Protective effect of pretreatment with ferrocene carbamate or physostigmine and diazepam in combination with HI-6 and atropine therapy against soman poisoning in guinea pig Treatment None
LD50 of soman (lag&g) 22 (18-
Protective ratio
27)
1
Ther
101 (74- 138)
5
Diaz + Ther
115 (75- 175)
5
FClow + Ther Diaz + FClow+ Ther
249 (203- 305) 413 (310- 551)
11 19
FChigh + Ther Diaz + FChigh + Ther
413 (310- 551) 716 (173-3343)" 930 (668 - 1294) a
19 35 42
Physo + Ther Diaz + Physo + Ther
197 (129- 300) 374 (274- 509)
9 17
Ther = HI-6, 50 mg/kg and atropine, 15 mg/kg, i. m. 1 min after soman Diaz = diazepam, 2 mg/kg, i.p., 60 min before soman FClow = ferrocene carbarnate, 10 mg/kg, i.p., 30 min before soman FChigh = ferrocene carbamate, 20 mg/kg, i.p., 30 rain before soman Physo = physostigmine sulphate, 0.16 mg/kg, i.p., 30 min before soman Soman was given s. c. a Two separate experiments
631 Table2. Inhibition of cholinesterasein blood and brain after administra-
tionof ferrocene-carbamateand physostigmine Blood (25 min)a
Brain (30 rain)a
Ferrocene-carbamate 10mg/kg,i.p. Ferrocene-carbamate 20mg/kg, i.p.
25.1___ 8.1
17.2+ 4.0
48.9 -+- 7.9
43.9-4- 6.0
Physostigminesulphate 0.16mg/kg, i.p.
64.5 -+-10.0
36.0 + 12.3
Thedataare given as mean values of percentageinhibition, with standard deviations,4 animals in each group. The blood values were calculatedin relationto blood sample taken from the same animal before carbamate administration,while the brain values are in relation to the ones obtained fromthree control animals a 25 and 30 min is the time elapsed between administration and withdrawalof blood and decapitation,respectively
Cholinesterase activities in blood and brain tissue In preliminary experiments it was found that, independent of the dose of ferrocene carbamate, maximum inhibition of ChE activity in the blood occurred around 30 min after administration (data not shown). From these experiments two doses of the ferrocene carbamate were chosen for later experiments, namely 10 and 20 mg/kg body weight, corresponding to 1/20 and 1/10 of the 24 h LD50 of the compound. We also compared the inhibition of cholinesterase caused by the ferrocene carbamate with that caused by physostigmine. As shown in Table 2, the degree of inhibition of the ChE activity with the ferrocene carbamate is roughly the same in blood and brain tissue, while with physostigmine this inhibition is significantly higher in the blood.
Clinical signs in soman treated animals All animals receiving doses of soman higher than 1 x LDs0 showed signs of cholinergic overstimulation, i.e. at least one of the following symptoms: chewing, tremor, incoordination, excess salivation, urination, defecation, convulsions, prostration, etc. The time till appearance of signs of poisoning was inversely proportional to the dose of soman. At doses of 1.5 x and 2 x LDs0, symptoms occurred within 1 0 - 2 0 min, when no treatment was given (i. e. in controls). At doses above 4.5 x LD50, symptoms generally appeared within 2 - 3 min, irrespective of supportive treatment. At doses above 10xLD50, signs of poisoning were seen within 1 min or less. In animals treated with diazepam in combination with therapy, clinical signs of poisoning occurred earlier than in animals given therapy only, at comparable doses of soman. This finding was unexpected, and to the best of our knowledge similar observations have not been reported in the literature. In animals pretreated with ferrocene carbamate the severity of the symptoms observed was noticeably lower than in other animals. In accordance with the findings of Gordon et al. (1978) the signs of poisoning were much less pronounced by 2 h. In six out of the nine experimental groups there was a tendency to an inverse relation between the dose of soman and the time until the animal died. It was also observed that the addition of diazepam resulted in a prolonged survival time after poisoning with soman. A similar effect of the carbamates could not be seen in this study, because the doses of soman were much higher than in comparable control groups. Surviving animals in the control group and animals given therapy showed no signs of poisoning 24 h after the administration of soman (doses up to 3 x LD50). All animals pretreated with the high dose of the ferrocene carbamate, diazepam and given therapy, that survived I0 x and 15 x LD50 of soman, did not show evident signs of poisoning at the end of the observation period. With the low dose of ferrocene carbamate or with physostigmine, most animals showed some remaining sign of poisoning.
