Medical Aspects of Chemical Warfare (2008) - The Black Vault

Medical DiagnosticsTable 22-5Red Blood Cell and Acetylcholinesterase protection studies using pyridostigminebromide and Huperzine A after ex-vivo exposure to somanProcedure* †Techniques and Rationale1) Obtain blood samples from human volunteers Aliquot blood from subjects administered oral dose of 30 mg pyridostigminebromide, 200 µg huperzine A, or no drug2) Ex-vivo exposure to soman incubated blood with 1 µM soman for 10 min at room temperature;incubate control samples (no soman) with saline3) Remove free drug and soman centrifuge samples through a C 18chromatography spin column tobind drug and remove from the blood4) Allow time for AChE decarbamylation (PB) Maintain postcolumn samples at room temperature for up to 24 hoursor dissociation (Hup A)5) Monitor time for regeneration of AChE after Aliquot samples collected for AChE activity assay at indicated timesdecarbamylation of PB or reversible inhibition postcolumnby Hup A6) Measure AChE activity use WRAIR whole blood ChE assay to determine recovered enzymeactivity*Acetylcholinesterase and pyridostigmine bromide or huperzine A yield an acetylcholinesterase-drug complex in procedures 1 and 2. Thisreaction demonstrates sequestered enzyme, which temporarily inhibits the active site.† Over time, the acetylcholinesterase-drug complex becomes acetylcholinesterase plus pyridostigmine bromide (the decarbamylated formof acetylcholinesterase) or dissociated huperzine A in procedures 3 and 4. These reactions demonstrate the sequestered and then restoredenzyme activity.AChE: acetylcholinesteraseChE: cholinesterasePB: pyridostigmine bromideHup A: huperzine Awith atropine and an oxime) may represent a superiortreatment strategy for protection against chemicalwarfare agent exposure and should be investigatedfurther.Animal studies have been used to examine the efficacyof high doses of Hup A against OP nerve agenttoxicity. A dose of Hup A (500 µg/kg) significantlyreduces soman lethality (protective ratios of 2–3 whenused alone) and inhibits blood and brain AChE by 60%to 70%. 74 Unlike PB, Hup A can cross the blood-brainbarrier and in rats protects brain AChE from inhibitionby soman, thus preventing build-up of excessiveacetylcholine leading to seizures and associated neuropathologicaldamage. In guinea pig hippocampus,natural Hup A in subchronic doses (yielding 20%–30%inhibition of RBC-AChE) has little or no affinity formuscarinic, nicotinic, or N-methyl-d-aspartate excitatoryamino acid receptors, and does not induceneuropathological damage.Because Hup A reversibly binds to AChE, thusprotecting the enzyme from reaction with OPs, theactivity of the Hup-A–protected but inhibited AChE isrestored once the drug-AChE complex spontaneouslydissociates, which occurs after soman is cleared fromthe blood. To illustrate this, Hup-A– inhibited blood(drawn 1.5 h after human volunteers were given a doseof 200 µg Hup A; Figure 22-5) was exposed ex vivo tosoman. 62 Blood samples were then rapidly centrifugedthrough a small column to remove any free soman andHup A, the latter binding to the column matrix whileallowing AChE and BChE to pass through the column.Under these circumstances, any RBC-AChE not protectedby Hup A would be irreversibly inhibited bysoman. In contrast, the RBC-AChE protected by HupA would dissociate over time, and the AChE activitywould eventually be restored. In one study, after somantreatment, no AChE activity was observed by theWalter Reed Army Institute of Research Whole BloodAssay in either the placebo- or drug-treated volunteers(see Figure 22-5). After the spin column removal offree Hup A and soman, and a 4-hour period to allowfor complete dissociation, the Hup-A–inhibited AChEwas restored to the level that was initially inhibitedby the drug (about 60% inhibition by the drug beforethe column compared to 54% returned AChE activityafter the column). AChE inhibition and sequestering705