Recombinant human butyrylcholinesterase from milk of transgenic animals to protect against organophosphate poisoning

Abstract

Dangerous organophosphorus (OP) compounds have been used as insecticides in agriculture and in chemical warfare. Because exposure to OP could create a danger for humans in the future, butyrylcholinesterase (BChE) has been developed for prophylaxis to these chemicals. Because it is impractical to obtain sufficient quantities of plasma BChE to treat humans exposed to OP agents, the production of recombinant BChE (rBChE) in milk of transgenic animals was investigated. Transgenic mice and goats were generated with human BChE cDNA under control of the goat ␤-casein promoter. Milk from transgenic animals contained 0.1-5 g/liter of active rBChE. The plasma half-life of PEGylated, goat-derived, purified rBChE in guinea pigs was 7-fold longer than non-PEGylated dimers. The rBChE from transgenic mice was inhibited by nerve agents at a 1:1 molar ratio. Transgenic goats produced active rBChE in milk sufficient for prophylaxis of humans at risk for exposure to OP agents.organophosphorus nerve agent ͉ recombinant protein expression ͉ transgenic production H uman plasma butyrylcholinesterase (huBChE) (EC 3.1.1.8) is a globular, tetrameric serine esterase with a molecular mass of Ϸ340 kDa that is stable in plasma with a half-life of Ϸ12 days (1, 2). Although the physiological function of huBChE is unclear, the enzyme prevents intoxication of animals exposed to organophosphorus (OP) compounds (3, 4). The huBChE enzyme also hydrolyzes many ester-containing drugs, such as cocaine and succinylcholine (5). The toxicity of OP agents is due to irreversible inhibition of acetylcholinesterase and the subsequent continuous stimulation of neurons by acetylcholine (6). Administration of exogenous huBChE, which irreversibly binds OP agents to prevent inactivation of acetylcholinesterase and continuous cholinergic stimulation, is a potential strategy for preventing toxicity from OP agents (4). Although huBChE has been obtained from human plasma by a large scale purification technique, this procedure is severely limited by the volume of human plasma needed (7). It is unlikely that a sufficient amount of enzyme could be purified commercially by this technique. Because of the 1:1 stoichiometry required for protection against exposure to OP agents (8), large quantities of huBChE are needed for effective prophylaxis and treatment of exposure. Compared with other potential enzymatic bioscavengers of OP agents, huBChE has a broad spectrum of activity, a relatively long half-life, and limited, if any, physiological side effects (9). Producing recombinant BChE (rBChE) is an alternative to purification of the enzyme from human plasma. Recombinant huBChE has been expressed in Escherichia coli (10), albeit in a nonfunctional form; mammalian 293T (11); and CHO (12) cells. However, these expression systems cannot economically produce sufficient quantities of rBChE with a residence time similar to native huBChE that would allow development of the enzyme as an agent for prophylaxis against OP poisoning.The production of recombinant proteins by the mammary g...

Adenovirus-mediated human paraoxonase1 gene transfer to provide protection against the toxicity of the organophosphorus pesticide toxicant diazoxon

“…Currently, plasma-derived human BChE [9][10][11] and Protexia, a recombinant BChE produced in the milk of transgenic goats, are under investigation. 12,13 BChE is a stoichiometric bioscavenger, requiring large quantities (200 mg per 70 kg person) of the enzyme to provide protection against 2LD 50 of soman. 14 More recently, enzymes that can inactivate OP without being consumed are being investigated for their potential as catalytic bioscavengers.…”

Pseudocholinesterase Deficiency in an Indian Community

“…Behringwerke, a German pharmaceutical company, created a purified, injectable form of human pseudocholinesterase. [30,23] Another, USA-based pharma company PharmAthene has begun using genetically modified goats to create pseudocholinesterase in large quantities. [11] Treatment or management involves the avoidance of following class of drugs like drugs containing succinylcholine: quelicin, anectine; drugs containing mivacurium: mivacron; drugs containing pilocarpine: salagen; drugs containing butyrylcholine; drugs containing huperzine A and donepezil; drugs containing propionylcholine and acetylcholine; drugs containing parathion; procaine drugs: novocaine.…”

Organophosphorus compounds as chemical warfare agents: a review

“…134,135 Several enzymes are being evaluated for use as scavengers, such as human serum BChE, recombinant human BChE expressed in the milk of transgenic goats, genetic variants of AChE and human paraoxonase. [134][135][136][137][138] Oximes can be employed for pretreatment, improving the post-treatment by atropine and other oximes 132,139 (see below), but some questions should be addressed for their use, such as timing, duration and achievement of adequate concentrations after administration. 132,133 The chemotherapy employed for the treatment of intoxication with OPCs includes the use of three types of drugs: 1,3,13,128 (i) an anticholinergic substance, to antagonize the effects of ACh accumulation in the cholinergic receptors; (ii) a central nervous system (CNS) depressor, which acts as an anticonvulsive; and (iii) an oxime to reactivate inhibited AChE.…”