Chunyuan Luo

Bioscavenger for protection from toxicity of organophosphorus compounds.

Current antidotal regimens for organophosphorus compound (OP) poisoning consist of a combination of pretreatment with a spontaneously reactivating AChE inhibitor such as pyridostigmine bromide, and postexposure therapy with anticholinergic drugs such as atropine sulfate and oximes such as 2-PAM chloride (Gray, 1984). Although these antidotal regimens are effective in preventing lethality of animals from OP poisoning, they do not prevent postexposure incapacitation, convulsions, performance deficits, or, in many cases, permanent brain damage (Dunn and Sidell, 1989). These problems stimulated the development of enzyme bioscavengers as a pretreatment to sequester highly toxic OPs before they reach their physiological targets. Several studies over the last two decades have demonstrated that exogenously administered human serum butyrylcholinesterase (Hu BChE) can be used successfully as a safe, efficacious, and single prophylactic treatment to counteract the toxicity of OPs. It also has potential use for first responders (civilians) reacting to terrorist nerve gas release, pesticide overexposure, or succinylcholine-induced apnea. A dose of 200 mg of Hu BChE in humans is envisioned as a prophylactic treatment that can protect from exposure of 2-5 x LD50 of nerve agents (Ashani, 2000).

Comparison of oxime reactivation and aging of nerve agent-inhibited monkey and human acetylcholinesterases.

Non-human primates are valuable animal models that are used for the evaluation of nerve agent toxicity as well as antidotes and results from animal experiments are extrapolated to humans. It has been demonstrated that the efficacy of an oxime primarily depends on its ability to reactivate nerve agent-inhibited acetylcholinesterase (AChE). If the in vitro oxime reactivation of nerve agent-inhibited animal AChE is similar to that of human AChE, it is likely that the results of an in vivo animal study will reliably extrapolate to humans. Therefore, the goal of this study was to compare the aging and reactivation of human and different monkey (Rhesus, Cynomolgus, and African Green) AChEs inhibited by GF, GD, and VR. The oximes examined include the traditional oxime 2-PAM, two H-oximes HI-6 and HLo-7, and the new candidate oxime MMB4. Results indicate that oxime reactivation of all three monkey AChEs was very similar to human AChE. The maximum difference in the second-order reactivation rate constant between human and three monkey AChEs or between AChEs from different monkey species was 5-fold. Aging rate constants of GF-, GD-, and VR-inhibited monkey AChEs were very similar to human AChE except for GF-inhibited monkey AChEs, which aged 2-3 times faster than the human enzyme. The results of this study suggest that all three monkey species are suitable animal models for nerve agent antidote evaluation since monkey AChEs possess similar biochemical/pharmacological properties to human AChE.

Two possible orientations of the HI-6 molecule in the reactivation of organophosphate-inhibited acetylcholinesterase.

The inhibition of acetylcholinesterase (AChE) by organophosphorus compounds (OPs) causes acute toxicity or death of the intoxicated individual. One group of these compounds, the OP nerve agents, pose an increasing threat in the world due to their possible use in the battlefield or terrorist acts. Antidotes containing oxime compounds to reactivate the inhibited enzyme are highly valued for treatment against OP poisoning. One of these reactivators, HI-6, was shown to be significantly more effective in treating soman toxicity than other oximes, such as 2-PAM, TMB4, and obidoxime. However, HI-6 was less effective in reactivating AChE inhibited by the OP pesticide, paraoxon. In this study, the mechanism for HI-6-induced reactivation of OP-AChE conjugates was investigated using mouse mutant AChEs inhibited with different OPs including organophosphate paraoxon, and several methylphosphonates. Results indicate that the HI-6 molecule may assume two different orientations in the reactivation of AChE inhibited by organophosphate and Sp methylphosphonates. These conclusions were further corroborated by reactivation studies using an analog of HI-6 in which the bispyridinium moieties are linked by a methylene bridge rather than an ether oxygen.

Developing procedures for the large-scale purification of human serum butyrylcholinesterase.

Human serum butyrylcholinesterase (Hu BChE) is the most viable candidate for the prophylactic treatment of organophosphate poisoning. A dose of 200 mg/70 kg is predicted to protect humans against 2x LD(50) of soman. Therefore, the aim of this study was to develop procedures for the purification of gram quantities of this enzyme from outdated human plasma or Cohn Fraction IV-4. The purification of Hu BChE was accomplished by batch adsorption on procainamide-Sepharose-CL-4B affinity gel followed by ion-exchange chromatography on a DEAE-Sepharose column. For the purification of enzyme from Cohn Fraction IV-4, it was resuspended in 25 mM sodium phosphate buffer, pH 8.0, and fat was removed by decantation, prior to batch adsorption on procainamide-Sepharose gel. In both cases, the procainamide gel was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0, containing 0.05 M NaCl, and the enzyme was eluted with the same buffer containing 0.1 M procainamide. The enzyme was dialyzed and the pH was adjusted to 4.0 before loading on the DEAE column equilibrated in sodium acetate buffer, pH 4.0. The column was thoroughly washed with 25 mM sodium phosphate buffer, pH 8.0 containing 0.05 M NaCl before elution with a gradient of 0.05-0.2M NaCl in the same buffer. The purity of the enzyme following these steps ranged from 20% to 40%. The purity of the enzyme increased to >90% by chromatography on an analytical procainamide affinity column. Results show that Cohn Fraction IV-4 is a much better source than plasma for the large-scale isolation of purified Hu BChE.

