P450 glo

The luminescence based P450-Glo™ (Promega Corp.) assay was used in 384-well assay format.
The cytochrome P450 (CYP) panel included microsomal preparations of cytochromes P450 1A2, 2C9, 2C19, 2D6, and 3A4 (Corning) from baculovirus infected insect cells (BTI-TN-5B1−4) which express cytochromes P450 and cytochrome c reductase (and cytochrome b5 for 3A4). Compounds were added (100 nL/well in 1% DMSO v/v) using the Echo 550® Liquid Handler followed by addition of 5 μL/well of CYP450/substrate mixture. Following incubation for 30 min at 37 °C, the reaction was initiated by the addition of 5 μL/well of the NADPH regeneration mixture. By the end of a further 30 min incubation (37 °C), the CYP450 reaction was stopped and the luciferase reaction was initiated by the addition of 10 μL/well of the luciferin detection reagent, followed by an additional 30 min of incubation at 37 °C. The luminescence signal was detected using an Infinite® M1000 PRO plate reader. The negative controls yielded 0% inhibition (1% v/v DMSO) and standard CYP450 specific inhibitors were used as positive controls with 100% inhibition (CYP450 1A2, alpha-naphthoflavone; CYP450 2C9, sulfaphenazole; CYP450 2C19, troglitazone; CYP450 2D6, quinidine; CYP450 3A4, ketoconazole). The raw data were normalised relative to the positive and negative controls yielding the % inhibition for each compound.

The inhibition of CYP (3A4, 1A2 and 2D6) and induction of CYP were measured using the luminescence-based P450-Glo (Promega Corp., USA) assay system. Briefly, sc1o in 11-point concentration-response format in triplicate (100 nl of 1 mM solution in 100% v/v DMSO), positive controls (3A4, ketoconazole; 1A2, α-naphthoflavone; 2D6, quinidine with a final concentration of 1 µM and 1% v/v DMSO) yielding 100% inhibition, and negative controls (100 nl of 100% v/v DMSO) yielding 0% inhibition were added to each well of a 384-well microtitre plate by using the Echo 550 liquid handler. This was followed by adding the CYP/substrates (5 µl/well) and was incubated for 30 min at 37 °C. Reactions were initiated by adding an NADPH regeneration system (5 μl/well). The reactions were stopped by adding luciferin detection reagent (10 μl/well), followed by an additional 30 min incubation at 37 °C with the luminescence signal detected using an Infinite M1000 PRO plate reader (Tecan, Männedorf, Switzerland).

CYP activity was assessed using the luciferin-based P450-Glo assays (Promega). Cells were washed with basal HCM for 15 min prior to incubation with CYP substrates at the following concentrations: 100 μM for 1A2, 100 μM for 2C9, 10 μM for 2C19, 30 μM for 2D6, 3 μM for 3A4, and 150 μM for 3A7. Cells were incubated with substrates for 3.5 h except for the 3A4 assay that was incubated for 70 min. At the end of the incubation period, 50 µL supernatant was transferred into a 96-well plate in technical triplicates, and 50 µL of CYP-specific detection reagent was added to each well. The plate was covered in foil and mixed gently for 20 min, and absorbance was measured at 450 nm on a plate reader (SpectraMax i3, Molecular Devices). Samples included a negative control consisting of HCM only (no cells) that was used for background correction.

Cells were treated for 24 hr with prototypical inducers 50 μM omeprazole (CYP1A2) or rifampicin (CYP3A4), with or without the addition of CYP450 isoform‐specific inhibitors 25 μM fluvoxamine (CYP1A2) or ketoconazole (CYP3A4), in MEME (C3As) or hepatocyte induction medium (HepaRGs). The cells were then washed twice with HBSS. Specific CYP450 enzyme activity was subsequently assessed using a non‐lytic luminescence assay using specific kits for CYP1A2 and CYP3A4, following the manufacturer's instructions (P450‐Glo, Promega, Southampton, UK). Bioluminescent signals were detected with a GloMax‐Multi+ Microplate Multimode Reader (Promega). Individual luminescent assay readings were background‐corrected and normalized to cellular ATP content.

Metabolic activity was measured in fresh and cryopreserved hepatocytes. Phase I metabolism was measured using commercially available luciferin assays, P450-Glo™ (Promega Corporation, Wisconsin, U.S.), for cytochrome P450 1A1, 1A2, 2B6, 2C9, 3A4, and 3A7 as described previously¹⁶
. Luminescence was read directly in a multi-well plate luminometer (CLARIOstar, BMG Labtech, North Carolina, U.S.) and expressed as luminescent counting units (LCU)/min normalized to a million viable cells. Phase II activity was determined by the metabolism of the fluorescent compound resorufin according to a published protocol¹³
. Resorufin fluorescent signals were quantified by a spectrophotometer (CLARIOstar BMG Labtech, North Carolina, U.S. excitation: 535 ± 25, emission: 581 ± 20). Conjugation efficiency was quantified by measuring the decrease in the fluorescent signal and reported as the percentage of resorufin metabolized.