Bimolecular Rate Constants For Inhibition Research Articles

Physiologically based pharmacokinetic and pharmacodynamic model for the inhibition of acetylcholinesterase by diisopropyfluorophosphate

Organophosphate (OP) exposure can be lethal at high doses while lower doses may impair performance of critical tasks. The ability to predict such effects for realistic exposure scenarios would expedite OP risk assessment. To this end, a physiologically based model for diisopropylfluorophosphate (DFP) pharmacokinetics and acetylcholinesterase (AChE) inhibition was developed in mammals. DFP tissue:blood partition coefficients, rates of DFP hydrolysis by esterases, and DFP-esterase bimolecular inhibition rate constants were determined in rat tissue homogenates. Other model parameters were scaled for rats and mice using standard allometric relationships. These DFP-specific parameter values were used with the model to simulate expected in vivo pharmacokinetic data from mice and rats. Literature data were used for model validation. DFP concentrations in mouse plasma and brain were successfully simulated after a single iv injection ( B. R. Martin, 1985, Toxicol. Appl. Pharmacol. 77, 275–284). AChE inhibition and AChE resynthesis data from this study were also simulated. Effects of repeated, subcutaneous DFP dosing on AChE activity in rat plasma and brain ( H. Michalek, A. Meneguz, and G. M. Bisso, 1982, Arch. Toxicol., Suppl. 5, 116–119; M. E. Traina and L. A. Serpietri, 1984, Biochem. Pharmacol. 33, 645–653) were also simulated well, but the return of brain AChE activity to basal levels after cessation of repeated dosing was not as well described. The initial model structure returned brain AChE activity to the original level, while in the laboratory studies brain AChE never returned to basal levels, even at 35 days after the last dose. These data suggest modulation of AChE synthesis with prolonged DFP exposure. This study demonstrated the possibility of using a model based on mammalian physiology and biochemistry to simulate in vivo data on DFP pharmacokinetics and AChE inhibition. Scaling of the model between rats and mice was also successful. The approach holds promise for predictive simulation of organophosphate-mediated AChE inhibition in humans.

Spontaneous and oxime-induced reactivation of acetylcholinesterase inhibited by phosphoramidates.

Methamidophos (CH3O(NH2)P(O)SCH3) and phosphoramidates, with the general structure RO(NH2)P(O)OC6H4-p-NO2, in which R = C2H5, C1CH2CH2, FCH2CH2 and F3CCH2, as well as (NH2)2P(O)OC6H4-p-NO2 were synthesized to investigate the relationship between the rates of inhibition and of spontaneous reactivation of AChE inhibited by these organophosphates and their potential as prophylactics against nerve agent poisoning. The phosphoramidates inhibit electric eel acetylcholinesterase (EEAChE), the bimolecular inhibition rate constants ranging from 5 x 10(4) to 3 x 10(6) M-1.min-1 at pH 7.5, 25 degrees C. The inhibited enzymes reactivate spontaneously, with half-lives ranging from 1.3 to 15 h at pH 7.5, 25 degrees C. These half-lives increase 2-4 fold when the temperature is raised to 37 degrees C. Reactivation is accelerated by micromolar concentrations of oximes such as obidoxime and HI-6. Aging of the inhibited enzymes was not observed. Nevertheless, reactivation appears to be incomplete for some of the inhibited enzymes. The title compounds seem promising as prophylactic agents against nerve agent intoxication.

Kinetic investigations into the interactions of aprophen with cholinesterases and a carboxylesterase

Acetylcholinesterases, butyrylcholinesterases, and carboxylesterases appear to form kinetically a homologous enzyme series with respect to many substrates and inhibitors. The present paper evaluates the interaction of aprophen with acetylcholinesterases, butyrylcholinesterases, and carboxylesterases with respect to (1) protecting the enzyme from organophosphate and carbamate inhibition, (2) accelerating pralidoxime iodide (2-PAM) regeneration of the diisopropylphospho-enzyme, and (3) comparing the inhibition and regeneration kinetics of a soluble mammalian acetylcholinesterase with that of bovine erythrocyte acetylcholinesterase. The irreversible inhibition kinetics of diisopropyl fluorophosphate (DFP) and eserine inhibition of fetal bovine serum acetylcholinesterase were typical of other acetylcholinesterases as indicated by the bimolecular inhibition rate constants, k i , of 7.7 ± 1.3 × 10 4 M −1 and 2.9 ± 1.7 × 10 6 M −1, respectively. Similarly, the bimolecular regeneration rate constant, k r , for 2-PAM regeneration of the diisopropylphospho-acetylcholinesterase was 14.7 M −1 min −1. The bimolecular rate constants, k i and k r , were not statisticaly perturbed when the reaction was monitored in the presence of aprophen with the fetal bovine serum acetylcholinesterase. Human serum butyrylcholinesterase was partially protected from DFP inhibition by aprophen with no detectable change in the bimolecular inhibition rate constant, k i . The regeneration of the diisopropylphospho-butyrylcholinesterase by 2-PAM was accelerated in the presence of aprophen by a factor of 2.7 over that of 2-PAM alone (8.4 ± 2.2 M −1 to 23.1 ± 2.6 M −1 min −1respectively). Neither the inhibition (DFP) nor the regeneration (2-PAM) kinetics observed for the carboxylesterase was perturbed by the presence of aprophen.

