基于LC-MS/MS的超级细菌耐药酶NDM-1的检测、降解抗生素特性及YH-8药代动力学
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摘要
近十年来,G-杆菌耐药问题日益严重,突出表现在耐碳青霉烯类抗生素的肠杆菌科细菌(大肠埃希菌、肺炎克雷伯杆菌、阴沟肠杆菌、产气肠杆菌等)、非发酵糖细菌(鲍曼不动杆菌、铜绿假单胞菌等),其中肺炎克雷伯杆菌、鲍曼不动杆菌、铜绿假单胞菌耐药最为严重,临床治疗困难。产NDM-1超级细菌首先在印度新德里发现,主要见于大肠埃希菌、肺炎克雷伯杆菌、阴沟肠杆菌、产气肠杆菌等肠杆菌科细菌,对临床常用的大多数抗生素包括碳青霉烯类抗生素耐药,可广泛传播,已经引起全世界的广泛关注和高度重视。
产NDM-1超级细菌对临床大多数抗生素耐药,治疗困难,而且其临床表现与敏感菌没有明显差异,临床诊断困难,临床上对碳青霉烯治疗无效的阴性菌感染需要考虑这类细菌感染可能。目前产NDM-1超级细菌的实验室检查主要分三步,该方法存在周期长、操作复杂、准确性不高等缺点。因此,迫切需要建立产NDM-1超级细菌的早期、快速、准确的诊断方法。
本论文旨在应用高灵敏度、高分辨率、高精确度的蛋白质谱检测技术(Nano UPLC Orbitrap)和生物信息分析系统,在现有耐药菌诊断技术和方法基础上,建立产NDM-1超级细菌感染的早期、快速、准确的诊断方法,研究不同抗生素对超级细菌耐药酶NDM-1的稳定性,将为临床超级细菌的诊断和治疗提供重要依据和技术支撑。
首先,应用基因工程技术获得NDM-1、OXA-23、VIM-2等7个重组细菌耐药酶,建立并优化了其蛋白酶解条件以及LC-MS/MS分析的液相条件和质谱条件,对7种重组耐药酶进行了分析,并应用Mascot软件将分析结果进行数据库搜索,结果满意,其中NDM-1的匹配度达到90%以上。
其次,应用LC-MS/MS技术检测细菌培养后复杂样品中的目的蛋白。以产NDM-1肺炎克雷伯杆菌为研究对象,通过细菌培养、超声裂解等方法获得含NDM-1的混合液样品,用LC-MS/MS检测,共鉴定出500多种蛋白包括NDM-1。
为模拟临床样本的检测,进一步确证所建方法的实用性,建立了产NDM-1肺炎克雷伯杆菌小鼠全身感染模型,并对感染前和感染后1h、2h、4h、8h小鼠血清中NDM-1进行了检测。结果显示,感染前及感染后小鼠血清中均检测到种类丰富的蛋白,小鼠感染后不同时间血清均检测到NDM-1,而感染前血清没有检测到NDM-1。采用实验室三步法对蛋白质谱检测结果进行验证,表明结果准确、可靠。
最后,应用LC-MS/MS和酶标仪,研究了NDM-1对5种β-内酰胺类抗生素的降解特性。结果表明,美罗培南对NDM-1的稳定性最低,10min的相对水解率为100%,酶动力学参数Km为79.39士6.34s-1。厄他培南和新药艾帕培南的稳定性次之,相对水解率在50%以上,Km值分别为120.11士10.53s-1、113.81士11.59s-1；新药百纳培南的稳定性较高,相对水解率为13%,Km值为240.26士38.56s-1。NDM-1对氨曲南没有水解作用。酶动力学研究结果与稳定性实验结果一致。
综上所述,本研究在现有耐药菌诊断技术和方法基础上,探索性建立应用Nano UPLC Orbitrap液质联用技术检测细菌耐药酶的方法,并应用该技术对产NDM-1肺炎克雷伯杆菌的培养粗提物和动物感染血液中的NDM-1成功进行了检测。特别需要指出的是,在动物超级细菌感染早期不同时间血液中均检测到NDM-1,为临床超级细菌感染的早期、快速、准确诊断提供了重要依据和技术支撑。此外,研究了NDM-1对5种p-内酰胺类抗生素的降解特性,为指导临床合理用药提供了科学依据,对抗超级细菌药物的研发也具有重要的指导意义。
结核病(Tuberculosis, TB)是严重危害人类健康的呼吸道传染病,被列入我国法定重大传染病。我国是全球22个结核病高负担国家之一,世界卫生组织估计,目前我国结核病年发病人数位居全球第二位。同时,我国也是全球27个耐多药高负担国家之一。从20世纪80年代以来,随着耐药结核病(尤其是耐多药结核病)发病率的不断上升以及结核病与HIV/AIDS并发导致的结核病疫情再度上升,成为全球重大公共卫生问题和社会问题。然而,近40余年来几乎没有新作用机制的抗TB药物问世。
4-氧代-4-(4-甲氧基苯基)-2(E)-丁烯酸甲酯(YH-8)是我所研发的新一类抗结核化合物,具有较高的抗结核尤其是抗多药耐药结核的活性,安全性较好,具有良好的研发前景。本论文旨在研究YH-8在大鼠体内的吸收、分布,预测YH-8发生代谢性药物相互作用的潜在可能性,为该药物的进一步开发和应用奠定理论基础。
首先,我们建立了生物样本中YH-8的LC-MS/MS分析方法,并对方法学进行了验证,为之后的研究打下基础。
