2-pyridine Aldoxime Methiodide Research Articles

Calorimetric Determination of Phosphorylated Serum Cholinesterase and Its Reactivation by an Antidote

Abstract Serum cholinesterase, inhibited in vitro up to 92% by 0, 0 dimethyl-2, 2-dichlorovinyl phosphate (4.52 × 10−9 mole/ml of 40% serum), was reactivated up to 35% with 2-pyridine aldoxime methiodide (3.5 × 10−6 mole/ml of 40% serum). Evaluation was based on the reaction rate between cholinesterase and acetylcholine chloride (2.2 × 10−6 mole) measured with a microcalorimeter. It was thus possible to approximate the “normal” or pre-exposure level of serum cholinesterase, while simultaneously determining the extent of poisoning.

New fluorescent organophosphates as probes for studying aging-induced conformational changes in inhibited acetylcholinesterase

Aging of organophosphoryl-acetylcholinesterase (AChE) conjugates, involving dealkylation of the bound organophosphoryl group, renders AChE resistant to reactivation by 2-pyridinealdoxime methiodide (2-PAM). The fluorescent organophosphates 1-pyrenebutyl ethylphosphorochloridate (PBEPC) and 1-pyrenebutylphosphorodichloridate (PBPDC) react stoichiometrically with purified electric eel AChE. PBEPC forms a non-aged AChE conjugate readily reactivated by 2-PAM; PBPDC forms an aged conjugate which cannot be reactivated. There is no difference in the wavelengths of excitation and emission maxima between the aged and non-aged AChE conjugates. However, the fluorescence quantum yield of pyrene in the non-aged conjugate is reduced by ca. 50% compared to the aged conjugate and from the shortening of the fluorescence decay time in the non-aged conjugate, it is concluded that the quenching is primarily dynamic. It is suggested that in the aged conjugate the organophosphoryl moiety is less accessible to the external medium than in the non-aged conjugate.

Fluorescent organophosphates: novel probes for studying aging-induced conformational changes in inhibited acetylcholinesterase and for localization of cholinesterase in nervous tissue.

Aging of acetylcholinesterase (AChE) inhibited by certain organophosphates such as diisopropylfluorophosphate apparently involves dealkylation of the bound organophosphoryl moiety; this renders the inactive enzyme resistant to reactivation by quaternary oximes such as 2-pyridinealdoxime methiodide (2-PAM) which are used in therapy of organophosphate intoxication. The fluorescent pyrenyl organophosphates synthesized in this study were designed to detect putative conformational changes which might explain this resistance. The following inhibitors: 1-pyrenebutyl phosphorodichloride (PBPDC), 1-pyrenebutyl ethylphosphorochloridate (PBEPC), and 1-pyrenebutyl ethylphosphorofluoridate (PBEPF), react specifically with purified electric eel AChE (ki = 10(6)-10(7) M-1 min-1). AChE inhibited by PBEPC and PBEPF was readily reactivated by 2-PAM, while enzyme inhibited PBPDC could not be reactivated. Conjugates were prepared of both PBEPC and PBPDC with AChE, each containing one molecule of florophore per catalytic subunit. Thus two stoichiometric conjugates, PBEP-AChE (non-aged) and POBP-AChE (aged), were obtained. The two complexes exhibited identical absorption spectra, but differed in their steady-state fluorescence spectra. Although the wave-lenths of the excitation and emission spectra were similar, the pyrene fluorescence of the non-aged conjugate was ca. 50% quenched relative to the aged conjugate. Nanosecond fluorescence decay studies revealed two principal lifetime components of pyrene fluorescence. Both were longer for the aged (PBP-AChE) than for the non-aged (PBEP-AChE) conjugate and revealed a ca. 50% lower quantum yield for the non-aged as compared to the aged conjugate. A possible interpretation for these results is that in the aged conjugate the organophosphoryl moiety is less acessible to the external medium. Measurement of quenching of pyrene fluorescence in the aged and non-aged conjugates by the peripheral anionic site ligand propidium also indicated marked conformational differences between the two conjugates, and circular polarization of luminescence measurements revealed that propidium itself induced a substantial conformational change in both conjugates. Fluorescence lifetime measurements revealed that whereas propidium had little effect on the decay parameters for the non-aged conjugate it caused a decrease in lifetime and in relative quantum yield for the aged conjugate. PBEPF virtually eliminated cholinesterase activity in dissociated cord and brain cultures. Fluorescence microscopy reveals fine green fluorescent grains distinctly located throughout many neurons and glia. Labelling is much more pronounced in larger and older neurons. No specific fluorescence could be detected in cultures preincubated with nonfluorescent organophosphates.