The protective effect of ferrocene carbamate and supportive treatment against soman The protection against soman poisoning afforded by the different treatments is shown in Table 1. The pretreatment was always combined with therapy, consisting of HI-6 and atropine. The protective ratio for the HI-6/atropine treatment was around five. The addition o f diazepam as a pretreatment 60 min before soman did not improve the protective effect of HI-6/atropine. However, the combination ofpretreatment with a carbamate 30 rain before soman and therapy with HI-6/atropine significantly increased the protective effect and the protective ratio obtained was 9 for physostigmine and 11 and 19 for the two doses of ferrocene carbamate. When diazepam was added as a pretreatment to the carbamates and HI-6/atropine a further significant increase of the protective ratio was obtained, namely 17 for physostigmine and 19 for the low dose (10 mg/kg) of the ferrocene carbamate. With the high dose of the ferrocene carbamate the protective ratio was found to be 35 and 42, respectively, in two separate experiments.
Discussion
The main purpose of the present investigation was to assess the efficacy of a ferrocene carbamate as pretreatment against organophosphates. This type of compound has been shown to pass the blood brain barrier in mice (Karlsson et al. 1984). As shown in Table 2, this holds true also in guinea pigs. A dose-related inhibition of the acetylcholinesterase activity in blood and brain tissue was found with roughly the same degree of inhibition in blood and brain. This was, however, not the case with the tertiary carbamate physostigmine. As shown in the table, the degree of inhibition with this carbamate was two times lower in the brain than in the blood. The two doses of the ferrocene carbamate (1/20 and 1/10 of the LD50 dose of the compound) did not induce any clinical sign of poisoning in the animals and they were therefore considered as "sign free" doses.
632 In a study of the protective effect of physostigmine against soman poisoning in guinea pigs, Berry and Davies (1970) and Lennox et al. (1985) found that the protection correlated with the dose of the carbamate. This was also found to be the case in the present study with the ferrocene carbamate. Thus the lower dose of the compound gave a protective ratio of 19, while this value was 35 and 42 with the higher dose (Table 1). It is interesting to note that in spite of a significantly lower degree of inhibition of acetylcholinesterase, both in blood and brain tissue, the low dose of the ferrocene carbamate gave as good protection as that obtained with physostigmine. When the degree of enzyme inhibition was in the same order of magnitude the ferroeene carbamate afforded a better protection than physostigmine. In contrast to our data and the data cited above, Gordon et al. (1978) found that the dose of carbamate is not critical for the protection of guinea pigs against soman lethality. Carbamates differ in potency in their protective action against organophosphates (see, e.g. Berry and Davies 1970). Thus, both pyridostigmine and physostigmine provide good protection of peripheral cholinesterase against inhibition by soman. In addition to that, physostigmine gives protection of brain cholinesterase, since this tertiary carbamate penetrates the blood-brain barrier. Harris et al. (1984) showed that rats pretreated with physostigmine recovered more rapidly from the incapacitating effect of soman than did animals pretreated with pyridostigmine, although both carbamates provided equivalent protection against the lethal effects of soman. Similar findings were reported by Leadbeater et al. (1985). Our finding that animals surviving doses of 10 x and 15 x LDs0 of soman did not show signs of poisoning is probably due to a central action of the ferrocene carbamate. The same most probably also goes for the finding of a prolonged survival time for rats pretreated with physostigmine before the administration of DFP (Husain et al. 1990). Green (1983) has, in an excellent theoretical analysis, been able to correlate m a n y of the observations made in animal experiments to some simple kinetic parameters. He identifies two important factors for the protection. One is the "minimal essential cholinesterase" necessary for survival, which is low ( 0 . 1 - 1 0 % ) . The other is the ratio between the rate constant for decarbamylation (k2) and the rate of inhibition by the organophosphorous compound (i. e. the product of the inhibition constant and the concentration of the agent), a ratio called "enzyme conservation index". The best protection is obtained when the first factor is low and the second one is high. W e find that the "enzyme conservation index" should be the same for the two carbamates used in the present study, as both are monomethylcarbamates, thus giving the same k2. There was a difference in cholinesterase inhibition, caused by the carbamates, before soman challenge, but this difference is of minor importance for protection, as judged from Green's calculations. W e would rather attribute the more efficient protection from ferrocene carbamate pretreatment, as compared to physostigmine pretreatment, to the first-mentioned critical factor, i.e. " m i n i m u m essential cholinester-
ase". As Green points out, this can be lowered by the administration of anticholinergic drugs. It is well known that carbamates have direct effects on cholinergic receptors. These effects might be of much greater importance with the ferrocene carbamate than with physostigmine. This hypothesis finds some support in our earlier experiments (Karlsson et al. 1984), in which we showed that mice could be protected against 5 - 6 • doses of soman, when pretreated with only ferrocene carbamate and atropine. In summary, the ferrocene carbamate used in the present study was inferior to physostigmine as a cholinesterase inhibitor. In spite of this, the former compound gave better protection against soman. W e suggest that this might be explained by properties not related to the cholinesterase inhibiting activity, e. g. direct receptor effects.
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