Interactions between 7-hydroxymethotrexate and folinic acid in RAJI cells, in vitro

HPLC analysis of plasma samples obtained from patients included in a high-dose methotrexate-folinic acid Rescue (HD-MTX-CF) protocol, allowed the simultaneous determination of MTX, CF and their respective plasma metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 5-methyltetrahydrofolate (5-CH3-FH4). These 4 compounds interact at the cellular level to ensure the selective effectiveness of the HD-MTX-CF rescue protocol. An in vitro study has been investigated in RAJI cells to better describe the interference of CF on uptake, accumulation and metabolism of [3H]7-OH-MTX. Results about uptake and accumulation of CF were also obtained using [3H]CF, in the absence or the presence of unlabeled 7-OH-MTX. The rate of [3H]7-OH-MTX influx in RAJI cells (Km = 25.30 +/- 7.75 microM, n = 3) was competitively inhibited by the presence of 10 microM CF with a Ki value of 6.00 +/- 1.94 microM (n = 2). Intracellular 7-OH-MTX accumulation was decreased by approximately 30% when extracellular CF concentration was twice as high as that of 7-OH-MTX, and 70% when CF extracellular concentration was 5 times higher. The metabolism of 7-OH-MTX to its intracellular polyglutamyl derivatives was depressed by 90% when 10 microM CF were incubated for 2 h with equimolar [3H]7-OH-MTX, and it was completely abolished in the presence of 100 microM CF.