Formation of an unstable covalent intermediate during the inhibition of electric-eel acetylcholinesterase with 1,3,2-dioxaphosphorinane 2-oxides

The kinetics of interaction of eel acetylcholinesterase (EC 3.1.1.7) with 1,3,2-dioxaphosphorinane 2-oxides were investigated. It was demonstrated that the rate of spontaneous re-activation as well as the re-activation profile in the presence of 2-pyridine aldoxime methiodide of the inhibited enzyme are irrespective of the leaving group of three inhibitors and exhibit the same values. The dissociation constant of the corresponding Michaelis complex was evaluated by two independent methods and the results were found to be in close agreement. It was shown that the active site is essential for interaction between the enzyme and the various dioxaphosphorinanes. The mixed anhydride of diethyl phosphoric acid and 2-hydroxy-1,3,2-dioxaphosphorinane 2-oxide behaves exactly as would be predicted from a typical diethyl phosphate inhibitor. Enxyme that was incubated with the cyclic acid or the corresponding methyl ester recovered immediately upon extensive dilution. Inhibition of enzyme in the presence of high concentratasions of the corresponding 2-chloro and 2-fluoro derivatives decreased the regeneration rates as well as the maximal amount of the re-activated enzyme. This observation could not be explained in terms of a classical aging process. On the basis of the kinetics observations it is suggested that an unstable covalent phospho-enzyme intermediate is formed during the reaction between acetylcholinesterase and 1,3,2-dioxaphosphorinane 2-oxides.

Acetylcholinesterase in the fast extraocular muscle of the mouse by light and electron microscope autoradiography.

The distribution of acetylcholinesterase (ACHe) in the twitch fibers of the extraocular muscles of the mouse was examined by light and electron microscope autoradiography after labeling with radioactive diisopropyl fluorophosphate (DFP) with, and without, 2-pyridine aldoxime methiodide (2-PAM) reactivation. The values obtained were compared with those previously reported for the diaphragm and sternomastoid muscles. The extraocular muscles were studied because they differ from the other two muscles in that they are among the fastest of the mammalian muscles, yet their endplates have sparse junctional folds. They could thus provide information on the extent to which ACHe concentration is an invariant feature of endplate morphology and what, if any aspects may be related to their fast speed of response. We found, using light microscope autoradiography, that in the twitch fibers of the extraocular muscle, there is n average of 6.4 +/- 2.1 X 10(7) DFP-binding sites per endplate, of which 29% (1.8 X 10(7)) are reactivated by 2-PAM and are thus AChe. The morphology of the extraocular endplates allowed us to conclude, on statistical grounds, that the AChe site are probably localized not only along the surface area of the postjunctional membrane (PJM) but also along the surface of the presynaptic axonal membrane. Based on this localization, we calculate 7,800 DFP sites and 2,500 2-PAM-reactivated sites/micron 2 of surface area of pre-and postjunctional membrane. This stacking density of DFP-binding sites per surface area of membrane ( probably in the overlying sheets of basal lamina) is very similar to that in the diaphragm and sternomastoid muscles.

Effects of quaternary ligands on the inhibition of acetylcholinesterase by arsenite.

Arsenite inhibits acetylcholinesterase in a second-order reaction. The rate and equilibrium constants depend upon pH and have values on the order of 10(2) M-1 min-1 and 10(5) M (dissociation), respectively. Some quaternary ammonium ligands completely block the arsenite inhibition of the enzyme, others decrease the rate of the reaction and some, notably pyridine-2 aldoxime methiodide, greatly accelerate the rate of the reaction, up to 220-fold. Accelerators may bind at a separate enzyme site distinct form the anionic site involved in substrate binding. Although the kinetic data are consistent with a covalent reaction between arsenite and acetylcholinesterase, chemical evidence excludes the involvement of sulfhydryl groups which are usually implicated in arsenite inhibition.