进一步,我们通过建立的定量分析方法,研究了YH-8在大鼠体内的血浆药代动力学参数。用非房室模型对其进行分析,得到高、中、低三个剂量的YH-8在大鼠内体的半衰期分别为7.16士0.62、7.13±0.65、7.21士0.57h,三个剂量间的半衰期没有显著差异。三个剂量的药时曲线下面积(AUCo-t)分别为473.70士200.63、1145.40±466.62、2101.66±270.92ng-h/mL。YH-8在大鼠体内呈线性动力学过程,给药剂量与药时曲线下面积呈正相关。
在组织分布实验中,单次灌胃给予大鼠YH-8后,采用LC-MS/MS方法测定各组织中原型药物的含量,结果显示,在小肠中YH-8含量最高,而在心脏、脾中的含量低于其他被检测的组织。YH-8在主要脏器的药物分布顺序为：肠>肝>肺>肾>心>脾。在肺组织间液及肺巨噬细胞中YH-8也具有相当的含量。
为考察YH-8是否为受害药(Victim drug),被单一的CYP450酶代谢,我们开展了YH-8的生物转化研究。YH-8与人肝脏S9组分和人肝微粒体共同孵育不同时间后,原药浓度逐渐降低,证明YH-8在人肝脏中可被代谢。接下来,我们选取了最常见的10种重组表达的人源CYP450酶,分别与YH-8共同孵育,考察参与YH-8代谢的CYP450酶种类。结果显示,上述10种CYP450酶均能不同程度的对YH-8进行代谢,即YH-8可以被多种CYP450酶代谢,因此YH-8不是受害药,与CYP450酶抑制剂联用时,代谢不会受到干扰。
在此基础上,借助质谱的不同扫描模式,我们对YH-8的代谢产物进行了初步的探索和预测,寻找到了3种代谢产物M1-M3。同时,在动物实验中,也证实了大鼠血浆和肝脏中这3种代谢产物的存在。
随后,我们考察了YH-8对主要CYP450酶的抑制作用,评价其是否为凶手药(Perpetrator drug),干扰其他药物的代谢。结果显示,YH-8对主要CYP450酶的半数抑制率(IC50)除CYP1A2外均大于50μM,比对YH-8在大鼠体内的血药浓度,我们认为YH-8对主要CYP450酶无强烈的抑制作用。
综上所述,我们对YH-8的药代动力学参数及组织分布进行了研究,并使用体外代谢方法预测了YH-8与其他药物联用后发生代谢性药物相互作用的潜在可能性。结果表明,YH-8半衰期合理,适合一日三次的给药方式。YH-8组织分布较广,尤其靶组织中药物原型含量较高,但各组织中药物消除均较快,没有蓄积产生。此外,YH-8被多种CYP450酶所代谢,不是受害药,与P450酶抑制剂联用时,代谢不会受到干扰；YH-8对主要CYP450酶无强烈抑制作用,不是凶手药,不会干扰其他药物的代谢。
During the past decade, drug resistance has become more and more serious in Gram-negative bacilli, especially in the carbapenem-resistant Enterobacteriaceae including Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes as well as non-fermentable sugars bacteria such as Acinetobacter baumanni, Pseudomonas aeruginosa, etc. The drug-resistance caused by Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa are the most serious, to which the clinical treatment is difficult. The New Delhi metallo-beta-lactamase (NDM-1)-producing superbug was first found in New Delhi, India. NDM-1mainly exists in Enterobacteriaceae, such as Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Enterobacter aerogenes, which are resistant to most antibiotics commonly used in clinical settings including carbapenems. NDM-1can widely spread and have drawn great concern all over the world.
