Ashima Saxena

Walter Reed Army Institute of Research, Silver Spring, Maryland, United States

Are you Ashima Saxena?

Claim your profile

Publications (72)208.64 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: 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. Published by Elsevier Ltd.
    Toxicology in Vitro 11/2014; DOI:10.1016/j.tiv.2014.11.001 · 3.21 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In order to identify novel Alzheimer’s modifying pharmacological tools, we developed bis-tacrines bearing a peptide moiety for specific interference with surface sites of human acetylcholinesterase (hAChE) binding amyloid-beta (Aβ). Accordingly, compounds 2a–c proved to be inhibitors of hAChE catalytic and noncatalytic functions, binding the catalytic and peripheral sites, interfering with Aβ aggregation and with the Aβ self-oligomerization process (2a). Compounds 2a–c in complex with TcAChE span the gorge with the bis-tacrine system, and the peptide moieties bulge outside the gorge in proximity of the peripheral site. These moieties are likely responsible for the observed reduction of hAChE-induced Aβ aggregation since they physically hamper Aβ binding to the enzyme surface. Moreover, 2a was able to significantly interfere with Aβ self-oligomerization, while 2b,c showed improved inhibition of hAChE-induced Aβ aggregation.
    ACS Medicinal Chemistry Letters 10/2013; 4(12). DOI:10.1021/ml4002908 · 3.07 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Acetylcholinesterase (AChE) in the serum of fetal cow is a tetramer. The related enzyme, butyrylcholinesterase (BChE), in the sera of humans and horse requires polyproline peptides for assembly into tetramers. Our goal was to determine whether soluble tetrameric AChE includes tetramer organizing peptides in its structure. Fetal bovine serum AChE was denatured by boiling to release non-covalently bound peptides. Bulk protein was separated from peptides by filtration and by high performance liquid chromatography. Peptide mass and amino acid sequence of the released peptides were determined by MALDI-TOF-TOF and LTQ-Orbitrap mass spectrometry. Twenty polyproline peptides, divided into 5 families, were identified. The longest peptide contained 25 consecutive prolines and no other amino acid. Other polyproline peptides included one non-proline amino acid, for example serine at the C-terminus of 20 prolines. A search of the mammalian proteome database suggested that this assortment of polyproline peptides originated from at least 5 different precursor proteins, none of which were the ColQ or PRiMA of membrane-anchored AChE. To date, AChE and BChE are the only proteins known that include polyproline tetramer organizing peptides in their tetrameric structure.
    Biochimica et Biophysica Acta 01/2013; DOI:10.1016/j.bbapap.2013.01.009 · 4.66 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Exogenously administered human serum butyrylcholinesterase (Hu BChE) was demonstrated to function as a bioscavenger of highly toxic organophosphorus (OP) compounds in several animal species. Since the enzyme is isolated from human serum, it is currently the most suitable pretreatment for human use. A dose of 200-300 mg/70 kg human adult is projected to provide protection from 2 X LD(50) of soman. Due to the limited supply of Hu BChE, strategies aimed at reducing the dose of enzyme are being explored. In this study, we investigated the effect of modification with polyethylene glycol (PEG) on the in vivo stability of Hu BChE. Mice were given two injections of either Hu BChE or Hu BChE modified with PEG-5K or PEG-20K, six weeks apart. Pharmacokinetic parameters, such as mean residence time (MRT), maximal concentration (C(max)), elimination half-life (T(1/2)), and area under the plasma concentration time curve extrapolated to infinity (AUC), were determined. For the first injection, values for MRT, T(1/2), C(max), and AUC for PEG-5K-Hu BChE and PEG-20K-Hu BChE were similar to those for Hu BChE. These values for the second injection of Hu BChE as well as PEG-Hu BChEs were lower as compared to those for the first injections, likely due to antibody-mediated clearance.
