Donald M. Maxwell

The specificity of carboxylesterase protection against the toxicity of organophosphorus compounds

The ability of endogenous carboxylesterase (CaE) to protect against the lethal effects of a variety of organophosphorus (OP) compounds was examined in rats. The in vivo protection provided by endogenous CaE was measured by the difference in the LD50 values of OP compounds in control rats and rats whose CaE activity had been inhibited by sc injection with 2 mg/kg of 2-(O-cresyl)-4H-1,3,2-benzodioxaphosphorin-2-oxide. Endogenous CaE provided significant protection against the in vivo toxicity of soman, sarin, tabun, and paraoxon, but not against dichlorvos, diisopropyl fluorophosphate, or ethoxymethyl-S-[2-(diisopropylamino)ethyl] thiophosphonate (VX). The relationship between the in vivo CaE protection against OP compounds and their relative reactivities with CaE and acetylcholinesterase (AChE) was evaluated by measuring the in vitro bimolecular rate constants (ki) for inhibition of plasma CaE and brain AChE. Except for VX, ki values for CaE inhibition varied less than 10-fold while ki values for AChE inhibition varied 10(5)-fold. The degree of in vivo inhibition of CaE by equitoxic doses of the OP compounds increased as the CaE/AChE ki ratio increased. However, the protective ratio of the LD50 values in control vs CaE-inhibited rats decreased as the CaE/AChE ki ratio increased. This inverse relationship between in vivo CaE protection and relative in vitro reactivity for CaE suggested that CaE detoxication is more important for highly toxic OP compounds (i.e., compounds with high AChE ki values and low LD50 values) than for less toxic compounds.

The effect of carboxylesterase inhibition on interspecies differences in soman toxicity

Subcutaneous administration of 2 mg/kg cresylbenzodioxaphosphorin oxide (CBDP) produced complete inhibition of carboxylesterase activity in plasma and lung of mice, rats, guinea pigs and rabbits, without inhibition of acetylcholinesterase activity in either brain or diaphragm. This CBDP treatment also reduced the subcutaneous soman LD50 in these species by 48-90% in comparison to the soman LD50 in control animals. The interspecies differences in the soman LD50 values that were seen in control animals were absent in CBDP-treated animals. The soman LD50 values in control animals were 125 micrograms/kg (mouse), 116 micrograms/kg (rat), 32.3 micrograms/kg (guinea pig) and 22.8 micrograms/kg (rabbit), whereas the soman LD50 values in CBDP-treated animals from these species were clustered in a narrow dose range (11.8-15.6 micrograms/kg) and were not significantly different. This suggests that the amount of CBDP-sensitive carboxylesterase available for detoxification of soman in each species may be an important determinant of interspecies differences in soman toxicity.

The effects of blood flow and detoxification on in vivo cholinesterase inhibition by soman in rats

The in vivo time course of cholinesterase inhibition was measured in brain, lung, spleen, hind limb skeletal muscle, diaphragm, intestine, kidney, heart, liver, and plasma of rats receiving 90 micrograms/kg soman, im. This dose of soman produced severe respiratory depression and transient hypertension, but no significant changes in the cardiac output or heart rate of anesthetized rats. The rate and maximal extent of in vivo cholinesterase inhibition by soman varied widely among the tissues. Although cardiac output was unchanged by soman administration, the blood flow in heart, brain, and lung (bronchial arterial flow and arteriovenous shunts) was increased, whereas blood flow in spleen, kidney, and skeletal muscle was decreased. The relative importance of tissue blood flow, tissue levels of cholinesterase and acetylcholinesterase, and tissue levels of soman-detoxifying enzymes (diisopropyl-fluorophosphatase and carboxylesterase) in determining the in vivo rate and maximal extent of cholinesterase inhibition was examined by multiple regression analysis. The best multiple regression model for the maximal extent of cholinesterase inhibition could explain only 63% of the observed variation. The best multiple regression model for the in vivo rate of cholinesterase inhibition contained three independent variables (blood flow, carboxylesterase, and cholinesterase) and could account for 94% of the observed variation. Of these three variables blood flow was the most important, accounting for 79% of the variation in the in vivo rate of cholinesterase inhibition. This suggests that it may be possible to use a flow-limited physiological pharmacokinetic model to describe the kinetics of in vivo cholinesterase inhibition by soman.

