Doctor BP

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Full name : Doctor Bhupendra P

First name : Bhupendra P

Mail : Division of Biochemistry\; Walter Reed Army Institute of Research\; 503 Robert Grant Road\; Silver Spring\; MD 20910

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Country : USA

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References (163)

Title : Prophylaxis with human serum butyrylcholinesterase protects Gottingen minipigs exposed to a lethal high-dose of sarin vapor - Saxena_2015_Chem.Biol.Interact_238_161
Author(s) : Saxena A , Hastings NB , Sun W , Dabisch PA , Hulet SW , Jakubowski EM , Mioduszewski RJ , Doctor BP
Ref : Chemico-Biological Interactions , 238 :161 , 2015
Abstract : Serum-derived human butyrylcholinesterase (Hu BChE) is a stoichiometric bioscavenger that is being developed as a potential prophylactic nerve agent countermeasure. Previously, we reported the prophylactic efficacy of Hu BChE in Gottingen minipigs against a whole-body exposure to 4.1mg/m3 of sarin (GB) vapor, which produced lethality over 60min. Since the toxicity of nerve agent is concentration-dependent, in the present study, we investigated the toxic effects of an almost 3-fold higher rate of GB vapor exposure and the ability of Hu BChE to protect minipigs against this exposure. Male minipigs were subjected to: (1) air exposure; (2) GB vapor exposure; or (3) pretreatment with 7.5mg/kg of Hu BChE by i.m. injection, 24h prior to whole-body exposure to 11.4mg/m3 of GB vapor for 10min. Electrocardiogram, electroencephalogram, and pupil size were monitored throughout exposure. Blood drawn before and throughout exposure was analyzed for blood gases, electrolytes, metabolites, acetylcholinesterase and BChE activities, and amount of GB bound to red blood cells and plasma. A novel finding was that saline-treated animals exposed to GB vapor did not develop any seizures, but manifested a variety of cardiac and whole blood toxic signs and rapidly died due to respiratory failure. Strikingly, pre-treatment with 7.5mg/kg of Hu BChE not only prevented lethality, but also avoided all cardiac toxic signs manifested in the non-treated cohort. Thus, Hu BChE alone can serve as an effective prophylactic countermeasure versus a lethal high-dose exposure to GB vapor.
ESTHER : Saxena_2015_Chem.Biol.Interact_238_161
PubMedSearch : Saxena_2015_Chem.Biol.Interact_238_161
PubMedID: 26145887

Title : A combination of [+] and [-]-Huperzine A improves protection against soman toxicity compared to [+]-Huperzine A in guinea pigs - Wang_2013_Chem.Biol.Interact_203_120
Author(s) : Wang Y , Wei Y , Oguntayo S , Doctor BP , Nambiar MP
Ref : Chemico-Biological Interactions , 203 :120 , 2013
Abstract : The neuropathologic mechanisms after exposure to lethal doses of nerve agent are complex and involve multiple biochemical pathways. Effective treatment requires drugs that can simultaneously protect by reversible binding to the acetylcholinesterase (AChE) and blocking cascades of seizure related brain damage, inflammation, neuronal degeneration as well as promoting induction of neuroregeneration. [-]-Huperzine A ([-]-Hup A), is a naturally occurring potent reversible AChE inhibitor that penetrates the blood-brain barrier. It also has several neuroprotective effects including modification of beta-amyloid peptide, reduction of oxidative stress, anti-inflammatory, anti-apoptotic and induction and regulation of nerve growth factor. Toxicities at higher doses restrict the neuroporotective ability of [-]-Hup A for treatment. The synthetic stereoisomer, [+]-Hup A, is less toxic due to poor AChE inhibition and is suitable for both pre-/post-exposure treatments of nerve agent toxicity. [+]-Hup A block the N-methyl-d-aspartate (NMDA)-induced seizure in rats, reduce excitatory amino acid induced neurotoxicity and also prevent soman induced toxicity with minimum performance decrement. Unique combinations of two stereo-isomers of Hup A may provide an excellent pre/post-treatment drug for the nerve agent induced seizure/status epilepticus. We investigated a combination of [+]-Hup A with a small dose of [-]-Hup A ([+] and [-]-Hup A) against soman toxicity. Our data showed that pretreatment with a combination [+] and [-]-Hup A significantly increased the survival rate and reduced behavioral abnormalities after exposure to 1.2xLD50 soman compared to [+]-Hup A in guinea pigs. In addition, [+] and [-]-Hup A pretreatment inhibited the development of high power of EEG better than [+]-Hup A pretreatment alone. These data suggest that a combination of [+] and [-]-Hup A offers better protection than [+]-Hup A and serves as a potent medical countermeasure against lethal dose nerve agent toxicity in guinea pigs.
ESTHER : Wang_2013_Chem.Biol.Interact_203_120
PubMedSearch : Wang_2013_Chem.Biol.Interact_203_120
PubMedID: 23123250

Title : Structural analogs of huperzine A improve survival in guinea pigs exposed to soman - Gunosewoyo_2013_Bioorg.Med.Chem.Lett_23_1544
Author(s) : Gunosewoyo H , Tipparaju SK , Pieroni M , Wang Y , Doctor BP , Nambiar MP , Kozikowski AP
Ref : Bioorganic & Medicinal Chemistry Lett , 23 :1544 , 2013
Abstract : Chemical warfare nerve agents such as soman exert their toxic effects through an irreversible inhibition of acetylcholinesterase (AChE) and subsequently glutamatergic function, leading to uncontrolled seizures. The natural alkaloid (-)-huperzine A is a potent inhibitor of AChE and has been demonstrated to exert neuroprotection at an appropriate dose. It is hypothesized that analogs of both (+)- and (-)-huperzine A with an improved ability to interact with NMDA receptors together with reduced AChE inhibition will exhibit more effective neuroprotection against nerve agents. In this manuscript, the tested huperzine A analogs 2 and 3 were demonstrated to improve survival of guinea pigs exposed to soman at either 1.2 or 2xLD(50).
ESTHER : Gunosewoyo_2013_Bioorg.Med.Chem.Lett_23_1544
PubMedSearch : Gunosewoyo_2013_Bioorg.Med.Chem.Lett_23_1544
PubMedID: 23395652

Title : Amino acid residues at the N- and C-termini are essential for the folding of active human butyrylcholinesterase polypeptide - Naik_2013_Chem.Biol.Interact_203_24
Author(s) : Naik RS , Pattabiraman N , Patel KA , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 203 :24 , 2013
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 2xLD50 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.
ESTHER : Naik_2013_Chem.Biol.Interact_203_24
PubMedSearch : Naik_2013_Chem.Biol.Interact_203_24
PubMedID: 23044488

Title : Effect of polyethylene glycol conjugation on the circulatory stability of plasma-derived human butyrylcholinesterase in mice - Sun_2013_Chem.Biol.Interact_203_172
Author(s) : Sun W , Luo C , Tipparaju P , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 203 :172 , 2013
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 200-300mg/70kg human adult is projected to provide protection from 2 X LD50 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 (Cmax), elimination half-life (T1/2), and area under the plasma concentration time curve extrapolated to infinity (AUC), were determined. For the first injection, values for MRT, T1/2, Cmax, 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.
ESTHER : Sun_2013_Chem.Biol.Interact_203_172
PubMedSearch : Sun_2013_Chem.Biol.Interact_203_172
PubMedID: 23220586

Title : Treatment with endotracheal therapeutics after sarin microinstillation inhalation exposure increases blood cholinesterase levels in guinea pigs - Che_2012_Toxicol.Mech.Methods_22_250
Author(s) : Che MM , Song J , Oguntayo S , Doctor BP , Rezk P , Perkins MW , Sciuto AM , Nambiar MP
Ref : Toxicol Mech Methods , 22 :250 , 2012
Abstract : Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in the blood and tissues of animals that are treated with a number of endotracheally aerosolized therapeutics for protection against inhalation toxicity to sarin. Therapeutics included, aerosolized atropine methyl bromide (AMB), scopolamine or combination of AMB with salbutamol, sphingosine 1-phosphate, keratinocyte growth factor, adenosine A1 receptor antisense oligonucleotide (EPI2010), 2,3-diacetyloxybenzoic acid (2,3 DABA), oxycyte, and survanta. Guinea pigs exposed to 677.4 mg/m(3) or 846.5 mg/m(3) (1.2 LCt(50)) sarin for 4 min using a microinstillation inhalation exposure technique and treated 1 min later with the aerosolized therapeutics. Treatment with all therapeutics significantly increased the survival rate with no convulsions throughout the 24 h study period. Blood AChE activity determined using acetylthiocholine as substrate showed 20% activity remaining in sarin-exposed animals compare to controls. In aerosolized AMB and scopolamine-treated animals the remaining AChE activity was significantly higher (45-60%) compared to sarin-exposed animals (p < 0.05). Similarly, treatment with all the combination therapeutics resulted in significant increase in blood AChE activity in comparison to sarin-exposed animals although the increases varied between treatments (p < 0.05). BChE activity was increased after treatment with aerosolized therapeutics but was lesser in magnitude compared to AChE activity changes. Various tissues showed elevated AChE activity after therapeutic treatment of sarin-exposed animals. Increased AChE and BChE activities in animals treated with nasal therapeutics suggest that enhanced breathing and reduced respiratory toxicity/lung injury possibly contribute to rapid normalization of chemical warfare nerve agent inhibited cholinesterases.
ESTHER : Che_2012_Toxicol.Mech.Methods_22_250
PubMedSearch : Che_2012_Toxicol.Mech.Methods_22_250
PubMedID: 22145985

Title : Hydrolysis potential of recombinant human skin and kidney prolidase against diisopropylfluorophosphate and sarin by in vitro analysis - Costante_2012_Toxicol.In.Vitro_26_182
Author(s) : Costante M , Biggemann L , Alamneh Y , Soojhawon I , Short R , Nigam S , Garcia G , Doctor BP , Valiyaveettil M , Nambiar MP
Ref : Toxicol In Vitro , 26 :182 , 2012
Abstract : Human prolidase (PROL), which has structural homology to bacterial organophosphate acid anhydrolase that hydrolyze organophosphates and nerve agents has been proposed recently as a potential catalytic bioscavenger. To develop PROL as a catalytic bioscavenger, we evaluated the in vitro hydrolysis efficiency of purified recombinant human PROL against organophosphates and nerve agents. Human liver PROL was purified by chromatographic procedures, whereas recombinant human skin and kidney PROL was expressed in Trichoplusia ni larvae, affinity purified and analyzed by gel electrophoresis. The catalytic efficiency of PROL against diisopropylfluorophosphate (DFP) and nerve agents was evaluated by acetylcholinesterase back-titration assay. Partially purified human liver PROL hydrolyzed DFP and various nerve agents, which was abolished by specific PROL inhibitor showing the specificity of hydrolysis. Both the recombinant human skin and kidney PROL expressed in T. ni larvae showed approximately 99% purity and efficiently hydrolyzed DFP and sarin. In contrast to human liver PROL, both skin and kidney PROL showed significantly low hydrolyzing potential against nerve agents soman, tabun and VX. In conclusion, compared to human liver PROL, recombinant human skin and kidney PROL hydrolyze only DFP and sarin showing the substrate specificity of PROL from various tissue sources.
ESTHER : Costante_2012_Toxicol.In.Vitro_26_182
PubMedSearch : Costante_2012_Toxicol.In.Vitro_26_182
PubMedID: 22120822

Title : Aerosolized delivery of oxime MMB-4 in combination with atropine sulfate protects against soman exposure in guinea pigs - Perkins_2012_Inhal.Toxicol_24_539
Author(s) : Perkins MW , Pierre Z , Sabnekar P , Sciuto AM , Song J , Soojhawon I , Oguntayo S , Doctor BP , Nambiar MP
Ref : Inhal Toxicol , 24 :539 , 2012
Abstract : We evaluated the efficacy of aerosolized acetylcholinesterase (AChE) reactivator oxime MMB-4 in combination with the anticholinergic atropine sulfate for protection against respiratory toxicity and lung injury following microinstillation inhalation exposure to nerve agent soman (GD) in guinea pigs. Anesthetized animals were exposed to GD (841 mg/m(3), 1.2 LCt(50)) and treated with endotracheally aerosolized MMB-4 (50 micromol/kg) plus atropine sulfate (0.25 mg/kg) at 30 sec post-exposure. Treatment with MMB-4 plus atropine increased survival to 100% compared to 38% in animals exposed to GD. Decreases in the pulse rate and blood O(2) saturation following exposure to GD returned to normal levels in the treatment group. The body-weight loss and lung edema was significantly reduced in the treatment group. Similarly, bronchoalveolar cell death was significantly reduced in the treatment group while GD-induced increase in total cell count was decreased consistently but was not significant. GD-induced increase in bronchoalveolar protein was diminished after treatment with MMB-4 plus atropine. Bronchoalveolar lavage AChE and BChE activity were significantly increased in animals treated with MMB-4 plus atropine at 24 h. Lung and diaphragm tissue also showed a significant increase in AChE activity in the treatment group. Treatment with MMB-4 plus atropine sulfate normalized various respiratory dynamics parameters including respiratory frequency, tidal volume, peak inspiratory and expiratory flow, time of inspiration and expiration, enhanced pause and pause post-exposure to GD. Collectively, these results suggest that aerosolization of MMB-4 plus atropine increased survival, decreased respiratory toxicity and lung injury following GD inhalation exposure.
ESTHER : Perkins_2012_Inhal.Toxicol_24_539
PubMedSearch : Perkins_2012_Inhal.Toxicol_24_539
PubMedID: 22860999

Title : Characterization of human serum butyrylcholinesterase in rhesus monkeys: behavioral and physiological effects - Myers_2012_Neurotoxicol.Teratol_34_323
Author(s) : Myers TM , Sun W , Naik RS , Clark MG , Doctor BP , Saxena A
Ref : Neurotoxicology & Teratology , 34 :323 , 2012
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.
ESTHER : Myers_2012_Neurotoxicol.Teratol_34_323
PubMedSearch : Myers_2012_Neurotoxicol.Teratol_34_323
PubMedID: 22402122

Title : Neuroprotective effects of imidazenil against chemical warfare nerve agent soman toxicity in guinea pigs - Wang_2012_Neurotoxicol_33_169
Author(s) : Wang Y , Oguntayo S , Wei Y , Wood E , Brown A , Jensen N , Auta J , Guiodotti A , Doctor BP , Nambiar MP
Ref : Neurotoxicology , 33 :169 , 2012
Abstract : The chemical warfare nerve agent, soman irreversibly inhibits acetylcholinesterase (AChE) leading to hypercholinergy and seizures which trigger glutamate toxicity and status epilepticus ultimately resulting in neuropathology and neurobehavioral deficits. The standard emergency treatment comprising of anticholinergic, AChE reactivator and anticonvulsant does not completely protect against soman toxicity. We have evaluated imidazenil, a new anticonvulsant imidazo benzodiazepine with high affinity and intrinsic efficacy at alpha5-, alpha2-, and alpha3- but low intrinsic efficacy at alpha1-containing GABA(A) receptors and is devoid of cardiorespiratory depression, sedative/hypnoitc and amnestic actions and does not elicit tolerance and dependence liabilities unlike diazepam, for protection against soman toxicity. Guinea pigs implanted with bipotential radiotelemetry probes for recording EEG and ECG were administered with 26 mug/kg pyridostigmine bromide 30 min prior to 2x LD(50) soman exposure and 1 min later treated with a combination of 2mg/kg atropine sulfate and 25mg/kg 2-pralidoxime and various doses of imidazenil. Intramuscular administration of imidazenil, dose-dependently protected against 2x LD(50) of soman toxicity up to 1mg/kg. Further increase in the dose of imidazenil to 2.5mg/kg was less effective than 1mg/kg probably due to non-specific actions at sites other than GABA(A) receptors. Compared to vehicle group, 1mg/kg imidazenil treatment showed optimal increase in survival rate, reduction in behavioral manifestations and high power of EEG spectrum as well as neuronal necrosis. These data suggest that imidazenil is an effective anticonvulsant for medical countermeasure against soman-induced toxicity.
ESTHER : Wang_2012_Neurotoxicol_33_169
PubMedSearch : Wang_2012_Neurotoxicol_33_169
PubMedID: 22245390

Title : In vitro efficacy of paraoxonase 1 from multiple sources against various organophosphates - Valiyaveettil_2011_Toxicol.In.Vitro_25_905
Author(s) : Valiyaveettil M , Alamneh Y , Biggemann L , Soojhawon I , Farag HA , Agrawal P , Doctor BP , Nambiar MP
Ref : Toxicol In Vitro , 25 :905 , 2011
Abstract : Paraoxonase 1 (PON1) has been described as a potential catalytic bioscavenger due to its ability to hydrolyze organophosphate (OP) insecticides and nerve agents. In vitro catalytic efficiency of purified human and rabbit serum PON1 against different OP substrates was compared to human recombinant PON1, expressed in Trichoplusia ni larvae. Highly purified human and rabbit serum PON1s were prepared by multiple chromatography methods. Purified enzymes showed higher catalytic activity with the substrate p-nitrophenyl acetate compared to diethyl paraoxon. The hydrolyzing potential of PON1s against multiple OPs was evaluated by using an in vitro acetylcholinesterase back-titration assay. Significant differences in the catalytic efficiency of all the three PON1s with regard to various OP substrates were observed. Purified PON1s showed higher catalytic activity towards diisopropylfluorophosphate followed by diethylparaoxon compared to dimethyl paraoxon. Heat inactivation or incubation of PON1 with specific inhibitor resulted in complete loss of the enzyme catalytic activity indicating that OP hydrolysis was intrinsic to PON1. In conclusion, purified PON1s from multiple sources show significant differences in the catalytic activity against several OP substrates. These results underscore the importance of systematic analysis of candidate PON1 molecules for developing as an effective catalytic bioscavenger against toxic OPs and chemical warfare nerve agents.
ESTHER : Valiyaveettil_2011_Toxicol.In.Vitro_25_905
PubMedSearch : Valiyaveettil_2011_Toxicol.In.Vitro_25_905
PubMedID: 21382471

Title : Recombinant paraoxonase 1 protects against sarin and soman toxicity following microinstillation inhalation exposure in guinea pigs - Valiyaveettil_2011_Toxicol.Lett_202_203
Author(s) : Valiyaveettil M , Alamneh Y , Rezk P , Perkins MW , Sciuto AM , Doctor BP , Nambiar MP
Ref : Toxicol Lett , 202 :203 , 2011
Abstract : To explore the efficacy of paraoxonase 1 (PON1) as a catalytic bioscavenger, we evaluated human recombinant PON1 (rePON1) expressed in Trichoplusia ni larvae against sarin and soman toxicity using microinstillation inhalation exposure in guinea pigs. Animals were pretreated intravenously with catalytically active rePON1, followed by exposure to 1.2 X LCt(5)(0) sarin or soman. Administration of 5 units of rePON1 showed mild increase in the blood activity of the enzyme after 30 min, but protected the animals with a significant increase in survival rate along with minimal signs of nerve agent toxicity. Recombinant PON1 pretreated animals exposed to sarin or soman prevented the reduction of blood O(2) saturation and pulse rate observed after nerve agent exposure. In addition, rePON1 pretreated animals showed significantly higher blood PON1, acetylcholinesterase (AChE), and butyrylcholinesterase activity after nerve agent exposure compared to the respective controls without treatments. AChE activity in different brain regions of rePON1 pretreated animals exposed to sarin or soman were also significantly higher than respective controls. The remaining activity of blood PON1, cholinesterases and brain AChE in PON1 pretreated animals after nerve agent exposure correlated with the survival rate. In summary, these data suggest that human rePON1 protects against sarin and soman exposure in guinea pigs.
ESTHER : Valiyaveettil_2011_Toxicol.Lett_202_203
PubMedSearch : Valiyaveettil_2011_Toxicol.Lett_202_203
PubMedID: 21329748

Title : Pretreatment with human serum butyrylcholinesterase alone prevents cardiac abnormalities, seizures, and death in Gottingen minipigs exposed to sarin vapor - Saxena_2011_Biochem.Pharmacol_82_1984
Author(s) : Saxena A , Sun W , Dabisch PA , Hulet SW , Hastings NB , Jakubowski EM , Mioduszewski RJ , Doctor BP
Ref : Biochemical Pharmacology , 82 :1984 , 2011
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 Gottingen 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.
ESTHER : Saxena_2011_Biochem.Pharmacol_82_1984
PubMedSearch : Saxena_2011_Biochem.Pharmacol_82_1984
PubMedID: 21968035

Title : Protective effects of aerosolized scopolamine against soman-induced acute respiratory toxicity in guinea pigs - Perkins_2011_Int.J.Toxicol_30_639
Author(s) : Perkins MW , Pierre Z , Rezk P , Song J , Oguntayo S , Morthole V , Sciuto AM , Doctor BP , Nambiar MP
Ref : Int J Toxicol , 30 :639 , 2011
Abstract : The protective efficacy of the antimuscarinic agent scopolamine was evaluated against soman (o-pinacolyl methylphosphonofluoridate [GD])-induced respiratory toxicity in guinea pigs. Anesthetized animals were exposed to GD (841 mg/m(3)) by microinstillation inhalation exposure and treated 30 seconds later with endotracheally aerosolized scopolamine (0.25 mg/kg) and allowed to recover for 24 hours. Treatment with scopolamine significantly increased survival and reduced clinical signs of toxicity and body weight loss in GD-exposed animals. Analysis of bronchoalveolar lavage (BAL) fluid showed normalization of GD-induced increased cell death, total cell count, and protein following scopolamine treatment. The BAL fluid acetylcholinesterase and butyrylcholinesterase levels were also increased by scopolamine treatment. Respiratory dynamics parameters were normalized at 4 and 24 hours post-GD exposure in scopolamine-treated animals. Lung histology showed that scopolamine treatment reduced bronchial epithelial and subepithelial inflammation and multifocal alveolar septal edema. These results suggest that aerosolized scopolamine considerably protects against GD-induced respiratory toxicity.
ESTHER : Perkins_2011_Int.J.Toxicol_30_639
PubMedSearch : Perkins_2011_Int.J.Toxicol_30_639
PubMedID: 21960666

Title : Prophylaxis with human serum butyrylcholinesterase protects guinea pigs exposed to multiple lethal doses of soman or VX - Saxena_2011_Biochem.Pharmacol_81_164
Author(s) : Saxena A , Sun W , Fedorko JM , Koplovitz I , Doctor BP
Ref : Biochemical Pharmacology , 81 :164 , 2011
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 2xLD(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 approximately 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 5.5xLD(50) of soman or 8xLD(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.
ESTHER : Saxena_2011_Biochem.Pharmacol_81_164
PubMedSearch : Saxena_2011_Biochem.Pharmacol_81_164
PubMedID: 20846507

Title : Protective efficacy of catalytic bioscavenger, paraoxonase 1 against sarin and soman exposure in guinea pigs - Valiyaveettil_2011_Biochem.Pharmacol_81_800
Author(s) : Valiyaveettil M , Alamneh Y , Rezk P , Biggemann L , Perkins MW , Sciuto AM , Doctor BP , Nambiar MP
Ref : Biochemical Pharmacology , 81 :800 , 2011
Abstract : Human paraoxonase 1 (PON1) has been portrayed as a catalytic bioscavenger which can hydrolyze large amounts of chemical warfare nerve agents (CWNAs) and organophosphate (OP) pesticides compared to the stoichiometric bioscavengers such as butyrylcholinesterase. We evaluated the protective efficacy of purified human and rabbit serum PON1 against nerve agents sarin and soman in guinea pigs. Catalytically active PON1 purified from human and rabbit serum was intravenously injected to guinea pigs, which were 30 min later exposed to 1.2 x LCt50 sarin or soman using a microinstillation inhalation exposure technology. Pre-treatment with 5 units of purified human and rabbit serum PON1 showed mild to moderate increase in the activity of blood PON1, but significantly increased the survival rate with reduced symptoms of CWNA exposure. Although PON1 is expected to be catalytic, sarin and soman exposure resulted in a significant reduction in blood PON1 activity. However, the blood levels of PON1 in pre-treated animals after exposure to nerve agent were higher than that of untreated control animals. The activity of blood acetylcholinesterase and butyrylcholinesterase and brain acetylcholinesterase was significantly higher in PON1 pre-treated animals and were highly correlated with the survival rate. Blood O saturation, pulse rate and respiratory dynamics were normalized in animals treated with PON1 compared to controls. These results demonstrate that purified human and rabbit serum PON1 significantly protect against sarin and soman exposure in guinea pigs and support the development of PON1 as a catalytic bioscavenger for protection against lethal exposure to CWNAs.
ESTHER : Valiyaveettil_2011_Biochem.Pharmacol_81_800
PubMedSearch : Valiyaveettil_2011_Biochem.Pharmacol_81_800
PubMedID: 21219877

Title : Systemic administration of the potential countermeasure huperzine reversibly inhibits central and peripheral acetylcholinesterase activity without adverse cognitive-behavioral effects - Myers_2010_Pharmacol.Biochem.Behav_94_477
Author(s) : Myers TM , Sun W , Saxena A , Doctor BP , Bonvillain AJ , Clark MG
Ref : Pharmacol Biochem Behav , 94 :477 , 2010
Abstract : Huperzine A is potentially superior to pyridostigmine bromide as a pretreatment for nerve agent intoxication because it inhibits acetylcholinesterase both peripherally and centrally, unlike pyridostigmine, which acts only peripherally. Using rhesus monkeys, we evaluated the time course of acetylcholinesterase and butyrylcholinesterase inhibition following four different doses of -(-)huperzine A: 5, 10, 20, and 40 microg/kg. Acetylcholinesterase inhibition peaked 30 min after intramuscular injection and varied dose dependently, ranging from about 30% to 75%. Subsequently, cognitive-behavioral functioning was also evaluated at each dose of huperzine A using a six-item serial-probe recognition task that assessed attention, motivation, and working memory. Huperzine did not impair performance, but physostigmine did. The results demonstrate that huperzine A can selectively and reversibly inhibit acetylcholinesterase without cognitive-behavioral side effects, thus warranting further study.
ESTHER : Myers_2010_Pharmacol.Biochem.Behav_94_477
PubMedSearch : Myers_2010_Pharmacol.Biochem.Behav_94_477
PubMedID: 19909771

