The synergism of atropine and the cholinesterase reactivator HI-6 in counteracting lethality by organophosphate intoxication in the rat

TOXICOLOGY AND APPLIED PHARMACOLOGY 107,47-53 (I 99 1)

The Synergism Counteracting

of Atropine and the Cholinesterase Reactivator HI-6 in Lethality by Organophosphate Intoxication in the Rat DAVID A. LIGTENSTEIN’ AND GERRIT W. H. MOES

Prim

Mauri&

Laboratory

Received

TNO,

May

P.O. Box 45, 2280 AA Rijswijk,

14. 1990; accepted

September

The Netherlands

7. 1990

The Synergism of Atropine and the Cholinesterase Reactivator HI-6 in Counteracting Lethality by Organophosphate Intoxication in the Rat. LIGTENSTEIN, D. A., AND MOES, G. W. H. (199 1). Toxicol. Appl. Pharmacol. 107, 47-53. Rats were intoxicated with two different Saminoalkylphosphonothioate cholinesterase inhibitors, viz. I-l (S-diethylaminoethyl-O-cyclohexyl-methylphosphonothioate), which has a mixed central/peripheral mode of action, and I-2, the methiodide derivative of I-l, which acts almost solely peripherally. It was found that atropine did not have any beneficial effect on lethality in the caseofan I-2 intoxication but did so, although only slightly, in the case of I-l. Therefore, the effect of atropine against I-l intoxications must be mediated through central mechanisms, the peripheral parasympatholytic effect being negligible in counteracting lethality. Furthermore atropine antagonized the convulsions caused by intoxication with I- I. The oxime used as a reactivator of inhibited acetylcholinesterase, HI-6, was more effective than atropine against either organophosphate. In the case of an I-2 intoxication HI-6 proved extremely active. It is, therefore, concluded that HI-6 acts mainly peripherally. It was also found that HI-6 has a slight anticonvulsive action. The combination of HI-6 and atropine had a large synergistic effect in the case of I-l, but in the case of I-2 hardly any synergism was observed. Obviously. the combination of the oxime and atropine is particularly effective when the toxicant has a mixed central/peripheral action. In such intoxications the acetylcholinesterase reactivation in the respiratory neuromuscular synapse by the oxime is supplemented by the central action of atropine, which improves respiratory control at the level of the central nervous system. o 1991 Academic Press. Inc.

In clinical practice, as well as in laboratory experiments, the treatment of organophosphate intoxications is primarily performed with large doses of atropine, sometimes combined with the application of an AChE2 reactivator such as pralidoxime or obidoxime. It is generally assumed that oximes without coadministration of atropine are of little benefit (Goodman and Gilman 1985; Daunderer and Weger, 1980; Klimmek et al., 1983).

However, adequate respiratory muscle activity cannot be obtained without a sufficient peripheral nicotinic neuromuscular transmission. Since atropine is a muscarinic blocker, with no influence on neuromuscular transmission, such a doctrine may well be incorrect. In order to investigate the efficacy of the treatment of organophosphate intoxications by atropine and the pyridinium-aldoxime HI-6 (for structures see Fig. 1) as single compounds, as well as in combination, a series of toxicity ’ To whom correspondence should be addressed. studies in rats was performed. ’ Abbreviations used: ACh, acetyl choline; AChE, aceTo be able to distinguish between central tylcholinesterase; BBB, blood brain barrier; CNS, central and peripheral effects, we designed and synnervous system; GLC, gas-liquid chromatography; ip, inthesized two organophosphate AChE inhibitraperitoneally; PML/TNO, Prins Maurits Laboratory TNO; SC,subcutaneously. tors. I- 1 (S-diethylaminoethyl-O-cyclohexyl41

OO41-008X/91 $3.00 Copyright 0 1991 by Academic Prey, Inc. All rights of reproduction in any form reserved.

