Fig. 4: Replacing NADPH with NADH as cofactor in HLM produces rates of metabolism that are more representative of HH. | Communications Biology

Fig. 4: Replacing NADPH with NADH as cofactor in HLM produces rates of metabolism that are more representative of HH.

From: A mismatch in enzyme-redox partnerships underlies divergent cytochrome P450 activities between human hepatocytes and microsomes

Fig. 4

A Comparison of scaled CLint,u values for 64 literature and AstraZeneca compounds determined in HH and in HLM using NADH or NADPH as cofactor. The fraction unbound used to calculate the scaled CLint,u was measured or estimated by in silico models of Fuinc76. Each point represents the scaled CLint,u for an individual compound from a single determination highlighting compounds with good permeability in Caco-2 cells Papp > 7 × 10−6 cm/s (blue dots), compounds with poor permeability in Caco-2 cells, Papp < 0.5 × 10−6 cm/s (red dots), permeability could not be determined for 11 compounds (black cross), the scaled CLint,u values for AZ compound (AZ15), savolitinib and the prototypic cytochrome P450 3A4 substrate midazolam all with good cell permeability (green dots), the major nonCYP3A4 substrates i.e. CYPs 1A2, 2C8, 2C9, 2C19 and 3A5 (orange crosses). The solid red line represents the line of unity and the dotted lines, the three-fold difference. Compounds with non-CYP metabolic pathways predominating in HH would be expected to lie in the region highlighted in yellow and those with metabolism-limiting permeability in white. B Structure of AstraZeneca compound AZ15. C A comparison of metabolite profiles (UV chromatograms and insert showing relative contributions of CYP versus non-CYP metabolism) after incubating 5 µM of AZ15 in HH and HLM with NADH or NADPH as cofactor for 1 h at 37 °C. The metabolites at retention times (4.53 and 3.58) are products of the cytosolic aldehyde oxidase (some cytosol ‘contaminating’ or carried over into microsomes during preparation by centrifugation yields the aldehyde oxidase metabolite in the NADH and NADPH profiles). Also shown as part of C are the structures of metabolites (products of oxidation by aldehyde oxidase and CYP, N-demethylation by CYP) that together, account for over 90% of metabolism in the respective matrices. D A comparison of the scaled CLint,u values between HLM with NADH and NADPH as cofactors. The solid black line represents the line of regression (\(y=0.86x-0.34\), r2 = 0.79, P < 0.0001).

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