Acetylcholinesterase-Fc Fusion Protein (AChE-Fc): A Novel Potential Organophosphate Bioscavenger with Extended Plasma Half-Life.

Acetylcholinesterase (AChE) is the physiological target of organophosphate nerve agent compounds. Currently, the development of a formulation for prophylactic administration of cholinesterases as bioscavengers in established risk situations of exposure to nerve agents is the incentive for many efforts. While cholinesterase bioscavengers were found to be highly effective in conferring protection against nerve agent exposure in animal models, their therapeutic use is complicated by short circulatory residence time. To create a bioscavenger with prolonged plasma half-life, compatible with biotechnological production and purification, a chimeric recombinant molecule of HuAChE coupled to the Fc region of human IgG1 was designed. The novel fusion protein, expressed in cultured cells under optimized conditions, maintains its full enzymatic activity, at levels similar to those of the recombinant AChE enzyme. Thus, this novel fusion product retained its binding affinity toward BW284c5 and propidium, and its bioscavenging reactivity toward the organophosphate-AChE inhibitors sarin and VX. Furthermore, when administered to mice, AChE-Fc exhibits exceptional circulatory residence longevity (MRT of 6000 min), superior to any other known cholinesterase-based recombinant bioscavengers. Owing to its optimized pharmacokinetic performance, high reactivity toward nerve agents, and ease of production, AChE-Fc emerges as a promising next-generation organophosphate bioscavenger.

Preclinical and first-in-human evaluation of PRX-105, a PEGylated, plant-derived, recombinant human acetylcholinesterase-R.

PRX-105 is a plant-derived recombinant version of the human 'read-through' acetylcholinesterase splice variant (AChE-R). Its active site structure is similar to that of the synaptic variant, and it displays the same affinity towards organophosphorus (OP) compounds. As such, PRX-105 may serve as a bio-scavenger for OP pesticides and chemical warfare agents. To assess its potential use in prophylaxis and treatment of OP poisoning we conducted several preliminary tests, reported in this paper. Intravenous (IV) PRX-105 was administered to mice either before or after exposure to an OP toxin. All mice who received an IV dose of 50nmol/kg PRX-105, 2min before being exposed to 1.33×LD50 and 1.5×LD50 of toxin and 10min after exposure to 1.5×LD50 survived. The pharmacokinetic and toxicity profiles of PRX-105 were evaluated in mice and mini-pigs. Following single and multiple IV doses (50 to 200mg/kg) no deaths occurred and no significant laboratory and histopathological changes were observed. The overall elimination half-life (t½) in mice was 994 (±173) min. Additionally, a first-in-human study, to assess the safety, tolerability and pharmacokinetics of the compound, was conducted in healthy volunteers. The t½ in humans was substantially longer than in mice (average 26.7h). Despite the small number of animals and human subjects who were assessed, the fact that PRX-105 exerts a protective and therapeutic effect following exposure to lethal doses of OP, its favorable safety profile and its relatively long half-life, renders it a promising candidate for treatment and prophylaxis against OP poisoning and warrants further investigation.

Mitigation of Acetylcholine Esterase Activity in the 1,7-Diazacarbazole Series of Inhibitors of Checkpoint Kinase 1.

Checkpoint kinase 1 (ChK1) plays a key role in the DNA damage response, facilitating cell-cycle arrest to provide sufficient time for lesion repair. This leads to the hypothesis that inhibition of ChK1 might enhance the effectiveness of DNA-damaging therapies in the treatment of cancer. Lead compound 1 (GNE-783), the prototype of the 1,7-diazacarbazole class of ChK1 inhibitors, was found to be a highly potent inhibitor of acetylcholine esterase (AChE) and unsuitable for development. A campaign of analogue synthesis established SAR delineating ChK1 and AChE activities and allowing identification of new leads with improved profiles. In silico docking using a model of AChE permitted rationalization of the observed SAR. Compounds 19 (GNE-900) and 30 (GNE-145) were identified as selective, orally bioavailable ChK1 inhibitors offering excellent in vitro potency with significantly reduced AChE activity. In combination with gemcitabine, these compounds demonstrate an in vivo pharmacodynamic effect and are efficacious in a mouse p53 mutant xenograft model.

