Intracellular CD3⁺ T-lymphocyte teriflunomide concentration is poorly correlated with and has greater variability than unbound plasma teriflunomide concentration

Abstract

Leflunomide's active metabolite teriflunomide inhibits dihydro-oroate dehydrogenase (DHODH), an enzyme essential to proliferation of T-lymphocytes. As teriflunomide must reach the target site to have this effect, this study assessed the distribution of teriflunomide into T-lymphocytes, as intracellular concentrations may be a superior response biomarker to plasma concentrations. CD3 MicroBeads (MiltenyiBiotec) were used to extract CD3(+) T-cells from the peripheral blood of patients with rheumatoid arthritis whom were taking a stable dose of leflunomide. Unbound plasma and intra-CD3(+) T-cell teriflunomide concentrations were quantified using liquid chromatography-mass spectrometry (LC-MSMS). Concentration (log transformed) and partition differences were assessed through paired Student's t-tests. Sixteen patients provided plasma steady-state teriflunomide samples, and eight provided a sample 6-12 weeks later. At time-point one, the geometric mean teriflunomide concentration (range) in CD3(+) T-cells was 18.12 ug/L (6.15 - 42.26 ug/L) compared to 69.75 ug/L (32.89 - 263.1 ug/L) unbound in plasma (p<0.001). The mean partition coefficient (range) for unbound plasma teriflunomide into CD3(+) T-cells was 0.295 (0.092 - 0.632), which was significantly different from unity (p<0.001). The median (range) change in teriflunomide concentration between the two time points was 14% (-10% to 40%) in unbound plasma and -29% (-69 to 138%) for CD3(+) T-cells. Since teriflunomide concentrations in CD3(+) T-cells were lower and displayed a higher intra-individual variability than the unbound plasma concentrations, its applicability as a therapeutic drug monitoring marker may be limited.