The NDM-1-producing superbugs are resistant to the majority of clinical antibiotics and are difficult to treat. There are no significant differences on the clinical manifestations caused by NDM-1-producing strains and carbapenem-susceptible strains. As a result, the clinical diagnosis of NDM-1-producing bacteria is difficult. This type of bacteria should be considered if ineffective treatment of Gram-negative bacterial infections with carbapenems occurs in clinical settings. Currently, laboratory identification of the NDM-1-producing superbugs includes three steps. The method has many shortcomings, such as time-consuming, complex operations and low accuracy. Therefore, it is urgent to establish an early, rapid and accurate diagnostic method to detect the NDM-1-producing superbugs.
One aim of this study is to establish an early, rapid and accurate diagnostic method of NDM-1-producing superbug by using the high-sensitivity, high-resolution and high-accuracy protein spectrum detection technology (Nano UPLC Orbitrap) on the basis of the existing drug-resistant diagnostic techniques and methods. The other aim is to study the stability of antibiotics to NDM-1. These will provide an important basis and technical support for diagnosis of clinical superbugs.
Firstly, we expressed and purified7recombinant proteins including NDM-1, OXA-23and VIM-2etc. Next, we established and optimized the methods and conditions of trypsin digestion as well as the LC-MS/MS detection methods and conditions of these proteins. The LC-MS/MS detection results were compared with the database. The results are satisfactory and the match degree of NDM-1is greater than90%.
After that, we detected the target proteins in complex samples using LC-MS/MS technology. After obtaining lysate of NDM-1-producing Klebsiella pneumoniae ATCC BAA2146strain, we analyzed the lysate using LC-MS/MS method. There were more than500kinds of mixed ingredients identified including NDM-1.
In order to simulate the detection of clinical samples and make further confirmation of the practicality of the method, we developed Klebsiella pneumoniae ATCC BAA2146mouse systemic infection model and tested NDM-1in serum samples from the infected mouse at1h,2h,4h and8h postinfection. The serum was detected by the established LC-MS/MS method. The results showed that a variety of proteins were detected in the serums. NDM-1was identified in the infected serum but was not detected in the non-infected serum. The LC-MS/MS detection results of the infected serum were verified with laboratory three-step method and results of the validation were accurate and satisfactory.
Finally, we studied the antibiotics-hydrolyzing characterization of NDM-1to five beta-lactam antibiotics. The results showed that meropenem had the lowest stability to NDM-1with the relative hydrolysis rate at10min of100%and the kinetic parameter Km of79.39±6.34s-1. The relative hydrolysis rate of ertapenem and apapenem to NDM-1were higher with hydrolysis rate of50%and Km of120.11±10.53s-1、113.81±11.59s-1, respectively. However, benapenem was with relatively higher stability, which has a relative hydrolysis rate of13%and the kinetic parameter Km of240.26±38.56s-1.Aztreonam cannot be hydrolyzed by NDM-1. The results of enzyme kinetics study were consistent with the results of stability study.
