Search

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/m3, 1.2 LCt50) and treated with endotracheally aerosolized MMB-4 (50 µmol/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 O2 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. [ABSTRACT FROM AUTHOR]

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/m3 or 846.5 mg/m3 (1.2 LCt50) 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. [ABSTRACT FROM AUTHOR]

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/m3) 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. [ABSTRACT FROM PUBLISHER]

Sarin is a volatile nerve agent that has been used in the Tokyo subway attack. Inhalation is predicted to be the major route of exposure if sarin is used in war or terrorism. Currently available treatments are limited for effective postexposure protection against sarin under mass casualty scenario. Nasal drug delivery is a potential treatment option for mass casualty under field conditions. We evaluated the efficacy of endotracheal administration of muscarinic antagonist scopolamine, a secretion blocker which effectively crosses the blood-brain barrier for protection against sarin inhalation toxicity. Age and weight matched male Hartley guinea pigs were exposed to 677.4 mg/m3 or 846.5 mg/ m3 (1.2 ×× LCt50) sarin by microinstillation inhalation exposure for 4 min. One minute later, the animals exposed to 846.5 mg/ m3 sarin were treated with endotracheally aerosolized scopolamine (0.25 mg/kg) and allowed to recover for 24 h for efficacy evaluation. The results showed that treatment with scopolamine increased the survival rate from 20% to 100% observed in untreated sarin-exposed animals. Behavioral symptoms of nerve agent toxicity including, convulsions and muscular tremors were reduced in sarin-exposed animals treated with scopolamine. Sarin-induced body weight loss, decreased blood O2 saturation and pulse rate were returned to basal levels in scopolamine-treated animals. Increased bronchoalveolar lavage (BAL) cell death due to sarin exposure was returned to normal levels after treatment with scopolamine. Taken together, these data indicate that postexposure treatment with aerosolized scopolamine prevents respiratory toxicity and protects against lethal inhalation exposure to sarin in guinea pigs. [ABSTRACT FROM AUTHOR]

Barometric whole-body plethysmography (WBP) was used to examine pulmonary functions at 4 and 24 hours postexposure to soman (GD) in guinea pigs without therapeutics to improve survival. Endotracheal aerosolization by microinstillation was used to administer GD (280, 561, and 841 mg/m3) or saline to anesthetized guinea pigs. Significant increases in respiratory frequency (RF), tidal volume (TV), and minute volume (MV) were observed with 841 mg/m3 GD at 4 hours and that were reduced at 24 hours postexposure. A dose-dependent increase in peak inspiration flow and peak expiration flow was present at 4-hour post-GD exposure that was reduced at 24 hours. Time of inspiration and expiration were decreased in all doses of GD exposure at 4 and 24 hours, with significant inhibition at 841 mg/m3. End-expiratory pause (EEP) increased at 280 and 561 mg/m3, but decreased in animals exposed 841 mg/m3 at 24 hours postexposure. Pseudo-lung resistance (Penh) and pause followed similar patterns and increased at 4 hours, but decreased at 24 hours postexposure to 841 mg/m3 of GD compared to control. These studies indicate GD exposure induces dose-dependent changes in pulmonary function that are significant at 841 mg/m3 at 4 hours and remains 24 hours postexposure. Furthermore, at 4 hours, GD induces bronchoconstriction possibly due to copious airway secretion and ongoing lung injury in addition to cholinergic effects, while at 24 hours GD induces bronchodilation a possible consequence of initial compensatory mechanisms. [ABSTRACT FROM PUBLISHER]

