| Summary: | 博士 === 國立臺灣大學 === 藥學研究所 === 107 === Cancer is one of the leading causes of death globally. To fight this disease, the researches such as the risk factors of cancer, cancer detection methods, and cancer treatment are all growing rapidly in recent years. Biomarker discovery was one of the potential strategies for early detection of cancer. In addition, due to the promising efficacy of therapeutic monoclonal antibodies (mAbs), these types of drugs are frequently used in the cancer treatment. Many studies are still investigating the pharmacokinetic (PK) and pharmacodynamic (PD) properties of therapeutic mAbs. Therefore, an accurate and precise quantification method would facilitate these studies. This study used both gas-chromatography mass spectrometry (GC-MS) and liquid- chromatography mass spectrometry (LC-MS) platforms to develop bio-analytical methods for cancer detection and quantification of therapeutic monoclonal antibodies (mAbs) drugs, and hopefully to improve cancer treatment and also to facilitate precision medicine.
This thesis is divided into two parts. In the first part, we used a targeted metabolomics approach to investigate the potential fatty acids as the biomarkers for breast cancer (BC) detection on GC-MS. Detect BC at early stage could not only reduce the cancer mortality but also improve the treatment outcome. To investigate potential fatty acid biomarkers, we developed a fatty acid profiling method for plasma metabolomic studies by GC-MS. The plasma samples were derivatized by acetyl chloride, and then analyzed by GC-MS. This analytical method was validated with accuracy within 100 ± 15%, and the intraday and interday precision were lower than 15% RSD for over 90% analytes. We further analyzed fatty acid profiles in both healthy volunteers and BC patients. To consider that the fatty acid profiles would be affected by the menopausal status, we divided the samples into pre- menopausal and post- menopausal. In post-menopausal group, the concentrations of three fatty acids including C22:0, C24:0, C18:2n6 were significantly lower in BC patients. In addition to C22:0, C24:0, C18:2n6, seven fatty acids, including C16:0, C18:0, C18:1n9c, C20:0, C20:4n6, C22:6n3, and C24:1n9, showed significant differences in pre-menopausal patients. We further applied these potential BC biomarkers to build the prediction model. The optimal prediction model for post-menopausal group was built by C18:2n6, C22:0 and C24:0 with the area under the curve (AUC) value of 0.72 (70.6% sensitivity and 70.4% specificity). In pre-menopausal group, the prediction model was built by C22:0 and C24:1n9, with the AUC value of 0.78 (73.3% sensitivity and 70.8% specificity). In the second part of this thesis, we developed two analytical methods for therapeutic mAb quantification in human plasma samples. In the first study, we developed a protein G purification method to trap the target analyte, bevacizumab, from human plasma, and applied another immunoglobulin G (IgG) based drug tocilizumab as the internal standard to correct the potential loss during sample preparation procedure. The method was validated, and then used to quantify the bevacizumab concentrations in patients’ plasma samples. The quantification results revealed that bevacizumab concentrations fluctuated significantly between individuals even though the dosage of bevacizumab and the collection time point were both the same for the tested individuals. This method could be applied to investigate the PK and PD properties of bevacizumab, and also for conducting therapeutic drug monitoring (TDM) of bevacizumab.
IgG represents a high percentage of mAb drugs that have been approved by the Food and Drug Administration (FDA). To facilitate therapeutic drug monitoring and PK/PD studies, we developed a general LC-MS/MS method to quantify the concentration of IgG-based mAbs in human plasma. Three IgG-based drugs (bevacizumab, nivolumab and pembrolizumab) were selected to demonstrate our method. Protein G beads were used for sample pretreatment, combined with a two internal standard (IS) calibration method that includes the IgG-based drug-IS tocilizumab and post-column infused IS. The results indicated that the accuracy for three demonstration drugs were all within 100 ±15%. The intraday and interday precision were lower than 15% RSD. Using two internal standards was found to effectively improve quantification accuracy. The successful application of the method to clinical samples at trough levels demonstrated its’ applicability in clinical analysis
This thesis used a targeted metabolomic approach to investigate the potential fatty acids as the BC detection biomarkers, and developed LC-MS/MS methods for quantifying therapeutic mAbs concentrations in human plasma. All of these methods were validated, and applied to clinical samples to investigate the method applicability. It is anticipated that these methods could be applied to clinical practice, and to achieve the goals of personalized medicine.
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