Enfermedad de Alzheimer: Tratamientos actuales y futuros / Alzheimer's Disease: Current and Future Treatments

Unas 25 millones de personas padecen enfermedad de Alzheimer en el mundo, y probablemente en los práximos 20 años, se registrarán unos 70 millones de nuevos casos. Caracterizaada por una pérdida progresiva de la memoria, el déficit de la capacidad cognitiva es proporcional a la densidad de placas seniles, a la acumulación de la proteína beta amiloide, degeneraciones neuríticas y ovillos neurofibrilares, particularmente en el hipocampo y en la corteza cerebral. Este cuadro histopatológico se asocia a otro neuroquímico, caracterizado por una disminución de las enzimas colina acetiltransferasa y acetilcolinesterasa, y una menor densidad de los receptores colinérgicos muscarínicos y nicotínicos. Ello ha generado la teoría colinérgica del Alzheimer, que ha dado lugar a una aproximación racional al tratamiento de la enfermedad. Hoy disponemos de 3 anticolinesterásicos, galantamina, donepecilo y rivastigmina aprobados por FDA, que se recomiendan en EA leves y moderadas y de un antagonista no competitovo de los receptores NMDA memantina EA. El beneficio es modesto en relación a lo cogitivo y conductual. Se incluyen estudios sobre recomendaciones, farmacología, farmacognética, eficacia, tolerancia, características de los pacientes respondedores a los diferentes anticolinesterásicos, sus reemplazos el uso de comprimidos, parches, beneficiios, efectos adversos y de las nuevas terapéuticas que están en desarrollo como estanercept, NP12, resveratrol PBT2 y vacuna nasal, entre otras...

About 25 millions of individuals in the world suffer from Alzheimer's disease. The next 20 years shall probably register 70 millions of new cases. Characterized by a progressive loss of memory, cognitive deficit is proportional to the density of senile plaques, accumulation of beta amyloid, neuritic degeneration and neurofibrillary tangles, particularly in the hippocampus and cerebral cortex. This histopathological condition is associated with another neurochemical, chracterized by a decrease in choline acetyltransferase and acetylcholinesterase enzymes, and a lower density of muscrinic and nicotinic cholinergic receptors. This results in the cholinergic theory of Alzheimer's, which has led to a rational approach to treatment of disease. Today we have 3 anticholinesterase Galantamine, Donepzil and Rivastigmine approved by FDA recommended in mild to moderate AD and an uncompetitive antagonist of NMDA receptors Memantine is recommended in mild Alzheimer's disease for people who can not take ACE inhibitors, and severe AD The benefit is modest in realtion to the cognitive and behavioral. Studies are included on therapeutic recommendations, pharmacology, pharmacogenetic, efficiency, tolerance, characteristics of responders to different anticholinesterases, their relacements, the use of pills, patches, benefits, side effects and new therapeutic under development as Etanercept, NP12, PBT 2, Resveratrol, Nasal Vaccine, among others...

Next generation OP-bioscavengers: a circulatory long-lived 4-PEG hypolysine mutant of F338A-HuAChE with optimal pharmacokinetics and pseudo-catalytic characteristics.

We have shown previously that conjugation of polyethylene glycol (PEG) chains to recombinant human acetylcholinesterase (rHuAChE) results in the extension of its residence time in the circulation of mice and monkeys [1,2]. By profiling the pharmacokinetic behavior of an array of well-defined hypolysine human mutant AChE molecules following PEGylation, we now determine that the duration of these enzyme forms in the circulation of rhesus macaques correlates with their number of appended PEG moieties, and is influenced by the actual location of the PEG chains at the molecule surface, as well. These findings, which concur with those we have previously established in mice, indicate that a common set of rules dictates the circulatory fate of PEGylated HuAChEs in rodents and non-human primates. In addition to its effect on circulatory residence, PEGylation reduces the ability of the rHuAChE bioscavenger to elicit an immune response in the heterologous mouse animal system. Thus, an inverse relationship between anti-AChE antibody production and PEG loading was observed following repeated administration of the different PEGylated hypolysine human AChEs to mice. We note however, that in rhesus macaques, the essentially homologous (human) AChE does not induce specific anti-AChE antibodies after repeated administration of high doses of the enzyme in its PEGylated form, and even in its non-PEGylated form. Taken together, these findings indicate that PEG acts by veiling enzyme-related epitopes, which would otherwise interact with host circulatory elimination pathways and immune system. The barring of such interactions by obstructive PEGs, confers the enzyme molecule with both extended circulatory residence and mitigated immunogenic properties. Further modulation by incorporation of the F338A mutation into the PEGylated hypolysine rHuAChE enzyme mold, resulted in the generation of an OP-bioscavenger that displayed reduced aging rates and could effectively protect mice against repeated exposure to CW agents. This selected 4-PEG F338A-AChE can serve as a paradigm for new generation OP-bioscavengers, specifically tailored for prophylactic treatment against toxic OP-agents.

