Matej Maksimović

Interaction of organophosphorus compounds with carboxylesterases in the rat

Abstract Carboxylesterases (CarbE) are involved in detoxication of organophosphorus compounds (OPC) through two mechanisms: hydrolysis of ester bonds in OPC which contain them and binding of OPC at the active site of CarbE which reduces the amount of OPC available for acetylcholinesterase inhibition. This study of the interaction of rat plasma and liver CarbE with dichlorvos, soman and sarin in vitro and in vivo was undertaken in order to contribute to better understanding of the role of CarbE in detoxication of OPC. The results obtained have shown that inhibitory potency (I50) of dichlorvos, sarin and soman towards rat liver CarbE was 0.2 μM, 0.5 μM and 4.5 μM, respectively, for 20-min incubation at 25°C. Second-order rate constants (ka) for liver CarbE inhibition were 2.3×105 M-1 min-1, 6.9×104 M-1 min-1 and 1.1× 104 M-1 min-1 for dichlorvos, sarin and soman, respectively. The corresponding values for plasma CarbE could not be calculated because of dominant spontaneous reactivation of inhibited CarbE. CarbE inhibited with these OPC in vitro spontaneously reactivate with half-times of 18, 143 and 497 min for sarin, dichlorvos and soman in plasma and 111, 163 and 297 min for sarin, soman and dichlorvos in liver, respectively. These results were also confirmed in experiments in vivo in which rats were subcutaneously treated with 0.5 LD50 of these agents. The half-times of spontaneous reactivation of rat plasma CarbE in vivo were 1.2, 2.0 and 2.7 h for dichlorvos, sarin and soman, respectively. These findings have changed current understanding of the mechanism of interaction of CarbE with OPC and involvement of the enzymes in detoxication of OPC, suggesting an active and important role of the enzymes in metabolic conversions of OPC to their less toxic metabolites.

Oxime-induced reactivation of acetylcholinesterase inhibited by phosphoramidates

The reaction of human erythrocyte acetylcholinesterase (AChE) with a set of structurally related phosphoramidates was studied in order to investigate the properties of phosphorylated enzyme and the effects of 4 oximes PAM-2, TMB-4, HI-6 and BDB-106 on the reactivation of inhibited AChE. Second-order rate constant of the phosphorylation reaction of the compounds towards the active site of AChE range between 5.0 x 10(2) and 4.9 x 10(6) M-1min-1 and their inhibitory power (I50) was from 7.3 x 10(-5) to 5.7 x 10(-9) M for 20 min incubation at 37 degrees C. The oximes used were weak reactivators of inhibited AChE except for (C4H9O)(NH2)P(O)DCP (DCP, -O-2,5-dichlorphenyl group) and (C6H13O)(NH2)P(O)SCH3 where we have obtained good reactivation. Imidazole oxime BDB-106 proved to be a potent reactivator of tabun-inhibited AChE.

A comparison of trimedoxime, obidoxime, pralidoxime and HI-6 in the treatment of oral organophosphorus insecticide poisoning in the rat

This study summarizes the results of examination of acute oral toxicity of 26 organophosphorus insecticides in rats. The effectiveness of trimedoxime, obidoxime, pralidoxime and HI-6, given with atropine and diazepam, was tested in the treatment of poisoning with 2 LD50 of the insecticides. It was shown that the oximes were potent antidotes in poisoning with phosphate insecticides. Obidoxime, pradidoxime and HI-6 had low effectiveness in the treatment of poisoning with phosphonates and phosphorothiolates. However, none of the oximes was an effective antidote in poisoning with dimethoate and pyridafenthion. Trimedoxime was the most effective oxime in the treatment of insecticide poisoning, being successful especially at the lowest tested doses.

Interaction of phosphamidon with neuropathy target esterase and acetylcholinesterase of hen brain

Phosphamidon (PSM) is an organophosphorus insecticide widely used in agriculture. This study was undertaken to examine the interaction of PSM with acetylcholinesterase (AChE) and neuropathy target esterase (NTE) of hen brain in vitro and in vivo. PSM was a potent inhibitor of AChE, with an I50 of 2.9μM and second-order rate constant (ka) of 1.2×104 M−1 min−1 at 37°C. PSM-inhibited AChE aged rapidly (t1/2=1.9h). Pyridinium oximes pralidoxime, trimedoxime, obidoxime and HI-6 were effective reactivators of PSM-inhibited AChE, providing up to 75% reactivation. PSM was one of the weakest inhibitors of NTE among organophosphorus compounds, with an I50 of 19 mM and ka of 1.8 M−1 min−1 at 37°C. Inhibited NTE did not reactivate spontaneously and KF-induced reactivation was not obtained even at the earliest tested moments, so it was not clear whether aging of PSM-inhibited NTE occurred very quickly or the KF molecule could not affect the stability of phosphoryl-NTE bond. From the ratio of kas for NTE and AChE (0.00015) it was predicted that delayed neuropathic effects of PSM in vivo would appear only at doses far above the acute LD50. The LD50 value of PSM p.o. for hens was 9 mg/kg. Hens were treated with a single oral dose of PSM, combined with standard antidotal treatment which included atropine, physostigmine, pralidoxime and anticonvulsant midazolam. Doses of 90 and 250 mg/kg caused up to 27% and 45% NTE inhibition 48h after poisoning, respectively. Clinical signs of neuropathy were not seen up to 25 days after treatment, which could be expected, since the proposed level (>70%) of NTE inhibition necessary for the occurrence of delayed neuropathy was not achieved. The results suggest that PSM, at doses tested, has no ability to cause delayed neuropathy in hens without showing signs of severe cholinergic intoxication.

