TY - JOUR
T1 - Oxidative mechanisms for the biotransformation of 1-methyl-1,6- dihydropyridine-2-carbaldoxime to pralidoxime chloride
AU - Khan, Farhat A.
AU - Campbell, Amy J.
AU - Hoyt, Benjamin
AU - Herdman, Christine
AU - Ku, Therese
AU - Thangavelu, Sonia
AU - Gordon, Richard K.
N1 - Funding Information:
We thank Julian R. Haigh, Ph.D. and Shahza Somerville, Ph.D. for critical reading of this manuscript. This work was supported by NIH CounterACT U01NS058166 . The opinions or assertions contained herein are private views of the authors, and are not to be construed as official views of the Department of the Army or the Department of Defense.
PY - 2011/12/19
Y1 - 2011/12/19
N2 - Aims: Due to pralidoxime chloride's (2-PAM) positive charge, it's penetration through the blood brain barrier (BBB) and reactivation of organophosphate (OP) inhibited central nervous system (CNS) acetylcholinesterase (AChE) is poor. The results of CNS inhibited AChE are seizures. Pro-2-PAM (1-methyl-1,6-dihydropyridine-2-carbaldoxime), a pro-drug of 2-PAM, due to higher hydrophobicity, penetrates the BBB better but must be oxidized to 2-PAM, the active form of the oxime to reactivate CNS AChE in order to abrogate seizures. In this study, we characterize the in vivo mechanism of pro-2-PAM oxidation. Main methods: A high pressure liquid chromatography (HPLC) assay was developed to quantify the conversion of pro-2-PAM to 2-PAM. NADPH oxidase activity was measured by a photo-luminescence assay using lucigenin substrate. Upon analysis, the rate of NADPH induced oxidation suggested that an alternate mechanism may be involved. Therefore, various enzyme co-factors of oxidation-reduction enzyme systems were evaluated, including nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD), riboflavin 5′-phosphate (FMN), and riboflavin. Next, a spectrophotometric assay was developed to measure the conversion of pro-2-PAM to 2-PAM in the presence of riboflavin. Key findings: In guinea pig brain homogenate, diphenyleneiodonium (DPI), a specific NADPH oxidase inhibitor, reduced pro-2-PAM to 2-PAM conversion to less than 25%. In contrast, riboflavin, FAD, and FMN rapidly oxidized all pro-2-PAM to 2-PAM in an in vitro assay. Riboflavin oxidized pro-2-PAM reactivated diisopropyl- fluorophosphate (DFP) inhibited AChE. Significance: The present study shows that pro-2-PAM was rapidly oxidized by riboflavin to 2-PAM, which reactivated organophosphate (OP)-inhibited AChE.
AB - Aims: Due to pralidoxime chloride's (2-PAM) positive charge, it's penetration through the blood brain barrier (BBB) and reactivation of organophosphate (OP) inhibited central nervous system (CNS) acetylcholinesterase (AChE) is poor. The results of CNS inhibited AChE are seizures. Pro-2-PAM (1-methyl-1,6-dihydropyridine-2-carbaldoxime), a pro-drug of 2-PAM, due to higher hydrophobicity, penetrates the BBB better but must be oxidized to 2-PAM, the active form of the oxime to reactivate CNS AChE in order to abrogate seizures. In this study, we characterize the in vivo mechanism of pro-2-PAM oxidation. Main methods: A high pressure liquid chromatography (HPLC) assay was developed to quantify the conversion of pro-2-PAM to 2-PAM. NADPH oxidase activity was measured by a photo-luminescence assay using lucigenin substrate. Upon analysis, the rate of NADPH induced oxidation suggested that an alternate mechanism may be involved. Therefore, various enzyme co-factors of oxidation-reduction enzyme systems were evaluated, including nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD), riboflavin 5′-phosphate (FMN), and riboflavin. Next, a spectrophotometric assay was developed to measure the conversion of pro-2-PAM to 2-PAM in the presence of riboflavin. Key findings: In guinea pig brain homogenate, diphenyleneiodonium (DPI), a specific NADPH oxidase inhibitor, reduced pro-2-PAM to 2-PAM conversion to less than 25%. In contrast, riboflavin, FAD, and FMN rapidly oxidized all pro-2-PAM to 2-PAM in an in vitro assay. Riboflavin oxidized pro-2-PAM reactivated diisopropyl- fluorophosphate (DFP) inhibited AChE. Significance: The present study shows that pro-2-PAM was rapidly oxidized by riboflavin to 2-PAM, which reactivated organophosphate (OP)-inhibited AChE.
KW - 1-Methyl-1,6-dihydropyridine-2-carbaldoxime
KW - Acetylcholinesterase
KW - Blood brain barrier
KW - NADPH oxidase
KW - Pralidoxime chloride
KW - Pro-drug
KW - Riboflavin
UR - http://www.scopus.com/inward/record.url?scp=84855723753&partnerID=8YFLogxK
U2 - 10.1016/j.lfs.2011.09.019
DO - 10.1016/j.lfs.2011.09.019
M3 - Article
C2 - 21989207
AN - SCOPUS:84855723753
SN - 0024-3205
VL - 89
SP - 911
EP - 917
JO - Life Sciences
JF - Life Sciences
IS - 25-26
ER -