Department of Regulated Laboratories, Division of Regulated Activities, Silver Spring, MD 20910, United States.
Life Sci. 2011 Dec 19;89(25-26):911-7. doi: 10.1016/j.lfs.2011.09.019. Epub 2011 Oct 4.
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.
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.
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.
The present study shows that pro-2-PAM was rapidly oxidized by riboflavin to 2-PAM, which reactivated organophosphate (OP)-inhibited AChE.
由于氯解磷定（2-PAM）带正电荷，其穿透血脑屏障（BBB）和重新激活有机磷（OP）抑制中枢神经系统（CNS）乙酰胆碱酯酶（AChE）的能力较差。CNS 抑制 AChE 的结果是癫痫发作。前体-2-PAM（1-甲基-1,6-二氢吡啶-2-甲酰肟），2-PAM 的前体药物，由于更高的疏水性，更好地穿透 BBB，但必须被氧化为 2-PAM，肟的活性形式，以重新激活 CNS AChE 以消除癫痫发作。在这项研究中，我们描述了前体-2-PAM 氧化的体内机制。
开发了一种高效液相色谱（HPLC）测定法来定量测定前体-2-PAM 转化为 2-PAM。使用荧光素底物通过光致发光测定法测量 NADPH 氧化酶活性。分析表明，NADPH 诱导的氧化速率表明可能涉及替代机制。因此，评估了氧化还原酶系统的各种酶辅助因子，包括烟酰胺腺嘌呤二核苷酸（NAD）、烟酰胺腺嘌呤二核苷酸磷酸（NADP）、黄素腺嘌呤二核苷酸（FAD）、核黄素 5'-磷酸（FMN）和核黄素。接下来，开发了一种分光光度法测定法来测量在核黄素存在下前体-2-PAM 转化为 2-PAM。
在豚鼠脑匀浆中，二苯基碘鎓（DPI），一种特异性 NADPH 氧化酶抑制剂，将前体-2-PAM 转化为 2-PAM 的转化率降低至 25%以下。相比之下，核黄素、FAD 和 FMN 在体外测定中迅速将所有前体-2-PAM 氧化为 2-PAM。核黄素氧化前体-2-PAM 重新激活了二异丙基氟磷酸酯（DFP）抑制的 AChE。
本研究表明，前体-2-PAM 被核黄素迅速氧化为 2-PAM，后者重新激活了有机磷（OP）抑制的 AChE。
