Karen M. Brecht

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 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.

Characterization and Treatment of the Toxicity of O-isobutyl S-[2-(diethylamino)ethyl]methylphosphonothioate, a Structural Isomer of VX, in Guinea Pigs

O-isobutyl S-[2-(diethylamino)ethyl]methylphosphonothioate (VR), a structural isomer of the chemical warfare agent O-ethyl S-[2(diisopropylamino)ethyl] methylphosphonothioate (VX) was characterized with respect to its toxicity, acetylcholinesterase (AChE) inhibition, oxime reactivation, and antidotal treatment. VR was highly toxic in guinea pigs, with an s.c. LD 50 of 11.3 μLg/kg. The bimolecular rate constant for inhibition of AChE by VR was 1.4 x 10 8 M -1 min -1 , which is similar to the AChE inhibition rate constants of VX and of other chemical warfare agents whose toxicity is attributed to their inhibition of AChE. Complete in vitro reactivation of VR-inhibited AChE was produced by oximes, but reactivation with the bispyridinium oximes 1-(((4-(aminocarbonyl)pyridino)methoxy methyl)-2-((hydroximino)-methyl)pyridinium dichloride (HI-6), 1-(((4-(aminocarbonyl)pyridino)methoxy)-methyl)-2,4-bis((hydroxyimino)methyl)pyridinium dimethanesulfonate (HLo7), and toxogonin dichloride (TOXO) occurred at a much higher rate than with the monopyridinium oxime 2-pyridine aldoxime methylchloride (2-PAM). The reactivation rates with HLo7, HI-6, and TOXO were, respectively, 21-, 5-, and 3-fold greater than with 2-PAM. The aging of VR-inhibited AChE to an oxime-resistant state occurred slowly with a half-life of 43 h. Treatment of guinea pigs with equimolar (145 μLmol/kg) amounts of HI-6 (55 mg/kg) or 2-PAM (25 mg/kg) in combination with atropine (16 mg/kg) i.m. 1 min after VR exposure produced protective ratios of 43.9 for HI-6 and 6.5 for 2-PAM. Carbamate pretreatment prior to these oxime/atropine treatments caused no increase in their protective ratios against VR. These observations suggest that VR is a highly toxic anticholinesterase agent that can be effectively treated with an appropriate oxime/atropine regimen.

A Common Mechanism for Resistance to Oxime Reactivation of Acetylcholinesterase Inhibited by Organophosphorus Compounds

Administration of oxime therapy is currently the standard approach used to reverse the acute toxicity of organophosphorus (OP) compounds, which is usually attributed to OP inhibition of acetylcholinesterase (AChE). Rate constants for reactivation of OP-inhibited AChE by even the best oximes, such as HI-6 and obidoxime, can vary >100-fold between OP-AChE conjugates that are easily reactivated and those that are difficult to reactivate. To gain a better understanding of this oxime specificity problem for future design of improved reactivators, we conducted a QSAR analysis for oxime reactivation of AChE inhibited by OP agents and their analogues. Our objective was to identify common mechanism(s) among OP-AChE conjugates of phosphates, phosphonates and phosphoramidates that result in resistance to oxime reactivation. Our evaluation of oxime reactivation of AChE inhibited by a sarin analogue, O-methyl isopropylphosphonofluoridate, or a cyclosarin analogue, O-methyl cyclohexylphosphonofluoridate, indicated that AChE inhibited by these analogues was at least 70-fold more difficult to reactivate than AChE inhibited by sarin or cyclosarin. In addition, AChE inhibited by an analogue of tabun (i.e., O-ethyl isopropylphosphonofluoridate) was nearly as resistant to reactivation as tabun-inhibited AChE. QSAR analysis of oxime reactivation of AChE inhibited by these OP compounds and others suggested that the presence of both a large substituent (i.e., ≥ the size of dimethylamine) and an alkoxy substituent in the structure of OP compounds is the common feature that results in resistance to oxime reactivation of OP-AChE conjugates whether the OP is a phosphate, phosphonate or phosphoramidate.

Comparison of Cholinesterases and Carboxylesterase as Bioscavengers for Organophosphorus Compounds

Inasmuch as the traditional pharmacological approaches to protection against organophosphorus (OP) compounds may be near their practical limits, a new approach using enzymes as bioscavengers has recently been examined. The feasibility of using bioscavengers, such as acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CaE), to provide protection against OP compounds has been demonstrated in rodents as well as nonhuman primates (1–5). The major advantages of bioscavengers for protection against OP toxicity are their rapid removal of OP compounds from circulation, their selective reactivities with the toxic stereoisomers of chiral compounds, and their relatively slow clearance from circulation. In comparison to other pharmacological approaches, the appeal of bioscavenger protection is that bioscavenger-protected survivors of exposure to OP compounds do not exhibit the postexposure incapacitation and toxic effects that are commonly observed with survivors protected by traditional antidotal approaches, such as oximes, anticholinergics, or carbamates (6).