In summary, on the basis of the existing drug-resistant diagnostic techniques and methods, this study made exploratory study on application of Nano UPLC Orbitrap technology for diagnosis of drug-resistance enzymes of bacteria. NDM-1extracted from Klebsiella pneumoniae and in infected mice's serum was successfully detected. Notably, the NDM-1in infected mice's serum was detected as early as1h postinfection. This provided an important basis and technical support for the early, rapid and accurate diagnosis of clinical superbug infection. In addition, we studied the stability of five antibiotics to NDM-1and the enzyme kinetics of NDM-1to these five antibiotics. The findings provided theoretical basis to guide clinical drug use and had a certain degree of significance for the research and development of anti-superbug drugs.
Tuberculosis (TB) is a serious respiratory diseases endangering people's health. It is included in the statutory major infectious diseases in China. China is one of the22high TB burden countries. The World Health Organization estimated that the number of annual incidence of TB in China was second largest in the world. Since the1980s, with the rising incidence of drug-resistant TB (MDR-TB), TB has become a major global public health and social problems. However, in the past40years, almost no new anti-TB drug was approved for marketing.
(E)-Methyl4-(4-methoxyphenyl)-4-oxabut-2-enoate (YH-8) is an anti-TB compound of a novel class which was developed by our institute. It has anti-TB especially MDR-TB activity and was relatively safe. The drug has prospects for research and development. The purpose of this work was to study the absorption and to distribution of YH-8in rats and predict the potential drug metabolic interactions of YH-8with other drugs. In one word, the study laid the theoretical foundation for the further development and application of this compound.
First, we established LC-MS/MS analysis methods of YH-8in biological samples, and the methodology was validated to lay the foundation for the following research.
Next, we studied the plasma pharmacokinetic of YH-8using the established quantitative analysis method. The pharmacokinetic properties of YH-8were characterized by noncompartment model and the parameters were found to be T1/2values of7.16±0.62,7.13±0.65,7.21±0.57h and AUC0-t values of473.70±200.63,1145.40±466.62,2101.66±270.92ng-h/ml according to the doses of50,100and200mg/kg, respectively. The doses and the AUCo-t values over the range of oral dosages (50-200mg/kg) were positively correlated.
In the tissue distribution experiment, we used the LC-MS/MS method to determine the prototype drug content in all the tissues after single oral administration to rats. Results of tissue distribution study indicated that the intestines had a higher concentration of YH-8than other tissues. The heart and spleen have the lower content than other tissues examined. The order of drug distribution in the main organs was:intestine> liver> lung> kidney> heart> spleen. YH-8also had a considerable content in the lung epithelial lining fluid and alveolar macrophages.
In order to investigate whether YH-8is a "Victim Drug", which is metabolized by a single CYP450enzyme, we carried out the biotransformation research. The original drug concentration decreased when YH-8was incubated with human liver S9fraction and human liver microsomes, which proved that the YH-8could be metabolized by human liver. Then,10commonly used recombinant human CYP450enzymes were selected and incubated with YH-8respectively to examine which CYP450enzymes were involved in the metabolism of YH-8. The results showed that YH-8could be metabolized by all the tested CYP450isoenzymes at varying speeds and degrees. YH-8, which can be metabolized by a variety of CYP450enzymes, was proved not to be the victim drug. Therefore, the metabolism of YH-8would not be disturbed when co-administrated with CYP450enzyme inhibitors.
Based on different scan mode of the mass spectrometry, the metabolites of YH-8were predicted and three metabolites (M1-M3) were found. The animal experiments also confirmed the presence of the three metabolites in rats'plasma and livers.
Next, we investigated the inhibitory effect of YH-8on main CYP450enzymes to evaluate whether it was a perpetrator drug. The results showed that IC50values of YH-8were greater than50μM towards the main CYP450enzymes except CYP1A2. Taking the plasma concentration of YH-8in rats into account, we considered that YH-8did not inhibit the main CYP450enzymes.
