Inhibitor Report for: Ambenonium

Edrophonium (ethyl(m-hydroxyphenyl)dimethylamine) acutely modifies carnitine levels in different rat tissues, increasing hepatic and reducing blood and renal levels. After 2 h edrophonium treatment, the total serum carnitine levels were decreased by 16 (P < 0.001) and 33 (P < 0.001) percent in fed and fasted rats respectively compared to control, and in kidney the levels decreased by 11 (P < 0.05) and 34 (P < 0.001) percent whereas in liver the edrophonium treatment increased the levels by 43 (P < 0.001) and 59 (P < 0.001) percent. The edrophonium action does not depend on the route of administration or on the nutritional state of the animal. Its activity on carnitine levels is neither accompanied by significant variation of serum parameters of carbohydrate, fat and protein metabolism nor of insulin levels. The edrophonium activity is not related to cholinergic action, as physostigmine and ambenonium at concentrations known to increase cholinergic activity do not modify carnitine distribution in tissues. Trimethylphenylammonium (TPA) and trimethyl(p-aminophenyl)ammonium (TPA.NH2), compounds structurally similar to edrophonium, are on the contrary active on levels of carnitine and this effect is not related to their cholinergic potency. In 24 h fasted rats after the TPA and TPA. NH2 treatment, the total serum carnitine levels were decreased by 32 (P < 0.001) and 13 (n.s.) percent respectively compared to control, and in kidney the levels decreased by 15 (P < 0.02) and 5 (n.s.) percent, whereas in liver the treatment increased the levels by 72 (P < 0.001) and 45 (P < 0.01) percent. Moreover atropine, an acetylcholine antagonist, affects carnitine distribution in a way similar to edrophonium. Edrophonium activity on carnitine distribution, probably affects (inter)cellular carnitine transport by direct action on plasma membrane. Effect on capillary endothelium may be responsible for its observed action on muscle contraction force in imminent ischemia.

Steady state patterns of inhibition of purified human erythrocyte acetylcholinesterase by three inhibitors were analyzed. Edrophonium acted essentially as a competitive inhibitor, whereas tacrine and ambenonium gave mixed competitive and uncompetitive inhibition with acetylthiocholine as substrate. Inhibition constants for the competitive components were 470 microM for edrophonium, 65 microM for tacrine, and 0.12 nM for ambenonium. The extremely high affinity of ambenonium permitted analysis of the rates of approach to steady state inhibition. These rates were characterized by a single exponential time course with rate constants, kexp, that showed a linear dependence when plotted against ambenonium concentration, at fixed substrate concentration. The intercepts of these plots were independent of the substrate concentration and indicated an ambenonium dissociation rate constant of 0.013 +/- 0.002 sec-1. The slope of the plot at the lowest substrate concentration approximated the ambenonium bimolecular or association rate constant and gave a value of 5.2 +/- 0.6 x 10(7) M-1 sec-1. Three models were examined to account for the nearly linear dependence of the slopes of these plots on the substrate concentration. These models indicated that ambenonium and acetylthiocholine competed for a peripheral anionic site in the acetyl-enzyme intermediate formed during substrate hydrolysis. The apparent equilibrium dissociation constant of acetylthiocholine for this peripheral site (1.2-1.4 mM) was significantly different from that calculated from substrate inhibition data (20.1 +/- 2.8 mM). We propose that acetylthiocholine can interact with the acetyl-enzyme both at the peripheral site and at the active site but that only the latter interaction inhibits substrate hydrolysis.

Edrophonium (ethyl(m-hydroxyphenyl)dimethylamine) acutely modifies carnitine levels in different rat tissues, increasing hepatic and reducing blood and renal levels. After 2 h edrophonium treatment, the total serum carnitine levels were decreased by 16 (P < 0.001) and 33 (P < 0.001) percent in fed and fasted rats respectively compared to control, and in kidney the levels decreased by 11 (P < 0.05) and 34 (P < 0.001) percent whereas in liver the edrophonium treatment increased the levels by 43 (P < 0.001) and 59 (P < 0.001) percent. The edrophonium action does not depend on the route of administration or on the nutritional state of the animal. Its activity on carnitine levels is neither accompanied by significant variation of serum parameters of carbohydrate, fat and protein metabolism nor of insulin levels. The edrophonium activity is not related to cholinergic action, as physostigmine and ambenonium at concentrations known to increase cholinergic activity do not modify carnitine distribution in tissues. Trimethylphenylammonium (TPA) and trimethyl(p-aminophenyl)ammonium (TPA.NH2), compounds structurally similar to edrophonium, are on the contrary active on levels of carnitine and this effect is not related to their cholinergic potency. In 24 h fasted rats after the TPA and TPA. NH2 treatment, the total serum carnitine levels were decreased by 32 (P < 0.001) and 13 (n.s.) percent respectively compared to control, and in kidney the levels decreased by 15 (P < 0.02) and 5 (n.s.) percent, whereas in liver the treatment increased the levels by 72 (P < 0.001) and 45 (P < 0.01) percent. Moreover atropine, an acetylcholine antagonist, affects carnitine distribution in a way similar to edrophonium. Edrophonium activity on carnitine distribution, probably affects (inter)cellular carnitine transport by direct action on plasma membrane. Effect on capillary endothelium may be responsible for its observed action on muscle contraction force in imminent ischemia.