LIGTENSTEIN

48

AND MOES

0 O;PT H3C

,CZH5 S-CHZ

-CH2

-N ‘QH5

I-1 HI -6 0 O-P/ /\ H3C

m:2H5 S-CHZ

-LY-~-CH~

1’

C2Hs I-2 iI -1 methybdldel

FIG. I. The chemical structures of I- I, I-2, HI-6. and atropine.

methylphosphonothioate)3*4 is a tertiary amine, the unprotonated form of which rapidly penetrates the CNS, and therefore can be expected to exhibit central as well as peripheral cholinesterase inhibiting properties. I-2 is the methiodide derivative of I-l, of which the strong and permanent ionic character will hamper penetration through the BBB. Identical cyclohexyl methylphosphonylated AChE’s are formed upon inhibition with either compound, since I-l and I-2 only differ in structure of their leaving groups. Consequently, there are no differences in enzyme reactivation by an oxime, or in rates of spontaneous reactivation and aging.5 METHODS Chemicals. HI-6 dichloride monohydrate (M = 377.22) was synthesized and purified by recrystallization according to the methods described by Schoene (1967). The product was homogeneous on TLC analysis, and its identity was confirmed by NMR. ir, and elemental analyses. Di(atropine) sulfate monohydrate was purchased from ’ Alkylaminothioates are often referred to as V compounds. 4 I-1 and 1-2 are extremely toxic compounds, requiring special safety procedures for appropriate laboratory handling. 5 The inhibition constants for I-l and l-2 of electric eel AChE, in 6.6 mM barbitone buffer, pH 7.7, at 25°C were 5.1* 10s and 4.8* lo8 mol-’ * min-‘, respectively. For conditions see Benschop et al. (1984).

Brocacef (Maarssen, The Netherlands). Iodomethane (Aldrich) was dried (0.01% H20) over anhydrous magnesium sulfate, distilled. and stored over copper turnings. CyclohexanoI (Aldrich) was dried by retluxing with sodium and distilled (bp 160°C 0.0 1% HzO). Methylphosphonous dichloride was prepared according to published methods (Fen-on et al.. 1960; Komkov et al., 1958: Reesor et a!.. 1960). and cyclohexyl methylphosphonothioate as described by Pelchowicz and Leader (1963). I-l (M = 293.44) was synthesized from 2-diethylaminoethylchloride hydrochloride and cyclohexyl methylphosphonothioate according to Boter and Platenburg (1967) and Gregg et al. (1961). I-2 iodide (M = 435.35) was then synthesized by treating I-l with iodomethane in ether. I-l and 1-2 were homogeneous on TLC analysis and their identities were confirmed by NMR, ir, MS, and elemental analysis. All other reagents were of analytical grade and dried or used as received. Animal procedures. Male “Small Wistar” rats, strain WAG/MBL, weighing 180-210 g, bred under SPF conditions at the Medical Biological Laboratory TNO, were used. Prior to the experiment the animals were fasted for 24 hr. but had accessto water ad libitum. They were each placed in a separate cage and allowed to remain there for at least 1 hr. After administration of the compounds (vide infra) the number of deaths per group was scored after 24 hr. During the first 2 hr, the occurrence of muscle spasms and convulsive seizures was assessedby visual observation. LD plots were determined by probit analysis, according to a method modified from Finney (1980) and Weber (1980) performed on a DEC PDP 1l/45 digital computer, and plotted on “probability paper” (Miller and Tainter, 1944). Administration of the compounds. HI-6 dichloride monohydrate was dissolved in water in a concentration of 50 mg *ml-’ (0. I3 M) and administered ip in a dose of 50 mg * kg-’ (0. I3 mmol . kg-‘).

INTERACTION

OF HI-6 AND ATROPINE

Di(atropine) sulfate monohydrate was dissolved to a concentration of 37.5 mg . ml-’ (54 mM = 108 mM atropine base) in 0.5% sodium chloride solution and administered ip at a dose of 37.5 mg. kg-’ (0.108 mmol *kg-’ atropine base). This and similarly high doses of atropine are commonly used in toxicity studies with organophosphates in rats (Kepner and Wolthuis, 1978; Van Helden and Wolthuis, 1983; Wolthuis et al., 1976, 198 la). In order to increase its rate of dissolution in water, I-l was dissolved, prior to further dilution, in I ml of waterfree isopropanol. A stock was prepared by dilution of this solution 50 times with saline and was stored in ice. Isotonic injection fluids were prepared from this stock by dilution with saline in such a way that the dose required could be injected in a volume of 0.25 to 0.50 ml. The final solution never contained more than 0.04% isopropanol. l-2 was treated the same way. Both I- I and I-2 were administered SCin the neck. The following regimes were applied: (I) LD50 values of I-l and I-2 were determined after SCadministration. (2) Atropine was injected ip together with a separate injection of the same volume of saline as a sham injection for HI-6 in the procedure described in (4). Five minutes later I-1 or I-2 was administered SC. (3) HI-6 was injected ip together with a separate injection of the same volume of saline as a sham injection for atropine in the procedure as described in (4). Five minutes later I-l or I-2 was administered SC. (4) HI-6 and atropine were injected ip in separate injections at the same time. Five minutes later I-l or l-2 was administered SC.