Aging-resistant organophosphate bioscavenger based on polyethylene glycol-conjugated F338A human acetylcholinesterase.

The high reactivity of cholinesterases (ChEs) toward organophosphorus (OP) compounds has led to propose recombinant ChEs as bioscavengers of nerve agents. The bioscavenging potential of recombinant ChEs can be enhanced by conjugation of polyethylene glycol (PEG) moieties, to extend their circulatory residence. However, the ability of exogenously administered ChEs to confer long-term protection against repeated exposures to nerve agents is still limited due to the aging process, whereby organophosphate-ChE adducts undergo irreversible dealkylation, which precludes oxime-mediated reactivation of the enzyme. To generate an optimal acetylcholinesterase (AChE)-based OP bioscavenger, the F338A mutation, known to decelerate the rate of aging of AChE-OP conjugates, was incorporated into polyethylene glycol-conjugated (PEGylated) human AChE. The PEGylated F338A-AChE displayed unaltered rates of hydrolysis, inhibition, phosphylation, and reactivation and could effectively protect mice against exposure to soman (pinacolylmethyl phosphonofluoridate), sarin (O-isopropyl methylphosphonofluoridate), or O-ethyl-S-(2-isopropylaminoethyl) methylphosphonothioate (VX). Unlike PEGylated wild-type (WT)-AChE, the PEGylated F338A-AChE exhibits significantly reduced aging rates after soman inhibition and can be efficiently reactivated by the 1-[[[4(aminocarbonyl)-pyridinio]methoxy]methyl]-2(hydroxyimino)methyl]pyridinium dichloride (HI-6) oxime, both in vitro and in vivo. Accordingly, oxime administration to PEG-F338A-AChE-pretreated mice enabled them to withstand repeated soman exposure (5.4 and 4 LD(50)/dose), whereas same regime treatment of non-PEGylated F338A-AChE- or PEGylated WT-AChE-pretreated mice failed to protect against the second challenge, due to rapid clearance or irreversible aging of the latter enzymes. Thus, judicious incorporation of selected mutations into the AChE mold in conjunction with its chemical modification provides means to engineer an optimal ChE-based OP bioscavenger in terms of circulatory longevity, resistance to aging, and reduced doses required for protection, even against repeated exposures to nerve agents, such as soman.

Controlled concealment of exposed clearance and immunogenic domains by site-specific polyethylene glycol attachment to acetylcholinesterase hypolysine mutants.

Cholinesterases are efficient scavengers of organophosphates and are currently being developed as drugs for treatment against poisoning by such compounds. Recombinant ChE bioscavengers have very short circular longevity, a limitation that can be overcome by complex post-translation manipulations or by chemical modification such as polyethylene glycol conjugation. Series of multiple Lys-Ala mutants of human acetylcholinesterase were prepared allowing the generation of homogenous and well defined polyethylene-glycol conjugated AChEs with either one, two, three, four, or five appended polyethylene glycol (PEG) moieties/molecule. The rank order of circulatory longevity of these molecules was dependent on the number of PEG appendages up to a certain threshold: 5 = 4 > 3 > 2 > 1 > 0. Hypolysine acetylcholinesterases (AChEs) carrying the same number of PEGs, and therefore with identical masses, allowed us to demonstrate that circulatory longevity correlates with the predicted extent of concealment of the AChE surface. Furthermore, circulatory profiles of high number and low number PEG-AChEs differing in their sialic acid contents demonstrate a direct relationship between PEG loading and the effective seclusion of AChE from the hepatic asialoglycoprotein receptor clearance system. Finally, an inverse relationship is found between the extent of PEG loading and the ability of the human acetylcholinesterase to elicit specific anti-HuAChE antibodies. In conclusion, these findings suggest that for the extension of circulatory longevity, protein surface domain concealment exerted by polyethylene glycol attachment is at least as important as its effect on size enlargement and highlights the role of PEG attachment in masking interactions between biomolecules and their cognate receptors.