Pharmacokinetics and immunologic consequences of exposing macaques to purified homologous butyrylcholinesterase

Exposure to organophosphorus compounds (OPs), in the form of nerve agents and pesticides poses an ever increasing military and civilian threat. In recent years, attention has focused on the use of exogenously administered cholinesterases as an effective prophylactic treatment for protection against OPs. Clearly, a critical prerequisite for any potential bioscavenger is a prolonged circulatory residence time, which is influenced by the size of protein, the microheterogeneity of carbohydrate structures, and the induction (if any) of anti-enzyme antibodies following repeated injections of the enzyme. Previously, it was demonstrated that multiple injections of equine butyrylcholinesterase (BChE) into rabbits, rats, or rhesus monkeys, resulted in a mean residence time spanning several days, and variable immune responses. The present study sought to assess the pharmacokinetics and immunological consequences of administration of purified macaque BChE into macaques of the same species at a dose similar to that required for preventing OP toxicity. An i.v. injection of 7,000 U of homologous enzyme in monkeys demonstrated much longer mean residence times in plasma (MRT = 225 +/- 19 h) compared to those reported for heterologous Hu BChE (33.7 +/- 2.9 h). A smaller second injection of 3,000 U given four weeks later, attained predicted peak plasma levels of enzyme activity, but surprisingly, the MRT in the four macaques showed wide variation and ranged from 54 to 357 h. No antibody response was detected in macaques following either injection of enzyme. These results bode well for the potential use of human BChE as a detoxifying drug in humans.

Role of edrophonium in prevention of the re-inhibition of acetylcholinesterase by phosphorylated oxime.

We examined the role of edrophonium in the acceleration phenomenon using mouse wild-type and mutant D74N AChE inhibited with 7-(O,O-diethyl-phosphinyloxy)-1-methylquinolinium methylsulfate (DEPQ). With DEPQ-inhibited wild-type mouse acetylcholinesterase (AChE), the reactivation kinetic profile demonstrated one-phase exponential association only when 2-[hydroxyimino methyl]-1-methylpyridinium chloride (2-PAM) and 1-(2-hydroxy-iminomethyl-1-pyridinium)-1-(4-carboxy-aminopyridi nium)-dimethyl ether hydrochloride (HI-6) were used as reactivators. When 1,1[oxybis-methylene)bis[4-(hydroxyimino)methyl] pyridinium dichloride (LüH6) and 1,1-trimethylene bis(4-hydroxyimino methyl) pyridinium dichloride (TMB4) were used, the reactivation kinetic profile was biphasic in nature. Edrophonium had no effect on reactivation by 2-PAM and HI-6, but significantly accelerated LüH6- and TMB4-induced reactivation of DEPQ-inhibited wild-type mouse AChE. Comparison of the initial and overall reactivation rate constants with five oximes indicated that acceleration by edrophonium may be due to the prevention of re-inhibition of the reactivated enzyme by the phosphorylated oxime (POX) produced during the reactivation. With LüH6 and TMB4, about 2.5-fold increase in the reactivation rate constants was observed in the presence of edrophonium, but little or no effect was observed with the other three oximes. The initial reactivation rate constants were 5.4- and 4.2-fold of the overall rate constants with LüH6 and TMB4 as reactivators respectively, however, very little change was found between the initial and overall rate constants with the other three oximes. In experiments with D74N AChE, for which the inhibition potency of charged organophosphate (OP) was two to three orders less than wild-type enzyme, edrophonium had no effect on the reactivation by LüH6 and TMB4 and the time courses of reactivation were monophasic. The data from mutant enzyme substantiate the involvement of edrophonium in protecting POX re-inhibition of reactivated enzyme formed during the reactivation of OP-inhibited AChE.

Y124 at the peripheral anionic site is important for the reactivation of nerve agent-inhibited acetylcholinesterase by H oximes

The toxicity of organophosphorus (OP) nerve agents is manifested through irreversible inhibition of acetylcholinesterase (AChE) at the cholinergic synapses, which stops nerve signal transmission, resulting in a cholinergic crisis and eventually death of the poisoned person. Oxime compounds used in nerve agent antidote regimen reactivate nerve agent-inhibited AChE and halt the development of this cholinergic crisis. Due to diversity in structures of OP nerve agents, none of the currently available oximes is able to reactivate AChE inhibited by different nerve agents. To understand the mechanism for the differential activities of oximes toward AChE inhibited by diverse nerve agents in order to aid the design of new broad-spectrum AChE reactivators, we undertook site-directed mutagenesis and molecular modeling studies. Recombinant wild-type and mutant bovine (Bo) AChEs were inhibited by two bulky side-chain nerve agents, GF and VR, and used for conducting reactivation kinetics with five oximes. A homology model for wild-type Bo AChE was built using the recently published crystal structure of human AChE and used to generate models of 2-PAM and HI-6 bound to the active-sites of GF- and VR-inhibited Bo AChEs before nucleophilic attack. Results revealed that the peripheral anionic site (PAS) of AChE as a whole plays a critical role in the reactivation of nerve agent-inhibited AChE by all 4 bis-pyridinium oximes examined, but not by the mono-pyridinium oxime 2-PAM. Of all the residues at the PAS, Y124 appears to be critical for the enhanced reactivation potency of H oximes.