Title : Safety of administration of human butyrylcholinesterase and its conjugates with soman or VX in rats - Genovese_2010_Basic.Clin.Pharmacol.Toxicol_106_428
Author(s) : Genovese RF , Sun W , Johnson CC , diTargiani RC , Doctor BP , Saxena A
Ref : Basic Clin Pharmacol Toxicol , 106 :428 , 2010
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.
ESTHER : Genovese_2010_Basic.Clin.Pharmacol.Toxicol_106_428
PubMedSearch : Genovese_2010_Basic.Clin.Pharmacol.Toxicol_106_428
PubMedID: 20050840

Title : Gastrointestinal acetylcholinesterase activity following endotracheal microinstillation inhalation exposure to sarin in guinea pigs - Chanda_2010_Chem.Biol.Interact_187_309
Author(s) : Chanda S , Song J , Rezk P , Sabnekar P , Doctor BP , Sciuto AM , Nambiar MP
Ref : Chemico-Biological Interactions , 187 :309 , 2010
Abstract : The goal of this study was to assess acetylcholinesterase (AChE) inhibition at different regions of the gastrointestinal (GI) tract following inhalation exposure to nerve agent sarin. Seven major regions of the GI tract were removed from saline control animals (n=3) and 677.4 mg/m(3) sarin-exposed animals at 4h (n=4) and 24h (n=4) post-exposure. AChE activity was determined in blood and homogenized tissue supernatant by specific Ellman's assay using Iso-OMPA, a BChE inhibitor, and expressed as activity/optical density of hemoglobin for blood and activity/mg protein for tissues. Our data showed that the AChE activity was significantly decreased for groups both 4h and 24h post-sarin exposure. Among the seven chosen regions of the guinea pig GI tract, duodenum showed the highest AChE activity in control animals. The AChE activity was significantly decreased in the stomach (p=0.03), duodenum (p=0.029), jejunum (p=0.006), and ileum (p=0.006) 4h following sarin exposure. At 24h post-sarin exposure the AChE activity of duodenum (p=0.029) and ileum (p=0.006) was significantly inhibited. Esophagus showed no inhibition following sarin exposure at both 4h and 24h groups. These results suggest that the AChE activity is different in different regions of the GI tract and highest levels of AChE inhibition following sarin exposure were seen in regions exhibiting higher overall AChE activity and cholinergic function.
ESTHER : Chanda_2010_Chem.Biol.Interact_187_309
PubMedSearch : Chanda_2010_Chem.Biol.Interact_187_309
PubMedID: 20227400

Title : Demonstration of in vivo stability and lack of immunogenicity of a polyethyleneglycol-conjugated recombinant CHO-derived butyrylcholinesterase bioscavenger using a homologous macaque model - Rosenberg_2010_Chem.Biol.Interact_187_279
Author(s) : Rosenberg YJ , Saxena A , Sun W , Jiang X , Chilukuri N , Luo C , Doctor BP , Lee KD
Ref : Chemico-Biological Interactions , 187 :279 , 2010
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.
ESTHER : Rosenberg_2010_Chem.Biol.Interact_187_279
PubMedSearch : Rosenberg_2010_Chem.Biol.Interact_187_279
PubMedID: 20211615

Title : Efficient hydrolysis of the chemical warfare nerve agent tabun by recombinant and purified human and rabbit serum paraoxonase 1 - Valiyaveettil_2010_Biochem.Biophys.Res.Commun_403_97
Author(s) : Valiyaveettil M , Alamneh Y , Biggemann L , Soojhawon I , Doctor BP , Nambiar MP
Ref : Biochemical & Biophysical Research Communications , 403 :97 , 2010
Abstract : Paraoxonase 1 (PON1) has been described as an efficient catalytic bioscavenger due to its ability to hydrolyze organophosphates (OPs) and chemical warfare nerve agents (CWNAs). It is the future most promising candidate as prophylactic medical countermeasure against highly toxic OPs and CWNAs. Most of the studies conducted so far have been focused on the hydrolyzing potential of PON1 against nerve agents, sarin, soman, and VX. Here, we investigated the hydrolysis of tabun by PON1 with the objective of comparing the hydrolysis potential of human and rabbit serum purified and recombinant human PON1. The hydrolysis potential of PON1 against tabun, sarin, and soman was evaluated by using an acetylcholinesterase (AChE) back-titration Ellman method. Efficient hydrolysis of tabun (100 nM) was observed with approximately 25-40 mU of PON1, while higher concentration (80-250 mU) of the enzyme was required for the complete hydrolysis of sarin (11 nM) and soman (3 nM). Our data indicate that tabun hydrolysis with PON1 was approximately 30-60 times and approximately 200-260 times more efficient than that with sarin and soman, respectively. Moreover, the catalytic activity of PON1 varies from source to source, which also reflects their efficiency of hydrolyzing different types of nerve agents. Thus, efficient hydrolysis of tabun by PON1 suggests its promising potential as a prophylactic treatment against tabun exposure.
ESTHER : Valiyaveettil_2010_Biochem.Biophys.Res.Commun_403_97
PubMedSearch : Valiyaveettil_2010_Biochem.Biophys.Res.Commun_403_97
PubMedID: 21040699

Title : Acute respiratory toxicity following inhalation exposure to soman in guinea pigs - Perkins_2010_Toxicol.Appl.Pharmacol_245_171
Author(s) : Perkins MW , Pierre Z , Rezk P , Sabnekar P , Kabra K , Chanda S , Oguntayo S , Sciuto AM , Doctor BP , Nambiar MP
Ref : Toxicol Appl Pharmacol , 245 :171 , 2010
Abstract : Respiratory toxicity and lung injury following inhalation exposure to chemical warfare nerve agent soman was examined in guinea pigs without therapeutics to improve survival. A microinstillation inhalation exposure technique that aerosolizes the agent in the trachea was used to administer soman to anesthetized age and weight matched male guinea pigs. Animals were exposed to 280, 561, 841, and 1121 mg/m(3) concentrations of soman for 4 min. Survival data showed that all saline controls and animals exposed to 280 and 561 mg/m(3) soman survived, while animals exposed to 841, and 1121 mg/m(3) resulted in 38% and 13% survival, respectively. The microinstillation inhalation exposure LCt(50) for soman determined by probit analysis was 827.2mg/m(3). A majority of the animals that died at 1121 mg/m(3) developed seizures and died within 15-30 min post-exposure. There was a dose-dependent decrease in pulse rate and blood oxygen saturation of animals exposed to soman at 5-6.5 min post-exposure. Body weight loss increased with the dose of soman exposure. Bronchoalveolar lavage (BAL) fluid and blood acetylcholinesterase and butyrylcholinesterase activity was inhibited dose-dependently in soman treated groups at 24h. BAL cells showed a dose-dependent increase in cell death and total cell counts following soman exposure. Edema by wet/dry weight ratio of the accessory lung lobe and trachea was increased slightly in soman exposed animals. An increase in total bronchoalveolar lavage fluid protein was observed in soman exposed animals at all doses. Differential cell counts of BAL and blood showed an increase in total lymphocyte counts and percentage of neutrophils. These results indicate that microinstillation inhalation exposure to soman causes respiratory toxicity and acute lung injury in guinea pigs.
ESTHER : Perkins_2010_Toxicol.Appl.Pharmacol_245_171
PubMedSearch : Perkins_2010_Toxicol.Appl.Pharmacol_245_171
PubMedID: 20206646

Title : Direct correlation between molecular dynamics and enzymatic stability: a comparative neutron scattering study of native human butyrylcholinesterase and its aged soman conjugate - Gabel_2009_Biophys.J_96_1489
Author(s) : Gabel F , Masson P , Froment MT , Doctor BP , Saxena A , Silman I , Zaccai G , Weik M
Ref : Biophysical Journal , 96 :1489 , 2009
Abstract : An incoherent elastic neutron scattering study of the molecular dynamics of native human butyrylcholinesterase and its "aged" soman-inhibited conjugate revealed a significant change in molecular flexibility on an angstrom-nanosecond scale as a function of temperature. The results were related to the stability of each state as established previously by differential scanning calorimetry. A striking relationship was found between the denaturation behavior and the molecular flexibility of the native and inhibited enzymes as a function of temperature. This was reflected in a quantitative correlation between the atomic mean-square displacements on an angstrom-nanosecond scale determined by neutron spectroscopy and the calorimetric specific heat. By the application of a simple two-state model that describes the transition from a folded to a denatured state, the denaturation temperatures of the native and the inhibited enzyme were correctly extracted from the atomic mean-square displacements. Furthermore, the transition entropy and enthalpy extracted from the model fit of the neutron data were, within the experimental accuracy, compatible with the values determined by differential scanning calorimetry.
ESTHER : Gabel_2009_Biophys.J_96_1489
PubMedSearch : Gabel_2009_Biophys.J_96_1489
PubMedID: 19217865

Title : Adenovirus-transduced human butyrylcholinesterase in mouse blood functions as a bioscavenger of chemical warfare nerve agents - Chilukuri_2009_Mol.Pharmacol_76_612
Author(s) : Chilukuri N , Duysen EG , Parikh K , diTargiani RC , Doctor BP , Lockridge O , Saxena A
Ref : Molecular Pharmacology , 76 :612 , 2009
Abstract : Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents. We have showed previously that recombinant (r) Hu BChE can be expressed at very high levels, 400 to 600 U/ml in mouse blood, by delivering the Hu BChE gene using adenovirus (Ad). Here, we report the biochemical properties of the Ad-expressed full-length and truncated rHu BChE in mouse blood. The molecular sizes of the full-length rHu BChE subunit and its oligomers were similar to those of native Hu BChE, although only a small portion of the full-length rHu BChE subunit underwent assembly into dimers and tetramers. As expected, Ad containing the truncated Hu BChE gene transduced the expression of monomeric rHu BChE only. Compared with 415 U of rHu BChE per milliliter in blood, tissues including liver, lung, heart, brain, kidney, muscle, intestine, diaphragm, salivary gland, and fat expressed <10 U/g of rHu BChE activity. Ad-expressed rHu BChE in mouse blood neutralized soman and O-ethyl S-2-N,N-diisopropylaminoethyl methylphosphonothiolate at rates similar to those of native Hu BChE and rHu BChE expressed in vitro. Because the expression of rHu BChE rapidly decreased 6 days after virus administration, sera were assayed for the presence of anti-Hu BChE antibodies. Anti-Hu BChE antibodies were detected on day 7 and in increased amounts thereafter, which coincided with the loss of Hu BChE expression in sera. In conclusion, the delivery of Hu BChE gene using Ad can be a promising strategy that can provide protection against multiple lethal doses of chemical warfare nerve agents in vivo.
ESTHER : Chilukuri_2009_Mol.Pharmacol_76_612
PubMedSearch : Chilukuri_2009_Mol.Pharmacol_76_612
PubMedID: 19542320

Title : Pharmacokinetics and immunologic consequences of repeated administrations of purified heterologous and homologous butyrylcholinesterase in mice - Sun_2009_Life.Sci_85_657
Author(s) : Sun W , Luo C , Naik RS , Doctor BP , Saxena A
Ref : Life Sciences , 85 :657 , 2009
Abstract : 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.
ESTHER : Sun_2009_Life.Sci_85_657
PubMedSearch : Sun_2009_Life.Sci_85_657
PubMedID: 19772863

Title : The IXth International Meeting on Cholinesterases, Suzhou, People's Republic of China, 6 to 10 May 2007 -
Author(s) : Doctor BP , Tsim KWK , Siow NL
Ref : Chemico-Biological Interactions , 175 :1 , 2008
PubMedID: 18582448

Title : A repeated injection of polyethyleneglycol-conjugated recombinant human butyrylcholinesterase elicits immune response in mice - Chilukuri_2008_Toxicol.Appl.Pharmacol_231_423
Author(s) : Chilukuri N , Sun W , Parikh K , Naik RS , Tang L , Doctor BP , Saxena A
Ref : Toxicol Appl Pharmacol , 231 :423 , 2008
Abstract : Human serum butyrylcholinesterase (Hu BChE) serves as an efficacious bioscavenger of highly toxic organophosphorus (OP) compounds. Since there is a concern that the supply of native Hu BChE may be limited, monomeric and tetrameric forms of recombinant Hu BChE (rHu BChE) were evaluated as replacements and found that they lacked sufficient stability in vivo. However, their in vivo stability could be significantly prolonged by conjugation with polyethyleneglycol-20K (PEG) suggesting that monomeric and tetrameric PEG-rHu BChE could function as bioscavengers. Here, the immunogenicity of PEG-rHu BChE was evaluated in mice following two injections given four weeks apart. In addition to pharmacokinetic parameters, such as mean residence time, maximal concentration, time to reach the maximal concentration, elimination half-life and area under the plasma concentration-time curve extrapolated to infinity, the presence of circulating anti-rHu BChE antibodies was also determined. Although the pharmacokinetic parameters were significantly improved for the first injection of monomeric and tetrameric PEG-rHu BChEs, they were much lower for the second injection. Anti-rHu BChE antibodies were detected in the blood of mice following the first and second enzyme injections and their levels were approximately higher by 5-fold and 2-fold in mice injected with monomeric and tetrameric PEG-rHu BChEs as compared to mice injected with unconjugated enzymes. The findings that the rapid clearance of a repeat injection of PEG-rHu BChEs in mice which coincides with the presence of circulating anti-rHu BChE antibodies suggest that PEG conjugation prolonged the circulatory stability of rHu BChE but failed to eliminate its immunogenicity in mice.
ESTHER : Chilukuri_2008_Toxicol.Appl.Pharmacol_231_423
PubMedSearch : Chilukuri_2008_Toxicol.Appl.Pharmacol_231_423
PubMedID: 18586293

Title : Developing procedures for the large-scale purification of human serum butyrylcholinesterase - Saxena_2008_Protein.Expr.Purif_61_191
Author(s) : Saxena A , Luo C , Doctor BP
Ref : Protein Expr Purif , 61 :191 , 2008
Abstract : 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.
ESTHER : Saxena_2008_Protein.Expr.Purif_61_191
PubMedSearch : Saxena_2008_Protein.Expr.Purif_61_191
PubMedID: 18602477

Title : Long-term effects of human butyrylcholinesterase pretreatment followed by acute soman challenge in cynomolgus monkeys - Sun_2008_Chem.Biol.Interact_175_428
Author(s) : Sun W , Doctor BP , Lenz DE , Saxena A
Ref : Chemico-Biological Interactions , 175 :428 , 2008
Abstract : Human serum butyrylcholinesterase (Hu BChE) was demonstrated previously to be an effective prophylaxis that can protect animals from organophosphate nerve agents. However, in most of those studies, the maximum dose used to challenge animals was low (<2x LD(50)), and the health of these animals was monitored for only up to 2 weeks. In this study, six cynomolgus monkeys received 75 mg of Hu BChE followed by sequential doses (1.5, 2.0, 2.0 x LD(50)) of soman 10h later for a total challenge of 5.5x LD(50). Four surviving animals that did not show any signs of soman intoxication were transferred to WRAIR for the continuous evaluation of long-term health effects for 14 months. Each month, blood was drawn from these monkeys and analyzed for serum chemistry and hematology parameters, blood acetylcholinesterase (AChE) and BChE levels. Based on the serum chemistry and hematology parameters measured, no toxic effects or any organ malfunctions were observed up to 14 months following Hu BuChE protection against exposure to 5.5x LD(50) of soman. In conclusion, Hu BChE pretreatment not only effectively protects monkeys from soman-induced toxicity of the immediate acute phase but also for a long-term outcome.
ESTHER : Sun_2008_Chem.Biol.Interact_175_428
PubMedSearch : Sun_2008_Chem.Biol.Interact_175_428
PubMedID: 18674756

Title : [+]-Huperzine A treatment protects against N-methyl-D-aspartate-induced seizure\/status epilepticus in rats - Coleman_2008_Chem.Biol.Interact_175_387
Author(s) : Coleman BR , Ratcliffe RH , Oguntayo SA , Shi X , Doctor BP , Gordon RK , Nambiar MP
Ref : Chemico-Biological Interactions , 175 :387 , 2008
Abstract : The toxicity of organophosphorous (OP) nerve agents is attributed to their irreversible inhibition of acetylcholinesterase (AChE), which leads to excessive accumulation of acetylcholine (ACh) and is followed by the release of excitatory amino acids (EAA). EAAs sustain seizure activity and induce neuropathology due to over-stimulation of N-methyl-d-aspartate (NMDA) receptors. Huperzine A (Hup A), a blood-brain barrier permeable selective reversible inhibitor of AChE, has been shown to reduce EAA-induced cell death by interfering with glutamate receptor-gated ion channels in primary neuronal cultures. Although [-]-Hup A, the natural isomer, inhibits AChE approximately 38-fold more potently than [+]-Hup A, both [-]- and [+]-Hup A block the NMDA channel similarly. Here, we evaluated the protective efficacy of [+]-Hup A for NMDA-induced seizure in a rat model. Rats implanted with radiotelemetry probes to record electroencephalography (EEG), electrocardiography (ECG), body temperature, and physical activity were administered various doses of [+]-Hup A (intramuscularly) and treated with 20 microg/kg NMDA (intracerebroventricular) 20-30 min later. For post-exposure, rats were treated with [+]-Hup A (3 mg/kg, intramuscularly) 1 min after NMDA (20 microg/kg). Our data showed that pre- and post-exposure, [+]-Hup A (3 mg/kg) protects animals against NMDA-induced seizures. Also, NMDA-administered animals showed increased survival following [+]-Hup A treatment. [+]-Hup A has no visible effect on EEG, heart-rate, body temperature, or physical activity, indicating a reduced risk of side effects, toxicity, or associated pathology. Our results suggest that [+]-Hup A protects against seizure and status epilepticus (SE) by blocking NMDA-induced excitotoxicity in vivo. We propose that [+]-Hup A, or a unique combination of [+]- and [-]-Hup A, may prove to be effective for pre- and post-exposure treatment of lethal doses of OP-induced neurotoxicity.
ESTHER : Coleman_2008_Chem.Biol.Interact_175_387
PubMedSearch : Coleman_2008_Chem.Biol.Interact_175_387
PubMedID: 18588864

Title : Adenovirus-mediated gene transfer of human butyrylcholinesterase results in persistent high-level transgene expression in vivo - Chilukuri_2008_Chem.Biol.Interact_175_327
Author(s) : Chilukuri N , Duysen EG , Parikh K , Sun W , Doctor BP , Lockridge O , Saxena A
Ref : Chemico-Biological Interactions , 175 :327 , 2008
Abstract : Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents, pesticide intoxication, and cocaine overdose. However, its widespread application is hampered by difficulties in large-scale production of the native protein from human plasma and/or availability as a recombinant protein suitable for use in vivo. This limitation may be resolved by in vivo delivery and expression of the Hu BChE gene. In this study, recombinant (r) adenoviruses (Ads) encoding full-length and truncated rHu BChEs were tested for in vivo expression in mice. Mice injected with these rAds intraperitoneally failed to express rHu BChE. However, a single tail vein injection of both rAds resulted in persistent high serum levels of rHu BChE in BChE knockout mice, which peaked on days 4/5 at 377+/-162U/ml for full-length rHu BChE and 574+/-143U/ml for truncated rHu BChE. These activity levels are orders of magnitude higher than 1.9U/ml of mouse BChE present in wild-type mouse serum. Thereafter, rHu BChE levels dropped rapidly and very little or no activity was detected in the serum 10 days post-virus administration. In conclusion, the present study demonstrates the potential of rAd-mediated Hu BChE gene therapy to counteract multiple lethal doses of chemical warfare nerve agent toxicity.
ESTHER : Chilukuri_2008_Chem.Biol.Interact_175_327
PubMedSearch : Chilukuri_2008_Chem.Biol.Interact_175_327
PubMedID: 18499092

Title : Effect of polyethylene glycol modification on the circulatory stability and immunogenicity of recombinant human butyrylcholinesterase - Chilukuri_2008_Chem.Biol.Interact_175_255
Author(s) : Chilukuri N , Sun W , Naik RS , Parikh K , Tang L , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 175 :255 , 2008
Abstract : The therapeutic value of human serum butyrylcholinesterase (Hu BChE) as a bioscavenger of chemical warfare agents is due to its high reactivity with organophosphorus compounds and prolonged circulatory stability. Native Hu BChE is mostly tetrameric in form while the enzyme produced using molecular cloning technology is a mixture of tetramers, dimers, and monomers. Previous studies revealed that monomers and dimers of recombinant human (rHu) BChE cleared rapidly from the circulation of mice compared to tetrameric rHu BChE and native Hu BChE, which have mean residence times (MRTs) of 18h and 45h, respectively. It was also shown that polyethylene glycol-20K (PEG) modification of tetrameric rHu BChE prolonged its circulatory stability and bioavailability in vivo. The goal of this study was to determine if modification with PEG could prolong the circulatory stability and eliminate the immunogenicity of monomeric rHu BChE. Monomeric rHu BChE was expressed in human 293A cells using a cDNA lacking the 45 amino acid tetramerization domain from the carboxyl terminus and the adenovirus expression system. The catalytic and inhibitory properties of purified monomeric rHu BChE were similar to those for native Hu BChE and were not affected by PEG modification. As expected, monomeric rHu BChE rapidly cleared from the circulation of mice (MRT=3.2+/-0.3h) while monomeric PEG-rHu BChE demonstrated significant improvement in its bioavailability and circulatory stability in blood (MRT=31.4+/-5.4h). However, a second injection of monomeric PEG-rHu BChE, 28 days after the first, displayed a much shorter MRT=11.6+/-0.4h, and circulating anti-monomeric PEG-rHu BChE antibodies were detected in the blood of mice. These results suggest that PEG modification increased the circulatory stability of monomeric rHu BChE but failed to reduce or eliminate its immunogenicity.
ESTHER : Chilukuri_2008_Chem.Biol.Interact_175_255
PubMedSearch : Chilukuri_2008_Chem.Biol.Interact_175_255
PubMedID: 18603232

Title : Comparison of methods used for the determination of cholinesterase activity in whole blood - Naik_2008_Chem.Biol.Interact_175_298
Author(s) : Naik RS , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 175 :298 , 2008
Abstract : Cholinesterases (ChEs) are classified as either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) based on their substrate and inhibitor specificity. Organophosphate and carbamate compounds commonly represented by herbicides, pesticides, and nerve gases irreversibly inhibit ChEs. Therefore, exposure to organophosphates and carbamates is normally assessed by measuring ChE activity in blood. There are two approaches for measuring AChE and BChE activity present in whole blood: (1) separating blood into erythrocytes, which contain only AChE, and plasma which contains only BChE, to measure their activity individually, or (2) use a BChE-specific inhibitor to measure the activity of AChE in whole blood. A number of studies have reported the use of different inhibitors for the simultaneous measurement of AChE and BChE activities. However, the inhibitors used for completely inhibiting BChE activity also inhibited AChE activity leading to errors in reported values. The goal of this study was to find the most accurate and simple method for the simultaneous determination of AChE and BChE activity in animal whole blood. Solutions containing human AChE and BChE in various proportions were prepared and AChE and BChE activities were measured using three reported methods. Results demonstrate that ethopropazine and (-) huperzine A appear to be the most specific ChE inhibitors. Preliminary results with human and animal whole blood suggest that 20 microM ethopropazine and 500 nM (-) huperzine A can be used for measuring AChE and BChE activities across species.
ESTHER : Naik_2008_Chem.Biol.Interact_175_298
PubMedSearch : Naik_2008_Chem.Biol.Interact_175_298
PubMedID: 18555980

Title : Resveratrol induces catalytic bioscavenger paraoxonase 1 expression and protects against chemical warfare nerve agent toxicity in human cell lines - Curtin_2008_J.Cell.Biochem_103_1524
Author(s) : Curtin BF , Seetharam KI , Dhoieam P , Gordon RK , Doctor BP , Nambiar MP
Ref : Journal of Cellular Biochemistry , 103 :1524 , 2008
Abstract : Current advances in enzyme bioscavenger prophylactic therapy against chemical warfare nerve agent (CWNA) exposure are moving towards the identification of catalytic bioscavengers that can degrade large doses of organophosphate (OP) nerve agents without self destruction. This is a preferred method compared to therapy with the purified stoichiometric bioscavenger, butyrylcholinesterase, which binds OPs 1:1 and would thus require larger doses for treatment. Paraoxonase-1 (PON-1) is one such catalytic bioscavenger that has been shown to hydrolyze OP insecticides and contribute to detoxification in animals and humans. Here we investigated the effects of a common red wine ingredient, Resveratrol (RSV), to induce the expression of PON-1 in the human hepatic cell line HC04 and evaluated the protection against CWNA simulants. Dose-response curves showed that a concentration of 20 microM RSV was optimal in inducing PON-1 expression in HC04 cells. RSV at 20 microM increased the extracellular PON-1 activity approximately 150% without significantly affecting the cells. Higher doses of RSV were cytotoxic to the cells. Resveratrol also induced PON-1 in the human lung cell line A549. RSV pre-treatment significantly (P = 0.05) protected the hepatic cells against exposure to 2x LD(50) of soman and sarin simulants. However, lung cells were protected against soman simulant exposure but not against sarin simulant exposure following RSV treatment. In conclusion, these studies indicate that dietary inducers, such as RSV, can up-regulate PON-1, a catalytic bioscavenger, which can then hydrolyze and protect against CWNA-induced toxicity, providing a prospective new method to protect against CWNA exposure.
ESTHER : Curtin_2008_J.Cell.Biochem_103_1524
PubMedSearch : Curtin_2008_J.Cell.Biochem_103_1524
PubMedID: 17879943

