利用質譜儀建立 Sitagliptin 簡單又快速之分析方法及鑑定其藥物引發之蛋白質表現

Sitagliptin 為一新型且具高選擇性之口服降血糖藥，屬於 dipeptidyl peptidase-4 抑制劑 (DPP-4 inhibitors)，用於治療第二型糖尿病。其作用機轉是透過抑制 DPP-4 酵素來增加腸泌素 glucagon-like peptide-1 (GLP-1) 和 glucose-dependent insulinotropic polypeptide (GIP) 的濃度，刺激胰島素分泌使病患得以控制血糖。在2009年美國 FDA 發佈 sitagliptin 藥品仿單應增加上市後不良反應報告急性胰臟炎 (acute pancreatitis)。因 sitagliptin 為較新的降血糖用藥，上市時間短，故服藥產生不良反應及服用過量之反應尚無足夠相關研究報告。然而，藥物血中濃度監控可用來判斷病人在臨床上所呈現的藥效反應，依每個病患的不同狀況透過藥物血中濃度監測的結果調整治療方式。因此，藥物血中濃度之檢測方法極為重要，故此研究利用介質輔助雷射脫附游離飛行時間質譜儀 (matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS) 建立一套快速之臨床監控方法，以及藥物品質管理之檢測方式。此分析方法有良好的精密度與準確度，其 RSD 和 RE 值皆小於 10 %，適用於臨床藥物血中濃度監控與藥物品管，此技術進行專利申請中 (申請案號：104119824)。近年來有文獻指出，除了用於治療第二型糖尿病外，sitagliptin 為一潛力藥物於其他疾病治療。故此研究選用人類肝癌細胞株 (Huh-7) 與人類近端腎小管上皮細胞株 (HK-2) 作為模型，進行 nanoUPLC-MS/MS 分析並以生物資訊軟體鑑定其藥物引發之蛋白質表現。結果顯示，在 Huh-7 中，sitagliptin誘導之蛋白與癌症、生物體損傷等途徑相關，在 HK-2 中，sitagliptin誘導之蛋白與癌症、DNA 複製/重組等途徑相關。

關鍵字

西他列汀；介質輔助雷射脫附游離飛行時間質譜儀；高效能液相層析串聯式質譜儀

並列摘要

Sitagliptin is a novel and highly selective oral hypoglycemic agents belonging to the DPP-4 inhibitors. The mechanism of DPP-4 inhibitors is to increase incretin levels (GLP-1 and GIP), which stimulates insulin secretion and improves glycaemia. Based on case reports, the U.S. Food and Drug Administration (FDA) has added warnings about acute pancreatitis to the labeling information of sitagliptin in 2009. However, their long-term safety remains inconclusive. It’s a relatively new drug that studies characterizing sitagliptin overdose have not been performed. Therefore, analytical methods for the analysis of sitagliptin in biological samples are required for therapeutic drug monitoring. Therapeutic drug monitoring can be used to determine the relationship between plasma drug concentration and the pharmacological effect. In this study, a sensitive MALDI-TOF MS method was developed and validated for the analysis of sitagliptin in human plasma and in the pharmaceutical tablets. These results show great precision and accuracy. The intra-day and inter-day relative standard deviation (RSD) were less than 10% and the relative error (RE) were all within 10%. The method was successfully applied to quantify the concentrations of sitagliptin in a clinical study. Since sitagliptin has already been proved clinically useful for type 2 diabetes, this drug may represent a potential new therapeutic strategy for other diseases. Therefore, cell lines used in the study are Huh-7 and HK-2. The tryptic peptides from sitagliptin-induced protein fractions were identified using nanoUPLC-MS/MS and the protein categorization and the network prediction were analyzed using bioinformatics programs. In Huh-7, network analysis shows an involvement of cancer, organismal injury and etc. In HK-2, network analysis shows an involvement of cancer, DNA replication /recombination and etc.

並列關鍵字

Sitagliptin ； MALDI-TOF MS ； nanoUPLC-MS/MS

參考文獻

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利用質譜儀建立 Sitagliptin 簡單又快速之分析方法及鑑定其藥物引發之蛋白質表現

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