Human Serum Butyrylcholinesterase: A Bioscavenger for the Protection of Humans from Organophosphorus Exposure

Human serum butyrylcholinesterase (Hu BChE) is currently under advanced development as a pretreatment drug for organophosphate (OP) poisoning in humans. Toward this effort, a procedure for the large-scale purification of Hu BChE from Cohn fraction IV-4 was developed, its pharmacokinetic properties were established, its shelf life was evaluated at various temperatures, and its safety and efficacy were examined. It was shown to protect mice, rats, guinea pigs, and monkeys against multiple LD50 challenges of OP nerve agents by i.v. or s.c. bolus injections. Since inhalation is the most likely route of exposure 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. The study was conducted in minipigs because the pig offers many similarities in anatomy and physiology to humans. Male Göttingen minipigs were subjected to one of the following treatments: (1) Air exposure; (2) GB vapor exposure; and (3) pretreatment with 7.5 mg/kg of Hu BChE followed by GB vapor exposure. Hu BChE was administered by i.m. injection, 24 h prior to whole-body exposure to GB vapor at a concentration of 4.1 mg/m3 for 60 min or 11.4 mg/m3 for 10 min. 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. Baseline blood AChE and BChE activities were 2.5 ± 0.1 U/ml and 0.14 ± 0.01 U/ml, respectively. Animals pretreated with 7.5 mg/kg of Hu BChE showed peak blood BChE activity of 41.2 ± 0.9 U/ml with a mean residence time of 287 ± 53 h. Similar retention times of 225 ± 19 h for monkey BChE in macaques and 8-11 days for Hu BChE in humans reported previously suggest that there is a high homology between human and pig enzymes. All animals exposed to GB vapor for 60 min showed signs of cardiac and neurological toxicity and died following exposure. Animals exposed to GB vapor for 10 min also died, but showed signs of cardiac toxicity only. All animals pretreated with 7.5 mg/kg of Hu BChE survived the GB exposure. Additionally, 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, and preventing it from inhibiting CNS AChE. Pretreatment with Hu BChE prevented cardiac abnormalities and seizure activity observed in untreated animals. These results provide convincing data that the use of Hu BChE would provide a capability for extended protection against a wide spectrum of nerve agents and would eliminate the need for extensive post-exposure therapy. In addition, the objective force will be able to take the advantage of these technologies, which will provide sustained maximum Report Documentation Page Form Approved OMB No. 0704-0188 Public reporting burden for the collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for Information Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arlington VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to a penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. 1. REPORT DATE OCT 2009 2. REPORT TYPE N/A 3. DATES COVERED 4. TITLE AND SUBTITLE Human Serum Butyrylcholinesterase: A Bioscavenger for the Protection of Humans from Organophosphorus Exposure 5a. CONTRACT NUMBER 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) Divisions of Bacterial & Rickettsial Diseases1 and Biochemistry Walter Reed Army Institute of Research Silver Spring, MD 20910-7500 USA 8. PERFORMING ORGANIZATION REPORT NUMBER 9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 12. DISTRIBUTION/AVAILABILITY STATEMENT Approved for public release, distribution unlimited 13. SUPPLEMENTARY NOTES See also ADA562561. RTO-MP-HFM-181 Human Performance Enhancement for NATO Military Operations (Science, Technology and Ethics) (Amelioration des performances humaines dans les operations militaires de l’OTAN (Science, Technologie et Ethique)). RTO Human Factors and Medicine Panel (HFM) Symposium held in Sofia, Bulgaria, on 5-7 October 2009., The original document contains color images. 14. ABSTRACT Human serum butyrylcholinesterase (Hu BChE) is currently under advanced development as a pretreatment drug for organophosphate (OP) poisoning in humans. Toward this effort, a procedure for the large-scale purification of Hu BChE from Cohn fraction IV-4 was developed, its pharmacokinetic properties were established, its shelf life was evaluated at various temperatures, and its safety and efficacy were examined. It was shown to protect mice, rats, guinea pigs, and monkeys against multiple LD50 challenges of OP nerve agents by i.v. or s.c. bolus injections. Since inhalation is the most likely route of exposure 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. The study was conducted in minipigs because the pig offers many similarities in anatomy and physiology to humans. Male Göttingen minipigs were subjected to one of the following treatments: (1) Air exposure; (2) GB vapor exposure; and (3) pretreatment with 7.5 mg/kg of Hu BChE followed by GB vapor exposure. Hu BChE was administered by i.m. injection, 24 h prior to whole-body exposure to GB vapor at a concentration of 4.1 mg/m3 for 60 min or 11.4 mg/m3 for 10 min. 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. Baseline blood AChE and BChE activities were 2.5 ± 0.1 U/ml and 0.14 ± 0.01 U/ml, respectively. Animals pretreated with 7.5 mg/kg of Hu BChE showed peak blood BChE activity of 41.2 ± 0.9 U/ml with a mean residence time of 287 ± 53 h. Similar retention times of 225 ± 19 h for monkey BChE in macaques and 8-11 days for Hu BChE in humans reported previously suggest that there is a high homology between human and pig enzymes. All animals exposed to GB vapor for 60 min showed signs of cardiac and neurological toxicity and died following exposure. Animals exposed to GB vapor for 10 min also died, but showed signs of cardiac toxicity only. All animals pretreated with 7.5 mg/kg of Hu BChE survived the GB exposure. Additionally, 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, and preventing it from inhibiting CNS AChE. Pretreatment with Hu BChE prevented cardiac abnormalities and seizure activity observed in untreated animals. These results provide convincing data that the use of Hu BChE would provide a capability for extended protection against a wide spectrum of nerve agents and would eliminate the need for extensive post-exposure therapy. 15. SUBJECT TERMS 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT SAR 18. NUMBER OF PAGES 12 19a. NAME OF RESPONSIBLE PERSON a. REPORT unclassified b. ABSTRACT unclassified c. THIS PAGE unclassified Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 Human Serum Butyrylcholinesterase: A Bioscavenger for the Protection of Humans from Organophosphorus Exposure 36 2 RTO-MP-HFM-181 protection to soldiers under the most adverse battlefield condition, without performance degradation or interruption of OPTEMPO.