Nerve agents belong to a class of compounds known as organophosphate (OP) agents. OP nerve agents, such as sarin gas, soman, tabun or VX, enter the blood stream via inhalation or absorption through the skin. The nerve agents travel in the circulatory system to the brain and muscles inhibiting the Acetylcholinesterase (AChE) enzyme, thereby causing the nerves to become over-stimulated leading to massive convulsions and death. Available pre-and post-treatment options for OP-intoxication are inadequate and there is a clear need for more efficacious countermeasures.
Several studies have demonstrated that exogenous administration of recombinant AChE as prophylactic therapy removes nerve agents directly from the bloodstream before they reach their targets. Yet, the therapeutic use of recombinant AChE (or any other recombinant cholinesterase) is complicated by short circulatory residence time [e.g., Kronman C. et al. (1995), Biochem. J. 311: 959-967; Chitlaru T. et al. (1998) Biochem. J. 336: 647-658].
To overcome the limitation of circulatory life-time of recombinant AChE, we conjugated polyethylene glycol (PEG) moieties to the C-terminal of recombinant human AChE (PEG-rHuAChE). Controlled conjugation of PEG to the recombinant enzyme resulted in fully active enzyme which is retained in the circulation. The plasma mean residual time (MRT) of PEG- rHuAChE increased as high as 50-fold when compared to the MRT of nonmodified rHuAChE in a murine animal model system [Cohen O. et al. (2001) Biochem. J. 357: 795-802]. The prophylactic activity of PEG-rHuAChE toward various nerve agents' intoxications was demonstrated in several animal model systems [Kronman C. et al. (2007) Toxicology. 233: 40-46; Mazor O. et al. (2008) Mol Pharmacol. 74: 755-763]. We have further demonstrated the increase of circulatory residence of PEGylated rHuAChE in rhesus macaques [Cohen O. et al. (2004) Biochem. J. 378: 117-128-802]. Moreover, we have demonstrated that PEG-rHuAChE retained in the circulation conferring superior prophylactic ability compared with native serum-derived Human butyrylcholinesterase [Cohen O. et al. (2006) Mol Pharmacol. 70: 1121-1131].
The substantial increase in the pharmacological stability of ChEs, mentioned above, has removed an obstacle on the way toward utilizing serine hydrolases as organophosphates scavengers. However, an enzyme-based detoxifying agent for pharmaceutical uses should exhibit, beside improved stability and retained activity, preferably also a well-defined composition. However, PEGylation of a protein provides a heterogeneous product corresponding to a mixture of various structures comprising randomly formed linkages of PEG with reactive sites. By examining a series of recombinant AChE hypolysine mutant, we have created a uniformly PEG-conjugated rHuAChE, in which the PEG chains are preferably conjugated at predetermined sites of the enzyme [Cohen O. et al. (2007) J. Biol. Chem. 282: 35491-35501].
Taking advantage of the fact that human IgG type antibodies exhibit very long circulatory half-life, a chimeric recombinant molecule of human AChE coupled to the Fc region of human IgG1 was designed. The novel fusion protein maintains its full enzymatic activity and sequesters nerve-agents such as sarin and VX [Noy-Porat. et al. (20015) Bioconj. Chem. 26: 1753-8]. As hypothesized, AChE-Fc exhibits exceptionally circulatory residence longevity in murine model, superior to any other known cholinesterase-based recombinant bioscavengers (including the previously developed PEG-rHuAChE). Owing to its optimized pharmacokinetic performance, high reactivity toward nerve agents, ease of production and compatibility with biotechnological production and purification, AChE-Fc emerges as a promising next-generation organophosphate bioscavenger.
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