AB0236 Development and validation of a sensitive lc-ms/ms-based method for analysis of enzymatic activity of folylpolyglutamate synthetase and methotrexate polyglutamates in peripheral blood mononuclear cells of rheumatoid arthritis patients

Background Methotrexate (MTX) is a widely applied anti-rheumatic and anti-leukemic drug. For its intracellular retention and pharmacologic activity, MTX relies on the enzymatic activity of folylpolyglutamate synthetase (FPGS) to convert MTX into its polyglutamate forms (MTX-PG 2–6 ). Loss of FPGS activity is associated with reduced MTX activity and although red blood cell (RBC) MTX-PG n levels correlate with disease activity in RA patients, 1 it is anticipated to be more relevant to measure MTX-PG n in peripheral blood mononuclear cells (PBMCs). Thus, the aim of our study was to develop a LC-MS/MS method to 1) measure FPGS activity replacing laborious radioactive assays, and 2) to measure MTX-PG n in PBMCs. 2 Objectives To validate a rapid, sensitive and non-radioactive assay to measure FPGS activity and MTX-PG n in PBMCs based on LC-MS/MS technology. Methods Protein extracts (n=5) of PBMCs of MTX-treated RA patients were incubated for 2 hours at 37°C in FPGS assay buffer (pH8.8) containing 250 µM MTX and 4 mM l-glutamic acid as substrates. Next, MTX-PG 2 formation was analysed with AB Sciex 4000 Q Trap tandem mass spectrometer coupled to an Acquity Ultra Performance LC system. Measurement of PBMC-MTX-PG n (n=5) was performed by extraction of MTX-PG n from PBMCs by perchloric acid precipitation. Quantification was performed with 13 C 5 15 N-labelled MTX-PG 1–5 internal standards. In FPGS activity and MTX-PG validation studies, human CCRF-CEM leukaemia cells, CEM/R30dm (a FPGS-deficient, MTX-resistant subline of CCRF-CEM), and human acute lymphoblastic leukemic (ALL) cells served as reference. Results In CCRF-CEM, the FPGS enzymatic assay showed linearity with protein input (10–250 µg) and incubation time (0.5–3 hours). Substrate affinity parameters (Km) for MTX (65 µM) and l-glutamic acid (2.2 mM) were consistent with earlier reports. 3 FPGS activity in CEM/R30dm was n in RA patient PBMCs were 22,1% (range: 8.2%–36.2%) for MTX-PG 2 , 32.8% (27.1%–43.6%) for MTX-PG 3 , 34.4% (30.4%–41.3%) for MTX-PG 4 and 10.6% (0.0%–28.4%) for MTX-PG 5 . Average total MTX-PG n levels per number of RA patient PBMCs were 30–50 fold higher than matched numbers of erythrocytes, and 6–9 fold lower than ALL blasts incubated for 24 hours with 1 µM MTX. Conclusions A sensitive LC-MS/MS based method was developed for the measurement of FPGS activity and MTX-PG n levels in PBMCs of RA patients. This method holds promise to guide future MTX-therapy response evaluations. References [1] de Rotte MC, et al. Ann Rheum Dis2015;74:408–414. [2] Jansen G, et al. Oncology Res1992;4:299–305. [3] Liani E, et al. Int. J. Cancer2003;103:587–599. Disclosure of Interest None declared