Title : Efficacy of human serum butyrylcholinesterase against sarin vapor - Saxena_2008_Chem.Biol.Interact_175_267
Author(s) : Saxena A , Sun W , Dabisch PA , Hulet SW , Hastings NB , Jakubowski EM , Mioduszewski RJ , Doctor BP
Ref : Chemico-Biological Interactions , 175 :267 , 2008
Abstract : Human serum butyrylcholinesterase (Hu BChE) is currently under advanced development as a pretreatment drug for organophosphate (OP) poisoning in humans. It was shown to protect mice, rats, guinea pigs, and monkeys against multiple LD(50) challenges of OP nerve agents by i.v. or s.c. bolus injections. Since inhalation is the most likely route of exposure to OP nerve agents on the battlefield or in public places, the aim of this study was to evaluate the efficacy of Hu BChE against whole-body inhalation exposure to sarin (GB) vapor. Male Gottingen minipigs were subjected to one of the following treatments: (1) air exposure; (2) GB vapor exposure; (3) pretreatment with 3 mg/kg of Hu BChE followed by GB vapor exposure; (4) pretreatment with 6.5 mg/kg of Hu BChE followed by GB vapor exposure; (5) pretreatment with 7.5 mg/kg of Hu BChE followed by GB vapor exposure. Hu BChE was administered by i.m. injection, 24h prior to whole-body exposure to GB vapor at a concentration of 4.1 mg/m(3) for 60 min, a dose lethal to 99% of untreated exposed pigs (LCt99). EEG, ECG, and pupil size were monitored throughout exposure, and blood drawn from a surgically implanted jugular catheter before and throughout the exposure period, was analyzed for acetylcholinesterase (AChE) and BChE activities, and the amount of GB present in plasma. All animals exposed to GB vapor alone or pretreated with 3 or 6.5 mg/kg of Hu BChE, died following exposure to GB vapor. All five animals pretreated with 7.5 mg/kg of Hu BChE survived the GB exposure. The amount of GB bound in plasma was 200-fold higher compared to that from plasma of pigs that did not receive Hu BChE, suggesting that Hu BChE was effective in scavenging GB in blood. Additionally, pretreatment with 7.5 mg/kg of Hu BChE prevented cardiac abnormalities and seizure activity observed in untreated animals and those treated with lower doses of Hu BChE.
ESTHER : Saxena_2008_Chem.Biol.Interact_175_267
PubMedSearch : Saxena_2008_Chem.Biol.Interact_175_267
PubMedID: 18597747

Title : Protection of red blood cell acetylcholinesterase by oral huperzine A against ex vivo soman exposure: next generation prophylaxis and sequestering of acetylcholinesterase over butyrylcholinesterase - Haigh_2008_Chem.Biol.Interact_175_380
Author(s) : Haigh JR , Johnston SR , Peppernay A , Mattern PJ , Garcia GE , Doctor BP , Gordon RK , Aisen PS
Ref : Chemico-Biological Interactions , 175 :380 , 2008
Abstract : As part of a phase Ib clinical trial to determine the tolerability and safety of the highly specific acetylcholinesterase (AChE) inhibitor huperzine A, twelve (12) healthy elderly individuals received an escalating dose regimen of huperzine A (100, 200, 300, and 400 microg doses, twice daily for a week at each dose), with three (3) individuals as controls receiving a placebo. Using the WRAIR whole blood cholinesterase assay, red blood cell AChE and plasma butyrylcholinesterase (BChE) were measured in unprocessed whole blood samples from the volunteers following each dose, and then for up to 48h following the final and highest (400 microg) dose to monitor the profile of inhibition and recovery of AChE. Significant inhibition of AChE was observed, ranging from 30-40% after 100 microg to >50% at 400 microg, and peaking 1.5h after the last dose. Gradual recovery of AChE activity then occurs, but even 48 h after the last dose red blood cell AChE was about 10% below control (pre-dose) values. Huperzine A levels in plasma peaked 1.5h after the final 400 microg dose (5.47+/-2.15 ng/mL). Plasma BChE was unaffected by huperzine A treatment (as expected). Aliquots of huperzine A-containing (from three individuals) and placebo blood samples were exposed ex vivo to the irreversible nerve agent soman (GD) for 10 min, followed by removal of unbound huperzine and soman from the blood by passing through a small C(18) reverse phase spin column. Eluted blood was diluted in buffer, and aliquots taken at various time intervals for AChE and BChE activity measurement to determine the time taken to achieve full return in activity of the free enzyme (dissociation from the active site of AChE by huperzine A), and thus the proportion of AChE that can be protected from soman exposure. Huperzine A-inhibited red blood cell (RBC) AChE activity was restored almost to the level that was initially inhibited by the drug. The increased doses of huperzine A used were well tolerated by these patients and in this ex vivo study sequestered more red blood cell AChE than has been previously demonstrated for pyridostigmine bromide (PB), indicating the potential improved prophylaxis against organophosphate (OP) poisoning.
ESTHER : Haigh_2008_Chem.Biol.Interact_175_380
PubMedSearch : Haigh_2008_Chem.Biol.Interact_175_380
PubMedID: 18572153

Title : Advantages of the WRAIR whole blood cholinesterase assay: comparative analysis to the micro-Ellman, Test-mate ChE, and Michel (DeltapH) assays - Haigh_2008_Chem.Biol.Interact_175_417
Author(s) : Haigh JR , Lefkowitz LJ , Capacio BR , Doctor BP , Gordon RK
Ref : Chemico-Biological Interactions , 175 :417 , 2008
Abstract : Red blood cell AChE (RBC-AChE) and plasma BChE can be used as sensitive biomarkers to detect exposure to OP nerve agents, pesticides, and cholinergic drugs. In a comparative study, RBC-AChE and serum BChE activities in whole blood was obtained from forty seven healthy male and female human volunteers, and then exposed separately ex vivo to three OP nerve agents (soman (GD), sarin (GB) and VX) to generate a wide range of inhibition of AChE and BChE activity (up to 90% of control). These samples were measured using four different ChE assays: (i) colorimetric microEllman (using DTNB at 412 nm), (ii) Test-mate ChE field kit (also based on the Ellman assay), (iii) Michel (delta pH), and (iv) the Walter Reed Army Institute of Research Whole Blood (WRAIR WB) cholinesterase assay. The WRAIR assay is a modified Ellman method using DTP at 324 nm (which minimizes hemoglobin interference and improves sensitivity), and determines AChE and BChE in a small whole blood sample simultaneously. Scatter plots of RBC-AChE activities were determined using the WRAIR ChE assay versus the micro-Ellman, Test-mate and Michel after exposure to varying concentrations of soman, sarin and VX. Regression analyses yielded mostly linear relationships with high correlations (r2 = 0.83-0.93) for RBC-AChE values in the WRAIR assay compared to the alternate methods. For the plasma BChE measurements, individual human values were significantly more variable (as expected), resulting in lower correlations using WRAIR ChE versus the alternate assays (r2 values 0.5 - 0.6). To circumvent the limitations of simple correlation analysis, Bland and Altman analysis for comparing two independent measurement techniques was performed. For example, a Bland and Altman plot of the ratio of the WRAIR whole blood AChE and Michel AChE (plotted on the y-axis) vs. the average of the two methods (x-axis) shows that the majority of the individual AChE values are within +/- 1.96 S.D. of the mean difference, indicating that the two methods may be used interchangeably with a high degree of confidence. The WRAIR ChE assay can be thus be used as a reliable inter-conversion assay when comparing results from laboratory-based (Michel) and field-based (Test-mate ChE kit), which use different methodology and report in different units of AChE activity.
ESTHER : Haigh_2008_Chem.Biol.Interact_175_417
PubMedSearch : Haigh_2008_Chem.Biol.Interact_175_417
PubMedID: 18555983

Title : Acute toxic effects of nerve agent VX on respiratory dynamics and functions following microinsillation inhalation exposure in guinea pigs - Rezk_2007_Inhal.Toxicol_19_291
Author(s) : Rezk PE , Graham JR , Moran TS , Gordon RK , Sciuto AM , Doctor BP , Nambiar MP
Ref : Inhal Toxicol , 19 :291 , 2007
Abstract : Exposure to a chemical warfare nerve agent (CWNA) leads to severe respiratory distress, respiratory failure, or death if not treated. We investigated the toxic effects of nerve agent VX on the respiratory dynamics of guinea pigs following exposure to 90.4 mug/m3 of VX or saline by microinstillation inhalation technology for 10 min. Respiratory parameters were monitored by whole-body barometric plethysmography at 4, 24, and 48 h, 7 d, 18 d, and 4 wk after VX exposure. VX-exposed animals showed a significant decrease in the respiratory frequency (RF) at 24 and 48 h of recovery (p value .0329 and .0142, respectively) compared to the saline control. The tidal volume (TV) slightly increased in VX exposed animals at 24 and significantly at 48 h (p = .02) postexposure. Minute ventilation (MV) increased slightly at 4 h but was reduced at 24 h and remained unchanged at 48 h. Animals exposed to VX also showed an increase in expiratory (Te) and relaxation time (RT) at 24 and 48 h and a small reduction in inspiratory time (Ti) at 24 h. A significant increase in end expiratory pause (EEP) was observed at 48 h after VX exposure (p = .049). The pseudo lung resistance (Penh) was significantly increased at 4 h after VX exposure and remained slightly high even at 48 h. Time-course studies reveal that most of the altered respiratory dynamics returned to normal at 7 d after VX exposure except for EEP, which was high at 7 d and returned to normal at 18 d postexposure. After 1 mo, all the monitored respiratory parameters were within normal ranges. Bronchoalveolar lavage (BAL) 1 mo after exposure showed virtually no difference in protein levels, cholinesterase levels, cell number, and cell death in the exposed and control animals. These results indicate that sublethal concentrations of VX induce changes in respiratory dynamics and functions that over time return to normal levels.
ESTHER : Rezk_2007_Inhal.Toxicol_19_291
PubMedSearch : Rezk_2007_Inhal.Toxicol_19_291
PubMedID: 17365032

Title : Abdominal bloating and irritable bowel syndrome like symptoms following microinstillation inhalation exposure to chemical warfare nerve agent VX in guinea pigs - Katos_2007_Toxicol.Ind.Health_23_231
Author(s) : Katos AM , Conti ML , Moran TS , Gordon RK , Doctor BP , Sciuto AM , Nambiar MP
Ref : Toxicol Ind Health , 23 :231 , 2007
Abstract : 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.
ESTHER : Katos_2007_Toxicol.Ind.Health_23_231
PubMedSearch : Katos_2007_Toxicol.Ind.Health_23_231
PubMedID: 18429383

Title : Bioscavenger for protection from toxicity of organophosphorus compounds - Saxena_2006_J.Mol.Neurosci_30_145
Author(s) : Saxena A , Sun W , Luo C , Myers TM , Koplovitz I , Lenz DE , Doctor BP
Ref : Journal of Molecular Neuroscience , 30 :145 , 2006
Abstract : 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).
ESTHER : Saxena_2006_J.Mol.Neurosci_30_145
PubMedSearch : Saxena_2006_J.Mol.Neurosci_30_145
PubMedID: 17192662

Title : Celastrus paniculatus seed oil and organic extracts attenuate hydrogen peroxide- and glutamate-induced injury in embryonic rat forebrain neuronal cells - Godkar_2006_Phytomedicine_13_29
Author(s) : Godkar PB , Gordon RK , Ravindran A , Doctor BP
Ref : Phytomedicine , 13 :29 , 2006
Abstract : Seed oil of Celastrus paniculatus Willd. (CP) has been reported to improve memory and the methanolic extract (ME) of CP was shown to exhibit free-radical-scavenging properties and anti-oxidant effects in human non-immortalized fibroblasts. In the present study, we have investigated the free-radical-scavenging capacity of CP seed oil (CPO) and two extracts, an ethanolic extract (EE) and a ME. CPO and EE showed dose-dependent, free-radical-scavenging capacity, but to a lesser degree than observed for ME. Oxidative stress involves the generation of free radicals and free radical scavenging is one of the mechanisms of neuroprotection. We therefore investigated the effects of CPO, ME, and EE for protection against hydrogen peroxide (H(2)O(2))- and glutamate-induced neurotoxicity in embryonic rat forebrain neuronal cells (FBNC). Pre-treatment of neuronal cells with CPO dose-dependently attenuated H(2)O(2)-induced neuronal death. Pre-treatment with ME and EE partially attenuated H(2)O(2)-induced toxicity, but these extracts were less effective than CPO for neuronal survival. In H(2)O(2)-treated cells, cellular superoxide dismutase (SOD) activity was unaffected, but catalase activity was decreased and levels of malondialdehyde (MDA) were increased. Pre-treatment with CPO, ME, or EE increased catalase activity and decreased MDA levels significantly. Also, CPO pre-treatment attenuated glutamate-induced neuronal death dose-dependently. The activity of cellular acetylcholinesterase (AChE) was not affected by CPO, ME, or EE, suggesting that the neuroprotection offered by CPO was independent of changes in AChE activity. Taken together, the data suggest that CPO, ME, and EE protected neuronal cells against H(2)O(2)-induced toxicity in part by virtue of their antioxidant properties, and their ability to induce antioxidant enzymes. However, CPO, which exhibited the least antioxidant properties, was the most effective in preventing neuronal cells against H(2)O(2)- and glutamate-induced toxicities. Thus, in addition to free-radical scavenging attributes, the mechanism of CP seed component (CP-C) neuroprotection must be elucidated.
ESTHER : Godkar_2006_Phytomedicine_13_29
PubMedSearch : Godkar_2006_Phytomedicine_13_29
PubMedID: 16360930

Title : Polyethylene glycosylation prolongs the circulatory stability of recombinant human butyrylcholinesterase - Chilukuri_2005_Chem.Biol.Interact_157-158_115
Author(s) : Chilukuri N , Parikh K , Sun W , Naik R , Tipparaju P , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 157-158 :115 , 2005
Abstract : Previous studies in rodents and non-human primates have demonstrated that pretreatment of animals with cholinesterases could provide significant protection against organophosphate (OP) nerve agent toxicity. Gene delivery/therapy is emerging as an approach to achieve high-level expression of proteins in vivo that are very similar to their native counterparts. Recently, adenoviral (Ad) vectors have proven to be excellent vehicles for delivering genes to cells in vitro and in vivo. In this study, we explored the use of the newly designed AdenoVATOR system for the expression of recombinant human butyrylcholinesterase (rHu BChE) in human embryonic kidney 293A (HEK-293A) cells. In these cells, rHu BChE was expressed as mostly tetrameric form by the simultaneous expression of proline-rich attachment domain. By optimizing the culture conditions, 1.5-2.0 U/ml of rHu BChE could be expressed in HEK-293A cells. Recombinant Hu BChE was purified to homogeneity by ammonium sulfate fractionation followed by affinity column chromatography using procainamide Sepharose and cobalt Sepharose gels. The enzymatic and physico-chemical properties of purified rHu BChE were similar to those of native serum-derived Hu BChE. To determine the suitability of this preparation for use as an antidote against highly toxic nerve agents, its pharmacokinetics were evaluated in mice. Recombinant Hu BChE exhibited a mean residence time of 18.3 h which was 2.5-fold shorter than that observed for native Hu BChE in mice. However, rHu BChE chemically modified with polyethyleneglycol (PEG) displayed a mean residence time of 36.2 h suggesting that PEG-modification can prolong the circulatory stability of rHu BChE. The efficacy of Ad-Hu BChE to induce the production of therapeutic levels of bioscavenger in vivo is under evaluation.
ESTHER : Chilukuri_2005_Chem.Biol.Interact_157-158_115
PubMedSearch : Chilukuri_2005_Chem.Biol.Interact_157-158_115
PubMedID: 16253215

Title : Safety and pharmacokinetics of human serum butyrylcholinesterase in guinea pigs - Sun_2005_Chem.Biol.Interact_157-158_428
Author(s) : Sun W , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 157-158 :428 , 2005
Abstract : Human serum butyrylcholinesterase (Hu BChE) has been demonstrated to be a highly effective detoxifying enzyme for counteracting the acute toxicity of organophosphorus (OP) nerve agents. In order to initiate an investigational new drug (IND) application for human use, the safety and pharmacokinetic properties of the enzyme were assessed in guinea pigs. Sixty milligrams per kilogram of Hu BChE was administered to guinea pigs by either i.p. or i.m. injection. Blood was drawn at various time points for up to 2 weeks following enzyme injection for the measurement of blood BChE activity. Hu BChE displayed a mean residence time of 110 h, regardless of the route of administration and the enzyme activity remained almost 10-fold above baseline level even after 2 weeks post enzyme injection. Fourteen days post Hu BChE administration, all animals were subjected to 20 panel serum chemistry, hematology, and complete gross/histopathology examination. Results showed no toxic effects as measured by general observation, serum chemistry, hematology, and gross and histological tissue changes. In conclusion, Hu BChE displays a long-lasting stability in the circulation of guinea pigs, and is devoid of any toxic side effects. These results provide convincing data for the safe and effective use of Hu BChE as a bioscavenger to protect humans against all OP nerve agents.
ESTHER : Sun_2005_Chem.Biol.Interact_157-158_428
PubMedSearch : Sun_2005_Chem.Biol.Interact_157-158_428
PubMedID: 16429577

Title : In vitro and in vivo characterization of recombinant human butyrylcholinesterase (Protexia) as a potential nerve agent bioscavenger - Cerasoli_2005_Chem.Biol.Interact_157-158_363
Author(s) : Cerasoli DM , Griffiths EM , Doctor BP , Saxena A , Fedorko JM , Greig NH , Yu QS , Huang Y , Wilgus H , Karatzas CN , Koplovitz I , Lenz DE
Ref : Chemico-Biological Interactions , 157-158 :363 , 2005
Abstract : Previous studies in rodents and nonhuman primates have demonstrated that pretreatment with cholinesterases can provide significant protection against behavioral and lethal effects of nerve agent intoxication. Human butyrylcholinesterase (HuBuChE) purified from plasma has been shown to protect against up to 5 x LD50s of nerve agents in guinea pigs and non-human primates, and is currently being explored as a bioscavenger pretreatment for human use. A recombinant form of HuBuChE has been expressed in the milk of transgenic goats as a product called Protexia. Protexia was supplied by Nexia Biotechnologies (Que., Canada) as a purified solution with a specific activity of 600 U/mg. Initial in vitro studies using radiolabeled 3H-soman or 3H-DFP (diisopropyl fluorophosphate) demonstrated that these inhibitors specifically bind to Protexia. When Protexia was mixed with soman, sarin, tabun or VX using varying molar ratios of enzyme to nerve agent (8:1, 4:1, 1:1 and 1:4, respectively), the data indicated that 50% inhibition of enzyme activity occurs around the 1:1 molar ratio for each of the nerve agents. Protexia was further characterized for its interaction with pyridostigmine bromide and six unique carbamate inhibitors of cholinesterase. IC50 and Ki values for Protexia were determined to be very similar to those of HuBuChE purified from human plasma. These data suggest that Protexia has biochemical properties very similar to those HuBuChE when compared in vitro. Together these data the continued development of the goat milk-derived recombinant HuBuChE Protexia as a potential bioscavenger of organophosphorus nerve agents.
ESTHER : Cerasoli_2005_Chem.Biol.Interact_157-158_363
PubMedSearch : Cerasoli_2005_Chem.Biol.Interact_157-158_363
PubMedID: 16429486

Title : Effects of physostigmine and human butyrylcholinesterase on acoustic startle reflex and prepulse inhibition in C57BL\/6J mice - Clark_2005_Pharmacol.Biochem.Behav_81_497
Author(s) : Clark MG , Sun W , Myers TM , Bansal R , Doctor BP , Saxena A
Ref : Pharmacol Biochem Behav , 81 :497 , 2005
Abstract : The use of exogenously administered cholinesterases as bioscavengers of highly toxic organophosphorus nerve agents is a viable prophylactic against this threat. To use this strategy, cholinesterases must provide protection without disrupting behavior when administered alone. To assess behavioral safety, the acoustic startle reflex and prepulse inhibition (PPI) of C57BL/6J mice were investigated following administration of human plasma-derived butyrylcholinesterase (HuBChE). Two hours before testing, four groups of mice (n=10 per group) were pretreated with saline or HuBChE (2000 U, ip). Fifteen minutes before testing, subjects received either saline or the carbamate physostigmine (0.4 mg/kg, sc). Mice exposed to physostigmine exhibited a significant attenuation of the startle reflex, an increased time to peak startle amplitude, and significantly increased PPI. This effect was partially mitigated in mice pretreated with HuBChE. HuBChE alone did not change startle behavior or PPI significantly compared to saline controls. The circulatory time-course of butyrylcholinesterase was assessed in a separate group of mice and revealed levels approximately 600 times the physiological norm 2-4 h post administration. Thus, HuBChE does not appear to significantly alter startle or PPI behavior at a dose 30-fold higher than that estimated to be necessary for protection against 2LD50 of soman in humans.
ESTHER : Clark_2005_Pharmacol.Biochem.Behav_81_497
PubMedSearch : Clark_2005_Pharmacol.Biochem.Behav_81_497
PubMedID: 15913750

Title : Transcriptional induction of cholinesterase expression and protection against chemical warfare nerve agents - Nambiar_2005_Chem.Biol.Interact_157-158_409
Author(s) : Nambiar MP , Curtin BF , Pal N , Compton JR , Doctor BP , Gordon RK
Ref : Chemico-Biological Interactions , 157-158 :409 , 2005
Abstract : We investigated whether transcriptional inducers could enhance the expression of acetylcholinesterase (AChE) in cell lines to achieve protection against organophosphate (OP) poisoning. Trichostatin A (TSA), an inhibitor of histone deacetylase that de-condenses chromatin and increases the binding of transcription factors and mRNA synthesis, induced three- to four-fold extracellular and 8-10-fold intracellular AChE expression at the optimal dose of 165-333 nM in Neuro 2A cells. Pre-treatment with TSA protected against OP exposure. Thus, transcriptional inducers, such as TSA, up-regulate AChE, which then can scavenge the OP and protect the cells from OP-induced toxicity, and are potential novel ways to treat chemical warfare nerve agent (CWNA) exposure.
ESTHER : Nambiar_2005_Chem.Biol.Interact_157-158_409
PubMedSearch : Nambiar_2005_Chem.Biol.Interact_157-158_409
PubMedID: 16429504

Title : Histone acetylase inhibitor trichostatin A induces acetylcholinesterase expression and protects against organophosphate exposure - Curtin_2005_J.Cell.Biochem_96_839
Author(s) : Curtin BF , Tetz LM , Compton JR , Doctor BP , Gordon RK , Nambiar MP
Ref : Journal of Cellular Biochemistry , 96 :839 , 2005
Abstract : The biological effects of organophosphorous (OP) chemical warfare nerve agents (CWNAs) are exerted by inhibition of acetylcholinesterase (AChE), which prevents the hydrolysis of the neurotransmitter acetylcholine, leading to hypercholinergy, seizures/status epilepticus, respiratory/cardiovascular failure, and potentially death. Current investigations show that bioscavenger therapy using purified fetal bovine AChE in rodents and non-human primates and the more recently tested human butyrylcholinesterase, is a promising treatment for protection against multiple LD(50) CWNA exposures. Potential impediments, due to the complex structure of the enzyme, purification effort, resources, and cost have necessitated alternative approaches. Therefore, we investigated the effects of transcriptional inducers to enhance the expression of AChE to achieve sufficient protection against OP poisoning. Trichostatin A (TSA), an inhibitor of histone deacetylase that de-condenses the chromatin, thereby increasing the binding of transcription factors and mRNA synthesis, was evaluated for induction of AChE expression in various neuronal cell lines. Dose-response curves showed that a concentration of 333 nM TSA was optimal in inducing AChE expression. In Neuro-2A cells, TSA at 333 nM increased the extracellular AChE activity approximately 3-4 fold and intracellular enzyme activity 10-fold. Correlating with the AChE induction, TSA pre-treatment significantly protected the cells against exposure to the organophosphate diisopropylfluorophosphate, a surrogate for the chemical warfare agents soman and sarin. These studies indicate that transcriptional inducers such as TSA up-regulate AChE, which then can bioscavenge any organophosphates present, thereby protecting the cells from OP-induced cytotoxicity. In conclusion, transcriptional inducers are prospective new methods to protect against CWNA exposure.
ESTHER : Curtin_2005_J.Cell.Biochem_96_839
PubMedSearch : Curtin_2005_J.Cell.Biochem_96_839
PubMedID: 16149071

Title : Protection against soman or VX poisoning by human butyrylcholinesterase in guinea pigs and cynomolgus monkeys - Lenz_2005_Chem.Biol.Interact_157-158_205
Author(s) : Lenz DE , Maxwell DM , Koplovitz I , Clark CR , Capacio BR , Cerasoli DM , Federko JM , Luo C , Saxena A , Doctor BP , Olson C
Ref : Chemico-Biological Interactions , 157-158 :205 , 2005
Abstract : Human butyrylcholinesterase (HuBuChE), purified from outdated human plasma, is being evaluated for efficacy against nerve agents in guinea pigs and cynomolgus monkeys. Previous studies in rodents and nonhuman primates demonstrated that pretreatment of animals with enzymes that can scavenge nerve agents could provide significant protection against behavioral and lethal effects of nerve agent intoxication. In preparation for evaluation of efficacy of HuBuChE prior to initiating an investigational new drug (IND) application, the pharmacokinetics of HuBuChE were evaluated in guinea pigs and in cynomolgus monkeys. HuBuChE was injected intramuscularly (i.m.) at two doses, and blood samples were taken to follow the time-course of HuBuChE in blood for up to 168 h after administration. In guinea pigs, the two doses of HuBuChE, 19.9 and 32.5 mg/kg, produced similar times of maximal blood concentration (T(max) of 26.0 and 26.8 h, respectively) and similar elimination half-times (t(1/2) of 64.6 and 75.5 h, respectively). Enzyme levels were still 10-fold over baseline at 72 h. Based on these data, guinea pigs were administered 150 mg/kg of enzyme i.m. and challenged at T(max). Soman or VX doses were approximately 1.5, 2.0 and 2.0 x LD50 administered subcutaneously (s.c.) in sequence at 90-120 min apart. None of the animals displayed signs of organophosphorus (OP) anticholinesterase intoxication at any of the challenge levels, and all survived for the 14-day duration of the experiment. Similar experiments were carried out with cynomolgus monkeys to determine the pharmacokinetics of HuBuChE and its efficacy against soman. The complete survival of nearly all animals tested to date, coupled with the maximal blood concentration and half-life elimination profile obtained for HuBuChE after i.m. injection, provides strong support for the continued development of HuBuChE as a product to protect against nerve agents.
ESTHER : Lenz_2005_Chem.Biol.Interact_157-158_205
PubMedSearch : Lenz_2005_Chem.Biol.Interact_157-158_205
PubMedID: 16289064

