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  • As noted earlier while exogenously administered BChE can pro

    2020-06-29

    As noted earlier, while exogenously administered BChE can protect against OP toxicity, its efficacy is limited by clearance of the protein from the circulation within a few days. A number of investigations have attempted to increase the circulation time of ChEs by chemical modification, gene transfer, and “nanoformulation.” Studies using AChE as a bioscavenger [167], [168], [169], [170], [171] led to the conclusion that the degree of AChE sialylation has a direct influence on the enzyme’s circulatory residence time. Chemical modification of recombinant BChE by polysialylation led to a 6-fold increase in mean residence time after iv dosing in mice, and such enzyme proved to be a bioscavenger offering protection against several OP nerve agents [172], [173]. Modifying proteins with polyethylene glycol (PEG, i.e., PEGylation) has been used for decades to reduce their clearance in vivo [174], [175], [176], [177]. Cohen and coworkers [171] reported that PEGylation of rhesus and human AChE led to large increases in mean residence times of recombinant rhesus and human AChE given to rhesus monkeys. PEGylation also doubled the mean circulatory residence time of recombinant tetrameric BChE from 18.3 to 36.2 h [178]. An almost 10-fold increase in circulatory time was gained by PEGylating recombinant monomeric BChE [179]. Thus, modifying ChEs by PEG generally leads to increased circulation times, but there are reports of antibody development and more rapid clearance of enzyme activity with subsequent doses of PEGylated proteins [179], [180]. Some studies have evaluated gene transfer as an approach to increase and prolong BChE activity for protection against OP toxicants. Chilukuri and coworkers [179], [181] showed that BChE−/− mice treated (iv but not ip) with recombinant adenoviruses encoding rHu-BChE showed elevated blood BChE levels (about 200-fold higher than wild-type controls) peaking about 5 days after treatment, but returning to baseline by 10 days post-inoculation. Antibodies to the native protein were detected in the serum. In a similar approach, Parikh and coworkers [182] reported up to 3400-fold increase in crizotinib synthesis BChE activity and transient protection against high (5–30-fold LD50) doses of the OP anti-ChEs echothiophate and VX (O-ethyl-S-2-N,N-diisopropylaminoethyl methylphosphonothiolate). Again BChE activity returned to baseline by day 10, however. Nanoformulations of BChE that might be useful as bioscavengers have been reported. Gaydess and coworkers [183] described a polyionic complex made of BChE with a poly-L-lysine and poly(ethylene glycol) copolymer, with an estimated diameter of about 15 nm. Fluorescence-labeled BChE-copolymer complexes injected into mice showed a small amount of BChE entered the brain. In 2015, Pope and colleagues [184] reported on a series of BChE-copolymer complexes synthesized following the general approach of Gaydess et al., [183]. A subset of these complexes was spherical, with a median diameter of about 35 nm [185]. In vitro sensitivity to the OP anti-ChE paraoxon, resistance to proteases and heat-inactivation, and in vitro “bioscavenging” activity against paraoxon were all equivalent or enhanced compared to native BChE [184], [185]. Recombinant dimeric BChE has also been conjugated with CdSe/CdZnS quantum dots [186]. These conjugates retained partial enzyme activity and showed similar sensitivity to paraoxon. Sokolov and colleagues [187] reported that human BChE conjugated with gold nanoparticles had a diameter about 15 nm and showed an interesting increase in BChE activity. In another recent study, equine serum BChE was coated with a zwitterionic polymer gel [188], leading to nanoparticles of 15–30 nm diameter that resisted inactivation by heat and trypsin. They also showed about a 3-fold increase in circulating time versus the free enzyme, and importantly no immune sensitization with repeated dosing. Rahhal and colleagues [189] reported an interesting formulation of equine BChE, laminating a film of protein onto a mold to produce 1 µm BChE microparticles. After purification, the preparation was administered by insufflation, with BChE retention times similar to free enzyme following oro-tracheal administration (48–72 h). Optimization of such formulations holds promise for further development of bioscavengers for OP and possibly other toxic chemicals.