In vitro and in vivo protein binding of methotrexate assessed by microdialysis

The purpose of the study was firstly to estimate methotrexate protein binding using microdialysis, and secondly to determine the influence of the protein content in the dialyzed medium on the dialysis membrane recovery. In vitro recovery was estimated by both water recovery method and concentration difference method. The relative recovery was independent of methotrexate concentration: 39.3% ± 2.86% and 39.2 ± 1.27% for 50 μM and 300 μM, respectively. A significant influence of the presence of proteins on the dialysis membrane recovery was observed : 55.9% ± 2.7%, 42.3% ± 7.5% and 45.5% ± 0.1% for buffer, human serum albumin (HSA) (600 μM) and human plasma, respectively. Methotrexate binding to human and rat plasma proteins showed a nonsaturable phenomenon. The bound percentages and corresponding total binding capacities were 58.7% ± 3.13% and 1.44 ± 0.033 mM -1 and 71.7% ± 4.38% and 2.18 ± 0.09 mM -1 for the human and rat plasma, respectively. For HSA this binding was saturable with an affinity constant of 4.75 ± 0.66 mM -1 . After intravenous administration (250 mg/kg) the in vivo rat plasma binding of methotrexate was roughly 20% higher (93.6%) than the in vitro methotrexate plasma binding. The in vivo relative recovery of methotrexate was found to be 12.6% ± 1.8% versus 25.4% ± 3.3% in vitro. This study showed that the protein content can directly affect microdialysis probe recovery. However, by taking into account this recovery, microdialysis allows to measure the protein binding of methotrexate.