Inhibition of cholinesterase activity induced by pyridaphenthion

Inhibitory effects on cholinesterase (ChE) activity in blood of the rabbit induced by pyridaphenthion(PD), an organophosphorus compound, and effects of 2-pyridine aldoxime methiodide(PAM) on this inhibition were examined. 1) Experimental results within 24 hr: An oral administration of each dose of PD(100 approximately 750 mg/kg) gradually decreased ChE activity and ChE activity value decreased to 20.5% in the erythrocytes and 21.5% in the plasma 24 hr after administration of 500 mg/kg of PD. However, this value recovered remarkably with an intravenous injection of PAM. The ChE activity value was 55.5% in the erythrocytes and 41.4% in the plasma with a single injection, and respectively 67.8% and 59.1% when PAM was injected three times. 2) Experimental results for 14 days: Following an increase of the administered dose (100 approximately 400 mg/kg) of PD, a decrease in ChE activity was apparent and a recovery to normal values was delayed. This inhibition could be ameliorated quickly with an injection of PAM. Considering that PAM does not have remarkable detoxicative effects on organophosphorus compounds which have a low toxicity, PAM appears to be a promising antidote against PD.

Biochemical basis for the toxic effects of triethyl lead.

<b>Galzigna, L., Ferraro, M. V., Manani, G., and Viola, A. (1973).</b><i>British Journal of Industrial Medicine,</i><b>30,</b> 129-133. <b>Biochemical basis for the toxic effects of triethyl lead.</b> The effects of triethyl lead (PbEt₃) have been studied <i>in vitro</i> on the cholinesterase activity of rat diaphragm and <i>in vivo</i> on serum cholinesterase in the dog. PbEt₃ dramatically increases the duration of succinylcholine-induced myoneural block and pyridine-2-aldoxime methiodide (PAM) is able to counteract both cholinesterase inhibition and the effects on neuromuscular transmission. On the other hand, PbEt₃ catalyses the rearrangement of catecholamines to aminochromes <i>in vitro</i> and inhibits catecholamine effect on smooth muscle contraction. The toxicity of PbEt₃ and particularly its action on the central nervous system can be explained by a combination of effects which might result from an upset of cholinergic and adrenergic central pathways due to the formation of endogenous psychotogenic complexes.

Inhibitory effect of triethyl lead on serum cholinesterase in vitro

The inhibitory effect of triethyl lead, which is the main toxic metabolite of tetraethyl lead responsible for industrial intoxication, is demonstrated in vitro with a purified enzyme preparation of horse serum cholinesterase. Pyridine-2-aldoxime methiodide (PAM) and pralidoxime methylsulfate are able to restore the activity of the inhibited enzyme up to 80%. Such a reactivation allows us to consider both substances as potential antidotes for tetraethyl lead poisoning.

Absorption and Excretion of Drugs. XLI. Studies on the Gastrointestinal Absorption of 2-Pyridine Aldoxime Methiodide and Its Derivatives.

The gastrointestinal absorption of pyridine aldoxime methiodides has been studied in rats using single loop and perfusion techniques. Contrary to their apparent physicochemical properties, these compounds were absorbed from the intestine to an appreciable extent but not from the stomach and the rectum within one hour. The absorption from the intestine became saturated when the concentration of the drugs increased and one will inhibit the absorption of the other. The presence of a metabolic inhibitor in the drug solution had no effect on the absorption of these compounds. Despite the complexity of the systems used, a straight line relationship between the reciprocal of absorption rate and the reciprocal of initial concentration was obtained for each compound studied.

Possible reactivating and sensitizing action of neuromyally acting agents.

The effects of a bisquaternary oxamide, methoxyambenonium (Meamb), d-tubocurarine ( d-tbc ), and of an oxime, pyridine-2-aldoxime methiodide (P-2-AM), upon the frog neuromyal junction, treated by organophosphorus anticholinesterases, TEPP and DFP, were studied. The oxamide and d-tbc resembled P-2-AM and other reactivators in that they caused a return of the sustained tetanic response and of the post-tetanic potentiation, after these were blocked by TEPP or DFP. Ringer's solution was did not reverse these actions of the oxamides and of d-tbc . In these actions, these drugs resembled two types of reactivators, NaF and P-2-AM. The well-known marked prolongation of the endplate potential (e.p.p.) by TEPP or DFP was markedly shortened by the oxamide and by d-tbc . The former also increased the amplitude of the e.p.p. These effects were obtained whether the transmission was blocked by Mg or by d-tbc itself. The finding of earlier investigators that d-tbc lowers the amplitude but does not shorten the duration of the e.p.p. was confirmed. P-2-AM antagonized the d-tbc block of the twitch response, increased the amplitude but not the duration of the e.p.p. and augmented but did not prolong acetylcholine depolarization of the e.p. This effect, described in this laboratory as sensitization, and distinct from anticholinesterase action, is shown by several reactivators, oxamides and several other neuromyal facilitators. It is suggested that the actions of the oxamide and of d-tbc upon TEPP or DFP response of the neuromyal junction may be explained by their reactivation capacity. Conversely, reactivators may exhibit sensitizing and facilitating actions.

