Analysis of Concentrations of Monomethyl Fumarate in Patients with Multiple Sclerosis: Result from Routine Health Care.

BACKGROUND: Dimethyl fumarate is used to treat patients with relapsing-remitting multiple sclerosis. After ingestion, it is rapidly hydrolyzed to the active primary metabolite monomethyl fumarate. OBJECTIVE: The main objective of our study was to analyze serum concentrations of monomethyl fumarate during routine health care in patients with multiple sclerosis treated with a fixed dose of dimethyl fumarate. METHODS: In the pilot cross-sectional study, data from 42 patients treated with dimethyl fumarate at a dose of 240 mg twice daily were collected. Concentrations of the active metabolite monomethyl fumarate were determined at 1-8 h (median, 3 h) or 10-14 h (median, 13 h) after taking the dose. The relationship between monomethyl fumarate concentrations and absolute lymphocyte count was evaluated. RESULTS: Concentrations of monomethyl fumarate ranged from 2.5-3177.9 µg/L, with most concentrations being undetectable approximately 10 hours after administration. In the 1-8 h (median, 3 h) post-dose subgroup, the concentration/dose ratio ranged widely from 0.04-6.62. The median concentration of monomethyl fumarate in the group with the absolute lymphocyte count <0.8 x 10^9/l was more than four times higher than in the group with the absolute lymphocyte count ≥0.8 x 10^9/l (median 440.1 µg/L versus 98.4 µg/L). CONCLUSION: The wide interindividual variability in monomethyl fumarate pharmacokinetics could contribute to the differential response to dimethyl fumarate in multiple sclerosis patients. A nonsignificant but noticeable trend was observed in the relationship of higher serum monomethyl fumarate concentrations to absolute lymphocyte counts.

Liquid chromatography-tandem mass spectrometry for determination of fingolimod and its active metabolite fingolimod phosphate in whole blood of patients with multiple sclerosis.

Fingolimod is an oral drug for the escalation of treatment of relapsing-remitting multiple sclerosis in patients with persistent disease activity on first-line drugs or in patients with rapidly progressive severe relapsing-remitting multiple sclerosis. An ultra-high-performance liquid chromatography-tandem mass spectrometry method for determining the concentrations of fingolimod and its active metabolite fingolimod phosphate in whole blood has been developed and validated. The advantages of this method are the easy, fast and cheap sample preparation using protein precipitation from blood with a mixture of acetonitrile-methanol (40:60, v/v). Chromatographic separation was performed on a ultra-high performance liquid chromatography BEH C18 1.7 µm (100 × 2.1 mm) column. Two modes of ionization, electrospray ionization and atmospheric pressure chemical ionization, were tested and compared. For validation, the electrospray ionization mode was chosen. As internal standard, isotopically labeled fingolimod-D4 was used to quantify the analytes. The method was validated according to the rules of the European Medicines Agency. The coefficients of variation for fingolimod were in the range of 1.13-11.88%, and the recovery was 98.80-106.00%. The coefficients of variation for fingolimod phosphate were in the range of 2.73-9.31%, and the recovery was 90.08-107.00%. The method is quite easy and fast and can be used for routine analysis.

Serum teriflunomide concentrations in routine multiple sclerosis therapy: A cross-sectional pilot study.

BACKGROUND: Teriflunomide is administered orally to treat relapsing-remitting multiple sclerosis. In this prospective pilot study, the free and total serum concentrations of teriflunomide obtained during routine health care were measured and their relationship with disease activity was evaluated. METHODS: Eighty-nine patients were included in this study. Blood samples were collected from April 2021 to February 2022, and free and total teriflunomide serum concentrations were measured. Patient assessment involved monitoring of blood counts and potential adverse effects of teriflunomide. RESULTS: In the steady-state group, total teriflunomide concentrations ranged from 14.7 to 144.2 mg/L, while free concentrations from 31.1 to 389.7 µg/L. In the non-steady-state group, the total concentration ranged from 2.2 to 59.3 mg/L, with free concentrations ranging from 6.8 to 143.5 µg/L. In the steady-state group, a significant inverse correlation was found between absolute peripheral blood lymphocyte count and both total and free teriflunomide serum concentrations. CONCLUSION: Although all patients were treated with the same dose, up to a 10-fold difference in total and free teriflunomide serum concentrations, and up to a 5-fold difference in steady-state trough concentrations were observed. This vast interindividual variability can potentially lead to toxicity or, conversely, to suboptimal therapeutic concentrations of teriflunomide, with the risk of further worsening of multiple sclerosis compensation.

Liquid chromatography-tandem mass spectrometry method for determination of total and free teriflunomide concentration in serum of patients with multiple sclerosis.

Teriflunomide belongs to disease-modifying drugs and is used in treatment of multiple sclerosis. According to in vitro studies more than 99.4 % of drug is binding to plasma proteins and only less than 1 % is free for clinical activity. The rapid and simple ultra-performance liquid chromatography-tandem mass spectrometry method (UPLC-MS/MS) was developed and validated for determination of total and free teriflunomide (TFM) in serum of patients with multiple sclerosis. To determine the total teriflunomide samples were precipitated with a precipitation reagent consisting of 11 % solution of ZnSO4 in acetonitrile/methanol (40:60, v/v). To determine the free fraction of teriflunomide, an ultracentrifugation method was used. The analysis was performed on a UPLC system connected to a XEVO TQ-XS mass spectrometer. Chromatographic separation was carried out on an Acquity UPLC BEH C18 1.7 µm (100 × 2.1 mm) column heated to 30 °C and teriflunomide-D4 was used as an internal standard. Ionization was performed by electrospray in negative ion mode. The developed methods were validated according to the rules of the European Medicines Agency (EMA) for the analytical method validation of bioanalytical methods. The coefficients of variation were in the range of 0.53-14.84 % and the recovery 97.92-108.33 %, respectively. Share of free teriflunomide was 0.15-0.40 % (mean 0.25 ± 0.05 %) of total teriflunomide and there was a significant correlation between free and total teriflunomide r2 = 0.9083 (p < 0.0001). This newly developed method allows the rapid and easy determination of the teriflunomide concentration with high sensitivity and can be applied to clinical samples of patients with multiple sclerosis.