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    • Some studies have evaluated gene transfer as an approach

    2020-07-30

    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 kn to nm 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. It has long been known that native BChE is a major factor in the inactivation of cocaine, an ester-type drug of abuse [190]. But recently some researchers conceived the idea that BChE mutations could improve that function to a point that would favorably impact cocaine overdose. Rapid progress was made in different laboratories approaching this goal, assisted by computer-based models of cocaine docking in the enzyme’s active site, which led to successful predictions to improve drug binding and hydrolysis [29]. At present, near-optimal BChE versions that enhance the rate of cocaine inactivation by more than a thousand-fold have been generated [191]. Efficacy with exogenous administration of such cocaine-hydrolyzing BChE variants can also suffer because they are more rapidly cleared from the circulation than native BChE. However, site-directed mutagenesis introducing disulfide bonds between two mutant BChE subunits led to an approximate doubling of circulatory half-life in rats [192]. In our hands, mice and rats treated with a cocaine hydrolase showed no reaction whatsoever to doses of cocaine that would ordinarily have been lethal within 1–2 min. In contrast, they merely continued normal cage-side activities, eating and grooming, just like vehicle-controls [72].