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

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 LD(50)) 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.

Abdominal bloating and irritable bowel syndrome like symptoms following microinstillation inhalation exposure to chemical warfare nerve agent VX in guinea pigs.

While assessing the methylphosphonothioic acid S-(2-(bis(1-methylethyl)amino)ethyl)O-ethyl ester (VX) induced respiratory toxicity and evaluating therapeutics against lung injury, we observed that the animals were experiencing abnormal swelling in the abdominal area. Nerve agent has been known to increase salivary, nasal and gastrointestinal secretion and cause diarrhea. This study was initiated to investigate the effect of VX on the gastrointestinal tract (GI) since abdominal pathology may affect breathing and contribute to the on going respiratory toxicity. The mid-abdominal diameter and the size of the lower left abdomen was measured before and after 27.3 mg/m3 VX exposure by microinstillation and at 30 min intervals up to 2 h post-VX exposure. Both VX and saline exposed animals exhibited a decrease in circumference of the upper abdomen, although the decrease was slightly higher in VX-exposed animals up to 1 h. The waist diameter increased slightly in VX-exposed animals from 60 to 90 min post-VX exposure but was similar to saline controls. The lower left abdomen near to the cecum, 6 cm below and 2cm to the right of the end of the sternum, showed an increase in size at 30-60 min that was significantly increased at 90-120 min post-VX exposure. In addition, VX-exposed animals showed loose fecal matter compared to controls. Necropsy at 24h showed an increased small intestine twisting motility in VX-exposed animals. Body tissue AChE assay showed high inhibition in the esophagus and intestine in VX-exposed animals indicating that a significant amount of the agent is localized to the GI following microinstillation exposure. These results suggest that microinstillatipn inhalation VX exposure induces gastrointestinal disturbances similar to that of irritable bowel syndrome and bloating.

Comparison of polyethylene glycol-conjugated recombinant human acetylcholinesterase and serum human butyrylcholinesterase as bioscavengers of organophosphate compounds.

Comparative protection studies in mice demonstrate that on a molar basis, recombinant human acetylcholinesterase (rHuAChE) confers higher levels of protection than native human butyrylcholinesterase (HuBChE) against organophosphate (OP) compound intoxication. For example, mice challenged with 2.5 LD50 of O-isopropyl methylphosphonofluoridate (sarin), pinacolylmethyl phosphonofluoridate (soman), and O-ethyl-S-(2-isopropylaminoethyl) methylphosphonothiolate (VX) after treatment with equimolar amounts of the two cholinesterases displayed 80, 100, and 100% survival, respectively, when pre-treatment was carried out with rHuAChE and 0, 20, and 60% survival, respectively, when pretreatment was carried out with HuBChE. Kinetic studies and active site titration analyses of the tested OP compounds with acetylcholinesterases (AChEs) and butyrylcholinesterases (BChEs) from different mammalian species demonstrate that the superior in vivo efficacy of acetyl-cholinesterases is in accordance with the higher stereoselectivity of AChE versus BChE toward the toxic enantiomers comprising the racemic mixtures of the various OP agents. In addition, we show that polyethylene glycol-conjugated (PEGy-lated) rHuAChE, which is characterized by a significantly extended circulatory residence both in mice and monkeys ( Biochem J 357: 795-802, 2001 ; Biochem J 378: 117-128, 2004 ), retains full reactivity toward OP compounds both in vitro and in vivo and provides a higher level of protection to mice against OP poisoning, compared with native serum-derived HuBChE. Indeed, PEGylated rHuAChE also confers superior prophylactic protection when administered intravenously or intramuscularly over 20 h before exposure of mice to a lethal dose of VX (1.3-1.5 LD50). These findings together with the observations that the PEGylated rHuAChE exhibits unaltered biodistribution and high bioavailability present a case for using PEGylated rHuAChE as a very efficacious bioscavenger of OP agents.