49

1 +H1-6

.HI-6

1-1+HI-6+Atrq5ne LD so .>5,000

I 10

1

I

50

100

RESULTS The LD50 of I- 1 was determined to be 16.24 pg. kg-’ (55.2 nmol * kg-‘; 95% confidence limits: 15.1-17.2 pg.kg-‘; Fig. 2). I-l produced severe convulsions in all animals. After pretreatment with 37.5 mg. kg-’ atropine, the LD line of I- 1 shifted to the right, by a factor of approximately 1.5. The LD50 increased to 25.1 Kg* kg-’ (85.6 nmol * kg-‘; 2 1.6-28.7 pg. kg-‘). Convulsions were absent, except at the highest dose of I-l where two of eight animals showed convulsions. Muscle

I

I

I

500

1000

5000

dose 01 I - 1 (w&g) FIG. 2. LD plots in the rat for I-l (sc) and the effects thereon of HI-6 (ip), atropine (ip), and the combination of these two compounds. The combination of atropine and HI-6 shows such a large synergism that below 5 mg * kg-’ no deaths occurred. The bracket indicates the two solid lines to form the plot of I-l counteracted by HI-6.

contractions and fasciculations were seen in all animals. After pretreatment with 50 mg . kg-’ HI-6 the LD line of I-l shifts to the right with a factor of at least 5. With doses of I- 1 increasing from 60 to 90 pg kg-‘, death scores increased. At doses of 1-I between 90 and 110 pg - kg-‘, however, no reproducible scores could be obtained. At 110 pg. kg-’ I- 1 the score decreased to a value significantly below that after 90 and from this point with increasing a-kg-‘, doses of I- 1, a new LD plot could be calculated (Fig. 2). We reproduced this phenomenon in three independent series of experiments. HI6 significantly suppressed convulsions in the lower dose range of I-1 (Table 1). Muscle contractions were almost invariably present, but were clearly milder than those with I-l alone or in the case of I-l pretreated with atropine solely. The combination of HI-6 and atropine as a pretreatment to I- 1 intoxication produced a large synergistic effect. No deaths were detected for doses of I- 1 up to 5 mg . kg-‘. Only at the highest dose, one animal convulsed. Muscle contractions and fasciculations were l

All experiments were carried out in groups ofeight rats, and with at least 5 groups of animals per LD plot. The 5min interval was chosen on the ground of a pilot study, showing that HI-6 concentrations in blood after this period of time had reached a considerable level.

/ &g

LIGTENSTEIN

50 TABLE I CONVULSION

SCORES AFTER SC ADMINISTRATION PRECEDED BY 50 mg. kg-’ HI-6 ip

Dose of I- I (a.k-‘)