In summary, we studied the pharmacokinetic parameters and tissue distribution of YH-8and predicted the potential drug-drug interactions of YH-8in vitro. The results indicated that the half-life of YH-8in rats was reasonable and was suitable for the mode of administration for three times a day. YH-8was widely distributed and had high levels in the target tissue. Meanwhile, the elimination of YH-8in all tissues was fast and not likely to accumulate. Moreover, the results showed that the YH-8is not a victim drug and can be metabolized by many CYP450enzymes. So the metabolism of YH-8will not be disturbed when co-administrated with a CYP450enzyme inhibitor. Moreover, YH-8did not exhibit inhibitory effect on main CYP450enzymes, so it was not a perpetrator drug, and would not disturb the metabolism of other drugs.
产NDM-1超级细菌对临床大多数抗生素耐药,治疗困难,而且其临床表现与敏感菌没有明显差异,临床诊断困难,临床上对碳青霉烯治疗无效的阴性菌感染需要考虑这类细菌感染可能。目前产NDM-1超级细菌的实验室检查主要分三步,该方法存在周期长、操作复杂、准确性不高等缺点。因此,迫切需要建立产NDM-1超级细菌的早期、快速、准确的诊断方法。
本论文旨在应用高灵敏度、高分辨率、高精确度的蛋白质谱检测技术(Nano UPLC Orbitrap)和生物信息分析系统,在现有耐药菌诊断技术和方法基础上,建立产NDM-1超级细菌感染的早期、快速、准确的诊断方法,研究不同抗生素对超级细菌耐药酶NDM-1的稳定性,将为临床超级细菌的诊断和治疗提供重要依据和技术支撑。
首先,应用基因工程技术获得NDM-1、OXA-23、VIM-2等7个重组细菌耐药酶,建立并优化了其蛋白酶解条件以及LC-MS/MS分析的液相条件和质谱条件,对7种重组耐药酶进行了分析,并应用Mascot软件将分析结果进行数据库搜索,结果满意,其中NDM-1的匹配度达到90%以上。
其次,应用LC-MS/MS技术检测细菌培养后复杂样品中的目的蛋白。以产NDM-1肺炎克雷伯杆菌为研究对象,通过细菌培养、超声裂解等方法获得含NDM-1的混合液样品,用LC-MS/MS检测,共鉴定出500多种蛋白包括NDM-1。
为模拟临床样本的检测,进一步确证所建方法的实用性,建立了产NDM-1肺炎克雷伯杆菌小鼠全身感染模型,并对感染前和感染后1h、2h、4h、8h小鼠血清中NDM-1进行了检测。结果显示,感染前及感染后小鼠血清中均检测到种类丰富的蛋白,小鼠感染后不同时间血清均检测到NDM-1,而感染前血清没有检测到NDM-1。采用实验室三步法对蛋白质谱检测结果进行验证,表明结果准确、可靠。
最后,应用LC-MS/MS和酶标仪,研究了NDM-1对5种β-内酰胺类抗生素的降解特性。结果表明,美罗培南对NDM-1的稳定性最低,10min的相对水解率为100%,酶动力学参数Km为79.39士6.34s-1。厄他培南和新药艾帕培南的稳定性次之,相对水解率在50%以上,Km值分别为120.11士10.53s-1、113.81士11.59s-1；新药百纳培南的稳定性较高,相对水解率为13%,Km值为240.26士38.56s-1。NDM-1对氨曲南没有水解作用。酶动力学研究结果与稳定性实验结果一致。
综上所述,本研究在现有耐药菌诊断技术和方法基础上,探索性建立应用Nano UPLC Orbitrap液质联用技术检测细菌耐药酶的方法,并应用该技术对产NDM-1肺炎克雷伯杆菌的培养粗提物和动物感染血液中的NDM-1成功进行了检测。特别需要指出的是,在动物超级细菌感染早期不同时间血液中均检测到NDM-1,为临床超级细菌感染的早期、快速、准确诊断提供了重要依据和技术支撑。此外,研究了NDM-1对5种p-内酰胺类抗生素的降解特性,为指导临床合理用药提供了科学依据,对抗超级细菌药物的研发也具有重要的指导意义。
结核病(Tuberculosis, TB)是严重危害人类健康的呼吸道传染病,被列入我国法定重大传染病。我国是全球22个结核病高负担国家之一,世界卫生组织估计,目前我国结核病年发病人数位居全球第二位。同时,我国也是全球27个耐多药高负担国家之一。从20世纪80年代以来,随着耐药结核病(尤其是耐多药结核病)发病率的不断上升以及结核病与HIV/AIDS并发导致的结核病疫情再度上升,成为全球重大公共卫生问题和社会问题。然而,近40余年来几乎没有新作用机制的抗TB药物问世。