Title : An ex vivo approach for the evaluation of reversible inhibitors as potential pretreatments against organophosphate toxicity - Tonduli_2005_Chem.Biol.Interact_157-158_426
Author(s) : Tonduli LS , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 157-158 :426 , 2005
Abstract : Several studies demonstrated that pretreatment with reversible acetylcholinesterase (AChE) inhibitor, such as (pyridostigmine) PYR, improved the survival of animals intoxicated by organophosphate nerve agents (OP). These compounds temporarily inhibited a fraction of the enzyme and protected it from inactivation by nerve agents. An important criterion for effective pretreatment is that it must ensure the recovery of the protected fraction of the enzyme. We thus designed a simple ex vivo method to investigate the recovery of AChE activity, that was protected by PYR, prior to irreversible inhibition by an OP, using a modified Ellman assay. Results show that our approach is suitable for routine use and can successfully predict the potential use of various AChE inhibitors as pretreatment drugs against OP nerve agent intoxication.
ESTHER : Tonduli_2005_Chem.Biol.Interact_157-158_426
PubMedSearch : Tonduli_2005_Chem.Biol.Interact_157-158_426
PubMedID: 16429574

Title : Inhibition of guinea pig hemi-diaphragm acetylcholinesterase activity by pyridostigmine bromide and protection against soman toxicity -
Author(s) : Haigh JR , Johnston SR , Peters BM , Doctor BP , Gordon RK , Adler M , Gall KJ , Deshpande SS
Ref : Chemico-Biological Interactions , 157-158 :381 , 2005
PubMedID: 16429510

Title : Species-related differences in the oxime-induced reactivation of organophosphate-inhibited acetylcholinesterases -
Author(s) : Luo C , Dawson M , Chambers C , Chilukuri N , Radic Z , Taylor P , Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 157-158 :393 , 2005
PubMedID: 16429528

Title : Oral administration of pyridostigmine bromide and huperzine A protects human whole blood cholinesterases from ex vivo exposure to soman - Gordon_2005_Chem.Biol.Interact_157-158_239
Author(s) : Gordon RK , Haigh JR , Garcia GE , Feaster SR , Riel MA , Lenz DE , Aisen PS , Doctor BP
Ref : Chemico-Biological Interactions , 157-158 :239 , 2005
Abstract : Cholinesterases (ChEs) are classified as acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) according to their substrate specificity and sensitivity to selected inhibitors. The activities of AChE in red blood cells (RBC-AChE) and BChE in serum can be used as potential biomarkers of suppressed and/or heightened activity in the central and peripheral nervous systems. Exposure to organophosphate (OP) chemical warfare agents (CWAs), pesticides, anesthetics, and a variety of drugs such as cocaine, as well as some neurodegenerative and liver disease states, selectively reduces AChE or BChE activity. In humans, the toxicity of pesticides is well documented. Therefore, blood cholinesterase activity can be exploited as a tool for confirming exposure to these agents and possible treatments. Current assays for measurement of RBC-AChE and serum BChE require several labor-intensive processing steps, suffer from wide statistical variation, and there is no inter-laboratory conversion between methods. These methods, which determine only the serum BChE or RBC-AChE but not both, include the Ellman, radiometric, and deltapH (modified Michel) methods. In contrast, the Walter Reed Army Institute of Research Whole Blood (WRAIR WB, US Patent #6,746,850) cholinesterase assay rapidly determines the activity of both AChE and BChE in unprocessed (uncentrifuged) whole blood, uses a minimally invasive blood sampling technique (e.g., blood from a finger prick), and is semi-automated for high-throughput using the Biomek 2000 robotic system. To date, the WRAIR whole blood assay was used to measure AChE and BChE activities in human blood from volunteers in FDA clinical trials. In the first FDA study, 24 human subjects were given either 30 mg PB orally (n = 19) or placebo (n = 5). Blood samples were obtained pre-dosing and 2.5, 5, 8, and 24 h post-dosing. The samples were analyzed for AChE and BChE activity using the WRAIR WB robotic system, and for PB concentration by HPLC. We found that maximal inhibition of AChE (26.2%) and concentration of PB (17.1 ng/mL) occurred at 2.5 h post-PB dosing. AChE activity returned to almost 100% of pre-dose values by 6 h. A dose-dependent linear correlation was found between the amount of PB measured in the blood and the inhibition of AChE. Following soman (GD) exposure, recovered AChE activity was similar to levels that were reversibly protected by the PB administration. Therefore, the WRAIR ChE WB data clearly supports the conclusion that PB is an effective pre-treatment drug for nerve agent exposure (GD). In the second FDA human study for the treatment of Alzheimer's disease, the WRAIR ChE WB assay was used to determine the RBC-AChE and serum BChE profile of healthy elderly volunteers receiving Huperzine A. Huperzine A is a plant-derived reversible and selective AChE inhibitor compared to BChE, and is a more potent inhibitor of AChE than PB. Huperzine A is available as a nutraceutical, a natural supplement reported to improve memory, and has a variety of neuroprotective effects. Individuals received an increasing dose regimen of huperzine A (final dose 200 microg after 4 weeks), which produced more than 50% inhibition of RBC-AChE. Huperzine A was well tolerated by these patients at doses that sequestered more RBC-AChE than PB, and thus warrants further study as a prophylaxis for OP poisoning in addition to Alzheimer's therapy. Due to the documented use of OPs by terrorists and in warfare around the globe, Federal, State, and local authorities need a reliable, fast, inexpensive, and standard method for confirming such an assault in order to initiate appropriate containment, decontamination, and treatment measures. This assay is ideal for prescreening military personnel for atypical ChE activities that would preclude their deployment to areas of potential CWA exposure. The WRAIR WB ChE assay will fulfill the requirement for rapid and reliable monitoring of such exposure in military and civilian populations.
ESTHER : Gordon_2005_Chem.Biol.Interact_157-158_239
PubMedSearch : Gordon_2005_Chem.Biol.Interact_157-158_239
PubMedID: 16256090

Title : Effects of soman inhibition and of structural differences on cholinesterase molecular dynamics: a neutron scattering study - Gabel_2005_Biophys.J_89_3303
Author(s) : Gabel F , Weik M , Masson P , Renault F , Fournier D , Brochier L , Doctor BP , Saxena A , Silman I , Zaccai G
Ref : Biophysical Journal , 89 :3303 , 2005
Abstract : Incoherent elastic neutron scattering experiments on members of the cholinesterase family were carried out to investigate how molecular dynamics is affected by covalent inhibitor binding and by differences in primary and quaternary structure. Tetrameric native and soman-inhibited human butyrylcholinesterase (HuBChE) as well as native dimeric Drosophila melanogaster acetylcholinesterase (DmAChE) hydrated protein powders were examined. Atomic mean-square displacements (MSDs) were found to be identical for native HuBChE and for DmAChE in the whole temperature range examined, leading to the conclusion that differences in activity and substrate specificity are not reflected by a global modification of subnanosecond molecular dynamics. MSDs of native and soman-inhibited HuBChE were identical below the thermal denaturation temperature of the native enzyme, indicating a common mean free-energy surface. Denaturation of the native enzyme is reflected by a relative increase of MSDs consistent with entropic stabilization of the unfolded state. The results suggest that the stabilization of HuBChE phosphorylated by soman is due to an increase in free energy of the unfolded state due to a decrease in entropy.
ESTHER : Gabel_2005_Biophys.J_89_3303
PubMedSearch : Gabel_2005_Biophys.J_89_3303
PubMedID: 16100272

Title : Human serum butyrylcholinesterase: in vitro and in vivo stability, pharmacokinetics, and safety in mice - Saxena_2005_Chem.Biol.Interact_157-158_199
Author(s) : Saxena A , Sun W , Luo C , Doctor BP
Ref : Chemico-Biological Interactions , 157-158 :199 , 2005
Abstract : The use of exogenously administered cholinesterases (ChEs) as bioscavengers of highly toxic organophosphate (OP) nerve agents is now sufficiently well documented to make them a highly viable prophylactic treatment against this potential threat. Of the ChEs evaluated so far, human serum butyrylcholinesterase (HuBChE) is most suitable for human use. A dose of 200 mg (3 mg/kg) of HuBChE is envisioned as a prophylactic treatment in humans that can protect from an exposure of up to 2 x LD50 of soman. In addition to its use as a prophylactic for a variety of wartime scenarios, including covert actions, it also has potential use for first responders (civilians) reacting to terrorist nerve gas release. We recently, developed a procedure for the large-scale purification of HuBChE, which yielded approximately 6 g of highly purified enzyme from 120 kg of Cohn fraction IV-4. The enzyme had a specific activity of 700-750 U/mg and migrated as a single band on SDS-PAGE. To provide data for initiating an investigational new drug (IND) application for the use of this enzyme as a bioscavenger in humans, we established its pharmacokinetic properties, examined its safety in mice, and evaluated its shelf life at various temperatures. In mice administered various doses up to 90 mg/kg, enzyme activity reached peak levels in circulation at 10 and 24 h following i.p. and i.m. injections, respectively. The enzyme displayed a mean residence time (MRT) of 40-50 h, regardless of the route of administration or dose of injected enzyme. Mice were euthanized 2 weeks following enzyme administration and tissues were examined grossly or microscopically for possible toxic effects. Results suggest that HuBChE does not exhibit any toxicity in mice as measured by general observation, serum chemistry, hematology, gross or histologic tissue changes. The shelf life of this enzyme stored at 4, 25, 37, and 45 degrees C was determined in lyophilized form. The enzyme was found to be stable when stored in lyophilized form at -20, 4, 25, or 37 degrees C to date (2 years), as measured by specific activity and SDS polyacrylamide gel electrophoresis. The effect of storage on circulatory stability was determined by measuring MRT in mice; there was no change in the MRT of lyophilized enzyme stored at -20 degrees C to date (2 years). These results provide convincing data that HuBChE is a safe bioscavenger that can provide protection against all OP nerve agents. Efforts are now underway to prepare the required documentation for submission of an IND application to the United States Food and Drug Administration (USFDA).
ESTHER : Saxena_2005_Chem.Biol.Interact_157-158_199
PubMedSearch : Saxena_2005_Chem.Biol.Interact_157-158_199
PubMedID: 16263104

Title : Bioscavengers for the protection of humans against organophosphate toxicity - Doctor_2005_Chem.Biol.Interact_157-158_167
Author(s) : Doctor BP , Saxena A
Ref : Chemico-Biological Interactions , 157-158 :167 , 2005
Abstract : Current antidotes for organophosphorus compounds (OP) poisoning consist of a combination of pretreatment with carbamates (pyridostigmine bromide), to protect acetylcholinesterase (AChE) from irreversible inhibition by OP compounds, and post-exposure therapy with anti-cholinergic drugs (atropine sulfate) to counteract the effects of excess acetylcholine and oximes (e.g., 2-PAM chloride) to reactivate OP-inhibited AChE. These antidotes are effective in preventing lethality from OP poisoning, but they do not prevent post-exposure incapacitation, convulsions, seizures, performance decrements, or in many cases permanent brain damage. These symptoms are commonly observed in experimental animals and are likely to occur in humans. The problems intrinsic to these antidotes stimulated attempts to develop a single protective drug, itself devoid of pharmacological effects, which would provide protection against the lethality of OP compounds and prevent post-exposure incapacitation. One approach is the use of enzymes such as cholinesterases (ChEs), beta-esterases in general, as single pretreatment drugs to sequester highly toxic OP anti-ChEs before they reach their physiological targets. This approach turns the irreversible nature of the OP: ChE interaction from disadvantage to an advantage; instead of focusing on OP as an anti-ChE, one can use ChE as an anti-OP. Using this approach, it was shown that administration of fetal bovine serum AChE (FBSAChE) or equine serum butyrylcholinesterase (EqBChE) or human serum BChE (HuBChE) protected the animals from multiple LD50s of a variety of highly toxic OPs without any toxic effects or performance decrements. The bioscavengers that have been explored to date for the detoxification of OPs fall into three categories: (A) those that can catalytically hydrolyze OPs and thus render them non-toxic, such as OP hydrolase and OP anhydrase; (B) those that stoichiometrically bind to OPs, that is, 1 mol of enzyme neutralizes one or 2 mol of OP inactivating both, such as ChEs and related enzymes; and (C) and those generally termed as "pseudo catalytic", e.g., a combination of ChE and an oxime pre-treatment such that the catalytic activity of OP-inhibited ChE can rapidly and continuously be restored in the presence of an oxime. Since the biochemical mechanism underlying prophylaxis by exogenous esterases such as ChEs is established and tested in several animal species, including non-human primates, this concept should allow a reliable extrapolation of results from animal experiments to human application. Having being extensively investigated by several groups, plasma derived HuBChE is judged to be the most suitable bioscavenger for its advancement for human use. The program is being developed at the present time for conducting a safety clinical trial in human volunteers. Several other candidate bioscavengers will follow; e.g., recombinant HuBChE expressed in the milk of transgenic goats, pseudo catalytic scavenger(s), e.g., a combination of ChE and oxime, and possibly PON 1 as a catalytic scavenger in the future.
ESTHER : Doctor_2005_Chem.Biol.Interact_157-158_167
PubMedSearch : Doctor_2005_Chem.Biol.Interact_157-158_167
PubMedID: 16293236

Title : Poster (81) Interaction of beta-amyloid and cholinesterases by fluorescent polarization -
Author(s) : Nigam SV , Doctor BP , Gordon RK
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :363 , 2004
PubMedID:

Title : The influence of solvent composition on global dynamics of human butyrylcholinesterase powders: a neutron-scattering study - Gabel_2004_Biophys.J_86_3152
Author(s) : Gabel F , Weik M , Doctor BP , Saxena A , Fournier D , Brochier L , Renault F , Masson P , Silman I , Zaccai G
Ref : Biophysical Journal , 86 :3152 , 2004
Abstract : A major result of incoherent elastic neutron-scattering experiments on protein powders is the strong dependence of the intramolecular dynamics on the sample environment. We performed a series of incoherent elastic neutron-scattering experiments on lyophilized human butyrylcholinesterase (HuBChE) powders under different conditions (solvent composition and hydration degree) in the temperature range from 20 to 285 K to elucidate the effect of the environment on the enzyme atomic mean-square displacements. Comparing D(2)O- with H(2)O-hydrated samples, we were able to investigate protein as well as hydration water molecular dynamics. HuBChE lyophilized from three distinct buffers showed completely different atomic mean-square displacements at temperatures above approximately 200 K: a salt-free sample and a sample containing Tris-HCl showed identical small-amplitude motions. A third sample, containing sodium phosphate, displayed highly reduced mean-square displacements at ambient temperature with respect to the other two samples. Below 200 K, all samples displayed similar mean-square displacements. We draw the conclusion that the reduction of intramolecular protein mean-square displacements on an Angstrom-nanosecond scale by the solvent depends not only on the presence of salt ions but also on their type.
ESTHER : Gabel_2004_Biophys.J_86_3152
PubMedSearch : Gabel_2004_Biophys.J_86_3152
PubMedID: 15111428

Title : Interaction of beta-amyloid and cholinesterases by fluorescent polarization -
Author(s) : Nigam SV , Doctor BP , Gordon RK
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :171 , 2004
PubMedID:

Title : Poster (15) Molecular recognition in AChE catalysis - the view from near and far. -
Author(s) : Moussavi-Harami F , Sikorski RS , Quinn DM , Feaster SR , Doctor BP
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :328 , 2004
PubMedID:

Title : Detection and identification of chemical warfare Agents polyurethane immobilized enzymes -
Author(s) : Gordon RK , Gunduz AT , Doctor BP
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :243 , 2004
PubMedID:

Title : Strategy for reactivation of organophosphate-inhibited human butyrylcholinesterase -
Author(s) : Luo C , Dawson M , Maxwell DM , Doctor BP , Saxena A
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :251 , 2004
PubMedID:

Title : Human serum butyrylcholinesterase: A future generation antidote for organophosphate chemical warfare agent toxicity . -
Author(s) : Saxena A , Luo C , Bansal R , Sun W , Clark M , Ashani Y , Ross M , Doctor BP
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :269 , 2004
PubMedID:

Title : Poster (30) Detection and identification of chemical warfare agents polyurethane immobilized enzymes -
Author(s) : Gordon RK , Gunduz AT , Doctor BP
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :336 , 2004
PubMedID:

Title : Poster (32) Bioscavengers: antidotes for organophosphate chemical warfare agent toxicity. -
Author(s) : Doctor BP , Saxena A , Ashani Y , Ross M
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :337 , 2004
PubMedID:

Title : Poster (33) Strategy for the reactivation of organophosphate-inhibited human butyrylcholinesterase -
Author(s) : Luo C , McKissic D , Doctor BP , Saxena A
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :338 , 2004
PubMedID:

Title : Poster (36) Human serum butyrylcholinesterase: a future generation antidote for organophosphate chemical warfare agent toxicity -
Author(s) : Saxena A , Luo C , Bansal R , Sun W , Clark M , Ashani Y , Ross M , Doctor BP
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :339 , 2004
PubMedID:

Title : Protective effect of equine butyrylcholinesterase in inhalation intoxication of rats with sarin: determination of blood and brain cholinesterase activities - Sevelova_2004_Inhal.Toxicol_16_531
Author(s) : Sevelova L , Bajgar J , Saxena A , Doctor BP
Ref : Inhal Toxicol , 16 :531 , 2004
Abstract : The effect of pretreatment with equine butyrylcholinesterase (EqBuChE) on cholinesterase inhibition in the blood and brain of rats following inhalation intoxication with low concentrations (1.25 microg/L for 60 min) of sarin were studied. Animals pretreated with different doses of equine butyrylcholinesterase showed significant increases in plasma butyrylcholinesterase activity. However, erythrocyte acetylcholinesterase activity was unchanged. The decrease in acetylcholinesterase and butyrylcholinesterase activity after inhalation intoxication was dependent on the dose of equine butyrylcholinesterase used for pretreatment and was always greater for erythrocyte acetylcholinesterase. Acetylcholinesterase activity in different brain regions was unchanged following pretreatment with equine butyrylcholinesterase. After inhalation exposure to sarin, acetylcholinesterase activity was diminished markedly in the pontomedullar area (51.5% of normal activity) and frontal cortex (72.0% of normal activity), and slightly in basal ganglia (91.4% of normal activity). Plasma levels of sarin were determined using fluoride-induced reactivation of inhibited enzyme. As expected, the amounts of sarin in plasma were almost identical in rats pretreated with EqBuChE as well as in untreated rats. In pretreated animals, the plasma amount of sarin did not depend on the dose of equine butyrylcholinesterase used for pretreatment. Our results demonstrate that equine butyrylcholinesterase pretreatment can be considered as an effective prophylaxis against nerve agents (at least with sarin) and seems to be an alternative or superior to prophylaxis provided by reversible cholinesterase inhibitors.
ESTHER : Sevelova_2004_Inhal.Toxicol_16_531
PubMedSearch : Sevelova_2004_Inhal.Toxicol_16_531
PubMedID: 15204744

Title : Polyurethane immobilized enzymes. -
Author(s) : Gordon RK , Gunduz AT , Doctor BP , Cronin T
Ref : Cholinergic Mechanisms, CRC Press :265 , 2004
PubMedID:

Title : Two possible orientations of the HI-6 molecule in the reactivation of organophosphate-inhibited acetylcholinesterase. -
Author(s) : Luo C , Leader H , Radic Z , Maxwell DM , Taylor P , Doctor BP , Saxena A
Ref : Cholinergic Mechanisms, CRC Press :627 , 2004
PubMedID:

Title : Natural monomeric form of fetal bovine serum acetylcholinesterase lacks the C-terminal tetramerization domain - Saxena_2003_Biochemistry_42_15292
Author(s) : Saxena A , Hur RS , Luo C , Doctor BP
Ref : Biochemistry , 42 :15292 , 2003
Abstract : Acetylcholinesterase isolated from fetal bovine serum (FBS AChE) was previously characterized as a globular tetrameric form. Analysis of purified preparations of FBS AChE by gel permeation chromatography revealed the presence of a stable, catalytically active, monomeric form of this enzyme. The two forms could be distinguished from each other based on their molecular weight, hydrodynamic properties, kinetic properties, thermal stability, and the type of glycans they carry. No differences between the two forms were observed for the binding of classical inhibitors such as edrophonium and propidium or inhibitors that are current or potential drugs for the treatment of Alzheimer's disease such as (-) huperzine A and E2020; tacrine inhibited the monomeric form 2-3-fold more potently than the tetrameric form. Sequencing of peptides obtained from an in-gel tryptic digest of the monomer and tetramer by tandem mass spectrometry indicated that the tetramer consists of 583 amino acid residues corresponding to the mature form of the enzyme, whereas the monomer consists of 543-547 amino acid residues. The subunit molecular weight of the protein component of the monomer (major species) was determined to be 59 414 Da and that of the tetramer as 64 239 Da. The N-terminal of the monomer and the tetramer was Glu, suggesting that the monomer is not a result of truncation at the N-terminal. The only differences detected were at the C-terminus. The tetramer yielded the expected C-terminus, CSDL, whereas the C-terminus of the monomer yielded a mixture of peptides, of which LLSATDTLD was the most abundant. These results suggest that monomeric FBS AChE is trimmed at the C-terminus, and the results are consistent with the involvement of C-terminal amino acids in the assembly of monomers into tetramers.
ESTHER : Saxena_2003_Biochemistry_42_15292
PubMedSearch : Saxena_2003_Biochemistry_42_15292
PubMedID: 14690439

Title : Inhibition of cholinesterases with cationic phosphonyl oximes highlights distinctive properties of the charged pyridine groups of quaternary oxime reactivators - Ashani_2003_Biochem.Pharmacol_66_191
Author(s) : Ashani Y , Bhattacharjee AK , Leader H , Saxena A , Doctor BP
Ref : Biochemical Pharmacology , 66 :191 , 2003
Abstract : Oxime-induced reactivation of phosphonylated cholinesterases (ChEs) produces charged phosphonyl pyridine oxime intermediates (POXs) that are most potent organophosphate (OP) inhibitors of ChEs. To understand the role of cationic pyridine oxime leaving groups in the enhanced anti-ChE activity of POXs, the bimolecular rate constants for the inhibition (k(i)) of acetylcholinesterases (AChE) and butyrylcholinesterases (BChE), and the rate of decomposition (k(d)) of authentic O-alkyl methylphosphonyl pyridine oximes (AlkMeP-POXs) and N,N-dimethylamidophosphoryl pyridine oximes (EDMP-POXs), were studied. Stability ranking order in aqueous solutions correlated well with the electronic features and optimized geometries that were obtained by ab initio calculations at 6-31G(**) basis set level. AlkMeP-POXs of the 2-pyridine oxime series were found to be 4- to 8-fold more stable (t(1/2)=0.7 to 1.5 min) than the homologous O,O-diethylphosphoryl (DEP) oxime. Results suggest that re-inhibition of enzyme activity by POX is less likely during the reactivation of DEP-ChEs (obtained by use of DEP-containing pesticides) by certain oximes, compared to nerve agent-inhibited ChEs. The greatest inhibition was observed for the O-cyclohexyl methylphosphonyl-2PAM derivative (4.0 x 10(9)M(-1)min(-1); mouse AChE) and is 10-fold higher than the k(i) of cyclosarin. Increasing the size of the O-alkyl substituent of AlkMeP-POXs had only a small to moderate effect on the k(i) of ChEs, signifying a major role for the cationic pyridine oxime leaving group in the inhibition reaction. The shape of plots of logk(i) vs. pK(a) of the leaving groups for AlkMeP-PAMs and DEP-PAMs, could be used as a diagnostic tool to highlight and rationalize the unique properties of the cationic moiety of pyridine oxime reactivators.
ESTHER : Ashani_2003_Biochem.Pharmacol_66_191
PubMedSearch : Ashani_2003_Biochem.Pharmacol_66_191
PubMedID: 12826262

Title : Two possible orientations of the HI-6 molecule in the reactivation of organophosphate-inhibited acetylcholinesterase - Luo_2003_Biochem.Pharmacol_66_387
Author(s) : Luo C , Leader H , Radic Z , Maxwell DM , Taylor P , Doctor BP , Saxena A
Ref : Biochemical Pharmacology , 66 :387 , 2003
Abstract : 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.
ESTHER : Luo_2003_Biochem.Pharmacol_66_387
PubMedSearch : Luo_2003_Biochem.Pharmacol_66_387
PubMedID: 12907237

