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While non-reactivability of cholinesterase from their phosphyl conjugates (aging) is attributed to an unimolecular process involving loss of alkyl group from the phosphyl moiety, no conclusive evidence is available that this is the only reaction path and involvement of other post-inhibitory processes cannot be ruled out, To address this issue, molecular masses of the bacterially expressed recombinant human acetylcholinesterase and of its conjugates with a homologous series of alkyl methyl-phosphonofluoridates, were measured by electrospray-ionization mass spectrometry (ESI-MS). The measured mass of the free enzyme was 64700 De (calculated 64 695 Da) and those of the methylphosphono-HuAChE adducts, bearing isopropyl, isobutyl, 1,2-dimethylpropyl and 1,2,2-trimethylpropyl substituents, were 64 820, 64 840, 64 852 and 64 860 Da, respectively. These values reflect both the addition of the phosphonyl moiety and the gradual mass increase due to branching of the alkoxy substituent, The composition of these adducts change with time to yield a common product with molecular mass of 64780 Da which is consistent with dealkylation of the phosphonyl moieties, Furthermore, in the case of 1,2,2-dimethylpropyl methylphosphono-HuAChE, the change in the molecular mass and the kinetics of non-reactivability appear to occur in parallel indicating that dealkylation is indeed the predominant molecular transformation leading to 'aging' of phosphonyl-AChE adducts.

We report the first toxicokinetic studies of (±)-sarin. The toxicokinetics of the stereoisomers of this nerve agent were studied in anesthetized, atropinized, and restrained guinea pigs after intravenous bolus administration of a dose corresponding to 0.8 LD50 and after nose-only exposure to vapor concentrations yielding 0.4 and 0.8 LCt50 in an 8-min exposure time. During exposure the respiratory minute volume and frequency were monitored. Blood samples were taken for gas chromatographic analysis of the nerve agent stereoisomers and for measurement of the activity of blood acetylcholinesterase (ACHE). In all experiments, the concentration of (+)-sarin was below the detection limit <5 pg/ml). The concentration-time profile of the toxic isomer, i.e., (-)-sarin, after an intravenous bolus was adequately described with a two-exponential equation. (-)-Sarin is distributed ca. 10-fold faster than C(-)P(-)-soman, whereas its elimination proceeds almost 10-fold slower. During nose-only exposure to 0.4 and 0.8 LCt50 of (±)-sarin in 8 min, (-)-sarin appeared to be rapidly absorbed. The blood AChE activity decreased during the exposure period to ca. 15 and 70% of control activity, respectively. There were no effects on the respiratory parameters. A significant nonlinearity of the toxico-kinetics with dose was observed for the respiratory experiments. © 2000 Academic Press.

The stereoselectivity of the phosphonylation reaction and the effects of adduct configuration on the aging process were examined for human acetylcholinesterase (HuAChE) and its selected active center mutants, using the four stereomers of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman). The reactivity of wild type HuAChE toward the P(S)-soman diastereomers was 4.0-7.5 x 104-fold higher than that toward the P(R)- diastereomers. Aging of the P(S)C(S)-somanyl-HuAChE conjugate was also > 1.6 x 104-fold faster than that of the corresponding P(R)C(S)-somanyl adduct, as shown by both reactivation and electrospray mass spectrometry (ESI/MS) experiments. On the other hand, both processes exhibited very limited sensitivity to the chirality of the alkoxy group C(α) of either P(S)- or P(R)-diastereomers. These stereoselectivities presumably reflect the relative participation of the enzyme in stabilization of the Michaelis complexes and in dealkylation of the respective covalent conjugates, and therefore could be utilized for further probing of the HuAChE active center functional architecture. Reactivities of HuAChE enzymes carrying replacements at the acyl pocket (F295A, F297A, and F295L/F297V) indicate that stereoselectivity with respect to the soman phosphorus chirality depends on the structure of this binding subsite, but this stereoselectivity cannot be explained only by limitation in the capacity to accommodate the P(R)-diastereomers. In addition, these acyl pocket enzyme mutants display some (5-10-fold) preference for the P(R)C(R)-soman over the P(R)C(S)-stereomer, while reactivity of the hydrophobic pocket mutant enzyme W86F toward the P(R)C(S)- soman resembles that of the wild type HuAChE. Residue substitutions in the H- bond network (E202Q, E450A, Y133F, and Y133A) and the hydrophobic pocket (F338A, W86A, W86F, and Y337A) result in a limited stereoselectivity for the P(S)C(S)- over the P(S)C(R)-stereomer. Aging of the P(S)-somanyl conjugates with all the HuAChE mutant enzymes tested practically lacked stereoselectivity with respect to the C(α) of the alkoxy moiety. Thus, the inherent asymmetry of the active center does not seem to affect the rate- determining step of the dealkylation process, possibly because both the P(S)C(S)- and the P(S)C(R)-somanyl moieties yield the same carbocationic intermediate.

In continuation of our investigations on the toxicokinetics of the volatile nerve agents C(±)P(±)-soman and (±)-sarin, we now report on the toxicokinetics of the rather nonvolatile agent (±)-VX. A validated method was developed to determine blood levels of (±)-VX by means of achiral gas chromatography at blood levels ≥10 pg/ml. The ratio of the two enantiomers of VX in blood could be measured at levels ≥1 ng/ml by using chiral HPLC in combination with off-line gas chromatographic analysis. In order to obtain basic information on the toxicokinetics of (±)-VX, i.e., under conditions of 100% bioavailability, the blood levels of this agent were measured in hairless guinea pigs at iv doses corresponding with 1 and 2 LD50. The derived AUCs indicate a reasonable linearity of the toxicokinetics with dose. Also, the toxicokinetics in marmoset primates was studied at an absolute iv dose corresponding with 1 LD50 in the hairless guinea pig which led to approximately the same levels of (±)-VX in blood as observed at 2 LD50 in the hairless guinea pig. Finally, the toxicokinetics of (±)-VX were measured in hairless guinea pigs via the most relevant porte d' entrée for this agent, which is the percutaneous route at a dose corresponding with 1 LD50 (pc). Large variations were observed between individual animals in the rate of penetration of (±)-VX and in concomitant progression of AChE inhibition in blood of these animals. Blood levels of (±)-VX increased gradually over a 6-h period of time. After a 7-h penetration period, the total AUC corresponded with 2.5% bioavailability relative to iv administration. In contrast with the G-agents C(±)P(±)-soman and (±)-sarin, stereospecificity in the sequestration of the two enantiomers of (±)-VX is not a prominent phenomenon. It appears that (±)-VX is substantially more persistent in vivo than the two G-agents. This persistence may undermine the efficacy of pretreatment with carbamates of percutaneous intoxication in particular due to gradual replacement of carbamate on AChE by (±)-VX, whereas classical treatment of intoxication with oximes is hampered by the short persistence of oximes relative to the agent. © 2003 Elsevier Science (USA). All rights reserved.