    Chemico-biological interactions 12/2012; 203(1). DOI:10.1016/j.cbi.2012.11.021 · 2.46 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human serum butyrylcholinesterase (HuBChE) is currently the most suitable bioscavenger for the prophylaxis of highly toxic organophosphate (OP) nerve agents. A dose of 200mg of HuBChE is envisioned as a prophylactic treatment that can protect humans from an exposure of up to 2×LD(50) of soman. The limited availability and administration of multiple doses of this stoichiometric bioscavenger make this pretreatment difficult. Thus, the goal of this study was to produce a smaller enzymatically active HuBChE polypeptide (HBP) that could bind to nerve agents with high affinity thereby reducing the dose of enzyme. Studies have indicated that the three-dimensional structure and the domains of HuBChE (acyl pocket, lip of the active center gorge, and the anionic substrate-binding domain) that are critical for the binding of substrate are also essential for the selectivity and binding of inhibitors including OPs. Therefore, we designed three HBPs by deleting some N- and C-terminal residues of HuBChE by maintaining the folds of the active site core that includes the three active site residues (S198, E325, and H438). HBP-4 that lacks 45 residues from C-terminus but known to have BChE activity was used as a control. The cDNAs for the HBPs containing signal sequences were synthesized, cloned into different mammalian expression vectors, and recombinant polypeptides were transiently expressed in different cell lines. No BChE activity was detected in the culture media of cells transfected with any of the newly designed HBPs, and the inactive polypeptides remained inside the cells. Only enzymatically active HBP-4 was secreted into the culture medium. These results suggest that residues at the N- and C-termini are required for the folding and/or maintenance of HBP into an active stable conformation.
    Chemico-biological interactions 10/2012; 203(1). DOI:10.1016/j.cbi.2012.09.018 · 2.46 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human prolidase is a binuclear metalloenzyme, which can potentially function as a catalytic bioscavenger for organophosphorus (OP) nerve agents. Although the biochemical properties of native prolidase purified from human erythrocytes, liver, kidney, and fibroblast cells are well known, it is very poorly characterized with regard to its OP hydrolyzing activity. Also, the high cost of purification of large quantities of native enzyme limits its use as a bioscavenger. Thus, recombinant human prolidase with similar biochemical properties to those of native enzyme would be more suitable as a catalytic bioscavenger. In this study, we established an Escherichia coli expression system, which produced a large amount of tagged human liver prolidase that was purified to over 95% purity from the soluble fraction of cell lysate by affinity chromatography on Streptavidin-agarose resin. The catalytic properties of the recombinant enzyme were compared in vitro with those of highly purified prolidase I isolated from human erythrocytes. The catalytic properties of recombinant prolidase overlap with those of the erythrocyte-derived native enzyme. Both enzymes efficiently hydrolyzed diisopropylfluorophosphate, sarin, soman, tabun and cyclosarin, but were much less efficient at hydrolyzing paraoxon and methyl paraoxon. These results suggest that human prolidase expressed in E. coli is suitable for further development as a catalytic bioscavenger for OP nerve agents.
    Toxicology in Vitro 06/2012; DOI:10.1016/j.tiv.2012.05.012 · 3.21 Impact Factor
  • Ramachandra S Naik, Weiyi Liu, Ashima Saxena
    [Show abstract] [Hide abstract]
    ABSTRACT: Current methods for measuring acetylcholinesterase (AChE) activities in whole blood use butyrylcholinesterase (BChE)-selective inhibitors. However, the poor selectivity of these inhibitors results in the inhibition of AChE activity to some degree, leading to errors in reported values. The goal of this study was to develop and validate a simple assay for measuring AChE and BChE activities in whole blood from humans as well as experimental animals. Blood was fractionated into plasma and erythrocytes, and cholinesterase activities were titrated against ethopropazine and (-)-huperzine A to determine the lowest concentration of ethopropazine that inhibited BChE completely without affecting AChE activity and the lowest concentration of (-)-huperzine A that inhibited AChE completely without interfering with BChE activity. Results indicate that 20 µm ethopropazine can be successfully used for the accurate measurement of AChE activity in blood from humans as well as animals. Use of (-)-huperzine A is not required for measuring BChE activity in normal or 'exposed' blood samples. The method was validated for blood from several animal species, including mice, rats, guinea pigs, dogs, minipigs, and African green, cynomolgus and rhesus monkeys. This method is superior to all reported methods, does not require the separation of erythrocyte and plasma fractions, and is suitable for measuring cholinesterase activities in fresh or frozen blood from animals that were exposed to nerve agents or those that were administered high doses of BChE. The method is simple, direct, reproducible, and reliable and can easily be adapted for high-throughput screening of blood samples. Published 2012. This article is a US Government work and is in the public domain in the USA.
