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

Nerve Agent Bioscavenger: Development of a New Approach to Protect Against Organophosphorus Exposureand it is readily absorbed from injection sites. Third,because the enzyme is from a human source, it shouldnot produce adverse immunological responses uponrepeated administration to humans. Fourth, becausethe enzyme has no known physiological function in thebody, it is unlikely to produce any physiological side effects.Because the biochemical mechanism underlyingprophylaxis by exogenous pHu BChE was establishedand tested in several species, including nonhuman primates,results can be reliably extrapolated from animalexperiments and applied to humans. A dose of 200 mgof pHu BChE is envisioned as prophylaxis for humansexposed to 2 to 5 times the LD 50of soman.The foremost requirement to advance pHu BChE asa bioscavenger for human use was to obtain sufficientamounts of purified enzyme with which to conductanimal and clinical studies. Although a procedure forthe purification of pHu BChE from human plasma,which contains ~ 2 mg of enzyme per liter of plasma,was described, this source is not suitable for producinggram quantities of pHu BChE. 31 Cohn FractionIV-4 paste (a by-product of human plasma generatedduring the production of human blood proteins, suchas γ-globulin and clotting factors), was identified asa rich source of pHu BChE. Cohn Fraction IV-4 pastecontains ~ 150 mg of enzyme per kg, which is muchhigher than human plasma, and contains much lowerquantities of other plasma proteins because of the fractionationsteps employed in the production process. Aprocedure for the large-scale purification of pHu BChEfrom Cohn Fraction IV-4 paste was developed usingbatch procainamide affinity chromatography followedby anion exchange chromatography. Approximately6 g of purified enzyme was obtained from 120 kg ofCohn fraction IV-4. 32Pharmacokinetics and the Safety of Plasma-Derived Human ButyrylcholinesterasePurified pHu BChE displayed high bioavailability inthe circulation of mice, guinea pigs, and nonhuman primateswhen administered by IV, IM, or intraperitoneal(IP) injections. The enzyme displayed a mean residencetime (MRT) of about 48 hours in mice (IM, IP), 109 to110 hours in guinea pigs (IM, IP) and 72 to 74 hours inrhesus (IV) or cynomolgus monkeys (IM). 32–34 The circulatorystability profiles were similar to those previouslyobserved for enzyme purified from human plasma inrats and mice, 17–19 guinea pigs, 35 and rhesus monkeys. 19Because the major envisaged use of bioscavengersis prophylactic, it was important to demonstrate thatpHu BChE was devoid of side effects of its own. Miceand guinea pigs with circulating levels of pHu BChE ashigh as 300 U/mL did not display any signs of clinicaltoxicity. Results of necropsy performed on animals,together with the examination of hematology andserum chemistry parameters, did not reveal any clinicalsigns of pathology following the administration oflarge doses of pHu BChE. 32,33In Vitro and In Vivo Stability of Plasma-DerivedHuman ButyrylcholinesteraseThe thermal stability of purified pHu BChE storedat various temperatures has been evaluated. 32 Enzymeactivity was stable when stored in lyophilized form at4°, 25°, 37°, or 45°C for 2 years. The enzyme was alsostable when stored in liquid form at 4° and 25°C for1 year. The circulatory (in vivo) stability of enzymestored in lyophilized form at −20°C was evaluated bymeasuring pharmacokinetic parameters in mice. 32 Thepharmacokinetic properties of the enzyme were notaffected upon storage at −20°C for 3 years.Efficacy of Plasma-Derived HumanButyrylcholinesteraseThe efficacy of pHu BChE was evaluated in guineapigs and cynomolgus monkeys against multiple LD 50challenges of nerve agents. 34 Guinea pigs were protectedagainst a cumulative dose of 5 times the LD 50s of eithersoman or VX, and there was a decrease in molar concentrationof exogenously administered circulating pHuBChE equivalent to the amount of OP administered in agiven time period. 36 For example, guinea pigs administeredHu BChE equivalent to 8 times the LD 50of somanattained peak blood BChE levels of approximately300 U/mL. After challenge with 5.5 times the LD 50ofsoman, the enzyme level decreased to approximately100 U/mL. This approximate 200 U/mL decrease inblood BChE level is equivalent to around 5 to 5.5 timesthe LD 50of soman. With VX challenge, proportionatelyless enzyme was administered because the LD 50of VXis smaller. No signs of poisoning were observed in theexperimental animals during the efficacy studies. Animalswere subjected to necropsy 7 or 14 days followingnerve agent challenge and all tissues were normal uponlight microscopic examination. In nonhuman primates,cynomolgus monkeys were protected against a cumulativechallenge of 5.5 times the LD 50of soman. Of the sixanimals challenged, one died after the final challengedose of soman (total 5.5 times the LD 50within 4 h) andone was euthanized 48 hours after the final dose ofsoman. The surviving animals displayed no signs ofpoisoning. Subsequent examination of these animalsdid not show any signs of delayed toxicity followingexaminations of blood chemistry and hematology parametersfor less than 20 months. 37247