Study of blood-brain barrier penetration of synthesized PAM analogues

本研究的目的是开发一种新的解毒剂，可以很容易地穿透血脑屏障（BBB）的有机磷中毒。我们合成了几种烷基化的PAM类似物作为VX或沙林中毒的解毒剂，并尝试用Ellman等人改进的方法测定它们的AChE再激活活性。然而，2-PAM剂量依赖性地水解碘化乙酰硫代胆碱（ASCh）。VX类似物抑制的AChE（0.3U）活性在5 mM 2-PAM作用下可恢复到正常水平的200%左右。HPLC分析进一步阐明了2-PAM通过水解由ASCh产生的乙酸转化为乙酰化的2-PAM。其他肟也表现出酯酶样活性，其强度与已知的抑制乙酰胆碱酯酶的再激活剂是一致的。这些结果表明，以前获得的数据与ASCh有关肟的影响，必须重新检查。接下来，选择了6种PAM类似物（2-PATB、3-PATB、4-PATB、4-PAPE、4-PAD和4-PAO），其对INMP暴露的人红细胞具有相对高的再活化活性。经大鼠尾静脉给药，测定其LO_<50>（mg/kg）值。它们的PAM类似物的毒性比2-PAM强。采用在体大鼠脑微透析技术结合LC-MS/MS测定PAM类似物的血脑屏障渗透性。静脉注射PAM类似物（10%LD 60）后，透析液中出现4-PAPE和4-PAO。这一发现表明，这些PAM类似物可以穿透血脑屏障。

The aim of this study is to develop a new antidote that can easily penetrate the blood-brain barrier (BBB) in organophosphate poisoning. We synthesized various alkylated PAM analogues as an antidote for VX or sarin intoxication and tried to determine their AChE reactivation activities by the method of Ellman et al. with modification. However, 2-PAM dose-dependently hydrolyzed an acetylthiocholine iodide (ASCh). The AChE (0.3U) activity inhibited by VX analog increased to approximately 200% of normal levels after a dosage of 5mM 2-PAM. HPLC analysis further clarified that 2-PAM was converted to acetylated 2-PAM with acetic acid produced from ASCh by hydrolysis. Other oximes also showed esterase-like activity, and their strengths were consistent with those of known reactivators of inhibited AChE. These results indicate that much of the previous data obtained with ASCh relating to the effects of oximes must be rechecked. Next, 6 PAM analogues (2-PATB, 3-PATB, 4-PATB, 4-PAPE, 4-PAD, and 4-PAO), which had relatively high reactivation activities to INMP-exposed human erythrocytes, were selected. Their PAM analogues were administered intravenously via the rat tail vein, and their LO_<50> (mg/kg) values were examined. The toxicities of their PAM analogues were stronger than that of 2-PAM. The in vivo rat brain microdialysis technique with LC-MS/MS was used to determine the BBB penetration of PAM analogues. After intravenous dosage of PAM analogues (10% concentration of LD6o), 4-PAPE and 4-PAO appeared in the dialysate. This finding indicates that these PAM analogues can penetrate across the BBB.