Enzymes as pretreatment drugs for organophosphate toxicity

We have successfully demonstrated that exogenously administered acetyl- or butyrylcholinesterase (AChE, BChE respectively) will sequester organophosphates (OPs) before they reach their physiological targets. In addition, a third enzyme, endogenous carboxylesterase is known to be capable of scavenging OPs. In these studies, we have administered AChE and BChE to three different species of animals (mice, marmosets and monkeys) which were challenged with three different OPs (VX, MEPQ and soman). Results obtained from these systematic studies demonstrate that: (a) a quantitative linear correlation exists between blood AChE levels and the protection afforded by exogenously administered ChEs in animals challenged with OP, (b) approximately one mole of either AChE or BChE sequesters one mole of OP, (c) such prophylactic measures are sufficient to protect animals against OPs without the administration of any supportive drugs. Thus the OP dose, the blood-level of esterase, the ratio of the circulating enzyme to OP challenge, and the rate of reaction between them determine the overall efficacy of an enzyme as a pretreatment drug. The biochemical mechanism underlying the sequestration of various OPs by the use of exogenously administered scavenging esterases is the same in all species of animals studied. Therefore, the extrapolation of the results obtained by the use of ChE prophylaxis in animals to humans should be more reliable and effective than extrapolating the results from currently used multidrug antidotal modalities.

A structure-activity analysis of the variation in oxime efficacy against nerve agents.

A structure-activity analysis was used to evaluate the variation in oxime efficacy of 2-PAM, obidoxime, HI-6 and ICD585 against nerve agents. In vivo oxime protection and in vitro oxime reactivation were used as indicators of oxime efficacy against VX, sarin, VR and cyclosarin. Analysis of in vivo oxime protection was conducted with oxime protective ratios (PR) from guinea pigs receiving oxime and atropine therapy after sc administration of nerve agent. Analysis of in vitro reactivation was conducted with second-order rate contants (k(r2)) for oxime reactivation of agent-inhibited acetylcholinesterase (AChE) from guinea pig erythrocytes. In vivo oxime PR and in vitro k(r2) decreased as the volume of the alkylmethylphosphonate moiety of nerve agents increased from VX to cyclosarin. This effect was greater with 2-PAM and obidoxime (>14-fold decrease in PR) than with HI-6 and ICD585 (<3.7-fold decrease in PR). The decrease in oxime PR and k(r2) as the volume of the agent moiety conjugated to AChE increased was consistent with a steric hindrance mechanism. Linear regression of log (PR-1) against log (k(r2)[oxime dose]) produced two offset parallel regression lines that delineated a significant difference between the coupling of oxime reactivation and oxime protection for HI-6 and ICD585 compared to 2-PAM and obidoxime. HI-6 and ICD585 appeared to be 6.8-fold more effective than 2-PAM and obidoxime at coupling oxime reactivation to oxime protection, which suggested that the isonicotinamide group that is common to both of these oximes, but absent from 2-PAM and obidoxime, is important for oxime efficacy.

The role of carboxylesterase in species variation of oxime protection against soman

Oxime protection against soman, a highly toxic anticholinesterase agent, was examined in mice and guinea pigs. The maximal protection produced by the oximes PAM and HI-6 varied as much as 6-fold between these species. Since endogenous carboxylesterase (CaE) is known to be an important determinant of species variation in soman toxicity, the protection of PAM and HI-6 against soman was also measured in animals whose endogenous CaE was inhibited with cresylbenzodioxaphosphorin oxide. In CaE-inhibited animals the soman LD50 values were similar in unprotected mice and guinea pigs (10.2 vs. 12.2 micrograms/kg) and oxime-protected mice and guinea pigs (38.1 vs. 40.3 micrograms/kg for PAM; 159 vs. 151 micrograms/kg for HI-6). The levels of oxime protection observed in CaE-inhibited animals agreed with previous experiments in other species that have no endogenous plasma CaE. The 4-5 times greater in vivo protection against soman of HI-6 vs. PAM in CaE-inhibited animals correlated with in vitro experiments in which HI-6 produced 3-5 times more oxime reactivation of soman-inhibited AChE than PAM.

Organophosphate skin decontamination using immobilized enzymes

We previously demonstrated that a combination of cholinesterase (ChE) pre-treatment with an oxime is an effective measure against soman and sarin. We describe here a novel approach for the preparation of covalently linked ChEs which are immobilized to a polyurethane matrix. Such preparation of ChE-sponges enhances the stability and usefulness of the enzymes in non-physiological environments. The ChE-sponges, which can be molded to any form, can effectively be used to remove and decontaminate organophosphates (OPs) from surfaces, biological (skin or wounds) or otherwise (clothing or sensitive medical equipment), or the environment. The ChE-sponges retained their catalytic activity under conditions of temperature, time, and drying where the native soluble enzyme would rapidly denature, and can be reused in conjunction with oximes many times. The ChE-sponge in the presence of oxime repeatedly detoxified OPs such as DFP or MEPQ. These developments in ChE technology have extended the applicability of OP scavengers from in vivo protection, to a variety of external detoxification and decontamination schemes. In addition to treatment of OP-contaminated soldiers, the ChE-sponge could protect medical personnel from secondary contamination while attending chemical casualties, and civilians exposed to pesticides or highly toxic nerve agents such as sarin.