Title : Aromatic amino-acid residues at the active and peripheral anionic sites control the binding of E2020 (Aricept) to cholinesterases - Saxena_2003_Eur.J.Biochem_270_4447
Author(s) : Saxena A , Fedorko JM , Vinayaka CR , Medhekar R , Radic Z , Taylor P , Lockridge O , Doctor BP
Ref : European Journal of Biochemistry , 270 :4447 , 2003
Abstract : E2020 (R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]methyl)piperidine hydrochloride is a piperidine-based acetylcholinesterase (AChE) inhibitor that was approved for the treatment of Alzheimer's disease in the United States. Structure-activity studies of this class of inhibitors have indicated that both the benzoyl containing functionality and the N-benzylpiperidine moiety are the key features for binding and inhibition of AChE. In the present study, the interaction of E2020 with cholinesterases (ChEs) with known sequence differences, was examined in more detail by measuring the inhibition constants with Torpedo AChE, fetal bovine serum AChE, human butyrylcholinesterase (BChE), and equine BChE. The basis for particular residues conferring selectivity was then confirmed by using site-specific mutants of the implicated residue in two template enzymes. Differences in the reactivity of E2020 toward AChE and BChE (200- to 400-fold) show that residues at the peripheral anionic site such as Asp74(72), Tyr72(70), Tyr124(121), and Trp286(279) in mammalian AChE may be important in the binding of E2020 to AChE. Site-directed mutagenesis studies using mouse AChE showed that these residues contribute to the stabilization energy for the AChE-E2020 complex. However, replacement of Ala277(Trp279) with Trp in human BChE does not affect the binding of E2020 to BChE. Molecular modeling studies suggest that E2020 interacts with the active-site and the peripheral anionic site in AChE, but in the case of BChE, as the gorge is larger, E2020 cannot simultaneously interact at both sites. The observation that the KI value for mutant AChE in which Ala replaced Trp286 is similar to that for wild-type BChE, further confirms our hypothesis.
ESTHER : Saxena_2003_Eur.J.Biochem_270_4447
PubMedSearch : Saxena_2003_Eur.J.Biochem_270_4447
PubMedID: 14622273

Title : Synthesis of more potent analogues of the acetylcholinesterase inhibitor, huperzine B - Rajendran_2002_Bioorg.Med.Chem.Lett_12_1521
Author(s) : Rajendran V , Saxena A , Doctor BP , Kozikowski AP
Ref : Bioorganic & Medicinal Chemistry Lett , 12 :1521 , 2002
Abstract : The synthesis and acetylcholinesterase inhibition activity of analogues of huperzine B are reported. These new racemic analogues show a better AChE inhibitory activity than the natural product huperzine B.
ESTHER : Rajendran_2002_Bioorg.Med.Chem.Lett_12_1521
PubMedSearch : Rajendran_2002_Bioorg.Med.Chem.Lett_12_1521
PubMedID: 12031333

Title : Pharmacokinetics and immunologic consequences of exposing macaques to purified homologous butyrylcholinesterase - Rosenberg_2002_Life.Sci_72_125
Author(s) : Rosenberg YJ , Luo C , Ashani Y , Doctor BP , Fischer R , Wolfe G , Saxena A
Ref : Life Sciences , 72 :125 , 2002
Abstract : 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.
ESTHER : Rosenberg_2002_Life.Sci_72_125
PubMedSearch : Rosenberg_2002_Life.Sci_72_125
PubMedID: 12417246

Title : The NMDA receptor ion channel: a site for binding of Huperzine A - Gordon_2001_J.Appl.Toxicol_21 Suppl 1_S47
Author(s) : Gordon RK , Nigam SV , Weitz JA , Dave JR , Doctor BP , Ved HS
Ref : J Appl Toxicol , 21 Suppl 1 :S47 , 2001
Abstract : Huperzine A (HUP-A), first isolated from the Chinese club moss Huperzia serrata, is a potent, reversible and selective inhibitor of acetylcholinesterase (AChE) over butyrylcholinesterase (BChE) (Life Sci. 54: 991-997). Because HUP-A has been shown to penetrate the blood-brain barrier, is more stable than the carbamates used as pretreatments for organophosphate poisoning (OP) and the HUP-A:AChE complex has a longer half-life than other prophylactic sequestering agents, HUP-A has been proposed as a pretreatment drug for nerve agent toxicity by protecting AChE from irreversible OP-induced phosphonylation. More recently (NeuroReport 8: 963-968), pretreatment of embryonic neuronal cultures with HUP-A reduced glutamate-induced cell death and also decreased glutamate-induced calcium mobilization. These results suggest that HUP-A might interfere with and be beneficial for excitatory amino acid overstimulation, such as seen in ischemia, where persistent elevation of internal calcium levels by activation of the N-methyl-D-aspartate (NMDA) glutamate subtype receptor is found. We have now investigated the interaction of HUP-A with glutamate receptors. Freshly frozen cortex or synaptic plasma membranes were used, providing 60-90% specific radioligand binding. Huperzine A (< or =100 microM) had no effect on the binding of [3H]glutamate (low- and high-affinity glutamate sites), [3H]MDL 105,519 (NMDA glycine regulatory site), [3H]ifenprodil (NMDA polyamine site) or [3H]CGS 19755 (NMDA antagonist). In contrast with these results, HUP-A non-competitively (Hill slope < 1) inhibited [3H]MK-801 and [3H]TCP binding (co-located NMDA ion channel PCP site) with pseudo K(i) approximately 6 microM. Furthermore, when neuronal cultures were pretreated with HUP-A for 45 min prior to NMDA exposure, HUP-A dose-dependently inhibited the NMDA-induced toxicity. Although HUP-A has been implicated to interact with cholinergic receptors, it was without effect at 100 microM on muscarinic (measured by inhibition of [3H]QNB or [3H]NMS binding) or nicotinic [3H]epibatidine binding) receptors; also, HUP-A did not perturb adenosine receptor binding [3H]PIA or [3H]NECA). Therefore, HUP-A most likely attenuates excitatory amino acid toxicity by blocking the NMDA ion channel and subsequent Ca2+ mobilization at or near the PCP and MK-801 ligand sites. Thus, on the one hand, HUP-A could be used as a pretreatment against OPs and it might also be a valuable therapeutic intervention in a variety of acute and chronic disorders by protecting against overstimulation of the excitatory amino acid pathway. By blocking NMDA ion channels without psychotomimetic side-effects, HUP-A may protect against diverse neurodegenerative states observed during ischemia or Alzheimer's disease.
ESTHER : Gordon_2001_J.Appl.Toxicol_21 Suppl 1_S47
PubMedSearch : Gordon_2001_J.Appl.Toxicol_21 Suppl 1_S47
PubMedID: 11920920

Title : Improvements in scavenger protection against organophosphorus agents by modification of cholinesterases - Maxwell_1999_Chem.Biol.Interact_119-120_419
Author(s) : Maxwell DM , Saxena A , Gordon RK , Doctor BP
Ref : Chemico-Biological Interactions , 119-120 :419 , 1999
Abstract : The ability of stoichiometric scavengers, such as ChEs, to protect against a variety of OP agents has been demonstrated in several in vivo models. To improve the detoxification of OP agents by ChEs, several approaches have been recently used to increase the stoichiometry, stability, and in vivo effectiveness of ChEs as OP scavengers. For example, the in vitro stoichiometric neutralization of sarin by AChE was increased from 1:1 to 3200:1 by the addition of the oxime HI-6, while the in vivo stoichiometry was increased to 57:1 in mice by HI-6. The aging rate of soman-inhibited mouse AChE was reduced 12-fold in a mutant AChE (E202Q) which resulted in a two-fold increase in oxime-assisted detoxification of soman. To improve the duration of scavenger protection provided by ChEs, the mean residence times of five tissue-derived and two recombinant ChEs injected i.v. in mice were compared with their oligosaccharide profiles. The mean residence times of these ChEs were found to increase with molecular weight and with the levels of oligosaccharide sialylation. The stability of AChE in non-physiological environments was improved by immobilizing it in a polyurethane foam matrix that allowed AChE to retain enzymatic activity at high temperature (75 degrees C) where soluble enzyme denatured. These developments in scavenger technology have improved the in vivo protection provided by OP scavengers and extended their applicability to provide external decontamination of chemical agents and pesticides.
ESTHER : Maxwell_1999_Chem.Biol.Interact_119-120_419
PubMedSearch : Maxwell_1999_Chem.Biol.Interact_119-120_419
PubMedID: 10421479

Title : Organophosphate skin decontamination using immobilized enzymes - Gordon_1999_Chem.Biol.Interact_119-120_463
Author(s) : Gordon RK , Feaster SR , Russell AJ , LeJeune KE , Maxwell DM , Lenz DE , Ross M , Doctor BP
Ref : Chemico-Biological Interactions , 119-120 :463 , 1999
Abstract : We previously demonstrated that a combination of cholinesterase (ChE) pre-treatment with an oxime is an effective measure against soman and sarin. We describe here a novel approach for the preparation of covalently linked ChEs which are immobilized to a polyurethane matrix. Such preparation of ChE-sponges enhances the stability and usefulness of the enzymes in non-physiological environments. The ChE-sponges, which can be molded to any form, can effectively be used to remove and decontaminate organophosphates (OPs) from surfaces, biological (skin or wounds) or otherwise (clothing or sensitive medical equipment), or the environment. The ChE-sponges retained their catalytic activity under conditions of temperature, time, and drying where the native soluble enzyme would rapidly denature, and can be reused in conjunction with oximes many times. The ChE-sponge in the presence of oxime repeatedly detoxified OPs such as DFP or MEPQ. These developments in ChE technology have extended the applicability of OP scavengers from in vivo protection, to a variety of external detoxification and decontamination schemes. In addition to treatment of OP-contaminated soldiers, the ChE-sponge could protect medical personnel from secondary contamination while attending chemical casualties, and civilians exposed to pesticides or highly toxic nerve agents such as sarin.
ESTHER : Gordon_1999_Chem.Biol.Interact_119-120_463
PubMedSearch : Gordon_1999_Chem.Biol.Interact_119-120_463
PubMedID: 10421484

Title : Phosphoryl oxime inhibition of acetylcholinesterase during oxime reactivation is prevented by edrophonium - Luo_1999_Biochemistry_38_9937
Author(s) : Luo C , Saxena A , Smith M , Garcia GE , Radic Z , Taylor P , Doctor BP
Ref : Biochemistry , 38 :9937 , 1999
Abstract : Reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) is a key objective in the treatment of OP poisoning. This study with native, wild-type, and mutant recombinant DNA-expressed AChEs, each inhibited by representative OP compounds, establishes a relationship between edrophonium acceleration of oxime-induced reactivation of OP-AChE conjugates and phosphoryl oxime inhibition of the reactivated enzyme that occurs during reactivation by pyridinium oximes LH6 and TMB4. No such recurring inhibition could be observed with HI-6 as the reactivator due to the extreme lability of the phosphoryl oximes formed by this oxime. Phosphoryl oximes formed during reactivation of the ethoxy methylphosphonyl-AChE conjugate by LH6 and TMB4 were isolated for the first time and their structures confirmed by (31)P NMR. However, phosphoryl oximes formed during the reactivation of the diethylphosphoryl-AChE conjugate were not sufficiently stable to be detected by (31)P NMR. The purified ethoxy methylphosphonyl oximes formed during the reactivation of ethoxy methylphosphonyl-AChE conjugate with LH6 and TMB4 are 10- to 22-fold more potent than MEPQ as inhibitors of AChE and stable for several hours at pH 7.2 in HEPES buffer. Reactivation of both ethoxy methylphosphonyl- and diethylphosphoryl-AChE by these two oximes was accelerated in the presence of rabbit serum paraoxonase, suggesting that organophosphorus hydrolase can hydrolyze phosphoryl oxime formed during the reactivation. Our results emphasize that certain oximes, such as LH6 and TMB4, if used in the treatment of OP pesticide poisoning may cause prolonged inhibition of AChE due to formation of phosphoryl oximes.
ESTHER : Luo_1999_Biochemistry_38_9937
PubMedSearch : Luo_1999_Biochemistry_38_9937
PubMedID: 10433700

Title : Role of edrophonium in prevention of the re-inhibition of acetylcholinesterase by phosphorylated oxime - Luo_1999_Chem.Biol.Interact_119-120_129
Author(s) : Luo C , Saxena A , Ashani Y , Leader H , Radic Z , Taylor P , Doctor BP
Ref : Chemico-Biological Interactions , 119-120 :129 , 1999
Abstract : 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 (LuH6) 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 LuH6- 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 LuH6 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 LuH6 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 LuH6 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.
ESTHER : Luo_1999_Chem.Biol.Interact_119-120_129
PubMedSearch : Luo_1999_Chem.Biol.Interact_119-120_129
PubMedID: 10421446

Title : Differences in active-site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase - Saxena_1999_Chem.Biol.Interact_119-120_61
Author(s) : Saxena A , Redman AM , Jiang X , Lockridge O , Doctor BP
Ref : Chemico-Biological Interactions , 119-120 :61 , 1999
Abstract : We examined the role of A328(F330) in the binding of various inhibitors to cholinesterases (ChEs) using human butyrylcholinesterase (BChE) mutants to determine if the conclusions drawn from studies with acetylcholinesterase (AChE) mutants could be extended to BChE. For huperzine A and edrophonium, the results obtained with AChE mutants could be directly correlated with those obtained with native ChEs and site-specific mutants of human BChE. Inhibition studies of ethopropazine with BChE mutants, where A328 was modified to either F or Y, suggested that A328 was not solely responsible for the selectivity of ethopropazine. Volume calculations for the active-site gorge showed that the poor inhibitory activity of ethopropazine towards AChE was due to the smaller dimension of the active-site gorge. The volume of the BChE active-site gorge is approximately 200 A3 larger than that of the AChE gorge, which allows the accommodation of ethopropazine in two different orientations as demonstrated by rigid-body refinement and molecular dynamics calculations. These results suggest that, although the overall scaffolding of the two enzymes may be highly similar, the dimensions and the micro-environment of the gorge play a significant role in determining the selectivity of substrate and inhibitors for ChEs.
ESTHER : Saxena_1999_Chem.Biol.Interact_119-120_61
PubMedSearch : Saxena_1999_Chem.Biol.Interact_119-120_61
PubMedID: 10421439

Title : Allosteric control of acetylcholinesterase activity by monoclonal antibodies -
Author(s) : Saxena A , Hur RS , Doctor BP
Ref : Biochemistry , 38 :15688 , 1999
PubMedID: 10569956

Title : Comparative Effects of Cholinesterase Inhibitors on Glutamate-Induced Neuronal Cell Death -
Author(s) : Ved HS , Dave JR , Nguyen T , Doctor BP
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :181 , 1998
PubMedID:

Title : Role of oligosaccharides in the pharmacokinetics of tissue-derived and genetically engineered cholinesterases - Saxena_1998_Mol.Pharmacol_53_112
Author(s) : Saxena A , Ashani Y , Raveh L , Stevenson D , Patel T , Doctor BP
Ref : Molecular Pharmacology , 53 :112 , 1998
Abstract : 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.
ESTHER : Saxena_1998_Mol.Pharmacol_53_112
PubMedSearch : Saxena_1998_Mol.Pharmacol_53_112
PubMedID: 9443938

Title : Acceleration of Oxime-Induced Reactivation of Organophosphate-Inhibited Acetylcholinesterase by Quaternary Ligands -
Author(s) : Luo C , Ashani Y , Saxena A , Leader H , Maxwell DM , Taylor P , Doctor BP
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :215 , 1998
PubMedID:

Title : A Comparison of Blood Cholinesterase Activities, Pyridostigmine Inhibition of Red Cell Acetylcholinesterase, and Butyrylcholinesterase Phenotypes in Gulf War Veterans and Normal Controls -
Author(s) : Gentry MK , Powell S , Bitsko N , Bartels CF , Bartko J , Chung R , Lockridge O , Ribas J , Roy M , Malone J , Doctor BP
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :300 , 1998
PubMedID:

Title : pH Dependence of Dealkylation in Soman-Inhibited Cholinesterases and Their Mutants -
Author(s) : Viragh C , Saxena A , Frazier DS , Kovach IM , Lockridge O , Doctor BP
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :247 , 1998
PubMedID:

Title : Synthesis and anticholinesterase activity of huperzine A analogues containing phenol and catechol replacements for the pyridone ring - Campiani_1998_Bioorg.Med.Chem.Lett_8_1413
Author(s) : Campiani G , Kozikowski AP , Wang S , Ming L , Nacci V , Saxena A , Doctor BP
Ref : Bioorganic & Medicinal Chemistry Lett , 8 :1413 , 1998
Abstract : Based upon modeling results obtained using the crystal structure of huperzine A in complex with acetylcholinesterase (AChE), two novel analogues of this potent AChE inhibitor were designed with phenol or catechol rings replacing the pyridone ring. From the modeling studies, the catechol analogue appeared capable of replacing one of the crystallographic waters bridging huperzine with Tyr 130 and Glu 199 of AChE. The synthesis of these materials by use of a palladium catalyzed bicycloannulation strategy is detailed together with the results of AChE inhibition assays.
ESTHER : Campiani_1998_Bioorg.Med.Chem.Lett_8_1413
PubMedSearch : Campiani_1998_Bioorg.Med.Chem.Lett_8_1413
PubMedID: 9871776

Title : The Role of Oligosaccharides in the Pharmacokinetics of Cholinesterases -
Author(s) : Saxena A , Ashani Y , Raveh L , Doctor BP
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :283 , 1998
PubMedID:

Title : Acceleration of oxime-induced reactivation of acetylcholinesterase-organophosphate conjugate and 31P NMR detection of phosporyl oxime from the conjugate -
Author(s) : Luo C , Ashani Y , Doctor BP
Ref : Journal de Physiologie (Paris) , 92 :461 , 1998
PubMedID:

Title : Stable Complexes Involving Acetylcholinesterase and Amyloid-beta Peptide Change the Biochemical Properties of the Enzyme and Increase the Neurotoxicity of Alzheimer's Fibrils - Alvarez_1998_J.Neurosci_18_3213
Author(s) : Alvarez A , Alarcon R , Opazo C , Campos EO , Munoz FJ , Calderon FH , Dajas F , Gentry MK , Doctor BP , de Mello FG , Inestrosa NC
Ref : Journal of Neuroscience , 18 :3213 , 1998
Abstract : Brain acetylcholinesterase (AChE) forms stable complexes with amyloid-beta peptide (Abeta) during its assembly into filaments, in agreement with its colocalization with the Abeta deposits of Alzheimer's brain. The association of the enzyme with nascent Abeta aggregates occurs as early as after 30 min of incubation. Analysis of the catalytic activity of the AChE incorporated into these complexes shows an anomalous behavior reminiscent of the AChE associated with senile plaques, which includes a resistance to low pH, high substrate concentrations, and lower sensitivity to AChE inhibitors. Furthermore, the toxicity of the AChE-amyloid complexes is higher than that of the Abeta aggregates alone. Thus, in addition to its possible role as a heterogeneous nucleator during amyloid formation, AChE, by forming such stable complexes, may increase the neurotoxicity of Abeta fibrils and thus may determine the selective neuronal loss observed in Alzheimer's brain.
ESTHER : Alvarez_1998_J.Neurosci_18_3213
PubMedSearch : Alvarez_1998_J.Neurosci_18_3213
PubMedID: 9547230

Title : Acceleration of Oxime-Induced Reactivation of Organophosphate-Inhibited Fetal Bovine Serum Acetylcholinesterase by Monoquaternary and Bisquaternary Ligands - Luo_1998_Mol.Pharmacol_53_718
Author(s) : Luo C , Ashani Y , Doctor BP
Ref : Molecular Pharmacology , 53 :718 , 1998
Abstract : Reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) by oximes is the primary reason for their effectiveness in the treatment of OP poisoning. Reactivation is reported to accelerate by quaternary ligands such as decamethonium, which is devoid of nucleophilicity. The mechanism of this enhancement is not known. To better understand the acceleration phenomenon, we examined ligand modulations of oxime-induced reactivation of methylphosphonylated AChE using 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide and fetal bovine serum AChE. Edrophonium, decamethonium, and propidium, three quaternary AChE ligands of different types, were tested as potential accelerators. Experiments were carried out with both soluble enzyme preparation and AChE conjugated to polyurethane. Kinetic measurements with oximes 2-[hydroxyiminomethyl]-1-methylpyridinium chloride, 1,1'-trimethylene bis-(4-hydroxyimino methyl)-pyridinium dibromide, and 1, 1'-[oxybis-methylene)bis[4-(hydroxyimino)methyl]pyridiniu um dichloride showed that in the presence of 50 microM edrophonium, the reactivation rate constants increased 3.3-12.0-fold; 200 microM decamethonium produced a 1.6-3.0-fold enhancement of reactivation rate constants by the same oximes. Reactivation of the inhibited enzyme by 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(4-carboxy-aminopyridinium )-d imethyl ether hydrochloride, 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(3-carboxy-aminopyridinium )-d imethyl ether hydrochloride, and 1-[[[4-(aminocarbonyl)pyridino]methoxy]methyl]-2, 4, -bis(hydroxyimino)methyl pyridinium dichloride was not affected by either ligand. Propidium slowed the reactivation of 7-(methylethoxyphosphinyloxy)-1- methylquinolinium iodide-inhibited AChE by all oximes. Results suggest that the accelerator site may reside inside the catalytic gorge rather than at its entrance and acceleration may be due to the prevention of reinhibition of the regenerated enzyme by the putative product, the phosphonylated oxime. In addition to the nucleophilic property of the oximate anion, some of the reactivators may carry an accelerating determinant, as characterized with respect to edrophonium and decamethonium. Results offer possible explanations for the superiority of 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(4-carboxy-aminopyridinium )-d imethyl ether hydrochloride over other oximes in the reactivation of specific AChE-OP conjugates.
ESTHER : Luo_1998_Mol.Pharmacol_53_718
PubMedSearch : Luo_1998_Mol.Pharmacol_53_718
PubMedID: 9547363

Title : Characterization of ChEs Immobilized on Polyurethane Foams -
Author(s) : Gordon RK , Feaster SR , Herron PC II , Lowe ER , LeJeune KE , Russell AJ , Lenz DE , Ross M , Doctor BP
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :307 , 1998
PubMedID:

Title : Inhibition of acetylcholinesterase and butyrylcholinesterase by chlorpyrifos-oxon - Amitai_1998_Biochem.Pharmacol_56_293
Author(s) : Amitai G , Moorad DR , Adani R , Doctor BP
Ref : Biochemical Pharmacology , 56 :293 , 1998
Abstract : Phosphorothionate insecticides such as parathion (O,O-diethyl O-p-nitrophenyl phosphorothioate) and chlorpyrifos (CPS; O,O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate; Dursban) are metabolically converted by oxidative desulfuration into paraoxon and chlorpyrifos-oxon (CPO). The insecticidal action of chlorpyrifos stems from inhibition of acetylcholinesterase (AChE) by CPO, resulting in severe cholinergic toxicity. Sensory peripheral neuropathy was observed in people exposed environmentally to chlorpyrifos sprayed in confined areas. We have examined the kinetics of inhibition of AChE and butyrylcholinesterase (BChE) by paraoxon and CPO. The bimolecular rate constants (ki) for inhibition by paraoxon of recombinant human (rH) AChE, recombinant mouse (rM) AChE, and fetal bovine serum (FBS) AChE were 7.0, 4.0, and 3.2 x 10(5) M(-1) min(-1). The ki values for the inhibition by CPO of rH AChE, fetal bovine serum AChE, human RBC AChE, Torpedo AChE, and recombinant mouse (rM) AChE were 9.3, 2.2, 3.8, 8.0, and 5.1 x 10(6) M(-1) min(-1), respectively. Inhibition of human serum BChE, rH BChE, and rM BChE by CPO yielded ki values of 1.65, 1.67, and 0.78 x 10(9) M(-1) min(-1), respectively. The ki values obtained for BChE from various species were 160- to 750-fold larger than those of AChE from parallel sources. Inhibition of the single-site mutant A328Y of rH BChE by CPO displayed a 21-fold lower rate than that of wild-type rH BChE (ki, 7.9 x 10(7) vs 1.67 x 10(9) M(-1) min(-1)). The double mutant of acyl pocket residues of rH AChE, F295L/F297V, was inhibited by CPO with a 150-fold larger ki than wild type (1.5 x 10(9) vs 1.0 x 10(7) M(-1) min(-1)). The increased rate obtained with the double mutant displaying characteristics of the BChE active center provides a rationale for higher efficacy of CPO scavenging by BChE, compared with AChE.
ESTHER : Amitai_1998_Biochem.Pharmacol_56_293
PubMedSearch : Amitai_1998_Biochem.Pharmacol_56_293
PubMedID: 9744565

Title : Allosteric control of acetylcholinesterase activity by monoclonal antibodies - Saxena_1998_Biochemistry_37_145
Author(s) : Saxena A , Hur RS , Doctor BP
Ref : Biochemistry , 37 :145 , 1998
Abstract : Previous studies showed that monoclonal antibodies raised against phosphorylated fetal bovine serum acetylcholinesterase appeared to modulate the catalytic activity of the enzyme by binding to a conformational epitope located at or near the region of the peripheral anionic site. The mechanism of inhibition of acetylcholinesterase by these monoclonal antibodies was further investigated by determining their effect on (i) substrate inhibition due to the binding of excess substrate to the peripheral anionic site and (ii) binding of peripheral anionic site ligands, such as propidium and fasciculin. Results of these experiments demonstrate that the accessibility of substrate to the peripheral anionic site in these complexes was restricted but not completely blocked, as none of the monoclonal antibodies eliminated the phenomenon of excess substrate inhibition. The results also show that propidium clearly slowed the inhibition of fetal bovine serum acetylcholinesterase by all six inhibitory monoclonal antibodies but to different levels. Complexation of fetal bovine serum acetylcholinesterase with monoclonal antibodies 25B1, 4E5, 6H9, and 5E8 interfered with the binding of fasciculin to the complexed enzyme, suggesting that part of their epitope overlapped with the fasciculin binding site. These monoclonal antibodies bind, in part, at the peripheral anionic site, since polyclonal anti-idiotypic antibodies generated against two monoclonal antibodies, 25B1 and 6H9, bound stoichiometric amounts of propidium. Like fasciculin, binding of these monoclonal antibodies in the vicinity of the peripheral anionic site at the rim of the active site gorge allosterically affects the orientation of W86 located at the base of the gorge, resulting in inhibition of enzyme activity.
ESTHER : Saxena_1998_Biochemistry_37_145
PubMedSearch : Saxena_1998_Biochemistry_37_145
PubMedID: 9425034