Host-regulated disposition of mammalian AChEs.

Primates are characterized by a paucity of soluble acetylcholinesterase (AChE) in the circulation at the adult stage, where the predominant circulating cholinesterase is butyrylcholinesterase. In recent years, we subjected recombinant human and bovine acetylcholinesterase to extensive pharmacokinetic studies in mice, an animal system which also displays very low levels of circulating AChE. In this system, a post-translation-related hierarchical pattern governing circulatory residence through AChE sialylation, subunit tetramerization and glycan loading was elucidated. Based on these studies, coordinated modulation of the sialic acid contents, state of subunit assembly and number of glycans allowed us to generate human or bovine AChE forms which reside in the circulation of mice for long periods of time, mimicking the pharmacokinetic behavior of native serum-derived cholinesterases. However, extension of the pharmacokinetic studies to primates, revealed an additional element, which affects circulatory residence of AChEs in this animal system. Unlike in the case of bovine AChE, optimization of subunit assembly and glycan loading of the primate versions of AChE (human or rhesus) did not increase their circulatory lifetime in rhesus macaques. This differential pharmacokinetic behavior of bovine and primate AChEs in macaques appears to be related to the 35 diverging bovine/primate AChE amino acids which are clustered within three defined domains at the enzyme surface, and thereby may facilitate the specific removal of "self" or "self-like" cholinesterases from the circulation of monkeys and thus provide an explanation for the absence of soluble AChE in the circulation of primates.

Kinetics study of Bungarus fasciatus venom acetylcholinesterase immobilised on a Langmuir-Blodgett proteo-glycolipidic bilayer.

This study deals with the kinetics properties of an enzyme immobilised in a defined orientation in a biomimetic environment. For this purpose, acetylcholinesterase (AChE) was captured at the surface of a nanostructured proteo-glycolipidic Langmuir-Blodgett film through specific recognition by a noninhibitor monoclonal antibody (IgG) inserted in a neoglycolipid bilayer. Modelling of this molecular assembly provided a plausible interpretation of the functional orientation of the enzyme. The AChE activity being stable for several weeks, the enzyme kinetics were investigated, and fitted perfectly with heterogeneous biocatalytic behaviour representative of cellular enzymatic catalysis. The AChE-IgG-glycolipid nanostructure was directly interfaced with an efficient optical device. Such an association, leading to an intimate contact between the nanostructure and the biochemical signal transducer, gives direct access to the intrinsic AChE behaviour. This study thus demonstrates the potential for direct investigation of the kinetic behaviour of an immobilised enzyme on a lipid bilayer through an efficient transduction system.

Evaluation of simplified kinetic analyses for measurement of brain acetylcholinesterase activity using N-[11C]Methylpiperidin-4-yl propionate and positron emission tomography.

The applicability of two reference tissue-based analyses without arterial blood sampling for the measurement of brain regional acetylcholinesterase (AChE) activity using N-[11C]methylpiperidin-4-yl propionate ([11C]MP4P) was evaluated in 12 healthy subjects. One was a linear least squares analysis derived from Blomqvist's equation, and the other was the analysis of the ratio of target-tissue radioactivity relative to reference-tissue radioactivity proposed by Herholz and coworkers. The standard compartment analysis using arterial input function provided reliable quantification of k3 (an index of AChE activity) estimates in regions with low (neocortex and hippocampus), moderate (thalamus), and high (cerebellum) AChE activity with a coefficient of variation (COV) of 12% to 19%. However, the precise k3 value in the striatum, where AChE activity is the highest, was not obtained. The striatum was used as a reference because its time-radioactivity curve was proportional to the time integral of the arterial input function. Reliable k3 estimates were also obtained in regions with low-to-moderate AChE activity with a COV of less than 21% by striatal reference analyses, though not obtained in the cerebellum. Shape analysis, the previous method of direct k3 estimation from the shape of time-radioactivity data, gave k3 estimates in the cortex and thalamus with a somewhat larger COV. In comparison with the standard analysis, a moderate overestimation of k3 by 9% to 18% in the linear analysis and a moderate underestimation by 2% to 13% in the Herholz method were observed, which were appropriately explained by the results of computer simulation. In conclusion, simplified kinetic analyses are practical and useful for the routine analysis of clinical [11C]MP4P studies and are nearly as effective as the standard analysis for detecting regions with abnormal AChE activity.

