Fig 2: Plasma concentration of S-warfarin and its hydroxy metabolites after the intravenous administration of S-warfarin (4.0 mg/kg) in non-humanized liver mice and humanized liver mice. The plasma concentrations of S-warfarin, 4'-hydroxywarfarin, 6-hydroxywarfarin, and 7-hydroxywarfarin in wild-type (circle) or POR cKO (triangle) mice with non-humanized or humanized livers were measured at 0.5, 1, 2, 4, 7, 24, 48, and 72 h after the intravenous administration of S-warfarin (4.0 mg/kg). Each group consists of two mice. The levels of 8- and 10-hydroxywarfarin were below the detection limit in all samples.

Fig 3: Schematic representation of deleted loci of Por gene and Flpo recombinase knocked-in loci of Cyp3a11 in the POR conditional knockout mice. pA poly A signal, HR homologous recombination.

Fig 4: Characterization of S-warfarin metabolism in POR cKO humanized liver mice. (A) Species-dependent regioselective S-warfarin hydroxylation in various animal liver microsomes. S-warfarin (10 µM) was incubated with pooled liver microsomes (0.20 mg/mL) at 37 °C for 20 min. (B) S-warfarin metabolism in pooled liver microsomes from wild-type (six males), humanized liver TK-NOG (Normal Hu-liver, four males) mice, POR cKO humanized liver (POR cKO Hu-liver, four males) mice, and humans. S-warfarin (10 µM) were incubated with the liver microsomes (0.20 mg/mL) at 37 °C for 20 min. (C) Cumulative urinary excretions of S-warfarin and its metabolites for 72 h after intravenous administration (4.0 mg/kg) in wild-type, POR cKO, Normal Hu-liver, and POR cKO Hu-liver mice. The 4'-, 6-, 7-, 8-, and 10-hydroxywarfarin were measured for urine samples treated with ß-glucuronidase. The data presented are from two animals.

Fig 5: Characterization of POR cKO humanized liver mice. (A) Immunohistochemical staining of human Mitochondria (hMIT), CYP1A2, CYP2C9, and CYP3A4 proteins in liver tissue from POR cKO humanized liver mice. Rectangular area on top panel was enlarged in lower panel. Dotted circles indicate the region comprising mouse hepatocytes (m). P, portal tract, C, central vein. Scale bar, 5 mm (top panel) and 500 µm (lower panel). (B) Expression level of human pharmacokinetics-related genes in the liver from humanized liver TK-NOG (Normal Hu-liver) mice (n = 3) and POR cKO humanized liver (POR cKO Hu-liver) mice (n = 3) were measured via qRT-PCR. Data are presented as the mean ± SD. Statistically significant difference when compared with that in normal-Hu-liver mice; ****p < 0.0001. (C) Drug-metabolizing activity in liver microsomes from TK-NOG humanized liver mice (Normal Hu-liver), POR cKO mice, POR cKO humanized liver mice (POR cKO Hu-liver), and humans. Phenacetin (50 µM), diclofenac (40 µM), omeprazole (10 µM), metoprolol (5 µM), and midazolam (5 µM) were incubated with pooled liver microsomes (0.20 mg/mL) from normal Hu-liver mice (n = 4), POR cKO mice (n = 5), POR cKO Hu-liver mice (n = 4), and humans (n = 50) at 37 °C for 10–20 min. Data are presented as the mean ± SD.