Characterization of midazolam metabolism using human hepatic musomal fractions and hepatocytes in suspension obtained by perfusing whole human livers

Abstract

Isolated human hepatocytes provide a useful model for studying xenobiotic metabolism. However, in vitro studies using human hepatocytes are scarce due to the limited availability of this material. A new methodology is described for obtaining hepatocytes from a whole adult human liver. This procedure is based on (i) the rapid and intense in situ washing step of the organ with Eurocollins then glucose supplemented HEPES buffer (10 mM, pH 7.4) at 4° in order to both minimize the warm ischemie period and remove erythrocytes, and (ii) a perfusion of collagenase solution (0.05% in 10 mM HEPES buffer at 37°) throughout the portal vein according to a recirculated model. All perfused buffers are oxygenized. Hepatocyte viability averaged 85% as determined by Trypan Blue dye exclusion. The ability of these hepatocytes to catalyze certain metabolic transformations such as Phase I and Phase II reactions has been particularly investigated using the benzodiazepine drug, midazolam, as a substance probe. Freshly isolated human hepatocytes in suspension retained the ability to metabolize midazolam to its different hydroxylated derivatives—mainly the 1-hydroxy-midazolam—which was further conjugated with glucuronic acid. For a better understanding of the cytochrome P-450 mediated reactions, we studied the metabolism of midazolam in musomal fractions prepared from twelve human livers. It was concluded that human musomes (i) exhibited a Type I binding spectrum upon midazolam addition ( K s = 3.3 μM) and (ii) intensively metabolized the drug to its different derivatives. Furthermore, and since we demonstrated that midazolam was predominantly transformed by a single cytochrome P-450 enzyme, we could attribute the large inter-individual variations in midazolam metabolism to differences in human liver cytochrome P-450 content.