title: Oxidative mechanisms for the biotransformation of 1-methyl-1,6-dihydropyridine-2-carbaldoxime to pralidoxime chloride.
abstract: OBJECTIVE
  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.
  
  
  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.
  
  
  RESULTS
  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 diisopropylfluorophosphate (DFP) inhibited AChE.
  
  
  CONCLUSIONS
  The present study shows that pro-2-PAM was rapidly oxidized by riboflavin to 2-PAM, which reactivated organophosphate (OP)-inhibited AChE.
authors: Department of Regulated Laboratories, Division of Regulated Activities, Silver Spring, MD 20910, United States. farhat.khan@amedd.army.mil
authors: Farhat A
authors: FA
authors: Khan
authors: Amy J
authors: AJ
authors: Campbell
authors: Benjamin
authors: B
authors: Hoyt
authors: Christine
authors: C
authors: Herdman
authors: Therese
authors: T
authors: Ku
authors: Sonia
authors: S
authors: Thangavelu
authors: Richard K
authors: RK
authors: Gordon
mesh_terms: D000110:Acetylcholinesterase / Q000378:metabolism*; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006168:Guinea Pigs; D019255:NADPH Oxidases / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D010084:Oxidation-Reduction; D011220:Pralidoxime Compounds / Q000378:metabolism*; D011355:Prodrugs; D051381:Rats; D012256:Riboflavin / Q000494:pharmacology; D013053:Spectrophotometry / Q000379:methods
chemical_list: D009861:Onium Compounds; D011220:Pralidoxime Compounds; D011355:Prodrugs; C011920:1-methyl-1,6-dihydropyridine-2-carbaldoxime; C007517:diphenyleneiodonium; D019255:NADPH Oxidases; D000110:Acetylcholinesterase; C028797:pralidoxime; D012256:Riboflavin