Amino acid domains control the circulatory residence time of primate acetylcholinesterases in rhesus macaques (Macaca mulatta).

An array of 13 biochemically well defined molecular forms of bovine, human and newly cloned rhesus macaque (Macaca mulatta) AChEs (acetylcholinesterases) differing in glycosylation and subunit assembly status were subjected to comparative pharmacokinetic studies in mice and rhesus macaques. The circulatory lifetimes of recombinant bovine, macaque and human AChEs in mice were governed by previously determined hierarchical rules; the longest circulatory residence time was obtained when AChE was fully sialylated and tetramerized [Kronman, Chitlaru, Elhanany, Velan and Shafferman (2000) J. Biol. Chem. 275, 29488-29502; Chitlaru, Kronman, Velan and Shafferman (2001) Biochem. J. 354, 613-625]. In rhesus macaques, bovine molecular forms still obeyed the same hierarchical rules, whereas primate AChEs showed significant deviation from this behaviour. Residence times of human and rhesus AChEs were effectively extended by extensive sialylation, but subunit tetramerization and N-glycan addition had a marginal effect on their circulatory longevity in macaques. It appears that the major factor responsible for the differential pharmacokinetics of bovine and primate AChEs in macaques is related to differences in primary structure, suggesting the existence of a specific mechanism for the circulatory clearance of primate AChEs in rhesus macaques. The 35 amino acids that differ between bovine and primate AChEs are clustered within three defined domains, all located at the enzyme surface, and may therefore mediate the facilitated removal of primate cholinesterases specifically from the circulation of monkeys. These surface domains can be effectively masked by poly(ethylene glycol) appendage, resulting in the generation of chemically modified human and macaque AChEs that reside in the circulation for extraordinarily long periods of time (mean residence time of 10000 min). This extended residence time is similar to that displayed by native macaque butyrylcholinesterase (9950 min), which is the prevalent cholinesterase form in the circulation of adult macaques.

Effect of chemical modification of recombinant human acetylcholinesterase by polyethylene glycol on its circulatory longevity.

Post-translational modifications were recently shown to be responsible for the short circulatory mean residence time (MRT) of recombinant human acetylcholinesterase (rHuAChE) [Kronman, Velan, Marcus, Ordentlich, Reuveny and Shafferman (1995) Biochem. J. 311, 959--967; Chitlaru, Kronman, Zeevi, Kam, Harel, Ordentlich, Velan and Shafferman (1998) Biochem. J. 336, 647--658; Chitlaru, Kronman, Velan and Shafferman (2001) Biochem. J. 354, 613--625], which is one of the major obstacles to the fulfilment of its therapeutic potential as a bioscavenger. In the present study we demonstrate that the MRT of rHuAChE can be significantly increased by the controlled attachment of polyethylene glycol (PEG) side chains to lysine residues. Attachment of as many as four PEG molecules to monomeric rHuAChE had minimal effects, if any, on either the catalytic activity (K(m)=0.09 mM and k(cat)=3.9 x 10(5) min(-1)) or the reactivity of the modified enzyme towards active-centre inhibitors, such as edrophonium and di-isopropyl fluorophosphate, or to peripheral-site ligands, such as propidium, BW284C51 and even the bulky snake-venom toxin fasciculin-II. The increase in MRT of the PEG-modified monomeric enzyme is linearly dependent, in the tested range, on the number of attached PEG molecules, as well as on their size. It appears that even low level PEG-conjugation can overcome the deleterious effect of under-sialylation on the pharmacokinetic performance of rHuAChE. At the highest tested ratio of attached PEG-20000/rHuAChE (4:1), an MRT of over 2100 min was attained, a value unmatched by any other known form of recombinant or native serum-derived AChE reported to date.

