Studies on the intracellular accumulation process of methotrexate and its correlation with the key protein using an LC-MS/MS method: a novel way to realize prospective individualized medication.

Abstract

High-dose methotrexate (HDMTX) combined with leucovorin (LV) is the first-line drug therapy for many kinds of malignant tumors. However, the specific treatment plans, such as dosage and duration of administration, are usually formulated according to the clinician's experience and therapeutic drug monitoring (TDM) of methotrexate in patients' plasma, which are responsible for strong individual differences of drug usage. A large number of studies have shown that methotrexate targets the inside of the cell. The key cytotoxic component is the methotrexate polyglutamates (MTXPGs) in the cell. The concentration of methotrexate in plasma does not reflect the efficacy and side effects well. Based on mass spectrometry technology, we developed and validated an accurate, sensitive, and stable method to quantify the intracellular MTX (MTXPG1) and its metabolites MTXPG2-7 simultaneously. The lower limit of quantification was 0.100ng/ml, and the run time was only 3min. Moreover, our team has already developed two LC-MS/MS-based methods to respectively quantify methotrexate in plasma samples and two key proteins (γ-glutamyl hydrolase [GGH] and folylpolyglutamate synthetase [FPGS]) in peripheral blood mononuclear cells (PBMC). Through these highly sensitive and accurate approaches, we have gained a deep understanding of the whole pharmacokinetic process of MTX and explored the key factors affecting the accumulation process of intracellular active components (MTXPGs). Based on this research, it is possible to find a more effective way to provide an accurate reference for clinical drug use than traditional therapeutic drug monitoring (TDM).