    Journal of Applied Toxicology 03/2012; 33(4). DOI:10.1002/jat.2730 · 3.17 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The effects of a large dose of human serum butyrylcholinesterase (HuBChE) were evaluated in rhesus monkeys using a serial-probe recognition (SPR) task designed to assess attention and short-term memory. Each monkey received an intravenous injection of 150 mg (105,000 U or 30 mg/kg) of HuBChE 60 min prior to testing on the SPR task. Concurrent with the cognitive-behavioral assessment, blood was collected at various time points throughout the study and was analyzed for acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities, anti-BChE antibody production and gross clinical pathology (i.e., complete blood count and clinical chemistry panel). HuBChE revealed a peak blood activity of 227 U/ml at 5 min after intravenous injection and a mean residence time of approximately 72 h. No cognitive-behavioral decrements of any kind in SPR performance and no toxic signs in clinical pathology were detected in any of the blood assays during the 5 weeks of observation. Anti-HuBChE antibodies peaked at about 14 days after injection, with no concomitant behavioral changes. These results demonstrate the behavioral and physiological safety of HuBChE in rhesus monkeys and support its development as a bioscavenger for the prophylaxis of chemical warfare agent toxicity in humans.
    Neurotoxicology and Teratology 02/2012; 34(3):323-30. DOI:10.1016/ · 3.22 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Senescence marker protein-30 (SMP-30) is a candidate enzyme that can function as a catalytic bioscavenger of organophosphorus (OP) nerve agents. We purified SMP-30 from mouse (Mo) liver and compared its hydrolytic activity towards various esters, lactones, and G-type nerve agents with that of human paraoxonase1 (Hu PON1) and squid diisopropylfluorophosphatase (DFPase). All three enzymes contain one or two metal ions in their active sites and fold into six-bladed β-propeller structures. While Hu PON1 hydrolyzed a variety of lactones, the only lactone that was a substrate for Mo SMP-30 was d-(+)-gluconic acid δ-lactone. Squid DFPase was much more efficient at hydrolyzing DFP and G-type nerve agents as compared to Mo SMP-30 or Hu PON1. The K(m) values for DFP were in the following order: Mo SMP-30>Hu PON1>squid DFPase, suggesting that the efficiency of DFP hydrolysis may be related to its binding in the active sites of these enzymes. Thus, homology modeling and docking were used to simulate the binding of DFP and selected δ-lactones in the active sites of Hu SMP-30, Hu PON1, and squid DFPase. Results from molecular modeling studies suggest that differences in metal-ligand coordinations, the hydrophobicity of the binding pockets, and limited space in the binding pocket due to the presence of a loop, are responsible for substrate specificities of these enzymes.
    Biochimica et Biophysica Acta 02/2012; 1824(5):701-10. DOI:10.1016/j.bbapap.2012.02.007 · 4.66 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We formulated a new gene delivery system based on targeted liposomes. The efficacy of the delivery system was demonstrated in in vitro and in vivo models. The targeting moiety consists of a high-affinity 7-amino-acid peptide, covalently and evenly conjugated to the liposome surface. The targeting peptide acts as an endothelin antagonist, and accelerates liposome binding and internalization. It is devoid of other biological activity. Liposomes with high phosphatidyl serine (PS) were specially formulated to help their fusion with the endosomal membrane at low pH and enable release of the liposome payload into the cytoplasm. A DNA payload, pre-compressed by protamine, was encapsulated into the liposomes, which directed the plasmid into the cell's nucleus. Upon exposure to epithelial cells, binding of the liposomes occurred within 5-10 min, followed by facilitated internalization of the complex. Endosomal escape was complete within 30 min, followed by DNA accumulation in the nucleus 2h post-transfection. A549 lung epithelial cells transfected with plasmid encoding for GFP encapsulated in targeted liposomes expressed significantly more protein than those transfected with plasmid complexed with Lipofectamine. The intra-tracheal instillation of plasmid encoding for GFP encapsulated in targeted liposomes into rat lungs resulted in the expression of GFP in bronchioles and alveoli within 5 days. These results suggest that this delivery system has great potential in targeting genes to lungs.