The efficacy of endotracheal aerosolization of atropine sulfate for protection against soman (GD)-induced respiratory toxicity was investigated using microinstillation technique in guinea pigs. GD (841 mg/m3, 1.3 LCt50 or 1121 mg/m3, 1.7 LCt50) was aerosolized endotracheally to anesthetized male guinea pigs that were treated with atropine sulfate (5.0 mg/kg) 30 s postexposure by endotracheal microinstillation. Animals exposed to 841 mg/m3 and 1121 mg/m3GD resulted in 31 and 13% while treatment with atropine sulfate resulted in 100 and 50% survival, respectively. Cholinergic symptoms and increased body weight loss were reduced in atropine-treated animals compared to GD controls. Diminished pulse rate and blood O2 saturation in GD-exposed animals returned to normal levels after atropine treatment. Increased cell death, total cell count and protein in the bronchoalveolar fluid (BALF) in GD-exposed animals returned to normal levels following atropine treatment. GD exposure increased glutathione and superoxide dismutase levels in BALF and that were reduced in animals treated with atropine. Respiratory parameters measured by whole-body barometric plethysmography revealed that treatment with atropine sulfate resulted in normalization of respiratory frequency, tidal volume, time of expiration, time of inspiration, end expiratory pause, pseudo lung resistance (Penh) and pause at 4 and 24 h post 841 mg/m3 GD exposure. Lung histopathology showed that atropine treatment reduced bronchial epithelial subepithelial inflammation and multifocal alveolar septal edema. These results suggest that endotracheal aerosolization of atropine sulfate protects against respiratory toxicity and lung injury induced by microinstillation inhalation exposure to lethal doses of GD. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We investigated the toxic effects of the chemical warfare nerve agent (CWNA) soman (GD) on the respiratory dynamics of guinea pigs following microinstillation inhalation exposure. Male Hartley guinea pigs were exposed to 841 mg/m3 of GD or saline for 4 min. At 24 and 48 h post GD exposure, respiratory dynamics and functions were monitored for 75 min after 1 h of stabilization in a barometric whole-body plethysmograph. GD-exposed animals showed a significant increase in respiratory frequency (RF) at 24 h postexposure compared to saline controls. The 24-h tidal volume (TV) increased in GD-exposed animals during the last 45 min of the 75-min monitoring period in the barometric whole-body plethysmograph. Minute ventilation also increased significantly at 24 h post GD exposure. The peak inspiratory flow (PIF) increased, whereas peak expiratory flow (PEF) decreased at 24 h and was erratic following GD exposure. Animals exposed to GD showed a significant decrease in expiratory (Te) and inspiratory time (Ti). Although end inspiratory pause (EIP) and end expiratory pause (EEP) were both decreased 24 h post GD exposure, EEP was more evident. Pause (P) decreased equally during the 75-min recording in GD-exposed animals, whereas the pseudo lung resistance (Penh) decreased initially during the monitoring period but was near control levels at the end of the 75-min period. The 48-h respiratory dynamics and function parameter were lower than 24 post GD exposures. These results indicate that inhalation exposure to soman in guinea pigs alters respiratory dynamics and function at 24 and 48 h postexposure. [ABSTRACT FROM AUTHOR]

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 2 cm 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 24 h 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 microinstillation inhalation VX exposure induces gastrointestinal disturbances similar to that of irritable bowel syndrome and bloating. [ABSTRACT FROM AUTHOR]

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 μg/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. [ABSTRACT FROM AUTHOR]

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 μg/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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Examines the toxicology and the binding site of huperzine A (HUP-A). Reduction of glutamate-induced cell death; Benefits of HUP-A for excitatory amino acid overstimulation; Interaction of HUP-A with glutamate receptors.

Butyrylcholinesterase (Eq-BChE) from equine (horse) serum has been used extensively to demonstrate the efficacy of cholinesterase as a single pretreatment drug (bioscavenger) for organophosphate toxicity in rodents and nonhuman primates. It has the longest mean retention time in animals among all ChE preparations examined. The authors describe the complete amino acid sequence of Eq-BChE. Like other mammalian BChEs, it is a glycosylated protein composed of four identical subunits. The apparent molecular weight of the subunit containing 574 amino acids and the estimated carbohydrate content of 26% is 84,551 Da. It has eight carbohydrate-modified sites at positions 57, 106, 241, 256, 341, 455, 481, and 486 as N-linked Asn residues. The active site charged triad aminoacids are Ser198, Glu325, and His438. The N-terminal aminoacids are GluGluAspIle, consistent with this being the mature form of the enzyme. In addition, a monomeric form of this enzyme that corresponds to 4% of the total BChE activity was isolated and purified. Comparison of Eq-BChE with other species shows that there is a high degree of primary sequence identity. There is a 90.1% identity with the human serum BChE sequence, although the equine enzyme has one less N-glycosylation site and Cys residue. If conserved amino acid substitutions are included then the proteins share 93.4% homology. [ABSTRACT FROM AUTHOR]

Human serum butyrylcholinesterase (Hu BChE) is currently under advance development for use as a pretreatment antidote against nerve agent toxicity. Due to the limited availability and cost of purification, the production of recombinant (r) Hu BChE is being pursued. Prior to testing in humans the safety of rHu BChE needs to be determined in animals. The problems associated with antibody production due to altered glycosylation patterns and sequence differences inherent to heterologous systems, highlight the need for developing homologous non-human primate models for conducting evaluations with rBChEs. In this study, recombinant macaque (Ma) BChE was cloned and expressed in CHO cells and the biochemical properties of both monomeric and tetrameric forms of rMa BChEs and their PEGylated forms were compared with native Hu and Ma BChE. Catalytic parameters were determined using butyrylthiocholine as the substrate and organophosphate scavenging capabilities were evaluated by measuring inhibitions of BChEs by di-isopropylfluorophosphate (DFP), and aging and reactivation of DFP-inhibited enzymes. Results show that the enzymatic and inhibitory properties of rMa BChEs are similar to those of native Ma and Hu BChE, and were not affected by PEGylation. However, the reactivation of DFP-inhibited Ma BChE was significantly slower than Hu BChE. Studies are underway to explain these differences. [ABSTRACT FROM AUTHOR]