The in vitro and in vivo reactivating power of 1,3-acetone-
bis-(4-hydroxyiminoformyl pyridinium) dibromide (MBM-3) was
compared with that of 1,3-trjmethylene~bis-(4-hydroxyjminoformyl
pynidinium) dibromide (TMB-4) and 1,3-dimethylether-bis-(4-hydroxyiminoformyl pyridinium) dibromide (Toxogonin). As a source of
choli\u27nesterase rat\u27s whole blood, plasma, erythrocytes, and brain ho~
mogenate, were u sed. The cholinesterase inhibition was performed
by ethyl N-dimethylaminophosphorocyanidate (Taibun); ethyl 4-
-nitrophenyl phosphonate (Armin), 0,0-diethyl-S-(2-diethylaminoethyl)
phosphorothioate (Amitone-3); pinacolyl-methyl-phosphorofluoridate
(Soman); and O-ethyl-S-(2-diethylamino ethyl phosphorothioate
(Edemo-3)

B. Bošković

M. Maksimović

Z . Binenfeld

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CR 0 AT IC A CHEMIC A ACT A 40 (1968) CCA-517 195 615.9:577.153 Note In vitro and in vivo Reactivation of Cholinesterases Inhibited by Highly Toxic Organophosphorus Compounds A Comparison Between 1,3-Acetone-bis-(4-hydroxyiminoformyl pyridini-um) dibromide, 1,3-Trimethylene-bis-(4-hydroxyiminoformyl pyridinium) dibromide, and 1,3-Dimethylether-bis-(4-hydroxyiminoformyl pyridinium) dibromide M .. Maksimovic, B. Boskovic, and Z. Binenfeld High Military Technical School, Zagreb, Yugoslavia Received April 24, 1968 The in vitro and in vivo reactivating power of 1,3-acetone--bis-(4-hydroxyiminoformyl pyridinium) dibromide (MBM-3) was compared with that of 1,3-trjmethylene~bis-(4-hydroxyjminoformyl pynidinium) dibromide (TMB-4) and 1,3-dimethylether-bis-(4-hydro-xyiminoformyl pyridinium) dibromide (Toxogonin). As a source of choli'nesterase rat's whole blood, plasma, erythrocytes, and brain ho~ mogenate, were used. The cholinesterase inhibition was performed by ethyl N-dimethylaminophosphorocyanidate (Taibun); ethyl 4--nitrophenyl phosphonate (Armin), 0,0-diethyl-S-(2-diethylamino-ethyl) phosphorothioate (Amitone-3); pinacolyl-methyl-phosphoro-fluoridate (Soman); and O-ethyl-S-(2-diethylamino ethyl phospho-rothioate (Edemo-3). The cholinesterase activities were measured by a modJified Michel electrometric method. The molar oxime concentrations which cause a 500/o reactivati0il1 of the phosphorylated enzyme (MRD50) were as follows a) erythrocytes : from 3.5 X 10-7 to 7.8 X 10-6 (TMB-4); from 1.5 X 10-1 to 7.5 X 10-s (Toxogonin); from 8 X 10-7 to 7.5 X 10-5 (MBM-3). b) plasma: from 1 X 10-6 (TMB-4); from 6.5 X 10-7 to 9.3 X X 10-s (Toxogonin); from 3.5 X 10-r. to > 1 X 10-4 (MBM-3), c) brain: from 1.5 X 10-7 to 1 X 10-s (TMB-4); from 1.3 X 10-1 to > 1 X 10-4 (Toxogonin); from 5.0 X 10-1 to > 1 X 10-4 (MBM-3). Cholinesterase inhibited by Soman could not be reacti'vated. In vivo experiments with Amitone-3 gave a variable percentage of reactivation of the whole blood cholinesterase, from 65 to 87°/0'. A statistically sigruficant difference was observed between Toxo-gontn and MBM-3 (P < 0.01). The results indicate that MBM-3 belongs to the class of the most powerful :reactivators of phosphorylated cholinesterase, e.g. TMB-4 or Toxogonin ." Continuing our earlier studies which have shown that 