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In vitro and in vivo protein binding of methotrexate assessed by microdialysis
M B Maia1, S Saivin, E Chatelut
1Laboratoire Cinétique des Xénobiotiques, Faculté des Sciences Pharmaceutiques, Hôpital Rangueil, Toulouse, France.
Abstract:
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 microM and 300 microM, 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 microM) 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.
Insights
Microdialysis can accurately measure methotrexate protein binding by accounting for how protein content affects probe recovery. This method is crucial for understanding drug distribution and efficacy.
Area of Science:
- Pharmacokinetics
- Analytical Chemistry
- Biochemistry
Background:
- Accurate measurement of drug protein binding is essential for understanding drug distribution and efficacy.
- Microdialysis is a technique used to measure unbound drug concentrations in biological fluids.
- The influence of protein content on microdialysis recovery requires careful consideration.
Purpose of the Study:
- To estimate methotrexate protein binding using microdialysis.
- To determine the impact of protein concentration on dialysis membrane recovery.
- To compare in vitro and in vivo methotrexate protein binding and recovery.
Main Methods:
- In vitro recovery estimation using water recovery and concentration difference methods.
- Assessment of methotrexate binding to human serum albumin (HSA) and plasma proteins.
- Intravenous administration in rats to determine in vivo binding and recovery.
Main Results:
- Methotrexate protein binding was independent of concentration in vitro.
- Protein presence significantly affected dialysis membrane recovery, with higher recovery in buffer than in HSA or plasma.
- In vivo plasma binding of methotrexate in rats was higher than in vitro, and in vivo recovery was lower than in vitro.
Conclusions:
- Protein content directly influences microdialysis probe recovery.
- Microdialysis can accurately measure methotrexate protein binding when recovery is accounted for.
- Understanding protein binding is critical for optimizing methotrexate therapy.