Effects of Various Media on the In Vitro Metabolism of Cytochrome P450 Substrates
In our previous work, we reported that neither cofactor availability nor membrane permeability accounted for the much slower in vitro clearance of midazolam in suspended cryopreserved Human hepatocytes (CHH) compared with human liver microsomes (HLM).
We posited the difference was possibly an effect of ionic strength (Kazmi et al., 2013a; Kazmi et al., 2013b). In the present study we evaluated the effects of buffer ionic strength and various media on CYP1A1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and multiple CYP3A4/5 activities in Human liver microsomes and cryopreserved Human hepatocytes. CYP activities were measured at three ionic strengths (5, 50 and 200 mM phosphate buffer) and/or in five commonly used cell culture media (KHB, MCM+, DMEM+HEPES, Waymouth’s and William’s E+HEPES) in both Human liver microsomes and cryopreserved Human hepatocytes. NADPH-fortified pooled HLM (n = 200) at 0.1mg/mL were incubated for 5 min with phenacetin, coumarin, bupropion, amodiaquine, diclofenac (tolbutamide for CHH), S‑mephenytoin, dextromethorphan, chlorzoxazone, midazolam, nifedipine, alfentanil, verapamil, testosterone and atorvastatin at their approximate Km values. In the case of pooled CHH (n = 50), incubations were conducted at 1 million cells/mL for 10-60 min. Reactions were terminated with an equal volume of organic solvent containing internal standard, followed by protein precipitation and analysis by LC/MS/MS.
In Human liver microsomes, phenacetin (CYP1A2), coumarin (CYP2A6), bupropion (CYP2B6), amodiaquine (CYP2C8), diclofenac (CYP2C9), S-mephenytoin (CYP2C19), and dextromethorphan (CYP2D6) activities all were highest at 50 mM phosphate buffer. For chlorzoxazone (CYP2E1) and midazolam (CYP3A4/5), the enzymatic activities were highest at 200 mM phosphate buffer. When incubations in human liver microsomes were conducted with various media, MCM+ medium was found to support the highest activity of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19 and CYP2D6.