    Journal of Controlled Release 11/2011; 160(2):217-24. DOI:10.1016/j.jconrel.2011.10.033 · 7.63 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human serum butyrylcholinesterase (Hu BChE) is a stoichiometric bioscavenger that is being developed as a prophylactic countermeasure against organophosphorus nerve agents. This study was designed to evaluate the efficacy of Hu BChE against whole-body inhalation exposure to a lethal dose of sarin (GB) vapor. Male Göttingen minipigs were subjected to: air exposure, GB vapor exposure, or pretreatment with Hu BChE followed by GB vapor exposure. Hu BChE was administered by i.m. injection 24 h prior to exposure to 4.1 mg/m(3) of GB vapor for 60 min. Electrocardiograms (ECG), electroencephalograms (EEG), and pupil size were recorded throughout exposure. Blood drawn before and throughout exposure was analyzed for blood gases, electrolytes, metabolites, acetylcholinesterase and BChE activities, and amount of GB present. Untreated animals exposed to GB vapor exhibited cardiac abnormalities and generalized seizures, ultimately succumbing to respiratory failure. Pretreatment with 3.0 or 6.5 mg/kg of Hu BChE delayed blood gas and acid-base disturbances and the onset of cardiac and neural toxic signs, but failed to increase survivability. Pretreatment with 7.5 mg/kg of Hu BChE, however, completely prevented toxic signs, with blood chemistry and ECG and EEG parameters indistinguishable from control during and after GB exposure. GB bound in plasma was 200-fold higher than plasma from pigs that did not receive Hu BChE, suggesting that Hu BChE scavenged GB in blood and prevented it from reaching other tissues. Thus, prophylaxis with Hu BChE alone not only increased survivability, but also prevented cardiac abnormalities and neural toxicity in minipigs exposed to a lethal dose of GB vapor.
    Biochemical pharmacology 09/2011; 82(12):1984-93. DOI:10.1016/j.bcp.2011.09.019 · 4.25 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Human serum butyrylcholinesterase (Hu BChE) is currently under advanced development as a bioscavenger for the prophylaxis of organophosphorus (OP) nerve agent toxicity in humans. It is estimated that a dose of 200mg will be required to protect a human against 2×LD(50) of soman. To provide data for initiating an investigational new drug application for the use of this enzyme as a bioscavenger in humans, we purified enzyme from Cohn fraction IV-4 paste and initiated safety and efficacy evaluations in mice, guinea pigs, and non-human primates. In mice, we demonstrated that a single dose of enzyme that is 30 times the therapeutic dose circulated in blood for at least four days and did not cause any clinical pathology in these animals. In this study, we report the results of safety and efficacy evaluations conducted in guinea pigs. Various doses of Hu BChE delivered by i.m. injections peaked at ∼24h and had a mean residence time of 78-103h. Hu BChE did not exhibit any toxicity in guinea pigs as measured by general observation, serum chemistry, hematology, and gross and histological tissue changes. Efficacy evaluations showed that Hu BChE protected guinea pigs from an exposure of 5.5×LD(50) of soman or 8×LD(50) of VX. These results provide convincing data for the development of Hu BChE as a bioscavenger that can protect humans against all OP nerve agents.
    Biochemical pharmacology 01/2011; 81(1):164-9. DOI:10.1016/j.bcp.2010.09.007 · 4.25 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 11/2010; 32(47). DOI:10.1002/chin.200147241
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Biochemical pharmacology 11/2010; 80(9):1427-36. DOI:10.1016/j.bcp.2010.07.020 · 4.25 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.
    ChemInform 10/2010; 32(44). DOI:10.1002/chin.200144219
  • ChemInform 09/2010; 33(38). DOI:10.1002/chin.200238211
  • [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Chemico-biological interactions 09/2010; 187(1-3):185-90. DOI:10.1016/j.cbi.2010.01.018 · 2.46 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Human serum butyrylcholinesterase (Hu BChE) is currently the most appropriate candidate for the prophylaxis of humans against organophosphate (OP) nerve agent toxicity. It is estimated that a dose of 200 mg will protect a human against 2× LD50 of soman, which means that gram quantities of enzyme are needed for human clinical studies. Toward this effort, we report the development of the first procedure that is suitable for the pilot-scale purification of Hu BChE from Cohn fraction IV-4 paste. This procedure involved resuspension of Cohn fraction IV-4 paste, followed by procainamide affinity and DEAE anion-exchange chromatography. The procedure yielded 6–7 g (4.3–5 million U) of purified enzyme from 80 kg of Cohn fraction IV-4 paste. The enzyme was >97% pure as judged by a specific activity of ∼700 U/mg and a major band with a subunit molecular weight of 85 kDa on SDS-PAGE. The high yield and purity obtained suggest that this manufacturing procedure is suitable for the pre-clinical production of Hu BChE.
