- 學位論文
利用質譜儀建立 Ethosuximide 之快速分析方法及篩選其在藥物合併下肝細胞之蛋白質表現
A fast method for the analysis of ethosuximide and detection of proteins in huh-7 cell after cotreatment with ethosuximide and other drugs by mass spectrometry
摘要
Ethosuximide (3-ethyl-3-methylpyrrolidine-2,5-dione) 為臨床上治療失神性癲癇 (absence seizure) 之首選用藥，其主要機轉為阻斷 T 型鈣離子通道，防止腦丘神經元接受鈣離子，進而達到抗癲癇之效果。大多數的癲癇患者須長期服藥預防癲癇發作，當患者癲癇發作時，需立即監控藥物濃度以調整用藥劑量，達到有效治療濃度及減少臨床肝臟毒性。已有研究證實 ethosuximide 有致畸作用 (teratogenic effect)，因此處於妊娠中的癲癇患者的藥物濃度監控的發展，變得更有其重要性，故本研究欲搭配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) 建立一個快速之臨床監控方式，以及藥物品質管理之檢驗方法。 此外，臨床研究上已知 ethosuximide 的藥物交互作用目前僅限於不同種類的抗癲癇藥物的並用，故本研究想利用液相層析串聯式質譜儀（liquid chromatography coupled with tandem mass spectrometry, LC-MS/MS）探討在抗癲癇以外的藥物如抑制胃酸的 cimetidine、抗組織胺的 ketotifen、控制血糖的 metformin及抗生素metronidazole，在合併ethosuximide使用下是否產生藥物交互作用及探討其蛋白質的表現。另外，這些藥物亦從肝臟進行代謝，故本研究以人類肝細胞株 (huh-7 cell line) 作為模型，探討ethosuximide與其他藥物並用下對於肝臟是否出現損傷，並且以生物資訊 (bioinformatics) 的方式整理蛋白質表現情形來進行肝臟毒性和副作用的評估。 目前已完成建立ethosuximide之快速分析方法，適用於藥物血中濃度監控及藥物品管，其精密度與準確度良好，且 relative standard deviation (RSD) 和 relative error (RE) 皆小於 13 %。蛋白質分析方面，預測ethosuximide合併 cimetidine 使用下，有可能會產生肝癌、肝臟纖維化、肝腫瘤以及腎炎，所以必須更加注意其安全性和毒性。 另外，ethosuximide合併不同藥物亦發現不斷重複出現的蛋白質跟腫瘤和癌症有關，並且影響其正常生理表現。最後，此研究結果對於藥物交互作用以及肝臟毒性的評估有進一步認知，希望未來可以協助肝臟毒性的評估並避免可能面臨的副作用。
並列摘要
Ethosuximide (3-ethyl-3-methylpyrrolidine-2,5-dione) is an antiepileptic drug with first select in the treatment of absence seizure. The mechanism by which ethosuximide blocks T-type calcium channels to prevent accepting calcium in thalamic neurons and gain the effect of anticonvulsant. Most of the patients keep taking ethosuximide to prevent seizure. When seizure duration, patients must monitor therapeutic drug of plasma concentration for regulating dose and gain the therapeutic concentration and reduce clinical hepatotoxicity. Previous studies have confirmed that ethosuximide has teratogenic effect, so it is important to develop a method for therapeutic drug monitoring of anticonvulsants for pregnancy. This study establishes a quick method for ethosuximide analysis by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). This method also could be applied for the purpose of quality control in pharmaceutical factory. Additionally, patients who take ethosuximide long time to prevent the activity of seizure may also take other drugs in clinical treatment , such as cimetidine (inhibition of gastric acid), ketotifen (antihistamine), metformin (control blood sugar) and metronidazole (antibiotic). This study discusses the effects of ethosuximide co-treatment with these drugs by huh-7 cell line and profiling their protein expressions by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and bioinformatics to predict hepatotoxicity and other side effects. The fast method has been established and is applicable for ethosuximide monitoring in human plasma and quality control of pharmaceutical formulation. The results show that good precision and accuracy are obtained the relative standard deviation (RSD) and relative error (RE) were < 13%. In protein analysis, co-treatment with ethosuximide and cimetidine may produce liver cancer, liver fibrosis, hepatocellular carcinoma, nephritis predicted by bioinformatics. Thus, most of the attention in safety and toxicity was necessary. Furthermore, we find some repeated proteins that are associated with cancer and carcinoma and disorders of their normal physiological responses. Ultimately, drug-drug interaction studies and assessment of hepatotoxicity could provide useful information to help to prevent hepatotoxicity and avoid harmful side effects.