Article Figures & Data
Figures
- Fig. 1.
Effect of compound 1 on enzyme activities (A) and mRNA expressions (B) of CYP1A2, CYP2B6, and CYP3A in human hepatocytes. Human hepatocytes were incubated with compound 1 or the vehicle control, which was replaced daily for 72 hours. Data represent the mean ± S.D. (n = 3). Dunnett’s multiple comparisons test was performed by comparing the compound 1–treated cells with the vehicle-treated cells (**P < 0.01; ***P < 0.001).
- Fig. 2.
Cell viability after treatment with compound 1. Human hepatocytes were incubated with several concentrations of compound 1, which was replaced daily for 72 hours. Cell viability was measured using the Cell Counting Kit-8. Data represent the mean ± S.D. (n = 4).
- Fig. 3.
Effects of the PHD2 inhibitors on CYP1A2 (A), CYP2B6 (B), and CYP3A4 (C) mRNA expressions. Human hepatocytes were incubated with the PHD2 inhibitors (compounds 2–5: 0.1, 1, and 10 μM; compound 6: 0.3, 3, and 30 μM) or a vehicle control, which were replaced daily for 72 hours. Data represent the mean ± S.D. (n = 3). The closed and open bars show the strong PHD2 inhibitors (compounds 2, 5, and 6) and weak PHD2 inhibitors (compounds 3 and 4), respectively. Dunnett’s multiple comparisons test was performed by comparing the PHD2 inhibitor–treated cells with the vehicle-treated cells (*P < 0.05; **P < 0.01; ***P < 0.001).
- Fig. 4.
Relationships of mRNA expressions of CYP1A2 (A and B), CYP2B6 (C and D), and CYP3A4 (E and F) with EPO production in human hepatocytes treated with the strong (A, C, and E) or weak (B, D, and F) PHD2 inhibitors. Compounds 1, 2, 5, and 6 are the strong PHD2 inhibitors. Compounds 3 and 4 are the weak PHD2 inhibitors. Human hepatocytes were incubated with the PHD2 inhibitors or a vehicle control, which were replaced daily for 72 hours. The concentrations tested were as follows: 1 and 10 μM (compound 1); 0.1, 1, and 10 μM (compounds 2–5); and 0.3, 3, and 30 μM (compound 6). Data represent the mean ± S.D. (n = 3).
- Fig. 5.
Correlations of mRNA repressions of CYP1A2, CYP2B6, and CYP3A4 with the corresponding transcription factors in human hepatocytes treated with the PHD2 inhibitors. Human hepatocytes were incubated with the individual PHD2 inhibitors (compounds 2–5: 0.1, 1, and 10 μM; compound 6: 0.3, 3, and 30 μM), which were replaced daily for 72 hours. The correlations between CYP1A2 and AhR (A), CYP1A2 and ARNT (B), CYP2B6 and CAR (C), CYP2B6 and RXR (D), CYP3A4 and PXR (E), and CYP3A4 and RXR (F) were analyzed. Data represent the mean (n = 3). Lines represent the best-fit estimates of least-squares liner regression analysis. A correlation coefficient (r) is shown for each plot.
Tables
- TABLE 2
LDH release after exposure to PHD2 inhibitors in human hepatocytes
The total amount of LDH released into the medium for 72 h from cells treated with tamoxifen (50 μM, a positive control for cell toxicity) was regarded as equivalent to 100% LDH in cells.
Compound Conc. (μM) LDH Release (%)a 0–24 h 24–48 h 48–72 h Control (0.1% DMSO) — 3.9 2.9 1.7 2 10 3.7 3.0 2.5 3 10 3.0 2.0 2.5 4 10 3.6 2.0 1.9 5 10 3.7 5.7 6.3 6 30 3.2 2.0 2.9 ↵a Each value represents the mean (n = 3).
- TABLE 3
Inhibitory effects of compound 1 (10 μM) on CYP1A2, CYP2B6, and CYP3A enzyme activities in both reversible and time-dependent inhibitions in human microsomes
Mechanism % Inhibitiona CYP1A2 CYP2B6 CYP3A Reversible inhibition 4.0 2.4 −3.2 Time-dependent inhibitionb −7.2 −2.3 −10.7 ↵a Each value represents the mean (n = 3).
b The time-dependent inhibition was indicated as the difference in the percent inhibition between with and without the first incubation.
Data Supplement
- Supplemental Data -
Supplementary Methods
Supplementary References
- Supplemental Data -