Hierarchy of post-translational modifications involved in the circulatory longevity of glycoproteins. Demonstration of concerted contributions of glycan sialylation and subunit assembly to the pharmacokinetic behavior of bovine acetylcholinesterase.

The tetrameric form of native serum-derived bovine acetylcholinesterase is retained in the circulation for much longer periods (mean residence time, MRT = 1390 min) than recombinant bovine acetylcholinesterase (rBoAChE) produced in the HEK-293 cell system (MRT = 57 min). Extensive matrix-assisted laser desorption ionization-time of flight analyses established that the basic structures of the N-glycans associated with the native and recombinant enzymes are similar (the major species (50-60%) are of the biantennary fucosylated type and 20-30% are of the triantennary type), yet the glycan termini of the native enzyme are mostly capped with sialic acid (82%) and alpha-galactose (12%), whereas glycans of the recombinant enzyme exhibit a high level of exposed beta-galactose residues (50%) and a lack of alpha-galactose. Glycan termini of both fetal bovine serum and rBoAChE were altered in vitro using exoglycosidases and sialyltransferase or in vivo by a HEK-293 cell line developed specifically to allow efficient sialic acid capping of beta-galactose-exposed termini. In addition, the dimeric and monomeric forms of rBoAChE were quantitatively converted to tetramers by complexation with a synthetic peptide representing the human ColQ-derived proline-rich attachment domain. Thus by controlling both the level and nature of N-glycan capping and subunit assembly, we generated and characterized 9 distinct bovine AChE glycoforms displaying a 400-fold difference in their circulatory lifetimes (MRT = 3.5-1390 min). This revealed some general rules and a hierarchy of post-translation factors determining the circulatory profile of glycoproteins. Accordingly, an rBoAChE was generated that displayed a circulatory profile indistinguishable from the native form.

Role of oligosaccharides in the pharmacokinetics of tissue-derived and genetically engineered cholinesterases.

To understand the role of glycosylation in the circulation of cholinesterases, we compared the mean residence time of five tissue-derived and two recombinant cholinesterases (injected intravenously in mice) with their oligosaccharide profiles. Monosaccharide composition analysis revealed differences in the total carbohydrate, galactose, and sialic acid contents. The molar ratio of sialic acid to galactose residues on tetrameric human serum butyrylcholinesterase, recombinant human butyrylcholinesterase, and recombinant mouse acetylcholinesterase was found to be approximately 1.0. For Torpedo californica acetylcholinesterase, monomeric and tetrameric fetal bovine serum acetylcholinesterase, and equine serum butyrylcholinesterase, this ratio was approximately 0.5. However, the circulatory stability of cholinesterases could not be correlated with the sialic acid-to-galactose ratio. Fractionation of the total pool of oligosaccharides obtained after neuraminidase digestion revealed one major oligosaccharide for human serum butyrylcholinesterase and three or four major oligosaccharides in other cholinesterases. The glycans of tetrameric forms of plasma cholinesterases (human serum butyrylcholinesterase, fetal bovine serum acetylcholinesterase, and equine serum butyrylcholinesterase) clearly demonstrated a reduced heterogeneity and higher maturity compared with glycans of monomeric fetal bovine serum acetylcholinesterase, dimeric tissue-derived T. californica acetylcholinesterase, and recombinant cholinesterases. T. californica acetylcholinesterase, recombinant cholinesterases, and monomeric fetal bovine serum acetylcholinesterase showed a distinctive shorter mean residence time (44-304 min) compared with tetrameric forms of plasma cholinesterases (1902-3206 min). Differences in the pharmacokinetic parameters of cholinesterases seem to be due to the combined effect of the molecular weight and charge- and size-based heterogeneity in glycans.

Structure of glycan moieties responsible for the extended circulatory life time of fetal bovine serum acetylcholinesterase and equine serum butyrylcholinesterase.