Title : Amino Acid Sequence of Horse Serum Butyrylcholinesterase -
Author(s) : Moorad DR , Luo C , Saxena A , Doctor BP , Garcia GE
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :145 , 1998
PubMedID:
Gene_locus related to this paper: horse-BCHE

Title : Comparison of Cholinesterases and Carboxylesterase as Bioscavengers for Organophosphorus Compounds -
Author(s) : Maxwell DM , Brecht KM , Saxena A , Feaster SR , Doctor BP
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :387 , 1998
PubMedID:

Title : The pH Dependence of Dealkylation in Soman-Inhibited Cholinesterases and Their Mutants: Further Evidence for a Push-Pull Mechanism - Saxena_1998_Biochemistry_37_15086
Author(s) : Saxena A , Viragh C , Frazier DS , Kovach IM , Maxwell DM , Lockridge O , Doctor BP
Ref : Biochemistry , 37 :15086 , 1998
Abstract : Bimolecular rate constants for the inactivation of recombinant (r) human (Hu) butyrylcholinesterase (BChE) with P(S)C(S)- and P(S)C(R)-2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman) are (92 +/- 7) x 10(6) M-1 min-1 and (13.7 +/- 0.8) x 10(6) M-1 min-1 at pH 7.4, mu = 0.1 M and 25 degreesC. Mutations of E197(199) to D or Q and W82(84) to A result in reductions in the rate constants for inactivation with P(S)C(S)-soman 4.3-, 11.8-, and 263-fold and with P(S)C(R)-soman by 6.5-, 47.3-, and 685-fold, respectively. The pH dependence of dealkylation (aging) in r mouse (Mo) acetylcholinesterase (AChE) and rHu BChE and their mutants inactivated with P(S)C(S)- and P(S)C(R)-soman was compared. Best-fit parameters for the asymmetric bell curves for the adducts of wild-type Mo AChE are pK1 = pK2 = 4.0-4.9 and pK3 = 5.2-6.6. These pKs are consistent with the involvement of two carboxylic acids, possibly E202(199) and either E334(327) or E450(443), and H447(440)H+ in the dealkylation of AChE. E202Q MoAChE inactivated with the soman diastereomers yielded pK3 = 5.5-5.8. Nearly symmetric pH curves for soman-inhibited wild-type and E197D Hu BChE gave pK2 = 3.7-4.6 and pK3 = 7.3-8.0, but much lower, pK3 approximately 5, for the corresponding adduct of the E197Q mutant. Dealkylation in soman-inhibited BChE is consistent with the participation of one carboxylic acid side chain and H438(440)H+. Maximal rate constants for dealkylation (kmax) are 1-6 min-1 for AChE and 2 min-1 for BChE at 25 degreesC. The W82 to A mutation in BChE results in the largest reduction, 2500-6000-fold, in the rate constant for dealkylation. The reduction in the rate constants for dealkylation in the E197 mutants is highly pH dependent. The solvent isotope effects at the pH maxima are 1.3-1.4, indicating unlikely preprotonation or proton in "flight" at the enzymic transition states. The new results support the push-pull mechanism of dealkylation in soman-inhibited cholinesterases proposed previously.
ESTHER : Saxena_1998_Biochemistry_37_15086
PubMedSearch : Saxena_1998_Biochemistry_37_15086
PubMedID: 9790671

Title : A monoclonal antibody against acetylcholinesterase inhibits the formation of amyloid fibrils induced by the enzyme - Reyes_1997_Biochem.Biophys.Res.Commun_232_652
Author(s) : Reyes AE , Perez DR , Alvarez A , Garrido J , Gentry MK , Doctor BP , Inestrosa NC
Ref : Biochemical & Biophysical Research Communications , 232 :652 , 1997
Abstract : A monoclonal antibody (mAb) 25B1 directed against fetal bovine-serum acetylcholinesterase (FBS AChE) was used to examine the ability of the cholinergic enzyme to promote the assembly of amyloid-beta peptides (A beta) into Alzheimer fibrils. This mAb binds to the peripheral anionic site of the enzyme and allosterically inhibits catalytic activity of FBS AChE. Several techniques, including thioflavine-T fluorescence, turbidity, and negative-staining at the electron microscopy level, were used to assess amyloid formation. Inhibition of amyloid formation was dependent on the molar ratio AChE:mAb 25B1, and at least 50% of the inhibition of the AChE promoting effect occurs at a molar ratio similar to that required for inhibition of the esterase activity. Our results suggest that mAb 25B1 inhibits the promotion of the amyloid fibril formation triggered by AChE by affecting the lag period of the A beta aggregation process.
ESTHER : Reyes_1997_Biochem.Biophys.Res.Commun_232_652
PubMedSearch : Reyes_1997_Biochem.Biophys.Res.Commun_232_652
PubMedID: 9126330

Title : Differences in active site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase - Saxena_1997_Biochemistry_36_14642
Author(s) : Saxena A , Redman AM , Jiang X , Lockridge O , Doctor BP
Ref : Biochemistry , 36 :14642 , 1997
Abstract : Amino acid sequence alignments of cholinesterases revealed that 6 of 14 aromatic amino acid residues lining the active center gorge of acetylcholinesterase are replaced by aliphatic amino acid residues in butyrylcholinesterase. The Y337 (F330) in mammalian acetylcholinesterase, which is replaced by A328 in human butyrylcholinesterase, is implicated in the binding of ligands such as huperzine A, edrophonium, and acridines and one end of bisquaternary compounds such as BW284C51 and decamethonium. Y337 may sterically hinder the binding of phenothiazines such as ethopropazine, which contains a bulky exocyclic substitution. Inhibition studies of (-)-huperzine A with human butyrylcholinesterase mutants, where A328 (KI = 194.6 microM) was modified to either F (KI = 0.6 microM, as in Torpedo acetylcholinesterase) or Y (KI = 0.032 microM, as in mammalian acetylcholinesterase), confirmed previous observations made with acetylcholinesterase mutants that this residue is important for binding huperzine A. Inhibition studies of ethopropazine with butyrylcholinesterase mutants, where A328 (KI = 0.18 microM) was modified to either F (KI = 0.82 microM) or Y (KI = 0.28 microM), suggested that A328 was not solely responsible for the selectivity of ethopropazine. Volume calculations for the active site gorge showed that the poor inhibitory activity of ethopropazine toward acetylcholinesterase was due to the smaller dimension of the active site gorge which was unable to accommodate the bulky inhibitor molecule. The volume of the butyrylcholinesterase active site gorge is approximately 200 A3 larger than that of the acetylcholinesterase gorge, which allows the accommodation of ethopropazine in two different orientations as demonstrated by rigid-body refinement and molecular dynamics calculations.
ESTHER : Saxena_1997_Biochemistry_36_14642
PubMedSearch : Saxena_1997_Biochemistry_36_14642
PubMedID: 9398183

Title : Structure of glycan moieties responsible for the extended circulatory life time of fetal bovine serum acetylcholinesterase and equine serum butyrylcholinesterase - Saxena_1997_Biochemistry_36_7481
Author(s) : Saxena A , Raveh L , Ashani Y , Doctor BP
Ref : Biochemistry , 36 :7481 , 1997
Abstract : 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.
ESTHER : Saxena_1997_Biochemistry_36_7481
PubMedSearch : Saxena_1997_Biochemistry_36_7481
PubMedID: 9200697

Title : Mutant acetylcholinesterases as potential detoxification agents for organophosphate poisoning - Saxena_1997_Biochem.Pharmacol_54_269
Author(s) : Saxena A , Maxwell DM , Quinn DM , Radic Z , Taylor P , Doctor BP
Ref : Biochemical Pharmacology , 54 :269 , 1997
Abstract : It has been demonstrated that cholinesterases (ChEs) are an effective mode of pretreatment to prevent organophosphate (OP) toxicity in mice and rhesus monkeys. The efficacy of ChE as a bioscavenger of OP can be enhanced by combining enzyme pretreatment with oxime reactivation, since the scavenging capacity extends beyond a stoichiometric ratio of ChE to OP. Aging has proven to be a major barrier to achieving oxime reactivation of acetylcholinesterase (AChE) inhibited by the more potent OPs. To further increase the stoichiometry of OP to ChE required, we have sought AChE mutants that are more easily reactivated than wild-type enzyme. Substitution of glutamine for glutamate (E199) located at the amino-terminal to the active-site serine (S200) in Torpedo AChE generated an enzyme largely resistant to aging. Here we report the effect of the corresponding mutation on the rate of inhibition, reactivation by 1-(2-hydroxyiminomethyl-1-pyridinium)-1(4-carboxyaminopyridinium)- dimethyl ether hydrochloride (HI-6), and aging of mouse AChE inhibited by C(+)P(-)- and C(-)P(-)-epimers of soman. The E202 to Q mutation decreased the affinity of soman for AChE, slowed the reactivation of soman-inhibited AChE by HI-6, and decreased the aging of mutant AChE. These effects were more pronounced with C(-)P(-)-soman than with C(+)P(-)-soman. In vitro detoxification of soman and sarin by wild-type and E202Q AChE in the presence of 2 mM HI-6 showed that, E202Q AChE was 2-3 times more effective in detoxifying soman and sarin than wild-type AChE. These studies show that these recombinant DNA-derived AChEs are a great improvement over wild-type AChE as bioscavengers. They can be used to develop effective methods for the safe disposal of stored OP nerve agents and potential candidates for pre- or post-exposure treatment for OP toxicity.
ESTHER : Saxena_1997_Biochem.Pharmacol_54_269
PubMedSearch : Saxena_1997_Biochem.Pharmacol_54_269
PubMedID: 9271331

Title : Reversal of Glycerol Ether-Stimulated Acetylcholinesterase Activity by c-fos Antisense Oligonucleotide in Primary Neuronal Cultures -
Author(s) : Dave JR , Fasnacht E , Tortella FC , Best JM , Doctor BP , Ved HS
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :289 , 1995
PubMedID:

Title : Different effects of two peripheral anionic site-binding ligands on acetylcholinesterase active-site gorge topography revealed by electron paramagnetic resonance - Grubic_1995_Biochim.Biophys.Acta_1249_155
Author(s) : Grubic Z , Stalc A , Sentjurc M , Pecar S , Gentry MK , Doctor BP
Ref : Biochimica & Biophysica Acta , 1249 :155 , 1995
Abstract : Both propidium and monoclonal antibody (mAb) 25B1 bind to the peripheral anionic site region of fetal bovine serum acetylcholinesterase (FBS AChE). Using electron paramagnetic resonance (EPR) with spin-labelled organophosphate specifically bound to the AChE active-site serine, we studied the effects of both ligands on the topography of the AChE active-site gorge. After incubation of FBS AChE with Fab fragments of mAb 25B1, freedom of motion of our spin label became more restricted, suggesting closing of the gorge. Stabilization against heat denaturation was also observed. No alterations in the freedom of motion or protection against heat denaturation could be detected after propidium binding. Our results demonstrate that two ligands binding to the peripheral anionic site region of AChE have different effects, suggesting a complex structure for this region of the molecule that allows various types of interactions with different ligands. We also demonstrate that EPR is a suitable tool for studying microtopographical alterations at the active sites of cholinesterases.
ESTHER : Grubic_1995_Biochim.Biophys.Acta_1249_155
PubMedSearch : Grubic_1995_Biochim.Biophys.Acta_1249_155
PubMedID: 7599168

Title : Characterization of monoclonal antibodies that inhibit the catalytic activity of acetylcholinesterases - Gentry_1995_J.Neurochem_64_842
Author(s) : Gentry MK , Moorad DR , Hur RS , Saxena A , Ashani Y , Doctor BP
Ref : Journal of Neurochemistry , 64 :842 , 1995
Abstract : Monoclonal antibodies were generated against fetal bovine serum acetylcholinesterase and fetal bovine serum acetylcholinesterase inhibited by diisopropyl fluorophosphate or 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide. Six monoclonal antibodies inhibited 70 to > 98% of the catalytic activity of fetal bovine serum acetylcholinesterase. Inhibition of serum acetylcholinesterase from several mammalia by four monoclonal antibodies showed broad cross-reactivity. In all cases, monoclonal antibodies bound to the native form of acetylcholinesterases. None reacted with serum butyrylcholinesterases from various species. Although all monoclonal antibodies inhibited catalytic activity of acetylcholinesterases, the site of interaction with acetylcholinesterase appeared to differ for several antibodies. Two types of acetylcholinesterase:monoclonal antibody complexes were formed: one between tetrameric forms and another between catalytic subunits within the tetramer. Monoclonal antibodies that inhibited acetylcholinesterase activity at > 98% also considerably slowed binding of diisopropyl fluorophosphate and other organophosphorus compounds to the acetylcholinesterase:monoclonal antibody complex. Binding of these monoclonal antibodies to acetylcholinesterase influenced function of the enzyme's peripheral anionic site. None of the antibodies bound to the esteratic site of acetylcholinesterase. Monoclonal antibodies caused changes in catalytic activity of acetylcholinesterase by interaction at a site remote from the catalytic site, presumably at the entrance to the active site gorge.
ESTHER : Gentry_1995_J.Neurochem_64_842
PubMedSearch : Gentry_1995_J.Neurochem_64_842
PubMedID: 7830078

Title : Horse Serum Butyrylcholinesterase Does Not Disrupt Passive Avoidance Learning or Spontaneous Motor Activity in Rats -
Author(s) : Genovese RF , Roberts AR , Fantegrossi WE , Larrison RW , Doctor BP
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :473 , 1995
PubMedID:

Title : Comparative Inhibition of Acetylcholinesterase by Tacrine, Physostigmine and Huperzine in the Adult Rat Brain -
Author(s) : Ved HS , Best JM , Dave JR , Doctor BP
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :477 , 1995
PubMedID:

Title : Amino Acid Residues that Control Mono- and Bisquaternary Oxime-Induced Reactivation of O-Ethyl Methylphosphonylated Cholinesterases -
Author(s) : Ashani Y , Radic Z , Tsigelny I , Vellom DC , Pickering NA , Quinn DM , Doctor BP , Taylor P
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :133 , 1995
PubMedID:

Title : Amino Acid Alignment of Cholinesterases, Esterases, Lipases, and Related Proteins -
Author(s) : Gentry MK , Doctor BP
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :493 , 1995
PubMedID:

Title : Comparison of Acetylcholinesterase, Pyridostigmine, and HI-6 as Antidotes against Organophosphorus Compounds -
Author(s) : Maxwell DM , Brecht KM , Saxena A , Taylor P , Doctor BP
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :353 , 1995
PubMedID:

Title : Structural Analysis of the Asparagine-Linked Oligosaccharides of Cholinesterases -
Author(s) : Saxena A , Doctor BP
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :105 , 1995
PubMedID:

Title : Alterations in the Topography of Acetylcholinesterase Active Site Gorge after Binding of Peripheral Anionic Site Ligands -
Author(s) : Stalc A , Grubic Z , Sentjurc M , Pecar S , Gentry MK , Doctor BP
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :125 , 1995
PubMedID:

Title : Amino acid residues controlling reactivation of organophosphonyl conjugates of acetylcholinesterase by mono- and bisquaternary oximes - Ashani_1995_J.Biol.Chem_270_6370
Author(s) : Ashani Y , Radic Z , Tsigelny I , Vellom DC , Pickering NA , Quinn DM , Doctor BP , Taylor P
Ref : Journal of Biological Chemistry , 270 :6370 , 1995
Abstract : Single and multiple site mutants of recombinant mouse acetylcholinesterase (rMoAChE) were inhibited with racemic 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide (MEPQ) and the resulting mixture of two enantiomers, CH3PR,S(O)(OC2H5)-AChE(EMPR,S-AChE), were subjected to reactivation with 2-(hydroxyiminomethyl)-1-methylpyridinium methanesulfonate (P2S) and 1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4"-carbamoyl-1"- pyridinium)-2-oxapropane dichloride (HI-6). Kinetic analysis of the reactivation profiles revealed biphasic behavior with an approximate 1:1 ratio of two presumed reactivatable enantiomeric components. Equilibrium dissociation and kinetic rate constants for reactivation of site-specific mutant enzymes were compared with those obtained for wild-type rMoAChE, tissue-derived Torpedo AChE and human plasma butyrylcholinesterase. Substitution of key amino acid residues at the entrance to the active-site gorge (Trp-286, Tyr-124, Tyr-72, and Asp-74) had a greater influence on the reactivation kinetics of the bisquaternary reactivator HI-6 compared with the monoquaternary reactivator P2S. Replacement of Phe-295 by Leu enhanced reactivation by HI-6 but not by P2S. Of residues forming the choline-binding subsite, the E202Q mutation had a dominant influence where reactivation by both oximes was decreased 16- to 33-fold. Residues Trp-86 and Tyr-337 in this subsite showed little involvement. These kinetic findings, together with energy minimization of the oxime complex with the phosphonylated enzyme, provide a model for differences in the reactivation potencies of P2S and HI-6. The two kinetic components of oxime reactivation of MEPQ-inhibited AChEs arise from the chirality of O-ethyl methylphosphonyl moieties conjugated with Ser-203 and may be attributable to the relative stability of the phosphonyl oxygen of the two enantiomers in the oxyanion hole.
ESTHER : Ashani_1995_J.Biol.Chem_270_6370
PubMedSearch : Ashani_1995_J.Biol.Chem_270_6370
PubMedID: 7890775

Title : Behavioral and pharmacological assessment of butyrylcholinesterase in rats - Genovese_1995_Pharmacol.Biochem.Behav_51_647
Author(s) : Genovese RF , Doctor BP
Ref : Pharmacology, Biochemistry & Behavior , 51 :647 , 1995
Abstract : Advances in the treatment of organophosphorus (OP) toxicity have focussed on the use of exogenous cholinesterases to act as scavengers for the OP agent. To further investigate the feasibility of the scavenger approach, we evaluated the effects of highly purified horse serum butyrylcholinesterase (HS-BChE) on performance in rats. HS-BChE (5000 U, IP) produced substantial increases in blood enzyme activity for up to 72 h after injection. HS-BChE (5000 U, IP) had no effect on acquisition or retention of a passive avoidance task. In contrast, atropine sulfate (10 mg/kg) impaired retention when tested 168 h after administration. When examined for 10 days following administration, HS-BChE (7500 U, IP) had no effect on either total daily motor activity or circadian pattern of activity. HS-BChE (5000 U, IM) also had no acute or prolonged effects on the rate of lever pressing maintained by a VI56 s schedule of food reinforcement. HS-BChE (7500 U, IM) was observed to confer significant, but partial, protection against response rate decreases produced by the OP, MEPQ, under the VI56 s schedule of reinforcement. These results suggest that, in rats, HS-BChE, at doses that attenuate OP toxicity, may be devoid of cognitive or motor effects.
ESTHER : Genovese_1995_Pharmacol.Biochem.Behav_51_647
PubMedSearch : Genovese_1995_Pharmacol.Biochem.Behav_51_647
PubMedID: 7675838

Title : Modulation of Catalysis and Inhibition of Fetal Bovine Serum Acetylcholinesterase by Monoclonal Antibodies -
Author(s) : Doctor BP , Gentry MK , Saxena A , Ashani Y
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :141 , 1995
PubMedID:

Title : Amplification of the effectiveness of acetylcholinesterase for detoxification of organophosphorus compounds by bis-quaternary oximes - Caranto_1994_Biochem.Pharmacol_47_347
Author(s) : Caranto GR , Waibel KH , Asher JM , Larrison RW , Brecht KM , Schutz MB , Raveh L , Ashani Y , Wolfe AD , Maxwell DM , Doctor BP
Ref : Biochemical Pharmacology , 47 :347 , 1994
Abstract : Pretreatment of rhesus monkeys with fetal bovine serum acetylcholinesterase (FBS AChE) provides complete protection against 5 LD50 of organophosphate (OP) without any signs of toxicity or performance decrements as measured by serial probe recognition tests or primate equilibrium platform performance (Maxwell et al., Toxicol Appl Pharmacol 115: 44-49, 1992; Wolfe et al., Toxicol Appl Pharmacol 117: 189-193, 1992). Although such use of enzyme as a single pretreatment drug for OP toxicity is sufficient to provide complete protection, a relatively large (stoichiometric) amount of enzyme was required in vivo to neutralize OP. To improve the efficacy of cholinesterases as pretreatment drugs, we have developed an approach in which the catalytic activity of OP-inhibited FBS AChE was rapidly and continuously restored, thus detoxifying the OP and minimizing enzyme aging by having sufficient amounts of appropriate oxime present. The efficacy of FBS AChE to detoxify several OPs was amplified by addition of bis-quaternary oximes, particularly 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(4-carboxyaminopyridinium) -dimethyl ether hydrochloride (HI-6). When mice were pretreated with sufficient amounts of FBS AChE and HI-6 and challenged with repeated doses of O-isopropyl methylphosphonofluridate (sarin), the OP was continuously detoxified so long as the molar concentration of the sarin dose was less than the molar concentration of AChE in circulation. The in vitro experiments showed that the stoichiometry of sarin:FBS AChE was higher than 3200:1 and in vivo stoichiometry with mice was as high as 57:1. Addition of HI-6 to FBS AChE as a pretreatment drug amplified the efficacy of enzyme as a scavenger of nerve agents.
ESTHER : Caranto_1994_Biochem.Pharmacol_47_347
PubMedSearch : Caranto_1994_Biochem.Pharmacol_47_347
PubMedID: 8304979

Title : Huperzine A as a pretreatment candidate drug against nerve agent toxicity - Grunwald_1994_Life.Sci_54_991
Author(s) : Grunwald J , Raveh L , Doctor BP , Ashani Y
Ref : Life Sciences , 54 :991 , 1994
Abstract : Huperzine A (HUP) is a naturally-occurring, potent, reversible inhibitor of acetylcholinesterase (AChE) that crosses the blood-brain barrier. To examine its ability to protect against nerve agent poisoning, HUP was administered i.p. to mice, and the s.c. LD50 of soman was determined at various time intervals after pretreatment. Results were compared to those obtained for animals treated with physostigmine. A protective ratio of approximately 2 was maintained for at least 6 hr after a single injection of HUP, without the need for any post-challenge drug therapy. By contrast, pretreatment with physostigmine increased the LD50 of soman by 1.4- to 1.5-fold for only up to 90 min. The long-lasting antidotal efficacy displayed by HUP correlated with the time course of the blood-AChE inhibition. The results suggest that the protection of animals by HUP from soman poisoning was achieved by temporarily sequestering the active site region of the physiologically important AChE.
ESTHER : Grunwald_1994_Life.Sci_54_991
PubMedSearch : Grunwald_1994_Life.Sci_54_991
PubMedID: 8139389

Title : Identification of amino acid residues involved in the binding of Huperzine A to cholinesterases - Saxena_1994_Protein.Sci_3_1770
Author(s) : Saxena A , Qian N , Kovach IM , Kozikowski AP , Pang YP , Vellom DC , Radic Z , Quinn DM , Taylor P , Doctor BP
Ref : Protein Science , 3 :1770 , 1994
Abstract : Huperzine A, a potential agent for therapy in Alzheimer's disease and for prophylaxis of organophosphate toxicity, has recently been characterized as a reversible inhibitor of cholinesterases. To examine the specificity of this novel compound in more detail, we have examined the interaction of the 2 stereoisomers of Huperzine A with cholinesterases and site-specific mutants that detail the involvement of specific amino acid residues. Inhibition of fetal bovine serum acetylcholinesterase by (-)-Huperzine A was 35-fold more potent than (+)-Huperzine A, with KI values of 6.2 nM and 210 nM, respectively. In addition, (-)-Huperzine A was 88-fold more potent in inhibiting Torpedo acetylcholinesterase than (+)-Huperzine A, with KI values of 0.25 microM and 22 microM, respectively. Far larger KI values that did not differ between the 2 stereoisomers were observed with horse and human serum butyrylcholinesterases. Mammalian acetylcholinesterase, Torpedo acetylcholinesterase, and mammalian butyrylcholinesterase can be distinguished by the amino acid Tyr, Phe, or Ala in the 330 position, respectively. Studies with mouse acetylcholinesterase mutants, Tyr 337 (330) Phe and Tyr 337 (330) Ala yielded a difference in reactivity that closely mimicked the native enzymes. In contrast, mutation of the conserved Glu 199 residue to Gln in Torpedo acetylcholinesterase produced only a 3-fold increase in KI value for the binding of Huperzine A.
ESTHER : Saxena_1994_Protein.Sci_3_1770
PubMedSearch : Saxena_1994_Protein.Sci_3_1770
PubMedID: 7849595

Title : Epitope mapping of form-specific and nonspecific antibodies to acetylcholinesterase - Wasserman_1993_J.Neurochem_61_2124
Author(s) : Wasserman L , Doctor BP , Gentry MK , Taylor P
Ref : Journal of Neurochemistry , 61 :2124 , 1993
Abstract : We have mapped the epitopes to which two monoclonal antibodies against acetylcholinesterase (AChE) from Torpedo californica are directed. One antibody, 2C9, has equivalent affinity for both the 5.6S (amphiphilic) and 11S (hydrophilic) enzyme forms; the other, 4E7, recognizes only the amphiphilic form and has been shown previously to require an N-linked oligosaccharide residue on the protein. Isolation of cyanogen bromide peptides from the amphiphilic form and assay by a competition ELISA for 2C9 and by a direct binding ELISA for 4E7 identified the same peptide, residues 44-82, as containing epitopes against both antibodies. The epitope for 4E7 includes the oligosaccharide conjugated to Asp59, an N-linked glycosylation site not present in mouse AChE. A 20-amino-acid synthetic peptide, RFRRPEPKKPWSGVWNASTY, representing residues 44-63, was synthesized and found to inhibit completely 2C9 binding to 5.6S enzyme at molar concentrations comparable to those of the cyanogen bromide peptide. It was unreactive with 4E7. Fractionation of the synthetic peptide further localized the 2C9 epitope. Peptides RFRRPEPKKPW and KPWSGVWNASTY both reacted but less so than the entire synthetic peptide at equivalent molar concentrations, whereas the peptide RPEPKKPWSGVWNASTY was as effective as the larger synthetic peptide. The crystal structure of AChE shows the peptide to be on the surface of the molecule as part of a convex hairpin loop starting before the first alpha-helix.
ESTHER : Wasserman_1993_J.Neurochem_61_2124
PubMedSearch : Wasserman_1993_J.Neurochem_61_2124
PubMedID: 7504082

