Background: Folylpolyglutamate synthetase (FPGS) is a crucial enzyme in both cellular folate homeostasis and the intracellular retention of folate analogue drugs such as methotrexate (MTX), which is commonly used for the treatment of (pediatric) leukemia and the anchor drug in rheumatoid arthritis (RA) treatment. To date, assessment of FPGS catalytic activity relies on assays using radioactive substrates that are labor-intensive and require relatively large numbers of cells. Here, we describe a nonradioactive, ultra–high-performance liquid chromatography–tandem mass spectrometer (UHPLC-MS/MS)–based method allowing for sensitive and accurate measurements of FPGS activity in low cell numbers (ie, 1–2 × 106) of biological specimens, including leukemic blast cells of acute lymphoblastic leukemia patients and peripheral blood mononuclear cells of patients with RA. Methods: The UHPLC-MS/MS assay was validated with 2 CCRF-CEM human leukemia cells, one proficient and one deficient in FPGS activity. Linearity of time and protein input were tested by measuring FPGS activity at 30–180 minutes of incubation time and 10–300 mcg protein extract. In addition, FPGS enzyme kinetic parameters were assessed. Results: The FPGS enzymatic assay showed a linear relation between FPGS activity and protein input (R2 ≥ 0.989) as well as incubation time (R2 ≥ 0.996). Moreover, the UHPLC-MS/MS method also allowed for evaluation of FPGS enzyme kinetic parameters revealing Km values for the substrates MTX and L-glutamic acid of 64 µmol/L and 2.2 mmol/L, respectively. The mean FPGS activity of acute lymphoblastic leukemia blast cells (n = 4) was 3-fold higher than that of CCRF-CEM cells and 44-fold and 88-fold higher than that of peripheral blood mononuclear cells from MTX-naive (n = 9) and MTX-treated RA patients (n = 6), respectively. Conclusions: Collectively, given its sensitivity with low cell numbers and avoidance of radioactive substrates, UHPLC-MS/MS–based analysis of FPGS activity may be eligible for routine therapeutic drug monitoring of MTX in RA and leukemia for therapy (non)response evaluations.

The psychedelic alkaloid ibogaine is increasingly used as an oral treatment for substance use disorders, despite being unlicensed in most countries and having reported adverse events. Using wild-type and genetically modified mice, we investigated the impact of mouse (m)Abcb1a/1b and Abcg2 drug efflux transporters, human and mouse OATP drug uptake transporters, and the CYP3A drug-metabolizing complex on the pharmacokinetics of ibogaine and its main metabolites. Following oral ibogaine administration (10 mg/kg) to mice, we observed a rapid and extensive conversion of ibogaine to noribogaine (active metabolite) and noribogaine glucuronide. Mouse Abcb1a/1b, in combination with mAbcg2, modestly restricted the systemic exposure (plasma AUC) and peak plasma concentration (Cmax) of ibogaine. Accordingly, we found a ∼2-fold decrease in the relative recovery of ibogaine in the small intestine with fecal content in the absence of both transporters compared to the wild-type situation. Ibogaine presented good intrinsic brain penetration even in wild-type mice (brain-to-plasma ratio of 3.4). However, this was further increased by 1.5-fold in Abcb1a/1b;Abcg2−/− mice, but not in Abcg2−/− mice, revealing a stronger effect of mAbcb1a/1b in restricting ibogaine brain penetration. The studied human OATP transporters showed no major impact on ibogaine plasma and tissue disposition, but the mOatp1a/1b proteins modestly affected the plasma exposure of ibogaine metabolites and the tissue disposition of noribogaine glucuronide. No considerable role of mouse Cyp3a knockout or transgenic human CYP3A4 overexpression was observed in the pharmacokinetics of ibogaine and its metabolites. In summary, ABCB1, in combination with ABCG2, limits the oral availability of ibogaine, possibly by mediating its hepatobiliary and/or direct intestinal excretion. Moreover, ABCB1 restricts ibogaine brain penetration. Variation in ABCB1/ABCG2 activity due to genetic variation and/or pharmacologic inhibition might therefore affect ibogaine exposure in patients, but only to a limited extent. The insignificant impact of human CYP3A4 and OATP1B1/1B3 transporters may be clinically advantageous for ibogaine and noribogaine use, as it decreases the risks of undesirable drug interactions or interindividual variation related to CYP3A4 and/or OATP activity.

BackgroundMethotrexate (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-PG2–6). Loss of FPGS activity is associated with reduced MTX activity and although red blood cell (RBC) MTX-PGn levels correlate with disease activity in RA patients,1 it is anticipated to be more relevant to measure MTX-PGn 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-PGn in PBMCs.2 ObjectivesTo validate a rapid, sensitive and non-radioactive assay to measure FPGS activity and MTX-PGn in PBMCs based on LC-MS/MS technology.MethodsProtein 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-PG2 formation was analysed with AB Sciex 4000 Q Trap tandem mass spectrometer coupled to an Acquity Ultra Performance LC system. Measurement of PBMC-MTX-PGn (n=5) was performed by extraction of MTX-PGn from PBMCs by perchloric acid precipitation. Quantification was performed with 13C515N-labelled MTX-PG1–5 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.ResultsIn 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 <1% of CCRF-CEM. FPGS activity in ALL blasts was similar to CCRF-CEM while FPGS activity in RA patient PBMCs was 1%–5% of CCRF-CEM, and was non-detectable in RBCs. Average individual fractions of total MTX-PGn in RA patient PBMCs were 22,1% (range: 8.2%–36.2%) for MTX-PG2, 32.8% (27.1%–43.6%) for MTX-PG3, 34.4% (30.4%–41.3%) for MTX-PG4 and 10.6% (0.0%–28.4%) for MTX-PG5. Average total MTX-PGn 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.ConclusionsA sensitive LC-MS/MS based method was developed for the measurement of FPGS activity and MTX-PGn 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 InterestNone declared