QSAR for Cholinesterase Inhibition by Organophosphorus Esters and CNDO/2 Calculations for Organophosphorus Ester Hydrolysis

AbstractQuantitative structure‐activity relationships were derived for acetyl‐ and butyrylcholinesterase inhibition by various organophosphorus esters. Bimolecular inhibition rate constants correlate well with hydrophobic substituent constants, and with the presence or absence of cationic groups on the inhibitor, but not with steric substituent constants. CNDO/2 calculations were performed on a separate set of organophosphorus esters, RR′P(O)X, where R and R′ are alkyl and/or alkoxy groups and X is fluorine, chlorine or a phenoxy group. For each subset with the same X, the CNDO‐derived net atomic charge at the central phosphorus atom in the ester correlates well with the alkaline hydrolysis rate constant. For the whole set of esters with different X, two equations were derived that relate either charge and leaving group steric bulk, or orbital energy and bond order to the hydrolysis rate constant.

Isolation, in vitro activity, and acute toxicity in mice of the four stereoisomers of soman

We report the isolation, on a 0.1- to 2-mg scale, of the four stereoisomers of the nerve agent pinacolyl methylphosphonofluoridate (soman), assigned as C(+)P(+), C(+)P(−), C(−)P(+), and C(−)P(−), with more than 99% optical purity. This result was realized by means of (i) complete optical resolution of pinacolyl alcohol, (ii) synthesis of C(+)- and C(−)-soman from the (+)- and (−)-enantiomers of the alcohol, (iii) optimalization of conditions for stereospecific inhibition of α-chymotrypsin with the P(−)-isomers of C(+)- and C(−)-soman, followed by isolation of the C(+)P(+)- and C(−)P(+)-isomers, (iv) isolation of the C(+)P(−)- and C(−)P(−)-isomers after incubation in rabbit plasma of C(+)- and C(−)-soman, respectively, which hydrolyzes stereospecifically the P(+)-isomers. Solutions (0.2 m m) of the steroisomers in organic solvents are optically stable for at least a month at −20°C. The bimolecular rate constants for inhibition of electric eel acetylcholinesterase (AChE) at pH 7.7, 25°C, are at least 3.6 × 10 4 larger for the P(−)- than for the P(+)-isomers. AChE from electric eel as well as from mouse erythrocytes inhibited with C(+)P(−)-soman is much more effectively reactivated with the oximes HI-6, HGG-42, and obidoxime than these enzymes inhibited with C(−)P(−)-soman. Similarly, the neuromuscular transmission (NMT) in mouse diaphragm strips poisoned with C(+)-soman is more easily restored with HI-6 than NMT of such muscle preparations poisoned with C(−)-soman. The LD50 values (mice, sc) are in accordance with the P(−) P(+) -ratio of inhibition rates of AChE, i.e. 99, 38, >5000, >2000, 214, 133, and 156 μg/kg for C(+)P(−)-, C(−)P(−)-, C(+)P(+)-, C(−)P(+), C(+)-, C(−)-soman, and “soman,” respectively. We suggest that mice challenged with a lethal dose of soman (sc) are killed primarily by the C(−)P(−)-isomer.

Isolation, anticholinesterase properties, and acute toxicity in mice of the four stereoisomers of the nerve agent soman

The four stereoisomers of the nerve agent pinacolyl methylphosphonofluoridate (soman), designated as C(+)P(+), C(+)P(−), C(−)P(+), and C(−)P(−), have different toxicologic properties due to stereospecific interactions in living organisms. We report the isolation of these stereoisomers with more than 99% optical purity. This result was realized by means of (i) complete optical resolution of pinacolyl alcohol, (ii) synthesis of C(+)- and C(−)-soman from the (+)- and (−)-enantiomers of the alcohol, (iii) optimalization of conditions for stereospecific inhibition of α-chymotrypsin with the P(−)-isomers of C(+)- and C(−)-soman, followed by isolation of the C(+)P(+)- and C(−)P(+)-isomers, (iv) isolation of the C(+)P(−)- and C(−)P(−)-isomers after incubation of C(+)- and C(−)-soman, respectively, in rabbit plasma, which hydrolyzes stereospecifically the P(+)-isomers. The bimolecular rate constants for inhibition of electric eel acetylcholinesterase (AChE) at pH 7.7, 25°C, are at least 3.6 × 10 4 larger for the P(−)- than for the P(+)-isomers. The enzyme inhibited with C(+)P(−)-soman is much more effectively reactivated with the oximes HI-6, HGG-42, and obidoxime than AChE inhibited with C(−)P(−)-soman. The LD50 values (sc, mice) are in accordance with the P(−) P(+) ratio of inhibition rates of AChE, i.e. 99, 38, >5000, >2000, 214, 133, and 156 μg/kg for C(+)P(−)-, C(−)P(−)-, C(+)P(+)-, C(−)P(+)-, C(+)-, C(−)-soman, and “soman,” respectively. The relative LD50 values of the C(−)P(−)- and C(+)P(−)-isomers do not correspond with the small differences in their rates of inhibition of AChE, indicating that such small rate ratios may be overruled by other stereospecific effects, e.g., in vivo rates of detoxification.