Design, synthesis, and characterization of novel, nonquaternary reactivators of GF-inhibited human acetylcholinesterase.

The goal of this research was to identify structurally novel, non-quaternarypyridinium reactivators of GF (cyclosarin)-inhibited hAChE that possess the capacity to mediate in vitro reactivation of GF-inhibited human acetylcholinesterase (hAChE). New compounds were designed, synthesized and assessed in GF-inhibited hAChE assays. Structure activity relationships for AChE binding and reactivation of GF-inhibited hAChE were developed. Lead compounds from two different chemical series, represented by compounds 17 and 38, displayed proficient in vitro reactivation of GF-inhibited hAChE, while also possessing low inhibition of native enzyme.

Toxicodynamic modeling of highly toxic organophosphorus compounds.

Although the in vitro effect of organophosphorus (OP) compounds on acetylcholine-esterase (AChE) has been studied extensively, the hypothesis that OP inhibition of AChE is the primary mechanism of acute in vivo OP toxicity has been controversial. For example, a recent review (Pope and Liu, 2004) suggested that OP compounds have direct toxic effects on other enzymes, ACh receptors, and receptor/ channel complexes that are independent of AChE inhibition. The purpose of this report is to examine the hypothesis that AChE inhibition is the mechanism of acute toxicity of OP compounds by mathematically modeling the in vivo lethal effects of highly toxic OP compounds and determining the amount of variation in OP toxicity that is explained by AChE inhibition.

Hepatic subcellular localization of cresylbenzodioxaphosphorin oxide (CBDP)-sensitive soman binding sites.

The toxicity of the organophosphorus poison soman (pinacolylmethylphosphonofluoridate) is attributable to its irreversible inhibition of the enzyme acetylcholinesterase. In addition, soman binds irreversibly to a number of noncholinesterase tissue binding sites which appear to be its major means of in vivo detoxification. This study was conducted to determine the hepatic subcellular localization of these sites. Subcellular fractions of liver from male Sprague-Dawley rats (200-250 g) were prepared by differential and isopycnic density gradient centrifugation. The binding of [14C]soman to these subcellular fractions was determined in the presence and absence of cresylbenzodioxaphosphorin oxide (CBDP), a compound that binds irreversibly to the noncholinesterase soman binding sites. Crude fractionation of liver homogenates into nuclear, mitochondrial, microsomal, and soluble fractions revealed that 78% of the total CBDP-sensitive binding activity was localized in the nuclear and microsomal fractions. Further purification of these fractions indicated that all of the homogenate binding activity could be accounted for in the purified microsomal fraction. When purified liver microsomes were solubilized and fractionated on linear sucrose gradients, 90% of the CBDP-sensitive soman binding activity cosedimented with carboxylesterase activity which suggests that these binding sites are carboxylesterase.

Comparison of Acetylcholinesterase, Pyridostigmine, and HI-6 as Antidotes against Organophosphorus Compounds

Conventional medical treatment against the toxicity of organophosphorus (OP) compounds consists of a regimen of anticholinergic drugs to counteract the accumulation of acetylcholine and oximes to reactivate OP-inhibited acetylcholinesterase (AChE) (Taylor, 1985). Reactivation of OP- inhibited AChE by oximes can generate enough active AChE in the peripheral nervous system, especially in the diaphragm, to restore normal cholinergic neurotransmission after exposure to many OP compounds. However, some OP compounds, such as soman (pinacolylmethylphosphonofluoridate), inhibit AChE and rapidly “age” into a form that cannot be reactivated by oximes (De Jong and Wolring, 1984), thereby reducing the ability of oximes to provide protection (Maxwell and Brecht, 1991). The inability of oximes to provide adequate protection against the toxicity of rapidly aging OP compounds stimulated the development of carbamate pretreatment in which carbamylation of AChE effectively protects it against inhibition by OP compounds (Leadbeater et al., 1985). Spontaneous decarbamylation of AChE after the OP compound has been detoxified then generates enough active AChE to allow normal cholinergic neurotransmission. Behavioral side effects from carbamate pretreatment in the absence of exposure to OP compounds have been avoided by the use of cationic pretreatment carbamates, such as pyridostigmine, which do not enter the central nervous system (Maxwell et al., 1988).