High resolution mass spectrometry-based methodologies for identification of Etravirine bioactivation to reactive metabolites: In vitro and in vivo approaches
High resolution mass spectrometry-based methodologies for identification of Etravirine bioactivation to reactive metabolites: In vitro and in vivo approaches
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DOI：
10.1016/j.ejps.2018.03.026
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发表时间：
2018-07-01
影响因子：
4.6
通讯作者：
Antunes, Alexandra M. M.
中科院分区：
文献类型：
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作者：
Godinho, Ana L. A.;Martins, Ines L.;Antunes, Alexandra M. M.
药物对能够与生物亲核体形成共价加合物的反应性代谢物的生物活化是药物诱导的不良反应的主要原因。因此，阐明反应性代谢物是必不可少的，以解开药物诱导的毒性机制，从而确定患者亚组在较高的risk.Etravirine（ETR）是第二代非核苷逆转录酶抑制剂（NNRTI）被批准，作为一种治疗选择的艾滋病毒感染的患者谁发展耐药的第一代NNRTI。此外，ETR进入市场的目的是克服与之前使用的依法韦仑（神经毒性）和奈韦拉平（肝毒性）疗法相关的一些不良反应。尽管如此，严重的ETR诱导的皮疹和超敏反应的上市后报告促使美国FDA发布了ETR的安全警报。考虑到ETR的使用在不久的将来可能会增加，由于可能使用药物治疗疟疾和HIV的合并感染，因此迫切需要开发早期风险/获益估计的可靠预后工具。在目前的研究中，基于高分辨率质谱的方法与MS 3实验相结合，用于鉴定反应性ETR代谢物/加合物：1）在I相和II相辅因子（包括谷胱甘肽）作为捕获生物亲核体的存在下，将药物与人和大鼠肝S9组分体外孵育;和2）在体内，使用来自HIV的尿液样本-我们获得了多种生物活化途径导致谷胱甘肽共价加合物形成的证据，和N-乙酰基-L-半胱氨酸。这些结果表明，蛋白质的半胱氨酸残基可能发生类似的反应，支持ETR生物活化在药物引起的毒性作用发作中的作用。此外，在体外和体内鉴定了来自胺氧化的具有潜在毒理学意义的ETR代谢物。同样值得注意的是，首次证明了葡萄糖醛酸代谢物的新代谢结合途径，这引发了对其潜在毒理学意义的质疑。总之，这些结果不仅对阐明药物的新代谢途径做出了贡献，而且也是阐明潜在毒性ETR途径的重要一步，对ETR途径的理解对于基于ETR的方案的可靠风险/获益估计可能至关重要。
Drug bioactivation to reactive metabolites capable of covalent adduct formation with bionucleophiles is a major cause of drug-induced adverse reactions. Therefore, elucidation of reactive metabolites is essential to unravel the toxicity mechanisms induced by drugs and thereby identify patient subgroups at higher risk.Etravirine (ETR) was the first second-generation Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI) to be approved, as a therapeutic option for HIV-infected patients who developed resistance to the first-generation NNRTIs. Additionally, ETR came into market aiming to overcome some adverse effects associated with the previously used efavirenz (neurotoxicity) and nevirapine (hepatotoxicity) therapies. Nonetheless, post-marketing reports of severe ETR-induced skin rash and hypersensitivity reactions have prompted the U.S. FDA to issue a safety alert on ETR. Taking into consideration that ETR usage may increase in the near future, due to the possible use of the drug for coinfection with malaria and HIV, the development of reliable prognostic tools for early risk/benefit estimations is urgent.In the current study, high resolution mass spectrometry-based methodologies were integrated with MS3 experiments for the identification of reactive ETR metabolites/adducts: 1) in vitro incubation of the drug with human and rat liver S9 fractions in the presence of Phase I and II co-factors, including glutathione, as a trapping bionucleophile; and 2) in vivo, using urine samples from HIV-infected patients on ETR therapy.We obtained evidence for multiple bioactivation pathways leading to the formation of covalent adducts with glutathione and N-acetyl-L-cysteine. These results suggest that similar reactions may occur with cysteine residues of proteins, supporting a role for ETR bioactivation in the onset of the toxic effects elicited by the drug. Additionally, ETR metabolites stemming from amine oxidation, with potential toxicological significance, were identified in vitro and in vivo. Also noteworthy is the fact that new metabolic conjugation pathways of glucuronide metabolites were demonstrated for the first time, raising questions about their potential toxicological implications. In conclusion, these results represent not only a contribution towards the elucidation of new metabolic pathways of drugs in general but also an important step towards the elucidation of potentially toxic ETR pathways, whose understanding may be crucial for reliable risk/benefit estimations of ETR-based regimens.