Number of convulsing animals

60 70 80 90 110 115 130

I/8 318 418 8/8

OF I-I

g/8 8/8

w

abundant. After 24 hr the animals seemed exhausted, but otherwise were in good condition, as suggested by the reappearance of eating and drinking behavior. All lines as depicted in Fig. 2 are parallel and at the same dosages of I- 1, death scores with the different regimes were significantly different, according to the test by Hald (1952). The LD50 value for I-2 was found to be 28.2 pg. kg-’ (64.8 nmol . kg-‘; 26.8-30.5 pg. kg-‘; Fig. 3). Convulsions were not observed, although severe muscle spasms occurred. On pretreatment with atropine, the line shifted slightly to the left. The shift is significant and parallel, the LD50 being 23.5 pg.kg-’ (54.8 nmol. kg-‘; 22.2-25.1 FLgkg-‘). No convulsions were observed but, as for I-2 alone, muscle spasms were abundant. Pretreatment with HI-6 provides a protection with a factor of 70. The LD50 was increased to 2.0 mg - kg-’ (4.6 nmol - kg-‘; 1.62.2 mg . kg-‘). Convulsions were seen only in two of eight animals receiving the highest dose. All animals suffered from muscle spasms. After 24 hr all surviving animals seemed extremely tired, but were in good condition, as indicated by active eating and drinking behavior. Pretreatment with both HI-6 and atropine yields a further shift to the right, the LD50 increasing to 2.7 mg. kg-’ (6.2 nmol- kg-‘; 2.3-3.0 mg. kg-‘).

AND MOES

As with I- 1, all lines are parallel and at the same dosages of I-2, death scores with the different regimes were significantly different, according to the test by Hald ( 1952). DISCUSSION Atropine, when given alone, only had a slight protective effect in the case of an I-l intoxication and no effect at all when the toxicant was I-2. In fact, an intoxication with the strongly peripherally acting I-2 is even worsened by atropine pretreatment. It should therefore be concluded that in the experiments described, the peripheral effects of atropine, being limited to its parasympatholytic action, contributed little to the effect of atropine on lethality. The deleterious effect of atropine on the lethality by I-2 can be explained by adapting the following functional concept. Animal experiments have shown that excess ACh, as caused by organophosphate intoxication, causes both nicotinic excitation and muscarinic inhibition of central respiratory activity, the muscarinic inhibition prevailing over the nicotinic effects (Bohmer et al., 1987). Atropine counteracts muscarinic inhibition, thereby restoring re-

10

I 50

I 100

I 600 1000 dase0l1-2($Qkg)

1 5000

FIG. 3. LD plots in the rat for I-2 (SC)and the effects thereon of HI-6 (ip), atropine. and the combination of these two compounds.

INTERACTION

OF

HI-6

AND

ATROPINE

51

spiratory activity after organophosphate in- (Wolthuis et al., 198 1b) it should be concluded toxication (Bohmer et al., 1987; Bradley and that restoration of neurotransmission at the Dray, 1976). If respiration is inhibited in the motor synapses of the respiratory musculature CNS this must result in a reduction of the ac- in such a mixed central/peripheral intoxicativity of peripheral respiratory motor neurons, tion is still more important than restoration of which the phrenic nerves are the most imof central respiratory regulating processes portant. Thus, in sublethal cases of I-l intoxalone. It can be easily inferred that without a ication, atropine will increase the phrenic sufficient restoration of neuromuscular transnerve activity, but not the neuromuscular mission, even if this process, as in the case of transmission, since postsynaptic receptors in a centrally acting inhibitor, is quantitatively the neuromuscular synapse are all nicotinic. less disordered than central respiratory reguIn the case of I-l, where the central action is lation, rhythmic respiratory muscle contracsignificant, the effect of decreased phrenic tions remain hampered, thereby limiting the nerve activity dominates that of the disturoverall efficiency of the central effects of atrobance of the neuromuscular transmission. pine. Here, the increase of phrenic nerve activity, The difficulty to obtain reproducible death as caused by atropine, and the resulting in- scores in the range 90 to 100 gg. kg-’ of I- 1 crease of ACh release in the neuromuscular in Hid-pretreated animals, where increasing synapse, improves muscle contractions since the dose of the toxicant reduces the death rate, the synapse still functions and therefore can is striking. I-l possibly alters the pharmacoabsorb the larger ACh release. However, in the kinetics of HI-6 in such a way that higher concase of I-2 the peripheral effect very much centrations of HI-6 are made available at esdominates the central inhibition, although the sential locations. Another possibility is that the latter is not completely absent. If atropine is BBB suffers from loss of integrity, leading to administered, and phrenic nerve activity in- higher concentrations of HI-6 in the CNS. This creases, the resulting extra ACh excreted into could be caused or at least reinforced by the the neuromuscular synapse where the ACh occurrence of excessive convulsions (Lorenzo level already is nearly maximally elevated leads and Barlow, 1967; Lorenzo et al., 1967; Ashani to a functional block of that synapse, thereby et al., 198 1; Ashani and Catravas, 198 1) in an further reducing the respiratory function. early stage of the intoxication, as seen with IThe experiments clearly show that HI-6 is 1. This might also account for the absence of therapeutically active on its own, without such a biphasic shape in the LD plot for I-2, atropine. The activity of HI-6 surpasses that since this compound did not cause significant of atropine, both in intoxications with I- 1 and convulsions. in those with I-2. Clement (198 1) found HI-6 Combination of HI-6 and atropine has a on its own to be active against intoxications marked synergistic effect on I-l intoxication. with soman. Clinically good results of a sole If indeed HI-6 acts mainly peripherally and treatment by the oxime pralidoxime have been the influence of atropine on lethality is brought reported by Namba and Hiraki ( 1958) and by about only via a central action, this combiXue et al. (1985). However, such practice has nation is extremely beneficial in the case of a never been generally accepted. mixed central/peripheral organophosphate It is obvious that HI-6 is very active in the intoxication. Under these conditions, contrary event of an intoxication with the mainly pe- to therapy with atropine without AChE reacripherally acting organophosphate I-2. Nev- tivation in neuromuscular synapses, the inertheless HI-6 is also much more active than crease of phrenic nerve activity, resulting in atropine in a combined central/peripheral in- an increase of ACh liberation into the respitoxication with I-l. Consequently, if we as- ratory motor synapses, is therapeutic since sume respiratory arrest to be the cause of death neurotransmission in these synapses has been