1,3-acetone-bis-(4-hydroxyiminoformyl pyridinium) dibromide (MBM-3) i:s an effective antidote in Paraoxon and Sarin poisoning1•2, we have measured the in vitro and in vivo reactivating activity of MBM-3 towards cholinesterases inhibited by a number of the most powerful anticholinesterases3• As reference reactivators we used 1,3-trimethylene-bis-(4-hydroxyiminoformyl pyridinium) dibromide (TMB-4), and 1,3-dimethylether-bis-(4-hydroxyiminoformyl pyridinium) dibromide (To:-xogonin). - _ 3 196 M. MAKSIMOVIC, B. BOSKOVIC, AND Z. BINENFELD MATERIALS AND METHODS Organophosphorus Compounds EthyI-4-nitrophenyl phosphonate (Armin); pinacolyl methyl phosphonofluoridate (Soman); 0,0-diethyl-S-(2-diethylaminoethyl) phosphorothioate (Amitone-3); ethyl-N-d.imethylphosphoro cyanidate (Tabun); 0-ethyl-S-(2-ddethylaminoethyl) phosphoro-thioate (Edemo-3) were of 98-990/o purity. All compounds were made up as 0.1 O/o stock solutions in propylene glycol and diluted to desired concentrations with isotoruc saline. The concentrations of the solutions were checked by spectrophotometric measurements4 and by determining their LD50 subcutaneously in white mice. Oximes TMB-4 (m. p. 235°), Toxogonin (m. p. 225°) and MBM-3 (m. p. 2190) were dissolved in redistilled water immediately before use. Cholinesterase Preparations As sources of enzyme we used: 1) Rat erythrocytes, washed three times with isotonic saline and haemolysed; the haemolysate volume was adjusted with distilled water to the corresponding blood volume. 2) Rat blood plasma. 3) Rat braiin homo-genate. 4) Rat whole blood. In vitro Cholinesterases Reactivation - Male wistar rats weighing between 200 and 250 g. were injected subcutaneously with 2/a of LD50 dose of a given organo-phosphorus compound, and the animals sacrificed half an hour later. The volume of the injected solution was 2 ml./kg. The activity of erythrocite, plasma and 'brain cholinesterases was about 10-200/o of the activity of untreated animals. The enzyme preparations were incubated for 10 min. with an oxime solution, and acetylcholine chloride (3 X 10-a M final) was then added. Cho1inesterases activity was measured electrometrically by the Michel's method, as modified by Stevanovic and .Jovic5• The final oxime concentrations before addition of substrate varied between 1 X 10-1 and 1 X 10-4 M. In vivo Cholinesterases Reactivation of Rat's Whole Blood. - Blood was taken from the rat's tail. One hour after the subcutaneous application of ""'!/2 LD50 dose of Amitone-3 the oximes (""' 1/s LD50) were given subcutaneously. The volume of the oxime and organophosphorus soLution together was 1 ml./kg. One hour after the administration of oxime the rats were sacrificed and the cho1inesterases activity was measured a'S described. Cholinesterases measurements were made on blood samples obtained before administration of the organophosphorus compounds, imme-diately before admQrustration of oximes and immediately after the rats had been sacrificed. The cholinesterase activity before oxime administration was about 35 to 45°/o of the normal value. The in vitro and in vivo degree of cho1inesterases reactivation