Article Figures & Data
Figures
- Fig. 1.
Substrate saturation curves for phenacetin O-deethylation (A), coumarin hydroxylation (B), bupropion hydroxylation (C), amodiaquine N-deethylation (D), diclofenac hydroxylation (E), mephenytoin hydroxylation (F), dextromethorphan O-demethylation (G), chlorzoxazone 6-hydroxylation (H), midazolam 1-hydroxylation (I), and testosterone 6-hydroxylation (J) in MMHHs. Each data point is the mean ± S.E.M. of triplicates from a single experiment.
- Fig. 2.
Comparison of IC50 values generated using isoform-specific marker probe reactions from CHHs and MMHHs. The solid line represents the line of unity, the dotted line represents a 2-fold deviation from the line of unity, and the dashed line represents a 3-fold deviation from the line of unity.
- Fig. 3.
The inhibition curves in MMHHs (●) and CHHs (○) for a set of representative inhibitors, including α-naphthoflavone (A), tranylcypromine (B), 2-phenyl-2-(1-piperidinyl)propane (PPP) (C), montelukast (D), sulfaphenazole (E), omeprazole (F), paroxetine (G), methylpyrazole (H), ketoconazole (I), and mibefradil (J), against CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 (midazolam hydroxylation), and CYP3A4 (testosterone hydroxylation) enzyme-mediated activities, respectively. Each data point is the mean ± S.E.M. of six replicates in a single experiment.
Tables
- TABLE 1
Ion mode application, mass transitions, and linearity range used for the quantification of marker metabolites of the isoform-specific pathways used for kinetic experiments in MMHHs and inhibition experiments in both MMHHs and CHHs
P450 Isozyme Marker Substrate Marker Metabolite Ion Mode Mass Transition Linearity Range m/z nM CYP1A2 Phenacetin Acetaminophen Positive 152.2→110 6.6–6622.5 CYP2A6 Coumarin Hydroxy coumarin Negative 160.8→132.7 6.2–6211.2 CYP2B6 Bupropion Hydroxy bupropion Positive 256→139 3.9–3921.6 CYP2C8 Amodiaquine Desethyl amodiaquine Positive 328.3→283.3 3.1–3058.1 CYP2C9 Diclofenac Hydroxy diclofenac Positive 311.927→266 3.2–3215.4 CYP2C19 Mephenytoin Hydroxy mephenytoin Positive 235.1→150.2 4.3–4273.5 CYP2D6 Dextromethorphan Dextrorphan Positive 258.3→157.2 3.9–3891.1 CYP2E1 Chlorzoxazone Hydroxy chlorzoxazone Negative 184→120.1 5.4–5434.8 CYP3A4 Midazolam Hydroxy midazolam Positive 342→203 2.9–2932.6 CYP3A4 Testosterone Hydroxy testosterone Positive 305.1→269.1 3.9–3289.5 - TABLE 2
Substrate concentrations, Vmax, and Km values determined for the marker reactions in MMHHs from two independent studies
Vmax units are picomoles per minute per million cells; Km units are micromolars.
P450 Marker Reaction Study Substrate Concentrations Vmax Mean Vmax Km Mean Km Kinetics µM CYP1A2 Phenacetin deethylationa 1 0, 15.625, 31.25, 62.5, 125, 250, 500, 1000 51 80 11.4 11 TS (atypical) 2 0, 7.81, 15.625, 31.25, 62.5, 125, 250, 500 109 10.7 TS (atypical) CYP2A6 Coumarin hydroxylation 1 0, 0.937, 1.875, 3.75, 7.5, 15, 30, 60 — 112 — 1.5 ND 2 0, 0.156, 0.312, 0.625,1.25, 2.5, 5,10 112 1.5 SI (atypical) CYP2B6 Bupropion hydroxylation 1 0, 15.625, 31.25, 62.5, 125, 250, 500, 1000 131 136 69 67 MM (typical) 2 0, 15.625, 31.25, 62.5, 125, 250, 500, 1000 140 66 MM (typical) CYP2C8 Amodiaquine deethylation 1 0, 0.937, 1.875, 3.75, 7.5, 15, 30, 60 758 517 1.4 1.4 MM (typical) 2 0, 0.937, 1.875, 3.75, 7.5, 15, 30, 60 277 1.4 MM (typical) CYP2C9 Diclofenac hydroxylation 1 0, 3.125, 6.25, 12.5, 25, 50, 100, 200 — 3273 — 2.4 ND 2 0, 0.781, 1.562, 3.125, 6.25, 12.5, 25, 50 3273 2.4 MM (typical) CYP2C19 Mephenytoin hydroxylation 1 0, 14.06, 28.12, 56.25, 112.5, 225, 450, 900 49 50 55 45 MM (typical) 2 0, 14.06, 28.12, 56.25, 112.5, 225, 450, 900 52 35 MM (typical) CYP2D6 Dextromethorphan O-demethylationa 1 0, 3.125, 6.25, 12.5, 25, 50, 100, 200 28 37 2.6 2.0 TS (atypical) 2 0,0.47, 0.937, 1.875, 3.75, 7.5, 15, 30 47 1.5 TS (atypical) CYP2E1 Chlorzoxazone hydroxylation 1 0, 31.25, 62.5, 125, 250, 500, 1000, 2000 693 462 469 465 MM (typical) 2 0, 15.62, 31.25, 62.5, 125, 250, 500, 1000 231 462 MM (typical) CYP3A4 Midazolam hydroxylation 1 0, 0.937, 1.875, 3.75, 7.5, 15, 30, 60 187 215 3.7 3.5 SI (atypical) 2 0, 0.312, 0.625,1.25, 2.5, 5,10, 20 243 3.3 MM (typical) CYP3A4 Testosterone hydroxylation 1 0, 15.62, 31.25, 62.5, 125, 250, 500, 1000 765 592 73b 70 HK (atypical) 2 0, 15.62, 31.25, 62.5, 125, 250, 500, 1000 418 67b HK (atypical) - TABLE 3
A comparison of Vmax and Km values determined using MMHHs from this study with those determined in the cryopreserved human hepatocytes and HLMs
The Vmax and Km values and the calculated ratio of the values for MMHH to that for hepatocytes and human liver microsomes are shown. Vmax values are in pmol/min per million cells for MMHH and hepatocytes; Km values are in µM.
