摘要
梭曼抑制乙酰胆碱酯酶(AChE)后,生成的膦酰化乙酰胆碱酯酶随时间延长将发生两种主要形式的转化:一种是自发或在药物作用下的脱膦酰基重活化过程,中毒酶恢复活力,重活化过程的主要产物是甲基膦酸嚬哪酯(PMPA);另一种是脱烷基老化过程,中毒酶失去重活化能力,主要产物是甲基膦酸(MPA)。梭曼膦酰化AChE的迅速老化和难重活化一直是梭曼难防难治的主要原因和抗毒药物研发的难点之一。即使像HI-6这样有效的重活化剂对梭曼膦酰化酶的重活化作用也是有限的。近年来,国外研究者发现一些季铵类化合物可显著提高肟类的重活化作用,与此同时,我所研究发现叔铵类药物L-1978和L-1655等也能增强肟类的重活化作用,因此,揭示药物增强重活化的作用机理将对梭曼抗毒药物的设计开发具有重大意义。
在评价梭曼抗毒药物的重活化效率时,通常人们采用酶活力测定法和同位素测定法等研究抗毒药物增强重活化的作用机理,但结果并不十分理想。本课题首次采用GC/MS等现代仪器分析技术,建立药物作用于梭曼膦酰化AChE后定量释放出PMPA和MPA的样品处理和检测方法,研究药物对梭曼膦酰化酶重活化过程和老化过程的影响,探寻新叔铵类药物对梭曼膦酰化AChE增强重活化的可能作用机理。
论文分为三章:
第一章前言,简要介绍了重活化剂发展的主要历程,影响膦酰化AChE重活化的主要因素,以及肟类重活化剂的几种作用机理;介绍了我所对梭曼膦酰化AChE重活化药物和作用机理的研究现状和存在问题,并在此基础上提出了论文的研究目的和研究内容。
After acetylcholinesterase (AChE) inhibited by pinacolyl methylphospho-nofluidate (soman), the phosphonylated AChE could go through two processes which may affect oxime effectiveness. One is that the phosphonylated AChE complex may undergo spontaneous dephosphonylation ("spontaneous reactivation"), a process which can be accelerated by several orders of magnitude by the addition of some oxime compounds. The activity of AChE would recover and the main degradation product of this process is pinacolyl methylphosphonate (PMPA). The other process is spontaneous dealkylation through alkyl-oxygen bond scission ("aging"), irreversibly resulting in an inactivated enzyme. The main degradation product of this process is methylphosphonate (MPA). The most difficulties in developing soman antidotes are the fast aging and resistance to the reactivation of soman-inhibited AChE. The high efficient oxime, 1-(((4-(aminocarbonyl) pyridino)-methoxy) methyl-2-(hydroxyimino methyl) pyridinium) dicholoride (HI-6), shows the better reactivation efficiency for phosphonylated AChE inhibited by most nerve agents, but gives only limit effect when treated soman inhibited AChE. However, some foreign researchers have discovered that the reactivation efficiency of oximes for the soman phosphonylated AChE could be significantly increased when they cooperated with quaternary ammonium compounds. Our institute has also found that tertiary ammonium compounds, such as L-1978 and L-1655 had the same ability to improve the reactivation effects, In order to exploit more powerful reactivators, it is very important to investigate the mechanism of
the enhanced reactivation of these drugs.Ellman colorimetric method and isotopic determination method have been widely used to evaluate the efficiency of reactivators. But the results obtained by these methods were indirect, and were greatly influenced by some experimental factors. In this dissertation, the gas chromatography-mass spectrometric method (GC/MS) and other modern techniques were used to quantitatively determine PMPA and MPA, which were released from the phosphonylated AChE complex after which was digested with trypsin and alkaline phosphatase calf intestinal (CIAP). The influence of the drugs on the course of reactivation and aging were studied. The accurate mechanisms of retardation aging or enhanced reactivation were explored.This dissertation consists of three chapters.Chapter one was the preface of the dissertation. The development of reactivators' drugs, the main influence factors of reactivation efficiency and several reactivation mechanisms were briefly d escribed. The progress of reactivators of soman inhibited AChE and their mechanism studied in our institute were also introduced. The research proposals and contents to be carried out were brought forward based on the present difficulties.In chapter two, a simple and accurate GC/MS method was developed to simultaneously determine the dissociative PMPA and MPA in water samples and human plasma samples, as their trimethylsilyl derivatives. Solid-phase extraction method was chosen as the sample preparation method after compared with liquid-liquid extraction method. The selectivity of different solid-phase extraction cartridges and efficiency of various deproteinization reagents were investigated. The loaded pH and concentration of eluent were also optimized.Based on the above method, we established the determination method of PMPA and MPA which were quantitatively released from phosphonylated AChE after
digestion. The quantity of soman inhibited AChE, the preparation method and digestion conditions of phosphonylated AChE were discussed. The influences of metal ions and residual drugs have also been investigated. At the optimum conditions, linear calibration curves were obtained over concentrations ranging from 50 ng/mL to 5 jig/mL in selected ion monitoring mode, and the quantification limits for PMPA and MPA were 10 ng/mL and 15 ng/mL, respectively. The within-day and between-day accuracy were less than 10% and 13%, respectively. This present method eliminated the influence of residual inhibitor and drugs and could offer relatively high sensitivity and reliable reproducibility of recoveries. Thus, it should become a powerful method for studying the mechanism of enhanced reactivation of drugs.In chapter three, the enhanced reactivation mechanism of novel tertiary ammonium drugs were studied. We proved that L-1978 and L-1655 did have the ability to augment the reactivation effect of obidoxime (LiiH6) by above established GC/MS method. The possible mechanism of enhanced reactivation was investigated on three aspects.Firstly, the influence of inhibition effect on the reactivation of soman phosphonylated AChE was discussed. Among the tested AChE inhibitors, only Huperzine A (8151) could significantly improve the reactivation ability of LuH6 at the concentration of 1 X 10" mol/L. This suggested that there was no necessary relationship between inhibition and reactivation action. The cholinesterase inhibitors were not necessarily having the ability in improving the reactivation effect.Secondly, the influence of the allosteric effect on the reactivation of soman phosphonylated AChE was investigated. Among the tested AChE allosteric agents, only decamethonium bromide (CIO) could significantly improve the reactivation ability of LiiH6 at the concentration of 1 X 10"3 mol/L. This indicated that allosteric agents maybe could augment the reactivation effect. But, it was not means that all the allosteric agents would improve the reactivation effect.
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