A pharmacodynamic model for soman in the rat

A pharmacodynamic model for inhibition of acetylcholinesterase (AChE) by soman was developed to describe the intertissue differences in AChE inhibition, the dose response of AChE to inhibition by soman, and the effect of differences in xenobiotic metabolism on soman toxicity. Based on the principles of physiological pharmacokinetics, this pharmacodynamic model consisted of a set of mass balance equations that included parameters for blood flow, tissue volumes, soman metabolism, tissue/plasma partition coefficients, initial AChE levels, and the rate constant for AChE inhibition. Sensitivity analysis of the model revealed that variation of the soman metabolism parameter in plasma was the most important determinant of variation in the inhibition of brain AChE by soman.

Mutant acetylcholinesterases as potential detoxification agents for organophosphate poisoning

It has been demonstrated that cholinesterases (ChEs) are an effective mode of pretreatment to prevent organophosphate (OP) toxicity in mice and rhesus monkeys. The efficacy of ChE as a bioscavenger of OP can be enhanced by combining enzyme pretreatment with oxime reactivation, since the scavenging capacity extends beyond a stoichiometric ratio of ChE to OP. Aging has proven to be a major barrier to achieving oxime reactivation of acetylcholinesterase (AChE) inhibited by the more potent OPs. To further increase the stoichiometry of OP to ChE required, we have sought AChE mutants that are more easily reactivated than wild-type enzyme. Substitution of glutamine for glutamate (E199) located at the amino-terminal to the active-site serine (S200) in Torpedo AChE generated an enzyme largely resistant to aging. Here we report the effect of the corresponding mutation on the rate of inhibition, reactivation by 1-(2-hydroxyiminomethyl-1-pyridinium)-1(4-carboxyaminopyridinium)- dimethyl ether hydrochloride (HI-6), and aging of mouse AChE inhibited by C(+)P(-)- and C(-)P(-)-epimers of soman. The E202 to Q mutation decreased the affinity of soman for AChE, slowed the reactivation of soman-inhibited AChE by HI-6, and decreased the aging of mutant AChE. These effects were more pronounced with C(-)P(-)-soman than with C(+)P(-)-soman. In vitro detoxification of soman and sarin by wild-type and E202Q AChE in the presence of 2 mM HI-6 showed that, E202Q AChE was 2-3 times more effective in detoxifying soman and sarin than wild-type AChE. These studies show that these recombinant DNA-derived AChEs are a great improvement over wild-type AChE as bioscavengers. They can be used to develop effective methods for the safe disposal of stored OP nerve agents and potential candidates for pre- or post-exposure treatment for OP toxicity.

Development of a physiologically based model for the toxicokinetics of C(±)P(±)-soman in the atropinized guinea pig

Abstract A physiologically based model was developed which describes the in vivo toxicokinetics of the highly reactive nerve agent C(±)P(±)-soman at doses corresponding to 0.8–6 LD50 in the atropinized guinea pig. The model differentiates between the summated highly toxic C(±)P(−)-soman stereoisomers at supralethal doses and the individual nontoxic C(±)P(+)-isomers. Several toxicant-specific parameters for the soman stereoisomers were measured in guinea pig tissue homogenates. Cardiac output and blood flow distribution were measured in the atropinized, anesthetized, and artificially ventilated guinea pig. The model was validated by comparison of the time courses for the blood concentrations of the two pairs of stereoisomers in the guinea pig after i.v. bolus administration with the blood concentrations predicted by the model. The predictions put forward for the summated C(±)P(−)-isomers are in reasonable agreement with the experimental data obtained after doses corresponding to 2 and 6 LD50. In view of large differences in the rates of hydrolysis of the C(±)P(+)-isomers, these two isomers had to be differentiated for satisfactory modeling of both isomers. In order to model the toxicokinetics of C(±)P(−)-soman at a dose of 0.8 LD50, the almost instantaneous elimination of the C(+)P(−)-isomer at that dose had to be taken into account. The sensitivity of the predictions of the model to variations in the parameters has been studied with incremental sensitivity analysis. The results of this analysis indicate that extension to a model involving four individual stereoisomers is desirable in view of large differences in the biochemical characteristics of the two C(±)P(−)- and C(±)P(+)-isomers.