The toxicity of haloxon to geese, ducks and hens, and its relationship to the stability of the DI-(2-chloroethyl) phosphoryl cholinesterase derivatives

Geese are very susceptible to the acute toxic effects ofhaloxon [di-(2-chloroethyl) 3-chloro-4-methylcoumarin-7-yl phosphate], 50 mg/kg being sufficient to elicit a lethal response in most birds. Haloxon is much less toxic to ducks than to geese, whilst hens can tolerate very large doses of the anthelmintic, 5000 mg/kg being the dose necessary to produce the typical symptoms of acute toxicity. Pyridine 2-aldoxime methiodide (2-PAM) is a very effective antidote to haloxon toxicity in geese, but has much less effect in ducks. The cholinesterase of goose brain differs from that of ducks and hens in forming a stable di-(2-chloroethyl) phosphoryl derivative after reaction with haloxon, and this stability virtually precludes spontaneous reactivation of the cholinesterase. However, the in vitro recovery of goose brain cholinesterase activity in the presence of 2-PAM is rapid, as is the response of the poisoned bird dosed with the oxime. The di-(2-chloroethyl) phosphoryl cholinesterase from duck brain is much less stable than that from goose brain, but more stable than that from hen brain cholinesterase This difference is reflected in the greater toxicity of haloxon to ducks than to hens, and also in the fact that 2-PAM speeds dephosphorylation of the di-(2-chloroethyl) phosphoryl cholinesterase formed by duck brain enzyme, but has no effect on the corresponding reaction involving hen brain cholinesterase. The plasma cholinesterases of all three species are similar in their susceptibility to inhibition by haloxon and in their rate of spontaneous reactivation (dephosphorylation) following inhibition.

Use of the chick embryo for testing the toxicity of cholinesterase-inhibiting compounds

The use of the chick embryo for screening cholinesterase-inhibiting compounds is suggested. Malathion and trichlorfon were injected into the air cell of 14-day-old incubated eggs and blood was drawn from the embryo 6–8 hours after injection of malathion, and 1 hour after injection of trichlorfon. The dosages necessary for 50% and 90% inhibition of whole-blood cholinesterase activity (IC 50 and IC 90, respectively) were determined. The IC 50 and IC 90 were 4.6 mg/egg and 16 mg/egg, respectively, for malathion, and 0.084 mg/egg and 0.77 mg/egg, respectively, for trichlorfon. Inhibition of cholinesterase by malathion was not antagonized by the injection of 2-pyridine aldoxime methiodide (2-PAM) at dosages of 2 mg/egg or 10 mg/egg given 30 minutes after drug administration, while such antagonizing effect was obtained with 2-PAM injected similarly into eggs treated with 0.1 mg/egg of trichlorfon.

2-pyridine aldoxime methiodide in the treatment of post-operative acute pulmonary edema: possible role of cholinesterase.

2-pyridine aldoxime methiodide in the treatment of post-operative acute pulmonary edema: possible role of cholinesterase.

The effects in sheep of quinuronium and amicarbalice and the influence of atropine pyridine 2-aldoxime methiodide (2-PAM). Adrenaline and mepyramine.

The effects in sheep of quinuronium and amicarbalice and the influence of atropine pyridine 2-aldoxime methiodide (2-PAM). Adrenaline and mepyramine.