4-氧代-4-(4-甲氧基苯基)-2(E)-丁烯酸甲酯(YH-8)是我所研发的新一类抗结核化合物,具有较高的抗结核尤其是抗多药耐药结核的活性,安全性较好,具有良好的研发前景。本论文旨在研究YH-8在大鼠体内的吸收、分布,预测YH-8发生代谢性药物相互作用的潜在可能性,为该药物的进一步开发和应用奠定理论基础。
首先,我们建立了生物样本中YH-8的LC-MS/MS分析方法,并对方法学进行了验证,为之后的研究打下基础。
进一步,我们通过建立的定量分析方法,研究了YH-8在大鼠体内的血浆药代动力学参数。用非房室模型对其进行分析,得到高、中、低三个剂量的YH-8在大鼠内体的半衰期分别为7.16士0.62、7.13±0.65、7.21士0.57h,三个剂量间的半衰期没有显著差异。三个剂量的药时曲线下面积(AUCo-t)分别为473.70士200.63、1145.40±466.62、2101.66±270.92ng-h/mL。YH-8在大鼠体内呈线性动力学过程,给药剂量与药时曲线下面积呈正相关。
在组织分布实验中,单次灌胃给予大鼠YH-8后,采用LC-MS/MS方法测定各组织中原型药物的含量,结果显示,在小肠中YH-8含量最高,而在心脏、脾中的含量低于其他被检测的组织。YH-8在主要脏器的药物分布顺序为：肠>肝>肺>肾>心>脾。在肺组织间液及肺巨噬细胞中YH-8也具有相当的含量。
为考察YH-8是否为受害药(Victim drug),被单一的CYP450酶代谢,我们开展了YH-8的生物转化研究。YH-8与人肝脏S9组分和人肝微粒体共同孵育不同时间后,原药浓度逐渐降低,证明YH-8在人肝脏中可被代谢。接下来,我们选取了最常见的10种重组表达的人源CYP450酶,分别与YH-8共同孵育,考察参与YH-8代谢的CYP450酶种类。结果显示,上述10种CYP450酶均能不同程度的对YH-8进行代谢,即YH-8可以被多种CYP450酶代谢,因此YH-8不是受害药,与CYP450酶抑制剂联用时,代谢不会受到干扰。
在此基础上,借助质谱的不同扫描模式,我们对YH-8的代谢产物进行了初步的探索和预测,寻找到了3种代谢产物M1-M3。同时,在动物实验中,也证实了大鼠血浆和肝脏中这3种代谢产物的存在。
随后,我们考察了YH-8对主要CYP450酶的抑制作用,评价其是否为凶手药(Perpetrator drug),干扰其他药物的代谢。结果显示,YH-8对主要CYP450酶的半数抑制率(IC50)除CYP1A2外均大于50μM,比对YH-8在大鼠体内的血药浓度,我们认为YH-8对主要CYP450酶无强烈的抑制作用。
综上所述,我们对YH-8的药代动力学参数及组织分布进行了研究,并使用体外代谢方法预测了YH-8与其他药物联用后发生代谢性药物相互作用的潜在可能性。结果表明,YH-8半衰期合理,适合一日三次的给药方式。YH-8组织分布较广,尤其靶组织中药物原型含量较高,但各组织中药物消除均较快,没有蓄积产生。此外,YH-8被多种CYP450酶所代谢,不是受害药,与P450酶抑制剂联用时,代谢不会受到干扰；YH-8对主要CYP450酶无强烈抑制作用,不是凶手药,不会干扰其他药物的代谢。
During the past decade, drug resistance has become more and more serious in Gram-negative bacilli, especially in the carbapenem-resistant Enterobacteriaceae including Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes as well as non-fermentable sugars bacteria such as Acinetobacter baumanni, Pseudomonas aeruginosa, etc. The drug-resistance caused by Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa are the most serious, to which the clinical treatment is difficult. The New Delhi metallo-beta-lactamase (NDM-1)-producing superbug was first found in New Delhi, India. NDM-1mainly exists in Enterobacteriaceae, such as Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Enterobacter aerogenes, which are resistant to most antibiotics commonly used in clinical settings including carbapenems. NDM-1can widely spread and have drawn great concern all over the world.
