Bioanalysis of EGFRm inhibitor osimertinib, and its glutathione cycle- and desmethyl metabolites by liquid chromatography-tandem mass spectrometry.
- 作者列表："Rood JJM","van Haren MJ","Beijnen JH","Sparidans RW
:Osimertinib is a "third-generation'' oral, irreversible, tyrosine kinase inhibitor. It is used in the treatment of non-small cellular lung carcinoma and spares wild-type EGFR. Due to its reactive nature, osimertinib is, in addition to oxidative routes, metabolized through GSH coupling and subsequent further metabolism of these conjugates. The extent of the non-oxidative metabolism of osimertinib is unknown, and methods to quantify this metabolic route have not been reported yet. To gain insight into this metabolic route, a sensitive bioanalytical assay was developed for osimertinib, the active desmethyl metabolite AZ5104, and the thio-metabolites osimertinibs glutathione, cysteinylglycine, and cysteine conjugates was developed. The ease of synthesis of these metabolites was a key-part in the development of this assay. This was done through simple one-step synthesis and subsequent LC-purification. The compounds were characterized by NMR and high-resolution mass spectrometry. Sample preparation was done by a simple protein crash with acetonitrile containing the stable isotopically labeled internal standards for osimertinib and the thio-metabolites, partial evaporation of solvents, and reconstitution in eluent, followed by UHPLC-MS/MS quantification. The assay was successfully validated in a 2-2000 nM calibration range for all compounds except the glutathione metabolite, where the LLOQ was set at 6 nM due to low accuracy at 2 nM. Limited stability was observed for osimertinib, AZ5104, and the glutathione metabolite. The clinical applicability of the assay was demonstrated in samples of patients treated with 80 mg osimertinib once daily, containing all investigated compounds at detectable and quantifiable levels.
: 奥希替尼是一种 “第三代” 口服、不可逆的酪氨酸激酶抑制剂。它用于治疗非小细胞肺癌，并保留野生型EGFR。由于其反应性，奥希替尼除了氧化途径之外，还通过GSH偶联和随后这些缀合物的进一步代谢进行代谢。奥希替尼的非氧化代谢的程度是未知的，并且定量该代谢途径的方法尚未报道。为了深入了解这一代谢途径，开发了对奥希替尼、活性去甲基代谢物AZ5104 和硫代代谢物奥希替尼谷胱甘肽、半胱氨酰甘氨酸和半胱氨酸缀合物的灵敏生物分析测定法。合成这些代谢物的容易性是该测定开发的关键部分。这通过简单的一步合成和随后的LC纯化来完成。通过NMR和高分辨质谱对化合物进行了表征。样品制备是通过简单的蛋白质碰撞，含有奥希替尼稳定的同位素标记内标物和硫代代谢物的乙腈，部分蒸发溶剂，并在洗脱液中重构，随后进行uhplc-ms/MS定量。对于除谷胱甘肽代谢物之外的所有化合物，该测定在 2-2000 nm校准范围内成功验证，其中由于在 2 nm处的低准确度，LLOQ设定在 6 nm。观察到奥希替尼、AZ5104 和谷胱甘肽代谢物的稳定性有限。在每天一次 80 mg奥希替尼治疗的患者样品中证明了该测定法的临床适用性，其中包含所有可检测和可定量水平的研究化合物。
METHODS::Pulmonary artery sling is a rare congenital anomaly of the origin and course of the left pulmonary artery. Patients with this condition typically present with respiratory failure in young infancy, and asymptomatic cases are uncommon. We describe the case of an adult patient with a lung adenocarcinoma of the right upper lobe, extending into the hilum and superior mediastinum, and with a previously unknown pulmonary artery sling anomaly. The local invasiveness of the tumor and the peculiar vascular anatomy contributed to a unique surgical scenario, wherein multiple reconstructive procedures were required.
METHODS::Patients with idiopathic pulmonary fibrosis (IPF) have higher risk of developing lung cancer, for example, squamous cell carcinoma (SCC), and show poor prognosis, while the molecular basis has not been fully investigated. Here we conducted DNA methylome analysis of lung SCC using 20 SCC samples with/without IPF, and noncancerous lung tissue samples from smokers/nonsmokers, using Infinium HumanMethylation 450K array. SCC was clustered into low- and high-methylation epigenotypes by hierarchical clustering analysis. Genes hypermethylated in SCC significantly included genes targeted by polycomb repressive complex in embryonic stem cells, and genes associated with Gene Ontology terms, for example, "transcription" and "cell adhesion," while genes hypermethylated specifically in high-methylation subgroup significantly included genes associated with "negative regulation of growth." Low-methylation subgroup significantly correlated with IPF (78%, vs. 17% in high-methylation subgroup, p = 0.04), and the correlation was validated by additional Infinium analysis of SCC samples (n = 44 in total), and data from The Cancer Genome Atlas (n = 390). The correlation between low-methylation subgroup and IPF was further validated by quantitative methylation analysis of marker genes commonly hypermethylated in SCC (HOXA2, HOXA9 and PCDHGB6), and markers specifically hypermethylated in high-methylation subgroup (DLEC1, CFTR, MT1M, CRIP3 and ALDH7A1) in 77 SCC cases using pyrosequencing (p = 0.003). Furthermore, low-methylation epigenotype significantly correlated with poorer prognosis among all SCC patients, or among patients without IPF. Multivariate analysis showed that low-methylation epigenotype is an independent predictor of poor prognosis. These may suggest that lung SCC could be stratified into molecular subtypes with distinct prognosis, and low-methylation lung SCC that significantly correlates with IPF shows unfavorable outcome.
METHODS::The role of Fyn-related kinase (FRK) in malignant tumors remains controversial. Our study investigated the function of FRK in lung cancer. Immunohistochemistry staining and generating a knockout of FRK by CRISPR/Cas9 in H1299 (FRK-KO-H1299) cells were strategies used to explore the role of FRK. Immunohistochemistry staining indicated that FRK expression was elevated in 223 lung cancer tissues compared to 26 distant normal lung tissues. FRK contributed to poor survival status in lung cancer patients and acted as a predictor for poor prognosis of lung cancer. Knockout of FRK by CRISPR/Cas9 markedly inhibited proliferation, invasion, colony formation and epithelial-mesenchymal transition (EMT) process in the lung cancer cell line H1299. Further exploration indicated that FRK-KO damaged the stemness phenotype of H1299 by inhibiting CD44 and CD133 expression. Seahorse detection and a U-13 C flux assay revealed that FRK-KO induced metabolism reprogramming by inhibiting the Warburg effect and changing the energy type in H1299 cells. Epidermal growth factor stimulation recovered the expression of FRK and biological functions, metabolic reprogramming and stemness phenotype of H1299 cells. FRK plays an oncogenic role in lung cancer cells via a novel regulation mechanism of enhancing the stemness of H1299 cells by inducing metabolism reprogramming, which finally promotes EMT and metastasis. Our study also indicates that FRK could be used as a potential therapeutic target for drug development.