The Anticholinesterase Activity of the Babesicidal Agents Quinuronium and Amicarbalide and the Influence of Pyridine 2-Aldoxime Methiodide

SUMMARY The piroplasmocidal agents quinuronium and amicarbalide were investigated for anticholinesterase activity in the blood of 9 species in vitro. Quinuronium was shown to be a potent inhibitor of circulating cholinesterases in all the species, whereas amicarbalide was much less active. In the sheep in vivo, quinuronium caused about 40% inhibition of cholinesterase and recovery of activity took place over a period of 24 to 48 hr. Eserine produced profound but transient inhibition and amicarbalide had a very small effect. Pyridine 2-aldoxime methiodide (2-PAM) failed to protect the enzyme from inhibition by quinuronium or eserine but temporarily relieved the inhibition produced by the organo-phosphorus compound octamethylpyrophosphoramide (OMPA). During all experiments in vivo in sheep, atropinisation (1 mg. per kg.) was used, and it was concluded that atropine provided the best antidote to quinuronium poisoning and the 2-PAM did not appear to alleviate circulating cholinesterase activity which had been inhibited by quinuronium.

Distribution and excretion of pyridine-2-aldoxime methiodide (PAM); atropine and PAM in sarin poisoning

Distribution and excretion of methyl-C 14 tagged PAM (pyridine-2-aldoxime methiodide) was studied in mice and dogs. The results show that, after intravenous injection, PAM penetrates readily into the liver and kidney of mice and into most soft tissues of the dog. No isotope was found in the brain of the mice, and only trace amounts of isotope were found in the brain of 1 dog. Isotope is retained in the tissues except for brain after it has been cleared from the blood. Approximately 50% of the isotope was recovered in the urine in 90 minutes. In a single human, 65% of intravenously injected isotope was recovered in 2 hours. Spontaneous motor activity of mice is decreased following small doses of sarin. This effect of sarin is antagonized by atropine but not by methylatropine or PAM. The mortality in mice injected intraperitoneally with 2 LD 50's of sarin was not influenced by prophylactic intraperitoneal injection of atropine, methylatropine, PAM, or methylatropine plus PAM. However, atropine plus PAM produced complete protection against the above dose of sarin. The effect of atropine when used in combined atropine-oxime therapy of anticholinesterase poisoning is ascribed to its ability to protect the central nervous system from effects of the antiesterase.

Oral Occupational Parathion Poisoning Treated with 2-PAM Iodide (2-Pyridine Aldoxime Methiodide)

Oral Occupational Parathion Poisoning Treated with 2-PAM Iodide (2-Pyridine Aldoxime Methiodide)

Oral occupational parathion poisoning treated with 2-PAM iodide (2-pyridine aldoxime methiodide).

OCCUPATIONAL poisonings in agricultural workers caused by organic phosphorus insecticides have become fairly common since the compounds were introduced. These compounds are potent cholinesterase inhibitors and produce symptoms that are due at least partly to this anticholinesterase action. Atropine has long been used in the treatment of intoxication from the organic phosphorus insecticides. It produces its action by pharmacologic blockade of the muscarinic effects of acetylcholine and as such is an incomplete antidote. With the demonstration that organic-phosphate-inhibited cholinesterase can be chemically reactivated by the use of certain hydroxamic acids or aldoxime-type compounds such reactivator therapy supplies a theoretically specific . . .

Ability of venoms to render squid axons sensitive to curare and acetylcholine

1. 1. Cottonmouth-moccasin venom is more potent than copperhead moccasin, fer-de-lance or Western diamondback rattlesnake venom in rendering (+)-tubocurarine (curare) capable of affecting conduction of the squid giant axon. 2. 2. Bee venom is more potent than the snake venoms. It irreversibly blocks axonal conduction in a concentration of 10 μg/ml. However, in lower concentrations (1–2 μg/ml) it does not render curare effective. 3. 3. Following exposure to cottonmouth venom, 8.8·10 −4 M acetylcholine markedly decreased axonal electrical activity. 4. 4. Physostigmine was antagonistic to the action of acetylcholine. An explanation for this apparently surprising effect is offered. 5. 5. Cottonmouth venom also rendered the following compounds active: decamethonium, 2-pyridine aldoxime methiodide and 2-benzoylpyridine oxime. The potency of atropine and ethanol was increased by cottonmouth venom. 6. 6. Carbamylcholine in 2.8·10 −2 M did not significantly affect conduction after pretreatment with cottonmouth venom while 1·10 −2 M nicotine had a weak effect.