Isolation, in Vitro Activity, and Acute Toxicity in Mice of the Four Stereoisomers of Soman

Isolation, in Vitro Activity, and Acute Toxicity in Mice of the Four Stereoisomers of Soman. BENSCHOP, H. P., KONINGS, C. A. G., VAN GENDEREN, J., AND DE JONG, L. P. A. (1984). Fundam. Appl. Toxicol. 4, S84–S95. We report the isolation, on a 0.1-to 2-mg scale, of the four stereoisomers of the nerve agent pinacolyl methylphosphonofluoridate (soman), assigned as C(+)P(+), C(+)P(−), C(−)P(+), and C(−)P(−), with more than 99% optical purity. This result was realized by means of (i) complete optical resolution of pinacolyl alcohol, (ii) synthesis of C(+)- and C(−)-soman from the (+)- and (−)-enantiomers of the alcohol, (iii) optimalization of conditions for stereospecific inhibition of α-chymotrypsin with the P(−)-isomers of C(+)- and C(−)-soman, followed by isolation of the C(+)P(+)- and C(−)P(+)-isomers, (iv) isolation of the C(+)P(−)- and C(−)P(−)-isomers after incubation in rabbit plasma of C(+)- and C(−)-soman, respectively, which hydrolyzes stereospecifically the P(+)-isomers. Solutions (0.2 mM) of the steroisomers in organic solvents are optically stable for at least a month at −20°C. The bimolecular rate constants for inhibition of electric eel acetylcholinesterase (AChE) at pH 7.7, 25°C, are at least 3.6 × 104 larger for the P(−)- than for the P(+)-isomers. AChE from electric eel as well as from mouse erythrocytes inhibited with C(+)P(−)-soman is much more effectively reactivated with the oximes HI-6, HGG-42, and obidoxime than these enzymes inhibited with C(−)P(−)-soman. Similarly, the neuro-muscular transmission (NMT) in mouse diaphragm strips poisoned with C(+)-soman is more easily restored with HI-6 than NMT of such muscle preparations poisoned with C(−)-soman. The LD50 values (mice, sc) are in accordance with the P(−)/P(+)-ratio of inhibition rates of AChE, i.e. 99, 38, >5000, >2000, 214, 133, and 156 μg/kg for C(+)P(−)-, C(−)P(−)-, C(+)P(+)-, C(−)P(+), C(+)-, C(−)-soman, and “soman,” respectively. We suggest that mice challenged with a lethal dose of soman (sc) are killed primarily by the C(−)P(−)-isomer.

Isolation, anticholinesterase properties and acute toxicity of the four stereoisomers of the nerve agent soman

Abstract The four stereoisomers of pinacolyl methylphospho-nofluoridate (soman) were isolated with more than 99% optical purity. The bimolecular rate constants for inhibition of electric eel acetylcholinesterase and the LD50-values (sc, mice) of the stereoisomers were determined.

Relationships between in vivo and in vitro inhibition of acetylcholinesterase (AChE) and impairment of neuromuscular transmission in the rat phrenic-nerve diaphragm by a tertiary anticholinesterase and its quaternary analogue

The tertiary anticholinesterase agent pinacolyl S-(2-dimethylaminoethyl) methylphosphonothioate (compound I) and its quaternary analogue (compound II), were administered subcutaneously to atropinized rats. The animals were killed 30 min later and the tension of the isolated phrenic nerve-diaphragm preparation, stimulated indirectly at 100 Hz for 10 sec, was compared with that of preparations from control animals. A dose of 1.5 μmole/kg of the quaternary compound II, which has a bimolecular rate constant of inhibition twice that of the tertiary compound I, reduced the tetanic response of the diaphragm by 50 per cent; the equivalent dose of the tertiary compound I was 5.2 μmole/kg. At these dose levels, compound II inhibited 65 per cent of the diaphragm acetylcholinesterase (AChE) activity, determined on homogenates, and compound 192 per cent. When the phrenic nerve-diaphragm preparation from untreated animals was incubated for 20 min with various concentrations of compound I or II, the rate of enzyme inhibition conformed approximately to first order kinetics and equimolar concentrations of the inhibitors reduced tetanic tension to 50 per cent in the same time. There was no discontinuity in the plot of per cent AChE inhibition vs logarithm of the concentration of compound I or II but the slope was much less with the quaternary compound II. The results provide additional evidence that quaternary compounds can reach all sites with AChE activity only in vitro and that their distribution when applied in vitro is not the same as that in vivo.