Human serum butyrylcholinesterase: in vitro and in vivo stability, pharmacokinetics, and safety in mice.
The use of exogenously administered cholinesterases (ChEs) as bioscavengers of highly toxic organophosphate (OP) nerve agents is now sufficiently well documented to make them a highly viable prophylactic treatment against this potential threat. Of the ChEs evaluated so far, human serum butyrylcholinesterase (HuBChE) is most suitable for human use. A dose of 200 mg (3 mg/kg) of HuBChE is envisioned as a prophylactic treatment in humans that can protect from an exposure of up to 2 x LD50 of soman. In addition to its use as a prophylactic for a variety of wartime scenarios, including covert actions, it also has potential use for first responders (civilians) reacting to terrorist nerve gas release. We recently, developed a procedure for the large-scale purification of HuBChE, which yielded approximately 6 g of highly purified enzyme from 120 kg of Cohn fraction IV-4. The enzyme had a specific activity of 700-750 U/mg and migrated as a single band on SDS-PAGE. To provide data for initiating an investigational new drug (IND) application for the use of this enzyme as a bioscavenger in humans, we established its pharmacokinetic properties, examined its safety in mice, and evaluated its shelf life at various temperatures. In mice administered various doses up to 90 mg/kg, enzyme activity reached peak levels in circulation at 10 and 24 h following i.p. and i.m. injections, respectively. The enzyme displayed a mean residence time (MRT) of 40-50 h, regardless of the route of administration or dose of injected enzyme. Mice were euthanized 2 weeks following enzyme administration and tissues were examined grossly or microscopically for possible toxic effects. Results suggest that HuBChE does not exhibit any toxicity in mice as measured by general observation, serum chemistry, hematology, gross or histologic tissue changes. The shelf life of this enzyme stored at 4, 25, 37, and 45 degrees C was determined in lyophilized form. The enzyme was found to be stable when stored in lyophilized form at -20, 4, 25, or 37 degrees C to date (2 years), as measured by specific activity and SDS polyacrylamide gel electrophoresis. The effect of storage on circulatory stability was determined by measuring MRT in mice; there was no change in the MRT of lyophilized enzyme stored at -20 degrees C to date (2 years). These results provide convincing data that HuBChE is a safe bioscavenger that can provide protection against all OP nerve agents. Efforts are now underway to prepare the required documentation for submission of an IND application to the United States Food and Drug Administration (USFDA).
Saxena
Ashima
A
Division of Biochemistry, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, MD 20910-7500, USA. ashima.saxena@na.amedd.army.mil
Sun
Wei
W
Luo
Chunyuan
C
Doctor
Bhupendra P
BP
D000818:Animals; D000931:Antidotes; D002091:Butyrylcholinesterase / Q000008:administration & dosage / Q000009:adverse effects* / Q000493:pharmacokinetics*; D004795:Enzyme Stability; D006801:Humans; D051379:Mice; D012999:Soman / Q000037:antagonists & inhibitors / Q000633:toxicity; D013696:Temperature
D000931:Antidotes; D012999:Soman; D002091:Butyrylcholinesterase