Reactivators of organophosphate-inhibited cholinesterase

Two isomeric cyclopentylcarbonyl and two cycloheptylcarbonyl derivatives of 2-hydroxyiminomethyl-1-[3-(1-pyridinio-2-oxapropyl] pyridinium diiodide and 4-hydroxyiminomethyl-1-[3-(1-pyridinio-2-oxapropyl] pyridinium diiodide were prepared and characterized by spectroscopic methods. The inhibitory power (I50) of the investigated oximes was determined using purified bovine erythrocyte AChE and human erythrocyte AChE. Percentage of reactivation after 30 min was estimated after inhibition of human erythrocyte AChE by sarin, VX, tabun, soman, and paraoxon. The in vitro protective indices (p.i. and P50) against inhibition by soman have been calculated using bovine erythrocyte AChE for p.i. and human erythrocyte AChE for P50. Their I50 for human erythrocyte AChE varied from 1.4–9.8 (10−4 mol · dm−3) and for bovine erythrocyte AChE in the range of 1.1–17 (10−5 mol · dm−3). With 2 × 10−5 mol · dm−3 oximes the percent of reactivation was: 0–17% for paraoxon-inhibited AChE, 9–49% for sarin-inhibited AChE, 16–65% for VX-inhibited AChE, 0–8% for tabun-inhibited AChE, and 0–4% for soman-inhibited AChE. The 2-hydroxyimino derivatives protect human erythrocyte AChE and purified bovine erythrocyte AChE from inhibition by soman.

Modification of the rate of aging of diisopropylfluorophosphate-inhibited neuropathy target esterase of hen brain

This study was aimed to investigate the possibility of modifying the rate of aging of diisopropylfluorophosphate-inhibited neuropathy target esterase (NTE) of hen brain. This reaction on NTE occurs with a half-time of 7.4 min. Atropine was effective in decreasing the rate of aging on DFP-inhibited NTE and this effect was time- and concentration-dependent. Atropine was also a weak but progressive inhibitor of NTE activity (I50 = 80 mM) and this reaction appears to be reversible at lower atropine concentrations. Among compounds containing oxime functional groups only OPAB, having longer methylene chain and being more lipophylic than other oximes usually used in acetylcholinesterase (AChE) reactivation studies, was effective in decreasing the rate of aging on DFP-inhibited NTE. However, when atropine and oximes were used together we have obtained a potentiating and/or synergistic effect which was most significant with combination of atropine and TMB-4 giving up to a 15-fold decrease in the rate of aging reaction. The efficacy of this particular combination was concentration-dependent. We have also discussed similarities and differences in aging reaction occurring on NTE and AChE.

Oral kinetics and bioavailability of the cholinesterase reactivator HI-6 after administration of 2 different formulations of tablets to dogs

A one-compartment open model with first-order absorption was used for comparing new oral formulations of the potent acetylcholinesterase reactivating oxime HI-6. Although mean peak plasma levels did not differ between retard and conventional tablets (21.38 and 20.74 mumol/l), the time for reaching peak levels was significantly longer (5.5 h) with retard than with conventional tablets (2.86 h). Among other pharmacokinetic estimates only absorption half-lives and areas under the concentration-time curve (AUC) were significantly different (P less than 0.05). The AUC with retard tablets was 8.07% and that of conventional tablets 5.42% of intravenous AUC, indicating low bioavailability of oral HI-6 formulations. Potential therapeutic use of HI-6 requires, therefore, further investigations in order to improve its gastrointestinal absorption.

Effects of atropine, trimedoxime and methylprednisolone on the development of organophosphate-induced delayed polyneuropathy in the hen

In this study we have examined the effects of atropine, trimedoxime (TMB-4) and methylprednisolone (MP) on the development of organophosphate-induced delayed polyneuropathy (OPIDP) in the hen. The birds were treated with standard neuropathic dose of diisopropylfluorophosphate (DFP) (1.1 mg/kg, sc), which produced OPIDP that could be graded as 5 on the 8-point scale, and the development of OPIDP was observed for the next 22 days. The results obtained have shown that atropine (20 mg/kg, ip), TMB-4 (15 mg/kg, im) and MP (2 or 10 mg/kg, ip) either alone or in different combinations are able to improve the condition of the birds. The most potent effect was obtained with atropine, TMB-4 (given 20 min before DFP) and MP (2 mg/kg, sc, given 20 min before and at 48 hour intrevals after poisoning) since the signs of OPIDP could hardly be seen (grade 1 at the 8-point scale). When TMB-4 and MP were given 15 or 40 min after DFP the protective/therapeutic effects of these drugs appeared to be diminished since walking disorders were more serious and graded as 2 or 4, respectively. The possible mechanisms of the action of the drugs in respect to OPIDP are discussed. In conclusion, the results of this study have shown that it is possible to prevent the development of DFP-induced OPIDP in the hen by treatment with atropine, trimedoxime and methylprednisolone when they were given before or soon after DFP.