Cholinesterases are serine hydrolases that can potentially be used as pretreatment drugs for organophosphate toxicity, as drugs to alleviate succinylcholine-induced apnea, and as detoxification agents for environmental toxins such as heroin and cocaine. The successful application of serum-derived cholinesterases as bioscavengers stems from their relatively long residence time in the circulation. To better understand the relationship between carbohydrate structure and the stability of cholinesterases in circulation, we determined the monosaccharide composition, the distribution of various oligosaccharides, and the structure of the major asparagine-linked oligosaccharides units present in fetal bovine serum acetylcholinesterase and equine serum butyrylcholinesterase. Our findings indicate that 70-80% of the oligosaccharides in both enzymes are negatively charged. This finding together with the molar ratio of galactose to sialic acid clearly suggests that the beta-galactose residues are only partially capped with sialic acid, yet they displayed a long duration in circulation. The structures of the two major oligosaccharides from fetal bovine serum acetylcholinesterase and one major oligosaccharide from equine serum butyrylcholinesterase were determined. The three carbohydrate structures were of the biantennary complex type, but only the ones from fetal bovine serum acetylcholinesterase were fucosylated on the innermost N-acetylglucosamine residue of the core. Pharmacokinetic studies with native, desialylated, and deglycosylated forms of both enzymes indicate that the microheterogeneity in carbohydrate structure may be responsible, in part, for the multiphasic clearance of cholinesterases from the circulation of mice.

Cholinesterases as scavengers for organophosphorus compounds: protection of primate performance against soman toxicity.

The present treatment for poisoning by organophosphates consists of multiple drugs such as carbamates, antimuscarinics, and reactivators in pre- and post-exposure modalities. Recently an anticonvulsant, diazapam, has been included as a post-exposure drug to reduce convulsions and increase survival. Most regimens are effective in preventing lethality from organophosphate exposure but do not prevent toxic effects and incapacitation observed in animals and likely to occur in humans. Use of enzymes such as cholinesterases as pretreatment drugs for sequestration of highly toxic organophosphate anticholinesterases and alleviation of side effects and performance decrements was successful in animals, including non-human primates. Pretreatment of rhesus monkeys with fetal bovine serum acetylcholinesterase protected them against lethal effects of soman (up to 5 LD50) and prevented signs of OP toxicity. Monkeys pretreated with fetal bovine serum acetylcholinesterase were devoid of behavioral incapacitation after soman exposure, as measured by serial probe recognition or primate equilibrium platform performance tasks. Use of acetylcholinesterase as a single pretreatment drug provided greater protection against both lethal and behavioral effects of potent organophosphates than current multicomponent drug treatments that prevent neither signs of toxicity nor behavioral deficits. Although use of cholinesterases as single pretreatment drugs provided complete protection, its use for humans may be limited, since large quantities will be required, due to the approximately 1:1 stoichiometry between organophosphate and enzyme. Bisquaternary oximes, particularly HI-6, have been shown to reactivate organophosphate-inhibited acetylcholinesterase at a rapid rate. We explored the possibility that enzyme could be continually reactivated in animals pretreated with fetal bovine serum acetylcholinesterase, followed by an appropriate dose of reactivator, and challenged with repeated doses of sarin. In in vitro experiments, stoichiometry greater than 1:400 for enzyme:sarin was achieved; in vivo stoichiometry in mice was 1:65. Pretreatment of mice with fetal bovine serum acetylcholinesterase and HI-6 amplified the effectiveness of exogenous enzyme as a scavenger for organophosphate.

Comparative efficacy of exogenous acetylcholinesterase administration on soman and dichlorvos toxicity in rats.

Cholinesterase (ChE) activity in the blood serum of rats was elevated to 15, 25, and 45 times by the sc administration of 1000, 2000 and 3000 electric eel acetylcholinesterase (AChE) units respectively. Apparently no ill-effect to animals was observed. The maximal activity of the enzyme occurred in 90 min after its administration and was directly proportional to the administered dose. The increase activity of ChE in the serum on the exogenous administration of AChE persisted for 18 hr. The exogenously raised serum ChE, protected rats against lethal dose of dichlorvos, but not against lethal dose of soman. The possible mechanism of differential response in discussed.