Butyrylcholinesterase as a therapeutic drug for protection against percutaneous VX

doi:10.1016/j.cbi.2010.05.014

Chemico-Biological Interactions

Volume 187, Issues 1–3, 6 September 2010, Pages 249-252

https://doi.org/10.1016/j.cbi.2010.05.014 Get rights and content

Abstract

The administration of purified human plasma-derived butyrylcholinesterase (HuBuChE) as a pretreatment has been demonstrated to enhance survival and protect against decreased cognitive function after exposure to organophosphorus poisons (OPs). Based on efficacy data obtained with guinea pigs and non-human primates and the lack of behavioral side effects, plasma-derived HuBuChE has been granted investigational new drug status by the US Food and Drug Administration. The recent availability of a recombinant form of HuBuChE (rHuBuChE) from the milk of transgenic goats has now allowed us to determine the pharmacokinetics of that material in guinea pigs and use it as a therapy following exposure to the VX. The rHuBuChE was expressed as a dimer and following intramuscular (i.m.) administration had more a rapid adsorption and clearance profile in guinea pigs than the plasma-derived material. Based on those data, we administered rHuBuChE i.m. 1 h after a percutaneous exposure of guinea pigs to either 2xLD₅₀ or 5xLD₅₀ of VX. Post-exposure therapy with rHuBuChE provided improved survival at both challenge levels, 90% and 33% respectively versus 20% or 0% respectively for animals that did not receive therapy. These studies showed that BuChE can be efficacious as a therapy against percutaneous exposure to VX.

Introduction

The efficacy of acetyl- and butyrylcholinesterase as protein drugs capable of providing protection against in vivo exposure to highly toxic organophosphorus compounds such as the chemical warfare agents soman, sarin or VX has been amply demonstrated over the past 15 years in a variety of animal model systems [1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11], [12], [13]. While mouse and macaque acetylcholinesterase and human butyrylcholinesterase have been used in pharmacokinetic studies and immunologic studies, plasma-derived human butyrylcholinesterase (HuBuChE, 7) and recombinant human butyrylcholinesterase (rHuBuChE) from the milk of transgenic goats [14], [15] have longer in vivo residence as determined by pharmacokinetic studies in either guinea pigs or monkeys. The capacity of this pretreatment approach to provide protection (assessed as survival after exposure to an otherwise lethal dose of agent) is readily apparent (Table 1).

While bioscavengers are efficacious in providing protection against nerve agent poisoning when used as pretreatments, their value as post-exposure therapeutics has received little attention. To address that deficit, we carried out a limited study in which rHuBuChE was administered to guinea pigs one hour after a multiple LD₅₀ percutaneous exposure to VX. The ability of BuChE to provide protection, as measured by enhanced survival, offers a clear in vivo demonstration of the therapeutic use of a bioscavenger to protect against an OP nerve agent.

Section snippets

Materials

The nerve agent VX (O-ethyl-S-(2-isopropylaminoethyl) methylphosphonothiolate) was obtained from the Research Development and Engineering Center, Aberdeen Proving Ground, MD. The compound was >97% pure by ³¹P NMR analysis. The goat milk derived rHuBuChE was supplied as a non-PEGylated purified solution in saline at 75 mg/mL as a gift from Nexia Biotechnologies (now PharmAthene Inc., Annapolis, MD). The rHuBuChE was diluted in physiologic saline to the desired concentration immediately before

rHuBuChE pharmacokinetics

The pharmacokinetics of rHuBuChE in guinea pigs followed a one-compartment model with a single elimination phase (Fig. 1). The estimated T_max was ∼4.2 h with >60% of the circulatory concentration of the enzyme available within 2 h. The estimated T_1/2 was 15.2 h. These results are in marked contrast to plasma-derived BuChE, which in guinea pigs has a circulatory T_max of 20 h after i.m. administration and a T_1/2 of ∼72 h [7].

Therapy experiments

Guinea pigs exposed to a 2 × LD₅₀ percutaneous dose of VX (n = 20) all displayed

Discussion

Butyrylcholinesterases from a variety of sources have been shown to have similar kinetic properties with respect to substrates and inhibitors [3], [4], [7]. This enzyme has also been shown to provide in vivo protection against poisoning by several different chemical warfare nerve agents when given as a pretreatment [2], [6], [7], [8]. The extent of protection afforded is hypothesized to be directly related to simple stoichiometry, where 1 mole of enzyme will bind, and hence neutralize, 1 mole of

Conflict of interest

None.

Funding

Support for this work at USAMRICD was provided in part by the Defense Threat Reduction Agency (DTRA) and in part by the Joint Program Executive Office for Chemical and Biological Defense (JPEO-CBD) of the Department of Defense.

Acknowledgements

Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the U.S. Army.

Investigators adhered to the Guide for the Care and Use of Laboratory Animals (1996) by the Institute of Laboratory Animal Resources, National Research Council, in accordance with stipulations for AAALAC accredited facilities. Principles of laboratory animal care (NIH publication No. 85-23, revised 1985) were followed.

References (21)

Y. Ashani et al.
Butyrylcholinesterase and acetylcholinesterase prophylaxis against soman poisoning in mice
Biochem. Pharmacol.
(1991)
B.P. Doctor et al.
Cholinesterases as scavengers for organophosphorous compounds: protection of primate performance against soman toxicity
Chem. Biol. Interact.
(1993)
B.P. Doctor et al.
Bioscavengers for the protection of humans against organophosphate toxicity
Chem. Biol. Interact.
(2005)
D.E. Lenz et al.
Protection against soman or VX poisoning by human butyrylcholinesterase in guinea pigs and cynomolgus monkeys
Chem. Biol. Interact.
(2005)
L. Raveh et al.
Human butyrylcholinesterase as a general prophylactic antidote for nerve agent toxicity
Biochem. Pharmacol.
(1993)
L. Raveh et al.
The stoichiometry of protection against soman and VX toxicity in monkeys pretreated with human butyrylcholinesterase
Toxicol. Appl. Pharmacol.
(1997)
A.D. Wolfe et al.
Acetylcholinesterase prophylaxis against organophosphate toxicity
Fund. Appl. Toxicol.
(1987)
A.D. Wolfe et al.
Use of cholinesterases as pretreatment drugs for the protection of rhesus monkeys against soman toxicity
Toxicol. Appl. Pharmacol.
(1992)
G.L. Ellman et al.
A new and rapid colorimetric determination of acetylcholinesterase activity
Biochem. Pharmacol.
(1961)
C.A. Castro et al.
Behavioral decrements persist in rhesus monkeys trained on a serial probe recognition task despite protection against soman lethality by butyrylcholinesterase
Neurotoxicol. Teratol.
(1994)

There are more references available in the full text version of this article.

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Butyrylcholinesterase as a therapeutic drug for protection against percutaneous VX

Abstract

Introduction

Section snippets

Materials

rHuBuChE pharmacokinetics

Therapy experiments

Discussion

Conflict of interest

Funding

Acknowledgements

Biochem. Pharmacol.

Chem. Biol. Interact.

Chem. Biol. Interact.

Chem. Biol. Interact.

Biochem. Pharmacol.

Toxicol. Appl. Pharmacol.

Fund. Appl. Toxicol.

Toxicol. Appl. Pharmacol.

Biochem. Pharmacol.

Neurotoxicol. Teratol.