Title : Comparison of antidote protection against soman by pyridostigmine, HI-6 and acetylcholinesterase - Maxwell_1993_J.Pharmacol.Exp.Ther_264_1085
Author(s) : Maxwell DM , Brecht KM , Doctor BP , Wolfe AD
Ref : Journal of Pharmacology & Experimental Therapeutics , 264 :1085 , 1993
Abstract : Carbamate, oxime and enzyme scavenger approaches to protection against the highly toxic organophosphorus compound, soman, were compared by using the most prominent example of each type of antidote. Pyridostigmine in combination with atropine, HI-6 [1-(2-(hydroxyimino)methyl))pyridinium-2-(4-(aminocarbonyl)p yridinium) dimethylether] in combination with atropine and fetal bovine serum acetylcholinesterase (FBS-AChE) without atropine were used as examples of oxime, carbamate and enzyme scavenger antidotes, respectively. Each antidotal regimen produced approximately equal maximal protection against the lethal effects of 952 to 1169 nmol/kg (LD50, 8-10) of soman in mice whose carboxylesterase had been inhibited with 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide. FBS-AChE was much better than either pyridostigmine-atropine or HI-6-atropine in reducing postexposure incapacitation from soman as measured by lacrimation, motor dysfunction, activity level and the inverted screen test. A lower dose of pyridostigmine (566 nmol/kg) or FBS-AChE (1150 nmol/kg) was required to protect against 968 nmol/kg (LD50, 8) of soman than was required for HI-6 (200,000 nmol/kg). Inasmuch as the in vivo biological half-life of FBS-AChE (1550 min) was much greater than the biological half-lives of pyridostigmine (48 min) or HI-6 (11 min), the ability of FBS-AChE to produce better protection against the postexposure incapacitation from soman suggests that it should be considered as an alternative to either pyridostigmine-atropine or HI-6-atropine antidotal regimens.
ESTHER : Maxwell_1993_J.Pharmacol.Exp.Ther_264_1085
PubMedSearch : Maxwell_1993_J.Pharmacol.Exp.Ther_264_1085
PubMedID: 8450452

Title : Direct observation and elucidation of the structures of aged and nonaged phosphorylated cholinesterases by 31P NMR spectroscopy - Segall_1993_Biochemistry_32_13441
Author(s) : Segall Y , Waysbort D , Barak D , Ariel N , Doctor BP , Grunwald J , Ashani Y
Ref : Biochemistry , 32 :13441 , 1993
Abstract : 31P NMR spectroscopy of butyrylcholinesterase (BChE), acetylcholinesterase (AChE), and chymotrypsin (Cht) inhibited by pinacolyl methylphosphonofluoridate (soman), methylphosphonodifluoridate (MPDF), and diisopropyl phosphorofluoridate (DFP) allowed direct observation of the OP-linked moiety of aged (nonreactivatable) and nonaged organophosphorus (OP)-ChE conjugates. The 31P NMR chemical shifts of OP-ChE conjugates clearly demonstrated insertion of a P-O- bond into the active site of aged OP-ChE adducts. The OP moiety of nonaged OP-ChEs was shown to be uncharged. The OP-bound pinacolyl moiety of soman-inhibited and aged AChE was detached completely, whereas only partial dealkylation of the pinacolyl group was observed for soman-inhibited BChEs. This suggests that the latter enzyme reacted with the less active stereoisomer(s) of soman. In the case of soman-inhibited Cht, no dealkylation could be experimentally detected for any of the four stereoisomers of OP-Cht adducts. Results are consistent with the contention that the phenomenon of enzyme-catalyzed dealkylation of OP adducts of serine hydrolases strongly depends on the orientation of both the catalytic His and the carboxyl side chain of either Glu or Asp positioned next to the catalytic Ser. The denatured protein of aged OP-ChE or OP-Cht is a convenient leaving group in nucleophilic displacements of tetrahedral OP compounds despite the presence of a P-O- bond. This indicates that the unusual resistance to reactivation of the aged enzyme cannot be ascribed to simple electrostatic repulsion of an approaching nucleophile. The broadening of the 31P NMR signal of native OP-ChEs relative to that of OP-Cht is in agreement with the crystal structure of AChE, showing that the active site region of ChEs in solution resides in a deep, narrow gorge.
ESTHER : Segall_1993_Biochemistry_32_13441
PubMedSearch : Segall_1993_Biochemistry_32_13441
PubMedID: 8257680

Title : Monoclonal antibody AE-2 modulates carbamate and organophosphate inhibition of fetal bovine serum acetylcholinesterase - Wolfe_1993_Mol.Pharmacol_44_1152
Author(s) : Wolfe AD , Chiang PK , Doctor BP , Fryar N , Rhee JP , Saeed M
Ref : Molecular Pharmacology , 44 :1152 , 1993
Abstract : The monoclonal antibody AE-2, raised against the human erythrocyte acetylcholinesterase (AChE) dimer (acetylcholine acetylhydrolase, EC 3.1.1.7), binds to other mammalian AChEs, including the tetramer that occurs in fetal bovine serum (FBS). AE-2 partially inhibited the rate of hydrolysis of the charged substrate acetylthiocholine by FBS AChE, whereas it increased the rate of hydrolysis of the neutral substrate indophenyl acetate. Present results show that AE-2 decreases the rate of inhibition of FBS AChE by the positively charged organophosphate amiton-p-toluene sulfonate and the positively charged carbamates pyridostigmine and neostigmine but accelerates inhibition of FBS AChE by the neutral organophosphates paraoxon and diisopropylfluorophosphate. Results suggest that AE-2 may allosterically modulate an anionic site in the catalytic center of FBS AChE.
ESTHER : Wolfe_1993_Mol.Pharmacol_44_1152
PubMedSearch : Wolfe_1993_Mol.Pharmacol_44_1152
PubMedID: 8264551

Title : Immunochemical characterization of anti-acetylcholinesterase inhibitory monoclonal antibodies - Gentry_1993_Chem.Biol.Interact_87_227
Author(s) : Gentry MK , Saxena A , Ashani Y , Doctor BP
Ref : Chemico-Biological Interactions , 87 :227 , 1993
Abstract : Monoclonal antibodies (mAbs) were prepared against native or DFP-inhibited Torpedo californica acetylcholinesterase and native or DFP-, MEPQ-, and soman-inhibited fetal bovine serum acetylcholinesterase. The cross reactivity of these antibodies with acetylcholinesterases from various species and their ability to inhibit catalytic activity were determined. Eight antibodies were found to inhibit catalytic activity of either Torpedo or fetal bovine serum enzyme. In all cases the antibodies bound to the native form of the enzymes and in some cases even to the denatured form. None of the antibodies recognized human or horse serum butyrylcholinesterase. Sucrose density gradient centrifugation of enzyme-antibody complexes provided two types of profiles, one with multiple peaks, indicating numerous complexes between tetrameric forms of the enzyme, and the other with single peaks, demonstrating complex formation within the tetrameric form. Different antibodies appeared to interact with slightly different regions, but in all cases the binding encompassed the peripheral anionic site. Decrease in catalytic activity of the enzyme was most likely caused by conformational changes in the enzyme molecule resulting from interaction with these mAbs.
ESTHER : Gentry_1993_Chem.Biol.Interact_87_227
PubMedSearch : Gentry_1993_Chem.Biol.Interact_87_227
PubMedID: 7688272

Title : Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins - Cygler_1993_Protein.Sci_2_366
Author(s) : Cygler M , Schrag JD , Sussman JL , Harel M , Silman I , Gentry MK , Doctor BP
Ref : Protein Science , 2 :366 , 1993
Abstract : Based on the recently determined X-ray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase and on their three-dimensional superposition, an improved alignment of a collection of 32 related amino acid sequences of other esterases, lipases, and related proteins was obtained. On the basis of this alignment, 24 residues are found to be invariant in 29 sequences of hydrolytic enzymes, and an additional 49 are well conserved. The conservation in the three remaining sequences is somewhat lower. The conserved residues include the active site, disulfide bridges, salt bridges, and residues in the core of the proteins. Most invariant residues are located at the edges of secondary structural elements. A clear structural basis for the preservation of many of these residues can be determined from comparison of the two X-ray structures.
ESTHER : Cygler_1993_Protein.Sci_2_366
PubMedSearch : Cygler_1993_Protein.Sci_2_366
PubMedID: 8453375

Title : The role of glutamate-199 in the aging of cholinesterase - Saxena_1993_Biochem.Biophys.Res.Commun_197_343
Author(s) : Saxena A , Doctor BP , Maxwell DM , Lenz DE , Radic Z , Taylor P
Ref : Biochemical & Biophysical Research Communications , 197 :343 , 1993
Abstract : Aging of organophosphate-conjugated acetylcholinesterase results from the loss of an alkoxy group with concomitant stabilization of the conjugate to spontaneous or nucleophile-induced deacylation. We have examined the kinetics of aging in a pinacolylmethylphosphonofluoridate (soman)-inhibited mutant enzyme in which the glutamate (E199) located at the amino-terminal to the active-site serine (S200) was converted to glutamine (Q). For wild type enzyme, the soman-acetylcholinesterase conjugate aged immediately, giving rise to a form of enzyme resistant to reactivation by oximes. In contrast, the E199Q mutant enzyme was largely resistant to aging and could be reactivated by oximes. Since the pH dependence for aging was not altered appreciably, the primary influence of the loss of charge appears to be on the intrinsic rate of aging. The negative charge on E199 likely imparts an inductive effect on the conjugated organophosphate to facilitate removal of the alkoxy group.
ESTHER : Saxena_1993_Biochem.Biophys.Res.Commun_197_343
PubMedSearch : Saxena_1993_Biochem.Biophys.Res.Commun_197_343
PubMedID: 7902714

Title : Cholinesterases as scavengers for organophosphorus compounds: protection of primate performance against soman toxicity - Doctor_1993_Chem.Biol.Interact_87_285
Author(s) : Doctor BP , Blick DW , Caranto G , Castro CA , Gentry MK , Larrison RW , Maxwell DM , Murphy MR , Schutz MB , Waibel KH , Wolfe AD
Ref : Chemico-Biological Interactions , 87 :285 , 1993
Abstract : 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.
ESTHER : Doctor_1993_Chem.Biol.Interact_87_285
PubMedSearch : Doctor_1993_Chem.Biol.Interact_87_285
PubMedID: 8343986

Title : Mechanism of inhibition of cholinesterases by huperzine A - Ashani_1992_Biochem.Biophys.Res.Commun_184_719
Author(s) : Ashani Y , Peggins JO, 3rd , Doctor BP
Ref : Biochemical & Biophysical Research Communications , 184 :719 , 1992
Abstract : Huperzine A, an alkaloid isolated from Huperzia serrata was found to reversibly inhibit acetylcholinesterases (EC 3.1.1.7) and butyrylcholinesterases (EC 3.1.1.8) with on- and off-rates that depend on both the type and the source of enzyme. Long-term incubation of high concentrations of purified cholinesterases (1-8 microM) with huperzine A did not show any chemical modification of huperzine A. A low dissociation constant KI was obtained for mammalian acetylcholinesterase-huperzine (20-40 nM) compared to mammalian butyrylcholinesterase-huperzine (20-40 microM). This indicates that the thermodynamic stability of huperzine-cholinesterase complex may depend on the number and type of aromatic amino acid residues in the catalytic pocket region of the cholinesterase molecule.
ESTHER : Ashani_1992_Biochem.Biophys.Res.Commun_184_719
PubMedSearch : Ashani_1992_Biochem.Biophys.Res.Commun_184_719
PubMedID: 1575745

Title : Protection of rhesus monkeys against soman and prevention of performance decrement by pretreatment with acetylcholinesterase - Maxwell_1992_Toxicol.Appl.Pharmacol_115_44
Author(s) : Maxwell DM , Castro CA , De La Hoz DM , Gentry MK , Gold MB , Solana RP , Wolfe AD , Doctor BP
Ref : Toxicology & Applied Pharmacology , 115 :44 , 1992
Abstract : The ability of acetylcholinesterase from fetal bovine serum (FBS AChE) to protect against soman, a highly toxic organophosphorus (OP) compound, was tested in rhesus monkeys. Intravenous administration of FBS AChE produced a minimal behavioral effect on the serial probe recognition task, a sensitive test of cognitive function and short-term memory. Pharmacokinetic studies of injected FBS AChE indicated a plasma half-life of 40 hr for FBS AChE in monkeys. Both in vitro and in vivo titration of FBS AChE with soman produced a 1:1 stoichiometry between organophosphate-inhibited FBS AChE and the cumulative dose of the toxic stereoisomers of soman. Administration of FBS AChE protected monkeys against the lethal effects of up to 2.7 LD50 of soman and prevented any signs of organophosphate intoxication, e.g., excessive secretions, respiratory depression, muscle fasciculations, or convulsions. In addition, monkeys pretreated with FBS AChE were devoid of any behavioral incapacitation after soman challenge, as measured by the serial probe recognition task. Compared to the current multicomponent drug treatment against soman, which does not prevent the signs or the behavioral deficits resulting from OP intoxication, use of FBS AChE as a single pretreatment drug provides significantly effective protection against both the lethal and the behavioral effects of soman.
ESTHER : Maxwell_1992_Toxicol.Appl.Pharmacol_115_44
PubMedSearch : Maxwell_1992_Toxicol.Appl.Pharmacol_115_44
PubMedID: 1631892

Title : A microtiter assay for determining protein, acetylcholinesterase activity, and G418 (Neomycin) resistance in cultured cells - Elson_1992_Anal.Biochem_200_268
Author(s) : Elson HF , Gentry MK , Doctor BP
Ref : Analytical Biochemistry , 200 :268 , 1992
Abstract : The Coomassie brilliant blue assay for the determination of protein has been extended to rapidly and conveniently measure the protein concentration of cells growing in culture in a 96-well microtiter format. Modifications of the standard assay include sodium hydroxide to solubilize the cells and ovalbumin, instead of bovine serum albumin, as a protein standard. The procedure allows a large number of small samples to be assayed simultaneously. Two examples of its use, enzyme-specific activity and drug resistance, are shown. An assay for acetylcholinesterase activity in the same culture plate is demonstrated. G418, an inhibitor of cell protein synthesis, is frequently used to select for cells transfected with the neomycin resistance gene. The required concentration of G418 can be easily determined with this protein assay.
ESTHER : Elson_1992_Anal.Biochem_200_268
PubMedSearch : Elson_1992_Anal.Biochem_200_268
PubMedID: 1378703

Title : Acetylcholinesterase: A Pretreatment Drug for Organophosphate Toxicity -
Author(s) : Doctor BP , Blick DW
Ref : In Multidisciplinary approaches to cholinesterase functions - Proceedings of Fourth International Meeting on Cholinesterases , (Shafferman, A. and Velan, B., Eds) Plenum Press, New York :277 , 1992
PubMedID:

Title : Use of cholinesterases as pretreatment drugs for the protection of rhesus monkeys against soman toxicity - Wolfe_1992_Toxicol.Appl.Pharmacol_117_189
Author(s) : Wolfe AD , Blick DW , Murphy MR , Miller SA , Gentry MK , Hartgraves SL , Doctor BP
Ref : Toxicology & Applied Pharmacology , 117 :189 , 1992
Abstract : Purified fetal bovine serum acetylcholinesterase (FBS AChE) and horse serum butyrylcholinesterase (BChE) were successfully used as single pretreatment drugs for the prevention of pinacolyl methylphosphonofluoridate (soman) toxicity in nonhuman primates. Eight rhesus monkeys, trained to perform Primate Equilibrium Platform (PEP) tasks, were pretreated with FBS AChE or BChE and challenged with a cumulative level of five median lethal doses (LD50) of soman. All ChE-pretreated monkeys survived the soman challenge and showed no symptoms of soman toxicity. A quantitative linear relation was observed between the soman dose and the neutralization of blood ChE. None of the four AChE-pretreated animals showed PEP task decrements, even though administration of soman irreversibly inhibited nearly all of the exogenously administered AChE. In two of four BChE-pretreated animals, a small transient PEP performance decrement occurred when the cumulative soman dose exceeded 4 LD50. Performance decrements observed under BChE protection were modest by the usual standards of organophosphorus compound toxicity. No residual or delayed performance decrements or other untoward effects were observed during 6 weeks of post-exposure testing with either ChE.
ESTHER : Wolfe_1992_Toxicol.Appl.Pharmacol_117_189
PubMedSearch : Wolfe_1992_Toxicol.Appl.Pharmacol_117_189
PubMedID: 1471150

Title : Membrane-bound acetylcholinesterase: an early differentiation marker for skeletal myoblasts - Elson_1992_Biochim.Biophys.Acta_1156_78
Author(s) : Elson HF , Gentry MK , Doctor BP
Ref : Biochimica & Biophysica Acta , 1156 :78 , 1992
Abstract : Cell-bound acetylcholinesterase (AChE) was found to be an early differentiation marker on embryonic chick skeletal myoblasts in mixed primary cell cultures. AChE biosynthesis was detected and characterized by (a) a sensitive microtiter assay, (b) use of selective inhibitors, and (c) with mono- and polyclonal antibodies. Both secreted and cell-bound AChE appeared on the first day in culture, at a time when no muscle cell fusion was observed. Characterization of this enzyme revealed that true AChE was bound and secreted by myoblasts. BW284c51, which permeates cell membranes poorly, inhibited all the cell-associated AChE activity on myoblasts, suggesting that the activity measured was on the outer cell surface. On the other hand, fibroblasts appeared to have no or very little bound enzyme and the low level of secreted enzyme activity had the characteristics of pseudo-, or butyrylcholinesterase. Polyclonal anti-Torpedo californica electroplax AChE antibody and several monoclonal antibodies were found to bind specifically to chick myoblasts. Since the cells had not been made permeable before antibody binding, a membrane-bound form of the enzyme was most likely being detected. The cell-bound true AChE was present in identifiable quantities from the first day of culture. Membrane-bound AChE can thus serve as an early differentiation marker for embryonic chick myoblasts in mixed primary cultures.
ESTHER : Elson_1992_Biochim.Biophys.Acta_1156_78
PubMedSearch : Elson_1992_Biochim.Biophys.Acta_1156_78
PubMedID: 1472543

Title : Bovine brain acetylcholinesterase primary sequence involved in intersubunit disulfide linkages - Roberts_1991_J.Biol.Chem_266_7481
Author(s) : Roberts WL , Doctor BP , Foster JD , Rosenberry TL
Ref : Journal of Biological Chemistry , 266 :7481 , 1991
Abstract : Three distinct classes of membrane-bound acetylcholinesterases (AChEs) have been identified. A12 AChE is composed of 12 catalytic subunits that are linked to noncatalytic collagen-like subunits through intersubunit disulfide bonds. G2 AChE is localized in membranes by a glycoinositol phospholipid covalently linked to the C-terminal amino acid. Brain G4 AChE involves two catalytic subunits linked by a direct intersubunit disulfide bond while the other two are disulfide-linked to a membrane-binding 20-kDa noncatalytic subunit. Molecular cloning studies have so far failed to find evidence of more than one AChE gene in any organism although alternative splicing of torpedo AChE mRNA results in different C-terminal sequences for the A12 and G2 AChE forms. Support for a single bovine AChE gene is provided in this report by amino acid sequencing of the N-terminal domains from the G2 erythrocyte, G4 fetal serum, and G4 brain AChE. Comparison of the 38-amino acid sequences reveals virtually complete identity among the three AChE forms. Additional extensive identity between the fetal serum and brain AChEs was demonstrated by sequencing several brain AChE peptides isolated by high performance liquid chromatography after trypsin digestion of nitrocellulose blots of brain AChE catalytic subunits. Cysteines involved in intersubunit disulfide linkages in brain AChE were reduced selectively with dithiothreitol in the absence of denaturants and radioalkylated with iodoacetamide. The observed sequence of the major radiolabeled tryptic peptide was C*SDL, where C* was the radioalkylated cysteine residue. This sequence is precisely the same as that observed at the C terminus of fetal bovine serum AChE and shows close homology to the C-terminal sequence of torpedo A12 AChE. We conclude that the mammalian brain G4 AChEs utilize the same exon splicing pattern as the A12 AChEs and that factors other than the primary sequence of the AChE catalytic subunits dictate assembly with either the collagen-like or the 20-kDa noncatalytic subunits.
ESTHER : Roberts_1991_J.Biol.Chem_266_7481
PubMedSearch : Roberts_1991_J.Biol.Chem_266_7481
PubMedID: 2019579

Title : APPENDIX Alignment of Amino Acid Sequences of Acetylcholinesterases and Butyrylcholinesterases -
Author(s) : Gentry MK , Doctor BP
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :394 , 1991
PubMedID:

Title : Poster: Esterases as organophosphate scavengers -
Author(s) : Wolfe AD , Ashani Y , Doctor BP , Maxwell DM
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :306 , 1991
PubMedID:

Title : Bovine Brain Acetylcholinesterase Sequence Involved in Intersubunit Disulfide Linkages -
Author(s) : Rosenberry TL , Roberts WL , Doctor BP
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :17 , 1991
PubMedID:

Title : Fetal Bovine Serum Acetylcholinesterase: Structure-Function Correlation -
Author(s) : Doctor BP , Gentry MK , Wu SJ , Ashani Y , De La Hoz DM
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :37 , 1991
PubMedID:

Title : Butyrylcholinesterase and acetylcholinesterase prophylaxis against soman poisoning in mice - Ashani_1991_Biochem.Pharmacol_41_37
Author(s) : Ashani Y , Shapira S , Levy D , Wolfe AD , Doctor BP , Raveh L
Ref : Biochemical Pharmacology , 41 :37 , 1991
Abstract : Human butyrylcholinesterase (BChE, EC 3.1.1.8) or acetylcholinesterase (AChE, EC 3.1.1.7) from fetal bovine serum (FBS), administered i.v. in mice, sequestered at approximately 1:1 stoichiometry the highly toxic anti-ChE organophosphate, 1,2,2-trimethylpropyl methyl-fluorophosphonate (soman). A quantitative linear correlation was demonstrated between blood-ChE levels and the protection conferred by exogeneously administered ChE. Results presented here demonstrate that either human BChE or FBS-AChE is an effective prophylactic measure sufficient to protect mice from multiple LD50S of soman without the administration of post-treatment supportive drugs.
ESTHER : Ashani_1991_Biochem.Pharmacol_41_37
PubMedSearch : Ashani_1991_Biochem.Pharmacol_41_37
PubMedID: 1986743

Title : Cholinesterase and Carboxyl esterase as Scavengers for Organophosphorus Agents -
Author(s) : Maxwell DM , Wolfe AD , Ashani Y , Doctor BP
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :206 , 1991
PubMedID:

Title : Enzymes as pretreatment drugs for organophosphate toxicity - Doctor_1991_Neurosci.Biobehav.Rev_15_123
Author(s) : Doctor BP , Raveh L , Wolfe AD , Maxwell DM , Ashani Y
Ref : Neurosci Biobehav Rev , 15 :123 , 1991
Abstract : We have successfully demonstrated that exogenously administered acetyl- or butyrylcholinesterase (AChE, BChE respectively) will sequester organophosphates (OPs) before they reach their physiological targets. In addition, a third enzyme, endogenous carboxylesterase is known to be capable of scavenging OPs. In these studies, we have administered AChE and BChE to three different species of animals (mice, marmosets and monkeys) which were challenged with three different OPs (VX, MEPQ and soman). Results obtained from these systematic studies demonstrate that: (a) a quantitative linear correlation exists between blood AChE levels and the protection afforded by exogenously administered ChEs in animals challenged with OP, (b) approximately one mole of either AChE or BChE sequesters one mole of OP, (c) such prophylactic measures are sufficient to protect animals against OPs without the administration of any supportive drugs. Thus the OP dose, the blood-level of esterase, the ratio of the circulating enzyme to OP challenge, and the rate of reaction between them determine the overall efficacy of an enzyme as a pretreatment drug. The biochemical mechanism underlying the sequestration of various OPs by the use of exogenously administered scavenging esterases is the same in all species of animals studied. Therefore, the extrapolation of the results obtained by the use of ChE prophylaxis in animals to humans should be more reliable and effective than extrapolating the results from currently used multidrug antidotal modalities.
ESTHER : Doctor_1991_Neurosci.Biobehav.Rev_15_123
PubMedSearch : Doctor_1991_Neurosci.Biobehav.Rev_15_123
PubMedID: 2052184

Title : Changes in the Catalytic Activity of Acetylcholinesterase upon Complexation with Monoclonal Antibodies -
Author(s) : Ashani Y , Bromberg A , Levy D , Gentry MK , Brady DR , Doctor BP
Ref : In: Cholinesterases: Structure, Function, Mechanism, Genetics, and Cell Biology , (Massoulie J, Barnard EA, Chatonnet A, Bacou F, Doctor BP, Quinn DM) American Chemical Society, Washington, DC :235 , 1991
PubMedID:

