Elsevier

Toxicology

Volume 233, Issues 1–3, 20 April 2007, Pages 40-46
Toxicology

Polyethylene-glycol conjugated recombinant human acetylcholinesterase serves as an efficacious bioscavenger against soman intoxication

https://doi.org/10.1016/j.tox.2006.08.036Get rights and content

Abstract

Extensive pharmacokinetic studies in both mice and rhesus macaques, with biochemically well defined forms of native and recombinant AChEs from bovine, rhesus and human origin, allowed us to determine an hierarchical pattern by which post-translation-related factors and specific amino-acid epitopes govern the pharmacokinetic performance of the enzyme molecule. In parallel, we demonstrated that controlled conjugation of polyethylene-glycol (PEG) side-chains to lysine residues of rHuAChE also results in the generation of active enzyme with improved pharmacokinetic performance. Here, we show that equally efficient extension of circulatory residence can be achieved by specific conditions of PEGylation, regardless of the post-translation-modification state of the enzyme. The masking effect of PEGylation, which is responsible for extending circulatory lifetime, also contributes to the elimination of immunological responses following repeated administration of AChE. Finally, in vivo protection studies in mice allowed us to determine that the PEGylated AChE protects the animal from a high lethal dose (2.5 LD50) of soman. On a mole basis, both the recombinant AChE and its PEGylated form provide higher levels of protection against soman poisoning than the native serum-derived HuBChE.

The findings that circulatory long-lived PEGylated AChE can confer superior protection to mice against OP-compound poisoning while exhibiting reduced immunogenicity, suggest that this chemically modified version of rHuAChE may serve as a highly effective bioscavenger for prophylactic treatment against OP-poisoning.

Introduction

The primary role of acetylcholinesterase (acetylcholine acetylhydrolase 3.1.1.7, AChE) is the termination of impulse transmission in cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine (ACh). Some organophosphate (OP) compounds, such as the nerve agent sarin and soman, inhibit AChE irreversibly by rapid phosphonylation of the serine residue in the enzyme active site. The high reactivity of ChEs towards OP-agents led to propose these biomolecules as exogenous scavengers for sequestration of toxic OP-agents before they reach their physiological target (Raveh et al., 1993, Raveh et al., 1997, Lenz et al., 2005 and references therein). Since ChEs react on a molar basis with the OP agents, the amounts of ChE required for sequestration of these compounds are high. This requirement encouraged the development of production systems for the generation of recombinant ChEs at large-scale (Kronman et al., 1992, Fischer et al., 1993, Saxena et al., 1998). However, pharmacokinetic studies (Kronman et al., 1992, Mendelson et al., 1998) have shown that recombinant enzymes generated by these systems, relying on either bacterial or mammalian cells, are retained in the circulation of experimental animals for much shorter periods of time than native fetal bovine serum acetylcholinesterase (FBS-AChE) or human serum butyrylcholinesterase (BChE). Therefore, deciphering the mechanisms involved in clearance of cholinesterases from the bloodstream is of importance for the development of enzyme-based bioscavengers for treatment of organophosphorous poisoning.

To allow a comprehensive appreciation of the structural basis for the circulatory residence of AChE, we conducted a series of studies in which the post-translation-related differences between the circulatory long-lived serum-derived native bovine AChE and its circulatory short-lived recombinant HEK-293-expressed counterpart were determined. These studies, in conjunction with extensive pharmacokinetic profiling of various AChE forms in mice, allowed us to demonstrate that both sialic acid occupancy and enzyme oligomerization contribute to the circulatory longevity of bovine AChE (Kronman et al., 2000). Further studies displayed that the circulatory residence of the human form of AChE in mice is regulated by the same set and pattern of rules (Chitlaru et al., 2001) and that N-glycan loading operates as a third component together with sialylation and tetramerization, within a hierarchical set of rules governing the circulatory lifetime of AChEs in mice. Thus, the effect of N-glycan addition on the pharmacokinetics of AChE was fully manifested only in the case of efficiently sialylated tetrameric forms of the enzyme (Chitlaru et al., 2002). In line with these studies, we could determine that by optimizing sialic acid occupancy, enzyme tetramerization and glycan loading, bovine and human recombinant AChEs can be converted into circulatory long-lived enzyme forms exhibiting mean residence time values which are similar to those of native serum-derived cholinesterases (Kronman et al., 2000, Chitlaru et al., 2002).