was calculated according to the method of Childs et al.6 ; Statistical calculations were carried out by the Eramens method7 • RESULTS AND DISCUSSION The results of the in vitro cholinesterases reactivation are presented in Table I. From these experiments and results obtained in our previous expe-riments with Sarin2 the following conclusions can be drawn. MBM-3 possesses a reactivating property similar to that observed with other oximes used so far and its efficacy varies from one inhibitor to the other. From these results it is not possible to draw a theoretical conclusion or to predict the antidotal activity of the three oximes studied. MBM-3, like Toxogonin and TMB-4, is very effective in reactivating brain cholinesterases in vitro. Recent experiments of Milosevic and Andelkovic9 and Hobbiger and Vojvodic9 have shown that it is also possible to reactivate cholinesterases inhibited by organophosphorus compounds in different brain areas with oximes in vivo. It seems likely that MBM-3 would also reactivate TABLE I In Vitro Reactivation of Rat Erythrocyte, Plasma, and Brain Cholinesterases MRDso (~tM) Inhibitor Erythrocyte I Plasma I Toxogonin J TMB-4 I MBM-3 J Toxogonin J TMB-4 I MBM-3 J Toxogonin J Ta bun 75 7.8 75 8.0 4.3 > 100 Soman none none none none none none Armin 1.0 0.75 0.95 8.0 5.0 20 Amitone-3 0.15 0.35 0.80 0.65 4.0 6.3 Edemo-3 8.0 4.8 6.8 9.3 1.0 3.5 MRD50 = µmolar concentration of oximes which causes a 50'/o reactivation of inhibited enzyme. TABLE II In Vivo Reactivation of Rat's Whole Blood Cholinesterase > 100 none 0.80 0.17 4.3 Inhibitor Reactivation of-cholinesterase (0 /o) x ± 2cr Amitone-3 TMB-4 78 ± 15 P> 0.1 Toxogonin 87 ± 11 p < 0.01 MBM-3 65.0 ± 4.2 I Brain TMB-4 MBM-3 10 > 100 none none 0.45 0.83 0.15 0.50 0.83 3.8 ~ l'.1 > (') J-j g ...... 0 z 0 "'] (') ::r: 0 t-< z l'.1 Ul J-j l'.1 ~ > Ul l'.1 Ul ...... (0 ~ 198 M. MAKSIMOVIC, B. BOSKOVIC, AND Z. BINENFELD brain cholinesterases in vivo, since its chemical structure is similar to that of TMB-4 and Toxogonin1• The results of the in vivo reactivation of whole blood cholinesterases are presented in Table II. These results are only preliminary and each number presents the mean obtained on 4 animals: The in vivo experiments with Amitone-3 showed good agreement with the in vitro results, although they were performed with whole blood prepa-rations which contain both erythrocyte and plasma cholinesterase. The sta-tistically significant difference between Toxogonin and MBM-3 in the in vivo experiments is probably due to the 10 and 5 fold respectively, higher reactivating power of Toxogonine as compared to MBM-3 in reactivating plasma and ery-throcyte cholinesterase inhibited by Amitone-3. In the case of TMB-4, where this difference was only 1.5 and 2.3 fold, respectively, no statistically significant difference was observed. The promissing results obtained with MBM-3 as an antidote in Sarin and Paraoxon poisoning1•2 were confirmed in the present experiments where MBM-3 showed equally effective reactivating pr.operties as TMB-4 and Toxogonine against cholinesterases inhibited by some of the most toxic organophosphorus compounds. REFERENCES 1. Z. B'inenfeid, D. Andelkovic, M. Milojevic, M. Orlov, D. Rakin, M. Te r z i c, and B. Va s i c, Arh. hig. rada 18 (1967) 139. 