The NDM-1-producing superbugs are resistant to the majority of clinical antibiotics and are difficult to treat. There are no significant differences on the clinical manifestations caused by NDM-1-producing strains and carbapenem-susceptible strains. As a result, the clinical diagnosis of NDM-1-producing bacteria is difficult. This type of bacteria should be considered if ineffective treatment of Gram-negative bacterial infections with carbapenems occurs in clinical settings. Currently, laboratory identification of the NDM-1-producing superbugs includes three steps. The method has many shortcomings, such as time-consuming, complex operations and low accuracy. Therefore, it is urgent to establish an early, rapid and accurate diagnostic method to detect the NDM-1-producing superbugs.
One aim of this study is to establish an early, rapid and accurate diagnostic method of NDM-1-producing superbug by using the high-sensitivity, high-resolution and high-accuracy protein spectrum detection technology (Nano UPLC Orbitrap) on the basis of the existing drug-resistant diagnostic techniques and methods. The other aim is to study the stability of antibiotics to NDM-1. These will provide an important basis and technical support for diagnosis of clinical superbugs.
Firstly, we expressed and purified7recombinant proteins including NDM-1, OXA-23and VIM-2etc. Next, we established and optimized the methods and conditions of trypsin digestion as well as the LC-MS/MS detection methods and conditions of these proteins. The LC-MS/MS detection results were compared with the database. The results are satisfactory and the match degree of NDM-1is greater than90%.
After that, we detected the target proteins in complex samples using LC-MS/MS technology. After obtaining lysate of NDM-1-producing Klebsiella pneumoniae ATCC BAA2146strain, we analyzed the lysate using LC-MS/MS method. There were more than500kinds of mixed ingredients identified including NDM-1.
In order to simulate the detection of clinical samples and make further confirmation of the practicality of the method, we developed Klebsiella pneumoniae ATCC BAA2146mouse systemic infection model and tested NDM-1in serum samples from the infected mouse at1h,2h,4h and8h postinfection. The serum was detected by the established LC-MS/MS method. The results showed that a variety of proteins were detected in the serums. NDM-1was identified in the infected serum but was not detected in the non-infected serum. The LC-MS/MS detection results of the infected serum were verified with laboratory three-step method and results of the validation were accurate and satisfactory.
Finally, we studied the antibiotics-hydrolyzing characterization of NDM-1to five beta-lactam antibiotics. The results showed that meropenem had the lowest stability to NDM-1with the relative hydrolysis rate at10min of100%and the kinetic parameter Km of79.39±6.34s-1. The relative hydrolysis rate of ertapenem and apapenem to NDM-1were higher with hydrolysis rate of50%and Km of120.11±10.53s-1、113.81±11.59s-1, respectively. However, benapenem was with relatively higher stability, which has a relative hydrolysis rate of13%and the kinetic parameter Km of240.26±38.56s-1.Aztreonam cannot be hydrolyzed by NDM-1. The results of enzyme kinetics study were consistent with the results of stability study.
In summary, on the basis of the existing drug-resistant diagnostic techniques and methods, this study made exploratory study on application of Nano UPLC Orbitrap technology for diagnosis of drug-resistance enzymes of bacteria. NDM-1extracted from Klebsiella pneumoniae and in infected mice's serum was successfully detected. Notably, the NDM-1in infected mice's serum was detected as early as1h postinfection. This provided an important basis and technical support for the early, rapid and accurate diagnosis of clinical superbug infection. In addition, we studied the stability of five antibiotics to NDM-1and the enzyme kinetics of NDM-1to these five antibiotics. The findings provided theoretical basis to guide clinical drug use and had a certain degree of significance for the research and development of anti-superbug drugs.