Title : Differences in conformational stability between native and phosphorylated acetylcholinesterase as evidenced by a monoclonal antibody - Ashani_1990_Biochemistry_29_2456
Author(s) : Ashani Y , Gentry MK , Doctor BP
Ref : Biochemistry , 29 :2456 , 1990
Abstract : Monoclonal antibody 25B1 generated against diisopropyl phosphorofluoridate inhibited fetal bovine serum acetylcholinesterase has been extensively characterized with respect to its anticholinesterase properties. This antibody demonstrated considerably different properties from previously reported inhibitory antibodies raised against acetylcholinesterase in terms of the degree of inhibition (greater than 98%), the high degree of specificity, and the stability of the antigen-antibody complex. Monoclonal antibody 25B1 appears to be directed against a conformational epitope located in close proximity to the catalytic center of the enzyme and was found to be most suitable for studying the stabilization of the active site of acetylcholinesterase against denaturation by heat or guanidine following phosphorylation by organophosphorus anticholinesterase compounds. This approach allowed the determination of stability rank order of various phosphorylated acetylcholinesterases. Among all the organophosphates tested, the combination of a methyl group and a negatively charged oxygen attached to the P atom, CH3P(O)(O-)-AChE, conferred the greatest protection to the active site of aged or nonaged organophosphoryl conjugates of acetylcholinesterase.
ESTHER : Ashani_1990_Biochemistry_29_2456
PubMedSearch : Ashani_1990_Biochemistry_29_2456
PubMedID: 1692236

Title : Studies on the topography of the catalytic site of acetylcholinesterase using polyclonal and monoclonal antibodies - Ogert_1990_J.Neurochem_55_756
Author(s) : Ogert RA , Gentry MK , Richardson EC , Deal CD , Abramson SN , Alving CR , Taylor P , Doctor BP
Ref : Journal of Neurochemistry , 55 :756 , 1990
Abstract : Polyclonal and monoclonal antibodies were generated against a synthetic peptide (25 amino acid residues) corresponding to the amino acid sequence surrounding the active site serine of Torpedo californica acetylcholinesterase (AChE). Prior to immunization, the peptide was either coupled to bovine serum albumin or encapsulated into liposomes containing lipid A as an adjuvant. To determine whether this region of AChE is located on the surface of the enzyme and thus accessible for binding to antibodies, or located in a pocket and thus not accessible to antibodies, the immunoreactivity of the antibodies was determined using enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, Western blots, and competition ELISA. The polyclonal antibody and several of the monoclonal antibodies failed to react with either Torpedo or fetal bovine serum AChE in their native conformations, but showed significant cross-reactivity with the denatured enzymes. Human serum butyrylcholinesterase, which has a high degree of amino acid sequence homology with these AChEs, failed to react with the same antibodies in either native form or denatured form. Chymotrypsin also failed to react with the monoclonal antibodies in either form. Eighteen octapeptides spanning the entire sequence of this region were synthesized on polyethylene pins, and epitopes of representative monoclonal antibodies were determined by ELISA. The reactivity of peptides suggest that a portion of the 25 mer peptide in AChE containing the active site serine is the primary epitope. It is not exposed on the surface of the enzyme and is most likely sequestered in a pocket-like conformation in the native enzyme.
ESTHER : Ogert_1990_J.Neurochem_55_756
PubMedSearch : Ogert_1990_J.Neurochem_55_756
PubMedID: 1696619

Title : Complete amino acid sequence of fetal bovine serum acetylcholinesterase and its comparison in various regions with other cholinesterases - Doctor_1990_FEBS.Lett_266_123
Author(s) : Doctor BP , Chapman TC , Christner CE , Deal CD , De La Hoz DM , Gentry MK , Ogert RA , Rush RS , Smyth KK , Wolfe AD
Ref : FEBS Letters , 266 :123 , 1990
Abstract : The complete amino acid sequence of a mammalian acetylcholinesterase from fetal bovine serum (FBS AChE) is presented. This enzyme has a high degree of sequence identity with other cholinesterases, liver carboxyesterases, esterase-6, lysophospholipase, and thyroglobulin. The locations of 191 amino acids in 10 regions of the FBS enzyme were compared with corresponding sequences of Torpedo, human, and Drosophila AChEs and human serum butyrylcholinesterase (BChE). In one region there is a marked difference in both the number of amino acids and their sequence between mammalian AChE and other AChEs and the human serum BChE. The amino acid sequence of FBS AChE showed overall homologies of 90% with human AChE, 60% with T. california AChE, 50% with human serum BChE, and 39% with Drosophila AChE in these regions.
ESTHER : Doctor_1990_FEBS.Lett_266_123
PubMedSearch : Doctor_1990_FEBS.Lett_266_123
PubMedID: 2365060
Gene_locus related to this paper: bovin-ACHE

Title : Structural and immunochemical properties of fetal bovine serum acetylcholinesterase -
Author(s) : Doctor BP , Smyth KK , Gentry MK , Ashani Y , Christner CE , De La Hoz DM , Ogert RA , Smith SW
Ref : Progress in Clinical & Biological Research , 289 :305 , 1989
PubMedID: 2471207

Title : Differences in structure and distribution of the molecular forms of acetylcholinesterase - Abramson_1989_J.Cell.Biol_108_2301
Author(s) : Abramson SN , Ellisman MH , Deerinck TJ , Maulet Y , Gentry MK , Doctor BP , Taylor P
Ref : Journal of Cell Biology , 108 :2301 , 1989
Abstract : Two structurally distinct molecular forms of acetylcholinesterase are found in the electric organs of Torpedo californica. One form is dimensionally asymmetric and composed of heterologous subunits. The other form is hydrophobic and composed of homologous subunits. Sequence-specific antibodies were raised against a synthetic peptide corresponding to the COOH-terminal region (Lys560-Leu575) of the catalytic subunits of the asymmetric form of acetylcholinesterase. These antibodies reacted with the asymmetric form of acetylcholinesterase, but not with the hydrophobic form. These results confirm recent studies suggesting that the COOH-terminal domain of the asymmetric form differs from that of the hydrophobic form, and represent the first demonstration of antibodies selective for the catalytic subunits of the asymmetric form. In addition, the reactive epitope of a monoclonal antibody (4E7), previously shown to be selective for the hydrophobic form of acetylcholinesterase, has been identified as an N-linked complex carbohydrate, thus defining posttranslational differences between the two forms. These two form-selective antibodies, as well as panselective polyclonal and monoclonal antibodies, were used in light and electron microscopic immunolocalization studies to investigate the distribution of the two forms of acetylcholinesterase in the electric organ of Torpedo. Both forms were localized almost exclusively to the innervated surface of the electrocytes. However, they were differentially distributed along the innervated surface. Specific asymmetric-form immunoreactivity was restricted to areas of synaptic apposition and to the invaginations of the postsynaptic membrane that form the synaptic gutters. In contrast, immunoreactivity attributable to the hydrophobic form was selectively found along the non-synaptic surface of the nerve terminals and was not observed in the synaptic cleft or in the invaginations of the postsynaptic membrane. This differential distribution suggests that the two forms of acetylcholinesterase may play different roles in regulating the local concentration of acetylcholine in the synapse.
ESTHER : Abramson_1989_J.Cell.Biol_108_2301
PubMedSearch : Abramson_1989_J.Cell.Biol_108_2301
PubMedID: 2472404

Title : Binary antidotes for organophosphate poisoning: aprophen analogues that are both antimuscarinics and carbamates - Leader_1989_J.Med.Chem_32_1522
Author(s) : Leader H , Smejkal RM , Payne CS , Padilla FN , Doctor BP , Gordon RK , Chiang PK
Ref : Journal of Medicinal Chemistry , 32 :1522 , 1989
Abstract : Prophylaxis against organophosphate poisoning can be achieved by pretreatment with physostigmine or pyridostigmine, which are carbamates, and aprophen, which is an anticholinergic agent. Thus, a series of aprophen analogues was synthesized with carbamyl substitutions on the phenyl rings (carbaphens). The rationale behind this design is that such compounds might exhibit most of the therapeutic characteristics of aprophen, as well as the ability to protect prophylactically by chemically masking cholinesterase enzymes. Compounds 4 (dimethylhydroxycarbaphen), 15 (dimethylcarbaphen), and 16 (monomethylcarbaphen) were found to inactivate human butyrylcholinesterase in a time-dependent manner with potencies similar to those of physostigmine or pyridostigmine, and the latter two exhibited almost the same antimuscarinic profile as aprophen. In contrast to the potent inactivation of butyrylcholinesterase by these compounds, marginal inactivation of acetylcholinesterase activity was observed, and only at much higher drug concentrations. The noncarbamylated analogues had no effect on the activity of either cholinesterase. The carbaphen compounds are hence prototype drugs that can interact with either muscarinic receptors or butyrylcholinesterase. Furthermore, these compounds are prodrugs, since after carbamylation of the cholinesterase, the leaving group 14 (hydroxyaprophen) is a potent antimuscarinic itself.
ESTHER : Leader_1989_J.Med.Chem_32_1522
PubMedSearch : Leader_1989_J.Med.Chem_32_1522
PubMedID: 2738887

Title : Acetylcholinesterase prophylaxis against organophosphate poisoning. Quantitative correlation between protection and blood-enzyme level in mice - Raveh_1989_Biochem.Pharmacol_38_529
Author(s) : Raveh L , Ashani Y , Levy D , De La Hoz DM , Wolfe AD , Doctor BP
Ref : Biochemical Pharmacology , 38 :529 , 1989
Abstract : Fetal bovine serum acetylcholinesterase (FBS-AChE, EC 3.1.1.7) was titrated, both in vitro and in vivo, with a highly toxic anti-ChE organophosphate, 7-(methylethoxyphosphinyloxy)-1-methyl-quinolinium iodie (MEPQ). Approximately 1:1 stoichiometry was obtained for the sequestration of MEPQ by FBS-AChE in mice. A quantitative, linear correlation was demonstrated between blood-AChE levels and the protection afforded by exogenously administered AChE in mice when challenged with anti-ChE MEPQ. The results presented in this report demonstrate that such prophylactic measures are indeed sufficient to protect animals against poisoning by as high as an 8 x LD50 dose of organophosphate without the administration of any supportive drug. Despite the relatively large toxic dose, most of the mice that survived the challenge did not show any classical clinical signs of severe anti-ChE poisoning. MEPQ may be considered a suitable model compound for studying the quantitative aspects of the scavenger prophylactic approach described here.
ESTHER : Raveh_1989_Biochem.Pharmacol_38_529
PubMedSearch : Raveh_1989_Biochem.Pharmacol_38_529
PubMedID: 2917010

Title : Desethylaprophen: a metabolite of aprophen with antimuscarinic activities - Brown_1988_J.Pharm.Sci_77_145
Author(s) : Brown ND , Smejkal RM , Breuer E , Doctor BP , Chiang PK
Ref : J Pharm Sci , 77 :145 , 1988
Abstract : The metabolic fate of aprophen hydrochloride (2-diethylaminoethyl 2,2-diphenylpropionate) was studied in rats after intravenous administration. Both 14C-labeled and unlabeled aprophen were used in these studies. Blood samples were collected and analyzed to determine the identities of the metabolites formed. Utilizing high-performance liquid chromatography, desethylaprophen was identified as a major metabolite in ether-extracted samples from rats, and could be detected in blood samples 1 min after intravenous administration. It was most likely formed by N-de-ethylation of aprophen by a cytochrome P-450-dependent monooxygenase. Synthetic desethylaprophen was found to possess cholinolytic activity (i.e., it functioned as a muscarinic antagonist by blocking the contraction of acetylcholine-stimulated guinea pig ileum, the release of alpha-amylase from pancreatic acinar cells stimulated by carbachol, and also by inhibiting the binding of [3H]N-methyl scopolamine to the muscarinic receptors of guinea pig ileum). It was interesting that although the biological effects of desethylaprophen were 100-fold lower than those of aprophen, it was equally able to compete for the binding sites of muscarinic receptors of the guinea pig ileum.
ESTHER : Brown_1988_J.Pharm.Sci_77_145
PubMedSearch : Brown_1988_J.Pharm.Sci_77_145
PubMedID: 3258910

Title : Acetylcholinesterase prophylaxis against organophosphate toxicity - Wolfe_1987_Fundam.Appl.Toxicol_9_266
Author(s) : Wolfe AD , Rush RS , Doctor BP , Koplovitz I , Jones D
Ref : Fundamental & Applied Toxicology , 9 :266 , 1987
Abstract : Fetal bovine serum acetylcholinesterase (FBS-AChE) protected mice from multiple LD50 doses of organophosphorus (OP) nerve agents. Mice were injected intraperitoneally (ip) with up to 3.3 mg (11,000 U) of FBS-AChE which exhibited a relatively long serum half-life and appeared well tolerated. The enzyme protected mice from the OP ethyl-S-2-diisopropylamino-ethylmethylphosphonothiolate (VX) with a stoichiometry equal to approximately 2 moles of enzyme active site per mole of VX. FBS-AChE, at a lower enzyme OP ratio, protected mice from 2 LD50s of the nerve agent methylphosphonofluoridic acid 1,2,2,-trimethylpropyl ester (soman) when used in conjunction with atropine and 2[(hydroxyimino)methyl]-1-methylpyridinium chloride. It is concluded that sequestration of highly toxic OPs by administration of AChE occurs in mice and suggests a new approach to treatment of OP intoxication.
ESTHER : Wolfe_1987_Fundam.Appl.Toxicol_9_266
PubMedSearch : Wolfe_1987_Fundam.Appl.Toxicol_9_266
PubMedID: 3653568

Title : Microtiter assay for acetylcholinesterase - Doctor_1987_Anal.Biochem_166_399
Author(s) : Doctor BP , Toker L , Roth E , Silman I
Ref : Analytical Biochemistry , 166 :399 , 1987
Abstract : A microtiter plate adaptation of the classical Ellman colorimetric procedure for measurement of acetylcholinesterase activity is described. This method permits use of an enzyme-linked immunosorbent assay plate reader for rapid analysis of multiple samples and is particularly suitable for analysis of acetylcholinesterase activity on sucrose gradients. The novel procedure is rapid and sensitive and does not require use of radioactive material.
ESTHER : Doctor_1987_Anal.Biochem_166_399
PubMedSearch : Doctor_1987_Anal.Biochem_166_399
PubMedID: 3434781

Title : Kinetic investigations into the interactions of aprophen with cholinesterases and a carboxylesterase - Rush_1986_Biochem.Pharmacol_35_4167
Author(s) : Rush RS , Doctor BP , Wolfe AD
Ref : Biochemical Pharmacology , 35 :4167 , 1986
Abstract : Acetylcholinesterases, butyrylcholinesterases, and carboxylesterases appear to form kinetically a homologous enzyme series with respect to many substrates and inhibitors. The present paper evaluates the interaction of aprophen with acetylcholinesterases, butyrylcholinesterases, and carboxylesterases with respect to protecting the enzyme from organophosphate and carbamate inhibition, accelerating pralidoxime iodide (2-PAM) regeneration of the diisopropylphospho-enzyme, and comparing the inhibition and regeneration kinetics of a soluble mammalian acetylcholinesterase with that of bovine erythrocyte acetylcholinesterase. The irreversible inhibition kinetics of diisopropyl fluorophosphate (DFP) and eserine inhibition of fetal bovine serum acetylcholinesterase were typical of other acetylcholinesterases as indicated by the bimolecular inhibition rate constants, ki, of 7.7 +/- 1.3 X 10(4) M-1 min-1 and 2.9 +/- 1.7 X 10(6) M-1 min-1, respectively. Similarly, the bimolecular regeneration rate constant, kr, for 2-PAM regeneration of the diisopropylphospho-acetylcholinesterase was 14.7 M-1 min-1. The bimolecular rate constants, ki and kr, were not statistically perturbed when the reaction was monitored in the presence of aprophen with the fetal bovine serum acetylcholinesterase. Human serum butyrylcholinesterase was partially protected from DFP inhibition by aprophen with no detectable change in the bimolecular inhibition rate constant, ki. The regeneration of the diisopropylphospho-butyrylcholinesterase by 2-PAM was accelerated in the presence of aprophen by a factor of 2.7 over that of 2-PAM alone (8.4 +/- 2.2 M-1 min-1 to 23.1 +/- 2.6 M-1 min-1 respectively). Neither the inhibition (DFP) nor the regeneration (2-PAM) kinetics observed for the carboxylesterase was perturbed by the presence of aprophen.
ESTHER : Rush_1986_Biochem.Pharmacol_35_4167
PubMedSearch : Rush_1986_Biochem.Pharmacol_35_4167
PubMedID: 3098245

Title : Enzymatic synthesis of a false cholinergic neurotransmitter: diethylaminoethyl acetate as a muscarinic agonist analog of acetylcholine - Miura_1986_Gen.Pharmacol_17_477
Author(s) : Miura GA , Chiang PK , Gordon RK , Doctor BP , Twine CE , Philip A , Kepler JA , Carroll FI
Ref : General Pharmacology , 17 :477 , 1986
Abstract : Diethylaminoethyl acetate, an acetylcholine analog, was formed upon the incubation of diethylaminoethanol and acetyl-CoA with bovine brain choline acetyltransferase (acetyl-CoA: choline O-acetyltransferase; EC 2.3.1.6). The new product co-chromatographed with authentic diethylaminoethyl acetate on thin layer plates, and its formation was proportional to the duration of incubation and enzyme concentrations. When tested on guinea-pig ileum, diethylaminoethyl acetate was found to be an agonist with an ED50 of 1.3 X 10(-4) M, compared to an ED50 of 2.0 X 10(-7) M for acetylcholine. The contraction of guinea-pig ileum induced by diethylaminoethyl acetate was blocked by atropine. Moreover, diethylaminoethyl acetate induced a secretion of alpha-amylase from isolated pancreatic acini cells; this effect was also blocked by atropine. It is entirely possible that diethylaminoethyl acetate can be a false cholinergic transmitter generated in vivo when drugs such as aprophen or procaine are administered to animals, since either of these drugs can undergo enzymatic hydrolysis to generate diethylaminoethanol. A method for the synthesis of radioactive diethylamino [1,2-14C]ethyl acetate was also described.
ESTHER : Miura_1986_Gen.Pharmacol_17_477
PubMedSearch : Miura_1986_Gen.Pharmacol_17_477
PubMedID: 2875921

Title : A simplified procedure for the purification of large quantities of fetal bovine serum acetylcholinesterase - De La Hoz_1986_Life.Sci_39_195
Author(s) : De La Hoz DM , Doctor BP , Ralston JS , Rush RS , Wolfe AD
Ref : Life Sciences , 39 :195 , 1986
Abstract : A simple procedure has been developed for the large scale purification of fetal bovine serum acetylcholinesterase (AChE) (EC 3.1.1.7). The procedure involves two steps: batch adsorption of the AChE from 250 L of serum onto a procainamide affinity Sepharose 4B gel; and analytical procainamide affinity chromatography of the step-1 product. Over 100 mg of AChE was purified in 10 days to apparent homogeneity with this procedure.
ESTHER : De La Hoz_1986_Life.Sci_39_195
PubMedSearch : De La Hoz_1986_Life.Sci_39_195
PubMedID: 3736320

Title : Disposition of aprophen in rats - Aarbakke_1986_J.Pharm.Pharmacol_38_928
Author(s) : Aarbakke J , Miura GA , Brown ND , Gray RR , Gordon RK , Doctor BP , Chiang PK
Ref : J Pharm Pharmacol , 38 :928 , 1986
Abstract : The pharmacokinetics of [14C]aprophen and its distribution were determined after intravenous administration to rats. The drug was distributed rapidly with a t1/2 (alpha) of 4 min to highly perfused organs like the brain, kidney and adrenals. An elimination phase was apparent 10 min after injection with a t1/2 (beta) of 23.5 min. The high plasma clearance of the drug was due both to a large volume of distribution and to a high metabolic rate. Aprophen could be hydrolysed to diphenylpropionic acid and diethylaminoethanol in-vivo and in-vitro. Diethylaminoethanol competed with [3H]QNB binding to muscarinic receptors of N4TG1 cells, whereas diphenylpropionic acid did not. The lower plasma concentrations and lower binding activity of diethylaminoethanol compared with aprophen indicate that unchanged aprophen is largely responsible for the in-vivo actions.
ESTHER : Aarbakke_1986_J.Pharm.Pharmacol_38_928
PubMedSearch : Aarbakke_1986_J.Pharm.Pharmacol_38_928
PubMedID: 2880971

Title : Acetylcholinesterase from fetal bovine serum. Purification and characterization of soluble G4 enzyme - Ralston_1985_J.Biol.Chem_260_4312
Author(s) : Ralston JS , Rush RS , Doctor BP , Wolfe AD
Ref : Journal of Biological Chemistry , 260 :4312 , 1985
Abstract : Acetylcholinesterase (EC 3.1.1.7) from fetal bovine serum (FBS) was purified to electrophoretic homogeneity. The procedure involved procainamide affinity chromatography with native FBS, followed by chromatography on Sepharose 6B and DEAE-Sephadex. The acetylcholinesterase was purified approximately 44,000-fold, and 13 mg was obtained corresponding to an overall yield of about 45%. The purified acetylcholinesterase was stable at 4 degrees C for at least 8 weeks but was labile to freezing; however, in 50% glycerol the enzyme was stable at -20 degrees C for at least 12 weeks. FBS acetylcholinesterase exhibited typical substrate inhibition, had a Km of 120 microM, and a turnover number of 5300 s-1 with the substrate acetylthiocholine. The enzyme was highly sensitive to the specific acetylcholinesterase inhibitor 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one. FBS acetylcholinesterase was characterized as a G4 form of acetylcholinesterase and was distinguished from bovine erythrocyte acetylcholinesterase on the basis of lectin gel binding, [3H] Triton X-100 binding, amino acid composition, number of catalytic subunits/molecule, and hydrodynamic properties. FBS acetylcholinesterase had a Stokes radius of 76 A as judged by gel filtration, and from this a molecular weight of 340,000 daltons was calculated. The enzyme had a subunit weight of approximately 83,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; paraoxon titration indicated a relative active site mass of 75,000 daltons. The amino acid composition of FBS acetylcholinesterase was similar to the human erythrocyte acetylcholinesterase (Rosenberry, T. L., and Scoggin, D. M. (1984) J. Biol. Chem. 259, 5643-5652). A monoclonal antibody directed against human erythrocyte acetylcholinesterase, AE-2, (Fambrough, D. M., Engel, A. G., and Rosenberry, T. L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1078-1082) cross-reacted with FBS acetylcholinesterase.
ESTHER : Ralston_1985_J.Biol.Chem_260_4312
PubMedSearch : Ralston_1985_J.Biol.Chem_260_4312
PubMedID: 3980478

Title : Stability study of HI-6 dichloride in various anticholinergic formulations -
Author(s) : Brown ND , Gray RR , Stermer-Cox MG , Doctor BP , Hagedorn I
Ref : Journal of Chromatography , 315 :389 , 1984
PubMedID: 6526905

Title : Poster 8. Antigenic and structural differences in the catalytic subunits of the molecular forms of acetylcholinesterase -
Author(s) : Doctor BP , Taylor P
Ref : In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases , (Brzin M, Barnard EA, Sket D, Eds) De Gruyter , 1984
PubMedID:

Title : Molecular aspects of the biosynthesis and disposition of multiple forms of acetylcholinesterase -
Author(s) : Taylor P , Camp S , Lee SL , Amitai G , Taylor SS , Doctor BP
Ref : In: Cholinesterases, fundamental and applied aspects : proceedings of the Second International Meeting on Cholinesterases , (Brzin M, Barnard EA, Sket D, Eds) De Gruyter :145 , 1984
PubMedID:

Title : Antigenic and structural differences in the catalytic subunits of the molecular forms of acetylcholinesterase - Doctor_1983_Proc.Natl.Acad.Sci.U.S.A_80_5767
Author(s) : Doctor BP , Camp S , Gentry MK , Taylor SS , Taylor P
Ref : Proceedings of the National Academy of Sciences of the United States of America , 80 :5767 , 1983
Abstract : A mixture of the 5.6S hydrophobic dimer and the asymmetric, tail-containing (17 + 13)S forms of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) from Torpedo californica was used to immunize mice, and spleen cells from these mice were used to produce nine hybridoma lines secreting antibodies against acetylcholinesterase. Antibodies from one of the lines showed a 100-fold greater affinity for the 5.6S species when compared with the catalytic subunits of the (17 + 13)S species. This difference in specificity was retained after denaturation of the two acetylcholinesterase species. Another line produced antibody directed only to structural subunits of the (17 + 13)S species, whereas the remaining seven antibodies exhibited nearly equivalent crossreactivity for all of the forms of acetylcholinesterase. Tryptic peptides were generated from the catalytic subunits of the 5.6S and tail-containing acetylcholinesterase species, and high-pressure liquid chromatographic profiles show at least two distinct peptides in the catalytic subunits for each enzyme species. Some of these peptides exhibit retention times different from those of the identified glycopeptides. Thus, it is likely that the catalytic subunits of two molecular forms of acetylcholinesterase differ in primary structure and sites of antigenicity.
ESTHER : Doctor_1983_Proc.Natl.Acad.Sci.U.S.A_80_5767
PubMedSearch : Doctor_1983_Proc.Natl.Acad.Sci.U.S.A_80_5767
PubMedID: 6193524

Title : Antimuscarinic activity of aprophen -
Author(s) : Gordon RK , Padilla FN , Moore E , Doctor BP , Chiang PK
Ref : Biochemical Pharmacology , 32 :2979 , 1983
PubMedID: 6626268

Title : A radioactive assay for acetylcholinesterase using anion-exchange disk -
Author(s) : Gordon RK , Doctor BP , Chiang PK
Ref : Analytical Biochemistry , 124 :333 , 1982
PubMedID: 6756205

Title : Inhibition of ribonucleic acid degradation in bacteria by spermine -
Author(s) : Herbst EJ , Doctor BP
Ref : Journal of Biological Chemistry , 234 :1497 , 1959
PubMedID: 13654404