52

LIGTENSTEIN

restored by reactivation of the inhibited enzyme. Addition of atropine to HI-6 in the pretreatment of an I-2 intoxication causes only a marginal increase of the LD50. This may very well be explained by assuming that, despite its quaternary ammonium structure, small amounts of I-2 do penetrate into the CNS, thus providing the ability for atropine to act on lethality. It is clear that without pretreatment I-l causes severe convulsive seizures, whereas I-2 lacks such an activity. This is another argument supporting the assumption as made in the introduction that the central action of I-2 is negligible compared with that of I- 1. Atropine antagonizes convulsions in the case of I- 1. This anticonvulsive effect of atropine in the case of organophosphate intoxications has been observed by several investigators (Ashani and Catravas, 1981; Wadia et al., 1974). Despite its quaternary ammonium structure, hampering penetration into the CNS, HI6 had a measurable anticonvulsive action. If an I-l intoxication is pretreated with HI-6 alone, a decrease in the occurrence of convulsions in the lower dose ranges is observed relative to no pretreatment. The penetration of HI-6 into the CNS as described by Ligtenstein and co-workers (Ligtenstein and Kossen, 1983; Ligtenstein, 1985) and the distribution of HI-6 in the CNS as illustrated by autoradiography (Ligtenstein et al., 1988) whereby small, but measurable, amounts of radioactivity were found in the cerebrum and cerebellum, together with the important role played by acetylcholine in the development of convulsions (Wood et al.. 1979; Cavalhiero et al., 1983), support the possibility of such an anticonvulsant action. Hauser and Weger ( 1979) have also found an anticonvulsant effect of bispyridinium oximes in organophosphate intoxication. From the results of these experiments we conclude that ( 1) the oxime HI-6 in the model studied is certainly therapeutically effective, also without atropine, both in intoxications

AND MOES

with I-l and in those with I-2; (2) the peripheral effects of atropine, being limited to its parasympathetic action, play no role in reducing lethality of the organophosphates; and (3) the synergism of HI-6 and atropine is brought about by the combination of the peripheral effect of HI-6, restoring neuromuscular transmission and the central effect of atropine on counteracting respiratory disregulation. Indeed the adage that, also in animal experiments, oximes are of no value without coadministration of atropine, and are of less importance than the latter, is belied by the results of our experiments. In the model applied, atropine should be regarded as an adjunct to the oxime, and not vice versa.

ACKNOWLEDGMENTS The authors thank Hendrik P. Benschop. Ph.D., and Leo P. A. de Jong, Ph.D., of the Prins Maurits Laboratory TN0 for their many contributions to this study. The technical assistance of Mr. Simon P. Kossen is also acknowledged.

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INTERACTION

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