2. Z. Binenfeld, B. Boskovic, and M. Maks'imovic, Acta Pharm. Jug. 17 (1967) 35. 3. B. Ho 1 ms t e d t, Pharmacol. Rev. 11 (1959) 567. 4. B. Ge ha u f and J. Go 1 dens on, Anal. Chem. 29 (1957) 276. 5. M. Stevanovic .and R. Jovic, Arh. Farm. (Beograd) 12 (1962) 327. 6. A. F. Chi 1 d s, D. R. Davies, L. A. Green, and J. P . Rutland, Brit. J. Pharmacol. 10 (1955) 462. 7. W. C. Er amens, Principles of Biological Assay, Chapman and Hall Ltd. London 1948. p. 18. 8. M. Milosevic and D. AndelkoviC, Nature 210 (1966) 206. 9. F. Hobb i g er and V. Vo j v o di c, Biochem. Pharmacol. 15 (1966). IZVOD In vitro i in vivo reaktivacija kolinesteraza inhibiranih najtoksicnijim organofosfornim spojevima ' Aktivnost 1,3-aceton-bis-(4-hidroksiiminoformilpiridinijum) dibromida u usporedbi s aktivnoscu 1,3-trimetilen-bis-(4-hidroksiiminoformilpiridinijum) dibromida i 1,3-dimetileter-bis-(4-hidroksiiminoformilpiridinijum) dibromida M. Maksimovic, B . Boskovic i Z. Binenfeld Ispitana je in vitro reaktivatorska moc 1,3-aceton-bis-(4-hidroks.iiminoformilpinidi-nijum) dibromida (MBM-3), 1,3-trimetilen~bis-(4-hidroksi.iminoformilpiridinijum) di-bromida (TMB-4) i 1,3-dimetJileter-bis-(4-hidroksi.iminoformilpiridirujum) d'icbromida (Toxogonin) na kolinesteraze erJtrocita, plazme i mozga stako1·a inhibirane etiletoksi--p-nitro-fenolatom (Armfo), dimetilamidoetoksifosfarilC'ijanidom (Tabun), pinakol-oksimetilfluorofosfatom (Soman), 0,0-d!~etoks,i-S-(2-dietil-aminoetil)-tiofosfatom (Ami-ton-3), i O-etoksi-S-(2-dietilaminoetil)-metiltiofosfo:natom (Edemo-3). Za mjerenje upotrebljena je modificirana lVIichelova elektrometrijska metoda a kao izvor enzima sluzili su eritrociti, nativne plazme i homogenizati mozga stakora prethodno otrovanih navedenim spojevima. Molarna koncentracija oksima, koja do-vodi do 500/o reaktivacije inhibiranog enzima _krece , se ovisno od alkilfosfatu za eri-REACTIVATION OF CHOLINESTERASES 199 trocitne kolinesteraze: od 3,5 X 10-7 do 7,8 X 10-6 (TMB-4), od 1,5 X 10-1 do 7,5 X 10-5 (Toxogonin); od 8 X 10-1 do 7.5 X 10-5 (MBM-3) ; za kolinesterazu plazme: od 1X10-6 do 5 X 10-6 (TMB-4) ; od 6,5 X 10-1 do 9,3 X 10-6 (Toxogonin); od 3,5 X 10-6 do> 1X10·4 (MBM-3); za koLinesterazu mozga: od 1,5 X 10-7 do 1 X 10-5 (TMB-4); od 1,3 X 10-7 do > 1X10-4 (Toxogonin); od 5 X 10 7 do> 1 X 10-4 (MBM-3); osim za Soman gdje ni u jednom slucaju nije doslo do reaktivacije. U in vivo eksperimentima sa Amitonom-3 procenat reaktivacije inhibirane koli-nesteraze pune krvi stakora kretao se od 65 do 87°/o s tim da se statisticki znacajna razlika pokazala izmedu Toxogonina i MBM-3 (P < 0,01). Na temelju ovih rezultata zakljuceno je da MBM-3 spada u grupu najefikasnijih oksima reaktivatora kolinesteraza inhibiranih alkilfosfatima u koje spadaju i TMB-4 i Toxogonin. VISOKA TEHNICKA SKOLA JNA ZAGRGEB Primljeno 24. travnja 1968. 

In vitro and in vivo Reactivation of Cholinesterases Inhibited by Highly Toxic Organophosphorus Compounds

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