Abstract
KRAS (Kirsten Rat Sarcoma virus) is the most frequently mutated oncogene in human cancers. Mutation of the KRAS protein was prevalent in patients with pancreatic cancer, colorectal cancer, lung cancer, and multiple myeloma. The predominate KRAS mutation is a missense mutation located at codon 12 of this protein. The mutated
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KRASG12C protein occurs in approximately 11 - 16% of lung adenocarcinoma and around 4% of colorectal adenocarcinoma. Moreover, lung adenocarcinoma patients who harbor the KRASG12C mutation have been associated with a poor prognosis. Lung cancer and colorectal cancer were reported in 2020 as the first and second leading causes of global cancer death, with an estimated 1.8 and 0.9 million deaths, respectively. Therefore, targeting KRAS is promising to alleviate the global cancer burden. Since the first identification of the KRAS protein in human cancer in 1982, many efforts have been made to target this protein. However, the first KRASG12C inhibitor, sotorasib, was approved by the FDA and EMA only in 2021 and 2022, respectively. Adagrasib, the second approved KRASG12C inhibitor, was approved by the FDA in 2022 and by EMA in 2023. Both inhibitors were developed to specifically target the KRASG12C-mutant via a covalent bond between their acrylamide moiety and the C12 of KRASG12C. Following the approval of sotorasib and adagrasib, many KRASG12C inhibitors are currently being investigated in both preclinical and clinical stages. One of the promising candidates currently in clinical investigation is opnurasib. Opnurasib was developed to have a novel binding mechanism that may lead to different tolerability and resistance profiles. This may offer implications for its future use in combination therapies with other anticancer drugs. Opnurasib demonstrated an acceptable safety profile at a dose of 200 mg BID with clinical activity in patients with non-small cell lung carcinoma (NSCLC).
This thesis describes research on the bioanalysis of novel KRASG12C inhibitors in supporting preclinical mouse studies of sotorasib, adagrasib, and opnurasib, investigating their pharmacokinetics. We employ state-of-the-art LC (liquid chromatography) tandem MS (mass spectrometry) for this bioanalytical research thesis. Primarily, we use the triple-quadrupole MS coupled with reverse-phase LC to quantify sotorasib, adagrasib, and opnurasib in various mouse matrices. The LC‒triple-quadrupole MS provides a superior sensitivity required for the quantification of drugs, which is often only present in a low concentration, especially in the least-distributed and best protected tissues or in the circulation in the late elimination phase. This method is also employed to quantify sotorasib and its major circulating metabolite (M24) in a subset of preclinical samples of sotorasib. Validation is performed to ensure the reliability of the bioanalytical method before it is used to quantify more than hundreds of preclinical samples. Furthermore, LC tandem hybrid triple-quadrupole linear-ion-trap MS (QTRAP) is utilized to perform metabolite screening of opnurasib in wild-type mice after oral administration of opnurasib. Although triple-quadrupole MS is considered a low-resolution MS less suitable for high-scan speed experiments such as metabolite screening, the QTRAP instrument offers a predictive multiple reaction monitoring-information dependent analysis-enhance product ion (pMRM-IDA-EPI) mode. This mode provides a higher sensitivity to perform metabolite screening than triple-quadrupole MS. The developed qualitative and quantitative bioanalytical methods of the three novel KRASG12C inhibitors and their application are presented in the content of this thesis.
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