The Reactivation by Oximes and Hydroxamic Acids of Cholinesterase Inhibited by Organo‐phosphorus Compounds

Childs, A. F.; Davies, D. R.; Green, A. L.; Rutland, J. P.

doi:10.1111/j.1476-5381.1955.tb00106.x

Cited by 87 publications

(72 citation statements)

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“…The experiments on human plasma cholinesterase were therefore extended to human and bovine true cholinesterases which had been inhibited by an organophosphate containing either a diethyl-or diisopropylphosphato group and the findings are reported in this paper. In addition to NHAM, ammonium molybdate and pyridine-2-aldoxime methiodide, a potent reactivator of phosphorylated electric eel cholinesterase and phosphorylated human and rat true ch6lin-esterases (Wilson and Ginsburg, 1955;and Childs, Davies, Green, and Rutland, 1955), were used for enzyme reactivation. The results obtained with all these reactivators show that the organophosphates form two types of phosphorylated enzyme with human and bovine true cholinesterases which thus behave like plasma cholinesterase.…”

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confidence: 99%

Chemical Reactivation of Phosphorylated Human and Bovine True Cholinesterases

Hobbiger

1956

British Journal of Pharmacology and Chemotherapy

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Organophosphates, such as tetraethyl pyrophosphate (TEPP) and diisopropyl phosphorofluoridate (DFP), which have a dialkylphosphato group, form with human plasma cholinesterase (also called pseudo-or butyro-cholinesterase) two types of phosphorylated enzyme which possess different stabilities of the enzyme-phosphorus linkage (Hobbiger, 1955). Only phosphorylated enzyme J, which is formed initially, can be readily reactivated by nucleophilic reagents such as nicotinhydroxamic acid methiodide (NHAM). Phosphorylated enzyme I then changes into phosphorylated enzyme II, most probably by transphosphorylation. With DFP or diisopropyl p-nitrophenylphosphate (D 600) the rate of transphosphorylation is faster than with TEPP or diethyl p-nitrophenylphosphate (E 600) and at 370 C. and a pH of 7.45 approximately 90% of the inhibited enzyme is converted into phosphorylated enzyme II within 1 hour.Since the hydrolysis of acetylcholine in vivo is mainly accomplished by true cholinesterase (also called aceto-cholinesterase) the formation of a phosphorylated true cholinesterase which cannot be reactivated by nucleophilic reagents could seriously affect the therapeutic usefulness of NHAM and other more potent substances. The experiments on human plasma cholinesterase were therefore extended to human and bovine true cholinesterases which had been inhibited by an organophosphate containing either a diethyl-or diisopropylphosphato group and the findings are reported in this paper. In addition to NHAM, ammonium molybdate and pyridine-2-aldoxime methiodide, a potent reactivator of phosphorylated electric eel cholinesterase and phosphorylated human and rat true ch6lin-esterases (Wilson and Ginsburg, 1955;and Childs, Davies, Green, and Rutland, 1955), were used for enzyme reactivation. The results obtained with all these reactivators show that the organophosphates form two types of phosphorylated enzyme with human and bovine true cholinesterases which thus behave like plasma cholinesterase. Only the initial form (phosphorylated enzyme l) can readily be reactivated by the nucleophilic reagents. Transphosphorylation-formation of the truly irreversible phosphorylated enzyme Il-occurs both in vitro and in vivo, and the rate of transphosphorylation is dependent upon pH. METHODSAU experiments were carried out in the Warburg apparatus.Cholinesterase activity was determined by the manometric technique using 0.01 M-acetylcholine chloride as substrate for human and bovine true cholinesterases and 0.02 M-(±)-acetyl-f-methylcholine chloride as substrate for the true cholinesterases of whole rabbit blood. The procedures adopted for determination of enzyme activity and reactivation were the same as those described in an earlier publication (Hobbiger, 1955).Except when otherwise stated, the gas phase consisted of 95% N2+5% CO2 and a solution containing 0.025 M-NaHCO3, 0.075 M-NaCl, 0.075 M-KCl, 0.04 M-MgCl2 and 0.1% bovine plasma albumin (Armour Laboratories) was used for all dilutions and as the medium during inhibition, reactivation and determination ...