    PROCESS BIOCHEMISTRY 08/2010; 45(8):1313-1318. DOI:10.1016/j.procbio.2010.04.021 · 2.52 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We evaluated the effects of conjugated enzyme-nerve agent product resulting from the inhibition of bioscavenger human serum butyrylcholinesterase (Hu BChE) by nerve agents soman or VX. Rats were trained on a multiple Fixed-Ratio 32, Extinction 30 sec. (FR32, Ext30) schedule of food reinforcement and then injected (i.m.) with Hu BChE (30 mg/kg), equivalent amounts of Hu BChE-soman conjugate (GDC), Hu BChE-VX conjugate, oxotremorine (OXO) (0.316 mg/kg) or vehicle (n = 8, each group). On the day of injection and on 10 subsequent daily sessions, performance was evaluated on the FR32, Ext30 schedule. Neither conjugates nor Hu BChE produced a performance deficit under the schedule. OXO produced a substantial decrease in responding on the day of administration, with complete recovery observed on subsequent sessions. None of the treatments affected circulating acetylcholinesterase (AChE) activity when evaluated 24-72 hr after injection. The dose of Hu BChE produced a 20,000-fold increase above baseline in circulating BChE activity. Pathological evaluation of organ systems approximately 2 weeks following administration of conjugates or Hu BChE alone did not show toxicity. Taken together, these results suggest that Hu BChE - nerve agent conjugates produced following bioscavenger protection against nerve agents soman and VX do not appear to be particularly toxic. These results add to the safety assessment of Hu BChE as a bioscavenger countermeasure against nerve agent exposure.
    Basic & Clinical Pharmacology & Toxicology 05/2010; 106(5):428-34. DOI:10.1111/j.1742-7843.2009.00508.x · 2.18 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Human serum and recombinant butyrylcholinesterase (rHuBChE) are the most advanced prophylactics against organophosphate (OP) toxicity due to nerve agent or insecticide exposure. For ethical reasons, such potential multi-use treatments cannot be tested in humans and will require extensive testing in animal models and the "Animal Rule" 21 (21 CFR 601.90) for regulatory approval. This will involve multiple injections of rHuBChE into heterologous animals, e.g. macaques, rodents with inevitable immunogenicity and subsequent elimination of the enzyme on repeat injections. In order to accurately assess pharmacokinetics, efficacy and safety of a candidate rBChE in an "antibody free" system, a homologous macaque (Ma) model has been developed. In these studies, macaques received single or multiple intravenous injections of native MaBChE as well as unmodified or PEG-conjugated forms of rMaBChE produced in CHO cells. Compared to the poor plasma retention of unmodified rBChE (MRT: <10h), three injections of 1.5-2.3mg/kg of PEG-conjugated tetrameric rBChE resulted in high circulatory stability (MRT: >134h) and lack of immunogenicity similar to native MaBChE. PEG-conjugation of the monomeric rMaBChE form also exhibited pharmacokinetic profiles comparable to the tetrameric form (MRT: >113h). However, despite the increased bioavailability of PEG-rBChE, antigenicity studies using sandwich ELISA showed that while macaque BChE was not immunogenic in macaques, PEGylation of rMaBChE did not prevent binding to anti-BChE antibodies, suggesting PEGylation may not be sufficient to mask non-human epitopes on rBChE. This homologous model can provide necessary preclinical protection data for the use of PEG-rHuBChE in humans and bodes well for a safe and efficacious CHO-derived rHuBChE therapeutic.
    Chemico-biological interactions 03/2010; 187(1-3):279-86. DOI:10.1016/j.cbi.2010.02.042 · 2.46 Impact Factor

Publication Stats

1k Citations
208.64 Total Impact Points


  • 1994–2014
    • Walter Reed Army Institute of Research
      • Center for Military Psychiatry and Neuroscience Research
      Silver Spring, Maryland, United States
  • 2002–2010
    • Georgetown University
      • Division of Neurology
      Washington, Washington, D.C., United States
  • 1996–2010
    • Università degli Studi di Siena
      • Department of Medicine, Surgery and Neuroscience
      Siena, Tuscany, Italy
  • 2003–2005
    • University of Naples Federico II
      Napoli, Campania, Italy