應用代謝體學於研究voriconazole引發之肝臟毒性

Abstract

Voriconazole屬三唑類廣效抗黴菌藥品，被應用於侵入性麴菌感染。目前已知可能造成的藥物不良反應包含紅疹，腸胃不適，視毒性及肝毒性。其中肝功能指數上升為常見之不良反應，可能干擾此藥本身及其他併用藥物之代謝，進一步加重肝臟毒性，並導致治療效果不彰、停藥，於危急患者甚至有致命風險。目前研究對於voriconazole引起的肝臟毒性機轉仍未釐清，臨床上亦無可專一性診斷、治療的方式。代謝體學為近年新興之研究領域，藉由廣泛地研究體內小分子代謝物的變化，推測出可能導致不同表現型的機轉及具有潛力的生物指標，以做為發展精準醫學的工具之一。本研究利用本實驗室先前開發之液相層析串聯式質譜儀之藥物肝毒性研究平台，將標的代謝體學應用於研究voriconazole引起之肝臟毒性，共分為兩個部分。 第一部份以C57BL/6品系之小鼠，研究voriconazole引發之肝臟毒性代謝體變化。給藥組 (n = 8) 使用劑量40 mg/ kg之voriconazole以尾靜脈注射重複給藥三次誘發肝臟毒性，控制組 (n = 10) 則未投與藥物。本部分研究同時收集分析血漿與肝臟樣品，以探討造成voriconazole肝臟毒性的機轉，及血中可反映與voriconazole肝臟毒性相關之代謝物。 第二部份以臨床病人血漿檢體，驗證小鼠實驗中推測之voriconazole肝臟毒性機轉。納入研究之病人皆接受voriconazole治療，並依照肝功能指數、電子病歷紀錄及Naranjo scale和RUCAM scale評估，分為肝功能正常之控制組 (n = 89) 以及voriconazole引發之肝臟毒性組 (n = 21)。 研究結果發現，在小鼠模型與臨床病人中皆觀察到與抗氧化相關之代謝物發生變化，許多與體內重要的抗氧化成分glutathione之生合成有關。此外，glutamine與glutamate之比值在voriconazole引發肝臟毒性的組別與控制組相比，都有顯著的降低，顯現出glutamine可能被大量轉換成glutamate，以增加生成glutathione調節氧化壓力。結合兩部分實驗結果，我們推測voriconazole引起的肝臟毒性與氧化壓力的形成有關而造成細胞功能損傷，並影響能量代謝、尿素循環與膽酸合成等。此為第一個應用代謝體方法研究voriconazole於小鼠及臨床病人引發之肝臟毒性機轉的研究。為闡明voriconazole誘發氧化壓力的分子機轉，並探討可用於臨床之生物指標，未來更多的研究將是必要的。

Voriconazole (VCZ) is a triazole-antifungal agent with broad spectrum, which has been widely used in invasive Aspergillosis infections. The adverse drug effects associated with VCZ include rash, gastrointestinal uncomfortable, visual disturbance and hepatotoxicity. Abnormality in liver function test (LFT) is common to VCZ-treated patients, and the abnormal liver function might affect drug metabolism involving itself and other co-administrated drugs, further progress the hepatotoxicity, which might lead to treatment-failure, withdrawal, and even death. The mechanism of VCZ-induced hepatotoxicity is unclear, and no specific marker can be used for diagnosis or treatment in clinic. Metabolomics, an emerging filed recently, is viewed as one of the tools to develop precision medicine. Through globally analyzing the alterations of small molecules in the living system, the mechanism leading to different phenotype and potential biomarkers could be proposed. This research applied the targeted metabolomics approach to study VCZ-induced hepatotoxicity by our previously developed analytic platform for hepatotoxicity-detection via liquid chromatography-triple quadrupole mass spectrometry (LC-QqQ MS). The thesis is divided into two parts. In the first part, the metabolic patterns of VCZ-induced hepatotoxicity in C57BL/6 mice were analyzed. Mice treated with three repeated dose of 40 mg/ kg by tail vein injection to induce hepatotoxicity (VCZ-induced hepatotoxicity group, n = 8) were compared with the mice without treatment (control group, n = 10). Both liver tissue and plasma were collected and analyzed to propose mechanisms associated VCZ-induced hepatotoxicity and the potential markers which could reflect the VCZ-induced hepatotoxicity in plasma. In the second part, plasma samples collected from clinical patients were analyzed for validation of the proposed mechanism of VCZ-induced hepatotoxicity. All of the recruited patients had received VCZ treatment, and were divided into control group (n = 89) and VCZ-induced hepatotoxicity group (n = 21) by their liver function. VCZ-induced hepatotoxicity was defined by the evaluation of Naranjo scale and RUCAM scale based on the information from electro-medical record. Results indicated that the metabolites associated with oxidative stress had altered, and most of the altered metabolites were involved in glutathione biosynthesis, which is an important antioxidant in vivo. In addition, the ratios of glutamine and glutamate showed significantly reduction in VCZ-induced hepatotoxicity group compared to control group in both studies, suggested that glutamine might be transformed into glutamate for glutathione biosynthesis. Combining the results from two parts, we proposed that VCZ-induced hepatotoxicity is associated with oxidative stress to cause cell dysfunction, leading to the alterations in energy metabolism, urea cycle, and bile acids metabolism. To our knowledge, this is the first study to apply metabolomics for the investigation of the mechanism of VCZ-induced hepatotoxicity. Future work is required to clarify the specific molecular mechanism of oxidative stress induced by VCZ and investigate the biomarkers for clinical practice.