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Chemical Reactivation of Phosphorylated Human and Bovine True Cholinesterases

Hobbiger

1956

British Journal of Pharmacology and Chemotherapy

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“…It has recently been found that relatively small amounts of certain oximes speedily restore the activity of freshly phosphorylated true cholinesterases in vitro (Childs et al, 1955;Wilson and Ginsburg, 1955). The results presented in this paper show that in the mouse pyridine-2-aldoxime methiodide (P-2-AM), which was the most active oxime found so far, was less effective in reactivating phosphorylated true cholinesterases in vivo than was indicated by the in vitro experiments.…”

Section: Discussionmentioning

confidence: 42%

“…Reactivation of phosphorylated cholinesterase in aqueous solution is determined by the dialkylphosphoryl group and can be markedly enhanced in vitro by nucleophilic reagents which remove the dialkylphosphoryl group from the enzyme by virtue of their high affinity for the phosphorus. One of the most powerful reactivators of phosphorylated true cholinesterase is pyridine-2-aldoxime methiodide (P-2-AM) (Childs, Davies, Green, and Rutland, 1955;Wilson and Ginsburg, 1955). The concentrations of P-2-AM which are required to restore 50% of the activity of phosphorylated human true cholinesterase within 1 hr.…”

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Protection Against the Lethal Effects of Organo‐phosphates by Pyridine‐2‐aldoxime Methiodide

Hobbiger

1957

British Journal of Pharmacology and Chemotherapy

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The mechanism responsible for the protection against lethal organophosphate poisoning by pyridine-2-aldoxime methiodide (P-2-AM) was studied in the mouse. Two types of organophosphates were used: ethyl pyrophosphate (TEPP), E 600, Ro 3-0340, and Ro 3-0422 which form with true cholinesterase a diethylphosphoryl enzyme (1) and DFP, D 600, and Ro 3-0351 which form with true cholinesterase a diisopropylphosphoryl enzyme (2).In vitro and under the experimental conditions used more than 50% reactivation of (1) was obtained within 1 hr. by concentrations of P-2-AM ranging from 0.5 to 1 x I0D-M; 30 times higher concentrations of the oxime were required to achieve the same effect with (2). In vivo reactivation of phosphorylated true cholinesterases in blood amounted to 10 to 24% within the first 30 min. if 25 mg./kg. P-2-AM was injected (i.p.) 5 min. before a sublethal dose of TEPP, E 600, Ro 3-0340, or Ro 3-0422 and reactivation reached a maximum within I to 2 hr. after the injection of the oxime. P-2-AM was more effective when given 30 min. after the organophosphate. The effect of 25 mg./kg. P-2-AM on the phosphorylated true cholinesterase in brain (experiments with TEPP and E 600) was negligible. A dose of 25 mg./kg. P-2-AM had no consistent effect on the phosphorylated true cholinesterases in blood and brain of mice injected with sublethal doses of DFP, D 600, or Ro 3-0351.The protection by 25 mg./kg. P-2-AM against lethal doses of TEPP, E 600, Ro 3-0422, and Ro 3-0340 was greater than that obtained with 50 mg./kg. atropine sulphate, but the degree of protection was determined by the organophosphate itself and not its dialkylphosphoryl group. Protection by 25 mg./kg. P-2-AM against lethal doses of DFP, D 600, and Ro 3-0351 was negligible. The antidotal effect of P-2-AM was potentiated by atropine. Mice which were injected with atropine and P-2-AM were protected to a greater extent against DFP than against Ro 3-0422, and protection against DFP was only slightly less than protection against TEPP. This is difficult to reconcile with a specific action of P-2-AM on phosphorylated cholinesterases.

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“…55,56 Estudos identificaram uma relação entre a capacidade de reativação e a acidez do grupo oxima. Reativadores em geral apresentam pKa entre 7,0 e 8,0.…”

Section: Reativação Da Acetilcolinesteraseunclassified