P450 Isoforms Marker Reaction MMHH Hepatocytes HLM Reference for Hepatocytes Reference for Human Liver Microsomes MMHH/Hepatocyte Ratio MMHH/HLM Ratio Vmax Km Vmax Km Km Vmax Km Vmax CYP1A2 Phenacetin deethylation 80 11 NA NA 112.7 NA Li et al. (2015) NA NA 0.10 CYP2A6 Coumarin hydroxylation 112 2 NA NA 1.9 NA Hosono et al. (2017) NA NA 1.05 CYP2B6 Bupropion hydroxylation 136 67 21 41 130 Li and Schlicht (2014) Faucette et al. (2000) 6.5 1.6 0.52 CYP2C8 Amodiaquine deethylation 517 1 334 10 1 Li and Schlicht (2014) Li et al. (2015) 1.5 0.1 1.00 CYP2C9 Diclofenac hydroxylation 3273 2 290 7 22.4 Brown et al. (2007) Siu and Lai (2017) 11.3 0.3 0.09 CYP2C19 Mephenytoin hydroxylation 50 45 16 13 56.8 Brown et al. (2007) Siu and Lai (2017) 3.1 3.5 0.79 CYP2D6 Dextromethorphan demethylation 37 2 50 1 2.9 Brown et al. (2007) Li et al. (2015) 0.7 2.0 0.69 CYP2E1 Chlorzoxazone hydroxylation 462 465 NA NA 149.8 NA Li et al. (2015) NA NA 3.10 CYP3A4 Midazolam hydroxylation 215 4 20 4 8.4 Li and Schlicht (2014) Siu and Lai (2017) 10.8 1.0 0.48 CYP3A4 Testosterone hydroxylation 592 70 1800 24 10.2 Brown et al. (2007) Siu and Lai (2017) 0.3 2.9 6.86 NA, not Available.
- TABLE 4
Absolute IC50 values of standard inhibitors for nine major P450 enzymes determined in MMHHs and CHHs and their corresponding fold difference values
P450 Isoform Maker Reaction Inhibitors Absolute IC50 Fold Difference MMHH/CHH MMHH CHH µM µM 1A2 Phenacetin O-deethylation Fluvoxamine 0.007 0.025 0.28a Furafylline 0.81 1.2 0.67 Methoxsalen 0.015 0.025 0.60 α-Naphthoflavone 0.029 0.047 0.62 Propranolol 0.57 0.39 1.46 2A6 Coumarin hydroxylation Ketoconazole 91.4 100 0.91 Methoxsalen 0.10 0.03 3.33a Phenelzine 2.1 2.0 1.07 Tranylcypromine 0.059 0.007 8.43a 2B6 Bupropion hydroxylation Ketoconazole 2.7 3.8 0.72 Methoxsalen 4.5 5.9 0.76 Phenelzine 11.2 5.6 2.01 PPP 4.8 5.5 0.87 Thiotepa 4.3 17.7 0.24a Ticlopidine 22.4 12.3 1.82 2C8 Amodiaquine N-deethylation Ketoconazole 11.9 10.2 1.17 Methoxsalen >100 >100 NC Montelukast 4.1 4.3 0.95 Tranylcypromine 77.8 58 1.35 2C9 Diclofenac hydroxylation Fluconazole 23.2 82.0 0.28a Ketoconazole 7.2 13.0 0.55 Methoxsalen 34.2 36.7 0.93 Sulfaphenazole 0.4 0.63 0.59 2C19 Mephenytoin hydroxylation Fluconazole 4.6 3.2 1.46 Fluoxetine 1.2 4.8 0.24a Fluvoxamine 0.02 0.04 0.64 Methoxsalen 14.5 12.1 1.19 Omeprazole 4.6 2.6 1.80 Ticlopidine 13.8 20.1 0.69 2D6 Dextromethorphan O-demethylation Cinacalcet 3.3 11.5 0.29a Fluoxetine 0.63 0.92 0.68 Methoxsalen 39.6 43.1 0.92 Paroxetine 0.37 0.75 0.49 Propafenone 0.08 0.07 1.14 Quinidine 0.04 0.02 2.33 2 E1 Chlorzoxazone hydroxylation Methoxsalen 75.7 81.5 0.93 Methylpyrazole 1.4 1.1 1.24 3A4 Midazolam hydroxylation Erythromycin 5.3 4.1 1.31 Ketoconazole 0.09 0.09 1.11 Methoxsalen >100 >100 NC Mibefradil 0.32 0.32 1.00 Troleandomycin 2.2 1.1 1.98 Verapamil 18.8 5.97 3.15a 3A4 Testosterone hydroxylation Erythromycin 13.9 22.4 0.62 Ketoconazole 0.045 0.12 0.38 Methoxsalen 10.3 29.8 0.35 Mibefradil 0.25 0.75 0.33 Troleandomycin 3.0 7.4 0.40 Verapamil 4.4 8.8 0.50 NC, fold difference not calculated; PPP, 2-phenyl-2-(1-piperidinyl)propane.
↵a Fold difference of ≥3 or ≤0.3 between MMHHs and CHHs.