Pharmacokinetics of human AChE in the rhesus animal system deviate from the classical hierarchical rules found for the same enzyme in mice due to the operation of a process which specifically promotes elimination of the human enzyme, but not the bovine enzyme, from the circulation of monkeys (Cohen et al., 2004). This allowed us to identify a species-dependent amino-acid-related elimination process that operates as a fourth element in determining the circulatory fate of AChEs, overriding the positive contribution of post-translation-related processes (i.e. enzyme assembly and glycan loading) to circulatory retention.

In parallel, studies carried out in our laboratory (Cohen et al., 2001) demonstrated that controlled conjugation of polyethylene-glycol (PEG) side-chains to lysine residues of rHuAChE as well as of rHuBChE, can generate bioactive ChE enzymes exhibiting an improved pharmacokinetic profile (Fig. 1). Under a defined set of conditions, 4 PEG molecules may be appended to monomeric rHuAChE with minimal effect on the catalytic activity of the enzyme or on the reactivity towards active center inhibitors. Further studies (Cohen et al., 2006) allowed us also to determine that appendage of the PEG chains did not alter the ability of the enzyme to interact in an efficient manner with different OP agents.

Pharmacokinetic profiling of PEGylated AChE in mice allowed us to determine that the circulatory retention rates of the chemically modified enzyme (mean residence time = 2100 min), surpasses that of native serum-derived enzymes such the fetal bovine serum AChE (Cohen et al., 2001). In the rhesus animal model, PEGylated AChE demonstrated an unprecedented circulatory retention time value (MRT ∼7 days; Cohen et al., 2004), suggesting that AChE PEGylation bestows the enzyme with pharmacological properties suitable for its application as a prophylactic bioagent against OP intoxication.

Here we report on the effect of PEGylation on the pharmacokinetic performance of various rHuAChE species, differing in their post-translation modification state. In addition, we examine the effect of PEGylation on the immunogenic properties of the enzyme. Finally, we show that the PEGylated version of rHuAChE can efficiently protect mice against OP agent intoxication.

Section snippets

Enzymes

Procedures of transfection of the human embryonal kidney derived cell line (HEK-293) with the expression vector of the C-terminus truncated HuAChE enzymes (Cohen et al., 2001) or of the rHuBChE enzyme and the generation of stable cell clones expressing high levels of recombinant ChE products, were described previously (Kronman et al., 1992, Kronman et al., 1995). The generation of fully sialylated AChE by HEK-293ST cells, and enzymatic desialylation of AChE were described previously (Chitlaru

Effect of PEGylation on the circulatory residence of AChE forms differing in their state of sialylation

The finding that AChE circulatory residence may be extended by either post-translation modification (Kronman et al., 2000, Chitlaru et al., 2001, Chitlaru et al., 2002) or by PEG-conjugation (Cohen et al., 2001, Cohen et al., 2004), prompted us to examine the interrelationship between PEGylation and enzyme processing, with regard to the overall effect on the pharmacokinetic performance of the enzyme. Specifically, we asked whether PEGylation can convert AChE molecular species varying in their

Acknowledgement

We thank Ms. Shirley Lazar, Ms. Nehama Zeliger and Ms. Dana Stein for excellent technical assistance.

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This work was supported in part by the U.S. Army Research and Development Command Contract DAMD17-03-C-0012 (to AS) and by a grant from Life Science Research Israel (Ltd.)

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