In vitro and in vivo protection of acetylcholinesterase against organophosphate poisoning by pretreatment with a novel derivative of 1,3,2-dioxaphosphorinane 2-oxide.

Covalent molecular combinations of a cyclic phosphate (dioxaphosphorinane) and a potential leaving group, such as 3-(trimethylammonio)phenol iodide (TMPH), suggested the synthesis of O-[3-(trimethylammonio)phenyl]-1,3,2-dioxaphosphorinane 2-oxide iodide (TDPI). TDPI inhibited acetylcholinesterase (AChE) (ki = 8.4 x 10(3) M-1 min-1) via the formation of an unstable covalent intermediate. TDPI-inhibited AChE hydrolyzed spontaneously with t1/2 approximately equal to 10 min. Butyrylcholinesterase (BuChE) was also inhibited by TDPI (ki = 1.8 x 10(4) M-1 min-1), but the inhibited BuChE was more stable (greater than 10 times) than the corresponding AchE-TDPI conjugate. Pretreatment of mice with TDPI conferred protection against 22 LD50's of paraoxon and 5 LD50's of soman, provided that treatment with anticholinergics and an oxime followed administration of these anticholinesterase poisons. Correlation between in vitro and in vivo observations suggests that the main protection of AChE conferred by TDPI results from temporary masking of the active site of the enzyme. The acute toxicity of TDPI was found to be 444 mg/kg (sc, mice), whereas analogous carbamates and a noncyclic phosphate also displaying antidotal properties are greater than 170 times more toxic.

A preclinical study of EO-122, a new lidocaine-like antiarrhythmic drug.

The 2,6-dimethylanilide of quinuclidine-3-carboxylic acid hydrochloride (EO-122), a new structural analog of lidocaine, has been shown to possess potent antiarrhythmic activity in experimentally induced arrhythmias in animals. Restoration of normal sinus rhythm and suppression of ouabain-induced arrhythmia in cats and dogs, and of coronary occlusion-induced arrhythmia in dogs, followed a single IV injection of 1--3 mg/kg, with an onset of 2 minutes and a duration of 20--240 minutes. Occlusion-induced arrhythmia was likewise suppressed after an oral dose of 10--20 mg/kg, with an onset of 11--65 minutes and a duration of 25--120 minutes. Under similar conditions, lidocaine was either totally ineffective or of ultra-short duration. The bioavailability of EO0122 by the oral route exceeded 80% of the oral dose. Therapeutic blood concentrations were in the range 0.5--7 microgram/ml. At about 5 microgram/ml there was a slight depression of cardiac function in the anesthetized cat, but not in the conscious dog. In cats, complete A-V block occurred at concentrations of 60--70 microgram/ml. The IV LD50 in mice was 22 mg/kg, and in rabbits 8.5 mg/kg. No overt signs of neurotoxicity could be observed at any dose of EO-122. The pharmacokinetic profile of the drug fits a two-compartment open model, with t1/2 congruent to 150 min and Vd (SS) congruent to 1.5 l/kg.

The muscarinic antagonists aprophen and benactyzine are noncompetitive inhibitors of the nicotinic acetylcholine receptor.

Certain muscarinic antagonists (e.g., atropine, aprophen, and benactyzine) are used as antidotes for the treatment of organophosphate poisoning. We have studied the interaction of aprophen and benactyzine, both aromatic esters of diethylaminoethanol, with nicotinic acetylcholine receptor (AChR) in BC3H-1 intact muscle cells and with receptor-enriched membranes of Torpedo californica. Aprophen and benactyzine diminish the maximal carbamylcholine-elicited sodium influx into muscle cells without shifting Kact (carbamylcholine concentration eliciting 50% of the maximal 22Na+ influx). The concentration dependence for the inhibition of the initial rate of 22Na+ influx by aprophen and benactyzine occurs at lower concentrations (Kant = 3 and 50 microM, respectively) than those needed to inhibit the initial rate of [125I]-alpha-bungarotoxin binding to the agonist/antagonist sites of the AChR (Kp = 83 and 800 microM, respectively). The effective concentration for atropine inhibition of AChR response (Kant = 150 microM in BC3H-1 cells) is significantly higher than those obtained for aprophen and benactyzine. Both aprophen and benactyzine interact with the AChR in its desensitized state in BC3H-1 cells without further enhancing agonist affinity. Furthermore, these ligands do not alter the value of Kdes (equilibrium concentration of agonist which diminishes 50% of the maximal receptor response) in BC3H-1 muscle cells. The affinity of aprophen and benactyzine for the allosterically coupled noncompetitive inhibitor site of the AChR in Torpedo was determined using [3H]phencyclidine as a probe. Both compounds were found to preferentially associate with the high affinity (desensitized) state rather than the resting state of Torpedo AChR. There is a 14- to 23-fold increase in the affinity of aprophen and benactyzine for the AChR (KD = 0.7 and 28.0 microM in the desensitized state compared to 16.4 and 384 microM in the resting state, respectively). These data indicate that aprophen and benactyzine binding are allosterically regulated by the agonist sites of Torpedo AChR. Thus, aprophen and benactyzine are effective noncompetitive inhibitors of the AChR at concentrations of 1-50 microM, in either Torpedo or mammalian AChR. These concentrations correspond very well with the blood level of these drugs found in vivo to produce a therapeutic response against organophosphate poisoning.