Tuberculosis (TB) is a serious respiratory diseases endangering people's health. It is included in the statutory major infectious diseases in China. China is one of the22high TB burden countries. The World Health Organization estimated that the number of annual incidence of TB in China was second largest in the world. Since the1980s, with the rising incidence of drug-resistant TB (MDR-TB), TB has become a major global public health and social problems. However, in the past40years, almost no new anti-TB drug was approved for marketing.
(E)-Methyl4-(4-methoxyphenyl)-4-oxabut-2-enoate (YH-8) is an anti-TB compound of a novel class which was developed by our institute. It has anti-TB especially MDR-TB activity and was relatively safe. The drug has prospects for research and development. The purpose of this work was to study the absorption and to distribution of YH-8in rats and predict the potential drug metabolic interactions of YH-8with other drugs. In one word, the study laid the theoretical foundation for the further development and application of this compound.
First, we established LC-MS/MS analysis methods of YH-8in biological samples, and the methodology was validated to lay the foundation for the following research.
Next, we studied the plasma pharmacokinetic of YH-8using the established quantitative analysis method. The pharmacokinetic properties of YH-8were characterized by noncompartment model and the parameters were found to be T1/2values of7.16±0.62,7.13±0.65,7.21±0.57h and AUC0-t values of473.70±200.63,1145.40±466.62,2101.66±270.92ng-h/ml according to the doses of50,100and200mg/kg, respectively. The doses and the AUCo-t values over the range of oral dosages (50-200mg/kg) were positively correlated.
In the tissue distribution experiment, we used the LC-MS/MS method to determine the prototype drug content in all the tissues after single oral administration to rats. Results of tissue distribution study indicated that the intestines had a higher concentration of YH-8than other tissues. The heart and spleen have the lower content than other tissues examined. The order of drug distribution in the main organs was:intestine> liver> lung> kidney> heart> spleen. YH-8also had a considerable content in the lung epithelial lining fluid and alveolar macrophages.
In order to investigate whether YH-8is a "Victim Drug", which is metabolized by a single CYP450enzyme, we carried out the biotransformation research. The original drug concentration decreased when YH-8was incubated with human liver S9fraction and human liver microsomes, which proved that the YH-8could be metabolized by human liver. Then,10commonly used recombinant human CYP450enzymes were selected and incubated with YH-8respectively to examine which CYP450enzymes were involved in the metabolism of YH-8. The results showed that YH-8could be metabolized by all the tested CYP450isoenzymes at varying speeds and degrees. YH-8, which can be metabolized by a variety of CYP450enzymes, was proved not to be the victim drug. Therefore, the metabolism of YH-8would not be disturbed when co-administrated with CYP450enzyme inhibitors.
Based on different scan mode of the mass spectrometry, the metabolites of YH-8were predicted and three metabolites (M1-M3) were found. The animal experiments also confirmed the presence of the three metabolites in rats'plasma and livers.
Next, we investigated the inhibitory effect of YH-8on main CYP450enzymes to evaluate whether it was a perpetrator drug. The results showed that IC50values of YH-8were greater than50μM towards the main CYP450enzymes except CYP1A2. Taking the plasma concentration of YH-8in rats into account, we considered that YH-8did not inhibit the main CYP450enzymes.
In summary, we studied the pharmacokinetic parameters and tissue distribution of YH-8and predicted the potential drug-drug interactions of YH-8in vitro. The results indicated that the half-life of YH-8in rats was reasonable and was suitable for the mode of administration for three times a day. YH-8was widely distributed and had high levels in the target tissue. Meanwhile, the elimination of YH-8in all tissues was fast and not likely to accumulate. Moreover, the results showed that the YH-8is not a victim drug and can be metabolized by many CYP450enzymes. So the metabolism of YH-8will not be disturbed when co-administrated with a CYP450enzyme inhibitor. Moreover, YH-8did not exhibit inhibitory effect on main CYP450enzymes, so it was not a perpetrator drug, and would not disturb the metabolism of other drugs.
引文
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