Mechanism for potent reactivation ability of H oximes analyzed by reactivation kinetic studies with cholinesterases from different species

Oxime-induced reactivation of organophosphorus (OP) nerve agent-inhibited acetylcholinesterase (AChE) is a very important step for the treatment of nerve agent toxicity. Therefore, extensive efforts are being made to develop more efficient and broad-spectrum oximes to replace the currently used oximes 2-PAM or obidoxime. In the 1970s and 1980s, several H oximes (such as HI-6 and HLo-7) were found to be very potent reactivators of non-aged soman-inhibited AChE. Later these oximes were shown to rapidly reactivate GF- and VR-inhibited AChE as well. However, the mechanism for the high potency of these H oximes is still unknown. In this study, the relationship between the reactivation rate constant of nerve agent-inhibited rhesus monkey AChE, human AChE and guinea pig AChE and the size of the alkoxyl (OR) group of nerve agents was analyzed. Results demonstrate that for nerve agent-inhibited rhesus monkey and human AChEs, reactivation by H oximes accelerated as the size of the OR group was increased. But with guinea pig AChE, reactivation by H oximes declined as the size of the OR group was increased. Reactivation kinetic study using GF- and VR-inhibited wild-type and mutant bovine AChEs has shown that mutations of Y124Q and W286A particularly reduced reactivation by these H oximes. Since these 2 amino acid residues are highly conserved in all AChEs sequenced to date, it is unlikely that the remarkable reduction observed in H oxime reactivation with guinea pig AChE is caused by a change in these two amino acid residues.

Pharmacokinetics and immunologic consequences of repeated administrations of purified heterologous and homologous butyrylcholinesterase in mice

AIM To assess the consequences of repeated administrations of purified human serum butyrylcholinesterase (Hu BChE) and mouse serum (Mo) BChE into mice. MAIN METHODS Purified Hu BChE and Mo BChE isolated from the sera of CD-1 mice were administered into Balb/c or CD-1 mice. The enzymes were delivered by i.m. injections of approximately 100U (0.15mg) on day 1 and on day 28, respectively. The effects of two injections were monitored by following blood BChE and anti-BChE IgG levels. KEY FINDINGS Hu BChE displayed a mean residence time (MRT) of 50h, and an area under the curve (AUC) of 1220U/ml.h in Balb/c or CD-1 mice. Mo BChE exhibited an MRT of 78h and an AUC of 1815U/ml.h in Balb/c mice; the AUC increased to 2504U/ml.h in CD-1 mice. A second injection of Hu BChE in both strains exhibited a marked reduction in circulatory stability. The circulatory stability of the second injection of Mo BChE was reduced in Balb/c mice, but was almost identical to the first injection in CD-1 mice. Consistent with these observations, circulating anti-BChE IgGs were observed in mice injected with Hu BChE; low levels of anti-BChE IgGs were observed only in Balb/c mice injected with Mo BChE. No antibody response was detected in CD-1 mice following either injection of homologous Mo BChE. SIGNIFICANCE The identical pharmacokinetic profiles and the absence of an immunologic response following a second administration of homologous BChE support the development of Hu BChE as a detoxifying drug in humans.

Probing the role of amino acids in oxime-mediated reactivation of nerve agent-inhibited human acetylcholinesterase

In this study, we employed site-directed mutagenesis to understand the role of amino acids in the gorge in oxime-induced reactivation of nerve agent-inhibited human (Hu) acetylcholinesterase (AChE). The organophosphorus (OP) nerve agents studied included GA (tabun), GB (sarin), GF (cyclosarin), VX, and VR. The kinetics of reactivation were examined using both the mono-pyridinium oxime 2-PAM and bis-pyridinium oximes MMB4, HI-6, and HLö-7. The second-order reactivation rate constants were used to compare reactivation of nerve agent-inhibited wild-type (WT) and mutant enzymes. Residues including Y72, Y124 and W286 were found to play important roles in reactivation by bis-pyridinium, but not by mono-pyridinium oximes. Residue Y124 also was found to play a key role in reactivation by HI-6 and HLö-7, while E202 was important for reactivation by all oximes. Residue substitutions of F295 by Leu and Y337 by Ala showed enhanced reactivation by bis-pyridinium oximes MMB4, HI-6, and HLö-7, possibly by providing more accessibility of the OP moiety associated at the active-site serine to the oxime. These results are similar to those observed previously with bovine AChE and demonstrate that there is significant similarity between human and bovine AChEs with regard to oxime reactivation.