The rate of thermal inactivation of Torpedo AChE at pH 8.5 was increased by the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). At 30 degrees C or 37 degrees C, inactivation rates with 0.3 mM DTNB increased about 5-fold for the wild-type enzyme and for two site-specific mutants, D72S and V129R. The reversible active site inhibitor, ambenonium, completely stabilized the wild type enzyme and partially stabilized the D72S mutant. However, ambenonium did not protect against the destabilization introduced by DTNB, which still accelerated inactivation of D72S 5-fold. When the only free sulfhydryl group in AChE was removed by replacing cysteine 231 with serine, increased rates of thermal inactivation were observed. The inactivation rate increased by a factor of 2 to 3 for the single mutant (C231S) and by a factor of 5 for the double mutant V129R/C231S. Even in the C231S mutants, DTNB still had an additional effect. It increased the inactivation rate for C231S and V129R/C231 by a factor of about 1.5 to 3 beyond the rates seen in the absence of DTNB. Therefore, at least part of the destabilization seen with DTNB in enzymes that retain C231 does not involve reaction of DTNB with C231.

Pharmacokinetics of a very short-acting, a short-acting and two long-acting cholinesterase (ChE) inhibitors, edrophonium, neostigmine, pyridostigmine and ambenonium, respectively, were compared to elucidate the major determinant of their pharmacokinetics. No dose-dependency in pharmacokinetic behavior was observed within the range of 2-10 mumol/kg for edrophonium, 0.5-2 mumol/kg for pyridostigmine, 0.1-0.5 mumol/kg for neostigmine and 0.3-3 mumol/kg for ambenonium, respectively. Neostigmine has the shortest elimination half-life, and edrophonium, pyridostigmine and ambenonium follow in that. Four ChE inhibitors have similar Vdss values within the range of 0.3-0.7 l/kg, which is similar to the muscle/plasma concentration ratio of these drugs. The liver or kidney to plasma concentration ratio of all ChE inhibitors at 20min after i.v. administration ranged from 5 to 15. Small distribution volumes estimated from the plasma concentration profiles may reflect the distribution to muscle and to the extracellular space of other organs/tissues, while the rapid disappearance of ChE inhibitors from plasma may reflect the concentrative uptake to the liver and kidney.

A sensitive analytical method for the determination of ambenonium, a selective acetylcholinesterase inhibitor, in plasma was developed. The procedure involves ultrafiltration to remove endogenous plasma cholinesterase, followed by colorimetric measurement of the inhibitory activity to acetylcholinesterase by the thiocholine method. Coefficient of variation of within-day triplicate analysis is less than 20% at the concentration of 5 nM. Detection limit of this method is 1 nM, which is twice lower than the most sensitive HPLC method reported previously. This assay procedure is applied to the pharmacokinetic study of ambenonium after intravenous administration of low dose (10-20 nmol/kg) to rat. This new method is rapid and simple and makes it possible to determine the ambenonium concentration in plasma with good accuracy.

Steady state patterns of inhibition of purified human erythrocyte acetylcholinesterase by three inhibitors were analyzed. Edrophonium acted essentially as a competitive inhibitor, whereas tacrine and ambenonium gave mixed competitive and uncompetitive inhibition with acetylthiocholine as substrate. Inhibition constants for the competitive components were 470 microM for edrophonium, 65 microM for tacrine, and 0.12 nM for ambenonium. The extremely high affinity of ambenonium permitted analysis of the rates of approach to steady state inhibition. These rates were characterized by a single exponential time course with rate constants, kexp, that showed a linear dependence when plotted against ambenonium concentration, at fixed substrate concentration. The intercepts of these plots were independent of the substrate concentration and indicated an ambenonium dissociation rate constant of 0.013 +/- 0.002 sec-1. The slope of the plot at the lowest substrate concentration approximated the ambenonium bimolecular or association rate constant and gave a value of 5.2 +/- 0.6 x 10(7) M-1 sec-1. Three models were examined to account for the nearly linear dependence of the slopes of these plots on the substrate concentration. These models indicated that ambenonium and acetylthiocholine competed for a peripheral anionic site in the acetyl-enzyme intermediate formed during substrate hydrolysis. The apparent equilibrium dissociation constant of acetylthiocholine for this peripheral site (1.2-1.4 mM) was significantly different from that calculated from substrate inhibition data (20.1 +/- 2.8 mM). We propose that acetylthiocholine can interact with the acetyl-enzyme both at the peripheral site and at the active site but that only the latter interaction inhibits substrate hydrolysis.

The inhibition of human motor endplate cholinesterase by anticholinesterase compounds was studied using isolated muscle membrane preparation. Ambenonium was most potent, and edrophonium was least potent in inhibiting motor endplate cholinesterase. The slope of the regression line for inhibition of motor endplate cholinesterase was greatest for ambenonium, and smallest for neostigmine and edrophonium. These compounds were less potent inhibitors of plasma cholinesterase. Ambenonium was more specific, and other compounds were less specific inhibitors of motor endplate cholinesterase. In myasthenic patients, these compounds produced adequate inhibition of motor endplate cholinesterase even in the presence of relatively mild plasma cholinesterase inhibition.